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Prevalence and antimicrobial resistance of Arcobacter species isolated from poultry meat in Iran a
Ebrahim Rahimi a
Department of Food Hygiene and Public Health, College of Veterinary Medicine, Islamic Azad University, Shahrekord Branch, Shahrekord, Iran Accepted author version posted online: 10 Jan 2014.Published online: 16 Jun 2014.
To cite this article: Ebrahim Rahimi (2014) Prevalence and antimicrobial resistance of Arcobacter species isolated from poultry meat in Iran, British Poultry Science, 55:2, 174-180, DOI: 10.1080/00071668.2013.878783 To link to this article: http://dx.doi.org/10.1080/00071668.2013.878783
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British Poultry Science, 2014 Vol. 55, No. 2, 174–180, http://dx.doi.org/10.1080/00071668.2013.878783
Prevalence and antimicrobial resistance of Arcobacter species isolated from poultry meat in Iran EBRAHIM RAHIMI
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Department of Food Hygiene and Public Health, College of Veterinary Medicine, Islamic Azad University, Shahrekord Branch, Shahrekord, Iran
Abstract 1. The objective of this study was to determine the prevalence and antimicrobial resistance of Arcobacter spp. isolated from different species of retail poultry meat in Iran. 2. From August 2012 to April 2013, a total of 540 raw poultry meat samples from chicken (n = 100), turkey (n = 100), quail (n = 100), partridge (n = 80), duck (n = 50), ostrich (n = 60) and geese (n = 50) were purchased from randomly selected retail outlets in Shahrekord, Isfahan, Sari and Rasht, Iran. 3. Using culture techniques, 71 of 540 poultry meat samples (13.1%) were positive for Arcobacter spp. The highest prevalence of Arcobacter spp. was found in chicken meat (28.0%), followed by quail (12.0%), duck (11.4%), turkey (11.0%), geese (8.0%), partridge (7.5%) and ostrich (3.3%) meat. The number of A. butzleri isolated from poultry meat samples (90.1%) was significantly higher than A. cryaerophilus (7.1%) and A. skirrowii (2.8%). Significantly more poultry meat samples were found to contain Arcobacter spp. by the PCR assay than by the culture method. 4. Susceptibilities of Arcobacter isolates were determined for 14 antimicrobial drugs using the disk diffusion method. All of the 71 Arcobacter isolates tested were resistant to one or more antimicrobial agents. Resistance to cephalothin and vancomycin (95.8%) was the most common finding, followed by resistance to methicillin, azithromycin and ampicillin. All Arcobacter isolates were susceptible to gentamicin, streptomycin, tetracyclin and kanamycin. 5. The results of this study indicated the importance of poultry meat, especially chicken meat, as potential sources of Arcobacter spp. infection in people. Furthermore, the strains indicated resistance to a broad spectrum of antibiotics.
INTRODUCTION Arcobacter species are Gram-negative spiral-shaped organisms belonging to the family Campylobacteraceae that can grow microaerobically or aerobically. The Arcobacter organisms also have the ability to be grown at 15°C, which is a distinctive feature that differentiates Arcobacter species from Campylobacter species (Lehner et al., 2005; Figueras et al., 2011). The genus Arcobacter has become increasingly important in recent years because its members have been considered as emergent enteropathogens and potential zoonotic agents (Ho et al., 2006). Until 2005, the genus comprised six species, including Arcobacter butzleri,
A. cryaerophilus, A. skirrowii, A. nitrofigilis, A. cibarius (Houf et al., 2005) and A. halophilus (Donachie et al., 2005; Debruyne et al., 2008). However, since then the genus has been extended with 12 additional species (Collado and Figueras, 2011; Figueras et al., 2011; Sasi Jyothsna et al., 2013). At present, A. butzleri, A. cryaerophilus and A. skirrowii are considered the most common species of the genus being regarded as zoonotic and emerging food-borne pathogens that could be associated with bacteraemia and human diarrhoeic illness. These have also been isolated from food of animal origin, especially from poultry, carcasses and offal, milk, mussels, as well as from water bodies, sewage and faecal samples of
Correspondence to: Ebrahim Rahimi, Department of Food Hygiene and Public Health, College of Veterinary Medicine, Islamic Azad University, Shahrekord Branch, Shahrekord, Iran. E-mail: [email protected] Accepted for publication 5 November 2013.
© 2014 British Poultry Science Ltd
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different animal species (Fernández et al., 2007; Pianta et al., 2007; Arias et al., 2011; Collado and Figueras, 2011). Food contamination with antibiotic-resistant bacteria can also be a major threat to public health, as the antibiotic resistance determinants can be transferred to other pathogenic bacteria, potentially compromising the treatment of severe bacterial infections. The prevalence of antimicrobial resistance among food-borne pathogens has increased during recent decades (Van et al., 2007) and is attributed to the selection pressure created by using antimicrobials in food-producing animals, in addition to unregulated use of antibiotics by humans in developing countries (Van den Bogaard and Stobberingh, 2000). Currently, there is limited information regarding the prevalence and antimicrobial susceptibility patterns of Arcobacter in poultry products in Iran. Therefore, the present study was conducted to determine the prevalence and antimicrobial resistance of Arcobacter spp. isolated from different retail poultry meat in Iran.
MATERIALS AND METHODS Sample collection From August 2012 to April 2013, a total of 540 raw poultry meat samples from chicken (n = 100), turkey (n = 100), quail (n = 100), partridge (n = 80), duck (n = 50), ostrich (n = 60) and geese (n = 50) were purchased from randomly selected retail outlets in Shahrekord, Isfahan, Sari and Rasht, Iran. All samples were placed in separate sterile plastic bags to prevent spilllage and cross-contamination and were immediately transported to the laboratory in a cooler with ice packs. Detection and identification Arcobacter spp. by PCR The samples were processed immediately upon arrival using aseptic techniques. Of each meat sample, 10 g was homogenised and transferred to 90 ml of Arcobacter broth (Oxoid) supplemented with CAT supplement (Cefoperazone, Teicoplanin, Amphotericin B, Oxoid). All enrichment samples were incubated for 48 h at 30°C under microaerophilic conditions. DNA from a total of 540 samples was extracted from Arcobacter broth after the enrichment step using a Genomic DNA purification kit (Fermentas, GmbH, Germany) according to the manufacturer’s protocol. The 50 μl polymerase chain reaction (PCR) reaction mixture contained 25 ng of DNA template, 50 pmol each of PCR primers N. cry and ARCO-U, 10 pmol each of N. butz and N. ski, 0.5 U of Taq DNA polymerase (Fermentas,
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GmbH, Germany), 0.8 mmol of dNTPs (Fermentas, GmbH, Germany), 10 mmol Tris– HCl (pH 8.3), 50 mmol KCl and 1.5 mmol MgCl2. Nuclease-free water was added to adjust the final reaction volume to 50 μl. The sequences (5′–3′) of the primers used in this PCR were: ARCO-U (Arcobacter): TTCGCTTGCGCTGCATCAT N. butz (A. butzleri): AGCGTTCTATTCAGCGT AGAAGATGT N. cry (A. cryaerophilus): ACCGAAGCTTTAGATT CGAATTTATTCG N. ski (A. skirrowii): CGAGGTCACGGATGGAA GTG.
Amplification was performed in a thermal cycler (Master Cycle Gradient, Eppendrof, Germany) using the following programme: initial denaturation at 94°C for 10 min, followed by 30 amplification cycles consisting of denaturation for 30 s at 94°C, annealing for 1 min at 64°C and elongation for 1 min at 72°C. The final elongation was performed at 72°C for 7 min. The samples were held at 4°C until the PCR products were analysed. The amplified DNA products were electrophoresed on 2% agarose gels at 90 V for 6 h using 1× TBE (0.89 M Tris borate, 0.02 M EDTA, pH 8.3) as the running buffer, then stained with ethidium bromide. Gels were visualised using a UV gel documentation system (Uvitech, UK). A set of molecular weight standards (Fermentas, GmbH, Germany) ranging from 100 bp to 3000 bp was included on each gel.
Isolation of Arcobacter spp. For isolation of bacteria, 50 μl of each enrichment sample was dropped on a cellulose-nitrate membrane filter (0.65 μm) which was placed on blood agar plates (Brain Heart Infusion Agar, Oxoid, plus 5% horse blood) supplemented with CAT (Cefoperazone, Teicoplanin, Amphotericin B, Oxoid). After 1 h incubation at 30°C in air, the filters were removed, the filtrates were evenly distributed over the agar surface with a sterile spreader and the plates were incubated for 48 h at 30°C under microaerophilic conditions. Plates with no growth were incubated for a further 2 d. Three to five colonies from each plate suspected to be Arcobacter spp. (small, nonpigmented or grey on blood agar plate and Gram stain) were transferred to blood agar plates without antimicrobial supplement and incubated for 48 h at 30°C under microaerophilic conditions. Isolates were purified by passing onto blood agar plates at least three times. They were confirmed to be Arcobacter spp. by the m-PCR.
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Inc., Chicago, IL, USA), a chi-square test and Fisher’s exact two-tailed test analysis was performed, and differences were considered significant at values of P < 0.05.
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Antimicrobial susceptibility testing Antimicrobial susceptibility testing was done by the Kirby–Bauer disc diffusion method using Mueller–Hinton agar (HiMedia Laboratories, Mumbai, India, MV1084) supplemented with 5% defibrinated sheep blood, according to the Clinical and Laboratory Standards Institute (CLSI, 2006) guidelines. The antimicrobial agents tested and their corresponding concentrations were as follows: ampicillin (10 μg), azithromycin (10 µg), chloramphenicol (30 µg), ciprofloxacin (15 µg), clindomycin (15 µg), erythromycin (15 µg), gentamicin (10 µg), kanamycin (30 µg), methicillin (30 µg), nalidixic acid (30 µg), streptomycin (30 µg), tetracycline (15 µg) and vancomycin (30 µg). After incubating the inoculated plate aerobically at 37°C for 72 h in a microaerophilic atmosphere, the susceptibility of the Arcobacter spp. to each antimicrobial agent was measured and the results were interpreted in accordance with interpretive criteria provided by CLSI (2006). Staphylococcus aureus and Escherichia coli were used as quality control organisms in antimicrobial susceptibility determination.
RESULTS Using cultural techniques, 71 of 540 poultry meat samples (13.1%) were positive for Arcobacter spp. (Table 1). The highest prevalence of Arcobacter spp. was found in chicken meat (28.0%), followed by quail (12.0%), duck (11.4%), turkey (11.0%), geese (8.0%), partridge (7.5%) and ostrich (3.3%) meat. Arcobacter spp. was isolated from only two ostrich meat samples and they were identified as A. butzleri. There were significant differences (P < 0.05) in the level of contamination with Arcobacter between chicken and goose meat, chicken and partridge meat, and chicken and ostrich meat samples. The number of A. butzleri isolated from poultry meat samples (90.1%) was significantly higher than A. cryaerophilus (7.1%) and A. skirrowii (2.8%, P < 0.05). Significantly more poultry meat samples were found to contain Arcobacter spp. by the PCR assay than by the culture method (P < 0.05, Table 2). Overall, 71 carcass samples were positive for Arcobacter spp. using both the cultural method and the PCR assay. The PCR assay could identify
Statistical analysis Data were transferred to a Microsoft Excel spreadsheet (Microsoft Corp., Redmond, WA, USA) for analysis. Using SPSS 16.0 statistical software (SPSS
Table 1. Prevalence of Arcobacter spp. in chicken carcasses on the processing line using culture techniques Positive samples (%) using cultural technique Sources of samples
No. of samples
Chicken Turkey Quail Partridge Duck Ostrich Geese Total
Arcobacter spp.
100 100 100 80 50 60 50 540
Table 2.
