Analysis of drug resistance, virulence phenotype, and genetic sequences in microevolutionary strains of Pseudomonas aeruginosa MPAO1

The Pseudomonas aeruginosa PAO1 strain is a foundation of research on bacterial virulence and antibiotic resistance. However, its tendency for microevolution during laboratory culture can lead to genetic and phenotypic divergence, potentially compromising experimental reproducibility. This study aimed to systematically characterize such variations in laboratory-maintained MPAO1 sublines to assess their genetic stability and suitability for research. We identified two distinct MPAO1 sublines (MPAO1-P and MPAO1-M) with divergent phenotypes. MPAO1-M exhibited markedly increased antimicrobial susceptibility to multiple antibiotics including ciprofloxacin, imipenem, gentamicin and chloramphenicol, while concurrently displaying enhanced production of key virulence factors, including pyocyanin, rhamnolipids, elastase, and twitching motility. Whole-genome resequencing uncovered a novel missense mutation in the mexT gene of MPAO1-M. Consistent with this finding, quantitative reverse transcription PCR analysis revealed a significant downregulation of the mexEF-oprN efflux pump operon and a marked upregulation of the quorum-sensing genes rhlI and pqsA. Our findings confirm the critical impact of microevolution on MPAO1 genotype and phenotype, underscoring the necessity of strain verification in experimental design. We further identify a novel mexT mutation as a potential mechanistic driver of these changes, providing new insights into the genetic basis of adaptive evolution in laboratory P. aeruginosa strains.