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This study aimed to determine the prevalence of V. parahaemolyticus in oysters from the northwestern coast of Mexico and to identify the serotypes, virulence factors, and antibiotic resistance of the strains. Oyster samples were collected from 2012 to 2020 from the northwest coast of Mexico; biochemical and molecular methods were used to identify V. parahaemolyticus from oysters; antiserum reaction to determine V. parahaemolyticus serotypes, and PCR assays were performed to identify pathogenic (tdh and/or trh) or pandemic (toxRS/new, and/or orf8) strains and antibiotic resistance testing. A total of 441 oyster samples were collected and tested for V. parahaemolyticus. Forty-seven percent of oyster samples were positive for V. parahaemolyticus. Ten different O serogroups and 72 serovars were identified, predominantly serotype O1:KUT with 22.2% and OUT:KUT with 17.3%. Twenty new serotypes that had not been previously reported in our region were identified. We detected 4.3% of pathogenic clones but no pandemic strains. About 73.5% of strains were resistant to at least one antibiotic, mainly ampicillin and ciprofloxacin; 25% were multi-drug resistant. In conclusion, the pathogenic strains in oysters and antibiotic resistance are of public health concern, as the potential for outbreaks throughout northwestern Mexico is well established., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Vibrio parahaemolyticus is a leading cause of human gastroenteritis associated with seafood consumption. The present study aimed to investigate the occurrence and risk assessment of V. parahaemolyticus isolated from live Indian black clams, sediment, and water samples collected from shellfish harvesting areas located along the south-west coast of India. Out of the total 72 samples collected, 55.6% revealed the presence of V. parahaemolyticus ; the highest occurrence was observed in shellfish samples. The presence of tdh and trh virulence genes was screened by multiplex PCR. Virulence genes could be detected in 25.8% of the strains; 19.35% of them were trh positive and 3.2% were tdh positive, while 3.2% of strains exhibited the coexistence of both virulence genes. Antimicrobial resistance (AMR) determined by the disk diffusion method revealed that 87% of the strains were multiple drug resistant and exhibited 21 diverse resistance patterns. The overall multiple antibiotic resistance (MAR) index values ranged from 0 to 0.8. To the best of our knowledge, this is the first report to document the presence of pathogenic and multidrug-resistant V. parahaemolyticus in shellfish harvesting areas of the Indian sub-continent. The study reveals possible health hazards associated with consuming shellfish harvested from the study area., Competing Interests: The authors declare there is no conflict., (© 2024 The Authors This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying, adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/).)

This study evaluated the occurrence and distribution of total and pathogenic V. parahaemolyticus in oyster culture environments in Taiwan. V. parahaemolyticus levels in oysters, seawater, and sediment were quantified using the most probable number (MPN) method combined with a qualitative polymerase chain reaction (PCR). Total V. parahaemolyticus was determined based on the presence or absence of tlh gene, whereas pathogenic V. parahaemolyticus was determined based on the detection of tdh and/or trh gene. The results showed that: 1) V. parahaemolyticus was detected in 93% of the collected samples, 2) the mean concentrations of total V. parahaemolyticus in oysters, seawater, and sediment were 4.1 log MPN/g, 2.1 log MPN/mL, and 4.2 log MPN/g, respectively, and 3) variations in the abundance of V. parahaemolyticus was significantly associated with sea surface temperature (SST). Findings in this study could be used to improve the accuracy of the risk assessment model for V. parahaemolyticus in oysters in Taiwan., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Vibrio parahaemolyticus is one of the leading causes of diarrhoea and gastroenteritis in human on consumption of raw or insufficiently cooked seafood. This study was aimed at isolating and characterizing the pathogenic and pandemic V. parahaemolyticus from oysters (n = 90) in coastal parts of West Bengal, India; their antibiotic resistance and potential for involvement in the food chain. During bacteriological culture, typical V. parahaemolyticus colony was recovered in 88.9% samples followed by presumptive identification in 71 (78.9%) samples by characteristic biochemical (K/A) test. All the presumptive isolates (n = 71) were confirmed by species specific Vp-toxR PCR assay. Of these, 10 (14.08%) were tdh + and none for the trh. Further, 5 (50%) of these tdh + isolates were found to carry the pandemic potential gene in PGS-PCR assay; however, none in GS-PCR. Majority (80%) of these pathogenic (tdh + ) isolates belonged to pandemic serovars (OUT: KUT; OUT: K24; O1: KUT; O1:K25; O10: KUT) and only 20% to non-pandemic serovars (OUT: K15; O9:K17). All the isolates (100%) exhibited resistance to cefpodoxime followed by ampicillin and cefotaxime (90%), ceftizoxime (60%), tetracycline (50%), ceftriaxone (40%), ciprofloxacin and nalidixic acid (10% each). Overall, the study findings suggested that 11.1% (10/90) of commonly marketed oysters in this area were harbouring pathogenic V. parahaemolyticus. Moreover, 5.5% (5/90) of the oyster population were harbouring pandemic strains of this pathogen. Besides, the pathogenic isolates from oysters were exhibiting a considerable genetic relatedness (53 to 70%) to human clinical isolates in PFGE analysis that relates to a substantial public health risk. Further, their multidrug resistance added gravity to the antimicrobial resistance (AMR), a globally growing public health threat and this is a critical area of concern especially during the treatment of foodborne gastroenteritis., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Vibrio parahaemolyticus is the most common pathogen causing seafood-borne illnesses in Korea. The present study evaluated the occurrence, virulence, and antimicrobial resistance of V. parahaemolyticus in seawater and bivalves obtained in 2016 from the southern coast of Korea, an important region for commercial aquaculture industries, especially the Korean raw seafood culture. V. parahaemolyticus was detected in 87 of 160 (54.4%) bivalve samples and in 32 of 130 (24.5%) seawater samples. Especially high levels were detected during summer to early autumn. All the seawater and bivalves contained less than 2 and 5% of the tdh and trh genes of the isolates, respectively, and seawater isolates possessed two fewer genes than the bivalve isolates. Of 23 antimicrobials tested, three agents (ofloxacin, norfloxacin, and trimethoprim/sulfamethoxazole) effectively treated V. parahaemolyticus illness due to the sensitivity of the isolates. The isolates were highly resistant to ampicillin, however, excluding it as a treatment option. More than half of the isolates exhibited resistance to at least three antimicrobials. These findings indicate the importance of an integrated monitoring and surveillance program noting the occurrence, virulence, and antimicrobial resistance patterns of V. parahaemolyticus in various aquatic sources for preventing human health risks from seafood consumption.

This study investigated the influences of seawater to oyster ratio on depuration for decontaminating V. parahaemolyticus in raw oysters with a goal of identifying the proper ratio of oyster to seawater capable of improving the efficacy of the depuration process. The water to oyster ratios tested in this study ranged from 1.0 to 2.5 L of artificial seawater (ASW) per oyster (40 oysters in 40, 60, 80 and 100 L ASW). The depuration efficacy for purging V. parahaemolyticus from oysters was highest when we applied a 2:1, followed by 1.5:1, 2.5:1, and 1:1 L of ASW/oyster. Further studies of depuration with 2:1 L of ASW/oyster found that the concentration of V. parahaemolyticus in oysters decreased in a nonlinear manner. The depuration curve was fitted to a one phase decay model with a coefficient of determination (R 2 ) of 0.933. The time for a 3 log reduction was 1.75 days with a 95% confidence interval from 1.65 to 1.85 days, which meets the FDA's requirement of larger than a 3.0 log (MPN/g) reduction as a post-harvest process for V. parahaemolyticus control. After 4 days levels in all trials were <100 MPN/g meeting performance standards established by Japan and Canada. Furthermore, the time for a 3.52 log reduction was 3.17 days with a 95% confidence interval from 2.92 to 3.54 days but it took 5 days to reduce levels to <30 MPN/g, which satisfies FDA's requirement as a post-harvest control process (>3.52 log MPN/g reduction) for the purpose of making safety added labeling claims for V. parahaemolyticus., (Copyright © 2018. Published by Elsevier Ltd.)

We determined the abundance and virulence of Vibrio parahaemolyticus in seawater and bivalves from the Gyeongnam coast in Korea, a major area for the seafood industry, during 2004-2016. V. parahaemolyticus is one of the most common pathogen causing seafood-borne illnesses in Korea, and increases during the summer. Its occurrence in seawater and bivalve samples was seasonally dependent, with high levels during the summer to early autumn. There were more strains in the area of sea continually exposed to inland wastewater. Only 5.1% and 3.5% of V. parahaemolyticus isolates from seawater and bivalves, respectively, had the trh gene, and only the bivalve isolates produced the tdh gene at levels below 2%. Continuous monitoring is clearly needed to reduce seafood-borne outbreaks of disease caused by V. parahaemolyticus, and to reveal the occurrence patterns and the presence of toxic genes of the strains in different marine environments., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The objective of this study was to determine the effect of high hydrostatic pressure (HHP) on the inactivation of Vibrio sp. in pure cultures and mussel homogenates. Four Vibrio strains including V. alginolyticus, V. cholerae, V. parahaemolyticus and V. vulnificus were used. HHP treatments were performed with both pure Vibrio sp. cultures in alkaline peptone water (2% NaCl) and artificially inoculated mussel homogenates at pressure levels of 250, 350 and 450 MPa for 1 and 3 min at 25°C. Counts of Vibrio were determined before and after treatment using drop plating method. The effect of high pressure on the reduction level significantly differed among the respective Vibrio species. Vibrio vulnificus was the most susceptible species to HHP. To achieve a >5 log reduction in mussel homogenates, pressure treatment needs to be (i) 350-450 MPa for ≥1 min at 25°C for both V. alginolyticus and V. cholerae, (ii) 250 MPa for ≥3 min or 350-450 MPa for ≥1 min for V. vulnificus and (iii) 350 MPa for ≥3 min or 450 MPa for ≥1 min for V. parahaemolyticus., Significance and Impact of the Study: High hydrostatic pressure (HHP) has been applied to inactivate spoilage and pathogenic micro-organisms in a variety of food products, including seafood. Vibrio sp. are frequently reported as the main cause of foodborne illness associated with consumption of raw or undercooked seafood particularly shellfish worldwide. To date, data on the inactivation of Vibrio sp. via HHP are still limited and most of the trials only investigated HHP application in oysters and clams. This study demonstrates the efficacy of HHP inactivating Vibrio sp. in both pure culture and mussel homogenates., (© 2018 The Society for Applied Microbiology.)

