2009～2011年无锡市副溶血性弧菌分离菌株的毒力检测及PFGE分析

目的了解无锡市2009～2011年副溶血性弧菌分离株携带的主要毒力因子的流行状况,并对同血清型菌株进行脉冲场凝胶电泳(PFGE)分析。方法应用PCR方法检测分离的35株副溶血性弧菌耐热性溶血毒素基因(tdh)、耐热性溶血毒素相关的溶血毒素基因(trh)和不耐热溶血毒素基因(tlh)。根据美国CDC PulseNet实验方法,用限制性内切酶SfiⅠ对O3﹕K6血清型菌株的染色体进行酶切,通过PFGE获得电泳图谱,利用BioNumerics软件对图谱进行聚类分析。结果 35株副溶血性弧菌tdh、trh及tlh基因的携带率分别为85.7%、8.6%和100%,77.1%的副溶血性弧菌携带的毒力基因为tdh+、trh-、tlh+。PFGE图谱显示,19株O3﹕K6血清型的副溶血性弧菌共有9种PFGE带型,带型100%相同的菌株几乎都出现在同一年代相近的时间点,但也出现了跨年代菌株。结论无锡市副溶血性弧菌致病性较强,具有潜在的O3﹕K6型副溶血性弧菌暴发流行可能,需进一步加强监测管理。     

脉冲场凝胶电泳用于副溶血性弧菌的分子流行病学研究

目的运用脉冲场凝胶电泳技术和荧光PCR技术对成都市2008-2009年分离的副溶血性弧菌进行分子分型和毒素基因检测,分析感染来源。方法利用PCR检测副溶血性弧菌分离株耐热直接溶血素(tdh)基因和耐热相关溶血素(trh)基因。用脉冲场凝胶电泳(PFGE)对菌株进行分型,所得结果用BioNumerics V 4.0软件UPGMA方法进行聚类分析。结果所试38株分离菌tdh基因阳性有31株,2株为trh阳性。以SfiⅠ酶切后PFGE分型,38株菌被分成了24个带型,大多数暴发有各自优势型别,其中有两起暴发优势型别一致;环境分离株菌型分散。结论多起暴发事件分离株之间PFGE型别差异大,成都市副溶血性弧菌暴发的感染来源复杂。     

宁波地区海产品及环境中副溶血弧菌主要毒力及耐药性分析

目的了解浙江省宁波地区海产品和环境中副溶血弧菌主要毒力和耐药性。方法采用生化反应、抗菌药物敏感性试验及PCR方法对分离的宁波地方副溶血弧菌进行毒力及耐药性测定。结果 90株分离株中,耐热直接溶血素基因（tdh）阳性率为2.2%,与神奈川溶血表型（KP,Kanagawa phenomenon）一致,但在tdh＋和KP阳性的分离株中未检测到Ⅲ型分泌系统2（T3SS2）;trh与ureC基因携带率分别为20.0%和12.2%,而在11株trh＋-ureC＋菌株中未显示尿素酶表型阳性,2株尿素酶阳性的菌株未携trh或ureC基因;Ⅵ型分泌系统的携带率为17.8%。所有的分离株对氟喹诺酮类、氯霉素类、四环素类药物敏感;72%以上分离株对青霉素类、磺胺类及氨基糖苷类中链霉素和卡那霉素耐药;分离株中至少耐2种药物,最多耐10种药物,超过82%的分离株对6种以上药物耐药。耐药基因tetB的检出率为0,bla TEM、sul2和strB的携带率分别为91.7%、16.7%和43.3%。结论宁波地区相当比例的菌株携带毒力,并呈现不同程度耐药。     

引起食物中毒副溶血性弧菌的病原学鉴定

目的分离食物中毒患者粪便及食物加工工具样本中的病原菌,对分离菌株进行表型和毒力基因鉴定。方法采用TCBS平板法分离食物中毒样本中的病原菌。采用细菌系统鉴定方法,确定所分离的副溶血性弧菌(Vibrio parahaemolyticus,Vp)生化反应特性和血清型。采用PCR检测Vp分离株的种属特异基因、直接耐热溶血素毒力基因(tdh)和直接耐热相关溶血素毒力基因(trh)。采用K-B纸片法检测Vp分离株对14种抗生素的敏感性。结果从1例携带者和3例病人粪便标本中分离出4株Vp,从2份食物加工工具样本中分离出2株Vp。6株Vp分离株均属于含Vp种属特异基因的O1∶K56血清型,tdh基因阳性但trh基因阴性。6株Vp分离株生物学性状和药敏试验结果一致。结论tdh+/trh-O1∶K56血清型Vp是引起本次食物中毒的病原菌。     

