Fourteen-genome comparison identifies DNA markers for severe-disease-associated strains of Clostridium difficile.

Abstract

Clostridium difficile is a common cause of infectious diarrhea in hospitalized patients. A severe and increased incidence of C. difficile infection (CDI) is associated predominantly with the NAP1 strain; however, the existence of other severe-disease-associated (SDA) strains and the extensive genetic diversity across C. difficile complicate reliable detection and diagnosis. Comparative genome analysis of 14 sequenced genomes, including those of a subset of NAP1 isolates, allowed the assessment of genetic diversity within and between strain types to identify DNA markers that are associated with severe disease. Comparative genome analysis of 14 isolates, including five publicly available strains, revealed that C. difficile has a core genome of 3.4 Mb, comprising ∼ 3,000 genes. Analysis of the core genome identified candidate DNA markers that were subsequently evaluated using a multistrain panel of 177 isolates, representing more than 50 pulsovars and 8 toxinotypes. A subset of 117 isolates from the panel had associated patient data that allowed assessment of an association between the DNA markers and severe CDI. We identified 20 candidate DNA markers for species-wide detection and 10,683 single nucleotide polymorphisms (SNPs) associated with the predominant SDA strain (NAP1). A species-wide detection candidate marker, the sspA gene, was found to be the same across 177 sequenced isolates and lacked significant similarity to those of other species. Candidate SNPs in genes CD1269 and CD1265 were found to associate more closely with disease severity than currently used diagnostic markers, as they were also present in the toxin A-negative and B-positive (A-B+) strain types. The genetic markers identified illustrate the potential of comparative genomics for the discovery of diagnostic DNA-based targets that are species specific or associated with multiple SDA strains.

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JOURNAL OF CLINICAL MICROBIOLOGY, June 2011, p. 2230–2238
0095-1137/11/$12.00doi:10.1128/JCM.00391-11
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 6
Fourteen-Genome Comparison Identifies DNA Markers for
Severe-Disease-Associated Strains of Clostridium difficile?†
Vincenzo Forgetta,1Matthew T. Oughton,2Pascale Marquis,3Ivan Brukner,2Ruth Blanchette,2
Kevin Haub,4Vince Magrini,4Elaine R. Mardis,4Dale N. Gerding,5Vivian G. Loo,6
Mark A. Miller,2Michael R. Mulvey,7Maja Rupnik,8
Andre Dascal,2and Ken Dewar1,3*
McGill University, Department of Human Genetics, Montreal, Canada1; Sir Mortimer B. Davis Jewish General Hospital, Montreal,
Canada2; McGill University and Ge ´nome Que ´bec Innovation Center, Montreal, Canada3; The Genome Center at Washington
University School of Medicine, St. Louis, Missouri4; Infectious Disease Section and Research Service, Department of
Medicine, Hines Veterans Affairs Hospital, and Loyola University Chicago Stritch School of Medicine, Hines, Illinois5;
McGill University Health Centre, Montreal, Canada6; National Microbiology Laboratory, Winnipeg, Manitoba,
Canada7; and Institute of Public Health Maribor and University of Maribor, Faculty of Medicine, Maribor, Slovenia8
Received 23 February 2011/Returned for modification 5 April 2011/Accepted 7 April 2011
Clostridium difficile is a common cause of infectious diarrhea in hospitalized patients. A severe and increased
incidence of C. difficile infection (CDI) is associated predominantly with the NAP1 strain; however, the
existence of other severe-disease-associated (SDA) strains and the extensive genetic diversity across C. difficile
complicate reliable detection and diagnosis. Comparative genome analysis of 14 sequenced genomes, including
those of a subset of NAP1 isolates, allowed the assessment of genetic diversity within and between strain types
to identify DNA markers that are associated with severe disease. Comparative genome analysis of 14 isolates,
including five publicly available strains, revealed that C. difficile has a core genome of 3.4 Mb, comprising
?3,000 genes. Analysis of the core genome identified candidate DNA markers that were subsequently evaluated
using a multistrain panel of 177 isolates, representing more than 50 pulsovars and 8 toxinotypes. A subset of
117 isolates from the panel had associated patient data that allowed assessment of an association between the
DNA markers and severe CDI. We identified 20 candidate DNA markers for species-wide detection and 10,683
single nucleotide polymorphisms (SNPs) associated with the predominant SDA strain (NAP1). A species-wide
detection candidate marker, the sspA gene, was found to be the same across 177 sequenced isolates and lacked
significant similarity to those of other species. Candidate SNPs in genes CD1269 and CD1265 were found to
associate more closely with disease severity than currently used diagnostic markers, as they were also present
in the toxin A-negative and B-positive (A-B?) strain types. The genetic markers identified illustrate the
potential of comparative genomics for the discovery of diagnostic DNA-based targets that are species specific
or associated with multiple SDA strains.
Clostridium difficile is the most common cause of infectious
diarrhea in hospitalized patients in the industrialized world (3,
23). With symptoms ranging from self-limited diarrhea to life-
threatening fulminant colitis, C. difficile infection (CDI) has
affected hundreds of thousands of patients worldwide and sub-
stantially burdens health care resources (29). In particular,
CDI has caused increased patient morbidity and mortality in
hospitals throughout the world since 2003, when outbreaks
with increased disease incidence and severity first emerged
(62), and from 2004 to 2007 contributed to almost 1,000 deaths
in the province of Quebec, Canada (16). Investigations of these
and other outbreaks across Canada, the United States, and
western Europe led to the recognition of a severe-disease-
associated (SDA) strain predominantly responsible for this
epidemic (27, 32). This strain has been classified as North
American pulse field type 1 (NAP1), ribotype 027, toxinotype
III, or restriction-endonuclease type BI (36), which we refer
to here as NAP1. Outbreaks have also been associated with
other SDA strains, such as the NAP7/toxinotype V/ribotype
078 (NAP7) strain found in cases of human and animal
disease (17, 38) and multiple toxin A-negative B-positive
(A-B?) pulsotypes and ribotypes responsible for CDI out-
breaks in Ireland, the United Kingdom, the United States,
and Canada (1, 58).
To date, the monitoring of C. difficile infection in a hospital
setting has been a reactive process in which patients are tested
after symptoms emerge, usually by employing methods that are
time-consuming and expensive and/or lack sufficient sensitivity
(11, 26). Improved tests are sought in which all at-risk patients
can be tested in a rapid, reliable, and cost-effective manner
(45). To address this need, DNA-based diagnostic tests have
been previously developed (2, 55, 56, 63), but they rely primar-
ily on targets from previously known genomic regions, such as
the genes tcdC (55, 63) and gyrA and gyrB (56). The available
tests include numerous commercially available assays (Xpect
toxin A/B test, from Remel, Inc., Lenexa, KS; BD GeneOhm
Cdiff assay, from BD Diagnostics, San Diego, CA; ProGastro
Cd assay, from Prodesse Inc., Waukesha, WI; Cepheid Xpert
* Corresponding author. Mailing address: McGill University, 740
Avenue Dr. Penfield, Room 7214, Montreal, Quebec, Canada H3A
1A4. Phone: (514) 398-3311, ext. 00089. Fax: (514) 398-1268. E-mail:
ken.dewar@mcgill.ca.
?Published ahead of print on 20 April 2011.
† The authors have paid a fee to allow immediate free access to
this article.
2230

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C. difficile, from Cepheid, Sunnyvale, CA; and illumigene C.
difficile, from Meridian Bioscience, Inc.).
With the advent of massively parallel DNA sequencing
(MPS) technology (35), bacterial whole-genome sequencing
has become rapid and affordable, and there are now sequences
from well over 5,000 completed or in-progress microbial ge-
nomes available in public databases such as NCBI Entrez.
MPS-based genome sequencing has been used in studies of C.
difficile and other human pathogens, including a comparative
genome analysis of 25 isolates that was performed to provide
insight into the molecular evolution of C. difficile (20, 53) and
a study of genome comparisons of 3 isolates that was used to
identify potential virulence mechanisms in the NAP1 strain
(59). These studies have confirmed the mobile nature of the C.
difficile genome (54) and that genetic diversity among strains is
high. In several studies (20, 22, 53), it has been shown that the
NAP7-NAP8 strain type is highly divergent from other strain
types and that the C. difficile core genome may be composed of
only ?1,000 genes (20, 22, 53, 58). To date, MPS and compar-
ative genome analyses of C. difficile have not been applied to
the search for additional DNA-based diagnostic targets, as has
been done for other human pathogens (6, 14, 15, 28).
In this report, we describe the comparative analysis of the
genomes of 14 isolates of C. difficile. The genomes of 9 isolates,
including 6 eastern Canadian and 3 reference isolates, were
sequenced as part of this study, and the genome sequences
from 5 additional publicly available isolates were also used in
the analyses. Our main objective was to identify DNA targets
for use in potentially addressing two major clinical issues: (i)
determining whether a given patient is infected with C. difficile
and, if so, (ii) determining whether the patient is infected with
a strain associated with severe disease. We thus identified
DNA-based diagnostic sequences that could be used to detect
any isolate of C. difficile, as well as targets able to discriminate
between SDA and non-SDA strain types. We also used com-
parative genome analysis to study the genetic diversity between
strain types, estimate the size of the C. difficile core genome,
and begin to investigate the existence of additional loci respon-
sible for virulence. Candidate targets identified in the 14-ge-
nome analysis were reconfirmed with a larger panel of 177
isolates. Clinical records available for 117 of these isolates
were cross-referenced by the use of target alleles to ascertain
their association with disease severity.
MATERIALS AND METHODS
Isolates for whole-genome sequencing. Within the available hospital collec-
tions, six isolates (QCD-32g58, QCD-66c26, QCD-97b34, QCD-37x79, QCD-
76w55, and CIP107932) of the predominant SDA strain of C. difficile (NAP1)
were selected to emphasize variation across geographic locations and times of
isolation. The remaining three of the nine isolates were a NAP2 strain (QCD-
63q42), an SDA NAP7 strain (QCD-23m63), and reference strain VPI10463
(ATCC 43255). Isolates were incubated anaerobically at 37°C on 5% Columbia
sheep’s blood agar in pure culture and identified by standard phenotypic criteria.
Genomic DNA from each isolate was extracted using standard commercial
column-based extraction (QIAamp DNA Minikit; Qiagen, Mississauga, CA),
stored in 1? Tris-EDTA (TE) buffer (pH 8.0), quantitated by spectrophotometry
(SmartSpec 3000 UV visible spectrophotometer; Bio-Rad, Mississauga, CA),
visualized by 1% agarose gel electrophoresis, and stored at ?20°C until further
use.
Whole-genome sequencing, gap closure, and comparative genome analysis. All
DNA extractions and sequencing experiments were performed at separate times
to minimize the risk of cross-contamination. Isolate QCD-32g58 was sequenced
using a first-generation Roche-454 GS system (GS20). The remaining isolates
were sequenced later using a second-generation Roche-454 GS system (GS-
FLX). Contigs (minimum size, 500 bases) were ordered and oriented based on
alignment to the finished genome of C. difficile strain 630 (54). Contigs that could
not to be ordered and oriented were placed into separate scaffolds. Assembly
improvement was accomplished by designing primers flanking each gap and by
performing conventional or long-range PCR (as required by predicted gap size)
using the genomic DNA. Bidirectional sequencing of the resulting amplicons was
performed with ABI 3730xl systems. Gaps were closed by aligning genomic and
gap-directed amplicon sequences with Consed software (18). Genes were pre-
dicted using GLIMMER 3.01 software (7). Gene functions were predicted by
aligning protein sequences using the NCBI nonredundant (nr) database (e-value
? 1.0e-20). A whole-genome multiple alignment was created using Multiz-TBA
software (4). DNA variations were catalogued when nucleotide quality (Q) was
?63 and fell within alignment blocks of ?80% pairwise identity present in all 14
genomes. The bootstrap consensus tree was inferred from 100 replicates and
constructed using MEGA 4 software (60). The pairwise genome comparison of
QCD-66c26 and QCD-23m63 was computed using lastz software (19), and a
histogram showing percent identity was generated using a custom Python script.
Validation via targeted resequencing. Within available hospital collections,
177 isolates of C. difficile were selected to assess genetic variation for each
candidate locus within the NAP1 strain and between strains of various pulsed-
field gel electrophoresis (PFGE) types. Of the 177 isolates, 170 (91%) were
successfully PCR amplified and DNA sequenced. We also included the 9 isolates
from the whole-genome sequencing as sequencing accuracy controls. We also
included a species-level control (C. spiroforme [ATCC 29900]) and a phylum-
level control (Mycobacterium intracellulare [kindly provided by Marcel Behr,
McGill University Health Centre]). Genomic DNA was extracted from isolates
by the use of standard lysis-based column extraction, and DNA yields were
estimated by spectrophotometry. For each candidate locus, primers were de-
signed using Primer3 software (51) to amplify regions of roughly 700 to 1,000 bp.
PCR and bidirectional sequencing of the resulting amplicons were performed
using an ABI 377 platform. Sequence chromatograms were base called using
Phred software (12, 13), and polymorphisms (Q ? 39) were catalogued on the
basis of C. difficile VPI10463 analyses.
RESULTS
Genome sequencing and analysis. We performed whole-
genome sequencing and assembly for nine isolates of C. difficile
(Table 1). Six isolates were of the predominant SDA strain
type (NAP1), and collection dates ranged from 1984 to 2007.
The international CIP 107932 reference isolate (isolated in
1984) and the BI-1 isolate (isolated in Minnesota in 1988 [47])
represented NAP1 isolates predating the CDI epidemic of
2003 to 2007. The other four NAP1 isolates (QCD-32g58,
QCD-66c26, QCD-37x79, and QCD-97b34) were collected
during the CDI epidemic from three locations across Canada.
In addition to the six NAP1 isolates, we sequenced and assem-
bled QCD-23m63, an SDA NAP7/toxinotype V/ribotype 078
(NAP7) isolate collected in 2007. The two non-SDA isolates
sequenced were international reference strain VPI10463
(ATCC 43255) and a NAP2 isolate (QCD-63q42), collected in
1980 and 2005, respectively.
We also used the publicly available genome assemblies of
another five isolates, including two additional NAP1 isolates
(R20291 and CD196 [59]), NAP7 and NAP8 isolates (Human
Microbiome Project [HMP]), and strain 630 (54) (Table 1).
Genome assembly details and the NCBI and GenBank acces-
sion numbers for all 14 isolates are given in Table 2.
The nine genomes sequenced in this study ranged in size
from 3.94 Mb (QCD-23m63) to 4.44 Mb (QCD-63q42) (Table
2), which corresponds to the range of genome sizes (3.90 to
4.29 Mb) observed in other C. difficile genome sequencing
projects (Table 2). We generated 9 draft genome assemblies in
which the contig numbers in the assemblies ranged from 16
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(QCD-32g58) to 66 (BI-1). However, the largest assembled
contigs were ?350 kb for all isolates, and the contig N80
calculation indicated that 80% of the assembled genomes was
found in contigs of ?59 kb (BI-1) to contigs of ?300 kb
(QCD-32g58). Given that C. difficile has a typical bacterial
gene density (80 to 85%) and typical bacterial gene lengths
(500 to 2,000 nucleotides [nt]), our genome assemblies pro-
vided contigs that could support synteny analyses combining
TABLE 1. Characteristics of C. difficile isolates used in this study
IsolateYr
PFGE
type
Location SourceAdditional characteristic(s)
Genome
size
(Mb)
Contig
no.
