Analyzing the 2024 WHO Priority Pathogen Lists: A Strategic Guide for AMR Research and Pandemic Preparedness

Camila Jenkins Nov 26, 2025 118

This analysis provides researchers, scientists, and drug development professionals with a comprehensive examination of the World Health Organization's updated 2024 pathogen lists.

Analyzing the 2024 WHO Priority Pathogen Lists: A Strategic Guide for AMR Research and Pandemic Preparedness

Abstract

This analysis provides researchers, scientists, and drug development professionals with a comprehensive examination of the World Health Organization's updated 2024 pathogen lists. It covers the revised Bacterial Priority Pathogens List (BPPL), which ranks 24 antibiotic-resistant bacteria, and the broader list of over 30 pathogens with pandemic potential. The article delves into the refined multi-criteria prioritization methodology, explores the critical threats posed by pathogens like carbapenem-resistant Klebsiella pneumoniae and Nipah virus, and compares the evolution of these lists from their 2017 counterparts. It further addresses the strategic shift towards a 'family approach' for R&D, the challenges in the antibacterial pipeline, and the integrated strategies—spanning novel drug development, infection prevention, and equitable access—required to mitigate these urgent public health threats.

Understanding the WHO Priority Pathogen Lists: Scope, Evolution, and Critical Threats

The World Health Organization (WHO) employs distinct pathogen prioritization lists as strategic tools to confront different facets of the global infectious disease threat landscape. Within the context of antimicrobial resistance (AMR) and epidemic preparedness research, two lists are of paramount importance: the Bacterial Priority Pathogens List (BPPL) and the list of priority diseases for pandemic and epidemic preparedness (often referred to as the Pandemic-Preparedness List). While they share the common goal of guiding research and development (R&D) to safeguard public health, their specific objectives, targets, and applications differ significantly [1] [2]. This whitepaper provides an in-depth technical analysis of these two critical lists, delineating their unique characteristics to inform the work of researchers, scientists, and drug development professionals. The BPPL focuses exclusively on the escalating crisis of antibiotic-resistant bacterial pathogens, guiding the development of new antibacterial agents and diagnostics [1] [3]. In contrast, the Pandemic-Preparedness List emphasizes viral pathogens with epidemic or pandemic potential, aiming to accelerate the development of vaccines, therapeutics, and non-pharmaceutical interventions before a crisis emerges [2] [4].

The Bacterial Priority Pathogens List (BPPL): A Framework for Combating Antimicrobial Resistance

Objective and Scope

The WHO BPPL is a critical instrument in the global fight against antimicrobial resistance (AMR). Its primary objective is to guide and prioritize R&D for new antibacterial medicines, diagnostics, and other strategies to control the spread of resistant bacterial infections [1] [5]. The list is inherently reactive and evolutionary, designed to address the ongoing and continuously evolving challenge of pathogens developing resistance to existing treatments. It focuses solely on bacterial pathogens, particularly those that have developed resistance to antibiotics, including last-resort treatments [1].

The 2024 BPPL is an update of the 2017 list and serves as a global public health tool to align research efforts and investments. It is targeted at a specific audience that includes developers of antibacterial medicines, academic and public research institutions, research funders, and public-private partnerships investing in AMR R&D [1].

Prioritization Methodology and Criteria

The development of the 2024 BPPL employed a rigorous, evidence-based, and transparent multi-criteria decision analysis (MCDA) framework. This methodology ensured that the prioritization was systematic and accounted for the multifaceted nature of the AMR threat [5].

Key Methodological Steps [5]:

  • Criteria Selection: Eight key criteria were defined to evaluate each pathogen:
    • Mortality
    • Non-fatal health burden (e.g., disability)
    • Incidence of resistant infections
    • 10-year trends of resistance
    • Preventability (ease of implementing effective infection prevention and control measures)
    • Transmissibility
    • Treatability (considering current treatment options and their effectiveness)
    • Status of the antibacterial R&D pipeline (number and innovativeness of agents in development)
  • Evidence Synthesis and Expert Judgement: For each of the 24 shortlisted pathogens, available evidence was gathered and scored against the eight criteria. Expert judgement was used to fill evidence gaps and ensure practical relevance.
  • Preference Weighting: An international survey of 78 AMR experts was conducted to determine the relative importance (weights) of each criterion through a pairwise comparison exercise. This step introduced a quantitative measure of value into the model.
  • Scoring and Ranking: A total score (0-100%) was calculated for each pathogen by combining its criterion-specific scores with the global preference weights.
  • Tiering and Validation: Pathogens were grouped into three priority tiers—critical, high, and medium—based on a quartile analysis of their total scores. The final list was reviewed by an independent advisory group to ensure robustness.

Table 1: Selected Pathogens from the 2024 WHO Bacterial Priority Pathogens List (BPPL)

Priority Tier Pathogen Key Resistance Traits Rationale for Prioritization
Critical Klebsiella pneumoniae Carbapenem-resistant Top-ranked pathogen; high burden in healthcare settings, limited treatment options [5].
Critical Acinetobacter baumannii Carbapenem-resistant Associated with high mortality in hospital-acquired infections; significant treatment challenges [5].
Critical Mycobacterium tuberculosis Rifampicin-resistant (and MDR-TB) Persistent global threat, complex and lengthy treatment regimens, significant public health burden [1] [5].
High Salmonella enterica Serotype Typhi Fluoroquinolone-resistant High burden in community settings, particularly in areas with inadequate sanitation [5].
High Neisseria gonorrhoeae Third-generation cephalosporin and/or fluoroquinolone-resistant Rising resistance threatens single-dose therapy; potential for untreatable infections [1] [5].
High Staphylococcus aureus Methicillin-resistant (MRSA) High prevalence in both healthcare and community settings, causing a wide range of infections [1] [5].

Impact and Strategic Application

The BPPL directly influences global policy and R&D investments in AMR. It highlights the stark reality of the antibacterial pipeline, which in 2025 contained only 90 agents in clinical development, with just 15 classified as innovative and only 5 effective against at least one "critical" priority pathogen [3]. This list is used to:

  • Steer funding for fundamental research and drug discovery toward the most urgent threats.
  • Inform regulatory pathways and incentive models for developing products targeting BPPL pathogens.
  • Guide the development of essential diagnostics to rapidly identify resistant infections and inform treatment decisions, addressing critical gaps in low-resource settings [3].

G Figure 1: WHO BPPL 2024 Development Workflow cluster_0 Methodology Inputs cluster_1 Multi-Criteria Decision Analysis (MCDA) Process cluster_2 Output: Priority Tiers A 24 Bacterial Pathogens Shortlisted D 1. Evidence Synthesis & Pathogen Scoring A->D B 8 Prioritization Criteria B->D C Global Expert Survey (78 respondents) E 2. Preference Weighting (Criteria Weights) C->E F 3. Total Score Calculation (0-100%) D->F E->F G Critical Priority (Highest Quartile) F->G H High Priority (Middle Quartiles) F->H I Medium Priority (Lowest Quartile) F->I

The Pandemic-Preparedness List: A Strategy for Epidemic and Pandemic Readiness

Objective and Scope

The WHO's R&D Blueprint for Action to Prevent Epidemics prioritizes diseases for research and development in emergency contexts [2]. The core objective of this list is to focus limited R&D resources on diseases and pathogens that pose the greatest public health risk due to their epidemic or pandemic potential and/or the lack of sufficient medical countermeasures (e.g., vaccines, therapeutics). The list is inherently proactive and preparatory, aiming to build tools and capabilities before a major outbreak occurs [2] [4].

Unlike the BPPL, this list focuses primarily on viruses (and one infectious agent of unknown origin, "Disease X") known to cause severe outbreaks with high mortality or rapid spread. It is a tool for the global health community, including vaccine developers, therapeutic researchers, and public health emergency planners, to ensure cross-cutting R&D preparedness [2].

Prioritization Methodology and Criteria

The methodology for the Pandemic-Preparedness List differs from the highly quantitative MCDA approach of the BPPL. It relies on a qualitative expert tool to identify diseases with the highest public health risk profile.

Key Methodological Principles [2] [6]:

  • Epidemic/Pandemic Potential: The primary driver is the assessed capacity of a pathogen to cause explosive, large-scale outbreaks, often facilitated by respiratory transmission or other efficient transmission routes.
  • Countermeasure Gaps: Diseases are prioritized where no or insufficient vaccines, therapeutics, or diagnostics exist, leaving populations vulnerable.
  • A "Family" Approach: The most recent updates (July 2024) have shifted focus from individual pathogens to broader virus families. This acknowledges that the next major threat may come from an unknown member of a high-risk family (e.g., Filoviridae, Coronaviridae) [6].
  • Incorporation of "Disease X": The list explicitly includes "Disease X," representing the knowledge that a serious international epidemic could be caused by a pathogen currently unknown to cause human disease. This forces preparedness for unknown threats [2].

Table 2: WHO Priority Diseases for Research & Development in Emergency Contexts (Pandemic-Preparedness List)

Disease Pathogen (Family) Transmission Key Characteristics & Rationale
COVID-19 SARS-CoV-2 (Coronaviridae) Respiratory Demonstrates pandemic potential of coronaviruses; ongoing evolution requires updated countermeasures [2].
"Disease X" Unknown (potentially from any high-risk family) Unknown Represents an unknown pathogen with epidemic potential; emphasizes need for platform technologies & flexible preparedness [2].
Ebola Virus Disease Ebola virus (Filoviridae) Contact with bodily fluids High case fatality rate; history of large outbreaks in West Africa and DRC; highlights threat from filoviruses [2] [6].
Marburg Virus Disease Marburg virus (Filoviridae) Contact with bodily fluids Similar high fatality rate to Ebola; underscores threat from the broader Filoviridae family [2] [6].
Nipah virus infection Nipah virus (Paramyxoviridae) Zoonotic/Respiratory High mortality; capacity for human-to-human transmission; identified as a pathogen of concern with high pandemic potential [2] [7] [8].
Crimean-Congo haemorrhagic fever CCHF virus (Nairoviridae) Tick-borne/Contact Emerging geographic spread; nosocomial transmission; high fatality rate [2].

Impact and Strategic Application

This list drives preparedness by focusing R&D on platform technologies (e.g., mRNA platforms, viral vectors) that can be rapidly adapted for new pathogens within a virus family [4]. It underpins the mission of organizations like the Coalition for Epidemic Preparedness Innovations (CEPI), which funds vaccine development against these priority pathogens and for "Disease X" [9]. Its strategic applications include:

  • Guiding fundamental virology and immunology research on high-risk virus families.
  • Informing the development of prototype vaccines and reagents that can be quickly modified.
  • Stimulating the creation of clinical trial networks (e.g., the VACCELERATE network) that can be activated immediately at the onset of an outbreak [7].
  • Supporting the PRET (Preparedness and Resilience for Emerging Threats) Initiative by highlighting pathogen families for which surveillance and response planning should be enhanced [6].

Comparative Analysis: BPPL vs. Pandemic-Preparedness List

Table 3: Comparative Analysis of WHO Pathogen Priority Lists

Feature Bacterial Priority Pathogens List (BPPL) Pandemic-Preparedness List
Primary Objective Combat antimicrobial resistance by guiding development of new antibiotics and diagnostics [1] [3]. Prepare for and enable rapid response to viral epidemics and pandemics [2] [4].
Pathogen Type Bacteria (antibiotic-resistant) [1]. Primarily Viruses (and "Disease X") [2].
Temporal Focus Present/Continuous threat (endemic AMR). Future/Emerging threat (epidemic/pandemic potential) [2] [4].
Core Driver Drug Resistance & Treatment Failure [5]. Epidemic Potential & Lack of Countermeasures [2].
Key Audiences Antibacterial drug developers, diagnostic firms, AMR researchers [1]. Vaccine developers, public health emergency planners, virologists [2] [9].
Prioritization Method Quantitative Multi-Criteria Decision Analysis (MCDA) with expert weighting [5]. Qualitative expert assessment of epidemic risk and countermeasure gaps [2] [6].
"Disease X" Concept Not applicable. Central component, representing an unknown pathogen [2].
Example R&D Output New small-molecule antibiotics; rapid antimicrobial susceptibility tests [3]. Broadly protective coronavirus vaccines; rapid-response mRNA platform technologies [9] [4].

G Figure 2: Logical Framework for WHO Pathogen Prioritization cluster_0 Key Decision Factor cluster_1 Key Decision Factor Start Global Infectious Disease Threat A Is the primary concern Antimicrobial Resistance (AMR)? Start->A B WHO Bacterial Priority Pathogens List (BPPL) A->B Yes D Is the primary concern Epidemic/Pandemic Potential? A->D No C Focus: Antibiotic-Resistant Bacteria Goal: Guide antibacterial R&D Method: Quantitative MCDA B->C E WHO Pandemic-Preparedness List (R&D Blueprint) D->E Yes F Focus: Viruses & 'Disease X' Goal: Enable rapid outbreak response Method: Qualitative expert assessment E->F

The Scientist's Toolkit: Essential Reagents and Materials for Priority Pathogen Research

Research into the pathogens highlighted by these WHO lists requires specialized reagents and tools. The following table details key solutions for both bacterial and viral priority pathogens.

Table 4: Key Research Reagent Solutions for Priority Pathogen R&D

Reagent / Material Category Function & Application Example Use Cases
Reference Genomic DNA & Strain Panels Serves as gold-standard controls for assay development (e.g., PCR, sequencing) and antimicrobial susceptibility testing (AST) [3]. BPPL: Characterizing resistance mechanisms (e.g., carbapenemase genes in K. pneumoniae). Pandemic-Preparedness: Confirming detection of novel coronaviruses or filoviruses.
Monoclonal & Polyclonal Antibodies Essential for immunoassays (ELISA, lateral flow), serosurveillance, therapeutic development, and as positive controls for in vitro diagnostics (IVDs) [3]. BPPL: Detecting specific bacterial antigens (e.g., S. aureus Protein A). Pandemic-Preparedness: Developing serological tests for Nipah virus or Lassa fever virus.
Recombinant Viral Antigens & Proteins Enable serological assay development (to detect host antibodies) and vaccine immunogenicity testing without handling live virus [9]. Pandemic-Preparedness: ELISA development for MERS-CoV spike protein; screening vaccine candidates for Ebola virus glycoprotein.
CRISPR-Based Assay Components Provide highly sensitive and specific nucleic acid detection for point-of-care (POC) diagnostics; can be designed for specific resistance mutations or viral sequences [3]. BPPL: Rapid detection of MRSA (mecA gene). Pandemic-Preparedness: Developing field-deployable tests for "Disease X" once sequenced.
Cell Culture Models (incl. 3D/Organoids) Mimic human physiology for pathogenicity studies, antiviral/antibacterial screening, and vaccine response evaluation [4]. BPPL: Studying host-pathogen interactions of M. tuberculosis. Pandemic-Preparedness: Investigating the tropism and cytopathic effects of Nipah virus.
Vaccine Platform Technologies (e.g., mRNA, VLP) Allow for rapid development of vaccine candidates against known or novel ("Disease X") pathogens by swapping genetic sequences [9] [4]. Pandemic-Preparedness: Rapid development of COVID-19 mRNA vaccines; CEPI's strategy for a "just-in-time" response to future outbreaks.
Bicyclo[2.2.2]octane-2-carbonitrileBicyclo[2.2.2]octane-2-carbonitrile, CAS:6962-74-9, MF:C9H13N, MW:135.21 g/molChemical Reagent
DidsDids, CAS:152216-76-7, MF:C16H10N2O6S4, MW:454.5 g/molChemical Reagent

The WHO's Bacterial Priority Pathogens List and the Pandemic-Preparedness List are complementary but fundamentally distinct tools in the global health security arsenal. The BPPL addresses the persistent, simmering crisis of AMR by directing efforts toward overcoming bacterial treatment failures. In contrast, the Pandemic-Preparedness List prepares for the acute, explosive threat of viral epidemics by fostering the development of platform technologies and countermeasures for known high-risk virus families and the inevitable "Disease X." For researchers and drug developers, a clear understanding of the objectives, methodologies, and target pathogens of each list is crucial for aligning R&D strategies with the most pressing public health needs. Success in mitigating these dual threats hinges on sustained investment, international collaboration, and the intelligent application of these prioritization frameworks to guide scientific innovation from the bench to the frontline.

The World Health Organization's (WHO) Bacterial Priority Pathogens List (BPPL) serves as a critical tool in the global fight against antimicrobial resistance (AMR). The 2024 BPPL update, building on the 2017 edition, responds to the evolving challenges of antibiotic resistance by refining the prioritization of antibiotic-resistant bacterial pathogens to guide research and development (R&D) and public health interventions [1]. This update arrives at a crucial time—AMR was directly responsible for 1.27 million global deaths in 2019 and contributed to 4.95 million deaths, establishing itself as one of the top global public health threats [10]. The BPPL's prioritization directly influences global policy, funding allocations, and research agendas, making its methodology and updates of paramount importance to researchers, scientists, and drug development professionals working at the forefront of this silent pandemic [5].

Methodological Framework of the 2024 BPPL

The 2024 WHO BPPL employed a sophisticated, evidence-based methodology to ensure a systematic and transparent prioritization process.

Multicriteria Decision Analysis Framework

The list development utilized a multicriteria decision analysis (MCDA) framework, similar to the 2017 approach but enhanced with newer data and evidence [5]. This rigorous methodology scored 24 antibiotic-resistant bacterial pathogens against eight predefined criteria:

  • Mortality: The direct fatality burden caused by the resistant pathogen
  • Non-fatal burden: Morbidity and disability impacts
  • Incidence: Frequency of new infections
  • 10-year resistance trends: Historical trajectory of resistance development
  • Preventability: Potential for prevention through vaccines, hygiene, or other measures
  • Transmissibility: Capacity for spread within communities and healthcare settings
  • Treatability: Current availability of effective therapeutic options
  • Antibacterial pipeline status: Number and innovativeness of agents in development [5]

Expert Evaluation and Weighting

A preferences survey using pairwise comparison was administered to 100 international experts, with 78 completing the survey to determine the relative weights of the criteria [5]. The survey demonstrated strong inter-rater agreement (Spearman's rank correlation coefficient and Kendall's coefficient of concordance both at 0.9), indicating robust expert consensus on priority-setting [5]. The final ranking was determined by calculating a total score from 0-100% for each pathogen, with subgroup and sensitivity analyses confirming high stability across experts' backgrounds and geographical origins [5].

Evidence Base and Data Integration

The 2024 revision relied on 92 systematic reviews covering literature from 2017-2022 to assess pathogen risk [11]. An independent advisory group reviewed the final list, and pathogens were subsequently streamlined and grouped into three priority tiers based on a quartile scoring system: critical (highest quartile), high (middle quartiles), and medium (lowest quartile) [5].

BPPL_Methodology Start Identify 24 Antibiotic-Resistant Bacterial Pathogens Criteria Score Against 8 Criteria: • Mortality • Non-fatal burden • Incidence • 10-year resistance trends • Preventability • Transmissibility • Treatability • Antibacterial pipeline Start->Criteria Expert Expert Preference Survey (78 international experts) Determine criteria weights Criteria->Expert Scoring Calculate Total Score (0-100%) for each pathogen Expert->Scoring Analysis Subgroup & Sensitivity Analysis Verify ranking stability Scoring->Analysis Tiering Group into Priority Tiers: Critical (Highest Quartile) High (Middle Quartiles) Medium (Lowest Quartile) Analysis->Tiering Final Final 2024 BPPL Tiering->Final

Figure 1: The 2024 WHO BPPL Development Workflow. This diagram illustrates the systematic, multi-stage methodology used to develop the 2024 Bacterial Priority Pathogens List, from initial pathogen identification through final priority tier assignment.

Key Changes in the 2024 BPPL

Priority Tiers and Pathogen Ranking

The 2024 BPPL categorizes pathogens into three priority tiers, with Gram-negative bacteria dominating the critical priority category alongside newly included rifampicin-resistant Mycobacterium tuberculosis.

Table 1: 2024 WHO Bacterial Priority Pathogens List - Priority Tiers and Key Pathogens

Priority Tier Pathogens Key Changes from 2017 List
Critical Priority Carbapenem-resistant Klebsiella pneumoniaeRifampicin-resistant Mycobacterium tuberculosisCarbapenem-resistant Acinetobacter baumanniiThird-generation cephalosporin-resistant & Carbapenem-resistant Escherichia coli Inclusion of rifampicin-resistant M. tuberculosis as a new addition
High Priority Fluoroquinolone-resistant Salmonella enterica serotype TyphiThird-generation cephalosporin-resistant & Fluoroquinolone-resistant Neisseria gonorrhoeaeMethicillin-resistant Staphylococcus aureus (MRSA)Clindamycin-resistant & Macrolide-resistant Streptococcus pneumoniaeFluoroquinolone-resistant Shigella spp. S. pneumoniae retained despite controversy
Medium Priority Penicillin-non-susceptible Group A streptococcusAmpicillin-resistant & Fluoroquinolone-resistant Haemophilus influenzaeFluoroquinolone-resistant & Macrolide-resistant Campylobacter spp.Clarithromycin-resistant Helicobacter pyloriFluconazole-resistant Candida albicans Campylobacter and H. pylori downgraded from 2017 list

The pathogens' total scores ranged from 84% for carbapenem-resistant Klebsiella pneumoniae (top-ranked) to 28% for penicillin-resistant group B streptococci (bottom-ranked) [12]. Among bacteria commonly responsible for community-acquired infections, the highest rankings were for fluoroquinolone-resistant Salmonella enterica serotype Typhi (72%), Shigella spp. (70%), and Neisseria gonorrhoeae (64%) [5].

Additions and Removals: Controversies and Rationale

Significant Additions

The most notable addition to the 2024 BPPL is rifampicin-resistant Mycobacterium tuberculosis in the critical priority category [13]. This inclusion reflects the significant challenges RR-TB poses in diagnosis, treatment, clinical management, and public health response beyond those of drug-susceptible TB [13]. The update also added three other new families of antibiotic-resistant bacterial pathogens to reflect the evolving AMR landscape [13].

Controversial Removals and Downgrading

The 2024 update removed five clinically significant pathogens that were included in the 2017 list, generating concern within the scientific community [11]. These include:

  • Fluoroquinolone-resistant Campylobacter jejuni
  • Clarithromycin-resistant Helicobacter pylori
  • Penicillin-non-susceptible Streptococcus pneumoniae
  • Third-generation cephalosporin-resistant Providencia spp.
  • Vancomycin-intermediate/resistant Staphylococcus aureus [11]

An open letter from researchers highlighted that the report offered little explanation for these removals, noting only that decisions were based on evidence and expert consensus, despite the significant public health impact of these pathogens [11]. This lack of transparency is particularly concerning given that fluoroquinolone resistance in Campylobacter continues to rise—from 5% in 1998 to 45% in 2018 in the UK, with recent reports of 48% and 90% resistance in Chile and Peru, respectively [11].

Similarly, clarithromycin-resistant H. pylori exhibits resistance prevalence of 30-50% in South America, the Middle East, and China, with rising trends in Australia and Europe [11]. Given H. pylori's role in gastric cancer (a leading cause of cancer deaths in low- and middle-income countries), its downgrading appears difficult to justify from a public health perspective [11].

The methodology has been criticized for potentially underestimating the burden of pathogens like Campylobacter due to limited surveillance data in resource-limited settings, where diagnostics are often unavailable [11]. Rather than justifying de-prioritization, researchers argue that data scarcity should prompt better monitoring, not removal from priority lists [11].

The AMR Crisis: Burden and Impact

Global Health and Economic Burden

Antimicrobial resistance represents a growing serious threat to global health security, with the prevalence of antimicrobial-resistant bacteria attaining incongruous levels worldwide [14]. The comprehensive burden of AMR includes:

Table 2: Global Burden of Antimicrobial Resistance - Health and Economic Impact

Category Statistics Source
Mortality Burden 1.27 million deaths directly attributable to AMR in 20194.95 million deaths associated with AMR in 2019 [10]
Projected Mortality 10 million annual deaths expected by 2050 without intervention [14]
Regional Burden Inequalities significant, with sub-Saharan Africa and South Asia most affected [15]
U.S.-Specific Data 2.8 million antimicrobial-resistant infections annually35,000+ resulting deaths48,000+ deaths when including C. difficile [16]
Economic Impact $1 trillion additional healthcare costs by 2050 (World Bank)$1-3.4 trillion GDP losses per year by 2030>$4.6 billion annual U.S. treatment costs for six resistant pathogens [10] [16]

Resistance Mechanisms and Historical Context

The AMR crisis has deep historical roots, with resistance emerging relatively quickly after the introduction of most antibiotics. The period from the 1940s to the 1960s is regarded as the "Golden Age" of antibiotic discovery, but since the 1980s, there has been a dramatic decrease in the speed of discovery [14]. This has created a dangerous imbalance between drug-resistant pathogens and available treatments.

Bacteria develop resistance through multiple mechanisms, including:

  • Genetic mutations that occur naturally under selective pressure
  • Enzymatic inactivation of antibiotics (e.g., β-lactamases)
  • Efflux pumps that remove antibiotics from bacterial cells
  • Target site modifications that prevent antibiotic binding [14]

The transmission and acquisition of AMR occur primarily via a human-human interface both within and outside healthcare facilities, with multiple interdependent factors related to healthcare and agriculture governing development through various drug-resistance mechanisms [14].

Research and Development Landscape

The Antibacterial Pipeline Crisis

The WHO's 2025 analysis of the antibacterial pipeline reveals a system in crisis, with the number of antibacterial agents in clinical development decreasing from 97 in 2023 to 90 in 2025 [3]. This declining pipeline faces a dual crisis: scarcity and lack of innovation.

Of the 90 antibacterials currently in development:

  • Only 15 qualify as innovative
  • For 10 of these, available data are insufficient to confirm the absence of cross-resistance
  • Only 5 are effective against at least one of the WHO "critical" priority bacteria [3]

The pipeline remains heavily focused on Gram-negative bacteria, where innovation is most urgently needed, with the preclinical pipeline showing 232 programs across 148 groups worldwide [3]. However, 90% of companies involved are small firms with fewer than 50 employees, highlighting the fragility of the R&D ecosystem [3].

Since July 2017, only 17 new antibacterial agents against priority bacterial pathogens have obtained marketing authorization, with just two representing a new chemical class [3]. Significant gaps persist in pediatric formulations, oral treatments for outpatient use, and solutions to address escalating resistance.

Diagnostic Gaps and Needs

Diagnostics are equally critical to AMR control, especially in low- and middle-income countries. The WHO's landscape analysis identified persistent diagnostic gaps, including:

  • Absence of multiplex platforms suitable for use in intermediate referral laboratories to identify bloodstream infections directly from whole blood without culture
  • Insufficient access to biomarker tests (C-reactive protein, procalcitonin) to distinguish bacterial from viral infections
  • Limited simple, point-of-care diagnostic tools for primary and secondary care facilities [3]

These limitations disproportionately affect patients in low-resource settings, where most people first present at primary health-care facilities [3].

AMR_Response AMR_Threat AMR Crisis Prevention Infection Prevention • Vaccines • Clean water & sanitation • IPC measures AMR_Threat->Prevention Stewardship Antimicrobial Stewardship • Appropriate prescribing • AWaRE guidelines • Surveillance AMR_Threat->Stewardship RDD_Innovation R&D & Diagnostic Innovation • Novel antibacterial agents • Rapid diagnostics • Equity-focused tools AMR_Threat->RDD_Innovation Policy Policy & Investment • One Health approach • Market incentives • Global collaboration AMR_Threat->Policy Impact Reduced AMR Impact • Mortality • Morbidity • Economic costs Prevention->Impact Reduces infection rate Stewardship->Impact Preserves efficacy RDD_Innovation->Impact Provides new tools Policy->Prevention Enables & funds Policy->Stewardship Enables & funds Policy->RDD_Innovation Enables & funds

Figure 2: Comprehensive AMR Crisis Response Framework. This diagram outlines the multi-faceted approach required to address the antimicrobial resistance crisis, spanning prevention, stewardship, innovation, and policy domains.

Research Applications and Implementation

The Scientist's Toolkit: Essential Research Reagents and Platforms

Research into priority pathogens requires specialized reagents, platforms, and methodologies. The following toolkit outlines critical resources for AMR research and development.

Table 3: Essential Research Reagents and Platforms for AMR Priority Pathogen Research

Category Specific Tools/Platforms Research Applications
Genomic Surveillance Whole Genome Sequencing (WGS)PulseNet SystemMolecular typing reagents Track resistance emergence and spreadOutbreak investigationResistance mechanism identification [16]
Susceptibility Testing Antimicrobial susceptibility testing (AST)MIC determination panelsGradient diffusion strips Phenotypic resistance profilingAntibiotic efficacy assessmentResistance breakpoint establishment [3]
Diagnostic Development Multiplex PCR platformsBiomarker assays (CRP, procalcitonin)Lateral flow immunoassays Rapid pathogen detectionBiomarker quantificationPoint-of-care test development [3]
Animal Models Murine infection modelsGalleria mellonella modelsIn vivo imaging systems Therapeutic efficacy evaluationInfection pathogenesis studiesTreatment regimen optimization
Compound Screening High-throughput screening assaysBacterial cell-based assaysTarget-based inhibition assays Novel compound identificationMechanism of action studiesStructure-activity relationship analysis
Bta-188Bta-188, CAS:314062-80-1, MF:C21H28N4O2, MW:368.5 g/molChemical Reagent
(RS)-Butyryltimolol(RS)-Butyryltimolol, MF:C17H30N4O4S, MW:386.5 g/molChemical Reagent

Experimental Considerations for Priority Pathogens

Research on critical priority pathogens requires specialized methodological considerations:

  • Containment facilities: BSL-2 or BSL-3 laboratories for resistant pathogen work
  • Standardized protocols: CLSI or EUCAST guidelines for susceptibility testing
  • Quality control strains: Reference strains for assay validation and quality assurance
  • Resistance induction studies: In vitro passage experiments to predict resistance development
  • Combination therapy assessments: Checkerboard assays and time-kill studies for synergy evaluation

The 2024 WHO BPPL update represents a significant evolution in the global strategy to combat antimicrobial resistance, refining pathogen prioritization based on current evidence and expert consensus. The list underscores the persistent threat of Gram-negative bacteria and elevates rifampicin-resistant Mycobacterium tuberculosis to critical priority, reflecting its substantial global impact [1] [13] [5].

However, the controversial removal of several pathogens, including fluoroquinolone-resistant Campylobacter jejuni and clarithromycin-resistant Helicobacter pylori, highlights the need for greater transparency in WHO's methodology and criteria [11]. These removals risk halting research momentum and reducing investment in diagnostics, treatment, and surveillance for these still-relevant pathogens [11].

The AMR crisis continues to escalate, with the antibacterial pipeline proving insufficient to address the threat [3]. The decline in traditional antibiotic development, combined with inadequate innovation and fragile R&D ecosystems dominated by small firms, creates substantial vulnerabilities in our global defense system [3]. Furthermore, critical diagnostic gaps persist, particularly for resource-limited settings where the burden of AMR is highest [3].

Moving forward, coordinated global action incorporating One Health approaches—integrating human, animal, and environmental health—is essential [10]. Strategic priorities must include enhanced surveillance, strengthened infection prevention and control, optimized antimicrobial use, and increased investment across the R&D pipeline [15]. Without urgent, collaborative intervention across sectors and borders, the post-antibiotic era may transition from apocalyptic fantasy to devastating reality [14].

The World Health Organization (WHO) has classified carbapenem-resistant Gram-negative bacteria as among the most dangerous pathogens confronting global health, designating them as Critical Priority in its 2024 Bacterial Priority Pathogens List (BPPL). This classification highlights organisms for which new antibiotics and therapeutic strategies are urgently needed due to their significant global burden, ability to resist treatment, and capacity to spread resistance to other bacteria [17]. Among these critical threats, carbapenem-resistant Klebsiella pneumoniae (CRKP) stands out for its rapid dissemination and devastating impact on patient outcomes, particularly in healthcare settings.

The escalating crisis of antimicrobial resistance (AMR) represents a fundamental challenge to modern medicine. In 2019 alone, bacterial AMR was directly responsible for approximately 1.27 million deaths globally, with projections suggesting this number could reach 10 million annually by 2050 without effective intervention [18]. Gram-negative bacteria, with their intrinsic and acquired resistance mechanisms, contribute significantly to this burden, and their resistance to carbapenems – often considered last-line antibiotics – heralds a potential return to the pre-antibiotic era for some infections.

Global Epidemiology and Burden

The Scope of Carbapenem Resistance

The WHO's 2024 report reveals alarming resistance rates among key Gram-negative pathogens. Surveillance data indicates that more than 55% of Klebsiella pneumoniae isolates globally are now resistant to third-generation cephalosporins, first-line antibiotics for serious infections. Even more concerning is the rising resistance to carbapenems, which was previously rare but is becoming increasingly frequent, severely narrowing treatment options [19]. This resistance is not uniformly distributed, with the highest burdens reported in the WHO South-East Asian and Eastern Mediterranean Regions, where approximately one in three reported bacterial infections demonstrate antibiotic resistance [19].

In Europe, carbapenem-resistant Klebsiella pneumoniae has become particularly established in Southern European countries. A comprehensive study collecting 687 carbapenem-resistant strains from 41 hospitals across nine Southern European countries (2016-2018) identified 11 major clonal lineages circulating in the region [20]. The same study found that blaKPC-like was the most prevalent carbapenemase-encoding gene (46%), followed by blaOXA-48-like (39%) [20].

Mortality and Clinical Impact

Infections caused by carbapenem-resistant Enterobacterales (CRE) are associated with mortality rates ranging from 18% to 48%, significantly higher than infections caused by susceptible strains [21]. This increased mortality is multifactorial, resulting from delayed initiation of effective therapy, pharmacokinetic limitations of available antibiotics, and the frequently critical condition of patients who develop these infections. A study focusing on Korea reported a 30-day mortality rate of 38% for patients with KPC-producing K. pneumoniae or E. coli bacteremia, with inappropriate antibiotic use and APACHE II scores significantly influencing outcomes [22].

Table 1: Critical Priority Carbapenem-Resistant Pathogens (WHO BPPL 2024)

Pathogen Resistance Profile Key Global Concerns
Acinetobacter baumannii Carbapenem-resistant Major global threat; high burden; ability to resist treatment and spread resistance
Enterobacterales Third-generation cephalosporin-resistant High prevalence in low- and middle-income countries; spreading resistance genes
Enterobacterales Carbapenem-resistant Limited treatment options; associated with significant mortality
Mycobacterium tuberculosis Rifampicin-resistant Included after independent analysis with tailored criteria

Table 2: Regional Distribution of Major CRKP Clones in Southern Europe (2016-2018)

Country Dominant Clonal Lineage Predominant Carbapenemase Gene
Greece ST258/512, ST11 blaKPC-like, blaNDM
Italy ST258/512 blaKPC-like
Spain ST258, ST11, ST15, ST147 blaKPC-like, blaOXA-48-like
Serbia ST101 blaOXA-48-like
Türkiye ST14 blaOXA-48-like

Mechanisms of Resistance: A Multifaceted Threat

Carbapenem resistance in Gram-negative bacteria arises through diverse and often overlapping mechanisms that can be broadly categorized into enzymatic and non-enzymatic pathways.

