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Journal of Clinical Microbiology, August 1998, p. 2178-2182, Vol. 36, No. 8
Institute of Anaerobic Bacteriology,
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 Clostridium difficile is
a causative agent of pseudomembranous colitis and a principle pathogen
causing antibiotic-associated diarrhea (AAD) and antibiotic-associated
colitis (AAC). Strains of C. difficile produce two toxins,
toxin A and toxin B, which are involved in the pathogenicity of this
organism (3, 15). Toxin A has been referred to as an
enterotoxin in view of its fluid accumulation activity in animal
intestinal loop tests (17), and toxin B has been referred to
as a cytotoxin whose cytopathic effect is considerably much more potent
than that of toxin A. Because toxigenic strains of C. difficile are believed to release both toxins A and B and cell
culture assay for toxin B is the most sensitive method for the
detection of toxin B, the cell culture assay is considered the "gold
standard" for the diagnosis of C. difficile-associated
diarrhea and colitis.
A strain named C. difficile 8864 has been reported to be
toxin A-negative and toxin B-positive (toxin A Here we report the use of PCR to amplify a segment of the repeating
sequences of the toxin A gene to distinguish toxin A Bacterial strains.
The 427 strains of C. difficile used in this study were isolated at the following
facilities: Institute of Anaerobic Bacteriology, Gifu University School
of Medicine, Gifu, Japan; Gifu University Hospital, Gifu, Japan;
Nagoyashi Koseiin Geriatric Hospital, Nagoya, Japan; Department of
Pediatrics, Meitetsu Hospital, Nagoya, Japan; Tropical Disease Research
Centre, Airlangga University, Surabaya, Indonesia; and Centers for
Disease Control and Prevention (CDC), Atlanta, Ga.
Toxin assays.
C. difficile was cultured anaerobically
in brain heart infusion broth (Becton Dickinson and Company,
Cockeysville, Md.) for 5 to 7 days. Two ELISA kits, Tox-A TEST
(TechLab, Blacksburg, Va.) and VIDAS CDA (bioMerieux Vitek,
Hazelwood, Mo.), were used to detect toxin. Enterotoxicity was
determined by a rabbit intestinal loop assay (5) with
10-week-old female Japanese White rabbits. To detect a low level of
toxin A, the culture supernatants of toxin A PCR assay.
PCR assay was performed as described previously
(13), with modifications. A single colony was suspended in
50 µl of TES (50 mM Tris hydrochloride [pH 8.0], 5 mM EDTA, 50 mM
NaCl), and the suspension was heated at 95°C for 10 min and
centrifuged at 15,000 × g for 2 min. One microliter of
the resultant supernatant was added to 30 µl of a reaction buffer
consisting of 2.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl (pH
9.0), the four deoxynucleoside triphosphates (200 µM each), 45 ng of
each primer, and 0.75 U of Taq DNA polymerase (Pharmacia
Biotech, Uppsala, Sweden). Two primer sets were used to detect the
toxin A gene; primers NK3 and NK2 were derived from the nonrepeating
portion of the C. difficile toxin A gene, and primers NK11
and NK9 were derived from the repeating portion of the C. difficile toxin A gene (13). A segment of the toxin B gene was amplified by using primer NK104 (sequence,
5'-GTGTAGCAATGAAAGTCCAAGTTTACGC-3'; positions 2945 to 2972)
(4) and primer NK105 (sequence,
5'-CACTTAGCTCTTTGATTGCTGCACCT-3'; positions 3123 to 3148)
(4), which were derived from the nonrepeating sequence of
the C. difficile toxin B gene. The specificity of the PCR
product with primer set NK104-NK105 was confirmed by Southern blot
analysis as described previously (10). Probe NK106
(sequence, 5'-GACTTACTTCCTACATTATCTGAAGG-3'; positions 3058 to 3083) (4) was used and was 3' end labeled with
digoxigenin with a digoxigenin labeling kit (Boehringer Mannheim,
Mannheim, Germany). The thermal profile for primer pairs NK3-NK2 and
NK104-NK105 was 35 cycles comprising 95°C for 20 s and 55°C
for 120 s. At the conclusion of the PCR cycles, the tubes were
incubated at 74°C for 5 min. PCR amplification with primer pair
NK11-NK9 was performed for 35 cycles, consisting of 95°C for 20 s and 62°C for 120 s.
