Veterinary Microbiology 168 (2014) 124–130

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Genotypes and antibiotic resistance of canine Campylobacter jejuni isolates Chantal Amar a, Sonja Kittl a, David Spreng b, Andreas Thomann a, Boz˙ena M. Korczak a, Andre´ P. Burnens c, Peter Kuhnert a,* a b c

Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Bern, Switzerland Small Animal Clinic, Vetsuisse Faculty, University of Bern, Bern, Switzerland Unilabs, Bern, Switzerland

A R T I C L E I N F O

A B S T R A C T

Article history: Received 31 July 2013 Received in revised form 2 October 2013 Accepted 3 October 2013

Campylobacter jejuni is the most important cause of bacterial gastroenteritis in humans. It is a commensal in many wild and domestic animals, including dogs. Whereas genotypes of human and chicken C. jejuni isolates have been described in some detail, only little information on canine C. jejuni genotypes is available. To gain more information on genotypes of canine C. jejuni and their zoonotic potential, isolates from routine diagnostics of diarrheic dogs as well as isolates of a prevalence study in non-diarrheic dogs were analyzed. Prevalence of thermophilic Campylobacter among non-diarrheic dogs was 6.3% for C. jejuni, 5.9% for Campylobacter upsaliensis and 0.7% for Campylobacter coli. The C. jejuni isolates were genotyped by multi locus sequence typing (MLST) and flaB typing. Resistance to macrolides and quinolones was genetically determined in parallel. Within the 134 genotyped C. jejuni isolates 57 different sequence types (ST) were found. Five STs were previously unrecognized. The most common STs were ST-48 (11.2%), ST-45 (10.5%) and ST21 (6.0%). Whereas no macrolide resistance was found, 28 isolates (20.9%) were resistant to quinolones. ST-45 was significantly more prevalent in diarrheic than in non-diarrheic dogs. Within the common time frame of isolation 94% of the canine isolates had a ST that was also found in human clinical isolates. In conclusion, prevalence of C. jejuni in Swiss dogs is low but there is a large genetic overlap between dog and human isolates. Given the close contact between human and dogs, the latter should not be ignored as a potential source of human campylobacteriosis. ß 2013 Elsevier B.V. All rights reserved.

Keywords: Dog Zoonosis Prevalence Multi locus sequence typing Campylobacter Antibiotic resistance

1. Introduction In many industrialized countries Campylobacter jejuni is the most common cause of bacterial gastroenteritis in humans (European Food Safety Authority, 2013). Similar to other countries, in Switzerland C. jejuni is isolated in about 90% of human cases followed by Campylobacter coli, which

* Corresponding author at: Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Laenggassstr. 122, 3001 Bern, Switzerland. Tel.: +41 31 6312485; fax: +41 31 6312634. E-mail address: [email protected] (P. Kuhnert). 0378-1135/$ – see front matter ß 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetmic.2013.10.006

accounts for the remaining 10% (Niederer et al., 2012). Campylobacter lari, Campylobacter upsaliensis and Campylobacter fetus are rarely found in humans and their occurrence may vary among different regions of the world (Moore et al., 2005). The main risk factors for human campylobacteriosis include consumption of contaminated foodstuffs, particularly undercooked chicken meat, raw milk or untreated water, as well as foreign travel and close contact with carrier animals, including pets (Moore et al., 2005; Mughini Gras et al., 2013; Ross et al., 2013). Source attribution studies clearly indicate chicken as the main reservoir with up to 80% of human cases linked to it (Mullner et al., 2009; Sheppard et al., 2009b; European

