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.     

Biochemical and virulence characterization of viable but nonculturable cells of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a common foodborne pathogen frequently causing outbreaks in summer. Maintenance of virulence by the viable but nonculturable (VBNC) state of this pathogen would allow its threat to human health to persist. This study reports on the change in virulence and concomitant changes in activity of two enzymes and fatty acid profiles when V. parahaemolyticus ST550 entered the VBNC state in the modified Morita mineral salt-0.5% NaCl medium incubated at 4 degrees C. The major change in fatty acid composition occurred in the first week, with a rapid increase in C15:0 fatty acid and saturated/unsaturated ratio while a rapid decrease in C16:1 was observed. The activity level of the inducible protective enzyme superoxide dismutase became undetectable in the VBNC state, whereas that of constitutive glucose-6-phosphate dehydrogenase did not change in either the exponential phase or the VBNC state. Cytotoxicity against HEp-2 cells and a suckling mouse assay showed that virulence was lowered in the VBNC state compared with exponential-phase cells. Longer incubation times were required by the VBNC cells to achieve the same level of virulence as seen in exponential-phase cells. Culturable cells were recovered on selective agar medium from the VBNC cultures injected into suckling mice, probably as the result of in vivo resuscitation. Results of this study add to our understanding of the biochemical and physiological changes that have not been reported when V. parahaemolyticus enters into the VBNC state.     

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.     

Retention of virulence in viable but non-culturable halophilic Vibrio spp

The viable but non-culturable (VBNC) forms of two environmental strains of Vibrio alginolyticus 1 and Vibrio parahaemolyticus 66 and one strain of V. parahaemolyticus ATCC 43996 showing virulence characteristics (hemolysin production, adhesive and/or cytotoxic ability, in vivo enteropathogenicity) were obtained by culturing bacteria in a microcosm consisting of artificial sea water (ASW) and incubating at 5 degrees C with shaking. Every 2 days, culturability of the cells in the microcosm was monitored by spread plates on BHI agar and total count and the percentage of viable cells were determined by double staining with DAPI and CTC. When cell growth was not detectable (<0.1 CFU/ml), the population was considered non-culturable and, then, the VBNC forms were resuscitated in a murine model. For each strain, eight male Balb/C mice were intragastrically inoculated with 0.1 ml of concentrated ASW bacterial culture. Two mice from each group were sacrificed at 2, 4, 8, and 12 days after challenge for autopsy and re-isolation of the microorganisms from the intestinal tissue cultures. Isolation was obtained in 25% of the animals challenged with the VBNC V. alginolyticus strain, in 37.5% of those challenged with the VBNC V. parahaemolyticus strain of environmental origin and in 50% of the animals infected with VBNC V. parahaemolyticus ATCC 43996. The strains thus isolated were again subjected to biological assays to determine the retention of pathogenicity. The virulence characteristics that seemed to disappear after resuscitation in the mouse were subsequently reactivated by means of two consecutive passages of the strains in the rat ileal loop model. The results obtained indicate that VBNC forms of the strains examined can be resuscitated and retain their virulence properties.     

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.     

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.     

Effects of heat shock on the thermotolerance, protein composition, and toxin production of Vibrio parahaemolyticus

Vibrio parahaemolyticus, an important seafood-associated enteropathogen, usually encounters different adverse conditions in its native or food-processing environment, and the stresses resulting from these conditions may affect the survival of this pathogen and thus change its risk with regard to food hygiene. In this study, we investigated the thermotolerance of V. parahaemolyticus under sublethal heat shock and characterized this response by examining the changes in protein profiles and toxin production. Logarithmically grown cells heat shocked at 42 degrees C for 30 min were more resistant to thermal inactivation at 47 degrees C than were unshocked cells. After the 25 degrees C culture was heat shocked, 24 species of proteins were induced, while 13 species were inhibited, as indicated by polyacrylamide gel electrophoresis. DnaJ-, GroEL-, and GroES-like proteins with molecular sizes of 47, 62, and 12 kDa, respectively, were detected by immunoblotting with antibodies raised against the Escherichia coli proteins. During 1 to 8 h of heat shock, GroEL-like protein was produced in substantial amounts and was present in the periplasmic and extracellular fractions, while DnaJ- and GroES-like proteins were present mainly in the total cellular fraction. DnaK-like protein was not detected; nevertheless, the presence of the dnaK-like genetic element was revealed by Southern blotting. Production of thermostable direct hemolysin, the major virulence factor in V. parahaemolyticus, was enhanced in the cells heat shocked at 42 degrees C but not in those heat shocked at 37 degrees C.     

