Journal of Applied Microbiology ISSN 1364-5072

REVIEW ARTICLE

Dissemination of Clostridium difficile in food and the

environment: Significant sources of C. difficile
community-acquired infection?
K. Warriner1, C. Xu2, M. Habash3, S. Sultan3 and S.J. Weese4
1 Department of Food Science, University of Guelph, Guelph, ON, Canada
2 Shanghai Ocean University, Shanghai, China
3 School of Environmental Biology, University of Guelph, Guelph, ON, Canada
4 Pathobiology, University of Guelph, Guelph, ON, Canada

Keywords
CDI, Clostridium difficile, Clostridium
perfringens, community-associated,
endospores, food, germination, meat,
microbiome.
Correspondence
Keith Warriner, Department of Food Science,
University of Guelph, Guelph, ON N1G 2W1,
Canada.
E-mail: kwarrine@uoguelph.ca
2016/1640: received 27 July 2016, revised 25
October 2016 and accepted 27 October 2016
doi:10.1111/jam.13338
Summary
Clostridium difficile is a significant pathogen with over 300 000 cases reported
in North America annually. Previously, it was thought that C. difficile was
primarily a clinically associated infection. However, through the use of whole
genome sequencing it has been revealed that the majority of cases are
community acquired. The source of community-acquired C. difficile infections
(CDI) is open to debate with foodborne being one route considered.
Clostridium difficile fits the criteria of a foodborne pathogen with respect to
being commonly encountered in a diverse range of foods that includes meat,
seafood and fresh produce. However, no foodborne illness outbreaks have been
directly linked to C. difficile there is also no conclusive evidence that its spores
can germinate in food matrices. This does not exclude food as a potential
vehicle but it is likely that the pathogen is also acquired through zoonosis and
the environment. The most significant factor that defines susceptibility to CDI
is the host microbiome and functioning immune system. In this respect,
effective control can be exercised by reducing the environmental burden of C.
difficile along with boosting the host defences against the virulent enteric
pathogen.

source of C. difficile implicated in community-acquired

Introduction
Clostridium difficile is responsible for 300 000 cases of
infection within North America each year and is attributed
to over 25 000 deaths. Although initially confined to North
America, the pathogen has become distributed across the
globe. Clostridium difficile was for a long time thought to
be restricted to clinical settings such as hospitals were it
continues to be the leading cause of antimicrobial-asso-
ciated diarrhoea (McFarland 2016). Yet, with increased
surveillance and the advent of whole genome sequencing
(WGS), the proportion of C. difficile infections (CDI)
acquired from clinical settings is lower than 35% of the
total cases reported (Knight et al. 2015). The remainder
are placed under a large umbrella of community-acquired
CDI that essentially encompasses cases that have no recent
contact with clinical settings or antibiotic use. The actual
CDI remains open to speculation although a foodborne
link has been proposed given the faecaloral route of trans-
mission. Moreover, other clostridia such as Clostridium
perfringens and Clostridium botulinum are well established
foodborne pathogens that would lend support to C. difficile
following the same routes.
The classification of a foodborne pathogen is open to
interpretation although it has significant implications. Specif-
ically, pathogens designated as foodborne need to be consid-
ered in risk assessment and the means of prevention or
control to be then devised. The following provides a review
of our knowledge of C. difficile in relation to foodborne and
environmental sources. To provide context, the definition of
a foodborne pathogen will be A pathogen that can be
encountered or grow in foods at a sufficient level to cause ill-
ness and has had historical association with foodborne illness

542 Journal of Applied Microbiology 122, 542--553 2016 The Society for Applied Microbiology
K. Warriner et al.
outbreaks. This definition fits C. perfringens and C. botuli-
num both of which typically (but not exclusively) are
encountered and grow in foods along with being implicated
in foodborne illness outbreaks. In this context, C. difficile can
be classified within the group of unspecified agents that are
recovered in food but the ability to cause illness via this route
remains unknown (Scallen et al., 2011).
Background to Clostridium difficile
When first isolated from an infants stool sample back in
1935, C. difficile was named Bacillus difficile (Hall and
OToole 1935). Later, the pathogen was transferred to clos-
tridia on the basis that it was an obligate anaerobic spore
former. In some respects, C. difficile resembles C. perfrin-
gens in terms of its primary habitat such as the gastroin-
testinal tract, association with meat and diarrhoea being
the major symptom of infection. However, on a genetic
level the two clostridia are distantly related to the extent
that it has been proposed to move C. difficile to the pep-
tostreptococcaceae family, thereby renaming the pathogen
Peptoclostridium difficile (Yutin and Galperin 2013). More
recently, reclassifying C. difficile as Clostridioides difficile
was proposed based on phenotypic, chemotaxonomic and
phylogenetic analysis (Lawson et al. 2016). Although
renaming C. difficile is unlikely it does underline distinct
differences compared to other clostridia.
A further noteworthy feature of C. difficile is the large
genome size with a high proportion of mobile elements
that contributes to genetic plasticity (Knight et al. 2015).
Phenotypically this has supported rapid diversification
and adaptability to different host environments (Knight
et al. 2015). Genetically, C. difficile is comprised of a
large number of strains divided between six phylogenetic
clades. There has been a range of DNA typing techniques
applied to differentiate C. difficile, with ribotyping emerg-
ing as the most widely applied. From a clinical point of
view, the most significant C. difficile are from Clade 2
and Clade 5 that contain ribotype 027 and 078 respec-
tively (Knight et al. 2015). However, the high rate of evo-
lution within C. difficile gives rise to genetic lineages
from ribotypes 027 and 078. With so many strains it is
not unexpected to find that virulence and other pheno-
typic characteristics vary significantly within the same
ribotype (Knight et al. 2015). For example, a number of
ribotypes that evolved from 027 and 078 have missing or
nonfunctional virulence genes, while others have alterna-
tive virulence factors (Badger et al. 2012). This is more
so for ribotype 078 but has also been observed in dece-
dents of 027 (Knetsch et al. 2011). Although WGS is clar-
ifying the phylogenetic relationships between C. difficile
strains, the majority of literature to date has focused on
ribotypes 027 and 078 due to clinical significance. Yet, it
Journal of Applied Microbiology 122, 542--553 2016 The Society for Applied Microbiology
Clostridium difficile in food and environment
would be too simplistic to suggest that these two ribo-
types encompass all the virulent strains encountered.
Clostridium difficile mode-of-illness
Clostridium difficile is an obligate anaerobe that follows
the faecaloral route of infection. Endospores can be shed
in high levels by not only symptomatic patients but also
asymptomatic carriers, especially the young (Lees et al.
2016). The infectious dose of C. difficile is unknown but
considered to be low (1001000 spores) and strongly
dependent on host-related factors (Kansau et al. 2016).
Once ingested, the spore passes through the stomach and
then enters the duodenum where primary bile salts stim-
ulate the germination cascade (Bhattacharjee et al. 2016).
The cell undergoes outgrowth in the colon and caecum
where enterotoxins are produced. Virulent strains of
C. difficile express enterotoxin toxin A (TcdA) and the
cytotoxin toxin B (TcdB) (Lyras et al. 2009; Kuehne et al.
2010). Both toxins can disrupt the epithelial mucosal sur-
face and cause significant colonic inflammation (Badger
et al. 2012).
Toxin A and toxin B are encoded by tcdA and tcdB
genes that form part of the pathogenicity locus (PaLoc)
along with the regulatory genes tcdR (positive regulator)
and tcdC (negative regulator) (Martin-Verstraete et al.
2016). In hypervirulent strains, there is a deletion in the
latter that results in increased toxin production although
quorum sensing is also thought to be involved (Martin
et al. 2013). Although nontoxigenic stains do not harbour
PaLoc, it can be acquired through horizontal transfer
from toxigenic C. difficile (Janoir 2016). Variations in the
sequence of PaLoc are used to classify C. difficile into tox-
inotypes of which there are currently 31 identified
(Rupnik et al. 2009).
Binary toxin is another virulence factor associated with
hypervirulent strains that enhances the activity of Toxin
A and B in a mechanism that is thought to disrupt the
immune response of the host (Janoir 2016). C. difficile
strains harbouring the binary toxin but lacking Toxin A
and B have also been recovered from CDI patients sug-
gesting that the latter two toxins are not necessarily
required to cause symptoms of infection (Eckert et al.
2015).
Antimicrobial resistance of Clostridium difficile
The wide spread use of fluoroquinolone coincided with
the emergence of hypervirulent C. difficile (Huang et al.
2009). Resistance to antibiotics is encoded on trans-
posons, some of which are encoded for by bacteriophages
that facilitate widespread horizontal gene transfer (Roberts
et al. 2014). Antimicrobial resistance of clinical C. difficile
543
Clostridium difficile in food and environment K. Warriner et al.

