Inactivation of Vibrio parahaemolyticus in shucked raw oyster ( Grassostrea gigas) and clam ( Venerupis phillippinarum) by using a combination of NaClO and gamma irradiation.

This study investigated the synergistic effects of sodium hypochlorite (NaClO) and gamma irradiation combination against Vibrio parahaemolyticus in shucked oysters and clams. V. parahaemolyticus decreased to 1.1-5.6 log10 CFU/g in oysters and 1.1-5.7 log10CFU/g in clams by NaClO (20-80 ppm) + γ irradiation (0.1-2.0 kGy) combinations. V. parahaemolyticus was not detected by 60 or 80 ppm NaClO + 2.0 kGy. Synergistic reduction of >1 log was observed by 60 ppm NaClO + 0.3-2 kGy and 80 ppm NaClO + 0.5 or 2 kGy. Specifically, >2 log of the synergistic reduction was obtained by 60 or 80 ppm NaClO + 2 kGy. Furthermore, using the Weibull model, 5D values (5-log reductions) were calculated for 60 or 80 ppm NaClO + 0.5-0.9 kGy. No significant differences were observed for all sensory parameters between samples of 2.0 kGy + 0-80 ppm NaClO. This study suggests that 60 ppm NaClO + 2.0 kGy in reducing 7-log V. parahaemolyticus without any deteriorative changes of sensory qualities could be a potential strategy for post-harvest process in seafood processing and distribution to enhance the microbial safety of molluscan shellfish.

Photodynamic effect of curcumin on Vibrio parahaemolyticus.

Vibrio parahaemolyticus (V. parahaemolyticus) is currently a major cause of bacterial diarrhoea associated with seafood consumption. The objective of this study was to determine the inactivation effect of curcumin-mediated photodynamic action on V. parahaemolyticus. First of all, V. parahaemolyticus suspended in PBS buffer was irradiated by a visible light from a LED light source with an energy density of 3.6J/cm(2). Colony forming units (CFU) were counted and the viability of V. parahaemolyticus cells was calculated after treatment. Singlet oxygen ((1)O2) production after photodynamic action of curcumin was evaluated using 9,10-Anthracenediyl-bis (methylene) dimalonic acid (ADMA). Bacterial outer membrane protein was extracted and analyzed using electrophoresis SDS-PAGE. DNA and RNA of V. parahaemolyticus were also extracted and analyzed using agarose gel electrophoresis after photodynamic treatment. Finally, the efficacy of photodynamic action of curcumin was preliminarily evaluated in the decontamination of V. parahaemolyticus in oyster. Results showed that the viability of V. parahaemolyticus was significantly decreased to non-detectable levels over 6.5-log reductions with the curcumin concentration of 10 and 20µM. Photodynamic action of curcumin significantly increased the singlet oxygen level with the curcumin concentration of 10µM. Notable damage was found to bacterial outer membrane proteins and genetic materials after photodynamic treatment. Photodynamic action of curcumin reduced the number of V. parahaemolyticus contaminating in oyster to non-detectable level. Our findings demonstrated that photodynamic action of curcumin could be a potentially good method to inactivate Vibrio parahaemolyticus contaminating in oyster.

Combined treatment of UVA irradiation and antibiotics induces greater bactericidal effects on Vibrio parahaemolyticus.

The presence of antibiotics in the environment and their subsequent impact on the development of multi-antibiotic resistant bacteria has raised concerns globally. Consequently, much research is focused on a method to produce a better disinfectant. We have established a disinfectant system using UVA-LED that inactivates pathogenic bacteria. We assessed the bactericidal efficiency of a combination of UVA-LED and antibiotics against Vibrio parahaemolyticus. Combined use of antibiotic drugs and UVA irradiation was more bactericidal than UVA irradiation or antibacterial drugs alone. The bactericidal synergy was observed at low concentrations of each drug that are normally unable to kill the bacteria. This combination has the potential to become a sterilization technology.

