The zoonotic potential of Clostridium difficile from small companion animals and their owners
The zoonotic potential of Clostridium difficile from small companion animals and their owners
Denise Rabold 0 1
Werner Espelage 0
Muna Abu Sin 0
Tim Eckmanns 0
Alexander Schneeberg 0
Heinrich Neubauer 0
Nadine MoÈ bius 0
Katja Hille 0
Lothar H. Wieler 0 1
Christian Seyboldt 0
Antina LuÈ bke-Becker 0 1
0 Editor: Michel R. Popoff, Institut Pasteur , FRANCE
1 Institute of Microbiology and Epizootics, Department of Veterinary Medicine, Freie UniversitaÈ t Berlin , Berlin, Germany, 2 Robert Koch Institute, Berlin, Germany , 3 Institute of Bacterial Infections and Zoonoses, Federal Research Institute for Animal Health (Friedrich-Loeffler-Institut) , Jena, Germany , 4 Department of Biometry, Epidemiology and Information Processing, University of Veterinary Medicine Hannover, Foundation , Hannover , Germany
July 2012-August 2013. PCR ribotyping, Multilocus VNTR Analysis (MLVA) and PCR
detection of toxin genes were used to characterize isolated C. difficile strains. A database was
defined and logistic regression used to identify putative factors associated with fecal
Data Availability Statement: All relevant data re
within the paper and its Supporting Information
Funding: This work was supported by the
Bundesministerium fuÈr Bildung und Forschung
01KI1107 (Mrs Denise Rabold) /01KI1108 (FLI,
Jena) (https://www.bmbf.de/) and the Friedrich
Naumann Stiftung (Mr Alexander Schneeberg)
(https://www.freiheit.org/). The funders had no role
in study design, data collection and analysis,
decision to publish, or preparation of the
ding of C. difficile.
In total, 1,418 samples met the inclusion criteria. The isolation rates for small companion
animals and their owners within the community were similarly low with 3.0% (25/840) and
2.9% (17/578), respectively. PCR ribotyping revealed eight and twelve different RTs in
animals and humans, respectively, whereas three RTs were isolated in both, humans and
animals. RT 014/0, a well-known human hospital-associated lineage, was predominantly
detected in animal samples. Moreover, the potentially highly pathogenic RTs 027 and 078
were isolated from dogs. Even though, C. difficile did not occur simultaneously in animals
Competing interests: The authors have declared
that no competing interests exist.
and humans sharing the same household. The results of the epidemiological analysis of
factors associated with fecal shedding of C. difficile support the hypothesis of a zoonotic
Molecular characterization and epidemiological analysis revealed that the zoonotic risk for
C. difficile associated with dogs and cats within the community is low but cannot be
Clostridium difficile is the major cause of antibiotic and hospital-associated diarrhea in
humans. Since 2001, changes in incidence and epidemiology have fostered discussions about
the source of infection and possible transmission routes. Although C. difficile infections (CDI)
are mainly diagnosed in health-care settings, about one quarter of the CDI-cases is estimated
to occur within the community [
]. Moreover, the epidemiological link between the majority
of symptomatic patients suffering from CDI and a subsequent CDI-patient is still missing,
thus, suggesting a community acquisition [
]. In particular, with regard to
communityacquired CDI, the overlap of C. difficile strains isolated from humans and animals has
increasingly urged one to explore the significance of C. difficile isolation in various animal species and
its potential for zoonotic transmission [
]. RT 014/0 has been reported to be the most
common cause of (CDI-) diarrhea in humans in Europe [
]. Although, RT 014/0 is seldom
involved in severe epidemic outbreaks, it seems to have particular adaptive capabilities since it
can be found in a broad spectrum of animal species [
]. The third most prevalent RT in
humans in European countries is RT 078  which is also the most common RT in bovine and
porcine populations [
]. Identical isolates from human and livestock samples and a genetic
relatedness between human and porcine RT 078 strains have been described before [11±13].
