Establishment and Comparison of Pulsed-field Gel Electrophoresis,Multiple-locus Variable Number Tandem Repeat Analysis and Automated Ribotyping Methods for Subtyping of Citrobacter Strains

Objective To establish and compare the pulsed-field gel electrophoresis （PFGE）, multiple-locus variable number tandem repeat analysis （MLVA） and automated ribotyping for subtyping of Citrobacter strains. Methods PFGE protocol was optimized in terms of plug preparation procedure, restriction enzymes and configuration of electrophoretic parameters. MLVA method was evaluated by finding variable number tandem repeats in two genomes of Citrobacter strains. The ribotyping was performed by using the automated RiboPrinter system. Results We optimized the plug preparation procedure, focused on the cell suspension concentration （turbidity of 2.5 to 3.5）, SDS addition （no SDS needed） and lysis time （1 h）, and selected the appropriate restriction enzyme （Xbal） and the electrophoretic parameters （1.0 s-20.0 s for 19 h） of PFGE. There was nearly no discriminatory power of MLVA between Citrobacter strains. For 51 Citrobacter strains, automated ribotyping gave a D-value of 0.9945, while PFGE gave a D-value of 0.9969. Both PFGE and automated ribotyping clustered strains from the same sources （with the same species from the same place at the same time identified as the same source） and divided strains from different sources （from different years, places and hosts） into different subtypes. Conclusion PFGE protocol established in this paper and automated ribotyping are suitable for application in Citrobacter subtyping.

Optimization of Pulsed-field Gel Electrophoresis Procedure for Bacillus cereus

In order to develop a rapid and reliable method for B. cereus genotyping, factors influencing PFGE results, including preparation of bacterial cells embedded in agarose, lysis of embedded cells, enzymatic digestion of intact genomic DNA, and electrophoresis parameters allowing for reproducible and meaningful DNA fragment separation, were controlled. Optimal cellular growth （Luria-Bertani agar plates for 12-18 h） and lysis conditions （4 h incubation with 500 μg/mL lysozyme） produced sharp bands on the gel.

Antimicrobial resistance, genotypic characterization and pulsed-field gel electrophoresis typing of extended spectrum β-lactamases-producing clinical Escherichia coli strains in Macao, China

Background The rise of the production of CTX-M class extended spectrum β-lactamases （ESBLs） has been well documented in traveling countries but no data are found for Macao, an international travel city. The objectives of this study were to identify the antimicrobial resistance pattern, and determine the prevalence, genotype and clonal relationship of ESBLs in 209 clinical Escherichia coli strains from Macao, China. Methods Antimicrobial susceptibility test was performed to determine the resistance patterns of the isolates using the disk diffusion method with 17 antimicrobial agents. Phenotypic detection was screened and confirmed according to the Clinical and Laboratory Standards Institute. Genotypic characterization was detected by isoelectric focusing analysis, polymerase chain reaction and sequencing. The clonal relationship between the different ESBL isolates was studied by pulsed-field gel electrophoresis （PFGE）. Results Imipenem and meropenem exhibited 100% susceptible among 209 strains. Overall, 82.3%, 67.3%, 52.9%, 51.2% and 51.0% of the isolates displayed resistance to ampicillin, tetracylcline, ciprofloxacin, sulfamethoxazole trimethoprin and gentamycin. The prevalence rate of ESBLs was 30.1%. Antibiotic resistances were found to be significantly higher among the ESBL producing group compared to non-ESBL producing group. We detected CTX-M-14 to be the major genotypic characterization of ESBLs （76.2%）. Two strains showed indistinguishable patterns by PFGE. Conclusions The prevalence of antimicrobial resistance is alarming high in Macao. Antimicrobial resistance is significantly higher among the ESBL producing group. This study documented CTX-M-14 as the predominant ESBL type. Although indistinguishable pattern was found between two strains, it was too small to decide whether any of the investigated strains was epidemic. Our findings may be also pertinent for other geographic areas undergoing similar travel characteristics to understand the corresponding effects on bacterial populations.

