Influence of Water Temperature and Salinity on Vibrio vulnificus in Northern Gulf and Atlantic Coast Oysters (Crassostrea virginica)

This study investigated the temperature and salinity parameters associated with waters and oysters linked to food-borne Vibrio vulnificus infections. V. vulnificus was enumerated in oysters collected at three northern Gulf Coast sites and two Atlantic Coast sites from July 1994 through September 1995. Two of these sites, Black Bay, La., and Apalachicola Bay, Fla., are the source of the majority of the oysters implicated in V. vulnificus cases. Oysters in all Gulf Coast sites exhibited a similar seasonal distribution of V. vulnificus: a consistently large number (median concentration, 2,300 organisms [most probable number] per g of oyster meat) from May through October followed by a gradual reduction during November and December to ≤10 per g, where it remained from January through mid-March, and a sharp increase in late March and April to summer levels. V. vulnificus was undetectable (<3 per g) in oysters from the North and South Carolina sites for most of the year. An exception occurred when a late-summer flood caused a drop in salinity in the North Carolina estuary, apparently causing V. vulnificus numbers to increase briefly to Gulf Coast levels. At Gulf Coast sites, V. vulnificus numbers increased with water temperatures up to 26°C and were constant at higher temperatures. High V. vulnificus levels (>10³ per g) were typically found in oysters from intermediate salinities (5 to 25 ppt). Smaller V. vulnificus numbers (<10² per g) were found at salinities above 28 ppt, typical of Atlantic Coast sites. On 11 occasions oysters were sampled at times and locations near the source of oysters implicated in 13 V. vulnificus cases; the V. vulnificus levels and environmental parameters associated with these samples were consistent with those of other study samples collected from the Gulf Coast from April through November. These findings suggest that the hazard of V. vulnificus infection is not limited to brief periods of unusual abundance of V. vulnificus in Gulf Coast oysters or to environmental conditions that are unusual to Gulf Coast estuaries.     

Effects of Physicochemical Factors and Bacterial Colony Morphotype on Association of Vibrio vulnificus with Hemocytes of Crassostrea virginica

Vibrio vulnificus is a naturally occurring marine bacterium that causes invasive disease of immunocompromised humans following the consumption of raw oysters. It is a component of the natural microbiota of Gulf Coast estuaries and has been found to inhabit tissues of oysters, Crassostrea virginica (Gmelin 1791). The interaction of V. vulnificus with oyster host defenses has not been reported in detail. We examined the interaction of V. vulnificus with phagocytic oyster hemocytes as a function of time, temperature, bacterial concentration, pretreatment with hemolymph, and V. vulnificus translucent and opaque colony morphotypes. Within these experimental parameters, the results showed that the association of V. vulnificus with hemocytes increased with time, temperature, and initial V. vulnificus/hemocyte ratio. Pretreatment of V. vulnificus with serum or an increased serum concentration did not enhance V. vulnificus-hemocyte associations, a result suggesting the absence of opsonic activity. More than 50% of hemocytes bound the translucent, avirulent morphotype, whereas 10 to 20% were associated with the opaque, virulent form, a result indicating that the degree of encapsulation was related to resistance to phagocytosis, as previously described for mammalian phagocytes. Understanding these cellular interactions may, in part, explain the persistence of V. vulnificus in oyster tissues and the ecology of V. vulnificus in estuarine environments.     

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

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.     

Abundance of Vibrio cholerae,V. vulnificus,and V. parahaemolyticus in Oysters (Crassostrea virginica) and Clams (Mercenaria mercenaria) from Long Island Sound

Vibriosis is a leading cause of seafood-associated morbidity and mortality in the United States. Typically associated with consumption of raw or undercooked oysters, vibriosis associated with clam consumption is increasingly being reported. However, little is known about the prevalence of Vibrio spp. in clams. The objective of this study was to compare the levels of Vibrio cholerae, Vibrio vulnificus, and Vibrio parahaemolyticus in oysters and clams harvested concurrently from Long Island Sound (LIS). Most probable number (MPN)–real-time PCR methods were used for enumeration of total V. cholerae, V. vulnificus, V. parahaemolyticus, and pathogenic (tdh⁺ and/or trh⁺) V. parahaemolyticus. V. cholerae was detected in 8.8% and 3.3% of oyster (n = 68) and clam (n = 30) samples, with levels up to 1.48 and 0.48 log MPN/g in oysters and clams, respectively. V. vulnificus was detected in 97% and 90% of oyster and clam samples, with median levels of 0.97 and −0.08 log MPN/g, respectively. V. parahaemolyticus was detected in all samples, with median levels of 1.88 and 1.07 log MPN/g for oysters and clams, respectively. The differences between V. vulnificus and total and pathogenic V. parahaemolyticus levels in the two shellfish species were statistically significant (P < 0.001). These data indicate that V. vulnificus and total and pathogenic V. parahaemolyticus are more prevalent and are present at higher levels in oysters than in hard clams. Additionally, the data suggest differences in vibrio populations between shellfish harvested from different growing area waters within LIS. These results can be used to evaluate and refine illness mitigation strategies employed by risk managers and shellfish control authorities.     