C. Amar et al. / Veterinary Microbiology 168 (2014) 124–130

Food Safety Authority, 2011). C. upsaliensis and C. jejuni are the predominant species found in dogs (Wieland et al., 2005; Carbonero et al., 2012). Risk factors described for C. jejuni infection in dogs include age between seven and 12 months, male sex, living together with children, living in urban areas, being a stray dog, and contact with other dogs or birds (Hald and Madsen, 1997; Wieland et al., 2005; Tsai et al., 2007; Domingues et al., 2012; Carbonero et al., 2012). Dogs can be healthy carriers of Campylobacter spp., whereby animals under six month of age show a higher rate of carriage. Higher carriage rates were also observed in younger dogs with diarrhea, while in older dogs no difference in Campylobacter spp. shedding was reported between healthy and diseased animals (Burnens et al., 1992). Multi locus sequence typing (MLST) is a standardized method, which has proven useful in epidemiological studies for C. jejuni and C. coli (Dingle et al., 2001; Korczak et al., 2009). It is highly reproducible, precise and can be compared between laboratories worldwide via public databases. For further differentiation, sequencing of the short variable region of the flagellin-encoding genes flaA or flaB can be used (Mellmann et al., 2004; Dingle et al., 2008; Korczak et al., 2009). Macrolides and quinolones are mainly considered for therapy of severe Campylobacter infections, and increased antibiotic resistance of C. jejuni, especially to quinolones, has been observed worldwide (Alfredson and Korolik, 2007). The recognition of quinolone or macrolide resistant strains can be achieved in parallel to genotyping by sequencing gyrA and 23S rRNA gene fragments (Korczak et al., 2009). The point mutation C257T in gyrA leads to a high level of resistance to quinolones in C. jejuni and C. coli. Macrolide resistance is due to an A2075G or A2074G transition in the 23S rRNA gene (Alfredson and Korolik, 2007). As currently little information on genotypes and antibiotic resistance of canine C. jejuni/coli isolates is available, we performed MLST, fla-typing and antibiotic resistance testing by sequencing partial gyrA and 23S rRNA genes on a large collection of canine C. jejuni/coli isolates from diarrheic and non-diarrheic dogs in Switzerland. 2. Materials and methods 2.1. Sampling and sample preparation Fecal samples were collected from June to October 2012 in the Capital region of Bern, Switzerland, from 303 nondiarrheic pet household dogs. Only dogs with no gastrointestinal symptoms at the time of visit and not having received any antimicrobial treatment for at least two weeks prior to sampling were included in the study. Of the 303 dogs, 145 (48%) were sampled at the Small Animal Clinic, University of Bern, 35 at two pet obedience schools and a further 123 dogs were tested at six animal shelters. For each dog the date of sampling, age, sex and breed were recorded. Samples were taken by rectal swabs (Transwabß Amies, Medical Wire & Equipment, United Kingdom) and kept at 4 8C, for no longer than 4 h before starting

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culture isolation. Swabs were then inoculated in 6 ml of Preston Campylobacter Selective Enrichment Broth (Oxoid, Pratteln, Switzerland) and incubated under microaerobic conditions at 41.5 8C for 24 h. A loopful of each enriched sample was streaked on two Campylobacter selective agar plates: mCCDA with antibiotic and antifungal agents (Oxoid) and Campylosel (BioMe´rieux, Geneva, Switzerland) and incubated under the same conditions for 48 h. Presumptive Campylobacter colonies were identified using matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF) before sub cultivation for pure cultures on tryptone soy agar with 5% sheep blood (TSA; Becton Dickinson, Allschwil, Switzerland) for 24 h. For DNA template preparation, a few colonies from each plate were subsequently added to 500 ml of lysis buffer (0.1 M Tris–HCl, pH 8.5, 0.05% Tween 20, 240 mg/ml proteinase K) and incubated for 1 h at 60 8C followed by 15 min at 95 8C. The remaining colonies were transferred to freezing medium (T-soy-bouillon with 30% glycerin) and stored at 80 8C. The set of 118 C. jejuni and C. coli isolates from dogs with diarrhea had been collected at the diagnostics unit (ZOBA) of the Institute of Veterinary Bacteriology Bern between 2003 and 2012. The strains were obtained from fecal specimens submitted by the veterinarian from individual dogs suffering from diarrhea. The isolates were stored at 80 8C, were grown on TSA under microaerobic conditions at 41.5 8C for 24 h and lysates were prepared as described above. 2.2. Multiplex PCR amplification and sequencing A multiplex amplification and sequencing strategy according to Korczak et al. (2009) was used to genotype the strains. For MLST the sequences of internal fragments of seven housekeeping genes and in parallel partial flaB, 23S rRNA and gyrA gene sequences were determined for each isolate. The protocol was slightly modified according to Kittl et al. (2011). 2.3. Data analysis The allele number, sequence type (ST), and clonal complex (CC) were determined with the online-based MLST application module for Campylobacter provided by SmartGene (Zug, Switzerland), which uses an integrated link to the PubMLST database (http://pubmlst.org/Campylobacter/). The PubMLST database was directly queried for determining flaB alleles. The 23S rRNA gene fragments were examined for point mutations A2075G and A2074G, which determine macrolide resistance and the gyrA fragments were screened for the C257T transition, which confers quinolone resistance. Statistical analyses were performed with the NCSS 2008 software (NCSS, Kaysville, UT, USA) or STATA IC 12.1 (StataCorp LP, TX, USA). Differences between diarrheic and non-diarrheic dogs were analyzed with the level of significance set at p < 0.05 using Fisher’s exact test (two tailed), Chi-square test or Armitage test for trend in proportions. The exact binominal method was applied for calculating the confidence intervals (CI).

