Screening Of Actinomycetes And Its Mechanism Inhibiting Biofilm Formation By Staphylococcus Epidermidis

Staphylococcus epidermidis is one of the most important conditioned pathogens caused bovine subclinical mastitis during the productive practice of dairy farming. The biofilm, formed by S. epidermidis and other rennet-negative staphylococci, can enhance the adhesion colonization ability of the pathogenic bacteria in mammary epithelial cell surface and assist the pathogens to release virulence factors to the outside. Usually, Large doses of traditional antibiotics were used as the choice drug for the clinical treatment. However, the emerge of the multiple drug resistant strains was intensified due to the biofilm formation. The biofilm could provide a protective barrier to the pathogens and block the combination of the drugs and the cellular targets. Finally it leaded to hard curing and recurrent attacking of the mastitis.Thus, the development of new biofilm inhibitors was becoming the hot topic in the field of veterinary prevention gradually. Actinomycetes is capable of producing abundant metabolites and is the natural treasure house to dig the biologically active substances. New biofilm inhibitors isolated from actinomycetes had great value in the prevention and treatment of mastitis in cows.First, 185 actinomycetes isolated from the saline soil of southern Xinjiang were used as the research object and the micro-plate semiquantitative method was used to screen the active strains which could inhibit the biofilm formation of the strains 5-121-2 and ATCC 35984. At last, 3 actinomycetes were obtained which inhibited the biofilm formation by S. epidermidis without significant cell growth inhibition.After 16 S rDNA sequence analysis and comparison, 3 actinomycetes were belong to Nocardiopsis isolated from Baicheng Xinjiang salt water beach soil(TRM46200), Streptomyces isolated from Xinjiang xiaoerkule Lake soil(TRM41337) and Saccharomonospora isolated from Xinjiang Yanqi salt soil(TRM46814), respectively. The optical microscope was used to observe the changes of adhesion ability on the solid support surface of the biofilm formation by S. epidermidis after treatment with fermentation broth of the 3 actinomycetes. The results showed that the nonspecific adhesion ability of the S. epidermidis decreased significantly on the solid support surface after treatment. Meanwhile, the stability of the fermentation broth of active strains were detected and the activity was obviously weakened or lost after protease K and heat treatment.Secondly, the crude protein of the actinomycetes fermentation broth was obtained through the ammonium sulfate precipitation method. The related activities and the action period were also measured.The Bacterial Adhesion to Hydrocarbon(BATH) was used to detect the Cell Surface Hydrophobicity(CSH)of the S. epidermidis 5-121-2 and ATCC 35984 before and after treatment of the 3 actinomycetes fermentation broth protein. The inhibitory activity of the protein played a significant role in both primary attachment and accumulation phase of biofilm formation. The CSH of the S. epidermidis was also effectively reduced by the protein. This may be because the intercellular adhesion was mediated by the CSH. Once the CSH decreased, the nonspecific adhesion between the pathogens and pathogens, the pathogens and the host would be affected and then the biofilm formation was blocked.Thirdly, sodium periodate, α-amylase, β-glucanase, β-glucosidase, proteinase K and DNase I was used to deal with the biofilm of S. epidermidis 5-121-2 and ATCC 35984, separately. Among them, the biofilm formation of S. epidermidis ATCC 35984 could be effectively restrained by sodium periodate and it appeared that the biofilm may compose of reduced exopolysaccharides(EPS). After treating by α-amylase,β-glucanase, β-glucosidase, proteinase K and DNase I, the biofilm of S. epidermidis 5-121-2 was destroyed.It appeared that the component of S. epidermidis 5-121-2 was more complex. Non-reducing EPS,extracellular proteins and extracellular DNA participated in the composition of the biofilm structure. The agarose gel electrophoresis and pre-column derivatization HPLC were used to detected the changes of the extracellular DNA and EPS in the S. epidermidis 5-121-2 and ATCC 35984 biofilm after treatment with fermentation broth proteins of the 3 actinomycetes. Both of the types of polysaccharides in the EPS and the relative molar proportions of the EPS changed and the extracellular DNA was degraded.It illustrated that the protein of the actinomycetes fermentation broth achieved its biological activity mainly by changing the composition of bacterial biofilm.At last, the sensitivities of the S. epidermidis 5-121-2 and ATCC 35984 to the penicillin(lactam antibiotics), streptomycin(aminoglycoside antibiotics) and oxytetracycline(macrolide antibiotics) were detected. The results indicated that ATCC 35984 was resistant to penicillin and streptomycin and 5-121-2also showed a certain resistance to streptomycin. The biofilm increased the resistance of pathogenic bacteria to antibiotics in some degree. The chessboard method was used to realize the combination in vitro of the actinomycetes fermentation broth protein and the streptomycin to inhibit the S. epidermidis biofilm formation. The analysis and evaluation of the clinical strain 5-121-2 was performed and the most obvious groups of the synergistic action were obtained. The results showed that in the groups which had the most obvious synergistic effect, the effective dose of combination of antibiotics and protein was significantly lower than the dose alone. It demonstrated that the fermentation broth protein inhibited the biofilm formation of S. epidermidis and meanwhile increased the sensitivity to antibiotics.The aim of this study is to make full use of the abundant resources of actinomycetes in Xinjiang and obtain the non-toxic, highly effective material inhibited biofilm formation from actinomycetes. Then the effect of the active substance against the biofilm formation of S. epidermidis caused subclinical mastitis was explored for curing the cow mastitis and other related diseases caused by S. epidermidis. Finally reached the aim that the dependence on antibiotics and the production of resistant strains were eased to a certain degree and provided a significant theoretical foundation to prevent and restrain the bovine subclinical mastitis and related diseases caused by S. epidermidis.