Campylobacter in small ruminants at slaughter: Prevalence, pulsotypes and antibiotic resistance

International Journal of Food Microbiology 173 (2014) 54–61

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Campylobacter in small ruminants at slaughter: Prevalence, pulsotypes and antibiotic resistance Thomai Lazou a, Kurt Houf b, Nikolaos Soultos a, Chrysostomos Dovas c, Eleni Iossifidou a,⁎ a b c

Laboratory of Hygiene of Foods of Animal Origin, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki (AUTh), 54124 Thessaloniki, Greece Department of Veterinary Public Health and Food Safety, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium Laboratory of Microbiology and Infectious Diseases, School of Veterinary Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki (AUTh), 54124 Thessaloniki, Greece

a r t i c l e

i n f o

Article history: Received 9 July 2013 Received in revised form 22 October 2013 Accepted 15 December 2013 Available online 21 December 2013 Keywords: Campylobacter Sheep Goat PFGE Antimicrobial resistance Carcass

a b s t r a c t The present study aimed to address the prevalence, pulsotypes, and antimicrobial susceptibility patterns of Campylobacter species present in sheep and goat carcasses at slaughter. In total, 851 samples were collected (343 meat surfaces, 282 ileum contents, 226 liver surfaces) and 835 Campylobacter isolates were detected in 274 out of 343 carcasses (116 kids, 110 lambs, 63 goats and 54 sheep). The contamination rates per carcass category were 78.4% for kids, 94.5% for lambs, 63.5% for goats, and 72.2% for sheep. On average, 30% of the intestinal content samples and more than 70% of carcass and liver surfaces yielded the presence of campylobacters. Multiplex-PCR and RFLP analysis identified Campylobacter coli as the most prevalent species (76.2%) followed by Campylobacter jejuni (21.4%), albeit 2.4% of selected colonies yielded the concurrent presence of both these species. Macrorestriction profiling by pulsed-field gel electrophoresis (PFGE) was applied in order to characterise a subset of isolates. SmaI-PFGE successfully clustered 222 isolates in 82 SmaI-PFGE types indicating high heterogeneity among the campylobacter isolates (67 types among 174C. coli isolates and 15 types among 48C. jejuni isolates). No carcass-type (lamb, kid, sheep, and goat) specific PFGE clusters were recognised since there was a general overlapping of PFGE patterns regarding ovine and caprine isolates. Multiple pulsotypes were simultaneously present on single carcasses in the majority of tested animals. PFGE provided data regarding the potential routes of meat and liver contamination such as spillage of faecal material and cross-contamination during slaughter. Antimicrobial susceptibility patterns of Campylobacter isolates (n = 240), determined by disk diffusion method, revealed resistance to tetracycline (47.9%) followed by streptomycin (22.9%) and ciprofloxacin along with nalidixic acid (18.3%). Isolates exhibited low resistance to erythromycin (2.5%) and were susceptible to gentamicin. The findings of the present study confirm the contamination of sheep and goats at slaughter with thermophilic campylobacters and underline their potential input in the epidemiology of human campylobacteriosis. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Food of animal origin is related to the transmission of zoonotic organisms to humans. Of these pathogens, the thermophilic Campylobacter species (Campylobacter jejuni and Campylobacter coli) represent a major bacterial cause of food-borne gastroenteritis worldwide. According to the latest report by the European Food Safety Authority (EFSA), Campylobacter continues to be the most commonly reported gastrointestinal bacterial pathogen in humans in the European Union (EU) (EFSA, 2013). In 2011, the overall EU notification rate of human campylobacteriosis was 50.28 per 100,000 population (EFSA and ECDC, 2012a, 2013). However, due to underreporting, the actual number of human campylobacteriosis is estimated to be around 9 million cases each year in the EU with ⁎ Corresponding author. Tel.: +30 2310 999815; fax: +30 2310 999833. E-mail address: [email protected] (E. Iossifidou). 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.12.011

the corresponding total annual costs reaching 2.4 billion € (EFSA, 2011). In the US, around 2.4 million persons are infected each year with campylobacters, with an overall US campylobacteriosis incidence of 14.28 per 100,000 population in 2011 (CDC, 2012; NCEZID, 2013). Human campylobacteriosis is predominantly acquired through the consumption of contaminated foods, wherein foods of animal origin play a key role (Jacobs-Reitsma et al., 2008). Animals become colonised with Campylobacter spp. once exposed to a contaminated external environment but remain mostly asymptomatic intestinal carriers and are not condemned at ante-mortem inspection prior to slaughter (Humphrey et al., 2007). Poultry products comprise the major source of human infection, but consumption and/or handling of other raw or undercooked meats, unpasteurised milk, and water have also been identified as risk factors (Humphrey et al., 2007; Jacobs-Reitsma et al., 2008; Sheppard et al., 2009). Apart from the Campylobacter genotypes that have been characterised as common to humans and poultry, an unidentified percentage of human campylobacteriosis cases in

