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

Abstract

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.

Antibiotic Susceptibility Profiles of Lactic Acid Bacteria from the Human Vagina and Genetic Basis of Acquired Resistances

“…The only criterion that LAB strains have to fulfill to meet QPS requirements is the absence of AR genes against antimicrobials of clinical and veterinary importance [8]. Although LAB are non-pathogenic, they can act as reservoirs of AR genes which might eventually be transferred via HGT to pathogenic bacteria during food manufacture or after consumption [9].…”

“…In order to prevent the spread of AR in the vaginal ecosystem, any strain used as a probiotic must be free of acquired AR genes with transference capability. It is important to note that some LAB strains, including some from the human vagina, have already been reported to carry transferable AR genes [9,[14][15][16][17].…”

Scientific Opinion on the update of the list of QPS‐recommended biological agents intentionally added to food or feed as notified to EFSA†

“…Flórez et al. () determined the MIC for 16 antimicrobials in 34 isolates of dairy origin. Atypical resistances were found for several antimicrobials.…”

Genomic Characterisation of Starter Cultures and Probiotic Bacteria

“…Recently, genome analysis of three Le. mesenteroides strains isolated from Italian soft cheese samples provided a better understanding of the genetic bases of AR in such species and its transference capability among food-borne bacteria (Flórez et al, 2016 displaying atypical resistance to this antibiotic (MIC of 32 μg/ml) (EFSA, 2012), thus suggesting a new mechanism of resistance which can be due either to acquired genes or to a mutation of indigenous genes. Interestingly, analysis of the AR genes and their flanking regions revealed the potential of some determinants to be horizontally transferred.…”

“…Indeed, the erythromycin resistance was transferred by conjugation between Le. mesenteroides and E. faecalis both in vitro and in cheese (Flórez et al, 2016), supplying novel proof that LAB can act as a reservoir of acquired AR genes, for which reason their safety should be carefully examined.…”