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.

A pH ultra-sensitive hydrated iridium oxyhydroxide films electrochemical sensor for label-free detection of Vibrio parahaemolyticus.

Vibrio parahaemolyticus (V. parahaemolyticus) is a major foodborne pathogen, which can cause serious foodborne illnesses like diarrhoea. Rapid on-site detection of foodborne pathogens is an ideal way to respond to foodborne illnesses. Herein, we provide an electrochemical sensor for rapid on-site detection. This sensor utilized a pH-sensitive metal-oxide material for the concurrent isothermal amplification and label-free detection of nucleic acids. Based on a pH-sensitive hydrated iridium oxide oxyhydroxide film (HIROF), the electrode transforms the hydrogen ion compound generated during nucleic acid amplification into potential, so as to achieve a real-time detection. The results can be transmitted to a smartphone via Bluetooth. Moreover, HIROF was applied in nucleic acid device detection, with a super-Nernst sensitivity of 77.6 mV/pH in the pH range of 6.0-8.5, and the sensitivity showed the best results so far. Detection of V. parahaemolyticus by this novel method showed a detection limit of 1.0 × 103 CFU/mL, while the time consumption was only 30 min, outperforming real-time fluorescence loop-mediated isothermal amplification (LAMP). Therefore, the characteristics of compact, portable, and fast make the sensor more widely used in on-site detection.

Serodiversity, antibiotic resistance, and virulence genes of Vibrio parahaemolyticus in oysters collected in coastal areas of northwestern Mexico between 2012 and 2020.

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.

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.

Machine learning to predict the relationship between Vibrio spp. concentrations in seawater and oysters and prevalent environmental conditions.

Vibrio parahaemolyticus and Vibrio vulnificus are bacteria with a significant public health impact. Identifying factors impacting their presence and concentrations in food sources could enable the identification of significant risk factors and prevent incidences of foodborne illness. In recent years, machine learning has shown promise in modeling microbial presence based on prevalent external and internal variables, such as environmental variables and gene presence/absence, respectively, particularly with the generation and availability of large amounts and diverse sources of data. Such analyses can prove useful in predicting microbial behavior in food systems, particularly under the influence of the constant changes in environmental variables. In this study, we tested the efficacy of six machine learning regression models (random forest, support vector machine, elastic net, neural network, k-nearest neighbors, and extreme gradient boosting) in predicting the relationship between environmental variables and total and pathogenic V. parahaemolyticus and V. vulnificus concentrations in seawater and oysters. In general, environmental variables were found to be reliable predictors of total and pathogenic V. parahaemolyticus and V. vulnificus concentrations in seawater, and pathogenic V. parahaemolyticus in oysters (Acceptable Prediction Zone >70 %) when analyzed using our machine learning models. SHapley Additive exPlanations, which was used to identify variables influencing Vibrio concentrations, identified chlorophyll a content, seawater salinity, seawater temperature, and turbidity as influential variables. It is important to note that different strains were differentially impacted by the same environmental variable, indicating the need for further research to study the causes and potential mechanisms of these variations. In conclusion, environmental variables could be important predictors of Vibrio growth and behavior in seafood. Moreover, the models developed in this study could prove invaluable in assessing and managing the risks associated with V. parahaemolyticus and V. vulnificus, particularly in the face of a changing environment.

Antibiotic resistance trends among Vibrio vulnificus and Vibrio parahaemolyticus isolated from the Chesapeake Bay, Maryland: a longitudinal study.