High salinity relay of Eastern oysters (Crassostrea virginica) was evaluated as a post-harvest processing (PHP) method for reducing Vibrio vulnificus. This approach relies on the exposure of oysters to natural high salinity waters and preserves a live product compared to previously approved PHPs. Although results of prior studies evaluating high salinity relay as a means to decrease V. vulnificus levels were promising, validation of this method as a PHP following approved guidelines is required. This study was designed to provide data for validation of this method following Food and Drug Administration (FDA) PHP validation guidelines. During each of 3 relay experiments, oysters cultured from 3 different Chesapeake Bay sites of contrasting salinities (10-21 psu) were relayed without acclimation to high salinity waters (31-33 psu) for up to 28 days. Densities of V. vulnificus and densities of total and pathogenic Vibrio parahaemolyticus (as tdh positive strains) were measured using an MPN-quantitative PCR approach. Overall, 9 lots of oysters were relayed with 6 exhibiting initial V. vulnificus >10,000/g. As recommended by the FDA PHP validation guidelines, these lots reached both the 3.52 log reduction and the <30 MPN/g densities requirements for V. vulnificus after 14 to 28 days of relay. Densities of total and pathogenic V. parahaemolyticus in relayed oysters were significantly lower than densities at the sites of origin suggesting an additional benefit associated with high salinity relay. While relay did not have a detrimental effect on oyster condition, oyster mortality levels ranged from 2 to 61% after 28 days of relay. Although the identification of the factors implicated in oyster mortality will require further examination, this study strongly supports the validation of high salinity relay as an effective PHP method to reduce levels of V. vulnificus in oysters to endpoint levels approved for human consumption., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Vibrio parahaemolyticus is a major pathogen that is mainly associated with seafood and is a global concern of food safety. With high prevalence of contamination in food, efficient strategy is needed to decontaminate those contaminated foods and control the emergence of vibriosis. In the present study, a V. parahaemolyticus-specific phage vB&#95;VpaS&#95;OMN (designated as phage OMN) was isolated from oyster. Phage OMN had good pH (5-9) and temperature tolerance (<50 °C). Phage OMN exhibited broad host range against isolates of V. parahaemolyticus (20/31). After treatment with phage OMN in the liquid condition for 7 h, the number of V. parahaemolyticus was reduced significantly compared to control treatment. When phage OMN was applied to oyster samples for 48 and 72 h, 90% and 99%, respectively, of V. parahaemolyticus was inactivated on Oyster meat surface. Sequence analysis showed that phage OMN had a 42.202 bp genome and revealed about 59.04% homology with Cronobacter phage vB&#95;CsaP&#95;Ss1. Only 10 CDSs can be predicted based on the GenBank database, while 42% of the CDSs were unique to OMN and had no known function, indicating that phage OMN is a new lytic phage. Fully understanding of the function for the phage genes and the properties of the phage is important for the development of strategies to control V. parahaemolyticus contamination in oysters and disease in aquaculture., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Tetrodotoxins (TTX) are a potent group of natural neurotoxins putatively produced by symbiotic microorganisms and affecting the aquatic environment. These neurotoxins have been recently found in some species of bivalves and gastropods along the European Coasts (Greece, UK, and The Netherlands) linked to the presence of high concentrations of Vibrio , in particular Vibrio parahaemolyticus . This study is focused on the evaluation of the presence of Vibrio species and TTX in bivalves (mussels, oysters, cockles, clams, scallops, and razor clams) from Galician Rias (northwest of Spain). The detection and isolation of the major Vibrio spp. and other enterobacterial populations have been carried out with the aim of screening for the presence of the pathways genes, poliketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) possibly involved in the biosynthesis of these toxins. Samples containing Vibrio spp. were analyzed by biochemical (API20E-galery) and genetic tests (PCR-RT). These samples were then screened for TTX toxicity by a neuroblastoma cell-based assay (N2a) and the presence of TTX was further confirmed by LC-MS/MS. TTX was detected in two infaunal samples. This is the first confirmation of the presence of TTX in bivalve molluscs from the Galician Rias., Competing Interests: The authors declare no conflict of interest.

Vibrio parahaemolyticus and Vibrio vulnificus are naturally occurring estuarine bacteria and are the leading causes of seafood-associated infections and mortality in the United States. Though multiple-antibiotic-resistant V. parahaemolyticus and V. vulnificus strains have been reported, resistance patterns in vibrios are not as well documented as those of other foodborne bacterial pathogens. Salinity relaying (SR) is a postharvest processing (PHP) treatment to reduce the abundances of these pathogens in shellfish harvested during the warmer months. The purpose of this study was to evaluate the antimicrobial susceptibility (AMS), pathogenicity, and genetic profiles of V. parahaemolyticus and V. vulnificus recovered from oysters during an oyster relay study. Isolates ( V. parahaemolyticus [ n = 296] and V. vulnificus [ n = 94]) were recovered from oysters before and during the 21-day relaying study to detect virulence genes ( tdh and trh ) and genes correlated with virulence ( vcgC ) using multiplex quantitative PCR (qPCR). AMS to 20 different antibiotics was investigated using microbroth dilution, and pulsed-field gel electrophoresis (PFGE) was used to study the genetic profiles of the isolates. Twenty percent of V. vulnificus isolates were vcgC + , while 1 and 2% of V. parahaemolyticus were tdh + and trh + , respectively. More than 77% of the V. vulnificus isolates and 30% of the V. parahaemolyticus isolates were resistant to at least one antimicrobial. Forty-eight percent of V. vulnificus and 8% of V. parahaemolyticus isolates were resistant to two or more antimicrobials. All isolates demonstrated a high genetic diversity, even among those isolated from the same site and having a similar AMS profile. No significant effects of the relaying process on AMS, virulence genes, or PFGE profiles of V. vulnificus and V. parahaemolyticus were observed. IMPORTANCE Analysis of the antibiotic resistance profiles of V. vulnificus and V. parahaemolyticus isolated from oysters during this study indicated that more than 48% of V. vulnificus isolates were resistant to two or more antimicrobials, including those recommended by the CDC for treating Vibrio infections. Also, the V. parahaemolyticus isolates showed high MICs for some of the Vibrio infection treatment antibiotics. Monitoring of AMS profiles of this bacterium is important to ensure optimal treatment of infections and improve food safety. Our study showed no significant differences in the AMS profiles of V. vulnificus ( P = 0.26) and V. parahaemolyticus ( P = 0.23) isolated from the oysters collected before versus after relaying. This suggests that the salinity of the relaying sites did not affect the AMS profiles of the Vibrio isolates, although it did reduce the numbers of these bacteria in oysters (S. Parveen et al., J Food Sci 82:484-491, 2017, https://doi.org/10.1111/1750-3841.13584)., (Copyright © 2018 American Society for Microbiology.)

The human pathogen Vibrio parahaemolyticus is a leading cause of seafood-borne illness in the USA, and infections with V. parahaemolyticus typically result from eating raw or undercooked oysters. V. parahaemolyticus has been shown to be highly resistant to oyster depuration, suggesting that the bacterium possesses specific mechanisms or factors for colonizing oysters and persisting during depuration. In this study, we characterized eight different V. parahaemolyticus strains for differences in resistance to oyster depuration, biofilm formation, and motility. While each strain exhibited distinct phenotypes in the various assays, we determined that biofilm formation on abiotic surfaces, such as glass or plastic, does not directly correlate with bacterial retention in oysters during depuration. However, we did observe that the motility phenotype of a strain appeared to be a better indicator for persistence in the oyster. Further studies examining the molecular mechanisms underlying the observed colonization differences by these and other V. parahaemolyticus strains may provide beneficial insights into what critical factors are required for proficient colonization of the Pacific oyster.