Isolates sequenced
in this study
QCD-66c262007NAP1a
Montreal, Quebec, Canada 56-yr-old male with severe CDI BT?h; tcdC ?117; 18-bp
deletion
BT?; tcdC ?117; 18-bp del
BT?; tcdC ?117; 18-bp del
Reference strain for binary
toxin
4.13 45
QCD-32g58
BI-1c
CIP 107932
2004
1988
1984
NAP1
NAP1a
NAP1a
Montreal, Quebec, Canada 70-yr-old male with CDI
Minneapolis, MN
Reims, Marne, France
4.11
4.4
4.04
18
89
69
Nonepidemic strain
28-yr-old female with PMCi
QCD-37x79
QCD-97b34
2005
2004
NAP1f
NAP1g
London, Ontario, Canada
St. John’s, Newfoundland,
Canada
Quebec, Quebec, Canada
67-yr-old patient with severe CDI BT?; tcdC ?117; 18-bp deletion
70-yr-old with severe CDI
4.33
4.07
59
74 BT?; tcdC ?117; 18-bp deletion
QCD-63q42
VPI 10463
QCD-23m63
2005
1980b
2007
NAP2 67-yr-old male with severe CDIToxinotype 0
Reference strain from ATCC
Toxinotype V/ribotype 078
4.44
4.21
3.94
87
88
80 NAP7 Montreal, Quebec, Canada Male with severe CDI
Publicly available
isolates
CD196
R20291
Strain 630
NAP07
NAP08
1985
2006e
1980
2008dNAP7
2008dNAP8
NAP1
NAP1
Paris, France
Stoke Mandeville, England Outbreak associated
Zurich, Switzerland
Unknown
Unknown
Nonepidemic strain4.11
4.19
4.29
3.90
4.08
1
1
2CDI and PMC
Human feces
Human feces
33
24
aPredicted from sequence similarity.
bEstimated year of isolation.
cN. Razaq et al. (47).
dEstimated from date of sequencing.
eEstimated from publication.
fSubtype b/006.
gSubtype a/001.
hBT?, binary toxin positive.
iPMC, pseudomembranous colitis.
TABLE 2. Characteristics of C. difficile genome assemblies used in this study
Isolate Technology
Genome
assembly
status
Genome size
(nt)a
No. of
contigs
Largest
contig (kb)
Contig N80
(kb)
NCBI or GenBank
accession no.
Reference
Isolates sequenced
in this study
QCD-66c26
QCD-32g58
BI-1
CIP 1079324
QCD-37x79
QCD-97b34
QCD-63q42
VPI 104634
QCD-23m63
GS-FLX
GS-20
GS-FLX
GS-FLX
GS-FLX
GS-FLX
GS-FLX
GS-FLX
GS-FLX
Draft
Draft
Draft
Draft
Draft
Draft
Draft
Draft
Draft
4,126,050
4,108,089
4,392,595
4,032,580
4,329,888
4,059,010
4,440,437
4,204,780
3,936,085
32
16
66
55
45
60
60
55
61
937.3
1,247.0
356.4
354.2
559.0
366.6
1,027.2
1293.1
440.0
232.5
302.3
59.6
81.1
128.7
76.7
101.2
138.5
93.8
NZ_ABFD00000000
NZ_AAML00000000
NZ_ABHE00000000
NZ_ABKK00000000
NZ_ABHG00000000
NZ_ABHF00000000
NZ_ABHD00000000
NZ_ABKJ00000000
NZ_ABKL00000000
This study
This study
This study
This study
This study
This study
This study
This study
This study
Publicly available
isolates
CD196
R20291
Strain 630
NAP07
GS-FLX
GS-20
ABI377
GS-FLX
Complete
Complete
Complete
Draft
4,110,554
4,191,339
4,290,252
3,862,058
1
1
1
4,110.5
4,191.3
4,290.2
269.2
4,110.5
4,191.3
4,290.2
35.7
FN538970
FN545816
NC_009089
NZ_ADVM00000000
Stabler et al. (59)
Stabler et al. (59)
Sebaihia et al. (54)
Human Microbiome
Projectb
Human Microbiome
Projectb
100
NAP08GS-FLX Draft4,022,033111 169.732.3NZ_ADNX00000000
ant, number of nucleotides.
bhttp://www.hmpdacc.org/.
2232FORGETTA ET AL.J. CLIN. MICROBIOL.

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sequence similarities in the context of information on gene
order and orientation. Draft assemblies from the Human Mi-
crobiome Project also provided contigs of lengths (contig
N80 ? 30 kb) useful for gene annotation and the derivation of
local synteny relationships.
We used whole-genome alignments to determine that a core
of 3.4 Mb of orthologous genomic sequence was present in all
of the 14 genomes. Within the core genome, the NAP7-NAP8
group of isolates tended to be the most divergent of all the
groups, with an average percent identity of 97% compared to
the NAP1 group, although this included regions of lower per-
cent identity embedded within the syntenic regions (Fig. 1).
The noncore genome sequences represented strain- and iso-
late-specific insertions and deletions often due to mobile ge-
netic elements and possible extrachromosomal plasmids (data
not shown).
