Study On The SCCmec Typing And Antibiotic-resistance Of MRSA

Staphylococcus aureus is the main entogenous and exogenous pathogen of the infections which acquired in hospital or society. It has not only vast pathogenicity but also a rapidly increasing of antibiotic-resistance. Methicillin had been successfully used to treat the infections with Pencillin-resistant S. aureus in 1959, but soon, the Methicillin- resistant S. aureus (MRSA) was found in 1961. In recent years, the positive-rate of MRSA is quickly rising and the resistant-spectrum is quickly extending, too. Together with the unknown mechanism of multi-drug resistance transferring and extending,MRSA has become the difficult problem of the world.SCCmec, stand for Staphylococcus cassette chromosome mec, is the resistant genomic island of MRSA and closely connect with gene transfer, multi-drug resistance. The MRSA molecule typing based on the structure of SCCmec is a research mean for exploring the relationship between the structure and the mechanism of multi-drug resistance transferring and extending, Because the structure of SCCmec is different among the country and area, Typing and polymorphism analyzing of it is the base for exploring the characteristics of molecule epidemiologic and multi-drug resistance, and for finding the effective means of preventing and curing infection with MRSA in an area.This paper collected 1174 strains of Staphylococcus aureus from clinical and, with the PBP2a test, confirmed 686 strains of MRSA, the distributing characteristics of it been analyzed . Using the Kirby-Bauer and minimal inhibitory concentration (MIC) test, the resistance to 7 kind's antibiotics of MRSA had been detected. The multi-drug resistant pattern, and the resistance development tend of them had been analyzed too.In this paper, the multiplex PCR typing method had been established and optimized. This technique can detect 9 loci (A~H and M) in a time. The A, B, E, F loci are special to the types reported in past, and G, H are the distinguish loci using them some sub-types can discriminated, and C, D are the indication loci of some resistant-plasmid, and M is a positive-control locus. Using the orthogonal design, the multiplex PCR are tested repeatedly in many dilution of reagents that primarily affect it and got the optimized reaction-system of multiplex PCR as follows: the total volume is 50μl, including 10×buffer: 5μl, TaqDNA: 6U, 4×dNTP: 0.2mmol/each, MgCl2: 1.5mmol, Template DNA: 1μl,Primers of B,E,F,H loci: 200nM/each, Primers of A,C,G,M loci: 400nM/each, Primers of D locus: 800nM/each. The initial denaturation: 94℃4 min, denaturation: 94℃30 sec, annealing: 52℃30sec,extention: 72℃1min,30 cycles,final extention: 72℃4min. Using the BLAST, 10 loci sequences of MRSA been aligned with the sequences of correlative loci and bacteria in sequence databases and using the parallel test, the results of multiplex PCR and single-locus PCR typing had been compared, The results Confirmed that the loci been exactly detected and the homology of sequences between them is 93% to 100% and the multiplex PCR is speediness, high efficiency and repeatable.The multiplex PCR technique been used to typing 58 strains of MRSA which be sampled randomly from the 686 strains. The result show that there are 9 kinds of new SCCmec types (new 1~9) been detected which possessed by 26 strains(44.8%) of MRSA, 20 strains(34.5%) possessing type-Ⅲof SCCmec and 12 strains(20.7%) possessing type-Ⅳ-dcs of SCCmec. In Haikou area, the type-Ⅲis the main type and the type-Ⅳ-dcs is the second, The SCCmec type of MRSA is high polymorphisms.Most of the strains with the new types had been isolated from the urinary and genital tract sample of out-patient. There is much difference among the strains in resistant rate, resistibility and multi-drug resistant pattern. Besides the same M locus, the gene structure of the new types/sub-types is different from the old ones. Type-New1 (6 strains) possess B and F loci which are special to the type-Ⅱand -Ⅲrespectively. Type-New2 (3 strains) possess A, B loci, which are special to type-ⅠandⅡrespectively, and