In addition, identical RT 078 strains shared by farmers and their pigs have also been identified
. These findings have triggered concerns about the zoonotic transmission of this important
pathogen. Moreover, the emergence of RT 027 has been particularly linked to elevated rates of
CDI in humans in Europe and Northern America [
]. Interestingly, RT 027 has also been
previously isolated from cattle and horses  for example, though data about RT 027 in
companion animals are rare.
Recent reports of C. difficile colonization and infection in dogs indicate that C. difficile has
also potentially emerged as a pathogen of small companion animals [
epidemiological data concerning companion animals are scarce. In Germany, surveys addressing
the occurrence of C. difficile in dogs and cats are restricted to an investigation in animal
shelters  and the reports about dogs and cats originating from veterinary clinics published
nearly 30 years ago [
While age, hospitalization and prior antibiotic exposure are confirmed risk factors for CDI
in humans [
], factors associated with the isolation of C. difficile in small companion animals
(dogs and cats) are widely unknown. Therefore, we aimed to comparatively determine the
isolation rates for C. difficile in dogs, cats and their owners, to describe the molecular
characteristics of the isolates and analyze the putative impact of demographic factors and variables such
as health status, prior medication, diet/feeding, and intensity of contact between humans and
animals on the occurrence of C. difficile.
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Materials and methods
Convenience samples from dog- and/or cat-owning households were acquired between July
2012 and August 2013, through personal contacts, kennel clubs, veterinarians, advertisements
in professional journals and social media. To participate in the study, at least one dog or cat
and at least one person (animal owner), living in the same household had to meet the inclusion
criteria which were residency in Germany, signed consent forms, filled in questionnaires and
one fecal sample per participating household member. For each participating household one
person was defined as a contact person and was personally instructed about the study (by
proxy for other participating household members if applicable), and was sent a package
containing individually labeled stool containers for self-sampling, information and instruction
sheets, consent forms, as well as a self-reporting questionnaire for each participating
household member. All samples originating from the same household were collected on the same
day. Prior to sampling, the study protocol was approved by the Ethical Committee of the
ChariteÂ, Campus Virchow-Clinic Berlin.
The questionnaire (S1 and S2 Figs) for animal participants covered demographic factors
such as breed, age, sex, and whether the animal was neutered as well as details of husbandry
(keeping inside/free roaming), and stay in different sites (e.g., sanctuary, animal shows). The
questionnaire for animal owners requested demographic data like age, gender, profession,
residence and residential environment (e.g., city, countryside). Additional questions assessed the
presence of other household members such as children and individuals with chronic disease,
confirmed CDI or persons who underwent chemotherapy. For both, animals and their owners,
additional information was collected about contact to other animals, intensity of contact
between the participating animal and owner (e.g., frequency and type of dog handling, including
physical contact), food/feed consumption, status of health (e.g. diarrhea in previous months),
prior hospitalization, intake of medication (e.g. antibiotics), and contact to other individuals
suffering from diarrhea or with a recent hospital stay.
Statistical analysis was performed using the software STATA1 (StataCorp. 2013. Release 13.
College Station, TX: StataCorp LP). Univariate analysis examined the association (odds ratio)
between potential exposure and outcome using logistic regression with dichotomous and
categorial independent variables at significance level of p 0.05. Odds ratios (OR) were calculated
with a 95% confidence interval (CI).
Sensitivity analyses were obtained to detect potential clustering. For this, (1) only
households participating with one animal and one pet owner were included, (2) all households were
considered, though, only one data set (ratio animal-pet owner 1:1) was included, (3) all
households were considered, though, only data sets with the ratio animal-pet owner n:n were
included, (4) all households were included with complete data sets (ratio animal-pet owner n:
m). No significant efficiency differences or increase in performance compared to the
univariate analysis of the whole data set were detected; thus, model (4) was selected for the statistical
For the multivariate analysis variables with p 0.2 associated with isolation of C. difficile
from the univariate analysis were considered as potential risk factors. To select the variables
which entered in the final multivariate logistic model a stepwise backward removal procedure
with a threshold p-value 0.05 was used as implemented in STATA.
