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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 nontoxigenic strains have been reported to produce neither of these toxins. Recent
studies indicate that it is not always true. We established a 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-linked immunosorbent assay (ELISA). By
using the PCR primer set NK11 and NK9 derived from the repeating
sequences of the toxin A gene, a shorter segment (ca. 700 bp) was
amplified from toxin A
, toxin B+ strains compared to the size of the
segment amplified from toxin A+, toxin B+ strains (ca. 1,200 bp), and
no product was amplified from toxin A
, toxin B
strains. We examined
a total of 421 C. difficile isolates by PCR. Of these, 48 strains showed a shorter segment by the PCR, were negative by ELISAs
for the detection of toxin A, and were positive by cell culture assay. Although the cytotoxin produced by the toxin A
, toxin B+ strains was
neutralized by anti-toxin B serum, the appearance of the cytotoxic effects on Vero cell monolayers was distinguishable from that of toxin
A+, toxin B+ strains. By immunoblotting, the 44 toxin A
, toxin B+
strains were typed to serogroup F and the remaining four strains were
serogroup X. Pulsed-field gel electrophoresis separated the 48 strains
into 19 types. The PCR assay for the detection of the repeating
sequences combined with PCR amplification of the nonrepeating sequences
of either the toxin A or the toxin B 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 role of 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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