Prevalence and molecular characteristics of colistin-resistant isolates among clinically isolated carbapenem-resistant Klebsiella pneumoniae in China

Colistin resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP) poses health challenges. To in-vestigate the prevalence and molecular characteristics of colistin-resistant CRKP, 708 isolates were col-lected consecutively from 28 tertiary hospitals in China from 2018 to 2019, and 14 colistin-resistant CRKP were identified. Two-component systems (TCSs) related to colistin resistance (PmrA/B, PhoP/Q, and CrrA/B), the negative regulator mgrB gene and mcr genes, were analysed using genomic sequencing. The relative expression of TCSs genes along with their downstream pmrC and pmrK genes was determined us-ing quantitative real-time PCR (qRT-PCR). A novel point mutation in PhoQ was confirmed by site-directed mutagenesis, and the subsequent transcriptome changes were analysed by RNA sequencing (RNA-Seq). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to detect modifications in lipid A. The results showed that only one isolate carried the mcr -8.1 gene, nine exhibited MgrB inactivation or absence, and three exhibited mutations in PmrB. One novel point mutation, L247P, in PhoQ was found to lead to a 64-fold increase in the minimum inhibitory concen-tration (MIC) of colistin. qRT-PCR revealed overexpression of phoP/Q and pmrK in isolates with or with-out MgrB inactivation, and pmrB mutation resulted in overexpression of pmrA and pmrC. Furthermore, transcriptome analysis revealed that the PhoQ L247P novel point mutation caused upregulated expres-sion of phoP/Q and its downstream operon pmrHFIJKLM. Meanwhile, the pmrA/B regulatory pathway did not evolve colistin resistance. Mass spectrometry analysis showed the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to lipid A in colistin-resistant isolates with absence of MgrB. These findings illustrate that the molecular mechanisms of colistin resistance in CRKP isolates are complex, and that MgrB inacti-vation or absence is the predominant molecular mechanism. Interventions should be initiated to monitor and control colistin resistance.& COPY; 2023 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )