Prevalence of Clostridium difficile in raw beef, cow, sheep, goat, camel and buffalo meat in Iran

Background Clostridium difficile has been shown to be a nosocomial pathogen associated with diarrhoea and pseudomembranous colitis in hospitalised patients and the infection is believed to be acquired nosocomially. Recent studies have shown the occurrence of C. difficile in food animals which may act as a source of infection to humans.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. Method From April to October 2012, a total of 660 raw meat samples from beef, cow, sheep, goat, camel and buffalo were purchased from 49 butcheries in Isfahan and Khuzestan provinces, Iran, and were evaluated for the presence of C. difficile using a method including selective enrichment in C. difficile broth, subsequent alcohol shock-treatment and plating onto C. difficile selective medium. C. difficile isolates were tested for the presence of toxin genes and were typed using PCR ribotyping. Results In this study, 13 of 660 meat samples (2%) were contaminated with C. difficile. The highest prevalence of C. difficile was found in buffalo meat (9%), followed by goat meat (3.3%), beef meat (1.7%), cow (0.94%) and sheep meat (0.9%). Seven of the 13C. difficile strains (53.9%) were positive for tcdA, tcdB and cdtB toxin genes and were classified as ribotype 078. Four strains (30.8%) were positive tcdA, and tcdB, and one strain (7.7%) was possessed only tcdB. The remaining isolate was non-toxigenic. Susceptibilities of 13C. difficile isolates were determined for 11 antimicrobial drugs using the disk diffusion assay. Resistance to clindamycin, gentamycin, and nalidixic acid was the most common finding. Conclusions To our knowledge, the present study is the first report of the isolation of C. difficile from raw buffalo meat. This study indicates the potential importance of food, including buffalo meat, as a source of transmission of C. difficile to humans.


Background
Clostridium difficile is a Gram-positive, anaerobic, sporeforming 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][5][6]. Food animals are an important source of enteropathogens, and C. difficile has been isolated from food animals such as poultry and sheep [4][5][6][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][11][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.

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.

Results and discussion
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 nontoxigenic. Our finding of C. difficile and its toxigenic strains in meat are supported by similar reports from other counters [9,15,16,[20][21][22][23]. The low prevalence of C. difficile in cow, beef, goat and sheep meat samples are comparable with those reported by others [16,[20][21][22][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.

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][30][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.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
The supporting of project were performed by ER, MJ and JSW, DNA extraction, PCR techniques were performed by ER and MJ and samples collection, culture, statistical analysis and writing of manuscript were performed by ER. All authors read and approved the final manuscript.