The supply of clean water for human consumption is being challenged by the appearance of anthropogenic pollutants in groundwater ecosystems. Because many plasmids can transfer horizontally between members of bacterial communities, they comprise promising vectors for the dissemination of pollutant-degrading genetic determinants within water purification plants.

The quantification and identification of new plasmid-acquiring bacteria in representative mating conditions is critical to characterize the risk of horizontal gene transfer in the environment. This study aimed to quantify conjugation events resulting from manure application to soils and identify the transconjugants resulting from these events. Conjugation was quantified at multiple time points by plating and flow cytometry, and the transconjugants were recovered by fluorescence-activated cell sorting and identified by 16S rRNA sequencing. Overall, transconjugants were only observed within the first 4 days after manure application and at values close to the detection limits of this experimental system (1.00–2.49 log CFU/g of manured soil, ranging between 10 –5 and 10 –4 transconjugants-to-donor ratios). In the pool of recovered transconjugants, we found amplicon sequence variants (ASVs) of genera whose origin was traced to soils ( Bacillus and Nocardioides ) and manure ( Comamonas and Rahnella ). This work showed that gene transfer from fecal to soil bacteria occurred despite the less-than-optimal conditions faced by manure bacteria when transferred to soils, but these events were rare, mainly happened shortly after manure application, and the plasmid did not colonize the soil community. This study provides important information to determine the risks of AMR spread via manure application.

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19The supply of clean water for human consumption is being challenged by the appearance 20 of pesticide pollutants in groundwater ecosystems. Biological rapid sand filtration is a 21 commonly employed method for the removal of organic and inorganic impurities in water 22 32 RSF1010, pKJK5 and TOL (pWWO), using a dual-fluorescent bioreporter platform 33 combined with FACS and 16S rRNA gene amplicon sequencing. Our results reveal that 34 plasmids can transfer at high frequencies and across distantly related taxa from rapid 35 sand filter communities, emphasizing their suitability for introducing pesticide degrading 36 determinants in the microbiomes of underperforming water purification plants. 37 38 65 129 meta-parental mating setup described previously (Klümper et al., 2015). Following this 130 approach, plasmids are tracked through a inserted gfp marker controlled by a lacIq 131 repressible promoter. The donor strain additionally harbors a chromosomal lacIq-Plpp-132

Horizontal gene transfer via plasmids is important for the dissemination of antibiotic resistance genes among medically relevant pathogens. Specifically, the transfer of IncHI1A plasmids is believed to facilitate the spread of antibiotic resistance genes, such as carbapenemases, within the clinically important family Enterobacteriaceae.The microbial community of urban wastewater treatment plants has been shown to be highly permissive towards conjugal transfer of IncP1 plasmids. Here, we tracked the transfer of the P1 plasmid pB10 and the clinically relevant HI1A plasmid R27 in the microbial communities present in urban residential sewage entering full-scale wastewater treatment plants. We found that both plasmids readily transferred to these communities and that strains in the sewage were able to further disseminate them. Furthermore, R27 has a broad potential host range, but a low host divergence.Interestingly, although the majority of R27 transfer events were to members of Enterobacteriaceae, we found a subset of transfer events to other families, even other phyla. This indicates that HI1A plasmids facilitate horizontal gene transfer both within Enterobacteriaceae, but also across families of, in particular, Gammaproteobacteria, such as Moraxellaceae, Pseudomonadaceae and Shewanellaceae. pB10 displayed a similar potential host range to R27. In contrast to R27, pB10 had a high host divergence. By culture enrichment of the transconjugant communities, we show that sewage strains of Enterobacteriaceae and Aeromonadaceae can stably maintain R27 and pB10, respectively. Our results suggest that dissemination in the urban residual water system of HI1A plasmids may result in an accelerated acquisition of antibiotic resistance genes among pathogens.