Bacterial strains and biofilms growth conditions
Methicillin-resistant S. aureus strain ATCC43300 provided by the Department of Laboratory Medicine (West China Hospital, Sichuan University, Chengdu, China) was cultured in tryptic soy broth (TSB). After overnight incubation at (37ºC, 5% CO2). Five hundred microfilters of S. aureus overnight suspension was inoculated into 10 mL fresh TSB medium to mid-logarithmic phase with OD600 value at 0.5. For the formation of biofilm, the sterilized glass disks (diameter in 14 mm) were applied for 24 hours biofilms for further analyses according to our previous study .
Construction of S. aureus antisense yycF/yycG overexpression strains
Recombination of plasmid pDL278 containing antisense yycF or yycG was conducted by inserting antisense yycF (ASyycF) or yycG (ASyycG) sequence into restriction sites between BamHI and EcoRI by Sangon Biotech (Shanghai, China). According to our previous study, an ASyycF overexpression methicillin-resistant S. aureus (ASyycG mutants) or an ASyycG overexpression methicillin-resistant S. aureus (ASyycF mutants) was constructed by adding recombinant pDL278 ASyycG or ASyycF plasmid. The empty pDL278 plasmid did not exert any effects on the viability of S. aureus .
Analysis of gene expression using quantitative real-time PCR (qRT-PCR)
MRSA, ASyycG and ASyycF strains were cultured to the midlogarithmic phase. Then, we treated the S. aureus planktonic cultures with 100mM H2O2 for 60 min, washed and resuspended in PBS (pH 7.2). The total RNAs were extracted and purified with the MasterPureTM RNA Purification Kit (Epicentre Technologies, Epicentre, Madison, WI, USA) including MRSA + H2O2 group, ASyycG + H2O2 group, and ASyycF + H2O2 group. The purified RNA was reverse transcribed to cDNA with the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). The quantitative real-time PCR assays were applied with LightCycler 480 system (Roche, Basel, Switzerland) with the primers listed in Table 1 with the 16Sr RNA gene as an internal control. Each sample was analyzed in triplicate, and the threshold cycle values (CT) were quantified .
Sequences of primers in this study
sequence 5’-3’ (Forward/Reverse)
5’- GATTATGTAATGTGCTTGGA − 3’/
5’- ACTACTGCTGCGTTAATAAT − 3’
5’ - TGGCGAAAGAAGACATCA − 3’/
5’ – AACCCGTTACAAATCCTG- 3’
5’ - CGGGGCGTTCAAAAGACTTT − 3’/
5’ - TCTGAACCTTTGAACACACGT − 3’
5’ - ATGGTCAAGCCCAGACAGAG-3’/
5’ –CGTGTTTTCAACATTTAATGCAA − 3’
5’ - GTAGGTGGCAAGCGTTATCC − 3’/
For protein extraction, the bacterial cells including MRSA + H2O2 _group, ASyycG + H2O2 group, and ASyycF + H2O2 group were mechanically disrupted and collected by centrifugation as previously described . For western blot analysis, the collected protein was probed with purified YycG- and YycF- specific antibodies (1:1000, HuaBio Biotechnology, Hangzhou, China). A BioRad GS-700 Imaging Densitometer was used to determine the signal density of Western blot bands for comparation.
Biofilms exposure to hydrogen peroxide
To determine the susceptibility of S. aureus ATCC43300, ASyycG mutants and ASyycF mutants to oxidants in biofilms conditions, all groups were treated with 3% H2O2 (for clinical irrigation, H2O2 is usually 3%) for 30 min at 37°C. Residual H2O2 was removed by diluting biofilms samples in PBS buffer. The treated biofilms samples were cultured in TSB medium for another 24 hours for further investigation, including MRSA + H2O2 group, ASyycG + H2O2 group, and ASyycF + H2O2 group.
The microtiter dish assay and epifluorescence staining for biofilm biomass
The microtiter dish assay was applied to evaluate the biomass of treated biofilm after cultured with another 24 hours with crystal violet (CV) following previous protocol . The dye bound to the biofilms was transferred into a new plate and the absorbance was measured with a microplate reader (ELX800, Gene) under OD600 nm.
For epifluorescence staining, the biofilms were labeled with SYTO9 (LIVE/DEAD Bacterial Viability Kit reagent; BacLight, Invitrogen, Grand Island, NY, USA); live cells were stained green, while dead cells appeared red. The cells were visualized using epifluorescence microscopy (Nikon Eclipse TE-2000S, Melville, NY) at 40×magnification. Notably, three random fields in each specimen were selected.
Characterizing biofilm morphologies
The 24 hours biofilms of MRSA + H2O2 group, ASyycG + H2O2 group, and ASyycF + H2O2 group were labeled with SYTO9 (Invitrogen; Thermo Fisher Scientific, Inc.). The observation was implemented using epifluorescence microscopy (Nikon Eclipse TE-2000S, Melville, NY) at 40 ×magnification. Notably, five random fields in each specimen were selected. To assess the structure of biofilms, the scanning electron microscopy (Inspect, Hillsboro, OR, USA; SEM) was conducted. Briefly, the treated biofilm samples were diluted twice using PBS and fixed with 2.5% glutaraldehyde for 4 hours. Then the fixed samples were serially dehydrated and dried using a critical point dryer. After coated with gold powder, the micrographs of biofilm samples were evaluated.
All statistical data were analyzed in SPSS 16.0 (SPSS Inc., Chicago, IL, USA). The Shapiro–Wilk test was used to analyze the distribution of data, and the Bartlett test was used to determine the homogeneity of variances. For parametric testing, we adopted a one-way ANOVA analysis to assess the statistical significance of variables followed by the Tukey test. The significant differences of data were set at P < 0.05.