Prevalence and antimicrobial profile of Campylobacter isolates from free-range and conventional farming chicken meat during a 6-year survey

Food Control 56 (2015) 161e168

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Prevalence and antimicrobial profile of Campylobacter isolates from free-range and conventional farming chicken meat during a 6-year survey Vangelis Economou a, b, Nikolaos Zisides a, Panagiota Gousia a, Stefanos Petsios a, Hercules Sakkas a, Nikolaos Soultos b, Chrissanthy Papadopoulou a, * a

Food-Water Microbiology Group, Department of Microbiology, Faculty of Medicine, School of Health Sciences, University of Ioannina, Ioannina 45110, Greece Department of Hygiene and Technology of Foods of Animals Origin, Faculty of Veterinary Medicine, School of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 September 2014 Received in revised form 27 February 2015 Accepted 7 March 2015 Available online 4 April 2015

The present study aimed to investigate the prevalence of Campylobacter spp. in the meat of free-range and conventional farming broilers and to assess the respective antimicrobial susceptibility of the isolates. Three hundred and sixty nine fresh chicken meat samples were collected from different farms at slaughter (60 from free-range poultry farms and 309 from conventional farms) in North Western Greece. Susceptibility tests against 12 antimicrobials (amikacin, gentamicin, imipenem, cefamandole, cefotaxime, cefoxitin, imipenem, erythromycin, ampicillin, amoxicillin e clavulanic acid, ciprofloxacin, tetracycline) were performed. Campylobacter spp. was isolated from 91 (29.4%) of the free-range chicken meat and from 106 (28.7%) of the conventional farming chicken meat samples. The annual incidence of Campylobacter spp. among the free-range and conventional broilers was not statistically significant. However, a reducing trend was observed in the overall prevalence of Campylobacter spp. from 2005 to 2010. Variable resistance rates were observed towards the tested antimicrobials: erythromycin (76%), tetracyclines (71%), ampicillin (66%), ciprofloxacin (51%), cefamendole (41%) cefoxitin (27%), amikacin (15%), and cefotaxime (5%). No differences were observed in the antimicrobial susceptibility of isolates from free-range and conventional farming samples. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Campylobacter Susceptibility Chicken Broilers Free-range Conventional farming

1. Introduction Campylobacter spp. is one of the major causes of bacterial gastroenteritis, inducing an acute selfelimiting diarrheic disease. Also, it is implicated in extra-intestinal infections that include bacteremia, reactive arthritis, hemolytic uremic syndrome, men€ lund-Karlsson, & ingitis, and septicemia (Foley, 2012; Ge, Wang, Sjo McDermott, 2013). Furthermore Campylobacter jejuni infection has been reported to be implicated to the onset of 30e40% of the  syndrome cases (McCarthy & Giesecke, 2001; Nyati GuillaineBarre & Nyati, 2013). Although Campylobacter is reported to be the most commonly isolated pathogenic agent of human diarrhea (45%), it

* Corresponding author. Department of Microbiology, Faculty of Medicine, School of Health Sciences, University of Ioannina, Ioannina 45110, Greece. Fax: þ30 2651007886. E-mail address: [email protected] (C. Papadopoulou). http://dx.doi.org/10.1016/j.foodcont.2015.03.022 0956-7135/© 2015 Elsevier Ltd. All rights reserved.

has attracted less attention compared to other enteric pathogens such as Salmonella, Shigella and STEC Escherichia coli (EFSA & ECDC, 2013). In the European Union Campylobacter is the most common cause of enteritis in humans, with 190,566 cases being reported in 2008 (Bahrndorff, Rangstrup-Christensen, Nordentoft, & Hald, 2013). The overall prevalence is supposed to be much higher since it is estimated that only 2.1% of all cases are reported. Therefore the true incidence of campylobacteriosis in the European Union is approximately 9 million cases per year, with the incidence differing among European countries (EFSA, 2011; Bahrndorff et al., 2013). The Campylobacter infection has been linked to the consumption of raw or undercooked poultry meat. Contaminated poultry meat is the cause of 20%e30% of all cases, with 50%e80% of them attributed to the chicken reservoir as a whole (EFSA, 2011; Bahrndorff et al., 2013). Broiler carcasses are usually contaminated during defeathering and evisceration by contaminated faeces leaking from the

