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. 2017 Apr 24;61(5):e02578-16. doi: 10.1128/AAC.02578-16

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

David A Boyd a, Laura F Mataseje a, Ross Davidson b, Johannes A Delport c, Jeff Fuller d,*, Linda Hoang e, Brigitte Lefebvre f, Paul N Levett g, Diane L Roscoe h, Barbara M Willey i, Michael R Mulvey a,✉
PMCID: PMC5404547  PMID: 28223374

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 blaNMC-A or blaIMI-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, blaNMC-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, blaIMI-5 and blaIMI-6, were harbored on different IncFII-type plasmids. Enterobacter cloacae complex isolates harboring blaNMC-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

INTRODUCTION

Enterobacter species bacteria are facultative Gram-negative rods belonging to the Enterobacteriaceae family and are ubiquitous in the environment in soil, sewage, and water and as commensal enteric bacteria in animals and humans (1). Enterobacter cloacae can also cause clinically relevant human infections. The genetically related group “E. cloacae complex” consists of several species, subspecies, and genetic clusters that have been characterized by sequence analysis of the hsp60 gene, multilocus sequence analysis, or comparative genomic hybridization (2–4). Enterobacter spp. are intrinsically resistant to first-generation cephalosporins and variably express an AmpC β-lactamase that confers resistance to third-generation cephalosporins and narrower-spectrum β-lactams. AmpC overproduction via derepression coupled with mutations in porins can lead to carbapenem resistance and thus limit treatment options (5). More worrisome is that, over the last decade, multidrug-resistant (MDR) Enterobacter spp. have emerged due to the acquisition of mobile elements that carry multiple antimicrobial resistance genes, including extended-spectrum β-lactamases (ESBL), such as blaCTX-M-15, and carbapenem-hydrolyzing β-lactamases (i.e., carbapenemases), including KPC, NDM, and OXA-48 types (1). Based on a multilocus sequence typing (MLST) scheme used in multiple studies, a number these MDR E. cloacae isolates from human clinical and animal origins have been assigned to clonal complexes that have appeared in multiple countries and continents (6–8). Less often described are Enterobacter isolates harboring the class A chromosomally encoded NMC-A or IMI-type carbapenemases, although NMC-A from E. cloacae NOR-1, isolated in 1990, was one of the first carbapenemases described in Enterobacteriaceae (9). The NMC-A/IMI group contains a small number of variants that usually vary by only one to a few amino acids (www.lahey.org/studies, www.ncbi.nlm.nih.gov/pathogens/beta-lactamase-data-resources/). Two variants, blaIMI-2 and blaIMI-6 (incorrectly designated blaIMI-3 in reference 10), have been found on self-transferable plasmids in E. cloacae and Escherichia coli (10–13). Recently the blaNMC-A region in Enterobacter ludwigii AOUC-8/14 was characterized and determined to be harbored on a 29-kb element called EludIMEX-1, an integrative mobile element exploiting the Xer recombinases of the host for integration (14). This study describes the characterization of E. cloacae complex isolates harboring an NMC/IMI-type enzyme that were submitted to a national reference laboratory since 2010. Whole-genome sequencing (WGS) was used to characterize the regions harboring the blaNMC/IMI genes.

RESULTS

Enterobacter isolates, gene variant, and patient information.

A total of 1,110 Enterobacter isolates were submitted to the National Microbiology Laboratory (NML) for carbapenemase gene testing between 2010 and 2015. Seventy-eight were submitted as Enterobacter spp., 71 as E. cloacae complex, 782 as E. cloacae, 14 as E. asburiae, 5 as E. amnigenus, and 160 as E. aerogenes. After removing five duplicates, 1.7% of isolates (n = 19) received as E. cloacae, E. cloacae complex, or Enterobacter sp. were found to harbor a blaNMC/IMI-type gene: 1 from 2010, 2 from 2011, 3 from 2012, 2 from 2013, 5 from 2014, and 6 from 2015 (see Table S2 in the supplemental material). None of these 19 isolates harbored any other acquired β-lactamase gene as tested using PCR and confirmed by WGS. Nine hundred three isolates (81.7%) from all other Enterobacter isolates received were negative for all β-lactamase genes tested, while 135 (12.2%) were positive for one or more of the common β-lactamase genes SHV, TEM, CTX-M, OXA-1, and CMY-2 types. Carbapenemase genes (other than blaNMC-A/IMI types) were found in 48 isolates (4.3%); 22 harbored blaKPC, 21 harbored blaNDM, 3 harbored blaVIM, and 2 harbored blaIMP. Thus, 6.04% of all submitted carbapenem-nonsusceptible Enterobacter isolates produced a common carbapenemase as tested. We cannot rule out that some of the other 1,038 isolates harbored a more rare carbapenemase not tested for by PCR. Whole-gene sequencing of the NMC-A/IMI amplicons revealed that 10 isolates harbored blaNMC-A, 5 harbored blaIMI-1, 2 harbored blaIMI-9, and 1 harbored a new variant, designated blaIMI-6 (Table S2). IMI-6 had a single amino acid difference from IMI-3, a proline at position 7 instead of a glutamine. The whole gene could not be amplified from 1 isolate, N13-1531, but this isolate did produce a faint product in PCR with the NMC1/2 primer set. Sequencing of this product revealed a novel variant subsequently named blaIMI-5 after the complete gene sequence was obtained from the WGS data (see below). Among these 19 isolates, 10 were from western Canada (British Columbia [n = 8], Alberta [n = 1], and Saskatchewan [n = 1]), 7 were from central Canada (Ontario [n = 4] and Quebec [n = 3]), and 2 were from eastern Canada (New Brunswick [n = 1] and Nova Scotia [n = 1]). Patient ages ranged from 39 to 92 years (average, 65 years), and 51% were female. Sites of isolation were urine (n = 6), blood (n = 6), rectal swab (n = 3), wound (n = 2), bile drainage (n = 1), and unknown (n = 1).

