| Dairy cow mastitis is caused by a variety of pathogenic bacteria,and it is one of the most serious diseases of dairy industry,it reduces the quality of dairy products,causing huge economic losses to the dairy industry.Staphylococcus aureus is one of its main pathogenic bacteria,and it can usually cause chronic infection in cow breasts by forming a biofilm.Antibiotics are the main means to control S.aureus infection.However,non-standard use of antibiotics for a long time aggravated resistance of S.aureus,especially the emergence of methicillin-resistant Staphylococcus aureus(MRSA),making prevention and treatment of Staphylococcus aureus is facing enormous challenges.In addition,Staphylococcus aureus usually has a strong ability to form biofilms,and its biofilm can make it more resistant to antibiotics and shows multi-drug resistance.Extracellular polysaccharide intercelluar adhesion(PIA)is a major component of bacterial biofilm.The formation of PIA is responsible for the protein encoded by the ica operon,ica operon encoding four functional genes of icaADBC and a regulate gene of icaR,icaR can inhibit the expression of icaADBC to reduce the synthesis of PIA,lessened ability of biofilm formation.The production of MRSA strains makes the treatment of antibiotics challenging,while natural,low price,little side effects of traditional Chinese medicine components can be used in the prevention and treatment of pathogenic bacteria in dairy cow mastitis,the research on the inhibition of biofilm formation of S.aureus by Chinese medicine components have a little reported,but the lack of research on the mechanism of traditional Chinese medicine inhibiting the formation of biofilm in S.aureus.In this study,we explored the effects of extracts from traditional Chinese medicine sanguisorba and stigmata maydis on biofilm formation of MRSA strains S A3,and its effect on icaADBC and icaR of film-forming genes of SA3.Furthermore,we explored the effect of aqueous extract of stigmata maydis was also investigated on vancomycin susceptibility of the MRSA strains under planktonic-cultured and biofilm-cultured conditions.The possibility and conditions of the combination therapy were explored preliminarily.The main research results are divided into the following sections:1.In this study,two-fold dilution method was used to detect the minimum inhibitory concentration(MIC)of MRSA strains SA3 to the ethanol extract of sanguisorba and aqueous extracts of stigmata maydis.As a result,the MIC of ethanol extract of sanguisorba on SA3 was 12.5mg/mL,while the high concentration of aqueous extracts of stigmata maydis had no inhibitory effect on the growth of strain SA3.The MIC of vancomycin on SA3 was 1 μg/mL.2.In this study,the effect of two traditional Chinese medicine that extracts on the growth of MRSA strains SA3,was detected by turbidimetry and colony counting methods.The results showed that high concentration of sanguisorba ethanol extract inhibited the growth of SA3;and high concentration of aqueous extract of stigmata did not affect the growth of SA3.3.In this study,96-well plates were used to detect the effect of different concentrations of Chinese herbal extracts on the formation of SA3 biofilm.The results showed that under culture conditions of the best time of biofilm formation for 16 h，if adding to lower concentrations of ethanol extract of sanguisorba and aqueous extracts of stigmata maydis can significantly inhibit the formation of SA3 biofilm.The minimum concentration of ethanol extract of sanguisorba which inhibits the biofilm formation of SA3 is 2.5mg/mL,and the minimum concentration of aqueous extracts of stigmata maydis which inhibits SA3 biofilm formation is lmg/mL.4.In this study,biofilms of strain SA3 containing green fluorescent protein plasmid were monitored by laser confocal scanning microscope under different conditions.The results showed that SA3 could form a complete and dense biofilm without adding Chinese herbal extract.However,and we can also find that biofilm formation was barely detectable after added the 2.5 mg/mL of ethanol extract of sanguisorba and the aqueous extract of stigmata maydis at a concentration of 1 mg/mL.5.In this study,we used PCR amplification and sequencing technologys to clone and accurately sequence the precise sequences of the membrane-forming genes icaADBC and icaR of the biofilm of MRSA strain SA3.6.In this study,real-time fluorescence quantitative PCR was used to detect the transcriptional levels of icaADBC and icaR.The results showed that after adding the ethanol extract of sanguisorba with the concentration of 1,3mg/ml and the aqueous extracts of stigmata maydis with the concentration of 0.5,1,2mg/ml,the transcriptional levels of icaA,icaD,icaB,icaC were significantly reduced,and with the increase of concentration,S.officinalis and Stigmata maydis inhibited the icaADBC more effectively.However,the transcription level of icaR gene increased with the increase of the concentration of ethanol extract of sanguisorba and aqueous extract of stigmata maydis.7.This study explored the level of change of growth state of strain SA3 under different culture conditions(planktonic-cultured and biofilm-cultured)in the combination therapy of aqueous extracts of stigmata maydis and vancomycin.The results showed that the aqueous extract of stigmata maydis could increase the sensitivity of bacteria to vancomycin under biofilm cultured conditions through inhibited the formation of biofilm.8.In summary,this study confirmed that although the need to add a higher concentration of Chinese medicine extract to inhibit bacterial growth,but low concentrations of extract of S.officinalis and Stigmata maydis could significantly inhibit the formation of bacterial biofilm.Further study found that the above important components could down-regulate the expression of icaADBC operon by up-regulating the transcription level of transcription factor icaR to inhibit the synthesis of PIA,thereby inhibiting the formation of biofilm.To sum up,this study confirmed that low concentrations of S.officinalis and Stigmata maydis can reduce the expression of ica operon by regulating the transcription level of icaR to inhibit the formation of biofilm.In addition,the stigmata maydis could enhance the vacomycin suspectibility of the biofilm-condition cultured MRSA strains,suggesting that this important component could be used as an ancillary aid to vancomycin treatment of MRSA,providing new clues for the prevention and control of bovine mastitis caused by MRSA strains. |
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