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.     

Effects of heat-degraded sugars on survival and growth of Vibrio parahaemolyticus and other bacteria

We studied the effects of autoclaved (121 degrees C, 15 min) sugar solutions on the survival and growth of Vibrio parahaemolyticus and other bacteria. The growth and survival of V. parahaemolyticus in Luria-Bertani media and phosphate buffer, respectively, were inhibited by the addition of D-glucose autoclaved in pH 8.0 phosphate buffer. The bactericidal effect of autoclaved D-glucose was very small when autoclaved in pH 7.0 phosphate buffer, but larger effects were observed when autoclaved in the buffer at an alkaline pH. The autoclaving of D-glucose in CH3COONa, NaHCO3, and Na2HPO4 solutions at pH 7.6 to 8.5 also generated bactericidal effects, but it was not the case when D-glucose was autoclaved in Na2SO4, (NH4)2SO4, or NH4Cl solution at pH 8.0. The same effects as autoclaved D-glucose were observed in autoclaved lactose, D-fructose, and D-ribose. The bactericidal effects of autoclaved D-glucose were also noted in Salmonella Enteritidis, Listeria monocytogenes, and E. coli strains, but the effects were smaller than those seen in V. parahaemolyticus and V. vulnificus. The growth of V. parahaemolyticus in clam extracts was also inhibited by the addition of autoclaved D-glucose, indicating that heat-treated reduced sugars can exert bactericidal effects in foods.     

Characterization of low salinity stress in Vibrio parahaemolyticus

Vibrio parahaemolyticus is a marine foodborne pathogenic bacterium commonly found in seawater or seafood. This bacterium often encounters low salinity stress when the contaminated seafood is washed with fresh water during food processing. This study was conducted to investigate the response of exponential- and stationary-phase cells of V. parahaemolyticus ST550 to lethal or sublethal low salinity. Tolerance to lethal low salinity (0.25% NaCl) was enhanced in V. parahaemolyticus cells in the exponential phase by previous adaptation in sublethal low salinity (0.6% NaCl). Low salinity-adapted cells in the exponential phase were also cross-protected against the challenge of lethal low pH, indifferent to heat, and sensitized to bile, acetic acid, and lactic acid stress. The adapted cells in the stationary phase were significantly protected against heat treatment at 44°C for 10 and 15 min, sensitized to bile and acetic acid treatment, and indifferent to low pH and lactic acid.     

Growth and survival of Vibrio parahaemolyticus in postharvest American oysters

Oysters at the retail stage of distribution generally contain greater densities of Vibrio parahaemolyticus than do oysters at harvest. The objective of this study was to determine the effects of postharvest storage at 26 and 3 degrees C on the growth and survival of naturally occurring V. parahaemolyticus in shellstock American oysters (Crassostrea virginica). Oysters were collected monthly from May 1998 through April 1999 from Mobile Bay, Alabama, and their V. parahaemolyticus densities were determined after 0, 5, 10, and 24 h of postharvest storage at 26 degrees C. After 24 h of storage at 26 degrees C, oysters were transferred to a refrigerator at 3 degrees C and analyzed 14 to 17 days later. V. parahaemolyticus numbers were determined by a direct plating method involving an alkaline-phosphatase-labeled DNA probe that targets the species-specific thermolabile hemolysin gene (tlh-AP) to identify suspect isolates. From April to December, when water temperatures at harvest were >20 degrees C, the geometric mean harvest density of V. parahaemolyticus was 130 CFU/g. When water temperatures were <20 degrees C, the geometric mean harvest density was 15 CFU/g. After harvest, V. parahaemolyticus multiplied rapidly in live oysters held at 26 degrees C, showing a 50-fold increase (1.7 log CFU/g) at 10 h and a 790-fold increase (2.9 log CFU/g) at 24 h (April through December). Average V. parahaemolyticus numbers showed a sixfold decrease (0.8 log CFU/g) after approximately 14 days of refrigeration. These results indicate that V. parahaemolyticus can grow rapidly in unrefrigerated oysters.     

