Conditions for high pressure inactivation of Vibrio parahaemolyticus in oysters

The objective of this study was to identify the high pressure processing conditions (pressure level, time, and temperature) needed to achieve a 5-log reduction of Vibrio parahaemolyticus in live oysters (Crassostrea virginica). Ten strains of V. parahaemolyticus were separately tested for their resistances to high pressure. The two most pressure-resistant strains were then used as a cocktail to represent baro-tolerant environmental strains. To evaluate the effect of temperature on pressure inactivation of V. parahaemolyticus, Vibrio-free oyster meats were inoculated with the cocktail of V. parahaemolyticus and incubated at room temperature (approximately 21 degrees C) for 24 h. Oyster meats were then blended and treated at 250 MPa for 5 min, 300 MPa for 2 min, and 350 MPa for 1 min. Pressure treatments were carried out at -2, 1, 5, 10, 20, 30, 40, and 45 degrees C. Temperatures >/=30 degrees C enhanced pressure inactivation of V. parahaemolyticus. To achieve a 5-log reduction of V. parahaemolyticus in live oysters, pressure treatment needed to be >/=350 MPa for 2 min at temperatures between 1 and 35 degrees C and >/=300 MPa for 2 min at 40 degrees C.     

Conditions for a 5-log reduction of Vibrio vulnificus in oysters through high hydrostatic pressure treatment

Vibrio vulnificus is frequently associated with oysters, and since oysters are typically consumed raw on a half shell, they can pose a threat to public health due to ingestion of this pathogenic marine microorganism. Oysters should be processed to reduce the number of this pathogen. High pressure processing is gaining more and more acceptance among oyster processors due to its ability to shuck oysters while keeping the fresh-like characteristics of oysters. Nine strains of V. vulnificus were tested for their sensitivities to high pressure. The most pressure-resistant strain of V. vulnificus, MLT 403, was selected and used in the subsequent experiments to represent a worst case scenario for evaluation of the processing parameters for inactivation of V. vulnificus in oysters. To evaluate the effect of temperature on pressure inactivation of V. vulnificus, oyster meats were inoculated with V. vulnificus MLT 403 and incubated at room temperature for 24 h. Oyster meats were then blended and treated at 150 MPa for 4 min, and 200 MPa for 1 min. Pressure treatments were carried out at -2, 1, 5, 10, 20, 30, 40, and 45 degrees C. Cold temperatures (<20 degrees C) and slightly elevated temperatures (>30 degrees C) substantially increased pressure inactivation of V. vulnificus. For example, a 4-min treatment of 150 MPa at -2 and 40 degrees C reduced the counts of V. vulnificus by 4.7 and 2.8 log, respectively, while at 20 degrees C the same treatment only reduced counts by 0.5 log. Temperatures of -2 and 1 degrees C were used to determine the effect of pressure level, temperature, and treatment time on the inactivation of V. vulnificus infected to live oysters through feeding. To achieve a >5-log reduction in the counts of V. vulnificus in a relatively short treatment time (or=250 MPa at -2 or 1 degrees C.     

Ethanol shock changes the fatty acid profile and survival behavior of Vibrio parahaemolyticus in various stress conditions

Vibrio parahaemolyticus 690, a clinical strain, was subjected to ethanol shock in the presence of 5% ethanol for a period of 30 and 60 min. Survival behaviors of the ethanol shocked and control cells of V. parahaemolyticus in the presence of H(2)O(2) (20 ppm), crystal violet (3 ppm), NaCl (20%), and low pH solution (pH 4.4) containing various organic acids including lactic acid, acetic acid, citric acid and tartaric acid (25 mM) were compared. In addition, the effects of ethanol shock on the fatty acid profile and recovery of V. parahaemolyticus on tryptic soy agar (TSA) plus various amounts of NaCl were also investigated. After ethanol shock, it was found that the proportion of vaccenic acid (18:1) increased, while the proportion of palmitic acid (16:0) and ratio of saturated fatty acid to unsaturated fatty acid decreased in cells of V. parahaemolyticus. The recovery of the ethanol-shocked cells on TSA plus 6.0% or 7.5% NaCl was significantly less than the control cells. Furthermore, ethanol shock increased the survival of V. parahaemolyticus in the presence of H(2)O(2), while made the test organism less resistant to crystal violet, high NaCl and organic acids. The degree of decreased acid tolerance observed on the ethanol-shocked cells of test organism varied with the organic acid examined. Finally, ethanol shock for 60 min exhibited either a higher or similar degree of effect on the test organism (depending on the type of stress encountered) than did ethanol shock for 30 min. Data obtained from the present study does provide useful information that is indispensable when control measure of V. parahaemolyticus is to be performed efficiently and adequately.     