Bacterial typing and phylogenetic analysis.

Macrorestriction analysis revealed that the isolates exhibited high diversity, with 18 of 19 strains having unique fingerprints (<100% similarity to another) (see Fig. S1 in the supplemental material). Only 2 isolates showed indistinguishable fingerprints; these were isolated from specimens from 2 patients at the same hospital, collected 2 weeks apart, and were confirmed as sequence type 260 (ST260), indicating nosocomial transmission. There were two other pairs of isolates whose fingerprints were >80% similar in each pair and did have the same ST: 2 isolates were ST258 and harbored blaNMC-A, where 1 isolate was from British Columbia and 1 from Quebec with no known patient linkage, and 2 isolates were ST373, harbored blaIMI-1, and were collected from the same hospital but at least 18 months apart and from different patients. There were a total of 16 STs, with 14 being novel at time of submission and 2 being extant in the E. cloacae MLST database, ST12 and ST13. Phylogenetic analysis using the concatenated MLST target sequences (Fig. 1A) was carried out on all blaNMC-A/IMI-harboring strains along with various E. cloacae complex strains belonging to several species or subspecies with known hsp60 cluster types (2) and also with concatenated sequences from the recently identified MDR clonal complex STs ST114, ST171, ST74-78, and ST234 (6–8). Interestingly, no E. cloacae isolate harboring blaNMC-A/IMI clustered with these MDR clonal complex STs, which all clustered in an E. hormaechei branch. Six STs from 7 isolates all harboring blaNMC-A, ST12, ST13, ST258 (n = 2), ST282, ST374, and ST748, clustered in an E. ludwigii branch which included the type strain EN119 and the EludIMEX-1 isolate E. ludwigii AOUC-8/14 (both ST714) and E. cloacae EcWSU1 (ST2). Two isolates, both ST373 harboring blaIMI-1, can be considered E. cloacae subsp. cloacae, as they clustered with the type strain ATCC 13047. Three isolates, 2 ST260 harboring blaNMC-A and 1 ST283 harboring blaIMI-6, clustered with E. asburiae ATCC 35953 (ST807). Five isolates clustered on their own branch, 2 harboring blaIMI-1 (ST259 and ST499) and 2 harboring blaIMI-9 (ST496 and ST498), with the latter clustering closely with ST635 isolate E. cloacae 50588862, which harbors blaIMI-9 (15). The isolate harboring blaIMI-5 (ST261) clustered closet to E. cloacae UCI39 (ST743), and an isolate harboring blaIMI-1 (ST497) clustered between this branch and E. kobei SMART_635 (ST810). For comparison, we also carried out phylogenetic analysis using partial sequences of the hsp60 gene (Fig. 1B) (2). The resultant tree closely matched the tree produced using the MLST sequences, with the exception of the reference hsp60 cluster III strain E. cloacae MGH85, which did not cluster with the E. hormaechei strains. This analysis also showed that the MLST branch consisting of ST259, -496, -498, and -499 belongs to hsp60 cluster IX. Overall, diverse strains can harbor blaNMC-A/IMI genes, though in this study blaNMC-A was mostly associated with E. ludwigii isolates.

FIG 1.

FIG 1

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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.

Antimicrobial susceptibility.

Antimicrobial data for the 19 isolates harboring blaNMC-A/IMI-type genes are shown in Table S2. Except for the isolate harboring blaIMI-5, all others were resistant to imipenem, meropenem, and ertapenem. All isolates were also resistant to ampicillin, amoxicillin-clavulanic acid, cefazolin, and cefoxitin, and all isolates were susceptible to amikacin, gentamicin, tobramycin, trimethoprim-sulfamethoxazole, tigecycline, and ciprofloxacin. Ten isolates were susceptible to nitrofurantoin, and 8 had intermediate resistance. Results for third-generation cephalosporins and piperacillin-tazobactam were variable, though there was an indication that isolates harboring NMC-A were mostly susceptible (80% susceptible). In contrast, those harboring IMI-1 were mostly resistant (75% resistant), but the results were not statistically significant (P = 0.9), likely due to the small number of isolates. Of the two isolates harboring blaIMI-9, one, N15-0261, was susceptible to third-generation cephalosporins and piperacillin-tazobactam, whereas the other, N15-1378, was resistant to these agents. The blaIMI-9 promoters (see Fig. S2 in the supplemental material) and the IMI-R proteins were identical in these two isolates. MIC values indicate that even in these carbapenemase producers, other strain-specific factors may play a role in β-lactam resistance, such as ampC expression, porin mutations, and/or efflux. The isolate harboring IMI-5 was susceptible to meropenem and imipenem but was ertapenem intermediate by Etest (MIC = 1 μg/ml) or resistant using Vitek 2 (MIC = 4 μg/ml). E. coli harboring the cloned blaIMI-5 was fully susceptible to carbapenems and cefotaxime but resistant to ceftazidime, aztreonam, and piperacillin-tazobactam. Thus, IMI-5 appears to be an inhibitor-resistant ESBL with low activity against carbapenems. It also may be poorly expressed, as it has a nonoptimal −35 box, TAGATT (consensus, TTGACA) (Fig. S2).

