Growth-Inhibitory Effect of d-Tryptophan on Vibrio spp. in Shucked and Live Oysters

Oysters are the primary transmission vehicles for human Vibrio infections. Raw oyster consumption is frequently associated with gastroenteritis. The current postharvest methods, such as high-pressure processing, used to control Vibrio spp. in fresh oysters are still insufficient because of limited facilities, high cost, and potential adverse effects on production. We demonstrate that adding a small amount of d-tryptophan (d-Trp) inhibits the growths of Vibrio parahaemolyticus and Vibrio vulnificus in a high-salt environment at even ambient temperature. We further investigated the d-Trp treatment conditions and clarified the relationship between salt and d-Trp concentrations for optimal growth-inhibitory effect of Vibrio spp. The results will be useful for enhancing the effectiveness of d-Trp by increasing salinity levels. Furthermore, in a nutrientfree environment (artificial seawater), a stronger inhibitory effect could be observed at relatively lower salinity levels, indicating that d-Trp may be regarded as effective food preservation in terms of salinity reduction. Therefore, we suggest the use of exogenous d-Trp in a seawater environment as a novel and effective strategy not only for controlling Vibrio in live oysters at even ambient temperature but also for effectively retarding spoilage bacterial growth and extending the shelf life of shucked oysters at refrigeration temperature.

ABSTRACT

Vibrio vulnificus and Vibrio parahaemolyticus are important human pathogens that are frequently transmitted via consumption of contaminated raw oysters. A small amount of d-tryptophan (d-Trp) inhibits some foodborne pathogenic bacteria in high-salt environments. In this study, we aimed to evaluate the antibacterial effect of d-Trp on V. vulnificus and V. parahaemolyticus in culture media, artificial seawater, and shucked and live oysters. The effectiveness of d-Trp in growth inhibition of Vibrio spp. was highly dependent on environmental NaCl concentrations. Higher levels of NaCl (>4.0%) with d-Trp (>20 mM) resulted in higher and more consistent growth inhibition of both Vibrio spp. Treatment with 40 mM d-Trp significantly (P < 0.05) reduced viable V. parahaemolyticus cell counts in tryptic soy broth (TSB) with >4.0% NaCl at 25°C. In contrast, V. vulnificus was more sensitive to d-Trp (20 mM) than V. parahaemolyticus. d-Trp (40 mM) treatment with NaCl (>4.5%) significantly (P < 0.05) inhibited the growth of V. parahaemolyticus and V. vulnificus in shucked oysters immersed in peptone water at 25°C throughout a 48-h incubation period. In artificial seawater, d-Trp exhibited a stronger growth-inhibitory effect on V. vulnificus and V. parahaemolyticus at 25°C than in TSB at the same level of salinity and inhibited the growth of both V. parahaemolyticus and V. vulnificus in live oysters at 25°C for 48 h. Furthermore, we tested the synergistic effect of d-Trp and salinity on the inhibition of total viable bacterial counts (TVC) at refrigeration temperature. d-Trp (40 mM) inhibited the growth of TVC in shucked oysters immersed in artificial seawater at 4°C. Therefore, these results revealed that d-Trp will serve as a novel and alternative food preservative to control Vibrio spp. in live oysters at ambient temperature and to extend the shelf-life of shucked oysters at refrigeration temperature.

IMPORTANCE Oysters are the primary transmission vehicles for human Vibrio infections. Raw oyster consumption is frequently associated with gastroenteritis. The current postharvest methods, such as high-pressure processing, used to control Vibrio spp. in fresh oysters are still insufficient because of limited facilities, high cost, and potential adverse effects on production. We demonstrate that adding a small amount of d-tryptophan (d-Trp) inhibits the growths of Vibrio parahaemolyticus and Vibrio vulnificus in a high-salt environment at even ambient temperature. We further investigated the d-Trp treatment conditions and clarified the relationship between salt and d-Trp concentrations for optimal growth-inhibitory effect of Vibrio spp. The results will be useful for enhancing the effectiveness of d-Trp by increasing salinity levels. Furthermore, in a nutrientfree environment (artificial seawater), a stronger inhibitory effect could be observed at relatively lower salinity levels, indicating that d-Trp may be regarded as effective food preservation in terms of salinity reduction. Therefore, we suggest the use of exogenous d-Trp in a seawater environment as a novel and effective strategy not only for controlling Vibrio in live oysters at even ambient temperature but also for effectively retarding spoilage bacterial growth and extending the shelf life of shucked oysters at refrigeration temperature.

