The Research Of Regulatory Mechanisms Of Biofilm Formation In Etiological Agents Of Bovine Mastitis

Mastitis is an important disease and is the most common cause of death in adult dairy cows.The economic losses arising from bovine mastitis treatment and death as well as due to decreased milk production are significant.A total of 150 microbial species,subspecies and serovars have been isolated from the bovine mammary gland.The most common species which cause mastitis are Staphylococcus,Escherichia and Streptococcus.The formation of a biofilm is characterized by the structure of a population of bacteria encased within a self-produced extracellular matrix of exopolysaccharide,proteins and some micromolecules,such as DNA.The presence of glycocalyx layers protects the enclosed bacteria from host defenses and resists the access of antibiotics.It has been reported that biofilms can resist antibiotic concentration 10-10,000 fold higher than those required to inhibit the growth of their planktonic counterparts.Therefore,biofilm formation brings great challenges for the infection treatment,eventually leading to chronic infections,which can be difficult to eradicate.Until now,the regulatory mechanisms of biofilm formation in etiological agents of bovine mastitis have not been intensively studied.In this study,we explored the biofilm associated genes and regulatory mechanisms of S.aureus and E.coli strains isolated from dairy cow with mastitis.We aim to find some pre-molecules which might be effective in mastitis treatment and find the ways of combined utilization of these molecules and antibiotics.This study will provide some new clues to the prevention and control of mastitis in dairy cows.The main results are as follows:1.Lactose increased biofilm formation predominantly by inducing PIA production,whereas milk increased biofilm formation through PIA as well as by increasing the production of other biofilm-associated proteins,which might be mediated by the transcriptional regulators intercellular adhesion regulator(ica R)and repressor of biofilm(rbf).2.Ethanol extract of S.officinalis strongly inhibited the biofilm formation of MRSA.With a confocal laser scanning microscope system,we observed that the biofilm structure of the test group with the addition of S.officinalis appeared looser and had less biomass compared with the control group without S.officinalis.Furthermore,we found that the transcript levels of the ica ADBC operon remarkably decreased upon addition of the ethanol extract of S.officinalis,indicating that S.officinalis inhibits biofilm formation of MRSA in an ica-dependent manner.3.AI-2 increases biofilm formation by enhancing the transcription of the ica operon,which is a known component in the AI-2-regulated biofilm pathway.In addition,we first observed that the transcript level of bhp,which encodes a biofilm-associated protein,was also increased following the addition of AI-2.Furthermore,we found that,among the known biofilm regulator genes(ica R,sig B,rbs U,sar A,sar X,sar Z,clp P,agr A,abf R,arl RS,sae RS),only ica R can be regulated by AI-2,suggesting that AI-2 may regulate biofilm formation by an ica R-dependent mechanism in S.epidermidis RP62 A.4.The aqueous extracts of stigmata maydis inhibit the biofilm formation ability of MRSA strains and increase the vancomycin susceptibility of the strains under biofilm-cultured conditions.This indicates that stigmata maydis might be used as an adjuvant ingredient in the vancomycin treatment of MRSA infections.5.QA NPs exhibit antibacterial and anti-biofilm properties in a multi-drug resistant E.coli strain isolated from a dairy cow with mastitis,indicating that QA NPs might be used as a potential antimicrobial agent against bovine mastitis.