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Antimicrobial resistance of 114 porcine isolates of Campylobacter coli
International Journal of Food Microbiology 118 (2007) 223 – 227 www.elsevier.com/locate/ijfoodmicro
Short communication
Antimicrobial resistance of 114 porcine isolates of Campylobacter coli E. Shin, Y. Lee ⁎ Culture Collection of Antimicrobial Resistant Microbes, Department of Biology, Seoul Women's University, Seoul 139-774, Republic of Korea Received 26 January 2007; received in revised form 7 April 2007; accepted 22 July 2007
Abstract Campylobacter species were isolated from 24 pig farms in 10 different regions of Korea, and were assayed with regard to their susceptibility to eight antimicrobial agents. A total of 114 Campylobacter isolates from 572 intestinal samples were all identified as C. coli via both classical methods and molecular methods, including 16S rDNA sequence analysis and polymerase chain reactions (PCR) using specific primer sets for the hippuricase gene and the aspartokinase gene, designed to differentiate C. coli from C. jejuni. Minimal inhibitory concentrations of seven antimicrobial agents were determined via agar dilution: the MIC90s were 64 μg/ml for ampicillin, 8 μg/ml for chloramphenicol, 64 μg/ml for ciprofloxacin, 16 μg/ml for enrofloxacin, ≥ 128 μg/ml for erythromycin, ≥ 128 μg/ml for gentamicin, and ≥ 128 μg/ml for tetracycline. The proportion of isolates resistant to each antimicrobial agent was as follows: 28.9% for ampicillin, 2.6% for chloramphenicol, 84.2% for ciprofloxacin, 83.3% for enrofloxacin, 46.5% for erythromycin, 20.2% for gentamicin, and 56.1% for tetracycline. All 114 isolates were found to be resistant to at least one antimicrobial agent, and 61 isolates (53.5%) were found to be multi-drug resistant (resistant to more than three antimicrobial agents in different classes). © 2007 Elsevier B.V. All rights reserved. Keywords: Campylobacter coli; Swine; Antimicrobial resistance
1. Introduction The zoonoses which occur most frequently in the industrialized world today include food-borne infections induced by bacteria enzootic to food animals. Most notable among these bacterial species are Salmonella, Campylobacter, Yersinia, Listeria, and enterohaemorrhagic Escherichia coli species. In recent years, human campylobacteriosis has shown a dramatic increase in industrialized countries, and currently represents one of the principal causes of bacterial food-borne disease (Taylor and Blaser, 1991; Tam et al., 2003). Campylobacter infections tend to be self limiting, and do not normally require treatment. However, in immunocompromised patients, such infections can result in bacteremia. Campylobacter bacteremia can have a fairly high mortality rate (N30%). Fatalities due to treatment failures have also been reported (Pasternack, 2002). Food animals are considered to be the primary reservoirs of the Campylobacter species which induce infections in humans (WHO, 1997; Harvey et al., 1999). Campylobacter jejuni appears ⁎ Corresponding author. Tel.: +82 2 970 5664; fax: +82 2 970 5901. E-mail address: [email protected] (Y. Lee). 0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2007.07.040
to predominate among cattle and broiler chickens, but is only found at a low frequency among pigs, in which C. coli predominates (Cabrita et al., 1992; Aarestrup and Wegener, 1999). An increase in Campylobacter resistance, especially to fluoroquinolones and erythromycin, as well as to other antimicrobial agents, has also been observed (Aarestrup and Engberg, 2001; Bae et al., 2005; Moore et al., 2006). As the potential exists for the transmission of antimicrobial-resistant animal isolates to humans (Engberg et al., 2001; Angulo et al., 2004; Kim et al., 2006), the presence of antimicrobial-resistant isolates in the food chain has raised concerns that the treatment of human infections will be compromised (Bodhidatta et al., 2002). The development of resistance in zoonotic bacteria constitutes a public health risk, principally as the result of an increased risk of treatment failures (FDA, 2001; Helms et al., 2005). In addition, development of resistance, notably via the acquisition of transmissible genetic elements (Velazquez et al., 1995), may affect other bacterial properties, most notably the ability to colonize an animal host or to persist within a farm or food-processing environment. In order to gather information regarding the antimicrobial resistance of Campylobacter in animals, Campylobacter were
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isolated from swine, and the antimicrobial resistance of the isolates was investigated. 2. Materials and methods 2.1. Isolation and identification of Campylobacter During a four-month period from July to October 2004, samples were collected from 24 pig farms located in 10 different regions in the Kyung-gi and Chungcheong Provinces of Korea. Intestinal samples (n = 572) were obtained at the abattoir immediately after slaughter, and were immediately transported to the laboratory in insulated containers. The samples were washed twice in sterile phosphate buffered saline (PBS, pH 7.0) in order to remove intestinal contents. The washed intestinal membranes were then smeared onto Brucella solid media (BBL, Sparks, MD, USA) supplemented with 10% horse serum, amphotericin B (2.5 μg/ml; Fungizone, Sigma, St. Louis, MO, USA), Skirrow's supplement (polymyxin B, 2.5 IU/ ml; vancomycin, 10 μg/ml; trimethoprim, 5 μg/ml) and campy blood-free selective medium (CCDA, Acumedia, Baltimore, MA, USA); samples were then incubated for 48 h under microaerophilic conditions at 42 °C. Catalase-positive, oxidasepositive colonies with the characteristic translucent morphology of Campylobacter were isolated and microscopically examined after Gram staining. After the identification of each isolate via 16S rRNA gene sequencing, one isolate per sample was stored at − 70 °C for further examination. 