Assessment of the Sanitary Effectiveness of Holding Temperatures on Beef Cooked at Low Temperature

Beef cubes cooked at low temperature were surface inoculated and incubated at 43.3 through 53.3 C to establish temperature limits for growth of staphylococci, salmonellae, and Clostridium perfringens. A greater than 99% reduction in staphylococci was achieved after 6 hr at 48.8 C, of salmonellae at 51.1 C, and of C. perfringens at 53.3 C. There were no survivors of a mixed inoculum of Staphylococcus aureus, Salmonella enteritidis, and C. perfringens after 12 hr at 51.1 C.     

ADP-ribosylation of actin isoforms by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin

The substrate specificities of the actin-ADP-ribosylating toxins, Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin were studied by using five different preparations of actin isoforms: alpha-skeletal muscle actin, alpha-cardiac muscle actin, gizzard gamma-smooth muscle actin, spleen beta- and gamma-cytoplasmic actin, and aortic smooth muscle actin containing alpha- and gamma-smooth muscle actin isoforms. C. perfringens iota toxin ADP-ribosylated all actin isoforms tested, whereas C. botulinum C2 toxin did not modify alpha-skeletal muscle actin or alpha-cardiac muscle actin. Spleen beta/gamma-cytoplasmic actin and gizzard gamma-smooth muscle actin were substrates of C. botulinum C2 toxin. In the aortic smooth muscle actin preparation, gamma-smooth muscle actin but not alpha-smooth muscle actin was ADP-ribosylated by C. botulinum C2 toxin. The data indicate that, in contrast to C. perfringens iota toxin, C. botulinum C2 toxin ADP-ribosylates only beta/gamma-cytoplasmic and gamma-smooth muscle actin and suggest that the N-terminal region of actin isoforms define the substrate specificity for ADP-ribosylation by C. botulinum C2 toxin.     

Effect of Vacuum Packaging on Growth of Clostridium botulinum and Staphylococcus aureus in Cured Meats

Incrimination of vacuum-packaged smoked fish in outbreaks of botulism has raised questions about the safety of this process in comparison with other methods of packaging foods. It has been suggested, for example, that Clostridium botulinum may grow better in a vacuum-packaged product than in one that is packaged without vacuum. To evaluate this possibility, sliced bologna was inoculated with spores of C. botulinum type A, packaged in transparent plastic film with and without vacuum, and stored at temperatures within the growth range of the organism. There was no detectable difference in the rate of toxin development in the two types of packages. In contrast, vacuum packaging markedly inhibited the growth of Staphylococcus aureus on sliced ham. The results indicate that vacuum packaging has little if any effect on the ability of C. botulinum to grow in cured meats, but it may reduce the likelihood of staphylococcal food poisoning.     

ADP-ribosylation of skeletal muscle and non-muscle actin by Clostridium perfringens iota toxin

The enzymatically active component ia of Clostridium perfringens iota toxin ADP-ribosylated actin in human platelet cytosol and purified platelet beta/gamma-actin, in a similar way to that been reported for component I of botulinum C2 toxin. ADP-ribosylation of cytosolic and purified actin by either toxin was inhibited by 0.1 mM phalloidin indicating that monomeric G-actin but not polymerized F-actin was the toxin substrate. Perfringens iota toxin and botulinum C2 toxin were not additive in ADP-ribosylation of platelet actin. Treatment of intact chicken embryo cells with botulinum C2 toxin decreased subsequent ADP-ribosylation of actin in cell lysates by perfringens iota or botulinum C2 toxin. In contrast to botulinum C2 toxin, perfringens iota toxin ADP-ribosylated skeletal muscle alpha-actin with a potency and efficiency similar to non-muscle actin. ADP-ribosylation of purified skeletal muscle and non-muscle actin by perfringens iota toxin led to a dose-dependent impairment of the ability of actin to polymerize.     