Antibiotics are often used to treat severe Vibrio infections, with third-generation cephalosporins and tetracyclines combined or fluoroquinolones alone being recommended by the US Centers for Disease Control and Prevention. Increases in antibiotic resistance of both environmental and clinical vibrios are of concern; however, limited longitudinal data have been generated among environmental isolates to inform how resistance patterns may be changing over time. Hence, we evaluated long-term trends in antibiotic resistance of vibrios isolated from Chesapeake Bay waters (Maryland) across two 3-year sampling periods (2009-2012 and 2019-2022). Vibrio parahaemolyticus (n = 134) and Vibrio vulnificus (n = 94) toxR-confirmed isolates were randomly selected from both sampling periods and tested for antimicrobial susceptibility against eight antibiotics using the Kirby-Bauer disk diffusion method. A high percentage (94%-96%) of V. parahaemolyticus isolates from both sampling periods were resistant to ampicillin and only 2%-6% of these isolates expressed intermediate resistance or resistance to third-generation cephalosporins, amikacin, tetracycline, and trimethoprim-sulfamethoxazole. Even lower percentages of resistant V. vulnificus isolates were observed and those were mostly recovered from 2009 to 2012, however, the presence of multiple virulence factors was observed. The frequency of multi-drug resistance was relatively low (6%-8%) but included resistance against antibiotics used to treat severe vibriosis in adults and children. All isolates were susceptible to ciprofloxacin, a fluoroquinolone, indicating its sustained efficacy as a first-line agent in the treatment of severe vibriosis. Overall, our data indicate that antibiotic resistance patterns among V. parahaemolyticus and V. vulnificus recovered from the lower Chesapeake Bay have remained relatively stable since 2009.IMPORTANCEVibrio spp. have historically been susceptible to most clinically relevant antibiotics; however, resistance and intermediate-resistance have been increasingly recorded in both environmental and clinical isolates. Our data showed that while the percentage of multi-drug resistance and resistance to antibiotics was relatively low and stable across time, some Vibrio isolates displayed resistance and intermediate resistance to antibiotics typically used to treat severe vibriosis (e.g., third-generation cephalosporins, tetracyclines, sulfamethoxazole-trimethoprim, and aminoglycosides). Also, given the high case fatality rates observed with Vibrio vulnificus infections, the presence of multiple virulence factors in the tested isolates is concerning. Nevertheless, the continued susceptibility of all tested isolates against ciprofloxacin, a fluoroquinolone, is indicative of its use as an effective first-line treatment of severe Vibrio spp. infections stemming from exposure to Chesapeake Bay waters or contaminated seafood ingestion.

A new simplified sequence-dependent loop-mediated isothermal amplification (LAMP) detection method.

Fluorescence dye-based loop-mediated isothermal amplification (LAMP) is a sensitive nucleic acid detection method, but is limited to single-plex detection and may yield non-specific signals. In this study, we propose a bifunctional probe-based real-time LAMP amplification method for single-plexed or multiplexed detection. The bifunctional probe is derived by modifying the 5' end of the fluorophore and an internal quencher on one of the LAMP primers; therefore, it can simultaneously be involved in the LAMP process and signal amplification. The fluorescence intensity undergoes a cumulative exponential increase during the incorporation of the bifunctional probe into double-stranded DNA amplicons. The bifunctional probe-based LAMP method is simplified and cost-effective, as the primer design and experimental operations align entirely with the ordinary LAMP. Different from other current probe-based methods, this method does not require additional enzymes, sequences, or special probe structures. Also, it is 10 min faster than several other probe-based LAMP methods. The bifunctional probe-based LAMP method allows the simultaneous detection of the target Vibrio parahaemolyticus DNA and the internal amplification control in a one-pot reaction, demonstrating its potential for multiplexed detection.

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.

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.

Tracking Vibrio: population dynamics and ecology of Vibrio parahaemolyticus and V. vulnificus in an Alabama estuary.

