Multiplex Multilocus Variable-Number Tandem-Repeat Analysis for Typing of Pandemic Vibrio parahaemolyticus O1:KUT Isolates.

Pandemic O3:K6 Vibrio parahaemolyticus emerged in 1996. Since then, this strain of pathogen and its serovariants (predominantly O1:KUT [untypable], O1:K25 and O4:K68) have caused gastroenteritis worldwide. Owing to the limitation in established K antisera, tracking the sources of KUT for epidemiological investigation is difficult. Therefore, the effective molecular typing is required to discriminate the strains. The aim of this study was to develop a multiplex multilocus variable-number tandem-repeat analysis (MLVA) assay for typing pandemic V. parahaemolyticus, including various O1:KUT isolates. The assay was based on the analysis of four variable number tandem repeat loci. Forty-six pandemic isolates, including O1:KUT, O1:K25, and O3:K6, were investigated. MLVA generated 38 distinct MLVA profiles, whereas only 16 types were obtained from pulsed-field gel electrophoresis (PFGE). In this work, MLVA resolved the 12 isolates of O1:KUT obtained in 2001-2005 with identical PFGE patterns into unique profiles. Our data indicated that multiplex MLVA developed in this study has high discriminatory power (D = 0.99), and is superior to PFGE for distinct pandemic V. parahaemolyticus, including O1:KUT isolates.

Genetic heterogeneity among Vibrio alginolyticus strains, and design of a PCR-based identification method using gyrB gene sequence.

Vibrio alginolyticus, a pathogen among humans and marine animals, is ubiquitous in marine environments. The aims of this study were to analyze the relationships between genetic diversity and origins, and to develop new primers based on the gyrB sequence to identify V. alginolyticus isolated from various sources. To determine the genetic diversity of this bacterium, an arbitrarily primed polymerase chain reaction (AP-PCR) technique was performed on 36 strains of V. alginolyticus isolated from diarrhea patients and from diseased marine animals and environments in southern Thailand. The results showed distinct DNA fingerprints of all strains, indicating that they are genetically heterogeneous. For species-specific identification of V. alginolyticus, primers targeting the gyrB gene of V. alginolyticus were developed. Thirty reference Vibrio spp., 13 non-Vibrio spp., and 160 strains of V. alginolyticus isolated from various sources in southern Thailand were used to evaluate the specificity of these primers. Our results showed that the gyrB primers could specifically identify V. alginolyticus from all sample types. In addition, the detection limit of the PCR was at least 95 pg of DNA template. Therefore, we concluded that the newly designed gyrB primers are rapid, highly sensitive, and specific to identify V. alginolyticus isolated from various sources.

Impact of human Campylobacter infections in Southeast Asia: The contribution of the poultry sector.

Campylobacter is globally recognized as a major cause of foodborne infection in humans, whilst the development of antimicrobial resistance and the possibility of repelling therapy increase the threat to public health. Poultry is the most frequent source of Campylobacter infection in humans, and southeast Asia is a global leader in poultry production, consumption, and exports. Though three of the world's top 20 most populated countries are located in southeast Asia, the true burden of Campylobacter infection in the region has not been fully elucidated. Based on published data, Campylobacter has been reported in humans, animals, and food commodities in the region. To our knowledge, this study is the first to review the status of human Campylobacter infection in southeast Asia and to discuss future perspectives. Gaining insight into the true burden of the infection and prevalence levels of Campylobacter spp. in the southeast Asian region is essential to ensuring global and regional food safety through facilitating improvements in surveillance systems, food safety regulations, and mitigation strategies.

CHARACTERIZATION OF MALARIA INFECTION AT TWO BORDER AREAS OF THAILAND ADJOINING WITH MYANMAR AND MALAYSIA.

During 2009 to 2010, a total of 408 blood samples collected from malaria patients in Ranong (149) and Yala (259) Provinces, Thailand were investigated for Plasmodium spp using microscopic examination. There are no statistical differences in the prevalence of P. falciparum and P. vivax in samples collected from Ranong and Yala (46% vs 52%, and 54% vs 45%, respectively). Single nucleotide polymorphism of codon 86 in pfmdr1 (encoding P. falciparum multidrug resistance protein 1) was investigated among 75 samples of P. falciparum and 2 samples of P. knowlesi. A pfmdr1 N86Y mutation was detected in 1 out of 29 samples and 45 out of 46 samples obtained from Ranong and Yala Provinces, respectively. It is interesting that pfmdr1 was detected in two P. knowlesi DNA samples obtained previously from Ranong Province which was 99% homologous to pfmdr1 obtained from falciparum parasites in the same area but the mutation was not observed. The difference in multidrug resistance protein in Plasmodium obtained from those two border areas of Thailand will be of use in monitoring drug resistance in these border regions of the country.

