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Applied and Environmental Microbiology, November 2006, p. 7205-7211, Vol. 72, No. 11
0099-2240/06/$08.00+0     doi:10.1128/AEM.01091-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Validation of a Green Fluorescent Protein-Labeled Strain of Vibrio vulnificus for Use in the Evaluation of Postharvest Strategies for Handling of Raw Oysters^,

S. L. Drake, D. Elhanafi, W. Bang, M. A. Drake, D. P. Green, and L. A. Jaykus^*

Food Science Department, North Carolina State University, Raleigh, North Carolina 27695-7624

Received 11 May 2006/ Accepted 4 September 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this paper we describe a biological indicator which can be used to study the behavior of Vibrio vulnificus, an important molluscan shellfish-associated human pathogen. A V. vulnificus ATCC 27562 derivative that expresses green fluorescent protein (GFP) and kanamycin resistance was constructed using conjugation. Strain validation was performed by comparing the GFP-expressing strain (Vv-GFP) and the wild-type strain (Vv-WT) with respect to growth characteristics, heat tolerance (45°C), freeze-thaw tolerance (–20^o and –80°C), acid tolerance (pH 5.0, 4.0, and 3.5), cold storage tolerance (5°C), cold adaptation (15°C), and response to starvation. Levels of recovery were evaluated using nonselective medium (tryptic soy agar containing 2% NaCl) with and without sodium pyruvate. The indicator strain was subsequently used to evaluate the survival of V. vulnificus in oysters exposed to organic acids (citric and acetic acids) and various cooling regimens. In most cases, Vv-GFP was comparable to Vv-WT with respect to growth and survival upon exposure to various biological stressors; when differences between the GFP-expressing and parent strains occurred, they usually disappeared when sodium pyruvate was added to media. When V. vulnificus was inoculated into shellstock oysters, the counts dropped 2 log₁₀ after 11 to 12 days of refrigerated storage, regardless of the way in which the oysters were initially cooled. Steeper population declines after 12 days of refrigerated storage were observed for both iced and refrigerated products than for slowly cooled product and product held under conservative harvest conditions. By the end of the refrigeration storage study (22 days), the counts of Vv-GFP in iced and refrigerated oysters had reached the limit of detection (10² CFU/oyster), but slowly cooled oysters and oysters stored under conservative harvest conditions still contained approximately 10³ and >10⁴ CFU V. vulnificus/oyster by day 22, respectively. The Vv-GFP levels in the oyster meat remained stable for up to 24 h when the meat was exposed to acidic conditions at various pH values. Ease of detection and comparability to the wild-type parent make Vv-GFP a good candidate for use in studying the behavior of V. vulnificus upon exposure to sublethal stressors that might be encountered during postharvest handling of molluscan shellfish.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The consumption of contaminated seafood is a significant cause of food-borne disease in the United States, and raw molluscan shellfish is one of the most important commodities associated with disease transmission (7). Bivalves are filter feeders that concentrate microorganisms in their digestive tracts and can serve as passive carriers of food-borne pathogens, especially since they are often consumed whole and raw. Vibrio vulnificus is an environmentally ubiquitous marine bacterium, and high numbers of this species can be isolated from United States Gulf Coast bivalve molluscan shellfish harvested during warm summer months. This organism is associated with several disease syndromes, the most serious of which is primary septicemia, which occurs in at-risk molluscan shellfish consumers at a frequency of about 30 cases per year in the United States and is often fatal. As a major public health objective, the Interstate Shellfish Sanitation Conference (ISSC) has proposed that V. vulnificus infections should be reduced by 60% by the year 2007. To achieve this reduction, the ISSC suggests that oysters contain <30 V. vulnificus cells per g of oyster meat (29).

Resiliency and adaptability in the face of exposure to sublethal stresses, such as temperature shifts or reduced pH and/or water activity, have been reported for many bacterial species, and members of the family Vibrionaceae are no exception (6). For example, the acid stress response (18, 20, 31, 32) and the cold adaptive response (5, 9, 24) have been at least partially characterized for members of this family. It has long been known that sublethally injured cells have increased sensitivity to hydrogen peroxide, which is a common by-product of medium preparation. By supplementing media with the peroxide-degrading compound catalase or sodium pyruvate, investigators have facilitated the recovery of sublethally injured cells (4), including V. vulnificus cells induced into the viable but nonculturable (VBNC) state (8).

