Resistome and mobilome in surface runoff from manured soil as affected by setback distance.

Land application of livestock manure introduces antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) into the soil environment. The objectives of this study were to examine the changes of resistome and mobilome in runoff and soil as a function of setback distance, i.e., the distance between manured soil and surface water, and to quantify the contributions of manure and background soil to the ARGs and MGEs in surface runoff. The resistome and mobilome in runoff and soil from a field-scale plot study were characterized using a high throughput quantitative polymerase chain reaction (HT-qPCR) array. It was estimated that a setback distance of ~40 m is required to reduce the total abundance of ARGs and MGEs in runoff from amended plots to that in control runoff. The resistome and mobilome of the soil in the setback region was not affected by manure-borne ARGs and MGEs. SourceTracker analyses revealed that background soil gradually became the predominant source of the ARGs and MGEs in runoff as setback distance increased. The results demonstrate how manure-borne ARGs and MGEs dissipated in agricultural runoff with increasing setback distance and had limited impacts on the resistome and mobilome of soil within the setback region.

Differential Survival of Non-O157 Shiga Toxigenic Escherichia coli in Simulated Cattle Feedlot Runoff.

Environmental survival time is important when evaluating adverse health outcomes from foodborne pathogens. Although outbreaks associated with manure-impacted irrigation or runoff water are relatively infrequent, their broad scope, regulatory importance, and severe health outcomes highlight the need to better understand the environmental survival of manure-borne pathogens. Shiga toxigenic Escherichia coli (STEC) are excreted in feces and persist in the environment until they die or recolonize a new host. Surface waters contaminated with manure-borne STEC can infect humans through drinking and recreational water use or irrigated crops that are minimally cooked. In this study, manure-impacted water microcosms mimicking beef cattle feedlot runoff were used to assess survival of STEC strains representing seven STEC serotypes (O26, O45, O103, O111, O121, O145, and O157) and persistence of target O antigen genes. Microcosms were sampled over the course of 1 year, and the entire experiment was repeated in a second year. Culture and polymerase chain reaction (PCR)-based techniques were used for detection and enumeration. Serotype-specific survival results were observed. Both STEC O26 and O45 declined slowly and remained culturable at 24 months. In contrast, STEC O121 and O145 decreased rapidly (-0.84 and -1.99 log10 abundance per month, respectively) and were unculturable by months 4 and 5, but detectable by PCR for a mean of 4.5 and 8.3 months, respectively. STEC O103, O111, and O157 remained culturable for a mean of 11.6, 5.5, and 15 months and detectable by PCR for a mean of 12, 13.8, and 18.6 months after inoculation, respectively. Results document that some STEC serotypes have the biological potential to survive in manure-impacted waters for extended periods of time when competing microflora are eliminated. Serotype-specific differences in survival of target bacteria and persistence of target genes were observed in this sample set, with STEC O26 and O45 strains appearing the most robust in these microcosm studies.

Antibiotic resistance genes in swine manure slurry as affected by pit additives and facility disinfectants.

Manure storage facilities are critical control points to reduce antibiotic resistance genes (ARGs) in swine manure slurry before the slurry is land applied. However, little is known about how exogenous chemicals entering the manure storage facilities may affect the fate of ARGs. The objective of this study was to analyze the impact of six commonly used pit additives and four facility disinfectants on the concentration of ARGs in swine manure slurry. Bench scale reactors, each containing approximately 50 L of liquid swine manure, were dosed with additives or disinfectants and were sampled for 40 days. Seven antibiotic resistance genes along with the intI1 gene and the 16S rRNA gene were monitored. Out of the six additives tested, Sludge Away significantly reduced the time-averaged absolute abundance of erm(C), erm(F), tet(Q), and the 16S rRNA gene as compared to the no additive control. Out of the four disinfectants tested, Tek-Trol significantly reduced the time-averaged absolute abundance of erm(B), erm(C), erm(F), intI1, tet(Q), and tet(X) than did the no-disinfectant control. According to Spearman's rank correlation, three genes erm(F), tet(Q), and tet(X) showed a strong to perfectly positive correlation and the two genes erm(B) and tet(O) showed a moderate to strong correlation in both the additive and disinfectant tests. Overall, the disinfectants were more effective in controlling the absolute abundance of ARGs than were the pit additives.

Antibiotic resistance gene profile changes in cropland soil after manure application and rainfall.

