Seaweeds as a reservoir for diverse Vibrio parahaemolyticus populations in Japan

Gastroenteritis caused by Vibrio parahaemolyticus has recently been associated with foods prepared with seaweeds, but little is known about the bacterium's abundance and diversity among seaweeds in coastal environment. Therefore, we determined its phenotypic and genotypic diversity in relation to its seasonal abundance in seawater and seaweed samples from three areas of Kii Channel, Japan during June 2003 to May 2004. Isolates were obtained by selective enrichment of samples and detection of V. parahaemolyticus by colony hybridization with a species-specific probe. A total of 128 isolates comprising 16 from each source in each season were characterized by serotyping and ribotyping. V. parahaemolyticus was more abundant in seaweeds (3,762 isolates) than in water samples (2,238 isolates). Twenty and 17 serotypes were found among the selected seaweed and seawater isolates, respectively. Cluster analysis revealed 19, 11, 7 and 9 ribotypes during summer, autumn, winter and spring, respectively. Seaweeds supported a diverse V. parahaemolyticus population throughout the year and thus seaweeds are a reservoir for the organism. However, V. parahaemolyticus occurrence had positive correlation with water temperature and its abundance in seaweeds was at least 50 times higher during summer than in winter.     

Real-time PCR quantification of Vibrio parahaemolyticus in oysters using an alternative matrix

This study examined the relationship between levels of total Vibrio parahaemolyticus found in oyster tissues and mantle fluid with the goal of using mantle fluid as a template matrix in a new quantitative real-time PCR assay targeting the thermolabile hemolysin (tlh) gene for the enumeration of total V. parahaemolyticus in oysters. Oysters were collected near Mobile Bay, Ala., in June, July, and September and tested immediately after collection and storage at 26 degrees C for 24 h. Initial experiments using DNA colony hybridization targeting tlh demonstrated that natural V. parahaemolyticus levels in the mantle fluid of individual oysters were strongly correlated (r = 0.85, P < 0.05) with the levels found in their tissues. When known quantities of cultured V. parahaemolyticus cells were added to real-time PCR reactions that contained mantle fluid and oyster tissue matrices separately pooled from multiple oysters, a strong linear correlation was observed between the real-time PCR cycle threshold and the log concentration of cells inoculated into each PCR reaction (mantle fluid: r = 0.98, P < 0.05; and oyster: r = 0.99, P < 0.05). However, the mantle fluid exhibited less inhibition of the PCR amplification than the homogenized oyster tissue. Analysis of natural V. parahaemolyticus populations in mantle fluids using both colony hybridization and real-time PCR demonstrated a significant (P < 0.05) but reduced correlation (r = -0.48) between the two methods. Reductions in the efficiency of the real-time PCR that resulted from low population densities of V. parahaemolyticus and PCR inhibitors present in the mantle fluid of some oysters (with significant oyster-to-oyster variation) contributed to the reduction in correlation between the methods that was observed when testing natural V. parahaemolyticus populations. The V. parahaemolyticus-specific real-time PCR assay used for this study could estimate elevated V. parahaemolyticus levels in oyster mantle fluid within 1 h from sampling time.     

Seasonal distribution of total and pathogenic Vibrio parahaemolyticus in Chesapeake Bay oysters and waters

