小肠结肠炎耶尔森菌生物学特性和分子分型研究
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摘要
【研究背景】
小肠结肠炎耶尔森菌(Yersinia enterocolitica)是一种革兰氏阴性无芽孢杆菌,属于耶尔森菌属,是该菌属的三大致病菌之一。小肠结肠炎耶尔森菌广泛存在于自然环境中,主要通过污染食物进入人体。人感染小肠结肠炎耶尔森菌后,主要表现为胃肠道症状,如呕吐、腹痛、腹泻等,多具有自限性。但是,部分患者可出现一系列严重的并发症,如心内膜炎、反应性关节炎、组织脓肿等,甚至引起败血症导致死亡。在欧洲,由该菌和假结核耶尔森菌引起的耶氏菌病在常见的人畜共患病中占第四位；在美国,由该菌引起的疾病每年发病约11.7万例；在我国,文献可查的小肠结肠炎耶尔森菌暴发有两次。该菌可分为6个生物型(1A,1B和2～5)和60个血清型,主要通过检测ail、ystA、ystB、yadA和virF5个毒力基因来判断其致病性。小肠结肠炎耶尔森菌对青霉素和一代头孢天然耐药,对其他种类的抗菌药物具有不同程度的耐药率。脉冲场凝胶电泳(Pulsed Field Gel Electrophoresis,PFGE)分型是现阶段小肠结肠炎耶尔森菌分子分型最常用的方法,主要用于菌株的溯源。
山东省自2006年开始,设立高密市和五莲县两个监测点对小肠结肠炎耶尔森菌开展监测,至2011年共从鸡、牛、羊、猪等动物粪便和生熟肉、苍蝇中分离到小肠结肠炎耶尔森菌237株。这些菌株的动力、生物型、血清型、毒力基因和抗菌药物敏感性等生物学特性尚缺乏系统性的研究分析,分子分型也尚未开展。
【研究目的】
1、系统分析分离到的小肠结肠炎耶尔森菌的动力、生物型、血清型、毒力基因和抗菌药物敏感性等生物学特性及其时间、地区和宿主分布特点。
2、对分离到的小肠结肠炎耶尔森菌进行PFGE分型,了解菌株的亲缘关系,探讨表型分型和PFGE分型的相互关系。
3、初步建立山东省小肠结肠炎耶尔森菌的基础数据库,为该菌的长期监测奠定基础,为耶氏菌病的预测、预警提供技术支持。
【研究方法】
1、动力试验采用半固体穿刺法,生物分型根据生化反应进行判断,血清分型采用玻片凝集法,毒力基因检测采用PCR法,抗菌药物敏感性检测采用VITEK2COMPACT系统和AST-GN16药敏卡。
2、PFGE分型参照PulseNet China推荐的标准方法进行,电泳图谱处理及聚类分析采用BioNumerics6.1软件。
3、资料的录入、整理和简单分析采用Excel2007软件,数据的统计学分析采用SPSS16.0软件。
【研究结果】
1、动力为“中”的菌株占4.2%(10/237),动力为“差”的菌株占3.0%(7/237),其余菌株动力良好。动力为“中”或“差”的菌株在各年份、各地区和多种宿主中均有分布。
2、98.7%(234/237)的菌株为生物1A型；其余3株菌为生物4型,均从2006年高密市的猪粪样本中分离到。
3、49.8%(118/237)的菌株凝出相应血清型,以0：5(21.9%,52/237)和O：8(21.1%,50/237)型为主。2011年可分血清型最多(5种)；高密分离株0：5型最多(23.9%,39/163),五莲分离株0：8型最多(33.8%,25/74)；各宿主分离株均以O：5和O：8型为主。
4、ystB基因的阳性率最高,为81.4%(193/237)；ail和ystA基因仅在3株菌中检测到,阳性率为1.3%(3/237)；未检测到yadA和virF基因。ystB基因阳性菌株在不同时间、地区和宿主中均为优势菌株。
5、共有235株菌获得抗菌药物敏感性试验的结果。菌株对头孢曲松、头孢吡肟、阿米卡星、庆大霉素和替加环素的敏感率最高,为100%；对氨苄西林的耐药率最高,为94.5%(222/235)。菌株的多重耐药率为12.3%(29/235)。2007和2011年分离株对呋喃妥因的敏感情况有差异(P<0.05)；高密市分离株对复方新诺明的耐药率(10.4%,17/163)高于五莲县分离株(1.4%,1/72)(P<0.05)；毒力基因A型(ail-ystA-ystB+yadA-virF-)菌株对氨苄西林、阿莫西林-克拉维酸、头孢唑林、头孢西丁的耐药率和对呋喃妥因的中介耐药率(依次为99.0%,190/192；89.6%,172/192；94.8%,182/192；80.2%,154/192；94.8%,182/192)高于毒力基因B型(ail-ystA-ystB-yadA-virF-)菌株(依次为72.5%,29/40；65.0%,26/40；67.5%,27/40；42.5%,17/40；65.0%,26/40)(P<0.05)。
6、237株菌中的231株被分为144个PFGE型别,相似性系数在61.1%～100.0%,无明显优势型别。五莲分离株的PFGE聚集性高于高密分离株,含7株及以上菌株的PFGE型别均出现在五莲县。其余6株菌未获得PFGE条带。144个PFGE型别中有15个型别在中国疾控中心数据库中可找到相同或相近的型别。
