Trh Genes Research Articles

Multiplexed real-time PCR amplification of tlh, tdh and trh genes in Vibrio parahaemolyticus and its rapid detection in shellfish and Gulf of Mexico water

In this study, we have developed a SYBR Green I-based real-time multiplexed PCR assay for the detection of Vibrio parahaemolyticus in Gulf of Mexico water (gulf water), artificially seeded and natural oysters targeting three hemolysin genes, tlh, tdh and trh in a single reaction. Post-amplification melt-temperature analysis confirmed the amplification of all three targeted genes with high specificity. The detection sensitivity was 10 cfu (initial inoculum) in 1 ml of gulf water or oyster tissue homogenate, following 5 h enrichment. The results showed 58% of the oysters to be positive for tlh, indicating the presence of V. parahaemolyticus; of which 21% were positive for tdh; and 0.7% for trh, signifying the presence of pathogenic strains. The C(t) values showed that oyster tissue matrix had some level of inhibition, whereas the gulf water had negligible effect on PCR amplification. The assay was rapid (approximately 8 h), specific and sensitive, meeting the ISSC guidelines. Rapid detection using real-time multiplexed PCR will help reduce V. parahaemolyticus-related disease outbreaks, thereby increasing consumer confidence and economic success of the seafood industry.

Clinical isolates of Aeromonas veronii biovar veronii harbor a nonfunctional gene similar to the thermostable direct hemolysin-related hemolysin (trh) gene of Vibrio parahaemolyticus

Thermostable direct hemolysin-related hemolysin encoded by the trh gene is considered a major virulence factor in the pathogenesis of Vibrio parahaemolyticus infections. In this study, we report the presence of a trh homolog in three clinical isolates of Aeromonas veronii biovar veronii. The presence of a trh homolog in these strains of A. veronii was confirmed by PCR, followed by cloning, sequencing and colony hybridization using a digoxigenin-labelled probe. DNA sequence analysis revealed that the A. veronii trh gene had an identity of 99% and 84% to the trh1 and trh2 genes of V. parahaemolyticus, respectively. However, the expression of a trh-like gene in A. veronii could not be detected by reverse transcription PCR. Hence, the role of the gene product in the virulence of A. veronii strains is not clear. Further, these A. veronii isolates were negative for the ure gene encoding urease and the transposase gene by PCR. These genes are part of the trh gene cluster in V. parahaemolyticus. However, the presence of a trh homolog in a pathogen other than V. parahaemolyticus points to the fact that detection of the trh gene in stool samples, seafood enrichments or environmental samples does not always imply that trh-carrying V. parahaemolyticus are present.

Species-specific PCR detection of the food-borne pathogen Vibrio parahaemolyticus using the irgB gene identified by comparative genomic analysis

Vibrio parahaemolyticus is an enteric pathogen, which can cause acute gastroenteritis in humans after consumption of raw or partially cooked seafood, and specific molecular markers are necessary for its accurate identification by PCR methods. In the present study, 23 protein-coding sequences were identified by the comparative genomics method as V. parahaemolyticus-specific candidate markers. We targeted the irgB gene (vp2603), coding for iron-regulated virulence regulatory protein IrgB, in order to develop a PCR method for the detection of V. parahaemolyticus. PCR specificity was identified by amplification of 293 V. parahaemolyticus templates and by the loss of a PCR product with 11 strains from other Vibrio species and 35 non-Vibrio bacterial strains. The PCR assay had the 369-bp fragment and the sensitivity of 0.17 pg purified genomic DNA from V. parahaemolyticus. Furthermore, a multiplex PCR assay for the detection of total and virulent strains of V. parahaemolyticus was developed by targeting irgB, tdh and trh genes. These data indicated that the irgB gene is a new and effective marker for the detection of V. parahaemolyticus. In addition, this study demonstrates that genome sequence comparison has a powerful application in identifying specific markers for the detection and identification of bacterial pathogens.