INTRODUCTION

Vibrio parahaemolyticus and Vibrio vulnificus are two of the most notable pathogenic Vibrio species and are known to contaminate ready-to-eat seafood, particularly raw oysters (Crassostrea spp.) (1, 2). V. parahaemolyticus is implicated as the primary source of seafood-associated human gastroenteritis in the United States (3), whereas V. vulnificus is associated with a high fatality rate (approximately 50%) and is responsible for 95% of all the seafood-related deaths in the United States (4, 5).

Oysters are the most abundantly harvested shellfish worldwide. Raw oyster consumption is associated with a high risk of Vibrio infections, even after proper handling during harvest (6). Recently, postharvest treatments such as use of electrolyzed water (7), heat shock (8), rapid chilling (9), irradiation (10), and hydrostatic high-pressure processing (11, 12) have been studied for reducing the level of Vibrio spp. in oysters. However, these treatments have detrimental effects on oyster quality, and the residual bacterial populations may lead to Vibrio contamination during transport, storage, and retail. Therefore, a novel and economical postharvest treatment with continuous effectiveness is required for controlling Vibrio spp. in oysters during postharvest periods.

d-Amino acids are considered to be absent in higher organisms, particularly in the mammalian brain (13). In bacteria, d-amino acid synthesis is a strategy for adapting to changing environmental conditions (14). Recently, some d-amino acids, such as d-tryptophan (d-Trp), were found to exhibit antibacterial activities against foodborne bacteria under osmotic stress. Our previous study reported that d-Trp adversely affected the growth of foodborne bacteria under high-salt conditions (14). One possible explanation for the antibacterial effect of d-Trp might be attributed to d-amino-acid-induced inhibition of biofilm formation, which plays a crucial role in bacterial multicellular communities and protects bacteria from environmental insults (15, 16). For example, exposure to 10 mM d-Trp causes biofilm disassembly in Cronobacter sakazakii by reducing the initial adhesion between cells and changing the properties of the extracellular matrix (17). Therefore, d-Trp may be used as a novel preservative for controlling bacterial growth in foods, particularly in seafood, because of its high effectiveness in environments with high sodium chloride concentration (NaCl concentration, > 3%).

In this study, we aimed to evaluate the inhibitory effects of d-Trp on V. parahaemolyticus and V. vulnificus under conditions with various NaCl and d-Trp concentrations in shucked and live oysters. This study serves as a foundation to highlight the potential application of d-Trp as a postharvest technique for reducing Vibrio contamination in live oysters and extending the shelf life of shucked oysters.

RESULTS

Effects of d-Trp on the growth of Vibrio spp. in culture media.

Growth inhibition of Vibrio spp. after d-Trp treatment was evaluated under various combinations of NaCl and d-Trp stress conditions (Fig. 1 and 2). Compared with the untreated control, samples treated with d-Trp at 25°C showed significantly reduced (P < 0.05) populations of V. parahaemolyticus (Fig. 1) and V. vulnificus (Fig. 2). Exposure to 40 mM d-Trp resulted in a more than 4.0 log CFU/ml decrease in V. vulnificus and V. parahaemolyticus numbers at NaCl concentrations of ≥4.5% during incubation at 25°C. An initial bactericidal effect was observed during the first 24 h, which resulted in a more than 4.0 log CFU/ml decrease in V. vulnificus and V. parahaemolyticus numbers at NaCl concentrations of ≥4.5% during incubation at 25°C, followed by a bacteriostatic effect. Although there was a slight increase in Vibrio cells after 24 h of incubation at low NaCl concentrations, a more gradual bacteriostatic effect was observed with an increase in NaCl concentration. When NaCl was increased by 5.0%, d-Trp exerted the greatest overall bacteriostatic effect. Furthermore, V. vulnificus and V. parahaemolyticus showed different sensitivities to d-Trp. Under 20 mM d-Trp treatment, the population of V. vulnificus was significantly reduced, whereas a similar reduction was not observed in V. parahaemolyticus, regardless of NaCl concentrations. Hence, higher concentrations of NaCl and d-Trp were required to achieve the same bactericidal effect on V. parahaemolyticus as on V. vulnificus.

FIG 1.

Effects of d-tryptophan on the changes in viable numbers of Vibrio parahaemolyticus in tryptic soy broth containing different concentrations of d-tryptophan (○, 0 mM; △, 20 mM; and □, 40 mM) under different sodium chloride concentrations of (a) 3.5%, (b) 4.0%, (c) 4.5%, and (d) 5.0% at 25°C. Results are the means ± standard deviations of three independent experiments. Values with different letters on the same sampling time between different conditions indicate significant difference (P < 0.05).

FIG 2.

Effects of d-tryptophan on the changes in viable numbers of Vibrio vulnificus in tryptic soy broth containing different concentrations of d-tryptophan (○, 0 mM; △, 20 mM; and □, 40 mM) under different sodium chloride concentrations of (a) 3.5%, (b) 4.0%, (c) 4.5%, and (d) 5.0% at 25°C. Results are the means ± standard deviations of three independent experiments. Values with different letters on the same sampling time between different conditions indicate significant difference (P < 0.05).

Effects of d-Trp on the survival of Vibrio spp. in shucked oysters immersed in peptone water.

Considering the inhibitory effects of d-Trp on Vibrio spp. in culture media, we further examined the inhibitory effects of d-Trp on the survival of V. vulnificus and V. parahaemolyticus in sterile freshly shucked oysters immersed in peptone water supplemented with various NaCl concentrations (3.5, 4.0, 4.5, and 5.0% [wt/vol]). Treatment with 40 mM d-Trp significantly inhibited the growth of V. parahaemolyticus (Fig. 3) and V. vulnificus (Fig. 4) at 25°C compared to that in the untreated control samples (without d-Trp). Similarly to the previous experiments, d-Trp exhibited a stronger growth-inhibitory effect on V. vulnificus than on V. parahaemolyticus at 25°C. The mean bacterial number of V. vulnificus in d-Trp-treated samples decreased with each NaCl concentration, compared to that in the respective control samples. In contrast, a comparable reduction in V. parahaemolyticus required relatively higher concentrations of NaCl. A greater decrease in bacterial viability was observed with increasing concentrations of NaCl in the environment. The greatest inhibition was observed at 5.0% NaCl for both V. vulnificus and V. parahaemolyticus.

FIG 3.

Effects of d-tryptophan on the survival of Vibrio parahaemolyticus in experimentally inoculated shucked oysters stored in peptone water under different sodium chloride concentrations of (a) 3.5%, (b) 4.0%, (c) 4.5%, and (d) 5.0% with 0 mM (○) and 40 mM (△) d-tryptophan at 25°C. Results are the means ± standard deviations of three independent experiments. Values with different letters on the same sampling time between different conditions indicate significant difference (P < 0.05).

FIG 4.

Effects of d-tryptophan on the survival of Vibrio vulnificus in experimentally inoculated shucked oysters stored in peptone water under different sodium chloride concentrations of (a) 3.5%, (b) 4.0%, (c) 4.5%, and (d) 5.0% with 0 mM (○) and 40 mM (△) d-tryptophan at 25°C. Results are the means ± standard deviations of three independent experiments. Values with different letters on the same sampling time between different conditions indicate significant difference (P < 0.05).

Effects of d-Trp on the growth of Vibrio spp. in artificial seawater.

