Yeast and bacterial diversity, dynamics and fermentative kinetics during small-scale tequila spontaneous fermentation.

The study of microbial communities associated with spontaneous fermentation of agave juice for tequila production is required to develop starter cultures that improve both yield and quality of the final product. Quantification by HPLC of primary metabolites produced during the fermentations was determined. A polyphasic approach using plate count, isolation and identification of microorganisms, denaturing gradient gel electrophoresis and next generation sequencing was carried out to describe the diversity and dynamics of yeasts and bacteria during small-scale spontaneous fermentations of agave juice from two-year samplings. High heterogeneity in microbial populations and fermentation parameters were observed, with bacteria showing higher diversity than yeast. The core microorganisms identified were Saccharomyces cerevisiae and Lactobacillus fermentum. Practices in tequila production changed during the two-year period, which affected microbial community structure and the time to end fermentation. Bacterial growth and concomitant lactic acid production were associated with low ethanol production, thus bacteria could be defined as contaminants in tequila fermentation and efforts to control them should be implemented.

Influence of agitation, inoculum density, pH, and strain on the growth parameters of Escherichia coli O157:H7--relevance to risk assessment.

Foods may differ in at least two key variables from broth culture systems typically used to measure growth kinetics of enteropathogens: initial population density of the pathogen and agitation of the culture. The present study used nine Escherichia coli O157:H7 strains isolated from beef and associated with human illness. Initial kinetic experiments with one E. coli O157:H7 strain in brain-heart infusion (BHI) broth at pH 5.5 were performed in a 2 x 2 x 3 factorial design, testing the effects of a low (ca. 1-10 colony-forming units [CFU]/ml) or high (ca. 1000 CFU/ml) initial population density, culture agitation or no culture agitation, and incubation temperatures of 10, 19, and 37 degrees C. Kinetic data were modeled using simple linear regression and the Baranyi model. Both model forms provided good statistical fit to the data (adjusted r(2)>0.95). Significant effects of agitation and initial population density were identified at 10 degrees C but not at 19 or 37 degrees C. Similar growth patterns were observed for two additional strains tested under the same experimental design. The lag, slope, and maximum population density (MPD) parameters were significantly different by treatment. Further tests were conducted in a 96-well microtiter plate system to determine the effect of initial population density and low pH (4.6-5.5) on the growth of E. coli O157:H7 strains in BHI at 10, 19, and 37 degrees C. Strain variability was more apparent at the boundary conditions of growth of low pH and low temperature. This study demonstrates the need for growth models that are specific to food products and environments for plausible extrapolation to risk assessment models.

Pathogenesis of infection by clinical and environmental strains of Vibrio vulnificus in iron-dextran-treated mice.

Vibrio vulnificus is an opportunistic pathogen that contaminates oysters harvested from the Gulf of Mexico. In humans with compromising conditions, especially excess levels of iron in plasma and tissues, consumption of contaminated seafood or exposure of wounds to contaminated water can lead to systemic infection and disfiguring skin infection with extremely high mortality. V. vulnificus-associated diseases are noted for the rapid replication of the bacteria in host tissues, with extensive tissue damage. In this study we examined the virulence attributes of three virulent clinical strains and three attenuated oyster or seawater isolates in mouse models of systemic disease. All six V. vulnificus strains caused identical skin lesions in subcutaneously (s.c.) inoculated iron dextran-treated mice in terms of numbers of recovered CFU and histopathology; however, the inocula required for identical frequency and magnitude of infection were at least 350-fold higher for the environmental strains. At lethal doses, all strains caused s. c. skin lesions with extensive edema, necrosis of proximate host cells, vasodilation, and as many as 10(8) CFU/g, especially in perivascular regions. These data suggest that the differences between these clinical and environmental strains may be related to growth in the host or susceptibility to host defenses. In non-iron dextran-treated mice, strains required 10(5)-fold-higher inocula to cause an identical disease process as with iron dextran treatment. These results demonstrate that s.c. inoculation of iron dextran-treated mice is a useful model for studying systemic disease caused by V. vulnificus.

Uptake and retention of Vibrio cholerae O1 in the Eastern oyster, Crassostrea virginica.

