Evaluation of Anaerobic Digestion Amended with Micro-Aeration and/or Sound Treatment on the Resistome and Virulence Factor Gene Profiles in Poultry Litter

<b>Background</b>: Commercial broiler farms produce a large amount of litter that must be removed. Anaerobic digestion (AD) is animal manure management technology with the added benefit of producing reusable energy. Our team previously showed that the micro-aeration and sound treatment of animal manure during AD increase biogas production. However, their influence on antimicrobial resistance genes (ARGs) and bacterial virulence factor genes (VFGs) is unknown. Therefore, the objective of this study was to evaluate the effect of AD on the resistome and VFGs in poultry litter (PL) and see if the effect is modified by micro-aeration and/or sound treatments. <b>Methods</b>: A field experiment was conducted in four anaerobic digesters that consisted of a control (a standard AD system with no air or sound), micro-aeration, sound, and combined micro-aeration and sound treatments. Overall, 21 samples were collected and analyzed with shotgun metagenomic sequencing. The samples included digestate samples (n = 12) from the four digesters obtained at 6 (baseline, i.e., before beginning of micro-aeration and sound treatments), 23 and 42 weeks, raw PL samples (n = 4), two disks comprised of the same wood as the bedding material, an initial digestate seed sample, and two initial week 0 mix samples. <b>Results</b>: Across all sequence reads (n = 3190) obtained from 21 samples, over 80% of the resistome was composed of four antimicrobial classes: macrolides-lincosamides-streptogramins, tetracyclines, aminoglycosides, and glycopeptides. While the total number of ARGs declined in the control digestor, it increased over time in micro-aerated or sound-treated digesters, and their combination greatly increased the number of ARGs detected. This is a new finding, and it clearly shows that micro-aeration, sound, and their combination treatment during the anaerobic digestion of PL enriches ARGs. In contrast, sound-treated AD by itself significantly (<i>p</i> = 0.035) reduced the mean total ARG abundance compared to the control. The number and abundance of ARGs detected in the initial digestate and PL were lower than those in the AD samples, indicating their enrichment during the AD process. On the other hand, although the AD samples had a lower frequency and abundance of VFGs than the PL, AD did not completely remove the VFGs, and their detection frequency increased over time. While micro-aeration increased the abundance of VFGs compared to the control, this effect was countered by its combination with sound treatment, offering a good animal manure treatment strategy to reduce bacterial VFGs. <b>Conclusions</b>: Although additional research may be required, it was shown that while sound treatment may enrich the occurrence of ARGs, it seems promising to reduce the abundance of ARGs and VFGs during the AD of PL. On the other hand, micro-aeration, alone or when combined with sound treatment, increases the abundance of both ARGs and VFGs. Moreover, the study showed that AD, with or without micro-aeration and sound treatment, is not effective for the complete removal of ARGs and VFGs from poultry litter. Rather, AD systems may act as a hotspot for ARGs, and post-AD treatments such as composting need to be evaluated.