This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Carlier, J.-P.
Right arrow Articles by Mory, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Carlier, J.-P.
Right arrow Articles by Mory, F.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, January 2004, p. 484-486, Vol. 42, No. 1
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.1.484-486.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Osteosynthesis-Associated Bone Infection Caused by a Nonproteolytic, Nontoxigenic Clostridium botulinum-Like Strain

Jean-Philippe Carlier,1* Guylène K'ouas,1 Alain Lozniewski,2 François Sirveaux,3 Philippe Cailloux,3 and Francine Mory2

Centre National de Référence des Bactéries Anaérobies et du Botulisme, Institut Pasteur, Paris,1 Laboratoire de Bactériologie, Centre Hospitalier et Universitaire,2 Clinique de Traumatologie et d'Orthopédie, Nancy, France3

Received 24 July 2003/ Returned for modification 10 September 2003/ Accepted 28 September 2003


arrow
ABSTRACT
 
A nonproteolytic, nontoxigenic Clostridium botulinum strain identified by conventional and molecular techniques as type B-, E-, or F-like (BEF-like) was isolated from a human postsurgical wound. All previous reports of such strains have been from environmental sources. Since toxin production is the main taxonomic denominator for C. botulinum, a new name is needed for nonproteolytic, nontoxigenic BEF-like clinical isolates.


arrow
CASE REPORT
 
A 50-year-old man was hospitalized in June 2002 for an open supracondylar fracture of the right humerus. His past medical history included several fractures between 1984 and 1999, psoriatic arthritis, and ankylosing spondylitis treated by corticotherapy up to 2001. The fracture was treated by open reduction and internal fixation with a Lecestre-type plate. The following week, the patient presented an inflammatory scar with fistula formation. Laboratory investigations revealed an erythrocyte sedimentation rate of 105 mm/h and a C-reactive protein value of 68 mg/liter. Surgical excision of infected tissues and drainage were performed. The patient was treated with intravenous ofloxacin (200 mg twice daily) and rifampin (1 g twice daily). Biopsy specimens of the triceps and articular capsule revealed after culture the presence of Clostridium perfringens and Sphingomonas paucimobilis. The patient was then treated with intravenous ceftriaxone (2 g once daily) and oral ofloxacin (200 mg twice daily) and metronidazole (200 mg twice daily) for 1 week. The symptoms gradually resolved, and the patient left the hospital on day 29 with trimethoprim-sulfamethoxazole (two tablets three times daily), ofloxacin (200 mg twice daily), and metronidazole (500 mg three times daily) prescribed. One month later, the patient developed an osteosynthesis-associated bone infection. Extensive debridement was performed, and all purulent-appearing bone was resected. The plate was removed and reinserted by using a gentamicin- and vancomycin-loaded spacer. The same antibiotic therapy was continued, and the patient was discharged on day 74. A strictly anaerobic, gram-positive, motile, spore-forming rod, designated AIP 355.02, was the only bacterium isolated from all samples obtained during surgery. It was found to be susceptible to all antibiotics used except for trimethoprim-sulfamethoxazole and gentamicin.

Strain AIP 355.02 was characterized according to conventional tests (13). Metabolic end products were assayed by quantitative gas chromatography as described previously (5). The organism was lecithinase negative and lipase positive on egg yolk agar plates. Gelatin was liquefied, and milk was not modified. Production of urease and indole was not detected. Acid was produced from glucose, fructose, maltose, mannose, ribose, starch, and sorbitol. Acid was not produced from arabinose, cellobiose, esculin, galactose, glycerol, inositol, lactose, mannitol, melezitose, melibiose, raffinose, rhamnose, sucrose, salicin, trehalose, and xylose. The strain hydrolyzes starch but not esculin. Abundant gas was produced. The major metabolic end products were acetic acid and butyric acid. These cultural and biochemical properties were consistent with those of nonproteolytic Clostridium botulinum (group II). However, a mouse toxicity test for botulinum toxin was negative.