Carba-NP testing of NMC-A/IMI isolates.

A modified Carba-NP test with all isolates harboring blaNMC-A, blaIMI-1, and blaIMI-9 gave positive reactions for carbapenemase activity, indicating utility in detecting this group of carbapenemases. Nonetheless, the carbapenem-sensitive blaIMI-5 isolate (N13-1531) and, surprisingly, the carbapenem-resistant blaIMI-6 isolate (N14-0444) were both negative for carbapenemase activity using the Carba-NP test. However, whereas the E. coli transformant harboring the cloned blaIMI-5 gene was also negative, the E. coli transformant harboring blaIMI-6 was positive for carbapenemase activity. The Carba-NP negative result for the blaIMI-6 clinical isolate may be due to weak expression due to a nonoptimal −10 box, TACAAT (consensus, TATAAT) (Fig. S2), whereas the cloned gene was most likely of a high enough copy number that carbapenemase activity was detected by this test.

Genetic context of blaNMC-A, blaIMI-1, and blaIMI-9 as determined by WGS.

E. ludwigii AOUC-8/14 blaNMC-A was found on an ∼29-kb putatively mobile element designated EludIMEX-1, which presumably uses a XerC/XerD recombination mechanism to integrate as postulated based on the xerC/xerD-like sequence motifs at the chromosomal-IMEX junctions (14). Integration of EludIMEX-1 was in a small gene which in the E. ludwigii P101 genome was designated locus tag M942_09480. This gene encodes a cysteine-rich protein belonging to the CxxCxxCC family (pfam03692) and which may possibly chelate zinc or iron ions. A different CxxCxxCC family gene is encoded within EcloIMEX-1. Here we used the E. cloacae EcWSU1 genome (accession no. CP002886) as a reference to order the assembled contigs of the study isolates. For all isolates harboring blaNMC-A (n = 10), we found the carbapenemase gene on contigs that contained IMEX elements with >99% identity to EludIMEX-1, and in all cases the element was inserted into the identical chromosomal location as in E. ludwigii AOUC-8/14 (Fig. 2). In E. cloacae EcWSU1 this gene, yieW (locus tag EcWSU1_03080), was located, as in E. ludwigii P101, between the sugar efflux transporter B gene setB (locus tag EcWSU1_03079) and the yeiP gene, encoding a elongation factor P-like protein (locus tag EcWSU1_03081). In two isolates a copy of an ISEhe3 variant was found inserted into the IMEX, in the recombinase gene in N14-2080 and in a hypothetical protein gene in N14-1704. In both instances ISEhe3 was flanked by 3-bp direct repeats, suggesting insertion by a transposition event. Analysis of read coverage for both isolates suggests that ISEhe3 was present in multiple copies in these two isolates (data not shown). Though there appears to be an association with E. ludwigii isolates, blaNMC-A was found on EludIMEX-1 elements in diverse E. cloacae complex strains with multiple sequence types. Thus, here we designated these elements EcloIMEX-1, as they may be associated with diverse E. cloacae complex strains, not just E. ludwigii isolates.

FIG 2.

FIG 2

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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.

Analysis of the genome in isolates harboring blaIMI-1 and blaIMI-9 revealed those genes to also be found on EcloIMEX-like elements (EcloIMEX-2 to EcloIMEX-6) located in the identical chromosomal location. However, in contrast to isolates harboring blaNMC-A, these elements show considerable diversity in at least one region, though they do share some homology with EcloIMEX-1s. The five isolates that harbor blaIMI-1 share homologous regions of ∼17.6 kb with EcloIMEX-1 which exhibit 90 to 99% sequence identity. The two isolates harboring blaIMI-9 share much of this region; however, a 3.4-kb region immediately upstream of the recombinase gene, which encodes a glycosyltransferase, a hypothetical protein, and a highly degenerate insertion sequence in EcloIMEX-1, has been replaced in N15-0261 by a defective ISEhe3-like element (1.33 kb) and in N15-1378 by this element associated with a hypothetical protein gene (2.73 kb). EcloIMEX-6 found in E. cloacae N15-1378 shares >99% identity with the EcloIMEX containing blaIMI-9 found in E. cloacae 50588862 isolated from a patient in Norway (15). The 17.6-kb conserved region is followed by diverse nonconserved regions of differing sizes in the various IMEXs which only share one common feature here, i.e., related resolvase genes where the proteins share between 86 and 100% identity. The nonconserved region is followed by second conserved region (∼1.6 kb) harboring a CxxCxxCC protein gene and a hypothetical protein gene.

