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Proceedings of the 22nd Annual Congress of Veterinary Association Malaysia. 30th July -1st August 2010, Seri Pacific Hotel, Kuala Lumpur, Malaysia. A new spa type t5696 MRSA Isolated from Veterinary Medicine Student at Universiti
Putra Malaysia
Aklilu, E1, Zunita, Z1*, Hassan, L.1, and Chen, H. C.1
Universiti Putra Malaysia, 43400 UPM Serdang, Malaysia Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) has been spreading worldwide since its emergence. Swab samples were collected from the nostrils and oral cavity of veterinary medicine student at Universiti Putra Malaysia. Phenotypic identification of MRSA was made by employing broth enrichment, catalase and cogulase tests, mannitol fermentation test on mannitol salt agar (MSA), slide latex agglutination test, growth on selective media, Oxacillin-resistant screening agar base (ORSAB). Intense blue colonies on ORSAB presumptively confirmed MRSA. Antibiotic sensitivity tests using 15 antibiotics revealed that the isolate was resistant against Amikacin 30 µg (AK30), Amoxicillin 25 µg (AML25), Methicillin 10 µg (MET10), Oxacillin 1 µg (OX1) Streptomycin 10 µg (S10), and Tetracycline 10 µg (TE10) while being intermediately resistant Oxacillin (OX1) and susceptible to other antibiotics tested. The minimum inhibitory concentration (MIC) of the isolate was 8 µg/mL. However, detection of the resistance gene mecA was proved negative. This discrepancy between phenotypic and mecA detection methods is attributed to the fact that there are other non-mecA dependent Oxacillin resistance factors in S. aureus. Amplification of the polymorphic X region of the staphylococcal protein A gene was done by PCR. Purified PCR product was sequenced, aligned and repeat numbers were assigned. Repeat sequence pattern was assigned a unique spa type, t5696. The finding hints the importance of further studies by focusing on the occurrence of MRSA in veterinary professionals and students with effective molecular typing techniques such as spa typing. This will in turn helps in understanding the similarities and uniqueness of local isolates in reference to MRSA isolates reported anywhere in the world Keywords: MRSA, antibiotic sensitivity test, MIC, mecA gene, spa typing Proceedings of the 22nd Annual Congress of Veterinary Association Malaysia. 30th July -1st August 2010, Seri Pacific Hotel, Kuala Lumpur, Malaysia. Introduction
Staphylococcus aureus features incredibly diverse pathogenic and resistance mechanisms, and MRSA has emerged as the major bacterial pathogen of the 21st century (Bartlett and Stenger, 2008). Since its emergence in 1961 in UK, the incidence of MRSA infections has been increasing steadily (Moodley et al., 2006). Methicillin resistance in S. aureus is mainly attributed to the presence of mecA gene which encodes the production of an additional protein-binding protein (PBP) named PBP2’/PBP2a. The PBP2a has been shown to be present in 90% of MRSA isolates (Ohwada et al., 1999). There are numerous techiniques available to differentiate S. aureus and specifically MRSA. Phenotypical methods such as antibiogram and phage typing were previously being used as the sole typing methods. However, DNA-based techniques such as spa typing, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) became popular and more reliable tools (Shopsin et al., 1999). The staphylococcal protein A gene (spa) contains a polymorphic region X of the spa gene consists of a variable number of tandem repeats, generally 24 bp but repeats of 21 and 27 bp in length have been identified (Moodley et al., 2006). The technique combines a number of technical advantages, such as rapidity, reproducibility, and portability. Moreover, due to its repeat structure, the spa locus simultaneously indexes genetic variations that accumulate both rapidly and slowly (micro-and macro-variations), enabling the use of spa typing in both local and global epidemiological studies (Hallin et al., 2009). In Malaysia, there were molecular typing studies conducted to assess epidemiology of MRSA in major hospitals in the country by using PFGE, MLST, and SCCmec typing as the main tools (Sam et al., 2008; Norazah et al., 2003). However, there was no data as to the occurrence and molecular typing of MRSA isolated from veterinary professionals, particularly veterinary medicine students. In this study we report a new spa sequence type for an MRSA isolated from Proceedings of the 22nd Annual Congress of Veterinary Association Malaysia. 30th July -1st August 2010, Seri Pacific Hotel, Kuala Lumpur, Malaysia. Methodology
