MLVA Genotype Of Methicillin-resistant Staphylococcus Aureus And The Status Of The Epidemiology

[Objective]MRSA is an important pathogen of nosocomial infection. Effective homology analysis method is the basis of analysis of the popular traceability for nosocomial infection caused by the bacteria. This study aims to construct MRSA strains homology MLVA classification method for clinical laboratory routine application. By using the method to type111MRSA strains separated from our hospital clinical microbiology lab from2010to2013.Establish MLVA genotype basic database for local clinical isolated MRSA strains. To analyze of MRSA prevalence in hospital combined with corresponding clinical data. It can provide objective information for the effective control measures of hospital infection caused by MRSA.[Method](1)Methods of separation and cultivation of staphylococcus aureus is by reference to clinical pathogenic microorganism separation cultivation methods of third edition of national clinical laboratory procedures. According to colony characteristics, plasma coagulase test, staphylococcus aureus SPA monoclonal antibody detection and Microscan walkaway40SI automatic bacteria identification and drug susceptibility system to make strain identification and antibacterial drug sensitivity test. Determination of MRSA is according to Oxacillin minimal inhibitory concentrations (mic) to staphylococcus aureus and cefoxitin Kirby-Bauer method detect the diameter of bacteriostasis circle. In the process, using ATCC25923as methicillin sensitive strains of bacteria in quality control. Using ATCC43300as methicillin resistant quality control strains in drugs susceptibility test results of quality monitoring.(2)By reference to Leo M. Schouls et in S.aureus strain N315genome sequence for sequence tagged loci of VNTR. After using BLASTN for primer sequence alignment and specificity verification, choose seven loci well in discrimination and classification ability. To compound of the flanking sequence specific primers, and optimize these PCR amplification reaction system and amplification condition. To establish PCR amplification detection methods for each VNTR sequence loci polymorphism. Then the repeatability and accuracy of PCR detection method can be verified by amplification products sequencing analysis.(3)Using verified PCR amplification detection methods for VNTR sequence loci polymorphism to test the111strains MRS A separated from clinical samples of our hospital clinical microbiology lab from October2010to December2013on seven VNTR sequence loci polymorphism respectively.(4)To encode the seven VNTR sequence loci polymorphism (motif repetitions)of each MRSA respectively, and then get the strain’s MLVA genotype, classify each bacteria strain’s genotype. Establish a MLVA genotype based database of local clinical isolated MRSA strains.(5) According to MLVA genotype database of the111strains of clinical isolated MRSA classified their types, combined with its appearance time and department, analysis the prevalence of MRSA in hospital during October2010to December2013.[Results](1) MRSA separation rate and drug resistance In October2010to December2013, the first people’s hospital of Kunming in clinical microbiology laboratory, a total of870strains staphylococcus aureus isolated and MRSA separation rate to an average of50.23%. MRSA in β-lactams, erythromycin, clindamycin, moxifloxacin, tetracycline, gentamycin, ciprofloxacin, azithromycin, ofloxacin, rifampicin and cotrimoxazole, drug resistance rate were100%,80.2%,78.4%,80.2%,64.9%,60.4%,55.0%,53.2%,45.9%,26.1%,45.9%, only sensitive to vancomycin, linezolid.(2) Sequence of VNTR loci polymorphism PCR amplification method optimized reaction system and amplification conditionSeven VNTR loci polymorphism PCR amplification system are in accordance with the lOxtaq Buffer5μl; dNTP Mix (2mm each)4μl; The Forward primer1.5μl, Reverse primerl.5μl;25mM MgC124μl; The Template DNA2μl;1.4μl Taq DNA Polymerase; Water (nuclease-free)30.6μl. Amplification conditions:95℃pre degeneration3min;95℃degeneration30sec, modified their primers optimum annealing temperature annealing30sec,72℃extension45sec,30cycle;72℃always extend10min, for PCR amplification.(3) Seven sequences of VNTR loci polymorphisms of111strains of MRSA detection results and polymorphism111strains of clinical isolated MRSA after verified and polymerase chain reaction (PCR) amplification, electrophoresis showed different strains of different loci showed polymorphism of the similarities and differences.VNTR61-01loci amplification product size from210bp to695bp,0to8repetitions;VNTR61-02loci amplification product size from233bp to366bp,0to2repetitions;VNTR67-01loci amplification product size from181bp to605bp,0to6repetitions;VNTR24-01loci amplification product size from265bp to410bp,4-10repetitions;VNTR63-01loci amplification product size from232bp to554bp,0to5repetitions;VNTR81-01loci amplification product size from222bp to568bp,1-5repetitions. VNTR09-01loci its amplification product size difference is very small, sequencing results showed that9bp repetitive regularity is not strong, easy to mutation.(4)The results of111strains MRSA’s MLVA genotype111strains clinical isolated MRSA can be divided into25MLVA genotypes (A-Y).Among them, G, A, B type as the main, there were53,15,9strains of bacteria respectively. Type D has four strains. F, I, R each genotype respectively with three strains, the rest of the each genotype only1-2strains.(5)From October2010to December2013, the prevalence of MRSA in hospital G type77.36%(41/53) concentrate in April2011to October2010,Mainly in the respiratory, neurosurgery and ICU three departments. During this period most ICU (13/14) separation of MRSA is MLVA genotype G. Type A (8/15) centralize in May2013in mammary gland. Type B (7/9) concentration distribution in the respiratory ward. Type D (3/4) strains in mammary gland ward.[Conclusion](1)This research selected six loci from seven VNTR loci (VNTR61-01, VNTR61-02, VNTR67-01, VNTR24-01, VNTR63-01, VNTR81-01) for MRSA homologous analysis has better resolution and classification ability. MLVA genotype can be used in routine clinical laboratory for MRS A. Loci VNTR09-01because of its small base sequence (9bp), repetitive regularity is not strong, easy to mutation, should not be used for the MRS A strains’MLVA genotype.(2)In this study builded six locus’(VNTR61-01, VNTR61-02, VNTR67-01, VNTR24-01, VNTR63-01, VNTR81-01) DNA fragments of PCR amplification method is simple, the amplification effect and repeatability is good, those locus can be applied in the VNTR polymorphisms detection.(3)In this study, through the six locus’(VNTR61-01, VNTR61-02, VNTR67-01, VNTR24-01, VNTR63-01, VNTR81-01) polymorphism information building of MRSA homologous MLVA genotype method has higher resolution, the classification result well repeatability, operating simply than the PFGE classification method, easy, fast, has good feasibility in clinical laboratory, is worthy of popularization and application.(4)This study found that in October2010to December2010.Our parts department has MRSA homologous bacteria concentration prevalence, it’s worthy of attention.