Horizontal gene transfer of an IncP1 plasmid to soil bacterial community introduced by Escherichia coli through manure amendment under realistic conditions. Manured soil

Authors:Goncalo Macedo, Asmus Kalckar Olesen, Lorrie Maccario, Lucia Hernandez-Leal, Peter van der Maas, Dick Heederik, Dik Mevius, Soren J. Sorensen, Heike Schmitt.Abstract:Horizontal gene transfer (HGT) is of great concern because it allows for the rapid dissemination of antibiotic resistance genes. This study quantified conjugation of an IncP1 plasmid carried by an Escherichia coli host to the indigenous microbial community in agricultural soil after manure application. Furthermore, new plasmid hosts were identified under conditions resembling environmental conditions in microcosms. Conjugation was quantified by plating and flow cytometry. Transconjugants were recovered by fluorescence-activated cell sorting and identified by 16S rRNA gene sequencing. Transconjugants were only observed within the first four days of incubation, and higher temperature enhanced the number of transconjugants detected. Still, transconjugant numbers were in all cases close to the detection limits of this experimental system (1.00 - 2.49 log CFU/g of transconjugants in manured soil). In the pool of recovered transconjugants, we found amplicon sequence variants (ASVs) of genera from soils (Bacillus and Nocardioides), as well as genera present in manure and soils after manuring (Comamonas and Rahnella). Acinetobacter and Pseudomonas were also identified in the transconjugant pool, but their abundance was probably below the detection limit in the microcosms, as it was not possible to track their specific ASVs in the microcosms. This work highlights the importance of environmental conditions and farm practices on the spread of AMR, and it shows that gene transfer is occurring at detectable levels also under realistic conditions in the soil.