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Table 1 Sampling season, age and sex of Campylobacter spp. positive dogs. Variable

Category

Number of samples

C. jejuni

C. coli

C. upsaliensis

Campylobacter spp.

Season of sampling

Summer Autumn

145 158

11 (7.6%) 8 (5.1%)

2 (1.4%) 0 (0%)

10 (6.9%) 8 (5.1%)

23 (15.9%) 16 (10.1%)

Age of dog

0–2 >2–4 >4–6 >6–8 >8

60 58 37 41 102

Sex of dog

Male Female

172 130

6 3 2 3 5

(10%) (5.2%) (5.4%) (7.3%) (4.9%)

11 (6.4%) 8 (6.2%)

1 1 0 0 0

(1.7%) (1.7%) (0%) (0%) (0%)

0 (0%) 2 (1.5%)

7 5 2 1 3

(11.7%) (8.6%) (5.4%) (2.4%) (2.9%)

7 (4.1%) 11 (8.5%)

14 9 4 4 8

(23.3%) (15.5%) (10.8%) (9.8%) (7.8%)

18 (10.5%) 21 (16.2%)

For five animals age was unknown and for one animal sex was not recorded.

A total of 558 genotyped isolates collected from human cases during 2008 and 2009 (Kittl et al., 2011; Niederer et al., 2012) were compared to the canine isolates. For pairwise comparisons of groups of isolates, the Czekanowski index (also known as proportional similarity index or PSI) was calculated. The standard deviation for the estimation of CIs was calculated using the bootstrap method with 1000 resampling steps. 3. Results 3.1. Prevalence of Campylobacter in non-diarrheic dogs Among the 145 fecal swabs taken from non-diarrheic animals at the Small Animal Clinic in summer, 11 (7.6%; 95% CI: 4.0–13.5%) were positive for C. jejuni, 2 (1.4%; 95% CI: 0.2– 5.4%) for C. coli and 10 (6.9%; 95% CI: 3.5–12.7%) for C. upsaliensis. Isolation rates were similar to these in the 158 samples taken at animal shelters and obedience schools in autumn [eight (5.1%; 95% CI: 2.4–10.1%) were positive for C. jejuni, eight (5.1%; 95% CI: 2.4–10.1%) for C. upsaliensis, no C. coli were detected]. The overall prevalence amounted to 6.3% (95% CI: 3.8–9.6%) for C. jejuni, 5.9% (95% CI: 3.6–9.2%) for C. upsaliensis, and 0.7% (95% CI: 0.0–2.4%) for C. coli. Four out of six dogs from a single breeder were positive for C. jejuni, however each carried a different strain. A significantly higher prevalence (17.4%; 95% CI: 5.7–39.5%) of C. jejuni was found in one animal shelter compared to the rest. There was no significant difference in C. jejuni prevalence between age groups or between males and females (Table 1). However, C. upsaliensis was significantly more prevalent in dogs younger than four years (10.2%; 95% CI: 5.6–17.4%) compared to dogs older than eight years (2.9%; 95% CI: 0.8–9.0%) and was slightly more prevalent in female dogs (8.5%; 95% CI: 4.5–15.0%), than in males (4.1%; 95% CI: 1.8–8.5%) but the difference was not significant. Carriage rates of total Campylobacter spp. was highest in young dogs and declined with age. This trend was significant with the score test for trend of odds giving a p < 0.005. 3.2. Genotyping All C. jejuni and C. coli strains could be successfully genotyped (Table 2). Species identification using MALDI-TOF concurred with the species specific genotype in all cases.

