Characterization of Vibrio parahaemolyticus isolated from clinical specimens and oysters in Thailand.

INTRODUCTION: Vibrio parahaemolyticus is a common pathogen that can cause seafood-borne gastroenteritis in humans. We determined the prevalence and characteristics of V. parahaemolyticus isolated from clinical specimens and oysters in Thailand. METHODOLOGY: Isolates of V. parahaemolyticus from clinical specimens (n = 77) and oysters (n = 224) were identified by biochemical testing, polymerase chain reaction (PCR) assays, and serotyping. The toxin genes, antimicrobial resistance, and ß-lactamase production were determined. RESULTS: A total of 301 isolates were confirmed as V. parahaemolyticus by PCR using specific primers for the toxR gene. The majority of clinical isolates carried the tdh+/trh- genotype (82.1%), and one of each isolate was tdh-/trh+ and tdh+/trh+ genotypes. One isolate from oyster contained the tdh gene and another had the trh gene. Twenty-six serotypes were characterized among these isolates, and O3:K6 was the most common (37.7%), followed by OUT:KUT, and O4:K9. In 2010, most clinical and oyster isolates were susceptible to antibiotics, with the exception of ampicillin. In 2012, clinical isolates were not susceptible to cephalothin (52.4%), streptomycin (95.2%), amikacin (66.6%), kanamycin (61.9%), and erythromycin (95.2%), significantly more frequently than in 2010. More than 95% of isolates that were not susceptible to ampicillin produced ß-lactamase enzymes. CONCLUSIONS: We found toxin genes in two oyster isolates, and the clinical isolates that were initially determined to be resistant to several antibiotics. Toxin genes and antimicrobial susceptibility profiles of V. parahaemolyticus from seafood and environment should be continually monitored to determine the spread of toxin and antimicrobial resistance genes.

Antimicrobial susceptibility profile and molecular characterization of Vibrio parahaemolyticus strains isolated from imported shrimps.

Vibrio parahaemolyticus is a threat to human health and one of the leading bacterial causes of seafood-borne infection worldwide. This pathogen is autochtonous in the marine environment and is able to acquire antimicrobial resistance (AMR) mechanisms, which is a global concern. However, the emergence of AMR V. parahaemolyticus strains in seafood is still understudied, as interpretation criteria for this species for antimicrobial susceptibility tests are limited in the literature. In this study, we investigated the susceptibility profiles to clinically important antibiotics and the associated genetic determinants of V. parahaemolyticus isolates cultured from imported shrimps. Based on the analysis of the resistance phenotypes of 304 V. parahaemolyticus isolates, we have defined experimental epidemiological cutoff values (COWT) for 14/15 antibiotics tested. We observed that 19.1% of the bacterial isolates had acquired resistance to at least one antibiotic class. The highest number of resistance was associated with tetracycline (14.5% of the strains) and trimethoprim-sulfamethoxazole (3.6%). Moreover, seven strains were multidrug-resistant (MDR, resistant to at least three antibiotic classes). The most frequently identified genes in these strains were aph(3″)-Ib/aph(6)-Id (aminoglycoside resistance), sul2 (sulfonamide), tet(59) (tetracycline), and floR (chloramphenicol). The SXT/R391 family ICE and class 1 integron-integrase genes were detected by PCR in three and one MDR V. parahaemolyticus strains, respectively. Consequently, V. parahaemolyticus in seafood can act as a reservoir of AMR, constituting a health risk for the consumer.IMPORTANCEOur study on "Antimicrobial Resistance Profiles and Genetic Determinants of Vibrio parahaemolyticus Isolates from Imported Shrimps" addresses a critical gap in understanding the emergence of antimicrobial resistance (AMR) in this seafood-associated pathogen. Vibrio parahaemolyticus is a major cause of global seafood-borne infections, and our research reveals that 19.1% of isolates from imported shrimps display resistance to at least one antibiotic class, with multidrug resistance observed in seven strains. Importantly, we establish experimental epidemiological cutoff values for antibiotic susceptibility, providing valuable criteria specific to V. parahaemolyticus. Our findings underscore the potential risk to consumers, emphasizing the need for vigilant monitoring and intervention strategies. This study significantly contributes to the comprehension of AMR dynamics in V. parahaemolyticus, offering crucial insights for global public health. The dissemination of our research through Microbiology Spectrum ensures broad accessibility and impact within the scientific community and beyond.

