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

• DOI: 10.1371/journal.pone.0145203
• Publication Date: 2016-01-04T18:39:00Z
• Authors: Ana Belén Flórez; Ilenia Campedelli; Susana Delgado; Ángel Alegría; Elisa Salvetti; Giovanna E. Felis; Baltasar Mayo; Sandra Torriani
• Keywords: 0301 basic medicine; Antibiotic resistance; Science; Microbial Sensitivity Tests; Antimicrobial resistance; 03 medical and health sciences; Antibiotics; Enterococcus faecalis; Animals; 2. Zero hunger; Q; R; Leuconostoc; Leuconostoc mesenteroides; lactic acid bacteria; starters; adjunct cultures; cheese; antibiotic resistance; erm(B) gene; tet(S) gene; genome sequencing; genome analysis; horizontal gene transfer; Polymerase chain reaction; Erythromycin; Anti-Bacterial Agents; 3. Good health; Dairying; Chloramphenicol; Tetracyclines; Genes, Bacterial; Medicine; Cattle; Leuconostoc; Research Article

In spite of a global concern on the transfer of antibiotic resistances (AR) via the food chain, limited information exists on this issue in species of Leuconostoc and Weissella, adjunct cultures used as aroma producers in fermented foods. In this work, the minimum inhibitory concentration was determined for 16 antibiotics in 34 strains of dairy origin, belonging to Leuconostoc mesenteroides (18), Leuconostoc citreum (11), Leuconostoc lactis (2), Weissella hellenica (2), and Leuconostoc carnosum (1). Atypical resistances were found for kanamycin (17 strains), tetracycline and chloramphenicol (two strains each), and erythromycin, clindamycin, virginiamycin, ciprofloxacin, and rifampicin (one strain each). Surprisingly, L. mesenteroides subsp. mesenteroides LbE16, showed resistance to four antibiotics, kanamycin, streptomycin, tetracycline and virginiamycin. PCR analysis identified tet(S) as responsible for tetracycline resistance in LbE16, but no gene was detected in a second tetracycline-resistant strain, L. mesenteroides subsp. cremoris LbT16. In Leuconostoc mesenteroides subsp. dextranicum LbE15, erythromycin and clindamycin resistant, an erm(B) gene was amplified. Hybridization experiments proved erm(B) and tet(S) to be associated to a plasmid of ≈35 kbp and to the chromosome of LbE15 and LbE16, respectively. The complete genome sequence of LbE15 and LbE16 was used to get further insights on the makeup and genetic organization of AR genes. Genome analysis confirmed the presence and location of erm(B) and tet(S), but genes providing tetracycline resistance in LbT16 were again not identified. In the genome of the multi-resistant strain LbE16, genes that might be involved in aminoglycoside (aadE, aphA-3, sat4) and virginiamycin [vat(E)] resistance were further found. The erm(B) gene but not tet(S) was transferred from Leuconostoc to Enterococcus faecalis both under laboratory conditions and in cheese. This study contributes to the characterization of AR in the Leuconostoc-Weissella group, provides evidence of the genetic basis of atypical resistances, and demonstrates the inter-species transfer of erythromycin resistance.