Thermostable Direct Hemolysin Research Articles

Innovative Multiplex PCR Assay for Detection of tlh, trh, and tdh Genes in Vibrio parahaemolyticus with Reference to the U.S. FDA's Bacteriological Analytical Manual (BAM).

Vibrio parahaemolyticus is an important foodborne bacterium that causes severe gastroenteritis following the consumption of contaminated seafood. To identify V. parahaemolyticus and determine its pathogenicity, the U.S. Food and Drug Administration (FDA)'s Bacteriological Analytical Manual (BAM) recommends a multiplex polymerase chain reaction (PCR) protocol to simultaneously detect the species-specific thermolabile hemolysin (tlh) gene and the pathogenic thermostable-related hemolysin (trh) and thermostable-direct hemolysin (tdh) genes. However, this assay has shown two limitations: difficulty in separating the amplicons of the trh (486 bp) and tlh (450 bp) genes due to their highly similar sizes, and the weaker band exhibited by the tdh gene amplicon (270 bp). The present study aimed to improve the BAM's multiplex PCR assay by separating the three amplicons with similar intensity. A new primer set was applied for the tlh gene (369 bp) alongside the existing primers for the trh and tdh genes. The amplicons for the three genes were effectively separated by electrophoresis on a 2% tris-borate-EDTA (TBE) agarose gel within 45 min. Primer concentrations of 0.25 µM for three genes produced a significant amount of amplicons among various combinations of primer concentrations with 35 PCR cycles. This assay exhibited a detection limit of 10 pg of bacterial DNA, demonstrating its high sensitivity. It did not display amplicons from nine Vibrio species known to be human pathogens or from 18 well-documented foodborne pathogens. Therefore, the present multiplex PCR protocol could help overcome the limitations of existing assays and provide a more reliable method for detecting the three genes of V. parahaemolyticus.

Public health aspects of Vibrio spp. related to the consumption of seafood in the EU.

Vibrio parahaemolyticus, Vibrio vulnificus and non-O1/non-O139 Vibrio cholerae are the Vibrio spp. of highest relevance for public health in the EU through seafood consumption. Infection with V. parahaemolyticus is associated with the haemolysins thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH) and mainly leads to acute gastroenteritis. V. vulnificus infections can lead to sepsis and death in susceptible individuals. V. cholerae non-O1/non-O139 can cause mild gastroenteritis or lead to severe infections, including sepsis, in susceptible individuals. The pooled prevalence estimate in seafood is 19.6% (95% CI 13.7-27.4), 6.1% (95% CI 3.0-11.8) and 4.1% (95% CI 2.4-6.9) for V. parahaemolyticus, V. vulnificus and non-choleragenic V. cholerae, respectively. Approximately one out of five V. parahaemolyticus-positive samples contain pathogenic strains. A large spectrum of antimicrobial resistances, some of which are intrinsic, has been found in vibrios isolated from seafood or food-borne infections in Europe. Genes conferring resistance to medically important antimicrobials and associated with mobile genetic elements are increasingly detected in vibrios. Temperature and salinity are the most relevant drivers for Vibrio abundance in the aquatic environment. It is anticipated that the occurrence and levels of the relevant Vibrio spp. in seafood will increase in response to coastal warming and extreme weather events, especially in low-salinity/brackish waters. While some measures, like high-pressure processing, irradiation or depuration reduce the levels of Vibrio spp. in seafood, maintaining the cold chain is important to prevent their growth. Available risk assessments addressed V. parahaemolyticus in various types of seafood and V. vulnificus in raw oysters and octopus. A quantitative microbiological risk assessment relevant in an EU context would be V. parahaemolyticus in bivalve molluscs (oysters), evaluating the effect of mitigations, especially in a climate change scenario. Knowledge gaps related to Vibrio spp. in seafood and aquatic environments are identified and future research needs are prioritised.

Virulence phenotypes differ between toxigenic Vibrio parahaemolyticus isolated from western coasts of Europe

Vibrio parahaemolyticus is the leading bacterial cause of gastroenteritis associated with seafood consumption worldwide. Not all members of the species are thought to be pathogenic, thus identification of virulent organisms is essential to protect public health and the seafood industry. Correlations of human disease and known genetic markers (e.g. thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH)) appear complex. Some isolates recovered from patients lack these factors, while their presence has become increasingly noted in isolates recovered from the environment. Here, we used whole-genome sequencing in combination with mammalian and insect models of infection to assess the pathogenic potential of V. parahaemolyticus isolated from European Atlantic shellfish production areas. We found environmental V. parahaemolyticus isolates harboured multiple virulence-associated genes, including TDH and/or TRH. However, carriage of these factors did not necessarily reflect virulence in the mammalian intestine, as an isolate containing TDH and the genes coding for a type 3 secretion system (T3SS) 2α virulence determinant, appeared avirulent. Moreover, environmental V. parahaemolyticus lacking TDH or TRH could be assigned to groups causing low and high levels of mortality in insect larvae, with experiments using defined bacterial mutants showing that a functional T3SS1 contributed to larval death. When taken together, our findings highlight the genetic diversity of V. parahaemolyticus isolates found in the environment, their potential to cause disease and the need for a more systematic evaluation of virulence in diverse V. parahaemolyticus to allow better genetic markers.

