Glossary of Antimicrobial Resistance

If you are just starting to learn about antimicrobials and resistance, first off welcome to the club, we are so happy to have more microbe obsessives! Second, we guessed you might have been encountering some words or concepts that you haven’t heard before. So, we’ve put together this visual glossary for you to explore. Search the table below for a word or unfamiliar phrase and you’ll find a definition AND videos or other websites where you can learn more about that concept. 

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Development of a Cost-Effective Treatment Process for Removing Antimicrobials from Agricultural Wastewater

Much of the antimicrobials (AM) used therapeutically and prophylactically pass through the animal and enter the environment through irrigation with beef runoff wastewater (WW).  There are concerns repeated low-level AM loading of soils through irrigation will alter the natural biota resulting in increased resistance; thereby, making AM critical for human and animal health less effective.

What Did We Do?

Several studies are summarized describing a cost-effective removal process of AM from beef wastewater before being applied as irrigation. Study 1 investigated three radiolabeled AM, ([14C]-erythromycin (ERY), [3H]-chlortetracycline (CTC), and [3H]-monensin (MON)) to quantify their partitioning in the aqueous and solids fractions of a beef wastewater (WW) containing suspended solids (SS).  Following this, several flocculants were evaluated for removing SS with sorbed AM from WW. Study 2 evaluated the sorption properties of tylosin (TYL) with diatomaceous earth (DE) added to WW as a binding agent. Study 3 evaluated the proposed treatment process using pre-treatment of WW with flocculants then adding a binding agent to remove aqueous phase AM before being used an irrigation water.

What Have We Learned?

Study 1) After 48 hours, more ERY sorbed to the SS fraction than the aqueous. CTC partitioning occurred in three phases: a rapid sorption to the SS fraction between 0.5 and 8 hours with desorption into the aqueous fraction at 24 hours followed by short steady state at 48 hours with further desorption at 96 hours. The most lipophilic antibiotic, MON, quickly sorbed into the SS fraction and remained in equilibrium with the aqueous fraction after 48 hours.  Calculated partitioning coefficients, Kd, for WW were very different from published soil-water values illustrating wastewater uniqueness. A follow up study determined alum and ferric chloride removed some ceftiofur (CEF) and chlorotetracycline (CTC); however, they were ineffective for removing TYL.  Therefore, a process was needed to remove aqueous phase AM (figure 1). Study 2 evaluated three DE sources for binding aqueous TYL in WW. Raw (DER) contained organic carbon (OC) (3% g g-1), clays (20%) and amorphous silica (65%). Kieselguhr (DEK) had no OC (<1% g g-1), no clay (2%) and amorphous silica (96%).   had nearly 3.5 times greater maximum sorption capacity when compared to DEK. Sorption of TYL to DEK and DER at different pH values showed cationic and hydrogen bonding interactions are important. Higher sorption of TYL to DER compared to DEK suggested clays-DE matrix was important for TYL sorption. Removing OC improved TYL sorption and decreased the separation time for DE/AM removal to complete treatment. Study 3 found CEF and CTC would bind to DE in controlled, neutral aqueous solution. When AMs were spiked into WW collected from a beef feedlot runoff pond, DE successfully removed TYL and CTC, but not CEF. Wastewater treated with excess alum to remove SS followed by DE treatment showed similar removal rates for TYL. Pretreatment of water with alum also resulted in CEF removal if larger amounts of DE are needed. The reason for change in CEF binding when pretreated WW with alum is still under investigation. Alum treatment appeared to remove most of the CTC spiked into WW and therefore no assessment of how CTC binding to DE after treatment.

Future Plans

Future work will include: evaluating the proposed treatment process on other AM and their metabolites. Development of modified DE matrices specific for non-polar AM and metabolites.  These matrices could be combined to remove both polar and non-polar contaminants. Finally, the process needs to be evaluated for effectiveness of contaminant removal with municipal wastewater treatment systems.

Authors

Bryan L. Woodbury, Agricultural Engineer, USDA-U.S. Meat Animal Research Center, Clay Center, NE

bryan.woodbury@ars.usda.gov

Bobbi S. Stromer, Chemist Post Doc., USDA-U.S. Meat Animal Research Center, Clay Center, NE

Clinton. F. Williams, Lead Soil Scientist, USDA-US Arid-Land Agric. Research Center, Maricopa, AZ.

