Transcriptional regulation drives heteroresistance in <i>Pseudomonas aeruginosa</i> : evidence from genomic and targeted gene expression analysis

ABSTRACT Bacterial heteroresistance, characterized by the presence of antibiotic-resistant subpopulations within phenotypically susceptible bacterial colonies, poses significant challenges to clinical antimicrobial susceptibility testing and treatment outcomes. While heteroresistance has been increasingly recognized across diverse bacterial species, the underlying mechanisms in Pseudomonas aeruginosa remain incompletely understood, particularly regarding carbapenem and monobactam resistance. In this study, we employed a multi-omics approach that integrates phenotypic screening, whole-genome sequencing, core genome multilocus sequence typing, and quantitative transcriptomic analysis to systematically investigate the phenotypic characteristics and mechanistic basis of heteroresistance in 38 non-duplicative clinical Pseudomonas aeruginosa samples. The findings reveal a high prevalence of heteroresistance among clinical isolates, and it is difficult to detect using currently commonly used automated detection methods, such as the VITEK 2 system. The comparative genomic analysis demonstrated that resistant and non-resistant subpopulations exhibit identical resistance gene repertoires, with no detectable genetic mutations, gene amplification, or plasmid acquisition. Instead, we identify differential expression of key resistance genes ( mexA , ampC, and oprD ) as the primary driver of heteroresistance, with the two-component system ParS/ParR negatively correlating with oprD expression ( r = −0.797, P &lt; 0.05). Furthermore, phenotypic validation using efflux pump and AmpC inhibitors substantiated the functional contribution of these mechanisms. Our findings indicate that Pseudomonas aeruginosa utilizes dynamic transcriptional regulation rather than genetic alteration to evade antibiotic pressure, representing a distinct evolutionary strategy for survival under antimicrobial stress. IMPORTANCE The clinical implications of our findings are profound. The stable inheritance of HR phenotypes implies that resistant subpopulations can persist as cryptic reservoirs within susceptible populations, evading routine antimicrobial susceptibility testing and potentially leading to relapse or treatment failure. Moreover, the fact that HR is driven by transcriptional rather than genetic mechanisms means that standard molecular diagnostics targeting resistance genes would fail to identify these strains. This highlights the urgent need for diagnostic approaches that capture functional resistance phenotypes or transcriptional signatures.