Enzymatic Resistance: Carbapenemase Production

The production of carbapenemases – enzymes that hydrolyze carbapenem antibiotics – represents the most significant mechanism of resistance. These enzymes are typically encoded by genes located on mobile genetic elements, facilitating rapid horizontal transfer between bacterial strains and species [23]. The Ambler classification system categorizes these critically important enzymes into several classes:

Class A (Serine β-lactamases)

  • KPC (Klebsiella pneumoniae carbapenemase): Initially identified in the United States, KPC has become a globally disseminated enzyme, particularly associated with the high-risk clone ST258 in K. pneumoniae [24] [20]. KPC enzymes hydrolyze penicillins, cephalosporins, and carbapenems, with variable inhibition by available β-lactamase inhibitors [21].

Class B (Metallo-β-lactamases, MBLs)

  • NDM (New Delhi metallo-β-lactamase), VIM (Verona integron-encoded metallo-β-lactamase), IMP (Imipenemase): These zinc-dependent enzymes hydrolyze a broad spectrum of β-lactams including carbapenems, but not aztreonam. They are not inhibited by conventional β-lactamase inhibitors like avibactam or vaborbactam, posing significant treatment challenges [21] [22].

Class D (OXA-type enzymes)

  • OXA-48-like: These enzymes hydrolyze penicillins and carbapenems at low levels but have weak activity against extended-spectrum cephalosporins. Their detection can be challenging, contributing to their silent spread [21]. OXA-48 is often associated with a promiscuous plasmid, explaining its wide dispersal across various genetic lineages [20].

CarbapenemResistance CRGNB Carbapenem-Resistant Gram-Negative Bacteria Enzymatic Enzymatic Resistance (Carbapenemase Production) CRGNB->Enzymatic NonEnzymatic Non-Enzymatic Resistance CRGNB->NonEnzymatic ClassA Class A Carbapenemases (Serine-based) • KPC Enzymatic->ClassA ClassB Class B Carbapenemases (Metallo-β-lactamases) • NDM, VIM, IMP Enzymatic->ClassB ClassD Class D Carbapenemases (OXA-type) • OXA-48-like Enzymatic->ClassD Porin Porin Mutations Reduced antibiotic entry NonEnzymatic->Porin Efflux Efflux Pump Overexpression Enhanced antibiotic removal NonEnzymatic->Efflux Target Target Modification Altered penicillin-binding proteins NonEnzymatic->Target

Diagram 1: Mechanisms of carbapenem resistance in Gram-negative bacteria

Non-Enzymatic Resistance Mechanisms

Beyond carbapenemase production, bacteria employ additional strategies to evade carbapenem activity:

  • Reduced Membrane Permeability: Modifications to outer membrane porins (e.g., OmpK35/36 in K. pneumoniae) limit the intracellular accumulation of antibiotics [25]. For instance, P. aeruginosa is intrinsically resistant to many antibiotics due to its reduced expression of high-permeability porins [25].

  • Efflux Pump Overexpression: Activation of multidrug efflux systems (e.g., AcrAB-TolC in E. coli, MexAB-OprM in P. aeruginosa) actively exports antibiotics from the cell, reducing intracellular concentrations to subtherapeutic levels [25] [23]. These pumps are often regulated by complex genetic systems that can be induced by antibiotic exposure.

  • Target Modification: Alterations in penicillin-binding proteins (PBPs), the molecular targets of β-lactams, through genetic mutations or post-translational modifications can reduce antibiotic binding affinity and confer resistance [25].

Experimental Methodologies for CRKP Research

Genomic Surveillance and Molecular Epidemiology

Tracking the emergence and spread of CRKP requires sophisticated genomic approaches:

Core Genome Multilocus Sequence Typing (cgMLST)

  • Purpose: High-resolution typing of CRKP isolates for outbreak investigation and global surveillance.
  • Methodology: Sequencing and analysis of hundreds to thousands of core genes present in ≥95% of K. pneumoniae isolates. The pan-genome of K. pneumoniae comprises approximately five to six Mbp, encoding five to six thousand genes, with about 1,700 recognized as core genes [24].
  • Application: Identification of high-risk clones (e.g., ST258, ST11, ST101, ST307) and tracking of their international spread [20]. This approach has revealed that CG258 (clonal complex 258) is the predominant global CRKP strain with 43 sequence type members [24].

Capsule Typing (K-typing)

  • Purpose: Determination of capsular polysaccharide type, a key virulence factor.
  • Methodology: Traditionally performed through serological methods, now largely replaced by wzi gene sequencing or whole genome sequencing-based analysis [24].
  • Significance: Hypervirulent strains often express specific capsule types (e.g., K1, K2) associated with increased virulence.

Carbapenemase Detection Methods

Rapid and accurate identification of carbapenemase production is essential for infection control and treatment guidance:

Phenotypic Methods

  • Modified Carbapenem Inactivation Method (mCIM): A culture-based test that detects carbapenemase activity.
  • Immunochromatographic Assays: Rapid tests (e.g., NG-Test CARBA-5) that detect the five major carbapenemase families (KPC, NDM, VIM, IMP, OXA-48-like) from pure colonies in approximately 15 minutes [22]. These tests provide rapid results without requiring molecular infrastructure.

Molecular Methods

  • PCR and Sequencing: The gold standard for precise identification of carbapenemase genes, allowing for detection of variants. When in-house testing is unavailable, samples can be sent to reference laboratories, though turnaround times may be longer [22].
  • Real-time PCR (e.g., Xpert Carba-R): Automated systems that provide rapid (∼1 hour) detection of common carbapenemase genes (KPC, NDM, VIM, IMP, OXA-48) directly from clinical specimens or bacterial colonies [22].

ExperimentalWorkflow cluster_1 Carbapenemase Detection cluster_2 Advanced Characterization Start Clinical Isolate Collection Culture Culture and Identification Start->Culture AST Antimicrobial Susceptibility Testing Culture->AST Screening Carbapenem Resistance Screening AST->Screening Phenotypic Phenotypic Methods • mCIM • Immunochromatography Screening->Phenotypic Molecular Molecular Methods • PCR/Sequencing • Real-time PCR Screening->Molecular Genotyping Genotype Result Phenotypic->Genotyping Molecular->Genotyping WGS Whole Genome Sequencing Genotyping->WGS Analysis Bioinformatic Analysis • cgMLST • Virulence Genes • Resistance Genes WGS->Analysis Surveillance Molecular Epidemiology & Surveillance Analysis->Surveillance

Diagram 2: Experimental workflow for CRKP characterization

Table 3: Research Reagent Solutions for CRKP Investigation

Reagent/Kit Application Function Key Features
Xpert Carba-R Molecular detection Identifies KPC, NDM, VIM, IMP, OXA-48 genes Automated, <1 hour turnaround, minimal hands-on time
NG-Test CARBA-5 Immunochromatography Detects 5 major carbapenemases 15-minute procedure, no specialized equipment needed
Multilocus Sequence Typing (MLST) Primers Molecular epidemiology Amplifies 7 housekeeping genes for ST assignment Standardized classification scheme for global comparisons
Whole Genome Sequencing Kits Genomic analysis Comprehensive characterization of resistance and virulence Identifies novel resistance mechanisms and transmission events
Porin Detection Antibodies Protein analysis Identifies porin loss/modifications Confirms non-enzymatic resistance mechanisms

Therapeutic Challenges and Emerging Solutions

Current Treatment Landscape

The treatment of CRKP infections remains challenging due to limited therapeutic options and the propensity for emerging resistance during therapy. Traditional approaches have relied on older, often more toxic antibiotics:

  • Polymyxins (colistin, polymyxin B): Once abandoned due to nephrotoxicity and neurotoxicity, polymyxins have been reintroduced as last-line options for CRKP infections. However, resistance to polymyxins is increasingly reported, further limiting their utility [21].

  • Aminoglycosides (amikacin, gentamicin, tobramycin): These agents may retain activity against some CRKP isolates, but susceptibility varies regionally and by strain type. Their use is limited by toxicity concerns and poor penetration at certain infection sites [21].

  • Tigecycline: A glycylcycline antibiotic with activity against many multidrug-resistant Gram-positive and Gram-negative pathogens, including some CRKP. Limitations include poor serum levels and concerns about efficacy in bloodstream infections [21].

  • Fosfomycin: An old antibiotic rediscovered for its activity against multidrug-resistant Gram-negative bacteria. It is primarily used for uncomplicated urinary tract infections, with variable availability of intravenous formulations in different countries [21].

Novel β-Lactam–β-Lactamase Inhibitor Combinations

Recent advances have introduced new β-lactam–β-lactamase inhibitor combinations that address some carbapenemase-mediated resistance:

  • Ceftazidime-avibactam: Effective against KPC-producing and OXA-48-like Enterobacterales, but not metallo-β-lactamases (MBLs) [22]. The identification of the carbapenemase genotype is therefore crucial for determining appropriate therapy [22].

  • Meropenem-vaborbactam: A combination of meropenem with a boronic acid β-lactamase inhibitor that potently inhibits KPC enzymes, restoring meropenem activity against KPC-producing CRKP [24].

  • Imipenem-cilastatin-relebactam: Similar to meropenem-vaborbactam, this combination inhibits class A (including KPC) and class C β-lactamases, but not MBLs or most class D enzymes [22].

Emerging Therapeutic Approaches

The pipeline of novel agents active against CRKP includes:

  • Cefiderocol: A siderophore cephalosporin that exploits bacterial iron transport systems to penetrate the outer membrane, evading many common resistance mechanisms including some porin mutations and efflux pump overexpression [24].

  • Eravacycline: A novel fluorocycline antibiotic with broad-spectrum activity against Gram-positive and Gram-negative pathogens, including carbapenem-resistant Enterobacterales [22].

  • Plazomicin: A next-generation aminoglycoside designed to evade common aminoglycoside-modifying enzymes, maintaining activity against many multidrug-resistant Enterobacterales [24].

Virulence and Pathogenesis: The Emergence of Hypervirulent Strains

Klebsiella pneumoniae strains are classified into two major pathotypes: classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp). While classical strains are frequent causes of healthcare-associated infections with limited virulence, hypervirulent strains can cause severe, invasive community-acquired infections in healthy individuals [24] [26]. Alarmingly, these pathotypes are converging, with reports of hypervirulent strains acquiring carbapenem resistance plasmids, and multidrug-resistant strains acquiring virulence plasmids [24].

The key virulence factors of K. pneumoniae include:

  • Capsular Polysaccharide: The primary virulence determinant, protecting against phagocytosis. Hypervirulent strains often produce excessive capsule material, resulting in a hypermucoviscous phenotype [24].

  • Siderophores: Iron-acquisition systems (e.g., aerobactin, enterobactin, yersiniabactin, salmonchelin) that enable bacterial growth in iron-limited environments in the host. Hypervirulent strains typically possess the aerobactin synthesis locus (iuc) [24].

  • Adhesive Fimbriae: Facilitate attachment to host cells and surfaces [24].

  • Lipopolysaccharide (LPS): Contributes to inflammation and septic shock [24].

The emergence of carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP), particularly sequence type ST23 carrying carbapenemase genes, represents a particularly dangerous development that combines multidrug resistance with enhanced pathogenicity [26]. As of 2024, this convergence has been reported in at least 16 countries across all six WHO regions [26].

Prevention and Control Strategies

Containing the spread of CRKP requires a comprehensive approach integrating multiple strategies:

  • Infection Prevention and Control: Strict adherence to contact precautions, including glove and gown use, dedicated patient care equipment, and isolation of colonized or infected patients [26]. Environmental cleaning and disinfection are crucial as CRKP can persist on environmental surfaces.

  • Antimicrobial Stewardship: Judicious use of antibiotics, particularly carbapenems and other broad-spectrum agents, to reduce selective pressure that drives resistance emergence and dissemination [19].

  • Active Surveillance Screening: Targeted screening of high-risk patients (e.g., those with previous healthcare exposures in endemic regions) allows for early detection of colonization and implementation of preventive measures [26].

  • Laboratory Capacity Building: Strengthening diagnostic capabilities, particularly in regions with limited resources, to enable rapid detection and characterization of CRKP isolates [19] [26]. The WHO recommends that member states progressively increase laboratory diagnostic capacity to allow for early and reliable identification of hypervirulent strains in addition to resistance genes [26].

Carbapenem-resistant Klebsiella pneumoniae and other Gram-negative bacteria represent a critical threat to global health, with the potential to undermine decades of medical progress. The convergence of resistance mechanisms, enhanced virulence, and global dissemination creates a perfect storm that demands urgent, coordinated action.

Addressing this complex challenge requires:

  • Enhanced global surveillance systems to track the emergence and spread of high-risk clones
  • Rapid diagnostic technologies that can quickly identify resistance mechanisms and guide targeted therapy
  • Sustained investment in novel antimicrobial development with innovative mechanisms of action
  • Implementation of comprehensive infection prevention and antimicrobial stewardship programs
  • International collaboration and data sharing to understand and contain cross-border transmission

The recently updated WHO Bacterial Priority Pathogens List serves as both a warning and a roadmap, highlighting the critical need for research and intervention strategies targeting carbapenem-resistant pathogens. As the scientific community responds to this challenge, integrated approaches combining basic science, clinical research, and public health implementation will be essential to preserve the efficacy of existing antibiotics and ensure the development of new therapeutic options for future generations.

The World Health Organization's 2024 Bacterial Priority Pathogens List (WHO BPPL) represents a critical advancement in the global strategy to combat antimicrobial resistance (AMR), building upon the foundation laid by the 2017 edition to address evolving challenges. This updated list incorporates new evidence and expert insights to guide research and development (R&D) priorities and public health interventions against antibiotic-resistant bacterial pathogens [1] [17]. The 2024 WHO BPPL underscores the persistent threat of AMR, which erodes the efficacy of numerous antibiotics and jeopardizes many gains of modern medicine [17]. By mapping the global burden of drug-resistant bacteria and assessing their impact on public health, this list is key to guiding investment and addressing the antibiotics pipeline and access crisis, serving as an essential tool for prioritizing R&D investments and informing global public health policies [17] [5].

The list is strategically designed to target multiple stakeholders, including developers of antibacterial medicines, academic and public research institutions, research funders, public-private partnerships investing in AMR R&D, and policy-makers responsible for developing and implementing AMR policies and programs [1]. The BPPL 2024 emphasizes the need for a comprehensive public health approach to addressing AMR, including universal access to quality and affordable measures for prevention, diagnosis, and appropriate treatment of infections [17]. This is crucial for mitigating AMR's impact on public health and the economy.

The 2024 WHO Bacterial Priority Pathogens List: A Detailed Analysis

Categorization and Prioritization Methodology

The 2024 WHO BPPL followed a rigorous, evidence-based methodology to prioritize bacterial pathogens. The process employed a multicriteria decision analysis framework in which 24 antibiotic-resistant bacterial pathogens were scored based on eight criteria: mortality, non-fatal burden, incidence, 10-year resistance trends, preventability, transmissibility, treatability, and antibacterial pipeline status [5]. A preferences survey using a pairwise comparison was administered to 100 international experts (with 78 completing the survey) to determine the relative weights of these criteria [5]. Applying these weights, the final ranking of pathogens was determined by calculating a total score ranging from 0-100% for each pathogen.

The pathogens were subsequently streamlined and grouped into three priority tiers based on a quartile scoring system: critical (highest quartile), high (middle quartiles), and medium (lowest quartile) [5]. The results demonstrated high stability, with subgroup and sensitivity analyses showing that clustering of experts based on backgrounds and geographical origins did not result in substantial changes to the ranking [5]. This methodological rigor ensures that the list reliably reflects the most pressing global threats from antibiotic-resistant bacteria.

Critical Priority Pathogens: The Most Urgent Threats

Critical priority pathogens represent the most severe global threats due to their high burden, ability to resist treatment, and potential to spread resistance to other bacteria [17]. Gram-negative bacteria with built-in abilities to find new ways to resist treatment and pass along genetic material are particularly concerning.

Table 1: Critical Priority Bacterial Pathogens (WHO BPPL 2024)

Pathogen Antibiotic Resistance Profile Key Characteristics & Global Impact
Acinetobacter baumannii Carbapenem-resistant Major threat in healthcare settings; built-in abilities to develop and transfer resistance.
Enterobacterales Third-generation cephalosporin-resistant High-burden pathogens; emphasized as standalone item due to significant impact, especially in LMICs.
Enterobacterales Carbapenem-resistant Pose severe treatment challenges; limited therapeutic options available.
Mycobacterium tuberculosis Rifampicin-resistant Assessed with independent analysis and adapted matrix; devastating global burden.

The inclusion of rifampicin-resistant Mycobacterium tuberculosis in the critical priority category, assessed through an independent analysis with parallel tailored criteria, underscores its persistent devastating global burden [17] [5]. The standalone listing of third-generation cephalosporin-resistant Enterobacterales emphasizes their substantial burden and the need for targeted interventions, especially in low- and middle-income countries [17].

High and Medium Priority Pathogens: Significant Challenges

High priority pathogens include those with particularly high burden in low- and middle-income countries, as well as those posing significant challenges in healthcare settings [17]. These pathogens present unique public health challenges, including persistent infections and resistance to multiple antibiotics, necessitating targeted research and public health interventions.

Table 2: High and Medium Priority Bacterial Pathogens (WHO BPPL 2024)

Priority Tier Pathogen Antibiotic Resistance Profile
High Salmonella Typhi Fluoroquinolone-resistant
Shigella spp. Fluoroquinolone-resistant
Enterococcus faecium Vancomycin-resistant
Pseudomonas aeruginosa Carbapenem-resistant
Non-typhoidal Salmonella Fluoroquinolone-resistant
Neisseria gonorrhoeae Third-generation cephalosporin- and/or fluoroquinolone-resistant
Staphylococcus aureus Methicillin-resistant
Medium Group A streptococci Macrolide-resistant
Streptococcus pneumoniae Macrolide-resistant
Haemophilus influenzae Ampicillin-resistant
Group B streptococci Penicillin-resistant

Among the high priority pathogens, fluoroquinolone-resistant Salmonella enterica serotype Typhi (score: 72%), Shigella spp. (score: 70%), and Neisseria gonorrhoeae (score: 64%) were ranked as the highest among bacteria commonly responsible for community-acquired infections [5]. The transition of carbapenem-resistant Pseudomonas aeruginosa (CRPA) from critical to high priority in BPPL 2024 mirrors recent reports of decreases in global resistance, though investment in R&D and other prevention and control strategies remains important given its significant burden in some regions [17].

Medium priority pathogens include Group A and B Streptococci (both new to the 2024 list), Streptococcus pneumoniae, and Haemophilus influenzae, which present a high disease burden and require increased attention, especially in vulnerable populations including paediatric and elderly populations, particularly in resource-limited settings [17].

The Viral Threat Landscape: Pandemic Preparedness and the "Viral Most Wanted"

The Prototype Pathogen and Pathogen X Approach

In response to the ever-evolving nature of viral threats, the WHO's updated list of emerging pathogens represents a paradigm shift from focusing on specific pathogens to adopting a broader family-focused approach [6]. This new list recognizes the shortcomings of previous lists and adopts a more forward-thinking, proactive, and flexible approach to dealing with familiar and unfamiliar pandemic risks, now incorporating 'Prototype Pathogens' and 'Pathogen X' into its risk classification [6]. This approach acknowledges that while hundreds of viruses have 'human outbreak potential,' it is feasible to prepare for future threats by understanding entire viral families rather than individual pathogens.

The viral family strategy enables scientists to rapidly prepare and develop vaccines for new Disease X pathogens by applying knowledge gained from representative or exemplar viruses within each family [27]. Using speed-enhancing technologies such as artificial intelligence and computational science, this approach allows scientists to reduce vaccine development time from years to mere weeks or months [27]. This strategy is fundamental to global efforts in preparing for unknown future pathogens that could cause pandemics.

Priority Viral Families and Representative Pathogens

Scientific consensus identifies approximately 25 viral families that historically have put humanity at greatest risk, containing viruses with the ability to infect people and significant potential to produce the next pandemic threat [27].

Table 3: Priority Viral Families and Representative Pathogens

Viral Family Representative Pathogens Key Characteristics & Threats
Coronaviridae SARS-CoV-1, MERS-CoV, SARS-CoV-2 Cause severe respiratory illnesses; known for rapid global spread and significant mortality.
Paramyxoviridae Nipah virus, Measles virus Include both highly infectious (Measles) and highly deadly (Nipah) pathogens; significant spillover potential.
Filoviridae Ebola virus, Marburg virus Cause severe hemorrhagic fevers with high case fatality rates; extended geographic reach in recent outbreaks.
Arenaviridae Lassa virus Include lethal hemorrhagic fevers; pose significant threats in endemic regions with potential for wider spread.
Phenuiviridae Rift Valley fever virus Significant threat to both people and livestock; causes serious disease and dangerous outbreaks.
Orthomyxoviridae Influenza viruses (H5N1, H7N9) Responsible for deadliest pandemics in history; continuous evolution and zoonotic spillover potential.
Poxviridae Mpox virus Includes fearsome contagious diseases; recent transnational spread to non-endemic nations.
Flaviviridae Zika virus, Dengue virus, Yellow fever virus Primarily vector-borne; causing reemerging outbreaks with expanding geographic range due to climate change.

Recent outbreaks of Mpox, Dengue fever, Avian influenza (H5N2), Nipah virus disease, and Oropouche virus necessitate intensifying regional disease surveillance and research prioritization [6]. The inclusion of Pathogen X in the WHO's classification acknowledges the very real threat of currently unknown viruses that could emerge from any of these families to cause a future pandemic [6].

Experimental Frameworks and Research Methodologies

Multicriteria Decision Analysis for Pathogen Prioritization

The WHO BPPL 2024 development followed a systematic methodology to ensure robust and evidence-based pathogen prioritization. The multicriteria decision analysis framework provided a structured approach to evaluate complex interrelationships between multiple criteria affecting pathogen priority.

G Start 24 Bacterial Pathogens Initial Candidate List Criteria 8 Evaluation Criteria: Mortality, Burden, Incidence, Resistance Trends, Preventability, Transmissibility, Treatability, R&D Pipeline Start->Criteria Expert International Expert Panel (100 invited, 78 completed) Preferences Survey & Pairwise Comparison Criteria->Expert Scoring Pathogen Scoring (0-100% range) Based on Weighted Criteria Expert->Scoring Ranking Final Pathogen Ranking Quartile-based Categorization Scoring->Ranking Output Priority Tiers: Critical, High, Medium Ranking->Output

Diagram 1: WHO Pathogen Prioritization Methodology

This methodological framework incorporated several crucial elements that enhanced its robustness. The preferences survey demonstrated strong inter-rater agreement, with Spearman's rank correlation coefficient and Kendall's coefficient of concordance both at 0.9, indicating remarkable consensus among experts despite diverse backgrounds [5]. The sensitivity analyses confirmed high stability in the final ranking, with clustering of pathogens based on experts' backgrounds and origins not resulting in any substantial changes to the ranking [5]. Additionally, specific pathogens like Mycobacterium tuberculosis underwent independent analysis with parallel tailored criteria and subsequent application of an adapted multi-criteria decision analysis matrix to account for its unique characteristics and surveillance data [5].

The Viral Family Research Framework

The viral family approach to pandemic preparedness represents a strategic shift from reactive to proactive defense against emerging viral threats. This methodology leverages representative viruses within families to develop broad-spectrum countermeasures.

G cluster_tech Enhancing Technologies ViralFamilies 25 High-Risk Viral Families Identified Exemplar Select Representative (Exemplar) Viruses from Each Family ViralFamilies->Exemplar Research Comprehensive Research: Viral Structure, Replication, Transmission, Pathogenesis, Host Interactions Exemplar->Research Platform Develop Platform Technologies: mRNA, Vector-Based, AI-Assisted Design Research->Platform Readiness Preparedness for Disease X & Novel Pathogens Platform->Readiness Tech1 Artificial Intelligence Tech1->Research Tech2 Computational Science Tech2->Research Tech3 Structural Biology Tech3->Research

Diagram 2: Viral Family Research Framework

This framework operationalizes several key concepts. Exemplar viruses like Nipah for Paramyxoviruses or SARS-CoV-2 for Coronaviruses serve as research models to understand features shared across their viral families [27]. Platform technologies such as mRNA and computational vaccine design enable rapid development of vaccines against newly emerging viruses within prepared families [27]. The approach specifically prepares for Disease X - the unknown pathogen with pandemic potential - by building defenses that can be rapidly adapted when new threats emerge [6] [27].

Essential Research Tools and Reagents

Advancing research on priority pathogens requires specialized reagents and tools that enable scientists to study these dangerous microorganisms safely and effectively. The following table summarizes key research solutions necessary for investigating the pathogens highlighted in the WHO 2024 lists.

Table 4: Essential Research Reagent Solutions for Priority Pathogen Research

Research Reagent Category Specific Examples Application & Function in Research
Antibiotic Resistance Detection Carbapenemase activity assays, ESBL confirmation tests, Antibiotic gradient strips (Etest) Phenotypic confirmation of resistance mechanisms; determination of minimum inhibitory concentrations (MICs).
Molecular Characterization PCR kits for resistance genes (e.g., blaKPC, blaNDM, mecA), Whole Genome Sequencing kits Detection and characterization of specific resistance markers; comprehensive genetic analysis of resistant strains.
Cell Culture Models Polarized epithelial cells, Macrophage cell lines, Three-dimensional tissue models Study of host-pathogen interactions; investigation of invasion mechanisms and immune response.
Animal Models Transgenic mice expressing human receptors, Syrian hamsters for virus studies, Non-human primates for critical pathogens Evaluation of pathogenesis and therapeutic efficacy in complex organisms; preclinical testing.
Serological Assays ELISA kits for antibody detection, Neutralization assay components, Protein arrays Measurement of immune responses; vaccine evaluation; seroprevalence studies.
Viral Entry Tools Pseudotyped virus systems, Viral-like particles (VLPs), Cell entry reporters Safe study of viral entry mechanisms without requiring high containment; antibody neutralization assessment.
Antiviral Screening Compound libraries, High-content screening systems, Reporter virus constructs Identification of potential therapeutic compounds; mechanism of action studies.
Protein Expression Recombinant viral proteins, Surface antigen preparations, Crystallization kits Structural studies; vaccine immunogen design; diagnostic assay development.

These research tools enable the detailed investigation of resistance mechanisms, pathogenicity, and potential therapeutic targets for both bacterial and viral priority pathogens. The development and standardization of these reagents across laboratories facilitate comparable results and accelerate progress toward countermeasures.

The 2024 WHO Bacterial Priority Pathogens List and the complementary approach to viral threats through the prototype pathogen framework represent complementary, integrated strategies for global pandemic preparedness. The BPPL 2024 addresses the ongoing crisis of antimicrobial resistance, emphasizing Gram-negative bacteria and rifampicin-resistant M. tuberculosis as critical priorities, while the viral family approach prepares for potential future outbreaks and pandemics from known viral families and unknown Disease X candidates [17] [5] [6].

Beyond guiding research and development, effectively addressing these pathogens requires expanding equitable access to existing drugs, enhancing vaccine coverage, and strengthening infection prevention and control measures [5]. As noted by Dr. Jérôme Salomon, WHO's Assistant Director-General for Universal Health Coverage, Communicable and Noncommunicable Diseases, "Antimicrobial resistance jeopardizes our ability to effectively treat high burden infections, such as tuberculosis, leading to severe illness and increased mortality rates" [17]. The updated lists serve as critical tools for focusing global efforts, investments, and innovations to protect humanity against the most threatening infectious agents known today and those yet to emerge tomorrow.

Antimicrobial resistance (AMR) represents one of the most significant threats to global public health, food security, and development today. Less than a century after the revolutionary discovery of penicillin, the world faces a critical health crisis as many commonly used antimicrobial drugs are losing effectiveness against evolving pathogens [28]. The phenomenon of AMR occurs when microbes—bacteria, fungi, parasites, and viruses—evolve to the point where antimicrobial drugs that previously controlled them are no longer effective, allowing infections to spread and become increasingly difficult to treat [28]. This escalating crisis not only jeopardizes modern medical procedures that rely on effective antibiotics—including cancer care, transplants, caesarean sections, and diabetic care—but also imposes staggering economic costs on healthcare systems and economies worldwide [28].

Understanding the dual burden of AMR—encompassing both its devastating mortality impact and substantial economic consequences—is essential for guiding research priorities, directing funding allocations, and shaping effective public health policies. This whitepaper provides a comprehensive technical analysis of AMR's global burden, with particular focus on the World Health Organization's priority pathogen list, surveillance methodologies, and the critical research tools needed to combat this escalating crisis within the framework of a broader thesis on WHO priority pathogen analysis.

Global Mortality Burden of AMR

Comprehensive Mortality Statistics

The mortality burden of AMR has reached alarming proportions, with recent data revealing that drug-resistant infections directly caused 1.27 million deaths globally in 2019, with an additional 4.95 million deaths associated with AMR factors [28] [29]. This makes AMR a larger killer than HIV/AIDS or malaria, positioning it as a leading cause of death worldwide [28]. Forecasts suggest that without urgent intervention, AMR-related deaths will be 70% higher by 2050 than they were in 2022, underscoring the accelerating nature of this crisis [28].

Table 1: Global Mortality Burden of Antimicrobial Resistance (2019 Data)

Metric Figure Context
Direct AMR deaths 1.27 million Deaths directly attributable to antibiotic-resistant infections
Associated AMR deaths 4.95 million Deaths where AMR was a contributing factor
Projected increase by 2050 70% Compared to 2022 levels
Potential lives saved with intervention 92 million With improved healthcare access and effective antibiotics (2025-2050)

Regional Variations and Surveillance Gaps

The mortality burden of AMR affects all countries, though significant disparities exist between regions. Available evidence suggests that Sub-Saharan Africa may bear a disproportionately heavy burden, though data limitations in low-income areas currently hinder precise quantification [28]. The World Health Organization's Global Antimicrobial Resistance and Use Surveillance System (GLASS) has made substantial progress in generating standardized data, drawing on more than 23 million bacteriologically confirmed cases of infections reported by 110 countries between 2016 and 2023 [30]. Despite this progress, critical surveillance gaps remain, particularly in resource-limited settings where the burden may be highest.

WHO Bacterial Priority Pathogens List: 2024 Update

The 2024 BPPL Framework

The World Health Organization's 2024 Bacterial Priority Pathogens List (BPPL) represents a critical tool in the global fight against antimicrobial resistance, building upon and refining the initial 2017 edition [1]. This updated list categorizes 24 antibiotic-resistant bacterial pathogens spanning 15 families into three priority groups—critical, high, and medium—to strategically guide research and development (R&D) efforts and public health interventions [1]. The BPPL serves as an essential guide for prioritizing R&D and investments in AMR, emphasizing the need for regionally tailored strategies to effectively combat resistance [1].

The methodology employed for the 2024 update expanded upon the factors used to evaluate each pathogen, incorporating more robust quantitative data to assess pathogens based on multiple criteria [31]. Pathogens were evaluated using eight key criteria: mortality, nonfatal burden, incidence, 10-year resistance trends, preventability, transmissibility, treatability, and antibacterial pipeline status [31]. Pathogens were then categorized into priority levels based on their composite scores, with the highest quartile classified as critical priority [31].

Critical Priority Pathogens

Among the critical priority pathogens, carbapenem-resistant Klebsiella pneumoniae scored highest with 84%, positioning it as the most threatening antibiotic-resistant bacterium [31]. This pathogen, along with other antibiotic-resistant gram-negative bacteria including Acinetobacter spp. and Escherichia coli, as well as rifampicin-resistant Mycobacterium tuberculosis, ranked in the highest quartile [31]. These critical priority pathogens represent the most urgent targets for drug development and infection control measures due to their combination of high mortality, transmissibility, and limited treatment options.

Table 2: WHO Bacterial Priority Pathogens List 2024 - Critical and High Priority Pathogens

Priority Level Pathogens Key Resistance Mechanisms
Critical Carbapenem-resistant Klebsiella pneumoniae Carbapenemase production
Rifampicin-resistant Mycobacterium tuberculosis Genetic mutations affecting drug targets
Antibiotic-resistant Acinetobacter spp. Multiple drug efflux pumps, enzymatic modification
Antibiotic-resistant Escherichia coli Extended-spectrum beta-lactamases (ESBLs)
High Fluoroquinolone-resistant Salmonella enterica serotype Typhi DNA gyrase mutations
Shigella spp. Multiple antibiotic resistance genes
Neisseria gonorrhoeae Chromosomal mutations, plasmid-mediated resistance
Pseudomonas aeruginosa Efflux pumps, enzymatic inactivation
Staphylococcus aureus Methicillin resistance (MRSA)

Community-Acquired Infection Pathogens

Among bacteria commonly responsible for community-acquired infections, the highest scores were observed for fluoroquinolone-resistant Salmonella enterica serotype Typhi (72%), Shigella spp. (70%), and Neisseria gonorrhoeae (64%) [31]. These pathogens represent significant challenges for public health systems worldwide due to their potential for rapid dissemination in community settings and the limited treatment options available for resistant strains.

Economic Burden of Antimicrobial Resistance

Comprehensive Economic Impact Analysis

The economic burden of AMR extends far beyond healthcare costs, affecting multiple sectors of the global economy. Recent comprehensive analyses indicate that ABR was associated with a median value of US$693 billion (IQR: US$627 bn–US$768 bn) in hospital costs globally [29]. When productivity losses are factored in, the economic impact becomes even more staggering, with productivity losses quantified at almost US$194 billion [29]. These figures underscore AMR as not merely a health crisis but a significant economic threat with potential to reduce global GDP by $3.4 trillion and drive an additional 24 million people into extreme poverty without effective intervention [28].

The economic impact varies significantly by pathogen and resistance profile. Multidrug-resistant tuberculosis had the highest mean hospital cost attributable to ABR per patient, ranging from US$3000 in lower-income settings to US$41,000 in high-income settings [29]. Similarly, carbapenem-resistant infections were associated with a high cost-per-case of US$3000–US$7000 depending on syndrome and geographic region [29].

Potential Economic Benefits of Intervention

Investment in AMR mitigation strategies represents significant economic opportunity. Analyses suggest that US$207 billion (IQR: US$186 bn–US$229 bn) of hospital costs could potentially be avertable by vaccines, with an additional US$76 billion in productivity losses avertable by the same interventions [29]. Improving access to healthcare and effective antibiotics could save 92 million lives between 2025 and 2050, representing not only an enormous public health achievement but also substantial economic preservation [28].

Table 3: Global Economic Burden of Antibiotic-Resistant Infections (2019)

Economic Category Estimated Cost (US$) Potential Avertable by Vaccines (US$)
Hospital Costs $693 billion (median) $207 billion
Productivity Losses $194 billion $76 billion
Pathogen-Specific Hospital Cost per Case
Multidrug-resistant tuberculosis $3,000 (LIC) - $41,000 (HIC) -
Carbapenem-resistant infections $3,000 - $7,000 -
Projected Global GDP Reduction (without action) $3.4 trillion -

Surveillance and Diagnostic Methodologies

Integrated AMR Surveillance Approaches

Effective AMR surveillance requires sophisticated methodological approaches that can detect and quantify resistance across diverse settings. Environmental compartments such as treated wastewater and biosolids can substantially improve monitoring efforts through integrated surveillance strategies [32]. A key challenge in this domain is the diversity of available protocols, which complicates comparability for the concentration and detection of antibiotic resistance genes (ARGs), particularly in complex matrices [32].

