Computational protocol: Antibiotic Susceptibility Profiles of Dairy Leuconostoc, Analysis of the Genetic Basis of Atypical Resistances and Transfer of Genes In Vitro and in a Food Matrix

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Protocol publication

[…] Identification of strains at species and subspecies level was carried out using molecular biology based methods and selected phenotypic tests.Amplification and sequencing of the 16S rRNA gene and three protein-coding genes, i.e. the genes encoding the α-subunit of ATP synthase (atpA), RNA polymerase α-subunit (rpoA) and phenylalanyl-tRNA synthase α-subunit (pheS), were carried out according to the indications of Rizzotti et al. [] and De Buyne et al. [], respectively. Primer sequences, PCR conditions and sequencing used in each case were those described by the corresponding reference (Table C in ).The atpA and pheS sequences of the isolates and type strains of species within the Leuconostoc genus (Table D in ) were used for phylogenetic analyses using MEGA version 6 software [].Leuconostoc mesenteroides strains were tested for their ability to ferment some carbohydrates using the protocol reported by Bjorkroth and Holzapfel []. Briefly, strains were grown in Basal MRS-medium (pH 6.5) supplemented with a selected carbohydrate to give a concentration of 1%. After incubation at 30°C for 7 days, acid production was indicated by a change from purple to yellow in the colour of the chlorophenol red indicator dye. [...] Sequences surrounding the AR genes from the strains L. mesenteroides LbE15, LbE16 and LbT16 were analysed by retrieving those contigs carrying antibiotic determinants from the published whole genome sequencing data []. The GenBank accession numbers for LbE15, LbE16, and LbT16 genomes are LAYN00000000, LAYU00000000, and LAYV00000000, respectively. The Comprehensive Antibiotic Resistance Database (CARD) at http://arpcard.mcmaster.ca/, CLC Bioinformatics Database software package (CLC bio, Aarhus, Denmark), RAST annotation system (http://rast.nmpdr.org/) and Basic Local Search Tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi) were consulted for detail description of AR determinants and their flanking regions. […]

Pipeline specifications

Software tools MEGA, RAST
Databases CARD NMPDR
Applications Phylogenetics, WGS analysis
Organisms Leuconostoc mesenteroides, Leuconostoc citreum, Leuconostoc lactis, Leuconostoc carnosum, Leuconostoc mesenteroides subsp. mesenteroides, Enterococcus faecalis
Chemicals Aminoglycosides, Chloramphenicol, Ciprofloxacin, Clindamycin, Erythromycin, Kanamycin, Rifampin, Streptomycin, Tetracycline, Virginiamycin