Vibrio parahaemolyticus and its specific bacteriophages as an indicator in cockles (Anadara granosa) for the risk of V. parahaemolyticus infection in Southern Thailand.

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

First report in Thailand of a stx-negative Escherichia Coli 0157 strain from a patient with diarrhea.

E. coli serotype 0157 is well known to cause serious illnesses in humans. However, there has been no case report to date of this serotype in Thailand. In this study, we report for the first time E. coli 0157 (designated as PSU120) isolated from a stool sample among 228 diarrheal swab samples at Hat Yai Hospital, Songkhla Province, Thailand. This PSU120 was identified as being stx-negative and lacked eae but carried escV, a marker for the locus of enterocyte effacement. Of the five reported integration sites frequently occupied by stx phages, the sbcB and yehV loci were occupied, suggesting that PSU120 is active in horizontal genetic transfer. Antimicrobial susceptibility assay revealed that E. coli 0157 strain PSU120 was resistant to cephalothin, erythromycin, methicillin and vancomycin. Using pulsed- field gel-electrophoresis to compare the genetic relatedness of E. coli 0157 strain PSU120 to two other E. coli 0157 strains, namely, the well-established EHEC strain EDL933 and PSU2, a surrogate of E. coli 0157:H7 whose genotype stx1-, stx2+, eae+ is frequently obtained from the environment in this area during the last decade, revealed 88.6% in similarity. We suggest that PSU120 was originally stx+ but lostthe gene after establishing infection.

Human Plasmodium knowlesi infection in Ranong province, southwestern border of Thailand.

BACKGROUND: Plasmodium knowlesi, a simian malaria parasite, has been reported in humans in many Southeast Asian countries. In Thailand, most of the limited numbers of cases reported so far were from areas near neighbouring countries, including Myanmar. METHODS: Blood samples collected from 171 Thai and 248 Myanmese patients attending a malaria clinic in Ranong province, Thailand, located near the Myanmar border were investigated for P. knowlesi using nested PCR assays. Positive samples were also investigated by PCR for Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale, and were confirmed by sequencing the gene encoding the circumsporozoite protein (csp). RESULTS: Two samples, one obtained from a Thai and the other a Myanmese, were positive for P. knowlesi only. Nucleotide sequences of the csp gene derived from these two patients were identical and phylogenetically indistinguishable from other P. knowlesi sequences derived from monkeys and humans. Both patients worked in Koh Song, located in the Kawthoung district of Myanmar, which borders Thailand. CONCLUSION: This study indicates that transmission of P. knowlesi is occurring in the Ranong province of Thailand or the Kawthoung district of Myanmar. Further studies are required to assess the incidence of knowlesi malaria and whether macaques in these areas are the source of the infections.

[Food poisoning--importance of international perspective].

It is important to obtain the information on food security in the countries other than Japan since more than 60 % of the food consumed come from these countries. Food security is now considered as a global issue. A global trend persuading us to provide safe food to humans is based on the concept of human security development associated with a sense of human mission to sustain one's life. Another global tendency pushing us to secure safety and hygiene of food is driven by the economic pressure coming from the rules in international trade established by Codex Committee under FAO/WHO. In contrast to these trends under globalization requesting safe and hygienic food, food habits based on tradition or religion are maintained locally in various parts of the world. These local habits include eating raw or improperly cooked foods, which may become a risk of being exposed to food poisoning pathogens. This issue may be adequately solved by a risk assessment approach based on the concept of appropriate level of protection (ALOP). Like or not, people in some local areas live in the unhygienic environment where they are unintentionally and frequently exposed to enteric pathogens or immunologically cross-reacting microorganisms through which they may acquire specific immunity to the pathogens and escape from infection by the pathogens. There are therefore many areas in the world where people understand the necessity to provide safe food at the international level (globalization) but actually consume food in varying hygienic conditions from area to area due in part to traditional food habits or living environments (localization); we call this situation as glocalization (global+local).