Vibrio is a genus of halophilic, gram-negative bacteria found in estuaries around the globe. Integral parts of coastal cultures often involve contact with vectors of pathogenic Vibrio spp. (e.g., consuming raw shellfish). High rates of mortality from certain Vibrio spp. infections demonstrate the need for an improved understanding of Vibrio spp. dynamics in estuarine regions. Our study assessed meteorological, hydrographic, and biological correlates of Vibrio parahaemolyticus and V. vulnificus at 10 sites in the Eastern Mississippi Sound System (EMSS) from April to October 2019. During the sampling period, median abundances of V. parahaemolyticus and V. vulnificus were 2.31 log MPN/L and 2.90 log MPN/L, respectively. Vibrio spp. dynamics were largely driven by site-based variation, with sites closest to freshwater inputs having the highest abundances. The E-W wind scalar, which affects Ekman transport, was a novel Vibrio spp. correlate observed. A potential salinity effect on bacterial-particle associations was identified, where V. vulnificus was associated with larger particles in conditions outside of their optimal salinity. Additionally, V. vulnificus abundances were correlated to those of harmful algal species that did not dominate community chlorophyll. Correlates from this study may be used to inform the next iteration of regionally predictive Vibrio models and may lend additional insight to Vibrio spp. ecology in similar systems. IMPORTANCE: Vibrio spp. are bacteria found in estuaries worldwide; some species can cause illness and infections in humans. Relationships between Vibrio spp. abundance, salinity, and temperature are well documented, but correlations to other environmental parameters are less understood. This study identifies unique correlates (e.g., E-W wind scalar and harmful algal species) that could potentially inform the next iteration of predictive Vibrio models for the EMSS region. Additionally, these correlates may allow existing environmental monitoring efforts to be leveraged in providing data inputs for future Vibrio risk models. An observed correlation between salinity and V. vulnificus/particle-size associations suggests that predicted environmental changes may affect the abundance of Vibrio spp. in certain reservoirs, which may alter which vectors present the greatest vibrio risk.

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.

Determination of Whole Molecular of Thermostable Direct Hemolysins in Milk Powder by HPLC-ESI-TOF.

Although Vibrio parahaemolyticus (V. parahaemolyticus) is a pathogen frequently found in seafood, there is a possibility of its presence in other foods, such as dairy products. The main virulence factors of V. parahaemolyticus are thermostable direct hemolysins (TDHs) which are lethal toxins, so it is necessary to establish qualitative and quantitative methods for determining TDHs. HPLC-ESI-TOF was employed to establish a method for identifying TDHs. The identification and quantification ions of TDHs were confirmed by HPLC-ESI-TOF. The method was developed for detecting TDHs in milk powder using HPLC-ESI-TOF in this paper, and limits of detection (were between 0.20 and 0.40 mg/kg, limits of quantitation were between 0.5 and 1.0 mg/kg and recoveries of all TDHs were between from 78% to 94% with relative standard deviation lower than 10%. This research will provide a reference for developing methods of HPLC-MS/MS to detect TDHs in food samples, which can provide a tool for the government to monitor TDHs contamination in foods.

Quantitative microbial risk assessment of the gastrointestinal risks to swimmers at Southeast Asian urban beaches using site-specific and combined autochthonous and fecal bacteria exposure data.

Recreational exposure to microbial pollution at urban beaches poses a health risk to beachgoers. The accurate quantification of such risks is crucial in managing beaches effectively and establishing warning guidelines. In this study, we employed a quantitative microbial risk assessment (QMRA) framework to assess marine water quality and estimate the risks associated with Vibrio parahaemolyticus, an autochthonous pathogen that causes gastrointestinal illnesses, and enterococci, a traditional fecal bacteria indicator. The microbial contamination levels of V. parahaemolyticus and enterococci were determined from 48 water samples collected at two beaches in Thailand during dry and wet seasons. The accidentally ingested water volumes were obtained through a survey involving 438 respondents. The probability of illness (Pill) was estimated using dose-response models and Monte Carlo simulation. The results revealed that enterococci posed a higher risk of illness than V. parahaemolyticus at all seven study sites. The median combined gastrointestinal (GI) risk from both bacteria at all sites met the US EPA risk benchmark of 0.036 and the 0.05 benchmark set by the WHO, but the 95th percentile risk data at all sites exceeded the benchmarks. This emphasizes the need for the continuous monitoring and management of microbial pollution at these sites. The site-specific exposure data showed higher estimated risks with increased variations compared to the WHO-referenced values, which highlights the significance of locally measured microbial concentrations and survey exposure data to avoid underestimation. Estimating the risks from recreational exposure to waterborne bacteria can inform beach management policies aimed at reducing public health risks to swimmers. The study findings improve the understanding of the risks associated with water recreation activities at Southeast Asian beaches and offer valuable insights for the development of water quality guidelines, which are crucial for the sustainable development of the blue economy.