Antimicrobial susceptibility and genomic characterization of Vibrio parahaemolyticus isolated from aquatic foods in 15 provinces, China, 2020.

Prevalent in marine, estuarine and coastal environments, Vibrio parahaemolyticus is one of the major foodborne pathogens which can cause acute gastroenteritis through consumption of contaminated food. This study encompassed antimicrobial resistance, molecular characteristics and phylogenetic relationships of 163 V. parahaemolyticus isolated from aquatic foods across 15 provinces in China. The isolates showed high resistance rates against ampicillin (90.80 %, 148/163) and cefazolin (72.39 %, 118/163). Only 5 isolates demonstrated multi-drug resistance (MDR) phenotypes. A total of 37 different antibiotic resistance genes (ARGs) in correlation with seven antimicrobial categories were identified. tet(34) and tet(35) were present in all 163 isolates. Other most prevalent ARGs were those conferring resistance to ß-lactams, with prevalence rate around 18.40 % (30/163). The virulence genes tdh and trh were found in 17 (10.43 %) and 9 (5.52 %) isolates, respectively. Totally 121 sequence types (STs) were identified through whole genome analysis, among which 60 were novel. The most prevalent sequence type was ST3 (9.20 %, 15/163), which shared the same genotype profile of trh_, tdh+ and blaCARB-22+. Most of the tdh+V. parahaemolyticus isolates was clustered into a distinctive clade by the phylogenetic analysis. Our study showed that the antimicrobial resistance of V. parahaemolyticus in aquatic foods in China was moderate. However, the emerging of MDR isolates implicate strengthened monitoring is needed for the better treatment of human V. parahaemolyticus infections. High genetic diversity and virulence potential of the isolates analyzed in this study help better understanding and evaluating the risk of V. parahaemolyticus posed to public health.

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.

Epidemiological and Genomic analysis of Vibrio parahaemolyticus isolated from imported travelers at the port of Shanghai, China (2017-2019).

BACKGROUND: Vibrio parahaemolyticus is the predominant etiological agent of seafood-associated foodborne illnesses on a global scale. It is essential to elucidate the mechanisms by which this pathogen disseminates. Given the existing research predominantly concentrates on localized outbreaks, there is a pressing necessity for a comprehensive investigation to capture strains of V. parahaemolyticus cross borders. RESULTS: This study examined the frequency and genetic attributes of imported V. parahaemolyticus strains among travelers entering Shanghai Port, China, between 2017 and 2019.Through the collection of 21 strains from diverse countries and regions, Southeast Asia was pinpointed as a significant source for the emergence of V. parahaemolyticus. Phylogenetic analysis revealed clear delineation between strains originating from human and environmental sources, emphasizing that underlying genome data of foodborne pathogens is essential for environmental monitoring, food safety and early diagnosis of diseases. Furthermore, our study identified the presence of virulence genes (tdh and tlh) and approximately 120 antibiotic resistance-related genes in the majority of isolates, highlighting their crucial involvement in the pathogenesis of V. parahaemolyticus. CONCLUSIONS: This research enhanced our comprehension of the worldwide transmission of V. parahaemolyticus and its antimicrobial resistance patterns. The findings have important implications for public health interventions and antimicrobial stewardship strategies, underscoring the necessity for epidemiological surveillance of pathogen at international travel hubs.

Genomic stability among O3:K6 V. parahaemolyticus pandemic strains isolated between 1996 to 2012 in American countries.

