Survival of Vibrio parahaemolyticus serovar O3:K6 strains under acidic conditions

The survival of Vibrio parahaemolyticus serovar O3:K6 strains and other serovars in the presence of acetic, citric and hydrochloric acids were studied. There were no differences in resistance to these acids between serovar O3:K6 and the other serovars. At pH 5.6, citric acid was more effective in reducing the number of viable cells of V. parahaemolyticus than acetic acid. However, at pH 4.5, acetic acid was more effective than citric acid. The number of viable cells decreased quickly in the presence of rice vinegar or wine vinegar at pH 4.0.     

Origin of Vibrio parahaemolyticus O3:K6 pandemic clone

O3:K6 pandemic clone of Vibrio parahaemolyticus has caused outbreaks in coastal countries since 1996. Mutilocus sequence typing (MLST) is an important tool to trace the source and analysis the evolution of bacteria. Based on MLST, the first pandemic clonal complex (CC) of V. parahaemolyticus has been confirmed. In this study, 57 pandemic strains, 27 pathogenic strains (tdh or trh positive) and 36 nonpathogenic strains isolated from China were analyzed with MLST. Forty-seven unique sequence types, one clonal complex (CC) and one doublet (D) were identified by eBURST and Mega4 analyses. CC corresponded to not only the known O3:K6 pandemic clone (including ST-3, ST-192, ST-227) but nonpathogenic clone (including ST-3, S-T2, ST-196, ST-220, ST-226). ST-3 was the founder of the complex. STs of the isolates were not inevitably associated with the presence or number of the accessory genes or the serotypes of the isolates. The ancestor strain of O3:K6 pandemic clone was originated from an environmental nonpathogenic O3:K6, ST-3 strain. The pandemic O3:K6 clone was developed from this strain in approximately 1996 by laterally transferring large fragments of genes including systematic functional genes and genomic islands.     

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.     

A PCR Assay for Specific Detection of the Pandemic Vibrio parahaemolyticus O3:K6 Clone from Shellfish

: The current standard method for identifying Vibrio parahaemolyticus serotype O3:K6, an emerging pathogen with apparent enhanced virulence characteristics, typically takes 4 to 6 d to complete and requires serotyping. To provide a more rapid strategy, we optimized a polymerase chain reaction (PCR)‐based assay for specific detection of V. parahaemolyticus O3:K6. Of 78 V. parahaemolyticus isolates and other related species; only strains classified into the V. parahaemolyticus O3:K6 clonal group (n= 39) showed positive results in the PCR assay. The assay detected 2.3 cells/PCR reaction and 310 cells/g using bacterial cultures and inoculated oyster samples, respectively. Sensitive and specific detection of V. parahaemolyticus O3:K6 was possible following a 6‐h enrichment.     

不同寡营养条件下副溶血性弧菌的生长异质性

为更准确地探讨副溶血性弧菌(Vibrio parahaemolyticus,Vp)菌株在寡营养条件(100％、75％、50％和25％标准TSB+ (3％ NaCl)占比的培养基稀释液)下的生长异质性对食品安全风险的影响,本实验选取20株分离自淡水和海水养殖虾中的Vp菌株,通过对其在不同寡营养条件下的生长数据进行拟合,得...     

Effects of pH, temperature and NaCl concentration on the growth kinetics, proteolytic activity and biogenic amine production of Enterococcus faecalis

In this work, the combined effects of temperature, pH and NaCl concentration on the growth dynamics of Enterococcus faecalis EF37, its proteolytic activity and its production of biogenic amines have been studied. The effects of the selected variables have been analysed using a Central Composite Design. The production of biogenic amines, under the adopted conditions, was found to be mainly dependent on the extent of growth of E. faecalis. Its proteolytic activity was not a limiting factor for the final amine production, because in the system studied (skim milk) an excess of precursors was guaranteed. Quantitatively, the most important biogenic amine produced was 2-phenylethylamine but substantial amounts of tyramine were detected in all the samples. This work confirms that the main biological feature influencing the biogenic amine formation is the extent of growth of microorganisms, like E. faecalis, characterised by decarboxylase activity. In the traditional and artisanal cheeses produced using raw milk, enterococci usually reach levels of 10(7) cells/g. With this perspective, it is important that the presence of biogenic amines due to the activities of these microorganisms is maintained within safe levels, without affecting the positive effects of enterococci on the final organoleptic characteristics of the cheese.     