Background: Vibrio parahaemolyticus is an emerging foodborne pathogen in the Mediterranean, usually associated with shellfish consumption. The increase in the number of outbreaks in Europe is primarily associated with the global warming of the ocean that has a great impact on the spread and genetic selection of waterborne pathogens. The primary role of Italy in Europe's mollusk production, together with the fact that cases of infections with V. parahaemolyticus are not always notified to the European community, highlighted the necessity of acquiring new information about the epidemiological involvement of shellfish products., Objective: The aim of the study was to provide useful insights into the first steps of the Risk Assessment associated with V. parahaemolyticus through the molecular characterization of isolates from commercialized mollusks., Materials and Methods: A total of 102 strains identified as V. parahaemolyticus were investigated as part of a larger sampling (1-year survey) from several shellfish species collected from the Venice lagoon and the North Adriatic sea. All strains were characterized by multilocus sequence typing and tested for the presence of virulence genes (trh and tdh). The study of sampling/environmental factors and epidemiological analyses was performed to describe the behaviors of the different genetic populations., Results: The population structure analysis highlighted three genetic clusters that could be subject to temperature selection during cold (≤15°C) and warm (>16°C) seasons. Moreover, other factors, such as molluscan species (clams/mussels), probably played a role in the distribution of genetic clusters. Although few strains carried the virulence factors (n = 6 trh + ), epidemiological links with clinical isolates and a local dissemination of some sequence types were underlined., Conclusion: This work provides a useful background on the genotype spread as a first step in the Hazard Identification in light of future climate changes.

Monitoring of Vibrio vulnificus and V. parahaemolyticus abundance is pertinent due to the ability of these species to cause disease in humans through aquatic vectors. Previously, we performed a multiyear investigation tracking Vibrio spp. levels in five sites along the southeastern North Carolina coast. From February 2013 to October 2015, total V. vulnificus and V. parahaemolyticus abundance was measured in water, oysters and clams. In the current study, pathogenic subpopulations were identified in these isolates using molecular markers, revealing that 5.3% of V. vulnificus isolates possessed the virulence-correlated gene (vcgC), and 1.9% of V. parahaemolyticus isolates harbored one or both of the virulence-associated hemolysin genes (tdh and trh). Total V. parahaemolyticus abundance was not sufficient to predict the abundance of pathogenic subpopulations. Specifically, pathogenic V. parahaemolyticus isolates were more often isolated in cooler waters and were sometimes isolated when no other V. parahaemolyticus strains were detectable. Vibrio vulnificus clinical (C-) genotypes correlated with total V. vulnificus; however, salinity, water depth and total suspended solids influenced C- and E-genotypes differently. Lastly, we documented individual oysters harboring significantly higher V. vulnificus levels for which there was no ecological explanation, a phenomenon that deserves closer attention due to the potentially elevated health hazard associated with these 'hot' shellfish., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Microbiological quality of 206 raw ready-to-eat seafood samples was investigated according to species (gizzard shad, halibut, rockfish, tuna, oyster and squid) and distribution channels (fishery, hyper and online market). Enumeration of aerobic plate count and total coliforms (TC) and pathogenic bacteria (Bacillus cereus, Staphylococcus aureus and Vibrio parahaemolyticus) was performed, and level of microbiological quality was classified into four groups: satisfactory, acceptable, unsatisfactory and unacceptable. Qualitative analysis was also performed for Escherichia coli and eight foodborne pathogens (B. cereus, E. coli O157:H7, Listeria monocytogenes, Salmonella spp., S. aureus, Vibrio cholerae, V. parahaemolyticus, and Vibrio vulnificus). Raw ready-to-eat seafood products revealed 0·5% at an unsatisfactory level and 4·9% at an unacceptable level due to ≥4 log CFU g -1 of TC in squid and ≥3 log CFU g -1 of V. parahaemolyticus in gizzard shad respectively. Gizzard shad was shown to be potentially hazardous, as its sashimi is eaten with its skin attached. Bacillus cereus, E. coli, S. aureus, V. parahaemolyticus and V. vulnificus were qualitatively detected. Samples from the fishery market showed higher detection rate especially in V. parahaemolyticus (21·6%) and V. vulnificus (1·7%) which indicates the need to improve microbiological safety of raw ready-to-eat seafood products in fishery market., Significance and Impact of the Study: Raw ready-to-eat seafood products like sashimi can be easily contaminated with various bacteria from aquatic environments and human reservoirs, which subsequently bring about a risk in food poisoning due to no heating process before consumption. The results of this study provide comprehensive microbiological data on various species of raw ready-to-eat seafood from various distribution channels. It may contribute to establish reasonable standard and effective strategies to ensure a good microbiological quality of raw ready-to-eat seafood for the safety of meals, like sashimi and sushi., (© 2016 The Society for Applied Microbiology.)

Background: Vibrio parahaemolyticus is an important gastroenteritis pathogen contaminating seafood in China. In this study a total of 992 seafood samples from major hypermarkets in Shanghai were monitored for prevalence and burden of V. parahaemolyticus from January 2011 to December 2012. Additionally, appropriate probability distributions for describing V. parahaemolyticus concentrations were assessed based on these surveillance data., Results: Seventeen of 992 samples were positive for V. parahaemolyticus and the geometric mean was 0.1581 most probable number (MPN) g -1 . The variation in prevalence of V. parahaemolyticus was seasonal and the burden of contamination in August (0.1942 MPN g -1 ) was significant (P < 0.01) between 2011 and 2012. Also, the prevalence of V. parahaemolyticus was higher in shellfish and cephalopods than in other seafood (P < 0.05). By comparison, the lognormal distribution and integrated distribution showed no obvious difference for characterizing V. parahaemolyticus contamination., Conclusion: The low prevalence and burden found indicated that seafood from hypermarkets may not be an important risk source for V. parahaemolyticus infection in Shanghai, and more attention should be paid to other areas for selling seafood, such as farmlands or farmers' markets. The simple and effective lognormal distribution is recommended as a better choice for describing V. parahaemolyticus contamination in future risk assessment studies. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

Filter feeding shellfish can concentrate pathogenic bacteria, including Vibrio vulnificus and Vibrio parahaemolyticus, as much as 100-fold from the overlying water. These shellfish, especially clams and oysters, are often consumed raw, providing a route of entry for concentrated doses of pathogenic bacteria into the human body. The numbers of foodborne infections with these microbes are increasing, and a better understanding of the conditions that might trigger elevated concentrations of these bacteria in seafood is needed. In addition, if bacterial concentrations in water are correlated with those in shellfish, then sampling regimens could be simplified, as water samples can be more rapidly and easily obtained. After sampling of oysters and clams, either simultaneously or separately, for over 2 years, it was concluded that while Vibrio concentrations in oysters and water were related, this was not the case for levels in clams and water. When clams and oysters were collected simultaneously from the same site, the clams were found to have lower Vibrio levels than the oysters. Furthermore, the environmental parameters that were correlated with levels of Vibrio spp. in oysters and water were found to be quite different from those that were correlated with levels of Vibrio spp. in clams., Importance: This study shows that clams are a potential source of infection in North Carolina, especially for V. parahaemolyticus These findings also highlight the need for clam-specific environmental research to develop accurate Vibrio abundance models and to broaden the ecological understanding of clam-Vibrio interactions. This is especially relevant as foodborne Vibrio infections from clams are being reported., (Copyright © 2016 American Society for Microbiology.)

Oysters can accumulate potentially pathogenic water bacteria. The objective of this study was to compare two procedures to quantify Vibrio species present in oysters to determine the most sensitive method. We analyzed oyster samples from the Gulf of Mexico, commercialized in Mexico City. The samples were inoculated in tubes with alkaline peptone water (APW), based on three tubes and four dilutions (10 -1 to 10 -4 ). From these tubes, the first quantification of Vibrio species was performed (most probable number (MPN) from tubes) and bacteria were inoculated by streaking on thiosulfate-citrate-bile salts-sucrose (TCBS) petri dishes. Colonies were isolated for a second quantification (MPN from dishes). Polymerase chain reaction (PCR) was used to determine species with specific primers: ompW for Vibrio cholerae, tlh for Vibrio parahaemolyticus, and VvhA for Vibrio vulnificus. Simultaneously, the sanitary quality of oysters was determined. The quantification of V. parahaemolyticus was significantly higher in APW tubes than in TCBS dishes. Regarding V. vulnificus counts, the differences among both approaches were not significant. In contrast, the MPNs of V. cholerae obtained from dishes were higher than from tubes. The quantification of MPNs through PCR of V. parahaemolyticus and V. vulnificus obtained from APW was sensitive and recommendable for the detection of both species. In contrast, to quantify V. cholerae, it was necessary to isolate colonies on TCBS prior PCR. Culturing in APW at 42 °C could be an alternative to avoid colony isolation. The MPNs of V. cholerae from dishes was associated with the bad sanitary quality of the samples.

