Viability of Salmonella in bone meal.

The ability of Salmonella to survive varying concentrations of sodium metabisulphite incorporated into bone meal (rendered animal by-product) was investigated. No viable Salmonella was detected after 4 days of exposure of the micro-organisms to 5,000 ppm of sodium metabisulphite. The minimum killing concentration of metabisulphite was also established at 4,000 ppm. However, the killing effect of the metabisulphite was moisture-dependent, being most potent at a moisture level of 8 to 12%.

Evaluation of the Potential for Botulinal Toxigenesis in Reduced-Sodium Processed American Cheese Foods and Spreads.

Process cheese foods and spreads manufactured to contain low sodium concentrations (ca 320 mg/100 g or 90 mg/28 g, one serving) and added delta-gluconolactone (0.33%) were resistant to toxigenesis by Clostridium botulinum inoculated at a rate of 1000 spores/g and held at 30°C for 84 d. Low pH values (5.26 or less) provided by delayed acidity development from delta-gluconolactone were influential in the C. botulinum inhibition observed. Disodium phosphate, trisodium citrate, dipotassium phosphate, tripotassium citrate, and sodium aluminum phosphate used in screening tests as single emulsifiers (2.5%) in process cheese foods and spreads allowed toxin formation in many samples prepared. However, some inhibition of toxin formation was indicated for samples emulsified with disodium phosphate, and possibly trisodium citrate.

Attachment of Salmonella to mammalian cells in vitro.

The attachment of Salmonella typhimurium strain PHL67342 to several mammalian tissue culture cell lines was investigated. Strain PHL67342 failed to attach in significant numbers to the Buffalo green monkey (BGM), swine testicular (ST), and HeLa cell lines. Significant attachment was observed with the Henle intestinal cell line. Log-phase cells of strain PHL67342 attached in greatest numbers to the Henle cells after 45 min of incubation at 37 degrees C. Attachment to the Henle cells was not affected by D-mannose or D-galactose, but was markedly inhibited by high concentrations of alpha-methyl-D-mannoside. Also, Salmonella lipopolysaccharide had no effect on the attachment of strain PHL67342 to the Henle cells. Fimbriae were not detected on the bacterial cells used in the adherence experiments. These results suggest that some bacterial factor(s) other than fimbriae and lipopolysaccharide mediate the attachment of strain PHL67342 to the Henle cells.

Effect of lipopolysaccharide mutations on the pathogenesis of experimental Salmonella gastroenteritis.

Lipopolysaccharide mutants of Salmonella typhimurium provoked diminished amounts of fluid in rabbit ileal loops as compared with the response to the wild type. The responses elicited by these mutants ranged from 0 to 60% of that caused by the parent strain. Two completely rough mutants and one leaky rough mutant were chosen for further study. Purified lipopolysaccharide from the parent and the mutant strains failed to stimulate fluid exsorption in ileal loop experiments. Histological studies revealed that the three lipopolysaccharide mutants were less invasive than wild type and were less able to generate an inflammatory reaction in the rabbit ileum. A Salmonella enterotoxin was present in culture filtrates from one rough mutant and the wild type; however, the rough mutant appeared to produce less toxin. Enterotoxic activity was absent in culture filtrates from the two other rough mutants. These results suggest that reductions in both invasiveness and the ability to produce Salmonella enterotoxin decreased the ability of these mutants to provoke fluid exsorption. Also, the results indicate that lipopolysaccharide mutations can have a profound effect on the enteropathogenic properties of S. typhimurium.

Effects of potassium sorbate and other antibotulinal agents on germination and outgrowth of Clostridium botulinum type E spores in microcultures.