Meta-Analysis and Systematic Review of Phenotypic and Genotypic Antimicrobial Resistance and Virulence Factors in Vibrio parahaemolyticus Isolated from Shrimp.

This systematic review and meta-analysis investigates the prevalence of Vibrio parahaemolyticus, its virulence factors, antimicrobial resistance (AMR), and its resistance determinants in shrimp. This study was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines, to identify and select relevant peer-reviewed articles published between January 2020 and December 2022. The search strategy involved multiple online databases, including Google Scholar, PubMed, ScienceDirect, and Scopus. The inclusion criteria focused on studies that examined V. parahaemolyticus prevalence, virulence factors, and AMR in shrimp from farms to retail outlets. A total of 32 studies were analyzed, revealing a pooled estimate prevalence of V. parahaemolyticus in shrimp at 46.0%, with significant heterogeneity observed. Subgroup analysis highlighted varying prevalence rates across continents, emphasizing the need for further investigation. Virulence factor analysis identified thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh) as the most common. Phenotypic AMR analysis indicated notable resistance to glycopeptides, nitrofurans, and beta-lactams. However, the correlation between antimicrobial usage in shrimp farming and observed resistance patterns was inconclusive. Funnel plots suggested potential publication bias, indicating a need for cautious interpretation of findings. This study underscores the urgency of coordinated efforts to address AMR in V. parahaemolyticus to safeguard public health and to ensure sustainable aquaculture practices.

Environmental magnesium ion affects global gene expression, motility, biofilm formation and virulence of Vibrio parahaemolyticus

Vibrio parahaemolyticus is widely distributed in marine ecosystems. Magnesium ion (Mg2+) is the second most abundant metal cation in seawater, and plays important roles in the growth and gene expression of V. parahaemolyticus, but lacks the detailed mechanisms. In this study, the RNA sequencing data demonstrated that a total of 1494 genes was significantly regulated by Mg2+. The majority of the genes associated with lateral flagella, exopolysaccharide, type III secretion system 2, type VI secretion system (T6SS) 1, T6SS2, and thermostable direct hemolysin were downregulated. A total of 18 genes that may be involved in c-di-GMP metabolism and more than 80 genes encoding putative regulators were also significantly and differentially expressed in response to Mg2+, indicating that the adaptation process to Mg2+ stress may be strictly regulated by complex regulatory networks. In addition, Mg2+ promoted the proliferative speed, swimming motility and cell adhesion of V. parahaemolyticus, but inhibited the swarming motility, biofilm formation, and c-di-GMP production. However, Mg2+ had no effect on the production of capsular polysaccharide and cytoxicity against HeLa cells. Therefore, Mg2+ had a comprehensive impact on the physiology and gene expression of V. parahaemolyticus.

Development of Multienzyme Isothermal Rapid Amplification (MIRA) Combined with Lateral-Flow Dipstick (LFD) Assay to Detect Species-Specific tlh and Pathogenic trh and tdh Genes of Vibrio parahaemolyticus.

Vibrio parahaemolyticus causes severe gastroenteritis in humans after consuming contaminated raw or undercooked seafood. A species-specific marker, the thermolabile hemolysin (tlh) gene, and two pathogenic markers, thermostable-related hemolysin (trh) and thermostable-direct hemolysin (tdh) genes, have been used to identify V. parahaemolyticus and determine its pathogenicity using both PCR and qPCR assays. To enable testing in field conditions with limited resources, this study aimed to develop a simple and rapid method to detect the species-specific (tlh) and pathogenic (trh and tdh) genes of V. parahaemolyticus using multienzyme isothermal rapid amplification (MIRA) combined with a lateral-flow dipstick (LFD). The amplification of the tlh, trh, and tdh genes could be completed within 20 min at temperatures ranging from 30 to 45 °C (p < 0.05). The test yielded positive results for V. parahaemolyticus but produced negative results for nine Vibrio species and eighteen foodborne pathogenic bacterial species. MIRA-LFD could detect 10 fg of DNA and 2 colony-forming units (CFU) of V. parahaemolyticus per reaction, demonstrating a sensitivity level comparable to that of qPCR, which can detect 10 fg of DNA and 2 CFU per reaction. Both MIRA-LFD and qPCR detected seven tlh-positive results from thirty-six oyster samples, whereas one positive result was obtained using the PCR assay. No positive results for the trh and tdh genes were obtained from any oyster samples using MIRA-LFD, PCR, and qPCR. This study suggests that MIRA-LFD is a simple and rapid method to detect species-specific and pathogenic genes of V. parahaemolyticus with high sensitivity.