Katherine A. Woodward, Ph.D. Candidate, Tufts University, Civil & Environmental Eng., Medford, MA

Heldur Hakk, Research Chemist, USDA-Edward T. Schafer Agricultural Research Center, Fargo, ND.

Sara Lupton, Research Chemist, USDA-Edward T. Schafer Agricultural Research Center, Fargo, ND.

Additional Information

Stromer, B.S., B.L. Woodbury and C.F. Williams. 2018. Tylosin sorption to diatomaceous earth described by Langmuir isotherm and Freundlich isotherm models. Chemosphere.  193:912-920.

Figure 1.  Illustration of a wastewater treatment process to reduce the antimicrobials load applied through irrigation of agricultural wastewater.  Loading soils with antimicrobials may cause increased antimicrobial resistance. This resistance may make antimicrobials less effective to treat humans.
Figure 1.  Illustration of a wastewater treatment process to reduce the antimicrobials load applied through irrigation of agricultural wastewater.  Loading soils with antimicrobials may cause increased antimicrobial resistance. This resistance may make antimicrobials less effective to treat humans.

Acknowledgements

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The authors are solely responsible for the content of these proceedings. The technical information does not necessarily reflect the official position of the sponsoring agencies or institutions represented by planning committee members, and inclusion and distribution herein does not constitute an endorsement of views expressed by the same. Printed materials included herein are not refereed publications. Citations should appear as follows. EXAMPLE: Authors. 2019. Title of presentation. Waste to Worth. Minneapolis, MN. April 22-26, 2019. URL of this page. Accessed on: today’s date.

Manure Land Application Strategies to Mitigate Antibiotics and Antibiotic Resistance Genes in the Agricultural Environment

When it comes to the land application of livestock manure, a major environmental concern is the loss of manure-borne nutrients to surface runoff. Management practices and regulations focus on how nutrients travel in runoff as functions of the timing and method of land application as well as proximity to water sources. When manure is applied to soil, manure constituents other than nutrients are also introduced to soil, such as heavy metals, antibiotics, and antibiotic resistance genes. Knowledge about the behaviors of these manure constituents in the environment as a function of manure application strategies is limited.

In this article we will discuss the effects of various land application strategies on the fate and transport of manure borne antibiotics and antibiotic resistance genes in soil and runoff. This will be broken down into three sections: manure storage; land application methods; and vegetative barrier. Although our studies were conducted using swine manure slurry, it is expected that that the general conclusion would also apply to other types of manure. Prior to a detailed description of our findings, we will first present some background information about manure-borne antibiotics and antibiotic resistance genes.

Why Care About Antibiotics?

Antibiotics are often used in concentrated animal feeding operations (CAFOs) to prevent and treat diseases in livestock animals, increasing the density at which livestock can be kept. A substantial portion of these antibiotics can move thorough the digestive system of livestock, and end up in livestock urine and feces. These antibiotics can persist in the livestock manure and go on to alter the microbiome of soil and water. Bacteria exposed to these antibiotics may gain resistance to the antibiotics. This can be a major public health concern, because even the antibiotic resistant bacteria are harmless they may spread the resistance genes to pathogens. This, in turn, could impact human and animal health, as the antibiotics which we rely upon to treat infection will no longer be effective on treating resistant pathogens.

graphic showing antibiotic movement from adminstration to cropland to runoff

Manure Storage

Prior to land application, manure is usually stored in livestock waste management structures. In one study, the effects of anaerobic storage of manure on the fate of antibiotics and antibiotic resistance genes in manure were investigated. In this study, the levels of chlortetracycline and tylosin in manure slurry were monitored. The two antibiotics in swine manure degraded substantially over time under the anaerobic condition. The antibiotic resistance genes corresponding to chlortetracycline was also reduced substantially during manure storage.  In contrast, the resistance genes corresponding to tylosin did not decrease significantly.

