Detection and genotyping of Helicobacter pylori in saliva versus stool samples from asymptomatic individuals in Northeastern Thailand reveals intra-host tissue-specific H. pylori subtypes.

BACKGROUND: Two-thirds of the world's population is thought to be infected by Helicobacter pylori. Although most people infected with H. pylori are asymptomatic, this pathogen is associated with several gastric pathologies including cancer. The risk factors for colonization are still unclear and the genetic diversity within individual hosts has never been clearly investigated. RESULT: This study determined the prevalence of, and explored risk factors for, H. pylori infection directly from paired saliva (n = 110) and stool (n = 110) samples from asymptomatic persons in Northeast Thailand. Samples were subjected to indirect immunofluorescence assay (IFA), 16S rRNA-based real-time PCR and vacA-based semi-nested PCR. Partial vacA gene sequences of H. pylori were compared between saliva and stool samples. The overall prevalence of H. pylori infection in our asymptomatic study population was 64%. Age, gender, occupation and frequency of brushing teeth were not found to be associated with H. pylori colonization. The vacA gene was successfully sequenced from both saliva and stool samples of 12 individuals. For seven of these individuals, saliva and stool sequences fell into different clusters on a phylogenetic tree, indicating intra-host genetic variation of H. pylori. CONCLUSION: This study reports a high prevalence of H. pylori infection in asymptomatic persons in this region of Thailand and demonstrates that genotypes (vacA gene sequences) of H. pylori may differ between the oral cavity and intestinal tract.

Antimicrobial resistance and genetic diversity of the SXT element in Vibrio cholerae from clinical and environmental water samples in northeastern Thailand.

Multidrug resistance in V. cholerae has been increasing around the world including northeastern Thailand. The aquatic environment is a reservoir of V. cholerae and might be an important source of resistant strains. The aims of this study were to investigate the phylogenetic relationships of intSXT gene sequences from 31 clinical and 14 environmental V. cholerae O1 and non-O1/non-O139 isolates and 11 sequences amplified directly from environmental water samples. We also amplified class 1 integrons, the SXT elements (targeting the intSXT gene) and antimicrobial resistance genes directly from water samples. Phylogenetic analysis displayed two major distinct clusters (clusters 1 and 2). Most V. cholerae O1 (19/20, 95%) and non-O1/non-O139 isolates (8/11, 72.7%) from clinical sources, and all sequences obtained directly from water samples, belonged to cluster 1. Cluster 2 mostly comprised environmental non-O1/non-O139 isolates (10/12, 83.3%). We successfully amplified the SXT elements directly from17.5% of water samples. Associated resistance genes were also amplified as follows: sul2 (41.3% of water samples), dfrA1 (60%), dfr18 (33.8%), strB (70%) and tetA (2.5%). Class 1 integrons were not found in water samples, indicating that the SXT element was the major contributor of multidrug resistance determinants in this region. The SXT element and antimicrobial resistance genes could be transferred from clinical V. cholerae O1 to environmental V. cholerae non-O1/non-O139 was demonstrated by conjugation experiment. These findings indicate that there may have been cross dissemination and horizontal gene transfer (HGT) of the SXT element harbored by V. cholerae O1 and non-O1/non-O139 strains isolated from clinical and environmental water sources. Environmental water might be an important source of antimicrobial resistance genes in V. cholerae in this region. Direct detection of antimicrobial resistance genes in water samples can be used for monitoring the spread of such genes in the ecosystem.

Genetic characterization of Helicobacter pylori vacA and cagA genes in Thai gastro-duodenal and hepatobiliary patients.