阪崎肠杆菌脉冲场凝胶电泳分型的研究

目的对29株阪崎肠杆菌分离株和2株阪崎肠杆菌模式株进行脉冲场凝胶电泳(PFGE)分型研究。方法分别利用限制性内切酶XbaⅠ和SpeⅠ酶切阪崎肠杆菌染色体DNA进行PFGE分析,利用BioNumerics软件进行聚类分析。结果31株阪崎肠杆菌使用XbaⅠ和SpeⅠ酶切分别有30种PFGE带型。除ES004和ES005外,其他29株阪崎肠杆菌经XbaⅠ或SpeⅠ酶切后的PFGE条带之间均存在不同程度的差异。来源于同品牌同批次中不同包装的产品中的ES004和ES005分离株XbaⅠ和SpeⅠ酶切图谱完全一致(相似度为100%)。根据BioNumerics软件分析结果可知,除分离株ES004和ES005外,其他分离株的带型和样品来源之间无显著相关性。结论虽然XbaⅠ酶切的PFGE带型平均条带少于SpeⅠ,但对阪崎肠杆菌具有足够的分辨率。两种PFGE分型方法都可应用于阪崎肠杆菌的分子分型和溯源。

PFGE与16S rDNA对阪崎克罗诺杆菌分型的研究

研究脉冲场凝胶电泳(PFGE)与16S rDNA序列分析在阪崎克罗诺杆菌分型中的应用。对分离自不同来源的10株阪崎克罗诺杆菌进行16S rDNA序列扩增及测序,并构建系统发育树进行聚类分析。再利用限制性内切酶Xba I对这10株菌酶切后,进行PFGE电泳分型。16S rDNA序列分析显示,所有分离菌株与ATCC标准菌株在同一系统发育分支下,其序列相似性达到98.37%,并且不能进一步分型。而PFGE分型结果显示,10株分离菌株和2株标准菌株共分成11种PFGE基因型,说明PFGE的分型能力明显优于16S rDNA序列分析,部分菌株的基因型与分离来源具有一定的相关性,所以PFGE分型方法可用于阪崎克罗诺杆菌分子分型及溯源的研究。

恒场电泳检测DNA双链断裂及其应用

应用普通恒场琼脂糖凝胶电泳检测受γ射线照射后小鼠胸腺细胞ＤＮＡ双链断裂（ｄｓｂ）。在一定条件下，该法可以获得与脉冲凝胶电泳相似的结果。用此法观察了离体照射小鼠胸腺细胞ＤＮＡｄｓｂ修复作用，发现该法可以检测到ＤＮＡ“梯状”带，可做为一种研究细胞凋亡的新方法。

糖尿病性白内障晶状体蛋白二维电泳和质谱鉴定

目的应用二维电泳和质谱鉴定技术对糖尿病性白内障进行蛋白质组学研究,探讨蛋白质组学在糖尿病性白内障发病机制中的价值。方法取8例(8眼)1型糖尿病并发白内障晶状体组织,提取晶状体组织中蛋白质进行二维电泳,凝胶染色后,通过Gel Doc2000凝胶成像仪采集图像,利用PDQuest8.0凝胶分析软件对图像进行分析。使用基质辅助激光分析离子飞行时间质谱仪(MALAI-TOF-MS)对蛋白斑点进行鉴定。结果二维电泳图谱能检测到35～40个左右的蛋白斑点,大部分高丰度蛋白斑点相对分子质量处于(20～31)×103范围内,小部分低丰度蛋白斑点相对分子质量处于(35～45)×103范围内,等电点在5～9之间。利用MASCOT软件查询NCBInr数据库对2个蛋白斑点的肽质量指纹图谱进行了鉴定,分别为α-B晶状体蛋白和β-B1晶状体蛋白。结论二维电泳和质谱鉴定能够较好地分离和鉴定糖尿病性白内障晶状体蛋白质,为研究糖尿病性白内障形成机制提供了新的方法和途径。

人胃癌蛋白质组研究技术体系的建立

目的:对传统的双向凝胶电泳技术和计算机图像分析技术进行优化并将其应用于胃癌蛋白质组研究,提高实验数据分辨率和实验结果的可重复性。方法:提取胃癌组织中蛋白质,对以固相pH梯度为第一向双向电泳的关键因素与环节(如样品处理、电泳参数和凝胶浓度等)进行一系列优化。进一步采用双向凝胶电泳分离胃癌组和正常组总蛋白质,银染显色,并通过优化的计算机图像分析软件分析电泳图谱。结果:重复性实验结果显示同组样品在3次不同实验中所得蛋白质斑点数目相对标准差(变异系数平均值%):胃癌组和正常组分别为(23.00±10.11)、(20.33±9.90)。变异系数范围(%):胃癌组和正常组分别为3.80～60.10、2.70～56.89。同一蛋白质斑点在3次实验中等电点、分子量的相对标准差分别为(8.76±5.14)%,(13.00±4.22)%和(10.84±9.16)%。获得了优于传统实验方法的分辨率和可重复性的双向凝胶电泳图谱。结论:优化的双向凝胶电泳和图像分析技术适合于胃癌蛋白质组研究。

