Michael A. Watson

Genogroup I and II Noroviruses Detected in Stool Samples by Real-Time Reverse Transcription-PCR Using Highly Degenerate Universal Primers

ABSTRACT Genogroup I noroviruses from five genetic clusters and genogroup II noroviruses from eight genetic clusters were detected in stool extracts using degenerate primers and single-tube, real-time reverse transcription-PCR (RT-PCR) with SYBR Green detection. Two degenerate primer sets, designated MON 431-433 and MON 432-434, were designed from consensus sequences from the major clusters of norovirus based on the RNA-dependent RNA polymerase region of the norovirus genome. Viruses were extracted from stool samples within 20 min using a viral RNA extraction kit. Real-time RT-PCR for noroviruses generated semiquantitative results by means of the cycle threshold data and dilution endpoint standard curves. Presumptive product verification was achieved by evaluation of first-derivative melt graphs. Multiple clusters of noroviruses were identified simultaneously in a multiplex fashion by virtue of slight differences in melting temperature. The detection of 13 different genetic clusters suggests that the MON primers may serve as universal primers for most, if not all, of the noroviruses in a multiplex assay. Our technique provides a framework for broad application of real-time RT-PCR in clinical, environmental, and food testing laboratories for a wide range of noroviruses.

Shewanella and Photobacterium spp. in Oysters and Seawater from the Delaware Bay

ABSTRACT Shewanella algae, S. putrefaciens, and Photobacterium damselae subsp. damselae are indigenous marine bacteria and human pathogens causing cellulitis, necrotizing fasciitis, abscesses, septicemia, and death. Infections are rare and are most often associated with the immunocompromised host. A study was performed on the microbiological flora of oysters and seawater from commercial oyster harvesting sites in the Delaware Bay, New Jersey. From 276 water and shellfish samples tested, 1,421 bacterial isolates were picked for biochemical identification and 170 (12.0%) of the isolates were presumptively identified as S. putrefaciens, 26 (1.8%) were presumptively identified as P. damselae subsp. damselae, and 665 (46.8%) could not be identified using the API 20E identification database. Sequencing of the 16S rRNA genes of 22 S. putrefaciens-like isolates identified them as S. abalonesis, S. algae, S. baltica, S. hafniensis, S. marisflavi, S. putrefaciens, Listonella anguillarum, and P. damselae. Beta-hemolysis was produced by some S. algae and P. damselae isolates, while isolates of S. baltica and L. anguillarum, species perceived as nonpathogenic, also exhibited β-hemolysis and growth at 37°C. To our knowledge, this is the first time these beta-hemolytic strains were reported from shellfish or seawater from the Delaware Bay. Pathogenic Shewanella and Photobacterium species could pose a health threat through the ingestion of contaminated seafood, by cuts or abrasions acquired in the marine environment, or by swimming and other recreational activities.

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

ABSTRACT 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.

Complete Genome Sequence for the Shellfish Pathogen Vibrio coralliilyticus RE98 Isolated from a Shellfish Hatchery

ABSTRACT Vibrio coralliilyticus is a pathogen of corals and larval shellfish. Publications on strain RE98 list it as a Vibrio tubiashii; however, whole genome sequencing confirms RE98 as V. coralliilyticus containing a total of 6,037,824 bp consisting of two chromosomes (3,420,228 and 1,917,482 bp) and two megaplasmids (380,714 and 319,400 bp).

Development of a Simple and Rapid Fluorogenic Procedure for Identification of Vibrionaceae Family Members

