Comparative Genomic Hybridization Analysis Shows
Different Epidemiology of Chromosomal and Plasmid-
Borne cpe-Carrying Clostridium perfringens Type A
Pa ¨ivi Lahti*, Miia Lindstro ¨m, Panu Somervuo, Annamari Heikinheimo, Hannu Korkeala
Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland
Clostridium perfringens, one of the most common causes of food poisonings, can carry the enterotoxin gene, cpe, in its
chromosome or on a plasmid. C. perfringens food poisonings are more frequently caused by the chromosomal cpe-carrying
strains, while the plasmid-borne cpe-positive genotypes are more commonly found in the human feces and environmental
samples. Different tolerance to food processing conditions by the plasmid-borne and chromosomal cpe-carrying strains has
been reported, but the reservoirs and contamination routes of enterotoxin-producing C. perfringens remain unknown. A
comparative genomic hybridization (CGH) analysis with a DNA microarray based on three C. perfringens type A genomes
was conducted to shed light on the epidemiology of C. perfringens food poisonings caused by plasmid-borne and
chromosomal cpe-carrying strains by comparing chromosomal and plasmid-borne cpe-positive and cpe-negative C.
perfringens isolates from human, animal, environmental, and food samples. The chromosomal and plasmid-borne cpe-
positive C. perfringens genotypes formed two distinct clusters. Variable genes were involved with myo-inositol,
ethanolamine and cellobiose metabolism, suggesting a new epidemiological model for C. perfringens food poisonings.
The CGH results were complemented with growth studies, which demonstrated different myo-inositol, ethanolamine, and
cellobiose metabolism between the chromosomal and plasmid-borne cpe-carrying strains. These findings support a
ubiquitous occurrence of the plasmid-borne cpe-positive strains and their adaptation to the mammalian intestine, whereas
the chromosomal cpe-positive strains appear to have a narrow niche in environments containing degrading plant material.
Thus the epidemiology of the food poisonings caused by two populations appears different, the plasmid-borne cpe-positive
strains probably contaminating foods via humans and the chromosomal strains being connected to plant material.
Citation: Lahti P, Lindstro ¨m M, Somervuo P, Heikinheimo A, Korkeala H (2012) Comparative Genomic Hybridization Analysis Shows Different Epidemiology of
Chromosomal and Plasmid-Borne cpe-Carrying Clostridium perfringens Type A. PLoS ONE 7(10): e46162. doi:10.1371/journal.pone.0046162
Editor: Holger Bruggemann, Aarhus University, Denmark
Received February 6, 2012; Accepted August 30, 2012; Published October 19, 2012
Copyright: ? 2012 Lahti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Finnish Centre of Excellence in Microbial Food Safety Research of the Academy of Finland. The funder had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Enterotoxin gene-carrying (cpe-positive) Clostridium perfringens
type A is one of the most common causes of food poisoning in
the industrialized world, and the third leading cause of food
poisoning in USA . Limited knowledge of the reservoirs and the
contamination routes of cpe-positive C. perfringens complicates the
prevention of C. perfringens food poisonings.
C. perfringens is an anaerobic ubiquitous spore-forming bacte-
rium, frequently present in the normal intestinal microbiota of
humans and animals. C. perfringens strains are classified into
different types (A–E) based on their expression of alpha, beta,
epsilon, and iota toxins . C. perfringens can cause several
diseases in humans and animals due to the variety of toxins it
Fewer than 5% of C. perfringens type A strains carry the
enterotoxin gene cpe . The cpe can be located in the bacterial
chromosome or on a large plasmid [4–6]. The chromosomal cpe is
flanked by an insertion sequence (IS) element IS1470 (cpe-genotype
IS1470) , whereas the plasmid-borne cpe is flanked by either the
IS1470-like or IS1151 element (cpe-genotypes IS1470-like or
IS1151) [6,5]. Until recently, only the chromosomal cpe-carrying
strains were associated with food poisonings . This was
explained by their better tolerance to heating, low temperatures,
and preservatives than that of the plasmid-borne cpe-carrying
strains [8,9]. However, also the plasmid-borne genotypes were
found to cause food poisonings [10–12] and the cpe was carried on
a plasmid in 25% of food poisoning outbreaks investigated in
Finland and Germany .
Both chromosomal and plasmid-borne cpe-positive C. perfringens
genotypes were found in retail meat products [13,14], but the
contamination route remains unknown. The contamination of
meat by the intestinal contents of slaughtered animals has been
suggested to serve as the main source of cpe-positive C. perfringens
. However, no successful isolations of cpe-positive C. perfringens
strains have been reported from healthy production animals [16–
18]; thus, the role of animals as the main reservoir of cpe-positive C.
perfringens has been questioned .
