Improved fluorogenic assay for rapid detection of Vibrio parahaemolyticus in foods

An improved fluorogenic assay for the rapid detection of Vibrio parahaemolyticus was developed. In the improved assay, the enrichment of V. parahaemolyticus was carried out in arabinose-glucuronate medium (0.5% arabinose, 0.25% glucuronate, 0.1% polypeptone, 0.1% yeast extract, 0.1% ammonium sulfate, 2% NaCl, 2 micrograms of polymyxin B sulfate per ml, pH 8.5) at 37 degrees C. After the cultivation, the trypsinlike activity of the bacteria was measured by fluorescence with the fluorogenic substrate benzoyl-L-arginine-7-aminomethylcoumarin. Even in the presence of 3 x 10(5) cells of Vibrio alginolyticus, 20 cells of V. parahaemolyticus were clearly detected after a 6-h enrichment cultivation by the assay. Fifty contaminated samples of 14 seafoods were examined for V. parahaemolyticus by the fluorogenic assay after enrichment cultivation for 6 or 8 h. The results were then compared with those obtained by the conventional bromothymol blue Teepol agar assay and the most-probable-number method. There was a linear relationship between trypsinlike activity measured by the assay and the number of V. parahaemolyticus cells in seafood as determined by the bromothymol blue Teepol agar and most-probable-number methods. Correlation coefficients were 0.95 and 0.93 after a 6-h cultivation and an 8-h cultivation, respectively. The presence of 10 cells of V. parahaemolyticus per gram of seafood sample was detected after a 10-h total detection time by the fluorogenic assay.     

Comparison of a fluorogenic assay with a conventional method for rapid detection of Vibrio parahaemolyticus in seafoods.

A conventional method and a fluorogenic assay for the detection of Vibrio parahaemolyticus were compared. Among 29 seafood samples examined for the presence of V. parahaemolyticus, 17 samples harbored V. parahaemolyticus, and trypsinlike activity was noticed in 19 seafoods. The added fluorogenic substrate was cleaved in single samples of shrimp, turbo, and cuttlefish from which V. parahaemolyticus could not be isolated by the conventional method. Vibrio alginolyticus, in addition to V. parahaemolyticus, was found to exhibit intracellular trypsinlike activity. Trypsinlike activity in seafoods was observed after the most probable number for the initial density of V. parahaemolyticus-like organisms was found to have reached > 10(2) per g. A V. parahaemolyticus inoculum at 10(4) CFU/ml in arabinose-glucuronate medium was required to attain growth to 10(6) CFU/ml, which is the level necessary for the release of detectable amounts of fluorescent compound from the added substrate.     

Improved method for detection of Vibrio parahaemolyticus in seafood.

We have developed a new, effective procedure for detecting Vibrio parahaemolyticus in seafoods using enrichment and plating onto a chromogenic agar medium. Samples were cultured in salt Trypticase soy broth, which is a nonselective medium, and then a portion of the culture was cultured with salt polymyxin broth, which is a selective medium for V. parahaemolyticus. This two-step enrichment was more effective than the one-step enrichment in salt polymyxin broth alone. The enrichment cultures were then plated onto a new chromogenic agar containing substrates for beta-galactosidase. The V. parahaemolyticus colonies developed a purple color on this growth medium that distinguished them from other related bacterial strains. V. parahaemolyticus was isolated more frequently from naturally contaminated seafood samples using the chromogenic agar than thiosulfate citrate bile salts sucrose agar medium, which is currently used for the isolation of V. parahaemolyticus. Our findings suggest that this new enrichment and isolation scheme is more sensitive and accurate for identifying V. parahaemolyticus in seafood samples than previously used methods.     

