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Indian Journal of Medical Microbiology, Vol. 29, No. 3, July-September, 2011, pp. 249-253 Original Article Development of TaqMan real-time polymerase chain reaction for the detection of the newly emerging form of carbapenem resistance gene in clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii V Manchanda1, S Rai2, S Gupta3, RS Rautela4, R Chopra1, DS Rawat4, N Verma1, NP Singh2, IR Kaur2, P Bhalla5 1 Clinical Microbiology and Infectious Diseases, Chacha Nehru Bal Chikitsalaya, (Affiliated to Maulana Azad Medical College), Govt of NCT of Delhi, Geeta Colony, Delhi - 110 031, India Date of Submission: 16-Jun-2011 Code Number: mb11061 PMID: 21860104 DOI: 10.4103/0255-0857.83907 Abstract Purpose: The newly emerging form of the so-called New Delhi Metallo-beta-lactamases (NDM-1) has been reported recently from patients worldwide and broadly thought as a potential source for the major global health problem. Thus, it is important to study the epidemiology of the so-called NDM-1 harbouring bacteria to prevent its further spread and to place effective control measures. The present study describes the use of the real-time polymerase chain reaction (PCR) assay for the detection of the bla NDM-1 gene using TaqMan probes among clinical isolates. Materials and Methods: Clinical isolates of Escherichia coli (11 strains), Klebsiella pneumoniae (17 strains) and Acinetobacter baumannii (six strains) that were resistant to either of the carbapenems (meropenem or imipenem) were included in the study. The presence of carbapenemases in such strains was confirmed using the modified Hodge test. A real-time PCR assay was optimized for the detection of NDM-1 using a cloned synthetic gene fragment followed by testing of the clinical isolates. The findings were further confirmed using PCR and gene sequencing. Results: TaqMan probe assay displayed a good detection limit with analytical sensitivity of the assay up to 10 copies of bla NDM-1 gene per reaction. The isolates of E. coli and K. pneumoniae revealed narrow range crossing point values (Cp values) between (12-17) cycles (mean Cp value 14), indicating number of bla NDM-1 gene copies of 106-108. The wider range of Cp values (15-34) cycles with a higher mean Cp value (23.6) was observed in A. baumannii with number of bla NDM-1 gene copies of 103-108. Conclusions: The study demonstrates that real-time PCR assay based on TaqMan chemistry is a useful technique for the detection of bla NDM-1 harbouring clinical isolates of E. coli, K. pneumoniae and A. baumannii. The assay has great precision in measuring the number of bla NDM-1 gene copies per specimen of DNA.Keywords: Carbapenemases, metallo-beta-lactamases, modified Hodge test, multi-drug resistant organisms, NDM-1 Introduction Carbapenemases are a diverse group of beta-lactamases that hydrolyze carbapenems, the Klebsiella pneumoniae carbapenemase (KPC) being the most common member. The so-called New Delhi Metallo-beta-lactamase (NDM-1) is a newer type of metallo-beta-lactamase first described in 2009 in K. pneumoniae. [1] It is similar to KPC in hydrolyzing carbapenems and making them ineffective. However, unlike the former, which are Ambler class B metallo-beta-lactamases and are inhibited by EDTA, KPC are class A, serine carbapenemases that are inhibited by boronic acid. Bacteria harbouring bla NDM-1 are also found to be resistant to large group of antimicrobials, including all beta-lactams, carbapenems, fluoroquinolones and aminoglycosides, leaving only polymyxins as therapy of choice among patients infected with such bacteria. [2],[3] The bla NDM-1 gene is present on plasmids and other mobile genetic elements, enabling its spread rapidly among other bacterial strains and species. [1],[4] Thus, it is important to study the epidemiology of NDM-1 harbouring bacteria to prevent its further spread and to place effective control measures. The present study describes the use of the real-time polymerase chain reaction (PCR) assay for the detection of the bla NDM-1 gene using Taq Man probes. Recently, a study has described the use of this technology in laboratory using Escherichia coli strain transformed with cloned plasmid carrying bla NDM-1 gene. [5] This is the first study to evaluate the performance of a real-time PCR assay in the detection of NDM-1 in clinical strains of E. coli, K. pneumoniae and Acinetobacter baumannii. Materials and Methods A total of 34 clinical strains isolated from blood, urine, cerebrospinal fluid, pus and tracheal aspirates of patients admitted to the hospital during June 2009 to November 2010 were included in the study. These bacterial strains, including E. coli (11 strains), K. pneumoniae (17 strains) and A. baumannii (six strains), were found to be resistant to meropenem and/or imipenem. Isolates were identified using the Vitek-2 compact automated system (BioMerieux, Marcy, France). Antimicrobial susceptibility to ampicillin + sulbactam, ceftriaxone, cefotaxime, ceftazidime, cefepime, piperacillin + tazobactam, gentamicin, amikacin, netilmicin, ciprofloxacin, levofloxacin, meropenem, imipenem and colistin were determined using the E test (BioMerieux). Interpretation was carried out using CLSI guidelines. [6] Screening for the presence of