Comp Clin Pathol (2013) 22:467–473 DOI 10.1007/s00580-012-1434-5
ORIGINAL ARTICLE
Prevalence and antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolated from raw camel, beef, and water buffalo meat in Iran Ebrahim Rahimi & Mehrdad Ameri & Mohammad Alimoradi & Ali Chakeri & Ahmad Reza Bahrami
Received: 5 January 2012 / Accepted: 2 February 2012 / Published online: 16 February 2012 # Springer-Verlag London Limited 2012
Abstract This study was conducted to determine the prevalence and antimicrobial resistance of Campylobacter spp. isolated from retail raw meats in Iran. From August 2009 to August 2010, a total of 379 raw meat samples from camel (n0130), beef (n0207), and water buffalo (n042) were purchased from randomly selected retail outlets in Chaharmahal va Bakhtiari and Khuzestan provinces in Iran. The samples were evaluated for the presence of Campylobacter using traditional bacteriological tests and a nested polymerase chain reaction. Overall, 31 of 379 meat samples (8.2%) were contaminated with Campylobacter. The highest prevalence of Campylobacter spp. was found in water buffalo meat (21.4%), followed by beef (9.2%), and camel (2.3%) meat. The most prevalent Campylobacter species isolated
E. Rahimi (*) Department of Food Hygiene, College of Veterinary Medicine, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran e-mail:
[email protected] E. Rahimi e-mail:
[email protected] M. Ameri Drug Safety Research and Development, Pfizer Inc., Groton, CT, USA M. Alimoradi College of Pharmacy, Jondishapour University of Medical Sciences, Ahvaz, Iran A. Chakeri : A. R. Bahrami College of Veterinary Medicine, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran
from meat samples was Campylobacter jejuni (77.4%); the remaining isolates were Campylobacter coli (22.6%). Susceptibilities of 31 Campylobacter isolates were determined for ten antimicrobial drugs using the disk diffusion assay. Of 31 Campylobacter isolates, 27 (87.1%) were resistant to one or more antimicrobial agents. Nine strains (29.0%) were resistant to one single antimicrobial agent, and eight strains (25.8%) showed resistance to two antimicrobial agents. Multidrug resistance was found in 32.3% of Campylobacter strains. Resistance to tetracycline was the most common finding (67.7%), followed by resistance to ciprofloxacin (32.7%), and nalidixic acid (32.7%). To the authors' knowledge, the present study is the first report of the isolation of Campylobacter spp. from raw water buffalo meat in Iran. Keywords Antimicrobial resistance . Beef . Campylobacter . Camel . Raw meat . Water buffalo
Introduction Campylobacter species are the most common cause of foodborne bacterial gastroenteritis worldwide (Friedman et al. 2000). The family Campylobacteraceae comprises small, spiral form, gram-negative bacteria with 18 species, 6 subspecies, and 2 biovars. They are essentially microaerophilic, growing best in an atmosphere containing approximately 10% CO2 and approximately 5% O2. The most important Campylobacter species associated with human illness are Campylobacter jejuni and Campylobacter coli (Wesley et al. 2000). Campylobacter species are widely distributed in nature and have been associated with poultry, pigs, cattle, sheep, shellfish, dogs, and cats (Skirrow 1977). Consumption
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of undercooked meat, unpasteurized milk, and contaminated drinking water is considered an important risk factor for campylobacteriosis (Eberhart-Phillips et al. 1997; Kapperud et al. 2003; Humphrey et al. 2007). Cross-contamination of ready to eat foods during food preparations with Campylobacter spp. as well as direct contact with animals have been reported (Mead et al. 1999; Hussain et al. 2007). Food animals may act as asymptomatic intestinal carriers of Campylobacter, and animal food products can become contaminated by this pathogen during slaughter and carcass dressing (Zanetti et al. 1996; Jorgensen et al. 2002; Whyte et al. 2004). Most patients with Campylobacter infections have a selflimited illness and do not require antimicrobial drugs except in cases with severe or prolonged symptoms, or in immunocompromised patients (Ketley 1997). Several studies have demonstrated that massive use of antimicrobial agents in animal production is linked to the development of resistance in zoonotic bacteria (Aarestrup and Wegener 1999), and foods of animal origin can be a source of transmission of resistant Campylobacter strains to people (Endtz et al. 1991; Nielsen et al. 1997; Taremi et al. 2006; Parisi et al. 2007). Although Campylobacter with resistance to antimicrobial agents has been reported worldwide (Van Looveren et al. 2001; Isenbarger et al. 2002; Wang et al. 2011), the situation seems to deteriorate more rapidly in developing countries, where there is widespread and uncontrolled use of antibiotics (Hart and Kariuki 1998). Surveillance and timely reporting of antimicrobial resistance patterns in C. jejuni and C. coli may provide important information to support actions directed at reducing the occurrence of resistance. Currently, there is limited information regarding the prevalence and antimicrobial susceptibility patterns of Campylobacter in raw meat in Iran. The present study was conducted to determine the prevalence and antimicrobial resistance of Campylobacter spp. isolated from retail raw camel, beef, and water buffalo meat in Chaharmahal va Bakhtiari and Khuzestan provinces in Iran.