Land application of manure introduces gastrointestinal microbes into the environment, including bacteria carrying antibiotic resistance genes (ARGs). Measuring soil ARGs is important for active stewardship efforts to minimize gene flow from agricultural production systems; however, the variety of sampling protocols and target genes makes it difficult to compare ARG results between studies. We used polymerase chain reaction (PCR) methods to characterize and/or quantify 27 ARG targets in soils from 20 replicate, long-term no-till plots, before and after swine manure application and simulated rainfall and runoff. All samples were negative for the 10 b-lactamase genes assayed. For tetracycline resistance, only source manure and post-application soil samples were positive. The mean number of macrolide, sulfonamide, and integrase genes increased in post-application soils when compared with source manure, but at plot level only, 1/20, 5/20, and 11/20 plots post-application showed an increase in erm(B), sulI, and intI1, respectively. Results confirmed the potential for temporary blooms of ARGs after manure application, likely linked to soil moisture levels. Results highlight uneven distribution of ARG targets, even within the same soil type and at the farm plot level. This heterogeneity presents a challenge for separating effects of manure application from background ARG noise under field conditions and needs to be considered when designing studies to evaluate the impact of best management practices to reduce ARG or for surveillance. We propose expressing normalized quantitative PCR (qPCR) ARG values as the number of ARG targets per 100,000 16S ribosomal RNA genes for ease of interpretation and to align with incidence rate data.

Influence of Setback Distance on Antibiotics and Antibiotic Resistance Genes in Runoff and Soil Following the Land Application of Swine Manure Slurry.

The environmental spread of antibiotics and antibiotic resistance genes (ARGs) from the land application of livestock wastes can be a potential public health threat. The objective of this study was to assess the effects of setback distance, which determines how close manure may be applied in relation to surface water, on the transport of antibiotics and ARGs in runoff and soil following land application of swine manure slurry. Rainfall simulation tests were conducted on field plots covered with wheat residues, each of which contained an upslope manure region where slurry was applied and an adjacent downslope setback region that did not receive slurry. Results show that all three antibiotics (chlortetracycline, lincomycin, and tiamulin) and seven out of the ten genes tested (erm(B), erm(C), intI1, tet(O), tet(Q), tet(X), and the 16S rRNA gene) decreased significantly in runoff with increased setback distance. Only blaTEM, chlortetracycline, and tiamulin decreased significantly in surface soil with increased setback distance, while the other analytes did not exhibit statistically significant trends. By using linear regression models with field data, we estimate that a setback distance between 34-67 m may allow manure-borne antibiotics and ARGs in runoff to reach background levels under the experimental conditions tested.

Swine slurry characteristics as affected by selected additives and disinfectants.

Current swine industry practice is to house animals in confinement facilities which capture and store feces and urine as slurry in pits below the production area. Additives and disinfectants may be introduced into the manure pits. This study was conducted to measure the effects of additives and disinfectants on temporal changes in swine slurry characteristics. Slurry from a commercial swine production facility in southeast Nebraska, USA was collected and transferred to 57 L reactors located within a greenhouse. Selected additives and disinfectants were added to the reactors and physical properties, chemical characteristics, and antibiotic concentrations were monitored for 40 days. Concentrations of dry matter (DM), total nitrogen (TN), phosphorus pentoxide (P2O5), calcium (Ca), magnesium (Mg), zinc (Zn), iron (Fe), manganese (Mn), and copper (Cu) were significantly greater than the Control in each of the reactors containing additives. The reactors in which the additives MOC-7, More Than Manure®, Sludge Away, and Sulfi-Doxx were introduced had significantly greater values of chemical oxygen demand (COD), total volatile solids (TVS), total suspended solids (TSS), total solids (TS), dry matter (DM), TN, P2O5, Ca, Mg, Zn, Fe, Mn, Cu and chlortetracycline than the other additive treatments. Concentrations of TVS and TSS were significantly lower in the reactors containing Clorox® and Virkon™ than the other disinfectant treatments. The total dissolved solids (TDS) concentration of 26,500 mg L-1 and pH value of 7.27 obtained for the reactors containing Tek-Trol were significantly greater than measurements obtained for the other treatments. Concentrations of chlortetracycline and tiamulin of 8840 and 28.8 ng g-1, respectively, were significantly lower for the treatments containing Tek-Trol. The sodium (Na) concentration of 1070 mg L-1 measured in the reactors containing Clorox® was significantly greater than values for the other disinfectant treatments. The introduction of selected additives and disinfectants may influence certain physical properties, chemical characteristics, and antibiotic concentrations of swine slurry.

Fate and transport of antibiotics and antibiotic resistance genes in runoff and soil as affected by the timing of swine manure slurry application.