几种天然食用成分对副溶血性弧菌抑菌作用研究

副溶血性弧菌是引起我国特别是沿海地区细菌性食物中毒危害的首要食源性致病菌。本实验通过单因素试验和正交试验研究醋酸、酒精与茶多酚3 种天然食用成分对菌悬液中副溶血性弧菌存活率的影响。单因素试验结果表明：经体积分数高于50%酒精或质量浓度为0.5mg/mL的茶多酚处理5min能完全杀灭菌悬液中的副溶血性弧菌;经pH2 的醋酸溶液处理也能使菌悬液中副溶血性弧菌降低4.79lg(CFU/mL)。正交试验结果表明：酒精体积分数、茶多酚质量浓度和酸度对副溶血性弧菌的抑制效果具有明显的协同作用;pH2.8、酒精体积分数20% 及茶多酚质量浓度0.125mg/mL 或pH2.4、酒精体积分数12% 及茶多酚质量浓度0.125mg/mL 的混合体系均能有效抑制副溶血性弧菌的生长。这些研究结果说明食用酒精、醋酸和茶叶提取物的适当配合将有可能作为复合杀菌剂用于水产食品中,从而降低副溶血性弧菌感染的风险。     

Hydrophobicity, cell adherence, cytotoxicity, and enterotoxigenicity of starved Vibrio parahaemolyticus

Vibrio parahaemolyticus is a ubiquitous gram-negative enteropathogenic bacterium that may encounter starvation or other environmental stresses during food processing or human infection. Pathogenic V. parahaemolyticus ST550 cultures starved in modified Morita mineral salt solution with 3 or 0.5% NaCl exhibited similar resistance against challenges of environmental stresses. Changes in virulence of the starved V. parahaemolyticus was determined using HEp-2 cell culture and suckling mouse assay. The starved cells exhibited greater cell adherence and hydrophobicity than did the cells in exponential growth phase. Expression of virulence in terms of cytotoxicity and mouse lethality was lower in the starved cells than in the exponential-phase cells at the same postinfection time. An additional 1 h of in vitro or in vivo incubation was required to enable these starved cells to reach the same cytotoxicity and mouse lethality levels as exhibited by the exponential-phase cells.     

Vibrio parahaemolyticus growth under low-iron conditions and survival under high-magnesium conditions

Since 1996, Vibrio parahaemolyticus serotype O3:K6 and closely related strains have been associated with an increased incidence of V. parahaemolyticus gastroenteritis worldwide, suggesting the emergence of strains with enhanced abilities to cause disease. One hypothesis for the recent emergence of V. parahaemolyticus O3:K6 and related strains is an enhanced capacity for environmental survival relative to other strains, which might result in increased human exposure to these organisms. Therefore, the objective of this study was to test the hypothesis that survival or growth characteristics of clinical V. parahaemolyticus isolates differ from those of nonclinical isolates under different environmental conditions. Twenty-six V. parahaemolyticus isolates selected to represent either clinical or food sources were monitored for either survival following exposure to high magnesium (300 mM) or growth under iron-limited conditions. Isolates in each category (clinical or food) differed widely in survival capabilities following 24 h of exposure to 300 mM Mg2+. Although 4 of 15 clinical isolates grew better at approximately 0.96 microM Fe2+ (iron-limited conditions) than at 50 microM Fe2+ (iron-rich conditions), as an entire group clinical isolates in this study were not more effective at growing under iron-limited conditions than were strains not associated with disease. Within the diverse collection of strains examined in these experiments, neither growth characteristics in low-iron environments nor survival capabilities following exposure to high magnesium concentrations were uniformly different between clinical and nonclinical V. parahaemolyticus isolates. Therefore, neither phenotypic characteristic can be used to reliably differentiate potentially pathogenic V. parahaemolyticus strains.     

Impact of acid on survival of Vibrio vulnificus and Vibrio vulnificus phage

Three strains of Vibrio vulnificus and V. vulnificus phages were tested for acid sensitivity at 21 degrees C. V. vulnificus strain 304 was more resistant to pH 4.0 than strains CVD-1 and A-9, whereas acid sensitivities of V. vulnificus strains at pH 3.0 and 2.0 were similar. V. vulnificus phage strain 110A-7 was more resistant to pH 4.0 than strain 153A-7, whereas acid sensitivities of phage strains at pH 3.5 and 3.0 were similar. Numbers of V. vulnificus and its phage were close to the limit of detection after 100 s at pH 2.0 and after 24 min at pH 3.0. Acid D-values at 21 degrees C decreased as pH decreased for both V. vulnificus and phages. D-values of phage strains at pH 3.5 were 10-fold greater than those of host strain at pH 4.0. D-values of phage strains were slightly greater than those of host strain at pH 3.0. These results suggest that V. vulnificus and its phage were very sensitive to pH of less than 3.0, although V. vulnificus phages were more resistant to acid than their host.     

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.     

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.     