2.2. Identification of Campylobacter by PCRs of 16S rRNA, hippuricase, and aspartokinase genes PCRs of 16S rRNA, hippuricase, and aspartokinase genes were performed for species identification. PCR was performed with cah primers (cah-1, 5′-AAT ACA TGC AAG TCG AAC GA-3′; cah-2, 5′-TTA ACC CAA CAT CTC ACG AC-3′) as designed by others (Marshall et al., 1999) to amplify a 1004-bp fragment within the coding region of the 16S rRNA gene in Campylobacter, Arcobacter and Helicobacter species. Primers (hip-1, 5′-ATG ATG GCT TCT TCG GAT AG-3′; hip-2, 5′GCT CCT ATG CTT ACA ACT GC-3′) designed by Hani and Chan (1995) were used to amplify a portion of the hippuricase gene found only in C. jejuni. Primers (CC18F, 5′-GGT ATG ATT TCT ACA AAG CGA G-3′; CC519R, 5′-ATA AAA GAC TAT CGT CGC GTG-3′) designed by Linton et al. (1997) were used to amplify an aspartokinase gene in C. coli. Template DNA was prepared by cetyltrimethylammonium bromide (CTAB) method (Honore-Bouakline et al., 2003) and PCR amplifications were performed as described in previous papers. C. jejuni ATCC 33560 and C. coli ATCC 33559 were included as controls. For sequencing, DNA fragment was purified using the QIAquick Gel extraction Kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer's and sequenced using an ABI 3100 automated sequencer. DNA sequences, using the online BLAST algorithm at the National Center for Biotechnology Information web server (www.ncbi.nlm.nih.gov) were compared.
2.3. Minimal inhibitory concentration (MIC) MICs were assayed on Muller–Hinton agar plates (Beckton Dickinson, MD, USA) containing 5% sheep blood, via standard agar dilution, in accordance with the recommendations of the Clinical Laboratory Standards Institute (CLSI; NCCLS, 2005). The isolates were grown microaerobically for 48 hours on Brucella agar containing Skirrow's supplement or Trypticase soy agar containing 5% sheep blood (BBL) at 42 °C. Campylobacter jejuni ATCC 33560 and C. coli ATCC 33559 were included in each batch of the agar dilution tests, and CLSI approved MIC quality control limits for these strains were used for the control of agar dilution performance. MICs were assayed using erythromycin, ciprofloxacin (Korea Research Institute of Chemical Technology), enrofloxacin (Dr Ehrenstofer, GmbH, Germany), ampicillin, chloramphenicol (Fluka, Buchs, Switzerland), gentamicin, and tetracycline at a variety of concentrations ranging from 0.5 μg/ml to 128 μg/ml. All chemicals used in this study were purchased from Sigma (St. Louis, MO, USA) unless otherwise stated. The MIC interpretive standards of Staphylococcus spp. and veterinary pathogens (NCCLS, 1999) were employed as breakpoints for Campylobacter resistance for erythromycin and enrofloxacin, respectively. For breakpoints of other antimicrobial agents, MIC interpretive standards for members of the Enterobacteriaceae family were utilized (NCCLS, 2005). 3. Results and discussion 3.1. Isolation of Campylobacter During a four-month period, 572 samples of porcine intestine were collected from 24 pig farms located in 10 different regions in Kyung-gi and Chungcheong Provinces where more than half of Korean population lives and a total of 114 Campylobacter isolates (20.1%) were acquired. When the isolates were identified via classical methods including the catalase test, oxidase test, and Gram staining, as well as molecular techniques including 16S rRNA gene sequencing, and PCRs with specific primers for the hippuricase gene present in C. jejuni, and aspartokinase gene present in C. coli, all of these isolates were identified as C. coli. 3.2. MIC of C. coli MICs of ampicillin, chloramphenicol, ciprofloxacin, enrofloxacin, erythromycin, gentamicin, and tetracycline for C. coli are shown in Table 1. MICs of erythromycin, gentamicin, and tetracycline fell into two separate groups. For example, erythromycin-resistant isolates showed MICs of higher than 64 μg/ml whereas erythromycin-susceptible isolates had MICs of less than 16 μg/ml, and there were no isolates with MICs between 64 μg/ml and 16 μg/ml. The MIC50s of ciprofloxacin, enrofloxacin, and tetracycline were higher than the limit for resistance criteria, whereas the MIC90s of every antimicrobial agent except chloramphenicol were found to be higher than the limits for resistance criteria. In this study, the proportion of
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Table 1 Minimal inhibitory concentrations of 114 porcine isolates of Campylobacter coli MIC (μg/ml)
Ampicillin Chloramphenicol Ciprofloxacin Enrofloxacin Erythromycin Gentamicin Tetracycline
(μg/ml)
≤0.5
1
2
4
8
16
32
64
≥128
MIC50
MIC90
MICR
6 1 12 13 8 12 5
6 21 1 6 10 38 0
24 38 5 15 16 41 2
33 39 10 11 27 1 0
7 9 19 40 3 0 4
5 3 26 19 1 0 12
21 3 27 6 0 0 26
7 0 13 4 1 1 24
5 0 1 0 48 21 41
4 2 16 8 4 2 64
64 8 64 16 ≥128 ≥128 ≥128
32 32 4 2 8 8 8
Number of resistant isolates (resistance rate) 33 (28.90%) 3 (2.60%) 96 (83.3%) 95 (83.3%) 53 (46.50%) 23 (20.20%) 64 (56.10%)
MICR, criteria limit for resistance according to NCCLS.
