Emergence of bla NDM-5 in Enterobacteriaceae Isolates from Companion Animals in Guangzhou,China

Introduction

Carbapenemase-producing isolates particularly New Delhi Metallo-β-lactamase (NDM)-encoding Enterobacteriaceae have been increasingly reported worldwide and have become a global challenge. ¹ Although NDM-producing Enterobacteriaceae are mainly described in clinical samples, they have also been identified in food-producing animals, wildlife, and animal-derived food products.^2–4 To date, some cases of NDM-producing Enterobacteriaceae have been described in companion animals in several countries, such as Korea, Egypt, and the United Kingdom, with NDM-5–encoding IncX3 being the major carriers.^5–8 However, the bla_NDM gene has been rarely report in companion animals in China, although bla_NDM-1–carrying Escherichia coli ST167 was previously reported in a diseased dog, and bla_NDM-5 was detected in three E. coli isolates (2.36%) from diseased dogs in Beijing.^9,10 In this study, we report six Enterobacteriaceae isolates producing NDM-5 recovered from healthy and diseased companion animals in Guangzhou, China.

A total of 257 Enterobacteriaceae isolates, including E. coli (n = 203), Klebsiella pneumoniae (n = 36), Enterobacter cloacae (n = 16), and two Citrobacter freundii (n = 2), were obtained from healthy and diseased companion animals at four animal hospitals located in four districts in Guangzhou, China, during 2016–2017. Enterobacteriaceae isolates were obtained and species identification was performed as previously described. ¹¹ Among them, 153 Enterobacteriaceae isolates collected in 2016 were previously described. ¹¹ Details of the source and origin of the 104 isolates collected in 2017 are given in Supplementary Table S1. All 257 Enterobacteriaceae isolates were detected for the presence of carbapenemase genes by PCR and sequencing. ¹² We found that one E. cloacae, one C. freundii, and four E. coli isolates carried bla_NDM-5 (Table 1). The minimal inhibitory concentrations (MICs) of 12 antimicrobial agents were determined by the agar dilution method or broth microdilution method (limited to colistin). ¹³ All NDM-producing strains exhibited MIC of 2–8 mg/L to imipenem, and showed resistance to ampicillin, ceftazidime, cefotaxime, gentamicin, and sulfamethoxazole/trimethoprim, but susceptibility to colistin; five exhibited resistance to tetracycline and ciprofloxacin. In addition, two, one, and one isolates displayed resistance to amikacin, florfenicol, and fosfomycin, respectively (Table 1 and Supplementary Table S3). The NDM-5–encoding isolates were confirmed to carry other resistance genes by PCR and sequencing with primers listed in Supplementary Table S2, including bla_CTX-M (four bla_CTX-M-55 and one bla_CTX-M-3), bla_CMY-2 (n = 4), rmtB (n = 1), armA (n = 1), floR (n = 1), and fosA3 (n = 1) (Table 1).

The genetic diversity of bla_NDM-5–positive isolates was analyzed by pulse field gel electrophoresis (PFGE), and was characterized by multilocus sequence typing (limited to E. coli).^14,15 GZ7DS11 belonged to ST9437, and the remaining three E. coli isolates obtained from the same hospital belonged to ST410 (Table 1). Three ST410 NDM-5–producing strains exhibited identical PFGE patterns, with identical antimicrobial susceptibility profiles and resistance genes, indicating that clonal spread of bla_NDM-5–positive E. coli had occurred among companion animals within the same hospital in Guangzhou (Table 1 and Supplementary Fig. S1). E. coli ST410 has been increasingly reported as NDM-5 carriers in companion animals,^5–8 this lineage has been identified worldwide and has emerged as new international successful high-risk clones associated with resistance to carbapenems. ¹⁶

Conjugation experiments were performed using streptomycin-resistant E. coli C600 as the recipient strain as previously described, ¹⁷ selected using 1 mg/L imipenem and 3,000 mg/L streptomycin. Transformation was carried out by heat shock ¹⁸ using E. coli strain DH5α as the recipient strain, selected by 0.5 mg/L imipenem. The presence of bla_NDM in the transconjugants/transformants was determined by PCR and sequencing. ¹² The bla_NDM-5 gene in six isolates was successfully transferred to E. coli strain C600 with frequencies of 10^–5 to 10^–2 transconjugants/recipient and DH5α with 10³–10⁵ CFU/mg (Supplementary Table S4). Transformants with a single bla_NDM-positive plasmid were selected for further study, confirmed by S1-PFGE ¹⁹ and Southern blot hybridization according to the manufacturer's instruction. The results showed that bla_NDM-5 was located on ∼46 kb plasmid (n = 5), or ∼104.5 kb plasmid (n = 1). Five ∼46 kb plasmids were classified as IncX3 plasmids, ²⁰ whereas the replicon type of ∼104.5 kb could not be determined by PCR-based replicon typing. ²¹ The transfer of bla_NDM-5–carrying plasmid caused resistance to ampicillin, ceftazidime, and cefotaxime, and increased MICs for imipenem (1–2 mg/L; four–eightfold). Resistance to sulfamethoxazole/trimethoprim was only observed in the transformant GZ7DS11-1T. Other resistance genes were not detected in any transformants.

