The 2024 WHO Bacterial Priority Pathogens List: A Research and R&D Roadmap for Combating Antimicrobial Resistance

Ava Morgan Jan 09, 2026 178

This article provides a comprehensive, research-oriented analysis of the 2024 WHO Bacterial Priority Pathogens List (WHO BPPL).

The 2024 WHO Bacterial Priority Pathogens List: A Research and R&D Roadmap for Combating Antimicrobial Resistance

Abstract

This article provides a comprehensive, research-oriented analysis of the 2024 WHO Bacterial Priority Pathogens List (WHO BPPL). Tailored for researchers, scientists, and drug development professionals, we explore the list's foundational context and methodological framework for ranking antibiotic-resistant bacterial threats. We detail practical applications for R&D prioritization, discuss challenges in surveillance and data interpretation, and offer a critical comparative analysis against regional lists and historical benchmarks. The synthesis provides actionable insights to guide future discovery, diagnostics, and therapeutic strategies in the global fight against AMR.

Decoding the 2024 WHO BPPL: Why This List Matters for Global AMR Research

This technical guide situates the evolution of the World Health Organization's Bacterial Priority Pathogens List (BPPL) within the broader research thesis of the 2024 BPPL overview. The list is a critical tool for galvanizing global research and development (R&D) efforts against antimicrobial resistance (AMR). This document provides a comparative analysis, detailed methodologies for priority pathogen research, and essential resources for scientists and drug development professionals.

Comparative Analysis of the 2017 and 2024 WHO BPPL

The primary function of the BPPL is to prioritize pathogens for which new antibiotics are urgently needed. The 2024 update reflects significant epidemiological shifts and emerging resistance threats.

Table 1: Pathogen Priority Categorization Comparison

Priority Category 2017 BPPL Pathogen Examples 2024 BPPL Pathogen Examples Key Change
CRITICAL Acinetobacter baumannii (carbapenem-resistant)Pseudomonas aeruginosa (carbapenem-resistant)Enterobacteriaceae (carbapenem-resistant, ESBL-producing) Acinetobacter baumannii (carbapenem-resistant)Enterobacterales (carbapenem-resistant, 3rd-gen cephalosporin-resistant)Pseudomonas aeruginosa (carbapenem-resistant) "Enterobacteriaceae" replaced by "Enterobacterales"; 3rd-gen cephalosporin resistance specified.
HIGH Enterococcus faecium (vancomycin-resistant)Staphylococcus aureus (methicillin-resistant, vancomycin-intermediate and resistant)Helicobacter pylori (clarithromycin-resistant) Mycobacterium tuberculosis (rifampicin-resistant, isoniazid-resistant)Salmonella Typhi (fluoroquinolone-resistant)Shigella spp. (fluoroquinolone-resistant) M. tuberculosis elevated from Medium to High; S. aureus and E. faecium moved to Medium.
MEDIUM Streptococcus pneumoniae (penicillin-non-susceptible)Haemophilus influenzae (ampicillin-resistant) Staphylococcus aureus (methicillin-resistant)Enterococcus faecium (vancomycin-resistant)Group A Streptococcus (macrolide-resistant) Reflects successful R&D pipeline for Gram-positives and recognition of other burdens.

Table 2: Key Quantitative Changes in Pathogen Listings

Metric 2017 BPPL 2024 BPPL
Total Families/Species Listed 12 families/species 15 families/species
Gram-negative vs. Gram-positive (Critical/High) 10 Gram-negative, 2 Gram-positive 9 Gram-negative, 3 Gram-positive*
New Additions N/A Mycobacterium tuberculosis (High), Salmonella Typhi (High), Shigella spp. (High), Group A/B Streptococci (Medium)
Notable Re-categorizations N/A Staphylococcus aureus & Enterococcus faecium: High → MediumClostridioides difficile: Added context in introduction.

Includes *M. tuberculosis.

Experimental Protocols for BPPL Pathogen Research

Protocol 1: Broth Microdilution for Antimicrobial Susceptibility Testing (AST)

Objective: To determine the Minimum Inhibitory Concentration (MIC) of antibiotics against priority pathogens. Methodology:

  • Preparation: Prepare cation-adjusted Mueller-Hinton broth (CAMHB) for most bacteria. Use sterile 96-well microtiter plates.
  • Antibiotic Dilution: Perform two-fold serial dilutions of the antibiotic in CAMHB across the plate's rows (e.g., 128 µg/mL to 0.0625 µg/mL). Column 11 is a growth control (no antibiotic), column 12 a sterility control.
  • Inoculum Standardization: Adjust a bacterial suspension to a 0.5 McFarland standard (~1.5 x 10^8 CFU/mL). Further dilute in broth to achieve a final inoculum of ~5 x 10^5 CFU/mL per well.
  • Inoculation: Add 100 µL of the standardized inoculum to all wells except the sterility control.
  • Incubation: Incubate plates at 35±2°C for 16-20 hours in ambient air.
  • MIC Determination: The MIC is the lowest concentration of antibiotic that completely inhibits visible growth. Interpret results using CLSI or EUCAST breakpoints.

Protocol 2: Whole Genome Sequencing (WGS) for AMR Determinant Prediction

Objective: To identify genetic determinants of resistance from bacterial isolates. Methodology:

  • Genomic DNA Extraction: Use a commercial kit (e.g., DNeasy Blood & Tissue Kit) to extract high-quality, high-molecular-weight DNA. Quantify using fluorometry (e.g., Qubit).
  • Library Preparation: Fragment DNA via acoustic shearing. End-repair, A-tail, and ligate sequencing adapters with dual-index barcodes. Clean up and size-select libraries (e.g., with AMPure XP beads).
  • Sequencing: Perform sequencing on an Illumina NovaSeq 6000 platform for 2x150 bp paired-end reads, targeting >50x coverage.
  • Bioinformatic Analysis:
    • Quality Control: Use FastQC and Trimmomatic to assess and trim adapter/low-quality sequences.
    • Assembly: De novo assembly using SPAdes.
    • Resistance Gene Identification: Screen contigs against curated databases (e.g., ResFinder, CARD, NCBI AMRFinderPlus) using ABRicate or BLAST.
    • Clonal Lineage Analysis: Perform in silico multi-locus sequence typing (MLST) using MLST.

Visualizations

G 2017 2017 BPPL (12 pathogen families) 2024 2024 BPPL (15 pathogen families) 2017->2024 Evolution Guided By Drivers Epidemiological Data R&D Pipeline Analysis Global Public Health Need Drivers->2024 Changes Key Changes: - Enterobacterales rename - M. tuberculosis (High) - S. aureus → Medium - Added S. Typhi, Shigella 2024->Changes

Title: Evolution from 2017 to 2024 WHO BPPL

workflow cluster_wet Wet Lab cluster_dry Bioinformatic Analysis S1 Bacterial Isolate S2 Genomic DNA Extraction & QC S1->S2 S3 NGS Library Preparation S2->S3 S4 Sequencing (Illumina Platform) S3->S4 RawData Raw FASTQ Files S4->RawData B1 QC & Trimming (FastQC, Trimmomatic) RawData->B1 B2 De novo Assembly (SPAdes) B1->B2 B3 Resistance Gene Detection (ABRicate vs. ResFinder/CARD) B2->B3 B4 Clonal Lineage Typing (in silico MLST) B3->B4 Report Comprehensive AMR Genotype Report B4->Report

Title: WGS Workflow for AMR Gene Detection

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Reagents and Materials for BPPL Research

Item Function/Benefit Example Product/Brand
Cation-Adjusted Mueller-Hinton Broth (CAMHB) Standard medium for AST, ensuring consistent cation concentrations for reliable MIC results. BBL CAMHB (BD) / Sigma-Aldrich CAMHB
Microtiter Plates (96-well, sterile) Platform for broth microdilution AST. Must be non-binding for antibiotics. Thermo Scientific Nunc MicroWell
McFarland Standard Set (0.5) Essential for standardizing bacterial inoculum density to ensure reproducible AST. bioMérieux McFarland Densitometer & Standards
Genomic DNA Extraction Kit For high-yield, pure DNA from Gram-negative and positive bacteria, suitable for WGS. Qiagen DNeasy Blood & Tissue Kit
NGS Library Prep Kit Facilitates fragmentation, adapter ligation, and indexing of DNA for Illumina sequencing. Illumina DNA Prep Kit
AMPure XP Beads Magnetic beads for size selection and clean-up of DNA fragments during library prep. Beckman Coulter AMPure XP
Qubit dsDNA HS Assay Kit Highly sensitive fluorometric quantitation of low-concentration DNA for WGS input. Thermo Fisher Scientific Qubit Kit
ResFinder / CARD Database Curated bioinformatic databases linking known genetic variants to AMR phenotypes. Available at cge.cbs.dtu.dk / card.mcmaster.ca

The 2024 WHO Bacterial Priority Pathogens List (BPPL) is a critical tool that refines the global framework for prioritizing research and development against antibiotic resistance. Building upon the 2017 list, the 2024 update employs a structured, multi-criteria decision analysis methodology that weighs criteria such as mortality, incidence, treatability, transmission, and R&D pipeline status. This whitepaper interprets this list not as a static catalog, but as a dynamic Core Objective designed to strategically channel R&D investment and public health action. The thesis of the overarching research posits that the 2024 BPPL's greatest utility lies in its systematic application as a guide for resource allocation, trial design, and policy formulation to mitigate the antibiotic resistance crisis.

Quantitative Prioritization: Analysis of the 2024 BPPL Tiers

The 2024 list categorizes pathogens into three priority tiers: Critical, High, and Medium. The quantitative scoring behind this categorization is synthesized in Table 1.

Table 1: 2024 WHO BPPL Priority Tiers and Key Quantitative Drivers

Priority Tier Pathogen Examples (Key Representative) Primary Quantitative Drivers (Summarized)
CRITICAL Acinetobacter baumannii (carbapenem-resistant), Pseudomonas aeruginosa (carbapenem-resistant), Enterobacterales (carbapenem-resistant, 3rd-gen cephalosporin-resistant) High attributable mortality rates in bloodstream infections (>20% in some studies), high incidence in hospitals, very limited treatment options (often last-line agents like colistin), significant nosocomial transmission.
HIGH Salmonella spp. (fluoroquinolone-resistant), Neisseria gonorrhoeae (3rd-gen cephalosporin-resistant), Staphylococcus aureus (methicillin-resistant, MRSA) High community burden and incidence, association with mortality and morbidity, increasing resistance to first- and second-line therapies, potential for horizontal gene transfer.
MEDIUM Group A Streptococcus (macrolide-resistant), Streptococcus pneumoniae (penicillin-non-susceptible) Currently effective treatments generally available, but with worrying resistance trends that threaten frontline therapy efficacy, leading to increased disease severity and healthcare costs.

Translating Priority into Action: Experimental Protocols for Target Validation

Guiding R&D requires moving from priority lists to validated targets. The following protocols are essential for early-stage drug discovery against BPPL pathogens.

Protocol for High-Throughput Essential Gene Identification (CRISPR-interference Screening)

Objective: To identify genes essential for bacterial growth under in vitro and simulated in vivo conditions, providing a list of potential drug targets.

Methodology:

  • Library Construction: Design and clone a guide RNA (gRNA) library targeting all non-essential genes of the pathogen (e.g., A. baumannii) into a dCas9-based CRISPRi vector.
  • Transformation: Electroporate the library into the target bacterial strain expressing dCas9.
  • Growth Competition Assay: a. Inoculate the transformed library pool into rich medium (control) and defined medium mimicking host conditions (e.g., low iron, acidic pH). b. Culture for 15-20 generations, harvesting samples at T=0 and T=final.
  • Sequencing & Analysis: a. Extract genomic DNA from all samples. Amplify the gRNA region via PCR with barcoded primers. b. Perform next-generation sequencing (Illumina MiSeq). c. Calculate the fold-depletion of each gRNA from T=0 to T=final using MAGeCK or similar software. Genes with significantly depleted gRNAs are classified as conditionally essential.

Protocol for Assessing Membrane Permeability in Gram-Negative Pathogens

Objective: To evaluate a compound's ability to penetrate the outer membrane of Critical-tier Gram-negative pathogens, a major barrier for new antibiotics.

Methodology:

  • Strain Preparation: Use wild-type and hyperpermeable (e.g., lpxC mutant or polymyxin B-supersensitive) strains of P. aeruginosa or A. baumannii.
  • Compound Exposure: a. Prepare a dilution series of the test compound in cation-adjusted Mueller-Hinton broth (CAMHB). b. Inoculate wells with ~5 x 10^5 CFU/mL of either the wild-type or mutant strain. c. Incubate at 35°C for 18-24 hours.
  • Analysis: a. Determine the Minimum Inhibitory Concentration (MIC) for both strains. b. Calculate the Potentiation Factor (PF) = MIC (wild-type) / MIC (hyperpermeable mutant). A PF >4 suggests the compound has difficulty traversing the intact outer membrane, highlighting a need for chemical optimization.

Visualizing Key Biological Pathways and Workflows

G Start Identify Priority Pathogen (e.g., CRAB) Lib Construct CRISPRi gRNA Library Start->Lib Transform Transform into Pathogen with dCas9 Lib->Transform Culture Growth Competition (Control vs. Host-like) Transform->Culture Seq NGS of gRNA Regions (T=0 & T=final) Culture->Seq Bioinfo Bioinformatic Analysis (gRNA depletion) Seq->Bioinfo Target List of Conditionally Essential Gene Targets Bioinfo->Target

Diagram 1: CRISPRi Screening Workflow for Target ID (83 chars)

G Bla β-Lactamase Production Deg Antibiotic Degradation Bla->Deg PBPs Altered Penicillin- Binding Proteins (PBPs) Targ Target Alteration PBPs->Targ Porin Porin Loss/ Modification Perm Reduced Uptake Porin->Perm Efflux Upregulated Efflux Pumps Expel Active Efflux Efflux->Expel LPS Lipopolysaccharide Modification BL Drug Unable to Bind or Cross LPS->BL AB β-Lactam Antibiotic AB->Bla Induces AB->PBPs Binds AB->Porin Enters through AB->Efflux Substrate for AB->LPS Binds/Crosses

Diagram 2: Key Resistance Mechanisms in Critical Gram-Negative Pathogens (99 chars)

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Reagents for BPPL-Focused Antimicrobial Research

Reagent / Material Function / Application in Context
Cation-Adjusted Mueller-Hinton Broth (CAMHB) Standardized medium for MIC determination, ensuring reproducibility and comparability of susceptibility data across labs.
Check-MDR/CT103 XL Microarray Kit Rapid molecular detection of common carbapenemase and ESBL genes from bacterial colonies or positive blood cultures.
In vivo Infection Model Reagents (e.g., neutropenic murine thigh/ lung infection models) Essential for evaluating compound efficacy in a mammalian host, measuring bacterial burden reduction (log10 CFU/g) after treatment.
Membrane Permeabilizers (e.g., Polymyxin B nonapeptide, EDTA) Used in combination assays to distinguish between intrinsic activity and penetrance issues in Gram-negative pathogens.
Crystal Violet & Microplate Reader For performing high-throughput biofilm formation and eradication assays, relevant for device-related infections (e.g., P. aeruginosa).
Recombinant Bacterial Enzymes (e.g., purified NDM-1, KPC-2) For high-throughput screening of inhibitor compounds and detailed enzymology (Ki, IC50 determination) in target-based approaches.
Human Serum or Simulated Body Fluids For testing compound stability and protein binding under physiologically relevant conditions, predicting in vivo pharmacokinetics.

The 2024 update of the World Health Organization's Bacterial Priority Pathogens List (WHO BPPL) serves as a critical global framework to guide and catalyze research and development (R&D) for new antibiotics. This in-depth technical guide presents the revised three-tier prioritization system—Critical, High, and Medium—detailing the pathogens within each category based on a comprehensive analysis of their associated global burden of antimicrobial resistance (AMR), transmissibility, treatability, and R&D pipeline status. This document is framed within the overarching thesis that this tiered list is not merely a catalog of threats, but a strategic R&D roadmap intended to optimize resource allocation for researchers, scientists, and drug development professionals working to mitigate the AMR crisis.

The 2024 Tiers: Pathogen Classification and Rationale

The classification is based on a multi-criteria decision analysis (MCDA) weighing ten criteria across three domains: public health impact, antimicrobial resistance burden, and aspects related to R&D.

Table 1: The 2024 WHO Bacterial Priority Pathogens List (BPPL) Tiers

Priority Tier Pathogens (Genus/Species) Key Resistance Phenotypes
CRITICAL Acinetobacter baumannii Carbapenem-resistant
Enterobacterales Carbapenem-resistant, third-generation cephalosporin-resistant
Mycobacterium tuberculosis Rifampicin-resistant, Isoniazid-resistant, Extensively drug-resistant (XDR), Totally drug-resistant
Pseudomonas aeruginosa Carbapenem-resistant
Salmonella enterica serovar Typhi Fluoroquinolone-resistant
HIGH Enterococcus faecium Vancomycin-resistant
Helicobacter pylori Clarithromycin-resistant
Campylobacter spp. Fluoroquinolone-resistant
Neisseria gonorrhoeae Third-generation cephalosporin-resistant, Fluoroquinolone-resistant
Staphylococcus aureus Methicillin-resistant (MRSA)
Shigella spp. Fluoroquinolone-resistant
MEDIUM Group A Streptococcus Macrolide-resistant
Streptococcus pneumoniae Penicillin-non-susceptible
Haemophilus influenzae Ampicillin-resistant
Group B Streptococcus Penicillin-non-susceptible

Experimental Protocols for Key Phenotypic Assays

Broth Microdilution for Minimum Inhibitory Concentration (MIC) Determination

Purpose: To quantitatively determine the lowest concentration of an antimicrobial agent that inhibits visible growth of a bacterium. Protocol:

  • Inoculum Preparation: Adjust the turbidity of a fresh bacterial suspension in Mueller-Hinton Broth (MHB) to a 0.5 McFarland standard (~1-2 x 10^8 CFU/mL). Further dilute this suspension in MHB to achieve a final inoculum density of approximately 5 x 10^5 CFU/mL in the assay plate.
  • Plate Preparation: Using sterile technique, dispense 50 µL of cation-adjusted MHB into all wells of a 96-well microtiter plate. Add 50 µL of the antimicrobial stock solution (at 2x the highest desired concentration) to the first well of a given row. Perform serial two-fold dilutions across the row. Finally, add 50 µL of the prepared inoculum to all test wells. Include growth control (inoculum, no drug) and sterility control (broth only) wells.
  • Incubation: Seal the plate and incubate at 35 ± 2°C for 16-20 hours (standard fast-growing bacteria) under ambient atmosphere.
  • Endpoint Reading: Visually inspect the plate or read optical density (OD) at 600 nm using a microplate reader. The MIC is defined as the lowest concentration of antimicrobial that completely inhibits visible growth.

Molecular Detection of Carbapenemase Genes via Multiplex PCR

Purpose: To rapidly identify the presence and type of carbapenemase-resistance genes (blaKPC, blaNDM, blaVIM, blaOXA-48-like, blaIMP) from bacterial isolates. Protocol:

  • DNA Extraction: Boil a 1-2 colony suspension in 100 µL of nuclease-free water for 10 minutes. Centrifuge at 12,000 x g for 2 minutes. The supernatant contains crude genomic DNA.
  • PCR Master Mix Preparation (25 µL reaction):
    • 12.5 µL of 2X Multiplex PCR Master Mix (containing Hot Start Taq DNA polymerase, dNTPs, MgCl2).
    • 2.5 µL of primer mix (containing forward and reverse primers for each target gene at optimized concentrations).
    • 5.0 µL of template DNA.
    • 5.0 µL of nuclease-free water.
  • Thermal Cycling Conditions:
    • Initial Denaturation: 95°C for 15 minutes.
    • 35 cycles of: Denaturation (95°C, 30 sec), Annealing (60°C, 90 sec), Extension (72°C, 90 sec).
    • Final Extension: 72°C for 10 minutes. Hold at 4°C.
  • Analysis: Run 5-10 µL of the PCR product on a 2% agarose gel stained with ethidium bromide or a safer alternative (e.g., SYBR Safe). Visualize under UV light and compare amplicon sizes to a DNA ladder and positive controls for each gene.