BACKGROUND: The V. parahaemolyticus pandemic clone, results in the development of gastrointestinal illness in humans. Toxigenic strains of this species are frequently isolated from aquatic habitats and organisms such as mollusks and crustaceans. Reports on the isolation of the pandemic clone started in 1996, when a new O3:K6 clone was identified in Asia, that rapidly spread worldwide, becoming the predominant clone isolated from clinical cases. In this study whole genome sequencing was accomplished with an Illumina MiniSeq platform, upon six novel V. parahaemolyticus strains, that have been isolated in Mexico since 1998 and three representative genomes of strains that were isolated from reported outbreaks in other American countries, and were deposited in the GenBank. These nine genomes were compared against the reference sequence of the O3:K6 pandemic strain (RIMD 2210633), which was isolated in 1996, to determine sequence differences within American isolates and between years of isolation. RESULTS: The results indicated that strains that were isolated at different times and from different countries, were highly genetically similar, among them as well as to the reference strain RIMD 2210633, indicating a high level of genetic stability among the strains from American countries between 1996 to 2012, without significant genetic changes relative to the reference strain RIMD 2210633, which was isolated in 1996 and was considered to be representative of a novel O3:K6 pandemic strain. CONCLUSIONS: The genomes of V. parahaemolyticus strains isolated from clinical and environmental sources in Mexico and other American countries, presented common characteristics that have been reported for RIMD 2210633 O3:K6 pandemic strain. The major variations that were registered in this study corresponded to genes non associated to virulence factors, which could be the result of adaptations to different environmental conditions. Nevertheless, results do not show a clear pattern with the year or locality where the strains were isolated, which is an indication of a genomic stability of the studied strains.

Whole genome sequencing reveals great diversity of Vibrio spp in prawns at retail.

Consumption of prawns as a protein source has been on the rise worldwide with seafood identified as the predominant attributable source of human vibriosis. However, surveillance of non-cholera Vibrio is limited both in public health and in food. Using a population- and market share-weighted study design, 211 prawn samples were collected and cultured for Vibrio spp. Contamination was detected in 46 % of samples, and multiple diverse Vibrio isolates were obtained from 34 % of positive samples. Whole genome sequencing (WGS) and phylogenetic analysis illustrated a comprehensive view of Vibrio species diversity in prawns available at retail, with no known pathogenicity markers identified in Vibrio parahaemolyticus and V. cholerae. Antimicrobial resistance genes were found in 77 % of isolates, and 12 % carried genes conferring resistance to three or more drug classes. Resistance genes were found predominantly in V. parahaemolyticus, though multiple resistance genes were also identified in V. cholerae and V. vulnificus. This study highlights the large diversity in Vibrio derived from prawns at retail, even within a single sample. Although there was little evidence in this study that prawns are a major source of vibriosis in the UK, surveillance of non-cholera Vibrio is very limited. This study illustrates the value of expanding WGS surveillance efforts of non-cholera Vibrios in the food chain to identify critical control points for food safety through the production system and to determine the full extent of the public health impact.

Genetic and serological characterization of capsular antigen untypeable Vibrio parahaemolyticus strains reveal novel K serotypes and epidemiological characteristics in Shandong, China.

Vibrio parahaemolyticus, which is commonly found in marine and estuarine environments worldwide and isolated from aquatic products, is one of the most important food-borne pathogens. Among the various typing methods, serotyping is widely accepted and utilized by infectious disease specialists and infection control agencies for the detection and epidemiological investigation of this pathogen. Thus far, 13 O serotypes and 71 K serotypes have been defined; however, untypeable strains are frequently isolated during routine detection, and some new O and/or K antigens have been identified and characterized. During a serotyping survey in Shandong province, China from 2016 to 2018, we collected 411 clinical V. parahaemolyticus strains and found that nine of them are untypeable K antigen strains. In this study, we identified three K serotypes of V. parahaemolyticus through in-depth genetic analysis of the K antigen gene cluster, serological tests, and the production of antisera. Among the nine strains, seven possess K untypeable 2 (KUT2) antigens, which have been reported recently by another group. However, two new O and K combinations (O3:KUT2 and O11:KUT2) were first characterized by us, with the remaining two each representing a novel K serotype. Moreover, through comparative genomic analysis, we showed that the Shandong KUT2 strains exhibit different virulence profiles compared to their identical K serotype partners from Zhejiang province, another Chinese coastal province; however, strains from these two regions are clustered into the same linage and may have evolved from a recent common ancestor. Additionally, one isolate, SD2016062, was phylogenetically similar to the strains associated with several local gastroenteritis outbreaks, with similar toxin patterns, suggesting its potential to cause sporadic occurrences of disease or even local pandemics. Finally, we developed a sero-specific PCR assay targeting the three novel K serotypes, which can monitor the V. parahaemolyticus spectrum for clinical and epidemiological purposes. Thus, we identified and characterized novel strains of V. parahaemolyticus and proposed a new technique for tracking the diversity of strains, which can help manage this food-borne pathogen.

