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Journal of Clinical Microbiology, March 2004, p. 1035-1041, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1035-1041.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Characterization of Toxin A-Negative, Toxin B-Positive Clostridium difficile Isolates from Outbreaks in Different Countries by Amplified Fragment Length Polymorphism and PCR Ribotyping

Renate J. van den Berg,1* Eric C. J. Claas,1 Duddy H. Oyib,1 Corné H. W. Klaassen,2 Lenie Dijkshoorn,1,3 Jon S. Brazier,4 and Ed J. Kuijper1

Departments of Medical Microbiology,1 Infectious Diseases, Center of Infectious Diseases, Leiden University Medical Center, Leiden,3 Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands,2 Anaerobe Reference Unit, Public Health Laboratory, University Hospital of Wales, Cardiff, United Kingdom4

Received 8 October 2003/ Returned for modification 20 November 2003/ Accepted 4 December 2003

Clinical Clostridium difficile isolates of patients with diarrhea or pseudomembranous colitis usually produce both toxin A and toxin B, but an increasing number of reports mention infections due to toxin A-negative, toxin B-positive (A-/B+) strains. Thirty-nine clinical toxin A-/B+ isolates, and 12 other unrelated isolates were obtained from Canada, the United States, Poland, the United Kingdom, France, Japan, and The Netherlands. The isolates were investigated by high-resolution genetic fingerprinting by use of amplified fragment length polymorphism (AFLP) and two well-described PCR ribotyping methods. Furthermore, the toxin profile and clindamycin resistance were determined. Reference strains of C. difficile representing 30 known serogroups were also included in the analysis. AFLP discriminated 29 types among the reference strains, whereas the two PCR ribotyping methods distinguished 25 and 26 types. The discriminatory power of AFLP and PCR ribotyping among 12 different unrelated isolates was similar. Typing of 39 toxin A-/B+ isolates revealed 2 AFLP types and 2 and 3 PCR ribotypes. Of 39 toxin A-/B+ isolates, 37 had PCR ribotype 017/20 and AFLP type 20 (95%). A deletion of 1.8 kb was seen in 38 isolates, and 1 isolate had a deletion of approximately 1.7 kb in the tcdA gene, which encodes toxin A. Clindamycin resistance encoded by the erm(B) gene was found in 33 of 39 toxin A-/B+ isolates, and in 2 of the 12 unrelated isolates (P < 0.001, chi-square test). We conclude that clindamycin-resistant C. difficile toxin A-/B+ strain (PCR ribotype 017/20, AFLP type 20, serogroup F) has a clonal worldwide spread.

* Corresponding author. Mailing address: Department of Medical Microbiology, E4-62, Center of Infectious Diseases, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Phone: 31-71-5265154. Fax: 31-71-5248148. E-mail: r.j.van_den_berg.mm{at}lumc.nl.

Journal of Clinical Microbiology, March 2004, p. 1035-1041, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.1035-1041.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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