Bactericidal effects of wine on Vibrio parahaemolyticus in oysters

The bactericidal effects of wines on Vibrio parahaemolyticus in oysters were studied to evaluate potential inactivation of V. parahaemolyticus in contaminated oysters by wine consumption. Shucked whole oyster and oyster meat homogenate were inoculated with V. parahaemolyticus and mixed with red or white wine. Survivals of V. parahaemolyticus in inoculated oysters were determined at 7 and 25 degrees C. Populations of V. parahaemolyticus in inoculated whole oysters (5.52 log most probable number [MPN] per g) decreased slightly to 4.90 log MPN/g (a 0.62-log reduction) after 24 h at 7 degrees C but increased to 7.37 log MPN/g over the same period at 25 degrees C. However, the populations in wine-treated whole oysters decreased by >1.7 and >1.9 log MPN/g after 24 h at 7 and 25 degrees C, respectively. Both red and white wines were more effective in inactivating V. parahaemolyticus in oyster meat homogenate than in whole oyster. Populations of V. parahaemolyticus in oyster meat homogenate (7.8 x 10(3) MPN/g) decreased rapidly to nondetectable levels (< 3 MPN/g) after 30 min of mixing with wine at 25 degrees C (a 3.89-log MPN/g reduction). These results suggest that chewing oysters before swallowing when eating raw oysters may result in greater inactivation of V. parahaemolyticus if wine is consumed. More studies are needed to determine the bactericidal effects of wine on V. parahaemolyticus in the complicated stomach environment.     

Influence of prior growth conditions, pressure treatment parameters, and recovery conditions on the inactivation and recovery of Listeria monocytogenes, Escherichia coli, and Salmonella Typhimurium in turkey meat

The relatively high prevalence of Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella enterica serovar Typhimurium in various food products is of great concern to the food industry. The objective of this study was to determine the pressure-inactivation of the pathogens in a representative food model as affected by prior growth temperature, physiological age of the culture, pressure level and treatment temperature. The effect of post-treatment conditions (incubation temperature and gas atmosphere) on the bacterial recovery was also determined. The pathogens being studied were inoculated into sterile turkey breast meat to a final level of ca. 3 logCFU/g and then grown to two stages, the early stage (representative of exponential phase) and late stage (representative of stationary phase), at 15, 25, 35, and 40 °C. Turkey meat samples were pressure-treated at 400 and 600 MPa for 2 min at initial sample temperatures of 4, 20 and 40 °C. Following treatment, bacterial counts in the samples were determined aerobically or anaerobically at incubation temperatures of 15, 25, 35, and 40 °C. Pressure inactivation of the bacterial pathogens increased as a function of the pressure levels and treatment temperatures. Generally speaking, early stage cells were more resistant than late stage cells (P<0.05). The incubation gas atmosphere did not affect bacterial recovery. Bacteria grown at 15-35 °C underwent higher population reductions than those grown at 40 °C. With regard to recovery temperatures, low temperatures promoted greater recovery of injured early and late stage cells than higher temperatures (P<0.05). This study indicates the importance of environmental conditions to which bacteria are exposed prior to pressure treatment and recovery conditions of the bacteria after pressure treatment when considering the adequacy of pressure treatments to enhance the microbiological safety of foods.     

Growth inhibition of Vibrio parahaemolyticus in seafood by tabletop dry ice cooler

Tabletop dry ice coolers (three types; dome model, cap model and tripod model), which are used in kitchens and hotel banquet halls to refrigerate fresh seafood, were investigated to determine whether growth of Vibrio parahaemolyticus was inhibited by their use. On TSA plates containing 1.8% NaCl and fresh seafood (fillets of squid, pink shrimp and yellowtail), V. parahaemolyticus (O3:K6, TDH+) inoculated at 4 to 5 log CFU/sample and left at ambient temperature (25 degrees C) grew by 1.0 to 2.8 orders in 4 hours. In contrast, with tabletop coolers no significant increase in viable count occurred in 3 to 4 hours, confirming that tabletop coolers inhibited the growth of V. parahaemolyticus. The temperature in each tabletop cooler was kept below 10 degrees C for 80 to 135 min, though the CO2 gas concentration in them remained high for only a short time (0 to 75 min). It was presumed that the refrigeration function mainly contributed to growth inhibition. Our results indicate that tabletop dry ice coolers are helpful for prevention of food-borne disease due to V. parahaemolyticus in food-service locations, such as kitchens and banquet halls.     

Modeling time to inactivation of Listeria monocytogenes in response to high pressure, sodium chloride, and sodium lactate

A mathematical model was developed to predict time to inactivation (TTI) by high pressure processing of Listeria monocytogenes in a broth system (pH 6.3) as a function of pressure (450 to 700 MPa), inoculum level (2 to 6 log CFU/ml), sodium chloride (1 or 2%), and sodium lactate (0 or 2.5%) from a 4°C initial temperature. Ten L. monocytogenes isolates from various sources, including processed meats, were evaluated for pressure resistance. The five most resistant strains were used as a cocktail to determine TTI and for model validation. Complete inactivation of L. monocytogenes in all treatments was demonstrated with an enrichment method. The TTI increased with increasing inoculum level and decreasing pressure magnitude, from 1.5 min at 700 MPa and 2 log CFU/ml, to 15 min at 450 MPa and 6 log CFU/ml. Neither NaCl nor sodium lactate significantly influenced TTI. The model was validated with ready-to-eat, uncured, Australian retail poultry products, and with product specially made at a U.S. Department of Agriculture, Food Safety and Inspection Service (FSIS)-inspected pilot plant in the United States. Data from the 210 individual product samples used for validation indicate that the model gives "fail-safe" predictions (58% with response as expected, 39% with no survivors where survivors expected, and only 3% with survivors where none were expected). This model can help manufacturers of refrigerated ready-to-eat meats establish effective processing criteria for the use of high pressure processing as a postlethality treatment for L. monocytogenes in accordance with FSIS regulations.     

Response of Vibrio parahaemolyticus 03:K6 to a hot water/cold shock pasteurization process.

Vibrio vulnificus and V. parahaemolyticus are natural inhabitants of estuarine environments world wide. Pathogenic strains of these bacteria are often transmitted to humans through consumption of raw oysters, which flourish in the same estuaries. Previous studies reported the effective use of hot water pasteurization followed by cold shock to eliminate from raw oysters naturally and artificially incurred environmental strains of V. vulnificus and V. parahaemolyticus common to the Gulf of Mexico. The present study focused on the use of the same pasteurization method to reduce a highly process resistant Vibrio strain, V. parahaemolyticus O3:K6 to non-detectable levels. Oysters were artificially contaminated with 10(4) and 10(6) V. parahaemolyticus 03:K6 cfu g(-1) oyster meat. Contaminated oysters were pasteurized between 50 and 52 degrees C for up to 22 min. Samples of processed oysters were enumerated for V. parahaemolyticus O3:K6 at 2-min intervals beginning after the 'come-up time' to achieve an oyster internal temperature of at least 50 degrees C. The D value (D(52)deg C) was 1.3-1.6 min. V. parahaemolyticus O3:K6 proved more process resistant than non-pathogenic environmental strains found in Gulf of Mexico waters. A total processing time of at least 22 min at 52 degrees C was recommended to reduce this bacterium to non-detectable levels (< 3 g(-1) oyster meat).     

Response of heat-shocked Vibrio parahaemolyticus to subsequent physical and chemical stresses

Vibrio parahaemolyticus foodborne strains 405, 556, and 690 and a V. parahaemolyticus chopping board isolate were heat shocked at 42 degrees C for 15, 30, or 45 min. Heat shock, regardless of heating periods tested, caused an increased demand for NaCl during recovery from heat injury. Further study with strain 690 and the chopping board isolate also revealed that heat shock generally increased the survival of the test organism during subsequent exposure to 47 degrees C, 20 ppm H202, and 8% ethanol and reduced the tolerance of the test organism to low temperatures (5 and -18 degrees C). The extent of the heat shock response of V. parahaemolyticus varied with strain and the duration of treatment. Furthermore, heat shock treatments in the present study caused the leakage of nucleic acids from V. parahaemolyticus cells. This effect was most pronounced with cells heat shocked at 42 degrees C for 45 min.     