Comparative genomic analysis of Vibrio parahaemolyticus: serotype conversion and virulence.

BACKGROUND: Vibrio parahaemolyticus is a common cause of foodborne disease. Beginning in 1996, a more virulent strain having serotype O3:K6 caused major outbreaks in India and other parts of the world, resulting in the emergence of a pandemic. Other serovariants of this strain emerged during its dissemination and together with the original O3:K6 were termed strains of the pandemic clone. Two genomes, one of this virulent strain and one pre-pandemic strain have been sequenced. We sequenced four additional genomes of V. parahaemolyticus in this study that were isolated from different geographical regions and time points. Comparative genomic analyses of six strains of V. parahaemolyticus isolated from Asia and Peru were performed in order to advance knowledge concerning the evolution of V. parahaemolyticus; specifically, the genetic changes contributing to serotype conversion and virulence. Two pre-pandemic strains and three pandemic strains, isolated from different geographical regions, were serotype O3:K6 and either toxin profiles (tdh+, trh-) or (tdh-, trh+). The sixth pandemic strain sequenced in this study was serotype O4:K68. RESULTS: Genomic analyses revealed that the trh+ and tdh+ strains had different types of pathogenicity islands and mobile elements as well as major structural differences between the tdh pathogenicity islands of the pre-pandemic and pandemic strains. In addition, the results of single nucleotide polymorphism (SNP) analysis showed that 94% of the SNPs between O3:K6 and O4:K68 pandemic isolates were within a 141 kb region surrounding the O- and K-antigen-encoding gene clusters. The "core" genes of V. parahaemolyticus were also compared to those of V. cholerae and V. vulnificus, in order to delineate differences between these three pathogenic species. Approximately one-half (49-59%) of each species' core genes were conserved in all three species, and 14-24% of the core genes were species-specific and in different functional categories. CONCLUSIONS: Our data support the idea that the pandemic strains are closely related and that recent South American outbreaks of foodborne disease caused by V. parahaemolyticus are closely linked to outbreaks in India. Serotype conversion from O3:K6 to O4:K68 was likely due to a recombination event involving a region much larger than the O-antigen- and K-antigen-encoding gene clusters. Major differences between pathogenicity islands and mobile elements are also likely driving the evolution of V. parahaemolyticus. In addition, our analyses categorized genes that may be useful in differentiating pathogenic Vibrios at the species level.

Occurrence of potentially pathogenic vibrios and related environmental factors in Songkhla Lake, Thailand.

Vibrios are halophilic bacteria that are ubiquitous in marine environments. Their occurrence in tropical lakes has rarely been investigated. In this study, the predominance and diversity of Vibrio spp. was investigated over a 12-month period in a coastal lagoon, Songkhla Lake, in southern Thailand. Water samples were collected at 2 stations in the estuary near Yor Island in Songkhla Lake. The predominant vibrios were detected by a culture-based method, using thiosulfate-citrate-bile salt-sucrose agar and CHROMagar Vibrio. The diversity of Vibrio spp. was evaluated using denaturant density gradient electrophoresis (DGGE). The highest numbers of total vibrios and Vibrio parahaemolyticus in both areas were observed during the summer. There was no significant correlation between the numbers of vibrios, including V. parahaemolyticus, and either the water temperature or plankton density. Variations in Vibrio species were observed with changes in salinity. Vibrio parahaemolyticus and V. cholerae non-O1/non-O139 were detected during the rainy season when the salinity dropped to nearly 0 parts per thousand. In both areas, V. alginolyticus was the most prominent species detected by the culture method, whereas Vibrio parahaemolyticus was detected by DGGE, every month. Other Vibrio spp. of potential public health concern were also detected by the culture method; they included V. vulnificus , V. fluvialis , and V. mimicus .

Corrigendum: Development and Improvement of Methods to Disinfect Raw Beef Using Calcium Hydroxide-Ethanol-Lactate-Based Food Disinfectant for Safe Consumption.

[This corrects the article DOI: 10.3389/fmicb.2020.537889.].

Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of the tdh and trh genes of Vibrio parahaemolyticus and related Vibrio species.

Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the major virulence determinants of Vibrio parahaemolyticus. TRH is further differentiated into TRH1 and TRH2 on the basis of genetic and phenotypic differences. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for sensitive and rapid detection of the tdh, trh1, and trh2 genes of V. parahaemolyticus. The LAMP assay was designed for both combined and individual detection of the tdh, trh1, and trh2 genes and combined detection of the trh1 and trh2 genes. Our results showed that it gave the same results as DNA probes and conventional PCR assays for 125 strains of V. parahaemolyticus, 3 strains of Grimontia hollisae, and 2 strains of Vibrio mimicus carrying the tdh, trh1, and trh2 genes in various combinations. No LAMP products were detected for any of the 20 bacterial strains lacking the tdh, trh1, and trh2 genes. The sensitivities of the LAMP assay for detection of tdh-, trh1-, and trh2-carrying V. parahaemolyticus strains in spiked shrimp samples were 0.8, 21.3, and 5.0 CFU per LAMP reaction tube, respectively. Starting with DNA extraction from a single colony and from spiked shrimp samples, the LAMP assay required only 27 to 60 min and less than 80 min, respectively. This is the first report of a rapid and specific LAMP assay for detection and differentiation of the tdh, trh1, and trh2 genes of V. parahaemolyticus and related Vibrio species.

Prevalence and quantification of Vibrio parahaemolyticus in raw salad vegetables at retail level.

The purpose of this study was to investigate the biosafety of Vibrio parahaemolyticus in raw salad vegetables at wet market and supermarket in Malaysia. A combination of Most Probable Number - Polymerase Chain Reaction (MPN-PCR) method was applied to detect the presence of V. parahaemolyticus and to enumerate their density in the food samples. The study analyzed 276 samples of common vegetables eaten raw in Malaysia (Wild cosmos = 8; Japanese parsley = 21; Cabbage = 30; Lettuce = 16; Indian pennywort = 17; Carrot = 31; Sweet potato = 29; Tomato = 38; Cucumber = 28; Four winged bean = 26; Long bean = 32). The samples were purchased from two supermarkets (A and B) and two wet markets (C and D). The occurrence of V. parahaemolyticus detected was 20.65%, with higher frequency of V. parahaemolyticus in vegetables obtained from wet markets (Wet market C = 27.27%Wet Market D = 32.05%) compared to supermarkets (Supermarket A = 1.64%; Supermarket B = 16.67%). V. parahaemolyticus was most prevalent in Indian pennywort (41.18%). The density of V. parahaemolyticus in all the samples ranged from <3 up to >2400 MPN/g, mostly <3 MPN/g concentration. Raw vegetables from wet markets contained higher levels of V. parahaemolyticus compared to supermarkets. V. parahaemolyticus were present in raw vegetables although in low numbers. The results suggest that raw vegetables act as a transmission route for V. parahaemolyticus. This study will be the first biosafety assessment of V. parahaemolyticus in raw vegetables in Malaysia.

Development and Improvement of Methods to Disinfect Raw BeefUsing Calcium Hydroxide-Ethanol-Lactate-Based Food Disinfectant for Safe Consumption.

The enterohemorrhagic Escherichia coli (EHEC) group is responsible for outbreaks and sporadic cases around the world annually. EHEC produces a potent protein known as Shiga toxin in the human intestine causing mild to bloody diarrhea. Some cases of EHEC infections may develop life-threatening symptoms, which may lead to human death. It also has other virulent factors that enable the EHEC cells to adhere to a target tissue and invade to some extent to crave more nutrition and escape the external extreme conditions, such as disinfection treatment. For those reasons, beef is not permitted for raw consumption unless guaranteed free of harmful bacteria, including EHEC, or the invading bacterial cells are completely removed or reduced to a safe level. A heat treatment that guarantees a sufficiently high temperature to reach inside the tissue of meat through the surface was established in Japan. This treatment may allow the core part of the meat to be consumed raw. However, it seemed to have some limitations. We aimed at developing a disinfection method with, hypothetically, nutrition-preserving property that is equivalent to the heat treatment or even superior. A combination of calcium hydroxide-ethanol-lactate-based food disinfectant and two proposed physical sterilization methods, assisted with microbial detection methods, exerted sufficient bactericidal activities against EHEC cells adhering to and/or invading the beef. These physical methods showed great usefulness in disinfecting fresh full-size boneless Round-beef of around 12 kg including fat on the outside. The first method applied a commercially available wide-drum washing machine (WM method) while the second method applied a specially designed plastic bag and a commercially available vibration machine (VV method). After trimming out the fat and the denatured surface of the beef (1 cm from the surface), the remaining meat mass showed no signs of denaturation and a significant reduction of viable EHEC cells by a factor of >104 CFU/ml. However, in the WM method, the disinfection process required a large amount of the disinfectant (150 L). The improved method, VV method, implemented a system that consumes a smaller amount of the disinfectant (50 L) while ensuring the targeted disinfection power degree.