Six NDM-5–encoding plasmids were extracted from their transformants using QIAGEN^® Plasmid Midi Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions, and were sequenced by Illumina Hiseq 2500 (Illumina, San Diego, CA, USA). Sequence reads were assembled into contigs with SOAPdenovo version 2.04. Plasmid contigs were assembled into the complete plasmid sequence with PCR and Sanger sequencing (limited to pHN7DS11; Supplementary Table S5). Analysis and annotation of plasmids were performed using the RAST server, ²² ISfinder, ResFinder, and BLAST. Integrons were analyzed using INTEGRALL. Three IncX3 plasmids (pHN7CS2, pHN7DH6, and pHN7DH7) obtained from three ST410 NDM-5–producing isolates were 46,161 bp long with identical nucleotide sequences, and pHN6DS3 differed from them by only one nucleotide at position 5934 resulting in amino acid change in ISKox3 transposase. The remaining IncX3 plasmid pHN7DS4 had a size of 45,907 bp, and was similar to other IncX3 plasmids in our study, differed by two nucleotide changes within the plasmid backbone (pHN6DS3, position 361, and 42579) or three nucleotide changes (pHN7CS2, pHN7DH6, and pHN7DH7, position 361, 5934, and 42579), and by deletions of 254 bp of the ΔTn2 that was truncated by IS3000 (Fig. 1A).

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FIG. 1.

Sequence comparison of (A) IncX3 plasmids pHN6DS3, pHN7CS2, pHN7DH6, pHN7DH7, and pHN7DS4 in this study with other NDM-5–producing plasmids pNDM5-SSH006 (46,253 bp, Salmonella Typhimurium, human, China, CM007781), pNDM-QD28 (46,161 bp, Escherichia coli, human, China, KU167608), pP855-NDM5 (46,161 bp, E. coli, swine, China, MF547508), pNDM-5_A0917122 (46,163 bp, E. coli, companion animal, Korea, MH094148), pNDM5_090011 (46,161 bp, Enterobacter hormaechei, human, China, CP036312), pNDM_MGR194 (46,253 bp, Klebsiella pneumoniae, human, India, KF220657), pK516_NDM5 (46,149 bp, Klebsiella michiganensis, human, China, CP022351), and pCRCB-101_1 (46,161 bp, Citrobacter freundii, human, South Korea, CP024820) (B) IncK2 plasmid pHN7DS11 with using pDV45 (85,963 bp, bla_CMY-2, Switzerland, poultry meat, KR905384), p1106-NDM (bla_NDM-5, MG825375), p92944-NDM (118,644 bp, bla_NDM-9, China, clinics, MG838260), pEC1107-NDM-116K (116,042 bp, bla_NDM-5, China, food, MG601057), pHNSD138–1 (103,506 bp, bla_NDM-9, China, chicken, MG271839), pHNTH02-1 (118,337 bp, bla_NDM-9, China, chicken meat, MG196294), and pMb1488 (101,065 bp, mcr-1, Switzerland, chicken retail meta, KY565558). The outer circle in red with annotation is the reference plasmid pHN6DS3 or pHN7DS11 obtained in this study. Color images are available online.

The five IncX3 plasmids in this study were identical or highly similar to other NDM-5–producing IncX3 plasmids obtained from several members of Enterobacteriaceae of various origins worldwide (99.9–100% identity and 100% coverage) (Fig. 1A), as well as plasmids encoding other NDM alleles, for example, pM216_X3 (NDM-4, AP018146), pJN05NDM7 (NDM-7, MH523639), and pAD-19R (NDM-17, KX833071). The observation of identical or similar IncX3 plasmids found in our study further highlights the important role of the epidemic IncX3 plasmids in carbapenem resistance transmission, but also the potential for IncX3 plasmids to horizontally transfer between diverse species and hosts.

