title: A standardized conjugation protocol to asses antibiotic resistance transfer between lactococcal species.
abstract: Optimal conditions and a standardized method for conjugation between two model lactococcal strains, Lactococcus lactis SH4174 (pAMbeta1-containing, erythromycin resistant donor) and L. lactis Bu2-60 (plasmid-free, erythromycin sensitive recipient), were developed and tested in a inter-laboratory experiments involving five laboratories from different countries. The ultimate goal of the study was to assess the microbial potential of antibiotic resistance transfer among Lactic Acid Bacteria (LAB). The influence of culture age (various OD values) and ratios of donor and recipient cultures as well as filter, solid and liquid mating techniques, were examined in order to optimize the conjugation protocol. In the result of these studies, we concluded that the donor-to-recipient ratio appear to be important; the most efficient technique for conjugation was filter mating and the optimal conditions for gene transfer were observed when late logarithmic cultures of both donor and recipient were used. Comparison of conjugal transfer frequencies between five partner laboratories showed that results are sufficiently intra-laboratory repeatable and inter-laboratory comparable. This is the first study of this kind, in which a standardized protocol of conjugal mating for testing antibiotic resistance dissemination among LAB was established and validated.
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawinskiego 5a, 02-106 Warsaw, Poland.
authors.forename: Joanna
authors.initials: J
authors.lastname: Lampkowska
authors.forename: Louise
authors.initials: L
authors.lastname: Feld
authors.forename: Aine
authors.initials: A
authors.lastname: Monaghan
authors.forename: Niamh
authors.initials: N
authors.lastname: Toomey
authors.forename: Susanne
authors.initials: S
authors.lastname: Schjørring
authors.forename: Bodil
authors.initials: B
authors.lastname: Jacobsen
authors.forename: Hilko
authors.initials: H
authors.lastname: van der Voet
authors.forename: Sigrid Rita
authors.initials: SR
authors.lastname: Andersen
authors.forename: Declan
authors.initials: D
authors.lastname: Bolton
authors.forename: Henk
authors.initials: H
authors.lastname: Aarts
authors.forename: Karen A
authors.initials: KA
authors.lastname: Krogfelt
authors.forename: Andrea
authors.initials: A
authors.lastname: Wilcks
authors.forename: Jacek
authors.initials: J
authors.lastname: Bardowski
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015169:Colony Count, Microbial; D003227:Conjugation, Genetic; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D005516:Food Microbiology; D015964:Gene Expression Regulation, Bacterial; D022761:Gene Transfer, Horizontal; D013294:Lactococcus lactis / Q000235:genetics* / Q000254:growth & development*; D008826:Microbial Sensitivity Tests; D010957:Plasmids / Q000235:genetics*; D011156:Population Density; D013045:Species Specificity
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial