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The prevalence of carbapenem-resistant  Enterobacteriaceae  (CRE) is increasing globally, with different molecular mechanisms described. Here we studied the molecular mechanisms of carbapenem resistance, including clonal and plasmid dissemination, of 67 CRE isolates collected between 2012 and 2016 from a tertiary hospital in Eastern China, an CRE endemic region. Species identification and susceptibility testing were performed using the BD Phoenix Automated Microbiology System. Isolates were characterized by PCR (for carbapenemases, ESBLs, AmpC and porin genes), multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and conjugation transfer experiments. Selected  bla  KPC-2  -harboring plasmids were subjected to next-generation sequencing using the Illumina Miseq platform. Among the 67 CRE isolates, 42  Klebsiella pneumoniae , 10  Serratia marcescens , 6  Enterobacter cloacae , 2  Raoultella ornithinolytica , 2  K. oxytoca , 1  K. aerogenes , and 4  Escherichia coli  isolates were identified. Six different carbapenemases were detected, including  bla  KPC-2  ( n  = 45),  bla  KPC-3  ( n  = 1),  bla  NDM-1  ( n  = 6),  bla  NDM-5  ( n  = 1),  bla  IMP-4  ( n  = 2), and  bla  VIM-1  ( n  = 2);  bla  OXA-48 -like genes were not detected. One  E. cloacae  strain possessed both  bla  NDM-1  and  bla  KPC-3 , while two  E. cloacae  isolates harbored  bla  NDM-1  and  bla  VIM-1 . ESBLs (CTX-M, SHV, and TEM) and/or AmpC (CMY, DHA, and ACT/MIR) genes were also identified in 59 isolates, including 13 strains that lacked carbapenemases. Several insertions or stop codon mutations were found within porin genes of  K. pneumoniae, E. coli  and  S. marcescens  isolates, both with and without carbapenemases. The 42  K. pneumoniae  isolates belonged to 12 different sequence types (ST), with ST11 being the most common, while the 6  E. cloacae  isolates comprised 4 different STs. The 10  S. marcescens  all shared the same PFGE pulsotype, suggestive of clonal spread. Complete plasmid sequencing and PCR screening revealed both intra-strain and inter-species spread of a common  bla  KPC-2 -harboring plasmid in our hospital. Taken together, our study revealed extensive genetic diversity among CRE isolates form a single Chinese hospital. CRE isolates circulating in the hospital differ significantly in their species, STs, porin genes, carbapenemase genes, and their plasmid content, highlighting the complex dissemination of CRE in this endemic region.

Genetic Diversity of Carbapenem-Resistant Enterobacteriaceae (CRE) Clinical Isolates From a Tertiary Hospital in Eastern China