INTRODUCTION: H. pylori has been detected in patients with hepatobiliary diseases. It is currently unclear whether the H. pylori detected in hepatobiliary patients are genetically similar to those in gastro-duodenal patients. The aim of this study was to determine H. pylori vacA and cagA genotypes in Thai patients with gastro-duodenal and hepatobiliary diseases. METHODOLOGY: H. pylori DNA was extracted from samples from gastric biopsies of gastro-duodenal patients (n=100) and from bile samples of hepatobiliary patients (n=80). The vacA and cagA genotypes were performed via polymerase chain reaction (PCR) followed by DNA sequencing. RESULTS: The vacA m1 was found in Thai hepatobiliary patients (90%) at a higher rate compared with gastro-duodenal patients (50%).The combined vacA s1a+c/m1 were mostly found in Thai gastro-duodenal and hepatobiliary patients. The cagA gene was detected in 94% of patients with gastro-duodenal diseases compared with 28.8% in those with hepatobiliary diseases (p<0.05). On the other hand, the Western type cagA was more prominent among hepatobiliary patients (100%) than gastro-duodenal patients (57.4%), and this type was grouped into same cluster with Thai gastro-duodenal patients via phylogenetic analysis. CONCLUSIONS: Based on vacA and cagA analysis, we conclude that infection with H. pylori in gastro-duodenal and hepatobiliary patients may be caused by the different H. pylori strains.

Coinfection with Helicobacter pylori and Opisthorchis viverrini Enhances the Severity of Hepatobiliary Abnormalities in Hamsters.

Persistent infection with Opisthorchis viverrini causes hepatobiliary abnormalities, predisposing infected individuals to cholangiocarcinoma (CCA). In addition, Helicobacter pylori is highly prevalent in most countries and is a possible risk factor for CCA; however, its role in enhancing hepatobiliary abnormality is unclear. Here, we investigated the effects of coinfection with H. pylori and O. viverrini on hepatobiliary abnormality. Hamsters were divided into four groups: (i) normal, (ii) H. pylori infected (HP), (iii) O. viverrini infected (OV), and (iv) O. viverrini and H. pylori infected (OV+HP). At 6 months postinfection, PCR and immunohistochemistry were used to test for the presence of H. pylori in the stomach, gallbladder, and liver. In the liver, H. pylori was detected in the following order: OV+HP, 5 of 8 (62.5%); HP, 2 of 5 (40%); OV, 2 of 8 (25%). H. pylori was not detected in normal (control) liver tissues. Coinfection induced the most severe hepatobiliary abnormalities, including periductal fibrosis, cholangitis, and bile duct hyperplasia, leading to a significantly decreased survival rate of experimental animals. The greatest thickness of periductal fibrosis was associated with a significant increase in fibrogenesis markers (expression of alpha smooth muscle actin and transforming growth factor beta). Quantitative reverse transcription-PCR revealed that the highest expression levels of genes for proinflammatory cytokines (interleukin-1 [IL-1], IL-6, and tumor necrosis factor alpha) were also observed in the OV+HP group. These results suggest that coinfection with H. pylori and O. viverrini increased the severity of hepatobiliary abnormalities to a greater extent than either single infection did.

Chronic Opisthorchis viverrini Infection Changes the Liver Microbiome and Promotes Helicobacter Growth.

Adults of Opisthorchis viverrini reside in the biliary system, inducing inflammation of bile ducts and cholangitis, leading to hepatobiliary disease (HBD) including cholangiocarcinoma. O. viverrini infection also has major implications for the bacterial community in bile ducts and liver. To investigate this in chronic O. viverrini infection (≥ 8 months p.i.), bacterial genomic DNA from livers of hamsters and from worms was investigated using culture techniques, PCR for Helicobacter spp. and high-throughput next-generation sequencing targeting the V3-V4 hypervariable regions of prokaryotic 16S rRNA gene. Of a total of 855,046 DNA sequence reads, 417,953 were useable after filtering. Metagenomic analyses assigned these to 93 operational taxonomic units (OTUs) consisting of 80 OTUs of bacteria, including 6 phyla and 42 genera. In the chronic O. viverrini-infected group, bacterial community composition and diversity were significantly increased compared to controls. Sequences of Fusobacterium spp. were the most common (13.81%), followed by Streptococcus luteciae (10.76%), Escherichia coli (10.18%), and Bifidobacterium spp. (0.58%). In addition, Helicobacter pylori (0.17% of sequences) was also identified in the liver of chronic O. viverrini infections, but not in normal liver. The presence of H. pylori was confirmed by PCR and by use of an antibody against bacterial antigen, supporting the metagenomics data. The identities of bacteria cultured for enrichment suggested that chronic O. viverrini infection changes the liver microbiome and promotes Helicobacter spp. growth. There may be synergy between O. viverrini and the liver microbiome in enhancing immune response-mediated hepatobiliary diseases.

