The establishment of a duplex real-time PCR assay for rapid and simultaneous detection of blaNDM and blaKPC genes in bacteria

The latest threat of multidrug-resistant Gram-negative bacteria corresponds to the emergence of carbapenemase New Delhi metallo-β-lactamase (NDM) and Klebsiella pneumoniae carbapenemase (KPC) producers. Rapid molecular detection is essential to limit their spread. In this study, a duplex real-time polymerase chain reaction (PCR) that was specific for the detection of blaNDM and blaKPC with the same limit of detection of ten plasmid copies was developed. The assay was linear over eight log dilutions for blaNDM (R2 = 0.971; slope, -3.273) and blaKPC (R2 = 0.992; slope, -2.997) with efficiencies of 102% and 115%, respectively. The assay was validated with 157 clinical isolates and showed 100% concordance with conventional PCR. The excellent performance of the duplex PCR assay makes it a powerful tool for surveillance of the carbapenemases NDM and KPC.

The increasingly common class A enzyme KPCs and the latest metallo-β-lactamase NDMs are able to hydrolyze nearly all β-lactams; moreover, almost all of their producers are broadly resistant to other drug classes in addition to βlactams due to a diversity of other resistance mechanisms (e.g. to aminoglycosides and fluoroquinolones), leaving physicians with limited antibiotic choices for treating infected patients [1,6,7]. KPC enzymes have been reported in numerous Enterobacteriaceae genera and even in nonfermenting bacteria, although KPCs are mostly identified from K. pneumoniae. In addition, KPC-producing isolates have disseminated worldwide, including several countries across Asia, America and Europe. The bla KPC genes are almost present on transferable plasmids and are flanked by transposable elements [8][9][10], which allows for potential dissemination among different species. Similarly, the bla NDM genes are primarily located on plasmids belonging to several incompatibility groups, and the diversity of genetic features associated with the bla NDM genes may explain its current high worldwide spread rate [11,12]. NDM enzymes have been recognized among different Enterobacteriaceae species as well as in non-fermenters and Vibrionaceae and have been reported worldwide in Asia, Europe, North America, Australia and the Middle East since 2008. To date, thirteen bla KPC gene variants (classified in sequential numeric order from bla KPC-1/2 to bla KPC-15 ) have been described and six minor NDM variants (bla NDM-2 to bla NDM-8 ) have been identified (http:// www.lahey.org/studies/).
The carbapenemases KPC and NDM have already become a worldwide public health issue due to their widespread distribution, broad range of activity against β-lactams and aggressive hazards to human beings. As a result, the sensitive and rapid detection of the bla NDM and bla KPC genes is essential for implementation of the infection control procedures that are required to limit their spread and to help clinicians guide individual patient management. In this study, we developed a duplex TaqMan probe-based real-time polymerase chain reaction (PCR) assay for the prompt and simultaneous detection of the bla NDM and bla KPC genes in bacteria in a single tube.

Primer and probe design
The details of the reference genes used in this assay are provided at http://www.lahey.org/studies/. The reference gene sequences for the NDM and KPC enzyme families were assembled from GenBank (http://www.ncbi.nlm.nih. gov/GenBank) as NDM accession numbers FN396876, JF703135, JQ734687, JQ348841, JN104597, JN967644, JX262694 and AB744718 representing alleles 1-8 and as KPC accession numbers AY034847, AF395881, AY700571, EU400222, EU555534, EU729727, FJ234412, FJ624872, GQ140348, HM066995, HQ641421, HQ342890, JX524191 and KC433553 representing alleles 2-15. Based on the comprehensive analyses and alignments of both gene families, primers and probes for the duplex real-time PCR assay were specifically designed to amplify all alleles of each gene family described above using Beacon Designer software (Premier Biosoft, Palo Alto, CA, USA) ( Table 1). All of the primers and probes were synthesized by TaKaRa Bio, Inc. (Dalian, China).

Bacterial strains and antibiotic susceptibility testing
A total of 157 Enterobacteriaceae isolates were collected at Nanfang Hospital from June 2010 to September 2012, including 17 resistant to imipenem or meropenem isolates and 140 randomly collected isolates with susceptible to imipenem and meropenem. All the isolates had previously been tested for bla KPC , bla IMP , bla VIM , bla NDM and bla OXA-48 using conventional PCR and sequencing [13][14][15]. Of them, 17 carbapenem-resistant isolates harbored the carbapenemase genes bla KPC , bla IMP or bla NDM , including nine bla KPC-2 -positive K. pneumoniae, one K. pneumoniae carrying bla KPC-2 and bla IMP-4 , three bla NDM-1 -positive K. pneumoniae, one Klebsiella oxytoca and one each bla NDM-1 -positive Enterobacter hormaechei, Enterobacter cloacae and Enterobacter aerogenes. The other 140 Enterobacteriaceae isolates were negative for the five tested carbapenemase genes. In addition, ten NDM/KPC non-producers, including Escherichia coli ATCC 25922, K. pneumoniae ATCC 700603, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853 and six known clinical strains without any carbapenemase gene(two Enterococcus spp., two Streptococcus spp. and two Staphylococcus spp.), were used to evaluate the specificity of the duplex qPCR assay.
All bacterial identification and susceptibility testing was performed using a BD Phoenix 100 Automated Microbiology System (Benton, Dickinson and Co., Franklin Lakes, NJ, USA). The results were interpreted according to the Clinical and Laboratory Standards Institute guidelines. The isolates were stored at −80°C in nutrient broth containing 30% (v/v) glycerol.

