Influence of curli expression by Escherichia coli 0157:H7 on the cell's overall hydrophobicity, charge, and ability to attach to lettuce.

Curli fibers are produced by some Escherichia coli cells in response to environmental stimuli. These extracellular proteins enhance the cell's ability to form biofilms on various abiotic surfaces. E. coli 0157:H7 cells readily attach to a variety of fruit and vegetable surfaces. It is not known whether the expression of curli influences the cell's ability to attach to produce surfaces. In this study, the effect of curli expression on the cell's overall hydrophobicity, charge, and ability to attach to cut and whole iceberg lettuce surfaces was examined. All strains, regardless of curli expression, attached preferentially to the cut edges of lettuce (P < 0.05). The curli-producing cells of E. coli 0157:H7 strain E0018 attached in significantly greater numbers to both cut and whole lettuce pieces than did the non-curli-producing E0018 cells (P < 0.05); however, no significant attachment differences were observed between the curli-producing and non-curli-producing cells of E. coli 0157:H7 strains 43894 and 43895. All curli-producing E. coli 0157:H7 strains were significantly more hydrophobic (P < 0.01); however, no association between the cells' hydrophobic characteristics and lettuce attachment was observed. Overall surface charge of the cells did not differ among strains or curli phenotypes. Results indicate that overall hydrophobicity and cell charge in E. coli 0157:H7 strains do not influence attachment to iceberg lettuce surfaces. The presence of curli may not have any influence on attachment of E. coli 0157:H7 cells to produce items. Additional factorsmay influence the attachment of E. coli 0157:H7 to plant surfaces and should be further examined.

Effects of modified atmosphere packaging on toxin production by Clostridium botulinum in raw aquacultured summer flounder fillets (Paralichthys dentatus).

Packaging fishery products under vacuum atmosphere packaging (VAC) and modified atmosphere packaging (MAP) conditions can significantly extend the shelf life of raw, refrigerated fish products. There is considerable commercial interest in marketing VAC and MAP refrigerated (never frozen) raw fish fillets. The objective of this study was to determine if Clostridium botulinum toxin development precedes microbiological spoilage in raw, refrigerated flounder fillets. Aquacultured flounder (Paralichthys dentatus) individual fish fillets either were packed with a film having an oxygen transmission rate (OTR) of 3000 cm3 m(-2) 24 h(-1) at 22.8 degrees C or were vacuum packaged or packaged under 100% CO2 with a film having an OTR of 7.8 cm3 m(-2) 24 h(-1) at 21.1 degrees C and were stored at 4 and 10 degrees C. Samples were analyzed by aerobic plate count (APC) for spoilage and qualitatively for botulinum toxin with a mouse bioassay. The results demonstrate that flounder fillets (4 degrees C) packaged with a film having an OTR of 3,000 were microbiologically spoiled (APC, > 10(7) CFU/g) on day 15, but there was no toxin formation, even after 35 days of storage. However, at 10 degrees C, toxin production occurred (day 8), but it was after microbial spoilage and absolute sensory rejection (day 5). Vacuum-packaged fillets and 100% CO2 fillets (4 degrees C) packaged with a film having an OTR of 7.8 were toxic on days 20 and 25, respectively, with microbial spoilage (APC, >10(7) CFU/g) not occurring during the tested storage period (i.e., >35 days). At 10 degrees C, in vacuum-packaged flounder, toxin formation coincided with microbiological spoilage (days 8 to 9). In the 100% CO2-packaged fillets, toxin formation occurred on day 9, with microbial spoilage occurring on day 15. This study indicates that films with an OTR of 3,000 can be used for refrigerated fish fillets and still maintain the safety of the product.

Efficacy of detergents in removing Salmonella and Shigella spp. from the surface of fresh produce.

