Detection of Malaysian methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates using simplex and duplex real-time PCR

Editorial Manager(tm) for World Journal of Microbiology and Biotechnology Manuscript Draft Manuscript Number: Title: Simplex Real-time PCR SYBR Green I assay for detection of Methicillin-resistance Staphylococcus aureus (MRSA) and antibiotic susceptibility profiles from Malaysian clinical isolates Article Type: Full-length Paper Keywords: Key words: Antibiotic-susceptibility, methicillin-resistant Staphylococcus aureus (MRSA), simplex real-time PCR, coa, nuc, and mecA genes. Corresponding Author: Mr zarizal suhaili, Bsc Biotech Corresponding Author's Institution: Universiti Darul Iman Malaysia First Author: zarizal suhaili, Bsc Biotech Order of Authors: zarizal suhaili, Bsc Biotech; Mohammad Hailmi Sajili, Bsc., Msc.; Saiful Azmi Johari, Bsc., Msc.; Mastura Mohtar, BSc., MSc.; Ahmad Rushdi Tan Abdullah, BSc.; Affandi Ahmad, BSc; Abdul Manaf Ali, BSc., MSC., Phd Abstract: The aims of this study is to compare methicillin-resistance Staphylococcus aureus (MRSA) detection methods and to generate antibiogram profile of selected S. aureus clinical isolates from east cost public hospital in Malaysia including characterized S. aureus clinical isolates from two teaching hospitals in Malaysia as well as reference isolates from American Type Culture Collection (ATCC). The simplex real-time PCR SYBR green amplification targeting three selected genes marker coa, nuc and mecA together with antibiotics disc diffusion test were applied to compare its MRSA detection abilities. No disagreement between the two methods was observed. From 19 bacterial isoaltes (including ATCC strains) tested, 11 isolates were confirmed as methicillin-resistance S. aureus with exhibiting multidrug-resistance. All isolates are still confer susceptible to vancomycin as indicated by antibiotic susceptibility test. Current antibiotic sesceptibility test are comparable with the simplex real-time PCR detection for coagulase positive MRSA while multidrug-resistance traits are present only in MRSA clinical isolates. Simplex real-time PCR assay was used for the simultaneos detection of nuc, coa (species-specific) and mecA (methicillin-resistance) genes from clinical isolates tested. Evaluation were based on the melting temperature (Tm) analysis of the amplicons using 32 bacterial isolates (including ATCC strains). Simples real-time PCR amplification products with melting peaks at 76.14 ± 0.8 ºC, 78.59 ± 0.4 ºC and 74.41 ± 0.6 ºC were detected for coa, nuc and mecA genes, respectively. Therefore, including these three primer pairs facilitates the definite identification of S. aureus isolates as well as the determination of their meticillin resistance genotypes supported with antibiograms profiles. Manuscript Click here to download Manuscript: MRSA antibiotic susceptibility profiles and simplexClick real time here PCR.doc to view linked References Simplex Real-time PCR SYBR Green I assay for detection of Methicillinresistance Staphylococcus aureus (MRSA) and antibiotic susceptibility profiles from Malaysian clinical isolates. Zarizal Suhaili, 1* Mohd Hailmi Sajili1, Saiful Azmi Johari2, Mastura Mohtar2, Ahmad Rushdi Tan Abdullah3, Affandi Ahmad3 and Abdul Manaf Ali1. 1 Faculty of Agriculture and Biotechnology, Universiti Darul Iman Malaysia (UDM), Kampus Kota, Jalan Sultan Mahmud, 20400 Kuala Terengganu, Terengganu, Malaysia 2 Antimicrobial Laboratory, Medicinal Plants Programme, Forest Biotechnology Division, Forest Research Institute Malaysia (FRIM), 52109 Kepong, Selangor, Malaysia 3 Department of Pathology, Hospital Sultanah Nur Zahirah, Jalan Sultan Mahmud, 20400 Kuala Terengganu, Terengganu, Malaysia *Corresponding author: Zarizal Suhaili Faculty of Agriculture and Biotechnology, Universiti Darul Iman Malaysia, Kampus Kota, Jalan Sultan Mahmud, 20400 Kuala Terengganu, Terengganu Tel: +609-627 5612 Fax: +609-627 5582 E-mail : zarizal@udm.edu.my 1 Abstract The aims of this study is to compare methicillin-resistance Staphylococcus aureus (MRSA) detection methods and to generate antibiogram profile of selected S. aureus clinical isolates from east cost public hospital in Malaysia including characterized S. aureus clinical isolates from two teaching hospitals in Malaysia as well as reference isolates from American Type Culture Collection (ATCC). The simplex real-time PCR SYBR green amplification targeting three selected genes marker coa, nuc and mecA together with antibiotics disc diffusion test were applied to compare its MRSA detection abilities. No disagreement between the two methods was