The identity of strain AIP 355.02 was subsequently confirmed by the sequence of the 16S rRNA gene. A PCR using universal 16S ribosomal DNA (rDNA) primers p584 (5'-AGAGTTTGATCATGGCTCAG-3'; 8-27f by the Escherichia coli numbering system) and p995 (5'-TACGGCTACCTTGTTACGACTT-3'; 1492-1513r) was performed. The PCR product was sequenced on an Applied Biosystems automatic sequencer (Genome Express, Meylan, France) in both directions by using forward and reverse primers. The 1,323-nucleotide sequence was compared with all eubacterial 16S rRNA gene sequences available in the GenBank database by using the multisequence Advanced BLAST comparison software from the National Center for Biotechnology Information (1). The alignment was done with CLUSTAL W (18). The highest sequence similarity value (100%) was obtained with the16S rDNA sequence of the nonproteolytic C. botulinum type B strain (Eklund 17B; ATCC 25765T). Lower percentages of identity (99%) were observed with the other C. botulinum strains belonging to group II, namely, C. botulinum type E (ATCC 9564T and Iwanai strains) and C. botulinum type F (ATCC 23387T), after which, the more closely related species was Clostridium ulginosum, which exhibited a more distant affinity (97%). From the distance matrix values, a dendrogram was constructed by using the Fich and Margoliash algorithm (9). The phylogenetic tree revealed that strain AIP 355.02 belongs to group II (nonproteolytic C. botulinum types B, E, and F) and is genetically highly related to the toxigenic C. botulinum type B strain, ATCC 25765T (Eklund 17B) (Fig. 1). This close genetic affinity is consistent with the phenotypic characters. Although the very high levels of rDNA sequence similarities (99 to 100% identity) do not allow definition with certainty of the toxigenic type to which this bacterium is related, these phylogenetic and biochemical characteristics clearly demonstrate that AIP 355.02 is an authentic nonproteolytic C. botulinum strain despite its nontoxigenic property. The possible presence of unexpressed neurotoxin genes as reported for C. botulinum type A (7, 10) was evaluated by PCR using specific primers (8). No type A, B, E, F, and G neurotoxin genes were detected.



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 1. Dendrogram showing the phylogenetic position of strain AIP 355.02 within group II C. botulinum based on 16S rDNA gene sequences. The sequence of Clostridium butyricum was used as an outgroup. The numbers above the branches are bootstrap percentages from 100 resampled data sets. The reference sequences were obtained from the GenBank and EMBL databases. Accession numbers are given in parentheses. The bar represents a 1% sequence difference.

Currently the species C. botulinum is defined on the basis of production of botulinal neurotoxins (types A to G) and encompasses four distinct metabolic groups (I to IV). Groups I and II are toxic to humans. Group I includes C. botulinum toxin type A and proteolytic strains of C. botulinum toxin types B and F. Group II contains all toxin type E strains and the nonproteolytic strains of toxin types B and F (12). Clostridium sporogenes displays very high 16S rRNA sequence homology and DNA relatedness with proteolytic C. botulinum toxin types A, B, and F and is considered as the nontoxigenic counterpart of group I (14). This species has been isolated from environmental sources in addition to multiple human infections, such as bacteremia, cutaneous abscesses, postsurgical wounds, leg or foot ulcers, and other pyogenic infections (2, 6). Similarly, the isolation of nontoxigenic Clostridium strains phenotypically highly related to group II has been frequently reported (4, 16, 17). All strains described hitherto were obtained from fish and environmental samples. So far, none had been isolated from human clinical specimens, except for one not documented isolate, obtained from an ear (11). Here we report one case of postoperative osteoarticular infection caused by a nonproteolytic, nontoxigenic C. botulinum-like strain.

For the clinician, the results described above create a problem for bacterial identification. They also highlight the nomenclatural problem evoked by Campbell et al. (3) for defining nonproteolytic C. botulinum and closely related nontoxigenic strains. Presently, there is no species name for these bacteria, which are often designated as nontoxigenic, nonproteolytic C. botulinum-like. Although unsatisfactory from a taxonomic point of view, this status can be sufficient for the environmental strains. However, the occurrence of this organism in a postoperative infection creates an urgent need to give a name to these nontoxic variants, to avoid confusion for the medical staff and microbiologists. In the present classification, the strains belonging to group I (proteolytic strains) are designated by two species names. The name C. botulinum must be conserved for toxigenic strains, while C. sporogenes is retained for nontoxigenic strains (15). A similar distinction between toxigenic strains and their nontoxigenic counterparts would be very useful within metabolic group II.

Nucleotide sequence accession number. Strain AIP 355.02 has been deposited in Collection de l'Institut Pasteur under accession no. CIP 107861. The GenBank accession no. for the 16S rDNA sequence is AY303799.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Centre National de Référence des Bactéries Anaérobies et du Botulisme, Institut Pasteur, 25-28 Rue du Dr. Roux, 75724 Paris Cedex 15, France. Phone: (33) 1 40 61 34 47. Fax: (33) 1 40 61 31 23. E-mail: jcarlier{at}pasteur.fr. Back


