Vibrio parahaemolyticus in granulated ark shell clam (Tegillarca granosas): accumulation from water and survival during cold storage and thermal process

This study investigated accumulation of Vibrio parahaemolyticus in granulated ark shell clam (Tegillarca granosas) exposed to contaminated water and survival of V. parahaemolyticus in the clams during cold storage and heating processes. Vibrio parahaemolyticus could be accumulated in clams to a level similar to that of contaminated water within 12 h of exposure of clams to contaminated water at temperatures between 9 and 33 °C. Keeping clams stored at 5 and 0 °C for 10 days resulted in 1.98 and 2.32 log MPN g^?1 reductions of V. parahaemolyticus, respectively, in the clams. Frozen storage at ?18 °C for 15 days or at ?30 °C for 30 days were capable of reducing V. parahaemolyticus from 4.05 log MPN g^?1 to non‐detectable levels (< 3 MPN g^?1). A heating process in hot water at 80 °C or higher for 1 min also reduced V. parahaemolyticus in the clams to non‐detectable levels.     

Comparison of a Chromogenic Medium with Thiosulfate-Citrate-Bile Salts-Sucrose Agar for Detecting Vibrio parahaemolyticus

ABSTRACT: The widely used most probable number (MPN) method for detecting Vibrio parahaemolyticus cannot differentiate growth of V. parahaemolyticus from Vibrio vulnificus or Vibrio mimicus on the thiosulfate-citrate-bile salts-sucrose agar (TCBS). Presumptive positive colonies grown onTCBS need to be confirmed with lengthy biochemical tests. This study compared a chromogenic medium, Bio-Chrome Vibrio medium (BCVM), with TCBS for detecting V. parahaemolyticus in seawater, sediment, and oysters using a 3-tube MPN method. Among the 296 samples tested, 136 and 92 samples produced presumptive positive results on TCBS and BCVM, respectively. Biochemical tests and a multiplex polymerase chain reaction (PCR) assay confirmed 74 of 83 samples that were presumptive positive on both TCBS and BVCM as V. parahaemolyticus . Although false-positive results were reported when either medium was used, there were 62 reported for TCBS whereas only 15 were reported for BCVM. The specificities of TCBS and BCVM for V. parahaemolyticus detection were determined to be 77% and 94%, respectively. The accuracies of detecting V. parahaemolyticus were 54% for TCBS and 84% for BCVM. The Bio-Chrome Vibrio medium can be used in the MPN method to reduce the number of biochemical tests needed for V. parahaemolyticus confirmation.     

Prevalence of Vibrio parahaemolyticus in seafoods and their processing environments as detected by duplex PCR

A duplex polymerase chain reaction (PCR) procedure targeting the genes gyrB and tl was established for specific identification of Vibrio parahaemolyticus from seafoods and processing environments. It could detect as few as 2.5 × 10² colony‐forming units mL^?1 in pure cultures. Direct detection of V. parahaemolyticus was also possible from artificially contaminated shrimp samples if combined with proteinase treatment. The homogeneous colonies on thiosulfate/citrate/bile salts/sucrose agar suspected to be V. parahaemolyticus or closely related species (n = 37) out of 259 samples of seafoods and processing environments were identified using the conventional method and duplex PCR. Both methods identified 17 isolates as V. parahaemolyticus from among the suspected isolates. Only one of the 17 V. parahaemolyticus isolates possessed the thermostable direct haemolysin gene (tdh) fragment as detected by different primer pairs in single PCR. Copyright © 2006 Society of Chemical Industry     

Occurrence and distribution of Vibrio parahaemolyticus in retail oysters in Sao Paulo State, Brazil

Vibrio parahaemolyticus is a potentially pathogenic bacterium that occurs naturally in estuarine environments worldwide, and is often associated with gastroenteritis in humans following consumption of raw bivalve mollusks, especially raw oysters. The occurrence of total and pathogenic V. parahaemolyticus in 74 samples of raw oysters collected in restaurants, supermarkets, groceries and beach huts in Sao Paulo State, was monitored between February 2006 and January 2007. Enumeration of V. parahaemolyticus was performed according to the most probable number (MPN) procedure. Five to ten typical colonies were selected from thiosulfate-citrate-bile salts-sucrose (TCBS) agar plates for confirmation by the presence of the species-specific gene tlh and the virulence genes tdh and trh by multiplex PCR. V. parahaemolyticus was detected in 100% of samples. The densities of total V. parahaemolyticus varied from 1.78 to 6.04 log₁₀ (MPN/g), with higher densities being detected in fall and summer, and lower densities in winter (P < 0.05). There was no statistical difference among densities of V parahaemolyticus regarding the site of collection. None of the 1943 V. parahaemolyticus isolates contained tdh and/or trh. These data provide information for the assessment of exposure to V. parahaemolyticus in oysters consumed in Sao Paulo, State, Brazil.     

