Kang Xia

Effect of manure application on abundance of antibiotic resistance genes and their attenuation rates in soil: field-scale mass balance approach.

The development of models for understanding antibiotic resistance gene (ARG) persistence and transport is a critical next step toward informing mitigation strategies to prevent the spread of antibiotic resistance in the environment. A field study was performed that used a mass balance approach to gain insight into the transport and dissipation of ARGs following land application of manure. Soil from a small drainage plot including a manure application site, an unmanured control site, and an adjacent stream and buffer zone were sampled for ARGs and metals before and after application of dairy manure slurry and a dry stack mixture of equine, bovine, and ovine manure. Results of mass balance suggest growth of bacterial hosts containing ARGs and/or horizontal gene transfer immediately following slurry application with respect to ermF, sul1, and sul2 and following a lag (13 days) for dry-stack-amended soils. Generally no effects on tet(G), tet(O), or tet(W) soil concentrations were observed despite the presence of these genes in applied manure. Dissipation rates were fastest for ermF in slurry-treated soils (logarithmic decay coefficient of -3.5) and for sul1 and sul2 in dry-stack-amended soils (logarithmic decay coefficients of -0.54 and -0.48, respectively), and evidence for surface and subsurface transport was not observed. Results provide a mass balance approach for tracking ARG fate and insights to inform modeling and limiting the transport of manure-borne ARGs to neighboring surface water.

Fate and effect of antibiotics in beef and dairy manure during static and turned composting

Manure composting has general benefits for production of soil amendment, but the effects of composting on antibiotic persistence and effects of antibiotics on the composting process are not well-characterized, especially for antibiotics commonly used in dairy cattle. This study provides a comprehensive, head-to-head, replicated comparison of the effect of static and turned composting on typical antibiotics used in beef and dairy cattle in their actual excreted form and corresponding influence on composting efficacy. Manure from steers (with or without chlortetracycline, sulfamethazine, and tylosin feeding) and dairy cows (with or without pirlimycin and cephapirin administration) were composted at small scale (wet mass: 20-22 kg) in triplicate under static and turned conditions adapted to represent US Food and Drug Administration guidelines. Thermophilic temperature (>55°C) was attained and maintained for 3 d in all composts, with no measureable effect of compost method on the pattern, rate, or extent of disappearance of the antibiotics examined, except tylosin. Disappearance of all antibiotics, except pirlimycin, followed bi-phasic first-order kinetics. However, individual antibiotics displayed different fate patterns in response to the treatments. Reduction in concentration of chlortetracycline (71-84%) and tetracycline (66-72%) was substantial, while near-complete removal of sulfamethazine (97-98%) and pirlimycin (100%) was achieved. Tylosin removal during composting was relatively poor. Both static and turned composting were generally effective for reducing most beef and dairy antibiotic residuals excreted in manure, with no apparent negative impact of antibiotics on the composting process, but with some antibiotics apparently more recalcitrant than others.

Development and Validation of a UPLC-MS/MS Method to Monitor Cephapirin Excretion in Dairy Cows following Intramammary Infusion

Cephapirin, a cephalosporin antibiotic, is used by the majority of dairy farms in the US. Fecal and urinary excretion of cephapirin could introduce this compound into the environment when manure is land applied as fertilizer, and may cause development of bacterial resistance to antibiotics critical for human health. The environmental loading of cephapirin by the livestock industry remains un-assessed, largely due to a lack of appropriate analytical methods. Therefore, this study aimed to develop and validate a cephapirin quantification method to capture the temporal pattern of cephapirin excretion in dairy cows following intramammary infusion. The method includes an extraction with phosphate buffer and methanol, solid-phase extraction (SPE) clean-up, and quantification using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The LOQ values of the developed method were 4.02 µg kg−1 and 0.96 µg L−1 for feces and urine, respectively. This robust method recovered >60% and >80% cephapirin from spiked blank fecal and urine samples, respectively, with acceptable intra- and inter-day variation (<10%). Using this method, we detected trace amounts (µg kg−1) of cephapirin in dairy cow feces, and cephapirin in urine was detected at very high concentrations (133 to 480 µg L−1). Cephapirin was primarily excreted via urine and its urinary excretion was influenced by day (P = 0.03). Peak excretion (2.69 mg) was on day 1 following intramammary infusion and decreased sharply thereafter (0.19, 0.19, 0.08, and 0.17 mg on day 2, 3, 4, and 5, respectively) reflecting a quadratic pattern of excretion (Quadratic: P = 0.03). The described method for quantification of cephapirin in bovine feces and urine is sensitive, accurate, and robust and allowed to monitor the pattern of cephapirin excretion in dairy cows. This data will help develop manure segregation and treatment methods to minimize the risk of antibiotic loading to the environment from dairy farms.

