Wagatsuma Agar Research Articles

Detection of tlh and tdh genes in Vibrio Parahaemolyticus inhabiting farmed water ecosystem used for L. Vannamei aquaculture

Vibrio parahaemolyticus is hallophilic gram-negative bacteria that live as natural inhabitant in aquatic environment. All Vibrio parahaemolyticus strain known to have thermolabile hemolysin encoded by tlh gene as species marker. Thermostable direct hemolysin encoded by tdh gene is responsible for regulating virulence factor in Vibrio parhaemolyticus. Aim of this research is to detect tlh and tdh gene from water of L. vannamei aquaculture in Rembang regency. Colonies of green-blueish bacteria grew from isolation of L. vannamei aquaculture water in CD-VP media which was identified as Vibrio parahaemolyticus. Colonies of V. parahaemolyticus grew to be small and green-blueish bacteria colonies in TCBS agar. Result of molecular analysis showed that bacteria isolated from water sample are specifically identified as Vibrio parahaemolyticus bacteria by the detection of tlh gene. Vibrio parahaemolyticus isolated from water of L. vannamei aquaculture detected as tdh negative that indicates tdh gene is not present in isolated bacteria. Vibrio parahaemolyticus isolate were cultured in Wagatsuma agar for tdh gene confirmation test that showed Kanagawa negative result, which indicated that V. parahaemolyticus did not produce thermostable direct hemolysin. These results showed that Vibrio parahaemolyticus isolated from aquatic environment of L. vannamei aquaculture in Rembang regency did not show virulence factors.

Vibrio parahaemolyticus CalR down regulates the thermostable direct hemolysin (TDH) gene transcription and thereby inhibits hemolytic activity

TDH, encoded by tdh gene, is a major virulent determinant of V. parahaemolyticus that controls various biological activities, such as hemolytic activity, cytotoxicity, and enterotoxicity. The hemolytic activity on Wagatsuma agar ascribed to TDH is called Kanagawa phenomenon (KP). All KP positive strains contain tdh1 and tdh2 genes, but tdh2 is predominantly responsible for KP. CalR is a regulatory protein that was originally identified as a repressor of swarming motility and T3SS1 gene expression in V. parahaemolyticus. In the present study, the regulation of tdh2 by CalR was investigated using a set of experiments including qRT-PCR, primer extension, LacZ fusion, hemolytic phenotype, EMSA, and DNase I footprinting assays. The results showed that His-CalR protected a single region from 224bp to 318bp upstream of tdh2 against DNase I digestion, and a transcriptional start site located at 42bp upstream of tdh2 was detected and its transcribed activity was inhibited by CalR. Moreover, the KP test results showed that the hemolytic activity of V. parahaemolyticus is also under negative control of CalR. The data demonstrated that CalR is a repressor of the tdh2 transcription and thereby inhibits the hemolytic activity of V. parahaemolyticus.

Prevalence of urease in Vibrio parahaemolyticus from the Mississippi Sound

Vibrio parahaemolyticus is a pathogenic marine bacterium that causes food-borne gastroenteritis and, less commonly, wound infections. As is the case for many pathogens, all V. parahaemolyticus strains possess at least one erythrocyte-lysing haemolysin. In addition, many V. parahaemolyticus also possess the enzyme urease. We tested 206 environmental V. parahaemolyticus isolates from Mississippi coastal waters for urease and haemolytic activity using urea agar with added salt and Wagatsuma agar, respectively. The relative abundance of haemolysin-producing V. parahaemolyticus was consistently high throughout the sampling period. In contrast, the number of urease-positive organisms increased from 36% in 2006 to 80% in 2007 and 97% in 2009. We then tested the ability of four strains representing each of the three sample years along with seven other bacterial strains for their ability to grow in seawater urea and raise the pH of this seawater broth. Finally, one of the 4 strains was tested for its ability to form an alkaline microhabitat immediately above its biofilm. The results of this study illustrate that V. parahaemolyticus has the ability to create alkaline microhabitats that could enhance virulence, including virulence from haemolysins. This finding could have both clinical and ecological impact as to how V. parahaemolyticus can modify its habitat.

