title: Demonstrating plasmid-based horizontal gene transfer in complex environmental matrices: a practical approach for a critical review.
abstract: Plasmid-based dissemination of antibiotic resistance genes in environmental microbial communities is a matter of concern for public health, but it remains difficult to study for methodological reasons. In this study, we used the broad host range plasmid pB10 to compare and to point out the main drawbacks of the three different approaches currently used to evaluate plasmid transfer in natural communities. Culture-based selection of transconjugants appeared to be compromised by high prevalence of antibiotic resistances among natural communities, unless high loads of initial pB10-donor inocula were used. Fluorescence-based detection of transconjugants reached a dead-end consequently to the narrow host range of bacteria expressing fluorescent proteins from a genetically modified pB10 plasmid, in addition to the relatively high background level of fluorescence exhibited by some environmental matrices. The molecular-based approach was the only one to provide a mean to detect rare plasmid transfer events following a low but realistic initial pB10-donor inoculation. Whatever the method, culture-based or molecular-based, the detection of successful transfer events in a given environmental matrix seemed to be linked to the initial stability of the donor inoculum. Depending on the matrix considered, eukaryotic predation plays a significant role in either limiting or promoting the plasmid transfer events.
authors.affiliation: Université de Lorraine and CNRS, LCPME, UMR 7564, 15 Avenue du Charmois, F-54500 Vandoeuvre-lès-Nancy, France. Electronic address: xavier.bellanger@univ-lorraine.fr.
authors.forename: Xavier
authors.initials: X
authors.lastname: Bellanger
authors.affiliation: Université de Lorraine and CNRS, LCPME, UMR 7564, 15 Avenue du Charmois, F-54500 Vandoeuvre-lès-Nancy, France. Electronic address: helene.guilloteau@univ-lorraine.fr.
authors.forename: Hélène
authors.initials: H
authors.lastname: Guilloteau
authors.affiliation: Université de Lorraine and CNRS, LCPME, UMR 7564, 15 Avenue du Charmois, F-54500 Vandoeuvre-lès-Nancy, France. Electronic address: bonot@lippmann.lu.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Bonot
authors.affiliation: Université de Lorraine and CNRS, LCPME, UMR 7564, 15 Avenue du Charmois, F-54500 Vandoeuvre-lès-Nancy, France. Electronic address: christophe.merlin@univ-lorraine.fr.
authors.forename: Christophe
authors.initials: C
authors.lastname: Merlin
mesh_terms: D001419:Bacteria / Q000235:genetics*; D004269:DNA, Bacterial; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004777:Environment; D022761:Gene Transfer, Horizontal; D010957:Plasmids
chemical_list: D004269:DNA, Bacterial
keywords: Antibiotic resistance dissemination; Environmental bacterial communities; Eukaryotic predation; Plasmid transfer; pB10