observed. From 19 bacterial isoaltes (including ATCC strains) tested, 11 isolates were confirmed as methicillin-resistance S. aureus with exhibiting multidrug-resistance. All isolates are still confer susceptible to vancomycin as indicated by antibiotic susceptibility test. Current antibiotic sesceptibility test are comparable with the simplex real-time PCR detection for coagulase positive MRSA while multidrug-resistance traits are present only in MRSA clinical isolates. Simplex real-time PCR assay was used for the simultaneos detection of nuc, coa (species-specific) and mecA (methicillinresistance) genes from clinical isolates tested. Evaluation were based on the melting temperature (Tm) analysis of the amplicons using 32 bacterial isolates (including ATCC strains). Simples real-time PCR amplification products with melting peaks at 76.14 ± 0.8 ºC, 78.59 ± 0.4 ºC and 74.41 ± 0.6 ºC were detected for coa, nuc and mecA genes, respectively. Therefore, including these three primer 2 pairs facilitates the definite identification of S. aureus isolates as well as the determination of their meticillin resistance genotypes supported with antibiograms profiles. Key words: Antibiotic-susceptibility, methicillin-resistant Staphylococcus aureus (MRSA), simplex real-time PCR, coa, nuc, and mecA genes. 3 Introduction Staphylococcus aureus is well known as a major pathogen, causing a variety of nosocomial and community-acquired infections (Hiramatsu et al., 2001), it’s also reputedly known as one of the most problematic clinically relevant pathogens and ranks as one of the most difficult bacteria to treat in patients and eradicate in a hospital environment (Oluwatuyi et al., 2004). Coagulase production is the principle criterion used in the clinical microbiology laboratory for the identification of Staphylococcus aureus. The coagulase status of an isolates is not always easily established in a timely fashion, increasing delay in definitive identification of S. aureus (Schmitz et al., 1998). Usually, misidentification of S. aureus as coagulase-negative staphylococcus (CoNS) can result in a costly search for other pathogens or unwarranted broad-spectrum empiric antimicrobial coverage (Papasian and Garrison, 1999). Although the tube coagulase test for the detection of free coagulase is generally the standard test for differentiating S. aureus from CoNS, the time-consuming nature of the test (incubation for 4 to 24 h is required) often forces clinical microbiology laboratories to use more rapid alternatives (Griethuysen et al., 2001). The slide coagulase test, which detects bound coagulase (clumping factor) is rapid (<1 min), but 10 to 15% of S. aureus strains may yield a false-negative result (Kloos and Bannerman, 1994). In addition, methicillin resistance can be found both in S. aureus and in coagulasenegative staphylococci (CoNS) (Griethuysen et al., 2001). On the other hand, methicillin resistance mediated by PBP2a is often heterogeneously expressed in 4 staphylococci, polymerase chain reaction (PCR) detection of mecA gene is more accurate than the standard susceptibility methods, especially for coagulase negative staphylococci (CoNS) and S. aureus isolates with low-level resistance due to other mechanisms (Carrol et al., 1996). Molecular methods of oxacillin/methicillin-resistance S. aureus (MRSA) are generally based on detection of mecA gene and S. aureus species-specific gene (Rushdy et al., 2007; Saiful et al., 2006). Currently, there are several types of antimicrobial susceptibility tests that are available in laboratories across the world. One of the most convenient, low-budget and highly reproducible test is the antibiotic disc diffusion assay that uses standardized antibiotic-coated paper disc and placed onto an appropriate susceptibility test media as recommended by international antimicrobial susceptibility guidelines organizations such as the Clinical and Laboratory Standards Institute (CLSI) formerly known as the National Committee for Clinical Laboratory Standards (NCCLS) in the US or the British Society for Antimicrobial Chemotherapy (BSAC) in the UK. The disc diffusion method continues to be adequate for confirming susceptibility in bacteria, including S. aureus, as compared to the minimum inhibitory concentration (MIC) method as previously reported (Potz et al., 2004; Eloff, 1998). Although recent publications have shown several rapid and accurate molecular methods of antimicrobial susceptibility techniques being developed such as the real-time PCR and DNA Microarray [Miller et al., 2005; Perreten et al., 2005), the conventional microbiology based techniques is still preferred