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V. Economou et al. / Food Control 56 (2015) 161e168

cloaca or by visceral rupture of the caeca carrying a high Campylobacter load, from within-flock or from previously processed flocks (Allen et al., 2007; Ghareeb, Awad, Mohnl, € hm, 2013). Another issue of concern relating to Schatzmayr, & Bo the Campylobacter presence in the food chain is the emergence of resistance to certain antimicrobials such as macrolides (erythromycin, clarithromycin and azithromycin) and fluoroquinolones, which are commonly used in treating human infections (Ge et al., 2013). In the European Union report for 2012 (EFSA & ECDC, 2013) the C. jejuni isolates from broilers exhibit high resistance to ciprofloxacin (57.2%), nalidixic acid (55.5%) and tetracycline (40.6%), and low resistance to erythromycin (1.6%) and gentamicin (0.9%). Higher resistance was exhibited by the Campylobacter coli broiler isolates to ciprofloxacin (76.6%), nalidixic acid (70.2%) and tetracycline (74.6%), and moderate to low resistance to erythromycin (15.5%) and gentamicin (3.8%). During the recent years the observed consumer preference to foods produced with biological or environmental friendly procedures has increased the demand for free-range chicken meat and eggs (Castellini, Berri, Bihan-Duval, & Martino, 2008). According to Colles et al. (2008) biosecurity measures are supposed to reduce C. jejuni shedding rates of housed chickens. Nevertheless, broilers from free-range farms are reported to be more easily contaminated by a number of pathogens, including Campylobacter, because they are in close contact with a potentially tainted environment. Avrain et al. (2003) reported that the type of husbandry influenced the frequency of Campylobacter isolation, the C. jejuni/C. coli ratio and €ther, Alter, the antimicrobial resistance pattern. According to Na Martin, and Ellerbroek (2009) the risk of Campylobacter colonization in free range flocks was significantly higher. Desmonts, DufourGesber, Avrain, and Kempf (2004) reported that isolates collected from conventional flocks were often more resistant than isolates from free-range chickens. Still there is a controversy on the correlation between breeding practices and Campylobacter colonization. The present study aimed to investigate the prevalence of Campylobacter spp. in the meat of free-range and conventionally produced broilers and to assess the respective susceptibility of the isolates. 2. Materials and methods 2.1. Sample collection A total of 369 raw chicken meat samples (breast or thigh) were collected at slaughter during a six year period (2005e2010). The samples were collected throughout the year randomly from one slaughterhouse and consisted of 60 samples originating from six free-range poultry farms and 309 samples originating from 20 conventional poultry farms. The total number of samples per year

for each bird raising system is presented in Table 1. The birds from free range farms and conventional farms were processed in the same slaughterhouse but independently. The samples were collected from flocks slaughtered first in the processing day so as to prevent cross-contamination (Allen et al., 2007). A portion of meat with skin-on weighting approximately 50 g was collected with the use of sterile forceps and scalpel from the breast or thigh of the carcass. We sampled breast and thighs, instead of the typical -for the campylobacters-neck-skin samples, because breast and thighs are the most widely consumed parts, generally separately packetized and sold in the retail market (the breasts usually with no skin on). We have chosen this kind of sampling in order to be closer to the consumers' risk of contamination. The samples were transported to the laboratory in sterile containers into portable coolers and were processed within 4 h after collection. 2.2. Poultry farms and processing plant The poultry farms and the slaughterhouse belonged to a large vertically integrated company located at Northwestern Greece within a radius of approximately 100 km from the laboratory. It has to be underlined that almost half (45.74%) of the broiler population in Greece is being bred in this area according to the statistical data provided by the Hellenic Ministry of Rural Development and Food (HMRDF), 2010). The processing plant had an automated processing line passing through a stunner, neck cutter, bleed out, scalding tanks, a bank of disc plucking machines, eviscerator, vent opener, cropper, neck cracker and puller, and lung remover, followed by spiral chilling. The biosecurity measures routinely taken at the poultry farms were providing protection against poultry pathogens and foodborne pathogens such as Salmonella spp. as stated in the Greek Ministerial decision 133362. These involved a) control of movement of wild animals by fencing the whole perimeter of the poultry farm, b) an all in e all out system, c) prohibition of the access of domestic animals, d) cleaning of the manure and disinfection each time broilers were evacuated for culling, e) control of rodents and insects with the appropriate vermicide and insecticide programs, f) use of potable water, g) disinfection of every vehicle entering and leaving the poultry farm, and h) hygiene facilities, appropriate clothing and cleaning procedures for the personnel and the visitors entering or leaving the poultry farm. In average the population of the conventional farms was 17.800 broilers, whereas the average population of the free-range farms was 12.500 broilers. The broilers populating the poultry farms were Ross and Cobb broilers from Greek breeding flocks. The chickens were placed in the farm as day e old chicks. In the free range poultry farms, they had access to open air yards after the 28th day after hatching. Access to feed and water was unlimited, whereas the diet was certified as plant-based (mainly containing soya and corn)

Table 1 Incidence of Campylobacter spp. in poultry meat from free range and conventional broiler flocks (incidence in italics). Year

