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.     

Clostridium perfringens in the Environment

Clostridium perfringens was isolated from samples collected in Puget Sound in the state of Washington and areas considered as possible sources of these organisms to Puget Sound. The distribution of C. perfringens in the total Clostridium population was determined for fish gut contents and sediments collected in highly polluted and less polluted areas, sewage samples, freshwater sediments, and soils. The greatest numbers of C. perfringens were obtained from marine sediments collected near the sewage outfall at West Point. Fewer isolates were made from fish collected from less polluted stations, although the number of C. perfringens remained high in sediments from other Puget Sound stations. The proportion of C. perfringens in the total Clostridium populations varied between 56 and 71% for sewage samples and only 0.4 to 4.1% for freshwater sediments and soil samples. Only 25 C. perfringens isolates out of 137 from fish guts, or 18%, were identifiable serologically and these fell into 12 groups. C. perfringens were fed to fish and the fish were sacrificed after varying lengths of time. The number of C. perfringens increased slightly in the gut during the first 24 h and then the numbers decreased rapidly for the next 120 h.     

Stable L-forms of Clostridium perfringens and their growth on glass surfaces.

L-forms of Clostridium perfringens were induced in brain heart infusion broth containing 10% sucrose and 2 units of penicillin. After a few hours of growth, spheroplasts, granules, and elongated bacilli were apparent. At 24-h intervals, serial subcultures were made in the above medium which resulted in a culture composed entirely of spheroplasts (or protoplasts) and granules. Upon the withdrawal of penicillin these L-form cultures grew well and, after 100 passages, there was no reversion to the bacillary form. Sucrose could also be withdrawn from the medium. The effects of centrifugation, osmotic stabilizer, ultraviolet light, temperature, pH, and lyophilization upon stable L-forms were examined. L-forms were found to attach to the walls of culture tubes during trowth and sheets of L-form growth were obtained on cover slips in Leighton tubes and on the sides of medicine bottles.     

CDP-diacylglycerol synthase activity in Clostridium perfringens.

CTP:phosphatidate cytidylyltransferase (CDP-diacylglycerol synthase; EC 2.7.7.41) was identified in the cell envelope fraction of the gram-positive anaerobe Clostridium perfringens. The association of this enzyme with the cell envelope fraction of cell extracts was demonstrated by glycerol density gradient centrifugation and by activity sedimenting with the 100,000 x g pellet. The enzyme exhibited a broad pH optimum between pH 6.5 and pH 7.5. Enzyme activity was dependent on magnesium (5 mM) or manganese (1 mM) ions. Activity was also dependent on the addition of the nonionic detergent Triton X-100 (5 mM). The apparent Km values for CTP and phosphatidic acid were 0.18 mM and 0.22 mM, respectively. Thioreactive agents inhibited activity, indicating that a sulfhydryl group is essential for activity. Maximal enzyme activity was observed at 50 degrees C.     

Empyema caused by Clostridium perfringens.

Trauma, chest surgery or other invasive procedures and underlying lung disease are often found to precede clostridial empyema. A case is described in which empyema caused by Clostridium perfringens was not associated with any of these factors.     

A new growth and in vitro sporulation medium for Clostridium perfringens

Growth and in vitro sporulation capabilities of three related Clostridium perfringens strains (NCTC 8798, 8-6 and R3) were followed in a new sporulation medium (NSM), with notable changes from a maintenance medium originally designed for strictly anaerobic bacteria. Compared with thioglycollate (FTG) medium, the new sporulation medium promoted growth of Cl. perfringens with a shorter lag phase and a 20% higher biomass production. The age of inoculum did not change Cl. perfringens growth kinetics. When compared with reference conditions, in vitro spore production kinetics were different in the new sporulation medium, but both conditions led routinely to 100% sporulation and spore counts of approximately 10(8) ml-1. The ease of preparation of the NSM, and the use of the same culture medium for good growth, high sporulation yields and spore production, represent an attractive alternative to the complex media routinely used for in vitro studies of Cl. perfringens physiology.     

Characterization of the autolytic enzymes of Clostridium perfringens.

Clostridium perfringens and isolated walls of this organism autolysed rapidly when incubated in buffer at pH 7.0 with the release of free-reducing groups but no N-terminal amino acids. The predominant autolytic enzyme was an endo-beta-N-acetylglucosaminidase, and an endo-beta-N-acetylmuramidase was also present. The autolytic enzymes could be solubilized by extraction of the organisms with 5 M-LiCl and would then subsequently bind to and rapidly lyse walls of Micrococcus luteus and, more slowly, formamide-extracted walls of C. perfringens and walls of Bacillus subtilis. Lysis of C. perfringens walls by these extracted enzymes could not be demonstrated.     

Clostridium perfringens in retail chicken

Clostridium perfringens isolates were recovered by enrichment from retail grocery chicken samples (n = 88) in Ontario, Canada, with one sample per site. The gene associated with necrotic enteritis in chickens, netB, was found in 21% of the isolates. The tpeL gene was found in 2% and the cpb2 gene in 68% (95% “atypical” genes) of isolates. This study suggests that netB-positive C. perfringens can reach people through retail chicken.     

