Genomic characterization of colistin resistance in spp. under the pressure of colistin.

Carbapenem-resistant (CRKP) has become a serious threat to global public health. Colistin is regarded as the last-resort antibiotic for CRKP infections, but colistin resistance among CRKP is increasingly being reported, making clinical treatment for CRKP infections more difficult. The molecular mechanisms of colistin resistance in spp. under the pressure of colistin have not been fully investigated. We aimed to investigate the phenotypic and genetic variation in two colistin-susceptible spp. strains under selective pressure of colistin. One hundred microlitres of overnight cultures of the CRKP clinical strain CRKP12-130 and of ATCC 700603 was spread on five Mueller-Hinton Agar (MHA) plates with colistin concentrations of 2, 4, 8, 16 and 32 µg ml, and growth of colonies was observed for five consecutive days. Colonies collected from plates were passaged daily for 10 days on MHA plates without colistin and susceptibility testing of colistin was performed by broth microdilution. Thirty-four colistin-resistant strains randomly selected were submitted to whole genome sequencing (WGS). Transcriptional levels of genes involved in colistin resistance (, , , , , , and ) were measured by quantitative real-time PCR. A total of 114 and 119 colistin-resistant colonies were obtained from CRKP12-130 and ATCC 700603 in this study, among which 16 and 18 colonies were submitted to WGS, respectively. Among these 34 sequenced isolates, mutation in (13/16, 81.25 %) was the main genetic factor mediating colistin resistance in strains from CRKP12-130, while for strains from ATCC 700603, mutation associated with (8/18, 44.44 %) was found to be the commonest. Mutation of led to a significant increase in the MIC for colistin (from 64 to >128 µg ml), and a novel mutation C28R in was first reported in this study. Colistin-resistant spp. could be easily selected under pressure of different concentrations of colistin. Mutations of , , and genes were the main mechanisms leading to chromosomally mediated colistin resistance in spp.