双毒力基因副溶血性弧菌引起食物中毒病原学分析

目的分析引起食物中毒的病原菌,掌握分离株生化、血清学、毒力基因、耐药性状况。方法采集患者肛拭子标本,进行分离鉴定、耐药性分析、血清学分型,实时荧光PCR法检测毒力基因tdh、trh。结果 3份肛拭子中分离出O3、O1 2株不同血清型的副溶血性弧菌;2株菌株均对氨苄西林耐药;1株同时携带毒力基因tdh和trh。结论这是一起由2种血清型副溶血性弧菌引起的食物中毒,菌株携带双毒力基因可能是导致此次食物中毒患者临床表现较重的原因。     

烟台市不同来源副溶血性弧菌的病原学特征及脉冲场凝胶电泳分型分析

目的分析烟台市不同来源副溶血性弧菌的优势血清型、耐药、毒力基因携带等病原学特征和分子分型特点。方法对2010-2015年分离于食源性疾病事件和主动监测中的病例、食品安全风险监测中的海产品及海水外环境的77株副溶血性弧菌测定14种抗菌药物的最低抑菌浓度,通过实时荧光PCR检测tlh、trh、tdh、orf8 4种毒力基因,并进行脉冲场凝胶电泳(PFGE)分型。结果77株副溶血性弧菌中仅1株氨苄西林耐药株,为海产品分离株(1.9%),且为AMP+FAZ双重耐药,对头孢唑啉的耐药率以海产品分离株较高(占44.2%),其次是腹泻患者分离株(5.0%),海水分离株100.0%中度敏感;携带≥1种毒力基因菌株占29.9%,全部病例分离株以4种组成形式携带毒力基因,其优势的血清型为O3∶K6(占50.0%);根据PT相似系数90.0%将77株副溶血性弧菌分成6个聚类群(A-F群),A群为优势群(占18.2%),A群内以SDYTVP001为代表的分子型别是优势带型(占64.3%),为引起食源性疾病的主要带型。结论烟台市副溶血性弧菌总体耐药情况较轻,病例分离株均携带毒力基因,PFGE型呈现多态性分布,存在优势分子型,为副溶血性弧菌导致的食源性疾病的早期预警、溯源提供了技术支撑。     

广东省猪链球菌2型菌株毒力基因及分子分型分析

目的 掌握广东地区猪链球菌2型菌株毒力因子及分子分型情况,为诊断、治疗和制定防控措施提供科学依据。方法 选取荚膜多糖(cps2J)、溶菌酶释放蛋白(mrp)、溶血素(sly)、谷氨酸脱氢酶(gdh)、胞外因子(ef)等5个猪链球菌2 型主要毒力基因,分别设计引物对分离自病人、病猪的22株猪链球菌2 型菌株进行PCR检测,并对菌株进行MLST分型分析。结果 22株菌分为5种毒力基因型,其中cps2J+/mrp+/sly+/gdh+/ef+型别为病人及病猪菌株所共有,cps2J+/mrp+/sly-/gdh-/ef+、cps2J+/mrp+/sly-/gdh+/ef+为病人菌株所特有,cps2J+/mrp-/sly+/gdh+/ef+、cps2J+/mrp-/sly-/gdh+/ef-为病猪菌株所特有;MLST分型结果显示,22株分为ST1、ST7和ST28三个型别,其中ST1、ST7型为病人和病猪菌株所共有,均同属于ST1克隆复合物,ST28型为病猪所特有。结论 广东地区病人、病猪的猪链球菌2 型菌株毒力基因型及MLST分子分型均呈现多样化,且部分型别为两者所共有,为阐明猪传染给人提供了直接的证据。     

