Comparative Immunology, Microbiology and Infectious Diseases 39 (2015) 47–52
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Comparative Immunology, Microbiology and Infectious Diseases journal homepage: www.elsevier.com/locate/cimid
Higher resistance of Campylobacter coli compared to Campylobacter jejuni at chicken slaughterhouse Alicia Torralbo a , Carmen Borge a , Ignacio García-Bocanegra a , Guillaume Méric b , Anselmo Perea a , Alfonso Carbonero a,∗ a b
Department of Animal Health, Campus de Excelencia Internacional Agroalimentario ceiA3, University of Cordoba, Cordoba, Spain College of Medicine, Swansea University, Swansea SA2 8PP, United Kingdom
a r t i c l e
i n f o
Article history: Received 5 April 2014 Received in revised form 21 February 2015 Accepted 26 February 2015 Keywords: Broiler Campylobacter Chicken meat MIC Slaughterhouse Spain
a b s t r a c t In order to compare the prevalence of Campylobacter coli and Campylobacter jejuni during the processing of broilers at slaughterhouse a total of 848 samples were analyzed during 2012 in southern Spain. Four hundred and seventy six samples were collected from cloaca, carcass surfaces and quartered carcasses. Moreover, 372 environmental swabs from equipment and scalding water were collected. Minimum inhibitory concentration (MIC) to ciprofloxacin, erythromycin, streptomycin, tetracycline and gentamicin was determined for isolates from chicken meat. The general prevalence of Campylobacter was 68.8% (40.2% of C. coli and 28.5% of C. jejuni). The relative prevalence of C. coli increased from loading dock area (41.5%) to packing area (64.6%). In contrast, the relative prevalence of C. jejuni decreased from 58.5% to 35.4%. These differences between species from initial to final area were significant (p = 0.02). The highest antimicrobial resistance for C. jejuni and C. coli was detected to tetracycline (100%) and ciprofloxacin (100%), respectively. Campylobacter coli showed an antimicrobial resistance significantly higher than C. jejuni to streptomycin (p = 0.002) and erythromycin (p < 0.0001). © 2015 Elsevier Ltd. All rights reserved.
1. Introduction With 220,209 human cases in the European Union, campylobacteriosis was the most commonly reported zoonosis in 2011 [1]. Undercooked poultry meat is the main source of campylobacteriosis for humans [2]. Furthermore, cross contamination from raw chicken meat through knives, cutting board or hands has been reported as a major risk factor [3]. Even though different authors have described the prevalence of Campylobacter in retail products [4,5], few studies include the entire production chain as far as retail [6].
∗ Corresponding author. Tel.: +34 650952630; fax: +34 957218725. E-mail address: [email protected] (A. Carbonero). http://dx.doi.org/10.1016/j.cimid.2015.02.003 0147-9571/© 2015 Elsevier Ltd. All rights reserved.
Campylobacter can be found at all steps along the poultry production chain [7]. The evisceration process, the contact with equipment and the scalding water containing Campylobacter can cause multiple cross-contaminations in broiler carcasses [8,9]. In contrast, it has been reported that the number of Campylobacter in broiler carcasses could be reduced but not eliminated from carcasses by scalding or chilling process [10]. The prevalence of Campylobacter in broiler carcasses in Europe was 75.8% [11]. About two thirds of the Campylobacter isolates from broiler carcasses were identified as Campylobacter jejuni, while one third was Campylobacter coli. However, C. coli was the most frequent species isolated in Spain, Italy and Bulgaria [11]. The calculation of the minimum inhibitory concentration (MIC) by agar plate dilution method is the technique
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recommended to determine the antibiotic susceptibility for Campylobacter species [12]. Antimicrobial resistance has been described in Campylobacter isolated from dressed chickens [13] or retail meat products [7]. Campylobacter species have been found to be resistant to macrolides, tetracyclines and fluroquinolones [14–16], which are the main antimicrobial used to treat severe cases in human [17]. In this study, our principal aim was to determine the prevalence of C. coli compared to C. jejuni during processing at slaughterhouse. In addition, the antibiotic susceptibility for isolates from chicken meat was evaluated. 2. Materials and methods 2.1. Study design and sampling The study was carried out along 2012 in a slaughterhouse located in southern Spain. About 60,000 chickens are slaughtered each day, with modern equipments to perform each process, as well as its own quartering room. The slaughterhouse was divided in 6 areas to collect the samples: loading dock, scalding (water temperature: 52 ◦ C), evisceration, classified (after air chilling for 2 h at 4 ◦ C), quartering and final meat product/packing (room temperature: 1 ◦ C). Swab samples were collected for 15 weeks in a row, one day each week, along the entire processing chain, from cloaca, carcass surfaces (breast and back area), quartered carcasses surfaces (breast, wing, leg and back) and slaughterhouse environment (equipment and scalding water). A total of 848 swab samples were obtained and analyzed: 476 were taken from broilers, carcass surfaces and quartered carcasses surfaces, and 372 from equipment and scalding water (Table 1). Samples were collected in all cases from the last flock slaughtered of the sampling day by systematic random sampling in each stage. Samples were collected using sterile swabs placed in tubes containing a transport medium (Amies, Eurotubo® ). Swabs were kept refrigerated until arrival at the laboratory and then processed within 24 h. 2.2. Isolation and identification of Campylobacter Every swab was streaked onto a plate with Campylobacter Blood-Free Selective Agar Base (Oxoid® CM0739) added with CCDA Selective Supplement (Oxoid® SR0155). After 48 h of incubation at 42 ◦ C in a CO2 -enriched atmosphere achieved by AnaeroGen sachets (Oxoid® ), 15–20 colonies of each plate which showed morphology compatible with Campylobacter were streaked onto blood agar and incubated under the same conditions as previously described. All selected isolates were confirmed by examination of colonial morphology, Gram staining, motility in dark field microscopy, oxidase and catalase testing. Afterward, DNA extraction from bacterial cultures for isolates phenotypically classified as Campylobacter was performed according to the method described by Sambrook and Russell [18]. Thus, bulk colonies for each isolate in blood agar plate were taken with a loopful of 10 l to carry out DNA extraction.