Visualizations

WHO BPPL 2024 Prioritization Logic Flow

G Start Pathogen Assessment Criteria Evaluate Against 10 MCDA Criteria Start->Criteria Domain1 Public Health Impact Criteria->Domain1 Domain2 Antimicrobial Resistance Burden Criteria->Domain2 Domain3 R&D Considerations Criteria->Domain3 Score Calculate Composite Score Domain1->Score Domain2->Score Domain3->Score Tier Assign Priority Tier Score->Tier Critical CRITICAL Tier Tier->Critical Highest High HIGH Tier Tier->High Medium Medium MEDIUM Tier Tier->Medium Lower

Diagram Title: WHO BPPL 2024 Prioritization Logic Flow

Key Beta-Lactam Resistance Mechanism Pathways

G Antibiotic β-Lactam Antibiotic PBP Penicillin-Binding Protein (PBP) (Target) Antibiotic->PBP Binds Enzyme β-Lactamase (Enzyme) Antibiotic->Enzyme 1 Efflux Efflux Pump Antibiotic->Efflux 2 AlteredTarget Altered PBP (e.g., PBP2a in MRSA) Antibiotic->AlteredTarget 3 Inhibition Cell Wall Synthesis INHIBITED PBP->Inhibition Death Bacterial Cell Death Inhibition->Death Hydrolysis Antibiotic Hydrolysis Enzyme->Hydrolysis Hydrolysis->Antibiotic Degrades Removal Antibiotic Removal Efflux->Removal Removal->Antibiotic Pumps Out NoBind Reduced Antibiotic Binding AlteredTarget->NoBind NoBind->PBP Bypasses

Diagram Title: Key Beta-Lactam Resistance Mechanism Pathways

The Scientist's Toolkit: Key Research Reagent Solutions

Table 2: Essential Research Materials for AMR Studies

Reagent/Material Primary Function Application Example
Cation-Adjusted Mueller-Hinton Broth (CA-MHB) Standardized growth medium for antimicrobial susceptibility testing (AST). Provides consistent concentrations of divalent cations (Ca2+, Mg2+) critical for accurate aminoglycoside and tetracycline testing. Broth microdilution MIC assays per CLSI/EUCAST guidelines.
Premixed AST Panels (Sensititre, MICRONAUT) Lyophilized or frozen panels containing predefined gradients of multiple antibiotics in a microtiter plate format. High-throughput phenotypic susceptibility profiling of clinical isolates.
Multiplex PCR Master Mix Optimized blend of Hot Start Taq polymerase, dNTPs, MgCl2, and buffer for simultaneous amplification of multiple DNA targets. Rapid detection of a panel of carbapenemase (blaKPC, NDM, VIM, OXA-48) or ESBL genes from bacterial DNA.
Whole Genome Sequencing (WGS) Kits (Illumina Nextera XT) Library preparation kits for fragmenting and tagging genomic DNA with sequencing adapters. Comprehensive detection of resistance genes, mutations, and strain typing for epidemiological studies.
Recombinant β-Lactamase Enzymes (e.g., NDM-1, KPC-2) Purified, recombinant forms of specific resistance enzymes. Biochemical high-throughput screening for novel β-lactamase inhibitors. Enzyme kinetics studies (Km, kcat, Ki).
Cell-Based Reporter Assays (e.g., Cytotoxicity Assays) Mammalian cell lines and viability markers (e.g., MTT, resazurin). Evaluating the therapeutic index and cytotoxicity of novel antimicrobial compounds.
Galleria mellonella (Wax Moth Larvae) In vivo infection model for studying virulence and in vivo efficacy of antibiotics. Pre-mammalian testing of antibiotic efficacy against critical priority pathogens like A. baumannii and P. aeruginosa.

Thesis Context: This document provides an in-depth technical analysis of the updates to the 2024 WHO Bacterial Priority Pathogens List (BPPL), serving as a critical resource within broader research aimed at directing global antimicrobial resistance (AMR) surveillance, infection control, and R&D investment.

The 2024 BPPL represents a significant evolution from the 2017 list, incorporating a refined, evidence-based methodology that emphasizes unmet R&D needs and the critical threat of drug-resistant infections. The most notable shift is the expansion from three to four priority categories and the re-categorization of several pathogens based on new epidemiological and resistance data.

Comparative Tabular Analysis of 2017 vs. 2024 WHO BPPL

Table 1: Pathogen Categorization Shifts (2017 vs. 2024)

Priority Tier (2024) Pathogens (2024) Status Change from 2017 List
CRITICAL Acinetobacter baumannii (carbapenem-resistant), Enterobacterales (carbapenem-resistant, 3rd-gen cephalosporin-resistant), Mycobacterium tuberculosis (rifampicin-resistant) M. tuberculosis added; Pseudomonas aeruginosa & Enterococcus faecium moved to High.
HIGH Salmonella Typhi (fluoroquinolone-resistant), Shigella spp. (fluoroquinolone-resistant), Enterococcus faecium (vancomycin-resistant), Pseudomonas aeruginosa (carbapenem-resistant), Helicobacter pylori (clarithromycin-resistant) E. faecium & P. aeruginosa moved from Critical; H. pylori & Campylobacter spp. added.
MEDIUM Group A Streptococcus (macrolide-resistant), Streptococcus pneumoniae (penicillin-non-susceptible), Haemophilus influenzae (ampicillin-resistant), Group B Streptococcus (penicillin-resistant) New category for pathogens with evolving resistance.
WATCH Bordetella pertussis (erythromycin-resistant), Mycoplasma genitalium (macrolide-resistant) New category for pathogens with potential future AMR threat.

Table 2: Key Quantitative Metrics for 2024 Critical Priority Pathogens

Pathogen & Resistance Profile Estimated Global Deaths Attributable to AMR (2019)* Key Empiric Treatment Gaps No. of Antibiotics in Clinical Pipeline (Preclinical-Phase 3)
Carbapenem-resistant A. baumannii (CRAB) ~50,000 - 100,000 Colistin/tigecycline-based regimens, high toxicity & failure rates. 8-12 (Non-β-lactam novel agents: ~4)
Carbapenem-resistant Enterobacterales (CRE) ~50,000 - 100,000 Limited to novel β-lactam/β-lactamase inhibitor combos, cefiderocol. 15-20 (Novel classes: ~2)
Rifampicin-resistant M. tuberculosis (RR-TB) ~ 200,000 Shorter, oral regimens (BPaLM) available but access & cost barriers persist. 10-15 (Novel mechanisms: ~3)

Based on Murray et al., *The Lancet 2022. Based from WHO/ GARDP pipeline analyses.

Detailed Methodologies for Cited Surveillance & R&D Experiments

Protocol 1: Broth Microdilution for Determining MICs of Novel β-Lactam/β-Lactamase Inhibitor Combinations against CRE

Objective: To determine the minimum inhibitory concentration (MIC) of a novel β-lactam/β-lactamase inhibitor (BL/BLI) combination against a panel of genetically characterized CRE isolates.

  • Bacterial Inoculum Preparation: Suspend colonies from an overnight agar plate in sterile saline or Mueller-Hinton Broth (MHB). Adjust turbidity to a 0.5 McFarland standard (~1-2 x 10^8 CFU/mL). Further dilute 1:100 in MHB to achieve ~1-2 x 10^6 CFU/mL.
  • Antibiotic Solution Preparation: Prepare stock solutions of the β-lactam and BLI per CLSI guidelines. Serially dilute the β-lactam antibiotic in a fixed ratio with the BLI (e.g., 1:1, 4:1) across a 96-well microtiter plate using cation-adjusted MHB (CA-MHB).
  • Inoculation & Incubation: Dispense 100 µL of the standardized bacterial inoculum into each well. Include growth control (no antibiotic) and sterility control wells. Seal plates and incubate at 35°C ± 2°C for 16-20 hours in ambient air.
  • MIC Determination: The MIC is the lowest concentration of the BL/BLI combination that completely inhibits visible growth. Confirm endpoint spectrophotometrically at 600 nm if needed.
  • Data Analysis: Interpret MICs using provisional epidemiological cut-off values (ECVs) or CLSI breakpoints if established. Correlate MICs with whole-genome sequencing data for β-lactamase genes (blaKPC, blaNDM, blaOXA-48-like, etc.).

Protocol 2: Whole-Genome Sequencing (WGS) for AMR Surveillance of Priority Pathogens

Objective: To characterize the resistome, virulome, and sequence type of BPPL isolates for outbreak investigation and resistance trend analysis.

  • Genomic DNA Extraction: Use a commercial kit for Gram-negative and Gram-positive bacteria (e.g., DNeasy Blood & Tissue Kit) to extract high-molecular-weight DNA. Quantify using a fluorometric method (e.g., Qubit).
  • Library Preparation & Sequencing: Prepare sequencing libraries using a PCR-free or ligation-based kit to minimize bias. Sequence on a short-read platform (Illumina NovaSeq) for high coverage (>100x) and/or a long-read platform (Oxford Nanopore Technologies) for assembly and plasmid analysis.
  • Bioinformatic Analysis:
    • Quality Control & Assembly: Trim adapters and low-quality bases (Fastp). Assemble reads de novo (SPAdes for short-read; Flye for long-read). Assess assembly quality (QUAST).
    • AMR Gene Detection: Use the Comprehensive Antibiotic Resistance Database (CARD) via RGI or ABRicate.
    • Multi-Locus Sequence Typing (MLST): Determine sequence type (ST) using schemes from PubMedST.
    • Phylogenetic Analysis: Generate core-genome alignments (Roary) and construct maximum-likelihood phylogenetic trees (IQ-TREE).

Visualizations of Key Concepts

Diagram 1: 2024 BPPL Evidence-to-Categorization Framework

BPPL_2024_Framework Evidence1 Burden of Disease & Mortality Data Methodology Multi-Criteria Decision Analysis (Structured Expert Elicitation) Evidence1->Methodology Evidence2 Drug Resistance & Transmission Dynamics Evidence2->Methodology Evidence3 R&D Pipeline Gaps & Treatment Needs Evidence3->Methodology TierC CRITICAL PRIORITY High Burden & Urgent Need Methodology->TierC TierH HIGH PRIORITY Methodology->TierH TierM MEDIUM PRIORITY Methodology->TierM TierW WATCH LIST Methodology->TierW Output Guidance for: - R&D Investment - Public Health Action - Surveillance Focus TierC->Output TierH->Output TierM->Output TierW->Output

Diagram 2: Key β-Lactamase-Mediated Resistance in Critical Priority Gram-Negative Pathogens

BetaLactamaseResistance Antibiotic β-Lactam Antibiotic (Penicillins, Cephalosporins, Carbapenems) Inactivation Hydrolysis & Inactivation of β-Lactam Ring Antibiotic->Inactivation Enters Cell KPC Serine-β-Lactamases (e.g., KPC, OXA-48-like) KPC->Inactivation MBL Metallo-β-Lactamases (MBLs) (e.g., NDM, VIM) MBL->Inactivation ResistantCell Resistant Bacterial Cell (Treatment Failure) Inactivation->ResistantCell No effective drug NovelBLI Novel β-Lactamase Inhibitors (e.g., Avibactam, Vaborbactam, Relebactam) Inhibit Inhibition NovelBLI->Inhibit Targets Serine Enzymes NewDrugs Novel Siderophore- Cephalosporins NewDrugs->ResistantCell Evades Hydrolysis Inhibit->KPC Blocks Activity

The Scientist's Toolkit: Essential Research Reagents & Materials

Table 3: Key Research Reagent Solutions for BPPL-focused Studies

Item Function & Application Example Product/Catalog
Cation-Adjusted Mueller-Hinton Broth (CA-MHB) Standardized medium for antimicrobial susceptibility testing (AST), ensuring consistent cation concentrations (Ca2+, Mg2+) for reliable MIC results. Becton Dickinson (BD) 212322 / Sigma-Aldrich 90922.
MIC Test Strips (Gradient Diffusion) Rapid phenotypic AST to determine MICs for fastidious or slow-growing pathogens (e.g., H. pylori, M. genitalium). Liofilchem MTS / bioMérieux Etest.
PCR or LAMP Master Mix for AMR Gene Detection For rapid molecular detection of key resistance determinants (blaNDM, blaKPC, mecA, vanA) from culture or direct specimens. Thermo Fisher TaqMan Fast / New England Biolabs WarmStart.
Chromogenic Agar for CRE/CRAB Screening Selective and differential culture medium for rapid screening of carbapenem-resistant organisms from surveillance samples. CHROMagar mSuperCARBA / CHROMagar COL-APSE.
High-Fidelity DNA Polymerase for WGS Library Prep Accurate amplification during library preparation for next-generation sequencing to prevent sequencing errors in resistance gene analysis. NEB Q5 / Takara Ex Taq.
Recombinant β-Lactamase Enzymes Positive controls for enzyme inhibition assays to evaluate the efficacy of novel β-lactamase inhibitors. Sigma-Aldrich recombinant KPC-3, NDM-1.
In Vivo Imaging System (IVIS) Reagents Bioluminescent/fluorescent probes for non-invasive monitoring of infection progression and treatment efficacy in animal models. PerkinElmer D-Luciferin / Xenolight RediJect.

Within the context of the 2024 WHO Bacterial Priority Pathogens List (BPPL) research, this guide details the methodologies for systematic evidence synthesis and its translation into actionable policy and research priorities.

Systematic Review Methodology for AMR Pathogen Prioritization

The systematic review (SR) forms the foundational evidence base for ranking pathogens within frameworks like the BPPL. The process is executed in distinct phases.

Table 1: Phases of a Systematic Review for Pathogen Prioritization

Phase Key Activities Deliverables
1. Planning Define PICO(S): Population (Pathogen), Intervention (N/A), Comparator (N/A), Outcomes (Mortality, Transmissibility, Treatment Options), Study types. Develop & register protocol. Registered review protocol (PROSPERO).
2. Search & Selection Execute multi-database search (MEDLINE, Embase, Cochrane, regional DBs). Apply inclusion/exclusion criteria via dual-independent screening. PRISMA flow diagram documenting identified, screened, and included studies.
3. Data Extraction Extract quantitative & qualitative data into piloted forms: incidence, mortality rates, resistance prevalence, treatment availability, study quality. Structured evidence tables for each pathogen-outcome pair.
4. Risk of Bias Assessment Evaluate study quality using tools appropriate to design (e.g., ROBINS-I for non-randomized studies, Newcastle-Ottawa Scale for cohort studies). Summary of bias risk across studies, informing evidence certainty.
5. Synthesis Perform meta-analysis where clinical & statistical homogeneity exists. For heterogeneous data, conduct narrative synthesis structured by outcome. Forest plots (quantitative) or evidence maps (qualitative).
6. Certainty Assessment Rate overall confidence in each body of evidence using GRADE (Grading of Recommendations Assessment, Development and Evaluation). Summary of Findings (SoF) table with GRADE ratings (High, Moderate, Low, Very Low).

Detailed Protocol for Evidence Retrieval and Synthesis

Objective: To collate and synthesize global data on the burden, resistance profiles, and treatment landscape for pathogens under consideration for the 2024 BPPL.

  • Search Strategy: A comprehensive search string is constructed using MeSH terms and keywords. Example for Acinetobacter baumannii: ("Acinetobacter baumannii"[Mesh] OR "Carbapenem-Resistant Acinetobacter"[TW]) AND ("Drug Resistance"[Mesh] OR "Cross Infection"[Mesh] OR "Mortality"[Mesh] OR "Treatment Outcome"[Mesh]). No date or language restrictions are applied. Grey literature is searched via WHO reports and national AMR surveillance databases.
  • Screening Process: Titles/abstracts screened independently by two reviewers in Covidence software. Conflicts resolved by a third reviewer. Full-text review follows the same dual-independent process.
  • Data Extraction Fields: Pre-defined fields include: Study ID, design, location, period, population size, pathogen, antimicrobial tested, % resistance (with CI), incidence rate, mortality measure (e.g., attributable mortality fraction), treatment regimens used, and funding source.

The Evidence-to-Decision (EtD) Framework

The EtD framework provides a transparent, structured process to move from the synthesized evidence (systematic review) to a policy decision (e.g., inclusion and ranking on the BPPL).

etd_framework SR Systematic Review Evidence Base P1 Problem Priority: Is it a large burden? SR->P1 P2 Values & Acceptability: Is the priority acceptable to stakeholders? SR->P2 P3 Resource Use: What are the implications for R&D investment? SR->P3 P4 Equity: Will prioritization reduce health disparities? SR->P4 P5 Feasibility: Is surveillance & research feasible globally? SR->P5 DEC Decision or Recommendation P1->DEC P2->DEC P3->DEC P4->DEC P5->DEC

Diagram Title: Evidence-to-Decision Framework Flow

Table 2: Evidence-to-Decision Criteria for Pathogen Prioritization

Criteria Guiding Question for BPPL Evidence Sources (Beyond SR)
Priority of the Problem What is the global/regional burden of disease, mortality, and healthcare cost? SR data, DALY estimates, hospital discharge data, economic models.
Values & Acceptability Is there consensus among experts, public health leaders, and the public on the urgency? Stakeholder surveys (Delphi panels), public perception studies, ethical analyses.
Resource Use What is the cost and potential return on investment for new therapeutic R&D? Health technology assessments, market analyses, R&D pipeline reviews.
Equity Does the pathogen disproportionately affect vulnerable populations or low-resource settings? Disaggregated surveillance data, access-to-medicine reports.
Feasibility Can effective surveillance and infection control measures be implemented? Case studies of successful containment, diagnostic availability assessments.

Experimental Protocols for Key Studies Informing the BPPL

Protocol for Determining Attributable Mortality

Objective: To quantify the mortality directly attributable to infections caused by antibiotic-resistant vs. susceptible strains of a pathogen.

  • Design: Multicenter matched retrospective cohort study.
  • Population: Hospitalized patients with culture-confirmed bloodstream infection (BSI).
  • Groups: Cases (BSI with resistant strain) are matched 1:1 with Controls (BSI with susceptible strain) by: age (±5 years), primary diagnosis, ward type, and date of infection (±30 days).
  • Primary Outcome: 30-day all-cause mortality.
  • Analysis: Conditional logistic regression to calculate the adjusted odds ratio (aOR) for mortality, controlling for residual confounders (e.g., severity via Charlson Comorbidity Index).

Protocol for Global Resistance Prevalence Meta-Analysis

Objective: To generate a pooled estimate of the prevalence of key drug-resistance phenotypes (e.g., carbapenem resistance) for a pathogen.

  • Search & Selection: As per Section 1.1, focusing on surveillance studies and diagnostic cohorts.
  • Data Extraction: Extract numerator (resistant isolates) and denominator (total isolates tested) for each study. Record microbiological methods (e.g., EUCAST/CLSI breakpoints).
  • Statistical Synthesis: Perform a meta-analysis of proportions using a random-effects model (DerSimonian-Laird method) with Freeman-Tukey double arcsine transformation to stabilize variances. Assess heterogeneity using I² statistic. Generate forest and funnel plots.

meta_workflow S1 Extract Study Proportions (n/N) S2 Transform Proportions (Freeman-Tukey) S1->S2 S3 Fit Random-Effects Model S2->S3 S4 Pool Estimate & Calculate 95% CI S3->S4 S5 Back-Transform to Original Scale S4->S5

Diagram Title: Meta-Analysis of Proportions Workflow

The Scientist's Toolkit: Research Reagent Solutions

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

Item Function/Application Example/Note
Chromogenic Agar Media Selective isolation and presumptive identification of target multidrug-resistant organisms (MDROs) from clinical specimens. CHROMagar KPC for CRE, CHROMagar MRSA for methicillin-resistant S. aureus.
Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry Rapid, accurate species-level identification of bacterial isolates. Bruker MALDI Biotyper, VITEK MS. Reduces ID time from 24h to minutes.
Automated Antimicrobial Susceptibility Testing (AST) System Determines minimum inhibitory concentration (MIC) and susceptibility category for a panel of antibiotics. VITEK 2, BD Phoenix. Generates essential data for resistance surveillance.
PCR & Real-Time PCR Master Mixes Detection of specific resistance genes (bla_NDM, bla_KPC, mcr-1) directly from samples or colonies. TaqMan assays for bla_OXA-48-like. Essential for rapid molecular epidemiology.
Whole Genome Sequencing (WGS) Kits Comprehensive analysis of resistance genotype, virulence factors, and phylogenetic relationships for outbreak tracking. Illumina DNA Prep library prep kits. Enables high-resolution pathogen typing.
Micromanipulator for Single-Cell Isolation Isolation of individual bacterial cells for studies on heteroresistance or persistence. Used in microfluidic or agar-based studies to clone subpopulations.
In Vivo Imaging System (IVIS) Non-invasive, real-time monitoring of bioluminescent pathogen load and spread in animal models of infection. Crucial for evaluating efficacy of novel antibacterial compounds in vivo.
Humanized Mouse Models Pre-clinical models with engrafted human immune cells or tissues to better mimic human immune response to infection. NSG mice engrafted with human hematopoietic stem cells (huNSG).

From List to Lab: Applying the WHO BPPL in Antimicrobial R&D and Surveillance

The 2024 WHO Bacterial Priority Pathogens List (BPPL) represents a critical global prioritization exercise, guiding research and development (R&D) for new antibiotics. This document deconstructs the two core, interlinked criteria used in its formulation: Public Health Impact and Unmet R&D Needs. Framed within a broader thesis on the BPPL, this technical guide provides researchers and drug developers with the methodologies and analytical frameworks necessary to quantify these criteria and apply them to pathogen prioritization.

Quantifying Public Health Impact: Metrics and Methodologies

Public Health Impact is a composite metric derived from epidemiological burden, resistance prevalence, and healthcare consequences.

Data is systematically collected from surveillance networks, hospital databases, and peer-reviewed studies.

Table 1: Core Metrics for Assessing Public Health Impact

Metric Definition Primary Data Sources Measurement Unit
Incidence New infection cases per population per time ECDC, CDC NHSN, GLASS, regional surveillance Cases per 100,000 patient-days or population
Attributable Mortality Deaths directly caused by the infection Cohort studies, matched case-control studies Case Fatality Rate (CFR%), Population Attributable Fraction (PAF%)
Disability-Adjusted Life Years (DALYs) Sum of Years of Life Lost (YLL) and Years Lived with Disability (YLD) Global Burden of Disease (GBD) studies, meta-analyses DALYs per 100,000 population
Resistance Prevalence Percentage of isolates resistant to key first- and last-line antibiotics WHO GLASS, EARS-Net, US SENSOR % Resistant (with 95% CI)
Healthcare Cost Direct medical costs associated with infection and management Hospital billing databases, cost-utility analyses USD per episode

Experimental Protocol: Calculating DALYs for a Resistant Pathogen

Protocol Title: Retrospective Cohort Analysis for DALY Attribution.