7、17株动力为“中”或“差”的菌株中的14株形成3个聚类簇；52株0：5血清型菌株中的31株形成4个聚类簇,50株0：8血清型菌株中的19株形成1个聚类簇；41株毒力基因B型(ail-ystA-ystB-yadA-virF-)菌株中的20株形成1个聚类簇；药敏型别在PFGE聚类中分布分散,无特异性。
8、动力、生物分型、血清分型、毒力基因分型、药敏分型和PFGE分型的Simpson指数分别为0.1362、0.0251、0.6564、0.3081、0.7214和0.9982。
[结论]
1、高密市和五莲县的小肠结肠炎耶尔森菌存在动力突变株；菌株几乎全为生物1A型；血清型以0：5和0：8型为主,存在地区差异；菌株携带的毒力基因主要为ystB基因,无传统致病性菌株；耐药谱广泛,大部分菌株对青霉素类和一、二代头孢耐药,存在一定的多重耐药现象,vstB基因阳性的生物1A型菌株与ystB基因阴性的生物1A型菌株耐药性不同。
2、高密市和五莲县的小肠结肠炎耶尔森菌存在多克隆系,基因多态性明显,部分克隆株长期存在,五莲地区可能存在相对优势克隆系；部分山东分离株可能与其他省份菌株存在联系。
3、小肠结肠炎耶尔森菌的PFGE分型可能与其动力有一定相关,与生物型、血清型、毒力基因型和药敏型别不具有相关性。
4.PFGE具有强大的分型能力,但在菌株溯源时需与表型分型联合使用。
[Background]
Yersina enterocolitica (Y. enterocolitica) is a gram-negative budless bacillus, which belongs to Yersina genus and is one of the three pathogenic bacteria for humans and animals of this genus. It distributes widely in the natural environment, and mainly infects humans by contaminated food. Y. enterocolitica infections are mainly characterized with gastrointestinal symptoms, such as vomit, abdominal pain and diarrhea, which are usually self-limiting. However, the infection could induce serious complications, such as endocarditis, reactive arthritis, abscess and fatal septicemia. Yersiniosis, which is caused by Y. enterocolitica and Y. pseudotuberculosis, is the fourth most commonly reported zoonosis in Europe. There are about117,000infections caused by Y. enterocolitica annually in America. Two outbreaks of Y. enterocolitica infection were reported in literatures in China. It has been differentiated into six biotypes (1A,1B, and2-5) and60serotypes. The pathogenicity of Y. enterocolitica is usually determined by testing five virulence genes, which are ail, ystA, ystB, yadA and virF. Y. enterocolitica is resistant to penicillins and first generation cephalosporins naturally, and has different resistant rates to other antimicrobial agents. Pulsed field gel electrophoresis (PFGE) typing is the most frequently used method for Y. enterocolitica molecular typing nowadays and is mainly used for tracing to the strain's source.
