Influence of environmental conditions on extracellular and intracellular antibiotic resistance genes in manure-amended soil: A microcosm study

Dungan, R.S. and McKinney, C.W. (2020) Influence of environmental conditions on extracellular and intracellular antibiotic resistance genes in manure-amended soil: A microcosm study. Soil Science Society of America Journal. 84(3):747-759. 3 February 2020.

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Abstract

Extracellular and intracellular antibiotic resistance genes (eARGs and iARGs) possess significant differences with respect to their mobility and availability to bacteria, thus it is important to understand their partitioning, persistence, and fate in the environment. The objectives of this study were to: (i) modify the protocol of a commercial DNA extraction kit to sequentially extract extracellular and intracellular DNA (eDNA and iDNA) from the same soil sample; and (ii) determine the effect of temperature (5, 20, and 35C), water holding capacity (25, 50, and 75% of WHCmax), and freeze-thaw (-20/20C) on the abundance of two ARGs [sul1 and tet(M)], class 1 integron-integrase gene (intI1), and 16S rRNA gene in soils treated with dairy manure. To assess the efficiency of the eDNA/iDNA method, we performed spiking and recovery experiments with a gene that codes for a green fluorescent protein (gfp). When soils were spiked with a whole-cell preparation of gfp-containing E. coli, the recovery of the gfp gene was on average 0.2% and 1.2% for eDNA and iDNA, respectively. Soils were also spiked with the gfp gene itself, which showed that nearly 80% of the DNA could not be recovered. Results from the microcosm experiments indicate that extracellular and intracellular sul1, tet(X), intI1, and 16S rRNA genes are resilient and not readily affected by temperature, WHC, or freeze-thaw cycles. The intracellular gene levels decreased only slightly during the 56-day incubation period. The extracellular gene levels (when detectable) decreased dramatically by day 7, then leveled off thereafter, which may have been caused by adsorption to soil particles and/or degradation. Absolute gene abundances (per g of dry soil) were one to two orders of magnitude higher in iDNA than in eDNA fractions, but were similar when normalized to the 16S rRNA gene throughout the incubation period, indicating that enrichment of eARGs and iARGs did not occur. It is important for the scientific community to standardize total, as well as, extracellular/intracellular DNA extractions in order to readily compare results between laboratories.

Item Type:	Article
NWISRL Publication Number:	1673
Subjects:	Irrigated crops > Sugarbeet > Root rots > Bacteria Research methodology
Depositing User:	Users 6 not found.
Date Deposited:	21 Jul 2020 21:51
Last Modified:	21 Jul 2020 21:51
Item ID:	1714
URI:	https://eprints.nwisrl.ars.usda.gov/id/eprint/1714