resistance was found to be 28.9% for ampicillin, 2.6% for chloramphenicol, 84.2% for ciprofloxacin, 83.3% for enrofloxacin, 46.5% for erythromycin, 20.2% for gentamicin, and 56.1% for tetracycline. In general, the prevalence of antimicrobial resistance in the porcine isolates of C. coli in this study was high (N 40%) for erythromycin, quinolone (ciprofloxacin and enrofloxacin) and tetracycline. Among the seven antimicrobial agents evaluated in this study, chloramphenicol showed the best inhibitory activity. Only three of the isolates were found to be resistant to chloramphenicol, and both the MIC50 and MIC90 of chloramphenicol were found to be lower than the MIC for resistance. With the exception of chloramphenicol, the proportion of isolates resistant to every antimicrobial agent was in excess of 20%, and both the MIC50 and MIC90 values were higher than the resistance criteria (Table 2). The resistance rates of Campylobacter to each antimicrobial agent have varied substantially in various countries — Canada (Larkin et al., 2006), Denmark (Aarestrup et al., 1997; Bywater et al., 2004), France (Payot et al., 2004), Germany in 1991 (Luber et al., 2003), Japan (Aquino et al., 2002), Spain (Reina et al., 1994; Saenz et al., 2000), Sweden (Sjögren et al., 1992), and USA (Thakur and Gebreyes, 2005). For example, in the case of tetracycline, resistance varied from 0% in human isolates in Japan to 100% in turkey isolates in Germany in 1991. In the case of gentamicin, the proportion of human isolates resistant in Denmark, Germany, and Sweden was 0%, whereas it was higher than 20% in Japan, Spain, and France. Low resistance to chloramphenicol was observed in Denmark, Spain, Canada except USA where the resistance rate to chloramphenicol was reported as 66.2%. Resistance rates of porcine C. coli isolates vary widely in each country and they are quite different from those of human isolates even in the same country. Ampicillin resistance varied from 15% in France to 65.7% in Spain, and resistance to tetracycline varied from 1% in Denmark to 94.4% in Spain. Ciprofloxacin resistance varied from 13% in Denmark to 100% in Spain. Gentamicin resistance ranged from 0% in Denmark to 34% in France while ciprofloxacin resistance ranged from 0.6% in USA to 100% in Spain. Antimicrobial resistance to erythromycin and gentamicin in Korea in this study was found to be similar to or less than the resistance reported in other countries. Chloramphenicol resistance was quite low: all but three C. coli isolates in the current
study were found to be susceptible to chloramphenicol. The varying resistance profiles in different countries have been attributed to different preferences with regard to the use of antimicrobial agents in each country. In Korea, the three most frequently employed antimicrobial agents in swine production are antimicrobial agents in the tetracycline, sulfa, and penicillin classes, as reported by the Veterinary Research and Quarantine Service in 2005 (http://www.nvrqs.go.kr/). 3.3. Multi-drug resistance among C. coli isolates The number of isolates resistant to quinolone and tetracycline (88 isolates, 77.2%) was higher than the number resistant to any other antibiotic combination in this study (Table 2). The results of this study indicate that at least 64 isolates (56.1%) of porcine C. coli isolates were multi-drug resistant (MDR). Table 2 Number of isolates of each resistance pattern Number of antimicrobial agent
Resistant to
No. of isolates
No. of isolates
1
Tc Gm EmGm CipTc EmTc AmGm AmCip AmTc CipEm AmCipTc CipEmTc CipGmTc AmCmTc AmEmTc AmCipEm AmCipTc AmCipEmTc AmCipEmGm CmCipEmTc CipEmGmTc AmCipEmGm CmCipGmTc AmEmGmTc AmCipEmGmTc
9 1 1 30 2 1 2 1 3 1 20 4 1 1 1 14 5 1 1 9 1 1 1 3
10
2
3
4
5
40
42
19
3
Am, Ampicillin; Cip, Ciprofloxacin; Em, Erythromycin; Gm, Gentamicin; Tc, Tetracycline.