Incidence and Enumeration of Vibrio parahaemolyticus in Shellfish from Two Retail Sources and the Genetic Diversity of Isolates as Determined by RAPD-PCR Analysis

A total of 83 shellfish samples from two local retail sources (A and B) yielded 38 samples positive for the presence of Vibrio parahaemolyticus based on 3 tube MPN enrichments and isolation from thiosulfate-citrate-bile salts-sucrose agar (TCBS) and biochemical tests. The 38 positive samples yielded 133 biochemically presumptive isolates of V. parahaemolyticus. Among these 133 presumptive isolates, 104 were confirmed by the polymerase chain reaction (PCR), which yielded more reliable identification results than the biochemical tests. The 38 biochemically presumptive samples yielded 29 samples that were confirmed by PCR to be positive for the presence of V. parahaemolyticus. RAPD analysis with three random primers was performed to examine the genetic diversity of 64 strains among the PCR confirmed V. parahaemolyticus isolates from both retail sources. 52 of 56 composite RAPD types consisted of single strains, indicating that most of the V. parahaemolyticus isolates were genetically quite heterogeneous. No strains representing the same RAPD type occurred in both retail outlets, implying that contamination of the shellfish by V. parahaemolyticus from the 2 retail sources was from different environmental locals and shellfish harvesting areas. Eight genomic clusters were generated at the 25% similarity level in a dendrogram based on RAPD profiles. With few exception, isolates with close genetic relationships grouping into an individual cluster tended to be derived from the same retail source.     

双层平板法对海产食品中副溶血性弧菌定量效果评价

评价了双层平板(DLAP)计数法对海产食品中副溶血性弧菌的定量分析效果。通过在混合菌悬液和鱼、虾、贝等生物体中人为模拟染菌,并进行冷冻受伤减菌处理,采用DLAP计数法对副溶血性弧菌处理前后的菌量变化进行分析,同时采用MPN法、BCVM平板计数法和TCBS平板计数法3种方法作为对照,运用SPSS软件分析4种方法测定结果的显著性差异(P<0.05)。试验结果表明,在理想的控制条件下,4种方法对不同基质条件中副溶血性弧菌的测定结果不存在明显差异;在菌体活力不强或受伤状态下,DLAP计数法对该菌的测定结果与MPN法在同一水平上且缩短了5d的检验周期,其准确性优于BCVM平板计数法和TCBS平板计数法,具有省时、准确的特点,适合广普应用。     

Selectivity and specificity of a chromogenic medium for detecting Vibrio parahaemolyticust

The thiosulfate-citrate-bile salts-sucrose agar (TCBS) used in the most-probable-number method for detecting Vibrio parahaemolyticus cannot differentiate growth of V. parahaemolyticus from Vibrio vulnificus or Vibrio mimicus. This study examined the selectivity and specificity of Bio-Chrome Vibrio medium (BCVM), a chromogenic medium that detects V. parahaemolyticus on the basis of the formation of distinct purple colonies on the medium. A panel consisting of 221 strains of bacteria, including 179 Vibrio spp. and 42 non-Vibrio spp., were examined for their ability to grow and produce colored colonies on BCVM. Growth of Salmonella, Shigella, Escherichia coli, Enterobacter cloacae, Yersinia enterocolitica, and Aeromonas was inhibited by both BCVM and TCBS. All 148 strains of V. parahaemolyticus grew on BCVM, and 145 of them produced purple colonies. The remaining 31 Vibrio spp., except one strain of Vibrio fluvialis, were either unable to grow or produced blue-green or white colonies on BCVM. Bio-Chrome Vibrio medium was capable of differentiating V. parahaemolyticus from other species, including V. vulnificus and V. mimicus. Further studies are needed to evaluate the sensitivity and specificity of BCVM for detecting V. parahaemolyticus in foods.     

