Canada IPAP: Evolutionary dynamics of antimicrobial resistance in human impacted soils: does the genie go back into the bottle?

Antimicrobial resistance (AMR) has evolved in environmental bacteria over billions of years, producing a vast reservoir of AMR genes that can potentially be transferred to clinical pathogens. Agricultural soils are exposed to antibiotics through the use of manures or sewage sludge as fertiliser, or irrigation with reclaimed water. There is a concern that such exposure may promote AMR in food producing environments, increasing the likelihood of AMR transmission to the human microbiome via contaminated crops or environmental matrices. There is a growing body of research using in vitro models to establish the minimal selective concentrations (MSCs) of antibiotics in the environment, but there is very little evidence relating to selection by antibiotic residues jn situ in soils. The field scale trials undertaken at Agriculture and Agri-Food Canada (AAFC) over the last 10-20 years offers a unique opportunity to investigate the effects of antibiotic application on soil communities and AMR, and how soils recover from long-term application of antibiotics. In this project, state of the art metagenomic and bioinformatic approaches will be applied to determine the evolutionary effects of antibiotic residues on the soil resistome. This will be investigated at three timepoints: short-term exposure (7 & 30 days after application); long-term exposure (after 10 years of annual exposure); and after cessation of antibiotic application (1, 2, and 3 years post-exposure). Macrolide and fluoroquinolone antibiotics will be studied, which have very different environmental fates and which have both been flagged as priority substances of concern by the EU Water Framework Directive's Hazardous Compound Watch List. Furthermore, we will assess whether or not in vitro experiments are predictive of selection in soils in situ. This work will provide the most comprehensive assessment of AMR evolution in antibiotic amended soils to date.

The exposure of agricultural soils to antibiotics, via use of manures or sewage sludge as fertiliser or irrigation with reclaimed water, can select for and promote antimicrobial resistance (AMR) in food producing environments. Soils could then act as a reservoir of AMR genes which could then be transferred to humans through contaminated crops or other environmental matrices. While there is a growing body of evidence using in vitro models to establish the minimal selective concentrations (MSCs) of antibiotics in the environment, there is very little evidence relating to selection by antibiotic residues in situ in soils. This project will leverage a unique long-term experiment run by Agriculture and Agri-Food Canada (AAFC) over the last 10-20 years examining the effects of antibiotic exposure on agricultural soils. Short- and long-read metagenome sequencing will be used to examine the effects on AMR, resilience and recovery of soils to antibiotic exposure at three timepoints: 1) short-term exposure (7 & 30 days after application); 2) long-term exposure (after 10 years of annual exposure); and 3) after cessation of antibiotic application (1, 2, and 3 years post-exposure). Bioinformatic analyses include hybrid assemblies, identification of AMR genes, classification of microbial taxa, statistical modelling and ecological diversity metrics to compare the unique types and abundances of AMR genes at the different timepoints. The effects of macrolides and fluoroquinolones, two different classes of antibiotics with different environmental fates, will be compared. Finally, we will assess whether or not in vitro experiments are predictive of selection in soils in situ. This work will provide the most comprehensive assessment of AMR evolution in antibiotic amended soils to date.