In2006, Shandong Province set up two surveillance points, Gaomi City and Wulian County, for Y. enterocolitica monitoring. Until the end of2011,237strains of Y. enterocolitica had been isolated from meat, flies and feces of chickens, swines, sheeps, cattles and other animals. The motility, biotype, serotype, virulence gene and antimicrobial agent susceptibility of these strains had not been analyzed systematically, and the molecular typing had also not been studied.
[Objectives]
1. To systematically analyze the motility, biotype, serotype, virulence gene and antimicrobial agent susceptibility of the Y. enterocolitica isolates and the distribution of time, geography and host for each biological characteristic.
2. To classify the Y. enterocolitica isolates by PFGE, in order to explore the genetic relationship of these strains and discuss the relations between phenotyping and PFGE typing.
3. To establish a Y. enterocolitica database of Shandong Province, in order to lay a foundation for the future long-term surveillance and provide technical support for the prediction and alert of yersiniosis.
[Methods]
1. Motility was tested by stabbing culture in semi-solid medium. Biotype was determined by the results of biochemical reactions. Serotype was determined by serum agglutination test on a glass slide. Virulence gene was tested by polymerase chain reaction. Antimicrobial agent susceptibility was tested by VITEK2COMPACT system and AST-GN16card.
2. PFGE typing was operated according to the standard method recommended by PulseNet China. BioNumerics6.1was used for PFGE spectrum processing and clustering analysis.
3. Excel2007was used for data entering, compilation and simple analysis. SPSS16.0was used for data statistic analysis.
[Results]
1. Among the strains,4.2%(10/237) had medium-active motility,3.0%(7/237) had poor-active motility, and the others all had well-active motility. The strains of medium-and poor-active motility were isolated from most kinds of hosts from the two districts every year.
2. Two hundred and thirty-four of the237strains (98.7%) were biotype1A, and the other three strains were all biotype4, which were isolated from swine feces samples in Gomi City, in2006.
3. One hundred and eighteen of the237strains could be classified by the antiserum, and O:5(21.9%,52/237) and0:8(21.1%,20/237) were the dominant serotypes. All the five serotypes which could be calssified were tested in the strains isolated in2011. Gaomi isolates were dominated by O:5serotype (23.9%,39/163) while Wulian isolates were dominated by O:8serotype (33.8%,25/74); isolates from each kind of hosts were all dominated by O:5and0:8serotypes.
4. The positive rate of ystB gene was the highest, which was81.4%(193/237); ail and ystA gene were found only in three strains, and the positive rate was1.3%(3/237) each; yadA and virF gene were negative in all the strains. The ystB gene positive strains were dominant in each year, either district and each kind of hosts.
5. Two hundred and thirty-five strains were tested for antimicrobial agent susceptibility. All the strains (235strains) were sensitive to ceftriaxone, cefepime, amikacin, gentamicin and tigecycline; the resistant rate to ampicillin was highest, which was94.5%(222/235). The multiple resistant rate was12.3%(29/235). The susceptibility to nitrofurantoin between2007and2011was significantly different (P <0.05); the resistant rate to trimethoprim/sulfamethoxazole between Gaomi City (10.4%,17/163) and Wulian County (1.4%,1/72) was significantly different (P<0.05); the resistant rate to ampicillin, amoxicillin/clavulanic acid, cefazolin and cefoxitin and the intermediate resistant rate to nitrofurantoin of virulence genotype A (ail-ystA-ystB+yadA+virF-) strains, which was99.0%(190/192),89.6%(172/192),94.8%(182/192),80.2%(154/192) and94.8%(182/192) respectively, was significantly higher than that of virulence genotype B (ail-ystA-ystB-yadA-virF-) strains (P<0.05), which was72.5%(29/40),65.0%(26/40),67.5%(27/40),42.5%(17/40) and65.0%(26/40) respectively.