Humans are a rich reservoir for plasmid-borne cpe-carrying
strains [19,20] and were thus suggested to introduce a contam-
ination risk into foods through handling . However, only a few
chromosomal strains were found in human feces . Plasmid-
borne cpe-positive strains were also detected in soil and sediments
PLOS ONE | www.plosone.org1 October 2012 | Volume 7 | Issue 10 | e46162
[21,22]. For better prevention of C. perfringens food poisonings, the
reservoirs of the cpe-positive C. perfringens strains and the potentially
different epidemiology of C. perfringens type A food poisonings
caused by the chromosomal and plasmid-borne cpe-carrying strains
need to be elucidated.
Comparative genomic hybridization (CGH) with DNA micro-
arrays was performed to shed light on the epidemiology of the
chromosomal and plasmid-borne cpe-carrying and cpe-negative C.
perfringens type A strains of food, human, or animal origin. The
results of the CGH analysis were complemented with growth
studies, which demonstrated different metabolism between the
chromosomal and plasmid-borne cpe-carrying strains. The results
suggest different epidemiology of the cpe-positive C. perfringens
groups, which is relevant when designing prevention of C.
perfringens food poisonings.
To assess genetic relatedness and possible metabolic differences
between the chromosomal and plasmid-borne cpe-positive and
cpe-negative C. perfringens strains, a DNA microarray was designed
based onthree sequenced
ATCC13124, strain 13 and SM101. A wide collection of C.
perfringens strains (n=83) from food and feces associated with food
poisonings, feces of healthy humans, feces of healthy production
animals, soil and sludge, were studied (Table S1). The strains
represented different cpe-positive genotypes and cpe-negative
strains, the latter including the reference strains ATCC13124
and 13 which were used as positive controls. A two-color labeling
system was used and the differently labeled DNA sample pairs to
be hybridized into one of the eight subarrays on each array slide
were randomly selected. Reproducibility of the hybridizations
was controlled by hybridizing 20 samples in duplicate and the
control strains in quadruplicate. The DNA samples of the
reference strains 13 and ATCC 13124 hybridized 99.9% with
their own gene probes. The putative metabolic differences
suggested by the CGH analysis were further confirmed by
metabolic tests using minimal growth medium. All strains tested
grew in the minimal medium with glucose as the sole carbon
source and failed to grow in minimal medium without any source
of carbon, demonstrating that the medium supported the growth
of C. perfringens.
The 54 cpe-positive C. perfringens type A strains formed two
distinct clusters, one consisting of the chromosomal cpe-carrying
genotypes and the other of the plasmid-borne cpe-carrying
genotypes (Figure S1). The similarity between strains, based on
Pearson’s correlation on a scale from 21 to 1, was 0.85 in the
chromosomal cpe group and 0.76 in the plasmid-borne cpe group.
The similarity between the two groups of cpe-positive C. perfringens
strains was 0.59 (Figure 1). When the 29 cpe-negative strains were
included in the analysis, the chromosomal strains still clustered
separately, and the plasmid-borne cpe-carrying strains and the cpe-
negative strains were evenly distributed in the other cluster. The
chromosomal cluster was homogeneous, whereas the cluster
consisting of plasmid-borne cpe-carrying or plasmid-borne cpe-
carrying and cpe-negative strains was more heterogeneous
In general, the plasmid-borne cpe-carrying strains shared more
CDSs (75,6%–87,4%) with the cpe-negative reference strains
ATCC 13124 and 13 than with the chromosomal cpe-carrying
reference strain SM101 (71,8%–84,4%) (Table 1). By contrast, the
chromosomal cpe-positive C. perfringens strains shared more CDSs
with the reference strain SM101 (86,2%-94,9%) than with the two
other reference strains (63,8%–81,4%) (Table 1). Altogether 372
CDSs were exclusively present in the plasmid-borne cpe-positive
strains, and 242 CDSs were exclusively present in the chromo-
somal cpe-carrying strains.
When the CDSs of the reference strains were divided into
functional groups based on J. Craig Venter Institute Comprehen-
sive Microbial Resource (CMR) annotations, the plasmid-borne
cpe-positive strains carried more CDSs than chromosomal strains
of all except two functional groups: transposable elements, and
protein synthesis and electron transport (Table S2). Marked
differences were present in the numbers of CDSs without specific
annotation (Table S2).
The major differences between the chromosomal and plasmid-
borne cpe-carrying strains were in the presence of the operons
related to myo-inositole and ethanolamine utilization; a gene cluster
encoding phosphotransferases and beta-glucanases, including
laminarinase and cellobiose phosphotransferase; and a gene
cluster encoding biotin synthesis.
