Association of vegetative antigens with the spores of Clostridium sporogenes

Clean spores of Clostridium sporogenes stored for two years continued to elicit the production of specific antibodies in immunized rabbits. Disintegrated spores stimulated not only antibodies specific for the spore component, but also those directed against 'H' and 'O' antigens of the sporangium cells.     

Effects of high hydrostatic pressure on Clostridium sporogenes spores

Spores of Clostridium sporogenes were found to be resistant to ultra high pressure, with treatments of 600 MPa for 30 min at 20 °C causing no significant inactivation. Combination treatments including heat and pressure applied simultaneously (e.g. 400 MPa at 60 °C for 30 min) or sequentially (e.g. 80 °C for 10 min followed by 400 MPa for 30 min) proved more effective at inactivating spores. Pressure cycling (e.g. 60 MPa followed by 400 MPa at 60 °C) also reduced spore numbers. Overall, these pressure treatments resulted in less than a 3 log reduction, and it was concluded that the spores could not be inactivated by pressure alone. This could indicate that for the effective inactivation of bacterial spores, high pressure technology may have to be used in combination with other preservation methods.     

Selective enumeration of spores of Clostridium species in dried foods

The suitability of a variety of media and procedures for the enumeration of sulphite-reducing clostridia in food was investigated. The most suitable procedure was pasteurization of the 1/10 macerate for at least 1 min at 80°C; followed by culture at 30°C for up to 3 d in a sulphite-based, differential reinforced clostridium medium, without bicarbonate or lactate but with an increased iron concentration, and sulphite and iron added after sterilization. Black sulphite-reducing colonies were finally tested for sensitivity to metronidazole and confirmation of their failure to grow on agar slopes under aerobic conditions.     

Selective enumeration of spores of Clostridium species in dried foods

The suitability of a variety of media and procedures for the enumeration of sulphite-reducing clostridia in food was investigated. The most suitable procedure was pasteurization of the 1/10 macerate for at least 1 min at 80 degrees C; followed by culture at 30 degrees C for up to 3 d in a sulphite-based, differential reinforced clostridium medium, without bicarbonate or lactate but with an increased iron concentration, and sulphite and iron added after sterilization. Black sulphite-reducing colonies were finally tested for sensitivity to metronidazole and confirmation of their failure to grow on agar slopes under aerobic conditions.     

Effect of heat treatment on the performance of tryptose-sulfite-cycloserine agar for enumeration of Clostridium perfringens.

Dissolving dehydrated tryptose-sulfite-cycloserine agar by only boiling or microwaving was found to inhibit Clostridium perfringens colony development in pour plates when compared with C. perfringens recovery in tryptose-sulfite-cycloserine agar prepared by autoclaving.     

Effect of heat treatment on the performance of tryptose-sulfite-cycloserine agar for enumeration of Clostridium perfringens.

Dissolving dehydrated tryptose-sulfite-cycloserine agar by only boiling or microwaving was found to inhibit Clostridium perfringens colony development in pour plates when compared with C. perfringens recovery in tryptose-sulfite-cycloserine agar prepared by autoclaving.     

Effect of microwave radiation on inactivation of Clostridium sporogenes (PA 3679) spores.

Three techniques for studying effects of microwave radiation on microorganisms were introduced. Spores of Clostridium sporogenes (PA 3679) were chosen as a test organism because the kinetic parameters for thermal inactivation are well known and because of the importance of the genus Clostridium to the food industry. For the first technique, a specially designed kinetics vessel was used to compare inactivation rates of microwave-heated and conventionally heated spores at steady-state temperatures of 90, 100, and 110 degrees C. Rates were found to be similar at the 95% confidence level. The second and third techniques were designed to study the effect of relatively high power microwave exposure at sublethal temperatures. In the second approach, the suspension was continuously cooled via direct contact with a copper cooling coil in a well-mixed vessel, outside the microwave oven. The suspension was pumped through a Teflon loop in the oven, where it continuously absorbed approximately 400 W of microwave power. Inactivation occurred in both irradiated and unirradiated samples. It was suspected that copper ions entered the suspension from the copper coil and were toxic to the spores. The fact that the results were similar, however, implied the absence of nonthermal microwave effects. In the third approach, the copper coil was replaced with a silicone tubing loop in a microwave transparent vessel. The suspension was continuously irradiated at 150 W of microwave power. No detectable inactivation occurred. Results indicated that the effect of microwave energy on viability of spores was indistinguishable from the effect of conventional heating.     

Recovery of spores of Clostridium botulinum in yeast extract agar and pork infusion agar after heat treatment.

Yeast extract agar, pork infusion agar, and modifications of these media were used to recover heated Clostridium botulinum spores. The D- and z-values were determined. Two type A strains and one type B strain of C. botulinum were studied. In all cases the D-values were largest when the spores were recovered in yeast extract agar, compared to the D-values for spores recovered in pork infusion agar. The z-values for strains 62A and A16037 were largest when the spores were recovered in pork infusion agar. The addition of sodium bicarbonate and sodium thioglycolate to pork infusion agar resulted in D-values for C. botulinum 62A spores similar to those for the same spores recovered in yeast extract agar. The results suggest that sodium bicarbonate and sodium thioglycolate should be added to recovery media for heated C. botulinum spores to obtain maximum plate counts.     

Recovery of spores of Clostridium botulinum in yeast extract agar and pork infusion agar after heat treatment.