Subsequently, the QIAGEN® Multiplex PCR Kit was used for the molecular identification as previously described [19]. 2.3. Minimum inhibitory concentration (MIC) For determination of the MIC, the agar dilution method was used following the protocol described by the European Committee for Antimicrobial Susceptibility Testing [20]. The cut off values used for the interpretation of the MIC results are developed by EUCAST [21]. The following antimicrobial were tested: ciprofloxacin, erythromycin, streptomycin, tetracycline and gentamicin (Sigma–Aldrich® ). Eighteen double dilutions, with antimicrobial concentration obtained from 10,240 mg/L to 0.0781 mg/L, were performed. Due to restraint budget, only a total of 60 randomly selected isolates (30 C. coli and 30 C. jejuni) from chicken meat were studied. The reference strains C. coli DSMZ 4689T and C. jejuni ATCC 33560 were used as positive controls. Plates were incubated under microaerobic atmosphere at 42 ◦ C. MIC values were read after 24 h of incubation. 2.4. Statistical analysis Prevalence of Campylobacter species in each area as well as frequencies of susceptible isolates and comparison of susceptibility between Campylobacter species was calculated using SPSS v15.0 software (SPSS Inc., Chicago, IL, USA). Chi squared test was used to evaluate frequencies of Campylobacter species in different areas during the processing and differences in antimicrobial susceptibility between C. coli and C. jejuni. 3. Results and discussion 3.1. Changes in C. coli and C. jejuni prevalence during processing in the slaughterhouse The general prevalence of Campylobacter was 68.8% (583/848), 40.2% (341/848) of C. coli and 28.5% (242/848) of C. jejuni. Campylobacter was isolated in all processing areas, agreeing with results reported by other authors in Spain [7] and other European countries [6]. The highest prevalence of Campylobacter was found in the evisceration area (92.8%) (Table 1). According to Allen et al. [9] and Frederick and Huda [22], this area is a critical control point where is required to maximize the cleaning and disinfection process. Moreover, to avoid the accidental breaking of gastrointestinal tracts and, in consequence, to decrease the contamination of carcasses, it would be necessary that the evisceration equipment is adapted to different carcass sizes [10]. However, it has been described that the most promising control strategy is to keep colonized and non-colonized flocks separate during slaughter. Furthermore, to reduce cases of campylobacteriosis in humans, meat from Campylobacter-positive flocks could be treated by freezing or chemical decontamination [23]. Most of the studies about the genus Campylobacter in broiler slaughterhouses were focussed on C. jejuni species
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Table 1 Number of samples in each area, relative prevalence of C. jejuni and C. coli isolates, and p-value. Area Loading dock Scalding
No. total samples 96 88
Evisceration
153
Classified
117
Quartering
311
Final meat product/packing a b c d e
83
No. specific samples Cloaca Whole carcasses Scalding water Total Whole carcasses Equipment Total Whole carcasses Equipment Total Quartered carcasses Equipment Total Quartered carcasses
96 39 49 88 67 86 153 46 71 117 145 166 311 83
Campylobacter No. (%)a
C. coli No. (%)b
C. jejuni No. (%)b
53 (55.2%) 26 (66.7%) 28 (57.1%) 54 (61.4%) 62 (92.5%) 80 (93.0%) 142 (92.8%) 30 (65.2%) 43 (60.6%) 73 (62.4%) 113 (77.9%) 100 (60.2%) 214 (68.5%) 48 (57.8%)
22 (41.5%) 16 (61.5%) 16 (57.1%) 32 (59.3%) 36 (58.1%) 45 (56.3%) 81 (57.0%) 13 (43.3%) 29 (67.4%) 42 (57.5%) 64 (56.6%) 69 (69.0%) 133 (62.4%) 31 (64.6%)
31 (58.5%) 10 (38.5%) 12 (42.9%) 22 (40.7%) 26 (41.9%) 35 (43.7%) 61 (43.0%) 17 (56.7%) 14 (32.6%) 31 (42.5%) 49 (43.4%) 31 (31.0%) 80 (37.6%) 17 (35.4%)
pc d
0.07
0.05e
0.08
0.006e
0.02e
General prevalence. Relative prevalence. p-value Chi-Square test. Reference category. Significant p-value (95% confidence level).
[7,24–26], although some authors have studied the prevalence of both C. jejuni and C. coli [27]. Some researchers have reported a higher prevalence of C. jejuni compared to C. coli along the entire production chain [28,29]. In contrast, C. coli was the species most frequently isolated during the whole processing at the slaughterhouse in Spain, Italy and Bulgaria [11]. On the other hand, Garin et al. [27] found that the Campylobacter species with highest prevalence was different depending on the slaughterhouse studied. Both species have been isolated in all flocks examined, with an increase in the trend of C. coli in the most of them (Table 2). It is striking in our study the relative prevalence variation during the processing, since C. coli almost doubles its percentage in the final product compared to C. jejuni, having found a lower percentage of C. coli species in the initial area. Thus, a lower relative prevalence of C. coli (41.5%) compared to C. jejuni (58.5%) was found in the loading dock area. In contrast, a significatly higher prevalence of C. coli (64.6%) compared to C. jejuni (35.4%) in the final product was observed (p = 0.02) (Table 1 and Fig. 1). In accordance with our results, percentage of C. coli isolates was slightly increased at the final processing area in Ireland [11]. In addition, a lower frequency of C. jejuni isolates compared to C. coli was observed during the scalding, quartering and final meat product/packing area (Table 1 and Fig. 1). This result could be related to a higher resistance to environmental conditions of C. coli compared to C. jejuni, such as temperature, desiccation or exposition to oxygen. Oyarzabal et al. [30] found that survival of C. jejuni was lower than for C. coli on retail broiler meat stored at 4 and 12 ◦ C. Significant differences in the prevalence between Campylobacter species were also found in loading dock area compared to evisceration (p = 0.05) and quartering areas (p = 0.006). In contrast, Hue et al. [29] did not found significant differences between both species. To the best of our knowledge, there are no other studies which have found significant differences between Campylobacter species in the slaughterhouse. For this reason, this research
results different from surveys performed at the slaughterhouse level before. Our findings would show that there is a variation in the survival of Campylobacter species, indicating that performance of control strategies to reduce this bacteria could be improved by collection of data to better define the survival response along the processing at slaughterhouse. Variations between studies may be due to the different sampling and detection methods as well as the existence of C. jejuni and C. coli strains which show different adherence or environmental resistance during poultry processing [31]. Several studies have reported an important reduction in the number of genotypes occurs during processing [7,32–34]. In addition, some authors have indicated that the environmental resistance (to high or low temperature, pH or oxygen) in the genus Campylobacter varies according to the strain or the genotype [33,35,36]. Therefore, the increasing of the relative prevalence of C. coli during the processing detected in our study could be linked with genetic differences among Campylobacter strains. The existence of a large antigenic variability in the surface of flagella or lipooligosaccharides of the cell membrane which determines the adhesion capacity of these bacteria to carcasses and inert surfaces in the slaughterhouse has been described [37]. Additional studies are required to clarify this point and to determine the specific mechanisms associated with the increasing of the relative prevalence of C. coli along the processing at slaughterhouse. 3.2. Antibiotic susceptibility Table 3 shows MICMode , MIC50 , MIC90 and percentage of C. jejuni and C. coli resistant isolates. The MICMode is the MIC that occurs most often, the MIC50 and MIC90 is the MIC required to inhibit growth by 50% and 90% respectively. The highest resistance was detected to tetracycline (100%) and ciprofloxacin (100%) for C. jejuni and C. coli, respectively. The antimicrobial which showed a lowest percentage of resistance were erythromycin for C. jejuni
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Table 2 Number of samples of C. jejuni and C. coli isolates in every week. Week
Total samples
Species
Loading dock
Scalding
Evisceration
Classified
1
56
2
56
3
56
4
56
5
58
6
56
7
56
8
56
9
58
10
60
11
56
12
56
13
56
14
56
15
56
C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni
2 1 4 3 1 0 4 3 4 3 2 2 3 0 0 0 1 1 1 1 1 0 1 1 1 1 3 3 3 3 31
0 0 2 4 0 0 2 3 4 2 0 2 3 0 2 0 0 5 0 1 2 2 2 2 0 1 4 5 1 5 22
0 1 2 7 0 1 7 12 7 7 4 5 5 1 3 3 4 6 9 9 9 7 6 4 2 5 3 5 0 8 61
0 0 1 0 0 0 1 2 2 3 0 1 4 4 3 3 6 8 2 6 2 2 3 2 3 3 3 5 1 3 31
3 9 6 8 0 0 4 8 12 12 1 6 14 17 7 5 8 9 5 7 8 8 7 8 2 10 0 14 3 12 80
0 1 1 2 0 0 0 0 1 0 1 2 2 3 2 3 2 2 2 3 4 5 0 1 0 1 1 4 1 4 17
C. coli
22
32
81
42
133
31
TOTAL positive samples
(10%) and gentamicin for C. coli (16.7%). Previous results obtained in Europe showed a lower percentage of resistant isolates against these antimicrobial agents [15,38,39]. This could be due to the high incidence of pathogens such us Escherichia coli or Mycoplasma gallisepticum in the study area, which makes frequently necessary the use of antimicrobials during the rearing period. This fact
Quartering
Final meat product/packing
could cause that other bacteria like Campylobacter species increase their resistance to antimicrobials. The reason why antimicrobial resistance is higher than other European countries could be due to a lack of awareness of the impact of indiscriminate use of antimicrobials. We found a significant higher number of resistant C. coli isolates compared to C. jejuni isolates for streptomycin
Fig. 1. Relative prevalence of C. coli and C. jejuni isolates in each processing area.
