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Prevalence and distribution of Clostridium difficile PCR ribotypes in cats and dogs from animal shelters in Thuringia, Germany
Anaerobe 18 (2012) 484e488
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Clinical microbiology
Prevalence and distribution of Clostridium difficile PCR ribotypes in cats and dogs from animal shelters in Thuringia, Germany Alexander Schneeberg a, *, Maja Rupnik b, c, d, Heinrich Neubauer a, Christian Seyboldt a a
Institute of Bacterial Infections and Zoonoses at the Federal Research Institute for Animal Health (Friedrich-Loeffler-Institut), Naumburger Strasse 96a, 07743 Jena, Germany Institute of Public Health Maribor, Maribor, Slovenia c University of Maribor, Faculty of Medicine, Maribor, Slovenia d Center of Excellence for Integrated approaches in Chemistry and Biology of Proteins, Ljubljana, Slovenia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 March 2012 Received in revised form 22 June 2012 Accepted 12 August 2012 Available online 24 August 2012
Clostridium difficile is an important cause of nosocomial diarrhoea in humans. Pet animals and livestock are discussed as potential natural reservoirs and sources of infection. In this study faecal samples from dogs and cats were collected at 10 animal shelters in Thuringia, Germany. C. difficile was isolated from 9 out of 165 (5.5%) canine and 5 out of 135 (3.7%) feline samples. Five PCR ribotypes (010, 014/020, 039, 045, SLO 066) were identified. PCR ribotypes 010 and 014/020 were detected in more than one shelter and PCR ribotypes 014/020 and 045 were isolated from dogs and cats. MLVA profiles of strains of a PCR ribotype from one shelter were identical or closely related, while strains of the same PCR ribotype from different shelters showed significant differences. This study shows that dogs and cats kept in animal shelters are a reservoir of C. difficile PCR ribotypes which can infect also humans. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Clostridium difficile Dog Cat Shelter Capillary gel electrophoresis Zoonosis
1. Introduction Clostridium difficile is the most common cause of antibioticassociated diarrhoea in humans [1]. The main virulence factors are two high-molecular-weight toxins, toxin A (enterotoxin; TcdA) and toxin B (cytotoxin; TcdB) [1]. Some strains additionally produce the binary toxin CDT [1]. During the last decade, clinical presentation and epidemiology of C. difficile infection (CDI) changed towards an increased morbidity and mortality [1]. Health-careassociated C. difficile infection incidence rates from 0 to 36.3 (mean: 4.1) per 10.000 patient-days have been reported for European hospitals [3]. Although CDI is still a nosocomial infection in general it is increasingly recognized as cause of community acquired diarrhoea and there is some evidence that animals might be reservoirs of virulent C. difficile and a possible source of infection for community acquired cases [2]. Numerous wild animals (e.g. primates, ostriches and prairie dogs), companion animals (horses, rodents) and livestock (especially pigs) can be affected by CDI [4]. Human pathogenic PCR ribotypes were found in several mammals including cattle, horses and pigs [5e7]. Furthermore, several
* Corresponding author. Tel.: þ49 3641 804 2341; fax: þ49 3641 804 2228. E-mail address: [email protected] (A. Schneeberg). 1075-9964/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.anaerobe.2012.08.002
studies demonstrated a significant contamination of food (meat and salads) with pathogenic C. difficile [2]. However, the zoonotic potential of the pathogen is still discussed controversially [2,6]. The relevance of C. difficile as a cause of disease in dogs and cats is not well understood yet. Intestinal colonization with C. difficile has been described to range from 1 to 57% in dogs and from 2 to 38% in cats (Table 1). Prevalences investigated in diarrhoeic dogs vary from 2 to 25% and were 7% and 16% in cats respectively (Table 1). Some studies showed a correlation between colonization and diarrhoea in dogs and cats [8e11]. However, the attempt to provoke CDI in healthy adult dogs by administering C. difficile with and without antibiotics failed [13]. Strains isolated from canines, felines and humans are often of the same PCR ribotypes [5,7,14]. Most studies concerning the prevalence of C. difficile in dogs and cats so far focus on veterinary hospitals (Table 1). Weese et al. [14] survey the prevalence of C. difficile in dogs and cats in the household environment in Canada. They isolated C. difficile from 14/139 dogs (10%) and from 3/14 cats (21%). McKenzie et al. [12] found 61/135 racing sled dogs (45%) to be positive for C. difficile. The situation in animal shelters has been rarely considered. Struble et al. [15] could not detect C. difficile in faecal specimens of 42 dogs collected in an unspecified number of shelters in the USA. Perrin et al. [16] showed that 1/74 dogs (1%) entering a kennel in Switzerland harboured C. difficile. Al Saif et al. [17] collected stool samples from 2 veterinary
A. Schneeberg et al. / Anaerobe 18 (2012) 484e488
485
Table 1 Reported prevalences of Clostridium difficile in dogs and cats. Country Canine Netherlands USA Canada
Canada USA
Canada USA USA Canada UK
Sample origin
Total individuals
Culture positive
Diarrhoeic
Culture positive
Non-diarrhoeic
Culture positive
Used methods for typing and characterization
Reference
Diagnostic samples Racing sled dogs Household environment
116
29 (25%)
116
29 (25%)
0
0
[7], 2011
135
61 (45%)
35
n.a.
94
n.a.
PCR ribotyping; MLVA; PCR toxin genes A, B, CDT ELISA toxins A, B
139
14 (10%)
0
0
139
14 (10%)
360
70 (19%)
n.a.
143
33 (23%)
100
102
58 (57%)
n.a.
334 132
52 (16%) 17 (13%)
260 32
47 (18%) 5 (16%)
74 100
5 (7%) 12 (12%)
142 100
2 (1%) 10 (10%)
87 n.a.
2 (2%)
55 n.a.
0
Vet. hospital, ICU Vet. hospital
Hospital visiting dogs Vet. hospital Vet. hospital
n.a. 20 (20%)
43
13 (30%)
n.a.
PCR ribotyping; PCR toxin genes A, B, CDT; Toxinotyping PCR ribotyping; PCR toxin genes A, B PCR toxin genes A, B; ELISA toxin A, B; Cytotoxicity PCR ribotyping; PCR toxin genes A, B, CDT ELISA toxin A ELISA toxin A; PCR toxin genes A, B ELISA toxin A, B EIA toxin A
[12], 2010 [14], 2009
[26], 2008 [27], 2006
[28], 2006 [29],a 2002 [10], 2002
194
28 (14%)
42
7 (17%)
110
21 (19%)
PCR toxin genes A, B
[15], 1994
Switzerland
Vet. hospital Vet. hospital, shelter Vet. hospital, shelter Litters, shelter
158
73 (46%)
11
1 (9%)
147
72 (49%)
[16], 1993
Australia Germany UK
Vet. hospital Vet. practices Vet. hospital
60 150 52
24 (40%) 9 (6%) 11 (21%)
n.a. 75 n.a.
2 (3%)
n.a. 75 n.a.
7 (9%)
GLC; Enzymatic activities; Cytotoxicity; ELISA toxin A Cytotoxicity Cytotoxicity Cytotoxicity, Hamster bioassay
Diagnostic samples Household environment Vet. hospital, ICU Vet. hospital
115
18 (16)
115
18 (16%)
0
0
0
14
3 (21%)
USA
Feline Netherlands Canada Canada USA UK Australia Germany UK a
Vet. hospital, shelter Vet. hospital Vet. practices Vet. hospital
14
3 (21%)
0
42
3 (7%)
n.a.