among researchers. This is due to the fact that conventional antibiotic susceptibility methods could test for several 5 antibiotics at the same time as compared to molecular methods (e.g. PCR) that requires numerous genetic assays (e.g. gene identification, primer design) to test for resistance for two or more antibiotics. Additionally, molecular testing methods may overestimate the presence of antibiotic resistance as they do not test for expression of the relevant resistant gene/s (Tan, 2003). A number of conventional gel-based PCR methods in the multiplex format have been published (Strommenger et al., 2003). Because methicillin resistance mediated by PBP2a (PBP2’) is often heterogeneously expressed in staphylococci, PCR detection mecA gene is more accurate than the standard susceptibility methods, especially for Coagulase-negative Staphylococci and S. aureus isolates with low-level resistance due to other mechanisms (Carrol et al., 1996). Molecular methods for rapid identification of MRSA are generally based on detection of mecA gene and S. aureus species-specific gene (Unal et al., 1992). There are several genes that have been used to identify S. aureus (e.g. nuc, gyrA, femA, and coa and Sa442) DNA fragment as a popular DNA target for identification of S. aureus by PCR (Grisold et al., 2002; Tan et al., 2001; Reischl et al., 2000; Martineau et al., 1998). Furthermore, several other PCR-based assays targeting this DNA fragment alone or in combination with a mecA for identification of MRSA also have been described (Saiful et al., 2006; Reischl et al., 2000; Martineau et al., 1998). The presence of MRSA in hospitals as a very important nosocomial pathogen is paralleled by an increase in the rate of bacteraemia associated with a high mortality rate (Cosgrove, et al., 2003). 6 Therefore, there is a need for rapid determination of the antibiotic profile of MRSA isolates in order to institute infection control measures and appropriate regiment. In this study, we investigate the antibiotics profiles by using antibiotics disc diffusions supported together with the simultaneous species-specific identification and detection of Coagulase Positive MRSA and MSSA by using simplex real-time PCR targeting three correlated genes coa, nuc, and mecA among selected Malaysia clinical isolates S. aureus. Materials and Methods Bacterial strains used All of the 32 bacterial strains as well as different genera used are listed in Table 1. This included sixth teen clinical isolates [HN1, HN 2, HN 3, HN 4, HN 5, HN 6, HN 7, HN 8, HN 9, HN 10, HN 11, HN 12, HN 13, HN 14, HN 15 and HN 16] taken from east coast public hospital at Kuala Terengganu; five [N 391, N 441, N 829, N 850 and N 1406] were from Hospital Universiti Kebangsaan Malaysia (HUKM), Cheras and five [UM 1, UM 2, UM 3, UM 6, and UM 7] were taken from Universiti Malaya Medical Center (UMMC), Kuala Lumpur. Standard methicillin-resistance S. aureus (MRSA) (ATCC 33591) and methicillin-sensitive S. aureus (MSSA) (ATCC 25923; ATCC 29213) strains were also employed as 7 reference and Staphylococcus epidermidis (ATCC 12228), Escherichia coli (ATCC 35218) and E. coli (ATCC 700728) were used as negative control strains for specificity of real-time PCR amplification in this study. Characterised clinical isolates from both HUKM and UMMC was used as additional PCR amplification control. Bacterial strains isolates were maintained on Protect Bacterial Preservers (Technical Service Consultants, Heywood, and Lancashire, England) and cultured in tripticase soy broth (TSB) (Difco Laboratories, Detroit, USA) at 37°C. Phenotypic identification and antimicrobial susceptibility testing of all strains (excluding the strains from east coast public Hospital from east coast, HN and negative control MSSA) used in this study was as previously reported by Saiful et al., (2006). Antibiotic Susceptibility Test The disc diffusion assay was performed by using the automated zone size reader; Osiris automated susceptibility detection systems (Bio-Rad, USA) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines and details as described (Suhaili et al., 2009). Discs containing 10 selected antibiotics were distributed on to the plates according to a predefined scheme using the Osiris Systems disc dispenser (Bio-Rad, USA). After incubation at 37°C for 18-24 h, the plates were read with the Osiris video instrument and the results were interpreted with the Osiris Extended Expert Systems (EES) according to the instructions of 8 the manufacturer. Variation in zone size measurements of ± 3 mm between the automated and manual readings were defined as tolerable (Sanchez