Effects of acid stress on Vibrio parahaemolyticus survival and cytotoxicity

For several foodborne bacterial pathogens, an acid tolerance response appears to be an important strategy for counteracting acid stress imposed either during food processing or by the human host. The acid tolerance response enhances bacterial survival of lethal acid challenge following prior exposure to sublethal acidic conditions. Previous studies have revealed relationships between a foodborne pathogen's ability to survive acid challenge and its infectious dose. Vibrio parahaemolyticus is capable of causing gastroenteritis when sufficient cells of pathogenic strains are consumed. This study was designed to characterize acid sensitivities and to compare the effects of sublethal acid exposure (adaptation) on survival capabilities and cytotoxicities of different V. parahaemolyticus strains. Survival of acid challenge by stationary-phase cells differed by up to 3 log CFU/ml among the 25 isolates tested. No differences in acid resistance were found between strains when they were grouped by source (clinical isolates versus those obtained from food). Survival at pH 3.6 for log-phase cells that had been previously exposed to sublethal acidic conditions (pH 5.5) was enhanced compared with that for cells not previously exposed to pH 5.5. However, for stationary-phase cells, exposure to pH 5.5 impaired both subsequent survival at pH 3.6 and cytotoxicity to human epithelial cells. Relative cytotoxicities of nonadapted stationary-phase cells were 1.2- to 4.8-fold higher than those of adapted cells. Sublethal acid exposure appears to impose measurable growth phase-dependent effects on subsequent lethal acid challenge survival and cytotoxicity of V. parahaemolyticus.     

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.     

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。     

耐药性副溶血性弧菌生长异质性比较研究

目的在不同温度(10、20、30、37℃)下,比较17株不同耐药性、耐药表型及基因型不一致的副溶血性弧菌在碱性蛋白胨水(3%NaCl)中最大比生长速率及生长快慢之间的差异。方法应用Bioscreen C全自动微生物生长曲线分析仪测定副溶血性弧菌的最大比生长速率,构建不同温度下生长曲线,采用修正后的Gompertz模型进行拟合得出生长动力学参数。结果不同耐药性副溶血性弧菌μmax存在一定差异,差异随着温度的降低而增加,抗生素耐药种类越多,其μmax越大;同一温度下,耐药表型与基因型不一致的副溶血性弧菌其生长参数存在一定的差异,但是菌株的生长参数的差异与菌株耐药表型与基因型不一致之间没有明显的对应关系。结论菌株之间的微生物生长动力学参数的异质性将会对风险评估的准确性带来影响,此结果可为多重耐药副溶血性弧菌风险评估提供基础数据支持。     

副溶血性弧菌鉴定能力验证样品的制备

目的 研制适用于副溶血性弧菌(Vibrio Parahaemolyticus,VP)能力验证(PT)项目的VP菌及干扰菌的冷冻干燥样品,建立有效的样品制备与评估流程.方法 通过不同比例混匀的VP和干扰菌拟态弧菌(Vibrio minicus,VM)经冻干后在鉴定培养基上菌落生长优势比,确定制备PT样品的混合菌VP和VM的浓度比例;通过系统抽样和鉴定以评估样品的均一性;通过观察样品在180 d保存期内菌量的变化情况以评估样品的稳定性;根据188间检测实验室反馈的鏊定结果以评估和计算样品的实际合格率.结果 选择5 000:1比例制备的VP和VM混合PT样品能在鉴定培养基有效分离两种菌落;抽检的40份样品均能有效鉴定,显示PT样品具有较好的一致性;通过保存温度和时间的稳定性实验显示,PT样品在36℃下能稳定保存5d,冻干后PT样品于4℃保存180 d后VP菌仍能检出;188家参与实验室检测结果显示,PT样品合格率为97.3％.结论 本研究制备的冻干PT样品及实验流程控制适用于副溶血性弧菌及类似的微生物鉴定能力验证项目.     

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。     

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.     

不同致病性副溶血性弧菌在南美白对虾中的生长动力学参数比较研究

为了探讨不同致病性副溶血性弧菌(pathogenic Vibrioparahaemolyticus,pVp)在南美白对虾及其他生长基质(培养基和三文鱼)中生长动力学参数的差异.测定了12℃和35℃下,三株pVp(含有tdh基因):ATCC33847、F13、临床分离株(Clinical Vibrio parahaemolyticus strain,CV)在南美白对虾中的生长曲线,采用不同一级模型拟合得出生长动力学参数并与同类研究进行比较.结果显示,Baranyi模型对三株致病性副溶血性弧菌生长曲线的拟合效果最好.通过将本研究结果与同类研究相比可知,血清型为O3:K6的致病性副溶血性弧菌CV在12℃和35℃下在南美白对虾中的μmax均大于同类研究中的pVp在培养基和三文鱼中的μmax.不同的pVp菌株在12℃、不同生长基质中的生长动力学参数存在较大差异.由此可见,在构建副溶血性弧菌的生长预测模型及进行风险评估时,应充分考虑不同菌株之间以及不同生长基质之间的差异对微生物生长动力学参数的影响.     