Real-Time PCR Detection of Vibrio vulnificus in Oysters: Comparison of Oligonucleotide Primers and Probes Targeting vvhA

We compared three sets of oligonucleotide primers and two probes designed for Vibrio vulnificus hemolysin A gene (vvhA) for TaqMan-based real-time PCR method enabling specific detection of Vibrio vulnificus in oysters. Two of three sets of primers with a probe were specific for the detection of all 81 V. vulnificus isolates by TaqMan PCR. The 25 nonvibrio and 12 other vibrio isolates tested were negative. However, the third set of primers, F-vvh1059 and R-vvh1159, with the P-vvh1109 probe, although positive for all V. vulnificus isolates, also exhibited positive cycle threshold (C_T) values for other Vibrio spp. Optimization of the TaqMan PCR assay using F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and the P-vvh874 probe detected 1 pg of purified DNA and 10³ V. vulnificus CFU/ml in pure cultures. The enriched oyster tissue homogenate did not exhibit detectable inhibition to the TaqMan PCR amplification of vvhA. Detection of 3 × 10³ CFU V. vulnificus, resulting from a 5-h enrichment of an initial inoculum of 1 CFU/g of oyster tissue homogenate, was achieved with F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and P-vvh875 probe. The application of the TaqMan PCR using these primers and probe, exhibited detection of V. vulnificus on 5-h-enriched natural oysters harvested from the Gulf of Mexico. Selection of appropriate primers and a probe on vvhA for TaqMan-PCR-based detection of V. vulnificus in post-harvest-treated oysters would help avoid false-positive results, thus ensuring a steady supply of safe oysters to consumers and reducing V. vulnificus-related illnesses and deaths.     

Real-Time PCR Analysis of Vibrio vulnificus from Oysters

Vibrio vulnificus is an opportunistic human pathogen commonly found in estuarine environments. Infections are associated with raw oyster consumption and can produce rapidly fatal septicemia in susceptible individuals. Standard enumeration of this organism in shellfish or seawater is laborious and inaccurate; therefore, more efficient assays are needed. An oligonucleotide probe derived from the cytolysin gene, vvhA, was previously used for colony hybridizations to enumerate V. vulnificus. However, this method requires overnight growth, and vibrios may lack culturability under certain conditions. In the present study, we targeted the same locus for development of a TaqMan real-time PCR assay. Probe specificity was confirmed by amplification of 28 V. vulnificus templates and by the lack of a PCR product with 22 non-V. vulnificus strains. Detection of V. vulnificus in pure cultures was observed over a 6-log-unit linear range of concentration (10² to 10⁸ CFU ml⁻¹), with a lower limit of 72 fg of genomic DNA μl of PCR mixture⁻¹ or the equivalent of six cells. Similar sensitivity was observed in DNA extracted from mixtures of V. vulnificus and V. parahaemolyticus cells. Real-time PCR enumeration of artificially inoculated oyster homogenates correlated well with colony hybridization counts (r² = 0.97). Numbers of indigenous V. vulnificus cells in oysters by real-time PCR showed no significant differences from numbers from plate counts with probe (t test; P = 0.43). Viable but nonculturable cells were also enumerated by real-time PCR and confirmed by the BacLight viability assay. These data indicate that real-time PCR can provide sensitive species-specific detection and enumeration of V. vulnificus in seafood.     

A Persistent, Productive, and Seasonally Dynamic Vibriophage Population within Pacific Oysters (Crassostrea gigas)

In an effort to understand the relationship between Vibrio and vibriophage populations, abundances of Vibrio spp. and viruses infecting Vibrio parahaemolyticus (VpVs) were monitored for a year in Pacific oysters and water collected from Ladysmith Harbor, British Columbia, Canada. Bacterial abundances were highly seasonal, whereas high titers of VpVs (0.5 × 10⁴ to 11 × 10⁴ viruses cm⁻³) occurred year round in oysters, even when V. parahaemolyticus was undetectable (<3 cells cm⁻³). Viruses were not detected (<10 ml⁻¹) in the water column. Host-range studies demonstrated that 13 VpV strains could infect 62% of the V. parahaemolyticus strains from oysters (91 pairings) and 74% of the strains from sediments (65 pairings) but only 30% of the water-column strains (91 pairings). Ten viruses also infected more than one species among V. alginolyticus, V. natriegens, and V. vulnificus. As winter approached and potential hosts disappeared, the proportion of host strains that the viruses could infect decreased by ~50% and, in the middle of winter, only 14% of the VpV community could be plated on summer host strains. Estimates of virus-induced mortality on V. parahaemolyticus indicated that other host species were required to sustain viral production during winter when the putative host species was undetectable. The present study shows that oysters are likely one of the major sources of viruses infecting V. parahaemolyticus in oysters and in the water column. Furthermore, seasonal shifts in patterns of host range provide strong evidence that the composition of the virus community changes during winter.     

Real-Time PCR Assays for Quantification and Differentiation of Vibrio vulnificus Strains in Oysters and Water

Vibrio vulnificus is an autochthonous estuarine bacterium and a pathogen that is frequently transmitted via raw shellfish. Septicemia can occur within 24 h; however, isolation and confirmation from water and oysters require days. Real-time PCR assays were developed to detect and differentiate two 16S rRNA variants, types A and B, which were previously associated with environmental sources and clinical fatalities, respectively. Both assays could detect 10² to 10³ V. vulnificus total cells in seeded estuarine water and in oyster homogenates. PCR assays on 11 reference V. vulnificus strains and 22 nontarget species gave expected results (type A or B for V. vulnificus and negative for nontarget species). The relationship between cell number and cycle threshold for the assays was linear (R² = >0.93). The type A/B ratio of Florida clinical isolates was compared to that of isolates from oysters harvested in Florida waters. This ratio was 19:17 in clinical isolates and 5:8 (n = 26) in oysters harvested from restricted sites with poor water quality but was 10:1 (n = 22) in oysters from permitted sites with good water quality. A substantial percentage of isolates from oysters (19.4%) were type AB (both primer sets amplified), but no isolates from overlying waters were type AB. The real-time PCR assays were sensitive, specific, and quantitative in water samples and could also differentiate the strains in oysters without requiring isolation of V. vulnificus and may therefore be useful for rapid detection of the pathogen in shellfish and water, as well as further investigation of its population dynamics.     

Detection and Enumeration of Vibrio vulnificus in Oysters from Two Estuaries along the Southwest Coast of India, Using Molecular Methods

This study was conducted to understand the seasonal distribution of Vibrio vulnificus in oysters from two estuaries and the effect of environmental factors on the abundance of V. vulnificus in tropical waters. V. vulnificus was detected in 56.6% of the samples tested by colony hybridization with an alkaline phosphatase-labeled oligonucleotide probe (VV-AP), and the counts ranged from <10/g during the summer months to 10³/g in the monsoon season at both sites. The density of V. vulnificus appeared to be controlled more by salinity than by temperature. A nested PCR used in this study detected V. vulnificus in 85% of the samples following 18 h of enrichment in alkaline peptone water.     

Rapid Detection of Vibrio vulnificus in Shellfish and Gulf of Mexico Water by Real-Time PCR

In this paper we describe optimization of SYBR Green I-based real-time PCR parameters and testing of a large number of microbial species with vvh-specific oligonucleotide primers to establish a rapid, specific, and sensitive method for detection of Vibrio vulnificus in oyster tissue homogenate and Gulf of Mexico water (gulf water). Selected oligonucleotide primers for the vvh gene were tested for PCR amplification of a 205-bp DNA fragment with a melting temperature of approximately 87°C for 84 clinical and environmental strains of V. vulnificus. No amplification was observed with other vibrios or nonvibrio strains with these primers. The minimum level of detection by the real-time PCR method was 1 pg of purified genomic DNA or 10² V. vulnificus cells in 1 g of unenriched oyster tissue homogenate or 10 ml of gulf water. It was possible to improve the level of detection to one V. vulnificus cell in samples that were enriched for 5 h. The standard curves prepared from the real-time PCR cycle threshold values revealed that there was a strong correlation between the number of cells in unenriched samples and the number of cells in enriched samples. Detection of a single cell of V. vulnificus in 1 g of enriched oyster tissue homogenate is in compliance with the recent Interstate Shellfish Sanitation Conference guidelines. The entire detection method, including sample processing, enrichment, and real-time PCR amplification, was completed within 8 h, making it a rapid single-day assay. Rapid and sensitive detection of V. vulnificus would ensure a steady supply of postharvest treated oysters to consumers, which should help decrease the number of illnesses or outbreaks caused by this pathogen.     

