Characterization of Vibrio parahaemolyticus and its specific phage from shrimp pond in Palk Strait, South East coast of India.

Phage therapy is an alternative and eco-friendly biocontrol agent to prevent and control multidrug resistant bacteria in the aquatic system. The aim of this study is to isolate and characterize the Vibrio parahaemolyticus and its potential lytic phage from Penaeus monodon growing-out by rearing in shrimp ponds in Palk Strait, South East coast of India. The conventional phenotypic characteristics and molecular identification was confirmed using 16S rRNA sequence and to determine the antibiotic resistant profiles. The V. parahaemolyticus phage was effective against V. parahaemolyticus through one-step growth experiments, phage survival was determined by long-term storage at various temperatures and pH. Further, transmission electron microscope (TEM) revealed that the lytic phage belongs to the Myoviridae family. The isolated lytic phage (VVP1) was more specific against N1A V. parahaemolyticus and was able to infect N7A V. parahaemolyticus, N3B and N13B Vibrio alginolyticus strains. Evaluation of microcosm studies with P. monodon larvae infected with V. parahaemolyticus showed the survival of larvae in the presence of phage treatment at 2.3 × 1010 PFU/mL-1 was enhanced when compared with the control. This study provides the application of phage as a useful strategy to prevent and eliminate or reduce shrimp pathogenic V. parahaemolyticus in the aquaculture system.

Proteomic identification of responsive proteins of Vibrio parahaemolyticus under high hydrostatic pressure.

BACKGROUND: High hydrostatic pressure (HHP) processing is currently being used as a treatment for certain foods to inhibit spoilage organisms and control the presence of foodborne pathogens. In this study proteome profiles were performed by two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF/TOF identification to determine the effects of HHP (50, 100, 150 and 200 MPa, each for 10 min) on Vibrio parahaemolyticus ATCC 17802 (∼8 log CFU mL⁻¹) in order to understand how it responds to mechanical stress injury. RESULTS: Multiple comparisons of 2-DE revealed that the majority of changes in protein abundance occurred in a pressure-dependent fashion. A total of 18 differentially expressed protein spots were successfully identified by MALDI-TOF/TOF analysis. Moreover, quantitative RT-PCR and immunoblotting also substantiated the changes of transcriptional and translational levels of representative proteins. CONCLUSIONS: Our results suggested that V. parahaemolyticus may respond to HHP treatment through suppression of membrane stability and functionality (PfaC, Alr2, MltA, PLA2 and PatH), depression of biosynthesis and cellular processes (NadB, PyrB and ArgB), decreased levels of transcription (RpoD) and translation (RpsA, RplJ and PheS), and effective activation of protein folding and stress-related elements (GroES, DnaK and GroEL). This study may provide insight into the nature of the cellular targets of high pressure and in high-pressure resistance mechanisms in V. parahaemolyticus.

Selective sterilization of Vibro parahaemolyticus from a bacterial mixture by low-amperage electric current.

The objective of this study was to investigate the possibility of using low-amperage electrical treatment (LAET) as a selective bacteriocide. Mixtures containing Escherichia coli, Staphylococcus aureus, and Vibrio parahaemolyticus were treated with different electric current intensities and for different times. The results showed that at 263 mA, treating bacteria for 100 ms eliminated all V. parahaemolyticus colonies. Although LAET reduced the populations of the three microorganisms, V. parahaemolyticus was more injured by LAET than S. aureus and E. coli when treated at the same processing conditions.

Antibacterial activity of a novel Forsythia suspensa fruit mediated green silver nanoparticles against food-borne pathogens and mechanisms investigation.

In the present study, novel silver nanoparticles (AgNPs) were synthesized via a green method by using Forsythia suspensa fruit water extract. The synthesized AgNPs showed antibacterial activities against all the tested food-borne pathogens, including Listeria monocytogenes, Vibrio parahaemolyticus, Escherichia coli O157:H7, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhimurium. Furthermore, the S. aureus and V. parahaemolyticus were introduced as Gram-positive and Gram-negative model strains to explore the antibacterial mechanism of AgNPs. Minimal inhibitory concentrations (MICs) of V. parahaemolyticus and S. aureus were 6.25 µg/mL and 50 µg/mL, respectively, and the minimum bactericidal concentrations (MBCs) of V. parahaemolyticus and S. aureus were 12.5 µg/mL and 100 µg/mL, respectively. Results indicated that the AgNPs caused morphological alterations and damaged the membrane integrity of strains S. aureus and V. parahaemolyticus. In addition, the AgNPs induced the release of nucleic acids of V. parahaemolyticus cells, resulting in disrupting of cells reproduction.