As previously described, the EcloIMEXs are flanked by imperfect 29-bp inverted repeats homologous to XerC/XerD binding sites (14), i.e., dif-like sites (16), here called dif-L and dif-R (Fig. 2). It was postulated that the low promiscuity of these elements is due to the dependence on the dif/XerC-XerD recombination mechanism (14, 16). All EcloIMEXs harbor a serine recombinase (89.7 to 99.7% identity) and a resolvase, and it remains to be determined if they are functional and involved in mobility of the element. Serine recombinases are often located at one extremity of mobile genetic elements (MGEs) and so are flanked by one of the recognition sites for integration. In fact, we detected a 29-bp sequence (dif-Lrec) with high identity to the dif-L sequence located upstream of the recombinase gene. Thus, it is tempting to speculate that the EcloIMEXs might have arisen as a result of recombination between the dif-L sequences of the conserved regions harboring the carbapenemase gene and the dif-Lrec/dif-R sequences of diverse MGEs harboring related serine recombinases.

blaIMI-5 and blaIMI-6 are located on IncFII-type plasmids.

In E. cloacae N13-1531 and N14-0444, no IMEX elements were detected in their sequence assemblies. In the Illumina sequencing assemblies, blaIMI-5 was located on a 17.5-kb contig and blaIMI-6 was located on a 2.8-kb contig. However, using the reads from MinION sequencing assembled with the Illumina reads, blaIMI-5 was found on an 89,451-bp contig and blaIMI-6 was found on a 163,767-bp contig. Each of these contigs was finished and closed to a circle by standard PCR.

The blaIMI-5 gene was harbored on an 89,970-bp plasmid (pIMI-5) containing a RepA protein with 97% identity to the IncFII RepA gene product from pECL-A found in E. cloacae ATCC 13047 (accession no. CP001919) (Fig. 3). Nonetheless, pIMI-5 showed only limited identity to the much larger pECL-A (199,562 bp). RepA proteins with >95% identity were found in numerous Enterobacter genome sequencing projects and/or draft genomes using BLAST (data not shown). The blaIMI-5 gene was the only antimicrobial resistance gene identified on pIMI-5. The complete blaIMI-5 gene was the same length as other IMI/NMC-type genes (879 bp) but was the most divergent, showing only 79 to 80% identity to the others, with the IMI-5 protein showing the highest identity, 81%, to IMI-8. An alignment of the enzymes from the isolates in this study is shown in Fig. S3 in the supplemental material. Antimicrobial data (see above) indicated that IMI-5 was a weak carbapenemase though it contained the cysteines at positions 69 and 238, as well as other residues that are conserved in class A carbapenemases (16–18, 33) (Fig. S3). Of note, however, is an asparagine in place of a conserved histidine at position 105, a position shown to have a high information content and therefore importance in structure and function in the related class A carbapenemase SME-1 (17). The blaIMI-5 promoter region is also divergent from the other blaNMC-A/IMI promoters, which are relatively conserved (Fig. S2). A putative weak −35 box, TAGATT, with the consensus being TTGACA, may lead to low expression of blaIMI-5. There was a short remnant of an imiR gene upstream from blaIMI-5, so it is unlikely to be regulated. The blaIMI-5 gene was located within an 8-kb region bracketed by two different IS3 family insertion sequences (ISs), one putatively defective, which may have played a role in its mobilization. The plasmid contains two other IS3 family ISs and short remnants of two others. Also of note in pIMI-5 is a 13-kb region harboring numerous genes involved in pilus biogenesis, which may play a role in the infection and pathogenesis capacity of the host bacterium. A 16-kb transfer region is present, but an alignment with the transfer region from pECL-A reveals that multiple transfer genes have been partially or fully deleted, likely rendering pIMI-5 non-self-transmissible (see Fig. S4 in the supplemental material). In fact, we were unable to obtain pIMI-5 transconjugants by conjugative transfer or electroporation experiments with an E. coli recipient.

FIG 3.

FIG 3

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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.

In E. cloacae N14-0444, blaIMI-6 was found on a 165,469-bp plasmid (Fig. 4). Plasmid pIMI-6 contained a RepA protein having 100% identity to the IncFIIY RepA proteins from two blaIMI-6 containing plasmids, the 166,620-bp pRJ46C (accession no. KT225520) isolated from Raoultella ornithinolytica RJ46C and the 140,698-bp pGA45 (accession no. KT780723) isolated from an uncultured bacterium from river sediment (10). The three plasmids are essentially syntenic and share large regions of high sequence identity (≥98%) (see Fig. S5 in the supplemental material). These include the IncFIIY 1.2-kb repA2-tap-repA replication region, a 31.6-kb IncFII conjugal transfer region, a 26-kb region carrying genes for a type VI secretion system (T6SS), and an 18.3-kb stability region. In contrast to pRJ46C and pGA4, pIMI-6 has an additional stability region (6 kb) that also carries another parA-parB locus and relE-relB toxin-antitoxin system genes. The rest of pIMI-6 can be considered accessory regions, as these regions are rich in ISs, some putatively defective or partially deleted (remnants). A divergently transcribed imiR gene was located 142 bp upstream of the start of the blaIMI-6 gene, the typical arrangement for the NMC-A/IMI family of genes. The imiR-blaIMI-6 region was bracketed by a putatively defective ISEc36-like IS and an ISEclI-like IS, one or both of which may have been involved in the mobilization of the carbapenemase gene. The only other resistance-related genes were a copper resistance copA-like transporter gene and a copC-like periplasmic copper binding protein gene, though they are separated by an IS. Two separate F1-like pilus operons are present, though they may be incomplete due to interruption by nearby ISs. The pilus operons (if functional) and the T6SS may increase the virulence potential of the host bacterium. As mentioned above, pIMI-6 contains a large conjugal transfer region carrying at least 30 genes, most with products with homology to cognate IncFII-type conjugal proteins. In fact, we were able to obtain pIMI-6 transconjugants with an E. coli recipient, demonstrating that the conjugal transfer region is functional.