Fitness and transcriptomic analysis of pathogenic Vibrio parahaemolyticus in seawater at different shellfish harvesting temperatures

ABSTRACT To better characterize the population dynamics of Vibrio parahaemolyticus ( Vp ) containing different virulence genes, two Vp strains were inoculated into seawater separately and incubated at temperatures (10°C and 30°C) mimicking winter and summer pre-harvest shellfish rearing seasons. The cellular responses of two Vp strains, one containing the thermostable direct hemolysin ( tdh +) gene and the other one containing tdh -related hemolysin ( trh +) gene, were studied at the transcriptomic level. Results showed that, at 30°C, tdh + and trh + strains reached 6.77 ± 0.20 and 6.14 ± 0.07 log CFU/mL, respectively, after 5 days. During this time, a higher growth rate was observed in the tdh + strain than in the trh + strain. When being kept at 10°C, both Vp strains persisted at ca. 3.0 log CFU/mL in seawater with no difference observed between them. Survival and growth models were then established based on the Baranyi equation. The goodness-of-fit scores ranged from 0.674 to 0.950. RNA sequencing results showed that downregulated central energy metabolism and weakened degradation of branched-chain amino acid were observed only in the trh + strain not in the tdh + strain at 30°C. This might be one reason for the lower growth rates of the trh + strain at 30°C. Histidine metabolism and biofilm formation pathways were significantly downregulated in both strains at 10°C. No significant difference was observed for virulence-associated gene expression between 10°C and 30°C, regardless of the strains. IMPORTANCE Given the involvement of Vibrio parahaemolyticus ( Vp ) in a wide range of seafood outbreaks, a systematical characterization of Vp fitness and transcriptomic changes at temperatures of critical importance for seafood production and storage is needed. In this study, one of each virulent Vp strain ( tdh + and trh +) was tested. While no difference in survival behavior of the two virulent strains was observed at 10°C, the tdh + strain had a faster growth rate than the trh + strain at 30°C. Transcriptomic analysis showed that a significantly higher number of genes were upregulated at 30°C than at 10°C. The majority of differentially expressed genes of Vp at 30°C were annotated to functional categories supporting cellular growth. At 10°C, the downregulation of the biofilm formation and histidine metabolism indicates that the current practice of storing seafood at low temperatures not only protects seafood quality but also ensures seafood safety.

Transcriptomic Profiles of Vibrio parahaemolyticus During Biofilm Formation.

Vibrio parahaemolyticus, the leading cause of bacterial seafood-associated gastroenteritis, can form biofilms. In this work, the gene expression profiles of V. parahaemolyticus during biofilm formation were investigated by transcriptome sequencing. A total of 183, 503, and 729 genes were significantly differentially expressed in the bacterial cells at 12, 24 and 48h, respectively, compared with that at 6h. Of these, 92 genes were consistently activated or repressed from 6 to 48h. The genes involved in polar flagellum, chemotaxis, mannose-sensitive haemagglutinin type IV pili, capsular polysaccharide, type III secretion system 1 (T3SS1), T3SS2, thermostable direct hemolysin (TDH), type VI secretion system 1 (T6SS1) and T6SS2 were downregulated, whereas those involved in V. parahaemolyticus pathogenicity island (Vp-PAI) (except for T3SS2 and TDH) and membrane fusion proteins were upregulated. Three extracellular protease genes (vppC, prtA and VPA1071) and a dozen of outer membrane protein encoding genes were also significantly differentially expressed during biofilm formation. In addition, five putative c-di-GMP metabolism-associated genes were significantly differentially expressed, which may account for the drop in c-di-GMP levels after the beginning of biofilm formation. Moreover, many putative regulatory genes were significantly differentially expressed, and more than 1000 putative small non-coding RNAs were detected, suggesting that biofilm formation was tightly regulated by complex regulatory networks. The data provided a global view of gene expression profiles during biofilm formation, showing that the significantly differentially expressed genes were involved in multiple cellular pathways, including virulence, biofilm formation, metabolism, and regulation.

Vibrio parahaemolyticus thermostable direct haemolysin induces non-classical programmed cell death despite caspase activation.

