外膜蛋白OmpR对副溶血弧菌致病特性的影响

[目的]探究OmpR在副溶血弧菌生物学特性和致病性中发挥的作用。[方法]利用同源重组技术构建了副溶血弧菌ompR基因缺失株(ΔompR)和互补株(CΔompR),分析各菌株的生长特性、运动性和生物被膜形成能力的差异;比较各菌株对细胞黏附、细胞毒性和小鼠致病性的影响。[结果]ompR基因缺失对副溶血弧菌的生长特性、运动性以及细胞毒性无显著影响。但与野生株相比,ΔompR的生物被膜的形成能力显著降低;感染ΔompR的小鼠存活率升高了25%,病变程度更低;ΔompR在小鼠心脏、肝脏和肾脏中的载菌量显著低于野生株,互补株毒力基本恢复至野生株水平。[结论]OmpR参与副溶血弧菌生物被膜形成和致病过程,是副溶血弧菌潜在的毒力因子。     

荧光定量PCR法检测副溶血弧菌tlh和tdh基因的表达差异

副溶血弧菌是广泛存在于近海区域,盐湖和海产品中的食源性致病菌,会引起大规模的食物中毒。TLH(不耐热溶血毒素)和TDH(耐热直接溶血毒素)是副溶血弧菌最主要的毒力基因,通过比较毒力基因的表达量可以间接比较同种菌株在不同应激条件下以及不同菌株之间的毒力差异。本文以在不同条件下培养的3株Vp为材料,分别提取其总RNA,以16S rRNA为内标基因,运用荧光定量PCR技术检测副溶血弧菌TLH和TDH基因在不同应激条件下的表达差异。结果表明:不同菌株和同种菌株在不同应激条件下tlh、tdh基因表达差异均显著;tlh的最适表达条件分别为5%盐度和20°C;tdh的最适表达条件分别为1%盐度和25°C。运用SPSS软件对实验结果进行统计学分析表明:菌株对tlh表达的影响大于盐度大于温度;菌株对tdh表达的影响大于温度大于盐度。     

宁波地区环境中副溶血弧菌血清学及毒力相关基因研究

目的了解宁波地区环境来源海产品中副溶血弧菌血清学特点及毒力相关基因分布。方法采集并分离2013年6-10月宁波地区海产品中副溶血弧菌,对其进行O、K抗原血清学分型;并采用PCR或多重PCR的方法来检测溶血素基因(tlh、tdh、trh)、大流行群遗传标志基因(toxRS/new、orf8)和Ⅲ型分泌系统(T3SS1、T3SS2α、T3SS2β)基因。结果从海产品样本中分离鉴定到44株副溶血弧菌的菌株,分属于20种血清型,型别多样,未见优势血清型;溶血素基因检测发现3株tdh+trh-致病性菌株,遗传标志基因检测发现1株tdh+trh-toxRS/new+大流行株,其血清型为O3:K6型;Ⅲ型分泌系统基因检测发现T3SS1基因存在于所有的副溶血弧菌菌株中,而T3SS2α基因则主要分布在tdh+的菌株中。结论宁波地区环境中副溶血弧菌致病性菌株和大流行株的检出,说明该地区具有潜在的食源性疾病爆发的风险。     