Isolation, Detection and Genomic Differentiation of Vibrio cholerae and Vibrio parahaemolyticus in Bachok, Kelantan

The objective of this study was to determine the genetic diversity and virulence factors of Vibrio cholerae and V. parahaemolyticus from selected aquatic environments in Bachok, Kelantan, Malaysia. Out of 50 water samples, 33 were confirmed with the presence of presumptive V. cholerae and V. parahaemolyticus based on biochemical tests. Primers targeting toxR gene specific for V. cholerae (800bp) and V. parahaemolyticus (368bp), tdh (251bp) and trh (250bp) virulence genes were used in the PCR. Results of PCR showed that only 25 isolates of V. parahaemolyticus and 3 of V. cholerae had the toxR gene. None of the V. parahaemolyticus isolates had the trh and tdh genes. Repetitive extragenic palindromic PCR (REP-PCR) was used to determine the relatedness of these strains at the genomic level. Cluster analysis for REP-PCR showed 2 major clusters A-B, with several isolates from different locations grouped together indicating the geographical relatedness due to interconnected waterways. REPPCR showed that the species of Vibrios are genetically diverse.

An efficient approach to isolate STAT regulated enhancers uncovers STAT92E fundamental role in Drosophila tracheal development

The ventral veinless (vvl) and trachealess (trh) genes are determinants of the Drosophila trachea. Early in development both genes are independently activated in the tracheal primordia by signals that are ill defined. Mutants blocking JAK/STAT signaling at any level do not form a tracheal tree suggesting that STAT92E may be an upstream transcriptional activator of the early trachea determinants. To test this hypothesis we have searched for STAT92E responsive enhancers activating the expression of vvl and trh in the tracheal primordia. We show that STAT92E regulated enhancers can be rapidly and efficiently isolated by focusing the analysis on genomic regions with clusters of putative STAT binding sites where at least some of them are phylogenetically conserved. Detailed analysis of a vvl early tracheal enhancer shows that non-conserved sites collaborate with conserved sites for enhancer activation. We find that STAT92E regulated enhancers can be located as far 60kb from the promoters. Our results indicate that vvl and trh are independently activated by STAT92E which is the most important transcription factor required for trachea specification.

The TRH Gene Is Regulated by Thyroid Hormone at the Level of Transcription in Vivo

ABSTRACT The expression of the TRH gene in the paraventricular nucleus (PVH) of the hypothalamus is required for the normal production of thyroid hormone (TH) in rodents and humans. In addition, the regulation of TRH mRNA expression by TH, specifically in the PVH, ensures tight control of the set point of the hypothalamic-pituitary-thyroid axis. Although many studies have assumed that the regulation of TRH expression by TH is at the level of transcription, there is little data available to demonstrate this. We used two in vivo model systems to show this. In the first model system, we developed an in situ hybridization (ISH) assay directed against TRH heteronuclear RNA to measure TRH transcription directly in vivo. We show that in the euthyroid state, TRH transcription is present both in the PVH and anterior/lateral hypothalamus. In the hypothyroid state, transcription is activated in the PVH only and can be shut off within 5 h by TH. In the second model system, we employed transgenic mice that express the Cre recombinase under the control of the genomic region containing the TRH gene. Remarkably, TH regulates Cre expression in these mice in the PVH only. Taken together, these data affirm that TH regulates TRH at the level of transcription in the PVH only and that genomic elements surrounding the TRH gene mediate its regulation by T3. Thus, it should be possible to identify the elements within the TRH locus that mediate its regulation by T3 using in vivo approaches.

Foodborne Pathogens in Retail Oysters in South China

To investigate the occurrence of important foodborne pathogens in shellstock Pacific oysters in the food markets in South China. From July 2007 to June 2008, retail oysters were collected in different seasons from South China and analyzed for the prevalence and levels of Listeria monocytogenes, Vibrio vulnificus and Vibrio parahaemolyticus. None of L. monocytogenes could be detected in any of the 202 oyster samples tested, while E vulnificus and V. parahaemolyticus could be detected in 67 (54.9%) and 109 (89.3%) of the 122 oyster samples analyzed, respectively, with an MPN (most probable number) value greater than or equal to 3. V. vulnificus and V. parahaemolyticus with a more than 102 MPN/g were found in 36 (29.5%) and 59 (48.4%) of the 122 oyster samples, respectively. The tdh and trh genes were detected in 4 (0.3%) and 8 (0.6%) of the 1 349 V parahaemolyticus isolates, respectively. Of the 122 samples, 4 (3.3%) was positive for either tdh or trh. The levels of V. vulnificus and total V. parahaemolyticus in oysters in South China varied in different seasons. V. vulnificus and pathogenic V. parahaemolyticus are frequently found in oysters in south China, which may pose a potential threat to public health. Data presented here will be useful for the microbiological risk assessment in oysters in China.