Thermostable direct haemolysin (TDH) is the key virulence factor secreted by the human gastroenteric bacterial pathogen Vibrio parahaemolyticus. TDH is a membrane-damaging pore-forming toxin. It evokes potent cytotoxicity, the mechanism of which still remains under-explored. Here, we have elucidated the mechanistic details of cell death response elicited by TDH. Employing Caco-2 intestinal epithelial cells and THP-1 monocytic cells, we show that TDH induces some of the hallmark features of apoptosis-like programmed cell death. TDH triggers caspase-3 and 7 activations in the THP-1 cells, while caspase-7 activation is observed in the Caco-2 cells. Interestingly, TDH appears to induce caspase-independent cell death. Higher XIAP level and lower Smac/Diablo level upon TDH intoxication provide plausible explanation for the functional inability of caspases in the THP-1 cells, in particular. Further exploration reveals that mitochondria play a central role in the TDH-induced cell death. TDH triggers mitochondrial damage, resulting in the release of AIF and endonuclease G, responsible for the execution of caspase-independent cell death. Among the other critical mediators of cell death, ROS is found to play an important role in the THP-1 cells, while PARP-1 appears to play a critical role in the Caco-2 cells. Altogether, our work provides critical new insights into the mechanism of cell death induction by TDH, showing a common central theme of non-classical programmed cell death. Our study also unravels the interplay of crucial molecules in the underlying signalling processes. Our findings add valuable insights into the role of TDH in the context of the host-pathogen interaction processes.

Trend of Pathogenic Vibrio parahaemolyticus Occurrences in Bivalve Molluscs Harvested in Sardinian Coastal Environments Between 2011 and 2018

The aim of the present study was to evaluate Vibrio parahaemolyticus occurrences in bivalve molluscs harvested from Sardinian coastal environments between 2013 and 2015. The prevalence of pathogenic V. parahaemolyticus isolates is based on the detection of the two major virulence genes thermostable direct hemolysin (tdh) and thermolabile hemolysin (trh) To assess changes between 2011 and 2018 in the prevalence of V. parahaemolyticus in bivalve molluscs, we compared our results with those of previous investigations. In total, 2,933 samples were collected: 1,079 in 2013, 1,288 in 2014, and 566 in 2015. The mean prevalence of V. parahaemolyticus in shellfish was 3.5% in 2013, 1.7% in 2014, and 3.5% in 2015. The highest percentage of positive samples in 2013 and 2014 was observed in clams (3.5% and 2.7%, respectively), whereas in 2015, it was reported in oysters (15.1%). By comparing the sampling period of 2011–2014 with that of 2015–2018, an increase in the prevalence of V. parahaemolyticus was observed in shellfish (p < 0.05). In parallel, 208 potentially enteropathogenic V. parahaemolyticus strains were identified through the years 2011–2018 and, in particular, 10 trh+ and six tdh+ isolates. Our present study provides information regarding trends of V. parahaemolyticus occurrences in bivalve molluscs harvested from Sardinian coastal environments between 2011 and 2018 suggesting that the prevalence varies depending on the sampling period and shellfish species.

Predator-Prey Interactions between Halobacteriovorax and Pathogenic Vibrio parahaemolyticus Strains: Geographical Considerations and Influence of Vibrio Hemolysins.

Halobacteriovorax is a genus of naturally occurring marine predatory bacteria that attack, replicate within, and lyse vibrios and other bacteria. This study evaluated the specificity of four Halobacteriovorax strains against important sequence types (STs) of clinically relevant Vibrio parahaemolyticus, including pandemic strains ST3 and ST36. The Halobacteriovorax bacteria were previously isolated from seawater from the Mid-Atlantic, Gulf of Mexico, and Hawaiian coasts of the United States. Specificity screening was performed using a double agar plaque assay technique on 23 well-characterized and genomically sequenced V. parahaemolyticus strains isolated from infected individuals from widely varying geographic locations within the United States. With few exceptions, results showed that Halobacteriovorax bacteria were excellent predators of the V. parahaemolyticus strains regardless of the origins of the predator or prey. Sequence types and serotypes of V. parahaemolyticus did not influence host specificity, nor did the presence or absence of genes for the thermostable direct hemolysin (TDH) or the TDH-related hemolysin, although faint (cloudy) plaques were present when one or both hemolysins were absent in three of the Vibrio strains. Plaque sizes varied depending on both the Halobacteriovorax and Vibrio strains evaluated, suggesting differences in Halobacteriovorax replication and/or growth rates. The very broad infectivity of Halobacteriovorax toward pathogenic strains of V. parahaemolyticus makes Halobacteriovorax a strong candidate for use in commercial processing applications to enhance the safety of seafoods. IMPORTANCE Vibrio parahaemolyticus is a formidable obstacle to seafood safety. Strains pathogenic to humans are numerous and difficult to control, especially within molluscan shellfish. The pandemic spread of ST3 and ST36 has caused considerable concern, but many other STs are also problematic. The present study demonstrates broad predatory activity of Halobacteriovorax strains obtained along U.S. coastal waters from the Mid-Atlantic, Gulf Coast, and Hawaii toward strains of pathogenic V. parahaemolyticus. This broad activity against clinically relevant V. parahaemolyticus strains suggests a role for Halobacteriovorax in mediating pathogenic V. parahaemolyticus levels in seafoods and their environment as well as the potential application of these predators in the development of new disinfection technologies to reduce pathogenic vibrios in molluscan shellfish and other seafoods.