Expression of superoxide dismutase, catalase and thermostable direct hemolysin by, and growth in the presence of various nitrogen and carbon sources of heat-shocked and ethanol-shocked Vibrio parahaemolyticus

Vibrio parahaemolyticus 690 was subjected either to heat shock at 42 degrees C or ethanol shock in the presence of 5% ethanol. The effects of those shocks on superoxide dismutase (SOD) and catalase (CAT) activities, and thermostable direct hemolysin (TDH) production were examined. In addition, the growth behaviors of the stressed and unstressed cells of V. parahaemolyticus in the presence of various nitrogen and carbon sources were compared. Both heat shock and ethanol shock reduced the levels of SOD and CAT activities in V. parahaemolyticus. Gel activity staining assay failed to detect the expression of CAT, while one SOD enzyme with an electrophoretic mobility greater than the [Mn]SOD and [Fe]SOD of Escherichia coli was detected in the unstressed, heat-shocked and ethanol-shocked cells of V. parahaemolyticus. Heat shock for 15-60 min and ethanol shock for 45-60 min were found to enhance the synthesis of TDH. Ethanol-shocked and unstressed cells of V. parahaemolyticus grew similarly and produced similar amounts of TDH when they were grown in TSB-3% NaCl, but slower growth and less production of TDH occurred with heat-shocked cells until after 200 min of cultivation. The growth rate and maximum growth of the unstressed, heat-shocked and ethanol-shocked cells varied with the nitrogen and carbon sources used. With the same nitrogen or carbon source, the growth patterns of the ethanol-shocked and unstressed cells were similar while the heat-shocked cells exhibited an extended lag period.     

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.     

Virulence and stress susceptibility of clinical and environmental strains of Vibrio vulnificus isolated from samples from Taiwan and the United States

Vibrio vulnificus is an estuarine bacterium that causes severe wound infection and septicemia with high mortality. It also can be transmitted through the consumption of raw contaminated seafood and is an important foodborne pathogen. A total of 40 environmental and clinical V. vulnificus strains isolated from the United States and Taiwan were analyzed for virulence in animals, the presence of virulence-associated factors, and susceptibility to environmental stresses. Virulence in mice was exhibited by 85% of the environmental strains and 95% of the clinical strains. Strains from environmental or clinical sources were similar in virulence-associated phenotypes (protease activity, utilization of transferrin-bound iron, hemolysis, and inactivation in serum) and susceptibility to various stresses (4 and 52 degrees C, 0.1 and 10% NaCl, and pH 3.2), except freeze-thaw treatment. The clinical strains killed experimental animals after a shorter incubation time than did the environmental strains. Most of the 15 virulence-associated genes examined were present in most of the strains, regardless of their sources or virulence, with the exception of vvh, flgF, and purH. vvh was significantly more common in clinical strains than in environmental strains, and vvh, flgF, and purH were more common in virulent strains than in nonvirulent strains. These data may be helpful in devising strategies to manage or reduce the presence of V. vulnificus in foods.     

胁迫生长条件下副溶血性弧菌的生物特性分析

为了解副溶血性弧菌在不同环境应激条件下的生长情况,以副溶血性弧菌标准菌株（Vibrio parahaemolyticus,CICC21617）为研究对象,使其在低盐度（0.9?g/100?mL?NaCl）、低pH值（pH?5.0）和高温（50?℃）条件下适应性传代培养后,通过测定菌株生存能力、生物被膜生成能力、溶血素活力和胞外蛋白酶产量等指标分析胁迫条件下副溶血性弧菌的生物特性。结果显示,经过胁迫传代的菌株较原始菌株,其生存能力和生物被膜生成能力有所增强,而溶血素活力和胞外蛋白酶产量却明显下降,外膜蛋白的种类和表达量亦有所不同。结果说明副溶血性弧菌可能是通过改变自身生物学特性来适应外界的不利生存环境,且菌株经应激适应后对逆境生存环境的耐受性明显增强。     

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。     

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.     