High Salinity Relaying to Reduce Vibrio parahaemolyticus and Vibrio vulnificus in Chesapeake Bay Oysters (Crassostrea virginica)

Cases of Vibrio infections in the United States have tripled from 1996 to 2009 and these infections are most often associated with the consumption of seafood, particularly oysters (Crassostrea virginica). Information is needed on how to reduce numbers of Vibrio parahaemolyticus and Vibrio vulnificus in bi‐valve molluscan shellfish (for example, oysters). The purpose of this study was to evaluate the effectiveness of high salinity relaying or treatment in recirculating aquaculture systems (RASs) as methods to reduce the abundance of V. parahaemolyticus and V. vulnificus in oysters. For relaying field trials, oysters were collected from approved harvest waters, temperature abused outside under a tarp for 4 h, and then transferred to high (29 to 33 ppt.) and moderate (12 to 19 ppt.) salinities. For RAS treatment trial, oysters were transferred to 32 to 34 ppt. salinity at 15 °C. After 7, 14, 21, and in some instances 28 d, oysters were collected and analyzed for V. parahaemolyticus and V. vulnificus levels using multiplex real‐time PCR. Initial levels of V. parahaemolyticus and V. vulnificus ranged from 3.70 to 5.64 log₁₀ MPN/g, and were reduced by 2 to 5 logs after 21 to 28 d in high salinity water (29 to 34 ppt.). Oyster mortalities averaged 4% or less, and did not exceed 7%. Relaying of oysters to high salinity field sites or transfer to high salinity RAS tanks was more effective in reducing V. vulnificus compared with V. parahaemolyticus. These results suggest that high salinity relaying of oysters is more effective in reducing V. vulnificus than V. parahaemolyticus in the oyster species used in this study.     

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.     

Detection,Identification, and Prevalence of Pathogenic Vibrio parahaemolyticus in Fish and Coastal Environment in Jordan

Vibrio parahaemolyticus is widely distributed in the marine environments and considered the leading cause of human gastroenteritis in Asian countries. A total of 150 marketed fish and 50 water and sediment samples from the Gulf of Aqaba were examined for the prevalence of pathogenic strains of V. parahaemolyticus. A total of 132 typical isolates obtained from the primary selective medium (thiosulfate‐citrate bile salt sucrose agar) and showed positive biochemical properties were subjected to confirmation by polymerase chain reaction targeting the gyrB and toxR genes. These genes were confirmed at rates of 82% (108 isolates) and 72% (95 isolates), respectively. The toxR positive isolates were tested for the presence of thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and tdh‐related hemolysin (trh) virulence genes. Accordingly, the prevalence rates of pathogenic V. parahaemolyticus were 4%, 8%, and 12% in sediment, water, and fish samples, respectively. The 16S rRNA amplification and sequences were conducted for confirmation of the isolates and showing the relatedness among these isolates. The results showed that both 16S rRNA and toxR assays had same sensitivity and tested isolates had high nucleotide similarity irrespective of their sources.     

A review of the current status of cultural and rapid detection of Vibrio parahaemolyticus

Vibrio parahaemolyticus is ubiquitous in estuarine environments and can be commonly found in seafood products. This bacterial pathogen continues to emerge as an important cause of foodborne illness, and several foodborne disease outbreaks caused by V. parahaemolyticus have been linked to the consumption of contaminated seafood, in particular those consumed raw such as oysters. In response to these outbreaks, especially during the 1990s, several cultural, immunological‐based and molecular detection methods have been developed, which allow for rapid detection and quantification of total and pathogenic V. parahaemolyticus. The development of molecular methodology has allowed for clinical and environmental isolates of V. parahaemolyticus to be subtyped, thus providing the framework for risk‐based strategies aimed at controlling foodborne outbreaks cause by this pathogen. It is important that the detection and typing methods strive to accomplish detection and differentiation of the pathogenic strains from environmental (non‐pathogenic) ones, as well as to detect the presence of the organism and not just the presence of V. parahaemolyticus produced toxins, which can also be produced by closely related species. This review covers the current status of detection and typing methodology for identification and characterisation of V. parahaemolyticus from seafood.     

Validation of high pressure processing for inactivating Vibrio parahaemolyticus in Pacific oysters (Crassostrea gigas)

This study identified and validated high hydrostatic pressure processing (HPP) for achieving greater than 3.52-log reductions of Vibrio parahaemolyticus in the Pacific oysters (Crassostrea gigas) and determined shelf life of processed oysters stored at 5 °C or in ice. Raw Pacific oysters were inoculated with a clinical strain of V. parahaemolyticus 10293 (O1:K56) to levels of 10^4-5 cells per gram and processed at 293 MPa (43 K PSI) for 90, 120, 150, 180 and 210 s. Populations of V. parahaemolyticus in oysters after processes were analyzed with the 5-tube most probable number (MPN) method. Negative results obtained by the MPN method were confirmed with a multiplex PCR detecting genes encoding thermolabile hemolysin (tl), thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). A HPP of 293 MPa for 120 s at groundwater temperature (8 ± 1 °C) was identified capable of achieving greater than 3.52-log reductions of V. parahaemolyticus in Pacific oysters. Oysters processed at 293 MPa for 120 s had a shelf life of 6-8 days when stored at 5 °C or 16-18 days when stored in ice. This HPP can be adopted by the shellfish industry as a post harvest process to eliminate V. parahaemolyticus in raw oysters.     