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Table 3 MIC results (mg/L) and susceptibility of Campylobacter species isolated from chicken meat. Antibiotic
Campylobacter species
MICMode
MIC50
MIC90
Resistant isolates (%)
Ciprofloxacin
C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli
32, 16 32 4 128 2 128 64 64 1 1
32 32 2 64 2 64 64 64 1 1
128 128 4 128 256 128 512 256 4 32
96.7 (MIC > 0.5) 100 (MIC > 0.5) 10.0 (MIC > 4) 73.3 (MIC > 8) 36.7 (MIC > 4) 76.7 (MIC > 4) 100 (MIC > 2) 96.7 (MIC > 2) 13.3 (MIC > 2) 16.7 (MIC > 2)
Erythromycin Streptomycin Tetracycline Gentamicin
Table 4 Multidrug resistance determined for 30 isolated of C. jejuni and 30 C. coli from chicken meat. Multidrug resistant isolates
Resistant to 3 antimicrobial Resistant to 4 antimicrobial Resistant to 5 antimicrobial Resistant to 3 o more antimicrobial
C. jejuni
C. coli
antimicrobial-resistant isolates in food may have important implications for public health. Conflict of interest
n
%
n
%
5 5 1 11
16.7 16.7 3.3 36.7
6 14 5 25
20.0 46.7 16.7 83.3
None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Acknowledgements
(p = 0.002) and erythromycin (p < 0.0001), which is consistent with the previously reported [40,41]. However, Tang et al. [42] did not find significant differences between C. coli and C. jejuni to both antimicrobial. Moreover, 11 from 30 (36.7%) C. jejuni isolates and 25 from 30 (83.3%) C. coli isolates were multidrug resistant (resistant to 3 or more antimicrobial) (Table 4). In contrast, Van Looveren et al. [38] reported a lower proportion of multidrug resistant isolates in poultry and pigs (20.3% and 33.9% for C. jejuni and C. coli, respectively). Nowadays, Campylobacter is the most important zoonotic agent, so the emergence of resistant isolates shows important implications for public health. Resistant Campylobacter isolates have been increasing over the years, especially for fluoroquinolones which are the treatment of choice in patients with campylobacteriosis [43]. If the increasing of antimicrobial resistance is not stopped, it might be necessary to search alternative treatments for humans with this disease. For this reason, the surveillance of antimicrobial resistance and multidrug resistance in Campylobacter isolates from chicken meat and the cautious use of these antimicrobial in broilers must have the highest priority [44]. The prevalence of C. coli was nearly twice the prevalence of C. jejuni in the final area at slaughterhouse, although C. jejuni was the most frequent Campylobacter species isolated in the initial area. The results suggest a higher resistance or adherence of C. coli isolates during the processing. Therefore, the development of control measures at slaughterhouse level should be carried out depending on the survival response of the different Campylobacter isolates in the poultry processing. Furthermore, the high proportion of antimicrobial-resistant isolates denotes the importance of controlling the emergence of antibiotic resistance. Since campylobacteriosis is transmitted primarily through chicken meat, the presence of
This study was supported by the Research Group AGR149 of the Junta de Andalucía and by the Centre for Industrial Technological Development of the Spanish Government through the research project: “Integravi: Improving the quality of poultry meat by production and health strategies”. Authors thank the companies and veterinaries for their collaboration with this study, to Miguel Ángel Díaz, José Miguel Montero and Mercedes Campo for their support during the sampling and to Alfonso Lara for his technical assistance. References [1] European Food Safety Authority. European Centre for Disease Prevention and Control. The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks in 2011. EFSA J 2013;11(4):3129. [2] Sheppard SK, Dallas JF, Strachan NJC, MacRae M, McCarthy ND, Wilson DJ, et al. Campylobacter genotyping to determine the source of human infection. Clin Infect Dis 2009;48:1072–8. [3] Luber P, Brynestad S, Topsch D, Scherer K, Bartelt E. Quantification of campylobacter species cross-contamination during handling of contaminated fresh chicken parts in kitchens. Appl Environ Microb 2006;72:66–70. [4] Mateo E, Cárcamo J, Urquijo M, Perales I, Fernández-Astorga A. Evaluation of a PCR assay for the detection and identification of Campylobacter jejuni and Campylobacter coli in retail poultry products. Res Microbiol 2005;156:568–74. [5] Praakle-Amin K, Roasto M, Korkeala H, Hänninen ML. PFGE genotyping and antimicrobial susceptibility of Campylobacter in retail poultry meat in Estonia. Int J Food Microbiol 2007;114: 105–12. [6] Damjanova I, Jakab M, Farkas T, Mészáros J, Galántai Z, Turcsányi I, et al. From farm to fork follow-up of thermotolerant campylobacters throughout the broiler production chain and in human cases in a Hungarian county during a ten-months period. Int J Food Microbiol 2011;150:95–102. [7] Melero B, Juntunen P, Hänninen M, Jaime I, Rovira J. Tracing Campylobacter jejuni strains along the poultry meat production chain from farm to retail by pulsed-field gel electrophoresis, and the antimicrobial resistance of isolates. Food Microbiol 2012;32: 124–8.
