We determined the presence of four virulence genes: (cna, seh, PVL, TSST-1) using multiplex PCR in 880 isolates of

methicillin-susceptible Staphylococcus aureus (MSSA) collected from the Universiti Kebangsaan Malaysia Medical

Centre (UKMMC) in 2009. We found that 51.59% (454/880) of the strains had cna; 21.82% (192/880) possessed seh;

10.23% (90/880) had PVL and 6.82% (60/880) haboured TSST-1. Although methicillin-susceptible, MSSA carries

important virulence genes which could affect patient’s clinical course

Ainihayati Noordin,

Hassriana Fazilla Sapri,

Hui-min Neoh,

Nurul Azirah Mohamad Sani,

Salasawati Hussin,

English

UKM Journal Article Repository

Asia-Pacific Journal of Molecular Medicine 2011, 1:4Virulence Gene Typing of Methicillin-susceptible Staphylococcus aureus (MSSA)Isolates in Universiti Kebangsaan Malaysia Medical Centre (UKMMC)1Hassriana Fazilla Sapri, 1Nurul Azirah Mohamad Sani, 1Ainihayati Noordin, 2Hui-min Neoh* and 1Salasawati Hussin*1Department of Medical Microbiology and Immunology, Faculty of Medicine, Universiti Kebangsaan Malaysia;2UKM Medical Molecular Biology Institute (UMBI), Universiti Kebangsaan MalaysiaReceived on 09/11/2010 / Accepted on 23/05/2011ABSTRACTWe determined the presence of four virulence genes: (cna, seh, PVL, TSST-1) using multiplex PCR in 880 isolates ofmethicillin-susceptible Staphylococcus aureus (MSSA) collected from the Universiti Kebangsaan Malaysia MedicalCentre (UKMMC) in 2009. We found that 51.59% (454/880) of the strains had cna; 21.82% (192/880) possessed seh;10.23% (90/880) had PVL and 6.82% (60/880) haboured TSST-1. Although methicillin-susceptible, MSSA carriesimportant virulence genes which could affect patient’s clinical course.INTRODUCTIONStaphylococcus aureus is a major nosocomial pathogenthat causes a wide range of diseases including endocarditis,osteomyelitis, pneumonia, toxic-shock syndrome, foodpoisoning, carbuncle and boils [1]. Its pathologicalimportance makes it a longstanding subject ofepidemiological investigation. In recent years, such studieshave typically involved genotyping via a variety ofmolecular techniques. In one such effort, researchers haveused the presence or absence of virulence elements tocharacterize isolates [2], where a multiplex-PCR protocol wasdeveloped for virulence gene detection of S. aureus [3].Epidemiological studies are usually carried out formethicillin-resistant S. aureus (MRSA) due to its importancein drug resistance. In comparison, less emphasis has beenplaced on methicillin-susceptible S. aureus (MSSA) isolates,even though they are often more genetically variable andhave a higher hospital prevalence (20-40%) than MRSA (5-10%) [4,5,6]. In this pilot study on MSSA isolates in ouruniversity hospital, we collected the index (first isolate fromeach patient) MSSA isolates of all MSSA infections reportedin the hospital in 2009 and detected the presence of fourclinically important virulence genes in these isolates.MATERIALS AND METHODSBACTERIAL STRAINSThe Universiti Kebangsaan Malaysia Medical Centre(UKMMC) is a teaching hospital with a capacity of 900beds located in the centre of Kuala Lumpur. From Januaryto December 2009, a total of 880 cases of MSSA relatedinfections from various wards of the hospital were reported.We collected the index MSSA isolate of each case andestablished these as strains. Most strains were isolatedfrom tissue and wound swabs. Species identification ofstaphylococci was performed by the Department of MedicalMicrobiology and Immunology, UKMMC. Strains werestocked from time of isolation in Brain Heart Infusion brothwith 40% glycerol at -70oC.GENOMIC DNA ISOLATIONChromosomal DNA of each MSSA strain was extractedusing DNeasy Blood & Tissue Kit (Qiagen) according tothe manufacturer’s instruction. The quantity and qualityof DNA were determined spectrophotometrically. ExtractedDNA samples were stored at -20 oC.VIRULENCE GENE TYPINGThe presence of four virulence genes, cna, seh, PVL andTSST-1 in our collection of MSSA strains were determinedby a modified multiplex-PCR (M-PCR) protocol [7]. M-PCRwas performed in 25 µl reaction volumes containing thefollowing: 2x PCR buffer (PROMEGA), 3 mM MgCl2, 400µM of each dNTP, 2.5 U Taq DNA Polymerase, 1 µl DNAtemplate and 10 mM of each primer. Primer sequences forthe genes are shown in Table 1. The cycle program for M-PCR was as follows: 1 cycle of pre-denaturation for 4 min at94°C; 30 cycles of 94°C for 2 min (denaturation), 58°C for 1min (annealing) and 72°C for 2 min (extension); and a final* Correspondence to: sawati@ppukm.ukm.myhui-min@ppukm.ukm.my12Hassriana Fazilla Sapri et al. Asia. Pac. J. Mol. Med.extension at 72°C for 5 min. M-PCR products were analysedby gel electrophoresis with a 1.5% agarose gel (Sigma). S.aureus strains 81/108 (PVL), WIS (TSST) and clinical isolateMSSA 251 (cna, seh) were used as positive controls for M-PCR. 81/108 and WIS were kindly provided by KeiichiHiramatsu and Teruyo Ito (Juntendo University, Japan).RESULTS AND DISCUSSIONMore attention has been focused towards MRSA as theyare usually multidrug resistant and pose a challenge forantibiotic prescription [8]. Nevertheless, MSSA has a higherprevalence than MRSA in most hospitals, and they werereported to bring certain degree of morbidity and mortalityif not properly monitored [9]. Even though they are