浙江省副溶血性弧菌03：K6血清型菌株多位点序列分型研究

目的掌握浙江省不同来源的副溶血性弧菌O3：K6血清型菌株的分子分型特征,为副溶血性弧菌食源性疾病的预防控制提供技术支持。方法选择副溶血性弧菌的7个管家基因dnaE、gyrB、recA、dtdS、pntA、pyrC及tnaA,对62株不同来源的副溶血性弧菌03：K6血清型菌株样本进行PCR扩增、测序,Chromas软件和DNAStar软件分析,核酸序列上传至MLST数据库进行比对,获得每株菌的序列型,绘制多位点序列分型遗传进化树并进行亲缘性分析。结果62株副溶血性弧菌03：K6血清型菌株中,59株菌为ST-3型（3,4,19,4,29,4,22）,1株菌为ST-121型（3,2,82,52,4,78,66）,1株菌为新的ST型（5,10,34,27,77,49,23）,1株菌仅gyrB基因第562位点由C突变为T,其余基因序列与ST4型（3,5,22,12,20,22,25）一致。结论浙江省副溶血性弧菌03：K6血清型菌株的主要MLST型别为ST-3型。     

青岛市售养殖海水虾中副溶血性弧菌的分离及耐药性分析

目的 对青岛市售养殖海水虾中副溶血性弧菌进行分离鉴定,并对分离菌株进行耐药性分析。方法 以常规培养法分离,全自动细菌鉴定仪进行鉴定,PCR扩增法检测毒力基因,K-B法进行药敏试验。结果 样品的副溶血性弧菌检出率为78.1% (50/64),所有菌株均不含tdh基因,其中3株菌含trh基因,所有菌株对头孢拉啶耐受,96%菌株对氨苄西林和阿莫西林耐受,部分菌株对头孢呋新钠、链霉素、四环素、土霉素和复方新诺明耐受,少量菌株出现耐受3类抗生素以上的多重耐药性。结论 青岛市售养殖海水虾中副溶血性弧菌存在较严重的污染和一定程度的耐药情况。     

引起一起食物中毒的副溶血性弧菌病原学检测和分子分型溯源研究北大核心CSCD

目的对引起一起食物中毒的副溶血性弧菌进行实验室鉴定,分析菌株间的相关性。方法按照国标GB4789和有关规范对采自患者的粪便肛拭标本及砧板涂抹标本进行肠道致病菌检测,同时采用实时荧光PCR对耐热直接溶血素(TDH)毒力基因进行鉴定。提取副溶血性弧菌分离株基因组DNA,经限制性内切酶SfiⅠ酶切后进行脉冲场凝胶电泳(PFGE),获得指纹图谱,利用BioNumerics6.6软件进行聚类分析。结果从病人和砧板标本共分离出8株O1血清型副溶血性弧菌,其TDH毒力基因为阳性。聚类分析显示,分离自中毒病人和砧板的8株副溶血性弧菌的指纹图谱相似性高达100%。结论引起该起食物中毒的病原菌为携带TDH毒力基因的O1血清型副溶血性弧菌,且来自同一污染源。     

Characterization of Vibrio parahaemolyticus isolated from oysters and mussels in São Paulo, Brazil

Vibrio parahaemolyticus is a marine bacterium, responsible for gastroenteritis in humans. Most of the clinical isolates produce thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) encoded by tdh and trh genes respectively. In this study, twenty-three V. parahaemolyticus, previously isolated from oysters and mussels were analyzed by PCR using specific primers for the 16S rRNA and virulence genes (tdh, trh and tlh) and for resistance to different classes of antibiotics and PFGE. Nineteen isolates were confirmed by PCR as V. parahaemolyticus. The tlh gene was present in 100% of isolates, the tdh gene was identified in two (10.5%) isolates, whereas the gene trh was not detected. Each isolate was resistant to at least one of the nine antimicrobials tested. Additionally, all isolates possessed the blaTEM-116 gene. The presence of this gene in V. parahaemolyticus indicates the possibility of spreading this gene in the environment. Atypical strains of V. parahaemolyticus were also detected in this study.     

