Quantitative prevalence and characterization of Campylobacter from chicken and duck carcasses from poultry slaughterhouses in South Korea

Quantitative prevalence and characterization of Campylobacter from chicken and duck carcasses... Abstract The objective of this study was to assess the quantitative prevalence, antibiotic resistance, and molecular subtyping pattern of Campylobacter isolates from chicken and duck products from poultry slaughterhouses in South Korea. A total of 240 chicken (n = 120) and duck (n = 120) carcass samples collected from 12 poultry slaughterhouses between June 2014 and February 2015 in 12 South Korean cities was tested, and 131 samples were positive for Campylobacter. Duck samples showed a higher prevalence (P < 0.05; 93 out of 120) compared to chicken samples (38 out of 120), whereas Campylobacter cell populations from positives were lower (P < 0.05) in ducks (mean count: 183.8 CFU/mL) than in chicken samples (mean count: 499.7 CFU/mL). Most isolates were resistant to nalidixic acid (93.9%), ciprofloxacin (95.4%), tetracycline (72.5%), or enrofloxacin (88.5%), but only a few strains were resistant to chloramphenicol (0.8%) or erythromycin (3.1%). Most of the tested strains were classified into diverse pulsotypes according to repetitive element sequence-based-PCR banding patterns, indicating the diversity of Campylobacter isolates present in chicken and duck samples from poultry slaughterhouses. The emergence of Campylobacter contamination and antibiotic-resistant strains in food animals poses a potential risk to public health and should be regularly monitored for developing proper control measures. INTRODUCTION Campylobacter spp. are among the most common causes of human bacterial enteritis worldwide (Alfredson and Korolik, 2007). Meats, especially poultry meats, are the most frequent source of Campylobacter, although it can be found in other foods (Oyarzabal et al. 2005; Williams et al. 2009; Chon et al., 2011). Campylobacter in raw or undercooked poultry products can be directly ingested by humans; this is a serious public health concern, and trends in the prevalence of Campylobacter should be monitored periodically (Nachamkin and Blaser, 2000; Belanger and Shryock, 2007). Campylobacter spp. are a natural part of the intestinal flora in poultry, and the number of Campylobacter in a carcass rinse can vary when present (Line et al., 2001; Josefsen et al., 2003; Line and Berrang, 2005; Oyarzabal et al., 2005; Belanger and Shryock, 2007). Although the enumeration of Campylobacter on chicken carcasses has been a major goal for many food quality-control authorities (Josefsen et al., 2003), there have been very few reports providing quantitative data of Campylobacter in South Korea because Campylobacter is regulated with zero tolerance by Korean food authorities in all types of foods (Korea Food and Drug Administration, 2014). Considering the goal of total elimination of Campylobacter from poultry, the quantitative evaluation of Campylobacter in poultry samples is important. The emergence of antibiotic-resistant strains among food animals and humans is of great public health concern in both developed and developing countries and thus should be monitored (Alfredson and Korolik, 2007). Pathogens resistant to antibiotics may transfer from animal products to humans through the food-chain or by direct contact (Barton, 2000). The heavy use of antibiotics in animal husbandry and veterinary medicine has been suggested as a cause of antibiotic resistance in human isolates (Hong et al., 2007). In South Korea, both antibiotic use and antibiotic resistance in Campylobacter isolates from meats are higher than in other countries (Bardon et al., 2009; Lim et al., 2014). Although most people recover without treatment, campylobacteriosis requires intensive care, especially in seniors, children, and immunodeficient patients (Nachamkin and Blaser, 2000; Feodoroff et al., 2011). However, elevated antibiotic resistance makes campylobacteriosis in humans more difficult to treat (Kang et al., 2006; Alfredson and Korolik, 2007). The molecular characterization using subtyping methods for epidemiological Campylobacter tracing is important in surveillance for potential bacterial pathogens in various sample types. The automated repetitive element sequence-based PCR (rep-PCR) system (DiversiLab) has been shown to offer superior typeability and similar discriminatory power when compared with pulsed field gel electrophoresis, while saving time and labor, when identifying Campylobacter strains from various sources (Healy et al., 2005; Abay et al., 2014). The objective of this study was to quantitatively determine the prevalence of Campylobacter isolates from chicken and duck products from poultry slaughterhouses in South Korea. The antibiotic-resistance patterns of isolated strains were also analyzed. In addition, molecular characterization using automated rep-PCR was conducted to assess the genetic similarities and intraspecific biodiversities of Campylobacter isolates from various poultry slaughterhouses. MATERIALS AND METHODS Sample Collection A total of 240 chicken and duck carcass samples were collected between June 2014 and February 2015 from 12 authorized poultry slaughterhouses, each located in 12 different cities of South Korea [chicken slaughterhouses (A to F; A, Dongducheon-si; B, Jincheon-gun; C, Yangju-si; D, Iksan-si; E, Taegue-si; F, Gunsan si) and duck slaughterhouses (G to L; G, Naju-si; H, Paju-si; I, Jangheung-gun; J, Buan-gun; K, Eumseong-gun; L, Naju-si). The processing capacities of slaughterhouse A, B, C, D, E, F, G, H, I, J, K, and L are 27,000, 195,000, 25,000, 400,000, 60,000, 64,000, 20,000, 12,000, 65,000, 13,000, 24,000, and 30,000 poultry per day, respectively. From June to August 2014 (summer season), 10 samples each from 12 slaughterhouses were collected. With a 6-mo interval, from December 2014 to February 2015 (winter season), we revisited the 12 slaughterhouses and collected an additional 120 samples (10 samples from each slaughterhouse). The fully processed and packed poultry product samples were collected. The samples were transported to the laboratory on ice within 1 d and stored at 4°C. All samples were analyzed within 24 h. Enumeration and Isolation of Campylobacter from Poultry Carcass Rinses All experimental procedures for the enumeration and isolation of Campylobacter were performed as described in the detection protocols of the Korea Food and Drug Administration (2014) and the U.S. Food Safety and Inspection Service (2013) with minor modifications for the qualitative and quantitative detection, respectively. Briefly, carcasses were rinsed with 400 mL of buffered peptone water (Difco, Sparks, MD) by gentle shaking for up to 1 min. After shaking, 1 mL of the rinsed samples was inoculated onto 4 modified charcoal-cefoperazone-deoxycholate agar (mCCDA; Oxoid, Hampshire, UK) plates (0.25 mL each), and 0.1 mL was inoculated onto 2 additional selective agars for quantitative Campylobacter detection. The plates were incubated at 42°C for 48 h microaerobically (5% O2, 10% CO2, and 85% N2), and then the number of suspected colonies on each plate was counted. For the qualitative detection of Campylobacter, the rinse fluid (25 mL) was enriched with an equal volume of 2 × blood-free Bolton enrichment broth (Oxoid), followed by incubation at 42°C for 48 h. A loopful of enrichment broth was inoculated onto a mCCDA plate and then incubated at 42°C for 48 h under microaerobic conditions. Small, round, grey, and translucent colonies were regarded as Campylobacter, otherwise it was considered as non-Campylobacter. To screen for Campylobacter colonies, suspected colonies (up to 5 per plate) from each plate were subcultured under aerobic and microaerobic conditions on blood agar (bioMérieux, Marcy l’Etoile, France). Colonies grown only under microaerobic conditions were selected and Campylobacter spp. were finally confirmed by colony PCR, according to the methods described by Yamazaki-Matsune et al. (2007). The number of Campylobacter per millimeter of chicken carcass rinse was calculated after considering the number of positive colonies and the inoculation level. Both qualitative and quantitative detection were concurrently conducted with the same sample portion, and the difference in the number of positives between 2 methods was also compared. To further characterize the isolates, at least 1 isolate from each Campylobacter-positive sample was identified at the species level by using colony PCR. The identified strains were stocked at −70°C using bead stock. Antibiotic Susceptibility Testing To test the antibiotic susceptibilities of Campylobacter isolates, we used the agar dilution method with nalidixic acid (Sigma-Aldrich), tetracycline (Sigma-Aldrich), chloramphenicol (Sigma-Aldrich), enrofloxacin (Sigma-Aldrich), ciprofloxacin (Sigma-Aldrich), and erythromycin (Sigma-Aldrich) according to the guidelines of Clinical and Laboratory Standards Institute (CLSI, 2010). Because the guidelines of CLSI provides breakpoints for erytromycin, ciprofloxacin, tetra-, and doxycycline, the breakpoints of nalidixic acid, chloramphenicol, and enrofloxacin were defined according to other published studies (Hong et al., 2007; Ge et al., 2013; Kim et al., 2013). The 7 antibiotics were individually combined with Mueller–Hinton agar (MHA) supplemented with 5% lysed horse blood (Oxoid). The final concentrations of all antibiotics in MHA ranged from 0.5 to 128 μg/mL. Isolates stored at −70°C were streaked onto blood agar, followed by suspension in Mueller–Hinton broth (MHB) to achieve a MacFarland turbidity of 0.5 for antibiotic-susceptibility testing. Bacteria suspended in MHB were inoculated onto MHA plates using a sterile swab and incubated under microaerobic conditions. The lowest antibiotic concentrations inhibiting bacterial growth were taken as the minimum inhibitory concentrations of the tested strains. Campylobacter jejuni ATCC 33,560 was used as the reference. rep-PCR DNA Fingerprinting For molecular subtyping, we selected 40 strains (25 Campylobacter jejuni and 15 Campylobacter coli isolates; 13 from chickens and 27 from ducks) among 131 strains. To get an overview of molecular character of isolates, 40 out of 131 isolates were selected for DiversiLab subtyping in consideration of their source of contamination, source of animals, and bacterial species. Campylobacter strains were incubated on blood agar overnight, and genomic DNA was extracted from colonies using the NucliSENS easyMAG platform (bioMérieux). The extracted genomic DNA was amplified using the DiversiLab Campylobacter PCR Kit (bioMérieux) according to the manufacturer's instructions. The amplified products were placed in a microfluidics chip, and electrophoresis was conducted using a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Dendrograms and subtyping banding patterns were generated by determining phylogenetic distances using the unweighted pair-group method in combination with the arithmetic mean method. The web-based DiversiLab software automatically analyzed and compared the subtyping patterns by using extended Jaccard coefficient. Based on cut-off value provided by Similarity Matrix of DiversiLab software, isolates having >95% similarity regarded genetically similar. RESULTS AND DISCUSSION Quantitative Prevalence of Campylobacter Our results regarding the enumeration and isolation of Campylobacter from chicken and duck carcasses from 12 poultry slaughterhouses are summarized in Table 1. Of the 240 chicken and duck samples, 131 (54.5%) were positive for Campylobacter. The prevalence of Campylobacter was higher (P < 0.05) in ducks (93 out of 120, 77.5%), compared to that of chickens (38 out of 120, 31.7%; Table 1). However, the Campylobacter cell populations from positive samples were lower in abundance (P < 0.05) in duck samples (mean count, 183.8 CFU/mL) than in chicken samples (mean count, 499.7 CFU/mL; Table 1). Chicken slaughterhouses B and D showed the highest prevalence (both 10 out of 20, 50%) and cell population of Campylobacter (B, 657.0 CFU/mL; D, 877.5 CFU/mL), whereas no positive sample for Campylobacter was observed from slaughterhouse E (Table 1). The prevalence and contamination level of Campylobacter in duck slaughterhouses ranged from 60 to 90% and from 22.9 to 312.3 CFU/mL, respectively (Table 1). The differences in the prevalence and levels of Campylobacter between the first (in summer season) and the second (in winter season) visits were more significant in chicken than in duck samples. The prevalence of Campylobacter in chickens and ducks decreased from the first to the second visit (58 to 0.5%, and from 91.7 to 61.7%). The cell population in chickens dropped from 541.8 CFU/mL in the first visit to 8.7 CFU/mL in the second visit, whereas the cell population increased from 154.1 to 226.9 CFU/mL in ducks in the same period. At least 1 isolate from each Campylobacter-positive sample was identified at the species level and stocked for the further characterization the isolates as described above. The 131 strains were identified at the species level, and 43 stains were identified as Campylobacter coli whereas 88 were Campylobacter jejuni. Table 1. The number of Campylobacter-positive samples and cells populations from chicken and duck carcasses in 12 poultry slaughterhouses. Number of positives1/number of total samples (%) Cell population in Campylobacter-positive samples (CFU/mL, Mean±SD2) Sample Slaughter house First visit (Jun to Aug, 2014) Second visit (Dec to Feb, 2014) Total3 First visit (Jun to Aug, 2014) Second visit (Dec to Feb, 2014) Total Chicken A 8/10 (80) ND 8/20 (40) 160.5 ± 238.2 ND 160.5 ± 90.0 B 10/10 (100) ND 10/20 (50) 657.0 ± 592.2 ND 657.0 ± 197.4 C 7/10 (70) ND 7/20 (35) 333.3 ± 623.2 ND 333.3 ± 254.4 D 10/10 (100) ND 10/20 (50) 877.5 ± 714.3 ND 877.5 ± 238.1 E ND ND ND ND4 ND ND F ND 3/10 (15) 3/20 (15) ND 8.7 ± 6.6 8.7 ± 4.7 Total 35/60 (58.3) 3/60 (0.5) 38/120 (31.7) A 541.8 ± 643.8 8.7 ± 6.6 499.7 ± 634.4 Duck G 10/10 (100) 7/10 (70) 17/20 (85) 459.3 ± 435.8 50.7 ± 24.2 291.1 ± 390.4 H 10/10 (100) 6/10 (60) 16/20 (80) 146.8 ± 183.8 260.7 ± 458.5 189.5 ± 320.9 I 9/10 (90) 9/10 (90) 18/20 (90) 55.2 ± 84.5 227.1 ± 360.3 141.2 ± 275.4 J 8/10 (80) 4/10 (40) 12/20 (60) 8.9 ± 8.5 51.0 ± 50.9 22.9 ± 36.2 K 8/10 (80) 10/10 (100) 18/20 (90) 64.3 ± 65.2 203.9 ± 268.3 141.8 ± 216.1 L 10/10 (100) 2/10 (20) 12/20 (60) 133.2 ± 118.2 1208.0 ± 4.0 312.3 ± 414.8 Total 55/60 (91.7) 38/60 (61.7) 93/120 (77.5) B 154.1 ± 260.5 226.9 ± 378.4 183.8 ± 316.1 Number of positives1/number of total samples (%) Cell population in Campylobacter-positive samples (CFU/mL, Mean±SD2) Sample Slaughter house First visit (Jun to Aug, 2014) Second visit (Dec to Feb, 2014) Total3 First visit (Jun to Aug, 2014) Second visit (Dec to Feb, 2014) Total Chicken A 8/10 (80) ND 8/20 (40) 160.5 ± 238.2 ND 160.5 ± 90.0 B 10/10 (100) ND 10/20 (50) 657.0 ± 592.2 ND 657.0 ± 197.4 C 7/10 (70) ND 7/20 (35) 333.3 ± 623.2 ND 333.3 ± 254.4 D 10/10 (100) ND 10/20 (50) 877.5 ± 714.3 ND 877.5 ± 238.1 E ND ND ND ND4 ND ND F ND 3/10 (15) 3/20 (15) ND 8.7 ± 6.6 8.7 ± 4.7 Total 35/60 (58.3) 3/60 (0.5) 38/120 (31.7) A 541.8 ± 643.8 8.7 ± 6.6 499.7 ± 634.4 Duck G 10/10 (100) 7/10 (70) 17/20 (85) 459.3 ± 435.8 50.7 ± 24.2 291.1 ± 390.4 H 10/10 (100) 6/10 (60) 16/20 (80) 146.8 ± 183.8 260.7 ± 458.5 189.5 ± 320.9 I 9/10 (90) 9/10 (90) 18/20 (90) 55.2 ± 84.5 227.1 ± 360.3 141.2 ± 275.4 J 8/10 (80) 4/10 (40) 12/20 (60) 8.9 ± 8.5 51.0 ± 50.9 22.9 ± 36.2 K 8/10 (80) 10/10 (100) 18/20 (90) 64.3 ± 65.2 203.9 ± 268.3 141.8 ± 216.1 L 10/10 (100) 2/10 (20) 12/20 (60) 133.2 ± 118.2 1208.0 ± 4.0 312.3 ± 414.8 Total 55/60 (91.7) 38/60 (61.7) 93/120 (77.5) B 154.1 ± 260.5 226.9 ± 378.4 183.8 ± 316.1 1Positives by either detection with enrichment or without enrichment. 2Mean ± SD was determined only with Campylobacter-positive samples. 3The numbers of samples positive for Campylobacter were compared in pairs using Fisher's exact test. Different upper letters within a column (A and B) indicate a difference (P < 0.05). 4ND, Not detected. View Large Table 1. The number of Campylobacter-positive samples and cells populations from chicken and duck carcasses in 12 poultry slaughterhouses. Number of positives1/number of total samples (%) Cell population in Campylobacter-positive samples (CFU/mL, Mean±SD2) Sample Slaughter house First visit (Jun to Aug, 2014) Second visit (Dec to Feb, 2014) Total3 First visit (Jun to Aug, 2014) Second visit (Dec to Feb, 2014) Total Chicken A 8/10 (80) ND 8/20 (40) 160.5 ± 238.2 ND 160.5 ± 90.0 B 10/10 (100) ND 10/20 (50) 657.0 ± 592.2 ND 657.0 ± 197.4 C 7/10 (70) ND 7/20 (35) 333.3 ± 623.2 ND 333.3 ± 254.4 D 10/10 (100) ND 10/20 (50) 877.5 ± 714.3 ND 877.5 ± 238.1 E ND ND ND ND4 ND ND F ND 3/10 (15) 3/20 (15) ND 8.7 ± 6.6 8.7 ± 4.7 Total 35/60 (58.3) 3/60 (0.5) 38/120 (31.7) A 541.8 ± 643.8 8.7 ± 6.6 499.7 ± 634.4 Duck G 10/10 (100) 7/10 (70) 17/20 (85) 459.3 ± 435.8 50.7 ± 24.2 291.1 ± 390.4 H 10/10 (100) 6/10 (60) 16/20 (80) 146.8 ± 183.8 260.7 ± 458.5 189.5 ± 320.9 I 9/10 (90) 9/10 (90) 18/20 (90) 55.2 ± 84.5 227.1 ± 360.3 141.2 ± 275.4 J 8/10 (80) 4/10 (40) 12/20 (60) 8.9 ± 8.5 51.0 ± 50.9 22.9 ± 36.2 K 8/10 (80) 10/10 (100) 18/20 (90) 64.3 ± 65.2 203.9 ± 268.3 141.8 ± 216.1 L 10/10 (100) 2/10 (20) 12/20 (60) 133.2 ± 118.2 1208.0 ± 4.0 312.3 ± 414.8 Total 55/60 (91.7) 38/60 (61.7) 93/120 (77.5) B 154.1 ± 260.5 226.9 ± 378.4 183.8 ± 316.1 Number of positives1/number of total samples (%) Cell population in Campylobacter-positive samples (CFU/mL, Mean±SD2) Sample Slaughter house First visit (Jun to Aug, 2014) Second visit (Dec to Feb, 2014) Total3 First visit (Jun to Aug, 2014) Second visit (Dec to Feb, 2014) Total Chicken A 8/10 (80) ND 8/20 (40) 160.5 ± 238.2 ND 160.5 ± 90.0 B 10/10 (100) ND 10/20 (50) 657.0 ± 592.2 ND 657.0 ± 197.4 C 7/10 (70) ND 7/20 (35) 333.3 ± 623.2 ND 333.3 ± 254.4 D 10/10 (100) ND 10/20 (50) 877.5 ± 714.3 ND 877.5 ± 238.1 E ND ND ND ND4 ND ND F ND 3/10 (15) 3/20 (15) ND 8.7 ± 6.6 8.7 ± 4.7 Total 35/60 (58.3) 3/60 (0.5) 38/120 (31.7) A 541.8 ± 643.8 8.7 ± 6.6 499.7 ± 634.4 Duck G 10/10 (100) 7/10 (70) 17/20 (85) 459.3 ± 435.8 50.7 ± 24.2 291.1 ± 390.4 H 10/10 (100) 6/10 (60) 16/20 (80) 146.8 ± 183.8 260.7 ± 458.5 189.5 ± 320.9 I 9/10 (90) 9/10 (90) 18/20 (90) 55.2 ± 84.5 227.1 ± 360.3 141.2 ± 275.4 J 8/10 (80) 4/10 (40) 12/20 (60) 8.9 ± 8.5 51.0 ± 50.9 22.9 ± 36.2 K 8/10 (80) 10/10 (100) 18/20 (90) 64.3 ± 65.2 203.9 ± 268.3 141.8 ± 216.1 L 10/10 (100) 2/10 (20) 12/20 (60) 133.2 ± 118.2 1208.0 ± 4.0 312.3 ± 414.8 Total 55/60 (91.7) 38/60 (61.7) 93/120 (77.5) B 154.1 ± 260.5 226.9 ± 378.4 183.8 ± 316.1 1Positives by either detection with enrichment or without enrichment. 