Inhibition of type A and type B (proteolytic) Clostridium botulinum by sorbic acid

The effect of sorbic acid in the pH range 4.9 to 7.0 on the probability P of growth of a single vegetative bacterium of proteolytic strains of Clostridium botulinum has been determined by comparison of the most probable number count of the bacteria in media at pH 4.9 to 7.0 containing a series of concentrations of potassium sorbate and in a nutrient medium at pH 6.8 to 7.0. The media were maintained under strictly anaerobic conditions at a redox potential equivalent to lower than -350 mV at pH 7. In medium adjusted to the required pH with HCl, P for strain ZK3 (type A) at pH 5.1 or 5.5 after 2 days at 30 degrees C was similar to that at pH 6.8 to 7.0 but was slightly lower at pH 4.9. Potassium sorbate inhibited growth, the inhibition being a function of the concentration of undissociated sorbic acid. A calculated undissociated sorbic acid concentration of 156 mg/liter delayed growth of strain ZK3 (type A) but did not result in a significant decrease in P after an incubation time of 14 days. Higher concentrations of undissociated sorbic acid caused longer delays before maximum most probable number counts developed, and a calculated undissociated sorbic acid concentration of 282 mg/liter decreased log P for strain ZK3 after an incubation time of 14 days by a factor of 5.5 to 7.5. Four additional type A strains and five type B strains were inhibited to an extent comparable to inhibition of strain ZK3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of type A and type B (proteolytic) Clostridium botulinum by sorbic acid.

The effect of sorbic acid in the pH range 4.9 to 7.0 on the probability P of growth of a single vegetative bacterium of proteolytic strains of Clostridium botulinum has been determined by comparison of the most probable number count of the bacteria in media at pH 4.9 to 7.0 containing a series of concentrations of potassium sorbate and in a nutrient medium at pH 6.8 to 7.0. The media were maintained under strictly anaerobic conditions at a redox potential equivalent to lower than -350 mV at pH 7. In medium adjusted to the required pH with HCl, P for strain ZK3 (type A) at pH 5.1 or 5.5 after 2 days at 30 degrees C was similar to that at pH 6.8 to 7.0 but was slightly lower at pH 4.9. Potassium sorbate inhibited growth, the inhibition being a function of the concentration of undissociated sorbic acid. A calculated undissociated sorbic acid concentration of 156 mg/liter delayed growth of strain ZK3 (type A) but did not result in a significant decrease in P after an incubation time of 14 days. Higher concentrations of undissociated sorbic acid caused longer delays before maximum most probable number counts developed, and a calculated undissociated sorbic acid concentration of 282 mg/liter decreased log P for strain ZK3 after an incubation time of 14 days by a factor of 5.5 to 7.5. Four additional type A strains and five type B strains were inhibited to an extent comparable to inhibition of strain ZK3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of a Clostridium perfringens predictive model, developed under isothermal conditions in broth, to predict growth in ground beef during cooling

Proper temperature control is essential in minimizing Clostridium perfringens germination, growth, and toxin production. The U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) offers two options for the cooling of meat products: follow a standard time-temperature schedule or validate that alternative cooling regimens result in no more than a 1-log(10) CFU/g increase of C. perfringens and no growth of Clostridium botulinum. A mathematical model developed by Juneja et al. (Food Microbiol. 16:335-349, 1999) may be helpful in determining if the C. perfringens performance standard has been achieved, but this model has not been extensively validated. The objective of this study was to validate the Juneja 1999 model in ground beef under a variety of changing temperature and temperature abuse situations. The Juneja 1999 model consistently underpredicted growth of C. perfringens during exponential cooling of ground beef. The model also underpredicted growth of C. perfringens in ground beef cooled at two different rates. The results presented here show generally good agreement with published data on the growth of C. perfringens in similar products. The model error may be due to faster-than-expected exponential growth rates in ground beef during cooling or an error in the mathematical formulation of the model.     

Clostridium perfringens: I. Sporulation in a Biphasic Glucose-Ion-Exchange Resin Medium

A biphasic culture medium suitable for cultivation and sporulation of Clostridium perfringens, C. botulinum, and C. sporogenes was devised. The medium designed for use in a disposable, compartmented, plastic film container contained peptones, yeast extract, minerals, an anion exchange resin, and glucose in 4% agar as the solid phase and (NH(4))(2)SO(4) and 0.1% agar as the liquid phase. With the biphasic system, it was not necessary to use active cultures as inocula. Growth was at least equal to that obtained in conventional media, and spore production of 9 out of 12 strains of C. perfringens equalled or usually exceeded that of conventional media.     