carbapenemases was performed by the modified Hodge test and disk synergy test (EDTA with imipenem). [7],[8] DNA extraction DNA extraction for all bacterial strains was performed using the MagnaPure compact automated nucleic acid extraction system (Roche Diagnostics, Basel, Switzerland) as per the manufacturer′s protocol for isolation of bacterial whole cell nucleic acid. Additionally, DNA was extracted from bacterial strains of E. coli ATCC 25922, K. pneumoniae ATCC 700603, A. baumannii 19609 and Pseudomonas aeruginosa ATCC 27853 (Microbiologics, St. Cloud, Minnesota, USA) were also included in the testing panel as negative control strains. Strains harbouring bla NDM-1 gene, identified by PCR and gene sequencing, were included as positive controls. Conventional polymerase chain reaction assay for the detection of the NDM-1 gene Detection of bla NDM-1 gene was performed by the conventional PCR assay. A 475 bp region of NDM -1 gene was amplified through the conventional PCR assay using NDM-1-specific primers F-(5′-GGG CAG TCG CTT CCA ACG GT-3′) and R-(5′-GTA GTG CTC AGT GTC GGC AT-3′) (synthesised at NCDC, Delhi, India). The PCR mix of 50 μl contained 100 ng total DNA as template, 1x PCR buffer with 1.5 mM MgCl 2 , 0.2 mM of each dNTP, 0.2 μM of each primer, 1.25 U Ampli Taq DNA polymerase (Applied Biosystems, Foster City, CA, USA) and nuclease-free water to make up the final volume. The initial step of the reaction was denaturation of DNA at 94 o C for 3 min, followed by 30 cycles of denaturation, annealing and extension at 94°C, 60°C and 72°C, respectively, for 30 s each. The final extension step was performed for 3 min at 72°C. The PCR product containing amplicons was analyzed in 1.5% agarose gel in 1x TAE buffer at 90 V for 1 h and was visualized with ethidium bromide under a gel documentation system (Biometra GmbH, Goettingen, Germany). Sequencing polymerase chain reaction The PCR products were purified using the PCR 96 clean up kit (Millipore Corporation, Bedford, MA, USA) as per the manufacture′s protocol before sequencing. The sequencing was carried out using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) as per the standard protocol. Briefly, each 10 μl sequencing reaction contains 1 μl big dye, 2 μl 5x sequencing buffer, 1 μl primer (10 μM) and 1 μl purified PCR product. The cycle sequencing parameters were: 25 cycles at 96°C for 10 s, 50°C for 5 s and 60°C for 4 min. The reaction mixture was purified with a Montage 96 well purification kit (Millipore Corporation, Billerica, MA, USA) as the manufacture′s protocol. The DNA pellet was re-suspended in 10 ul of hi-di formamide, heated at 95°C for 2 min and immediately chilled on ice, mixed after a brief spin and finally loaded on the ABI 3130x1 genetic analyser (Applied Biosystems) for sequencing. The sequences of the study specimens were compared with the previously reported sequences in the NCBI database using the BLAST search. [9] Real-time polymerase chain reaction assay Real-time PCR assay was performed using the primer and probe sequences derived from the Genbank sequence AB571289. Primer sequence F-(5′-GCT GGC GGT GGT GAC TC-3′), R-(5′-GGC AAG CTG GTT CGA CAA C-3′) and TaqMan probe sequence ′FAM-TGG CAT AAG TCG CAA TCC CCG C-BBQ′ were used (kindly provided by TIB MOLBIOL, Berlin, Germany). The assay contained 5 μl (10 ng of whole cell DNA) along with 0.5 μM of the primers and 0.2 μM of the probe was used along with the LightCycler 480 probe master mix (Roche Diagnostics). The PCR cycles were performed running 10 min denaturation at 95°C followed by 45 cycles each at 10 s at 95°C, 1 min at 58°C and 1 s at 72°C (LightCycler 480 instrument). The assay was optimized using a cloned synthetic gene fragment covering the region 35-366 (AB571289). This was followed by assay consisting of clinical strains. E. coli ATCC 25922, K. pneumoniae ATCC 700603, A. baumannii ATCC 19609 and P. aeruginosa ATCC 27853 were included in the testing panel as negative control strains. Strains harbouring bla NDM-1 gene, identified by PCR and gene sequencing, were included as positive controls. Load of bla NDM-1 gene was calculated through a standard curve plotted using log10 dilutions of synthetic peptide. Results All the isolates were found to be resistant to ampicillin + sulbactam, ceftriaxone, cefotaxime, ceftazidime, cefepime, piperacillin + tazobactam, gentamicin, netilmicin, ciprofloxacin and levofloxacin. Resistance to amikacin, imipenem and meropenem was observed in 88%, 90% and 71% of the isolates, respectively. Resistance to both carbapenems (meropenem and imipenem) was observed among 19 (56%) of the strains. Resistance to imipenem with susceptibility to meropenem was observed in 10 (29%) of the isolates. None of the isolates were found to be resistant to colistin. MIC90 for both the carbapenems (imipenem and meropenem), among A. baumannii, E. coli and K. pneumoniae were >32 mcg/ml, 16 mcg/ml and >32 mcg/ml, respectively. The modified Hodge test confirmed the presence of carbapenemases in all the clinical strains. The amplification products of the conventional PCR assay revealed a 475 bp amplified product [Figure - 1]. The sequences of the present study revealed 100% homology with most of the NDM-1 sequences reported from other parts of the world (accession number: HQ2529057.1, AB571289.1, HQ162469.1, FN396876.1, HQ171206.1, HM853678.1 and HQ256747.1) available in the global genome data base, earlier reported in E. coli strains having mettallo-beta-lactamase-1 gene from India and Australia, in K. pneumoniae strains having bla NDM-1 gene from India and Sweden and also in Entercoccus faecium strain ZW031-2 having bla NDM-1 gene from China. TaqMan probe assay was established and validated using a synthetic gene fragment. The assay displayed a very good detection limit, with analytical sensitivity (limit of detection) of the assay up to 10 copies per reaction. The linear range covers 10 6 to ten copies, reaching 100% of the fluorescent signal over the entire range [Figure - 2]. The assay was analysed for its specificity, running a panel of known NDM-1 harbouring clinical strains and panel of ATCC strains found negative for NDM-1 gene by conventional PCR and gene sequencing. The assay did not show interference with other clinically important bacterial species nor with any isolates known to contain other beta-lactamase genes (ESBL, AmpC; data not shown), allowing a rapid analysis among clinical bacterial strains. Replicates of varied concentrations of synthetic peptide and few clinical isolates were run to determine the precision of the assay. The standard deviation of the concentration value was found to be <0.0834. The isolates of E. coli and K. pneumoniae revealed Crossing point values (Cp values) between 12 and 17 cycles (mean Cp value 14; [Figure - 3], [Table - 1]), indicating the similar number of bla NDM-1 gene copies (10 6 -10 8 ). However, a wider range of Cp values (15-34) with a higher mean Cp value (23.6) was observed in A. baumannii. This may be attributable to the variability in number of the copies of bla NDM-1 gene among A. baumannii (10 3 -10 8 ). Discussion Infections due to bacteria harbouring the so-called NDM-1 gene poses great therapeutic challenge as these bacteria are resistant to almost all tested antimicrobials, except colistin. The so-called NDM-1 gene was first described in 2009 for a Swedish patient. [1] Within 2 years, NDM-1 harbouring bacterial isolates has been described worldwide. [1],[10],[11],[12],[13] Many of the carbapenem-resistant bacteria isolated from patients from several parts of India carried the bla NDM-1 gene. [2],[14],[15] Till date, the two methods that have been described for the detection of bla NDM-1 gene include conventional PCR assay and DNA probes. [1],[14],[15] In the present study, TaqMan probe assay for the detection of bla NDM-1 gene was first established and validated with PCR and gene sequencing using a synthetic gene fragment. The TaqMan assay displayed a very good detection limit, with analytical sensitivity (limit of detection) of the assay up to 10 copies per reaction. Low standard deviation (<0.08) among the results of gene copies and among replicate assays indicates that the assay has great precision in measuring the number of bla NDM-1 gene copies per specimen of DNA. The Cp values of the three bacterial species tested in the present study revealed that E. coli and K. pneumoniae have a narrow range of Cp values and similar mean Cp values, indicating similar number of bla NDM-1 gene copies. This may be due to the clonal nature of the plasmids or similar number of plasmids carrying bla NDM-1 gene among the two bacterial species. Lower Cp values in the two bacterial species also indicated a higher number of bla NDM-1 gene copies. However, wider range of Cp values with higher mean Cp values in A. baumannii revealed variability of the number of copies of bla NDM-1 gene among the bacterial species. This observation can be attributed to the multiclonal nature of the strains and/or plasmids such strains carry. The study also demonstrates that all the NDM-1 harbouring bacterial species were positive for the modified Hodge test. Thus, like KPC, the test can be used to detect NDM-1 harbouring bacteria, but will not be able to differentiate between the type of carbapenemases. [16] The study successfully demonstrates the utility of the real-time PCR assay based on TaqMan chemistry as a useful technique in the detection of NDM-1 harbouring clinical isolates of E. coli, K. pneumoniae and A. baumannii. The technique being lesser labour-intensive than conventional PCR assay and lesser time-consuming will allow laboratories to detect such strains in a more efficient manner. Additionally, when performed as quantitative real-time PCR, the bla NDM-1 gene load can also be determined with great precision using this technique, and is helpful in bringing more insights to the epidemiology of bacterial strains harbouring such genes. Because of the difficulty in treating patients infected with NDM-1 harbouring bacterial pathogens, it is necessary to identify such strains as soon as possible. Moreover, studying the epidemiology of such resistant strains helps to limit the spread of such strains in hospital settings as well as in the community, and also helps in initiating specific hospital infection control measures. References
Copyright 2011 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb11061f1.jpg] [mb11061f3.jpg] [mb11061t1.jpg] [mb11061f2.jpg] |
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