Response of Vibrio parahaemolyticus to ethanol shock

In this study, the growth and survival of Vibrio parahaemolyticus in the presence of 0.0-8.0% ethanol was first examined. V. parahaemolyticus was then exposed to a sub-lethal dose of 5.0% ethanol for 30 and 60 min (ethanol shock). Morphological changes and alterations in cell leakage, thermal tolerance at 47 degrees C, and susceptibility to 8% ethanol and low temperature (4 and -18 degrees C) of V. parahaemolyticus caused by ethanol shock were investigated. In addition, recoveries of the ethanol-shocked cells of V. parahaemolyticus on thiosulfate-citrate-bile salts-sucrose agar (TCBS) and TSA-3.0% NaCl were also compared. The findings revealed that the presence of ethanol in TSB-3.0% NaCl at 6.0-8.0% and 5.0% or less, exerted bactericidal and partial growth inhibition effect, respectively, on V. parahaemolyticus. Recovery of ethanol-shocked cells of V. parahaemolyticus was significantly (P<0.05) less on TCBS than on TSA-3.0% NaCl. A significantly (P<0.05) marked increase of protein and nucleic acid material in the supernatant of cell suspension was found after cells of V. parahaemolyticus were exposed to ethanol shock. Extensive cell disruption, wrinkling and cell-wall pitting, indicative of cell-surface damage were also noted on the ethanol-shocked cells. Ethanol-shocked cells of V. parahaemolyticus exhibited a similar yet higher susceptibility at 4 and -18 degrees C compared with the control cells. Moreover, there was a marked increase in the thermal tolerance and resistance to 8.0% ethanol with cells of V. parahaemolyticus after ethanol shock. Finally, the duration of ethanol shock testing did not affect the extent of increased thermal tolerance. While cells of V. parahaemolyticus subjected to ethanol shock for 60 min showed an increase in their resistance to 8.0% ethanol, they also showed an increase in susceptibility at -18 degrees C, than those ethanol shocked for 30 min.     

耐超高压胁迫副溶血性弧菌的逆境耐受性

目的：探讨水产品中耐超高压胁迫的副溶血性弧菌对高压及其他逆境环境的耐受性。方法：以80～250 MPa超高压处理原始敏感菌株（Vibrio. parahaemolyticus ZJGSMC001）,筛选得到耐高压菌株（V. parahaemolyticus ZJGSPR001）,以其他逆境处理,分析二者对高压和其他逆境的耐受性差异。结果：以250 MPa压力胁迫处理,耐压菌株存活量比原始菌株高2（lg（CFU/mL））。3 ℃以下原始菌株生长速率为负值,45 ℃以上原始菌株不能存活,耐压菌株则生长良好,1 ℃和48 ℃时仍可存活。在NaCl质量浓度高达11.5 g/100 mL时,原始菌株为负生长,耐压菌株仍能生长。耐压菌株对有机溶剂和有机酸的耐受性增强,其中乙醇体积分数12%、丙酮体积分数9%、甲苯体积分数0.75%、柠檬酸3 mg/mL和乳酸体积分数1.5%时,原始菌株全部致死,耐压菌株仍存活。结论：原始菌株对压力较敏感,而耐压菌株经超高压胁迫处理后,除对高压的耐受能力提高外,对其他逆境（如温度、氯化钠、有机溶剂、有机酸）的耐受性也有所增强。     

Response of heat-shocked Vibrio parahaemolyticus to subsequent physical and chemical stresses

Vibrio parahaemolyticus foodborne strains 405, 556, and 690 and a V. parahaemolyticus chopping board isolate were heat shocked at 42 degrees C for 15, 30, or 45 min. Heat shock, regardless of heating periods tested, caused an increased demand for NaCl during recovery from heat injury. Further study with strain 690 and the chopping board isolate also revealed that heat shock generally increased the survival of the test organism during subsequent exposure to 47 degrees C, 20 ppm H202, and 8% ethanol and reduced the tolerance of the test organism to low temperatures (5 and -18 degrees C). The extent of the heat shock response of V. parahaemolyticus varied with strain and the duration of treatment. Furthermore, heat shock treatments in the present study caused the leakage of nucleic acids from V. parahaemolyticus cells. This effect was most pronounced with cells heat shocked at 42 degrees C for 45 min.     