Isolation and identification of Vibrio parahaemolyticus from seawater and sediment samples in the southern coast of the Caspian Sea

The objectives of this study were to investigate the occurrence of Vibrio parahaemolyticus in the seawater and its sediment by molecular techniques and conventional microbiological methods. Of 300 samples analyzed, 20.3 % was recorded positive for V. parahaemolyticus. Of the 62 strains isolated, 26 (8.3 %) were obtained from the seawater samples, and 36 (12 %); from sediments. Only three strains (4.83 %) showed hemolytic activity in Wagatsuma agar. The results of this study demonstrated the presence of V. parahaemolyticus in the southern coast of the Caspian Sea (Northern Iran). Furthermore, the PCR approach proved useful for reliable confirmation of species identification. V. parahaemolyticus is an important human pathogen responsible for food-borne gastroenteritis worldwide. These findings indicated the potential sanitary risk associated with the presence of pathogenic V. parahaemolyticus in the Caspian Sea.

Kanagawa-Negative, tdh- and trh-Positive Vibrio parahaemolyticus Isolated from Fresh Oysters Marketed in Fortaleza, Brazil

Between October 2008 and June 2009, 15 samples of 10 live oysters each (Crassostrea rhizophorae) measuring 8.31-10.71cm were purchased from a restaurant on the seashore of Fortaleza, Brazil. The Vibrio count ranged from 75 (estimated) to 43,500CFU/g. Fourteen species were identified among the 56 isolated Vibrio strains, with V. parahaemolyticus as the most prevalent. Two of the 17V. parahaemolyticus strains were urease-positive and tdh- and trh-positive on multiplex PCR, but neither produced β-hemolysis halos in Wagatsuma agar. Thus, fresh oysters served in natura in Fortaleza, Brazil, were found to contain Vibrio strains known to cause gastroenteritis in humans.

Comparative evaluation of a chromogenic agar medium – PCR protocol with a conventional method for isolation of Vibrio parahaemolyticus strains from environmental and clinical samples

Screening for pathogenic Vibrio parahaemolyticus has become routine in certain areas associated with food-borne outbreaks. This study is an evaluation of the CHROMagar Vibrio (CV) medium-PCR protocol and the conventional method (TCBS (thiosulfate-citrate-bile salts-sucrose) agar plus biochemical and Wagatsuma agar tests) for detection of V. parahaemolyticus in shrimp, water, sediment, and stool samples collected for biosurveillance in an endemic area of northwestern Mexico. A total of 131 environmental and clinical samples were evaluated. The CV medium-PCR protocol showed a significantly improved ability (P < 0.05) to isolate and detect V. parahaemolyticus, identifying isolates of this bacteria missed by the conventional method. Although some other bacteria, distinct from pathogenic V. parahaemolyticus, produced violet colonies similar to that of V. parahaemolyticus on CV medium, we were able to detect a superior number of samples of V. parahaemolyticus with the CV medium-PCR protocol than with the conventional method. The Kanagawa phenomenon is routinely determined on Wagatsuma agar for the diagnosis of V. parahaemolyticus (pathogenic) positive for thermostable direct hemolysin (TDH) in developing countries. In our results, Wagatsuma agar showed low sensitivity (65.4% at 24 h and 75.6% at 48 h) and specificity (52.4% at 48 h) for identifying V. parahaemolyticus positive for TDH. Overall, our data support the use of the CV medium-PCR protocol in place of the conventional method (TCBS-biochemical tests-Wagatsuma agar) for detection of pathogenic V. parahaemolyticus, both in terms of effectiveness and cost efficiency.

High potential of adhesion to abiotic and biotic materials in fish aquaculture facility byVibrio alginolyticusstrains

The ability of Vibrio alginolyticus strains isolated from Sparus aurata and Dicentrarchus labrax nursery to adhere to epithelial cell lines (Hep-2 and Caco-2), fish mucus and their ability to form a biofilm on different surfaces (glass, polystyrene, polyethylene and polyvinyl-chloride) was investigated in this study. The extracellular products were rich in enzymes and the strains were haemolytic on Wagatsuma agar and possessed several hydrolytic exoenzymes such as proteases, DNase and lipases. Most strains tested were multiresistant to the 17 antibiotics tested including those used in the farm to treat vibriosis. These bacteria were able to form a biofilm on all the surfaces tested and the cell density was the highest on the PVC surface followed by that on the glass slides, polystyrene and the polyethylene surface. More than 50% of the tested strains were adhesive to the epithelial cell lines (Hep-2 and Caco-2). These properties allow these bacteria to survive, proliferate and persist in all stages of fish rearing nursery even after seawater treatment with UV light.

