青岛地区医院内感染耐甲氧西林金黄色葡萄球菌分子分型研究

Molecular typing of methicillin-resistant Staphylococcus aureus isolated from hospitalized patients in Qingdao

目的探讨青岛地区医院内感染的耐甲氧西林金黄色葡萄球菌（MRSA）分子流行病学特征及脉冲场凝胶电泳（PFGE）型别与菌株表型、一般临床资料间的关系。方法收集2003—2007年间青岛地区主要医院内感染MRSA360株，SmaⅠ酶切菌株染色体DNA后，进行PFGE电泳，用Bionumericus2．0软件对电泳图谱进行比较和聚类分析，绘制进化树。同时对患者的性别、年龄、菌株来源等进行多变量统计分析。应用纸片扩散法测定分离菌株的药物敏感谱，并与PFGE型别进行比较分析。PCR扩增不同PFGE型别MRSA代表株25株分离株的7个管家基因进行序列测定和多位点测序分型分析（MLST）。结果所有菌株经PFGE电泳后共分为5型（M0～M4型），其中M1型为优势菌型，M2型次之，M4型相对少见，M0为独特型，明显不同于其他已知PFGE型别。统计学分析发现5种PFGE型别在患者性别、年龄分布上的差异无统计学意义，但在菌株分离部位、来源有统计学的差异：M2型多分离自伤口感染，而M3型菌株多来自ICU病房，5种PFGE型在不同医院间及医院内的分布存在差异。M1与M2两型构成各医院分离菌株的主要型别。抗生素敏感性测定中未发现万古霉素耐药菌株，亦未发现某种PFGE型别与某种特定抗生素抗性之间的直接相关性。MLST分型发现优势型M1与M3共属于国内常见ST239型，M2型则归类于ST5，M4型属于ST240，独特型中的2种PFGE谱型则分属于ST45及ST398。结论ST239菌株为青岛地区医院内感染MRSA优势菌株；医院内MRSA的PFGE分型与菌株来源明显相关，与患者年龄、性别无关，MRSA感染普遍存在于各年龄人群中。

Objective To explore the molecular types of methicillin-resistant Staphylococcus aureus （MRSA） strains present in major hospitals in Qingdao area, using pulsed-field gel electrophoresis （PFGE） and multilocus sequence typing （MLST） methods, trying to find out the epidemiological characteristics of these MRSA isolates. Correlation of the PFGE types with microbiological phenotypes and clinical data was also studied. Methods 360 isolates of MRSA were procured during 2003 to 2007 from major hospitals in Qingdao. PFGE technology was applied to comparatively analyze the chromosomal DNA digested with endonuclease Sma Ⅰ . Comparison of DNA fragments patterns from each MRSA strain and cluster analysis were performed with the Bionumericus version ＇ 2.0＇ software. A dendogram was generated using PFGE macrorestriction fragments on gel images. Data was used to predict the possibility of each PFGE type via SPSS software version 11.0, using the variables as predictors including groups on patient＇ s age, gender, source and the site where MRSA was isolated. Antibiotic sensitivity patterns of these MRSA isolates were determined by K-B tests, and a correlation between these patterns and PFGE types was investigated. Housekeeping genes were amplified with PCR and sequenced in representative strains of variant PFGE types to identify their allelic profile. Results 5 types of PFGE patterns （M0-M4） were identified with M1 being the predominant and M2 next to it which was significantly correlated to the isolates from wounds. M3 type strains were mainly isolated from ICU wards and there were a few cases complied with M4 type with no correlated variant factors found in this study. A unique pattern of MRSA isolates with its M0 distinct from other types had not been reported. No significant association was found between PFGE individual types,gender or age groups. M1 and M2 types were the major proportional PFGE patterns among different hospitals. No vaneomycin-resistant isolates were detected among 360 MRSA strains. No significant association was found between individual antibiotic resistance and specific PFGE types. Data from MLST analysis showed that the allelic profiles of M1 and M3 type strain had the same ST239 linage which was commonly present in China. For M2 and M4 representative strains, the allelic profiles were ST5 and ST240, respectively. ST45 and ST398 were corresponding to two PFGE patterns clustered as M0 type. Conclusion Nosocomial infection due to MRSA was evenly distributed among different age groups and no gender bias was observed. The PFGE types of MRSA strains isolated in major hospitals in Qingdao were highly correlated with the sources of isolates and ST239 isolate seemed the prevalent and widespread one. Strategies should be designed to further monitor and prevent or minimize the spread of ST5 MRSA isolates and the like, in Qingdao area.