Flow cytometric evaluation of Vibrio parahaemolyticus adhesion inhibition to human epithelial cells.

BACKGROUND: The present report demonstrates the usefulness of flow cytometry for a quantitative assessment of adhesion inhibition of a Vibrio parahaemolyticus strain to human epithelial cells to acquire more information about the nature of its adhesins. METHODS: The inhibition of the adhesive process to Hep-2 was assayed by adding several monosaccharides to infected cells monolayers. The quantification of the adherent bacteria, labeled with a specific primary antibody plus a secondary fluorescein isothiocyanate-conjugated antibody, was performed by flow cytometry in comparison with light microscopy. The adherence was quantified in terms of the proportion of cells with adherent V. parahaemolyticus and as the mean of adherent bacteria per cell. RESULTS: The adhesion showed a percentage of 98% with a mean fluorescence channel of 331 comparable to those obtained by light microscopy. The addition of monosaccharides resulted in a D-mannose and N-acetyl-galactosamine sensitive adherence. Even if this environmental strain also showed a mannose-sensitive cell-associated hemoagglutination that could mediate V. parahaemolyticus adherence, our results suggest that different sites for an irreversible adherence to host cell are involved. CONCLUSIONS: Flow cytometry in combination with indirect immunofluorescence is an effective tool to investigate the adhesive process of bacteria to epithelial cells because it is more sensitive and reproducible than visual counting of bacteria performed in light microscopy.

Fatty acid composition, cell morphology and responses to challenge by organic acid and sodium chloride of heat-shocked Vibrio parahaemolyticus.

Vibrio parahaemolyticus 690, a clinical strain, was subjected to heat shock at 42 degrees C for 45 min. The fatty acid profile and recovery of the heat-shocked cells of V. parahaemolyticus on TSA-3.0% NaCl, APS agar (Alkaline peptone salt broth supplemented with 1.5% agar) and TCBS (Thiosulfate-citrate-bile salts-sucrose agar) were compared with those of the nonheat-shocked cells. Furthermore, the morphology of V. parahaemolyticus and survival in the presence of various organic acids (25 mM acetic acid, lactic acid, citric acid or tartaric acid) and NaCl (0.1% and 20.0%) as influenced by heat shock treatment were also investigated. It was found that heat shock caused a change in the proportions of the unsaturated and saturated fatty acid. The ratio of saturated fatty acids to unsaturated fatty acids observed on heat-shocked V. parahaemolyticus cells was significantly (p<0.05) higher than that on the control cells. Extensive cell-wall pitting and cell disruption, representing cell-surface damage, were also observed on the cells which were subjected to heat shock treatment. Recovery of heat-shocked cells of V. parahaemolyticus was significantly less on TCBS and APS agar than on TSA-3.0% NaCl. Heat shock decreased the tolerance of V. parahaemolyticus to organic acids. The extent of decreased acid tolerance observed on heat-shocked cells varied with the organic acid tested. While heat shock increased the survival of V. parahaemolyticus in the presence of 0.1% NaCl and made the test organism more susceptible to 20.0% NaCl than the control cells.

Resuscitation of viable but nonculturable cells of Vibrio parahaemolyticus induced at low temperature under starvation.

Vibrio parahaemolyticus is known to exist in a viable but nonculturable state when incubated at low temperature under starvation. It has long been debated whether the culturable cells which appear after temperature upshift are the result of true resuscitation or regrowth of a few residual culturable cells. Starved V. parahaemolyticus cells at 4 degrees C reached the nonculturable stage in about 12 days. The true resuscitation of nonculturable cells of V. parahaemolyticus occurred after spreading them onto an agar medium supplemented with H(2)O(2)-degrading compounds such as catalase or sodium pyruvate. The proposed method may be applicable to detecting the enteropathogen from environmental samples.