Due to the fact that seawater is the native environment for Vibrio growth, we further examined the inhibitory effect of d-Trp on Vibrio survival in artificial seawater with salinities ranging from 3.5 to 5.0% (Fig. 5 and 6). Interestingly, 40 mM d-Trp exhibited a much stronger inhibitive effect on growth of both V. vulnificus and V. parahaemolyticus in artificial seawater than in tryptic soy broth (TSB). As expected, treatment of salinity alone had little or no effect on suppression of the growth of both V. vulnificus and V. parahaemolyticus in artificial seawater, with the exception of samples containing 4.5% and 5.0% seawater, in which the bacterial growth of V. vulnificus was inhibited. However, after adding d-Trp to artificial seawater, there were significant log reductions (P < 0.05) in V. vulnificus and V. parahaemolyticus counts. The sensitivity of Vibrio spp. to d-Trp was consistent with the results of culture media. V. vulnificus tended to be more sensitive than V. parahaemolyticus to d-Trp. The growth of V. parahaemolyticus was significantly inhibited by 40 mM d-Trp, and a more gradual growth decrease was observed with the increased salinity level, whereas the population of V. vulnificus was reduced to less than 1.0 log CFU/ml (detected by plate count on tryptic soy agar [TSA] with 2% NaCl [TSA-2%] after 24 h) during d-Trp treatment at each salinity level. Similarly to previous results, the effectiveness of d-Trp tended to be enhanced at high salinity levels. At high levels of salinity (5%), even V. parahaemolyticus, which was found to be more resistant than V. vulnificus to d-Trp in TSB, was also greatly inhibited and declined continually from 5.8 to 3.1 log CFU/ml.

FIG 5.

Effects of d-tryptophan on the survival of Vibrio parahaemolyticus in artificial seawater under different sodium chloride concentrations of (a) 3.5%, (b) 4.0%, (c) 4.5%, and (d) 5.0% with 0 mM (○) and 40 mM (△) d-tryptophan at 25°C. Results are the means ± standard deviations of three independent experiments. Values with different letters on the same sampling time between different conditions indicate significant difference (P < 0.05).

FIG 6.

Effects of d-tryptophan on the survival of Vibrio vulnificus in artificial seawater under different sodium chloride concentrations of (a) 3.5%, (b) 4.0%, (c) 4.5%, and (d) 5.0% with 0 mM (○) and 40 mM (△) d-tryptophan at 25°C. Results are the means ± standard deviations of three independent experiments. ND, not detected; the detection limit is 10 CFU/ml.

Effects of d-Trp on the survival of Vibrio spp. in experimentally inoculated live oysters.

Since highly inhibitory efficiency of d-Trp in artificial seawater was observed, we further evaluated the effect of d-Trp on internal Vibrio growth in live oysters with shells (Fig. 7). Thirty oysters were immersed in artificial seawater inoculated with V. parahaemolyticus or V. vulnificus (approximately 10⁵ CFU/ml) and the oysters were allowed to internalize the bacteria via filter feeding. The bacterial numbers in individual oysters (n = 3) were determined via plate counts on Vibrio CHROMagar after 0, 24, and 48 h, with or without d-Trp exposure. As expected, after exposure to 40 mM d-Trp at 25°C, the growth of V. vulnificus and V. parahaemolyticus was significantly (P < 0.05) inhibited and yielded 2.9-log CFU/g and 2.2-log CFU/g reductions, respectively, after 48 h of treatment, compared to that in the untreated controls.

FIG 7.

Effects of d-tryptophan (○, 0 mM, and △, 40 mM) on the survival of (a) Vibrio vulnificus and (b) Vibrio parahaemolyticus in experimentally inoculated live oysters. Results are the means ± standard deviations of three independent experiments. Values with different letters on the same sampling time between different conditions indicate significant difference (P < 0.05).

Combined efficacy of d-Trp and salinity on total viable bacterial count (TVC) in shucked oyster culture during refrigerated storage.

Because shucked oysters are normally maintained under constant refrigeration to prevent spoilage, we subsequently examined the combined efficacy of d-Trp and salinity on total bacterial population in sterile freshly shucked oysters immersed in artificial seawater at 4°C. The mean initial population of total viable bacteria in shucked oyster culture was 3.3 log CFU/ml. In the untreated control (without d-Trp) the total bacterial count increased by approximately 2.6 log CFU/ml during 5 days of incubation, whereas total bacterial population in artificial seawater with 3.5% salinity remained at the initial level during the first 3 days of incubation, followed by a slight increase of approximately 1 log CFU/ml (Fig. 8). Consistent with previous experiments, a stronger inhibitive effect was observed at high salinity levels (5%). The total bacterial population in d-Trp-treated samples remained the same throughout the 5-day incubation period.

FIG 8.

