Enhancing Food Safety Through Control of Food-Borne Disease Agents

NON-TECHNICAL SUMMARY: Every year during the summer months the oyster industry is threatened by closure of water ways and recall of oysters due to V. vulnificus and V. parahaemolyticus contamination. This proposed project could lead to the destruction of V. parahaemolyticus and V. vulnificus in shell stock oysters by using bacteriophages. By destroying these human pathogens in shell stock oysters it will reduce the economic loss due to recalls, protect the oyster industries reputation along with the health and welfare of the consumers.

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APPROACH: Naturally occurring bacteriophages will be isolated from oysters and seawater collected from the Gulf of Mexico during the spring (April), summer (July), fall (October) and winter (January) to determine if there is variability in the presence of possible bacteriophages that are more inhibitory or virulent towards V. parahaemolyticus or V. vulnificus strains. Bacteriophage enumeration will be done using the soft agar overlay technique. To increase the numbers of bacteriophages in seawater 5 ml of double strength CPM broth will be added to 45 ml of seawater along with 5 ml of V. parahaemolyticus or V. vulnificus strains and the samples will be incubated at 37 degree C for 24 hours. To isolate the bacteriophages from oysters we will remove the mantle, hemolymph, adductor, gill, digestive tissue, and combine the remaining tissues of the oyster. Each tissue sample will be ground with a glass mortal and pestal in 5 ml of CMP and then transferred to 45 ml of CMP inoculated with 5 ml of V. parahaemolyticus or V. vulnificus and incubated for 42 hours at 37 degree C. After incubation, bacteriophage stock samples will be prepared. In addition, the effects of pH, temperature, and salt content on the bacteriophages inactivation will be evaluated. The bacteriophages that have the highest resistance to the physiochemical conditions will be utilize further in the experiments involving the life oysters. Transmission electron microscopy will be used to observe the morphology of the newly isolated bacteriophages. Next we will determine the concentration of nisin, chelating agents, lauric acid or lysozyme with or without the bacteriophages needed to reduce V. parahaemolyticus and V. vulnificus in vitro. The bacterial cultures will be grown up over-night, decimally diluted and 1.0-ml placed into microcentrifuge tubes. Then the samples will be centrifuged and the supernatant discarded. The different treatments will be suspended in sterile seawater and 1.0-ml added to the bacterial cultures. Bacteriophages will be added to the bacterial cultures with and without added nisin (1000 IU/ml, 2000 IU/ml or 4000 IU/ml) EDTA (15 or 30 mM), lysozyme (33, 66 or 99 mg/g), or lauric acid (4 or 8 percent (w/w)). The samples will be incubated at 37C for 2 hours and bacterial and bacteriophage counts determined. Finally, this research project will examine how effective the developed antimicrobial formulation and bacteriophages are in reducing or eliminating V. parahaemolyticus or V. vulnificus in shell stock oysters over 10 days at 4, 8, or 25 degree C. The oysters will be maintained in 20-liter tanks that contain 10 liters of aerated and continuous flowing sterile seawater with a salinity of 14 ppt. Each tank will contain 20 oysters. Mutant strains of V. parahaemolyticus or V. vulnificus that are nalidixic acid resistance will be used to evaluate the effect that the bacteriophages and other treatments have on the reduction or elimination of these foodborne pathogens in live oysters.

