Putative Integrative Mobile Elements That Exploit the Xer Recombination Machinery Carrying bla_IMI-Type Carbapenemase Genes in Enterobacter cloacae Complex Isolates in Singapore

ABSTRACT

Whole-genome sequencing was performed on 16 isolates of the carbapenemase-producing Enterobacter cloacae complex to determine the flanking regions of bla_IMI-type genes. Phylogenetic analysis of multilocus sequence typing (MLST) targets separated the isolates into 4 clusters. The bla_IMI-type genes were all found on Xer-dependent integrative mobile elements (IMEX). The IMEX elements of 5 isolates were similar to those described in Canada, while the remainder were novel. Five isolates had IMEX elements lacking a resolvase and recombinase.

TEXT

Enterobacter cloacae complex (ECC) isolates producing the carbapenemase IMI-1 were isolated in the United States as early as 1984. However, the flanking regions of the bla_IMI-1 gene, and the mechanism of its mobilization into this species, have remained elusive until only recently (1).

First, Antonelli et al. characterized the Xer-dependent integrative mobile elements (IMEX) responsible for inserting the gene for the closely related carbapenemase NMC-A in members of the ECC (2). Then, Boyd et al., studying isolates in Canada, found that similar IMEX were responsible for mobilizing bla_IMI-type genes into ECC (3).

As a result of an extensive screening program for carbapenemase-producing Enterobacteriaceae, 16 bla_IMI-type-positive ECC were isolated from patients in a large teaching hospital from April 2013 to March 2015. All were isolated from stool or rectal swabs except sample DU20756, which was isolated from urine. MICs to a range of antimicrobials were determined using Etest (bioMérieux, Marcy l'Etoile, France).

DNA was extracted and whole-genome sequencing was performed to determine the flanking regions of the bla_IMI-type genes. The sequencing libraries were prepared using the NEBNext Ultra DNA library prep kit for Illumina (NEB E7370L; NEB Inc., Singapore) and custom primers with an 8-bp barcode index and sequenced on an Illumina HiSeq 4000 (Illumina Inc., San Diego, CA). De novo genome assemblies were created using the Velvet assembler (version 1.2.10) with parameters optimized by Velvet Optimizer (k-mer length ranging from 81 to 127), scaffolded with Opera (version 1.4.1), and finished with FinIS (version 0.3) (4, 5, 6). Genomes were initially annotated with rapid annotation of microbial genomes using subsystems technology (RAST) with default parameters (7). An additional assembly was done using SPAdes (version 3.11.0) using the “-cov-cutoff auto” and “-careful” parameters to validate the junction of two scaffolds for strain DS36577 (8). The Velvet assembly genome sequences (annotated by the GenBank Prokaryotic Annotation Pipeline) and raw sequencing reads have been deposited at GenBank under BioProject PRJNA393769.

Fifteen isolates had bla_IMI-1 and one isolate (DS2686) had bla_IMI-4. The rpoB and hsp60 genes of all sequenced isolates had ≥96% nucleotide identity to the respective ECC genes (2). Detection of antimicrobial resistance genes and multilocus sequence typing (MLST) based on the ECC database (www.pubmlst.org) (9) was performed by SRST2 analysis of the raw sequencing reads (10). Cluster analysis using partial hsp60 sequences (Fig. 1) of the ECC in this study (and selected strains from GenBank) was performed as described by Boyd et al. (3).

FIG 1.

Phylogenetic analysis of the E. cloacae complex isolates in this study with selected reference strains based on concatenated partial hsp60 sequences. Scale bars are nucleotide substitutions per site.

Phylogenetic analysis of the MLST profiles divided the isolates into four groups, A, B, C, and D (Fig. 2). The MICs of antimicrobials tested were consistent with the resistance gene profiles.

FIG 2.

Neighbor-joining tree of isolates with bla_IMI-like genes based on phylogenetic analysis of MLST profiles showing resistance gene profiles and MICs to a range of antimicrobials. AMK, amikacin; TET, tetracycline; CIP, ciprofloxacin; GEN, gentamicin; SXT, sulfamethoxazole-trimethoprim; CRO, ceftriaxone; IPM, imipenem.

The bla_IMI-type genes of isolates in this study were all found within IMEX elements with conserved features similar to those previously described (Fig. 3) (3). The initial Velvet-based assembly of DS36577 placed the IMEX sequence on two different contigs. We mapped the raw reads back to this assembly and found 60 reads that supported joining these two contigs; furthermore, a SPAdes-based assembly resulted in the IMEX element from DS36577 on a single contig that was consistent with the joining of the two Velvet-based contigs. Finally, SPAdes-based assemblies were concordant with the Velvet-based assemblies for all the other strains. Therefore, diagrams in Fig. 4 are drawn based on the SPAdes-based assembly for consistency. The IMEX sequences of strains DS36577, DS15987, DS2686, and DS18454 from cluster D and strain DS05262 from cluster C were more than 94% similar to that of EcloIMEX-2 (3). The IMEX elements of the remaining Singapore isolates were more divergent from those found in Canadian and Japanese isolates, with higher levels of sequence variation found between the resolvase and elongation factor P-like protein (yeiP) genes.

FIG 3.

The xerC/xerD recombination sites defining the left (dif-L) and right (dif-R) chromosomal/IMEX junctions and the internal dif-Lrec site, with nucleotides identical to those for EcloIMEX-1 indicated as dashes.

FIG 4.

Comparison of the genomic region of isolates carrying the bla_IMI-like gene in this study and the corresponding regions of Enterobacter ludwigii AOUC-8/14 EludIMEX-1(KR919803) and E. cloacae EcloIMEX-2 (KR057494). Homologous regions to EcloIMEX-2 are indicated by gray shading, with the percent identity in the gray box on the right.

Notably, five isolates (all three from cluster A, one from cluster B, and one from cluster C) had IMEX elements that lacked a resolvase and recombinase. Importantly, the entire IMEX element in these strains was contained within a single assembled contig, suggesting that the lack of these genes was not due to breaks in the assembly.

In conclusion, we have shown that bla_IMI-type genes in ECC in Singapore are also found within IMEX elements, and we believe this is only the second publication to demonstrate this association. Together with the results of the Canadian study, the chromosomal diversity of the isolates and the heterogeneity of their IMEX elements hint at a potentially large and/or widely distributed environmental source of bla_IMI-type genes.

Accession number(s).

Raw sequencing reads have been deposited at GenBank under Bioproject PRJNA393769.