Response of Vibrio parahaemolyticus 03:K6 to a hot water/cold shock pasteurization process.

Vibrio vulnificus and V. parahaemolyticus are natural inhabitants of estuarine environments world wide. Pathogenic strains of these bacteria are often transmitted to humans through consumption of raw oysters, which flourish in the same estuaries. Previous studies reported the effective use of hot water pasteurization followed by cold shock to eliminate from raw oysters naturally and artificially incurred environmental strains of V. vulnificus and V. parahaemolyticus common to the Gulf of Mexico. The present study focused on the use of the same pasteurization method to reduce a highly process resistant Vibrio strain, V. parahaemolyticus O3:K6 to non-detectable levels. Oysters were artificially contaminated with 10(4) and 10(6) V. parahaemolyticus 03:K6 cfu g(-1) oyster meat. Contaminated oysters were pasteurized between 50 and 52 degrees C for up to 22 min. Samples of processed oysters were enumerated for V. parahaemolyticus O3:K6 at 2-min intervals beginning after the 'come-up time' to achieve an oyster internal temperature of at least 50 degrees C. The D value (D(52)deg C) was 1.3-1.6 min. V. parahaemolyticus O3:K6 proved more process resistant than non-pathogenic environmental strains found in Gulf of Mexico waters. A total processing time of at least 22 min at 52 degrees C was recommended to reduce this bacterium to non-detectable levels (< 3 g(-1) oyster meat).     

Ethanol shock changes the fatty acid profile and survival behavior of Vibrio parahaemolyticus in various stress conditions

Vibrio parahaemolyticus 690, a clinical strain, was subjected to ethanol shock in the presence of 5% ethanol for a period of 30 and 60 min. Survival behaviors of the ethanol shocked and control cells of V. parahaemolyticus in the presence of H(2)O(2) (20 ppm), crystal violet (3 ppm), NaCl (20%), and low pH solution (pH 4.4) containing various organic acids including lactic acid, acetic acid, citric acid and tartaric acid (25 mM) were compared. In addition, the effects of ethanol shock on the fatty acid profile and recovery of V. parahaemolyticus on tryptic soy agar (TSA) plus various amounts of NaCl were also investigated. After ethanol shock, it was found that the proportion of vaccenic acid (18:1) increased, while the proportion of palmitic acid (16:0) and ratio of saturated fatty acid to unsaturated fatty acid decreased in cells of V. parahaemolyticus. The recovery of the ethanol-shocked cells on TSA plus 6.0% or 7.5% NaCl was significantly less than the control cells. Furthermore, ethanol shock increased the survival of V. parahaemolyticus in the presence of H(2)O(2), while made the test organism less resistant to crystal violet, high NaCl and organic acids. The degree of decreased acid tolerance observed on the ethanol-shocked cells of test organism varied with the organic acid examined. Finally, ethanol shock for 60 min exhibited either a higher or similar degree of effect on the test organism (depending on the type of stress encountered) than did ethanol shock for 30 min. Data obtained from the present study does provide useful information that is indispensable when control measure of V. parahaemolyticus is to be performed efficiently and adequately.     

An Overview of Vibrio vulnificus and Vibrio parahaemolyticus

ABSTRACT: The Vibrionaceae are environmentally ubiquitous to estuarine waters. Two species in particular, V. vulnificus and V. parahaemolyticus, are important human pathogens that are transmitted by the consumption of contaminated molluscan shellfish. This document provides a comprehensive review of the current state of knowledge about these important foodborne disease agents. Topics include the epidemiology of human disease; biotypes and virulence factors; cultural and molecular‐based detection methods; phenotyping and genotyping approaches; microbial ecology; and candidate control strategies. Recent international risk assessment efforts are also described. The reader will gain an understanding of why these organisms pose a public health risk and how improving our understanding of their behavior in the environment and the host can aid in reducing that risk in the future.     