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Although Campylobacter is not considered a major pathogen associated with food-borne disease in Korea, this study revealed the prevalence of antimicrobial resistance in Campylobacter from swine which is the mostly consumed food animal in Korea and showed the possibility of transmission of antimicrobial resistance to humans. Preventive measures should be urgently implemented, including the establishment of a surveillance system for human campylobacteriosis, and the prudent use of antimicrobial agents in food animals. The results of this study suggest that urgent monitoring of antimicrobial resistance in Campylobacter should be carried out to prevent the occurrence of untreatable food-borne disease. Acknowledgments The authors would like to express thanks to Dr Won Jun Whang for sampling, and to the Korea Research Institute of Chemical Technology for providing antimicrobial agents. This study was funded by the Ministry of Health and Welfare (No. 03PG1-CH03-0002) and the Ministry of Agricultural and Forestry (201102-3). References Aarestrup, F.M., Engberg, J., 2001. Antimicrobial resistance of thermophilic Campylobacter. Veterinary Research 32, 311–321. Aarestrup, F.M, Wegener, H.C., 1999. The effects of antibiotic usage in food animals on the development of antimicrobial resistance of importance for humans in Campylobacter and Escherichia coli. Microbes and Infection 1, 639–644. Aarestrup, F.M., Nielsen, E.M., Madsen, M., Engberg, J., 1997. Antimicrobial susceptibility patterns of thermophilic Campylobacter spp. from humans, pigs, cattle, and broilers in Denmark. Antimicrobial Agents and Chemotherapy 41, 2244–2250. Angulo, F.J., Baker, N.L., Olsen, S.J., Anderson, A., Barrett, T.J., 2004. Antimicrobial use in agriculture: controlling the transfer of antimicrobial resistance to humans. Seminars in Pediatric Infectious Diseases 15, 78–85. Aquino, M.H.C., Filgueiras, A.L.L., Ferreira, M.C.S., Oliveira, S.S., Bastos, M.C., Tibana, A., 2002. Antimicrobial resistance and plasmid profiles of Campylobacter jejuni and Campylobacter coli from human and animal sources. Letters in Applied Microbiology 34, 149–153. Bae, W., Kaya, K.N., Hancock, D.D., Call, D.R., Park, Y.H., Besser, T.E., 2005. Prevalence and antimicrobial resistance of thermophilic Campylobacter spp. from cattle farms in Washington State. Applied and Environmental Microbiology 71, 169–174. Bodhidatta, L., Vithayasai, N., Eimpokalarp, B., Pitarangsi, C., Serichantalergs, O., Isenbarger, D.W., 2002. Bacterial enteric pathogens in children with acute dysentery in Thailand: increasing importance of quinolone-resistant Campylobacter. Southeast Asian Journal of Tropical Medicine and Public Health 33, 752–757. Bywater, R., Deluyker, H., Deroover, E., de Jong, A., Marion, H., McConville, M., Rowan, T., Shryock, T., Shuster, D., Thomas, V., Valle, M., Walters, J., 2004. A European survey of antimicrobial susceptibility among zoonotic and commensal bacteria isolated from food-producing animals. Journal of Antimicrobial Chemotherapy 54, 744–754. Cabrita, J., Rodrigues, J., Braganca, F., Morgado, C., Pires, I., Goncales, A.P., 1992. Prevalence, biotypes, plasmid profile and antimicrobial resistance of Campylobacter isolated from wild and domestic animals from Northeast Portugal. Journal of Applied Bacteriology 73, 279–285. Engberg, J., Aarestrup, F.M., Tayler, D.E., Gerner-Smidt, P., Nachamkin, I., 2001. Quinolone and macrolide resistance in Campylobacter jejuni and C. coli: resistance mechanisms and trends in human isolates. Emerging Infectious Diseases 7, 24–34.