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Isolation and molecular characterization of C. difficile
For C. difficile cultivation, isolation and identification 2±3 g of each fecal sample was
inoculated in 10 ml C. difficile moxalactam-norfloxacin broth (CDMN, SR173, Oxoid Ltd.,
Hampshire, United Kingdom) and then underwent direct plating and enrichment culturing as
described by Schneeberg et al. [
]. Genomic DNA extraction, toxin gene detection, seq-PCR
ribotyping, and Multilocus Variable Number of Tandem Repeat Analysis (MLVA) were
performed as previously reported by Schneeberg et al. [
We declare that we comply with the terms of service for the websites from which we
C. difficile isolation rates
In total, 415 households were included in the study; these households were geographically
distributed throughout Germany with participants from all 16 federal states. Twelve households
did not meet the inclusion criteria for the following reasons: missing questionnaires and/or
fecal sample(s) of participating household members, incomplete consent forms, or mixed up
ID numbers by different household members. Almost half of the households (46%; 190/415)
contributed one animal and one human fecal sample with corresponding questionnaires.
Other participant compositions from the same household were manifold, with two animal
participants and one owner (11%; 45), one animal and two owners (10%; 40), and two animals
and two owners (9%; 36) being the most common.
In total, 1,447 fecal samples were collected, of which 29 samples did not meet the inclusion
criteria either due to the provision of more than one sample from the same participant, or the
above mentioned reasons. A total of 1,418 fecal samples with 59.2% (840) of animal and 40.8%
(578) of human origin were included. C. difficile was isolated from 42/1418 (3.0%) fecal
samples with isolation rates of 3.0% (25/840) for animal and 2.9% (17/578) for human samples
Characterization of C. difficile isolates
Animal C. difficile isolates were assigned to eight different PCR ribotypes (RTs) (001/5/FLI01,
009, 010, 014/0, 014/0/FLI01, 027, 039, 078) (Table 2). From one canine sample two C. difficile
strains corresponding to different RTs were isolated (RTs 010 and 039). The predominant RT
was RT 014/0, which was determined for 10/26 (38.5%) isolates. RT 010 was found in 5/26
(19.2%) isolates, whereas RTs 001/5/FLI01 and 039 were each detected in 3/26 (11.5%)
samples. RTs 014/0/FLI01, 027 and 078 were each isolated from 1/26 (3.8%) isolates. RTs 027 and
h: two different RTs were isolated in sample 0748M2
a: two different RTs were isolated in sample 0934T1
Authors’ comment: three RTs were isolated in humans and animals but did not originate from the same household
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078 are often described as highly pathogenic for humans, here they originated from canine
Within two households identical RTs were isolated from two animals (in both cases cats
which harbored RT 014/0). Further characterization applying MLVA proved that the strains
isolated within the same household were clonally related, with a STDR 2 (Table 3). (The
complete results of MLVA-analysis of all isolated strains are available in S1 Table.)
Human C. difficile isolates belonged to 12 different RTs (003, 003/FLI02, 009/FLI01, 010,
010/FLI01, 014/0, 014/5, 020, 070, 078, 087, 441/FLI01). In one human sample two different
RTs (RTs 003 and 078) were detected. The most prevalent human RT was RT 014/0, which
was ascertained in 22.2% (4/18) of the human isolates. RT 078 was isolated from 3/18 (16.7%)
and RT 003 from 2/18 (11.1%) isolates. RTs 003/FLI02, 009/FLI01, 010, 010/FLI01, 014/5, 020,
070, 087 and 441/FLI01 were each determined in 1/18 (5.6%) isolates. No parallel occurrence
of C. difficile in dogs or cats and their owners sharing the same household was detected.
