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Establishment and Comparison of pulsed-field gel electrophoresis,Multiple-locus Variable Number Tandem Repeat Analysis and Automated Ribotyping Methods for Subtyping of Citrobacter Strains

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Biomedical and Environmental Sciences 2012年第6期25卷 653-662页

作者： ZHANG Xiao Ai BAI Xue Mei YE Chang Yun REN Zhi Hong XU Jian Guo State Key Laboratory for Infectious Disease Prevention and Control National Institute for Communicable Disease Control and Prevention Chinese Center for Disease Control and Prevention Beijing 102206 China Beijing Center for Disease Prevention and Control Beijing 100013 China

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.

关键词： Citrobacter pulsed-field gel electrophoresis Multiple-locus variable number tandem repeatanalysis Ribotyping Molecular subtyping

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

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Chinese Medical Journal 2011年第17期000卷 2701-2707页

作者： YE Qian-hong LAU Ying LIANG Bin TIAN Su-fei School of Health Sciences Macao Polytechnic Institute Macao Special Administrative Region (SAR) China School of Health Sciences China Medical University Shenyang Liaoning 110001 China Department of Laboratory Medicine First Affiliated Hospital China Medical University Shenyang Liaoning 110001 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 eff

关键词： antimicrobial resistance Escherichia coli pulsed-field gel electrophoresis extended spectrum β-lactamases Macao

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

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2009年第10期10卷 753-760页

作者： Jian-ping LI Hai-jian ZHOU Lin YUAN Ting HE Song-hua HU Department of Veterinary Medicine College of Animal Science Zhejiang University Hangzhou 310029 China National Institute for Communicable Disease Control and Prevention State Key Laboratory for Infectious Disease Prevention and Control Chinese Center for Disease Control and Prevention Beijing 102206 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.

关键词： Staphylococcus aureus Bovine mastitis Antimicrobial resistance Genetic diversity pulsed-field gel electrophoresis

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

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Journal of Integrative Agriculture 2012年第1期11卷 116-121页

作者： GUO Li-li ZHANG Jian-min XU Cheng-gang REN Tao ZHANG Bin CHEN Ji-dang LIAO Ming Key Laboratory of Animal Disease Control and Prevention Ministry of Agriculture/Key Laboratory of Zoonoses Prevention and Control of GuangdongCollege of Veterinary MedicineSouth China Agricultural UniversityGuangzhou 510642P.R.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.

关键词： Haemophilus parasuis β-lactamase genes resistance pulsed-field gel electrophoresis

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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

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Biomedical and Environmental Sciences 2013年第5期26卷 331-337页

作者： ZHANG Ji ZHOU Hai Jian XU Li HU Guang Chun ZHANG Xue Hua XU Sheng Ping LIU Zun Yu SHAO Zhu Jun Jinan Municipal Center for Disease Control and Prevention State Key Laboratory for Infectious Disease Prevention and Control National Institute for Communicable Disease Control and PreventionChinese Center for Disease Control and Prevention

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.

关键词： pulsed-field gel electrophoresis Multilocus sequence typing Neisseria meningitidis ST-4821 Serogroup C

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

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Chinese Medical Journal 2012年第13期125卷 2289-2294页

作者： ZOU Ming-xiang ZHOU Rong-rong WU Wen-jun ZHANG Ning-jie LIU Wen-en HU Fu-ping FAN Xue-gong Department of Clinical LaboratoryXiangya Hospital Central South University ChangshaHunan 410008 China Department of Infectious DiseasesXiangya Hospital Central South University ChangshaHunan 410008 China Institute of Antibiotics Huashan Hospital Fudan UniversityShanghai 200040 China

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.

关键词： staphylococcus aureus antimicrobial resistance pulsed-field gel electrophoresis molecular epidemiological characteristics

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

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Chinese Medical Journal 2009年第3期122卷 301-306页

作者： JIN Hui XU Xiao-min MI Zu-huang MOU Yi LIU Pei Department of Epidemiology and Health Statistics Southeast University Nanjing Jiangsu 210009 China Department of Microbiology Laboratory Second Affiliated Hospital Ningbo Zhejiang 315010 China Institute of Gene and Clone Technology Wuxi Jiangsu 214026 China China West Second University Hospital Sichuan University Chengdu Sichuan 610041 China

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.

