Acquired mutations in transcriptional regulator <i>hns</i> and lipopolysaccharide biosynthesis and modification genes impact colistin resistance and fitness of <i>mcr</i> -carrying <i>E. coli</i>

ABSTRACT Gram-negative bacteria can develop resistance to the last-resort antimicrobial colistin through the acquisition of mobile colistin resistance ( mcr ) genes or chromosomal mutations. Bacteria carrying mcr variants do not always have colistin minimum inhibitory concentrations (MICs) above the clinical breakpoint, and there is a gap in understanding how mcr -carrying bacteria adapt to colistin selective pressure. To address this gap, we propagated Escherichia coli strains carrying mcr-3 or mcr-9 , which have been reported to confer different levels of colistin resistance and fitness costs, and a control strain lacking an mcr gene in the presence of increasing concentrations of colistin. While the mcr-9 and control strains maintained their baseline colistin MICs, the colistin MIC of the strain expressing mcr-3 increased, alongside acquisition of nonsynonymous mutations in genes involved in lipopolysaccharide biosynthesis and modification. Analysis of individual mcr-3 strains that evolved during the experiment revealed that mutants exhibited improved competitive performance under colistin selection, but showed reduced fitness in standard media, or in the presence of human serum. Nonsynonymous lpxC and hns mutations were significant predictors of overall fitness and bacterial growth, but were infrequent among wild-type E. coli isolates, and even less frequent in mcr- positive wild-type E. coli . Our results suggest that mcr -expressing E. coli can evolve to show increased colistin MICs; however, fitness costs may prevent these mutations from being established in natural populations. Moreover, E. coli might control mcr-3 expression through the nucleoid protein H-NS, providing novel insights into bacterial regulation of mcr expression.