52
A. Torralbo et al. / Comparative Immunology, Microbiology and Infectious Diseases 39 (2015) 47–52
[8] Chantarapanont W, Berrang ME, Frank JF. Direct microscopic observation of viability of Campylobacter jejuni on chicken skin treated with selected chemical sanitizing agents. J Food Protect 2004;67:1146–52. [9] Allen VM, Bull SA, Corry JE, Domingue G, Jorgensen F, Frost JA, et al. Campylobacter spp. contamination of chicken carcasses during processing in relation to flock colonisation. Int J Food Microbiol 2007;113:54–61. [10] Rosenquist H, Sommer HM, Nielsen NL, Christensen BB. The effect of slaughter operations on the contamination of chicken carcasses with thermotolerant Campylobacter. Int J Food Microbiol 2006;108:226–32. [11] European Food Safety Authority. Analysis of the baseline survey on the prevalence of Campylobacter in broiler batches and of Campylobacter and Salmonella on broiler carcasses in the EU, 2008. Part A: Campylobacter and Salmonella prevalence estimates. EFSA J 2010;8(3):1503. [12] NCCLS, National Committee for Clinical Laboratory Standards (NCCLS) document M31-A2 Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; approved standard. 2nd ed. Wayne, PA: National Committee for Clinical Laboratory Standards; 2002. [13] Osaili TM, Alaboudi AR, Al-Akhras RR. Prevalence and antimicrobial susceptibility of Campylobacter spp. in live and dressed chicken in Jordan. Foodborne Pathog Dis 2012;9:54–8. ˜ [14] Sáenz Y, Zarazaga M, Lantero M, Gastanares MJ, Baquero F, Torres C. Antibiotic resistance in Campylobacter strains isolated from animals, foods, and humans in Spain in 1997–1998. Antimicrob Agents Chemother 2000;44:267–71. [15] Luber P, Wagner J, Hahn H, Bartelt E. Antimicrobial resistance in Campylobacter jejuni and Campylobacter coli strains isolated in 1991 and 2011–2002 from Poultry and Humans in Berlin, Germany. Antimicrob Agents Chemother 2003;47:3825–30. [16] Pezzotti G, Serafín A, Luzzi I, Mioni R, Milan M, Perin R. Occurrence and resistance to antibiotics of Campylobacter jejuni and Campylobacter coli in animals and meat in northeastern Italy. Int J Food Microbiol 2003;82:281–7. [17] Silva J, Leite D, Fernandes M, Mena C, Gibbs PA, Teixeira P. Campylobacter spp. as a foodborne pathogen: a review. Front Microbiol 2011;2:200. [18] Sambrook J, Russell DW. Molecular cloning: a laboratory manual. 3rd ed. Cold Spring Harbor Laboratory Press; 2001. [19] Yamazaki-Matsune W, Taguchi M, Seto K, Kawahara R, Kawatsu K, Kumeda Y, et al. Development of a multiplex PCR assay for identification of Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis. J Med Microbiol 2007;56:1467–73. [20] EUCAST, EUCAST definitive document E. Def 3.1 Determination of minimum inhibitory concentrations (MICs) of antibacterial agents by agar dilution. European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID); 2000. [21] EUCAST. European Committee on antimicrobial susceptibility testing. European Society of Clinical Microbiology and Infectious Diseases; 2012 http://www.eucast.org/ [22] Frederick A, Huda N. Campylobacter in poultry: incidences and possible control measures. Res J Microbiol 2011;6:182–92. [23] Wagenaar JA, Mevius DJ, Havelaar AH. Campylobacter in primary animal production and control strategies to reduce the burden of human campylobacteriosis. Rev Sci Tech Off Int Epiz 2006;25:581–94. [24] Kudirkiene E, Buneviciene J, Brondsted L, Ingmer H, Olsen JE, Malakauskas M. Evidence of broiler meat contamination with postdisinfection strains of Campylobacter jejuni from slaughterhouse. Int J Food Microbiol 2011;145:116–20. [25] Kudirkiene E, Cohn MT, Stabler RA, Strong PC, Serniene L, Wren BW, et al. Phenotypic and genotypic characterizations of Campylobacter jejuni isolated from the broiler meat production process. Curr Microbiol 2012;65:398–406.
[26] Jong AEI, Van Asselt ED, Zwietering MH, Nauta MJ, Jonge R. Extreme heat resistance of food borne pathogens Campylobacter jejuni, Escherichia coli, and Salmonella typhimurium on chicken breast fillet during cooking. Int J Food Microbiol 2012:1–10. [27] Garin B, Gouali M, Wouafo M, Perchec AM, Thu PM, Ravaonindrina N, et al. Prevalence, quantification and antimicrobial resistance of Campylobacter spp. on chicken neck-skins at points of slaughter in 5 major cities located on 4 continents. Int J Food Microbiol 2012;157:102–7. [28] Wirz SE, Overesch G, Kuhnert P, Korczak BM. Genotype and antibiotic resistance analyses of Campylobacter isolates from ceca and carcasses of slaughtered broiler flocks. Appl Environ Microb 2010;76: 6377–86. [29] Hue O, Allain V, Laisney M, Le Bouquin S, Lalande F, Petetin I, et al. Campylobacter contamination of broiler caeca and carcasses at the slaughterhouse and correlation with Salmonella contamination. Food Microbiol 2011;28:862–8. [30] Oyarzabal OA, Oscar TP, Speegle L, Nyati H. Survival of Campylobacter jejuni and Campylobacter coli on retail broiler meat stored at −20, 4 or 12 ◦ C development of weibull models for survival. J Food Protect 2010;73:1438–46. [31] Nguyen VT, Turner MS, Dykes GA. Influence of cell surface hydrophobicity on attachment of Campylobacter to abiotic surfaces. Food Microbiol 2011;28:942–50. [32] Hiett KL, Stern NJ, Fedorka-Cray P, Cox NA, Musgrove MT, Ladely S. Molecular subtype analyses of Campylobacter spp. from Arkansas and California poultry operations. Appl Environ Microb 2002;68:6220–36. [33] Klein G, Beckmann L, Vollmer HM, Bartelt E. Predominant strains of thermophilic Campylobacter spp. in a German poultry slaughterhouse. Int J Food Microbiol 2007;117:324–8. [34] Kudirkiene E, Malakauskas M, Malakauskas A, Bojesen AM, Olsen JE. Demonstration of persistent strains of C. jejuni within broiler farms over one year period in Lithuania. J Appl Microbiol 2010;108:868–77. [35] Newell DG, Shreeve JE, Toszeghy M, Domingue G, Bull S, Humphrey T, et al. Changes in the carriage of Campylobacter strains by poultry carcasses during processing in abattoirs. Appl Environ Microb 2001;67:2636–40. [36] Alter T, Gaull F, Froeb A, Fehlhaber K. Distribution of Campylobacter jejuni strains at different stages of a turkey slaughter line. Food Microbiol 2005;22:345–51. [37] Fouts DE, Mongodin EF, Mandrell RE, Miller WG, Rasko DA, Ravel J, et al. Major structural differences and novel potential virulence mechanisms from the genomes of multiple Campylobacter species. PLoS Biol 2005;3:72–85. [38] Van Looveren M, Daube G, De Zutter L, Dumont J, Lammens C, Wijdooghe M, et al. Antimicrobial susceptibilities of Campylobacter strains isolated from food animals in Belgium. J Antimicrob Chemother 2001;48:235–40. [39] Wilson IG. Antibiotic resistance of Campylobacter in raw retail chickens and imported chicken portions. Epidemiol Infect 2003;131:1181–6. [40] Ge B, White DG, McDermott PF, Girard W, Zhao S, Hubert S, et al. Antimicrobial-resistant Campylobacter species from retail raw meats. Appl Environ Microb 2003;69:3005–7. [41] Angelovski L, Sekulovski P, Jankuloski D, Ratkova M, Prodanov M, Kostova S. Antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolates from broiler flocks. Maced Vet Rev 2011;34:15–8. [42] Tang JYH, Mohamad Ghazali F, Saleha AA, Nishibuchi M, Son R. Comparison of thermophilic Campylobacter spp. occurrence in two types of retail chicken samples. Int Food Res J 2009;16:277–88. [43] Luber P, Wagner J, Hahn H, Bartelt E. Antimicrobial resistance in Campylobacter jejuni and Campylobacter coli strains isolated in 1991 and 2001–2002 from poultry and humans in Berlin, Germany. Antimicrob Agents Chemother 2003;47:3825–30. [44] Moore JE, Barton MD, Blair IS, Corcoran D, Dooley JSG, Fanning S, et al. The epidemiology of antibiotic resistance in Campylobacter. Microbes Infect 2006;8:1955–66.