Food and Drug Administration (FDA), 2001. Hot topics. Antibiotic resistance a growing threat[On line] http://www.fda.gov/oc/opacom/hottopics/ anti_resist2001. Hani, E.K., Chan, V.L., 1995. Expression and characterization of Campylobacter jejuni benzoylglycine amidohydrolase (Hippuricase) gene in Escherichia coli. Journal of Bacteriology 177, 2396–2402. Harvey, R.B., Young, C.R., Ziprin, R.L., Hume, M.E., Genovese, K.J., Anderson, R.C., Droleskey, R.E., Stanker, L.H., Nisbet, D.J., 1999. Prevalence of Campylobacter species isolated from the intestinal tract of pigs raised in an integrated swine production system. Journal of the American Veterinary Medical Association 215, 1601–1604. Helms, M., Simonsen, J., Olsen, K.E., Molbak, K., 2005. Adverse health events associated with antimicrobial drug resistance in Campylobacter species: a registry-based cohort study. Journal of Infectious Diseases 191, 1050–1055. Honore-Bouakline, S., Vincensini, J.P., Giacuzzo, V., Lagrange, P.H., Herrmann, J.L., 2003. Rapid diagnosis of extrapulmonary tuberculosis by PCR: impact of sample preparation and DNA extraction. Journal of Clinical Microbiology 41, 2323–2329. Kim, J.-S., Carver, D.K., Kathariou, S., 2006. Natural transformation-mediated transfer of erythromycin resistance in Campylobacter coli strains from turkeys and swine. Applied and Environmental Microbiology 72, 1316–1321. Larkin, C., Van Donkersgoed, C., Mahdi, A., Johnson, P., McNab, B., Odumeru, J., 2006. Antibiotic resistance of Campylobacter jejuni and Campylobacter coli isolated from hog, beef, and chicken carcass samples from provincially inspected abattoirs in Ontario. Journal of Food Protection 69, 22–26. Linton, D., Lawson, A.J., Owen, R.J., Stanley, J., 1997. PCR detection, identification to species level, and fingerprinting of Campylobacter jejuni and Campylobacter coli direct from diarrheic samples. Journal of Clinical Microbiology 35, 2568–2572. Luber, P., Wagner, J., Hahn, H., Bartelt, E., 2003. Antimicrobial resistance in Campylobacter jejuni and Campylobacter coli strains isolated in 1991 and 2001–2002 from poultry and humans in Berlin, Germany. Antimicrobial Agents and Chemotherapy 47, 3825–3830. Marshall, S.M., Melito, P.L., Woodward, D.L., Johnson, W.M., Rodgers, F.G., Mulvey, M.R., 1999. Rapid identification of Campylobacter, Arcobacter, and Helicobacter isolates by PCR-restriction fragment length polymorphism analysis of 16S rRNA gene. Journal of Clinical Microbiology 37, 4158–4160. Moore, J.E., Barton, M.D., Blair, I.S., Corcoran, D., Dooley, J.S., Fanning, S., Kempf, I., Lastovica, A.J., Lowery, C.J., Matsuda, M., McDowell, D.A., McMahon, A., Millar, B.C., Rao, J.R., Rooney, P.J., Seal, B.S., Snelling, W.J., Tolba, O., 2006. The epidemiology of antibiotic resistance in Campylobacter. Microbes and Infection 8, 1955–1966. National Committee for Clinical Laboratory Standards (NCCLS), 1999. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals: Approved Standard M31-A. National Committee for Clinical Laboratory Standards, Wayne, Pennsylvania. National Committee for Clinical Laboratory Standards (NCCLS), 2005. Performance Standards for Antimicrobial Susceptibility Testing; Fifteenth Informational Supplement. M100-S15. Clinical and Laboratory Standards Institute, Wayne, Pennsylvania. Pasternack, M.S., 2002. Impact and management of Campylobacter in human medicine — US perspective. International Journal of Infectious Diseases 6, S37–S43. Payot, S., Dridi, S., Laroche, M., Federighi, M., Magras, C., 2004. Prevalence and antimicrobial resistance of Campylobacter coli isolated from fattening pigs in France. Veterinary Microbiology 101, 91–99. Reina, J., Ros, M.J., Serra, A., 1994. Susceptibilities to 10 antimicrobial agents of 1,220 Campylobacter strains isolated from 1987 to 1993 from feces of pediatric patients. Antimicrobial Agents and Chemotherapy 38, 2917–2920. Saenz, Y., Zarazaga, M., Lantero, M., Gastanares, M.J., Baquero, F., Torres, C., 2000. Antibiotic resistance in Campylobacter strains isolated from animals, foods, and humans in Spain in 1997–1998. Antimicrobial Agents and Chemotherapy 44, 267–271. Sjögren, E., Kaijser, B., Werner, M., 1992. Antimicrobial susceptibilities of Campylobacter jejuni and Campylobacter coli isolated in Sweden: a 10-year follow-up report. Antimicrobial Agents and Chemotherapy 36, 2847–2849.
E. Shin, Y. Lee / International Journal of Food Microbiology 118 (2007) 223–227 Tam, C.C., O'Brien, S.J., Adak, G.K., Meakins, S.M., Frost, J.A., 2003. Campylobacter coli — an important foodborne pathogen. Journal of Infection 47, 28–32. Taylor, D.N., Blaser, M.J., 1991. Campylobacter infections. In: Evans, A.S., Brachmann, P.S. (Eds.), Bacterial infections in humans. Plenum Publishing Corp., New York, N.Y., pp. 151–172. Thakur, S., Gebreyes, W.A., 2005. Prevalence and antimicrobial resistance of Campylobacter in antimicrobial-free and conventional pig production systems. Journal of Food Protection 68, 2402–2410.
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Velazquez, J.B., Jimenez, A., Chomon, B., Villa, T.G., 1995. Incidence and transmission of antibiotic resistance in Campylobacter jejuni and Campylobacter coli. Journal of Antimicrobial Chemotherapy 35, 173–178. World Health Organization (WHO), 1997. The medical impact of the use of antimicrobials in food animals. Report of a WHO meeting. World Health Organization, Geneva, Switzerland.