F, H loci, which are special to type-Ⅲ. Type-New3 (4 strains) lack B locus comparing with the type-New2. Type-New4 has the special F and H loci which are special to type-Ⅲ, but lack C, E loci of type-Ⅲ. Type- New5 (2 strains) is lesser two loci of C and G than type-Ⅱ. Type-New6 (2 strains) lack F, H loci comparing with type-New2. Type-New7 ( 3 strains ) is lesser F locus than type-Ⅲ. Type-New8 (2 strains) lack D locus comparing with type-Ⅰ. Type-New9 (1 strain) has more C locus than type-New8.Most of the strains with type-Ⅲbeen isolated from the respiratory tract sample of in-patient. All of them are frequently resistant to those antibiotics which usually used to treat the infection with MRSA in clinic, and also, the resistibility of them is very strong. The strains with type-Ⅳ-dcs are often isolated from the urinary and genital tract sample of out-patient, but the resistant rate, resistibility and multi-drug resistant spectrum of them are obviously lower than strains with type-Ⅲ.Using Multivariate Statistical Package version 3.1(MVSP 3.1), the new and old types or sub-types been clustered into 7 small classes, which made up 4 medial classes, and then, 2 big classes. The analysis result reveal that type-New2 is closely related to–New3, which make up a small class with type-New4, and then make up a medial class with type-New1. Type-New7 is closely related to type-Ⅲand -ⅢA, which make up a medial class. Type-New6 and–New8 are closely related to type-Ⅳ-dcs andⅣC, which make up a medial class. Type-New9 is closely related to type-ⅢB and -ⅡD, which make up a small class. Type-New5 is closely related to type-ⅡE,-ⅡC and -ⅣA, which make up a medial class.The epidemiologic analysis results of 686 strains of MRSA reveal that the positive-rate of out- and in-patient is similar in 75.8% to 78.2% respectively (P>0.05) and the susceptible population is greatly wide (P>0.05). The positive-rate of MRSA in Summer (April-June) is the highest (89.2%) and in Autumn (October-December) is the lowest (64.4%) (Ρ<0.01). During the years of 2000-2004, the annual positive-rate is uptrend. There is endemic prevalence took place in 2001(Ρ<0.01). The infection sites mainly locate at respiratory, genital and urinary tract (Ρ<0.01).Antibiotic susceptibility test results show that, among 7 classes of antibiotic which routine-used in the clinical, the resistant-rate to Erythromycin (EM) is highest (84.8%), the resistibility to it is very strong and its MIC25 is over 160 times to upper limiter of sensitivity. The resistant-rate to Amikacin (AK) is the lowest (39.4%), and to Doxycycline (DOX) is 49.7%, similarly, the resistant-rate to AK quickly rise from 24.2% in 2000 to the 60.6% in 2004 (Ρ<0.01), and so does to DOX, which rise from 33.3% to 70.2% (Ρ<0.01). Also, the resistant-rates to Chloramphenicol (CL) and Ciprofloxacin (CIP) are visibly uptrend during the years (Ρ<0.01). MRSA has high resistance- heterogeneity to Oxacillin (OX) whose MIC range from 8μg/ml to≥256μg/ml, but all of the strains of MRSA are susceptible to Vancomycin (VA). In Haikou area, the multi-drug resistant pattern is very complex, among the 7 classes of antibiotics, there are 19 kinds of pattern that resistant to more than three classes of antibiotic, the proportion is 96.0%. Except VA, the proportion that resistant to all of 6 classes of antibiotic is 20.0%.It is the first time that typing the SCCmec with multiplexes PCR in our country. Not only the kinds, composing of SCCmec types, the distribute characteristics, multi-drug resistant pattern and the developing trend of types were found out, but also 9 kinds of new SCCmec type or subtype were found. The paper elucidates the prevalence status, distribute characteristics, multi-drug resistant pattern and the developing trend of MRSA. It also provides more useful science data for health officer and manager to make out some individual and effective measure, policy to control and prevent the MRSA expanding and breaking out. Furthermore, it has established a base for a further researching on the mechanism of multi-drug resistance transferring and extending in aftertime.