食品与临床分离的致病性副溶血性弧菌耐药性比较

目的比较食品与临床分离的致病性副溶血性弧菌耐药性差异,为该菌耐药机制研究提供基础。方法运用K-B纸片法,对上海市28株临床菌株、18株食品源菌株的副溶血性弧菌进行耐药性监测,以PCR分析菌株的耐药基因。再用多位点测序技术揭示46株副溶血性弧菌的遗传多样性,针对相同进化分支中不同来源的分离株进行耐药性比较。结果28株临床菌株的耐药率(100%)明显高于18株食品源分离株(88.9%),而且临床菌株全部为多重耐药性菌株(n=28),而食品源中仅有2株具有多重耐药性。临床菌株所携带的耐药基因数量比食品源分离菌株更多、种类更为丰富。多位点测序分型结果也显示相同的趋势,在同一进化分支中,临床菌株的耐药性也显著高于食品分离株。结论本研究系统地比较食品与临床分离的致病性副溶血性弧菌耐药表型与耐药基因型的差异,分析了耐药性形成的原因,为副溶血性弧菌耐药性的起源、传播与控制提供依据。     

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。     

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。     

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。     

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。     

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。     

丽水市贝类产品中副溶血性弧菌的血清分型及耐药性研究

目的了解丽水市贝类产品中副溶血性弧菌的血清型分布及耐药性,为防治副溶血性弧菌引起的食源性疾病提供依据。方法从丽水市区农贸市场等采集贝壳类水产品,常规方法分离副溶血性弧菌,参照GB/T 4789.7—2003方法,用标准血清进行分型,用纸片扩散法进行药敏试验。结果检出阳性标本28份,检出率为26.67%(28/105)。分离得到的28株副溶血性弧菌分属于7个血清群,分别为O:4群占39.29%(11/28)、O:3群占14.29%(4/28)、O:2群占14.29%(4/28)、O:11群占14.29%(4/28)、O:1群占7.14%(2/28)、O:7群占7.14%(2/28)、O:10群占3.57%(1/28)。28株副溶血性弧菌中有25株对氨苄西林耐药,1株对四环素耐药。结论从丽水市贝类产品分离的副溶血性弧菌具有血清分群多样化和耐药性简单的特点。     

MALDI-TOF MS用于副溶血性弧菌检测初探北大核心CSCD

目的评价基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS)对副溶血性弧菌的识别能力,并将该方法用于副溶血性弧菌的同源性分析。方法运用标准菌株判断MALDI-TOF MS方法用于检测副溶血性弧菌的重复性及特异性,将从5种贝类中收集到的65株菌株运用MALDI-TOF MS做鉴定,PCA同源性分析。结果 MALDI-TOFMS对65株副溶血性弧菌的质谱鉴定结果与生化鉴定及PCR鉴定结果一致,即能有效区分副溶血性弧菌、创伤弧菌、蜡样芽胞杆菌、金黄色葡萄球菌、大肠埃希菌等5种细菌,其中在副溶血性弧菌,3%NaCl TSA培养24h后的结果鉴定效果更好。10次重复检测副溶血性弧菌的分值均在2.3分以上,PCA聚类分析将65株菌株被分成了4个大类,大多数菌株的PC1、PC2、PC3相对集中靠拢。结论 MALDI-TOF MS检测副溶血性弧菌具有较好的稳定性、特异性,对不同型别的细菌具有较强的分辨能力,且重复性好,同源性分析具有高通量、快速、简单、低成本等优点。     