南美白对虾养殖系统中氨氧化菌多样性研究

[目的]为解决养殖水体的氨氮污染问题提供依据。[方法]研究南美白对虾养殖池塘底泥中的氨氧化菌的多样性和种群分布及其amoA基因的多样性,并与池塘周边土壤中的氨氧化菌群落结构进行比较。[结果]不同样品中氨氧化菌数量差异显著,其中池塘周边土壤中最多,达到4.5×105cfu/g,其次是池塘底泥和水体。来自土壤和底泥两个DNA样品均扩增到了预期长度(491 bp,517 bp)的amoA基因片段。底泥样品的DGGE条带数明显多于池塘周边土壤样品。土壤和底泥样品仅有1个共同的氨氧化菌种属,其序列与Nitrosospira sp.相似性达96%。池塘周边土壤的氨氧化菌种属序列与Nitrosospira sp.都有高度同源性。池塘底泥中Nitrosomonas sp.为氨氧化菌的优势菌群。[结论]构建的amoA基因克隆文库能较好反映南美白对虾养殖系统中氨氧化菌的多样性。

Molecular Characteristics of Neisseria meningitidis Isolated during an Outbreak in a Jail: Association with the Spread and Distribution of ST-4821 Complex Serogroup C Clone in China

Objective To characterize the meningococcal strains isolated from cases and close contacts with meningococcal disease associated with an outbreak in a jail in May 2010 by investigating the national distribution of hyperinvasive ST-4821 serogroup C clone associated with this outbreak. Methods The cases were described based on the clinical symptoms and laboratory results. Pharyngeal swabs were cultured for N. meningitidis from men in the jail. Meningococcal isolates were identified by serogrouping, pulsed-field gel electrophoresis （PFGE） and multilocus sequence typing （MLST）, respectively. Four hundred and sixteen serogroup C N. meningitidis strains were collected from 27 provinces between 2003 and 2010 for a nationwide survey and analyzed by PFGE and MLST. Results Three persons in a jail system were infected with invasive N. meningitidis serogroup C. All isolates tested had matching PFGE patterns and belonged to the multilocus sequence type （ST） 4821 clonal complex. All 47 N. meningitidis strains were identified from the pharyngeal swabs of 166 peoples in the jail, and 26 of them belonged to ST-4821 serogroup C clone, and 90.14% （375/416） serogroup C strains identified in the nationwide survey belonged to the ST-4821 complex. The ST-4821 serogroup C clone was spread nationwide, distributed in 24 provinces, especially in eastern provinces between 2003 and 2010. Conclusion Endemic transmission and carriage rate of ST-4821 serogroup C clone are high in this jail system. The ST-4821 serogroup C clone is spreading in China and nationwide distributed despite the existence of some effective vaccines.

Detection and Characterization of β-Lactam Resistance in Haemophilus parasuis Strains from Pigs in South China

To characterize the β-lactam resistance in veterinary clinical isolates of Haemophilus parasuis, 115 isolates were examined for the β-lactam resistance, the possession of β-lactamase, and the presence of β-lactamase genes. The genetic relationship among isolates was evaluated by pulsed-field gel electrophoresis （PFGE）. Overall, the commonly detected resistance phenotypes were resistant to ampicillin （26.09%）, penicillin （22.61%）, amoxicillin （21.74%）, cefazolin （14.78%）, cefaclor （12.17%）, and cefotaxime （6.96%）. These strains showed high minimal inhibitory concentration （MICs） to oxacillin. 20.87% strains produced β-lactamase, and 4.35% strains showed extended-spectrum b-lactamase （ESBL） phenotype. Moreover, 19 strains harboured bla genes including TEM-1 （n=5）, TEM-116 （n=10）, and ROB-1 （n=5）. Significantly, one strain possessed both TEM-1 and ROB-1, and displayed resistance to cefotaxime （MIC=8 mg L-1）. The epidemiological analysis of PFGE revealed high genetic diversity among bla-positive isolates. This work shows that TEM- and ROB-type β-lactamases are prevalent in H. parasuis isolates in China.