Phenotypic and genotypic typings.
The C. difficile strains were analyzed by Western immunoblotting
(11) and pulsed-field gel electrophoresis (PFGE) typing (12). Immunoblot typing was performed with EDTA-extracted
proteins of bacterial cells, followed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, as described previously
(11), with antisera against reference strains of the 10 different serogroups established by a slide agglutination test
(7). For PFGE analysis, the DNA in the inserts was digested
with SacII (New England Biolabs, Beverly, Mass.), and the
resultant restriction fragments were separated at a constant voltage of
200 V with 25-s pulses for 5 h followed by 50-s pulses for 19 h (12).
First, we examined the specificity of a PCR assay for the
detection of a segment of the toxin B gene using primer set
NK104-NK105. The specificities of the PCR products were eventually
confirmed by Southern hybridization with the digoxigenin-labeled NK106
probe. A total of 273 randomly selected C. difficile strains
were tested by both cell culture and PCR. All 204 toxin B+ strains were
PCR positive, and the remaining 69 toxin B Second, six strains of C. difficile were examined for the
production of toxins A and B and the presence of the toxin A and B
genes. The results are summarized in Table
1. Five of the six strains tested were
PCR positive for the segment of the nonrepeating region of the toxin A
gene (Fig. 1A, lanes 2 to 6) and the
toxin B gene (Fig. 1B, lanes 2 to 6) and were cell culture positive for
toxin B. However, there were three strains from which a shorter segment
of approximately 700 bp (Fig. 1C, lanes 2 to 4) was amplified by PCR
for the repeating region of the toxin A gene, whereas for toxin A+,
toxin B+ a PCR product of approximately 1,200 bp in size (Fig. 1C,
lanes 5 and 6) was generated. The three strains were toxin A
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Identification of Toxin A-Negative, Toxin
B-Positive Clostridium difficile by PCR
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
, 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.
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
, toxin B+) (5, 14). The strain fails to produce toxin A but shows enterotoxic activity in a rabbit ileal loop test. The activity may be due to the
fact that toxin B has a potent enterotoxin activity(s) (14)
or that this strain produces additional factors unrelated to toxin A
(5). Serogroup F strains of C. difficile which
were often isolated from asymptomatic infants were toxin A by
enzyme-linked immunosorbent assay (ELISA) and toxin B+ by cell culture
assay and generated no diarrhea in hamsters and axenic mice (6,
8). However, little regarding the clinical significance,
pathogenicity, prevalence, and genetic backgrounds of the toxin A,
toxin B+ strains of C. difficile is known. A simple and
rapid method is needed to identify toxin A, toxin B+ strains to
characterize them.
, toxin B+
strains from toxin A+, toxin B+ and toxin A, toxin B strains as an
alternative to cell culture assay for toxin B and ELISA for toxin A. We
examined 421 clinically isolated C. difficile strains and
found an unexpectedly high incidence of toxin A, toxin B+ strains not
only in children but also in adults.
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
, toxin B+ strains were
concentrated 10 times by ultrafiltration with Molcut II (NMWL 10,000;
Millipore, Marlborough, Mass.). Toxin B was detected by cell culture
assay with Vero cells (5). For detection of toxin B, the
culture supernatant was filtered through Millex-GP (Millipore) filters
before it was applied to cell monolayers. C. difficile goat
anti-toxin B serum (TechLab) was used for the cytotoxin neutralization
assay.
RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
strains were PCR negative. Southern hybridization gave results consistent with the PCR results (data not shown).
by two
ELISAs and a rabbit ileal loop test. Thus, we designated them toxin
A, toxin B+ strains.
TABLE 1.
Results of toxin production assays and PCR for the
repeating and nonrepeating regions of toxin A and B genes for
six strains
View larger version (78K):
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FIG. 1.
Polyacrylamide gel electrophoresis of PCR products of
six C. difficile strains. Three primer pairs, NK3-NK2
derived from the nonrepeating portion of the toxin A gene (A),
NK104-NK105 derived from the nonrepeating portion of the toxin B gene
(B), and NK11-NK9 derived from the repeating portion of the toxin A
gene (C), were used for PCR. Lane 1, toxin A , toxin B strain; lanes
2 to 4, toxin A, toxin B+ strains; lanes 5 and 6, toxin A+, toxin B+
strains. The size markers used were a 100-bp ladder (A and B) and
pBR322 digested with AvaII and
AvaII-EcoRI (C).