Serogroup, virulence, and molecular traits of Vibrio parahaemolyticus isolated from clinical and cockle sources in northeastern Thailand.

Vibrio parahaemolyticus is responsible for seafood-borne gastroenteritis worldwide. Isolates of V. parahaemolyticus from clinical samples (n=74) and cockles (Anadara granosa) (n=74) in Thailand were analyzed by serotyping, determination of virulence and related marker genes present, response to antimicrobial agents, and genetic relatedness. Serological analysis revealed 31 different serotypes, 10 of which occurred among both clinical and cockle samples. The clinical isolates commonly included the pandemic serogroup O3:K6, while a few of the cockle isolates exhibited likely pandemic serovariants such as O3:KUT and O4:KUT, but not O3:K6. The pandemic (orf8 gene-positive) strains were more frequently found among clinical isolates (78.4%) than cockle isolates (28.4%) (p<0.001). Likewise, the virulence and related marker genes were more commonly detected among clinical than cockle isolates; i.e., tdh gene (93.2% versus 29.7%), vcrD2 (97.3% versus 23.0%), vopB2 (89.2% versus 13.5%), vopT (98.6% versus 36.5%) (all p<0.001) and trh (10.8% versus 1.4%) (p<0.05). Pulsed-field gel electrophoresis of NotI-digested genomic DNA of 41 randomly selected V. parahaemolyticus isolates representing different serotypes produced 33 pulsotypes that formed 5 different clusters (clonal complexes) (A-E) in a dendrogram. Vibrio parahaemolyticus O3:K6 and likely related pandemic serotypes were especially common among the numerous clinical isolates in cluster C, suggesting a close clonal link among many of these isolates. Most clinical and cockle isolates were resistant to ampicillin. This study indicates that O3:K6 and its likely serovariants based on the PFGE clusters, are causative agents. Seafoods such as cockles potentially serve as a source of virulent V. parahaemolyticus, but further work is required to identify possible additional sources.

SXT ELEMENT, CLASS 1 INTEGRON AND MULTIDRUG-RESISTANCE GENES OF VIBRIO CHOLERAE ISOLATED FROM CLINICAL AND ENVIRONMENTAL SOURCES IN NORTHEAST THAILAND.

Emergence of multiple drug resistance in Vibrio cholerae has been increasing around the world including Northeast Thailand. In this study, 92 isolates of V. cholerae (50 O1 and 42 non-O1/non-O139 isolates) from clinical and environmental sources in Northeast Thailand were randomly selected and investigated for the presence of SXT element, class 1 integron and antimicrobial resistance genes. Genotypic-phenotypic concordance of antimicrobial resistance was also determined. Using PCR-based assays, 79% of V. cholerae isolates were positive for SXT element, whereas only 1% was positive for class 1 integron. SXT element harbored antimicrobial resistance genes, dfrA1 or dfr18, floR, strB, sul2, and tetA. Overall phenotypic-genotypic concordance of antimicrobial resistance was 78%, with highest and lowest value being for trimethoprim (83%) and chloramphenicol (70%), respectively. Ninety-two percent of V. cholerae O1 strains isolated from clinical sources harbored both dfrA1 (O1-specific trimethoprim resistance gene) and dfr18 (non-O1-specific trimethoprim resistance gene), whereas only 5% of V. cholerae non-O1/non-O139 strains harbored both genes. All V. cholerae O1 isolated from environmental source harbored dfr18 but 48% of V. cholerae non-O1/non-O139 harbored dfrA1. This study indicates that SXT element was the main contributor to the circulation of multiple-drug resistance determinants in V. cholerae strains in Northeast Thailand and that genetic exchange of SXT element can occur in both V. cholerae O1 and non-O1/non-O139 strains from clinical and environmental sources.

DETECTION OF HELICOBACTER PYLORI AND VIRULENCE-ASSOCIATED GENES IN SALIVA SAMPLES OF ASYMPTOMATIC PERSONS IN NORTHEAST THAILAND.

