corresponding author’: zunita@upm.edu.my DETECTION OF fem FACTORS AND PHENOTYPIC VANCOMYCIN RESISTANCE IN Staphylococcus aureus AND METHICILLIN RESISTANT Staphylococcus aureus ISOLATED FROM ANIMALS AND HUMANS Asinamai Athliamai Bitrus1, Zakaria Zunita1, Siti Khairani Bejo1, Sarah Othman2, Nur Adilah Ahmad Nadzir1 1Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Malaysia 2Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Malaysia corresponding author’: zunita@upm.edu.my ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of nosocomial infection and an emerging zoonosis. The magnitude of phenotypic resistance in MRSA strain does not correlate with the quantity of penicillin binding protein (PBP2a) produced by the organism. This surprising observation led to the suggestions that intrinsic factors called factors essential for methicillin resistance (fem) and not the mec gene, might be responsible for the expression of methicillin resistance. This study was designed to detect the factors essential for methicillin resistance and phenotypic vancomycin resistance in S. aureus and MRSA. In this study, a total of 28 S. aureus isolates were used. PCR assay was used to detect for factors essential for methicillin resistance (femA) and tetracycline resistance gene (tet-L and O). E-test agar diffusion was used to determine susceptibility of eight mecA positive S. aureus to Amoxicillin-256 µg/mL ,Oxacillin -256 µg/mL, Vancomycin-256 µg/mL, Erythromycin-256 µg/mL, Linezolid-256 µg/mL, Levofloxacin-256 µg/mL and Tetracycline-256 µg/mL. The result obtained showed that 82% (23/28) of the isolates were positive for the factors essential for methicillin resistance (femA), none of the isolates were positive for tet-O and L respectively. Phenotypic antibiotics susceptibility showed that all isolates were susceptible to levofloxacin, amoxicillin, oxacillin and linezolid (MIC of ≤ 2 µg/mL). Susceptibility to vancomycin was observed in seven isolates with only one (SH2) showing intermediate resistance (MIC of 2 µg/mL). However, phenotypic resistance using disk diffusion method as previously published showed that four isolates were resistant to vancomycin 30 µg with diameter breakpoint of < 17mm. In addition 37.5% (3/8) (SH1, SH3 and SH4) and 25% (2/8) (SH1 and SDG2) of the MRSA isolates were resistant to erythromycin and tetracycline. The finding of this study affirms the importance of fem factors and MIC in determining the expression of phenotypic methicillin and vancomycin resistance in S. aureus and MRSA isolates. INTRODUCTION RESULTS Resistance to methicillin in staphylococci is an inherent resistance of the cells to almost all beta lactam antibiotics including the carbenem and cephalosporin. This however does not involve destruction of the drug (Henze et al.,1993). Methicillin resistance is mediated by the acquisition of a methicillin resistance determinants (mec) that codes for another penicillin binding protein with reduced susceptibility to methicillin (Maidhof et al., 1991). Initially, mec was thought to be the only agent responsible for methicillin resistance development in S. aureus. However, chromosomal factors, otherwise called aux or fem factors were later observed to play a role in the expression of methicillin resistance in S. aureus (Berger-Bächi et al., 1992). This new factors make up the normal array of genes found in both MRSA and S. aureus, and can be identified by transposon 551 (Tn551)-mediated insertional activation of methicillin resistance (Fontana, 1985). For more than five decades, antibiotics resistance development in S. aureus has become a major public health problem. In addition, quiet a large number of MRSA clones even though resistant to more than one class of antibiotics were susceptible to glycopeptides. However, at present, the emergence of MRSA clones with low level vancomycin resistance has been reported (Rajaduraipandi et al., 2006). The emergence of vancomycin resistant and vancomycin intermediate resistant S. aureus as a result of increased resistance to vancomycin a strategic drug for the treatment of MRSA and S. aureus has also been reported (Perichon and Courvalin,2009). For this reasons, this study was designed to detect the factors responsible for methicillin resistance and phenotypic vancomycin resistance in S. aureus and MRSA isolated from animals and human. The findings of this study showed that; 82% (23/28) of all the isolates were positive for factor responsible for