Samples (N) FG

2005 2006 2007 2008 2009 2010 Total

13 9 11 12 8 7 60

C. jejuni C

75 46 71 68 29 20 309

C. coli

FG

C

C. lari

FG

C

FG

n

(%)

n

(%)

n

(%)

n

(%)

n

3 2 2 1 0 0 8

(23.1) (22.2) (18.2) (8.3)

27 8 7 7 4 2 55

(36.0) (17.4) (9.9) (10.3) (13.8) (10.0) (17.8)

2 1 2 0 1 0 6

(15.4) (11.1) (18.2)

12 8 5 6 2 2 35

(16.0) (17.4) (7.0) (8.8) (6.9) (10.0) (11.3)

0 0 0 1 0 0 1

FG: meat samples from free range flocks. C: meat samples from conventional flocks.

(13.3)

(12.5) (10.0)

C (%)

n

(%)

(8.3)

0 0 0 0 0 1 1

(5.0) (0.3)

(1.7)

V. Economou et al. / Food Control 56 (2015) 161e168

for both free range and conventional broilers. No antibiotics were used as growth promoters, since there is a ban on the use of antibiotics as growth promoters issued from January 2006. The litter used was wood shavings, chopped straw or rice hulls. The broilers were slaughtered at age 44e48 days for conventional broilers and 68e72 days for free range broilers.

163

2.5. Statistical analysis The chi square test was utilized to assess the prevalence difference between samples from free-range and conventional broilers. 3. Results

2.3. Sample examination ®

For the detection of Campylobacter the mini VIDAS automated rieux S.A., France) was employed. immunoassay analyzer (BioMe The detection was performed with the use of the enzyme linked rieux S.A., France) acfluorescent assay kit VIDAS® CAM (BioMe cording to the instructions of the manufacturer. In brief, 25 g of meat were aseptically removed and suspended in 225 ml Bolton broth (Oxoid, UK). The emulsion was homogenized in a stomacher (Вag Mixer-Interscience) for 1.5 min. 25 ml of each homogenized sample were incubated under microaerophilic conditions (5% O2, 10% CO2, kai 85% N2) at 42  C for 48 h under microaerophilic conditions (GENbag Microaer e 45532, Biomerieux, France). Following incubation, 2 ml were heated in test tubes in a water bath at the temperature of 95e100  C for 15 min. A portion of 500 ml was placed in a VIDAS® CAM strip and examined in the mini VIDAS® automated immunoassay analyzer. For the Campylobacter isolation 10 ml from the incubated Bolton broth suspension prior to heating was inoculated onto CampyFood rieux S.A., France). The plates were incubated at agar plates (BioMe 42  C for 24e48 h under microaerophilic conditions (GENbag Microaer e 45532, Biomerieux, France). The suspected colonies were pure cultured onto Columbia blood agar plates with 5% lysed rieux S.A., France) and identified to the species horse blood, (BioMe level according to their morphology, catalase and oxidase reaction rieux S.A., and with the use of the АPI Campy system (BioMe France). 2.4. Antimicrobial susceptibility Antimicrobial susceptibility testing was performed for all the strains isolated from 2007 to 2010 (23 C. jejuni isolates and 18 C. coli isolates). The examination was performed by the KirbyeBauer disk diffusion method as described by the Clinical Laboratory Standard Institute (CLSI, 2013). Pure cultures of C. jejuni and C. coli were grown overnight in Mueller-Hinton broth (Oxoid, Basingstoke, UK) at 42  C under microaerophilic conditions. Muller-Hinton 2 agar plates with 5% lysed horse blood were inoculated using sterile cotton swabs and standard antimicrobial susceptibility test discs [aminoglycosides: amikacin (AK, 30 mg/disc), gentamicin (CN, 10 mg/disc); cephalosporins: cefamandole (МА, 30 mg/disc), cefotaxime (CTX, 30 mg/disc), cefoxitin (FOX, 30 mg/disc); carbapenems: imipenem (IPM, 10 mg/disc); macrolides: erythromycin (E, 15 mg/ disc); penicillins: ampicillin (AMP, 10 mg/disc), amoxicillin e clavulanic acid (AMC, 20e10 mg/disc); quinolones: ciprofloxacin (CIP, 5 mg/disc); tetracyclines: tetracycline (TE, 30 mg/disc)] were applied onto the surface of the plate using a disc dispenser. The plate was inverted and incubated at 42  C for 24 h under microaerophilic conditions. The diameter of the growth inhibition zones was measured with an electronic caliper and interpreted as resistant or susceptible, according to EUCAST breakpoints concerning ciprofloxacin, erythromycin and tetracycline (EUCAST, 2014) and the breakpoints of CLSI for Enterobacteriaceae concerning ampicillin, amikacin, amoxicillin e clavulanic acid, cefamandole, cefotaxime, cefoxitin, gentamicin, and imipenem (CLSI, 2013). The characterization of the antimicrobial resistance levels was performed according to EFSA/ECDC (2012b).