Polymorphism discovery. Within the 3.4-Mb core genome,
we identified 127,442 single nucleotide polymorphisms (SNPs).
Although other types of genomic variation, including inser-
tions and deletions, were present, they were not analyzed in
this study, as they accounted for less than 3% (3,211) of all
instances of nucleotide-level variations. A phylogenetic tree
constructed using the SNPs clustered the isolates into three
distinct groups: the eight NAP1 isolates, the three NAP7-
NAP8 isolates, and the three remaining isolates (ATCC 43255,
630, and QCD-63q42), which we refer here to as the R group
(Fig. 2a). Levels of genetic variations among these three
groups are greater than the level of variation within any one
group (Fig. 2b). The NAP1 isolates differ from the NAP7-
NAP8 group at 104,853 SNP positions and differ from the
three remaining isolates (R group) at 17,076 SNP positions.
The NAP7-NAP8 isolates differ from the R group of isolates at
96,302 SNP positions.
The members of the NAP1 group consisting of 8 isolates are
highly identical within the core genome, with identities for any
two members ranging from 9 to 62 polymorphic SNP positions
(Fig. 2b). We identified 11 nonsynonymous nucleotide substi-
tutions that distinguished members of a subset of Canadian
NAP1 isolates that consists of QCD-32g58 (isolated in Quebec
in 2004), QCD-66c26 (Quebec, 2007), and QCD-37x79 (On-
tario, 2005) as well as the United Kingdom outbreak strain
R20291 (Stoke Mandeville) from the others, namely, QCD-
97b34 (Newfoundland, Canada, 2004), BI-1 (Minnesota,
1988), CIP107932 (France, 1984), and CD196 (France, 1985).
This set of 11 SNPs includes the previously described mutation
responsible for resistance to fluoroquinolones (10).
The members of the group of three NAP7-NAP8 isolates are
also highly similar to each other, with at most 1,851 SNPs
separating QCD-23m63 (NAP 7) from the Human Micro-
FIG. 1. Percent identity plot (top) and dot plot (bottom) depicting
the whole-genome pairwise alignments of a NAP1 isolate (QCD-
66c26) and a NAP7 isolate (QCD-23m63). The dot plot in the bottom
panel depicts the colinearity of the two genomes, and the percent
identity plot in the upper panel depicts the levels of nucleotide-level
similarity of the two genomes (the red line indicates average percent
identity).
FIG. 2. (a) Phylogenetic tree of 14 C. difficile genomes constructed using SNP data. Branches corresponding to partitions reproduced in less
than 50% of replicate bootstrap determinations are collapsed. The percentages of replicate trees in which the associated taxa clustered together
in the bootstrap test (100 replicates) are shown next to the branches. Genomes cluster into 3 distinct groups: NAP1 isolates (red), NAP7-NAP8
isolates (blue), and the 3 remaining isolates (black [R group]). (b) Numbers of polymorphic SNPs observed for the three groups (large boxes), as
well as variations observed between isolates within each group (remaining values). SNPs (text at center bottom) that uniquely identify the members
of each group are also indicated.
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biome Project (HMP) NAP07 isolate. However, the two HMP
isolates are very similar, with only 297 SNPs. The number of
variations distinguishing the three isolates in the R group is
greater than the number of variations within the other two
groups, with each of the three isolates in the group having over
11,000 SNPs.
Genetic variations consistently present in members of a sin-
gle group are candidate DNA markers of potential diagnostic
value. We found 10,683 SNPs that distinguish the NAP1 group
from the other two groups (Fig. 2b). There were also 89,954
SNPs that separated the NAP7-NAP8 set from all others and
6,342 SNPs that separated the remainder (R group) from the
NAP1 and NAP7-NAP8 groups.
The 10,683 SNPs that are specific to the NAP1 group have a
genome-wide distribution (Fig. 3a) and include a prominent
cluster overlapping the tcdB cytotoxin gene (Fig. 3a). Among
the other prominent clusters, 545 SNPs were observed in a
5.8-kb region of 6 genes (CD1265 to CD1270), annotated as a
two-component system (TCS) adjacent to an ABC transporter
(TCS-ABC) (Fig. 3b). The predicted protein sequences across
this TCS-ABC locus showed no premature stop codons, pro-
viding preliminary evidence that these remain functional genes
despite the increased level of genetic variation. Other promi-
nent clusters include an ABC transporter (CD0336 to
CD0337), and a hypothetical protein adjacent to a transcrip-
tional regulator (CD3143 to CD3144).