294
23 (8%)
n.a.
100
2 (2%)
n.a.
21 175 20
8 (38%) 14 (8%) 6 (30%)
n.a. 75 n.a.
n.a. 10
80
0
n.a.
5 (7%)
n.a. 100 n.a.
9 (9%)
PCR ribotyping; MLVA; PCR toxin genes A, B, CDT PCR ribotyping; PCR toxin genes A, B, CDT; Toxinotyping PCR ribotyping; PCR toxin genes A, B PCR toxin genes A, B; AP-PCR (Genotyping) EIA toxin A Cytotoxicity Cytotoxicity Cytotoxicity, Hamster bioassay
[8], 2001 [17], 1996
[30], 1991 [31], 1989 [32], 1983
[7], 2011 [14], 2009 [26], 2008 [33], 1999 [17], 1996 [30], 1991 [31], 1989 [32], 1983
Data shown in the abstract differ from the presented results.
clinics and an animal shelter. They isolated C. difficile from 10/100 dog samples and from 2/100 cat samples. However, they do not distinguish between samples from the clinics and the shelter. All these studies considering animal shelters are from the nineties and were using EIA/ELISA of toxin A and cytotoxicity assays or toxin gene PCR for detecting C. difficile. Information on the PCR ribotype diversity in shelter animals is so far not available [Table 1].
groups; H: 8 cats in 4 groups; I: 25 cats in 10 groups; J: 48 cats in 2 groups). Canine faecal samples were taken from the floor of the kennels. Each sample could be assigned to an individual animal. Feline faecal specimens were usually taken from litter trays (up to 4/litter tray, mostly 1e2) which made an assignment of samples to individuals impossible. We assumed that all feline faecal samples originated from different cats. The samples were transported on ice and processed within 2e4 h.
2. Materials and methods 2.2. Isolation and identification 2.1. Sampling Twenty five shelters of the state Thuringia, Germany, were invited to attend this investigation. The shelters differed in size and in their proportional composition of animal species. A total of 10 shelters volunteered to participate. Reasons for non-participation were not requested. Faecal specimens of dogs and cats were collected between January and March 2010. In all shelters dogs were kept individually or in groups of 2. Cats were mostly kept in groups up to 60 animals (Shelter A: approx. 60 cats in 1 group; B: approx. 60 cats in 6 groups; C: 50 cats in 7 groups; D: no cats; E: 2 cats kept individually; F: 31 cats in 13 groups; G: 16 cats in 7
Isolation of C. difficile was performed using direct plating and enrichment culture in parallel. Depending on its consistency 1e4 inoculation loops of each sample (approximately 0.5 g) were resuspended in 10 ml C. difficile moxalactam/norfloxacin broth (CDMN, Oxoid, SR173) containing 0.1% sodium-taurocholate (SigmaeAldrich, 86339). 100 ml of this mixture was immediately plated onto CDMN agar. Plates were incubated for 1e3 days, the enrichment culture for 14e21 days at 37 C under anaerobic conditions. In order to select spores, 900 ml of each enrichment culture was mixed with the same volume of 99% ethanol and left at room
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temperature for 30 min. After centrifugation at 5000 g for 10 min, the pellet was re-suspended in 200 ml of 0.8% NaCl. 100 ml were transferred on CDMN agar and incubated anaerobic for 1e3 days at 37 C. Presumptive C. difficile colonies were identified based on their irregular margin and finely branched morphology, weak-green fluorescence at 360 nm illumination and positive L-prolinee aminopeptidase test. 2.3. Toxinotyping, PCR ribotyping and MLVA C. difficile-like colonies were subcultured at least 3 times on CDMN agar plates to obtain pure cultures. Bacterial DNA was isolated using a Qiagen DNeasy Blood & Tissue KitÔ. All strains were tested by cdd3-PCR (species proof), tcdA- and tcdB-PCR (detection of fragment A3 of the toxin A gene, respectively B1 of the toxin B gene) as well as cdtA- and cdtB-PCR (binary toxin genes) as described elsewhere [18]. Capillary gel electrophoresis based PCR ribotyping was performed according to the protocol of Indra et al. [19] using an Applied Biosystems 3130 Genetic Analyzer with GeneScanTM-600LizÒ size marker (Capillary: 36 cm, Gel: POP7). Resulting peak sizes and height were imported to Webribo database (http://webribo.ages.at) for assignment of Webribo PCR ribotypes. Conventional PCR ribotyping was performed on 24e48 h cultures (Columbia blood agar) using primers and amplification conditions previously described [20]. PCR ribotype profiles were analysed by BioNumericsÔ software and PCR ribotypes were designated either with standard Cardiff nomenclature (010, 014/ 020, 039, 045) if the profile corresponded to one of 25 reference PCR ribotype strains, including current most frequent PCR ribotypes (e.g. 001, 002, 014/020, 015, 023, 027, 078, 106, 126) or with internal nomenclature (SLO 066). Multilocus Variable Number of Tandem Repeat Analysis (MLVA) was performed as described by van den Berg et al. [21] with two modified primers of locus C6Cd [22]. All fluorescent dye labelled forward primers were purchased from Applied Biosystems (Life Technologies Cooperation). Forward primer A6Cd was labelled with 6-FAM, B7Cd with VIC, C6Cd with 6-FAM, E7Cd with PETÒ, F3Cd with NED, G8Cd with NED and H9Cd with PETÒ. PCR fragments were analysed using the same system and size marker as described above for capillary gel electrophoresis based PCR ribotyping. Repeat copy numbers were determined on the basis of sequencing results. For each loci A6Cd, B7Cd, C6Cd, E7Cd and G8Cd two PCR products of different sizes were sequenced (Eurofins MWG Operon, Germany). One PCR product was sequenced for loci F3Cd and H9Cd because of its low variation in length. A correlation between the lengths determined by capillary gel electrophoresis and the number of repeats was set by linear regression. Repeat units were imported to BioNumericsÔ software (Version 6.6) and the cluster analysis was carried out based on the categorical coefficient and unweighted pair group method using arithmetic averages (UPGMA). 3. Results and discussion This is the first study on the PCR ribotype diversity of C. difficile in dogs and cats from animal shelters. By using MLVA, this study also provides information on intra- and interspecies transmission. C. difficile was detected in 7 out of 10 shelters (Table 2). Nine of 165 (5.5%) canine faecal samples and 5 of 135 (3.7%) feline faecal samples (5/52 cat groups, 9.6%) were positive for C. difficile after enrichment culture. The overall shelter prevalence was high (70%) but the animal prevalence was low when compared to isolation rates reported in literature (Table 1). Struble et al. [15] and Perrin et al. [16] found low isolation rates (0% and 1% respectively) of C. difficile in dogs from animal shelters as well. This might indicate a generally lower prevalence of C. difficile in shelter animals. The
Table 2 Isolation and typing of Clostridium difficile from dog and cat faecal samples. Shelters
A B C D E F G H I J Total a b c d
Samples
Test-positive animals/identified PCR ribotypes
Dogs
Cats
Dogs
11 12 15 21 8 21 28 11 29 9 165
11 12 31 0 2 19 10 6 16 28 135
3 1 1 0 1 1 0 0 2 0 9
Ribotypes
Cats
(3c/2d) (1c) (1c)
SLO 066 010 045
(1c) (1c,a)
010 014/020
0 0 2 0 0 0 1 0 2 0 5
(2c/1d)
010
(9c/3d)
Ribotypes
(2c,b)
045
(1c)
014/020
(2c,b)
014/020, 039
(5c)
culture with large amount of colony forming units. recovered from animals of different groups. enrichment culture. direct plating.
