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Journal of Clinical Microbiology, April 2005, p. 2024-2025, Vol. 43, No. 4
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.4.2024-2025.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Clostridium tetani Is a Phospholipase (Lecithinase)-Producing Bacterium


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LETTER
 
Many Clostridium spp. produce phospholipases (lecithinases). The phospholipase of Clostridium perfringens is known as alpha-toxin (Cpa), which is the best characterized of all clostridial phospholipases. C. tetani, the causative organism of tetanus, has been recognized as a phospholipase-negative clostridium. In the course of our research on clostridial phospholipases (2, 3), a BLAST homology search revealed that the C. tetani E88 genome (1) contained a cpa-related gene (gene identifier, 1058703; locus tag, CTC00990. Cpa consists of two domains, the N-terminal and C-terminal domains (2). The N-terminal domain has phospholipase activity, while the C-terminal domain is a calcium-dependent putative phospholipid binding domain. According to the E88 genome sequence, the phospholipase gene of C. tetani E88 comprising 242 amino acid residues has similarity with the N-terminal domain of Cpa and does not have a domain corresponding to the C-terminal domain. The presence of this gene prompted us to examine phospholipase production by C. tetani.

All tested strains—NCTC 279 (the type strain of C. tetani), NCTC 5404, CN 655 (Pasteur Institute strain 105554), Ramon (Pasteur Institute strain 60.28), KZ 1180, KZ 1186, KZ 1189, and KZ 1199—had been identified as C. tetani. The strains KZ 1180, KZ 1186, KZ 1189, and KZ 1199 were isolated from soil. In this study we confirmed these strains as C. tetani by using a 16S rRNA gene bacterial sequencing kit (Microseq 500; Applied Biosystems). C. perfringens KZ 221 (3) and C. butyricum NCTC 7423 were used for phospholipase-positive and -negative controls. An anaerobic culture using mixed gases (85% N2, 10% CO2, and 5% H2) was carried out at 37°C for 2 days.

We found that C. tetani NCTC 5404 expressed a positive phospholipase reaction, characterized by opacity around a colony, on a medium, the basal formula of which had been used for spore formation of C. tetani in our laboratory. We therefore examined medium conditions to enhance phospholipase production. The alterations were based on the knowledge that C. tetani possesses an extensive sodium ion bioenergetics and a sodium-glucose or galactose cotransporter (gene identifier, 1059145; locus tag, CTC01237 (1) and that several C. tetani strains are also able to utilize glucose (4, 5). Finally, we developed a peptone-rich yeast extract glucose (PRYG) agar medium containing 5% (vol/vol) egg yolk. PRYG agar medium consisted of the following per liter of water: 40 g of Bacto proteose peptone 2 (Difco Laboratories), 10 g of yeast extract (Difco Laboratories), 1 g of glucose, and 20 g of agar (pH 7.4). In brief, PRYG agar medium contained a high concentration of peptone and a small amount of glucose but no sodium chloride. All strains clearly showed the phospholipase reaction on PRYG agar medium containing 5% egg yolk (Fig. 1). It was noted that not all colonies produced phospholipase. Research on the regulation system of the gene expression is awaited. Additionally, we tested a commercial brain heart infusion agar (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) for NCTC 279, NCTC 5404, KZ 1180, KZ 1186, KZ 1189, and KZ 1199. Growth of all strains on brain heart infusion agar containing 5% egg yolk was poor, but the NCTC 5404, KZ 1186, KZ 1189, and KZ 1199 strains exhibited the phospholipase reaction after 4 days of incubation.



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  		FIG. 1. Phospholipase production by C. tetani strains on PRYG agar medium containing 5% egg yolk. (A to D) A phospholipase reaction characterized by opacity around a colony is observed. Yellow arrowheads indicate representative phospholipase-positive colonies. (E to H) A phospholipase reaction mixture is distributed on the medium plates. (A) Ramon; (B) KZ 1189; (C) NCTC 5404; (D) CN 655; (E) NCTC 279; (F) KZ 1180; (G) KZ 1186; (H) KZ 1199; (I) C. perfringens KZ 221 (phospholipase-positive control); (J) C. butyricum NCTC 7423 (phospholipase-negative control). For C. butyricum, 0.5% glucose was added to the medium.

These results demonstrated that under the appropriate culture conditions, C. tetani is a phospholipase-producing bacterium. C. tetani phospholipase may be produced in vivo and contribute indirectly to tetanus by helping nutrient acquisition for cell growth and multiplication.


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ACKNOWLEDGMENTS
 
We are grateful to S. Nakamura for providing C. tetani strains; to X. Wang and Y. Sakaguchi for critical discussion; to C. Ishiyama, Y. Ohiwa, and C. Nabeta for technical assistance; and to J. J. Ferretti for reviewing the manuscript.


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REFERENCES
 
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  1. Brüggemann, H., S. Bäumer, W. F. Fricke, A. Wiezer, H. Liesegang, I. Decker, C. Herzberg, R. Martinez-Arias, R. Merkl, A. Henne, and G. Gottschalk. 2003. The genome sequence of Clostridium tetani, the causative agent of tetanus disease. Proc. Natl. Acad. Sci. USA 100:1316-1321.[Abstract/Free Full Text]
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  2. Clark, G. C., D. C. Briggs, T. Karasawa, X. Wang, A. R. Cole, T. Maegawa, P. N. Jayasekera, C. E. Naylor, J. Miller, D. S. Moss, S. Nakamura, A. K. Basak, and R. W. Titball. 2003. Clostridium absonum alpha-toxin: new insights into clostridial phospholipase C substrate binding and specificity. J. Mol. Biol. 333:759-769.[CrossRef][Medline]
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  3. Karasawa, T., X. Wang, T. Maegawa, Y. Michiwa, H. Kita, K. Miwa, and S. Nakamura. 2003. Clostridium sordellii phospholipase C: gene cloning and comparison of enzymic and biological activities with those of Clostridium perfringens and Clostridium bifermentans phospholipase C. Infect. Immun. 71:641-646.[Abstract/Free Full Text]
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  4. Society of American Bacteriologists. 1923. Bergey's manual of determinative bacteriology, 1st ed. Williams & Wilkins, Baltimore, Md.
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  5. Yamagishi, T. 1969. Biological properties of Clostridium tetani: (II) glucose utilization by Clostridium tetani. J. Juzen Med. Soc. 5:504-508 (In Japanese with English abstract.)
Emi Temaru
Satoshi Shimura
Tadahiro Karasawa*
Department of Laboratory Sciences
School of Health Sciences
Kanazawa University
Kodatsuno
Kanazawa, Japan

* Phone: 81 (76) 265-2597Fax: 81 (76) 234-4369 E-mail: karasawa{at}mhs.mp.kanazawa-u.ac.jp

Journal of Clinical Microbiology, April 2005, p. 2024-2025, Vol. 43, No. 4
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.4.2024-2025.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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