Characterization of Salmonella Enterica Serotype Typhimurium from Outpatients of 28 Hospitals in Henan Province in 2006

Objective To characterize the diarrheal patients with Salmonella typhimurium （S. typhimurium） infections and to set up the first baseline for S. typhimuri_um pulsed-field gel electrophoresis （PFGE） patterns in Henan province, thus laying a foundation for comprehensive surveillance of Salmonella in human as well as foods. Methods S. typhimurium isolates recovered from outpatients with diarrhea in Henan province from May to October of 2006 were characterized. Antimicrobial susceptibility tests of 8 antimicrobial agents and PFGE were carried out to analyze the S. typhimurium isolates. Results Twenty-four （0.9%） S. typhimurium isolates were identified from 2661 stool specimens of diarrheal cases. Eighty-eight percent of isolates showed resistance to at least one antimicrobial agent. The resistance to chloramphenicol （79%） was most common. Fifty-eight percent of isolates were resistant to ciprofloxacin. All the 14 ciprofloxacin-resistant isolates were resistant to more than five antimicrobial agents. Thirty-three percent of S. typhimurium isolates were resistant to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline （R-type ACSSuT）. Eight antimicrobia-resistant phenotypes were found among the 24 isolates in 16 PFGE patterns. Conclusion The rate of multidrug-resistant S. typhimurium is relatively high in S. typhimurium PFGE patterns of Henan province. Multidrug-resistant S. typhimurium should be considered a public health threat.

一起由阿贡纳沙门菌引起的食物中毒的病原学研究及溯源分析

目的 对引起2022年夏季绍兴市某区一起食物中毒事件的沙门菌进行血清型、耐药表型及分子分型分析,研究分析其相关性。方法 使用玻片凝集法对6株沙门菌进行血清分型;采用微量肉汤稀释法测定分离株对14种抗生素的耐药性;采用脉冲场凝胶电泳(PFGE)法对分离株进行分子分型和同源性分析,并与数据库中其他沙门菌比较。结果 6份患者肛拭子样本中检出1株阿贡纳沙门菌,7份从业人员肛拭子样本中检出2株阿贡纳沙门菌、1株婴儿沙门菌及1株阿姆德尼斯沙门菌,7份餐馆环境涂抹样本中检出1株阿贡纳沙门菌。对6株沙门菌进行脉冲场凝胶电泳分型,患者肛拭子样本、从业人员肛拭子样本、环境涂抹样本检出的3株阿贡纳沙门菌PFGE条带完全一致。6株分离菌株耐药谱相同。在绍兴市沙门菌PFGE数据库中未发现相同带型的菌株。结论 该事件是绍兴市首起由阿贡纳沙门菌感染引起的食物中毒事件,PFGE分型揭示分离株之间的相关性,为事件的分析和溯源提供了依据。

浙江省伤寒副伤寒沙门菌耐药及分子分型

目的了解浙江省伤寒副伤寒沙门菌耐药及分子分型特点,为伤寒副伤寒防制工作提供依据。方法采用WHO推荐的改良K-B纸片法,对209株甲型副伤寒沙门菌和5株伤寒沙门菌进行14种抗生素敏感性试验;运用脉冲场凝胶电泳分型(PFGE)方法对119株甲型副伤寒沙门菌进行分子分型及流行病学特征分析。结果209株甲型副伤寒沙门菌对红霉素、萘啶酸和利福平100%耐药,对阿米卡星100%敏感,对环丙沙星的敏感率仅为58%;5株伤寒沙门菌对红霉素和利福平100%耐药,对头孢噻吩、头孢噻肟、庆大霉素、环丙沙星、氨苄西林、阿米卡星和阿莫西林/克拉维酸100%敏感;不同地区间的甲型副伤寒沙门菌对大部分药物敏感率一致,只有多西环素存在较大差异;119株甲型副伤寒沙门菌共产生20种PFGE带型,并主要集中在2种同源性较高的型别。结论2008年浙江省伤寒副伤寒以甲型副伤寒菌株为优势株,不同地区甲型副伤寒沙门菌对大部分抗生素敏感率一致;高发地区(台州)的菌株PFGE带型比较分散,可能与菌株变异有关。

