Vibrio Parahaemolyticus Strains Research Articles

Transoceanic spreading of pathogenic strains of Vibrio parahaemolyticus with distinctive genetic signatures in the recA gene.

Vibrio parahaemolyticus is an important human pathogen whose transmission is associated with the consumption of contaminated seafood. Consistent multilocus sequence typing for V. parahaemolyticus has shown difficulties in the amplification of the recA gene by PCR associated with a lack of amplification or a larger PCR product than expected. In one strain (090–96, Peru, 1996), the produced PCR product was determined to be composed of two recA fragments derived from different Vibrio species. To better understand this phenomenon, we sequenced the whole genome of this strain. The hybrid recA gene was found to be the result of a fragmentation of the original lineage-specific recA gene resulting from a DNA insertion of approximately 30 kb in length. This insert had a G+C content of 38.8%, lower than that of the average G+C content of V. parahaemolyticus (45.2%), and contained 19 ORFs, including a complete recA gene. This new acquired recA gene deviated 24% in sequence from the original recA and was distantly related to recA genes from bacteria of the Vibrionaceae family. The reconstruction of the original recA gene (recA3) identified the precursor as belonging to ST189, a sequence type reported previously only in Asian countries. The identification of this singular genetic feature in strains from Asia reveals new evidence for genetic connectivity between V. parahaemolyticus populations at both sides of the Pacific Ocean that, in addition to the previously described pandemic clone, supports the existence of a recurrent transoceanic spreading of pathogenic V. parahaemolyticus with the corresponding potential risk of pandemic expansion.

Photorhabdus insect-related (Pir) toxin-like genes in a plasmid of Vibrio parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND) of shrimp.

The 69 kb plasmid pVPA3-1 was identified in Vibrio parahaemolyticus strain 13‑028/A3 that can cause acute hepatopancreatic necrosis disease (AHPND). This disease is responsible for mass mortalities in farmed penaeid shrimp and is referred to as early mortality syndrome (EMS). The plasmid has a GC content of 45.9% with a copy number of 37 per bacterial cell as determined by comparative quantitative PCR analyses. It consists of 92 open reading frames that encode mobilization proteins, replication enzymes, transposases, virulence-associated proteins, and proteins similar to Photorhabdus insect-related (Pir) toxins. In V. parahaemolyticus, these Pir toxin-like proteins are encoded by 2 genes (pirA- and pirB-like) located within a 3.5 kb fragment flanked with inverted repeats of a transposase-coding sequence (1 kb). The GC content of these 2 genes is only 38.2%, substantially lower than that of the rest of the plasmid, which suggests that these genes were recently acquired. Based on a proteomic analysis, the pirA-like (336 bp) and pirB-like (1317 bp) genes encode for 13 and 50 kDa proteins, respectively. In laboratory cultures of V. parahaemolyticus 13-028/A3, both proteins were secreted into the culture medium. We developed a duplex PCR diagnostic method, with a detection limit of 10(5) CFU ml(-1) and targeting pirA- and pirB-like genes in this strain of V. parahaemolyticus. This PCR protocol can reliably detect AHPND-causing strains of V. parahaemolyticus and does not cross react with non-pathogenic strains or with other species of Vibrio isolated from shrimp ponds.

Genetic variation in Vibrio parahaemolyticus isolated from the aquacultural environments.

Environmental strains of Vibrio parahaemolyticus, a foodborne pathogen, were isolated from milkfish and grouper aquaculture facilities in southern Taiwan and characterized by pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction targeting on the virulence-associated and genomic island (VPaI) marker genes. Analyses of 62 environmental isolates, including two putative pathogenic isolates, by NotI-PFGE revealed 11 pulse-type clusters with a similarity of 85%. Some of the T3SS2α-associated genes (vopB2, vopC and vopT) were not present in all of these two putative pathogenic isolates. Marker genes of VPaI-1 (MTase gene), VPaI-2 (VP0636) and VPaI-3 (VP1073 and VP1077) were detected in 14-100% of the environmental isolates examined, and the VP1073 and VP1077 of VPaI-3 marker genes were not detected. This study confirmed the high genetic variability of the environmental isolates including the putative pathogenic strains and identified some marker genes of VPaI in the environmental V. parahaemolyticus isolates. Vibrio parahaemolyticus is a prevalent seafood-borne enteropathogen with the appearance of pandemic O3:K6 strains in 1996. This study characterized the environmental nontoxigenic and toxigenic isolates by pulsed-field gel electrophoresis and the presence of marker genes of genomic islands. Results showed that the T3SS2α-associated genes are not present in all environmental tdh(+) isolates, and the presence of movable elements may contribute to genetic variation in the environmental V. parahaemolyticus isolates.