The Role of Clostridium perfringens in the Growth Response of Chicks to Dietary Penicillin

S ummary . The previous observation that Clostridium perfringens is responsible for penicillin-relieved growth depression in chicks has been reassessed. The administration of Cl. perfringens to germfree chicks, to birds harbouring a non-growth depressing gut flora and to birds reared in an environment free from Cl. perfringens was without effect on growth rate. No correlation was observed in conventional chicks between the incidence of growth depression and the presence of Cl. perfringens in the gut of the chicks one day after food was first given.     

De-ADP-ribosylation actin by Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin

The reverse reaction of the ADP-ribosylation of actin by Clostridium botulinum C2 toxin and Clostridium perfringens iota-toxin was studied. In the presence of nicotinamide (30-50 mM) C2 toxin and iota-toxin decreased the radioactive labeling of [32P]ADP-ribosylated actin and catalyzed the formation of [32P]NAD. The pH optima for both reactions were 5.5-6.0. Concomitant with the removal of ADP-ribose, the ability of actin to polymerize was restored and actin ATPase activity increased. Neither ADP-ribosylation nor removal of ADP-ribose was observed after treatment of actin with EDTA, indicating that the native structure of actin is required for both reactions. ADP-ribosylation of platelet actin by C2 toxin was reversed by iota-toxin, confirming recent reports that both toxins modify the same amino acid in actin. However, C. botulinum C2 toxin was not able to cleave ADP-ribose from skeletal muscle actin which had been incorporated by iota-toxin, corroborating the different substrate specificities of both toxins.     

Mechanisms of sorbate inhibition of Bacillus cereus T and Clostridium botulinum 62A spore germination.

The mechanism by which potassium sorbate inhibits Bacillus cereus T and Clostridium botulinum 62A spore germination was investigated. Spores of B. cereus T were germinated at 35 degrees C in 0.08 M sodium-potassium phosphate buffers (pH 5.7 and 6.7) containing various germinants (L-alanine, L-alpha-NH2-n-butyric acid, and inosine) and potassium sorbate. Spores of C. botulinum 62A were germinated in the same buffers but with 10 mM L-lactic acid, 20 mM sodium bicarbonate, L-alanine or L-cysteine, and potassium sorbate. Spore germination was monitored by optical density measurements at 600 nm and phase-contrast microscopy. Inhibition of B. cereus T spore germination was observed when 3,900 micrograms of potassium sorbate per ml was added at various time intervals during the first 2 min of spore exposure to the pH 5.7 germination medium. C. botulinum 62A spore germination was inhibited when 5,200 micrograms of potassium sorbate per ml was added during the first 30 min of spore exposure to the pH 5.7 medium. Potassium sorbate inhibition of germination was reversible for both B. cereus T and C. botulinum 62A spores. Potassium sorbate inhibition of B. cereus T spore germination induced by L-alanine and L-alpha-NH2-n-butyric acid was shown to be competitive in nature. Potassium sorbate was also a competitive inhibitor of L-alanine- and L-cysteine-induced germination of C. botulinum 62A spores.     

Effect of Soy Proteins on the Growth of Clostridium perfringens

Proteins that are used to fabricate imitation foods such as synthetic meats were evaluated for stimulative or inhibitory effects on the growth of Clostridium perfringens. Growth rate and extent were measured in thioglycolate medium without dextrose. This liquid medium contains Trypticase (BBL) which served as the protein control. For comparison, various soy proteins, synthetic meats, beef, turkey, sodium caseinate, and combinations of each were substituted for Trypticase. Meat loaf systems were also employed to determine the effects of protein additives to meat under actual meat loaf conditions. Growth of C. perfringens type A, strain S40, was measured in the respective media at 45 C at a pH of 7.0 and an E(h) of below -300 mv. Viable populations were enumerated by agar plate techniques on Trypticase-sulfite-yeast-citrate-agar incubated anaerobically (90% N(2)-10% CO(2)) for 18 hr at 35 C. When compared to Trypticase, some soy proteins had stimulative effects on the growth of C. perfringens, whereas sodium caseinate and some soy proteins were inhibitory. In liquid medium in which meat or soy meat was the source of protein, there was a marked stimulation by beef, chicken, and soy beef. Soy chicken supported growth at a rate less than observed with Trypticase. Under actual meat loaf conditions, the addition of soy meat or protein additives to beef did not affect the growth of C. perfringens. The addition of protein additives to turkey meat loaves significantly enhanced the rate of growth of C. perfringens. The stimulative effects of some soy proteins are significant in relation to control of foodborne disease.     

