Colistin heteroresistance in <i>Enterobacter</i> due to base heterozygosity at certain <i>phoP</i> and <i>phoQ</i> locations

Colistin heteroresistance (CHR) is a growing concern in clinical settings. We aimed to investigate CHR for its prevalence in <i>Enterobacter</i> strains, its impact on colistin treatment, and its genetic mechanisms. We analyzed 109 non-duplicated <i>Enterobacter</i> strains isolated from blood cultures. Minimum inhibitory concentrations (MICs) of colistin for each strain were determined using microdilution and CHR was assessed by population analysis profile (PAP) assays. <i>In vitro</i> time-killing assays and <i>in vivo</i> murine intra-abdominal infection models were conducted to evaluate whether CHR contributes to colistin treatment failure. Whole genome sequencing and single nucleotide polymorphism (SNP) analysis were performed to uncover the genetic mechanisms associated with CHR, which were verified using cloning experiments. About 30% of colistin-susceptible <i>Enterobacter</i> strains exhibited CHR, which indeed increased treatment failures. Novel base alterations in the two-component system gene <i>phoP</i> or <i>phoQ</i> were identified as the mechanism for colistin resistance. The presence of such colistin-resistance-mediated base alterations in minor subpopulations of the same strain was detected, which generates base heterozygosity resulting in heterogeneity of colistin resistance. Colistin resistance can be mediated by various mutations in the same strain after exposure to colistin, which provides the flexibility to accommodate antimicrobial selection pressure. In conclusion, these findings allow us to disclose the genetic heterogeneity in CHR <i>Enterobacter</i> strains, which is consistent with "phenotypic heterogeneity" and provides genetic explanations for the dynamic heterogeneous resistance. Our study underscores the clinical significance of CHR and provides important insights and new perspectives into the mechanisms for the heterogeneity of antimicrobial resistance.