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

Authors: Dungan, Robert - Rob; McKinney, Chad

Submitted to: Soil Science Society of America Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/27/2020
Publication Date: 2/3/2020
Citation: Dungan, R.S., 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. https://doi.org/10.1002/saj2.20049.
DOI: https://doi.org/10.1002/saj2.20049

Interpretive Summary: Antibiotic resistance genes (ARGs) are responsible for causing antibiotic resistance in bacteria. The ARGs are found inside bacteria, but can also be released from the bacteria upon death, and are respectively known as intracellular and extracellular genes. Both intracellular and extracellular ARGs (iARGs and eARGs) can make their way into bacteria and potentially cause them to resist drug treatment. Because these ARG fractions may behave differently in the environment, it is important to have methods available to extract extracellular and intracellular DNA (eDNA and iDNA). As a result, we modified the method of a commercial DNA extraction kit to sequentially extract eDNA and iDNA, then used it to determine the influence of temperature and moisture on ARGs in extracellular and intracellular fractions in manure-amended. During a 56-day incubation period it was found that temperature and moisture generally did not affect the eARG and iARG levels, thus are quite resilient. The persistence of the ARGs could mean that they are available for transfer to or uptake by bacteria for an extended period of time in manure-amended soil. A common eDNA/iDNA extraction method should be used among researchers to ensure results generated from different ARG studies can be more easily compared by reducing the bias associated with the use of various DNA extraction procedures.

Technical 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.