双组分系统EnvZ/OmpR促进副溶血弧菌抵抗碱胁迫的作用机制

【目的】探究双组分系统(two-component system,TCS)EnvZ/OmpR对副溶血弧菌(Vibrio parahaemolyticus,VP)抵抗碱胁迫的作用机制。【方法】用SMART在线工具(https://smart.embl.de/)鉴定出副溶血弧菌基因组中的双组分系统EnvZ/OmpR,再利用同源重组技术将envZ和ompR基因分别进行缺失,构建相应回补株,比较各菌株的生长曲线来检测相应基因对细菌适应高渗透胁迫和碱胁迫的作用,并结合qRT-PCR及荧光检测系统,筛选参与EnvZ/OmpR抵抗碱胁迫的下游靶基因,鉴定该双组分系统对下游基因的调控机制。【结果】在副溶血弧菌基因组中鉴定出vp0155/vp0154编码EnvZ/OmpR双组分系统同源蛋白。△ompR菌株在高渗透胁迫和碱胁迫中的生长能力明显弱于野生株,而回补株C△ompR、△envZ和C△envZ菌株生长能力与野生株类似。在△ompR菌株中,孔道蛋白基因vp1218、vp0493、vpa1745、vpa0085与vpa1308的转录水平均明显低于野生株,并且发现这些孔道蛋白基因缺失株(△vpa1308除外)在碱性环境中生长能力均明显弱于野生株。OmpR蛋白可直接抑制调控因子AphB基因转录,而△aphB菌株在碱胁迫中的生长能力明显强于野生株。此外,AphB蛋白可直接抑制孔道蛋白基因vp0493和vpa0085转录。【结论】双组分系统EnvZ/OmpR促进副溶血弧菌抵抗碱胁迫,其中OmpR蛋白可通过抑制调控因子AphB的表达,以促进部分孔道蛋白的表达,从而增强副溶血弧菌抵抗碱胁迫的能力。     

VPA1500基因缺失对副溶血弧菌生物学特性和致病性的影响

Ⅵ型分泌系统（T6SS）是细菌的一种毒力因子分泌系统,通过分泌蛋白参与调控细菌的环境适应性和毒力。副溶血弧菌具有两个T6SS系统（T6SS1和T6SS2）。[目的] 前期通过差异蛋白质组学技术筛选到副溶血弧菌T6SS1相关的分泌蛋白,本文选择其中的VPA1500为研究对象,研究其基因缺失对副溶血弧菌的生物学特性及致病性的影响。[方法] 利用同源重组技术构建缺失株ΔVPA1500和互补株CΔVPA1500;分析各菌株生长特性、在体外的细菌竞争能力、运动性、细菌鞭毛相关基因的转录水平及生物被膜形成能力的差异,比较各菌株对细胞毒性、小鼠毒力、动物组织载菌量以及组织病理学变化的影响。[结果] 与野生株相比,VPA1500基因缺失后不影响细菌的生长能力、生物被膜形成能力和群集运动,然而ΔVPA1500的浮泳运动能力显著下降;进一步通过透射电镜观察和实时定量PCR检测发现,VPA1500缺失影响副溶血弧菌鞭毛的形成;细菌竞争实验显示缺失VPA1500基因降低了副溶血弧菌野生株体外对大肠杆菌的杀伤能力;ΔVPA1500对细胞毒性、小鼠毒力以及在动物组织的定殖能力均显著低于野生株,互补株毒力基本恢复至野生株水平;组织病理学结果进一步表明,缺失VPA1500基因能够降低副溶血弧菌对小鼠组织的损伤。[结论] VPA1500参与副溶血弧菌的体外细菌竞争能力、浮游运动能力和致病性。     

