Population Genomics of Antibiotic Resistance Spread in Agricultural Runoff Bacteria
DOI:
https://doi.org/10.5281/zenodo.22687831Keywords:
antimicrobial resistance, agricultural runoff, population genomics, plasmid transfer, whole-genome sequencing, Escherichia coliAbstract
Agricultural runoff carrying manure-associated bacteria into surface waterways is widely suspected as a route by
which antibiotic resistance genes move between livestock and environmental, and ultimately human-adjacent,
bacterial populations, but the population-genomic structure of this transfer has rarely been characterised at a
resolution sufficient to distinguish genuine cross-compartment gene flow from independent acquisition of the same
resistance elements. We whole-genome sequenced 312 Escherichia coli isolates recovered over eighteen months
from paired sampling of manure storage lagoons, adjacent field-edge runoff channels, and downstream stream
water at four mixed dairy-and-arable farms, and combined core-genome phylogenetics with plasmid-resolved
long-read assembly to reconstruct resistance gene movement across the manure-to-stream continuum.
Core-genome multi-locus sequence typing identified extensive strain sharing between manure and adjacent runoff
samples (78% of runoff sequence types also recovered from paired manure) but markedly less sharing between
runoff and downstream stream isolates (31%), suggesting that most resistant strains originating in manure fail to
establish persistently in the stream environment. Despite this attenuation in strain-level sharing, plasmid-resolved
assembly identified an IncF-family multidrug-resistance plasmid, pAR117, carrying blaCTX-M-15, sul2 and tet(A)
in near-identical form across genetically distinct E. coli backgrounds recovered from all three compartments,
indicating that horizontal plasmid transfer, rather than strain persistence, is the dominant mechanism by which
resistance genes cross from manure into the stream bacterial community. Resistance gene abundance in stream
water was significantly associated with recent heavy rainfall events (P=0.004) but not with distance downstream of
the runoff entry point within the 2 km sampled reach. These findings indicate that agricultural runoff functions
primarily as a conduit for mobile genetic elements rather than for the resistant strains that originally carried them,
with direct implications for how runoff-focused antimicrobial resistance mitigation efforts should be prioritised.
References
Larsson DGJ, Flach CF (2022) Antibiotic resistance in the environment. Nature Reviews Microbiology 20(5):257-269. https://doi.org/10.1038/s41579-021-00649-x
Berendonk TU, Manaia CM, Merlin C, et al. (2015) Tackling antibiotic resistance: the environmental framework. Nature Reviews Microbiology 13(5):310-317. https://doi.org/10.1038/nrmicro3439
Heuer H, Schmitt H, Smalla K (2011) Antibiotic resistance gene spread due to manure application on agricultural fields. Current Opinion in Microbiology 14(3):236-243. https://doi.org/10.1016/j.mib.2011.04.009
Zhu YG, Johnson TA, Su JQ, et al. (2013) Diverse and abundant antibiotic resistance genes in Chinese swine farms. PNAS 110(9):3435-3440. https://doi.org/10.1073/pnas.1222743110
Wichmann F, Udikovic-Kolic N, Andrew S, Handelsman J (2014) Diverse antibiotic resistance genes in dairy cow manure. mBio 5(2):e01017-13. https://doi.org/10.1128/mBio.01017-13
Garcia-Alonso J, Ferrer C, Garcia-Rodriguez JA (2020) Environmental spread of antimicrobial resistance from farm to stream: a review. Science of the Total Environment 741:140477. https://doi.org/10.1016/j.scitotenv.2020.140477
von Wintersdorff CJH, Penders J, van Niekerk JM, et al. (2016) Dissemination of antimicrobial resistance in microbial ecosystems through horizontal gene transfer. Frontiers in Microbiology 7:173. https://doi.org/10.3389/fmicb.2016.00173
San Millan A (2018) Evolution of plasmid-mediated antibiotic resistance in the clinical context. Trends in Microbiology 26(12):978-985. https://doi.org/10.1016/j.tim.2018.06.007
Partridge SR, Kwong SM, Firth N, Jensen SO (2018) Mobile genetic elements associated with antimicrobial resistance. Clinical Microbiology Reviews 31(4):e00088-17. https://doi.org/10.1128/CMR.00088-17
Carattoli A (2013) Plasmids and the spread of resistance. International Journal of Medical Microbiology 303(6-7):298-304. https://doi.org/10.1016/j.ijmm.2013.02.001