The results concerning the incidence of Campylobacter spp. in chicken meat are presented in Table 1 and in Fig. 1. During 2007 a marked decrease of the annual incidence of Campylobacter spp. is observed in the total number of samples obtained from both raising systems and from then onwards the incidence remains stable. C. jejuni was isolated from 13.3% of the meat samples from freerange broilers and from 17.8% of the meat samples from conventional broilers. C. coli was isolated from 10.0% of the free-range broilers and from 11.3% of the conventional broilers. C. lari was isolated twice, with one isolate obtained from free-range broilers (1.7%) and one from conventional broilers (0.3%). In total, Campylobacter was isolated in 25.0% of the samples from free-range broilers and in 29.4% of the samples from conventional broilers. The difference of the annual incidence of Campylobacter spp. between free-range and conventional broilers was not statistically significant (p > 0.05). A reducing trend was observed concerning the incidence of Campylobacter spp. from 2005 to 2010. This observation was particularly evident for C. jejuni, where the total annual incidence decreased from 34.1 % to 7.4 % during the six years of this study. A less marked decrease was observed for C. coli decreasing from 15.9 % to 7.4 %, and for the total annual incidence decreasing from 50.0 % to 18.5 %. Concerning the antimicrobial susceptibility of the tested isolates the resistance profiles and the occurrence rates are shown in Table 2 and Table 3 respectively. No different trends were observed in the antimicrobial susceptibility of isolates from free-range and conventional farming samples. The resistance profiles demonstrate the existence of multiresistant isolates of C. jejuni (31.7%) and C. coli (29.3%). 4. Discussion In the present study the prevalence and antimicrobial resistance of Campylobacter spp. in free-range and conventional farming chicken was investigated. The prevalence of Campylobacter isolation was in average 28.73% and ranged from 18.51% to 50%. According to EFSA and ECDC (EFSA/ECDC, 2007; EFSA/ECDC, 2010; EFSA/ECDC, 2011a; EFSA/ECDC, 2012a; EFSA/ECDC, 2013) (Table 4) the average prevalence of Campylobacter spp. in broiler meat in the

Fig. 1. Prevalence of Campylobacter spp. in poultry meat from free range, conventional and total broiler flocks.

164

V. Economou et al. / Food Control 56 (2015) 161e168

birds coming first in the processing line from either system were sampled in order to avoid contamination problems at slaughter (Allen et al., 2007), it may be assumed that the raising system potentially influenced the incidence. Similar studies from other countries have yielded variable results. Nobile, Costantino, Bianco, Pileggi, and Pavia (2013) examined 113 chicken meat samples at retail from Italy and found a Campylobacter spp. prevalence rate of 18.6%. Hansson et al. (2007) reported that the annual incidence of Campylobacter-positive slaughter batches in Sweden between 2002 and 2005 ranged from to 6%e9%, an incidence rate much lower than that in this study, probably attributed to the different climatic conditions. Ghafir, China, Dierick, de Zutter, and Daube (2007) reported that in broiler samples taken during 2000 and 2003 in Belgium, 30.9% (28.2%e33.7%) of broiler carcasses were positive for Campylobacter spp. In a latter study in Belgium, Habib et al. (2012) sampled 389 broiler carcasses during 2008 and found the prevalence of Campylobacter spp. to be 51.9%. In this study the most commonly isolated Campylobacter species was C. jejuni (59.43%) followed by C. coli (38.68%) and C. lari (1.89%). This observation is in accordance with the results reported by Нue et al. (2011) who have found that among Campylobacter spp. isolated from broiler meat from France, the most frequent was C. jejuni (57.1%), followed by C. coli (42.5%). A similar observation was reported by Petridou and Zdragas (2009), who out of 44 Campylobacter spp. isolates from chicken samples at retail shops in Greece, identified 39 as C. jejuni (88.7%) and five as C. coli (11.4%). In contrast, Lazou, Dovas, Houf, Soultos, and Iossifidou (2014); Lazou, Houf, Soultos, Dovas, and Iossifidou (2014) report that among Campylobacter spp. isolated form sheep and goat carcasses, an increased occurrence of C. coli (76.2%) was observed compared to C. jejuni (21.4%), an observation attributed to a possible cross contamination with the swine slaughtering line in the abattoirs sampled. This finding though cannot be directly related to the results of this study since reports from birds and swine grown at close proximity do not necessarily imply larger infestation by C. coli (Wright et al., 2008). Analogous results to the ones observed in this study have been reported from other researchers elsewhere. Moran, Scates, and Madden (2009) reported that among 652 samples of broiler meat collected at retail level, the identified Campylobacter isolates were C. jejuni (64.6%), C. coli (27.4%) and C. lari (1%). Deckert et al. (2010) reported that the majority of isolated Campylobacter from 1256 broiler meat samples from Ontario, Canada, were C. jejuni (90%), followed by a small percentage of C. coli (9%) and C. lari (1%). In the European Union in the years 2008e2010, C. jejuni was detected from the majority of the positive

Table 2 Antimicrobial resistance profile of the investigated Campylobacter isolates.