The objectives of this study were to investigate the seasonal distribution of total and pathogenic Vibrio parahaemolyticus in the Chesapeake Bay oysters and waters, and to determine the degree of association between V. parahaemolyticus densities and selected environmental parameters. Oyster and water samples were collected monthly from three sites in Chesapeake Bay, Maryland from November 2004 through October 2005. During collection of samples, water temperature, salinity, turbidity, dissolved oxygen, pH, chlorophyll a, and fecal coliform levels in oysters were also determined. V. parahaemolyticus levels were enumerated by a quantitative direct-plating method followed by DNA colony hybridization; presence/absence was further determined by overnight broth enrichment followed by either standard colony isolation or real-time PCR. The thermolabile hemolysin (tlh) gene and thermostable direct hemolysin (tdh) gene were targeted for detection of total and pathogenic V. parahaemolyticus, respectively, for both direct plating and enrichment. The thermostable related hemolysin (trh) gene, which is a presumptive pathogenicity marker, was targeted only for the enrichment approach. By direct plating, colonies producing tlh signals were detected in 79% of oyster samples at densities ranging from 1.5x10(1) to 6.0x10(2) CFU/g. Pathogenic V. parahaemolyticus (tdh+) was detected in 3% (level was 10 CFU/g) of oyster samples while no V. parahaemolyticus was detected in water samples. By the enrichment approach with standard colony isolation, 67% of oyster and 55% of water samples (n=33) were positive for total V. parahaemolyticus, and all samples were negative for pathogenic V. parahaemolyticus. In contrast, enrichment followed by real-time PCR detected tlh, tdh and trh in 100%, 20% and 40% of oyster and 100%, 13% and 40% of water enrichments collected from June to October 2005, respectively. V. parahaemolyticus densities in oysters varied seasonally and were found to be positively correlated with water temperature, turbidity, and dissolved oxygen.     

Pathogenetic characterization of Vibrio parahaemolyticus isolates from clinical and seafood sources

A total of 216 Vibrio parahaemolyticus isolates from seafood and clinical samples in eastern China were investigated for their hemolytic and urea-producing phenotypes, presence of putative virulence genes tdh and trh. Twenty-one clinical isolates (84%, 21/25) and 3 seafood isolates (1.57%, 3/191) were tdh-positive while only 3 clinical isolates (12%) and 7 seafood isolates (3.66%) were positive for trh gene. We further examined the pathogenicity of selected V. parahaemolyticus isolates in in vitro and in vivo systems. The clinical isolates were apparently more enteropathogenic (74.26 per thousand vs 62.07 per thousand expressed as intestine/body weight ratio, P<0.01) and more virulent than their seafood counterparts to mice (log LD(50) 6.86 vs 7.40 via orogastric route, P<0.05). They were also more adherent to in vitro cultured cells and of higher cytotoxicity as measured by LDH release of the HeLa cells although there were no statistical differences. The tdh-positive V. parahaemolyticus isolates were of higher enteropathogenicity (P<0.05, 74.24 per thousand vs 60.55 per thousand) and more virulent (log LD(50) 6.55 vs 7.21 via intraperitoneal route, P<0.05) than tdh-negative isolates. The tdh-positive isolates were generally more cytotoxic and adhesive to the cultured cell lines as well. From the in vitro and in vivo pathogenicity profiles, trh-positive isolates seemed to line between tdh-positive isolates and those without tdh and trh. There were two isolates H8 and H10 from clinical cases having moderate enteropathogenicity and virulence to mice, but were tdh-negative yet trh-positive. These results seem to suggest that hemolysins TDH and/or TRH may not be necessarily the only virulence factors of pathogenic V. parahaemolyticus isolates.     

Populations of Vibrio parahaemolyticus in retail oysters from Florida using two methods

Vibrio parahaemolyticus is a naturally occurring estuarine bacterium that is often associated with gastroenteritis in humans following consumption of raw molluscan shellfish. A number of studies have investigated the environmental distribution of V. parahaemolyticus, but little is known about the levels of this organism during distribution of oysters or at the point of consumption. Duplicate samples of shellstock oysters were collected monthly (September 1997 to May 1998) from the same four restaurants and three wholesale seafood markets in the Gainesville, Fla. area and analyzed for total V. parahaemolyticus densities using two methods: a standard MPN method (BAM-MPN) and a new direct plating procedure (direct-VPAP). Both methods employed an alkaline phosphatase-labeled DNA probe (VPAP) targeting the species-specific thermolabile hemolysin (tlh) gene to confirm suspect colonies as V. parahaemolyticus. The highest monthly geometric mean V. parahaemolyticus density was observed in October of 1997 (approximately 3,000/g) with similarly high values during September and November of 1997. From December 1997 to May 1998 mean densities were generally less than 100/g, falling to approximately 10/g in February and March. A strong correlation (r = 0.78) between the direct-VPAP and BAM-MPN methods for determining V. parahaemolyticus densities in market-level oysters was observed. The direct-VPAP method was more rapid and precise while the BAM-MPN was more sensitive and may better recover stressed cells. The utilization of the VPAP probe for identification of V. parahaemolyticus sharply reduced the labor for either method compared to biochemical identification techniques used in earlier V. parahaemolyticus surveys.     