T. Lazou et al. / International Journal of Food Microbiology 173 (2014) 54–61

the developed world may be attributed to as yet unspecified sources (O'Leary et al., 2011). The presence of Campylobacter in small ruminants has not been as extensively investigated as in other farm animals. However, campylobacters have been isolated from sheep carcasses, in retail ovine meat and liver, gallbladder, gut contents, and faeces (Stanley et al., 1998; Ertaş et al., 2003; Whyte et al., 2004; Zweifel et al., 2004; Açik and Çetinkaya, 2006; Oporto et al., 2007; Dadi and Asrat, 2008; Little et al., 2008; Milnes et al., 2008; Horrocks et al., 2009). Campylobacters have been more frequently isolated from lamb offal than swine and beef offal (Devane et al., 2005; Little et al., 2008) and the relation between strains isolated from sheep liver and those from human diarrheal cases has been demonstrated (Cornelius et al., 2005; Strachan et al., 2012). Data regarding the prevalence of campylobacters in goats and products thereof are scarce. Nevertheless, raw goat's milk has been associated with human C. jejuni enteritis cases in the US (Newell & Davison, 2003) and Dadi and Asrat (2008) detected campylobacters in 9% of goat meat samples. Antimicrobial resistance among zoonotic pathogens represents a hazard to the effectiveness of antimicrobial treatment. Though campylobacteriosis manifests as a self-limiting gastroenteritis, it may appear with systemic sequelae that require antimicrobial treatment, especially in high-risk groups (Payot et al., 2006). On these occasions, the macrolide erythromycin is usually the first-choice drug whereas fluoroquinolones and, to a lesser extent, tetracycline represent alternative options (Allos, 2001; Solomon & Hoover, 1999). According to EFSA, existing data on Campylobacter in goats and sheep are primarily from clinical investigations since no surveillance so far has been carried out. Moreover, the largest goat population and the fourth largest sheep population in the EU are reared reared in Greece (EFSA, 2012a), and the contamination of sheep and goat meat with campylobacters is a matter of national public health interest. The present study aimed to address the lack of data regarding the occurrence and evaluation of the presence of different pulsotypes of Campylobacter species present in sheep and goat carcasses. The objectives were to assess the prevalence of Campylobacter species on carcasses of young and adult sheep and goats, and to determine their genotypes in order to identify probable contamination routes. A last objective was to assess antimicrobial susceptibility patterns of Campylobacter isolates originating from small ruminants. 2. Materials and methods 2.1. Collection of samples The total sample size was 343 carcasses consisting of 116 kids, 110 lambs, 63 goats, and 54 sheep. Animals originated from 53 farms (two to 12 animals sampled per farm) located in 5 regions in Northern Greece. In total, 851 samples were collected (343 meat surfaces, 282 ileum contents, 226 liver surfaces) from August 2007 to February 2009 from two abattoirs in Central Macedonia in Greece (in total, 27 samplings). The age of animals at slaughter was two to five months for kids and lambs and four to six years for goats and sheep. Carcasses were sampled successively in the exact order as they were processed by the technicians at the slaughter line prior to chilling. The meat surface and a proportion of ileum content were sampled for each animal. The liver surface was sampled for each lamb and kid only, since the livers of adult sheep and goats are usually rejected during the veterinary inspection. In brief, instantly following evisceration, approximately 40 ml of ileum content per carcass was collected in a sterile bottle according to the sampling scheme applied by Stanley et al. (1998). Polyurethane sponges (Whirl-Pak, Nasco, Fort Atkinson, WI, USA) moistened with sterile 0.1% peptone water (Bacteriological peptone, Oxoid Ltd., Basingstoke, Hampshire, United Kingdom) were used to swab the meat and liver surfaces after carcass washing. One sponge was used to sample the whole exterior surface of each carcass. In the case of kids and lambs, a second sponge was used to sample the

