Genomic Characterization of Enterobacter cloacae Isolates from China That Coproduce KPC-3 and NDM-1 Carbapenemases

Abstract

c Here, we report two Enterobacter cloacae sequence type 231 isolates coproducing KPC-3 and NDM-1 that have caused lethal infections in a tertiary hospital in China. The bla NDM-1 -harboring plasmids carry IncA/C 2 and IncR replicons, showing a mosaic plasmid structure, and the bla NDM-1 is harbored on a novel class I integron-like element. bla KPC-3 is located on a Tn3-⌬bla TEM-1 -bla KPC-3 -⌬Tn1722 element, flanked by two 9-bp direct-repeat sequences and harbored on an IncX6 plasmid. Carbapenemase-producing Enterobacteriaceae (CRE) strains have spread worldwide and have become a significant public health threat (1, 2). It is alarming that CRE isolates coproducing multiple carbapenemases recently emerged and pose significant challenges to our limited treatment strategies, as these bacteria tend to be extremely highly resistant (3-10). To date, the coexistence of Klebsiella pneumoniae carbapenemase 2 (KPC-2) and New Delhi metallo-␤-lactamase-1 (NDM-1) in single clinical isolates from different species, including K. pneumoniae, Enterobacter cloacae, Enterobacter hormaechei, and Citrobacter freundii, has been reported in Brazil, India, Pakistan,8,9). However, knowledge regarding the genetic content and plasmid structure of these multicarbapenemase-producing bacteria remains limited (3,6). In this study, we used next-generation sequencing to characterize the genomes of two KPC-3-and NDM-1-coproducing E. cloacae strains and their plasmids. Our analysis revealed distinct plasmid structures that, to our knowledge, have not been described previously.Two carbapenem-resistant E. cloacae strains (SZECL1 and SZECL2) were identified in a retrospective study on CRE at a tertiary-care hospital in China. They were isolated from sputum samples from two female patients (63 and 57 years old) admitted to the same intensive care unit in 2012. The two isolates were collected 11 days apart. The patients died 13 and 18 days after the isolation of the organisms as a result of severe pulmonary infections. Antimicrobial susceptibility testing showed that the two isolates, SZECL1 and SZECL2, had the same resistance profile (Table 1). They were resistant to all ␤-lactams and inhibitors, including imipenem, meropenem, ertapenem, and ceftazidime-avibactam, and were resistant to amikacin, gentamicin, tobramycin, ciprofloxacin, levofloxacin, moxifloxacin, and trimethoprim-sulfamethoxazole but remained susceptible to tetracycline, tigecycline, and colistin.Genotyping results showed that SZECL1 carries bla KPC-3 , bla NDM-1 , and bla , while SZECL2 harbors the same ␤-lactamase genes (bla , bla NDM-1 , and bla ) as SZECL1, with an additional bla CTX-M-15 gene (11,12). Multilocus sequence typing indicated that the two isolates were of the same sequence type, ST231 (13), and they displayed indistinguishable patterns in pulsed-field gel electrophoresis analysis, suggesting that these two isolates are closely related and may have evolved from the same ancestor.The bla KPC-3 -harboring plasmids in SZECL1 and SZECL2 were successfully transferred to...

Identification of IncA/C Plasmid Replication and Maintenance Genes and Development of a Plasmid Multilocus Sequence Typing Scheme

“…ST1 is strongly associated with the carriage of bla NDM (15/20 plasmids; 75%), while only two plasmids outside ST1 carry bla NDM (one each from ST3 and ST6). Comparison of the bla NDM genetic location has shown multiple genetic organizations (41)(42)(43)(44)(45)(46). Within our database, we observed a total of seven distinct bla NDM structures (see Fig.…”

The rapid spread of carbapenem-resistant Enterobacteriaceae

“…A K. pneumoniae isolate obtained in the United States in 2013 from the high risk ST258 clade expressed plasmid borne bla KPC-2 and bla VIM-4 [Castanheira 2016]. An E. cloacae ST231 from China in 2012 was found to harbor bla NDM-1 on IncA/C2 mosaic plasmid and bla KPC-3 on a novel IncX6 plasmid [Du 2016]. These same resistance genes were found on two different IncF plasmids in an E. cloacae isolate from China in April 2014, further increasing the likelihood that a recombination event could create a dual carbapenemase plasmid [Wu 2015].…”

“…The first characterization of a bla IMI-1 producing, colistin resistant E. cloacae from the United States in February 2015 showed absence of mcr-1 and canonical chromosomal modifications, suggesting that Enterobacteriaceae contain other colistin resistance mechanisms [Norgan 2016]. A retroactive study on colistin-resistant Enterobacteriaceae isolates obtained from January 2013 to November 2015 at a hospital in China found two K. pneumoniae harboring mcr-1 and bla NDM-5 [Du 2016]. Four bla KPC-2 producing K. pneumoniae ST101 from a cohort of nosocomial CRE obtained in Italy from November 2013 to August 2014 had colistin resistance (MIC 16–125 μg/ml) through an unidentified mechanism [Del Franco 2015].…”

IncX2 and IncX1-X2 Hybrid Plasmids Coexisting in a FosA6-Producing Escherichia coli Strain

“…Recently, Chen et al and Du et al reported bla KPC -bearing IncX5 and IncX6 plasmids (pBK31567, GenBank accession number JX193302.1; pKPC3_SZ, KU302800.1), classified by taxC genes and conserved backbone sequences, which are now included in PlasmidFinder as IncX5 and IncX6 plasmids on the basis of pir genes (Fig. S2) (4,6). We therefore performed a phylogenetic analysis to compare the backbone genes taxC, pir, and bis of 33 IncX plasmids, including pYD786-4, pYD786-3, plasmid C, pMCR1_IncX4, pNDM5_IncX3, pCQ02-121, pESTMCR (IncX4; GenBank accession number KU743383.1), pHS696_34 (IncX4; JX258654.1), pJARS35 (IncX7; NC_015054.1), pHI4320 (IncX8; AM942760.1), and 23 IncX plasmids, as described by Du et al (6).…”

“…S2) (4,6). We therefore performed a phylogenetic analysis to compare the backbone genes taxC, pir, and bis of 33 IncX plasmids, including pYD786-4, pYD786-3, plasmid C, pMCR1_IncX4, pNDM5_IncX3, pCQ02-121, pESTMCR (IncX4; GenBank accession number KU743383.1), pHS696_34 (IncX4; JX258654.1), pJARS35 (IncX7; NC_015054.1), pHI4320 (IncX8; AM942760.1), and 23 IncX plasmids, as described by Du et al (6). All of them were subtyped based on the criteria of PlasmidFinder, with four reference bis genes for the IncX1 subgroup and nine reference pir genes for the subgroups IncX2 to IncX8.…”