Isolation and characterization of Vibrio parahaemolyticus from seafoods along the southwest coast of India

The work was aimed to study the microbial quality of the seafood sold in the domestic markets and incidence of Vibrio parahaemolyticus. Samples comprising of shellfish, finfish, and cephalopods were collected from various fish markets in and around Cochin. Presumed V. parahaemolyticus were identified by standard biochemical tests, and further confirmed by polymerase chain reaction targeting species-specific tl gene (450 bp). About 81% of the samples were found to exceed the limits specified for total plate count while total presumptive V. parahaemolyticus count was above the limit in 71% of the samples ranging from 5.5 × 105 to 9.7 × 107 and 0.31 × 102 to 7.8 × 106 cfu/g, respectively. Pathogenicity of the identified isolates was confirmed by Kanagawa phenomenon and urease activity. A total of 10% of the isolates exhibited weak haemolysis on Wagatsuma agar, and 1% of the isolates showed urease activity using Christensen’s urea agar. Random amplified polymorphic DNA analysis revealed two major clusters based on the species rather than seasonality. The gel pattern revealed 8–10 bands ranging from 0.45 to 3.0 kb. Antibiogram results revealed 85% of the strains sensitive to chloramphenicol and nitrofurantoin. Multiple antibiotic resistance index was found to be 0.4 thus suggesting the risk potential involved in consuming seafoods. The present study has clearly demonstrated the need to adopt seafood safety measures for the products meant for human consumption.

Comparative Genomic Analysis Using Microarray Demonstrates a Strong Correlation between the Presence of the 80-Kilobase Pathogenicity Island and Pathogenicity in Kanagawa Phenomenon-Positive Vibrio parahaemolyticus Strains

Vibrio parahaemolyticus is a gram-negative marine bacterium. A limited population of the organisms causes acute gastroenteritis in humans. Almost all of the clinical V. parahaemolyticus isolates exhibit beta-type hemolysis on Wagatsuma agar, known as the Kanagawa phenomenon (KP). KP is induced by the thermostable direct hemolysin produced by the organism and has been considered a crucial marker to distinguish pathogenic strains from nonpathogenic ones. Since 1996, so-called "pandemic clones," the majority of which belong to serotype O3:K6, have caused worldwide outbreaks of gastroenteritis. In this study, we used a DNA microarray constructed based on the genome sequence of a pandemic V. parahaemolyticus strain, RIMD2210633, to examine the genomic composition of 22 strains of V. parahaemolyticus, including both pathogenic (pandemic and nonpandemic) and nonpathogenic strains. More than 86% of the RIMD2210633 genes were conserved in all of the strains tested. Many variably present genes formed gene clusters on the genome of RIMD2210633 and were probably acquired through lateral gene transfer. At least 65 genes over 11 loci were specifically present in the pandemic strains compared with any of the nonpandemic strains, suggesting that the difference between pandemic and nonpandemic strains is not due to a simple genetic event. Only the genes in the 80-kb pathogenicity island (Vp-PAI) on chromosome II, including two tdh genes and a set of genes for the type III secretion system, were detected only in the KP-positive pathogenic strains. These results strongly suggest that acquisition of this Vp-PAI was crucial for the emergence of V. parahaemolyticus strains that are pathogenic for humans.

