Bacterial strains and materials

In this study, three reported strains commonly found in voice prosthesis biofilms, Staphylococcus aureus (ATCC 25923), Streptococcus faecalis (ATCC 13419) and Candida albicans (SC 5314), were used to construct an in vitro model of voice prosthesis biofilms [17,18,19]. These three strains were cultured in 70% yeast extract/peptone/dextrose medium (Sigma‒Aldrich) + 30% fetal bovine serum (Gibco) (YPDF medium) and incubated overnight at 37 °C, after which plaques were clearly visible [20]. Medical-grade silicone membranes (thickness: 1 mm) were purchased from Suzhou Shoucheng Electronics Co., Ltd., China, and were sterilized under high pressure at 121 °C before use.

Construction of mature biofilms on medical silicone membranes

The construction of mature biofilms on medical silicone membranes was carried out as previously reported [20, 21]. Appropriate amounts of mature colonies were picked and cultured in 5 ml of YPDF medium at 37 °C for 6–8 h, until the OD600 was approximately 0.6. These bacterial cultures were diluted and mixed in equal volumes, inoculated into YPDF medium at a 1% ratio, then added to 96-well plates (Corning, 3599) with sterilized medical silicone membranes. The cells were cultured at 37 °C for 48 h.

Determination of the minimum mature mixed biofilm inhibitory concentration (BIC) and the minimum mature mixed biofilm eradication concentration (BEC) on voice prostheses

Three groups were set up for this experiment, a blank control group (medium only in well, without biofilm or SSD), a negative control group (medium and biofilm in the well, without SSD), and experimental groups (medium, biofilms and SSD at different concentrations). Sterilized medical silicone membranes were placed obliquely in the wells of a 96-well deep-well plate, and 2 ml of YPDF medium was added to each well. Bacterial cultures of each species diluted to 1% were mixed in equal volumes and inoculated into each well. Silver sulfadiazine (Sigma‒Aldrich 481,181-5G) at different concentrations was added, with an equal volume of H2O used as a blank control, followed by incubation at 37 °C for 24 h. The number of colonies in each well was measured by the plate counting method. The lowest SSD concentration that reduced the number of colonies in the voice prosthesis biofilms by 50% compared to the blank control was taken as BIC50. Compared with the negative control group (SSD concentration: 0 µg/ml), the lowest SSD concentration that reduced the number of colonies in the voice prosthesis biofilms by 30% was taken as BEC30, the lowest SSD concentration at which the number of colonies was reduced by 50% was taken as BEC50, and the lowest SSD concentration at which the number of colonies was reduced by 70% was taken as BEC70.

Plate counting of biofilms

The biofilms on the medical silicone membranes were eluted by ultrasonication. The bacterial cultures were diluted to different concentrations with phosphate-buffered saline (PBS) (Gibco™, 70011044), and 100 µl of each dilution was spread on a YPDF plate. The cells were incubated at 37 °C for approximately 18 h and counted by taking pictures with an automatic colony counter (Interscience, SCAN1200). The percentage of colonies removed by SSD from the mature voice prosthesis biofilms was calculated by Formula (1):

$$}\;}\;} = \left[ }\;}\;}\;}\;}\;}}}}\;}\;}\;}\;}\;}\;}}}} \right] \times 100}$$

(1)

Real-time fluorescence quantitative PCR

Standard plasmids for S. aureus, S. faecalis and C. albicans were constructed, the concentration of each standard plasmid was adjusted to 109 copies/µl, and each standard plasmid was diluted fivefold to draw a standard curve. A bacterial genome extraction kit (Vazyme, DC103) was used to extract DNA from the bacterial strains in the biofilms, and the SYBR Green Master Mix (Vazyme, Q111-02) kit was used to carry out qPCR on the obtained DNA and plasmids. The final reaction system volume was 10 µl. The reaction conditions were as follows: 50 °C for 2 min and 95 °C for 5 min, followed by 40 cycles of 95 °C for 15 s, 56 °C for 20 s, and 72 °C for 40 s. The real-time fluorescence quantitative PCR primers are shown in Table 1.

Table 1 Primers for real-time fluorescence quantitative PCR

In this experiment, a blank control group, a negative control group, and experimental groups (SSD concentrations: BEC30, BEC50 and BEC70 and 5 µg/ml, 10 µg/ml, 15 µg/ml, 20 µg/ml, and 30 µg/ml) were prepared. According to the method described above, a mature mixed bacterial voice prosthesis biofilm was constructed, the medium and different concentrations of SSD were added to the 96-well plate according to the experimental grouping. The cells were cultured at 37 °C for 24 h. Real-time fluorescence quantitative PCR was used to determine the copy numbers of the three strains in the biofilms of each group to evaluate the corresponding inhibitory effects of SSD on the three strains in the biofilms. The percentage of strain copies removed from the biofilms by SSD was calculated by Formula (2).

