We used standard strains of staphylococcus aureus (ACTT25923 Microbiology Laboratory of Fuzhou General Hospital of Nanjing Military Command),titanium-alloy plates (Ti6Al4V, diameter 12.7 mm, thickness 4.7 mm, Shenzhen Smarl Equipment Co., Ltd.),phosphate buffer (PBS), 24-well tissue culture plates, standard agar culture dishes, an electronic confocal laser scanning microscope (CLSM; FVI000,Olympus Corporation), CLSM professional dishes, and a Quanta 450 scanning electron microscope (SEM; Thermo Fisher Scientific).
1.2 Experimental Methods
1.2.1 Preparation of Bacterial Suspension
Standard staphylococcus aureusstrains were grown on slants in 10 ml trypticsoy broth (TSB) and were placed in a constant temperature incubator(37°C, 5%CO2) for overnight anabiosis. A trace amount of bacteria solution was taken, grown in blood plate medium using the four-zone streaking method, and cultivated to obtain bacterial colonies. A single bacterial colony was dissolved, resulting in a turbidity of 0.5 (measuring using aturbidimeter). One ml of bacteria solution was diluted in 149 ml of sterile TSB medium, yielding afinal concentration of 1×106CFU/ml, which was used for further applications.
1.2.2 Preparation of Bacterial Biofilms (BBF) on a Titanium Alloy Surface
The titanium alloy plates were pretreated by sterilization using the moulding method. They were then put into 24-well plates and 1mlof bacterial suspension at a concentration of 1×106CFU/ml was added to each well. The plate was placed in a constant temperature incubator to cultivate for 7,14,21, or 28d.
1.2.3 Treatment of Biofilms with PVP-I
After surface biofilm growth, the titanium alloy plates were washed 3 times with PBS to remove non-adherent floating bacteria and then put into a new sterile 24-well plate. One ml of 0.5% PVP-I solution was added to each well and incubated at 37°C for 5min, or10min. Additionally, some plates were treated with PBS only.
1.2.4 Experimental Groups
A total of 480 titanium alloy plates were used to establish biofilms in 4 phases (120 plates at each time point). The cultivated biofilm in each phase was randomly divided into 3 treatment groups with 40 plates per group. After PVP-I treatment for 0, 5,or 10 min, 10 plates from each group were randomly selected for scanning electron microscopy, 20 plates were imaged using CLSM, and 10 plates were used for CFU counts.
1.2.5 CLSM Imaging of Bacterial Biofilm Structure
After different cultivation times, titanium alloy plates with surface biofilm were washed with PBS. 40 plates were selected randomly from each cultivation phase group and treated with PVP-I for 5 min or10 min. Another 20 plates from each group were treated with PBS only. 10 plates from each PVP-I treatment group (30 plates per cultivation phase) were selected randomly, stained using fluorescein isothiocyanate-conjugated concanavalinA (FITC-ConA) in the dark for 30 min, and then stained using propidium iodide (PI) in the dark for 15 min. The other 10 plates from each PVP-I treatment group were stained using PI and SYTO™ 9 green fluorescent nucleic acid stain(SYTO 9) in the dark for 20 min. After staining, the plates were washed with PBS, dried, and placed in an observation dish. The biofilm morphology and bacteria vitality were imaged used CLSM.
1.2.6 SEM Imaging
After different cultivation times, titanium alloy plates with surface biofilm were washed with PBS. 20plates were selected randomly from each cultivation phase group and treated by PVP-I for 5 min or10 min. Another 10 plates from each group were treated with PBS only. The plates were fixed in glutaraldehyde for more than2 hours and then dehydrated with different concentrations ofethyl alcohol. The plates were then dried using aK850 critical point dryer and placed into a Q 150R S/E/ES sample preparation system for metal spraying. The morphological structure of iodine-treated bacterial biofilms indifferent cultivation phases was then observed using SEM. Random fields were selected for imaging.
1.2.7 Viability Counts
After different cultivation times, titanium alloy plates with surface biofilm were washed with PBS. 20 plates were selected randomly from each cultivation phase group and treated by PVP-I for 5 min or 10 min. Another 10 plates from each group were treated with PBS only. The plates were washed again using PBS and then placed into a culture flask with 10 ml of PBS. The plates were then treated with an ultrasonic cleaning machine and vortex oscillator and diluted with eluent. 100μlof eluent was added dropwise to the center of a general nutrition agar plate and applied homogeneously using a triangle push rod. The plate was placed in a constant temperature incubator at5% CO2and 37°C for 48 h, and then CFU counts were performed.
1.3 Statistical Analysis
We performed a normality test of the CFU counts using SPSS20.0 software and found that the test results were not normally distributed. Thus, we performed a Kruskal-Wallis H non-parametric test on multiple independent samples and a Mann-Whitney U test for comparison among groups. The test level was set to 0.05 and P<0.05 was considered statistically significant.