The effects of potassium sorbate, sodium hypophosphite, sodium tripolyphosphate, sodium nitrite, and linoleic acid on the germination and outgrowth of Clostridium botulinum type E spores were studied in microcultures. At pH 5.8 to 6.0 in liver veal agar, the germination rate was decreased to nearly zero with 1.0, 1.5, or 2.0% sorbate. At pH 7.0 t 7.2, these levels of sorbate afforded germination and outgrowth of abnormally shaped cells that were defective in cell division. At the high pH range, 0.5 or 1.0% hypophosphite had effects similar to those of sorbate. The use of 0.05% sodium nitrite with sorbate enhanced the lysis of outgrowing cells at pH 7.2 or lower. Emergence and elongation were inhibited by 0.05% linoleic acid with or without 1.0% sorbate at pH 7.0 to 7.2. The addition of 0.5% tripolyphosphate to media containing 1.5% sorbate at pH 7.1 prevented normal cell growth to an extent greater than with sorbate alone.

A comparative study of the lethal effect of metabisulphite on the viability of three bacterial species in bone meal and gelatin.

An investigation into the effect of sodium metabisulphite on the viability of three bacterial species, Salmonella typhimurium 59143, Escherichia coli ES-1 and Pseudomonas aeruginosa 8602, in bone meal and gelatin has been reported in this paper. Methods used in detecting the viability of the bacteria after exposure to metabisulphite-treated bone meal and gelatin have been presented. Results have shown that the bacterial species were all susceptible to metabisulphite in both rendered by-products but in all cases, the micro-organisms were far more susceptible in metabisulphite-treated gelatin than in metabisulphite-treated bone meal. Reasons for the difference in susceptibility were also adduced.

Viability of Salmonella typhimurium in metabisulphite-treated bone meal: effect of inocula and moisture variation on the lethal potency of metabisulphite.

The susceptibility of different inocula of Salmonella typhimurium to the antimicrobial action of various concentrations of metabisulphite in rehydrated bone meal has been investigated. Furthermore, the lethal potency of metabisulphite in the presence of various metabisulphite and moisture concentrations has been examined. It has been shown that the lethal potency of metabisulphite was dependent upon the inocula used in the simulated contamination and the moisture content of the bone meal. A study of the longevity of the potency of metabisulphite has revealed that such a potency could be maintained without a decrease in efficacy for 14--28 days depending on the metabisulphite level in the bone meal.

New medium for rapid screening and enumeration of Clostridium perfringens in foods.

A rapid and sensitive procedure for estimating low numbers of Clostridium perfringens has been investigated and compared to methods used currently in the food industry. The new liquid medium, RPM (rapid perfringens medium), was compared with sulfite-polymyxin-sulfadiazine agar and tryptose-sulfite-cycloserine agar in recovery studies with naturally contaminated and with inoculated foods. The medium consists of a mixture of litmus milk and fluid thioglycolate medium fortified with glucose, peptone, gelatin, yeast extract, sodium chloride, and ferrous sulfate. Selectivity is based on an antibiotic system (polymyxin B sulfate and neomycin sulfate) incorporated into the medium, coupled with an incubation temprature of 46 to 48 degrees C for 24 h. Tubes were scored as positive if a stormy fermentation was observed. All tubes demonstrating stormy fermentation were confirmed as containing C. perfringens. Of a total of 774 naturally contaminated food samples, 546 samples (71%) were found to contain C. perfringens with RPM, whereas only 168 (22%) of the samples were positive using sulfite-polymyxin-sulfadiazine agar. C. perfringens was isolated from 71% of 85 other samples using RPM as compared to 14% with tryptose-sulfite-cycloserine agar. Enumeration studies on 14 individual samples using the most probable number technique also demonstrated greater sensitivity with RPM.

Rapid qualitative method for detecting staphylococcal nuclease in foods.

A rapid method for the detection of heat-stable staphylococcal nuclease in foods is described. The procedure consists of an acid precipitation, boiling, and centrifugation followed by enzyme detection in an agar plate containing deoxyribonucleic acid. To test the efficacy of the procedure, purified Staphylococcus aureus nuclease was added to various foods and recovery experiments were performed. Additionally, foods were inoculated and incubated with S. aureus, and the staphylococcal counts were compared with nuclease activity. The results indicate that the procedure possesses merit for a rapid method that can be incorporated into quality control programs. The procedure requires approximately 2.5 h, and it will detect nuclease levels as low as 10 ng/g of food.