Vibrio cholerae 01, the causative agent of cholera, is known to persist in estuarine environments as endogenous microflora. The recent introduction of V. cholerae 01 into estuaries of the North and South American continents has stimulated the need to determine the effect of controlled purification on reducing this pathogen in edible molluscan shellfish. Experiments defined parameters for the uptake and retention of V. cholerae 01 in tissues of Crassostrea virginica, and these parameters were compared with those for Escherichia coli and Salmonella tallahassee, bacteria which are usually eliminated from moderately contaminated shellfish within 48 h. Oysters accumulated greater concentrations of V. cholerae 01 than E. coli and S. tallahassee. When V. cholerae 01 was exposed to controlled purification at 15, 19 and 25 degrees C over 48 h, it persisted in oysters at markedly higher levels than E. coli and S. tallahassee. The concentration of a V. cholerae 01-specific agglutinin did not positively correlate with the uptake or retention of V. cholerae 01. These data show that state and federally approved controlled purification techniques are not effective at reducing V. cholerae 01 in oysters.

Enzyme immunoassay for identification of Vibrio vulnificus in seawater, sediment, and oysters.

Historically, methods used to identify Vibrio vulnificus in environmental samples have been inadequate because isolation and identification procedures are time-consuming and fail to separate V. vulnificus from other bacterial species. We describe an enzyme immunoassay (EIA) and culture techniques which identified V. vulnificus in seawater, sediment, and oysters. The EIA used monoclonal antibody FRBT37 to a species-specific epitope of V. vulnificus. No cross-reactions were observed among 72 non-V. vulnificus strains comprising 34 species and 15 genera. In field trials, the EIA identified correctly 99.7% of 348 biochemically confirmed V. vulnificus isolates. The epitope corresponding to FRBT37 was found in cells lysed by Triton X-100, deionized H2O, and ultrasonication but was not found in culture supernatants, indicating that its location was intracellular. In addition, electron micrographs of V. vulnificus labeled with FRBT37-biotin-avidin-gold showed that epitope FRBT37 reacted with fragments of lysed cells but not whole cells. FRBT37 was expressed when V. vulnificus was cultured in different growth media. The minimum level of detection of the EIA was approximately 2,000 V. vulnificus cells per EIA well. Epitope FRBT37 was labile at 70 degrees C for 30 min. Immunoblot and EIA plate formats reduced assay time and facilitated handling large numbers of test samples.

Occurrence and characteristics of agglutination of Vibrio cholerae by serum from the eastern oyster, Crassostrea virginica.

Cell-free hemolymph (serum) of the eastern oyster, Crassostrea virginica, agglutinated Vibrio cholerae, including all O1 serovars and biovars. Seventy-nine other strains of bacteria, including 14 genera and 26 species, were not agglutinated. The A, B, and C factors of O1 antigen were not involved in agglutination. Bacterial agglutinating (BA) activity was demonstrated for oysters inhabiting different environments of the U.S. Atlantic and Gulf coasts. Oyster serum BA titers showed high individual variation. The serum component(s) involved in BA was inhibited by 80 degrees C heat, pronase, EDTA, mucin, and fetuin treatments. N-Acetylneuraminic acid (10 mg/ml) weakly inhibited BA activity. Ligands of V. cholerae were sensitive to neuraminidase and resistant to 80 degrees C and pronase. High salinities (24 and 30%) enhanced BA. Cross-adsorption tests with V. cholerae and human O+ erythrocytes indicated that BA and hemagglutinating activities may involve different serum components. These results imply that the ecology of V. cholerae in C. virginica is influenced by agglutinating activity of oyster serum.

A tissue culture assay for tetrodotoxin, saxitoxin and related toxins.

In the presence of ouabain, veratridine enhances sodium influx in the mouse neuroblastoma cell line Neuro-2A (ATCC, CCL131), causing cellular swelling and subsequent death. Tetrodotoxin (puffer fish toxin) or saxitoxin (paralytic shellfish poison), both of which block the sodium channel of excitable membranes, antagonize this effect, enabling cell growth to continue. This phenomenon was used as the basis of a new assay for these toxins. It is also possible to estimate the quantity of TTX from the relationship between TTX concentration and percentage of living cells. This new method is simple, inexpensive, and sensitive, and may replace the conventional mouse bioassay.

Vibrio vulnificus resists phagocytosis in the absence of serum opsonins.

Invasive disease caused by Vibrio vulnificus may result partially from resistance to phagocytic host defense mechanisms. The present studies show that V. vulnificus resists phagocytosis by murine peritoneal macrophages in the absence of serum opsonins and extracellular bacterial products, apparently through the anti-phagocytic properties of the bacterial surface.