Effects of intrinsic characteristics of Salmonella enterica strains isolated from foods and humans, and their interaction with food matrices during simulated gastric conditions.

The stomach's acidic pH is a crucial barrier against foodborne pathogens such as Salmonella enterica. This study investigated the survival of S. enterica under simulated oral and gastric conditions (SGC; pH 2 for 120 min) as a function of intrinsic pathogen characteristics and food matrix. Fifty-seven S. enterica strains isolated from food and human infections (previously characterized by serotype, virulotype, multi-drug resistance, isolation source, and isolation season) were subjected to SGC using water as a vehicle. Population reduction among the 57 isolates ranged from 2.7 to 4.7 log CFU, revealing that human isolates were inactivated less than food isolates (p = 0.0008). Among food strains, strains isolated during the cold season (food sampled from December to February) displayed the highest reduction (p = 0.00002). Six representatives of the 57 S. enterica strains were selected according to their virulotype and antimicrobial profile. They were further used to evaluate their survival under SGC in four food matrices (water, mango, tomato, and chicken), measuring S. enterica at 30 min intervals. The strains in chicken showed the lowest reduction and inactivation rate (1.42 ± 0.35 log CFU; 0.03 ± 0.005 min-1), followed by tomato (3.75 ± 0.57 log CFU; 0.15 ± 0.02 min-1), water (4.23 ± 0.27 log CFU; 0.17 ± 0.02 min-1), and mango (4.49 ± 0.39 log CFU; 0.17 ± 0.03 min-1). These data suggest that not all S. enterica strains have the same ability to survive under SGC, influencing the probability of arriving into the small intestine.

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.

Predictive model for the growth of spoilage bacteria on modified atmosphere packaged Atlantic salmon produced in Australia.

Most existing models for the spoilage of modified atmosphere packed Atlantic salmon are based on the growth of the spoilage organism Photobacterium phosphoreum. However, there is evidence that this organism is not the specific spoilage organism on salmon produced and packaged in Australia. We developed a predictive model for the growth of bacteria in Australian-produced Atlantic salmon stored under modified atmosphere conditions (30-98% carbon dioxide in nitrogen) at refrigeration temperatures (0-10 °C). As expected, both higher levels of carbon dioxide and lower temperatures decreased the observed growth rates of the total population. A Belehrádek-type model for growth rate fitted the data best with an acceptably low root mean square error. At low temperatures (∼0 °C) the growth rates in this study were similar to those predicted by other models but at higher temperatures (∼10 °C) the growth rates were significantly lower in the current study.

Pyrosequencing-based characterization of gastrointestinal bacteria of Atlantic salmon (Salmo salar L.) within a commercial mariculture system.

AIMS: The relationship of Atlantic salmon gastrointestinal (GI) tract bacteria to environmental factors, in particular water temperature within a commercial mariculture system, was investigated. METHODS AND RESULTS: Salmon GI tract bacterial communities commercially farmed in south-eastern Tasmania were analysed, over a 13-month period across a standard commercial production farm cycle, using 454 16S rRNA-based pyrosequencing. Faecal bacterial communities were highly dynamic but largely similar between randomly selected fish. In postsmolt, the faecal bacteria population was dominated by Gram-positive fermentative bacteria; however, by midsummer, members of the family Vibrionaceae predominated. As fish progressed towards harvest, a range of different bacterial genera became more prominent corresponding to a decline in Vibrionaceae. The sampled fish were fed two different commercial diet series with slightly different protein, lipid and digestible energy level; however, the effect of these differences was minimal. CONCLUSIONS: The overall data demonstrated dynamic hind gut communities in salmon that were related to season and fish growth phases but were less influenced by differences in commercial diets used routinely within the farm system studied. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides understanding of farmed salmon GI bacterial communities and describes the relative impact of diet, environmental and farm factors.

Dynamics of seawater bacterial communities in a shellfish hatchery.

