Effect of metal ions on growth and sporulation of Clostridium perfringens in a synthetic medium.

All cells, whatever their origin, function in an essentially inorganic environment. In this environment, metal cations play an important role. Attempts were made in the present study to determine if different amounts of five metal ions (MG++, Fe++, Mn++, Zn++, Ca++) are needed for secondary metabolism of Clostridium perfringens than are needed for primary metabolism. Both the vegetative growth stage (primary metabolic stage) and spore stage (secondary metabolic stage) of Clostridium perfringens were studied. Endeavors were made to detect the effects of metal ions, if any, on growth and sporulation of the organisms. Mg++ was required for growth of vegetative cells and maintenance of normal cellular morphology, Fe++ was needed for sporulation as well as for vegetative growth, but the amount needed for spore formation was higher than that needed for growth. Ca++ was essential to refractility and heat-resistance of spores. Zn++ inhibited both growth and sporulation, if the Mg++ concentration was low. Mn++ was required for neither growth nor sporulation. For maximum heat-resistance of the spores, Ca++ plus unidentified organic substances were required.     

Theta-toxin of Clostridium perfringens. I. Purification and some properties.

Theta-Toxin, an oxygen-labile hemolysin produced by Clostridium perfringens, was purified 3300 fold from culture filtrate by successive chromatography on DEAE-Sephadex A-50 and Sephadex G-150. The purified toxin gave two distinct bands in disc electrophoresis, while the same material, after mild reduction with dithiothreitol, yielded a single band, indicating that the purified theta-toxin contained, as well as a reduced, active form, an oxidized, inactive form of toxin. These two forms of the toxin had a similar, if not identical molecular size. The purified preparation gave a single band in a sodium dodecyl sulfate polyacrylamide gel electrophoresis and formed a single precipitin line with National Standard gas gangrene (C. perfringens) antitoxin. By sodium dodecyl sulfate polyacrylamide gel electrophoresis, the molecular weight of theta-toxin was estimated to be 51 000, the value being in exact accordance with that obtained by amino acid analysis. The amino acid composition of theta-toxin was very close to that of cereolysin, an oxygen-labile hemolysin produced by Bacillus cereus. The amino-terminal residue of theta-toxin was lysine as determined by the Dansyl method.     

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.     

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.     

The effects of Clostridium perfringens enterotoxin on morphology, viability, and macromolecular synthesis in Vero cells.

Vero (African green monkey kidney) cells grown in tissue culture monolayer were sensitive to Clostridium perfringens enterotoxin. Within 30 minutes of exposure to the enterotoxin gross morphological damage was observed and within 40 minutes approximately 75% of the cells had detached. Nearly half of the cells were nonviable following 35 to 40 minutes incubation with the enterotoxin. Doses as low as 0.1 ng caused small but detectable inhibition of plating efficiency of the cells while more than 100 ng caused the inhibition to approach 100%. Total inhibition of DNA, RNA, and protein synthesis occurred within 30 minutes exposure to enterotoxin. Heat inactivated enterotoxin had no apparent effects upon cellular morphology, detachment, viability, plating efficiency, or incorporation. We propose that the enterotoxin induces structural damage to the cytoplasmic membrane which results in loss of electrolytes and other essential substances from the cells. The outcome of this process is shut down of macromolecular synthesis, gross morphological damage, and eventual death of the cell.     

"Phoenix phenomenon" in the growth of Clostridium perfringens.

The "Phoenix phenomenon" was observed with Clostridium perfringens Hobbs' serological type 9 (HT9) in a cooked-meat medium at 81.7 degrees C by a decrease in plate count (phase I), followed by an increase in count to the intiial level (phase II) and a continued increase above the initial count (phase III). The effects of sporulation, age of inoculum, assay medium, anaerobiosis, diluent, and growth inhibitor were studied. This phenomenon was reproduced in experiments with sporulation-negative mutants derived from HT9 inocula of various cell ages, and different assay media (sulfite-iron agar, tryptose-soytone-yeast extract agar, prereduced peptone-yeast extract agar, prereduced veal agar, and veal agar). When strict anaerobic conditions were employed, it was necessary to increase the heating temperature to 52.3 degrees C to observe the phenomenon. The phenomenon was eliminated at 52.3 degrees C when a combination of strict anaerobic conditions, prereduced media, and prereduced veal diluent was employed. The addition of nalidixic acid at the minimum point of the growth curve (end of phase I) had no effect on the appearance of phase II; however, phase III was completely inhibited. This indicated that phases I and II were an injury-recovery process.     

