Bacterial Strains and Growth Conditions
In this study, 42 clinical S. aureus isolates were collected from urology research center of Sina hospital, Iran. All isolates were confirmed as S. aureus according to phenotypic and biochemical method including Gram-stain, catalase, coagulase, DNase, and mannitol fermentation tests. All isolates were stored in Tryptic Soy Broth (TSB) (Merck, Germany) with 20% glycerol at -80℃ for additional study.
Methicillin-resistant Staphylococcus aureus (MRSA) identification
For detection of MRSA isolates, bacterial suspensions of S. aureus isolates equivalent to 0.5 McFarland were prepared and cultured on Mueller-Hinton agar. Then, a cefoxitin disk (30μg; MAST Diagnostics, Merseyside, U.K) were placed on the plate and incubated overnight at 37 °C. The growth inhibition zones were analyzed by the Clinical and Laboratory Standards Institute (CLSI) guidelines [11]. As a quality control, S. aureus ATCC 25923 was used. Moreover, for the molecular recognition of MRSA, PCR assay for the mecA gene was done [12].
DNA extraction
S. aureus isolates were grown overnight at 37℃ on Trypticase soy agar (TSA) (Merck; Germany). Then genomic DNA was isolated using the High Pure PCR Template Preparation kit (Roche, Germany)[13, 14]. Evaluation of quality and concentration of each extracted DNA was examined using NanoDrop (Thermo Fisher Scientific; USA).
Identification of type II TA systems
The whole genome of S. aureus MU50 was retrieved from the NCBI database to identify the type II TA genes in S. aureus isolates. Type II TA systems were determined by TADB database (Toxin-Antitoxin database) and specific primers were designed using OLIGO software V. 7.56 [15]. The presence of gene coding ClpP and type II TA system (mazE/mazF, relE1/relB1, relE2/relB2, immA/irrA and omega/epsilon-zeta) was evaluated by PCR. The PCR was performed in a DNA thermal cycler (Bio-Rad, USA) in a final volume of 25 μl. The PCR program comprised of an initial denaturation step at 94 °C for 4 min; 35 (94 °C for 35 s; annealing for 45 s (Table 1); with a final extension at 72 °C for 20 s) [15]. Finally, two MRSA and MSSA (Methicillin-sensetive Staphylococcus aureus) isolates were selected to evaluate the expression levels of mRNA of Type II TA and ClpP protease in thermal and oxidative stresses.
Heat and cold shock responses
Overnight cultures of MRSA and MSSA cells were diluted 1:100 in 50 ml fresh TSB medium and incubated at 37°C at 200 rpm. Cultures of S. aureus were grown to mid-log phase (optical density at 600 nm, 0.25). For induction of the cold shock and heat shock responses, were then incubated for an additional 30 min with aeration at 10°C and 42°C, respectively. The control sample was placed in the incubator at 37 °C [16].
Growth curve and viability assessment
The Growth curve and the viability of MRSA and MSSA isolates were determined in the presence of 5mM and 10 mM H2O2. Overnight cultures were diluted 100 – fold in 50 ml TSB in 250 ml Erlenmeyer flasks. Cultures were placed in an incubator with shaking at 200 rpm at 37 ℃ until reaching an optical density of 0.4 at 600 nm (OD600). Then, cultures were diluted 3-fold to reach an OD of 0.08-0.1 at 600 nm. These cultures were divided and H2O2 was added to the two concentration of 5 mM and 10 mM of H2O2. Following incubation of each concentration, 1 ml of cell was removed at 0, 1, 2, 3, 4 and 24h, then growth curves were assessed turbidimetrically. Moreover, in each time point, 1 ml of cells were washed twice with 0.9% sterile saline solution and serial dilution was prepared and plated onto TSA plates and incubated in 37 ℃. Colony count was done after 24h incubation. Untreated bacterial cells were used as controls at different time intervals. All experiments were done independently at least three times.
RNA isolation and quantitative real-time reverse-transcriptase PCR (qRT-PCR)
The expression levels of gene coding ClpP protease and toxin of type II TA systems (mazF, relE1, relE2 and immA) upon oxidative and thermal stresses were evaluated by qRT-PCR method. RNA extraction was performed using a high pure RNA isolation kit (Roche; Germany) 1 hour after adding H2O2 and 30 min after the heat and cold shock.
Bacteria were pelleted by centrifugation at 7000×g for 10 min, the supernatant was discarded and the pellet was resuspended in Tris-EDTA (TE) buffer (pH 8). Then 50 μg/ml lysozyme (Sigma-Aldrich; Germany) and 250 μg/ml lysostaphin (Sigma-Aldrich; Germany) were added followed by incubation at 37 °C for 10 min and 15 min, respectively[13]. RNA extraction was performed using a high pure RNA isolation kit (Roche; Germany) according to the manufacturer's instructions. The quantity and quality were evaluated by NanoDrop spectrophotometer (Thermo Fisher Scientific, USA) and gel electrophoresis. DNA contamination was resolved with the addition of an extra DNase I (Thermo Fisher Scientific, USA) treatment. The absence of DNA contamination in RNA samples was confirmed by PCR amplification of the housekeeping 16S rRNA gene. Reverse transcription was performed using the FIREScript RT cDNA synthesis kit (Solis BioDyne; South Korea) according to the manufacturer's instructions. Finally, quantitative real-time PCR was carried out in a Rotor-Gene thermal cycler (Corbett 6000; Germany) using SYBR Green method (Amplicon; Denmark) by specific primers. A total volume of 20 μl reaction containing 2 μl of cDNA, 12.5 μl SYBR Green master mix, 3.5 μl nuclease-free water, and 1 μl of each primer (5 pmol) was performed according to the following program: an initial activation step at 94 °C for 10min, 45 cycles of denaturation at 95 °C for 30 s, and one cycle of 60 ℃ for 45 s. the 16s rRNA was used as an internal control to normalization of mRNA levels and fold changes. Expression was calculated by the 2 - Δ ΔCT method [8, 15].
Statistical analysis
Data obtained from growth curves and viability assessment were expressed as the mean of the three independent examinations. Data of the mRNA expression analysis were presented as means ± standard error of three independent experiments. All the statistics were performed by Graph pad prism 8 (GraphPad Software, Inc). Student’s t-test (for two groups) and analysis of variance (two-way ANOVA) were used.