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Journal of Clinical Microbiology, March 2003, p. 1118-1125, Vol. 41, No. 3
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.3.1118-1125.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

New Types of Toxin A-Negative, Toxin B-Positive Strains among Clostridium difficile Isolates from Asia

Maja Rupnik,1* Naoki Kato,2 Miklavz Grabnar,1 and Haru Kato3

Department of Biology, University of Ljubljana, Ljubljana, Slovenia,1 Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Diseases, Tokyo,3 Institute of Anaerobic Bacteriology, School of Medicine Gifu University, Gifu, Japan2

Received 19 August 2002/ Returned for modification 23 October 2002/ Accepted 29 November 2002

A total of 56 C. difficile strains were selected from 310 isolates obtained from different hospitals in Japan and Korea and from healthy infants from Indonesia. Strains that had been previously typed by pulsed-field gel electrophoresis and PCR ribotyping, were characterized by toxinotyping and binary toxin gene detection. When toxinotyped, 35 strains were determined to be toxinotype 0, whereas 21 strains showed variations in toxin genes and could be grouped into 11 variant toxinotypes. Six of the toxinotypes had been described before (I, III, IV, VIII, IX, and XII). In addition, five new toxinotypes were defined (XVI to XX). Three of the new toxinotypes (XVIII, XIX, and XX) vary only in repetitive regions of tcdA and produce both toxins. In two strains from toxinotypes XVI and XVII, the production of TcdA could not be detected with commercial immunological kits. Strain J9965 (toxinotype XVII) was in PaLoc similar but not identical to another known A-B+ strain, C. difficile 8864. Strain SUC 36 (toxinotype XVI), on the other hand, was similar to well-defined group consisting of toxinotypes V, VI, and VII, which thus far includes only A+B+ strains. Toxinotypes XVI and XVII represent two new groups of A-B+ strains. Strains of the well-known A-B+ group from toxinotype VIII have a nonsense mutation at the beginning of tcdA gene, and the introduction of a stop codon at amino acid position 47 results in nonproduction of TcdA. The 5'-end sequence of tcdA in two newly described A-B+ strains does not contain an identical mutation. The prevalence of variant C. difficile strains varied greatly among nine hospitals. Only five strains from four different hospitals were positive in PCR for amplification of the binary toxin gene.

* Corresponding author. Mailing address: University of Ljubljana, Biotechnical Faculty, Department of Biology, Vecna pot 111, 1000 Ljubljana, Slovenia. Phone: 386-1-2565-584. Fax: 386-1-257-33-90. E-mail: maja.rupnik{at}uni-lj.si.

Journal of Clinical Microbiology, March 2003, p. 1118-1125, Vol. 41, No. 3
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.3.1118-1125.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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