Bacterial disease is a significant issue for larviculture of several species of shellfish, including oysters. One source of bacteria is the seawater used throughout the hatchery. In this study carried out at a commercial oyster hatchery in Tasmania, Australia, the diversity of the bacterial community and its relationship with larval production outcomes were studied over a 2-year period using terminal restriction fragment length polymorphism and tag-encoded pyrosequencing. The bacterial communities were very diverse, dominated by the Alphaproteobacteria, Gammaproteobacteria, Flavobacteria and Cyanobacteria. The communities were highly variable on scales of days, weeks and seasons. The difference between the intake seawater and treated clean seawater used in the hatchery was smaller than the observed temporal differences in the seawater throughout the year. No clear correlation was observed between production outcomes and the overall bacterial community structure. However, one group of Cyanobacterial sequences was more abundant when mass mortality events occurred than when healthy spat were produced although they were always present.

Molecular analysis of the bacterial communities in the live Pacific oyster (Crassostrea gigas) and the influence of postharvest temperature on its structure.

AIMS: To evaluate the effect of postharvest temperature on bacterial communities in live Pacific oysters (Crassostrea gigas) using nonculture-based methods. METHODS AND RESULTS: Live oysters were compared before and after storage at 4, 6, 15, 20 and 30°C using terminal restriction fragment length polymorphism (T-RFLP). Bacterial communities in freshly harvested (control) vs stored oysters were significantly different. Changes in bacterial communities at 4, 15 and 30°C observed by T-RFLP were further investigated by clone library analysis. Members of the Proteobacteria predominated (43·0-57·0% of clones) in control oysters, while storage altered the bacterial profile. At 4°C, Psychrilyobacter spp. (phylum Fusobacteria) predominated (43·8% of clones), while at 15 and 30°C, members of the phylum Bacteroidetes represented 63·0 and 60·2% of clones, respectively. High microbial diversity in oysters was observed, with at least 73 different genera-related clones among all samples. CONCLUSIONS: Changes in the overall bacterial community of Pacific oysters were influenced by storage temperature and would likely not be detected by standard culture-based methods currently used to assess oyster quality. Certain dominant genera, such as Psychrilyobacter, Polynucleobacter and a bacterial group related to Alkaliflexus, should be further studied as possible indicators for postharvest temperature control. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is the first report describing the effect of different storage temperatures on bacterial diversity in postharvest live Pacific oysters using molecular-based methods.

Microbial communities on Australian modified atmosphere packaged Atlantic salmon.

The role of specific spoilage organisms (SSO) in products such as Atlantic salmon has been well documented. However, little is known about what other micro-organisms are present and these organisms may indirectly influence spoilage by their interactions with the SS0. We used a combination of culture-based and DNA-based methods to explore the microbial communities found on Atlantic salmon fillets packed in a modified atmosphere of carbon dioxide and nitrogen. After 15 days the communities were dominated by Shewanella spp. or Carnobacterium spp. and a variety of other genera were present in smaller numbers. Variability in the microbial community composition in packages processed on the same day was also observed. This was mostly due to differences in the presence of minor members of the community including species from genera such as Iodobacter, Serratia, Morganella and Yersinia. The combination of culture-based and culture-independent methods provided greater insight into the development of microbial communities on Atlantic salmon than would have been possible using only one method. This work highlights the potential importance of lactic acid bacteria (LAB) in fresh Atlantic salmon stored under modified atmosphere conditions.

Evaluation of thermal inactivation of Escherichia coli using microelectrode ion flux measurements with osmotic stress.