shelter prevalence of 60% (6 out of 10) regarding dogs only is much higher than the shelter prevalence of 30% (3 out of 9) regarding cats only but this difference is not statistically significant by Fisher’s exact test. However, the comparison of data reported so far indicate that dogs might be more often colonized with C. difficile than cats (Table 1). For the isolation of C. difficile direct plating and enrichment culture was used. Enrichment culture is a sensitive method for the detection of viable cells or spores of C. difficile in a sample. Direct plating provides additional information on the number of the pathogen. Direct plating resulted in 4 positive individuals (dogs only) with respective isolates. Animals which were positive by direct plating were all positive in enrichment culture, too. Three dogs and 2 cats (in quarantine) were suffering from diarrhoea at time of sampling. One cat with diarrhoea harbouring PCR ribotype 014/020 was negative by direct plating. It has to be questioned that C. difficile was the cause of disease. A high number of C. difficile colony forming units was solely found in one canine faecal sample (PCR ribotype 014/020). The respective animal showed neither symptoms of diarrhoea nor had it been treated with antibiotics. It could not be determined whether this high CFU (colony forming units) reflected the extensive colonization of this dog. However, no correlation between the number of CFU and diarrhoea could be demonstrated. From all 14 positive samples altogether 18 isolates were obtained and characterized by PCR ribotyping and MLVA (Fig. 1). The isolates could be assigned to 5 different PCR ribotypes 010 (SLO 010), 014/020, 039, 045 and SLO 066 (Fig. 1) using conventional PCR ribotyping. The capillary gel electrophoresis based PCR ribotyping in combination with the Webribo database analyses approved the strains of PCR ribotype 010 belonging to 010 and strains of PCR ribotype 014/020 belonging to 014/subgroup 0. Two strains (sample 249 and 331) had differences in one peak to corresponding reference profiles [Supplementary data]. These differences could hardly be displayed or are even not present in conventional PCR ribotyping confirming the higher discriminatory power of the capillary gel electrophoresis based method [19]. The same applies on PCR ribotype 045. Its profile has similarities to Webribo profile 045 but show differences in minor peaks [Supplementary data]. Strains of SLO 066 show high similarities to Webribo profile AI-60 whereas the isolate of sample 449 (PCR ribotype 039) is most similar to AI34 (related to Webribo type strain for 039). PCR ribotypes 014/020 and 010 were present in 3 shelters. In 6 of 7 shelters a single PCR ribotype was found, whereas one shelter harboured 3 PCR ribotypes (Table 2). PCR ribotype 010 was detected only in dog samples whereas PCR ribotypes 014/020 and 045 were isolated from both species. Interestingly, strains of the PCR ribotype
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Fig. 1. MLVA and toxin gene PCR results of isolates in this study. Horizontal lines separate PCR ribotypes. Samples were obtained by enrichment culture (e) or direct plating (p).
014/020 are the most often cause of C. difficile associated human diarrhoea in the European Union [3]. This PCR ribotype was also frequently recovered from animal samples (dogs, cat, horses and poultry) in the Netherlands [7]. Koene et al. [7] found the majority of isolates originating from pet animals belong to the non-toxinogenic PCR ribotypes 010 and 039. One strain of PCR ribotype 039 was also isolated in our study. Both PCR ribotypes were previously recovered from dogs, cats, horses and poultry (PCR ribotype 010 only) but rarely from humans [7,23,24]. The toxinogenic PCR ribotype 045 found in this study was previously isolated from humans and a horse [7]. Information for the host range of PCR ribotype SLO 066 is so far not available. Our results are in good agreement with those studies highlighting that PCR ribotypes isolated from dogs and cats are often corresponding to those found in humans [5,7,14]. The majority of strains of this study were positive in toxin A and toxin B PCR (ribotypes 014/020, 045 and SLO 066). Both exotoxins are main virulence factors of C. difficile. At least one of it is necessary to provoke diarrhoea [25]. Strains of PCR ribotype 045 were also positive for the binary toxin CDT gene. CDT has cytotoxic and enterotoxic effects. However C. difficile strains producing only CDT colonize but do not kill hamsters (CDI model) [1]. The intended purpose of MLVA analysis is outbreak investigation. Identical MLVA patterns were found for PCR ribotype 045 isolates (dog and cats) from shelter C and PCR ribotype 010 strains in dogs of shelter I. The strains of PCR ribotype SLO 066 from shelter A however differed in locus B7Cd (Fig. 1, Samples 10, 9) and Sample 1 additional in locus A6Cd. Furthermore, our study includes 4 pairs of isolates from dogs obtained by direct plating and enrichment culture (Fig. 1, Samples 273, 10, 9, 432). Two pairs had an identical MLVA pattern (273, 432). MLVA subtypes of SLO 066 strains from one shelter differed in two loci (A6Cd and B7Cd). Thus, the MLVA profiles of the PCR ribotypes which were isolated from one shelter or from one animal were identical or had a mutation in one locus only. van den Berg et al. [21] concluded that a difference of only one repeat unit between strains should not be interpreted as indicative of separate types or subtypes. Consequently, MLVA analysis of the strains isolated in our study indicated that C. difficile had circulated between animals of one shelter (Fig. 1, PCR ribotype 045 in shelter
C, PCR ribotypes SLO 066 in shelter A and PCR ribotypes 010 in shelter I). In contrast, strains with identical PCR ribotype originating from different shelters showed differences in 3e5 loci in their MLVA profile, e.g. PCR ribotypes 010 and 014/020 (Fig. 1). 3.1. Conclusions This study showed that C. difficile could be recovered from canine and feline faeces collected in Thuringian animal shelters. The same PCR ribotypes can also be isolated from humans. PCR ribotype 014/020 was isolated in 3 shelters from one dog and cats. This PCR ribotype is often provoking diarrhoea in humans. In 3 shelters strains of one particular PCR ribotype belonged to the same MLVA subtype showing intra- and interspecies transmission of C. difficile. The presence of C. difficile, however, was not associated with disease. In summary, the results presented here provide evidence that dogs and cats in animal shelters are a reservoir of human pathogenic C. difficile. A threat to human health by companion animals can therefore not be excluded. Authors’ contributions AS participated in the design of the study, carried out the sampling, isolation and molecular typing (except gel based PCR ribotyping) and drafted the manuscript. MR carried out the gel based PCR ribotyping, proved the typing results by own molecular analyses and helped to draft the manuscript. HN helped to draft the manuscript. CS conceived the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Acknowledgements We are grateful to all attending animal shelters and their staffs for the provision of faecal material and for the assistance in sampling. We thank Gernot Schmoock for his assistance in capillary gel electrophoresis and BioNumericsÔ software. A. Kocuvan is acknowledged for assistance in molecular characterization.