Abalone is a popular seafood in South Korea; however, because it contains various microorganisms, its ingestion can cause food poisoning. Therefore, analysis of the microbiota on abalone can improve understanding of outbreaks and causes of food poisoning and help to better manage seafood products. In this study, we collected a total of 40 abalones from four different regions in March and July, which are known as the maximum abalone production areas in Korea. The microbiota were analyzed using high-throughput sequencing, and bacterial loads on abalone were quantified by real-time PCR. Over 2700 species were detected in the samples, and Alpha- and Gammaproteobacteria were the predominant classes. The differences in microbiota among regions and at each sampling time were also investigated. Although Psychrobacter was the dominant genus detected on abalone in both March and July, the species compositions were different between the two sampling times. Five potential pathogens (Lactococcus garvieae, Yersinia kristensenii, Staphylococcus saprophyticus, Staphylococcus warneri, and Staphylococcus epidermidis) were detected among the abalone microbiota. In addition, we analyzed the influence of Vibrio parahaemolyticus infection on shifts in abalone microbiota during storage at different temperatures. Although the proportion of Vibrio increased over time in infected and non-infected abalone, the shifts of microbiota were more dynamic in infected abalone. These results can be used to better understand the potential of food poisoning caused by abalone consumption and manage abalone products according to the microbiota composition., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The development of procedures for the efficient removal or inactivation of noroviruses from contaminated oysters is of great interest in oyster production. However, there is a critical limitation for evaluating the depuration efficacy of presently available procedures, as no suitable cell culture system currently exists to cultivate noroviruses. Thus, we applied a next-generation sequencing (NGS) technique to characterize norovirus genotypes in pre- and post-depurated oysters. As a result, we revealed the diversity of noroviruses in pre- and post-depurated oysters. Although the applied depuration procedure could reduce the number of bacterial agents to the level recommended by the Japanese Ministry of Health, Labour and Welfare, no significant changes were observed in the detection rate and the proportion of norovirus group (G) I and GII genotypes. To our knowledge, this is the first report to evaluate the profile of noroviruses in pre- and post-depurated oysters, specifically with respect to norovirus removal, using NGS; the findings imply that the removal of noroviruses from oysters through depuration is not presently sufficient. Further studies are needed to develop a more suitable depuration procedure for removing and/or inactivating noroviruses from contaminated oysters.

Vibrio parahaemolyticus is a causative agent of human serious seafood-borne gastroenteritis disease and even death. Shrimps, often eaten raw or undercooked, are an important reservoir of the bacterium. In this study, we isolated and characterized a total of 400 V. parahaemolyticus strains from commonly consumed fresh shrimps (Litopenaeus vannamei, Macrobrachium rosenbergii, Penaeus monodon, and Exopalaemon carinicauda) in Shanghai fish markets, China in 2013-2014. The results revealed an extremely low occurrence of pathogenic V. parahaemolyticus carrying two major toxic genes (tdh and trh, 0.0 and 0.5 %). However, high incidences of antibiotic resistance were observed among the strains against ampicillin (99 %), streptomycin (45.25 %), rifampicin (38.25 %), and spectinomycin (25.50 %). Approximately 24 % of the strains derived from the P. monodon sample displayed multidrug resistant (MDR) phenotypes, followed by 19, 12, and 6 % from the E. carinicauda, L. vannamei, and M. rosenbergii samples, respectively. Moreover, tolerance to heavy metals of Cr(3+) and Zn(2+) was observed in 90 antibiotic resistant strains, the majority of which also displayed resistance to Cu(2+) (93.3 %), Pb(2+) (87.8 %), and Cd(2+)(73.3 %). The pulsed-field gel electrophoresis (PFGE)-based genotyping of these strains revealed a total of 71 distinct pulsotypes, demonstrating a large degree of genomic variation among the isolates. The wide distribution of MDR and heavy-metal resistance isolates in the PFGE clusters suggested the co-existence of a number of resistant determinants in V. parahaemolyticus population in the detected samples. This study provided data in support of aquatic animal health management and food safety risk assessment in aquaculture industry.

Vibrio parahaemolyticus can cause serious human seafoodborne gastroenteritis and even death. In this study, we isolated and characterized 208 V. parahaemolyticus strains from 10 species of commonly consumed crustaceans and shellfish available in fish markets in Shanghai, People's Republic of China, in 2014. Most of these aquatic species had not been detected previously. The results revealed an extremely low occurrence of pathogenic V. parahaemolyticus carrying the toxin gene trh (1.9%). However, a high level of resistance to the antibiotics ampicillin (94.2%), rifampin (93.3%), and streptomycin (77.9%) was found. Approximately 74.5% of the isolates had multidrug-resistant phenotypes. Tolerance to the heavy metals Cu(2+), Pb(2+), and Cd(2+) was detected in the majority of antibiotic resistant isolates. The resistance patterns differed depending on the tested samples. The crustaceans Penaeus monodon and Marsupenaeus japonicus harbored more antibiotic-resistant bacteria, whereas the isolates from the crustacean Litopenaeus vannamei and the shellfish Busycon canaliculatus had high tolerance to eight heavy metals tested. In contrast to the wide distribution of multidrug resistance and tolerance to heavy metals, lower percentages of plasmid DNA (22.6%) and SXT/R391-like integrative and conjugative elements (4.8%) were detected in the isolates, suggesting that V. parahaemolyticus in these aquatic species may have adopted some other molecular mechanisms that mediated the high prevalence of resistance determinants. The results of this study support the need for food safety risk assessment of aquatic products.

Unlabelled: Vibrio parahaemolyticus and Vibrio vulnificus can grow rapidly in shellfish subjected to ambient air conditions, such as during intertidal exposure. In this study, levels of total and pathogenic (tdh(+) and/or trh(+)) V. parahaemolyticus and total V. vulnificus were determined in oysters collected from two study locations where intertidal harvest practices are common. Samples were collected directly off intertidal flats, after exposure (ambient air [Washington State] or refrigerated [New Jersey]), and after reimmersion by natural tidal cycles. Samples were processed using a most-probable-number (MPN) real-time PCR method for total and pathogenic V. parahaemolyticus or V. vulnificus In Washington State, the mean levels of V. parahaemolyticus increased 1.38 log MPN/g following intertidal exposure and dropped 1.41 log MPN/g after reimmersion for 1 day, but the levels were dependent upon the container type utilized. Pathogenic V. parahaemolyticus levels followed a similar trend. However, V. vulnificus levels increased 0.10 log MPN/g during intertidal exposure in Washington but decreased by >1 log MPN/g after reimmersion. In New Jersey, initial levels of all vibrios studied were not significantly altered during the refrigerated sorting and containerizing process. However, there was an increase in levels after the first day of reimmersion by 0.79, 0.72, 0.92, and 0.71 log MPN/g for total, tdh(+) and trh(+) V. parahaemolyticus, and V. vulnificus, respectively. The levels of all targets decreased to those similar to background after a second day of reimmersion. These data indicate that the intertidal harvest and handling practices for oysters that were studied in Washington and New Jersey do not increase the risk of illness from V. parahaemolyticus or V. vulnificus, Importance: Vibrio parahaemolyticus and Vibrio vulnificus are the leading causes of seafood-associated infectious morbidity and mortality in the United States. Vibrio spp. can grow rapidly in shellfish subjected to ambient air conditions, such as during periods of intertidal exposure. When oysters are submersed with the incoming tide, the vibrios can be purged. However, data on the rates of increase and purging during intertidal harvest are scarce, which limits the accuracy of risk assessments. The objective of this study was to help fill these data gaps by determining the levels of total and pathogenic (tdh(+) and/or trh(+)) V. parahaemolyticus and V. vulnificus in oysters from two locations where intertidal harvest practices are common, using the current industry practices. The data generated provide insight into the responses of Vibrio spp. to relevant practices of the industry and public health, which can be incorporated into risk management decisions., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Vibrio parahaemolyticus is a marine bacterium that is considered as one of the major causes of bacterial food-borne outbreaks at a global scale. A total of 114 samples including mussel (n = 42), seawater (n = 22) and fish (n = 50) samples were collected and subjected to investigation. Vibrio parahaemolyticus was detected in 45 (39%) of 114 samples with an occurrence in mussel, seawater and fish samples of 76, 40·9 and 8% respectively. A total of 96 isolates were positive for the species-specific genes toxR and tlh and confirmed as V. parahaemolyticus. Presence of the virulence marker gene tdh was not identified in any of the strains investigated; however, four of strains were positive for the trh gene. Serological analysis of eight randomly selected trh-negative isolates identified three different serotypes: O4:K untypeable (KUT), O2:KUT, O3:KUT. Conversely, all four trh-positive strains belonged to a single serotype (O1:K1) and share an undistinguishable genetic profile by PFGE analysis, suggesting the existence of a dominant clone for the trh-positive strains in the region., Significance and Impact of the Study: Vibrio parahaemolyticus is the most prevalent food-poisoning bacterium associated with seafood consumption. The number of infections is increasing worldwide and is being reported in areas with no previous incidence. This study provides the first instance of the occurrence of V. parahaemolyticus strains with virulence traits in the Black Sea, contributing to gain a better understanding about potential risk associated with this pathogen in the region., (© 2016 The Society for Applied Microbiology.)