原儿茶酸对副溶血弧菌的抑菌和减毒作用

该研究旨在探讨原儿茶酸对副溶血弧菌的抑菌活性和减毒作用,揭示原儿茶酸抑菌的作用机制。通过测定最小抑菌浓度（Minimal Inhibitory Concentration,MIC）、生长曲线、核酸与蛋白质泄漏量、丙二醛（MDA）含量,利用扫描电镜观察副溶血弧菌形态的变化,来评估原儿茶酸对副溶血弧菌的抑菌活性及其对细胞膜完整性和通透性的影响。同时,通过检测亚抑菌浓度（Sub-inhibitory Concentrations,SICs）下原儿茶酸对副溶血弧菌毒力因子合成的影响,研究原儿茶酸对副溶血弧菌的毒力衰减作用。实验结果表明,原儿茶酸的MIC为2 mg/mL,经MIC浓度原儿茶酸处理后,副溶血弧菌发生严重内陷和破裂,上清液中核酸、蛋白质、MDA含量分别是对照组的2.65倍、1.94倍和10.05倍。此外,原儿茶酸在浓度为1/4 MIC时对胞外多糖、胞外蛋白酶、生物被膜的抑制率分别为40.57%、19.79%和26.04%,在浓度为1/2 MIC时的抑制率分别为52.85%、28.38%和34.69%。原儿茶酸主要作用于细胞膜,通过影响细胞膜的完整性和通透性抑制副溶血弧菌的生长,在亚抑菌浓度下便可有效减弱副溶血弧菌毒力。     

细胞壁缺失对副溶血性弧菌生物学特性的影响

副溶血性弧菌是沿海地区重要的食源性致病菌之一,该菌的抗药性问题逐渐凸显,急需开发安全、有效、具有新作用位点的抑菌剂。本文以细胞壁为潜在靶点,采用肉桂醇作为抑菌剂研究细胞壁缺失对副溶血性弧菌生物学特性的影响。通过测定去壁菌体在最小抑菌浓度、生长曲线、细胞膜完整性、内膜通透性和外膜蛋白竞争试验中的变化,探寻肉桂醇作用于菌体的部位和抑制方式,并通过药敏试验验证获得的去壁细胞。结果表明：细胞壁缺失使肉桂醇对副溶血性弧菌的MIC降低为未处理组的1/8。肉桂醇对不同状态的副溶血性弧菌均有很强的抑制作用,可造成细胞膜损伤,引起胞内特征大分子外漏。硫酸粘杆菌素和去壁处理能够使肉桂醇失去作用靶点。外膜蛋白竞争试验进一步证实,肉桂醇能与外膜蛋白产生相互作用。去壁处理使原本作用于细胞壁的抗生素失去药效,表明去壁操作无误。结论：肉桂醇可以作用于副溶血性弧菌的细胞膜和外膜,可能与外膜蛋白发生靶向结合。外膜是小分子进入阴性菌的第1道屏障,可作为抑菌剂开发的新靶点。     

Evaluation of a tissue culture-based approach for differentiating between virulent and avirulent Vibrio parahaemolyticus strains based on cytotoxicity

The ability of only a subset of Vibrio parahaemolyticus strains to cause human infection underscores the need for an analytical method that can effectively differentiate between pathogenic strains and those that do not cause disease. We tested the feasibility of a tissue culture-based assay to determine whether clinical isolates could be differentiated from nonclinical isolates based on relative isolate cytopathogenicity. To screen for cytotoxic capability, we measured relative extracellular lactate dehydrogenase as an indicator of host cell damage in five different mammalian cell lines in the presence of V. parahaemolyticus. Isolates originating from clinical sources exhibited 15.5 to 59.3% relative cytotoxicity, whereas those originating from food sources exhibited 4.4 to 54.9% relative cytotoxicity. In the presence of -1.2 x 10(6) cells, cytotoxicity was 1.6- to 3.5-fold higher (P < 0.05) for clinical isolates than for nonclinical isolates in L2, Henle 407, and Caco-2 cell lines. V. parahaemolyticus serotype O3:K6 clinical isolates had 1.6- to 2.1-fold higher cytotoxicity than did the non-O3:K6 clinical isolates, with significantly higher cytotoxicity in HeLa, J774A.1, and Henle 407 cells than in L2 and Caco-2 cells. Because V. parahaemolyticus often is found in oysters, the effect of the presence of an oyster matrix on assay efficacy was also tested with L2 cells. The cytotoxicity elicited by a highly cytotoxic V. parahaemolyticus isolate was not affected by the presence of oyster tissue, suggesting that an oyster matrix will not interfere with assay sensitivity. In the present format, this assay can detect the presence of > 10(5) cells of a virulent V. parahaemolyticus strain in an oyster matrix.     

