Pathogenic potential of Vibrio parahaemolyticus isolated from tropical estuarine environments in Ceará, Brazil

Vibrio parahaemolyticus is a potentially pathogenic bacterium that occurs naturally in estuarine environments worldwide. This research aimed to investigate the occurrence of V. parahaemolyticus in estuarine environments and determine the virulence profile in an aquaculture environment by molecular techniques and conventional microbiological methods. Sampling was conducted in four estuaries in the State of Ceará (Pacoti, Choró, Pirangi and Jaguaribe), Brazil, between January and April 2009. The analysis included 64 samples of water (n=32) and sediment (n=32) collected from the estuaries. The samples yielded 64 isolates suspected to be V. parahaemolyticus. The isolates were submitted to biochemical identification using a dichotomous key and PCR for the detection of the species-specific tlh gene. Virulence was assessed by testing for urea hydrolysis and ß-hemolysis in erythrocytes (Kanagawa phenomenon) and simultaneous detection of the tdh and trh genes. All but one of the isolates (63/64) were confirmed to be V. parahaemolyticus by genotypic detection of tlh gene. The tdhand trh genes were detected in 57 and 19 isolates, respectively. The Kanagawa test was positive for 51 isolates. Only one isolate was positive for urease. The incidence of tdh/trh-positivity was very high in isolates recovered from the environment. The present study demonstrates the need to increase knowledge of the ecology and pathogeny of V. parahaemolyticus

Virulence gene detection and multi-locus sequence typing of Vibrio parahaemolyticus from patients in Ningbo,China / 中国人兽共患病学报

To investigated the toxin genes distribution and molecular characteristics of Vibrio parahaemolyticus from pa‐tients in Ningbo ,V .parahaemolyticus strains were collected from patients with food poisoning and diarrhea .Thermostable di‐rect hemolysin gene (tdh) and TDH‐related hemolysin gene (trh) were detected by polymerase chain reaction (PCR) .Molecu‐lar characteristics were acquired by multi‐locus sequence typing (MLST ) .Of 248 clinical strains were isolated from 2006 to 2012 .Forty‐eight strains were selected to detect virulence genes and MLST genotyping .Forty‐two isolates were detected as tdh+ and 11 isolates were detected as trh+ .There were 9 STs and one undifferentiated type in Ningbo clinical strains .Thirty‐two strains were classified into ST3 ,5 strains into ST265 and 3 strains into ST120 .ST265 was found in Ningbo strains com‐pared with strains from other regions of China .Strains with tdh+ accounted for the majority in Ningbo clinical strains .Twen‐ty‐five strains of ST3 clone were tdh+ /trh‐.There were 9 STs coexsited in Ningbo clinical strains .ST3 clone was dominant , followed by ST265 and ST120 .Strains with tdh+ /trh‐were dominant in the ST3 clone .The unique ST262 was found in Ning‐bo clinical strains .

Characterization of Vibrio Parahaemolyticus isolated from oysters and mussels in São Paulo, Brazil / Caracterização de Vibrio parahaemolyticus isolados de ostras e mexilhões em São Paulo, Brasil

Vibrio parahaemolyticus is a marine bacterium, responsible for gastroenteritis in humans. Most of the clinical isolates produce thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) encoded by tdh and trh genes respectively. In this study, twenty-three V. parahaemolyticus, previously isolated from oysters and mussels were analyzed by PCR using specific primers for the 16S rRNA and virulence genes (tdh, trh and tlh) and for resistance to different classes of antibiotics and PFGE. Nineteen isolates were confirmed by PCR as V. parahaemolyticus. The tlh gene was present in 100 percent of isolates, the tdh gene was identified in two (10.5 percent) isolates, whereas the gene trh was not detected. Each isolate was resistant to at least one of the nine antimicrobials tested. Additionally, all isolates possessed the blaTEM-116 gene. The presence of this gene in V. parahaemolyticus indicates the possibility of spreading this gene in the environment. Atypical strains of V. parahaemolyticus were also detected in this study.