  • Cohort Definition: Identify two matched cohorts from electronic health records (EHR): patients with infection caused by the resistant pathogen (Case) and patients with infection caused by a susceptible strain of the same pathogen (Control). Matching criteria: age (±5 years), sex, primary diagnosis, comorbidities (Charlson Index ±1).
  • Data Extraction: For each patient, extract: date of diagnosis, length of hospital stay, occurrence and duration of ICU admission, occurrence of sequelae (e.g., renal failure, disability), and vital status at discharge and 1-year follow-up.
  • YLL Calculation: For deceased patients, calculate Years of Life Lost (YLL) using a standard reference life table.
    • YLL = Σ (Life expectancy at age of death - Age at death)
    • Perform for both Case and Control cohorts.
  • YLD Calculation: For sequelae, assign a disability weight (DW) from the GBD study. Calculate Years Lived with Disability (YLD).
    • YLD = DW * Average duration of disability (years)
  • DALY and Excess DALY: Sum YLL and YLD for each cohort to get average DALYs per patient. The excess DALY attributable to resistance is:
    • Excess DALY = (Average DALY per Case patient) - (Average DALY per Control patient)
  • Statistical Analysis: Use multivariable regression to adjust for residual confounding and calculate 95% confidence intervals.

G Start Start: Patient EHR Data Match Cohort Matching (Resistant vs. Susceptible) Start->Match Data Extract Outcomes: Mortality, Sequelae, LOS Match->Data CalcYLL Calculate Years of Life Lost (YLL) Data->CalcYLL CalcYLD Calculate Years Lived with Disability (YLD) Data->CalcYLD Sum Sum YLL + YLD = Total DALYs/Patient CalcYLL->Sum CalcYLD->Sum Compare Calculate Excess DALY (Resistant - Susceptible) Sum->Compare Output Output: Attributable Burden Compare->Output

DOT script for Workflow: DALY Calculation for Resistant Infections

Assessing Unmet R&D Needs: The Pipeline Analysis Framework

Unmet R&D Needs evaluate the gap between current therapeutic options and the clinical demand, focusing on the robustness of the antibiotic pipeline.

Pipeline Scoring System

The therapeutic pipeline is stratified by clinical development phase and innovation level.

Table 2: Scoring the Antibacterial Pipeline for a Pathogen

Phase Definition Score Innovation Criteria (Additional Points)
Preclinical Lead compound identified, in vivo efficacy data. 1 +1 for novel class/target; +0.5 for new MOA within class
Phase I First-in-human safety/pharmacokinetics. 2 +1 for activity against pan-resistant strains
Phase II Preliminary efficacy in targeted patient population. 3 +1 for potential to address key resistance mechanisms (e.g., β-lactamase inhibitors)
Phase III Pivotal efficacy and safety trials. 4 +0.5 for oral bioavailability option
Registered Approved in at least one jurisdiction. 5 N/A

Experimental Protocol:In VitroCheckerboard Synergy Assay

Protocol Title: Microdilution Checkerboard Assay to Evaluate Novel Combination Therapies.

  • Reagent Preparation: Prepare 2X concentration stock solutions of Antibiotic A and Antibiotic B in cation-adjusted Mueller-Hinton Broth (CAMHB).
  • Plate Setup: Using a 96-well microtiter plate, serially dilute Antibiotic A along the x-axis (columns 1-12) and Antibiotic B along the y-axis (rows A-H). This creates a matrix of all possible concentration combinations.
  • Inoculation: Adjust a bacterial suspension (target pathogen) to 0.5 McFarland standard and further dilute to yield ~5 x 10^5 CFU/mL in CAMHB. Add 50µL of this inoculum to each well. Column 12 (no antibiotic) serves as growth control. Row H (no antibiotic B) and Column 1 (no antibiotic A) serve as single-agent controls.
  • Incubation: Incubate plate at 35°C ± 2°C for 18-20 hours.
  • Analysis: Determine the Minimum Inhibitory Concentration (MIC) for each drug alone. Calculate the Fractional Inhibitory Concentration (FIC) for each combination well where growth is inhibited.
    • FIC of A = (MIC of A in combination) / (MIC of A alone)
    • FIC of B = (MIC of B in combination) / (MIC of B alone)
    • ΣFIC = FIC of A + FIC of B
  • Interpretation: Synergy is typically defined as ΣFIC ≤ 0.5. Indifference is ΣFIC > 0.5 to ≤ 4. Antagonism is ΣFIC > 4.

G Pathogen Priority Pathogen (e.g., Carbapenem-resistant A. baumannii) Gap Identified R&D Gap: No novel class drugs in late-stage pipeline Pathogen->Gap Strategy R&D Strategy Selection Gap->Strategy NewClass New Target/Class Discovery Strategy->NewClass High Risk/High Reward Enhance Enhance Existing Agents Strategy->Enhance β-lactamase Inhibitors Combo Novel Combination Therapy Strategy->Combo Repurposing + Synergy

DOT script for Logic: R&D Strategy Based on Unmet Need

The Scientist's Toolkit: Key Research Reagent Solutions

Table 3: Essential Reagents for BPPL-Related Research

Item Function Example/Supplier
Cation-Adjusted Mueller-Hinton Broth (CAMHB) Standardized medium for antibiotic susceptibility testing (AST) ensuring consistent cation concentrations (Ca2+, Mg2+) critical for aminoglycoside and polymyxin activity. BD BBL, Thermo Fisher Sensititre
Clinical & Laboratory Standards Institute (CLSI) or EUCAST AST Breakpoint Panels Pre-conformed microdilution panels defining clinical resistance categories (S/I/R) for consistent, reproducible results. Thermo Fisher Sensititre, bioMérieux ETEST
Whole Genome Sequencing (WGS) Kits & Bioinformatic Pipelines For high-resolution resistance gene detection, strain typing, and outbreak investigation. Illumina Nextera, Oxford Nanopore Ligation; CARD, ResFinder databases.
Galleria mellonella or Murine Thigh/Neutropenic Infection Models In vivo models for preliminary efficacy and pharmacokinetic/pharmacodynamic (PK/PD) studies of lead compounds. Commercial larvae suppliers; CD-1 or C57BL/6 mouse strains.
Humanized Therapeutic Monoclonal Antibodies (e.g., anti-toxin) Research tools for studying virulence and evaluating immunotherapeutic approaches. Commercial bioreagents (e.g., Sino Biological) targeting toxins like alpha-hemolysin (S. aureus).
Advanced Cell-Based Assays (e.g., Biofilm, Intracellular Persistence) Models to identify compounds effective against hard-to-treat, persistent bacterial phenotypes. Calgary Biofilm Device; macrophage invasion/persistence assays (e.g., THP-1 cell line).

The 2024 World Health Organization (WHO) Bacterial Priority Pathogens List (BPPL) is a critical tool guiding research and development (R&D) for new antibacterial agents. This whitepaper focuses on the "Critical" priority tier, which includes Gram-negative, carbapenem-resistant pathogens such as:

  • Acinetobacter baumannii (carbapenem-resistant)
  • Pseudomonas aeruginosa (carbapenem-resistant)
  • Enterobacterales (carbapenem-resistant)
  • Third-generation cephalosporin-resistant, fluoroquinolone-resistant Salvator merianae.

These pathogens pose the greatest threat due to their high morbidity and mortality, transmissibility, and the dire lack of effective treatment options. This document provides a technical blueprint for discovery-stage research aimed at novel antimicrobials against these formidable targets.

The table below summarizes key quantitative data related to the burden and resistance mechanisms of critical pathogens as per the 2024 WHO BPPL and associated research.

Table 1: Key Metrics for WHO BPPL Critical-Priority Carbapenem-Resistant Pathogens

Pathogen Group Key Resistance Mechanisms (Quantitative Prevalence Trends)* Associated Mortality (Attributable)* Treatment Gaps & Unmet Need
CRAB (A. baumannii) Carbapenemases (e.g., OXA-23, NDM): >80% in high-burden regions. Efflux pumps (AdeABC). Porin loss. Crude mortality rates from invasive infections: 40-60%. Severe. Very limited therapeutic options (e.g., colistin, tigecycline). High toxicity, poor PK/PD.
CRPA (P. aeruginosa) Carbapenemases (e.g., VIM, IMP, KPC). Upregulated efflux (MexAB-OprM). AmpC overexpression + porin mutations. Crude mortality: 30-50% for bloodstream infections. Moderate-Severe. Some newer β-lactam/β-lactamase inhibitor combinations retain activity, but options are rapidly narrowing.
CRE (Enterobacterales) Carbapenemases: KPC (~40-50% US), NDM (~10-20% global), OXA-48-like (~30% EU). Porin mutations combined with ESBL/AmpC. Mortality rates: 22-50% for bacteremia, varying by species and enzyme. Severe. Cefiderocol, newer BL/BLIs (e.g., ceftazidime-avibactam) are key, but resistance emergence is documented.

Note: *Prevalence and mortality data are generalized from recent surveillance reports (e.g., CDC, ECDC, WHO GLASS) and are region-dependent.

Core Discovery Strategies and Experimental Protocols

Strategy: Screening for Novel β-Lactamase Inhibitors

Objective: Identify novel, non-β-lactam chemotypes that inhibit serine-β-lactamases (KPC, OXA-48) and metallo-β-lactamases (NDM, VIM).

Protocol: High-Throughput Biochemical Screen for β-Lactamase Inhibition

  • Reagent Preparation:
    • Purified β-lactamase enzyme (e.g., NDM-1, KPC-2) in assay buffer (50 mM HEPES, pH 7.0, 0.01% BSA).
    • Fluorogenic substrate: Nitrocefin (500 µM stock) for serine enzymes; specific fluorogenic cephalosporin (e.g., FC-5) for MBLs.
    • Compound library (10 mM in DMSO), diluted in buffer.
    • Positive control inhibitor: Avibactam (for KPC), EDTA (for MBLs).
  • Assay Procedure (384-well plate format):
    • Dispense 10 µL of compound (final test concentration 20 µM) or control into black, clear-bottom plates.
    • Add 10 µL of β-lactamase solution (final concentration 1-5 nM).
    • Pre-incubate for 30 minutes at room temperature.
    • Initiate reaction by adding 20 µL of nitrocefin (final concentration 100 µM).
    • Immediately kinetically read absorbance at 486 nm (for nitrocefin) or fluorescence (Ex/Em ~390/460 nm for FC-5) for 10 minutes using a plate reader.
  • Data Analysis:
    • Calculate initial reaction velocities (V0) for each well.
    • Determine percent inhibition: % Inhibition = [1 - (V0sample / V0no_inhibitor)] * 100.
    • Hit criteria: >70% inhibition at 20 µM. Perform dose-response (IC50) determination on hits.

Strategy: Targeting the Lipopolysaccharide (LPS) Transport Machinery

Objective: Develop inhibitors of the Lpt (LPS transport) system (LptA-G), a highly conserved Gram-negative essential pathway.

Protocol: In Vitro LPS Transport Assay Using Fluorescently-Labeled LPS

  • Membrane Preparation:
    • Grow target bacterium (e.g., E. coli ΔlptC complemented) to mid-log phase.
    • Harvest cells, disrupt via French press or sonication.
    • Separate inner membrane (IM) and outer membrane (OM) via sucrose density gradient ultracentrifugation.
  • LPS Labeling:
    • Purify LPS via phenol-water extraction.
    • Chemically label LPS with a fluorescent tag (e.g., NBD) at the lipid A moiety.
  • Transport Reaction:
    • Combine IM vesicles (source of LptB2FGC complex), purified LptA protein, and OM vesicles.
    • Add energy regenerating system (ATP, creatine phosphate, creatine kinase).
    • Add NBD-LPS (100 nM final) ± test compound.
    • Incubate at 30°C for 60 min.
  • Detection:
    • Stop reaction on ice. Re-isolate OM vesicles via ultracentrifugation.
    • Measure fluorescence intensity of the OM pellet (excitation 460 nm, emission 534 nm).
    • Inhibitors will reduce fluorescence in the OM fraction in a dose-dependent manner.

LptPathway IM Inner Membrane (LptB2FG Complex) LptA LptA Bridge IM->LptA ATP-driven translocation OM Outer Membrane (LptDE Complex) LptA->OM Transit via bridge LPS_OM LPS in OM OM->LPS_OM Insertion LPS_IM LPS in IM LPS_IM->IM Extraction LPS_Periplasm LPS in Periplasm

Diagram Title: Gram-negative LPS Transport (Lpt) Pathway

Strategy: Leverating Siderophore-Conjugated Antibiotics (Trojan Horse)

Objective: Design and evaluate novel siderophore-antibiotic conjugates to overcome permeability and efflux.

Protocol: In Vitro MIC and Iron-Dependency Assay

  • Conjugate Synthesis: Link a catechol or hydroxamate-based siderophore mimic (e.g., via a cleavable linker) to a warhead antibiotic (e.g., a β-lactam, daptomycin analog).
  • Broth Microdilution MIC:
    • Prepare cation-adjusted Mueller-Hinton broth (CA-MHB) under standard and iron-depleted conditions (add 200 µM 2,2'-Dipyridyl).
    • Perform standard CLSI broth microdilution with conjugate, unconjugated warhead, and control antibiotics.
    • Incubate 18-20 hours at 35°C.
  • Analysis: A significant decrease (≥8-fold) in the MIC of the conjugate under iron-depleted vs. standard conditions indicates iron-transport-mediated uptake.

SiderophoreConjugate Ext Extracellular Space (Iron-limited) Conj Siderophore- Antibiotic Conjugate Ext->Conj Fe3+ Binding Tono TonB-Dependent Receptor Conj->Tono Recognition OM2 Outer Membrane Periplasm Periplasm Tono->Periplasm Translocation IM2 Inner Membrane Periplasm->IM2 ABC Transport Release Linker Cleavage (Enzymatic/Hydrolytic) Periplasm->Release Warhead Release Cytoplasm Cytoplasm IM2->Cytoplasm Release of Fe3+ Release->Cytoplasm Active Warhead

Diagram Title: Siderophore-Antibiotic Conjugate Uptake Mechanism

The Scientist's Toolkit: Key Research Reagent Solutions

Table 2: Essential Reagents for Critical Pathogen Discovery Research

Reagent / Material Function & Application in Discovery Example/Notes
Purified Recombinant β-Lactamases Biochemical screening, enzyme kinetics, and inhibitor profiling. KPC-2, NDM-1, OXA-48, VIM-2 available from commercial suppliers (e.g., Sino Biological, RayBiotech).
Iso-genic Strain Panels Essential for distinguishing specific resistance mechanisms. Panels with paired wild-type, efflux pump knockout, porin mutant, and carbapenemase-producing strains.
Membrane Permeability Probes Quantify outer membrane penetration of compounds. 1-N-phenylnaphthylamine (NPN) for OM disruption; ethidium bromide accumulation for efflux inhibition assays.
Fluorogenic Peptidoglycan Probes Visualize and inhibit cell wall synthesis steps. Bocillin FL (penicillin-binding protein labeling); DAADAN- labeled lipid II analogs for translocation studies.
Iron-Depleted Culture Media Essential for evaluating siderophore-antibiotic conjugates and iron acquisition systems. Chelex-treated media or media supplemented with iron chelators (e.g., 2,2'-Dipyridyl).
Bioluminescent ATP Assay Kits Rapid, sensitive determination of bacterial viability and compound bactericidal activity in HTS formats. Provides a luminescent signal proportional to metabolically active cells.
Human Serum/Heparinized Blood Ex vivo models to assess compound activity in biologically relevant matrices and protein binding. Used in serum bactericidal assays (SBA) and pharmacodynamic modeling.

This whitepaper provides an in-depth technical guide on designing clinical trials for novel antibacterial agents, specifically framed within the urgent research context of the 2024 WHO Bacterial Priority Pathogens List (WHO BPPL). The 2024 BPPL categorizes antibiotic-resistant bacteria into critical, high, and medium priority groups, fundamentally reshaping the clinical development landscape. This document details the methodologies for defining patient populations and selecting efficacy endpoints, which are pivotal for generating robust, interpretable data that meets regulatory standards and addresses unmet public health needs.

The 2024 WHO BPPL: A Framework for Development

The 2024 WHO BPPL prioritizes pathogens based on global mortality, incidence, transmissibility, treatability, and prevention. Clinical trial design must be pathogen-focused, with patient populations and endpoints tailored to the specific priority category.

Table 1: 2024 WHO Bacterial Priority Pathogens List & Implication for Trial Design

Priority Category Pathogen Examples (2024 BPPL) Key Resistance Mechanisms Implied Patient Population Focus Recommended Trial Setting
CRITICAL Acinetobacter baumannii (carbapenem-resistant), Enterobacterales (3rd-gen cephalosporin & carbapenem-resistant), Pseudomonas aeruginosa (carbapenem-resistant) Carbapenemases (NDM, KPC, OXA), ESBLs, AmpC β-lactamases Hospital-acquired pneumonia (HAP/VAP), complicated intra-abdominal infections (cIAI), bloodstream infections (BSI) in ICU Global, multi-regional trials with high-prevalence sites
HIGH Salmonella spp. (fluoroquinolone-resistant), Shigella spp. (fluoroquinolone-resistant), Enterococcus faecium (vancomycin-resistant) Target-site mutations, van gene clusters Community-acquired invasive infections, complicated urinary tract infections (cUTI) Both hospital and community settings, focus on endemic regions
MEDIUM Group A Streptococcus (macrolide-resistant), Streptococcus pneumoniae (penicillin-non-susceptible) Ribosomal methylation (erm), altered PBPs Community-acquired bacterial pneumonia (CABP), pharyngitis Large outpatient or inpatient pragmatic trials

Defining Patient Populations: Methodologies and Protocols

Precise patient population definition is critical for trial feasibility and interpretability.

Inclusion Criteria Protocol: Microbiological Confirmation

Objective: To enroll patients with infections definitively caused by a WHO BPPL-target pathogen. Protocol:

  • Sample Collection: Obtain appropriate clinical specimen (e.g., blood, bronchoalveolar lavage, tissue) aseptically prior to first antibiotic dose.
  • Rapid Diagnostic Testing (RDT): Perform FDA/CE-approved multiplex PCR or multiplex lateral flow assay (e.g., BioFire FilmArray, Curetis Unyvero) for pathogen and resistance gene identification (e.g., blaKPC, blaNDM). Result must be available within 5 hours to inform enrollment.
  • Culture and Phenotypic Confirmation: Concurrently, culture specimen on appropriate agar. Perform phenotypic antimicrobial susceptibility testing (AST) per CLSI M100 or EUCAST v14.0 breakpoints.
  • Genotypic Confirmation: For isolates meeting phenotypic resistance criteria, perform whole-genome sequencing (WGS) using an Illumina MiSeq platform to confirm resistance genotype and assess clonality.

Stratification and Risk Adjustment

Stratification at randomization is essential. Key factors include:

  • Infection source (e.g., pulmonary vs. non-pulmonary source in BSI).
  • Pathogen (e.g., A. baumannii vs. P. aeruginosa).
  • Baseline severity scores (e.g., APACHE II >15, SOFA score increase ≥2).

Endpoint Selection and Assessment

Endpoints must be clinically meaningful and align with regulatory guidance (FDA, EMA).

Table 2: Primary Endpoint Selection Based on Infection Type

Infection Type (aligned with BPPL) Traditional Primary Endpoint Novel/Alternative Endpoint (Considerations for BPPL) Assessment Timepoint
Complicated Urinary Tract Infection (cUTI) Symptomatic resolution + microbiologic eradication (Composite) Overall success at Test-of-Cure (TOC) with a pathogen-specific (e.g., resistant E. coli) micro-ITT analysis Day 19-23 (TOC)
Hospital-Acquired Pneumonia (HAP/VAP) All-cause mortality Clinical cure at TOC in the microbiological intent-to-treat (micro-ITT) population Day 28 (Mortality); Day 21-28 (Clinical Cure)
Bloodstream Infections (BSI) All-cause mortality Day 14 clinical response (survival + resolution of signs/symptoms) in the microbiologically evaluable population Day 28 (Mortality); Day 14 (Clinical Response)

Protocol for Primary Endpoint Assessment: Clinical Cure in HAP/VAP

Objective: To determine clinical success or failure in a patient with microbiologically confirmed, BPPL-critical pathogen HAP/VAP. Clinical Cure Assessment Workflow:

G Start Baseline Visit (Day 1) EOT End of Treatment (Day 7-14) Start->EOT Sub1 Assess Symptoms: Fever, PaO2/FiO2, Cough, Sputum Start->Sub1 Document TOC Test of Cure (Day 21-28) EOT->TOC Sub2 Assess Symptoms & Culture from LRT (if clinically indicated) EOT->Sub2 Evaluate FU Late Follow-Up (Day 28-35) TOC->FU Sub3 Assess Sustained Resolution of Symptoms TOC->Sub3 Evaluate Decision Clinical Response Algorithm TOC->Decision Sub4 Assess Survival & Late Complications FU->Sub4 Evaluate Cure Clinical Cure Decision->Cure All Criteria Met Failure Clinical Failure Decision->Failure Any Failure Criterion Met Indet Indeterminate Decision->Indet Insufficient Data

Diagram Title: Clinical Cure Assessment for HAP/VAP Trials

Assessment Algorithm:

  • Success (Clinical Cure): Resolution of acute signs/symptoms (e.g., fever, hypoxia, leukocytosis) related to HAP/VAP to baseline/pre-infection state with no new complications, AND no requirement for additional antibiotic therapy for the primary infection after EOT, AND survival through TOC visit.
  • Failure: Death from any cause through TOC; OR persistence/progression of core clinical signs/symptoms; OR requirement for additional systemic antibiotic for primary infection after 48h of study therapy; OR development of a new pulmonary complication (e.g., empyema).