The TRH Gene Is Regulated by Thyroid Hormone at the Level of Transcriptionin Vivo

The TRH Gene Is Regulated by Thyroid Hormone at the Level of Transcription<i>in Vivo</i>

Etiologic and molecular characteristics of Vibrio parahaemolyticus strains isolated from diarrheal patients in Shenzhen, in 2007 - 2008

To study the infection status and the molecular characteristics of Vibrio parahaemolyticus isolated from diarrheal patients in Shenzhen, in 2007 to 2008 and to provide evidence for the prevention and control of diarrheal diseases caused by Vibrio parahaemolyticus. More than 80 fecal specimens from four sentinel surveillance hospitals were collected and cultured each month. A total of 361 isolates of Vibrio parahaemolyticus were sero-typed and examined by real-time PCR for the presence of two major virulence genes, tdh and trh. Of 361 strains, 60 O3: K6 strains isolated from six suspected outbreaks in August, 2007 and in September, 2008 were typed by pulsed-field gel electrophoresis (PFGE). 4384 stool samples were detected in four sentinel surveillance hospitals and with 361 Vibrio parahaemolyticus strains isolated that belonged to 28 serotypes. Serotype O3:K6, O4:K8 and O1:KUT accounted for 67.90%, 7.50% and 6.10%, respectively. Of 361 strains, 337 strains belonged to tdh+trh-, 11 strains were tdh-trh- and 13 strains were tdh+trh+. The most prevalent serotype which caused diarrheal diseases was tdh+trh in Shenzhen. The 60 isolates were discriminated into twenty different PFGE patterns, which belonged to three clones. Among the 60 isolates, most of the PFGE patterns of isolates from the suspected outbreak locations were identical and some strains isolated from different year were different. Vibrio parahaemolyticus isolates in Shenzhen were dominated by O3:K6 strains. Most of these isolates carried tdh gene and few carried trh gene. Meanwhile, the identical patterns of isolates from 6 suspected outbreaks locations demonstrated that Vibrio parahaemolyticus outbreaks occurred in July 2007 and in September 2008 in Shenzhen. However, the dominated strains' PFGE patterns were different each year, indicating that the sources of Vibrio parahaemolyticus had a multiplex nature and the multiplex sources such as water, sea food and pickled products should be integrated monitored. Laboratory based surveillance of diarrheal diseases could contribute in establishing early warning system for the better prevention and control of diarrheal diseases.

The Thyrotropin-Releasing Hormone Gene Is Regulated by Thyroid Hormone at the Level of Transcription in Vivo

The expression of the TRH gene in the paraventricular nucleus (PVH) of the hypothalamus is required for the normal production of thyroid hormone (TH) in rodents and humans. In addition, the regulation of TRH mRNA expression by TH, specifically in the PVH, ensures tight control of the set point of the hypothalamic-pituitary-thyroid axis. Although many studies have assumed that the regulation of TRH expression by TH is at the level of transcription, there is little data available to demonstrate this. We used two in vivo model systems to show this. In the first model system, we developed an in situ hybridization (ISH) assay directed against TRH heteronuclear RNA to measure TRH transcription directly in vivo. We show that in the euthyroid state, TRH transcription is present both in the PVH and anterior/lateral hypothalamus. In the hypothyroid state, transcription is activated in the PVH only and can be shut off within 5 h by TH. In the second model system, we employed transgenic mice that express the Cre recombinase under the control of the genomic region containing the TRH gene. Remarkably, TH regulates Cre expression in these mice in the PVH only. Taken together, these data affirm that TH regulates TRH at the level of transcription in the PVH only and that genomic elements surrounding the TRH gene mediate its regulation by T(3). Thus, it should be possible to identify the elements within the TRH locus that mediate its regulation by T(3) using in vivo approaches.