FIG 4.

FIG 4

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

DISCUSSION

Among carbapenem-resistant Enterobacter strains, Enterobacter cloacae complex isolates harboring blaNMC-A/IMI-type carbapenemase genes are relatively rare in Canada, which mirrors global epidemiology (19). Results from the Canadian Nosocomial Infection Surveillance Program showed only four NMC-A/IMI-type producers isolated in participating acute care hospitals between 2010 and 2014 (20). One limitation of that study, however, is that isolates were submitted voluntarily and NMC-A/IMI prevalence may be underestimated. The mechanism of resistance to carbapenems exhibited by the majority of Enterobacter isolates submitted is presumably due to overexpression of AmpC with concomitant porin mutations (1). Most isolates harboring blaNMC-A/IMI were detectable by the Carba-NP test, but low enzyme expression contributed to presumed false-negative results. From a clinical perspective, aminoglycosides, fluoroquinolones, or tigecycline may remain viable treatment options for infections caused by an Enterobacter strain harboring a blaNMC-A/IMI-type carbapenemase, as our results indicate that these isolates remain susceptible to these drugs. As previously demonstrated for blaNMC-A and supported here, other types in this group, namely, blaIMI-1 and blaIMI-9, are also found chromosomally integrated on EludIMEX-1-like elements (14), called here EcloIMEX-1, though the non-blaNMC-A-harboring variants are more diverse than the EludIMEX-1 elements. As previously stated (14), a site-specific and Xer recombination-dependent mechanism of mobility may limit the diffusion of this group of carbapenemases in comparison to other types of mobile genetic elements such as transposons and genetic islands. More worrisome is that some blaIMI types have been acquired by IncFII plasmids and are associated with ISs (10–13), as we found here for blaIMI-5 and blaIMI-6, thus making them potentially more amenable to transfer. Despite this, however, blaNMC-A/IMI-type genes have rarely been found outside Enterobacter, which is likely due to their rarity overall, the low likelihood of being mobilized to a plasmid (via insertion sequences), and potential plasmid instability in other genera, as putatively shown for pIMI-5 in E. coli. In previous studies of isolates from human infections using hsp60 partial gene sequencing for identification (2), E. hormaechei isolates in clusters VI and VIII and E. cloacae isolates in cluster III were the main species identified (21–24). More recently, MLST of MDR isolates has identified several clonal groups which cluster with E. hormaechei (Fig. 1) (6–8). In contrast, we found that none of the E. cloacae isolates harboring blaNMC-A/IMI types clustered with E. hormaechei by MLST or hsp60 analysis. Instead, blaNMC-A was highly associated (8/10) with E. ludwigii isolates (cluster V) or E. asburiae (cluster I). The isolates harboring blaIMI types were more widely distributed among multiple clusters. It has been hypothesized that the higher prevalence of E. hormaechei and cluster III isolates in a nosocomial environment is due to niche adaptation (21). The paucity of Enterobacter isolates harboring blaNMC-A/IMI types from hospitalized patients may reflect poor adaptation to this environment for these genetic clusters and hence a barrier to a more widespread distribution here, such as outbreaks. The substantial increase in whole-genome sequencing among bacteria, including enterobacters (http://www.ncbi.nlm.nih.gov/genomes/MICROBES/microbial_taxtree.html), in the last decade will greatly assist future studies to discern the pathogenic and virulence potential among different E. cloacae complex isolates.

MATERIALS AND METHODS

Bacterial isolates.

Enterobacter strains nonsusceptible to a carbapenem (as determined by the submitting site) isolated from patients between 2010 and 2015 were voluntarily submitted to the National Microbiology Laboratory (NML) for detection of carbapenemase genes. Isolates were identified by the submitting site and were received as Enterobacter sp., E. cloacae, E. cloacae complex, E. asburiae, E. amnigenus, or E. aerogenes. A basic set of patient information (gender, age, and site of isolation) was obtained when possible. Electrocompetent Escherichia coli DH10B (ThermoFisher Scientific, Burlington, ON, Canada) was used in transformations, and E. coli J53AzR (sodium azide resistant) was used as the recipient in liquid mating experiments. Selection for transformants was with ampicillin (100 μg/ml), and that for transconjugants was with ampicillin and sodium azide (150 μg/ml).