珠江三角洲地区副溶血性弧菌遗传多样性和致病性相关研究

副溶血性弧菌(Vibiro parahaemolyticus)是一种常见的食源性致病菌,本文研究了珠江三角洲地区副溶血性弧菌的遗传多样性。从54株副溶血性弧菌出发,研究了它们的API20E生化反应、抗生素耐药性、O抗原血清型,进行了ERIC-PCR分子分型,并检测了两种毒力基因tdh和trh的分布。54株副溶血性弧菌可被分为6个生化反应类群,主要类群为Biochem-A;菌株对萘啶酮酸、环丙沙星、氯霉素均不耐药,而对氨苄青霉素耐药率最高,耐药率0.88;O抗原血清型分别为O1、O2、O3、O4、O5、O6、O8、O10、O11,O2为主要血清型,O3为临床主要血清型;ERIC-PCR分子分型将54株菌分成47个型别,ERIC-PCR图谱相似性大于0.80的类群有12个,没有明显的优势类群;有12株副溶血性弧菌为tdh阳性,阳性率为0.22,其中10株为临床来源菌株;有2株副溶血性弧菌为trh阳性,阳性率为0.04,均为食品分离株。珠三角地区食品和临床来源的副溶血性弧菌具有丰富的遗传多样性。     

Evaluation of nonisotopic DNA hybridization methods for detection of the tdh gene of vibrio parahaemolyticus

Production of the thermostable direct hemolysin (TDH) by Vibrio parahaemolyticus is associated with pathogenicity of the organism and is encoded by the tdh gene. The timely resolution of seafood-associated outbreaks requires rapid and accurate detection of pathogenic V. parahaemolyticus. The specificity of alkaline phosphatase- and digoxigenin-labeled tdh gene probes was evaluated against 61 strains of V. parahaemolyticus (including isolates from recent outbreaks involving oysters from the Pacific Northwest, Texas, and New York), 85 strains of other vibrios, and 7 strains of non-vibrio species from clinical and environmental sources. The probes were specific for detection of the V. parahaemolyticus tdh gene.     

食品中副溶血性弧菌toxR和tdh基因双色荧光PCR检测方法的建立

目的 建立对副溶血性弧菌（Vibrio parahaemolyticus）特异性检测toxR（跨膜转录激活蛋白）基因和tdh（热稳定性直接溶血素）毒力基因的Taqman探针双色荧光PCR检测方法。方法 根据副溶血性弧菌toxR基因和tdh基因,分别设计引物和探针,建立Taqman探针双色荧光PCR扩增体系,进行特异性、灵敏度试验;对副溶血性弧菌分离菌株实施检测,了解其tdh基因和tdh基因分布情况。结果 结果表明,副溶血性弧菌标准菌株和3株从食物中毒患者中分离获得的分离株均出现toxR基因和tdh扩增曲线,而溶藻弧菌、单增李斯特菌等31株弧菌属其他菌株和肠杆菌科的菌株未见扩增曲线。从食品中分离的37株副溶血性弧菌分离株均未携带tdh毒力基因。副溶血性弧菌检测灵敏度可达到3.6×102 cfu/mL。结论 该方法可用于同时检测食品中副溶血性弧菌的特异性和毒力基因。     