Prevalence of pandemic thermostable direct hemolysin-producing Vibrio parahaemolyticus O3:K6 in seafood and the coastal environment in Japan

Although thermostable direct hemolysin (TDH)-producing Vibrio parahaemolyticus has caused many infections in Asian countries, the United States, and other countries, it has been difficult to detect the same pathogen in seafoods and other environmental samples. In this study, we detected and enumerated tdh gene-positive V. parahaemolyticus in Japanese seafoods with a tdh-specific PCR method, a chromogenic agar medium, and a most-probable-number method. The tdh gene was detected in 33 of 329 seafood samples (10.0%). The number of tdh-positive V. parahaemolyticus ranged from <3 to 93/10 g. The incidence of tdh-positive V. parahaemolyticus tended to be high in samples contaminated with relatively high levels of total V. parahaemolyticus. TDH-producing strains of V. parahaemolyticus were isolated from 11 of 33 tdh-positive samples (short-necked clam, hen clam, and rock oyster). TDH-producing strains of V. parahaemolyticus were also isolated from the sediments of rivers near the coast in Japan. Representative strains of the seafood and sediment isolates were examined for the O:K serovar and by the PCR method specific to the pandemic clone and arbitrarily primed PCR and pulsed-field gel electrophoresis techniques. The results indicated that most O3:K6 tdh-positive strains belonged to the pandemic O3:K6 clone and suggested that serovariation took place in the Japanese environment.     

Enrichment of Vibrio parahaemolyticus in a Simple Medium

A medium which contained 3% NaCl and 0.2% Teepol in 1/15 M phosphate buffer was prepared and was evaluated to be a useful enrichment medium for the isolation of Vibrio parahaemolyticus in marine specimens. Glucose salt Teepol broth produced a poorer result than direct culture.     

Recovery of chill-stressed Vibrio parahaemolyticus from oysters with enrichment broths supplemented with magnesium and iron salts.

The effects of magnesium and iron salts on the recovery and growth of chill-stressed cells of Vibrio parahaemolyticus were studied. Supplementation of glucose salt Teepol (GST) broth with 20 to 100 mM of Mg2+ significantly (P less than or equal to 0.05) increased the number of cells recovered from oyster homogenate stored at 3 degrees C. Populations detected with supplemented GST were comparable to those obtained with Horie arabinose ethyl violet (HAE) broth, with or without Mg2+. Recovery of V. parahaemolyticus from homogenates stored at -18 degrees C was also improved when enrichment broths supplemented with Mg2+ were used. Ferric iron (added as FeCl3) at 240 microM in GST and 240 or 960 microM in HAE significantly enhanced the extent of recovery of chilled cells. Ferrous iron was generally less effective. Teepol did not influence the growth of nonchilled cells, but significantly reduced the viable population in suspensions of chilled cells when used at a level of 0.4% in GST. The relatively high pH (9.0) of HAE caused a significant reduction in the number of viable, chill-stressed cells of V. parahaemolyticus. The overall results indicated that HAE broth is superior to GST for recovering V. parahaemolyticus from refrigerated and frozen oyster homogenates.     

Application of real-time PCR for quantitative detection of Vibrio parahaemolyticus from seafood in eastern China

Vibrio parahaemolyticus is recognized as a leading human food-borne pathogen. A TaqMan PCR assay based on the gyrase B gene (gyrB) sequence of V. parahaemolyticus was developed for quantitative detection of V. parahaemolyticus in seafood. The study involving 27 V. parahaemolyticus and 10 strains of other species indicated that the real-time PCR test was highly specific. The sensitivity of the assay was approximately a single CFU per PCR in pure culture and six to eight CFU per PCR in spiked raw oyster, respectively. Real-time PCR values of artificially inoculated oyster homogenates correlated well with plate counts determined using culture methods. A total of 300 seafood samples were analyzed and 78 (26%) of these samples were positive for V. parahaemolyticus using a conventional culture method and 97 (32.3%) using the real-time PCR assay. All culture-positive samples were PCR-positive. However, 19 samples positive by PCR were culture-negative. The results show that retail seafood is commonly contaminated with V. parahaemolyticus in harvest season in eastern China. These data also indicate that real-time PCR can provide sensitive species-specific detection and quantification of V. parahaemolyticus in seafood without prior isolation and characterization of the bacteria by traditional microbiological methods.     

