In vitro exposure to non-antipseudomonal antibiotics (NAPA) induces Pseudomonas aeruginosa resistance to antipseudomonal antibiotics (APA)

Background Pseudomonas aeruginosa readily evolves antimicrobial resistance through regulatory plasticity and stress-adaptive pathways. Clinically, antibiotics lacking intrinsic antipseudomonal activity are often favored with the assumption that they avoid selective pressure on P. aeruginosa . Whether subinhibitory exposure to such “non-antipseudomonal antibiotics” (NAPA) can nevertheless select for canonical resistance pathways remains incompletely defined. Methods: Three P. aeruginosa strains (ATCC 27853 and two bloodstream isolates) were serially passaged over 14 days in the presence of ertapenem, ceftriaxone, or moxifloxacin at one-third the baseline minimum inhibitory concentration (MIC). MICs for antipseudomonal antibiotics (meropenem, ceftazidime, ciprofloxacin) were measured at serial time points and after a 3-day antibiotic-free recovery (day 14). Whole-genome sequencing was performed longitudinally to identify mutations. Results NAPA exposure led to reproducible elevations in antipseudomonal MICs: ertapenem triggered up to a 29-fold increase in meropenem MIC, ceftriaxone up to a 31-fold rise in ceftazidime MIC, and moxifloxacin up to a 12-fold increase in ciprofloxacin MIC. Elevated MICs persisted on day 14 despite absence of further antibiotic pressure. Genomic analysis revealed convergent evolution of mutations in efflux regulator genes ( nfxB, nalC, nalD, amrR ) and the β-lactamase–regulating gene dacB , emerging during periods of MIC escalation and mapping to regulatory pathways governing efflux and AmpC expression. Conclusion Subinhibitory exposure to antibiotics without intrinsic antipseudomonal activity reproducibly selected for heritable multidrug-resistant phenotypes in P. aeruginosa . Resistance arose through convergent evolution in regulatory genes classically associated with direct antipseudomonal antibiotic pressure, demonstrating that resistance architectures can be selected independent of target engagement and underscoring the inevitability of collateral resistance under antibiotic stress. Importance Pseudomonas aeruginosa is a major cause of hospital-acquired infections and is well known for its ability to develop antibiotic resistance. Clinicians often assume that antibiotics without activity against P. aeruginosa do not meaningfully influence its resistance behavior and are therefore safe choices when this organism is not the primary target. Our study challenges this assumption. We show that low-level exposure to such antibiotics is associated with increased resistance to key antipseudomonal drugs, even after the initial antibiotic exposure ends. Rather than arising from a single stable genetic change, resistance was accompanied by shifting genetic alterations in regulatory pathways that control drug efflux and β-lactamase expression. These findings highlight how antibiotic exposure can broaden the evolutionary pathways available for resistance in unintended pathogens. Recognizing these indirect and population-level effects of antibiotic use may help inform more cautious antimicrobial prescribing strategies in clinical settings.