产气荚膜梭菌促进黑腹果蝇的生长和发育

【目的】黑腹果蝇Drosophila melanogaster肠道中栖生着众多微生物,通过分离和研究其内共生菌,以研究肠道菌群的多态性和作用。【方法】利用Hungate滚管技术从黑腹果蝇成虫肠道分离厌氧细菌;通过记录果蝇的发育历期和生长速率,检测该细菌对果蝇发育和生长的影响。【结果】首次从黑腹果蝇肠道内分离到一株产气荚膜梭菌Clostridium perfringens。该菌能够有效地定植到果蝇肠道内,是果蝇肠道共生菌。产气荚膜梭菌显著地缩短无菌果蝇的发育历期,将无菌果蝇成蛹天数由20 d缩短到8.1 d,羽化天数由30 d缩短到12.7 d。该菌还可以提高果蝇生长速率。【结论】本研究揭示了产气荚膜梭菌是果蝇的内共生菌,可以通过提高生长速率而有效地促进果蝇的生长和发育。     

Changes in lipid metabolism and cell morphology following attack by phospholipase C (Clostridium perfringens) on red cells or lymphocytes.

When intact human erythrocytes were treated with phospholipase C (Clostridium perfringens), up to 30% of the membrane phospholipids were broken down without significant cell lysis. Only phosphatidylcholine and sphingomyelin were attacked. Ceramide (derived from sphingomyelin) accumulated, but 1,2-diacylglycerol (derived from phosphatidylcholine) was largely converted into phosphatidate. Up to 12% of the cell phospholipid could be converted into phosphatidate in this way. Pig erythrocytes and lymphocytes showed a similar but smaller synthesis of phosphatidate after phospholipase C attack. Phospholipase C also caused a marked morphological change in erythrocytes, giving rise to spherical cells containing internal membrane vesicles. This change appeared to be due to ceramide and de and diacylglycerol accumulation rather than to increased phosphatidate content of the cells.     

Complex transcriptional regulation of citrate metabolism in Clostridium perfringens

A Gram-positive, spore-forming bacterium, Clostridium perfringens, possesses genes for citrate metabolism, which might play an important role in the utilization of citrate as a sole carbon source. In this study, we identified a chromosomal citCDEFX-mae-citS operon in C. perfringens strain 13, which is transcribed on three mRNAs of different sizes. Expression of the cit operon was significantly induced when 5 mM extracellular citrate was added to the growth medium. Most interestingly, three regulatory systems were found to be involved in the regulation of the expression of cit genes: 1) the two upstream divergent genes citG and citI; 2) two different two-component regulatory systems, CitA/CitB (TCS6 consisted of CPE0531/CPE0532) and TCS5 (CPE0518/CPE0519); and 3) the global two-component VirR/VirS-VR-RNA regulatory system known to regulate various genes for toxins and degradative enzymes. Our results suggest that in C. perfringens the citrate metabolism might be strictly controlled by a complex regulatory system.     

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.     

Effect of Clostridium perfringens epsilon toxin on MDCK cells

Epsilon toxin is one of the major lethal toxins produced by Clostridium perfringens type D and B. It is responsible for a rapidly fatal disease in sheep and other farm animals. Many facts have been published about the physical properties and the biological activities of the toxin, but the molecular mechanism of the action inside the cells remains unclear. We have found that the C. perfringens epsilon toxin caused a significant decrease of the cell numbers and a significant enlargement of the mean cell volume of MDCK cells. The flow cytometric analysis of DNA content revealed the elongation of the S phase and to a smaller extent of the G2+M phase of toxin-treated MDCK cells in comparison to untreated MDCK cells. The results of ultrastructural studies showed that the mitosis is disturbed and blocked at a very early stage, and confirmed the toxin influence on the cell cycle of MDCK cells.