Intracellular survival of virulence and low-virulence strains of Vibrio parahaemolyticus in Epinephelus awoara macrophages and peripheral leukocytes.

In this study, we examined the virulence factors and pathogenesis of Vibrio parahaemolyticus in Epinephelus awoara. The chemotactic motility of V. parahaemolyticus for phagocytosis and intracellular survival in fish macrophages was determined using virulence strains and low-virulence strains of V. parahaemolyticus. We found that the intracellular mean number of virulence strains of V. parahaemolyticus ranged from 0-180 min after co-incubation with macrophages and peripheral leukocytes, was relatively low, and decreased steadily over the observation period. Low-virulence strains of V. parahaemolyticus were unable to survive in peripheral leukocytes and macrophages. Cell viability in response to V. parahaemolyticus was assessed using the MTT assay. Low-virulence V. parahaemolyticus strains exhibited lower cytotoxicity compared to virulent strains. The average percent of live macrophages and peripheral leukocytes infected by V. parahaemolyticus ranged from 13.50-79.20%. These results indicate that V. parahaemolyticus in E. awoara is a facultative intracellular bacterium that may be involved in virulence.

Efficacy of low-temperature high hydrostatic pressure processing in inactivating Vibrio parahaemolyticus in culture suspension and oyster homogenate

Culture suspensions of five clinical and five environmental Vibrio parahaemolyticus strains in 2% NaCl solution were subjected to high pressure processing (HPP) under various conditions (200–300MPa for 5 and 10min at 1.5–20°C) to study differences in pressure resistance among the strains. The most pressure-resistant and pressure-sensitive strains were selected to investigate the effects of low temperatures (15, 5 and 1.5°C) on HPP (200 or 250MPa for 5min) to inactivate V. parahaemolyticus in sterile oyster homogenates. Inactivation of V. parahaemolyticus cells in culture suspensions and oyster homogenates was greatly enhanced by lowering the processing temperature from 15 to 5 or 1.5°C. A treatment of oyster homogenates at 250MPa for 5min at 5°C decreased the populations of V. parahaemolyticus by 6.2logCFU/g for strains 10290 and 100311Y11 and by >7.4logCFU/g for strain 10292. Decreasing the processing temperature of the same treatment to 1.5°C reduced all the V. parahaemolyticus strains inoculated to oyster homogenates to non-detectable (<10CFU/g) levels. Factors including pressure level, processing temperature and time all need to be considered for developing effective HPP for eliminating pathogens from foods. Further studies are needed to validate the efficacy of the HPP (250MPa for 5min at 1.5°C) in inactivating V. parahaemolyticus cells in whole oysters.

Alteration of structure and cellular protein profiles of Vibrio parahaemolyticus cells by high pressure treatment

Effects of high pressure processing on changes of cell morphology and cellular proteins were investigated using two Vibrio parahaemolyticus strains 10290 and 10292 subjected to pressure treatments (200 or 300 MPa for 5 min) at 20 °C. Cells with or without pressure treatments were studied for structural damage by leakage of internal cellular substances and scanning electron microscopy (SEM) and for cellular proteins profiles by total sulfhydryl contents and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Increase in leakage of cellular substances was observed as the pressure of a treatment increased. Scanning electron microscopy images revealed cell damage of both V. parahaemolyticus strains after the pressure treatments, particularly at 300 MPa for 5 min. Total protein and sulfhydryl contents in cellular protein extracts of both strains decreased as the pressure of a treatment increased. SDS-PAGE analysis of cellular proteins extracted from V. parahaemolyticus cells after the pressure treatments revealed changes of cellular protein profiles with a few protein fragments of 150–250 kDa being affected most. These data suggest that inactivation of V. parahaemolyticus cells by high pressure processing is associated with cell membrane damage and changes of cellular protein profiles, which appear to be dependent on the strain and pressure applied in a treatment.

