Background

Clostridium difficile is a Gram-positive, anaerobic, spore-forming bacterium that has come to the forefront as an important human pathogen. It was initially dismissed as commensal in healthy infants, but was recognized as an important cause of antimicrobial-associated with diarrhoea in the 1970s. It is, now, the most commonly diagnosed cause of antimicrobial-associated and hospital-associated diarrhoea, and the cause of virtually all cases of pseudomembranous colitis [1]. C. difficile infection (CDI) more recently was described in non-hospitalized patients without underlying disease or a predisposing risk factor such as antimicrobial exposure, advanced age or significant comorbidities [2, 3].

C. difficile also appears to be an important cause of enteric disease or a commensal in a wide variety of animal species [4–6]. Food animals are an important source of enteropathogens, and C. difficile has been isolated from food animals such as poultry and sheep [4–7], pigs [8, 9], chickens, goats and cattle [6] and calves [10]. The types of C. difficile found in animals and humans are often indistinguishable [10–12] raising concerns that C. difficile might be a zoonotic pathogen [9, 11]. In particular, ribotype 078 is commonly found in food animals [5, 13] and an increasingly reported cause of community-associated CDI in humans [5, 14].

The epidemiology of CDI in Iran is poorly understood. The recent finding of ribotype 078 as the leading ribotype in a small study of CDI in humans in Iran [14] raised concern about the potential for food as a source of infection, but the prevalence of C. difficile in food in Iran has never been reported. The aim of this study was to determine the occurrence of C. difficile in retail raw beef, cow, sheep, goat, camel and buffalo meat in Iran.

Methods

Sample collection

From April to October 2012, a total of 660 raw meat samples from beef (young cattle) (n = 121), cow (adult dairy cow) (n = 106), sheep (n = 150), goat (n = 92), camel (n = 124) and buffalo (n = 67) were purchased unpacked from 49 butcheries in Isfahan and Khuzestan provinces, Iran. These cities are the most prominent national cultural and tourist centers located in the center and south of the country, respectively. From each city 40–55 samples (about 0.5 kg / sample; two sections of meat (10 cm × 10 cm × 3 cm) from neck of each carcasses were aseptically removed) were purchased monthly. All samples were placed in separate sterile plastic bags to prevent from spilling and cross contamination and were immediately transported to the laboratory in a cooler with ice packs and processed within 6 h.

Isolation and identification of C. difficile

The samples were processed immediately upon arrival using aseptic techniques. The detection and isolation method used were based on the method described by Rodriguez-Palacios et al. [15] and de Boer et al. [16]. Briefly, 5 g of each sample was transferred to 20 mL of C. difficile broth (CDB; Oxoid SR0048) containing 40 g/l proteose peptone, 5.0 g/l, disodium hydrogen phosphate, 0.1 g/l magnesium sulphate, 2.0 g/l sodium chloride, 6.0 g/lfructose and 1.0 g/l sodium taurocholate supplemented with C. difficile selective supplement (Oxoid, UK, Code: SR0173) and 5% (v/v) defibrinated sheep blood. After incubation at 37°C for 10 to 15 days under anaerobic conditions 2 mL of the enrichment broth was added to 2 mL of 96% ethanol in a centrifuge tube and homogenized for 50 min on a shaker at room temperature. After centrifugation (3800 × g for 10 min), a loopful of the sediment was streaked onto C. difficile agar base (Oxoid, UK, Code: CM0601) supplemented with an antibiotic supplement for the selective isolation of C. difficile (Oxoid, UK, Code: SR0173) and 7% (v/v) defibrinated sheep blood and the plates were incubated for 48 h at 37°C, under anaerobic conditions. Three colonies per plate were subcultured onto tryptone soya agar (Oxoid, UK, Code: CM0131) and tested by standard microbiological and biochemical procedures including odour, Gram stain morphology and L-proline aminopeptidase test [4]. Crudely extracted DNA [boiling method: One colony was suspected in 500 μl distilled water and after heating for 10 min at 95°C, the suspension was centrifuged (5 min, 10000 × g)] was used for PCR confirmation (tpi gene detection), determination of toxin gene (tcdA, tcdB and cdtB), and PCR ribotyping of isolates as performed in previous studies [17, 18]. For assurance managed at the lab positive and negative controls were included in each batch.

The limitations of the study include the small number of C. difficile isolates which were analysed in the study and the impossibility of sampling from other areas of Iran.