No resistance a AMP E TE þ E TE þ E þ AMP TE þ E þ CIP þ TE þ E þ CIP þ TE þ E þ CIP þ TE þ E þ CIP þ TE þ E þ CIP þ a

AMP MA þ AMP CT þ FOX þ MA þ AK þ AMP FOX þ MA þ AK þ AMP FOX þ MA þ AMP

C. jejuni (n ¼ 23)

C. coli (n ¼ 18)

4 2 2 2 2 4 2 1 3 1

4

(22.%)2

2 2 1 3

(11.1%) (11.1%) (5.6%) (16.7%)

2 4

(11.1%) (22.2%)

(17.4%) (8.7%) (8.7%) (8.7%) (8.7%) (17.4%) (8.7%) (4.3%) (13.0%) (4.3%)

No resistance exhibited to the antibiotics tested.

European Union between 2005 and 2010 was 29.6% which was rather stable during these years (standard deviation ¼ 1.8%). Therefore the prevalence reported in this study is quite close to the average prevalence of the European Union. Nevertheless, when comparing the reported prevalence of Campylobacter spp. among the Mediterranean countries (Spain, Italy, France, Greece e Table 4) during these years, the results of the present study are considerably lower. Regarding previous studies in Greece, Petridou and Zdragas (2009) examined samples from 60 retail meat places and found 73.3% of the samples positive for Campylobacter spp.. Marinou et al. (2012) reported that among 150 carcass samples and 830 poultry farm samples examined, only 16 strains were isolated with none of them found in carcass samples. Regarding Campylobacter spp. occurrence in Greece, Lazou, Dovas, Houf, Soultos, and Iossifidou (2014); Lazou, Houf, Soultos, Dovas, and Iossifidou (2014) report that the contamination rate of sheep and goat carcasses between 2007 and 2009 was 69.41% and 76.88%, indicating increased occurrence of Campylobacter spp.. According to the results presented in Fig. 1, in 2007 a marked decrease of the annual incidence of Campylobacter spp., is observed in the total number of birds examined and from then onwards the incidence remains stable. However, when assessing separately the results from the two bird-raising systems a different trend is observed; in 2007 the Campylobacter spp. incidence is at decrease in the conventional bird samples but at increase in the free-range bird samples. Whether the difference in the incidence trends was due to biosecurity at the farm or HACCP implementation at the slaughterhouse level cannot be determined since no caecal samples were collected to verify infection rates at the farm level. Nevertheless, considering that at each visit at the slaughterhouse, only

Table 3 Antimicrobial resistance rates of Campylobacter spp. isolated and comparison with results reported by selected publications (number of resistant isolates in bold, percentage in parentheses in italics).

Present study

European Union 2010 (EFSA & ECDC, 2012b) European Union 2009 (EFSA & ECDC, 2011b) European Union 2008 (EFSA, 2010) Bostan et al. (2009) Marinou et al. (2012) Nobile et al. (2013)

C. jejuni C. coli Total C. jejuni C. coli b C. jejuni C. coli b C. jejuni Total Totalb Total

n

AKa

CNa

CTXa

FOXa

MAa

IPMa

Ea

AMCa

AMPa

CIPa

TEa

23 18 41

3 (13) 3 (17) 6 (15)

0 0 0 (0.7) (0) (6) (7) (13) 51 (27) 2 (12) 4 (19)

2 (9) 0 2 (5)

6 (26) 5 (28) 11 (27)

7 (30) 10 (56) 17 (41)

0 0 0

17 (74) 14 (78) 31 (76) (2) (12) (3) (13) (6) 104 (55) 13 (81) 12 (37)

0 0 0

15 (65) 12 (67) 27 (66)

0 8 (38)

13 (87) 13 (62)

11 (48) 10 (56) 21 (51) (50) (72) (44) (67) (38) 88 (46) 0 8 (38)

15 (65) 14 (78) 29 (71) (22) (62) (40) (46) (50) 125 (66) 0 18 (86)

b

c

b

c

b

c

c

c

190 16 21

0

a Aminoglycosides: AK:amikacin, CN:gentamicin. Cephalosporins: CTX:cefotaxime, FOX:cefoxitin, МА:cefamandole. Carbapenems: IPM: imipenem. Macrolides: E: erythromycin. Penicillins: AMC: amoxicillin e clavulanic acid, AMP: ampicillin. Quinolones: CIP: ciprofloxacin. TE: tetracycline. b Samples examined by a minimum inhibitory concentration method. c The number of samples examined varied between different antibiotics.