Thin agar layer method for recovery of heat-injured Listeria monocytogenes

A thin agar layer (TAL) method was developed to recover heat-injured Listeria monocytogenes. Modified Oxford medium (MOX), a selective plating medium, inhibits heat-injured L. monocytogenes from growing, whereas tryptic soy agar (TSA), a nonselective medium, does not. In order to facilitate recovery of heat-injured L. monocytogenes cells while providing selectivity of isolation of L. monocytogenes from other bacteria in the sample, a unique TAL procedure was developed by overlaying 5 ml of nonselective medium (TSA) onto prepoured and solidified MOX medium in an 8.5-cm-diameter petri dish. The injured L. monocytogenes repaired and started to grow in the TSA during the first few hours after incubation of the plate. During the resuscitation of injured cells, the selective agents from MOX diffused to the TSA top layer to inhibit other microorganisms. L. monocytogenes showed a typical reaction (black colonies) on TAL after 24 h of incubation at 37 degrees C. The recovery rate for heat-injured L. monocytogenes with the TAL method was compared with those rates associated with TSA, MOX, and the traditional overlay method (OV; pouring selective agar on top of resuscitated cells on TSA agar after 3 h incubation). Milk and 0.1% peptone water that were inoculated with L. monocytogenes (4 to 5 log CFU/ml) were heated for 15 min at 55 degrees C. L. monocytogenes was enumerated on TSA, MOX, OV, and TAL media and procedures. No significant difference occurred among TSA, OV, and TAL (P > 0.05) in terms of enumeration of heat-injured L. monocytogenes, but these media recovered significantly higher numbers than did MOX agar (P < 0.05)-in both samples. The TAL method involves only one step, whereas OV is a more cumbersome two-step procedure.     

Inactivation of vibrio parahaemolyticus and vibrio vulnificus in phosphate-buffered saline and in inoculated whole oysters by high-pressure processing

Inactivation studies for Vibrio parahaemolyticus TX-2103 (serotype O3:K6) and Vibrio vulnificus MO-624 (clinical isolate) were conducted in phosphate-buffered saline (PBS) and in inoculated oysters under high-pressure processing conditions. V. parahaemolyticus was more resistant than V. vulnificus in PBS at all pressures and times. A 6-log reduction of V. parahaemolyticus and V. vulnificus in PBS at 241 MPa required 11 and 5 min, respectively, which included a 3-min pressure come-up time. A 4.5-log reduction of V. parahaemolyticus in oysters at 345 MPa required 7.7 min, which included a 6.7-min pressure come-up time. More than a 5.4-log reduction of V. vulnificus in oysters at 345 MPa occurred during the 6-min pressure come-up time. Both V. parahaemolyticus and V. vulnificus in PBS and in oysters were reduced to nondetectable numbers at 586 MPa during the 8- and 7-min pressure come-up times, respectively.     

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.     

Characterization of low salinity stress in Vibrio parahaemolyticus

Vibrio parahaemolyticus is a marine foodborne pathogenic bacterium commonly found in seawater or seafood. This bacterium often encounters low salinity stress when the contaminated seafood is washed with fresh water during food processing. This study was conducted to investigate the response of exponential- and stationary-phase cells of V. parahaemolyticus ST550 to lethal or sublethal low salinity. Tolerance to lethal low salinity (0.25% NaCl) was enhanced in V. parahaemolyticus cells in the exponential phase by previous adaptation in sublethal low salinity (0.6% NaCl). Low salinity-adapted cells in the exponential phase were also cross-protected against the challenge of lethal low pH, indifferent to heat, and sensitized to bile, acetic acid, and lactic acid stress. The adapted cells in the stationary phase were significantly protected against heat treatment at 44°C for 10 and 15 min, sensitized to bile and acetic acid treatment, and indifferent to low pH and lactic acid.     

Use of linear, Weibull, and log-logistic functions to model pressure inactivation of seven foodborne pathogens in milk

Survival curves of six foodborne pathogens suspended in ultra high-temperature (UHT) whole milk and exposed to high hydrostatic pressure at 21.5 degrees C were obtained. Vibrio parahaemolyticus was treated at 300 MPa and other pathogens, Listeria monocytogenes, Escherichia coli O157:H7, Salmonella enterica serovar Enteritidis, Salmonella enterica serovar Typhimurium, and Staphylococcus aureus were treated at 600 MPa. All the survival curves showed a rapid initial drop in bacterial counts followed by tailing caused by a diminishing inactivation rate. A linear model and two nonlinear models were fitted to these data and the performances of these models were compared using mean square error (MSE) values. The log-logistic and Weibull models consistently produced better fits to the inactivation data than the linear model. The mean MSE value of the linear model was 6.1, while the mean MSE values were 0.7 for the Weibull model and 0.3 for the log-logistic model. There was no correlation between pressure resistance and the taxonomic group the bacteria belong to. The order, most to least pressure-sensitive, of the single strains tested was: V. parahaemolyticus (gram negative)相似文献   