On the basis of the results that were obtained, a total of 421 C. difficile strains isolated from 190 children ranging in age from 0 and 14 years and 231 adults older than 14 years of age were examined by the PCR assay. Of the 421 strains tested, 246 were PCR positive and 175 were PCR negative for the nonrepeating region of the both the toxin A and the toxin B genes. Of the 246 PCR-positive strains tested, 48 generated a PCR product of approximately 700 bp and 198 yielded a PCR product of approximately 1,200 bp by PCR for the repetitive region of the toxin A gene.
The VIDAS CDA test was performed for the detection of toxin A with 100 of the 198 strains for which the PCR for the repetitive region of the
toxin A gene generated a longer PCR product and with all 48 strains for
which the PCR for the repetitive region of the toxin A gene generated a
shorter PCR product, resulting in the former strains being positive for
toxin A and the latter strains being negative for toxin A. Furthermore,
the 48 toxin A, toxin B+ strains were tested by the Tox-A TEST, with
negative results. To examine the strains for a low level of production of toxin A, if any such strains existed, the culture supernatants of
the 48 strains were concentrated 10 times and were subjected to the
Tox-A TEST, with negative results.
Eventually, the bioassay results completely agreed with those of the
PCR assay. Toxin A, toxin B strains were negative by PCR with the
NK3-NK2 and NK104-NK105 primer sets, toxin A+, toxin B+ strains were
positive by PCR with these two primer sets and generated an
approximately 1,200-bp product by PCR with the NK11-NK9 primer set, and
toxin A, toxin B+ strains were positive by PCR with these two primer
sets and yielded an approximately 700-bp product by PCR with the
NK11-NK9 primer set.
Although the cytotoxin produced by all toxin A, toxin B+ strains was
neutralized by C. difficile anti-toxin B serum, the cytotoxic effect of toxin A, toxin B+ strains on Vero cell monolayers was apparently distinguishable from that of toxin A+, toxin B+ strains.
Culture filtrates of toxin A+, toxin B+ strains caused cell rounding
and disrupted cell-to-cell contact; in contrast, Vero cells incubated
with culture filtrates of toxin A, toxin B+ strains appeared as
discrete clusters (Fig. 2).
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Western immunoblot typing with 48 toxin A, toxin B+ strains showed
that 44 strains belonged to serogroup F (subserogroup F-0) and that
four strains belonged to serogroup X (subserogroup X-2). All four
strains of serogroup X were originally from Indonesia. PFGE analysis
after SacII digestion demonstrated a variety of banding
patterns among the toxin A, toxin B+ strains. Overall, PFGE analysis
separated the 48 toxin A, toxin B+ strains into 19 types. No evidence
of the monoclonal spread of a toxin A, toxin B+ strain was found for
any cohort studied.
For the 333 subjects whose clinical backgrounds were recorded, we
examined whether there was a relation between the symptoms of the
diarrheal disorder and the toxigenic patterns of the C. difficile strain (Table 2). Toxin
A, toxin B+ strains were isolated from 6.7 and 12.5% of symptomatic
and asymptomatic children, respectively. For adults, no toxin A,
toxin B+ strain was recovered from symptomatic patients, but 12.5% of
asymptomatic adults were colonized or infected with toxin A, toxin B+
strains. Among the 88 subjects whose data are not listed in Table 2 due
to their unclear clinical background, 31.8% were positive for toxin
A, toxin B+ strains.