The aims of the study were to develop nested-PCR (targeting vacA and cagA), SYBR green quantitative PCR (targeting 16S rDNA) tests and compared them with indirect fluorescent-monoclonal antibody (IFA) method for determination of the prevalence of Helicobacter pylori in 118 saliva samples from asymptomatic individuals in Khon Kaen, Thailand. Detection limit of both PCR-based assays was one cell. Prevalence of H. pylori in saliva samples was 55% based on the criterion of positivity of IFA test and one of the PCR-based methods or positivity of both PCR assays. Forty-nine percent of H. pylori detected carried cagA, encoding a cytotoxin associated with severe clinical outcomes. These results imply that the mouth may be an important reservoir for H. pylori, with nearly 50% of the virulent type that could possibly lead to gastroduodenal disease.

PHENOTYPIC AND GENOTYPIC DETECTION OF ENTEROTOXINS, TOXIC SHOCK SYNDROME TOXIN-1 AND OF METHICILLIN RESISTANCE IN STAPHYLOCOCCUS AUREUS ISOLATED FROM RETAIL READY-TO-EAT FOODS IN NORTHEASTERN THAILAND.

Toxigenic Staphylococcus aureus contamination of ready-to-eat (RTE) foods is a leading cause of foodborne illness in Thailand. From 151 RTE food samples randomly collected from food vendors and food shops in Khon Kaen municipality, Thailand and after culture-based identification of S. aureus isolates, pentaplex PCR was used for simultaneous detection of super-antigenic toxin (SE) genes (sea, seb, sec, sed and tst-1) and presence of their toxins by reversed passive latex agglutination assay. S. aureus was identified in 57 isolates, of which 60% and 25% was positive for presence of super-antigenic toxin genes and toxins, respectively; and among the former isolates sea was the most common (46%), as well as its product (SEA) (14%) among the latter group. Methicillin resistance S. aureus mecA was not found in any of the isolates using both PCR and disk diffusion methods. These results showed that pentaplex PCR is a useful tool for detection of SE-encoding genes in S. aureus isolates from RTE food.

The carcinogenic liver fluke Opisthorchis viverrini is a reservoir for species of Helicobacter.

There has been a strong, positive correlation between opisthorchiasis-associated cholangiocarcinoma and infection with Helicobacter. Here a rodent model of human infection with Opisthorchis viverrini was utilized to further investigate relationships of apparent co-infections with O. viverrini and H. pylori. A total of 150 hamsters were assigned to five groups: i) Control hamsters not infected with O. viverrini; ii) O. viverrini-infected hamsters; iii) non-O. viverrini infected hamsters treated with antibiotics (ABx); iv) O. viverrini-infected hamsters treated with ABx; and v) O. viverrini-infected hamsters treated both with ABx and praziquantel (PZQ). Stomach, gallbladder, liver, colonic tissue, colorectal feces and O. viverrini worms were collected and the presence of species of Helicobacter determined by PCR-based approaches. In addition, O. viverrini worms were cultured in vitro with and without ABx for four weeks, after which the presence of Helicobacter spp. was determined. In situ localization of H. pylori and Helicobacter-like species was performed using a combination of histochemistry and immunohistochemistry. The prevalence of H. pylori infection in O. viverrini-infected hamsters was significantly higher than that of O. viverrini-uninfected hamsters (p≤0.001). Interestingly, O. viverrini-infected hamsters treated with ABx and PZQ (to remove the flukes) had a significantly lower frequency of H. pylori than either O. viverrini- infected hamsters treated only with ABx or O. viverrini-infected hamsters, respectively (p≤0.001). Quantitative RT-PCR strongly confirmed the correlation between intensity H. pylori infection and the presence of liver fluke infection. In vitro, H. pylori could be detected in the O. viverrini worms cultured with ABx over four weeks. In situ localization revealed H. pylori and other Helicobacter-like bacteria in worm gut. The findings indicate that the liver fluke O. viverrini in the biliary tree of the hamsters harbors H. pylori and Helicobacter-like bacteria. Accordingly, the association between O. viverrini and H. pylori may be an obligatory mutualism.