Contents lists available at ScienceDirect
Comparative Immunology, Microbiology and Infectious Diseases journal homepage: www.elsevier.com/locate/cimid
Higher resistance of Campylobacter coli compared to Campylobacter jejuni at chicken slaughterhouse Alicia Torralbo a , Carmen Borge a , Ignacio García-Bocanegra a , Guillaume Méric b , Anselmo Perea a , Alfonso Carbonero a,∗ a b
Department of Animal Health, Campus de Excelencia Internacional Agroalimentario ceiA3, University of Cordoba, Cordoba, Spain College of Medicine, Swansea University, Swansea SA2 8PP, United Kingdom
a r t i c l e
i n f o
Article history: Received 5 April 2014 Received in revised form 21 February 2015 Accepted 26 February 2015 Keywords: Broiler Campylobacter Chicken meat MIC Slaughterhouse Spain
a b s t r a c t In order to compare the prevalence of Campylobacter coli and Campylobacter jejuni during the processing of broilers at slaughterhouse a total of 848 samples were analyzed during 2012 in southern Spain. Four hundred and seventy six samples were collected from cloaca, carcass surfaces and quartered carcasses. Moreover, 372 environmental swabs from equipment and scalding water were collected. Minimum inhibitory concentration (MIC) to ciprofloxacin, erythromycin, streptomycin, tetracycline and gentamicin was determined for isolates from chicken meat. The general prevalence of Campylobacter was 68.8% (40.2% of C. coli and 28.5% of C. jejuni). The relative prevalence of C. coli increased from loading dock area (41.5%) to packing area (64.6%). In contrast, the relative prevalence of C. jejuni decreased from 58.5% to 35.4%. These differences between species from initial to final area were significant (p = 0.02). The highest antimicrobial resistance for C. jejuni and C. coli was detected to tetracycline (100%) and ciprofloxacin (100%), respectively. Campylobacter coli showed an antimicrobial resistance significantly higher than C. jejuni to streptomycin (p = 0.002) and erythromycin (p < 0.0001). © 2015 Elsevier Ltd. All rights reserved.
1. Introduction With 220,209 human cases in the European Union, campylobacteriosis was the most commonly reported zoonosis in 2011 [1]. Undercooked poultry meat is the main source of campylobacteriosis for humans [2]. Furthermore, cross contamination from raw chicken meat through knives, cutting board or hands has been reported as a major risk factor [3]. Even though different authors have described the prevalence of Campylobacter in retail products [4,5], few studies include the entire production chain as far as retail [6].
∗ Corresponding author. Tel.: +34 650952630; fax: +34 957218725. E-mail address: [email protected] (A. Carbonero). http://dx.doi.org/10.1016/j.cimid.2015.02.003 0147-9571/© 2015 Elsevier Ltd. All rights reserved.
Campylobacter can be found at all steps along the poultry production chain [7]. The evisceration process, the contact with equipment and the scalding water containing Campylobacter can cause multiple cross-contaminations in broiler carcasses [8,9]. In contrast, it has been reported that the number of Campylobacter in broiler carcasses could be reduced but not eliminated from carcasses by scalding or chilling process [10]. The prevalence of Campylobacter in broiler carcasses in Europe was 75.8% [11]. About two thirds of the Campylobacter isolates from broiler carcasses were identified as Campylobacter jejuni, while one third was Campylobacter coli. However, C. coli was the most frequent species isolated in Spain, Italy and Bulgaria [11]. The calculation of the minimum inhibitory concentration (MIC) by agar plate dilution method is the technique
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recommended to determine the antibiotic susceptibility for Campylobacter species [12]. Antimicrobial resistance has been described in Campylobacter isolated from dressed chickens [13] or retail meat products [7]. Campylobacter species have been found to be resistant to macrolides, tetracyclines and fluroquinolones [14–16], which are the main antimicrobial used to treat severe cases in human [17]. In this study, our principal aim was to determine the prevalence of C. coli compared to C. jejuni during processing at slaughterhouse. In addition, the antibiotic susceptibility for isolates from chicken meat was evaluated. 2. Materials and methods 2.1. Study design and sampling The study was carried out along 2012 in a slaughterhouse located in southern Spain. About 60,000 chickens are slaughtered each day, with modern equipments to perform each process, as well as its own quartering room. The slaughterhouse was divided in 6 areas to collect the samples: loading dock, scalding (water temperature: 52 ◦ C), evisceration, classified (after air chilling for 2 h at 4 ◦ C), quartering and final meat product/packing (room temperature: 1 ◦ C). Swab samples were collected for 15 weeks in a row, one day each week, along the entire processing chain, from cloaca, carcass surfaces (breast and back area), quartered carcasses surfaces (breast, wing, leg and back) and slaughterhouse environment (equipment and scalding water). A total of 848 swab samples were obtained and analyzed: 476 were taken from broilers, carcass surfaces and quartered carcasses surfaces, and 372 from equipment and scalding water (Table 1). Samples were collected in all cases from the last flock slaughtered of the sampling day by systematic random sampling in each stage. Samples were collected using sterile swabs placed in tubes containing a transport medium (Amies, Eurotubo® ). Swabs were kept refrigerated until arrival at the laboratory and then processed within 24 h. 2.2. Isolation and identification of Campylobacter Every swab was streaked onto a plate with Campylobacter Blood-Free Selective Agar Base (Oxoid® CM0739) added with CCDA Selective Supplement (Oxoid® SR0155). After 48 h of incubation at 42 ◦ C in a CO2 -enriched atmosphere achieved by AnaeroGen sachets (Oxoid® ), 15–20 colonies of each plate which showed morphology compatible with Campylobacter were streaked onto blood agar and incubated under the same conditions as previously described. All selected isolates were confirmed by examination of colonial morphology, Gram staining, motility in dark field microscopy, oxidase and catalase testing. Afterward, DNA extraction from bacterial cultures for isolates phenotypically classified as Campylobacter was performed according to the method described by Sambrook and Russell [18]. Thus, bulk colonies for each isolate in blood agar plate were taken with a loopful of 10 l to carry out DNA extraction.