Correlation between cell-associated mannose-sensitive hemagglutination by Vibrio parahaemolyticus and adherence to a human colonic cell line Caco-2.

Cell-associated hemagglutination (cHA) activity with human erythrocytes was examined for 468 clinical and 71 environmental strains of Vibrio parahaemolyticus. Approximately 95% of the strains tested were cHA positive irrespective of source or Kanagawa phenomenon. 75% of clinical strains showed relatively strong mannose-sensitive hemagglutination (MSHA), whereas 88% of the environmental strains showed relatively weak mannose-resistant hemagglutination (MRHA). Adherence of V. parahaemolyticus to Caco-2 cells was also determined. A clear positive correlation between cell-associated MSHA and adherence to Caco-2 cells was observed.

In vitro attachment of Vibrio parahaemolyticus to hemocytes of two gastropod molluscs.

Vibrio parahaemolyticus D-3 was observed to attach to hemocytes of a marine gastropod mollusc, Nerita albicilla, regardless of the presence of N. albicilla serum. The organism attached to hemocytes of an estuarine gastropod, Clithon retropictus, in the presence of C. retropictus serum while the attachment to the hemocytes was decreased significantly in the absence of the serum. These evidences suggest that N. albicilla hemocytes would facilitate the clearance of V. parahaemolyticus from the alimentary tract of the mollusc and that C. retropictus hemocytes would protect C. retropictus against the invasion of V. parahaemolyticus to hemocoel of the mollusc.

Characterization of the pili isolated from Vibrio parahaemolyticus O3:K6.

Pilus of Vibrio parahaemolyticus O3:K6 strain LVP9 belonging to the newly identified clone was purified and characterized. The molecular mass of the pilin was estimated to be about 18 kDa by SDS-PAGE, and the isoelectric point of the pilin was 5.0 +/- 0.2. The LVP9 pili were antigenically different from the other V. parahaemolyticus Na2 pili and Ha7 pili as previously reported, nevertheless all three had indistinguishable morphology and shared a high degree of homology in their N-terminal amino acid sequences. Strain LVP9 and its purified pili did not agglutinate human and rabbit erythrocytes. The LVP9 organisms and the purified pili were adhesive to the rabbit intestine. The adhesion was inhibited by pretreatment of the rabbit intestine with the purified pili or by pretreatment of the organisms with the Fab fractions of anti-pilus antibody. These results indicate that the LVP9 pilus is an adherent factor to the rabbit intestine.

Biofilms, flagella, and mechanosensing of surfaces by bacteria.

Formation of a bacterial biofilm is a developmental process that begins when a cell attaches to a surface, but how does a bacterial cell know it is on or near a surface in the first place? The phase of this 'swim-or-stick' switch is determined by a sensory transduction mechanism referred to as surface sensing, which involves the rotating bacterial flagellum. This review explores six bacterial species as models of flagellar mechanosensing of surfaces to understand the current state of our knowledge and the challenges that lie ahead. A common link between these bacteria is a requirement for the proper function of the flagellar motor stators that channel ions into the cell to drive flagellar rotation. Conditions that affect ion flow act as a signal that, ultimately, controls the master transcriptional regulatory circuits controlling the flagellar hierarchy and biofilm formation.

Changes of ultrastructure and stress tolerance of Vibrio parahaemolyticus upon entering viable but nonculturable state.

This study examined the change of ultrastructure and stress tolerance of the marine foodborne pathogen, Vibrio parahaemolyticus 1137, when incubated under viable but nonculturable (VBNC) state induction conditions for different time intervals. The rod-shaped V. parahaemolyticus cells in the exponential phase became coccoid cells in the VBNC state, with aberrantly shaped cells formed in the initial stage. In the aberrantly shaped cells, the cell wall was loosened, flexible and allowed the cell to bulge, and the formation of new and thin cell wall or the expansion of cell wall was also discerned primarily at the polar position, enclosing an empty cellular space. The thickness of the cell wall increased with the VBNC induction time, and was increased in cultures that were removed from the induction conditions and whose temperature was upshifted to 25°C for 1 or 2days. The incubation of V. parahaemolyticus under the VBNC induction conditions significantly enhanced its tolerance to heat, H(2)O(2) and low salinity, but sensitized it to bile salts. Tolerance to heat, bile salts and low salinity was significantly higher in the temperature upshifted cultures than in the corresponding unheated cultures, and the heated cultures were also more susceptible to H(2)O(2). The V. parahaemolyticus cultures that were incubated in the VBNC state induction conditions and the corresponding temperature-upshifted cultures exhibited unique changes in ultrastructure and tolerance to various stresses, unlike the nutrient-starved cells.

