Simulated winter incubation of soil with swine manure differentially affects multiple antimicrobial resistance elements

Authors: Miller, Daniel; JURGENS, MADISON - University Of Nebraska; Durso, Lisa; SCHMIDT, AMY - University Of Nebraska

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/25/2020
Publication Date: 12/16/2020
Citation: Miller, D.N., Jurgens, M., Durso, L.M., Schmidt, A. 2020. Simulated winter incubation of soil with swine manure differentially affects multiple antimicrobial resistance elements. Frontiers in Microbiology. (11):3235. https://doi.org/10.3389/fmicb.2020.611912.
DOI: https://doi.org/10.3389/fmicb.2020.611912

Interpretive Summary: Antibiotic resistance genes (ARG) in animal manures is a major concern when manure nutrients are applied to row crops. Slow decomposition after Fall manure applications helps retain nutrients for crops, but the affect of cold soil temperatures on antibiotic resistance in soil and manure microbes is unexplored. A laboratory soil incubation study was conducted mimicking fall swine manure application to soils, and selected ARG were monitored during simulated 120-day winter incubation with multiple freeze-thaw events. Two soil moistures (10% and 30% water holding capacity) and two manure treatments [untreated versus hydrated lime alkaline stabilization (HLAS)] were also tested. Fourteen tetracycline resistance genes (tet) were initially evaluated; tet(D), tet(G), and tet(L) were detected in background soil while swine manure contained tet(A), tet(B), tet(C), tet(G), tet(M), tet(O), tet(Q), and tet(X). By day 120, the tet(M) and tet(O) in manure were still present (100% of samples) while tet(C), tet(D), tet(L), and tet(X) genes were detected less frequently (<44% of samples). Other tet resistance genes [tet(E), tet(L), and tet(S)] were detected rarely, if at all. The diversity of tet genes decreased over the incubation from an average of 8.9 to 3.8. Four resistance elements [intI1, blactx-m-32, sul(I), erm(B)] and 16s rRNA genes were measured using quantitative PCR. ARG abundances relative to 16S abundance were initially 100- to 1000-fold greater in the manure compared to soil but decreased 10- to 100-fold relative to the abundance of 16S during the incubation due imainly to an increase in 16S rRNA abundance--microorganisms grew during the simulated winter incubation. Over the winter incubation, the total abundance of intI1, blactx-m-32, sul(I), and erm(B) in HLAS treated manure was lower than untreated manure. Under low initial soil moisture conditions, HLAS treatment reduced the abundance of intI1, and resulted in a more rapid loss of blactx-m-32, sul(I), and erm(B)]. Although one might expect antibiotic resistance to be relatively unchanged during simulated winter soil application, a variety of changes in diversity and relative abundance can be expected.

Technical Abstract: Gastrointestinal bacteria that harbor antibiotic resistance genes (ARG) become enriched with antibiotic use. Livestock manure application to cropland for soil fertility presents a concern that ARG and bacteria may proliferate and be transported in the environment. In the United States, manure applications typically occur during autumn with slow mineralization until spring planting season. A laboratory soil incubation study was conducted mimicking autumn swine manure application to soils with concentrations of selected ARG monitored during simulated 120-day winter incubation with multiple freeze-thaw events. Additionally, the effects of two soil moistures (10% and 30% water holding capacity) and two manure treatments [raw versus hydrated lime alkaline stabilization (HLAS)] were assessed. Fourteen tetracycline resistance genes were evaluated; tet(D), tet(G), and tet(L) were detected in background soil while swine manure contained tet(A), tet(B), tet(C), tet(G), tet(M), tet(O), tet(Q), and tet(X). By day 120, the manure-borne tet(M )and tet(O) were still detected while tet(C), tet(D), tet(L), and tet(X) genes were detected less frequently. Other tet resistance genes were detected rarely, if at all. The sum of unique tet resistance genes among all treatments decreased during the incubation from an average of 8.9 to 3.8. Four resistance elements [intI1, blactx-m-32, sul(I), erm(B)] and 16s rRNA genes were measured using quantitative PCR. ARG abundances relative to 16S abundance were initially greater in the raw manure compared to background soil (-1.53 to -3.92 log abundance in manure; -4.02 to <-6.7 log abundance in soil). In the mixed manure/soil, relative abundance of the four resistance elements decreased (0.87 to 1.94 log abundance) during the incubation largely because 16S rRNA genes increased by 1.21 log abundance. Throughout the incubation, the abundance of intI1, blactx-m-32, sul(I), and erm(B) per gram in soil amended with HLAS-treated manure was lower than in soil amended with raw manure. Under low initial soil moisture conditions, HLAS treatment reduced the abundance of intI1 and resulted in loss of blactx-m-32, sul(I), and erm(B)] compared to other treatment-moisture combinations. Although one might expect antibiotic resistance to be relatively unchanged after simulated winter manure application to soil, a variety of changes in diversity and relative abundance can be expected.