VHH/TLH溶血素基因在海洋弧菌中分布的研究

哈维氏弧菌(V.harveyi)的VHH溶血素是对海水养殖鱼类的潜在致病因子。哈维氏弧菌的VHH溶血素基因与副溶血弧菌(V.parahaemolyticus)的TLH热不稳定性溶血素基因具有高度相似性,其氨基酸序列的相似性达到85.6 %。根据哈维氏弧菌vhhA溶血素基因序列,合成一个地高辛标记的VHH基因探针,利用其进行Southern Blot ,检测VHH溶血素基因在57株弧菌(包括26株国际标准菌株,20株哈维氏弧菌,11株副溶血弧菌)中的分布情况。结果显示,VHH基因探针与13株弧菌标准菌株有强杂交信号,包括2株溶藻胶弧菌(V.alginolyticus) ,2株哈维氏弧菌以及1株霍氏格里蒙菌(Grimontia hollisae) ,坎贝氏弧菌(V.campbellii) ,辛辛那提弧菌(V.cincinatiensis) ,费氏弧菌(V.fischeri) ,拟态弧菌(V.mimicus) ,飘浮弧菌(V.natriegens) ,副溶血弧菌,解蛋白弧菌(V.proteolyticus)和火神弧菌(V.logei)。与6株弧菌标准菌株有弱杂交信号,包括鳗弧菌(V.anguillarum) ,河口弧菌(V.aestuarianus) ,美人鱼发光杆菌(Photobacterium damselae subsp.damselae) ,河弧菌(V.fluvialis) ,弗尼斯弧菌(V.furnissii)和创伤弧菌(V.vulnificus) ,而另外7株弧菌标准菌株中无杂交信号。所有的哈维氏弧菌菌株至少含有一条杂交带,其中菌株VIB645 , VIB 648和SF-1分别含有2条杂交带。11株副溶血弧菌中均含有一条杂交带。上述数据表明,vhh/tlh溶血素基因广泛分布于弧菌中,尤其是哈维氏弧菌相关菌株和费氏弧菌相关菌株中。另外对鳗弧菌VIB 72 ,坎贝氏弧菌VIB 285 ,飘浮弧菌VIB 299和哈维氏弧菌VIB 647的vhh/tlh溶血素基因进行克隆并测序,其氨基酸序列与VHH溶血素和TLH溶血素氨基酸序列的同源性分别为67 %~99 %和69 %~91 %。对vhh/tlh溶血素基因在弧菌中的分布研究,将有助于进一步确定这类溶血素基因在病原弧菌致病性中的作用。     

肽酶PepT参与副溶血弧菌的浮游运动和生物被膜形成

pepT基因编码一种金属依赖性肽酶T (peptidase T,PepT),能特异性催化三肽N端氨基酸,因此也称为氨肽酶T。研究发现大多数氨肽酶参与细菌蛋白质新陈代谢和调节三肽活性,但关于PepT在细菌毒力及致病性方面的报道较少。[目的]本文选取PepT为研究对象,研究其对副溶血弧菌生物学特性及致病性的影响。[方法]通过构建缺失株ΔpepT和回补株CΔpepT,比较菌株在运动性、生物被膜、环境耐受、细胞毒性等方面的差异。[结果]与野生株相比,ΔpepT缺失株的极性鞭毛转录水平极显著下降,浮游运动能力降低;同时生物被膜形成能力减弱,而细菌群集运动及环境耐受能力无显著差异。此外,缺失pepT基因会导致副溶血弧菌的细胞毒性和小鼠毒力作用显著下降。[结论]pepT基因与副溶血弧菌浮游运动和生物被膜形成能力相关,并且影响其致病性。     

ToxR负调控副溶血弧菌中c-di-GMP的合成

副溶血弧菌(Vibrio parahaemolyticus)是世界范围内引起海产品相关食物中毒的主要致病菌,具有很强的生物膜形成能力。ToxR是一种膜结合调控蛋白,对副溶血弧菌生物膜形成具有一定的调控作用,但具体机制尚未见报道。c-di-GMP是一种普遍存在于细菌中重要的第二信使,参与调控细菌的多种生物学行为包括生物膜的形成。本文探究ToxR对副溶血弧菌中c-di-GMP代谢的调控作用。利用酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)测定副溶血弧菌野生株(wild-type,WT)和toxR突变株(ΔtoxR)中c-di-GMP水平的差异。挑选c-di-GMP代谢相关基因scrA、scrG和vpa0198为进一步研究的靶标,采用实时定量qPCR实验检测靶基因在WT和ΔtoxR中的转录水平差异;将靶基因调控区DNA序列克隆入pHRP309质粒中无启动子的β半乳糖苷酶基因上游,采用lacZ报告基因融合实验进一步研究ToxR对靶基因的转录调控关系;将重组质粒分别导入含有pBAD33或pBAD33-toxR的EC100lpir中,采用lacZ报告基因融合实验研究ToxR是否能在异体宿主中调控靶基因的表达;PCR扩增靶基因上游调控区DNA序列,并纯化His-ToxR蛋白,用凝胶阻滞实验(electrophoresis mobility shift assay,EMSA)研究His-ToxR与靶基因启动子区DNA序列是否具有结合作用。ELISA结果显示ΔtoxR中c-di-GMP含量显著性高于WT中的,说明ToxR抑制c-di-GMP的产生;实时定量qPCR结果表明WT中scrA、scrG和vpa0198的转录水平显著性高于ΔtoxR中的,表明ToxR抑制它们的转录;lacZ报告基因融合实验结果表明ToxR可抑制副溶血弧菌和EC100lpir中scrA、scrG和vpa0198的启动子区活性;EMSA实验显示His-ToxR能特异性地结合到scrA和scrG的上游调控区DNA序列上,而对vpa0198的上游调控区DNA序列无结合作用。综上所述,ToxR通过直接调控相关酶蛋白基因的转录来抑制副溶血弧菌内c-di-GMP的合成,从而有助于精确调控生物膜形成等细菌行为。     