For each positive sample in the prevalence study two to three primary colonies were analyzed to test for multiple strains being present. In all but one case the same strain as determined by identical ST, flaB-type, and antibiotic resistance markers was detected in an individual. One dog carried two different STs (ST-441 and ST-21) and flaBtypes (34 and 103, respectively) with one of them being quinolone resistant (ST-441). Isolates with matching genotypes from the same dog were treated as one isolate in all subsequent statistical analyses. Among the 114 C. jejuni isolates from diarrheic dogs and the 20 C. jejuni isolates from non-diarrheic dogs a total of 57 different STs was found (Table 2). Five of these were new and submitted to the PubMLST database for allele number and ST designation. From these five, three showed a new allele with ST-6192 having the new atpA-374, whereas ST-6193 and ST-6194 showed the new alleles tkt-510 and tkt-511, respectively. The other two new STs, ST-6190 and ST-6191, resulted from new combinations of previously described alleles. Three of the new STs could be assigned to an already known clonal complex, ST-6192 to CC41, ST-6193 to CC45 and ST-6194 to CC443. The ST-6190 and ST-6191 could not be assigned to any existing CC. The most frequent STs identified in dogs were ST-48 (n = 15, 11.2%) followed by ST-45 (n = 14, 10.5%), ST-21 (n = 8, 6.0%), ST-22 (n = 7, 5.2%) and ST-441 (n = 6, 4.5%). Twenty-one isolates had a ST, which could not be assigned to any known clonal complex. The three predominant clonal complexes were represented by CC21 (17.9%), CC48 (16.4%) and CC45 (14.9%). With flaB-typing 43 different types were determined, all of them were previously known. The most common flaB types were 103 (19.4%), 34 (6.0%), 8 (4.5%), 5 (3.7%) and 36 (3.7%). Two out of four positive dogs from one poodle breeder carried strains with the same genotype (ST-1073, flaB type 208 and sensitive to quinolones and macrolides). The same was true for isolates from two out of four positive dogs from one shelter (ST-441, flaB type 34, resistant to quinolones and sensitive to macrolides). From the diarrheic dogs four C. coli isolates were obtained, all belonging to CC828 with ST-827, ST-1058, ST2142 and ST-3336. The two C. coli from non-diarrheic dogs were also CC828, with ST-829 and ST-4950 (Table 2).

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Table 2 Distribution of clonal complexes (CC), sequence types (ST) and flaB types among C. jejuni and C. coli strains isolated from non-diarrheic and diarrheic dogs. NF indicates STs for which no clonal complex is defined. Species

CC

ST

flaB

Number of isolates (non-diarrheic/diarrheic)

Quinolone-resistant isolates (non-diarrheic/diarrheic)

C.jejuni

21

19 19 21 21 50 50 50 53 148 262 883 917 22 22 1947 6192 42 42 45 45 45 45 45 45 45 137 137 233 418 2219 6193 48 48 475 918 52 775 61 122 572 572 257 257 267 383 574 353 400 878 1073 403 1775 51 6194 132 607 904 658 1044 1395 1967 2180 677 441 448 586 881 995

36 53 103 414 36 103 429 103 36 137 103 96 309 442 442 1256 177 301 8 22 31 66 307 463 506 36 441 22 307 32 307 8 103 105 1123 57 57 1179 395 96 260 16 301 177 177 57 45 67 34 208 51 51 21 21 26 36 34 5 5 5 5 402 864 34 1514 402 321 787

1 1 6 2 1 3 1 1 1 2 3 2 2 5 1 1 2 1 4 2 1 1 1 4 1 1 1 1 2 1 1 2 13 5 2 1 1 2 3 1 1 1 1 1 1 1 2 1 1 3 1 2 1 1 1 1 1 2 1 1 1 1 3 6 1 3 1 1

1 0 0 0 0 3 1 0 1 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 1 1 0 0 0 1 0 0 0 0 1 0 0 1 0 1 0 1 0 0 0 1 0 0 0 0 1 0 6 0 0 1 0