Identification and validation of targets for SDA strains. To
investigate the potential of NAP1 SNPs in the TCS-ABC locus
as diagnostic targets associated with disease severity, we se-
lected 2 candidate genes, CD1265 and CD1269, for compari-
son to 9 putative or known virulence genes (tcdA, tcdB, tcdC,
tcdE, codY, fliC, groEL, gyrB, and mviN). These genes were
PCR amplified and resequenced using a panel of 177 isolates
of C. difficile. Isolates were selected to assess genetic variation
for each candidate locus within the NAP1 strain and between
strains of various PFGE types. The 177 isolates were collected
predominantly from Canadian hospital, provincial, and na-
tional reference collections and chosen to sample variations in
PFGE types. The isolates were also selected to encompass
different collection sites and years of isolation. A second set of
international strains facilitated another survey of C. difficile
diversity based on toxinotyping (52). A total of 20 contributing
sites were represented, including 17 Canadian, 2 American,
and one European institution. There were 163 isolates col-
lected in or after 2002, with 22 isolated prior to 2002. Sixty-
seven isolates were classified as NAP1, 12 as NAP2, and 109
into more than 50 other pulsovars. Ten isolates were from the
reference panel of toxinotyping strains, and 50 were from 9
hospitals in the province of Quebec, Canada. A total of 117
isolates were obtained from The Canadian Nosocomial Infec-
tion Surveillance Program (CNISP) and had available patient
data. Seventy of the 117 clinical records reported severe CDI,
which was defined as death associated with CDI or intensive
care units or colectomy due to CDI (all measured at least 30
days after diagnosis).
Our set included 10 A-B? isolates, all of which were of
FIG. 3. (a) Genome-wide distribution of SNPs that uniquely identify the NAP1 group of isolates. Prominent clusters overlap known genes (e.g.,
tcdB in PaLoc) and unknown loci (indicated with arrows). (b) Genomic intervals of 5 loci with prominent clusters of NAP1 SNPs. From top to
bottom: CD0366-CD0337, tcdB, TCS-ABC, and CD3143-CD3144. Annotations for each genomic interval are indicated as follows (from top to
bottom): a scale, the genomic position, the pileup of NAP1 SNPs, and gene annotations (arrows indicate gene direction).
2234FORGETTA ET AL. J. CLIN. MICROBIOL.

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PFGE type 00065 (NAP9; personal communication) and cor-
related with toxinotype VIII (22). In the patient data available
for 100 of the isolates that were resequenced, SNP genotypes
in two genes (CD1269 and CD1265) were seen to be associated
with 70% (44/63) of severe-disease cases (comprising the three
PFGE types NAP1, NAP7, and NAP9), whereas analysis by
existing typing methods, such as PFGE NAP1 classification or
determination of deletions in tcdC, accounted for 52% (33/63)
or 62% (39/63), respectively, and was limited to two PFGE
types (NAP1 and NAP7). For example, the G allele for the
SNP at genomic location 1,387,666 in CD1269 (Fig. 4) was
associated with more severe-disease cases than the single base
deletion at position 117 (?117) in tcdC, as revealed by the
observation that the A-B? strains have the G allele but lack
the tcdC deletion. In contrast, analysis focusing on the ?117
deletion in tcdC detected 33 of 63 severe-disease cases, and the
larger deletions of at least 18 bp in the 3? end of tcdC ac-
counted for 39 of 63 of severe-disease cases. Deletions in tcdC
were diagnostic markers for two of three strain types previ-
ously associated with severe disease (NAP1 and NAP7),
whereas analysis of the genotypes for each of 18 SNPs in
CD1269 (1/18) and CD1265 (17/18) (the TCS-ABC locus) de-
tected 44 of 63 severe-disease cases and specific alleles were
associated with three SDA strain types (NAP1, NAP7, and
A-B? [NAP9/toxinotype VIII]) (Fig. 4). In addition, 5 of the
SNPS in CD1265 are triallelic and partition the SDA strains
into two groups (NAP1 versus NAP7 and A-B? [NAP9/toxi-
notype VIII]) (Fig. 4). For example, the SNP at genomic lo-
cation 1,383,226 has three alleles, C, T, and A. The A allele is
associated with the NAP1 strain, the T allele with the NAP7
and A-B? strains, and the C allele with all the others.
Identification and validation of targets for species-level de-
tection. We selected a set of 12 genes (spoIIIAG, CD0596,
CD0117, CD3014, CD0279, CD1795, CD2251, CD0017, cdd1,
srlE, prdB, and sspA) that displayed a high level of nucleotide
identity across all 14 sequenced genomes as species-wide de-
tection candidates (Table 3). After the panel of 177 isolates
was resequenced, sspA was found to be 100% conserved at the
nucleotide level for all isolates analyzed (Table 3). The remain-
ing 11 candidates contained a few polymorphisms and did not
deviate substantially from what was originally observed in the
14-genome analysis (Table 3). To further investigate whether
these gene sequences could be used as DNA-based markers for
specific detection of C. difficile (regardless of strain type) and
yet remain specific to C. difficile and to no other species, we
aligned the genomic sequence from each candidate gene re-
FIG. 4. Correlation of disease severity with SNPs from the TCS-
ABC locus or with existing diagnostic methodologies. The incidence of
severe CDI outcome (SDA column [gray boxes]) was higher for the
NAP1 strain and occurred in 7/10 and 2/2 cases for the A-B? and
NAP7 strains (plus one closely related strain, 11ACD0028), respec-
tively (genome-sequenced isolates were not phenotyped and are indi-
cated with an “x”). Molecular markers such as the PFGE type of strain
NAP1 and the presence of binary toxin are diagnostic markers for
NAP1, with deletions in tcdC additionally capturing the NAP7 strain.
Genotypes of SNPs identified in the TCS-ABC locus are identifiers for
three SDA strain types (the NAP1 strain, NAP7, and A-B? strains)
and include 5 triallelic SNPs (the third allele is boxed).