28 11 12 6 8 2 4 71
(28.0) (11.0) (12.0) (7.5) (11.4) (3.3) (8.0) (13.1)
A. butzleri 26 11 9 5 8 2 3 64
(92.9) (100) (75.0) (83.3) (100) (100) (75.0) (90.1)
A. cryaerophilus 2 0 1 1 0 0 1 5
(17.1) (-) (8.3) (26.7) (-) (-) (25.0) (7.1)
A. skirrowii 0 0 2 0 0 0 0 2
(-) (-) (16.7) (-) (7.1) (-) (-) (2.8)
Prevalence of Arcobacter spp. in poultry meat using a PCR assay Positive samples (%) using PCR assay
Meat samples Chicken Turkey Quail Partridge Duck Ostrich Geese Total
No. of samples 100 100 100 80 50 60 50 540
Arcobacter spp. 31 12 12 9 14 2 5 85
(31.0) (12.0) (12.0) (11.3) (28.0) (3.3) (10.0) (15.7)
A. butzleri 28 12 9 6 10 2 4 71
(90.3) (100) (75.0) (66.7) (71.4) (100) (80.0) (83.5)
A. cryaerophilus 2 0 1 1 3 0 1 8
(6.5) (-) (8.3) (11.1) (21.4) (-) (20.0) (9.4)
A. skirrowii 1 0 2 2 1 0 0 6
(3.2) (-) (16.7) (22.2) (7.1) (-) (-) (7.1)
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Table 3. Antimicrobial resistance profiles of Arcobacter strains isolated from poultry meat Antimicrobial agent
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Ampicillin Azithromycin Cephalothin Chloramphenicol Ciprofloxacin Clindomycin Erythromycin Gentamicin Kanamycin Nalidixic acid Methicillin Streptomycin Tetracycline Vancomycin
Arcobacter spp. (N = 71) 43 62 68 7 1 50 2 0 0 16 63 0 0 68
(60.6%) (87.3%) (95.8%) (9.9%) (1.4%) (70.4%) (2.8%) (-) (-) (22.5%) (88.7%) (-) (-) (95.8%)
14 Arcobacter-contaminated carcass samples that were negative using the culture method. Table 3 summarises the resistance pattern of Arcobacter isolates to 14 antimicrobial agents tested in this study. All of the 71 Arcobacter isolates tested were resistant to one or more antimicrobial agents. A high proportion of the Arcobacter isolates was resistant to cephalothin, vancomycin, methicillin, azithromycin, clindomycin and ampicillin and to a lesser extent to nalidixic acid, chloramphenicol, erythromycin and ciprofloxacin. All Arcobacter isolates were susceptible to gentamicin, streptomycin, tetracyclin and kanamycin. A. skirrowii isolates were susceptible to azithromycin, clindamycin, chloramphenicol, nalidixic acid and ciprofloxacin.
DISCUSSION Overall, 13.1% (71 of 540) of all poultry meat samples were contaminated with Arcobacter spp. In this study, Arcobacter spp. were isolated from approximately 30% of raw chicken meat samples. A previous study in Iran has reported prevalence rates of Arcobacter spp. in chicken meat of 37.5% in Isfahan (Rahimi et al., 2012). The prevalence of Arcobacter spp. in retail chicken meat was reported to be 32% in Nigeria (Adesiji Yemisi et al., 2011), 39.2% in Malaysia (Amare et al., 2011), 23% in Japan (Kabeya et al., 2004), 0–100% in The Netherlands (Ho et al., 2008), 68% in Turkey (Aydin et al., 2007), 23.2% in Korea (Lee et al., 2010), 62% in Northern Ireland (Scullion et al., 2006), 43% in Germany (Atanassova et al., 2008) and 64.3% in Spain (Collado et al., 2009). Arcobacter spp. are frequently found in the intestinal tract of poultry where colonisation leads to contamination of carcasses during processing, especially at the defeathering, evisceration and
A. butzleri (N = 64) 37 61 64 6 1 48 1 0 0 14 60 0 0 63
(57.8%) (95.3%) (100%) (9.4%) (1.6%) (75.0%) (1.6%) (-) (-) (21.9%) (93.8%) (-) (-) (98.4%)
A. cryaerophilus (N = 5) 4 1 2 1 0 2 0 0 0 2 2 0 0 3
(80.0%) (20.0%) (40.0%) (20.0%) (-) (40.0%) (-) (-) (-) (40.0%) (40.0%) (-) (-) (60.0%)
A. skirrowii (N = 2) 2 0 1 0 0 0 1 0 0 0 1 0 0 2
(100%) (-) (50.0%) (-) (-) (-) (50.0%) (-) (-) (-) (50.0%) (-) (-) (100%)
chilling stages (Son et al., 2007a; Collado and Figueras, 2011). The contamination rate of turkey meat samples observed in this study (11.0%) was in agreement with those reported by Atanassova et al. (2008) in Germany (18.2%) and Aydin et al. (2007) in Turkey (4.0%). However, Manke et al. (1998) and Collado et al. (2009) reported a higher prevalence of Arcobacter in turkey meat in Ireland (77%) and Spain (33.3%), respectively. Also, in a study conducted in the United States, Arcobacter species were isolated from 93% of turkey carcass swabs (139 of 150) samples (Andersen et al., 2007). Variation in the prevalence of Arcobacter isolates from raw poultry meat samples reported in other studies may be a result of different sampling techniques employed (meat sample, carcass swab or carcass rinse fluid sample), seasonal effects, sample size and distribution, the types and concentrations of antimicrobials used in the media and/or laboratory methodologies employed in different studies (bacteriological and biochemical testing vs. PCR assays). According to relative increase in the consumption of ostrich, quail, partridge, duck and goose meat in Iran, we included 340 ostrich, quail and partridge meat samples in this study. A prevalence rate of Arcobacter of 40.0% (2 of 5) has been reported in duck meat in Spain (Collado et al., 2009). No extensive study was found on the prevalence of Arcobacter in quail, partridge, ostrich and goose meat. In a recent study conducted in Turkey, Arcobacter spp. were isolated from 16 (18%) of the 90 cloacal swabs of healthy geese (Atabay et al., 2008). Also, in an earlier study from Denmark (Atabay et al., 2006) ducks were reported to have a 75% prevalence of Arcobacter spp. at the flock level. According to our knowledge, the present study is the first report of the isolation of Arcobacter spp. from raw ostrich, quail, partridge, duck and goose meat in Iran; however,
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more research is needed to confirm the prevalence rate of Arcobacter in these meats. Multiple contaminations with different Arcobacter species were observed in 2 of 540 poultry meat samples (0.04%) in the present study. These findings suggest that Arcobacter may be easily spread among carcasses during poultry processing stages such as scalding and result in high prevalence among poultry meat. In our study, A. butzleri was more common than A. cryaerophilus and A. skirrowii. Studies for other countries also reported A. butzleri as the predominant species isolated from poultry and other meat at rates of up to 100% (Corry and Atabay, 2001; Kabeya et al., 2004; Ho et al., 2008; Collado et al., 2009; Amare et al., 2011). However, Son et al. (2007a) reported that A. cryaerophilus was the most frequent species recovered from poultry carcasses. The low recovery of A. skirrowii from poultry samples is in accordance with previous reports (Atabay et al., 1998; Houf et al., 2002). The low recovery of A. skirrowii may represent two possibilities: the low prevalence of the species among poultry samples, and the difficulty in the isolation of the species at the presence of other organisms. In addition, some changes for the isolation methods including the cultivation under microaerobic conditions or the use of blood agar plates without any antibiotics may improve the recovery rates of A. skirrowii and A. cryaerophilus since these species are more fastidious than A. butzleri (Kabeya et al., 2004). In this study, Arcobacter was better detected by the PCR assays than the culture method. This could be due to the higher analytical and diagnostic sensitivities of the PCR assays. However, care must be taken to avoid false-positive results arising from DNA contamination, as well as false-negative results caused by inhibitory substances in foods or enrichment broths. As the possibilities for amplicon contamination were minimised by separation of the preparation and amplification/detection laboratories and the blank controls were negative, false-positive results due to amplicants are unlikely to have occurred. Antibiotic susceptibility test results indicated that there was an overall increase in the resistance of Arcobacter to methicillin, vancomycin, cephalothin and azithromycin. Results of this study are comparable to those reported by others (Atabay and Aydin, 2001; Houf et al., 2002; Kabeya et al., 2004; Son et al., 2007b). In the present study, all the strains of A. butzleri were found to be resistant against cephalothin which is comparable to those reported by Atabay and Aydin (2001) and Kabeya et al. (2004). However, resistance to this antibiotic was lower than in A. cryaerophilus and A. skirrowii. These findings suggest that attention will be necessary when selective medium containing cephalothin for the isolation of A. cryaerophilus and A.