et al., 2002). This test was carried out to phenotypically verify the presence of methicillinresistance in the HSNZ S. aureus isolates since both UMMC and HUKM isolates have been verified previously using conventional disc-diffusions test (Saiful et al., 2006). Genomic DNA extraction Whole genomic DNA extractions of S. aureus isolates and references strain were done using the Wizard Genomic DNA extraction kit (Promega Inc., Madison, WI, USA) without using lysostaphin. Colonies were grown overnight at 37 oC in TSB and centrifuged at 13,000 X g for 2 min. Cell pellets were resuspended in 500 μl of distilled water with 700 μl of lysozyme (30 mg/ml) and the mixture were incubated at 37 oC for 1 h. Other prepared solutions from the extraction kit were added accordingly to the manufacturer’s recommendations. Extracted DNA was stored at -20oC until PCR was performed. 9 Simplex real-time PCR SYBR Green Real-time PCR amplification with SYBR Green I dye was performed using RotorGene RG6000 cycler (Corbett Research, Mortlake, Australia) in the 36carousel rotor. Simplex real-time PCR primers and concentration used are listed in Table 2. The nuc and mecA primers used were previously described by Saiful et al., (2006) and Suhaili et al., (2009) respectively. Meanwhile primer for coa gene was newly used in this study. Amplification conditions were optimized for each of the three single gene targets, before proceeding to further characterize the detection assay. For each reaction, 12.5 µl of QuantiTect® SYBR Green PCR master mix (Qiagen) and all templates DNA in a final volume of 25 µl were employed. Cycling conditions began with an initial hold at 95 °C for 2 min, followed by 40 cycles consisting of 95 °C for 50 s, 58 °C for 50 s and 72 °C for 50 s. All isolates and negative control samples were tested in triplicate. 10 Results Antibiotic disc diffusion test The results of the susceptibility test for the 19 S. aureus clinically isolates including three ATCC reference strains against 10 selected antibiotics are shown in Table 1. All antibiotics inhibition zone diameters against the control strains S. aureus ATCC 33591(MRSA), ATCC 25923 and ATCC 29213 (MSSA) are in compliance with the NCCLS and BSAC guidelines. In this study, the pattern of antibiotic susceptibility of S. aureus differed significantly between MRSA and MSSA isolates. Resistance frequencies of S. aureus clinical isolates towards 10 antibiotics used in this study were: Penicillin (93.75%), Oxacillin and cefoxitin (68.75%), Gentamycin (56.75%), Erythromycin (50.00%), Clindamycin and Fusidic acid (37.50%), Trimethoprim (31.25%), and Rifampin (18.75%) meanwhile Vancomycin does not shows any resistance activity against all isolates. Multidrug-resistance frequency among oxacillin/methicillin resistance S. aureus isolates was; 80% (HN 13 and HN 16), 70% (HN 4, HN 5, HN 14, and HN 15), 60% (HN 3 and HN 12), 50% (HN 6), and the least resistance pattern against 10 antibiotics used was 30 % (HN 1 and HN 2).The most resistance isolates was HN 13 and HN 16 being resistant towards 8 out of 10 antibiotics used in this study meanwhile the most sensitive isolate was HN 7 which is susceptible to all the antimicrobial agents used. 11 Simplex real-time PCR assay Representative results obtained by simplex real-time PCR for the clinical isolates as well as ATCC reference strains that were tested simultaneously for coa, nuc and mecA genes are shown in Figure 1. The results for screening of 26 clinical isolates of S. aureus including 3 ATCC S. aureus reference strains as well as 2 non-staphylococci by using simplex real-time PCR SYBR Green I assay have been summarized in Table 3. MRSA and MSSA isolates were correctly identified as observed by each representative positive and distinct melting peak with specific temperatures. For coa and nuc amplification, all S. aureus clinical isolates including three ATCC S. aureus references strains produced single melting peak profiles at 76.18 ± 0.8 °C and Tm=78.39 ± 0.4°C, respectively. There were no amplification produced on negative strains control S. epidermidis (ATCC 12228), E. coli (ATCC 35218) and E. coli (ATCC 700728). Meanwhile, for mecA amplification, 19 out of 27 clinical isolates including 8 positive MRSA isolates from UMMC and HUKM as well as MRSA ATCC 33591 were positively identified as MRSA by a melting peak profile at Tm= 74.41 ± 0.6°C . No melting peaks were observed in the three ATCC negative control strains as described in nuc and coa genes amplification. As shown in Table 1 the presence of mecA in each of the 27 MRSA isolates is in total agreement with the disc diffusion test results. 