Sample collection
In this study, 100 non-duplicate S. aureus isolates were collected during 10 months (March to December 2019) from different clinical specimens (blood, urine, ulcer, pus, body fluids, trachea, sputum, etc.) of patients admitted to Zare hospital of Sari city (a burn center) and Imam Khomeini hospital of Behshahr city (a general center).The isolates were transferred to the Microbiology Laboratory and cultured in Blood Agar (Merck, Germany) and incubated at 37°C for 24 hours. Then, all isolates were identified by standard microscopic and biochemical methods such as gram staining, catalase and coagulase assays, mannitol fermentation and DNase assay(13), and were confirmed by PCR using nuc gene-specific primers. S. aureus ATCC 25923 was used as a control strain for diagnostic tests.
Antimicrobial susceptibility testing (AST)
The antibiotic susceptibility pattern of the isolates against 6 antibiotics including penicillin (10 μg), vancomycin (30 μg), erythromycin (15 μg), tetracycline (30 μg), ciprofloxacin (5 μg), and clindamycin (2 μg) (Roscoe, Denmark) was determined by Kirby-Bauer method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI)(15). We chose the S. aureus ATCC 25923 as a control strain in AST. Also, micro broth dilution method based on the CLSI guidelines was used to evaluate the minimum inhibitory concentration (MIC) of vancomycin, and S. aureus ATCC 29213 was used as a standard strain in this study(15).
Phenotypic assessment of biofilm production
The capability of biofilm formation in the isolates was investigated by microtiter plate method(16). Briefly, 180 µl of TSB (trypticase soy broth) containing 1% glucose was poured into 96 well microplate wells.Then, 20 µl of 0. 5 McFarland's equivalent bacterial suspension was added to TSB medium in each well. Next, the microplates were incubated at 37°C for 20 h. After the contents were thoroughly discharged from the wells and washed three times with 0.15M PBS (phosphate-buffered saline), the microplates were completely air-dried. Then, the wells were stained with 0.1% crystal violet.After dye evacuation, we washed the wells three times with distilled water and added 200 µl alcohol-acetone (1:4 ethanol to acetone) to the wells in order to release the dye on the wall of bacteria producing biofilm and attached to the well. The amount of dye released at each well was evaluated using the ELISA reader (Biotech, USA) at 590 nm. The OD of the samples were then compared with the OD of the control (ODC) and the results were analyzed using the cut-off method. The isolates which showed OD ≤ ODC, were considered as no biofilm producers, while the results as ODC < OD ≤ 2×ODC, 2×ODC < OD ≤ 4×ODC, and 4×ODC < OD were reflected as weak, moderate, and strong biofilm producer isolates, respectively. The TSB medium containing 1% glucose was used as the negative control, while S. aureus ATCC 35556 was used as a positive control (biofilm-producing strain) in this test(16).
Molecular analysis of nuc, fnbA and fnbB genes
Genomic DNAs were extracted from clinical isolates of S. aureus using a DNA extraction kit (SinaClon, Iran) according to manufacturer’s instructions. To confirm the purity of the extracted DNAs, a Nanodrop machine (Thermo Scientific, USA) at 260 nm was used, and the DNAs were electrophoresed on 1.5% agarose gel (Wizbiosolutions, South Korea). PCR was used to identify the nuc gene (for the final confirmation of S. aureus isolates) and to detect the presence of fnbA and fnbBgenes in clinical isolates. Primers sequences for the identification of target genes have been described previously (8, 14). The PCR reaction was performed in a final volume of 25 µl, consisting of 12.5 µl of premix (Denmark, Ampliqon), 10 picomoles of each primer, 1 µl of Taq DNA polymerase, 5 µl of distilled water, and 300 ng of template DNA. A thermal cycler (SensoQuest GmbH, Germany) was used to amplify the mentioned genes. The PCR reaction consisted of an initial denaturation step at 95°C for 2 minutes and 30 cycles of denaturation at 95°C for 25 seconds, followed by 30 s of the annealing stage at 53°C for the nuc gene, 52°C for the fnbA gene, and 55°C for the fnbB gene, and extension at 72°C for 30 s, along with a final amplification step at 72°C for 5 min. PCR products were electrophoresed on a 1.5% agarose gel (Wizbio, Korea) along with a DNA fragment length marker (GeneDireX, Taiwan) to investigate the presence of the target genes.
Statistical Analysis
Data were analyzed by SPSS software (version 22) and mean of quantitative data was analyzed using Descriptive software and were presented as Mean ± SD. Also, the significance level was evaluated by two-tailed and chi-square tests and P-value< 0.05 was considered statistically significant.