The Molecular Characteristic And Virulence Factor Of Community-acquired Methicillin-resistant Staphylococcus Aureus

Objective The present study is aimed to investigate the distribution of Panton-Valentine leucocidin(PVL) gene, staphylococcal cassette chromosome mec (SCCmec) type, multilocus sequence types(MLST) type and the antimicrobial susceptibility of clinical isolates of Methicillin-resistant Staphylococcus aureus (MRSA) in three children's hospitals (Shanghai Children's Medical Center, Shanghai Children's Hospital, Children's Hospital of Fudan University) in 2008, and comparing the prevalence of PVL gene and antimicrobial susceptibility of methicillin-susceptible Staphylococcus aureus(MSSA)in the same periods, to analyze the molecular characteristic, the virulence factors and antimicrobial resistance of Community-acquired MRSA(CA-MRSA).Methods ninety eight non-duplicate strains of MRSA and forty nine non-duplicate stains of MSSA isolated from the three Children's hospitals in Shanghai in 2008 were investigated. PVL genes were detected by polymerase chain reaction (PCR). The genotypes of SCCmec of the MRSA isolates were confirmed by multiplex PCR. The allelic profile of each strain was obtained by sequencing internal fragments of seven housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, and yqiL) and entering them on the MLST home page (http://saureus.mlst.net), where seven numbers depicting the allelic profile were assigned which defined an ST, at last the algorithm eBURST was used to identify groups of clonal complex (CC) and to predict the ancestral genotype of each group and the most parsimonious patterns of descent from the corresponding ancestor. The minimal inhibitory concentrations (MICs) of fourteen antibiotics for all isolates were determined by agar dilution method.Results Among ninety eight isolates of MRSA, the PVL genes were found in 6.1%(6/98) of the MRSA strains, in which one isolate was from Shanghai Children's Medical Center, four isolates from Shanghai Children's Hospital, one isolate from Children's Hospital of Fudan University. In contrast, the PVL genes were found in 4.1%(2/49) of the MSSA strains, two isolates carring PVL genes were from Shanghai Children's Hospital, Whereas all the other isolates were negative for PVL genes. Among ninety eight isolates of MRSA,4.1%(4/98),23.5%(23/98),53%(52/98) and 15.3%(15/98) of the strains harboured SCCmec typesⅡ,Ⅲ,ⅣandⅤ, respectively, the remaining four isolates (4.1%) presented a unique SCCmec pattern that could not be classified to any known types by the employed typing assays. The SCCmec typeⅣand SCCmec type V were predominant in Shanghai Children's Medical Center and Shanghai Children's Hospital, while the SCCmec typeⅢwas predominant in Children's Hospital of Fudan University. Among six PVL gene-positive isolates of MRSA, one isolate which was from Shanghai Children's Hospital harbored SCCmec typeⅣ, the others were SCCmec typeⅤ. Eleven sequence types (STs) were identified among ninety eight isolates. The ST59 and ST239 isolates predominated in three Children's hospital in Shanghai and accounted for 33.7%(33/98) and 29.6%(29/98) respectively, ST5, ST88 and ST1 were not uncommon and accounted for 12.2%(12/98),9.2%(9/98) and 8.2%(8/98) respectively, the rest of the STs, such as ST8, ST7, ST9, ST22, ST45, ST910 were found in 1%-2% of the strains.ST59 was prevalent in Shanghai Children's Medical Center and Shanghai Children's Hospital, while ST239 was dominated in Children's Hospital of Fudan University. In addition, ST239 was not found in Shanghai Children's Hospital. Among six PVL gene-positive isolates of MRSA, three, two and one belonged to ST88-SCCmecⅤ, ST59-SCCmecⅤand ST59-SCCmecⅣrespectively. Combining the ST and SCCmec type, fourteen clone were identified, the predominant clone was ST59-SCCmecⅣ(thirty strains) and ST239-SCCmecⅢ(twenty tree strains), followed by ST5-SCCmecⅣand STl-SCCmecⅣ(eight strains for each clone), ST239-SCCmecⅤ(six strains), ST88-SCCmecⅤ(five strains), ST5-SCCmecⅡ(four strains), ST59-SCCmecⅤ(three strains), ST8-SCCmecⅣand ST88-SCCmecⅣ(two strains for each clone), ST22-SCCmecⅣ, ST910-SCCmecⅣand ST45-SCCmecⅤ(one strain for each clone). The eBURST analyses distributed the MRSA isolates into several CCs. ST8 and ST239 belonged to ST8 CC, ST1 belonged to ST15 CC, ST910 belonged to ST 30 CC, ST59, ST5, ST88, ST45, ST22, ST9 and ST7 were the origin of their own CCs. The results of MIC showed that the MSSA isolates were more susceptible than the MRSA isolates to fourteen antimicrobial agents tested, All strains including MSSA and MRSA were susceptible to vancomycin, teicoplanin and linezolid, no vancomycin-resistant strain was found. PVL gene-positive strains were much more susceptible to non-β-lactam antibiotics than those without PVL genes, both of them were highly resistant toβ-lactam antibiotics, macrolides and clindamycin, they had fairly susceptibility to gentamicin and tetracycline. Among sixty seven strains of MRSA harboring SCCmec type IV or SCCmec type V, the resistant rates to erythromycin and clindamycin were high, with 92.5% and 76.1% respectively. The resistant rates to other non-β-lactam antibiotics of sixty seven strains of MRSA were as follows: gentamicin 26.9%, rifampicin 9%, tetracycline 34.3%, levofloxacin 14.9%, ciprofloxacin 22.4%, which were more susceptible than twenty seven strains of MRSA harboring SCCmec typeⅡor SCCmec typeⅢ, both of them remained highly sensitivety to SMZ-TMP.Conclusions Among three children's hospitals in Shanghai, the PVL gene-positive rate of MRSA isolates was relatively low, however,a considerable part of CA-MRSA strains might be undetected if PVL gene was treated as a sign of CA-MRSA. The genotypes of SCCmec could effectively distinguish CA-MRSA from HA-MRSA. SCCmec typeⅣand SCCmec typeⅤbelonged to CA-MRSA, which could spread among hospitals to form a small scale epidemic, even outbreaks. The STs of SCCmec type Hand SCCmec typeⅢstrains showed a high degree of consistency, while the SCCmec typeⅣand SCCmec type V strains had a variety of STs, furthermore, the smaller the SCCmec the more STs were associated with it. CA-MRSA was susceptible to various non-β-lactam antibiotics, with the low incidence of multidrug resistance. No vancomycin-resistant strain was found.