Subsequently, the QIAGEN® Multiplex PCR Kit was used for the molecular identification as previously described [19]. 2.3. Minimum inhibitory concentration (MIC) For determination of the MIC, the agar dilution method was used following the protocol described by the European Committee for Antimicrobial Susceptibility Testing [20]. The cut off values used for the interpretation of the MIC results are developed by EUCAST [21]. The following antimicrobial were tested: ciprofloxacin, erythromycin, streptomycin, tetracycline and gentamicin (Sigma–Aldrich® ). Eighteen double dilutions, with antimicrobial concentration obtained from 10,240 mg/L to 0.0781 mg/L, were performed. Due to restraint budget, only a total of 60 randomly selected isolates (30 C. coli and 30 C. jejuni) from chicken meat were studied. The reference strains C. coli DSMZ 4689T and C. jejuni ATCC 33560 were used as positive controls. Plates were incubated under microaerobic atmosphere at 42 ◦ C. MIC values were read after 24 h of incubation. 2.4. Statistical analysis Prevalence of Campylobacter species in each area as well as frequencies of susceptible isolates and comparison of susceptibility between Campylobacter species was calculated using SPSS v15.0 software (SPSS Inc., Chicago, IL, USA). Chi squared test was used to evaluate frequencies of Campylobacter species in different areas during the processing and differences in antimicrobial susceptibility between C. coli and C. jejuni. 3. Results and discussion 3.1. Changes in C. coli and C. jejuni prevalence during processing in the slaughterhouse The general prevalence of Campylobacter was 68.8% (583/848), 40.2% (341/848) of C. coli and 28.5% (242/848) of C. jejuni. Campylobacter was isolated in all processing areas, agreeing with results reported by other authors in Spain [7] and other European countries [6]. The highest prevalence of Campylobacter was found in the evisceration area (92.8%) (Table 1). According to Allen et al. [9] and Frederick and Huda [22], this area is a critical control point where is required to maximize the cleaning and disinfection process. Moreover, to avoid the accidental breaking of gastrointestinal tracts and, in consequence, to decrease the contamination of carcasses, it would be necessary that the evisceration equipment is adapted to different carcass sizes [10]. However, it has been described that the most promising control strategy is to keep colonized and non-colonized flocks separate during slaughter. Furthermore, to reduce cases of campylobacteriosis in humans, meat from Campylobacter-positive flocks could be treated by freezing or chemical decontamination [23]. Most of the studies about the genus Campylobacter in broiler slaughterhouses were focussed on C. jejuni species
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Table 1 Number of samples in each area, relative prevalence of C. jejuni and C. coli isolates, and p-value. Area Loading dock Scalding
No. total samples 96 88
Evisceration
153
Classified
117
Quartering
311
Final meat product/packing a b c d e
83
No. specific samples Cloaca Whole carcasses Scalding water Total Whole carcasses Equipment Total Whole carcasses Equipment Total Quartered carcasses Equipment Total Quartered carcasses
96 39 49 88 67 86 153 46 71 117 145 166 311 83
Campylobacter No. (%)a
C. coli No. (%)b
C. jejuni No. (%)b
53 (55.2%) 26 (66.7%) 28 (57.1%) 54 (61.4%) 62 (92.5%) 80 (93.0%) 142 (92.8%) 30 (65.2%) 43 (60.6%) 73 (62.4%) 113 (77.9%) 100 (60.2%) 214 (68.5%) 48 (57.8%)
22 (41.5%) 16 (61.5%) 16 (57.1%) 32 (59.3%) 36 (58.1%) 45 (56.3%) 81 (57.0%) 13 (43.3%) 29 (67.4%) 42 (57.5%) 64 (56.6%) 69 (69.0%) 133 (62.4%) 31 (64.6%)
31 (58.5%) 10 (38.5%) 12 (42.9%) 22 (40.7%) 26 (41.9%) 35 (43.7%) 61 (43.0%) 17 (56.7%) 14 (32.6%) 31 (42.5%) 49 (43.4%) 31 (31.0%) 80 (37.6%) 17 (35.4%)
pc d
0.07
0.05e
0.08
0.006e
0.02e
General prevalence. Relative prevalence. p-value Chi-Square test. Reference category. Significant p-value (95% confidence level).
[7,24–26], although some authors have studied the prevalence of both C. jejuni and C. coli [27]. Some researchers have reported a higher prevalence of C. jejuni compared to C. coli along the entire production chain [28,29]. In contrast, C. coli was the species most frequently isolated during the whole processing at the slaughterhouse in Spain, Italy and Bulgaria [11]. On the other hand, Garin et al. [27] found that the Campylobacter species with highest prevalence was different depending on the slaughterhouse studied. Both species have been isolated in all flocks examined, with an increase in the trend of C. coli in the most of them (Table 2). It is striking in our study the relative prevalence variation during the processing, since C. coli almost doubles its percentage in the final product compared to C. jejuni, having found a lower percentage of C. coli species in the initial area. Thus, a lower relative prevalence of C. coli (41.5%) compared to C. jejuni (58.5%) was found in the loading dock area. In contrast, a significatly higher prevalence of C. coli (64.6%) compared to C. jejuni (35.4%) in the final product was observed (p = 0.02) (Table 1 and Fig. 1). In accordance with our results, percentage of C. coli isolates was slightly increased at the final processing area in Ireland [11]. In addition, a lower frequency of C. jejuni isolates compared to C. coli was observed during the scalding, quartering and final meat product/packing area (Table 1 and Fig. 1). This result could be related to a higher resistance to environmental conditions of C. coli compared to C. jejuni, such as temperature, desiccation or exposition to oxygen. Oyarzabal et al. [30] found that survival of C. jejuni was lower than for C. coli on retail broiler meat stored at 4 and 12 ◦ C. Significant differences in the prevalence between Campylobacter species were also found in loading dock area compared to evisceration (p = 0.05) and quartering areas (p = 0.006). In contrast, Hue et al. [29] did not found significant differences between both species. To the best of our knowledge, there are no other studies which have found significant differences between Campylobacter species in the slaughterhouse. For this reason, this research
results different from surveys performed at the slaughterhouse level before. Our findings would show that there is a variation in the survival of Campylobacter species, indicating that performance of control strategies to reduce this bacteria could be improved by collection of data to better define the survival response along the processing at slaughterhouse. Variations between studies may be due to the different sampling and detection methods as well as the existence of C. jejuni and C. coli strains which show different adherence or environmental resistance during poultry processing [31]. Several studies have reported an important reduction in the number of genotypes occurs during processing [7,32–34]. In addition, some authors have indicated that the environmental resistance (to high or low temperature, pH or oxygen) in the genus Campylobacter varies according to the strain or the genotype [33,35,36]. Therefore, the increasing of the relative prevalence of C. coli during the processing detected in our study could be linked with genetic differences among Campylobacter strains. The existence of a large antigenic variability in the surface of flagella or lipooligosaccharides of the cell membrane which determines the adhesion capacity of these bacteria to carcasses and inert surfaces in the slaughterhouse has been described [37]. Additional studies are required to clarify this point and to determine the specific mechanisms associated with the increasing of the relative prevalence of C. coli along the processing at slaughterhouse. 3.2. Antibiotic susceptibility Table 3 shows MICMode , MIC50 , MIC90 and percentage of C. jejuni and C. coli resistant isolates. The MICMode is the MIC that occurs most often, the MIC50 and MIC90 is the MIC required to inhibit growth by 50% and 90% respectively. The highest resistance was detected to tetracycline (100%) and ciprofloxacin (100%) for C. jejuni and C. coli, respectively. The antimicrobial which showed a lowest percentage of resistance were erythromycin for C. jejuni
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Table 2 Number of samples of C. jejuni and C. coli isolates in every week. Week
Total samples
Species
Loading dock
Scalding
Evisceration
Classified
1
56
2
56
3
56
4
56
5
58
6
56
7
56
8
56
9
58
10
60
11
56
12
56
13
56
14
56
15
56
C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni
2 1 4 3 1 0 4 3 4 3 2 2 3 0 0 0 1 1 1 1 1 0 1 1 1 1 3 3 3 3 31
0 0 2 4 0 0 2 3 4 2 0 2 3 0 2 0 0 5 0 1 2 2 2 2 0 1 4 5 1 5 22
0 1 2 7 0 1 7 12 7 7 4 5 5 1 3 3 4 6 9 9 9 7 6 4 2 5 3 5 0 8 61
0 0 1 0 0 0 1 2 2 3 0 1 4 4 3 3 6 8 2 6 2 2 3 2 3 3 3 5 1 3 31
3 9 6 8 0 0 4 8 12 12 1 6 14 17 7 5 8 9 5 7 8 8 7 8 2 10 0 14 3 12 80
0 1 1 2 0 0 0 0 1 0 1 2 2 3 2 3 2 2 2 3 4 5 0 1 0 1 1 4 1 4 17
C. coli
22
32
81
42
133
31
TOTAL positive samples
(10%) and gentamicin for C. coli (16.7%). Previous results obtained in Europe showed a lower percentage of resistant isolates against these antimicrobial agents [15,38,39]. This could be due to the high incidence of pathogens such us Escherichia coli or Mycoplasma gallisepticum in the study area, which makes frequently necessary the use of antimicrobials during the rearing period. This fact
Quartering
Final meat product/packing
could cause that other bacteria like Campylobacter species increase their resistance to antimicrobials. The reason why antimicrobial resistance is higher than other European countries could be due to a lack of awareness of the impact of indiscriminate use of antimicrobials. We found a significant higher number of resistant C. coli isolates compared to C. jejuni isolates for streptomycin
Fig. 1. Relative prevalence of C. coli and C. jejuni isolates in each processing area.