浙江沿海地区海产品及环境中副溶血弧菌的分离与主要毒力基因分析

2007~2008年间, 我们调查了浙江沿海地区海产品和养殖环境中副溶血弧菌的污染状况, 并分析了不同来源副溶血弧菌中主要毒力相关基因tdh、trh、ureC和T3SS2(vscC2、vcrD2)的分布特征及溶血表型与尿素酶表型。结果显示, 566份样品中共分离到395株副溶血弧菌, 检出率高达70%, 毒力相关基因分析结果发现, tdh基因阳性率为10.1%, trh与ureC基因阳性率分别为 20.0%与 11.1%, 40株tdh+菌中组成T3SS2的vscC2基因阳性率为32.5%, 其中38株tdh+菌的神奈川试验亦呈阳性; 但在44株trh+-ureC+菌株中, 尿素酶表型阳性只有6株。试验表明, 浙江沿海地区海产品及其养殖环境中副溶血弧菌污染状况比较严重, 且有相当比例的菌株携带毒力或疑似毒力基因。研究结果为深入探索副溶血弧菌的致病性、基因结构与功能(或表型)及其分子演化提供基础。     

副溶血弧菌VP2918基因缺失株的构建及功能研究

[目的] 以副溶血弧菌VP2918为研究对象,研究其对副溶血弧菌的生物学特性和致病性的影响。[方法] 利用同源重组技术构建了vp2918基因的基因缺失株（Δvp2918）和互补株（CΔvp2918）,并对野生株、缺失株和互补株的细菌生长曲线、运动性、生物被膜形成能力、对HeLa细胞的黏附能力、细胞毒性、对小鼠的致死率和组织载菌量进行分析。[结果] 缺失vp2918基因不影响副溶血弧菌的生长特性、运动性、生物被膜形成能力以及对HeLa细胞的黏附能力。但与野生株相比,Δvp2918对HeLa细胞的毒性作用显著降低;感染Δvp2918的小鼠症状明显减轻,存活率更高;Δvp2918在小鼠脾脏和肝脏中的载菌量显著低于野生株,互补株毒力基本恢复至野生株水平。[结论] vp2918不参与副溶血弧菌的运动性和生物被膜形成能力等过程,但与该菌的致病性相关,为潜在的毒力因子。     

Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh.