Effects of d-tryptophan (○, 0 mM, and △, 40 mM) under different salinities of (a) 3.5% and (b) 5.0% on total bacterial population in freshly shucked oysters in artificial seawater at 4°C. Results are the means ± standard deviations of three independent experiments. Values with different letters on the same sampling time between different conditions indicate significant difference (P < 0.05).

DISCUSSION

Recently, d-amino acids have been proven to exist in higher organisms, including humans, in a considerable amount and to possess specific biological functions. For example, d-serine was identified to be present in the mammalian brain and contribute to neurotransmission (13, 18), whereas d-aspartate was shown to be implicated in development and endocrine function (19). Besides their therapeutic potential, d-amino acids such as d-proline (20), d-alanine (21), and d-serine (22) were shown to inhibit bacterial growth under osmotic stress. Moreover, in our previous study, we found that d-Trp significantly inhibited the growth of Escherichia coli O157:H7 and Salmonella spp. in the presence of salt (14). Therefore, utilizing exogenous d-amino acids, free or incorporated in peptides and proteins, might be regarded as a novel strategy for food protection.

In this study, interestingly, we found that adding exogenous d-Trp (20 to 40 mM) was effective in inhibiting Vibrio growth in TSB supplemented with ≥3.5% NaCl at 25°C. Compared with the negative control (without d-Trp), 1-day treatment with 40 mM d-Trp significantly reduced V. parahaemolyticus and V. vulnificus levels by 4.6 log CFU/ml and 3.7 log CFU/ml, respectively, at 3.5% NaCl. We further increased the salinity from 3.5% to 5.0% to examine the effect of d-Trp on bacterial inactivation. Consistently, the populations of V. parahaemolyticus and V. vulnificus increased significantly, even at the higher salinity level of 5%. However, when 40 mM d-Trp was added, the extent of salinity enhanced the bacterial growth inhibition. Exposure to 40 mM d-Trp inhibited the growth of V. vulnificus and delayed the growth of V. parahaemolyticus at NaCl concentrations of ≥3.5%. When the NaCl concentration was increased to 5%, d-Trp strongly inhibited the growth of these two bacterial species. Thus, higher levels of NaCl are required for consistent and efficient growth inhibition for both V. parahaemolyticus and V. vulnificus.

In general, V. vulnificus is more sensitive than V. parahaemolyticus to various stresses, such as gamma irradiation (23), electrolyzed water (24), X rays (10), and high hydrostatic pressure (11). In our study, V. vulnificus was found to be more sensitive to d-Trp than V. parahaemolyticus was. Although the treatment with 20 mM d-Trp significantly (P < 0.05) reduced V. vulnificus levels by approximately 4 log CFU/ml after 24 h of exposure at NaCl concentrations of >3.5%, higher concentrations of d-Trp (such as 40 mM) were necessary for efficient growth inhibition of V. parahaemolyticus. The results of the difference in stress sensitivity of both Vibrio spp. are consistent with previous reports.

Raw, freshly shucked oysters are one of the main products in the oyster processing industry (25, 26). After oyster shucking, the oyster meats are generally packed in flexible pouches merged in oyster culture. This closed culture environment poses a high risk of microbial contamination because if even one shucked oyster meat carries Vibrio, during transport, storage, and retail, the residual Vibrio populations will multiply markedly in oyster tissues, and large numbers will be released into the surroundings, which may lead to Vibrio cross-contamination. In this regard, we also investigated whether d-Trp could exhibit strong inhibitory effects in experimentally inoculated shucked oyster culture. Consistent with the present culture medium experiments, after d-Trp was added to the shucked oyster culture, we observed considerable inhibition of the growths of both V. parahaemolyticus and V. vulnificus. In addition, elevated levels of NaCl increased the inhibition efficiency, leading to a more gradual reduction of Vibrio populations in the oyster culture. Therefore, d-Trp may be considered a novel preservative to control Vibrio contamination, even at room temperature, in freshly shucked oysters supplemented with seawater or sterile water at high levels of salinity.

