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.     

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.     

Sporicidal Action of Auto-oxidized Ascorbic Acid for Clostridium

Neutralized ascorbic acid (AA), buffered or unbuffered and autoclaved or filter-sterilized, was sporicidal for Clostridium. A 0.2% concentration of AA was generally employed, and spore counts were made in a soft-agar modification of Wynne's medium in Prickett tubes. Spores of Clostridium botulinum 115B were less susceptible than those of C. sporogenes PA 3679, whereas C. bifermentans spores were by far the most sensitive. At 75 C, spores of PA 3679 were killed at a rate of about 9% at 0 min (warm-up) to 99+% at 100 min. The lower the temperature, the longer the time needed for a given lethality. The percentage of killing increased with increasing concentrations of AA, and the rate of killing was lower at a higher concentration of spores. At least two mechanisms were operative: a major mechanism involving a product(s) of AA auto-oxidation, and a minor mechanism involving copper-ascorbate toxicity. AA reduced in natural gas was not sporicidal after 18.5 hr at 25 C, whereas 92% of the spores were killed by oxidized AA. Although H(2)O(2) per se was sporicidal, catalase did not reverse lethality of fresh or oxidized AA. Dehydroascorbate was as sporicidal as any AA preparation. Added copper (0.00001%) increased the rate of lethality of freshly prepared AA from 66 to 83% but was not effective with thoroughly oxidized AA. Ethylenediaminetetraacetic acid, NH(4) (+), and phosphate partially reversed AA toxicity, deionized water had no effect, and complex media, as well as thioglycolate, eliminated AA lethality. Since the percentage of killing was affected by spore concentration, AA did not seem to stimulate "lethal germination."     

Development of a Selective Medium for the Isolation of Clostridium sporogenes and Related Organisms

An attempt was made to develop a selective isolation medium for Clostridium sporogenes and related organisms based on the ability of these organisms to obtain their energy for growth by means of coupled oxidation-reduction reactions between appropriate pairs of amino acids (Stickland reaction). Using a semi-defined basal medium containing various combinations of amino acids, it was found that Cl. sporogenes utilized a wider range of amino acid pairs than strains of five other species of clostridia known to carry out a Stickland-type fermentation.
With alanine and proline as the principal energy sources and the medium solidified with agar. it was shown that reference strains of Cl. sporogenes and proteolytic Cl. botulinum types A, B and F could be recovered almost quantitatively, with or without prior heating at 80 °C for 10 min. By contrast, growth of test strains of Streptococcus faecalis, Strep. faecium , 'saccharolytic' Cl. botulinum types B, C, D, E and F and 'proteolytic' strains of types C and D was suppressed on this medium, as were strains of 26 other species of clostridia.
Addition of 50 μg/ml of polymyxin to the agar medium had no detectable effect on the recovery of Cl. sporogenes or Cl. botulinum. When samples of soil and mud were plated on the antibiotic-containing medium, 63.1% of 225 isolates thus obtained were identified as Cl. sporogenes/botulinum.     

New media for detection and counting of clostridia in foods

The growth of pure cultures of Clostridium perfringens (ATCC 12922) and Cl. sporogenes (PA 3679) in five non-selective media, fluid thioglycollate medium (FTM), rapid perfringens medium (RPM), Columbia broth Malthus (CBM), reinforced clostridial medium (RCM) and lactose sulphite (LS), was monitored using conductance measurements with a Malthus analyser. Only FTM and CBM gave useful results. The correlation of log₁₀ plate counts on blood agar of the pure strain of Cl. sporogenes with detection times in FTM was highly significant ( r = 0·96, n = 73), and with detection times in CBM less so ( r = −0·909, n = 33). The correlation of log₁₀ counts on tryptose sulphite neomycin medium (TSN) of wild strains of Cl. sporogenes and Cl. perfringens with detection times with FTM in meat was also highly significant ( r = 0·933, n = 54).     