Triplex reverse transcription-PCR for detecting viable toxigenic Vibrio cholerae in water samples in Thailand.

Abstract. Detection of toxigenic Vibrio cholerae O1/O139 in aquatic environment is difficult to achieve using the culture method. For direct detection of viable toxigenic V. cholerae in aquatic environment, we developed a triplex reverse transcription (RT)-PCR, targeting genes for the outer membrane protein (ompW), cholera toxin A (ctxA) and toxin-coregulated pilli (tcpA) and compared the assay with the culture method. After enrichment of the bacteria-containing filters in alkaline peptone water for 6 hours, the sensitivity of triplex RT-PCR for detecting V. cholerae was 7 cfu/ml. Of the 80 environmental water samples collected from various regions in Northeast Thailand, triplex RT-PCR detected 15 toxigenic and 20 non-toxigenic V. cholerae, whereas the culture method detected only 3 toxigenic V. cholerae--containing water samples. These results show that this triplex RT-PCR method could be used as an alternative tool for rapid and sensitive identification of viable toxigenic V cholerae in environmental water samples.

Molecular analysis of Vibrio vulnificus isolated from cockles and patients in Thailand.

Vibrio vulnificus can cause septicemia, wound infection and gastroenteritis. The most severe infections are related to consumption of raw or undercooked seafood. Virulence genes, biomarkers, antimicrobial resistance, and genetic relationships among V vulnificus isolated from clinical and environmental sources in Thailand have not hitherto been investigated. ViuB encoding vulnibactin siderophore was detected in 33% and 50% of clinical and environmental (cockle) V. vulnificus isolates, respectively, and capsular polysaccharide allele 1 in 67% and 75% of clinical and environmental isolates, respectively. Analysis of the 16 S rDNA gene revealed that type B was the most frequent in both clinical and environmental isolates (67%) whereas the non type-able (30%) was detected only in environmental isolates. The virulence-correlated gene (vcg) with both type C and E together was the most frequently found among the clinical (67%) and environmental (72%) isolates. Pulsed-field gel electrophoresis differentiated V vulnificus into 2 clusters; most cockle samples (83%) and all clinical isolates grouped into cluster II, indicating a possible clonal relationship between V. vulnificus isolated from patients and cockles. Only 20% of environmental isolates were resistant to ampicillin. These studies suggest that V vulnificus isolated from cockles has virulence genes similar to those in clinical isolates and thus may have the potential of causing disease.

GENOTYPE AND DRUG RESISTANCE OF CLINICAL AND ENVIRONMENTAL VIBRIO CHOLERAE NON-O1/NON-O139 IN NORTHEASTERN THAILAND.

A total of 124 V cholerae non-O1/non-O139 isolates were collected in Khon Kaen, Thailand from diarrheal patients, asymptomatic carriers and environmental water. The presence of virulence-associated and regulatory genes including ctxA, tcpA, zot, ace, ompU, stn, hlyA and toxR) were examined using multiplex PCR. The genomic diversity of the various V. cholerae isolates were differentiated using the random amplified polymorphic DNA (RAPD) method. Antimicrobial susceptibility was tested using disk diffusion. All of V. cholerae non-O/non-O139 isolates carried hlyA and toxR and none carried ctxA and tcpA. The zot, ace and both genes together were found in 1.6%, 4.7% and 4.7% of 64 clinical V. cholerae non-O1 isolates, respectively, while the environmental ones did not. The stn gene was found in 3.1% (2/64) of the clinical and 3.3% (2/60) of the environmental isolates. The RAPD patterns were differentiated into 45 types (A to 2S). RAPD type A (32.3%) was the most frequently found in both clinical and environmental V cholerae non-O1 strains (34.4% and 30.0%, respectively); indicating that there was a clonal relationship between some clinical and environmental isolates whereas almost all of the environmental isolates belonged to different clones. All strains were sensitive to ciprofloxacin and norfloxacin. The environmental isolates (30%) were more resistant than the clinical ones (21.9%). Resistance to sulfamethoxazole/trimethoprim and tetracycline among the clinical isolates occurred in 9.4% (6/64) in 2007, during which period the prevalence of V cholerae O1 increased. We conclude that V. cholerae non-O1/non-O139 from the aquatic environment are potentially pathogenic and this same aquatic environment may be a source of antimicrobial resistance in V. cholerae.