V. Economou et al. / Food Control 56 (2015) 161e168

165

Table 4 Prevalence rates of Campylobacter spp. during 2005e2010 in the Mediterranean countries according to reports by EFSA and ECDC (2007), EFSA and ECDC (2010), EFSA and ECDC (2011)a; EFSA and ECDC (2012)a; EFSA and ECDC (2013) (positive samples in bold, prevalence rates in italics). 2010

EU Mediterranean countries

Italy

Spain

France

Greece

Total Slaughter Plants Retail NS Total Slaughter Plants NS Total Slaughter Plants Retail Total Slaughter Retail Total Slaughter Total

AV

MIN

MAX

DEV

n

%

n

%

n

%

n

%

n

%

n

%

%

%

%

%

7413 169 178 126

29.6 35.7 74.7 25.4

6142 420 50 165 92 727

30.1 86.2 58. 13.3 5.4 28.9

26 71.2 20.5 30.8

7826

30.4

12,730

30.5

37.5 26.7

31 83 70.7 62.9 8.5 56.7

7598 339 491 208

473 30

7312 119 99 634 167 1019

1038

45.6

424 424

19.8 19.8

323

11.8

8.4 8.5 95.8 70.7 49.5 72

92 92 420 50 165 635

5.3 5.4 86.2 58 13.3 52.5

323 147 168 208 523 192

11.8 55.8 29.2 30.8 38.6 86.5

424 424

19.8 19.8

361 361 47 47

76.2 76.2 70.2 70.2

192

86.5

26 35.7 20.5 13.3 5.4 19.8 26.7 11.8 5.4 5.4 44.6 29.2 13.3 38.6 86.5 76.2 76.2 70.2 70.2

31 86.2 74.7 62.9 19.8 56.7 26.7 11.8 19.8 26.7 95.8 74.7 49.5 72 86.5 76.2 86.5 70.2 70.2

1.8 23.2 24.7 21.1 7.6 14.3

167 167 72 99 273 444

29.6 69 5 33.1 11.2 37.7 26.7 11.8 11.2 14.4 70.6 58.2 29.8 52.8 86.5 76.2 81.4 70.2 70.2

30 139 178 126 443

2009

26.7 44.6 74.7 25.4 48.2

2008

2007

2006

0

2005

7.6 8.7 24.3 20.6 15.1 14

7.3

NS: single. AV: average. MIN: minimum value. MAX: maximum value. DEV: standard deviation.

for Campylobacter spp. broiler meat samples (34.2e41.2%), followed by C. coli (19.7%e27.3%) and C. lari (0.1e0.2 %) (EFSA & ECDC, 2011a; EFSA & ECDC, 2012a; EFSA & ECDC, 2013). It is interesting though that the unspecified Campylobacter spp. comprised a large percentage (35.4e46.9%) of the overall Campylobacter spp. reported. However, there a couple of studies reporting C. coli to be the prevailing Campylobacter species. In the study by Marinou et al. (2012) the most frequently isolated species was C. coli (87.5%), while C. jejuni accounted for 12.5% of their isolates. Also, Nobile et al. (2013) reported the most frequent isolates from chicken meat samples at retail level in Italy to be C. coli (42.9%), C. sputorum fecalis (23.8%), and C. jejuni (19%). In this survey a decrease in the overall incidence of Campylobacter spp. has been observed throughout the period of study (2005e2010). A similar observation was made by Hansson et al. (2007) reporting the annual incidence of Campylobacter-positive slaughter batches in Sweden to be progressively decreasing from 9% in 2001 to 6% in 2005 after the implementation of a Campylobacter monitoring program. During the period of our study the legislation concerning Salmonella control in broilers has come into force with the Commission Regulation (EC) No 646/2007 implementing Regulation (EC) No 2160/2003. Salmonella presence was not studied in this paper; however it is assumed that the control measures taken for the Salmonella eradication in the flocks might have contributed to the overall decrease of Campylobacter incidence, due to the application of stricter hygiene rules and official inspections. Furthermore, Salmonella and Campylobacter share some common characteristics concerning transmission and contamination of chicken meat. Nevertheless, there is a controversy concerning the correlation between Salmonella spp. and Campylobacter presence in broiler carcasses, with Hue et al. (2011) reporting that no correlation exists, while Rasschaert, Houf, van Hende, and de Zutter (2007) and Franz, van der Fels-Klerx, Thissen, and van Asselt (2012) reporting the opposite. This controversy is more pronounced since Smulders et al. (2011) report that among flocks tested positive for Salmonella spp. and Campylobacter spp. presence, the percentage of individual carcasses tested positive after