Draft Genome Sequences of Four Strains of Vibrio parahaemolyticus, Three of Which Cause Early Mortality Syndrome/Acute Hepatopancreatic Necrosis Disease in Shrimp in China and Thailand.

We sequenced four Vibrio parahaemolyticus strains, three of which caused serious acute hepatopancreatic necrosis disease. Sequence analysis of the virulent strains revealed not only genes related to cholera toxin and the type IV pilus/type IV secretion system but also a unique, previously unreported, large extrachromosomal plasmid that encodes a homolog to the insecticidal Photorhabdus insect-related binary toxin PirAB.

High-Quality Draft Genomes of Two Vibrio parahaemolyticus Strains Aid in Understanding Acute Hepatopancreatic Necrosis Disease of Cultured Shrimps in Mexico.

The high-quality draft genomes of two Vibrio parahaemolyticus strains, one that causes the acute hepatopancreatic necrosis disease (AHPND) in cultured shrimps (FIM-S1708+), and another that does not (FIM-S1392−) are reported. A chromosome-scale assembly for the FIM-S1392− genome is reported here. The analysis of the two genomes gives some clues regarding the genomic differences between the strains.

Draft Genome Sequences of Clinical Vibrio parahaemolyticus Strains Isolated in Maryland (2010 to 2013)

Vibrio parahaemolyticus is the leading cause of food-borne illnesses associated with the consumption of raw shellfish worldwide. Here, we report 45 draft genomes of V. parahaemolyticus. Thirty-five of them are strains that were isolated from clinical cases in the state of Maryland from 2010 to 2013. The remaining 10 strains were historical isolates, isolated mostly from the West Coast of the United States during the period of 1988 to 2004. The availability of these genomes will allow for future phylogenetic analyses with other V. parahaemolyticus strains.

Occurrence of virulence genes among Vibrio cholerae and Vibrio parahaemolyticus strains from treated wastewaters.

Pathogenic Vibrio species are an important cause of foodborne illnesses. The aim of this study was to describe the occurrence of potentially pathogenic Vibrio species in the final effluents of a wastewater treatment plant and the risk that they may pose to public health. During the 1-year monitoring, a total of 43 Vibrio strains were isolated: 23 Vibrio alginolyticus, 1 Vibrio cholerae, 4 Vibrio vulnificus, and 15 Vibrio parahaemolyticus. The PCR investigation of V. parahaemolyticus and V. cholerae virulence genes (tlh, trh, tdh, toxR, toxS, toxRS, toxT, zot, ctxAB, tcp, ace, vpi, nanH) revealed the presence of some of these genes in a significant number of strains. Intraspecies variability and genetic relationships among the environmental isolates were analyzed by random amplified polymorphic DNA-PCR (RAPD-PCR). We report the results of the first isolation and characterization of an environmental V. cholerae non-O1 non-O139 and of a toxigenic V. parahaemolyticus strain in Tunisia. We suggest that non-pathogenic Vibrio might represent a marine reservoir of virulence genes that can be transmitted between strains by horizontal transfer.

Seasonal Abundance of Total and Pathogenic Vibrio parahaemolyticus Isolated from American Oysters Harvested in the Mandinga Lagoon System, Veracruz, Mexico: Implications for Food Safety

