Co-infections of two carbapenemase-producing Enterobacter hormaechei clinical strains isolated from the same diabetes individual in China

Abstract

<b>Introduction.</b> Since <i>mcr-1</i> was first reported in China, there have been ten variants of MCR appearing nationwide so far. Multidrug-resistant <i>Enterobacteriaceae</i> bacteria carrying both NDM and MCR have become a serious threat to global public health.<b>Hypothesis/Gap Statement.</b> The genetic structure of <i>mcr-9</i> needs to be better understood in order to better prevent and control the transmission of drug-resistant genes.<b>Aims.</b> The aim of this study was to characterize the presence of two <i>Enterobacter hormaechei</i> isolates, which carries <i>bla</i> _NDM-5 CME2 and the coexistence of <i>mcr-9</i> and <i>bla</i> _NDM-1 strain CMD2, which were isolated from a patient with diabetes in Sichuan, China.<b>Methodology.</b> The microbroth dilution method was used for antibiotic susceptibility. Conjugation experiment was used to investigate the transferability of <i>bla</i> _NDM-1, <i>bla</i> _NDM-5 and <i>mcr-9</i>. Whole-genome sequencing was performed on Illumina HiSeq platform. The ability of biofilm formation was detected by crystal-violet staining, the virulence of the bacteria was measured by <i>Galleria mellonella</i> killing assay.<b>Results.</b> <i>bla</i> _NDM-5 carrier CME2 and CMD2 with <i>bla</i> _NDM-1 and <i>mcr-9</i> were resistant to carbapenems, β-lactam, aminoglycoside, quinolone and tetracycline, while CMD2 was also resistant to colistin. Conjugation assay and plasmid replicon typing further demonstrated that both <i>bla</i> _NDM-1 and <i>bla</i> _NDM-5 were respectively present on the self-transferrable IncX3 plasmid, <i>mcr-9</i> was located on the self-transferrable IncHI2 plasmid. Through the analysis of <i>mcr-9</i> gene context, the structure was <i>DUF4942-rcnR-rcnA-copS</i>-IS<i>903-mcr-9-wbuC-qseC-qseB-IS1R</i>-Δ<i>silR</i>-IS<i>903</i>, <i>bla</i> _NDM-1 context was IS<i>3000</i>-ΔIS<i>Aba125</i>-IS<i>5-bla</i> _NDM-1-<i>ble-trpF-groS-groL-insE</i>-ΔIS<i>26</i> structure, <i>bla</i> _NDM-5 structure was IS<i>3000-bla</i> _NDM-5-<i>ble-trpF-dsbC</i>-ΔIS<i>26-umuD</i>-IS<i>Kox3-tnpR-parA</i>. Biofilm formation of CME2 was stronger than CMD2. There was no significant difference in virulence between the two strains.<b>Conclusion.</b> This study reveals two multiple drug-resistant <i>E. hormaechei</i> isolates from diabetes patient samples. <i>E. hormaechei</i> carrying two NDM-resistant genes is already a serious threat, where MCR is an important cause of treatment failure in bacterial infections. This study is a reminder not only to prevent infection in patients with diabetes, but also to constantly monitor the epidemic and spread of the drug-resistant gene.