Materials and methods Sample collection From August 2009 to August 2010, a total of 379 raw meat samples from camel (n0130), beef (n0207), and water buffalo (n042) were purchased unpacked from randomly selected butcheries in Chaharmahal va Bakhtiari and Khuzestan provinces in Iran. All samples were placed in separate sterile plastic bags to prevent spilling and cross-contamination and were immediately transported to the laboratory in a cooler with ice packs.
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Microbiological analysis The samples were processed immediately upon arrival using aseptic techniques. Of each meat sample, 25 g was homogenized and transferred to 225 mL of Preston enrichment broth base containing Campylobacter selective supplement IV (HiMedia Laboratories, Mumbai, India) and 5% (v/v) defibrinated sheep blood. After inoculation at 42°C for 24 h in a microaerophilic condition (85% N2, 10% CO2, 5% O2), 0.1 mL of the enrichment was then streaked onto Campylobacter selective agar base (HiMedia Laboratories, Mumbai, India) supplemented with an antibiotic supplement for the selective isolation of Campylobacter species (HiMedia Laboratories, Mumbai, India) and 5% (v/v) defibrinated sheep blood and was incubated at 42°C for 48 h under the same condition. One presumptive Campylobacter colony from each selective agar plate was subcultured, and identification of presumptive Campylobacter species was performed using standard microbiological and biochemical procedures including Gram staining, production of catalase, oxidase, hippurate hydrolysis, urease activity, indoxyl acetate hydrolysis, and susceptibility to cephalotin (Bolton et al. 1992; Whyte et al. 2004). DNA extraction and PCR condition Only Campylobacter spp. isolates identified by bacteriological methods were tested by PCR. The PCR procedures used in this study have been described previously (Denis et al. 1999). Briefly, 1 mL of pure culture of Campylobacter was centrifuged at 13,000×g for 5 min at room temperature. The DNA was then extracted using a genomic DNA purification kit (Fermentas, GmbH, Germany, K0512) according to the manufacturer's protocol. Three genes selected for the identification of the Campylobacter spp., C. jejuni and C. coli were the 16S rRNA gene (Linton et al. 1997), the mapA gene (Stucki et al. 1995), and the ceuE gene (Gonzalez et al. 1997), respectively. The sequences of the three sets of primers used for gene amplification are presented in Table 1. Amplification reactions were performed in a 30-μL mixture containing 0.6 U Taq polymerase (Fermentas, GmbH, Germany), 100 μmol 1−1 of each dNTP, 0.11 μmol 1−1 of MD16S1 and MD16S2 primers, and 0.42 μmol 1−1 of MDmapAl, MDmapA2, COL3, and MDCOL2 primers in the Fermentas buffer (Fermentas, GmbH, Germany). Amplification reactions were carried out using a DNA thermal cycler (Master Cycle Gradiant, Eppendrof, Germany) with the following program: one cycle of 10 min at 95°C, 35 cycles each consisting of 30 s at 95°C, 1 min and 30 s at 59°C, 1 min at 72°C, and a final extension step of 10 min at 72°C. The amplification generated 857 bp, 589 bp, and 462 bp DNA fragments corresponding to the Campylobacter genus, C. jejuni and C. coli, respectively. C. coli (ATCC 33559) and C. jejuni (ATCC 33560) were used as the positive controls and DNase free water was used as the
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Table 1 Prevalence of Campylobacter spp. isolated from camel, beef, and water buffalo meat in Chaharmahal va Bakhtiari and Khuzestan provinces, Iran Meat sample
No. of samples
Campylobacter spp. positivea (%)
C. jejuni (%)
Camel Beef Water buffalo Total
130 207 42 379
3 (2.3)a 19 (9.2)b 9 (21.4)c 31 (8.2)
1 (33.3)a 17 (89.5)a 6 (66.7)a 24 (77.4)
C. coli (%) 2 2 3 7
(66.7)a (10.5)a (33.3)a (22.6)
Values in the same column with different lowercase letters are significantly different (p