不同副溶血性弧菌菌株成膜能力及成膜影响因子的研究

目的:研究不同来源副溶血性弧菌的生物膜形成及粘附情况,并对多因素影响副溶血性弧菌成膜进行探究。方法:采用改良试管法和微孔板法对不同影响因子(p H 6.5~9.5;Na Cl 2%~4%)下21株副溶血性弧菌的生物膜粘附程度和生物膜量进行测定,然后运用改良微孔板法对成膜效果较好的菌株以及标准菌株Vp BJ1.1616的成膜特性进行初步研究。结果:21株菌在实验条件下成膜能力与菌株来源和细菌致病性并没有表现出明显相关性,且Vp F29成膜效果最好;在p H6.5~9.5,Na Cl 2%~4%条件下,不同菌株平均OD570nm值变异系数(CV=标准偏差/平均值)分别从0.357变化到0.447,从0.383变化到0.472;在胰蛋白胨大豆肉汤(TSB)浓度为2.4%~9.6%时,Vp F29在32~37℃条件以及Vp BJ1.1616在37℃条件下都能形成大量生物膜。结论:不同环境条件下的成膜情况说明,副溶血弧菌成膜能力的强弱具有菌株多样性以及特性规律,为研究生物膜引起的食品安全问题提供理论依据。      

应用三种方法观察副溶血性弧菌生物被膜

采用三种方法(结晶紫染色法、荧光显微镜法、扫描电镜法)观察了两株分离自食品中的副溶血性弧菌Vp8和Vp32在4℃条件下的生物被膜形成情况,旨在为进一步研究低温条件下副溶血性弧菌生物被膜提供依据。结果表明:三种观察方法所得结果基本一致,Vp8有大量生物被膜形成,而Vp32几乎没有生物被膜形成。结论:在副溶血性弧菌生物被膜的研究中,各种观察方法各有利弊,可根据不同实验室条件或不同研究需求选择合适的观察方法。      

应用三种方法观察副溶血性弧菌生物被膜

采用三种方法(结晶紫染色法、荧光显微镜法、扫描电镜法)观察了两株分离自食品中的副溶血性弧菌Vp8和Vp32在4℃条件下的生物被膜形成情况,旨在为进一步研究低温条件下副溶血性弧菌生物被膜提供依据。结果表明:三种观察方法所得结果基本一致,Vp8有大量生物被膜形成,而Vp32几乎没有生物被膜形成。结论:在副溶血性弧菌生物被膜的研究中,各种观察方法各有利弊,可根据不同实验室条件或不同研究需求选择合适的观察方法。     

Survival of Vibrio parahaemolyticus during freezing

The ability of Vibrio parahaemolyticus to survive on frozen rock lobster tails was investigated. Rock lobster tails were artificially inoculated with the organism and were stored at ? 18°C. Vibrio died off within 1 week at ?18°C in lightly contaminated tails but a heavy infection survived for a few months at this temperature. Recovery depended on the degree of infection, the presence of other bacteria and the selectivity of the medium.     

PCR-焦磷酸测序检测副溶血性弧菌研究

目的：建立用DNA碱基序列检测副溶血性弧菌(Vibrio parahaemolyticus,VP )的方法。方法：根据副溶血性弧菌toxR基因设计扩增引物和测序引物,先用PCR特异性地扩增目的片段,再制备单链模版在测序引物引导下用焦磷酸测序法检测DNA碱基序列,检测的序列为CCAA GGATTCACAGCAGAAGCCACAGGTGCTTTT,则判断为副溶血性弧菌。结果：扩增引物和测序引物表现出良好的特异性,PCR扩增结果,20株VP均扩增出大小137bp的DNA片段,而创伤弧菌等对照菌株未扩增出DNA条带。焦磷酸测序结果,20株副溶血性弧菌均测出与预期相符的DNA碱基序列,而其他对照菌株未测出DNA 碱基序列或检测结果与测序引物后的序列不匹配。VP标准菌株ATCC 17802试验菌株发生A-T突变,密码子CCU变成CCA,而两个密码子都是脯氨酸的密码子。结论：建立的方法特异性高, 整个试验可在21h-27h完成,是快速检测VP有效手段。     