All plasmid-borne cpe-carrying strains carried the myo-inositol
operon, whereas all chromosomal cpe-positive strains lacked this
operon (Figure 2, Table S4). Accordingly, all tested plasmid-borne
cpe-positive C. perfringens strains and none of the tested chromo-
somal cpe-positive strains utilized myo-inositol (Table S3). In the
reference strain ATCC13124, the myo-inositol operon is located in
the chromosome and consists of 13 CDSs (locus CPF0079–
CPF0092). iolR upstream of the cluster is predicted to encode a
All the 21 chromosomal cpe-carrying strains lacked the operon
predicted to encode ethanolamine utilization, whereas 23 of the
33 plasmid-borne cpe-carrying strains, including all strains
representing genotype IS1151-cpe and ten of 20 strains repre-
senting genotype IS1470-like-cpe, had this operon (Figure 2,
Table S4). Again, the result was verified by all tested plasmid-
borne cpe-positive C. perfringens strains and none of the tested
chromosomal cpe-positive strains utilizing myo-inositol (Table S3).
The ethanolamine utilization operon is found in the genomes of
the reference strains ATCC13124 and 13, and it contains 17
Nearly all (19 of 21) chromosomal cpe-carrying strains had a
gene cluster predicted to encode cellobiose phosphotransferase,
laminarinase, and beta-glucanases, whereas all plasmid-borne cpe-
carrying strains lacked this gene cluster. In support of the CGH
data, all the chromosomal cpe-positive strains tested utilized
cellobiose as the only carbon source (Table S3). Of the 12
plasmid-borne cpe-positive strains that lacked this gene cluster, nine
failed to grow in minimal medium with cellobiose as the sole
carbon source, and three grew in the minimal medium with
cellobiose despite lacking the gene cluster. The gene cluster
predicted to encode phosphotransferases and beta-glucanases
contains 11 CDSs and is located in the chromosome of the cpe-
positive SM101 (locus CPR2599–CPR2609) (Figure 2, Table S4).
Moreover, all chromosomal cpe-positive C. perfringens strains lacked
a gene cluster containing nine CDSs, of which bioB and bioD
encode biotin synthesis (locus CPF1787–1795 in ATCC 13124).
All 83 strains carried bioY (CPF1796), predicted to encode biotin
The genomic content of the cpe-negative strains resembled that
of the the plasmid-borne cpe-gene carrying strains. All cpe-negative
strains carried an operon for myo-inositol utilization, most (26 of
29) carried the operon encoding ethanolamine utilization, all
carried the gene cluster encoding biotin synthesis, and all lacked
the gene cluster encoding phosphotransferases and beta-
CGH Analysis of cpe-Positive C. perfringens Type A
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The chromosomal and the plasmid-borne cpe-carrying C.
perfringens type A strains differed in their gene composition and
clustered separately in the CGH analysis. The microarray results
were confirmed by functional metabolic studies. The main
differences were related to genes involved in the utilization of
myo-inositole, ethanolamine, and cellobiose, and the synthesis of
biotin. Accordingly, different ability of the chromosomal and
plasmid-borne cpe-positive strains to utilize myo-inositole, ethanol-
amine, and cellobiose as the only source of energy was
demonstrated. This suggests that the chromosomal and plasmid-
borne cpe-carrying C. perfringens strains are differently adapted to
various environments, and thus, the epidemiology of C. perfringens
food poisoning caused by the two strain populations may be
The plasmid-borne cpe-carrying and the cpe-negative strains
formed a heterogeneous group, with some plasmid-borne cpe-
carrying and cpe-negative strains being very similar. This supports
horizontal transfer of the cpe plasmid between C. perfringens strains,
as proposed in previous studies [19,23,24].
The chromosomal cpe-positive strains formed a homogeneous
cluster, which is in agreement with an earlier study using multi-
locus sequence typing . It seems plausible that the chromo-
somal cpe-positive strains have diverged from the remaining C.
perfringens population, which is ubiquitous in nature and consists of
a heterogeneous group of cpe-negative but also plasmid-borne cpe-
carrying strains. Although the chromosomal cpe-carrying strains
appear to better survive in certain extreme conditions [8,9], the
present results suggested that the plasmid-borne cpe-carrying and
cpe-negative strains have specific properties by which they are
better adapted to diverse environments than the chromosomal cpe-
The property of both the plasmid-borne cpe-positive and cpe-
negative strains, utilizing myo-inositol, suggests that these strains
are similarly adapted to multiple habitats. Apart from being
abundant in the soil and environment, myo-inositol is a component
of the eukaryotic cell wall and has been reported to be used by C.
perfringens as an alternative carbon source in the absence of glucose
. Several microorganisms inhabiting the soil can utilize myo-
inositol . The absence of this operon in all chromosomal cpe-
carrying strains may limit their ubiquitous occurrence considered
typical for C. perfringens, which suggests that the chromosomal cpe-
carrying strains have their own, an as-yet unidentified narrow
niche in the environment.