Isolation and Identification of MRSA
Nasal and oral swab samples were obtained from the student (PJ404) on voluntary basis in which the student signed a consent form following briefing about the objectives and procedures of the study. Swabs were placed into 10 mL Tryptone Soya Broth (TSB) for 48 hours to enrich the growth after which streaking was done onto blood agar with 7% horse blood. The inoculated plates were incubated aerobically at 37oC for 18-24 h. Gram-staining, catalase and coagulase tests, mannitol fermentation test on MSA and slide latex agglutination were carried out to identify S. aureus colonies. The presumptive S. aureus colonies (yellow colonies) were then streaked onto ORSAB (Oxoid, UK) supplemented with ORSAB selective supplement consisting of 1 mg oxacillin and 25,000 IU polymyxin B incorporated into 500 mL of the agar solution. Antibiotic Sensitivity Test
Antibiotic sensitivity tests were carried out for MRSA isolates according to CLSI standards (2006). The antimicrobials tested were: Amikacin 30 µg (AK30), Amoxicillin 25 µg (AML25), Methicillin 10 µg (MET10), Oxacillin 1 µg (OX1), Cefoxitin 30 µg (FOX30), Streptomycin 10 µg (S10), Vancomycin 30 µg (VA30), Minocycline 30 µg (MH30), Rifampicin 15 µg (RD15), Doxycycline Hydrochloride 30 µg (DO30), Amoxycillin-Clauvulanic acid 30 µg (AMC30), Gentamicin 10 µg (CN10), Impenem 10 µg (IPM 10), Tetracycline 10 µg (TE10), Erythromycin Minimum Inhibitory Concentration (MIC) By Etest
Determination of MIC was done by using Oxacillin Etest (AB Biodisc, Solana, Sweden) strips according the manufacturer’s recommendations. According to the break points defined (CLSI, 2006), isolates with MIC ≥ 4 µg/mL were considered oxacillin resistant. PCR Amplification of mecA Gene
The PCR amplification was done by using mecA primers (Bio Basic Inc., Canada) which were previously designed by Murakami et al.( 1991), which amplifies the 533 bp segment of the mecA Proceedings of the 22nd Annual Congress of Veterinary Association Malaysia. 30th July -1st August 2010, Seri Pacific Hotel, Kuala Lumpur, Malaysia. gene and with the following sequences: mecA1, 5’-AAA ATC gAT ggT AAA ggT Tgg C-3’ and mecA2, 5’-AgT TCT gCA gTA CCg gAT TTg C-3’. spa typing
The polymorphic x region of S. aureus protein A was amplified using forward and reverse CAGCAGTAGTGCCGTTTGCTT-3’) respectively as described previously by Harmsen et al. (2003). The PCR products were purified by QIAquick PCR Purification Kit (Qiagen) and the purified products were sent to MACROGEN (S. Korea) for sequencing. Identification of MRSA
Typical intense blue colonies of the isolate on ORSAB showed that the isolate was MRSA. However, PCR amplification for detection of the mecA gene proved negative result. Antibiotic Sensitivity Test and MIC determination by Etest
The isolate was resistant against AK30, AML25, MET10, S10, and TE10 while being intermediately resistant Oxacillin (OX1). Whereas the isolate was detected to be susceptible to all the remaining antibiotics tested including cefoxitine (FOX30) and vancomycin (VA30) and Impenem (IMP10). The MIC value for the isolate was 8 µg/mL, which is above the 4µg/mL breakpoint recommended for MRSA according to the CLSI standards. spa Typing
Amplification of the polymorphic X region of the spa gene gave a 240 bp product. Upon sequence alignment and analysis, the repeat sequences were identified, assigned repeat numbers and spa type by the help of the online database (http://www.ridom.de). Accordingly the repeat pattern was 07-16-12-23-02-02-02-02-02-34. The repeat patterns along with the chromatogram Proceedings of the 22nd Annual Congress of Veterinary Association Malaysia. 30th July -1st August 2010, Seri Pacific Hotel, Kuala Lumpur, Malaysia. were sent to the spa curator to be recognized and assigned a new spa type. Hence, the repeat pattern was assigned a new spa type, (t5696), GeneBank accession number: GU229647. Discussions
