Molecular antibiotic resistance mechanisms and co-transmission of the mcr-9 and metallo-β-lactamase genes in carbapenem-resistant Enterobacter cloacae complex

Abstract

Carbapenem-resistant <i>Enterobacter cloacae</i> complex (CRECC) has increasingly emerged as a major cause of healthcare-associated infections, with colistin being one of the last-resort antibiotics of treatment. Mobile colistin resistance (<i>mcr</i>)<i>-9</i> is a member of a growing family of <i>mcr</i> genes and has been reported to be an inducible gene encoding an acquired phosphoethanolamine transferase. Here, we collected 24 ECC strains from Chongqing, China from 2018 to 2021. Subsequently, antibiotic resistance genes and the transmission dynamics of the strains were determined by PCR, whole-genome sequencing, and bioinformatic analysis. The <i>mcr-9</i> was identified in IncHI2/2A or IncHI2/2A + IncN plasmids from six CRECC strains and was co-located with <i>bla</i> _NDM-1 or <i>bla</i> _IMP-4 in 2/6 plasmids. The genetic environment of <i>mcr-9.1</i> was composed of IS<i>903B</i>-<i>mcr-9.1</i>-<i>wbuC</i>-IS<i>26</i> in the five <i>mcr-9.1</i>-harboring-plasmid, but IS<i>1B</i> was located downstream of <i>mcr-9.2</i> in the pECL414-1 sequence. We also found that the pNDM-068001 plasmid carrying <i>mcr-9.1</i> could be a hybrid plasmid, formed by a Tn<i>6360</i>-like <i>bla</i> _NDM-1 region inserted into an <i>mcr-9.1</i>-positive IncHI2/2A plasmid. A conjugation assay showed that plasmids mediated the co-dissemination of <i>mcr-9</i> and metallo-β-lactamase (MBL) genes. In addition, we performed induction assays with sub-inhibitory concentrations of colistin and found an increase in the relative expression levels of the <i>mcr-9.2</i>, <i>qseC</i>, and <i>qseB</i> genes, as well as an increase in the minimum inhibitory concentration values of colistin in the CRECC414 strain. These findings provide a basis for studying the regulatory mechanisms of <i>mcr-9</i> expression and highlight the importance of effective monitoring to assess the prevalence of MBL and <i>mcr-9</i> co-existing plasmids.