Recent comparative studies have evaluated concentration methods including filtration–centrifugation (FC) and aluminum-based precipitation (AP), with results indicating that the AP method provided higher ARG concentrations than FC, particularly in wastewater samples [32]. For detection techniques, comparisons between quantitative PCR (qPCR) and droplet digital PCR (ddPCR) revealed that ddPCR demonstrated greater sensitivity than qPCR in wastewater, whereas in biosolids, both methods performed similarly [32]. These methodological insights are crucial for developing standardized surveillance protocols.

Diagnostic Gaps and Innovations

Significant diagnostic gaps persist in AMR surveillance, particularly affecting low-resource settings. Current limitations include the absence of multiplex platforms suitable for use in intermediate referral laboratories to identify bloodstream infections directly from whole blood without culture, insufficient access to biomarker tests to distinguish bacterial from viral infections, and limited simple, point-of-care diagnostic tools for primary and secondary care facilities [3]. These limitations disproportionately affect patients in low-resource settings, where most people first present at primary health-care facilities [3].

The WHO stresses the urgent need for affordable, robust, and easy-to-use diagnostic platforms, including sample-in/result-out systems that work with multiple sample types (blood, urine, stool, respiratory specimens) [3]. Such innovations are essential for strengthening global capacity for AMR surveillance and enabling timely, appropriate treatment decisions.

Research and Development Pipeline Analysis

Current State of Antibacterial Development

The pipeline for new antibacterial agents faces a dual crisis: scarcity and lack of innovation [3]. According to WHO's 2025 analysis, the number of antibacterials in the clinical pipeline decreased from 97 in 2023 to 90 in 2025 [3]. Among these, only 15 qualify as innovative, and for 10 of these, available data are insufficient to confirm the absence of cross-resistance [3]. Most alarmingly, only 5 of the antibacterials in development are effective against at least one of the WHO "critical" priority pathogens [3].

The preclinical pipeline remains somewhat more active, with 232 programmes across 148 groups worldwide, though the ecosystem is fragile with 90% of companies involved being small firms with fewer than 50 employees [3]. This highlights the vulnerability of the antibacterial development landscape and the need for sustained investment and support.

Vaccine Development for AMR Control

Vaccines represent a promising approach to tackling AMR by preventing infections outright, thereby reducing the need for antibiotics and limiting opportunities for resistance development. Recent analyses indicate that vaccines against Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae would avert a substantial portion of the economic burden associated with ABR [29]. Under utilized product-profile assumptions, bacterial vaccines have the potential to avert 30%–40% of hospital and labor productivity costs associated with ABR [29].

The recent development of GanLum, a new antimalarial drug with over 97% effectiveness in recent trials, demonstrates the potential for innovative antimicrobial development, even in the face of rising resistance [28]. Such successes provide important models for future antibacterial development efforts.

Experimental Approaches and Research Toolkit

Key Methodologies for AMR Research

AMR_Research_Workflow SampleCollection Sample Collection (Water, Biosolids, Clinical) Concentration Sample Concentration SampleCollection->Concentration Filtration Filtration-Centrifugation (FC) Method Concentration->Filtration Precipitation Aluminum-based Precipitation (AP) Method Concentration->Precipitation DNAExtraction DNA Extraction ARGDetection ARG Detection DNAExtraction->ARGDetection qPCR Quantitative PCR (qPCR) ARGDetection->qPCR ddPCR Droplet Digital PCR (ddPCR) ARGDetection->ddPCR DataAnalysis Data Analysis & Quantification Filtration->DNAExtraction Precipitation->DNAExtraction qPCR->DataAnalysis ddPCR->DataAnalysis

Figure 1: AMR Research Experimental Workflow. This diagram illustrates the key methodological approaches for detecting and quantifying antibiotic resistance genes (ARGs) in environmental and clinical samples, highlighting two concentration methods (FC and AP) and two detection techniques (qPCR and ddPCR).

Essential Research Reagents and Tools

Table 4: Research Reagent Solutions for AMR Surveillance Studies

Reagent/Tool Application Technical Function
Aluminum-based precipitation reagents Sample concentration Concentrates bacterial cells and ARGs from large liquid samples through chemical flocculation
Membrane filters (0.45 µm, 0.22 µm) Sample concentration and phage purification Separates microbial fractions based on size; removes bacteria for phage isolation
DNA extraction kits (Maxwell RSC) Nucleic acid purification Isolves high-quality DNA from complex matrices for downstream molecular analysis
qPCR and ddPCR master mixes ARG detection and quantification Enables amplification and detection of target resistance genes with high sensitivity
Chloroform treatment Phage purification Removes bacterial cell debris while preserving phage particles and associated DNA
CTAB buffer DNA extraction Lyses cells and removes contaminants during nucleic acid purification
Proteinase K DNA extraction Digests proteins and nucleases that could degrade DNA during extraction
ATP synthase inhibitor 1ATP synthase inhibitor 1, MF:C17H18ClN3O3S2, MW:411.9 g/molChemical Reagent
1-(2-Methoxyethyl)2-nitrobenzene1-(2-Methoxyethyl)2-nitrobenzene, CAS:102871-91-0, MF:C9H11NO3, MW:181.19 g/molChemical Reagent

Quantitative Evaluation Methods for Antimicrobial Use

Accurate assessment of antimicrobial use patterns is essential for AMR stewardship and research. Two primary metrics are employed for quantitative evaluation: the defined daily dose (DDD) and days of therapy (DOT) [33]. The DDD represents the average daily dose administered to adults for treating infectious diseases when a specific antimicrobial is the primary indication, while DOT measures the sum of the number of days a patient was given antimicrobials [33]. Each approach has distinct advantages and limitations, with DDD being easier to collect but not applicable to children, while DOT is more intuitive but requires patient-specific data [33].

The WHO's Access, Watch, and Reserve (AWaRe) system categorizes antimicrobials according to the associated risk of developing resistant bacteria, providing a crucial framework for monitoring and evaluating antimicrobial use patterns in research and clinical settings [33]. This classification system helps standardize surveillance data and facilitates comparisons across different regions and healthcare settings.

The rising burden of antimicrobial resistance presents a complex and multidimensional challenge that demands coordinated global action across the research, clinical, and public health sectors. The staggering mortality figures—1.27 million direct deaths annually—coupled with the profound economic impact—approximately $693 billion in hospital costs—underscore the urgent need for sustained intervention [28] [29]. The WHO Bacterial Priority Pathogens List provides a crucial framework for targeting research and development efforts toward the most threatening resistant pathogens, with carbapenem-resistant Klebsiella pneumoniae, rifampicin-resistant Mycobacterium tuberculosis, and other critical priority pathogens representing the most pressing challenges [1] [31].

Addressing the AMR crisis will require multifaceted strategies including enhanced surveillance with standardized methodologies, accelerated development of innovative antibacterial agents and vaccines, strengthened antimicrobial stewardship programs, and improved infection prevention and control measures [31] [3]. The promising potential of vaccines to avert 30%-40% of AMR-associated economic burden highlights the importance of preventive approaches [29]. As the global community confronts this escalating threat, sustained investment in research, coordinated international collaboration, and equitable access to effective treatments will be essential to protect the foundations of modern medicine and secure a healthier future for generations to come.

Decoding the WHO's Prioritization Framework and Its Application in R&D Strategy

Multi-Criteria Decision Analysis (MCDA) represents a structured, transparent approach to complex decision-making that systematically evaluates alternatives across multiple, often competing criteria. Within public health, particularly for prioritizing pathogens and guiding research and development (R&D) investments, MCDA has emerged as a critical tool for global health organizations. This whitepaper provides a technical examination of MCDA methodologies, with a specific focus on the application of criteria such as mortality, transmissibility, and treatability in the context of the World Health Organization's (WHO) priority pathogen lists. We detail experimental protocols for data collection, weighting procedures, and model aggregation, supported by quantitative data tables and visualized workflows. This guide aims to equip researchers, scientists, and drug development professionals with the frameworks necessary to implement MCDA in their own pathogen prioritization and antimicrobial resistance (AMR) mitigation strategies.

The systematic prioritization of pathogens is a cornerstone of effective pandemic preparedness and antimicrobial resistance (AMR) containment. The World Health Organization (WHO) and other global health bodies have adopted Multi-Criteria Decision Analysis (MCDA) as a robust methodology to navigate the complex landscape of threats posed by various infectious agents. MCDA moves beyond single-metric assessments, enabling decision-makers to integrate diverse quantitative and qualitative evidence into a coherent, transparent, and defensible ranking [34]. This structured approach is particularly valuable in contexts characterized by high uncertainty, scarce evidence, and the need for rapid, yet reasoned, action—hallmarks of public health emergencies [34].

The 2024 WHO Bacterial Priority Pathogens List (BPPL), which updates the 2017 list, is a prime example of MCDA in action. It categorizes 24 antibiotic-resistant bacterial pathogens across 15 families into critical, high, and medium priority groups to guide R&D and public health interventions [1]. The development of this list required the careful balancing of multiple criteria, including the core factors of mortality, transmissibility, and treatability, to provide a nuanced understanding of each pathogen's overall public health threat [31]. Similarly, MCDA has been deployed to identify global priority endemic pathogens for vaccine R&D, aligning with the objectives of the Immunization Agenda 2030 (IA2030) [35]. The following sections deconstruct the MCDA process, detailing the operationalization of key criteria and the methodologies for synthesizing them into actionable priorities for the research and drug development community.

Core Criteria in Pathogen Prioritization

The efficacy of an MCDA framework hinges on the careful selection and definition of its evaluation criteria. For pathogen prioritization, criteria are chosen to collectively capture the overall burden, potential for spread, and challenges associated with clinical management. The criteria of mortality, transmissibility, and treatability are frequently identified as central pillars in these assessments.

Mortality and Disease Burden

Mortality serves as a primary indicator of a pathogen's severity. Within MCDA frameworks, it is often quantified as the number of deaths attributable to an infection, sometimes further refined into metrics like the infection fatality ratio (IFR). However, the assessment of burden frequently extends beyond mortality alone to include nonfatal consequences, such as long-term disability, healthcare system utilization, and economic impact. For example, the 2024 WHO BPPL explicitly used "Mortality" and "nonfatal burden" as two of its eight key criteria to evaluate bacterial pathogens [31]. Quantitative data is essential here; a study analyzing SARS-CoV-2 variants in England provided variant-specific IFR estimates, finding the Alpha variant had the highest basic infection fatality ratio at 3.0%, compared to 0.7% for Omicron (BA.1) [36]. This kind of precise, quantitative data is crucial for scoring pathogens on the mortality criterion.

Transmissibility and Transmission Dynamics

Transmissibility refers to the capacity of a pathogen to spread within a host population. The basic reproduction number (Râ‚€), defined as the average number of secondary infections generated by one primary case in a fully susceptible population, is a fundamental metric for this criterion. The intrinsic transmissibility of a pathogen is a key driver of outbreak potential and pandemic risk. Research on SARS-CoV-2 variants has demonstrated sequential increases in Râ‚€, from 2.6 for the wild-type virus to 8.4 for the Omicron BA.1 variant, illustrating how viral evolution selects for enhanced transmissibility [36]. Beyond Râ‚€, transmissibility assessments may also incorporate qualitative aspects of transmission dynamics, such as the multiplicity of transmission routes (e.g., respiratory, fecal-oral, bloodborne) and the potential for asymptomatic spread, which complicates public health control measures [31].

Treatability encompasses the availability, efficacy, and accessibility of therapeutic countermeasures. This criterion directly influences clinical management and patient outcomes. A key component of treatability is the current state and trajectory of antimicrobial resistance (AMR). The WHO BPPL is inherently focused on this facet, highlighting pathogens like carbapenem-resistant Klebsiella pneumoniae and rifampicin-resistant Mycobacterium tuberculosis [1] [31]. The evaluation of treatability includes an analysis of the antibacterial R&D pipeline. A 2025 WHO report revealed a fragile and insufficient pipeline, with only 90 antibacterials in clinical development and a mere 5 of these being effective against at least one "critical"-priority BPPL pathogen [3]. This quantitative assessment of the treatment landscape is vital for identifying the most pressing gaps and prioritizing pathogens for which therapeutic options are dwindling or nonexistent.

Table 1: Quantitative Metrics for Core MCDA Criteria in Pathogen Prioritization

Criterion Key Quantitative Metrics Example from Literature
Mortality & Burden - Infection Fatality Ratio (IFR)- Annual deaths (e.g., in children under 5)- Disability-Adjusted Life Years (DALYs)- Hospitalization rate Alpha variant SARS-CoV-2 IFR: 3.0% (95% CrI 2.8-3.2) [36]. "Mortality" was a top-weighted criterion in 5/6 WHO regions for vaccine R&D prioritization [35].
Transmissibility - Basic Reproduction Number (Râ‚€)- Effective Reproduction Number (Reff)- Secondary attack rate SARS-CoV-2 Râ‚€ estimates: Wildtype: 2.6; Alpha: 4.2; Delta: 7.2; Omicron (BA.1): 8.4 [36].
Treatability & Resistance - Proportion of isolates with resistance to key antibiotics- 10-year resistance trends- Number of active drug classes available- Agents in clinical/preclinical R&D pipeline Only 5 of 90 antibacterials in clinical development target a WHO "critical" priority pathogen [3]. Carbapenem-resistant K. pneumoniae scored highest (84%) on the WHO BPPL [31].

MCDA Methodologies and Experimental Protocols

Implementing MCDA requires a structured, multi-stage process that transforms raw data and expert judgment into a prioritized list. The methodology can be broken down into sequential stages: problem structuring, scoring, weighting, and aggregation.

Problem Structuring and Criteria Selection

The initial phase involves defining the decision problem and selecting a coherent set of criteria. For the WHO BPPL 2024, this resulted in eight criteria: Mortality, nonfatal burden, incidence, 10-year resistance trends, preventability, transmissibility, treatability, and antibacterial pipeline status [31]. A similar process was used for the IA2030 vaccine R&D prioritization, which also considered "Contribution to antimicrobial resistance" as a key criterion [35]. This stage requires a thorough review of scientific literature and surveillance data to ensure all relevant aspects of the public health threat are captured. The identified criteria form the axes upon which all alternative pathogens will be evaluated.

Data Collection and Scoring Protocols

Each pathogen is then scored against the selected criteria. This requires gathering robust quantitative and qualitative data.

  • Quantitative Data Sources: Data is sourced from global surveillance systems, epidemiological studies, and clinical trials. For mortality and burden, this includes vital statistics, hospital admission records, and cohort studies [36] [37]. Transmissibility estimates (Râ‚€) often require sophisticated mathematical modeling of incidence data, as demonstrated in the analysis of SARS-CoV-2 variants in England [36]. Treatability and resistance data are gleaned from antimicrobial resistance surveillance networks (e.g., GLASS, EARS-Net) and clinical microbiology reports.
  • Qualitative Data and Expert Elicitation: For criteria less amenable to pure quantification (e.g., ease of prevention, operational challenges in treatment delivery), structured expert elicitation is used. Surveys of stakeholders, such as the survey of 412 Romanian healthcare professionals used to identify risks and opportunities for healthcare resilience, are a common tool [38]. The WHO IA2030 MCDA survey targeted policymakers and immunization stakeholders in each WHO region to weight the prioritization criteria [35].

Weighting and Aggregation Techniques

Weighting reflects the relative importance of each criterion. The Simple Multiple Attribute Rating Technique (SMART) is a common linear additive model used for this purpose [38]. In this protocol, stakeholders are asked to allocate 100 points across the criteria based on their perceived importance. The IA2030 exercise used MCDA surveys in each WHO region to generate region-specific criterion weights [35].

Once weights (wi) and pathogen scores (si) for each criterion are established, an overall value score for each pathogen is calculated. The formula for a linear additive model is:

Overall Pathogen Score = Σ (wi * si)

where the sum is taken over all criteria (i). The pathogens are then ranked based on their overall scores. The 2024 WHO BPPL categorized pathogens into critical (highest quartile), high (middle quartile), and medium (lowest quartile) priority groups based on this aggregated score [31].

MCDA_Workflow Start Define Decision Problem Structuring 1. Problem Structuring - Identify Alternatives (Pathogens) - Define Evaluation Criteria Start->Structuring DataCollection 2. Data Collection & Scoring - Gather quantitative data (e.g., R₀, IFR) - Conduct expert elicitation - Score each pathogen per criterion Structuring->DataCollection Weighting 3. Criterion Weighting - Survey stakeholders (e.g., SMART method) - Assign weights to reflect importance DataCollection->Weighting Aggregation 4. Aggregation & Ranking - Calculate overall score: Σ(weight × score) - Rank pathogens based on total score Weighting->Aggregation Output Priority List Aggregation->Output

Diagram 1: MCDA Workflow for Pathogen Prioritization. This flowchart outlines the key stages in a typical Multi-Criteria Decision Analysis, from problem definition to the generation of a ranked priority list.

The Scientist's Toolkit: Essential Research Reagents and Materials

The execution of MCDA and the underlying research supporting pathogen criteria evaluation rely on a suite of specific reagents, assays, and data tools. The following table details key components of the methodological toolkit.

Table 2: Research Reagent Solutions for Pathogen Prioritization and MCDA

Item/Tool Function in MCDA & Pathogen Research
Vero E6 Cell Line A mammalian kidney cell line used as the gold standard for isolating and titrating SARS-CoV-2 and other viruses to determine infectiousness, a key factor in transmissibility assessments [39].
Real-Time PCR (RT-PCR) The primary method for detecting and quantifying viral RNA load in respiratory specimens. Used for diagnosis and as a proxy for infectiousness in shedding kinetics studies [39].
Antigen-Detecting Rapid Diagnostic Tests (Ag-RDTs) Lateral flow tests used to quickly detect viral proteins. Positivity correlates better with infectious virus presence than RT-PCR and is useful for informing isolation policies [39].
International Standard for SARS-CoV-2 RNA An inactivated SARS-CoV-2 isolate provided by the WHO to calibrate nucleic acid amplification techniques, ensuring comparability of viral load data between different labs and assays [39].
Bayesian Inference Modeling A robust mathematical framework used to analyze epidemiological surveillance data and estimate key parameters like Râ‚€ and IFR for different pathogen variants [36].
SMART (Simple Multi-Attribute Rating Technique) A specific MCDA weighting technique where stakeholders distribute a fixed sum of points (e.g., 100) across criteria to determine their relative importance [38].
Losartan-d9Losartan-d9, CAS:1030937-18-8, MF:C22H23ClN6O, MW:432 g/mol
PallidolPallidol

Case Study: Application to the WHO Priority Pathogens List

The 2024 WHO Bacterial Priority Pathogens List (BPPL) serves as a canonical case study for the practical application of MCDA in global health. The update process, building on the 2017 list, employed a refined MCDA methodology to evaluate 24 antibiotic-resistant bacterial pathogens against eight defined criteria [1] [31].

The outcome of this process was the categorization of pathogens into three priority tiers. Carbapenem-resistant Klebsiella pneumoniae emerged as the highest-scoring pathogen, with a score of 84%, landing it in the critical priority group [31]. This result was driven by its high mortality, significant nonfatal burden, and severe challenges in treatability due to resistance to last-resort antibiotics like carbapenems. Other pathogens in the highest quartile included antibiotic-resistant Gram-negative bacteria like Acinetobacter baumannii and Escherichia coli, as well as rifampicin-resistant Mycobacterium tuberculosis [31]. The MCDA framework also effectively highlighted priorities among community-acquired pathogens, with fluoroquinolone-resistant Salmonella enterica serotype Typhi (72%), Shigella spp. (70%), and Neisseria gonorrhoeae (64%) scoring highly [31].

This structured, transparent prioritization directly informs global R&D strategy. The accompanying WHO report on the antibacterial pipeline analyzed the alignment between R&D activity and the BPPL, finding a critical lack of innovative agents targeting the most threatening pathogens [3]. Thus, the MCDA output provides a powerful evidence-based guide for directing research investments and policy measures.

WHO_BPPL Criteria WHO BPPL 2024 Evaluation Criteria C1 Mortality Criteria->C1 C2 Nonfatal Burden Criteria->C2 C3 Incidence Criteria->C3 C4 10-Year Resistance Trends Criteria->C4 C5 Preventability Criteria->C5 C6 Transmissibility Criteria->C6 C7 Treatability Criteria->C7 C8 Antibacterial Pipeline Criteria->C8 Output Pathogen Priority Tier C1->Output C2->Output C4->Output C6->Output C7->Output C8->Output High Critical Priority (Highest Quartile) Output->High Medium High Priority (Middle Quartile) Output->Medium Low Medium Priority (Lowest Quartile) Output->Low P1 Carbapenem-resistant Klebsiella pneumoniae (84%) High->P1 P2 Rifampicin-resistant Mycobacterium tuberculosis High->P2 P3 Fluoroquinolone-resistant Salmonella Typhi (72%) Medium->P3 P4 Penicillin-resistant Group B Streptococci (28%) Low->P4 Example Example Pathogens

Diagram 2: WHO BPPL 2024 MCDA Logic. This diagram visualizes how key criteria, particularly mortality, transmissibility, and treatability/resistance, drive the classification of bacterial pathogens into critical, high, and medium priority tiers in the WHO BPPL.

Multi-Criteria Decision Analysis provides an indispensable, systematic framework for navigating the complex and high-stakes landscape of global health security. By explicitly weighing critical factors such as mortality, transmissibility, and treatability, MCDA transforms multifaceted and often conflicting data into clear, actionable priority lists, as exemplified by the WHO BPPL and IA2030 vaccine R&D priorities. The rigorous, protocol-driven approach—encompassing structured problem definition, robust data collection, stakeholder-weighted criteria, and transparent aggregation—ensures that resulting priorities are both evidence-based and defensible.

For researchers, scientists, and drug development professionals, understanding and utilizing MCDA methodologies is paramount. The outputs of these analyses directly shape the R&D agenda, highlighting pathogens for which new antibiotics, vaccines, and diagnostics are most urgently needed. As the threat of AMR continues to escalate and the risk of future pandemics persists, the continued refinement and application of MCDA will be critical for focusing finite resources on the most dangerous pathogens, ultimately safeguarding global public health.

The World Health Organization's (WHO) Bacterial Priority Pathogens List (BPPL) serves as a critical tool in the global fight against antimicrobial resistance (AMR), guiding research, development, and public health strategies. The 2024 edition represents a significant evolution from its 2017 predecessor, incorporating a more sophisticated multicriteria decision analysis framework to evaluate and rank bacterial threats [1]. This systematic approach recognizes that assessing the threat level of antibiotic-resistant pathogens requires consideration of multiple interrelated factors beyond simple incidence rates. The BPPL aims to strategically direct limited resources toward the most pressing AMR challenges, informing priorities for antibiotic development, infection control measures, and public health interventions across human, animal, and environmental health sectors.

The prioritization methodology balances both quantitative epidemiological metrics and qualitative expert assessment to create a comprehensive threat evaluation system. This technical guide examines the specific criteria employed in the 2024 WHO BPPL development process, detailing the data requirements, methodological approaches, and analytical frameworks used to rank 24 pathogens across 15 families of antibiotic-resistant bacteria [1]. For researchers, scientists, and drug development professionals, understanding these criteria is essential for aligning research agendas with global public health priorities and addressing the most critical gaps in our antimicrobial arsenal.

Comprehensive Analysis of Prioritization Criteria

The Eight Core Criteria for Pathogen Evaluation

The WHO BPPL development process employed eight specific criteria to evaluate and rank bacterial pathogens, each capturing a distinct dimension of the threat posed by antimicrobial resistance. These criteria were selected through a rigorous methodology development process to ensure comprehensive assessment of each pathogen's public health impact, transmission dynamics, and tractability to interventions. The criteria incorporate both quantitative data-driven metrics and qualitative expert judgment to create a balanced evaluation framework.

The complete set of criteria includes: (1) mortality impact, measuring infection-associated fatality rates; (2) non-fatal burden, encompassing disability and long-term sequelae; (3) incidence and prevalence rates across different populations and geographic regions; (4) 10-year resistance trends, tracking the evolution of resistance patterns over time; (5) preventability potential through non-pharmaceutical interventions; (6) transmissibility between humans, animals, and environments; (7) treatability with existing therapeutic options; and (8) antibacterial pipeline status, assessing development of new countermeasures [5]. Each criterion was operationalized with specific metrics and data requirements to enable systematic comparison across diverse bacterial pathogens.

Table 1: Core Criteria for Pathogen Prioritization in the 2024 WHO BPPL

Criterion Measurement Approach Data Sources Weight in Overall Score
Mortality Infection-associated fatality rates; attributable mortality National surveillance systems; clinical studies; burden estimates High (exact weighting not specified)
Non-fatal Burden Disability-adjusted life years (DALYs); long-term sequelae Global Burden of Disease studies; clinical cohort studies High
Incidence Case numbers; prevalence rates; laboratory-confirmed infections WHO GLASS; national surveillance programs; laboratory networks Medium
10-Year Resistance Trends Annual percentage change in resistance rates; emerging resistance patterns Longitudinal surveillance data; resistance monitoring programs High
Preventability Effectiveness of infection control; vaccine availability; hygiene measures Intervention studies; outbreak investigations; systematic reviews Medium
Transmissibility Reproductive number (R0); outbreak potential; spread in healthcare settings Transmission modeling; genomic epidemiology; outbreak reports Medium
Treatability Available treatment options; access to effective antibiotics; reserve antibiotics Treatment guidelines; therapeutic guidelines; essential medicines lists High
Antibacterial Pipeline Number of candidates in development; innovation level; projected availability Clinical trial registries; drug development reports; regulatory databases Medium

The quantitative dimensions of the prioritization framework rely heavily on robust surveillance data and standardized measurement approaches. Incidence and mortality metrics primarily draw from the WHO Global Antimicrobial Resistance and Use Surveillance System (GLASS), which compiled data from over 23 million bacteriologically confirmed cases across 110 countries between 2016 and 2023 [30]. This unprecedented dataset enables comparative analysis of resistance patterns across different geographic regions and healthcare settings, providing essential input for the incidence and resistance trends criteria.

The 10-year resistance trends criterion represents a particularly sophisticated quantitative metric, tracking the temporal evolution of resistance across pathogen-antibiotic combinations. According to recent WHO analyses, antibiotic resistance rose in over 40% of monitored pathogen-antibiotic combinations between 2018 and 2023, with an average annual increase of 5-15% [19]. This trend analysis reveals the alarming acceleration of resistance across multiple bacterial species and antibiotic classes, with Gram-negative bacteria showing particularly concerning trajectories. The GLASS report provides specific data on resistance patterns, such as the finding that over 40% of E. coli and more than 55% of K. pneumoniae globally are now resistant to third-generation cephalosporins, first-line treatments for serious infections [19].

Mortality assessments incorporate both direct attribution models and statistical estimates of excess mortality associated with resistant infections. The 2024 BPPL development process utilized data from the Global Burden of Disease studies, which provide systematic estimates of AMR-associated mortality across different regions and populations [5]. These analyses revealed stark disparities in mortality impact, with critical priority pathogens such as carbapenem-resistant K. pneumoniae and rifampicin-resistant Mycobacterium tuberculosis associated with significantly higher case fatality rates, particularly in resource-limited settings where access to second-line treatments may be constrained.

Methodological Framework and Experimental Protocols

Multicriteria Decision Analysis Methodology

The WHO BPPL development employed a structured multicriteria decision analysis (MCDA) framework to synthesize complex, multidimensional data into a coherent prioritization system. The MCDA approach followed a systematic process beginning with criterion definition and proceeding through evidence collection, expert weighting, pathogen scoring, and final ranking. This methodology enables transparent and reproducible decision-making when faced with multiple competing objectives and complex trade-offs between different types of evidence.

The specific MCDA protocol involved several distinct phases. First, the eight evaluation criteria were defined through a structured literature review and consultation with technical experts. Second, a comprehensive evidence collection process gathered published and unpublished data for each pathogen-criterion combination, with systematic documentation of data sources and quality assessments. Third, a preferences survey using pairwise comparisons was administered to 100 international experts (with 78 complete responses) to determine the relative weights of each criterion [5]. This expert panel represented diverse geographical backgrounds and technical expertise to minimize regional or disciplinary biases.

Table 2: Research Reagent Solutions for AMR Priority Pathogen Studies

Research Reagent Primary Application Function in Experimental Protocols
Culture Media for Carbapenem-resistant Enterobacteriaceae Isolation and identification Selective isolation of target pathogens from clinical samples; antibiotic susceptibility testing
Molecular Typing Kits (PCR, Whole Genome Sequencing) Resistance mechanism characterization Detection of resistance genes (e.g., blaKPC, blaNDM); phylogenetic analysis; outbreak investigation
Antibiotic Susceptibility Testing Panels Phenotypic resistance profiling Determination of minimum inhibitory concentrations (MICs); detection of emerging resistance patterns
Animal Infection Models Pathogenesis and therapeutic studies Evaluation of virulence mechanisms; efficacy testing of new antibacterial agents
Immunoassays for Biomarker Detection Host response characterization Measurement of inflammatory markers (e.g., C-reactive protein); differentiation of bacterial vs. viral infections
Microbial Strain Repositories Reference materials Quality control; method validation; comparative studies across laboratories
Biofilm Formation Assays Treatment resistance studies Assessment of biofilm-associated resistance; evaluation of anti-biofilm agents

The scoring system assigned values for each pathogen on each criterion based on available evidence, with standardized approaches for handling data gaps or conflicting information. The final score for each pathogen was calculated using a weighted sum model, combining the criterion-specific scores with the expert-derived weights. The resulting total scores ranged from 84% for the top-ranked pathogen (carbapenem-resistant Klebsiella pneumoniae) to 28% for the lowest-ranked pathogen (penicillin-resistant group B streptococci) [5]. The high concordance among experts (Spearman's rank correlation coefficient and Kendall's coefficient of concordance both at 0.9) demonstrated strong consensus on the relative importance of the different criteria.

The preference weighting process followed a structured expert elicitation protocol to ensure methodological rigor and minimize cognitive biases. The pairwise comparison approach required experts to repeatedly choose between two criteria, indicating which they considered more important for pathogen prioritization in the context of AMR threat assessment. This forced-choice methodology produces more reliable weight estimates than simple rating scales, as it reduces scale use biases and facilitates more consistent judgment.

The expert panel was carefully composed to represent diverse perspectives, including clinical microbiology, infectious diseases, epidemiology, pharmaceutical development, public health policy, and veterinary medicine. Geographical representation was also prioritized, with experts from all WHO regions participating in the weighting exercise. The high response rate (79 out of 100 invited experts responded, with 78 completing the survey) indicates strong engagement with the process and enhances the legitimacy of the resulting weights [5].

Statistical analysis of expert responses included consistency checks to identify potentially unreliable respondents, though the high overall concordance suggests generally consistent judgment patterns across the expert panel. Sensitivity analyses tested the stability of the final rankings to variations in criterion weights, confirming that the priority groupings remained robust across different weighting scenarios. This methodological rigor ensures that the resulting pathogen list reflects collective expert judgment rather than the biases of a particular disciplinary or regional perspective.

Visualization of the Prioritization Framework

WHO Pathogen Prioritization Logic Flow

who_prioritization cluster_criteria Evaluation Criteria Application cluster_scoring Scoring and Weighting cluster_output Priority Tier Assignment Start Start: 24 Bacterial Pathogens Mortality Mortality Impact Start->Mortality Burden Non-fatal Burden Start->Burden Incidence Incidence Rates Start->Incidence Trends 10-Year Resistance Trends Start->Trends Preventability Preventability Start->Preventability Transmissibility Transmissibility Start->Transmissibility Treatability Treatability Start->Treatability Pipeline Antibacterial Pipeline Start->Pipeline DataSynthesis Data Synthesis and Scoring Mortality->DataSynthesis Burden->DataSynthesis Incidence->DataSynthesis Trends->DataSynthesis Preventability->DataSynthesis Transmissibility->DataSynthesis Treatability->DataSynthesis Pipeline->DataSynthesis MCDA Multicriteria Decision Analysis DataSynthesis->MCDA ExpertWeighting Expert Preference Weighting (n=78) ExpertWeighting->MCDA Critical Critical Priority (Highest Quartile: 84%-71%) MCDA->Critical High High Priority (Middle Quartiles: 70%-42%) MCDA->High Medium Medium Priority (Lowest Quartile: 41%-28%) MCDA->Medium

AMR Research and Development Workflow

amr_workflow cluster_data_inputs Data Inputs cluster_methods Methodological Approaches cluster_outputs Research Outputs Surveillance Disease Surveillance and Burden Estimation Epidemiology Epidemiological Modeling Surveillance->Epidemiology Mechanism Resistance Mechanism Characterization Laboratory Laboratory Methods (AST, WGS, MIC) Mechanism->Laboratory Diagnostic Diagnostic Tool Development Trials Clinical Development Programs Diagnostic->Trials Therapeutic Therapeutic Development Therapeutic->Trials Prevention Prevention Strategy Implementation IPC Infection Prevention and Control Prevention->IPC Glass WHO GLASS Data (23M+ infections) Glass->Surveillance Clinical Clinical Trial Data Clinical->Surveillance Genomic Genomic Surveillance Genomic->Mechanism Pipeline R&D Pipeline Analysis Pipeline->Therapeutic BPPL WHO BPPL Priority Pathogens Epidemiology->BPPL Diagnostics Novel Diagnostics Laboratory->Diagnostics Guidelines Treatment Guidelines Trials->Guidelines Therapeutics New Therapeutics Trials->Therapeutics Policies Public Health Policies IPC->Policies

Critical Priority Pathogens and Their Characteristics

The 2024 WHO BPPL identified several bacterial pathogens in the critical priority category, representing the highest quartile of threat scores (84%-71%). These pathogens share several concerning characteristics, including rapid spread of resistance mechanisms, limited treatment options, and significant mortality impacts. Gram-negative bacteria with carbapenem resistance features prominently in this category, reflecting the particular challenges posed by these pathogens due to their structural characteristics and resistance gene transfer capabilities.

Carbapenem-resistant Klebsiella pneumoniae ranked as the top priority pathogen with a score of 84%, reflecting its global dissemination, association with healthcare-associated infections, and limited therapeutic alternatives [5]. Other critical priority pathogens include carbapenem-resistant Acinetobacter baumannii, rifampicin-resistant Mycobacterium tuberculosis, and third-generation cephalosporin-resistant Escherichia coli. These pathogens demonstrate the increasing threat of Gram-negative bacteria, which possess outer membranes that limit antibiotic penetration and efflux pump systems that enhance resistance mechanisms.

The critical priority pathogens exhibit alarming resistance trends over the past decade, with particularly rapid escalation of resistance to last-resort antibiotics. The GLASS report notes that carbapenem resistance, once rare, is becoming more frequent, dramatically narrowing treatment options and forcing reliance on last-resort antibiotics that are often costly, difficult to access, and unavailable in many low- and middle-income countries [19]. This trend is especially concerning for critically ill patients in intensive care settings, where these pathogens cause life-threatening bloodstream infections and pneumonia.

Global and Regional Resistance Patterns

The WHO surveillance data reveals significant geographic variation in resistance patterns, with the highest burdens concentrated in specific regions. Antibiotic resistance is most prevalent in the WHO South-East Asian and Eastern Mediterranean Regions, where approximately one in three reported infections were resistant to first-line antibiotics [19]. The African Region shows similarly concerning patterns, with one in five infections demonstrating resistance, compounded by limited diagnostic capacity and treatment access.

Table 3: Regional Variation in Antibiotic Resistance Patterns for Key Pathogens

Pathogen Antibiotic Class Global Resistance Rate Highest Resistance Region Regional Resistance Rate
Klebsiella pneumoniae Third-generation cephalosporins >55% African Region >70%
Escherichia coli Third-generation cephalosporins >40% African Region >70%
Acinetobacter spp. Carbapenems Increasing South-East Asia Not specified
Neisseria gonorrhoeae Extended-spectrum cephalosporins Widespread Multiple regions Not specified
Staphylococcus aureus Methicillin (MRSA) Variable All regions Not specified
Salmonella spp. Fluoroquinolones Rising South-East Asia Not specified

The surveillance data also highlights disparities in diagnostic capacity and reporting completeness that potentially affect these estimates. Approximately 48% of countries did not report data to GLASS in 2023, and about half of reporting countries lacked the systems to generate reliable data [19]. This surveillance gap is particularly pronounced in regions facing the largest AMR challenges, creating a vicious cycle where the areas most affected by antimicrobial resistance have the least capacity to monitor and respond to the threat.

Implications for Research and Public Health Strategy

Guiding Antibacterial Development Priorities

The WHO BPPL serves as a strategic roadmap for directing antibacterial research and development toward the most pressing public health needs. Current analysis of the clinical development pipeline reveals significant gaps between public health priorities and actual R&D activities. According to WHO's assessment, the number of antibacterial agents in clinical development decreased from 97 in 2023 to 90 in 2025, with only 15 of these qualifying as truly innovative compounds [3]. This declining and insufficiently innovative pipeline threatens our ability to address currently untreatable infections.

The pipeline assessment reveals particularly concerning gaps for critical priority Gram-negative pathogens. Only five of the antibacterial agents in development demonstrate effectiveness against at least one of the WHO "critical" priority bacteria [3]. Additionally, significant gaps exist in specific product profiles essential for clinical utility, including pediatric formulations, oral treatments for outpatient use, and solutions for escalating resistance through combination strategies with non-traditional agents. The preclinical pipeline shows somewhat more activity, with 232 programs across 148 research groups worldwide, though approximately 90% of these efforts are driven by small firms with fewer than 50 employees, highlighting the fragility of the R&D ecosystem [3].

The WHO calls for enhanced investment and innovation in both traditional and non-traditional approaches to address these gaps. Non-traditional approaches include bacteriophage therapy, monoclonal antibodies, microbiome-modulating agents, and immunotherapies that offer potential alternatives or adjuncts to conventional antibiotics. For drug development professionals, the BPPL provides clear direction for target selection and development priorities, emphasizing pathogens with limited treatment options and rapidly evolving resistance mechanisms.

Diagnostic Development and Infection Prevention Strategies

The prioritization criteria have important implications for diagnostic development and infection prevention strategies beyond therapeutic development. The preventability criterion specifically evaluates the potential for reducing infections through non-pharmaceutical interventions, highlighting the importance of infection prevention and control (IPC) measures in comprehensive AMR management. WHO data indicates that a large proportion of healthcare-associated infections could be prevented with basic IPC measures and water, sanitation, and hygiene (WASH) services, with a high return on investment [40].

Diagnostic gaps remain a critical challenge, particularly in resource-limited settings. WHO's analysis identifies persistent needs including the absence of multiplex platforms suitable for intermediate referral laboratories to identify bloodstream infections directly from whole blood without culture, insufficient access to biomarker tests to distinguish bacterial from viral infections, and limited simple, point-of-care diagnostic tools for primary and secondary care facilities [3]. These limitations disproportionately affect patients in low-resource settings, where most people first present at primary healthcare facilities.

Infection prevention and control represents a cornerstone of AMR mitigation, with WHO establishing a Global Action Plan for IPC (2024-2030) that provides clear actions, indicators, and targets to support member states in improving national and facility-level IPC programs [41]. Effective IPC requires constant action at all levels of the health system and is universally relevant to every health worker and patient, at every healthcare interaction [42]. The preventability scores in the BPPL prioritization framework reflect the potential impact of these non-pharmaceutical interventions in reducing the burden of resistant infections across different pathogen types and healthcare settings.

The quantitative and qualitative criteria underlying the WHO Bacterial Priority Pathogens List represent a sophisticated framework for evaluating the multifaceted threat of antimicrobial resistance. By integrating metrics ranging from incidence and mortality to 10-year resistance trends and preventability, the 2024 BPPL provides a comprehensive assessment that guides global research, development, and public health strategies. The methodology balances empirical data with expert judgment to create a transparent, evidence-based prioritization system that reflects both current burden and future threat trajectories.

For researchers, scientists, and drug development professionals, understanding these criteria is essential for aligning activities with global public health priorities. The stark findings regarding escalating resistance patterns, particularly among Gram-negative pathogens in specific geographic regions, underscore the urgency of targeted action. Similarly, the concerning gaps in the antibacterial development pipeline and diagnostic tools highlight areas requiring intensified investment and innovation. As antimicrobial resistance continues to evolve, the systematic application of these prioritization criteria will remain essential for directing resources to the most pressing challenges and mitigating the devastating impact of drug-resistant infections worldwide.

The escalating frequency of emerging and re-emerging infectious disease threats necessitates a paradigm shift in pandemic preparedness strategies. This technical guide delineates a novel, integrated framework that combines two complementary approaches: the 'Family Approach' to pathogen categorization and the 'Prototype Pathogen' methodology for countermeasure development. Framed within the context of the World Health Organization's (WHO) priority pathogen list analysis, this paradigm aims to transition global health efforts from reactive to proactive preparedness. The 2024 WHO Bacterial Priority Pathogens List (BPPL) and the updated list of emerging pathogens exemplify this strategic shift, organizing pathogens by taxonomic families and highlighting critical gaps in our therapeutic and diagnostic arsenals [1] [6]. This whitepaper provides researchers, scientists, and drug development professionals with the technical rationale, methodological protocols, and implementation pathways for this consolidated framework, which is essential for building a resilient defense against future pandemic threats.

The Conceptual Foundation: Integrating Two Proactive Paradigms

The "Prototype Pathogen" Approach

The Prototype Pathogen Approach is a systematic strategy for pandemic preparedness that involves conducting in-depth research on representative viruses from each family of viral pathogens known to infect humans. This approach is predicated on the biological principle that viruses within the same family or genus share fundamental structural and functional properties, meaning that vaccine and therapeutic solutions developed for one member can often be rapidly adapted for closely related family members [43].

This methodology involves four discrete activities:

  • Targeted Basic Research: Using structural biology to define atomic-level details of surface proteins, understanding replication mechanisms, and establishing animal models.
  • Translational Research and Product Development: Developing candidate vaccines and therapeutics through to phase 1 clinical evaluation for representative pathogens.
  • Clinical Trial Infrastructure: Establishing a global network for the rapid testing and deployment of medical countermeasures during an outbreak [43].

The expedited development of SARS-CoV-2 vaccines was a validation of this approach, as prior research on other coronaviruses like MERS-CoV provided the foundational knowledge and technological platforms [43].

The "Family Approach" to Pathogen Prioritization

The "Family Approach" is a strategic evolution in how global health institutions prioritize pathogens for research and preparedness. Moving beyond focusing on specific, known pathogen species, this approach categorizes and assesses threats at the level of viral or bacterial families. This acknowledges that the next pandemic may be caused by a previously unknown virus from a known family of risky pathogens [6].

In July 2024, the WHO updated its list of emerging pathogens through a global process that evaluated the evidence related to 28 Viral Families and one core group of Bacteria, encompassing 1,652 individual pathogens [44]. This "family-focused" method is more forward-thinking, proactive, and flexible, as it prepares for both known ("Prototype Pathogens") and unknown ("Pathogen X") members within high-risk families [6].

Table 1: WHO Priority Pathogen Lists Exemplifying the Family-Focused Approach

List Name Publication Date Scope Key Categorization
WHO Bacterial Priority Pathogens List (BPPL) [1] May 2024 24 antibiotic-resistant bacterial pathogens across 15 families Critical, High, and Medium Priority
WHO List of Emerging Pathogens [44] [6] July 2024 28 Viral Families, 1 core Bacterial group (1,652 pathogens) Focus on entire viral families of concern

Technical Implementation: Methodologies and Workflows

Pathogen Prioritization and Categorization Methodology

The development of the WHO BPPL 2024 demonstrates a rigorous, multi-criteria methodology for pathogen prioritization within the Family Approach. Pathogens are evaluated using a combination of quantitative and qualitative criteria to ensure a comprehensive assessment of their public health impact and associated risks [1] [31].

Quantitative and Qualitative Criteria for Prioritization:

  • Mortality & Nonfatal Burden: Assesses the direct and indirect death toll and disability caused by the pathogen.
  • Incidence & 10-Year Resistance Trends: Evaluates current case rates and historical data on the development of antimicrobial resistance (AMR).
  • Transmissibility & Treatability: Analyzes how easily the pathogen spreads and the current availability and effectiveness of treatments.
  • Preventability & R&D Pipeline Status: Considers existing preventive measures (e.g., vaccines) and the number of innovative countermeasures in development [31].

Based on this scoring system, pathogens are categorized into three priority levels: Critical (highest quartile), High (middle quartile), and Medium (lowest quartile) [31]. For example, in the 2024 BPPL, Gram-negative bacteria like carbapenem-resistant Klebsiella pneumoniae and rifampicin-resistant Mycobacterium tuberculosis ranked in the highest quartile [31].

Table 2: Top Priority Pathogens from the WHO 2024 BPPL and their Key Characteristics

Pathogen Priority Level Key Resistance Traits Notable R&D Gaps
Carbapenem-resistant Klebsiella pneumoniae [31] Critical Resistance to last-resort carbapenem antibiotics Only 5 innovative agents in development are effective against critical priority pathogens [3]
Rifamp-resistant Mycobacterium tuberculosis [31] Critical Resistance to first-line antibiotic rifampicin 18 of 50 traditional antibiotics in development target drug-resistant TB [3]
Fluoroquinolone-resistant Salmonella Typhi [31] High Resistance to fluoroquinolone antibiotics Focus needed on equitable access to existing medications and vaccines [31]
Multidrug-resistant Shigella spp. [31] High Resistance to multiple first-line therapies Need for oral treatments for outpatient use [3]
Antibiotic-resistant Neisseria gonorrhoeae [31] High Escalating resistance to multiple drug classes Gaps in the preclinical and clinical pipeline [3]

The Prototype Pathogen Research and Development Pipeline

The R&D pipeline for prototype pathogens is fragile and requires strategic reinforcement. A 2025 WHO analysis reveals a concerning scarcity and lack of innovation in the antibacterial pipeline, which is a critical component of pandemic preparedness [3].

Analysis of the Current R&D Pipeline:

  • Clinical Pipeline: As of 2025, there are only 90 antibacterial agents in clinical development, down from 97 in 2023. Of these, only 15 are classified as innovative, and a mere 5 are effective against at least one "critical" priority pathogen [3].
  • Preclinical Pipeline: The preclinical pipeline is more active, with 232 programs across 148 groups worldwide. However, the ecosystem is fragile, as 90% of the involved companies are small firms with fewer than 50 employees [3].
  • Non-Traditional Agents: Of the 90 agents in clinical development, 40 are non-traditional antibacterial approaches, including bacteriophages, antibodies, and microbiome-modulating agents [3]. These represent a promising avenue for tackling AMR.

The following workflow diagram illustrates the integrated research and development pathway for a prototype pathogen, from initial discovery to preparedness for outbreak response.

G Start Start: Viral Family Identification P1 1. Discovery & Surveillance Start->P1 P2 2. Targeted Basic Research P1->P2 D1 Virus discovery in reservoir hosts P1->D1 P3 3. Translational Research & Product Development P2->P3 D2 Structural biology, Animal models, Reagents P2->D2 P4 4. Clinical Evaluation & Trial Infrastructure P3->P4 D3 Candidate vaccines & therapeutics P3->D3 D4 Phase 1 clinical trials, Global network P4->D4 Outcome Outcome: Pandemic Preparedness P4->Outcome

The Scientist's Toolkit: Essential Research Reagents and Materials

Implementing the Prototype Pathogen Approach requires a standardized set of research tools and reagents. The table below details key materials essential for experimental work in this field.

Table 3: Key Research Reagent Solutions for Prototype Pathogen Research

Research Reagent / Material Function & Application Technical Specifications & Considerations
Monoclonal Antibodies [43] Used for passive immunization studies, therapeutic development, and structural biology to define epitopes. Requires isolation from convalescent patients or engineered based on structural data; key for defining protective correlates.
Recombinant Viral Proteins [43] Serve as immunogens for vaccine design, antigens for immunoassays, and reagents for structural studies (e.g., X-ray crystallography, Cryo-EM). Typically surface proteins (e.g., spike glycoproteins); must be stabilized in pre-fusion conformation for optimal immune response.
Animal Models [43] [45] Critical for in vivo assessment of pathogenicity, immune responses, and efficacy of vaccines/therapeutics. Must reflect human disease; includes wild-type, transgenic (e.g., hACE2 mice for coronaviruses), and gnotobiotic models for microbiota studies.
Genomic Sequencing Libraries [43] Enable virus discovery, surveillance, and tracking of evolutionary trends and resistance markers within pathogen families. Next-generation sequencing (NGS) platforms; essential for building the "periodic table of viruses" and defining the virome.
Microbiota Consortiums [45] Defined microbial communities used to study colonization resistance against antibiotic-resistant pathogens and to develop live biotherapeutic products. Can be derived from human donor samples; used in gnotobiotic animals to elucidate protective mechanisms against pathogens like K. pneumoniae and VRE.
Sikokianin ASikokianin A, CAS:106293-99-6, MF:C31H24O10, MW:556.5 g/molChemical Reagent
Kuguacin NKuguacin N, CAS:1141453-73-7, MF:C30H46O4, MW:470.7 g/molChemical Reagent

A New Frontier: The Microbiota as a Family-Level Protective Barrier

An advanced application of the "family" concept involves leveraging the host's microbiota—the community of commensal microorganisms—as a broad-spectrum, protective barrier against pathogenic families. The microbiota mediates protection through several mechanisms: priming immune defenses, metabolic exclusion of pathogens from their preferred niches, and direct antimicrobial antagonism [45].

Experimental Protocols for Microbiota Research:

  • Gnotobiotic Mouse Models: Use germ-free mice colonized with a defined human microbiota consortium to study specific host-microbe-pathogen interactions in a controlled setting [45].
  • Antibiotic Perturbation Model: Treat mice with a cocktail of broad-spectrum antibiotics (e.g., ampicillin, vancomycin, neomycin, metronidazole) in drinking water for 1-2 weeks to deplete the indigenous microbiota, thereby increasing susceptibility to pathogen colonization [45].
  • Fecal Microbiota Transplantation (FMT): Transfer fecal material from a resistant donor mouse (or human) to an antibiotic-treated, susceptible recipient to test the restoration of colonization resistance against a specific priority pathogen, such as vancomycin-resistant Enterococci (VRE) or Clostridioides difficile [45].
  • Mechanistic Validation: Use of knockout mice (e.g., IL-17R⁻/⁻, MyD88⁻/⁻) or antibodies to deplete specific immune cells (e.g., neutrophils) to dissect the precise immune pathways by which the microbiota confers protection [45].

The following diagram maps the key signaling pathways through which the microbiota orchestrates innate immune defense, a core mechanism of family-level protection against a wide range of pathogens.

G Microbiota Microbiota & Microbiota-Derived Signals (e.g., SCFAs, PSA) ImmuneCell Innate Immune Cells Microbiota->ImmuneCell SCFAs IL-17A Induction IL-36 Induction Epithelium Epithelial Barrier Microbiota->Epithelium TLR Signaling Metabolites Lymphocyte Lymphocytes (Th17/Tregs) Microbiota->Lymphocyte PSA → IL-10 SCFAs Effect2 ↑ Neutrophil Production & Bactericidal Activity ImmuneCell->Effect2 Effect3 ↑ Macrophage/Dendritic Cell Antimicrobial Activity ImmuneCell->Effect3 Effect1 ↑ Antimicrobial Peptides (AMPs) ↑ Mucus Production Epithelium->Effect1 Effect4 Balanced Inflammatory Response (IL-10, Treg induction) Lymphocyte->Effect4 Outcome Enhanced Clearance of Priority Pathogens Effect1->Outcome Effect2->Outcome Effect3->Outcome Effect4->Outcome

Synthesis and Future Directions: Integrating Paradigms for Enhanced Resilience

The integration of the Family Approach and the Prototype Pathogen paradigm represents the most comprehensive and scientifically-grounded strategy for achieving global pandemic resilience. This synthesis allows for a more efficient allocation of R&D resources, targeting entire families of pathogens based on their pandemic potential and AMR risk, as classified by the WHO [1] [44] [6]. The Prototype Pathogen methodology within these families ensures that foundational knowledge and platform technologies are pre-established, shaving critical time off the development of medical countermeasures when a new threat emerges from a known family [43].

Critical Gaps and Strategic Recommendations for Researchers and Funders:

  • Reinforce the R&D Pipeline: Address the dire scarcity of innovative antibacterial agents, especially those targeting critical priority Gram-negative bacteria. This requires sustained investment and novel funding models to support the small and medium-sized enterprises that drive this research [31] [3].
  • Bridge the Diagnostic Divide: Develop affordable, robust, and easy-to-use diagnostic platforms suitable for primary and secondary care facilities in low-resource settings. Key gaps include multiplex platforms for bloodstream infections and point-of-care tools to distinguish bacterial from viral infections [3].
  • Optimize Prevention and Access: While R&D for new countermeasures is crucial, parallel efforts must focus on optimizing infection prevention and control (e.g., hand hygiene) and expanding equitable access to existing vaccines and treatments [31].
  • Leverage Non-Traditional Therapies: Intensify research into non-traditional agents, such as bacteriophages and microbiome-modulating therapies, which offer promising pathways to combat AMR and restore colonization resistance against priority pathogens [3] [45].

The "world on fire" metaphor used to describe the COVID-19 pandemic underscores the insufficiency of reactive responses [43]. The future of pandemic preparedness lies in building fire-resistant systems. By adopting this integrated, proactive paradigm—guided by WHO's evolving pathogen prioritization—the scientific community can transform the global capacity to anticipate, prevent, and decisively respond to the infectious disease threats of the 21st century.

The World Health Organization (WHO) Bacterial Priority Pathogens List (BPPL) serves as a critical roadmap in the global fight against antimicrobial resistance (AMR), categorizing antibiotic-resistant bacterial pathogens into critical, high, and medium priority groups to guide research and development (R&D) and public health interventions. The 2024 edition updates and refines the prioritization of pathogens to address evolving antibiotic resistance challenges, covering 24 pathogens across 15 families of antibiotic-resistant bacteria [1]. This list underscores the global impact of these pathogens in terms of burden, transmissibility, treatability, and prevention options, while also reflecting the R&D pipeline of new treatments and emerging resistance trends [1]. In the antiviral domain, while no formal priority list is detailed in the search results, the lessons from rapid drug discovery efforts during the COVID-19 pandemic have highlighted the need for proactive target selection against viruses of pandemic concern [46].

The strategic importance of these priority lists is magnified by the alarming weaknesses in the current antimicrobial pipeline. According to recent WHO analyses, the number of antibacterials in clinical development has decreased from 97 in 2023 to 90 in 2025, with only 15 qualifying as innovative and a mere 5 being effective against at least one of the WHO's "critical priority" pathogens [3] [47]. This scarcity and lack of innovation creates a dual crisis in the antibacterial pipeline, leaving few options to address the escalating threat of drug-resistant infections that contribute to nearly 5 million deaths annually [47]. This whitepaper provides a comprehensive technical guide for researchers and drug development professionals on target selection strategies informed by WHO priority pathogens, offering detailed methodologies and frameworks to accelerate the discovery of novel antibacterial and antiviral agents.

The Current Development Landscape: Critical Gaps and Opportunities

Analysis of the Antibacterial Pipeline

The global pipeline for new antibacterial agents reveals significant vulnerabilities that threaten the medical community's ability to combat resistant infections. Table 1 summarizes the current state of antibacterial agents in clinical development, highlighting the disproportionate focus relative to WHO priority pathogens.

Table 1: WHO Bacterial Priority Pathogens List (2024) and Corresponding R&D Coverage

Priority Category Pathogen Examples No. of Pathogens Innovative Antibacterials in Development Key Resistance Mechanisms
Critical Carbapenem-resistant Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacterales 4 Only 5 agents effective against ≥1 critical pathogen Enzymatic degradation, efflux pumps, target modification
High Salmonella, Shigella, Neisseria gonorrhoeae, methicillin-resistant Staphylococcus aureus 8 Limited innovative candidates Membrane permeability barriers, enzymatic inactivation
Medium Drug-resistant Streptococcus pneumoniae, Haemophilus influenzae 12 Scattered coverage across pathogens Altered drug binding sites, efflux mechanisms

The preclinical pipeline shows slightly more promise with 232 programs in development, but this ecosystem remains fragile, with 90% of involved companies being small firms with fewer than 50 employees, creating significant economic vulnerability in the R&D landscape [3]. Beyond the sheer numbers, critical gaps persist in specific therapeutic areas, including pediatric formulations, oral treatments for outpatient use, and solutions to address escalating resistance through combination strategies with non-traditional agents [3]. Since July 2017, only 17 new antibacterial agents against priority bacterial pathogens have obtained marketing authorization, with just two representing a new chemical class – highlighting the profound innovation gap [3].

Analysis of the Antiviral Pipeline and Preparedness

The antiviral development landscape presents different but equally concerning challenges. The COVID-19 pandemic revealed the precarious state of broad-spectrum antiviral preparedness, with most existing antivirals being narrowly targeted and effective against only a small set of related viruses [48]. While the pandemic stimulated unprecedented rapid drug discovery efforts, leading to the development of novel therapeutics within a 2-year timeframe, this success was built upon previous research on coronaviruses following the 2003 SARS outbreak [46].

Significant advances in target-based drug design have emerged as crucial strategies to overcome resistance to antiviral agents. Structure-based approaches using protein structures acquired through crystallographic techniques and homology modeling have become central to modern antiviral discovery [49]. These methods facilitate drug discovery through practical design, virtual screening, and optimization of known ligands, with particular promise in addressing the challenge of resistance mutations that impact drug binding through stereochemical irregularities or diminished interactions between target and drug [49].

Target Selection Frameworks for Antibacterial Development

Criteria for Ideal Antibacterial Targets

Selecting targets with the highest potential for successful antibacterial development requires systematic evaluation against defined criteria. The ideal antibacterial target should be: (1) essential for bacterial survival and pathogenesis; (2) absent in humans to minimize host toxicity; (3) conserved across bacterial strains and species; (4) tractable to chemical inhibition; and (5) possess a high fitness barrier to resistance development [50]. Additionally, targets should have assayable functions with available or developable screening assays to enable drug discovery campaigns.

The essentiality criterion is particularly crucial, as inhibition of non-essential targets may produce bacteriostatic effects rather than the desired bactericidal activity needed for treating serious infections, especially in immunocompromised patients. Conservation across bacterial species expands the potential spectrum of new agents, making them more valuable from a public health perspective, while sequence variability at the binding site may predict rapid resistance development [50]. The fitness barrier refers to the cost to bacterial growth and transmission imposed by resistance mutations – targets where resistance causes significant fitness defects are preferred as they may limit the spread of resistance in bacterial populations [51].

Prioritized Antibacterial Targets Based on WHO BPPL

Table 2 showcases promising antibacterial targets aligned with WHO priority pathogens, along with experimental methodologies for their validation.

Table 2: Promising Antibacterial Targets and Validation Methodologies

Target Category Specific Target Priority Pathogens Key Assays for Validation Advantages/Rationale
Cell wall biosynthesis DapE enzyme ESKAPE pathogens Molecular docking, enzymatic inhibition assays, MIC determination Absent in humans; essential for peptidoglycan biosynthesis
Riboswitches T-box riboswitch Staphylococcus aureus In silico screening, biofilm inhibition assays, synergy studies Regulates multiple amino acid metabolism genes; unique to bacteria
Membrane integrity Undisclosed membrane targets MDR Gram-negative bacteria Chelocardin derivative testing against efflux-prone strains Overcomes common resistance mechanisms like efflux pumps
tRNA-dependent pathways Aminoacyl-tRNA synthetases Drug-resistant Gram-positive pathogens In vivo biofilm growth inhibition, combination studies with standard antibiotics Simultaneously inhibits multiple genes; reduces resistance emergence

Novel Strategies for Overcoming Antibacterial Resistance

Beyond traditional target selection, innovative strategies are emerging to address the resistance crisis. One promising approach involves targeting bacterial biofilms, which are associated with numerous persistent and drug-resistant infections. Research has identified small molecules that inhibit biofilm growth by targeting tRNA-dependent regulated T-box genes that modulate the expression of aminoacyl-tRNA synthetases and amino acid metabolism genes [51]. These compounds demonstrated a 10-fold greater potency in inhibiting Staphylococcus aureus biofilm growth compared to vancomycin, along with synergistic effects when administered with gentamicin and rifampin [51].

Another strategy focuses on bypassing common resistance mechanisms. For instance, amidochelocardin, a biosynthetic derivative of the natural product chelocardin, acts as a broad-spectrum antibacterial active against the ESKAPE group of clinically relevant bacteria while demonstrating the ability to overcome known resistance mechanisms, particularly efflux processes [51]. This molecule represents a promising candidate for development against multidrug-resistant uropathogenic clinical isolates.

The exploration of allosteric inhibition represents a third innovative approach. For β-lactamases – bacterial enzymes representing a major resistance mechanism to β-lactam antibiotics in Gram-negative bacteria – targeting allosteric sites could overcome the frequent occurrence of mutations that diminish drug effectiveness [51]. Allosteric inhibitors could work synergistically with traditional inhibitors, increasing the chances of restoring bacterial susceptibility to available antibiotics.

Target Selection Frameworks for Antiviral Development

Criteria for Ideal Antiviral Targets

The selection of optimal antiviral targets requires consideration of virological, chemical, and clinical factors. Ideal antiviral targets should possess: (1) essential function in the viral life cycle; (2) high sequence conservation across variants and related viruses; (3) druggability with small molecules; (4) high genetic barrier to resistance; and (5) clinical validation where possible [46]. Additionally, the availability of quality chemical probes provides critical validation of target tractability before embarking on extensive drug discovery campaigns.

During the COVID-19 pandemic, the most successful antiviral programs targeted the SARS-CoV-2 main protease (Mpro) and RNA-dependent RNA polymerase (RdRp). These targets benefited from target-class confidence based on successful therapeutics against similar targets in other viruses, mechanistic understanding of their essential functions, rapid availability of protein structures to facilitate structure-based drug design, and pre-existing chemical probes from previous coronavirus research [46].

Emerging Antiviral Strategies and Targets

Broad-Spectrum Antiviral Approaches

A groundbreaking development in antiviral therapy is the emergence of truly broad-spectrum approaches. Recent research has identified synthetic carbohydrate receptors (SCRs) that target viral envelope glycans – sugar molecules structurally conserved across unrelated viral families [48]. This strategy represents a paradigm shift from the traditional narrow-spectrum antiviral model to one more analogous to broad-spectrum antibiotics.

Researchers screened 57 SCRs and identified four lead compounds that successfully blocked infection from seven different viruses across five unrelated families, including Ebola, Marburg, Nipah, Hendra, SARS-CoV-1, and SARS-CoV-2 [48]. In animal studies, one SCR compound resulted in 90% survival of mice infected with SARS-CoV-2, compared to no survival in the control group [48]. This approach offers potential as a first-line defense against emerging viral threats while specific vaccines and therapeutics are developed.

Host-Targeting Antivirals (HTAs)

Another strategic approach involves targeting host proteins essential for viral replication rather than viral proteins themselves. Host-targeting antivirals (HTAs) offer potential advantages including a higher barrier to resistance (since host proteins do not mutate rapidly like viral proteins) and broad-spectrum activity if the host pathway is utilized by multiple viruses [49] [46].

However, HTAs present distinct challenges, including possible host pathway-mediated (on-target) toxicity, lower efficacy if viruses utilize redundant entry pathways, and poor translation from in vivo models to human clinical benefit [46]. Despite these challenges, successful HTAs have been developed, including CCR5 antagonists for HIV and interferon for HCV and HBV treatment [46].

Structure-Based Strategies to Overcome Antiviral Resistance

Target-based drug design strategies are increasingly important for overcoming resistance to existing antiviral agents. When resistance-associated mutations impact drug binding by creating stereochemical clashes or diminishing interactions, structure-based approaches offer solutions:

  • Increasing protein-ligand interactions: Designing compounds that form additional favorable interactions with the target, potentially including regions unaffected by resistance mutations [49].
  • Reducing interactions with mutated binding pockets: Designing more flexible inhibitors that can accommodate mutations without significant loss of binding affinity [49].
  • Targeting alternative sites: Identifying and targeting cryptic or allosteric sites that may be more conserved than the primary active site [49].

These approaches benefit from advances in structural biology techniques, including cryo-electron microscopy, which has enabled the determination of complex viral protein structures that were previously challenging to characterize [46].

Experimental Methodologies for Target Validation

Comprehensive Workflow for Target Identification and Validation

The transition from bioinformatic target identification to experimentally validated targets requires a systematic workflow. The following diagram illustrates a comprehensive pipeline for target identification and validation, integrating computational and experimental approaches:

G cluster_0 Target Identification Phase cluster_1 Experimental Validation Phase cluster_2 Lead Development Phase BioinformaticAnalysis Bioinformatic Analysis GenomicSequencing Pathogen Genomic Sequencing BioinformaticAnalysis->GenomicSequencing ProteomeMapping Proteome-Wide Mapping GenomicSequencing->ProteomeMapping Essentiality Essentiality Validation (Gene Knockout) ProteomeMapping->Essentiality Druggability Druggability Assessment Essentiality->Druggability Screening High-Throughput Screening Druggability->Screening Resistance Resistance Propensity Testing Screening->Resistance Optimization Lead Optimization Resistance->Optimization Efficacy In Vivo Efficacy Studies Optimization->Efficacy Toxicology Preclinical Toxicology Efficacy->Toxicology

Target Identification and Validation Workflow

Advanced Methodologies for Protein-Protein Interaction Mapping

For antiviral targets specifically, mapping virus-host protein-protein interactions (PPIs) provides valuable targets for intervention. The following diagram details an advanced pipeline for systematic identification of host-directed antiviral targets:

G ViralSeq Viral Genome Sequencing ORFIdentification ORF Identification and Cloning ViralSeq->ORFIdentification InteractomeMapping Interactome Mapping (AP-MS, Y2H) ORFIdentification->InteractomeMapping DomainMapping Interacting Domain Mapping InteractomeMapping->DomainMapping VirtualScreening Ultra-Large Virtual Screening DomainMapping->VirtualScreening ExperimentalValidation Experimental Validation (ITC, SPR, NMR) VirtualScreening->ExperimentalValidation StructuralStudies Structural Studies (X-ray, Cryo-EM) ExperimentalValidation->StructuralStudies BiologicalAssays Biological Assays and Resistance Testing ExperimentalValidation->BiologicalAssays

Virus-Host Protein Interaction Mapping Pipeline

The Scientist's Toolkit: Essential Research Reagents and Platforms

Table 3 catalogues essential research reagents, computational platforms, and experimental systems critical for implementing the target validation workflows described in this section.

Table 3: Essential Research Reagent Solutions for Target Validation

Category Specific Tool/Platform Application in Target Validation Key Features/Benefits
Computational Platforms VirtualFlow Ultra-large virtual screening of compound libraries Capable of screening billions of compounds; freely accessible
Protein Structure Prediction AlphaFold Protein Structure Database Predicting 3D structures of viral and bacterial targets High-accuracy structure predictions for targets without experimental structures
Interaction Assays NanoLuc Two-Hybrid (N2H), Luminescence-based Two-Hybrid (LuTHy) Identifying direct protein-protein interactions Quantitative measurement of binary interactions; higher throughput than traditional Y2H
Binding Assays Isothermal Titration Calorimetry (ITC), Surface Plasmon Resonance (SPR) Validating small molecule binding to targets Provides quantitative binding affinity measurements
Structural Biology Cryo-Electron Microscopy (Cryo-EM) Determining complex structures of target-inhibitor complexes Can handle larger complexes than X-ray crystallography
Metabolic Modeling Recon3D Predicting metabolic perturbations following target inhibition Comprehensive human metabolic network with >3,200 ORFs
Compound Libraries ZINC, Enamine REAL Source of compounds for virtual and high-throughput screening Billions of commercially available compounds in ready-to-dock format
Finasteride-d9Finasteride-d9, CAS:1217547-06-2, MF:C23H36N2O2, MW:381.6 g/molChemical ReagentBench Chemicals

The escalating crisis of antimicrobial resistance demands a paradigm shift in how we approach antibacterial and antiviral development. The WHO Bacterial Priority Pathogens List provides a crucial roadmap for focusing limited R&D resources on the most threatening resistant infections, while lessons from recent pandemics highlight the need for proactive antiviral development against emerging threats [1] [46]. The current pipeline remains dangerously inadequate, with only 5 innovative antibacterial agents in development targeting critical priority pathogens and a continued reliance on narrow-spectrum antiviral approaches [3] [47].

Moving forward, successful antimicrobial development will require integration of artificial intelligence throughout the drug discovery pipeline. AI is already transitioning from hype to practical application, enabling target discovery through mining of pathogen genomes, predicting resistance patterns, and generating initial inhibitor scaffolds with optimized pharmacological properties [52]. Furthermore, AI-driven clinical trial design can accelerate the translation of promising candidates by predicting enrollment sites with appropriate resistant isolates and simulating inclusion criteria against historical patient populations [52].

The economic challenges of antimicrobial development must also be addressed through innovative business models and public-private partnerships. The fragility of the current ecosystem – with 90% of preclinical antibacterial development driven by small firms – necessitates new funding mechanisms and market incentives to ensure viable antimicrobial pipelines [3]. Simultaneously, greater emphasis on real-world evidence generation can support appropriate use of novel agents and justify their placement in treatment guidelines, enhancing both commercial viability and patient access [52].

By leveraging the frameworks and methodologies outlined in this technical guide, researchers and drug development professionals can strategically navigate the complex landscape of antimicrobial discovery, prioritizing targets with the greatest potential to impact global health and overcome the mounting challenge of antimicrobial resistance.

The World Health Organization's (WHO) Bacterial Priority Pathogens List (BPPL) serves as a critical framework for guiding the global response to antimicrobial resistance (AMR), one of the most pressing public health challenges of our time. The 2024 WHO BPPL categorizes 24 antibiotic-resistant bacterial pathogens across 15 families into critical, high, and medium priority groups to strategically direct research and development (R&D) and public health interventions [1]. This technical guide examines evidence-based strategies for infection prevention, control, and vaccination, providing a comprehensive framework for researchers, scientists, and drug development professionals working to counteract these priority pathogens. The persistent threat requires a multifaceted approach, combining traditional infection control with innovative vaccine development and diagnostic tools to effectively address both current and emerging challenges in AMR.