Background: Vibrio parahaemolyticus is a major foodborne pathogen, particularly in Asian countries. Increased occurrence of outbreaks of V. parahaemolyticus gastroenteritis in China indicates the need to evaluation of the prevalence of this pathogenic species. V. parahaemolyticus distribution in shellfish from the eastern coast of China has been reported previously. However, to date, the prevalence of V. parahaemolyticus in retail aquatic products in North China has not been determined. To investigate the prevalence of V. parahaemolyticus in aquatic products in North China, 260 aquatic product samples were obtained from retail markets in 6 provinces of North China from November to December in 2012 and July to August in 2013., Results: V. parahaemolyticus was detected in 94 (36.2%) of the samples by the most probable number method. The density of V. parahaemolyticus ranged from 1.50 to 1100 MPN/g. V. parahaemolyticus was detected at a rate of 50.0% and 22.7% in summer and in winter, respectively. The density of V. parahaemolyticus was significantly higher in summer than in winter, with mean levels of 16.5 MPN/g and 5.0 MPN/g, respectively. Among 145 V. parahaemolyticus isolates examined, none of the isolates possessed tdh and trh. In multiplex PCR-based O-antigen serotyping of these 145 isolates, all serotypes, other than O6, O7, and O9, were detected, and serotype O2 was found to be the most prevalent (detected in 54 isolates). The 145 isolates were grouped into 7 clusters by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) at a similarity coefficient of 0.66. The antimicrobial resistance patterns of these 145 isolates to 12 antimicrobial agents revealed that most of the isolates were resistant to streptomycin (86.2%), while fewer were resistant to ampicillin (49.6%), cefazolin (43.5%), cephalothin (35.9%), and kanamycin (22.1%). All of the examined isolates were susceptible to azithromycin and chloramphenicol., Conclusions: The findings of this study will help in defining appropriate monitoring programs, understanding of the dissemination of antibiotic resistant strains, and providing information for the assessment of exposure to this microorganism at the consumption level.

The aim of the study was to investigate the prevalence and the antimicrobial resistance patterns of Vibrio (V.) spp. isolated from retail shrimp in Hanoi, Vietnam A total of 202 shrimp samples were collected from retail markets located in ten urban districts of Hanoi. Among those, 201 (99.5%) samples were positive for Vibrio spp. The most common species detected was V parahaemolyticus (96.5%), followed by V. alginolyticus (56.4%), V. cholerae (2%) and V. vulnificus (1.5%). Multiple Vibrio spp. were found in 114 (56.4%) samples. None of the V. parahaemolyticus isolates carried the virulence-associated tdh (thermostable direct haemolysin) and trh (tdh-related haemolysin) genes. In total, 195 V. parahaemolyticus isolates, four V. cholerae isolates and three V. vulnificus isolates were tested for resistance to eight antimicrobial agents. V. parahaemolyticus isolates showed high rates of resistance against ampicillin (87.2%), while a moderate rate was observed for sulfamethoxazole/trimethoprim (18.5%) and intermediate resistance towards tetracycline (24.6%). Low resistance rates (0.5%) were recorded against both ciprofloxacin and cefalothin. Only one V. cholerae isolate with resistance to ampicillin and two V. cholerae isolates with resistance to sulfamethoxazole/trimethoprim were found. All V. vulnificus isolates were susceptible to the eight antimicrobial agents tested. However, the number of V. vulnificus and V. cholerae was small. Multi-resistant isolates were found in V. parahaemolyticus with a low frequency (16.9%). The results of this study revealed the ubiquitous nature of Vibrio spp. in shrimp at retail. To reduce the potential risk of Vibrio infections due to handling or consumption of undercooked seafood, good manufacturing practice as well as safe handling and processing should be encouraged.

The aim of this study was to investigate the prevalence of Vibrio spp. in shrimp at retail and in shrimp farms in Ecuador and to determine the antimicrobial agent resistance patterns of farm isolates. The presence of genes linked to early mortality syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) also was evaluated. Vibrio spp. were isolated from retail shrimps in Cuenca, Ecuador, and farm shrimps originating from provinces El Oro and Guayas, Ecuador. A total of 229 shrimp samples were collected, of which 71 originated from retail markets in Cuenca and 158 came from shrimp farms. Overall, 219 (95.6%) samples tested positive for Vibrio spp. Vibrio parahaemolyticus (80.8%) was the most common species detected, followed by Vibrio alginolyticus (50.2%), Vibrio cholerae (11.3%), and Vibrio vulnificus (3.5%). None of the V. parahaemolyticus isolates carried the virulence-associated tdh and trh genes. In V. parahaemolyticus shrimp farm isolates, high resistance was found to ampicillin (92.2%), and intermediate resistance was found to tetracycline (51.3%) and amikacin (22.1%). Of the V. parahaemolyticus strains, 68 were resistant to at least three antimicrobial agents, and 2 were resistant to seven antimicrobial agents simultaneously. Up to 18 resistant isolates were found for V. alginolyticus, whereas V. vulnificus and V. cholerae isolates were more susceptible. None of the V. parahaemolyticus isolates carried the EMS-AHPND plasmid. The results of this study revealed the ubiquitous occurrence of Vibrio spp. in shrimps at retail and on shrimp farms in Ecuador.

A magnetic nanoprobe labeled immunochromatographic test strip (MNP/ICTS) was developed to detect food-borne pathogen Vibrio parahaemolyticus. Specific antibody against V. parahaemolyticus was used as test line by coating onto the nitrocellulose membrane. Magnetic nanoprobe was prepared by immobilizing the specific antibody onto the surface of superparamagnetic nanoparticles. Specificity and sensitivity of the MNP/ICTS system were verified by artificially contaminated shrimp homogenate samples. Reliability and application feasibility of the MNP/ICTS system were demonstrated by using seafood samples (n=36). Comparing with polymerase chain reaction (PCR) and traditional culture methods, the MNP/ICTS system is found to be not only a rapid qualitative analysis (~10 min), but also an accurately quantitative detection platform. Through its rapid magnetic separation property, the MNP/ICTS system is capable to flexibly combine with a sample enrichment and pre-incubation process. This combination makes the qualitative sensitivity for the food samples surged more than 100-fold. A naked-eye observation of 1.58×10(2) CFU/g V. parahaemolyticus was realized. This sensitivity could meet the V. parahaemolyticus test threshold value in many countries. Also, the total sample pre-treatment plus MNP/ICTS assay only needs about 4.5h. Namely, we can get test results in a day. Hence, the developed MNP/ICTS assay platform is simple, rapid and highly sensitive. It is a flexible test platform for pathogen detection. The favorable comparison with PCR and culture methods further proves that the developed MNP/ICTS is applicable into food-borne pathogen or other areas where a simple, rapid, sensitive and point-of-care analysis is desirable., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Unlabelled: Vibrio parahaemolyticus (Vp) and Vibrio vulnificus (Vv) are associated with the consumption of raw oysters and cause illnesses ranging from simple gastroenteritis to life-threatening septicaemia. These halophilic bacteria are frequently found in marine and estuarine systems, accumulating within the tissues of a number of aquatic organisms and passing on to humans after consumption, through contaminated water, or via open wounds. As benthic organisms capable of filtering 40 gallons of water per hour, sediment is an important source of potentially pathogenic vibrios in oysters destined for raw consumption. This research used off-bottom oyster culture to reduce vibrio concentrations in oysters. Colony hybridization was used to enumerate Vp and Vv in bottom and suspended oysters. Vv and Vp concentrations were generally lower in oysters suspended off-bottom, and suspension decreased vibrio loads in oysters by an average of 13%. Suspension of oysters reduced vibrio concentrations., Significance and Impact of the Study: This study found that oyster suspension significantly reduced some populations of potentially pathogenic vibrios. These results indicate that oyster suspension could be a viable approach for preharvest treatment to reduce illness in consumers of raw oysters., (© 2015 The Society for Applied Microbiology.)

Unlabelled: Vibrios are natural inhabitants of estuarine ecosystems and some species may pose public health problem as agents of sporadic or collective food-borne infections associated with the consumption of fish or shellfish. Samples of raw shrimp (n = 299), fished in coastal areas of the city of Agadir, Morocco, and collected from its fish marketplace, were examined for the presence of pathogenic vibrios. Microbiological analysis was carried out according to a protocol using thiosulphate citrate bile sucrose (TCBS) agar and CHROMagar Vibrio (CV) as selective media. Presumptive-positive colonies were identified by biochemical and species-specific PCR systems, and further tested by PCR for the presence of pathogenicity factors. The overall prevalence of Vibrio spp. in shrimps was 55·8%. Vibrio parahaemolyticus was recovered from 25 of 299 samples (8·4%), Vibrio cholerae from six samples (2%) and Vibrio alginolyticus from 161 samples (53·8%). No virulence genes were found among V. parahaemolyticus and V. cholerae isolates. The CHROMagar Vibrio plating medium was found to be more efficient than the Thiosulphate Citrate Bile salt Sucrose (TCBS) Agar in the isolation of Vibrio organisms., Significance and Impact of the Study: A significant proportion of shrimps marketed and consumed in Morocco are caught in the coastal region of the city of Agadir. This study provides interesting data of prevalence of Vibrio spp. in raw shrimps as well as better understanding of their potential virulence. It is apparent from this study that genes and primers used in multiplex PCR for identification and detection of virulence factors, can be used to monitor shrimps for the presence of potentially pathogenic strains of Vibrio cholerae and Vibrio parahaemolyticus. The results highlight the added value of using a chromogenic medium for research and isolation of pathogenic Vibrio in seafood, more specific and accurate than TCBS., (© 2015 The Society for Applied Microbiology.)