6. Two hundred and thirty-one of the237strains were divided into144PFGE types with no one significantly dominant, and the similarity was61.1%to100.0%. The clustering of strains isolated from Wulian County was more obvious than that from Gaomi City, and the PFGE types, which had seven or more than seven strains, were all in Wulian County. The other six strains couldn't be classified by PFGE. Fifteen of the144PFGE types could find their same or similar types in the database of Chinese center for disease control and prevention.
7. Fourteen of all the17medium-or poor-active motility strains were grouped into three clusters. Thirty-one of all the52O:5serotype strains were grouped into four clusters. Nineteen of all the50O:8serotype strains were grouped into one cluster. Twenty of all the41virulence genotype B (ail-ystA-ystB-yadA-virF-) strains were grouped into one cluster. The antimicrobial agent susceptibility types were distributed irregularly in the PFGE clustering.
8. The Simpson's diversity index of motility, biotyping, serotyping, virulence genotyping, antimicrobial agent susceptibility typing and PFGE typing was0.1362,0.0251,0.6564,0.3081,0.7214and0.9982respectively.
[Conclusions]
1. The Y. enterocolitica strains isolated from Gaomi City and Wulian County had motility mutant strains. Almost all the strains isolated were biotype1A. The serotypes were dominated by O:5and O:8and had a difference between the two districts. The main virulence gene that the strains had was ystB and there was no pathogenic strain traditionally defined. The isolates were resistant to many antimicrobial agents, but most of the strains were resistant to penicillins and first and second generation cephalosporins. There were multiple resistant strains. The antimicrobial agent susceptibility of ystB-positive biotype1A strains were significantly different to that of ystB-negative biotype1A strains.
2. The Y. enterocolitica strains isolated from Gaomi City and Wulian County had many clones, and the gene heterogeneity was obvious. Some of the clones existed persistently. There might be relative dominant clones in Wulian County. Some of the clones might have relations with strains from other provinces.
3. The PFGE types of Y. enterocolitica might have relations with motility, but no relations with biotypes, serotypes, virulence genotypes or antimicrobial agent susceptibility types were observed.
4. PFGE was a typing method with high discrimination power, but a combined use with phenotyping was necessary in microbial source tracking
小肠结肠炎耶尔森菌(Yersinia enterocolitica)是一种革兰氏阴性无芽孢杆菌,属于耶尔森菌属,是该菌属的三大致病菌之一。小肠结肠炎耶尔森菌广泛存在于自然环境中,主要通过污染食物进入人体。人感染小肠结肠炎耶尔森菌后,主要表现为胃肠道症状,如呕吐、腹痛、腹泻等,多具有自限性。但是,部分患者可出现一系列严重的并发症,如心内膜炎、反应性关节炎、组织脓肿等,甚至引起败血症导致死亡。在欧洲,由该菌和假结核耶尔森菌引起的耶氏菌病在常见的人畜共患病中占第四位；在美国,由该菌引起的疾病每年发病约11.7万例；在我国,文献可查的小肠结肠炎耶尔森菌暴发有两次。该菌可分为6个生物型(1A,1B和2～5)和60个血清型,主要通过检测ail、ystA、ystB、yadA和virF5个毒力基因来判断其致病性。小肠结肠炎耶尔森菌对青霉素和一代头孢天然耐药,对其他种类的抗菌药物具有不同程度的耐药率。脉冲场凝胶电泳(Pulsed Field Gel Electrophoresis,PFGE)分型是现阶段小肠结肠炎耶尔森菌分子分型最常用的方法,主要用于菌株的溯源。
