Identification And Susceptibility Test Of Streptococcus Agalactiae Biofilm Of Dairy Cow In Subclinical Mastitis

Purpose: Streptococcus agalactiae is one of the major pathogenic bacterias of dairy cow in subclinical mastitis. The pathogenic bacterias exist in mammary gland tissue with free and biofolms state. Biofilms are bacterial communities which formatted by excyst polymer and bacterias when sticking with object surface. The free state bactericas often cause acute infection, while the chronic infection are induced by biofilms. At present ,the free state bacterias are study object in the susceptibility test of dairy cow in subclinical mastitis. In this experiment, use the SEM to observe the S. agalactiae biofilms in mammary gland tissue; compare the drug resistance with free and biofilms state. of S. agalactiae. Offer the correlate data to clinic treat and diagnosis. Method: Collect the milk from subclinical mastitis cow, isolate and identify the bacterias. Take the mammary gland tissue from cow in which infected by S. agalactiae ; through 2.5%GAPBS fixation, alcohol gradient anhydration, dry, plating film, observed by SEM. Use MTT to determine the S. agalactiae viable count standard curve. Use sewing needle as carrier, added in MH bouillon which have S. agalactiae to make vitro biofilms model. Then cultivate 8 days, dislodge the carrier and use SEM to observe. Use test tube double dilution to measure MIC of S. agalactiae with OF, EM and RK, put the carriers into two groups of test tube, adding antibacterials cultivate 8 hours, then take out of carriers from the first group, use SEM to observe ,while the carriers of the other group, affected by ultrasonator and whirlpooler to make float bacterium liquor. Use MTT to measure optical density(OD). Calculate the viable count compare with standard curve and denote by logarithm. Quantitative analysis the discard effect of antibacterials to S. agalactiae statistically.Result: Dissociate the S. agalactiae from milk sample; certificate the existence of S.agalactiae biofilms; in vitro cultivate S. agalactiae biofilms is possible. The MIC of OF, EM and RK are0.5μg/ml, 1μg/ml and 2g/ml. The effect of antibacterials to S. agalactiae biofilms viable count indicate that to compare with NS: when the concentration of OF, EM, and RK are 1 MIC, not significant (P>0.05), the biofilms' structure are integrity and distribute lamellarly. When the concentration of OF, EM, and RK are 50 MIC, significant (P<0.05), the biofilms' structure are loosen and thin.Conclusion: The biofilms are exist in mammary gland tissue of dairy cow in subclinical mastitis. It is possible in vitro cultivate S. agalactiae biofilms Drug resistance of biofilms state of S agalactiae is far exceed to free state. The association of OF, EM and RK can reduce the viable count of S. galactiae in biofilms state.