12 To evaluate the specificity of three primers used, extracted DNA from 32 bacterial isolates including three negative control ATCC references strains were examined as templates (Table 3). All 29 S. aureus strains tested were positively identified as coagulase positive S. aureus by using real-time PCR targeting nuc and coa genes. On the other hand, 19 isolates included MRSA ATCC 33591 reference strain were also positive in real-time PCR targeting mecA genes. Specificity of primers and absence of unspecific products of primer dimers were also tested by analysing the reaction in 1.8 % (w/v) agarose gel stained with ethidium bromide. The molecular weight of amplified coa (Figure 2.1a, and 2.1b), nuc (Figure 2.2a and 2.2b), and mecA (Figure 2.3a and 2.3b) product was as expected, and no other non-specific amplification observed. Interestingly, there was no primer-dimer formation observed in all lanes of electrophoresis real-time PCR amplification products. Insilico PCR (data not shown) also performed in order to double confirm the specificity of the primers design. Thus, the primers used here conclusively specific against S. aureus clinical isolates as well as S. aureus ATCC references strains. Discussions All of the clinical isolates displayed multidrug-resistance towards 9 (90%) out of 10 antibiotics used except for the vancomycin. Multidrug-resistant (resist three or more group of antibiotics) S. aureus isolates was identified as a serious cause of 13 nosocomial infections, and more recently, community-acquired MRSA was recognized as an emerging problem in a number countries (Malik et al., 2006). From this study, the least effective antibiotics was Penicillin with 15 (93.75%) isolates out of 16 collected show the significant resistance pattern, and this results same as control strains from ATCC 33591 (MRSA), and both MSSA strains (ATCC 25213 and ATCC 25923). In agreement with the current data, while studying 87 strains of S. aureus isolated from nose of two teaching hospitals personnel against 14 different antibiotics in Tehran, Iran found that a large proportion (90.8%) of the S. aureus isolates was resistance against penicillin (Saderi et al., 2005). Reports from some Asian countries indicated that only MetRSA would exhibit multidrug-resistance (Saiful et al., 2006; Tambyah et al., 2003; Alborzi et al., 2000; Wongwanich et al., 2000). Several studies also noted the presence of non-multidrug resistant MetRSA strains often associated with community-acquired MetRSA (Watson et al., 2003; Ala’Aldeen 2002). Isolate HN 8, a methicillin-sensitive S. aureus (MetSSA) strain, shows multipleresistance trait agains gentamycin, fusidic acid, and penicillin. This observation concurs with the finding which implying MetSSA with multidrug-resistance traits may exist in a small percentage of hospital community (Fluit et al., 2001). Alternatively, other MetSSA strains HN 9, did not exhibit any multidrugresistance apart from fusidic acid except for HN 7. These findings suggest that the MetSSA isolates has been exposed to fusidic acid may developed if the drug is used alone (Chambers, 1997). In Malaysia practice, a fusidic acid-rifampin combination is used as an alternative oral regimen for the treatment of 14 bacteraemia musculoskeletal and cardiovascular infections caused by MRSA (Norazah et, al., 2002). Luckily all of the MetSSA isolates used in this study are still susceptible to rifampin. The data shows that the resistance of erythromycin (50%) was higher than of clindamycin (37.5%) even though it was not too significant among all MRSA and MSSA isolates. Staphylococal strains which posses the erm gene prevent macrolides and lincosamides (erythromycin and clindamycin) from binding to their target site (Fluit et al., 2001). Clindamycin is a poor inducer of the erm gene compared to erythromycin (Joseph and Kosinki. 2005), which explains the higher resistance rate to erythromycin compare with that to clindamycin observed in our study. Macrolides and lincosamide (clindamycin) resistance most commonly results an altered 23S ribosomal RNA resulting in the MLSB phenotype (macrolide-lincosamide-streptogramin Bresistant). The resistance gene can be either constitutively expressed or inducible. In the latter case, erythromycin is a more potent inducer of resistance results in S. aureus isolates that are sensitive to clindamycin but resistance to erythromycin (Johnigan et al., 2003). This is in agreement with the data for three clinical isolates (HN 3, HN, 6 and HN 12) which is showing susceptibility against clindamycin but resistance against erythromycin. On the other hand, 11 (68.75%) out of 16 