Evaluation of loop-mediated isothermal amplification assay along with conventional and real-time PCR assay for sensitive detection of pathogenic Vibrio parahaemolyticus from seafood sample without enrichment.

The primary reason for foodborne illness is improper seafood safety testing, and hence, an appropriate tool for testing is the key to control the outbreaks. The current study aimed to develop a loop-mediated isothermal amplification (LAMP) assay to detect pathogenic Vibrio parahaemolyticus, important foodborne pathogen, targeting tdh, and trh genes. The specificity of the LAMP assay was good without any false-positive and false-negative results. The assay was highly sensitive and could detect the pathogenic V. parahaemolyticus as low as 1 CFU/reaction in spiked seafood samples and 1 pg of extracted DNA. Out of 62 seafood samples from India's southwest coastal region tested with LAMP assay, eight (12.9%) were positive for trh, and seven (11.29%) samples were positive tdh gene. LAMP-based on tdh and trh was found to be significantly more sensitive (p < 0.05) than conventional PCR and nearly equal sensitive as real-time PCR (RT-PCR) for the detection of pathogenic V. parahaemolyticus. Our study shows that LAMP assay can be a better approach as a point-of-care (POC) diagnostic tool and could detect pathogenic V. parahaemolyticus on seafood samples directly without enrichment and isolation. The high sensitivity and simplicity make LAMP assay a better alternative method than the conventional method and RT-PCR for the detection of pathogens. LAMP assay can be considered as a good alternative to PCR for the routine detection of pathogenic V. parahaemolyticus in seafood.

Characterization of Vibrio parahaemolyticus strains isolated from clinically asymptomatic seafood workers.

Vibrio parahaemolyticus (VP) is a major cause of gastroenteritis outbreaks in Thailand and other countries due to the consumption of contaminated and undercooked seafood. However, there have been few reports of the molecular epidemiology of VP isolates from asymptomatic seafood handlers. Here, we report the phenotypic and genetic characterization of 61 VP isolates obtained from asymptomatic workers in two seafood-processing plants. We found 24 O:K serotypes, of which O11:KUT, O1:KUT and O3:KUT were the dominant serotypes. Analysis by PCR showed that 12 isolates harbored either tdh or trh genes with the potential to be pathogenic VP strains. The presence of T3SS2α and T3SS2ß genes was correlated with the presence of tdh and trh, respectively. Four tdh+ isolates were positive for pandemic marker. In this study, VP isolates were commonly resistant to ampicillin, cephazolin, fosfomycin and novobiocin. Phylogenetic analysis of VP1680 loci in 35 isolates from 17 asymptomatic workers, 6 gastroenteritis patients, 7 environmental samples and 5 genomes from a database showed 22 different alleles. Gene VP1680 was conserved in tdh+ isolates and pandemic strains, while that of trh + isolates was diverse. Asymptomatic workers carrying VP were the most likely source of contamination, which raises concerns over food safety in seafood-processing plants.

A database for risk assessment and comparative genomic analysis of foodborne Vibrio parahaemolyticus in China.