The abundance of total and pathogenic Vibrio parahaemolyticus (Vp) strains in American oysters (Crassostrea virginica) harvested in two different harvest sites from the Mandinga lagoon System was evaluated monthly for 1 year (January through December 2012). Frequencies of species-specific genes and pathogenic genes exhibited a seasonal distribution. The annual occurrence of Vp with the species-specific tlh gene (tlh+) was significantly higher during the winter windy season (32.50%) and spring dry season (15.0%), with the highest densities observed during spring dry season at 283.50 most probable number (MPN)/ g (lagoon bank A, near human settlements), indicating the highest risk of infection during warmer months. Pathogenic Vp tlh+/tdh+ frequency was significantly higher during the winter windy and the spring dry seasons at 22.50 and 10.00%, respectively, with highest densities of 16.22 and 41.05 MPN/g (bank A), respectively. The tlh/trh and tdh/trh gene combinations were also found in Vp isolates during the spring dry season at 1.25 and 1.3%, respectively, with densities of 1.79 and 0.4 MPN/g (bank A), respectively. The orf8 genes were detected during the winter windy season (1.25%) with highest densities of 5.96 MPN/g (bank A) and 3.21 MPN/g (bank B, near mangrove islands and a heron nesting area). Densities of Vp tdh+ were correlated (R2 = 0.245, P < 0.015) with those of Vp orf8+. The seasonal dynamics of Vp harboring pathogenic genes varied with seasonal changes, with very high proportions of Vp tdh+ and Vp orf8+ isolates in the winter windy season at 46.2 and 17.0%, respectively, which suggests that environmental factors may differentially affect the abundance of pathogenic subpopulations. Although all densities of total Vp (Vp tlh+) were lower than 104 MPN/g, thus complying with Mexican regulations, the presence of pathogenic strains is a public health concern. Our results suggest that total Vp densities may not be appropriate for assessing oyster contamination and predicting the risk of infection. Evaluation of the presence of pathogenic strains would be a better approach to protecting public health.

Trends in the epidemiology of pandemic and non-pandemic strains of Vibrio parahaemolyticus isolated from diarrheal patients in Kolkata, India.

A total of 178 strains of V. parahaemolyticus isolated from 13,607 acute diarrheal patients admitted in the Infectious Diseases Hospital, Kolkata has been examined for serovar prevalence, antimicrobial susceptibility and genetic traits with reference to virulence, and clonal lineages. Clinical symptoms and stool characteristics of V. parahaemolyticus infected patients were analyzed for their specific traits. The frequency of pandemic strains was 68%, as confirmed by group-specific PCR (GS-PCR). However, the prevalence of non-pandemic strains was comparatively low (32%). Serovars O3:K6 (19.7%), O1:K25 (18.5%), O1:KUT (11.2%) were more commonly found and other serovars such as O3:KUT (6.7%), O4:K8 (6.7%), and O2:K3 (4.5%) were newly detected in this region. The virulence gene tdh was most frequently detected in GS-PCR positive strains. There was no association between strain features and stool characteristics or clinical outcomes with reference to serovar, pandemic/non-pandemic or virulence profiles. Ampicillin and streptomycin resistance was constant throughout the study period and the MIC of ampicillin among selected strains ranged from 24 to >256 µg/ml. Susceptibility of these strains to ampicillin increased several fold in the presence of carbonyl cyanide-m-chlorophenyldrazone. The newly reported ESBL encoding gene from VPA0477 was found in all the strains, including the susceptible ones for ampicillin. However, none of the strains exhibited the β-lactamase as a phenotypic marker. In the analysis of pulsed-field gel electrophoresis (PFGE), the pandemic strains formed two different clades, with one containing the newly emerged pandemic strains in this region.

Draft Genome Sequences of Six Strains of Vibrio parahaemolyticus Isolated from Early Mortality Syndrome/Acute Hepatopancreatic Necrosis Disease Shrimp in Thailand.

Some strains of Vibrio parahaemolyticus cause acute hepatopancreatic necrosis disease (AHPND) in shrimp. We sequenced 3 AHPND and 3 non-AHPND strains and found that all of them lacked the pathogenicity island relevant to human infection. A unique sequence encoding a type IV pilus/type IV secretion system was found in 3 AHPND strains.

PandemicVibrio parahaemolyticus, Maryland, USA, 2012

To the Editor: Since 1996, an increasing number of infections caused by Vibrio parahaemolyticus strains belonging to a pandemic clonal complex (CC), CC3, typically O3:K6, have been observed worldwide (1–3); most of these strains are sequence type (ST) 3. In the summer of 1998, outbreaks linked to O3:K6 occurred in Galveston Bay, Texas, and Oyster Bay, New York, USA; the illnesses were associated with oyster consumption (4). Strains belonging to CC36 are the leading cause of V. parahaemolyticus infections in the United States. These strains are endemic to the West Coast (2) and have been historically linked to outbreak-associated V. parahaemolyticus infections caused by consumption of raw oysters harvested from the region (5).