Since ethanolamine is abundant in the human intestine , the
presence of the operon encoding ethanolamine utilization in 70%
of the plasmid-borne cpe-carrying strains and in 90% of the cpe-
negative strains probably provides an advantage for survival and
colonization of the intestine for these strains [19,27]. The
clostridial ethanolamine operon resembles that of Enterobacteriacae
, among which ethanolamine utilization is common. Due to
their ability to utilize ethanolamine, the plasmid-borne cpe-
carrying strains could be adapted to the intestinal environment,
unlike the chromosomal cpe-carrying strains, that lacked the
ethanolamine utilization operon.
Biotin is involved in the central pathways of cell metabolism,
and bacteria unable to synthesize biotin need to acquire it from
external sources . The lack of this gene cluster in the
chromosomal cpe-positive strains may indicate that the habitat of
these strains is rich in free biotin.
The ability of the chromosomal cpe-positive C. perfringens strains
to utilize cellobiose obtained by enzymatic or acidic hydrolysis of
cellulose and laminarin common in plant cell walls may indicate
that these polysaccharides are available in the yet unknown habitat
of the chromosomal cpe-positive strains. Despite lacking the gene
cluster predicted to encode utilization of cellobiose, three plasmid-
borne cpe-carrying strains utilized cellobiose, which may indicate
that cellobiose utilization is encoded by multiple loci, not
necessarily represented in our microarrays.
Based on the currently available genome sequences, we expect
the cpe-positive C. perfringens strain population to contain hundreds
of genes not present in the reference genomes and thus not
represented on the microarrays. For example, majority of the 73
and 62 genes of the cpe-containing plasmids pCPF5603 and
pCPF4969 , respectively, are specific to plasmids, since they
share only 10 and 7 genes with SM101 according to BLAST.
Therefore one should bear in mind that the differential gene pool
observed in this study is likely to be larger and warrants future
The chromosomal cpe-carrying C. perfringens strains seem unable
to utilize myo-inositol or ethanolamine or to synthesize biotin,
Figure 1. Chromosomal cpe-carrying C. perfringens strains clustered separately from the plasmid-borne cpe-carrying and cpe-
negative strains. The figure was constructed using the MEV software .
Table 1. Minimum and maximum percentage of CDSs in the
three reference strains (SM101, ATCC13124, and 13) carried by
chromosomal and plasmid-borne cpe-carrying and cpe-
negative C. perfringens strains.
SM101 ATCC13124Strain 13
minmax minmax min max
Chromosomal86.2 94.970.7 81.4 63.874.8
Plasmid-borne71.8 84.482.891.575.6 87.4
cpe-negative73.4 85.3 80.4 99.974.599.0
CGH Analysis of cpe-Positive C. perfringens Type A
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which are important for soil and intestinal bacteria competing in
complex environments. The majority of the chromosomal cpe-
carrying strains in this study also lacked the fucose and sialidase
encoding genes, which further diminishes the territory of the
chromosomal cpe-positive strains . Presumably, the chromo-
somal cpe-positive strains are not ubiquitous and soil or intestines
are not the habitat of these strains, although the chromosomal
strains may compensate for some of the aforementioned deficien-
cies by producing toxins or by acquiring appropriate genes from
the environment. This is supported by the presence of many IS-
elements suggestive of gene transfer .
In light of our results, the habitat of the chromosomal cpe-
carrying C. perfringens strains appears to be rich in biotin, and the
ability to utilize cellobiose and laminarin may be beneficial.
Cellobiose, laminarin, and biotin are available in environments
where bacteria decompose plant material, such as composts. In
composts, the temperature may be high, allowing only the most
heat-tolerant strains, such as the chromosomal cpe-carrying strains,
to survive. Access of the spores of chromosomal cpe-carrying C.
perfringens to the food chain via the compost soil on the surface of
vegetables should be investigated.
Other environments rich in biotin and cellobiose include sewage
and sludge , where the chromosomal cpe-carrying C. perfringens
strains may end up via the excretions of food-poisoning patients.
The heat-resistant spores of the chromosomal cpe-positive strains
could also tolerate heat treatments and drying [8,9], which are
usually included in the waste water treatment procedures. Thus,
the role of sewage and sludge as a reservoir of chromosomal cpe-
positive C. perfringens should be addressed, as the spores surviving
the waste water treatment procedures may return to the food
chain via sludge used as fertilizer.