Mechanism of actin filament nucleation by the bacterial effector VopL.

Vibrio parahaemolyticus protein L (VopL) is an actin nucleation factor that induces stress fibers when injected into eukaryotic host cells. VopL contains three N-terminal Wiskott-Aldrich homology 2 (WH2) motifs and a unique VopL C-terminal domain (VCD). We describe crystallographic and biochemical analyses of filament nucleation by VopL. The WH2 element of VopL does not nucleate on its own and requires the VCD for activity. The VCD forms a U-shaped dimer in the crystal, stabilized by a terminal coiled coil. Dimerization of the WH2 motifs contributes strongly to nucleation activity, as do contacts of the VCD to actin. Our data lead to a model in which VopL stabilizes primarily lateral (short-pitch) contacts between actin monomers to create the base of a two-stranded filament. Stabilization of lateral contacts may be a common feature of actin filament nucleation by WH2-based factors.

Mechanism of actin filament nucleation by Vibrio VopL and implications for tandem W domain nucleation.

Pathogen proteins targeting the actin cytoskeleton often serve as model systems to understand their more complex eukaryotic analogs. We show that the strong actin filament nucleation activity of Vibrio parahaemolyticus VopL depends on its three W domains and on its dimerization through a unique VopL C-terminal domain (VCD). The VCD shows a previously unknown all-helical fold and interacts with the pointed end of the actin nucleus, contributing to the nucleation activity directly and through duplication of the W domain repeat. VopL promotes rapid cycles of filament nucleation and detachment but generally has no effect on elongation. Profilin inhibits VopL-induced nucleation by competing for actin binding to the W domains. Combined, the results suggest that VopL stabilizes a hexameric double-stranded pointed end nucleus. Analysis of hybrid constructs of VopL and the eukaryotic nucleator Spire suggest that Spire may also function as a dimer in cells.

Not without cause: Vibrio parahaemolyticus induces acute autophagy and cell death.

Vibrio parahaemolyticus (V. parahaemolyticus) is a gram-negative halophillic bacterium that causes worldwide seafood-borne gastroenteritis. The prevalence of V. parahaemolyticus in the environment and incidence of infection have been linked to rising water temperatures caused by global warming. Among its virulence factors, V. parahaemolyticus harbors two type III secretion systems (T3SS). Recently, we have shown that T3SS1 induces rapid cellular death that initiates with acute autophagy, as measured by LC3 lipidation and accumulation of early autophagosomal vesicles. While not the first characterized pathogen to usurp autophagy, this is the first example of an extracellular pathogen that exploits this pathway for its own benefit. Here we discuss possible roles for the induction of autophagy during infection and discuss how V. parahaemolyticus-induced autophagy provides insight into key regulatory steps that govern the decision between apoptosis and autophagy.

Bactericidal effect of lactoferrin and lactoferrin chimera against halophilic Vibrio parahaemolyticus.

Infections caused by Vibrio parahaemolyticus, an halophilic member of the genus Vibrio, have increased globally in the last 5 years. Diarrhea caused by V. parahaemolyticus results from eating raw or undercooked seafood. The aim of this work was to investigate whether lactoferrin and some lactoferrin-peptides have bactericidal activity against Vibrio parahaemolyticus ATCC 17802, the pandemic strain O3:K6, and the multidrug resistant isolate 727, as well as against Vibrio cholerae strains O1 and non-O1. Whereas both peptides lactoferricin (17-30) and lactoferrampin (265-284) did not have bactericidal activity, 40 microM of lactoferrin chimera (a fusion of the two peptides) inhibited the growth of all Vibrio tested to the same extent as the antibiotic gentamicin. The cidal effect of LFchimera showed a clear concentration response in contrast to bovine lactoferrin which showed higher inhibition at 10 microM than at 40 microM. FITC-labeled LFchimera bound to the bacterial membranes. Moreover LFchimera permeabilized bacterial cells and membranes were seriously damaged. Finally, in experiments with the multidrug resistant isolate 727, sub-lethal doses of LFchimera strongly reduced the concentrations of ampicillin, gentamicin or kanamicin needed to reach more than 95% growth inhibition, suggesting synergistic effects. These data indicate that LFchimera is a potential candidate to combat the multidrug resistant pathogenic Vibrio species.