The WHO Priority Pathogens List: A Strategic Framework for Public Health Action

The WHO BPPL represents a refined tool for prioritizing R&D efforts and investments in the AMR landscape, emphasizing the necessity for regionally tailored strategies to combat resistance effectively. The list highlights the global impact of pathogens based on criteria including disease burden, transmissibility, treatability, and available prevention options [1]. Notably, the 2024 update underscores several key bacterial threats:

  • Gram-negative bacteria resistant to last-resort antibiotics
  • Drug-resistant mycobacterium tuberculosis
  • Other high-burden resistant pathogens including Salmonella, Shigella, Neisseria gonorrhoeae, Pseudomonas aeruginosa, and Staphylococcus aureus [1]

The list specifically targets developers of antibacterial medicines, academic and public research institutions, research funders, and public-private partnerships investing in AMR R&D, providing a strategic roadmap for focusing limited resources on the most threatening pathogens [1].

Table 1: WHO Bacterial Priority Pathogens List 2024 - Critical Priority Pathogens

Pathogen Family Specific Pathogens Key Resistance Threats
Enterobacteriaceae Klebsiella pneumoniae, E. coli, Enterobacter spp. Carbapenem-resistant, ESBL-producing
Pseudomonas Pseudomonas aeruginosa Carbapenem-resistant
Acinetobacter Acinetobacter baumannii Carbapenem-resistant

Infection Prevention and Control in Healthcare Settings

Infection prevention and control (IPC) represents a practical, evidence-based approach to preventing patients and health workers from avoidable infections during healthcare delivery. Effective IPC requires constant action at all levels of the health system and is universally relevant to every health worker and patient, at every health care interaction [42]. The fundamental framework for IPC includes standardized precautions applied to all patients, supplemented by transmission-based protocols for specific pathogens.

Transmission-Based Precautions and Protocols

Healthcare facilities must implement a tiered approach to infection prevention, beginning with standard precautions for all patient interactions and adding specific measures based on pathogen characteristics and transmission routes:

  • Standard Precautions: Include hand hygiene, use of personal protective equipment (PPE), appropriate patient placement, cleaning and disinfection of patient care equipment, management of textiles and laundry, safe injection practices, and proper disposal of needles and other sharp objects [53].

  • Contact Precautions: Used for patients with known or suspected infections transmitted by direct or indirect contact. Requires standard precautions plus use of dedicated equipment, thorough cleaning strategies, and limited patient transport. Indicated for C. difficile, multidrug-resistant organisms, and draining wounds [53].

  • Droplet Precautions: For pathogens transmitted through respiratory droplets during coughing, sneezing, or talking. Patients should wear masks and transport should be limited. Used for respiratory infections, meningitis, and other specified conditions [53].

  • Airborne Precautions: Necessary for infections transmitted via airborne droplet nuclei that remain suspended in the air. Requires negative pressure isolation rooms, respirators, and sterile gowns/gloves. Essential for tuberculosis, measles, chickenpox, and disseminated zoster [53].

The following diagram illustrates the decision framework for implementing these core infection control precautions within a healthcare setting:

InfectionControlFramework Start Patient Presentation StandardPrec Implement Standard Precautions Start->StandardPrec Assessment Assess Transmission Risk StandardPrec->Assessment Contact Contact Precautions Assessment->Contact Known/suspected contact transmission Droplet Droplet Precautions Assessment->Droplet Known/suspected droplet transmission Airborne Airborne Precautions Assessment->Airborne Known/suspected airborne transmission MRSA Multidrug-resistant organisms Draining wounds C. difficile Contact->MRSA Resp Respiratory infections Meningitis in infants Droplet->Resp TB Tuberculosis Measles Chickenpox Airborne->TB

Healthcare-Associated Infection Surveillance and Outbreak Management

Effective IPC programs require robust surveillance systems to monitor healthcare-associated infection (HAI) rates and identify outbreaks. Surveillance should prioritize high-risk areas including intensive care units, hematology/oncology units, and surgical departments, though hospital-wide surveillance is increasingly mandated [53]. Modern electronic health records enable sophisticated algorithmic surveillance, identifying patients at highest risk for HAIs. When infection rates exceed established thresholds or clusters are identified, outbreak investigations should be initiated, utilizing molecular techniques such as pulsed-field gel electrophoresis or whole-genome sequencing for precise pathogen tracking [53].

The Society for Healthcare Epidemiology of America (SHEA) Compendium of Strategies provides evidence-based guidance for preventing specific HAIs, including central line-associated bloodstream infections (CLABSI), catheter-associated urinary tract infections (CAUTI), Clostridium difficile infections (CDI), methicillin-resistant Staphylococcus aureus (MRSA) infections, and surgical site infections (SSI) [54]. Implementation of bundled interventions for these infections has demonstrated significant reductions in HAI rates across diverse healthcare settings.

Vaccine Development and Deployment Strategies

Vaccines represent a crucial tool in preventing infections and reducing antibiotic use, thereby combating AMR. However, vaccine development faces substantial challenges including long timelines (up to 15 years), high costs (approximately $1 billion per licensed vaccine), and low probability of market entry (approximately 10%) [55].

De-risking Vaccine Development Through Correlates of Protection

A critical strategy for accelerating vaccine development involves identifying and utilizing correlates of protection (CoPs) – immune markers that predict protection against clinical disease. CoPs can de-risk and reduce the size of late-stage clinical studies by supporting dose selection, pivotal data generation, and immuno-bridging between populations [55]. Established examples include:

  • Meningococcal vaccines: Serum bactericidal activity (SBA) assays served as the basis for licensure of MenC conjugate vaccines, with a defined titer threshold (<1:4) associated with protection [55].

  • Pneumococcal vaccines: An aggregate CoP (serotype-specific IgG level of 0.35 μg/mL) was established from efficacy studies and incorporated into WHO guidance for licensure of higher-valency vaccines [55].

  • RSV vaccines: Neutralizing antibody titers defined through monoclonal antibody studies provided thresholds for assessing maternal vaccine responses and establishing proof-of-concept [55].

The workflow below illustrates how CoPs are identified and applied throughout the vaccine development process:

VaccineDevelopment Preclinical Preclinical Studies EpiStudies Seroepidemiological Studies Preclinical->EpiStudies ImmuneResp Immune Response Analysis Preclinical->ImmuneResp Challenge Human Challenge Studies Preclinical->Challenge CoP Correlate of Protection Established EpiStudies->CoP ImmuneResp->CoP Challenge->CoP Application CoP Application CoP->Application Dose Dose/Regimen Selection Application->Dose Bridging Immuno-bridging Studies Application->Bridging Licensure Accelerated Licensure Application->Licensure

Vaccination in Special Populations: Immunocompromised Patients

Immunocompromised patients represent a critical population for targeted vaccination strategies due to their increased susceptibility to severe outcomes from respiratory infections. The Infectious Diseases Society of America (IDSA) 2025 guidelines provide specific recommendations for this population:

Table 2: IDSA 2025 Recommendations for Respiratory Virus Vaccination in Immunocompromised Patients

Vaccine Type Recommendation Certainty of Evidence Key Considerations
COVID-19 Strong recommendation for age-appropriate 2025-2026 vaccination Moderate certainty Second dose likely extends protection; household contacts should also be vaccinated
Influenza Strong recommendation for age-appropriate 2025-2026 vaccination Moderate certainty High-dose or adjuvanted vaccines preferred; contraindicate live-attenuated vaccines
RSV Strong recommendation for age-appropriate vaccination Moderate certainty For patients <18 years, use shared decision-making; vaccinate household contacts

For COVID-19 vaccination, immunocompromised patients show vaccine effectiveness of 33-56% against hospitalization, 40% against critical illness, and 61% against COVID-19-related mortality, though protection wanes over time [56]. The 2025-2026 COVID-19 vaccination schedule varies by age and prior vaccination status, with specific recommendations for different age groups [57].

Diagnostic Innovations for Targeted Therapy

Rapid and accurate diagnostics are essential components of effective infection control, enabling appropriate antibiotic use and early implementation of transmission-based precautions. WHO's landscape analysis of diagnostics for priority bacterial pathogens identifies persistent gaps, particularly in low-resource settings [3]. Critical diagnostic needs include:

  • Multiplex platforms suitable for intermediate referral laboratories to identify bloodstream infections directly from whole blood without culture
  • Biomarker tests (e.g., C-reactive protein, procalcitonin) to distinguish bacterial from viral infections
  • Point-of-care tools for primary and secondary care facilities that are affordable, robust, and easy-to-use [3]

The ideal diagnostic development pathway addresses testing needs across the healthcare system, from community settings to tertiary hospitals, as illustrated below:

DiagnosticPathway Primary Primary Care Point-of-care tests Rapid results Minimal training Secondary Secondary Care Multiplex platforms Biomarker tests Moderate complexity Primary->Secondary Tertiary Tertiary Care Advanced molecular platforms Whole-genome sequencing High complexity Secondary->Tertiary Sample Sample Types: Blood, urine, stool, respiratory specimens Need Unmet Need: Culture-independent sample-in/result-out systems

Experimental Protocols and Research Methodologies

Quantitative Benefit-Risk Assessment of Medical Countermeasures

Multi-criteria decision analysis (MCDA) provides a structured framework for quantitatively evaluating the benefit-risk profiles of vaccines and therapeutics, particularly important during public health emergencies. The IMI-PROTECT Benefit-Risk Group has established methodology for quantitative benefit-risk assessment [58]. The protocol includes:

  • Establishing the assessment objective (e.g., compare BR profiles of COVID-19 vaccines)
  • Identifying key benefits and risks in a value tree structure
  • Selecting and extracting relevant data from clinical trials, observational studies, and adverse event monitoring
  • Scoring and weighting each criterion based on expert input
  • Evaluating the benefit-risk balance and conducting sensitivity analyses [58]

For COVID-19 vaccine assessment, key benefit criteria include prevention of infection in adults ≥18 years, prevention in seniors ≥60 years, and prevention of severe COVID-19. Risk criteria include adverse events and serious adverse events [58]. This methodology enables systematic comparison of medical products and supports transparent decision-making for policymakers and health professionals.

Research Reagent Solutions for AMR Research

Table 3: Essential Research Reagents for Antimicrobial Resistance Studies

Reagent Category Specific Examples Research Applications
Molecular Typing Reagents Pulsed-field gel electrophoresis (PFGE) reagents, Whole-genome sequencing kits Outbreak investigation, pathogen tracking, transmission dynamics
Susceptibility Testing Antimicrobial gradient strips, Broth microdilution panels, PCR assays for resistance genes Phenotypic and genotypic AMR profiling, MIC determination
Immunological Assays ELISA kits for antibody detection, SBA assay components, Cytokine panels Correlate of protection studies, vaccine immunogenicity evaluation
Cell Culture Models Human cell lines, Growth media, Antimicrobial agents In vitro efficacy testing, mechanism of action studies
Animal Challenge Models Specialized rodent models, Inoculation materials, Pathogen strains In vivo efficacy evaluation, pathogenesis studies

Addressing the threat posed by WHO priority pathogens requires an integrated approach combining robust infection prevention, strategic vaccination, rapid diagnostics, and antimicrobial stewardship. The declining pipeline of innovative antibacterial agents – with only 90 in clinical development in 2025, down from 97 in 2023, and only 5 effective against critical priority pathogens – underscores the urgent need for enhanced R&D investment and innovation [3]. Furthermore, coordination across sectors and implementation of regionally tailored strategies are essential to effectively combat AMR. By leveraging the WHO BPPL as a strategic roadmap, researchers, public health officials, and drug development professionals can align efforts to address the most pressing threats and mitigate the growing impact of antimicrobial resistance on global health.

Navigating R&D Challenges and Optimizing Strategies for Priority Pathogen Countermeasures

Antimicrobial resistance (AMR) is a grave global health threat, directly causing an estimated 1.91 million deaths annually and associated with nearly 5 million more [59]. Projections indicate that without effective intervention, AMR could lead to 10 million deaths per year by 2050, imposing a catastrophic burden on healthcare systems and the global economy [59]. The World Health Organization (WHO) has responded by establishing a Bacterial Priority Pathogens List (BPPL), a critical tool to guide research and development (R&D) against the most dangerous drug-resistant bacteria [1]. The 2024 BPPL categorizes 24 pathogens across 15 families into critical, high, and medium priority groups, highlighting those for which new antibiotics are most urgently needed [1].

Despite this clear and present danger, the antibacterial drug development pipeline is both insufficient and fragile. A significant disconnect exists between global public health needs and the economic incentives that drive pharmaceutical innovation. This has resulted in a market failure—often described as a "broken market"—where the scientific imperative to develop new antibiotics is not matched by a viable commercial model. Major pharmaceutical companies have largely exited the field; since the 1990s, 18 major firms have abandoned antibacterial R&D [59] [60]. This exodus is primarily driven by a combination of scientific challenges, regulatory hurdles, and profound economic barriers that make sustained investment in antibiotic development unsustainable under current market conditions [59]. This whitepaper analyzes these economic hurdles within the context of the WHO BPPL and outlines the coordinated global response required to revitalize the pipeline.

Analysis of the Current Antibacterial Development Landscape

The WHO Priority Pathogens List and the Clinical Pipeline

The WHO BPPL is designed to direct R&D efforts toward the most threatening drug-resistant bacteria. The list is categorized as follows [1]:

  • Critical Priority: Includes Gram-negative bacteria resistant to last-resort antibiotics, such as carbapenem-resistant Acinetobacter baumannii (CRAB), Pseudomonas aeruginosa, and Enterobacterales.
  • High and Medium Priority: Includes other high-burden resistant pathogens such as Salmonella, Shigella, Neisseria gonorrhoeae, and Staphylococcus aureus.

An analysis of the clinical pipeline against these priorities reveals a state of crisis. As of 2025, the number of antibacterial agents in clinical development has decreased to 90, down from 97 in 2023 [3]. Among these 90 agents, only 50 are traditional antibacterial drugs, with the remainder being non-traditional agents like bacteriophages and antibodies [3]. Most alarmingly, the pipeline suffers from a critical lack of innovation. Of the traditional agents in development, only 15 are considered innovative, and for 10 of these, data are insufficient to confirm no cross-resistance exists [3]. A mere five of the agents in development are effective against at least one "critical" priority pathogen [3].

Table 1: Analysis of the Antibacterial Clinical Pipeline (2025)

Pipeline Metric Number Context and Implication
Total Clinical Agents 90 Down from 97 in 2023, indicating a shrinking pipeline.
Traditional Antibacterial Agents 50 The core of new chemical entities being developed.
Innovative Agents 15 Only a small fraction represent truly novel approaches.
Agents Targeting WHO "Critical" Pathogens 5 Grossly inadequate for the most urgent threats.
New Antibiotics Approved (Since 2017) 17 Only two of these represented a new chemical class.

The preclinical pipeline, while more active with 232 programs, is fragile as it is dominated by small firms (90% have fewer than 50 employees), which are highly vulnerable to financial instability [3].

The Prevailing Economic Hurdles and Market Failures

The collapse of private-sector investment in antibiotics is a direct consequence of several interconnected economic realities that define the "broken market."

  • Low Return on Investment (ROI): Unlike medications for chronic conditions (e.g., hypertension, diabetes) that patients may take for years, antibiotics are typically prescribed for short courses, lasting days or weeks. This fundamental usage pattern severely limits the revenue potential of any single antibiotic product [59] [61]. Consequently, the net present value of a new antibiotic is often close to zero or negative, making it impossible to justify the high costs of R&D compared to other therapeutic areas [61].

  • Stewardship vs. Commercial Success: To preserve the efficacy of new antibiotics for as long as possible, they are rightly placed under strict antimicrobial stewardship protocols and often classified as "Watch" or "Reserve" drugs by the WHO. This means they are used sparingly and only as a last resort [59] [61]. While critical for public health, this practice directly undermines commercial viability. As one expert noted, it is akin to selling a car that can only be driven on Sundays between 8 a.m. and 10 a.m. [61].

  • High Development Costs and Attrition: The cost of discovering, developing, and securing regulatory approval for a new drug is extraordinarily high, often exceeding $1 billion. This cost is compounded in the antibiotic field by high rates of failure and the scientific difficulty, particularly in overcoming Gram-negative bacterial defenses [59] [50].

  • Pricing and Reimbursement Challenges: Despite the high societal value of effective antibiotics, health systems and payers have been resistant to paying premium prices for them. This is partly because the economic value of an antibiotic—preventing hospitalizations, enabling complex surgeries, and saving lives—is not fully captured in the price of the drug itself [59].

Table 2: Key Economic Hurdles in Antibacterial Drug Development

Economic Hurdle Impact on Development Consequence
Poor Revenue Model Short-duration therapy limits sales volume and revenue. Inability to recoup R&D costs or generate profit.
Antimicrobial Stewardship Deliberately limited use of new, effective agents. Suppressed market demand even for breakthrough drugs.
High R&D Costs & Risk Significant upfront investment with high risk of failure. Deters investment; capital flows to less risky areas.
Inadequate Pricing Models Prices do not reflect full societal value of effective antibiotics. Perpetuates the low-return business model.

Innovative Strategies and Methodologies to Overcome Economic Hurdles

Addressing the broken antibiotic market requires a multi-pronged approach that de-risks R&D, creates viable commercial pathways, and fosters scientific innovation.

Pull Incentives and New Commercial Models

A critical strategy recognized by global health authorities is the implementation of "pull incentives." These are rewards granted upon the successful development and approval of a new antibiotic, designed to create a predictable and sufficient return on investment, independent of sales volume.

  • Examples of Pull Incentives:
    • Market Entry Rewards: Large, upfront payments from governments or international consortia to developers of high-priority antibiotics. This delinks profit from volume sold and ensures the drug is available while being used sparingly.
    • Subscription Models: Governments or health systems pay an annual subscription fee for access to a portfolio of novel antibiotics, regardless of how much is used. The UK and Sweden are pioneers of this model [60].
    • Transferable Exclusivity Vouchers: Granting a voucher for extended market exclusivity on another, more profitable drug in a company's portfolio as a reward for developing a new antibiotic.

The 2024 UN High-Level Meeting on AMR endorsed the need for such incentives and set a target of mobilizing $100 million in catalytic financing [59] [62]. However, beyond pilots, widespread adoption of pull incentives has been slow [60].

Leveraging Artificial Intelligence and Novel Discovery Platforms

Scientific innovation that lowers the cost and increases the efficiency of early-stage R&D is crucial. Artificial Intelligence (AI) is emerging as a transformative tool in this domain.

  • AI in Drug Discovery: Companies like Atomwise use deep learning models to virtually screen billions of chemical compounds to predict which will effectively inhibit bacterial targets, dramatically reducing the time and cost of initial candidate identification [63].
  • Partnership Models: Collaborations between big pharma and AI specialists are growing. For instance, Pfizer extended its partnership with CytoReason, investing $20 million to use AI for its drug development programs, including for infectious diseases [63].

Public Funding and Global Coordination

Given the market failure, public and philanthropic funding remains the bedrock of early-stage antibacterial research. However, this funding is vulnerable to political shifts and budget cuts, as seen with recent U.S. NIH funding terminations that jeopardized promising discoveries like the new antibiotic class lariocidin [61].

Global coordination is essential to optimize finite resources. A recent ReAct policy brief recommends [62]:

  • Strengthening the AMR Multi-Partner Trust Fund (MPTF): As the only dedicated multilateral fund for AMR, it must be expanded and better resourced.
  • Establishing an AMR Financing Coordination Hub: To provide guidance and improve coherence among existing funding mechanisms.
  • Enhancing Domestic Ownership: Countries must integrate AMR into national health budgets and plans to ensure long-term sustainability.

The following diagram illustrates the interconnected economic challenges and the multi-level solutions required to address them.

G cluster_problems Economic Hurdles (The Broken Market) cluster_solutions Integrated Solutions Framework P1 Low Return on Investment (ROI) P5 Major Pharma Exodus P1->P5 P2 High R&D Cost & Attrition P2->P5 P3 Stewardship Limits Sales P3->P1 P4 Inadequate Pricing P4->P1 P6 Fragile SME Pipeline P5->P6 S1 Pull Incentives: Market Entry Rewards, Subscriptions O1 Revitalized & Sustainable Antibacterial Pipeline S1->O1 Creates Market S2 Push Funding: Public Grants, R&D Subsidies S2->O1 De-risks R&D S3 Scientific Innovation: AI, Novel Discovery Platforms S3->O1 Lowers Cost S4 Global Coordination: MPTF, Financing Hubs S4->O1 Optimizes Resources S5 Policy & Pricing Reform: Value-based Pricing S5->O1 Aligns Incentives

Figure 1: Mapping the broken antibiotic market and its potential solutions. Economic hurdles (red) create a vicious cycle that pushes major players out of the field. A coordinated framework of solutions (green) is required to rebuild a sustainable pipeline.

The Scientist's Toolkit: Key Reagents and Methodologies

Advancing novel antibacterial agents requires a sophisticated toolkit to identify and validate new targets, particularly against priority pathogens. The following table details essential reagents and their applications in modern antibacterial discovery.

Table 3: Key Research Reagent Solutions for Antibacterial Discovery

Research Reagent / Tool Function in Antibacterial Discovery
Structure-Based Drug Design (SBDD) Enables precise inhibitor design by visualizing drug-target interactions at the atomic level. Critical for developing novel β-lactam/β-lactamase inhibitor combinations [63].
AI-Driven Compound Screening Platforms Uses deep learning to predict molecule-bacteria interactions, virtually screening billions of compounds to identify high-probability candidates (e.g., Atomwise platform) [63].
Microfluidic Point-of-Care (POC) Diagnostic Devices Rapidly identifies causative pathogens and their resistance profiles directly from patient samples, enabling targeted therapy and supporting antimicrobial stewardship [63].
Advanced Animal Infection Models Preclinical models (e.g., mouse thigh or lung infection) are essential for validating the in vivo efficacy of lead compounds against WHO priority pathogens like CRAB [60].
Novel Target Validation Assays High-throughput in vitro assays to test compound activity against newly identified, essential bacterial targets (e.g., the LptB2FGC transport complex in CRAB) [60] [50].

Experimental Protocol: In Vitro and In Vivo Efficacy Assessment

A typical workflow for evaluating a novel antibacterial compound, such as the recently discovered lariocidin or Roche's zosurabalpin, involves a multi-stage experimental process [60] [61].

1. Target Identification and Validation:

  • Method: Employ genetic (e.g., CRISPRi), biochemical, and bioinformatic approaches to identify a target that is essential for bacterial survival and not shared by humans.
  • Example: For zosurabalpin, the target was validated as the LptB2FGC complex, which transports lipopolysaccharide in Gram-negative bacteria.

2. In Vitro Characterization of Lead Compounds:

  • Minimum Inhibitory Concentration (MIC) Assay:
    • Procedure: Serial dilutions of the test compound are prepared in a broth medium in a 96-well plate. A standardized inoculum (~5 x 10^5 CFU/mL) of the target pathogen (e.g., a CRAB strain) is added to each well. The plate is incubated at 35°C for 16-20 hours.
    • Analysis: The MIC is the lowest concentration of the antibiotic that completely prevents visible growth. This determines the compound's potency.
  • Cytotoxicity Assay:
    • Procedure: The lead compound is tested against mammalian cell lines (e.g., HEK-293 or HepG2) using a method like MTT or Alamar Blue. Cells are exposed to a range of compound concentrations for 24-72 hours.
    • Analysis: The concentration that kills 50% of mammalian cells (CC50) is calculated. A high selectivity index (CC50/MIC) indicates a safe therapeutic window.

3. In Vivo Efficacy Studies:

  • Animal Model: Typically a murine (mouse) model of infection.
  • Procedure:
    • Infection Induction: Mice are rendered neutropenic via cyclophosphamide and then infected via intramuscular (thigh) or intranasal (lung) inoculation with a defined CFU of the target pathogen.
    • Treatment: Animals are randomized into groups receiving the test compound (at various doses), a vehicle control, and a standard-of-care antibiotic. Treatment is administered subcutaneously or intravenously at defined intervals post-infection.
    • Endpoint: After ~24 hours, animals are euthanized, and the infected tissue (thigh or lung) is harvested, homogenized, and plated for bacterial CFU enumeration.
  • Analysis: The log10 reduction in bacterial burden in treated groups compared to the vehicle control group is the primary measure of in vivo efficacy.

The development of new antibacterial drugs to combat WHO priority pathogens is paralyzed by a profound and persistent market failure. The economic realities of low returns, high costs, and necessary stewardship have stifled the private investment required to sustain a robust pipeline. While scientific innovation from small and medium-sized enterprises and academia continues, it is not enough to overcome these structural economic hurdles.

A sustainable future for antibacterial medicine depends on a fundamental restructuring of the market. This requires the urgent, coordinated implementation of pull incentives, increased and stable public funding, and global policy coordination that collectively value antibiotics as essential societal goods. The 2024 UN High-Level Meeting's commitment to catalytic funding is a positive step, but it must be rapidly translated into concrete action. Without a new economic model that realigns commercial viability with public health necessity, the world risks regressing to a pre-antibiotic era, where routine infections and medical procedures once again become life-threatening.

The Global Burden of Gram-Negative Resistance

Antimicrobial resistance (AMR), particularly from Gram-negative pathogens, represents one of the most pressing global public health crises of our time. In 2019 alone, bacterial AMR was associated with approximately 4.95 million deaths worldwide, with Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii accounting for a substantial proportion of this mortality [18]. Projections indicate that without effective intervention, AMR could cause over 10 million deaths annually by 2050 [18]. The World Health Organization's 2024 report reveals alarming trends: one in six laboratory-confirmed bacterial infections globally were resistant to antibiotic treatments in 2023, with resistance rising in over 40% of monitored pathogen-antibiotic combinations between 2018 and 2023 [19].

The challenge is particularly acute for Gram-negative bacteria due to their complex cell envelope structure, which includes an outer membrane that acts as a formidable barrier to antimicrobial entry [64]. Additionally, these pathogens possess and rapidly acquire sophisticated resistance mechanisms, including enzymatic drug inactivation through expanded-spectrum β-lactamases (ESBLs) and carbapenemases, efflux pumps, target site modifications, and biofilm formation [18] [65]. The situation is further exacerbated by the decline in antibiotic discovery and development, with many large pharmaceutical companies de-investing from this area due to scientific and economic challenges [64].

WHO Priority Pathogens: A Framework for Action

The World Health Organization's 2024 Bacterial Priority Pathogens List (WHO BPPL) strategically categorizes antibiotic-resistant bacterial pathogens to guide research and development (R&D) and public health interventions [1]. This list categorizes 24 pathogens across 15 families into critical, high, and medium priority groups based on criteria including global burden, transmissibility, treatability, and prevention options [1] [18].

Table 1: WHO Bacterial Priority Pathogens List 2024 - Gram-Negative Bacteria

Priority Category Pathogens Key Resistance Profiles
Critical Acinetobacter baumannii Carbapenem-resistant [18]
Enterobacterales Carbapenem-resistant, third-generation cephalosporin-resistant [18]
High Pseudomonas aeruginosa Carbapenem-resistant [18]
Salmonella Typhi Fluoroquinolone-resistant [18]
Shigella spp. Fluoroquinolone-resistant [18]
Neisseria gonorrhoeae Third-generation cephalosporin-resistant, fluoroquinolone-resistant [18]
Non-typhoidal Salmonella Fluoroquinolone-resistant [18]

The critical priority pathogens, notably carbapenem-resistant Acinetobacter baumannii and carbapenem-resistant Enterobacterales, are characterized by extremely limited or absent therapeutic options, high associated morbidity and mortality, rapidly increasing resistance trends, and difficult-to-control transmission mechanisms [18]. Surveillance data from the WHO GLASS report indicates that more than 40% of E. coli and over 55% of K. pneumoniae globally are now resistant to third-generation cephalosporins, which are first-line treatments for serious bloodstream infections [19]. Furthermore, resistance to last-resort carbapenem antibiotics, once rare, is becoming increasingly frequent, narrowing treatment options to a dangerous degree [19].

Established and Emerging Resistance Mechanisms in Gram-Negative Pathogens

Gram-negative bacteria employ a diverse and sophisticated array of resistance mechanisms, often in combination, leading to multidrug-resistant (MDR), extensively drug-resistant (XDR), and even pan-drug-resistant (PDR) phenotypes [18]. Understanding these mechanisms is fundamental to developing effective countermeasures.

Enzymatic Inactivation

The production of β-lactamases remains the most prevalent mechanism of resistance to β-lactam antibiotics, which are the most widely used antibiotic class.

  • Extended-Spectrum β-Lactamases (ESBLs): These enzymes, predominantly of the TEM, SHV, and CTX-M types, hydrolyze penicillins, cephalosporins, and aztreonam but are generally inhibited by β-lactamase inhibitors like clavulanate [65]. They are commonly found in E. coli and K. pneumoniae.
  • AmpC β-Lactamases: These are typically chromosomally encoded in organisms like Enterobacter spp., Citrobacter freundii, and Serratia marcescens but can be plasmid-mediated, facilitating spread. AmpC enzymes confer resistance to penicillins, cephalosporins, cephamycins, and β-lactam/β-lactamase inhibitor combinations (with the exception of the newer combinations like ceftazidime-avibactam) [65]. Derepression of AmpC production can lead to treatment failures.
  • Carbapenemases: These enzymes hydrolyze carbapenems, last-resort antibiotics, and are categorized into three main classes:
    • Class A (KPC): Common in K. pneumoniae, often treatable with novel β-lactam/β-lactamase inhibitor combinations like ceftazidime-avibactam [66].
    • Class B (Metallo-β-Lactamases, MBLs): Including NDM, VIM, and IMP variants, these use zinc ions for catalysis and are not inhibited by avibactam, vaborbactam, or relebactam. They represent a major treatment challenge [66].
    • Class D (OXA-type): OXA-48-like enzymes are common in Enterobacterales, while OXA-23 and OXA-58 are associated with Acinetobacter baumannii [66].

Non-Enzymatic Mechanisms

  • Efflux Pumps: Overexpression of multi-component efflux systems (e.g., AcrAB-TolC in Enterobacterales, MexAB-OprM in P. aeruginosa) can extrude multiple, structurally unrelated drug classes, contributing to MDR phenotypes [18] [65].
  • Reduced Permeability: Modifications to outer membrane porins, such as loss or downregulation of OmpF and OmpC in Enterobacterales or OprD in P. aeruginosa, limit the intracellular accumulation of antibiotics [65].
  • Target Modification: Mutations in genes encoding penicillin-binding proteins (PBPs), DNA gyrase, and topoisomerase IV can reduce the binding affinity of β-lactams and fluoroquinolones, respectively [18].
  • Biofilm Formation: The formation of structured microbial communities encased in a polymeric matrix provides a physical and physiological barrier that significantly increases resistance to antimicrobial agents and host immune responses [67].

The following diagram illustrates the interplay of these primary resistance mechanisms in a Gram-negative bacterial cell.

GramNegativeResistance cluster_outer Outer Membrane cluster_periplasm Periplasm cluster_cytoplasm Cytoplasm/Cell Membrane BacterialCell Gram-Negative Bacterial Cell PorinLoss Porin Loss/Alteration (Reduced Permeability) PorinLoss->BacterialCell EnzymaticInactivation Enzymatic Inactivation (e.g., β-lactamases) EnzymaticInactivation->BacterialCell EffluxPump Efflux Pump Overexpression EffluxPump->BacterialCell TargetModification Target Site Modification TargetModification->BacterialCell Biofilm Biofilm Formation Biofilm->BacterialCell Antibiotic Antibiotic Antibiotic->PorinLoss Blocked Antibiotic->EnzymaticInactivation Inactivated Antibiotic->EffluxPump Extruded Antibiotic->TargetModification No Binding Antibiotic->Biofilm Trapped

The Pipeline of Novel Therapeutic Strategies

The fight against Gram-negative resistance is being waged on multiple fronts, encompassing both novel antibiotic classes and non-traditional antimicrobial therapies.

Recently Approved and Late-Stage Antibiotics

In response to the MDR crisis, several new antimicrobials and combinations have been approved or are in advanced clinical development. These agents are designed to overcome specific, prevalent resistance mechanisms.

Table 2: Novel Antimicrobial Agents for Resistant Gram-Negative Infections

Agent(s) Drug Class Mechanism of Action Target Spectrum & Key Resistances
Cefiderocol [66] Siderophore cephalosporin Trojan horse strategy; uses bacterial iron transport systems to cross outer membrane Broad GNB; stable against all β-lactamases (KPC, MBL, OXA). Not effective against GPB or anaerobes.
Imipenem-Cilastatin-Relebactam [66] Carbapenem + BLI Relebactam inhibits Class A/C β-lactamases; imipenem kills cell GNB, GPB, anaerobes. Active vs. KPC, AmpC, ESBL. Not active vs. MBL, OXA.
Meropenem-Vaborbactam [66] Carbapenem + BLI Vaborbactam inhibits Class A/C β-lactamases; meropenem kills cell GNB, GPB, anaerobes. Active vs. KPC, AmpC, ESBL. Not active vs. MBL, OXA.
Aztreonam-Avibactam [66] Monobactam + BLI Avibactam protects aztreonam from Ambler Class A/C/D enzymes; Aztreonam stable to MBLs Crucial for MBL-producing Enterobacterales.
Sulbactam-Durlobactam [68] BL/BLI Durlobactam inhibits Class A/C/D β-lactamases & Penicillin-Binding Proteins (PBPs); Sulbactam also targets PBPs Specifically developed for CRAB.
Cefepime-Taniborbactam [66] Cephalosporin + BLI Taniborbactam inhibits Class A/C β-lactamases & some Class B (MBLs) Broad-spectrum; potentially active against some KPC & MBL co-producers.
TGV-49 [67] Membrane-targeting polymer Positively charged groups bind & disrupt microbial membrane, causing leakage & cell lysis Broad-spectrum; low resistance rate in experimental evolution.

Non-Antibiotic Therapeutic Approaches

Beyond traditional small molecules, innovative non-antibiotic therapies are emerging, many of which have progressed to compassionate use or clinical trials.

  • Bacteriophage (Phage) Therapy: Phages are viruses that specifically infect and lyse bacteria. They offer a highly targeted approach, can disrupt biofilms, and are often used in combination with antibiotics [64]. Two primary development models exist: 1) On-demand personalized therapy, where phages are selected from large banks based on the patient's isolate susceptibility (e.g., the Magistral Phage system in Belgium), and 2) Fixed-composition phage products for specific pathogens or conditions, which have a clearer regulatory pathway but cannot adapt to new strains as easily [64]. Phage therapy has shown efficacy in compassionate use cases for a variety of infections, including those caused by P. aeruginosa, E. coli, K. pneumoniae, and A. baumannii [64].
  • Anti-Virulence Agents: These compounds target bacterial pathogenicity factors (e.g., toxins, secretion systems, quorum-sensing) without directly killing the bacteria, potentially reducing selective pressure for resistance [64].
  • Antimicrobial Peptides (AMPs): These are short, naturally occurring peptides that disrupt microbial membranes and have immunomodulatory activities. Their development is challenging due to issues with stability, toxicity, and production cost [64].
  • Immunotherapy and Monoclonal Antibodies: These strategies aim to enhance or modulate the host immune response to clear bacterial infections. This includes antibodies that neutralize specific bacterial toxins or virulence factors [64].

The workflow for evaluating a novel antimicrobial candidate, from discovery to resistance profiling, integrates both standard and advanced methodologies.