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。     

Characteristics of virulent vibrio parahaemolyticus isolated from Oregon and Washington

Thirty-four virulent strains of Vibrio parahaemolyticus containing tdh and/or trh genes isolated from Oregon and Washington coastal water were analyzed for O-group antigens and urease activity, and by pulsed-field gel electrophoresis. Six O serotypes (O1, O3, O4, O5, O10, and O11) were identified among the isolates, with the O5 group (19 isolates) being the most prevalent, followed by the 01 group (9 isolates). Nearly all (33 of 34) isolates were capable of producing urease, which reaffirmed the correlation between urease production and virulence factors of V. parahaemolyticus strains isolated from the Pacific Northwest. Pulsed-field gel electrophoresis analysis with NotI and SfiI digestions of the 34 V. parahaemolyticus isolates plus five clinical strains revealed 22 patterns (NlS1 to N20S22), with NIS1 (25.6%) being the most common, followed by N2S2 (10.3%). Nine Oregon isolates were grouped with a 1997 Oregon outbreak strain (027-1C1) with the same serotype (O5), virulence factors (tdh+ and trh+), and genotype (N S 1). Three Washington isolates were found to share the same serotype (O1), virulence factors (tdh' and trh'), and genotype (N2S2) with a 1997 Washington outbreak strain (10293). The repetitive isolation of virulent strains of V. parahaemolyticus identical to clinical strains involved in previous outbreaks indicates potential hazards associated with oyster consumption. These data may be useful in risk assessment of V. parahaemolyticus infections associated with raw oyster consumption in Oregon and Washington.     

副溶血性弧菌毒性相关因子及其外分泌蛋白的研究进展

副溶血性弧菌(Vibrio parahaemolyticus)是一种重要的食源性致病菌,已成为我国沿海地区微生物性食物中毒事件的首要风险因子。该菌主要通过分泌胞外蛋白和毒性因子进入胞外环境或者直接注入宿主靶细胞内,从而使宿主致病。本文主要从副溶血性弧菌毒性相关因子及其分泌系统相关分泌蛋白进行概述,概括阐明近年来针对该菌毒性及分泌蛋白的相关研究进展,并对今后分泌蛋白的研究方向进行了初步探讨。     

Antimicrobial resistance of Vibrio parahaemolyticus and Vibrio alginolyticus strains isolated from farmed fish in Korea from 2005 through 2007

The antimicrobial resistance patterns to 15 antimicrobial agents of Vibrio parahaemolyticus and Vibrio alginolyticus isolated from farmed fishes, including olive flounder (Paralichthys olivaceus), black rockfish (Sebastes schlegeli), red sea bream (Pagrus major), and sea bass (Lateolabrax japonicus), were investigated from 2005 through 2007. A total of 218 V. parahaemolyticus isolates and 153 V. alginolyticus isolates were obtained from the 180 fish samples collected from fish farms located along the southern coast of Korea. We found that 65.1% of V. parahaemolyticus and 85.6% of V. alginolyticus isolates showed antimicrobial resistance against more than one antimicrobial agent. The prevalence of resistance in V. parahaemolyticus isolates to ampicillin was highest (57.8%), followed by resistance to rifampin (11.9%), streptomycin (8.7%), and trimethoprim (6.4%). V. alginolyticus isolates were also most resistant to ampicillin (75.2%), followed by tetracycline (15.0%), trimethoprim (12.4%), and rifampin (9.8%). The prevalence of multiresistance to four or more antimicrobials was higher in V. alginolyticus (11.1%) than in V. parahaemolyticus (5%). Antimicrobial resistance rates per isolate of V. parahaemolyticus and V. alginolyticus possessing virulence genes were not different from those of the rest of the isolates.     

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.     

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).