skirrowii is used because cephalothin may cause low recovery of these species (Kabeya et al., 2004). A high level of resistance in Arcobacter species was found to clindamycin (70.3%), a drug recommended as an alternative treatment for Campylobacter gastroenteritis in humans. Other authors have also reported a high level of resistance to clindamycin in A. butzleri from human and animal isolates (Fera et al., 2003). In contrast, resistance to erythromycin, chloramphenicol and ciprofloxacin was very low in A. butzleri and A. cryaerophilus while all the A. skirrowii isolates were susceptible to these antibiotics. This result is in agreement with the results reported by Atabay and Aydin (2001), Son et al. (2007b) and Fera et al. (2003). However, Otth et al. (2004) reported that 98% of A. butzleri isolated form foodstuffs were resistant to chloramphenicol. Especially the resistance to erythromycin and the decreased susceptibility to ciprofloxacin may have human health implications, as the two antimicrobials are generally prescribed as firstline drugs for the treatment of infections with Campylobacteraceae. In the present study, 60.6% of strains were resistant to ampicillin. These results are in agreement with those reported by other authors for ampicillin (Kiehlbauch et al., 1992; Harrass et al., 1998; Atabay and Aydin, 2001; Shah et al., 2013). However, some differences have been observed. While Kiehlbauch et al. (1992) reported 90% resistance, all the strains studied by Kabeya et al. (2004) were susceptible to this antibiotic. The differences in susceptibility patterns could be due to the frequency of the use of drug in animals for treatment and/or prophylaxis. Lack of gold-standard sensitivity methods and break points of antibiotics has made the comparison of results of antibiotic resistance patterns more difficult. However, compared with our data, a lower prevalence of resistance against nalidixic acid was also reported by Son et al. (2007b) and Atabay and Aydin (2001), while a high prevalence of resistance to nalidixic acid was reported in other studies (Kabeya et al., 2004). All Arcobacter strains included in our study were susceptible to the aminoglycoside gentamicin, streptomycin and kanamycin. Other authors have also reported that Arcobacter was susceptible to aminoglycosides, including kanamycin, amikacin gentamicin and streptomycin (Fera et al., 2003; Kabeya et al., 2004; Otth et al., 2004; Son et al., 2007b). In addition, all the Arcobacter isolates examined have been demonstrated to be susceptible to tetracycline as reported previously (Atabay and Aydin, 2001; Kabeya et al., 2004; Vandenberg et al., 2006; Son et al., 2007b). Thus, aminoglycosides and tetracycline would be suitable antibiotics for the treatment of disease caused by Arcobacter with veterinary and human medicine.
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In conclusion, the results of this study indicated the importance of poultry meats, especially chicken meat, as potential sources of Arcobacter spp. infection in people. Since high levels of contamination with Arcobacter spp. can occur in slaughterhouses (Rahimi et al., 2012), the maintenance of slaughter hygiene and regular microbiological monitoring of carcasses are essential to minimise the risk of contaminating when a danger of cross-contamination exists, especially when other poultry species with lower prevalence levels are slaughtered on the same slaughtering line. Furthermore, to ensure food safety, poultry meats must be cooked properly before consumption. The increased level of drug resistance, as encountered in this study, is important in terms of both animal and public health. Aminoglycosides and tetracycline can be used as first-choice drugs to fight Arcobacter infections.
ACKNOWLEDGEMENTS I would like to thank Dr Hassan Momtaz, Dr Amir Shakerian, Manouchehr Momeni and Majid Riahi for help in performing technical parts of the project. I am also grateful to Dr Mazear Rafei and Dr Morteza Mosavean for assistance with sampling.
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mechanically separated turkey. Journal of Food Protection, 61: 1623–1628. OTTH, L., WILSON, M., CANCINO, R. & FERNÁNDEZ, H. (2004) In vitro susceptibility of Arcobacter butzleri to six antimicrobial drugs. Archivos De Medicina Veterinaria, 2: 207–210. PIANTA, C., THOMPSEN PASSOS, D., HEPP, D. & DE OLIVEIRA, D.J. (2007) Isolation of Arcobacter spp. from the milk of dairy cows in Brazil. Ciência Rural, 37: 171–174. RAHIMI, E., HORMOZIPOOR, H., GHOLAMI AHANGARAN, M. & YAZDI, F. (2012) Prevalence of Arcobacter species on chicken carcasses during processing in Iran. The Journal of Applied Poultry Research, 21: 407–412. SASI JYOTHSNA, T.S., RAHUL, K., RAMAPRASAD, E.V., SASIKALA, C. & RAMANA, C.V. (2013) Arcobacter anaerophilus sp. nov., isolated from an estuarine sediment and emended description of the genus Arcobacter. International Journal of Systematic and Evolutionary Microbiology, 63: 4619–4625. SCULLION, R., HARRINGTON, C.S. & MADDEN, R.H. (2006) Prevalence of Arcobacter spp. in raw milk and retail raw meats in Northern Ireland. Journal of Food Protection, 69: 1986–1990. SHAH, A.H., SALEHA, A.A., ZUNITA, Z., MURUGAIYAH, M., ALIYU, A.B. & JAFRI, N. (2013) Prevalence, distribution and antibiotic resistance of emergent Arcobacter spp. from clinically
healthy cattle and goats. Transboundary and Emerging Diseases, 60: 9–16. SON, I., ENGLEN, M.D., BERRANG, M.E., FEDORKA-CRAY, P.J. & HARRISON, M.A. (2007a) Prevalence of Arcobacter and Campylobacter on broiler carcasses during processing. International Journal of Food Microbiology, 113: 16–22. SON, I., ENGLEN, M.D., BERRANG, M.E., FEDORKA-CRAY, P.J. & HARRISON, M.A. (2007b) Antimicrobial resistance of Arcobacter and Campylobacter from broiler carcasses. International Journal Antimicrobial Agents, 29: 451–455. VAN, T.H., MOUTAFIS, G., TRAN, L.T. & COLOE, P.J. (2007) Antibiotic resistance in food-borne bacterial contaminants in Vietnam. Applied and Environmental Microbiology, 73: 7906–7911. VAN DEN BOGAARD, A.E. & STOBBERINGH, E.E. (2000) Epidemiology of resistance to antibiotics: links between animals and humans. International Journal Antimicrobial Agents, 14: 327–335. VANDENBERG, O., HOUF, K., DOUAT, N., VLAES, L., RETORE, P., BUTZLER, J.P. & DEDISTE, A. (2006) Antimicrobial susceptibility of clinical isolates of non-jejuni/coli campylobacters and arcobacters from Belgium. Journal of Antimicrobial Chemotherapy, 57: 908–913.