Antibacterial efficacy of 405, 460 and 520 nm light emitting diodes on Lactobacillus plantarum, Staphylococcus aureus and Vibrio parahaemolyticus.

AIMS: Little information is available on a direct comparison of the antibacterial efficacy of light emitting diode (LEDs) of different peak wavelengths. Thus, the objective of this study was to evaluate the effect of LEDs of three different wavelengths on bacterial inactivation. METHODS AND RESULTS: Lactobacillus plantarum, Staphylococcus aureus and Vibrio parahaemolyticus were illuminated with 405, 460 and 520 nm LEDs at 4, 10 and 25°C respectively. Inactivation curves were plotted and fitted using Gompertz Model. Illumination with 405 and 460 nm LED produced significant inactivation (P < 0·05) in the population of V. parahaemolyticus (>4 log) while Lact. plantarum and Staph. aureus showed relatively less susceptibility to the LED illumination. The 520 nm LED produced negligible inactivation. CONCLUSIONS: The 405 and 460 nm LEDs proved more effective in inactivating the selected foodborne bacteria in this study compared to 520 nm LED. The 405 nm LED showed the greatest antibacterial effect at the same level of energy dose. SIGNIFICANCE AND IMPACT OF THE STUDY: The results in this study demonstrated the antibacterial efficacy of 405 nm LED on Lact. plantarum and V. parahaemolyticus, suggesting its potential for use in food industry for the control of these micro-organisms.

Inactivation of Vibrio parahaemolyticus by antimicrobial photodynamic technology using methylene blue.

BACKGROUND: Vibrio parahaemolyticus is the leading causative pathogen of gastroenteritis often related to contaminated seafood. Photodynamic inactivation has been recently proposed as a strategy for killing cells and viruses. The objective of this study was to verify the bactericidal effects caused by photodynamic inactivation using methylene blue (MB) over V. parahaemolyticus via flow cytometry, agarose gel electrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Vibrio parahaemolyticus counts were determined using the most probable number method. A scanning electron microscope and a transmission electron microscope were employed to intuitively analyze internal and external cell structure. RESULTS: Combination of MB and laser treatment significantly inhibited the growth of V. parahaemolyticus. The inactivation rate of V. parahaemolyticus was >99.99% and its counts were reduced by 5 log10 in the presence of 0.05 mg mL(-1) MB when illuminated with visible light (power density 200 mW cm(-2)) for 25 min. All inactivated cells showed morphological changes, leakage of cytoplasm and degradation of protein and DNA. CONCLUSION: Results from this study indicated that photodynamic technology using MB produced significant inactivation of V. parahaemolyticus mainly brought about by the degradation of protein and DNA.

Changes in global gene expression of Vibrio parahaemolyticus induced by cold- and heat-stress.

BACKGROUND: Vibrio (V.) parahaemolyticus causes seafood-borne gastro-intestinal bacterial infections in humans worldwide. It is widely found in marine environments and is isolated frequently from seawater, estuarine waters, sediments and raw or insufficiently cooked seafood. Throughout the food chain, V. parahaemolyticus encounters different temperature conditions that might alter metabolism and pathogenicity of the bacterium. In this study, we performed gene expression profiling of V. parahaemolyticus RIMD 2210633 after exposure to 4, 15, 20, 37 and 42 °C to describe the cold and heat shock response. METHODS: Gene expression profiles of V. parahaemolyticus RIMD 2210633 after exposure to 4, 15, 20, 37 and 42 °C were investigated via microarray. Gene expression values and RT-qPCR experiments were compared by plotting the log2 values. Moreover, volcano plots of microarray data were calculated to visualize the distribution of differentially expressed genes at individual temperatures and to assess hybridization qualities and comparability of data. Finally, enriched terms were searched in annotations as well as functional-related gene categories using the Database for Annotation, Visualization and Integrated Discovery. RESULTS: Analysis of 37 °C normalised transcriptomics data resulted in differential expression of 19 genes at 20 °C, 193 genes at 4 °C, 625 genes at 42 °C and 638 genes at 15 °C. Thus, the largest number of significantly expressed genes was observed at 15 and 42 °C with 13.3 and 13%, respectively. Genes of many functional categories were highly regulated even at lower temperatures. Virulence associated genes (tdh1, tdh2, toxR, toxS, vopC, T6SS-1, T6SS-2) remained mostly unaffected by heat or cold stress. CONCLUSION: Along with folding and temperature shock depending systems, an overall temperature-dependent regulation of expression could be shown. Particularly the energy metabolism was affected by changed temperatures. Whole-genome gene expression studies of food related pathogens such as V. parahaemolyticus reveal how these pathogens react to stress impacts to predict its behaviour under conditions like storage and transport.

Strain-level visualized analysis of cold-stressed Vibrio parahaemolyticus based on MALDI-TOF mass fingerprinting.

In this study, strain-level visualized analysis of cold-stressed Vibrio parahaemolyticus based on matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass fingerprinting was investigated. All the peptide mass fingerprinting profiles obtained were analyzed by self-organized map (SOM) and cluster analysis. Our results showed that the peptide mass fingerprinting profiles of V. parahaemolyticus substantially changed under cold stress at strain level. The cold-stressed V. parahaemolyticus strains were distributed to 14 neurons by SOM classification, almost totally different from the controls. This is the first time that so many strains had been chosen to study bacterial cold stress responses, which can help promote an overall understanding to stress responses of cold-stressed strains.

Simultaneous irradiation with different wavelengths of ultraviolet light has synergistic bactericidal effect on Vibrio parahaemolyticus.

Ultraviolet (UV) irradiation is an increasingly used method of water disinfection. UV rays can be classified by wavelength into UVA (320-400 nm), UVB (280-320 nm), and UVC (<280 nm). We previously developed UVA sterilization equipment with a UVA light-emitting diode (LED). The aim of this study was to establish a new water disinfection procedure using the combined irradiation of the UVA-LED and another UV wavelength. An oxidative DNA product, 8-hydroxy-2'-deoxyguanosine (8-OHdG), increased after irradiation by UVA-LED alone, and the level of cyclobutane pyrimidine dimers (CPDs) was increased by UVC alone in Vibrio parahaemolyticus. Although sequential irradiation of UVA-LED and UVC-induced additional bactericidal effects, simultaneous irradiation with UVA-LED and UVC-induced bactericidal synergistic effects. The 8-OHdG and CPDs production showed no differences between sequential and simultaneous irradiation. Interestingly, the recovery of CPDs was delayed by simultaneous irradiation. The synergistic effect was absent in SOS response-deficient mutants, such as the recA and lexA strains. Because recA- and lexA-mediated SOS responses have crucial roles in a DNA repair pathway, the synergistic bactericidal effect produced by the simultaneous irradiation could depend on the suppression of the CPDs repair. The simultaneous irradiation of UVA-LED and UVC is a candidate new procedure for effective water disinfection.

Depuration of Oysters (Crassostrea gigas) contaminated with Vibrio parahaemolyticus and Vibrio vulnificus with UV light and chlorinated seawater.

The efficacy of depuration using UV light and chlorinated seawater for decontaminating Vibrio parahaemolyticus and Vibrio vulnificus from oysters was investigated. Oysters were contaminated with a five-strain cocktail of V. parahaemolyticus or V. vulnificus to levels of 10(4) to 10(5) CFU ml(-1) for bioaccumulation. The depuration was conducted in a closed system in which 350 liters of seawater was recirculated at a rate of 7 liters/min for 48 h at room temperature. Counts of V. parahaemolyticus or V. vulnificus were determined at 0, 6, 18, 24, and 48 h. Three treatments were conducted: T1, control treatment; T2, UV treatment; and T3, UV plus chlorine treatment. After 48 h of depuration of V. parahaemolyticus, T3 reduced the count by 3.1 log most probable number (MPN) g(-1) and T2 reduced the count by 2.4 log MPN g(-1), while T1 reduced the count by only 2.0 log MPN g(-1). After 48 h of depuration of V. vulnificus, T2 and T3 were efficient, reducing the counts by 2.5 and 2.4 log MPN g(-1), respectively, while T1 reduced the count by only 1.4 log MPN g(-1). The UV light plus chlorine treatment was more efficient for controlling V. parahaemolyticus in oysters. Both UV light and UV light plus chlorine were efficient for V. vulnificus. The present study is the first report showing the efficacy of depuration systems for decontaminating V. parahaemolyticus and V. vulnificus in oysters cultivated on the Brazilian coast. This study provides information on processes that can contribute to controlling and preventing such microorganisms in oysters and could be used for effective postharvest treatment by restaurants and small producers of oysters on the coast of Brazil.

Control of the potential health hazards of smoked fish by gamma irradiation.

This study aims to control the presence of Listeria monocytogenes and Vibrio parahaemolyticus and the formation of biogenic amines in cold-smoked salmon by gamma irradiation. Irradiation at doses of 3 and 1 kGy inactivated 6.59 and 6.05 log cfu/g of L. monocytogenes and V. parahaemolyticus in the inoculated samples, respectively. Furthermore, irradiation of the un-inoculated samples significantly decreased their microbial populations of mesophilic aerobic bacteria, anaerobic bacteria, psychrophilic bacteria, lactic acid bacteria, and molds and yeasts. The Enterobacteriaceae were almost undetectable in samples irradiated at 2 kGy dose. The concentrations of biogenic amines significantly decreased in the irradiated samples due to microbial inactivation. However, irradiation of samples had no significant effects on their moisture and salt contents as well as on their pH values, total volatile base nitrogen, and trimethylamine nitrogen contents, but significantly decreased their amounts of phenolic compounds and increased their levels of thiobarbituric acid reactive substances. Moreover, irradiation treatments at doses up to 3 kGy showed no significant effect on the sensory acceptability of samples. Therefore, gamma irradiation at dose of 3 kGy can be successfully applied to provide significant improvement in the safety of cold smoked salmon with respect to L. monocytogenes, V. parahaemolyticus, and biogenic amines without adverse effects on chemical or sensory quality attributes of the product.

Predictive models for the effect of storage temperature on Vibrio parahaemolyticus viability and counts of total viable bacteria in Pacific oysters (Crassostrea gigas).

Vibrio parahaemolyticus is an indigenous bacterium of marine environments. It accumulates in oysters and may reach levels that cause human illness when postharvest temperatures are not properly controlled and oysters are consumed raw or undercooked. Predictive models were produced by injecting Pacific oysters (Crassostrea gigas) with a cocktail of V. parahaemolyticus strains, measuring viability rates at storage temperatures from 3.6 to 30.4°C, and fitting the data to a model to obtain parameter estimates. The models were evaluated with Pacific and Sydney Rock oysters (Saccostrea glomerata) containing natural populations of V. parahaemolyticus. V. parahaemolyticus viability was measured by direct plating samples on thiosulfate-citrate-bile salts-sucrose (TCBS) agar for injected oysters and by most probable number (MPN)-PCR for oysters containing natural populations. In parallel, total viable bacterial counts (TVC) were measured by direct plating on marine agar. Growth/inactivation rates for V. parahaemolyticus were -0.006, -0.004, -0.005, -0.003, 0.030, 0.075, 0.095, and 0.282 log10 CFU/h at 3.6, 6.2, 9.6, 12.6, 18.4, 20.0, 25.7, and 30.4°C, respectively. The growth rates for TVC were 0.015, 0.023, 0.016, 0.048, 0.055, 0.071, 0.133, and 0.135 log10 CFU/h at 3.6, 6.2, 9.3, 14.9, 18.4, 20.0, 25.7, and 30.4°C, respectively. Square root and Arrhenius-type secondary models were generated for V. parahaemolyticus growth and inactivation kinetic data, respectively. A square root model was produced for TVC growth. Evaluation studies showed that predictive growth for V. parahaemolyticus and TVC were "fail safe." The models can assist oyster companies and regulators in implementing management strategies to minimize V. parahaemolyticus risk and enhancing product quality in supply chains.

A study of bactericidal effect and optimization of pathogenic bacteria using TiO2 photocatalyst.

The photocatalytic degradation of Salmonella choleraesuis subsp. and Vibrio parahaemolyticus in water by TiO2 catalysts was investigated in a batch reactor. After 30 min of irradiation with UV light in the presence of 1 mg/ml of TiO2, death ratio of S. choleraesuis subsp. and V. parahaemolyticus was 60% and 83%, respectively. And complete killing of the cells was achieved after 3 h of illumination in the presence of TiO2. We established the response surface methodology to investigate the effect of principal parameters on the pathogenic bacteria sterilization such as TiO2 concentration, pH and temperature. By applying response surface analysis to the bactericidal effect of S. almonella choleraesuis subsp. and V. parahaemolyticus, we found that the cell death ratio was influenced significantly by the first order term of TiO2 concentration.

Identification of outer membrane proteins of Vibrio parahaemolyticus and Vibrio alginolyticus altered in response to γ-irradiation or long-term starvation.

Vibrio parahaemolyticus and Vibrio alginolyticus were subjected to γ-irradiation (0.5 kGy) or starvation by incubation for 8 months in seawater to study modifications in their outer membrane protein patterns. After treatment, outer membrane protein profiles of starved or γ-irradiated bacteria were found to be altered when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Altered proteins were identified by mass spectrometry (MS and MS/MS) and analyses revealed that OmpU can be considered a starvation stress-induced protein. In addition, expression of OtnA, OmpW, OmpA and peptidoglycan-associated lipoprotein decreased to non-detectable levels in starved cells. Furthermore, MltA-interacting protein MipA appeared under γ-irradiation or starvation conditions. Thus, it can be considered to be a γ-irradiation, long-term starvation stress protein in some vibrios.

Photoinactivation of Staphylococcus aureus and Vibrio parahaemolyticus in the model aquatic microcosm: effect of light intensity and dissolved biodegradable organic compound.

The impact of light (1,000 × 100,000 lx) on the inactivation of S. aureus and V. parahaemolyticus has been assessed under different concentrations of dissolved biodegradable organic compound (BOC) at pH 7.0. First, a gradual decrease in the number of cultivable cells was observed. Secondly, a cell reactivation was observed and it was marked in the absence of BOC. In the absence of BOC, the lowest value of cell inhibition rate (CIR) during the first 3 h was 0.138 h(-1) for S. aureus and 0.218 h(-1) for V. parahaemolyticus. In the presence of 10,100 and 1,000 mg/l of BOC, it was 0.196 h(-1), 0.243 h(-1) and 0.257 h(-1) for S. aureus respectively, and 0.285 h(-1), 0.306 h(-1) and 0.409 h(-1) for V. parahaemolyticus respectively. The CIRs values of each bacterial species significantly varied (P<0.001) with the changes in BOC concentration. In most cases, no significant difference was noted in the CIRs of both species when they were under the same light intensity and BOC. Nevertheless, it seems important to consider the impact of dissolved BOC during the treatment of bacterial polluted water.

Modulation of responses of Vibrio parahaemolyticus O3:K6 to pH and temperature stresses by growth at different salt concentrations.

Vibrio parahaemolyticus inhabits marine, brackish, and estuarine waters worldwide, where fluctuations in salinity pose a constant challenge to the osmotic stress response of the organism. Vibrio parahaemolyticus is a moderate halophile, having an absolute requirement for salt for survival, and is capable of growth at 1 to 9% NaCl. It is the leading cause of seafood-related bacterial gastroenteritis in the United States and much of Asia. We determined whether growth in differing NaCl concentrations alters the susceptibility of V. parahaemolyticus O3:K6 to other environmental stresses. Vibrio parahaemolyticus was grown at a 1% or 3% NaCl concentration, and the growth and survival of the organism were examined under acid or temperature stress conditions. Growth of V. parahaemolyticus in 3% NaCl versus that in 1% NaCl increased survival under both inorganic (HCl) and organic (acetic acid) acid conditions. In addition, at 42 degrees C and -20 degrees C, 1% NaCl had a detrimental effect on growth. The expression of lysine decarboxylase (encoded by cadA), the organism's main acid stress response system, was induced by both NaCl and acid conditions. To begin to address the mechanism of regulation of the stress response, we constructed a knockout mutation in rpoS, which encodes the alternative stress sigma factor, and in toxRS, a two-component regulator common to many Vibrio species. Both mutant strains had significantly reduced survival under acid stress conditions. The effect of V. parahaemolyticus growth in 1% or 3% NaCl was examined using a cytotoxicity assay, and we found that V. parahaemolyticus grown in 1% NaCl was significantly more toxic than that grown in 3% NaCl.

Effect of X-ray treatments on inoculated Escherichia coli O157: H7, Salmonella enterica, Shigella flexneri and Vibrio parahaemolyticus in ready-to-eat shrimp.

This study was conducted to evaluate the inactivation effect of X-ray treatments on Escherichia coli O157: H7, Salmonella enteric (S. enterica), Shigella flexneri (S. flexneri) and Vibrio parahaemolyticus (V. parahaemolyticus) artificially inoculated in ready-to-eat (RTE) shrimp. A mixed culture of three strains of each tested pathogen was used to inoculate RTE shrimp. The shrimp samples were inoculated individually with selected pathogenic bacteria then aseptically placed in sterile plastic cups and air-dried at 22 degrees C for 30 min (to allow bacterial attachment) in the biosafety cabinet prior to X-ray treatments. The inoculated shrimp samples were then placed in sterilized bags and treated with 0.1, 0.2, 0.3, 0.5, 0.75, 1.0, 2.0, 3.0 and 4.0 kGy X-ray at ambient temperature (22 degrees C and 60% relative humidity). Surviving bacterial populations were evaluated using a non-selective medium (TSA) with the appropriate selective medium overlay for each bacterium; CT-SMAC agar for E. coli O157: H7, XLD for S. enterica and S. flexneri and TCBS for V. parahaemolyticus. More than a 6 log CFU reduction of E. coli O157: H7, S. enterica, S. flexneri and V. parahaemolyticus was achieved with 2.0, 4.0, 3.0 and 3.0 kGy X-ray, respectively. Furthermore, treatment with 0.75 kGy X-ray significantly reduced the initial microflora on RTE shrimp samples from 3.8 +/- 0.2 log CFU g(-1) to less than detectable limit (<1.0 log CFU g(-1)).

Alterations of outer membrane proteins and virulence genes expression in gamma-irradiated Vibrio parahaemolyticus and Vibrio alginolyticus.

Gamma-irradiation technology sterilizes microorganisms and thereby prevents decay and improves the safety and shelf stability of food products. In this study we treated the foodborne pathogens Vibrio parahaemolyticus and Vibrio alginolyticus with gamma-irradiation (0.5 kGy) to evaluate their adaptative response. Outer membrane protein patterns of irradiated bacteria were found altered when analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. These modifications were manifested by the appearance and/or disappearance of bands as well as in the expression level of certain proteins. In addition, we searched for the presence of eight Vibrio cholerae virulence genes, toxR, toxS, toxRS, ctxA, zot, ace, toxT, and virulence pathogenicity island (VPI), in the genome of investigated strains. The expression of toxR, toxS, VPI, and ace genes in gamma-irradiated bacteria, studied by reverse transcriptase polymerase chain reaction, was altered. These variations were manifested by an increase and/or a decrease in the expression level of tested virulence genes.

Inactivation of Vibrio parahaemolyticus in pure culture, whole live and half shell oysters (Crassostrea virginica) by X-ray.