Vibrio vulnificus (Vv) and Vibrio parahaemolyticus (Vp) are the two leading causes of bacterial illnesses associated with raw shellfish consumption. Levels of these pathogens in oysters can increase during routine antifouling aquaculture practices involving dry storage in ambient air conditions. After storage, common practice is to resubmerge these stored oysters to reduce elevated Vv and Vp levels, but evidence proving the effectiveness of this practice is lacking. This study examined the changes in Vv and in total and pathogenic (thermostable direct hemolysin gene and the tdh-related hemolysin gene, tdh+ and trh+) Vp levels in oysters after 5 or 24 h of dry storage (28 to 32°C), followed by resubmersion (27 to 32°C) for 14 days. For each trial, replicate oyster samples were collected at initial harvest, after dry storage, after 7 days, and after 14 days of resubmersion. Oysters not subjected to dry storage were collected and analyzed to determine natural undisturbed vibrio levels (background control). Vibrio levels were measured using a most-probable-number enrichment followed by real-time PCR. After storage, vibrio levels (excluding tdh+ and trh+ Vp during 5-h storage) increased significantly (P < 0.001) from initial levels. After 7 days of resubmersion, Vv and total Vp levels (excluding total Vp in oysters stored for 5 h) were not significantly different (P < 0.1) from levels in background oysters. Vv and total and pathogenic Vp levels were not significantly different (P > 0.1) from levels in background oysters after 14 days of resubmersion, regardless of dry storage time. These data demonstrate that oyster resubmersion after dry storage at elevated ambient temperatures allows vibrio levels to return to those of background control samples. These results can be used to help minimize the risk of Vv and Vp illnesses and to inform the oyster industry on the effectiveness of routine storing and resubmerging of aquaculture oysters.

Raw oyster consumption is the most common route of exposure for Vibrio spp. infections in humans. Vibriosis has been increasing steadily in the United States despite efforts to reduce the incidence of the disease. Research has demonstrated that ice is effective in reducing postharvest Vibrio spp. growth in oysters but has raised concerns of possible contamination of oyster meat by filth (as indicated by the presence of fecal coliform bacteria or Clostridium perfringens). This study examined the use of ice slurries (<4.5°C) to reduce Vibrio growth. Ice slurries showed rapid internal cooling of oysters, from 23.9°C (75°F) to 10°C (50°F) within 12 min. The initial bacterial loads in the ice slurry waters were near the limits of detection. Following repeated dipping of oysters into ice slurries, water samples exhibited significant (P < 0.05) increases in median levels of fecal coliforms (9.5 most probable number [MPN]/100 ml), C. perfringens (280 MPN/100 ml), Vibrio vulnificus (11,250 MPN/ml), and total Vibrio parahaemolyticus (3,900 MPN/ml). The microbial load in oyster meat, however, was unchanged after 15 min of submergence, with no significant differences (P < 0.05) in levels of filth indicator (range, 250 to 720 MPN/100 g) or Vibrio spp. (range, 9,000 to 20,000 MPN/g) bacteria. These results support the use of ice slurries as a postharvest application for rapid cooling of oysters to minimize Vibrio growth.

We investigated the abundance of total and pathogenic Vibrio parahaemolyticus in shellfish sampled from four provinces in China during May 2013 and March 2014 using the most probable number-polymerase chain reaction (MPN-PCR) method. Total V. parahaemolyticus was detected in 67.7% of 496 samples. A total of 38.1% and 10.1% of samples exceeded 1,000 MPN g(-1) and 10,000 MPN g(-1), respectively. V. parahaemolyticus densities followed a seasonal and geographical trend, with Guangxi and Sichuan shellfish possessing total V. parahaemolyticus levels that were 100-fold higher than those of the Liaoning and Shandong regions. Moreover, the levels of V. parahaemolyticus were at least 10-fold higher in the summer and autumn than in the cooler seasons. Pathogenic V. parahaemolyticus levels were generally lower than total V. parahaemolyticus levels by several log units and tended to be high in samples contaminated with high total V. parahaemolyticus levels. The aqua farms had a lower prevalence but higher abundance of total V. parahaemolyticus compared to retail markets. The catering markets showed the lowest levels of total V. parahaemolyticus, but 20.0% of samples exceeded 1,000 MPN g(-1). The levels of both total and pathogenic V. parahaemolyticus in oysters were higher than in clams. The log-transformed abundance of V. parahaemolyticus was significantly correlated with both water temperature and air temperature but not water salinity. These results provide baseline contamination data of V. parahaemolyticus in shellfish in China, which can be applied to local risk assessments to prioritize risk control to key sectors and evaluate the effectiveness of future control measures.

The following study aimed to determine the antimicrobial susceptibility profile of Vibrio parahaemolyticus strains from fresh and frozen oysters Crassostrea rhizophorae sold in Fortaleza-Brazil. An antibiogram was performed on 87 isolates using nine antibiotics: gentamicin (Gen 10 µg), ampicillin (Amp 10 µg), penicillin G (Pen 10U), ciprofloxacin (Cip 5 µg), chloramphenicol (Chl 30 µg), nalidixic acid (Nal 30 µg), tetracycline (Tet 30 µg), vancomycin (Van 30 µg) and erythromycin (Ery 15 µg). All strains were resistant to at least one antibiotic, and 85 (97.7%) were multi-resistant, with predominance of the Van+ Pen+Amp resistance profile (n = 46). Plasmid resistance to Pen, Amp and Ery was detected. Thus, the risk that raw oyster consumption poses to the health of consumers is highlighted, due to the fact that these bivalves may host antibacterial-resistant microorganisms.

Vibrio parahaemolyticus is a marine bacterium recognized as an important cause of gastroenteritis in humans consuming contaminated shellfish. In recent years, increasing resistance to ampicillin and aminoglycosides has been observed among V. parahaemolyticus isolates. However, the first-line antimicrobials such as tetracyclines and fluoroquinolones remained highly effective against these bacteria. The aim of this study was to evaluate the occurrence of V. parahaemolyticus in live bivalve molluscs available on the Polish market and to determine the antimicrobial resistance of the recovered isolates. A total of 400 shellfish samples (mussels, oysters, clams, and scallops) from 2009 to 2012 were tested using the International Organization for Standardization standard 21872-1 method and PCR for the species-specific toxR gene. Antimicrobial susceptibility of the isolates was determined using a microbroth dilution method. V. parahaemolyticus was identified in 70 (17.5%) of the 400 samples, and the toxR gene was confirmed in 64 (91.4%) of these isolates. Most of the isolates were recovered from clams (31 isolates; 48.4% prevalence) followed by mussels (17 isolates; 26.6% prevalence). More V. parahaemolyticus-positive samples were found between May and September (22.7% prevalence) than between October and April (11.4% prevalence). Antibiotic profiling revealed that most isolates were resistant to ampicillin (56 isolates; 87.5%) and to streptomycin (45 isolates; 70.3%), but all of them were susceptible to tetracycline and chloramphenicol. Forty-one isolates (64.1%) were resistant to two or more antimicrobials; however, only one isolate (1.6%) was resistant to three antimicrobial classes. The antimicrobials used in treatment of human V. parahaemolyticus infection had high efficacy against the bacterial isolates tested. This study is the first concerning antibiotic resistance of V. parahaemolyticus isolates in Poland, and the results obtained indicate that these bacteria may pose a health risk to consumers.

Objective: To quantify Vibrio parahaemolyticus densities in American oyster (Crassostrea virginica) under cold storage., Materials and Methods: 320 oysters were stored at 7°C for nine days and total and pathogenic densities were determined by the NMP-PCR methodology., Results: V. parahaemolyticus tlh+ densities were observed on 0,3, and 6 days of storage at 1.134, 2.764 and 0.785 log10NMP/g, respectively, and pathogenic density trh+ on 0 and 3 days at 0.477 and 0.519 log10NMP/g, respectively; the pathogenic densities tdh+ (0.519 log10NMP/g), tdh+/trh+ (0.519 log10NMP/g), and tdh+orf8+ (-0.444 log10NMP/g) were detected on day 3 of storage., Conclusion: The results suggest that V. parahaemolyticus growth and pathogenic genes occurrence at 7°C involve changes in the genetic expression as a cold shock response, favoring V. parahaemolyticus survival and virulence, representing a health risk.

SUMMARY To determine risk factors for sporadic Vibrio parahaemolyticus gastroenteritis, we conducted a population-based case-control study in sentinel hospital surveillance areas of Shanghai and Jiangsu province, China. Seventy-one patients with diarrhoea and confirmed V. parahaemolyticus infections were enrolled, and they were matched with 142 controls for gender, age and residential area. From the multivariable analysis, V. parahaemolyticus infections were associated with antibiotics taken during the 4 weeks prior to illness [odds ratio (OR) 7·6, 95% confidence interval (CI) 1·1–54·4)], frequent eating out (OR 3·3, 95% CI 1·0–10·4), and shellfish consumption (OR 3·4, 95% CI 1·0–11·1), with population-attributable fractions of 0·09, 0·24, and 0·14, respectively. Protective factors included keeping the aquatic products refrigerated (OR 0·4, 95% CI 0·2–1·0) and pork consumption (OR 0·2, 95% CI 0·1–0·9) [corrected] .