Short communication
Antimicrobial resistance of 114 porcine isolates of Campylobacter coli E. Shin, Y. Lee ⁎ Culture Collection of Antimicrobial Resistant Microbes, Department of Biology, Seoul Women's University, Seoul 139-774, Republic of Korea Received 26 January 2007; received in revised form 7 April 2007; accepted 22 July 2007
Abstract Campylobacter species were isolated from 24 pig farms in 10 different regions of Korea, and were assayed with regard to their susceptibility to eight antimicrobial agents. A total of 114 Campylobacter isolates from 572 intestinal samples were all identified as C. coli via both classical methods and molecular methods, including 16S rDNA sequence analysis and polymerase chain reactions (PCR) using specific primer sets for the hippuricase gene and the aspartokinase gene, designed to differentiate C. coli from C. jejuni. Minimal inhibitory concentrations of seven antimicrobial agents were determined via agar dilution: the MIC90s were 64 μg/ml for ampicillin, 8 μg/ml for chloramphenicol, 64 μg/ml for ciprofloxacin, 16 μg/ml for enrofloxacin, ≥ 128 μg/ml for erythromycin, ≥ 128 μg/ml for gentamicin, and ≥ 128 μg/ml for tetracycline. The proportion of isolates resistant to each antimicrobial agent was as follows: 28.9% for ampicillin, 2.6% for chloramphenicol, 84.2% for ciprofloxacin, 83.3% for enrofloxacin, 46.5% for erythromycin, 20.2% for gentamicin, and 56.1% for tetracycline. All 114 isolates were found to be resistant to at least one antimicrobial agent, and 61 isolates (53.5%) were found to be multi-drug resistant (resistant to more than three antimicrobial agents in different classes). © 2007 Elsevier B.V. All rights reserved. Keywords: Campylobacter coli; Swine; Antimicrobial resistance
1. Introduction The zoonoses which occur most frequently in the industrialized world today include food-borne infections induced by bacteria enzootic to food animals. Most notable among these bacterial species are Salmonella, Campylobacter, Yersinia, Listeria, and enterohaemorrhagic Escherichia coli species. In recent years, human campylobacteriosis has shown a dramatic increase in industrialized countries, and currently represents one of the principal causes of bacterial food-borne disease (Taylor and Blaser, 1991; Tam et al., 2003). Campylobacter infections tend to be self limiting, and do not normally require treatment. However, in immunocompromised patients, such infections can result in bacteremia. Campylobacter bacteremia can have a fairly high mortality rate (N30%). Fatalities due to treatment failures have also been reported (Pasternack, 2002). Food animals are considered to be the primary reservoirs of the Campylobacter species which induce infections in humans (WHO, 1997; Harvey et al., 1999). Campylobacter jejuni appears ⁎ Corresponding author. Tel.: +82 2 970 5664; fax: +82 2 970 5901. E-mail address: [email protected] (Y. Lee). 0168-1605/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2007.07.040
to predominate among cattle and broiler chickens, but is only found at a low frequency among pigs, in which C. coli predominates (Cabrita et al., 1992; Aarestrup and Wegener, 1999). An increase in Campylobacter resistance, especially to fluoroquinolones and erythromycin, as well as to other antimicrobial agents, has also been observed (Aarestrup and Engberg, 2001; Bae et al., 2005; Moore et al., 2006). As the potential exists for the transmission of antimicrobial-resistant animal isolates to humans (Engberg et al., 2001; Angulo et al., 2004; Kim et al., 2006), the presence of antimicrobial-resistant isolates in the food chain has raised concerns that the treatment of human infections will be compromised (Bodhidatta et al., 2002). The development of resistance in zoonotic bacteria constitutes a public health risk, principally as the result of an increased risk of treatment failures (FDA, 2001; Helms et al., 2005). In addition, development of resistance, notably via the acquisition of transmissible genetic elements (Velazquez et al., 1995), may affect other bacterial properties, most notably the ability to colonize an animal host or to persist within a farm or food-processing environment. In order to gather information regarding the antimicrobial resistance of Campylobacter in animals, Campylobacter were
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isolated from swine, and the antimicrobial resistance of the isolates was investigated. 2. Materials and methods 2.1. Isolation and identification of Campylobacter During a four-month period from July to October 2004, samples were collected from 24 pig farms located in 10 different regions in the Kyung-gi and Chungcheong Provinces of Korea. Intestinal samples (n = 572) were obtained at the abattoir immediately after slaughter, and were immediately transported to the laboratory in insulated containers. The samples were washed twice in sterile phosphate buffered saline (PBS, pH 7.0) in order to remove intestinal contents. The washed intestinal membranes were then smeared onto Brucella solid media (BBL, Sparks, MD, USA) supplemented with 10% horse serum, amphotericin B (2.5 μg/ml; Fungizone, Sigma, St. Louis, MO, USA), Skirrow's supplement (polymyxin B, 2.5 IU/ ml; vancomycin, 10 μg/ml; trimethoprim, 5 μg/ml) and campy blood-free selective medium (CCDA, Acumedia, Baltimore, MA, USA); samples were then incubated for 48 h under microaerophilic conditions at 42 °C. Catalase-positive, oxidasepositive colonies with the characteristic translucent morphology of Campylobacter were isolated and microscopically examined after Gram staining. After the identification of each isolate via 16S rRNA gene sequencing, one isolate per sample was stored at − 70 °C for further examination. 