Rapid molecular characterization of Clostridium difficile and assessment of populations of C. difficile in stool specimens.
ABSTRACT Our laboratory has developed testing methods that use real-time PCR and pyrosequencing analysis to enable the rapid identification of potential hypervirulent Clostridium difficile strains. We describe a real-time PCR assay that detects four C. difficile genes encoding toxins A (tcdA) and B (tcdB) and the binary toxin genes (cdtA and cdtB), as well as a pyrosequencing assay that detects common deletions in the tcdC gene in less than 4 h. A subset of historical and recent C. difficile isolates (n = 31) was also analyzed by pulsed-field gel electrophoresis to determine the circulating North American pulsed-field (NAP) types that have been isolated in New York State. Thirteen different NAP types were found among the 31 isolates tested, 13 of which were NAP type 1 strains. To further assess the best approach to utilizing our conventional and molecular methods, we studied the populations of C. difficile in patient stool specimens (n = 23). Our results indicated that 13% of individual stool specimens had heterogeneous populations of C. difficile when we compared the molecular characterization results for multiple bacterial isolates (n = 10). Direct molecular analysis of stool specimens gave results that correlated well with the results obtained with cultured stool specimens; the direct molecular analysis was rapid, informative, and less costly than the testing of multiple patient stool isolates.
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ABSTRACT: Clostridium difficile (CD)-associated diarrhea is a well-recognized complication of antibiotic use. Historically, diagnosing CD had been difficult as antigen assays are insensitive and culture-based methods require several days to yield results. Nucleic acid amplification tests (NAAT) are quickly becoming the standard of care. We compared the performance of two automated Investigational/Research-Use-Only (IUO/RUO) NAAT for the detection of CD toxin genes, the IMDx C. difficile for Abbott m2000 Assay (IMDx) and the BD MAX™ Cdiff Assay (MAX). A prospective analysis of 111 stool specimens received in the laboratory for CD testing by the laboratory's test of record (TOR), BD GeneOhm™ Cdiff Assay, and a retrospective analysis of 88 specimens previously determined to be positive for CD, were included in this study. One prospective specimen was excluded due to loss to follow-up discrepancy analysis. Of the remaining 198 specimens, 90 were positive by all three methods; 9 were positive by TOR and MAX, and 3 were positive by TOR only. One negative specimen was initially inhibitory by MAX. The remaining 95 specimens were negative by all methods. Toxigenic CD culture was performed on the 12 discrepant samples. True CD positive was defined as either positive by all three amplification assays or positive by toxigenic culture. Based on this definition, the sensitivity and specificity were 96.9% and 95% for MAX and 92.8% and 100% for IMDx. In summary, both highly automated systems demonstrated excellent performance and each have individual benefits, which will ensure that they will both have a niche in clinical laboratories.Journal of clinical microbiology 02/2014; · 4.23 Impact Factor
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ABSTRACT: The incidence and severity of Clostridium difficile infection (CDI) has been increasing and long-term care facility (LTCF) residents are at high risk given their age, co-morbidities, and high antibiotic exposure. Infection control policies are crucial for controlling CDI, but there are currently no regulatory guidelines in the United States. Therefore, we evaluated infection control policies in local LTCFs to define the CDI-specific policies and the administrative and staff understanding of CDI, so as to identify perceived barriers for compliance.International Journal of Clinical Medicine 04/2014; 5(7):414-419.
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ABSTRACT: The presence of gene 16S rRNA and genes encoding toxin A (tcdA), toxin B (tcdB), and binary toxin (cdtA/cdtB) of Clostridium difficile in stool samples from children with (110) and without (150) diarrhea was determined by using a TaqMan system. Fifty-seven (21.9%) out of 260 stool samples harbored the 16S rRNA gene. The genetic profile of tcdA+/tcdB- and cdtA+/cdtB+ was verified in one C. difficile-positive diarrhea sample and of tcdA+/tcdB+ in three C. difficile-positive nondiarrhea samples. The presence of tcdA+/tcdB+ in stools obtained from children without diarrhea, suggests that they were asymptomatic carriers of toxigenic strains.Scientifica. 01/2014; 2014:594014.
JOURNAL OF CLINICAL MICROBIOLOGY, July 2009, p. 2142–2148
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 47, No. 7
Rapid Molecular Characterization of Clostridium difficile and
Assessment of Populations of C. difficile in Stool Specimens?
