Biofilm Formation And Influencing Factors Of Staphylococcus Epidermidis Isolated From The Sourthern Xinjiang

Staphylococcus epidermidis is a conditional pathogenic bacterium which can easily cause bovine subclinical mastitis. The infection caused by S. epidermidis is hard to cure and recurrent attacks after cows are breasted, and then results in significant economic losses. Biofilm formation is the key reason for its hard curing and recurrent attacking. However, when the disease treated with antibiotic, the persister would appear and meanwhile the antibiotic residue is harmful to human health. The Chinese medicinal herbs are harmfulless and have a rich resourch. It is substantial meaning to prevent and restrain bovine subclinical mastitis with Chinese medicinal herbs.In this study, we detected a total of189milk samples, which were collected from2dairy farms in southern Xinjiang, using LMT (Lanzhou Mastitis Test). Then molecular methods were combined to isolate and identify S. epidermidis from subclinical mastitis cow milk samples. Micro-plate semiquantitative method was used to separate biofilm-positive S. epidermidis and to invest the effect of glycyrrhiza herb decoction and glabrous crazyweed herb decoction on the S. epidermidis biofilm formation. And at last, thin layer chromatography (TLC) experiments and quantitative real-time PCR assay were used to study the mechanism of the glabrous crazyweed herb decoction inhibiting biofilm formation.1) The results showed that the amount of positive subclinical mastitis milk in tested189samples was128, which occupied67.7%, and56strains of S. epidermidis were isolated.22strains of S. epidermidis are biofilm positive or weakly positive and all of those are ica independent form.2) The results of micro-plate semiquantitative method detecting different external factors influcing S. epidermidis biofilm formation showed:The external factors closely related to the S. epidermidis biofilm formation. The oligotrophic conditions can significantly inhibited the biofilm formation and in the neutral pH value conditions, NaCl had obvious inhibiting effect on S. epidermidis biofilm formation. When the pH value is greater than7, the ability of biofilm formation apparently weakened. In the simulation of environmental factors in dairy cow, a certain concentration of Ca2+(0.2%and above) and high concentrations of lactose (8%and above) had visible inhibition on biofilm formation. But there were no significant effects on S. epidermidis biofilm formation when the exogenous casein was added.3) The results of the Micro-plate semiquantitative method of glycyrrhiza herb decoction and glabrous crazyweed herb decoction on the S. epidermidis biofilm formation showed that a high concentration of15%or above of glycyrrhiza herb decoction inhibited cell growth and then attenuated biofilm formation by S. epidermidis. However, a low concentration of7.5%of glabrous crazyweed herb decoction decreased significantly biofilm formation by S. epidermidis without significant cell growth inhibition. The thin layer chromatography (TLC) Experiments and quantitative real-time PCR assay results showed that the alkaloids in glabrous crazyweed herb decoction inhibited the synthesis of the polysaccharide intercellular adhesin (PIA), which is one of the important compositions in biofilm formation, through down-regulating icaB and aap genes or up-regulating icaR gene so as to inhibite the biofilm formation. And the glabrous crazyweed herb in different areas had different effect on the biofilm formation.The research provides a significant theoretical foundation for using characteristic Chinese medicinal herb from sourthern Xinjiang to prevent and restrain the bovine subclinical mastitis.