Serodiversity, Pandemic O3:K6 Clone, Molecular Typing, and Antibiotic Susceptibility of Foodborne and ClinicalVibrio parahaemolyticusIsolates in Jiangsu, China

Vibrio parahaemolyticus is a major foodborne pathogen in China, Japan, and other Asian countries. In this study, we collected 437 strains of V. parahaemolyticus and investigated their serotypes, distribution of virulence genes, and presence of pandemic O3:K6 clone strains. A total of 327 strains were isolated from food and 110 strains were isolated from active surveillance hospitals or food outbreaks during 2005 to 2008. Presence of the tdh and trh genes is the key characteristic of virulent strains. Positive for both the tdh gene and group-specific polymerase chain reaction is the key characteristic of pandemic strains. A total of 9 O serogroups and 62 serovars were identified in all strains. Nine O serogroups and 56 serovars existed in 327 foodborne strains, and 6 O serogroups and 20 serovars existed in 110 clinical strains. Among the 327 food isolates, 6 isolates belonged to the pandemic clone with the orf8 gene (1 isolate was O1:KUT (untyped) and 5 isolates were O3:K6) and 4 isolates carried the trh gene (2 isolates belonged to O1:KUT and 2 isolates belonged to O5:KUT and O5:K17). Seventy-nine percent of the clinical isolates were pandemic strains, 9.4% of which lacked the orf8 gene. O3:K6 was the main serovar of the pandemic strains accounting for 83.5% of the clinical pandemic strains. Pandemic clonal serovars included O3:K6, O1:KUT, O1:K25, O1:K26, and O4:K68, and the newly emerging serovars O1:K36, O3:K25, and O3:K68 identified in the current study. O3:K6 was the dominant serovar in pandemic strains. All pandemic isolates had identical arbitrarily primed polymerase chain reaction fragment patterns, but did not share similar antibiotic sensitivity patterns. These results suggest that high serodiversity of V. parahaemolyticus was present in foodborne strains. Pathogenic isolates, especially pandemic isolates, were present in high-priced iced seafood and became the potential risk factor in food.

Detection of thetdhandtrhgenes inVibrio parahaemolyticusisolates in fish and mussels from Middle Black Sea Coast of Turkey

The aim of this study was to demonstrate the occurrence of potential pathogenic Vibrio parahaemolyticus in seafoods using DNA-based techniques in comparison with bacteriological methods. From 120 fresh and processed fish and mussel samples collected from Middle Black Sea, 32 isolates were identified as V. parahaemolyticus by bacteriological methods and confirmed by tl gene-based conventional PCR. Of them, 13 isolates were found positive for only tdh gene, six isolates for only trh gene and 13 isolates for both genes by multiplex PCR. It is the first report demonstrating the presence of potential pathogenic V. parahaemolyticus isolates from the Black Sea seafoods by PCR detection of tl, trh and tdh genes that was found more rapid than bacteriological methods. This study confirmed the previous reports that characterization of potential pathogenic V. parahaemolyticus isolates based on the PCR techniques was reliable and cost-effective. These results suggest that molecular detection methods should be included in Turkish Standards of seafood control in addition to bacteriological methods.

Serogroup, Virulence, and Genetic Traits of Vibrio parahaemolyticus in the Estuarine Ecosystem of Bangladesh

Forty-two strains of Vibrio parahaemolyticus were isolated from Bay of Bengal estuaries and, with two clinical strains, analyzed for virulence, phenotypic, and molecular traits. Serological analysis indicated O8, O3, O1, and K21 to be the major O and K serogroups, respectively, and O8:K21, O1:KUT, and O3:KUT to be predominant. The K antigen(s) was untypeable, and pandemic serogroup O3:K6 was not detected. The presence of genes toxR and tlh were confirmed by PCR in all but two strains, which also lacked toxR. A total of 18 (41%) strains possessed the virulence gene encoding thermostable direct hemolysin (TDH), and one had the TDH-related hemolysin (trh) gene, but not tdh. Ten (23%) strains exhibited Kanagawa phenomenon that surrogates virulence, of which six, including the two clinical strains, possessed tdh. Of the 18 tdh-positive strains, 17 (94%), including the two clinical strains, had the seromarker O8:K21, one was O9:KUT, and the single trh-positive strain was O1:KUT. None had the group-specific or ORF8 pandemic marker gene. DNA fingerprinting employing pulsed-field gel electrophoresis (PFGE) of SfiI-digested DNA and cluster analysis showed divergence among the strains. Dendrograms constructed using PFGE (SfiI) images from a soft database, including those of pandemic and nonpandemic strains of diverse geographic origin, however, showed that local strains formed a cluster, i.e., "clonal cluster," as did pandemic strains of diverse origin. The demonstrated prevalence of tdh-positive and diarrheagenic serogroup O8:K21 strains in coastal villages of Bangladesh indicates a significant human health risk for inhabitants.