Surveillance of Vibrio parahaemolyticus pathogens recovered from ready-to-eat foods.

This study examined the occurrence of V. parahaemolyticus from ready-to-eat (RTE) food in Delta State, Nigeria. It also characterized antibiotic resistance and virulence gene profile patterns to determine the associated health risk hazard. Food samples total of 380 were collected randomly and assessed for V. parahaemolyticus. V. parahaemolyticus isolates were characterized for their virulence and antibiogram potentials using a phenotypic and polymerase chain reaction (PCR) approach. A total of 42 (11.1%) samples were contaminated with V. parahaemolyticus. In 17/42 (40.5%) of the V. parahaemolyticus-positive samples, the densities were < 10 MPN/g. However, 19/42 (45.2%) and 6/42 (14.3%) of the samples had densities of 10 - 102 and > 102 MPN/g, respectively. A total of 67 V. parahaemolyticus isolates were identified using PCR; 54(80.6%) isolates were multidrug resistant. A total of 22 (32.8%), 39 (58.2%), and 67 (100%) of the V. parahaemolyticus harbored the tdh, trh, and tlh toxin genes, respectively. The T3SS1 gene (vcrD1) was detected in 67 (100%) of the isolates. The T3SS2α genes which were vcrD2, vopB2, and vopT were detected in 21 (31.3%), 11 (16.4%) and 30 (44.8%) of the isolates respectively. Some of the V. parahaemolytics strains harbored the orf8 gene 20 (29.9%), and a combination of orf8 + tdh genes 12 (17.9%), categorized as pandemic strains. The antibiotic resistance genes detected in this study include blaTEM 33 (49.3), tetM 19 (28.4), cmlA 32(47.8) and sul1 14 (20.9). The concentration levels and prevalence of V. parahaemolyticus in RTE foods indicate contamination of ready-to-eat foods, particularly street foods consumed in the Delta State of Nigeria, threatening public health and consumer safety.

Outbreak of seafood-related food poisoning from undetectable Vibrio parahaemolyticus-like pathogen, Chiang Mai Province, Thailand, December 2020.

OBJECTIVE: On 1 December 2020, the Department of Disease Control of Thailand was notified of a cluster of food poisoning cases among participants at a church festival in Mae Ai district, Chiang Mai province. We conducted an outbreak investigation to confirm diagnosis, describe the epidemiological characteristics of the outbreak, identify possible sources of the outbreak and provide appropriate control measures. METHODS: We reviewed medical records of the food poisoning cases from the health care centres. Active case finding was conducted among participants who had consumed food and water at the festival. An environmental survey was done in the village where the festival was held. A case-control study was conducted to identify the source of the outbreak. Samples for laboratory analysis included rectal swabs and fresh stool specimens from the cases and food handlers, surface swabs of cooking equipment, food, water and ice samples. RESULTS: Among 436 participants surveyed, 368 (84.4%) cases of food poisoning were identified. The most common clinical manifestation was abdominal pain (89.7%), followed by watery diarrhoea (45.7%), nausea (43.5%), vomiting (38.9%), fever (18.5%) and bloody diarrhoea (4.6%). None died in this outbreak. The case-control study showed that mixed spicy seafood salad served in the festival was significantly associated with the disease by both univariable and multivariable analyses. However, the causative agent could not be identified. The environmental investigation suggested this seafood might have been undercooked. CONCLUSION: Clinical manifestations of the cases, incubation period and the suspected seafood salad suggested seafood-related food poisoning. Grimontia hollisae, the organism causing illness similar to Vibrio parahaemolyticus and commonly undetectable in the laboratory with routine testing, might be the pathogen that caused this outbreak. G. hollisae should be in differential diagnosis and identified in seafood-associated outbreaks.