DNA extraction
Fresh, well-isolated colonies were used for total bacterial DNA extraction using a TaKaRa MiniBEST DNA Fragment

Standard curve and sensitivity test on recombinant plasmids
To determine the efficiency of the duplex real-time PCR assay, Ct values obtained from a series of template DNA dilutions were graphed on the y axis versus the log of the dilution on the x axis. The slope of this line was used to determine efficiency (E) according to this equation: E = 10 (−1/slope) . Assay sensitivity and reproducibility were estimated by serial ten-fold dilution experiments using the mixture of recombinant plasmids pNDM-1 and pKPC-2.

Results
The assay was initially validated using the recombinant plasmids pNDM-1 and pKPC-2 carrying the bla NDM-1 and bla KPC-2 genes. Clearly defined amplification curves were observed for the two positive recombinant plasmids in their predicted fluorescence channels. Primer and probe specificities were evaluated at the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov). No matches to the primer or probe sequences of bla NDM and bla KPC were found other than those for corresponding bla NDM and bla KPC genes. The analytical specificity was also applied on clinically representative bacteria with no known antibiotic resistance gene, including E. coli ATCC 25922, K. pneumoniae ATCC 700603, S. aureus ATCC 25923, P. aeruginosa ATCC 27853, Enterococcus spp. (two isolates), Staphylococcus spp. (two isolates) and Streptococcus spp. (two isolates). The recombinant plasmids pNDM-1 and pKPC-2 were used as positive controls. No amplification of bla NDM or bla KPC was observed with DNA extracted from any of the ten organisms on our specificity test panel. Thus, the specificity of our duplex PCR is considered satisfactory.
Assay linearity and limit of detection were determined by performing serial ten-fold dilutions of a mixture of the recombinant plasmids pNDM-1 and pKPC-2 from 10 to 10 8 copies/μL (Figure 1). The assay correlated well with bla NDM (R 2 = 0.971) and bla KPC (R 2 = 0.992) over the entire copy range with an efficiency of 102% and 115%, respectively. The limit of detection for both targets was 10 copies per 20-μL reaction volume.
We have also shown that the assay was highly stable and precise as evidenced by the performance of the recombinant plasmid pNDM-1 (10 5 copies of bla NDM-1 -carrying plasmid) and pKPC-2 (10 5 copies of bla KPC-2 -carrying plasmid), which were stable over five consecutive runs for both bla NDM (mean Ct, 22.8; standard deviation, 0.4; coefficient of variation, 1.8%) and bla KPC (mean Ct, 19.3; standard deviation, 0.1; coefficient of variation, 0.5%).
Finally, when tested against the 157 previously characterized carbapenem-resistant Enterobacteriaceae isolates, including seven with NDM-1 metallo-carbapenemases, ten with KPC-2 serine carbapenemases and 140 strains without bla NDM and bla KPC carbapenemases, the duplex PCR assay showed 100% concordance with conventional PCR previously identified. In our opinion, the 100% sensitivity and specificity of the duplex PCR validated against clinical isolates of carbapenem-resistant Gramnegative bacilli with well-defined carbapenemase genes make it a useful tool for the screening and surveillance of isolates carrying the carbapenemase genes bla NDM and bla KPC .

Discussion
The co-occurrence of bla NDM-1 and bla KPC-2 in a clinical isolate of K. pneumoniae from India was recently reported [7], suggesting that the growing emergence of these powerful resistance mechanisms should be given great attention. Considering the difficulty in preventing their emergence due to the potential for rapid horizontal and vertical transmission determined by genetics and treating patients infected with bla NDM and bla KPC harboring bacterial pathogens, active surveillance and early detection are mandatory for preventing their further spread.
Molecular assays have been reported to identify carbapenemase genes in Gram-negative bacteria. Several real-time PCR assays targeting NDM-1 or KPC have been described [16][17][18][19][20], but each targets only one kind of carbapenemase gene. Assays that target more than one class of carbapenemase (A, B, D) have been developed [15,21], but each of those uses conventional PCR requiring post-amplification analysis. Real-time PCR requiring less time and labor for the simultaneous detection of multiple carbapenemase genes including NDM and KPC is rare. Monteiro et al. described a multiplex real-time PCR for the rapid detection of KPC, GES, NDM, IMP, VIM and OXA-48 carbapenemase genes using melt curve analysis [22], however, no details of the assay design for the detection of all bla NDM types other than bla NDM-1 were provided. The presently described duplex PCR assay is capable of simultaneously detecting all of the minor bla NDM-1 variants since the nucleotide changes between the variants are located outside the primer-and probe-binding sequences. Likewise, in the case of bla KPC , the assay described here is expected to amplify all known bla KPC types. Although our duplex PCR assay was validated with Gram-negative bacteria, it could amplify the expected product from all bla KPC -and bla NDM -carrying isolates, including Gram-positive bacteria. Recently, Cunningham SA et al. described a FRET hybridization probe-based real-time PCR assay that targets bla KPC and bla NDM in a single assay [23]. Although the assay performed equivalently to our assay, the FRET hybridization probe is more expensive than ours TaqMan probe. Additionally, the FRET hybridization probe-based real-time PCR assay needs LightCycler instrument(Roche Applied Science, Indianapolis, IN) to match it, which limited its wide application; the classic TaqMan probe in our assay is suitable for any real-time PCR detection system, which has wider applicability.
In conclusion, we successfully established a novel duplex real-time PCR assay for the prompt and simultaneous screening of the bla NDM and bla KPC genes in a single reaction that had good sensitivity and specificity and excellent agreement with conventional PCR and sequencing. The excellent performance of duplex PCR makes it an important tool for guiding the appropriate choice of antimicrobial therapy and helping limit spread of the resistance genes through aggressive infection control measures. Its use would be especially suitable for national epidemiological purposes in an outbreak situation thanks to its comprehensive ability to detect all known bla NDM and bla KPC variants.