Vibrio parahaemolyticus é uma bactéria marinha, responsável por gastroenterite em humanos. A maioria dos isolados clínicos produzem hemolisina termoestável direta (TDH) e hemolisina TDH-relacionada (TRH) codificadas por genes tdh e trh, respectivamente. Neste estudo, vinte e três V. parahaemolyticus, previamente isolados de ostras e mexilhões foram analisados por PCR utilizando indicadores específicos para o gene 16S rRNA, genes de virulência (tdh, trh e tlh), resistência a diferentes classes de antibióticos, e PFGE. Dezenove isolados foram confirmados por PCR, como V. parahaemolyticus. O gene tlh estava presente em 100 por cento dos isolados, o gene tdh foi identificado em dois (10,5 por cento) dos isolados, enquanto que o gene trh não foi detectado. Cada isolado foi resistente a pelo menos um dos nove antibióticos testados. Além disso, todos os isolados apresentaram resultado positivo para o gene blaTEM-116. A presença deste gene em V. parahaemolyticus indica a possibilidade de propagação desse gene no ambiente. Cepas atípicas de V. parahaemolyticus foram também detectadas neste estudo.

The effects of heat and osmotic stress on vibrio parahaemolyticus survival rates, cellular morphology and TDH production.

Vibrio parahaemolyticus is an emerging enteric pathogen often associated with the consumption of improperly cooked seafood. The major virulence factor of this pathogen is represented by the termostable direct hemolysin (TDH). When entering the host organism, the vibrion has to face a series of environmental changes (such as temperature and osmolarity) which determine various cellular responses among which the heat shock response has an important role. The purpose of this study was to analyze the survival profiles, the morphological changes and the TDH production in V. parahaemolyticus strains grown under sub-lethal heat and osmotic stress. Our study has demonstrated the thermolotolerance in V. parahaemolyticus strains and the absence of cross-protection between the osmotic and heat stress. We have also observed that the osmotic stress induced important changes to the bacterial cell morphology. The production of the thermostable direct hemolysin, the major virulence factor in V. parahaemolyticus, was increased in cells remained viable after thermal inactivation at 47oC, these results demonstrating the risk of virulence conservation in insufficiently heat treated food.

Prevalence and Molecular Genetic Characteristics of Vibrio parahaemolyticus New 03:K6 and 04:K68 / 병원감염관리

BACKGROUND: Vibrio parahaemolyticus belonged to the new 03:K6 serotype was demonstrated an unusual potential to spread and an enhanced propensity to cause infection in the worldwide. Recently, increased numbers of V. parahaemolyticus 03:K6 had been isolated in Korea, and we analyzed the prevalence and molecular genetic characteristics of V. parahaemolyticus 03:K6 isolates. METHODS: V. parahaemolyticus were isolated from clinical specimens of patients with diarrhea in Hanyang University Hospital, Seoul, Korea, from 1998 to 2005. The serovars of isolates were determined by slide agglutination test with specific antisera. All isolates were examined for the presence of tdh/trh genes. AGS-PCR method detecting the new 03:K6 clone was used in this study. We analyzed clonality of these isolates by infrequent restriction site-polymerase chain reaction (IRS-PCR). RESULTS: Thirty-six strains were isolated from 1998 to 2005. The main serotype of isolates were 03:K6 (24/36: 67%), All of them have tdh gene but not trh gene and an unique toxRS gene of the new 03:K6. The morphotypes of 03:K6 isolates show a same pattern of IRS-PCR, but can easily be differentiated from non-03:K6 and 04:K68 isolates. CONCLUSION: The 03:K6 was a main serotype of V. parahaemolyticus isolated from 1998 to 2005 and they show same molecular characteristics.