2Mean ± SD was determined only with Campylobacter-positive samples. 3The numbers of samples positive for Campylobacter were compared in pairs using Fisher's exact test. Different upper letters within a column (A and B) indicate a difference (P < 0.05). 4ND, Not detected. View Large The detection rates in the present study are in general agreement with other published studies conducted in South Korea that showed that the prevalence commonly ranged from 37.4 to 90% in chicken products (Kang et al., 2006; Hong et al., 2007; Kim et al., 2010; Park et al., 2010). When Campylobacter spp. are present in a chicken carcass rinses, their numbers per milliliter in the carcass rinses can vary from 0 to 3 log CFU (Line, 2001; Line et al., 2001; Line and Berrang, 2005; Oyarzabal et al., 2005; Stern et al., 2007; Chon et al., 2014). The cell populations of Campylobacter from our study were somewhat lower than that of Kang et al. (2006), who reported a mean value of 335.6 CFU/g from retail chicken samples in South Korea. Duck production, accounting for 13.4% of animal husbandry, has increased more rapidly than that of any other meat product in South Korea since 2001 (Food and Agriculture Organization, 2014; Wei et al., 2014). Although duck products are frequently consumed in Asian countries, including South Korea, only limited number of reports on Campylobacter contamination in duck products are available (Wei et al., 2014). Kim et al. (2013) reported that 32.9% of duck cecum samples collected in the slaughterhouse were positive for Campylobacter. Wei et al. (2014) also investigated duck cloacal swabs collected from 58 farms, and reported that 96.6% of samples were Campylobacter-positive. However, few reports have provided quantitative data regarding presence of Campylobacter in duck products in South Korea, making comparison with the present quantitative data difficult. Thus, the results from the current study provide useful data for quantitative risk assessment of Campylobacter contamination in poultry slaughterhouses of South Korea. Considering the difference in the cell population of Campylobacter observed between the 2 visits, the potential risk of consuming duck products would be higher in the winter season than in the summer season (Table 1). The prevalence and levels of Campylobacter varied between poultry slaughterhouses, especially in 6 chicken slaughterhouses. The discrepancy between slaughterhouses might be related to differences in avian species, slaughtering processing, the hygiene and sanitation levels of each slaughterhouse, and the related farms supplying poultries to the slaughterhouses, as well as seasonal difference (Kim et al., 2012; Wei et al., 2014; Chon et al., 2015). In this study, both qualitative and quantitative detection were concurrently conducted with the same sample portion. The difference in the number of positives between qualitative and quantitative detection is presented in Table 2. Interestingly, the qualitative detection, involving an enrichment step, showed much lower isolation rate than quantitative detection in both chickens (10 vs. 38 out of 120 samples) and ducks (48 vs. 91 out of 120 samples, Table 2). Our finding is consistent with the results of previous studies that showed a higher prevalence rate with direct plating than with selective enrichment during the isolation of Campylobacter from chicken samples (Musgrove et al., 2001; Habib et al., 2008). Although the enrichment step is critical for recovering sub-lethal and injured cells, overgrowth of competing microflora in broth culture highly inhibited the detection of target bacteria (Jasson et al., 2009; Moran et al., 2011). This indicates that the selectivity of broth media during the enrichment step should be improved to effectively exclude competing microbiota that may concurrently grow in broth culture along with Campylobacter. Table 2. The number of Campylobacter-positive samples with or without enrichment step from chicken and duck carcasses in 12 poultry slaughterhouses. No. of positives/No. of total samples (%) Sample w enrichment wo enrichment Total1 Chicken2 10/120 (8.3) A 38/120 (31.7) B 38/120 (31.7) Duck2 48/120 (40.0) A 91/120 (75.8) B 93/120 (77.5) No. of positives/No. of total samples (%) Sample w enrichment wo enrichment Total1 Chicken2 10/120 (8.3) A 38/120 (31.7) B 38/120 (31.7) Duck2 48/120 (40.0) A 91/120 (75.8) B 93/120 (77.5) 1Positives by either detection with enrichment or without enrichment. 2The numbers of samples positive for Campylobacter were compared in pairs using Fisher's exact test. Different upper letters within a row (A and B) indicate a difference (P < 0.05). View Large Table 2. The number of Campylobacter-positive samples with or without enrichment step from chicken and duck carcasses in 12 poultry slaughterhouses. No. of positives/No. of total samples (%) Sample w enrichment wo enrichment Total1 Chicken2 10/120 (8.3) A 38/120 (31.7) B 38/120 (31.7) Duck2 48/120 (40.0) A 91/120 (75.8) B 93/120 (77.5) No. of positives/No. of total samples (%) Sample w enrichment wo enrichment Total1 Chicken2 10/120 (8.3) A 38/120 (31.7) B 38/120 (31.7) Duck2 48/120 (40.0) A 91/120 (75.8) B 93/120 (77.5) 1Positives by either detection with enrichment or without enrichment. 2The numbers of samples positive for Campylobacter were compared in pairs using Fisher's exact test. Different upper letters within a row (A and B) indicate a difference (P < 0.05). View Large Antibiotic-Resistance Profiles of Isolates The antibiotic-resistance profiles of the isolates are shown in Table 3. Most isolates were resistant to ciprofloxacin (95.4%), enrofloxacin (88.5%), nalidixic acid (93.9%), or tetracycline (72.5%), but only less than 4% of strains were resistant to erythromycin (3.1%) or chloramphenicol (0.8%), as shown in Table 3. No notable differences were observed in terms of antibiotic resistance between Campylobacter jejuni and Campylobacter coli (Table 3) and the isolates from chicken and duck samples (data not shown). Table 3. Comparison of antibiotic-resistance profiles and minimum inhibitory concentrations (MICs) for 131 Campylobacter isolates from chicken and duck carcasses in 12 poultry slaughterhouses. Number of isolates with each indicated MIC Antimicrobial agents Speices 0.5< 1 2 4 8 16 32 64 128 >256 n1 R (%) Ciprofloxacin jejuni 0 0 0 0 1 31 46 9 1 0 88 100 coli 6 0 0 0 4 20 5 5 0 3 37 86.0 Total 6 0 0 02 5 51 51 14 1 3 125 95.4 Enrofloxacin jejuni 2 1 3 16 44 19 3 0 0 0 82 93.2 coli 6 0 3 15 9 3 4 1 1 1 34 79.1 Total 8 1 6 312 53 22 7 1 1 1 116 88.5 Nalidixic acid jejuni 0 0 0 0 0 0 1 1 32 54 87 98.9 coli 0 0 0 0 2 4 1 0 17 19 36 83.7 Total 0 0 0 0 2 4 2 12 49 73 123 93.9 Tetracycline jejuni 12 10 1 1 0 0 0 0 28 36 64 72.7 coli 5 5 0 0 2 0 0 1 5 25 31 72.1 Total 17 15 1 1 2 02 0 1 32 62 95 72.5 Erythromycin jejuni 15 25 48 0 0 0 0 0 0 0 0 0 coli 19 4 13 3 0 0 0 3 0 1 4 9.3 Total 34 29 61 3 0 0 02 3 0 1 4 3.1 Chloramphenicol jejuni 10 2 26 19 23 7 1 0 0 0 1 1.1 coli 4 2 13 20 1 3 0 0 0 0 0 0 Total 14 4 39 39 24 10 12 0 0 0 1 0.8 Number of isolates with each indicated MIC Antimicrobial agents Speices 0.5< 1 2 4 8 16 32 64 128 >256 n1 R (%) Ciprofloxacin jejuni 0 0 0 0 1 31 46 9 1 0 88 100 coli 6 0 0 0 4 20 5 5 0 3 37 86.0 Total 6 0 0 02 5 51 51 14 1 3 125 95.4 Enrofloxacin jejuni 2 1 3 16 44 19 3 0 0 0 82 93.2 coli 6 0 3 15 9 3 4 1 1 1 34 79.1 Total 8 1 6 312 53 22 7 1 1 1 116 88.5 Nalidixic acid jejuni 0 0 0 0 0 0 1 1 32 54 87 98.9 coli 0 0 0 0 2 4 1 0 17 19 36 83.7 Total 0 0 0 0 2 4 2 12 49 73 123 93.9 Tetracycline jejuni 12 10 1 1 0 0 0 0 28 36 64 72.7 coli 5 5 0 0 2 0 0 1 5 25 31 72.1 Total 17 15 1 1 2 02 0 1 32 62 95 72.5 Erythromycin jejuni 15 25 48 0 0 0 0 0 0 0 0 0 coli 19 4 13 3 0 0 0 3 0 1 4 9.3 Total 34 29 61 3 0 0 02 3 0 1 4 3.1 Chloramphenicol jejuni 10 2 26 19 23 7 1 0 0 0 1 1.1 coli 4 2 13 20 1 3 0 0 0 0 0 0 Total 14 4 39 39 24 10 12 0 0 0 1 0.8 1Number of resistant strains. 