VOL. 49, 2011 DNA MARKERS FOR STRAINS OF C. DIFFICILE 2235

Page 7

gion to sequences in the NCBI nonredundant DNA database.
Database hits for all 12 gene regions were well below 90%
identity and/or 90% query coverage (Table 3). Furthermore,
experiments performed using C. difficile PCR primers for all 12
genes did not lead to amplification products from Clostridium
spiroforme and Mycobacterium intracellulare (data not shown).
DISCUSSION
The primary objectives of this study was to identify DNA
markers for C. difficile that could be used to test stool samples
of patients and to determine (i) whether they are infected with
C. difficile and, if they are infected, (ii) whether the particular
strain is associated with severe disease. Our strain selection,
which was influenced by the availability of isolates in well-
characterized hospital collections, such as the Canadian Nos-
ocomial Infection Surveillance Program, included an emphasis
on isolates of the predominant epidemic strain (NAP1) as well
as an isolate of another SDA strain (NAP7) previously re-
ported in the literature (17, 38). Our strain choices comprised
the majority of SDA strain types (21, 32, 46, 61) as well as two
widely used research reference isolates (CIP107932 and
VPI10463). Our testing of an extended panel of isolates, from
an even wider diversity of strain types than those described
here, further demonstrated that we have analyzed C. difficile
representing a wide spectrum of naturally occurring genetic
diversity.
In agreement with previous studies, we observed variations
in genome size that are largely attributable to mobile genetic
element activity (20, 53, 54, 59). Mobile genetic elements in C.
difficile have been found to carry virulence factors (59); there-
fore, a more detailed comparative genome analysis of the
NAP1 isolates from this study and others might provide further
insight into the differential virulence characteristics observed
within this strain. We observed 3.4 Mb of conserved sequence
present in all 14 genomes, comprising 3,063 genes, a determi-
nation that differs substantially from observations in other
studies, which estimated a smaller core genome of less than
1,000 genes (20, 22, 53, 58). We do not believe that this is a
reflection of strain choices, as our most divergent pairs (NAP1
versus NAP7) were also recognized in other studies as being
the most highly divergent (20). Rather, we believe the differ-
ences in interpretations of core genome size are due to differ-
ences in methodologies and analysis techniques. Given the
high quality of the 3 completed genomes and 11 draft genomes
used in our analyses, we were able to combine sequence com-
parisons and syntenic relationships to determine gene or-
thologs and observed that, compared to NAP1 references, the
members of the NAP7-NAP8 group displayed a higher level of
genetic diversity, with an average of 97% identity. We believe
that in comparative genome hybridization (CGH) studies, for
example, where probe mismatches can affect hybridization (41,
49) and where NAP1 or strain 630 genomes have been used as
the reference, even a very low number of mismatches between
the probe and target DNA can increase the false-negative rate
(34, 58). This leads us to suggest that the C. difficile CGH
arrays, which were designed using either strain 630 (R group)
or NAP1 isolate QCD-32g58, may produce numerous false-
negative hybridizations when tested using more a more dis-
tantly related strain (e.g., NAP7) and have resulted in the
calculation of a smaller core genome size. Other whole-ge-
nome sequence-based studies have used differing analytical
techniques to determine the core genome size. For example,
one study determined the core genome to consist of 622 genes,
but only after the researchers stringently considered the non-
recombining genes in the genome (20). Another study esti-
mated the core genome to be comprised of 947 genes (53);
however, as it was a gene-centered analysis based on sequence
identity without synteny evaluation, that study may have clas-
sified genes of lower-than-average sequence identity, such as
those present in the NAP7-NAP8 group, as nonorthologous.
Our whole-genome multiple-alignment-based approach al-
lowed the identification of orthologous regions of lower se-
quence identity which might not have been detectable using
CGH arrays or might have been classified as below similarity
thresholds by the use of a gene-centered approach.
At this time, our catalogue of genetic variation does not
include insertions or deletions. Future work may reveal inser-
tions or deletions in SDA strains in addition to those previ-
ously identified in tcdC (5, 32, 33, 36). Indeed, whereas dele-
tions in tcdC have previously been hypothesized to lead to
increased toxin production (33, 57, 62), findings from a recent
study (39) indicated that deletions in tcdC do not predict the
biological activity of the PaLoc toxin genes, providing further
TABLE 3. Targets for species-level detection of C. difficile
Gene or locus
Number of SNPs
Most similar species by BLASTn analysis (nr)
Query
coverage (%)
Maximum
identity (%)
14 genomes
Resequencing
result
sspA
prdB
CD0017
cdd1
srlE
CD2251
CD1795
CD0596
CD0117
CD3014
CD0279
spoIIIAG
0
2
2
2
6
3
4
6
6
6
8
8
0
5
6
6
6
8
9
Alkaliphilus oremlandii OhILAs
Clostridium sticklandii
Clostridium botulinum E3 strain Alaska E43
Leptospira interrogans serovar lai strain 56601
Clostridium botulinum B strain Eklund 17B
Trichomonas vaginalis
Polistes sp. MD1 mitochondrion
Brassica rapa subsp. Pekinensis
Alkaliphilus metalliredigens QYMF
Listeria welshimeri serovar 6b strain SLCC5334
Clostridium acetobutylicum ATCC 824
Mycoplasma mycoides subsp. mycoides SC strain PG1
94
96
83
39
97
13
16
22
96
96
45
8
74
76
73
74
83
85
84
81
76
86
67
84
10
10
10
10
14
2236FORGETTA ET AL. J. CLIN. MICROBIOL.

