HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Fernandez-Miyakawa, M. E. Articles by Uzal, F. A. Search for Related Content PubMed PubMed Citation Articles by Fernandez-Miyakawa, M. E. Articles by Uzal, F. A.

Clostridium perfringens type A toxin production in 3 commonly used culture media

Correspondence: ¹Corresponding Author: Francisco A Uzal, California Animal Health and Food Safety Laboratory, 105 West Central Avenue, San Bernardino, CA 92408

	Abstract

TOP Sources and manufacturers Abstract References

 
In vitro toxin production is an important tool not only for diagnostic purposes but also for the study of pathogenesis of Clostridium perfringens infections. The present study was carried out to compare the level of toxin production by several strains of C. perfringens type A, isolated from the intestine of animals, when cultured in 3 different conventional culture media. Six strains of C. perfringens type A isolated from the small intestine of healthy sheep were cultured in commercial cooked meat medium (CMM), brain heart infusion (BHI), and tryptone glucose yeast (TGY). Intravenous lethality in mice and phospholipase C (PLC) activity were measured in filtered culture supernatants. Lethality of culture supernatants was highest for all isolates when grown in BHI, followed by CMM. No supernatants from any isolates grown in TGY produced lethality in mice. Phospholipase C activity was highest when the isolates were grown in BHI and CMM and significantly lower when grown in TGY.

Key Words: Clostridium perfringens • culture medium • intestine • lethality • phospholipase C

Clostridium perfringens is a Gram positive, spore-forming, anaerobic rod that is classified into 5 types (A, B, C, D, and E) according to the production of 4 major toxins (, ß, , ).4 This microorganism is widely distributed in the environment and is responsible for several diseases of animals and humans.1,8 Although several reports describe the isolation and toxinotyping of C. perfringens from the intestine of healthy animals,1,5,10 information about the lethal potential of C. perfringens strains isolated from the intestine of nondiseased animals is scant. In vitro toxin production evaluation is an important tool not only for diagnostic purposes but also for the study of pathogenesis of C. perfringens infections. The present study was carried out to define which culture media could optimize toxin production by C. perfringens type A obtained from clinically healthy sheep.

Six strains of C. perfringens type A (Table 1) isolated from the small intestine of healthy sheep at slaughter were used. The intestinal contents were cultured on 5% sheep blood agar^a at 37°C in anaerobiosis for 24 hours. Colonies compatible with C. perfringens were Gram stained and then processed by a multiplex polymerase chain reaction (PCR)Author. Text, paragraph 2. PCR has been defined as "polymerase chain reaction." Please verify accuracy. technique to amplify the genes that encode the 4 major toxins of C. perfringens plus the genes that encode beta 2 toxin and enterotoxin as previously described.6 The 6 isolates were then freeze-dried and stored for 6 years until they were reconstituted in cooked meat medium (CMM)^b before being cultured in 10 ml of commercial CMM, brain heart infusion (BHI),^b or tryptone glucose yeast (TGY)^b anaerobically at 37°C to late log phase. Monitoring and determination of the growth phase was achieved by measuring the optical density at 600 nm (OD_600 nm) Author, text, paragraph 2, sentence beginning "Monitoring." Table 1 has the OD at 620 nm, but here the OD is at 600 nm. Should they be the same?of the cultures at hourly intervals. Bacteria were then removed from the cultures by centrifugation at 3,000 x g for 30 minutes at 4°C, and supernatants were filter sterilized through 0.45-µm filters. The toxic activity of the filtered culture supernatants was evaluated by several assays as follows.

1. Surviving time was recorded in groups of 3 mice inoculated intravenously with 0.5 ml of a 1/2 dilution of culture supernatant in 1% peptone water. The survival time until assay endpoint (see definition of assay endpoint below) was recorded for each mouse, and the average survival time of each group was calculated.
   
2. Lethal dose of 50 (LD₅₀)/ml was determined as previously described.2 In brief, pairs of mice received IV injections containing 2-fold dilutions (between 1/2 and 1/80) of filtered culture supernatants in 1% peptone water. LD₅₀ was calculated as the reciprocal doses that produced a transition from total survival to death in mice within 48 hours.
   
3. To prove that lethality was due to alpha toxin, 0.6-ml volume of undiluted type A culture supernatants were mixed with 0.1 ml of a solution containing 2 mg/ml of an alpha toxin-neutralizing monoclonal antibody (MAb),^c brought to 1.2 ml with 1% peptone water and incubated for 30 minutes at room temperature. A 0.5-ml aliquot of each mixture was injected intravenously into 2 mice, while an additional pair of mice received a similar IV injection of the same supernatant that had been identically prepared, except for the omission of alpha toxin MAb. The mice were observed until assay endpoint (see definition of assay endpoint below).
   

For all experiments involving mice, assay endpoint was defined as severe clinical signs necessitating euthanasia, spontaneous death, or survival without clinical alterations for 48 hours when the animals were euthanized. Euthanasia was performed with CO₂.Author. Text, list, paragraph following section 3). Should this paragraph, defining assay endpoint, be moved to follow section 4) so that the numbered sections are not interrupted?

1. To determine phospholipase C (PLC) activity, an egg yolk solution (10% vol/vol) was prepared by diluting freshly obtained egg yolk in phosphate buffered solution (PBS) pH 7.4 and by filtering the solution through 0.45-µm filters. Samples of filtered supernatants were diluted 2-fold in 100 µl of PBS; and after the addition of 10% (vol/vol) egg yolk solution, they were incubated at 37°C for 3 hours. After incubation, samples were diluted 1/100 in PBS and optical density at 620 nm was measured in a Spectronic D20 spectrophotometerAuthor. Text, list, section 4). Should the company information for "Spectronic D20 spectrophotometer" be added to the Sources of materials section?. A positive control consisting of semipurified C. perfringens alpha toxin^d at 3 different concentrations (2, 20, and 200 LD₅₀, respectively) and a negative control consisting of sterile, nontoxic media culture were included in the assay.
   