isolates have been found to vary between geographical proximity. However, this view was questioned with the
locations (Huang et al. 2009). For example, erythromycin WGS of C. difficile isolates from clinical settings (Eyre
resistance is more common in UK isolates with cefotax- et al. 2013). Eyre et al. (2013) sequenced over 1200 iso-
one resistance being more frequently encountered in lates collected over a period of 4 years at a University
North America (Huang et al. 2009). Different antibiotic- hospital. The key conclusions from the study were that
resistant profiles have also been observed in isolates only 35% of the cases could be attributed to direct
derived from animal production. For example, clin- patient-to-patient transmission. Over 45% of the isolates
damycin resistance is more commonly encountered in were genetically distinct and most likely originated out-
cattle C. difficile isolates compared to swine, with line- side the hospital environment (Eyre et al. 2013). This
zolid resistance being more frequently found in pigs finding again underlined the increased significance of
(Thitaram et al. 2016). Linezoild resistance has also been community-associated CDI that was for many years con-
encountered in cattle which is significant considering the sidered secondary to those of hospital-acquired
antibiotic is not commonly used in animal production infections.
(Rodriguez-Palacios et al. 2011). It has been speculated The risk factors for community-acquired CDI differ to
that linezolid-resistant strains could be derived from the those of hospital-acquired infections. Specifically, com-
environment although selective pressure based on munity-acquired CDI occurs in young persons without
production practices cannot be discounted (Rodriguez- contact with hospital environments or a recent history of
Palacios et al. 2011; Bandelj et al. 2016). In addition to antibiotic but frequent use of proton inhibitors (Freeman
resistance, genes for antibiotic biosynthesis have been et al. 2010).
identified in strains of C. difficile ribotype 078 that, if
expressed, could potentially confer a competitive advan-
Incidence of Clostridium difficile in food animals
tage within the gastrointestinal tract environment
(Knetsch et al. 2011). The carriage of C. difficile in animals and foods has been
subjected to significant attention in a bid to confirm that
community-acquired CDI has origins in a zoonotic and/
Hospital-acquired infection vs community-acquired
or foodborne source. Clostridium difficile is frequently
infections
encountered in a diverse range of animals that includes
Community-acquired infections are essentially those per- both pets and those destined for meat production
sons who have not been discharged from a healthcare (Table 1). Young animals typically have the highest
facility in the previous 12 weeks and a patient who had prevalence of C. difficile with higher carriage being associ-
symptom onset during the first 48 h after hospitalization ated with herds being provided medicated feed, or those
(Cohen et al. 2010). In theory at least, foodborne acquisi- carrying underlying infections such as mastitis in the case
tion can take place in both hospitals and the community of dairy farms (Bandelj et al. 2016).
given foods can become contaminated at any point from Although ribotype 027 has been recovered in animals
production through consumption. Yet, historically food- (pigs, cattle and poultry) the main ribotype 078 accounts
borne transmission is commonly linked to community- for 7590% of isolates (Hawken et al. 2013b). From
acquired infection.
Another point of differentiation between hospital- and Table 1 The prevalence of Clostridium difficile ribotype 078 in food
community-acquired infections relates to the implicated animals
genotype. Specifically, hospital-acquired infections are
Animal Prevalence (%) Country Source
commonly associated with the hypervirulent strain ribo-
type 027/North American pulsotype (NAP) 1/toxinotype Piglets 66
7 Belgium Rodriguez et al. (2012)
III (Cohen 2009). The success of ribotype 027 is consid- Piglets 100 Holland Hopman et al. (2011)
ered to be due to the hypertoxin production, antibiotic Piglets 94
4 Spain Pel

aez et al. (2013)
Pig 91 Canada Weese et al. (2010c)
resistance, along with high sporulation and germination
Pig 83 Canada Keel et al. (2007)
rates (Akerlund et al. 2008). The hypervirulent ribotype Pig 67 Canada Weese et al. (2011)
027 was thought to emerge under a background of heavy Pig 78 Holland Koene et al. (2012)
fluoroquinolone that imposed selective pressure to Pig 31 Holland Keessen et al. (2011)
increase antibiotic resistance and virulence. Through Calves 75 Belgium Rodriguez et al. (2012)
using typing techniques (MLST and ribotyping) along Calves 67 Canada Costa et al. (2011)
with epidemiology, it was demonstrated that the main Calves 94 Canada Keel et al. (2007)
Calves 17 Germany Schneeberg et al. (2013)
transmission route of ribotype 027 was via horizontal
Calves 94 United States Hammitt et al. (2008)
transmission from symptomatic patients to those in close

544 Journal of Applied Microbiology 122, 542--553 2016 The Society for Applied Microbiology
K. Warriner et al. Clostridium difficile in food and environment

DNA sequence studies it has been established that 078
strains recovered in pigs show high similarity to those
isolated from clinical cases thereby confirming transmis-
sion from animal-to-human but also human-to-animal
(Knight et al. 2015). The result could be interpreted as
evidence that C. difficile populations associated with food
animals have a wide spectrum of different hosts or may
simply reflect lack of diversity within ribotype 078 (Bak-
ker et al. 2010; Knight et al. 2015).
Incidence of Clostridium difficile in wild animals
Wild animals have been considered as a vector for dissemi-
nating enteric pathogens between farms and urban centres.
It has been hypothesized that wild animals could act as vec-
tors for C. difficile which is supported by the recovery of
the pathogen from raccoons (8%, 4/52) and shrews (1/2)
(Jardine et al. 2013). Yet, no toxigenic C. difficile have been
recovered from deer mice, house mice, skunks, rats, voles,
opossums and groundhogs suggesting low carriage in wild
animals although more data are required to support such a
conclusion (Jardine et al. 2013).
Incidence in meat and other foods
The high prevalence of C. difficile in animals would translate
into carriage of the pathogen on meat through cross-con-
tamination events during the slaughter process. Hawken
et al. (2013a) undertook a sampling study within a pork
slaughter house, whereby samples were taken within the
holding area and from carcasses at each point of the slaugh-
ter process. Clostridium difficile was recovered from 80% of
manure samples taken from the holding area and 15% of
carcasses at postbleed, in addition to 1
5% at posteviscera-
tion (Hawken et al. 2013a). In a similar study performed in
the United States, C. difficile was recovered from only three
carcasses (post chill) from over 1500 tested samples despite
up to 30% prevalence in pigs at the production level
(Susick et al. 2012). Yet, the C. difficile recovered were
antibiotic resistant and toxigenic. In a comparable study in
a Korean pork slaughter house, two carcasses from 659
sampled tested positive for C. difficile representing a 0
2%
prevalence (Cho et al. 2015). Both isolates recovered were
identified as 078 and expressed antibiotic resistance.
Sampling performed in a Belgium slaughter house
recovered toxigenic C. difficile from 8% of beef and 7%
pork carcasses (Rodriguez et al. 2013). Through typing of
the isolates, 19 ribotypes were identified that included the
hypervirulent 078. More significantly, ribotypes recovered
from carcasses could be matched to clinical cases within
Belgium (Rodriguez et al. 2013).
From studies performed within slaughter houses, it can
be concluded that carriage of C. difficile on carcasses is
low. This could be attributed to the lack of cross-contam-
ination events during the slaughter process or effective-
ness of carcass decontamination methods. Yet, the
carriage of C. difficile on carcasses is likely to reflect the
sanitary practices with meat processing facilities.
Several studies have been performed to determine the
incidence of C. difficile in meat products, in addition to
other foods (Table 2). A common finding was the recov-
ery of toxigenic C. difficile in both ground and intact
meat from various species (Table 2). The data presented
cannot be considered as true prevalence given the limited