Bevan ER, Jones AM, Hawkey PM (2017) Global epidemiology of CTX-M beta-lactamases: temporal and geographical shifts in genotype. Journal of Antimicrobial Chemotherapy 72(8):2145-2155. https://doi.org/10.1093/jac/dkx146
Woolhouse M, Ward M, van Bunnik B, Farrar J (2015) Antimicrobial resistance in humans, livestock and the wider environment. Philosophical Transactions of the Royal Society B 370(1670):20140083. https://doi.org/10.1098/rstb.2014.0083
Manaia CM (2017) Assessing the risk of antibiotic resistance transmission from the environment to humans: non-direct proportionality between abundance and risk. Trends in Microbiology 25(3):173-181. https://doi.org/10.1016/j.tim.2016.11.014
Wirth T, Falush D, Lan R, et al. (2006) Sex and virulence in Escherichia coli: an evolutionary perspective. Molecular Microbiology 60(5):1136-1151. https://doi.org/10.1111/j.1365-2958.2006.05172.x
Pitout JDD, Nordmann P, Poirel L (2015) Carbapenemase-producing Klebsiella pneumoniae, a key pathogen set for global nosocomial dominance. Antimicrobial Agents and Chemotherapy 59(10):5873-5884. https://doi.org/10.1128/AAC.01019-15
Zurfluh K, Wang J, Klumpp J, Nuesch-Inderbinen M, Fanning S, Stephan R (2014) Vertical transmission of highly similar bla(CTX-M-1)-harboring IncI1 plasmids in Escherichia coli. BMC Microbiology 14:307. https://doi.org/10.1186/s12866-014-0307-4
Pehrsson EC, Tsukayama P, Patel S, et al. (2016) Interconnected microbiomes and resistomes in low-income human habitats. Nature 533(7602):212-216. https://doi.org/10.1038/nature17672
Baker S, Thomson N, Weill FX, Holt KE (2018) Genomic insights into the emergence and spread of antimicrobial-resistant bacterial pathogens. Science 360(6390):733-738. https://doi.org/10.1126/science.aar3777
Wick RR, Judd LM, Gorrie CL, Holt KE (2017) Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLOS Computational Biology 13(6):e1005595. https://doi.org/10.1371/journal.pcbi.1005595
Zankari E, Hasman H, Cosentino S, et al. (2012) Identification of acquired antimicrobial resistance genes. Journal of Antimicrobial Chemotherapy 67(11):2640-2644. https://doi.org/10.1093/jac/dks261
Alcock BP, Raphenya AR, Lau TTY, et al. (2020) CARD 2020: antibiotic resistome surveillance with the comprehensive antibiotic resistance database. Nucleic Acids Research 48(D1):D517-D525. https://doi.org/10.1093/nar/gkz935
Carattoli A, Zankari E, Garcia-Fernandez A, et al. (2014) In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrobial Agents and Chemotherapy 58(7):3895-3903. https://doi.org/10.1128/AAC.02412-14
Zhou Z, Alikhan NF, Mohamed K, et al. (2020) The EnteroBase user guide, with case studies on Salmonella transmissions, Yersinia pestis phylogeny, and Escherichia core genomic diversity. Genome Research 30(1):138-152. https://doi.org/10.1101/gr.251678.119
Stamatakis A (2014) RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30(9):1312-1313. https://doi.org/10.1093/bioinformatics/btu033
Bankevich A, Nurk S, Antipov D, et al. (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. Journal of Computational Biology 19(5):455-477. https://doi.org/10.1089/cmb.2012.0021
Larranaga O, Brown-Jaque M, Quiros P, et al. (2018) Phenotypic and genotypic characterization of antibiotic resistance in Escherichia coli isolates from surface waters. Environment International 121:1247-1257. https://doi.org/10.1016/j.envint.2018.10.012
Amos GCA, Gozzard E, Carter CE, et al. (2015) Validated predictive modelling of the environmental resistome. ISME Journal 9(6):1467-1476. https://doi.org/10.1038/ismej.2014.237
McEwen SA, Collignon PJ (2018) Antimicrobial resistance: a one health perspective. Microbiology Spectrum 6(2):ARBA-0009-2017. https://doi.org/10.1128/microbiolspec.ARBA-0009-2017
Franz E, Veenman C, van Hoek AHAM, de Roda Husman A, Blaak H (2015) Pathogenic Escherichia coli producing extended-spectrum beta-lactamases isolated from surface water and wastewater. Scientific Reports 5:14372. https://doi.org/10.1038/srep14372
Blaak H, van Hoek AH, Veenman C, et al. (2015) Extended spectrum ss-lactamase- and constitutively AmpC-producing Enterobacteriaceae on fresh produce and in the agricultural environment. International Journal of Food Microbiology 168-169:8-16. https://doi.org/10.1016/j.ijfoodmicro.2013.10.006
Vikesland PJ, Pruden A, Alvarez PJJ, et al. (2017) Toward a comprehensive strategy to mitigate dissemination of environmental sources of antibiotic resistance. Environmental Science & Technology 51(22):13061-13069. https://doi.org/10.1021/acs.est.7b03623