ABSTRACT We describe a simple colony overlay procedure for peptidases (COPP) for the rapid fluorogenic detection and quantification of Vibrionaceae from seawater, shellfish, sewage, and clinical samples. The assay detects phosphoglucose isomerase with a lysyl aminopeptidase activity that is produced by Vibrionaceae family members. Overnight cultures are overlaid for 10 min with membranes containing a synthetic substrate, and the membranes are examined for fluorescent foci under UV illumination. Fluorescent foci were produced by all the Vibrionaceae tested, including Vibrio spp., Aeromonas spp., and Plesiomonas spp. Fluorescence was not produced by non-Vibrionaceae pathogens. Vibrio cholerae strains O1, O139, O22, and O155 were strongly positive. Seawater and oysters were assayed, and 87 of 93 (93.5%) of the positive isolates were identified biochemically as Vibrionaceae, principally Vibrio vulnificus, Vibrio parahaemolyticus, Aeromonas hydrophila, Photobacterium damselae, and Shewanella putrefaciens. None of 50 nonfluorescent isolates were Vibrionaceae. No Vibrionaceae were detected in soil, and only A. hydrophila was detected in sewage. The COPP technique may be particularly valuable in environmental and food-testing laboratories and for monitoring water quality in the aquaculture industry.

Flavor of Fresh Blueberry Juice and the Comparison to Amount of Sugars, Acids, Anthocyanidins, and Physicochemical Measurements

Six cultivars of southern highbush (SHB) and rabbiteye (RE) blueberry samples were harvested on 2 different dates. Each treatment combination was pressed 2 times for repeated measures. Fresh juice was characterized for 18 flavor/taste/feeling factor attributes by a descriptive flavor panel. Each sample was measured for sugars, acids, anthocyanidins, Folin-Ciocalteu, soluble solids (BRIX), titratable acidity (TA), and antioxidant capacity (ORACFL ). Flavors were correlated with the composition and physicochemical data. Blueberry flavor correlated with 3 parameters, and negatively correlated with 2. Strawberry correlated with oxalic acid and negatively correlated with sucrose and quinic acid. Sweet aroma correlated with oxalic and citric acid, but negatively correlated with sucrose, quinic, and total acids. Sweet taste correlated with 11 parameters, including the anthocyanidins; and negatively correlated with 3 parameters. Neither bitter nor astringent correlated with any of the antioxidant parameters, but both correlated with total acids. Sour correlated with total acids and TA, while negatively correlating with pH and BRIX:TA. Throat burn correlated with total acids and TA. Principal component analysis negatively related blueberry, sweet aroma, and sweet to sour, bitter, astringent, tongue tingle, and tongue numbness. The information in this component was related to pH, TA, and BRIX:TA ratio. Another principal component related the nonblueberry fruit flavors to BRIX. This PC, also divided the SHB berries from the RE. This work shows that the impact of juice composition on flavor is very complicated and that estimating flavor with physicochemical parameters is complicated by the composition of the juice.

Seasonal Levels of the Vibrio Predator Bacteriovorax in Atlantic, Pacific, and Gulf Coast Seawater

Bacteriovorax were quantified in US Atlantic, Gulf, and Pacific seawater to determine baseline levels of these predatory bacteria and possible seasonal fluctuations in levels. Surface seawater was analyzed monthly for 1 year from Kailua-Kona, Hawaii; the Gulf Coast of Alabama; and four sites along the Delaware Bay. Screening for Bacteriovorax was performed on lawns of V. parahaemolyticus host cells. Direct testing of 7.5 mL portions of seawater from the Atlantic, Pacific, and Gulf coasts gave mean annual counts ≤12.2 PFU. Spikes in counts were observed at 3 out of 4 sites along the Delaware Bay 1 week after Hurricane Sandy. A comparison of summer versus winter counts showed significantly more Bacteriovorax (P ≤ 0.0001) in the Delaware Bay during the summer and significantly more (P ≤ 0.0001) in the Gulf during the winter, but no significant seasonal differences (P > 0.05) for Hawaiian seawater. Bacteriovorax counts only correlated with seawater salinity and temperature at one Delaware site (r = 0.79 and r = 0.65, resp.). There was a relatively strong negative correlation between temperature and Bacteriovorax levels (r = −0.585) for Gulf seawater. Selected isolates were sequenced and identified by phylogenetic analysis as Bacteriovorax clusters IX, X, XI, and XII.