In conclusion, the results suggest the plasmid-borne cpe-carrying
strains and cpe-negative strains to be ubiquitous and adaptated to
the mammalian intestine. By contrast, the chromosomal cpe-
carrying strains appear to have a narrow niche in environments
containing degrading plant material. Thus, the plasmid-borne cpe-
carrying strains are proposed to contaminate foods by human due
to poor hygiene, whereas the chromosomal cpe-carrying strains
Figure 2. Genes differentiating the chromosomal cpe-carrying C. perfringens strains from the plasmid-borne cpe-carrying and cpe-
negative strains. The figure was constructed using the MEV software .
CGH Analysis of cpe-Positive C. perfringens Type A
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could spread to the food chain through ingredients of plant origin.
Further research is needed to elucidate the habitat of these strains.
Materials and Methods
A total of 83 C. perfringens type A strains isolated from foods
(n=19) and feces (n=9) associated with food poisonings, feces of
healthy (n=21) and ill (n=6) people, feces of healthy production
animals (pigs n=7, cattle n=5, broiler chickens n=5), soil (n=5),
and sludge (n=6) during 1986–2007 included 54 cpe-positive
strains and 29 cpe-negative strains (Table S1). Of the cpe-positive
strains, a chromosomal cpe was carried by 21 strains while 33
carried the cpe on a plasmid. Of the plasmid-borne cpe-carrying
strains, 20 represented genotype IS1470-like and 13 represented
genotype IS1151. The cpe-negative C. perfringens strains ATCC
13124 and 13, and the chromosomal cpe-positive strain SM101
were used as hybridization references. Genomic DNA of all strains
was isolated as described by Keto-Timonen et al. (2005) .
The DNA microarrays, based on the genomes of C. perfringens
type A strains 13 , ATCC13124, and SM101 , contained
two 60-mer probes for all protein coding sequences (CDSs)
annotated in the three genomes. The probes were designed using
the OligoArray2.1 software . Upon the probe design,
OligoArray2.1 software utilizes the BLAST algorithm for checking
the specificity of a probe. There are 2170 conserved genes (core
genome) in all the three reference genomes and over 2000 strain-
specific genes or genes present only in two of the reference strains.
First, probe design was done for each individual strain, and then
the results were combined. Up to five candidate probes were
designed for each CDS. Finally a maximum of two probes per
CDS were selected, not accepting any duplicate probes. Moreover,
four probes for each IS element (IS1469, IS1470, IS1470-like, and
IS1151) [6,4,5] were included for genotyping of cpe-positive C.
perfringens. Each of the eight sub-arrays of Agilent 8*15K custom
arrays (Agilent, Santa Clara, CA, USA) contained an equal set of
15 744 probes.
Hybridization and washes
A total of 0.5 mg of genomic DNA from each C. perfringens strain
was fluorescently labeled using the BioPrime labeling kit (Invitro-
gen, Carlsbad, CA, USA). The 26-ml labeling reaction contained
11.5 ml of diluted DNA, 10 ml of random octamer primers
(Invitrogen), 2.5 ml of 106 dCTP Nucleotide mix (Invitrogen),
1.5 ml of either Cy3 or Cy5-dCTP (GE Healthcare, Buckingham-
shire, UK), and 0.5 ml of Exo-Klenow fragment solution
(Invitrogen). The reactions were incubated at 37uC for 2 hours
and stopped by adding 2.5 ml of stop buffer (EDTA, Invitrogen).
For each hybridization, one Cy3-labeled and one Cy5-labeled
DNA sample were combined; thus two samples were hybridized
on each subarray and 16 samples on each array slide. The mixture
was purified with a DNA purification kit (QIAquick PCR
Purification Kit, Qiagen, Hilden, Germany) according to the
manufacturer’s instructions. The concentration of DNA and the
incorporation of the dye were checked with the Nanodrop device
(Nanodrop Technologies, Wilmington, MA, USA) before and after
labeling. A volume of 2.2 ml salmon sperm DNA (1 mg/ml) was
added to 17.8 ml of labeled combined sample solution, and the
mixture was heated at 95uC for 2 minutes for denaturation. A
volume of 5 ml of 106 blocking agent (Agilent) and 25 ml 2xGE
(HI-RPI) hybridization buffer (Agilent) were added. A total of
45 ml of the solution was hybridized to each microarray at 65uC
for 16 hours. The arrays were washed for 261 minute with Wash
Buffer 1 (Agilent) and for 1 minute with Wash Buffer 2 (Agilent),
pre-warmed to 37uC.
