Sir,
Carbapenemases are the most common mechanism of carbapenem resistance and include β-lactamases of Ambler classes A, D and B (metallo-β-lactamases). Among the carbapenemases, the New Delhi metallo-β-lactamase-1 (NDM-1) has gained particular attention.1 Since its initial identification in Klebsiella pneumoniae and Escherichia coli from a patient of Indian descent in 2008 in Sweden,2 the blaNDM-1 gene has been reported in many countries.2,3 Most reports showed that blaNDM-1 is frequently located on mobile genetic elements that often carry additional resistance genes.4–6 So far, data regarding the presence of blaNDM-1 in food-producing animals have been very limited; only one NDM-1-producing Acinetobacter lwoffii has been detected in chickens in China.7 In that strain, the blaNDM-1 gene was located on an ∼270 kb plasmid, of which 6451 bp are deposited in GenBank (accession number JN616388).
To investigate how widespread the blaNDM-1 gene is in bacteria of food animal origin, a survey of Gram-negative bacteria from pigs, chickens and ducks was conducted in South China (Guangdong province) from November 2011 to May 2012. Samples (n = 1293) were collected from 5 commercial pig farms (313 lungs, 240 livers, 133 maxillary lymph nodes and 127 pericardial fluid swabs), 3 commercial chicken farms (155 lungs, 119 livers and 87 intestinal lymph nodes) and 1 duck farm (64 lungs, 32 livers and 23 intestinal lymph nodes). Each sample originated from an individual animal. Non-susceptibility to carbapenems was assessed by growth on brain–heart infusion agar plates supplemented with 2 mg/L of meropenem for 18 h at 37°C. A total of 276 Gram-negative bacilli (135 from pigs, 96 from chickens and 96 from ducks) that grew on these selective plates were screened for the presence of the blaNDM-1 and other carbapenemase genes by PCR.3,8
A single A. baumannii isolate, designated GF216, from the lung sample of a pig with pneumonia and sepsis was positive for blaNDM-1. Treatment records of the farm from which the pig originated revealed the use of different β-lactams (alone or in combination with β-lactamase inhibitors), third- and fourth-generation cephalosporins (ceftriaxone and ceftiofur), aminoglycosides and quinolones for the prevention or treatment of bacterial infections. MIC determination by broth microdilution according to CLSI document M100-S229 showed that A. baumannii GF216 was resistant to all tested β-lactams, and also to ciprofloxacin, chloramphenicol, tetracycline, kanamycin, tilmicosin and erythromycin, while it was susceptible to gentamicin and colistin (Table S1, available as Supplementary data at JAC Online).
S1 nuclease PFGE and subsequent Southern blot hybridization identified the blaNDM-1 gene in A. baumannii GF216 on an ∼47 kb plasmid, designated pNDM-AB (Figure S1, available as Supplementary data at JAC Online). This plasmid was transferred by conjugation at a high frequency of 1.15 × 10−2 per donor cell into E. coli J53. Moreover, it was transferred by electrotransformation into A. baylyi ADP1. Attempts to identify the replicon type of pNDM-AB were not successful.10 This result was consistent with the replicon typing approaches conducted for pNDM-BJ01 from A. lwoffii and suggest that pNDM-BJ01 and pNDM-AB may represent a novel incompatibility group.
The complete sequence of plasmid pNDM-AB was obtained using the 454-Genome sequencer FLX procedure (Roche). The gaps between the different contigs were closed by PCR and sequencing. Plasmid pNDM-AB has a length of 47 098 bp. Forty-three predicted coding sequences (CDS) for proteins of ≥100 amino acids were identified in pNDM-AB. Of these, 38 CDS were closely related if not identical to genes found on the blaNDM-1-carrying plasmid pNDM-BJ01 from A. lwoffii of human origin.4 Similar to pNDM-BJ01, pNDM-AB also had three different functional regions: (i) a region for conjugative transfer and plasmid replication; (ii) a region that included a bacterial type IV secretion system; and (iii) a resistance gene region that encompassed the resistance genes blaNDM-1, aphA6, ble and msr(E)-mph(E) and one copy of an ISAba125 element (Figure 1). The MICs for transformant ADP1-GF216 and transconjugant J53-GF216 were in agreement with the resistance genes detected on pNDM-AB, except that the aphA6 gene seems not to be active in E. coli J53 (Supplementary Data).
Schematic representation of the alignment of the sequences of plasmids pNDM-AB (GenBank accession number KC503911) and pNDM-BJ01. Relevant genes with reference to the description of pNDM-BJ014 are presented as arrows, with the arrowhead indicating the direction of transcription. Grey shading indicates ≥99.9% nucleotide sequence identity. ORF, open reading frame.
Schematic representation of the alignment of the sequences of plasmids pNDM-AB (GenBank accession number KC503911) and pNDM-BJ01. Relevant genes with reference to the description of pNDM-BJ014 are presented as arrows, with the arrowhead indicating the direction of transcription. Grey shading indicates ≥99.9% nucleotide sequence identity. ORF, open reading frame.
Sequence comparisons revealed two regions that distinguished pNDM-AB from pNDM-BJ01. First, a 6200 bp region that contains the genes groS, groEL and insE and a copy of the insertion sequence ISAba125 in pNDM-BJ01 (nucleotides 12 037–18 236) was replaced by a 2492 bp insert, which contains the genes msr(E)-mph(E) in pNDM-AB (nucleotides 12 037–14 530). These genes code for an ABC transporter and a phosphotransferase, respectively, and have recently been shown to confer resistance against macrolides and triamilides, including gamithromycin and tildipirosin, the newest members of these classes approved in the EU, USA and Canada in 2011 for use in food-producing animals.11 Second, a 3530 bp segment (nucleotides 43 569–47 098) containing the genes traD, insB and a gene encoding a type I restriction-modification system was present in pNDM-AB but absent in pNDM-BJ01.
In summary, this study showed that the blaNDM-1 is present in bacteria of food animal origin, although currently at a very low prevalence. Nevertheless, further monitoring of the presence of the blaNDM-1 gene and other carbapenemase genes in bacteria of animal origin is warranted.
Funding
This study was supported by grants from the National ‘973’ programme (no. 2012CB518801) and the National Science Foundation of China (no. NSFC31201862).
Transparency declarations
None to declare.
Acknowledgements
We thank the Commissariat à l'Energie Atomique/Direction des Sciences du Vivant for kindly providing the A. baylyi ADP1 strain.
