Adhesion and biofilm inhibiting properties of Chinese Herbal Formula SanHuang decoction against antibiotic resistant staphylococcal strains

Abstract

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.