isolates were considered resistance towards oxacillin and cefoxitin while the other 5 were remain susceptible to β-lactam antibiotics (oxacillin and penicillin). Traditionally, methicillin or oxacillin has been tested and results are representative of all β-lactams agents (Huang et al., 2004). Even though oxacillin has been used to replace methicillin since it is no longer the agent of choice for 15 testing or treatment of S. aureus infections (NCCLS 2004), the name methicillinresistance S. aureus (MetRSA) is still being used to describe any β-lactam resistance S. aureus instead of using oxacillin-resistance S. aureus (ORSA) which may reduce any confusion especially for those that who are not familiar with this pathogenic microorganism. However the acronym ORSA has been use in several refereed journals (Rutala et al., 2006; Anderegg et al., 2005; Christiansen et al., 2004). Lately, a lot of controversies have been raised regarding the effectiveness of the oxacillin disc diffusion method for the detection of methicillin-resistance S. aureus isolates as several studies have shown that the usage of cefoxitin disc diffusion method preferred over the oxacillin disc diffusion test for mecAmediated oxacillin resistance in S. aureus [Palazzo and Darini, 2006; Swenson et al., 2005). Cefoxitin has recently been investigated as an alternative agents for detection of resistance by disc diffusion and all studies indicate that tests are more reliable that those with oxacillin (Cauwelier et al., 2004; Felten et al., 2002; Maugeot et al., 2001; Skov et al., 2003). The cefoxitin disk test was first approved by the Clinical and Laboratory Standard Institute (CLSI) for predicting mecAmediated resistance in Staphylococcus spp in 2004 (CLSI, 2006). This antibiotic also initially proposed for S. aureus by investigator in France (Felten et al., 2002; Maugeot et al., 2001). Usually, cefoxitin disk diffusion (or another cefoxitinbased test) alone as a surrogate test for determination of oxacillin resistance is adequate for routine testing in the clinical laboratory (Salimnia and Brown 2005, 16 Swenson et al., 2005). The current data also showed that 72.7% (8 out of 11) MRSA isolates were susceptible to gentamycin (aminoglycosides group). Even though the small numbers of isolates (27.3%) were still susceptible towards gentamycin, this also indicated that despite aminoglycosides resistance among MRSA isolates being widespread, gentamycin remains active against most MRSA strains in European countries (Jacqueline et al., 2004). Moreover, aminoglycosides are known as bactericidal agents possessing rapid lethal activity on susceptibility MRSA strains both in vitro and in vivo (Grohs et al., 2003). On the other hand, about 43.75% (7 out of 16) S. aureus isolates tested were susceptible to erythromycin and clindamycin including both MRSA and MSSA. Nevertheless, multidrug-resistance S. aureus (MRSA) isolates have been detected in Malaysia and it should raise the paramount concern amongst both medical and the public community. Oxacillin has been used to replace methicillin since the latter is no longer the agent of choice for testing or treatment of S. aureus infections (NCCLS 2004). The detection of mecA for MRSA has been considered to be the “gold standard” method because of its simplicity and reproducibility (Saiful et al., 2006, Strommenger et al., 2003; Fluit et al., 2001). Susceptibility testing of methicillin resistance in S. aureus may be problematic due to heterogeneous resistance displayed by many clinical isolates (Smyth et al., 2001; Unal et al., 1992). Some isolate may contain mecA gene, but however they may appear phenotypic susceptibility if they are exposed to antistaphylococcal penicillins (Smyth et al., 17 2001; Wallet et al., 1996). After exposition, they become methicillin-resistant. Furthermore, standard susceptibility testing requires an additional 24-h incubation period compared to time required for assays for mecA or PBP2a (Wallet et al., 1996). In a clinical laboratory setting, S. aureus is identified by growth characteristics followed by detection of catalase and coagulase activities [Brown 2001; Smyth et al., 2001; Wallet et al., 1996). Subsequently, these conventional culture methods are time consuming and misinterpretation sometimes occurs when methicillin resistance is simulated by the hyper-production of β-lactamase (Chambers, 1997; Bignardi et al., 1996), or a weak coagulase activity (Chapin and Musgnug, 2003; Shopsin, 2000). While conventional antibiotic susceptibility testing of S. aureus was carried out using agar dilution tests, disc diffusion tests, or agar screening