Scanning, image processing and data analysis
The slides were scanned (Axon GenePix Autoloader 4200 AL,
Westburg, Leusden, The Netherlands) using 5 mm pixel resolution.
Image processing was performed with the GenePix Pro 6.0
software. All hybridizations were normalized to the reference
strains after background correction. Since the probes were
designed based on three genomes, the location of the main mode
of log2-ratio distribution was calculated between the hybridized
strain and all reference strains, and the median value was used for
The distribution of logarithmic signal intensities formed two
clear peaks in each hybridization. A threshold was set between the
peaks based on replicated hybridizations of the two reference
strains ATCC13124 and 13; signal intensities from the probes
designed for the reference strain were above the threshold. The
selected threshold divided the probes into two groups: The peak
with greater values corresponded to probes with specific hybrid-
ization and genes predicted to be present, and the peak below the
threshold corresponded to probes predicting a gene to be absent/
divergent or yiedling unspecific hybridization and. The data
analysis was done using the R software , and visualization and
clustering were conducted using MEV . The data discussed in
this publication are compliant with the MIAME guidelines and
were deposited in NCBI’s Gene Expression Omnibus and are
accessible through GEO Series accession number GSE30954 (http://
To validate the DNA microarray results, the intensity of the IS
element, plc (encoding the alpha toxin), and the cpe probe spots was
compared with results of PCR assays showing the IS elements
downstream of cpe [4,5,23] and the presence of plc and cpe .
The signal intensity values of all validated probe spots were in
concordance with the PCR results.
Myo-inositol, cellobiose, and ethanolamine utilization of
the cpe-positive C. perfringens strains
The minimal medium was prepared according to Sebald and
Costilow (1975) . The growth of 10, 10, and 8 chromosomal,
and 11, 12, and 7 plasmid-borne cpe-carrying C. perfringens strains
was examined in the minimal medium using myo-inositol,
cellobiose, and ethanolamine, respectively, as the sole source of
energy. For controls, the growth of each strain was also examined
in minimal medium with glucose and in a plain minimal medium.
In brief, 25 ml of a 56104cfu/ml cell suspension of each strain was
inoculated into 2.5 ml of fresh minimal medium containing 1% of
either myo-inositol, cellobiose, or ethanolamine and incubated at
37uC overnight under anaerobic conditions. Growth in the
presence of myo-inositol and cellobiose was studied in an
automated turbidity reader (Bioscreen C Microbiology Reader,
Growth Curves, Helsinki, Finland). To demonstrate ethanolamine
utilization, 0.05% adenocylcobalamine, which is considered
essential for ethanolamine consumption , was added to the
media together with bromothymol blue as an indicator. Growth in
the presence of ethanolamine was studied in 10-ml aliquots. A
change of the indicator colour suggested ethanolamine utilization.
of chromosomal and plasmid-borne cpe-carrying C.
perfringens strains. The similarity between the chromosomal
Similarity between the strains in the clusters
CGH Analysis of cpe-Positive C. perfringens Type A
PLOS ONE | www.plosone.org5 October 2012 | Volume 7 | Issue 10 | e46162
cpe-carrying strains is 0.85 (Pearson’s correlation) The similarity
between the plasmid-borne cpe-carrying strains (IS1470-like and
IS1151) is 0.76, and the similarity between the two clusters is 0.59.
isolated from various sources.
Characterization of Clostridium perfringens type A strains
C. perfringens strains related to plasmid-borne cpe-carrying strains.
Variable CDSs (probes) in chromosomal cpe-carrying
biose of chromosomal and plasmid-borne cpe-carrying C. perfringens
Utilization of myo-inositole, ethanolamine, and cello-
clusters encoding the metabolic traits differentiating between the
chromosomal and plasmid-borne cpe-carrying C. perfringens strains.
The presence (+) and absence (2) of operons and gene
We thank Jari Aho and Kirsi Ristkari for technical assistance.
Conceived and designed the experiments: PL HK ML PS AH. Performed
the experiments: PL PS. Analyzed the data: PL PS ML HK. Contributed
reagents/materials/analysis tools: PL AH PS. Wrote the paper: PL ML
1. Scallan E, Hoekstra RM, Angulo FJ, Tauxe RV, Widdowson MA, et al. (2011)
Foodborne illness acquired in the united states–major pathogens. Emerg Infect
Dis 17(1): 7–15.
2. Smedley JG 3rd, Fisher DJ, Sayeed S, Chakrabarti G, McClane BA (2004) The
enteric toxins of Clostridium perfringens. Rev Physiol Biochem Pharmacol 152:
3. Kokai-Kun JF, Songer JG, Czeczulin JR, Chen F, McClane BA (1994)
Comparison of western immunoblots and gene detection assays for identification
of potentially enterotoxigenic isolates of Clostridium perfringens. J Clin Microbiol
4. Brynestad S, Synstad B, Granum PE (1997) The Clostridium perfringens enterotoxin
gene is on a transposable element in type A human food poisoning strains.