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PROGRESS: 2002/10 TO 2006/09<BR>
Transmission electron microscope observations showed that the predominant morphology of bacteriophages active against V. vulnificus and V. parahaemolyticus were icosahedra with thin flexible tails. The bacteriophages V. vulnificus opaque (Vvo), V. vulnificus translucent (Vvt) and V. parahaemolyticus (Vp) retained their activity at a pH of 7 and 8 after 36 days at all temperatures tested. All of the bacteriophages immediately lost activity at an acidic pH of 3 and 4 for all temperatures tested. Furthermore, the bacteriophages lost activity by day 28 when stored at 45degreeC in all salt concentrations tested. However, when the bacteriophages Vvo and Vvt were stored at 4degreeC in salt concentrations of 6 to 22% and at 30degreeC in salt concentrations of 6 to 10% they had retained activity by day 52. Bacteriophage Vp remained active in salt concentrations of 6 to 30% at 4degreeC and in salt concentrations of 6 to 22% at 30degreeC by day 52. Our results showed that bacteriophages Vvo, Vvt, and Vp lost activity at an acidic pH. Also, these bacteriophages were sensitive to elevated temperatures. We also investigated the effect of bacteriophages treatments on Vvo, Vvt and Vp inoculated into raw oysters with or without UV exposure for 5, 15 and 30 min. The Vvo phage treatment reduced V. vulnificus opaque counts by 2.00 log CFU/g for all treatment times in raw oysters. However, non-UV treatments were not significantly different from the UV treatments. In addition there were no significant change between samples exposed for 5, 15 and 30 min. We found that the activity of the Vvt phages was increased against V. vulnificus translucent with UV treatment in raw oysters. When V. vulnificus translucent was exposed to the Vvt phage, the bacterial counts were reduction to 3.30 log CFU/g compared to 6.24 log CFU/g for the control samples treated with UV light for 30 min in raw oysters. Vp phage treated V. parahaemolyticus counts in oysters were significantly lower then the non-treated controls by 2.00 log CFU/g. UV light exposure had no effect on Vp phage activity against V. parahaemolyticus in raw oysters.
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IMPACT: 2002/10 TO 2006/09<BR>
Every year during the summer months the oyster industry is threatened by closure of water ways and recall of oysters due to V. vulnificus and V. parahaemolyticus contamination. Bacteriophages have the potential to control foodborne pathogens in seafood or could be used for the indication of Vibrio vulnificus and Vibrio parahaemolyticus contamination of oysters. Our results indicate that bacteriophages active against the virulent Vibrio vulnificus and Vibrio parahaemolyticus are naturally found in oysters and could possibly be used as a processing aid to control this pathogen in live oysters. Bacteriophage treatments alone or in combination with UV exposure were very effective in reducing V. vulnificus and V. parahaemolyticus population in raw oysters. By destroying these human pathogens in shell stock oysters it will reduce the economic loss due to recalls, protect the oyster industries reputation along with the health and welfare of the consumers.
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PROGRESS: 2005/01/01 TO 2005/12/31<BR>
The purpose of this study was to investigate the effectiveness of bacteriophages in reducing V. parahaemolyticus and V. vulnificus in raw oysters during storage with or without UV-light exposure. Bacteriophage stock samples were prepared as previously described. Oysters were harvested from the Gulf of Mexico and upon arrival to our lab, 10 to 12 oysters were randomly selected and the surface microflora and mud were removed by scrubbing the oysters under running water. The oysters were then shucked with a sterile knife and placed with their liquor into sterile sampling bags. One oyster per treatment was inoculated with 1 ml of a decimally diluted V. vulnificus or V. parahaemolyticus cultures then the oysters were treated with 1 ml of Vvo, Vvt or Vp phages. Samples were divided into 2 groups, one group was not treated with UV-light and the other was treated with UV-light for 5, 15 or 30 min then stored at 4degreeC for 24 h. The oysters were transferred into stomacher bags, weighted and then equivalent amounts (ml) of PBS (1:1 dilution) were added to each bag. The samples were stomached for 2 min; serial dilutions in sterile PBS were made then samples were spread-plated in duplicate on TCBS agar. All plates were incubated at 35degreeC for 18 to 24h. Yellow (V. vulnificus) and green (V. parahaemolyticus) colonies were counted and log CFU/g calculated. All experiments were repeated 3 times. The initial bacterial counts for V. vulnificus opaque, V. vulnificus translucent and V. parahaemolyticus control samples with or with out UV-light treatments were about 7.0 log CFU/g. The phage treatment reduced V. vulnificus opaque counts in raw oysters by 2.0 log CFU/g for all treatment times as compared to the controls. However, non-UV light treatments were not significantly different from the UV-light treatments. When oysters contaminated with V. vulnificus translucent were treated with Vvt phage and UV-light for 30 min, the bacterial counts were reduced to 3.3 log CFU/g compared to 6.3 log CFU/g for the control samples. Furthermore, the antimicrobial activity of the Vvt phages with UV-light treatment was significantly different from the V. vulnificus translucent counts that were treated with Vvt phages with out UV-light exposure. Vp phage treated oysters inoculated with V. parahaemolyticus counts were 4.83, 4.96 and 5.16 log CFU/g for non-UV and 4.97, 4.71 and 4.68 log CFU/g for UV-exposed at 5, 15 and 30 min exposure, respectively. There was no significant difference in V. parahaemolyticus counts between the UV-light exposed and non-exposed Vp phage treated oysters.
