Objective: The spread of the plasmid-mediated colistin resistance gene mcr-1  poses a significant public health threat. Little information is available on the development of high-level colistin-resistant mutants (HLCRMs) in MCR-1-producing Escherichia coli  (MCRPEC). The present study was designed to evaluate the impact of chromosomal modifications in pmrAB, phoPQ , and mgrB  combined with mcr-1  on colistin resistance in E. coli .
Methods: Five MCRPEC and three non-MCRPEC (E. coli  ATCC25922 and two plasmid-curing) strains were used. The HLCRMs were selected through multi-stepwise colistin exposure. Moreover, two E. coli  C600-pMCRs were constructed and used for selection of HLCRMs. Further analysis included mutation rates and DNA sequencing. Transcripts of pmrABC, phoP, mgrB , and mcr-1  were quantified by real-time quantitative PCR.
Results: All tested HLCRMs were successfully isolated from their parental strains. Non-MCRPEC strains had higher minimum inhibitory concentrations (MICs) and mutation rates than MCRPEC strains. Nineteen amino acid substitutions were identified: seven in PmrA, six in PmrB, one in PhoP, three in PhoQ, and two in MgrB. Most were detected in non-MCRPEC strains. Sorting Intolerant From Tolerant predicted that four substitutions, PmrA Gly15Arg, Gly53Arg, PmrB Pro94Gln, and PhoP Asp86Gly, affected protein function. Two HLCRM isolates did not show amino acid substitutions in contrast to their parental MCRPEC isolates. No further mutations were detected in the second- and third-step mutants. Further transcriptional analysis showed that the up-regulation of pmrCAB  expression was greater in the mutant of E. coli  C600 than in E. coli  C600-pMCR.
Conclusion: Acquisition of the mcr-1  gene had a negative impact on the development of HLCRMs in E. coli , but was associated with low-level colistin resistance. Thus, colistin-based combination regimens may be effective against infections with MCR-1-producing isolates.
Keywords: mutation, mcr-1 , chromosomal resistance mechanisms, high-level colistin resistance, Escherichia coli