Land application of swine manure slurry is a common practice to supplement nutrients to soil for crop production. This practice can introduce antibiotic residues and antibiotic resistance genes (ARGs) into the environment. Field testing is critical in identifying manure management practices effective in minimizing the environmental impacts of manure-borne antibiotic and ARGs. The objective of this study was to determine how the timing of swine manure application relative to rainfall events impacts the fate and transport of antibiotics and ARGs in surface runoff and manure-amended soil. Swine manure slurry was either broadcast or injected on test plots in the field. A set of three 30-min simulated rainfall events, 24 h apart, were initiated on manured plots 1 day, 1 week, 2 weeks, or 3 weeks after the manure application. Results showed that an interval longer than 2 weeks between application and rainfall often significantly reduced the levels of antibiotics and ARGs tested in runoff with the exception of tet(X). For soil samples from broadcast plots, concentrations of two of the three antibiotics tested (lincomycin and tiamulin) decreased substantially in the first two weeks after manure application. In contrast, concentrations of most of the ARGs tested (tet(Q), tet(X), and erm(A)) in soil did not change significantly during the test period. Information obtained from the study can be beneficial in designing manure management practices and estimating the environmental loading of antibiotics and ARGs resulting from manure application.

Assessment of Selected Antibiotic Resistances in Ungrazed Native Nebraska Prairie Soils.

The inherent spatial heterogeneity and complexity of antibiotic-resistant bacteria and antibiotic resistance (AR) genes in manure-affected soils makes it difficult to sort out resistance that can be attributed to human antibiotic use from resistance that occurs naturally in the soil. This study characterizes native Nebraska prairie soils that have not been affected by human or food-animal waste products to provide data on background levels of resistance in southeastern Nebraskan soils. Soil samples were collected from 20 sites enumerated on tetracycline and cefotaxime media; screened for tetracycline-, sulfonamide-, ß-lactamase-, and macrolide-resistance genes; and characterized for soil physical and chemical parameters. All prairies contained tetracycline- and cefotaxime-resistant bacteria, and 48% of isolates collected were resistant to two or more antibiotics. Most (98%) of the soil samples and all 20 prairies had at least one tetracycline gene. Most frequently detected were (D), (A) (O), (L), and (B). Sulfonamide genes, which are considered a marker of human or animal activity, were detected in 91% of the samples, despite the lack of human inputs at these sites. No correlations were found between either phenotypic or genotypic resistance and soil physical or chemical parameters. Heterogeneity was observed in AR within and between prairies. Therefore, multiple samples are necessary to overcome heterogeneity and to accurately assess AR. Conclusions regarding AR depend on the gene target measured. To determine the impacts of food-animal antibiotic use on resistance, it is essential that background and/or baseline levels be considered, and where appropriate subtracted out, when evaluating AR in agroecosystems.

Narrow grass hedges reduce tylosin and associated antimicrobial resistance genes in agricultural runoff.

Agricultural runoff from areas receiving livestock manure can potentially contaminate surface water with antimicrobials and antimicrobial resistance genes (ARGs). The objective of this study was to investigate the effectiveness of narrow grass hedges (NGHs) on reducing the transport of antimicrobials and ARGs in runoff after land application of swine manure slurry. Plot-scale rainfall simulation tests were conducted on 0.75 m by 4.0 m plots designed to test three treatment factors: manure amendment (control plots receiving no manure vs. amended plots receiving manure based on 3 times N requirement), NGH (plots with a NGH vs. plots without a NGH), and rainfall events (days 1-3). Runoff generated during three 30-min simulated rainfall events was sampled and analyzed for antimicrobials and ARGs. Manure amendment was responsible for the presence of antimicrobial tylosin ( < 0.0001) and tylosin resistance gene (B) ( < 0.0001) in runoff. Narrow grass hedges proved to be effective in reducing tylosin ( < 0.0001) and (B) ( < 0.0347) in runoff. Manure amendment was responsible for the introduction of tylosin ( < 0.0482) and (B) ( = 0.0128) into the soil; however, it had no significant impact on the abundance of the 16S rRNA gene in soil. Results from this study suggest that NGHs could be a best management practice to control the transport of antimicrobials and ARGs in agricultural runoff.

Fate of antimicrobials and antimicrobial resistance genes in simulated swine manure storage.

The behavior of three antibiotics (bacitracin, chlortetracycline, and tylosin) and two classes of antibiotic resistance genes (ARGs), tet and erm, were monitored in swine manure slurry under anaerobic conditions. First-order decay rates were determined for each antibiotic with half-lives ranging from 1 day (chlortetracycline) to 10 days (tylosin). ARGs were monitored in the swine manure slurry, and losses of approximately 1 to 3 orders of magnitude in relative abundance were observed during the 40 day storage period. First-order degradation profiles were observed for chlortetracycline and its corresponding resistance genes, tet(X) and tet(Q). Tylosin was degraded to approximately 10% of the starting concentration by day 40; however, the relative abundance of erm(B) remained at 50-60% of the initial relative abundance while the relative abundance of erm(F) decreased by 80-90%, consistent with tylosin. These results indicate that tet resistance genes respond primarily to chlortetracycline antimicrobials, and may be lost when the parent tetracycline compound is degraded. In contrast, erm(B) resistance gene may respond to a range of antimicrobials in animal manure, and may persist despite losses of tylosin.