关键词： Acinetobacter baumannii drug-resistance gene typing pulsed-field gel electrophoresis amplified fragment length polymorphism fingerprinting

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

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Biomedical and Environmental Sciences 2009年第2期22卷 136-140页

作者： MIN ZHU ZHI-QIANG XIE LI-SHI ZHANG SHENG-LI XIA WEI-ZHONG YANG LU RAN AND ZI-JUN WANG Huaxi School of Public Health Sichuan University Chengdu 610041 Sichuan China Henan Center for Disease Control and Prevention Zhengzhou 450016 Henan China Chinese Center for Disease Control and Prevention Beijing 100050 China

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.

关键词： Salmonella typhimurium Outpatients Ciprofloxacin Multidrug-Resistant pulsed-field gel electrophoresis

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中美有机磷杀虫药剂抗性库蚊复合种群酯酶基因扩增的研究(英文)

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Insect Science 1996年第2期3卷 165-171页

作者：乔传令中国科学院动物研究所北京100080

用脉冲电场凝胶电泳技术,从北京和美国加州的有机磷杀虫药剂抗性库蚊复合品系中分离出一条49kb的酯酶基因扩增片段。该片段可能是一个完整的扩增单元,也是迄今分离出的最大的一条昆虫酯酶基因扩增片段,扩增基因结构的研究正在进行之中... 详细信息

用脉冲电场凝胶电泳技术,从北京和美国加州的有机磷杀虫药剂抗性库蚊复合品系中分离出一条49kb的酯酶基因扩增片段。该片段可能是一个完整的扩增单元,也是迄今分离出的最大的一条昆虫酯酶基因扩增片段,扩增基因结构的研究正在进行之中。本文还重点讨论了DNA大片段的制备方法和脉冲电场电泳分离技术。

关键词： Culex pipiens mosquitoes pulsed-field gel electrophoresis insecticide resistance

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Dissemination of carbapenem-resistant Acinetobacter baumannii in a Chinese university hospital and analysis of infected patients' prognosis

Dissemination of carbapenem-resistant Acinetobacter baumanni...

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第十届全国化疗药理暨抗感染药理高峰论坛

作者：周庆涛贺蓓姚贝刘振英张捷北京大学第三医院呼吸科北京大学第三医院检验科

Objectives:To assess the genetic relatedness of clinical isolates of carbapenem-resistant *** (resistant to both imipenem and meropenem) during January 2007 to March 2008 in Peking University Third Hospital in order to take certain measures to decrease the isolates;to investigate the characteristics of patients with carbapenem-resistant *** colonization or infection and to evaluate antibiotic treatment for health care-associated infections caused by carbapenem-resistant ***. Methods:The medical records of patients with carbapenem-resistant *** colonization or infection were *** susceptibilities of the isolates were determined by the standardized disk-diffusion method and the clonal relationship of the isolates was analyzed by pulsed-field gel electrophoresis. Results:A total of 49 carbapenem-resistant *** strains were isolated from the 49 patients hospitalized during the study period and pulsed-field gel electrophoresis typing yielded 7 different patterns.A total of 45(91.8%) genotyped strains showed clonal *** most frequently identified predisposing factors were intensive care unit stay,invasive procedures,and hypoalbuminemia. Chronic obstructive pulmonary disease(12 cases) and cerebrovascular disease(10 cases) were the most common comorbid *** mortality of patients with carbapenem-resistant *** infection was 38.1%(8 of 21 patients),and the acute physiology and chronic health evaluationⅡscore was found to be the independent risk factor for mortality(p=0.02,OR=1.7,95%CI 1.1-2.5).Combination therapy regimens had higher success rates than monotherapy(11/13,84.6%vs.3/17,17.6%),especially cefoperazone-sulbactam plus levofloxacin. Conclusions:There has been clonal spread of carbapenem-resistant *** strains among patients in our hospital since *** risk factors for acquisition of carbapenem-resistant *** were intensive care unit stay,invasive procedures,hypoalbumine

关键词： Acinetobacter baumannii Combination therapy Drug resistance Health care-associated infections pulsed-field gel electrophoresis

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