2Breakpoint for each antibiotic. View Large Table 3. Comparison of antibiotic-resistance profiles and minimum inhibitory concentrations (MICs) for 131 Campylobacter isolates from chicken and duck carcasses in 12 poultry slaughterhouses. Number of isolates with each indicated MIC Antimicrobial agents Speices 0.5< 1 2 4 8 16 32 64 128 >256 n1 R (%) Ciprofloxacin jejuni 0 0 0 0 1 31 46 9 1 0 88 100 coli 6 0 0 0 4 20 5 5 0 3 37 86.0 Total 6 0 0 02 5 51 51 14 1 3 125 95.4 Enrofloxacin jejuni 2 1 3 16 44 19 3 0 0 0 82 93.2 coli 6 0 3 15 9 3 4 1 1 1 34 79.1 Total 8 1 6 312 53 22 7 1 1 1 116 88.5 Nalidixic acid jejuni 0 0 0 0 0 0 1 1 32 54 87 98.9 coli 0 0 0 0 2 4 1 0 17 19 36 83.7 Total 0 0 0 0 2 4 2 12 49 73 123 93.9 Tetracycline jejuni 12 10 1 1 0 0 0 0 28 36 64 72.7 coli 5 5 0 0 2 0 0 1 5 25 31 72.1 Total 17 15 1 1 2 02 0 1 32 62 95 72.5 Erythromycin jejuni 15 25 48 0 0 0 0 0 0 0 0 0 coli 19 4 13 3 0 0 0 3 0 1 4 9.3 Total 34 29 61 3 0 0 02 3 0 1 4 3.1 Chloramphenicol jejuni 10 2 26 19 23 7 1 0 0 0 1 1.1 coli 4 2 13 20 1 3 0 0 0 0 0 0 Total 14 4 39 39 24 10 12 0 0 0 1 0.8 Number of isolates with each indicated MIC Antimicrobial agents Speices 0.5< 1 2 4 8 16 32 64 128 >256 n1 R (%) Ciprofloxacin jejuni 0 0 0 0 1 31 46 9 1 0 88 100 coli 6 0 0 0 4 20 5 5 0 3 37 86.0 Total 6 0 0 02 5 51 51 14 1 3 125 95.4 Enrofloxacin jejuni 2 1 3 16 44 19 3 0 0 0 82 93.2 coli 6 0 3 15 9 3 4 1 1 1 34 79.1 Total 8 1 6 312 53 22 7 1 1 1 116 88.5 Nalidixic acid jejuni 0 0 0 0 0 0 1 1 32 54 87 98.9 coli 0 0 0 0 2 4 1 0 17 19 36 83.7 Total 0 0 0 0 2 4 2 12 49 73 123 93.9 Tetracycline jejuni 12 10 1 1 0 0 0 0 28 36 64 72.7 coli 5 5 0 0 2 0 0 1 5 25 31 72.1 Total 17 15 1 1 2 02 0 1 32 62 95 72.5 Erythromycin jejuni 15 25 48 0 0 0 0 0 0 0 0 0 coli 19 4 13 3 0 0 0 3 0 1 4 9.3 Total 34 29 61 3 0 0 02 3 0 1 4 3.1 Chloramphenicol jejuni 10 2 26 19 23 7 1 0 0 0 1 1.1 coli 4 2 13 20 1 3 0 0 0 0 0 0 Total 14 4 39 39 24 10 12 0 0 0 1 0.8 1Number of resistant strains. 2Breakpoint for each antibiotic. View Large The high antibiotic resistance observed with Campylobacter isolates from poultry samples to certain antibiotics is in agreement with previous studies conducted in South Korea, which reported resistances of 68 to 97% to ciprofloxacin, 58 to 84% to enrofloxacin, 71 to 100% to nalidixic acid, and 60 to 95% to tetracycline (Kang et al., 2006; Hong et al., 2007; Kim et al., 2010; Chae et al., 2011; Kim et al., 2013; Wei et al., 2014). Although the rates of antibiotic resistance found in this study agree with other studies performed in South Korea, antibiotic resistance, especially to fluoroquinolone, is much higher than in studies conducted in other countries (Gyles, 2008; Bardon et al., 2009; Bernadette et al., 2012; Garin et al., 2012; Wieczorek and Osek, 2015; Mäesaar et al., 2016). Fluoroquinolones, such as nalidixic acid, ciprofloxacin, and enrofloxacin are important antibiotics because they are the primary choice for treating campylobacteriosis (Ge et al., 2003; Kang et al., 2006; Alfredson and Korolik, 2007). These agents have been used for many years in veterinary medicine and poultry husbandry in Korea (Hong et al., 2007). Enrofloxacin, in particular, has been widely used since 1987 to treat Escherichia coli infection in poultry husbandry, and such resistance is related to enhanced resistance to other quinolones, such as ciprofloxacin, because they have closely related structures (McDermott and Bodeis, 2002; Hong et al., 2007; Pallo-Zimmerman et al., 2010). The resistance of pathogenic bacteria to antibiotics limits the available therapeutic options (Alfredson and Korolik, 2007). Tetracycline has been the most widely used antibiotic agent in animal husbandry in South Korea, leading to high rates of resistance (Lim et al., 2014). Macrolide resistance was relatively rare compared with resistance to other agents, such as fluoroquinolones or tetracycline. Although only 3.1% of the isolates were resistant to erythromycin, this should be continually monitored because these are the antibiotics of choice for treating campylobacteriosis, which is unresponsive to fluoroquinolones (Ge et al., 2003; Kang et al., 2006; Alfredson and Korolik, 2007). Although the use of specific antibiotics such as fluoroquinolones as a feed supplement has been banned in South Korea since 2011 (Kim et al., 2012; Lim et al., 2014), resistance to fluoroquinolones does not appear to have declined. The use of antibiotics in animals is associated with resistant Campylobacter isolates in humans (Alfredson and Korolik, 2007). Both duck and chicken may transmit Campylobacter having multidrug-resistance to human through a food-chain (Wei et al., 2014). The results from the current study show that it is necessary to strengthen the implementation of specific procedures in South Korea to reduce antibiotic resistance in Campylobacter. Molecular Characterization of Isolates using Rep-PCR Fingerprinting The collected Campylobacter isolates subtyped using an automated rep-PCR system to identify the relatedness among the isolates from poultry slaughterhouses. As we stated above, we selected 40 strains from 131 strains in consideration of their source of contamination, source of animals, and bacterial species. From 2 to 10 Campylobacter isolates were isolated from 17 sampling trials in 11 slaughterhouses [A (first), B (first), C (first), D (first), F (second), G (first/second), H (first/second), I (first/second), J (first/second), K (first/second), and L (first/second)], and at least two strains from each sampling were included in the subtyping test. The computer-generated virtual gel images, dendrogram analysis patterns, and profiles of each strain following rep-PCR are presented in Figure 1. Figure 1. View largeDownload slide Computer-generated virtual gel images and a dendrogram of 40 Campylobacter jejuni and Campylobacter coli strains isolated from chicken and duck carcasses in 12 poultry slaughterhouses prepared by DiversiLab rep-PCR fingerprinting. The isolates having > 95% similarity (cut-off bar) in their rep-PCR banding patterns were classified into the same category. First, first visit-samples collected from June to August 2014 (summer season);second, second visit-samples collected from December 2014 to February 2015 (winter season) Figure 1. View largeDownload slide Computer-generated virtual gel images and a dendrogram of 40 Campylobacter jejuni and Campylobacter coli strains isolated from chicken and duck carcasses in 12 poultry slaughterhouses prepared by DiversiLab rep-PCR fingerprinting. The isolates having > 95% similarity (cut-off bar) in their rep-PCR banding patterns were classified into the same category. First, first visit-samples collected from June to August 2014 (summer season);second, second visit-samples collected from December 2014 to February 2015 (winter season) All strains were classified into 36 subtypes with less than 95% similarity according to the rep-PCR banding patterns (Figure 1). These results indicated the diversity of Campylobacter isolates present in chicken and duck samples from poultry slaughterhouses. Except for Key numbers 7 and 8, the banding patterns of 2 species, Campylobacter jejuni and Campylobacter coli, were well distinguished from each other by rep-PCR (Figure 1). Most strains originated from same slaughterhouses were considered genetically unrelated. However, some strain pairs such as Key numbers 5 to 6, 7 to 8, 9 to 10, and 18 to19 showed similar subtyping patterns with more than 95% similarity, despite the use of individually packed products (Figure 1). Each strain pair was not distinguished by antibiotic resistance. Those genetically related strains were obtained from the same slaughterhouses in the same visit, suggesting that they may have originated from either the same farm that supplied live poultry to a specific slaughterhouse or from cross-contamination during the slaughter processing step (Chon et al., 2015). Strains from the second visit were also distinguished from strains from first visit, indicating that no strain persisted for a long time (6 mo) in the slaughterhouse environments. In this study, the quantitative prevalence, antibiotic resistance, and molecular subtyping patterns in Campylobacter were surveyed in chickens and ducks from poultry slaughterhouses in South Korea. 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This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Quantitative prevalence and characterization of Campylobacter from chicken and duck carcasses from poultry slaughterhouses in South Korea

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Oxford University Press
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© 2018 Poultry Science Association Inc.
