ABSTRACT
ObjectiveTo study the virulence and biofilm inhibition effect of Fufang Huangbai Fluid Paint （FFHBFP） on methicillin-resistant Staphylococcus aureus （MRSA）， and to explore the antibacterial effect of FFHBFP on MRSA， which provides a theoretical basis and reference for clinical medication. MethodFirstly， the microdilution method and time–growth curve were used to determine the minimum inhibitory concentration （MIC） of FFHBFP and vancomycin （VAN） against MRSA and the effect on bacterial growth. The effects of FFHBFP and VAN on the inhibition of MRSA virulence factor lipase and restoration of hydrogen peroxide （H2O2） sensitivity were detected under sub-minimum inhibitory concentration （sub-MIC）. The inhibitory effect of FFHBFP and VAN on MRSA biofilm formation and maturation was detected by the microplate method. The morphological changes of mature biofilms before and after administration were observed under a scanning electron microscope （SEM）. Real-time polymerase chain reaction （Real-time PCR） was utilized to detect the effect of 50.600 g·L-1 concentration of FFHBFP on the expression of MRSA virulence gene crtM and biofilm-forming genes fnbA and icaA. Finally， molecular docking technology was used to predict the mechanism of potential antibacterial active ingredients of FFHBFP in inhibiting the virulence and biofilm of MRSA. ResultThe MIC of VAN was 2 mg·L-1， and VAN below 1 mg·L-1 exerted no effect on MRSA growth. The MIC of FFHBFP was not determined， while the 101.200-202.400 g·L-1 original solution inhibited MRSA growth. Compared with the blank group and the VAN group， sub-MIC （25.300-50.600 g·L-1 original solution） inhibited lipase and recovered MRSA sensitivity to H2O2 （P<0.01）. The results of the microplate method showed that FFHBFP （25.300-202.400 g·L-1 original solution） inhibited biofilm formation and maturation （P<0.05， P<0.01）. The SEM exhibited that FFHBFP made the structure of biofilm loose and the size of the bacteria varied. FFHBFP at 50.600 g·L-1 concentration can inhibit the expression of related virulence genes and biofilm-forming genes （P<0.05， P<0.01）， and molecular docking results also showed that the main antibacterial active ingredients in FFHBFP have good binding ability to the target. ConclusionFFHBFP that cannot directly kill MRSA exerts clinical efficacy by impairing virulence expression， biofilm formation， and other pathogenic properties.
ABSTRACT
Objective:To study whether Tanreqing injection （TRQ） can alleviate the body injury in the process of infection by inhibiting the production and release of <italic>α</italic>-hemolysin of <italic>Staphylococcus aureus</italic> under sub-minimal inhibitory concentration， and to provide experimental basis for better guidance of clinical medication. Method:The effects of TRQ on the minimum inhibitory concentration （MIC） and bacterial growth of <italic>S.aureus</italic> were determined firstly by microplate method and time-growth curve. The different sub-minimal inhibitory concentrations of TRQ were co-cultured with bacteria or bacterial supernatants， and then co-incubated with defibrillated rabbit blood to detect the inhibitory and neutralizing effects of TRQ on <italic>S.aureus</italic> <italic>α</italic>-hemolysin. Cell counting kit-8 （CCK-8） cell viability assay was used to detect the protective effect of TRQ on <italic>S. aureus</italic>-mediated damage to human alveolar epithelial cells （A549）. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the effect of sub-minimal inhibitory concentration of TRQ on the mRNA expression of <italic>S.aureus</italic> <italic>α</italic>-hemolysin regulatory genes hla and agrA. Result:The MIC of TRQ to <italic>S.aureus </italic>was 1/8 of the stock solution， and the sub-minimal inhibitory concentration （1/64MIC-1/16MIC） TRQ used in this study did not affect the growth of bacteria. 1/64MIC-1/16 MIC TRQ had the effect of inhibiting and neutralizing the hemolytic activity of <italic>α</italic>-hemolysin， with a protective effect on <italic>S.aureus</italic> supernatant-mediated A549 cell damage， and its inhibitory effect on <italic>α</italic>-hemolysin was closely related to the inhibition of hla and agrA mRNA expression. Conclusion:The sub-minimal inhibitory concentration TRQ can inhibit and neutralize the hemolytic activity of <italic>α</italic>-hemolysin of <italic>S.aureus</italic>， with a protective effect on A549 cell damage mediated by <italic>S.aureus</italic> infection， and its mechanism of inhibiting <italic>α</italic>-hemolysin is closely related to the interference with agr regulatory system.