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was done by the Kirby–Bauer disc diffusion method using Mueller–Hinton agar (HiMedia Laboratories, Mumbai, India) according to the Clinical Laboratory Standards Institute (CLSI) [19] as has been previously described [4]. The antimicrobial agents tested and their corresponding concentrations were as follows: nalidixic acid (30 μg), ciprofloxacin (5 μg), erythromycin (15 μg), tetracycline (30 μg), doxycycline (30 μg), gentamicin (10 μg), metronidazol (5 μg), ampicillin (10 μg), chloramphenicol (30 μg), vancomycin (30 μg), and clindamycin (2 μg). After incubating the inoculated plate for 48 h at 37°C, under anaerobic conditions, the susceptibility of the C. difficile to each antimicrobial agent was measured and the results were interpreted in accordance with interpretive criteria provided by CLSI [19].

Results and discussion

Table 1 shows the prevalence of C. difficile isolated from beef, cow, sheep, goat, camel and buffalo meat in two provinces, Iran. C. difficile was isolated 13/660 meat samples (Table 1). There were no significant differences (P > 0.05) in the frequency of positive samples among the meat samples or between Isfahan (4/315, 1.3%) and Khuzestan (9/355, 2.3%) (P > 0.05).

Table 1 Prevalence of Clostridium difficile detected in beef, cow, sheep, goat, camel and buffalo meat samples in Iran
Full size table

The highest prevalence of C. difficile was found in buffalo meat samples (6/67), followed by goat (3/92), beef (2/121) and sheep (1/150) (Table 1). All the camel meat samples found to be negative. Toxigenic C. difficile strains (tcdA, tcdB and cdtB toxin genes) were detected in 12/13 of isolates. Seven of the 13C. difficile strains (53.9%) were positive fortcdA, tcdB and cdtB toxin genes. Four strains (30.8%) were positive tcdA, and tcdB, and one strain (7.7%) was possessed only tcdB. The remaining isolate was non-toxigenic. Our finding of C. difficile and its toxigenic strains in meat are supported by similar reports from other counters [9, 15, 16, 20–23]. The low prevalence of C. difficile in cow, beef, goat and sheep meat samples are comparable with those reported by others [16, 20–23]. However, higher contamination rates (20% to 50%) have also been reported [9, 15]. In contrast, Von Abercron et al. [24] did not detect C. difficile in meat samples other than beef. Whether this reflects a true different prevalence or is due to differences in sampling techniques employed (meat sample, carcass swab or carcass rinse fluid sample), seasonal effects [20] and/or laboratory methodologies employed in different studies is not clear.

The source of C. difficile in food products is unclear. Contamination of meat might be due to C. difficile residing in the gastrointestinal tract of animals, but could also originate from the hands of personnel working in the slaughterhouse, meat processing equipment or the slaughterhouse environment during the slaughtering process [5, 25, 26]. The prolonged survival of C. difficile spores in the environment increases the possibilities for contamination of animals and foods. Another potential source of infection that requires investigation is the presence of C. difficile spores in healthy muscle tissue in living animals [1].

Seven of the 13C. difficile strains were positive for tcdA, tcdB and cdtB toxin genes and were classified as ribotype 078. The predominance of ribotype 078 is consistent with other studies of food animals and food [8, 23, 27]. Given the presence of this strain in humans in the same region with CDI, consideration must be given as to whether food might be the source. However, further study is required to determine whether food is a reasonable source of infection.

Antimicrobial susceptibility data are presented in Table 2. Resistance of C. difficile to clindomycin, gentamycin, nalidixic acid, ciprofloxacin, erythromycin, ampicillin, and tetracycline was high. These results are comparable to those reported by other investigators [6, 22, 28]. All the C. difficile isolates were susceptible to metronidazole, and vancomycin as was observed in other studies [6, 15, 22]. These two drugs are the most commonly used to treat C. difficile diarrhea in humans but are not used in food animals. The results of antimicrobial resistance found in this study are correlated with antibiotics usage to treat infections in food animals in Iran. In contrast, many of the drugs to which the isolates were resistant (i.e. gentamicin) are commonly used in food animals.

Table 2 Antimicrobial resistance of 13 Clostridium difficile isolated from beef, cow, sheep, goat, camel and buffalo meat in, Iran
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Conclusions

This study indicates that the potential importance of food, including buffalo meat, as a source of transmission of C. difficile to humans. Slaughterhouses can be heavily contaminated with foodborn pathogens [29–31], the maintenance of slaughter hygiene, regular microbiological monitoring of carcasses, implementation of good manufacturing practices and a food safety system such as the HACCP system are essential to minimize the risk to the consumer. To the author’s knowledge, the present study is the first report of the isolation of C. difficile from raw beef, cow, goat, sheepand buffalo meat in Iran. Further studies are required to determine the prevalence of C. difficile in meat in Iran and to explore the potential risk of human infection with C. difficile via consumption of meat.