Study On The Effect And Mechanism Of Ceftazidime At Sub-MIC On Biofilm Formation Of Escherichia Coli

Background and objectivesEscherichia coli(E.coli)is a clinical common pathogen,whose separation rate among gram negative bacteria is in the first place.Besides,it is common in hospital and community acquired infections.Once biofilm froms,E.coli performs high drug resistance and is able to escape from the attack of immune system.The infection of E.coli is easy to be chronic and difficult to control,and currently there is no effective clinical treatment.Therefore,it has become a hot topic to find out the anti-infective drugs which deal with biofilm as target.Screening from existing drugs whose anti-biofilm can be directly used in clinic has important research value and clinical significance.In the previous studies,our group found that sub-MIC of ceftazidime(CAZ)was able to inhibit the biofilm formation of E.coli,but the mechanism of this inhibition is not yet clear.CAZ is a kind of beta-lactam antibiotics and widely used in clinical infectious treatment.CAZ can block the synthesis of bacterial cell wall through combining with the penicillin binding proteins(PBPs)and lead to bacteria dissolution and death.PBPs are kinds of membrane proteins which are widespread on the surface of bacteria.They participate in the synthesis of cell wall,maintenance of the morphology and adjustment of glycopeptide structure.Previous researches show that the biofilm formation of bacteria like Streptococcus gordonii and Streptococcus mutants is associated with PBPs.However,the relationship between PBPs and biofilm formation has not been cleared in E.coli,but the expression of PBPs of the E.coli can affect the structure of cell wall and thus affect flagellum.Flagellum,as a motor organ of E.coli,plays an important role both in bacteria initial adhesion and in formation by motility.Thus,it suggests that CAZ may inhibit flagella and motility by inhibiting PBPs,and decrased the adhesion and the ability of biofilm formation of E.coli.To prove this speculate,we selected E.coli laboratory standard strain MG1655 as the research object,firstly investigated the effect of CAZ at sub-MIC on biofilm formation,bacterial adhesion and motility of E.coli.Then we constructed the PBPs deleted strains by homologous recombination to measure the differences of ability of biofilm formation,bacterial adhesion,flagellum and motility between knockout and wild strains,so that we could clarify which PBP is associated with biofilm formation of E.coli.And then we compared the inhibition of CAZ at sub-MIC on biofilm formation,adhesion,motility,flagellum and the expression of related genes between wild and knockout strains to further analyze the role of PBPs in inhibiting MG1655 biofilm formation,and understand the mechanism of CAZ at sub-MIC on biofilm formation of E.coli.It will provide basis and reference for more scientifically formulate effective treatment plans in clinical practice.Methods1.Effect of CAZ at sub-MIC on motility,adhesion and biofilm formation of E.coli1.1 Drug sensitivity testThe minimum inhibitory concentration(MIC)of CAZ on E.coli was tested by the double dilution methods.1.2 Dose-effect analysis of CAZ at sub-MICs on biofilm formation of E.coliWe used E.coli MG1655 as the research object and crystal violet staining as the method.100μl bacteria and 100μl CAZ solution were added into each well.The experiment was divided into 6 groups: control group,group of CAZ1/32 MIC,1/16 MIC,1/8MIC,1/4MIC,1/2MIC,respectively.After incubation for 24 h at 37 °C,the medium was removed and the wells were washed 2times with PBS.The microtiter plate was dried before adding 1% crystal violet for 15 min at room temperature.After staining,the dye was removed and the wells were washed 3 times with tap water.The microtiter plate was dried prior to the addition of 30% glacial acetic acid to solubilize the dye bound to the biofilm.The absorbance was measured at 590 nm.1.3 Effect of sub-MIC CAZ on growth of E.coli MG1655MG1655 was incubated at 