slaughter varied from 84% to 100% for Campylobacter spp. and only 11.1%e24.2% for Salmonella spp.. Still it is acknowledged that the stricter biosecurity measures in the farm level are the most efficient measure to reduce Campylobacter spp. contamination (Franz et al., 2012). Habib et al. (2012) reported that after the examination of nine slaughterhouses in Belgium, the ranking based on slaughterhouses inspection scores was statistically correlated with their ranking based on the incidence of Campylobacter spp. Campylobacteriosis is not a reportable disease in Greece, the available epidemiological data are very limited (HCDCP, 2014) and no surveillance program is recorded in ECDC reports (EFSA/ECDC, 2013). Published studies on human foodborne, waterborne or sporadic Campylobacter infections in Greece are scarce. In a study by Gkogka, Reij, Havelaar, Zwietering, and Gorris (2011) it is reported that during 1996e2006 it is estimated that the incidence of total foodborne Campylobacter illnesses per million inhabitants in Greece was 3571 illnesses, while the estimated reported illnesses were only 13, indicating that campylobacteriosis is an underreported foodborne disease in Greece; also the same authors ranked campylobacteriosis fifth among the top five causes of foodborne illness in Greece. According to recent data available through the Hellenic Center for Disease Control and Prevention System on Voluntary Reporting, (HCDCP, 2014), the Campylobacter human isolates for the year 2013 were 632, which is almost double of the obligatory reported Salmonella isolates (326), demonstrating the true presence of Campylobacter and ranking the first foodborne infection in Greece. In 2011 and 2012, 676 and 623 stool samples were tested positive for Campylobacter in 28 and 37 Greek hospitals respectively (HCDCP, 2014), whereas between 2000 and 2009, the average number of positive stool samples voluntary reported by less than 15 hospital laboratories were 285 (Mellou & Velonakis, 2011). Іn a study of Campylobacter jejuni infections in children in Attica, Greece, between 2004 and 2006, chicken meat consumption was recognized as the most important risk factor (Mellou, Sourtzi, Tsakris, Saroglou, & Velonakis, 2010). There is only one recorded outbreak, reported in Chania, Crete, involving 60 cases between May 1-June 24, 2009, being attributed to contaminated tap water,

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although no Campylobacter was isolated from the drinking water (Karagiannis et al., 2010). From the available epidemiological data on human foodborne campylobacteriosis, and the data reported in our study and in other Greek studies relating to the incidence of campylobacter in productive animals and food samples it is well justified that Campylobacteriosis is present in Greece, but it is an extensively under-reported foodborne infection (Gousia, Economou, Sakkas, Leveidiotou, & Papadopoulou, 2011; Lazou, Dovas, Houf, Soultos, & Iossifidou, 2014; Lazou, Houf, Soultos, Dovas, & Iossifidou, 2014; Marinou et al., 2012; Petridou & Zdragas, 2009). According to the results of this study, no significant difference was observed between the annual incidence of both C. jejuni and C. coli between free-range and conventional broilers. There is an ongoing controversial discussion concerning the role of the freerange environment to Campylobacter spp. transmission and particularly of the possible contamination of free-range broilers by wild or migrating birds. Newell and Fearnley (2003) and Sopwith et al. (2003) state that wild birds are an important source of infection for housed birds. On the other hand, Colles et al. (2008) report that no evidence of contamination was found after studying C. jejuni populations from chickens and wild birds on the same farm. Still, Rivoal, Ragimbeau, Salvat, Colin, and Ermel (2005) report that chickens stayed Campylobacter e free until they had access to the open area and that they were rapidly colonized by the Campylobacter strains isolated from the soil, highlighting its possible involvement in Campylobacter spp. transmission to chickens. Also Miraglia et al. (2007) report that the rearing system did not influence the prevalence of the Campylobacter spp., partly due to the length of the productive cycle which increased the animal exposure time to the various contamination sources. Concerning the prevalence of Campylobacter spp. in free-range farms, in a study carried out in Denmark, the prevalence in organic free-range flocks was 100.0% compared to 36.7% in conventional flocks (Heuer, Pedersen, Andersen, & Madsen, 2001). Esteban, Oporto, Aduriz, Juste, and Hurtado (2008) report a prevalence of 70.6% in broilers from organic farms of the Basque country, which is higher than the findings of this study, while they state C. coli to be more prevalent than C. jejuni. Rosenquist, Boysen, Krogh, Jensen, and Nauta (2013) report that organic broiler carcasses were more frequently contaminated with thermotolerant Campylobacter spp. than conventional broilers with the yearly mean prevalence being 54.2% for the organic and 19.7% for the conventional broilers. In the present study the observed incidence for conventional broiler meat was similar to the findings reported by Rosenquist et al. (2013), but the incidence in free-range broiler meat was lower. It is likely that the lower incidence observed in this study, is due to the common husbandry practices applied in both bird raising systems, and the common slaughtering process, the only difference being in the housing conditions. However, since we have not included a parallel sampling of caeca, which can indentify infection rates at the farm level reliably, the role of the different raising system can be assumed but not robustly proved, Hence, the gain of this study is that in overall a decrease of Campylobacter spp. incidence was recorded, which is of significant public health concern meaning reduced risk for the consumers. Diverse resistance rates are recorded in the available relevant literature, thus making hard to compare the findings from this and other studies (Table 3). No resistance to gentamycin was observed in our study and low to moderate resistance ranging from 0 to 27% was observed in other studies (Bostan, Aydin, & Ang, 2009; EFSA, 2010; EFSA & ECDC, 2011b; EFSA & ECDC, 2012b; Nobile et al. 2013), including one study from Greece where low resistance (12%) is reported as well (Marinou et al., 2012).