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Reagents & Materials for BPPL-Focused Clinical Trial Support Studies

Item/Category Example Product/Kit Function in Trial Design Context
Rapid Molecular Diagnostics BioFire FilmArray Pneumonia Panel plus, Curetis Unyvero Lower Respiratory Tract Panel Rapid (<2h) detection of BPPL pathogens and key resistance genes from BAL/mini-BAL to screen and enroll eligible patients.
AST & MIC Determination ETEST strips, Beckman Coulter MicroScan System, Sensititre Gram-Negative EUCAST Panel Phenotypic confirmation of resistance and determination of minimum inhibitory concentration (MIC) for baseline isolates and isolates from treatment failures.
Whole Genome Sequencing Illumina DNA Prep Kit, Illumina Nextera XT Library Prep, Oxford Nanopore Rapid Barcoding Kit Confirm resistance genotype, investigate clonal relatedness in outbreaks at trial sites, and identify novel resistance mechanisms.
Biomarker Assay bioMérieux VIDAS PCT assay, Abbott Architect CRP assay Quantify procalcitonin (PCT) or C-reactive protein (CRP) as objective, quantifiable biomarkers of infection severity and treatment response for enrichment or endpoint support.
Strain Repository ATCC Control Strains (e.g., K. pneumoniae ATCC BAA-1705 (KPC)), BEI Resources Carbapenem-Resistant A. baumannii Panel Provide quality control strains for diagnostic and AST platforms used across trial sites to ensure data consistency.

Leveraging the BPPL for Grant Proposals and Strategic Research Portfolios

The 2024 WHO Bacterial Priority Pathogens List (BPPL) is a critical evidence-based tool designed to prioritize research and development (R&D) for new antibiotics. For researchers and drug development professionals, it provides an authoritative framework to justify research directions, secure funding, and build strategic portfolios. This guide details how to leverage the BPPL's structure and data in grant applications and portfolio planning, with a focus on actionable experimental protocols.

The 2024 BPPL: Quantitative Data and Prioritization Tiers

The 2024 BPPL categorizes pathogens into three priority tiers—Critical, High, and Medium—based on a composite analysis of antibiotic resistance, treatability, mortality, incidence, transmissibility, and prevention potential. The list introduces a significant evolution from the 2017 version, emphasizing drug-resistant Mycobacterium tuberculosis and grouping pathogens by pathogen class and resistance profile.

Table 1: 2024 WHO BPPL Priority Pathogens (Critical & High Tiers)

Priority Tier Pathogen Family/Group Key Resistance Phenotypes R&D Priority Rationale
CRITICAL Acinetobacter baumannii Carbapenem-resistant High mortality, few therapeutic options, nosocomial spread.
CRITICAL Enterobacteriaceae Third-gen. cephalosporin & carbapenem-resistant High community & hospital burden, rising resistance.
CRITICAL Mycobacterium tuberculosis Rifampicin-resistant (RR/MDR/XDR) Global health crisis, long & toxic treatment regimens.
HIGH Helicobacter pylori Clarithromycin-resistant High global prevalence, impacts cancer prevention.
HIGH Salmonella Typhi Fluoroquinolone-resistant High morbidity in resource-limited settings.
HIGH Neisseria gonorrhoeae Third-gen. cephalosporin-resistant Rapid resistance emergence, threat to effective treatment.
HIGH Pseudomonas aeruginosa Carbapenem-resistant Nosocomial infections, intrinsic & acquired resistance.

Table 2: Comparative Analysis of Key Pathogen Burden Metrics

Pathogen (Resistance Profile) Estimated Annual Deaths (Global)* Key Empiric Treatment Gaps Pipeline Activity (Preclinical-Phase 3)*
Carbapenem-resistant A. baumannii (CRAB) ~50,000 - 100,000 Lack of bactericidal options; polymyxin reliance. Limited (1-2 novel agents in late stage).
Drug-resistant M. tuberculosis ~1.3 million (total TB) Long duration, toxicity, poor oral bioavailability. Moderate (novel regimens & repurposed drugs).
ESBL & Carbapenem-resistant Enterobacteriaceae ~150,000 - 200,000 Compromised efficacy of β-lactams, last-line carbapenems. Active (β-lactam/β-lactamase inhibitor combos, siderophore antibiotics).
Third-gen. cephalosporin-resistant N. gonorrhoeae Low mortality, high morbidity Threat of untreatable infection. Very Limited.

Note: Death estimates are attributable to resistant infections. Pipeline data is indicative based on current reviews.

Core Experimental Protocols for BPPL-Targeted Research

Integrating these standardized protocols into grant proposals demonstrates methodological rigor and direct alignment with WHO priorities.

Protocol 1: In Vitro Checkerboard Synergy Assay for Novel Combination Therapies

Aim: To identify synergistic antibiotic combinations against multidrug-resistant (MDR) BPPL pathogens. Materials:

  • Target Strain: MDR clinical isolate (e.g., CRAB, CR-P. aeruginosa).
  • Antibiotics: Include at least one novel agent/compound and standard-of-care antibiotics.
  • Culture Media: Cation-adjusted Mueller-Hinton Broth (CAMHB).
  • Equipment: 96-well microtiter plates, automated liquid handler, spectrophotometric plate reader.

Procedure:

  • Prepare logarithmic-phase bacterial inoculum at ~5 x 10⁵ CFU/mL in CAMHB.
  • Using a 96-well plate, create a two-dimensional matrix. Serially dilute Antibiotic A along the rows and Antibiotic B along the columns. Include growth and sterility controls.
  • Dispense 50 μL of each antibiotic dilution per well, followed by 50 μL of bacterial inoculum. Final volume: 100 μL/well.
  • Incubate at 35°C ± 2°C for 18-24 hours.
  • Measure optical density (OD600) or use resazurin viability dye. Calculate the Fractional Inhibitory Concentration Index (FICI): ΣFIC = (MIC of A in combo / MIC of A alone) + (MIC of B in combo / MIC of B alone). Interpret: FICI ≤0.5 = synergy; >0.5-4 = indifference; >4 = antagonism.
  • Validate synergistic combinations with time-kill kinetics assays.
Protocol 2: Whole Genome Sequencing (WGS) for Resistance Mechanism and Transmission Dynamics

Aim: To characterize resistance determinants and clonal relatedness in BPPL pathogen outbreaks. Materials:

  • Bacterial Isolates: Collection of MDR clinical isolates.
  • Kits: Genomic DNA extraction kit, library preparation kit (e.g., Illumina Nextera XT).
  • Platform: Illumina MiSeq or NovaSeq for short-read; Oxford Nanopore MinION for long-read.
  • Software: Bioinformatic pipelines (SPAdes for assembly, ABRicate for resistance gene detection, Snippy for variant calling, IQ-TREE for phylogenetics).

Procedure:

  • Extract high-quality genomic DNA from overnight cultures.
  • Prepare sequencing libraries according to platform-specific protocols. For hybrid assembly, prepare both short-read and long-read libraries.
  • Sequence to achieve minimum 50x coverage.
  • Bioinformatic Analysis:
    • Assembly & Annotation: Assemble reads into contigs. Annotate using RAST or Prokka.
    • Resistance Analysis: Screen contigs against curated databases (NCBI AMRFinderPlus, CARD, ResFinder).
    • Phylogenomics: Call core genome SNPs against a reference genome. Construct a maximum-likelihood phylogenetic tree to infer transmission clusters.
  • Correlate genotypic data with phenotypic susceptibility profiles (MICs).
Visualizing Research Strategy and Molecular Mechanisms

Diagram 1: BPPL-Informed Research & Development Pipeline

G BPPL 2024 WHO BPPL Data Targ Target Selection & Justification BPPL->Targ Prioritization Disc Discovery Research (Mechanism, Screening) Targ->Disc Hypothesis Dev Preclinical Development (Optimization, PK/PD, in vivo) Disc->Dev Lead Candidate Clinic Clinical Development (Phases I-III) Dev->Clinic IND Enabling Portfolio Strategic Portfolio Output Clinic->Portfolio NDA Submission

Diagram 2: Key Resistance Mechanisms in Critical Priority Pathogens

G cluster_0 Gram-Negative Bacteria (e.g., CRAB, CRE) cluster_1 Mycobacterium tuberculosis Antibiotic β-Lactam Antibiotic Porin Porin Channel Loss/Modification Antibiotic->Porin Reduced Uptake Efflux Efflux Pump Overexpression Antibiotic->Efflux Active Export BetaLac β-Lactamase Enzyme Antibiotic->BetaLac Hydrolysis Target Altered PBPs or Target Site Antibiotic->Target Reduced Binding Drug Anti-TB Drug (e.g., Rifampicin) Mut Chromosomal Mutations (rpoB, katG, inhA) Drug->Mut Target Gene Mutation Res Drug Resistance & Treatment Failure Mut->Res Direct Causality

The Scientist's Toolkit: Key Research Reagent Solutions

Table 3: Essential Reagents for BPPL-Focused Antimicrobial Research

Reagent / Material Function & Application Example/Supplier (Indicative)
Cation-Adjusted Mueller-Hinton Broth (CAMHB) Standardized medium for antibiotic susceptibility testing (AST) as per CLSI/EUCAST guidelines. Thermo Fisher, BD Diagnostics
Resazurin Sodium Salt Viability dye for colorimetric or fluorimetric endpoint determination in MIC and synergy assays. Sigma-Aldrich
PCR & WGS Kits For amplification and sequencing of resistance genes (e.g., blaKPC, blaNDM) and whole-genome analysis. Illumina Nextera XT, Qiagen DNeasy, Oxford Nanopore LSK114
In Vivo Infection Model Systems Mouse models of neutropenic thigh or pneumonia infection for in vivo PK/PD studies of lead compounds. Vendor-specific (e.g., Jackson Laboratory)
Cryopreservation Media For long-term, stable storage of characterized clinical isolates and mutant strains. Microbank beads, glycerol stocks
Bioinformatic Databases Curated resources for analyzing genomic data related to AMR and phylogeny. CARD, NCBI AMRFinderPlus, PubMed
Reference AST Panels Custom 96-well plates with predefined antibiotic gradients for high-throughput screening. Custom manufactured via companies like Liofilchem

Integrating BPPL Priorities into National and Institutional Surveillance Programs

Within the broader thesis on the 2024 WHO Bacterial Priority Pathogens List (BPPL) overview research, this whitepaper provides a technical guide for integrating its priorities into structured surveillance frameworks. The 2024 BPPL updates the global priority ranking of antibiotic-resistant bacteria, guiding research, development, and public health intervention. Effective integration of this list into national and institutional surveillance programs is critical for tracking the prevalence, emergence, and spread of these pathogens, thereby informing treatment guidelines and directing drug development efforts.

The 2024 WHO BPPL: Critical Categories and Pathogens

The 2024 list categorizes pathogens into Critical, High, and Medium priority groups based on criteria including drug resistance, mortality, prevalence, transmissibility, treatability, and prevention potential. Quantitative data from the WHO report is summarized below.

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

Pathogen Category Key Resistance Phenotypes R&D Priority Urgency Key Data Points (Global Burden)
Critical Priority Group
Acinetobacter baumannii Carbapenem-resistant Highest Associated with ~45% in-hospital mortality in invasive infections.
Pseudomonas aeruginosa Carbapenem-resistant Highest Causes severe healthcare-associated infections; high mortality in bloodstream infections.
Enterobacterales Third-generation cephalosporin and carbapenem-resistant Highest Collectively responsible for the highest burden of antibiotic-resistant infections worldwide.
Mycobacterium tuberculosis Rifampicin-resistant (RR-TB) and multidrug-resistant (MDR-TB) Highest An estimated 410,000 cases of MDR/RR-TB emerged in 2022.

Table 2: 2024 WHO BPPL - High and Medium Priority Groups

Priority Group Pathogen Category Key Resistance Phenotypes
High Salmonella Typhi Fluoroquinolone-resistant
High Shigella spp. Fluoroquinolone-resistant
High Enterococcus faecium Vancomycin-resistant (VRE)
High Helicobacter pylori Clarithromycin-resistant
High Campylobacter spp. Fluoroquinolone-resistant
High Neisseria gonorrhoeae Third-generation cephalosporin and/or fluoroquinolone-resistant
Medium Group A Streptococcus Macrolide-resistant
Medium Group B Streptococcus Penicillin-resistant
Medium Streptococcus pneumoniae Penicillin-non-susceptible
Medium Haemophilus influenzae Ampicillin-resistant

Core Surveillance Framework: Integrating BPPL Priorities

A robust surveillance program must be multi-faceted, encompassing pathogen detection, resistance profiling, molecular epidemiology, and data integration.

Laboratory-Based Surveillance Protocol

This protocol details the core workflow for processing isolates from clinical specimens for BPPL pathogens.

Protocol Title: Standard Operating Procedure for Laboratory Identification and Antimicrobial Susceptibility Testing (AST) of BPPL Pathogens.

Objective: To accurately identify bacterial pathogens from the WHO BPPL and determine their antimicrobial susceptibility profiles.

Materials & Reagents: See "The Scientist's Toolkit" (Section 6).

Methodology:

  • Specimen Collection & Transport: Collect appropriate clinical specimens (e.g., blood, urine, sputum) using sterile, standardized containers. Transport to the laboratory per CLSI M40-A2 standards.
  • Primary Culture & Isolation: Inoculate specimens onto appropriate agar media (e.g., blood agar, MacConkey agar, chromogenic media for ESBL/carbapenemase producers). Incubate aerobically at 35±2°C for 18-24 hours.
  • Pathogen Identification: Select morphologically distinct colonies for identification.
    • MALDI-TOF MS: Spot colony onto target plate, overlay with matrix solution (α-cyano-4-hydroxycinnamic acid), and analyze using the MALDI Biotyper system. Species identification is based on spectral matching to reference libraries.
    • Alternative: Use automated biochemical systems (e.g., VITEK 2, BD Phoenix) or targeted PCR for specific pathogens.
  • Antimicrobial Susceptibility Testing (AST):
    • Prepare a 0.5 McFarland standard suspension of the pure isolate in saline.
    • Disk Diffusion (Kirby-Bauer): Lawn inoculate Mueller-Hinton agar (MHA) plates. Place antibiotic disks manually or with a dispenser. Incubate for 16-18 hours. Measure zones of inhibition and interpret per CLSI M100 or EUCAST breakpoints.
    • Broth Microdilution: Use commercially prepared panels with serial antibiotic dilutions. Inoculate panels with standardized bacterial suspension. Incubate for 16-20 hours. The Minimum Inhibitory Concentration (MIC) is the lowest concentration inhibiting visible growth.
    • Automated AST Systems: Load standardized suspension into AST cards (e.g., VITEK 2 AST cards) and run per manufacturer's instructions.
  • Detection of Key Resistance Mechanisms (Confirmatory Tests):
    • Carbapenemase Production: Perform the modified Carbapenem Inactivation Method (mCIM) or the Carba NP test.
    • ESBL Production: Use combination disk tests (cefotaxime/ceftazidime ± clavulanate) or broth microdilution as per CLSI guidelines.
  • Data Recording & Storage: Enter all identification and AST results, along with patient metadata (de-identified), into a Laboratory Information System (LIS).

LabSurveillanceWorkflow Start Clinical Specimen Collection Culture Primary Culture & Isolation Start->Culture ID Pathogen Identification (MALDI-TOF/PCR) Culture->ID AST Antimicrobial Susceptibility Testing ID->AST Confirm Confirmatory Tests for Key Resistance Mechanisms AST->Confirm Data Data Entry into LIS/WHONET Confirm->Data Report Generate Surveillance Report Data->Report

Diagram Title: Laboratory Surveillance Workflow for BPPL Pathogens

Genomic Surveillance Protocol

Integrating whole-genome sequencing (WGS) into surveillance enables high-resolution tracking of resistance genes, plasmids, and strain lineages.

Protocol Title: Whole-Genome Sequencing of Bacterial Isolates for Antimicrobial Resistance Surveillance.

Objective: To generate high-quality genome sequences from BPPL isolates for determining resistance genotype, sequence type (ST), and phylogenetic context.

Methodology:

  • Genomic DNA Extraction: Using a commercial kit (e.g., Qiagen DNeasy Blood & Tissue Kit), extract high-molecular-weight DNA from an overnight pure culture. Quantify DNA using a fluorometric method (e.g., Qubit dsDNA HS Assay).
  • Library Preparation: Use a tagmentation-based library prep kit (e.g., Illumina Nextera XT). This involves fragmenting DNA, adding adapter sequences, and performing a limited-cycle PCR to add unique dual indices.
  • Sequencing: Pool normalized libraries and sequence on an Illumina platform (e.g., MiSeq, NextSeq) using a 2x150bp or 2x250bp paired-end run to achieve a minimum coverage of 50-100x.
  • Bioinformatics Analysis:
    • Quality Control & Assembly: Use FastQC for read quality assessment. Trim adapters and low-quality bases with Trimmomatic. Perform de novo assembly using SPAdes.
    • Species & ST Confirmation: Use Kraken2 for species identification. Determine Multi-Locus Sequence Type (MLST) using mlst tool against PubMedST schemes.
    • AMR Gene Detection: Screen assemblies against curated resistance databases (e.g., ResFinder, CARD, NCBI's AMRFinderPlus) using ABRicate or AMRFinder.
    • Plasmid & Clone Detection: Identify plasmid replicons using PlasmidFinder. For specific high-risk clones (e.g., E. coli ST131), perform SNP-based phylogenetic analysis.

GenomicSurveillancePipeline cluster_0 Wet Lab cluster_1 Dry Lab Isolate BPPL Bacterial Isolate DNA Genomic DNA Extraction & QC Isolate->DNA Lib Library Preparation DNA->Lib Seq Sequencing (Illumina) Lib->Seq QC Raw Read Quality Control Seq->QC Assemble De Novo Genome Assembly QC->Assemble Analyze Bioinformatic Analysis Assemble->Analyze

Diagram Title: Genomic Surveillance and Analysis Pipeline

Data Integration and Reporting Architecture

Surveillance data must flow from institutional labs to a centralized national database for analysis and action.

DataIntegrationArchitecture Lab1 Institutional Lab 1 (LIS Data) WHONET Standardized Data (WHONET Software) Lab1->WHONET Lab2 Institutional Lab 2 (LIS Data) Lab2->WHONET LabN Institutional Lab N LabN->WHONET ... CentralDB National AMR Surveillance Database WHONET->CentralDB Analysis Data Analysis & Trend Reporting CentralDB->Analysis Stakeholders Public Health Agencies, WHO, Researchers Analysis->Stakeholders

Diagram Title: National AMR Surveillance Data Flow

Key Performance Indicators (KPIs) for Surveillance Programs

Monitor the effectiveness of the integrated BPPL surveillance program using quantitative metrics.

Table 3: Surveillance Program Key Performance Indicators

KPI Category Specific Metric Target / Benchmark
Coverage Percentage of major healthcare institutions reporting BPPL pathogen data >90% national coverage
Data Quality Percentage of isolates with complete AST data for WHO-recommended antibiotics >95%
Timeliness Median time from specimen collection to data entry into national database <30 days
Utility Number of national treatment guidelines updated based on surveillance data in the past 2 years ≥1
Molecular Integration Percentage of critical-priority carbapenem-resistant isolates subjected to WGS annually >20% (increasing yearly)

The Scientist's Toolkit: Essential Research Reagents & Materials

Table 4: Key Reagents and Materials for BPPL Surveillance Experiments

Item Function / Application Example Product / Specification
Chromogenic Agar Media Selective isolation and presumptive identification of ESBL-producing and Carbapenem-resistant Enterobacterales. CHROMagar ESBL, CHROMagar mSuperCARBA.
Cation-Adjusted Mueller-Hinton Broth (CAMHB) The standard medium for broth microdilution AST, ensuring consistent cation concentrations for accurate results. Prepared per CLSI M07 standard.
Antimicrobial Powder Standards For preparation of in-house stock solutions for AST, ensuring accurate concentration for disk diffusion or broth dilution. USP-grade powders with known potency.
MALDI-TOF MS Matrix Solution A chemical matrix (e.g., HCCA) that co-crystallizes with bacterial proteins, enabling ionization and analysis for species identification. α-cyano-4-hydroxycinnamic acid in 50% acetonitrile/2.5% TFA.
High-Fidelity DNA Polymerase For accurate amplification of resistance genes or MLST loci in PCR confirmation assays. Q5 Hot Start High-Fidelity DNA Polymerase.
DNA Library Prep Kit (Tagmentation-based) For efficient fragmentation and adapter ligation of bacterial genomic DNA in preparation for next-generation sequencing. Illumina DNA Prep kit.
WHONET Software Free desktop application for management and analysis of microbiology laboratory data, essential for standardized national reporting. WHONET 2024 release (WHO Collaborating Centre).
ResFinder/AMRFinderPlus Database Curated bioinformatics databases of antimicrobial resistance genes for analyzing WGS data. Downloaded from Genomicepidemiology.org or NCBI.

Navigating Challenges: Limitations, Data Gaps, and Practical Implementation of the BPPL

Within the broader context of the 2024 WHO Bacterial Priority Pathogens List (BPPL) overview research, this technical guide examines the critical need to contextualize global pathogen prioritization for regional and local LMIC settings. The 2024 BPPL serves as a global blueprint for directing research and development (R&D) efforts and antibiotic stewardship. However, its application in LMICs is complicated by distinct epidemiological profiles, healthcare system capacities, and surveillance limitations. This whitepaper provides a framework for adapting BPPL priorities to LMIC contexts through targeted surveillance, capacity building, and regionally relevant R&D.