山东省自2006年开始,设立高密市和五莲县两个监测点对小肠结肠炎耶尔森菌开展监测,至2011年共从鸡、牛、羊、猪等动物粪便和生熟肉、苍蝇中分离到小肠结肠炎耶尔森菌237株。这些菌株的动力、生物型、血清型、毒力基因和抗菌药物敏感性等生物学特性尚缺乏系统性的研究分析,分子分型也尚未开展。
【研究目的】
1、系统分析分离到的小肠结肠炎耶尔森菌的动力、生物型、血清型、毒力基因和抗菌药物敏感性等生物学特性及其时间、地区和宿主分布特点。
2、对分离到的小肠结肠炎耶尔森菌进行PFGE分型,了解菌株的亲缘关系,探讨表型分型和PFGE分型的相互关系。
3、初步建立山东省小肠结肠炎耶尔森菌的基础数据库,为该菌的长期监测奠定基础,为耶氏菌病的预测、预警提供技术支持。
【研究方法】
1、动力试验采用半固体穿刺法,生物分型根据生化反应进行判断,血清分型采用玻片凝集法,毒力基因检测采用PCR法,抗菌药物敏感性检测采用VITEK2COMPACT系统和AST-GN16药敏卡。
2、PFGE分型参照PulseNet China推荐的标准方法进行,电泳图谱处理及聚类分析采用BioNumerics6.1软件。
3、资料的录入、整理和简单分析采用Excel2007软件,数据的统计学分析采用SPSS16.0软件。
【研究结果】
1、动力为“中”的菌株占4.2%(10/237),动力为“差”的菌株占3.0%(7/237),其余菌株动力良好。动力为“中”或“差”的菌株在各年份、各地区和多种宿主中均有分布。
2、98.7%(234/237)的菌株为生物1A型；其余3株菌为生物4型,均从2006年高密市的猪粪样本中分离到。
3、49.8%(118/237)的菌株凝出相应血清型,以0：5(21.9%,52/237)和O：8(21.1%,50/237)型为主。2011年可分血清型最多(5种)；高密分离株0：5型最多(23.9%,39/163),五莲分离株0：8型最多(33.8%,25/74)；各宿主分离株均以O：5和O：8型为主。
4、ystB基因的阳性率最高,为81.4%(193/237)；ail和ystA基因仅在3株菌中检测到,阳性率为1.3%(3/237)；未检测到yadA和virF基因。ystB基因阳性菌株在不同时间、地区和宿主中均为优势菌株。
5、共有235株菌获得抗菌药物敏感性试验的结果。菌株对头孢曲松、头孢吡肟、阿米卡星、庆大霉素和替加环素的敏感率最高,为100%；对氨苄西林的耐药率最高,为94.5%(222/235)。菌株的多重耐药率为12.3%(29/235)。2007和2011年分离株对呋喃妥因的敏感情况有差异(P<0.05)；高密市分离株对复方新诺明的耐药率(10.4%,17/163)高于五莲县分离株(1.4%,1/72)(P<0.05)；毒力基因A型(ail-ystA-ystB+yadA-virF-)菌株对氨苄西林、阿莫西林-克拉维酸、头孢唑林、头孢西丁的耐药率和对呋喃妥因的中介耐药率(依次为99.0%,190/192；89.6%,172/192；94.8%,182/192；80.2%,154/192；94.8%,182/192)高于毒力基因B型(ail-ystA-ystB-yadA-virF-)菌株(依次为72.5%,29/40；65.0%,26/40；67.5%,27/40；42.5%,17/40；65.0%,26/40)(P<0.05)。
6、237株菌中的231株被分为144个PFGE型别,相似性系数在61.1%～100.0%,无明显优势型别。五莲分离株的PFGE聚集性高于高密分离株,含7株及以上菌株的PFGE型别均出现在五莲县。其余6株菌未获得PFGE条带。144个PFGE型别中有15个型别在中国疾控中心数据库中可找到相同或相近的型别。
7、17株动力为“中”或“差”的菌株中的14株形成3个聚类簇；52株0：5血清型菌株中的31株形成4个聚类簇,50株0：8血清型菌株中的19株形成1个聚类簇；41株毒力基因B型(ail-ystA-ystB-yadA-virF-)菌株中的20株形成1个聚类簇；药敏型别在PFGE聚类中分布分散,无特异性。
8、动力、生物分型、血清分型、毒力基因分型、药敏分型和PFGE分型的Simpson指数分别为0.1362、0.0251、0.6564、0.3081、0.7214和0.9982。
[结论]
1、高密市和五莲县的小肠结肠炎耶尔森菌存在动力突变株；菌株几乎全为生物1A型；血清型以0：5和0：8型为主,存在地区差异；菌株携带的毒力基因主要为ystB基因,无传统致病性菌株；耐药谱广泛,大部分菌株对青霉素类和一、二代头孢耐药,存在一定的多重耐药现象,vstB基因阳性的生物1A型菌株与ystB基因阴性的生物1A型菌株耐药性不同。
2、高密市和五莲县的小肠结肠炎耶尔森菌存在多克隆系,基因多态性明显,部分克隆株长期存在,五莲地区可能存在相对优势克隆系；部分山东分离株可能与其他省份菌株存在联系。
3、小肠结肠炎耶尔森菌的PFGE分型可能与其动力有一定相关,与生物型、血清型、毒力基因型和药敏型别不具有相关性。
4.PFGE具有强大的分型能力,但在菌株溯源时需与表型分型联合使用。
[Background]
Yersina enterocolitica (Y. enterocolitica) is a gram-negative budless bacillus, which belongs to Yersina genus and is one of the three pathogenic bacteria for humans and animals of this genus. It distributes widely in the natural environment, and mainly infects humans by contaminated food. Y. enterocolitica infections are mainly characterized with gastrointestinal symptoms, such as vomit, abdominal pain and diarrhea, which are usually self-limiting. However, the infection could induce serious complications, such as endocarditis, reactive arthritis, abscess and fatal septicemia. Yersiniosis, which is caused by Y. enterocolitica and Y. pseudotuberculosis, is the fourth most commonly reported