Fresh produce has been implicated in several foodborne disease outbreaks. Produce surfaces can be primary sites of contamination during production and handling. One approach to reduce contamination is to treat fresh produce with rinsing agents. In this study, different detergent agents were used at 22 and 40 degrees C to determine their efficacy in removing Salmonella and Shigella spp. from the surfaces of strawberries, tomatoes, and green-leaf lettuce. Produce was inoculated at 22 degrees C with a cocktail of nalidixic acid-resistant organisms (6 to 6.5 log CFU/ml). After air drying for 1 h, samples were rinsed with either 0.1% Tween 80, 0.1% sodium lauryl sulfate (SLS), or water (control) at 22 or 40 degrees C. Rinse solutions were spiral plated onto tryptic soy agar supplemented with 50 mg of nalidixic acid per liter. In trials involving strawberries and lettuce, Salmonella and Shigella were removed at levels of 4 and 3 log CFU/ml, respectively, except from Salmonella-inoculated strawberries rinsed with SLS, for which minimal removal rates were 1.5 log CFU/ml at 22 degrees C and < 1 log CFU/ml at 40 degrees C. When whole strawberries were analyzed after rinsing with SLS, few organisms were recovered. This result suggests that SLS may have a lethal or sublethal effect on Salmonella, especially when a 40 degrees C solution is used. Salmonella and Shigella removal rates for tomatoes were 1 and 1.5 log CFU/ml lower, respectively, than those for strawberries or lettuce. Overall, detergents were no more effective in removing organisms from produce than water was. The detergents examined would not constitute effective overall produce rinse treatments.

Effect of organic acids and hydrogen peroxide on Cryptosporidium parvum viability in fruit juices.

Cryptosporidium parvum has historically been associated with waterborne outbreaks of diarrheal illness. Foodborne cryptosporidiosis has been associated with unpasteurized apple cider. Infectious oocysts are shed in the feces of common ruminants like cattle and deer in and near orchards. In this study, the ability of organic acids and hydrogen peroxide (H2O2) added to fruit juice to inhibit the survival of C. parvum was analyzed. Oocyst viability was analyzed by a cell culture infectivity assay with the use of a human ileocecal cell line (HCT-8) whose infectivity pattern is similar to that for human oral infectivity. Cell monolayers were infected with 10(6) treated oocysts or a series of 10-fold dilutions. Parasitic life stages were visualized through immunohistochemistry with 100 microscope fields per monolayer being counted. In vitro excystation assays were also used to evaluate these treatments. Organic acids and H2O2 were added to apple cider, orange juice, and grape juices on a weight/volume basis. Malic, citric, and tartaric acids at concentrations of 1 to 5% inhibited C. parvum's infectivity of HCT-8 cells by up to 88%. Concentrations ranging from 0.025 to 3% H2O2 were evaluated. The addition of 0.025% H2O2 to each juice resulted in a >5-log reduction of C. parvum infectivity as determined with a most-probable-number-based cell culture infectivity assay. As observed with differential interference contrast and scanning electron microscopy, reduced infectivity may be mediated through effects on the oocyst wall that are caused by the action of H2O2 or related oxygen radicals. The addition of low concentrations of H2O2 can represent a valuable alternative to pasteurization.

Survivability of Salmonella and Shigella spp. in sodium lauryl sulfate and tween 80 at 22 and 40 degrees C.

Fresh produce has been implicated in several foodborne disease outbreaks. A primary site of contamination during production and handling is the surface of produce. One approach to reducing contamination is to treat fresh produce with rinsing agents. Studies have examined the efficacies of detergents and other rinses in recovering pathogens from produce surfaces. The determination of how these detergents affect bacterial cells may aid in understanding the mechanisms behind their removal. This study examines the survivability of Salmonella and Shigella in two detergents. A 0.1% sodium lauryl sulfate (SLS) solution, a 0.1% Tween 80 solution, and water were inoculated with a cocktail of stationary-phase organisms (3 log CFU/ml) and incubated for up to 32 h at 22 degrees C and 40 degrees C. Samples were taken over time and plated on tryptic soy agar supplemented with 50 ppm of nalidixic acid. Salmonella survived in all solutions and exhibited significant growth in water (0.8 log CFU/ml at 22 degrees C and 1.9 log CFU/ml at 40 degrees C) and Tween 80 (1.0 log CFU/ml at 40 degrees C). Shigella survived in all solutions at 22 degrees C and exhibited a growth level of 2.0 log CFU/ml in SLS. Shigella also survived in all solutions at 40 degrees C, although its populations decreased significantly in Tween 80 over time. Elevated temperatures may allow Tween 80 to kill Shigella spp. over time. Overall, the detergents tested were not detrimental to the cells; therefore, if these solutions were to be used as produce rinse agents, they would aid in removal of organisms from surfaces rather than kill the cells.