Kraemer SA, Ramachandran A, Perron GG (2019) Antibiotic pollution in the environment: from microbial ecology to public policy. Microorganisms 7(6):180. https://doi.org/10.3390/microorganisms7060180
Bengtsson-Palme J, Kristiansson E, Larsson DGJ (2018) Environmental factors influencing the development and spread of antibiotic resistance. FEMS Microbiology Reviews 42(1):fux053. https://doi.org/10.1093/femsre/fux053
Rothrock MJ, Min BR, Castro-Prieto V, Dyer L, Pieroni A, Estep A (2020) A review of the microbiology and management of antibiotic resistance in agricultural water. Journal of Environmental Quality 49(3):485-501. https://doi.org/10.1002/jeq2.20037
Suzuki S, Ogo M, Miller TW, Shimizu A, Takada H, Siringan MAT (2013) Who possesses drug resistance genes in the aquatic environment? Sulfamethoxazole resistance genes among the bacterial community in tropical marine sediments. Frontiers in Microbiology 4:102. https://doi.org/10.3389/fmicb.2013.00102
Djordjevic SP, Stokes HW, Chowdhury PR (2013) Mobile elements, zoonotic pathogens and commensal bacteria: conduits for the delivery of resistance genes into humans, livestock and soil microbiota. Frontiers in Microbiology 4:86. https://doi.org/10.3389/fmicb.2013.00086
Pruden A, Larsson DGJ, Amezquita A, et al. (2013) Management options for reducing the release of antibiotics and antibiotic resistance genes to the environment. Environmental Health Perspectives 121(8):878-885. https://doi.org/10.1289/ehp.1206446
Karkman A, Do TT, Walsh F, Virta MPJ (2018) Antibiotic-resistance genes in waste water. Trends in Microbiology 26(3):220-228. https://doi.org/10.1016/j.tim.2017.09.005
Sivalingam P, Pote J, Prabakar K (2020) Environmental prevalence, hotspots and sink of antibiotic resistance genes: a review. Applied Sciences 10(23):8577. https://doi.org/10.3390/app10238577
Card RM, Cawthraw SA, Nunez-Garcia J, et al. (2017) An in vitro chicken gut model demonstrates transfer of a multidrug resistance plasmid from Salmonella to commensal Escherichia coli. mBio 8(4):e00777-17. https://doi.org/10.1128/mBio.00777-17
Woodford N, Turton JF, Livermore DM (2011) Multiresistant Gram-negative bacteria: the role of high-risk clones in the dissemination of antibiotic resistance. FEMS Microbiology Reviews 35(5):736-755. https://doi.org/10.1111/j.1574-6976.2011.00268.x
Hooban B, Joyce A, Fitzhenry K, Chique C, Morris D (2020) The role of the natural aquatic environment in the dissemination of extended spectrum beta-lactamase and carbapenemase encoding genes: a scoping review. Water Research 180:115880. https://doi.org/10.1016/j.watres.2020.115880
Leonard AFC, Zhang L, Balfour AJ, Garside R, Gaze WH (2015) Human recreational exposure to antibiotic resistant bacteria in coastal bathing waters. Environment International 82:92-100. https://doi.org/10.1016/j.envint.2015.02.013
Skurnik D, Ruimy R, Andremont A, et al. (2006) Effect of human vicinity on antimicrobial resistance and integrons in animal faecal Escherichia coli. Journal of Antimicrobial Chemotherapy 57(6):1215-1219. https://doi.org/10.1093/jac/dkl122
Croucher NJ, Page AJ, Connor TR, et al. (2015) Rapid phylogenetic analysis of large samples of recombinant bacterial whole genome sequences using Gubbins. Nucleic Acids Research 43(3):e15. https://doi.org/10.1093/nar/gku1196
Page AJ, Cummins CA, Hunt M, et al. (2015) Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics 31(22):3691-3693. https://doi.org/10.1093/bioinformatics/btv421
Treangen TJ, Ondov BD, Koren S, Phillippy AM (2014) The Harvest suite for rapid core-genome alignment and visualization of thousands of intraspecific microbial genomes. Genome Biology 15:524. https://doi.org/10.1186/s13059-014-0524-x
Kluytmans-van den Bergh MFQ, Huizinga P, Bonten MJM, et al. (2016) Presence of mcr-1-positive Enterobacteriaceae in retail chicken meat but not in humans in the Netherlands since 2009. Eurosurveillance 21(9):30149. https://doi.org/10.2807/1560-7917.ES.2016.21.9.30149
World Health Organization (2021) Global antimicrobial resistance and use surveillance system (GLASS) report. WHO Technical Report Series. https://doi.org/10.2471/B09876
European Food Safety Authority, European Centre for Disease Prevention and Control (2021) The European Union summary report on antimicrobial resistance in zoonotic and indicator bacteria from humans, animals and food. EFSA Journal 19(4):e06490. https://doi.org/10.2903/j.efsa.2021.6490
Seemann T (2014) Prokka: rapid prokaryotic genome annotation. Bioinformatics 30(14):2068-2069. https://doi.org/10.1093/bioinformatics/btu153