pHN7DS11 was 115,619 bp in size with an average GC content of 54.41%, and was identified as IncK2 plasmid. The backbone of pHN7DS11 was highly similar in organization to other IncK2 plasmids (96.9–99.9% identity and 66–94% coverage), possessing a complete array of genes involved in replication, conjugation, maintenance, and stability, such as pDV45 (bla_CMY-2, 85,963 bp, KR905384), p1106-NDM (bla_NDM-5, 113,687 bp, and MG825375), p92944-NDM (bla_NDM-9, 118,644 bp, and MG838260), pEC1107-NDM-116K (bla_NDM-5, 116,042 bp, and MG601057), pHNTH02–1 (bla_NDM-9, 118,337 bp, and MG196294), pHNSD138–1 (bla_NDM-9, 103,506 bp, and MG271839), and pMb1488 (mcr-1, 101,065 bp, and KY565558) (Fig. 1B). These plasmids mainly differed by the presence of a putative group II intron inserted upstream of the ssb gene (pHN7DS11) and variable shufflon region (Fig. 1B and Supplementary Fig. S2). The shufflon is a site-specific recombination system identified in the IncI1/I2 plasmids as well as IncK plasmids.^23,24 It contains different DNA segments flanked by conserved 19-bp repeats (recombination sites), resulting in the diversity of the C-terminal end of the PilV protein. ²⁴ The shufflon region of these IncK2 plasmids is highly diverse, with different combination of repeat sequences (previously designated as repeats e, f, g, and h), ²³ such as pHN7DS11 (g-g-e-e-g-f), p1106-NDM (e-e-g-f-g-g), pEC1107-NDM-116K (h-g-g-g-e-e), and pDV45 (e-e-e-e-g-g-h-f) (Supplementary Fig. S2).

In addition to companion animal, similar IncK2 plasmids carrying bla_NDM genes (bla_NDM-5/-9) have been detected in E. coli isolates from food-producing animal ²⁵ and food ²⁶ and human clinic in China (Fig. 1B). However, the multiresistance regions (MRRs) of these IncK2 plasmids were varied, which may have resulted from the acquisition, deletion, and rearrangement events mediated by mobile elements (e.g., IS1294, IS26). In general, MRRs in NDM-producing IncK2 plasmids were all bounded by inverted repeat of ΔTn1721 and ΔTn5393 and inserted at the same location, which differed from other IncK2 plasmids (Fig. 2). The MRR in pHN7DS11 was related to those of other NDM-encoding IncK2 plasmids. In these plasmids, bla_NDM-5/-9 gene was embedded within the bla_NDM module associated with an ISCR1 complex class 1 integron and flanked by IS26 (IS26-intI1-dfrA12-gcuF-aadA2-qacEΔ1-sul1-ISCR1-cutA-tat-trpF-ble_MBL-bla_NDM-ΔISAba125-IS26) (Fig. 2). However, the class 1 integron was truncated by IS26, resulting in the deletion of a 4,109-bp segment in pHN7DS11 (Fig. 2). In pHNSD138-1, the whole bla_NDM-9 segment was located in inverse orientation, one copy of IS1294 was inserted into the tat gene, and 765-bp of IS26 upstream of ΔISAba125 and 418-bp of 5′-CS of the class 1 integron along with 20-bp spacer were absent (Fig. 2). The MRRs of these IncK2 plasmids were further differed by the presence of the 3,317-bp fosA3 segment (Δorf3-orf2-orf1-fosA3-IS26) (pHNTH02-1 and p92944-NDM), by the loss of the 5,010-bp macrolides resistance module mph(A)-mrx-mphR(A)-IS6100-IS26 (p92944-NDM), by the insertion of IS1294 within ΔTn1721 and ΔTn21 (pHN7DS11), and by the acquisition of a 3,338-bp resistance segment (aac(3)-IV-aph(4)-Ia-ISEc59) and the deletion involving ΔIS5075, ΔTn21, the macrolides region and ΔTn2, and a 165-bp segment of ΔTn5393 (pHNSD138–1) (Fig. 2). In addition, pHNSD138-1 had a 301-bp shorter Tn1721 (Fig. 2). In addition to bla_NDM genes, IncK2 plasmids were also described as vectors for bla_CMY and mcr-1 in E. coli isolates from calves, poultry, poultry meat, and human in Europe.^23,27–29 It highlighted the potential of IncK2 plasmids to become efficient vectors for bla_NDM dissemination and to capture more resistance genes during horizontal spread.

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FIG. 2.

Genetic organization of the multiresistance region of plasmid pHN7DS11, and structural comparison with other IncK2 plasmids. The extents and directions of antibiotic resistance (thick arrows) and other genes are indicated. Δ indicates a truncated gene or mobile element. ISs are shown as boxes labeled with their name. Labeled vertical arrows with IS box indicate the insertion site of IS element. Tall bars represent the inverted repeats of transposon. The collection years of plasmids pEC1107-NDM-116K, pHNTH02-1, pHNSD138-1 are provided in Figure 2. IS, insertion sequence. Color images are available online.

In this study, the dissemination of bla_NDM-5 in companion animals may be mainly associated with clonal spread of E. coli ST410 isolates within the same hospital, involving with the global successful pandemic bla_NDM-carrying IncX3 plasmids. The horizontal transfer of IncK2 plasmid also facilitates the bla_NDM-5 dissemination, and may further spread to other Enterobacteriaceae.