Examination of attachment and survival of Toxoplasma gondii oocysts on raspberries and blueberries.

The consumption of Toxoplasma gondii oocysts on fresh produce may be a means of its transmission to humans. Cats shed T. gondii oocysts, which contaminate produce directly or contaminate water sources for agricultural irrigation and pesticide and fertilizer applications. Cyclospora cayetanensis is a related coccidial parasite, and outbreaks of diarrhea caused by C. cayetanensis have been associated with the ingestion of contaminated raspberries. The oocysts of these coccidians are similar in size and shape, indicating that they may attach to and be retained on produce in a similar manner. In the present study the attachment and survival of T. gondii oocysts on 2 structurally different types of berries were examined. Raspberries and blueberries were inoculated individually with 1.0 x 10(1) to 2.0 x 10(4) oocysts of sporulated T. gondii. Berries inoculated with 2.0 x 10(4) oocysts were stored at 4 C for up to 8 wk. Oocyst viability and recovery were analyzed by feeding processed material to mice. Mice fed T. gondii-inoculated berries stored at 4 C for 8 wk developed acute infections. In other experiments mice fed raspberries inoculated with > or = 1.0 x 10(1) oocysts became infected, whereas only mice fed blueberries inoculated with > or = 1.0 x 10(3) oocysts became infected. This study demonstrates that T. gondii oocysts can adhere to berries and can be recovered by bioassays in mice and that raspberries retain more inoculated oocysts than do blueberries. The results suggest that T. gondii may serve as a model for C. cayetanensis in food safety studies.

Antibacterial Effects of Hydrogen Peroxide and Methods for Its Detection and Quantitation †.

Hydrogen peroxide is responsible for certain bactericidal effects observed in biological systems, such as growth inhibition of one bacterial species by another and killing of invading microorganisms by activated phagocytic cells. H2O2 might be generated in bacteriological media by their exposure to light and/or oxygen and become an important mediator of toxic effects. H2O2 cytotoxicity is apparently due to its capacity-generally mediated by transition metal ions-to generate more reactive and cytotoxic oxygen species such as the hydroxyl radical, which is a powerful oxidant, and which can initiate oxidation of biomolecules. The conversion of H2O2 into more cytotoxic compounds may be potentiated by reducing agents and by peroxidases. Cells may protect themselves against H2O2 toxicity either by the action of catalases or, in the case of DNA damage, by repairing the damage after it has taken place. Assays for the detection and quantitation of H2O2 in cell cultures include those based on (i) catalase-dependent oxidation of formate to CO2, (ii) generation of fluorescent products due to a H2O2- mediated oxidative reaction, (iii) the loss of fluorescence upon the oxidation of scopoletin, (iv) change in absorbance upon oxidation of phenol red, or (v) formation of complexes with peroxidases. Some possible antimicrobial uses of H2O2 in the food industry are presented.

Listeria monocytogenes Occurrence and Growth at Refrigeration Temperatures in Fresh Blue Crab ( Callinectes sapidus ) Meat.