2.2. Identification of Campylobacter by PCRs of 16S rRNA, hippuricase, and aspartokinase genes PCRs of 16S rRNA, hippuricase, and aspartokinase genes were performed for species identification. PCR was performed with cah primers (cah-1, 5′-AAT ACA TGC AAG TCG AAC GA-3′; cah-2, 5′-TTA ACC CAA CAT CTC ACG AC-3′) as designed by others (Marshall et al., 1999) to amplify a 1004-bp fragment within the coding region of the 16S rRNA gene in Campylobacter, Arcobacter and Helicobacter species. Primers (hip-1, 5′-ATG ATG GCT TCT TCG GAT AG-3′; hip-2, 5′GCT CCT ATG CTT ACA ACT GC-3′) designed by Hani and Chan (1995) were used to amplify a portion of the hippuricase gene found only in C. jejuni. Primers (CC18F, 5′-GGT ATG ATT TCT ACA AAG CGA G-3′; CC519R, 5′-ATA AAA GAC TAT CGT CGC GTG-3′) designed by Linton et al. (1997) were used to amplify an aspartokinase gene in C. coli. Template DNA was prepared by cetyltrimethylammonium bromide (CTAB) method (Honore-Bouakline et al., 2003) and PCR amplifications were performed as described in previous papers. C. jejuni ATCC 33560 and C. coli ATCC 33559 were included as controls. For sequencing, DNA fragment was purified using the QIAquick Gel extraction Kit (Qiagen, Valencia, CA, USA) in accordance with the manufacturer's and sequenced using an ABI 3100 automated sequencer. DNA sequences, using the online BLAST algorithm at the National Center for Biotechnology Information web server (www.ncbi.nlm.nih.gov) were compared.
2.3. Minimal inhibitory concentration (MIC) MICs were assayed on Muller–Hinton agar plates (Beckton Dickinson, MD, USA) containing 5% sheep blood, via standard agar dilution, in accordance with the recommendations of the Clinical Laboratory Standards Institute (CLSI; NCCLS, 2005). The isolates were grown microaerobically for 48 hours on Brucella agar containing Skirrow's supplement or Trypticase soy agar containing 5% sheep blood (BBL) at 42 °C. Campylobacter jejuni ATCC 33560 and C. coli ATCC 33559 were included in each batch of the agar dilution tests, and CLSI approved MIC quality control limits for these strains were used for the control of agar dilution performance. MICs were assayed using erythromycin, ciprofloxacin (Korea Research Institute of Chemical Technology), enrofloxacin (Dr Ehrenstofer, GmbH, Germany), ampicillin, chloramphenicol (Fluka, Buchs, Switzerland), gentamicin, and tetracycline at a variety of concentrations ranging from 0.5 μg/ml to 128 μg/ml. All chemicals used in this study were purchased from Sigma (St. Louis, MO, USA) unless otherwise stated. The MIC interpretive standards of Staphylococcus spp. and veterinary pathogens (NCCLS, 1999) were employed as breakpoints for Campylobacter resistance for erythromycin and enrofloxacin, respectively. For breakpoints of other antimicrobial agents, MIC interpretive standards for members of the Enterobacteriaceae family were utilized (NCCLS, 2005). 3. Results and discussion 3.1. Isolation of Campylobacter During a four-month period, 572 samples of porcine intestine were collected from 24 pig farms located in 10 different regions in Kyung-gi and Chungcheong Provinces where more than half of Korean population lives and a total of 114 Campylobacter isolates (20.1%) were acquired. When the isolates were identified via classical methods including the catalase test, oxidase test, and Gram staining, as well as molecular techniques including 16S rRNA gene sequencing, and PCRs with specific primers for the hippuricase gene present in C. jejuni, and aspartokinase gene present in C. coli, all of these isolates were identified as C. coli. 3.2. MIC of C. coli MICs of ampicillin, chloramphenicol, ciprofloxacin, enrofloxacin, erythromycin, gentamicin, and tetracycline for C. coli are shown in Table 1. MICs of erythromycin, gentamicin, and tetracycline fell into two separate groups. For example, erythromycin-resistant isolates showed MICs of higher than 64 μg/ml whereas erythromycin-susceptible isolates had MICs of less than 16 μg/ml, and there were no isolates with MICs between 64 μg/ml and 16 μg/ml. The MIC50s of ciprofloxacin, enrofloxacin, and tetracycline were higher than the limit for resistance criteria, whereas the MIC90s of every antimicrobial agent except chloramphenicol were found to be higher than the limits for resistance criteria. In this study, the proportion of
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225
Table 1 Minimal inhibitory concentrations of 114 porcine isolates of Campylobacter coli MIC (μg/ml)
Ampicillin Chloramphenicol Ciprofloxacin Enrofloxacin Erythromycin Gentamicin Tetracycline
(μg/ml)
≤0.5
1
2
4
8
16
32
64
≥128
MIC50
MIC90
MICR
6 1 12 13 8 12 5
6 21 1 6 10 38 0
24 38 5 15 16 41 2
33 39 10 11 27 1 0
7 9 19 40 3 0 4
5 3 26 19 1 0 12
21 3 27 6 0 0 26
7 0 13 4 1 1 24
5 0 1 0 48 21 41
4 2 16 8 4 2 64
64 8 64 16 ≥128 ≥128 ≥128
32 32 4 2 8 8 8
Number of resistant isolates (resistance rate) 33 (28.90%) 3 (2.60%) 96 (83.3%) 95 (83.3%) 53 (46.50%) 23 (20.20%) 64 (56.10%)
MICR, criteria limit for resistance according to NCCLS.