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(2)

Crystal violet staining

The cultured medical silicone membranes were removed, washed with PBS twice, fixed with 0.10 ml of 10% methanol solution (BBI, A601617) for 15 min, and stained with 0.1% crystal violet staining solution (KeyGEN BioTECH, KGA229) after air-drying. The membranes were then placed at room temperature for 10 min, rinsed three times with PBS, and dried at 37 °C; 33% glacial acetic acid (Sangon Biotech (Shanghai) Co., Ltd., A501931) was then added to dissolve the crystal violet, and 200 µl of the solution was taken to measure the absorbance at 590 nm on a spectrophotometer (Beckman, AD340). The degree of inhibition of biofilm formation ability by SSD was calculated by Formula (3):

$$}\;}\;} = \left[ }\;}\;}\;}\;} - }\;}\;}\;}\;}\;}}}}\;}\;}\;}\;} - }\;}\;}\;}\;}\;}}}} \right] \times 100}$$

(3)

XTT reduction assay

The cultured silicone membranes were washed three times with PBS and transferred into the wells of a 96-well deep-well plate, 500 µl of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) (XTT) assay working solution (KeyGEN BioTECH, KGA313) was added, and the plate was incubated at 37 °C for 4 h in the dark. The absorbance at 450 nm was measured by a spectrophotometer. The degree of inhibition of biofilm metabolic activity by SSD was calculated using Formula (2).

Scanning electron microscopy (SEM)

The cultured medical silicone membrane was rinsed three times with PBS, fixed overnight at 4 °C in 2.5% glutaraldehyde phosphate buffer (BBI, A600875), washed twice with 0.15% glutaraldehyde phosphate buffer, and then dehydrated with an ethanol series (40%, 70%, 90%, 100%) for 15 min with each concentration. Then, the cells were dried in a critical-point desiccator. After gold was sprayed with a vacuum coating device, the changes in the ultraspatial structure of the voice prosthesis biofilms in each group were observed under scanning electron microscopy (SEM) (ZEISS, GeminiSEM 360).

Laser confocal microscopy

Live and dead bacteria on the biofilms were stained according to the instructions of the LIVE/DEAD BACLIGHT BACTERIAL C 1 KIT (Invitrogen, L7012). The culture medium was aspirated, and 0.85% NaCl was added to the well plate to rinse the medical silicone membranes three times. Equal volumes of component A (SYTO 9 dye, 3.34 mM) and component B (propidium iodide, 20 mM) were mixed thoroughly, and 3 µl of the mixed dye solution was added to each well. The plate was incubated for 15 min at room temperature in the dark. The distribution of live and dead bacteria in the biofilms in each group was observed under a laser confocal microscope (Olympus, FV3000).

Polysaccharide‒phenol‒sulfuric acid method

The cultured medical silicone membranes were washed with PBS, transferred into a centrifuge tube, and 1 mm glass beads washed with concentrated hydrochloric acid were added. The samples were then placed into an automatic rapid sample grinder with 100 µl of PBS. The biofilms were eluted by shaking four times at 60 Hz for 60 s. A 5% phenol solution (Sinopharm Shanghai Test, 10015328) and 98% concentrated sulfuric acid (Sinopharm Shanghai Test, 10021608) were mixed to prepare the chromogenic solution at a ratio of 1:5. A total of 180 µl of the chromogenic solution was added to 60 µl different concentrations of glucose solution (Amresco, 0188) and the biofilm eluate. After thorough mixing, the mixture was heated in a metal bath at 100 °C for 25 min, after which 100 µl of the solution was removed to measure the absorbance at 490 nm on a spectrophotometer.

Bicinchoninic acid (BCA) method

Protein determination was performed with the abovementioned eluates. CST RIPA buffer (Biyuntian, P0013B) was used to lyse each group of biofilms and collect proteins. Different concentrations of bovine serum albumin (BSA) standard and bicinchoninic acid (BCA) working solution were prepared according to the instructions of the BCA protein concentration determination kit (Sangon Biotech, C503021). Fifty microlitres of sample lysis buffer or 500 µl of BSA standard was mixed quickly with 500 µl of BCA working solution. The mixture was placed in a water bath at 37 °C for 30 min and then cooled to room temperature, and the A560 value was measured on a spectrophotometer.

Effect of SSD on the mature biofilm on patients’ voice prostheses

To further verify the inhibitory and removal effects of SSD on mature biofilms on voice prostheses, we tested the effects on voice prosthesis biofilms from patients in vitro. We collected isolated, nonfunctional voice prostheses from three patients. For collection and sampling of patients’ voice prostheses, we obtained ethical certification and approval from the Ethics Committee of Peking Union Medical College Hospital (ethics number: JS2084). The biofilms on the voice prostheses were eluted by ultrasonication, and the bacterial strains in the biofilms were collected. The sterilized medical silicone membranes and 2 ml of the bacterial solution collected above were placed obliquely in the wells of a 96-well deep-well plate and incubated at 37 °C for 48 h. Different concentrations of SSD were added, with a medium control was established at the same time, and the cells were cultured at 37 °C for 24 h. The plate counting method was used to determine the effect of different concentrations of SSD on the number of bacterial colonies in the mature biofilms from the patients’ voice prostheses, and the XTT method was used to verify the inhibitory effect of SSD on the metabolic activity of the biofilms.

Data analysis

Experiments in each group were repeated at least three times. The measurement data are expressed as the mean ± standard deviation. Differences among multiple groups were analysed by one-way analysis of variance (ANOVA). Comparison between the experimental group and the negative control group was performed by Dunnett’s t test. The data were analysed using GraphPad Prism 9 software. All tests were two-sided, and P < 0.05 indicated that the difference was statistically significant.