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DISCUSSION |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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In this study, we successfully discriminated toxin A+, toxin B+,
toxin A, toxin B+, and toxin A, toxin B strains of C. difficile by PCR using two primer sets derived from the
nonrepeating sequence of the toxin A and the toxin B genes and a primer
set derived from the repetitive sequence of the toxin A gene. PCR for
the nonrepeating sequence of the toxin A gene always gave results that
were the same as those of PCR for the nonrepeating sequence of the
toxin B gene. PCR for the repetitive sequence of the toxin A gene with
primer set NK11-NK9 was able to differentiate toxin A, toxin B+
strains from toxin A+, toxin B+ strains. Thus, PCR with primer set
NK11-NK9 combined with either one of the PCR assays detecting the
nonrepeating sequence of the toxin A or the toxin B gene can be used to
differentiate three types of toxin production. In a previous
investigation, Depitre et al. (8) failed to differentiate
toxin A, toxin B+ strains (serogroup F) from toxin A+, toxin B+
strains using PCR with primer set P31-P60 directed at the repeating
region of the toxin A gene. We tried to use several primer sets to
amplify a portion of the repetitive sequences of the toxin A gene;
however, all but primer set NK11-NK9 failed to provide distinct PCR
results.
All except four of the toxin A, toxin B+ strains tested in this study
were typed into serogroup F by immunoblotting; the exceptions were four
strains recovered in Indonesia that belonged to serogroup X. These
findings are consistent with those presented in a previous report of
Depitre et al. (8) that serogroup F strains produce toxin B
but not toxin A. These observations indicate that C. difficile strains belonging to serogroup F have a unique feature
in their toxigenicity. Interestingly, polyclonal antisera against
serogroups F and X had a cross-reaction to each other by a dot blot
test (data not shown), suggesting that serogroups F and X share some
immunological features. PFGE analysis of the toxin A, toxin B+
strains tested in this study provided no evidence that a single clone
was spread within each hospital.
Depitre et al. (8) reported that C. difficile
serogroup F strains in Belgium were often isolated from asymptomatic
children and that they were all toxin A, toxin B+ strains. Valenzuela Montero et al. (18) demonstrated that 7 (12.1%) of the 58 cytotoxic strains from healthy Chilean infants were toxin A by a
commercial ELISA. In this study toxin A, toxin B+ strains were
isolated from 6.7% of symptomatic and asymptomatic children (younger
than 15 years old) in Japan and 12.5% of healthy children (younger than 2 years old) in Indonesia. Taken together, the studies indicate that, globally, some children, especially younger ones, carry toxin
A, toxin B+ strains of C. difficile in their intestines.
In adults, although the clinical background was unclear for a portion
of the subjects studied, toxin A, toxin B+ strains were recovered at
unexpectedly high rates from geographically separate facilities in
Japan, indicating that intestinal carriage of these strains is not rare
among Japanese adults. In contrast, none of the 100 C. difficile strains recovered from patients with AAD or AAC in the
United States were toxin A, toxin B+. A recent study in the United
States by Lyerly et al. (16) showed a low incidence of toxin
A, toxin B+ strains (0.2%). Altaie et al. (2) recovered
no toxin A, toxin B+ strains from 300 stool specimens obtained from
patients who were clinically suspected of having AAD. Toxin A, toxin
B+ strains are likely to be of low prevalence in the United States or
uncommon, at least in patients with AAD or AAC. So far no information
regarding the prevalence of toxin A, toxin B+ strains in other
countries is available.
Although the cytotoxin produced by toxin A, toxin B+ strains was
neutralized by anti-toxin B serum, the appearance of cytotoxic effects
on Vero cell monolayers was distinguishable between toxin A+, toxin B+
strains and toxin A, toxin B+ strains. Interestingly, the toxin B
produced by strain 8864 caused a clumping of rounding cells within the
cell sheet on Vero cells (5), which was similar to the
cytotoxic effects of the toxin A, toxin B+ strains observed in this
study. A difference in the N-terminal region of the toxin B gene
between the reference strain of serogroup F (strain 1470) and the toxin
A+, toxin B+ strain (strain VPI 10463) has been documented
(19). In addition, the morphological response of endothelial
cells of the pig pulmonary artery to toxin B of strain 1470 was
reported to be different from that to toxin B of toxin A+, toxin B+
strains but indistinguishable from that to the lethal toxin of
Clostridium sordellii (19). These results suggest
that the structure of toxin B of toxin A, toxin B+ C. difficile strains may be similar to that of the lethal toxin of
C. sordellii and different in part from that of the toxin B
of toxin A+, toxin B+ C. difficile strains.
