Pathways of antibiotics, and antimicrobial resistance genes and organisms from manure to soil

In response to the increasing human health concerns regarding antimicrobial resistance, it is helpful to understand that there is a large reservoir of beneficial microbes in a healthy soil containing active organic matter is crucial in reducing the potential harm resulting from antimicrobial use in humans and animals.

In order to understand the risks, and the important role of the soil organic matter, we need to understand some of the important pathways of antibiotics, antibiotic resistant organisms, and antibiotic resistant genes. The graphic below provides a summary of the products excreted with manure that are important in our discussions of antimicrobial resistance. It also illustrates the importance of soil organic matter in managing antimicrobial resistance resulting from animal manure. We will work through literature research documenting each of the pathways and their significance.

Pathways of antimicrobial resistant microbes, genes, and antimicrobial residue from animal manure into the environment.

There are three potential streams of concern related to antibiotic use in agriculture and specifically manure management.

1. animals excrete antibiotics or their metabolites that may favor selection of antibiotic resistant organisms in the soil or receiving environment.

2. manure may contain potentially pathogenic organisms that are resistant to antibiotics.

3. manure may contain antibiotic resistant genes, which may be transferred to other organisms in the receiving environment.

Manure also contains readily available carbon that stimulates microbial activity in soil. We think that this is a good thing in terms of maintaining a healthy soil, but we also need to understand the dynamics of antimicrobial resistance in the soil microbial population.

Manure may contain antibiotic residue

Livestock excrete 17-90% of antibiotics into the urine or feces, either unchanged or as an active metabolite (Masse et al. 2014). In a review, EPA (2013) reported that up to 80% of tetracyclines fed to swine and poultry was excreted in the manure. They reported that up to 67% of tylosin was excreted from animals used for food production.

Masse et al. (2014) suggested that: “most of the antibiotic residues in manure form complexes with soluble organics and remain stable during manure storage.”

Furtula et al. (2010) found that antibiotics present in poultry litter on nine commercial farms in BC reflected the antibiotics in the feed. Ratsak et al. (2013) reported measured for tetracyclines, sulfonamides and fluoroquinolones in 69 manure and digestate samples in Germany. They observed that 62% of the manure samples and 80% of the digestate samples contained antibiotics. They found that pig and poultry manure contained more antibiotics than cattle manure. Zhang et al. (2015) measured the presence of seven antibiotics in pig manure in China.

It is important to note that antibiotic residue in manure decomposes likely due to the significant microbial population in manure. Joy et al. (2014) observed that the half-lives of three antimicrobials used in hog production (bacitracin, chlorotetracycline and tylosin) ranged from 1 to 10 days. Masse et al. (2014) also included a review of the persistence and biological decomposition of antibiotics in manure and soil.

Manure may contain antibiotic resistant genes

In the Fraser Valley of British Columbia, Keen (2009) measured four tetracycline resistance genes in poultry manure during storage and following field application. The resistance genes were present even though the potentially pathogenic bacteria were no longer viable. Diarrassouba (2008) found antibiotic resistant and virulence genes in E. coli and salmonella isolated from nine commercial poultry broiler farms in BC. He et al. (2014) found resistance genes in poultry litter for chloramphenicols, sulfonamides, and tetracyclines in 11 broiler operations.

Zhang et al. (2015) found 10 antibiotic resistant genes in swine manure and concluded that chlorotetracycline demonstrated the highest ecological risk. Whichmann et al. (2014) identified 80 antibiotic resistant genes in cow manure from five different cows with or without a history of antibiotic use in the US, but found that cow manure carried a lower frequency of resistance genes than poultry litter. Brooks, et al. (2014) found antibiotic resistance to Salmonella and Campylobacter spp in the manure lagoon from all 37 swine manure farms tested in the US.

Not all of the antimicrobial resistant genes are found in potentially harmful bacteria. Nandi et al. (2004) reported that antimicrobial resistant genes were found in Gram positive bacteria that constituted > 85% of the microbes in poultry litter and concluded that the antimicrobial resistance genes appear to move relatively freely among the microbial population.

Manure may contain potentially harmful antibiotic resistant microbes

In a survey of dairy cattle manure from 23 farms, Sawant et al. (2009) found that E. coli isolates were resistant to ampicillin (48%), ceftiofur (11%), chloramphenicol (20%), florfenicol (78%), spectinomycin (18%), and tetracycline (93%). They found multidrug resistance in 40% of the E.coli from healthy lactating dairy cattle.