Prevalence and antibiotic resistance patterns of Vibrio parahaemolyticus isolated from different types of seafood in Selangor, Malaysia.

Vibrio parahaemolyticus is a foodborne bacterial pathogen that may cause gastroenteritis in humans through the consumption of seafood contaminated with this microorganism. The emergence of antimicrobial and multidrug-resistant bacteria is another serious public health threat worldwide. In this study, the prevalence and antibiotic susceptibility test of V. parahaemolyticus in blood clams, shrimps, surf clams, and squids were determined. The overall prevalence of V. parahaemolyticus in seafood was 85.71% (120/140), consisting of 91.43% (32/35) in blood clam, 88.57% (31/35) in shrimps, 82.86% (29/35) in surf clams, and 80% (28/35) in squids. The majority of V. parahaemolyticus isolates from the seafood samples were found to be susceptible to most antibiotics except ampicillin, cefazolin, and penicillin. The MAR indices of V. parahaemolyticus isolates ranged from 0.04 to 0.71 and about 90.83% of isolates were found resistant to more than one antibiotic. The high prevalence of V. parahaemolyticus in seafood and multidrug-resistant isolates detected in this study could pose a potential risk to human health and hence appropriate control methods should be in place to minimize the potential contamination and prevent the emergence of antibiotic resistance.

Characterization of proteins secreted from a type III secretion system of Edwardsiella tarda and their roles in macrophage infection.

The Type III secretion system is essential for intracellular replication of Edwardsiella tarda in phagocytes of fish and mammals. We identified the secreted proteins of the Type III secretion system by comparing the wild-type strain and the Type III mutant mET1229. The wild-type strain secreted 55, 25, and 22 kDa proteins into the culture supernatant, whereas the Type III mutant did not. These proteins were identified as EseB, EseC, and EseD and are similar in sequence to Salmonella SseB, SseC, and SseD that function as a translocon. The EseB, EseC, and EseD knockout mutants did not replicate in murine macrophages, suggesting that these proteins are essential for intracellular replication of E. tarda. Highest secretion of EseBCD proteins was observed when bacterial cells were cultured in neutral and alkaline pHs but not in acidic pH. When the pH of the phagosomes was examined using an acidotropic probe, the phagosomes containing the wild-type strain showed neutral pH, whereas those containing the Type III mutant exhibited acidic pH. These results suggest that the Type III-dependent interference with formation of the acidic environment in phagosomes is essential for intracellular replication of bacteria in murine macrophages.

Detection and characterization of a functional insertion sequence, ISVpa2, in Vibrio parahaemolyticus.

PCR analysis of the pandemic strain of Vibrio parahaemolyticus, KX-V237 (total genome sequenced) showed a subculture where the size of the amplicons had increased. The purpose of this study was to analyze the mechanism of this change. We found a 1,243-bp DNA sequence inserted in one of the pandemic marker genes in this strain. The inserted DNA sequence possessed the genetic structures shared by insertion sequences (ISs) of the IS3 family. This IS had 26-bp imperfect terminal inverted repeats (IRs) and two partially overlapping reading frames, orfA and orfB. OrfA codes for a helix-turn-helix, OrfA and OrfAB produced by translational frameshifting code for leucine zipper motifs, and OrfB codes for a DDE motif. orfA and orfB were homologous to those in the IS3 family. This IS was named ISVpa2. Southern blot analysis showed the copy number of ISVpa2 in our stock culture and its subculture of KX-V237 was three and four, respectively; whereas it was only one in the reported genome sequence. Analysis of the flanking sequences for seven ISVpa2 copies showed ISVpa2 is capable of inserting at multiple sites and ISVpa2 causes genetic rearrangements including insertional inactivation of the target gene and adjacent deletion. ISVpa2 created 3-base duplications upon insertion. PCR, hybridization, and nucleotide sequence analyses showed ISVpa2 homologs were detected in all of the 62 other strains of V. parahaemolyticus examined; and in some strains of Vibrio vulnificus (98% identity), Vibrio penaeicida (86% identity), and Vibrio splendidus (87% identity); but was not in 25 other species in the genus Vibrio. The data demonstrate that ISVpa2 is a transpositionally active IS discovered for the first time in V. parahaemolyticus and suggest that ISVpa2 may be transferred among the species of the genus Vibrio.