Manure Injection Affects the Fate of Pirlimycin in Surface Runoff and Soil.

Antibiotics used in animal agriculture are of increasing environmental concern due to the potential for increased antibiotic resistance after land application of manure. Manure application technology may affect the environmental behavior of these antibiotics. Therefore, rainfall simulations were conducted on plots receiving three manure treatments (surface application, subsurface injection, and no manure control) to determine the fate and transport of pirlimycin, an antibiotic commonly used in dairy production. Rainfall simulations were conducted immediately and 7 d after application of dairy manure spiked with 128 ng g (wet weight) pirlimycin. Soil samples were collected from all plots at two depths (0-5 and 5-20 cm). For injection plots, soil was collected from injection slits and between slits. Pirlimycin concentrations were higher in soil within the injection slits compared with surface application plots at 0 and 7 d. Pirlimycin concentrations in the 0- to 5-cm depth decreased by 30, 55, and 87% in the injection slit, between injection slits, and surface application plots 7 d after application. Pirlimycin concentrations were 106 ng g in sediment and 4.67 ng mL in water from the surface application plots, which were 21 and 32 times that of the injection plots, respectively. After 7 d, pirlimycin levels in runoff sediment and water decreased 80 to 98%. Surface application resulted in six and three times higher pirlimycin concentrations in water and sediment than injection. These results indicate that pirlimycin is most susceptible to loss immediately after manure application. Thus, injection could be considered a best management practice to prevent loss of antibiotics in surface runoff.

Transport of a neonicotinoid pesticide, thiamethoxam, from artificial seed coatings

Neonicotinoid insecticides coat the seeds of major crops worldwide; however, the high solubility of these compounds, combined with their toxicity to non-target organisms, makes it critical to decipher the processes by which they are transported through soils and into aquatic environments. Transport and distribution of a neonicotinoid (thiamethoxam, TMX) were investigated by growing TMX-coated corn seeds in coarse-textured and fine-textured soil columns (20 and 60cm lengths). To understand the influence of living plants, corn plants were terminated in half of the columns (no plant treatment) and allowed to grow to the V5 growth stage (33days of growth) in the other half (with plant treatment). TMX was analyzed in leachate 12 times over 33days and in bulk soil after 8, 19, and 33days of corn growth. All 20cm columns leached TMX at levels exceeding the United States Environmental Protection Agency benchmark for aquatic invertebrates (17.5μgL-1). TMX migrated from seeds to adjacent bulk soil by the eighth day and reached deeper soil sections in later growth stages (e.g., 30-45cm depth by Day 33). Fine-particle soils transported over two orders of magnitude more TMX than coarse-textured soils (e.g., 29.9μg vs 0.17μg, respectively), which was attributed to elevated evapotranspiration (ET) rates in the sandy soil driving a higher net retention of the pesticide and to structural flow occurring in the fine-textured soil. Living plants increased TMX concentrations at depth (i.e., 30-60cm) compared to the no plant treatment, suggesting that corn growth may drive preferential transport of TMX from coated seeds. Altogether, this study showed that neonicotinoid seed coatings can be mobilized through soil leachate in concentrations considered acutely toxic to aquatic life.