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IMPACT: 2005/01/01 TO 2005/12/31<BR>
Every year during the summer months the oyster industry is threatened by closure of water ways and recall of oysters due to V. vulnificus and V. parahaemolyticus contamination. Our results indicate that bacteriophages active against the Vibrio vulnificus and V. parahaemolyticus are naturally found in oysters and could possibly be used as a processing aid to control these pathogens in live oysters. Bacteriophage treatments alone or in combination with UV-light exposure were very effective in reducing V. vulnificus and V. parahaemolyticus population in raw oysters.
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PROGRESS: 2005/01/01 TO 2005/12/31<BR>
The purpose of this study was to investigate the effectiveness of bacteriophages in reducing V. parahaemolyticus and V. vulnificus in raw oysters during storage with or without UV-light exposure. Bacteriophage stock samples were prepared as previously described. Oysters were harvested from the Gulf of Mexico and upon arrival to our lab, 10 to 12 oysters were randomly selected and the surface microflora and mud were removed by scrubbing the oysters under running water. The oysters were then shucked with a sterile knife and placed with their liquor into sterile sampling bags. One oyster per treatment was inoculated with 1 ml of a decimally diluted V. vulnificus or V. parahaemolyticus cultures then the oysters were treated with 1 ml of Vvo, Vvt or Vp phages. Samples were divided into 2 groups, one group was not treated with UV-light and the other was treated with UV-light for 5, 15 or 30 min then stored at 4degreeC for 24 h. The oysters were transferred into stomacher bags, weighted and then equivalent amounts (ml) of PBS (1:1 dilution) were added to each bag. The samples were stomached for 2 min; serial dilutions in sterile PBS were made then samples were spread-plated in duplicate on TCBS agar. All plates were incubated at 35degreeC for 18 to 24h. Yellow (V. vulnificus) and green (V. parahaemolyticus) colonies were counted and log CFU/g calculated. All experiments were repeated 3 times. The initial bacterial counts for V. vulnificus opaque, V. vulnificus translucent and V. parahaemolyticus control samples with or with out UV-light treatments were about 7.0 log CFU/g. The phage treatment reduced V. vulnificus opaque counts in raw oysters by 2.0 log CFU/g for all treatment times as compared to the controls. However, non-UV light treatments were not significantly different from the UV-light treatments. When oysters contaminated with V. vulnificus translucent were treated with Vvt phage and UV-light for 30 min, the bacterial counts were reduced to 3.3 log CFU/g compared to 6.3 log CFU/g for the control samples. Furthermore, the antimicrobial activity of the Vvt phages with UV-light treatment was significantly different from the V. vulnificus translucent counts that were treated with Vvt phages with out UV-light exposure. Vp phage treated oysters inoculated with V. parahaemolyticus counts were 4.83, 4.96 and 5.16 log CFU/g for non-UV and 4.97, 4.71 and 4.68 log CFU/g for UV-exposed at 5, 15 and 30 min exposure, respectively. There was no significant difference in V. parahaemolyticus counts between the UV-light exposed and non-exposed Vp phage treated oysters.
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IMPACT: 2005/01/01 TO 2005/12/31<BR>
Every year during the summer months the oyster industry is threatened by closure of water ways and recall of oysters due to V. vulnificus and V. parahaemolyticus contamination. Our results indicate that bacteriophages active against the Vibrio vulnificus and V. parahaemolyticus are naturally found in oysters and could possibly be used as a processing aid to control these pathogens in live oysters. Bacteriophage treatments alone or in combination with UV-light exposure were very effective in reducing V. vulnificus and V. parahaemolyticus population in raw oysters.