Effect of thermal treatments in oils on bacterial spore survival

The heat resistance of Bacillus cereus F4165/75, Clostridium sporogenes PA 3679 and Cl. botulinum 62A spores suspended in buffer (pH 7.2), olive oil and a commercial oil (a mixture of rapeseed oil and soy oil) was investigated. Linear survivor curves were obtained with B. cereus spores in the three menstrua and with 62A and PA 3679 spores suspended in buffer. However, the inactivation kinetics of the clostridial spores suspended in oils were concave upward with a characteristic tailing-off for 62A spores suspended in olive oil. These deviations from the semi-log model could not be ascribed to a heterogeneity in heat resistance of the spore population or to the variation of aw during heating. Spore resistance to heat increased in the order: buffer much less than commercial oil less than olive oil. The greater heat resistance of oil-suspended spores was ascribed to the low aw (0.479 and 0.492 for commercial oil and olive oil, respectively) and to the composition of the oils. The difference in z values (ca 28 degrees C in oils and 10 degrees-12 degrees C in buffer) suggested that the mechanism of inactivation differs for spores suspended in lipids and in aqueous systems. The thermodynamic data were consistent with this hypothesis.     

Nisin dissipates the proton motive force of the obligate anaerobe Clostridium sporogenes PA 3679.

The influence of nisin on the proton motive force (delta p) generated by glucose-energized cells of the obligate putrefactive anaerobe Clostridium sporogenes PA 3679 was determined. The components of delta p, the transmembrane potential (delta psi) and the pH gradient (delta pH), were determined from the distributions of the lipophilic cation [3H]TPP+ ([3H]tetraphenylphosphonium bromide) and [14C]salicylic acid, respectively. The cells maintained a constant delta p of -111 mV, consisting of a delta pH of 0.4 to 1.0 pH units at an external pH of 5 to 7 and a delta psi of -60 to -88 mV. Nisin, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and N,N'-dicyclohexylcarbodiimide (DCCD) at pH 6.0 elicited the complete release of preaccumulated [3H]tetraphenylphosphonium bromide and [14C]salicylic acid, with a concomitant depletion of delta psi and delta pH. Nisin and DCCD caused rapid drops in intracellular ATP levels from 1.2 to 0.01 and 0.06 nmol/mg of cells (dry weight), respectively. Cells exposed to nisin and DCCD lost the ability to form colonies, thus suggesting that delta psi and delta pH are necessary for cell viability. The data suggest that depletion of delta p and exhaustion of cellular ATP reserves are the basis for nisin inhibition of C. sporogenes PA 3679.     

Effect of thermal treatments in oils on bacterial spore survival

The heat resistance of Bacillus cereus F4165/75, Clostridium sporogenes PA 3679 and Cl. botulinum 62A spores suspended in buffer (pH 7˙2), olive oil and a commercial oil (a mixture of rapeseed oil and soy oil) was investigated. Linear survivor curves were obtained with B. cereus spores in the three menstrua and with 62A and PA 3679 spores suspended in buffer. However, the inactivation kinetics of the clostridial spores suspended in oils were concave upward with a characteristic tailing-off for 62A spores suspended in olive oil. These deviations from the semi-log model could not be ascribed to a heterogeneity in heat resistance of the spore population or to the variation of a_w during heating. Spore resistance to heat increased in the order: buffer ⋖ commercial oil < olive oil. The greater heat resistance of oil-suspended spores was ascribed to the low a_w (0˙479 and 0˙492 for commercial oil and olive oil, respectively) and to the composition of the oils. The difference in z values ( ca 28°C in oils and 10°-12°C in buffer) suggested that the mechanism of inactivation differs for spores suspended in lipids and in aqueous systems. The thermodynamic data were consistent with this hypothesis.     

Nisin dissipates the proton motive force of the obligate anaerobe Clostridium sporogenes PA 3679.

The influence of nisin on the proton motive force (delta p) generated by glucose-energized cells of the obligate putrefactive anaerobe Clostridium sporogenes PA 3679 was determined. The components of delta p, the transmembrane potential (delta psi) and the pH gradient (delta pH), were determined from the distributions of the lipophilic cation [3H]TPP+ ([3H]tetraphenylphosphonium bromide) and [14C]salicylic acid, respectively. The cells maintained a constant delta p of -111 mV, consisting of a delta pH of 0.4 to 1.0 pH units at an external pH of 5 to 7 and a delta psi of -60 to -88 mV. Nisin, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and N,N'-dicyclohexylcarbodiimide (DCCD) at pH 6.0 elicited the complete release of preaccumulated [3H]tetraphenylphosphonium bromide and [14C]salicylic acid, with a concomitant depletion of delta psi and delta pH. Nisin and DCCD caused rapid drops in intracellular ATP levels from 1.2 to 0.01 and 0.06 nmol/mg of cells (dry weight), respectively. Cells exposed to nisin and DCCD lost the ability to form colonies, thus suggesting that delta psi and delta pH are necessary for cell viability. The data suggest that depletion of delta p and exhaustion of cellular ATP reserves are the basis for nisin inhibition of C. sporogenes PA 3679.     