State transitions of Vibrio parahaemolyticus VBNC cells evaluated by flow cytometry.

BACKGROUND: Vibrio parahaemolyticus, in response to environmental conditions, may be present in a viable but nonculturable state (VBNC), which can still be responsible for cases of infectious diseases in humans. METHODS: The characterization of the cellular states of V. parahaemolyticus during entry into, persistence in, and resuscitation from the VBNC state, was assessed through plate culture method and epifluorescence microscope evaluation of actively respiring cells. Flow cytometry (FCM) in combination with SYBR Green I (SG) and propidium iodide allowed us to distinguish between viable, dead, and damaged-cells. Immunofluorescence labeling detected by FCM was used to study changes in antibody affinity. RESULTS: Two groups of bacteria, one with High Nucleic Acid (HNA) and one having Low Nucleic Acid (LNA) content, were differentiated using SG and FCM and each was correlated with cell viability. With the aging of the microcosm, the LNA bacteria population increased while the HNA population gradually disappeared. Cytofluorimetric immunofluorescence analyses showed that the bacterial cell levels dropped from 95% at day 0 to 40% at day 26 and by day 29, antibody affinity was virtually lost. FCM analyses of light scatter signals expressed by cell population highlighted morphological changes indicating a reduction in cell size, as also shown by scanning electron microscopy images and variations in cell structure. CONCLUSIONS: The methodology used has provided useful data in relation to the state transitions of V. parahaemolyticus regarding cell viability, antigenic surface components, and the quantification of morphological variations during its entry into the VBNC state.

Response of Vibrio parahaemolyticus to ethanol shock.

In this study, the growth and survival of Vibrio parahaemolyticus in the presence of 0.0-8.0% ethanol was first examined. V. parahaemolyticus was then exposed to a sub-lethal dose of 5.0% ethanol for 30 and 60 min (ethanol shock). Morphological changes and alterations in cell leakage, thermal tolerance at 47 degrees C, and susceptibility to 8% ethanol and low temperature (4 and -18 degrees C) of V. parahaemolyticus caused by ethanol shock were investigated. In addition, recoveries of the ethanol-shocked cells of V. parahaemolyticus on thiosulfate-citrate-bile salts-sucrose agar (TCBS) and TSA-3.0% NaCl were also compared. The findings revealed that the presence of ethanol in TSB-3.0% NaCl at 6.0-8.0% and 5.0% or less, exerted bactericidal and partial growth inhibition effect, respectively, on V. parahaemolyticus. Recovery of ethanol-shocked cells of V. parahaemolyticus was significantly (P<0.05) less on TCBS than on TSA-3.0% NaCl. A significantly (P<0.05) marked increase of protein and nucleic acid material in the supernatant of cell suspension was found after cells of V. parahaemolyticus were exposed to ethanol shock. Extensive cell disruption, wrinkling and cell-wall pitting, indicative of cell-surface damage were also noted on the ethanol-shocked cells. Ethanol-shocked cells of V. parahaemolyticus exhibited a similar yet higher susceptibility at 4 and -18 degrees C compared with the control cells. Moreover, there was a marked increase in the thermal tolerance and resistance to 8.0% ethanol with cells of V. parahaemolyticus after ethanol shock. Finally, the duration of ethanol shock testing did not affect the extent of increased thermal tolerance. While cells of V. parahaemolyticus subjected to ethanol shock for 60 min showed an increase in their resistance to 8.0% ethanol, they also showed an increase in susceptibility at -18 degrees C, than those ethanol shocked for 30 min.