Detection of Five <i>mcr-9</i> -Carrying <i>Enterobacterales</i> Isolates in Four Czech Hospitals

Abstract

The aim of this study was to report the characterization of the first <i>mcr</i>-positive <i>Enterobacterales</i> isolated from Czech hospitals. In 2019, one <i>Citrobacter freundii</i> and four <i>Enterobacter</i> isolates were recovered from Czech hospitals. The production of carbapenemases was examined by a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) imipenem hydrolysis assay. Additionally, bacteria were screened for the presence of carbapenemase-encoding genes and plasmid-mediated colistin resistance genes by PCR. To define the genetic units carrying <i>mcr</i> genes, the genomic DNAs of <i>mcr</i>-carrying clinical isolates were sequenced on the PacBio Sequel I platform. Results showed that all isolates carried <i>bla</i>_VIM- and <i>mcr</i>-like genes. Analysis of whole-genome sequencing (WGS) data revealed that all isolates carried <i>mcr-9</i>-like alleles. Furthermore, the three sequence type 106 (ST106) <i>Enterobacter hormaechei</i> isolates harbored the <i>bla</i>_VIM-1 gene, while the ST764 <i>E. hormaechei</i> and ST95 <i>C. freundii</i> included <i>bla</i>_VIM-4 Analysis of plasmid sequences showed that, in all isolates, <i>mcr-9</i> was carried on IncHI2 plasmids. Additionally, at least one multidrug resistance (MDR) region was identified in each <i>mcr-9</i>-carrying IncHI2 plasmid. The <i>bla</i>_VIM-4 gene was found in the MDR regions of p48880_MCR_VIM and p51929_MCR_VIM. In the three remaining isolates, <i>bla</i>_VIM-1 was localized on plasmids (∼55 kb) exhibiting <i>repA</i>-like sequences 99% identical to the respective gene of pKPC-CAV1193. In conclusion, to the best of our knowledge, these 5 isolates were the first <i>mcr-9</i>-positive bacteria of clinical origin identified in the Czech Republic. Additionally, the carriage of the <i>bla</i>_VIM-1 on pKPC-CAV1193-like plasmids is described for the first time. Thus, our findings underline the ongoing evolution of mobile elements implicated in the dissemination of clinically important resistance determinants.<b>IMPORTANCE</b> Infections caused by carbapenemase-producing bacteria have led to the revival of polymyxins as the "last-resort" antibiotic. Since 2016, several reports describing the presence of plasmid-mediated colistin resistance genes, <i>mcr</i>, in different host species and geographic areas were published. Here, we report the first detection of <i>Enterobacterales</i> carrying <i>mcr</i>-9-like alleles isolated from Czech hospitals in 2019. Furthermore, the three ST106 <i>Enterobacter hormaechei</i> isolates harbored <i>bla</i>_VIM-1, while the ST764 <i>E. hormaechei</i> and ST95 <i>Citrobacter freundii</i> isolates included <i>bla</i>_VIM-4 Analysis of WGS data showed that, in all isolates, <i>mcr-9</i> was carried on IncHI2 plasmids. <i>bla</i>_VIM-4 was found in the MDR regions of IncHI2 plasmids, while <i>bla</i>_VIM-1 was localized on pKPC-CAV1193-like plasmids, described here for the first time. These findings underline the ongoing evolution of mobile elements implicated in dissemination of clinically important resistance determinants. Thus, WGS characterization of MDR bacteria is crucial to unravel the mechanisms involved in dissemination of resistance mechanisms.