Danielle Wroblewski,1George E. Hannett,1Dianna J. Bopp,1Ghinwa K. Dumyati,2Tanya A. Halse,1
Nellie B. Dumas,1and Kimberlee A. Musser1*
Wadsworth Center, New York State Department of Health, Albany, New York,1and University of Rochester Medical Center,
Rochester, New York2
Received 29 December 2008/Returned for modification 15 February 2009/Accepted 20 April 2009
Our laboratory has developed testing methods that use real-time PCR and pyrosequencing analysis to enable
the rapid identification of potential hypervirulent Clostridium difficile strains. We describe a real-time PCR
assay that detects four C. difficile genes encoding toxins A (tcdA) and B (tcdB) and the binary toxin genes (cdtA
and cdtB), as well as a pyrosequencing assay that detects common deletions in the tcdC gene in less than 4 h.
A subset of historical and recent C. difficile isolates (n ? 31) was also analyzed by pulsed-field gel electro-
phoresis to determine the circulating North American pulsed-field (NAP) types that have been isolated in New
York State. Thirteen different NAP types were found among the 31 isolates tested, 13 of which were NAP type
1 strains. To further assess the best approach to utilizing our conventional and molecular methods, we studied
the populations of C. difficile in patient stool specimens (n ? 23). Our results indicated that 13% of individual
stool specimens had heterogeneous populations of C. difficile when we compared the molecular characterization
results for multiple bacterial isolates (n ? 10). Direct molecular analysis of stool specimens gave results that
correlated well with the results obtained with cultured stool specimens; the direct molecular analysis was rapid,
informative, and less costly than the testing of multiple patient stool isolates.
Clostridium difficile is one of the leading causes of infectious
antibiotic-associated diarrhea and pseudomembranous colitis
worldwide (2, 16). This is illustrated by the increased incidence
and severity of C. difficile infection, suggesting the emergence
of a new hypervirulent strain (5, 13–15, 17, 25, 32).
While TcdB, a cytotoxin, is the known established virulence
factor of C. difficile, toxin A (TcdA), a cytotoxic enterotoxin,
works synergistically with TcdB, causing damage to the intes-
tinal mucosa in cases of C. difficile infection (17). The genes
that encode these toxins are located on the pathogenicity locus
of C. difficile (4, 10, 24). Additionally, several deletions in the
tcdC gene, a putative negative regulator of the expression of
the toxin A (tcdA) and the toxin B (tcdB) genes, have been
identified, and these deletions result in higher levels of cyto-
toxin expression (11). Furthermore, research has shown that
some C. difficile strains produce another toxin, known as the
binary toxin (19, 22, 28). The genes that encode this toxin, cdtA
and cdtB, together produce an actin-specific ADP-ribosyltrans-
ferase that induces damage to the actin skeleton, leading to
cytopathic effects in cell lines (1). It has been suggested that
the binary toxin genes and deletions in the tcdC gene are
potential virulence factors in the recent emerging hyperviru-
lent strain (22, 29).
The “gold standard” for the detection of C. difficile toxin
production is a cytotoxin assay with stool specimens or isolates
from anaerobic culture. The cytotoxin assay is difficult to per-
form and time-consuming, and it is often less sensitive than
molecular assays (20, 23, 26). Enzyme immunoassays (EIAs)
are used most often, and recent reports suggest that manufac-
turers have improved the performance of EIA kits since their
introduction; however, the disadvantages of EIAs include the
lower levels of sensitivity and specificity compared to those of
the gold standard methods. More importantly, culture is not
specific for the identification of toxigenic strains. The labora-
tory at the Wadsworth Center has developed a multiplex real-
time PCR assay and a tcdC gene pyrosequencing assay that
rapidly identify potential virulence factors of C. difficile strains
and that can be used to directly test patient stool specimens for
(Part of this report was presented at the 107th American
Society for Microbiology General Meeting in 2007 [Toronto,
MATERIALS AND METHODS
Bacterial strains, clinical specimens, and specimen processing. The bacterial
strains utilized in this study included a specificity panel of 55 Clostridium difficile
isolates, gastrointestinal flora, and other bacterial pathogens, which are listed
with their sources in Table 1. Included in this panel were 30 strains of C. difficile
with known toxin gene profiles. Ten strains were positive for only the clostridial
toxins (tcdA, tcdB), 10 strains were positive for all four toxin genes (tcdA, tcdB,
cdtA, cdtB), and 10 were nontoxigenic. Archived frozen specimens from several
hospitals were also tested. Thirty-six isolates from stool specimens received by
our laboratory between 1990 and 1992 were from a Veterans Affairs facility in
Albany, NY (18, 21), and 113 stool specimens from 2005 to the present came
from several different hospitals in New York State. In total, 149 specimens were
C. difficile strains were plated on CDC anaerobe agar and grown at 35°C under
anaerobic conditions for 48 h. For molecular testing, isolated colonies were
suspended in 1? PCR buffer (Applied Biosystems, Foster City, CA) at a level
equivalent to a 1 McFarland standard and heat treated at 95°C for 20 min. The
specimens were either used directly in the PCR or stored at ?20°C until use.