Effects of electrolyzed oxidizing water treatment on reducing Vibrio parahaemolyticus and Vibrio vulnificus in raw oysters

Contamination of Vibrio parahaemolyticus and Vibrio vulnificus in oysters is a food safety concern. This study investigated effects of electrolyzed oxidizing (EO) water treatment on reducing V. parahaemolyticus and V. vulnificus in laboratory-contaminated oysters. EO water exhibited strong antibacterial activity against V. parahaemolyticus and V. vulnificus in pure cultures. Populations of V. parahaemolyticus (8.74 x 10(7) CFU/ml) and V. vulnificus (8.69 x 10(7) CFU/ml) decreased quickly in EO water containing 0.5% NaCl to nondetectable levels (> 6.6 log reductions) within 15 s. Freshly harvested Pacific oysters were inoculated with a five-strain cocktail of V. parahaemolyticus or V. vulnificus at levels of 10(4) and 10(6) most probable number (MPN)/g and treated with EO water (chlorine, 30 ppm; pH 2.82; oxidation-reduction potential, 1131 mV) containing 1% NaCl at room temperature. Reductions of V. parahaemolyticus and V. vulnificus in oysters were determined at 0 (before treatment), 2, 4, 6, and 8 h of treatment. Holding oysters inoculated with V. parahaemolyticus or V. vulnificus in the EO water containing 1% NaCl for 4 to 6 h resulted in significant (P < 0.05) reductions of V. parahaemolyticus and V. vulnificus by 1.13 and 1.05 log MPN/g, respectively. Extended exposure (> 12 h) of oysters in EO water containing high levels of chlorine (> 30 ppm) was found to be detrimental to oysters. EO water could be used as a postharvest treatment to reduce Vibrio contamination in oysters. However, treatment should be limited to 4 to 6 h to avoid death of oysters. Further studies are needed to determine effects of EO water treatment on sensory characteristics of oysters.     

水飞蓟素对副溶血性弧菌的抑制作用

目的 以两种副溶血性弧菌为研究对象,探究水飞蓟素对于副溶血性弧菌的抑制及其作用机制。方法 通过琼脂板稀释法和肉汤稀释法确定水飞蓟素对两株菌的最小抑制浓度（MIC）,然后进一步通过研究水飞蓟素在不同培养基上处理后的副溶血性弧菌的生长曲线、生物膜形成情况、细胞膜完整性及钾离子外流情况的变化,探究水飞蓟素在不同培养基上对副溶血性弧菌的抑制作用及可能的作用机制。结果 水飞蓟素能够抑制生物膜的形成,损害细胞膜,导致细胞变形,显著增加钾离子外流,来抑制副溶血性弧菌的生长,并且不同培养基的抑制作用是不一样的。结论 水飞蓟素能够对副溶血性弧菌产生较强的抑制作用,有作为天然的抗菌物质应用于食品工业的潜力。     

副溶血弧菌的磁珠捕获及检测

建立结合免疫磁珠和PCR快速检测副溶血性弧菌的新方法。本研究选用副溶血弧菌作为抗原免疫日本大耳兔制备了多克隆抗体,用ELISA法检测抗体效价。利用辛酸硫酸铵沉淀结合逆向吸附对多抗血清进行纯化,经SDS-PAGE和斑点杂交检测验证纯化后抗体的特异性。将纯化后的抗体偶联于羧基化修饰的纳米磁珠表面,用来进行菌体的捕获。结果表明,抗体的效价达到106,纯化获得只与副溶血弧菌发生反应的高纯度抗体。制备的免疫磁珠捕获率在55%～70%之间,且稳定性较好,盐离子浓度（3.5%NaCl、4.5%NaCl）对捕获率的影响不大,在pH5～9之间,磁珠捕获差异也较小。在对纯培养物的检测实验中,本方法最低检测限为102cfu/mL;而在鱼肉糜样本中的检测限为103cfu/mL,且样品中高浓度杂菌的存在不影响检测灵敏度。免疫磁珠结合PCR用于副溶血性弧菌的检测具有很高的灵敏度和特异性,具有广阔的前景。      

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.     