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Table 3 MIC results (mg/L) and susceptibility of Campylobacter species isolated from chicken meat. Antibiotic
Campylobacter species
MICMode
MIC50
MIC90
Resistant isolates (%)
Ciprofloxacin
C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli C. jejuni C. coli
32, 16 32 4 128 2 128 64 64 1 1
32 32 2 64 2 64 64 64 1 1
128 128 4 128 256 128 512 256 4 32
96.7 (MIC > 0.5) 100 (MIC > 0.5) 10.0 (MIC > 4) 73.3 (MIC > 8) 36.7 (MIC > 4) 76.7 (MIC > 4) 100 (MIC > 2) 96.7 (MIC > 2) 13.3 (MIC > 2) 16.7 (MIC > 2)
Erythromycin Streptomycin Tetracycline Gentamicin
Table 4 Multidrug resistance determined for 30 isolated of C. jejuni and 30 C. coli from chicken meat. Multidrug resistant isolates
Resistant to 3 antimicrobial Resistant to 4 antimicrobial Resistant to 5 antimicrobial Resistant to 3 o more antimicrobial
C. jejuni
C. coli
antimicrobial-resistant isolates in food may have important implications for public health. Conflict of interest
n
%
n
%
5 5 1 11
16.7 16.7 3.3 36.7
6 14 5 25
20.0 46.7 16.7 83.3
None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Acknowledgements
(p = 0.002) and erythromycin (p < 0.0001), which is consistent with the previously reported [40,41]. However, Tang et al. [42] did not find significant differences between C. coli and C. jejuni to both antimicrobial. Moreover, 11 from 30 (36.7%) C. jejuni isolates and 25 from 30 (83.3%) C. coli isolates were multidrug resistant (resistant to 3 or more antimicrobial) (Table 4). In contrast, Van Looveren et al. [38] reported a lower proportion of multidrug resistant isolates in poultry and pigs (20.3% and 33.9% for C. jejuni and C. coli, respectively). Nowadays, Campylobacter is the most important zoonotic agent, so the emergence of resistant isolates shows important implications for public health. Resistant Campylobacter isolates have been increasing over the years, especially for fluoroquinolones which are the treatment of choice in patients with campylobacteriosis [43]. If the increasing of antimicrobial resistance is not stopped, it might be necessary to search alternative treatments for humans with this disease. For this reason, the surveillance of antimicrobial resistance and multidrug resistance in Campylobacter isolates from chicken meat and the cautious use of these antimicrobial in broilers must have the highest priority [44]. The prevalence of C. coli was nearly twice the prevalence of C. jejuni in the final area at slaughterhouse, although C. jejuni was the most frequent Campylobacter species isolated in the initial area. The results suggest a higher resistance or adherence of C. coli isolates during the processing. Therefore, the development of control measures at slaughterhouse level should be carried out depending on the survival response of the different Campylobacter isolates in the poultry processing. Furthermore, the high proportion of antimicrobial-resistant isolates denotes the importance of controlling the emergence of antibiotic resistance. Since campylobacteriosis is transmitted primarily through chicken meat, the presence of
This study was supported by the Research Group AGR149 of the Junta de Andalucía and by the Centre for Industrial Technological Development of the Spanish Government through the research project: “Integravi: Improving the quality of poultry meat by production and health strategies”. Authors thank the companies and veterinaries for their collaboration with this study, to Miguel Ángel Díaz, José Miguel Montero and Mercedes Campo for their support during the sampling and to Alfonso Lara for his technical assistance. References [1] European Food Safety Authority. European Centre for Disease Prevention and Control. The European Union Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Food-borne Outbreaks in 2011. EFSA J 2013;11(4):3129. [2] Sheppard SK, Dallas JF, Strachan NJC, MacRae M, McCarthy ND, Wilson DJ, et al. Campylobacter genotyping to determine the source of human infection. Clin Infect Dis 2009;48:1072–8. [3] Luber P, Brynestad S, Topsch D, Scherer K, Bartelt E. Quantification of campylobacter species cross-contamination during handling of contaminated fresh chicken parts in kitchens. Appl Environ Microb 2006;72:66–70. [4] Mateo E, Cárcamo J, Urquijo M, Perales I, Fernández-Astorga A. Evaluation of a PCR assay for the detection and identification of Campylobacter jejuni and Campylobacter coli in retail poultry products. Res Microbiol 2005;156:568–74. [5] Praakle-Amin K, Roasto M, Korkeala H, Hänninen ML. PFGE genotyping and antimicrobial susceptibility of Campylobacter in retail poultry meat in Estonia. Int J Food Microbiol 2007;114: 105–12. [6] Damjanova I, Jakab M, Farkas T, Mészáros J, Galántai Z, Turcsányi I, et al. From farm to fork follow-up of thermotolerant campylobacters throughout the broiler production chain and in human cases in a Hungarian county during a ten-months period. Int J Food Microbiol 2011;150:95–102. [7] Melero B, Juntunen P, Hänninen M, Jaime I, Rovira J. Tracing Campylobacter jejuni strains along the poultry meat production chain from farm to retail by pulsed-field gel electrophoresis, and the antimicrobial resistance of isolates. Food Microbiol 2012;32: 124–8.