Direct, uncultured stool specimens were subjected to nucleic acid extraction with
* Corresponding author. Mailing address: Wadsworth Center, New
York State Department of Health, P.O. Box 22002, Albany, NY 12201-
2002. Phone: (518) 474-4177. Fax: (518) 486-7971. E-mail: musser
?Published ahead of print on 29 April 2009.
the QIAamp DNA stool minikit by use of a QIAcube (Qiagen, Valencia, CA),
according to the manufacturer’s instructions.
Real-time PCR primer and probe design. Unique primer and probe sets
targeting C. difficile enterotoxin A (tcdA), cytotoxin B (tcdB), and binary toxin
genes A and B (cdtA and cdtB, respectively) were designed by using Primer
Express (version 2.0) software (Applied Biosystems) on the basis of the se-
quences of strains Clostridium difficile VPI 10463 (NCBI accession number
X92982, ATCC 43255) and Clostridium difficile CD196. The tcdA and tcdB
primers and probes were selected so that they did not overlap known variable
regions. The cdtA and cdtB primers and probes were designed to bind to the full
4.3-kb fragment which encodes the functional binary toxin gene, avoiding areas
of homology to box 1, box 2, and box 3 found in all C. difficile strains (22). The
primers and probe sequences are summarized in Table 2. BLAST (the Basic
Local Alignment Search Tool) searches were performed with all selected poten-
tial primer and probe oligonucleotides to test that no cross-reaction would occur
with other human or microbial pathogens. The Amplify (version 3.0) program
was utilized to determine any possible primer-dimer interactions among the
oligonucleotides. Probes TcdA-P, CdtB-P, and CdtA-P and all primers were
synthesized by Integrated DNA Technologies (Coralville, IA). Probes TcdB-P
and Inhib-P were obtained from Applied Biosystems.
Real-time multiplex PCR assay. All isolates were tested in duplicate, and the
DNA from stool specimens was tested in triplicate. The real-time PCR assay was
performed on an ABI 7500 Fast real-time PCR system (Applied Biosystems) in
a 96-well optical plate format with a LightCycler-FastStart DNA master hybrid-
ization probes kit (Roche Diagnostics Corp, Indianapolis, IN). For testing of the
isolates, each 25-?l reaction mixture consisted of 1? LightCycler-FastStart DNA
master hybridization probes, 3.0 mM MgCl2, 900 nM of all eight primers (Table
2), 250 nM of all four probes (Table 2), sterile water, and 10 ?l DNA template.
For the testing of stool samples, probes TcdA-P, TcdB-P, and CdtA-P were
initially added to the mixture. The master mixture was then split, and probe
CdtB-P was added to half of the master mixture for the testing of the DNA
isolated from the stool and probe Inhib-P was added to the other half of the
master mixture; the latter portion was then seeded with the internal inhibition
control, for the assessment of inhibition. The thermal cycling conditions were as
follows: 1 cycle of 95°C for 10 min, followed by 40 cycles (45 cycles for testing of
stool specimens) at 95°C for 15 s and 60°C for 1 min. The data analysis was
performed according to the manufacturer’s instructions, with no passive refer-
ence dye being used.
Sensitivity and specificity. The analytical sensitivity of the real-time PCR was
determined for each target through the use of 10-fold dilutions of DNA from a
hypervirulent strain of C. difficile (strain NYS BAC-05-3671) with known densi-
ties and by the generation of standard curves. The sensitivity of detection in stool
specimens was determined by spiking stool specimens known to be negative with
known densities of C. difficile cells, processing them according to the protocol,
and testing the purified DNA by real-time PCR. The specificity of the assay was
determined by testing the DNA from 55 organisms, as shown in Table 1.