Vibrio parahaemolyticus is a major foodborne pathogen worldwide. The increasing number of cases of V. parahaemolyticus infections in China indicates an urgent need to evaluate the prevalence and genetic diversity of this pathogenic bacterium. In this paper, we introduce the Foodborne Vibrio parahaemolyticus genome database (FVPGD), the first scientific database of foodborne V. parahaemolyticus distribution and genomic data in China, based on our previous investigations of V. parahaemolyticus contamination in different kinds of food samples across China from 2011 to 2016. The dataset includes records of 2,499 food samples and 643 V. parahaemolyticus strains from supermarkets and marketplaces distributed over 39 cities in China; 268 whole-genome sequences have been deposited in this database. A spatial view on the risk situations of V. parahaemolyticus contamination in different food types is provided. Additionally, the database provides a functional interface of sequence BLAST, core genome multilocus sequence typing, and phylogenetic analysis. The database will become a powerful tool for risk assessment and outbreak investigations of foodborne pathogens in China.

Molecular characterization of clinical and environmental Vibrio parahaemolyticus isolates in Huzhou, China.

Vibrio parahaemolyticus is responsible for seafood-borne gastroenteritis worldwide. Isolates of V. parahaemolyticus from clinical samples (n = 54) and environmental samples (n = 38) in Huzhou were analyzed by serological typing, virulence gene detection, antibiotic resistance testing, and pulsed-field gel electrophoresis (PFGE) for molecular typing. O3:K6 was the main serotype and tlh+tdh+trh- was the most frequently detected virulence genotype in clinical strains. O2:Kut was the main serotype and tlh+tdh-trh- was the most frequently detected virulence genotype in environmental strains. Antibiotic resistance testing indicated that the isolates were highly resistant to ampicillin (90.76%), followed by gentamicin and tetracycline. Following the restriction enzyme NotI digestion, the 91 strains yielded 81 PFGE patterns, and 16 clones had similarity values of > 85.00%, indicating a high level of diversity. Finally, there may be cross-contamination between freshwater and seawater products, so it is necessary to strengthen supervision of food processing.

Seasonal and Geographical Differences in Total and Pathogenic Vibrio parahaemolyticus and Vibrio vulnificus Levels in Seawater and Oysters from the Delaware and Chesapeake Bays Determined Using Several Methods.

Oyster and seawater samples were collected from five sites in the Chesapeake Bay, MD, and three sites in the Delaware Bay, DE, from May to October 2016 and 2017. Abundances and detection frequencies for total and pathogenic Vibrio parahaemolyticus and Vibrio vulnificus were compared using the standard most-probable-number-PCR (MPN-PCR) assay and a direct-plating (DP) method on CHROMagar Vibrio for total (tlh+ ) and pathogenic (tdh+ and trh+ ) V. parahaemolyticus genes and total (vvhA) and pathogenic (vcgC) V. vulnificus genes. The colony overlay procedure for peptidases (COPP) assay was evaluated for total Vibrionaceae DP had high false-negative rates (14 to 77%) for most PCR targets and was deemed unsatisfactory. Logistic regression models of the COPP assay showed high concordances with MPN-PCR for tdh+ and trh+V. parahaemolyticus and vvhA+V. vulnificus in oysters (85.7 to 90.9%) and seawater (81.1 to 92.7%) when seawater temperature and salinity were factored into the model, suggesting that the COPP assay could potentially serve as a more rapid method to detect vibrios in oysters and seawater. Differences in total Vibrionaceae and pathogenic Vibrio abundances between state sampling sites over different collection years were contrasted for oysters and seawater by MPN-PCR. Abundances of tdh+ and trh+V. parahaemolyticus were ∼8-fold higher in Delaware oysters than in Maryland oysters, whereas abundances of vcgC+V. vulnificus were nearly identical. For Delaware oysters, 93.5% were both tdh+ and trh+, compared to only 19.2% in Maryland. These results indicate that pathogenic V. parahaemolyticus was more prevalent in the Delaware Bay than in the Chesapeake Bay.IMPORTANCE While V. parahaemolyticus and V. vulnificus cause shellfish-associated morbidity and mortality among shellfish consumers, current regulatory assays for vibrios are complex, time-consuming, labor-intensive, and relatively expensive. In this study, the rapid, simple, and inexpensive COPP assay was identified as a possible alternative to MPN-PCR for shellfish monitoring. This paper shows differences in total Vibrionaceae and pathogenic vibrios found in seawater and oysters from the commercially important Delaware and Chesapeake Bays. Vibrio parahaemolyticus isolates from the Delaware Bay were more likely to contain commonly recognized pathogenicity genes than those from the Chesapeake Bay.