In this study, 126 samples of freshly cooked and picked blue crab ( Callinectes sapidus ) meat collected from different processing facilities were analyzed for the presence of Listeria spp. Thirteen samples (10%) were positive for Listeria , with 10 samples positive for L. monocytogenes and 3 samples positive for L. innocua . Fraser broth was used in a 5-tube most probable number (MPN) enumeration, in duplicate, of Listeria in 25-g samples incubated at 36°C for 24 h and plated in modified Oxford agar and blood agar with API strip confirmation. The levels of Listeria cells found in fresh blue crab meat were always less than 100/g with only one exception, in which the MPN index was 1,100/g. A L. monocytogenes strain (168) isolated from fresh blue crab meat was inoculated (less than 50 CFU/g) into pasteurized crab meat and incubated at 1.1, 2.2, and 5°C for 21 days. Growth curves were obtained by analyzing 2-g samples at intervals of 0, 8, 10, 12, 14, 16, 19, and 21 days. When inoculated into pasteurized blue crab meat, L. monocytogenes had an increased growth rate as the storage temperature increased, with approximately a 7-log10-unit increase in population at 5°C and only a 2.5-log10-unit increase in population at 1.1 °C after the 21 days of incubation.

Improved Recovery of Stressed Bifidobacterium from Water and Frozen Yogurt.

A roll-tube repair-detection procedure was developed to enumerate injured and noninjured cells of Bifidobacterium species from water and food samples. This procedure combined the Virginia Polytechnic Institute and State University's anaerobic roll-tube procedure and the repair-detection technique for detecting stressed cells. Mara and Oragui's human bifid sorbitol agar medium was modified for use in the roll-tube procedure by replacing the indicator bromocresol purple with phenyl red (0.027 g/l), adding 0.0006 g of methylene blue per 1, increasing the agar content to 25 g/l and adjusting the pH of the medium to 7.1 ± 0.1. The repair-detection roll-tube technique was shown to recover Bifidobacterium cells significantly (P < 0.01) better than pour plates, using the same medium incubated in anaerobe jars, even when a repair-detection system was used. Most repair in the roll tubes occurred in the first hour. B. adolescentis had a poor survival rate after 96 hours in water. Glucose was substituted for sorbitol in the medium used for enumeration of B. longum added to frozen yogurt, because this organism cannot utilize sorbitol. This medium, when used as part of a repair detection system, significantly (P < 0.01) recovered more cells than anaerobic pour plate techniques and was able to separate Bifidobacterium species and Lactobacillus acidophilus by colony morphology. Bifidobacterium cells were 1mm or larger, round and yellow, while the L. acidophilus colonies were so small (< 1/4 mm) their detection and enumeration was difficult.

Effect of Heat Shock and Incubation Atmosphere on Injury and Recovery of Escherichia coli O157:H7.

Escherichia coli serotype O157:H7 cells were grown at 30°C for 6 h and subjected to a heat stress, or heat shock, at 42°C for 5 min. Heat-shocked and nonheat-shocked controls were heat treated at 55°C for up to 60 min. The number of injured cells was significantly higher in heat-shocked cells than in controls, and the rate of release of cell components was higher in heat-shocked cells. Anaerobic plating resulted in higher recovery of injured cells, when compared with aerobic plating, regardless of whether the cells were heat shocked or not. In addition, heat shocking resulted in lower catalase and superoxide dismutase activities when compared with controls. It also resulted in greater survivability after exposure to hydrogen peroxide, suggesting that heat shocking somehow enables the cells to survive exposure to toxic substances in addition to heat. The heat-shock response, coupled with anaerobic conditions, increased the ability of E. coli O157:H7 cells to recover after a heat treatment. Thus, heat shock did not afford protection to the cells against injury, but rather enhanced their ability to recover during storage.

Effect of Heat Shock and Growth Atmosphere on the Heat Resistance of Escherichia coli 0157:H7.