Internal inhibition control. The DNA isolated from stool specimens was also
tested for the presence of PCR inhibitors. An internal control plasmid was
constructed from a PCR product that contained a portion of heterologous DNA
flanked by the primer binding sites of the cdtA gene. This PCR product was
cloned into a plasmid vector (pCR2.1-TOPO) with a TOPO TA cloning kit
(Invitrogen Life Technologies, Carlsbad, CA). The plasmid was diluted to yield
a threshold cycle (CT) value of approximately 30. The control plasmid was added
to the PCR mixtures containing DNA from the stool specimen and was detected
by the use of a different fluorescent probe (Table 2). The CTvalue obtained from
this reaction was compared with that obtained from a second reaction containing
only the control plasmid. The reaction was considered to be inhibited if the CT
value of the reaction was 3 CTunits or more greater than that for the reaction
containing only the control plasmid or if no amplification was found.
tcdC sequence analysis by Sanger sequencing. To determine whether deletions
were present or absent, we performed a conventional PCR and then sequenced
the amplified products. A primer pair was adapted to target the tcdC gene (24);
it is shown in Table 2. Each 100-?l amplification reaction mixture consisted of
1? PCR buffer; 2.5 mM MgCl2; 10 mM each of dATP, dCTP, dTTP, and dGTP;
sterile water; 50 ?M of primers C1 and C2; and 2.5 U of AmpliTaq Gold
(Applied Biosystems). The PCR products were analyzed on a 2% E-gel (Invitro-
gen Life Technologies) with a 1-kb DNA marker (Invitrogen Life Technologies)
and were visualized under UV light by using a Bio-Rad Laboratories (Hercules,
CA) Gel Doc XR apparatus. The PCR products were purified with ExoSAP-IT
(USB, Cleveland, OH) and were sequenced by the Applied Genomic Technol-
ogies Core facility at the Wadsworth Center. The resultant sequences were
compared to the sequence of the tcdC gene (NCBI accession number X92982 for
C. difficile strain VPI 10463) to determine the presence or absence of tcdC
tcdC sequence analysis by pyrosequencing. A pyrosequencing protocol was
developed to determine whether the 1-bp, 18-bp, and/or 39-bp deletions within
a portion of the tcdC gene were present or absent. Amplification and sequencing
primers were designed with Assay Design software (Qiagen; previously Biotage).
Briefly, a 378-bp PCR product was amplified with forward primer tcdC-F, which
was labeled with biotin for immobilization onto streptavidin-coated Sepharose
beads. Two separate sequencing primers (primers TcdC-S1 and TcdC-S2) were
used to assess the regions where the 1-bp and 18-bp deletions may be present. If
the 18-bp pyrosequencing reaction failed, a third sequencing primer (primer
TcdC-S3) was used to assess the sample for the 39-bp deletion, since the binding
region of the 18-bp sequencing primer falls within the region of the 39-bp
deletion. Primers TcdC-F and TcdC-R were synthesized by Integrated DNA
Technologies and are shown in Table 2. The locations of the sequences of the
PCR primers and the sequencing primers within the tcdC gene are shown in Fig.
1. The amplification reaction mixtures consisted of 1? PCR buffer; 2.0 mM
MgCl2; 10 mM each of dATP, dCTP, dTTP, and dGTP; sterile water; 200 nM of
primers TcdC-F and TcdC-R; 2.5 U of AmpliTaq Gold (Applied Biosystems);
and 10 ?l of template DNA in a final volume of 100 ?l. The thermocycling
conditions in a Bio-Rad Laboratories iCycler apparatus were as follows: 1 cycle
of 5 min at 95°C, followed by 50 cycles of 30 s at 95°C, 30 s at 50°C, and 30 s at
72°C and a final extension for 5 min at 72°C. Pyrosequencing was performed with
Pyro Gold reagents on a PyroMark vacuum prep workstation and a PyroMark ID
instrument according to the manufacturer’s instructions (Qiagen). The specific
dispensation order for the 1-bp, 18-bp, and 39-bp deletions was GCTGAATAT,
GTTAGCTCTCAGCTAGCT, and GCTCTCTCTCTCT, respectively, with sin-
gle nucleotide polymorphism analysis of the nucleotides.