ExperimentalWorkflow InVitro In Vitro Susceptibility Testing Checkerboard Synergy Testing (Checkerboard Assay) InVitro->Checkerboard TimeKill Time-Kill Kinetics Assay InVitro->TimeKill BiofilmAssay Biofilm Eradication Assay InVitro->BiofilmAssay Morbidostat Experimental Evolution (Morbidostat) InVitro->Morbidostat AnimalModel In Vivo Animal Models TimeKill->AnimalModel BiofilmAssay->AnimalModel WGS Whole Genome Sequencing (WGS) Morbidostat->WGS Mechanism Mechanism of Action Studies WGS->Mechanism Mechanism->AnimalModel

Experimental Protocols for Resistance Assessment

Morbidostat-Driven Experimental Evolution

Purpose: To quantitatively measure the rate and genetic pathways of spontaneous resistance development to a novel antimicrobial agent under controlled, escalating drug pressure [67].

Methodology:

  • Setup: A computer-controlled continuous culturing bioreactor (morbidostat) is inoculated with the bacterial strain of interest (e.g., Acinetobacter baumannii ATCC 17978).
  • Culture Monitoring: The optical density (OD~600~) of the bacterial culture is continuously monitored in real-time.
  • Feedback Loop: The morbidostat algorithm performs automated dilutions based on bacterial growth:
    • Drug-containing media is added to increase drug concentration when bacterial growth exceeds a set threshold.
    • Drug-free media is added to decrease drug concentration when growth is inhibited due to excessive drug pressure.
  • Outcome: This dynamic process applies gradual and sustained selective pressure, driving the evolution of resistant subpopulations. Isolates are periodically sampled from the morbidostat for further analysis [67].

Downstream Analysis:

  • Minimum Inhibitory Concentration (MIC) Determination: The broth macrodilution method, as per CLSI guidelines (e.g., M27-A3), is used to track MIC changes over time in evolved isolates [67].
  • Whole Genome Sequencing (WGS): Clonal isolates from different time points are sequenced to identify mutations (single nucleotide polymorphisms, indels, amplifications) conferring resistance. This links genotype to phenotype [67].

Broth Macrodilution for MIC Determination

Purpose: To determine the minimum inhibitory concentration (MIC) of an antimicrobial agent, defined as the lowest concentration that inhibits visible bacterial growth [67].

Methodology:

  • Preparation: Prepare a standardized bacterial inoculum (e.g., 5 x 10^5 CFU/mL) in Mueller-Hinton Broth (MHB).
  • Dilution Series: Create a two-fold serial dilution of the antimicrobial agent in tubes containing MHB.
  • Inoculation: Add the standardized bacterial inoculum to each tube.
  • Incubation: Incubate the tubes at the appropriate temperature (e.g., 37°C for most human pathogens) for 16-20 hours.
  • Reading Results: The MIC is the lowest concentration of antimicrobial agent that shows no turbidity (OD~570~ increase ≤ 0.05) [67]. Interpretive categories (susceptible, intermediate, resistant) are assigned based on CLSI or EUCAST clinical breakpoints.

The Scientist's Toolkit: Essential Research Reagents and Solutions

Table 3: Key Research Reagents for Antimicrobial Resistance Studies

Reagent / Material Function & Application Example from Search Results
Reference Bacterial Strains Essential controls for susceptibility testing and experimental evolution studies. A. baumannii (ATCC BAA-1605, BAA-1710), K. pneumoniae (ATCC BAA-1705), P. aeruginosa (ATCC BAA-2108) [67].
Clinical Isolate Collections Provide genetically diverse, clinically relevant isolates for evaluating drug efficacy and prevalence of resistance. Collections from healthcare institutions; used to test TGV-49 against pathogens like K. pneumoniae (VT-2646), P. aeruginosa (VT-7530) [67].
Morbidostat Device Advanced bioreactor for conducting experimental evolution studies under precise, escalating drug pressure to probe resistance development. Custom-engineered device used to evolve A. baumannii under TGV-49 pressure, revealing minimal resistance development [67].
Culture Media Standardized growth media for susceptibility testing and culturing. Essential for reproducibility. Mueller-Hinton Broth (MHB) and Columbia Blood Agar Base, as per CLSI standards [67].
Comparative Antimicrobial Agents Benchmark compounds for comparing the activity of novel agents against current standard-of-care treatments. Ampicillin, meropenem, ciprofloxacin, polymyxin B, etc. (all from Sigma-Aldrich) [67].

The battle against Gram-negative bacterial resistance is at a critical juncture. The convergence of a deepening AMR crisis, the nuanced prioritization by the WHO, and the emergence of innovative therapeutic strategies defines the current landscape. While the pipeline of new antibiotics, particularly those combining novel β-lactamase inhibitors with established β-lactams, has provided crucial new options, the continued evolution of resistance—especially via metallo-β-lactamases—demands relentless innovation. The most promising path forward lies in a multi-pronged approach that includes: 1) the development of agents with truly novel, non-β-lactam mechanisms of action, such as membrane-targeting polymers like TGV-49; 2) the clinical validation of non-traditional therapies like bacteriophages; and 3) the implementation of robust global surveillance and stewardship programs to preserve the efficacy of both existing and new therapies. Success will require sustained collaboration between researchers, clinicians, industry, and public health agencies worldwide to ensure that the scientific advances in the laboratory translate into effective treatments for patients facing these formidable infections.

The World Health Organization's 2024 Bacterial Priority Pathogens List (WHO BPPL) categorizes 15 families of antibiotic-resistant bacteria into critical, high, and medium priority groups, representing some of the most significant threats to global public health [1]. These pathogens, including Gram-negative bacteria resistant to last-resort antibiotics and drug-resistant Mycobacterium tuberculosis, underscore the evolving challenges of antimicrobial resistance (AMR) [5]. Despite their recognized danger, a critical gap persists in our ability to rapidly and affordably detect these pathogens, particularly in low-resource settings where the burden of infectious diseases is often highest [69]. The COVID-19 pandemic demonstrated the transformative value of accessible testing while simultaneously revealing stark disparities in diagnostic access between high-income and low- to middle-income countries (LMICs) [70].

This technical guide examines the current landscape of rapid diagnostic tests (RDTs) for priority pathogens, analyzes persistent technological gaps, details emerging diagnostic platforms, and outlines essential experimental protocols and reagent solutions. The development of low-cost, rapid diagnostics that meet REASSURED criteria (Real-time connectivity, Ease of specimen collection, Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free, and Deliverable to end users) represents a critical component of comprehensive AMR control strategies and pandemic preparedness efforts [69] [70]. For researchers and drug development professionals working at the intersection of public health and diagnostic innovation, addressing these gaps requires both technical excellence and deep understanding of the implementation contexts where these tools will be deployed.

The WHO Priority Pathogens List: A Diagnostic Targeting Framework

The 2024 WHO BPPL builds upon the 2017 edition and refines the prioritization of antibiotic-resistant bacterial pathogens through a multicriteria decision analysis framework [5]. The list serves as a crucial guide for prioritizing research and development (R&D) and investments in AMR, emphasizing the need for regionally tailored strategies to effectively combat resistance [1]. Pathogens were scored based on eight evidence-based criteria: mortality, non-fatal burden, incidence, 10-year resistance trends, preventability, transmissibility, treatability, and antibacterial pipeline status [5].

Table 1: 2024 WHO Bacterial Priority Pathogens List - Categorization and Key Examples

Priority Tier Pathogen Examples Key Resistance Phenotypes
Critical Klebsiella pneumoniae, Acinetobacter baumannii, Mycobacterium tuberculosis Carbapenem resistance, Rifampicin resistance
High Salmonella enterica serotype Typhi, Shigella spp., Neisseria gonorrhoeae Fluoroquinolone resistance, Third-generation cephalosporin resistance
Medium Group A streptococci, Haemophilus influenzae, Group B streptococci Macrolide resistance, Ampicillin resistance

The list highlights the particular threat of Gram-negative bacteria, which dominate the critical priority category, along with rifampicin-resistant Mycobacterium tuberculosis [5]. Among bacteria commonly responsible for community-acquired infections, fluoroquinolone-resistant Salmonella enterica serotype Typhi, Shigella spp., and Neisseria gonorrhoeae received high rankings [5]. The inclusion of these pathogens underscores their global impact in terms of burden, as well as issues related to transmissibility, treatability, and prevention options, and reflects the R&D pipeline of new treatments and emerging resistance trends [1].

Beyond bacterial pathogens, the WHO R&D Blueprint also identifies high priority viral diseases requiring urgent diagnostic attention, including COVID-19, Crimean-Congo haemorrhagic fever, Ebola virus disease, Marburg virus disease, Lassa fever, MERS-CoV, Nipah virus, Rift Valley fever, Zika, and "Disease X" (representing a pathogen currently unknown to cause human disease) [69]. The 100 Days Mission, a global initiative embraced by the G7 and G20, aims to prepare the world for the next pandemic by driving the development of diagnostics, vaccines, and therapeutics within 100 days of recognition of a novel Disease X threat, with RDTs playing a pivotal role in this ambitious timeline [69].

The Diagnostic Gap Analysis: Current Limitations and Unmet Needs

Landscape of Available Diagnostics

According to WHO analyses, the current pipeline for both antibacterial agents and diagnostics remains insufficient to address the growing threat of antimicrobial resistance [3]. The 2025 WHO report on antibacterial agents in clinical and preclinical development revealed that the number of antibacterials in the clinical pipeline has decreased from 97 in 2023 to 90 in 2025, with only 15 qualifying as innovative and merely 5 being effective against at least one of the WHO "critical" priority bacteria [3]. This scarcity and lack of innovation in the therapeutic pipeline increases the pressure on diagnostic systems to enable optimal use of existing antibiotics through antimicrobial stewardship.

Complementary WHO analyses of diagnostics have identified persistent gaps, particularly concerning the detectability of BPPL pathogens in low-resource settings [3]. These limitations include the absence of multiplex platforms suitable for use in intermediate referral (level II) laboratories to identify bloodstream infections directly from whole blood without culture, insufficient access to biomarker tests to distinguish bacterial from viral infections, and limited simple, point-of-care diagnostic tools for primary and secondary care facilities [3]. These limitations disproportionately affect patients in low-resource settings, where most people first present at primary health-care facilities [3].

Limitations of Current Rapid Diagnostic Tests

Currently available RDTs for high priority pathogens face significant challenges related to clinical sensitivity and specificity, inadequate validation, and insufficient investment in development [69]. For many high priority viral pathogens such as Nipah, MERS-CoV, and Lassa fever, highly sensitive, specific, and validated RDTs simply do not exist [69]. This diagnostic void severely compromises early case detection and management, impedes effective surveillance, and undermines outbreak response capabilities.

The COVID-19 pandemic demonstrated that even when RDTs become available, significant disparities exist in their global distribution and implementation. Many people in LMICs found themselves unable to obtain testing—either home or lab-based—a problem that existed long before COVID-19 for diseases like HIV and tuberculosis [70]. As noted by diagnostics specialist Tivani Mashamba-Thompson, "We were actually living with a pandemic already. We were living with the HIV pandemic, with a tuberculosis pandemic—these needed the same systems, but nothing was ready" [70].

Table 2: Diagnostic Gaps for Selected WHO Priority Pathogens

Pathogen Category Example Pathogens Current Diagnostic Status Major Gaps
Critical Priority Bacteria Carbapenem-resistant K. pneumoniae, A. baumannii Culture-based AST (days); limited molecular tests Lack of rapid phenotypic AST at point-of-care
Diarrheal Pathogens V. cholerae, Shigella spp., ETEC Culture (2-3 days); some recent RDT developments Limited use of rapid tests in outbreak settings
High Priority Viruses Nipah, Lassa, MERS-CoV PCR at reference labs only; no commercial RDTs No validated RDTs available for frontline use

Emerging Technological Solutions and Platforms

Molecular Diagnostics for Resource-Limited Settings

Innovative approaches are emerging to address the diagnostic gap for priority pathogens. Researchers from Johns Hopkins Bloomberg School of Public Health developed a Rapid Loop-mediated Isothermal Amplification (LAMP) based Diagnostic Test (RLDT) that detects cholera from a stool or water sample in minutes at a fraction of the cost of lab-based tests [71]. This platform addresses critical limitations of traditional diagnostic methods through several key advantages: testing performed directly from wet or dried stool samples or contaminated water; complete testing process within approximately 1 hour; high specificity and sensitivity detecting both O1 and cholera toxin genes; easy-to-use self-contained kit with all reagents; no cold-chain or electricity requirements; and point-of-care capability in endemic settings [71].

The RLDT platform has demonstrated successful implementation for enterotoxigenic E. coli (ETEC) and Shigella in Zambia, where the diseases are endemic, and is currently being evaluated in multiple South Asian and African countries [71]. The technology shows particular promise for phase III Shigella vaccine trials because it is a rapid, simple, and sensitive assay that can be used for case detection and treatment as a point-of-care tool in health care facilities [71].

Advanced Biosensing Platforms

At Arizona State University, researchers have developed a breakthrough diagnostic tool called NasRED (Nanoparticle-Supported Rapid Electronic Detection) that uses just a single drop of blood, costs a couple of dollars, and delivers results in only 15 minutes [72]. The platform utilizes gold nanoparticles engineered to detect extremely small amounts of disease-related proteins, achieving sensitivity that the developers claim is "even better than lab-based tests" [72].

The core technology employs nanoparticles coated with special molecules designed to detect specific diseases—some carrying antibodies that stick to proteins released by viruses or bacteria, others carrying antigens that attract antibodies produced by the body to fight infections [72]. The device shines a small beam of LED light through liquid containing the sample and nanoparticles, with a custom electronic detector sensing how much light passes through to determine disease presence [72]. The platform is highly modular, allowing researchers to easily swap in different proteins to adapt the same platform for many different diseases, including Shiga toxin-producing E. coli, cancer biomarkers, Alzheimer's-related proteins, Lyme disease, and African swine fever [72].

Machine Learning-Enhanced Detection

A novel integration of machine learning with molecular agglutination assays has demonstrated potential for rapid pathogen detection. This approach utilizes a wash-free molecular agglutination assay with straightforward mixing and incubation steps that significantly simplify molecular testing procedures [73]. By targeting the 16S rRNA of pathogens (which exists in more than 10,000 copies per bacterial cell), researchers achieve rapid pathogen identification within 30 minutes on a dark-field imaging microfluidic cytometry platform [73].

The innovation applies machine learning algorithms to deconvolute topological features of agglutinated clusters and thus quantify bacterial abundance [73]. In clinical studies for urinary tract infections, this approach distinguished Escherichia coli positive from other E. coli negative among 50 clinical samples with 96% sensitivity and 100% specificity [73]. The platform also applied the same protocols to achieve rapid antimicrobial susceptibility testing within 3 hours, offering potential for clinical treatment guidance [73].

G Rapid LAMP Diagnostic Test (RLDT) Workflow SampleCollection Sample Collection (Stool/Water) SampleProcessing Sample Processing (<5 minutes, minimal hands-on) SampleCollection->SampleProcessing LAMPAmplification Isothermal Amplification (LAMP, 45-60°C) SampleProcessing->LAMPAmplification Detection Pathogen Detection (Visual or electronic readout) LAMPAmplification->Detection Result Result Interpretation (~1 hour total process) Detection->Result

Diagram 1: Rapid LAMP Diagnostic Test (RLDT) Workflow

Experimental Protocols for Diagnostic Development

Loop-Mediated Isothermal Amplification (LAMP) Assay Protocol

The RLDT platform employs a carefully optimized LAMP protocol suitable for low-resource settings [71]:

Sample Preparation:

  • Collect stool samples (0.5-1g) in sterile containers or 50-100mL water samples
  • For dried stool samples, use specialized collection cards with sample preservation matrix
  • Prepare samples by resuspending in 1mL of lysis buffer (20 mM Tris-HCl, 2 mM EDTA, 1.2% Triton X-100)
  • Heat sample at 95°C for 5 minutes to lyse cells and release nucleic acids
  • Centrifuge at 12,000 × g for 2 minutes to pellet debris
  • Transfer supernatant to fresh tube for immediate use or storage at -20°C

LAMP Reaction Setup:

  • Prepare lyophilized reaction pellets containing:
    • 1.6 µM each of inner primers (FIP/BIP)
    • 0.2 µM each of outer primers (F3/B3)
    • 0.8 µM each of loop primers (LF/LB)
    • 1.4 mM dNTPs
    • 0.8 M betaine
    • 20 mM Tris-HCl
    • 10 mM (NH4)2SO4
    • 10 mM KCl
    • 8 mM MgSO4
    • 0.1% Tween 20
    • Bst DNA polymerase (8 units)
  • Rehydrate pellets with 23 µL of nuclease-free water
  • Add 2 µL of prepared sample template
  • Include positive and negative controls in each run

Amplification and Detection:

  • Incubate reaction at 65°C for 45-60 minutes
  • Visual results: Observe color change from blue to sky blue with hydroxynaphthol blue
  • Electronic readout: Use handheld battery-operated device for fluorescence measurement
  • Specificity validation: Test against related pathogen strains to confirm no cross-reactivity

Molecular Agglutination Assay with Machine Learning

This protocol enables rapid pathogen detection without nucleic acid amplification [73]:

Probe Design and Immobilization:

  • Design two specific oligonucleotide probes (20-25 bases each) complementary to target 16S rRNA
  • Synthesize probes with C12-linker and biotinylated ends to reduce steric hindrance
  • Immobilize probes on magnetic microparticles (1-2μm diameter) using streptavidin-biotin interaction
  • Wash probe-coated microparticles with hybridization buffer to remove unbound probes
  • Resuspend in storage buffer at 4°C until use

Sample Processing and Hybridization:

  • Lysate bacterial cells using enzymatic lysis buffer (lysozyme + proteinase K)
  • Centrifuge at 5,000 × g for 5 minutes to remove debris
  • Mix 50μL lysate with equal volume of probe-coated microparticles
  • Add 100μL high-stringency hybridization buffer (5× SSC, 0.1% SDS)
  • Incubate at 55°C for 15 minutes with gentle shaking

Image Acquisition and Analysis:

  • Load mixture into microfluidic channel with capillary action
  • Use narrow beam scanning dark-field microscopy for image acquisition
  • Capture 30 frames per second using CMOS imager (e.g., smartphone camera)
  • Apply machine learning algorithm for cluster analysis:
    • Preprocessing: Background subtraction and noise reduction
    • Feature extraction: Cluster size, shape, texture, and scattering intensity
    • Classification: Random forest algorithm trained on known positive/negative samples
    • Quantification: Correlation between cluster features and bacterial concentration

Validation and Quality Control:

  • Test with serial dilutions of target bacteria to establish limit of detection
  • Validate specificity against non-target bacterial species
  • Include internal control particles in each assay
  • Establish threshold values for positive/negative classification

G Molecular Agglutination Detection Workflow cluster_sample Sample Processing cluster_detection Detection & Analysis Lysate Bacterial Lysate (16S rRNA release) Hybridization Solution Hybridization (15-30 minutes, 55°C) Lysate->Hybridization Probes Probe-Coated Magnetic Particles Probes->Hybridization Microfluidic Microfluidic Flow (Capillary action) Hybridization->Microfluidic Imaging Dark-Field Imaging (CMOS/smartphone) Microfluidic->Imaging MLAnalysis Machine Learning Cluster Analysis Imaging->MLAnalysis Result Quantitative Result (Pathogen ID & Concentration) MLAnalysis->Result

Diagram 2: Molecular Agglutination Detection Workflow

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Research Reagents for Diagnostic Development

Reagent/Material Function Implementation Example Considerations
Bst DNA Polymerase Isothermal amplification LAMP-based assays [71] High strand displacement activity, thermal stability
Lyophilized Reagents Stability without cold chain RLDT for cholera, ETEC, Shigella [71] Pre-mixed master mixes, long-term storage
Gold Nanoparticles Signal generation and amplification NasRED platform [72] Tunable optical properties, surface functionalization
Magnetic Microparticles Target capture and separation Molecular agglutination assays [73] Surface chemistry for probe immobilization
Loop Primers Accelerate LAMP amplification RLDT platform [71] Design specificity for target pathogen
CMOS Imagers Signal detection and readout Smartphone-based diagnostics [73] Cost-effectiveness, widespread availability
Paper Substrates Fluidic control without instrumentation Lateral flow assays [70] Wickling properties, reagent immobilization
Cluster Analysis Algorithms Quantitative agglutination interpretation Machine learning-enhanced detection [73] Training dataset requirements, validation

The development of low-cost, rapid diagnostics for WHO priority pathogens represents both an urgent public health need and a formidable technological challenge. Current gaps in the diagnostic pipeline disproportionately affect low-resource settings where the burden of infectious diseases is highest [3] [69]. Emerging technologies such as isothermal amplification platforms, nanoparticle-enhanced detection, and machine learning-assisted interpretation offer promising paths forward, particularly when designed with REASSURED criteria in mind [71] [72] [73].

Success in this field requires multidisciplinary collaboration across microbiology, engineering, data science, and public health. Future development should prioritize: (1) multiplexed platforms capable of detecting multiple pathogens and resistance markers simultaneously; (2) enhanced connectivity for real-time surveillance and data reporting; (3) simplified sample processing to minimize hands-on time; and (4) robust validation in diverse field settings to ensure reliability across implementation contexts [3] [70]. Additionally, sustainable diagnostic development must address not only technical performance but also manufacturing scalability, regulatory approval pathways, and implementation frameworks that ensure equitable access [69] [70].

As the global community strengthens its preparedness for future epidemics and pandemics through initiatives like the 100 Days Mission, rapid diagnostics will play an increasingly critical role in the early detection and containment of outbreaks [69]. By focusing development efforts on the pathogens identified in the WHO priority lists and designing for the constraints of low-resource settings, researchers and drug development professionals can contribute significantly to global health security and antimicrobial resistance control.

The 2024 World Health Organization (WHO) Bacterial Priority Pathogens List (BPPL) outlines 24 antibiotic-resistant bacterial pathogens that pose a critical threat to global public health, with carbapenem-resistant Klebsiella pneumoniae identified as the highest priority [1] [31]. This technical guide proposes a novel paradigm: the repurposing of the "Family Approach," a proven model from addiction therapy, to optimize the research and development (R&D) pipeline for medical countermeasures. This whitepaper provides a detailed framework for implementing this approach, including quantitative pathogen analysis, structured experimental protocols, and visualization of integrated workflows, to accelerate the delivery of therapeutics against the most urgent antimicrobial resistance (AMR) threats.

Antimicrobial resistance represents one of the most pressing public health challenges of our time, directly or indirectly contributing to an estimated 4.95 million global deaths annually [31]. The 2024 WHO BPPL is a critical tool for guiding the global response, categorizing pathogens into three priority tiers—critical, high, and medium—based on eight criteria, including mortality, nonfatal burden, incidence, 10-year resistance trends, preventability, transmissibility, treatability, and the status of the antibacterial R&D pipeline [1] [31].

Despite the approval of 13 new antibiotics since the 2017 BPPL, resistance trends continue to outpace development [31]. The traditional, siloed model of drug discovery is ill-suited to this complex, evolving threat. This paper introduces the "Family Approach" as a transformative strategy for pipeline optimization. In its original context, this approach involves actively engaging family members in substance use disorder treatment, creating a collaborative, non-blaming system focused on shared outcomes and holistic support [74]. When transposed to pathogen R&D, this translates to a deeply integrated, cross-disciplinary pipeline where information, resources, and strategies are shared across projects targeting pathogen "family" members, thereby eliminating redundancies and accelerating discovery.

The following table synthesizes the key pathogens from the 2024 WHO BPPL, providing a structured overview for targeting R&D efforts.

Table 1: 2024 WHO Bacterial Priority Pathogens List (Critical and High Priority)

Priority Tier Pathogen Key Resistance Phenotype Quantitative Score (%)
Critical Klebsiella pneumoniae Carbapenem-resistant 84 [31]
Critical Acinetobacter baumannii Carbapenem-resistant N/A [1]
Critical Escherichia coli Carbapenem-resistant N/A [31]
Critical Mycobacterium tuberculosis Rifampicin-resistant N/A [31]
High Salmonella enterica Serotype Typhi Fluoroquinolone-resistant 72 [31]
High Shigella spp. Antibiotic-resistant 70 [31]
High Neisseria gonorrhoeae Antibiotic-resistant 64 [31]
High Pseudomonas aeruginosa Antibiotic-resistant N/A [1]
High Staphylococcus aureus Antibiotic-resistant N/A [1]

Note: N/A indicates that a specific quantitative score was not provided in the source material, though the pathogen is confirmed in the stated priority tier.

The "Family Approach": From Behavioral Health to Biodefense

Core Principles and Their R&D Analogues

The Family Approach in behavioral health is built on systems theory, viewing the individual as part of an interconnected system. Its core principles, when adapted for pathogen R&D, offer a powerful new operational model [74].

Table 2: Translating Core Principles from Behavioral Health to R&D

Principle in Behavioral Health [74] Translated Principle for Pathogen R&D
Involving family members in treatment Creating integrated, cross-disciplinary project teams
A non-blaming, collaborative approach Fostering pre-competitive data and resource sharing consortia
Expanding outcome measures to family well-being Defining success by portfolio-wide metrics and public health impact
Valuing relationships and social networks Leveraging open-source databases, AI tools, and shared biorepositories
Adapting methods to family culture and community Tailoring therapeutic strategies to regional pathogen prevalence and resistance patterns

Repurposed Workflow: An Integrated Pipeline Model

The following diagram visualizes the optimized, repurposed R&D pipeline, which replaces traditional linear stages with a parallel, integrated system.

family_approach_pipeline Integrated R&D Pipeline Using Family Approach cluster_shared Shared Foundation (Pathogen Family Core) SharedDB Shared Data & AI Platform (Genomics, Resistome, HTS) Critical Critical Priority Pathogen Project SharedDB->Critical High High Priority Pathogen Project SharedDB->High Medium Medium Priority Pathogen Project SharedDB->Medium SharedRepo Reagent & Compound Library (Common Targets & Chemotypes) SharedRepo->Critical SharedRepo->High SharedRepo->Medium Critical->High Shared Learnings Output Accelerated Candidate Therapeutics Critical->Output High->Critical Shared Learnings High->Medium Shared Learnings High->Output Medium->High Shared Learnings Medium->Output

Experimental Protocol: A Cross-Pathogen Screening Cascade

This section details a standardized, high-throughput screening (HTS) protocol designed to evaluate compound libraries against multiple priority pathogens simultaneously, embodying the "Family Approach."

Detailed HTS Methodology

Objective: To identify broad-spectrum lead compounds with activity against critical and high-priority Gram-negative pathogens. Primary Strains:

  • Critical: Carbapenem-resistant K. pneumoniae (CRKP), A. baumannii (CRAB), P. aeruginosa (CRPA) [1].
  • Control: Pan-susceptible E. coli (ATCC 25922).

Procedure:

  • Inoculum Preparation: Grow bacterial strains to mid-log phase (OD₆₀₀ ≈ 0.5) in cation-adjusted Mueller-Hinton Broth (CA-MHB). Dilute to a final density of 5 × 10⁵ CFU/mL in the assay plate.
  • Compound Dispensing: Pin-transfer 50 nL of compounds (from a 10 mM DMSO stock) from the shared library into sterile 384-well polypropylene plates. Final test concentration is 10 µg/mL.
  • Assay Incubation: Add 50 µL of the bacterial inoculum to each well. Include growth control (bacteria + DMSO) and sterility control (media only) wells. Seal plates and incubate at 35°C for 18 hours.
  • Viability Readout: Add 10 µL of resazurin indicator (0.01% w/v) to each well. Incubate for 2-4 hours and measure fluorescence (Ex560/Em590). A ≥90% reduction in fluorescence compared to the growth control is considered a positive hit.
  • Hit Confirmation: Retest all primary hits in a 10-point, 2-fold dilution series (64 to 0.125 µg/mL) in duplicate to determine minimum inhibitory concentration (MIC). A compound is advanced if MIC ≤ 8 µg/mL for at least two of the three target pathogens.

The Scientist's Toolkit: Key Research Reagent Solutions

Table 3: Essential Reagents for Cross-Pathogen Screening

Reagent / Material Function / Rationale Example Specification
Cation-Adjusted Mueller-Hinton Broth (CA-MHB) Standardized medium for reproducible antimicrobial susceptibility testing (AST). Compliant with CLSI M07 guidelines.
Resazurin Sodium Salt Cell-permeant oxidation-reduction indicator for measuring cell viability in HTS. Ready-to-use solution at 0.01% (w/v) in sterile water.
384-Well Assay Plates Microtiter plates for high-density, low-volume screening. Black-walled, clear-bottom, tissue culture-treated.
Clinical Isolate Panel Characterized, multidrug-resistant strains of WHO priority pathogens. Panels should include CRKP, CRAB, CRPA, and ESBL-positive E. coli.
Frozen "Ready-To-Pin" Compound Library A centralized, curated collection of diverse chemical entities for screening. 100,000+ compounds in 10 mM DMSO, pre-plated in 384- or 1536-well format.

Data Integration and Analysis Framework

A centralized data platform is the backbone of the Family Approach, enabling the synthesis of information from disparate projects.

Centralized Data Analysis Workflow

The following diagram outlines the flow of data from primary assays to shared knowledge, facilitating machine learning and predictive modeling.

data_workflow Centralized Data Analysis Workflow HTS HTS & MIC Data CentralAI Centralized AI/ML Platform (Structure-Activity-Relationship Model) HTS->CentralAI Genomics Genomic & Transcriptomic Data Genomics->CentralAI Chemo Chemical Structure & ADMET Chemo->CentralAI SAR Validated SAR Hypotheses CentralAI->SAR Candidates Prioritized Lead Candidates CentralAI->Candidates Models Predictive Models for Compound Efficacy CentralAI->Models Models->HTS Informs New Library Design

The 2024 WHO BPPL presents a clear and urgent call to action. Optimizing the R&D pipeline through the repurposed Family Approach—emphasizing integrated workflows, shared resources, and collaborative data analysis—offers a transformative path forward. By treating the set of priority pathogens as an interconnected system rather than a collection of discrete targets, the global research community can accelerate the delivery of critically needed medical countermeasures. This strategy promises to enhance efficiency, reduce redundant costs, and ultimately, mitigate the devastating global health impact of antimicrobial resistance.

The World Health Organization (WHO) updated its Bacterial Priority Pathogens List (BPPL) in 2024, providing a critical framework for guiding the global response to antimicrobial resistance (AMR), which was associated with an estimated 4.95 million deaths worldwide in 2019 [31]. This technical guide addresses a pivotal yet often undervalued front in this battle: the integration of non-R&D interventions. While the clinical pipeline for new antibacterial agents has decreased from 97 in 2023 to 90 in 2025, with a concerning lack of innovation, the strategic implementation of hand hygiene, antimicrobial stewardship, and measures to ensure equitable access to existing tools presents a powerful, immediate strategy to mitigate the impact of priority pathogens and preserve the efficacy of existing antimicrobials [3]. This document provides researchers, scientists, and drug development professionals with the data, protocols, and implementation frameworks necessary to advance these essential non-R&D pillars within the context of WHO BPPL analysis.

Table 1: 2024 WHO Bacterial Priority Pathogens List (BPPL) - Critical Priority Level

Pathogen Key Resistance Phenotype Rationale for Priority Tier
Klebsiella pneumoniae Carbapenem-resistant Highest score (84%) in evaluation; high burden, treatability challenges, and resistance trends [31].
Acinetobacter spp. Carbapenem-resistant Gram-negative bacteria with high mortality and resistance to most newer antibiotics [31].
Escherichia coli Carbapenem-resistant Gram-negative bacterium ranking in the highest quartile for burden and resistance [31].
Mycobacterium tuberculosis Rifampicin-resistant Remains a critical priority pathogen due to global disease burden and treatment challenges [31].

The Foundation: Hand Hygiene in Healthcare Settings

Quantitative Evidence and Impact on AMR

Hand hygiene is the most effective and feasible infection prevention and control measure within healthcare facilities, capable of reducing healthcare-associated infections by up to 55% [75] [76]. Its impact extends directly to combating AMR by preventing infections in the first place, thereby reducing antibiotic use. The CDC reports that handwashing can prevent about 30% of diarrhea-related sicknesses and about 20% of respiratory infections, which are common drivers of antibiotic consumption. This reduction helps prevent the overuse of antibiotics—the single most important factor leading to antibiotic resistance globally [77].

Despite its proven efficacy, adherence among healthcare workers (HCWs) remains suboptimal, with rates ranging from 21% in Ethiopia to 84.3% in Australia, highlighting significant disparities and implementation challenges [76]. A 2025 systematic review of 28 qualitative studies identified four interconnected thematic barriers to adherence: behavioral, organizational, physical, and societal/interpersonal barriers [76].

Experimental Protocols and Workflow for Hand Hygiene Observation

Objective: To assess and quantify hand hygiene adherence among healthcare workers according to the WHO "My Five Moments for Hand Hygiene" framework.

Methodology: Direct observation using a structured checklist is the gold standard for measuring adherence [78] [76].

  • Pre-Observation Phase:

    • Train Observers: Observers must undergo standardized training to ensure consistent identification of the "Five Moments" and accurate data recording. Inter-rater reliability should be assessed.
    • Define Scope and Setting: Determine the clinical units for observation (e.g., ICU, surgical wards) and obtain necessary ethical approvals.
    • Develop Data Collection Tool: Create a checklist that records: HCW profession, specific moment for hand hygiene, type of procedure (clean/aseptic), and whether action was taken (soap and water vs. alcohol-based hand rub vs. not performed).

  • Observation Phase:

    • Positioning: The observer should be positioned to minimize the "Hawthorne effect" (where behavior changes due to awareness of being watched) while maintaining patient privacy.
    • Data Recording: For each observed patient-care activity, the observer records all opportunities for hand hygiene and the HCW's corresponding action or inaction.

  • Post-Observation Phase:

    • Data Analysis: Calculate adherence rates: (Number of times hand hygiene was performed / Total number of opportunities) x 100.
    • Feedback: Provide unit-specific and role-specific feedback to HCWs and management to inform targeted interventions.

G cluster_pre Pre-Observation Phase cluster_observation Observation Phase cluster_post Post-Observation Phase Start Hand Hygiene Observation Workflow Pre1 1. Train Observers Start->Pre1 Pre2 2. Define Scope & Setting Pre1->Pre2 Pre3 3. Develop Data Tool Pre2->Pre3 Obs1 4. Position Observer Pre3->Obs1 Obs2 5. Record Opportunities & Actions per WHO '5 Moments' Obs1->Obs2 Post1 6. Calculate Adherence Rate Obs2->Post1 Post2 7. Provide Feedback to Staff Post1->Post2

Hand Hygiene Observation Workflow

Research Reagents and Solutions for Hand Hygiene Studies

Table 2: Key Reagents and Materials for Hand Hygiene Research

Item Function/Application in Research
Alcohol-Based Hand Rub (ABHR) Primary intervention for hand hygiene when hands are not visibly soiled; used in studies comparing efficacy, adherence, and skin tolerance [78].
Antimicrobial Soap (e.g., Chlorhexidine) Used for handwashing when hands are visibly soiled or in scenarios involving spore-forming organisms (e.g., C. difficile); compared against plain soap and ABHR [78].
Adenosine Triphosphate (ATP) Bioluminescence Meters Provides quantitative, real-time measurement of organic matter on hands as a proxy for cleanliness; used for rapid feedback in intervention studies [76].
Structured Observation Checklists Standardized data collection tools based on the WHO "Five Moments" to ensure consistent and reliable measurement of adherence rates [78] [76].
Culture Media (e.g., Tryptic Soy Agar) Used for hand imprint or broth sampling techniques to quantify colony-forming units (CFUs) before and after hand hygiene, providing microbiological efficacy data [78].