Detection and Enumeration of Vibrio vulnificus by Direct Colony Immunoblot

ABSTRACT:  A direct colony immunoblot method (DCI) for the enumeration of Vibrio vulnificus was developed. Bacterial colonies were transferred from agar plates to membranes, which were then dried and blocked with bovine serum albumin. Subsequently, the membranes were treated with anti- V. vulnificus H antibodies, washed and incubated with peroxidase-conjugated goat anti-rabbit IgG. After a final wash, the membranes were exposed to a substrate mixture containing H₂O₂ which resulted in the development of a purple color by V. vulnificus colonies. The DCI detected all clinical and environmental V. vulnificus strains tested and did not cross-react with other Vibrio species including V. cholerae , V. parahaemolyticus , or V. fluvialis . The DCI was then compared to the DNA hybridization procedure (DNAH) using V. vulnificus agar plates inoculated with mixed cultures of V. vulnificus and V. parahaemolyticus and V. vulnificus -seeded oyster homogenates. Both DCI and DNAH detected 1 to 2 log colony forming units (CFU)/mL V. vulnificus mixed with 4 log CFU/mL V. parahaemolyticus . Both methods were comparable and demonstrated no significant statistical differences when enumerating V. vulnificus in mixed cultures or in oyster homogenates seeded with levels of V. vulnificus from 2 to 6 log CFU/mL. The DCI demonstrated clearer color development and was less time consuming than the DNAH.     

A rapid and improved method for the detection of Vibrio parahaemolyticus and Vibrio vulnificus strains grown on hydrophobic grid membrane filters

DNA probe-based detection methods were developed and characterized as an alternative to time-consuming and less specific conventional protocols. Digoxigenin-labeled probes were prepared by polymerase chain reaction amplification of the targeted sequences in the specific amplicons generated from genomic DNA. Specific probes with high yields were generated for the detection of the tlh gene of Vibrio parahaemolyticus and the cth gene of V. vulnificus. Colony (Southern) hybridization analyses were carried out using hydrophobic grid membrane filters (HGMFs) to allow biotype-specific differentiation of the two species. Eight strains of V. vulnificus and five strains of V. parahaemolyticus, including one standard (ATCC) strain of each biotype, were examined. Colony lysis, hybridization, and nonradioactive detection parameters were optimized for identification of the target biotypes arranged on the same HGMF and also on a conventional nylon membrane, thereby confirming the specificity of the probes and the comparative usefulness of the HGMFs. The experimental procedure presented here can be completed in 1 day. The protocol was designed specifically to identify the target Vibrio spp. and could potentially be used for the enumeration and differentiation of V. parahaemolyticus and V. vulnificus in foods.     

Development and evaluation of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Vibrio parahaemolyticus

Loop-mediated isothermal amplification (LAMP) assays targeting the rpoD and toxR genes were developed to detect Vibrio parahaemolyticus. All 78 tested V. parahaemolyticus strains yielded positive results within 40 min, while negative results were obtained for 69 strains of other organisms even at 60 min. For V. parahaemolyticus ATCC 17802 in pure culture, the detection limits of LAMP assays targeting rpoD and toxR were 3.7 and 450 CFU per test, respectively. Due to the higher sensitivity of rpoD-LAMP, it was further evaluated for the ability to detect V. parahaemolyticus in seafood samples. V. parahaemolyticus populations spiked in short-necked clams were enumerated by the most-probable-number (MPN) method combined with the rpoD-LAMP assay and the MPN method with a culture method using agar medium. The MPN-rpoD-LAMP method had better sensitivity and was more rapid than the conventional method. These results indicate that the MPN-LAMP assay targeting the rpoD gene is a specific, sensitive, and rapid method to enumerate V. parahaemolyticus organisms.     

Oyster-to-oyster variability in levels of Vibrio parahaemolyticus

This study examined the variability in the levels of total and pathogenic Vibrio parahaemolyticus in individual oysters. Twenty oysters were collected on three occasions (in June, July, and September 2001) from a site near Mobile Bay, Ala. Ten of these oysters were tested immediately, and 10 were tested after 24 h of storage at 26 degrees C. Levels of total and pathogenic V. parahaemolyticus were determined by alkaline phosphatase-labeled DNA probe procedures targeting the thermolabile hemolysin and thermostable direct hemolysin genes, respectively. Similar V. parahaemolyticus levels (200 to 2,000 CFU/g) were found in nearly 90% of the oysters (for all sampling occasions) prior to storage. The log-transformed densities (means +/- standard deviations) of V. parahaemolyticus in oysters immediately after harvest were 2.90 +/- 0.91, 2.88 +/- 0.36, and 2.47 +/- 0.26 log10 CFU/g for June, July, and September, respectively. After storage for 24 h at 26 degrees C, the mean V. parahaemolyticus densities increased approximately 13- to 26-fold. Before storage, pathogenic V. parahaemolyticus was detected in 40% (10 to 20 CFU/g) of the oysters collected in June and July but was not detected in any oysters collected in September. After storage, pathogenic V. parahaemolyticus was detected in some oysters at levels of > 100 CFU/g. These data should aid in the development of sampling protocols for oyster monitoring programs and in the determination of exposure distributions associated with raw oyster consumption.     