PFGE. A selection of 31 strains of C. difficile was additionally examined by
pulsed-field gel electrophoresis (PFGE) to preliminarily assess the North Amer-
ican pulsed-field (NAP) types in our collection and for comparison of the NAP
types to potential virulence characteristics. These strains were chosen from
TABLE 1. Bacterial organisms used for specificity testing of
multiplex real-time PCR for C. difficile toxin genes
Bacteroides fragilis...............................................................ATCC 25285
Clostridium botulinum type A ...........................................NYSDOH
Clostridium botulinum type B............................................NYSDOH
Clostridium botulinum type E............................................NYSDOH
Clostridium botulinum type F............................................ATCC 35415
Clostridium difficile (binary toxin gene negative)c..........NYSDOH
Clostridium difficile (nontoxigenic)d..................................NYSDOH
Clostridium perfringens........................................................ATCC 13124
Enterobacter aerogenes........................................................ATCC 13048
Enterobacter cloacae ...........................................................ATCC 13047
Enterobacter lentum ............................................................ATCC 43055
Enterococcus faecalis ..........................................................ATCC 51299
Enterococcus faecium..........................................................ATCC 12477
Escherichia coli....................................................................ATCC 12799
Klebsiella pneumoniae.........................................................ATCC 700603
Salmonella enterica serovar Enteritidis............................NYSDOH
Vibrio parahaemolyticus......................................................ATCC 275519
aATCC, American Type Culture Collection; NYSDOH, New York State
Department of Health.
bTen C. difficile strains containing all four toxin genes (tcdA, tcdB, cdtA, cdtB)
cTen C. difficile strains containing only the clostridial toxins (tcdA, tcdB) were
dTen nontoxigenic C. difficile strains were tested.
VOL. 47, 2009MOLECULAR CHARACTERIZATION OF CLOSTRIDIUM DIFFICILE2143
among strains submitted between 1980 and 2008 and included strains that we had
determined harbored the tcdA and tcdB toxin genes or the tcdA, tcdB, cdtA, and
cdtB toxin genes. DNA was prepared from C. difficile cells grown for 7 h in
reduced peptone-yeast-glucose broth at 37°C, according to established protocols
(12). DNA was digested with restriction endonuclease SmaI (New England
Biolabs, Beverly, MA), and the fragments were separated in 1.0% agarose gels
on a clamped homogeneous electric field apparatus (CHEF Mapper; Bio-Rad
Laboratories, Richmond, CA). The initial pulse time of 5.0 s was increased to
40.0 s over 19.5 h. The gels were stained with ethidium bromide, destained in
distilled water, and visualized with a Gel Doc 2000 gel analysis system (Bio-Rad
Laboratories). The PFGE patterns were analyzed with Bionumerics software
(Applied Maths, Belgium). Dendrograms were created by use of unweighted pair
group similarity and arithmetic mean with Dice coefficients, and the position
tolerance was set at 1.1%. NAP types were determined by comparison with
TABLE 2. Primers and probes used for real-time PCR, Sanger sequencing, and pyrosequencing
Probe sequence (5?–3?)a
aBHQ-2, Black Hole Quencher 2; MGB, Minor Groove Binder; FAM, 6-carboxyfluorescein; NED, 2?-chloro-5?-fluoro-7?,8?-fused phenyl-1,4-dichloro-6-carboxy-
bOn the basis of sequences in GenBank with accession number X92982 for tcdA, tcdB, and tcdC.
cOn the basis of sequences in GenBank with accession number L76081 for cdtA and cdtB.
dAs described previously (24).
eNA, not applicable.
FIG. 1. Design for amplification and pyrosequencing of the tcdC gene. A diagram of a portion of the tcdC gene used to detect the 1-bp, 18-bp,
and 39-bp tcdC gene deletions found in strains of Clostridium difficile is shown. The sequence from NCBI accession number DQ870674 (C. difficile
strain ATCC 43594) was used, and the forward and reverse primer locations are shaded in gray. Deletion locations are shown in boldface and in
brackets, with the 1-bp and 18-bp deletions being underlined. The 39-bp deletion is shown with a dotted line over it. Sequencing primers S1, S2,
and S3 are boxed and have arrows indicating the direction of sequencing.
2144WROBLEWSKI ET AL. J. CLIN. MICROBIOL.
known NAP types. An isolate with ?80% similarity to a known NAP type was
considered to belong to that NAP type (11).