Detection of a functional insertion sequence responsible for deletion of the thermostable direct hemolysin gene (tdh) in Vibrio parahaemolyticus.

The thermostable direct hemolysin coded by the tdh gene is a marker of virulent strains of Vibrio parahaemolyticus. The tdh genes are flanked by insertion sequences collectively named as ISVs or their remnants; but the ISVs so far examined have accumulated mutations in the transposase genes and underwent structural arrangements and their transposition activity could not be expected; the tdh gene was thus considered to have been acquired by V. parahaemolyticus through horizontal transfer in the past during evolution. We recently isolated from the same patient tdh+ strains and a tdh(-) strain (PCR examination) that were otherwise indistinguishable. The purpose of this study was to examine the hypothesis that the tdh(-) strain was derived from the tdh+ strain by a deletion of the tdh gene mediated by a functional ISV. Southern blot hybridization showed tdh+ sequences in the tdh(-) strain (PSU-1466). Nucleotide sequence analysis of the tdh and its flanking sequences revealed the tdh gene was split into two parts and they were located 3182-bp apart in PSU-1466. The two tdh sequences were flanked by one of the ISVs, named as ISVpa3, in PSU-1466. This genetic structure could be explained by an ISVpa3-mediated partial tdh deletion from a tdh+ strain followed by transposition of the duplicated ISVpa3 and the deleted tdh sequence into a neighboring location. The ISVpa3 of PSU-1466 coded for a full-length transposase and a DDE motif. We were able to demonstrate transposition activity of the ISVpa3 cloned from PSU-1466 using the replicon fusion assay with the conjugal transfer of a cointegrate from Escherichia coli to V. parahaemolyticus. Our data support ISVpa3-mediated partial tdh deletion resulted in the emergence of the tdh(-) strain.

On the origin and function of an insertion element VPaI-1 specific to post-1995 pandemic Vibrio parahaemolyticus strains.

Since 1995, new virulent strains of Vibrio parahaemolyticus have emerged and spread throughout the world. These "pandemic" strains have four strain specific genomic islands (GIs), which are considered to be potential factors of the pandemicity. We investigated the origin and function of 24 genes in the so-called VPaI-1, one of the four GIs, by searching homologs in various species in Bacteria and Archaea. Of these 24 genes, two are found only in Vibrio vulnificus CMCP6 and Shewanella sp. MR-7. The genomic segment (- 8 kb) encompassing the two genes shows the synteny among the three species. Since many of the Shewanella species can grow at 4 degrees C, these two genes may be candidates of adaptation to temperature stress. Further, we found a candidate for a swarming gene, which is reported as the V. cholerae virulence gene. Based on these findings, we hypothesized the emergence of pandemicity and discuss the mechanism for how these strains spread throughout the world.

Quantitative modeling for risk assessment of Vibrio parahaemolyticus in bloody clams in southern Thailand.

A risk assessment of Vibrio parahaemolyticus in bloody clams (Anadara granosa) consumed in southern Thailand was conducted. This study estimated the prevalence and concentration of pathogenic V. parahaemolyticus in bloody clams at harvest and retail stages; and during this process, methods to detect the total and pathogenic V. parahaemolyticus were investigated. Consumption of bloody clams and cooking efficiency were studied using interviews and on-site observation of consumers. A beta-Poisson dose-response model was used to estimate probability of illness applying estimation methods for the most likely parameter values presented by USFDA. Microbial and behavioral data were analyzed by developing a stochastic model and the simulation gave a mean number of times a person would get ill with V. parahaemolyticus by consuming bloody clams at 5.6 x 10(-4)/person/year. Sensitivity analysis demonstrated the fraction of people who did not boil the clams properly was the primary factor in increasing risk. This study serves as an example of how a microbiological risk assessment with limited data collection and international cooperation leads to valuable local insight.