Efflux pump inhibitors targeting MexAB-OprM restore carbapenem susceptibility in MDR Pseudomonas aeruginosa by reducing intracellular drug efflux, converting clinical resistance (MIC >8 µg/mL) to susceptibility (MIC ≤2 µg/mL)

In clinical MDR Pseudomonas aeruginosa isolates overexpressing the MexAB-OprM efflux operon (confirmed by qRT-PCR fold-change ≥3× relative to PAO1 wild-type reference), co-administration of a validated efflux pump inhibitor (EPI) (e.g., PAβN [phenylalanine-arginine β-naphthylamide] at 20–50 µg/mL, or a next-gen analog) with a carbapenem (meropenem or imipenem) will reduce the carbapenem MIC from a resistant baseline (>8 µg/mL, CLSI/EUCAST breakpoint) to a susceptible range (≤2 µg/mL) in ≥70% of isolates in which resistance is attributable primarily to efflux overexpression (i.e., excluding isolates where resistance is driven solely by carbapenemase production, e.g., KPC, NDM, VIM, or by OprD porin loss with no efflux contribution). The effect must be efflux-pump-specific, demonstrated by: (a) abrogation of the MIC shift in a ΔmexAB-oprM isogenic deletion mutant, and (b) dose-dependent reduction in intracellular carbapenem efflux measured by LC-MS/MS drug accumulation assay.

Minimum Viable Test (Phase 1, n=20 isolates, 3–4 weeks):

1. Select 20 clinical MDR P. aeruginosa isolates with confirmed carbapenem MIC >8 µg/mL (broth microdilution).
2. Genotype all isolates: whole-genome sequencing (WGS) for carbapenemase genes (blaKPC, blaNDM, blaVIM, blaIMP), oprD mutations/truncations, and mexR/nalC/nalD regulatory mutations.
3. Quantify mexB transcript levels via qRT-PCR (normalized to rpsL housekeeping gene) to confirm efflux overexpression (≥3-fold vs PAO1).
4. Perform checkerboard broth microdilution: meropenem (0.03–64 µg/mL) × PAβN (0–64 µg/mL), calculate FIC index.
5. Repeat MIC testing with EPI at fixed sub-inhibitory concentration (20 µg/mL) ± carbapenem.
6. Construct isogenic ΔmexAB-oprM deletion mutant in 3 representative overexpressor strains (allelic exchange, e.g., pEX18Ap suicide vector) as specificity control.
7. Measure intracellular meropenem accumulation via LC-MS/MS (radiolabeled or stable-isotope-labeled meropenem, 30-min uptake assay) ± EPI.

PIPELINE: EPI-Carbapenem-Synergy-Validation

1. isolate_bank = load_clinical_isolates(n=60, source="hospital_biobank/CDC_AR_bank")
2. for isolate in isolate_bank:
       wgs_data = sequence_genome(isolate)
       resistome = annotate_resistance_genes(wgs_data, db="CARD/ResFinder")
       mechanism_class = classify(resistome)  
           # {carbapenemase_positive, efflux_overexpressor, porin_loss, mixed}
       mexB_expression = qRT_PCR(isolate, target="mexB", ref_gene="rpsL")
       baseline_MIC = broth_microdilution(isolate, drug="meropenem")

3. checkerboard_results = {}
   for isolate in isolate_bank:
       FIC = checkerboard_assay(isolate, drug="meropenem", adjuvant="PAbetaN",
                                 drug_range=[0.03,64], adjuvant_range=[0,64])
       MIC_with_EPI = min_MIC_at_FIC_optimum(FIC)
       checkerboard_results[isolate] = {FIC, MIC_with_EPI, mechanism_class}

4. knockout_strains = construct_isogenic_mutants(
        strains=sample(efflux_overexpressors, n=5),
        target_operon="mexAB-oprM", method="allelic_exchange_pEX18Ap")
   validate_knockout(knockout_strains)  # PCR + qRT-PCR confirm no mexB expression
   repeat_checkerboard(knockout_strains)  # specificity control

5. accumulation_assay:
   for isolate in sample(isolate_bank, n=15):
       intracellular_drug = LC_MS_MS_uptake(isolate, drug="meropenem",
                                              condition=["±EPI"])
       correlate(intracellular_drug, MIC_shift)

6. statistical_model = logistic_regression(
        outcome="MIC_reversal_binary",
        predictors=["mechanism_class", "mexB_expression_foldchange", "ST_type"])

7. validate_success_thresholds(checkerboard_results, statistical_model)
8. external_replication(second_lab_cohort, protocol=identical)