ID: identification number; PCR-RT: PCR-ribotype; MLVA: Multilocus Variable Number of Tandem Repeat Analysis; STRD: Summed Tandem-Repeat Differences,
STRD relating to the previous isolate; tcdA: gene encoding for toxin A; tcdB: gene encoding for toxin B; cdtA and cdtB: genes encoding for the binary toxin called CDT;
cdd3: gene encoding an ABC-type transport system, cdd3-PCR performed as a species proof.
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In total, 61.5% (16/26) of the animal isolates were positive for toxin genes. Of those, 87.5%
(14/16) were tcdA- and tcdB-positive (genes encoding for toxins A and B) with an additional
12.5% (2/16) also yielding positive PCR results for genes cdtA and cdtB encoding for the binary
toxin (Table 2). In comparison, 77.7% (14/18) of the human isolates were toxigenic. Of these,
78.6% (11/14) were tcdA- and tcdB-positive with an additional 21.4% (3/14) positive for
cdtAand cdtB-genes. In contrast, non-toxigenic RTs accounted for 38.5% (10/26) and 22.2% (4/18)
in animal and human samples, respectively.
Significant factors associated with C. difficile-positivity are listed in Tables 4 and 5. (For the
complete univariate analysis see S2 and S3 Tables.)
In the multivariate analysis we included all animal variables with a p 0.2 in the univariate
analysis (Table 6). The regular intake of proton pump inhibitors (PPI) (OR 14.82, 95% CI
1.73±126.78, p = 0.014), antibiotic treatment during the preceding 3 months (OR 4.13; 95%
CI 1.44±11.84, p = 0.008), contact to a human with diarrhea (OR 2.94; 95% CI 1.01±8.60,
CD: Clostridium difficile isolation; Ref.: reference category; OR: odds ratio; CI: confidence interval. Authors’ comment: bracketed data indicate the number of participants
not applying to the variable in row.
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CD: Clostridium difficile isolation; Ref.: reference category; OR: odds ratio; CI: confidence interval
: excluding professions in agriculture, food production and health care. Authors’ comment: bracketed data indicate the number of participants not applying to the
variable in row.
This is the first systematic large-scale survey presenting data on the occurrence, molecular
characteristics and potential risk factors of C. difficile in small companion animals and their
corresponding owners within the community. Since C. difficile isolation rates in dogs and cats
have been reported to range between 1 and 58% [
], the isolation rate observed within
this study (3.0% (25/840)) is low despite of sensitive detection methods [
comparison is difficult as there is no uniform approach of study designs and populations [
example, most of the former studies focused on animal populations in veterinary settings or
shelters. There are hardly any data on C. difficile colonization rates from asymptomatic
nonhospitalized humans [
], and pet owners. Nonetheless, the results of our study are in line
with an estimated 3% asymptomatic carriage in humans within the community [
], and even
somewhat higher than in a recent cross-sectional population-based study conducted in the
Netherlands reporting a prevalence rate of 1.23% [
Among the 17 different isolated ribotypes RT 014/0 was the most prevalent in this survey,
detected in 38.5% of animal and 19.2% of human isolates. Interestingly, in two independent
households, four cats (two in each household) carried RT 014/0. The strains differed between
the households, whereas within the households the partner cats harbored clonally related 014/
0 strains as observed through MLVA. This indicates that transmission between individuals in
the same household is possible and confirms the supposed considerable endemic potential of
RT 014/0 [
]. However, whether a common source of infection or intra- and/or interspecies
transmission cause widespread dissemination of RT 014/0 remains unclear.
C. difficile RT 078, often discussed as a highly human pathogenic, hypervirulent strain, was
also detected in human and animal samples in this study. The detection of RT 078 in one
canine and three human samples in our study indicates that not just livestock but companion
animals might also play a role in interspecies transmission of this RT.
