Comparative in vivo evaluation of colistin resistance determinants in Escherichia coli clone ST131

Background Colistin is a last-resort antimicrobial for multidrug-resistant (MDR) Gram-negative infections. Health risks associated with colistin resistance are commonly inferred from resistance phenotypes, despite limited in vivo evidence linking resistance determinants to pathogenic outcomes. Whether distinct colistin resistance determinants confer differential clinical risks remains unknown. Methods Using the MDR Escherichia coli ST131 clone, we constructed isogenic mutants harbouring representative plasmid-mediated ( mcr -encoding plasmids) or chromosomal ( pmrAB ) colistin resistance determinants. We systematically evaluated antimicrobial susceptibility, in vivo fitness, virulence, and therapeutic efficacy using murine infection models in female BALB/c mice. Transcriptomic, biochemical, and genetic analyses were performed to identify determinant-specific pathogenic mechanisms. Findings Clinical risk associated with colistin resistance was highly determinant-specific. Acquisition of pIncI2_ mcr-1 , pIncP1_ mcr-3 , or pmrA mutations significantly attenuated in vivo fitness and virulence, whereas pIncFII_ mcr-5 and pmrB mutations preserved pathogenicity and caused marked colistin treatment failure. We identified two plasmid-encoded virulence attenuation factors, VafA and VafB, carried on pIncI2_ mcr-1 . These factors interacted with adenylate cyclase (CyaA), reprogrammed the ST131 transcriptome, suppressed purine biosynthesis, and reduced bacterial growth and virulence independently of colistin resistance itself. Interpretation Health risks of colistin-resistant E. coli cannot be inferred from resistance phenotypes alone. Determinant-specific in vivo evaluation reveals clinically important heterogeneity in pathogenic potential and treatment outcomes. Surveillance and risk assessment frameworks should prioritise resistance determinants that preserve virulence and compromise last-line therapy, rather than treating colistin resistance as a uniform clinical threat.