A Study of Virulence Factors and Pathogenicity of Vibrio parahaemolyticus Isolated from Diarrheal Patients and Environmental Sources in Gyeongbuk Province / 감염과화학요법

BACKGROUND: V. parahaemolyticus, an important seafood-borne pathogen, is the causative agent of gastroenteritis in humans. In this study, the occurrence and distribution of the thermostable hemolysin (tdh) gene and the tdh-related hemolysin (trh) gene were investigated, and as many as 72 different O:K serovar combinations were determined from diarrheal patients and environmental sources in Gyeongbuk provinceas during the 2000-2001. METHODS: A total of 50 strains of V. parahaemolyticus isolated from diarrheal patients and environmental sources were analyzed for the tdh gene and trh gene. The strains were analyzed for kanagawa phenomenon (KP) with the wagatsuma blood agar medium test. Furthermore, the strains belonging to O and K serotypes were determined. DNA sequence determination of the tdh gene were verified using GenBank and analyzed. RESULTS: In the KP experiments of V. parahaemolyticus, 80% of the strains isolated from diarrheal patients showed positive, but all of the strains isolated from environment sources were negative The distribution of O and K serotypes were O3:K29, O3:K6, and O3:K31, atc. isolated from diarrheal patients, and O2:K28, O3:K29, and 04:K34 atc. isolated from environmental sources. As for the tdh and trh gene of V. parahaemolyticus, the tdh gene was detected from 88.0% of diarrheal patients and 16.0% of environmental sources. The trh gene was not detected from diarrheal patients and 8.0 % of environmental sources, whereas all urease positive strains possessed the trh gene. The DNA sequence of tdh gene were verified using GenBank and analyzed the difference, tdh nucleotide sequence was found among the V. parahaemolyticus. CONCLUSION: V. parahaemolyticus is distributed with high frequency in the environmental sources such as sea water, fishes and shellfishes sampled at the Gyeongbuk coastal area, and the O3:K6 strains producing TDH were confirmed, which is prevalent throughout the world, from diarrheal patients. Thus, it is very important to establish a measure to prevent food poisoning by V. parahaemolyticus.

A Study of Virulence Factors and Pathogenicity of Vibrio parahaemolyticus Isolated from Diarrheal Patients and Environmental Sources in Gyeongbuk Province / 감염과화학요법

BACKGROUND: V. parahaemolyticus, an important seafood-borne pathogen, is the causative agent of gastroenteritis in humans. In this study, the occurrence and distribution of the thermostable hemolysin (tdh) gene and the tdh-related hemolysin (trh) gene were investigated, and as many as 72 different O:K serovar combinations were determined from diarrheal patients and environmental sources in Gyeongbuk provinceas during the 2000-2001. METHODS: A total of 50 strains of V. parahaemolyticus isolated from diarrheal patients and environmental sources were analyzed for the tdh gene and trh gene. The strains were analyzed for kanagawa phenomenon (KP) with the wagatsuma blood agar medium test. Furthermore, the strains belonging to O and K serotypes were determined. DNA sequence determination of the tdh gene were verified using GenBank and analyzed. RESULTS: In the KP experiments of V. parahaemolyticus, 80% of the strains isolated from diarrheal patients showed positive, but all of the strains isolated from environment sources were negative The distribution of O and K serotypes were O3:K29, O3:K6, and O3:K31, atc. isolated from diarrheal patients, and O2:K28, O3:K29, and 04:K34 atc. isolated from environmental sources. As for the tdh and trh gene of V. parahaemolyticus, the tdh gene was detected from 88.0% of diarrheal patients and 16.0% of environmental sources. The trh gene was not detected from diarrheal patients and 8.0 % of environmental sources, whereas all urease positive strains possessed the trh gene. The DNA sequence of tdh gene were verified using GenBank and analyzed the difference, tdh nucleotide sequence was found among the V. parahaemolyticus. CONCLUSION: V. parahaemolyticus is distributed with high frequency in the environmental sources such as sea water, fishes and shellfishes sampled at the Gyeongbuk coastal area, and the O3:K6 strains producing TDH were confirmed, which is prevalent throughout the world, from diarrheal patients. Thus, it is very important to establish a measure to prevent food poisoning by V. parahaemolyticus.