A multitude of potential processing control options are being evaluated in an effort to achieve the ISSC target for the reduction in V. vulnificus infections (1). As scientists evaluate the efficacy of these approaches, it will be important to consider the effects of sublethal injury on the recovery of this organism. A further complication for this bacterial species is that it is difficult to distinguish V. vulnificus from the background microflora associated with oyster meat. Consequently, time-consuming most-probable-number enrichment followed by selective plating and/or colony lift hybridization must be used for enumeration of the pathogenic Vibrio species (3). In an effort to facilitate evaluation of control options, in this study we constructed a biological indicator which can be used to study the behavior of V. vulnificus in response to a variety of sublethal stresses that might be encountered during the processing and storage of raw molluscan shellfish. In our initial work, we produced a strain of V. vulnificus that expressed green fluorescent protein (GFP) and could be readily differentiated from the natural microflora associated with the oyster matrix. In further experiments, we compared the growth and survival characteristics of the engineered strain to the growth and survival characteristics of the parent to ensure that the engineered strain is a useful surrogate for stress response studies. The growth and survival characteristics were evaluated in nutrient-rich medium both with and without sodium pyruvate. Finally, the engineered strain was used to evaluate the effects of different cooling treatments and combinations of pH and acid treatments on the survival of V. vulnificus in oysters.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacteriological media.
Most of the media used in this work have been described previously in the Food and Drug Administration Bacteriological Analytical Manual (3). The bacteriological media were obtained from Becton Dickinson and Company (Franklin Lakes, NJ) unless indicated otherwise and were prepared according to the manufacturer's recommendations or were altered based on requirements of the experimental design. In most cases cultures were incubated at 37°C for 24 h on nonselective media and at 40°C for 24 h on selective media, unless indicated otherwise. Trypticase soy broth supplemented with 2% NaCl (TSBN₂) was used for growth and as a diluent, and Trypticase soy agar supplemented with 2% NaCl (TSAN₂) was used for enumeration. The selective media included modified cellobiose-polymyxin B-colistin (mCPC) agar. Since the GFP-expressing strain was designed to also express kanamycin resistance, media were supplemented with this antibiotic (0.05 g/liter) (TSAN₂K and mCPCK), which ensured the stability of the GFP expression vector and prevented growth of the competitive microflora associated with the oyster matrix. Kanamycin was not added when the wild-type V. vulnificus strain was recovered. Phosphate-buffered saline was used a diluent for oyster homogenates, and alkaline peptone water was used for subsequent serial dilutions. To promote recovery of injured cells in some experiments, TSAN₂ was supplemented with sodium pyruvate (Sigma Chemical Co., St. Louis, MO). In preliminary experiments, media were supplemented with different concentrations of sodium pyruvate (0 to 320 mg/ml); consistent with the findings of other workers (8), we found that 40 mg/ml or more of pyruvate improved recovery, but we chose to supplement our media with 80 mg/ml (data not shown). All experiments except the growth rate experiments were conducted with and without sodium pyruvate.

Stock cultures of the wild-type and GFP-producing strains of V. vulnificus were maintained at room temperature on TSAN₂ and TSAN₂K slants, respectively, with sterilized mineral oil overlays (Sigma Chemical Co.). Stock strains were transferred monthly. Three consecutive transfers of each strain at 37°C for 12 h were done before experimental inoculation.