ROLE OF HLYA-POSITIVE VIBRIO CHOLERAE NON-O1/NON-O139 ON APOPTOSIS AND CYTOTOXICITY IN A CHINESE HAMSTER OVARY CELL LINE.

Vibrio cholerae non-O1/non-O139 is capable of producing sporadic outbreaks of cholera-like diarrhea; however, the pathogenic mechanisms of this bacterium remain unclear. The objectives of this study were to: 1) compare the apoptosis induction and cytotoxicity between hlyA-positive and hlyA-negative strains of V. cholerae non-O1/non-O139; 2) clarify the molecular mechanisms by which these strains induce apoptosis; and 3) compare clinical and environmental V. cholerae non-O1/non-O139 isolates with respect to cytotoxicity and ability to induce apoptosis. Using cytotoxicity and apoptosis assays, it was shown that hlyA-positive strains of V. cholerae non-O1/non-O139 had significantly higher cytotoxic activity (70.6%) and levels of apoptosis induction (59.6%) than hlyA- negative strains (37.0% and 37.5%, respectively). Western blot analyses revealed that hlyA-positive strains had significantly increased expression of Bax; active caspase-3 and -9; and significantly decreased expression of NF-κB and Bcl-2 relative to hlyA-negative strains. Expression of BID did not differ significantly between hlyA-positive and negative strains. The truncated BID was not found, indicating that V. cholerae non-O1/non-O139 induces apoptosis through a mitochondria- dependent apoptosis pathway and not an extrinsic pathway. V. cholerae non-O1/ non-O139 isolated from clinical sources exhibited significantly higher cytotoxic activity (79%) and levels of apoptosis induction (65.2%) than bacteria isolated from environmental sources (63% and 54.6%, respectively), suggesting that the clini- cal isolates may have other virulence-associated genes besides hlyA. Our results indicate that hlyA products play a role in cytotoxicity and apoptosis induction and that a mitochondria-dependent apoptosis pathway is involved.

Synergistic effects of isomorellin and forbesione with doxorubicin on apoptosis induction in human cholangiocarcinoma cell lines.

BACKGROUND: Chemotherapy for advanced cholangiocarcinoma (CCA) is largely ineffective, but innovative combinations of chemotherapeutic agents and natural compounds represent a promising strategy. In our previous studies, isomorellin and forbesione, caged xanthones isolated from Garcinia hanburyi, were found to induce cell cycle arrest and apoptosis in CCA cell lines. The subject of our inquiry is the synergistic effect(s) of these caged xanthones with doxorubicin on growth inhibition and apoptosis induction in human CCA cell lines. METHODS: KKU-100, KKU-M139 and KKU-M156 cell lines and Chang cells were treated with either isomorellin or forbesione alone or in combination with doxorubicin. Cell viability was determined using the sulforhodamine B assay. The combined effects of plant compounds with doxorubicin were analyzed using the isobologram and combination index method of Chou-Talalay. Apoptosis was determined by ethidium bromide/acridine orange staining. Protein expressions were determined by Western blot analysis. RESULTS: Isomorellin or forbesione alone inhibited the growth of these CCA cell lines in a dose-dependent manner and showed selective cytotoxicity against CCA cells but not against Chang cells. Isomorellin/doxorubicin combination showed a synergistic growth inhibitory effect on KKU-M139 and KKU-M156 cells, while the forbesione/doxorubicin combination showed a synergistic growth inhibitory effect on KKU-100 and KKU-M139 cells. The percentages of apoptotic cells were significantly higher in the combined treatments than in the respective single drug treatments. The combined treatments strongly enhanced the expression of Bax/Bcl-2, activated caspase-9 and caspase-3, while suppressing the expression of survivin, procaspase-9 and procaspase-3, compared with single drug treatments. The degree of suppression of NF-κB activation mediated by a decrease in the expression of NF-κB/p65, a reduction of the pIκB-α level and an increase in the IκB-α protein level, was significantly higher in the combined treatment groups than in the single drug treatment groups. The degree of suppression of MRP1 protein expression was also significantly higher in the combined treatment than in the single drug treatment groups. CONCLUSION: The combinations of isomorellin/doxorubicin and forbesione/doxorubicin showed significant synergistic effects on the growth inhibition and apoptosis induction in KKU-M156 and KKU-100 cells. Caged xanthones may be useful adjunct treatments with chemotherapy for Opisthorchis viverrini (OV)-associated CCA.