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。     

面包虾中副溶血性弧菌温度预测模型的建立

为探讨面包虾生产过程中副溶血性弧菌的生长规律,测定了不同温度(10、14、18、22、26、30、37℃)下副溶血性弧菌的生长曲线,运用Origin8.0软件和DMFit软件,分别采用修正Gompertz模型、修正Logistic模型、Baranyi模型对生长曲线进行拟合,建立初级模型。应用平方根模型对由最适生长模型得出的最大比生长速率进行拟合,建立二级生长预测模型。研究结果表明,通过模型检验比较,修正Gompertz模型模型对7种温度培养条件下的拟合度较好(决定系数=0.9917),进一步拟合所得二级模型拟合度较好(决定系数=0.9687),得到了最适生长方程,可用于10~37℃温度条件下的面包虾中副溶血性弧菌生长预测。      

不同耐药性致病性副溶血性弧菌的生长特性比较研究

用K-B纸片扩散法分析了8株致病性副溶血性弧菌菌株对10种抗生素的耐药性,发现6株菌株具有耐药性,其中,1株耐3种抗生素,1株耐2种抗生素,其余4株耐1种抗生素。比较了20、25、37℃下这8株菌株在TSB（含3%NaCl,pH8.0）中的生长特性。通过Bioscreen全自动微生物生长曲线分析仪建立了这8株菌的一级生长模型,并比较其最大比生长速率（μmax）,延滞期（λ）及最大生长量（a）。同时比较了这8株菌的ERIC-PCR图谱的差异性。结果显示,不同耐药性致病性副溶血性弧菌菌株的生长参数存在一定的差异性,但是菌株的各个生长参数的大小与菌株的耐药性之间没有明显的对应关系。并且发现ERIC-PCR图谱与菌株的耐药性之间也没有明显的对应关系。本研究为耐药性致病副溶血性弧菌的风险评估提供参考依据。      

副溶血性弧菌拮抗菌的筛选鉴定及抑菌物质特性研究

为获取既可抑制食源性致病菌又可抑制水产致病菌的益生菌株,以人畜共患致病菌副溶血性弧菌(Vibrio parahaemolyticus ATCC17802)为指示菌,采用点种法初筛以及打孔扩散法复筛,得到1株来源于上海东海海水的H19菌株,其抑菌物抑菌圈直径达(17.16±0.28)mm。通过形态观察及16SrDNA序列分析可知,菌株H19为枯草芽孢杆菌(Bacillus subtilis H19,GenBank登录号:MG383451)。将菌株H19进行抑菌谱试验,探究菌株H19抑菌物的理化性质,并且抑菌物通过硫酸铵沉淀及不同孔径透析袋截留粗提,粗提物经蛋白酶处理及Tricine-SDS-PAGE蛋白质凝胶电泳可知,该抑菌物具有广谱抑菌活性,既可抑制革兰氏阳性菌又可抑制大部分革兰氏阴性菌,且具有较好的热、pH以及紫外线稳定性,该抑菌物可能为2种新型抗菌肽,分子量分别在6.5~9.5kDa以及27.0~35.0kDa。由此可见,枯草芽孢杆菌H19所产抗菌肽具有防治食源性疾病以及水产疾病的应用潜力。     

不同致病性副溶血性弧菌在南美白对虾中的生长动力学参数比较研究

为了探讨不同致病性副溶血性弧菌(pathogenic Vibrio parahaemolyticus,pVp)在南美白对虾及其他生长基质(培养基和三文鱼)中生长动力学参数的差异。测定了12℃和35℃下,三株pVp(含有tdh基因):ATCC33847、F13、临床分离株(Clinical Vibrio parahaemolyticus strain,CV)在南美白对虾中的生长曲线,采用不同一级模型拟合得出生长动力学参数并与同类研究进行比较。结果显示,Baranyi模型对三株致病性副溶血性弧菌生长曲线的拟合效果最好。通过将本研究结果与同类研究相比可知,血清型为O3:K6的致病性副溶血性弧菌CV在12℃和35℃下在南美白对虾中的μmax均大于同类研究中的pVp在培养基和三文鱼中的μmax。不同的pVp菌株在12℃、不同生长基质中的生长动力学参数存在较大差异。由此可见,在构建副溶血性弧菌的生长预测模型及进行风险评估时,应充分考虑不同菌株之间以及不同生长基质之间的差异对微生物生长动力学参数的影响。      

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。     

模拟蟹肉中副溶血性弧菌预测生长预测模型的建立

为了控制模拟蟹肉中副溶血性弧菌的危害,运用预测食品微生物学的方法,本文分别研究了在温度10～37℃条件下模拟蟹肉中副溶血性弧菌的生长规律,建立了一级和二级预测模型。结果表明,副溶血性弧菌的生长曲线在此温度范围内都呈典型的S型,故本文采用Gompertz模型拟合,二级模型采用常用的平方根模型拟合,拟合方程为U=0·01578(T+7·294)。通过模型验证,预测值和实测值的残差在±0·04范围内,偏差因子和精准因子均在可接受范围内,模型具有可靠性。     

运用Real-time quantification PCR方法建立副溶血性弧菌在即食虾中的生长预测模型

运用Real-time quantification PCR(real-time qPCR)方法建立副溶血性弧菌在即食虾中生长预测模型.首先构建质粒标准品,梯度稀释后建立标准曲线,然后用Real-time qPCR方法检测虾中副溶血性弧菌的数量,最后建立37℃下即食虾中副溶血性弧菌生长预测模型,并与传统涂布计数方法进行比较.结果表明,Real-time qPCR方法和传统计数方法均可建立Gmopertz模型、Logistic模型和Richards模型,模型拟合的相关系数R2均在0.9以上.基于Real-time qPCR方法省时省力、特异性好等优点,用Real-time qPCR方法建立微生物预测模型是未来预测微生物学领域的一种发展趋势.     

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.