Table 2 Incidence of Clostridium difficile in meat, vegetables and seafood

% Positive for
Product Country C. difficile % Toxigenic Source

Ground beef Canada 6
1 80
0 Visser et al. (2012)

Ground beef Canada 12
2 100 Weese et al. (2009)
Ground veal USA 8
0 Not tested Houser et al. (2012)
Ground pork USA <1 0
3 Mooyottu et al. (2015)
Ground pork USA 9
5 100 Harvey et al. (2011)
Chicken meat USA 44
4 100 Songer et al. (2009)
Chicken meat Canada 12
5 100 Weese et al. (2010b)
Processed Canada 4
5 100 Metcalf et al. (2010)
vegetables
Raw vegetables UK 2
3 71
4 Bakri et al. (2009)
Leafy greens Iran 5
7 16
6 Yamoudy et al. (2015)
Lettuce USA 4
6 100 Rodriguez-Palacios et al. (2014)
Lettuce USA 13
8 100 Han (2016)
Lettuce France 2
9 100 Eckert et al. (2013)
Molluscs Italy 3
9 2 Troiano et al. (2015)
Shellfish USA 4
5 100 Norman et al. (2014)
Seafood (general) Canada 4
8 100 Metcalf et al. (2011)

Journal of Applied Microbiology 122, 542--553 2016 The Society for Applied Microbiology 545
Clostridium difficile in food and environment
sampling size used, along with different methodologies
and tendency for studies that failed to recover the patho-
gen not being published. With regard to the latter, it was
noted that in an extensive sampling trial that screened
over 1755 retail meat samples, no C. difficile was detected.
(Limbago et al. 2012). It should also be noted that in
those studies where C. difficile were recovered, there was
a need to be enriched indicating low levels of the patho-
gen. Nevertheless, in fulfilling the requirements to be
classed as a foodborne pathogen, it can be considered
that C. difficile can be potentially carried on meat prod-
ucts.
Additional foods have also been screened for the pres-
ence of C. difficile with a focus on those prone to be con-
taminated with faecal contamination. For example,
bivalve shellfish are filter feeders that can potentially
accumulate enteric pathogens when raised in polluted
waters. From the limited studies performed, C. difficile
have been isolated from both shellfish and fish suggesting
a potential source of toxigenic strains (Metcalf et al.
2011).
Fresh produce is another food group that has been
linked to outbreaks caused by enteric pathogens. Again,
sampling studies were limited and primarily sampled at
retail that raises questions with respect to the original
source of contamination. Yet, toxigenic, antibiotic-resis-
tant C. difficile strains have been recovered in vegetables
including leafy greens (Metcalf et al. 2010; Eckert et al.
2013; Han 2016).
The origins of C. difficile associated with vegetables
remain to be elucidated but is likely from water, manure,
compost and/or biosolids. This hypothesis is supported
by the finding that C. difficile can survive sewage treat-
ment and can be recovered in high prevalence in bioso-
lids along with effluent discharge (Xu et al. 2014). In the
aforementioned study, ribotype 078 was recovered from
19% of primary sludge (21/108), 8% of digested sludge
(8/106), 35% of biosolids (15/43) and 60% of down-
stream river samples (15/25). When biosolids harbouring
C. difficile was applied to land, the pathogen persisted
over a 9-month period with no overall decrease in levels
(Xu et al. 2016b). Indeed, evidence was found to suggest
that C. difficile could grow within the subsurface of bio-
soild-amended soil (Xu et al. 2016b). Given the combina-
tion of discharge or deposition of C. difficile into the
environment, along with long-term persistence, it can be
suggested that crops would be exposed to the enteric
pathogen.
Incidence of Clostridium difficile in the environment
Toxigenic C. difficile are commonly encountered in the
sediments from rivers and recreational lakes due to
546 Journal of Applied Microbiology 122, 542--553 2016 The Society for Applied Microbiology
K. Warriner et al.
constant input from land runoff or effluent derived from
wastewater treatments along with the longevity of endo-
spores. In a study performed within Southern Ontario,
92% of C. difficile recovered from river sediments were
found to be toxigenic (Xu et al. 2014). Of more concern
was the recovery of ribotype 078 from municipal water
samples taken from domestic housing that suggests the
C. difficile endospores can survive through drinking water
treatments (Bizaid 2013).
Studies to determine the incidence of C. difficile in soil
have been restricted to high-risk areas where manure has
been applied directly or indirectly to land. For example,
soil sampled within a stud farm recovered 14 isolates
from 132 samples screened (11% prevalence) compared
to 1% were only mature horses were kept (B averud et al.
2003). Clostridium difficile was recovered from 22% of
soil samples taken from a farmers market in Zimbabwe
(Simango and Mwakurudza 2008). In the same study, C.
difficile was found in environmental surface samples taken
from floors, tables, contact surfaces, windowsill, floor
drain, feed bowl and around watering devices (Simango
and Mwakurudza 2008).
Baverud et al. (2003) reported a 4% of soil samples
taken from rural area parks, playgrounds, gardens and
cultivated fields tested positive for C. difficile. Clostridium
difficile was also isolated from 21% (22/104) of the
suburb soil samples from public parks, gardens, play-
grounds and fields of South Wales (Al-saif and Brazier
1996).
Prevalence of Clostridium difficile in domestic homes
and residences
Within hospital environments there is significant person-
to-person or contact surface-to-person transmission of C.
difficile (Boyle et al. 2015). In principal, C. difficile could
also be acquired and transferred within enclosed spaces
such as domestic homes and residences. Homes for the
elderly are well acknowledged to have high risk of C. dif-
ficile due to a concentration of highly susceptible popula-
tions and challenges in sanitation (Shin et al. 2016). In a
small study performed within an elderly home a food
sample was found to be positive for C. difficile ribotype
078 demonstrating how foods can be contaminated dur-
ing preparation and handling (Rodriguez et al. 2015).
The incidence of C. difficile on domestic environments
(e.g. kitchens) has not been studied to any great extent
despite the risk of contaminating foods. From studies
performed in private residences, C. difficile was isolated
from 32% of the surfaces sampled (Alam et al. 2014).
The highest prevalence of C. difficile was from shoes with
the pathogen also being recovered from bathrooms in
addition to floors. Of the 30 households visited, C.
K. Warriner et al.
difficile was recovered from 25 illustrating the wide distri-
bution of the pathogen within the domestic setting (Alam
et al. 2014).
The original source of C. difficile within domestic
homes remains unclear although domestic pets have been
identified as a significant source. Studies have reported a
range of carriage levels of 5% for dogs and 3% for cats
although carriage depends on the pets age (Weese et al.
2010b). The source of C. difficile could be through envi-
ronmental exposure, pet food and water. However, the
transfer from humans to pets is also thought to be signif-
icant. This was underlined by the fact that dogs tested
more frequently for C. difficile when living in a household
with immune-comprised persons who carried the patho-
gen (Weese et al. 2010a).
The incidence of C. difficile in high population envi-
ronments such as schools, cruise ships, restaurants or
gymnasiums has not been studied. However, a study per-
formed to assess the sanitary status of hotel rooms has
been undertaken. Here, samples were taken from the liv-
ing area and bathroom surfaces within hotels across
Canada (Xu et al. 2015). Two toxigenic C. difficile isolates
were recovered from over 300 samples screened
representing <1% prevalence. Although low, the recovery
of C. difficile again underlines the wide distribution of
the enteric pathogen.
Growth of Clostridium difficile in foods
The growth of C. perfringens and C. botulinum in foods
is commonly, but not exclusively, a prerequisite for
both pathogens to cause foodborne illness. The extent
to which this applies to C. difficile remains unknown,
although it should be noted that spores rather than
vegetative cells need to be ingested to cause CDI. Nev-
ertheless, there remain large knowledge gaps with
respect to growth ranges of C. difficile, especially in
foods. It has been reported that C. difficile can metabo-
lize a broad range of carbohydrates and acids and
hence is adaptable to utilize the energy sources within
the gastrointestinal tract (Scaria et al. 2015). A compar-
ative study has been performed to determine the
growth limits of ribotype 078 and 027. Both ribotypes
grew within the pH range of 79 although greatly
reduced growth rates at pH values <6
5 and salt con-
centrations >4% was observed. No growth occurs at
4C although it could at 22 with an optimum of
37C (unpublished results). The growth of C. difficile is