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。     

同时检测海产品中副溶血弧菌和霍乱弧菌的合检方法评价研究

目的建立水产品中副溶血弧菌和霍乱弧菌合检方法,并对合检方法对比进行评价。方法利用副溶血弧菌和霍乱弧菌阳性菌株,制备纯菌液、不同浓度梯度的人工污染样品。通过对30份纯菌液、30份人工污染样品和250份实际样品的检测,将建立的合检方法与行业标准(SN/T 1022-2010进出口食品中霍乱弧菌检验方法和SN/T 0173-2010进出口食品中副溶血性弧菌检验方法)进行比较,对合检方法进行效果评价。结果实验结果表明副溶血弧菌和霍乱弧菌合检方法,与行业标准检测结果完全一致。结论该方法可靠,对实验仪器和操作人员的要求低,具有良好的实用性,适合基础检测实验室。     

Effects of heat, acid, and freeze-thaw challenges on survival of starved Vibrio parahaemolyticus in minimal salt medium, tryptic soy broth, and filtered oyster homogenate medium

Vibrio parahaemolyticus is a ubiquitous gram-negative enteropathogenic bacterium. To evaluate the risk of stress-adapted V. parahaemolyticus cells in food, we investigated the survivability of starvation-adapted and starvation-low salinity-adapted cells of this pathogen in different media against different stresses. Logarithmically grown bacterial cells were starved at 25 degrees C in a minimal salt medium with 0.5 or 3.0% NaCl for 24 h. Resistances against challenges of heat, acid, and freeze-thaw treatment exhibited by the starvation-adapted cells were similar to those exhibited by the starvation-low salinity-adapted cells but substantially higher than those of the unadapted control cells. The increased stress resistance of the adapted cells against freeze-thaw challenge was lower in tryptic soy broth than in the starving medium. Resistance of the adapted bacteria against heat and freeze-thaw treatment was completely eliminated in filter-sterilized oyster homogenate medium. Practically, these results help to assess the risk of stress-adapted V. parahaemolyticus in food.     

Antimicrobial effect of spices and herbs on Vibrio parahaemolyticus

The antimicrobial effects of spices and herbs from 18 plant species were examined on a foodborne pathogen, Vibrio parahaemolyticus, with the use of combinations of temperatures and nutrient levels. Basil, clove, garlic, horseradish, marjoram, oregano, rosemary, and thyme exhibited antibacterial activities at incubation of 30 degrees C, while with the exception of horseradish, the same spices and additional 7 species exhibited the activities at 5 degrees C. The lowest MIC (minimum inhibitory concentration) was 0.125% observed in clove and marjoram at 30 degrees C in a nutrient rich medium. Lowering of incubation temperature produced little effect on the MICs except for turmeric. The decreasing of the MIC in turmeric appeared to be basically attributed to the sensitivity of the bacterium to coldness. In nutrient poor medium, the lowest was 0.001 and 0.00025% in marjoram at 30 degrees C and at 5 degrees C, respectively. The sensitivity to several spices and herbs was similar among different clinical serotypes including the emerging strain O3:K6. These results suggest that the spices and herbs can be practical for protecting seafood from the risk of contamination by V. parahaemolyticus and used in hurdle technology with low temperature.     

Temperature Effects on the Depuration of Vibrio parahaemolyticus and Vibrio vulnificus from the American Oyster (Crassostrea virginica)

ABSTRACT:  This study investigated temperature effects on depuration for reducing Vibrio parahaemolyticus and Vibrio vulnificus in American oyster ( Crassostrea virginica ). Raw oysters were inoculated with 5-strain cocktail of V. parahaemolyticus or V. vulnificus to levels of 10⁴ to 10⁵ MPN (most probable number)/g and depurated in artificial seawater (ASW) at 22, 15, 10, and 5 °C. Depuration of oysters at 22 °C had limited effects on reducing V. parahaemolyticus or V. vulnificus in the oysters. Populations of V. parahaemolyticus and V. vulnificus were reduced by 1.2 and 2.0 log MPN/g, respectively, after 48 h of depuration at 22 °C. Decreasing water temperature to 15 °C increased the efficacy of depuration in reducing V. parahaemolyticus and V. vulnificus in oysters. Reductions of V. parahaemolyticus and V. vulnificus in oysters increased to 2.1 and 2.9 log MPN/g, respectively, after 48 h of depuration at 15 °C. However, depurations at 10 and 5 °C were less effective than at 15 °C in reducing the Vibrio spp. in oysters. Extended depuration at 15 °C for 96 h increased reductions of V. parahaemolyticus and V. vulnificus in oysters to 2.6 and 3.3 log MPN/g, respectively.     

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。      

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.