55

liver surface of each corresponding carcass. All samples were placed in insulated containers with ice packs and transported to the laboratory within 4 h after collection for analysis. 2.2. Microbiological analysis The ileum contents were directly inoculated onto a modified Charcoal Cefoperazone Deoxycholate agar (mCCDA, Merck, Darmstadt, Germany) and a Karmali (Oxoid) agar plate using sterile swabs. For the sponge samples, enrichment medium (50 ml) Bolton Broth (Merck), supplemented with 5% lysed horse blood (Oxoid) was added to the sample bags containing the sponges. Both enrichment media and selective agar plates were incubated at 41.5 °C for 44 h ± 4 h under microaerophilic conditions in a jar (Genbox jar, Genbox Microaer Generator, Biomérieux, Lyon, France). After incubation, the cultures obtained in the enrichment media were inoculated onto the two selective solid isolation media that were incubated under the same conditions. The mCCDA and Karmali agar plates were examined for typical colonies of Campylobacter species, and, due to the large scale of the study, one colony per plate was selected for further analysis. Each selected colony was subcultured onto a Columbia blood agar (CBA) plate (Biomérieux) and incubated at 41.5 °C for 44 h ± 4 h under microaerophilic conditions. In this way, the maximum number of selected colonies for each carcass was six for kids and lambs (two selective agar plates × three samples) and four for the goats and sheep (two selective agar plates × two samples). The isolates were then examined for morphology, motility, and catalase and oxidase activity, sensitivity to nalidixic acid and cephalothin, hippurate and indoxyl acetate hydrolysis. Isolates were stored at −80 °C in Nutrient Broth No. 2 (Oxoid) supplemented with 5% lysed horse blood (Oxoid) and 20% glycerol (BDH Laboratory Suppliers, Poole, England) until further analysis. 2.3. Identification of Campylobacter species DNA was extracted using a commercial kit (Nucleospin® Blood, Macherey-Nagel, Düren, Germany) modified properly to increase DNA recovery from Campylobacter cells. More specifically, one loop of cells was dispersed in 100 μl “dispersal buffer” (50 mM Tris–HCl, 50 mM EDTA, 1% v/v Triton x-100, pH 7.5). Volumes of 100 μl “lysis buffer I” (50 mM Tris–HCl, 50 mM EDTA, 4 M GuHCl, 10 mM CaCl2, 1% v/v Triton x-100, 2% N-Lauroyl-Sarcosine, pH 7.5) and 25 μl of a Proteinase K solution (22.4 mg/ml, Merck) were added, followed by incubation at 56 °C for 1 h. 250 μl of “Lysis Buffer II” (50 mM Tris–HCl, 25 mM EDTA, 8 M GuHCl, 3% v/v Triton x-100, 3% N-Lauroyl-Sarcosine, pH 6.3) was added and the lysates were incubated at 70 °C for 10 min. Finally 250 μl of absolute ethanol was added and the mixture was then applied to nucleospin columns. The DNA extraction procedure was further continued using the washing and elution buffers contained in the Nucleospin® Blood kit according to the manufacturer's instructions. The multiplex PCR assay developed by Wang et al. (2002) was used to identify Campylobacter at species level. The assay simultaneously detects genes from the five major clinically relevant Campylobacter species: the hipO and 23S rRNA genes for C. jejuni; the glyA gene for C. coli, Campylobacter lari, and Campylobacter upsaliensis; and the sapB2 gene for Campylobacter fetus subsp. fetus. Specific identification was achieved by applying Restriction Fragment Length Polymorphism (RFLP) digestion using the restriction endonucleases BsrDI, AluI, ApoI, DdeI, BclI and HhaI, resulting in specific restriction fragments for each Campylobacter species (Wang et al., 2002). 2.4. PFGE typing Macrorestriction profiling by Pulsed Field Gel Electrophoresis (PFGE) was applied on isolates originating from samplings that yielded the highest prevalence of Campylobacter contamination among all the corresponding collected samples (ileum contents, meat and liver

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surfaces). In particular, PFGE was applied to 256 Campylobacter isolates from ileum contents (n = 64), meat surfaces (n = 106) and liver surfaces (n = 86) from 91 animals processed in one abattoir, which originated from 16 farms and 13 sampling dates. PFGE was performed using SmaI-digested fragments of bacterial chromosomal DNA as described by Ribot et al. (2001). Subsequent KpnI-digestion was applied on representative isolates within a same cluster displaying similar SmaI profiles and on all isolates that resisted restriction by SmaI. Gel patterns were analysed using GelCompar software (Applied Maths, Kortrijk, Belgium) with a 1.7% band position tolerance and 1.75% optimisation. 2.5. Antimicrobial susceptibility testing The disk diffusion method according to Bauer et al. (1966) was used to test the antimicrobial susceptibility profile of 240 Campylobacter isolates (74C. jejuni and 166C. coli) that were selected in order to represent samples from all 27 samplings (37 ileum contents, 44 liver and 86 meat surfaces) and 121 animals (42 kids, 34 lambs, 20 goats, and 25 sheep), against ciprofloxacin (CIP 5 μg), erythromycin (E 15 μg), gentamicin (GM 10 μg), nalidixic acid (NA 30 μg), streptomycin (S 10 μg) and tetracycline (TE 30 μg). The inhibition zones were measured with callipers and interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2013) guidelines for Campylobacter as regards erythromycin, ciprofloxacin and tetracycline, and to CLSI guidelines for Enterobacteriaceae (CLSI, 2008) as regards gentamicin, streptomycin and nalidixic acid. Isolates exhibiting phenotypic resistance to more than two antibiotics from different classes were regarded as multidrug resistant. 2.6. Statistical analysis Statistical analysis was performed using SPSS statistics 19.0. Variances in prevalence and antimicrobial resistance patterns were tested between Campylobacter isolates using the Pearson's chi-square and the Fisher's exact two-tailed test. For all statistical comparisons an ‘α’ level of significance of 0.05 was used. 3. Results 3.1. Prevalence and speciation of Campylobacter isolates In total, 835 Campylobacter isolates were detected in 274 out of 343 domestic small ruminants (453 isolates in 143 ovine carcasses and 382 isolates in 131 caprine carcasses) yielding an overall contamination rate of approximately 80%. The contamination rates per animal category (= at least one positive sample per carcass) were 78.4% (91/116) for kids, 94.5% (104/110) for lambs, 63.5% (40/63) for goats, and 72.2% (39/54) for sheep. The prevalence of Campylobacter species varied according to sample and carcass type (Table 1). On average, 30% of the