37℃ for 24 h,measured at OD590 nm per 2 h,and the growth curve was plotted.The experiments were divided into control group,1/2MIC CAZ group,1/4MIC CAZ group,and 1/8MIC CAZ group.1.4 Effect of 1/4 MIC CAZ on adhesion of E.coli MG1655The experiments were divided into the control group and 1/4MIC CAZ group.The plates were rinsed twice with PBS to remove the non-adherent bacteria after incubation at 37 °C for 6 h.1ml 0.9% Na Cl was added to each well to dissolve the adherent bacteria,and the flat colony counting method was used to observe.1.5 Effect of 1/4 MIC CAZ on motility of E.coli MG1655Swimming motility was assayed on agar plates containing specialized medium in the presence or absence of sub-MIC CAZ.The plates were point-inoculated from an overnight culture with a sterile toothpick and incubated at 37 °C for 24 h.The diameter of the zone was measured to assess the motility.1.6 Statistical analysisIndependent sample t test was used for the two sample mean comparison and SPSS 13.0 for statistics.2.Mechanism of CAZ at sub-MIC inhibited E.coli biofilm formation by PBPs2.1 Construction of gene knockout strain of mrc A,mrc B,fts I in E.coli MG1655Using plasmid p KD3 as template,we constructed targeting sequences which contain the chloramphenicol resistance gene by PCR,and transformed it into MG1655/p KD46 and screening positive clones through chloramphenicol resistant plate.The p CP20 plasmid was used to remove the chloramphenicol resistance gene,and the mrc A,mrc B and fts I gene deletion strains were obtained respectively.2.2 The effects of knocking of mrc A,mrc B,fts I genes on growth,biofilm formation,adhesion and motility2.2.1 Growth curveSpectrophotometric detection was used for bacterial growth determination.Strains were cultured at 37℃ for 24 h and detected at OD590 nm per 2h.2.2.2 Biofilm analysisThe MG1655 wild-type and mrc A,mrc B,fts I gene knockout strains were cultured in 96 well plates at 37℃ for 24 h and then crystal violet staining was used to measure the absorbance at 590 nm.2.2.3 Adhesion assaysThe MG1655 wild-type mrc A,mrc B,fts I gene knockout strains were cultured in 96 well plates respectively at 37℃ for 6h,the plate colony counting method was used to observe the difference between wild and knockout strains.2.2.4 Motility assaysThe MG1655 wild-type and mrc A,mrc B,fts I gene knockout strains were cultured in LB agar plate respectively at 37℃ overnight.The plates were point-inoculated from an overnight culture with a sterile toothpick and incubated at 37 °C for 24 h.The diameter of the zone was measured to assess the motility.2.3 Effect of sub-MIC CAZ on the change of the ability of biofilm formation,adhesion,motility,flagellum and the expression of related genes in wild strain and gene knockout strain of MG16552.3.1 MICs determinationDouble dilution method was used to value the MICs of CAZ with E.coli wild and gene knockout strains.2.3.2 Effect of 1/4MIC CAZ on growth of E.coli wild and gene knockout strainsEach strain was cultured at 37℃ for 24 h with or without 1/4MIC CAZ,respectively.Growth curve was plotted by detecting OD590 nm per 2h.2.3.3 Effect of 1/4MIC CAZ on biofilm formation of E.coli wild and gene knockout strainsWild and mrc A,mrc B and fts I gene knockout strains of MG1655 were studied,the experiments were divided into control group and 1/4MIC group,and the absorbance was measured by crystal violet staining at 590 nm.2.3.4 Effect of 1/4MIC CAZ on adhesion of E.coli wild and gene knockout strainsEach strain was divided into blank control group and 1/4MIC CAZ group,and the effects of CAZ at sub-MIC on adhesion of the four strains were measured by colony plate counting method.2.3.5 Effect of 1/4MIC CAZ on motility of E.coli wild and gene knockout strainsEach strain was divided into control group and 1/4MIC CAZ group.Toothpicks were selected for single colony vertical dip to the surface of the medium of swimming motility.Observing the swimming motility and measuring the range of motility after culturing at 37℃ for 24 h.2.3.6 Effect of mrc B gene