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

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.

作者：贡萨洛·马塞多、阿斯穆斯·卡尔克·奥勒森、洛里·马卡里奥、露西娅·埃尔南德斯-莱尔、彼得·范德马斯、迪克·赫德里克、迪克·梅维乌斯、索伦·J·索伦森、海克·施密特。
摘要：水平基因转移（Horizontal gene transfer, HGT）因可快速传播抗生素抗性基因而备受关注。本研究量化了粪肥施用后，宿主为大肠杆菌（Escherichia coli）的IncP1型质粒向农田土壤土著微生物群落的接合转移效率；此外，在模拟环境条件的微宇宙培养体系中，鉴定出了新的质粒宿主。研究通过平板涂布法与流式细胞术定量接合转移事件，通过荧光激活细胞分选术回收转接合子，并通过16S rRNA基因测序对其进行鉴定。结果显示，转接合子仅在培养的前4天内被检出，且更高的培养温度可提升检出的转接合子数量。但在所有实验组中，转接合子数量均接近本实验体系的检测限（施用粪肥的土壤中转接合子为1.00~2.49 log 菌落形成单位（colony-forming unit, CFU）/g）。在回收的转接合子菌群中，我们检出了土壤来源菌属的扩增子序列变异体（amplicon sequence variants, ASVs）：芽孢杆菌属（Bacillus）与诺卡氏菌属（Nocardioides），以及粪肥及粪肥施用后土壤中常见的菌属：丛毛单胞菌属（Comamonas）与拉恩菌属（Rahnella）。研究还在转接合子菌群中检出了不动杆菌属（Acinetobacter）与假单胞菌属（Pseudomonas），但由于无法在微宇宙培养体系中追踪其特异性扩增子序列变异体，推测二者的丰度低于检测限。本研究凸显了环境条件与农田耕作方式对抗生素耐药性（Antimicrobial Resistance, AMR）传播的重要影响，并证实了在土壤真实环境中，基因转移仍可达到可检测水平。