Application Method

land application with manure tankerManure and manure slurry may be applied to fields using application methods such as broadcast, injection, and incorporation. These land application methods have varying effects on the spread of antibiotics and antibiotic resistance genes in croplands. A study was conducted to investigate how these land application methods may affect the concentrations of antibiotics and antibiotic resistance genes in runoff and soil following the land application of swine slurry. Results show that land application methods had no statistically significant effect on the aqueous concentrations of antibiotics in the runoff.  However, among the three land application methods tested, broadcast resulted in the highest total mass load of antibiotics in runoff from the three simulated rainfall events. Similarly, broadcast resulted in higher concentrations of antibiotic resistance genes in runoff than did injection and incorporation. In manure amended soils, the effects of land application on the concentration of antibiotics were compound specific. No clear trend was observed in the antibiotic resistance gene levels in soil.

Vegetative Barrier

grasses in a vegetative bufferVegetative barriers are strips of densely growing perennial plants seeded downslope on cropland adjacent to surface water. Vegetative barriers can stabilize the soil in local areas and reduce dissolved and sediment bound compounds in runoff, such as nutrients and particulates. The barriers are often used as an erosion control measure and some states regulate the use of vegetative barriers next to bodies of water and in areas with high slopes. One study tested whether vegetative barriers are effective in reducing antibiotics and antibiotic resistance genes. Results show that stripes of switchgrass (panicum virgatum L.) can effectively reduce antibiotic tylosin and its corresponding resistance gene erm(B) in runoff. Hence, vegetative barriers can be used as a low-cost option to reduce the spread of antibiotic and antibiotic resistance gene through runoff.

Conclusions

The control of manure-borne antibiotics and antibiotic resistance genes in the environment is complicated. Different antibiotic compounds have different properties, such as vulnerability to photo degradation and tendency to adsorb to soil particles. Hence, it is hard to use one land application strategy to effectively manage multiple antibiotics in both runoff and soil. Hence, knowing the dominant antibiotic compounds in the manure can be important. Similarly, different antibiotic resistance genes may be hosted in different bacterial species. These bacteria differ in their metabolisms and consequently respond differently to various land application strategies. Like the case for antibiotics, it is difficult to develop one land application strategy that would be effective to all classes of antibiotic resistance genes in manure. So, in addition to land application strategies, attention should also be given to develop manure storage strategies to reduce antibiotics and antibiotic resistance genes prior to land application.

For more information about this article, contact Xu Li.

Sensitivity of Soil Microbial Processes to Livestock Antimicrobials

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Purpose

Many of the antimicrobials administered to livestock are excreted in manure where they may undergo natural breakdown, become more tightly associated with the manure and soil, or become mobilized in wastewater/runoff. Both liquid and solid manure is usually applied to nearby crop fields as a manure fertilizer, recycling the nutrients in the manure. Public concerns about the overuse of antimicrobials leading to greater antibiotic resistance and potentially greater risk for human health have led to new regulations limiting the use of antimicrobials in animal production. However, there are several significant research questions that need to be explored in order to determine how important the links are between antimicrobial use in livestock production and increased antibiotic resistance in humans.

One important issue involves how important soil processes (decomposition, nutrient transformation, and gas emissions) could be altered by antimicrobial compounds in manures and wastewater. In a previous study at a cattle feedlot in central Nebraska, we found typical antimicrobial concentrations in feedlot runoff at low part per billion (ppb) levels and were detected infrequently (<20% of the time). One exception, monensin, was usually detected with an average concentration of 87 ppb and peak concentrations above 200 ppb. Adding complexity to this issue is that soils may experience a variety of conditions ranging from fully aerobic, to denitrifying (using nitrate as a terminal electron acceptor), to anaerobic, and a diverse variety of microbes may predominate in these various conditions. How might soil functions be affected under a range of conditions experiencing differing concentrations of antibiotic? Are there clear very high concentration thresholds that completel! y inhibit specific soil functions? The purpose of this study was to determine the effects of three common livestock antibiotics at multiple concentrations on decomposition, nutrient transformation, and gas production in pasture soil under aerobic, denitrifying, and anaerobic conditions.

What did we do?

