title: Functional screening of antibiotic resistance genes from a representative metagenomic library of food fermenting microbiota.
abstract: Lactic acid bacteria (LAB) represent the predominant microbiota in fermented foods. Foodborne LAB have received increasing attention as potential reservoir of antibiotic resistance (AR) determinants, which may be horizontally transferred to opportunistic pathogens. We have previously reported isolation of AR LAB from the raw ingredients of a fermented cheese, while AR genes could be detected in the final, marketed product only by PCR amplification, thus pointing at the need for more sensitive microbial isolation techniques. We turned therefore to construction of a metagenomic library containing microbial DNA extracted directly from the food matrix. To maximize yield and purity and to ensure that genomic complexity of the library was representative of the original bacterial population, we defined a suitable protocol for total DNA extraction from cheese which can also be applied to other lipid-rich foods. Functional library screening on different antibiotics allowed recovery of ampicillin and kanamycin resistant clones originating from Streptococcus salivarius subsp. thermophilus and Lactobacillus helveticus genomes. We report molecular characterization of the cloned inserts, which were fully sequenced and shown to confer AR phenotype to recipient bacteria. We also show that metagenomics can be applied to food microbiota to identify underrepresented species carrying specific genes of interest.
authors.affiliation: CRA-NUT, Food & Nutrition Research Center, Agricultural Research Council, Via Ardeatina 546, 00178 Rome, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Devirgiliis
authors.affiliation: CRA-NUT, Food & Nutrition Research Center, Agricultural Research Council, Via Ardeatina 546, 00178 Rome, Italy.
authors.forename: Paola
authors.identifier: 0000-0002-3176-7007
authors.initials: P
authors.lastname: Zinno
authors.affiliation: CRA-NUT, Food & Nutrition Research Center, Agricultural Research Council, Via Ardeatina 546, 00178 Rome, Italy ; Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185 Rome, Italy.
authors.forename: Mariarita
authors.identifier: 0000-0001-8620-0190
authors.initials: M
authors.lastname: Stirpe
authors.affiliation: CRA-NUT, Food & Nutrition Research Center, Agricultural Research Council, Via Ardeatina 546, 00178 Rome, Italy.
authors.forename: Simona
authors.initials: S
authors.lastname: Barile
authors.affiliation: CRA-NUT, Food & Nutrition Research Center, Agricultural Research Council, Via Ardeatina 546, 00178 Rome, Italy.
authors.forename: Giuditta
authors.initials: G
authors.lastname: Perozzi
mesh_terms: D002611:Cheese / Q000382:microbiology; D003611:Dairy Products / Q000382:microbiology; D004352:Drug Resistance, Microbial / Q000235:genetics*; D005285:Fermentation / Q000235:genetics*; D005516:Food Microbiology; D015723:Gene Library; D005820:Genetic Testing; D016680:Genome, Bacterial; D054892:Metagenome / Q000235:genetics*; D064307:Microbiota / Q000235:genetics*