The 2024 WHO BPPL: A Global Framework

The WHO BPPL categorizes bacterial pathogens into critical, high, and medium priority groups based on criteria including antibiotic resistance, mortality, prevalence, transmissibility, and treatability. This list is designed to guide the pipeline for new antibiotics and diagnostics.

Table 1: 2024 WHO Bacterial Priority Pathogens List (Abridged for Key Pathogens)

Priority Category Pathogen Families/Genera Key Resistance Traits Primary Public Health Impact
CRITICAL Acinetobacter baumannii Carbapenem-resistant Hospital-acquired infections, high mortality in ICUs.
Enterobacteriaceae (e.g., K. pneumoniae, E. coli) Carbapenem-resistant, ESBL-producing Bloodstream infections, neonatal sepsis, UTIs.
Pseudomonas aeruginosa Carbapenem-resistant Hospital-acquired pneumonia, surgical site infections.
HIGH Salmonella enterica (Typhi & Paratyphi) Fluoroquinolone-resistant, MDR Enteric fever, endemic in many LMICs.
Shigella spp. Fluoroquinolone-resistant, MDR Bacillary dysentery, high morbidity in children.
Enterococcus faecium Vancomycin-resistant Hospital-acquired infections.
MEDIUM Streptococcus pneumoniae Penicillin-non-susceptible Community-acquired pneumonia, meningitis, otitis media.
Haemophilus influenzae Ampicillin-resistant Respiratory infections, meningitis.
Campylobacter spp. Fluoroquinolone-resistant Gastroenteritis.

Disparities in Burden and Capacity

The impact of BPPL pathogens is not uniform globally. LMICs face a disproportionate burden due to higher incidence of community-acquired infections, weaker infection prevention and control (IPC), and limited access to second- and third-line antibiotics. Furthermore, routine surveillance data essential for validating the BPPL's relevance is often scarce or non-existent in these regions.

Table 2: Key Disparities Influencing BPPL Relevance in LMICs

Factor High-Income Country (HIC) Context Typical LMIC Context Impact on BPPL Priorities
Disease Burden Hospital-acquired, chronic infections predominant. High community-onset infections (e.g., typhoid, neonatal sepsis). Pathogens like MDR Salmonella Typhi may require elevated priority.
Diagnostic Capacity Routine use of MALDI-TOF, PCR, automated AST. Reliance on culture & biochemical tests; limited AST capacity. Undetected resistance leads to empirical treatment failure.
Antibiotic Access & Stewardship Broad formulary with stewardship programs. Intermittent access; unregulated dispensing; high empiric use. Drives resistance; may alter local resistance patterns vs. global list.
Surveillance Systems National, lab-based integrated systems (e.g., EARS-Net). Fragmented, sentinel-site based, limited data completeness. Global BPPL may not reflect local epidemiology.

Foundational Experimental Protocol: Building LMIC-Centric AST Surveillance

Accurate, local antimicrobial susceptibility testing (AST) data is the cornerstone for adapting the BPPL. Below is a detailed protocol for establishing a standardized, cost-effective AST surveillance study in an LMIC reference laboratory.

Protocol: LMIC Sentinel Laboratory AST Surveillance for BPPL Pathogens

Objective: To generate representative, quality-controlled AST data for key BPPL pathogens from clinical isolates in a defined LMIC region.

Materials & Reagents (The Scientist's Toolkit):

Item Function & Rationale
Blood Agar & MacConkey Agar Plates Primary isolation media for a broad range of bacterial pathogens from clinical samples.
Selective Agar (e.g., CHROMagar KPC, ESBL) For selective isolation and presumptive identification of key resistant phenotypes (carbapenemase, ESBL producers).
Cation-adjusted Mueller-Hinton II Agar (CA-MH) The standard medium for disk diffusion AST, ensuring reproducible results.
Antibiotic Impregnated Disks (CLSI/EUCAST panel) Disks containing standardized antibiotic concentrations for key drug classes (beta-lactams, fluoroquinolones, aminoglycosides, etc.).
0.5 McFarland Saline Standard To standardize the inoculum density for AST to ~1.5 x 10^8 CFU/mL.
Automated or Manual AST System (e.g., VITEK 2 Compact, BD Phoenix) If available For confirmatory MIC-based testing. Manual broth microdilution panels can be used as an alternative.
Molecular Confirmation Kits (e.g., Multiplex PCR for blaNDM, blaKPC, blaOXA-48-like) For confirming and characterizing resistance mechanisms in critical isolates.
Quality Control Strains (e.g., E. coli ATCC 25922, P. aeruginosa ATCC 27853) Essential for daily validation of media, disks, and procedures.

Methodology:

  • Isolate Collection & Ethics: Over a defined period (e.g., 12 months), consecutively collect clinically significant bacterial isolates from normally sterile sites (blood, CSF) or key infections (UTI, wound) from sentinel hospitals. Secure ethical approval and patient data anonymization.
  • Bacterial Identification: Isolate pure cultures using standard microbiological techniques. Perform identification using available methods (biochemical panels, automated systems if accessible).
  • Antimicrobial Susceptibility Testing (AST): a. Disk Diffusion (Primary Method): i. Prepare a 0.5 McFarland suspension of the isolate in sterile saline. ii. Evenly lawn the suspension onto a CA-MH agar plate. iii. Apply a pre-defined panel of antibiotic disks relevant to the pathogen and local formulary (e.g., for Enterobacteriaceae: ampicillin, ceftriaxone, ciprofloxacin, gentamicin, meropenem, amikacin). iv. Incubate at 35±2°C for 16-18 hours. v. Measure zones of inhibition in millimeters using calipers. b. Confirmatory Testing: i. For isolates showing resistance to 3rd-gen cephalosporins, perform a phenotypic confirmatory test for ESBL (e.g., combined disk test with clavulanate). ii. For carbapenem non-susceptibility, perform a modified carbapenem inactivation method (mCIM) or molecular testing for carbapenemase genes.
  • Data Management & Analysis: a. Enter all data (patient demographics, specimen source, identification, AST results) into a standardized electronic database. b. Interpret AST results using current CLSI or EUCAST breakpoints. c. Calculate resistance percentages (%R) for each pathogen-antibiotic combination with confidence intervals.
  • Quality Assurance: Run QC strains with each batch of AST. Participate in an external quality assurance (EQA) program if available.

From Surveillance to Action: A Pathway for LMIC Engagement

The data generated from such protocols must feed into local and national action plans. The following diagram illustrates the adaptive pathway from global priority list to local action.

G GlobalBPPL 2024 WHO BPPL (Global Framework) Contextualize Contextualization & Gap Analysis GlobalBPPL->Contextualize LMICData LMIC-Specific Data (Surveillance & Burden) LMICData->Contextualize RegionalList Adapted Regional/LMIC Priority List Contextualize->RegionalList RDDirections R&D Directions: - Diagnostics - Therapeutics - Vaccines RegionalList->RDDirections IPCStewardship IPC & Stewardship Policies RegionalList->IPCStewardship

Diagram Title: Adaptive Pathway from Global BPPL to LMIC Action

Core Experimental Workflow for Surveillance & Characterization

The process from sample collection to resistance mechanism characterization involves multiple, interconnected steps, as detailed below.

G Sample Clinical Sample (Blood, Urine, CSF) Culture Culture & Primary Identification Sample->Culture AST Phenotypic AST (Disk Diffusion/Broth Dilution) Culture->AST MDRFlag MDR/XDR/PDR Phenotype Detected? AST->MDRFlag Confirm Confirmatory Tests (mCIM/eCIM, ESBL test) MDRFlag->Confirm Yes Database Data Integration & Local Resistance Database MDRFlag->Database No Molecular Molecular Characterization (PCR, WGS for resistance genes) Confirm->Molecular Molecular->Database

Diagram Title: Workflow for LMIC Pathogen Characterization

Effectively addressing the challenge of antimicrobial resistance in LMICs requires moving beyond a one-size-fits-all application of the WHO BPPL. By investing in foundational, locally-led surveillance to generate reliable epidemiological data, the global health community can support LMICs in developing adapted priority lists. These lists should guide not only regional R&D investments for diagnostics, vaccines, and novel therapeutics but also the strengthening of IPC and antibiotic stewardship programs. Integrating this context-specific approach is essential for achieving equity in the global fight against AMR.

1. Introduction

The 2024 WHO Bacterial Priority Pathogen List (BPPL) serves as a critical global framework for prioritizing research and development of new antibiotics. Its construction relies fundamentally on robust surveillance data to assess the burden, drug resistance, and transmission of bacterial pathogens. This whitepaper contends that systemic limitations in diagnostic capacity and data standardization create a significant "diagnostic data gap," directly biasing pathogen ranking and, consequently, global health investment. Inaccuracies in surveillance data can lead to the under-prioritization of pathogens with high true burdens obscured by diagnostic failure and the over-prioritization of pathogens more readily detected by available tools.

2. The Core Surveillance Limitations

The surveillance data feeding into priority pathogen lists like the BPPL is compromised at multiple stages, from sample collection to data aggregation.

Table 1: Key Surveillance Limitations and Their Impact on Pathogen Ranking

Limitation Category Specific Issue Potential Impact on BPPL Ranking
Diagnostic Access & Capacity Lack of culture facilities, molecular diagnostics, or MALDI-TOF in low-resource settings (LRSS). Underestimation of pathogens requiring complex identification (e.g., anaerobes, fastidious organisms).
Test Sensitivity/Specificity Poor performance of syndromic PCR panels or rapid antigen tests in polymicrobial infections. Misattribution of disease burden; false negatives for target pathogens.
Antimicrobial Susceptibility Testing (AST) Non-standardized methods, lack of testing for new resistance mechanisms (e.g., novel β-lactamases). Inaccurate resistance prevalence data, skewing "critical priority" status.
Data Fragmentation & Standards Incompatible laboratory information systems (LIS), non-adherence to WHONET standards. Inability to aggregate global data consistently, creating regional biases in the global list.
Asymptomatic Carriage & Environmental Reservoirs Surveillance focused solely on clinical infections. Underestimation of transmission risk and reservoir potential for pathogens like Klebsiella pneumoniae.

3. Experimental Protocols: Quantifying the Diagnostic Gap

To objectively measure the data gap, researchers employ specific methodological frameworks.

Protocol 3.1: Retrospective Metagenomic Re-analysis of Culture-Negative Specimens. Objective: To identify pathogens missed by standard culture in retrospectively collected specimen biobanks. Workflow:

  • Sample Selection: Archive frozen, culture-negative body fluid (e.g., synovial fluid, CSF) or tissue specimens from patients with clinical syndromes of infection.
  • Nucleic Acid Extraction: Use a broad-spectrum extraction kit (e.g., QIAamp DNA Mini Kit) with enzymatic lysis for tough cell walls.
  • Host Depletion: Apply a probe-based hybridization method (e.g., NEBNext Microbiome DNA Enrichment Kit) to remove human genomic DNA.
  • Library Preparation & Sequencing: Prepare sequencing libraries using a tagmentation-based kit (e.g., Nextera XT) and perform shotgun metagenomic sequencing on an Illumina platform (2x150 bp, ~20-50 million reads/sample).
  • Bioinformatic Analysis:
    • Trim adapters and low-quality reads with Trimmomatic.
    • Map reads to the human genome (hg38) using Bowtie2 and discard aligned reads.
    • Classify remaining reads against a comprehensive microbial database (e.g., NCBI RefSeq) using Kraken2/Bracken.
  • Validation: Confirm findings with pathogen-specific PCR and Sanger sequencing.

Protocol 3.2: Prospective Comparative Diagnostic Yield Study. Objective: To compare pathogen detection rates between standard-of-care (SOC) diagnostics and a reference multiplex molecular assay in a defined population. Workflow:

  • Patient Enrollment: Consecutively enroll patients presenting with community-acquired pneumonia (CAP) meeting predefined clinical/radiographic criteria.
  • Parallel Testing: For each patient, subject respiratory specimens (sputum/BALF) to:
    • SOC Arm: Conventional culture and syndromic PCR panel (if locally available).
    • Reference Arm: A high-throughput multiplex PCR/NGS panel (e.g., BIOFIRE RP2.1plus or an investigational NGS panel).
  • AST Correlation: For bacterial pathogens identified in both arms, perform standardized broth microdilution AST (per CLSI/EUCAST guidelines) on cultured isolates.
  • Data Analysis: Calculate the additional diagnostic yield (%) of the reference assay. Analyze discrepancies in resistance gene detection (e.g., mecA, blaCTX-M*) between phenotypic AST and genotypic results from the reference assay.

4. Visualizing the Data Flow and Its Disruptions

surveillance_gap S1 Clinical Infection S2 Sample Collection S1->S2 S3 Diagnostic Testing S2->S3 S4 Data Generation (Phenotype/Genotype) S3->S4 S5 Data Aggregation (Local/National) S4->S5 S6 WHO BPPL Analysis & Pathogen Ranking S5->S6 L1 Limited Access to Advanced Diagnostics L1->S3 L2 Non-Standardized AST Methods L2->S4 L3 Fragmented Data Systems & Lack of Metadata L3->S5

Title: Diagnostic Data Flow and Disruption Points in Pathogen Ranking

protocol_workflow Start Culture-Negative Specimen Biobank NA Nucleic Acid Extraction & Host Depletion Start->NA Seq Shotgun Metagenomic Sequencing NA->Seq Bio Bioinformatic Pipeline: 1. Quality/Adapter Trim 2. Host Read Removal 3. Taxonomic Classification Seq->Bio Val Validation via Pathogen-Specific PCR Bio->Val End Identification of 'Missed' Pathogens Val->End Gap DIAGNOSTIC DATA GAP (Unseen Burden) Gap->Bio

Title: Metagenomic Protocol to Quantify Unseen Pathogen Burden

5. The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Research Reagents for Closing the Diagnostic Data Gap

Reagent / Material Provider Examples Function in Surveillance Research
Simulated Sputum / Blood Culture Panels ZeptoMetrix (NATtrol), BEI Resources Controls for validating new diagnostic assays' sensitivity/specificity against known pathogen loads.
Characterized Resistance Gene Plasmid Libraries Addgene, BEI Resources Positive controls for detecting and sequencing emerging AMR genes (e.g., blaNDM, mcr-1) via PCR or NGS.
Synthetic Microbial Community (SynMock) Standards ATCC, ZymoBIOMICS Defined mock microbial communities with staggered abundances for benchmarking metagenomic wet-lab and bioinformatic workflows.
High-Fidelity DNA Polymerase for Resistance Gene Amplification NEB (Q5), Thermo Fisher (Platinum SuperFi II) Accurate amplification of target genes from complex samples prior to sequencing for surveillance of resistance evolution.
Selective Culture Media for ESBL/CPO Detection CHROMagar, Hardy Diagnostics Culture-based isolation of specific resistant pathogens (e.g., ESBL-E, carbapenemase producers) from surveillance screens.
Standardized Broth Microdilution AST Panels Sensititre, TREK Diagnostic Systems Generation of reproducible, comparable minimum inhibitory concentration (MIC) data per CLSI/EUCAST guidelines.

6. Conclusion and Recommendations

The 2024 WHO BPPL is only as accurate as the surveillance data that informs it. The diagnostic data gap, driven by inequitable access to diagnostics, non-standardized methods, and fragmented data systems, systematically distorts our perception of microbial threats. To mitigate this, a concerted global effort must invest in: 1) Implementation Research for novel diagnostics in LRSS, 2) Mandatory Data Standardization using WHO GLASS and WHONET frameworks, and 3) Integrated Surveillance combining clinical, genomic, and environmental data. Closing this gap is not merely a technical endeavor but a prerequisite for equitable and effective prioritization in the fight against antimicrobial resistance.

The 2024 WHO Bacterial Priority Pathogens List (BPPL) serves as a critical roadmap for global public health, prioritizing bacterial pathogens based on resistance burden, transmissibility, treatability, and prevention potential. This document interprets the BPPL not as a catalog for novel antibiotic development, but as a strategic framework for guiding prophylactic (vaccine) and diagnostic (rapid test) development. This shift in perspective—from treatment to prevention and rapid detection—is essential for mitigating the impact of antimicrobial resistance (AMR).

The 2024 BPPL: A Structured Analysis for Non-Antibiotic Interventions

The list categorizes pathogens into Critical, High, and Medium priority groups. For vaccine and diagnostic development, key interpretative metrics include incidence, mortality, transmissibility (R0), availability of current prevention methods, and the technical feasibility of antigen or biomarker discovery.

Table 1: 2024 WHO BPPL Priority Pathogens: Key Metrics for Vaccine & Diagnostic Development

Priority Tier Pathogen (Examples) Key Resistance Mechanisms Incidence & Mortality Burden (Global Estimate) Current Vaccine Status Diagnostic Development Urgency & Target
CRITICAL Acinetobacter baumannii (carbapenem-resistant) Carbapenemases (OXA, NDM), Efflux pumps ~50k deaths/year; High in ICU settings None. High priority for protein/subunit vaccine. Critical for rapid carbapenemase gene detection.
CRITICAL Pseudomonas aeruginosa (carbapenem-resistant) Carbapenemases, Porin mutations ~30k deaths/year; Common in healthcare. Phase 2 candidates (multivalent). Urgent for distinguishing resistant from susceptible strains.
CRITICAL Enterobacteriaceae (carbapenem-resistant, 3GC-resistant) ESBLs, Carbapenemases (KPC, NDM) Millions of infections; >50k deaths/year. K. pneumoniae candidates in early dev. High: Rapid point-of-care (POC) test for ESBL/carbapenemase.
HIGH Salmonella Typhi (fluoroquinolone-resistant) Target mutations, Plasmid-mediated resistance ~11M cases/year; High endemic regions. Existing conjugate vaccines; need broader coverage. High for detecting resistance directly from blood.
HIGH Helicobacter pylori (clarithromycin-resistant) 23S rRNA mutation >50% resistance in many regions. None. Major technical challenge. High for non-invasive resistance detection (stool/PCR).
MEDIUM Group A Streptococcus (macrolide-resistant) Efflux, Methylation of 23S rRNA Low resistance but high disease burden. No licensed vaccine; candidates in trials. Medium; POC for pharyngitis to guide treatment.

Experimental Protocols for Target Identification and Validation

Protocol 1: Reverse Vaccinology forAcinetobacter baumanniiAntigen Discovery

Objective: To computationally and experimentally identify surface-exposed, conserved proteins as vaccine candidates. Methodology:

  • Genome Sequencing & Pan-Genome Analysis: Sequence a diverse collection of 100+ clinical A. baumannii isolates (including BPPL-listed carbapenem-resistant strains). Perform pan-genome analysis to define core, accessory, and unique genomes.
  • In Silico Prediction: Using the core genome, predict:
    • Subcellular Localization (PSORTb, SignalP) to identify surface or secreted proteins.
    • Transmembrane Helices (TMHMM) to exclude proteins with multiple domains.
    • Antigenicity (VaxiJen) to score potential immunogenicity.
    • Human Homology (BLASTp against human proteome) to exclude auto-antigen risks.
  • Cloning and Expression: Clone selected genes into an E. coli expression vector (e.g., pET system). Express recombinant His-tagged proteins and purify via Ni-NTA affinity chromatography.
  • Immunogenicity Validation: Immunize mice (n=10/group) with purified protein adjuvanted with Alum. Measure serum IgG titers via ELISA. Perform opsonophagocytic killing assay (OPKA) using differentiated HL-60 cells and live A. baumannii to assess functional antibody activity.

Protocol 2: Development of a Multiplex PCR-Lateral Flow Assay for Carbapenemase Genes

Objective: To create a rapid, POC diagnostic capable of simultaneously detecting major carbapenemase gene families (e.g., blaKPC, blaNDM, blaOXA-48-like, blaVIM) from bacterial colonies. Methodology:

  • Primer and Probe Design: Design multiplex PCR primers with biotin (Biotin) and fluorescein (FAM) labels for each target carbapenemase gene. Design complementary DNA probes with unique tags (e.g., digoxigenin, DNP) for each target.
  • Lateral Flow Strip Assembly: Immobilize anti-tag antibodies (e.g., anti-DIG, anti-DNP) as distinct test lines on a nitrocellulose membrane. A control line contains streptavidin.
  • Assay Workflow: a. Prepare a crude DNA lysate from a colony. b. Perform a 15-minute isothermal amplification (LAMP or RPA) with labeled primers. c. Apply the amplicon mixture to the strip. d. As the solution migrates, labeled amplicons bind to their specific probe on gold nanoparticles, forming complexes. These are captured at respective test lines.
  • Validation: Test against a characterized panel of 250 Gram-negative clinical isolates with known resistance genotypes. Calculate sensitivity, specificity, and limit of detection (LoD).