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PROGRESS: 2004/01/01 TO 2004/12/31<BR>
In this study we investigated the effects that pH, temperature and salt content have on the activities of bacteriophages isolated from oysters against virulent Vibrio vulnificus, attenuated Vibrio vulnificus or Vibrio parahaemolyticus. Bacteriophage stock samples were prepared as previously described. Tryptone broth was adjusted to a pH of 3, 4, 5, 6, 7, 8, 9 and 10. Then 100 microliters of bacteriophages were added to 900 microliters of the different pH TB broths and stored at 4, 30, and 45degreesC for 36 days. Bacteriophages (100 microliters) were added to 900 microliters of sterile water with the following salt concentrations 0, 2, 6, 10, 14, 18, 22, 26, or 30% and stored at 4, 30, and 45degreesC. The bacteriophage activity was determined using the soft agar overlay technique using phages active against virulent Vibrio vulnificus (Vvo), attenuated Vibrio vulnificus (Vvt) or Vibrio parahaemolyticus (Vp). Retention of bacteriophages activity was determined at days 0, 1, 4, 7, 14, 19, 25, 28, 36, 52 for salt concentration and at days 0, 1, 4, 8, 12, 24, and 36 for pH. For transmission electron microscope observations the bacteriophages were negative stained with 2% phosphotungstic acid and observed under a transmission electron microscope. Transmission electron microscope observations showed the predominant morphology of bacteriophages active against V. vulnificus and V. parahaemolyticus were icosahedra with thin flexible tails. The bacteriophages Vvo, Vvt and Vp retained their activity at a pH of 7 and 8 after 36 days at all temperatures tested. All of the bacteriophages immediately lost activity at an acidic pH of 3 and 4 for all temperatures tested. Furthermore, the bacteriophages lost activity by day 28 when stored at 45degreesC in all salt concentrations tested. However, when the bacteriophages Vvo and Vvt were stored at 4degreesC in salt concentrations of 6 to 22% and at 30degreesC in salt concentrations of 6 to 10% they had retained activity by day 52. Bacteriophage Vp remained active in salt concentrations of 6 to 30% at 4degreesC and in salt concentrations of 6 to 22% at 30degreesC by day 52.
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IMPACT: 2004/01/01 TO 2004/12/31<BR>
Every year during the summer months the oyster industry is threatened by closure of water ways and recall of oysters due to V. vulnificus and V. parahaemolyticus contamination. Our results indicate that bacteriophages active against the virulent Vibrio vulnificus are naturally found in oysters and could possibly be used as a processing aid to control this pathogen in live oysters. In the future we plan on using the newly isolated bacteriophages against V. parahaemolyticus and V. vulnificus in contaminated shell stock oysters. By destroying these human pathogens in shell stock oysters it will reduce the economic loss due to recalls, protect the oyster industries reputation along with the health and welfare of the consumers.

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PROGRESS: 2003/01/01 TO 2003/12/31<BR>
Our results indicated that the bacteriophages found in the oyster tissue were more abundant against the virulent strain of Vibrio vulnificus compared to the attenuated Vibrio vulnificus strain and Vibrio parahaemolyticus. The highest counts for bacteriophages active against virulent Vibrio vulnificus were from April 2003 to October 2003 with August having the highest counts of 3.93 PFU/g of bacteriophages. The bacteriophages active against attenuated Vibrio vulnificus had higher counts (about 3.00 PFU/g) between April 2003 and August 2003 but the counts were at non-detectable levels by October 2003. The bacteriophages active against Vibrio parahaemolyticus were always below 2.50 PFU/g and were at non-detectable levels in December 2002 and August 2003. These results indicate that bacteriophages active against both Vibrio vulnificus strains have a seasonal distribution occurring mainly in the summer months when both of these bacteria are at their highest numbers in oysters.
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IMPACT: 2003/01/01 TO 2003/12/31<BR>
Bacteriophages have the potential to control foodborne pathogens in seafood or could be used for the indication of Vibrio vulnificus and Vibrio parahaemolyticus contamination of oysters. Our results indicate that bacteriophages active against the virulent Vibrio vulnificus and Vibrio parahaemolyticus are naturally found in oysters and could possibly be used as a processing aid to control this pathogen in live oysters.