22

41 42 45

48

52 61 206

257 283 305 353 354 403 443 508 607 658

677 NF NF NF NF NF

(0/1) (0/1) (2/4) (1/1) (0/1) (1/2) (0/1) (0/1) (0/1) (0/2) (0/3) (1/1) (0/2) (0/5) (0/1) (0/1) (1/1) (1/0) (0/4) (0/2) (0/1) (0/1) (0/1) (0/4) (0/1) (0/1) (0/1) (0/1) (0/2) (0/1) (0/1) (1/1) (1/12) (0/5) (0/2) (1/0) (0/1) (0/2) (0/3) (0/1) (0/1) (0/1) (1/0) (0/1) (0/1) (0/1) (1/1) (0/1) (0/1) (2/1) (0/1) (1/1) (0/1) (0/1) (0/1) (0/1) (0/1) (0/2) (0/1) (0/1) (0/1) (0/1) (0/3) (4/2) (0/1) (0/3) (0/1) (0/1)

(0/1) (0/0) (0/0) (0/0) (0/0) (1/2) (0/1) (0/0) (0/1) (0/0) (0/1) (0/0) (0/0) (0/0) (0/1) (0/0) (0/0) (0/0) (0/0) (0/0) (0/0) (0/0) (0/0) (0/0) (0/0) (0/0) (0/0) (0/0) (0/0) (0/0) (0/0) (0/0) (0/1) (0/0) (0/1) (1/0) (0/1) (0/0) (0/0) (0/0) (0/1) (0/0) (0/0) (0/0) (0/0) (0/1) (0/0) (0/0) (0/1) (0/0) (0/1) (0/0) (0/1) (0/0) (0/0) (0/0) (0/1) (0/0) (0/0) (0/0) (0/0) (0/1) (0/0) (4/2) (0/0) (0/0) (0/1) (0/0)

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128 Table 2 (Continued ) Species

CC

ST

flaB

C.coli

NF NF NF NF NF 828

1080 2258 3573 6190 6191 827 829 1058 2142 3336 4950

473 402 22 402 45 236 107 1459 66 1517 402

3.3. Antibiotic resistance For determining antibiotic resistance toward quinolones and macrolides the gyrA and the 23S rRNA genes were analyzed. The point mutations A2075G or A2074G in the 23S rRNA gene, which is associated with macrolide resistance, were not observed in any of the strains. For C. jejuni the mutation C257T in gyrA which leads to quinolone resistance was present in 28 isolates (20.9%; 95% CI: 14.3– 28.7%), whereas three out of the six C. coli were resistant to quinolones. Interestingly quinolone resistance was most frequently found with CC21 (29.2%) and in all 6 isolates of ST-441. In contrast, isolates of CC45 never showed the quinolone resistance specific mutation. A trend toward an increase in quinolone resistance over the years was observed (Armitage test for trend in proportions, p = 0.04). 3.4. Comparison of strains from non-diarrheic and diarrheic dogs The 20 C. jejuni isolates from non-diarrheic dogs were compared to the 17 strains available from diarrheic dogs collected during the same time period in 2012. There was a low amount of overlapping genotypes between those two groups, which might however be due to the small sample size. A significant difference was observed for CC45 in particular ST-45, which was more frequent in diarrheic dogs (four isolates compared to zero among the nondiarrheic dogs). There was no significant difference in quinolone resistance rates between strains from nondiarrheic and diarrheic dogs. 3.5. Comparison with human isolates For comparing dog and human isolates the 64 C. jejuni from dogs collected between 2007 and 2010 (covering the human collection time span) and 558 isolates from Swiss human cases of campylobacteriosis collected from 2008 to 2009 were compared (Kittl et al., 2011; Niederer et al., 2012). A total of 28 common STs were found with dog and human isolates, whereby 94% of dog isolates had a corresponding genotype in human isolates. The most frequent commonly found ST in dogs and humans were ST48 (10.4% in human, 11.2% in dog) and ST-21 (19.2% in human, 6.0% in dog). The PSI for ST comparison between human and dog isolates was 0.39 (95% CI: 0.31–0.48). The most common CCs seen in dogs and humans were CC21