Pandemic strains of O3:K6Vibrio parahaemolyticusin the aquatic environment of Bangladesh

A total of 1500 environmental strains of Vibrio parahaemolyticus, isolated from the aquatic environment of Bangladesh, were screened for the presence of a major V. parahaemolyticus virulence factor, the thermostable direct haemolysin (tdh) gene, by the colony blot hybridization method using a digoxigenin-labeled tdh gene probe. Of 1500 strains, 5 carried the tdh sequence, which was further confirmed by PCR using primers specific for the tdh gene. Examination by PCR confirmed that the 5 strains were V. parahaemolyticus and lacked the thermostable direct haemolysin-related haemolysin (trh) gene, the alternative major virulence gene known to be absent in pandemic strains. All 5 strains gave positive Kanagawa phenomenon reaction with characteristic beta-haemolysis on Wagatsuma agar medium. Southern blot analysis of the HindIII-digested chromosomal DNA demonstrated, in all 5 strains, the presence of 2 tdh genes common to strains positive for Kanagawa phenomenon. However, the 5 strains were found to belong to 3 different serotypes (O3:K29, O4:K37, and O3:K6). The 2 with pandemic serotype O3:K6 gave positive results in group-specific PCR and ORF8 PCR assays, characteristics unique to the pandemic clone. Clonal variations among the 5 isolates were analyzed by comparing RAPD and ribotyping patterns. Results showed different patterns for the 3 serotypes, but the pattern was identical among the O3:K6 strains. This is the first report on the isolation of pandemic O3:K6 strains of V. parahaemolyticus from the aquatic environment of Bangladesh.

Levels of thermostable direct hemolysin production by Vibrio parahaemolyticus strains carrying both tdh and trh genes

One hundred and twenty-five strains of Vibrio parahaemolyticus carrying both the tdh and trh genes were selected from the strains isolated from the travelers with diarrhea by an hybridization test using polynucleotide probes. The levels of TDH produced by these strains and the association between the TDH levels and related characteristics in these strains were analyzed. The TDH level varied greatly from strain to strain, but none of the levels was as high as that of the typical Kanagawa phenomenon-positive strains. The strains were classified into "TDH producer" (18 strains), "Low-level TDH producer" (85 strains), and "No TDH producer" (22 strains) based on the results of a modified Elek test and the hemolysis assay on Wagatsuma agar. The highest TDH level achieved by the "TDH producer" was twofold lower than that of the Kanagawa phenomenon-positive strains as assayed by the RPLA method. All strains possessed the toxR gene. The trh1 and trh2 genes were detected in, respectively, 105 and 20 strains, and no correlation existed between the type of the trh gene and the levels of TDH produced. Considerable restriction fragment length polymorphism was observed with the tdh gene-bearing HindIII DNA fragment in different strains, but it was not related with the TDH level.

Studies on adhesion, haemagglutination and other biological properties of Vibrio cholerae O139

The adhesive capabilities of eight Vibrio cholerae O139 epidemic strains to isolated rabbit intestinal epithelial cells (RIEC) were observed to be high similar to those observed with a Vibrio cholerae O1 strain isolated from patients. Toxin production by the strains, measured by accumulation of fluid in rabbit ileal loop model, was high and the toxin was lethal as the animal expired within 6 h. Culture filtrates of the strains exhibited the presence of vascular permeability factor which produce induration and necrosis in the adult rabbit and guinea pig skin. All the strains showed high to moderate haemagglutinin titres against chicken erythrocytes and produced El Tor-like haemolysin. SDS-PAGE of the outer membrane preparation of the strains showed the presence of major protein component at 38 kDa region. The lethality of the toxin, high adhesive activity, shifting of the major outer membrane protein band and production of thermolabile haemolysin on Wagatsuma agar were the major variations of these epidemic strains from V. cholerae O1 and V. cholerae non-O1 strains isolated previously.

Studies on adhesion, haemagglutination and other biological properties ofVibrio choleraeO139

The adhesive capabilities of eight Vibrio cholerae O139 epidemic strains to isolated rabbit intestinal epithelial cells (RIEC) were observed to be high similar to those observed with a Vibrio cholerae O1 strain isolated from patients. Toxin production by the strains, measured by accumulation of fluid in rabbit ileal loop model, was high and the toxin was lethal as the animal expired within 6 h. Culture filtrates of the strains exhibited the presence of vascular permeability factor which produce induration and necrosis in the adult rabbit and guinea pig skin. All the strains showed high to moderate haemagglutinin titres against chicken erythrocytes and produced El Tor-like haemolysin. SDS-PAGE of the outer membrane preparation of the strains showed the presence of major protein component at 38 kDa region. The lethality of the toxin, high adhesive activity, shifting of the major outer membrane protein band and production of thermolabile haemolysin on Wagatsuma agar were the major variations of these epidemic strains from V. cholerae O1 and V. cholerae non-O1 strains isolated previously.