intestinal content samples and more than 70% of meat and liver surfaces yielded the presence of campylobacters. Multiplex-PCR and RFLP analysis identified C. coli as the most prevalent species (636/835, 76.2%) followed by C. jejuni (179/835, 21.4%), albeit 2.4% (20/835) of the typical colonies selected from the mCCDA and Karmali agar plates yielded the concurrent presence of both these species. C. coli dominated in meat (88.7% of isolates) and liver samples (82.7% of isolates) whereas C. jejuni was specified as the most prevalent species in ileum content samples (69.7% of isolates). Lamb carcasses were significantly more contaminated with C. coli whereas kid and sheep carcasses were more contaminated with C. jejuni (p b 0.001). C. coli presence was significantly related to carcass surfaces in contrast to C. jejuni which was significantly more present in the ileum content (p b 0.001). No significant difference was observed (p N 0.05) in the isolation rate of Campylobacter spp. between the two selective agar plates used in this study. 3.2. PFGE typing SmaI-PFGE successfully typed 248 out of 256 selected Campylobacter isolates (typeability 96.9%) (Figs. 1 & 2). Macrorestriction by KpnI clustered in one pulsotype the eight remaining C. jejuni isolates that were resistant to restriction by SmaI and originated from three kid carcasses sampled on the same date. Single-fragment patterns were observed for 26 isolates (three C. coli and 23C. jejuni grouped in two different patterns, respectively) following SmaI and KpnI digestion, and were excluded from further analysis. The remaining 222 isolates (originating from 78 animals, 14 farms, and 11 sampling dates) were clustered in 82 SmaI-PFGE types (67 types among 174C. coli isolates and 15 types among 48C. jejuni isolates) each consisting of five to 17 fragments. Among these pulsotypes, 52 (47 for C. coli and five for C. jejuni) were represented only by a single isolate (indicated later in the text as ‘single-isolate pulsotypes’). Fifteen pulsotypes (nine for C. coli and six for C. jejuni) were represented by only two isolates. Isolates clustered in one group after SmaI restriction displayed also identical patterns when KpnI digestion was applied, representing identical genotypes. No carcass-type (lamb, kid, sheep, and goat) specific PFGE clusters were documented since there was a general overlapping between PFGE patterns of ovine and caprine isolates. Despite the overall heterogeneity among the tested C. coli isolates, two pulsotypes encompassed more than one third of the SmaI-restricted isolates (Table 2). The predominant C. coli pulsotype (CC-013) yielded a 12-fragment pattern and encompassed isolates mostly of meat and liver surfaces of lambs and kids. The corresponding animals originated from farms located in two adjacent regions in northern Greece and were sampled during four different occasions. The second common C. coli pulsotype (CC-047) displayed a distinctive six-fragment pattern and comprised isolates of meat and liver surfaces. The latter pulsotype was linked to three separate sampling dates and animals reared in farms located in three different regions (Table 2). More than one third of the C. coli isolates

Table 1 Presence of Campylobacter species per sample and carcass type. Sample type

Carcass type

No. of collected samples

No. of samples positive for C. jejuni

C. coli

C. jejuni & C. coli

Ileum content

Kid Lamb Goat Sheep Kid Lamb Goat Sheep Kid Lamb

100 80 52 50 116 110 54 63 116 110

23 11 3 16 11 – 4 4 18 2

5 17 4 1 54 91 30 30 46 81

2 3 1 – 13 3 6 5 10 3

Carcass surface

Liver surfacea a

Liver surfaces were sampled only in kids and lambs.

Total positive samples (%) 30 (30.0%) 31 (38.8%) 8 (15.4%) 17 (34.0%) 78 (67.2%) 94 (85.5%) 40 (74.1%) 39 (61.9%) 74 (63.8%) 86 (78.2%)

T. Lazou et al. / International Journal of Food Microbiology 173 (2014) 54–61 SmaI

100

95

90

85

80

75

70

65

Dice (Opt:1.75%) (Tol 1.7%-1.7%) (H>0.0% S>0.0%) [2.9%-88.8%] PFGE PFGE SmaI

57

Species

PFGE type

n

C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli C. coli

CC-001 CC-002 CC-003 CC-004 CC-013 CC-014 CC-015 CC-016 CC-017 CC-019 CC-020 CC-005 CC-006 CC-007 CC-022 CC-008 CC-053 CC-009 CC-010 CC-011 CC-012 CC-018 CC-046 CC-045 CC-037 CC-039 CC-035 CC-040 CC-036 CC-034 CC-042 CC-038 CC-043 CC-044 CC-027 CC-029 CC-028 CC-026 CC-041 CC-030 CC-031 CC-032 CC-024 CC-025 CC-023 CC-021 CC-033 CC-066 CC-067 CC-048 CC-059 CC-049 CC-058 CC-050 CC-047 CC-060 CC-061 CC-051 CC-052 CC-054 CC-055 CC-056 CC-057 CC-062 CC-063 CC-064 CC-065