arrow
REFERENCES
 
    1
  1. Altschul, S. F., T. L. Madden, A. A. Schaeffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.[Abstract/Free Full Text]
    2. 2
  2. Bowler, P. G., B. I. Duerden, and D. G. Armstrong. 2001. Wound microbiology and associated approaches to wound management. Clin. Microbiol. Rev. 14:244-269.[Abstract/Free Full Text]
    3. 3
  3. Campbell, K. D., A. K. East, D. E. Thompson, and M. D. Collins. 1993. Studies on the large subunit ribosomal RNA genes and intergenic spacer regions of non-proteolytic Clostridium botulinum types B, E and F. Res. Microbiol. 144:171-180.[Medline]
    4. 4
  4. Cann, D. C., B. B. Wilson, G. Hobbs, J. M. Shewan, and A. Johannsen. 1965. The incidence of Clostridium botulinum type E in fish and bottom deposits in the North Sea and off the coast of Scandinavia. J. Appl. Bacteriol. 28:426-430.[Medline]
    5. 5
  5. Carlier, J. P. 1985. Gas chromatography of fermentation products: its application in diagnosis of anaerobic bacteria. Bull. Inst. Pasteur 83:57-69.
    6. 6
  6. Cato, E. P., W. L. George, and S. M. Finegold. 1986. Genus Clostridium, Prazmowski, 1880, 23AL, p. 1141-1200. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 2. Williams & Wilkins, Baltimore, Md.
    7. 7
  7. Cordoba, J. J., M. D. Collins, and A. K. East. 1995. Studies on the gene encoding botulinum neurotoxin type A of Clostridium botulinum from a variety of sources. Syst. Appl. Microbiol. 18:13-22.
    8. 8
  8. Fach, P., M. Gibert, R. Griffais, J. P. Guillou, and M. R. Popoff. 1995. PCR and gene probe identification of botulinum neurotoxin A-, B-, E-, F-, and G-producing Clostridium spp. and evaluation in food samples. Appl. Environ. Microbiol. 61:389-392.[Abstract]
    9. 9
  9. Felsenstein, J. 1993. PHYLIP (Phylogeny Inference Package) version 3.5c. Department of Genetics, University of Washington, Seattle. (Distributed by the author.)
    10. 10
  10. Franciosa, G., J. L. Ferreira, and C. L. Hatheway. 1994. Detection of type A, B, and E botulism neurotoxin genes in Clostridium botulinum and other Clostridium species by PCR: evidence of unexpressed type B toxin genes in type A toxigenic organisms. J. Clin. Microbiol. 32:1911-1917.[Abstract/Free Full Text]
    11. 11
  11. Ghanem, F. M., A. C. Ridpath, W. E. C. Moore, and L. V. H. Moore. 1991. Identification of Clostridium botulinum, Clostridium argentinense, and related organisms by cellular fatty acid analysis. J. Clin. Microbiol. 29:1114-1124.[Abstract/Free Full Text]
    12. 12
  12. Hatheway, C. L. 1990. Toxigenic clostridia. Clin. Microbiol. Rev. 3:66-98.[Abstract/Free Full Text]
    13. 13
  13. Holdeman, L. V., E. P. Cato, and W. E. C. Moore. 1977. Anaerobe laboratory manual, 4th ed. Virginia Polytechnic Institute and State University, Blacksburg, Va.
    14. 14
  14. Hutson, R. A., D. E. Thompson, P. A. Lawson, R. P. Schocken-Itturino, E. C. Bottger, and M. D. Collins. 1993. Genetic interrelationships of proteolytic Clostridium botulinum types A, B, and F and other members of the Clostridium botulinum complex as revealed by small-subunit rRNA gene sequences. Antonie Leeuwenhoek 64:273-283.
    15. 15
  15. Judicial Commission of the International Committee on Systematic Bacteriology. 1999. Rejection of Clostridium putrificum and conservation of Clostridium botulinum and Clostridium sporogenes—opinion 69. Int. J. Syst. Bacteriol. 49:339.[Abstract/Free Full Text]
    16. 16
  16. Kautter, D. A., S. M. Harmon, R. K. Lynt, Jr., and T. Lilly, Jr. 1966. Antagonistic effect on Clostridium botulinum type E by organisms resembling it. Appl. Microbiol. 14:616-622.[Medline]
    17. 17
  17. Lindstrom, M. K., H. M. Jankola, S. Hielm, E. K. Hyytia, and H. J. Korkeala. 1999. Identification of Clostridium botulinum with API 20 A, rapid ID 32 A and RapID ANA II. FEMS Immunol. Med. Microbiol. 24:267-274.[Medline]
    18. 18
  18. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.[Abstract/Free Full Text]

Journal of Clinical Microbiology, January 2004, p. 484-486, Vol. 42, No. 1
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.1.484-486.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Carlier, J.-P., Manich, M., Loiez, C., Migaud, H., Courcol, R. J. (2006). First Isolation of Clostridium amygdalinum from a Patient with Chronic Osteitis.. J. Clin. Microbiol. 44: 3842-3844 [Abstract] [Full Text]  
  • Mory, F., Carlier, J.-P., Alauzet, C., Thouvenin, M., Schuhmacher, H., Lozniewski, A. (2005). Bacteremia Caused by a Metronidazole-Resistant Prevotella sp. Strain. J. Clin. Microbiol. 43: 5380-5383 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Carlier, J.-P.
Right arrow Articles by Mory, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Carlier, J.-P.
Right arrow Articles by Mory, F.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»