knockout on flagellumTransmission electron microscope was used to observe the flagella of MG1655 wild and mrc B gene knockout strain,and the difference between two groups after cultured with 1/4MIC CAZ.2.3.7 Effect of mrc B gene knockout on the expression of flagellum related genesReal-time PCR was used to determine the change of expression of flagellum related genes after mrc B knockout.2.4 Statistical analysisIndependent sample t test was used for the two sample mean comparison and SPSS 13.0 for statistics.The main results:1.Effect of CAZ at sub-MIC on motility,adhesion and biofilm formation of E.coli1.1 The MIC of CAZ on the E.coli MG1655 was o.5μg/ml.1.2 In the range of 1/2MIC to 1/8MIC,CAZ inhibited biofilm formation of MG1655 in a dose-dependent manner,and 1/2MIC performed the strongest inhibition(P<0.01).1.3 1/2MIC CAZ inhibited the growth of MG1655,and 1/4MIC was selected to use in follow-up experiments.1.4 The number of colonies in the flat plate was significantly reduced after the addition of 1/4MIC CAZ,which showed that sub-MIC CAZ was able to inhibit the adhesion of E.coli.1.5 The diameter of colony on the culture containing 1/4MIC CAZ was 1.5mm,significantly lower than the blank group(P <0.01),which showed that the sub-MIC CAZ could inhibit the motility of E.coli.2.Mechanism of CAZ at sub-MIC inhibited E.coli biofilm formation by PBPs2.1 After knocking the mrc A,mrc B,fts I genes,the length of PCR amplified products was 486 bp,611bp and 555 bp,respectively,in line with the expectations.2.2 The effect of genes knockout on growth,biofilm formation,adhesion and motility2.2.1 There was no significant difference in the growth of wild strain and mrc A,mrc B,fts I knockout strains of MG1655,indicating that the deletion of PBP1 a,PBP1b and PBP3 had no effect on the growth of MG1655.2.2.2 There was no significant difference in biofilm formation between mrc A,fts I gene knockout strains with wild strain,while mrc B gene knockout strain was significantly reduced(P <0.05).2.2.3 There was no significant difference in adhesion between mrc A,fts I gene knockout strains with wild strain,while mrc B gene knockout strain was significantly reduced(P <0.05).2.2.4 The average diameter of MG1655 wild strain,mrc A,fts I gene knockout strain was 0.92 cm,0.91 cm,0.90 cm respectively;the mrc B gene knockout strain was 0.84 cm,and the difference was statistically significant(P< 0.05).2.3 The effect of sub-MIC CAZ on biofilm formation,adhesion,motility and flagellum on the wild and knockout strains2.3.1 The MIC of MG1655 wild strain and mrc A,mrc B,fts I gene knockout strains was 0.5μg/ml,0.5μg/ml,0.125μg/ml,1μg/ml,respectively.2.3.2 1/4MIC CAZ had no effect on the growth of MG1655 wild and knockout strains,1/4MIC was selected for further study.2.3.3 1/4MIC CAZ only had no significant effect on E.coli biofilm formation with PBP1 b deletion.2.3.4 The inhibitory effect of sub-MIC of CAZ on the adhesion of PBP1 b gene deleted strains was weaker than that of other strains.2.3.5 The inhibitory effect of sub-MIC of CAZ on motility of the PBP1 b gene deleted strain was weaker than that of the other strains.2.3.6 Compared with the wild strain,the flagellum around the bacteria significantly reduced in the E.coli strains with PBP1 b gene knockout,which was consistent with the bacteria under the inhibitory of sub-MIC of CAZ.2.3.7 Compared with the wild strains,the expression of the gene related to flagellum was significantly reduced in the E.coli strains with PBP1 b gene knockout,which was consistent with the bacteria under the inhibition of sub-MIC of CAZ.Conclusions1.sub-MIC of CAZ can inhibit the motility,adhesion and biofilm formation of E.coli.2.The results of Gene knockout show that the E.coli with PBP1 b deletion causes the reduction of the gene expression which is associated with flagellum.The number of flagellum and the ability of motility is reduced,thereby reducing the adhesion and biofilm formation.3.PBP1 b is the main target for the inhibition of biofilm formation of E.coli by sub-MIC of CAZ.