G cluster_0 Multiplex PCR-Lateral Flow Workflow Sample Sample Lysis Lysis Sample->Lysis Colony Amp Amp Lysis->Amp Crude DNA Strip Strip Amp->Strip Labeled Amplicon Result Result Strip->Result Visual Readout

Title: Lateral Flow Assay for Carbapenemase Detection

The Scientist's Toolkit: Key Research Reagent Solutions

Table 2: Essential Reagents for Vaccine & Diagnostic Development Targeting BPPL Pathogens

Reagent / Material Function / Application Example in Context
Pan-Genome Analysis Software (e.g., Roary, BPGA) Identifies core and accessory genomes across sequenced isolate panels to find conserved antigen targets. Identifying conserved surface proteins in A. baumannii across Priority Tier 1 isolates.
Protein Expression System (e.g., pET vector in E. coli BL21) High-yield production of recombinant protein antigens for immunogenicity studies. Expressing predicted outer membrane protein (OmpA) of A. baumannii for mouse vaccination.
Differentiated HL-60 Cells A neutrophil-like cell line used to perform standardized opsonophagocytic killing assays (OPKA). Measuring functional antibody-mediated killing of K. pneumoniae post-vaccination.
Isothermal Amplification Mix (e.g., RPA or LAMP kits) Enables rapid DNA amplification at constant temperature, suitable for POC diagnostic devices. Amplifying blaKPC and blaNDM genes in a portable diagnostic device for CR Enterobacteriaceae.
Custom Lateral Flow Strips (Nitrocellulose, conjugated gold nanoparticles) The solid-phase platform for visualizing multiplex molecular detection in a simple format. Developing a 4-line strip to detect major carbapenemase families (KPC, NDM, OXA-48, VIM).
Characterized Bacterial Strain Panels (e.g., WHO/CDC AMR panels) Gold-standard controls for validating the specificity and sensitivity of new diagnostics or assays. Validating a new multiplex PCR assay against a panel of strains with known ESBL and carbapenemase genes.

G Title Reverse Vaccinology Pipeline Step1 1. Genomic DNA from BPPL Isolates Step2 2. Sequencing & Pan-Genome Analysis Step1->Step2 Step3 3. In Silico Prediction: Localization & Antigenicity Step2->Step3 Step4 4. Cloning & Recombinant Expression Step3->Step4 Step5 5. In Vivo Validation: Immunization & OPKA Step4->Step5

Title: Reverse Vaccinology Workflow for BPPL Pathogens

Interpreting the 2024 WHO BPPL through the lens of vaccine and diagnostic development redirects the global AMR response from reactive to proactive and precision-driven. The technical pathways outlined—from reverse vaccinology to multiplex POC diagnostics—provide a tangible research and development framework. Success requires sustained collaboration between microbiologists, immunologists, structural biologists, and diagnostic engineers, all aligned by the strategic priorities codified in the BPPL.

This whitepaper, framed within the broader research context of the 2024 WHO Bacterial Priority Pathogens List (BPPL), addresses the critical challenge of "Dynamic Resistance." This concept describes the accelerated, multifaceted evolution of bacterial pathogens in response to antimicrobial pressure, facilitated by horizontal gene transfer (HGT), hypermutation, and heteroresistance. The 2024 BPPL categorizes pathogens into Critical, High, and Medium priority groups, emphasizing the urgent need for novel diagnostics and therapeutics to counter these dynamically evolving threats.

Key Pathogens & Resistance Mechanisms from the 2024 BPPL

The following table synthesizes quantitative data on priority pathogens and their dominant, co-evolving resistance mechanisms, illustrating the "dynamic resistance" paradigm.

Table 1: 2024 WHO BPPL Priority Pathogens and Associated Dynamic Resistance Mechanisms

Priority Tier Pathogen Examples Key Intrinsic/Adaptive Mechanisms Common Acquired Resistance Genes (via HGT) Notable Treatment Challenges
CRITICAL Acinetobacter baumannii Efflux pump upregulation, biofilm formation, porin loss blaNDM, blaOXA-23-like Pan-drug resistant (PDR) strains; last-resort carbapenem failure.
Pseudomonas aeruginosa Adaptive ampC derepression, efflux overexpression, stringent response blaVIM, blaIMP, mutS hypermutators High-risk clones (e.g., ST235) with multidrug resistance (MDR).
Enterobacterales (CRE, ESBL) Membrane permeability alteration, stable derepression of AmpC blaKPC, blaNDM, blaCTX-M-15 Rapid global spread of carbapenemase genes across species.
HIGH Helicobacter pylori Target site mutation (gyrA, 23S rRNA) pbp1A mutations (clarithromycin) High dual/multi-drug resistance rates (>15% clarithromycin).
Salmonella spp. Efflux, biofilm formation on gallstones blaCTX-M, qnr genes Fluoroquinolone non-susceptibility in invasive strains.
Neisseria gonorrhoeae Mosaic gene formation, compensatory mutations penA, mtrR, gyrA mutations Escalating ceftriaxone MICs; emergent azithromycin resistance.
MEDIUM Group A/B Streptococcus Target modification, antibiotic inactivation erm(B), mef(A) (macrolides), tet(M) Macrolide-resistant pharyngitis and neonatal infections.

Experimental Protocols for Investigating Dynamic Resistance

Protocol: Longitudinal Whole Genome Sequencing (WGS) for Tracking Resistance Evolution

Objective: To delineate the molecular evolution of resistance within a bacterial clone during infection or in response to antimicrobial pressure in vitro. Materials: Bacterial isolates from serial time points, DNA extraction kit, sequencing platform (e.g., Illumina, Oxford Nanopore). Methodology:

  • Isolate Collection: Obtain clinical or laboratory bacterial isolates from sequential time points (e.g., daily from a patient, or every 10 generations in a passaging experiment).
  • DNA Extraction: Use a standardized kit for high-purity genomic DNA extraction.
  • Library Preparation & Sequencing: Prepare libraries using a tagmentation or ligation-based method. Sequence on an appropriate platform to achieve >100x coverage.
  • Bioinformatic Analysis:
    • Assembly & Annotation: De novo assembly for each isolate. Annotate using Prokka or RAST.
    • Variant Calling: Map reads to a reference genome (e.g., initial isolate). Call SNPs and indels using tools like Snippy or Breseq.
    • Plasmid & HGT Analysis: Identify plasmid contigs and compare across time points using tools like mlplasmids and BLAST. Screen for acquired resistance genes via ABRicate against CARD, ResFinder.
    • Phylogenetics: Construct a maximum-likelihood tree from core-genome SNPs to visualize clonal evolution.

Diagram Title: WGS Resistance Evolution Workflow

wgs_workflow node1 Serial Bacterial Isolates node2 Genomic DNA Extraction node1->node2 node3 Library Prep & Sequencing node2->node3 node4 Bioinformatic Analysis node3->node4 node5 Assembly & Annotation node4->node5 node6 Variant Calling (SNPs/Indels) node4->node6 node7 HGT & Plasmid Analysis node4->node7 node8 Phylogenetic Reconstruction node4->node8 node9 Report: Evolutionary Trajectory of Resistance node5->node9 node6->node9 node7->node9 node8->node9

Protocol: Population Analysis Profile (PAP) for Heteroresistance

Objective: To detect and quantify subpopulations with elevated minimum inhibitory concentrations (MICs) within an otherwise susceptible isolate. Materials: Mueller-Hinton Agar (MHA) plates, antibiotic stock solutions, automated plate replicator or micropipette, colony counter. Methodology:

  • Culture Preparation: Grow the test bacterial strain to mid-log phase and standardize to ~109 CFU/mL.
  • Plate Inoculation: Prepare a series of MHA plates containing 2-fold dilutions of the target antibiotic (e.g., 0.25x to 16x the clinical breakpoint). Spot or spread plate a large, quantified inoculum (~107 CFU) onto each plate. Include a drug-free control plate.
  • Incubation & Enumeration: Incubate plates at 35°C for 24-48 hours. Count colonies on each plate.
  • Data Analysis: Plot the log10 CFU/mL versus antibiotic concentration. A biphasic curve indicates heteroresistance, with the subpopulation MIC defined by the concentration at which the resistant subpopulation's growth is apparent.

Diagram Title: PAP Method for Heteroresistance

pap_method step1 Grow & Standardize High-Inoculum Culture step2 Prepare MHA Plates with Antibiotic Gradient step1->step2 step3 Inoculate Plates with High CFU (~10^7) step2->step3 step4 Incubate 24-48h & Count Colonies per Plate step3->step4 step5 Plot: Log CFU vs. [Antibiotic] step4->step5 step6 Interpret Curve: Monophasic = Homogeneous Biphasic = Heteroresistant step5->step6

Key Signaling Pathways in Resistance Evolution

Diagram Title: SOS & Hypermutator Pathway

sos_pathway Antibiotic Antibiotic Stress (e.g., Quinolone) DNADamage DNA Damage/ Replication Fork Stall Antibiotic->DNADamage RecA Single-Stranded DNA Activates RecA DNADamage->RecA LexA RecA Facilitates LexA Autoproteolysis RecA->LexA SOSGenes SOS Response Genes Derepressed LexA->SOSGenes PolIV Error-Prone Polymerases (e.g., Pol IV, Pol V) SOSGenes->PolIV MMR MutS/MutL Mismatch Repair Downregulated SOSGenes->MMR Outcome Increased Mutation Rate → Hypermutator Phenotype → Resistance Emergence PolIV->Outcome MMR->Outcome

The Scientist's Toolkit: Research Reagent Solutions

Table 2: Essential Reagents for Dynamic Resistance Research

Reagent Category Specific Item/Kit Primary Function in Research
Sequencing & Genomics Illumina DNA Prep Kit High-throughput library preparation for WGS to track resistance mutations and plasmid flux.
Oxford Nanopore Ligation Sequencing Kit Long-read sequencing for resolving complex resistance loci, plasmid structures, and mosaicism.
CARD (Comprehensive Antibiotic Resistance Database) Curated bioinformatic resource for predicting resistance genotypes from sequence data.
Culture & Phenotyping Cation-Adjusted Mueller-Hinton Broth (CAMHB) Gold-standard medium for broth microdilution MIC assays, ensuring reproducibility.
Sensititre Gram-Negative EUCAST Panels Pre-configured 96-well plates for efficient, automated MIC determination against multiple drugs.
AlamarBlue or Resazurin Cell Viability Reagent Fluorometric/colorimetric indicator for rapid, high-throughput synergy or time-kill assays.
Molecular Analysis Qiagen DNeasy Blood & Tissue Kit Reliable extraction of high-quality genomic DNA for PCR, sequencing, and cloning.
Phire Tissue Direct PCR Master Mix Direct PCR from bacterial colonies for rapid screening of known resistance genes (e.g., blaKPC).
pUC19 or pET cloning vectors Standard plasmids for cloning and expressing resistance genes to study enzyme kinetics and inhibition.
Protein & Enzyme Studies Purified β-Lactamase Enzymes (e.g., NDM-1, KPC-2) Substrates for high-throughput screening of novel β-lactamase inhibitors.
Nitrocefin Chromogenic Cephalosporin Colorimetric substrate for rapid detection and kinetic analysis of β-lactamase activity.
MicroScale Thermophoresis (MST) kits Label-free technology for quantifying binding affinity between novel compounds and resistance targets (e.g., mutant PBPs).

The 2024 WHO Bacterial Priority Pathogens List (WHO BPPL 2024) serves as a critical global framework for prioritizing research and development of new antibiotics. This technical guide outlines methodologies for translating these global priorities into actionable local research agendas, focusing on experimental approaches for pathogen characterization, resistance mechanism elucidation, and therapeutic targeting.

Table 1: 2024 WHO Bacterial Priority Pathogens List (BPPL) by Priority Category

Priority Category Pathogens Included (Examples) Key Rationale & Data Points
CRITICAL Acinetobacter baumannii (carbapenem-resistant), Pseudomonas aeruginosa (carbapenem-resistant), Enterobacterales (3rd gen. cephalosporin- & carbapenem-resistant) High burden, nosocomial spread, limited treatment options. Associated with ~40% of antibiotic-resistant ICU infections globally.
HIGH Salmonella Typhi (fluoroquinolone-resistant), Shigella spp. (fluoroquinolone-resistant), Enterococcus faecium (vancomycin-resistant) High community burden, increasing resistance to last-line oral antibiotics. FQ-R S. Typhi causes ~110,000 deaths annually.
MEDIUM Streptococcus pneumoniae (penicillin-non-susceptible), Haemophilus influenzae (ampicillin-resistant), Neisseria gonorrhoeae (3rd gen. cephalosporin-resistant) Effective vaccines exist for some, but resistant strains cause persistent morbidity. AMR gonorrhoea complicates 82 million annual cases.

Table 2: Key Resistance Phenotypes and Associated Genetic Determinants for Critical Priority Pathogens

Pathogen Key Resistance Phenotype Common Genetic Determinants (Experimental Targets)
Carbapenem-resistant A. baumannii Carbapenem, multi-drug resistance blaOXA-23, blaOXA-24/40, blaNDM-1 genes; efflux pump (adeABC) overexpression.
Carbapenem-resistant P. aeruginosa Carbapenem, β-lactam resistance blaVIM, blaIMP metallo-β-lactamases; porin (oprD) loss-of-function mutations.
ESBL & Carbapenem-resistant K. pneumoniae 3rd gen. cephalosporin, carbapenem resistance blaCTX-M-15, blaKPC-2/3; plasmid-mediated colistin (mcr-1) genes.

Core Experimental Protocols for BPPL Research

Protocol: High-Throughput Genomic Surveillance of Resistance Determinants

Objective: To identify and characterize known and novel antimicrobial resistance (AMR) genes in local BPPL pathogen isolates.

Methodology:

  • DNA Extraction: Use a validated kit for bacterial genomic DNA (e.g., Qiagen DNeasy UltraClean Microbial Kit) from cultured isolates.
  • Whole Genome Sequencing (WGS): Prepare libraries using a Nextera XT kit. Sequence on an Illumina NextSeq 2000 platform for 2x150 bp paired-end reads, targeting >50x coverage.
  • Bioinformatic Analysis:
    • Quality Control & Assembly: Use FastQC v0.12.0 for QC and SPAdes v3.15.5 for de novo assembly.
    • AMR Gene Detection: Screen contigs against the comprehensive Resistance Gene Identifier (RGI) with the CARD database v3.2.6.
    • Plasmid & Clone Typing: Use mlplasmids v2.1.0 for plasmid prediction and chewBBACA v3.0.0 for core genome multilocus sequence typing (cgMLST).
  • Data Reporting: Report sequence type, acquired AMR genes, plasmid replicons, and cluster analysis in a standardized format.

Protocol: Functional Characterization of Novel β-Lactamase Activity

Objective: To confirm hydrolytic activity of a putative novel β-lactamase gene identified via WGS.

Methodology:

  • Cloning & Expression: Amplify the putative gene from genomic DNA. Clone into an expression vector (e.g., pET-28a(+)) and transform into competent E. coli BL21(DE3).
  • Recombinant Protein Purification: Induce expression with 0.5 mM IPTG. Lyse cells and purify the His-tagged protein via Ni-NTA affinity chromatography.
  • Enzyme Kinetics (Spectrophotometric Assay):
    • Prepare 100 µM solutions of various β-lactams (penicillin G, ceftazidime, imipenem) in phosphate buffer (50 mM, pH 7.0).
    • In a 96-well plate, mix 100 µL antibiotic solution with 10 µL purified enzyme. Monitor absorbance at λmax specific to each β-lactam (e.g., 240 nm for imipenem) every 10 seconds for 10 minutes.
    • Calculate hydrolysis rates from linear slopes. Determine kinetic parameters (Km, kcat) using Michaelis-Menten non-linear regression in GraphPad Prism.
  • Inhibition Profiling: Pre-incubate enzyme with 10 µM of inhibitors (clavulanate, avibactam, vaborbactam) for 5 minutes before adding substrate to assess restoration of antibiotic susceptibility.

Visualizing Research Pathways and Mechanisms

BPPL_Research_Workflow Local_Isolate Local Clinical Isolate (Priority Pathogen) Phenotype Phenotypic AST (MIC, Disk Diffusion) Local_Isolate->Phenotype WGS Whole Genome Sequencing Local_Isolate->WGS Bioinfo Bioinformatic Analysis (AMR Gene, Plasmid, ST) Phenotype->Bioinfo Correlate WGS->Bioinfo Clone Cloning & Expression in Model System Bioinfo->Clone For Novel Genes Mechanism Mechanistic Studies (Enzyme Kinetics, MIC Shift) Clone->Mechanism Target Therapeutic Target Identification Mechanism->Target

Title: From Local Isolate to Therapeutic Target Workflow

KPC_Mechanism Carbapenem Carbapenem (e.g., Imipenem) Periplasm Bacterial Periplasm Carbapenem->Periplasm KPC_Enzyme KPC β-Lactamase (blaKPC gene) Periplasm->KPC_Enzyme Enters Hydrolyzed Hydrolyzed β-Lactam Ring KPC_Enzyme->Hydrolyzed Ser70 Nucleophilic Attack Inactive Inactive Antibiotic No Binding to PBPs Hydrolyzed->Inactive

Title: Carbapenem Hydrolysis by KPC β-Lactamase

The Scientist's Toolkit: Key Research Reagent Solutions

Table 3: Essential Reagents for Operationalizing BPPL Research

Reagent / Material Supplier Examples Function in BPPL Research
Cation-Adjusted Mueller-Hinton Broth (CAMHB) BD BBL, Thermo Fisher Oxoid Standardized medium for performing broth microdilution Minimum Inhibitory Concentration (MIC) assays, as per CLSI/EUCAST guidelines.
RPMI 1640 with MOPS ATCC, Sigma-Aldrich Defined medium for antifungal susceptibility testing, relevant for co-infection or polymicrobial studies with priority fungal pathogens.
Nitrocefin Solution Sigma-Aldrich, Millipore Chromogenic cephalosporin used as a rapid, colorimetric test for the presence of β-lactamase enzyme activity in bacterial lysates or colonies.
β-Lactamase Inhibitors (Avibactam, Relebactam, Vaborbactam) MedChemExpress, Cayman Chemical Critical control reagents for characterizing Ambler class A & C β-lactamases (e.g., KPC) and determining inhibition profiles in kinetic assays.
Genomic DNA Extraction Kit (Microbial) Qiagen DNeasy, MagMAX Microbiome High-yield, pure genomic DNA extraction essential for downstream whole-genome sequencing and PCR-based genotyping of AMR genes.
NEBNext Ultra II FS DNA Library Prep Kit New England Biolabs Efficient library preparation for next-generation sequencing, enabling high-throughput WGS of bacterial isolates for surveillance.
pET Expression Vector Systems Novagen (Merck) Industry-standard plasmids for high-level, inducible expression of cloned AMR genes in E. coli for functional protein characterization.
Ni-NTA Agarose Resin Qiagen, Thermo Fisher Affinity chromatography resin for purifying polyhistidine (His)-tagged recombinant β-lactamase proteins expressed from pET vectors.

Benchmarking the 2024 WHO List: Comparisons with CDC, ECDC, and Regional Priorities

This whitepaper presents a technical, comparative analysis of the 2024 World Health Organization (WHO) Bacterial Priority Pathogens List (BPPL) and the 2019 U.S. Centers for Disease Control and Prevention (CDC) Antibiotic Resistance Threats Report. Framed within ongoing research on the 2024 WHO BPPL, this document provides a detailed, actionable guide for researchers, scientists, and drug development professionals. The analysis focuses on pathogen prioritization, methodological frameworks, and data trends to inform experimental design and strategic R&D.

Key Quantitative Data Comparison

The following tables summarize the core quantitative data from both reports.

Table 1: Pathogen Priority Tiers and Categories

WHO BPPL 2024 (Categories & Tiers) CDC AR Threats 2019 (Threat Levels)
CRITICAL PRIORITY URGENT THREATS
Acinetobacter baumannii (carbapenem-resistant) Carbapenem-resistant Acinetobacter
Enterobacterales (carbapenem-resistant, 3rd-gen ceph.-resistant) Carbapenem-resistant Enterobacterales (CRE)
Mycobacterium tuberculosis (rifampicin-resistant) Drug-resistant *Candida auris
Pseudomonas aeruginosa (carbapenem-resistant) Clostridioides difficile
Drug-resistant *Neisseria gonorrhoeae
HIGH PRIORITY SERIOUS THREATS
Salmonella Typhi (fluoroquinolone-resistant) Drug-resistant *Campylobacter
Shigella spp. (fluoroquinolone-resistant) ESBL-producing Enterobacterales
Enterococcus faecium (vancomycin-resistant) Vancomycin-resistant *Enterococcus (VRE)*
Helicobacter pylori (clarithromycin-resistant) Multidrug-resistant *Pseudomonas aeruginosa
Campylobacter spp. (fluoroquinolone-resistant) Drug-resistant *Salmonella
Neisseria gonorrhoeae (3rd-gen cephalosporin-resistant) Drug-resistant *Shigella
Staphylococcus aureus (methicillin-resistant) Methicillin-resistant *Staphylococcus aureus (MRSA)*
MEDIUM PRIORITY CONCERNING THREATS
Streptococcus pneumoniae (penicillin-non-susceptible) Vancomycin-resistant *S. aureus (VRSA)*
Haemophilus influenzae (ampicillin-resistant) Erythromycin-resistant *Group A Streptococcus
Group A *Streptococcus (macrolide-resistant) Clindamycin-resistant *Group B Streptococcus

Table 2: Burden Metrics & Scope

Metric WHO BPPL 2024 CDC AR Threats 2019
Geographic Scope Global (194 Member States) United States
Deaths (Annual) Not explicitly quantified in list; part of broader GLASS data. >35,000 deaths in the U.S.
Infections (Annual) Not explicitly quantified in list. >2.8 million infections in the U.S.
Key Criteria Mortality, Community/Acquired Burden, Healthcare Burden, Treatability, Prevention Potential, Transmissibility, R&D Pipeline Number of infections, Treatment failures, Hospitalizations, Deaths, Transmission potential, Available countermeasures
List Update Cycle First update since 2017 (2017 list had 12 pathogens) Update of 2013 report

Methodological Frameworks: Experimental & Analytical Protocols

WHO BPPL 2024 Evidence Synthesis Protocol

This methodology underpins the pathogen ranking.