1 1 2 1 42 1 1 8 6 2 1 1 7 1 1 1 3 3 1 1 1 1 1 1 2 1 7 1 1 3 1 1 4 1 1 1 1 1 1 1 1 1 1 1 1 2 1 2 1 1 1 1 2 2 20 1 1 6 2 1 2 1 1 1 1 1 1

Fig. 1. Dendrogram of all representative SmaI PFGE types of the 174C. coli isolates examined in this study (n = number of isolates belonging to each pulsotype).

were distributed in 18 different pulsotypes that consisted of two to eight isolates (‘single-isolate pulsotypes’ excluded) (Fig. 1). Among the C. jejuni cohort, the most common SmaI-PFGE type (CJ-001) was represented exclusively by isolates of caprine carcasses originating from the same farm and slaughtered on the same date. Apart from the five C. jejuni

‘single-isolate pulsotypes’, the remaining C. jejuni isolates (n = 26) were scattered into nine pulsotypes that consisted of two to six isolates (Fig. 2). Regarding the number of pulsotypes detected in individual animals, in 27 of the 78 carcasses (34.6%), the corresponding Campylobacter-positive

58

T. Lazou et al. / International Journal of Food Microbiology 173 (2014) 54–61

100

95

90

85

80

75

70

65

Dice (Opt:1.75%) (Tol 1.7%-1.7%) (H>0.0% S>0.0%) [2.9%-88.8%] PFGE SmaI PFGE SmaI

Species

PFGE type

n

C. jejuni

CJ-001

17

C. jejuni

CJ-002

2

C. jejuni

CJ-003

4

C. jejuni

CJ-004

6

C. jejuni

CJ-007

1

C. jejuni

CJ-008

1

C. jejuni

CJ-009

2

C. jejuni

CJ-010

2

C. jejuni

CJ-011

2

C. jejuni

CJ-012

2

C. jejuni

CJ-013

1

C. jejuni

CJ-005

2

C. jejuni

CJ-006

1

C. jejuni

CJ-014

4

C. jejuni

CJ-015

1

Fig. 2. Dendrogram of all representative SmaI PFGE types of the 48C. jejuni isolates examined in this study (n = number of isolates belonging to each pulsotype).

samples (ranging from one to all three collected samples per animal) yielded isolates that displayed the same PFGE pattern (C. jejuni in nine cases and C. coli in 18 cases). Within these cases, the ileum of six animals was the only Campylobacter-positive sample and five carcasses were positive for both the ileum and meat and/or liver surfaces. Interestingly the remaining 16 carcases were Campylobacter-positive only on the surfaces indicating possible environmental contamination. In the majority of animals (n = 51), multiple pulsotypes were simultaneously present in the positive samples of a single carcass. Of note is the occurrence of four to six pulsotypes per carcass in eight animals. Nevertheless, two pulsotypes were the maximum number of profiles that could be detected in one sample (ileum content, meat surface or liver surface) since two were originally the maximum number of selected colonies for each sample during microbiological analysis. When carcasses were clustered per sampling date, 11 slaughter groups were generated. Within a single slaughter group, from two to 28 Campylobacter pulsotypes (‘singe-isolate pulsotypes’ included) were detected among carcasses. In six slaughter groups, all Campylobacter pulsotypes present in the ileum contents were detected on the meat and/or liver surfaces. Interestingly, the aforementioned surfaces yielded from two to six additional dissimilar pulsotypes. In addition, meat and/ or liver surfaces in three slaughter groups yielded Campylobacter isolates that did not share any pulsotype with the isolates detected in the corresponding ileum contents. Finally, in two slaughter groups, apart from the shared pulsotypes between ileum contents and meat/liver surfaces, there were additional pulsotypes, exclusively present either in the ileum contents or on the meat/liver surfaces.