非O157产志贺毒素大肠杆菌分离株的多位点序列分型研究

目的 对国内部分地区非O157产志贺毒素大肠杆菌分离株进行分子分型分析,了解菌株间的遗传进化关系。 方法 根据mlst.ucc.ie数据库提供的大肠杆菌多位点序列分型（Multilocus Sequence Typing, MLST）方案,对来自我国河南、黑龙江2省的29株产志贺毒素大肠杆菌分离株的7个管家基因进行PCR扩增并测序,通过序列比对确定其等位基因谱及菌株序列型（Sequence type, ST）,使用MEGA、eBURST生物信息软件分析不同ST序列群及菌株间的进化关系。结果 29株非O157产志贺毒素大肠杆菌呈现较大的遗传多态性,可分为13个ST型别,其中ST155为优势型别（34.48%）。同时研究发现2个新等位基因型（fumC376、recA214）和3个新序列型（ST2460、ST2467、ST2468）。结论 29株非O157产志贺毒素大肠杆菌菌株之间具有分子多态性,与国际流行菌株具有一定的亲缘关系。加强我国对这一类菌株的检测和监测具有重要的公共卫生意义。     

两株布鲁菌MLST新序列型(ST)的鉴定

目的 对北京地区分离自布病患者血液的2株布鲁菌进行多位点序列分型(Multilocus sequence typing,MLST)及其遗传学特征进行研究,为布病的预防和控制提供科学依据。方法 应用布鲁菌属特异性PCR(BCSP31-PCR)和种/型特异性PCR(AMOS-PCR)对分离的2株布鲁菌进行鉴定,采用MLST技术对2株布鲁菌分离株的19个管家基因、1个外膜蛋白基因及1个基因间区的序列进行测定,与MLST数据库中的等位基因序列进行比对,确定菌株的等位基因谱及9位点和21位点序列型(sequence type,ST)。采用聚类分析法研究2株菌ST型与各种布鲁菌已知ST型的遗传进化关系。结果 2株分离株经BCSP31-PCR鉴定为布鲁菌属细菌,AMOS-PCR鉴定为非羊种、非牛种1、2、4型、非猪种1型、非绵羊附睾种布鲁菌;MLST分析显示,2株菌的等位基因编号(gap/aroA/glk/dnaK/gyrB/trpE/cobQ/int-hyp/omp25/prpE/caiA/csdB/soxA/leuA/ mviM/fumC/fbaA/ddlA/putA/mutL/acnA)分别为2、1、2、2、1、3、1、4、1、13、2、2、2、2、1、1、3、7、6、2、3和2、1、2、2、1、3、1、4、29、13、2、2、2、2、1、1、3、7、6、2、3,比对MLST数据库,结果显示这2株细菌的等位基因编号组合未见报道,为新ST型。聚类分析显示这2种新的ST型与牛种布鲁菌ST型处于同一分支。结论 北京地区分离的2株布鲁菌为新ST型牛种布鲁菌,已被MLST数据库确认,分别命名为ST74和ST75(9位点序列型)与ST108和ST109(21位点序列型)。与同属牛种布鲁菌的ST型遗传关系最近,该结果为北京地区布病的预防和控制提供了科学依据。     

Detection of tdh and trh genes in Vibrio parahaemolyticus isolated from Corbicula moltkiana prime in West Sumatera, Indonesia

The occurrence of Vibrio parahaemolyticus in raw Corbicula moltkiana Prime from Lake Singkarak and Pasar Raya Padang market and in cooked samples in West Sumatera, Indonesia, was studied. Thirteen raw and seven cooked bivalve samples were positive using CHROMAgar Vibrio medium. All 47 V parahaemolyticus isolates were positive for toxR gene but negative for trh. However, 36% (17/47) of V parahaemolyticus strains were positive for tdh gene. Antibiotic profiling showed that 76% and 38% of isolates from raw and cooked bivalves respectively were resistant to ampicillin. Using RAPD-PCR analysis, most of the strains were clustered according to their source of isolation but some of the strains from raw and cooked samples were clustered together. These results indicate that pathogenic V parahaemolyticus isolates are present in Corbicula moltkiana Prime in West Sumatera, Indonesia, suggesting that V parahaemolyticus may also be present in seafood in other regions of Indonesia.     

O3:K6 serotype of Vibrio parahaemolyticus identical to the global pandemic clone associated with diarrhea in Peru.