MALDI-TOF MS用于副溶血性弧菌检测初探

目的评价基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS)对副溶血性弧菌的识别能力,并将该方法用于副溶血性弧菌的同源性分析。方法运用标准菌株判断MALDI-TOF MS方法用于检测副溶血性弧菌的重复性及特异性,将从5种贝类中收集到的65株菌株运用MALDI-TOF MS做鉴定,PCA同源性分析。结果 MALDI-TOFMS对65株副溶血性弧菌的质谱鉴定结果与生化鉴定及PCR鉴定结果一致,即能有效区分副溶血性弧菌、创伤弧菌、蜡样芽胞杆菌、金黄色葡萄球菌、大肠埃希菌等5种细菌,其中在副溶血性弧菌,3%NaCl TSA培养24h后的结果鉴定效果更好。10次重复检测副溶血性弧菌的分值均在2.3分以上,PCA聚类分析将65株菌株被分成了4个大类,大多数菌株的PC1、PC2、PC3相对集中靠拢。结论 MALDI-TOF MS检测副溶血性弧菌具有较好的稳定性、特异性,对不同型别的细菌具有较强的分辨能力,且重复性好,同源性分析具有高通量、快速、简单、低成本等优点。     

贵州省3株钩端螺旋体分离株PFGE分型与基因种鉴定

目的应用脉冲场电泳（PFGE）分型技术和16S rRNA基因测序分析技术分别对贵州省3株动物宿主钩 端螺旋体分离菌株进行分子分型和基因种鉴定,了解贵州省钩端螺旋体的分子流行病学特征。方法应用 DNA限制性内切酶Not I对钩端螺旋体染色体DNA酶切后,用PFGE将DNA片段分离,采用BionumericsV4.0将3 株钩体菌株PFGE图谱与中国15群15型参考菌株进行聚类分析;同时,应用PCR扩增几乎全长的钩体16S rRNA基因片段,并将扩增产物进行双向序列测定,并与GenBank数据库已注册的核酸序列进行同源性比对 、确定基因种、分析亲缘进化关系。结果来自贵州省的3株动物宿主分离钩体菌株PFGE带型命名为LepNot I 002和LepNot I 003,经聚类分析,3株菌株与黄疸出血群黄疸出血型赖株的相似性大于95%。16S rRNA 基因测序和分析表明,3株贵州分离钩体菌株之间的同源性为100%,与致病性钩体问号钩端螺旋体种（L. interrogans）不同血清型参考菌株的同源性达100%。结论3株贵州动物分离钩体菌株经PFGE分型鉴定与黄 疸出血群赖型赖株的相似性大于95%,经16S rRNA基因测序分析鉴定为L. interrogans种,上述两种方法 对贵州省钩体分离菌株的鉴定结果一致,有助于贵州省钩端螺旋体病的主动监测、暴发调查和传染源追踪 。     

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.     

Surveys on the contamination of marine fish with non-O1 Vibrio cholerae and Vibrio mimicus and food poisoning cases by these organisms

The present paper describes the relationship between the contamination with non-O1 Vibrio cholerae and Vibrio mimicus of marine fish, with special reference to the seasonal variation and the concentration of contamination, and the actual cases of domestic food poisoning by these organisms. A 10 year survey revealed that non-O1 Vibrio cholerae (non-O1 V. cholerae) strains were frequently isolated from fish during the summer season with some variations from one year to another, and isolates from fish showed similar biological properties to those of isolates from diarrhea cases of over-sea travellers. Experimentally enteropathogenic strains were included among these isolates. Vibrio mimicus (V. mimicus) strains were also isolated from fish, the frequency being not so high as in the case of non-O1 V. cholerae Strains of serovar O-41 which was most predominant among strains from diarrhea cases were also detected among the isolates from fish. The viable cell counts, however, were very small with regard to both non-O1 V. cholerae and V. mimicus From these observations, factors causing food poisoning by non-O1 V. cholerae or V. mimicus seemed to be essentially similar to those by Vibrio parahaemolyticus (V. parahaemolyticus); that is, the food poisoning by non-O1 V. cholerae or V. mimicus is apt to occur in the summer season and is caused by the consumption of raw fish, although the frequency might be significantly low in comparison to that of V. parahaemolyticus. The actual cases of the domestic food poisoning by non-O1 V. cholerae or V. mimicus were retrospectively surveyed by the literature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enteric infections in Singapore with special reference to typhoid.

