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To gain insight into the microbiological safety of food products routinely traded across international borders in Southeast Asian countries, beef imported from Malaysia to southern Thailand was examined for contamination with Escherichia coli O157 and its subsequent spread into the imported areas. We screened 31 samples exported from Malaysia and 36 domestic Thai samples. Isolation methods including an O157 antigen-targeted immunomagnetic separation technique, screening on CHROMagar O157 medium, and serotype confirmation of E. coli isolates by specific agglutination tests were employed. Fourteen strains of E. coli O157:H7 were isolated from eight Malaysian samples (25.8%) and six strains from four Thai samples (11.1%). These strains were of the stx1- stx2+ eae+ genotype except one Malaysian strain which was of the stx1- stx2- eae+ genotype. All 19 O157:H7 strains possessing the stx2 gene produced little or no Stx2 (reversed passive latex agglutination titer ≤ 4). Of the 19 strains, five Malaysian (38.5%) and two Thai (33.3%) strains exhibited resistance to a set of antibiotics. Finally, the results of two DNA fingerprinting analyses (O157 IS-printing targeted to IS629 and pulsed-field gel electrophoresis, PFGE) of the O157:H7 strains possessing the stx2 gene, indicated that the Malaysian and Thai strains are closely related. Therefore, E. coli O157:H7 might be transferred from Malaysia to southern Thailand through beef trade. [ABSTRACT FROM AUTHOR]

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.

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

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.

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., (© 2012 Sermwittayawong et al; licensee BioMed Central Ltd.)

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 .

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.

Infection by the pandemic clone of Vibrio parahaemolyticus is prevalent in southern Thailand. This study actively surveyed the incidence of V. parahaemolyticus infection in this area. A total of 865 isolates of V. parahaemolyticus was obtained from patients at Hat Yai Hospital, the main public hospital in Songkhla Province, Thailand, from 2000 to 2005. The isolates were examined by group-specific PCR (GS-PCR) specific for the pandemic clone, and for the presence of two major virulence genes, tdh and trh, and the O : K serotype. Representative isolates were also examined by antibiogram pattern and DNA fingerprinting using an arbitrarily primed PCR method to determine the clonal relationships between isolates. The total number of isolates was less in 2000 and more in 2004 and 2005 than in the years 2001-2003. The increase in the numbers of infections in 2004 and 2005 was not due to the emergence of a particular clone having unique characteristics, but was probably due to climate change. From 2000 to 2003, the percentages of pandemic strains of V. parahaemolyticus, defined as GS-PCR-positive tdh(+) trh(-), was stable at 64.1, 67.5, 69.7 and 67.7 % of the total isolates each year, respectively. However, in 2004 and 2005, the percentages decreased to 56.1 and 55.5 %, respectively. The O : K serotypes of the pandemic isolates remained unchanged. The proportional decrease in infections caused by the pandemic strains are probably due to the population in this area gradually developing immunity to the pandemic clone whilst continuing to be susceptible to other strains.

A total of 493 stool samples from diarrheal patients in Songklanagarind Hospital, in southern Thailand, were examined for Escherichia coli O157 by the culture method combined with an immunomagnetic separation (IMS) technique. E. coli O157 was not found, although the IMS-based method could detect 10(2)-10(3) CFU of artificially inoculated O157/g of stool samples. Polymerase chain reaction was also used for the detection and identification of diarrheagenic E coli from 530 stool samples. The target genes were eae for enteropathogenic E. coli (EPEC), stx for enterohemorrhagic E. coli (EHEC), elt and est for enterotoxigenic E. coli (ETEC), ipaH for enteroinvasive E. coli (EIEC), and aggR for enteroaggregative E. coli (EAggEC). Fifty-eight diarrheagenic E. coli strains were detected in 55 stool samples (10%) from 32 children and 23 adults. These included 31 EAggEC strains (5.8%), 13 ETEC strains (2.5%), 13 EPEC strains (2.5%), and one EIEC strain (0.2%). EHEC was not detected. The diarrheagenic E. coli strains were found mainly in children under 2 years of age (24 of 32 children). EAggEC strains and ETEC strains were susceptible to several antibiotics whereas the EPEC strains exhibited resistance to these antibiotics.

Infections by strains belonging to the O3:K6 pandemic clone of Vibrio parahaemolyticus are prevalent in southern Thailand, and serovariants of these strains have also been detected. V. parahaemolyticus strains lacking important virulence genes (tdh and trh) were isolated from 6.5 to 10.9% of clinical specimens during the period from 2000 to 2003. In order to understand whether changes to the characteristics of V. parahaemolyticus occur during infection, 10 isolates collected from each of 63 patients who presented with diarrhea at the Hat Yai hospital from 2003 to 2004 were examined for the presence of the tdh and trh genes, the O:K serotype, and genetic markers for the pandemic clone. A total of 42 patients (66.7%) yielded identical isolates (homogeneous populations), and 21 of the patients (33.3%) yielded isolates that differed in at least one character from the other isolates (heterogeneous populations). The DNA fingerprints (examined by arbitrarily primed PCR and pulsed-field gel electrophoresis) of some, but not all, of the heterogeneous populations from single patients were indistinguishable. The results indicated that some patients were infected with a unique strain and that in vivo changes (tdh deletion or serotype conversion) might have occurred in certain individuals. It is therefore important to bear in mind that epidemiological studies based on the analysis of a single colony from a single patient might lead to misleading conclusions. Finally, the present study did not rule out the possibility that isolates lacking tdh and trh have unknown virulence mechanisms other than the tdh and trh genes.