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Alexander Schneeberg is funded by the Friedrich Naumann Foundation for Freedom, Potsdam. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.anaerobe.2012.08.002. References [1] Rupnik M, Wilcox MH, Gerding DN. Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat Rev Microbiol 2009;7: 526e36. [2] Gould LH, Limbago B. Clostridium difficile in food and domestic animals: a new foodborne pathogen? Clin Infect Dis 2010;51:577e82. [3] Bauer MP, Notermans DW, Van Benthem BH, Brazier JS, Wilcox MH, Rupnik M, et al. Clostridium difficile infection in Europe: a hospital-based survey. Lancet 2011;377:63e73. [4] Keel MK, Songer JG. The comparative pathology of Clostridium difficile-associated disease. Vet Pathol 2006;43:225e40. [5] Arroyo LG, Kruth SA, Willey BM, Staempfli HR, Low DE, Weese JS. PCR ribotyping of Clostridium difficile isolates originating from human and animal sources. J Med Microbiol 2005;54:163e6. [6] Rupnik M. Is Clostridium difficile-associated infection a potentially zoonotic and foodborne disease? Clin Microbiol Infect 2007;13:457e9. [7] Koene MG, Mevius D, Wagenaar JA, Harmanus C, Hensgens MP, Meetsma AM, et al. Clostridium difficile in Dutch animals: their presence, characteristics and similarities with human isolates. Clin Microbiol Infect 2011;18:778e84. [8] Weese JS, Staempfli HR, Prescott JF, Kruth SA, Greenwood SJ, Weese HE. The roles of Clostridium difficile and enterotoxigenic Clostridium perfringens in diarrhea in dogs. J Vet Intern Med 2001;15:374e8. [9] Weese JS, Armstrong J. Outbreak of Clostridium difficile-associated disease in a small animal veterinary teaching hospital. J Vet Intern Med 2003;17:813e6. [10] Marks SL, Kather EJ, Kass PH, Melli AC. Genotypic and phenotypic characterization of Clostridium perfringens and Clostridium difficile in diarrheic and healthy dogs. J Vet Intern Med 2002;16:533e40. [11] Weese JS, Weese HE, Bourdeau TL, Staempfli HR. Suspected Clostridium difficile-associated diarrhea in two cats. J Am Vet Med Assoc 2001;218. 1436e9, 1421. [12] McKenzie E, Riehl J, Banse H, Kass PH, Nelson Jr S, Marks SL. Prevalence of diarrhea and enteropathogens in racing sled dogs. J Vet Intern Med 2010;24: 97e103. [13] Clooten JK, Kruth SA, Weese JS. Genotypic and phenotypic characterization of Clostridium perfringens and Clostridium difficile in diarrheic and healthy dogs. J Vet Intern Med 2003;17:123 [author reply]. [14] Weese JS, Finley R, Reid-Smith RR, Janecko N, Rousseau J. Evaluation of Clostridium difficile in dogs and the household environment. Epidemiol Infect 2009:1e5. [15] Struble AL, Tang YJ, Kass PH, Gumerlock PH, Madewell BR, Silva Jr J. Fecal shedding of Clostridium difficile in dogs: a period prevalence survey in a veterinary medical teaching hospital. J Vet Diagn Invest 1994;6:342e7.
[16] Perrin J, Buogo C, Gallusser A, Burnens AP, Nicolet J. Intestinal carriage of Clostridium difficile in neonate dogs. Zentralbl Veterinarmed B 1993;40:222e 6. [17] Al Saif N, Brazier JS. The distribution of Clostridium difficile in the environment of South Wales. J Med Microbiol 1996;45:133e7. [18] Zidaric V, Zemljic M, Janezic S, Kocuvan A, Rupnik M. High diversity of Clostridium difficile genotypes isolated from a single poultry farm producing replacement laying hens. Anaerobe 2008;14:325e7. [19] Indra A, Huhulescu S, Schneeweis M, Hasenberger P, Kernbichler S, Fiedler A, et al. Characterization of Clostridium difficile isolates using capillary gel electrophoresis-based PCR ribotyping. J Med Microbiol 2008; 57:1377e82. [20] Bidet P, Barbut F, Lalande V, Burghoffer B, Petit JC. Development of a new PCRribotyping method for Clostridium difficile based on ribosomal RNA gene sequencing. FEMS Microbiol Lett 1999;175:261e6. [21] van den Berg RJ, Schaap I, Templeton KE, Klaassen CH, Kuijper EJ. Typing and subtyping of Clostridium difficile isolates by using multiple-locus variablenumber tandem-repeat analysis. J Clin Microbiol 2007;45:1024e8. [22] Zaiss NH, Rupnik M, Kuijper EJ, Harmanus C, Michielsen D, Janssens K, et al. Typing Clostridium difficile strains based on tandem repeat sequences. BMC Microbiol 2009;9:6. [23] Terhes G, Brazier JS, Urban E, Soki J, Nagy E. Distribution of Clostridium difficile PCR ribotypes in regions of Hungary. J Med Microbiol 2006;55:279e82. [24] Pituch H, Brazier JS, Obuch-Woszczatynski P, Wultanska D, MeiselMikolajczyk F, Luczak M. Prevalence and association of PCR ribotypes of Clostridium difficile isolated from symptomatic patients from Warsaw with macrolide-lincosamide-streptogramin B (MLSB) type resistance. J Med Microbiol 2006;55:207e13. [25] Kuehne SA, Cartman ST, Minton NP. Both, toxin A and toxin B, are important in Clostridium difficile infection. Gut Microbes 2011;2:252e5. [26] Clooten J, Kruth S, Arroyo L, Weese JS. Prevalence and risk factors for Clostridium difficile colonization in dogs and cats hospitalized in an intensive care unit. Vet Microbiol 2008;129:209e14. [27] Chouicha N, Marks SL. Evaluation of five enzyme immunoassays compared with the cytotoxicity assay for diagnosis of Clostridium difficile-associated diarrhea in dogs. J Vet Diagn Invest 2006;18:182e8. [28] Lefebvre SL, Waltner-Toews D, Peregrine AS, Reid-Smith R, Hodge L, Arroyo LG, et al. Prevalence of zoonotic agents in dogs visiting hospitalized people in Ontario: implications for infection control. J Hosp Infect 2006;62: 458e66. [29] Cave NJ, Marks SL, Kass PH, Melli AC, Brophy MA. Evaluation of a routine diagnostic fecal panel for dogs with diarrhea. J Am Vet Med Assoc 2002;221: 52e9. [30] Riley TV, Adams JE, O’Neill GL, Bowman RA. Gastrointestinal carriage of Clostridium difficile in cats and dogs attending veterinary clinics. Epidemiol Infect 1991;107:659e65. [31] Weber A, Kroth P, Heil G. The occurrence of Clostridium difficile in fecal samples of dogs and cats. Zentralbl Veterinarmed B 1989;36:568e76. [32] Borriello SP, Honour P, Turner T, Barclay F. Household pets as a potential reservoir for Clostridium difficile infection. J Clin Pathol 1983;36:84e7. [33] Madewell BR, Bea JK, Kraegel SA, Winthrop M, Tang YJ, Silva Jr J. Clostridium difficile: a survey of fecal carriage in cats in a veterinary medical teaching hospital. J Vet Diagn Invest 1999;11:50e4.