also inhibited by preservatives commonly applied to
control clostridia, namely nitrite, nitrate, sodium
metabisulphite and nisin (Le Lay et al. 2016; Lim et al.
2016). Therefore, from the current evidence available it
can be concluded that the growth of C. difficile is rare
Journal of Applied Microbiology 122, 542--553 2016 The Society for Applied Microbiology
Clostridium difficile in food and environment
and those hurdles applied to control either C. perfrin-
gens and C. botulinum would also be effective against
the enteric pathogen (Lund and Peck 2015).
Germination of Clostridium difficile
Given that C. difficile exists in the endospore form out-
side the host environment there is a need to initiate ger-
mination prior to growth. Like most spores, germination
is enhanced through an initial heat shock applied for a
short period (10 min) between 60 and 100C (Ghosh
et al. 2009). It was naturally assumed that C. difficile
would follow the same germination mechanisms as other
clostridia such as C. perfringens. That is, receptors on the
inner spore membrane are activated to induce a cascade
of degradation reactions leading to eventual outgrowth.
In most spore formers the receptors sense the presence of
amino acids (L-alanine being a classic example) that initi-
ates the germination cascade. However, C. difficile is dif-
ferent in that germination is not triggered by amino acids
alone but requires bile or bile breakdown products (Bhat-
tacharjee et al. 2016). In evolutionary terms this would
make sense given the presence of bile would be an envi-
ronmental trigger so that the C. difficile spores germi-
nated within the gastrointestinal tract. By accident or
design, the presence of bile in the colon would also sig-
nify a disrupted microflora as microbes within the gas-
trointestinal tract would ordinarily breakdown bile
(Allegretti et al. 2016). Specifically, with a disrupted
microflora (for example, in the event of administration
of antibiotics) the bile acids, principally cholate, tauro-
cholate and glycocholate, are not metabolized thereby
activating the germination of C. difficile that then under-
goes out-growth with associated toxin production (Alle-
gretti et al. 2016). A further secondary bile by-product,
deoxycholate, can also stimulate germination but inhibits
the outgrowth of the vegetative cell, thereby having a duel
effect.
Given that the C. difficile endospore has a relatively
small time frame to germinate and grow in the lower gas-
trointestinal tract, it follows that those strains that germi-
nate quickly are more successful in causing infection. In
this regard, it is interesting to note that in hypervirulent
stains the inclusion of glycine or histidine, along with bile
salts, enhances the germination of spores and thereby the
ability to grow and produce toxin (Carlson et al. 2015).
Yet, it should be noted that strains within the same ribo-
type exhibit different responses to germinating agents.
Specifically, in some strains the proportion of superdor-
mancy is high. Superdormancy is a state where spores
experience a lag period prior to initiating germination
despite germinating agents being present. The lag period
can range from hours to weeks in some cases and is
547
Clostridium difficile in food and environment
significant in terms of returning false-negative results in
diagnostic tests (Rodriguez-Palacios and LeJeune 2011).
From an evolutionary standpoint, superdormancy allows
a proportion of the spores to remain in the dormant state
in the event the environmental conditions become
adverse for the germinating cells. Superdormancy has
been observed in C. difficile ribotype 078 but is strain
dependent (Xu et al. 2016a).
Given the essential need for bile salts and neutral to
alkaline pH for germination of C. difficile spores, it is
unlikely that this would occur in food systems. Indeed,
there have been no reports published in the literature on
C. difficile spore germination in food systems. In our own
studies, no germination of ribotype 027 and 078 occurred
in extracts of beef or fish without the addition of sodium
taurocholate bile salts (unpublished results). Yet, both
beef and fish extract did support the growth of both C.
difficile ribotypes when inoculated with vegetative cells.
Sporulation and endospore resistance in Clostridium
difficile
One of the notable features of sporulation in C. difficile is
a network that regulates sporulation which also controls
solventogenesis, biofilm formation and toxin production
(Burns and Minton 2011; Zidaric and Rupnik 2016).
Therefore, individual cells would respond differently to
the same signal with only a proportion entering into the
sporulation cycle (Saujet et al. 2014). In hypervirulent C.
difficile the yield of spores and faecal shedding is high,
thereby increasing the probability to be transferred to the
next host (Kansau et al. 2016).
Although the germination and growth of C. difficile in
foods is unlikely its endospores are encountered in food
products (Table 2). Reports have indicated that C. difficile
spores inoculated into meat can survive recommended
cooking practices (i.e. internal temperature of 73C)
(Redondo-Solono et al. 2016). Yet, it should be noted that
endospores would be expected to survive such temperatures
given the inherent thermal resistance (Setlow 2007; Rodri-
guez-Palacios et al. 2010; Paredes-Sabja et al., 2014). The
reported D values for C. difficile at 100C varies from 2.5 to
33 min depending on the strain, suspending medium and
methods of recovery (Lund and Peck 2015). With regard to
the latter, the apparent heat resistance of C. difficile can be
increased by inclusion of lysozyme in the recovery media.
Here, the lysozyme replaces the cortex-degrading enzyme
during spore germination in the event that the endogenous
enzymes were degraded (Lund and Peck 2015). In terms of
strain variation, there is no conclusive evidence that thermal
resistance correlates with virulence although it is interesting
to note that endospores derived from ribotype 078 have
enhanced heat tolerance compared to other types including
548 Journal of Applied Microbiology 122, 542--553 2016 The Society for Applied Microbiology
K. Warriner et al.
027 (Rodriguez-Palacios et al. 2016). In relative terms,
endospores of C. difficile spores show comparable or lower
resistance to C. perfringens although a high level of inter-
strain variation exists (Nakamura et al. 1985).
Like other endospores, those of C. difficile are resistant
to chemical antimicrobials with 5 log CFU reduction
requiring chlorine concentrations of over 3000 ppm for
15 min or 30% v/v hydrogen peroxide vapour (Perez
et al. 2005; Barbut et al. 2009). Therefore, C. difficile
spores are likely to survive standard sanitation regimes
applied in the industry.
Host-related factors increase susceptibility to Clostridium
difficile
The susceptibility of the host plays a significant role in
determining the outcomes of infection. This is most
evident for Listeria monocytogenes infection and is espe-
cially relevant in the case of C. difficile. Those within
the population most susceptible to CDI are the elderly
especially with most people suffering from CDI being
over the age of 65 although the young (<3 years old)
are also considered a high-risk group (Britton and
Young 2014).
The susceptibility towards C. difficile is dependent in
several factors that likely interplay to lead to CDI. For
example, the innate and humoral immune system is
thought to play a vital role in protecting against C. diffi-
cile along with neutrophiles and general gastrointestinal
physiology, all of which weaken with age (Shin et al.
2016). The microbiome is frequently identified as the
main factor on defining if C. difficile can germinate and
go on to produce toxin. Specifically, the decrease in the
proportion of bile acid degraders by exposure to antibi-
otics increases the concentration of germinants within the
colon thereby supporting C. difficile outgrowth and toxin
production (Allegretti et al. 2016).
Members of the microbiome can also directly inhibit
the growth and toxin production in C. difficile. These can
be naturally present or administered through probiotic
preparations or faecal transplants (McFarland 2016). Pro-
biotics can also produce antimicrobial compounds to
inhibit C. difficile growth or inhibit toxin production
(McFarland 2016).
In community-acquired CDI one of the most signifi-
cant risk factor is proton inhibitors that are taken to
reduce stomach acid (Clooney et al. 2016). The reduced
acidity remodel the microbiome to decrease bacteroide-
tes and increase firmicutes thereby increasing suscepti-
bility to CDI (Clooney et al. 2016). The microbiome-
modulating effects of proton inhibitors are not due to
changes in acidity but more with the types of nutrients
that reaches the lower gastrointestinal tract (Clooney
K. Warriner et al. Clostridium difficile in food and environment