Assessment of C. difficile populations in patient specimens. Twenty-three stool
specimens from cases of presumed community-associated C. difficile infection
were examined to determine whether the patients studied had homogeneous
populations of C. difficile. The definition of presumed community-associated C.
difficile infection was a positive EIA result for C. difficile toxin with a specimen
collected when the patient was as an outpatient or within 72 h of patient admis-
sion to a hospital and with no evidence of hospitalization in the preceding 3
months. “Hospitalization” was defined as an overnight stay in a hospital or other
skilled nursing facility. The patients were surveyed for antibiotic use preceding C.
difficile infection, but antibiotic use was not used as an exclusion criterion.
Specimens were collected and stored frozen until culture could be performed.
Twenty microliters of thawed stool specimen was inoculated onto in-house-
prepared Clostridium difficile selective agar and cycloserine-cefoxitin-fructose
agar with antibiotic concentrations of 480 ?g/ml for cycloserine and 15.4 ?g/ml
for cefoxitin (9). The media were not prereduced before they were plated. The
plates were incubated for 4 days at 37°C under anaerobic conditions. Up to 10
colonies from each specimen were subcultured to CDC anaerobe agar. One
colony from each specimen was identified as C. difficile by a battery of conven-
tional biochemical tests, including Gram staining and tests for anaerobic growth
requirement, motility, catalase production, indole reaction, nitrate reduction,
H2S production, growth in bile, lipase production, lecithinase production, and
urease production. The isolates were also assayed for their abilities to ferment
carbohydrates; the substrates included glucose, maltose, mannitol, lactose, su-
crose, xylose, salicin, arabinose, and glycerol. Cellular fatty acid analysis was also
performed by using the Microbial Identification System (Midi Inc., Newark, DE).
The remaining isolates from each specimen were identified only by Gram stain-
ing, a test for anaerobic growth requirement, and cellular fatty acid analysis.
Each isolate was then assessed for the presence of potential virulence factors by
the molecular methods described above. In a further prospective analysis, two
isolates from each of the 89 patient samples were examined in a continuation of
our assessment of C. difficile bacterial populations and virulence characteristics.
Sensitivity and specificity of real-time PCR assay. The an-
alytical results obtained with the isolated colonies indicate that
the real-time PCR assay evaluated in the present study has a
limit of detection of 1 CFU per reaction mixture. The sensi-
tivity of both the real-time PCR assay and pyrosequencing by
direct testing of stool specimens was determined to be 10 CFU
per reaction mixture (results not shown). In a study that com-
pared the results of the real-time PCR assay to those of the
gold standard cytotoxin assay, the real-time assay was found to
be 100% sensitive (results not shown). The specificity of the
real-time PCR assay was determined to be 100%, as the C.
difficile strains assessed in the multiplex real-time PCR were
positive for the respective genes (tcdA and tcdB or tcdA, tcdB,
cdtA, and cdtB) and all non-C. difficile bacterial isolates and
nontoxigenic C. difficile were negative for all four genes, as
Analysis of deletions in tcdC gene by Sanger sequencing and
pyrosequencing. The tcdC gene from 154 isolates was ampli-
fied and sequenced by both the pyrosequencing and the Sanger
sequencing methods to determine the presence or absence of
the 1-bp, 18-bp, and/or 39-bp deletions. Comparison of the
results obtained by the two tests revealed a 100% correlation
between the results of the two methods (results not shown).
Analysis of C. difficile isolates by PFGE. Figure 2 summa-
rizes the results of PFGE analysis of C. difficile isolates to
obtain a preliminary understanding of the NAP types circulat-
ing in New York State. Thirteen NAP type 1 (NAP1) types
were identified in the group and possessed all four toxin genes
and both the 1-bp and 18-bp tcdC gene deletions. Eight addi-
tional NAP types were also identified (NAP2, NAP3, NAP4,
NAP5, NAP8, NAP10, NAP11, NAP12). All the isolates with
NAP types 2, 3, 4, 5, and 10 and undesignated NAP types
CldS16015 and CldS16006 were C. difficile isolates that con-
tained no binary toxin genes or tcdC gene deletions. Interest-
ingly, NAP types 8, 11, and 12, as well as two others, types
CldS16002 and CldS16005, were found to have some or all of
the potential virulence characteristics. The NAP8 (2008) strain
was positive for all toxin genes and the tcdC gene 18-bp dele-
tion. The NAP11 (2006) and NAP12 (1990, 1992) strains were
negative for the binary toxin genes but positive for the tcdC
gene 18-bp deletion. Two strains of types CldS16002 and
CldS16005 (2006), respectively, differed from the strains des-
ignated NAP1 by four bands but were found to be positive for
all toxin genes and for the tcdC gene 1-bp and 18-bp deletions.
Assessment of C. difficile populations in patient specimens.
Twenty-three stool specimens were examined by culture, real-
time multiplex PCR, as well as Sanger sequencing and pyro-
sequence analysis for tcdC gene deletions. All stool specimens
were initially found to be positive for C. difficile by culture. One
hundred ninety-four colonies from the 23 specimens were ex-
amined. The data in Table 3 demonstrate that of the 194
strains isolated, 3 strains (1.5%) exhibited a molecular profile
different from that of the other strains isolated from the same
individual’s stool specimen. Analysis of the stool specimens
revealed that three stool specimens (13%) did not contain
homogeneous populations. In two of these stool specimens
(specimens 4 and 15), the difference was the presence of the
binary toxin genes in one or more of the isolates of the pa-
tient’s stool specimens. For the third specimen (specimen 1),
the difference was the absence of both the binary toxin genes
and the tcdC gene deletion in 1 of 10 isolates from the same
patient’s stool specimen. Overall, among the 89 stool speci-
mens tested in this prospective analysis (two isolates per stool
specimen), no differences in their molecular profiles were iden-
tified, as illustrated in Table 4.
Direct molecular analysis of patient stool specimens. The C.
difficile population data led us to evaluate the potential benefits
of direct molecular analysis of stool specimens. A subset of 24
stool specimens was directly tested by the multiplex real-time
PCR and pyrosequencing. The preliminary data showed a cor-
relation between the results of direct testing and those of
testing following culture. For 23 (95.8%) of the stool speci-
mens tested, identical results and molecular profiles were ob-
tained. However, one stool specimen was negative by direct
testing, while culture of that stool specimen identified two
colonies of C. difficile.
Although the binary toxin genes and the tcdC gene deletions
and their contribution to the virulence of C. difficile strains
causing infections are still poorly understood, characterization
may be important for infection control and a proper public
health response. Currently, only a few real-time PCR assays
are available for the detection of toxins A and B (3, 6, 23, 31).
We have developed a sensitive and specific one-tube, real-time
PCR assay capable of determining in less than 2 h whether
toxins A and B (tcdA and tcdB) and the binary toxins (cdtA and
cdtB) are present. Pyrosequencing is a novel technique that we
have implemented for the detection of the deletions in the
VOL. 47, 2009MOLECULAR CHARACTERIZATION OF CLOSTRIDIUM DIFFICILE2145
tcdC gene. Studies have previously shown that the deletions
that have been described in the tcdC gene (7, 17, 23, 27) may
be responsible for increases in toxin production. Two dele-
tions, a 39-bp deletion and an 18-bp deletion, were thought to
contribute to the enhancement of disease severity and high
rates of morbidity; however, newer investigations suggest that
a 1-bp deletion in the tcdC gene may be the cause of the
overproduction of the toxins (7). Utilization of these assays
may be informative and may help to provide an understanding
of the roles of these genetic factors in C. difficile infections.
Following several outbreaks in 2003, further characteriza-
tion of the hypervirulent strain of C. difficile determined that it
is PCR ribotype 027 (PCR ribotyping), NAP1 (PFGE), restric-
tion endonuclease analysis type BI, and toxinotype III (toxino-
typing) (5, 13, 16). In our analysis of the NAP types circulating
in New York State, we found that NAP1 strains were first
FIG. 2. Molecular characterization of Clostridium difficile from patient specimens (1980 to 2008) by multiplex real-time PCR, sequence analysis
of the tcdC gene, and PFGE analysis.a, the tcdA and tcdB genes were detected by real-time PCR;b, the cdtA and cdtB genes were detected by
real-time PCR;c, CldS is the abbreviation used in our database to designate a Clostridium difficile subtype;d, NAP is the abbreviation used for
North American pulsed-field type;e, this strain, received by our laboratory in 1980, was a control strain provided by the CDC for our C. difficile
2146 WROBLEWSKI ET AL. J. CLIN. MICROBIOL.
isolated in 2005. The NAP1 strains that we tested were isolated
in recent years, and most contained the four toxin genes in
combination with the 1-bp and 18-bp tcdC deletions. Retro-
spectively, we were also interested in assessing the molecular
profiles of strains that had caused major outbreaks at a local
facility in 1990 and 1992. We found several NAP strains that
contained the 18-bp tcdC deletion; the other two NAP types
were found to be negative for the binary toxin genes and tcdC
deletions. We also observed other NAP types that had subsets
of the genetic factors present; it is unclear whether the strains
identified as being unmatched to known NAP designations
have been recognized previously or whether they are novel.