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Anaerobe journal homepage: www.elsevier.com/locate/anaerobe
Clinical microbiology
Prevalence and distribution of Clostridium difficile PCR ribotypes in cats and dogs from animal shelters in Thuringia, Germany Alexander Schneeberg a, *, Maja Rupnik b, c, d, Heinrich Neubauer a, Christian Seyboldt a a
Institute of Bacterial Infections and Zoonoses at the Federal Research Institute for Animal Health (Friedrich-Loeffler-Institut), Naumburger Strasse 96a, 07743 Jena, Germany Institute of Public Health Maribor, Maribor, Slovenia c University of Maribor, Faculty of Medicine, Maribor, Slovenia d Center of Excellence for Integrated approaches in Chemistry and Biology of Proteins, Ljubljana, Slovenia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 March 2012 Received in revised form 22 June 2012 Accepted 12 August 2012 Available online 24 August 2012
Clostridium difficile is an important cause of nosocomial diarrhoea in humans. Pet animals and livestock are discussed as potential natural reservoirs and sources of infection. In this study faecal samples from dogs and cats were collected at 10 animal shelters in Thuringia, Germany. C. difficile was isolated from 9 out of 165 (5.5%) canine and 5 out of 135 (3.7%) feline samples. Five PCR ribotypes (010, 014/020, 039, 045, SLO 066) were identified. PCR ribotypes 010 and 014/020 were detected in more than one shelter and PCR ribotypes 014/020 and 045 were isolated from dogs and cats. MLVA profiles of strains of a PCR ribotype from one shelter were identical or closely related, while strains of the same PCR ribotype from different shelters showed significant differences. This study shows that dogs and cats kept in animal shelters are a reservoir of C. difficile PCR ribotypes which can infect also humans. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Clostridium difficile Dog Cat Shelter Capillary gel electrophoresis Zoonosis
1. Introduction Clostridium difficile is the most common cause of antibioticassociated diarrhoea in humans [1]. The main virulence factors are two high-molecular-weight toxins, toxin A (enterotoxin; TcdA) and toxin B (cytotoxin; TcdB) [1]. Some strains additionally produce the binary toxin CDT [1]. During the last decade, clinical presentation and epidemiology of C. difficile infection (CDI) changed towards an increased morbidity and mortality [1]. Health-careassociated C. difficile infection incidence rates from 0 to 36.3 (mean: 4.1) per 10.000 patient-days have been reported for European hospitals [3]. Although CDI is still a nosocomial infection in general it is increasingly recognized as cause of community acquired diarrhoea and there is some evidence that animals might be reservoirs of virulent C. difficile and a possible source of infection for community acquired cases [2]. Numerous wild animals (e.g. primates, ostriches and prairie dogs), companion animals (horses, rodents) and livestock (especially pigs) can be affected by CDI [4]. Human pathogenic PCR ribotypes were found in several mammals including cattle, horses and pigs [5e7]. Furthermore, several
* Corresponding author. Tel.: þ49 3641 804 2341; fax: þ49 3641 804 2228. E-mail address: [email protected] (A. Schneeberg). 1075-9964/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.anaerobe.2012.08.002
studies demonstrated a significant contamination of food (meat and salads) with pathogenic C. difficile [2]. However, the zoonotic potential of the pathogen is still discussed controversially [2,6]. The relevance of C. difficile as a cause of disease in dogs and cats is not well understood yet. Intestinal colonization with C. difficile has been described to range from 1 to 57% in dogs and from 2 to 38% in cats (Table 1). Prevalences investigated in diarrhoeic dogs vary from 2 to 25% and were 7% and 16% in cats respectively (Table 1). Some studies showed a correlation between colonization and diarrhoea in dogs and cats [8e11]. However, the attempt to provoke CDI in healthy adult dogs by administering C. difficile with and without antibiotics failed [13]. Strains isolated from canines, felines and humans are often of the same PCR ribotypes [5,7,14]. Most studies concerning the prevalence of C. difficile in dogs and cats so far focus on veterinary hospitals (Table 1). Weese et al. [14] survey the prevalence of C. difficile in dogs and cats in the household environment in Canada. They isolated C. difficile from 14/139 dogs (10%) and from 3/14 cats (21%). McKenzie et al. [12] found 61/135 racing sled dogs (45%) to be positive for C. difficile. The situation in animal shelters has been rarely considered. Struble et al. [15] could not detect C. difficile in faecal specimens of 42 dogs collected in an unspecified number of shelters in the USA. Perrin et al. [16] showed that 1/74 dogs (1%) entering a kennel in Switzerland harboured C. difficile. Al Saif et al. [17] collected stool samples from 2 veterinary
A. Schneeberg et al. / Anaerobe 18 (2012) 484e488
485
Table 1 Reported prevalences of Clostridium difficile in dogs and cats. Country Canine Netherlands USA Canada
Canada USA
Canada USA USA Canada UK
Sample origin
Total individuals
Culture positive
Diarrhoeic
Culture positive
Non-diarrhoeic
Culture positive
Used methods for typing and characterization
Reference
Diagnostic samples Racing sled dogs Household environment
116
29 (25%)
116
29 (25%)
0
0
[7], 2011
135
61 (45%)
35
n.a.
94
n.a.
PCR ribotyping; MLVA; PCR toxin genes A, B, CDT ELISA toxins A, B
139
14 (10%)
0
0
139
14 (10%)
360
70 (19%)
n.a.
143
33 (23%)
100
102
58 (57%)
n.a.
334 132
52 (16%) 17 (13%)
260 32
47 (18%) 5 (16%)
74 100
5 (7%) 12 (12%)
142 100
2 (1%) 10 (10%)
87 n.a.
2 (2%)
55 n.a.
0
Vet. hospital, ICU Vet. hospital
Hospital visiting dogs Vet. hospital Vet. hospital
n.a. 20 (20%)
43
13 (30%)
n.a.
PCR ribotyping; PCR toxin genes A, B, CDT; Toxinotyping PCR ribotyping; PCR toxin genes A, B PCR toxin genes A, B; ELISA toxin A, B; Cytotoxicity PCR ribotyping; PCR toxin genes A, B, CDT ELISA toxin A ELISA toxin A; PCR toxin genes A, B ELISA toxin A, B EIA toxin A
[12], 2010 [14], 2009
[26], 2008 [27], 2006
[28], 2006 [29],a 2002 [10], 2002
194
28 (14%)
42
7 (17%)
110
21 (19%)
PCR toxin genes A, B
[15], 1994
Switzerland
Vet. hospital Vet. hospital, shelter Vet. hospital, shelter Litters, shelter
158
73 (46%)
11
1 (9%)
147
72 (49%)
[16], 1993
Australia Germany UK
Vet. hospital Vet. practices Vet. hospital
60 150 52
24 (40%) 9 (6%) 11 (21%)
n.a. 75 n.a.
2 (3%)
n.a. 75 n.a.
7 (9%)
GLC; Enzymatic activities; Cytotoxicity; ELISA toxin A Cytotoxicity Cytotoxicity Cytotoxicity, Hamster bioassay
Diagnostic samples Household environment Vet. hospital, ICU Vet. hospital
115
18 (16)
115
18 (16%)
0
0
0
14
3 (21%)
USA
Feline Netherlands Canada Canada USA UK Australia Germany UK a
Vet. hospital, shelter Vet. hospital Vet. practices Vet. hospital
14
3 (21%)
0
42
3 (7%)
n.a.