A soil slurry incubation study was conducted with pasture soil where runoff from a nearby cattle feedlot was occasionally applied. Monensin, sulfamethazine, and lincomycin were amended (0, 5, 500, and 5000 ppb) to mason jars and serum bottles containing soil and simulated cattle feedlot runoff. The mason jars were flushed with air (aerobic) while serum bottles were flushed with nitrogen gas (anaerobic). Denitrifying conditions were established initially in a subset of anaerobic serum bottles which were supplemented with nitrate (100 mg NO3-N L-1). All antimicrobial amendments and conditions were replicated in triplicate and incubated at 20°C. Headspace gas composition and decomposition products were both measured using gas chromatography and monitored over several weeks.Table 1. Summary of the effects of various livestock antibiotics on decomposition under aerobic, anaerobic, and denitrifying conditions

What have we learned?

Soil processes were generally affected only at the highest antibiotic concentrations, which are 10x greater than observed levels in feedlot runoff. Furthermore, the effects on soil processes depended upon the antibiotic tested (Table 1). Monensin, a broad-range antimicrobial, had the greatest effect on a number of processes. At highest monensin concentrations tested (5000 ppb), both aerobic and anaerobic decomposition (including denitrification) were affected as shown by greater VFA concentrations and low to no gas production (CO2, N2O, and CH4). Even at 500 ppb, monensin had some effect—CO2, N2O, and CH4 gas production were reduced. Sulfamethazine at 5000 ppb inhibited full denitrification (no N2O produced), but there was no effect on other gases or VFA. At 500 ppb sulfamethazine, N2O production was reduced by half. Lincomycin’s only observable effect was lower (0.5x) N2O production at the 5000 ppb level under denitrification conditions.

These results show important soil processes can be blocked by high levels of antibiotics found in animal manures, but inhibition depends upon the antibiotic.  A general antimicrobial like monensin affected microbial processes far more than antimicrobials with a specific mode of action.  The highest antibiotic levels evaluated were 5 to 10 times higher than levels found in animal manures, so soils are likely not impacted under normal conditions where manures mixed and distributed into soils.  Antibiotic breakdown in the soil further helps reduce the potential for antibiotics to build up in the soils.

Future Plans

These incubations only assessed the effect of a one-time dose of antimicrobials. Future studies will examine how longer soil exposures affect soil processes. Additional studies will also compare how soils that have different manure exposure histories (cattle feedlot soil with heavy exposure versus protected prairie soils with very low manure exposure) would react to higher levels of antimicrobials.

Corresponding author, title, and affiliation

Dan Miller, Microbiologist, USDA-ARS

Corresponding author email

dan.miller@ars.usda.gov

Other authors

Matteo D’Alessio, Postdoctoral Researcher, Nebraska Water Center; Dan Snow, Director of Services, Water Sciences Laboratory

Additional information

151 Filley Hall, UNL East Campus, Lincoln, NE 68583

Ph: 402-472-0741

https://dl.sciencesocieties.org/publications/csa/pdfs/61/8/4?search-result=1

The authors are solely responsible for the content of these proceedings. The technical information does not necessarily reflect the official position of the sponsoring agencies or institutions represented by planning committee members, and inclusion and distribution herein does not constitute an endorsement of views expressed by the same. Printed materials included herein are not refereed publications. Citations should appear as follows. EXAMPLE: Authors. 2017. Title of presentation. Waste to Worth: Spreading Science and Solutions. Cary, NC. April 18-21, 2017. URL of this page. Accessed on: today’s date.

Ciprofloxacin residues in biosolids compost do not selectively enrich populations of resistant bacteria

Biosolids and livestock manure are valuable high-carbon soil amendments, but they commonly contain antibiotic residues that might persist after land application. While composting reduces the concentration of extractable antibiotics in these materials, if the starting concentration is sufficiently high then remaining residues could impact microbial communities in the compost and soil to which these materials are applied. To examine this issue we spiked biosolids compost feedstock with ciprofloxacin at a concentration (19 ppm), approximately 5-fold higher than normally detected by LC-MS/MS (1-3.5 ppm). This feedstock was  placed into mesh bags that were buried in aerated compost bays. Once a week a set of bags was removed and analyzed (treated and untreated, three replicates of each; 4 weeks). Addition of ciprofloxacin had no effect on recovery of resistant bacteria at any time point (P = 0.86), and a separate bioassay showed that aqueous extractions from materials with an estimated 59 ppm ciprofloxacin had no effect on the growth of a susceptible strain of E. coli (P = 0.28). Regression analysis showed that growth of the susceptible strain was diminished when compost was spiked with a wide range of ciprofloxacin (0-160 ppm; P<0.007), consistent with adsorption as the primary mechanism of antibiotic sequestration. Because bioassays reflect the bioavailability of residues whereas analytical assays do not, we recommend that similar bioassays be incorporated into studies of other antibiotic residues to better assess the risk that these residues pose for proliferating resistant populations of bacteria. 