Bacteriophage Therapy of a Vibrio parahaemolyticus Infection Caused by a Multiple-Antibiotic–Resistant O3:K6 Pandemic Clinical Strain

ABSTRACT Background. Recently isolated Vibrio parahaemolyticus strains have displayed multiple antibiotic resistance. Alternatives to conventional antibiotics are needed, especially for the multiple-antibiotic–resistant V. parahaemolyticus pandemic strain. Methods. A bacteriophage, designated pVp-1, showed effective infectivity for multiple-antibiotic–resistant V. parahaemolyticus and V. vulnificus, including V. parahaemolyticus pandemic strains. The therapeutic potential of the phage was studied in a mouse model of experimental infection using a multiple-antibiotic–resistant V. parahaemolyticus pandemic strain. We monitored the survivability and histopathological changes, quantified the bacterial and phage titers during phage therapy, and observed the immune response induced by phage induction. Results. Phage-treated mice displayed protection from a V. parahaemolyticus infection and survived lethal oral and intraperitoneal bacterial challenges. Conclusions. To the best of our knowledge, this is the first report of phage therapy in a mouse model against a multiple-antibiotic–resistant V. parahaemolyticus pandemic strain infection.

Effect of Acid Adaptation on the Environmental Stress Tolerance of Three Strains ofVibrio parahaemolyticus

Three strains of Vibrio parahaemolyticus (690, BCRC 13023, and BCRC 13025), involved in foodborne outbreaks in Taiwan, were subjected to acid adaptation at pH 5.5 for 90 min. The effects of acid adaptation on the tolerance of V. parahaemolyticus to various environmental stresses, including heat (47°C), cold (4°C and -20°C), ethanol (8%), high salt (20% NaCl), and hydrogen peroxide (20 ppm) were examined. Results showed that acid adaptation increased the thermal tolerance of the three test strains of V. parahaemolyticus, while it did not affect their cold tolerance. Acid adaptation also increased the ethanol tolerance in V. parahaemolyticus 690 and BCRC 13025, but not in BCRC 13023. Differences in the tolerance to high salts were noted among the three strains after prior acid adaptation. However, these acid-adapted V. parahaemolyticus strains were more susceptible to hydrogen peroxide than their nonadapted controls. These findings demonstrated that acid adaption responses of V. parahaemolyticus varied among strains and types of stress challenge.

Measurement of Haemolysin Activities in Vibrio vulnificus

VvhA produced by Vibrio vulnificus exhibits cytolytic activity to human cells including erythrocytes. Since haemolysis by VvhA may provide iron for bacterial growth and pathogenicity, we investigated the expression of VvhA to elucidate the regulatory roles of Fur, a major transcription factor controlling iron-homeostasis. Fur repressed the transcription of vvhBA operon via binding to the promoter region. However, haemolysin content and haemolytic activity were lowered in cell-free supernatant of fur mutant. This discrepancy between the levels of vvhA transcript and VvhA protein in fur mutant was caused by exoproteolytic activities of the elastase VvpE and another metalloprotease VvpM, which were also regulated by Fur. vvpE gene expression was repressed by Fur via binding to the Fur-box homologous region. Regulation of VvpM expression by Fur did not occur at the level of vvpM transcription. In vitro proteolysis assays showed that both proteases efficiently degraded VvhA. In addition, the extracellular levels of VvhA were higher in culture supernatants of vvpE or vvpM mutants than in the wild type. Thus this study demonstrates that Fur regulates haemolysin production at the transcription level of the vvhBA operon and at the post-translation level by regulating the expressions of two VvhA-degrading exoproteases, VvpE and VvpM. This protocol can be applied to other Vibrio strains with haemolysin activities, such as Vibrio parahaemolyticus (V. parahaemolyticus) or other human pathogen strains with similar heamolysin activities.