Vibrio parahaemolyticus is an important human pathogen which can cause gastroenteritis when consumed in raw or partially-cooked seafood. A multiplex PCR amplification-based detection of total and virulent strains of V. parahaemolyticus was developed by targeting thermolabile hemolysin encoded by tl, thermostable direct hemolysin encoded by tdh, and thermostable direct hemolysin-related trh genes. Following optimization using oligonucleotide primers targeting tl, tdh and trh genes, the multiplex PCR was applied to V. parahaemolyticus from 27 clinical, 43 seafood, 15 environmental, 7 strains obtained from various laboratories and 19 from oyster plants. All 111 V. parahaemolyticus isolates showed PCR amplification of the tl gene; however, only 60 isolates showed amplification of tdh, and 43 isolates showed amplification of the trh gene. Also, 18 strains showed amplification of the tdh gene, but these strains did not show amplification of the trh gene. However, one strain exhibited amplification for the trh but not the tdh gene, suggesting both genes need to be targeted in a PCR amplification reaction to detect all hemolysin-producing strains of this pathogen. The multiplex PCR approach was successfully used to detect various strains of V parahaemolyticus in seeded oyster tissue homogenate. Sensitivity of detection for all three target gene segments was at least between 10(1)-10(2) cfu per 10 g of alkaline peptone water enriched seeded oyster tissue homogenate. This high level of sensitivity of detection of this pathogen within 8 h of pre-enrichment is well within the action level (10(4) cfu per 1 g of shell stock) suggested by the National Seafood Sanitation Program guideline. Compared to conventional microbiological culture methods, this multiplex PCR approach is rapid and reliable for accomplishing a comprehensive detection of V. parahaemolyticus in shellfish.     

Rapid detection and enumeration of trh-carrying Vibrio parahaemolyticus with the alkaline phosphatase-labelled oligonucleotide probe

An alkaline phosphatase (AP)-labelled oligonucleotide probe was developed to detect and enumerate trh(+)Vibrio parahaemolyticus in seafood. The probe was evaluated using 40 isolates of V. parahaemolyticus, 45 isolates of other vibrios and 55 non-vibrio isolates. The probe reacted specifically with V. parahaemolyticus possessing either the trh1 or trh2 variant of the trh gene and was found to be 100% specific for trh(+)V. parahaemolyticus. Using the trh probe, V. parahaemolyticus carrying trh gene was targeted in 34 seafood samples by direct plating and colony hybridization procedure. The trh(+)V. parahaemolyticus could be detected in five of 34 (14.7%) samples and the levels ranged from 5.0 x 10(2) to 3.4 x 10(3) cfu g(-1). Colonies of trh(+)V.parahaemolyticus were isolated from the five positive samples. Forty seafood samples were analysed for trh(+)V. parahaemolyticus by colony hybridization following enrichment in alkaline peptone water. 16 samples (40%) were positive for trh gene and trh(+)V. parahaemolyticus was isolated from 15 samples (37.5%). To assess the sensitivity of the trh probe, seafood homogenates spiked with known concentrations of trh-positive V. parahaemolyticus were plated and hybridized. Counts obtained using the probe were similar to those of inocula. The results suggest that the AP-labelled trh probe is useful for the detection and enumeration of trh(+)V. parahaemolyticus in seafood.     

Enzyme-labeled oligonucleotide probes for detection of the genes for thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus.

Alkaline phosphatase conjugated oligonucleotide probes were developed to detect the genes (tdh and trh) coding for the thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) of Vibrio parahaemolyticus. Using dot blot hybridization, probes were tested with 94 clinical isolates of V. parahaemolyticus. Results agreed well with those obtained using radio-labeled recombinant DNA probes for the genes tdh and trh. Specificity and sensitivity of enzyme tdh probes for detection of the trh gene were 100 and 93%, respectively, and those of the trh probes for trh gene detection were 93 and 86%, respectively. The tdh probes also hybridized with tdh-like genes processed by all strains of V. hollisae, and some strains of V. mimicus and V. cholerae non-O1, but neither tdh nor trh probes reacted with other bacterial species isolated from diarrheal stools. However, some V. parahaemolyticus strains that were negative with the enzyme trh probe hybridized weakly with a radio-labeled trh DNA fragment probe at medium stringency, and a few strains that were negative in high stringency conditions with a radio-labeled trh DNA fragment probe hybridized with the enzyme trh probe. This suggests that some strains of V. parahaemolyticus may carry another gene resembling trh.     