Low temperature pasteurization to reduce the risk of vibrio infections from raw shell-stock oysters

Vibrio vulnificus and V. parahaemolyticus are natural inhabitants of estuarine environments and may be transmitted to humans by ingestion of raw oysters. This study focused on the use of low temperature pasteurization, to reduce these Vibrio spp. to nondetectable levels, thus reducing the risk of infection associated with raw oyster consumption. Artificially inoculated V. vulnificus and V. parahaemolyticus and naturally-contaminated V. vulnificus in live oysters were pasteurized at 50%deg;C for up to 15min. Samples of processed and unprocessed oysters were enumerated for V. vulnificus, V. parahaemolyticus, and aerobic spoilage bacteria for 0-14 days. Low temperature pasteurization was effective in reducing these pathogens from > 100000 to non-detectable levels in less than 10min of processing. Spoilage bacteria were reduced by 2-3 logs, thus increasing the shelf-life for up to 7 days beyond live unprocessed oysters. Vibrio vulnificus in control oysters was reduced by 102 during ice storage alone. Following pasteurization and during a temperature storage abuse study (24h at 22°C), V. vulnificus was not recovered. During this storage period spoilage bacteria exceeded 1 million/g oyster meat.     

Effects of heat, acid, and freeze-thaw challenges on survival of starved Vibrio parahaemolyticus in minimal salt medium, tryptic soy broth, and filtered oyster homogenate medium

Vibrio parahaemolyticus is a ubiquitous gram-negative enteropathogenic bacterium. To evaluate the risk of stress-adapted V. parahaemolyticus cells in food, we investigated the survivability of starvation-adapted and starvation-low salinity-adapted cells of this pathogen in different media against different stresses. Logarithmically grown bacterial cells were starved at 25 degrees C in a minimal salt medium with 0.5 or 3.0% NaCl for 24 h. Resistances against challenges of heat, acid, and freeze-thaw treatment exhibited by the starvation-adapted cells were similar to those exhibited by the starvation-low salinity-adapted cells but substantially higher than those of the unadapted control cells. The increased stress resistance of the adapted cells against freeze-thaw challenge was lower in tryptic soy broth than in the starving medium. Resistance of the adapted bacteria against heat and freeze-thaw treatment was completely eliminated in filter-sterilized oyster homogenate medium. Practically, these results help to assess the risk of stress-adapted V. parahaemolyticus in food.     

Comparison of methods for recovering Vibrio cholerae O1 from ice

Alkaline peptone water (1% peptone, 1% NaCl, pH 8.5) and Trypticase soy yeast extract broth (TSYB) supplemented with 2.5% NaCl (pH 8.5) or 1% NaCl (pH 7.5) were evaluated as enrichment broths for the isolation of Vibrio cholerae O1 from ice. Thirty samples of sterile and nonsterile mineral water were inoculated with cell suspensions of this bacterium, quickly frozen, and stored for 3 days at--18 degrees C. After thawing, samples were analyzed by a three-tube most-probable-number technique. Incubation in TSYB with 2.5% NaCl (pH 8.5) for 18 h at 37 degrees C yielded the highest recovery of V. cholerae O1 cells (P < 0.05), a result that might be attributable to the nutrients and to the NaCl concentration of the TSYB, both of which would promote V. cholerae O1 growth and prevent the growth of competitive microbiota.     