The food-borne pathogen Vibrio parahaemolyticus has been reported as being present in New Zealand (NZ) seawaters, but there have been no reported outbreaks of food-borne infection from commercially grown NZ seafood. Our study determined the current incidence of V. parahaemolyticus in NZ oysters and Greenshell mussels and the prevalence of V. parahaemolyticus tdh and trh strains. Pacific (235) and dredge (21) oyster samples and mussel samples (55) were obtained from commercial shellfish-growing areas between December 2009 and June 2012. Total V. parahaemolyticus numbers and the presence of pathogenic genes tdh and trh were determined using the FDA most-probable-number (MPN) method and confirmed using PCR analysis. In samples from the North Island of NZ, V. parahaemolyticus was detected in 81% of Pacific oysters and 34% of mussel samples, while the numbers of V. parahaemolyticus tdh and trh strains were low, with just 3/215 Pacific oyster samples carrying the tdh gene. V. parahaemolyticus organisms carrying tdh and trh were not detected in South Island samples, and V. parahaemolyticus was detected in just 1/21 dredge oyster and 2/16 mussel samples. Numbers of V. parahaemolyticus organisms increased when seawater temperatures were high, the season when most commercial shellfish-growing areas are not harvested. The numbers of V. parahaemolyticus organisms in samples exceeded 1,000 MPN/g only when the seawater temperatures exceeded 19°C, so this environmental parameter could be used as a trigger warning of potential hazard. There is some evidence that the total V. parahaemolyticus numbers increased compared with those reported from a previous 1981 to 1984 study, but the analytical methods differed significantly., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The influence of environmental parameters on the total and pathogenic Vibrio parahaemolyticus seasonal densities in American oysters (Crassostrea virginica) was evaluated for 1 year. Harvesting site A yielded the highest mean densities of V. parahaemolyticus tlh+, tdh+/trh-, tdh-/trh+ and tdh+/trh+ during spring season at 2.57, 1.74, 0.36, and -0.40 log10 MPN/g, respectively, and tdh+/orf8+ during winter season (0.90 log10 MPN/g). V. parahaemolyticus tlh+ densities were associated to salinity (R(2)=0.372, P<0.022), tdh+/trh+ to turbidity (R(2)=0.597, P<0.035), and orf8+ to temperature, salinity, and pH (R(2)=0.964, P<0.001). The exposure to salinity and temperature conditions during winter and spring seasons regulated the dynamics of V. parahaemolyticus harboring potentially pathogenic genotypes within the oyster. The adaptive response of V. parahaemolyticus to seasonal environmental changes may lead to an increase in survival and virulence, threatening the seafood safety and increasing the risk of illness., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Vibrio parahaemolyticus carrying the tdh gene, encoding the thermostable direct hemolysin (TDH), or the trh gene, encoding the TDH-related hemolysin (TRH), are both considered virulent strains. There are, however, disproportionally fewer reports of infections caused by seafood contaminated with trh-positive strains than by seafood contaminated with tdh-positive strains. Bivalves such as clams and oysters are the major seafood varieties associated with the infections. In this study, the prevalence of strains possessing the tdh and trh genes was investigated in Japan in 74 samples collected in 2007-2008 and in 177 samples collected in 2010 of domestic bivalves, bloody clams, hen clams, short-neck clams, and rock oysters. The tdh-positive and trh-negative, tdh-negative and trh-positive, and tdh-positive and trh-positive samples represented 5.4%, 12.2%, and 4.1% of all samples collected in 2007-2008, and 5.1%, 18.6%, and 5.6% of all samples collected in 2010, respectively. As determined by polymerase chain reaction, the prevalence of tdh negative and trh positive in all samples was two to four times higher than that of tdh positive and trh negative. In the samples collected in 2010, the tdh-negative and trh-positive V. parahaemolyticus (20 samples) was more often isolated than tdh-positive and trh-negative V. parahaemolyticus (7 samples). The most common serotype of tdh-positive isolates (22 of 24 strains) was pandemic O3:K6. The trh-positive isolates (61 strains) were various serotypes including OUT:KUT. In 330 V. parahaemolyticus outbreaks and sporadic infections in Japan, most outbreaks and sporadic infections were caused by tdh-positive and trh-negative strains (89.4%). The frequencies of infections caused by tdh-negative and trh-positive, and both tdh- and trh-positive strains were 1.2% and 3.0%, respectively. This finding suggests that the virulence of trh might be less than that of tdh, although trh-positive V. parahaemolyticus frequently contaminated bivalves.

Vibrio spp. have emerged as a serious threat to human health worldwide. Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus are of particular concern as they have been linked to gastrointestinal infections and septicemia associated with the consumption of raw or undercooked seafood. We developed hydrolysis probe-based real-time PCR systems with an internal amplification control for the detection of these species. We applied these systems to a total of 167 fresh or frozen crustacean, fish and shellfish samples consumed in France. Of them, 34.7% (n=58) were positive for Vibrio. V. parahaemolyticus was the most common, in 31.1% of samples, followed by V. vulnificus in 12.6% and V. cholerae in 0.6%. Furthermore, V. parahaemolyticus and V. vulnificus were present simultaneously in 9.6% of samples. Virulence genes (tdh and trh sequences) were present in 25% of the V. parahaemolyticus-positive samples. The V. cholerae strain detected was non toxigenic. The densities of V. parahaemolyticus and V. cholerae ranged from <10(2) to 10(4)bacteria/g of seafood. All samples positive for V. vulnificus displayed low-level contamination with fewer than 10(2)bacteria/g. Our findings indicate that seafood consumption presents a potential risk to human health in France and highlight the importance of tools for a preventive consumer protection policy., (Copyright © 2014. Published by Elsevier B.V.)

Vibrio parahaemolyticus is a marine microorganism, recognized as cause of gastroenteritis outbreaks associated with seafood consumption. In this study the development and the in-house validation of a colony hybridization method for the enumeration of total and potentially pathogenic V. parahaemolyticus is reported. The method included a set of three controls (process, hybridization and detection control) for the full monitoring of the analytical procedure. Four digoxigenin-labeled probes were designed for pathogenic strains enumeration (tdh1, tdh2, trh1 and trh2 probes) and one for total V. parahaemolyticus count (toxR probe). Probes were tested on a panel of 70 reference strains and 356 environmental, food and clinical isolates, determining the inclusivity (tdh: 96.7%, trh: 97.8%, toxR: 99.4%) and the exclusivity (100% for all probes). Accuracy and linearity of the enumeration were evaluated on pure and mixed cultures: slopes of the regression lines ranged from 0.957 to 1.058 depending on the target gene and R(2) was greater than or equal to 0.989 for all reactions. Evaluation was also carried on using four experimentally contaminated seafood matrices (shellfish, finfish, crustaceans and cephalopods) and the slopes of the curves varied from 0.895 (finfish) to 0.987 (cephalopods) for the counts of potentially pathogenic V. parahaemolyticus (R(2)≥0.965) and from 0.965 to 1.073 for total V. parahaemolyticus enumeration (R(2)≥0.981). Validation was performed on 104 naturally contaminated shellfish samples, analyzed in parallel by colony hybridization, ISO/TS 21872-1 and MPN enumeration. Colony hybridization and ISO method showed a relative accuracy of 86.7%, and a statistically significant correlation was present between colony hybridization enumeration and MPN results (r=0.744, p<0.001). The proposed colony hybridization can be a suitable alternative method for the enumeration of total and potentially pathogenic V. parahaemolyticus in seafood., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Vibrio parahaemolyticus is a marine microorganism, recognized as an important cause of foodborne illness particularly in Asia, South America and United States. Outbreaks are rarely reported in Europe, but they can occur unexpectedly in relation, among other reasons, to the spread of highly virulent strains. It is known that the risk is proportional to exposure levels to pathogenic V. parahaemolyticus (i.e. carrying the tdh and/or the trh genes) but currently there is a lack of occurrence data for pathogenic V. parahaemolyticus in shellfish production areas of the Member States. In this study a total of 147 samples of bivalve molluscs, from harvesting areas of two Italian regions (Sardinia and Veneto) were analyzed for Escherichia coli and salmonella, according to Reg 2073/2005, and for detection and enumeration of total and toxigenic V. parahaemolyticus strains using a new DNA colony hybridization method. Environmental parameters (water temperature and salinity) were also recorded. Results of E. coli were consistently in agreement with the legislation limits for the harvesting class of origin and Salmonella was detected only in one sample. The average contamination levels for total V. parahaemolyticus were 84 and 73 CFU/g respectively for Sardinia and Veneto, with the highest value reaching 8.7 × 10(3)CFU/g. Nineteen samples (12.9%) resulted positive for the presence of potentially pathogenic V. parahaemolyticus strains, with levels ranging between 10 and 120 CFU/g and most of the positive samples (n=17) showing values equal or below 20 CFU/g. A significant correlation (r=0.41) was found between water temperature and V. parahaemolyticus levels, as well as with isolation frequency. The data provided in this study on contamination levels of total and potentially pathogenic V. parahaemolyticus, seasonal distribution and correlation with water temperature, will help in defining appropriate monitoring programs and post-harvest policies for this hazard, improving the management of the harvesting areas and the safety of bivalve molluscs., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The abundance of total and pathogenic Vibrio parahaemolyticus (Vp) strains in American oysters (Crassostrea virginica) harvested in two different harvest sites from the Mandinga lagoon System was evaluated monthly for 1 year (January through December 2012). Frequencies of species-specific genes and pathogenic genes exhibited a seasonal distribution. The annual occurrence of Vp with the species-specific tlh gene (tlh(+)) was significantly higher during the winter windy season (32.50%) and spring dry season (15.0%), with the highest densities observed during spring dry season at 283.50 most probable number (MPN)/g (lagoon bank A, near human settlements), indicating the highest risk of infection during warmer months. Pathogenic Vp tlh(+)/tdh(+) frequency was significantly higher during the winter windy and the spring dry seasons at 22.50 and 10.00%, respectively, with highest densities of 16.22 and 41.05 MPN/g (bank A), respectively. The tlh/trh and tdh/trh gene combinations were also found in Vp isolates during the spring dry season at 1.25 and 1.3%, respectively, with densities of 1.79 and 0.4 MPN/g (bank A), respectively. The orf8 genes were detected during the winter windy season (1.25%) with highest densities of 5.96 MPN/g (bank A) and 3.21 MPN/g (bank B, near mangrove islands and a heron nesting area). Densities of Vp tdh(+) were correlated (R(2) = 0.245, P < 0.015) with those of Vp orf8(+). The seasonal dynamics of Vp harboring pathogenic genes varied with seasonal changes, with very high proportions of Vp tdh(+) and Vp orf8(+) isolates in the winter windy season at 46.2 and 17.0%, respectively, which suggests that environmental factors may differentially affect the abundance of pathogenic subpopulations. Although all densities of total Vp (Vp tlh(+)) were lower than 10(4) MPN/g, thus complying with Mexican regulations, the presence of pathogenic strains is a public health concern. Our results suggest that total Vp densities may not be appropriate for assessing oyster contamination and predicting the risk of infection. Evaluation of the presence of pathogenic strains would be a better approach to protecting public health.