240株O3血清型副溶血性弧菌tdh和trh毒力基因研究

目的掌握不同来源O3血清型副溶血性弧菌毒力基因携带状况,分析毒力基因与传统神奈川现象的相关性,为深入研究副溶血性弧菌致病性提供基础数据。方法用多重聚合酶链式反应(PCR)方法扩增tdh、trh毒力基因。按GB 4789.7—2013《食品安全国家标准食品微生物学检验副溶血性弧菌检验》方法进行神奈川试验。用χ~2检验方法确定tdh、trh基因与神奈川现象的相关性。结果 183株O3∶K6血清型的副溶血性弧菌中,有182株检出tdh基因,1株检出trh基因,检出率分别为99.45%(182/183)和0.55%(1/183),神奈川试验阳性率为100.00%(183/183)。57株O3非K6群血清型副溶血性弧菌tdh基因检出率为3.51%(2/57),未检出trh基因。神奈川试验结果阳性率为10.53%(6/57)。经χ~2检验tdh基因与神奈川现象有相关性(χ~2=1.78,P0.05),trh基因与神奈川现象无相关性(χ~2=186.01,P0.05)。结论 O3∶K6血清型的副溶血性弧菌tdh基因携带率极高,且与神奈川现象相关性有统计学意义。神奈川现象可作为副溶血性弧菌tdh基因和致病性的指标。     

副溶血性弧菌温度-pH双因素预警模型的建立

以副溶血性弧菌VpBJ1.1997为研究对象,采用均匀设计实验方法,建立并验证了温度范围为7～43℃,pH范围为5.5～10.0的生长动力学模型。结果表明,所选一级模型的拟合效果优劣依次为Logistic方程>Gompertz方程>Linear方程,以Logistic方程为一级模型计算生长参数;二级模型采用平方根模型进行拟合,得到模型相关系数r为0.9739,最低生长温度为11.7769℃,最低生长pH为7.38,经F检验和偏差因子、准确因子验证,模型可接受。     

副溶血弧菌在玻璃表面生物菌膜的生长特性及超声波法解离作用

本文研究了副溶血弧菌(Vibrio parahaemolyticus)在玻璃表面生物菌膜(biofilm,BF)的生长特性,生长过程中环境因素的影响及超声波对其的解离作用。采用结晶紫染色法观察生物菌膜在玻璃表面的生长形态;酶标仪在595 nm波长处测量不同培养条件下生物菌膜的生物量;平板菌落计数法衡量超声波对菌膜的解离效果。结果表明:结晶紫染色法可直观清晰观察副溶血弧菌在玻璃表面形成的生物菌膜,且随着培养时间的延长,副溶血弧菌生物菌膜形成的网状结构越来越致密。根据酶标仪测得的生物菌膜生物量的大小可得到在玻璃表面菌膜生长的最佳条件,当培养基盐度为3%,培养时间为24 h,培养时转速为70 r/min得到的副溶血弧菌生物菌膜已经成熟且菌体个数达到2.56×107 CFU/cm2。用50 k Hz超声波,采用间歇式超声波处理方法(每作用30 s间隔30 s)作用总时间4 min在保持菌体活性的同时能达到最佳解离效果。     

Effects of varying concentrations of sodium chloride and acidic conditions on the behavior of Vibrio parahaemolyticus and Vibrio vulnificus cold-starved in artificial sea water microcosms

Food Science and Biotechnology - There has been limited information available on the behavior of Vibrio parahaemolyticus and Vibrio vulnificus as a function of higher levels of NaCl in combination...     

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.     

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).     

Growth kinetics and cell morphology of Listeria monocytogenes Scott A as affected by temperature,NaCl, and EDTA

Growth kinetics and morphological characteristics of Listeria monocytogenes Scott A grown under stress conditions induced by increasing levels of NaCl and EDTA were studied as a function of temperature. L. monocytogenes Scott A was inoculated into brain heart infusion broth (pH 6) at 19, 28, 37, and 42 degrees C. Test cultures contained NaCl (at concentrations of 4.5, 6.0, and 7.5%) or EDTA (at concentrations of 0.1, 0.2, and 0.3 mM); control cultures contained 0.5% NaCl. Growth curves were fitted from plate count data by the Gompertz equation, and growth kinetics parameters were derived. Stationary-phase cells were examined by scanning and transmission electron microscopy. Generation times (GTs) and lag phase duration times (LPDs) increased as additive levels were increased. The bacterium grew at all NaCl levels. At 37 and 42 degrees C, growth was slow in media containing 7.5% NaCl, and no growth occurred in media containing 0.3 mM EDTA. Temperature was a major factor in certain stress conditions that led to cell elongation and loss of flagella. Cells in control media at 28 degrees C grew as short rods (0.5 by 1.0 to 2.0 microm), while at 42 degrees C most cells were 4 to 10 times as long. Higher levels of NaCl at higher temperatures resulted in longer and thicker cells. At 28 degrees C, 0.1 mM EDTA had little effect on growth kinetics and morphology; however, 0.3 mM EDTA caused a sixfold increase in GT and LPD and loss of flagellae, with most cells being two to six times as long as normal. Cell length did not correlate with growth kinetics. The results of this study suggest that the effect of altered morphological characteristics of L. monocytogenes cells grown under stress on the virulence and subsequent survival of these cells should be investigated.     

Automated ribotyping differentiates vibrio parahaemolyticus O3:K6 strains associated with a Texas outbreak from other clinical strains

Automated ribotyping with a Qualicon Riboprinter was used to determine whether clinical isolates of Vibrio parahaemolyticus O3:K6 recovered during two U.S. outbreaks of oyster-associated gastroenteritis in 1998 were related to each other and to a previously identified highly virulent Asian clone of this serotype. The patterns produced using the restriction enzymes Eco RI and Pst I suggest that the outbreak in the Northeastern United States was caused by a single strain closely related to the Asian clone. In contrast, it appears that multiple strains were involved in the Texas outbreak and that the predominant type was genetically distinct from the Northeastern and Asian clone.     

