Isolation and Characterization of Clostridium difficile in Farm Animals from Slaughterhouse to Retail Stage in Isfahan,Iran

Introduction

C lostridium difficile infection (CDI) has been identified as an important cause of hospital-associated diarrhea in healthcare settings and community. The epidemiology of community CDI is poorly understood, with a lack of clear information on routes of bacterium transmission. This has led to consideration of different potential sources in the community in specific food. The hypothesis that C. difficile is a foodborne pathogen has been highlighted due to the genotypic overlap of C. difficile from humans with CDI and those isolated from food animals and retail meat. Recently, C. difficile has been reported in retail meat and meat-processing plants in Iran, with slaughterhouses identified as a possible source of this organism (Esfandiari et al., 2014a, b; Rahimi et al., 2014). Therefore, the aim of this study was to evaluate the prevalence of C. difficile in cattle, camels, goats, and sheep in slaughterhouse and retail meat in Isfahan, Iran.

Materials and Methods

A large slaughterhouse of cattle, camels, goats, and sheep in Isfahan province, Iran was enrolled. A total of 750 samples were collected during 12 slaughterhouse and retail visits between December 2012 and July 2013. At each visit, two to five animals per species were sampled. Feces were collected before slaughtering (n = 150). Animals were followed through the slaughtering line so that further samples could be collected from the same animals' carcasses throughout processing. Samples of carcasses were taken at postevisceration (n = 150) and postwashing (n = 150) steps. A further 300 samples of meat were collected from 11 butcher stores supplied from the slaughterhouse under study. Presumptive colonies of C. difficile recovered from samples were screened by l-proline test. For molecular characterization, isolates were screened for the presence of genes encoding tpi, tcdA, tcdB, and cdtB. C. difficile isolates were also subjected to polymerase chain reaction (PCR) ribotyping (Bidet et al., 1999). The ribotypes were assessed visually and assigned using internal nomenclature.

Results and Discussion

C. difficile was isolated from 133 (17.7%) samples, but only 29 (3.8%) of these were toxigenic. Only toxigenic isolates are discussed further. At retail level, C. difficile was isolated from 5.6% (17/300) of meat taken from 4/11 (36%) of butcheries. One store accounted for 64% of retail isolates. All toxigenic isolates possessed both tcdA and tcdB but not cdtB. Twenty-one different ribotypes were identified among the 29 toxigenic isolates. PCR ribotypes including IR12, IR33, and IR37 were the most frequent (14.2% each). Ribotypes IR12, IR45, and IR46 were isolated from both feces and carcass samples of three animals. However, most strains found at the slaughterhouse differed from those found at the retail level (Table 1).

C. difficile was identified in food animals shortly before slaughter; however, the prevalence was low, which is consistent with report by Costa et al. (2011). Higher rates of C. difficile have been reported by Houser et al. (2012). Isolation of C. difficile from cattle, goat, and sheep species has been previously reported, yet data pertaining to camels are very limited (Hafiz and Oakley, 1976). Despite the presence of C. difficile in feces and initial carcass sampling, no C. difficile was isolated from washed carcasses, suggesting the efficiency of carcass washing with lactic acid on eliminating C. difficile spores. These results are consistent with a study that found no spores on carcasses postwashing by citric acid at an abattoir (Houser et al., 2012).

At a retail outlet, the difference in contamination of chopped and ground meat of beef and sheep was significant, perhaps reflective of the greater surface area of ground meats or more chances for contamination during the additional processing involved in grinding meat. All 29 strains possessed tcdA and tcdB but did not harbor cdtB. The large number of different ribotypes indicates marked heterogeneity in C. difficile in food animals in Iran. Three of the six ribotypes isolated from postevisceration carcass (IR12, IR45, and IR46) were also found in fecal samples, showing that animal's intestinal tract is the most likely source of contamination (Rahimi et al., 2014). However, given the hardy nature of spores, it is possible that environmental contamination of the animals or processing could also play a role in this issue. Ribotype IR12 found here was identified in ground beef in a previous Iranian study in a meat-packaging plant (Esfandiari et al., 2014a).

A limitation of the present study would include lack of a reference laboratory for Iranian ribotypes found in healthcare facilities and different sources of community as well as limited geographical area of sample collection.