Helicobacter pylori cag pathogenicity island (cagPAI) involved in bacterial internalization and IL-8 induced responses via NOD1- and MyD88-dependent mechanisms in human biliary epithelial cells.

Helicobacter pylori infection has been proposed to be associated with various diseases of the hepatobiliary tract, including cancer of the bile duct epithelial cells (cholangiocarcinoma, CCA). The ability of H. pylori bacteria to cause pathogenic effects in these cells has, however, yet to be investigated. Given that the cag pathogenicity island (cagPAI) is required for H. pylori pathogenesis in gastric epithelial cells, we investigated wild-type and cag mutant strains for their ability to adhere, be internalized and induce pro-inflammatory responses in two bile duct epithelial cell lines derived from cases of CCA. The findings from these experiments were compared to results obtained with the well-characterized AGS gastric cancer cell line. We showed that the cagPAI encodes factors involved in H. pylori internalization in CCA cells, but not for adhesion to these cells. Consistent with previous studies in hepatocytes, actin polymerization and α5ß1 integrin may be involved in H. pylori internalization in CCA cells. As for AGS cells, we observed significantly reduced levels of NF-κB activation and IL-8 production in CCA cells stimulated with either cagA, cagL or cagPAI bacteria, when compared with wild-type bacteria. Importantly, these IL-8 responses could be inhibited via either pre-treatment of cells with antibodies to α5ß1 integrins, or via siRNA-mediated knockdown of the innate immune signaling molecules, nucleotide oligomerization domain 1 (NOD1) and myeloid differentiation response gene 88 (MyD88). Taken together, the data demonstrate that the cagPAI is critical for H. pylori pathogenesis in bile duct cells, thus providing a potential causal link for H. pylori in biliary tract disease.

Multiplex PCR for detection of superantigenic toxin genes in methicillin-sensitive and methicillin-resistant Staphylococcus aureus isolated from patients and carriers of a hospital in northeast Thailand.

The aims of this study were to develop multiplex PCR for simultaneous detection of five superantigenic toxin genes (sea, seb, sec, sed and tst-1) in Staphylococcus aureus isolated from 149 clinical samples and nasal swabs from 201 healthy subjects in Thailand, and to compare prevalence and expression of those genes between methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA). The sensitivity of multiplex PCR was 10(3) CFU/ml (60 CFU/PCR reaction) for DNA templates extracted by both boiling and extraction methods. S. aureus strains from patients (65%) harbored more superantigenic toxin genes than healthy subjects (54%). MRSA (80%) isolated from patients harbored more superantigenic toxin genes than MSSA (52%). Sea was the most frequently found gene in S. aureus strains from patients and carriers. MRSAisolates harbored sea and produced SEA more frequently than MSSA isolates (p <0.05) and MRSA isolates (59%) from blood samples consisted of a higher number of superantigenic toxin producers than MSSA (9%) (p < 0.05). More S. aureus strains isolated from patients with severe septicemia contained superantigenic toxin genes (94%) and produced toxins (82%) than those from non-severe patients (64% and 57%, respectively). The multiplex PCR method described here offers a reliable tool for simultaneous detection of various staphylococcal toxin genes.

Application of tetraplex PCR for detection of Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus in cockle.

