Characterization of two exoU+/exoS+ carbapenem-non-susceptible Pseudomonas aeruginosa co-colonizing the lung of a bacterial pneumonia patient

Abstract Background Pseudomonas aeruginosa is a major cause of acute nosocomial infections, as well as chronic respiratory infections associated with cystic fibrosis (CF). In chronic lung infections, P. aeruginosa populations typically exhibit extensive phenotypic variation, a trait linked to their need to undergo pathoadaptive mutations to counteract host-derived selective pressures. Methods In this study, two clonally related P. aeruginosa isolates, SCPA07 and SCPA08, were identified to coexist in a single bronchoalveolar lavage fluid (BALF) sample from a patient with bacterial pneumonia. Whole-genome sequencing (WGS) was conducted to characterize their genetic background, as well as antimicrobial resistance and virulence gene profiles. A comprehensive analysis of phylogenetic relationships and comparative genomic features of the two isolates was conducted using a panel of bioinformatics tools. Their antimicrobial resistance mechanisms were elucidated via gene sequence analysis and quantitative reverse-transcription PCR (qRT-PCR). A series of phenotypic experiments, including growth, biofilm formation, environmental stress, and virulence assays, and others were performed to characterize their phenotypic traits. Results Antimicrobial susceptibility assay showed that both strains were carbapenem-non-susceptible (defined as intermediate or resistant according to the Clinical and Laboratory Standards Institute (CLSI) guidelines). Genomic analysis revealed that they are ‘hypermutator’ strains and harbor both the exoS and exoU virulence genes, indicating an increased propensity for persistent host infection and high virulence potential. Both strains harbored mutations in oprD , and exhibited elevated expression of AmpC β-lactamase and the efflux pump MexB, which most likely contributed to their non-susceptibility to carbapenems. Despite harboring nucleotide variations at only ten genetic loci, these two strains exhibited distinct phenotypic traits: SCPA07 showed rapid growth, strong biofilm formation, high virulence, and growth advantages under iron limitation and serum stress; in contrast, its variant SCPA08 had slow growth, poor motility, reduced pyocyanin production, low virulence, and increased tolerance to the host antimicrobials human cathelicidin LL-37 and hydrogen peroxide (H 2 O 2 ). These phenotypic variations are proposed to be primarily driven by genetic mutations affecting the O-antigen biosynthesis, iron utilization, Porin D, and other determinants. Conclusions This study elucidates the divergent adaptive evolutionary strategies of a single exoS + / exoU + P. aeruginosa clone within the host during bacterial pneumonia, as well as their critical role in shaping the bacterium’s virulence and adaptability, which sheds light on the within-host evolution dynamics of P. aeruginosa populations during their pathogenesis and persistence in the lung.