Page 8

motivation to identify all sources of genetic variation within the
C. difficile genomes that may correlate with disease severity.
There are 64 SNPs that discriminate isolates within the
NAP1 strain type, including 14 SNPs that separate eastern
Canadian isolates from the others, and these demonstrate the
potential of massively parallel sequencing for identification of
SNPs suitable for subsequent intrastrain typing and tracking.
The SNPs that distinguish the NAP1 strain from all other
strains show an uneven genome-wide distribution and cluster
in known pathogenicity genes as well as in genes with currently
unrecognized roles in CDI. The other genes with clusters of
NAP1 SNPs include a general stress gene (CD2599), a carbon
starvation gene (CD2600), and an ABC transporter. The most
prominent cluster of SNPs that discriminate the epidemic
strain was observed in a genetic locus consisting of a two-
component system (CD1269 and CD1270) adjacent to an ABC
transporter (CD1265 to CD1268). The adjacency of these two
loci has been shown to be functionally relevant in Bacillus and
is present in other low-GC-content Gram-positive bacteria,
including C. difficile (25). The function of the TCS-ABC system
has been investigated previously in Bacillus subtilis and in-
cludes detoxification of antimicrobial compounds (42). The
function of any particular TCS-ABC system is primarily dic-
tated by the protein domains present in the histidine kinase
gene (25). Sequence analysis of the histidine kinase gene
(CD1270) in this TCS-ABC system suggests that it plays a role
in detoxification (data not shown). In silico analysis indicates
that this locus is not comprised of pseudogenes and thus may
have a functional role. Future experiments are needed to con-
firm the expression of genes in the TCS-ABC system and
investigate their roles in CDI.
Enzyme-based strategies for the detection of C. difficile have
limited sensitivity and specificity (11). To address this, alterna-
tive gene targets, such as the tpi housekeeping gene, have been
investigated (8). While this assay is specific to C. difficile, it
requires the additional procedure of restriction fragment
length polymorphism (RFLP) to achieve its specificity, making
it less suitable in a clinical setting. Our genome analysis has
identified numerous highly conserved genes that may be suit-
able for PCR-based specific detection of C. difficile. The 12
candidate genes displayed a high level of sequence conserva-
tion across the 14 genomes as well as a diverse population of
177 isolates, representing major PFGE types and toxinotypes.
Moreover, our in silico analysis showed that many of these
candidates have low sequence identity to other bacterial spe-
cies and, upon further experimental validation and develop-
ment, may define a more rapid and specific clinical detection
assay.
The development of genetic markers to track severe-disease-
causing strains has previously relied on variations in known
pathogenicity genes, such as the detection of deletions in tcdC
(63). While deletions in tcdC are diagnostic markers for the
NAP1 strain and the SDA NAP7 strain, they are not diagnostic
markers for the A-B? isolates (NAP9/toxinotype VIII) that
have been found in food animals (44) and retail meats (50).
Using comparative genome analysis with candidate gene rese-
quencing, we have identified numerous SNPs that detect these
three SDA strain types, and this has in turn led to an increase
of almost 20% in the detection of severe-disease-causing cases.
However, strains containing these SNPs do not always lead to
CDI, and severe CDI can be observed in cases of infection with
non-SDA strains. Part of this phenomenon may be attributable
to host factors that alter disease severity, such as variation in
immune response (30, 31) and exposure to certain antibiotics
(32, 40, 43) or acid-reducing agents (9, 32, 43). Alternatively, as
our resequencing of candidate regions was limited, the rese-
quencing of additional genes or loci may uncover SNPs that are
markers for additional severe-disease-causing isolates. Also,
severe disease or outbreak occurrences and numbers of out-
break infections may result from a constellation of genetic loci,
such as those responsible for sporulation (37) or gut survival,
and investigation of these other loci may identify SNPs that
account for additional severe-disease cases.
This report demonstrates the utility of massively parallel
DNA sequencing to identify clinically relevant diagnostic
markers of C. difficile. As the cost of whole-genome sequencing
continues to decrease, we envision this approach being applied
to the study of other human pathogens.
ACKNOWLEDGMENTS
We thank the DNA sequencing teams at the Genome Center at
Washington University School of Medicine and the McGill Univer-
sity and Genome Quebec Innovation Centre for the sequencing of
the C. difficile isolates. We thank Manon Lorange for the isolation
of C. difficile QCD-63q42. We also thank Marcel Behr for providing
C. spiroforme and Mycobacterium intracellulare control DNAs. We
thank the Human Microbiome Project (HMP) and the HMP DACC
for prepublicationdatarelease
(ADVM00000000.1) and NAP08 (ADNX00000000.1) sequences.
This study was funded by Genome Canada and Genome Quebec
(K.D.) and the NHGRI (E.R.M.). V.F. was the recipient of a Canadian
Institutes of Health Research Doctoral Research Award. M.T.O. was
the recipient of an AMMI Canada/CIHR/CFID/Bayer Healthcare re-
search fellowship.
ofthe C.difficile NAP07
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