Table 1 shows the results of characterization of the 6 C. perfringens isolates grown in the 3 culture media. The levels of PLC in culture supernatants were inversely correlated to the survival times in mice (r = 0.8, P < 0.05). Survival times were shortest in BHI culture supernatants, followed by CMM, while no lethality was observed in TGY. Since BHI produced the shortest survival times, LD₅₀/ml was measured by growing these isolates in BHI culture medium only. Levels up to 40 LD₅₀/ml were detected in BHI culture supernatants (Table 1). The lethality of all culture supernatants was neutralized when the supernatants were previously incubated with anti-alpha toxin MAb, indicating that alpha toxin was responsible for the lethality observed in mice. Phospholipase C levels were similar in BHI and CMM culture supernatants, both of which were higher than in TGY. Culture supernatants were collected at late log phase because previous reports showed that for most C. perfringens toxins the highest level of production occurs at this stage.2,6

The analysis of toxicity of C. perfringens supernatants indicates that the culture medium is an important factor in determining the toxigenicity of this microorganism. Tryptone glucose yeast and CMM are recommended in different laboratory manuals for toxinotyping of C. perfringens.3,7,9 However, under the conditions used in this study neither TGY nor CMM were the ideal culture media for lethal toxin production by C. perfringens type A. Although it seems that BHI and CMM are the best media for PLC activity expression, BHI produced higher lethal activity than CMM. The results show that although CMM is a suitable medium, under our conditions BHI was the best of the tested media for the assessment of toxicity of most C. perfringens type A strains isolated from the intestine of healthy sheep.

The pathogenesis of C. perfringens infections is mostly related to powerful toxins, which are produced in variable levels by different strains. However, detailed information about the toxigenic potential of C. perfringens isolated Author. Sources and manufacturers section. Letter "a," "Hardy" has been expanded to "Hardy Diagnostics." Please verify accuracy. Also, items "c and d" have been deleted because they indicate the same manufacturer. In text, the citations have been changed to letter "b".from healthy animals is scant. It is possible that earlier attempts to evaluate C. perfringens toxicity established the absence or low toxic activity of many isolates based on the use of culture media that were not ideal for toxin production.Author. Acknowledgements section. Please provide an affiliation for Dr. McClane.

	Acknowledgments

 
This research was supported by Fondo Nacional para la Ciencia y la Tecnología (PICT-01-3591), Argentina, and Public Health Service Grant AI56177 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD. We thank Dr. Bruce McClane for his suggestions.

	Sources and manufacturers

TOP Sources and manufacturers Abstract References

 
From the California Animal Health and Food Safety Laboratory, San Bernardino Branch, Faculty of Veterinary Medicine, University of California Davis, San Bernardino, CA 92408 (Fernandez-Miyakawa, Uzal), and the National Institute of Agricultural Technology, CC 277, (8400) Bariloche, Argentina (Marcellino).

^a. Hardy Diagnostics, Santa Maria, CA.

^b. Anaerobe Systems, Morgan Hill, CA.

^c. Dr. P. Hauer, Center for Veterinary Biologics, Ames, IA.

^d. CSL Ltd., Melbourne, Victoria, Australia.

	References

TOP Sources and manufacturers Abstract References

 

1. Bullen J.J.: 1952, Enterotoxaemia of sheep: Clostridium Welchii type D in the alimentary tract of normal animals. J Path Bac 62:201–206.
2. Fisher D.J., Fernandez-Miyakawa M.E., Sayeed S., et al.: 2006, Dissecting the contributions of Clostridium perfringens type C toxins to lethality in the mouse intravenous injection model. Infect Immun 74:5200–5210.[Abstract/Free Full Text]
3. Levett P.N.: 1991, Anaerobic microbiology: a practical approach. pp. 169–171. Oxford University Press, New York, NY.
4. McClane B.A., Uzal F.A., Fernandez-Miyakawa M.E., et al.: 2004, The enterotoxic clostridia. In: The prokaryotes: an evolving electronic resource for the microbiological community, 3rd ed. pp. 1–52. Springer-Verlag, New York, NY.
5. Niilo L.: 1980, Clostridium perfringens in animal disease: a review of current knowledge. Can Vet J 21:141–148.[Medline]
6. Sayeed S., Fernandez-Miyakawa M.E., Fisher D.J., et al.: 2005, Epsilon-toxin is required for most Clostridium perfringens type D vegetative culture supernatants to cause lethality in the mouse intravenous injection model. Infect Immun 73:7413–7421.[Abstract/Free Full Text]
7. Sebald M., Petit J.C.: 1997, Laboratory methods for anaerobic bacteria and their identification, 2nd ed. Institute Pasteur, Paris, France.
8. Songer J.G.: 1996, Clostridial enteric diseases of domestic animals. Clin Microbiol Rev 69:216–234.
9. Sterne M., Batty I.: 1975, Pathogenic Clostridia. Butterworths, London, UK.
10. Tschirdewahn B., Notermans S., Wernars K., Untermann F.: 1991, The presence of enterotoxigenic Clostridium perfringens strains in faeces of various animals. Int J Food Microbiol 14:175–178.[Medline]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Fernandez-Miyakawa, M. E. Articles by Uzal, F. A. Search for Related Content PubMed PubMed Citation Articles by Fernandez-Miyakawa, M. E. Articles by Uzal, F. A.