A review of the epidemiological features of the more important enteric infections in Singapore was given. Enteric fevers (typhoid and paratyphoid), Salmonella gastroenteritis and Shigellosis remained endemic at a rather constant level in spite of vast improvement in environmental sanitation, but amoebic dysentery showed marked decline. Vibrio parahaemolyticus is an important cause of food poisoning since it was first reported in 1973. El tor cholera is believed to be introduced through regional trade and travel. Control measures directed mainly at typhoid include detection of typhoid carriers in the community responsible for transmission of infection, control and licensing of public food handlers and health education.     

A rare outbreak of food poisoning caused by Salmonella enterica serovar. Oranienburg--a case report and features of isolates

A rare food poisoning outbreak caused by S. Oranienburg occurred at a junior high school athletic meet in Kurashiki, Okayama, in September 2005. The 70 patients included junior high school students, teachers and other school staff, and their families. This bacillus was isolated from stools of two employees and another in catered sandwiches. The cause of the outbreak was determined by evidence and epidemiological investigation to be sandwiches served at the athletic meet. Biochemical features, sensitivity to 12 antibacterial agents, and DNA patterns determined by pulse field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic sequence PCR (ERIC2-PCR) agreed for all isolates from outbreak samples. Isolates resembled strains isolated from broilers and a patient stool in an outbreak involving cuttlefish chips from 1998 to 1999 in Okayama Prefecture. A number of differences in strains isolated from broilers, chicken appendix content, and feed were detected in 2004, so we concluded that few outbreaks of food poisoning occurred due to S. Oranienburg in Okayama, attention is required for food poisoning by S. Oranienburg in the future because the dissemination of S. Oranienburg strains showing different features has been confirmed.     

一起副溶血性弧菌食物中毒事件判定依据的探讨

目的对一起副溶血性弧菌食物中毒事件判定依据进行探讨。方法开展现场流行病学和卫生学调查方法,所得调查数据进行描述性统计分析;并组织专家论证,进行病因的因果推断。结果此事件中共确认病例7例,病例均出现恶心、呕吐、腹泻等症状。2名患者粪样中检出副溶血性弧菌。综合可疑食品调查、现场卫生学调查、暴露因素推断和实验室检测结果,经专家论证,定性为一起副溶血性弧菌食物中毒事件。结论流行病学调查在食物中毒事件调查中有确认地位,现场流行病学调查结果作为食物中毒判定主要依据应得到法律的支持。     

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。     

基于TaqMan探针的双重荧光定量PCR方法检测海产品中的副溶血弧菌

目的建立海产品中副溶血弧菌检测的双重荧光定量PCR体系。方法针对副溶血弧菌种特异性基因tlh和tdh设计引物和TaqMan探针,建立双重荧光定量PCR体系,进行特异性与敏感性研究;利用该体系检测海产品中的副溶血弧菌。结果副溶血弧菌可得到特异性扩增,而其它与副溶血弧菌共存于海产品中的细菌均未见扩增曲线。副溶血弧菌与其它细菌的混合DNA检测表明,其它细菌基因组的存在时并不干扰副溶血弧菌检测。副溶血弧菌典型菌株FJ14和BJ97的敏感性试验显示,该体系的最低检测DNA浓度分别为49.8pg与77.8pg,最低检测细菌浓度为56CFU/mL和371CFU/mL。对舟山菜市场采集的50份样本检测表明,32份为tlh基因阳性,3份为tdh基因阳性,与传统方法的检测结果相同。结论与传统检测方法相比,副溶血弧菌的双重荧光定量PCR检测方法快速准确,结果直观。     

A case of cervical abscess caused by Salmonella enteritidis after intestinal infection