Nevertheless, the enteropathogenicity of toxin A, toxin B+ strains
remains unclear. We tested seven toxin A, toxin B+ strains (three
from children and four from adults) for enterotoxic activities in a
rabbit ileal loop assay, but the result was no fluid accumulation. The
results suggest that toxin A, toxin B+ strains may not cause diarrheal disease in humans. Additional experiments including an
experiment with a hamster model and a study of the prevalence of toxin
A, toxin B+ strains among symptomatic and asymptomatic subjects will
be required to determine the role of toxin A, toxin B+ strains in
intestinal disorders.
In conclusion, PCR assay for the detection of the repeating sequence of
the toxin A gene is useful as a tool for discriminating toxin A+, toxin
B+, toxin A, toxin B+, and toxin A, toxin B strains. It is a
simple and rapid technique for the identification of toxin A, toxin
B+ strains and should be valuable in elucidating the pathogenic and
epidemic potentials of these strains.
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ACKNOWLEDGMENTS |
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We thank Kakuyo Sawa of Gifu University Hospital for sample collection and George E. Killgore of the CDC for editing this article.
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FOOTNOTES |
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* 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.
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REFERENCES |
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Abstract
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Materials & Methods
Results
Discussion
References
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| 1. | Allen, S. D., and E. J. Baron. 1991. Clostridium, p. 505-521. In A. Balows, W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.), Manual of clinical microbiology, 5th ed. American Society for Microbiology, Washington, D.C. |
| 2. | Altaie, S. S., P. H. Penque, S. Mookherjee, and D. T. Evans. 1996. Should laboratories test for toxin A-negative, toxin B-positive Clostridium difficile? Lab. Med. 27:468-471. |
| 3. | Banno, Y., T. Kobayashi, H. Kono, K. Watanabe, K. Ueno, and Y. Nozawa. 1984. Biochemical characterization and biologic actions of two toxins (D-1 and D-2) from Clostridium difficile. Rev. Infect. Dis. 6:S11-S20. |
| 4. |
Barroso, L. A.,
S. Z. Wang,
C. J. Phelps,
J. L. Johnson, and T. D. Wilkins.
1990.
Nucleotide sequence of Clostridium difficile toxin B gene.
Nucleic Acids Res.
18:4004 |
| 5. |
Borriello, S. P.,
B. W. Wren,
S. Hyde,
S. V. Seddon,
P. Sibbons,
M. M. Krishna,
S. Tabaqchali,
S. Manek, and A. B. Price.
1992.
Molecular, immunological, and biological characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile.
Infect. Immun.
60:4192-4199 |
| 6. |
Delmee, M., and V. Avesani.
1990.
Virulence of ten serogroups of Clostridium difficile in hamsters.
J. Med. Microbiol.
33:85-90 |
| 7. |
Delmee, M.,
Y. Laroche,
V. Avesani, and G. Cornelis.
1986.
Comparison of serogrouping and polyacrylamide gel electrophoresis for typing Clostridium difficile.
J. Clin. Microbiol.
24:991-994 |
| 8. |
Depitre, C.,
M. Delmee,
V. Avesani,
R. L'Haridon,
A. Roels,
M. Popoff, and G. Corthier.
1993.
Serogroup F strains of Clostridium difficile produce toxin B but not toxin A.
J. Med. Microbiol.
38:434-441 |
| 9. | Gumerlock, P. H., Y. J. Tang, F. J. Meyers, and J. Silva, Jr. 1991. Use of the polymerase chain reaction for the specific and direct detection of Clostridium difficile in human feces. Rev. Infect. Dis. 13:1053-1060[Medline]. |
| 10. | Jotwani, R., N. Kato, H. Kato, K. Watanabe, and K. Ueno. 1995. Detection of Bacteroides fragilis in clinical specimens by polymerase chain reaction amplification of the neuraminidase gene. Curr. Microbiol. 31:215-219[Medline]. |
| 11. |
Kato, H.,
J. J. Cavallaro,
N. Kato,
S. L. Bartley,
G. E. Killgore,
K. Watanabe, and K. Ueno.
1993.
Typing of Clostridium difficile by Western immunoblotting with 10 different antisera.
J. Clin. Microbiol.
31:413-415 |
| 12. |
Kato, H.,
N. Kato,
K. Watanabe,
K. Ueno,
H. Ushijima,
S. Hashira, and T. Abe.
1994.
