2. Material and methods
2.1 Sample collection
The following 14 samples of Staphylococcus aureus were used in this study. All samples were obtained from food contact surfaces at three food premises in University of Putra Malaysia (UPM). Samples were collected from the surface of each material according to standard swabbing techniques. The collection was carried out during weekdays (15:00-17:00) after finished the preparation of foods. Sterile swabs were removed from the coded test tubes that contain 5 ml of sterile phosphate buffered saline with pH 7.4±1 (Oxoid, Basingstoke, UK) and the targeted areas on the cutting board, tray, plate and wok were swabbed.
Sampling has been done by swabbing all of these surfaces horizontally, vertically and diagonally by using standard templates with the size of 10 cm x 10 cm. The whole procedure will be carried out aseptically to minimize the risk of contamination. After swabbing, samples were put back into their respective coded test tubes. The collected samples were stored in an insulated ice box filled with crushed ice. The storage temperature was around 0-4oC and transportation time taken back to UPM was around 15 mins. The analysis was carried out as soon as reaching the laboratory.
2.2 Isolation and identification of Staphylococcus aureus
A single colony of S. aureus for each sample from the food contact surfaces were isolated from their respective Petri film plates and enriched in universal bottles that contain 10ml of Tryptic Soy broth (TSB). The broth cultures then were incubated at 37oC for 24h. The colonies were identified through a series of biochemical tests. Prior to the biochemical test, the isolates were being revived on nutrient agar to obtain pure fresh cultures.
2.2.1 Biochemical test
The suspected S. aureus isolates then were identified through Mannitol Salt agar (MSA) test and catalase test. The isolates sample was tested by looking at the biochemical and the reaction towards the chemical. These have been confirmed by gram staining and biochemical test with Baird Parker agar (BPA) and coagulase test. The suspected bacterium was grown on MSA and look out for the golden-like sheen colonies. We might wish to confirm the colonies by emulsifying a bit of the growth on 2% hydrogen peroxide. Observe for bubbling and frothing in catalase test which is peculiar to Staphylococcus. To distinguish between the species, pick a bit of the growth from the MSA and grow with incubation at 37oC in rabbit citrated plasma and observe for coagulation of the plasma with coagulase test. This is positive for Staphylococcus aureus as shown in Table 1. Microbiological analysis was done on the food contact surfaces found in all food premises.
Gram-staining, biochemical test and culture characteristics on selective media
Culture characteristic of S. aureus
Black and shiny with narrow white margins and surrounded by clear zone.
Yellow colonies with yellow zones in the media.
Positive cocci in clusters
Appearance of gas bubbles
Clumping cocci within 5-10 seconds
2.3 Antimicrobial resistance test
All these identified Staphylococcus aureus were tested for their resistance against the antibiotic. 11 antibiotics from five different classes (Penicillin, Sulphonamide, Cephalosporins, Quinolones and Amino-glycosides) have been used against Staphylococcus aureus. The antibiotic was test from the disc diffusion test by observing and measuring the diameter presence from inhibition zone surrounds the bacteria.
2.3.1 Antibiotic test (disc diffusion)
Working cultures of the S. aureus were maintained on Tryptic Soy agar (TSA), following incubation at 37 oC for 24 h. Inocula were obtained from overnight fresh cultures adjusted to approximately log 108 CFU/ml, a turbidity equivalent to a 0.5 McFarland standard (Kroning et al., 2016). The inocula were spread on the surface of Mueller-Hinton agar (MHA) plates. The disk diffusion test, recommended by the Clinical and Laboratory Standards Institute (CLSI, 2015), was employed to determine the resistance to 11 antimicrobials: ciprofloxacin (5 µg), amoxicillin (10 µg), gentamicin (10 µg), penicillin G (10 µg), ceftriaxone (5 µg), sulphafurazole (300 µg), streptomycin (25 µg), ceftazidime (30 µg), cephalothin (30 µg), nalidixic acid (30 µg), cefotaxime (30 µg) purchased from the company Mayang Scientific®. The results were interpreted in accordance with the standards for inhibition zone diameters for Staphylococcus spp. (CLSI, 2015). Staphylococcus aureus ATCC 13565 was used as the positive control for the tests.
2.4 Statistical analysis
All experiment was performed at least three times and two results were examined in each replication. Data were analysed using the Microsoft Excel 2010 software. Resistance rates were computed by the average and standard deviation calculation.