PCR and DNA methodology.

Multiplex PCRs for carbapenemases and β-lactamases were as previously described (25, 26). PCR for blaNMC/IMI-type gene detection was conducted with in-house-designed primers NMC1 (5′-TGGTGTCACGCTTTAGACAC) and NMC2 (5′-ACCATGTCTGATAGGTTTCC) or NMC1 and NMC3 (5′-AGTTTTATCGCCAACTACCC). The entire blaNMC-A, blaIMI-1, and blaIMI-6 genes, including the promoter regions, were amplified with IMIRup (5′-TAAAGGTAATCTGGCACGCAT) and IMIstop (5′-ATCAYAATRAARTGATGGCTA), while blaIMI-5 with its promoter was amplified with IMIvarup (5′-TAATAGTAGTTTTGCTCGCAT) and IMIstop. Gene amplicons were cloned into the pCR-XL-TOPO vector with the TOPO XL PCR cloning kit (ThermoFisher Scientific). Macrorestriction/pulsed-field gel electrophoresis (PFGE) fingerprints using XbaI were analyzed using BioNumerics v5.1 (Applied Maths, Sint-Martens-Latem, Belgium). Genomic DNA isolation was with InstaGene Matrix (Bio-Rad, Mississauga, ON, Canada) or the Master Pure Complete DNA and RNA purification kit (EpiCentre, Madison, WI) for use in routine PCR or for whole-genome sequencing (WGS), respectively. Plasmid isolation was with plasmid miniprep kits (Qiagen Inc., Toronto, ON, Canada).

WGS and bioinformatics.

Genome sequencing was conducted using Illumina MiSeq (Illumina Inc., San Diego, CA) and/or MinION MK1B with R9 flow cells (Oxford Nanopore Technologies [ONT], Oxford, UK). The Fast5 read files generated by MinION were base-called by the 2D workflow of ONT Metrichor software and converted to fastq files by Poretools (27). De novo assembly of Illumina and MinION fastq reads was done with SPAdes 3.6.0 (28). ProgressiveMAUVE (29) was used to order the assembled contigs to the E. cloacae EcWSU1 genome (accession no. CP002886). Annotation was done manually using ORFinder (https://www.ncbi.nlm.nih.gov/orffinder/) and the BLAST suite for gene/protein identification (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The E. cloacae multilocus sequence typing (MLST) scheme (6) was carried out in silico (https://cge.cbs.dtu.dk/services/MLST/) using assembled contigs, with novel alleles confirmed by Sanger sequencing. Novel allele and sequence type (ST) assignment was done by the curator of the E. cloacae database (http://pubmlst.org/ecloacae). Phylogenetic analysis was carried out with MEGA 7.0.14 (30) using the concatenated sequences of the MLST targets (3511 bp) and also with partial sequences (272 bp) of the hsp60 gene (2). STs were obtained for reference E. cloacae complex strains by in silico MLST as described above with either their fully assembled or draft genomes.

Antimicrobial susceptibility and phenotypic testing.

Antimicrobial susceptibilities were determined using Vitek 2 (AST-GN219 card) and Etest (bioMérieux Canada Inc., St. Laurent, QC, Canada) and interpreted based on CLSI 2015 guidelines (31). A modified Carba-NP test was carried out to detect carbapenemase activity (32).

Accession number(s).

Nucleotide accession numbers as assigned by the GenBank database for the genomic regions harboring blaNMC-A/IMI and the partial sequence for the hsp60 genes for the isolates in this study are listed in Table S1 in the supplemental material. Plasmids pIMI-5 and pIMI-6 have been assigned accession no. KT599915 and KT599916, respectively.

Supplementary Material

Supplemental material
supp_61_5_e02578-16__index.html (1KB, html)

ACKNOWLEDGMENTS

We declare that we have no conflicts of interest.

We thank the Genomics Core Facility of the National Microbiology Laboratory for carrying out the Illumina sequencing. We gratefully acknowledge the expert technical assistance of Romeo Hizon and Ken Fakharuddin.

Footnotes

Supplemental material for this article may be found at https://doi.org/10.1128/AAC.02578-16.

