Clostridiodes difficilepresencia a lo largo de la cadena alimentaria

Resumo

This doctoral thesis is presented as a collection of five manuscripts whose main objective has been to expand the knowledge of C. difficile in the food environment and determine its occurrence at different steps of the food supply chain. The first study has focussed on a literature review that delves into the epidemiology of C. difficile associated with food. Highest C. difficile prevalence sources in the different stages of the food supply chain have analysed to define the goals for searching the microorganism in the food environment. The literature review reflects the lack of a standardized methodology for sampling and recovery techniques, that hinders the comparison of data from different studies. On the other hand, there are few data about the growth and resistance of vegetative forms and spores of C. difficile in food matrices, and the number of spores necessary or infectious dose that results in CDI has not been established. Currently there are no documented cases of CDI related with a food consumption pattern and the risk factors involved in acquiring CDI from food are not known. All these factors preclude the inclusion of this pathogen as a foodborne agent. In order to draw conclusions about the presence of the pathogen in primary production, in the second study a procedure to detect C. difficile from faecal specimens of piglets has been developed. A comparison of the C. difficile isolation rates after different treatments of the specimens, prior to culture in a selective medium, and an evaluation of the correlation of the isolation rates of C. difficile between faeces and rectal swab specimens has been studied. Combined pre-treatment of faecal specimens with enrichment broth followed by ethanol shock has been shown to achieve the highest recovery rate of C. difficile. Rectal swabs present sensitivity values equivalent to that obtained in faeces specimens after pre-treatment with the broth enrichment method, thus, the use of rectal swabs is proposed in C. difficile surveillance studies due to their easy collection, transport and handling. Swine has been selected as the prevalent source of C. difficile in slaughter animals to determine the presence of C. difficile in the first supply chain stage and to study the effect of the production cycle on its presence in animals. The results reflect a toxigenic C. difficile isolation rate of 16.9 % of the studied population, a rate that decreases with the animal age. In the third study, the data obtained in the slaughterhouse are presented as the next step in the food supply chain. The presence of C. difficile has been determined in poultry giblets obtained in the cutting room of a processing plant. The samples were subjected to a pre-treatment consisting of incubation in an enrichment broth supplemented with sodium taurocholate followed by an ethanol shock, before their isolation in a selective culture medium. The results reflect an isolation rate of C. difficile from gizzards and liver of 16.7 % showing the survival of the microorganism in giblets and its possible transmission to other pieces of meat during slaughter operations has been demonstrated. Additionally, in the third article, the presence of C. difficile in meat acquired in the retail stage has been determined. Fresh and processed poultry and pork meat have been selected due to their importance in the sector and their high consumption, and the same pre-treatment and culture protocol for the giblet samples has been followed. We conclude that the absence of C. difficile detection in fresh and processed poultry and pork meat samples indicates that the meat, and not giblets, does not appear to be a significant source of transmission of the pathogen to humans. The fourth study also provides data on the presence of C. difficile in molluscs acquired in the retail stage. The mussel samples were also processed with a pre-treatment consisting of incubation in an enrichment broth followed by an ethanol shock, before their isolation in solid medium. The C. difficile isolation in 8.5 % of the analysed mollusc samples indicates that the ingestion of contaminated raw or poorly cooked molluscs could be a vehicle to consider in the acquisition of the bacteria. In the fifth study, a first approach to evaluate the in vitro effect of natural substances, such as Moringa (Moringa oleífera), on the growth of spore-forming bacteria of the genus Clostridium spp. Broth microdilution method containing serial dilutions of extracts of the edible parts of the plant has been developed. It has been observed that the different presentations of the Moringa leaf and seed extracts present variable inhibitory effect depending on the extraction solvent used, showing the extracts of fresh red stem leaves the greatest inhibitory effect. As a whole, the thesis provides a first approach to the investigation of C. difficile in the food supply chain, contributing to understanding the risk of infection associated with food consumption and its transmission to human.