Rapid detection and enumeration of trh-carrying Vibrio parahaemolyticus with the alkaline phosphatase-labelled oligonucleotide probe

An alkaline phosphatase (AP)-labelled oligonucleotide probe was developed to detect and enumerate trh(+)Vibrio parahaemolyticus in seafood. The probe was evaluated using 40 isolates of V. parahaemolyticus, 45 isolates of other vibrios and 55 non-vibrio isolates. The probe reacted specifically with V. parahaemolyticus possessing either the trh1 or trh2 variant of the trh gene and was found to be 100% specific for trh(+)V. parahaemolyticus. Using the trh probe, V. parahaemolyticus carrying trh gene was targeted in 34 seafood samples by direct plating and colony hybridization procedure. The trh(+)V. parahaemolyticus could be detected in five of 34 (14.7%) samples and the levels ranged from 5.0 x 10(2) to 3.4 x 10(3) cfu g(-1). Colonies of trh(+)V.parahaemolyticus were isolated from the five positive samples. Forty seafood samples were analysed for trh(+)V. parahaemolyticus by colony hybridization following enrichment in alkaline peptone water. 16 samples (40%) were positive for trh gene and trh(+)V. parahaemolyticus was isolated from 15 samples (37.5%). To assess the sensitivity of the trh probe, seafood homogenates spiked with known concentrations of trh-positive V. parahaemolyticus were plated and hybridized. Counts obtained using the probe were similar to those of inocula. The results suggest that the AP-labelled trh probe is useful for the detection and enumeration of trh(+)V. parahaemolyticus in seafood.     

Soft-agar-coated filter method for early detection of viable and thermostable direct hemolysin (TDH)- or TDH-related hemolysin-producing Vibrio parahaemolyticus in seafood

A novel method for detecting viable and thermostable direct hemolysin (TDH)-producing or TDH-related hemolysin (TRH)-producing Vibrio parahaemolyticus in seafood was developed. The method involved (i) enrichment culture, selective for viable, motile cells penetrating a soft-agar-coated filter paper, and (ii) a multiplex PCR assay targeting both the TDH gene (tdh) and TRH gene (trh) following DNase pretreatment on the test culture to eradicate any incidental DNAs that might have been released from dead cells of tdh- or trh-positive (tdh+ trh+) strains and penetrated the agar-coated filter. A set of preliminary laboratory tests performed on 190 ml of enrichment culture that had been inoculated simultaneously with ca. 100 viable cells of a strain of tdh+ trh+ V. parahaemolyticus and dense populations of a viable strain of tdh- and trh-negative V. parahaemolyticus or Vibrio alginolyticus indicated that the method detected the presence of viable tdh+ trh+ strains. Another set of preliminary tests on 190 ml of enrichment culture that had been initially inoculated with a large number of dead cells of the tdh+ trh+ strain together with dense populations of the tdh- and trh-negative strains confirmed that the method did not yield any false-positive results. Subsequent quasi-field tests using various seafood samples (ca. 20 g), each of which was experimentally contaminated with either or both hemolysin-producing strains at an initial density of ca. 5 to 10 viable cells per gram, demonstrated that contamination could be detected within 2 working days.     

Survey of Media for the Resuscitation of Heat-stressed Vibrio parahaemolyticus

Eighteen agar media were compared with Thiosulphate–Citrate–Bile salts–Sucrose agar for their ability to permit colony formation by heated and non-heated Vibrio parahaemolyticus . Seven enrichment broths were tested for their ability to recover V. parahaemolyticus from homogenates of crab meat. Water Blue–Alizarin Yellow agar, Arabinose–Ammonium Sulphate–Cholate agar and Horie's Arabinose–Ethyl Violet broth were found to be most suitable for detecting non-heated and heat-stressed V. parahaemolyticus .     