AIMS: To elucidate the potential use of microelectrode ion flux measurements to evaluate bacterial responses to heat treatment. METHODS AND RESULTS: Escherichia coli K12 was used as a test bacterium to determine whether various heat treatments (55-70°C for 15 min) affected net ion flux across E. coli cell membranes using the MIFE™ system to measure net K(+) fluxes. No difference in K(+) fluxes was observed before and after heat treatments regardless of the magnitude of the treatment. Applying hyperosmotic stress (3% NaCl w/v) during flux measurement led to a net K(+) loss from the heat-treated E.coli cells below 65°C as well as from nonheated cells. In contrast, with E. coli cells treated at and above 65°C, hyperosmotic stress disrupted the pattern of K(+) flux observed at lower temperatures and resulted in large flux noise with random scatter. This phenomenon was particularly apparent above 70°C. Although E. coli cells lost the potential to recover and grow at and above 62°C, K(+) flux disruption was not clearly observed until 68°C was reached. CONCLUSIONS: No changes in net K(+) flux from heat-stressed E. coli cells were observed directly as a result of thermal treatments. However, regardless of the magnitude of heat treatment above 55°C, loss of viability indicated by enrichment culture correlated with disrupted K(+) fluxes when previously heated cells were further challenged by imposing hyperosmotic stress during flux measurement. This two-stage process enabled evaluation of the lethality of heat-treated bacterial cells within 2 h and may be an alternative and more rapid method to confirm the lethality of heat treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to confirm the lethality of thermal treatments and to specify minimal time/temperature combinations by a nonculture-dependent test offers an alternative system to culture-based methods.

Modeling the lag phase and growth rate of Listeria monocytogenes in ground ham containing sodium lactate and sodium diacetate at various storage temperatures.

Refrigerated ready-to-eat (RTE) meats contaminated with Listeria monocytogenes were implicated in several listeriosis outbreaks. Lactate and diacetate have been shown to control L. monocytogenes in RTE meats. The objective of this study was to examine and model the effect of lactate (1.0% to 4.2%) and diacetate (0.05% to 0.2%) in ground ham on the lag phase duration (LPD, h) and growth rate (GR, log CFU/h) of L. monocytogenes at a range of temperatures (0 to 45 degrees C). A 6-strain mixture of L. monocytogenes was inoculated into ground ham containing lactate and diacetate, and stored at various temperatures. The LPD and GR of L. monocytogenes in ham as affected by lactate, diacetate, and storage temperature were analyzed and accurately represented with mathematical equations. Resulting LPD and GR equations for storage temperatures within the range of 0 to 36 degrees C significantly represented the experimental data with a regression coefficient of 0.97 and 0.96, respectively. Significant factors (P < 0.05) that affected the LPD were temperature, lactate, diacetate, and the interactions of all three, whereas only temperature and the interactions between temperature and lactate and diacetate had a significant effect on GR. At suboptimal growth temperatures (< or = 12 degrees C) the increase of lactate and diacetate concentrations, individually or in combination, extended the LPD. The effect of higher concentrations of both additives on reducing the GR was observed only at temperatures that were more suitable for growth of L. monocytogenes, that is, 15 to 35 degrees C. These data may be used to assist in determining concentrations of lactate and diacetate in cooked ham products to control the growth of L. monocytogenes over a wide range of temperatures during manufacturing, distribution, and storage.

Effect of preinoculation growth media and fat levels on thermal inactivation of a serotype 4b strain of Listeria monocytogenes in frankfurter slurries.

Preinoculation growth conditions and fat levels were evaluated for effects on the heat resistance of Listeria monocytogenes strain MFS 102 in formulated frankfurter slurries and on frankfurter surfaces. Comparison of linear inactivation rates (D-values) for cells heated in frankfurter slurry showed that growth conditions were significant (P<0.05) factors affecting subsequent thermal resistance. The average D(60 degrees C)-values for the five preinoculation growth media tested from most resistant to least heat resistant were: tryptic soy broth with 0.6% yeast extract (TSBYE) (2.2 min) and 8.5% fat slurry (2.2 min), followed by 23% fat slurry (1.7 min) and 11% fat slurry (1.7 min), and then TSYBE with quaternary ammonium compounds added (TSBYE+Q) (1 min). The fat level in the frankfurter heating media also had a significant (P<0.05) effect on the thermal death rate of L. monocytogenes. Cells heated in 8.5% fat slurry had a significantly higher (P<0.05) D(60 degrees C)-value (2.2 min) than those heated in 11% fat (1.0 min) and 23% fat slurry (0.9 min). Growth media (TSBYE, 8.5% fat slurry, and TSBYE+Q), and fat level (15% and 20%), however, were not significant factors (P>0.05) affecting thermal inactivation rates on frankfurter surfaces. Heat inactivation rates were consistently higher on frankfurter surfaces compared to similar treatments done in frankfurter slurry. On frankfurter surfaces, a 2.3- to 5.1-log(10) reduction was achieved after 15 min depending on frankfurter surface type. The time necessary to achieve a 3-log(10) reduction using post-processing pasteurization of frankfurters in a hot water-bath at 60 degrees C almost doubled for cells grown in TSBYE and heated in 23% fat frankfurter slurry (19.6 min) versus cells grown and heated in 8.5% fat frankfurter slurry (10.8 min).