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0032-5791
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1525-3171
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10.3382/ps/pey120
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Abstract

Abstract The objective of this study was to assess the quantitative prevalence, antibiotic resistance, and molecular subtyping pattern of Campylobacter isolates from chicken and duck products from poultry slaughterhouses in South Korea. A total of 240 chicken (n = 120) and duck (n = 120) carcass samples collected from 12 poultry slaughterhouses between June 2014 and February 2015 in 12 South Korean cities was tested, and 131 samples were positive for Campylobacter. Duck samples showed a higher prevalence (P < 0.05; 93 out of 120) compared to chicken samples (38 out of 120), whereas Campylobacter cell populations from positives were lower (P < 0.05) in ducks (mean count: 183.8 CFU/mL) than in chicken samples (mean count: 499.7 CFU/mL). Most isolates were resistant to nalidixic acid (93.9%), ciprofloxacin (95.4%), tetracycline (72.5%), or enrofloxacin (88.5%), but only a few strains were resistant to chloramphenicol (0.8%) or erythromycin (3.1%). Most of the tested strains were classified into diverse pulsotypes according to repetitive element sequence-based-PCR banding patterns, indicating the diversity of Campylobacter isolates present in chicken and duck samples from poultry slaughterhouses. The emergence of Campylobacter contamination and antibiotic-resistant strains in food animals poses a potential risk to public health and should be regularly monitored for developing proper control measures. INTRODUCTION Campylobacter spp. are among the most common causes of human bacterial enteritis worldwide (Alfredson and Korolik, 2007). Meats, especially poultry meats, are the most frequent source of Campylobacter, although it can be found in other foods (Oyarzabal et al. 2005; Williams et al. 2009; Chon et al., 2011). Campylobacter in raw or undercooked poultry products can be directly ingested by humans; this is a serious public health concern, and trends in the prevalence of Campylobacter should be monitored periodically (Nachamkin and Blaser, 2000; Belanger and Shryock, 2007). Campylobacter spp. are a natural part of the intestinal flora in poultry, and the number of Campylobacter in a carcass rinse can vary when present (Line et al., 2001; Josefsen et al., 2003; Line and Berrang, 2005; Oyarzabal et al., 2005; Belanger and Shryock, 2007). Although the enumeration of Campylobacter on chicken carcasses has been a major goal for many food quality-control authorities (Josefsen et al., 2003), there have been very few reports providing quantitative data of Campylobacter in South Korea because Campylobacter is regulated with zero tolerance by Korean food authorities in all types of foods (Korea Food and Drug Administration, 2014). Considering the goal of total elimination of Campylobacter from poultry, the quantitative evaluation of Campylobacter in poultry samples is important. The emergence of antibiotic-resistant strains among food animals and humans is of great public health concern in both developed and developing countries and thus should be monitored (Alfredson and Korolik, 2007). Pathogens resistant to antibiotics may transfer from animal products to humans through the food-chain or by direct contact (Barton, 2000). The heavy use of antibiotics in animal husbandry and veterinary medicine has been suggested as a cause of antibiotic resistance in human isolates (Hong et al., 2007). In South Korea, both antibiotic use and antibiotic resistance in Campylobacter isolates from meats are higher than in other countries (Bardon et al., 2009; Lim et al., 2014). Although most people recover without treatment, campylobacteriosis requires intensive care, especially in seniors, children, and immunodeficient patients (Nachamkin and Blaser, 2000; Feodoroff et al., 2011). However, elevated antibiotic resistance makes campylobacteriosis in humans more difficult to treat (Kang et al., 2006; Alfredson and Korolik, 2007). The molecular characterization using subtyping methods for epidemiological Campylobacter tracing is important in surveillance for potential bacterial pathogens in various sample types. The automated repetitive element sequence-based PCR (rep-PCR) system (DiversiLab) has been shown to offer superior typeability and similar discriminatory power when compared with pulsed field gel electrophoresis, while saving time and labor, when identifying Campylobacter strains from various sources (Healy et al., 2005; Abay et al., 2014). The objective of this study was to quantitatively determine the prevalence of Campylobacter isolates from chicken and duck products from poultry slaughterhouses in South Korea. The antibiotic-resistance patterns of isolated strains were also analyzed. In addition, molecular characterization using automated rep-PCR was conducted to assess the genetic similarities and intraspecific biodiversities of Campylobacter isolates from various poultry slaughterhouses. MATERIALS AND METHODS Sample Collection A total of 240 chicken and duck carcass samples were collected between June 2014 and February 2015 from 12 authorized poultry slaughterhouses, each located in 12 different cities of South Korea [chicken slaughterhouses (A to F; A, Dongducheon-si; B, Jincheon-gun; C, Yangju-si; D, Iksan-si; E, Taegue-si; F, Gunsan si) and duck slaughterhouses (G to L; G, Naju-si; H, Paju-si; I, Jangheung-gun; J, Buan-gun; K, Eumseong-gun; L, Naju-si). The processing capacities of slaughterhouse A, B, C, D, E, F, G, H, I, J, K, and L are 27,000, 195,000, 25,000, 400,000, 60,000, 64,000, 20,000, 12,000, 65,000, 13,000, 24,000, and 30,000 poultry per day, respectively. From June to August 2014 (summer season), 10 samples each from 12 slaughterhouses were collected. With a 6-mo interval, from December 2014 to February 2015 (winter season), we revisited the 12 slaughterhouses and collected an additional 120 samples (10 samples from each slaughterhouse). The fully processed and packed poultry product samples were collected. The samples were transported to the laboratory on ice within 1 d and stored at 4°C. All samples were analyzed within 24 h. Enumeration and Isolation of Campylobacter from Poultry Carcass Rinses All experimental procedures for the enumeration and isolation of Campylobacter were performed as described in the detection protocols of the Korea Food and Drug Administration (2014) and the U.S. Food Safety and Inspection Service (2013) with minor modifications for the qualitative and quantitative detection, respectively. Briefly, carcasses were rinsed with 400 mL of buffered peptone water (Difco, Sparks, MD) by gentle shaking for up to 1 min. After shaking, 1 mL of the rinsed samples was inoculated onto 4 modified charcoal-cefoperazone-deoxycholate agar (mCCDA; Oxoid, Hampshire, UK) plates (0.25 mL each), and 0.1 mL was inoculated onto 2 additional selective agars for quantitative Campylobacter detection. The plates were incubated at 42°C for 48 h microaerobically (5% O2, 10% CO2, and 85% N2), and then the number of suspected colonies on each plate was counted. For the qualitative detection of Campylobacter, the rinse fluid (25 mL) was enriched with an equal volume of 2 × blood-free Bolton enrichment broth (Oxoid), followed by incubation at 42°C for 48 h. A loopful of enrichment broth was inoculated onto a mCCDA plate and then incubated at 42°C for 48 h under microaerobic conditions. Small, round, grey, and translucent colonies were regarded as Campylobacter, otherwise it was considered as non-Campylobacter. To screen for Campylobacter colonies, suspected colonies (up to 5 per plate) from each plate were subcultured under aerobic and microaerobic conditions on blood agar (bioMérieux, Marcy l’Etoile, France). Colonies grown only under microaerobic conditions were selected and Campylobacter spp. were finally confirmed by colony PCR, according to the methods described by Yamazaki-Matsune et al. (2007). The number of Campylobacter per millimeter of chicken carcass rinse was calculated after considering the number of positive colonies and the inoculation level. Both qualitative and quantitative detection were concurrently conducted with the same sample portion, and the difference in the number of positives between 2 methods was also compared. To further characterize the isolates, at least 1 isolate from each Campylobacter-positive sample was identified at the species level by using colony PCR. The identified strains were stocked at −70°C using bead stock. Antibiotic Susceptibility Testing To test the antibiotic susceptibilities of Campylobacter isolates, we used the agar dilution method with nalidixic acid (Sigma-Aldrich), tetracycline (Sigma-Aldrich), chloramphenicol (Sigma-Aldrich), enrofloxacin (Sigma-Aldrich), ciprofloxacin (Sigma-Aldrich), and erythromycin (Sigma-Aldrich) according to the guidelines of Clinical and Laboratory Standards Institute (CLSI, 2010). Because the guidelines of CLSI provides breakpoints for erytromycin, ciprofloxacin, tetra-, and doxycycline, the breakpoints of nalidixic acid, chloramphenicol, and enrofloxacin were defined according to other published studies (Hong et al., 2007; Ge et al., 2013; Kim et al., 2013). The 7 antibiotics were individually combined with Mueller–Hinton agar (MHA) supplemented with 5% lysed horse blood (Oxoid). The final concentrations of all antibiotics in MHA ranged from 0.5 to 128 μg/mL. Isolates stored at −70°C were streaked onto blood agar, followed by suspension in Mueller–Hinton broth (MHB) to achieve a MacFarland turbidity of 0.5 for antibiotic-susceptibility testing. Bacteria suspended in MHB were inoculated onto MHA plates using a sterile swab and incubated under microaerobic conditions. The lowest antibiotic concentrations inhibiting bacterial growth were taken as the minimum inhibitory concentrations of the tested strains. Campylobacter jejuni ATCC 33,560 was used as the reference. rep-PCR DNA Fingerprinting For molecular subtyping, we selected 40 strains (25 Campylobacter jejuni and 15 Campylobacter coli isolates; 13 from chickens and 27 from ducks) among 131 strains. To get an overview of molecular character of isolates, 40 out of 131 isolates were selected for DiversiLab subtyping in consideration of their source of contamination, source of animals, and bacterial species. Campylobacter strains were incubated on blood agar overnight, and genomic DNA was extracted from colonies using the NucliSENS easyMAG platform (bioMérieux). The extracted genomic DNA was amplified using the DiversiLab Campylobacter PCR Kit (bioMérieux) according to the manufacturer's instructions. The amplified products were placed in a microfluidics chip, and electrophoresis was conducted using a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Dendrograms and subtyping banding patterns were generated by determining phylogenetic distances using the unweighted pair-group method in combination with the arithmetic mean method. The web-based DiversiLab software automatically analyzed and compared the subtyping patterns by using extended Jaccard coefficient. Based on cut-off value provided by Similarity Matrix of DiversiLab software, isolates having >95% similarity regarded genetically similar. RESULTS AND DISCUSSION Quantitative Prevalence of Campylobacter Our results regarding the enumeration and isolation of Campylobacter from chicken and duck carcasses from 12 poultry slaughterhouses are summarized in Table 1. Of the 240 chicken and duck samples, 131 (54.5%) were positive for Campylobacter. The prevalence of Campylobacter was higher (P < 0.05) in ducks (93 out of 120, 77.5%), compared to that of chickens (38 out of 120, 31.7%; Table 1). However, the Campylobacter cell populations from positive samples were lower in abundance (P < 0.05) in duck samples (mean count, 183.8 CFU/mL) than in chicken samples (mean count, 499.7 CFU/mL; Table 1). Chicken slaughterhouses B and D showed the highest prevalence (both 10 out of 20, 50%) and cell population of Campylobacter (B, 657.0 CFU/mL; D, 877.5 CFU/mL), whereas no positive sample for Campylobacter was observed from slaughterhouse E (Table 1). The prevalence and contamination level of Campylobacter in duck slaughterhouses ranged from 60 to 90% and from 22.9 to 312.3 CFU/mL, respectively (Table 1). The differences in the prevalence and levels of Campylobacter between the first (in summer season) and the second (in winter season) visits were more significant in chicken than in duck samples. The prevalence of Campylobacter in chickens and ducks decreased from the first to the second visit (58 to 0.5%, and from 91.7 to 61.7%). The cell population in chickens dropped from 541.8 CFU/mL in the first visit to 8.7 CFU/mL in the second visit, whereas the cell population increased from 154.1 to 226.9 CFU/mL in ducks in the same period. At least 1 isolate from each Campylobacter-positive sample was identified at the species level and stocked for the further characterization the isolates as described above. The 131 strains were identified at the species level, and 43 stains were identified as Campylobacter coli whereas 88 were Campylobacter jejuni. Table 1. The number of Campylobacter-positive samples and cells populations from chicken and duck carcasses in 12 poultry slaughterhouses. Number of positives1/number of total samples (%) Cell population in Campylobacter-positive samples (CFU/mL, Mean±SD2) Sample Slaughter house First visit (Jun to Aug, 2014) Second visit (Dec to Feb, 2014) Total3 First visit (Jun to Aug, 2014) Second visit (Dec to Feb, 2014) Total Chicken A 8/10 (80) ND 8/20 (40) 160.5 ± 238.2 ND 160.5 ± 90.0 B 10/10 (100) ND 10/20 (50) 657.0 ± 592.2 ND 657.0 ± 197.4 C 7/10 (70) ND 7/20 (35) 333.3 ± 623.2 ND 333.3 ± 254.4 D 10/10 (100) ND 10/20 (50) 877.5 ± 714.3 ND 877.5 ± 238.1 E ND ND ND ND4 ND ND F ND 3/10 (15) 3/20 (15) ND 8.7 ± 6.6 8.7 ± 4.7 Total 35/60 (58.3) 3/60 (0.5) 38/120 (31.7) A 541.8 ± 643.8 8.7 ± 6.6 499.7 ± 634.4 Duck G 10/10 (100) 7/10 (70) 17/20 (85) 459.3 ± 435.8 50.7 ± 24.2 291.1 ± 390.4 H 10/10 (100) 6/10 (60) 16/20 (80) 146.8 ± 183.8 260.7 ± 458.5 189.5 ± 320.9 I 9/10 (90) 9/10 (90) 18/20 (90) 55.2 ± 84.5 227.1 ± 360.3 141.2 ± 275.4 J 8/10 (80) 4/10 (40) 12/20 (60) 8.9 ± 8.5 51.0 ± 50.9 22.9 ± 36.2 K 8/10 (80) 10/10 (100) 18/20 (90) 64.3 ± 65.2 203.9 ± 268.3 141.8 ± 216.1 L 10/10 (100) 2/10 (20) 12/20 (60) 133.2 ± 118.2 1208.0 ± 4.0 312.3 ± 414.8 Total 55/60 (91.7) 38/60 (61.7) 93/120 (77.5) B 154.1 ± 260.5 226.9 ± 378.4 183.8 ± 316.1 Number of positives1/number of total samples (%) Cell population in Campylobacter-positive samples (CFU/mL, Mean±SD2) Sample Slaughter house First visit (Jun to Aug, 2014) Second visit (Dec to Feb, 2014) Total3 First visit (Jun to Aug, 2014) Second visit (Dec to Feb, 2014) Total Chicken A 8/10 (80) ND 8/20 (40) 160.5 ± 238.2 ND 160.5 ± 90.0 B 10/10 (100) ND 10/20 (50) 657.0 ± 592.2 ND 657.0 ± 197.4 C 7/10 (70) ND 7/20 (35) 333.3 ± 623.2 ND 333.3 ± 254.4 D 10/10 (100) ND 10/20 (50) 877.5 ± 714.3 ND 877.5 ± 238.1 E ND ND ND ND4 ND ND F ND 3/10 (15) 3/20 (15) ND 8.7 ± 6.6 8.7 ± 4.7 Total 35/60 (58.3) 3/60 (0.5) 38/120 (31.7) A 541.8 ± 643.8 8.7 ± 6.6 499.7 ± 634.4 Duck G 10/10 (100) 7/10 (70) 17/20 (85) 459.3 ± 435.8 50.7 ± 24.2 291.1 ± 390.4 H 10/10 (100) 6/10 (60) 16/20 (80) 146.8 ± 183.8 260.7 ± 458.5 189.5 ± 320.9 I 9/10 (90) 9/10 (90) 18/20 (90) 55.2 ± 84.5 227.1 ± 360.3 141.2 ± 275.4 J 8/10 (80) 4/10 (40) 12/20 (60) 8.9 ± 8.5 51.0 ± 50.9 22.9 ± 36.2 K 8/10 (80) 10/10 (100) 18/20 (90) 64.3 ± 65.2 203.9 ± 268.3 141.8 ± 216.1 L 10/10 (100) 2/10 (20) 12/20 (60) 133.2 ± 118.2 1208.0 ± 4.0 312.3 ± 414.8 Total 55/60 (91.7) 38/60 (61.7) 93/120 (77.5) B 154.1 ± 260.5 226.9 ± 378.4 183.8 ± 316.1 1Positives by either detection with enrichment or without enrichment. 2Mean ± SD was determined only with Campylobacter-positive samples. 3The numbers of samples positive for Campylobacter were compared in pairs using Fisher's exact test. Different upper letters within a column (A and B) indicate a difference (P < 0.05). 4ND, Not detected. View Large Table 1. The number of Campylobacter-positive samples and cells populations from chicken and duck carcasses in 12 poultry slaughterhouses. Number of positives1/number of total samples (%) Cell population in Campylobacter-positive samples (CFU/mL, Mean±SD2) Sample Slaughter house First visit (Jun to Aug, 2014) Second visit (Dec to Feb, 2014) Total3 First visit (Jun to Aug, 2014) Second visit (Dec to Feb, 2014) Total Chicken A 8/10 (80) ND 8/20 (40) 160.5 ± 238.2 ND 160.5 ± 90.0 B 10/10 (100) ND 10/20 (50) 657.0 ± 592.2 ND 657.0 ± 197.4 C 7/10 (70) ND 7/20 (35) 333.3 ± 623.2 ND 333.3 ± 254.4 D 10/10 (100) ND 10/20 (50) 877.5 ± 714.3 ND 877.5 ± 238.1 E ND ND ND ND4 ND ND F ND 3/10 (15) 3/20 (15) ND 8.7 ± 6.6 8.7 ± 4.7 Total 35/60 (58.3) 3/60 (0.5) 38/120 (31.7) A 541.8 ± 643.8 8.7 ± 6.6 499.7 ± 634.4 Duck G 10/10 (100) 7/10 (70) 17/20 (85) 459.3 ± 435.8 50.7 ± 24.2 291.1 ± 390.4 H 10/10 (100) 6/10 (60) 16/20 (80) 146.8 ± 183.8 260.7 ± 458.5 189.5 ± 320.9 I 9/10 (90) 9/10 (90) 18/20 (90) 55.2 ± 84.5 227.1 ± 360.3 141.2 ± 275.4 J 8/10 (80) 4/10 (40) 12/20 (60) 8.9 ± 8.5 51.0 ± 50.9 22.9 ± 36.2 K 8/10 (80) 10/10 (100) 18/20 (90) 64.3 ± 65.2 203.9 ± 268.3 141.8 ± 216.1 L 10/10 (100) 2/10 (20) 12/20 (60) 133.2 ± 118.2 1208.0 ± 4.0 312.3 ± 414.8 Total 55/60 (91.7) 38/60 (61.7) 93/120 (77.5) B 154.1 ± 260.5 226.9 ± 378.4 183.8 ± 316.1 Number of positives1/number of total samples (%) Cell population in Campylobacter-positive samples (CFU/mL, Mean±SD2) Sample Slaughter house First visit (Jun to Aug, 2014) Second visit (Dec to Feb, 2014) Total3 First visit (Jun to Aug, 2014) Second visit (Dec to Feb, 2014) Total Chicken A 8/10 (80) ND 8/20 (40) 160.5 ± 238.2 ND 160.5 ± 90.0 B 10/10 (100) ND 10/20 (50) 657.0 ± 592.2 ND 657.0 ± 197.4 C 7/10 (70) ND 7/20 (35) 333.3 ± 623.2 ND 333.3 ± 254.4 D 10/10 (100) ND 10/20 (50) 877.5 ± 714.3 ND 877.5 ± 238.1 E ND ND ND ND4 ND ND F ND 3/10 (15) 3/20 (15) ND 8.7 ± 6.6 8.7 ± 4.7 Total 35/60 (58.3) 3/60 (0.5) 38/120 (31.7) A 541.8 ± 643.8 8.7 ± 6.6 499.7 ± 634.4 Duck G 10/10 (100) 7/10 (70) 17/20 (85) 459.3 ± 435.8 50.7 ± 24.2 291.1 ± 390.4 H 10/10 (100) 6/10 (60) 16/20 (80) 146.8 ± 183.8 260.7 ± 458.5 189.5 ± 320.9 I 9/10 (90) 9/10 (90) 18/20 (90) 55.2 ± 84.5 227.1 ± 360.3 141.2 ± 275.4 J 8/10 (80) 4/10 (40) 12/20 (60) 8.9 ± 8.5 51.0 ± 50.9 22.9 ± 36.2 K 8/10 (80) 10/10 (100) 18/20 (90) 64.3 ± 65.2 203.9 ± 268.3 141.8 ± 216.1 L 10/10 (100) 2/10 (20) 12/20 (60) 133.2 ± 118.2 1208.0 ± 4.0 312.3 ± 414.8 Total 55/60 (91.7) 38/60 (61.7) 93/120 (77.5) B 154.1 ± 260.5 226.9 ± 378.4 183.8 ± 316.1 1Positives by either detection with enrichment or without enrichment. 