Effect of composting and soil type on dissipation of veterinary antibiotics in land-applied manures

The objective of this study was to determine the fate of commonly used veterinary antibiotics in their naturally excreted form when manure-based amendments are applied to soil. Beef cattle were administered sulfamethazine, tylosin, and chlortetracycline and dairy cows were treated with pirlimycin. The resulting manure was composted for 42 d under static or turned conditions and applied at agronomic N rates to sandy, silt, and silty clay loam soils and compared with amendment with corresponding raw manures in sacrificial microcosms over a 120-day period. Antibiotic dissipation in the raw manure-amended soils followed bi-phasic first order kinetics. The first phase half-lives for sulfamethazine, tylosin, chlortetracycline, and pirlimycin ranged from 6.0 to 18, 2.7 to 3.7, 23 to 25, and 5.5-8.2 d, respectively. During the second phase, dissipation of sulfamethazine was negligible, while the half-lives for tylosin, chlortetracycline, and pirlimycin ranged from 41 to 44, 75 to 144, and 87-142 d, respectively. By contrast, antibiotic dissipation in the compost-amended soils followed single-phase first order kinetics with negligible dissipation of sulfamethazine and half-lives of tylosin and chlortetracycline ranging from 15 to 16 and 49-104 d, respectively. Pirlimycin was below the detection limit in the compost-amended soils. After incubating 120 d, antibiotics in compost-amended soils (up to 3.1 μg kg-1) were significantly lower than in manure-amended soils (up to 19 μg kg-1, p < .0001), with no major effect of soil type on the dissipation. Risk assessment suggested that composting can reduce antibiotic resistance selection potential in manure-amended soils.

Fe3 +-saturated montmorillonite effectively deactivates bacteria in wastewater

Existing water disinfection practices often produce harmful disinfection byproducts. The antibacterial activity of Fe3+-saturated montmorillonite was investigated mechanistically using municipal wastewater effluents. Bacterial deactivation efficiency (bacteria viability loss) was 92±0.64% when a secondary wastewater effluent was mixed with Fe3+-saturated montmorillonite for 30min, and further enhanced to 97±0.61% after 4h. This deactivation efficiency was similar to that when the same effluent was UV-disinfected before it exited a wastewater treatment plant. Comparing to the secondary wastewater effluent, the bacteria deactivation efficiency was lower when the primary wastewater effluent was exposed to the same dose of Fe3+-saturated montmorillonite, reaching 29±18% at 30min and 76±1.7% at 4h. Higher than 90% bacterial deactivation efficiency was achieved when the ratio between wastewater bacteria population and weight of Fe3+-saturated montmorillonite was at <2×103CFU/mg. Furthermore, 99.6-99.9% of total coliforms, E. coli, and enterococci in a secondary wastewater effluent was deactivated when the water was exposed to Fe3+-saturated montmorillonite for 1h. Bacterial colony count results coupled with the live/dead fluorescent staining assay observation suggested that Fe3+-saturated montmorillonite deactivated bacteria in wastewater through two possible stages: electrostatic sorption of bacterial cells to the surfaces of Fe3+-saturated montmorillonite, followed by bacterial deactivation due to mineral surface-catalyzed bacterial cell membrane disruption by the surface sorbed Fe3+. Freeze-drying the recycled Fe3+-saturated montmorillonite after each usage resulted in 82±0.51% bacterial deactivation efficiency even after its fourth consecutive use. This study demonstrated the promising potential of Fe3+-saturated montmorillonite to be used in applications from small scale point-of-use drinking water treatment devices to large scale drinking and wastewater treatment facilities.

Antibiotics and Antibiotic Resistance Genes in Bulk and Rhizosphere Soils Subject to Manure Amendment and Vegetable Cultivation

There is interest in understanding effects of amending soil with manure in a cultivation setting and if composting can provide benefits. Raw or composted manure from cattle administered with and without sulfamethazine, chlortetracycline, and tylosin was amended to loamy sand and silty clay loam soils, where lettuce ( L.), radish ( L.), and broccoli ( L. var. ) were cultivated and compared with those grown in soil amended with fertilizer as a control. Upon plant maturation, rhizosphere and bulk soils were analyzed for antibiotics, and 1, B, (W), and I1 genes were quantified. Antibiotic concentrations in compost-amended soils were below detection limits. For soils amended with manure containing antibiotics, sulfamethazine ranged from 1.1 to 3.1 μg kg in the bulk soils but was below detection limits in the rhizosphere soils. Chlortetracycline (2.8-9.3 μg kg) was two times lower in the rhizosphere than in the bulk soil. Levels of tylosin in the rhizosphere soil were similar to the bulk soil. Soil texture or vegetable type did not have significant influence on antibiotic concentration differences between the bulk and rhizosphere soils. Relative abundances of (W) and I1 in the fertilizer-amended soil were significantly lower than in those amended with manure or compost ( < 0.05), whereas B was not detected in any soils. Rhizosphere zone has no significant effect on the detected antibiotic resistance genes. It is suggested that plant roots may have a substantial effect on the fate of certain antibiotics in manure-amended fields, but less of an effect on antibiotic resistance and mobility genes.