methods according to the standards of the NCCLS, this technique will result in differences of inoculums size, incubation time, medium pH and medium salt concentrations (Brown 2001, Smyth et al., 2001). On the other hand, false-negative or even non-interpretable results may be observed when commercially available kits for coagulase testing are used (Edwards et al., 2001). Thus, we conclude that antibiotics profiles generated together with the simplex real-time PCR assay may represent a feasible and practical tool for use in routine diagnostic molecular microbiology laboratories as a rapid, specific and improved assay for accurate identification of Coagulase positive MRSA isolates with respect to specificity. 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Journal of Clinical Microbiology 41:867-872 Wongwanich S, Prapawadee T, Paisomboon S, Ohta T, Hayashi H (2000) Epidemiological analysis of methicillin resistant Staphylococcus aureus in 28 Thailand. Southeast Asian Journal of Tropical Medicine and Public Health 31:72-76. 29 Table 1. Antibiogram profile of S. aureus clinical isolates and ATCC reference strains against selected antibiotics. All Disc diffusion breakpoints are based on the NCCLS and BSAC guidelines. Antibiotics used / Diameter of inhibition zone (mm) Clinical Ioslates 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 HN 1 HN 2 HN 3 HN 4 HN 5 HN 6 HN 12 HN 13 HN 14 HN 15 HN 16 HN 7 HN 8 HN 9 HN 10 HN 11 ATCC 33591 MRSA ATCC 25923 MSSA ATCC 29213 MSSA CF R (13) R (13) R (6) R (6) R (13) R (12) S (30) S (28) S (28) S (26) S (28) R (13) R (12) R (6) R (8) R (8) R (13) S (28) S (26) CN S (23) S (23) R (6) R (6) R (6) R (9) S (21) R (9) S (21) S (23) S (25) S (24) R (9) R (9) R (9) R (9) S (21) S (23) S (23) DA S (28) S (27) S (31) R (14) R (14) S (30) S (26) S (26) S (26) S (28) S (34) S (27) R (13) R (13) R (10) R (10) R (14) S (26) S (28) ERY S (26) S (26) R (9) R (10) R (10) R (12) S (25) S (25) S (23) S (26) S (28) R (10) S (25) R (10) R (10) R (10) R (10) S (23) S (23) FD S (35) S (35) S (35) S (35) S (35) S (30) S (30) R (22) R (9) S (35) S (35) S (30) R (8) R (8) R (9) R (9) R (8) R (9) R (9) OX R (6) R (6) R (6) R (6) R (8) R (8) S (24) S (17) S (18) S (17) S (18) R (6) R (6) R (7) R (7) R (7) R (6) S (24) S (20) PN R (6) R (6) R (6) R (6) R (8) R (8) S (35) R (6) R (9) R (10) R (11) R (8) R (10) R (10) R (8) R (8) R (8) R (10) R (10) RD S (35) S (35) S (35) S (35) S (35) S (30) S (29) S (35) S (30) S (35) S (35) R (10) R (9) S (9) S (20) R (9) S (29) S (20) S (35) V S (18) S (17) S (18) S (20) S (20) S (16) S (16) S (17) S (15) S (17) S (19) S (16) S (16) S (16) S (16) S (19) S (16) S (19) S (17) W S (29) S (28) R (6) R (6) R (6) S (28) S (26) S (28) S (26) S (35) S (31) R (7) R (6) S (26) S (26) S (26) S (27) S (26) S (26) Degree of susceptibility: R= Resistance and S= Susceptible Antibiotics used in this study: CF: cefoxitin, 30 µg; GM: Gentamicin, 10 µg; DA: Clindamycin, 2 µg; ERY: Erythromycin, 15 µg; FD: Fusidic acid, 10 µg; W: Trimethoprim, 5 µg; OX: Oxacillin, 1 µg; RD: Rifampin, 5 µg; V: Vancomycin, 30 µg; PN: Penicillin, 10 units. 30 Table 2. Target Genes List of primers concentrations used in this study. Prime sequences (5’-3’) Amplicon size (bp) Primer conc. (µM) Nuc F (5’- GCG ATT GAT GGT GAT ACG GTT-3’) R (5’- AGC CAA GCC TTG ACG AAC TAA AGC-3’) 279 0.65 0.65 Coa F (5’-GTA GAT TGG GCA ATT ACA TTT TGG AGG -3’) R (5’-CGC ATC AGC TTT GTT ATC CCA TGT -3’) 117 0.45 0.45 mecA F (5’- AAA ACT AGG TGT TGG TGA AGA TAT ACC -3’) R (5’-GAA AGG ATC TGT ACT GGG TTA ATC AG-3’) 147 0.50 0.50 31 Table 3. Simplex real-time PCR amplification of bacterial isolates used in this study. Simplex real-time PCR Amplification Isolates coa gene nuc gene mecA gene Reference strains ATCC 25293 (MSSA)* ATCC 29213 (MSSA) ATCC 33591 (MRSA)** ATCC 12228 (MSSE)*** ATCC 35218 (E. coli) ATCC 700728 (E. coli) + + + - + + + - + - Clinical isolates HN 1 HN 2 HN 3 HN 4 HN 5 HN 6 HN 7 HN 8 HN 9 HN 10 HN 11 HN 12 HN 13 HN 14 HN 15 HN 16 N 391 N 441 N 829 N 850 N 1405 UM 1 UM 2 UM 3 UM 6 UM 7 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - Degree of amplification: Positive amplified (+); Negative amplified (-) *Methicillin-sensitive S. aureus; **Methicillin-resistance S. aureus; ***Methicillin-sensitive S. epidermidis 32 Figure 1. Representative melting curve analysis of simplex real-time PCR assay targeting coa, nuc, and mecA genes with melting temperature (Tm) peak profile at 76.16 ± 0.8 °C, 78.39 ± 0.4°C, and 74.41 ± 0.6°C respectively. 