柠檬醛对副溶血性弧菌的抑制作用

有效预防和控制副溶血性弧菌对食品的污染对于保障公众健康具有重要意义。本研究首先利用琼脂稀释法测定7?种植物源活性物质（丁香酸、阿魏酸、绿原酸、硫辛酸、原儿茶酸、原儿茶醛和柠檬醛）对副溶血性弧菌的最小抑菌浓度,在此基础上选择柠檬醛进行后续实验;通过检测经柠檬醛处理后的副溶血性弧菌生长曲线、细胞膜电位、胞内ATP浓度、细胞膜完整性以及细胞形态的变化,探究柠檬醛对副溶血性弧菌的抑制作用及可能的作用机理。结果表明：柠檬醛相比于其他6?种植物源活性物质对副溶血性弧菌有更好的抑菌效果,其对两株标准菌株和8?株分离菌株的最小抑菌浓度在0.10～0.60?mg/mL范围内;柠檬醛能够引起副溶血性弧菌细胞膜电位去极化、胞内ATP浓度降低及细胞膜完整性下降,同时可使细胞皱缩变形。本研究结果表明柠檬醛具有良好的抑菌功效,并有潜力作为天然的抗菌物质应用于食品工业。     

Reductions of Vibrio parahaemolyticus in Pacific oysters (Crassostrea gigas) by depuration at various temperatures

Consumption of raw oysters has been linked to several outbreaks of Vibrio parahaemolyticus infection in the United States. This study investigated effects of ice storage and UV-sterilized seawater depuration at various temperatures on reducing V. parahaemolyticus in oysters. Raw Pacific oysters (Crassostrea gigas) were inoculated with a mixed culture of five clinical strains of V. parahaemolyticus (10290, 10292, 10293, BE 98-2029 and 027-1c1) at levels of 10⁴⁻⁶ MPN/g. Inoculated oysters were either stored in ice or depurated in recirculating artificial seawater at 2, 3, 7, 10, 12.5, and 15 °C for 4–6 days. Holding oysters in ice or depuration of oysters in recirculating seawater at 2 or 3 °C for 4 days did not result in significant reductions (P > 0.05) of V. parahaemolyticus in the oysters. However, depuration at temperatures between 7 and 15 °C reduced V. parahaemolyticus populations in oysters by >3.0 log MPN/g after 5 days with no loss of oysters. Depuration at refrigerated temperatures (7–15 °C) can be applied as a post-harvest treatment for reducing V. parahaemolyticus in Pacific oysters.     

副溶血弧菌的多重PCR检测

副溶血弧菌是一种致病性弧菌,毒力因子主要有不耐热溶血毒素、耐热直接溶血毒素和相对耐热直接溶血毒素三种。本实验根据FDA标准推荐的引物对其进行了检验,发现副溶血弧菌环境分离株中没有耐热直接溶血毒素和相对耐热直接溶血毒素基因,仅有不耐热溶血毒素基因。     

双重DPO-PCR检测副溶血弧菌和霍乱弧菌

根据副溶血弧菌collagenase基因和霍乱弧菌omp W基因,分别设计特异性DPO（dual priming oligonucleotide）引物,建立一种快速检测这两种弧菌的多重DPO-PCR方法,并对其特异性和灵敏度进行了评价。结果显示,设计的DPO引物特异性较强,副溶血弧菌和霍乱弧菌DNA可分别扩增出307 bp与463 bp的特异性条带,检测灵敏度均达0.1 ng/μL。该检测方法对退火温度不敏感。利用该方法对69株疑似弧菌菌株进行鉴定,结果与生理生化鉴定结果一致。该方法特异性强、灵敏度高,适合于对食品、水产品等中副溶血弧菌和霍乱弧菌的进行快速筛检。