Incidence and Molecular Typing of Vibrio parahaemolyticus from Tiger Shrimp Culture Environments along the Southwest Coast of India

Vibrio parahaemolyticus is one of the most prevalent food-borne pathogens along the southwest coast of India, where marine foods are frequently consumed. Shrimp (Penaeus monodon) and environmental samples were collected from aquaculture farms located in and around Cochin. Confirmation of the biochemically identified strains with species-specific toxR gene and detection of virulent genes viz., tdh and trh was performed by polymerase chain reaction (PCR). The phenotypic markers for the presence of tdh and trh genes were assayed by Kanagawa phenomenon and urease activity, respectively. Protease activity was examined to identify other potential virulence factors. After phenotypic characterization of bacterial strains fingerprinting of genomic DNA was carried by various typing methods, viz., random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus sequence (ERIC), repetitive extragenic palindromic sequence (REP), and ribosomal gene spacer sequence (RS) PCR methods to assess the genetic diversity within the isolates. Eighteen percent of the samples were found positive for the incidence of V. parahaemolyticus by biochemical protocols and toxR (368 bp) targeted PCR. PCR analyses revealed 1% of the samples positive for tdh (269 bp) and trh (500 bp) gene. RAPD analysis revealed clustering of toxigenic strains into a single group. Cluster analysis revealed the conglomeration of isolates into two, five, and seven major groups using RS, ERIC, and REP PCR methods, respectively. RS PCR generated fewer amplified bands compared to REP and ERIC PCR methods, thus giving scope for higher discrimination. Moreover, RS PCR patterns were more discernible visually from other patterns, suggesting RS PCR as a considerably practical method for routine use.

Disruption of GPR39 Impairs Insulin Secretion In Vivo

GPR39 is a G protein-coupled receptor expressed in liver, gastrointestinal tract, adipose tissue and pancreas. We have recently shown that young GPR39 mice have normal body weight, food intake, and fasting glucose and insulin levels. In this study, we examined the role of GPR39 in aging and diet-induced obese mice. Body weight and food intake were similar in wild-type (WT) and GPR39 / mice as they aged from 12 to 52 weeks or when fed a low-fat/high-sucrose (LFHS) or high-fat/high-sucrose (HFHS) diet. 52-week-old GPR39 / mice showed a trend toward decreased insulin levels after oral glucose challenge. When fed either a LFHS or HFHS diet, GPR39 / mice had increased fed glucose levels and showed decreased serum insulin levels during an oral glucose tolerance test in the face of unchanged insulin tolerance. Pancreas morphology and glucose-stimulated insulin secretion in isolated islets from WT and GPR39 / mice were comparable, suggesting that GPR39 is not required for pancreas development or ex vivo insulin secretion. Small interfering RNA-mediated knockdown of GPR39 in clonal NIT-1 -cells revealed that GPR39 regulates the expression of IRS-2 and PDX-1 in a cell-autonomous manner; IRS-2 mRNA was also significantly decreased in the pancreas of GPR39 / mice. Taken together, our data indicate that GPR39 is required for the increased insulin secretion in vivo under conditions of increased demand, i.e. upon development of age-dependent and diet-induced insulin resistance. Thus, GPR39 agonists may have potential for the treatment of type 2 diabetes. Minireview: Thyrotropin-Releasing Hormone and the Thyroid Hormone Feedback Mechanism Maria Izabel Chiamolera and Fredric E. Wondisford (Endocrinology, published January 29, 2009, 10.1210/en.2008-1795) ABSTRACT Thyroid hormone (TH) plays a critical role in development, growth, and cellular metabolism. TH production is controlled by a complex mechanism of positive and negative regulation. Hypothalamic TRH stimulates TSH secretion from the anterior pituitary. TSH then initiates TH synthesis and release from the thyroid gland. The synthesis of TRH and TSH subunit genes is inhibited at the transcriptional level by TH, which also inhibits posttranslational modification and release of TSH. Although opposing TRH and TH inputs regulate the hypothalamic-pituitary-thyroid axis, TH negative feedback at the pituitary was thought to be the primary regulator of serum TSH levels. However, study of transgenic animals showed an unexpected, dominant role for TRH in regulating the hypothalamic-pituitary-thyroid axis and an unanticipated involvement of the thyroid hormone receptor ligand-dependent activation function (AF-2) domain in TH negative regulation. These results are summarized in the review. T R A N S L A T I O N A L H I G H L I G H T S F R O M E N D O C R I N O L O G YThyroid hormone (TH) plays a critical role in development, growth, and cellular metabolism. TH production is controlled by a complex mechanism of positive and negative regulation. Hypothalamic TRH stimulates TSH secretion from the anterior pituitary. TSH then initiates TH synthesis and release from the thyroid gland. The synthesis of TRH and TSH subunit genes is inhibited at the transcriptional level by TH, which also inhibits posttranslational modification and release of TSH. Although opposing TRH and TH inputs regulate the hypothalamic-pituitary-thyroid axis, TH negative feedback at the pituitary was thought to be the primary regulator of serum TSH levels. However, study of transgenic animals showed an unexpected, dominant role for TRH in regulating the hypothalamic-pituitary-thyroid axis and an unanticipated involvement of the thyroid hormone receptor ligand-dependent activation function (AF-2) domain in TH negative regulation. These results are summarized in the review. T R A N S L A T I O N A L H I G H L I G H T S F R O M E N D O C R I N O L O G Y