Total and pathogenic Vibrio parahaemolyticus in shrimp: Fast and reliable quantification by real-time PCR

Vibrio parahaemolyticus is found in aquatic environments and is the leading cause of gastroenteritis due to seafood consumption worldwide. We evaluated a quantitative real-time PCR (Q-PCR) assay with hydrolysis probes, to determine whether this method could be used for the efficient counting of total, tdh and trh-positive V. parahaemolyticus in shrimps. We assessed the specificity of this assay, using 62 strains from 12 non target bacterial species of the Vibrio, Photobacterium, Shewanella and Aeromonas genera. Only V. parahaemolyticus with the appropriate target gene generated a fluorescent signal. We assessed the robustness of this assay, by analyzing spiked shrimps by Q-PCR and traditional culture methods, using most probable number (MPN)-PCR. After a 6 h enrichment period, the assay successfully detected total and pathogenic V. parahaemolyticus in shrimps samples spiked with less than 5 V. parahaemolyticus cells/g of shrimp. The Q-PCR results obtained were compared with those obtained by most probable number (MPN)-PCR format. An excellent correlation between the two methods was observed in all cases (R² > 0.9742). The application of this Q-PCR assay to 85 natural shrimp samples also resulted in the successful quantification of V. parahaemolyticus in this matrix, and the counts obtained were correlated with those obtained by (MPN)-PCR (P = 0.2598). Thus, this rapid and sensitive Q-PCR can be used to quantify V. parahaemolyticus in natural shrimp samples. This assay could be proposed, in response to the demands of the European Commission, as a tool for testing for the presence of vibrios in crustaceans, making it possible to legislate in this domain.     

东南沿海地区零售海产品中创伤弧菌的监测

目的了解我国东南沿海地区零售海产品中创伤弧菌的污染状况。方法采用3%氯化钠碱性蛋白胨水增菌,改良纤维二糖-多粘菌素B-多粘菌素E(mCPC)琼脂和纤维二糖-多粘菌素E(CC)琼脂分离海产品试样中的创伤弧菌,可疑菌落的鉴定采用生化试验或PCR法。采用最可能数(MPN)法和PCR法检测牡蛎试样的创伤弧菌污染水平。结果天然污染海产品试样创伤弧菌的检出率为19.8%(20/101)。牡蛎试样中45%(55/122)创伤弧菌密度低于3MPN/g的最低检出限。牡蛎中创伤弧菌的污染水平存在季节性差异。结论未来应加强对我国海产品中创伤弧菌污染状况的监测,尤其是在温暖的季节。     

Comparison of molecular detection methods for Vibrio parahaemolyticus and Vibrio vulnificus

Pathogenic vibrios are a global concern for seafood safety and many molecular methods have been developed for their detection. This study compares several molecular methods for detection of total and pathogenic Vibrio parahaemolyticus and Vibrio vulnificus, in MPN enrichments from oysters and fish intestine samples. This study employed the DuPont Qualicon BAX^® System Real-Time PCR assay for detection of V. parahaemolyticus and V. vulnificus. Multiplex real-time PCR detection of total (tlh+), tdh+, and trh+ V. parahaemolyticus was conducted on the Cepheid SmartCycler II. Total (rpoD) and tdh+ V. parahaemolyticus were also detected using LAMP. V. vulnificus detection was performed using real-time PCR methods developed for the SmartCycler and the AB 7500 Fast. Recommended template preparations were compared to BAX^® lysis samples for suitability. There was no significant difference in detection of V. parahaemolyticus and V. vulnificus using the BAX^® or SmartCycler assays. The AB assay showed no difference from other methods in detection of V. vulnificus unless boiled templates were utilized. There was a significant difference in detection of tdh+ V. parahaemolyticus between SmartCycler and LAMP assays unless the total (tlh+) V. parahaemolyticus gene target was omitted from the SmartCycler assay; a similar trend was observed for trh+ V. parahaemolyticus.     

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.     

