Study On The Effect Of IcaA/D Genes On The Capacity To Form Biofilm And Antibiotic Resistances In MRSA Isolate

Methicillin resistant Staphylococcus aureus(MRSA)is an important zoonotic pathogen which can cause skin and soft tissue purulent inflammation,endocarditis,pneumonia,and other disease in human and animals.MRSA has severe multi-drug resistant and immune evasion when they form biofilms which making it easy to chronic infection and difficult to completely cure.So MRSA makes threat to health development and public health security.It has been demonstrated that Polysaccharide Imercellular Adhesin(PIA)plays an important role in the course of staphylococcal biofilms formation.PIA is the product of ica operon which contains a regulatory gene icaR and four functional genes(followed icaA,icaD,icaB,icaC),icaA and icaD genes co-expression can greatly improve the activity of N-acetyl glucosamine transferase,then promote PIA synthesis.For the current status of the lack of effective drug to MRSA infections,this study used gene knockout technology to build methicillin-resistant Staphylococcus aureus icaA/D genes deletion strains and detect antibiotic resistances and capacity of biofilm formation to clear the effect of icaA/D genes in MRSA biofilm formation and resistance,thereby laying the foundation for the development of effective treatments.The contents and results of this study include the following aspects:Firstly,in order to identify the resistance of MRSA isolates,we use KB disk diffusion method and the broth dilution method to measure the resistance of 41 MRSA to 14 kinds of antimicrobial drug,and use the PCR technique to detect the resistance genes which MRSA strains carryed.The results showed that the resistance rates of penicillin,oxacillin,ceftazidime,clindamycin,azithromycin,ciprofloxacin,doxycycline,tetracycline,cefotaxime,gentamicin,amikacin and chloramphenicol were 100%(MIC?0.25),100%(MIC?4),80.5%(MIC?32),78.1%(MIC?4),63.4%(MIC?8),63.4%(MIC?4),48.8%(MIC?16),46.3%(MIC?16),41.5%(MIC?64),41.5%(MIC?16),39.0%(MIC?64),12.2%(MIC?32);and 97.6%(40/41)of the test isolates were multidrug-resistant(?3 species),the strains of 3-resistant,4-resistance,5-resistance,6-resistant,7-resistan,8-resistant,9-resistant were 2,4,11,13,6,3,1;the test isolates carryed at least one kind of resistance genes investigated and the detection rates of mecA,blaTEM,aac(6 ')/ aph(2 "),aph(3')-?,ErmB,tetM and qacA were 92.7%,34.2%,92.7%,63.4%,12.2%,47.5% and 63.4%.The results show that the resistance phenomenon and multi-drug resistance in the 41 tested MRSA are more serious.Secondly,to clear the capacity to form biofilm in MRSA isolates,we use 96-well plate crystal violet staining and PCR methods to detect biofilm formation phenotype and biofilm-related genes in 41 MRSA isolates,then analysed the relation between phenotype and genes,and differences between strains of biofilm formation and non-formation rate of resistant strains.The results showed that 80.5%(33/41)of the test isolates could form different intensity biofilm;and the detection rate of five kind of biofilm-related genes sarA,icaA,icaD,sigB and agr respectively was 85.4%,82.9%,70.7%,80.5% and 31.7%.Ordinal variable correlation analysis showed that,sarA,icaA,icaD and sigB genes were significantly correlated to biofilm phenotypes(Sig <0.05);Chi-square analysis showed that the resistance rate of biofilm formation and biofilm unformation in MRSA isolates were significant differences(P <0.05).Then,the Knockout plasmid was constructed and screening and identification of icaA/D gene deletion strains.Two pairs of specific primers were designed according to the upstream and downstream sequences of icaA / D gene from GenBank reference strain N315.The upstream and downstream fragments of icaA / D gene of MRSA M5(icaAR,icaDL)by PCR,then the integration fragment(icaAL-icaDR,1477bp)was obtained by overlap extension PCR(SOE-PCR)and cloned into the plasmid pBT2.After sequencing,both the integration fragment and pBT2 were digested with restriction enzymes Sal I and EcoR I,the recombinant plasmid was obtained about the size of 6970 bp and 1477 bp DNA bands by two double digested,respectively,consistent with the size and integration pBT2 fragments.DNA sequencing results also showed that two DNA fragment sequences and joined order completely correct,explain knockout plasmid pBT2-icaAL-icaDR successfully constructed.The knockout plasmid was transferred into aureus strain RN4220 to modify recombinant plasmid then extracted transferred into MRSA M5 isolates.Using chloramphenicol resistance marker and PCR methods to obtain three icaA / D gene deletion strains suspected.Using icaA1 / icaD2,P1 / icaD2,icaA1 / S2,P1 / S2 primers suspected deletion mutant combination of PCR,the amplified size was 1469 bp,2202bp,2229 bp,2898bp and 1477 bp target gene,the results of sequencing identification also show that three icaA / D gene deletion mutant were successfully constructed.Finally,to clear the role of icaA / D genes in the capacity to form biofilm and antibiotic resistances in MRSA isolates,we use the broth dilution method to measure the MICs of 14 kinds of antibiotics to before and after gene deletion strains.While using coverslips fluorescence confocal to detect the capacity to form biofilm and dynamic formation.Compared with the wild-type strain,the resistance of 14 kinds of antibiotics to gene deletion strains decreased into sensitive or agency;the capacity to form biofilm of deletion mutant strain 60 decreased significantly,and deletion mutant strain 24 and 60 completely lost the capacity to form biofilms.In conclusion,the phenomenon of drug resistance and multiple drug in MRSA isolates are more serious,and most isolates have the capacity to form biofilms.icaA/D genes play an important role on the drug resistance and biofilm formation in MRSA islates.