Immunodiffusion method for detection of type A Clostridium botulinum

A simple gel immunodiffusion agar procedure was developed for detecting toxigenic strains of Clostridium botulinum type A. The method consisted of overlaying colonies grown on thin-layer tryptone-peptone-glucose-yeast extract agar with gel diffusion agar containing desired levels of C. botulinum type A antitoxin. Concentric precipitin zones formed around colonies of C. botulinum type A. Strains of C. botulinum type A were detected by this procedure. However, C. botulinum type B reacted to a lesser degree with this system. No reaction was noted with types E, F, Langeland, F8G, Clostridium perfringens, or with strains of nontoxigenic Clostridium sporogenes. Thickness of the plating medium, incubation time and temperature, environmental growth conditions, and levels of both agar an antitoxin were important factors affecting the efficiency of the procedure, whereas the age of the culture (used as inoculum) was not critical. Thin agar medium (5 ml per plate [15 by 100 mm]) containing 1.5% agar gave consistent results, but more agar limited diffusion, and lower levels encouraged spreaders. The optimal concentration of antitoxin incorporated in to the gel diffusion agar overlay was 1.2 IU/ml gel diffusion agar. Rabbit type A antitoxin prepared with purer immunizing agent gave similar reactions. The addition of type A antitoxin in tryptone-peptone-glucose-yeast extract agar medium before inoculation with type A C. botulinum showed promising results.     

Immunodiffusion method for detection of type A Clostridium botulinum.

A simple gel immunodiffusion agar procedure was developed for detecting toxigenic strains of Clostridium botulinum type A. The method consisted of overlaying colonies grown on thin-layer tryptone-peptone-glucose-yeast extract agar with gel diffusion agar containing desired levels of C. botulinum type A antitoxin. Concentric precipitin zones formed around colonies of C. botulinum type A. Strains of C. botulinum type A were detected by this procedure. However, C. botulinum type B reacted to a lesser degree with this system. No reaction was noted with types E, F, Langeland, F8G, Clostridium perfringens, or with strains of nontoxigenic Clostridium sporogenes. Thickness of the plating medium, incubation time and temperature, environmental growth conditions, and levels of both agar an antitoxin were important factors affecting the efficiency of the procedure, whereas the age of the culture (used as inoculum) was not critical. Thin agar medium (5 ml per plate [15 by 100 mm]) containing 1.5% agar gave consistent results, but more agar limited diffusion, and lower levels encouraged spreaders. The optimal concentration of antitoxin incorporated in to the gel diffusion agar overlay was 1.2 IU/ml gel diffusion agar. Rabbit type A antitoxin prepared with purer immunizing agent gave similar reactions. The addition of type A antitoxin in tryptone-peptone-glucose-yeast extract agar medium before inoculation with type A C. botulinum showed promising results.     

Common Mesophilic Anaerobes, Including Clostridium botulinum and Clostridium tetani, in 21 Soil Specimens

A relatively rich medium was markedly superior to a dilute medium for the isolation of anaerobic bacteria from soil. The obligate anaerobes isolated from 21 soil samples were all clostridia and the counts ranged from 2.7 X 10-2 to 3.3 X 10-6 per g. The organisms most frequently isolated were Clostridium subterminate, C. sordelii, C. sporogenes, C. indolis, C. bifermentans, C. mangenoti, and C. perfringens. Seventeen other species were also recognized but almost one-third of the isolates could not be identified with any known species of Clostridium. C. botulinum type A was demonstrated in six soil samples, and type B in one. These soils were neutral to alkaline in reaction (average pH 7.9) and low in organic matter content (1.4%). The association of C. botulinum types A and B with neutral to alkaline soils was statistically significant (P = 0.001) as was their association with soils low in organic matter (P = 0.005). C. botulinum types E and F were found in one soil sample, pH 4.5, with organic matter 13.7%. C. tetani was isolated from two soil samples, both of intermediate pH value and higher than average organic matter content.     