Factors affecting growth of Staphylococcus aureus L forms on semidefined medium

Banville, Robert R. (The Catholic University of America, Washington, D.C.). Factors affecting growth of Staphylococcus aureus L forms on semidefined medium. J. Bacteriol. 87:1192-1197. 1964.-A semidefined agar medium was found suitable for production and cultivation of the L form of Staphylococcus aureus. In semidefined liquid medium, growth of the L form took place in the form of a sediment containing large masses of cells, but heavy and diffuse growth occurred in the same medium with 0.05% agar. The optimal pH for L-colony formation on solid medium was 6.5. More L colonies developed on 0.75% agar than at higher agar concentrations. L colonies developed in greater numbers on pour plates than on streak plates, and in some cases more L colonies appeared under anaerobic incubation. L-colony formation appeared to be inhibited by sodium citrate. The vitamin requirements of the L forms studied were similar to those of the classical form.     

Characterization of a halo-acid-tolerant variant of Clostridium botulinum B-aphis.

Clostridium botulinum B-aphis spores plated on medium containing 4% salt at pH 6.0 yielded colonies at a frequency of ca. 1 in 10(6). A subculture of one of these colonies, designated strain Ba410, was compared with the parent strain, B-aphis, for a variety of traits. After 7 days of incubation at 37 degrees C, strain Ba410 grew in medium containing 7% NaCl, whereas strain B-aphis could not grow in salt concentrations greater than 5%. The strains also differed in cellular and colonial morphology. After exponential growth in the basal medium was completed, lysis of both strains was pH dependent; in media containing salt, lysis of Ba410 cells was pH independent. Strain Ba410 was more proteolytic than strain B-aphis in conditions of low pH and high salt, so that its toxin could be detected by the mouse assay. In a medium containing alanine and cysteine, the germination rate of B-aphis was 0.77% min-1, whereas that of Ba410 was 0.14% min-1; 2% salt inhibited the germination of Ba410 but not B-aphis.     

Characterization of a halo-acid-tolerant variant of Clostridium botulinum B-aphis

Clostridium botulinum B-aphis spores plated on medium containing 4% salt at pH 6.0 yielded colonies at a frequency of ca. 1 in 10(6). A subculture of one of these colonies, designated strain Ba410, was compared with the parent strain, B-aphis, for a variety of traits. After 7 days of incubation at 37 degrees C, strain Ba410 grew in medium containing 7% NaCl, whereas strain B-aphis could not grow in salt concentrations greater than 5%. The strains also differed in cellular and colonial morphology. After exponential growth in the basal medium was completed, lysis of both strains was pH dependent; in media containing salt, lysis of Ba410 cells was pH independent. Strain Ba410 was more proteolytic than strain B-aphis in conditions of low pH and high salt, so that its toxin could be detected by the mouse assay. In a medium containing alanine and cysteine, the germination rate of B-aphis was 0.77% min-1, whereas that of Ba410 was 0.14% min-1; 2% salt inhibited the germination of Ba410 but not B-aphis.     

Nitrite-induced germination of putefactive anaerobe 3679h spores

Sodium nitrite alone has been shown to stimulate germination of PA 3679h spores. The process was accelerated by using increased concentrations of sodium nitrite, a low pH, and a high temperature of incubation. At low concentrations of nitrite (0.01 to 0.2%), the delay of 36 to 48 hr occurred before germination commenced at 37 C. However, with 3.45% nitrite at 45 C and pH 6.0, most of the spores germinated within 1 hr. At pH 7.0, the germination rate decreased markedly, and at pH 8.0 it was nil. The greatest acceleration in germination rate occurred near 60 C. Hydroxylamine was completely inhibitory to nitrite-induced germination. Sodium nitrite, in turn, inhibited germination by l-alanine, the degree of inhibition being influenced by nitrite concentration and pH.     

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.     