methicillin resistance (femA), while none of the isolates were positive for tet-O and tet-L (Table 1). All isolates were phenotypically susceptible to oxacilin, amoxicillin, levofloxacin and linezolid (MIC of ≤ 2 µg/mL). Susceptibility to vancomycin was also observed in seven isolates with only one (SH2) showing intermediate resistance (MIC of 2 µg/mL). In addition 37.5% (3/8) of the isolates (SH1, SH3 and SH4) were resistant to erythromycin. (Figure 1). CONCLUSION In conclusion, the findings of this study validates the argument that mec gene alone is not responsible for the expression of methicillin resistance in S. aureus and that none of our isolates were vancomycin resistant REFERENCES Berger-Bächi, B, Strässle, A, Gustafson, John E, and Kayser, FRITZ H. (1992). Mapping and characterization of multiple chromosomal factors involved in methicillin resistance in Staphylococcus aureus. Antimicrobial agents and chemotherapy, 36(7), 1367-1373. Berger-Bächi B, Tschierske M: Role of fem factors in methicillin resistance. Drug Resistance Updates 1998, 1(5):325-335. Fontana, R. (1985). Penicillin-binging proteins and the intrinsic resistance to βlactams in Gram-positive cocci. Journal of Antimicrobial Chemotherapy, 16(4), 412-416. Henze, U, Sidow, T, Wecke, J, Labischinski, H, and Berger-Bächi, B. (1993). Influence of femB on methicillin resistance and peptidoglycan metabolism in Staphylococcus aureus. Journal of bacteriology, 175(6), 1612-1620. Maidhof, H, Reinicke, B, Blümel, P, Berger-Bächi, B, and Labischinski, H. (1991). femA, which encodes a factor essential for expression of methicillin resistance, affects glycine content of peptidoglycan in methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains. Journal of bacteriology, 173(11), 3507-3513 Périchon, Bruno, and Courvalin, Patrice. (2009). VanA-type vancomycin-resistant Staphylococcus aureus. Antimicrobial agents and chemotherapy, 53(11), 4580-4587. Rajaduraipandi, K, Mani, KR, Panneerselvam, K, Mani, M, Bhaskar, M, and Manikandan, P. (2006). Prevalence and antimicrobial susceptibility pattern of methicillin resistant Staphylococcus aureus: A multicentre study. Indian journal of medical microbiology, 24(1), 34.   OBJECTIVE The objective of this study is: to detect the factors essential for methicillin resistance and phenotypic vancomycin resistance in S. aureus and MRSA isolated from animals and humans MATERIALS AND METHODS DISCUSSION Development of resistance to antibiotics is a major global public health problem. Central to the development of resistance in methicillin- resistant S. aureus is the acquisition of a methicillin resistance determinants mecA and femA. In this study, 82% (23/28) of all the isolates were positive for the femA gene thus validating the argument that PBP2’ is not the only factor responsible for methicillin resistance. The absence of the gene in the remaining five isolates doesn’t invalidate the assertion that this isolates does not possessed fem factors; however, it indicates that the isolate might be harboring other fem factors other than femA. Studies have shown that, there are about four well characterized fem factors other than the mec gene which helps in the expression of methicillin resistance in S. aureus (Berger-Bachi et al., 1998; Henze et al., 1993) In this study, Etest agar diffusion test showed that none of the isolates were vancomycin resistant even though Kirby Bauer disc diffusion method performed with the same isolates in another study showed that four of the isolates were resistant to Vancomycin-30µg with breakpoint diameter of ≤ 17 mm. Thus, indicating that phenotypic resistance to antibiotics does not define the true resistance profile of an isolate, however, it gives a clue as to what antibiotics should be used in the treatment of infection. Resistance to erythromycin and tetracycline was also observed in 3/8 and 2/8 of the MRSA isolate respectively. This result is consistent with the resistance profile of the isolates using disc diffusion method. Thus, validating their resistance profile. However, PCR detection was negative for tet-O, tet-L and erythromycin methylases gene ermF respectively. ACKNOWLEDGEMENT The authors wish to thank the staff of Bacteriology Laboratory, Faculty of Veterinary Medicine, Universiti Putra Malaysia for providing the facilities and enabling environment to carry out this study. This research was funded by RUGS, UPM (9428800) and FRGS Ministry of Higher Education Malaysia. (5524643).