Moreover, here we report on the first RT 027-isolation from a dog outside Canada as this
human epidemic RT has, so far, only been described by Lefebvre et al. in two healthy hospital
visitation dogs [
]. This shows that epidemic RTs primarily associated with humans can
also occur in companion animals raising the question whether animals can serve as a reservoir
for humans or contrarily, whether humans rather pose an infectious risk for animals.
Nevertheless, whole genome data are needed to investigate the phylogeny of human and companion
We did not detect C. difficile in dogs or cats and their owners sharing the same household,
simultaneously. Yet, the overall low isolation rate within the community will have impaired
the chances of detecting isolate pairs. In a smaller scale survey examining the environment of
eight households of recurrent CDI-patients [
], none of the eight tested pets were positive.
However, the overlap in human and animal RTs has been described thoroughly and the
genetic relatedness of certain strains suggests that interspecies transmission as well as zoonotic
transmission is probably possible to occur [
]. Recently, Loo et al.  detected C. difficile in
dogs and cats in households of CDI-patients (index cases). Although the authors presented
indistinguishable C. difficile-profiles by pulsed-field gel electrophoresis (PFGE) originating
from animal-human isolate pairs, the discriminatory power of PFGE does not enable a full
molecular characterization of strains. Hence, zoonotic transmission of C. difficile remains to
be verified. Although the study populations described by Shaughnessy et al. [
] and Loo et al.
] consisted of human participants with a history of CDI in contrast to the participants in
this survey, here, we confirmed that clinically relevant RTs are shared between mainly
asymptomatic human carriers and small companion animals. Moreover, the high proportion of
toxigenic strains raises concerns that interspecies transmission would have a clinical impact.
In our statistical analysis we identified common risk factors associated with human CDI for
dogs and cats as well. Antibiotic treatment has been recognized as the major risk factor for
]. The disruption of healthy microbiota by antibiotics is indisputable, thus, allowing C.
difficile to cause disease; this is also applicable in human and animal participants in this survey.
In contrast, whether the use of PPI or AID increases the risk for CDI in humans has been
discussed controversially [35±38]. In this study, intake of PPI or AID was not a significant risk
factor for humans who were C. difficile-positive. In small companion animals the use of AID
may be associated with fecal shedding of C. difficile, with a p-value slightly greater than 0.05. In
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the study population presented here, we were able to show the significant association of
antibiotics and PPI with C. difficile detection in small companion animals. To our knowledge, we are
the first to report the association between PPI treatment and C. difficile-positivity in dogs and
cats. Within our study population, PPI was more often administered to dogs (9/10), suggesting
that medicating dogs with PPI severely increases the risk of C. difficile being present in fecal
samples. Additionally, not only certain pharmaceuticals but also inappetence could be linked
with C. difficile-positivity in the univariate analysis in this study. Yet, whether inappetence
leads to disruption of the intestinal flora and can thereby enable colonization, infection or
transient passage with C. difficile, or whether inappetence is caused by C. difficile remains
speculative. Here, dogs or cats suffering from inappetence or acute disease were six-fold more
often positively tested for C. difficile in the univariate model; besides, animals suffering from
acute disease were often treated with antibiotics.
The significant decrease in the likelihood of animals being C. difficile-positive when fed
with dry food was surprising. This variable was also confirmed in the final model as an
independent factor showing that pets fed on dry feedstuffs are approximately 10-times less likely
to harbor C. difficile. A possible explanation could be that high temperatures for desiccation
during food processing could impair the survival of vegetative cells and spores of C. difficile.
Commercial dry dog and cat feedstuffs are mainly produced using extrusion processes with
temperatures up to 200ÊC [
]. Although C. difficile spores are able to survive temperatures
of 71ÊC for at least 120 min , the extreme conditions during food processing could prevent
survival. Besides the composition of diet, the feeding strategy can also influence the microbiota
as dry feedstuffs are often fed ad libitum to dogs and cats. The combination of feeding strategy
and a probably low contamination of dry feedstuffs seem to have a positive effect on the
intestinal microenvironment of companion animals.