incidence of antimicrobial resistance was detected for tetracycline (47.9%) followed by streptomycin (22.9%) and quinolones (ciprofloxacin and nalidixic acid, 18.3%). Resistance to tetracycline was significantly more common in isolates from adult goats than from other animals (p b 0.05). A significant association (p b 0.05) was detected between resistance and susceptibility to streptomycin and isolates from lambs and kids, respectively. Other significant differences in resistance per host were not observed for the remaining antibiotics. 4. Discussion Campylobacter was recovered from the ileum contents of both goats (15.4 and 30.0% for goats and kids, respectively) and sheep (34.0% and 38.8% for sheep and lambs, respectively) in this study. These results indicate that not only sheep but also goats are a potential reservoir for this foodborne pathogen. The detection of Campylobacter-positive goat ileum contents is interesting as no Campylobacter bacteria were recovered from goats at farm level in Norway, Portugal, and Spain (Rosef et al., 1983; Cabrita et al., 1992; Cortés et al., 2006). Regarding the isolation of campylobacters from sheep intestinal contents, the prevalence (Table 1) falls within the range of previously reported findings even though the rate of Campylobacter contamination in this kind of matrix varies from 6.8 to 91.7% (Raji et al., 2000; Milnes et al., 2008; Açik & Çetinkaya, 2006; Stanley et al., 1998; Zweifel et al., 2004; Cabrita et al., 1992). However, the isolation methodology could have contributed to the lower percentage of contamination in ileum contents in comparison to carcass surfaces (Table 1) since direct plating (ileum samples) has been associated with lower isolation rates than those obtained after enrichment (meat and liver surface samples) (Stanley et al., 1998; Açik & Çetinkaya, 2006; Milnes et al., 2008; Habib et al., 2011). The overall prevalence of Campylobacter contamination detected on the meat surfaces was more than 70%. This demonstrates a significant contamination at the primary stage of production. Other studies have reported lower contamination rates in carcasses of small ruminant origin (Madden et al., 1998; Dadi and Asrat 2008; Duffy et al., 2001). Although

3.3. Antimicrobial susceptibility profile The antimicrobial resistance patterns of the 240 examined Campylobacter isolates against six critically important antimicrobials are presented in Table 3. All isolates were susceptible to gentamicin. Seventy isolates (29.2%) were resistant to a single antimicrobial whereas multidrug resistance was observed in 6.3% of isolates. The highest Table 2 Distribution of Campylobacter isolates within the most common PFGE types. Distribution of Campylobacter isolates SmaI PFGE type

CJ-001a CC-013b CC-047b a b c

Sheep

Kid

Ileum

Lamb Meat

Liver

Meat

Ileum

Meat

Liver

Ileum

Meat

– 2 –

– 14 2

– 9 –

– – 3

10 1 –

2 9 7

4 5 8

– 1 –

1 1 –

PFGE type of the Campylobacter jejuni cohort. PFGE type of the Campylobacter coli cohort. Thessaloniki (Th), Chalkidiki (Ch), Rodopi (Ro), Serres (Se).

Goat

Sampling code

Farm code

Region code

N

13 6, 13, 24, 25 10, 16, 17

G A, B, G, O, P F, H, I, J

Thc Chc, Thc Roc, Sec, Thc

17 42 20

T. Lazou et al. / International Journal of Food Microbiology 173 (2014) 54–61 Table 3 Antimicrobial resistance profile of Campylobacter isolates (n = 240). Antimicrobial resistance profilea

C. jejuni (n = 74)

C. coli (n = 166)

Total (n = 240)

TE S TE + S CIP + NA CIP + NA + TE CIP + NA + TE + S CIP + E + NA + TE + S Total

20 – 1 7 3 – – 31

39 11 28 10 9 9 6 112

59 11 29 17 12 9 6 143

(27%) – (1,4%) (9,5%) (4,1%) – – (41,9%)

(23.5%) (6.6%) (16.9%) (6%) (5.4%) (5.4%) (3.6%) (67.5%)

(24.6%) (4.6%) (12.1%) (7.1%) (5%) (3.8%) (2.5%) (59.6%)

a CIP: ciprofloxacin; E: erythromycin; NA: nalidixic acid; TE: tetracycline; S: streptomycin. All tested isolates were susceptible to gentamicin.

Campylobacter has been recovered more frequently from offal than meat originating from the same carcass (Kramer et al., 2000; Alter et al., 2005; Little et al., 2008), we detected campylobacters on the majority of lamb and kid livers (on average, 70.8%) but yet less frequently (p N 0.05) than on carcass surfaces (Table 1). A high prevalence (72.9–78%) of Campylobacter spp. in sheep raw livers has been a common finding, confirming their putative role as a source for human infection (Kramer et al., 2000; Scates et al., 2003; Cornelius et al. 2005; Strachan et al., 2012). Our findings suggest that not only lamb but also kid livers can serve as vehicles for this pathogen. In the present study, C. jejuni dominated in the ileum content samples, a finding that is in agreement with those reported by other authors (Cabrita et al., 1992; Stanley et al., 1998; Zweifel et al., 2004; Devane et al., 2005; Oporto et al., 2007; Milnes et al., 2008), and C. coli was the dominant species on meat and liver surfaces. However, other researchers have reported the dominance of C. jejuni over C. coli in various samples originating from small ruminants (Whyte et al., 2004; Devane et al., 2005; Hussain et al., 2007; Wong et al., 2007; Dadi & Asrat, 2008). In this study, small ruminants and swine shared the same slaughter line and equipment post-dressing and the processing of swine carcasses normally preceded the processing of small ruminant carcasses. Therefore, the C. coli isolates detected on ovine and caprine carcasses could, to some extent, originate from swine (Cabrita et al., 1992; van Looveren et al., 2001; Oporto et al., 2007). On the other hand, C. coli was the most common isolated species in poultry in the Balkan region (Uzunović-Kamberović et al., 2007) and was more frequently detected in sheep than cattle at farm level in Spain and Scotland (Oporto et al., 2007; Rotariu et al., 2009). The higher Campylobacter-contamination rate on meat and liver surfaces compared to ileum contents could be attributed to weak slaughter hygiene during slaughter. Such practice involves spillage of faecal material during evisceration and subsequent cross-contamination across the slaughter line due to the persistence of campylobacters on the equipment and/or hands of the technicians, and/or close contact between consecutively processed carcasses. Indeed, the variable ‘carcass touching’ in the slaughter line proved to be significant for the presence of Campylobacter on sheep carcasses (Garcia et al., 2010). PFGE analysis indicated the occurrence of cross-contamination events at the abattoir. In eight out of the 11 slaughter groups, the presence of several shared pulsotypes between ileum contents and meat/liver surfaces is a strong indication that faecal contamination at slaughter was frequent. For example, the CJ-001 pulsotype was present in all samples (ileum contents, meat and liver surfaces) originating from animals reared in the same farm and slaughtered on the same day (Table 2). Additionally, the KpnI pulsotype, representing the eight C. jejuni isolates resistant to restriction by SmaI, indicated the carriage of a strain in the ileum content of one preceding kid to the meat and liver surfaces of two other kid carcasses following in the slaughter line (data not shown). On the other hand, the majority of slaughter groups yielded meat and liver surfaces harbouring additional pulsotypes not detected in the ileum content samples, indicating either a shortcoming of the isolation methodology