Sediment and Vegetation as Reservoirs of Vibrio vulnificus in the Tampa Bay Estuary and Gulf of Mexico

The opportunistic pathogen Vibrio vulnificus occurs naturally in estuarine habitats and is readily cultured from water and oysters under warm conditions but infrequently at ambient conditions of <15°C. The presence of V. vulnificus in other habitats, such as sediments and aquatic vegetation, has been explored much less frequently. This study investigated the ecology of V. vulnificus in water by culture and quantitative PCR (qPCR) and in sediment, oysters, and aquatic vegetation by culture. V. vulnificus samples were taken from five sites around Tampa Bay, FL. Levels determined by qPCR and culture were significantly correlated (P = 0.0006; r = 0.352); however, V. vulnificus was detected significantly more frequently by qPCR (85% of all samples) compared to culture (43%). Culturable V. vulnificus bacteria were recovered most frequently from oyster samples (70%), followed by vegetation and sediment (∼50%) and water (43%). Water temperature, which ranged from 18.5 to 33.4°C, was positively correlated with V. vulnificus concentrations in all matrices but sediments. Salinity, which ranged from 1 to 35 ppt, was negatively correlated with V. vulnificus levels in water and sediments but not in other matrices. Significant interaction effects between matrix and temperature support the hypothesis that temperature affects V. vulnificus concentrations differently in different matrices and that sediment habitats may serve as seasonal reservoirs for V. vulnificus. V. vulnificus levels in vegetation have not been previously measured and reveal an additional habitat for this autochthonous estuarine bacterium.     

Environmental Investigations of Vibrio parahaemolyticus in Oysters after Outbreaks in Washington, Texas, and New York (1997 and 1998)

Total Vibrio parahaemolyticus densities and the occurrence of pathogenic strains in shellfish were determined following outbreaks in Washington, Texas, and New York. Recently developed nonradioactive DNA probes were utilized for the first time for direct enumeration of V. parahaemolyticus in environmental shellfish samples. V. parahaemolyticus was prevalent in oysters from Puget Sound, Wash.; Galveston Bay, Tex.; and Long Island Sound, N.Y., in the weeks following shellfish-associated outbreaks linked to these areas. However, only two samples (one each from Washington and Texas) were found to harbor total V. parahaemolyticus densities exceeding the level of concern of 10,000 g⁻¹. Pathogenic strains, defined as those hybridizing with tdh and/or trh probes, were detected in a few samples, mostly Puget Sound oysters, and at low densities (usually <10 g⁻¹). Intensive sampling in Galveston Bay demonstrated relatively constant water temperature (27.8 to 31.7°C) and V. parahaemolyticus levels (100 to 1,000 g⁻¹) during the summer. Salinity varied from 14.9 to 29.3 ppt. A slight but significant (P < 0.05) negative correlation (−0.25) was observed between V. parahaemolyticus density and salinity. Based on our data, findings of more than 10,000 g⁻¹ total V. parahaemolyticus or >10 g⁻¹ tdh- and/or trh-positive V. parahaemolyticus in environmental oysters should be considered extraordinary.     

Mortalities of Eastern and Pacific Oyster Larvae Caused by the Pathogens Vibrio coralliilyticus and Vibrio tubiashii