单核细胞增生李斯特菌脉冲场凝胶电泳分型技术的内切酶质量评估

目的对单核细胞增生李斯特菌脉冲场凝胶电泳(PFGE)分子分型的相关影响因素进行分析和探讨,并建立该菌的PFGE双酶切技术。方法分别利用限制性内切酶AscⅠ和ApaⅠ对1/2a、1/2b、1/2c、3a和4b型的单核细胞增生李斯特菌进行PFGE分析(包括1种品牌的AscⅠ和5种品牌的ApaⅠ),通过指纹图谱的比较,优化各种内切酶的实验参数。结果AscⅠ酶切产生11条左右清晰的DNA指纹图谱条带,ApaⅠ酶切产生的条带数量与之相似,但其中约10%的菌株(1/2a、1/2b血清型)均产生消化不完全的DNA片段。同时用5种品牌的ApaⅠ酶消化,并改变消化温度、提高酶浓度、延长酶消化时间均无法克服此现象。结论AscⅠ酶适于1/2a、1/2b、1/2c、3a和4b血清型的单核细胞增生李斯特菌的分型,但ApaI酶适于多数1/2a和1/2b血清型的分型。

阪崎肠杆菌脉冲场凝胶电泳分子分型分析

目的:对19株阪崎肠杆菌分离株和1株阪崎肠杆菌标准菌株进行脉冲场电泳(PFGE)分型分析。方法:利用限制性内切酶Xba1对阪崎肠杆菌基因组DNA进行酶切和PFGE分析,并利用BioNumerics软件进行聚类分析。结果:20株阪崎肠杆菌PFGE带型表现出多样性,在可分成16种PFGE型。BQ9、BQ11、BQ12、BQ13和BQ14相似性系数为100%,属于同一PFGE型别。BQ9、BQ11、BQ13和BQ14是同一生产厂家不同品牌的四种产品。其余各分离株和标准菌株的酶切图谱各不相同,相似性系数均在90%以下,被确定为不同PFGE型。结论:PFGE酶切带型与菌株来源有相关性,可应用于阪崎肠杆菌的分子分型和溯源。

2005―2014年佛山市霍乱弧菌病原学特征分析

目的分析比较分离自病例与环境的霍乱弧菌的病原学特征,为霍乱防治工作提供实验依据。方法选取分离自霍乱病例（患者和密切接触者）和环境（水体和海水产品）的霍乱弧菌,采用血清学、药物敏感性试验、多重PCR检测毒力基因和脉冲场凝胶电泳（PFGE）分子分型等方法,分析不同来源的霍乱弧菌在菌型分布、药物敏感性、毒素基因携带以及分子分型方面的异同。结果 2005―2014年佛山市共分离各类来源霍乱弧菌39株,其中病例来源菌株6株、环境来源菌株33株。2种来源菌株的菌型构成均以O1群稻叶型为主;病例来源菌株以产毒株为主,ctx A毒力基因携带率为66.67%,携带8种毒力基因阳性率为50.00%;环境来源菌株以非产毒株为主,ctx A毒力基因携带率为21.21%,携带8种毒力基因阳性率为6.06%。药物敏感性试验结果显示,不同来源的菌株对萘啶酸均有较高的耐药率（〉50.00%）;病例来源菌株对复方新诺明的耐药率（83.33%）高于环境来源菌株（18.18%）。PFGE聚类分析结果显示,39株霍乱弧菌可分为35个PFGE型别,菌株PFGE带型相似度为72.5%~100.0%。结论佛山市霍乱弧菌血清型、毒力基因和遗传特征复杂多样,霍乱防控形势严峻,需要加强霍乱弧菌常规药敏和菌株型别变异情况的监测。

Prevalence, genetic diversity, and antimicrobial susceptibility profiles of Staphylococcus aureus isolated from bovine mastitis in Zhejiang Province, China

