| Background and objectiveAt present, Staphylococcus aureus(staphylococcus aureus, SA) has been in a high separation rate both in hospital and in community acquired infections. With the extensive use of lactam drugs,multidrug-resistant Staphylococcus aureus(especially the methicillin-resistant staphylococcus aureus, MRSA) burst out in the past decades. Since the first case of drug-resistant strain was found in 1961, MRSA was reported and revealed significant growth trend throughout the world. MRSA has a very wide drug-resistant spectrum, it can hydrolyze almost all kinds of lactams, and the drug-resistant strains spread quickly, causing high mortality rate in infected patients and the serious overdraft of medical resources. Infections caused by MRSA have become one of the world’s top three serious infectious diseases, and the study of MRSA has become a hot sector of Clinical Microbiology.It is of great value to provide a distinct drug-resistant spectrum guiding a reasonable infection treatment and preventing the epidemic of strains. Due to the different habits of using antimicrobial drugs, MRSA strains resistant phenotypes appear to be different in different regions, and so there are differences in clinical antimicrobial drug selection. This is also one of the sections studied in MRSA. In addition, vancomycin is the last line of defense in treatments of MRSA. Currently, no vancomycin-resistant strain has been reported in Henan Province. Studies have shown that, the MIC vancomycin of MRSA increases year by year. It is of serious concern to identify vancomycin MIC in MRSA and its difference between MRSA and MSSA in Henan, in order to modify the clinical treatments. Nevertheless, no report has been published in Henan.Statistics have shown that MSSA has become MRSA after the acquiring of SCCmec, SCCmec is a mobile genetic element which contains mec A gene(the genetic basis of drug resistance leading to MRSA), and mec A gene encodes PBP2 a to hydrolyze β- lactam drugs. Studies have shown that, SCCmec elements have a variety of types, positioning on different plasmids or transposons, result in different resistant phenotype strains. In molecular typing of MRSA, clarifying the types of SCCmec elements in Henan is an important part of the MRSA study.We have designed this study to solve this problem. With the detection of MRSA strains in the First Affiliated Hospital of Zhengzhou University from 2013.8 to 2014.7, we clarified the clinical biological phenotype of MRSA strains, investigated the changes in vancomycin MIC of MRSA, and completed molecular typing of MRSA. The resistant status in Henan province eventually reflects the separation of methicillin-resistant Staphylococcus Aureus, providing sufficient evidence for the treatment of laboratory MRSA, making it easy to control the infection and the epidemic of MRSA in clinical fundamentally. Methods1. Strains were collected in the First Affiliated Hospital of Zhengzhou University from 2013.8 to 2014.7 from outpatients and inpatients, and various specimens were isolated from non-repeating 325 Staphylococcus aureus strains. All strains were confirmed by VITEK-II automated microbial analyzer to identify their species.2. Vitro drug sensitivity test was partly accomplished by French Merieux drug susceptibility testing and identification plates and VITEK-II automated microbial identification analyzer. The rest of drug sensitivity tests were completed using KB method, the results were judged by the American Association of Clinical and Laboratory Standards(Clinical and Laboratory Standards Institute, CLSI). And ATCC25923 strain was used as a control strain.3. Test of susceptibility to cefoxitin disk diffusion method was used as a screening test for MRSA. The results criteria: inhibition zone diameter ≥22mm for MSSA, inhibition zone diameter ≤21mm screening for MRSA.4. Molecular biology techniques were used to detect mec A gene as a confirmatory test for MRSA. It is the gold standard when mec A gene is positive in MRSA determination.5. E-Test experiment completed the test of vancomycin MIC of MRSA strains.6. Strains were divided into MRSA groups and MSSA groups with different concentrations of vancomycin MIC as an indicator of difference analysis, and differences in the use of vancomycin MIC rank correlation statistical analysis, SPSS 17.0 software was used in statistical analysis of the results, with P <0.05 was considered statistically significant7. Genomic DNA was extracted using lysostaphin against MRSA strains, after which strict accordance were carried to genomic DNA extraction kit instructions to get genomic DNA of MRSA strains.8. Multiplex PCR typing was used in SCCmec genotyping assay. Construction and annealing temperature of the system is the focus of the exploration. For five kinds of types other standard strains were designed specific primers, after many experiments to explore the conditions, 48 ℃ was the most suitable annealing temperature.9. Annealing temperature 54 ℃ is the most suitable temperature of PVL genes after gradient-PCR genetic testing, common PCR was used to detect PVL genes in all MRSA strains.10. Different SCCmec genotypes MRSA strains were used to test their antibiotic resistance, and were searched for the co-relationship between SCCmec genotypes and the results of antibiotic resistance. Results1. A total of 325 Staphylococcus aureus strains were collected, mainly from secretions(39.4%), followed by sputum(31.7%) and blood samples(12.3%).2. In 325 Staphylococcus aureus, MRSA were 180 strains(55.4%), MSSA were 145 strains(44.6%). The main sources were sputum(43.9%), discharge(30.6%) and blood samples(10.6%); these strains were from distributed department, mainly from the ICU department(32.8%), respiratory medicine department(8.8%), pediatrics department(7.8%), and neurosurgery department(7.8%).3. The susceptibility results show that, MRSA strains were highly resistant to internal lactams, especially resistance rate of penicillin and oxacillin were 100%. Non-β- lactam drug resistance rates are over 50%. Strains stabilize sensitive to vancomycin, tigecycline, linezolid, teicoplanin and other drugs.4. MRSA resistance rates were significantly higher than MSSA, and two kinds of strains had significant differences on vancomycin MIC values, MRSA was higher than MSSA.5.SCCmec molecular typing results of isolated MRSA strains showed that, SCCmec type III is the predominant type, a total of 143 strains, accounting for 85.1%; followed by 12 SCCmec type II strains, accounting for 7.1%; SCCmec type V were rare, only 3 strains were detected, 1.7%; and 10 strains, accounting for 5.9%,were not typed using this test method.6. Around 30 strains were detected PVL gene-positive in 168 MRSA strains, accounting for 17.9%.7. Susceptibility results showed that, SCCmec type Ⅱ and SCCmec type Ⅲ were resistant not only to β- lactams, but also to quinolones, macrolides, clindamycin and tetracycline. However, SCCmec type V were only resistant to erythromycin, clindamycin, tetracycline, besides β- lactam drug resistance; and all strains were nitrofurantoin, vancomycin, tigecycline and linezolid sensitive.Conclusions1. MRSA strains were isolated from various specimen species, and the strains distributed in different departments with a wide range of drug-resistant spectrum. MRSA strains may carry higher drug resistance than MSSA, and vancomycin MIC values of MRSA are higher than MSSA. Thus reasonable treatment options(based on strain identification) should be in consider in clinical treatment.2.SCCmec type III is the predominant strain in methicillin-resistant Staphylococcus aureus of Henan Province. So we recommend that quinolones, macrolides, clindamycin and tetracycline should be reasonably applied in clinical anti-infective therapy, in addition to lactams. |
PDF Full Text Request