Characterization of Multiple NDM-1-Producing Enterobacteriaceae Isolates from the Same Patient

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Carbapenem resistance in Enterobacteriaceae has emerged worldwide mainly by the production of carbapenemases (1). Carbapenemase genes are commonly carried on conjugative plasmids, representing a significant infection control challenge because of the potential horizontal transfer of resistance genes between bacterial isolates, species, and genera. The New Delhi metallo-β-lactamase (NDM), first described in 2008 (2), has been detected in different species and genera without a clear link to dominant plasmids or clones (3). In this study, we characterized three different NDM-producing enterobacterial species isolated from the same patient.

A 65-year-old man presented to the emergency department of a community hospital in Ontario, Canada, with fever and generalized weakness. He had been hospitalized in India with a urinary tract infection, and his stay was complicated by the development of a large sacral decubitus ulcer. The patient was admitted to the hospital after returning from India with a diagnosis of an infected sacral ulcer. His symptoms improved with the initial treatment (cefazolin and metronidazole), but he had a recurrence of fever. Blood cultures grew Escherichia coli GN568 resistant to all of the antibiotics tested in the hospital except tigecycline, with which the patient was treated for 14 days and recovered. However, 2 weeks after treatment, his fever recurred and blood and urine cultures were collected. His blood cultures grew an extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae strain. His urine grew Enterobacter cloacae GN574 sensitive only to tigecycline and Providencia stuartii GN576 sensitive to tigecycline, trimethoprim-sulfamethoxazole, and ciprofloxacin. The patient was asymptomatic for bacteriuria, and his urinary catheter was changed. He was diagnosed with an infected peripherally inserted central catheter, which was removed, and treated with ertapenem for 14 days against an ESBL-producing, carbapenem-susceptible K. pneumoniae strain detected in blood samples. He recovered from his infections and was discharged to a nursing home. These three carbapenem-resistant clinical isolates (E. coli GN568, E. cloacae GN574, and P. stuartii GN576) were submitted to the Public Health Ontario laboratories for further studies. They were positive for carbapenemase activity by the KPC/MBL Confirm kit (Rosco Diagnostica). They were resistant to all β-lactams and aminoglycosides, consistent with the detection of different β-lactamase-encoding genes (including bla_CTX-M-15 and bla_CMY-6 in two of the isolates, in addition to bla_NDM-1) and the 16S rRNA methyltransferase rmtC gene (Table 1). E. coli GN568 (assigned to sequence type 1289 by multilocus sequence typing) (4) was also resistant to quinolones and co-trimoxazole; E. cloacae GN574 was also resistant to quinolones, co-trimoxazole, and tigecycline; and P. stuartii GN576 was also resistant to tetracycline and colistin. IncA/C plasmids were identified in E. coli GN568 (IncFIA and Frep were also detected in this isolate) and P. stuartii GN576 by PCR (5). E. cloacae GN574 was negative for all of the Inc groups tested. In conjugative assays with E. coli J53 as the acceptor (6), bla_NDM-1 and bla_CMY-6 were cotransferred on the IncA/C plasmid to strains J-568 (derived from E. coli GN568) and J-576 (from P. stuartii GN576) (Table 1). The only β-lactamase-encoding gene transferred to E. coli J-574 (transconjugant strain derived from E. cloacae GN574) was bla_NDM-1, consistent with its low aztreonam MIC.

NDM-1-carrying plasmids were extracted from the transconjugant E. coli strains with the Qiagen Large-Construct kit (Qiagen, Valencia, CA) and sequenced with the Illumina compact MiSeq system. Assembly of the contigs obtained was done with the CLC Genomics Workbench software (CLC bio, Qiagen). Gaps were filled by PCR amplification and Sanger sequencing. Open reading frames (ORFs) were predicted and annotated by the RAST server (available at rast.nmpdr.org) (7), followed by manual comparative curation and sequence similarity searches directed against the NCBI (www.ncbi.nlm.nih.gov/BLAST).