MINIATURIZED ANAEROBIC CULTIVATION METHODS FOR RECOVERY OF CLOSTRIDIUM SPOROGENES FROM MEAT

Two new miniaturized methods (sandwiched microtiter plate [SMP] and mini-tube) for recovery of Clostridium sporogenes from food samples were developed and evaluated. One hundred microliter of SFP (Shahidi Ferguson Perfringens) agar and 10 üL of diluted food sample were used in the SMP anaerobic system. The samples were sandwiched between two sterile microtiter plates to create an anaerobic environment. Black colonies in the sandwiched wells were counted as C. sporogenes. In case of mini-tube system, 1 mL of SFP agar (45C) containing diluted food sample was aspirated into a 1.2 mL sterile pipet and allowed to solidify in place. The two ends are sealed to create an anaerobic environment. Black colonies were counted directly through the plastic tube. C. sporogenes was inoculated into ground beef samples for recovery study. The recovery rates in SFP agar, using the SMP and mini-tube method were compared with the double-tube method. Organisms were recovered more in the double-tube than with SMP method and mini-tube method after 24–30 h incubation. However, the final counts at 48 h were similar in all methods from the food samples. These new simple methods have potential use for recovery of Clostridium spp.     

Selective and differential medium for detecting Clostridium botulinum.

A selective and differential growth medium was developed for detection of Clostridium botulinum types A, B, and F. The medium consisted of peptone-glucose-yeast extract agar supplemented with cycloserine, 250 micrograms/ml; sulfamethoxazole, 76 micrograms/ml; and trimethoprim, 4 micrograms/ml as selective inhibitors and various types and levels of botulinal antibodies for type differentiation in the immunodiffusion reaction. Growth of proteolytic types of C. botulinum were not affected by the incorporation of the selective agents, but some nonproteolytic types were suppressed. Cross-reactions between types A and B were visually distinguishable, whereas cross-reactions between type F and Clostridium sporogenes did not occur at the optimum antibody titer. Optimum antibody titer varied with toxin type. The proposed selective differential medium should be valuable in isolating and typing of proteolytic C. botulinum types A, B, and F from samples containing mixed microbial populations.     

Selective and differential medium for detecting Clostridium botulinum

A selective and differential growth medium was developed for detection of Clostridium botulinum types A, B, and F. The medium consisted of peptone-glucose-yeast extract agar supplemented with cycloserine, 250 micrograms/ml; sulfamethoxazole, 76 micrograms/ml; and trimethoprim, 4 micrograms/ml as selective inhibitors and various types and levels of botulinal antibodies for type differentiation in the immunodiffusion reaction. Growth of proteolytic types of C. botulinum were not affected by the incorporation of the selective agents, but some nonproteolytic types were suppressed. Cross-reactions between types A and B were visually distinguishable, whereas cross-reactions between type F and Clostridium sporogenes did not occur at the optimum antibody titer. Optimum antibody titer varied with toxin type. The proposed selective differential medium should be valuable in isolating and typing of proteolytic C. botulinum types A, B, and F from samples containing mixed microbial populations.     

The Effect of Carbon Dioxide on Spore Germination in Some Clostridia

S ummary . The rate and extent of spore germination in 3 cultures of clostridia have been investigated under controlled gas environment conditions. Germination in each of them was markedly stimulated by carbon dioxide in a variety of media. Quantitative measurements on 2 of the cultures indicated that while Clostridium bifermentans gave rapid and complete germination with as little as 3 mM bicarbonate, Cl. sporogenes PA 3679/S₂ required higher concentrations for maximal response.     

Nitrite-induced Germination of Putrefactive Anaerobe 3679h Spores

Sodium nitrite alone has been shown to stimulate germination of PA 3679h spores. The process was accelerated by using increased concentrations of sodium nitrite, a low pH, and a high temperature of incubation. At low concentrations of nitrite (0.01 to 0.2%), the delay of 36 to 48 hr occurred before germination commenced at 37 C. However, with 3.45% nitrite at 45 C and pH 6.0, most of the spores germinated within 1 hr. At pH 7.0, the germination rate decreased markedly, and at pH 8.0 it was nil. The greatest acceleration in germination rate occurred near 60 C. Hydroxylamine was completely inhibitory to nitrite-induced germination. Sodium nitrite, in turn, inhibited germination by l-alanine, the degree of inhibition being influenced by nitrite concentration and pH.