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applied or environmental contamination during slaughter. The potential survival of some campylobacter strains present in pre-existing biofilms in the abattoir environment, resistant to disinfection, has been suggested as a cause of overlapping PFGE patterns detected on carcasses (Trachoo and Frank, 2002). Further indications of surface contamination with Campylobacter from the abattoir environment, in our study, are provided by the fact that the two predominant pulsotypes, CC-013 and CC-047, encompassed more than 35% of the C. coli isolates even though there was a period of two (CC-047) to up to 10 (CC-013) months intervening between sampling the corresponding animals (Table 2). The simultaneous detection of both C. coli and C. jejuni isolates in one sample may constitute a finding of special concern regarding food safety. In particular, as Melero et al. (2012) emphasised, the virulence of Campylobacter strains may fluctuate and the possibility of virulent strains being present on food increases as the number of strains being concurrently present on them increases as well. Interestingly, coinfection of humans with mixed Campylobacter strains is a common finding in outbreaks of foodborne campylobacteriosis (Frost et al., 2002; Forbes et al., 2009). Given that in the current study only one presumptive Campylobacter colony from each selective agar medium was chosen for further analysis, the undetected presence of even more multiple-strain contaminated samples cannot be disregarded. Even so, the fact that PCR/PFLP-based identification revealed the concurrent presence of C. jejuni and C. coli in 2.1% (6/282) of the ileum contents, and the incidence of two different pulsotypes among the same Campylobacter species in 9% (6/64) of the ileum content samples are indicative of multiple Campylobacter colonisations of small ruminants at farm level. Another feature that could explain the heterogeneity of the closely-related Campylobacter pulsotypes present on individual carcasses is the unsteadiness of the Campylobacter genome, which verifies a widespread horizontal genetic exchange within the genus and Campylobacter as ‘naturally transformable’ (de Boer et al., 2002; Ge et al., 2006; Zorman et al., 2006; Uzunović-Kamberović et al., 2007; Denis et al., 2011; O'Leary et al., 2011; Melero et al., 2012). Ge et al. (2006) suggested that the unstable nature of the Campylobacter genome can be detected by the presence of single-band different pulsotypes. Our results could support the aforementioned hypothesis: closely-related single-band different pulsotypes in three different pairwise comparisons regarding C. coli isolates were detected on the same or on two different surface samples originating from the same carcass (CC-008/ CC-053, CC-013/CC-014, and CC-043/CC-044, see Supplementary data). In all these three pairwise comparisons, one PFGE pattern was always a ‘singe-isolate pulsotype’ (Fig. 1), supporting the possibility of horizontal genetic exchange between campylobacter bacteria. Public health concerns are related to antimicrobial resistance of pathogenic bacteria against critically important antimicrobials (WHO, 2009), as the ones applied in the current study. None of the tested Campylobacter isolates exhibited resistance to gentamicin and resistance to erythromycin was low. Our findings are rather comforting as erythromycin is a drug of choice for treating enteritis caused by Campylobacter whereas gentamicin has been recommended for the treatment of patients with systemic infection. These findings are similar to (Zweifel et al., 2004; de Jong et al., 2009) or lower than (Bostan et al., 2009) those reported by other studies. According to the latest EFSA report, among Campylobacter isolated from meat and animals, resistance to ciprofloxacin, nalidixic acid, and tetracycline was generally at levels from 21% to 84%, whilst much lower levels of resistance to erythromycin and gentamicin were reported (EFSA, 2012b). The profile of quinoloneresistant campylobacters was exhibited by 18.3% of isolates screened in this study and cross-resistance to nalidixic acid and ciprofloxacin was always observed. The increase in fluoroquinolone-resistant C. jejuni strains of human origin coincided with the introduction of enrofloxacin for veterinary use (van Looveren et al., 2001). Tetracycline is considered an alternative choice for campylobacteriosis treatment (Engberg et al., 2006). In the present study, approximately 48% of the isolates displayed resistance to tetracycline. Within the EU, differences in the occurrence

