Anaerobic digestion of tetracycline spiked livestock manure and poultry litter increased the abundances of antibiotic and heavy metal resistance genes

Authors: Agga, Getahun; KASUMBA, JOHN - Western Kentucky University; Loughrin, John; CONTE, ERIC - Western Kentucky University

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/30/2020
Publication Date: 12/18/2020
Citation: Agga, G.E., Kasumba, J., Loughrin, J.H., Conte, E.D. 2020. Anaerobic digestion of tetracycline spiked livestock manure and poultry litter increased the abundances of antibiotic and heavy metal resistance genes. Frontiers in Microbiology. 11. Article 614424. https://doi.org/10.3389/fmicb.2020.614424.
DOI: https://doi.org/10.3389/fmicb.2020.614424

Interpretive Summary: Antibiotic resistance is a growing global public health problem. Antibiotics are used in food animal production to treat and control bacterial infections. Livestock and poultry produce large amount of manure that contains excreted antibiotics, pathogenic and antibiotic resistant bacteria, antibiotic resistance genes, and heavy metals. These can spread from the point of production into the environment when manure is land applied as soil amendment or through runoff from animal farms. Effective animal waste management strategies are required. Anaerobic digestion is degradation of organic materials by microorganisms contained in the animal manure with the production of a biofuel energy. However, the use of anaerobic digestion as a tool for the control of antibiotic resistance and environmental pollution from heavy metals has not been clearly elucidated. In this study we added tetracycline to cattle and swine manure and poultry litter and evaluated the impact of anaerobic digestion on antibiotic and heavy metal resistance genes and pathogenic bacteria. Although anaerobic digestion reduced the concentration of tetracycline, it did not have any effect on the concentrations of heavy metals copper and zinc. It increased pathogenic bacteria, and antibiotic and heavy metal resistance genes. Secondary treatments are required after anaerobic digestion of animal manure.

Technical Abstract: Anaerobic digestion is a promising technology for the treatment of manure generated from food animal production facilities while also producing useable biogas energy. However, it is important to evaluate its effectiveness as an antimicrobial resistance mitigation strategy. In a previous study that evaluated the effect of mesophilic anaerobic digestion of cattle manure (CM), swine manure (SM), and poultry litter (PL) on the concentrations of tetracyclines, we observed highest concentrations of copper and zinc in the SM followed by PL. Heavy metals, in addition to their environmental impacts, can co-select for antimicrobial resistance. Here we evaluated the impact of mesophilic anaerobic digestion of CM, SM and PL on the concentrations of seven tetracycline resistance genes tet(A), tet(B), tet(G), tet(M), tet(O), tet(Q) and tet(W), macrolide resistance gene erm(B), and copper (copB, pcoA, pcoD and tcrB) and zinc (czrC) resistance genes, and three bacterial genera (E. coli, Enterococcus and Staphylococcus) reported to carry these genes. The total abundance of seven tetracycline resistance genes significantly increased from baseline levels during anaerobic digestion of CM, SM and PL. The total bacterial population also increased during anaerobic digestion, suggesting a potential shift in the bacterial population composition, perhaps towards anaerobic bacteria. The highest concentration of tet(M) was observed in the SM followed by PL, and it was strongly correlated with the concentrations of erm(B) and Enterococcus species. As concentration of tetracyclines declined during anaerobic digestion, concentrations of four tet genes (A, B, Q and W) and 16S rRNA increased, that of tet(M) decreased, and that of tet(G) and tet(O) did not change. SM had the highest copB concentration, and PL did not have any detectable concentration. The pcoA gene concentration was highest in the PL and least in the CM. Anaerobic digestion did not have any significant effect on copB and pcoA; pcoD was highest in the PL and remained unaffected by anaerobic digestion, but it increased in the SM and CM following anaerobic digestion. While the concentration of enterococci was highest in the undigested CM and remained unchanged, it significantly increased in the PL and SM after digestion. The gram-positive copper resistance gene tcrB was highest in the PL and significantly increased after digestion in all three manure types. While concentrations of Staphylococcus species significantly increased in the CM and PL, that of SM was not affected by anaerobic digestion. Concentrations of Staphylococcus aureus did not differ by the manure type; however, it significantly increased after digestion. While the concentration of methicillin resistant S. aureus (MRSA) did not change from its pre-digested level in the SM, which also had the highest concentrations, concentrations in the CM and PL significantly increased after digestion with the highest concentration in the PL. In the pre-digested samples, SM had the highest concentration of zinc resistance gene, czrC; while concentrations in the CM remained low, that of the PL significantly increased but that of the SM significantly reduced. In conclusion, while mesophilic anaerobic digestion of food animal manure decreased concentration of tetracyclines, it increased the concentrations of total bacterial population, abundance of tetracycline resistance genes, E. coli, Enterococcus spp. and Staphylococcus spp. (generic, S. aureus and MRSA). Anaerobic digestion of animal manure did not have any effect on the concentrations of the heavy metals copper and zinc, while heavy metal resistance genes either increased or remained unaffected. This study showed the need for technologies such as composting that can remove bacteria, antibiotic resistance genes, heavy metals and their resistance genes after mesophilic anaerobic digestion of anima