Beneficial effect of catalase treatment on growth of Clostridium perfringens.

Several common plating media were tested for their ability to support growth of Clostridium perfringens after storage of the plates for 1 to 10 days at 4 and 25 degrees C with and without subsequent addition of catalase. Liver-veal (LV) agar and brain heart infusion (BHI) agar quickly become incapable of supporting growth after storage without added catalase, whereas Shahidi Ferguson perfringens (SFP) agar and Brewer anaerobic (BA) agar were less affected. Plate counts of C. perfringens on untreated LV and BHI agars stored 3 days at 25 degrees C showed a reduction of 98.2%, whereas counts on SFP and BA agars were reduced by 13.6% and 46.2%, respectively. Addition of 1,500 U of beef liver catalase to the surface of the 3-day-old agars before incubation resulted in substantial restoration of their growth-promoting ability. Counts of colonies on LV, GHI, SFP, and BA agars with added catalase were usually 20 to 90% higher than untreated controls. Similar results were obtained using purified catalase, fungal catalase, and horseradish peroxidase. These results suggest that inhibition may be due to peroxide formed during storage and incubation and that additon of catalase provides near optimum conditions for growth of C. perfringens on these media.     

Beneficial effect of catalase treatment on growth of Clostridium perfringens.

Several common plating media were tested for their ability to support growth of Clostridium perfringens after storage of the plates for 1 to 10 days at 4 and 25 degrees C with and without subsequent addition of catalase. Liver-veal (LV) agar and brain heart infusion (BHI) agar quickly become incapable of supporting growth after storage without added catalase, whereas Shahidi Ferguson perfringens (SFP) agar and Brewer anaerobic (BA) agar were less affected. Plate counts of C. perfringens on untreated LV and BHI agars stored 3 days at 25 degrees C showed a reduction of 98.2%, whereas counts on SFP and BA agars were reduced by 13.6% and 46.2%, respectively. Addition of 1,500 U of beef liver catalase to the surface of the 3-day-old agars before incubation resulted in substantial restoration of their growth-promoting ability. Counts of colonies on LV, GHI, SFP, and BA agars with added catalase were usually 20 to 90% higher than untreated controls. Similar results were obtained using purified catalase, fungal catalase, and horseradish peroxidase. These results suggest that inhibition may be due to peroxide formed during storage and incubation and that additon of catalase provides near optimum conditions for growth of C. perfringens on these media.     

The Growth and Toxin Production of Clostridium botulinum Type E in Certain Vacuum Packed Fish

The growth and toxin production of Clostridium botulinum type E in various types of vacuum packed fish products was tested, with particular reference to the time/temperature relationship of storage. Toxin production was most rapid in herring although smoking retarded its development. With as small an inoculum as 10² spores/pack, fresh herring became toxic after storage for 15 days at 5°. Irradiation of three species of fish after inoculation with Cl. botulinum type E showed that spores surviving radiation germinated and produced toxin more rapidly than an equivalent concentration of spores in nonirradiated fish.     

Benzylpenicillin-induced filament formation of Clostridium perfringens

Growth of Clostridium perfringens with low concentrations of benzylpenicillin inhibited septum formation and division of the organisms. This resulted in continued growth of the organisms as aseptate filaments. The effect was reversed on removal of the antibiotic. The composition of walls isolated from organisms grown with the antibiotic was similar to that of walls from untreated bacteria. In addition, both contained non-N-acetylated glucosamine residues in their peptidoglycan. No differences were detected in the degree of cross-linkage of peptidoglycan. Clostridium perfringens contains six membrane-associated penicillin-binding proteins (PBPs) which have different affinities for [3H]benzylpenicillin. Concentrations of the antibiotic which were sufficient to cause filamentation of apparently all organisms in a culture caused almost complete saturation of PBPs 3, 4, 5 and 6. At these concentrations there was no measurable interaction with PBPs 1 and 2. Thus interaction of the antibiotic with the lower molecular weight PBPs is correlated with the inhibition of septum formation in C. perfringens.