Temporal variability of microbiome in the different plankton hosts revealed distinct environmental persistence of Vibrio parahaemolyticus in shrimp farms

Plankton-bacteria interactions may play essential roles in maintaining the persistence of pathogenic Vibrio spp. However, the actual plankton host of the toxigenic Vibrio parahaemolyticus that harbors thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) remains unclear. To answer this question, we measured the abundance of toxigenic and nontoxigenic Vibrio parahaemolyticus in different types of plankton by most probable number (MPN)-PCR. We next investigated the dynamics of the microbiomes of rearing water, copepods, Chlorella, four predominant diatom genera (Nitzschia, Melosira, Skeletonema and Chaetoceros) and the gut of the shrimp in two recirculated shrimp farming systems from April to September using high-throughput 16 S rRNA amplicon sequencing. The survival of trh-positive and trh-negative V. parahaemolyticus in seawater and different plankton hosts was examined under low temperature and starvation conditions. The results showed that copepods harbored the highest proportion of trh-positive V. parahaemolyticus, followed by diatoms. Chitinous diatoms (CD) harbored a high proportion of Vibrionaceae, of which a high abundance of V. parahaemolyticus was found in summer. In contrast, Vibrio spp. is rarely found in Chlorella and nonchitinous diatoms. Taxon-specific associations were also observed, including a relatively high abundance of Vibrio and Halodesulfovibrio on copepods and covariation of Aeromonas and Bacillus inside the Chlorella. The survival assays showed that, in comparison to trh-negative V. parahaemolyticus, trh-positive V. parahaemolyticus showed better survival in copepods and CD under starvation conditions and maintained high persistence in the above hosts at low temperature. In conclusion, the results herein suggested that chitinous plankton might provide protection for V. parahaemolyticus, especially trh-positive V. parahaemolyticus, and improve their persistence under harsh conditions. Our study provided in-depth insights into the persistence of V. parahaemolyticus in the environment, which would promote targeted disease prevention measures.

Duplex electrochemical biosensor for the detection of the tdh and trh virulence genes of Vibrio parahaemolyticus in oysters

Vibrio parahaemolyticus is a bacterium present in estuarine environments. Since the first outbreak in Japan in 1950, it has been a dominant cause of foodborne infections throughout the world. Vibrio parahaemolyticus strains can accumulate in shellfish and cause gastroenteritis. The thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) encoded by tdh and trh genes, respectively, are considered major virulence factors in V. parahaemolyticus. Conventional methods used for V. parahaemolyticus monitoring in seafood, based on microbiological counts, are time consuming and laborious, and they cannot identify virulence genes. Therefore, a duplex electrochemical biosensor for the detection of the tdh and trh genes of V. parahaemolyticus in oysters was developed. Oyster homogenates were prepared, enriched for bacterial content, and DNA was extracted. Then, a duplex PCR with tailed primers was performed to amplify both genes. Afterwards, sandwich hybridisation assays were carried out on magnetised working electrodes of an array. The electrochemical biosensor was able to detect the tdh and trh genes from V. parahaemolyticus strains, and no cross-contaminations were observed between electrodes. The limit of detection for both genes was 10 pg/μL and no cross-reactivity was observed when using other Vibrio species and non-Vibrio pathogens. The biosensor was able to detect as low as 1 CFU of V. parahaemolyticus in oyster homogenate. Screening of oysters from an infectivity experiment and comparison with other techniques proved the proper performance of the biosensor and its applicability to the analysis of natural samples, with added advantages of specificity, duplexing capability, portability and provided virulence information.

Multiplex PCR-Lateral Flow Dipstick Method for Detection of Thermostable Direct Hemolysin (TDH) Producing V. parahaemolyticus.

