The Effect And Mechanism Of Mesna Against Biofilm Of Escherichia Coli

PartI Efects of mesna on Escherichia coli biofilm early adhesion andextracellular polymeric substances【Objective】 To investigate the effects of mesna on adhesion ofEscherichia coli biofilm at early stage and extracellular polymericsubstances (EPS).【Methods】The MIC of mesna against Escherichia coliATCC25922wastested by agar plate method;The standard colony counting method was usedto determine the effects of mesna on E.coli adhesion at differenttimepoints(2,4,6,8h); Polysaccharides and bacteria were labeled byimmunofluorescent technique, the adherence and polysaccharides of E.coli was qualitatively observed under confocal laser scanning microscope(CLSM); the phenol-sulfuric acid method was used to quantitate thepolysaccharides in each group;the extracellular protein was determined byBradford reagent。【Result】 The MIC of mesna against Escherichia coliATCC25922was10mg/ml;To compare with the normal saline control group, the number ofadhered bacteria was decreased in2h,4h,6h,8h groups after interventionwith mesna(F=31.038,11.180,80.686,10.362,respectively, P<0.05), andthe high-doses had positive effect than the low-doses(P <0.05)；Eeighthours after intervention with mesna, compared with normal saline control group, the colony distribution of E.coli became rarefactive, thePolysaccharides in biofilm was significantly decreased, especially withhigh doses,which was visualized by CLSM based on the FITC-ConA andPistaining;Quantificationof polysaccharides showed that totalpolysaccharides (μg)/dry weight of bacterial(mg) of high-dose group(13.57±0.59)and low-dose group (27.77±0.77)were significantlydecreased as compared with normal saline(35.73±0.44)(F=3332.150,P<0.05),and there was significant difference between high-dose group andlow-dose group(P <0.05);Quantification of extracellular protein showedthat total extracellular protein (μg)/dry weight of bacterial(mg) ofhigh-dose group(7.76±0.32)and low-dose group(17.59±0.86)weresignificantly decreased as compared with normal saline(23.31±1.36)(F=688.129,P<0.05),and there was significant difference betweenhigh-dose group and low-dose group(P <0.05).【Conclusion】 Mesna can inhibit the early adhesion of E. coli biofilm;Mesna can also decrease polysaccharides and extracellular proteinproducing ability of significantly. PartⅡ Research on effect of mesna alone,or in combination withciprofloxacin on Escherichia coli biofilm【Objective】 To observe the influence of mesna on the formation ofEscherichia coli biofilm, and study the effect of mesna or/andciprofloxacin on Escherichia coli biofilm．【Methods】Agar plate method was used to measure the MIC of mesna andciprofloxacin,then the appearance of the biofilm was detected by scanningelectron microscope (SEM),the biofilms were treated by mesna,ciprofloxacin alone or combined, and the number of the bacteria in biofilmwas measured by agar plate. Biofilm structure was observed under confocallaser scanning microscope (CLSM), and the parameters of biofilmstructure were analyzed though pictures from CLSM with Image StructureAnalyzer (ISA) software.【Result】The MIC of ciprofloxacin against Escherichia coliATCC25922was0.25μg/ml；Scanning electron microscope showed that the mucoidmaterials among biofilms was significantly redued and the thickness ofbiofilms was decreased in mesna groups;The single use of mesna at theconcentration of8mg/ml could significantly decline the viable cells inbiofilms, the individual use of1MIC ciprofloxacin could significantlydesease the total living number of bacteria, however, when1mg/ml mesnawas combined with1/2MIC ciprofloxacin,viable counts in biofilms wereless than they were when mesna or ciprofloxacin was given alone;The confocal laser scanning microscope showed biofilms were thinner and morescattered than control group;The results of ISA showed that with thetreatment of5mg/ml mesna,biofilms were decreased in thicknessF=67.880,P<0.05),average difusiondistance(ADD)(F=15.977,P<0.05)andtextualentropy(TE)(F=39.432,P<0.05)were decreased in comparison withthe group without mesna treatment,however areal porosity(AP) wasincreased (F=25.100,P<0.05).Whereas the same tendency of theseparameters was seen in the group treated by2mg/ml mesna though not soobvious.【Conclusion】Mesna can reduce Escherichia coli biofilm formation andaffect the structure of Escherichia coli biofilm; Mesna can also enhance theability of ciprofloxacin against Escherichia coli biofilm. Part Ⅲ Interfering effect of mesna on Escherichia coli biofilm onindwelling urethral catheter in vivo【Objective】To establish a animal model of E.coli biofilm on indwellingurethral catheter in vivo;Study the interfering influence of mesna on E.colibiofilm on indwelling urethral catheter and catheter-associated urinarytract infections in vivo.