Development of GFP-expressing strain of V. vulnificus (Vv-GFP).
The wild-type V. vulnificus ATCC 27562 strain (Vv-WT) was used for production of the GFP-expressing strain; strain ATCC 27562 was chosen as previous studies demonstrated that it survived better upon exposure to low temperature and acidic conditions than other strains (5, 6). Furthermore, ATCC 27562 is not a naturally fluorescing V. vulnificus strain. The plasmid pNKBOR system, an oriR6K-based suicide vector system that permits random insertion of a minitransposon into the chromosome of gram-negative bacteria (27), was used for conjugation and transposition. The minitransposon contains a conditional R6K plasmid origin of replication, a kanamycin resistance gene, and unique nuclease restriction sites (specifically, PstI and BamHI sites). pNKBOR can be propagated by replication in Escherichia coli strains containing the R6K replicase protein; in this study we used E. coli S17-1 pir (obtained from M. Izallalen, University of Laval, Canada). Efficient pNKBOR transposition is ensured by expression of an adjacent Tn10 transposase gene. To allow entry of plasmid pNKBOR into V. vulnificus, we cloned an 850-bp PstI fragment containing the oriT origin of transfer sequence from plasmid pCON-1 (23) into the PstI site of pNKBOR, yielding plasmid pMAD278. OriT allows transfer of pNKBOR from S17-1 pir to V. vulnificus by conjugation. The gfp gene was isolated from plasmid pGFP, in which it was overexpressed by the lactose operon promoter (CLONTECH Laboratories, Palo Alto, CA). Plasmid pGFP was digested with restriction endonucleases AseI (New England Biolabs, Beverly, MA) and EcoRI (Promega, Madison, WI), producing a Plac-gfp DNA fragment, the extremities of which were filled in with the Klenow enzyme (Promega, Madison, WI). This fragment was then inserted into the BamHI site of the transposon in plasmid pMAD278, generating plasmid pMAD281, in which the gfp gene was overexpressed by the Plac promoter present inside the pNKBOR transposon. To isolate a Plac::gfp insertion in the chromosome of V. vulnificus, an aliquot of an 8-h culture of S17-1 pir/pMAD281 was spotted on TSAN₂, dried, overlaid with an aliquot of an 8-h culture of V. vulnificus, and dried, and the plate was incubated at 37°C for 12 to 18 h. After incubation, the mixed culture was spread on TSAN₂K. V. vulnificus strains with a chromosomal insert of the Plac::gfp transposon were easily identified as large fluorescent colonies displaying kanamycin resistance due to their rapid growth compared to the growth of strain E. coli S17-1 pir, which grew very slowly and formed very small colonies on TSAN₂K. The GFP-producing strain, designated Vv-GFP, was used in subsequent studies.

Validation of the biological indicator.
Various growth and survival studies were done to validate the hypothesis that Vv-GFP was comparable to Vv-WT and to determine the usefulness of Vv-GFP as a biological indicator. In initial studies to compare plating and recovery efficiencies, 25-g samples of shucked oysters were homogenized and then inoculated with overnight cultures of Vv-WT or Vv-GFP to obtain an inoculum level of approximately 10⁵ to 10⁶ CFU/g; this was followed by serial dilution and plating on TSAN₂, TSAN₂K, mCPC, and mCPCK. Growth rate and plating efficiency studies were performed by inoculating Vv-WT and Vv-GFP into sterile TSBN₂ and plating samples for recovery on nonselective media (TSAN₂ and TSAN₂K, respectively) and selective media (mCPC and mCPCK, respectively). The exponential growth rate (EGR) was calculated as previously described (13), using data for 4 h to 10 h (inclusive) and for 4 h to 12 h (inclusive) for nonselective and selective media, respectively.

A series of experiments were done to compare the behavior of Vv-WT and the behavior of Vv-GFP under sublethal stress conditions. Overnight cultures of Vv-WT and Vv-GFP were inoculated into fresh TSBN₂ at a concentration of around 10⁶ CFU/ml. The cultures were then exposed to starvation, low-temperature, reduced-pH, and high-temperature conditions, and survival over time was monitored by direct plating on TSAN₂ and TSAN₂K both with and without sodium pyruvate. The specific treatments are described below.

(i) Refrigerated storage and starvation.
For starvation studies, overnight cultures of Vv-WT and Vv-GFP were transferred to prechilled sterile artificial seawater which was stored at 5°C, and samples were taken daily for up to 25 days. Refrigerated storage was evaluated in two ways. In the first analysis, TSBN₂ was prechilled to 5°C before addition of an overnight culture; after this, samples were stored at 5°C for 10 days and plated daily for recovery. For low-temperature adaptation studies, Vv-WT and Vv-GFP cells were stored at 15°C for 4 h (5) before they were inoculated into prechilled (5°C) TSBN₂ and stored for 15 days with daily sampling.

(ii) Frozen storage.
Two different types of freezing conditions were used, one to evaluate the survival of V. vulnificus during long-term, low-temperature (–80°C) storage and the other to evaluate survival after repetitive freeze-thaw cycling. For long-term, low-temperature storage, aliquots were removed every 5 days for 3 months. The aliquots were thawed at room temperature (approximately 5 min) and plated. To evaluate freeze-thaw survival, aliquots were stored at –20°C. One freeze-thaw cycle was defined as freezing at –20°C for 24 h, followed by thawing at 23°C for 30 min (22). Multiple freeze-thaw cycles with a single sample were performed daily for 5 days, and plating for recovery was performed after each freeze-thaw cycle.