Yeast extract agar, pork infusion agar, and modifications of these media were used to recover heated Clostridium botulinum spores. The D- and z-values were determined. Two type A strains and one type B strain of C. botulinum were studied. In all cases the D-values were largest when the spores were recovered in yeast extract agar, compared to the D-values for spores recovered in pork infusion agar. The z-values for strains 62A and A16037 were largest when the spores were recovered in pork infusion agar. The addition of sodium bicarbonate and sodium thioglycolate to pork infusion agar resulted in D-values for C. botulinum 62A spores similar to those for the same spores recovered in yeast extract agar. The results suggest that sodium bicarbonate and sodium thioglycolate should be added to recovery media for heated C. botulinum spores to obtain maximum plate counts.     

Effects of inoculum level and pressure pulse on the inactivation of Clostridium sporogenes spores by pressure-assisted thermal processing

The effects of initial concentration and pulsed pressurization on the inactivation of Clostridium sporogenes spores suspended in deionized water were determined during thermal processing (TP; 105 degrees C, 0.1 MPa) and pressure-assisted thermal processing (PATP; 105 degrees C and 700 MPa) treatments for 40 min and 5 min holding times, respectively. Different inoculum levels (10(4), 10(6), and 10(8) CFU/ml) of C. sporogenes spores suspended in deionized water were treated at 105 degrees C under 700 MPa with single, double, and triple pulses. Thermally treated samples served as control. No statistical significances (p > 0.05) were observed among all different inoculum levels during the thermal treatment, whereas the inactivation rates (k1 and k2) were decreased with increasing the initial concentrations of C. sporogenes spores during the PATP treatments. Double- and triple-pulsed pressurization reduced more effectively the number of C. sporogenes spores than single-pulse pressurization. The study shows that the spore clumps formed during the PATP may lead to an increase in pressure-thermal resistance, and multiple-pulsed pressurization can be more effective in inactivating bacterial spores. The results provide an interesting insight on the spore inactivation mechanisms with regard to inoculum level and pulsed pressurization.     

Application of the 'Inoculated Pack Test' method for the determination of the heat resistance of Clostridium sporogenes PA3679 spores in acidified mushrooms

The effect of pH in the range 5.2–6.7 on the thermal destruction of Clostridium sporogenes PA3679 spores suspended in mushrooms in brine acidified with citric acid was examined by the 'inoculated pack test' method. The results indicated that increasing acidity is accompanied by decreasing decimal reduction times at 121.1°C: D _121.1 at pH 6.0 and 5.2 was, respectively, 64% and 17.5% of that at pH 6.7, the pH of natural mushrooms ( D _121.1= 2.22 min). A linear model ( r = 0.988, α= 0.05) was developed where the D _121.1 value was a function of the pH over the range studied. The inoculated pack test seems to be the only method to evaluate the actual microbial heat resistance, whether of spore or of vegetative forms, in order to estimate within reasonably close limits a suitable process time required to eliminate health hazards and to prevent spoilage losses in a given food product.     

Thiaminase Activity Amongst Strains of Clostridium sporogenes

All the 28 strains of Clostridium sporogenes type I tested produced thiaminase. Only 2 of the 16 strains of Cl. sporogenes type II tested were positive for the enzyme; these gave a weak positive reaction. The single strain of Cl. sporogenes type III behaved in a manner similar to the strains of type I, giving a strong positive thiaminase reaction. Thiaminase production amongst the strains of Cl. sporogenes does in the main support the cultural, biochemical and immunological properties described earlier.     

Regulation of protease production in Clostridium sporogenes

The physiological and nutritional factors that regulate protease synthesis in Clostridium sporogenes C25 were studied in batch and continuous cultures. Formation of extracellular proteases occurred at the end of active growth and during the stationary phase in batch cultures. Protease production was inversely related to growth rate in glucose-excess and glucose-limited chemostats over the range D = 0.05 to 0.70 h-1. In pulse experiments, glucose, ammonia, phosphate, and some amino acids (tryptophan, proline, tyrosine, and isoleucine) strongly repressed protease synthesis. This repression was not relieved by addition of 4 mM cyclic AMP, cyclic GMP, or dibutyryl cyclic AMP. Protease formation was markedly inhibited by 4 mM ATP and ADP, but GTP and GDP had little effect on the process. It is concluded that protease production by C. sporogenes is strongly influenced by the amount of energy available to the cells, with the highest levels of protease synthesis occurring under energy-limiting conditions.     

Sheathed Cells in Cultures of Clostridium sporogenes

Many strains of Clostridium sporogenes were shown to contain two types of cells which exhibited strikingly different growth habits. Over 99% of the population of most strains were motile bacilli which occurred singly or in short chains. Infection by any of several C. sporogenes bacteriophages lysed most of these cells and revealed a minority population component consisting of cells which grew in extremely long chains. Each chain was surrounded by and contained in a long tubular polysaccharide sheath which was ultrastructurally quite separate and distinct from the cell walls of the enclosed cells. The sheathed cells were identical to "normal" cells of C. sporogenes in anaerobiosis, Gram reaction, sporulation, deoxyribonucleic base composition, general morphology, and ultrastructure. They differed from the "normal" cells in having a sheath, in being nonmotile, and in that they were infected by C. sporogenes bacteriophages but not usually lysed by them. The sheathed cells appeared spontaneously in cultures cloned from single colonies and were demonstrably present in cultures before bacteriophage infection. Thus, they were not contaminants but were normal, although inconspicuous, growth forms of C. sporogenes which were selected but not induced by bacteriophage infection.     

Draft genome sequence of Clostridium sporogenes PA 3679, the common nontoxigenic surrogate for proteolytic Clostridium botulinum

Clostridium sporogenes PA 3679 is widely used as a nontoxigenic surrogate for proteolytic strains of Clostridium botulinum in the derivation and validation of thermal processes in food. Here we report the draft assembly and annotation of the C. sporogenes PA 3679 genome. Preliminary analysis demonstrates a high degree of relatedness between C. sporogenes PA 3679 and sequenced strains of proteolytic C. botulinum.