Production and partial purification of Salmonella enterotoxin.

By using a strain of Salmonella typhimurium, we detected the presence of an enterotoxin, as determined by the rabbit ileal loop assay, in various complex and defined media. The enterotoxin was concentrated by ultrafiltration of culture supernatant fluids and eluted in and adjacent to the void volume of a Sephadex G-100 column. This suggested that the enterotoxic factor was of a relatively high molecular weight, and additional evidence indicated it was heterogeneous in size. Further chromatography, using a diethylaminoethyl-cellulose anion exchanger, facilitated at least a 50-fold purification of the Salmonella enterotoxin.

Detection of Salmonella enterotoxin using rabbit ileal loops.

The presence of an enterotoxin produced by Salmonella in broth culture has been demonstrated by using the rabbit ileal loop model. Response by the animal to enterotoxin in sterile culture supernatant fluids is enhanced when the intestinal lumen is washed with a mucolytic agent prior to the administration of toxin. Fluid secretion is untreated intestinal loops was also observed if enterotoxin was administered with a live, invasive Salmonella strain which did not evoke a secretory response. A limited survey of Salmonella isolated for clinical and food sources indicated the common occurrence of enterotoxin production, and stock cultures maintained the ability to produce the toxin. The host-adapted species which were tested varied in their ability to produce enterotoxin.

Rabbit intestinal fluid stimulation by an enterotoxigenic factor of Staphylococcus aureus.

An exoprotein of Staphylococcus aureus 100 that elicited a positive ileal loop response in the rabbit model was investigated in this study. The protein, as it occurred in the culture supernatant fluid, could be detected initially in the late log phase of growth under aerobic and anaerobic conditions. It was stable under acidic conditions to pH 2.0 after 24 h at 4 degrees C. Thirty-minute treatments at 80 degrees C destroyed the ileal loop activity whereas similar trials at 70 degrees C had no effect. Although preparations of staphylococcal enterotoxins purified by other investigators did not produce positive ileal loops, the enterotoxigenic activity of the S. aureus 100 supernatant fluid could be neutralized by antisera prepared against enterotoxins A and B. Throughout purification studies, the active moiety reacted serologically with antiserum A. Polyacrylamide gel electrophoresis of the partially purified protein produced migration patterns nearly identical to those of enterotoxin A.

Assay, characterization, and localization of an enterotoxin produced by Salmonella.

An enterotoxic factor isolated from cultures of Salmonella yielded reproducible results in the suckling mouse model in contrast to other animal models. The enterotoxin appears to possess properties similar to both the heat-stable and heat-labile enterotoxins of Escherichia coli. Preliminary results indicate that the toxin is a protein, is located in the cell wall or outer-membrane fraction, and is difficult to separate from other cell wall constituents.

Effect of sodium ascorbate and sodium nitrite on toxin formation of Clostridium botulinum in wieners.

Toxin production by Clostridium botulinum was inhibited by sodium nitrite levels above 50 mug/g of wiener. Sodium ascorbate at levels of 105 and 655 mug/g of product did not decrease the effectiveness of the sodium nitrite inhibition, nor did sodium ascorbate potentiate it. The results indicate that the use of sodium ascorbate in vacuum-packaged wieners does not appreciably alter the inhibition of C. botulinum toxin formation by sodium nitrite.

Effect of sodium nitrite and sodium nitrate on botulinal toxin production and nitrosamine formation in wieners.