These data illustrate that these molecular profiles have actually
been present in strains for a long time, yet it is only recently
that more virulent disease has been appreciated. More work
directed toward obtaining an understanding of the NAP types
circulating throughout the world and the morbidity and mor-
tality rates associated with each of the strains should continue
to shed light on the hypervirulent strain of C. difficile.
The assessment of the C. difficile populations within individ-
ual stool specimens demonstrated that the C. difficile isolates
recovered from a stool specimen are for the most part homo-
geneous in a patient with presumed community-associated C.
difficile infection (described above). However, the isolates in
13% of the stool specimens examined were not homogeneous
by our molecular methods, and this raises the question of
whether the determination of potential genetic factors from a
single C. difficile colony isolated from a stool specimen is the
best practice. We are not the first to show that more than one
strain can be present in a given stool specimen; however, con-
flicting data have been reported (30). In our analysis, we have
shown that for at least one case (stool specimen 1 in Table 3),
the possibility exists that the single colony possessing no binary
toxin genes or tcdC gene deletions could have been the one
isolated from the patient’s stool specimen, thus resulting in a
false-negative result for that patient. The further prospective
analysis of 89 additional stool specimens could have shed more
light on this question; however, no heterogeneity was identified
in that set of specimens.
The results of our testing have led us to conclude that char-
acterization of C. difficile directly from stool specimens is the
testing route of choice at the Wadsworth Center for the rapid
identification of putative genetic factors of C. difficile. The idea
of testing stool specimens directly is not a novel one (8). Our
preliminary data have shown that C. difficile could not be de-
tected by the direct method in 4% (1/24) of specimens. The
specimen in which C. difficile could not be detected by the
direct method was found, upon culture analysis, to have only
two colonies of C. difficile, which was below our limit of detec-
tion for stool specimens. We believe, on the basis of our data,
that direct testing of stool specimens combined with culture at
the start of testing is the most informative approach for labo-
ratories that have the ability to perform all of these methods.
If only PCR analysis of stool specimens is utilized, there is a
risk that a potentially highly virulent strain may fail to be
identified. If culture is routinely performed first, followed by
molecular analysis of one or even a few colonies from each
patient, there is still the possibility that the hypervirulent strain
will be missed among the typical C. difficile strains in a given
stool specimen. The latter approach can also lead to additional
costs because of the need to perform more than one molecular
test per specimen. An alternative approach, which we have
previously utilized for other pathogens, is to first perform mo-
lecular analysis as a screen and to then perform culture only
with the specimens with negative or inconclusive results.
In conclusion, the C. difficile multiplex real-time PCR assay
that we have developed is sensitive, specific, and rapid. Pyro-
sequencing of the tcdC gene is a novel and valuable tool for the
further characterization of C. difficile isolates that cause infec-
tions and that are detected in stool specimens. These molecu-
lar assays can be applied directly for the screening of patient
specimens and may provide valuable information that helps
provide an understanding of these genetic factors and the roles
that they may play in C. difficile infection.
TABLE 4. Prospective C. difficile stool specimen characterizationa
No. of stool
No. of specimens with the
following type of bacterial
tcdC deletions HomogeneousHeterogeneous
aTwo isolates from each stool specimen were analyzed.
TABLE 3. Testing of presumed community-associated C. difficile-
No. of strains
No. of strains with the following molecular profile:
aBoldface represents the results for the strains that exhibited a molecular
profile different from that of the other strains isolated from the same individual’s
VOL. 47, 2009MOLECULAR CHARACTERIZATION OF CLOSTRIDIUM DIFFICILE2147
We acknowledge the Wadsworth Center Applied Genomic Tech-
nologies Core Facility for Sanger sequencing of the PCR products and
Adriana Verschoor and Ron Limberger for critical reading of the
manuscript. We thank Angela Thompson from the CDC for her assis-
tance with determining the NAP types from our PFGE data and
Qiagen (Biotage) for technical assistance with the design of the pyro-
sequencing assay. We also acknowledge the technical support of
Yvette Khachadorian with PFGE analysis and the Wadsworth Center
Media Department for making the Clostridium difficile selective agar
and cycloserine-cefoxitin-fructose agar plates for stool culture.
Funding for this study was provided in part from the Emerging
Infections Program, CDC, Atlanta, GA.
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