(iii) Acid exposure.
In initial studies, the acid tolerance of Vv-GFP and Vv-WT was evaluated by acidifying TSBN₂ to obtain pH values of 5.0, 4.0, and 3.5 using 1 N HCl. The acidified media were inoculated and kept at 23°C on a bench top, and samples were removed and plated immediately (time zero control) and after 1, 3, 5, and 10 h for pH 5.0; after 10, 20, 30, 40, 50, and 60 min for pH 4.0; and after 5, 10, 15, 20, 25, and 30 min for pH 3.5. In a second set of studies, acetic and citric acids (Sigma Chemical Co.) were evaluated. A stock solution of acetic acid (5 M) was used to adjust the pH of TSBN₂ to 5.0 and 4.5, and the pH of TSBN₂ was adjusted to 5.0, 4.5, and 4.0 using a 1 M stock solution of citric acid. After inoculation of Vv-WT and Vv-GFP, the cultures were kept at 23°C on a bench top, and samples were removed and plated immediately after inoculation and after 1, 2, 3, 4, and 5 h for acetic acid-containing cultures at pH 5.0 and after 15, 30, 45, 60, 75, and 90 min for acetic acid-containing cultures at pH 4.5. The times used for citric acid-containing cultures were as follows: after 2, 4, 6, 8, and 10 h for pH 5.0; after 1, 2, 3, 4, and 5 h for pH 4.5; and after 15, 30, 45, 60, 75, 90, 105, and 120 min for pH 4.0.

(iv) Thermal inactivation.
For thermal inactivation experiments, the capillary tube method described by Foegeding and Leasor (15) was used. Briefly, 50 µl (approximately 1.0 x 10⁶ CFU) was added to capillary tubes, and the tubes were heat sealed and submerged in a water bath (45°C). At designated times, the tubes were removed and placed in an ice slurry. Specifically, capillary tubes were examined prior to heat treatment and at 10-min intervals when they were heated at 45°C. Immediately after cooling, the tubes were sanitized by dipping them in sodium hypochlorite (500 ppm, pH 6.5) and crushed, and the resulting preparations were serially diluted and plated.

Use of the biological indicator in oyster matrix studies.
Vv-GFP was used to evaluate the behavior of V. vulnificus when it was suspended in oyster matrix and subsequently exposed to organic acids and various refrigeration regimens. For the former experiments, whole shucked oysters were used; for the latter experiments, shellstock were used. In all cases, oysters (Crassostrea virginica) harvested from the United States Gulf Coast were obtained from local commercial sources and inoculated with an overnight culture of Vv-GFP at a level of approximately 10⁶ CFU/oyster. After exposure to the various conditions, samples were processed for enumeration of Vv-GFP by plating them on TSAN₂K with and without sodium pyruvate.

(i) Behavior of Vv-GFP in shellstock oysters stored under refrigeration conditions.
Shellstock oysters were washed with cold water to remove debris, and then holes were drilled in the shells, 50 µl of an overnight culture of Vv-GFP was injected, and the holes were sealed with epoxy resin, consistent with the method of Kaysner et al. (19). Three shellstock oysters for each sampling time were placed in an open Whirl-pak bag (to facilitate respiration) and stored in accordance with four treatments: (i) oysters were immediately placed on ice until the internal temperature was 5°C and then placed in refrigerated storage at 5°C (ice cooling); (ii) oysters were immediately placed in a refrigerator at 5°C (refrigeration); (iii) the temperature was allowed to drop from the ambient temperature (25°C) to 5°C slowly (over 8 h) (slow cooling); and (iv) oysters were held at 25°C for 8 h and then placed at 5°C, followed by extended refrigerated storage at 5°C (conservative harvest conditions). For each replicate of each treatment, thermocouples (TMQSS-032U-6; OMEGA Engineering Inc., Stamford, CT) were placed in the meat of six uninoculated oysters to monitor the internal temperature during the cooling phase. For all treatments, samples were taken on day 0 both immediately after inoculation and when the internal temperature reached 5°C; daily for days 1 to 8; and then every other day through day 22.

(ii) Survival of Vv-GFP in shucked oysters upon exposure to organic acids.
Shellstock oysters were washed, shucked, allowed to equilibrate to room temperature (23°C), and inoculated by injecting 50 µl of an overnight culture of Vv-GFP into the soft body tissues. In Whirl-pak bags, two shucked oysters per time were immersed (the flesh was completely covered) in 10 ml of fresh TSBN₂ acidified to the target pH (pH 5.0 and 4.0 for acetic acid and pH 5.0 and 3.5 for citric acid) using stock solutions of 5 M acetic acid and 1 M citric acid. During the experiments, oysters were kept at 23°C on a bench top so that any reduction in the Vv-GFP level could be attributed to the acidic conditions alone. Samples were plated for recovery on TSAN₂K with and without sodium pyruvate immediately following exposure (time zero); after 4, 8, 12, 16, 20, and 24 h for pH 5.0 (both acetic and citric acids); and after 2, 4, 6, and 8 h for pH 4.0 (acetic acid) or pH 3.5 (citric acid).

Statistical analysis.
In all cases, three replications of each treatment were done for each strain on all recovery media. The D value was defined as the time (in days or minutes) necessary to obtain a 1-log₁₀ reduction in the population size, and D values were determined using regression analysis (PROC REG; SAS Statistical Analysis Software, version 8.0; SAS Institute, Cary, NC). Statistical comparisons of D values were performed using analysis of variance (PROC MIXED), and the least-squares method was used to determine statistically significant differences (P < 0.05) (SAS).

Top Abstract Introduction Materials and Methods Results Discussion References

 
There was no difference between Vv-WT and Vv-GFP in terms of physiological and fermentation characteristics, including the Gram reaction; appearance on gelatin agar, gelatin agar supplemented with 2% NaCl, and triple sugar iron agar supplemented with 2% NaCl; motility; fermentation of cellobiose; and -nitrophenyl-ß-D-galactosidase activity (data not shown). Vv-WT colonies appeared to be more translucent and slightly smaller on TSAN₂ than Vv-GFP colonies, and Vv-GFP colonies clearly fluoresced upon exposure to UV light. On mCPCK, Vv-GFP did not fluoresce under UV light, but this could have been due to the natural color of mCPC (dark green); however, on mCPCK, Vv-GFP produced small, flat, yellow colonies with halos, whereas Vv-WT colonies had the same morphology but lacked the halos (data not shown).

To evaluate whether Vv-GFP could be recovered consistently from kanamycin-supplemented media, 12-h cultures of both Vv-GFP and Vv-WT were plated on mCPC and TSAN₂ both with and without kanamycin. The concentrations of the two organisms were similar (approximately 10⁷ CFU/ml) on TSAN₂ without kanamycin; Vv-WT was not recovered at all on medium supplemented with kanamycin, even though high levels (10⁷ CFU/ml) were obtained for Vv-GFP using TSAN₂K. Likewise, on selective medium (mCPC) without kanamycin both Vv-WT and Vv-GFP were detected, while kanamycin effectively suppressed the growth of Vv-WT (data not shown). When Vv-WT and Vv-GFP were inoculated into oyster homogenates and recovered using media with and without kanamycin, the supplemented media effectively reduced the level of the background microflora by 1 to 2 log₁₀ and suppressed the growth of Vv-WT without affecting the recovery of Vv-GFP (data not shown). Furthermore, Vv-GFP could be clearly discriminated from the background microflora, including Vv-WT, by visualization using UV illumination for TSAN₂ and incandescent (indoor) lighting for mCPC (data not shown). The nonselective media TSAN₂ and TSAN₂K were used in subsequent experiments to facilitate the recovery of sublethally injured cells of Vv-WT and Vv-GFP, respectively.

There was no statistically significant difference between the growth rates of the two strains when they were grown on TSBN₂ and plated on nonselective medium (Fig. 1A); the EGRs were 1.88 ± 0.11 h and 1.40 ± 0.52 h for Vv-GFP and Vv-WT, respectively, and both strains a reached maximum population density of approximately 8 log₁₀ CFU/ml within 12 h. When the strains were plated on selective medium (mCPC), the EGRs of Vv-GFP and Vv-WT were statistically significantly different (1.85 ± 0.12 and 2.33 ± 0.36 h, respectively) (Fig. 1B). As observed with nonselective media, both strains reached a maximum population density of approximately 8 log₁₀ CFU/ml within 12 h on mCPC. When selective and nonselective media were compared for both strains, there were no significant differences in recovery. Interestingly, on nonselective media, the survival of Vv-WT after 12 h declined more rapidly than the survival of Vv-GFP declined, but exploration of the implications of this observation was beyond the scope of this work. Vv-GFP did not lose its selective antibiotic resistance marker or its ability to be cultured on selective medium after numerous (>10) transfers to fresh media (data not shown). For the most part, the two strains behaved similarly when they were enumerated on nonselective or selective medium, demonstrating that either medium could be used for recovery.