Fate of Pirlimycin and Antibiotic-Resistant Fecal Coliforms in Field Plots Amended with Dairy Manure or Compost during Vegetable Cultivation

Identification of agricultural practices that mitigate the environmental dissemination of antibiotics is a key need in reducing the prevalence of antibiotic-resistant bacteria of human health concern. Here, we aimed to compare the effects of crop (lettuce [ L.] or radish [ L.]), soil amendment type (inorganic fertilizer, raw dairy manure, composted dairy manure, or no amendment), and prior antibiotic use history (no antibiotics during previous lactation cycles vs. manure mixed from cows administered pirlimycin or cephapirin) of manure-derived amendments on the incidence of culturable antibiotic-resistant fecal coliforms in agricultural soils through a controlled field-plot experiment. Antibiotic-resistant culturable fecal coliforms were recoverable from soils across all treatments immediately after application, although persistence throughout the experiment varied by antibiotic class and time. The magnitude of observed coliform counts differed by soil amendment type. Compost-amended soils had the highest levels of cephalosporin-resistant fecal coliforms, regardless of whether the cows from which the manure was derived were administered antibiotics. Samples from control plots or those treated with inorganic fertilizer trended toward lower counts of resistant coliforms, although these differences were not statistically significant. No statistical differences were observed between soils that grew leafy (lettuce) versus rooted (radish) crops. Only pirlimycin was detectable past amendment application in raw manure-amended soils, dissipating 12 to 25% by Day 28. Consequently, no quantifiable correlations between coliform count and antibiotic magnitude could be identified. This study demonstrates that antibiotic-resistant fecal coliforms can become elevated in soils receiving manure-derived amendments, but that a variety of factors likely contribute to their long-term persistence under typical field conditions.

Method of Dairy Manure Application and Time before Rainfall Affect Antibiotics in Surface Runoff

Although research has shown that manure soil subsurface injection reduces nutrient input to the aquatic environment, it is less known if it also reduces antibiotic surface runoff from manure-applied fields. Surface runoff of four dairy production antibiotics was monitored comparing (i) surface application and subsurface injection of manure and (ii) time gaps between manure application and a subsequent rain event. Liquid dairy manure spiked with pirlimycin, tylosin, chlortetracycline, and sulfamerazine was applied to 1.5-m × 2-m test plots at an agronomic N rate via surface application and subsurface injection. On the day of application (Day 0), and 3 and 7 d after manure application, a simulated rainfall (70 mm h) was conducted to collect 30 min runoff. Target antibiotics in runoff water and sediment were quantified using ultra-performance liquid chromatography tandem mass spectrometry. Results demonstrated that runoff was a significant route for transporting antibiotics off manure-applied fields, amounting to 0.45 to 2.62% of their initial input with manure. However, compared with manure surface application, subsurface injection reduced sulfamerazine, chlortetracycline, pirlimycin, and tylosin losses in runoff by at least 47, 50, 57, and 88%, respectively. Antibiotic distribution between aqueous and solid phases of runoff was largely determined by water solubility and partition capacity of antibiotics to soil particles. Masses in the aqueous phase were 99 ± 0.5, 94 ± 4, 91 ± 7, and 22 ± 15% of pirlimycin, sulfamerazine, tylosin, and chlortetracycline, respectively. Manure application 3 d or longer before a subsequent rain event reduced antibiotic runoff by 9 to 45 times. Therefore, using subsurface injection and avoiding manure application <3 d before rain would be a recommended manure land management best practice.