Lecithinase-negative variants of Clostridium perfringens; the identity of C. plagarum with C. perfringens.

Identity of Clostridium plagarum (Prévot) com. nov. (1938) with C. perfringens was demonstrated in the tests for the cultural and biochemical properties, DNA-DNA homology, susceptibility to C. perfringens-specific lysin, complementary synthesis of C. perfringens theta-toxin between C. plagarum and theta-toxin-negative mutants of C. perfringens, as well as the tests for gas-chromatographic patterns.     

Quantitation of Clostridium perfringens in Foods

A procedure is described for identifying and enumerating Clostridium perfringens in foods by means of a simplified agar plating method, followed by confirmation of black colonies in tubes of motility-nitrate medium and sporulation broth. The test is routinely completed within 48 hr. Under experimental conditions, the procedure has been used to quantitatively recover various levels of C. perfringens contamination in a variety of foods and has recovered as few as ten C. perfringens per g without interference from food constituents and associated flora. Under practical conditions of field application, the method has been used to investigate five food-poisoning outbreaks, and C. perfringens was implicated as the etiological agent in two of these outbreaks.     

Transformation of bile acids by Clostridium perfringens.

Thirty-five strains of Clostridium perfringens were examined for their ability to transform bile acids, both in growing cultures and by washed whole cells. All of the strains oxidized the 3 alpha-hydroxy group to an oxo group, and all except three converted the same alpha-hydroxy group into a beta-configuration. The oxidative 3 alpha-dehydrogenation was barely detectable under anaerobic cultural conditions but was clearly demonstrated in an aerated system using washed whole cells, with a pH optimum between 7.0 and 9.0. The epimerizing reaction amounting to 10 to 20% conversion was observed in anaerobic cultures and also with resting cells, irrespective of oxygen supply. Both reactions were carried out with seven conventional 3 alpha-hydroxy bile acids, thus producing a series of 3-oxo and 3 beta-hydroxy derivatives that could be examined for gas-liquid chromatographic and mass spectrometric behavior. No evidence for the occurrence of 7 alpha- and 12 alpha-hydroxysteroid dehydrogenase activities among the test strains was found. A highly potent deconjugating hydrolase was elaborated by all of the strains.     

Molecular genetics and pathogenesis of Clostridium perfringens.

Clostridium perfringens is the causative agent of a number of human diseases, such as gas gangrene and food poisoning, and many diseases of animals. Recently significant advances have been made in the development of C. perfringens genetics. Studies on bacteriocin plasmids and conjugative R plasmids have led to the cloning and analysis of many C. perfringens genes and the construction of shuttle plasmids. The relationship of antibiotic resistance genes to similar genes from other bacteria has been elucidated. A detailed physical map of the C. perfringens chromosome has been prepared, and numerous genes have been located on that map. Reproducible transformation methods for the introduction of plasmids into C. perfringens have been developed, and several genes coding for the production of extracellular toxins and enzymes have been cloned. Now that it is possible to freely move genetic information back and forth between C. perfringens and Escherichia coli, it will be possible to apply modern molecular methods to studies on the pathogenesis of C. perfringens infections.     

Sporulation-promoting ability of Clostridium perfringens culture fluids.

The culture supernatant fluids (CSFs) of 12 strains of Clostridium perfringens types A, B, C, and D stimulated sporulation of test strains NCTC 8238 and NCTC 8449 of this organism. The sporulation-promoting ability was present in vegetative and sporulating CSFs of both enterotoxin-positive (Ent+) and Ent- strains. The sporulation factor possessed a molecular weight between 1,000 and 5,000 and was heat and acid stable. This study suggests a potential role for Ent- strains in food-borne disease outbreaks caused by Ent+ strains of C. perfringens type A.     

Rapid extraction of plasmids from Clostridium perfringens.

Two rapid methods were evaluated for their extraction of plasmids from Clostridium perfringens. The first method involved lysis of 1 to 2 ml of C. perfringens culture by treatment with hyaluronidase, lysozyme, and sarcosyl. DNA, extracted with phenol-chloroform, was treated with RNase, boiled, and electrophoresed in a 1.2% agarose gel. The second method involved lysis of 2 ml of culture by lysozyme treatment and extraction with alkaline sodium dodecyl sulfate (SDS). Extracted DNA was treated with RNase, boiled, and electrophoresed in a 0.7% agarose gel. Of 57 strains of C. perfringens analyzed by both extraction procedures, 11 were shown to have plasmids by the alkaline SDS method which were missed by the phenol-chloroform extraction method. These new plasmids were of higher molecular mass and ranged up to 68 megadaltons. Use of the DNase inhibitor diethyl pyrocarbonate did not further improve the yield of plasmid DNA. An additional 159 isolates of C. perfringens screened by the alkaline SDS method revealed plasmids up to 80 megadaltons in mass and an overall plasmid carriage rate of 69%.