Despite the absence of animal-human isolate pairs, the results of the epidemiological
analysis of factors associated with fecal shedding of C. difficile in our univariate analysis support the
hypothesis of a zoonotic potential for C. difficile. In particular, we found that companion
animals tend to be C. difficile-positive more often when (1) the owner suffered from a chronic
disease (p = 0.006) or, (2) has recently suffered from diarrhea (p = 0.171), (3) the animal was in
contact with a diarrheic person (p = 0.022), (4) washed in the tub/shower (p = 0.057) and/or
(5) a chronically sick person lived in the same household (p = 0.062). The latter risk factor was
already previously described as contact to immunocompromised individuals [
indicates that sharing the environment with humans influences whether companion animals
harbor C. difficile. Even in our multivariate analysis, contact to a person with diarrhea was found
to be an independent risk factor, increasing the chances for dogs and cats of becoming
colonized or infected with C. difficile three-fold. This observation supports the findings by Lefebvre
et al. [
] describing hospital visiting dogs acquiring C. difficile in health-care facilities.
Thus, speculations of the impact of animals for human CDI should probably be reassessed as
humans might rather pose a risk for animals than the other way around. Nonetheless, they
might be a source for reinfection.
One limitation of the study is that sampling was restricted to one fecal sample per
individual. Repeated sampling could have increased isolation rates and would have increased the
chance to detect intermittent shedding, and thereby, finding animal-human isolate pairs.
In conclusion, well known human ribotypes like RT 010, the hospital-associated lineage RT
014/0 and the potentially virulent RTs 027 and 078 also occur in cats and dogs suggesting at
least a common source of infection. Moreover, previously described risk factors for C. difficile
colonization or infection in humans also apply in companion animals. To define possible
sources of C. difficile acquisition and to clarify its zoonotic character further studies involving
humans and animals are required.
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S1 Fig. C.difficile-questionnaire in German.
S2 Fig. C.difficile-questionnaire in English.
S1 Table. Complete characterization by means of MLVA for all C. difficile isolates.
S2 Table. Complete univariate analysis for fecal shedding of C. difficile in dogs and cats.
S3 Table. Complete univariate analysis for fecal shedding of C. difficile in animal owners.
We thank Nadine Pfaffendorf-Regler for her technical support within the laboratory of the
Federal Research Institute for Animal Health, Germany, and Felix Grzebin from the Robert
Koch Institute, Germany, for digitalizing the questionnaires. We also thank Esther-Maria
Antão from the Freie UniversitaÈt Berlin, Germany, for her support during the revision
This survey was funded by the Federal Ministry of Education and Research [No.
"Clostridium difficile", 01KI1107 (Mrs. Denise Rabold, IMT)/01KI1108 (FLI, Jena)]. Alexander
Schneeberg was funded by the Friedrich Naumann Foundation for Freedom, Potsdam, NY.
boldt, Antina LuÈbke-Becker.
Conceptualization: Tim Eckmanns, Heinrich Neubauer, Lothar H. Wieler, Christian
SeyData curation: Denise Rabold, Nadine MoÈbius, Katja Hille.
Formal analysis: Werner Espelage, Muna Abu Sin, Tim Eckmanns.
Investigation: Alexander Schneeberg.
Methodology: Denise Rabold, Alexander Schneeberg, Christian Seyboldt.
Supervision: Tim Eckmanns, Antina LuÈbke-Becker.
Writing ± original draft: Denise Rabold.
Writing ± review & editing: Denise Rabold, Werner Espelage, Alexander Schneeberg, Katja
Hille, Lothar H. Wieler, Christian Seyboldt, Antina LuÈbke-Becker.
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