Rapid and Visual Detection of Vibrio parahaemolyticus in Aquatic Foods Using blaCARB-17 Gene-Based Loop-Mediated Isothermal Amplification with Lateral Flow Dipstick (LAMP-LFD).

Vibrio parahaemolyticus is recognized as one of the most important foodborne pathogens responsible for gastroenteritis in humans. The blaCARB-17 gene is an intrinsic ß-lactamase gene and a novel species-specific genetic marker of V. parahaemolyticus. In this study, a loop-mediated isothermal amplification (LAMP) assay combined with a lateral flow dipstick (LFD) was developed targeting this blaCARB-17 gene. The specificity of LAMP-LFD was ascertained by detecting V. parahaemolyticus ATCC 17802 and seven other non-V. parahaemolyticus strains. Finally, the practicability of LAMP-LFD was confirmed by detection with V. parahaemolyticus-contaminated samples and natural food samples. The results showed that the optimized reaction parameters of LAMP are as follows: 2.4 mmol/l Mg2+, 0.96 mmol/l dNTPs, 4.8 U Bst DNA polymerase, and an 8:1 ratio of inner primer to outer primer, at 63°C for 40 min. The optimized reaction time of the LFD assay is 60 min. Cross-reactivity analysis with the seven non-V. parahaemolyticus strains showed that LAMP-LFD was exclusively specific for V. parahaemolyticus. The detection limit of LAMP-LFD for V. parahaemolyticus genomic DNA was 2.1 × 10-4 ng/µl, corresponding to 630 fg/reaction and displaying a sensitivity that is 100-fold higher than that of conventional PCR. LAMP-LFD in a spiking study revealed a detection limit of approximately 6 CFU/ml, which was similar with conventional PCR. The developed LAMP-LFD specifically identified the 10 V. parahaemolyticus isolates from 30 seafood samples, suggesting that this LAMP-LFD may be a suitable diagnostic method for detecting V. parahaemolyticus in aquatic foods.

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.

Molecular characterization and antibiotic resistance of Vibrio parahaemolyticus from Indian oyster and their probable implication in food chain.

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.

Establishment of a visualized isothermal nucleic acid amplification method for on-site diagnosis of acute hepatopancreatic necrosis disease in shrimp farm.

Acute hepatopancreatic necrosis disease (AHPND) is a significant deadly infectious disease in the shrimp farming industry, causing serious economic losses globally every year. Because of the rapid progress speed, lack of effective treatment and high mortality rate of AHPND, monitoring with frequent diagnostic tests is vital for a successful prevention. The conventional histopathological diagnosis fell far short of the requirement for efficient monitoring, and the polymerase chain reaction (PCR)-based molecular diagnostic methods that rely on sophisticated thermocycler and trained personnel are hardly applicable in the field. Combining the recombinase polymerase amplification (RPA) and the lateral flow strips (LFSs), a diagnostic method suitable for on-site everyday monitoring of AHPND has been established in this study. This RPA-LFS method targeted the binary toxic photorhabdus insect-related genes PirA and PirB on a virulence plasmid of the AHPND-causative Vibrio parahaemolyticus strains. The diagnostic test was completed within 30 min at 37°C and showed good specificity and good sensitivity of 20 fg DNA of the AHPND shrimp or one colony-forming unit of the causative bacterium per reaction, which was better than the administration-approved standard AP4 assay. Crude templates from sample boiling could be directly used. Tests of clinical samples showed 100% consistency of this method with the standard AP4 assay. This RPA-LFS method can be a good choice for on-site diagnosis of AHPND with quick response time, easy procedure and low demand for resources, and should have significant value for the control of spreading of this dangerous disease in farmed shrimp.

Influence of climatic factors on the temporal occurrence and distribution of total and pathogenic Vibrio parahaemolyticus in oyster culture environments in Taiwan.

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.