Due to the fact that seawater is the native environment for Vibrio growth, we further examined the inhibitory effects of d-Trp on the survival of V. vulnificus and V. parahaemolyticus in artificial seawater at various salinity levels (3.5, 4.0, 4.5, and 5.0% [wt/vol]). Under the same salinity conditions, d-Trp exhibited a stronger growth-inhibitory effect on V. vulnificus and V. parahaemolyticus at 25°C than in culture medium (TSB), indicating that the inhibitive effect of d-Trp can be achieved at even relative low salinity. Moreover, a more gradual growth decrease was observed with the salinity level increase. The reason d-Trp had an unexpectedly stronger inhibitory effect on Vibrio in artificial seawater than in culture medium is not clear. However, one possible explanation is that some ingredients in the artificial seawater increase bacterial sensitivity to d-Trp or give rise to the nutrientfree environment of artificial seawater that contributes to bacterial growth inhibition. The results indicate that lower concentrations of NaCl and d-Trp are needed for bacterial growth inhibition under low-nutrient conditions.

For live oysters, when postharvest temperatures are not properly controlled in supply chains, Vibrio spp. can multiply and reach potentially hazardous levels. For example, V. parahaemolyticus is capable of growing rapidly by 1.7 and 2.9 log CFU/g in oysters after being exposed to 26°C for 10 and 24 h, respectively (27). The population of V. vulnificus in oysters increases rapidly and reaches a peak during the first 12 h after the harvest of oysters at room temperature (28). In the present study, we conducted the experiments at 25°C, which is generally considered the optimal growth temperature for Vibrio spp. (29). Moreover, because d-trp could exhibit a strong inhibitory efficiency in artificial seawater and high levels of salinity might cause live oyster death, the artificial seawater (approximately 2.7% salinity) without extra NaCl added was used in this experiment. As expected, d-Trp treatment reduced the Vibrio load in live oysters, consistent with previous experiments. Additionally, V. vulnificus appeared to be more sensitive to d-Trp treatment than was V. parahaemolyticus, thus exhibiting a higher growth reduction.

Refrigeration is one of the most common methods to retard spoilage and extend the shelf life of fresh food (25). After oyster shucking, shucked oysters are normally maintained under constant refrigeration to effectively retard microbial growth and extend the shelf life of seafood during the postharvest process, particularly in the area of controlling Vibrio contamination (23). For example, depuration at refrigeration temperature (5 to 15°C) could effectively reduce V. parahaemolyticus and V. vulnificus contamination in American oysters (26). In this study, the survival numbers of both Vibrio spp. gradually decreased at refrigeration temperatures, regardless of d-Trp, as expected (data not shown).

Unlike Vibrio spp., the total bacterial load on the shucked oyster will increase gradually even during refrigeration storage, which is expected to cause bacterial spoilage in oysters. Some studies found that the total viable bacterial count (TVC) exceeded 10⁶ CFU/g after 14 days and 9 days at 0.5°C and 4°C storage, respectively (30, 31). Salinity, even at high levels, plays little role in controlling Vibrio numbers in oysters (29). Based on this fact, we further evaluated the combined efficacy of d-Trp and salinity for reducing the total bacterial population in shucked oysters supplemented with artificial seawater at 3.5% and 5.0% salinity. In the control (without d-Trp treatment), the TVC increased by 3 log units after storage at 4°C for 1 week, and the average growth rate was approximately 0.4 log units each day, which is in good agreement with results reported by Fernandez-Piquer (32), who developed a predictive model for TVC growth in oysters and the average growth rates at 3.6°C and 6.2°C. According to the U.S. Food and Drug Administration microbial standard, TVCs in fresh bivalve mollusks below 5 × 10⁵ CFU/g are generally considered to indicate good quality (33). Interestingly, we found that d-Trp could strongly inhibit TVC increase in shucked oysters surrounded with artificial seawater during storage at 4°C. Moreover, increase of the salinity level to 5% could further extend the shelf life of oysters by delaying the time until 5 × 10⁵ CFU/g is exceeded. These results demonstrate that d-Trp could reduce the TVC in oysters during refrigeration storage, indicating good potential for shelf-life extension of oysters during postharvest storage and transport at refrigeration temperature.

In conclusion, contamination of V. parahaemolyticus and V. vulnificus in raw oysters could be reduced by holding oysters in artificial seawater containing d-Trp at a certain predetermined, high level of NaCl. However, further studies are warranted to investigate the detrimental effect of d-Trp, as well as the combined effect of d-Trp and high salinity, on oysters. Nevertheless, we suggest d-Trp as a potential novel alternative food preservative to control Vibrio contamination in oysters at room temperature and to extend the shelf life of raw oysters at refrigeration temperatures.