Aberrant Histone Modifications at the Thyrotropin-Releasing Hormone Gene in Resistance to Thyroid Hormone: Analysis of F455S Mutant Thyroid Hormone Receptor

We reported a novel mutation of thyroid hormone receptor (TR)-beta, F455S, in a patient with pituitary resistance to thyroid hormone (RTH), who showed impaired release of nuclear receptor corepressor and abnormal histone deacetylation. In the present study, we further analyzed the histone modifications and the dynamics of TR and RNA polymerase II on the TRH gene. The lysine residues 9 (H3K9) and 14 (K14) of the histone H3 were acetylated in the absence of thyroid hormone (TH), and addition of TH caused a temporary deacetylation of both residues. Although H3K4 was di- and trimethylated in the absence of T(3), no methylation of H3K9 or K27 was detected. Long-term incubation with T(3) decreased the level of trimethylated H3K4, the amount of TR, and the level of phosphorylated RNA polymerase II but not dimethylated H3K4. Treatment with an inhibitor for H3K4 methyltransferase, 5'-deoxy-5'-methylthioadenosine, decreased basal promoter activity but did not affect the repression by TH. Conversely, overexpression of MLL, an H3K4-specific methyltransferase, caused an increase in basal activity. In the presence of F455S, methylation of H3K4 and the dynamics of TR were intact, but both H3K9 and H3K14 were hyperacetylated, and T(3)-induced deacetylation was impaired, resulting in a high transcriptional level. These findings demonstrated that 1) negative regulation of the TRH gene by TH involves both the acetylation and methylation of specific residues of histone tails and changing the amount of TR, and 2) the major impairment to histone modifications in F455S was hyperacetylation of the specific histone tails.

Molecular Epidemiological Characteristics of Vibrio parahaemolyticus Isolated from Diarrheal Patients in Jeonnam, Korea

To investigate the occurrence and distribution of serotype, specific virulence genes, and pulse field gel electrophoresis (PFGE) patterns in Vibrio parahaemolyticus isolates from Jeonnam, Korea, we tested 87 strains which were identified with V. parahaemolyticus from diarrheal episode patients in 2005. In this study, 16 different O:K serotype combinations of V. parahaemolyticus were determined. The distributions of O and K serotypes were O4:K68 (51.72%), O1:K70 (18.39%), O3:K6 (5.74%), O1:K68 (4.60%) and O3:K57 (4.60%) respectively. Serotype O4:K68 was the regional dominant specific serotype of V. parahaemolyticus in Sinan of Jeonnam, Korea. For the detection of thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh) gene of V. parahaemolyticus, PCR was performed. The tdh gene was detected in all of the V. parahaemolyticus isolates from diarrheal patients, but trh gene was not detected. Analysis of PFGE patterns of 30 V. parahaemolyticus isolates showed 3 groups and 20 types. Among 14 O4:K68 serotypes which were isolated in Sinan, PFGE patterns of 12 strains were closely related (100%), but 2 strains were related by 58.3% and 45.4%, respectively. Also two strains of O1:K4 serotype in Gurye and two strains of O3:K6 serotype in Yeosu were closely related (100%), respectively. Although serotypes (O1:K4, O1:K70, O3:K6 and O4:K68) were different, PFGE patterns were related for more than 80.9%. Therefore, the epidemiological surveillance of V. parahaemolyticus is required by PFGE typing scheme as a further diagnostic tool.