AIMS: To study the inactivation effect of different doses of X-ray on Vibrio parahaemolyticus in pure culture, inoculated whole live and half shell oysters and to evaluate the efficacy of X-ray doses on reduction of inherent microflora on oysters. METHODS AND RESULTS: X-ray was produced using RS 2400 generator system (Rad Source Technologies Inc.). Pure culture of V. parahaemolyticus, inoculated half and whole shell oysters with V. parahaemolyticus were treated with 0.0, 0.1, 0.5, 0.75, 1.0, 1.5, 2.0, 3.0 and 5.0 kGy X-ray. Surviving bacteria in the pure culture and inoculated oysters, before and after treatment, were enumerated using overlay plating (in TSA then TCBS) and most probable number (MPN) methods. A greater than 6.0 log reduction of V. parahaemolyticus was observed with 0.75, 2.0 and 5.0 kGy X-ray for pure culture, half shell and whole shell oysters, respectively. Treatment with 0.75, 2.0 and 5.0 kGy X-ray reduced the MPN to <3 for pure culture, half and whole shell oysters, respectively. Treatment with 1.0 kGy X-ray significantly (P < 0.05) reduced the inherent micro-organisms on whole shell oysters from 4.7 +/- 0.1 to less than the detectable limit (<1.0 log CFU g(-1)). CONCLUSIONS: X-ray (1-5 kGy) significantly (P < 0.05) reduced V. parahaemolyticus and inherent microflora on oysters to less than detectable limit (<1.0 log CFU g(-1)). SIGNIFICANCE AND IMPACT OF THE STUDY: Treatment with X-ray could control pathogenic bacteria and extend the shelf life of oysters.

Enhancement of UV light sensitivity of a Vibrio parahaemolyticus O3:K6 pandemic strain due to natural lysogenization by a telomeric phage.

The Vibrio parahaemolyticus O3:K6 pandemic clonal strain was first observed in southern Chile in 2004 and has since caused approximately 8,000 seafood-related diarrhea cases in this region. The massive proliferation of the original clonal population offers a unique opportunity to study the evolution of a bacterial pathogen in its natural environment by detection and characterization of emerging bacterial variants. Here, we describe a group of pandemic variants characterized by the presence of a 42-kb extrachromosomal DNA that can be recovered by alkaline extraction. Upon treatment with mitomycin C, these variants lyse with production of a myovirus containing DNA of equal size to the plasmid but which cannot be recovered by alkaline extraction. Plasmid and phage DNAs show similar restriction patterns corresponding to enzyme sites in a circular permutation. Sequenced regions showed 81 to 99% nucleotide similarity to bacteriophage VHML of Vibrio harveyi. Altogether these observations indicate that the 42-kb plasmid corresponds to a prophage, consisting of a linear DNA with terminal hairpins of a telomeric temperate phage with a linear genome. Bacteria containing the prophage were 7 to 15 times more sensitive to UV radiation, likely due to phage induction by UV irradiation as plasmid curing restored the original sensitivity. The enhanced UV sensitivity could have a significant role in reducing the survival and propagation capability of the V. parahaemolyticus pandemic strain in the ocean.

Bactericidal effect of TiO2 on the selected Vibrio parahaemolyticus and optimization using response surface methodology.

In this work, the bactericidal effect of TiO2 on selected typical food pathogenic bacteria, Vibrio parahaemolyticus was studied. V parahaemolyticus is an important pathogen of humans and aqua-cultured animals. We established the response surface methodology (Box-Behnken Design) to investigate the effect of principal parameters on the cell sterilization such as TiO2 concentration, UV illumination time, temperature, and pH. The sterilization rate reached maximum value at the TiO2 concentration of 1.0 mg/ml. During irradiation under the time of 30 min with UV light with the 1g-TiO2/l, the sterilization rate was greater than 85%, and 99% or more cell lost their viability with 3 hours of irradiation. Sterilization rate of the cell increased with decrease in the pH and temperature.