zoonosis in Europe. There are about117,000infections caused by Y. enterocolitica annually in America. Two outbreaks of Y. enterocolitica infection were reported in literatures in China. It has been differentiated into six biotypes (1A,1B, and2-5) and60serotypes. The pathogenicity of Y. enterocolitica is usually determined by testing five virulence genes, which are ail, ystA, ystB, yadA and virF. Y. enterocolitica is resistant to penicillins and first generation cephalosporins naturally, and has different resistant rates to other antimicrobial agents. Pulsed field gel electrophoresis (PFGE) typing is the most frequently used method for Y. enterocolitica molecular typing nowadays and is mainly used for tracing to the strain's source.
In2006, Shandong Province set up two surveillance points, Gaomi City and Wulian County, for Y. enterocolitica monitoring. Until the end of2011,237strains of Y. enterocolitica had been isolated from meat, flies and feces of chickens, swines, sheeps, cattles and other animals. The motility, biotype, serotype, virulence gene and antimicrobial agent susceptibility of these strains had not been analyzed systematically, and the molecular typing had also not been studied.
[Objectives]
1. To systematically analyze the motility, biotype, serotype, virulence gene and antimicrobial agent susceptibility of the Y. enterocolitica isolates and the distribution of time, geography and host for each biological characteristic.
2. To classify the Y. enterocolitica isolates by PFGE, in order to explore the genetic relationship of these strains and discuss the relations between phenotyping and PFGE typing.
3. To establish a Y. enterocolitica database of Shandong Province, in order to lay a foundation for the future long-term surveillance and provide technical support for the prediction and alert of yersiniosis.
[Methods]
1. Motility was tested by stabbing culture in semi-solid medium. Biotype was determined by the results of biochemical reactions. Serotype was determined by serum agglutination test on a glass slide. Virulence gene was tested by polymerase chain reaction. Antimicrobial agent susceptibility was tested by VITEK2COMPACT system and AST-GN16card.
2. PFGE typing was operated according to the standard method recommended by PulseNet China. BioNumerics6.1was used for PFGE spectrum processing and clustering analysis.
3. Excel2007was used for data entering, compilation and simple analysis. SPSS16.0was used for data statistic analysis.
[Results]
1. Among the strains,4.2%(10/237) had medium-active motility,3.0%(7/237) had poor-active motility, and the others all had well-active motility. The strains of medium-and poor-active motility were isolated from most kinds of hosts from the two districts every year.
2. Two hundred and thirty-four of the237strains (98.7%) were biotype1A, and the other three strains were all biotype4, which were isolated from swine feces samples in Gomi City, in2006.