Improved isolation and detection of pathogenic Vibrio parahaemolyticus from seafood using a new enrichment broth

The efficiency of a new enrichment broth containing bile salt, sodium taurocholate (ST broth) in detecting pathogenic Vibrio parahaemolyticus from seafood was compared with the traditional alkaline peptone water (APW). Both the enrichment broths were compared using 58 seafood samples harvested along the southwest coast of India using conventional isolation, colony hybridization following enrichment (CFE) and PCR. V. parahaemolyticus carrying tdh gene were isolated from 6.9% (4/58) and 3.4% (2/58) of the samples after enrichment in ST broth and APW, respectively. Pathogenic V. parahaemolyticus carrying trh gene were isolated from 20.7% (12/58) and 13.8% (8/58) of the samples after enrichment in ST broth and APW respectively. PCR using enrichment lysate as template could detect V. parahaemolyticus carrying tdh gene in 8 (13.8%) and 12 (20.7%) samples after enrichment in APW and ST broth, respectively. Positivity for trh gene based PCR was 24 (41.4%) for ST broth and 19 (32.8%) for APW. Total V. parahaemolyticus were detected and isolated from almost same number of samples using two enrichment broths by three methods used in this study. The results suggest that the ST broth is superior to APW for detection and isolation of pathogenic V. parahaemolyticus from seafood.

Characteristics of a pandemic clone of O3 : K6 and O4 : K68 Vibrio parahaemolyticus isolated in Beira, Mozambique

The genetic characteristics of Vibrio parahaemolyticus strains isolated in 2004 and 2005 in Mozambique were assessed in this study to determine whether the pandemic clone of V. parahaemolyticus O3 : K6 and O4 : K68 serotypes has spread to Mozambique. Fifty-eight V. parahaemolyticus strains isolated from hospitalized diarrhoea patients in Beira, Mozambique, were serotyped for O : K antigens and genotyped for toxR, tdh and trh genes. A group-specific PCR, a PCR that detects the presence of ORF8 of the filamentous phage f237, arbitrarily primed PCR, PFGE and multilocus sequence typing were performed to determine the pandemic status of the strains and their ancestry. All strains of serovars O3 : K6 (n=38) and O4 : K68 (n=4) were identified as a pandemic clonal group by these analyses. These strains are closely related to the pandemic reference strains of O3 : K6 and O4 : K68, which emerged in Asia in 1996 and were later found globally. The pandemic serotypes O3 : K6 and O4 : K68 including reference strains grouped into a single cluster indicating emergence from a common ancestor. The O3 : K58 (n=8), O4 : K13 (n=6), O3 : KUT (n=1) and O8 : K41 (n=1) strains showed unique characteristics different from the pandemic clone.

Antibiotic Resistance in the Shellfish Pathogen Vibrio parahaemolyticus Isolated from the Coastal Water and Sediment of Georgia and South Carolina, USA

Vibrio parahaemolyticus is a gram-negative pathogen commonly encountered in estuarine and marine environments, and a common cause of seafood-related gastrointestinal infections. We isolated 350 V. parahaemolyticus strains from water and sediment at three locations along the Atlantic coast of Georgia and South Carolina during various seasons. These isolates were tested for susceptibility to 24 antibiotics. Isolate virulence was determined through PCR of tdh and trh genes. The breadth of resistance to antibiotics was unexpectedly high, with 24% isolates demonstrating resistance to 10 or more agents. A significant fraction of isolates were resistant to diverse β-lactams, aminoglycosides, and other classes of antibiotics. Fifteen of the 350 strains possessed virulence genes, with no apparent correlation between virulence and site, sample type, or season of isolation. Antibiotic resistance was slightly reduced among the virulent strains. This study represents one of the largest surveys to date of the virulence and antibiotic resistance in environmental V. parahaemolyticus strains. The observed antibiotic susceptibility patterns suggest that current guidelines for the antibiotic treatment of non-cholerae Vibrio should be reevaluated and extended.