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FIG. 1. Growth rates in TSBN2 at 37°C as evaluated by recovery on nonselective media (TSAN2 and TSAN2K) (A) and selective media (mCPC and mCPCK) (B) for Vv-WT and Vv-GFP, respectively. The EGR was calculated using data for 4 h to 10 h (inclusive) and for 4 to 12 h (inclusive) for nonselective and selective media, respectively. EGR values followed by different superscript letters are statistically significantly different (P < 0.05).

Validation experiments to compare the survival of the two strains were done on TSAN₂ both with and without sodium pyruvate. When the responses of the two strains to temperature were compared (Table 1), statistically significant differences between survival of Vv-WT and survival of Vv-GFP were observed at 5°C (D values, 5.64 ± 2.95 and 2.15 ± 0.18 days, respectively), under cold adaptation conditions (D values, 7.44 ± 1.24 and 2.84 ± 0.28 days, respectively), and after starvation (D values, 2.83 ± 0.04 and 7.52 ± 0.86 days, respectively) when Vv-WT and Vv-GFP were recovered using TSAN₂ and TSAN₂K, respectively. Interestingly, on sodium pyruvate-supplemented media, the statistically significant differences between Vv-WT and Vv-GFP for storage at 5°C (D values, 6.54 ± 0.55 and 4.91 ± 0.13 days, respectively) and cold adaptation (D values, 6.07 ± 0.48 and 9.74 ± 1.44 days, respectively) disappeared, although there was a significant difference after starvation. Under frozen-storage conditions (–80°C) or under freeze-thaw conditions (–20°C), as well as after exposure to 45°C (thermal inactivation), there were not statistically significant differences between Vv-WT and Vv-GFP using TSAN₂ and TSAN₂K either with or without sodium pyruvate. When we compared the recovery data for each strain obtained with different media, addition of pyruvate improved the recovery of the wild-type strain only under –20°C freeze-thaw conditions, at pH 4.0 and 3.5 when the medium was acidified with HCl, and at pH 4.0 when the medium was acidified with citrate, while addition of pyruvate had a more profound effect on the recovery of Vv-GFP, for which statistically significant differences between supplemented and nonsupplemented media were observed in virtually all cold and starvation stress studies.

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TABLE 1. Comparison of D values for Vv-WT and Vv-GFP after exposure to various sublethal stresses as evaluated on TSAN2 and TSAN2K, respectively, with and without sodium pyruvate

The inactivation data for Vv-WT and Vv-GFP, as evaluated using TSAN₂ and TSAN₂K, respectively, were statistically similar for all acid treatments except the pH 5.0 sodium acetate treatment (D values, 2.09 ± 0.13 and 1.41 ± 0.16 h, respectively) (Table 1). Similar results were obtained using pyruvate-supplemented media, for which the survival data for Vv-WT and Vv-GFP were similar except for the pH 4.0 sodium citrate acidification treatment (D values, 38.68 ± 5.66 and <30.0 min, respectively). In general, the recovery of cells exposed to low pH was improved when pyruvate-supplemented media were used, an effect that was more pronounced for the GFP-expressing strain treated with inorganic acid.

The survival of Vv-GFP in shellstock oysters upon exposure to a low temperature was evaluated using TSAN₂K both with and without sodium pyruvate. When Vv-GFP was subjected to four different cooling treatments (iced, refrigerated, slowly cooled, and conservative harvest conditions) and plated on TSAN₂K without sodium pyruvate, there were no statistically significant differences in the D values when we compared iced, refrigerated, and slowly cooled oysters (D values, 4.95 ± 1.71, 4.51 ± 1.08, and 4.76 ± 0.51 days, respectively); however, the D values for the oysters treated under conservative harvest conditions were significantly different (8.40 ± 1.40 days) than the D values for the other temperature treatments (Fig. 2A). When we used TSAN₂K with pyruvate, a similar trend was seen (Fig. 2B). Regardless of the presence of sodium pyruvate or treatment, the V. vulnificus counts decreased 2 log₁₀ after 11 to 12 days of refrigerated storage. Steeper declines in population sizes after 12 days of refrigerated storage were observed for both the iced and refrigerated products than for the slowly cooled product and the product held under conservative harvest conditions (data not shown). By the end of the study (22 days), the counts of Vv-GFP in iced and refrigerated oysters had reached the limit of detection (10² CFU/oyster), but the slowly cooled oysters still contained approximately 10³ CFU/oyster. Furthermore, the oysters stored under conservative harvest conditions contained >10⁴ CFU V. vulnificus/oyster by the end of the 22-day study.