3. One hundred and eighteen of the237strains could be classified by the antiserum, and O:5(21.9%,52/237) and0:8(21.1%,20/237) were the dominant serotypes. All the five serotypes which could be calssified were tested in the strains isolated in2011. Gaomi isolates were dominated by O:5serotype (23.9%,39/163) while Wulian isolates were dominated by O:8serotype (33.8%,25/74); isolates from each kind of hosts were all dominated by O:5and0:8serotypes.
4. The positive rate of ystB gene was the highest, which was81.4%(193/237); ail and ystA gene were found only in three strains, and the positive rate was1.3%(3/237) each; yadA and virF gene were negative in all the strains. The ystB gene positive strains were dominant in each year, either district and each kind of hosts.
5. Two hundred and thirty-five strains were tested for antimicrobial agent susceptibility. All the strains (235strains) were sensitive to ceftriaxone, cefepime, amikacin, gentamicin and tigecycline; the resistant rate to ampicillin was highest, which was94.5%(222/235). The multiple resistant rate was12.3%(29/235). The susceptibility to nitrofurantoin between2007and2011was significantly different (P <0.05); the resistant rate to trimethoprim/sulfamethoxazole between Gaomi City (10.4%,17/163) and Wulian County (1.4%,1/72) was significantly different (P<0.05); the resistant rate to ampicillin, amoxicillin/clavulanic acid, cefazolin and cefoxitin and the intermediate resistant rate to nitrofurantoin of virulence genotype A (ail-ystA-ystB+yadA+virF-) strains, which was99.0%(190/192),89.6%(172/192),94.8%(182/192),80.2%(154/192) and94.8%(182/192) respectively, was significantly higher than that of virulence genotype B (ail-ystA-ystB-yadA-virF-) strains (P<0.05), which was72.5%(29/40),65.0%(26/40),67.5%(27/40),42.5%(17/40) and65.0%(26/40) respectively.
6. Two hundred and thirty-one of the237strains were divided into144PFGE types with no one significantly dominant, and the similarity was61.1%to100.0%. The clustering of strains isolated from Wulian County was more obvious than that from Gaomi City, and the PFGE types, which had seven or more than seven strains, were all in Wulian County. The other six strains couldn't be classified by PFGE. Fifteen of the144PFGE types could find their same or similar types in the database of Chinese center for disease control and prevention.
7. Fourteen of all the17medium-or poor-active motility strains were grouped into three clusters. Thirty-one of all the52O:5serotype strains were grouped into four clusters. Nineteen of all the50O:8serotype strains were grouped into one cluster. Twenty of all the41virulence genotype B (ail-ystA-ystB-yadA-virF-) strains were grouped into one cluster. The antimicrobial agent susceptibility types were distributed irregularly in the PFGE clustering.
8. The Simpson's diversity index of motility, biotyping, serotyping, virulence genotyping, antimicrobial agent susceptibility typing and PFGE typing was0.1362,0.0251,0.6564,0.3081,0.7214and0.9982respectively.
[Conclusions]
1. The Y. enterocolitica strains isolated from Gaomi City and Wulian County had motility mutant strains. Almost all the strains isolated were biotype1A. The serotypes were dominated by O:5and O:8and had a difference between the two districts. The main virulence gene that the strains had was ystB and there was no pathogenic strain traditionally defined. The isolates were resistant to many antimicrobial agents, but most of the strains were resistant to penicillins and first and second generation cephalosporins. There were multiple resistant strains. The antimicrobial agent susceptibility of ystB-positive biotype1A strains were significantly different to that of ystB-negative biotype1A strains.
2. The Y. enterocolitica strains isolated from Gaomi City and Wulian County had many clones, and the gene heterogeneity was obvious. Some of the clones existed persistently. There might be relative dominant clones in Wulian County. Some of the clones might have relations with strains from other provinces.
3. The PFGE types of Y. enterocolitica might have relations with motility, but no relations with biotypes, serotypes, virulence genotypes or antimicrobial agent susceptibility types were observed.
4. PFGE was a typing method with high discrimination power, but a combined use with phenotyping was necessary in microbial source tracking
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