The aim of this study was to explore the effect of Chinese herbal SanHuang decoction (SH) on biofilm formation of antibiotic-resistant Staphylococci on titanium surface, and to explore its mechanism. Biofilm-forming ATCC 35984, ATCC 43300 and MRSE 287 were used in this study. The MICs of SH and vancomycin against Staphylococci were determined by the broth microdilution method. Six groups were designed, namely control group (bacteria cultured with medium), 1/8MIC SH group (1MIC SH was diluted by 1/8 using TSB or saline), 1/4MIC SH group, 1/2MIC SH group, 1MIC SH group and vancomycin group (bacteria cultured with 1MIC vancomycin). The inhibitory effect on bacterial adhesion and biofilm formation were observed by the spread plate method, CV staining, SEM, and CLSM. Real-time PCR was performed to determine the effect of SH on the expression levels of ica AD and ica R gene in ATCC 35984 during the biofilm formation. The strains were found to be susceptible to SH and vancomycin with MIC of 38.75 mg/ml and 2.5 μg/ml, respectively. SH with 1 MIC and 1/2 MIC could inhibit the bacteria adhesion, showing only scattered adhesion from SEM. CLSM showed that SH with 1 MIC and 1/2 MIC inhibited the biofilm formation. The quantitative results of the spread plate method and CV staining showed that there was significant differences between the SH groups (P < 0.05). Further, with an increase in SH concentration, the inhibitory effect became more obvious when compared with control group. Among the groups, vancomycin had the strongest inhibitory effect on bacterial adhesion and biofilm formation (P < 0.01). With an increase in SH concentration, the expression levels of ica AD decreased, and the expression of ica R increased correspondingly (P < 0.05). In conclusions, SH can inhibit the biofilm formation of antibiotic-resistant Staphylococci. Its probable mechanistic activity may be through the inhibition of polysaccharide intercellular adhesin synthesis by down-regulating the expression of ica AD gene.

Background. External socking and washing with the Chinese herbal Sanhuang Jiedu decoction (SHJD) can effectively control local limb infections with bone and implant exposure. However, the antibiofilm activities of this decoction in vitro have not yet been investigated. Therefore, the aim of this study was to examine the effects and characteristics of SHJD on the mature biofilms of multidrug-resistant staphylococci on a titanium surface. Methods. Biofilm-forming methicillin-resistant Staphylococcus epidermidis ATCC 35984 and S. aureus ATCC 43330, and non-biofilm-forming S. epidermidis ATCC 12228 were selected as the experimental strains. The mature biofilms were prepared on titanium surfaces. The five experimental groups were based on dilution concentrations (DC) of SHJD: the control group (biofilm incubated with 0.85% NaCl solution), the SHJD (DC:1/8) group (initial SHJD solution was diluted 1/8), the SHJD (DC:1/4) group, the SHJD (DC:1/2) group, and the SHJD (DC:1/1) group (initial SHJD solution). The effects of SHJD on the mature biofilms were observed with the bacterial spread plate method, crystal violet (CV) staining, scanning electron microscopy, and confocal laser scanning microscopy. Results. After culture in tryptic soy broth for 72 h, ATCC 43300 and ATCC 35984 produced mature biofilms and ATCC 12228 did not. The optical density value of ATCC 12228 was 0.11 ± 0.02 , significantly lower than that of ATCC 35984 ( 0.42 ± 0.05 ) or ATCC 43300 ( 0.41 ± 0.03 ) ( P < 0.05 ). The mature biofilms of ATCC 43300 and ATCC 35984 clearly disintegrated when incubated for 12–24 h with SHJD (DC:1/1) or SHJD (DC:1/2), showing only scattered bacterial adhesion. In the SHJD (DC:1/4) group, although many residual bacterial colonies still clustered together, presenting a biofilm structure, it was very looser than that in the SHJD (DC:1/8) group in which the biofilm was similar to that in the control group. For ATCC 12228, only colony adhesion was observed, and the number of colonies decreased as the concentration of SHJD or the culture period increased. The quantitative results for the bacterial spread plate and CV staining showed significant differences between the SHJD groups ( P < 0.05 ). Conclusion. SHJD has antibiofilm activity against multidrug-resistant Staphylococcus strains. It weakens or disrupts already-formed mature biofilms on titanium surfaces in a concentration- and incubation time-dependent manner.

Clinical study has shown that external socking and washing with the Chinese herbal SanHuang decoction (SH) can control the orthopedic-biofilm related infections. However, the antibiofilm activities of SH in vitro have not been investigated. The aim of the current study was to explore the effect of SH on adhesion and biofilm formation of antibiotic-resistant staphylococci on titanium surface, and to explore its probable mechanistic effects on staphylococcal strains. Biofilm-forming ATCC 35984 (methicillin-resistant Staphylococcus epidermidis, MRSE) and ATCC 43300 (methicillin-resistant Staphylococcus aureus, MRSA) strains were used in this study. The minimum inhibitory concentrations (MICs) of SH and vancomycin against planktonic bacterial strains were determined by the broth microdilution method. Different sub-MIC of SH with TSB (Tryptic soy broth) were used as the basis for experimental grouping (SH group). TSB culture medium alone (TSB group) or TSB containing vancomycin (vancomycin group) incubated with bacteria were considered as the negative or positive control group, respectively. The inhibitory effect of different treatment on bacterial adhesion and biofilm formation were observed by the spread plate method, CV (crystal violet) staining, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). Real time PCR analysis was performed to determine the effect of SH on the expression levels of ica AD and ica R gene in ATCC 35984 during the biofilm formation. The strains were found to be susceptible to SH decoction with MIC and MBC values of 38.75 mg/ml and 77.5 mg/ml, respectively. The MIC values for vancomycin was 2.5 μg/ml. SH treatment with 1 MIC and 1/2 MIC could inhibit the bacteria adhesion on the titanium surface, showing only scattered bacterial adhesion from SEM. CLSM showed that SH with 1 MIC and 1/2 MIC could also inhibit the bacterial biofilm formation. The quantitative results of the spread plate method and CV staining showed that there was significant differences between the SH groups (P < 0.05). Further, with an increase in SH concentration, the inhibitory effect became more obvious at different culture time points, when compared with TSB control group (P < 0.05). Among the groups, vancomycin had the strongest inhibitory effect on bacterial adhesion and biofilm formation (P < 0.01). Meanwhile, with an increase in SH concentration, the expression levels of ica A and ica D decreased, and the expression of ica R increased correspondingly (P < 0.05). In conclusions, a certain concentration of SH can inhibit the adhesion and biofilm formation of antibiotic-resistant Staphylococcal strains on the titanium surface, but the effect was not as good as vancomycin. Its probable mechanistic activity may be through the inhibition of polysaccharide intercellular adhesin synthesis by down-regulating the expression of ica AD gene, thus inhibiting bacterial biofilm formation.