| BackgroundStaphylococcus aureus is one of the most prevalent human pathogens, causing a broad variety of diseases ranging from mild skin and soft tissue infections to infective endocarditis, osteomyelitis, bacteremia and necrotizing pneumonia. S. aureus isolated clinically from different specimens is polymorphic and can be divided into different clones. Whether the distribution of resistance and virulence genes are different in diverse clones, and whether the phenotypic characterization is associated with molecular characteristics. The molecular characterization analysis of S. aureus can help us solve these problems and them provide significant implications for establishing effective infection control measures, monitoring process of evolution and follow-up mechanism study.Molecular characterization analysis of S. aureus showed high prevalence of mupirocin resistance gene mupA. Mupirocin is mainly used to rapidly eradicate nasal carriage with S. aureus in populations including health-care workers and hospital inpatients. Nasal carriage of S. aureus in hospital inpatients is associated with an increased risk of nosocomial staphylococcal bloodstream infection, dialysis-associated infections, and surgical site infections. Therefore, the research of epidemiology and mechanism of mupirocin resistance is significant.Part1Molecular characterization of Staphylococcus aureusObjective To investigate the clonal types, including its dynamic changes and genotype-phenotype correlation of S. aureus recovered from different clinical specimens of inpatients over a six-year span at Huashan Hospital in Shanghai; To detect the variation in antimicrobial resistance and virulence-gene content in different strain types.Methods Between2005and2010, random samples of610unique S. aureus isolates were collected from different clinical samples of inpatients from Huashan Hospital in Shanghai. The standard disk diffusion method was used to test the antibiotic susceptibility of all isolates. Typing of S. aureus isolates was identified by using multilocus sequence typing (MLST) and S. aureus-specific staphylococcal protein A typing (spa typing). The Methicillin-resistant Staphylococcus aureus (MRSA) isolates were subjected to SCCmec typing, which was carried out by the multiplex PCR (M-PCR). PCR was used to detect the antimicrobial resistance and virulence-gene of108nonduplicate S. aureus isolates.Results Among the610S. aureus isolates,20MLST sequence types (STs; primarily ST239, ST5, ST7, ST188, and ST398) and52spa types (primarily t002, t037, t030, and t601) were found. In total,444isolates (72.8%) were MRSA and166(27.2%) were Methicillin-susceptible Staphylococcus aureus (MSSA). The ST239-MRSA-III-spa t037and ST5-MRSA-Ⅱ-spa t002were the most predominant MRSA clones. From2005to2010, spa t002, spa t037, and their corresponding STs (ST5and ST239) were the most frequent clones among all of the S. aureus isolates and showed the most resistant phenotypes to various antibiotics. Generally, the different genotypes showed different drug resistance rates, but no isolates were resistant to vancomycin, teicoplanin, or linezolid. The profiles of virulence and resistance genes differed by genetic background, with the ST239and ST5strains showing higher resistance rates to gentamicin, cefoxitin, ampicillin, cefazolin, erythromycin, clindamycin and levofloxacin than trains of other types. Moreover, the antiseptic resistance genes qacA/B were generally associated with these two types. The prevalence of STs were different among different clinical specimens, which also changed by year. Recently (2009to2010), the distribution of predominant MRSA clones decreased, while the prevalence of non-predominant MSSA clones increased, especially for the isolates causing bacteremia.Conclusions There are associations of phenotypes and the distribution of virulence and resistance genes with specific genotypes. The infections of non-predominant MSSA STs increased in the most recent two years (2009and2010). Continual monitoring of clinical isolates is necessary to develop and maintain an effective strategy against S. aureus infection in the hospital setting. Part2Phenotypes and mechanisms of mupirocin resistance of Staphylococcus aureusObjective To analyze phenotypes and mechanisms of mupirocin resistance of S. aureus collected from Huashan Hospital in Shanghai, Shandong Provincial Hospital, the First Affiliated Hospital of Wenzhou Medical College and Wenzhou community.Methods A total of796nonduplicated S. aureus isolates were recovered respectively from Huashan Hospital in Shanghai (270), Shandong Provincial Hospital (185), the First Affiliated Hospital of Wenzhou Medical College (241) and Wenzhou community (100). Mupirocin-resistant strains were screened by agar dilution method and confirmed by standard broth dilution method. PCR was used to detect mupirocin resistant gene (mupA) of621nonduplicate S. aureus isolates.17high level mupirocin-resistant (HLR-Mup) isolates,9low level mupirocin-resistant (LLR-Mup) isolates and13susceptible isolates were selected to amplify the entire coding sequence of the ileS gene. The sequences were compared with those of the published S. aureus ileS gene sequence.5HLR-Mup isolates were selected to extract plasmids. These plasmids were electrophoresed and transferred to a nylon membrane. The membrane were probed with mupA gene probe labelled by digaoxin (DIG). The extracted plasmids were electroporated into recipients (S. aureus RN4220). The recipients which were high level resistant to mupirocin were screened. Plasmids of These isolates were extracted, electrophoresed and transferred to a nylon membrane. The membrane were probed with mupA gene probe labelled by DIG.Results A total of40mupirocin-resistant isolates were screened with31HLR-Mup and9LLR-Mup strains. The prevalence of mupirocin resistance of Huashan Hospital, Provincial Hospital, and Affiliated Hospital of Wenzhou were9.6%(26/270),4.9%(9/185) and2.1%(5/241), respectively. All isolates of Wenzhou community were susceptible to mupirocin. The frequency of mupirocin resistance of Huashan Hospital in2004,2007and2010, were12.8%(12/94),9.6%(8/83) and6.5%(6/93), respectively, with a downward trend. For the detection of HLR-Mup of SI aureus, the amplification of mupA gene by PCR showed100%sensitivity and specificity of98.7%compared with the results of standard broth dilution method. Mupirocin-susceptible S. aureus acquired LLR-Mup through a single mutation, G1762T. Southern blotting analysis showed that mupA gene probe only hybridised with the plasmid about54kb.Conclusions The prevalence of mupirocin-resisitance of S. aureus from Huashan Hospital is higher than that from Shandong Provincial Hospital and the First Affiliated Hospital of Wenzhou Medical College, with a downward trend. Mupirocin-susceptible S. aureus acquired LLR-Mup through a single mutation, G1762T. mupA gene on plasmids about54kb mediate HLR-Mup of S. aureus. |
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