294
23 (8%)
n.a.
100
2 (2%)
n.a.
21 175 20
8 (38%) 14 (8%) 6 (30%)
n.a. 75 n.a.
n.a. 10
80
0
n.a.
5 (7%)
n.a. 100 n.a.
9 (9%)
PCR ribotyping; MLVA; PCR toxin genes A, B, CDT PCR ribotyping; PCR toxin genes A, B, CDT; Toxinotyping PCR ribotyping; PCR toxin genes A, B PCR toxin genes A, B; AP-PCR (Genotyping) EIA toxin A Cytotoxicity Cytotoxicity Cytotoxicity, Hamster bioassay
[8], 2001 [17], 1996
[30], 1991 [31], 1989 [32], 1983
[7], 2011 [14], 2009 [26], 2008 [33], 1999 [17], 1996 [30], 1991 [31], 1989 [32], 1983
Data shown in the abstract differ from the presented results.
clinics and an animal shelter. They isolated C. difficile from 10/100 dog samples and from 2/100 cat samples. However, they do not distinguish between samples from the clinics and the shelter. All these studies considering animal shelters are from the nineties and were using EIA/ELISA of toxin A and cytotoxicity assays or toxin gene PCR for detecting C. difficile. Information on the PCR ribotype diversity in shelter animals is so far not available [Table 1].
groups; H: 8 cats in 4 groups; I: 25 cats in 10 groups; J: 48 cats in 2 groups). Canine faecal samples were taken from the floor of the kennels. Each sample could be assigned to an individual animal. Feline faecal specimens were usually taken from litter trays (up to 4/litter tray, mostly 1e2) which made an assignment of samples to individuals impossible. We assumed that all feline faecal samples originated from different cats. The samples were transported on ice and processed within 2e4 h.
2. Materials and methods 2.2. Isolation and identification 2.1. Sampling Twenty five shelters of the state Thuringia, Germany, were invited to attend this investigation. The shelters differed in size and in their proportional composition of animal species. A total of 10 shelters volunteered to participate. Reasons for non-participation were not requested. Faecal specimens of dogs and cats were collected between January and March 2010. In all shelters dogs were kept individually or in groups of 2. Cats were mostly kept in groups up to 60 animals (Shelter A: approx. 60 cats in 1 group; B: approx. 60 cats in 6 groups; C: 50 cats in 7 groups; D: no cats; E: 2 cats kept individually; F: 31 cats in 13 groups; G: 16 cats in 7
Isolation of C. difficile was performed using direct plating and enrichment culture in parallel. Depending on its consistency 1e4 inoculation loops of each sample (approximately 0.5 g) were resuspended in 10 ml C. difficile moxalactam/norfloxacin broth (CDMN, Oxoid, SR173) containing 0.1% sodium-taurocholate (SigmaeAldrich, 86339). 100 ml of this mixture was immediately plated onto CDMN agar. Plates were incubated for 1e3 days, the enrichment culture for 14e21 days at 37 C under anaerobic conditions. In order to select spores, 900 ml of each enrichment culture was mixed with the same volume of 99% ethanol and left at room
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temperature for 30 min. After centrifugation at 5000 g for 10 min, the pellet was re-suspended in 200 ml of 0.8% NaCl. 100 ml were transferred on CDMN agar and incubated anaerobic for 1e3 days at 37 C. Presumptive C. difficile colonies were identified based on their irregular margin and finely branched morphology, weak-green fluorescence at 360 nm illumination and positive L-prolinee aminopeptidase test. 2.3. Toxinotyping, PCR ribotyping and MLVA C. difficile-like colonies were subcultured at least 3 times on CDMN agar plates to obtain pure cultures. Bacterial DNA was isolated using a Qiagen DNeasy Blood & Tissue KitÔ. All strains were tested by cdd3-PCR (species proof), tcdA- and tcdB-PCR (detection of fragment A3 of the toxin A gene, respectively B1 of the toxin B gene) as well as cdtA- and cdtB-PCR (binary toxin genes) as described elsewhere [18]. Capillary gel electrophoresis based PCR ribotyping was performed according to the protocol of Indra et al. [19] using an Applied Biosystems 3130 Genetic Analyzer with GeneScanTM-600LizÒ size marker (Capillary: 36 cm, Gel: POP7). Resulting peak sizes and height were imported to Webribo database (http://webribo.ages.at) for assignment of Webribo PCR ribotypes. Conventional PCR ribotyping was performed on 24e48 h cultures (Columbia blood agar) using primers and amplification conditions previously described [20]. PCR ribotype profiles were analysed by BioNumericsÔ software and PCR ribotypes were designated either with standard Cardiff nomenclature (010, 014/ 020, 039, 045) if the profile corresponded to one of 25 reference PCR ribotype strains, including current most frequent PCR ribotypes (e.g. 001, 002, 014/020, 015, 023, 027, 078, 106, 126) or with internal nomenclature (SLO 066). Multilocus Variable Number of Tandem Repeat Analysis (MLVA) was performed as described by van den Berg et al. [21] with two modified primers of locus C6Cd [22]. All fluorescent dye labelled forward primers were purchased from Applied Biosystems (Life Technologies Cooperation). Forward primer A6Cd was labelled with 6-FAM, B7Cd with VIC, C6Cd with 6-FAM, E7Cd with PETÒ, F3Cd with NED, G8Cd with NED and H9Cd with PETÒ. PCR fragments were analysed using the same system and size marker as described above for capillary gel electrophoresis based PCR ribotyping. Repeat copy numbers were determined on the basis of sequencing results. For each loci A6Cd, B7Cd, C6Cd, E7Cd and G8Cd two PCR products of different sizes were sequenced (Eurofins MWG Operon, Germany). One PCR product was sequenced for loci F3Cd and H9Cd because of its low variation in length. A correlation between the lengths determined by capillary gel electrophoresis and the number of repeats was set by linear regression. Repeat units were imported to BioNumericsÔ software (Version 6.6) and the cluster analysis was carried out based on the categorical coefficient and unweighted pair group method using arithmetic averages (UPGMA). 3. Results and discussion This is the first study on the PCR ribotype diversity of C. difficile in dogs and cats from animal shelters. By using MLVA, this study also provides information on intra- and interspecies transmission. C. difficile was detected in 7 out of 10 shelters (Table 2). Nine of 165 (5.5%) canine faecal samples and 5 of 135 (3.7%) feline faecal samples (5/52 cat groups, 9.6%) were positive for C. difficile after enrichment culture. The overall shelter prevalence was high (70%) but the animal prevalence was low when compared to isolation rates reported in literature (Table 1). Struble et al. [15] and Perrin et al. [16] found low isolation rates (0% and 1% respectively) of C. difficile in dogs from animal shelters as well. This might indicate a generally lower prevalence of C. difficile in shelter animals. The
Table 2 Isolation and typing of Clostridium difficile from dog and cat faecal samples. Shelters
A B C D E F G H I J Total a b c d
Samples
Test-positive animals/identified PCR ribotypes
Dogs
Cats
Dogs
11 12 15 21 8 21 28 11 29 9 165
11 12 31 0 2 19 10 6 16 28 135
3 1 1 0 1 1 0 0 2 0 9
Ribotypes
Cats
(3c/2d) (1c) (1c)
SLO 066 010 045
(1c) (1c,a)
010 014/020
0 0 2 0 0 0 1 0 2 0 5
(2c/1d)
010
(9c/3d)
Ribotypes
(2c,b)
045
(1c)
014/020
(2c,b)
014/020, 039
(5c)
culture with large amount of colony forming units. recovered from animals of different groups. enrichment culture. direct plating.
