The Inhibition Of Rifaximin On Staphylococcus Aureus And Biofilm Of Milk From Mastitis In Vitro

S.aureus is the main pathogenic bacteria of dairy cow mastitis,which brings great economic loss to the dairy farming industry.Biofilm formation is considered to be an important factor in the infection of S.aureus,bacteria more resistant,causing problems in the prevention and treatment of clinical gold and S.aureus.In this paper,firstly,we determined the MIC and FIC value of the common antibacterial drugs for the prevention and control of mastitis,and determined the PAE of rifaximin.Subsequently,the growth law of the biofilm was investigated,and the effect of rifaximin on the membrane elimination and molecular mechanism was further studied.The main research contents and results are as follows:1 Rifaximin and other antibacterial agents were used to determine S.aureus of MIC、FIC and PAEThe microbroth dilution method and checkerboard method were accepted,and the strain was diluted several times to be treated with S.aureus which was isolated from the milk of cows with mastitis.Chose rifaximin and antibacterial agents with dairy cows commonly used.ATCC25923 and 9 clinical strains of S.aureus in gram-positive bacteria were selected as the research object,and the interaction between agents was discussed.By the experimental results showed the MIC of ATCC25923 was 0.015625 μg/mL,and the MIC of 8 strains was 0.015625～0.03125 μg/mL.1 strain was 4 μg/mL.The MIC of other antibacterial agents was as follows:ampicillin was 0.5-4 μg/mL;amoxicillin was 0.0625～1 μg/mL;cloxacillin benzathine was 0.125～8 μg/mL;lincomycin was 2～64 μg/mL;kanamycin was 0.125～64 μg/mL;cefalexin was 0.25～4 μg/mL;ceftiofur was 0.25～8μg/mL;cefquinome was 0.125～2 μg/mL.The combined effect of rifaximin and other agents showed that there were 7 strains of rifaximin and ceftiofur combined,2 strains showed no correlation and 1 showed synergistic effect.Combined with cefquinome,2 strains were additive,and 8 strains irrelevant.Combined with cefalexin,4 strains were additive and 6 irrelevant.Combined with kanamycin,5 strains were additive and irrelevant.The combined effect of lincomycin and ampicillin was only 1,and the rest were irrelevant.There were 3 synergistic effects with cloxacillin benzathine,6 strains additive and 1 antagonistic effect.The combined use of amoxicillin was irrelevant.To study the antibacterial effect of rifaximin on S.aureus ATCC25923 in vitro which supports the basis for the clinical application of rifaximin.The MIC of rifaximin on S.aureus ATCC25923 was determined by microbroth dilution method,according to the PAE theory in vitro.Rifaximin was determined by the colony counting method of S.aureus ATCC25923 PAE in vitro,the results show that rifaxmiin in the MIC,2MIC,4MIC,8MIC concentration,For ATCC25923,a PAE of 0.473～2.995 h,with an average value of 1.800±0.852 h.PAE of ATCC25923 concrete values:agent exposure time is 1 h,0.473 h 1.720 h,1.859 h,2.781 h;The agent contact time of 2 h was 0.851 h,1.767 h,1.950 h,2.995 h.The PAE and concentration of rifaximin were in a certain range of dose dependent,compared with PAE in different concentrations,the difference was statistically significant(P<0.05).PAE showed a certain elongation as the formulation contact time increased,The difference between 1 h and 2 h was statistically significant(P<0.05).2 The molecular mechanism of biofilm growth in vitro and the effect of rifaximin on its eliminationAdopted the method of congo red plate,alcian blue-congo red stain method of identification of S.aureus can form biofilm,Through the crystal violet semi-quantitative method measuring S.aureus biofilm formation ability,shows that 10 test strains,5 strains including ATCC25923 of OD value in the range of the 40Dc-60Dc,means moderate capacity of producing biofilms,and the rest OD value were all>60Dc,indicating the strong strain of the film.Crystal violet staining method draw 7 days of biofilm growth curve,the results showed that s.aureus bacteria biofilm growth in 4 days to form a stable state,"S" pattern in biofilm growth curve,consistent results and film-forming ability to divide.Through the crystal violet method and the silver dyeing method,the growth process of the biofilm was observed in 7 days,which also showed that the growth of the biofilm was stable for the fourth day.When SEM observed the concentration of rifaximin at 1/2MIC,the structure and morphology of the bacteria remained normal after 24h.After 24 h at the concentration of rifaximin MIC,there was a network of tissue among the bacteria,and the damaged bacteria and some normal forms of bacteria were visible.When the concentration of rifaximin reached 2MIC for 24 h,there was almost no fibrous structure among the bacteria,and incomplete bacterial remains were seen.It is indicated that rifaximin has a certain inhibitory effect on the early biofilms of S.aureus at different concentrations.RT-PCR determination of rifaximin for quality control strains ATCC25923 icaA and agrC gene expression,the results showed that the increase of rifaximin can influence the icaA gene and agrC gene expression,and on its amount of gene expression had a significant inhibition(P<0.01),when the concentration of rifaximin reached 2MIC,its amount of gene expression was reduced by 60%and 30%respectively.