Application of typing by pulsed-field gel electrophoresis to the study of Clostridium difficile in a neonatal intensive care unit.
J. Clin. Microbiol.
32:2067-2070 |
| 13. |
Kato, N.,
C. Y. Ou,
H. Kato,
S. L. Bartley,
V. K. Brown,
V. R. Dowell, Jr., and K. Ueno.
1991.
Identification of toxigenic Clostridium difficile by the polymerase chain reaction.
J. Clin. Microbiol.
29:33-37 |
| 14. |
Lyerly, D. M.,
L. A. Barroso,
T. D. Wilkins,
C. Depitre, and G. Corthier.
1992.
Characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile.
Infect. Immun.
60:4633-4639 |
| 15. |
Lyerly, D. M.,
H. C. Krivan, and T. D. Wilkins.
1988.
Clostridium difficile: its disease and toxins.
Clin. Microbiol. Rev.
1:1-18 |
| 16. |
Lyerly, D. M.,
L. M. Neville,
D. T. Evans,
J. Fill,
S. Allen,
W. Greene,
R. Sautter,
P. Hnatuck,
D. J. Torpey, and R. Schealbe.
1998.
Multicenter evaluation of the Clostridium difficile TOX A/B TEST.
J. Clin. Microbiol.
36:184-190 |
| 17. |
Lyerly, D. M.,
K. E. Saum,
D. K. MacDonald, and T. D. Wilkins.
1985.
Effects of Clostridium difficile toxins given intragastrically to animals.
Infect. Immun.
47:349-352 |
| 18. | Valenzuela Montero, M. E., T. Lobos, A. Valenzuela, O. Roos, L. Rodriguez, P. Piemonte, and M. Soledad Giglio. 1995. Prevalence Clostridium difficile among healthy Chilean infants: evaluation by commercial enzyme immunoassay versus standard cytotoxin assay. Clin. Infect. Dis. 20:S259-S260. |
| 19. | von Eichel-Streiber, C., D. Meyer zu Heringdorf, E. Habermann, and S. Sartingen. 1995. Closing in on the toxic domain through analysis of a variant Clostridium difficile cytotoxin B. Mol. Microbiol. 17:313-321[Medline]. |
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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[Abstract] [Full Text] -
Amimoto, K., Noro, T., Oishi, E., Shimizu, M.
(2007). A novel toxin homologous to large clostridial cytotoxins found in culture supernatant of Clostridium perfringens type C. Microbiology
153: 1198-1206
[Abstract] [Full Text] -
van den Berg, R. J., Vaessen, N., Endtz, H. P., Schulin, T., van der Vorm, E. R., Kuijper, E. J.
(2007). Evaluation of real-time PCR and conventional diagnostic methods for the detection of Clostridium difficile-associated diarrhoea in a prospective multicentre study. J Med Microbiol
56: 36-42
[Abstract] [Full Text] -
Arroyo, L. G., Stampfli, H. R., Weese, J. S.
(2006). Potential role of Clostridium difficile as a cause of duodenitis-proximal jejunitis in horses.. J Med Microbiol
55: 605-608
[Abstract] [Full Text] -
Terhes, G., Brazier, J. S., Urban, E., Soki, J., Nagy, E.
(2006). Distribution of Clostridium difficile PCR ribotypes in regions of Hungary.. J Med Microbiol
55: 279-282
[Abstract] [Full Text] -
Kato, H., Yokoyama, T., Kato, H., Arakawa, Y.
(2005). Rapid and Simple Method for Detecting the Toxin B Gene of Clostridium difficile in Stool Specimens by Loop-Mediated Isothermal Amplification. J. Clin. Microbiol.
43: 6108-6112
[Abstract] [Full Text] -
Lemee, L., Bourgeois, I., Ruffin, E., Collignon, A., Lemeland, J.-F., Pons, J.-L.
(2005). Multilocus sequence analysis and comparative evolution of virulence-associated genes and housekeeping genes of Clostridium difficile. Microbiology
151: 3171-3180
[Abstract] [Full Text] -
Voth, D. E., Ballard, J. D.
(2005). Clostridium difficile Toxins: Mechanism of Action and Role in Disease. Clin. Microbiol. Rev.