Vibrio parahaemolyticus is usually found in seafood and causes acute gastroenteritis in humans. Therefore, a detection method of pathogenic V. parahaemolyticus is necessary. Multiplex PCR combined with lateral flow dipstick (LFD) assay was developed to detect pathogenic V. parahaemolyticus. Biotin-, FAM-, and Dig-conjugated primers targeting thermolabile hemolysin (TLH) and thermostable direct hemolysin (TDH) genes were used for multiplex PCR amplification. The condition of the method was optimized and evaluated by agarose gel electrophoresis and universal lateral flow dipstick. The specificity assay was evaluated using strains belonging to seven foodborne pathogen species. The sensitivity of the method was also evaluated using DNA in the concentration range of 0.39-100 ng/reaction. The artificial spiking experiment was performed using 10 g of shrimp samples with an enrichment time of 0, 4, and 8 h with 101, 102, and 103 CFU of V. parahaemolyticus. The developed multiplex PCR-LFD assay showed no non-specific amplification with a limit of the detection of 0.78 ng DNA/reaction visualized by agarose gel electrophoresis and 0.39 ng DNA with LFD assay. The artificial spiking experiment demonstrated that this method could detect pathogenic V. parahaemolyticus at 10 CFU/10 g shrimp samples following a 4 h of enrichment. Multiplex PCR-LFD assay was therefore established for detecting pathogenic V. parahaemolyticus with high sensitivity and specificity and might be a useful tool to develop a detection kit used in the food safety sector.

Comparative genomics uncovered differences between clinical and environmental populations of Vibrio parahaemolyticus in New Zealand.

Vibrio parahaemolyticus has been identified as an emerging human pathogen worldwide with cases undergoing a global expansion over recent decades in phase with climate change. New Zealand had remained free of outbreaks until 2019, but different outbreaks have been reported consecutively since then. To provide new insights into the recent emergence of cases associated with outbreak clones over recent years, a comparative genomic study was carried out using a selection of clinical (mostly outbreak) and environmental isolates of V. parahaemolyticus obtained in New Zealand between 1973 and 2021. Among 151 isolates of clinical (n=60) and environmental (n=91) origin, 47 sequence types (STs) were identified, including 31 novel STs. The population of environmental isolates generated 30 novel STs, whereas only 1 novel ST (ST2658) was identified among the population of clinical isolates. The novel clinical ST was a single-locus variant of the pandemic ST36 strain, indicating further evolution of this pandemic strain. The environmental isolates exhibited a significant genetic heterogeneity compared to the clinical isolates. The whole-genome phylogeny separated the population of clinical isolates from their environmental counterparts, clearly indicating their distant genetic relatedness. In addition to differences in ancestral profiles and genetic relatedness, these two groups of isolates exhibited a profound difference in their virulence profiles. While the entire population of clinical isolates harboured the thermostable direct haemolysin (tdh) and/or the thermostable-related haemolysin (trh), only a few isolates of environmental origin possessed the same virulence genes. In contrast to tdh and trh, adhesin-encoding genes, vpadF and MSHA, showed a significantly (P<0.001) greater association with the environmental isolates compared to the clinical isolates. The effectors, VopQ, VPA0450 and VopS, which belong to T3SS1, were ubiquitous, being present in each isolate regardless of its origin. The effectors VopC and VopA, which belong to T3SS2, were rarely detected in any of the examined isolates. Our data indicate that the clinical and environmental isolates of V. parahaemolyticus from New Zealand differ in their population structures, ancestral profiles, genetic relatedness and virulence profiles. In addition, we identified numerous unique non-synonymous single-nucleotide polymorphisms (nsSNPs) in adhesins and effectors, exclusively associated with the clinical isolates tested, which may suggest a possible role of these mutations in the overall virulence of the clinical isolates.

Comparative genomic analysis reveals the potential transmission of Vibrio parahaemolyticus from freshwater food to humans