Detection and molecular characterization of Vibrio parahaemolyticus isolated from seafood harvested along the southwest coast of India

The levels of total and tdh⁺ Vibrio parahaemolyticus were estimated in 83 seafood samples from southwest coast of India by colony hybridization. Conventional enrichment and isolation technique was also used to study the prevalence. Polymerase chain reaction (PCR) was performed on bacterial cell lyates for detection of total and pathogenic V. parahaemolyticus by amplification of specific genes. Of 83 samples tested, V. parahaemolyticus could be detected in 74 (89.2%) samples and tdh⁺ V. parahaemolyticus in 5 (6.0%) samples by colony hybridization. V. parahaemolyticus was detected in 68 (81.9%) of 83 samples after 18 h of enrichment by PCR, and isolated from 63 (75.9%) of 83 samples by conventional isolation. The virulence genes tdh and trh could be detected in 8.4% and 25.3%, respectively, in the sample enrichment broths by PCR. Use of colony hybridization following enrichment to achieve sensitive detection of tdh⁺ V. parahaemolyticus in seafood was evaluated using another set of 58 seafood samples. Thirty pathogenic V. parahaemolyticus strains isolated during the study were screened by PCR for genetic markers to be specific for the detection of the pandemic clone. Results of this study suggest that the GS-PCR may serve as a reliable genetic marker for the pandemic clone of V. parahaemolyticus.     

Development of enhanced selective media for detection of Vibrio parahaemolyticus in oysters

This study was undertaken to develop enhanced selective media for detection of Vibrio parahaemolyticus in oysters. Primarily, tryptic soy agar (TSA) was supplemented with 4.5–5% NaCl, 0.1–0.5% oxgall, and/or 1–2% sodium citrate, and adjusted to pH 8–9. A total of 21 Vibrio spp., 24 indicators, and 26 food-borne isolates were streaked on the modified media, followed by 24 h of incubation at 37 °C. While all the indicators and isolates failed to grow on TSA containing 5% NaCl, 0.5% oxgall, and 2% sodium citrate (TSA_OSS1; pH 9), V. parahaemolyticus was culturable on this selective medium. Particularly, the ability of TSA_OSS1 to quantify V. parahaemolyticus in oysters was superior to thiosulphate citrate bile salts sucrose (TCBS) agar. V. parahaemolyticus distinctly produced its white-yellowish, round, and edge-pointed colony on TSA_OSS1. TSAO_SS1 with high selectivity potentials over TCBS may be a promising alternative for detection of V. parahaemolyticus in seafoods or natural reservoirs. Supplementary InformationThe online version contains supplementary material available at (10.1007/s10068-021-00877-0)     

Managing the risk of Vibrio parahaemolyticus infections associated with oyster consumption: A review

Vibrio parahaemolyticus is a Gram‐negative bacterium that is naturally present in the marine environment. Oysters, which are water filter feeders, may accumulate this pathogen in their soft tissues, thus increasing the risk of V. parahaemolyticus infection among people who consume oysters. In this review, factors affecting V. parahaemolyticus accumulation in oysters, the route of the pathogen from primary production to consumption, and the potential effects of climate change were discussed. In addition, intervention strategies for reducing accumulation of V. parahaemolyticus in oysters were presented. A literature review revealed the following information relevant to the present study: (a) managing the safety of oysters (for human consumption) from primary production to consumption remains a challenge, (b) there are multiple factors that influence the concentration of V. parahaemolyticus in oysters from primary production to consumption, (c) climate change could possibly affect the safety of oysters, both directly and indirectly, placing public health at risk, (d) many intervention strategies have been developed to control and/or reduce the concentration of V. parahaemolyticus in oysters to acceptable levels, but most of them are mainly focused on the downstream steps of the oyster supply chain, and (c) although available regulation and/or guidelines governing the safety of oyster consumption are mostly available in developed countries, limited food safety information is available in developing countries. The information provided in this review may serve as an early warning for managing the future effects of climate change on the safety of oyster consumption.     

Rapid and sensitive detection of Vibrio parahaemolyticus in sea foods by electrochemiluminescence polymerase chain reaction method

Vibrio parahaemolyticus has been considered as one of the most important food-borne bacterial pathogens. Because of the safety concerns, detection and characterization of V. parahaemolyticus have attracted much attention. In this study, electrochemiluminescence polymerase chain reaction (ECL-PCR) method combined with universal probes hybridization technique was applied to rapid detection of V. parahaemolyticus, infected and uninfected sea foods for the first time. Whether the sea food samples were infected was discriminated by detecting the gyrase B (gyrB) gene. We detect V. parahaemolyticus both in artificially contaminated sea foods and natural samples. The experiment results show that the infected and uninfected sea food samples can be clearly identified and the detection limit for V. parahaemolyticus is 1.6 pg purified genomic DNA in the presence of 1 μg non-specific background DNA. The technique may provide a new means in V. parahaemolyticus detection due to its simplicity and high efficiency.