resistance was found to be 28.9% for ampicillin, 2.6% for chloramphenicol, 84.2% for ciprofloxacin, 83.3% for enrofloxacin, 46.5% for erythromycin, 20.2% for gentamicin, and 56.1% for tetracycline. In general, the prevalence of antimicrobial resistance in the porcine isolates of C. coli in this study was high (N 40%) for erythromycin, quinolone (ciprofloxacin and enrofloxacin) and tetracycline. Among the seven antimicrobial agents evaluated in this study, chloramphenicol showed the best inhibitory activity. Only three of the isolates were found to be resistant to chloramphenicol, and both the MIC50 and MIC90 of chloramphenicol were found to be lower than the MIC for resistance. With the exception of chloramphenicol, the proportion of isolates resistant to every antimicrobial agent was in excess of 20%, and both the MIC50 and MIC90 values were higher than the resistance criteria (Table 2). The resistance rates of Campylobacter to each antimicrobial agent have varied substantially in various countries — Canada (Larkin et al., 2006), Denmark (Aarestrup et al., 1997; Bywater et al., 2004), France (Payot et al., 2004), Germany in 1991 (Luber et al., 2003), Japan (Aquino et al., 2002), Spain (Reina et al., 1994; Saenz et al., 2000), Sweden (Sjögren et al., 1992), and USA (Thakur and Gebreyes, 2005). For example, in the case of tetracycline, resistance varied from 0% in human isolates in Japan to 100% in turkey isolates in Germany in 1991. In the case of gentamicin, the proportion of human isolates resistant in Denmark, Germany, and Sweden was 0%, whereas it was higher than 20% in Japan, Spain, and France. Low resistance to chloramphenicol was observed in Denmark, Spain, Canada except USA where the resistance rate to chloramphenicol was reported as 66.2%. Resistance rates of porcine C. coli isolates vary widely in each country and they are quite different from those of human isolates even in the same country. Ampicillin resistance varied from 15% in France to 65.7% in Spain, and resistance to tetracycline varied from 1% in Denmark to 94.4% in Spain. Ciprofloxacin resistance varied from 13% in Denmark to 100% in Spain. Gentamicin resistance ranged from 0% in Denmark to 34% in France while ciprofloxacin resistance ranged from 0.6% in USA to 100% in Spain. Antimicrobial resistance to erythromycin and gentamicin in Korea in this study was found to be similar to or less than the resistance reported in other countries. Chloramphenicol resistance was quite low: all but three C. coli isolates in the current
study were found to be susceptible to chloramphenicol. The varying resistance profiles in different countries have been attributed to different preferences with regard to the use of antimicrobial agents in each country. In Korea, the three most frequently employed antimicrobial agents in swine production are antimicrobial agents in the tetracycline, sulfa, and penicillin classes, as reported by the Veterinary Research and Quarantine Service in 2005 (http://www.nvrqs.go.kr/). 3.3. Multi-drug resistance among C. coli isolates The number of isolates resistant to quinolone and tetracycline (88 isolates, 77.2%) was higher than the number resistant to any other antibiotic combination in this study (Table 2). The results of this study indicate that at least 64 isolates (56.1%) of porcine C. coli isolates were multi-drug resistant (MDR). Table 2 Number of isolates of each resistance pattern Number of antimicrobial agent
Resistant to
No. of isolates
No. of isolates
1
Tc Gm EmGm CipTc EmTc AmGm AmCip AmTc CipEm AmCipTc CipEmTc CipGmTc AmCmTc AmEmTc AmCipEm AmCipTc AmCipEmTc AmCipEmGm CmCipEmTc CipEmGmTc AmCipEmGm CmCipGmTc AmEmGmTc AmCipEmGmTc
9 1 1 30 2 1 2 1 3 1 20 4 1 1 1 14 5 1 1 9 1 1 1 3
10
2
3
4
5
40
42
19
3
Am, Ampicillin; Cip, Ciprofloxacin; Em, Erythromycin; Gm, Gentamicin; Tc, Tetracycline.
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