Poultry manure was reported to contain Salmonella, E. coli and Enteroccoccus sp. that were resistant to both oxytetracycline and tetracycline (Keen 2009). It was noted that the viability of these organisms decreased during the manure storage/composting period before field application. Diarrassouba et al. (2007) found that 70% of the E. coli isolates and 14% of Salmonella isolates in the manure from nine different broiler chicken farms in the Fraser Valley of BC were multiresistant to at least nine antibiotics.   In the same study, Diarrassouba (2008) found that all of the E. coli strains isolated on the farms were resistant to penicillin, erythromycin, tylosin, clindamycin and novobiocin, and that although tetracycline is not used as a growth promoter in Canada, 74% of the E.coli were tetracycline resistant. She also found multiresistance in some of the Salmonella isolates. She concluded that:

“antibiotic resistant Salmonella carrying virulence genes can be isolated from healthy commercial broiler chickens fecal samples and letter, suggesting the potential to contaminate the environment including crops and runoff water, if litter is applied to soil as a fertilizer”

Gao et al. (2015) concluded that antibiotic resistant (extended spectrum beta-lactamase, or ESBL) E. coli contained in swine manure was the likely contributor to antibiotic resistant gene spread in the soil.  Laube et al. (2014)  measured antibiotic resistant (ESBL) E. coli in the manure and surrounding air from seven poultry broiler farms in Germany. Hartmann et al. (2012) reported ESBL producing E. coli in cattle feces during a survey of 182 farms in Burgundy, France.

Antimicrobial Genes are Everywhere 

An important fact to note is that antimicrobial genes are everywhere in nature and have been for much longer than the commercial development of antibiotics (Davies and Davies 2010). Davies and Davies (2010) also speculated whether some of these antimicrobial resistant genes have other important functions that we may not yet be aware of. Clemente et al. (2015) found antibiotic resistance genes in an isolated South American tribe with no known contact for more than 11,000 years.

Knapp et al. (2009) measured antibiotic resistant genes in soil samples that had been archived since 1940 from several experimental sites in the Netherlands. They found that the concentration of antibiotic resistant genes increased in all 18 genes studied, and that some antibiotic resistant gene concentrations increased 15 fold since 1940, likely due to increased commercial antibiotic use.

Popowska et al (2012) observed significant amounts of antimicrobial resistant genes in forest soil, compost, garden soil and farm soil. They observed that agricultural soils had a more diverse population of bacteria containing resistance genes. The composted and forest soils had lower levels of antibiotic resistance, and a lower presence of antibiotic resistant genes.

Keen (2009) measured tetracycline resistant genes in a forest soil that had not been fertilized with manure, as well as in the water from a mountain stream in the Lower Fraser Valley in British Columbia.

Manure application may stimulate growth of antibiotic resistant organisms in soil.

Udikovic-Kolic et al. (2014) found more B-lactam antibiotic resistant bacteria in soil (7.4%) than in manure (0.67%) from cows that had not been fed antibiotics. They observed higher populations of cephalothin-resistant bacteria in soil following manure application for the duration of the 94 day study. Marti et al. (2014) measured increased frequencies of antimicrobial resistance genes in soil following application of swine or dairy manure. Animal manure contains significant concentrations of readily available carbon which increases the microbial biomass population in soil (Paul and Beauchamp 1996), which are then likely to increase the number of antibiotic resistant genes in the soil.

Summary

Animal manure and how we manage it has an important impact on human health risk associated with antimicrobial resistance. You and Silbergeld (2014) summarized the concerns as follows:

“This waste contains complex constituents that are challenging to treat, including AMR determinants and low-dose antimicrobials. Unconfined storage or land deposition of a large volume of animal waste causes its wide contact with the environment and drives the expansion of the environmental resistome through mobilome facilitated horizontal genet transfer. The expanded environmental resistome, which encompasses both natural constituents and anthropogenic inputs, can persist under multiple stressors from agriculture and may re-enter humans, thus posing a public health risk to humans”.

When we add manure to our soil, our soil acts both as a reservoir for antimicrobial resistance as well as a purifier that destroys antimicrobial resistance organisms, and genes. We will explore the microbes and the soil further.

References

Brooks, J.P. A. Adeli, and M.R. McLaughlin. 2014. Microbial ecology, bacterial pathogens, and antibiotic resistant genes in swine manure wastewater as influenced by three swine management systems. Water Research 57: 96-103.

Clemente, J.C., E.C. Pehrsson, M.J. Blaser, K. Sandhu, Z. Gao, B.Wang, M. Magris, G. Hidalgo, M. Contreras, O. Noya Alarcon, O. Lander, J. McDonald, M. Cox, J. Walter, P.L. Oh, J.F. Ruiz, S. Rodriguez, N. Shen, S.J. Song, J. Metcalf, R. Knight, G. Dantas and M.G. Dominguez-Bello. 2015. The microbiome of uncontacted Amerindians. Science Advances 2015;1:e1500183.

Davies, J. and D. Davies. 2010. Origins and evolution of antibiotic resistance. Microbiology and Molecular Biology Reviews 74: 417-433.

Diarrassouba. 2008. Impact of antimicrobial growth promoters used in broiler chicken production on the emergence of antibiotic resistance in commensal E. coli and Salmonella. MSc. thesis. University of British Columbia.

Diarrassouba, F. M.S. Diarra, S.J. Bach, P.J. Pritchard, E. Topp and B.J. Skura. 2007. Antibiotic resistance and virulence genes in commensal Escherichia coli and salmonella isolates from commercial broiler chicken farms. Journal of Food Protection 70: 1316-1327.

