Parallel microevolution of multidrug resistance and hypermucoviscosity in two carbapenem-resistant Pseudomonas aeruginosa clinical isolates with strikingly divergent virulence outcomes

Multidrug-resistant (MDR), and mucoid Pseudomonas aeruginosa strains are relatively uncommon in clinical settings, and genomic data concerning such strains are particularly scarce, which limits the comprehensive understanding of this group of opportunistic pathogens. This study systematically characterized two clinically isolated, carbapenem-resistant, MDR, and highly mucoid P. aeruginosa strains, designated YL-1 and YL-2. We employed whole-genome sequencing, comparative genomics, and quantitative PCR (qPCR) to investigate the mechanisms of multidrug resistance and other phenotypic variations. Their pathogenicity was further compared using a Galleria mellonella infection model. Neither YL-1 nor YL-2 harbors plasmids; all resistance and virulence associated genes were located on their chromosomes. Only 9 genomic sites differ between the two strains, suggesting they derived from a common ancestor but have diverged evolutionarily. In the phylogenetic tree, both strains were located in the evolutionary branch containing the hypervirulent reference strain PA14. Mutations in oprD and AmpC regulatory element, and overexpressed efflux pumps contributed to their multidrug resistance, particularly to carbapenems. In YL-2, a frameshift mutation inactivated gacS , a key virulence regulator, which likely accounts for its significantly lower pyocyanin production compared to YL-1. The mortality rate of G. mellonella larvae infected with YL-1 was significantly higher than that of YL-2, indicating that YL-1 is more virulent. This study provides a comprehensive genomic and phenotypic analysis of two MDR hypermucoid P. aeruginosa strains, shedding light on their in vivo microevolution. These findings offer the theory for the clinical management and control of hypermucoid P. aeruginosa infections.