Research Projects

Abstract

The extensive use of antibiotics in humans and animals has accelerated the spread of antibiotic resistance among bacteria, which poses a significant threat to their effectiveness. The antibiotic resistance of foodborne pathogenic bacteria and their spread throughout the agro-food chain is a major food safety concern that has been recognized and studied for a long time. Only in recent years has there been increasing evidence that commensal bacteria, including lactic acid bacteria, can also serve as reservoirs of antibiotic resistance genes (ARGs). However, until recently, advanced and powerful Next Generation Sequencing technologies such as shotgun metagenomic sequencing, which enables the exploration of resistomes and mobilomes in a specific environment, including food, have not been used in the microbiota of fermented foods. The available data are therefore insufficient to address with certainty the safety of lactic acid bacteria and the fermented foods containing them with regard to antibiotic resistance and its transmission potential. In the proposed project, we will use advanced sequencing technologies to reveal the burden of mobile resistomes in the microbiota of fermented foods and evaluate their actual transferability. Various samples of fermented foods representing different groups depending on the raw material, such as fermented dairy products and fermented vegetable products, will be collected from local producers, retail outlets and markets. Using shotgun metagenomic sequencing technology, we will acquire whole bacterial metagenomes of these samples. After thorough bioinformatic analysis and de novo assembly, sequences will be mined for ARGs and mobile genetic elements using custom scripts and our non-redundant databases of ARGs and mobile genetic elements. Next, we will analyse the transferability of selected antibiotic resistance genes using in vitro and in situ experiments. First, a number of strains isolated from probiotic products, cheeses and other fermented dairy products carrying acquired ARGs will be used in filter mating experiments in which we will study the transfer of these genes to a selected recipient strain. Based on the results, the transfer potential of selected ARGs into a recipient strain will also be analysed in situ in a food matrix using the milk fermentation model. In both methods, we will confirm the transconjugants by phenotypic assessment and molecular methods, such as microdilution, PCR/qPCR, RAPD. In addition, we will monitor the transmission events of ARGs carried by up to five selected donor strains in situ in a food matrix during production and ripening of semi-soft cheese from raw milk by investigating the metagenomes of the microbiota of the cheese samples at different time points of cheese ripening. The metagenomes will be sequenced using Next Generation Sequencing and mined for ARGs and mobile genetic elements of donor strains. We will also perform a control experiment of fermentation without a donor strain. The approach presented in the submitted project, which includes an in silico survey of shotgun metagenomic sequences and an in situ analysis of the transferability of resistance genes in the food matrix, will reveal the burden of mobile resistomes in fermented foods and allow a better assessment of the risk of ARGs transmission along the food chain. We expect to close this knowledge gap, which will ultimately improve the safety of widely consumed fermented foods.

Researchers

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The phases of the project and their realization