usuallysusceptible to many antibiotics, MSSA has been reportedto harbor various virulence genes, such as PVL, whichcould cause potentially fatal necrotizing pneumonia [2].Our university hospital is a tertiary hospital which notonly accepts admission from patients residing in KualaLumpur, but also referrals from various hospitals in therural areas. In 2009, the percentage of MSSA infectionsamong S. aureus cases in UKMMC was as high as 73.4%(880 cases out of 1199 S. aureus infections). This is almostsimilar to another tertiary hospital in Spain (70.4%) [10]. Incontrast, MSSA prevalence in Australian and Nepalesetertiary hospitals were lower at 62.7 and 55.1%, respectively[8, 9].In this study, we determined the prevalence of fourvirulence genes, cna, seh, PVL and TSST-1 in index MSSAisolates collected from various wards of UKMMC in 2009.cna is a staphylococcal virulence factor involved inattachment of S. aureus to cells or extracellular matrices,where it codes for a collagen adhesin [11]. Collagen adhesinis an important step for the pathogenesis of staphylococcalarthritis and osteomyelitis; it mediates the attachment of S.aureus cells to cartilage [12]. We found 51.59% of the MSSAstrains in our study haboured the cna gene, which wasalso the most prevalent virulence factor among the fourgenes that we investigated (Figure 1). About 12% (105/880) of the MSSA cases in UKMMC in 2009 were from theorthopedic ward (unpublished observation), where cna wasdetected in 55.24% (58/105) of these strains.seh encodes for staphylococcal enterotoxin H (SEH),which is one of the newly reported staphylococcalTable 1. Primers used for detection of virulence genesPrimer Sequence Product size (bp) Referencecna-1 5’-ACACCAGACGGTGCAACAATTA-3’cna-2 5’-AGCAATACCGTTTGCATCTGTTA-3’ 815 7seh-F 5’-ATTCACATCATATGCGAAAGCAG 3’seh-R 5’-ATGTCGAATGAGTAATCTCTAG-3’ 555 7pvl-F 5’-ATGTCTGGACATGATCCAA-3’pvl-R 5’-AACTATCTCTGCCATATGGT-3’ 970 7TSST-1 5’-TGATATGTGGATCCGTCAT-3’TSST-1 5’-AAACACAGATGGCAGCAT-3’ 387 7Figure 1. The prevalence of 4 virulence genes (cna, seh, PVL and TSST-1) in index MSSA strains isolated from UKMMC, 20093Hassriana Fazilla Sapri et al. Asia. Pac. J. Mol. Med.enterotoxin [13]. Reports show that most seh-harboring S.aureus isolates were able to produce a significant amountof SEH, leading to food poisoning and staphylococcalsuperantigen-related diseases. In UKMMC, the prevalenceof the seh gene was 21.82%, which is higher than thosereported by Becker et al. (5%) [14] and Peacock et al. (15%)[15]. These differences in prevalence might be due to therole of seh in invasive illnesses [16]: 50% of the strainsused in the studies of Becker et al. and Peacock et al. wereisolated from the anterior nares of healthy individuals,therefore the prevalence of seh in their strains were lowercompared to our study, where all of our MSSA were isolatedfrom ill, hospitalized patients.Interestingly, the prophage sequence encodingPanton-Valentine leukocidin (PVL), a cytotoxin usuallyassociated with community-acquired MRSA, was detectedin 10.23% of MSSA strains and this prevalence was higherthan another report by Sila et al. (3%) [2]. PVL lysesleukocytes and several findings linked PVL positive strainsto necrotizing pneumonia and severe skin infections [17].Nevertheless, it is still controversial if the cytotoxin itselfis pathogenic [17].Toxic shock syndrome (TSS) is an acute systemicdisease characterised by fever, hypotension, myalgia, rash,multiple-organ failure, and late desquamation of the handsand feet [18]. It is caused by the Toxic Shock SyndromeToxin (TSST), a superantigen produced by S. aureusisolates carrying the tst gene, where TSST-1 is the commonform of exotoxin related to TSS. In our study, the TSST-1gene was present in 6.82% isolates, which is comparable tothat of Sila et al’s [2] investigation. Nevertheless, it is lowerthan those published by Becker et al. [14] who reported an18% and 22% prevalence of TSST-1 in strains isolated fromblood and nasal specimens, respectively.CONCLUSIONMost S. aureus isolates from UKMMC in 2009 were stillsusceptible to methicillin (73.4%). Nevertheless, this currentstudy on the prevalence of four important virulence genesin these isolates revealed that about half (58.75%) of theseMSSA isolates haboured virulence factors, where the mostcommonly detected virulence gene was cna (51.59%). Eventhough MSSA infections are generally easier to manage asthey are commonly susceptible to most available antibiotics,infections with MSSA should be treated be caution as theycould still serve as reservoirs of virulence factors andintroduce complications into patients’ clinical course.ACKNOWLEGDEMENTWe thank Professor Keiichi Hiramatsu and AssociateProfessor Teruyo Ito, Bacteriology Department, School ofMedicine, Juntendo University, Tokyo, Japan, for their kindgifts of reference strains.This research is supported by UKM ResearchUniversity Grant UKM-GUP-TKP-08-19-067.REFERENCES1. Shopsin B, Kreiswirth BN. Molecular epidemiology ofmethicillin-resistant Staphylococcus aureus. EmergingInfectious Diseases 2001; 7(2): 323-326.2. 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Virulence gene typing of Methicillin-susceptible Staphylococcus aureus (MSSA)isolates in Universiti Kebangsaan Malaysia Medical Centre (UKMMC)

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