Elsevier

Science of The Total Environment

Volume 635, 1 September 2018, Pages 1477-1483
Science of The Total Environment

Tracking antibiotic resistance genes in soil irrigated with dairy wastewater

https://doi.org/10.1016/j.scitotenv.2018.04.020Get rights and content

Highlights

  • Dairy wastewaters contain antibiotic resistance and integron-integrase genes.

  • Antibiotic resistance gene levels in soil increase after wastewater irrigation.

  • Repeated monthly applications of dairy wastewater sustain the gene levels.

  • Wastewater irrigation enlarges the reservoir of antibiotic resistance genes in soil.

Abstract

The application of dairy wastewater to agricultural soils is a widely used practice to irrigate crops and recycle nutrients. In this study, small-scale field plots were irrigated monthly (6 times) with dairy wastewater (100%), wastewater diluted to 50% with irrigation (canal) water, and diluted wastewater spiked with copper sulfate (50 mg Cu L−1), while control plots were irrigated with canal water. In addition, half of all plots were either planted with wheat or were left as bare soil. Biweekly soil samples were collected during this period and processed to determine the occurrence and abundance of antibiotic resistance genes [blaCTX-M-1, erm(B), sul1, tet(B), tet(M), and tet(X)] and a class 1 integron-integrase gene (intI1) via quantitative real-time PCR (qPCR). Only sul1 and tet(X) were detected in soil (3 out of 32 samples) before the wastewater treatments were applied. However, the occurrence and relative abundance (normalized to 16S rRNA gene copies) of most genes [erm(B), intI1, sul1, and tet(M)] increased dramatically after wastewater irrigation and levels were maintained during the entire study period. blaCTX-M-1 was the only gene not detected in wastewater-treated soils, which is likely related to its absence in the dairy wastewater. Relative gene levels in soil were found to be statistically similar among the treatments in most cases, regardless of the wastewater percentage applied and presence or absence of plants. The key result from this study is that dairy wastewater irrigation significantly enlarges the reservoir of ARGs and intI1 in soils, while detection of these genes rarely occurred in soil irrigated only with canal water. In addition, elevated levels of Cu in the wastewater and treated soil did not produce a concomitant increase of the ARG levels.

Introduction

Approximately 57 million kg of antibiotics were used in animal agriculture globally in 2010 (Van Boeckel et al., 2015). While antibiotics are a valuable tool to prevent and treat disease in food-producing animals, overuse of antibiotics can produce negative outcomes (Marshall and Levy, 2011). Antibiotics can select for antibiotic resistant bacteria (ARB) in the animal gut, which are then released into the farm environment via excreted feces. Antibiotic resistant bacteria can make their way from the farm environment to humans through occupational exposures on farms and at meat processing facilities, as well as by foodborne exposures among consumers, use of animal manures as crop fertilizers, and contamination of surface water and groundwater at animal production facilities (Koch et al., 2017). Antibiotic resistance genes (ARGs) are the genetic code ARB use to produce proteins that allow them to resist the effects of antibiotics. Antibiotic resistance genes can be distributed to similar, distantly related, and pathogenic bacteria through horizontal gene transfer mechanisms (Alekshun and Levy, 2007) and are considered to be emerging contaminants (Pruden et al., 2006).

There are nearly 9.2 million dairy cattle in the United States (FAO, 2014), with each animal producing about 50 to 70 kg of manure per day (Dungan and Leytem, 2014). At dairies, liquid manure (combined feces and urine) is generally subjected to a solid separation process, where the solid fraction is stockpiled, composted, or reused as bedding and the liquid fraction is sent to a wastewater storage pond. Due to limited capacity, the storage ponds require removal of the wastewater on a regular basis to accommodate new wastewater influxes. The application of dairy wastewaters to agricultural soils is a common method to irrigate and fertilize the soil to support growth of forages, and reuse the wastewater. Like other animal wastewaters, it is well known that dairy wastewaters contain ARB and ARGs (McKinney et al., 2010), as well as antibiotic residues and copper (Ippolito and Moore, 2013; Wei et al., 2011). In addition to applied nutrients, any contaminants or residues in the wastewater will accumulate in the soil during irrigation events (Chee-Sanford et al., 2009).

Most studies exploring the occurrence and fate of ARB and ARGs in soil agroecosystems focus on land application of animal manure solids or reclaimed wastewater from domestic wastewater treatment plants (Franklin et al., 2016). As a result, there are significant knowledge gaps regarding the impacts of animal wastewaters on antibiotic resistance in treated soils. The primary objective of this field study was to determine the effect of repeated applications of straight and diluted (50%) dairy wastewater on the occurrence and abundance of several ARGs [blaCTX-M-1, erm(B), sul1, tet(B), tet(M), and tet(X)] and a class 1 integron-integrase (intI1) in receiving soil (bare or with wheat) over one growing season. We hypothesized that the long-term application of dairy wastewater would significantly expand the soil resistome when compared to soil that received regular irrigation water.

Section snippets

Field site and treatments

The field site was located at the USDA–ARS Northwest Irrigation and Soils Research Laboratory in Kimberly, Idaho. Soil at the site is a Portneuf silt loam (coarse-silty, mixed, superactive, mesic Durinodic Xeric Haplocalcids). This field site received manure applications (type of manure was not recorded, but most likely beef cattle) in 1977 and 1981 at an amount of 112 and 45 Mg ha−1, respectively. Thirty-two aluminum borders were installed (extending 7.6 cm below and 2.5 cm above ground) to

Results and discussion

Antibiotics from eight classes were targeted for analysis in the wastewater samples, with 15 out of 23 detected (Table 2). The most frequently detected antibiotics were lincomycin, chlortetracycline, monensin, oxytetracycline, and sulfadimethoxine (6 out of 6 wastewater samples), followed by sulfamethazine and sulfamethoxazole (5 out of 6 samples). Antibiotics or their metabolites that were detected less frequently were erythromycin, anhydroerythromycin (metabolite of erythromycin), penillic

Conclusions

The application of dairy wastewater to agricultural soils is a practice that will surely continue to be used as a method to irrigate crops, recycle nutrients, and reuse wastewater. What is clear from the results of this study, is that dairy wastewater irrigation dramatically increases the occurrence and abundance of ARGs and intI1 in the treated soil. The gene levels in soil were found to be statistically similar in most cases among the treatments, regardless of the wastewater percentage

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