A predictive model to describe the effects of temperature, sodium lactate, and sodium diacetate on the inactivation of a serotype 4b strain of Listeria monocytogenes in a frankfurter slurry.

A modified Gompertz equation was used to model the effects of temperature (55, 60, and 65 degrees C), sodium lactate (0, 2.4, and 4.8%), and sodium diacetate (0, 0.125, and 0.25%) on inactivation of Listeria monocytogenes strain MFS 102 (serotype 4b) in frankfurter slurry. The effects of these factors were determined on the shouldering region (parameter A), maximum death rate (parameter B), and tailing region (parameter C) of microbial inactivation curves. Increased temperature or sodium diacetate concentrations increased the death rate, whereas increased sodium lactate concentrations decreased heat resistance. Complex two-way interactive effects were also observed. As both temperature and sodium lactate increased, the death rate decreased; however, as temperature and sodium diacetate increased, the death rate increased. The effect of the interaction between sodium lactate and sodium diacetate on the maximum death rate varied with temperature. Increases in both acidulants at temperatures above 56.7 degrees C decreased the death rate, whereas at temperatures below 56.7 degrees C, increases in both acidulants increased the death rate. To test for significant differences between treatments, D-values were calculated and compared. This comparison revealed that, in general, sodium lactate increased heat resistance and sodium diacetate decreased heat resistance of L. monocytogenes. This information is important for reducing and minimizing contamination during postprocessing thermal treatments.

Geographical variation in antibiotic resistance profiles of Escherichia coli isolated from swine, poultry, beef and dairy cattle farm water retention ponds in Florida.

AIMS: The aim of this study was to assess geographical variation in multiple antibiotic resistance (MAR) profiles of livestock Escherichia coli as well as to evaluate the ability of MAR profiles to differentiate sources of faecal pollution. METHODS AND RESULTS: More than 2000 E. coli isolates were collected from water retention ponds and manure of swine, poultry, beef and dairy farms in south, central and north Florida, and analysed for MAR using nine antibiotics. There were significant differences in antibiotic resistance of E. coli by season and livestock type for more than one antibiotic, but regional differences were significant only for ampicillin. Over the three regions, discriminant analysis using MAR profiles correctly classified 27% of swine, 49% of poultry, 56% of beef and 51% of dairy isolates. CONCLUSIONS: Regional variations in MAR combined with moderate discrimination success suggest that MAR profiles of E. coli may only be marginally successful in identifying sources of faecal pollution. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the existence of regional and seasonal differences in MAR profiles as well as the limited ability of MAR profiles to discriminate among livestock sources.

Quantifying the robustness of a broth-based model for predicting Listeria monocytogenes growth in meat and poultry products.