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of resistant campylobacters to tetracycline exist between Member States (EFSA, European Food Safety Authority and ECDC, European Centre for Disease Prevention and Control, 2012b). Nevertheless, the low frequency of multidrug resistance in Campylobacter (6.3%) observed in this study could be an alleviative finding in terms of public health considering the reported increasing trend in the multiple antibiotic resistance of campylobacters worldwide (Engberg et al., 2006). In conclusion, the majority of sheep and goats at slaughter carried Campylobacter, with multiple pulsotypes present on meat and liver. This indicates that meat and offal not only of sheep but also of goat origin are commonly contaminated with a diverse population of thermophilic campylobacters, and could serve as a potential vehicle for human infection with these food-related pathogens. Further research within the context of epidemiological studies comparing Campylobacter strains of small ruminant origin and human diarrheal cases is necessary in order to confirm the actual input of sheep and goat meat and offal to human campylobacteriosis. Acknowledgements The authors gratefully acknowledge Julie Baré, Sarah De Smet, Inge Van Damme, and Laid Douidah for their kind support during macrorestriction profiling by PFGE. Acknowledgements are also extended to Theofilos Papadopoulos for his valuable contribution during PFGE data processing. We thank Sandra Vangeenberghe and Carine Van Lancker for laboratory assistance. We also thank the administration and personnel of the abattoirs for participating in this study and for their valuable cooperation during sampling. Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.ijfoodmicro.2013.12.011. References Açik, M.N., Çetinkaya, B., 2006. Heterogeneity of Campylobacter jejuni and Campylobacter coli strains from healthy sheep. Vet. Microbiol. 115, 370–375. Allos, B.M., 2001. Campylobacter jejuni infections: update on emerging issues and trends. Clin. Infect. Dis. 32, 1201–1206. Alter, T., Gaull, F., Kasimir, S., Gurtler, M., Fehlhaber, K., 2005. Distribution and genetic characterization of porcine Campylobacter coli isolates. Berl. Munch. Tierarztl. Wochenschr. 118, 214–219. Bauer, A.W., Kirby, W.M.M., Sherris, J.C., Turk, M., 1966. Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol. 45, 493–496. Bostan, K., Aydin, A., Ang, M.K., 2009. Prevalence and antibiotic susceptibility of thermophilic Campylobacter species on beef, mutton, and chicken carcasses in Istanbul, Turkey. Microb. Drug Resist. 15 (2), 143–149. Cabrita, J., Rodrigues, J., Bragança, F., Morgado, C., Pires, I., Gonçalves, A.P., 1992. Prevalence, biotypes, plasmid profile and antimicrobial resistance of Campylobacter isolated from wild and domestic animals from northeast Portugal. J. Appl. Bacteriol. 73 (4), 279–285. CDC (Centers for Disease Control and Prevention), 2012. Foodborne Diseases Active Surveillance Network (FoodNet): FoodNet Surveillance Report for 2011 (Final Report). U.S. Department of Health and Human Services, Atlanta, Georgia. CLSI (Clinical and Laboratory Standards Institute), 2008. Performance standards for antimicrobial susceptibility testing. Eighteenth Informational Supplement. CLSI document. M100 -S18, Wayne, PA. Cornelius, A.J., Nicol, C., Hudson, J.A., 2005. Campylobacter spp. in New Zealand raw sheep liver and human campylobacteriosis cases. Int. J. Food Microbiol. 99, 99–105. Cortés, C., de la Fuente, R., Contreras, A., Sánchez, A., Corrales, J.C., Martínez, S., Orden, M.J., 2006. A survey of Salmonella spp. and Campylobacter spp. in dairy goat faeces and bulk tank milk in the Murcia region of Spain. Ir. Vet. J. 59 (7), 391–393. Dadi, L., Asrat, D., 2008. Prevalence and antimicrobial susceptibility profiles of thermotolerant Campylobacter strains in retail raw meat products in Ethiopia. Ethiop. J. Health Dev. 22 (2), 195–200. de Boer, P., Wagenaar, J.A., Archterberg, R.P., van Putten, P.M., Schouls, L.M., Duim, B., 2002. Generation of Campylobacter jejuni genetic diversity in vivo. Mol. Microbiol. 44 (2), 351–359. de Jong, A., Bywater, R., Butty, P., Deroover, E., Godinho, K., Klein, U., Marion, H., Simjee, S., Smets, K., Thomas, V., Vallé, M., Wheadon, A., 2009. A pan-European survey of antimicrobial susceptibility towards human-use antimicrobial drugs among zoonotic and commensal enteric bacteria isolated from healthy food-producing animals. J. Antimicrob. Chemother. 63 (4), 733–744.

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