Vibrio parahaemolyticus is an increasingly important foodborne pathogen that cause acute gastroenteritis in humans. However, the prevalence and transmission of this pathogen in freshwater food remains unclear. This study aimed to determine the molecular characteristics and genetic relatedness of V. parahaemolyticus isolates obtained from freshwater food, seafood, environmental, and clinical samples. A total of 138 (46.6%) isolates were detected from 296 food and environmental samples, and 68 clinical isolates from patients. Notably, V. parahaemolyticus was more prevalent in freshwater food (56.7%, 85/150) than in seafood (38.8%, 49/137). Virulence phenotype analyses revealed that the high motility of isolates from freshwater food (40.0%) and clinical isolates (42.0%) was higher than that of isolates from seafood (12.2%), whereas the biofilm-forming capacity of freshwater food isolates (9.4%) was lower than that of seafood (22.4%) and clinical isolates (15.9%). Virulence genes analysis showed that 46.4% of the clinical isolates contained the tdh gene encoding thermostable direct hemolysin (TDH) and only two freshwater food isolates contained the trh gene encoding TDH-related hemolysin (TRH). Multilocus sequence typing (MLST) analysis divided the 206 isolates into 105 sequence types (STs), including 56 (53.3%) novel STs. ST2583, ST469, and ST453 have been isolated from freshwater food and clinical samples. Whole-genome sequence (WGS) analyses revealed that the 206 isolates were divided into five clusters. Cluster II contained isolates from freshwater food and clinical samples, whereas the other clusters contained isolates from seafood, freshwater food, and clinical samples. In addition, we observed that ST2516 had the same virulence pattern, with a close phylogenetic relationship to ST3. The increased prevalence and adaption of V. parahaemolyticus in freshwater food is a potential cause of clinical cases closely related to the consumption of V. parahaemolyticus contaminated freshwater food.

A lanthanide-based high-sensitivity fluorescence method for the on-site rapid detection of thermostable direct hemolysin of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a common foodborne pathogen in seafood, which often causes seafood borne bacterial gastroenteritis or food poisoning. Thermostable direct hemolysin (TDH) is considered to be one of the main virulence factors involved in this pathogen. The most clinical V. parahaemolyticus isolates produce TDH. Therefore, high sensitivity and specificity detection of TDH are of great significance for food safety and early diagnosis of diseases caused by V. parahaemolyticus. In this study, we developed a rapid, sensitive immunochromatographic test paper assay for the quantitative detection of TDH in seafood samples using time-resolved fluorescence techniques. First, we completed the preparation of fluorescent detection antibodies by coupling lanthanide fluorescent nanospheres with homemade high-affinity polyclonal antibodies based on the principle of the double-antibody sandwich. The lanthanide fluorescent nanospheres used in this study are characterized by a large stokes shift and a long fluorescence lifetime, which effectively reduces background noise and improves detection sensitivity. In addition, the method can be completed within 15 min for the detection of TDH, has a detection limit below 50 ng/mL and good linearity in the range of 50–5000 ng/mL. Moreover, it has good specificity and no cross-reactivity with Vibrio vulnificus hemolysin (VVH), Clostridium perfringens α toxin (CPA) or C. perfringens ε toxin (ETX). Finally, the sensitivity of this method was unchanged when the three simulated samples of Patinopecten yessoensis, Ruditapes philippinarum, and Scapharca broughtonii tested, indicating that the method is not affected by samples in a complex matrix. In conclusion, this study establishes a practical new method for on-site rapid detection of TDH, which is easy to operate, fast response, easy to carry and can be implemented under the field conditions without expensive equipment and professional person.

Detection and pathogenicity of Vibrio parahaemolyticus strains in Penaeus vannamei

Background and purpose: The Vibrio spp. are indigenous bacteria in the marine environment and usually constitute the majority in normal microflora of farmed and wild penaeid shrimp; and are one of the most diverse and vital shrimp pathogens that cause heavy mortalities in aquaculture facilities worldwide. The present study aimed to isolate and characterize the causative agent concerned with mass mortality in Peneaus vannamei collected from the shrimp farms of Kakinada, Andhra Pradesh, India.Materials and methods: To isolate and characterize the pathogenic bacteria from the hepatic pancreatic tissue of moribund, P. vannamei performed conventional culture tests, physical and biochemical tests, and molecular analysis. In addition to that virulence gene study, bacterial pathogenicity with different salinity and antibacterial activity of the stem extract of Tinospora cordifolia against Vibrio parahaemolyticus were tested.Results: The phenotypic traits, 16s rRNA gene, and phylogenetic tree analysis showed that the isolated pathogen was V. parahaemolyticus strain KKD 02. A study on bacterial pathogenicity with different salinity 0, 5, 10, 15, 20, and 25 ppt showed that 5 ppt to 10 ppt was connected to the maximum resistance against bacterial infection in P. vannamei with higher CFU/shrimp value. Two genes encoding thermostable direct hemolysin (tdh) and the thermostable direct hemolysin-related hemolysin (trh) were present in the isolate. The antibacterial activity of the stem extract of T. cordifolia, tested against V. parahaemolyticus, revealed that both the ethanol extracts and the crude stem juice exhibited antibacterial activity against V. parahaemolyticus.Conclusion: The cause for the mass mortality of juvenile shrimp P. vannamei in Kakinada, Andhra Pradesh, India, was V. parahaemolyticus strain KKD 02. Further work is necessary to isolate and purify the active constituents in T. cordifolia stem extracts and examine the absorption pattern of the active ingredients of these plants, which will allow the scientific community to recommend their utilization as an accessible alternative to synthetic antibiotics.