Contribution of the tdh 1 gene of Kanagawa phenomenon-positive Vibrio parahaemolyticus to production of extracellular thermostable direct hemolysin

Kanagawa phenomenon-positive strains of Vibrio parahaemolyticus contain two copies of the tdh gene ( tdh1 and tdh2) encoding thermostable direct hemolysin (TDH). Previous studies suggested that the tdh2 gene, but not the tdh1 gene, was responsible for production of extracellular TDH. In this study, a tdh2-deficient isogenic mutant of Kanagawa phenomenon-positive strain AQ3815 was constructed by a suicide vector-mediated in vivo recombination method. The intact tdh1 gene in the mutant contributed little to Kanagawa phenomenon on Wagatsuma agar but produced TDH in broth media, accounting for 0.5–9.4% of total extracellular TDH of AQ3815.

Urease-positive, Kanagawa-negative Vibrio parahaemolyticus from patients and the environment in the Pacific Northwest.

We previously reported the occurrence of Vibrio parahaemolyticus in patients and the environment in the Pacific Northwest. The present studies compare the biochemical characteristics, Kanagawa hemolysin reactions, and plasmid profiles of 13 patient and 221 environmental isolates of the organism. Classical biochemical testing of the isolates revealed similar reactions for the clinical and environmental strains, and analysis in agarose gels revealed that 13 to 15% of the isolates had plasmids. The strains were tested for production of Kanagawa hemolysin on Wagatsuma agar, and 1.4% of environmental isolates and 23% of clinical isolates were positive. Clinical isolates from locally acquired extraintestinal infections were urease negative and Kanagawa hemolysin negative, isolates from locally acquired gastroenteritis cases were urease positive and Kanagawa negative, and isolates from traveler's diarrhea were urease negative and Kanagawa positive. Eight percent of the local environmental isolates were also urease positive and Kanagawa hemolysin negative. These findings suggest that expression of the Kanagawa hemolysin is not essential for the pathogenesis of V. parahaemolyticus infections. In addition, our findings suggest that V. parahaemolyticus gastroenteritis in the Pacific Northwest is associated with a urease-positive, Kanagawa-negative biotype of the organism.

Hemolytic activity of and lethal toxin production by environmental strains of Vibrio parahaemolyticus.

Repeated subculturing of Kanagawa-negative strains of Vibrio parahaemolyticus on Wagatsuma agar induced the production of a hemolysin which was not the thermostable direct hemolysin. Crude hemolysin exhibited a 30 to 40% lethal toxicity in mice after intraperitoneal injection. A 21-kilodalton protein band was observed with all the environmental isolates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results suggested that a certain percentage of environmental strains of V. parahaemolyticus is responsible for pathogenesis.

Effect of pH on the production of the Kanagawa hemolysin by Vibrio parahaemolyticus.

Production of the Kanagawa hemolysin by patient strains of Vibrio parahaemolyticus was found to respond to the pH rather than to the type of carbohydrate present in the growth medium. Regardless of the carbohydrate present, hemolysin production in peptone broth cultures occurred only when the pH was between 6.5 and 5.5. Mannitol, the sugar used in the Wagatsuma agar, lowered the pH to within this range, thus providing optimal conditions for hemolysin production. Glucose and mannose, although readily metabolized, lowered the pH below this range, inhibiting growth and hemolysin production. Alkaline cultures either without carbohydrates or containing non-metabolizable sugars showed little hemolytic activity because the pH always remained alkaline. In pH-stat cultures maintained at pH 6.2, higher hemolysin yields were produced irrespective of the presence or absence of mannitol. We conclude that the production of the Kanagawa hemolysin is under pH control. Marine strains of V. parahaemolyticus, which are Kanagawa negative, did not express detectable amounts of hemolysin under those conditions shown to stimulate hemolysin production by Kanagawa-positive strains.