Antimicrobial Stewardship and Advancing Health Equity

Antimicrobial stewardship (AMS) is inherently linked to health equity, defined as "the state in which everyone has a fair and just opportunity to attain their highest level of health" [79]. Health inequities, driven by social determinants of health (SDOH), lead to differential infection risks and antibiotic use [79]. For instance, disparities in access to quality healthcare and health literacy can result in delayed treatment seeking, higher rates of complicated infections, and consequently, inappropriate antibiotic use. Pharmacoequity—ensuring all individuals have access to high-quality medications regardless of race, ethnicity, or socioeconomic status—must be a core goal of AMS programs [79].

Operationalizing Equity in Stewardship: Diagnostic Access

A critical barrier to equitable AMS is the disparity in access to diagnostics. The WHO's 2025 landscape analysis highlights persistent gaps, including [3]:

  • Absence of multiplex platforms for identifying bloodstream infections directly from whole blood in intermediate referral laboratories.
  • Insufficient access to biomarker tests (e.g., C-reactive protein, procalcitonin) to distinguish bacterial from viral infections.
  • Limited simple, point-of-care tools for primary and secondary care facilities, which disproportionately affects patients in low-resource settings.

These gaps can lead to prolonged empirical antibiotic therapy, fueling resistance. Stewardship programs must therefore advocate for investments in affordable, robust, and easy-to-use diagnostic platforms suitable for resource-limited settings [3].

Innovative Models for Equitable Access to Healthcare

Mobile Medical Units (MMUs) as a Scalable Solution

To address access barriers, innovative service delivery models like Mobile Medical Units (MMUs) have proven effective. A 2024 qualitative study in India identified MMUs as a critical intervention for providing equitable and convenient access to healthcare, including vaccination services, for underserved populations [80]. The study of Jivika Healthcare's "VaccineOnWheels" initiative identified four critical components for a successful operational model [80]:

  • Scalability: The ability to expand services to cover larger populations or new regions.
  • Affordability: Providing low-cost or free services to ensure financial barriers do not prevent access.
  • Replicability: The model can be successfully implemented in different geographic and cultural contexts.
  • Sustainability: Ensuring long-term operational and financial viability, often through public-private partnership (PPP) models.

MMUs are a cost-effective and scalable healthcare delivery model that can be easily replicated in primary healthcare service delivery, directly supporting efforts to combat priority pathogens by improving preventive care and early intervention [80].

G Core Core MMU Operational Model S Scalability Expand coverage to new regions Core->S A Affordability Provide low-cost services Core->A R Replicability Adapt model to new contexts Core->R S2 Sustainability Ensure long-term viability via PPP Core->S2 Outcomes Key Outcomes for AMR Control S->Outcomes A->Outcomes R->Outcomes S2->Outcomes O1 Improved Preventive Care Outcomes->O1 O2 Increased Vaccination Uptake Outcomes->O2 O3 Early Infection Detection Outcomes->O3

Mobile Medical Unit Operational Logic

Protocol for Assessing Barriers to Equitable Access

Objective: To identify context-specific barriers (physical, financial, cultural) that limit access to infection prevention, diagnostics, and treatment services in a target population.

Methodology: Mixed-methods approach, combining qualitative and quantitative data collection [75] [80].

  • Study Design: Cross-sectional assessment.
  • Data Collection:
    • In-depth Interviews (Qualitative): Conduct semi-structured interviews with key stakeholders, including healthcare providers, policymakers, and community health workers. Questions should explore perceptions of barriers, resource availability, and community trust.
    • Knowledge, Attitudes, and Practices (KAP) Surveys (Quantitative): Administer structured surveys to healthcare workers and community members. Sections should cover demographics, knowledge of infections/AMR, attitudes towards health services, and self-reported practices.
  • Data Analysis:
    • Qualitative Analysis: Transcribe interviews verbatim. Use software (e.g., MaxQDA) for thematic analysis, coding responses into emergent themes (e.g., "lack of infrastructure," "cost of transportation," "language barriers").
    • Quantitative Analysis: Use statistical software (e.g., STATA) to generate descriptive statistics for survey responses. Knowledge scores can be tabulated and compared across different HCW groups using non-parametric tests like the Mann-Whitney U test.

This protocol allows for a comprehensive understanding of access barriers, enabling the design of tailored interventions like MMUs or targeted education campaigns [75] [80].

Synthesis and Integrated Framework for Action

The fight against WHO priority pathogens demands a multi-pronged approach that complements R&D for new antibacterials. The evidence is clear: strengthening health systems through rigorous hand hygiene, equity-focused antimicrobial stewardship, and innovative access models like MMUs is not merely supportive but foundational to curbing AMR. As noted by Sati and colleagues, "Focused efforts and sustained investments" are needed, and these must extend beyond drug discovery to include "equitable access to existing medications, improved vaccine availability, and strengthened infection-prevention measures" [31]. Researchers and drug developers have a critical role in advocating for, designing, and evaluating these non-R&D interventions to create a more resilient global health ecosystem capable of withstanding the threat of antimicrobial resistance.

Benchmarking and Validating the 2024 Lists Through Historical and Global Comparison

The World Health Organization (WHO) Bacterial Priority Pathogens List (BPPL) serves as a critical framework for guiding global research, development, and public health strategies against antimicrobial resistance (AMR). First published in 2017, this list categorized antibiotic-resistant bacteria based on their urgency of threat, directing investments and antibiotic development toward the most pressing challenges [81]. The 2024 update represents a significant evolution of this tool, incorporating seven years of new evidence, enhanced surveillance data, and lessons learned from a rapidly changing AMR landscape, including impacts from the COVID-19 pandemic [82] [83]. This analysis details the key changes between the 2017 and 2024 editions, examining the refined methodology, specific pathogen additions and removals, shifts in priority rankings, and the broader implications for researchers and drug development professionals engaged in AMR mitigation.

Methodological Evolution in the 2024 BPPL

The 2024 BPPL maintains a consistent overall framework but enhances the rigor and nuance of its prioritization process. Similar to the 2017 edition, the list was developed using a multi-criteria decision analysis (MCDA) methodology [5] [84]. This structured approach scores and ranks pathogens against a defined set of criteria to ensure an objective and transparent prioritization process.

The 2024 exercise evaluated 24 antibiotic-resistant bacterial pathogens against eight core criteria [5] [83] [84]:

  • Mortality
  • Incidence
  • Non-fatal health burden
  • Trend of resistance (over 10 years)
  • Transmissibility
  • Preventability (in both healthcare and community settings)
  • Treatability
  • Antibacterial pipeline status (number of developing antibiotics likely to be effective)

A key methodological step was a preferences survey administered to 100 international experts to determine the relative weights of these criteria. The survey revealed that the highest weight was assigned to treatability and mortality, with equal but lesser weight given to incidence, non-fatal burden, transmissibility, and preventability [5] [84]. This weighting directly influenced the final ranking, ensuring it reflects the most critical concerns for public health and clinical management. The final ranking was highly stable, with sensitivity analyses showing no substantial changes based on experts' backgrounds or geographical origins [5].

The following diagram illustrates the core methodological workflow for developing the 2024 BPPL:

cluster_0 2024 BPPL Development Methodology Node1 Identify 24 Candidate Pathogens Node2 Score Pathogens Against 8 MCDA Criteria Node1->Node2 Node3 Expert Survey to Weight Criteria (n=78) Node2->Node3 Node4 Calculate Final Score & Rank Pathogens Node3->Node4 Node5 Group into Priority Tiers (Critical, High, Medium) Node4->Node5

The evolution from the 2017 to the 2024 BPPL involved strategic additions, removals, and priority shifts, resulting in a list that more accurately reflects the current global burden of antimicrobial resistance.

Table 1: Comprehensive Comparison of the 2017 and 2024 WHO BPPL

Priority Tier 2017 WHO BPPL Pathogens 2024 WHO BPPL Pathogens Change Rationale & Key Context
Critical Acinetobacter baumannii, carbapenem-resistantPseudomonas aeruginosa, carbapenem-resistantEnterobacteriaceae, carbapenem-resistant, ESBL-producing Acinetobacter baumannii, carbapenem-resistantEnterobacterales, third-generation cephalosporin-resistantEnterobacterales, carbapenem-resistantMycobacterium tuberculosis, rifampicin-resistant Additions:• 3GCRE: High burden, especially in neonatal sepsis in LMICs [83].• RR-TB: Major global impact; complex treatment [17] [83].Movement:• CRPA moved to High priority due to lower transmission and decreasing resistance in some regions [83] [84].
High Enterococcus faecium, vancomycin-resistantStaphylococcus aureus, methicillin-resistant, vancomycin-intermediate and resistantHelicobacter pylori, clarithromycin-resistantCampylobacter spp., fluoroquinolone-resistantSalmonellae, fluoroquinolone-resistantNeisseria gonorrhoeae, cephalosporin-resistant, fluoroquinolone-resistant Salmonella Typhi, fluoroquinolone-resistantShigella spp., fluoroquinolone-resistantEnterococcus faecium, vancomycin-resistantPseudomonas aeruginosa, carbapenem-resistantNon-typhoidal Salmonella, fluoroquinolone-resistantNeisseria gonorrhoeae, third-generation cephalosporin- and/or fluoroquinolone-resistantStaphylococcus aureus, methicillin-resistant Additions:• Shigella: Elevated from Medium priority; high diarrheal mortality in children and emerging in MSM populations in HICs [83].Removals:• H. pylori and Campylobacter: Removed based on updated evidence [83] [84].Refinements:• Salmonella split into Typhi and non-typhoidal for clearer targeting.
Medium Streptococcus pneumoniae, penicillin-non-susceptibleHaemophilus influenzae, ampicillin-resistantShigella spp., fluoroquinolone-resistant Group A streptococci, macrolide-resistantStreptococcus pneumoniae, macrolide-resistantHaemophilus influenzae, ampicillin-resistantGroup B streptococci, penicillin-resistant Additions:• Group A & B Streptococci: New to the list; increasing resistance concerns in vulnerable populations in LMICs [17] [83].Changes:• S. pneumoniae criteria changed from penicillin-non-susceptible to macrolide-resistant, reflecting evolving resistance patterns [84].

Analysis of Key Changes and Strategic Shifts

Critical Priority: Refining the Highest Tier

The critical priority tier saw the most significant structural changes. A major update was the addition of rifampicin-resistant Mycobacterium tuberculosis (RR-TB) as a standalone critical priority pathogen [17] [83]. While TB was acknowledged in 2017, its explicit inclusion in 2024 underscores its massive global burden—1.3 million deaths in 2022—and the profound challenges in diagnosing and treating drug-resistant forms [83]. Another critical addition is third-generation cephalosporin-resistant Enterobacterales (3GCRE), highlighted due to its association with high rates of treatment failure and increased mortality, particularly in cases of neonatal sepsis in low- and middle-income countries (LMICs) [17] [83]. Conversely, carbapenem-resistant Pseudomonas aeruginosa (CRPA) was moved from critical to high priority. This demotion reflects surveillance data showing a lower transmission capability compared to other carbapenem-resistant pathogens and an actual decrease in resistance rates in at least one WHO region [17] [83] [84].

High & Medium Priority: Emphasizing Community Pathogens and Vulnerable Populations

The 2024 update demonstrates a heightened focus on the threat of AMR outside hospital settings. Fluoroquinolone-resistant Shigella was elevated from medium to high priority, recognizing its role as the second leading cause of diarrheal mortality globally, disproportionately affecting children in LMICs and emerging in men who have sex with men in high-income countries [83]. This "increase in the priority of community pathogens... reflects growing concern about these pathogens and their resistance to antibiotics" [83]. The medium-priority tier was refreshed with new additions relevant to community and vulnerable populations: macrolide-resistant Group A Streptococci and penicillin-resistant Group B Streptococci [17] [83]. These pathogens represent a significant and growing challenge in paediatric and elderly populations, especially in resource-limited settings [17].

Removals and Refinements: Reflecting Dynamic Resistance Landscapes

The 2024 BPPL removed five pathogen-antibiotic combinations present in the 2017 list: clarithromycin-resistant Helicobacter pylori, fluoroquinolone-resistant Campylobacter spp., penicillin-non-susceptible Streptococcus pneumoniae, third-generation cephalosporin-resistant Providencia spp., and vancomycin-intermediate and -resistant Staphylococcus aureus [82] [83] [84]. These removals are based on updated evidence and expert consensus, which, for some pathogens like Campylobacter, indicated a decrease in global resistance prevalence [83] [84]. The list also features refined terminology, such as the updated taxonomic classification from "Enterobacteriaceae" to "Enterobacterales" [84].

The following diagram summarizes the primary pathways for changes observed in the 2024 BPPL update:

Change Key Change Drivers for 2024 BPPL Sub1 Improved Surveillance Data Change->Sub1 Sub2 Evolving Resistance Trends Change->Sub2 Sub3 Focus on Community Burden Change->Sub3 Effect1 Pathogen Additions (e.g., RR-TB, Group A & B Streptococci) Sub1->Effect1 Effect3 Pathogen Removals/Priority Reductions (e.g., H. pylori, CRPA) Sub1->Effect3 Effect2 Priority Elevations (e.g., Shigella, 3GCRE) Sub2->Effect2 Sub2->Effect3 Sub3->Effect2

Essential Research Reagents and Methodologies

Research and development targeting the pathogens on the BPPL rely on a suite of specialized reagents, tools, and methodologies. The table below details key components of the "scientist's toolkit" essential for working in this field.

Table 2: Research Reagent Solutions for AMR R&D

Reagent / Tool Category Specific Examples Critical Function in AMR R&D
Reference Strains WHO BPPL pathogen strains (e.g., CRKP, MRSA, RR-TB) from repositories like ATCC, BEI Resources. Serve as quality controls and benchmarks for validating assays, testing drug efficacy, and comparing data across laboratories [85].
Culture Media & Supplements Cation-adjusted Mueller-Hinton Broth (CAMHB), specific media for fastidious bacteria (e.g., H. influenzae, Streptococci), antibiotic powders. Used in gold-standard broth microdilution assays to determine Minimum Inhibitory Concentrations (MICs) for antimicrobial susceptibility testing (AST) [82].
Molecular Biology Kits PCR/NGS kits for resistance gene detection (e.g., mecA, blaKPC, blaNDM), plasmid isolation kits, CRISPR-Cas systems. Enable genotypic resistance profiling, tracking plasmid-mediated resistance spread, and exploration of novel genetic countermeasures [86].
Antibiotic Standards WHO-endorsed chemical reference standards for antibiotics (e.g., carbapenems, fluoroquinolones). Essential for calibrating susceptibility tests, ensuring accurate and reproducible measurement of resistance in vitro [85].
Animal Model Reagents Immunocompromised mouse strains (e.g., neutropenic thigh infection model), specialized diets. Facilitate in vivo efficacy testing of lead compounds against BPPL pathogens in models simulating human infection [86].

Implications for Research and Public Health Strategy

The updated 2024 BPPL has profound implications for the global AMR R&D ecosystem. It serves as a definitive guide for prioritizing research funding and investments, helping to align the development of new antibiotics, rapid diagnostics, and vaccines with the most urgent public health needs [1] [17]. The list is explicitly targeted at "developers of antibacterial medicines, academic and public research institutions, research funders, and public–private partnerships investing in AMR R&D" [1]. The emphasis on community-acquired pathogens and those affecting vulnerable populations in LMICs calls for a reorientation of clinical trial networks and epidemiological studies to ensure these populations are adequately represented and that developed products are accessible and suitable for their use [17] [5].

Beyond steering R&D, the BPPL is a cornerstone for strengthening public health systems. It informs the development and implementation of national and regional AMR action plans, guides AMR surveillance networks like WHO's GLASS (Global Antimicrobial Resistance and Use Surveillance System), and underscores the need for robust infection prevention and control (IPC) measures in both healthcare and community settings [1] [83]. The list reinforces that a comprehensive, "people-centred approach"—including universal access to prevention, diagnosis, and treatment—is crucial to mitigating AMR's impact [17]. Finally, the changes between the 2017 and 2024 editions highlight the dynamic nature of AMR and the necessity for continuous surveillance, periodic list updates, and flexible, long-term funding commitments to keep pace with this evolving threat [5] [83].

The World Health Organization's (WHO) Bacterial Priority Pathogens List (BPPL) is a critical public health tool designed to steer the global response to antimicrobial resistance (AMR) by prioritizing the most threatening antibiotic-resistant bacteria. The 2017 WHO BPPL has been instrumental in guiding policy, research, and development investments. The 2024 WHO BPPL refines this effort, incorporating a robust, transparent, and evidence-based methodology to address previous limitations and respond to the evolving AMR landscape [5] [87]. This technical guide delves into the core of the 2024 list's development, with a specific focus on the validation of its rankings through formal expert consensus and comprehensive stability analyses. For researchers and drug development professionals, understanding this validation process is paramount, as it underpins the credibility and reliability of the list as a foundation for global health strategy and R&D target selection.

Methodological Framework of the 2024 BPPL

The 2024 BPPL was constructed using a multicriteria decision analysis (MCDA) framework, a systematic approach well-suited for complex prioritization problems involving multiple, competing criteria. This methodology ensures that the final list is not based on a single metric but reflects a balanced consideration of various factors that collectively define a pathogen's threat level [5] [87].

The process involved several key stages, which are also depicted in the workflow diagram below:

  • Pathogen and Criteria Selection: The study evaluated 24 antibiotic-resistant bacterial pathogens across 15 families. Each pathogen was scored against eight predefined criteria, which were selected to capture the multifaceted nature of the AMR threat [5] [87].
  • Expert Preference Weighting: A preferences survey was administered to a global panel of 100 experts to determine the relative importance of each criterion. This step ensured that the final ranking reflected the collective judgment of the international AMR community [5].
  • Scoring and Ranking: Pathogen scores on each criterion were combined with the expert-derived weights to calculate a total score out of 100% for each pathogen. These scores formed the basis of the initial ranking [5] [87].
  • Validation and Stability Analysis: The robustness of the ranking was rigorously tested through subgroup and sensitivity analyses to ensure it was not unduly influenced by the background or origin of the experts [5].
  • Final Tier Assignment: An independent advisory group reviewed the results, and pathogens were grouped into three priority tiers—critical, high, and medium—based on a quartile scoring system of the total scores [5] [87].

The following diagram illustrates the logical workflow and the key decision points in this methodology:

Start Start: Develop 2024 BPPL Step1 Select 24 Pathogens & 8 Criteria Start->Step1 Step2 Administer Preference Survey to 100 Experts Step1->Step2 Step3 Calculate Weighted Pathogen Scores Step2->Step3 Step2->Step3 Apply Weights Step4 Perform Stability Analysis Step3->Step4 Step3->Step4 Test Ranking Step5 Group into Priority Tiers (Quartiles) Step4->Step5 End Final 2024 BPPL Step5->End Subgraph1 Validation Core

The eight criteria used for prioritization were carefully chosen to provide a comprehensive assessment of each pathogen's threat level, as detailed in the table below.

Table 1: Criteria for Pathogen Prioritization in the 2024 BPPL

Criterion Description Rationale for Inclusion
Mortality The death rate associated with the infection. Directly measures the most severe health outcome.
Non-fatal Burden Morbidity and long-term disability caused by the infection. Captures the broader impact on quality of life and health systems.
Incidence The frequency of new infections. Indicates how common and widespread the threat is.
10-Year Resistance Trends The historical trajectory of resistance. Helps identify worsening threats and guides proactive strategies.
Preventability The feasibility of preventing infections. Informs the potential for non-treatment control measures.
Transmissibility The capacity for pathogen spread. Identifies pathogens with potential for outbreaks and rapid dissemination.
Treatability Current availability and effectiveness of treatment options. Highlights gaps where treatment options are limited or failing.
Antibacterial Pipeline Status Number and stage of new antibiotics in development. Assesses whether the R&D pipeline is addressing the threat.

Source: [5] [87]

A cornerstone of the 2024 BPPL's validity is its foundation on international expert consensus. The process moved beyond simple opinion gathering to a structured, quantitative elicitation of preferences.

A key step was a preferences survey using a pairwise comparison method. This survey was administered to 100 international AMR experts, with a high completion rate of 78%. In a pairwise comparison, experts are presented with two criteria at a time and asked to judge which is more important for pathogen prioritization. This method forces fine-grained distinctions and generates robust, reliable data on the relative value experts assign to each criterion [5].

The outcome of this survey was a set of weights for each of the eight criteria. These weights determined how much influence each criterion had on the final pathogen score. The study reported a strong inter-rater agreement among the experts, with both Spearman's rank correlation coefficient and Kendall's coefficient of concordance at 0.9. This high level of agreement indicates that the global expert community shares a common understanding of what makes an AMR pathogen a priority, thereby validating the weighting structure used in the MCDA model [5].

Stability Analysis of Pathogen Rankings

A critical test for any prioritization system is the stability of its results. A ranking that changes drastically with minor methodological adjustments or different subsets of experts would be of limited use for guiding long-term policy and investment. The 2024 BPPL development included a rigorous sensitivity analysis to address this very concern.

The investigators conducted subgroup analyses to assess the impact of the experts' consistency, professional background, and geographical origin on the final pathogen rankings. The results confirmed that the ranking possessed high stability. Clustering the pathogens based on these different expert demographics did not result in any substantial changes to the overall ranking. This finding underscores the robustness of the list and suggests that the prioritization is representative of a broad consensus within the global AMR community, rather than being skewed by a particular subgroup [5].

Quantitative Results and Pathogen Ranking

The application of the weighted MCDA framework yielded a clear quantitative ranking of the 24 pathogens. The total scores ranged from 84% for the top-ranked pathogen to 28% for the bottom-ranked pathogen. The pathogens were then grouped into three priority tiers based on a quartile system of these total scores [5] [87].

Table 2: 2024 WHO Bacterial Priority Pathogens List: Ranking and Priority Tiers

Priority Tier Pathogen Key Resistance Profile(s) Total Score (%)
Critical Klebsiella pneumoniae Carbapenem-resistant 84
Acinetobacter spp. Carbapenem-resistant 83
Escherichia coli Third-generation cephalosporin and carbapenem-resistant 82
Mycobacterium tuberculosis Rifampicin-resistant 82
High Salmonella enterica serotype Typhi Fluoroquinolone-resistant 72
Shigella spp. Fluoroquinolone-resistant 70
Neisseria gonorrhoeae Third-generation cephalosporin and fluoroquinolone-resistant 64
Pseudomonas aeruginosa Carbapenem-resistant 63
Staphylococcus aureus Meticillin-resistant (MRSA) 62
Medium ... (Other pathogens, e.g., Enterococcus faecium, Streptococcus pneumoniae, etc.) ... ...
Group B Streptococcus Penicillin-resistant 28

Note: This table shows a selection of key pathogens from the full list of 24. Total scores are approximate values derived from the source material. Source: [5] [87]

The results highlight the persistent critical threat of Gram-negative bacteria, particularly those resistant to last-resort antibiotics like carbapenems. The inclusion of rifampicin-resistant Mycobacterium tuberculosis in the critical tier also underscores its ongoing global burden. Among community-acquired infections, resistant strains of Salmonella Typhi, Shigella, and Neisseria gonorrhoeae received high rankings, reflecting their significant impact on public health [1] [5].

Essential Research Toolkit for AMR Prioritization Studies

For research teams aiming to conduct similar prioritization exercises in infectious diseases or AMR, the methodology of the 2024 BPPL serves as an excellent blueprint. The following table outlines key components of the "research toolkit" implied by this study.

Table 3: Research Reagent Solutions for AMR Pathogen Prioritization Studies

Tool / Component Function in the Research Process Example from 2024 BPPL
Multicriteria Decision Analysis (MCDA) Framework Provides a structured, quantitative model for evaluating multiple, competing criteria simultaneously. The core methodology used to score and rank the 24 pathogens [5] [87].
Expert Panel Provides specialized knowledge and judgment for criteria weighting and pathogen assessment. 100 international AMR experts from various backgrounds and geographies [5].
Pairwise Comparison Survey A robust psychometric method for eliciting consistent and reliable preference weights from experts. The instrument used to determine the relative importance of the 8 criteria [5].
Stability/Sensitivity Analysis Protocol A set of statistical methods to test the robustness of the model's outputs to changes in assumptions or inputs. Subgroup analysis based on expert demographics to confirm ranking stability [5].
Global Burden of Disease Data Provides standardized, quantitative estimates of mortality, morbidity, and incidence for pathogens. Key data source for populating the mortality, incidence, and non-fatal burden criteria [5].

Furthermore, the logical relationships between the core components of the BPPL methodology—from inputs and processes to outputs—can be visualized as a system. This systems-level view is crucial for understanding how the various elements interact to produce the final, validated list.

Input1 Epidemiological Data (Burden, Incidence) Process1 Multicriteria Decision Analysis (MCDA) Input1->Process1 Input2 Expert Judgment & Preferences Input2->Process1 Process2 Preference Weighting (Pairwise Survey) Input2->Process2 Input3 R&D Pipeline Data Input3->Process1 Process3 Stability Analysis (Sensitivity Tests) Process1->Process3 Preliminary Ranking Process2->Process1 Weights Output Validated & Stabilized Priority Pathogens List Process3->Output

The 2024 WHO BPPL represents a significant advancement in the science of pathogen prioritization. Its validation rests on a transparent and rigorous process that integrates quantitative data with structured expert consensus. The high degree of stability in the rankings, even when examined through the lens of different expert subgroups, provides strong confidence in the results. For the global research and public health community, this validated list is more than a catalog of threats; it is a strategic compass. It directs finite resources toward the most pressing challenges, guiding R&D for new antibiotics, diagnostics, and vaccines, while also reinforcing the importance of infection prevention, control measures, and equitable access to existing treatments. The methodologies detailed in this guide for consensus building and stability analysis offer a replicable model for future national and regional efforts to combat AMR.

The relentless evolution of antimicrobial resistance (AMR) and the persistent threat of emerging infectious diseases necessitate robust global preparedness strategies. A cornerstone of these strategies is the systematic prioritization of pathogens to guide research and development (R&D) efforts for new diagnostics, therapeutics, and vaccines (DTVs) [8]. Internationally, the World Health Organization's (WHO) Bacterial Priority Pathogens List (BPPL) serves as a critical reference point for directing these efforts [1]. Similarly, national public health agencies develop their own tailored lists to address specific regional threats and biosecurity concerns. The United Kingdom Health Security Agency (UKHSA) recently joined this effort by publishing its "Priority Pathogen Families" reference tool in March 2025 [8] [88].

This whitepaper provides an in-depth technical analysis of the global alignment and divergence between the 2024 WHO BPPL and the 2025 UKHSA Priority Pathogens list. Framed within a broader research thesis on WHO priority pathogen list analysis, this guide is intended for researchers, scientists, and drug development professionals. It aims to dissect the methodologies, structural frameworks, and strategic priorities underpinning these lists, offering a detailed comparison to inform future R&D investments and policy decisions. The analysis is supplemented with experimental protocols for genomic surveillance and a curated list of research reagents essential for working with these high-priority pathogens.

The WHO BPPL, updated in 2024, is a fundamental tool for combating antimicrobial resistance on a global scale [1]. Its primary purpose is to guide and prioritize R&D for new antibacterial treatments, with a specific focus on antibiotic-resistant bacteria [1] [3]. The list is unambiguously focused on bacterial pathogens, explicitly excluding viruses, fungi, and other microbial threats.

The 2024 list refines its 2017 predecessor, categorizing 24 antibiotic-resistant bacterial pathogens across 15 families into three priority tiers: critical, high, and medium [1]. This prioritization is based on a multi-factorial methodology that integrates data on global burden, mortality, transmissibility, treatability, and the current state of the R&D pipeline [1]. The list highlights the dire threat of Gram-negative bacteria resistant to last-resort antibiotics and reaffirms the persistent high burden of drug-resistant Mycobacterium tuberculosis [1].

A key function of the WHO BPPL is to act as a framework for assessing the global antibacterial pipeline. A 2025 WHO report revealed a concerning scarcity and lack of innovation in this pipeline. As of 2025, only 90 antibacterial agents were in clinical development, a decrease from 97 in 2023. Among these, a mere 15 were considered innovative, and only 5 were effective against at least one pathogen in the "critical" priority category [3]. This stark data underscores the list's role in highlighting critical gaps and urging increased investment.

The UKHSA's "Priority Pathogen Families reference tool," published in March 2025, represents a distinct yet complementary approach to pathogen prioritization [8] [88]. Its stated objective is to guide England-based funders and research institutions in their R&D investments for DTVs, thereby strengthening national biosecurity [8] [89].

A fundamental point of divergence from the WHO list is the scope of pathogens covered. The UKHSA tool adopts a family-level approach, encompassing 24 families of pathogens, which include viruses and bacteria [8]. This broader scope is designed to prepare for both known endemic diseases and future pandemic threats, including the concept of "Disease X"—a known or unknown pathogen with epidemic potential [8].

The UKHSA methodology relies on the collective knowledge and opinion of its internal subject matter experts [89]. For viral families, it provides an indicative rating of "pandemic and epidemic potential" (high, medium, or low), based on the historical precedent of pathogens within the family, their routes of transmission, and disease severity [8]. For bacterial families, such as Enterobacteriaceae, the tool explicitly highlights concerns related to AMR as a key reason for prioritization [8]. The agency emphasizes that the list is not a ranked threat assessment and is intended to be updated annually to reflect evolving threats and scientific progress [88].

Comparative Analysis: WHO BPPL vs. UKHSA List

A side-by-side comparison of the WHO and UKHSA lists reveals critical alignments and strategic divergences in the global fight against pathogenic threats. The table below summarizes the core characteristics of each list.

Table 1: Fundamental Characteristics of the WHO and UKHSA Pathogen Lists

Feature WHO BPPL (2024) UKHSA Priority Pathogens (2025)
Publisher World Health Organization (WHO) UK Health Security Agency (UKHSA)
Primary Objective Guide global R&D for antibacterial medicines; control AMR [1] Guide national R&D for diagnostics, therapeutics, and vaccines (DTVs); bolster UK biosecurity [8] [89]
Pathogen Scope Exclusively bacterial (15 families, 24 pathogens) [1] Viral and bacterial families (24 total families) [8]
Prioritization Criteria Global burden, mortality, transmissibility, treatability, R&D pipeline [1] Expert opinion on pandemic/epidemic potential, AMR concerns, relevance to UK population [8] [89]
Priority Categories Critical, High, Medium [1] High, Medium, Low (Pandemic/Epidemic Potential for viruses) [8]
Key Audiences Global drug developers, research institutions, funders, policymakers [1] England-based R&D funders, academic and industry scientists [8] [89]

Key Alignments

Despite their differences, the lists share significant common ground:

  • AMR as a Unifying Threat: Both entities recognize antimicrobial resistance as a paramount public health threat. The WHO list is entirely composed of antibiotic-resistant bacteria [1], while the UKHSA list explicitly flags AMR concerns within its included bacterial families, such as Enterobacteriaceae [8].
  • Overlap in Bacterial Pathogens: Several high-burden bacterial pathogens appear on both lists, either explicitly or within prioritized families. These include Salmonella, Shigella, Neisseria gonorrhoeae, Pseudomonas aeruginosa, and Staphylococcus aureus [1] [8].
  • Guidance for R&D Investment: Both lists are fundamentally designed to steer research funding and scientific effort towards the areas of greatest unmet need, preventing a scattered approach to R&D [1] [8].

Critical Divergences

The differences in the lists' construction highlight their distinct strategic focuses:

  • Pathogen Scope: Bacteria vs. Pathogen Families: The most significant divergence is the WHO's exclusive focus on antibiotic-resistant bacteria versus the UKHSA's inclusion of multiple viral families. The UKHSA list places a strong emphasis on viral families with high pandemic potential, such as Coronaviridae (which includes MERS and SARS-CoV-2), Paramyxoviridae (which includes Nipah virus), and Orthomyxoviridae (which includes avian influenza) [8] [88].
  • Geographical Perspective: Global vs. National: The WHO list is developed from a global health perspective, considering the worldwide burden of disease [1]. The UKHSA tool, while acknowledging global threats, is the first list specifically designed to also consider the diseases most relevant to the UK population, creating a nationally tailored preparedness strategy [8] [88].
  • Methodological Transparency: The WHO employs a documented methodology for its prioritization [1]. In contrast, the UKHSA's tool has been noted by experts to lack published references and a detailed methodology, which, as Dr. Catrin Moore from St. George's, University of London, points out, makes it difficult to assess the quality of the research and the basis for its conclusions [90].

The following diagram illustrates the conceptual relationship and overlap between the two lists.

G A WHO BPPL Scope B Antibiotic-Resistant Bacteria A->B C Global AMR Focus A->C D Quantitative & Qualitative Methodology A->D Overlap Alignment: - AMR Concern - Key Bacterial Pathogens - R&D Guidance A->Overlap E UKHSA Scope F Viral & Bacterial Families E->F G UK Biosecurity Focus E->G H Expert-Opinion Methodology E->H E->Overlap

Technical and Research Implications

Analysis of R&D Pipelines and Gaps

The pathogen priorities identified by the WHO and UKHSA must be evaluated against the current landscape of medical countermeasures. The WHO's 2025 pipeline analysis reveals a precarious situation. The clinical pipeline for antibacterial agents is both shrinking and lacking in innovation, with only 90 agents in development and a mere 15 deemed innovative [3]. Critically, only 5 of these target the "critical" priority pathogens on the BPPL [3]. This indicates a severe misalignment between the greatest threats and the commercial R&D focus.

The diagnostic landscape, as analyzed by WHO, also shows critical gaps, particularly affecting low- and middle-income countries. These include the absence of multiplex platforms for identifying bloodstream infections directly from blood and limited simple, point-of-care tools for primary care facilities [3]. The UKHSA list, with its inclusion of viral families, implicitly calls for a parallel assessment of R&D pipelines for broad-spectrum antiviral drugs and vaccines against known viral threats like Nipah virus and potential "Disease X" agents.

Expert Commentary and Critical Perspectives

The scientific community's reaction to the UKHSA list, as collated by the Science Media Centre, provides valuable critical insights [90]. While experts like Prof. Emma Thomson (University of Glasgow) and Prof. Miles Carroll (University of Oxford) praised the tool as an "important and valuable resource" that aligns with existing evidence, others raised substantive concerns [90].

  • Risk of Narrowed Research Focus: Prof. Jose Vazquez-Boland (University of Edinburgh) warned that prioritization is a "double-edged sword." In a context of scarce funding, it could lead to the neglect of research on non-priority pathogens, which is essential for a comprehensive understanding of infection biology and emerging threats [90].
  • Omissions and Lack of Methodology: Dr. Catrin Moore (St. George's, University of London) noted the omission of Mycobacterium tuberculosis and critiqued the lack of references and methodological transparency, making it difficult to validate the tool's conclusions [90].
  • Potential for Misdirection: Prof. Robert Read (University of Southampton) questioned the utility of such lists, stating they are "at best pointless, and at worst potentially harmful to the public health" because they could misdirect funding away from urgent, unforeseen problems [90].