Wieners were formulated and processed approximating commercial conditions as closely as possible. Twenty-four batches of product were made with the addition of six levels of sodium nitrite (0, 50, 100, 150, 200, and 300 mug/g), four levels of sodium nitrate (0, 50, 150, and 450 mug/g), and two levels of Clostridium botulinum (0 and 620 spores/g). After formulation, processing, and vacuum packaging, portions of each batch were incubated at 27 C or held for 21 days at 7 C followed by incubation at 27 C for 56 days. The latter storage condition approximated distribution of product through commercial channels and potential temperature abuse at the consumer level. Samples were analyzed for botulinal toxin, nitrite, and nitrate levels after 3, 7, 14, 21, 28, and 56 days of incubation. When nitrite was not added, toxic samples were detected after 14 days of incubation at 27 C. At the lowest level of nitrite added (50 mug/g), no toxic samples were observed until 56 days of incubation. Higher levels of nitrite completely inhibited toxin production throughout the incubation period. Nine uninoculated samples, representing various levels and combinations of nitrite and nitrate, were evaluated organoleptically. The flavor quality of wieners made with nitrite was judged significantly higher (P = 0.05) than of wieners made without nitrite. The nine samples were negative for 14 volatile nitrosamines at a sensitivity level of 10 ng/g. The results indicated that nitrite effectively inhibited botulinal toxin formation at commercially employed levels in wieners and that detectable quantities of nitrosamines were not produced during preparation and processing of the product for consumption.

Production and heat stability of staphylococcal nuclease.

No correlation existed between numbers of organisms and nuclease activity in laboratory-grown cultures of Staphylococcus aureus. Nuclease production was inhibited by anaerobic incubation and stimulated by aeration. Strains of S. aureus varied in the production of nuclease. The optimum pH for enzyme production was 8.3 and employment of a tris(hydroxymethyl)aminomethane buffer system resulted in increased production of the enzyme as compared with a phosphate buffer. The nuclease was extremely heat-stable and had a D value of 16.6 min at 130 C.

Turbidimetric assay of staphylococcal nuclease.

A simplified turbidimetric procedure was developed to assay staphylococcal nuclease activity. The ease of performance and sensitivity to nanogram quantities enhance the utilization of the method for the quantitative or qualitative estimation of the enzyme. Unlike plating methods, the turbidimetric procedure affords the differentiation between heat-stable and heat-labile nuclease activity.

Effect of lysozyme on enterococcal viability in low ionic environments.

The action of lysozyme on the enterococcal cell differed markedly as a function of the ionic strength of the environment. In high ionic environments (I approximately 0.3), the traditionally slow lytic response and decrease in viability were noted. In a low ionic environment the majority of the cell wall was hydrolyzed, but cellular integrity was preserved and almost all cellular protein, deoxyribonucleic acid and ribonucleic acid remained with the lysozyme-cell complex. However, under these conditions, lysozyme inactivated energy-yielding metabolism, and a rapid extensive loss of viability was observed. Some other basic compounds without lytic activity on the cell wall also effected a substantial reduction in viability. The data suggest that lysozyme acts on the cell membrane to effect disruption of cellular metabolism.

Effect of pH and oxygen tension on staphylococcal growth and enterotoxin formation in fermented sausage.

Commercial fermented 0sausages that contained significant numbers of viable coagulase-positive staphylococci were found to have the growth localized in the outermost areas of the sausage where oxygen tension was highest. Staphylococci were found to be more acid-tolerant aerobically than anaerobically. With chemical acidulation of sausage, growth could be controlled both aerobically and anaerobically with approximately 1.5% glucono delta lactone. Biological acidulation with a high inoculum of Pediococcus cerevisiae inhibited anaerobic staphylococcal growth but failed to suppress aerobic growth completely. A staphylococcal count of approximately 4 x 10(7) cells/g of sausage appeared to be necessary to produce detectable enterotoxin A within 24 hr in sausage. A minor difference existed in the relative rates of production of the different types of enterotoxin. Detectable enterotoxin A was produced in 24 hr in sausage held in atmospheres containing 10, 15, and 20% oxygen. In an atmosphere containing 5% oxygen, toxin was detected after 48 hr of incubation. No toxin was detected after 120 hr under anaerobic conditions. Most staphylococcal strains tested initiated growth and produced detectable enterotoxin aerobically at a pH of 5.1 in broth media. Anaerobically, however, most strains failed to produce detectable enterotoxin below pH 5.7.

Flagellar cross-repolymerization among different Salmonella serotypes and Escherichia coli.

Flagellar preparations from several serotypes of Salmonella and one strain of Escherichia coli were tested for their ability to cross-repolymerize. Repolymerization was found not to be dependent on the antigenic determinants of the flagella.