Virulence Factors and Genotyping of Vibrio parahaemolyticus in Sea Water and Clinical Isolates

Six strains of Vibrio parahemolyticus isolated from diarrheal patients and the 12 strains from sea water were serotyped and analyzed for biochemical characteristics, antibiotics sensitivity and detection of toxR, gyrB, tdh, and trh genes. Arbitrarily-primed polymerase chain reaction method were performed on the 6 strains from patients and the following results were obtained. 1. The Vibrio parahemolyticus isolated from patients were belonged to 5 different serotypes: 04:K8, 04:KUT, 06: K18, 010:K71 and 03:K6, but those isolated from sea water were belonged to 5 different serotypes: O1:KUT, 02:KUT, 03:K45, 04:K37 and OUT:KUT. All strains explained have different serotypes depending on the different source, 2. Three serotype (04:K8, 04:KUT, 06:K18) isolated from patients were positive for the urease hydrolysis, whereas only one strain of serotype O1:KUT isolated from sea water was positive to the same. Furthermore, the serotype 06:K18 (1 strain) was positive for the fermentation of dulcito1. Both toxR and gyrB genes were detected from all strains isolated. 3. As for control the 2 strains of serotype 03:K6 and 6 strains isolated from patients, serotype 03:K6 were resistant to oxacillin, penicillin, vancomycin. All strains were sensitive to chloramphenicol and tetracycline yet the antibiogram type showed 6 groups from I to VI. 4. DNA probe hybridization method was used to detect genes. The trh1 was detected both from serotype 04:KUT and 06:K18 isolated from patients and the trh2 was also detected from one strain from each 010:K71 and O1:KUT isolated from patients and sea-water respectively, The tdh gene only was detected from two strains of 03:K6 isolated from patients of 1998. The tdh, trh 1 and trh2 were not detected from 7 strains out of 12 strains isolated from sea water whereas the production titer of TDH isolated from patients showed from 2048 times to 4096 times. 5. Four strains of the serotype 03:K6 isolated from Korea, India and Japan as well as 3 strains from Korean patients were tested by AP-PCR to classify serotypes. As for its result the amplicon showed the same in the 4 strains of the serotype 03:K6 whereas the four strains of different serotype from patients are so difference as to explain no inter- relations at all. The result explains that the serotype 03:K6 is the same genes regardless from where it is isolated.

Detection of Thermostable Direct Hemolysin Gene and Thermostable Direct Hemolysin-Related Hemolysin Gene of Vibrio parahaemolyticus by Polymerase Chain Reaction / 대한임상병리학회지

BACKGROUND: Vibrio parahaemolyticus is one of the most important food-borne pathogens in Korea. Although the mechanism of its pathologic effect is still not clearly understood, epidemiological studies have suggested a very strong association of thermostable direct hemolysin (TDH) and thermostable direct hemolysin-related hemolysin (TRH) with the disease. We detected the tdh gene and the trh gene of V. parahaemolyticus isolates by polymerase chain reaction (PCR). METHODS: V. parahaemolyticus strains were isolated from clinical specimens of patients with diarrhea in different geographic areas of Seoul (16 cases), Inchon (27 cases) and Kwang Joo (2 cases) in Korea between 1998 and 2000. The colonies selected were identified by using the API 20E identification strip (API system, Analytab Products, Montalieu-Vercieu, France). PCR protocols were established for specific detection of the tdh and trh genes. Oligonucleotide primers were designed based on the reported nucleotide sequence of the tdh2 gene and the trh1 gene, respectively. RESULTS: The protocols established for the tdh and trh genes could detect 400 fg (100 colony-forming units) of cellular DNA carrying the respective gene. All strains of V. parahaemolyticus isolates in this study contained the tdh gene but not the trh gene. CONCLUSIONS: The detection of the tdh gene and trh gene of V. parahaemolyticus using the PCR is an easy and rapid method.