MATERIALS AND METHODS

Bacterial strains.

V. vulnificus (NBRC 103026) and V. parahaemolyticus (NBRC 12711) were obtained from the Biological Resource Center, NITE (NBRC), in Japan. The strains were stored at −80°C in brain heart infusion (BHI) broth (Merck, Darmstadt, Germany) with 2.0% NaCl and 50% glycerol. For each experiment, bacterial cells were collected from the frozen stock and plated on TSA (Merck) supplemented with 2% NaCl (TSA-2%). Individual colonies were selected and enriched in TSB with 2% NaCl (TSB-2%) at 25°C for 48 h to obtain a final concentration of approximately 10⁹ CFU/ml.

Effects of d-Trp on the growth of Vibrio spp. in culture media.

To evaluate the growth-inhibitory effects of d-Trp (Wako Pure Chemical Industries, Ltd., Osaka, Japan) on V. vulnificus and V. parahaemolyticus in TSB, the bacterial culture was prepared as described above and diluted in phosphate-buffered saline (PBS) to obtain a concentration of approximately 5 × 10⁷ log CFU/ml. A 0.1-ml aliquot of this diluted bacterial culture was added into 0.9 ml of TSB supplemented with various concentrations of NaCl (3.5, 4.0, 4.5, and 5.0% [wt/vol]) and d-Trp (0, 20, and 40 mM) and incubated at 25°C without shaking. Bacterial survival was determined via plate counts on TSA-2% after each 24 h of incubation. Successive 10-fold serial dilutions were performed using PBS (2% NaCl), and 100 μl aliquots of the diluted samples were then spread on TSA-2% plates and cultured at 25°C for 24 h.

Effects of d-Trp on survival of Vibrio spp. in shucked oysters immersed in peptone water.

This experiment was performed as described in a previous study (34), with some modifications. Freshly shucked oysters were obtained from a local seafood market (Akkeshi, Hokkaido, Japan) and transported immediately in a styrene foam box with crushed ice. Each oyster was artificially contaminated by pipetting 300 μl (approximately 5.0 × 10⁷ CFU/ml) of Vibrio culture and then air dried at 25°C for 30 min to allow bacterial attachment. Subsequently, each Vibrio-contaminated oyster meat was individually transferred into a sterile plastic bag (85 by 60 by 0.04 mm), followed by the addition of 30 ml of 0.1% peptone water with 40 mM d-Trp. The NaCl concentration in the peptone water ranged from 3.5% to 5.0%. Samples were incubated at 25°C for 48 h. The treatment without d-Trp was considered the control condition. To enumerate viable Vibrio spp., the culture and shucked oysters were aseptically placed in a sterile 400-ml filter stomacher bag and pummeled with a Stomacher 400-T (Seward, UK). The stomached samples were diluted in 10-fold serial dilutions with PBS and plated onto Vibrio CHROMagar (CHROMagar, Paris, France), a selective medium for Vibrio spp. V. parahaemolyticus appears as mauve colonies, whereas V. vulnificus appears as turquoise colonies (35). Although the sodium chloride concentration of the CHROMagar is as high as 5.14%, we previously confirmed that such a salinity does not significantly impact the recovery of viable Vibrio cell numbers compared with that from TSA-2% (data not shown). The CHROMagar only inhibits recovery of natural microflora in a sample other than Vibrio spp. Each condition was independently evaluated using three oysters, and the viable number of bacteria in the homogenates was determined after each 24-h interval.

Effects of d-Trp on the growth of Vibrio spp. in artificial seawater of various salinities.

The inhibitory effect of d-Trp in artificial seawater of various salinities (3.5 to 5.0%) was also evaluated by the pour plating method, as described above. Higher salinity was achieved by altering the amount of NaCl added. The final salinity was measured by a salt meter (B-721; HORIBA, Japan). All of the plates were incubated at 25°C for 48 h. Growth inhibition tests were performed three times. The colonies formed on the plates were counted and expressed as log CFU/ml. Results were recorded as the means ± standard deviations from three independent experiments.