British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Prevalence and antimicrobial resistance of Arcobacter species isolated from poultry meat in Iran a
Ebrahim Rahimi a
Department of Food Hygiene and Public Health, College of Veterinary Medicine, Islamic Azad University, Shahrekord Branch, Shahrekord, Iran Accepted author version posted online: 10 Jan 2014.Published online: 16 Jun 2014.
To cite this article: Ebrahim Rahimi (2014) Prevalence and antimicrobial resistance of Arcobacter species isolated from poultry meat in Iran, British Poultry Science, 55:2, 174-180, DOI: 10.1080/00071668.2013.878783 To link to this article: http://dx.doi.org/10.1080/00071668.2013.878783
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British Poultry Science, 2014 Vol. 55, No. 2, 174–180, http://dx.doi.org/10.1080/00071668.2013.878783
Prevalence and antimicrobial resistance of Arcobacter species isolated from poultry meat in Iran EBRAHIM RAHIMI
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Department of Food Hygiene and Public Health, College of Veterinary Medicine, Islamic Azad University, Shahrekord Branch, Shahrekord, Iran
Abstract 1. The objective of this study was to determine the prevalence and antimicrobial resistance of Arcobacter spp. isolated from different species of retail poultry meat in Iran. 2. From August 2012 to April 2013, a total of 540 raw poultry meat samples from chicken (n = 100), turkey (n = 100), quail (n = 100), partridge (n = 80), duck (n = 50), ostrich (n = 60) and geese (n = 50) were purchased from randomly selected retail outlets in Shahrekord, Isfahan, Sari and Rasht, Iran. 3. Using culture techniques, 71 of 540 poultry meat samples (13.1%) were positive for Arcobacter spp. The highest prevalence of Arcobacter spp. was found in chicken meat (28.0%), followed by quail (12.0%), duck (11.4%), turkey (11.0%), geese (8.0%), partridge (7.5%) and ostrich (3.3%) meat. The number of A. butzleri isolated from poultry meat samples (90.1%) was significantly higher than A. cryaerophilus (7.1%) and A. skirrowii (2.8%). Significantly more poultry meat samples were found to contain Arcobacter spp. by the PCR assay than by the culture method. 4. Susceptibilities of Arcobacter isolates were determined for 14 antimicrobial drugs using the disk diffusion method. All of the 71 Arcobacter isolates tested were resistant to one or more antimicrobial agents. Resistance to cephalothin and vancomycin (95.8%) was the most common finding, followed by resistance to methicillin, azithromycin and ampicillin. All Arcobacter isolates were susceptible to gentamicin, streptomycin, tetracyclin and kanamycin. 5. The results of this study indicated the importance of poultry meat, especially chicken meat, as potential sources of Arcobacter spp. infection in people. Furthermore, the strains indicated resistance to a broad spectrum of antibiotics.
INTRODUCTION Arcobacter species are Gram-negative spiral-shaped organisms belonging to the family Campylobacteraceae that can grow microaerobically or aerobically. The Arcobacter organisms also have the ability to be grown at 15°C, which is a distinctive feature that differentiates Arcobacter species from Campylobacter species (Lehner et al., 2005; Figueras et al., 2011). The genus Arcobacter has become increasingly important in recent years because its members have been considered as emergent enteropathogens and potential zoonotic agents (Ho et al., 2006). Until 2005, the genus comprised six species, including Arcobacter butzleri,
A. cryaerophilus, A. skirrowii, A. nitrofigilis, A. cibarius (Houf et al., 2005) and A. halophilus (Donachie et al., 2005; Debruyne et al., 2008). However, since then the genus has been extended with 12 additional species (Collado and Figueras, 2011; Figueras et al., 2011; Sasi Jyothsna et al., 2013). At present, A. butzleri, A. cryaerophilus and A. skirrowii are considered the most common species of the genus being regarded as zoonotic and emerging food-borne pathogens that could be associated with bacteraemia and human diarrhoeic illness. These have also been isolated from food of animal origin, especially from poultry, carcasses and offal, milk, mussels, as well as from water bodies, sewage and faecal samples of
Correspondence to: Ebrahim Rahimi, Department of Food Hygiene and Public Health, College of Veterinary Medicine, Islamic Azad University, Shahrekord Branch, Shahrekord, Iran. E-mail: [email protected] Accepted for publication 5 November 2013.
© 2014 British Poultry Science Ltd
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ARCOBACTER SPP. IN POULTRY MEAT
different animal species (Fernández et al., 2007; Pianta et al., 2007; Arias et al., 2011; Collado and Figueras, 2011). Food contamination with antibiotic-resistant bacteria can also be a major threat to public health, as the antibiotic resistance determinants can be transferred to other pathogenic bacteria, potentially compromising the treatment of severe bacterial infections. The prevalence of antimicrobial resistance among food-borne pathogens has increased during recent decades (Van et al., 2007) and is attributed to the selection pressure created by using antimicrobials in food-producing animals, in addition to unregulated use of antibiotics by humans in developing countries (Van den Bogaard and Stobberingh, 2000). Currently, there is limited information regarding the prevalence and antimicrobial susceptibility patterns of Arcobacter in poultry products in Iran. Therefore, the present study was conducted to determine the prevalence and antimicrobial resistance of Arcobacter spp. isolated from different retail poultry meat in Iran.