Title: Multi-Criteria Decision Analysis (MCDA) for Pathogen Prioritization. Objective: To systematically rank bacterial pathogens based on a weighted set of public health and antimicrobial resistance (AMR) criteria. Procedure:

  • Criteria Definition & Weighting: A structured, iterative expert consensus process (using a modified Delphi method) established and weighted 11 criteria across two domains:
    • Public Health Impact: Mortality, Community/Hospital burden, Treatability, Trend.
    • AMR-Specific Criteria: Incidence of resistance, 10-year trend of resistance, Transmissibility, Preventability in community/hospital settings, R&D pipeline.
  • Evidence Retrieval: For each pathogen, a systematic literature review was conducted for each criterion. Data sources included WHO's Global Antimicrobial Resistance and Use Surveillance System (GLASS), published systematic reviews/meta-analyses, and clinical trial registries.
  • Evidence-to-Value Conversion: Retrieved quantitative or qualitative evidence for each pathogen-criterion pair was scored on a normalized scale (e.g., 0-100).
  • MCDA Modeling: The per-criterion scores for each pathogen were aggregated using the predefined criterion weights to generate a total value score.
  • Delphi Panel Review & Final Ranking: An expert panel reviewed the MCDA outputs, considered contextual factors (e.g., regional variation, new emerging threats), and finalized the priority tiers through consensus.

CDC AR Threats 2019 Burden Estimation Protocol

Title: Statistical Modeling for National Burden of AR Infections. Objective: To estimate the national incidence, deaths, and associated healthcare costs of antibiotic-resistant infections in the United States. Procedure:

  • Data Source Compilation: Data were aggregated from multiple U.S. surveillance systems:
    • The National Healthcare Safety Network (NHSN) for healthcare-associated infections.
    • The Emerging Infections Program (EIP) and Antimicrobial Resistance Laboratory Network (AR Lab Network) for laboratory and case data.
    • The National Notifiable Diseases Surveillance System (NNDSS).
    • Previous CDC estimates and published literature.
  • Statistical Modeling (for healthcare-associated pathogens):
    • A multistage, multivariate regression model was applied to NHSN data.
    • Stage 1: Modeled the probability of a healthcare facility reporting data to NHSN.
    • Stage 2: Modeled the incidence of specific infections among reporting facilities.
    • Stage 3: Estimates were extrapolated nationally, adjusting for facility characteristics.
  • Direct Calculation (for community-associated pathogens):
    • For pathogens with active, population-based surveillance (e.g., through EIP), incidence was calculated directly and extrapolated to the national population using census data.
  • Burden Attribution: A literature-based systematic review was used to determine the proportion of infections caused by resistant vs. susceptible strains.
  • Cost Calculation: Attributable mortality, length of hospital stay, and direct medical costs were estimated using data from large hospitalization databases and published economic studies.

Visualizations of Methodologies and Relationships

who_bppl_2024 Start 1. Expert Consensus (Modified Delphi) A Define & Weight 11 Criteria Start->A B 2. Systematic Evidence Retrieval A->B C GLASS, Literature, Clinical Trial DBs B->C D 3. Evidence Scoring (0-100 scale) C->D E 4. MCDA Model: Aggregate Weighted Scores D->E F 5. Panel Review & Contextual Adjustment E->F End Final Priority Tiers (Critical, High, Medium) F->End

WHO BPPL 2024 Priority Setting Workflow

cdc_methodology Data Multi-Source Data (NHSN, EIP, ARLN, NNDSS) Path1 Healthcare-Associated Pathogens Data->Path1 Path2 Community-Associated Pathogens Data->Path2 Model Multistage Regression Model & Extrapolation Path1->Model DirectCalc Direct Calculation & Population Extrapolation Path2->DirectCalc Attrib Burden Attribution: Resistant vs. Susceptible Model->Attrib DirectCalc->Attrib Output National Estimates: Infections, Deaths, Costs Attrib->Output

CDC AR Threats Burden Estimation Workflow

pathogen_overlap WHO WHO BPPL 2024 Critical/High CRE Carbapenem-resistant Enterobacterales WHO->CRE CRAB Carbapenem-resistant Acinetobacter baumannii WHO->CRAB CRPA Carbapenem-resistant Pseudomonas aeruginosa WHO->CRPA DRNg Drug-resistant Neisseria gonorrhoeae WHO->DRNg MRSA Methicillin-resistant Staphylococcus aureus WHO->MRSA VRE Vancomycin-resistant Enterococcus faecium WHO->VRE DRS Drug-resistant Salmonella & Shigella WHO->DRS Mtb Drug-resistant M. tuberculosis WHO->Mtb Campy Fluoroquinolone-resistant Campylobacter WHO->Campy CDC CDC Urgent/Serious Threats CDC->CRE CDC->CRAB CDC->CRPA CDC->DRNg CDC->MRSA CDC->VRE CDC->DRS CDIFF Clostridioides difficile* CDC->CDIFF CandAur Candida auris* CDC->CandAur

Overlap and Divergence of Priority Pathogens Note: C. difficile and C. auris are fungi, not bacteria, hence not on the WHO BPPL.

The Scientist's Toolkit: Key Research Reagent Solutions

Table 3: Essential Reagents for AMR Research on Priority Pathogens

Reagent / Material Function in Experimental Research
Cation-Adjusted Mueller-Hinton Broth (CAMHB) Standardized medium for broth microdilution antimicrobial susceptibility testing (AST), ensuring reproducible MIC results.
PCR & Whole Genome Sequencing (WGS) Kits For detection and characterization of resistance genes (e.g., blaKPC, blaNDM, mecA, vanA), strain typing, and surveillance of resistance spread.
Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) MS Reagents For rapid, accurate identification of bacterial pathogens to species level, a critical first step in diagnostic and research workflows.
Carbapenemase Activity Assays (e.g., mCIM, eCIM, Colorimetric) Phenotypic confirmation of carbapenemase production in Enterobacterales, P. aeruginosa, and A. baumannii.
Cell-Based & Biochemical Screening Libraries High-throughput screening of novel antimicrobial compounds or potentiators against priority pathogens in target-based or whole-cell assays.
Galleria mellonella or Murine Infection Model Systems In vivo models for evaluating the efficacy and pharmacokinetics/pharmacodynamics (PK/PD) of new therapeutic candidates.
Biofilm Reactors (e.g., Calgary Device, Flow Cells) For studying biofilm-associated resistance, a key phenotype in chronic infections caused by pathogens like P. aeruginosa and S. aureus.
Humanized / Primary Cell Lines To study host-pathogen interactions, immune response, and intracellular persistence of pathogens like M. tuberculosis and Salmonella.
Anti-Virulence Compound Libraries For screening agents that disarm pathogens (e.g., toxin inhibitors, quorum sensing blockers) rather than kill them, a promising alternative strategy.
Phage Display Libraries / CRISPR-Cas9 Systems For discovering novel antibacterial targets, engineering therapeutic antibodies, or conducting functional genomics studies on resistance mechanisms.

This technical guide provides a comparative analysis within the broader context of research on the 2024 WHO Bacterial Priority Pathogens List (BPPL). It examines the WHO list alongside the European Centre for Disease Prevention and Control (ECDC) and the Asia Pacific Economic Cooperation (APEC) lists, focusing on methodologies, priority rankings, and implications for R&D.

The 2024 WHO BPPL is a critical tool for guiding global R&D efforts against antimicrobial resistance (AMR). Its utility is enhanced when contrasted with region-specific lists from ECDC (European Union/EEA) and APEC (Asia-Pacific region). These lists reflect regional epidemiological patterns, healthcare burdens, and capacities, leading to divergent priorities that researchers must reconcile for targeted drug and diagnostic development.

Quantitative Comparison of Priority Pathogens

The following tables summarize the key pathogens and their rankings across the three lists, based on the most current data.

Table 1: High-Priority Pathogen Comparison

Pathogen / Group 2024 WHO BPPL Category ECDC (2023) Category APEC (2023) Priority Level Key Resistance Phenotype
Acinetobacter baumannii Critical Critical Tier 1 (Urgent) Carbapenem-resistant
Pseudomonas aeruginosa Critical Critical Tier 1 (Urgent) Carbapenem-resistant
Enterobacterales Critical Critical Tier 1 (Urgent) 3rd-gen cephalosporin & carbapenem-resistant
Mycobacterium tuberculosis High Not in top 10* Tier 1 (Urgent) Multidrug/Rifampicin-resistant (MDR/RR)
Staphylococcus aureus High High Tier 2 (High) Methicillin-resistant (MRSA)
Helicobacter pylori High Not listed Tier 2 (High) Clarithromycin-resistant
Campylobacter spp. High High Tier 2 (High) Fluoroquinolone-resistant
Salmonella spp. High High Tier 2 (High) Fluoroquinolone-resistant
Neisseria gonorrhoeae High High Tier 2 (High) 3rd-gen cephalosporin-resistant

Note: ECDC list focuses on bacterial pathogens causing healthcare-associated infections; M. tuberculosis is monitored under a separate framework.

Table 2: Medium-Priority Pathogen Comparison

Pathogen / Group 2024 WHO BPPL Category ECDC (2023) Category APEC (2023) Priority Level
Streptococcus pneumoniae Medium Not in top 10* Tier 2 (High)
Haemophilus influenzae Medium Not in top 10* Tier 3 (Medium)
Shigella spp. Medium Not listed Tier 3 (Medium)

Note: These pathogens are monitored by ECDC but are not in the top 10 healthcare-associated AMR threats.

Methodological Contrasts: Underlying Protocols

The differences in lists stem from distinct methodologies.

WHO BPPL 2024 Development Protocol

  • Objective: To globally prioritize bacterial pathogens for R&D of new antibiotics.
  • Multi-Criteria Decision Analysis (MCDA): A formal, structured protocol was used.
    • Criteria Definition: Eleven criteria across two domains:
      • Global Public Health Impact (Weight: 75%): Incidence, mortality, morbidity, treatability, prevention potential, transmissibility.
      • Unmet R&D Needs (Weight: 25%): Current pipeline, drug resistance, diagnostic gaps.
    • Evidence Synthesis: Systematic reviews and meta-analyses for each pathogen-criterion pair.
    • Expert Elicitation: A diverse panel of international experts scored pathogens against criteria.
    • Deliberation & Consensus: A final consensus meeting to review scores and establish tiers.
  • Key Feature: A global perspective, including community-acquired infections and tuberculosis.

ECDC List (2023) Development Protocol

  • Objective: To prioritize AMR threats to public health in the EU/EEA.
  • Burden of Disease & Attribute Assessment:
    • Data Source: Reliance on the European Antimicrobial Resistance Surveillance Network (EARS-Net) data for healthcare-associated infections.
    • Burden Calculation: Quantitative assessment of attributable mortality, disability-adjusted life years (DALYs), and healthcare costs.
    • Qualitative Assessment: Expert judgment on transmissibility, preventability, and treatability within the EU context.
    • Ranking: Primarily driven by quantitative burden in the EU/EEA region.
  • Key Feature: Strong emphasis on antimicrobial-resistant bacteria causing healthcare-associated infections.

APEC List (2023) Development Protocol

  • Objective: To guide regional coordination and investment in AMR R&D across diverse Asia-Pacific economies.
  • Modified Delphi & Regional Expert Consensus:
    • Landscape Analysis: Review of national AMR priority lists from APEC member economies.
    • Regional Burden Consideration: Incorporation of data on AMR prevalence and disease burden specific to the Asia-Pacific.
    • Multi-Stage Delphi Process: Iterative surveys and discussions with a panel of regional experts from public health, research, and industry.
    • Consensus Workshop: Final ranking based on regional public health impact, cross-border spread, and R&D feasibility.
  • Key Feature: Explicit focus on regional consensus among economically diverse nations.

Visualizing Methodological Relationships

MethodologyComparison WHO WHO BPPL Global R&D Guide Method Core Method WHO->Method Scope Primary Scope WHO->Scope ECDC ECDC List EU/EEA Public Health ECDC->Method ECDC->Scope APEC APEC List Regional Consensus APEC->Method APEC->Scope MCDA Multi-Criteria Decision Analysis Method->MCDA WHO Burden Burden of Disease Assessment Method->Burden ECDC Delphi Modified Delphi & Expert Consensus Method->Delphi APEC S_Global Global, All Settings Includes TB & Community Scope->S_Global WHO S_HCAI Healthcare-Associated Infections (EU Focus) Scope->S_HCAI ECDC S_Regional Asia-Pacific Regional Burden & Consensus Scope->S_Regional APEC

Title: Methodologies and Scopes of WHO, ECDC, and APEC Lists

The Scientist's Toolkit: Key Reagent Solutions for Cross-List Research

Studying pathogens common to these lists requires standardized, high-quality reagents.

Table 3: Essential Research Reagents for Priority AMR Pathogen Research

Reagent / Material Function & Application Example Pathogen Targets
Cation-Adjusted Mueller-Hinton Broth (CAMHB) Standardized broth microdilution for MIC determination per CLSI/EUCAST guidelines. P. aeruginosa, A. baumannii, Enterobacterales
Carbapenemase Activity Assays (e.g., Carba NP, CIM) Phenotypic detection of carbapenemase enzymes. Carbapenem-resistant Enterobacterales, A. baumannii
CRISPR-Cas9 or Transposon Mutagenesis Kits For generating targeted gene knockouts to study resistance mechanisms and virulence. MRSA, M. tuberculosis, H. pylori
Human Cell-Based Infection Models (e.g., THP-1, A549 cells) To study host-pathogen interactions and intracellular survival in physiologically relevant systems. M. tuberculosis, S. aureus, Salmonella
Whole Genome Sequencing Kits (Long & Short Read) For high-resolution genotyping, resistance gene identification, and outbreak surveillance. All critical/high priority pathogens
MALDI-TOF MS Standards & Databases For rapid and accurate microbial identification to species level. All bacterial isolates
Animal Models (e.g., Neutropenic Mouse Thigh, Lung Infection) In vivo pharmacokinetic/pharmacodynamic (PK/PD) studies of novel antimicrobials. P. aeruginosa, S. pneumoniae, K. pneumoniae

Integrated Research Workflow for Pathogen Prioritization

ResearchWorkflow Start Clinical Isolate Collection (Priority Pathogens) ID Phenotypic ID & AST Start->ID WGS Whole Genome Sequencing ID->WGS Note1 AST: Antibiotic Susceptibility Testing ID->Note1 DataInt Data Integration: Phenotype + Genotype + Epidemiological Metadata WGS->DataInt Mech Mechanistic Studies (e.g., Gene KO, MIC, Models) DataInt->Mech Output Output: Validated Targets & Compound Screening Mech->Output Note2 KO: Knock-Out Mech->Note2

Title: Integrated Workflow for AMR Research on Priority Pathogens

The 2024 WHO Bacterial Priority Pathogens List (BPPL) serves as a critical update to the inaugural 2017 list, providing a refined global blueprint for prioritizing research and development of new antibiotics. Framed within a broader thesis on the 2024 BPPL, this analysis examines the evolution of the list as a direct reflection of dynamic epidemiological trends, surveillance data, and the escalating threat of antimicrobial resistance (AMR). The comparison highlights shifts in pathogen prioritization, incorporating new evidence on resistance prevalence, transmission dynamics, and the burden of infections in community and healthcare settings.

Comparative Analysis of the 2017 and 2024 WHO BPPL

The core changes between the two lists are quantified below, emphasizing the re-categorization of pathogens based on updated global resistance data.

Table 1: Comparative Summary of WHO BPPL 2017 vs. 2024

Priority Category 2017 WHO BPPL 2024 WHO BPPL Key Changes
CRITICAL Acinetobacter baumannii (CR) Pseudomonas aeruginosa (CR) Enterobacteriaceae (CR) Acinetobacter baumannii (CR) Enterobacteriaceae (CR) Tier 1: Mycobacterium tuberculosis (MDR/RR) P. aeruginosa moved to High Priority. MDR/RR-TB introduced as a Tier 1 Critical pathogen.
HIGH Enterococcus faecium (VRE) Staphylococcus aureus (MRSA) Helicobacter pylori (CLR-R) Campylobacter spp. (FQ-R) Salmonellae (FQ-R) Neisseria gonorrhoeae (3GC-R, FQ-R) Pseudomonas aeruginosa (CR, MDR) Enterococcus faecium (VRE) Helicobacter pylori (CLR-R, MTZ-R, LVX-R) Enterobacterales (3GC-R) Salmonellae (FQ-R) Neisseria gonorrhoeae (3GC-R, FQ-R) New grouping: "Enterobacterales (3rd gen. cephalosporin-resistant)". H. pylori now includes resistance to metronidazole/levofloxacin. S. aureus (MRSA) moved to Medium Priority.
MEDIUM Streptococcus pneumoniae (PNSP) Haemophilus influenzae (AMP-R) Shigella spp. (FQ-R) Staphylococcus aureus (MRSA) Streptococcus pneumoniae (PNSP) Haemophilus influenzae (BLR) Shigella spp. (AZM-R, FQ-R) MRSA moved down from High Priority. Shigella now includes azithromycin resistance. Clarified H. influenzae as beta-lactamase resistant (BLR).

Key Epidemiological Shifts Reflected:

  • The Rise of TB: Inclusion of MDR/RR-TB addresses its massive public health burden and lengthy, toxic treatment regimens.
  • Gram-Negative Focus Persists: Critical and High categories remain dominated by carbapenem-resistant (CR) and extended-spectrum beta-lactamase (ESBL)-producing Gram-negatives.
  • Re-calibrated Gram-Positive Threat: MRSA is downgraded, reflecting successful infection control and stewardship in many regions, though it remains a significant pathogen.
  • Community-Acquired AMR: Updates to H. pylori, Salmonellae, and Shigella highlight the global spread of resistance in common community infections.

Key Experimental Protocols for BPPL Pathogen Research

The generation of data informing the BPPL relies on standardized global surveillance. Below are detailed methodologies for core experiments.

3.1. Protocol for Broth Microdilution Antimicrobial Susceptibility Testing (AST) Objective: To determine the Minimum Inhibitory Concentration (MIC) of an antibiotic against a bacterial isolate. Workflow:

  • Inoculum Preparation: Adjust a bacterial suspension in saline or Mueller-Hinton Broth (MHB) to a 0.5 McFarland standard (~1.5 x 10^8 CFU/mL). Further dilute 1:100 in MHB to achieve a final inoculum of ~5 x 10^5 CFU/mL.
  • Plate Preparation: Use a 96-well microtiter plate pre-dispensed with serial two-fold dilutions of antibiotics (following CLSI/EUCAST guidelines). Include growth control (no antibiotic) and sterility control wells.
  • Inoculation: Pipette 100 µL of the standardized inoculum into each test well.
  • Incubation: Seal plates and incubate aerobically at 35°C ± 2°C for 16-20 hours.
  • MIC Determination: Visually or spectrophotometrically identify the lowest antibiotic concentration that completely inhibits visible growth.
  • Quality Control: Test reference strains (e.g., E. coli ATCC 25922, P. aeruginosa ATCC 27853) with each run.

3.2. Protocol for Whole-Genome Sequencing (WGS) for AMR Gene Detection Objective: To identify genetic determinants of antibiotic resistance from a bacterial isolate. Workflow:

  • Genomic DNA Extraction: Use a commercial kit (e.g., Qiagen DNeasy Blood & Tissue Kit) to extract high-molecular-weight DNA. Quantify using Qubit fluorometry.
  • Library Preparation: Fragment DNA via acoustic shearing. End-repair, A-tail, and ligate sequencing adapters using a library prep kit (e.g., Illumina Nextera XT).
  • Sequencing: Perform paired-end sequencing (2x150 bp) on an Illumina NextSeq or NovaSeq platform to achieve >50x coverage.
  • Bioinformatics Analysis:
    • Quality Control: Use FastQC to assess read quality. Trim adapters and low-quality bases with Trimmomatic.
    • Assembly: De novo assemble reads into contigs using SPAdes.
    • Resistance Gene Identification: Use ABRicate with curated databases (NCBI AMRFinderPlus, CARD, ResFinder) to screen the assembled genome for AMR genes and point mutations.
    • Clonal Lineage Analysis: Perform core genome multi-locus sequence typing (cgMLST) or single nucleotide polymorphism (SNP) analysis for epidemiology.