Watercourse

Seafood

Sewage Treatment Facilities

Vegetables

Biosolids Spreading

Animal Production

Community Acquired CDI

Wild Animals Meat

Figure 1 The cyling and recycling of Clostridium difficile from zoonotic (- - - -), environmental (. . .. . .. . ..) or foodborne (______) sources implicated in
community-associated infections. The main incubators for C. difficile are animal production and general population. Clostridium difficile associ-
ated with animals can contaminate meat during processing and survive up to the point of consumption. Manure produced in animal production
can contaminate crops directly or leach into irrigation water or watersheds. Treated sewage effluent and biosolids disposed in the environment
can contaminate crops and seafood when released into estuaries. In addition, potential zoonotic transfer from animals to humans and vice versa
can occur to further disseminate C. difficile. [Colour figure can be viewed at wileyonlinelibrary.com]

et al. 2016). There is also evidence that proton inhibi-
tors disrupt the host immune system that would ordi-
narily reduce the risk of CDI (Larcombe et al. 2016).
Conclusions
Community-acquired CDI is increasing and accounts for
65% of cases reported with actual numbers probably
being underestimated. Through various lines of research,
C. difficile satisfies a number of criteria that would desig-
nate the pathogen as foodborne. Specifically, C. difficile is
encountered in foods such as meat and can survive the
cooking process. The pathogen is also encountered in
fresh produce and seafood that are minimally processed
and thereby remain so up to the point of consumption.
However, in other ways C. difficile is not a typical food-
borne pathogen given that it cannot grow in foods due
to the lack of ability to germinate in the absence of bile
salts. A counter argument would be that there are numer-
ous examples of foodborne pathogens that can cause ill-
ness without growing in foods. Enteric viruses, protozoa
along with virulent pathogens such as Campylobacter
jejuni are classic examples. Yet, there is a large knowledge
gap on what the infectious dose of C. difficile actually is.
Regardless of this fact, the most significant piece of evi-
dence against C. difficile being classed as a foodborne
pathogen is that no outbreaks have been traced to foods
contaminated with the pathogen.
In reality, it is likely that C. difficile is foodborne but
this is one of several routes that includes zoonotic, water-
borne, environmental and person-to-person (Fig. 1). It is
reasonable to suggest that we are exposed to toxigenic C.
difficile on a daily basis but only leads to CDI when a
combination of events occurs to disrupt the microbiome

