Study Of Biofilm Formation In Staphylococcus Epidermidis Isolations And Affection Of Medications On Bacterial Biofilms

Staphylococcus epidermidis normally is considered commensal flora and lowvirulence.It has emerged as important pathogen for increasing prominence in aclinical setting resulted from the growing use of biomaterials.Its pathogenicity iscaused by the ability of forming biofilm by adhering to the surfaces of biomaterials orhuman mucous membranes.,treatment is extremely difficulty with biofilm formed,asmicrobial cells within the biofilm are protected from the action of antibiotics and theimmune system.Furthermore,a bacterial biofilm may continuously release bacteriainto the bloodstream on a chronic basis,which may lead to consequent bacteraemia aswell as seeding to any other indwelling devices.Ultimately,infected biomedicalimplants required surgical removal.Therefore,bacterial-related infections hasbecome an severe problem which has impacted and limited the medical applicationsof biological materials in clinical.Objective:1.To investigate MRSE fromnosocomial infections of S.epidermidis in order to help clinical therapy;and comparethe results between phenotype and genotype methods.2.To detect the ability offorming biofilms and analyse the relationship between biofilm formation and icaAgene in order to understand the molecular biological basis of biofilm formation.3.Todetect the expression of icaA gene and analyse relationship between the expressionlevels of icaA gene and the ability of forming biofilms.4.To search for medicinecombinations which can effectively affect on bacterial biofilms.Methods:1.205strains of S.epidermidis were isolated and collected from patients' specimens withnosocomial infections from 2006 to 2008.2.Detected MRSE using K-B method withcefoxitin (30μg)and mecA gene PCR method.3.Detected the ability of formingbiofilms with Congo Red Agar(CRA)method and adhesion assays,and amplifiedicaA gene with PC,observed biofilm morphous with scanning electronmicroscope(SEM).4.Dectected DNA sequence of icaA gene and contrasted equencesof icaA gene with that of ATCC35984 published in GenBank.5.Analyzed expressionlevels of icaA with RT-PCR and the relationship between expression of icaA gene andthe ability of biofilm formation.6.Combined vancomycin with erythromycin,ambroxol hydrochloride or DL-Lysine acetylsalicylate by chessboard methods,Using MTT methods and MHA plates to count bacteria after biofilms incubated inmedications for 24h,and compared the results affected by medicine combination.Results:1.Of all 205 strains of S.epidermidis,187 strians(91.2%)were MRSEdetected by mecA-PCR,K-B method with cefoxitin disk and mecA-PCR showed arelevance of statistical significance (P＜0.05)and a coincidence rate of 91.2%.2.24isolates(+)in CRA test,22 isolates (+)in adhesion assays and 28 isolates existenceicaA gene in all of 205 S.epidermidis isolates.19 strains(+)by all 3 methods.Itpresented correlations among 3 methods (P＜0.05).3.Biofilm morphous with SEMshowed microcolonies and slime substances surrounded bacterial cells.4.DNAsequence of icaA gene showed 97%-100% identified with ATCC35984.5.theexpression levels of icaA from 4 isolates with icaA(+)/BF(+)showed higher than thatof 2 isolates with icaA(+)/BF(-),it needs further research.6.Vancomycin combinedwith erythromycin,ambroxol Hydrochloride or DL-Lysine acetylsalicylate coulddecrease the densities in biofilm and the degree of reduction relied on concentrationsof medications.Conclutions:1.MRSE occupied a high proportion in S.epidermidisisolates,it reminded clinicians of choosing antimicrobial agents rationally accordingto the results of drug sensitivity,cefoxitin disk method can be used as conventionalscreening test in laboratory.2.icaA gene showed 13.7% positive in 205 isolates of S.epidermidis,combined biofilm morphous with SEM comfirmed that S.epidermidisisolates have ability of forming biofilms,and icaA maybe the molecular biologicalbasis.3.The phenotype methods and icaA-PCR showed statistical correlation andconsistency,and phenotype methods may be recommended for laboratoryapplications as conventional screening methods.4.The expression levels of icaAgene may correlated with the ability of forming biofilm,it meed further research.5.Combination of vancomycin with erythromycin,ambroxol hydrochloride orDL-Lysine acetylsalicylate showed synergistic effect on S.epidermidis biofilm,itsuggests that ambroxol Hydrochloride and DL-Lysine acetylsalicylate can enhancethe bactericidal action of vancomycin on biofilm.