Log phase cells of Escherichia coli 0157:H7 were grown aerobically at 30°C and heat shocked at various time/temperature combinations to determine the optimum conditions that would result in the highest number of survivors to a subsequent 55°C heat treatment. Heat shocking at 42°C for 5 min resulted in the largest increase in D55 value over nonheat-shocked controls of all heat-shock time/temperature combinations tested. Growth atmosphere significantly contributed to the heat resistance of both heat-shocked and nonheat-shocked cells, with anaerobically grown cells having D55 values higher than those of cells grown aerobically. Therefore, both heat shocking and anaerobic growth contributed to an increase in the number of survivors of E. coli 0157:H7 cells when compared with nonheat-shocked cells grown aerobically. Aerobically grown, heat-shocked cells, and anaerobically grown cells, both heat-shocked and nonheat-shocked, contained a 71,000 dalton protein not present in aerobically grown, nonheat-shocked controls. This protein was found to be immunologically similar to a sigma32 subunit of RNA polymerase, as evidenced by Western Blot using monoclonal antibodies specific for the sigma subunit. In addition to heat, anaerobic growth appeared to be a form of stress, since it resulted in the synthesis of heat-shock proteins and in an increased survival of cells to a heat treatment.

Sodium Hypophosphite Inhibition of the Growth of Selected Gram Negative Foodborne Pathogenic and Spoilage Bacteria.

Sodium hypophosphite was evaluated for inhibition of growth of selected gram negative foodborne pathogenic and spoilage bacteria in laboratory media. In addition, the effects of pH and sodium chloride alone and in combination with sodium hypophosphite were examined. All inhibition studies were performed with optimal or nearly optimal growth conditions for each bacterium. Growth was monitored by measuring optical density at 600 nm, and a time to significant growth determined in each test medium. Ratios of time to significant growth in controls over that in test media were used to evaluate the effect that sodium hypophosphite and other variables had on growth. Sodium hypophosphite was most effective in inhibiting growth of gram negative facultatively anaerobic bacteria, but generally ineffective against the aerobic Pseudomonas fluorescence and microaerophilic Campylobacter jejuni strains H-840 and Smith. Results from this investigation indicate the potential use of sodium hypophosphite as an antimicrobial food preservative.

Influence of pH and Phosphate Buffer on Inhibition of Clostridium botulinum by Antioxidants and Related Phenolic Compounds.

The compounds butylated hydroxyanisole (BHA); butylated hydroxytoluene (BHT); tert-butylhydroquinone (TBHQ); nor-dihydroguaiaretic acid (NDGA); 2', 4', 5'-trihydroxybutyrophenone (THBP); 8-hydroxyquinoline; isoamyl and isobutyl esters of gallic acid; ethyl, propyl, and butyl esters of p-hydroxybenzoic acid; thymol; 2-isopropylphenol; 2-tert-butylphenol; and 2-tert-butyl-4-methylphenol were tested for their antibotulinal activity in prereduced Thiotone-yeast extract-glucose (TYG) broth with and without hosphate buffer (0.05 M, pH 6.0 and 7.0). Isoamyl gallate, isobutyl gallate, propyl ester of p-hydroxybenzoic acid, NDGA, BHA, 8-hydroxyguinoline, and 2-tert-butylphenol were more inhibitory when phosphate buffer (pH 7.0(was added to prereduced TYG. The propyl and butyl esters of p-hydroxybenzoic acid and 2-isopropylphenol were more effective at pH 6.0 than at pH 7.0, whereas thymol and 8-hydroxyquinoline were slightly more active at pH 7.0 than at pH 6.0 NDGA at 50 µg/ml was the most effective compound tested for delaying growth and toxin production.

Inhibition and Control of Bacterial Spore Germination.

Factors affecting germination of bacterial spores as well as chemical inhibition of germination are reviewed. Current theories on the nature of initiation of the germination process are also presented. Transformation of a dormant bacterial spore into a metabolically active vegetative cell involves a myriad of complex biochemical events of which the "trigger reaction" is thought to be the prime event. The ability to control or prevent this complex sequence of events from occurring by manipulation of environmental factors or use of chemical inhibitors has been the objective of numerous research endeavors. A thorough understanding of these factors is important to both maintenance and future development of an adequate, safe and wholesome food supply.