These critiques underscore the necessity for list-developing agencies to couple their publications with detailed methodological documentation and to ensure that prioritization does not stifle fundamental, discovery-oriented research.

Experimental Protocols for Pathogen Surveillance and Characterization

Research on priority pathogens requires advanced genomic and bioinformatic techniques. The following protocol details a modern computational method for identifying Mobile Genetic Elements (MGEs), which are crucial for understanding the spread of antibiotic resistance genes.

Protocol: Identification of Mobile Genetic Elements (MGEs) Using Skandiver

The horizontal transfer of MGEs across bacterial species is a primary driver of AMR dissemination. Skandiver is a bioinformatics tool designed to efficiently detect MGEs from whole-genome assemblies without the need for gene annotation or curated databases, making it suitable for discovering novel elements [91].

Table 2: Key Reagents and Resources for Skandiver Analysis

Item Function/Description
Whole-Genome Assemblies Input data; metagenome-assembled genomes (MAGs) or isolate assemblies in FASTA format [91].
Skandiver Software The core algorithm for MGE detection, which operates on a divergence-based paradigm [91].
Skani A dependency of Skandiver; a fast and accurate tool for calculating Average Nucleotide Identity (ANI) between genome fragments [91].
GTDB Database The Genome Taxonomy Database; used as a reference set of representative genomes for the skani search [91].
TimeTree of Life A resource of species divergence times; used by Skandiver to annotate matches with evolutionary time [91].
Biopython Library A set of Python tools for computational biology; used for tasks like genome fragmentation [91].

Procedure:

  • Genome Fragmentation:
    • Input genome assemblies are fragmented into non-overlapping chunks of sequence, typically 10,000 base pairs in length, using a Python script leveraging the Biopython library [91].
    • Note: This step limits detection to MGEs >10 kb, with >20 kb needed for reliable detection due to edge effects. [91]

  • Skani Search:

    • The fragmented genome sequences are searched against a large database of representative genomes (e.g., GTDB) using skani search [91].
    • This step identifies all genome fragments in the database that have strong sequence similarity to the query fragments, as measured by high Average Nucleotide Identity (ANI) and Alignment Fraction (AF) [91].

  • Divergence Time Calculation:

    • The significant matches from the skani search are filtered and annotated with evolutionary divergence times (in million years ago, MYA) using the TimeTree of Life resource [91].

  • MGE Prediction and Output:

    • The core logic of Skandiver is applied: a genome fragment is predicted to be an MGE if it has strong matches to a set of bacterial species that have high mutual evolutionary divergence times [91].
    • In contrast, a fragment that is conserved (i.e., an essential gene) will only appear in closely related species with low divergence times [91].
    • The tool outputs a table of putative MGE sequences, their genomic locations, and the divergence data for the species they mapped to [91].

The workflow of this protocol is depicted in the following diagram.

G Input Whole-Genome Assembly (FASTA) Step1 1. Genome Fragmentation (10 kb chunks) Input->Step1 Step2 2. Skani Search vs. GTDB Step1->Step2 Step3 3. Calculate Divergence Times (TimeTree of Life) Step2->Step3 Step4 4. Predict MGEs Step3->Step4 Output Output: Table of Putative MGEs Step4->Output

The comparative analysis of the 2024 WHO BPPL and the 2025 UKHSA Priority Pathogens list reveals a strategic, multi-layered global health defense architecture. The WHO BPPL provides a specialized, deep focus on the global crisis of antibiotic-resistant bacteria, functioning as a universally accepted benchmark for antibacterial R&D. In contrast, the UKHSA list offers a broader, nationally-oriented strategy that encompasses both bacterial and high-consequence viral threats, thereby enhancing the UK's preparedness for a wider spectrum of potential epidemics and pandemics.

For the global research and drug development community, these lists are not contradictory but complementary. They collectively highlight the most pressing challenges: the critically anemic antibacterial pipeline flagged by the WHO and the enduring threat of viral spillover emphasized by the UKHSA. The divergence in scope between the lists should not be seen as a conflict but as a reflection of different, essential tiers of responsibility—global versus national.

The path forward requires a dual commitment. First, researchers and funders must leverage these prioritized lists to channel resources into the development of innovative DTVs for the pathogens of highest concern. Second, the scientific community must advocate for and engage in the continuous refinement of these tools, ensuring they remain dynamic, methodologically transparent, and responsive to the evolving pathogen landscape. Ultimately, aligning and acting upon these guiding documents is paramount to building a resilient global defense against the persistent and emerging threats of infectious diseases.

The concept of "Pathogen X" represents a critical acknowledgment of the limitations of our current infectious disease knowledge. Defined by the World Health Organization as an unknown pathogen with the potential to trigger a severe global epidemic, Pathogen X embodies the fundamental "known unknown" in pandemic preparedness planning [92]. This conceptual framework, first incorporated into the WHO's Blueprint list of priority diseases in 2018, serves as a placeholder for the significant probability that the next major pandemic threat will emerge from a pathogen not currently characterized by science [93] [94]. The COVID-19 pandemic, caused by the previously unknown SARS-CoV-2 virus, stands as a historic example of the first Disease X to manifest, demonstrating with devastating clarity the tangible reality behind this theoretical construct [95] [94].

The systematic analysis of Pathogen X is not speculative exercises but rather a necessary component of comprehensive epidemic readiness. By preparing for an unknown threat, the global health community aims to build resilient systems capable of mounting swift, effective countermeasures regardless of the specific pathogen identity [93]. This proactive approach recognizes that novel pathogens are emerging with increasing frequency, with research indicating approximately 1.67 million unknown viruses existing in nature, of which an estimated 631,000 to 827,000 possess the capacity to infect humans [96]. The strategic focus on Pathogen X thus represents a paradigm shift from reactive response to proactive preparedness, demanding sophisticated assessment frameworks and innovative research methodologies to address this ultimate unknown threat.

Pathogen Characteristics and Prioritization Frameworks

Defining Characteristics of High-Priority Threat Pathogens

While Pathogen X remains unidentified by definition, analysis of historical pandemics and emerging infectious diseases reveals common characteristics that inform prioritization frameworks. Pathogens with pandemic potential frequently demonstrate respiratory transmission capabilities, as airborne spread through talking, coughing, or sneezing presents significant challenges for public health intervention [93]. The reproductive number (R0) and case fatality rates serve as critical quantitative metrics, with pathogens exhibiting moderate to high values in both categories representing the greatest concern. Genomic features also contribute to pandemic risk, with RNA viruses being particularly concerning due to their high mutability from error-prone replication enzymes, facilitating rapid adaptation to new hosts [95] [97].

Transmission dynamics further refine threat assessment, with zoonotic spillover potential representing a key risk factor. Pathogens circulating in animal reservoirs, particularly mammals and birds, with ecological pressure driving cross-species transmission pose disproportionate threats [96]. The animal-human interface has been identified as the origin point for approximately 60% of emerging infectious diseases, with wildlife serving as the source for 71% of these zoonotic transmissions [96]. Additional concerning characteristics include pre-symptomatic or asymptomatic transmission that evades detection, environmental stability that prolongs infectious potential outside a host, and the absence of effective medical countermeasures such as vaccines, therapeutics, or diagnostics [93].

The WHO Blueprint and Pathogen Pyramid Frameworks

The World Health Organization employs systematic methodologies to categorize and prioritize pathogens with epidemic potential. The WHO R&D Blueprint for Epidemics, initially developed in 2015 and continuously refined, represents a comprehensive global research strategy and preparedness plan [92]. A cornerstone of this approach is the WHO pathogen priority list, which ensures research investments target diseases with limited or non-existent medical countermeasures. In a significant evolution from past practice, the current WHO approach focuses on entire classes of viruses or bacteria rather than individual pathogens, with approximately 200+ scientists from 53 countries independently evaluating evidence related to 30 viral families, one core group of bacteria, and Pathogen X [92].

Complementing this list-based approach, the pathogen pyramid framework provides an ecological model for understanding zoonotic disease emergence through progressive stages of adaptation [96]. This conceptual model outlines four critical transitions:

  • Level 1: Human exposure to environmental or animal pathogens without established infection or disease
  • Level 2: Zoonotic pathogens overcome species barriers to cause human disease but with minimal human-to-human transmission
  • Level 3: Pathogens achieve limited human-to-human transmission causing outbreaks that eventually diminish
  • Level 4: Efficient human-to-human transmission enables epidemic or pandemic spread

This pyramid structure illustrates the diminishing number of pathogens capable of achieving each successive level, with only a fraction of the estimated hundreds of thousands of potential human-infecting viruses reaching the pinnacle of pandemic capability [96]. The framework helps researchers identify intervention points to disrupt pathogen progression at earlier stages, potentially preventing full emergence.

Table 1: WHO Bacterial Priority Pathogens List 2024 Categorization

Priority Category Pathogen Examples Key Characteristics
Critical Gram-negative bacteria resistant to last-resort antibiotics High-burden resistant pathogens with limited treatment options
High Drug-resistant Salmonella, Shigella Significant public health impact with growing resistance patterns
Medium Other resistant bacterial pathogens with fewer therapeutic challenges Require monitoring but currently have more effective countermeasures

Source: WHO Bacterial Priority Pathogens List, 2024 [1]

Transmission Dynamics and Spillover Mechanisms

Ecological Drivers of Emergence

The emergence of novel pathogens follows recognizable ecological patterns, with specific environmental and anthropogenic factors creating favorable conditions for cross-species transmission. Climate change represents a significant driver of disease emergence, altering the suitability of various regions for specific biomes and causing migration of species to new areas [95]. This redistribution facilitates novel mixing patterns of pathogen, plant, and animal species, creating new interfaces for disease transmission [95]. The migration of wildlife to regions with dense human populations due to rising temperatures heightens the risk of viral transmission from animals to humans, potentially sparking the next pandemic [95].

Human activities similarly contribute to emergence risk through habitat destruction and encroachment into wildlife territories. Deforestation, agricultural expansion, and resource extraction increase contact between humans and wildlife, raising the probability of zoonotic spillover [96]. The densification of animals through practices such as factory farming, animal markets, and wildlife trade creates additional risks by enhancing opportunities for viral replication and mutation [96]. These activities, driven by economic imperatives and consumption patterns, often overlook the disease risk perspective in favor of cost-benefit rationalizations, creating blind spots in pandemic preparedness.

The Spillover Process and Adaptive Evolution

The transmission of pathogens from animal reservoirs to human populations involves complex biological processes influenced by pathogen genetics, host susceptibility, and environmental factors. Spillover events typically begin with direct or indirect contact between humans and infected animals, often through hunting, wildlife trade, or contamination of shared environments [96]. Following exposure, successful infection requires the pathogen to overcome species-specific barriers, including cellular receptors incompatible with binding, host immune defenses, and intracellular environments unsuitable for replication.

Genetic studies of historical emergence events suggest two primary pathways for adaptive evolution enabling human transmission: viral mutations in humans after initial zoonotic transmission, or potentially more efficient mutations in animal reservoirs before zoonotic transmission to humans [96]. The latter pathway represents a particular concern as it may allow for more optimized human adaptation prior to detection. RNA viruses pose special threats in this context due to their high mutation rates and ability to undergo recombination or reassortment, generating novel variants with altered host range, tissue tropism, or transmission efficiency [95].

G Pathogen Emergence and Spillover Process cluster_animal_reservoir Animal Reservoir cluster_human_interface Human-Animal Interface cluster_spillover Spillover Event A1 Pathogen Circulation in Wildlife A2 Genetic Diversity through Mutation A1->A2 Viral Evolution H1 Environmental Encroachment A2->H1 Ecological Pressure H2 Animal Agriculture & Markets A2->H2 Human Activities H3 Wildlife Trade A2->H3 Interface Expansion S1 Initial Zoonotic Transmission H1->S1 Exposure Event H2->S1 Direct Contact H3->S1 Cross-Species Jump S2 Limited Human-to-Human Transmission S1->S2 Adaptation S3 Efficient Human-to-Human Transmission S2->S3 Increased Fitness E Sustained Epidemic or Pandemic S3->E Sustained Transmission

Research Methodologies for Unknown Pathogen Preparedness

Surveillance and Discovery Approaches

Proactive surveillance represents the foundational methodology for early detection of potential Pathogen X candidates. The PREDICT project by the United States Agency for International Development (USAID) exemplifies this approach, implementing comprehensive surveillance programs to identify novel viruses before they spread extensively in human populations [95]. This initiative has collaborated with over 60 countries to enhance zoonotic disease surveillance since 2009, discovering at least 931 novel viruses from 145,000 samples of humans, animals, and wildlife [95]. Strategic surveillance focuses on geographic hotspots with high biodiversity and frequent human-animal interaction, particularly in regions of Asia, Africa, and Latin America, though emergence can occur globally as demonstrated by the 2009 H1N1 pandemic originating in Mexico [95] [93].

Modern discovery methodologies employ metagenomic sequencing of samples from wildlife, domestic animals, and humans with undiagnosed febrile illnesses, enabling identification of novel pathogens without prior knowledge of their sequences [92]. The representative virus approach within viral families serves as a pathfinder strategy, generating evidence and filling knowledge gaps that may apply to other threats within the same viral family [92]. This methodology allows researchers to develop prototype assays, animal models, and countermeasures for entire classes of pathogens rather than individual species, significantly enhancing preparedness efficiency. These surveillance networks form early warning systems that can detect signals of unusual disease clusters that might represent the initial emergence of Pathogen X.

Platform Technologies and Prototype Development

The development of adaptive platform technologies represents a cornerstone of Pathogen X preparedness, enabling rapid medical countermeasure development once a novel pathogen is identified. mRNA and adenovirus vaccine platforms have demonstrated particular utility, potentially reducing vaccine development timelines to less than a year, with organizations like the Coalition for Epidemic Preparedness Innovations (CEPI) advocating for a 100 Days Mission to develop new vaccines within three months of pandemic threat recognition [93] [94]. These platforms allow researchers to quickly insert genetic sequences from newly identified pathogens into established delivery systems, bypassing much of the traditional development process.

Complementing vaccine platforms, broad-spectrum antiviral development focuses on compounds targeting conserved viral proteins or host pathways essential for pathogen replication. This approach includes investigation of monoclonal antibodies with cross-reactive potential against related pathogens within viral families [3]. Diagnostic preparedness similarly employs multiplexed platforms capable of detecting signature patterns of novel pathogens while distinguishing them from known pathogens [3]. The WHO emphasizes the need for sample-in/result-out systems that work with multiple sample types (blood, urine, stool, respiratory specimens) and are suitable for deployment in low-resource settings [3].

Table 2: Essential Research Reagents and Platform Technologies for Pathogen X Preparedness

Research Tool Category Specific Examples Application in Pathogen X Preparedness
Vaccine Platforms mRNA, Adenovirus vector, Nanoparticle Rapid antigen switching for accelerated vaccine development
Antiviral Compounds Broad-spectrum antivirals, Monoclonal antibodies Targeting conserved viral mechanisms across pathogen families
Diagnostic Technologies Multiplex PCR, Metagenomic sequencing, CRISPR-based detection Pathogen-agnostic detection methods for novel agent identification
Animal Models Humanized mice, Ferret transmission models Evaluation of pathogenesis and transmission without prior pathogen knowledge
Computational Tools Machine learning prediction models, Phylogenetic analysis Forecasting spillover risk and evolutionary trajectories

Source: Adapted from WHO Consultations on Research Response to Pathogen X [92]

Assessment of Current Preparedness and Critical Gaps

Progress in Global Preparedness Initiatives

Significant advancements have been made in pandemic preparedness since the COVID-19 pandemic highlighted systemic vulnerabilities. The World Health Organization has launched new initiatives such as the Preparedness and Resilience for Emerging Threats (PRET) program to enhance pandemic awareness and planning [98]. This initiative incorporates lessons from past pandemics, with its initial module focusing on respiratory pathogen pandemics in recognition of their disproportionate epidemic potential [98]. The WHO Biohub system and similar pathogen sharing networks represent additional progress, facilitating rapid characterization of novel threats through global scientific collaboration.

The research and development ecosystem for medical countermeasures has demonstrated notable achievements, particularly in the acceleration of vaccine development platforms. The successful deployment of mRNA vaccines against COVID-19 within unprecedented timelines validated the prototype pathogen approach, wherein prior research on coronaviruses following the MERS epidemic enabled rapid stabilization of spike proteins and engendered appropriate antibody responses [93]. This experience proved that preparatory work on viral families with pandemic potential can significantly accelerate response times when novel pathogens emerge from those families. Additionally, regulatory frameworks have evolved to support more flexible approval mechanisms for platform technologies and adaptive licensure pathways.

Persistent Vulnerabilities and Research Gaps

Despite measurable progress, significant vulnerabilities remain in global preparedness for Pathogen X. The antibacterial development pipeline illustrates one concerning gap, with the number of antibacterials in clinical development decreasing from 97 in 2023 to 90 in 2025, of which only 15 qualify as truly innovative [3]. Particularly alarming is that only 5 of these agents demonstrate effectiveness against WHO "critical" priority bacteria, the highest risk category [3]. The fragility of this ecosystem is further highlighted by the composition of its contributors, with 90% of companies involved in the preclinical pipeline being small firms with fewer than 50 employees [3].

Diagnostic capabilities face similar challenges, with persistent gaps in point-of-care testing platforms suitable for primary health care facilities in low-resource settings [3]. The absence of multiplex platforms capable of identifying bloodstream infections directly from whole blood without culture represents a particular technological hurdle [3]. Beyond technological limitations, structural and coordination gaps persist, including insufficient integration of One Health approaches into governmental systems, limited cross-sectoral training programs, and inadequate funding mechanisms for preparedness in inter-epidemic periods [96] [98]. These vulnerabilities are compounded by manufacturing concentration for essential medical supplies, with most mask production having ceased after COVID-19 subsided, and ongoing challenges with hospital capacity limitations that would likely be overwhelmed during another severe pandemic [93].

G Integrated One Health Preparedness Framework cluster_human Human Health cluster_animal Animal Health cluster_environment Environmental Health H1 Surveillance Systems P Pathogen X Preparedness H1->P H2 Health Care Infrastructure H2->P H3 Medical Countermeasure Development H3->P A1 Wildlife Disease Monitoring A1->P A2 Livestock Health Surveillance A2->P A3 Interface Management A3->P E1 Climate Change Impact Assessment E1->P E2 Ecosystem Monitoring E2->P E3 Habitat Encroachment Tracking E3->P

The threat posed by Pathogen X represents one of the most complex challenges in global health security, demanding sustained scientific innovation, strategic investment, and unprecedented international cooperation. The known unknowns of future pandemic threats cannot be eliminated, but their potential impact can be substantially mitigated through systematic preparedness grounded in the One Health approach that integrates human, animal, and environmental health monitoring [96]. The increasing frequency of emerging infectious disease events suggests that the question is not whether another Pathogen X will emerge, but when it will appear and how effectively the global community will respond.

Building a resilient defense system requires addressing critical gaps in the medical countermeasure ecosystem, particularly the precarious state of antibacterial development and the insufficient diagnostic capabilities for low-resource settings [3]. Simultaneously, strengthening surveillance networks to enhance early detection and investing in platform technologies that can be rapidly adapted to novel pathogens must remain priorities [92]. Perhaps most importantly, the institutionalization of cross-sectoral collaboration mechanisms between human health, animal health, and environmental sectors remains essential, ensuring that relationships and communication channels established during crisis responses are maintained during inter-epidemic periods [96]. By embracing these strategies with sustained commitment, the global research community can transform the existential threat of Pathogen X into a manageable risk, potentially preventing the devastating human, social, and economic costs witnessed during the COVID-19 pandemic.

The global battle against antimicrobial resistance (AMR) and infectious diseases requires a precise understanding of regional pathogen priorities. While the World Health Organization (WHO) provides a global framework for categorizing antibiotic-resistant bacterial pathogens through its Bacterial Priority Pathogens List (BPPL), the actual burden of these pathogens exhibits significant geographical variation [1]. This technical guide examines the distinct patterns of pathogen prevalence and priority between high-income countries (HICs) and low- and middle-income countries (LMICs), providing researchers, scientists, and drug development professionals with evidence-based insights for targeted intervention strategies. Understanding these disparities is crucial for directing research resources, developing context-appropriate diagnostics and treatments, and ultimately mitigating the disproportionate burden of infectious diseases in vulnerable populations.

The analysis presented herein is framed within a broader thesis on WHO priority pathogen list analysis research, emphasizing how global guidelines must be interpreted through the lens of regional epidemiological realities. By synthesizing data from recent global burden studies, socioeconomic analyses, and antimicrobial resistance research, this document provides a comprehensive technical foundation for developing region-specific approaches to pathogen control.

Foundational Frameworks: Global Priority Pathogens

WHO Bacterial Priority Pathogens List (BPPL)

The WHO BPPL serves as the cornerstone for global efforts to combat antimicrobial resistance. The 2024 edition represents a significant update from the 2017 version, categorizing 24 pathogens across 15 families of antibiotic-resistant bacteria into critical, high, and medium priority groups [1]. This list is intentionally designed to guide research and development (R&D) and public health interventions at a global level, emphasizing the need for regionally tailored strategies to effectively combat resistance.

The list notably features Gram-negative bacteria resistant to last-resort antibiotics, drug-resistant Mycobacterium tuberculosis, and other high-burden resistant pathogens including Salmonella, Shigella, Neisseria gonorrhoeae, Pseudomonas aeruginosa, and Staphylococcus aureus [1]. The inclusion criteria for these pathogens encompass their global impact in terms of burden, alongside considerations of transmissibility, treatability, prevention options, and the R&D pipeline for new treatments. This framework provides the essential structure against which regional variations can be analyzed and understood.

Limitations of Global Guidelines in Regional Contexts

While international and national clinical practice guidelines are meant to contain pragmatic and standardized recommendations to improve quality of care, most guidelines from LMICs are adopted or adapted from existing HIC guidelines or international organization guidelines with little consideration for resource availability, contextual factors, logistical issues, and general feasibility [99]. This approach creates significant challenges for implementation fidelity and adherence in LMIC settings, where factors such as poor health literacy, limited health budgets, inadequate clinical expertise and personnel, poor drug supply, and out-of-pocket health expenditures predominate [99].

The disconnect between global priority lists and regional realities extends to diagnostic capabilities. Critical gaps persist, particularly in low- and middle-income countries, including the absence of multiplex platforms suitable for use in intermediate referral laboratories to identify bloodstream infections directly from whole blood without culture, insufficient access to biomarker tests to distinguish bacterial from viral infections, and limited simple, point-of-care diagnostic tools for primary and secondary care facilities [3]. These limitations disproportionately affect patients in low-resource settings and complicate both the measurement and management of pathogen priorities based on global frameworks.

Comparative Burden of Disease

The composition of disease burden among the world's poorest billion people, as measured by disability-adjusted life years (DALYs), demonstrates striking disparities compared to high-income populations. A comprehensive analysis revealed that the disease burden among the poorest billion is composed of 65% communicable, maternal, neonatal, and nutritional (CMNN) diseases, 29% non-communicable diseases (NCDs), and 6% injuries [100]. This stands in sharp contrast to the disease distribution patterns typically observed in HICs.

When examining age-standardized DALY rates, the disparities become even more pronounced. Rates from NCDs are 44% higher in the poorest billion (23,583 DALYs per 100,000) compared to high-income regions (16,344 DALYs per 100,000) [100]. However, the most dramatic disparities are observed in CMNN conditions, where age-standardized DALY rates are 2,147% higher (32,334 DALYs per 100,000) in the poorest billion, and injuries, which are 86% higher (4,182 DALYs per 100,000) compared to high-income regions [100]. These figures underscore the enormous inequality in infectious disease burden that persists between economic regions.

Urban-Rural Disparities Within Regions

Beyond the clear HIC-LMIC divisions, significant intra-regional disparities further complicate the pathogen priority landscape. A nine-year observational study in China examining 43 notifiable infectious diseases among individuals aged 4 to 24 years found a substantial and progressively widening urban-rural disparity [101]. Children, adolescents, and youths in urban areas experienced a higher average yearly incidence compared to their rural counterparts, with rates of 439 per 100,000 compared to 211 per 100,000, respectively [101].

This urban-rural disparity was primarily driven by higher incidences of specific pathogens in urban areas, including pertussis and seasonal influenza among vaccine-preventable diseases, tuberculosis and scarlet fever among bacterial diseases, infectious diarrhea and hand, foot, and mouth disease among gastrointestinal and enterovirus diseases, dengue among vectorborne diseases, and sexually transmitted infections including syphilis, gonorrhea, HIV/AIDS, and hepatitis C [101]. These findings challenge simplistic assumptions about disease burden being uniformly higher in either urban or rural settings and highlight the need for nuanced, sub-national prioritization of pathogens.

Table 1: Urban-Rural Disparities in Infectious Disease Incidence in China (2013-2021)

Disease Category Specific Pathogens Incidence Rate Ratio (Urban:Rural)
Vaccine-preventable Pertussis 1.782
Vaccine-preventable Seasonal Influenza 3.213
Bacterial Tuberculosis 1.011
Bacterial Scarlet Fever 2.942
Gastrointestinal & Enterovirus Infectious Diarrhea 1.932
Gastrointestinal & Enterovirus Hand, Foot, and Mouth Disease 2.501
Vectorborne Dengue 11.952
Sexually Transmitted & Bloodborne Syphilis 1.743
Sexually Transmitted & Bloodborne Gonorrhea 2.658
Sexually Transmitted & Bloodborne HIV/AIDS 2.269

Socioeconomic Determinants of Pathogen Burden

Poverty as a Determinant of Infection Risk

Low socioeconomic status (SES) exacerbates the risk for colonization or infection with priority bacterial pathogens through multiple mechanisms. A systematic review of literature from 14 countries found that low educational attainment, lower than average income levels, lack of healthcare access, residential crowding, and high deprivation scores were generally associated with higher risks of colonization or infection with antimicrobial-resistant pathogens [102]. These SES indicators function as proxies for the underlying biological, environmental, and social factors that directly increase exposure to pathogens and compromise host defenses.

The relationship between SES and infection risk is particularly pronounced for specific pathogens. Research has demonstrated that shorter duration of education was significantly associated with a greater risk of K. pneumoniae oropharyngeal tract colonization among community-dwelling adults and children in Vietnam, and with a higher incidence of S. aureus bacteremia, E. coli bacteremia, and community-acquired bacteremia among Danish patients [102]. Similarly, compared to participants who had a college-level education or higher, having no formal education was associated with a greater risk of bacteriuria among adults visiting an outpatient department in Ethiopia [102]. These findings consistently demonstrate how socioeconomic factors transcend national boundaries to create similar risk patterns across different economic contexts.

Structural Mechanisms Linking Poverty and Pathogen Exposure

The structural mechanisms through which poverty influences health outcomes are multifaceted, encompassing political and socioeconomic contexts—including governance, societal values, and economic, social, and public policies—that underlie individuals' socioeconomic positions [100]. An individual's socioeconomic status acts as a determinant of health inequities by patterning access to the flexible resources of knowledge, money, power, prestige, and social connections, which enable action to avoid health risks and minimize the impact of poor health [100].

The multidimensional nature of poverty is captured in indices that incorporate factors such as child school attendance, educational attainment, electricity access, sanitation, safe water, floor materials, cooking fuel, and asset ownership [100]. These indicators have direct theoretical links to disease risk: use of biomass fuels is associated with household air pollution; lack of sanitation and safe drinking water is linked to diarrhea and malnutrition; dirt floors provide environments for particular pathogens; and maternal and childhood education have well-established links to mortality [100]. The convergence of these deprivations in the world's most impoverished populations creates a syndemic of risk factors that amplify the burden of priority pathogens.

Research Methodologies for Regional Pathogen Surveillance

Comprehensive Burden Assessment Methodology

The Global Burden of Disease (GBD) study represents the most extensive effort to quantify health loss across locations and over time. The GBD methodology employs 607 billion+ highly standardized and comprehensive estimates to measure health outcomes and systems, tracking 463 health outcomes and risk factors across 204 countries and territories, plus numerous subnational locations [103]. This systematic approach enables comparisons of pathogen priorities across economic regions and informs resource allocation for research and development.

For analyzing disease burden specifically in impoverished populations, sophisticated methodological approaches are required to account for within-country variation. One established method involves:

  • Utilizing national-level disease burden estimates from the GBD study and adjusting these to account for within-country variation [100]
  • Examining the relationship between rates of extreme poverty and disease rates across countries
  • Incorporating expert opinion from disease experts regarding associations between household poverty and the incidence and fatality of conditions
  • Applying multidimensional poverty indices that include indicators such as educational access, infrastructure, and living standards [100]
  • Comparing results across multiple estimation approaches, including aggregating national-level burden from countries with the highest poverty rates

This methodological framework allows researchers to move beyond national averages to understand the precise distribution of pathogen burden across socioeconomic strata within and between countries.

Table 2: Key Methodological Approaches for Regional Pathogen Surveillance

Methodological Approach Key Components Applications
Global Burden of Disease Study 463 health outcomes and risk factors across 204 countries; Disability-Adjusted Life Years (DALYs) as metric Comparing pathogen priorities across regions; Tracking trends over time
Multidimensional Poverty Analysis Eight household-level indicators (education, infrastructure, living standards); Within-country variation assessment Identifying subpopulations at highest risk; Targeting interventions
Notifiable Disease Surveillance 43 notifiable infectious diseases; Urban-rural stratification; Incidence rate ratios Understanding intra-country disparities; Monitoring specific pathogen trends
AMR Intervention Context Analysis Social-ecological system framework; Thematic analysis of success factors Designing context-appropriate interventions; Identifying implementation barriers

Experimental Workflow for Regional Pathogen Priority Analysis

The following diagram illustrates the integrated methodological approach for analyzing regional variations in pathogen priorities:

G Pathogen Priority Analysis Methodology cluster_global Global Framework cluster_regional Regional Assessment cluster_integration Priority Setting WHO WHO Priority Pathogens List Burden Burden Quantification WHO->Burden GBD Global Burden of Disease Data GBD->Burden SES Socioeconomic Factor Analysis Context Contextual Factor Integration SES->Context UrbanRural Urban-Rural Disparity Assessment UrbanRural->Context AMR Antimicrobial Resistance Patterns AMR->Context Priority Regional Priority Pathogen List Burden->Priority Context->Priority

Research Reagents and Tools for Pathogen Surveillance

Table 3: Essential Research Reagents and Tools for Regional Pathogen Surveillance

Research Tool Category Specific Examples Technical Function Regional Application Considerations
Culture-Based Isolation Media Chromogenic agar for ESKAPE pathogens; Selective media for enteric pathogens Pathogen isolation and presumptive identification LMICs: Cost constraints, shelf-life stability, storage requirements
Molecular Detection Kits Multiplex PCR panels; Whole Genome Sequencing kits; AMR gene detection arrays Pathogen identification; Resistance mechanism detection HICs: High-throughput capabilities; LMICs: Need for simplified, point-of-care versions
Antimicrobial Susceptibility Testing Broth microdilution panels; Disk diffusion assays; Gradient diffusion strips Phenotypic resistance profiling LMICs: Need for cost-effective methods; Both: Quality control challenges
Immunoassay Platforms Lateral flow assays; ELISA for biomarker detection; Rapid diagnostic tests Pathogen detection; Host response measurement LMICs: Temperature stability, minimal equipment requirements
Biobanking Systems Cryopreservation media; Bacterial storage systems; Nucleic acid preservation solutions Sample preservation for future analysis LMICs: Reliable cold chain requirements; Both: Ethical considerations for sample sharing

Discussion and Research Implications

Intervention Success Factors Across Economic Contexts

The implementation of successful interventions to address priority pathogens varies significantly between HICs and LMICs, with distinct factors emerging as critical in each context. An exploratory study of 77 AMR interventions across multiple countries identified that themes of 'behavior', 'capacity and resources', 'planning', and 'information' were frequently reported as important for intervention success across all economic settings [104]. However, specific sub-themes demonstrated significant regional variation.

In LMICs, key success factors included 'funding and finances' and 'surveillance, antimicrobial susceptibility testing, and preventive screening' [104]. These sub-themes reflect the fundamental health system challenges that continue to plague resource-limited settings. In contrast, important success factors in HICs were more specific and detailed, including 'mandatory' enforcement, 'multiple profiles' (addressing various aspects of AMR), 'personnel', 'management', and 'design' [104]. This divergence highlights how HICs have progressed to addressing implementation nuances while LMICs continue to struggle with foundational health system requirements.

Diagnostic and Therapeutic Pipeline Disparities

Significant disparities exist in the development pipeline for new antibacterial agents and diagnostics, with concerning implications for addressing regional pathogen priorities. According to WHO's 2025 analysis, the number of antibacterials in the clinical pipeline has decreased from 97 in 2023 to 90 in 2025, with only 15 qualifying as innovative [3]. Most alarmingly, only 5 of these antibacterials are effective against at least one of the WHO "critical" priority pathogens [3].

The preclinical pipeline remains active, with 232 programs across 148 groups worldwide, but 90% of companies involved are small firms with fewer than 50 employees, highlighting the fragility of the R&D ecosystem [3]. Concurrently, critical diagnostic gaps persist, particularly affecting low-resource settings, including the absence of multiplex platforms suitable for intermediate referral laboratories, insufficient access to biomarker tests to distinguish bacterial from viral infections, and limited simple, point-of-care diagnostic tools for primary and secondary care facilities [3]. These gaps in both the therapeutic and diagnostic pipelines disproportionately affect LMICs and hinder effective management of priority pathogens in these settings.

The analysis of regional variations in pathogen priorities between high-income and low- and middle-income countries reveals a complex landscape shaped by socioeconomic factors, healthcare system capacities, and environmental conditions. While the WHO BPPL provides an essential global framework, its effective implementation requires significant regional adaptation to address the specific burden patterns and resource constraints present in different economic contexts.

The disproportionate burden of communicable diseases in LMICs, coupled with the unique urban-rural disparities within regions, demands a nuanced approach to pathogen priority setting. Research and development efforts must address the diagnostic and therapeutic gaps that particularly affect resource-limited settings, while accounting for the structural mechanisms linking poverty to pathogen exposure. Future strategies to combat priority pathogens must be informed by comprehensive burden assessments, contextualized implementation frameworks, and targeted R&D investments that specifically address the divergent needs of HICs and LMICs. Only through such regionally-tailored approaches can the global community effectively mitigate the threat of antimicrobial resistance and infectious diseases across all economic settings.

Conclusion

The 2024 WHO Priority Pathogen Lists represent a significant evolution in the global strategy to combat antimicrobial resistance and prepare for future pandemics. The critical placement of Gram-negative bacteria and rifampicin-resistant Mycobacterium tuberculosis in the BPPL underscores a persistent and severe threat that the current antibacterial pipeline is ill-equipped to address. Concurrently, the expanded pandemic list, employing a novel 'family approach,' enhances our agility against both known and unknown ('Disease X') threats. For researchers and drug developers, the key takeaways are clear: sustained, focused investment in novel antibacterials with new mechanisms of action is non-negotiable. Future success hinges on embracing the prototype pathogen model to develop broad-spectrum countermeasures, aggressively tackling the economic and scientific challenges of the antibiotic pipeline, and integrating R&D with robust infection prevention, improved diagnostics, and strategies for equitable access. The lists are not merely catalogs of threats but a strategic call to action for a coordinated, multi-faceted global research and public health response.

References