Growth Rates of Vibrio parahaemolyticus Sequence Type 36 Strains in Live Oysters and in Culture Medium.

The pathogenic marine bacterium Vibrio parahaemolyticus can cause seafood-related gastroenteritis via the consumption of raw or undercooked seafood. Infections originating from relatively cool waters in the northeast United States are typically rare, but recently, this region has shown an increase in infections attributed to the ecological introduction of pathogenic sequence type 36 (ST36) strains, which are endemic to the cool waters of the Pacific Northwest. A 2005 risk assessment performed by the Food and Drug Administration (FDA) modeled the postharvest growth of V. parahaemolyticus in oysters as a function of air temperature and the length of time the oysters remained unrefrigerated. This model, while useful, has raised questions about strain growth differences in oyster tissue and whether invasive pathogenic strains exhibit different growth rates than nonclinical strains, particularly at lower temperatures. To investigate this question, live eastern oysters were injected with ST36 clinical strains and non-ST36 nonclinical strains, and growth rates were measured using the most probable number (MPN) enumeration. The presence of V. parahaemolyticus was confirmed using PCR by targeting the thermolabile hemolysin gene (tlh), thermostable direct hemolysin (tdh), tdh-related hemolysin (trh), and a pathogenesis-related protein (prp). The growth rates of the ST36 strains were compared to the FDA model and several other data sets of V. parahaemolyticus growth in naturally inoculated oysters harvested from the Chesapeake Bay. Our data indicate that the growth rates from most studies fall within the mean of the FDA model, but with slightly higher growth at lower temperatures for ST36 strains injected into live oysters. These data suggest that further investigations of ST36 growth capability in oysters at temperatures previously thought unsuitably low for Vibrio growth are warranted. IMPORTANCE Vibrio parahaemolyticus is the leading cause of seafood-related gastroenteritis in the United States, with an estimated 45,000 cases per year. Most individuals who suffer from vibriosis consume raw or undercooked seafood, including oysters. While gastroenteritis vibriosis is usually self-limiting and treatable, V. parahaemolyticus infections are a stressor on the growing aquaculture industry. Much effort has been placed on modeling the growth of Vibrio cells in oysters in order to aid oyster growers in designing harvesting best practices and ultimately, to protect the consumer. However, ecological invasions of nonnative bacterial strains make modeling their growth complicated, as these strains are not accounted for in current models. The National Shellfish Sanitation Program (NSSP) considers 10°C (50°F) a temperature too low to enable Vibrio growth, where 15°C is considered a cutoff temperature for optimal Vibrio growth, with temperatures approaching 20°C supporting higher growth rates. However, invasive strains may be native to cooler waters. This research aimed to understand strain growth in live oysters by measuring growth rates when oysters containing ST36 strains, which may be endemic to the U.S. Pacific Northwest, were exposed to multiple temperatures postharvest. Our results will be used to aid future model development and harvesting best practices for the aquaculture industry.

Light response of Vibrio parahaemolyticus

Light is one of the most critical stimuli in the majority of living organisms. In the last two decades, blue light (BL) has become a major subject of attention because of developments in light-emitting diodes (LED). The effects of BL on eukaryotic organisms and phototrophic prokaryotes have been well studied, but the knowledge of its effects on non-phototrophic prokaryotes remains unclear. Since BL can penetrate seawater, it is expected that most prokaryotes living in the ocean possess molecular mechanisms which protect against BL. The aim of this study is to assess the molecular mechanisms of Vibrio parahaemolyticus cells against BL as a marine bacterial model compared to other wavelength light exposures. Physiological and transcriptomic analyses of BL-exposed cells compared to other light treated cells revealed the highest ROS fold change, the highest number of differentially expressed genes (DEGs), and up-regulation in the gene responsible to not only compatible solute such as glycine betaine and ectoine but also iron-sulfur biosynthesis related to ROS formation. Furthermore, red light (RL) up-regulated the expression of cryptochrome DASH, a protein known to be excited by BL, and orange light (OL) decreased the expression of thermostable direct hemolysin (TDH), suggesting that OL attenuates the virulence of V. parahaemolyticus. In addition, the expression of VtrA (V. parahaemolyticus type III secretion system 2 (T3SS2) regulator A) but not VtrB (V. parahaemolyticus T3SS2 regulator B) increased under both light treatments, indicating that light exposure is unlikely to be involved in T3SS2-mediated pathogenicity. These results expand our knowledge on unique light responses in non-phototrophic marine prokaryotes.