Enterobacter cloacae Complex Isolates Harboring blaNMC-A or blaIMI-Type Class A Carbapenemase Genes on Novel Chromosomal Integrative Elements and Plasmids

Abstract

Carbapenem-resistant Enterobacter cloacae complex isolates submitted to a reference laboratory from 2010 to 2015 were screened by PCR for seven common carbapenemase gene groups, namely, KPC, NDM, OXA-48, VIM, IMP, GES, and NMC-A/IMI. Nineteen of the submitted isolates (1.7%) were found to harbor Ambler class A bla_NMC-A or bla_IMI-type carbapenemases. All 19 isolates were resistant to at least one carbapenem but susceptible to aminoglycosides, trimethoprim-sulfamethoxazole, tigecycline, and ciprofloxacin. Most isolates (17/19) gave positive results with the Carba-NP test for phenotypic carbapenemase detection. Isolates were genetically diverse by pulsed-field gel electrophoresis macrorestriction analysis, multilocus sequence typing, and hsp60 gene analysis. The genes were found in various Enterobacter cloacae complex species; however, bla_NMC-A was highly associated with Enterobacter ludwigii Whole-genome sequencing and bioinformatics analysis revealed that all NMC-A (n = 10), IMI-1 (n = 5), and IMI-9 (n = 2) producers harbored the carbapenemase gene on EludIMEX-1-like integrative mobile elements (EcloIMEXs) located in the identical chromosomal locus. Two novel genes, bla_IMI-5 and bla_IMI-6, were harbored on different IncFII-type plasmids. Enterobacter cloacae complex isolates harboring bla_NMC-A/IMI-type carbapenemases are relatively rare in Canada. Though mostly found integrated into the chromosome, some variants are located on plasmids that may enhance their mobility potential.

Keywords: carbapenemase.

FIG 1

Phylogenetic analysis of the E. cloacae complex isolates in this study with other selected strains obtained from GenBank, based on concatenated MLST targets (A) or on the partial hsp60 gene sequences (B). New STs determined in this study are circled, and the cluster (roman numerals) as based on partial hsp60 sequences is in parentheses (A) or boxed (B). Concatenated MLST target sequences were obtained from directly from the E. cloacae MLST website (pubmlst.org/ecloacae/) or from the following sequences using that website: E. cloacae cloacae ATCC 13047 (CP001918), E. cloacae EcWSU1 (CP002886), E. cloacae MGH85 (LETE00000000), E. hormaechei oharae SMART_488 (LPPY00000000), E. hormaechei steigerwaltii SMART_503 (LPPX00000000), E. hormaechei ATCC 49162 (AFHR00000000), E. ludwigii EN119 (JTLO00000000), E. cloacae dissolvens SDM (CP003678), E. cloacae UCI 39 Hof_IV (JCKQ00000000), E. asburiae ATCC 35953 (CP0011863), E. kobei SMART_635 (LPPL00000000), and E. ludwigii AOUC-8/14 (LGIV00000000). hsp60 gene sequences were extracted from the WGS data generated for this study or from the above sequences and the Enterobacter sp. strain R7 hsp60 partial sequence (FJ595742) and the E. cloacae 50588862 draft genome (LNHM00000000). Scale bars are nucleotide substitutions per site.

FIG 2

Schematic diagram of the EcloIMEXs characterized from the isolates in this study. Homologous regions are indicated by gray shading, with the percent identity boxed. Putatively defective insertion sequences preceded by a “d.” The xerC/xerD recombination sites defining the left (dif-L) and right (dif-R) chromosomal/IMEX junctions and the internal dif-Lrec site are shown at the bottom, with nucleotides identical to those for EcloIMEX-1 indicated as dashes. See Table S1 in the supplemental material for accession numbers.

FIG 3

Schematic diagram of plasmid pIMI-5 (KX858825). Genes with a putative function are labeled with a gene designation or protein function (white boxes), insertion sequences or remnants of insertion sequences are depicted as black boxes, and hypothetical protein genes are depicted as gray boxes. Boxes on the outside are transcribed clockwise, and those on the inside are transcribed counterclockwise.

FIG 4

Schematic diagram of pIMI-6 (accession no. KX786187). Labeling is as for Fig. 3.