MATERIALS AND METHODS Sample collection From August 2012 to April 2013, a total of 540 raw poultry meat samples from chicken (n = 100), turkey (n = 100), quail (n = 100), partridge (n = 80), duck (n = 50), ostrich (n = 60) and geese (n = 50) were purchased from randomly selected retail outlets in Shahrekord, Isfahan, Sari and Rasht, Iran. All samples were placed in separate sterile plastic bags to prevent spilllage and cross-contamination and were immediately transported to the laboratory in a cooler with ice packs. Detection and identification Arcobacter spp. by PCR The samples were processed immediately upon arrival using aseptic techniques. Of each meat sample, 10 g was homogenised and transferred to 90 ml of Arcobacter broth (Oxoid) supplemented with CAT supplement (Cefoperazone, Teicoplanin, Amphotericin B, Oxoid). All enrichment samples were incubated for 48 h at 30°C under microaerophilic conditions. DNA from a total of 540 samples was extracted from Arcobacter broth after the enrichment step using a Genomic DNA purification kit (Fermentas, GmbH, Germany) according to the manufacturer’s protocol. The 50 μl polymerase chain reaction (PCR) reaction mixture contained 25 ng of DNA template, 50 pmol each of PCR primers N. cry and ARCO-U, 10 pmol each of N. butz and N. ski, 0.5 U of Taq DNA polymerase (Fermentas,
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GmbH, Germany), 0.8 mmol of dNTPs (Fermentas, GmbH, Germany), 10 mmol Tris– HCl (pH 8.3), 50 mmol KCl and 1.5 mmol MgCl2. Nuclease-free water was added to adjust the final reaction volume to 50 μl. The sequences (5′–3′) of the primers used in this PCR were: ARCO-U (Arcobacter): TTCGCTTGCGCTGCATCAT N. butz (A. butzleri): AGCGTTCTATTCAGCGT AGAAGATGT N. cry (A. cryaerophilus): ACCGAAGCTTTAGATT CGAATTTATTCG N. ski (A. skirrowii): CGAGGTCACGGATGGAA GTG.
Amplification was performed in a thermal cycler (Master Cycle Gradient, Eppendrof, Germany) using the following programme: initial denaturation at 94°C for 10 min, followed by 30 amplification cycles consisting of denaturation for 30 s at 94°C, annealing for 1 min at 64°C and elongation for 1 min at 72°C. The final elongation was performed at 72°C for 7 min. The samples were held at 4°C until the PCR products were analysed. The amplified DNA products were electrophoresed on 2% agarose gels at 90 V for 6 h using 1× TBE (0.89 M Tris borate, 0.02 M EDTA, pH 8.3) as the running buffer, then stained with ethidium bromide. Gels were visualised using a UV gel documentation system (Uvitech, UK). A set of molecular weight standards (Fermentas, GmbH, Germany) ranging from 100 bp to 3000 bp was included on each gel.
Isolation of Arcobacter spp. For isolation of bacteria, 50 μl of each enrichment sample was dropped on a cellulose-nitrate membrane filter (0.65 μm) which was placed on blood agar plates (Brain Heart Infusion Agar, Oxoid, plus 5% horse blood) supplemented with CAT (Cefoperazone, Teicoplanin, Amphotericin B, Oxoid). After 1 h incubation at 30°C in air, the filters were removed, the filtrates were evenly distributed over the agar surface with a sterile spreader and the plates were incubated for 48 h at 30°C under microaerophilic conditions. Plates with no growth were incubated for a further 2 d. Three to five colonies from each plate suspected to be Arcobacter spp. (small, nonpigmented or grey on blood agar plate and Gram stain) were transferred to blood agar plates without antimicrobial supplement and incubated for 48 h at 30°C under microaerophilic conditions. Isolates were purified by passing onto blood agar plates at least three times. They were confirmed to be Arcobacter spp. by the m-PCR.
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Inc., Chicago, IL, USA), a chi-square test and Fisher’s exact two-tailed test analysis was performed, and differences were considered significant at values of P < 0.05.
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Antimicrobial susceptibility testing Antimicrobial susceptibility testing was done by the Kirby–Bauer disc diffusion method using Mueller–Hinton agar (HiMedia Laboratories, Mumbai, India, MV1084) supplemented with 5% defibrinated sheep blood, according to the Clinical and Laboratory Standards Institute (CLSI, 2006) guidelines. The antimicrobial agents tested and their corresponding concentrations were as follows: ampicillin (10 μg), azithromycin (10 µg), chloramphenicol (30 µg), ciprofloxacin (15 µg), clindomycin (15 µg), erythromycin (15 µg), gentamicin (10 µg), kanamycin (30 µg), methicillin (30 µg), nalidixic acid (30 µg), streptomycin (30 µg), tetracycline (15 µg) and vancomycin (30 µg). After incubating the inoculated plate aerobically at 37°C for 72 h in a microaerophilic atmosphere, the susceptibility of the Arcobacter spp. to each antimicrobial agent was measured and the results were interpreted in accordance with interpretive criteria provided by CLSI (2006). Staphylococcus aureus and Escherichia coli were used as quality control organisms in antimicrobial susceptibility determination.
RESULTS Using cultural techniques, 71 of 540 poultry meat samples (13.1%) were positive for Arcobacter spp. (Table 1). The highest prevalence of Arcobacter spp. was found in chicken meat (28.0%), followed by quail (12.0%), duck (11.4%), turkey (11.0%), geese (8.0%), partridge (7.5%) and ostrich (3.3%) meat. Arcobacter spp. was isolated from only two ostrich meat samples and they were identified as A. butzleri. There were significant differences (P < 0.05) in the level of contamination with Arcobacter between chicken and goose meat, chicken and partridge meat, and chicken and ostrich meat samples. The number of A. butzleri isolated from poultry meat samples (90.1%) was significantly higher than A. cryaerophilus (7.1%) and A. skirrowii (2.8%, P < 0.05). Significantly more poultry meat samples were found to contain Arcobacter spp. by the PCR assay than by the culture method (P < 0.05, Table 2). Overall, 71 carcass samples were positive for Arcobacter spp. using both the cultural method and the PCR assay. The PCR assay could identify
Statistical analysis Data were transferred to a Microsoft Excel spreadsheet (Microsoft Corp., Redmond, WA, USA) for analysis. Using SPSS 16.0 statistical software (SPSS
Table 1. Prevalence of Arcobacter spp. in chicken carcasses on the processing line using culture techniques Positive samples (%) using cultural technique Sources of samples
No. of samples
Chicken Turkey Quail Partridge Duck Ostrich Geese Total
Arcobacter spp.