OBJECTIVES: To determine if the Vibrio parahaemolyticus O3:K6 global pandemic clone has spread into Peru. METHODS: A collection of 100 V. parahaemolyticus strains isolated from diarrhea cases in Peru were serotyped for O:K antigens and genotyped for the presence of the species-specific toxR gene and for the tdh and trh genes. In addition, the group-specific PCR (GS-PCR) and PCR for the presence of the open reading frame ORF8 of the filamentous phage f237 was performed to determine the pandemic status of the strains. RESULTS: Fifty strains of V. parahaemolyticus in this collection were identified as pandemic strains. Forty-six ORF8 and GS-PCR positive strains were identical to the global pandemic clone O3:K6, while four strains that also possessed the pandemic genotype and were ORF8 and GS-PCR positive belonged to serotypes O3:K68, O3:K58 and OUT (untypable):K6. One of the O3:K6 strains was isolated in 1996, indicating that the pandemic strain was present in Peru at about the same time that it caused the first outbreak in Calcutta in February 1996. CONCLUSIONS: Based on this first report in Peru of such strains, we recommend including V. parahaemolyticus in the differential diagnosis of the etiologic agents for diarrhea in this part of the world.     

Comparison of epidemiological markers for Vibrio parahaemolyticus isolated from food poisoning

Vibrio parahaemolyticus food poisoning, is the most prevalent among bacterial food poisoning in Japan. Study of epidemiologic markers is important in an attempt to trace the source of contamination. The purpose of this study was to compare seven different typing methods (serotyping, plasmid profile, antibiogram, phage susceptibility. TDH production, tdh and trh gene and pulsed-field gel electrophoresis [PFGE]) for V. parahaemolyticus. Outbreaks of V. parahaemolyticus food poisoning which occurred during the 13 years from 1981 to 1993 numbered 43 including 481 cases in Nagano Prefecture. Serovar O4:K8 was the most prlevalent serovar isolated, serovar O2:K3, O4:K63 and O3:K5 followed. Forty one strains of V. parahaemolyticus were used in this study. All of the strains were isolated from 12 food poisoning cases at Nagano Prefectual Research Institute for Health and Pollution. Of the 41 strains, twenty two strains (O4:K8, O4:K63) were sensitive to both phi VP 253 and phi VP 143 phages, six strains (O3:K5) to phage phi VP 143. Thirteen strains (O3:K29, O4:K11, O4:K12 and O5:KUT) were insensitive to both phages. CBPC, CBPC.CEZ and CBPC.CEZ.KM.SM resistant strains was determined in 22 strains out of 41 strains. Five strains of V. parahaemolyticus carried plasmid. Of the 41 strains, thirty nine strains were possessive to tdh gene and productive to TDH. Chromosomal DNA of the isolates from 12 different outbreaks was analysed by PFGE after Not I digestion. PFGE analysis of the digested DNA yielded 11 to 21 DNA fragments. Twelve distinctive fragment patterns were identified in 41 V. parahaemolyticus isolates from 12 different food poisonings. These results showed that the PFGE method is an useful tool to analyse an epidemiological survey for isolates of Vibrio parahaemolyticus food poisoning.     

Characterization of vibrio parahaemolyticus isolated from coastal seawater in peninsular Malaysia

Twenty-one Vibrio parahaemolyticus isolates representing 21 samples of coastal seawater from three beaches in peninsular Malaysia were found to be sensitive to streptomycin, norfloxacin and chloramphenicol. Resistance was observed to penicillin (100%), ampicillin (95.2%), carbenicilin (95.2%), erythromycin (95.2%), bacitracin (71.4%), cephalothin (28.6%), moxalactam (28.6%), kanamycin (19.1%), tetracycline (14.3%), nalidixic acid (9.5%) and gentamicin (9.5%). Plasmids of 2.6 to 35.8 mDa were detected among plasmid-containing isolates. All isolates carried the Vp-toxR gene specific to V. parahaemolyticus and were negative for the tdh gene, but only one isolate was positive for the trh gene. DNA fingerprinting of the isolates using ERIC-PCR and PFGE showed that the isolates belong to two major clonal groups, with several isolates from different locations in the same group, indicating the presence of similar strains in the different locations.