Oyster-to-oyster variability in levels of Vibrio parahaemolyticus

This study examined the variability in the levels of total and pathogenic Vibrio parahaemolyticus in individual oysters. Twenty oysters were collected on three occasions (in June, July, and September 2001) from a site near Mobile Bay, Ala. Ten of these oysters were tested immediately, and 10 were tested after 24 h of storage at 26 degrees C. Levels of total and pathogenic V. parahaemolyticus were determined by alkaline phosphatase-labeled DNA probe procedures targeting the thermolabile hemolysin and thermostable direct hemolysin genes, respectively. Similar V. parahaemolyticus levels (200 to 2,000 CFU/g) were found in nearly 90% of the oysters (for all sampling occasions) prior to storage. The log-transformed densities (means +/- standard deviations) of V. parahaemolyticus in oysters immediately after harvest were 2.90 +/- 0.91, 2.88 +/- 0.36, and 2.47 +/- 0.26 log10 CFU/g for June, July, and September, respectively. After storage for 24 h at 26 degrees C, the mean V. parahaemolyticus densities increased approximately 13- to 26-fold. Before storage, pathogenic V. parahaemolyticus was detected in 40% (10 to 20 CFU/g) of the oysters collected in June and July but was not detected in any oysters collected in September. After storage, pathogenic V. parahaemolyticus was detected in some oysters at levels of > 100 CFU/g. These data should aid in the development of sampling protocols for oyster monitoring programs and in the determination of exposure distributions associated with raw oyster consumption.     

Sensitivities of foodborne pathogens to pressure changes

Eight foodborne pathogens were suspended in ultrahigh-temperature whole milk and treated at pressure levels of 0.1 to 690 MPa at 21.5 degrees C for 10 min. There was no clear trend in pressure resistance between gram-negative and gram-positive organisms. The order of the single strains tested, from most to least pressure sensitive, was Vibrio parahaemolyticus < Yersinia enterocolitica < Listeria monocytogenes < Salmonella enterica serovar Typhimurium < S. enterica serovar Enteritidis < Escherichia coli O157:H7 approximately equal to Staphylococcus aureus < Shigella flexneri. For each organism there existed a pressure range in which log(number of survivors) had a near linear relationship when plotted versus treatment pressure level. In this study, a decimal reduction pressure (Dp) value was defined and used to measure the sensitivity of these pathogens to pressure changes. L. monocytogenes and V. parahaemolyticus were most sensitive to pressure changes, and S. flexneri was most resistant. The D(P) values were 16.3 MPa for L. monocytogenes, 21.7 MPa for V. parahaemolyticus, and 127.0 MPa for S. flexneri. The most pressure-resistant gram-negative bacterium, S. flexneri, and most pressure-resistant gram-positive bacterium, S. aureus, were treated at 50 degrees C and pressures of 0.1 to 650 MPa for 10 min. High temperature considerably enhanced pressure inactivation of these two organisms and affected their sensitivities to pressure changes. The effect of treatment time on the D(P) values of L. monocytogenes and V. parahaemolyticus was also determined, and it was found that it did not significantly affect their D(P) values.     

Real-time PCR quantification of Vibrio parahaemolyticus in oysters using an alternative matrix

This study examined the relationship between levels of total Vibrio parahaemolyticus found in oyster tissues and mantle fluid with the goal of using mantle fluid as a template matrix in a new quantitative real-time PCR assay targeting the thermolabile hemolysin (tlh) gene for the enumeration of total V. parahaemolyticus in oysters. Oysters were collected near Mobile Bay, Ala., in June, July, and September and tested immediately after collection and storage at 26 degrees C for 24 h. Initial experiments using DNA colony hybridization targeting tlh demonstrated that natural V. parahaemolyticus levels in the mantle fluid of individual oysters were strongly correlated (r = 0.85, P < 0.05) with the levels found in their tissues. When known quantities of cultured V. parahaemolyticus cells were added to real-time PCR reactions that contained mantle fluid and oyster tissue matrices separately pooled from multiple oysters, a strong linear correlation was observed between the real-time PCR cycle threshold and the log concentration of cells inoculated into each PCR reaction (mantle fluid: r = 0.98, P < 0.05; and oyster: r = 0.99, P < 0.05). However, the mantle fluid exhibited less inhibition of the PCR amplification than the homogenized oyster tissue. Analysis of natural V. parahaemolyticus populations in mantle fluids using both colony hybridization and real-time PCR demonstrated a significant (P < 0.05) but reduced correlation (r = -0.48) between the two methods. Reductions in the efficiency of the real-time PCR that resulted from low population densities of V. parahaemolyticus and PCR inhibitors present in the mantle fluid of some oysters (with significant oyster-to-oyster variation) contributed to the reduction in correlation between the methods that was observed when testing natural V. parahaemolyticus populations. The V. parahaemolyticus-specific real-time PCR assay used for this study could estimate elevated V. parahaemolyticus levels in oyster mantle fluid within 1 h from sampling time.