Vibrio tubiashii is reported to be a bacterial pathogen of larval Eastern oysters (Crassostrea virginica) and Pacific oysters (Crassostrea gigas) and has been associated with major hatchery crashes, causing shortages in seed oysters for commercial shellfish producers. Another bacterium, Vibrio coralliilyticus, a well-known coral pathogen, has recently been shown to elicit mortality in fish and shellfish. Several strains of V. coralliilyticus, such as ATCC 19105 and Pacific isolates RE22 and RE98, were misidentified as V. tubiashii until recently. We compared the mortalities caused by two V. tubiashii and four V. coralliilyticus strains in Eastern and Pacific oyster larvae. The 50% lethal dose (LD₅₀) of V. coralliilyticus in Eastern oysters (defined here as the dose required to kill 50% of the population in 6 days) ranged from 1.1 × 10⁴ to 3.0 × 10⁴ CFU/ml seawater; strains RE98 and RE22 were the most virulent. This study shows that V. coralliilyticus causes mortality in Eastern oyster larvae. Results for Pacific oysters were similar, with LD₅₀s between 1.2 × 10⁴ and 4.0 × 10⁴ CFU/ml. Vibrio tubiashii ATCC 19106 and ATCC 19109 were highly infectious toward Eastern oyster larvae but were essentially nonpathogenic toward healthy Pacific oyster larvae at dosages of ≥1.1 × 10⁴ CFU/ml. These data, coupled with the fact that several isolates originally thought to be V. tubiashii are actually V. coralliilyticus, suggest that V. coralliilyticus has been a more significant pathogen for larval bivalve shellfish than V. tubiashii, particularly on the U.S. West Coast, contributing to substantial hatchery-associated morbidity and mortality in recent years.     

Antimicrobial Susceptibilities of Vibrio parahaemolyticus and Vibrio vulnificus Isolates from Louisiana Gulf and Retail Raw Oysters

The antimicrobial susceptibilities of 168 Vibrio parahaemolyticus and 151 Vibrio vulnificus isolates recovered from 82 Louisiana Gulf and retail oysters in 2005 and 2006 were determined. Overall, the two vibrios remained susceptible to the majority of antimicrobials tested; reduced susceptibility was detected only in V. parahaemolyticus for ampicillin (81%; MIC ≥ 16 μg/ml). Additionally, V. parahaemolyticus displayed significantly higher MICs for cefotaxime, ciprofloxacin, and tetracycline than V. vulnificus.     

Preference Alters Consumptive Effects of Predators: Top-Down Effects of a Native Crab on a System of Native and Introduced Prey

Top-down effects of predators in systems depend on the rate at which predators consume prey, and on predator preferences among available prey. In invaded communities, these parameters might be difficult to predict because ecological relationships are typically evolutionarily novel. We examined feeding rates and preferences of a crab native to the Pacific Northwest, Cancer productus, among four prey items: two invasive species of oyster drill (the marine whelks Urosalpinx cinerea and Ocenebra inornata) and two species of oyster (Crassostrea gigas and Ostrea lurida) that are also consumed by U. cinerea and O. inornata. This system is also characterized by intraguild predation because crabs are predators of drills and compete with them for prey (oysters). When only the oysters were offered, crabs did not express a preference and consumed approximately 9 juvenile oysters crab⁻¹ day⁻¹. We then tested whether crabs preferred adult drills of either U. cinerea or O. inornata, or juvenile oysters (C. gigas). While crabs consumed drills and oysters at approximately the same rate when only one type of prey was offered, they expressed a strong preference for juvenile oysters over drills when they were allowed to choose among the three prey items. This preference for oysters might negate the positive indirect effects that crabs have on oysters by crabs consuming drills (trophic cascade) because crabs have a large negative direct effect on oysters when crabs, oysters, and drills co-occur.     

Distribution ofVibrio cholerae in two Florida estuaries

The distribution ofVibrio cholerae was examined in 2 Florida estuaries, Apalachicola and Tampa Bay.Vibrio cholerae serotype non-01 was the most abundant serotype, being isolated from 45% of the oyster samples, 30% of the sediments, 50% of the waters, and 75% of the blue crabs.Vibrio cholerae serotype 01 was isolated from only one oyster sample. Strong linear correlations betweenV. cholerae and temperature, salinity, or the other physical/chemical parameters measured,Escherichia coli, or fecal coliforms were not observed, but a range of temperatures and salinities appeared relevant to the distribution of the organism. The organism was present in the highest concentrations when salinities were 10‰–25‰ and temperatures were 20?C–35?C.In vitro growth curves of 95V. cholerae environmental isolates further supported that 10‰–25‰ was an ideal salinity range for the organisms. The results suggest thatV. cholerae is a widely distributed organism in the nutrient-rich warm waters of the Gulf Coast estuaries.     