Salmonella Enteritidis often causes food poisoning. In this study, an extremely rare case of cervical abscess caused by S. Enteritidis is reported. In January 2003, a 44-year-old man visited our hospital with swelling of the left submandibular region. He had been suffering from a severe diabetic condition but had neglected to seek medical attention. An incision was made at the abscess to drain the pus from which only S. Enteritidis was isolated. This finding led to the discovery that he had suffered from an episode of food poisoning 6 months earlier. However, the organism was not isolated from the stool. The patient recovered with the administration of panipenem/betamipron and gatifloxacin. The S. Enteritidis strain isolated from the pus obtained from this case and that detected from the samples originating from the other patients during the episode of food poisoning 6 months earlier were examined by using pulse-field gel electrophoresis (PFGE). The PFGE patterns of the strains were almost identical. The molecular epidemiological analysis by PFGE was useful in estimating the infection route. In an immuno-compromised host such as those suffering from diabetes mellitus, one must be reminded that unusual bacteria (including S. Enteritidis) may cause a cervical abscess.     

Isolation of thermostable direct hemolysin producing Vibrio parahaemolyticus from food using screening by PCR in food-borne outbreaks

The producibility of thermostable direct hemolysin (TDH) is the most important pathogenic factor in Vibrio parahaemolyticus. TDH (+) V. parahaemolyticus is usually isolated from patients having V. parahaemolyticus food-borne disease. TDH (+) V. parahaemolyticus is, however, very difficult to isolate from food and environmental samples. In the 5 years from 2000 to 2004 in Tokyo, V. parahaemolyticus was isolated from food samples related to 67 of 227 V parahaemolyticus food-borne outbreaks. In these outbreaks, TDH (+) strains were also tried to isolate using PCR as the screening methods. TDH (+) V. parahaemolyticus strains were able to isolate from enrichment broth in which toxR and tdh genes become positive in PCR. TDH (+) strains of the same serotype with patients were able to be isolated from 23 food samples related to 11 outbreaks (16.4%); 3 outbreaks in 2000, 2 in 2001, 2 in 2002, 1 in 2003, and 3 in 2004. The serotypes of V. parahaemolyticus isolated from food were O3 : K6 (10 samples), O3 : K5 (6 samples), O1 : K25 (4 samples), O3 : K29 (2 samples), O4 : K 8 (1 sample), and O4 : K11 (1 sample). The isolation rate of the TDH (+) strain from enrichment broth differed with samples. In several samples TDH (+) strains were isolated easily only by examining 3 colonies, hence no TDH (+) strains were isolated in spite of the examination of 250 colonies. No correlation was seen between the number of V. parahaemolyticus and the isolation rate of TDH (+) strains in food samples. Screening using PCR is very effective method for isolating TDH (+) V. parahaemolyticus from food samples.     

宁波副溶血性弧菌临床株毒力基因与多位点序列分型研究

目的 了解宁波地区副溶血性弧菌临床分离株毒力基因分布以及分子分型特征。方法 收集来源于食物中毒和散发腹泻患者副溶血弧菌菌株,利用聚合酶链式反应(polymerase chain reaction,PCR)检测耐热直接溶血素基因(tdh)和耐热直接溶血素相关溶血素基因(trh),利用多位点序列分型(multi-locus sequence typing, MLST)进行分子分型。结果 2006—2012年共分离临床株248株,选择48株进行毒力基因和MLST分型研究。42株tdh+,为93.75%;11株trh+,为22.92%。48株菌株可分为9个ST型和一个未分型,ST3有32株,占66.67%; ST265有5株,占10.42%;ST120有3株,占6.25%。ST3克隆群中tdh+/trh-菌株有25株,占78.16%。与全国其他地区比较,在宁波临床株中发现ST262。结论 tdh+型是宁波地区副溶血性弧菌优势菌株。有9种ST型,以ST3克隆为主,其次为ST265和ST120。ST3克隆中以tdh+/trh-型为主。另发现1个独特的ST262菌株。