Study area: The study was conducted in the laboratory of the Department of Microbiology and Hygiene, Bangladesh Agricultural University (BAU), Mymensingh during the period from January 2012 to June 2012.
Sources of samples A total of 100 samples were collected from humans (n=30), livestock (n=50) and poultry (n= 20) with proper aseptic precautions. In case of livestock, a total of 30 nasal swabs viz. 10 slaughter cattle, 10 dairy cattle and 10 breeding goat were collected from slaughter house of Mymensingh, BAU dairy farm and goat farm respectively. Ten abscess samples viz. 5 cattle and 5 goat/sheep from BAU veterinary clinic were collected aseptically for the study. For assessment the MRSA contaminated meat, 10 samples were collected from local market, Mymensingh. The samples was collected with proper aseptic precaution and carried to the laboratory for inoculation into culture media with specific identification marks. In case of Human, a total of 30 nasal swabs viz. 10 slaughter house employees, 10 dairy farm workers and 10 goat breeding farm workers were collected from slaughter house of Mymensingh, BAU dairy farm and goat breeding farm respectively. Immediately after collection the swab sticks were put into sterile test tubes and carried to the laboratory of the Department of Microbiology for inoculation into culture media with specific identification marks.
Experimental design The entire study was divided into three major steps: The first step included collection of samples from different areas, their transportation to the laboratory and inoculation into nutrient agar, blood agar and mannitol salt agar. In the second step isolation and identification of the Staphylococcus spp. was done on their cultural character including pigment production, haemolytic activity, Gram’s staining character and catalase activity. In the third step characterization of the organism was done using coagulase test and basic sugar fermentation test. Finally their antibiotic sensitivity test was also performed by using commercially available antibiotic discs (Oxoid, England) following the procedure described by Monica Chessbrough (1985).
Hemolytic activity Haemolytic activities of S. aureus were observed as per the method described by Chatterjee et al., (1990). All the strains were tested for the production of alpha (α) and beta (β) haemolysis by growing them on bovine BA plates and were then incubated at 37 °C for 24 hours to determine their hemolytic property. The colony developed on the BA was examined for various types of hemolysis. The hemolytic pattern of the bacteria was categorized according to the types of hemolysis produced on BA plates (Alpha (α) hemolysis: a zone of greenish discoloration around the colony manifested by partial hemolysis. Beta (β) hemolysis: complete clear zone of hemolysis around the colony: Gamma (γ) hemolysis: no detectable hemolysis).
Biochemical tests Sugar fermentation test, Indole and MR-VP test, catalase test, coagulase test according to the procedure described by Cowan (1985). Test for pigment production was performed according to the procedures described by Chatterjee et al., (1990).
Isolation and identification of Staphylococcus spp. For the isolation and identification of bacterial flora, the procedure suggested by Carter, 1979 was followed throughout the experiment. Isolation of Staphylococcus spp. from the collected samples was made by inoculating the samples on NA, BA and MSA. The inoculated media were then incubated aerobically at 37°C for 24 hours for growth. The isolates were identified on Staphylococcus based on their morphology and cultural characteristics and biochemical characters (Ellner 1978). The coagulase test was performed for the identification of the pathogenic Staphylococcus aureus from non-pathogenic ones. All the coagulase positive staphylococcal strains were further tested for pigment production, haemolysis on nutrient and blood agar, respectively. Stock culture was prepared and maintained for subsequent studies. To differentiate between Staphylococcus aureus and other Staphylococcus spp. it was considered the hemolysis, pigment production and coagulase test.
Antibiotic sensitivity test Staphylococci were tested for antimicrobial drug susceptibility against 02 commonly used antibiotics belonging to different groups by disc diffusion method or Kirby-Bauer method (Bauer et al., 1966). The antibiogram of isolates (Staphylococcus spp.) were determined on freshly prepared, dried up Mueller Hinton agar using by the Kirby-Bauer Disc Diffusion Method (Bauer et al., 1966) according to Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS, 2007) procedures. The tested antibiotics included: Methicillin (5 μg/ disc), Vancomycin (30 μg/ disc), and (Oxoid, England). Antimicrobial agents with their disc concentrations and zone diameter interpretive standards for Staphylococcus spp were followed as per recommendation by CLSI (2007). The isolates resistant to three or more antibiotics were considered as multi-drug resistant (MDR) strains. Turbidity standard for inoculums preparation To standardize the inoculum density for a susceptibility test, a BaSO4 turbidity standard, equivalent to a 0.5 McFarland standard or its optical equivalent (e.g., latex particle suspension) were used.