Number of isolates (non-diarrheic/diarrheic) 1 1 1 1 1 1 1 1 1 1 1

(0/1) (0/1) (0/1) (0/1) (1/0) (0/1) (1/0) (0/1) (0/1) (0/1) (1/0)

Quinolone-resistant isolates (non-diarrheic/diarrheic) 0 0 1 0 1 0 1 1 0 0 1

(0/0) (0/0) (0/1) (0/0) (1/0) (0/0) (1/0) (0/1) (0/0) (0/1) (1/0)

(35.9% in human, 17.9% in dog) and CC48 (10.9% in human, 16.4% in dog). Among all overlapping STs a significant difference in frequency was detected for ST-21, ST-257 and ST-50 (higher prevalence in humans) and ST-45 (more frequent in dogs). An overlap of 23 common types was seen for flaB with 91% of dog isolates having a corresponding flaB type in human isolates. The PSI for flaB types was 0.48 (95% CI: 0.38–0.57) slightly higher than for MLST. The prevalence of quinolone resistant C. jejuni isolated from dogs during this time span was 16.9%, significantly lower than in humans with 41.1%. 4. Discussion The prevalence determined in the framework of this investigation revealed 6.3% of dogs carrying C. jejuni. This is similar to results of a former Swiss study conducted in 2002 where a prevalence of 5.7% was found. It therefore seems that such prevalence remained relatively invariant over the last 10 years (Wieland et al., 2005). This prevalence is, however, rather low compared to findings reported from other countries. Studies in Denmark, Ireland and Norway found prevalences of 19–23%, and studies in the Netherlands, Spain and the United States found prevalences of 13–14% (Hald and Madsen, 1997; Sandberg et al., 2002; Lee et al., 2004; Koene et al., 2004; Acke et al., 2009; Carbonero et al., 2012). In Taiwan 20.6% of stray dogs were positive for C. jejuni, but the prevalence among household dogs was much lower (2.5%) (Tsai et al., 2007). The less hygienic environment to which stray dogs are exposed as well as the likely consumption of contaminated food waste might be the main reason for the higher prevalence of campylobacters observed in this group of dogs. No significant differences in prevalence between sexes were observed, and there was no seasonal change in Campylobacter prevalence. There was a higher prevalence observed in one animal shelter and among the dogs of one poodle breeder. Differences in the prevalence between shelters were also observed in the US study (Lee et al., 2004). Interestingly, all but two dogs from each of these two locations carried different genotypes, indicating only minor transmission of campylobacters between animals in either of these groups. With C. upsaliensis some studies show that it is the most often isolated species in dogs and especially in young dogs,

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while others found C. jejuni more prevalent than C. uppsaliensis (Burnens et al., 1992; Hald and Madsen, 1997; Hald et al., 2004; Carbonero et al., 2012). We found that C. upsaliensis is not significantly more often found than C. jejuni. However, compared to C. jejuni it was more prevalent in younger and female dogs. Other studies also found a higher prevalence of C. upsaliensis in young nondiarrheic and diarrheic dogs (Wieland et al., 2005; Parsons et al., 2009; Carbonero et al., 2012). Besides geographical differences also differences in isolation methods could explain the observed discrepancies. The prevalence of C. coli was very low (0.7%) which is consistent with the results of other studies (Burnens et al., 1992; Hald et al., 2004; Acke et al., 2009). Overall numbers of animals positive for thermophilic Campylobacter spp. was around 13% and carriage rates were dependent on the age with young dogs showing higher prevalence, which is decreasing with increasing age. Comparing the CCs and STs found in the non-diarrheic and diarrheic dogs from the same time period, i.e. 2012, a low overlap was found. This might indicate a difference in pathogenicity among the genotypes, however due to the small sample size this conclusion must be viewed with caution. The only significant difference was with CC45, which was only isolated from diarrheic dogs but not from non-diarrheic ones in 2012. CC45 and predominantly ST-45, was also often found in other years in diseased dogs. In the literature this CC is reported from various sources in different countries, i.e. in farm animals wild birds, rabbits, badgers, turkey chicks and soil (Dingle et al., 2001; Colles et al., 2003; French et al., 2005). As CC45 is also the CC most frequently isolated from water it has been suggested that it might be better adapted to survive outside a host and thus be crucial in the transmission of C. jejuni through the environment (Sopwith et al., 2008). In our study CC45 was detected very often in diarrheic dogs, but never in the nondiarrheic dogs. Possible reasons for the presence of strains of CC45 in only dogs suffering from diarrhea might be exposure to surface water as well as feeding poultry meat to dogs, since this CC was also often detected in Swiss broilers (Wirz et al., 2010). Interestingly, ST-45 was rarely found in infected humans in Switzerland, even though poultry is supposed to be the main source of campylobacteriosis (Kittl et al., 2011; Niederer et al., 2012). So it may be possible that this ST is more pathogenic for dogs than for humans. This does however not seem to be true for other countries since a British study frequently found ST-45 in non-diarrheic dogs and in the Netherlands ST-45 was found to be the predominant genotype in dogs, independently of symptoms (Parsons et al., 2009; Mughini Gras et al., 2013). Even though the prevalence of C. jejuni in dogs is low, they should not be discounted as a possible source of infection due to their close contact with humans. To investigate the zoonotic potential of canine C. jejuni we further analyzed genotypes of dog isolates and compared them to human isolates. Whereas many studies comparing isolates from various animals found a high overlap of human, poultry and ruminant isolates (Manning et al., 2003; Levesque et al., 2008; Wilson et al., 2008; Sheppard et al., 2009a), there are only few studies comparing C. jejuni genotypes in dogs and humans (Parsons et al., 2009; Mughini Gras et al., 2013).