pNDM-EcoGN568 resulted in a closed circular sequence of 166,750 bp (average GC content of 51.8%). Its annotation revealed 205 predicted ORFs ranging from 117 to 5,487 bp, 98 of which showed homology to proteins with known functions (Fig. 1; see Table S1 in the supplemental material). pNDM-EcoGN568 was almost identical to pNDM10-0505 (plasmid not yet published, GenBank accession no. JF503991), a 166,744-bp IncA/C plasmid detected in an E. coli isolate from a female patient hospitalized in Vancouver, British Columbia, Canada (8). Like the patient described here, that patient also received medical attention in India before being transferred to Canada. Moreover, pNDM-EcoGN568 also had ∼89% identity with pNDM-KN (GenBank accession no. JN157804), another IncA/C plasmid of 162,746 bp detected in a K. pneumoniae isolate from Kenya (9, 10) (Fig. 1A). Differences between these two plasmids were detected mainly immediately upstream of the bla_NDM-1 gene (Fig. 1B): the cassette content in a class I integron (four cassettes and qacEΔ1 deleted from the 3′ conserved region in pNDM-KN versus only one cassette plus qacEΔ1 in pNDM-EcoGN568) and a fragment of ∼10 kb flanked by two copies of ISKpn14 between the rmtC and bla_NDM-1 genes. Besides some transposase genes, this fragment also included two determinants of aminoglycoside resistance. One of these ISKpn14 copies in pNDM-EcoGN568 disrupts ISAba125, part of which was absent from pNDM-KN (Fig. 1). Upstream of this region, a copy of ISEc23 inserted into the sugE gene (close to the bla_CMY-6 gene) in pNDM-KN was not present in pNDM-EcoGN568.

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FIG 1

Comparison of the NDM plasmids characterized in this study with closely related IncA/C plasmid pNDM-KN (GenBank accession no. JN157804) (9). (A) Regions with high homology are shaded light blue. Arrows indicate the following predicted ORFs: conjugation, stability, and accessory genes (green, yellow), antimicrobial resistance genes (red, bla_NDM-1 is light green), transposon-related genes (blue), and hypothetical proteins (white). The replicase gene is pink. The orange-boxed segments are detailed in panel B. (B) Detail of the bla_NDM-1-flanking regions (those of pNDM-PstGN576 are the same as those of pNDM-EcoGN568). The orange circles represent the 256-bp direct repeats with similarities to MITEs identified in bla_NDM-1 and insA. A third MITE was found ∼11 kb upstream of the bla_NDM-1 gene, between a truncated copy of IS5 and a putative endonuclease III gene.

pNDM-PstGN576 had a circular sequence of 147,886 bp with a GC content of ∼52% and 190 predicted ORFs (from 117 to 5,487 bp in size) (Fig. 1; see Table S1 in the supplemental material). Compared to pNDM-EcoGN568, pNDM-PstGN576 showed a deletion of ∼19 kb corresponding to a Tn7-like transposon (the missing fragment was flanked by the inverted repeats IR-L and -R, which define the ends of the transposon) (Fig. 1A). The rest of both plasmid sequences showed 99% identity. These similarities suggest possible in vivo horizontal transfer between E. coli and P. stuartii clinical isolates with their subsequent evolution in the colonized patient, although their independent acquisition by each isolate cannot be ruled out.

pNDM-EclGN574 was 110,625 bp in length, with an average GC content of 54.8% and harboring 146 predicted ORFs (114 to 3,045 bp in size) (Fig. 1; see Table S1 in the supplemental material). pNDM-EclGN574 was identical in sequence to pKOX_NDM1 isolated from a Klebsiella oxytoca strain recovered in Taiwan from a patient who underwent renal transplantation in China (11, 12). pNDM-EclGN574 showed high identity with pNDM-EcoGN568 and pNDM-PstGN576 only in the region containing the bla_NDM-1 gene (Fig. 1B). As described for pKOX_NDM1, this region was flanked by 256-bp direct repeats (Fig. 1B, orange circles) with similarities to miniature inverted-repeat transposable elements (MITEs) suggested to be involved in the acquisition of this bla_NDM-1 region (11). The low sequence identity of pNDM-EclGN574 with the other two NDM plasmids recovered from the same patient suggests its independent acquisition by E. cloacae GN574. We were unable to identify the incompatibility group of plasmids from E. cloacae GN574 and its E. coli transconjugant by the standard plasmid replicon typing method described by Carattoli et al. (5). However, the repA gene on pNDM-EclGN574 had the highest identity with RepFIB replication protein A. Analysis of the primers used for amplification of this replicon type indicated that one of them (FIB F) was not identical to the repA sequence in this plasmid, and the reverse primer had two mismatches.

In conclusion, plasmid sequencing and comparative analysis suggest both lateral plasmid transfer and independent acquisition of the bla_NDM-1 gene in these three different enterobacterial genera recovered from the same patient. Plasmids with high sequence identity recovered from different bacterial species in different geographic areas support the idea of lateral dissemination as the main mechanism of the spread of this metallo-β-lactamase.