The intensive use of antibiotics in human and veterinary medicine in the past 50 years has brought the problem of resistance to antibiotics (AR) in pathogenic microorganisms and reduced the effectiveness of infections’ treatment which the World Health Organisation declared to be one of the greatest threats to health. It is well known that the antibiotic resistance genes (ARGs) can be horizontally transferred among bacteria of different species, and pathogenic bacteria with AR occur along the entire agro‐food chain. The fact that the commensal bacteria in food can present a reservoir of ARG have begun to draw attention of scientists years ago, but until recently there were not so advanced and powerful molecular methods and technologies available that enable the disclosure of whole bacterial genomes and microbiomes of complex samples, including the "resistome" ‐ collection of all AR genes present in a given environment. The study focuses on the bacterial representatives of starter cultures and probiotics, which are intentionally added to the food chain, as well as on their whole genomic sequences (WGS) ‐ either publicly available or those that were obtained in this study. Using next generation sequencing (Illumina technology), we acquired the WGSs of commercial or own bacterial isolates, representatives of starter and probiotic cultures. In the known or newly acquired WGS, we identified the ARGs, mutations and mobile elements in silico, by comparative genomic analysis, and determined the matching of the in silico AR predictions with the results of phenotypic detection of sensitivity to selected antibiotics. The latter is important from the standpoint of assessing the nature (intrinsic or acquired) of the observed phenotypic resistance and possible improvement of criteria (MIC cut‐off) for AR in lactic acid bacteria and bifidobacteria. We set up and validated the protocols based on polymerase chain reaction (PCR, qPCR) for the targeted screening of the resistomes of probiotic products and fermented foods, produced with commercial starter cultures or without them. Furthermore, selected food samples were examined by metagenomic sequencing in order to identify more ARGs and determine the relative abundances of these ARGs, and to assess the contribution of added starter and probiotic cultures to the resistomes of investigated food samples. The approach presented in the project, which includes in silico examination of whole bacterial genomes, metagenomic sequencing of complex food samples, and quantitative support by real‐time PCR (qPCR) enabled efficient detection of these features in different resistomes associated with foods, and better assessment of the risk of transmission of ARGs along the food chain. We covered the gap between previous studies, which were based on phenotypic testing and detection of a limited number of well‐known targets, and among those in the coming years, when the use of the most powerful tools for metagenomic analyses is expected to spread well into the food safety area.
Less...