shelter prevalence of 60% (6 out of 10) regarding dogs only is much higher than the shelter prevalence of 30% (3 out of 9) regarding cats only but this difference is not statistically significant by Fisher’s exact test. However, the comparison of data reported so far indicate that dogs might be more often colonized with C. difficile than cats (Table 1). For the isolation of C. difficile direct plating and enrichment culture was used. Enrichment culture is a sensitive method for the detection of viable cells or spores of C. difficile in a sample. Direct plating provides additional information on the number of the pathogen. Direct plating resulted in 4 positive individuals (dogs only) with respective isolates. Animals which were positive by direct plating were all positive in enrichment culture, too. Three dogs and 2 cats (in quarantine) were suffering from diarrhoea at time of sampling. One cat with diarrhoea harbouring PCR ribotype 014/020 was negative by direct plating. It has to be questioned that C. difficile was the cause of disease. A high number of C. difficile colony forming units was solely found in one canine faecal sample (PCR ribotype 014/020). The respective animal showed neither symptoms of diarrhoea nor had it been treated with antibiotics. It could not be determined whether this high CFU (colony forming units) reflected the extensive colonization of this dog. However, no correlation between the number of CFU and diarrhoea could be demonstrated. From all 14 positive samples altogether 18 isolates were obtained and characterized by PCR ribotyping and MLVA (Fig. 1). The isolates could be assigned to 5 different PCR ribotypes 010 (SLO 010), 014/020, 039, 045 and SLO 066 (Fig. 1) using conventional PCR ribotyping. The capillary gel electrophoresis based PCR ribotyping in combination with the Webribo database analyses approved the strains of PCR ribotype 010 belonging to 010 and strains of PCR ribotype 014/020 belonging to 014/subgroup 0. Two strains (sample 249 and 331) had differences in one peak to corresponding reference profiles [Supplementary data]. These differences could hardly be displayed or are even not present in conventional PCR ribotyping confirming the higher discriminatory power of the capillary gel electrophoresis based method [19]. The same applies on PCR ribotype 045. Its profile has similarities to Webribo profile 045 but show differences in minor peaks [Supplementary data]. Strains of SLO 066 show high similarities to Webribo profile AI-60 whereas the isolate of sample 449 (PCR ribotype 039) is most similar to AI34 (related to Webribo type strain for 039). PCR ribotypes 014/020 and 010 were present in 3 shelters. In 6 of 7 shelters a single PCR ribotype was found, whereas one shelter harboured 3 PCR ribotypes (Table 2). PCR ribotype 010 was detected only in dog samples whereas PCR ribotypes 014/020 and 045 were isolated from both species. Interestingly, strains of the PCR ribotype
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487
Fig. 1. MLVA and toxin gene PCR results of isolates in this study. Horizontal lines separate PCR ribotypes. Samples were obtained by enrichment culture (e) or direct plating (p).
014/020 are the most often cause of C. difficile associated human diarrhoea in the European Union [3]. This PCR ribotype was also frequently recovered from animal samples (dogs, cat, horses and poultry) in the Netherlands [7]. Koene et al. [7] found the majority of isolates originating from pet animals belong to the non-toxinogenic PCR ribotypes 010 and 039. One strain of PCR ribotype 039 was also isolated in our study. Both PCR ribotypes were previously recovered from dogs, cats, horses and poultry (PCR ribotype 010 only) but rarely from humans [7,23,24]. The toxinogenic PCR ribotype 045 found in this study was previously isolated from humans and a horse [7]. Information for the host range of PCR ribotype SLO 066 is so far not available. Our results are in good agreement with those studies highlighting that PCR ribotypes isolated from dogs and cats are often corresponding to those found in humans [5,7,14]. The majority of strains of this study were positive in toxin A and toxin B PCR (ribotypes 014/020, 045 and SLO 066). Both exotoxins are main virulence factors of C. difficile. At least one of it is necessary to provoke diarrhoea [25]. Strains of PCR ribotype 045 were also positive for the binary toxin CDT gene. CDT has cytotoxic and enterotoxic effects. However C. difficile strains producing only CDT colonize but do not kill hamsters (CDI model) [1]. The intended purpose of MLVA analysis is outbreak investigation. Identical MLVA patterns were found for PCR ribotype 045 isolates (dog and cats) from shelter C and PCR ribotype 010 strains in dogs of shelter I. The strains of PCR ribotype SLO 066 from shelter A however differed in locus B7Cd (Fig. 1, Samples 10, 9) and Sample 1 additional in locus A6Cd. Furthermore, our study includes 4 pairs of isolates from dogs obtained by direct plating and enrichment culture (Fig. 1, Samples 273, 10, 9, 432). Two pairs had an identical MLVA pattern (273, 432). MLVA subtypes of SLO 066 strains from one shelter differed in two loci (A6Cd and B7Cd). Thus, the MLVA profiles of the PCR ribotypes which were isolated from one shelter or from one animal were identical or had a mutation in one locus only. van den Berg et al. [21] concluded that a difference of only one repeat unit between strains should not be interpreted as indicative of separate types or subtypes. Consequently, MLVA analysis of the strains isolated in our study indicated that C. difficile had circulated between animals of one shelter (Fig. 1, PCR ribotype 045 in shelter
C, PCR ribotypes SLO 066 in shelter A and PCR ribotypes 010 in shelter I). In contrast, strains with identical PCR ribotype originating from different shelters showed differences in 3e5 loci in their MLVA profile, e.g. PCR ribotypes 010 and 014/020 (Fig. 1). 3.1. Conclusions This study showed that C. difficile could be recovered from canine and feline faeces collected in Thuringian animal shelters. The same PCR ribotypes can also be isolated from humans. PCR ribotype 014/020 was isolated in 3 shelters from one dog and cats. This PCR ribotype is often provoking diarrhoea in humans. In 3 shelters strains of one particular PCR ribotype belonged to the same MLVA subtype showing intra- and interspecies transmission of C. difficile. The presence of C. difficile, however, was not associated with disease. In summary, the results presented here provide evidence that dogs and cats in animal shelters are a reservoir of human pathogenic C. difficile. A threat to human health by companion animals can therefore not be excluded. Authors’ contributions AS participated in the design of the study, carried out the sampling, isolation and molecular typing (except gel based PCR ribotyping) and drafted the manuscript. MR carried out the gel based PCR ribotyping, proved the typing results by own molecular analyses and helped to draft the manuscript. HN helped to draft the manuscript. CS conceived the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Acknowledgements We are grateful to all attending animal shelters and their staffs for the provision of faecal material and for the assistance in sampling. We thank Gernot Schmoock for his assistance in capillary gel electrophoresis and BioNumericsÔ software. A. Kocuvan is acknowledged for assistance in molecular characterization.
