Influencing Factors Of In Vitro Biofilm Formation Of E.coli With Overexpression Of Maternal Membrane Protein A

Part I Effects of matrine on the formation of E.coli biofilms with overexpression of outer membrane proteins.OBJECTIVE: To construct an E.coli biofilm model with outer membrane protein A overexpression and investigate the effect of different concentrations of matrine on the biofilm formation capacity of E.coli.METHODS: A biofilm model of E.coli was constructed and the minimum effective inhibitory concentration(MIC)of matrine on E.coli was measured using a two-fold dilution method.The peritoneal dialysis external short tube was used as the carrier to construct the E.coli biofilm(Biofilm,BF)model.The carrier was placed in a 6-well plate and randomly divided into 3 groups: blank control group(negative control group),1/2 MIC matrine group,1/4 MIC matrine group.In the early and middle stages of E.coli biofilm formation(corresponding to 1 day,3 days and 7 days).Silver staining was used to observe the formation of biofilms in each group.Semi-quantitative method of crystal violet was used to perform relative quantification of each group of E.coli biofilms.RESULTS: The MIC of matrine to E.coli was 4096 μg/ml.Rapid silver staining showed that in the blank group and 1/4MIC matrine group,large amounts of black-brown bulk biofilm were observed on the surface of the carrier after silver staining in the early,middle,and late stages of biofilm formation.Formation,and 1/2MIC matrine group carrier surface dark brown lumps of material were scattered in a small number.Semiquantitative detection of crystal violet formed by biofilm showed that the semi-quantitative results of 1/2MIC matrine group and 1/4MIC matrine group were lower than those of the blank control group on the 3rd and 7th days.CONCLUSION: Omp A-overexpressing E.coli strains can form biofilms.Matrine inhibits the formation of Omp A over-expressed E.coli biofilms.Part II Effects of matrine on the formation of E.coli biofilms with overexpression of outer membrane protein A: effects on Omp A,Mic A,and σE.OBJECTIVE: To investigate the effects of different concentrations of matrine on the expression of Omp A-overexpression E.coli Omp A,Mic A and σE in the early,middle and late stages of E.coli biofilm formation.METHODS: E.coli strains with overexpression of outer membrane protein A was used as the research object.The external short tube of peritoneal dialysis was used as the carrier in the 6-well plate to construct the in vitro biofilm(Biofilm,BF)model of E.coli.The experiment was divided into 3 groups: blank control group(negative control group),1/2MIC matrine group,1/4MIC matrine group.Matrine was added at 0 hours to construct the biofilm model,and the carriers were tested at 1 day,3 days,and 7 days after the intervention(corresponding to the early,middle,and late stages of E.coli biofilm formation).The expression levels of E.coli Omp A,Omp A m RNA,Mic A,and σE in the biofilm were measured by Western blot and real-time fluorescence quantitative PCR.RESULTS: In the middle(3 days)and late(7 days)stages of E.coli biofilm formation,1/2 MIC matrine group and 1/4 MIC matrine group compared to the blank control group at the protein level.The expression of membrane protein A was decreased(P<0.05).In the early(1 day),middle(3 days)and late(7 days)stages of E.coli biofilm formation,1/2 MIC matrine group,1/4 MIC matrine group,compared with the blank control group,The expression of membrane protein A was reduced at m RNA transcription level(P<0.05).At the same time,the expression of Mic A and σE were up-regulated in the middle and late stages of E.coli biofilm formation(P<0.05).CONCLUSION: Matrine can inhibit the expression of Omp A at the proteinlevel and m RNA level in the middle and late stages of E.coli biofilm.Matrine can upregulate the expression of Mic A and σE in the late stage of E.coli biofilm formation.It was verified that matrine inhibited the formation of E.coli biofilm by inhibiting the expression of Omp A.