Methicillin-resistant S. aureus has recently been considered as important pathogen in animals and animal health practitioners as it has been increasingly reported in veterinary medicine (Moodley et al., 2008). Previously we reported the occurrence of MRSA among veterinary medicine students at Universiti Putra Malaysia in which 23.3% (24/103) were MRSA culture positive (Aklilu et al., 2008). A study conducted by Wulf et al. (2006) also revealed MRSA carriage among veterinary doctors and students in the Netherlands. On the other hand, a screening survey conducted on pre-clinical medical students at Melaka Manipal Medical College in Malaysia did not find any MRSA (Santhosh et al., 2007). In the current study, discrepant results in phenotypic and molecular identification have been observed. This can be ascribed to the absence of the resistant gene, mecA. Absence of the mecA gene can be attributed to several factors since there are other mechanisms that are non-mec- dependent that contribute individually or in combination towards antibiotic resistance in staphylococci strains (Berger-Bachi and Tschierske, 1998; Berger-Bachi, 1995). Overproduction of normal PBP with an altered binding capacity or other unidentified factors potentially contribute to the rise of methicillin resistance in mecA negative S. aureus strains (Chambers, 1997). Such features imply that these mecA negative isolates might be those MRSA strains showing border-line resistance towards oxacillin/methicillin (BORSAs). Strains with borderline or low-level methicillin resistance have MIC values of 1.5-12 mg/L for the majority of the cell population, but clinical strains without mecA and with methicillin MICs in the 4-16 mg/L range have also been reported (Bignardi et al., 1996). Borderline methicillin-resistant S. aureus may or may not carry mecA and are extremely heterogeneous or produce PBP2a or be penicillinase hyperproducers (Hall, 2003; Brown, 2001). Moreover, the MRSA selective agar (ORSAB) used in this study does not exclude growth of S. aureus that are hyperproducers of penicillase Proceedings of the 22nd Annual Congress of Veterinary Association Malaysia. 30th July -1st August 2010, Seri Pacific Hotel, Kuala Lumpur, Malaysia. Among the various sequence-based MRSA typing methods, spa typing has been shown to be an effective and rapid method for typing MRSA (Shopsin et al., 1999). In the current study, the MRSA isolate was designated as a new spa type. The differences in spa types among S. aureus isolates is attributed to the diversity of short sequence repeat(SSR) region arise from deletion and duplication of the repetitive units and also by point mutation (Shopsin et al., 1999; Brigido et al., 1991). Previous studies by Wulf et al. (2006) in the Netherlands and Moodley et al. (2006) in UK and Irelands also used spa typing for MRSA isolated from veterinary medicine students and staff. The discovery of a new spa type in the current study confers a unique identity to the isolate in question. This can further be used as a reference for future studies to be done on spa typing of S. aureus isolates. Moreover, the finding pin points the importance of further studies focusing on the occurrence of MRSA in veterinary professionals and students with effective molecular typing techniques such as spa typing. This will in turn helps in understanding the similarities and uniqueness of local isolates in reference to MRSA isolates reported anywhere in Acknowledgements
The authors would like thank all the students who voluntarily took part in the study. We also thank the laboratory technicians at the Bacteriology Laboratory of Faculty of Veterinary Medicine, Universiti Putra Malaysia for their cooperative contribution. This study was supported by Science Fund, MOSTI 02-01-04-SF0195 and partly by Nuffic Foundation. References
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