Rapid detection of the blaNDM-1 gene by real-time PCR

Sir, New Delhi metallo-β-lactamase-1 (NDM-1) is a recently reported novel plasmid-borne metallo-β-lactamase that represents an emerging public health threat.1 To date, the international spread of NDM-1 has already been reported to diverse locations such as the UK, the USA, Japan, Australia and most recently the Middle East.2 With the increasing need for efficient surveillance and detection of NDM-1,3 a rapid assay for the detection of this gene is also required. We describe a real-time PCR assay for the rapid detection of NDM-1 that was evaluated on 47 well-characterized carbapenem-resistant Enterobacteriaceae and Pseudomonas aeruginosa, 12 of which were positive for blaNDM-1. Enterobacteriaceae (Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Enterobacter cloacae and P. aeruginosa) were derived from ertapenem-resistant (MIC ≥ 1 mg/L) clinical isolates collected from three hospitals, with 89% also demonstrating resistance (MIC > 4 mg/L) to imipenem, meropenem or doripenem. All P. aeruginosa isolates were resistant to the four tested carbapenems (MIC ≥ 64 mg/L). Detection of the blaNDM-1 gene was initially performed by conventional PCR using forward (5′-GAA GCT GAG CAC CGC ATT AG-3′) and reverse (5′-GGG CCG TAT GAG TGA TTG C-3′) primers, and all positive PCR results were confirmed by sequencing the obtained amplicon. Study isolates were also screened for extended-spectrum β-lactamase (ESBL) genes (blaSHV, blaTEM and blaCTX-M),4,5 metallo-β-lactamase genes (blaIMP, blaVIM, blaSPM-1, blaGIM-1 and blaSIM-1),6 the blaKPC gene7 and plasmid-borne ampC genes.8 Twelve Enterobacteriaceae isolates were positive for the blaNDM-1 gene (eight K. pneumoniae, two Enterobacter cloacae, one E. coli and one P. mirabilis) with the remaining 30 Enterobacteriaceae possessing combinations of ESBL genes (blaCTX-M and/or blaSHV) (n = 30), blaDHA-like ampC genes (n = 20) and metallo-β-lactamase genes (blaIMP) (n = 4). The five P. aeruginosa isolates were positive for blaVIM.