Novel Podoviridae Family Bacteriophage Infecting Weissella cibaria Isolated from Kimchi

The first complete genome sequence of a phage infecting Weissella cibaria (Weissella kimchii) is presented. The bacteriophage ϕYS61 was isolated from kimchi, a Korean fermented vegetable dish. Bacteriophages are recognized as a serious problem in industrial fermentations; however, ϕYS61 differed from many virulent phages associated with food fermentations since it was difficult to propagate and was very susceptible to resistance development. Sequence analysis revealed that ϕYS61 resembles Podoviridae of the subfamily Picovirinae. Within the subfamily Picovirinae, the ϕ29-like phages have been extensively studied, and their terminal protein-primed DNA replication is well characterized. Our data strongly suggest that ϕYS61 also replicates by a protein-primed mechanism. Weissella phage ϕYS61 is, however, markedly different from members of the Picovirinae with respect to genome size and morphology. Picovirinae are characterized by small (approximately 20-kb) genomes which contrasts with the 33,594-bp genome of ϕYS61. Based on electron microscopy analysis, ϕYS61 was classified as a member of the Podoviridae of morphotype C2, similar to the ϕ29-like phages, but its capsid dimensions are significantly larger than those reported for these phages. The novelty of ϕYS61 was also emphasized by the low number of open reading frames (ORFs) showing significant similarity to database sequences. We propose that the bacteriophage ϕYS61 should represent a new subfamily within the family Podoviridae.     

Populations of Anaerobic Phototrophic Bacteria in a Spartina alterniflora Salt Marsh

Habitat-simulating media were used with the Hungate anaerobic roll tube technique to enumerate culturable anaerobic photosynthetic bacteria in sediment, tidal waters, and Spartina alterniflora plant samples collected from the salt marsh at Sapelo Island, Ga. No phototrophs were detected in samples of creekside (low marsh) sediment or in tidal waters in creekside regions. In the high marsh region, 90% of anaerobic phototrophic bacteria occurred in the top 5 mm of sediment and none were detected below 6 mm. There was a seasonal variation, with maximal populations occurring in summer and fall (mean, 4.4 × 10⁵ phototrophs g of dry sediment⁻¹) and minimal numbers occurring in winter (mean, 3.9 × 10³ phototrophs g of dry sediment⁻¹). During winter and late spring, phototrophs had a patchy distribution over the high marsh sediment surface. In contrast, during late summer they had a random uniform distribution. Tidal water collected over high marsh sediment contained an average of 8.7 × 10² phototrophs ml⁻¹, with no significant seasonal variation. Anaerobic phototrophic bacteria were also cultured from the lower stem tissue of S. alterniflora growing in both the high (4.3 × 10⁴ phototrophs g of dry tissue⁻¹) and creekside (4.9 × 10⁴ phototrophs g of dry tissue⁻¹) marsh regions. Chromatium buderi, Chromatium vinosum, Thiospirillum sanguineum, Rhodospirillum molischianum, and Chlorobium phaeobacteroides were the predominant anaerobic phototrophic species cultured from high marsh sediment. The two Chromatium species were dominant.     

Application of Chitosan Microparticles for Reduction of Vibrio Species in Seawater and Live Oysters (Crassostrea virginica)

Human Vibrio infections associated with consumption of raw shellfish greatly impact the seafood industry. Vibrio cholerae-related disease is occasionally attributed to seafood, but V. vulnificus and V. parahaemolyticus are the primary targets of postharvest processing (PHP) efforts in the United States, as they pose the greatest threat to the industry. Most successful PHP treatments for Vibrio reduction also kill the molluscs and are not suitable for the lucrative half-shell market, while nonlethal practices are generally less effective. Therefore, novel intervention strategies for Vibrio reduction are needed for live oyster products. Chitosan is a bioactive derivative of chitin that is generally recognized as safe as a food additive by the FDA, and chitosan microparticles (CMs) were investigated in the present study as a potential PHP treatment for live oyster applications. Treatment of broth cultures with 0.5% (wt/vol) CMs resulted in growth cessation of V. cholerae, V. vulnificus, and V. parahaemolyticus, reducing culturable levels to nondetectable amounts after 3 h in three independent experiments. Furthermore, a similar treatment in artificial seawater at 4, 25, and 37°C reduced V. vulnificus levels by ca. 7 log CFU/ml after 24 h of exposure, but 48 h of exposure and elevated temperature were required to achieve similar results for V. parahaemolyticus and V. cholerae. Live oysters that either were artificially inoculated or contained natural populations of V. vulnificus and V. parahaemolyticus showed significant and consistent reductions following CM treatment (5%) compared to the amounts in the untreated controls. Thus, the results strongly support the promising potential for the application of CMs as a PHP treatment to reduce Vibrio spp. in intact live oysters.