Soft-agar-coated filter method for early detection of viable and thermostable direct hemolysin (TDH)- or TDH-related hemolysin-producing Vibrio parahaemolyticus in seafood

A novel method for detecting viable and thermostable direct hemolysin (TDH)-producing or TDH-related hemolysin (TRH)-producing Vibrio parahaemolyticus in seafood was developed. The method involved (i) enrichment culture, selective for viable, motile cells penetrating a soft-agar-coated filter paper, and (ii) a multiplex PCR assay targeting both the TDH gene (tdh) and TRH gene (trh) following DNase pretreatment on the test culture to eradicate any incidental DNAs that might have been released from dead cells of tdh- or trh-positive (tdh+ trh+) strains and penetrated the agar-coated filter. A set of preliminary laboratory tests performed on 190 ml of enrichment culture that had been inoculated simultaneously with ca. 100 viable cells of a strain of tdh+ trh+ V. parahaemolyticus and dense populations of a viable strain of tdh- and trh-negative V. parahaemolyticus or Vibrio alginolyticus indicated that the method detected the presence of viable tdh+ trh+ strains. Another set of preliminary tests on 190 ml of enrichment culture that had been initially inoculated with a large number of dead cells of the tdh+ trh+ strain together with dense populations of the tdh- and trh-negative strains confirmed that the method did not yield any false-positive results. Subsequent quasi-field tests using various seafood samples (ca. 20 g), each of which was experimentally contaminated with either or both hemolysin-producing strains at an initial density of ca. 5 to 10 viable cells per gram, demonstrated that contamination could be detected within 2 working days.     

Serologic and molecular characterization of Vibrio parahaemolyticus strains isolated from seawater and fish products of the Gulf of Mexico

The thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the main virulence factors of Vibrio parahaemolyticus. We isolated V. parahaemolyticus from seawater, fish, and oysters obtained from the Pueblo Viejo Lagoon in Veracruz, determined the serogroups, phenotypically and genotypically characterized TDH and TRH, and investigated the presence of the toxR gene. A total of 46 V. parahaemolyticus strains were isolated, and all of them amplified the 368-bp toxR gene fragment. The trh gene was not identified in any of the strains; 4 of the 46 strains were Kanagawa phenomenon (KP) positive and amplified the 251-bp tdh gene fragment. The most frequent serogroup was serogroup O3. This is the first report of the presence of KP-positive tdh-positive environmental V. parahaemolyticus strains in Mexico.     

Molecular characterization of thermostable direct haemolysin-related haemolysin (TRH)-positive Vibrio parahaemolyticus from oysters in Mangalore, India

Pathogenic Vibrio parahaemolyticus strains producing either or both of a thermostable direct haemolysin (TDH) and a TDH-related haemolysin (TRH) encoded by tdh and trh genes, respectively, are isolated at a low rate from the environment. However, recently we observed that a considerable percentage of APW (alkaline peptone water) enrichment broths of oysters collected off Mangalore India, were trh(+), rather than tdh(+) by PCR. In order to further investigate the prevalence and genetic diversity of trh bearing V. parahaemolyticus in our coast, we attempted to isolate and characterize trh(+)V. parahaemolyticus from oysters. A total of 27 trh(+) strains were isolated during the period between March 2002 and February 2004, of which nine were also tdh(+). All the trh(+) isolates were positive for urease phenotype. The isolates belonged to diverse phenotypes. In order to explore the possible presence of heterogeneity in the trh gene region among trh(+)V. parahaemolyticus, a 1.5 kb region around trh gene was PCR amplified and restriction digested using selected restriction enzymes. The whole genome comparison of strains was performed by randomly amplified polymorphic DNA PCR (RAPD PCR). The PCR-RFLP results revealed fairly well conserved nature of the trh gene region studied in different serotypes. Though 11 strains were positive by PCR for a genomic fragment that has been reported to be amplified in pandemic strains, all strains were negative by group-specific PCR (GS-PCR), orf8 PCR and showed a different RAPD pattern compared with pandemic strains. The results suggest that genetically diverse V. parahaemolyticus carrying virulence genes are associated with the aquatic environment in this region.     