REFERENCES

  • 1.Davin-Regli A, Pages J-M. 2015. Enterobacter aerogenes and Enterobacter cloacae; versatile bacterial pathogens confronting antibiotic treatment. Front Microbiol doi: 10.3389/fmicb.2015.00392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Hoffmann H, Roggenkamp A. 2003. Population genetics of the nomenspecies Enterobacter cloacae. Appl Environ Microbiol 69:5306–5318. doi: 10.1128/AEM.69.9.5306-5318.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Hoffmann H, Stindl S, Ludwig W, Stumpf A, Mehlen A, Heesemann J, Monget D, Schleifer KH, Roggenkamp A. 2005. Reassignment of Enterobacter dissolvens to Enterobacter cloacae as E. cloacae subspecies dissolvens comb. nov. and emended description of Enterobacter asburiae and Enterobacter kobei. Syst Appl Microbiol 28:196–205. doi: 10.1016/j.syapm.2004.12.010. [DOI] [PubMed] [Google Scholar]
  • 4.Brady C, Cleenwerck I, Venter S, Coutinho T, De Vos P. 2013. Taxonomic evaluation of the genus Enterobacter based on multilocus sequence analysis (MLSA): proposal to reclassify E. nimipressuralis and E. amnigenus into Lelliottia gen. nov. as Lelliottia nimipressuralis comb. nov. and Lelliottia amnigena comb. nov., respectively, E. gergoviae and E. pyrinus into Pluralibacter gen. nov. as Pluralibacter gergoviae comb. nov. and Pluralibacter pyrinus comb. nov., respectively, E. cowanii, E. radicincitans, E. oryzae and E. arachidis into Kosakonia gen. nov. as Kosakonia cowanii comb. nov., Kosakonia radicincitans comb. nov., Kosakonia oryzae comb. nov. and Kosakonia arachidis comb. nov., respectively, and E. turicensis, E. helveticus and E. pulveris into Cronobacter as Cronobacter zurichensis nom. nov., Cronobacter helveticus comb. nov. and Cronobacter pulveris comb. nov., respectively, and emended description of the genera Enterobacter and Cronobacter. Syst Appl Microbiol 36:309–319. doi: 10.1016/j.syapm.2013.03.005. [DOI] [PubMed] [Google Scholar]
  • 5.Doumith M, Ellington MJ, Livermore DM, Woodford N. 2009. Molecular mechanisms disrupting porin expression in ertapenem resistant Klebsiella and Enterobacter spp. clinical isolates from the UK. J Antimicrob Chemother 63:659–667. doi: 10.1093/jac/dkp029. [DOI] [PubMed] [Google Scholar]
  • 6.Miyoshi-Akiyama T, Hayakawa K, Ohmagari N, Shimojima M, Kirikae T. 2013. Multilocus sequence typing (MLST) for characterization of Enterobacter cloacae. PLoS One 8:e66358. doi: 10.1371/journal.pone.0066358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Izdebski R, Baraniak A, Herda M, Fiett J, Bonten MJM, Carmeli Y, Goossens H, Hryniewicz W, Brun-Buisson C, Gniadkowski M, on behalf of the MOSAR WP2, WP3 and WP5 Study Groups. 2015. MLST reveals potentially high-risk international clones of Enterobacter cloacae. J Antimicrob Chemother 70:48–56. doi: 10.1093/jac/dku359. [DOI] [PubMed] [Google Scholar]
  • 8.Girlich D, Poirel L, Normann P. 2015. Clonal distribution of multidrug-resistant Enterobacter cloacae. Diagn Microbiol Infect Dis 81:264–268. doi: 10.1016/j.diagmicrobio.2015.01.003. [DOI] [PubMed] [Google Scholar]
  • 9.Nordmann P, Mariotte S, Naas T, Labia R, Nicolas MH. 1993. Biochemical properties of a carbapenem-hydrolyzing β-lactamase from Enterobacter cloacae and cloning of the gene into Escherichia coli. Antimicrob Agents Chemother 37:939–946. doi: 10.1128/AAC.37.5.939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Dang B, Mao D, Luo Y. 2016. Complete nucleotide sequence of pGA45, a 140,698 bp IncFIIY plasmid encoding blaIMI-3-mediated carbapenem resistance, from river sediment. Front Microbiol doi: 10.3389/fmicb.2016.00188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Aubron C, Poirel L, Ash RJ, Nordmann P. 2005. Carbapenemase-producing Enterobacteriaceae, US rivers. Emerg Infect Dis 11:260–264. doi: 10.3201/eid1102.030684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Yu YS, Zhou ZH, Chen YG, Li LJ. 2006. First isolation of blaIMI-2 in an Enterobacter cloacae clinical isolate from China. Antimicrob Agents Chemother 50:1610–1611. doi: 10.1128/AAC.50.4.1610-1611.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Rojo-Bezares B, Martín C, López M, Torres C, Sáenz Y. 2012. First detection of blaIMI-2 gene in a clinical Escherichia coli strain. Antimicrob Agents Chemother 56:1146–1147. doi: 10.1128/AAC.05478-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Antonelli A, D'Andrea MM, Di Pilato V, Viaggi B, Torricelli F, Rossolini GM. 2015. Characterization of a novel putative Xer-dependent integrative mobile element carrying the blaNMC-A carbapenemase gene, inserted into the chromosome of members of the Enterobacter cloacae complex. Antimicrob Agents Chemother 59:6620–6624. doi: 10.1128/AAC.01452-15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Di Luca MC, Skaare D, Aasnaes B, Sundsfjord A, Samuelsen O. 29 September 2016. Identification of a novel IMI carbapenemase (IMI-9) in Enterobacter cloacae complex. Int J Antimicrob Agents doi: 10.1016/j.ijantimicag.2016.09.004. [DOI] [PubMed] [Google Scholar]