Prevalence, antibiotic susceptibility and characterization of Vibrio parahaemolyticus isolates in China.

Vibrio parahaemolyticus is a marine and estuarine bacterium that poses a major threat to human health worldwide. In this study, from 2017 to 2019, we evaluated 900 food samples collected from China in 2017, with the aim of determining the incidence and features of V. parahaemolyticus in ready-to-eat (RTE) foods, shrimp and fish in China. The contamination rates in these were 3.67, 19.33 and 10.67%, respectively, and the prevalence of V. parahaemolyticus was higher in summer than in winter. In addition, 101 V. parahaemolyticus strains were isolated. Our results suggested that most of the isolates were resistant to aminoglycosides based on the antimicrobial resistance patterns of these aquatic product isolates against 14 antimicrobial agents. Furthermore, most of the isolates were multidrug-resistant. Serotyping showed that the isolates of the O2 serotype comprised the maximum proportion. Enterobacterial repetitive intergenic consensus sequence (ERIC)-PCR results indicated that the isolates (n = 101) could be classified into 12 clusters. There were 82 STs suggesting genetic variation and relatedness among these isolates. Our findings demonstrated the presence of V. parahaemolyticus in foods from Chinese retail markets and show that this methodology can be used for microbiological risk assessment in China.

Comparative genomic provides insight into the virulence and genetic diversity of Vibrio parahaemolyticus associated with shrimp acute hepatopancreatic necrosis disease.

Acute hepatopancreatic necrosis disease (AHPND) is an important shrimp disease of economic importance which causes mass mortality of cultivated penaeid shrimps in Southeast Asian countries, Mexico and South America. This disease was originally caused by Vibrio parahaemolyticus (VPAHPND) which is reported to harbour a transferable plasmid carrying the virulent PirAB-like toxin genes (pirABvp). However, little is known about the pathogenicity of VPAHPND. To extend our understanding, comparative genomic analyses was performed in this study to identify the genetic differences and to understand the phylogenetic relationship of VPAHPND strains. Seven Vibrio parahaemolyticus strains (five VPAHPND strains and two non-VPAHPND strains) were sequenced and 31 draft genomes of V. parahaemolyticus were retrieved from NCBI database and incorporated into the genomic comparison to elucidate their genomic diversity. The study showed that the genome sizes of the VPAHPND strains were approximately 5 Mbp. Ten sequence types (STs) were identified among the VPAHPND strains using in silico-Multilocus Sequence Typing analysis (MLST) and ST 970 was the predominant ST. Phylogenetic analysis based on MLST and single nucleotide polymorphisms (SNP) showed that the VPAHPND strains were genetically diverse. Based on the comparative genomic analysis, several functional proteins were identified from diiferent categories associated with virulence-related proteins, secretory proteins, conserved domain proteins, transporter proteins, and phage proteins. The CRISPR analysis showed that VPAHPND strains contained less number of CRISPRs elements than non-VPAHPND strains while six prophages regions were identified in the genomes, suggested the lack of CRISPR might promote prophage insertion. The genomic information in this study provide improved understanding of the virulence of these VPAHPND strains.

Genetic and virulence characterisation of Vibrio parahaemolyticus isolated from Indian coast.