18: 247-263
[Abstract] [Full Text] -
Goh, S., Chang, B. J, Riley, T. V
(2005). Effect of phage infection on toxin production by Clostridium difficile. J Med Microbiol
54: 129-135
[Abstract] [Full Text] -
Pituch, H., Rupnik, M., Obuch-Woszczatynski, P., Grubesic, A., Meisel-Mikolajczyk, F., Luczak, M.
(2005). Detection of binary-toxin genes (cdtA and cdtB) among Clostridium difficile strains isolated from patients with C. difficile-associated diarrhoea (CDAD) in Poland. J Med Microbiol
54: 143-147
[Abstract] [Full Text] -
Alonso, R, Martin, A, Pelaez, T, Marin, M, Rodriguez-Creixems, M, Bouza, E
(2005). Toxigenic status of Clostridium difficile in a large Spanish teaching hospital. J Med Microbiol
54: 159-162
[Abstract] [Full Text] -
Arroyo, L. G, Kruth, S. A, Willey, B. M, Staempfli, H. R, Low, D. E, Weese, J S.
(2005). PCR ribotyping of Clostridium difficile isolates originating from human and animal sources. J Med Microbiol
54: 163-166
[Abstract] [Full Text] -
Kato, H., Yokoyama, T., Arakawa, Y.
(2005). Typing by sequencing the slpA gene of Clostridium difficile strains causing multiple outbreaks in Japan. J Med Microbiol
54: 167-171
[Abstract] [Full Text] -
van den Berg, R. J, Ameen, H. A., Furusawa, T., Claas, E. C., van der Vorm, E. R, Kuijper, E. J
(2005). Coexistence of multiple PCR-ribotype strains of Clostridium difficile in faecal samples limits epidemiological studies. J Med Microbiol
54: 173-179
[Abstract] [Full Text] -
Lemee, L., Dhalluin, A., Testelin, S., Mattrat, M.-A., Maillard, K., Lemeland, J.-F., Pons, J.-L.
(2004). Multiplex PCR Targeting tpi (Triose Phosphate Isomerase), tcdA (Toxin A), and tcdB (Toxin B) Genes for Toxigenic Culture of Clostridium difficile. J. Clin. Microbiol.
42: 5710-5714
[Abstract] [Full Text] -
Snell, H., Ramos, M., Longo, S., John, M., Hussain, Z.
(2004). Performance of the TechLab C. DIFF CHEK-60 Enzyme Immunoassay (EIA) in Combination with the C. difficile Tox A/B II EIA Kit, the Triage C. difficile Panel Immunoassay, and a Cytotoxin Assay for Diagnosis of Clostridium difficile-Associated Diarrhea. J. Clin. Microbiol.
42: 4863-4865
[Abstract] [Full Text] -
Terhes, G., Urban, E., Soki, J., Hamid, K. A., Nagy, E.
(2004). Community-Acquired Clostridium difficile Diarrhea Caused by Binary Toxin, Toxin A, and Toxin B Gene-Positive Isolates in Hungary. J. Clin. Microbiol.
42: 4316-4318
[Abstract] [Full Text] -
Geric, B., Rupnik, M., Gerding, D. N., Grabnar, M., Johnson, S.
(2004). Distribution of Clostridium difficile variant toxinotypes and strains with binary toxin genes among clinical isolates in an American hospital. J Med Microbiol
53: 887-894
[Abstract] [Full Text] -
Lemee, L., Dhalluin, A., Pestel-Caron, M., Lemeland, J.-F., Pons, J.-L.
(2004). Multilocus Sequence Typing Analysis of Human and Animal Clostridium difficile Isolates of Various Toxigenic Types. J. Clin. Microbiol.
42: 2609-2617
[Abstract] [Full Text] -
van den Berg, R. J., Claas, E. C. J., Oyib, D. H., Klaassen, C. H. W., Dijkshoorn, L., Brazier, J. S., Kuijper, E. J.
(2004). Characterization of Toxin A-Negative, Toxin B-Positive Clostridium difficile Isolates from Outbreaks in Different Countries by Amplified Fragment Length Polymorphism and PCR Ribotyping. J. Clin. Microbiol.
42: 1035-1041
[Abstract] [Full Text] -
Blake, J. E., Mitsikosta, F., Metcalfe, M. A.
