High Prevalence of Metallo-β-Lactamase-Producing Enterobacter cloacae From Three Tertiary Hospitals in China

Abstract

<i>Enterobacter cloacae</i> has recently emerged as one of the most common carbapenem-resistant <i>Enterobacteriaceae</i>. The emergence and spread of metallo-β-lactamase-producing <i>E. cloacae</i> have posed an immediate threat globally. Here, we investigated the molecular characteristics of 84 carbapenem-resistant <i>Enterobacter cloacae</i> (CREL) collected from three tertiary hospitals in China between 2012 and 2016. Species identification and antimicrobial susceptibility testing were performed using a VITEK-2 system. Carbapenems, polymyxins B, and tigecycline were tested by broth microdilution method. The carbapenem in activation method (CIM) and cefoxitin three-dimensional test were used to detect carbapenemase and AmpC β-lactamase, respectively. Isolates were screened for β-lactam resistance genes by PCR, and expression of <i>ompC</i> and <i>ompF</i> was determined by qRT-PCR. Genetic relatedness was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), while selected isolates were subjected to whole-genome sequencing. Among the 84 CREL isolates, 50 (59.5%) were detected as carbapenemase producers. NDM-1 was the dominant carbapenemase (80.0%), followed by IMP-26 (8.0%) and IMP-4 (6.0%). Notably, we identified the first NDM-1 and IMP-1 co-producing <i>E. cloacae</i>, carrying plasmids of several incompatibility (Inc) groups, including IncHI2, IncHI2A, and IncN. Most isolates showed decreased expression of <i>ompC</i> and/or <i>ompF</i>, and contained a broad distribution of ESBLs and AmpC β-lactamases. These findings suggested that different molecular mechanisms, including carbapenemase, ESBL and/or AmpC plus loss of porins, have contributed to carbapenem resistance. The <i>bla</i> _NDM-1-harboring plasmids contained highly conserved gene environment around <i>bla</i> _NDM-1 (<i>bla</i> _NDM-1-<i>ble</i> _MBL-<i>trpF</i>-<i>dsbD</i>-<i>cutA1</i>-<i>groES</i>-<i>groEL</i>), which could be associated with the potential dissemination of <i>bla</i> _NDM-1. IMP-type MBL was located within a variety of integrons and usually contained various gene cassettes encoding multidrug resistance. These isolates produced 54 different pulsotypes, and were classified into 42 STs by MLST. Nineteen <i>bla</i> _NDM-1-positive <i>E. cloacae</i> isolates obtained from Ningxia had the same pulsotype (PFGE type 1), belonging to ST78 within clonal complex 74 (CC74). The plasmid-based replicon typing indicated that IncX3 plasmids mediated the dissemination of <i>bla</i> _NDM-1 among these homologous strains. This is the first report on the outbreak of NDM-1-producing <i>E. cloacae</i> ST78 with contribution of IncX3 plasmids in Northwestern China. There's an immediate need to intensify surveillance attentively to prevent and control the further spread of NDM-1 in China.