Distribution of pandemic strains of Vibrio parahaemolyticus in seafood, particularly in molluscan shellfish, and their serological and molecular relationships to clinical strains were examined from Hat Yai City in southern Thailand. During 2000 to 2002, virulent strains (tdh+ or trh+) were isolated from 13 of 230 molluscan shellfish samples using alkaline peptone water enrichment followed by immunomagnetic separation. The isolates included 12 pandemic strains (tdh+, trh-, group-specific PCR positive) from five Oriental hard clam samples, five green mussel samples, and one bloody clam sample. Among the pandemic strains, eight belonged to serogroup O3:K6, three belonged to O1:K25, and one was O1:K untypeable. One hundred eighty-seven strains of V. parahaemolyticus were isolated from clinical specimens obtained from a hospital in this city during 2000 to 2001. The pandemic strains comprised 64 and 68% of the isolates in 2000 and 2001, respectively. Among the serotypes of the pandemic strains, O3:K6 was dominant at 73% in 2000 and 76% in 2001 followed by O1:K25 at 20% in 2000 and 13% in 2001. Pulsed-field gel electrophoresis profiles of the pandemic strains from molluscan shellfish were indistinguishable or very similar to those of patient isolates. Similarity of the serotype distribution and DNA fingerprints occurring between the molluscan shellfish strains and clinical strains suggests that molluscan shellfish may be an important source of pandemic V. parahaemolyticus infection in southern Thailand. For public health, proper cooking of molluscan shellfish in this area is strongly recommended.

We collected diarrhoea specimens in two hospitals in southern Thailand in 1999 to examine whether infection by the Vibrio parahaemolyticus pandemic clone is prevalent. V. parahaemolyticus was isolated from 317 specimens. Seventy-six per cent of the isolated strains had the pandemic clone-specific characteristics (tdh+, trh-, and an unique toxRS sequence detectable by GS-PCR) and an associated characteristic (the ORF8 sequence of f237 phage). These strains belonged to the three pandemic servovars with the O3:K6 strains being dominant and three other serovars (O1:K25, O1:K41 and O4:K12). We also found O1:K25 and O1:K41 strains with the pandemic clone-specific characteristics among the strains isolated from the international travellers who left Thailand and three other Asian countries between 1998 and 1999, verifying pandemic potential of these strains. The results demonstrate prevalence of infection by the pandemic clone in southern Thailand and suggest emergence of various serovariants in this area and their implication in international spread.

An unusually high incidence of Vibrio cholerae O1 infection was observed in southern Thailand between late December 1997 and March 1998. Fifty-seven V. cholerae O1 strains were isolated in five provinces during this epidemic and were examined. They were El Tor Ogawa strains exhibiting similar antibiograms. All strains were resistant to tetracycline, which had not been reported in Thailand since 1993. The ribotypes. hybridization patterns with ctx and zot gene probes, arbitrarily primed PCR profiles, and pulsed-field gel electrophoresis profiles of the representative strains were compared with the clinical strains isolated from patients in India and Bangladesh in 1997 and 1998 and from international travellers originating from various Asian countries during the 1992-8 period. All southern Thailand strains and the 1998 international traveller strain of Thai origin showed indistinguishable genetic fingerprinting patterns that were distinct from those of other test strains. The results suggest that a tetracycline-resistant clone newly emerged in late December 1997 caused the large epidemic in southern Thailand and that the variants with a slightly different antibiogram appeared during the course of the spreading epidemic.

Active surveillance of Vibrio parahaemolyticus infection among hospitalized patients in Calcutta, India, showed the appearance of the O4:K68 serovar for the first time in March 1998 alongside the continued predominant incidence of the O3:K6 serovar. Strains belonging to both these serovars have been reported to possess pandemic potential. The genomes of O3:K6 and O4:K68 strains and for comparison, non-O3:K6 and non-O4:K68 strains isolated from two different countries, India and Thailand, were examined by different molecular techniques to determine their relatedness. The O3:K6 and O4:K68 strains from Calcutta and Bangkok carried the tdh gene but not the trh gene. Characterization of representative strains of these two serovars by ribotyping and by arbitrarily primed-polymerase chain reaction (AP-PCR) showed that the isolates had identical ribotype and DNA fingerprint. Pulsed-field gel electrophoresis (PFGE) performed with the same set of strains yielded nearly similar restriction fragment length polymorphism (RFLP) patterns for the O3:K6 and O4:K68 isolates from Calcutta and Thailand. Phylogenetic analysis of the NotI RFLP showed that the O3:K6 and O4:K68 strains formed a cluster with 78-91% similarity thus indicating close genetic relationship between the two different serovars isolated during the same time-frame but from widely separated geographical regions. The non-O3:K6 and non-O4:K68, in contrast, showed different ribotype, AP-PCR and PFGE patterns.

Application of an immunomagnetic enrichment method selective for Vibrio parahaemolyticus serovar K6 allowed isolation of a strain belonging to the pandemic O3:K6 clone of V. parahaemolyticus from fresh shellfish not implicated in a clinical case in southern Thailand. Arbitrarily primed PCR profiles of this strain, clinical O3:K6 strains isolated from sporadic diarrhea cases in the same area, and a standard pandemic O3:K6 strain were indistinguishable.

Antibody to Escherichia coli O157 lipopolysaccharide was detected in the sera of healthy individuals more frequently in Southern Thailand than in Japan. The result suggested possible exposure of Thai people to E. coli O157. E. coli O157:H7 or O157:H(-) was isolated from four of 95 retail beef and one of 55 bovine feces samples collected in Southern Thailand by enrichment culture followed by immunomagnetic bead separation. Four of the five strains carried the stx(2) gene alone or in combination with the stx(1) gene. The strains were shown to be genetically distinct by an arbitrarily primed PCR method.