Virulence Gene Profile of Methicillin Resistant Staphylococcus aureus (MRSA) and Methicillin Susceptible Staphylococcus aureus (MSSA) Isolated From Animals and Humans in Malaysia 1Asinamai Athliamai Bitrus., 1Zunita Zakaria*., 1Khairani-Bejo S., 2Sarah Othman, 1Nur Adilah Ahmad Nadzir & 1Abdul-Nasir Tijjani 1 Department of Pathology and Microbiology, Faculty of Veterinary Medicine, 2Faculty of Biotechnology and Biomolecular Science , Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Corresponding Author : zunita@upm.edu.my *Corresponding author email: Zunita@upm.edu.my Abstract This study determines the virulence gene profile of S. aureus and MRSA isolated from animals and humans using polymerase chain reaction (PCR). Amplification of ten (10) selected virulence gene coding for Staphylococcal exotoxin-like toxin 1(Set-1), Panton valentine leucocidin (PVL), haemolysin alpha and beta (hlα and β), exfoliative toxin A and B (et A and B), toxic shock syndrome toxin (tst-1), Staphylococcal enterotoxin u (Seu), Lipase and Protease encoding gene (geh and SspA) were tested on 29 isolates. The isolates comprised of humans (8), dogs (4), chickens (4), horses (11) , cats (1) and environmental surface (1). The result obtained reveals that 5(17.23%), 23(79.31%) and 3 (10.34%) of the isolates were positive for PVL, Set-1 and (etA). A total of 13(44.80%) of the isolates were positive for hlβ, 15(51.72%)for lipase encoding gene (geh) and 9(31.03%) for V8 protease (SspA). In addition, 3 (10.34%) of the isolates were positive for Seu and hlα while 2(6.9%) and 1(3.4%) were positive for etB and tst-1 respectively. In general, a higher carriage rate was observed in isolates from human source than isolates from animal sources.   Introduction: Staphylococcus aureus is a pathogen of veterinary and public health significance (Alfatemi et al., 2014). Methicillin resistant S. aureus is a major cause of nosocomial, community and livestock associated infection worldwide (Saleha & Zunita, 2010). It is a known producer of a number of putative virulence determinants and also an emerging zoonosis. The spectrum of disease caused by MRSA is complicated by its’ ability to acquire resistance and virulence determinants (Ghaznavi-Rad et al., 2010). Resistance to methicillin was first reported in the early 1960s when a strain of S.aureus acquired a genomic island carry methicillin resistance determinants mecA (Lindsay, 2014). Since then, MRSA has become a major global public health concern causing severe disease among individuals in the hospital and community and recently in livestock and pets (Chua et al., 2014). The successful adaption and difference in pathogenic potentials of MRSA is largely due to the presence of a variety of coordinated virulence gene (Shaw et al., 2004). Leading to the emergence of highly pathogenic strains causing severe disease in humans and animals. This study was designed to determine the virulence gene profile of MRSA isolated from human and animal using 10 selected virulence gene. Results & Discussion: The result obtained following screening of genes coding for virulence reveals that out of the twenty nine (29)S. aureus and MRSA isolates screened 5(17.23%), 23(79.31%) and 3 (10.34%) of the isolates were positive for PVL, Set-1 and (etA) respectively as shown in Figure 1. The detection of PVL toxin in five isolates of which one each is from dog and environment respectively might be due to cross infection. Since PVL is known to play a very significant role in the pathogenesis of community acquired - MRSA infection. However, this condition is never seen in hospital acquired-MRSA, indicating the possibility that most of the isolated MRSA strains in this study harboring Pvl gene might actually be community acquired. Figure 4: Distribution of Virulence gene based on source of isolates FIGURE 1: Virulence gene profile of S. aureus and MRSA Similarly, 13(44.80%) and 3 (10.34%) of the isolates were positive for hlβ and hlα while 15(51.72%)of the isolates were positive for lipase encoding gene (geh) and 9(31.03%) for V8 protease (SspA) respectively Figure 1. Hemolysin are known to play a very significant role in the laboratory diagnosis of S.aureus . In addition, both hemolysin hlα and hlβ are known have strong affinity for both rabbit and sheep erythrocytes, however, hlα is mostly seen in animal isolates (Dinges et al., 2000, Otto, 2014). This however, is consistent with the findings in this study, as only one isolate from cat have been shown to harbor hlα. Furthermore, 3(10.34%) of the isolates were positive for Staphylococcal enterotoxin like toxin u (Seu) while only 1(3.4%) each of the isolates were positive for both etA and tst-1 respectively as shown in Figure 1. Exfoliative toxin A and B are responsible for scalded skin syndromes in children and adults with underlying infections (Ladhani, 2003). Staphylococcal exfoliative toxins are mostly seen in human strains (Bukowski et al., 2010). This is in agreement with our findings as only our human isolates were observed to show expression of exfoliative toxins. Furthermore, association between exfoliative B and community acquired MRSA have been reported in Japan (Bukowski et al., 2010). This findings however, provides further evidence that this may be community associated MRSA since, it was observed that the isolate (SH4) harbors both etB and Pvl gene Figure 5: Distribution of Virulence gene based on carriage rate between MRSA and MSSA Conclusions: Carriage rate was observed more in human than animal isolates Exfoliative A and B as well as toxic shock syndrome toxin were only observed in human isolates PVL toxins were observed in both humans and animal isolates While alpha haemolysin was only observed in one animal isolate Methodology Acknowledgement This research has been supported by Universiti Putra Malaysia Research University Grant (9428800) and Fundamental Research Grant Scheme, Ministry of Higher Education Malaysia. (5524643). The authors thank the laboratory staff at Bacteriology laboratory, Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, for their kind cooperation throughout the course of this study . References: Alfatemi, S. M. H., Motamedifar, M., Hadi, N., & Saraie, H. S. E. (2014). Analysis of virulence genes among methicillin resistant Staphylococcus aureus (MRSA) strains. Jundishapur journal of microbiology, 7(6). Bukowski, Michal, Wladyka, Benedykt, & Dubin, Grzegorz. (2010). Exfoliative toxins of Staphylococcus aureus. Toxins, 2(5), 1148-1165. Chua KYL, BP Howden, JH Jiang, T Stinear, AY Peleget al., 2014. Population genetics and the evolution of virulence in Staphylococcus aureus. Infect, Gen and Evol, 21:554-562. doi: http://dx.doi.org/10.1016/j.meegid.2013.04.026 Dinges, Martin M, Orwin, Paul M, & Schlievert, Patrick M. (2000). Exotoxins of Staphylococcus aureus. Clinical microbiology reviews, 13(1), 16-34. 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Figure 2: Distribution of Virulence gene based on rate of carriage between MRSA and MSSA In general, high carriage rate of virulence gene was observed in human isolates rather than the each individual animal isolates Figure 3. This could be due to the fact that virulence determinants such as exfoliative toxin, toxic shock syndrome toxin and staphylococcus enterotoxin u are mostly associated with disease conditions such as scalded skin syndrome, toxic shock and staphylococcal food poisoning mostly seen in humans. Similarly, higher carriage rate was also observed in MSSA than in MRSA which could be due fitness cost as shown in Figure 2. INTERNATIONAL CONGRESS OF THE MALAYSIAN SOCIETY FOR MICROBIOLOGY (ICMSM 2015) 7TH TO 10TH DECEMBER 2015 (BAYVIEW BEACH RESORT PENANG, MALAYSIA)