This data report presents 111 metagenome-assembled genomes (MAGs) reconstructed from manure, soil and manured soil samples from microcosms after enriching for non-fermenting Gram-negative bacteria (NFGNB). Two independent microcosm experiments were conducted to investigate the spread of NFGNB from the fresh manure of dairy cows under antibiotic prophylaxis to the pasture soil of two organic farms. After sampling the microcosms on days 2, 14 and 28, the manure and soil samples were plated in duplicate on CHROMagar Acinetobacter medium for NFGNB enrichment and incubated at 28°C for 24 h. DNA was extracted from the cultures and sequenced using the Illumina NovaSeq 6000 platform with 150-bp paired-end reads. Reads were assembled with metaSPAdes both individually and by co-assembly. MAGs were reconstructed using MetaBAT, MaxBin, SemiBin2, COMEbin, and AVAMB, and then de-replicated at >95 % ANI (pairwise comparisons) using dRep. A total of 111 MAGs of at least medium quality (MIMAG standard) were obtained. These included 10 high-quality MAGs (>90 % completeness, <5 % contamination, rRNA genes and tRNA for at least 18 amino acids), 47 putative high-quality MAGs (>90 % completeness, <5 % contamination) and 54 medium-quality MAGs (>50 % completeness, <10 % contamination). The FASTA files of the MAGs as well as their taxonomic identifications, completeness and contamination, origin, genomic statistics and rRNA sequences are publicly available in a Zenodo dataset and the genomes in the NCBI database. The majority of MAGs (99) were assigned to Pseudomonadota, mainly Pseudomonas (28 MAGs), Stenotrophomonas (20 MAGs) and Acinetobacter (18 MAGs), while the remaining 12 MAGs belonged to Bacteroidota. Most MAGs (44) were of manure origin, followed by manured soil (38 MAGs) and soil (29 MAGs). High-quality MAGs were predominantly obtained from manure (6 high-quality, 21 putative high-quality), compared to manured soil (3 high-quality, 12 putative high-quality) and soil (1 high-quality, 14 putative high-quality). By providing their MAGs, this dataset offers a valuable resource for researchers investigating the genomic characteristics associated with the survival, environmental dispersal and ecological role of potentially hazardous NFGNB species in soil, particularly following the application of antibiotic-treated animal manure, and for comparative genomics studies in related environments.

INRAE University Bourgogne Institut Agro Dijon Department of Agroecology 17 rue de Sully Dijon 21000 France

Institute of Soil Biology and Biogeochemistry Biology Centre of the Czech Academy of Sciences Na Sádkách 7 České Budějovice 370 05 Czech Republic

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