A tetraplex PCR method was developed for simultaneous detection of Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus in cockle samples in comparison with conventional culture method. Specific primers targeting ompW of V. cholerae, tl of V. parahaemolyticus, hsp60 of V. vulnificus and sodB of V. mimicus were employed in the same PCR. Detection limit of the tetraplex PCR assay was 104 cfu/ml (400 cfu/PCR reaction) for pure cultures of all four species of Vibrio. In Vibrio spiked cockle samples, the limit of detection after 6 hours enrichment in alkaline peptone water was 1 cfu/10 g of cockle tissue for three Vibrio spp, except for V. mimicus that was 102 cfu/10 g of cockle tissue. When the tetraplex PCR and culture methods were applied to 100 cockle samples, V. parahaemolyticus, V. vulnificus, V. cholerae and V. mimicus were detected in 100, 98, 80 and 9% of the samples by tetraplex PCR and in 76, 42, 0 and 0% by the culture method, respectively. This developed tetraplex PCR method should be suitable for simultaneous and rapid detection of Vibrio species in food samples and for food safety assessment.

Drug response and genetic properties of Vibrio cholerae associated with endemic cholera in north-eastern Thailand, 2003-2011.

Cholera, caused by Vibrio cholerae, results in significant morbidity and mortality worldwide, including Thailand. Representative V. cholerae strains associated with endemic cholera (n = 32), including strains (n = 3) from surface water sources, in Khon Kaen, Thailand (2003-2011), were subjected to microbiological, molecular and phylogenetic analyses. According to phenotypic and related genetic data, all tested V. cholerae strains belonged to serogroup O1, biotype El Tor (ET), Inaba (IN) or Ogawa (OG). All of the strains were sensitive to gentamicin and ciprofloxacin, while multidrug-resistant (MDR) strains showing resistance to erythromycin, tetracycline, trimethoprim/sulfamethoxazole and ampicillin were predominant in 2007. V. cholerae strains isolated before and after 2007 were non-MDR. All except six diarrhoeal strains possessed ctxA and ctxB genes and were toxigenic altered ET, confirmed by MAMA-PCR and DNA sequencing. Year-wise data revealed that V. cholerae INET strains isolated between 2003 and 2004, plus one strain isolated in 2007, lacked the RS1 sequence (rstC) and toxin-linked cryptic plasmid (TLC)-specific genetic marker, but possessed CTX(CL) prophage genes ctxB(CL) and rstR(CL). A sharp genetic transition was noted, namely the majority of V. cholerae strains in 2007 and all in 2010 and 2011 were not repressor genotype rstR(CL) but instead were rstR(ET), and all ctx(+) strains possessed RS1 and TLC-specific genetic markers. DNA sequencing data revealed that strains isolated since 2007 had a mutation in the tcpA gene at amino acid position 64 (N→S). Four clonal types, mostly of environmental origin, including subtypes, reflected genetic diversity, while distinct signatures were observed for clonally related, altered ET from Thailand, Vietnam and Bangladesh, confirmed by distinct subclustering patterns observed in the PFGE (NotI)-based dendrogram, suggesting that endemic cholera is caused by V. cholerae indigenous to Khon Kaen.

Duplex PCR for detection of Salmonella and Shigella spp in cockle samples.

Salmonella and Shigella spp are important causative agents of foodborne diseases. A sensitive, specific and rapid method is essential for detection of these pathogens. In this study, a duplex PCR method was developed for simultaneous detection of Salmonella and Shigella spp in cockle samples and compared with the traditional culture method. Enrichment broths for Salmonella spp recovery were also compared. Sensitivity of the duplex PCR for simultaneous detection of Salmonella and Shigella spp from pure culture was 10(3) CFU/ml (40 CFU/PCR reaction), and that of sterile cockle samples spiked with these two pathogens was 1 CFU/10 g of cockle tissue after 9 hours enrichment [3 hours in buffered peptone water (BPW), followed by 6 hours in Rappaport Vasiliadis (RV) broth or tetrathionate (TT) broth for Salmonella spp and 6 hours enrichment in Shigella broth (SB) for Shigella spp]. There was no significant difference in detection sensitivity between enrichment in RV and TT broths. Salmonella spp detected in cockles in Khon Kaen, Thailand by duplex PCR and culture method was 17% and 13%, respectively but Shigella spp was not detected. The duplex PCR technique developed for simultaneous detection of Salmonella and Shigella spp in cockle samples was highly sensitive, specific and rapid and could serve as a suitable method for food safety assessment.