Evaluation of anaerobic digestion amended with micro- aeration and/or sound treatment on the resistome and virulence factor gene profiles in poultry litter

Authors: Agga, Getahun; Loughrin, John

Submitted to: Antibiotics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/27/2026
Publication Date: 2/2/2026
Citation: Agga, G.E., Loughrin, J.H. 2026. Evaluation of anaerobic digestion amended with micro- aeration and/or sound treatment on the resistome and virulence factor gene profiles in poultry litter. Antibiotics. 15(2). Article 153. https://doi.org/10.3390/antibiotics15020153.
DOI: https://doi.org/10.3390/antibiotics15020153

Interpretive Summary: Anaerobic digestion is a waste management technology with the added benefits of converting animal manure to biogas that can be used as energy source to farms. Anaerobic digestion reduces the volume of animal manure to be removed thus reducing transportation costs while also producing usable energy and digestate that can be used as organic fertilizer for crop production. Commercial broiler farms produce a large amount of litter and are faced with a challenge of its removal from the farms. The effect of anaerobic digestion was evaluated either alone, or when treated with micro-aeration, sound, or their combination for the removal of antibiotic resistance genes and virulence factor genes associated with disease causing bacteria. In general, the resistance gene pool was dominated by genes conferring resistance to four classes of antibiotics macrolides-lincosamides-streptogramins, tetracyclines, aminoglycosides, and glycopeptides. Total count and concentration of resistance genes in the initial digestate and poultry litter were lower than the anaerobic digester samples indicating their enrichment during anaerobic digestion process as bacteria share their genetic material in the system. While the traditional (control) digester decreases the total counts of antimicrobial resistance genes detected, micro-aeration, sound treatment and their combination increased the total count of the resistance genes. However, sound treatment by itself significantly reduced the total concentration of resistance genes compared to the control digester. Although anaerobic digestion did not completely remove the virulence factor genes, it significantly decreased their total counts and concentration compared to the original poultry litter. In conclusion, anaerobic digestion is not an effective treatment to completely remove antimicrobial resistance genes from poultry litter, and its effect was not improved by micro-aeration, sound, or their combination treatments.

Technical Abstract: Commercial broiler farms generate large amounts of litter that require proper management. Anaerobic digestion (AD) is a manure management technology that also produces renewable energy, and previous research has shown that micro-aeration and sound treatment during AD can increase biogas production. However, their impact on antimicrobial resistance genes (ARGs) and bacterial virulence factor genes (VFGs) has not been well understood. This study aimed to evaluate the effect of AD on the resistome and VFGs in poultry litter (PL) and determine whether micro-aeration and/or sound treatments modify these effects. A field experiment was conducted using four anaerobic digesters: a control (standard AD), micro-aeration, sound, and combined micro-aeration and sound treatments. Twenty-one samples, including digestate from different time points, raw PL, wood disks, and initial mixes, were analyzed using shotgun metagenomic sequencing. Results showed that over 80% of the resistome consisted of four antimicrobial classes: macrolides–lincosamides–streptogramins, tetracyclines, aminoglycosides, and glycopeptides. While ARGs declined in the control digester, they increased in digesters treated with micro-aeration or sound, and their combination led to the greatest increase. Interestingly, sound treatment alone significantly reduced ARG abundance compared to the control. Although AD samples had fewer VFGs than raw PL, AD did not completely eliminate them, and their frequency increased over time. Micro-aeration increased VFG abundance, but this effect was counteracted when combined with sound treatment, suggesting a potential strategy to reduce VFGs. Overall, the study indicates that while sound treatment may help reduce ARGs and VFGs, micro-aeration alone or combined with sound tends to increase them, and AD—regardless of treatment—does not fully remove these genes, highlighting the need for additional post-AD treatments. 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.