Effects of d-Trp on survival of Vibrio load on artificially inoculated live oysters.

Bacterial inoculum was prepared in broth culture, as described above. Stationary-phase Vibrio cells were suspended at an initial concentration of approximately 5 × 10⁴ CFU/ml in artificial seawater (2.7% salinity, Daigo's artificial seawater S; Nihon Pharmaceutical Co., Ltd., Osaka, Japan) containing 40 mM d-Trp. Serial 10-fold PBS dilutions were used to enumerate the Vibrio bacteria by spread plating on Vibrio CHROMagar. The survival of Vibrio spp. was determined after incubation for 24 and 48 h. All experiments were independently performed three times.

The growth inhibition effect of d-Trp on V. parahaemolyticus and V. vulnificus was further analyzed in experimental Vibrio-inoculated live oysters, as described in previous oyster studies (1, 28), with some modifications. Live oysters (Crassostrea gigas) were harvested from Akkeshi (Hokkaido, Japan), transported in coolers on ice packs, delivered to the laboratories within 48 to 72 h, and processed immediately. After 2 h of acclimation at 25°C, the oyster surface was washed with tap water to remove dirt or debris. Thirty oysters were placed in a sterile styrene foam box with 20 liters of artificial seawater containing V. vulnificus or V. parahaemolyticus (approximately 10⁵ CFU/ml) and incubated at 25°C for 24 h. Inoculated oysters were subsequently transferred to a new sterile styrene foam box with 5 liters of artificial seawater supplemented with or without 40 mM d-Trp, and were individually evaluated for the survival of V. vulnificus or V. parahaemolyticus after 0, 24, and 48 h. A laboratory water circulator (SM-05R; TAITEC) was used for Vibrio accumulation in oysters. The water jet pump was set on the sterile styrene foam box, and artificial seawater was circulated to keep dissolved oxygen levels favorable for oyster pumping and uptake of Vibrio spp.

To enumerate Vibrio spp. in oysters, three oysters were randomly selected from the styrene foam box at each sampling time point and shucked aseptically with a sterile shucking knife. The oyster meat was collected aseptically in a sterile 400-ml filter stomacher bag. Samples were weighed and homogenized using the Stomacher 400-T for 60 s at high speed (120 strokes per min) with an equal weight of PBS. The stomached samples were then serially diluted 10-fold using PBS and plated on Vibrio CHROMagar to enumerate the Vibrio spp. The counts were recorded as the number of log CFU/g.

Combined efficacy of d-Trp and salinity on total viable bacterial count in shucked oysters during refrigerated storage.

This experiment was performed as in a previous study (34), with some modifications. Freshly shucked oysters were obtained from a local seafood market (Akkeshi, Hokkaido, Japan) and transported immediately in a styrene foam box with crushed ice. Subsequently, each shucked oyster meat was individually transferred into a sterile plastic bag (85 by 60 by 0.04 mm), followed by the addition of 30 ml of artificial seawater with or without 40 mM d-Trp. The levels of salinity in the artificial seawater were 3.5% and 5.0%. Samples were incubated at 25°C for 48 h. The treatment without d-Trp was considered the control condition. To determine the total viable bacterial count, the culture and shucked oyster were aseptically placed in a sterile 400-ml filter stomacher bag and pummeled with a Stomacher 400-T (Seward, UK). The resultant samples were 10-fold serially diluted with PBS and plated onto TSA supplemented with 0.6% yeast extract (TSAYE). The plates were incubated at 25°C for 48 h. Each condition was independently evaluated using three oysters, and the number of viable bacteria in the homogenates was determined after 1, 3, 5, and 7 days of storage.

Statistical analysis.

Triplicate samples were collected at each sampling time. The colony-counting data for the triplicate samples of each bacterium at each sampling interval were transformed to log CFU/ml or log CFU/g, and the values of the triplicate samples were averaged to represent the number of viable cells at each sampling time. One-way analysis of variance (ANOVA) tests were performed to compare the differential degrees between treatments. Then, the viable counts under each condition were compared by Tukey-Kramer's multiple-comparison test to analyze the statistical difference (P < 0.05). Statistical data were evaluated using the commercial software KaleidaGraph 4.5 (Synergy Software, Reading, PA).