Application of polymerase chain reaction for detection of Vibrio parahaemolyticus associated with tropical seafoods and coastal environment

AIMS: To study the incidence of Vibrio parahaemolyticus in seafoods, water and sediment by molecular techniques vs conventional microbiological methods. METHODS AND RESULTS: Of 86 samples analysed, 28 recorded positive for V. parahaemolyticus by conventional microbiological method, while 53 were positive by the toxR-targeted PCR, performed directly on enrichment broth lysates. While one sample of molluscan shellfish was positive for tdh gene, trh gene was detected in three enrichment broths of molluscan shellfish. CONCLUSIONS: Direct application of PCR to enrichment broths will be useful for the rapid and sensitive detection of potentially pathogenic strains of V. parahemolyticus in seafoods. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrio parahaemolyticus is an important human pathogen responsible for food-borne gastroenteritis world-wide. As, both pathogenic and non-pathogenic strains of V. parahaemolyticus exist in the seafood, application of PCR specific for the virulence genes (tdh & trh) will help in detection of pathogenic strains of V. parahaemolyticus and consequently reduce the risk of food-borne illness.     

Suitability of some enrichment broths and diluents for enumerating cold- and heat-stressed Vibrio parahaemolyticus.

Vibrio parahaemolyticus cells were injured by chilling and heating, and their recovery was tested in glucose-salt-Teepol broth (GSTB), tryptic soy broth containing 7% NaCl (TSBS), Horie - arabinose - ethyl violet broth (HAEB), and water blue - alizarin yellow broth (WBAY). Exponential phase cells were more sensitive to cold shock than were stationary phase cells. Exposure of chill-injured V. parahaemolyticus to GSTB and TSBS resulted in 70 to 80% death; about 70% lethality was noted for heat-injured cells inoculated into TSBS. Neither HAEB nor WBAY enrichment media were lethal to stressed cells, although rates of growth were retarded. The 3% NaCl in 0.1 M potassium phosphate (pH 7.0) diluent proved to be most suitable for protecting against inactivation of cold- and heat-injured cells.     

Rapid Quantitative Detection of <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> in Seafood by MPN-PCR

This study aimed to adopt MPN-PCR (most probable number-polymerase chain reaction) for rapid detection of the quantity of Vibrio parahaemolyticus in seafood. V. parahaemolyticus in seafood could be quantitated by MPN statistics according to PCR products. The sensitivity of MPN-PCR was 100 times higher than that of direct PCR. Of 225 seafood samples from Qingdao, 165 were positive for the presence of V. parahaemolyticus, with an MPN value of >719 per gram, and about 41.5% of samples were positive for tdh gene-possessing cells. Eighty muscle tissues from the 225 seafood samples were investigated by direct PCR and MPN-PCR, but no V. parahaemolyticus was detected. The MPN-PCR test could be completed in less than 16 h from the time of sample preparation. It was rapid, sensitive, and reliable for comprehensive detection and quick quantitative determination of V. parahaemolyticus in seafood and it revealed the potential risk of illness associated with their consumption.     

Optimal enrichment time for isolation of Vibrio parahaemolyticus from seafood.

The growth of Vibrio parahaemolyticus in a liquid medium was compared with that of human fecal flora and estuarine flora. No marked differences were noted between growth at 25 and 37 degrees C for V. parahaemolyticus. However, the marine organisms were strongly inhibited when incubated at 37 degrees C. Incubation for 8 h in an enrichment broth yielded V. parahaemolyticus growth, even with a small inoculum, whereas the marine and fecal floras were inhibited. Therefore, enrichment for 8 h at 37 degrees C appears to be optimal for isolation of V. parahaemolyticus, permitting more rapid results in seafood analysis.     

An investigation into the changed physiological state of Vibrio bacteria as a survival mechanism in response to cold temperatures and studies on their sensitivity to heating and freezing