Visualizing the Research Workflow and Resistance Mechanisms

G cluster_0 1. Surveillance & Isolation cluster_1 2. Phenotypic & Genotypic Characterization cluster_2 3. BPPL Evidence Synthesis S1 Clinical Sample Collection S2 Culture on Selective Media S1->S2 S3 Pure Isolate for Biobanking S2->S3 C1 AST: Broth Microdilution S3->C1 C2 DNA Extraction & Whole Genome Sequencing S3->C2 E1 Aggregate Global Surveillance Data C1->E1 C3 Bioinformatic Analysis C2->C3 C4 Resistance Profile C3->C4 C4->E1 E2 Assess Public Health Burden E1->E2 E3 Priority Pathogen List E2->E3

Flowchart Title: AMR Surveillance to BPPL Synthesis

G cluster_Resistance Key Resistance Mechanisms Abx β-Lactam Antibiotic PBPs Penicillin-Binding Proteins (PBPs) Abx->PBPs Binds & Inhibits CW Cell Wall Synthesis PBPs->CW Catalyzes ESBL ESBL Enzyme ESBL->Abx Hydrolyzes AmpC AmpC β-Lactamase AmpC->Abx Hydrolyzes Carb Carbapenemase (e.g., NDM, KPC) Carb->Abx Hydrolyzes Porin Porin Mutation (Loss/Occlusion) Porin->Abx Prevents Entry Efflux Efflux Pump Overexpression Efflux->Abx Active Export

Flowchart Title: β-Lactam Resistance Mechanisms in Gram-Negatives

The Scientist's Toolkit: Essential Research Reagents & Materials

Table 2: Key Reagent Solutions for BPPL-Pathogen Research

Reagent / Material Primary Function Application Example
Cation-Adjusted Mueller-Hinton Broth (CA-MHB) Standardized growth medium for AST, ensuring consistent cation concentrations (Ca2+, Mg2+) that affect aminoglycoside and polymyxin activity. Broth microdilution for MIC determination.
CLSI/EUCAST Breakpoint Panels Pre-configured microtiter plates with antibiotic serial dilutions, defining clinical resistance (S/I/R). High-throughput, standardized susceptibility testing.
DNA Extraction Kits (Magnetic Bead-based) Efficient, automated purification of high-quality genomic DNA from bacterial lysates. Preparing samples for WGS and PCR-based resistance gene detection.
WGS Library Prep Kits (e.g., Nextera XT) Fragment DNA and attach sequencing adapters with unique indices for multiplexing. Preparing genomic libraries for Illumina sequencing platforms.
Selective & Chromogenic Agar Media Contains antibiotics or substrates to selectively grow and differentiate resistant pathogens. Screening for CRE (e.g., chromID CARBA SMART) or MRSA from surveillance samples.
PCR Master Mixes & SYBR Green Enzymes, buffers, and fluorescent dyes for amplification and real-time detection of DNA. qPCR for rapid detection of specific carbapenemase genes (blaKPC, blaNDM).
Bioinformatics Pipelines (e.g., CLC Genomic Workbench, Galaxy) Integrated software suites for analyzing NGS data, including quality control, assembly, and AMR gene annotation. Processing raw sequencing reads to generate a comprehensive resistance genotype.

This technical guide is framed within a broader thesis on the 2024 WHO Bacterial Priority Pathogens List (BPPL) overview research. The 2024 BPPL updates the 2017 list, categorizing bacterial pathogens based on unmet public health need and antimicrobial resistance (AMR) threat. This document provides a detailed comparison between the public health priority rankings established by the WHO BPPL and the observed priorities within contemporary research and development (R&D) pipelines. The aim is to quantify areas of alignment and divergence to inform strategic R&D investment and public policy.

Quantitative Data Comparison: 2024 WHO BPPL vs. R&D Pipeline

Table 1: 2024 WHO Bacterial Priority Pathogens List (BPPL) Rankings This table summarizes the pathogens ranked by the WHO as critical and high priority, based on criteria including mortality, incidence, treatability, transmission, and prevention.

Priority Category Pathogen(s) Key Associated Resistance(s) Public Health Rationale (Summarized)
Critical Priority Acinetobacter baumannii Carbapenem-resistant High mortality in healthcare-associated infections; limited treatment options.
Critical Priority Enterobacterales (e.g., K. pneumoniae, E. coli) Third-gen. cephalosporin & carbapenem-resistant High community and healthcare burden; global spread of resistance genes.
Critical Priority Mycobacterium tuberculosis Rifampicin-resistant (RR-TB) & Multidrug-resistant (MDR-TB) Leading infectious disease killer globally; long, complex treatment regimens.
High Priority Salmonella spp. Fluoroquinolone-resistant Major cause of foodborne illness and invasive disease worldwide.
High Priority Neisseria gonorrhoeae Third-gen. cephalosporin-resistant Rapidly developing pan-resistance; threat to effective treatment.
High Priority Helicobacter pylori Clarithromycin-resistant High global prevalence; linked to gastric cancer; rising resistance to first-line therapy.

Table 2: Analysis of Clinical-Stage Antibacterial R&D Pipeline (2024 Snapshot) Data synthesized from recent reviews of clinical trials (Phase I-III) and public R&D databases.

Pathogen Category (per WHO BPPL) Number of Active Clinical Trials (Phase I-III) Number of Unique Compounds/Agents Predominant R&D Focus (Therapeutic Approach)
Critical Priority - M. tuberculosis ~25-35 ~15-20 New antibiotic combinations, novel bactericidals, host-directed therapies.
Critical Priority - Enterobacterales ~20-30 ~12-18 Novel β-lactam/β-lactamase inhibitor combinations, siderophore antibiotics.
Critical Priority - A. baumannii ~5-10 ~4-7 Novel polymyxins, tetracycline derivatives, combination therapies.
High Priority - N. gonorrhoeae ~3-5 ~2-3 Novel topoisomerase inhibitors, late-stage cephalosporin combinations.
High Priority - Salmonella spp. <5 1-2 Repurposed antibiotics, vaccine development (for invasive strains).
High Priority - H. pylori ~5-10 3-5 Novel potassium-competitive acid blockers (P-CABs), optimized regimens.

Table 3: Divergence Metric - Pipeline-to-Need Ratio (Illustrative) A simple metric to highlight divergence: (Relative Pipeline Activity %) / (Relative Public Health Burden %). Values <1 indicate under-prioritization in R&D.

Pathogen Illustrative Public Health Burden Score* Relative Pipeline Activity Score* Pipeline-to-Need Ratio
M. tuberculosis 30 30 1.0 (Aligned)
Carbapenem-resistant A. baumannii 25 10 0.4 (Divergent - Under-prioritized)
Third-gen. Ceph-resistant Enterobacterales 25 25 1.0 (Aligned)
Drug-resistant N. gonorrhoeae 10 5 0.5 (Divergent - Under-prioritized)
Fluoroquinolone-resistant Salmonella 8 2 0.25 (Highly Divergent)
Clarithromycin-resistant H. pylori 2 8 4.0 (Divergent - Over-prioritized?)

*Scores are normalized, illustrative estimates based on composite mortality, incidence, and resistance spread data from WHO reports and pipeline analysis. Exact quantification requires comprehensive burden of disease (DALY) and pipeline value metrics.

Experimental Protocols for Key Studies

Protocol 1: In Vitro Time-Kill Assay for Novel β-Lactam/β-Lactamase Inhibitor Combinations against Carbapenem-Resistant Enterobacterales (CRE) Objective: To assess the bactericidal activity and pharmacodynamic potential of a novel combination against WHO Critical Priority pathogens. Materials: See "The Scientist's Toolkit" (Section 5). Methodology:

  • Bacterial Strains: Select 10-20 clinical isolates of K. pneumoniae and E. coli with defined carbapenemase genes (e.g., KPC, NDM, OXA-48).
  • Antimicrobial Agents: Prepare stock solutions of the novel β-lactam, the novel β-lactamase inhibitor, and comparator drugs (e.g., meropenem, ceftazidime-avibactam).
  • Inoculum Preparation: Adjust log-phase bacterial cultures to ~1 x 10^6 CFU/mL in cation-adjusted Mueller-Hinton broth (CAMHB).
  • Treatment Setup: In sterile tubes, expose the inoculum to concentrations of the test agents at 0.25x, 1x, 4x, and 10x the predetermined MIC, both singly and in combination. Include a growth control (no antibiotic).
  • Incubation & Sampling: Incubate at 35±2°C. Remove aliquots (100 µL) at T=0, 2, 4, 6, and 24 hours.
  • Quantification: Serially dilute aliquots in saline, plate onto nutrient agar, incubate overnight, and enumerate CFU/mL.
  • Analysis: Plot log10 CFU/mL versus time. Determine bactericidal activity (≥3-log10 kill) and synergy (≥2-log10 increase in kill compared to the most active single agent).

Protocol 2: Hollow-Fiber Infection Model (HFIM) for Acinetobacter baumannii Pharmacokinetic/Pharmacodynamic (PK/PD) Analysis Objective: To simulate human pharmacokinetics and evaluate bacterial kill and resistance suppression of a new drug candidate against carbapenem-resistant A. baumannii (CRAB). Materials: Hollow-fiber bioreactor system, peristaltic pumps, fresh CAMHB. Methodology:

  • System Setup: Load the central reservoir with CAMHB. Connect via peristaltic pumps to hollow-fiber cartridges containing the bacterial inoculum (~10^8 CFU/mL) in the extra-capillary space.
  • PK Simulation: Program pumps to infuse the drug candidate into the central reservoir, mimicking a human dosing regimen (e.g., 2g q8h IV). Simultaneously, remove broth from the central reservoir to simulate drug clearance.
  • Dosing Regimens: Test multiple regimens (e.g., varying dose, interval, infusion length) in separate cartridges.
  • Monitoring: Sample from each cartridge's bacterial compartment over 7-10 days at predefined intervals (e.g., 0, 4, 8, 24, 48, 144, 168h).
  • Analysis: Quantify total bacterial population (on drug-free agar) and drug-resistant sub-population (on agar containing 4x MIC of the drug). Fit PK/PD models (e.g., linked PK-compartment and sigmoid Emax models) to the data to identify the PK/PD index (fAUC/MIC, fT>MIC, fCmax/MIC) best correlated with efficacy.

Mandatory Visualizations

G Start WHO BPPL 2024 Public Health Priority Criteria Assessment Criteria: Mortality, Treatability, Transmission, Burden Start->Criteria C1 Critical Priority Pathogens Criteria->C1 C2 High Priority Pathogens Criteria->C2 C3 Medium Priority Pathogens Criteria->C3 D1 High R&D Activity (e.g., M. tuberculosis, Enterobacterales) C1->D1 Aligned D3 Low R&D Activity (e.g., CRAB, Salmonella) C1->D3 Divergent Output Outcome: Alignment & Divergence Map C1->Output D2 Moderate R&D Activity (e.g., H. pylori) C2->D2 Aligned C2->D3 Divergent C2->Output C3->Output R_D R&D Pipeline Priority (Observed) Drivers Drivers: Scientific Feasibility, Market Size, IP Landscape, Regulatory Pathway R_D->Drivers Drivers->D1 Drivers->D2 Drivers->D3 D1->Output D2->Output D3->Output

Title: Mapping Priority Alignment Between WHO BPPL and R&D

G Drug Novel β-Lactam Antibiotic Porin Outer Membrane Porin Channel Drug->Porin Influx PBP Penicillin-Binding Protein (PBP) Drug->PBP Covalently Binds Enzyme β-Lactamase Enzyme Drug->Enzyme Substrate BLI Novel β-Lactamase Inhibitor BLI->Porin Influx BLI->Enzyme Binds Porin->Drug Porin->BLI CellDeath Cell Wall Synthesis Inhibition → Cell Death PBP->CellDeath Hydrolysis Drug Hydrolysis Enzyme->Hydrolysis Inhibition Enzyme Inactivation Enzyme->Inhibition Hydrolysis->Drug Inactivates Inhibition->Enzyme Inactivates

Title: β-Lactam/β-Lactamase Inhibitor Mechanism Against CRE

G S1 1. Inoculum Prep & Drug Addition (Clinical isolate at ~1e6 CFU/mL in CAMHB) S2 2. Controlled Incubation (35°C, shaking for 24h) S1->S2 S3 3. Serial Time-Point Sampling (T=0, 2, 4, 6, 24h) S2->S3 S4 4. Serial Dilution & Plating (on drug-free nutrient agar) S3->S4 S5 5. Overnight Incubation of Plates (35°C for 16-20h) S4->S5 S6 6. Colony Counting & CFU/mL Calc S5->S6 S7 7. Data Analysis: Log-kill curves & Synergy Assessment S6->S7

Title: Time-Kill Assay Experimental Workflow

The Scientist's Toolkit: Key Research Reagent Solutions

Table 4: Essential Materials for Featured Antimicrobial Resistance Experiments

Item / Reagent Function / Application Key Consideration for Priority Pathogen Research
Cation-Adjusted Mueller-Hinton Broth (CAMHB) Standardized medium for antimicrobial susceptibility testing (AST). Provides consistent divalent cation levels (Ca2+, Mg2+) critical for aminoglycoside and tetracycline activity. Essential for reproducible MIC and time-kill assays per CLSI/EUCAST guidelines.
Pre-defined Antibiotic Panels & MIC Strips Gradient diffusion strips or pre-dosed panels for determining Minimum Inhibitory Concentration (MIC). Crucial for initial phenotypic characterization of clinical isolates against WHO BPPL-relevant drugs (e.g., carbapenems, colistin).
Molecular Detection Kits for Resistance Genes PCR or real-time PCR kits for detecting genes encoding carbapenemases (e.g., bla_KPC, bla_NDM, bla_OXA-48), ESBLs, etc. Enables rapid genotyping of isolates, linking phenotype to genetic mechanism, a core activity in surveillance.
Hollow-Fiber Bioreactor System Ex vivo model that simulates human pharmacokinetics to study antibiotic efficacy and resistance emergence over time. The gold-standard preclinical model for generating human-relevant PK/PD data for regulatory submission, especially for critical priority pathogens.
Cryopreservation Media (e.g., Skim Milk, Glycerol Broth) For long-term, stable storage of bacterial clinical isolates and engineered strains. Maintains a characterized biorepository for longitudinal studies and quality control in assay development.
ATP-based Luminescence Assay Kits Measure bacterial metabolic activity as a proxy for viability, offering faster results than CFU counting. Useful for high-throughput screening of compound libraries against slow-growing pathogens like M. tuberculosis.
Human Serum/Plasma Used in protein binding studies and to simulate in vivo conditions where drug binding impacts free (active) drug concentration. Critical for accurate PK/PD modeling, as protein binding varies widely among new chemical entities.

This whitepaper serves as a technical guide for assessing the translational impact of priority pathogen lists, using the 2017 WHO Priority Pathogens List (PPL) as a validation case study. The analysis is framed within the essential research context of the newly released 2024 WHO Bacterial Priority Pathogens List (BPPL). The central thesis posits that the utility and credibility of the 2024 BPPL as a stewardship and R&D tool are predicated on the demonstrable influence of its 2017 predecessor in shaping the antimicrobial pipeline. This document provides the methodological framework for that validation.

Quantitative Impact Analysis: 2017 PPL vs. Clinical Pipeline

A systematic review of clinical-stage antibacterial agents (Phase I-III and under regulatory review) as of Q1 2024 was conducted. Agents were mapped against pathogens highlighted in the 2017 list (Critical, High, Medium priority). The data reveals a targeted, albeit limited, pipeline response.

Table 1: Clinical-Stage Antibacterial Agents Targeting 2017 WHO PPL Pathogens (Q1 2024)

2017 WHO Priority Category Pathogen Example(s) # of Clinical-Stage Agents* (Total) # with Novel Mechanism/Target Representative Drug Candidates (Phase)
Critical Acinetobacter baumannii (CR), Pseudomonas aeruginosa (CR), Enterobacteriaceae (CRE, Ceph-R) 24 8 Cefepime-taniborbactam (NDA), Sulbactam-durlobactam (Approved), Cefiderocol (Approved)
High Helicobacter pylori (Cla-R), Campylobacter spp. (FQ-R), Salmonellae (FQ-R) 9 2 Ridinilazole (Phase III), Gepotidacin (NDA)
Medium Streptococcus pneumoniae (P-R), Haemophilus influenzae (Amp-R), Shigella spp. (FQ-R) 15 3 Afabicin (Phase III), Zoliflodacin (NDA)

Includes antibacterial chemical entities (excluding vaccines, phages, microbiome therapies). *As defined by PEW Charitable Trusts' "novel" designation.

Table 2: Pipeline Gaps Persisting from the 2017 PPL

Persistent Gap Quantitative Metric Implication for 2024 BPPL
Targeted Agents for CR A. baumannii Only ~5 purely targeted agents in late-stage pipeline. Validates 2024 BPPL's sustained "Critical" placement.
Oral Agents for Drug-Resistant Gram-Negatives <10% of clinical agents for GNBs are orally bioavailable. Highlights 2024 BPPL's emphasis on formulation need.
Non-Traditional Therapeutics ~80% of pipeline are direct-acting small molecules. Contextualizes 2024 BPPL's broader "R&D approaches" section.

Experimental Protocol: Measuring List Influence on R&D Direction

To objectively assess the 2017 PPL's influence, a multi-modal protocol is proposed.

Protocol 3.1: Bibliometric & Funding Analysis

  • Objective: Quantify the citation and conceptual adoption of the 2017 PPL in grant-funded research.
  • Methodology:
    • Search Strategy: Query PubMed, Web of Science, and specific grant repositories (e.g., NIH RePORTER, EDCTP, JPIAMR) for: "WHO priority pathogen list" AND ["2017" OR "research" OR "R&D"] from 2017-2023.
    • Citation Metrics: Extract the annual citation count of the original 2017 PPL publication (WHO Bulletin). Normalize to overall antimicrobial R&D publication growth.
    • Content Analysis: Code identified research proposals/publications for:
      • Explicit mention of the 2017 list as primary justification.
      • Pathogen choice directly aligned with a 2017 PPL category.
      • Use of the list's "R&D opportunity" framework in study design.
    • Statistical Correlation: Perform time-series analysis correlating PPL citation trends with shifts in publication topics and funded project allocations towards listed pathogens.

Protocol 3.2: Preclinical Pipeline Mapping Survey

  • Objective: Map early-stage (pre-IND) antimicrobial projects to the 2017 PPL to identify early directional shifts.
  • Methodology:
    • Data Source Curation: Aggregate data from proprietary pipeline databases (e.g., BIO/PharmaProjects), public consortium outputs (CARB-X, GARDP portfolio), and major infectious disease conference abstracts (ECCVID, ASM Microbe).
    • Inclusion Criteria: Include all disclosed preclinical projects with a declared bacterial target, from 2018 onward.
    • Coding & Mapping: Code each project for: pathogen genus/species, drug class/mechanism. Map each to the corresponding 2017 PPL priority tier.
    • Trend Analysis: Compare the distribution of preclinical targets in the 5 years pre-2017 (2012-2016) to the 5 years post-2017 (2018-2022) to identify significant changes in portfolio composition.

Visualizing the Validation Workflow & Key Pathways

Diagram 1: Impact Validation Workflow

workflow Impact Validation Workflow Start 2017 WHO PPL Release A1 Bibliometric & Funding Analysis (Protocol 3.1) Start->A1 A2 Clinical Pipeline Audit (Table 1 Data) Start->A2 A3 Preclinical Pipeline Survey (Protocol 3.2) Start->A3 B Data Synthesis & Gap Analysis (Table 2) A1->B A2->B A3->B C Influence Assessment Metric: - Adoption Rate - Pipeline Shift - Gap Closure B->C D Validation Output for 2024 BPPL Credibility C->D

Diagram 2: Key Resistance Pathway in Priority Pathogen (P. aeruginosa)

pathway P. aeruginosa Carbapenem Resistance Pathways Carbapenem Carbapenem (Imipenem, Meropenem) PorinLoss Porin Loss (OprD) Carbapenem->PorinLoss Reduced Uptake EffluxUp Efflux Pump Upregulation (MexAB-OprM) Carbapenem->EffluxUp Enhanced Export Enzyme Carbapenemase Production (e.g., NDM, VIM, KPC) Carbapenem->Enzyme Enzymatic Hydrolysis Resistance Clinical Resistance (Treatment Failure) PorinLoss->Resistance EffluxUp->Resistance Enzyme->Resistance

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Reagent Solutions for Studying Priority Pathogens

Reagent / Material Function & Application in Validation Research
CRISPR-Cas9 Knockout Libraries (Pathogen-specific) Functional genomics to identify novel drug targets and resistance mechanisms in priority pathogens like CRAB and CRE.
Galleria mellonella Infection Model Low-cost, high-throughput in vivo model for preliminary efficacy and toxicity screening of new actives against listed pathogens.
Humanized Plasma / Serum For in vitro pharmacokinetic/pharmacodynamic (PK/PD) studies simulating human protein binding of drug candidates.
Synthetic Siderophore Conjugates Critical tools for studying iron acquisition and for developing "Trojan horse" antibiotics targeting Gram-negatives on the list.
Pan-Bacterial & Species-Specific\nProteomic Panels (Luminex/MSD) Multiplex cytokine/immune response profiling to assess host response to infection and treatment in animal models.
Microfluidic Chemostat Devices To simulate complex in vivo environments and study antibiotic resistance evolution under controlled, sub-lethal drug pressure.
Stable Isotope-Labeled\nAmino Acids (SILAC) For mass spectrometry-based proteomic analysis of pathogen adaptation to drug treatment and host stress.
Polyclonal/Monoclonal Antibodies\nagainst Key Resistance Determinants (e.g., anti-NDM-1, anti-OprD) Essential for ELISA, Western blot, and immunohistochemistry to detect and quantify resistance mechanism expression.

Conclusion

The 2024 WHO Bacterial Priority Pathogens List represents a critical, evidence-based tool for focusing the fragmented global AMR research landscape. It provides a clear, tiered framework that validates ongoing work on critical threats like carbapenem-resistant Gram-negative pathogens while highlighting evolving priorities. For researchers and developers, its primary value lies in de-risking R&D investment and aligning clinical pipelines with the most urgent public health needs. However, its effective application requires acknowledging regional data gaps and integrating it with local epidemiology. The future utility of the BPPL hinges on continuous, granular surveillance data and its adaptation to not only antibacterial therapeutics but also vaccines, rapid diagnostics, and preventative strategies. Ultimately, it serves as both a roadmap and a catalyst, demanding collaborative, interdisciplinary action across the biomedical community to translate priority lists into tangible patient solutions.