BACKGROUND: V. parahaemolyticus is autochthonous to the marine environment and causes seafood-borne gastroenteritis in humans. Generally, V. parahaemolyticus recovered from the environment and/or seafood is thought to be non-pathogenic and the relationship between environmental isolates and acute diarrhoeal disease is poorly understood. In this study, we explored the virulence potential of environmental V. parahaemolyticus isolated from water, plankton and assorted seafood samples collected from the Indian coast. RESULTS: Twenty-two V. parahaemolyticus isolates from seafood harboured virulence associated genes encoding the thermostable-direct haemolysin (TDH), TDH-related haemolysin (TRH), and Type 3 secretion systems (T3SS) and 95.5% of the toxigenic isolates had pandemic strain attributes (toxRS/new+). Nine serovars, with pandemic strain traits were newly identified and an O4:K36 tdh-trh+V. parahaemolyticus bearing pandemic marker gene was recognised for the first time. Results obtained by reverse transcription PCR showed trh, T3SS1 and T3SS2ß to be functional in the seafood isolates. Moreover, the environmental strains were cytotoxic and could invade Caco-2 cells upon infection as well as induce changes to the tight junction protein, ZO-1 and the actin cytoskeleton. CONCLUSION: Our study provides evidence that environmental isolates of V. parahaemolyticus are potentially invasive and capable of eliciting pathogenic characteristics typical of clinical strains and present a potential health risk. We also demonstrate that virulence of this pathogen is highly complex and hence draws attention for the need to investigate more reliable virulence markers in order to distinguish the environmental and clinical isolates, which will be crucial for the pathogenomics and control of this pathogen.

Development of a monoclonal antibody for specific detection of Vibrio parahaemolyticus and analysis of its antigen.

Vibrio parahaemolyticus is a major foodborne pathogen worldwide. Contamination of V. parahaemolyticus in foods must be detected as quickly as possible because raw seafood, a major source of V. parahaemolyticus infection, is shipped immediately after production due to its short expiration date. In this study, we generated monoclonal antibodies (mAbs) against V. parahaemolyticus to develop a rapid and specific detection assay. Obtained mAbs were categorized into four groups according to their specificity. Of the groups, Group 1 (mAb VP7, VP11, and VP24) reacted to O1-O12 of V. parahaemolyticus without cross-reaction with human pathogenic Vibrio spp. (V. alginolyticus, V. cholerae, V. fluvialis, V. furnissii, V. mimicus, and V. vulnificus). We developed an immunochromatographic (IC) strip for the rapid detection of V. parahaemolyticus in the field using VP7 as a membrane-immobilized antibody and VP24 as a colloidal gold-conjugated antibody. The IC strip detected any and all serogroups (O1 to O12) or isolates (clinical, food, and environmental strains) of V. parahaemolyticus, regardless of the presence of virulence factors thermostable direct hemolysin (TDH) or TDH-related hemolysin (TRH). It did not cross-react with any other non-V. parahaemolyticus strains tested. To elucidate the target of the IC strip, we analyzed the antigen recognized by these mAbs. Group 1 mAbs showed two specific bands at molecular masses of approximately 11 and 16 kDa by western blotting analysis. Nano liquid chromatography mass spectrometry (LC-MS)/MS analysis revealed that the candidate antigen recognized by these mAbs was outer membrane (OM) lipoprotein Q87G48. We verified that mAb VP7 detected His-tagged OM lipoprotein synthesized by reconstituted cell-free protein synthesis reagent. Reactivity to an N-terminus deletion form and protease digestion form of the OM lipoprotein showed that the extent of epitope recognized by VP mAbs was 22nd-41st amino acids (AAs) from N-terminus of the OM lipoprotein, with the sequence "22SDDAATANAAKLDEL36." This region was also confirmed to be a V. parahaemolyticus-specific sequence by comparing putative orthologs of OM lipoprotein among Vibrio spp. The C-terminus deletion form (1st-39th AAs) including the sequence primarily recognized by VP mAbs (22nd-36th AAs) showed poor reactivity, indicating that the sequence after 40 residues of OM lipoprotein is also important for recognition by VP mAbs and VP mAbs recognize a conformational epitope. Bioinformatics research demonstrated that the OM lipoprotein is an ortholog of the lpp protein conserved throughout many bacteria. Lpp is an abundant and constitutively expressed protein and exists on the bacterial surface, suggesting it may be a good target for detection of V. parahaemolyticus.