2Mean ± SD was determined only with Campylobacter-positive samples. 3The numbers of samples positive for Campylobacter were compared in pairs using Fisher's exact test. Different upper letters within a column (A and B) indicate a difference (P < 0.05). 4ND, Not detected. View Large The detection rates in the present study are in general agreement with other published studies conducted in South Korea that showed that the prevalence commonly ranged from 37.4 to 90% in chicken products (Kang et al., 2006; Hong et al., 2007; Kim et al., 2010; Park et al., 2010). When Campylobacter spp. are present in a chicken carcass rinses, their numbers per milliliter in the carcass rinses can vary from 0 to 3 log CFU (Line, 2001; Line et al., 2001; Line and Berrang, 2005; Oyarzabal et al., 2005; Stern et al., 2007; Chon et al., 2014). The cell populations of Campylobacter from our study were somewhat lower than that of Kang et al. (2006), who reported a mean value of 335.6 CFU/g from retail chicken samples in South Korea. Duck production, accounting for 13.4% of animal husbandry, has increased more rapidly than that of any other meat product in South Korea since 2001 (Food and Agriculture Organization, 2014; Wei et al., 2014). Although duck products are frequently consumed in Asian countries, including South Korea, only limited number of reports on Campylobacter contamination in duck products are available (Wei et al., 2014). Kim et al. (2013) reported that 32.9% of duck cecum samples collected in the slaughterhouse were positive for Campylobacter. Wei et al. (2014) also investigated duck cloacal swabs collected from 58 farms, and reported that 96.6% of samples were Campylobacter-positive. However, few reports have provided quantitative data regarding presence of Campylobacter in duck products in South Korea, making comparison with the present quantitative data difficult. Thus, the results from the current study provide useful data for quantitative risk assessment of Campylobacter contamination in poultry slaughterhouses of South Korea. Considering the difference in the cell population of Campylobacter observed between the 2 visits, the potential risk of consuming duck products would be higher in the winter season than in the summer season (Table 1). The prevalence and levels of Campylobacter varied between poultry slaughterhouses, especially in 6 chicken slaughterhouses. The discrepancy between slaughterhouses might be related to differences in avian species, slaughtering processing, the hygiene and sanitation levels of each slaughterhouse, and the related farms supplying poultries to the slaughterhouses, as well as seasonal difference (Kim et al., 2012; Wei et al., 2014; Chon et al., 2015). In this study, both qualitative and quantitative detection were concurrently conducted with the same sample portion. The difference in the number of positives between qualitative and quantitative detection is presented in Table 2. Interestingly, the qualitative detection, involving an enrichment step, showed much lower isolation rate than quantitative detection in both chickens (10 vs. 38 out of 120 samples) and ducks (48 vs. 91 out of 120 samples, Table 2). Our finding is consistent with the results of previous studies that showed a higher prevalence rate with direct plating than with selective enrichment during the isolation of Campylobacter from chicken samples (Musgrove et al., 2001; Habib et al., 2008). Although the enrichment step is critical for recovering sub-lethal and injured cells, overgrowth of competing microflora in broth culture highly inhibited the detection of target bacteria (Jasson et al., 2009; Moran et al., 2011). This indicates that the selectivity of broth media during the enrichment step should be improved to effectively exclude competing microbiota that may concurrently grow in broth culture along with Campylobacter. Table 2. The number of Campylobacter-positive samples with or without enrichment step from chicken and duck carcasses in 12 poultry slaughterhouses. No. of positives/No. of total samples (%) Sample w enrichment wo enrichment Total1 Chicken2 10/120 (8.3) A 38/120 (31.7) B 38/120 (31.7) Duck2 48/120 (40.0) A 91/120 (75.8) B 93/120 (77.5) No. of positives/No. of total samples (%) Sample w enrichment wo enrichment Total1 Chicken2 10/120 (8.3) A 38/120 (31.7) B 38/120 (31.7) Duck2 48/120 (40.0) A 91/120 (75.8) B 93/120 (77.5) 1Positives by either detection with enrichment or without enrichment. 2The numbers of samples positive for Campylobacter were compared in pairs using Fisher's exact test. Different upper letters within a row (A and B) indicate a difference (P < 0.05). View Large Table 2. The number of Campylobacter-positive samples with or without enrichment step from chicken and duck carcasses in 12 poultry slaughterhouses. No. of positives/No. of total samples (%) Sample w enrichment wo enrichment Total1 Chicken2 10/120 (8.3) A 38/120 (31.7) B 38/120 (31.7) Duck2 48/120 (40.0) A 91/120 (75.8) B 93/120 (77.5) No. of positives/No. of total samples (%) Sample w enrichment wo enrichment Total1 Chicken2 10/120 (8.3) A 38/120 (31.7) B 38/120 (31.7) Duck2 48/120 (40.0) A 91/120 (75.8) B 93/120 (77.5) 1Positives by either detection with enrichment or without enrichment. 2The numbers of samples positive for Campylobacter were compared in pairs using Fisher's exact test. Different upper letters within a row (A and B) indicate a difference (P < 0.05). View Large Antibiotic-Resistance Profiles of Isolates The antibiotic-resistance profiles of the isolates are shown in Table 3. Most isolates were resistant to ciprofloxacin (95.4%), enrofloxacin (88.5%), nalidixic acid (93.9%), or tetracycline (72.5%), but only less than 4% of strains were resistant to erythromycin (3.1%) or chloramphenicol (0.8%), as shown in Table 3. No notable differences were observed in terms of antibiotic resistance between Campylobacter jejuni and Campylobacter coli (Table 3) and the isolates from chicken and duck samples (data not shown). Table 3. Comparison of antibiotic-resistance profiles and minimum inhibitory concentrations (MICs) for 131 Campylobacter isolates from chicken and duck carcasses in 12 poultry slaughterhouses. Number of isolates with each indicated MIC Antimicrobial agents Speices 0.5< 1 2 4 8 16 32 64 128 >256 n1 R (%) Ciprofloxacin jejuni 0 0 0 0 1 31 46 9 1 0 88 100 coli 6 0 0 0 4 20 5 5 0 3 37 86.0 Total 6 0 0 02 5 51 51 14 1 3 125 95.4 Enrofloxacin jejuni 2 1 3 16 44 19 3 0 0 0 82 93.2 coli 6 0 3 15 9 3 4 1 1 1 34 79.1 Total 8 1 6 312 53 22 7 1 1 1 116 88.5 Nalidixic acid jejuni 0 0 0 0 0 0 1 1 32 54 87 98.9 coli 0 0 0 0 2 4 1 0 17 19 36 83.7 Total 0 0 0 0 2 4 2 12 49 73 123 93.9 Tetracycline jejuni 12 10 1 1 0 0 0 0 28 36 64 72.7 coli 5 5 0 0 2 0 0 1 5 25 31 72.1 Total 17 15 1 1 2 02 0 1 32 62 95 72.5 Erythromycin jejuni 15 25 48 0 0 0 0 0 0 0 0 0 coli 19 4 13 3 0 0 0 3 0 1 4 9.3 Total 34 29 61 3 0 0 02 3 0 1 4 3.1 Chloramphenicol jejuni 10 2 26 19 23 7 1 0 0 0 1 1.1 coli 4 2 13 20 1 3 0 0 0 0 0 0 Total 14 4 39 39 24 10 12 0 0 0 1 0.8 Number of isolates with each indicated MIC Antimicrobial agents Speices 0.5< 1 2 4 8 16 32 64 128 >256 n1 R (%) Ciprofloxacin jejuni 0 0 0 0 1 31 46 9 1 0 88 100 coli 6 0 0 0 4 20 5 5 0 3 37 86.0 Total 6 0 0 02 5 51 51 14 1 3 125 95.4 Enrofloxacin jejuni 2 1 3 16 44 19 3 0 0 0 82 93.2 coli 6 0 3 15 9 3 4 1 1 1 34 79.1 Total 8 1 6 312 53 22 7 1 1 1 116 88.5 Nalidixic acid jejuni 0 0 0 0 0 0 1 1 32 54 87 98.9 coli 0 0 0 0 2 4 1 0 17 19 36 83.7 Total 0 0 0 0 2 4 2 12 49 73 123 93.9 Tetracycline jejuni 12 10 1 1 0 0 0 0 28 36 64 72.7 coli 5 5 0 0 2 0 0 1 5 25 31 72.1 Total 17 15 1 1 2 02 0 1 32 62 95 72.5 Erythromycin jejuni 15 25 48 0 0 0 0 0 0 0 0 0 coli 19 4 13 3 0 0 0 3 0 1 4 9.3 Total 34 29 61 3 0 0 02 3 0 1 4 3.1 Chloramphenicol jejuni 10 2 26 19 23 7 1 0 0 0 1 1.1 coli 4 2 13 20 1 3 0 0 0 0 0 0 Total 14 4 39 39 24 10 12 0 0 0 1 0.8 1Number of resistant strains. 