【作者】 曹江红；

【导师】 李光辉； 吴菊芳； 徐晓刚；

【作者基本信息】 复旦大学 ， 内科学， 2010， 硕士

【摘要】 [摘要]目的：了解2008年上海市儿童医学中心、上海市儿童医院、复旦大学附属儿科医院3家医院甲氧西林耐药金黄色葡萄球菌(MRSA)临床分离株中的PV杀白细胞素(PVL)基因分布、葡萄球菌mec盒式染色体(SCCmec)分型、多位点基因序列类型(MLST)分型及药物敏感性,并与同期甲氧西林敏感金黄色葡萄球菌(MSSA)的PVL基因分布及药物敏感性进行比较,分析社区获得性MRSA (CA-MRSA)的分子特征、毒力因子和药物敏感性。方法：对上海市三家儿童医院98株MRSA和49株MSSA临床分离株,用聚合酶链反应(PCR)方法筛选PVL基因阳性菌株并进行序列测定；用多重PCR方法对所有MRSA菌株进行SCCmec分型；用PCR方法通过金黄色葡萄球菌(SA)的7对看家基因(arcC、aroE、glpF、gmk、pta、tpi、yqi)对98株MRSA进行测序,通过MLST网站(http://www.mlst.net/)进行序列比对得出序列类型(STs)型别,eBURST软件分析各型在菌株克隆复合体(CC)内的相关性；用琼脂稀释法测定MRSA和MSSA对14种常用抗菌药物的敏感性(MIC)。结果：三家儿童医院的98株MRSA临床分离株中有6株PVL基因阳性,阳性率为6.1%(6／98),其中上海市儿童医学中心1株,上海市儿童医院4株,复旦大学附属儿科医院1株。49株MSSA临床分离株中有2株PVL基因阳性,阳性率为4.1%(2／49),该2株均分离自上海市儿童医院。所有MRSA临床分离株中发现4个SCCmec型别,其中Ⅱ型4株,占4.1%(4／98)；Ⅲ型23株,占23.5%(23／98)；Ⅳ型52株,占53%(52／98)；Ⅴ型15株,占15.3%(15／98)；4株(4.1%)未能分型。上海市儿童医学中心和上海市儿童医院以SCCmecⅣ型、Ⅴ型为主,复旦大学附属儿科医院则以SCCmecⅢ型多见。PVL基因阳性的6株MRSA中,有1株为SCCmecⅣ型,来自上海市儿童医院,余5株为SCCmecⅤ型。98株MRSA临床分离株中发现11个ST型别,其中ST59型33株,占33.7%(33／98)；ST239型29株,占29.6%(29／98)；ST5型12株,占12.2%(12／98)；ST88型9株,占9.2%(9／98)；STl型8株,占8.2%(8／98)；ST8型2株,占2%(2／98)；ST7、ST9、ST22、ST45、ST910各有一株,均占1%(1／98)。上海市儿童医学中心和上海市儿童医院多见ST59型,复旦大学附属儿科医院以ST239型为主。此外上海市儿童医院菌株中暂未发现ST239型存在。PVL基因阳性的6株MRSA中,3株为ST88-SCCmecⅤ,2株为ST59-SCCmecⅤ,1株为ST59-SCCmecⅣ。SCCmec分型中,4株Ⅱ型为ST5；23株Ⅲ型为ST239；52株Ⅳ型中30株为ST59,ST5和ST1各8株,ST8和ST88各2株,ST22和ST910各1株；15株Ⅴ型中6株为ST239、5株ST88、3株ST59、1株ST45。eBURST软件分析结果显示,ST8、ST239属于ST8 CC,ST1属于ST15 CC,ST910属于ST 30 CC,ST59、ST5、ST88、ST45、ST22、ST9、ST7均为各自CC的起源型别。MIC结果显示MSSA对药物的敏感性普遍高于MRSA,二者均对万古霉素、替考拉宁、利奈唑胺保持高度敏感,未发现万古霉素耐药株。PVL基因阳性株较阴性株对多种非β内酰胺类抗菌药更为敏感,但两者均对β内酰胺类、大环内酯类和克林霉素高度耐药,对庆大霉素、四环素的敏感性大致相仿。67株SCCmecⅣ、Ⅴ型MRSA对红霉素、克林霉素高度耐药,耐药率分别为92.5%和76.1%；对于其他非β内酰胺类抗菌药,67株SCCmecⅣ、V型MRSA的耐药率分别为：庆大霉素26.9%、利福平9%、四环素34.3%、左氧氟沙星14.9%、环丙沙星22.4%,均比27株SCCmecⅡ、Ⅲ型MRSA更为敏感；二者均对复方磺胺甲噁唑高度敏感。结论：在上海市三家儿童医院中,MRSA临床分离株的PVL基因阳性率相对较低,若仅以此作为CA-MRSA的标志可能漏检相当部分菌株。SCCmec分型可有效区分CA-MRSA和HA-MRSA。SCCmecⅣ型、V型菌株为CA-MRSA,可在院内传播,形成小范围内的流行,甚至暴发。SCCmecⅡ、Ⅲ型菌株的ST分型呈现高度克隆一致性,而SCCmecⅣ、Ⅴ型菌株中存在多种ST分型,并且分子量越小的SCCmec,相关的ST分型类别越多。CA-MRSA对多种非β内酰胺类抗菌药的敏感性显著高于HA-MRSA,多重耐药菌株的发生率较低,未发现万古霉素耐药株。更多还原

【Abstract】 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.更多还原