New Delhi metallo-beta-lactamase, Ontario, Canada.

To the Editor: The New Delhi metallo-β-lactamase (NDM-1) was first characterized in 2009 from Klebsiella pneumoniae and Escherichia coli isolated from a patient in Sweden who had received medical care in New Delhi, India (). Further studies have shown broad dissemination of this β-lactamase gene (blaNDM-1) in India, Pakistan, Bangladesh, and the United Kingdom (). Additional isolates have been detected in other countries, and many of the patients with NDM-1–producing Enterobacteriaceae reported receiving medical care in the Indian subcontinent (–). We describe detection and characterization of an NDM-1–producing K. pneumoniae isolated in Ontario, Canada.

In August 2010, a urinary tract infection was diagnosed in a 36-year-old woman in a hospital in Brampton, Ontario. An E. coli strain sensitive to multiple antibacterial drugs (including carbapenems) was isolated from a midstream urine sample; the patient was successfully treated with ciprofloxacin. One week after treatment, when the patient did not have a fever or other clinical signs, a urine culture was repeated, and a carbapenem-resistant K. pneumoniae isolate (GN529) was recovered. Travel history indicated that the patient had recently returned from India, where in mid-July she had had a miscarriage and had been hospitalized in Mumbai for 2 days. At that time, no antimicrobial drug treatment was prescribed.

Susceptibility profiles of K. pneumoniae GN529 and its E. coli transconjugant were obtained by using Etest (bioMérieux, Marcy l’Etoile, France) and the agar dilution method based on the Clinical and Laboratory Standards Institute guidelines (). Multilocus sequence typing (MLST) of isolate GN529 was performed as described (). The Pasteur Institute online database (www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html) was used to assign the allelic numbers and sequence type (ST).

To screen for the most commonly known β-lactamase genes in enterobacteria, we performed multiplex PCRs (). Primers were designed (NDM-F, 5′-AATGGAATTGCCCAATATTATGC-3′; NDM-R, 5′-CGAAAGTCAGGCTGTGTTG C-3′) for the specific detection of blaNDM-1 and included in 1 of the multiplex PCRs (multiplex V). Primers NDM-F and NDM-R2 (5′-TCAGCGCAGCTTGTCGGC-3′) were used to amplify and sequence the entire blaNDM-1 gene. The samples were screened for the presence of six 16S methylase genes (armA, rmtA–D, and npmA) by PCR. E. coli J53 transconjugants were selected on Luria-Bertani plates containing sodium azide and meropenem (100 µg/mL and 1 µg/mL, respectively). The plasmid harboring blaNDM-1 was identified by Southern blot analysis by using a specific digoxigenin-labeled blaNDM-1 probe (Roche Diagnostics, Indianapolis, IN, USA).

K. pneumoniae GN529 was highly resistant to all β-lactams, aminoglycosides, quinolones, tetracycline, nitrofurantoin, and co-trimoxazole. MICs of 0.5 µg/mL for colistin (European Committee on Antimicrobial Susceptibility Testing colistin breakpoint for Enterobacteriaceae: susceptibility <2 μg/mL) and 1 μg/mL for tigecycline (European Committee on Antimicrobial Susceptibility Testing and US Food and Drug Administration tigecycline breakpoint for Enterobacteriaceae: susceptibility <1 and <2 μg/mL, respectively) were also obtained (Table).

Table

Antibacterial drug susceptibility profiles and resistance genes of Klebsiella pneumoniae GN529 clinical isolate and its Escherichia coli transconjugant, Ontario, Canada, 2010*

Antibacterial drug or gene	MIC, μg/mL
	Kpn GN529	Eco J529	Eco J53
Ampicillin	>256	>256	6
Cefoxitin	>256	>256	8
Ceftazidime	>256	>256	0.19
Cefotaxime	>256	>256	0.094
Cefepime	48	48	0.064
Ertapenem	32	12	0.008
Meropenem	>32	4	0.023
Imipenem	>32	32	0.38
Amikacin	>256	>256	1.5
Gentamicin	>256	>256	1.5
Tobramycin	>256	>256	1
Ciprofloxacin	>32	0.012	0.012
Tetracycline	>16	0.78	1
Tigecycline	1	ND	ND
Nitrofurantoin	>512	ND	ND
Colistin	0.5	ND	ND
Co-trimoxazole	4/76	ND	ND
PCR† and sequencing
blaNDM-1	+	+	ND
blaCTX-M-15	+	−	ND
blaSHV-12	+	+	ND
blaSHV-11	+	–	ND
blaOXA-1	+	–	ND
blaTEM-1	+	–	ND
armA	+	+	ND

*Kpn, Klebsiella pneumoniae; Eco J529, Escherichia coli transconjugant strain; Eco J53, recipient E. coli J53; ND, not determined.
†PCR screening included blaTEM, blaSHV, blaOXA-1-like, blaCTX-M groups 1, 2, and 9, blaVEB, blaPER, blaGES, blaOXA-48-like, blaIMP, blaVIM, blaKPC, blaNDM-1, and 6 groups of blaAmpC genes.

Considering the travel history of the patient and the high level resistance to all β-lactams, molecular screening of β-lactamases in strain GN529 was initiated to identify possible carbapenemases (e.g., blaNDM-1) in that isolate. Five β-lactamases genes (blaNDM, blaSHV, blaTEM, group 1 blaCTX-M , and blaOXA) and one 16S rRNA methylase (armA) were detected. By using primers for amplification of complete genes, we obtained sequences of blaNDM-1, 2 extended-spectrum β-lactamases (blaCTX-M-15 and blaSHV-12), 3 broad-spectrum β-lactamases (blaSHV-11, blaTEM-1 and blaOXA-1), and methyltransferase armA. No AmpC β-lactamases were linked to this isolate. Southern blotting identified a plasmid of ≈150 kb harboring blaNDM-1 (data not shown). A transconjugant E. coli positive for blaNDM-1 (E. coli J529, Table) was resistant to all β-lactams and aminoglycosides tested. In addition, blaSHV-12 and armA were detected in strain J529 (Table), indicating the potential for the horizontal spread of these resistance genes.

K. pneumoniae GN529 was typed by MLST as ST147, the same type as a clinical NDM-1–producing strain isolated in Australia () but distinct from ST14 and ST16 strains described (,). There are insufficient MLST data to confirm polyclonal dissemination of NDM-1, but previous pulsed-field gel electrophoresis results support that hypothesis ().

K. pneumoniae GN529 was isolated from a patient who had recently received emergency medical care in India, suggesting importation of this clinical strain. In the United Kingdom, where Enterobacteriaceae containing blaNDM-1 are increasingly common, carriage of these organisms has been closely linked to receipt of medical care in the Indian subcontinent (). Similar association as a risk factor was observed in other regions, including blaNDM-1-positive clinical strains isolated in North America, Australia, and Africa (–,).

The NDM-1–producing enterobacteria described in this study previously had low MICs only for colistin and tigecycline (,,,). However, an NDM-1 isolate resistant to these antimicrobial drugs has also been described (). Early detection and implementation of infection control interventions is essential for preventing the spread of multidrug-resistant organisms such as these. It may be prudent to consider medical exposure in the Indian subcontinent as a risk factor for possible infection, colonization, or both with multidrug-resistant, NDM-1–producing Enterobacteriaceae.