52
A. Torralbo et al. / Comparative Immunology, Microbiology and Infectious Diseases 39 (2015) 47–52
[8] Chantarapanont W, Berrang ME, Frank JF. Direct microscopic observation of viability of Campylobacter jejuni on chicken skin treated with selected chemical sanitizing agents. J Food Protect 2004;67:1146–52. [9] Allen VM, Bull SA, Corry JE, Domingue G, Jorgensen F, Frost JA, et al. Campylobacter spp. contamination of chicken carcasses during processing in relation to flock colonisation. Int J Food Microbiol 2007;113:54–61. [10] Rosenquist H, Sommer HM, Nielsen NL, Christensen BB. The effect of slaughter operations on the contamination of chicken carcasses with thermotolerant Campylobacter. Int J Food Microbiol 2006;108:226–32. [11] European Food Safety Authority. Analysis of the baseline survey on the prevalence of Campylobacter in broiler batches and of Campylobacter and Salmonella on broiler carcasses in the EU, 2008. Part A: Campylobacter and Salmonella prevalence estimates. EFSA J 2010;8(3):1503. [12] NCCLS, National Committee for Clinical Laboratory Standards (NCCLS) document M31-A2 Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; approved standard. 2nd ed. Wayne, PA: National Committee for Clinical Laboratory Standards; 2002. [13] Osaili TM, Alaboudi AR, Al-Akhras RR. Prevalence and antimicrobial susceptibility of Campylobacter spp. in live and dressed chicken in Jordan. Foodborne Pathog Dis 2012;9:54–8. ˜ [14] Sáenz Y, Zarazaga M, Lantero M, Gastanares MJ, Baquero F, Torres C. Antibiotic resistance in Campylobacter strains isolated from animals, foods, and humans in Spain in 1997–1998. Antimicrob Agents Chemother 2000;44:267–71. [15] Luber P, Wagner J, Hahn H, Bartelt E. Antimicrobial resistance in Campylobacter jejuni and Campylobacter coli strains isolated in 1991 and 2011–2002 from Poultry and Humans in Berlin, Germany. Antimicrob Agents Chemother 2003;47:3825–30. [16] Pezzotti G, Serafín A, Luzzi I, Mioni R, Milan M, Perin R. Occurrence and resistance to antibiotics of Campylobacter jejuni and Campylobacter coli in animals and meat in northeastern Italy. Int J Food Microbiol 2003;82:281–7. [17] Silva J, Leite D, Fernandes M, Mena C, Gibbs PA, Teixeira P. Campylobacter spp. as a foodborne pathogen: a review. Front Microbiol 2011;2:200. [18] Sambrook J, Russell DW. Molecular cloning: a laboratory manual. 3rd ed. Cold Spring Harbor Laboratory Press; 2001. [19] Yamazaki-Matsune W, Taguchi M, Seto K, Kawahara R, Kawatsu K, Kumeda Y, et al. Development of a multiplex PCR assay for identification of Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis. J Med Microbiol 2007;56:1467–73. [20] EUCAST, EUCAST definitive document E. Def 3.1 Determination of minimum inhibitory concentrations (MICs) of antibacterial agents by agar dilution. European Committee for Antimicrobial Susceptibility Testing (EUCAST) of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID); 2000. [21] EUCAST. European Committee on antimicrobial susceptibility testing. European Society of Clinical Microbiology and Infectious Diseases; 2012 http://www.eucast.org/ [22] Frederick A, Huda N. Campylobacter in poultry: incidences and possible control measures. Res J Microbiol 2011;6:182–92. [23] Wagenaar JA, Mevius DJ, Havelaar AH. Campylobacter in primary animal production and control strategies to reduce the burden of human campylobacteriosis. Rev Sci Tech Off Int Epiz 2006;25:581–94. [24] Kudirkiene E, Buneviciene J, Brondsted L, Ingmer H, Olsen JE, Malakauskas M. Evidence of broiler meat contamination with postdisinfection strains of Campylobacter jejuni from slaughterhouse. Int J Food Microbiol 2011;145:116–20. [25] Kudirkiene E, Cohn MT, Stabler RA, Strong PC, Serniene L, Wren BW, et al. Phenotypic and genotypic characterizations of Campylobacter jejuni isolated from the broiler meat production process. Curr Microbiol 2012;65:398–406.
[26] Jong AEI, Van Asselt ED, Zwietering MH, Nauta MJ, Jonge R. Extreme heat resistance of food borne pathogens Campylobacter jejuni, Escherichia coli, and Salmonella typhimurium on chicken breast fillet during cooking. Int J Food Microbiol 2012:1–10. [27] Garin B, Gouali M, Wouafo M, Perchec AM, Thu PM, Ravaonindrina N, et al. Prevalence, quantification and antimicrobial resistance of Campylobacter spp. on chicken neck-skins at points of slaughter in 5 major cities located on 4 continents. Int J Food Microbiol 2012;157:102–7. [28] Wirz SE, Overesch G, Kuhnert P, Korczak BM. Genotype and antibiotic resistance analyses of Campylobacter isolates from ceca and carcasses of slaughtered broiler flocks. Appl Environ Microb 2010;76: 6377–86. [29] Hue O, Allain V, Laisney M, Le Bouquin S, Lalande F, Petetin I, et al. Campylobacter contamination of broiler caeca and carcasses at the slaughterhouse and correlation with Salmonella contamination. Food Microbiol 2011;28:862–8. [30] Oyarzabal OA, Oscar TP, Speegle L, Nyati H. Survival of Campylobacter jejuni and Campylobacter coli on retail broiler meat stored at −20, 4 or 12 ◦ C development of weibull models for survival. J Food Protect 2010;73:1438–46. [31] Nguyen VT, Turner MS, Dykes GA. Influence of cell surface hydrophobicity on attachment of Campylobacter to abiotic surfaces. Food Microbiol 2011;28:942–50. [32] Hiett KL, Stern NJ, Fedorka-Cray P, Cox NA, Musgrove MT, Ladely S. Molecular subtype analyses of Campylobacter spp. from Arkansas and California poultry operations. Appl Environ Microb 2002;68:6220–36. [33] Klein G, Beckmann L, Vollmer HM, Bartelt E. Predominant strains of thermophilic Campylobacter spp. in a German poultry slaughterhouse. Int J Food Microbiol 2007;117:324–8. [34] Kudirkiene E, Malakauskas M, Malakauskas A, Bojesen AM, Olsen JE. Demonstration of persistent strains of C. jejuni within broiler farms over one year period in Lithuania. J Appl Microbiol 2010;108:868–77. [35] Newell DG, Shreeve JE, Toszeghy M, Domingue G, Bull S, Humphrey T, et al. Changes in the carriage of Campylobacter strains by poultry carcasses during processing in abattoirs. Appl Environ Microb 2001;67:2636–40. [36] Alter T, Gaull F, Froeb A, Fehlhaber K. Distribution of Campylobacter jejuni strains at different stages of a turkey slaughter line. Food Microbiol 2005;22:345–51. [37] Fouts DE, Mongodin EF, Mandrell RE, Miller WG, Rasko DA, Ravel J, et al. Major structural differences and novel potential virulence mechanisms from the genomes of multiple Campylobacter species. PLoS Biol 2005;3:72–85. [38] Van Looveren M, Daube G, De Zutter L, Dumont J, Lammens C, Wijdooghe M, et al. Antimicrobial susceptibilities of Campylobacter strains isolated from food animals in Belgium. J Antimicrob Chemother 2001;48:235–40. [39] Wilson IG. Antibiotic resistance of Campylobacter in raw retail chickens and imported chicken portions. Epidemiol Infect 2003;131:1181–6. [40] Ge B, White DG, McDermott PF, Girard W, Zhao S, Hubert S, et al. Antimicrobial-resistant Campylobacter species from retail raw meats. Appl Environ Microb 2003;69:3005–7. [41] Angelovski L, Sekulovski P, Jankuloski D, Ratkova M, Prodanov M, Kostova S. Antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolates from broiler flocks. Maced Vet Rev 2011;34:15–8. [42] Tang JYH, Mohamad Ghazali F, Saleha AA, Nishibuchi M, Son R. Comparison of thermophilic Campylobacter spp. occurrence in two types of retail chicken samples. Int Food Res J 2009;16:277–88. [43] Luber P, Wagner J, Hahn H, Bartelt E. Antimicrobial resistance in Campylobacter jejuni and Campylobacter coli strains isolated in 1991 and 2001–2002 from poultry and humans in Berlin, Germany. Antimicrob Agents Chemother 2003;47:3825–30. [44] Moore JE, Barton MD, Blair IS, Corcoran D, Dooley JSG, Fanning S, et al. The epidemiology of antibiotic resistance in Campylobacter. Microbes Infect 2006;8:1955–66.
