The widespread use of Recycled Manure Solids (RMS) as cow bedding material is not without risks, since cattle manure may act as a vehicle for pathogenic and antimicrobial resistant bacteria dissemination. Thus, our aim was to evaluate RMS-supplemented with a pine biochar produced in Portugal as a new cow bedding material, since the use of biochar has been shown to have the potential to mitigate the impact of relevant bacterial species when added to animal manure microbiota. Our experimental setup consisted on fresh RMS samples that were collected on a commercial dairy farm and placed in naturally-ventilated containers for a total of 4 groups: 1-non-supplemented RMS; 2-RMS supplemented with 2.5% (wt/wt) of biochar; 3-RMS supplemented with 5% (wt/wt) of biochar; and 4-RMS supplemented with 10% (wt/wt) of biochar. Sampling was performed at 4 different incubation times (0, 5, 15 and 30 d) and in 2 distinct seasons: April-May (humid season) and June-July (dry season). The resulting 32 samples were subjected to DNA extraction and their microbiome profile determined through complete 16S rDNA gene sequencing using Nanopore next-generation sequencing. We observed that biochar supplementation clearly altered the microbiome of RMS, which was reflected in changes in populations' diversity and their relative abundance of relevant pathogenic bacteria. In particular, we found that long-term storage (30 d) was more beneficial than short-term storage, an effect that was more evident for samples supplemented with 2.5% or 5% biochar. In both seasons, those concentrations of biochar led to a decrease in the levels of several mastitis-causing agents (Enterobacteriaceae, streptococci, enterococci and staphylococci). In addition, we also observed a reduction in the levels of Salmonella spp. and Gram-positive bacilli in the biochar-supplemented samples. Unexpectedly, however, those same conditions yielded an increase in the abundance of Brucella spp., a group which includes important infectious agents, highlighting the need for a deeper evaluation of the impact of biochar supplementation of RMS to ensure the future safe and sustainable use of this environmental-friendly resource in animal production.

The use of recycled manure solids (RMS) as cow bedding in dairy farms poses concerns due to its potential to harbor pathogenic and antimicrobial-resistant bacteria. This study evaluated the impact of RMS supplementation with biochar at three concentrations (2.5%, 5%, and 10%) on bacterial counts and on the antimicrobial resistance and virulence profiles of Escherichia coli and Enterococcus isolates. The results show that biochar supplementation did not promote a significant reduction in bacterial numbers. Overall, there were no significant changes in the isolates’ virulence or resistance profiles, and none of the isolates obtained were classified as high or moderate threats based on their MAR and VIR indexes. The most pathogenic Enterococcus isolates found were present in the control samples. A phylogenetic analysis of E. coli isolates allowed us to identify phylogroup D strains, predominantly in RMS supplemented with 2.5% and 10% biochar, which are associated with higher virulence and resistance. These findings indicate that the biochar concentrations tested were not effective in significantly reducing the bacterial risks associated with RMS. Further research is needed to evaluate different biochar formulations and concentrations, aiming to optimize its potential use for RMS supplementation.