Analysis of the tdh gene cloned from a tdh gene- and trh gene-positive strain of Vibrio parahaemolyticus

A variant of the gene (tdh) encoding thermostable direct hemolysin (TDH) was cloned from the chromosome of Vibrio parahaemolyticus AQ3860, which gave positive results in the hybridization tests with the tdh gene probe and the trh (tdh-related hemolysin) gene probe and showed a low level of reaction in an enzyme-linked immunosorbent assay for TDH. Nucleotide sequence analysis of the cloned gene (tdh5) provided no evidence that tdh5 is evolutionally closer to the trh gene than the other tdh genes. The tdh5 gene was flanked by 40 base-pair sequences constituting perfect inverted repeats, which may suggest association of the tdh5 gene with insertion sequence-like structure. These results suggest that the tdh5 gene and the trh gene were not originally produced by gene duplication in AQ3860 but rather that one of the two genes moved into AQ3860 from an external source.     

Molecular,serological, and virulence characteristics of Vibrio parahaemolyticus isolated from environmental,food, and clinical sources in North America and Asia

Potential virulence attributes, serotypes, and ribotypes were determined for 178 pathogenic Vibrio parahaemolyticus isolates from clinical, environmental, and food sources on the Pacific, Atlantic, and Gulf Coasts of the United States and from clinical sources in Asia. The food and environmental isolates were generally from oysters, and they were defined as being pathogenic by using DNA probes to detect the presence of the thermostable direct hemolysin (tdh) gene. The clinical isolates from the United States were generally associated with oyster consumption, and most were obtained from outbreaks in Washington, Texas, and New York. Multiplex PCR was used to confirm the species identification and the presence of tdh and to test for the tdh-related hemolysin trh. Most of the environmental, food, and clinical isolates from the United States were positive for tdh, trh, and urease production. Outbreak-associated isolates from Texas, New York, and Asia were predominantly serotype O3:K6 and possessed only tdh. A total of 27 serotypes and 28 ribogroups were identified among the isolates, but the patterns of strain distribution differed between the serotypes and ribogroups. All but one of the O3:K6 isolates from Texas were in a different ribogroup from the O3:K6 isolates from New York or Asia. The O3:K6 serotype was not detected in any of the environmental and food isolates from the United States, and none of the food or environmental isolates belonged to any of the three ribogroups that contained all of the O3:K6 and related clinical isolates. The combination of serotyping and ribotyping showed that the Pacific Coast V. parahaemolyticus population appeared to be distinct from that of either the Atlantic Coast or Gulf Coast. The fact that certain serotypes and ribotypes contained both clinical and environmental isolates while many others contained only environmental isolates implies that certain serotypes or ribotypes are more relevant for human disease.     

Seasonal abundance of total and pathogenic Vibrio parahaemolyticus in Alabama oysters

Recent Vibrio parahaemolyticus outbreaks associated with consumption of raw shellfish in the United States focused attention on the occurrence of this organism in shellfish. From March 1999 through September 2000, paired oyster samples were collected biweekly from two shellfish-growing areas in Mobile Bay, Ala. The presence and densities of V. parahaemolyticus were determined by using DNA probes targeting the thermolabile hemolysin (tlh) and thermostable direct hemolysin (tdh) genes for confirmation of total and pathogenic V. parahaemolyticus, respectively. V. parahaemolyticus was detected in all samples with densities ranging from <10 to 12,000 g(-1). Higher V. parahaemolyticus densities were associated with higher water temperatures. Pathogenic strains were detected in 34 (21.8%) of 156 samples by direct plating or enrichment. Forty-six of 6,018 and 31 of 6,992 V. parahaemolyticus isolates from enrichments and direct plates, respectively, hybridized with the tdh probe. There was an apparent inverse relationship between water temperature and the prevalence of pathogenic strains. Pathogenic strains were of diverse serotypes, and 97% produced urease and possessed a tdh-related hemolysin (trh) gene. The O3:K6 serotype associated with pandemic spread and recent outbreaks in the United States was not detected. The efficient screening of numerous isolates by colony lift and DNA probe procedures may account for the higher prevalence of samples with tdh(+) V. parahaemolyticus than previously reported.