不同条件下副溶血弧菌生物膜形成规律及其天然抑制物质的研究

主要研究了副溶血弧菌在不同培养条件下生物膜形成规律以及天然产物茶多酚、生姜提取物、大蒜提取物对其生物膜形成的影响。结果表明,不同副溶血弧菌菌株形成生物膜能力不同,其中VP-S17形成量最高。一般在培养48⁷2h时,生物膜达到成熟阶段,之后形成量开始下降。高温、高盐有助于提高副溶血弧菌生物膜的形成,3%盐浓度比1%浓度形成生物膜能力更强;盐浓度相同时,37℃条件下副溶血弧菌形成生物膜含量明显高于25℃。茶多酚、大蒜提取物和生姜提取物能有效抑制副溶血弧菌生物膜的形成,较合适的抑制浓度分别为0.5%、1%和1%。      

Short communication: Urea hydrolysis in dairy cattle manure under different temperature,urea, and pH conditions

The objective of the study was to quantify the rate of urea hydrolysis in dairy cattle manure under different initial urea concentration, temperature, and pH conditions. In particular, by varying all 3 factors simultaneously, the interactions between them could also be determined. Fresh feces and artificial urine solutions were combined into a slurry to characterize the rate of urea hydrolysis under 2 temperatures (15°C and 35°C), 3 urea concentrations in urine solutions (500, 1,000, and 1,500 mg of urea-N/dL), and 3 pH levels (6, 7, and 8). Urea N concentration in slurry was analyzed at 0.0167, 1, 2, 4, 6, 8, 12, 16, 20, and 24 h after initial mixing. A nonlinear mixed effects model was used to determine the effects of urea concentration, pH, and temperature treatments on the exponential rate of urea hydrolysis and to predict the hydrolysis rate for each treatment combination. We detected a significant interaction between pH and initial urea level. Increasing urea concentration from 1,000 to 1,500 mg of urea-N/dL decreased the rate of urea hydrolysis across all pH levels. Across all pH and initial urea levels, the rate of urea hydrolysis increased with temperature, but the effect of pH was only observed for pH 6 versus pH 8 at the intermediate initial urea concentration. The fast rates of urea hydrolysis indicate that urea was almost completely hydrolyzed within a few hours of urine mixing with feces. The estimated urea hydrolysis rates from this study are likely maximum rates because of the thorough mixing before each sampling. Although considerable mixing of feces and urine occurs on the barn floor of commercial dairy operations from cattle walking through the manure, such mixing may be not as quick and thorough as in this study. Consequently, the urea hydrolysis rates from this study indicate the maximum loss of urea and should be accounted for in management aimed at mitigating ammonia emissions from dairy cattle manure under similar urea concentration, pH, and temperature conditions reported in this experiment.     

Model for the combined effects of temperature, pH and sodium chloride concentration on survival of Shigella flexneri strain 5348 under aerobic conditions

Shigella is recognized as a major foodborne pathogen; however, relatively few studies have been reported on its growth and survival characteristics, particularly under conditions relevant to food. A fractional factorial design was used to measure the effects and interactions of temperature (4-37 degrees C), pH (2-6) and NaCl (0.5-9%) on survival kinetics of Shigella flexneri strain 5348 in BHI broth. Stationary-phase cells were inoculated into sterile media to give initial populations of 6-7 log(10) CFU/ml and bacterial populations were determined periodically by aerobic plate counts. A total of 267 cultures, representing 83 variable combinations of temperature, pH and NaCl concentration, were analyzed. Survivor curves were fitted from plate count data by means of a two-phase linear model to determine lag times and slopes of the curves, from which decimal reduction times (D-values) and times to a 4-log10 inactivation (t 4D) were calculated. Second order response surface models in terms of temperature, initial pH and NaCl concentration were obtained for the inactivation kinetics parameters of S. flexneri using regression analysis. The use of log10 transformation of the inactivation kinetics parameters yielded models with R2 values of >0.8. These models can provide an estimate of Shigella inactivation. The data obtained suggest that Shigella is resistant to acid and salt and that low pH foods stored at low temperatures may serve as vehicles for gastrointestinal illness.     

A collagenase-targeted multiplex PCR assay for identification of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus

A multiplex PCR assay using three collagenase-targeted primer pairs for the species-specific detection of Vibrio alginolyticus, Vibrio cholerae, and Vibrio parahaemolyticus was developed. The results highlight the species specificity of the three primer sets designed. Because of the increasing importance of Vibrio spp. in human foodborne diseases, molecular approaches for routine microbial screening and monitoring of clinical, environmental, and food samples also have become more important. The results of this study indicate that the gene coding for collagenase should be used as an alternative molecular target to discriminate among the three Vibrio species.