Journal of Applied Microbiology 122, 542--553 2016 The Society for Applied Microbiology 549
Clostridium difficile in food and environment
and compromise the functioning of the immune system.
In addition to host-related factors, the virulence of C. dif-
ficile strains also contributes to the likelihood of acquir-
ing CDI. In this respect, the rapid evolution and
diversification of C. difficile means that the focus on ribo-
type 027 and 078 is too restrictive and virulence markers
would be more appropriate (Janoir 2016).
In terms of control, it would be unfeasible to inactivate
C. difficile in foods due to the inherent resistance of
endospores. A more productive approach is to minimize
the environmental burden of C. difficile through a One
Health approach. For example, it has been proposed to
administer vaccines against C. difficile to reduce carriage
in animals (Rodriguez-Palacios et al. 2013; Ghose and
Kelly 2015). Other initiatives including composting of
biosolids or thermophilic sludge digestion have proven to
be effective interventions to reduce the carriage of C. dif-
ficile in biosolids (Xu et al. 2016a,b). Increasing the resis-
tance of the host to CDI by tailoring the microbiome
would also be an effective protective approach.
Acknowledgements
We thank the Ontario Ministry of Agriculture, Food and
Rural Affairs for funding our work and scientists around
the globe for continuing to gain further insights into
Clostridium difficile.
Conflict of Interest
None declared.
References
Akerlund, T., Persson, I., Unemo, M., Noren, T., Svenungsson,
B., Wullt, M. and Burman, L.G. (2008) Increased
sporulation rate of epidemic Clostridium difficile Type 027/
NAP1. J Clin Microbiol 46, 15301533.
Alam, M.J., Anu, A., Walk, S.T. and Garey, K.W. (2014)
Investigation of potentially pathogenic Clostridium difficile
contamination in household environs. Anaerobe 27, 3133.
Allegretti, J.R., Kearney, S., Li, N., Bogart, E., Bullock, K.,
Gerber, G.K., Bry, L., Clish, C.B. et al. (2016) Recurrent
Clostridium difficile infection associates with distinct bile
acid and microbiome profiles. Aliment Pharmacol Therap
43, 11421153.
Al-saif, N. and Brazier, J.S. (1996) The distribution of
Clostridium difficile in the environment of South Wales.
J Med Microbiol 45, 133137.
Badger, V.O., Ledeboer, N.A., Graham, M.B. and Edmiston, C.E.
Jr (2012) Clostridium difficile: epidemiology, pathogenesis,
management, and prevention of a recalcitrant healthcare-
associated pathogen. JPEN 36, 645662.
550 Journal of Applied Microbiology 122, 542--553 2016 The Society for Applied Microbiology
K. Warriner et al.
Bakker, D., Corver, J., Harmanus, C., Goorhuis, A., Keessen,
E.C., Fawley, W.N., Wilcox, M.H. and Kuijper, E.J. (2010)
Relatedness of human and animal Clostridium difficile PCR
ribotype 078 isolates determined on the basis of
multilocus variable-number tandem-repeat analysis and
tetracycline resistance. J Clin Microbiol 48, 37443749.
Bakri, M.M., Brown, D.J., Butcher, J.P. and Sutherland, A.D.
(2009) Clostridium difficile in ready-to-eat salads, Scotland.
Emerg Infect Dis 15, 817818.
Bandelj, P., Blagus, R., Briski, F., Frlic, O., Rataj, A.V., Rupnik,
M., Ocepek, M. and Vengust, M. (2016) Identification of
risk factors influencing Clostridium difficile prevalence in
middle-size dairy farms. Vet Res 47, 11.
Barbut, F., Menuet, D., Verachten, M. and Girou, E. (2009)
Comparison of the efficacy of a hydrogen peroxide dry-
mist disinfection system and sodium hypochlorite solution
for eradication of Clostridium difficile spores. Infect Con
Hosp Epidemiol 30, 507514.
B
averud, V., Gustafsson, A., Franklin, A., Asp
an, A. and
Gunnarsson, A. (2003) Clostridium difficile: prevalence in
horses and environment, and antimicrobial susceptibility.
Equine Vet J 35, 465471.
Bhattacharjee, D., Francis, M.B., Ding, X., McAllister, K.N.,
Shrestha, R. and Sorg, J.A. (2016) Reexamining the
germination phenotypes of several Clostridium difficile
strains suggests another role for the CspC germinant
receptor. J Bacteriol 198, 777786.
Bizaid, F (2013). Distribution and sources of Clostridium
difficile present in water sources. MSc University of Guelph.
https://atrium.lib.uoguelph.ca/xmlui/bitstream/handle/
10214/5254/Bazaid_Fahad_201301_Msc.pdf?sequence=1
Boyle, M.L., Ruth-Sahd, L.A. and Zhou, Z. (2015) Fecal
microbiota transplant to treat recurrent Clostridium
difficile infections. Crit Care Nurse 35, 5164.
Britton, R.A. and Young, V.B. (2014) Role of the intestinal
microbiota in resistance to colonization by Clostridium
difficile. Gastroenterology 146, 15471553.
Burns, D.A. and Minton, N.P. (2011) Sporulation studies in
Clostridium difficile. J Microbiol Meth 87, 133138.
Carlson, P.E., Kaiser, A.M., McColm, S.A., Bauer, J.M., Young,
V.B., Aronoff, D.M. and Hanna, P.C. (2015) Variation in
germination of Clostridium difficile clinical isolates
correlates to disease severity. Anaerobe 33, 6470.
Cho, A., Byun, J.W., Kim, J.W., Oh, S.I., Lee, M.H. and Kim,
H.Y. (2015) Low prevalence of Clostridium difficile in
slaughter pigs in Korea. J Food Prot 78, 10341036.
Clooney, A.G., Bernstein, C.N., Leslie, W.D., Vagianos, K.,
Sargent, M., Laserna-Mendieta, E.J., Claesson, M.J. and
Targownik, L.E. (2016) A comparison of the gut
microbiome between long-term users and non-users of
proton pump inhibitors. Aliment Pharmacol Ther 43,
974984.
Cohen, M.B. (2009) Clostridium difficile infections: emerging
epidemiology and new treatments. J Pediatr Gastroenterol
Nut 48, 6365.
K. Warriner et al.
Cohen, S.H., Gerding, D.N., Johnson, S., Kelly, C.P., Loo,
V.G., McDonald, L.C., Pepin, J. and Wilcox, M.H. (2010)
Clinical practice guidelines for Clostridium difficile
infection in adults: 2010 update by the Society for
Healthcare Epidemiology of America (SHEA) and the
Infectious Diseases Society of America (IDSA). Inf Con
Hosp Epidemiol 31, 431455.
Costa, M.C., Stampfli, H.R., Arroyo, L.G., Pearl, D.L. and
Weese, J.S. (2011) Epidemiology of Clostridium difficile on
a veal farm: prevalence, molecular characterization and
tetracycline resistance. Vet Microbiol 152, 379384.
Eckert, C., Burghoffer, B. and Barbut, F. (2013)
Contamination of ready-to-eat raw vegetables with
Clostridium difficile in France. J Med Microbiol 62, 1435
1438.
Eckert, C., Emirian, A., Le Monnier, A., Cathala, L., de
Montclos, H., Goret, J., Berger, P., Petit, A. et al. (2015)
Prevalence and pathogenicity of binary toxin-positive
Clostridium difficile strains that do not produce toxins A
and B. New Micro New Infect 3, 1217.
Eyre, D.W., Cule, M.L., Wilson, D.J., Griffiths, D., Vaughan,
A., OConnor, L. and Ip, C.L.C. (2013) Diverse Sources of
C. difficile Infection Identified on Whole-Genome
Sequencing. N Engl J Med 369, 11951205.
Freeman, J., Bauer, M.P., Baines, S.D., Corver, J., Fawley,
W.N., Goorhuis, B., Kuijper, E.J. and Wilcox, M.H. (2010)
The changing epidemiology of Clostridium difficile
infections. Clin Microbiol Rev 23, 529549.
Ghose, C. and Kelly, C.P. (2015) The prospect for vaccines to
prevent Clostridium difficile infection. Infect Dis Clin N
Am 29, 145162.
Ghosh, S., Zhang, P., Li, Y.Q. and Setlow, P. (2009)
Superdormant spores of Bacillus species have elevated wet-
heat resistance and temperature requirements for heat
activation. J Bacteriol 191, 55845591.