This study was conducted to determine genetic diversity and antimicrobial susceptibility profiles of Staphylococcus aureus recovered from bovine mastiffs in Zhejiang Province, China. Out of 3178 quarter milk samples from 846 lactating cows, among which 459 cows （54.3%） were found HMT positive, 890 quarters （28%） were found having subclinical mastitis. From 75 representative S. aureus isolates, 16 distinct types were identified by pulsed-field gel electrophoresis （PFGE）. Four major PFGE types （A, B, C, and D） accounted for 82.7% of all isolates, and type A （41.3%） was observed in multiple herds across the studied areas. Each region was found to have a predominant type： Hangzhou type A （64.1%）, Ningbo type C （34.5%） and type B （23.1%）, Jinhua type D （53.3%）, and Taizhou type C （62.5%）. Results of antimicrobial susceptibility tests showed that 90.7% of the isolates were resistant to at least one antimicrobial. Resistance to penicillin and ampicillin （77.3%）, tetracycline （60.0%）, or erythromycin （48.0%） was observed. The bacteria resistant to multiple antibiotics such as penicillin, ampicillin, tetracycline, and erythromycin were commonly found. The information obtained from this study is useful for designing specific control programs for bovine S. aureus mastitis in this region.

Drug-resistant gene based genotyping for Acinetobacter baumannii in tracing epidemiological events and for clinical treatment within nosocomial settings

Background Acinetobacter baumannfi has emerged as an important pathogen related to serious infections and nosocomial outbreaks around the world. However, of the frequently used methods, pulsed-field gel electrophoresis （PFGE） and amplified fragment length polymorphism （AFLP） in Acinetobacter baumannfi genotyping lack the direct molecular proof of drug resistance. This study was conducted to establish a typing method based on drug resistant gene identification in contrast to traditional PFGE and AFLP in the period of nosocomial epidemic or outbreak. Methods From January 2005 to October 2005, twenty-seven strains of Acinetobacter species from Intensive Care Units, the Second Affiliated Hospital in Ningbo were isolated, including both epidemic and sporadic events. Susceptibility test, PFGE, AFLP and drug resistance gene typing （DRGT） were carried out to confirm the drug resistance and analyze the genotyping, respectively. PFGE was used as a reference to evaluate the typeability of DRGT and AFLP. Results Twenty-seven strains of Acinetobacter displayed multiple antibiotic resistance and drug resistant genes, and β-1actamase genes were detected in 85.2% strains. The result of DRGT was comparable to PFGE in Acinetobacter strains with different drug resistance though a little difference existed, and even suggested a molecular evolution course of different drug-resistant strains. AFLP showed great polymorphism between strains and had weak ability in distinguishing the drug resistance. Conclusion Compared to AFLP and PFGE, DRGT is useful to analyze localized molecular epidemiology of nosocomial infections and outbreaks, which would benefit clinical diagnosis and therapy.

Antimicrobial resistance and molecular epidemiological characteristics of clinical isolates of Staphylococcus aureus in Changsha area

Background Increasing prevalence of Staphylococcus aureus （S. aureus）, particularly methicillin-resistant S. aureus （MRSA） has been reported in China. In this study, we investigated the drug resistance characteristic, genetic background, and molecular epidemiological characteristic of S. aureus in Changsha. Methods Between January 2006 and December 2008, 293 clinical isolates of S. aureus were collected from 11 hospitals in Changsha and identified by the Vitek-2 system. All the isolates were verified as MRSA by PCR amplification of both femA and mecA genes. K-B disk method was used to test drug sensitivity of S. aureus to antibiotics. Pulsed-field gel electrophoresis （PFGE） was performed for genotypic and homologous analysis of 115 isolates randomly selected from the original 293 clinical S. aureus isolates. Results S. aureus was highly resistant to penicillin, ampicillin, erythromycin, and clindamycin with resistant rates of 96.6%, 96.6%, 77.1%, and 67.2% respectively. All the isolates were susceptible to tecoplanin, vancomycin, and linezolid. MRSA accounted for 64.8% （190/293） of all the S. aureus strains. The 115 S. aureus isolates were clustered into 39 PFGE types by PFGE typing, with 13 predominant patterns （designated types A to M） accounting for 89 isolates. The most prevalent PFGE type was type A （n=56, 48.7%） and 100.0% of type A strains were MRSA. PFGE type A included 13 subtypes, and the most prevalent subtype was subtype A1 （46.4%, 26/56）. Strains with PFGE type A were isolated from eight hospitals （8/11）, and both subtypes A1 and A4 strains were isolated in a university hospital. Conclusions Clinical isolates of S. aureus in Changsha were resistant to multiple traditional antibiotics. There was an outbreak of PFGE type A MRSA in this area and the A1 subtype was the predominant epidemic clone. Dissemination of the same clone was an important reason for the wide spread of MRSA.