Isolation, Molecular Characterization and Antibiotic Susceptibility Pattern of Vibrio parahaemolyticus from Aquatic Products in the Southern Fujian Coast, China.

Vibrio parahaemolyticus is a major gastroenteritis-causing pathogen in many Asian countries. Antimicrobial resistance in V. parahaemolyticus has been recognized as a critical threat to food safety. In this study, we determined the prevalence and incidence of antimicrobial resistance in V. parahaemolyticus in the southern Fujian coast, China. A total of 62 isolates were confirmed in retail aquatic products from June to October of 2018. The serotype O3:K6 strains, the virulence genes tdh and trh, antibiotic susceptibility and molecular typing were investigated. Then plasmid profiling analysis and curing experiment were performed for multidrug-resistant strains. The results showed that the total occurrence of V. parahaemolyticus was 31% out of 200 samples. Five strains (8.1%) out of 62 isolates were identified as the V. parahaemolyticus O3:K6 pandemic clone. A large majority of isolates exhibited higher resistance to penicillin (77.4%), oxacillin (71%), ampicillin (66.1%) and vancomycin (59.7%). Seventy-one percent (44/62) of the isolates exhibited multiple antimicrobial resistance. All 62 isolates were grouped into 7 clusters by randomly amplified polymorphic DNA, and most of the isolates (80.6%) were distributed within cluster A. Plasmids were detected in approximately 75% of the isolates, and seven different profiles were observed. Seventy-six percent (25/33) of the isolates carrying the plasmids were eliminated by 0.006% SDS incubated at 42°C, a sublethal condition. The occurrence of multidrug-resistant strains could be an indication of the excessive use of antibiotics in aquaculture farming. The rational use of antimicrobial agents and the surveillance of antibiotic administration may reduce the acquisition of resistance by microorganisms in aquatic ecosystems.

Improvement and evaluation of loop-mediated isothermal amplification combined with chromatographic flow dipstick assays for Vibrio parahaemolyticus.

Vibrio parahaemolyticus, a major food-borne pathogen, is a gram-negative rod-shaped halophilic bacterium which inhabits marine environments throughout the world. It can pose a threat to humans after the consumption of raw or undercooked seafood. Fast detection is crucial for hindering and controlling V. parahaemolyticus infection. Compared with traditional methods, loop-mediated isothermal amplification (LAMP) is a simple, rapid and versatile method. It can be performed at one temperature without the need for cycling. As a new method in recent years, LAMP combined with a chromatographic flow dipstick (LFD) meets the needs of point-of-care testing without the need for special instruments. It avoids the limitations of LAMP, reduces detection time and increases detection accuracy. Our previous studies have suggested that the optimized LFD method can improve the sensitivity of LAMP detection and shorten the isothermal amplification time during the detection process. In the present study, two LAMP assays were improved to LFD methods, and a LFD targeting 16S23S rRNA gene internal transcribed spacer (ITS) of V. parahaemolyticus was developed. The lower limit for tlh, toxR, ITS LFD assays were detected as 3.1 × 100, 3.1 × 101, and 3.1 × 100 CFU respectively, whether in pure cultures or artificially contaminated food samples. The shortest amplification times at the limit of each assay were determined as 20 min, 35 min and 25 min. A heating block was used to perform two (tlh and ITS) LFD assays to detect 20 food samples. Compared to a standard method (GB 4789.7-2013 National Food Safety Standards, Food Microbiology Inspection, Vibrio parahaemolyticus test), tlh and ITS LFD assays showed more MPN (most probable number) results than that of culture. It demonstrated that the improved LFD technology can provide a simple and rapid detection method with high sensitivity and specificity for detection of V. parahaemolyticus.