Ampicillin Fight E.coli From Mastitis And Vitro Study On Biofilm

In nature environment,most bacteria have an ability to form biofilm.Once bacteria form biofilm its drug-resistance ability will enchance dramatically.the antibacterial effect of agents will be deteriorated.E.coli isolates derive from mastitis of dairy and we test MIC of commonly used antibacterial agents in mastitis prevention,and then,we carry out FIC,which ampicillin combined with other mastitis antibacterial agents.And we make a further study to explore Chinese traditional medicine with different sub-MIC concentration,whether E.coli biofilm gene expression will be regulated or not.The main research contents and results are as followed:1 Ampicillin combined with other antibiotics to test MIC and FIC of clinical isolatesTwo-fold dilution method was used to test MIC of 30 isolates.The MIC results of 9 antibacterial agents showed that the MIC of cefquinome lay between 2 and 8?g/mL,and the MIC values of ceftiofur between 4 and 8?g/mL.The ampicillin MIC values ranged from 8 to 32 ?g/mL,the rifaximin MIC values ranged from 4 to 8 ?g/mL,the lincomycin MIC values ranged from 64 to 128 ?g/mL,and the cloxacillin MIC values 8 to 128 ?g/mL,amoxicillin MIC values from 8 to 32 ?g/mL,kanamycin MIC values from 16 to 64 ?g/mL,and cephalexin MIC values from 16 to 64 ?g/mL.Determination of ampicillin by micro checkerboard combined with other antibacterial drugs commonly used in mastitis for FIC.The ATCC25922 strain results showed that:ampicillin+ceftiofur synergy;ampicillin+cefquinome synergy;ampicillin+benzmethicillin addition;ampicillin+amoxicillinaddition;ampicillin+lincomycinhydrochloride antagonism;Ampicillin+kanamycin unrelated;ampicillin+cephalexin unrelated;ampicillin+rifaximin unrelated.The clinical isolates FIC results showed that:ampicillin+ceftiofur,ampicillin+cefquinome better synergistic bactericidal effect,a synergistic or additive effect on different clinical isolates;ampicillin+comet cloxacillin,ampicillin Xilin+amoxicillin showed synergistic,additive,and irrelevant effects on clinical isolates.The results of FIC showed that the combination of ampicillin+cefquinome,ampicillin+ceftiofur has a good synergistic bactericidal effect for clincial isolates.2 In vitro study of E.coli biofilm and preliminary study on mechanism of Sub-MIC of Chinese medicine on its biofilm genesBy crystal violet semi-quantitative experiments,we found 2 strong-abilty biofilm isolates,5 monderate-ability biofilm isolates,21 weak-abilty biofilm isolates,and 2 non-biofilm isolates.So as to make further study on biofilm,we test biofilm growth curve of isolates.We apply microplate method to cultivate biofilm model and use crystal violet to stain biofilm in different phase.By results,we found strong biofilm-abilty isolates can measure maximum OD in 12 h,monderate-ability biofilm isolates can test maximum OD in 24 h or before 36 h,weak-abilty biofilm isolates can observe maximum OD about 36 h.In order to acknowledge strong biofilm-ability structure and development in vitro,we apply silver staining to observe its biofilm change in 12 h,24 h,36 h,48 h,60 h,72 h,and we found black clusters within 12?24 h,but,we found black cluster become small and little after 24 h.According to biofilm study in vitro,we can judge strong biofilm-ability can produce mature biofim within 12 h.?24 h.The results of fluorescence quantitative PCR showed that Shuangding could inhibit the expression of biofilm genes at sub-MIC concentrations,rpoS,csgA,csgB,csgC,csgD((P<0.001;P<0.01;P<0.05).Our results found that Shuanghuanglian sub-MIC concentration could promote expression of csgC,while rpoS,csgA,csgB,csgD expression(P<0.001;P<0.01;P<0.05)decraseed remarkablely.In conclusion,both trditional Chinese medicine can inhibit biofilm genes expression.When Sub-MIC concentration become lower,these genes will have a tendency of recovery.