Study On The Effect Of Lactobacillus Plantarum CFS On The Formation Of Staphylococcus Epidermidis Biofilm

Objective:To explore the effect of Lactobacillus plantarum(Lb.plantarum)CICC 6257 cell-free supernatant(CFS)on the formation of Staphylococcus epidermidis(S.epidermidis)biofilm(BF),and provide experimental basis for the development of new anti-bacterial biofilm drugs.Methods:1.The crystal violet semi-quantitative method was used to determine the culture time of Lb.plantarum CFS with stable anti-S.epidermidis BF formation.The minimum inhibitory concentration(MIC),minimum biofilm inhibitory concentration(MBIC)and sub-inhibitory concentration of CFS were determined by the broth microdilution method and the crystal violet semi-quantitative method.2.In the S.epidermidis culture,with or without sub-inhibitory concentration of Lb.plantarum CFS as the experimental group and control group,respectively.To study the effect of Lb.plantarum CFS on the growth curve,hydrophobicity and self-aggregation of S.epidermidis.3.On the surface of polyvinyl chloride(PVC),add culture medium,S.epidermidis,S.epidermidis with different concentrations of Lb.plantarum CFS as the blank group,control group and experimental group.Detect the effect of Lb.plantarum CFS on the formation of S.epidermidis biofilm and the clearance rate of mature biofilm.Detect the growth dynamics of bacteria in S.epidermidis biofilm.Observe the microstructure of BF on PVC surface with confocal laser scanning microscope(CLSM)and scanning electron microscope(SEM).Results:1.The Lb.plantarum CFS extracted from 4,8,12,24,30,and 48h cultivation has an inhibitory effect on the formation of S.epidermidis biofilm.The inhibitory effect increases with the extension of the culture time,reaching a peak at 12h(inhibition rate 99%)and no significant difference from 24h,30h,48h(P>0.05).The concentration of MIC and MBIC of Lb.plantarum CFS against S.epidermidis is 50%(v/v),and the sub-inhibitory concentration is determined to be 60%MIC(v/v).2.The sub-inhibitory concentration(60%MIC,v/v)of Lb.plantarum CFS has an inhibitory effect on the growth of planktonic S.epidermidis,and significantly delays the logarithmic growth phase.The 60%MIC(v/v)of Lb.plantarum CFS significantly reduces the hydrophobicity and self-aggregation of S.epidermidis.3.The sub-inhibitory concentration(60%MIC,v/v)of Lb.plantarum CFS has an inhibitory effect on all stages(6h、12h、24h、30h)of BF formation of S.epidermidis on the PVC surface(P<0.05),and its inhibition rate is most significant in the early stage(6h)of BF formation(90.33%),and the inhibitory effect gradually weakens with the culture time.4.Bacterial growth dynamic test results:compared with the control group,the bacterial growth dynamic in the MIC and 60%MIC groups was significantly different at each stage(P<0.05),and the 60%MIC group had the most significant decrease at 12 hours of culture.5.Lactobacillus CFS has a clearing effect on mature S.epidermidis BF.The clearance rates of the MIC,2MIC,and 60%MIC groups are 63.08%,54.02%,and 35.15%,respectively.The MIC group has the largest clearance rate(P<0.05).6.SEM found that only a single colony was found on the PVC surface of the MIC group,and the surface of S.epidermidis was not smooth,and the cells were shrunk and broken.The BF structure of S.epidermidis in the 60%MIC group was poorer than the control group in terms of three-dimensionality,complexity,and number of colonies,and there were more "large cells" that were not completely isolated.However,there are still filamentous viscous connections between the bacteria.CLSM found that compared with the control group,there was no BF formation in the MIC group.The proportion of viable bacteria in the 60%MIC group was low at each culture time.Except for 30 hours,the thickness of BF in the 60%MIC group was lower than that in the control group,and the structure was sparse.Conclusions:1.The CFS of Lb.plantarum can inhibit S.epidermidis cell division,affect the structural integrity and permeability of cell membrane and cell wall,and inhibit the growth of planktonic S.epidermidis.2.The CFS of Lb.plantarum can reduce the hydrophobicity,self-aggregation of S.epidermidis and bacterial activity in the biofilm,inhibit the formation of S.epidermidis biofilm on the PVC surface.3.The CFS of Lb.plantarum has certain ability to break down mature S.epidermidis biofilm,and the clearance rate has no obvious dose-effect relationship.