双层平板直接定性定量法检测海产品中副溶血性弧菌

目的:建立一种直接定量定性检测海产品中副溶血性弧菌的方法。方法:参照国标菌落计数方法,筛选具备计数和显示副溶血性弧菌典型菌落颜色功能的培养基,将人工染菌样品分别置于50、4和-18℃温度下并分别进行测试。结果:在NaCl NA双层平板上副溶血性弧菌菌落呈淡紫色,在人工染菌样品实验中与NaCl NA平板计数无显著差异;NaCl NA双层平板直接定量检测4℃冷藏处理和-18℃冷冻的样品中副溶血性弧菌浓度分别是对照组的68.9%和21.7%。结论:NaCl NA双层平板法具备操作简便、检验周期短、结果稳定、灵敏度高等特点,适合应用于-18~50℃温度环境下放置的海产品中副溶血性弧菌直接定性定量检测,能如实反映样品受污染的情况。     

巢式PCR快速检测海产品中的副溶血弧菌

副溶血弧菌是一种世界范围性的食源性致病菌,食用了该菌污染的海产品可导致胃肠炎等疾病。为了建立一种可快速、特异地检测海产品中副溶血弧菌的方法,通过把副溶血弧菌基因组序列和其它不同种类弧菌的基因组序列进行比较分析,筛选出了一个副溶血弧菌特异性的标记基因-VP1331,根据该基因建立了副溶血弧菌的巢式PCR快速检测方法,并评估了其特异性、敏感性和稳定性。实验结果表明,该方法只有在以副溶血弧菌基因组DNA为模板时才能扩增出目的片段,而其它11种弧菌和非弧菌均不能扩增出目的片段。该方法的最低检测限为副溶血弧菌基因组DNA 10 fg、纯培养物6.6 CFU。人工污染实验表明,初始菌液浓度为25.7 CFU/100 mL时只需经过2 h的增菌培养即可检出。上述结果表明,VP1331基因可以作为副溶血弧菌种特异性标记,本方法可以用于污染海产品中该菌的检测与鉴定。     

Development of a quantitative real-time PCR method for estimation of the total number of Vibrio parahaemolyticus in contaminated shellfish and seawater

A real-time PCR method targeting the toxR gene of Vibrio parahaemolyticus was developed to quantify the number of V. parahaemolyticus cells, including those of both the hemolysin-producing and nonproducing strains. The specificity of the primer and probe set was confirmed using 25 strains of V. parahaemolyticus and 30 strains of other microbial species. We determined the threshold cycle number using the real-time PCR and the number of V. parahaemolyticus cells by plate count using serially diluted pure culture and developed a standard curve for quantification. Standard curves for V. parahaemolyticus in seawater and seafood were established using artificially inoculated samples. The threshold cycle number and the number of V. parahaemolyticus cells were correlated with 10(1) to 10(7) CFU/ml in pure culture, seawater, and shellfish homogenate. The real-time PCR method developed in this study was compared with the most-probable-number method in seafood samples that were naturally contaminated. The differences in the number of V. parahaemolyticus cells as determined by the culture method and the PCR method were less than 10-fold.     

Western 斑点印迹法技术检测致病性副溶血弧菌

目的：对副溶血弧菌(Vibrio parahaemolyticus,VP)的直接耐热溶血毒素(thermostable direct hemolysin,TDH)进行分离纯化,建立Western 斑点印迹法技术检测致病性副溶血弧菌的方法。方法：用直接耐热溶血毒素免疫小鼠得到特异性抗血清,利用棋盘方法确定免疫血清最佳工作浓度,并对致病性与非致病性副溶血弧菌分别进行特异性测定。结果：最佳免疫血清最佳工作浓度为1:3200;检测致病性副溶血弧菌为阳性,非致病性副溶血弧菌属呈阴性。结论：Western 斑点印迹法技术可以特异性检测致病性副溶血弧菌,灵敏度高、操作简单,可用肉眼判定结果。     