  • 16.Das B, Martínez E, Midonet C, Barre FX. 2013. Integrative mobile elements exploiting Xer recombination. Trends Microbiol 21:23–30. doi: 10.1016/j.tim.2012.10.003. [DOI] [PubMed] [Google Scholar]
  • 17.Majiduddin FK, Palzkill T. 2005. Amino acid residues that contribute to substrate specificity of class A β-lactamase SME-1. Antimicrob Agents Chemother 49:3421–3427. doi: 10.1128/AAC.49.8.3421-3427.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Jeon JH, Lee JH, Lee JJ, Park KS, Karim AM, lee C-R, Jeong BC, Lee SH. 2015. Structural basis for carbapenem-hydrolyzing mechanisms of carbapenemases conferring antibiotic resistance. Int J Mol Sci 16:9654–9692. doi: 10.3390/ijms16059654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Walther-Rasmussen J, Hɵiby N. 2007. Class A carbapenemases. J Antimicrob Chemother 60:470–482. doi: 10.1093/jac/dkm226. [DOI] [PubMed] [Google Scholar]
  • 20.Mataseje LF, Abdesselam K, Vachon J, Mitchell R, Bryce E, Roscoe D, Boyd DA, Embree J, Katz K, Kibsey P, Simor AE, Taylor G, Turgeon N, Langley J, Gravel D, Amaratunga K, Mulver MR. 2016. Results from the Canadian Nosocomial Infection Surveillance Program on carbapenemase-producing Enterobacteriaceae, 2010-2014. Antimicrob Agents Chemother 60:6787–6794. doi: 10.1128/AAC.01359-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Morand PC, Billoet A, Rottman M, Sivadon-Tardy V, Eyrolle L, Jeanne L, Tazi A, Anract P, Courpied JP, Poyart C, Dumaine V. 2009. Specific distribution within the Enterobacter cloacae complex of strains isolated from infected orthopedic implants. J Clin Microbiol 47:2489–2495. doi: 10.1128/JCM.00290-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Paauw A, Caspers P, Leverstein-van Hall MA, Schuren FH, Montijn RC, Verhoef J, Fluit AC. 2009. Identification of resistance and virulence factors in an epidemic Enterobacter hormaechei outbreak strain. Microbiology 155:1478–1488. doi: 10.1099/mic.0.024828-0. [DOI] [PubMed] [Google Scholar]
  • 23.Kremer A, Hoffmann H. 2012. Prevalences of the Enterobacter cloacae complex and its phylogenetic derivatives in the nosocomial environment. Eur J Clin Microbiol Infect Dis 31:2951–2955. doi: 10.1007/s10096-012-1646-2. [DOI] [PubMed] [Google Scholar]
  • 24.Akbar M, Bakhshi B, Najar S, Peerayeh N. 2016. Particular distribution of Enterobacter cloacae strains isolated from urinary tract infection within clonal complexes. Iranian Biomed J 20:49–55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Mataseje LF, Bryce E, Roscoe D, Boyd DA, Embree J, Gravel D, Katz K, Kibsey P, Kuhn M, Mounchili A, Simor A, Taylor G, Thomas E, Turgeon N, Mulvey MR, on behalf of the members of the Canadian Nosocomial Infection Surveillance Program. 2012. Carbapenem-resistant Gram-negative bacilli in Canada 2009-10: results from the Canadian Nosocomial Infection Surveillance Program (CNISP). J Antimicrob Chemother 67:1359–1367. doi: 10.1093/jac/dks046. [DOI] [PubMed] [Google Scholar]
  • 26.Mataseje LF, Boyd DA, Delport J, Hoang L, Imperial M, Lefebvre B, Kuhn M, Van Caeseele P, Willey BM, Mulvey MR. 2014. Serratia marcescens harbouring SME-type class A carbapenemases in Canada and the presence of blaSME on a novel genomic island, SmarGI1-1. J Antimicrob Chemother 69:1825–1829. doi: 10.1093/jac/dku040. [DOI] [PubMed] [Google Scholar]
  • 27.Loman NJ, Quinaln AR. 2014. Poretools: a toolkit for analyzing nanopore sequence data. Bioinformatics 30:3399–3401. doi: 10.1093/bioinformatics/btu555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comp Biol 19:455–477. doi: 10.1089/cmb.2012.0021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Darling AE, Mau B, Perna NT. 2010. progressiveMAUVE: multiple genome alignment with gene gain, loss and rearrangement. PLoS One 5:e1147. doi: 10.1371/journal.pone.0011147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Kumar S, Stechner G, Tamura K. 2016. Molecular evolutionary genetics analysis version 7 for bigger datasets. Mol Biol Evol 33:1870–1874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Clinical and Laboratory Standards Institute. 2015. Performance standards for antimicrobial susceptibility testing; 25th informational supplement. CLSI M100-S25. Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]
  • 32.Dortet L, Bréchard L, Poirel L, Nordmann P. 2014. Impact of the isolation medium for detection of carbapenemase-producing Enterobacteriaceae using an updated version of the Carba NP test. J Med Microbiol 63:772–776. doi: 10.1099/jmm.0.071340-0. [DOI] [PubMed] [Google Scholar]
  • 33.Philippon A, Slama P, Dény P, Labia R. 2016. A structure-based classification of class A β-lactamases, a broadly diverse family of enzymes. Clin Microbiol Rev 29:29–57. doi: 10.1128/CMR.00019-15. [DOI] [PMC free article] [PubMed] [Google Scholar]

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