100 100 100 80 50 60 50 540
Table 2.
28 11 12 6 8 2 4 71
(28.0) (11.0) (12.0) (7.5) (11.4) (3.3) (8.0) (13.1)
A. butzleri 26 11 9 5 8 2 3 64
(92.9) (100) (75.0) (83.3) (100) (100) (75.0) (90.1)
A. cryaerophilus 2 0 1 1 0 0 1 5
(17.1) (-) (8.3) (26.7) (-) (-) (25.0) (7.1)
A. skirrowii 0 0 2 0 0 0 0 2
(-) (-) (16.7) (-) (7.1) (-) (-) (2.8)
Prevalence of Arcobacter spp. in poultry meat using a PCR assay Positive samples (%) using PCR assay
Meat samples Chicken Turkey Quail Partridge Duck Ostrich Geese Total
No. of samples 100 100 100 80 50 60 50 540
Arcobacter spp. 31 12 12 9 14 2 5 85
(31.0) (12.0) (12.0) (11.3) (28.0) (3.3) (10.0) (15.7)
A. butzleri 28 12 9 6 10 2 4 71
(90.3) (100) (75.0) (66.7) (71.4) (100) (80.0) (83.5)
A. cryaerophilus 2 0 1 1 3 0 1 8
(6.5) (-) (8.3) (11.1) (21.4) (-) (20.0) (9.4)
A. skirrowii 1 0 2 2 1 0 0 6
(3.2) (-) (16.7) (22.2) (7.1) (-) (-) (7.1)
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Table 3. Antimicrobial resistance profiles of Arcobacter strains isolated from poultry meat Antimicrobial agent
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Ampicillin Azithromycin Cephalothin Chloramphenicol Ciprofloxacin Clindomycin Erythromycin Gentamicin Kanamycin Nalidixic acid Methicillin Streptomycin Tetracycline Vancomycin
Arcobacter spp. (N = 71) 43 62 68 7 1 50 2 0 0 16 63 0 0 68
(60.6%) (87.3%) (95.8%) (9.9%) (1.4%) (70.4%) (2.8%) (-) (-) (22.5%) (88.7%) (-) (-) (95.8%)
14 Arcobacter-contaminated carcass samples that were negative using the culture method. Table 3 summarises the resistance pattern of Arcobacter isolates to 14 antimicrobial agents tested in this study. All of the 71 Arcobacter isolates tested were resistant to one or more antimicrobial agents. A high proportion of the Arcobacter isolates was resistant to cephalothin, vancomycin, methicillin, azithromycin, clindomycin and ampicillin and to a lesser extent to nalidixic acid, chloramphenicol, erythromycin and ciprofloxacin. All Arcobacter isolates were susceptible to gentamicin, streptomycin, tetracyclin and kanamycin. A. skirrowii isolates were susceptible to azithromycin, clindamycin, chloramphenicol, nalidixic acid and ciprofloxacin.
DISCUSSION Overall, 13.1% (71 of 540) of all poultry meat samples were contaminated with Arcobacter spp. In this study, Arcobacter spp. were isolated from approximately 30% of raw chicken meat samples. A previous study in Iran has reported prevalence rates of Arcobacter spp. in chicken meat of 37.5% in Isfahan (Rahimi et al., 2012). The prevalence of Arcobacter spp. in retail chicken meat was reported to be 32% in Nigeria (Adesiji Yemisi et al., 2011), 39.2% in Malaysia (Amare et al., 2011), 23% in Japan (Kabeya et al., 2004), 0–100% in The Netherlands (Ho et al., 2008), 68% in Turkey (Aydin et al., 2007), 23.2% in Korea (Lee et al., 2010), 62% in Northern Ireland (Scullion et al., 2006), 43% in Germany (Atanassova et al., 2008) and 64.3% in Spain (Collado et al., 2009). Arcobacter spp. are frequently found in the intestinal tract of poultry where colonisation leads to contamination of carcasses during processing, especially at the defeathering, evisceration and
A. butzleri (N = 64) 37 61 64 6 1 48 1 0 0 14 60 0 0 63
(57.8%) (95.3%) (100%) (9.4%) (1.6%) (75.0%) (1.6%) (-) (-) (21.9%) (93.8%) (-) (-) (98.4%)
A. cryaerophilus (N = 5) 4 1 2 1 0 2 0 0 0 2 2 0 0 3
(80.0%) (20.0%) (40.0%) (20.0%) (-) (40.0%) (-) (-) (-) (40.0%) (40.0%) (-) (-) (60.0%)
A. skirrowii (N = 2) 2 0 1 0 0 0 1 0 0 0 1 0 0 2
(100%) (-) (50.0%) (-) (-) (-) (50.0%) (-) (-) (-) (50.0%) (-) (-) (100%)
chilling stages (Son et al., 2007a; Collado and Figueras, 2011). The contamination rate of turkey meat samples observed in this study (11.0%) was in agreement with those reported by Atanassova et al. (2008) in Germany (18.2%) and Aydin et al. (2007) in Turkey (4.0%). However, Manke et al. (1998) and Collado et al. (2009) reported a higher prevalence of Arcobacter in turkey meat in Ireland (77%) and Spain (33.3%), respectively. Also, in a study conducted in the United States, Arcobacter species were isolated from 93% of turkey carcass swabs (139 of 150) samples (Andersen et al., 2007). Variation in the prevalence of Arcobacter isolates from raw poultry meat samples reported in other studies may be a result of different sampling techniques employed (meat sample, carcass swab or carcass rinse fluid sample), seasonal effects, sample size and distribution, the types and concentrations of antimicrobials used in the media and/or laboratory methodologies employed in different studies (bacteriological and biochemical testing vs. PCR assays). According to relative increase in the consumption of ostrich, quail, partridge, duck and goose meat in Iran, we included 340 ostrich, quail and partridge meat samples in this study. A prevalence rate of Arcobacter of 40.0% (2 of 5) has been reported in duck meat in Spain (Collado et al., 2009). No extensive study was found on the prevalence of Arcobacter in quail, partridge, ostrich and goose meat. In a recent study conducted in Turkey, Arcobacter spp. were isolated from 16 (18%) of the 90 cloacal swabs of healthy geese (Atabay et al., 2008). Also, in an earlier study from Denmark (Atabay et al., 2006) ducks were reported to have a 75% prevalence of Arcobacter spp. at the flock level. According to our knowledge, the present study is the first report of the isolation of Arcobacter spp. from raw ostrich, quail, partridge, duck and goose meat in Iran; however,