Hall, I.C. and OToole, E. (1935) Intestinal flora in new-born
infants: with a description of a new pathogenic anaerobe,
Bacillus difficilis. Am J Dis Child 49, 390402.
Hammitt, M.C., Bueschel, D.M., Keel, M.K., Glock, R.D.,
Cuneo, P., DeYoung, D.W., Reggiardo, C., Trinh, H.T.
et al. (2008) A possible role for Clostridium difficile in the
etiology of calf enteritis. Vet Microbiol 127, 343352.
Han, Y. (2016) Detection of antibiotic resistant Clostridium
difficile in lettuce. MSc thesis. Louisiana State University.
http://etd.lsu.edu/docs/available/etd-07072016-015631/
unrestricted/YiHan.pdf (accessed October 2016).
Harvey, R.B., Norman, K.N., Andrews, K., Norby, B., Hume,
M.E., Scanlan, C.M., Hardin, M.D. and Scott, H.M.
(2011) Clostridium difficile in retail meat and processing
plants in Texas. J Vet Diagn Invest 23, 807811.
Hawken, P., Weese, J.S., Friendship, R. and Warriner, K.
(2013a) Carriage and dissemination of Clostridium difficile
and methicillin resistant Staphylococcus aureus in pork
processing. Food Cont 31, 433437.
Journal of Applied Microbiology 122, 542--553 2016 The Society for Applied Microbiology
Clostridium difficile in food and environment
Hawken, P., Weese, J.S., Friendship, R. and Warriner, K.
(2013b) Longitudinal study of Clostridium difficile and
methicillin-resistant Staphylococcus aureus associated with
pigs from weaning through to the end of processing.
J Food Prot 76, 624630.
Hopman, N.E.M., Keessen, E.C., Harmanus, C., Sanders, I.,
van Leengoed, L., Kuijper, E.J. and Lipman, L.J.A. (2011)
Acquisition of Clostridium difficile by piglets. Vet Microbiol
149, 186192.
Houser, B.A., Soehnlen, M.K., Wolfgang, D.R., Lysczek, H.R.,
Burns, C.M. and Jayarao, B.M. (2012) Prevalence of
Clostridium difficile toxin genes in the feces of veal calves
and incidence of ground veal contamination. Food Pathog
Dis 9, 3236.
Huang, H.H., Weintraub, A., Fang, H. and Nord, C.E. (2009)
Antimicrobial resistance in Clostridium difficile. Int J
Antimicrob Agents 34, 516522.
Janoir, C. (2016) Virulence factors of Clostridium difficile and
their role during infection. Anaerobe 37, 1324.
Jardine, C.M., Reid-Smith, R.J., Rousseau, J. and Weese, J.S.
(2013) Detection of Clostridium difficile in small and
medium-sized wild mammals in Southern Ontario,
Canada. J Wildlife Dis 49, 418421.
Kansau, I., Barketi-Klai, A., Monot, M., Hoys, S., Dupuy, B.,
Janoir, C. and Collignon, A. (2016) Deciphering
adaptation strategies of the epidemic Clostridium difficile
027 strain during infection through in vivo transcriptional
analysis. PLoS ONE 11, e0158204.
Keel, K., Brazier, J.S., Post, K.W., Weese, S. and Songer, J.G.
(2007) Prevalence of PCR ribotypes among Clostridium
difficile isolates from pigs, calves, and other species. J Clin
Microbiol 45, 19631964.
Keessen, E.C., Van den Berkt, A.J., Haasjes, N.H., Hermanus,
C., Kuijper, E.J. and Lipman, L.J.A. (2011) The relation
between farm specific factors and prevalence of
Clostridium difficile in slaughter pigs. Vet Microbiol 154,
130134.
Knetsch, C.W., Hensgens, M.P.M., Harmanus, C., van der Bijl,
M.W., Savelkoul, P.H.M., Kuijper, E.J., Corver, J. and van
Leeuwen, H.C. (2011) Genetic markers for Clostridium
difficile lineages linked to hypervirulence. Microbiology 157,
31133123.
Knight, D.R., Elliott, B., Chang, B.J., Perkins, T.T. and Riley,
T.V. (2015) Diversity and evolution in the genome of
Clostridium difficile. Clin Microbiol Rev 28, 721741.
Koene, M.G.J., Mevius, D., Wagenaar, J.A., Harmanus, C.,
Hensgens, M.P.M., Meetsma, A.M., Putirulan, F.F., van
Bergen, M.A.P. et al. (2012) Clostridium difficile in Dutch
animals: their presence, characteristics and similarities
with human isolates. Clin Microbiol Infect 18, 778784.
Kuehne, S.A., Cartman, S.T., Heap, J.T., Kelly, M.L.,
Cockayne, A. and Minton, N.P. (2010) The role of toxin
A and toxin B in Clostridium difficile infection. Nature
467, 711713.
551
Clostridium difficile in food and environment
Larcombe, S., Hutton, M.L. and Lyras, D. (2016) Involvement
of bacteria other than Clostridium difficile in antibiotic-
associated diarrhoea. Tren Microbiol 24,
463476.
Lawson, P.A., Citron, D.M., Tyrrell, K.L. and Finegold, S.M.
(2016) Reclassification of Clostridium difficile as
Clostridioides difficile (Hall and OToole 1935) Prevot
1938. Anaerobe 40, 9599.
Le Lay, C., Dridi, L., Bergeron, M.G., Ouellette, M. and Fliss,
I. (2016) Nisin is an effective inhibitor of Clostridium
difficile vegetative cells and spore germination. J Med
Microbiol 65, 169175.
Lees, E.A., Miyajima, F., Pirmohamed, M. and Carrol, E.D.
(2016) The role of Clostridium difficile in the paediatric
and neonatal gut a narrative review. Eur J Clin Microbiol
Infect Dis 35, 10471057.
Lim, S.C., Foster, N.F. and Riley, T.V. (2016) Susceptibility of
Clostridium difficile to the food preservatives sodium
nitrite, sodium nitrate and sodium metabisulphite.
Anaerobe 37, 6771.
Limbago, B., Thompson, A.D., Greene, S.A., MacCannell, D.,
MacGowan, C.E., Jolbitado, B., Hardin, H.D., Estes, S.R.
et al. (2012) Development of a consensus method for
culture of Clostridium difficile from meat and its use in a
survey of US retail meats. Food Microbiol 32, 448451.
Lund, B.M. and Peck, M.W. (2015) A possible route for
foodborne transmission of Clostridium difficile? Foodborne
Pathog Dis 12, 177182.
Lyras, D., OConnor, J.R., Howarth, P.M., Sambol, S.P.,
Carter, G.P., Phumoonna, T., Poon, R., Adams, V. et al.
(2009) Toxin B is essential for virulence of Clostridium
difficile. Nature 458, 11761181.
Martin, M.J., Clare, S., Goulding, D., Faulds-Pain, A., Barquist,
L., Browne, H.P., Pettit, L., Dougan, G. et al. (2013) The
agr locus regulates virulence and colonization genes in
Clostridium difficile 027. J Bacteriol 195, 36723681.
Martin-Verstraete, I., Peltier, J. and Dupuy, B. (2016) The
regulatory networks that control Clostridium difficile toxin
synthesis. Toxins 8, E153.
McFarland, L.V. (2016) Therapies on the horizon for
Clostridium difficile infections. Expert Opin Invest Drugs
25, 541555.
Metcalf, D.S., Costa, M.C., Dew, W.M.V. and Weese, J.S.
(2010) Clostridium difficile in vegetables, Canada. Lett Appl
Microbiol 51, 600602.
Metcalf, D., Avery, B.P., Janecko, N., Matic, N., Reid-Smith,
R. and Weese, J.S. (2011) Clostridium difficile in seafood
and fish. Anaerobe 17, 8586.
Mooyottu, S., Flock, G., Kollanoor-Johny, A., Upadhyaya, I.,
Jayarao, B. and Venkitanarayanan, K. (2015)
Characterization of a multidrug resistant C. difficile meat
isolate. Int J Food Microbiol 192, 111116.
Nakamura, S., Yamakawa, K., Izumi, J., Nakashio, S. and
Nishida, S. (1985) Germinability and heat resistance of
552 Journal of Applied Microbiology 122, 542--553 2016 The Society for Applied Microbiology
K. Warriner et al.
spores of Clostridium difficile strains. Microbiol Immun 29,
113118.
Norman, K.N., Harvey, R.B., Andrews, K., Hume, M.E.,
Callaway, T.R., Anderson, R.C. and Nisbet, D.J. (2014)
Survey of Clostridium difficile in retail seafood in College
Station, Texas. Food Addit Contam Part A Chem Anal
Control Expo Risk Assess 31, 11271129.
Paredes-Sabja, D., Shen, A. and Sorg, J.A. (2014) Clostridium
difficile spore biology: sporulation, germination, and spore
structural proteins. Trends in Microbiology 22, 406416.