Given the importance of Listeria monocytogenes as a risk factor in meat and poultry products, there is a need to evaluate the relative robustness of predictive growth models applied to meat products. The U.S. Department of Agriculture-Agricultural Research Service Pathogen Modeling Program is a tool widely used by the food industry to estimate pathogen growth, survival, and inactivation in food. However, the robustness of the Pathogen Modeling Program broth-based L. monocytogenes growth model in meat and poultry application has not, to our knowledge, been specifically evaluated. In the present study, this model was evaluated against independent data in terms of predicted microbial counts and covered a range of conditions inside and outside the original model domain. The robustness index was calculated as the ratio of the standard error of prediction (root mean square error of the model against an independent data set not used to create the model) to the standard error of calibration (root mean square error of the model against the data set used to create the model). Inside the calibration domain of the Pathogen Modeling Program, the best robustness index for application to meat products was 0.37; the worst was 3.96. Outside the domain, the best robustness index was 0.40, and the worst was 1.22. Product type influenced the robustness index values (P < 0.01). In general, the results indicated that broth-based predictive models should be validated against independent data in the domain of interest; otherwise, significant predictive errors can occur.

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.

Salmonella spp. and Escherichia coli biotype I on swine carcasses processed under the hazard analysis and critical control point-based inspection models project.

The present study examined the prevalence of Salmonella spp. and the prevalence and quantity of generic (biotype I) Escherichia coli on carcasses or in pig feces at a pork processing plant operating under the hazard analysis and critical control point-based inspection models project (HIMP) program. The surfaces of carcasses were sponged on 10 separate days over a 30-day period at two processing steps: (i) immediately following exsanguination (100 carcasses), and (ii) after the carcasses were washed, eviscerated, and chilled overnight (122 carcasses). Feces were also collected from 60 of the 100 sponged, postexsanguinated pigs. Salmonella spp. were detected on 73.0% of the 100 postexsanguinated pigs, in 33.3% of the 60 fecal samples, and on 0.7% of the 122 chilled carcasses. E. coli was found on 100.0% of the postexsanguinated pigs and on 30.1% of chilled carcasses tested. The mean concentration of E. coli on carcasses was 1,700 CFU/cm2 immediately after the exsanguination step and 1.1 CFU/cm2 at the chilled carcass stage. Previous studies at this processing plant showed that the pre-HIMP baseline level of Salmonella spp. on the chilled carcasses was 0.8%, indicating that the present HIMP inspection system produced an equivalent level of bacteriological performance.

Comparison of cultivation and PCR-hybridization for detection of Salmonella in porcine fecal and water samples.

A total of 150 fecal and water samples from four swine farms were tested for the presence of Salmonella enterica using different enrichment techniques as follows: (i) 92 fecal samples from nursery and farrowing barns at three swine farms were preenriched overnight in tryptic soy broth (TSB) at 37 degrees C followed by overnight enrichment in Rappaport-Vassiliadis 10 broth (RV10) at 42 degrees C; (ii) 24 water samples from the third farm were preenriched overnight in 3MC broth at 37 degrees C followed by overnight enrichment in RV10 at 42 degrees C; and (iii) 34 fecal samples from a fourth farm, a finishing farm, were enriched overnight in RV10 at 42 degrees C with no additional enrichment. Following each of the enrichment techniques, samples were subcultured onto modified semisolid Rappaport-Vassiliadis (MSRV) agar prior to transfer to Hektoen Enteric agar plates for the recovery of viable Salmonella bacteria. Presumptive Salmonella isolates were biochemically and serologically confirmed. For the PCR detection of Salmonella, a 1-ml portion was removed from each sample after the first overnight enrichment and the DNA was extracted using a Sepharose CL-6B spin column. Amplicons (457 bp) derived from primers to the invA and invE genes were confirmed as Salmonella specific on ethidium bromide-stained agarose gels by Southern hybridization with a 20-mer oligonucleotide probe specific for the Salmonella invA gene. Neither the standard microbiological method nor the molecular method detected all of the 65 samples that tested positive by both methods or either method alone. Salmonella bacteria were detected by both cultivation and PCR-hybridization in 68% (17 of 25) of the positive samples that were preenriched in TSB, in 73% (11 of 15) of the positive samples preenriched in 3MC broth, and in 24% (6 of 25) of the positive samples enriched in RV10. Agreement between Salmonella detection using cultivation with preenrichment and detection by PCR was 76% using the kappa statistic. However, agreement between Salmonella detection using cultivation without preenrichment and detection by PCR was about 6%; the PCR assay detected 80% (20 of 25) of the 25 positive samples, while Salmonella bacteria were recovered from only 44% (11 of 25) by cultivation. Our results indicate that the PCR-hybridization approach is equivalent to or better than cultivation for detecting Salmonella in swine feces or water samples from swine farms when using the medium combinations evaluated in this study.