【Methods】1. In the experiment of in vivo models of E.coli biofilm onindwelling urethral catheter,rabbits were randomly divided into two groups:control group,model group. rabbits were underwent catheterization,theninoculated with E. coli ATCC25922though catheters daily for4days, a invivo model of E.coli biofilm on indwelling urethral catheter was estimatedby scanning electron microscope (SEM)and dilution-plate method.2. In theintervention experiments, rabbits were randomly divided into fivegroups:control group, normal saline group(NS group), Mesna group,ceftazidime treatment group (CAZ group), combination therapy group(Mesna+CAZ group). After animal models of E.coli biofilm on indwellingurethral catheter were constructed, received bladder instillation of mesna(20mg/ml×5ml,bid) in Mesna group and normal salinein(5ml,bid) in NSgroup, CAZ group was treated with ceftazidime(50mg/kg,iv,bid),Mesna+CAZ group was given with instillation of mesna (20mg/ml×5ml, bid)and intravenous injection with ceftazidime(50mg/kg,bid),the number of thebacteria in urine bag were measured by agar plat,the rabbits were sacrificed on the3th day,the E.coli biofilms on indwelling urethral catheter wereestimated by SEM, the bacterial counts on indwelling urethral catheter andbladder wall were measured by dilution-plate method,histopathologicalchanges of bladders were investigated by HE stain, the levels of IL-1β,TNF-α andIL-6in blood plasma were determined by ELISA.【Results】In the experimental models, large numbers of bacterias werefound on the catheters in model group, which showed as pellets ormembrane adhesion growth. Uneven thickness of the mucus-like substancelinked into a large field. The average standard colonys (Log10CFU) in themodel group (4.76±0.29) were much more than that in control group (2.49±0.22),(t=17.44,P<0.01).2. In the intervention experiments, bacterialnumber in the urine bags were reduced on the2th day; Structure of BF wasmore obviously destroyed and bacterial number on indwelling urethralcatheter was less in Mesna group than in CAZ group; The average standardcolonys on bladder wall were significantly reduced in Mesna group and inCAZ group, combination therapy was more obvious;Mucosa damage, themucosal cells congestion, edema and inflammatory cell invasion weredetected in the bladder of the control group and the NS group,by contrast,intact mucosa, alleviated cells congestion and edema, less inflammatorycells were shown in the bladder of Mesna group and CAZ group;furthermore, the changes were further relieved in combination therapygroup;The serum IL-1β levels in control group,NS group,Mesna group, CAZ group,Mesna+CAZ group were397.97±100.75,402.87±109.81,287.35±65.05,257.81±78.36,138.61±55.71(pg/ml)respectively(F=12.650,P<0.05),the serum TNF-α levels were525.30±81.09,491.94±138.54,251.12±79.51,254.60±78.36,135.68±46.77respectively (F=28.207,P<0.05),the serum IL-6levels were840.60±106.92,837.39±120.67,556.17±108.57,446.32±129.33,188.54±43.65respectively(F=54.184,P<0.05), the IL-1β,TNF-a and IL-6levels were significantly reduced inMesna group and in CAZ group, combination therapy was more obvious.【Conclusion】A animal model of E.coli biofilm on indwelling urethralcatheter was constructed successfully in vivo; Mesna has a destructive effecton E.coli biofilm on indwelling urethral catheter, which could decrease thebacterial counts on indwelling urethral catheter and bladder wall, attenuatethe pathyologic change of urinary bladder, reduce the urinary tract immuneinjury caused by IL-1β,TNF-αand IL-6in the urinary tract,the interferingeffects of mesna combined with ceftazidime was better. Part IV The effects of mesna on the expression of Escherichia coliadhesion-related and Polysaccharide production-related gene genes【Objective】To study the interference effects of mesna on the mRNAexpression of flu, fimH, papC, glmS, glmU msbB andlpxA in the E. coli andexplore the new mechanism that mesna inhibit the formation of E. colibiofilm.【Methods】The samples were divided into four groups in the experiments:control group,0.5mg/ml mesna group,2mg/ml mesna group,5mg/ml mesnagroup. E. coli ATCC25922were diluted to OD (600)=0.05after overnightculture, then they were treated separately with the same volume of LBculture medium,0.5mg/ml,2mg/ml,5mg/ml of mesna, the bacterias werecollected after2h treatment. the relative expression level of the target genesin each samples were detect by real-time quantitative PCR.【Results】Compared with the control group, the relative expression levelsof the target genes were all reduced in the treatment groups. In0.5mg/mland2mg/ml,5mg/ml mesna groups, the relative expression levels of flugene level were0.587±0.058,0.334±0.054,0.480±0.075respectively,th e expression was the lowest in2mg/ml group,it was higher in5mg/mlgroup than in2mg/ml group (P <0.05);those of fimH gene were0.601±0.014,0.370±0.064,0.407±0.056respectively, they weresignificantly lower in2mg/ml and5mg/mlgroup(P <0.05); those of papCgene were0.628±0.06,0.278±0.072,0.30±0.0053respectively, they were decreased more significantly in2mg/ml and5mg/ml group than that in0.5mg/ml group(P <0.05); those of glmS gene were0.624±0.105,0.378±0.085,0.469±0.105respectively, the expression was the lowest in2mg/ml group and in5mg/ml group(P <0.05); those of glmU gene were0.585±0.062,0.285±0.075,0.172±0.007respectively, the inhibitoryeffection enhanced with increasing concentrations of mesna,. It was thelowest in5mg/ml group; those of msbB gene were0.697±0.096,0.52±0.088,0.36±0.045respectively,theinhibitory effection also improvedwith increasing concentrations of mesna, It was the lowest in5mg/ml group;those of lpxA gene were0.937±0.146,0.632±0.125,0.761±0.09relativelyexpression was respectively.Compared with the control group, there was nosignificant difference in0.5mg/ml group,however,they were significantlylower in2mg/ml and5mg/mlgroup(P <0.05),the expression was thelowest in2mg/ml group(P <0.05).【Conclusion】Mesna inhibited adhesion-related gene expression of flu,fimH, of papC in E. coli; moreover, inhibited gene expression of glmS, glmU,msbB, IpxA, which regulated polysaccharide production. This inhibitoryeffection was different in different concentrations and didn't enhance withincreasing concentrations of mesna.