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FIG. 2. Survival of Vv-GFP in shellstock oysters at various low temperatures as evaluated by plating on TSAN2K without sodium pyruvate (A) and TSAN2K with sodium pyruvate (B). The treatments included the following: the temperature was decreased from 25°C to 5°C over 8 h and then the oysters were kept at 5°C (slow cooling) (•); oysters were placed directly into a refrigerator at 5°C (); oysters were immediately iced and then refrigerated at 5°C (ice cooling) (); and oysters were held at 25°C for 8 h and then kept at 5°C (conservative harvest conditions) (). D values followed by different superscript letters are statistically significantly different (P < 0.05).

When Vv-GFP was inoculated directly into shucked oysters which were then exposed to a surface acid treatment and held at room temperature, the V. vulnificus populations remained stable, irrespective of the acid type (citrate or acetate), pH, or the presence of pyruvate in the media (data not shown). When a similar experiment was done at refrigeration temperatures, the Vv-GFP levels were stable for 24 h when the oysters were stored in acetic or citric acid at pH 5.0 and for 8 h when the oysters were stored in acetic acid at pH 4.0 or in citric acid at pH 3.5. As in the experiments described above, there was no difference between the recovery on pyruvate-supplemented media and the recovery on nonsupplemented media (data not shown).

Top Abstract Introduction Materials and Methods Results Discussion References

 
In our study, a V. vulnificus strain was engineered to express the green fluorescent protein and kanamycin resistance. This strain was then characterized as a potential biological indicator for evaluating the survival of the organism upon exposure to sublethal stresses encountered in oyster processing. In previous studies, researchers encountered difficulties when they attempted to isolate and identify V. vulnificus in shellfish because of the high background levels of the indigenous microflora (2). The use of the kanamycin-resistant and GFP-expressing strain of V. vulnificus allowed easy discrimination of the organism from the natural oyster microflora when seeding studies were performed.

In order to justify the use of a surrogate or indicator organism to evaluate the behavior of a pathogen, the surrogate or indicator must be comparable to the target organism in key biochemical, physiological, growth, survival, and inactivation characteristics. Interestingly, many previous reports provided limited evaluations of strain comparability. In some cases, no comparisons to parental strains were made (17, 25, 28); in other cases, only a few comparisons were made (16). The importance of comparisons was illustrated by Foegeding and Stanley (14), who found that plasmid-encoded antibiotic resistance resulted in lower growth rates of transformed Listeria monocytogenes than of the wild-type strain, presumably due to issues associated with the maintenance of the plasmid. Although Sun and Oliver (28) used a kanamycin-resistant strain to evaluate the effects of Tabasco or horseradish-based cocktail sauces on the survival of V. vulnificus in oyster meat, their antibiotic-resistant strain was not compared to the wild-type parent. In perhaps the most comprehensive comparison study, Dombroski et al. (13) found that their spontaneous nalidixic acid-resistant V. vulnificus strain was comparable to the wild-type strain in physiological and biochemical characteristics but differed in freeze tolerance, with the resistant V. vulnificus strain providing a more conservative estimate of total inactivation.

In our study, the physiological and biochemical characteristics of Vv-GFP and Vv-WT were frequently comparable. Overall, the most dramatic differences between the two strains were seen under starvation, cold adaptation, and low-temperature storage (5°C) conditions, although for the low-temperature storage conditions the differences disappeared when the Vv-GFP strain was recovered using pyruvate-supplemented TSAN₂K. Under frozen-storage conditions (–80°C) or under freeze-thaw conditions (–20°C), as well as upon exposure to 45°C (thermal inactivation), statistically significant differences between Vv-WT and Vv-GFP were not apparent, regardless of medium supplementation. Although in a few instances there were statistically significant differences in acid survival, these differences were generally minimal and disappeared when the recovery medium was supplemented with sodium pyruvate. In the few cases where there were statistically significant differences between strains Vv-WT and Vv-GFP on media both with and without pyruvate (e.g., cold adaptation and starvation), the use of sodium pyruvate resulted in a more conservative estimate of survival when Vv-GFP was compared to the wild-type parent. Based on this comprehensive analysis, we are comfortable with using Vv-GFP to evaluate the survival of V. vulnificus when it is exposed to a variety of processing-related stresses and believe that the indicator strain is equivalent to, or better than, Vv-WT as long as the recovery medium is supplemented with sodium pyruvate.