Pel
aez, T., Alcal
a, L., Blanco, J.L., Alvarez-P

erez, S., Mar
n, M.,
Mart
n-L
opez, A., Catal
an, P., Reigadas, E. et al. (2013)
Characterization of swine isolates of Clostridium difficile in
Spain: a potential source of epidemic multidrug resistant
strains? Anaerobe 22, 4549.
Perez, J., Springthorpe, V.S. and Sattar, S.A. (2005) Activity of
selected oxidizing microbicides against the spores of
Clostridium difficile: relevance to environmental control.
Am J Infect Control 33, 320325.
Redondo-Solono, M., Burson, D.E. and Thippareddi, H.
(2016) Thermal resistance of Clostridium difficile spores in
peptone and pork meat. J Food Prot 79, 14681474.
Roberts, A.P., Allan, E. and Mullany, P. (2014) The impact of
horizontal gene transfer on the biology of Clostridium
difficile. In Adv Microb Physiol, Vol 65: Advances in
Bacterial Pathogen Biology ed. Poole, R.K., pp 6382.
London: Academic Press Ltd-Elsevier Science Ltd.
Rodriguez, C., Taminiau, B., van Broeck, J., Avesani, V.,
Delm
ee, M. and Daube, G. (2012) Clostridium difficile in
young farm animals and slaughter animals in Belgium.
Anaerobe 18, 621625.
Rodriguez, C., Avesani, V., van Broeck, J., Taminiau, B.,
Delmee, M. and Daube, G. (2013) Presence of Clostridium
difficile in pigs and cattle intestinal contents and carcass
contamination at the slaughterhouse in Belgium. Int J
Food Microbiol 166, 256262.
Rodriguez, C., Korsak, N., Taminiau, B., Avesani, V., van
Broeck, J., Brach, P., Delmee, M. and Daube, G. (2015)
Clostridium difficile from food and surface samples in a
Belgian nursing home: an unlikely source of
contamination. Anaerobe 32, 8789.
Rodriguez-Palacios, A. and LeJeune, J.T. (2011) Moist-heat
resistance, spore aging, and superdormancy in Clostridium
difficile. Appl Environ Microbiol 77, 30853091.
Rodriguez-Palacios, A., Reid-Smith, R.J., Staempfli, H.R. and
Weese, J.S. (2010) Clostridium difficile survives minimal
temperature recommended for cooking ground meats.
Anaerobe 16, 540542.
Rodriguez-Palacios, A., Koohmaraie, M. and LeJeune, J.T.
(2011) Prevalence, enumeration, and antimicrobial agent
resistance of Clostridium difficile in cattle at harvest in the
United States. J Food Prot 74, 16181624.
Rodriguez-Palacios, A., Borgmann, S., Kline, T.R. and Lejeune,
J.T. (2013) Clostridium difficile in foods and animals:
K. Warriner et al.
history and measures to reduce exposure. Anim Health Res
Rev 14, 2935.
Rodriguez-Palacios, A., Ilic, S. and LeJeune, J.T. (2014)
Clostridium difficile with moxifloxacin/clindamycin
resistance in vegetables in Ohio, USA, and prevalence
meta-analysis. J Path 2014, 158601.
Rodriguez-Palacios, A., Ilic, S. and LeJeune, J.T. (2016)
Subboiling moist heat favors the selection of enteric
pathogen Clostridium difficile PCR ribotype 078 spores in
food. Can J Infect Dis Med Microbiol 8, 1462405.
Rupnik, M., Wilcox, M.H. and Gerding, D.N. (2009) Clostridium
difficile infection: new developments in epidemiology and
pathogenesis. Nature Rev Microbiol 7, 526536.
Saujet, L., Pereira, F.C., Henriques, A.O. and Martin-
Verstraete, I. (2014) The regulatory network controlling
spore formation in Clostridium difficile. FEMS Microbiol
Lett 358, 110.
Scallen, E., Griffin, P.M., Angulo, F.J., Tauxa, R.V. and
Hoekstra, M. (2011) Foodborne illness acquired in the
United States- unspecified agents. Emerg Inf Dis 17, 1622.
Scaria, J., Suzuki, H., Ptak, C.P., Chen, J.-W., Zhu, Y., Guo,
X.-K. and Chang, Y.-F. (2015) Comparative genomic and
phenomic analysis of Clostridium difficile and Clostridium
sordellii, two related pathogens with differing host tissue
preference. BMC Genom 16, 448.
Schneeberg, A., Neubauer, H., Schmoock, G., Grossmann, E. and
Seyboldt, C. (2013) Presence of Clostridium difficile PCR
ribotype clusters related to 033, 078 and 045 in diarrhoeic
calves in Germany. J Med Microbiol 62, 11901198.
Setlow, P. (2007) I will survive: DNA protection in bacterial
spores. Trends Microbiol 15, 172180.
Shin, J.H., High, K.P. and Warren, C.A. (2016) Older is not
wiser, immunologically speaking: effect of aging on host
response to Clostridium difficile infections. J Gerontol A
Biol Sci Med Sci 71, 916922.
Simango, C. and Mwakurudza, S. (2008) Clostridium difficile
in broiler chickens sold at market places in Zimbabwe and
their antimicrobial susceptibility. Int J Food Microbiol 124,
268270.
Songer, J.G., Trinh, H.T., Killgore, G.E., Thompson, A.D.,
McDonald, L.C. and Limbago, B.M. (2009) Clostridium
difficile in retail meat products, USA, 2007. Emerg Infect
Dis 15, 819821.
Susick, E.K., Putnam, M., Bermudez, D.M. and Thakur, S.
(2012) Longitudinal study comparing the dynamics of
Clostridium difficile in conventional and antimicrobial free
pigs at farm and slaughter. Vet Microbiol 157, 172178.
Thitaram, S.N., Frank, J.F., Siragusa, G.R., Bailey, J.S., Dargatz,
D.A., Lombard, J.E., Haley, C.A., Lyon, S.A. et al. (2016)
Antimicrobial susceptibility of Clostridium difficile isolated
from food animals on farms. Int J Food Microbiol 227, 15.
Troiano, T., Harmanus, C., Sanders, I., Pasquale, V.,
Dumontet, S., Capuano, F., Romano, V. and Kuijper, E.J.
Journal of Applied Microbiology 122, 542--553 2016 The Society for Applied Microbiology
Clostridium difficile in food and environment
(2015) Toxigenic Clostridium difficile PCR ribotypes in
edible marine bivalve molluscs in Italy. Int J Food
Microbiol 208, 3034.
Visser, M., Sepehrim, S., Olson, N., Du, T., Mulvey, M.R. and
Alfa, M.J. (2012) Detection of Clostridium difficile in retail
ground meat products in Manitoba. Can J Inf Dis Med
Microbiol 23, 2830.
Weese, J.S., Avery, B.P., Rousseau, J. and Reid-Smith, R.J.
(2009) Detection and enumeration of Clostridium difficile
spores in retail beef and pork. Appl Environ Microbiol 75,
50095011.
Weese, J.S., Finley, R., Reid-Smith, R.R., Janecko, N. and
Rousseau, J. (2010a) Evaluation of Clostridium difficile in
dogs and the household environment. Epidemiol Infect
138, 11001104.
Weese, J.S., Reid-Smith, R.J., Avery, B.P. and Rousseau, J.
(2010b) Detection and characterization of Clostridium
difficile in retail chicken. Lett Appl Microbiol 50, 362365.
Weese, J.S., Wakeford, T., Reid-Smith, R., Rousseau, J. and
Friendship, R. (2010c) Longitudinal investigation of
Clostridium difficile shedding in piglets. Anaerobe 16, 501
504.
Weese, J.S., Rousseau, J., Deckert, A., Gow, S.l. and Reid-
Smith, R.J. (2011) Clostridium difficile and methicillin-
resistant Staphylococcus aureus shedding by slaughter-age
pigs. BMC Vet Res 7, 17.
Xu, C., Weese, J.S., Flemming, C., Odumeru, J. and Warriner,
K. (2014) Fate of Clostridium difficile during wastewater
treatment and incidence in Southern Ontario watersheds.
J Appl Microbiol 117, 1219.
Xu, C., Weese, S.J., Namvar, A. and Warriner, K. (2015)
Sanitary status and incidence of methicillin-resistant
Staphylococcus aureus and Clostridium difficile within
Canadian hotel rooms. J Environ Health 77, 815.
Xu, C., Salsali, H., Weese, S. and Warriner, K. (2016a)
Inactivation of Clostridium difficile in sewage sludge by
anaerobic thermophilic digestion. Can J Microbiol 62, 1623.
Xu, C., Wang, D., Huber, A., Weese, S.J. and Warriner, K.
(2016b) Persistence of Clostridium difficile in wastewater
treatment-derived biosolids during land application or
windrow composting. J Appl Microbiol 120, 312320.
Yamoudy, M., Mirlohi, M., Isfahani, B.N., Jalali, M.,
Esfandiari, Z. and Hosseini, N.S. (2015) Isolation of
toxigenic Clostridium difficile from ready-to-eat salads by
multiplex polymerase chain reaction in Isfahan, Iran. Adv
Biomed Res 4, 87.
Yutin, N. and Galperin, M.Y. (2013) A genomic update on
clostridial phylogeny: Gram-negative spore formers and other
misplaced clostridia. Environ Microbiol 15, 26312641.
Zidaric, V. and Rupnik, M. (2016) Sporulation properties and
antimicrobial susceptibility in endemic and rare
Clostridium difficile PCR ribotypes. Anaerobe 39, 183188.
553