三重real-time PCR检测副溶血弧菌主要毒力基因

针对编码副溶血弧菌的特异性基因和主要毒力基因tlh、tdh、ureR进行引物及探针设计,通过优化反应条件,建立基于Taqman探针快速检测副溶血弧菌毒力基因的三重实时聚合酶链式反应（real-time polymerase chain reaction,real-time PCR）方法。利用10 株副溶血弧菌和22 株非副溶血弧菌对设计的引物及探针进行特异性验证,结果表明设计的引物及探针具有很高的特异性;优化后的引物浓度分别为tdh 0.15 μmol/L、tlh 0.15 μmol/L、ureR 0.80 μmol/L,探针浓度分别为HEX 0.50 μmol/L、FAM 0.50 μmol/L;该方法即使在高浓度的背景细菌存在下,DNA浓度与Ct值均呈良好的线性关系,检出限为1.8×102 拷贝/mL;以完整菌细胞的悬液为模板时,预变性时间为30 min,扩增检测效果与以基因组DNA（gDNA）为模板相当（ΔCt＜1）。本研究建立的三重real-time PCR方法能实现快速定量检测副溶血弧菌,并能有效区分致病性及非致病性副溶血弧菌,为副溶血弧菌的快速定量检测和风险评估提供快速、灵敏、准确的方法。     

Role of the rpoS gene in the survival of Vibrio parahaemolyticus in artificial seawater and fish homogenate

Vibrio parahaemolyticus is a foodborne pathogen isolated from coastal waters of the United States and from a variety of seafood, including fish. Seawater represents a nutrient-limiting environment for V. parahaemolyticus. During its persistence in seawater, V. parahaemolyticus is exposed to a variety of environmental stresses, including hyperosmolarity, fluctuations in temperature, and cold stress. The alternate sigma factor of RNA polymerase, designated as (RpoS), encoded by the gene rpoS has been shown to play a major role in bacterial adaptive responses to adverse environmental conditions. The present study was undertaken to investigate the role of rpoS in the survival of V. parahaemolyticus in seawater and fish. A V. parahaemolyticus rpoS mutant was constructed by the insertion of a chloramphenicol acetyltransferase gene cassette within the rpoS gene, and the wild and mutant strains were assayed for their ability to survive in artificial seawater (ASW) at 6 and 1 degrees C and in fish homogenate at 4 and 8 degrees C. The survival of the rpoS mutant of V. parahaemolyticus both in ASW and fish homogenate at either storage temperature was significantly (P < 0.05) lower than that of the wild strain. Further, the viability of V. parahaemolyticus, especially the mutant, was significantly reduced at lower storage temperatures of ASW and fish homogenate. Results of this study indicate that rpoS potentially plays an important role in the survival of V. parahaemolyticus under conditions of cold stress and hyperosmolarity.     

北部湾茅尾海副溶血性弧菌的分离鉴定与致病性分析

为解析北部湾海域及水产品中副溶血性弧菌的多样性特征与安全风险,本研究采集了北部湾茅尾海养殖区域海水和水产经济动物样品,利用硫代硫酸盐柠檬酸盐胆盐蔗糖琼脂培养基(TCBS)对所采样品进行海洋弧菌的分离和纯化,共分离获得109株疑似弧菌菌株。通过16S rDNA和特异功能基因toxR的PCR扩增并测序鉴定,共检出副溶血性弧菌20株,检出率为18.3%。此外,通过系统发育分析还发现副溶血性弧菌的toxR和tdh基因序列都存在水平基因转移现象,呈现出较大的多样性。对20个副溶血性弧菌菌株的毒力基因tdh进行分析,结果表明有4株携带了tdh毒力基因,检出率为20%,易引起食物中毒,对公共卫生造成的威胁较大。因此,本研究建议采用PCR技术开展副溶血性弧菌特异种属基因和毒力基因检测,准确评估北部湾区域海水及其水产品的卫生安全性,降低爆发水产养殖业病害和食源性疾病的风险。