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When comparing dog to human isolates covering the same time period 94% of the dog isolates had a ST found also in human, with ST-48 and ST-21 being the most frequent. It should however be noted that this overlap might also be due to dogs acquiring C. jejuni from the same sources as humans. In addition, the PSI between human and dog isolates (0.39) was lower than the PSI of human and chicken isolates (0.50) described by Kittl et al. (2011) which indicates that chickens are a more likely source for campylobacteriosis in humans than dogs. In Switzerland human C. jejuni isolates showed a significantly higher prevalence of quinolone resistance than chicken isolates with 37.5% and 18.9%, respectively (Wirz et al., 2010; Kittl et al., 2011). In comparison, the dog isolates showed a much lower level in quinolone resistance, with 16.9% between 2007 and 2010, the collection period of human isolates. Similar to Swiss broilers where no macrolide resistance was detected in C. jejuni (Wirz et al., 2010), no isolates resistant to macrolides were found in dogs and these results correspond to another study where only a low amount of erythromycin resistance was found (Carbonero et al., 2012). Interestingly, there is a relation between specific genotypes and resistance to quinolones. None of the CC45 isolates of dogs showed the mutation that confers quinolone resistance. In contrast CC21 showed a high rate of 29.2% resistant strains. Other studies found the same situation in chickens where CC45 isolates were also pansusceptible and a higher resistance rate was found in CC21, so this might be a general observation (Habib et al., 2009; Wirz et al., 2010). Quinolone resistance increased in C. jejuni from dogs over the last ten years. Similar observations were made for Swiss poultry, where the rate of quinolone resistance increased from 15% in 2006 to over 40% in 2011 (Bu¨ttner et al., 2012). This may be due to the continuing widespread use of these antibiotics in both human and veterinary medicine. If antibiotic treatment seems necessary gentamicin and erythromycin are now the drugs of choice for treatment of campylobacteriosis in dogs (Carbonero et al., 2012). In conclusion, prevalence of C. jejuni and more so C. coli in dogs was low. Nevertheless, dogs should not be ignored as a potential source of campylobacteriosis in humans as a relatively high overlap in STs between these two groups was found. The increasing resistance to quinolones among isolates from different sources is cause for concern as these drugs are important for treatment. Conflict of interest There are no conflicts of interest. Acknowledgement This research was supported by a Swiss Federal Veterinary Office grant 1.10.08. References Acke, E., McGill, K., Golden, O., Jones, B.R., Fanning, S., Whyte, P., 2009. Prevalence of thermophilic Campylobacter species in household cats and dogs in Ireland. Vet. Rec. 164, 44–47.

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Genotypes and antibiotic resistance of canine Campylobacter jejuni isolates.

Campylobacter jejuni is the most important cause of bacterial gastroenteritis in humans. It is a commensal in many wild and domestic animals, includin...
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