(2004). Immunological detection and cytotoxic properties of toxins from toxin A-positive, toxin B-positive Clostridium difficile variants. J Med Microbiol
53: 197-205
[Abstract] [Full Text] -
Ozaki, E., Kato, H., Kita, H., Karasawa, T., Maegawa, T., Koino, Y., Matsumoto, K., Takada, T., Nomoto, K., Tanaka, R., Nakamura, S.
(2004). Clostridium difficile colonization in healthy adults: transient colonization and correlation with enterococcal colonization. J Med Microbiol
53: 167-172
[Abstract] [Full Text] -
Johnson, S., Sambol, S. P., Brazier, J. S., Delmee, M., Avesani, V., Merrigan, M. M., Gerding, D. N.
(2003). International Typing Study of Toxin A-Negative, Toxin B-Positive Clostridium difficile Variants. J. Clin. Microbiol.
41: 1543-1547
[Abstract] [Full Text] -
Rupnik, M., Kato, N., Grabnar, M., Kato, H.
(2003). New Types of Toxin A-Negative, Toxin B-Positive Strains among Clostridium difficile Isolates from Asia. J. Clin. Microbiol.
41: 1118-1125
[Abstract] [Full Text] -
Wilkins, T. D., Lyerly, D. M.
(2003). Clostridium difficile Testing: after 20 Years, Still Challenging. J. Clin. Microbiol.
41: 531-534
[Full Text] -
Belanger, S. D., Boissinot, M., Clairoux, N., Picard, Francois. J., Bergeron, M. G.
(2003). Rapid Detection of Clostridium difficile in Feces by Real-Time PCR. J. Clin. Microbiol.
41: 730-734
[Abstract] [Full Text] -
Barbut, F., Lalande, V., Burghoffer, B., Thien, H. V., Grimprel, E., Petit, J.-C.
(2002). Prevalence and Genetic Characterization of Toxin A Variant Strains of Clostridium difficile among Adults and Children with Diarrhea in France. J. Clin. Microbiol.
40: 2079-2083
[Abstract] [Full Text] -
Johnson, S., Kent, S. A., O'Leary, K. J., Merrigan, M. M., Sambol, S. P., Peterson, L. R., Gerding, D. N.
(2001). Fatal Pseudomembranous Colitis Associated with a Variant Clostridium difficile Strain Not Detected by Toxin A Immunoassay. ANN INTERN MED
135: 434-438
[Abstract] [Full Text] -
KATO, H., KITA, H., KARASAWA, T., MAEGAWA, T., KOINO, Y., TAKAKUWA, H., SAIKAI, T., KOBAYASHI, K., YAMAGISHI, T., NAKAMURA, S.
(2001). Colonisation and transmission of Clostridium difficile in healthy individuals examined by PCR ribotyping and pulsed-field gel electrophoresis. J Med Microbiol
50: 720-727
[Abstract] [Full Text] -
Sambol, S. P., Merrigan, M. M., Lyerly, D., Gerding, D. N., Johnson, S.
(2000). Toxin Gene Analysis of a Variant Strain of Clostridium difficile That Causes Human Clinical Disease. Infect. Immun.
68: 5480-5487
[Abstract] [Full Text] -
Moncrief, J. S., Zheng, L., Neville, L. M., Lyerly, D. M.
(2000). Genetic Characterization of Toxin A-Negative, Toxin B-Positive Clostridium difficile Isolates by PCR. J. Clin. Microbiol.
38: 3072-3075
[Abstract] [Full Text] -
Alfa, M. J., Kabani, A., Lyerly, D., Moncrief, S., Neville, L. M., Al-Barrak, A., Harding, G. K. H., Dyck, B., Olekson, K., Embil, J. M.
(2000). Characterization of a Toxin A-Negative, Toxin B-Positive Strain of Clostridium difficile Responsible for a Nosocomial Outbreak of Clostridium difficile-Associated Diarrhea. J. Clin. Microbiol.
38: 2706-2714
[Abstract] [Full Text] -
Limaye, A. P., Turgeon, D. K., Cookson, B. T., Fritsche, T. R.
(2000). Pseudomembranous Colitis Caused by a Toxin A- B+ Strain of Clostridium difficile. J. Clin. Microbiol.
38: 1696-1697
[Abstract] [Full Text]
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