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Alexander Schneeberg is funded by the Friedrich Naumann Foundation for Freedom, Potsdam. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.anaerobe.2012.08.002. References [1] Rupnik M, Wilcox MH, Gerding DN. Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat Rev Microbiol 2009;7: 526e36. [2] Gould LH, Limbago B. Clostridium difficile in food and domestic animals: a new foodborne pathogen? Clin Infect Dis 2010;51:577e82. [3] Bauer MP, Notermans DW, Van Benthem BH, Brazier JS, Wilcox MH, Rupnik M, et al. Clostridium difficile infection in Europe: a hospital-based survey. Lancet 2011;377:63e73. [4] Keel MK, Songer JG. The comparative pathology of Clostridium difficile-associated disease. Vet Pathol 2006;43:225e40. [5] Arroyo LG, Kruth SA, Willey BM, Staempfli HR, Low DE, Weese JS. PCR ribotyping of Clostridium difficile isolates originating from human and animal sources. J Med Microbiol 2005;54:163e6. [6] Rupnik M. Is Clostridium difficile-associated infection a potentially zoonotic and foodborne disease? Clin Microbiol Infect 2007;13:457e9. [7] Koene MG, Mevius D, Wagenaar JA, Harmanus C, Hensgens MP, Meetsma AM, et al. Clostridium difficile in Dutch animals: their presence, characteristics and similarities with human isolates. Clin Microbiol Infect 2011;18:778e84. [8] Weese JS, Staempfli HR, Prescott JF, Kruth SA, Greenwood SJ, Weese HE. The roles of Clostridium difficile and enterotoxigenic Clostridium perfringens in diarrhea in dogs. J Vet Intern Med 2001;15:374e8. [9] Weese JS, Armstrong J. Outbreak of Clostridium difficile-associated disease in a small animal veterinary teaching hospital. J Vet Intern Med 2003;17:813e6. [10] Marks SL, Kather EJ, Kass PH, Melli AC. Genotypic and phenotypic characterization of Clostridium perfringens and Clostridium difficile in diarrheic and healthy dogs. J Vet Intern Med 2002;16:533e40. [11] Weese JS, Weese HE, Bourdeau TL, Staempfli HR. Suspected Clostridium difficile-associated diarrhea in two cats. J Am Vet Med Assoc 2001;218. 1436e9, 1421. [12] McKenzie E, Riehl J, Banse H, Kass PH, Nelson Jr S, Marks SL. Prevalence of diarrhea and enteropathogens in racing sled dogs. J Vet Intern Med 2010;24: 97e103. [13] Clooten JK, Kruth SA, Weese JS. Genotypic and phenotypic characterization of Clostridium perfringens and Clostridium difficile in diarrheic and healthy dogs. J Vet Intern Med 2003;17:123 [author reply]. [14] Weese JS, Finley R, Reid-Smith RR, Janecko N, Rousseau J. Evaluation of Clostridium difficile in dogs and the household environment. Epidemiol Infect 2009:1e5. [15] Struble AL, Tang YJ, Kass PH, Gumerlock PH, Madewell BR, Silva Jr J. Fecal shedding of Clostridium difficile in dogs: a period prevalence survey in a veterinary medical teaching hospital. J Vet Diagn Invest 1994;6:342e7.
[16] Perrin J, Buogo C, Gallusser A, Burnens AP, Nicolet J. Intestinal carriage of Clostridium difficile in neonate dogs. Zentralbl Veterinarmed B 1993;40:222e 6. [17] Al Saif N, Brazier JS. The distribution of Clostridium difficile in the environment of South Wales. J Med Microbiol 1996;45:133e7. [18] Zidaric V, Zemljic M, Janezic S, Kocuvan A, Rupnik M. High diversity of Clostridium difficile genotypes isolated from a single poultry farm producing replacement laying hens. Anaerobe 2008;14:325e7. [19] Indra A, Huhulescu S, Schneeweis M, Hasenberger P, Kernbichler S, Fiedler A, et al. Characterization of Clostridium difficile isolates using capillary gel electrophoresis-based PCR ribotyping. J Med Microbiol 2008; 57:1377e82. [20] Bidet P, Barbut F, Lalande V, Burghoffer B, Petit JC. Development of a new PCRribotyping method for Clostridium difficile based on ribosomal RNA gene sequencing. FEMS Microbiol Lett 1999;175:261e6. [21] van den Berg RJ, Schaap I, Templeton KE, Klaassen CH, Kuijper EJ. Typing and subtyping of Clostridium difficile isolates by using multiple-locus variablenumber tandem-repeat analysis. J Clin Microbiol 2007;45:1024e8. [22] Zaiss NH, Rupnik M, Kuijper EJ, Harmanus C, Michielsen D, Janssens K, et al. Typing Clostridium difficile strains based on tandem repeat sequences. BMC Microbiol 2009;9:6. [23] Terhes G, Brazier JS, Urban E, Soki J, Nagy E. Distribution of Clostridium difficile PCR ribotypes in regions of Hungary. J Med Microbiol 2006;55:279e82. [24] Pituch H, Brazier JS, Obuch-Woszczatynski P, Wultanska D, MeiselMikolajczyk F, Luczak M. Prevalence and association of PCR ribotypes of Clostridium difficile isolated from symptomatic patients from Warsaw with macrolide-lincosamide-streptogramin B (MLSB) type resistance. J Med Microbiol 2006;55:207e13. [25] Kuehne SA, Cartman ST, Minton NP. Both, toxin A and toxin B, are important in Clostridium difficile infection. Gut Microbes 2011;2:252e5. [26] Clooten J, Kruth S, Arroyo L, Weese JS. Prevalence and risk factors for Clostridium difficile colonization in dogs and cats hospitalized in an intensive care unit. Vet Microbiol 2008;129:209e14. [27] Chouicha N, Marks SL. Evaluation of five enzyme immunoassays compared with the cytotoxicity assay for diagnosis of Clostridium difficile-associated diarrhea in dogs. J Vet Diagn Invest 2006;18:182e8. [28] Lefebvre SL, Waltner-Toews D, Peregrine AS, Reid-Smith R, Hodge L, Arroyo LG, et al. Prevalence of zoonotic agents in dogs visiting hospitalized people in Ontario: implications for infection control. J Hosp Infect 2006;62: 458e66. [29] Cave NJ, Marks SL, Kass PH, Melli AC, Brophy MA. Evaluation of a routine diagnostic fecal panel for dogs with diarrhea. J Am Vet Med Assoc 2002;221: 52e9. [30] Riley TV, Adams JE, O’Neill GL, Bowman RA. Gastrointestinal carriage of Clostridium difficile in cats and dogs attending veterinary clinics. Epidemiol Infect 1991;107:659e65. [31] Weber A, Kroth P, Heil G. The occurrence of Clostridium difficile in fecal samples of dogs and cats. Zentralbl Veterinarmed B 1989;36:568e76. [32] Borriello SP, Honour P, Turner T, Barclay F. Household pets as a potential reservoir for Clostridium difficile infection. J Clin Pathol 1983;36:84e7. [33] Madewell BR, Bea JK, Kraegel SA, Winthrop M, Tang YJ, Silva Jr J. Clostridium difficile: a survey of fecal carriage in cats in a veterinary medical teaching hospital. J Vet Diagn Invest 1999;11:50e4.