Mechanisms for Pseudoalteromonas piscicida-induced Killing of Vibrios and Other Bacterial Pathogens

ABSTRACT Pseudoalteromonas piscicida is a Gram-negative gammaproteobacterium found in the marine environment. Three strains of pigmented P. piscicida were isolated from seawater and partially characterized by inhibition studies, electron microscopy, and analysis for proteolytic enzymes. Growth inhibition and death occurred around colonies of P. piscicida on lawns of the naturally occurring marine pathogens Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio cholerae, Photobacterium damselae, and Shewanella algae. Inhibition also occurred on lawns of Staphylococcus aureus but not on Escherichia coli O157:H7 or Salmonella enterica serovar Typhimurium. Inhibition was not pH associated, but it may have been related to the secretion of a cysteine protease with strong activity, as detected with a synthetic fluorogenic substrate. This diffusible enzyme was secreted from all three P. piscicida strains. Direct overlay of the Pseudoalteromonas colonies with synthetic fluorogenic substrates demonstrated the activity of two aminopeptidase Bs, a trypsin-like serine protease, and enzymes reactive against substrates for cathepsin G-like and caspase 1-like proteases. In seawater cultures, scanning electron microscopy revealed numerous vesicles tethered to the outer surface of P. piscicida and a novel mechanism of direct transfer of these vesicles to V. parahaemolyticus. Vesicles digested holes in V. parahaemolyticus cells, while the P. piscicida congregated around the vibrios in a predatory fashion. This transfer of vesicles and vesicle-associated digestion of holes were not observed in other bacteria, suggesting that vesicle binding may be mediated by host-specific receptors. In conclusion, we show two mechanisms by which P. piscicida inhibits and/or kills competing bacteria, involving the secretion of antimicrobial substances and the direct transfer of digestive vesicles to competing bacteria. IMPORTANCE Pseudoalteromonas species are widespread in nature and reduce competing microflora by the production of antimicrobial compounds. We isolated three strains of P. piscicida and characterized secreted and cell-associated proteolytic enzymes, which may have antimicrobial properties. We identified a second method by which P. piscicida kills V. parahaemolyticus. It involves the direct transfer of apparently lytic vesicles from the surface of the Pseudoalteromonas strains to the surface of Vibrio cells, with subsequent digestion of holes in the Vibrio cell walls. Enzymes associated with these vesicles are likely responsible for the digestion of holes in the cell walls. Pseudoalteromonas piscicida has potential applications in aquaculture and food safety, in control of the formation of biofilms in the environment, and in food processing. These findings may facilitate the probiotic use of P. piscicida to inactivate pathogens and may lead to the isolation of enzymes and other antimicrobial compounds of pharmacological value.

Complete Genome Sequence of the Larval Shellfish Pathogen Vibrio tubiashii Type Strain ATCC 19109

ABSTRACT Vibrio tubiashii is a larval shellfish pathogen. Here, we report the first closed genome sequence for this species (ATCC type strain 19109), which consists of two chromosomes (3,294,490 and 1,766,582 bp), two megaplasmids (251,408 and 122,808 bp), and two plasmids (57,076 and 47,973 bp).

Influence of Cooking Formulation on Flavor and Hydrophilic Oxygen Radical Absorption Capacity Values of Whole Grain Colored Rice

ABSTRACT Whole grain rice is rich in healthful polyphenolic compounds that can impart undesirable flavors. Rice is typically prepared with water and sometimes salt, oil, or both, which can influence flavor. This research examines the influences of cooking whole grain rice with salt, oil, or both on flavor and antioxidant capacity. Nine commercial rice samples (three brown, three red, and three black or purple) were cooked with water only, added salt (0.5%), oil (1.575% v/w), or salt with oil (same concentrations). Flavor was measured with descriptive flavor analysis, and antioxidant capacity was measured by the hydrophilic oxygen radical absorption capacity (H-ORAC) method. The addition of salt and salt with oil significantly reduced bitter, waterlike/metallic, and astringent flavors, whereas corn/popcorn/buttery flavor was increased. Cooking formulation significantly affected more flavor attributes in black/purple and brown bran samples than in the red bran samples. Salt, oil, or both can be added to ric...