Microbiology 143 (Pt 7): 2109–2115.
5. Miyamoto K, Chakrabarti G, Morino Y, McClane BA (2002) Organization of
the plasmid cpe locus in Clostridium perfringens type A isolates. Infect Immun 70(8):
6. Cornillot E, Saint-Joanis B, Daube G, Katayama S, Granum PE, et al. (1995)
The enterotoxin gene (cpe) of Clostridium perfringens can be chromosomal or
plasmid-borne. Mol Microbiol 15(4): 639–647.
7. Sparks SG, Carman RJ, Sarker MR, McClane BA (2001) Genotyping of
enterotoxigenic Clostridium perfringens fecal isolates associated with antibiotic-
associated diarrhea and food poisoning in North America. J Clin Microbiol
39(3): 883–888. 10.1128/JCM.39.3.883–888.2001.
8. Li J, McClane BA (2006) Further comparison of temperature effects on growth
and survival of Clostridium perfringens type A isolates carrying a chromosomal or
plasmid-borne enterotoxin gene. Appl Environ Microbiol 72(7): 4561–4568.
9. Li J, McClane BA (2006) Comparative effects of osmotic, sodium nitrite-
induced, and pH-induced stress on growth and survival of Clostridium perfringens
type A isolates carrying chromosomal or plasmid-borne enterotoxin genes. Appl
Environ Microbiol 72(12): 7620–7625.
10. Tanaka D, Kimata K, Shimizu M, Isobe J, Watahiki M, et al. (2007)
Genotyping of Clostridium perfringens isolates collected from food poisoning
outbreaks and healthy individuals in Japan based on the cpe locus. Jpn J Infect
Dis 60(1): 68–69.
11. Grant KA, Kenyon S, Nwafor I, Plowman J, Ohai C, et al. (2008) The
identification and characterization of Clostridium perfringens by real-time PCR,
location of enterotoxin gene, and heat resistance. Foodborne Pathog Dis 5(5):
12. Lahti P, Heikinheimo A, Johansson T, Korkeala H (2008) Clostridium perfringens
type A strains carrying a plasmid-borne enterotoxin gene (genotype IS1151-cpe
or IS1470-like-cpe) as a common cause of food poisoning. J Clin Microbiol 46(1):
13. Wen Q, McClane BA (2004) Detection of enterotoxigenic Clostridium perfringens
type A isolates in American retail foods. Appl Environ Microbiol 70(5): 2685–
14. Miki Y, Miyamoto K, Kaneko-Hirano I, Fujiuchi K, Akimoto S (2008)
Prevalence and characterization of enterotoxin gene-carrying Clostridium
perfringens isolates from retail meat products in Japan. Appl Environ Microbiol
15. Labbe R (2000) The microbial safety and quality of food. In: Anonymous
Gaithersburg, Md, , USA: Aspen Publishers. pp. 1110–1135.
16. Daube G, Simon P, Limbourg B, Manteca C, Mainil J, et al. (1996)
Hybridization of 2,659 Clostridium perfringens isolates with gene probes for seven
toxins (alpha, beta, epsilon, iota, theta, mu, and enterotoxin) and for sialidase.
Am J Vet Res 57(4): 496–501.
17. Miwa N, Nishina T, Kubo S, Honda H (1997) Most probable numbers of
enterotoxigenic Clostridium perfringens in intestinal contents of domestic livestock
detected by nested PCR. J Vet Med Sci 59(7): 557–560.
18. Lindstro ¨m M, Heikinheimo A, Lahti P, Korkeala H (2011) Novel insights into
the epidemiology of Clostridium perfringens type A food poisoning. Food Microbiol
28(2): 192–198. 10.1016/j.fm.2010.03.020.
19. Heikinheimo A, Lindstro ¨m M, Granum PE, Korkeala H (2006) Humans as
reservoir for enterotoxin gene–carrying Clostridium perfringens type A. Emerg
Infect Dis 12(11): 1724–1729.
20. Carman RJ, Sayeed S, Li J, Genheimer CW, Hiltonsmith MF, et al. (2008)
Clostridium perfringens toxin genotypes in the feces of healthy North Americans.
Anaerobe 14(2): 102–108.
21. Li J, Sayeed S, McClane BA (2007) Prevalence of enterotoxigenic Clostridium
perfringens isolates in Pittsburgh (Pennsylvania) area soils and home kitchens. Appl
Environ Microbiol 73(22): 7218–7224.