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more research is needed to confirm the prevalence rate of Arcobacter in these meats. Multiple contaminations with different Arcobacter species were observed in 2 of 540 poultry meat samples (0.04%) in the present study. These findings suggest that Arcobacter may be easily spread among carcasses during poultry processing stages such as scalding and result in high prevalence among poultry meat. In our study, A. butzleri was more common than A. cryaerophilus and A. skirrowii. Studies for other countries also reported A. butzleri as the predominant species isolated from poultry and other meat at rates of up to 100% (Corry and Atabay, 2001; Kabeya et al., 2004; Ho et al., 2008; Collado et al., 2009; Amare et al., 2011). However, Son et al. (2007a) reported that A. cryaerophilus was the most frequent species recovered from poultry carcasses. The low recovery of A. skirrowii from poultry samples is in accordance with previous reports (Atabay et al., 1998; Houf et al., 2002). The low recovery of A. skirrowii may represent two possibilities: the low prevalence of the species among poultry samples, and the difficulty in the isolation of the species at the presence of other organisms. In addition, some changes for the isolation methods including the cultivation under microaerobic conditions or the use of blood agar plates without any antibiotics may improve the recovery rates of A. skirrowii and A. cryaerophilus since these species are more fastidious than A. butzleri (Kabeya et al., 2004). In this study, Arcobacter was better detected by the PCR assays than the culture method. This could be due to the higher analytical and diagnostic sensitivities of the PCR assays. However, care must be taken to avoid false-positive results arising from DNA contamination, as well as false-negative results caused by inhibitory substances in foods or enrichment broths. As the possibilities for amplicon contamination were minimised by separation of the preparation and amplification/detection laboratories and the blank controls were negative, false-positive results due to amplicants are unlikely to have occurred. Antibiotic susceptibility test results indicated that there was an overall increase in the resistance of Arcobacter to methicillin, vancomycin, cephalothin and azithromycin. Results of this study are comparable to those reported by others (Atabay and Aydin, 2001; Houf et al., 2002; Kabeya et al., 2004; Son et al., 2007b). In the present study, all the strains of A. butzleri were found to be resistant against cephalothin which is comparable to those reported by Atabay and Aydin (2001) and Kabeya et al. (2004). However, resistance to this antibiotic was lower than in A. cryaerophilus and A. skirrowii. These findings suggest that attention will be necessary when selective medium containing cephalothin for the isolation of A. cryaerophilus and A.
skirrowii is used because cephalothin may cause low recovery of these species (Kabeya et al., 2004). A high level of resistance in Arcobacter species was found to clindamycin (70.3%), a drug recommended as an alternative treatment for Campylobacter gastroenteritis in humans. Other authors have also reported a high level of resistance to clindamycin in A. butzleri from human and animal isolates (Fera et al., 2003). In contrast, resistance to erythromycin, chloramphenicol and ciprofloxacin was very low in A. butzleri and A. cryaerophilus while all the A. skirrowii isolates were susceptible to these antibiotics. This result is in agreement with the results reported by Atabay and Aydin (2001), Son et al. (2007b) and Fera et al. (2003). However, Otth et al. (2004) reported that 98% of A. butzleri isolated form foodstuffs were resistant to chloramphenicol. Especially the resistance to erythromycin and the decreased susceptibility to ciprofloxacin may have human health implications, as the two antimicrobials are generally prescribed as firstline drugs for the treatment of infections with Campylobacteraceae. In the present study, 60.6% of strains were resistant to ampicillin. These results are in agreement with those reported by other authors for ampicillin (Kiehlbauch et al., 1992; Harrass et al., 1998; Atabay and Aydin, 2001; Shah et al., 2013). However, some differences have been observed. While Kiehlbauch et al. (1992) reported 90% resistance, all the strains studied by Kabeya et al. (2004) were susceptible to this antibiotic. The differences in susceptibility patterns could be due to the frequency of the use of drug in animals for treatment and/or prophylaxis. Lack of gold-standard sensitivity methods and break points of antibiotics has made the comparison of results of antibiotic resistance patterns more difficult. However, compared with our data, a lower prevalence of resistance against nalidixic acid was also reported by Son et al. (2007b) and Atabay and Aydin (2001), while a high prevalence of resistance to nalidixic acid was reported in other studies (Kabeya et al., 2004). All Arcobacter strains included in our study were susceptible to the aminoglycoside gentamicin, streptomycin and kanamycin. Other authors have also reported that Arcobacter was susceptible to aminoglycosides, including kanamycin, amikacin gentamicin and streptomycin (Fera et al., 2003; Kabeya et al., 2004; Otth et al., 2004; Son et al., 2007b). In addition, all the Arcobacter isolates examined have been demonstrated to be susceptible to tetracycline as reported previously (Atabay and Aydin, 2001; Kabeya et al., 2004; Vandenberg et al., 2006; Son et al., 2007b). Thus, aminoglycosides and tetracycline would be suitable antibiotics for the treatment of disease caused by Arcobacter with veterinary and human medicine.
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In conclusion, the results of this study indicated the importance of poultry meats, especially chicken meat, as potential sources of Arcobacter spp. infection in people. Since high levels of contamination with Arcobacter spp. can occur in slaughterhouses (Rahimi et al., 2012), the maintenance of slaughter hygiene and regular microbiological monitoring of carcasses are essential to minimise the risk of contaminating when a danger of cross-contamination exists, especially when other poultry species with lower prevalence levels are slaughtered on the same slaughtering line. Furthermore, to ensure food safety, poultry meats must be cooked properly before consumption. The increased level of drug resistance, as encountered in this study, is important in terms of both animal and public health. Aminoglycosides and tetracycline can be used as first-choice drugs to fight Arcobacter infections.
ACKNOWLEDGEMENTS I would like to thank Dr Hassan Momtaz, Dr Amir Shakerian, Manouchehr Momeni and Majid Riahi for help in performing technical parts of the project. I am also grateful to Dr Mazear Rafei and Dr Morteza Mosavean for assistance with sampling.
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