2Breakpoint for each antibiotic. View Large Table 3. Comparison of antibiotic-resistance profiles and minimum inhibitory concentrations (MICs) for 131 Campylobacter isolates from chicken and duck carcasses in 12 poultry slaughterhouses. Number of isolates with each indicated MIC Antimicrobial agents Speices 0.5< 1 2 4 8 16 32 64 128 >256 n1 R (%) Ciprofloxacin jejuni 0 0 0 0 1 31 46 9 1 0 88 100 coli 6 0 0 0 4 20 5 5 0 3 37 86.0 Total 6 0 0 02 5 51 51 14 1 3 125 95.4 Enrofloxacin jejuni 2 1 3 16 44 19 3 0 0 0 82 93.2 coli 6 0 3 15 9 3 4 1 1 1 34 79.1 Total 8 1 6 312 53 22 7 1 1 1 116 88.5 Nalidixic acid jejuni 0 0 0 0 0 0 1 1 32 54 87 98.9 coli 0 0 0 0 2 4 1 0 17 19 36 83.7 Total 0 0 0 0 2 4 2 12 49 73 123 93.9 Tetracycline jejuni 12 10 1 1 0 0 0 0 28 36 64 72.7 coli 5 5 0 0 2 0 0 1 5 25 31 72.1 Total 17 15 1 1 2 02 0 1 32 62 95 72.5 Erythromycin jejuni 15 25 48 0 0 0 0 0 0 0 0 0 coli 19 4 13 3 0 0 0 3 0 1 4 9.3 Total 34 29 61 3 0 0 02 3 0 1 4 3.1 Chloramphenicol jejuni 10 2 26 19 23 7 1 0 0 0 1 1.1 coli 4 2 13 20 1 3 0 0 0 0 0 0 Total 14 4 39 39 24 10 12 0 0 0 1 0.8 Number of isolates with each indicated MIC Antimicrobial agents Speices 0.5< 1 2 4 8 16 32 64 128 >256 n1 R (%) Ciprofloxacin jejuni 0 0 0 0 1 31 46 9 1 0 88 100 coli 6 0 0 0 4 20 5 5 0 3 37 86.0 Total 6 0 0 02 5 51 51 14 1 3 125 95.4 Enrofloxacin jejuni 2 1 3 16 44 19 3 0 0 0 82 93.2 coli 6 0 3 15 9 3 4 1 1 1 34 79.1 Total 8 1 6 312 53 22 7 1 1 1 116 88.5 Nalidixic acid jejuni 0 0 0 0 0 0 1 1 32 54 87 98.9 coli 0 0 0 0 2 4 1 0 17 19 36 83.7 Total 0 0 0 0 2 4 2 12 49 73 123 93.9 Tetracycline jejuni 12 10 1 1 0 0 0 0 28 36 64 72.7 coli 5 5 0 0 2 0 0 1 5 25 31 72.1 Total 17 15 1 1 2 02 0 1 32 62 95 72.5 Erythromycin jejuni 15 25 48 0 0 0 0 0 0 0 0 0 coli 19 4 13 3 0 0 0 3 0 1 4 9.3 Total 34 29 61 3 0 0 02 3 0 1 4 3.1 Chloramphenicol jejuni 10 2 26 19 23 7 1 0 0 0 1 1.1 coli 4 2 13 20 1 3 0 0 0 0 0 0 Total 14 4 39 39 24 10 12 0 0 0 1 0.8 1Number of resistant strains. 2Breakpoint for each antibiotic. View Large The high antibiotic resistance observed with Campylobacter isolates from poultry samples to certain antibiotics is in agreement with previous studies conducted in South Korea, which reported resistances of 68 to 97% to ciprofloxacin, 58 to 84% to enrofloxacin, 71 to 100% to nalidixic acid, and 60 to 95% to tetracycline (Kang et al., 2006; Hong et al., 2007; Kim et al., 2010; Chae et al., 2011; Kim et al., 2013; Wei et al., 2014). Although the rates of antibiotic resistance found in this study agree with other studies performed in South Korea, antibiotic resistance, especially to fluoroquinolone, is much higher than in studies conducted in other countries (Gyles, 2008; Bardon et al., 2009; Bernadette et al., 2012; Garin et al., 2012; Wieczorek and Osek, 2015; Mäesaar et al., 2016). Fluoroquinolones, such as nalidixic acid, ciprofloxacin, and enrofloxacin are important antibiotics because they are the primary choice for treating campylobacteriosis (Ge et al., 2003; Kang et al., 2006; Alfredson and Korolik, 2007). These agents have been used for many years in veterinary medicine and poultry husbandry in Korea (Hong et al., 2007). Enrofloxacin, in particular, has been widely used since 1987 to treat Escherichia coli infection in poultry husbandry, and such resistance is related to enhanced resistance to other quinolones, such as ciprofloxacin, because they have closely related structures (McDermott and Bodeis, 2002; Hong et al., 2007; Pallo-Zimmerman et al., 2010). The resistance of pathogenic bacteria to antibiotics limits the available therapeutic options (Alfredson and Korolik, 2007). Tetracycline has been the most widely used antibiotic agent in animal husbandry in South Korea, leading to high rates of resistance (Lim et al., 2014). Macrolide resistance was relatively rare compared with resistance to other agents, such as fluoroquinolones or tetracycline. Although only 3.1% of the isolates were resistant to erythromycin, this should be continually monitored because these are the antibiotics of choice for treating campylobacteriosis, which is unresponsive to fluoroquinolones (Ge et al., 2003; Kang et al., 2006; Alfredson and Korolik, 2007). Although the use of specific antibiotics such as fluoroquinolones as a feed supplement has been banned in South Korea since 2011 (Kim et al., 2012; Lim et al., 2014), resistance to fluoroquinolones does not appear to have declined. The use of antibiotics in animals is associated with resistant Campylobacter isolates in humans (Alfredson and Korolik, 2007). Both duck and chicken may transmit Campylobacter having multidrug-resistance to human through a food-chain (Wei et al., 2014). The results from the current study show that it is necessary to strengthen the implementation of specific procedures in South Korea to reduce antibiotic resistance in Campylobacter. Molecular Characterization of Isolates using Rep-PCR Fingerprinting The collected Campylobacter isolates subtyped using an automated rep-PCR system to identify the relatedness among the isolates from poultry slaughterhouses. As we stated above, we selected 40 strains from 131 strains in consideration of their source of contamination, source of animals, and bacterial species. From 2 to 10 Campylobacter isolates were isolated from 17 sampling trials in 11 slaughterhouses [A (first), B (first), C (first), D (first), F (second), G (first/second), H (first/second), I (first/second), J (first/second), K (first/second), and L (first/second)], and at least two strains from each sampling were included in the subtyping test. The computer-generated virtual gel images, dendrogram analysis patterns, and profiles of each strain following rep-PCR are presented in Figure 1. Figure 1. View largeDownload slide Computer-generated virtual gel images and a dendrogram of 40 Campylobacter jejuni and Campylobacter coli strains isolated from chicken and duck carcasses in 12 poultry slaughterhouses prepared by DiversiLab rep-PCR fingerprinting. The isolates having > 95% similarity (cut-off bar) in their rep-PCR banding patterns were classified into the same category. First, first visit-samples collected from June to August 2014 (summer season);second, second visit-samples collected from December 2014 to February 2015 (winter season) Figure 1. View largeDownload slide Computer-generated virtual gel images and a dendrogram of 40 Campylobacter jejuni and Campylobacter coli strains isolated from chicken and duck carcasses in 12 poultry slaughterhouses prepared by DiversiLab rep-PCR fingerprinting. The isolates having > 95% similarity (cut-off bar) in their rep-PCR banding patterns were classified into the same category. First, first visit-samples collected from June to August 2014 (summer season);second, second visit-samples collected from December 2014 to February 2015 (winter season) All strains were classified into 36 subtypes with less than 95% similarity according to the rep-PCR banding patterns (Figure 1). These results indicated the diversity of Campylobacter isolates present in chicken and duck samples from poultry slaughterhouses. Except for Key numbers 7 and 8, the banding patterns of 2 species, Campylobacter jejuni and Campylobacter coli, were well distinguished from each other by rep-PCR (Figure 1). Most strains originated from same slaughterhouses were considered genetically unrelated. However, some strain pairs such as Key numbers 5 to 6, 7 to 8, 9 to 10, and 18 to19 showed similar subtyping patterns with more than 95% similarity, despite the use of individually packed products (Figure 1). Each strain pair was not distinguished by antibiotic resistance. Those genetically related strains were obtained from the same slaughterhouses in the same visit, suggesting that they may have originated from either the same farm that supplied live poultry to a specific slaughterhouse or from cross-contamination during the slaughter processing step (Chon et al., 2015). Strains from the second visit were also distinguished from strains from first visit, indicating that no strain persisted for a long time (6 mo) in the slaughterhouse environments. In this study, the quantitative prevalence, antibiotic resistance, and molecular subtyping patterns in Campylobacter were surveyed in chickens and ducks from poultry slaughterhouses in South Korea. 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Poultry ScienceOxford University Press

Published: Jul 11, 2018

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