By and large, addition of pyruvate made a difference when cells were exposed to acidic conditions, starvation, or long-term storage at refrigeration temperatures. It has been suggested that the VBNC state and perhaps injury may be manifested by increased sensitivity to hydrogen peroxide. For instance, Bogosian et al. (8) recovered V. vulnificus on pyruvate-supplemented media even after the cells had entered the VBNC state. Our hypothesis was that addition of pyruvate would indeed promote the recovery of sublethally injured cells, and like Bogosian et al. (8), we observed better recovery, sometimes statistically significant and sometimes not, of both Vv-WT and Vv-GFP when we used sodium pyruvate-supplemented media. Interestingly, the degree to which addition of pyruvate enhanced the recovery of sublethally injured cells appeared to be greater for Vv-GFP than for Vv-WT. This may have been due to the exact location of the chromosomal insertion, although this explanation is speculative. The fact that addition of pyruvate was effective at some times and unnecessary at other times suggests that hydrogen peroxide sensitivity may be a consequence of exposure to some but not all sublethal stresses. Additional studies at the molecular level are necessary to better understand the relationship between catalase activity and bacterial cell culturability (21).

Refrigeration is an excellent method for controlling the multiplication of V. vulnificus (10, 11), and there has been great interest in the value of immediate or almost immediate postharvest chilling of oysters to control or even reduce the levels of V. vulnificus (26). Consistent with the findings of other workers (12), our temperature study using shellstock oysters artificially contaminated with Vv-GFP showed that there were differences between the survival of the organism in shellstock oysters held under slow-cooling conditions and the survival of the organism in shellstock oysters which were cooled more quickly. During the extended time that it took to cool the product, the organism may have undergone cold adaptation. This response has been reported by other workers (5, 9) and may have been responsible for the improved survival of Vv-GFP under long-term refrigerated storage conditions for the samples which were chilled more slowly. Additional studies are necessary to understand the importance of cold adaptation in the survival of V. vulnificus in shellfish.

Although V. vulnificus was sensitive to acid in a broth model, there was little reduction in the Vv-GFP levels in oyster meat after surface exposure to organic acids, regardless of the pH or acid type, over an 8- to 24-h period. We concluded that in this case the oyster meat provided a protective environment that prevented inactivation of the organism. Similarly, Sun and Oliver (28) observed no decrease in V. vulnificus levels in oyster meat when half-shell oysters were placed in Tabasco or horseradish-catsup-based sauces for 10 min. Indeed, there have been several studies with other foods that have suggested that the food matrix can protect bacteria (30), which appears to be the case with oysters.

In summary, we constructed a GFP-expressing strain of V. vulnificus for use as a biological indicator to measure the survival of this organism after exposure to representative processing controls that might be encountered during oyster harvesting, processing, and/or storage. Overall, the Vv-GFP strain showed similar or better resistance than Vv-WT, especially when sodium pyruvate-supplemented media were used. The organism could also be readily differentiated from the natural oyster microflora, meaning that evaluations could be done without using time-consuming most-probable-number enrichment protocols. Low-temperature studies confirmed that cooling methods alone cannot be relied upon to eliminate V. vulnificus from shellstock oysters. Consistent with the findings of other investigators, organic acids (acetic and citric acids) had no effect on the survival of V. vulnificus in oyster meat, which was probably protective, and hence, treatment of shucked oysters with lemon juice or other acidic condiments cannot be relied upon as an effective control. This study demonstrated the importance of validation of biological indicators. Ease of detection and comparability to the wild-type parent make Vv-GFP a good candidate for use in studying the behavior of V. vulnificus upon exposure to sublethal stressors that might be encountered during postharvest handling of molluscan shellfish.

 
This study was funded by North Carolina State University Sea Grant Program project R/SST-27 and by the U.S. Department of Agriculture Cooperative State Research, Education and Extension Service (CSREES) National Research Initiative Competitive Grants Program (Epidemiological Approaches to Food Safety grant 2004-35212-14882).

The use of trade names does not imply endorsement by the North Carolina Agricultural Research Service or criticism of similar products not mentioned.

 
* Corresponding author. Mailing address: Food Science Department, North Carolina State University, Raleigh, NC 27695-7624. Phone: (919) 513-2074. Fax: (919) 513-0014. E-mail: leeann_jaykus{at}ncsu.edu.

Paper number FSR 06-25 in the Journal Series of the Department of Food Science, North Carolina State University, Raleigh.

Published ahead of print on 15 September 2006.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, November 2006, p. 7205-7211, Vol. 72, No. 11
0099-2240/06/$08.00+0     doi:10.1128/AEM.01091-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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