Data from: Microbial source tracking for antibiotic resistance genes in southwest Wisconsin private wells

Authors: Opelt, Sarah; FIRNSTAHL, AARON - Us Geological Survey; Cook, Rachel; STOKDYK, JOEL - Us Geological Survey; Burch, Tucker

Submitted to: Ag Data Commons
Publication Type: Database / Dataset
Publication Acceptance Date: 8/18/2025
Publication Date: N/A
Citation: N/A

Interpretive Summary: Antibiotic-resistant bacterial infections complicate routine hospital treatments and contribute to more than 2 million infections per year in the United States. Antibiotic resistance is driven by antibiotic use in humans and livestock, and antibiotic-resistant bacteria in feces from humans and livestock can be transported via environmental routes, including groundwater. Consumption of groundwater from private wells may be a significant point of exposure to antibiotic-resistant bacteria, but the relative contributions of human and livestock fecal sources to antibiotic-resistant bacteria in groundwater is unknown for most areas of the United States. This study investigated antibiotic resistance genes (ARGs) – the genetic basis of resistance in bacteria – in private wells from a rural region of southwest Wisconsin where groundwater contains fecal material from residential septic systems and land-applied livestock manure. Of 138 wells studied, 117 were positive for one or more ARGs, but most individual ARGs were detected in few (= 10%) wells. Some ARGs were more frequently detected with markers for human fecal material, others with markers for livestock manure. Overall, more ARG detections co-occurred with human fecal markers than livestock fecal markers, consistent with the fact that more wells were positive for human fecal markers than livestock fecal markers. These data inform local resource managers on water quality and will be used by other researchers to understand the role of rural groundwater in transmission of antibiotic-resistant infections.

Technical Abstract: Groundwater was collected by dead-end ultrafiltration and small-volume grab sampling from 138 wells in southwest Wisconsin across Grant, Iowa, and Lafayette Counties. Samples were collected to assess occurrence of antibiotic resistance genes in private wells and investigate their association with microbial source tracking markers. For ultrafiltration samples, microbes were backflushed, desiccated beef extract was added to the eluate, and samples were concentrated by polyethylene glycol precipitation; concentrate was frozen at -80 degrees C. Small-volume grab samples were concentrated on 0.45-micron mixed cellulose ester filters, filters were eluted, and eluate was frozen at -80 degrees C following addition of beef extract. Nucleic acids were extracted from both sample types using a QIAcube and QIAamp DNA mini kit with buffer AVL and carrier RNA (Qiagen). Nucleic acids were extracted from 280 µL of sample concentrate and eluted into 140 µL AE Buffer (Qiagen). Nucleic acids were analyzed in duplicate using quantitative polymerase chain reaction (qPCR) on a Roche LightCycler 480 II using hydrolysis probes. Inhibition was assessed for every sample using Sketa DNA as inhibition control and mitigated by dilution with AE buffer as necessary. No-template negative controls were performed for all analysis steps: secondary concentration, nucleic acid extraction, and qPCR. For each assay with amplification in negative controls, the cycle of quantification in unknown samples must be >3 standard deviations below the average of controls to be accepted as positive. Positive controls (bovine herpes virus vaccine) for extraction were included with each analysis batch and evaluated qualitatively. Positive controls were run in duplicate reactions for all targets and had to be within 0.5 cycle of the expected cycle of quantification. Data are expressed as genomic copies per liter of groundwater sampled. Dataset consists of 1 spreadsheet file: SWIGG ARG Data Summary - Cleaned.csv. Variables in this file are described in the included data dictionary.