AIMS: To induce pathogenic Vibrio bacteria into a changed physiological state, in response to cold temperatures in sea water, and assess their sensitivity to heating and freezing, as compared with normal cells. METHODS AND RESULTS: Cells of exponential phase Vibrio vulnificus, V. cholerae and V. parahaemolyticus were washed and inoculated into flasks of sea water, which were stored at 20 and 4 degrees C. Cells stored at 20 degrees C could be recovered after 60 d on non-selective agar (heart infusion agar; HIA) and on the selective agar (thiosulphate citrate bile salts agar) which is used in most Vibrio detection methodology. At 4 degrees C cells became non-culturable on both agars over time. The non-culturable cells appeared to be metabolically active and maintained their membrane integrity, whilst undergoing a change in morphology from rod-shaped to coccoid cells. Resuscitation was possible, in some cases, by an upshift in temperature before plating and the addition of catalase to HIA plates was found to increase recovery. Studies were carried out to assess the sensitivity of the non-culturable cells to heating and freezing compared with the normal cells. Vibrio organisms, whether culturable or in the non-culturable form, were not inactivated by freezing to -20 degrees C. Heating studies showed that V. parahaemolyticus was very heat resistant at low temperatures. However, a pasteurization regime of 2 min at 70 degrees C was found to be effective against all three strains. Experiments showed that the non-culturable cells of all three strains were similar in their heat resistance or, in some cases, were more heat sensitive than cells in the normal form. CONCLUSIONS: Cells in the changed physiological form would not be detected in fish or seafood products by the current Vibrio detection methods. Freezing had no effect in reducing cell numbers. Vibrio parahaemolyticus was very heat resistant in the low temperature pasteurization studies. The higher pasteurization regime of 70 degrees C for 2 min was effective against all three pathogens. Non-culturable cells had similar heat sensitivity or were more heat sensitive than cells in the normal state. SIGNIFICANCE OF IMPACT OF THE STUDY: The study has highlighted a need for the development of better Vibrio detection methods. The low temperature pasteurization of oysters, which has been recommended in the USA, would not be adequate against the strain of V. parahaemolyticus used in this study. Heating regimes which were found to control cells in the normal form will also be effective for the control of the cells with changed physiology.     

Analysis of gyrB and toxR gene sequences of Vibrio hollisae and development of gyrB- and toxR-targeted PCR methods for isolation of V. hollisae from the environment and its identification

Isolation of Vibrio hollisae strains, particularly from the environment, is rare. This may be due, in part, to the difficulty encountered when using conventional biochemical tests to identify the microorganism. In this study, we evaluated whether two particular genes may be useful for the identification of V. hollisae. The two genes are presumed to be conserved among the bacterial species (gyrB) or among the species of the genus Vibrio (toxR). A portion of the gyrB sequence of V. hollisae was cloned by PCR using a set of degenerate primers. The sequence showed 80% identity with the corresponding Vibrio parahaemolyticus gyrB sequence. The toxR gene of V. hollisae was cloned utilizing a htpG gene probe derived from the V. parahaemolyticus htpG gene, which is known to be linked to the toxR gene in V. hollisae. The coding sequence of the cloned V. hollisae toxR gene had 59% identity with the V. parahaemolyticus toxR coding sequence. The results of DNA colony hybridization tests using the DNA probes derived from the two genes of V. hollisae indicated that these gene sequences could be utilized for differentiation of V. hollisae from other Vibrio species and from microorganisms found in marine fish. PCR methods targeting the two gene sequences were established. Both PCR methods were shown to specifically detect the respective target sequences of V. hollisae but not other organisms. A strain of V. hollisae added at a concentration of 1 to 10(2) CFU/ml to alkaline peptone water containing a seafood sample could be detected by a 4-h enrichment incubation in alkaline peptone water at 37 degrees C followed by quick DNA extraction with an extraction kit and 35-cycle PCR specific for the V. hollisae toxR gene. We conclude that screening of seafood samples by this 35-cycle, V. hollisae toxR-specific PCR, followed by isolation on a differential medium and identification by the above htpG- and toxR-targeted PCR methods, can be useful for isolation from the environment and identification of V. hollisae.     

Method for the detection of injured Vibrio parahaemolyticus in seafoods.

The sensitivity of Vibrio parahaemolyticus cells to refrigeration and frozen storage and the development of a method for detecting injured and uninjured V. parahaemolyticus cells were studied. Cell suspensions in different kinds of seafood homogenates were either regrigerated (4 degrees C) or frozen (-20 degrees C), stored, and examined for cell survival during storage. V. parahaemolyticus cells were sensitive to both storage temperatures. Many cells died, and many survivors were sublethally injured. In general, refrigeration storage appeared to be more injurious than frozen storage. The initial recovery of the sublethally injured cells was highest in a nutritionally rich, nonselective liquid medium such as Trypticase soy broth, whereas maximum cell multiplication was observed in Trypticase soy broth containing 3% NaCl. The sublethally injured V. parahaemolyticus cells demonstrated sensitivity to the selective enrichment medium, glucose salt teepol broth. From these findings, a new method (designated as the "repair-detection" method) was developed for the isolation and enumeration of V. parahaemolyticus. Comparative studies between the recommended and the repair-detection methods showed that injured V. parahaemolyticus cells were present in commercial seafoods and that the repair-detection method was definitely more effective for the detection of total numbers of V. parahaemolyticus cells.     