Although thermostable direct hemolysin-producing (tdh(+)) Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis, the enumeration of tdh(+) V. parahaemolyticus remains challenging due to its low densities in the environment. In this study, we developed a most-probable-number (MPN)-based procedure designated A-IS(1)-LAMP, in which an immunomagnetic separation (IMS) technique targeting as many as 69 established K antigens and a loop-mediated isothermal amplification (LAMP) assay targeting the thermostable direct hemolysin (tdh) gene were applied in an MPN format. Our IMS employed PickPen, an eight-channel intrasolution magnetic particle separation device, which enabled a straightforward microtiter plate-based IMS procedure (designated as PickPen-IMS). The ability of the procedure to quantify a wide range of tdh(+) V. parahaemolyticus levels was evaluated by testing shellfish samples in Japan and southern Thailand, where shellfish products are known to contain relatively low and high levels of total V. parahaemolyticus, respectively. The Japanese and Thai shellfish samples showed, respectively, relatively low (< 3 to 11 MPN/10 g) and considerably higher (930 to 110,000 MPN/10 g) levels of tdh(+) V. parahaemolyticus, raising concern about the safety of Thai shellfish products sold to domestic consumers at local morning markets. LAMP showed similar or higher performance than conventional PCR in the detection and quantification of a wide range of tdh(+) V. parahaemolyticus levels in shellfish products. Whereas a positive effect of PickPen-IMS was not observed in MPN determination, PickPen-IMS was able to concentrate tdh(+) V. parahaemolyticus 32-fold on average from the Japanese shellfish samples at an individual tube level, suggesting a possibility of using PickPen-IMS as an optional tool for specific shellfish samples. The A-IS(1)-LAMP procedure can be used by any health authority in the world to measure the tdh(+) V. parahaemolyticus levels in shellfish products.

Unlabelled: Vibrio vulnificus and Vibrio parahaemolyticus are Gram-negative marine bacteria naturally found in estuaries such as the Gulf of Mexico and can be pathogenic to humans. We quantified both of these organisms in fish, oyster, sediment, and water using culture-independent (quantitative PCR; qPCR) and culture-dependent (direct plating-colony hybridization; DP-CH) techniques during the transition period between winter and spring. We correlated these levels to environmental conditions and to abundance of total bacteria and total vibrio. By qPCR, fish intestine samples had the highest V. vulnificus densities and were 2·7, 3·5, and 4·2 logs greater than oyster, sediment and water samples, respectively. Densities of V. parahaemolyticus in fish samples by qPCR were 1·0, 2·1, and 3·1 logs greater than oyster, sediment and water samples, respectively. Similar differences between sample types were also observed by DP-CH. The difference between the more favourable and less favourable environmental conditions identified in this study was small (mean salinity 4·3 vs. 13 ppt). However, V. vulnificus and V. parahaemolyticus were consistently detected in fish intestines, but infrequently detected and at lower levels in oysters and during the less favourable period. This trend was observed by qPCR and DP-CH, indicating fish intestines are a significant source of pathogenic vibrios in the environment., Significance and Impact of the Study: This is the first study to report the densities of Bacteria 16S rRNA, Vibrio 16S rRNA, Vibrio vulnificus, and V. parahaemolyticus in fish intestine, oyster, sediment and water samples, as well as compare these values through culture-dependent and culture-independent methodology. Vibrio vulnificus and V. parahaemolyticus were detected in samples of fish intestines by qPCR and colony hybridization when conditions were less favourable for their occurrence in the environment. In contrast, V. vulnificus and V. parahaemolyticus were infrequently detected and at lower levels in other niches examined. These results indicate that fish intestinal tracts are a significant source of these pathogens., (© 2014 Society for Applied Microbiology. This article has been contributed to by US Government employees and their work is in the public domain in the US.)

Correlation between the numbers of Vibrio parahaemolyticus and its specific bacteriophages in cockles was investigated from June 2009 to May 2010 in Hat Yai, Songkhla, Thailand. Cockles obtained monthly from a local market were sampled to determine the numbers of V. parahaemolyticus and bacteriophages that could form plaques on ten strains of pandemic and nonpandemic V. parahaemolyticus. In addition, V. parahaemolyticus isolates from clinical samples from Hat Yai hospital over the same period were investigated. All 139 cockles sampled were positive for V. parahaemolyticus. However, only 76 of them were positive for bacteriophages. During the testing period, the number of bacteriophages was not significantly correlated with the incidence of V. parahaemolyticus-infected patients, but the numbers of V. parahaemolyticus isolates from the cockle samples were closely related to the number of infected patients. The bacteriophages isolated from V. parahaemolyticus also infected Vibrio alginolyticus and Vibrio mimicus, suggesting that the broad host range of phages may be a factor of providing the possibility of their participation in the processes of genetic exchange between V. parahaemolyticus and closely related Vibrio spp. In conclusion, this study indicated that the number of V. parahaemolyticus in cockles may be a useful tool for predicting the relative risk of infection by V. parahaemolyticus in this area of Thailand.

Objective: We evaluated the difference of three standard curves in quantifying viable Vibrio parahaemolyticus in samples by real-time reverse-transcriptase PCR (Real-time RT-PCR)., Methods: The standard curve A was established by 10-fold diluted cDNA. The cDNA was reverse transcripted after RNA synthesized in vitro. The standard curve B and C were established by 10-fold diluted cDNA. The cDNA was synthesized after RNA isolated from Vibrio parahaemolyticus in pure cultures (10(8) CFU/mL) and shrimp samples (10(6) CFU/g) (Standard curve A and C were proposed for the first time). Three standard curves were performed to quantitatively detect V. parahaemolyticus in six samples, respectively (Two pure cultured V. parahaemolyticus samples, two artificially contaminated cooked Litopenaeus vannamei samples and two artificially contaminated Litopenaeus vannamei samples). Then we evaluated the quantitative results of standard curve and the plate counting results and then analysed the differences., Results: The three standard curves all show a strong linear relationship between the fractional cycle number and V. parahaemolyticus concentration (R2 > 0.99); The quantitative results of Real-time PCR were significantly (p < 0.05) lower than the results of plate counting. The relative errors compared with the results of plate counting ranked standard curve A (30.0%) > standard curve C (18.8%) > standard curve B (6.9%); The average differences between standard curve A and standard curve B and C were - 2.25 Lg CFU/mL and - 0.75 Lg CFU/mL, respectively, and the mean relative errors were 48.2% and 15.9%, respectively; The average difference between standard curve B and C was among (1.47 -1.53) Lg CFU/mL and the average relative errors were among 19.0% - 23.8%., Conclusion: Standard curve B could be applied to Real-time RT-PCR when quantify the number of viable microorganisms in samples.

During the warmer summer months, oysters are conditioned to spawn, resulting in massive physiological efforts for gamete production. Moreover, the higher temperatures during the summer typically result in increased bacteria populations in oysters. We hypothesized that these animals are under multiple stresses that lead to possible immune system impairments during the summer months that can possibly lead to death. Here we show that in the summer and the fall animals exposed to a short heat stress respond similarly, resulting in a general trend of more bacteria being found in heat shocked animals than their non-heat shocked counterparts. We also show that naturally occurring bacterial populations are effected by a heat shock. In addition, oysters artificially contaminated with Vibrio parahaemolyticus were also affected by a heat shock. Heat shocked animals contained higher concentrations of V. parahaemolyticus in their tissues and hemolymph than control animals and this was consistent for animals examined during summer and fall. Finally, oyster hemocyte interactions with V. parahaemolyticus differed based on the time of the year. Overall, these findings demonstrate that seasonal changes and/or a short heat shock is sufficient to impact bacterial retention, particularly V. parahaemolyticus, in oysters and this line of research might lead to important considerations for animal harvesting procedures., (Copyright © 2013 Elsevier Ltd. All rights reserved.)