EPA 2013. Literature Review of Contaminants in Livestock and Poultry Manure and Implications for Water Quality. Office of Water (4304T) EPA 820-R-13-002 July 2013

Furtula, V., E.G. Farrell, F. Diarrassouba, H. Rempel, J. Pritchard and M.S. Diarra. 2010. Veterinary pharmaceuticals and antibiotic resistance of Escherichia coli isolates in poultry litter from commercial farms and controlled feeding trials. Poultry Science 89: 180-188.

Gao, L, J. Hu., X. Zhang, L. Wei, S. Li, Z. Miao, and T. Chai. 2015. Application of swine manure on agricultural fields contributes to extended spectrum B-lactamase-producing Esherichia coli spread in Tai’an, China. Frontiers in Microbiology. April 2015, Volume 6, Article 313.

Hartmann, A., A. Locatelli, L. Amoureux, G. Depret, C. Jolivet and E. Gueneau. 2012. Occurrence of CTX-M producing Escherichia coli in soils, cattle, and farm environment in France (Burgundy region). Frontiers in Microbiology Volume 3 Article 83.  doi: 10.3389/fmicb.2012.00083

He, L-Y., Y-S. Liu, H-C Su, J-L. Zhao, S-S Liu, J. Chen, W-R Liu and G-G Ying. 2014. Dissemination of antibiotic resistance genes in representative broiler feedlots environments: Identification of indicator ARGs and correlations with environmental variables. Environmental Science and Technology 48: 13120-13129

Joy, S.R., X. Li, D.D. Snow, J.E. Gilley, B. Woodbury, and S.L. Bartelt-Hunt. 2014. Fate of antimicrobials and antimicrobial resistance genes in simulated swine manure storage. Science of the Total Environment 481: 69-74.

Keen, P.L. 2009. Seasonal Dynamics of Tetracycline Resistant Genes and Antibiotics in a British Columbia Agricultural Watershed. PhD Thesis, University of British Columbia.

Knapp, C.W., J. Dolfing, P.A.I. Ehlert and D.W. Graham. 2009. Evidence of increasing antibiotic resistance gene abundances in archived soils since 1940. Environmental Science and Technology 44: 580-587.

Laube, H., A. Friese, C. von Salviati, B. Guerra and U. Rosler. 2014. Transmission of ESBL/Amp C-producing Escherichia coli from broiler chicken farms to surrounding areas. Veterinary Microbiology 172: 519-527.

Marti, R. Y-C Tien, R. Murray, A Scot, L. Sabourin and E. Topp. 2014. Safely coupling livestock and crop production systems: how rapidly do antibiotic resistance genes dissipate in soil following a commercial application of swine or dairy manure? Applied and Environmental Microbiology 80: 3258-3265.

Masse, D.I., N.M. Cata Saady and Y. Gilbert. 2014. Potential of biological processes to eliminate antibiotics in livestock manure: an overview. Animals 2014, 4, 146-163.

Munir, M., and I. Xagoraraki. 2011. Levels of antibiotic resistance genes in manure, biosolids and fertilized soil. Journal of Environmental Quality 40: 248-255.

Nandi, S., J.J. Maurer, C. Hofacre and A.O. Summers. 2004. Gram positive bacteria are a major reservoir of Class 1 antibiotic resistance integrons in poultry litter. PNAS www.pnas.orgcgidoi10.1073pnas.0306466101

Paul, J.W. and E.G. Beauchamp. 1996. Soil microbial biomass C, N mineralization, and N uptake by corn in dairy cattle slurry- and urea-amended soils. Canadian Journal of Soil Science 76: 469-472.

Popowska, M., M. Rzeczycka, A. Miernik, A. Krawcyk-Balska, F. Walsh and B. Duffy. 2012. Influence of soil use on prevalence of tetracycline, streptomycin, and erythromycin resistance and associated resistance genes. aac.asm.org March 2012 Vol. 56. No 3.

Ratsak, C., B. Guhl, S. Zuhlke and T. Delschen. 2013. Veterinary antibiotic residues in manure and digestates in Northrhein-Westfalia. Environmental Sciences Europe 2013, 25:7.

Udikovic-Kolic, N., F. Wichmann, N.A. Broderick and J. Handelsman. 2014. Bloom of resident antibiotic resistant bacteria in soil following manure fertilization. Proceedings of the National Academy of Sciences Vol 111. No. 42. pp 15202-15207.

Whichmann, F., N. Udikovic-Kolic, S. Andrew and J. Handelsman. 2014. Diverse antibiotic resistant genes in dairy cow manure. mBio Volume 5 Issue 2, e01017-13.

You, Y. and E.K. Silbergeld. 2014. Learning from agriculture: understanding low dose antimicrobials as drivers of resistome expansion. Frontiers in Microbiology June 2014, Volume 5, Article 284.

Zhang, S., J. Gu, C. Wang, P. Wang, A. Jiao, Z.L. He and B. Han. 2015. Characterization of antibiotics and antibiotic resistance genes on an ecological farm system. Journal of Chemistry Article ID 526143.