Detection of Vibrio parahaemolyticus in shellfish by use of multiplexed real-time PCR with TaqMan fluorescent probes

We developed a multiplexed real-time PCR assay using four sets of gene-specific oligonucleotide primers and four TaqMan probes labeled with four different fluorophores in a single reaction for detection of total and pathogenic Vibrio parahaemolyticus, including the pandemic O3:K6 serotype in oysters. V. parahaemolyticus has been associated with outbreaks of food-borne gastroenteritis caused by the consumption of raw or undercooked seafood and therefore is a concern to the seafood industry and consumers. We selected specific primers and probes targeting the thermostable direct hemolysin gene (tdh) and tdh-related hemolysin gene (trh) that have been reported to be associated with pathogenesis in this organism. In addition, we targeted open reading frame 8 of phage f237 (ORF8), which is associated with a newly emerged virulent pandemic serotype of V. parahameolyticus O3:K6. Total V. parahaemolyticus was targeted using the thermolabile hemolysin gene (tlh). The sensitivity of the combined four-locus multiplexed TaqMan PCR was found to be 200 pg of purified genomic DNA and 10(4) CFU per ml for pure cultures. Detection of an initial inoculum of 1 CFU V. parahaemolyticus per g of oyster tissue homogenate was possible after overnight enrichment, which resulted in a concentration of 3.3x10(9) CFU per ml. Use of this method with natural oysters resulted in 17/33 samples that were positive for tlh and 4/33 samples that were positive for tdh. This assay specifically and sensitively detected total and pathogenic V. parahaemolyticus and is expected to provide a rapid and reliable alternative to conventional detection methods by reducing the analysis time and obviating the need for multiple assays.     

Membrane filter procedure for enumeration of Vibrio parahaemolyticus.

A membrane filtration procedure has been developed for the enumeration of Vibrio parahaemolyticus in marine waters. Background microbial growth on the primary medium was decreased through the use of sodium cholate and copper sulfate, high pH, 3% NaCl, and an elevated incubation temperature. A series of in situ tests was employed to obviate the picking of colonies for identification; thereby, the enumeration of V. parahaemolyticus was accomplished within 30 h. Confirmation of typical colonies approached 95%. Relative to immediate plating on brain heart infusion agar spread plates, the recovery of V. parahaemolyticus cells suspended in phosphate-buffered saline or in seawater held for 24 h at 4 to 6 degrees C was about 90%. Assay variability did not exceed that expected by chance. Recoveries of V. parahaemolyticus from coastal and estuarine surface waters exceeded those obtainable by other methods examined.     

Vibrio parahaemolyticus and other halophilic vibrios associated with seafood in Hong Kong

The summer prevalence of Vibrio parahaemolyticus and other halophilic vibrios in seafood from Hong Kong markets was investigated. Halophilic vibrios were isolated from all seven types of seafood examined, and comprised 9.1%, 8% and 6.1% of contaminating aerobic heterotrophic bacteria from mussels, clams and oysters respectively. Sucrose-positive vibrios were more common than sucrose-negative varieties. Vibrio alginolyticus was the most frequently isolated species, followed by V. parahaemolyticus, V. harveyi, V. fluvialis, V. vulnificus, V. pelagius, V. campbellii, V. spendidus and V. marinus. Mussels contained the highest concentration of V. parahaemolyticus (4.6 x 10(4)/g); oysters and clams contained 3.4 x 10(4)/g and 6.5 x 10(3)/g respectively. The ubiquity and relatively high concentrations of V. parahaemolyticus and other pathogenic vibrios in shellfish is a potential public health hazard in Hong Kong and other subtropical Asian countries.