RP Substitution in the PmrB HAMP Domain Contributes to Colistin Heteroresistance in Escherichia coli Isolates from Swine.

Here, the mechanisms of colistin heteroresistance (CHR) were assessed in 12 isolates from swine in China. CHR was investigated by population analysis profile tests. CHR stability was studied by culturing isolates for five overnight incubation periods in colistin-free medium. Subsequently, the gene and mutations in PmrAB, PhoPQ, and MgrB were screened in parental isolates and resistant subpopulations. Additionally, the expression levels of , its target gene , and its negative regulator gene , as well as and its target genes and , were determined by real-time relative quantitative PCR. Eleven of the 12 isolates were confirmed to show CHR, with 17 resistant subpopulations. Also, 11 of the 17 subpopulations (64.71%) harbored point mutations in PmrB and/or PhoQ, differing from their parental isolates. However, only one stable resistant subpopulation (EPF42-4) was identified; it harbored an arginine-to-proline substitution at position 93 (RP) within the PmrB HAMP (histidine kinase, adenylyl cyclase, methyl-binding protein, and phosphatase) domain. Compared to the expression levels in the parental isolate EPF42 and K-12 MG1655, remarkable overexpression was observed in EPF42-4, which showed upregulated , , and expression. Structural analysis demonstrated that the RP substitution promotes conformational changes in the HAMP domain, leading to an acceleration in its signal transduction ability and the activation of PmrB expression. In conclusion, point mutations in PmrB and/or PhoQ were primarily associated with CHR. The RP substitution resulted in the establishment of stable resistant subpopulations in showing CHR.