Phylogenetic and in silico functional analyses of thermostable-direct hemolysin and tdh-related encoding genes in Vibrio parahaemolyticus and other Gram-negative bacteria.

Emergence and spread of pandemic strains of Vibrio parahaemolyticus have drawn attention to make detailed study on their genomes. The pathogenicity of V. parahaemolyticus has been associated with thermostable-direct hemolysin (TDH) and/or TDH-related hemolysin (TRH). The present study evaluated characteristics of tdh and trh genes, considering the phylogenetic and in silico functional features of V. parahaemolyticus and other bacteria. Fifty-two tdh and trh genes submitted to the GenBank were analyzed for sequence similarity. The promoter sequences of these genes were also analyzed from transcription start point to −35 regions and correlated with amino acid substitution within the coding regions. The phylogenetic analysis revealed that tdh and trh are highly distinct and also differ within the V. parahaemolyticus strains that were isolated from different geographical regions. Promoter sequence analysis revealed nucleotide substitutions and deletions at −18 and −19 positions among the pandemic, prepandemic, and nonpandemic tdh sequences. Many amino acid substitutions were also found within the signal peptide and also in the matured protein region of several TDH proteins as compared to TDH-S protein of pandemic V. parahaemolyticus. Experimental evidences are needed to recognize the importance of substitutions and deletions in the tdh and trh genes.

Multiple enzymatic profiles of Vibrio parahaemolyticus strains isolated from oysters

The enzymatic characterization of vibrios has been used as a virulence indicator of sanitary interest. The objective of this study was to determine the enzymatic profile of Vibrio parahaemolyticus strains (n=70) isolated from Crassostrea rhizophorae oysters. The strains were examined for the presence of gelatinase (GEL), caseinase (CAS), elastase (ELAS), phospholipase (PHOS), lipase (LIP), amilase (AML) and DNase. All enzymes, except elastase, were detected in more than 60% of the strains. The most recurrent enzymatic profiles were AML + DNase + PHOS + GEL + LIP (n=16; 22.9%) and AML + CAS + DNase + PHOS + GEL + LIP (n=21; 30%). Considering the fact that exoenzyme production by vibrios is closely related to virulence, one must be aware of the bacteriological risk posed to human health by the consumption of raw or undercooked oysters.

Antibiotic resistance and molecular typing among cockle (Anadara granosa) strains of Vibrio parahaemolyticus by polymerase chain reaction (PCR)-based analysis

Genomic DNA of Vibrio parahaemolyticus were characterized by antibiotic resistance, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis. These isolates originated from 3 distantly locations of Selangor, Negeri Sembilan and Melaka (East coastal areas), Malaysia. A total of 44 (n = 44) of tentatively V. parahaemolyticus were also examined for the presence of toxR, tdh and trh gene. Of 44 isolates, 37 were positive towards toxR gene; while, none were positive to tdh and trh gene. Antibiotic resistance analysis showed the V. parahaemolyticus isolates were highly resistant to bacitracin (92%, 34/37) and penicillin (89%, 33/37) followed by resistance towards ampicillin (68%, 25/37), cefuroxime (38%, 14/37), amikacin (6%, 2/37) and ceftazidime (14%, 5/37). None of the V. parahaemolyticus isolates were resistant towards chloramphenicol, ciprofloxacin, ceftriaxone, enrofloxacin, norfloxacin, streptomycin and vancomycin. Antibiogram patterns exhibited, 9 patterns and phenotypically less heterogenous when compared to PCR-based techniques using ERIC- and RAPD-PCR. The results of the ERIC- and RAPD-PCR were analyzed using GelCompare software. ERIC-PCR with primers ERIC1R and ERIC2 discriminated the V. parahaemolyticus isolates into 6 clusters and 21 single isolates at a similarity level of 80%. While, RAPD-PCR with primer Gen8 discriminated the V. parahaemolyticus isolates into 11 clusters and 10 single isolates and Gen9 into 8 clusters and 16 single isolates at the same similarity level examined. Results in the presence study demonstrated combination of phenotypically and genotypically methods show a wide heterogeneity among cockle isolates of V. parahaemolyticus.