Emission of volatile organic compounds after land application of cattle manure.

Beef cattle manure can serve as a valuable source of nutrients for crop production. However, emissions of volatile organic compounds (VOCs) after land application may pose an odor nuisance to downwind populations. This study was conducted to evaluate the effects of land application method, diet, soil moisture content, and time since manure application on VOC emissions. Manure was collected from feedlot pens where cattle were fed diets containing 0, 10, or 30% wet distillers grains with solubles (WDGS). Land application methods included surface-applying manure (i.e., no-tillage) or incorporating manure using disk tillage. The effects of soil moisture content on VOC emissions was determined by adding water to each of the plots approximately 24 h after manure application. Isovaleric acid, butyric acid, and 4-methylphenol contributed 28.9, 18.0, and 17.7%, respectively, of the total measured odor activity values. In general, the largest emissions of volatile fatty acids and aromatics were measured during the initial collection periods on the no-tillage plots under dry soil moisture conditions. Emissions of volatile fatty acids and aromatics were reduced after water additions because these compounds were stored in the soil-water matrix rather than released into the atmosphere. In contrast, sulfide emissions generally increased with the addition of the water, especially on the plots containing manure from the 30% WDGS diet. Sulfur content of manure increases with higher percentages of WDGS feed stock. Application method, diet, soil moisture content, and time since application should be considered when estimating VOC emissions.

Fate and transport of antimicrobials and antimicrobial resistance genes in soil and runoff following land application of swine manure slurry.

Due to the use of antimicrobials in livestock production, residual antimicrobials and antimicrobial resistance genes (ARGs) could enter the environment following the land application of animal wastes and could further contaminate surface and groundwater. The objective of this study was to determine the effect of various manure land application methods on the fate and transport of antimicrobials and ARGs in soil and runoff following land application of swine manure slurry. Swine manure slurries were obtained from facilities housing pigs that were fed chlortetracyline, tylosin or bacitracin and were land applied via broadcast, incorporation, and injection methods. Three rainfall simulation tests were then performed on amended and control plots. Results show that land application methods had no statistically significant effect on the aqueous concentrations of antimicrobials in runoff. However, among the three application methods tested broadcast resulted in the highest total mass loading of antimicrobials in runoff from the three rainfall simulation tests. The aqueous concentrations of chlortetracyline and tylosin in runoff decreased in consecutive rainfall events, although the trend was only statistically significant for tylosin. For ARGs, broadcast resulted in significantly higher erm genes in runoff than did incorporation and injection methods. In soil, the effects of land application methods on the fate of antimicrobials in top soil were compound specific. No clear trend was observed in the ARG levels in soil, likely because different host cells may respond differently to the soil environments created by various land application methods.

Effects of animal diet, manure application rate, and tillage on transport of microorganisms from manure-amended fields.

Manure from cattle fed distillers' grain or corn diets was applied to fields, and the fields were subjected to rainfall simulation tests. Manure was added at three rates on till and no-till plots. Correlations between microbial transport and runoff characteristics were identified. Results indicate that diet affects phage but not bacterial transport from manure-amended fields.

Microbial quality of runoff following land application of cattle manure and swine slurry.

Concentrations of human health-related microorganisms in runoff from agricultural plots (0.75 m x 2 m) treated with fresh and aged cattle manure, swine slurry and no manure (control) were determined. Three consecutive simulated rainfall events, producing 35 mm rainfall and separated by 24 h, were carried out for each plot. Fecal indicator (Escherichia coli, enterococci, Clostridium perfringens and coliphage) loads released in rainfall runoff from plots treated with fresh cattle manure, aged cattle manure and swine slurry treatments ranged from 5.52 x 10(5) to 4.36 x 10(9), 3.92 x 10(4) to 4.86 x 10(8), and 9.63 x 10(5) to 3.05 x 10(8), respectively. Plot runoff concentrations of protozoa (Cryptosporidium oocysts and Giardia cysts) ranged from 1.65 x 10(5) to 1.04 x 10(6), 2.93 x 10(3) to 2.75 x 10(5), and 9.12 x 10(4) to 3.58 x 10(6) for fresh cattle manure, aged cattle manure and swine slurry plot treatments, respectively. These results suggest that large microbial loads could be released via heavy precipitation events that produce runoff from livestock manure-applied agricultural fields, of even modest size, and could have a significant impact on water bodies within the watershed. Because of the lack of multiplication in the environment, highly elevated concentrations in manured land runoff, and correlation to protozoan parasite presence, Clostridium may be an alternative indicator for livestock manure contamination.