Elution, detection, and quantification of polio I, bacteriophages, Salmonella montevideo, and Escherichia coli O157:H7 from seeded strawberries and tomatoes.

This study compared the effect of different physical and chemical treatments of strawberries and tomatoes to determine their ability to recover seeded viral and bacterial pathogens from produce surfaces. Solutions of salts, amino acids, complex media, and detergents were compared as eluants. Phosphate-buffered saline (PBS) containing 0.1% Tween 80 eluted the highest number of seeded microorganisms. Elution with this defined solution was then compared under different conditions of physical agitation. Rotary shaking for 20 min at 36 degrees C eluted higher numbers of viruses and bacteria than did low- or high-speed stomaching. Commercially available and laboratory prepared bacteriological differential media were compared for their ability to recover and distinguish eluted Salmonella Montevideo and Escherichia coli O157:H7 strains from seeded produce. The recovery of seeded bacterial pathogens was low when differential media containing selective ingredients were used (MacConkey sorbitol agar, XLD agar, MacConkey agar). Highest recoveries were obtained on a medium consisting of tryptic soy agar supplemented with sodium thiosulfate and ferric ammonium citrate compared with selective media that inhibited up to 50% of the growth of the eluted microorganisms.

Phenotypic and genotypic characterization of human and nonhuman Escherichia coli.

Estuarine waters receive fecal pollution from a variety of sources, including humans and wildlife. Escherichia coli is one of several fecal coliform bacteria that inhabit the intestines of many warm-blooded animals that sometimes contaminate water. Its presence does not specifically implicate human fecal input, therefore it is necessary to differentiate contamination sources to accurately assess health risks. E. coli were isolated from human sources (HS) and nonhuman sources (NHS) in the Apalachicola National Estuarine Research Reserve and analyzed for fatty acid methyl ester (FAME), O-serogroup, and pulsed-field gel electrophoresis (PFGE) profiles. For FAME and PFGE analyses, there was no relationship between profile and isolate source. Human source PFGE profiles were less diverse than NHS isolates, and conversely for FAME. In contrast, O-serogrouping showed less diversity for HS vs. NHS isolates, and the predominant HS O-serogroups differed significantly (P < 0.01) from those of NHS isolates.

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.

Discriminant analysis of ribotype profiles of Escherichia coli for differentiating human and nonhuman sources of fecal pollution.

Estuarine waters receive fecal pollution from a variety of sources, including humans and wildlife. Escherichia coli is a ubiquitous bacterium in the intestines of warm-blooded animals and is used as an indicator of fecal pollution. However, its presence does not specifically differentiate sources of pollution. A total of 238 E. coli isolates from human sources (HS) and nonhuman sources (NHS) were collected from the Apalachicola National Estuarine Research Reserve, from associated sewage treatment plants, and directly from animals and tested for ribotype (RT) profile. HS and NHS isolates showed 41 and 61 RT profiles, respectively. At a similarity index of ca. 50%, HS and NHS isolates demonstrated four clusters, with the majority of HS and NHS isolates located in clusters C and D; isolates obtained directly from human and animal feces also could be grouped within these clusters. Discriminant analysis (DA) of RT profiles showed that 97% of the NHS isolates and 100% of the animal fecal isolates were correctly classified. The average rate of correct classification for HS and NHS isolates was 82%. We conclude that DA of RT profiles may be a useful method for identifying HS and NHS fecal pollution and may potentially facilitate management practices.