33 (a) M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 200 bp 100 bp (b) M 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 200 bp 100 bp Figure 2.1 An ethidium bromide-stained gel demonstrating the typical banding patterns of real-time PCR amplification products for selected strains used in this study. (a) Lanes: (1) HN 1, (2) HN 2, (3) HN 3, (4) HN 4, (5) HN 5, (6) HN 6, (7) HN 12, (8) HN 13, (9) HN 14, (10) HN 15, (11) HN 16, (12) HN 7, (13) HN 8, (14) HN 9, (15) HN 10, (16) HN 11. (b) Lanes: (17) N 391, (18) N 441, (19) N 829, (20) N 850, (21) N 1405, (22) UM 1, (23) UM 2, (24) UM 3, (25) UM 6, (26) UM 7, (27) MRSA ATCC 33591, (28) MSSA ATCC 29213, (29) MSSA ATCC 25293, (30) S. epidermidis ATCC 12228, (31) E. coli ATCC 35218, (32) E. coli ATCC 700728. Lane M: 100 bp DNA marker (Promega Inc., Madison, WI, USA). All S. aureus clinical isolates produces amplification products of coa (117 bp) gene. All negative control strains lane 30, 31 and 32 did not produced any amplification products. 34 (a) M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 300 bp 100 bp (b) M 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 300 bp 100 bp Figure 2.2 An ethidium bromide-stained gel demonstrating the typical banding patterns of real-time PCR amplification products for selected strains used in this study. (a) Lanes: (1) HN 1, (2) HN 2, (3) HN 3, (4) HN 4, (5) HN 5, (6) HN 6, (7) HN 12, (8) HN 13, (9) HN 14, (10) HN 15, (11) HN 16, (12) HN 7, (13) HN 8, (14) HN 9, (15) HN 10, (16) HN 11. (b) Lanes: (17) N 391, (18) N 441, (19) N 829, (20) N 850, (21) N 1405, (22) UM 1, (23) UM 2, (24) UM 3, (25) UM 6, (26) UM 7, (27) MRSA ATCC 33591, (28) MSSA ATCC 29213, (29) MSSA ATCC 25293, (30) S. epidermidis ATCC 12228, (31) E. coli ATCC 35218, (32) E. coli ATCC 700728. Lane M: 100 bp DNA marker (Promega Inc., Madison, WI, USA). All S. aureus clinical isolates produces amplification products of nuc (279 bp) gene. All negative control strains lane 30, 31 and 32 did not produced any amplification products. 35 (a) M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 300 bp 100 bp (b) M 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 300 bp 100 bp Figure 2.3 An ethidium bromide-stained gel demonstrating the typical banding patterns of real-time PCR amplification products for selected strains used in this study. (a) Lanes: (1) HN 1, (2) HN 2, (3) HN 3, (4) HN 4, (5) HN 5, (6) HN 6, (7) HN 12, (8) HN 13, (9) HN 14, (10) HN 15, (11) HN 16, (12) HN 7, (13) HN 8, (14) HN 9, (15) HN 10, (16) HN 11. (b) Lanes: (17) N 391, (18) N 441, (19) N 829, (20) N 850, (21) N 1405, (22) UM 1, (23) UM 2, (24) UM 3, (25) UM 6, (26) UM 7, (27) MRSA ATCC 33591, (28) MSSA ATCC 29213, (29) MSSA ATCC 25293, (30) S. epidermidis ATCC 12228, (31) E. coli ATCC 35218, (32) E. coli ATCC 700728. Lane M: 100 bp DNA marker (Promega Inc., Madison, WI, USA). 36 Cover letter for manuscript Click here to download attachment to manuscript: WJMB Cover letter.doc ZARIZAL SUHAILI Science Officer Faculty of Agricultural and Biotechnology University Darul Iman Malaysia Kampus Kota, Jln Sultan Mahmud 20400 Kuala Terengganu Malaysia Dear Prof/Dr/Sir/Madam, Submission of the manuscript entitled “Simplex real-time PCR SYBR Green I assay for detection of Methicillin-reistance Staphylococcus aureus (MRSA) and antibiotic susceptibility profiles from Malaysian clinical isolates”. The authors of the above mentioned manuscript have would like to share findings and new knowledge generated especially on Malaysian MRSA isolates based on our recent work. We listed herewith amongst the novelty and findings for your kind perusal. The communication is the first to report on: 1. The establishment of antibiogram profiles of Malaysian clinical MRSA isolates with ATCC culture strains (MRSA and MSSA) as comparison together with the usage of three selected genes (coa, nuc, and mecA) as species-specific targeted marker in simplex real-time PCR amplification. 2. The use of SYBR Green I dye in a simplex real-time PCR assay for the simultaneous detection of coa, nuc and mecA genes against selected Malaysian clinical isolates of S. aureus. 3. The first attempt to compare and analyzing the results by melting curve analysis of coa, nuc and mecA genes using Malaysian MRSA clinical isolates as well as ATCC reference strains. 4. A novel set of primers for the detection of coa gene in S. aureus that produces a melting peak at 76.14+0.8 ºC and an amplicon of 117-bp. All the authors were concurring with this submission and we believed that the work here has not been published elsewhere. As such, we do hope that due consideration will be given to publish the manuscript in your esteem journal. Thank you very much for your cooperation. Best regards ZARIZAL SUHAILI (Corresponding Author)