Author

Youngquist, Caitlin           caitlinmp@gmail.com     University of Wyoming

The authors are solely responsible for the content of these proceedings. The technical information does not necessarily reflect the official position of the sponsoring agencies or institutions represented by planning committee members, and inclusion and distribution herein does not constitute an endorsement of views expressed by the same. Printed materials included herein are not refereed publications. Citations should appear as follows. EXAMPLE: Authors. 2015. Title of presentation. Waste to Worth: Spreading Science and Solutions. Seattle, WA. March 31-April 3, 2015. URL of this page. Accessed on: today’s date.

Antibiotic Losses during Thermophilic Composting

Purpose

Residual antibiotics in land-applied manure and biosolids present a potential threat to public and ecological health, so it is important to determine antibiotic removal efficiencies for manure and biosolids waste management practices and to identify conditions that enhance antibiotic degradation.

What we did

Loss of the antibiotics florfenicol, sulfadimethoxine, sulfamethazine, and tylosin was studied during pilot-scale static pile thermophilic composting and the effects of temperature and feedstock particles on antibiotic removal rates were tested. The antibiotics were spiked into dairy manure solids and wastewater biosolids, and treatments included aerated and non-aerated manure and biosolids/wood-product (1:3 v/v) composting.

Figure 1. Applying antibiotic solution to biosolids

Figure 1. Applying antibiotic solution to biosolids

What have we learned

Results showed no significant differences between aerated and non-aerated treatments; on average ≥85%, ≥93%, and ≥95% antibiotic reduction was observed after 7, 14, and 21 d of composting. Greater antibiotic reduction was observed in manure compost compared to biosolids compost for florfenicol (7, 14, 21, 28 d) and tylosin (7, 14, 28 d); however, there was no significant difference for sulfadimethoxine and sulfamethazine. Peak temperatures were 66-73°C, and ≥55°C was maintained for 6-7 d in the biosolids compost and 17-20 d in the manure compost.

Bench-scale experiments conducted at 25, 55, and 60°C showed that lower temperature decreased removal of the sulfonamides and tylosin in both feedstocks and florfenicol in the biosolids. The presence of compost particles increased antibiotic loss, with time to 50% dissipation ≤ 2 d in the presence of solids (60°C), compared to no degradation in their absence. These results indicate that thermophilic composting effectively reduces residual antibiotics in manure and biosolids.

Figure 2. Mixing biosolids and wood shavings

Figure 2. Mixing biosolids and wood shavings

Figure 3. Mixing biosolids and wood shavings

Figure 3. Mixing biosolids and wood shavings.

 

Authors

A. Bary*, S.M. Mitchell*, J.L. Ullman**, C.G. Cogger*, A.L. Teel*, R.J. Watts*

Washington State University*, University of Florida**.

Andy Bary, bary@wsu.edu

 

The authors are solely responsible for the content of these proceedings. The technical information does not necessarily reflect the official position of the sponsoring agencies or institutions represented by planning committee members, and inclusion and distribution herein does not constitute an endorsement of views expressed by the same. Printed materials included herein are not refereed publications. Citations should appear as follows. EXAMPLE: Authors. 2015. Title of presentation. Waste to Worth: Spreading Science and Solutions. Seattle, WA. March 31-April 3, 2015. URL of this page. Accessed on: today’s date.

Figure 4. Compost bins

Figure 4. Compost bins

Antibiotic Resistance and Animal Agriculture

This webcast examines the veterinary feed directive and how it impacts animal agriculture as well as discusses the state of the science as it relates to antibiotic resistance. Finally, it busts some myths and reviews “facts” to keep in mind when chatting with livestock producers and the general public.This presentation was originally broadcast on March 25, 2016. More… Continue reading “Antibiotic Resistance and Animal Agriculture”