22. Mueller-Spitz SR, Stewart LB, Klump JV, McLellan SL (2010) Freshwater
suspended sediments and sewage are reservoirs for enterotoxin-positive
Clostridium perfringens. Appl Environ Microbiol 76(16): 5556–5562. 10.1128/
23. Miyamoto K, Wen Q, McClane BA (2004) Multiplex PCR genotyping assay
that distinguishes between isolates of Clostridium perfringens type A carrying a
chromosomal enterotoxin gene (cpe) locus, a plasmid cpe locus with an IS1470-
like sequence, or a plasmid cpe locus with an IS1151 sequence. J Clin Microbiol
24. Brynestad S, Sarker MR, McClane BA, Granum PE, Rood JI (2001)
Enterotoxin plasmid from Clostridium perfringens is conjugative. Infect Immun
69(5): 3483–3487. 10.1128/IAI.69.5.3483-3487.2001.
25. Deguchi A, Miyamoto K, Kuwahara T, Miki Y, Kaneko I, et al. (2009) Genetic
characterization of type A enterotoxigenic Clostridium perfringens strains. PLoS
ONE 4(5): e5598.
26. Kawsar HI, Ohtani K, Okumura K, Hayashi H, Shimizu T (2004) Organization
and transcriptional regulation of myo-inositol operon in Clostridium perfringens.
FEMS Microbiol Lett 235(2): 289–295.
27. Roof DM, Roth JR (1988) Ethanolamine utilization in Salmonella typhimurium.
J Bacteriol 170(9): 3855–3863.
28. Tsoy O, Ravcheev D, Mushegian A (2009) Comparative genomics of
ethanolamine utilization. J Bacteriol 191(23): 7157–7164. 10.1128/JB.00838-09.
29. Streit WR, Entcheva P (2003) Biotin in microbes, the genes involved in its
biosynthesis, its biochemical role and perspectives for biotechnological
production. Appl Microbiol Biotechnol 61(1): 21–31. 10.1007/s00253-002-
30. Miyamoto K, Fisher J, Li J, Sayeed S, Akimoto S, et al. (2006) Complete
sequencing and diversity analysis of the enterotoxin-encoding plasmids in
Clostridium perfringens type A non-food-borne human gastrointestinal disease
isolates. J Bacteriol 188(4): 1585–1598.
31. Myers GS, Rasko DA, Cheung JK, Ravel J, Seshadri R, et al. (2006) Skewed
genomic variability in strains of the toxigenic bacterial pathogen, Clostridium
perfringens. Genome Res 16(8): 1031–1040.
32. Ho KL, Lee DJ (2011) Harvesting biohydrogen from cellobiose from sulfide or
nitrite-containing wastewaters using Clostridium sp. R1. Bioresour Technol
102(18): 8547–8549. 10.1016/j.biortech.2011.04.031.
33. Keto-Timonen R, Nevas M, Korkeala H (2005) Efficient DNA fingerprinting of
Clostridium botulinum types A, B, E, and F by amplified fragment length
polymorphism analysis. Appl Environ Microbiol 71(3): 1148–1154. 10.1128/
34. Shimizu T, Ohtani K, Hirakawa H, Ohshima K, Yamashita A, et al. (2002)
Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater.
Proc Natl Acad Sci U S A 99(2): 996–1001. 10.1073/pnas.022493799.
35. Rouillard JM, Zuker M, Gulari E (2003) OligoArray 2.0: Design of
oligonucleotide probes for DNA microarrays using a thermodynamic approach.
Nucleic Acids Res 31(12): 3057–3062.
36. R Development Core Team (2009) R: A Language and Environment for Statistical
Computing. R Foundation for Statistical Computing, Vienna, Austria.
CGH Analysis of cpe-Positive C. perfringens Type A
PLOS ONE | www.plosone.org6 October 2012 | Volume 7 | Issue 10 | e46162
37. Saeed AI, Bhagabati NK, Braisted JC, Liang W, Sharov V, et al. (2006) TM4 Download full-text
microarray software suite. In: Kimmel A, Oliver B, editors. Methods in
Enzymology. San Diego, California, , USA: Elsevier Academic Press. pp. 134–
38. Heikinheimo A, Korkeala H (2005) Multiplex PCR assay for toxinotyping
Clostridium perfringens isolates obtained from Finnish broiler chickens. Lett Appl
Microbiol 40(6): 407–411. 10.1111/j.1472-765X.2005.01702.x.
39. Sebald M, Costilow RN (1975) Minimal growth requirements for Clostridium
perfringens and isolation of auxotrophic mutants. Appl Microbiol 29(1): 1–6.
CGH Analysis of cpe-Positive C. perfringens Type A
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