Macrolide (erythromycin) resistance rate reported in this and other studies (Bostan et al., 2009; Marinou et al., 2012; Nobile et al., 2013) was very high ranging from 55 to 81%, in contrast with the average EU rate (2e13%) reported by EFSA (2010), EFSA and ECDC (2011)b, EFSA and ECDC (2012)b. It has to be underlined that our resistance rates (74e78%) are similar to those (81%) reported by another Greek study for erythromycin (Marinou et al., 2012). Concerning penicillins, resistance to ampicillin was very high (62e87%) in our study and in another Greek study (Marinou et al., 2012) as well as in the study by Nobile et al. (2013). However, no resistance to the amoxicillin e clavulanic acid combination was observed in this study and another Greek study by Marinou et al. (2012), while 38% resistance rate is reported elsewhere (Nobile et al. 2013). The resistance rate to ciprofloxacin in this study and other studies was high to very high ranging from 38% to 72% (EFSA, 2010; EFSA & ECDC, 2011b; EFSA & ECDC, 2012b; Bostan et al., 2009; Nobile et al., 2013) except to one earlier Greek study where all strains were susceptible (Marinou et al., 2012), most likely due to the small number of isolates tested. Also, the tetracycline resistance rate reported in this and other studies (Bostan et al., 2009; Nobile et al., 2013) was very high (65%e86%), while the EU rate reported by EFSA is ranging from 22 to 62% (EFSA, 2010; EFSA & ECDC, 2011b; EFSA & ECDC, 2012b); however in another Greek study (Marinou et al. 2012) all strains were found susceptible. Resistance to cephalosporins is not reported by other researchers but it is recorded in the present study ranging from low (cefotaxime 5%) to moderate (cefoxitin 27%) to high (cefamandole 41%). As well, susceptibility data about imipenem are not reported by other researchers but in this study. Summarizing the above findings it is noted that in overall, high resistance rates are reported by this and other studies in relation to ampicillin, tetracycline, ciprofloxacin and erythromycin, which are commonly prescribed antibiotics in both medical and veterinary practice. The observed diversity of findings in Campylobacter resistance, can be attributed most likely to the different methodological approaches relating to the sample numbers, sample type, sample collection, breeding and slaughtering processes, geographic limitations, different antibiotics tested by the various researchers; for example in our study we tested for 12 antibiotics routinely used in the medical practice, while the majority of the researchers use a selection of less than six antibiotics. Due to these inconsistencies, it is difficult to compare the existing findings in a reliable and effective way. However, we believe that every result relating to antibiotic resistance whether it is for Campylobacter or other foodborne pathogen, is worth reporting and recording, because it provides information about the emergence and distribution of resistance, which is currently of foremost public health importance, particularly under the onehealth initiative.

5. Conclusions The most frequently isolated Campylobacter species were C. jejuni and C. coli. Contrary to the widespread belief of free-range poultry being more contaminated with pathogens compared to conventional farming poultry, in the present survey no such evidence was found for Campylobacter spp. The decreased incidence of Campylobacter spp. from 2005 to 2010 is most likely due to the increased biosecurity measures applied, although more research is needed towards this area. Conclusively, the exhibition of marked resistances of the Campylobacter spp. isolates calls for advanced vigilance concerning the prudent use of antimicrobials in the foodproducing animals.

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