Identification of Toxin A-Negative, Toxin B-Positive Clostridium difficile by PCR

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Journal of Clinical Microbiology, August 1998, p. 2178-2182, Vol. 36, No. 8
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Identification of Toxin A-Negative, Toxin B-Positive Clostridium difficile by PCR

Haru Kato,¹^, Naoki Kato,¹^,^* Kunitomo Watanabe,¹ Naoichi Iwai,² Haruhi Nakamura,² Toshinobu Yamamoto,³ Kanzo Suzuki,³ Shin-Moo Kim,⁴ Yunsop Chong,⁵ and Eddy Bagus Wasito⁶

Institute of Anaerobic Bacteriology, Gifu University School of Medicine, Gifu,¹ and Department of Pediatrics, Meitetsu Hospital,² and Department of Internal Medicine, Nagoyashi Koseiin Geriatric Hospital,³ Nagoya, Japan; Department of Clinical Pathology, Wonkwang Public Health Junior College, Iri,⁴ and Department of Clinical Pathology, Yonsei University College of Medicine, Seoul,⁵ Korea; and Tropical Disease Research Centre, Faculty of Medicine, Airlangga University, Surabaya, Indonesia⁶

Received 6 March 1998/Returned for modification 13 April 1998/Accepted 28 April 1998

Toxigenic strains of Clostridium difficile have been reported to produce both toxins A and B nearly always, and nontoxigenicstrains have been reported to produce neither of these toxins.Recent studies indicate that it is not always true. We establisheda PCR assay to differentiate toxin A-negative, toxin B-positive(toxin A $-$ , toxin B+) strains from both toxin-positive (toxin A+,toxin B+) strains and both toxin-negative (toxin A $-$ , toxin B $-$ )strains as an alternative to cell culture assay and enzyme-linkedimmunosorbent assay (ELISA). By using the PCR primer set NK11and NK9 derived from the repeating sequences of the toxin A gene,a shorter segment (ca. 700 bp) was amplified from toxin A $-$ , toxinB+ strains compared to the size of the segment amplified fromtoxin A+, toxin B+ strains (ca. 1,200 bp), and no product wasamplified from toxin A $-$ , toxin B $-$ strains. We examined a totalof 421 C. difficile isolates by PCR. Of these, 48 strains showeda shorter segment by the PCR, were negative by ELISAs for thedetection of toxin A, and were positive by cell culture assay.Although the cytotoxin produced by the toxin A $-$ , toxin B+ strainswas neutralized by anti-toxin B serum, the appearance of the cytotoxiceffects on Vero cell monolayers was distinguishable from thatof toxin A+, toxin B+ strains. By immunoblotting, the 44 toxinA $-$ , toxin B+ strains were typed to serogroup F and the remainingfour strains were serogroup X. Pulsed-field gel electrophoresisseparated the 48 strains into 19 types. The PCR assay for thedetection of the repeating sequences combined with PCR amplificationof the nonrepeating sequences of either the toxin A or the toxinB gene is indicated to be useful for differentiating toxin A $-$ ,toxin B+ strains from toxin A+, toxin B+ and toxin A $-$ , toxin B $-$ strains and will contribute to elucidation of the precise roleof toxin A $-$ , toxin B+ strains in intestinal diseases.

^* Corresponding author. Mailing address: Institute of Anaerobic Bacteriology, Gifu University School of Medicine, 40 Tsukasa-machi,Gifu 500-8705, Japan. Phone: 81-58-267-2342. Fax: 81-58-265-9001.E-mail: nk19{at}cc.gifu-u.ac.jp.

Present address: Department of Bacteriology, School of Medicine, Kanazawa University, 13-1 Takara-machi, Kanazawa 920-8640,Japan.

Journal of Clinical Microbiology, August 1998, p. 2178-2182, Vol. 36, No. 8
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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