This research project aims to investigate whether different pasture fertilization practices impact the selection and spread of antimicrobial resistance (AMR) within the livestock farming environment. 
A major objective of the work involved testing bacteria for resistance to a wide range of antimicrobial compounds. Around three hundred bacterial Escherichia coli isolates from soil, water, sheep faeces, organic fertilisers (slurry, manure and biosolids) and septic tank samples (farmers) were examined against a panel of antimicrobials representing multiple drug classes. Using standard laboratory thresholds to tell apart normal (wild-type) E. coli from strains that carry resistance mechanism that make them less sensitive to antibiotics, this analysis identified some differences in resistance patterns among bacteria in soil. This included a higher proportion of non-wild-type E. coli in slurry-treated fields, suggesting that slurry application may be associated with an increased presence of antimicrobial-resistant bacteria in soil. We found only a few bacteria producing compounds that break down commonly used antimicrobials, such as penicillins and cephalosporins, and these were only present in water samples from one farm. This suggests limited spread of this important form of antimicrobial resistance, with water acting as a potential local reservoir. 
Genetic analyses were also used to examine the Resistome—the collection of resistance genes. By measuring resistance genes and mobile genetic elements, which are segments of DNA that allow resistance genes to move between bacteria, this work provided insight into how resistance traits are distributed across fields, grazing animals, farm environment, including different points within farm water streams, and how these patterns vary over time in relation to fertiliser application and land management practices. These analyses help to build a more complete picture of how resistance circulates within agricultural systems.
Fresh sheep faecal samples collected from grazing pasture fields before and one month after fertiliser application were used to compare total viable bacterial counts (aerobic and anaerobic) and to culture AMR anaerobic bacteria. 67 cultured sheep bacterial isolates were whole-genome sequenced and analysed. The sheep isolates, from both sampling timepoints,  contained on average 16 AMR genes, ranging from 5 to 71, consistent with values reported in a related RESAS-funded project (RI-A2-02). Efflux pump genes were the most prevalent, reflecting their ability to confer resistance to multiple antimicrobials. Twelve different tetracycline resistance genes were identified, with tet(W) and tet(Q) present in 15% of isolates. A single type of mosaic tetracycline resistance gene, associated with higher tetracycline resistance, was detected in one sheep isolate. Such genes have been reported more frequently in intensively reared systems and were also detected in pig, calf and poultry isolates (related project RI-B6-01). Following the establishment of a collaboration agreement, between MRI and JHI, soil samples were prepared and shipped to JHI for analysis of antimicrobial and metal residues. 
 

Impact

This project has contributed to a better understanding of how antimicrobial resistance (AMR) develops and circulates within livestock farming systems, supporting Scotland’s wider efforts to mitigate AMR across the animal–environment–human interface. By generating integrated phenotypic and genomic data from soils, animals, water, and organic fertilisers, the research has strengthened the evidence base needed to inform responsible antimicrobial use and sustainable land management.
Policy impact The project has contributed policy-relevant evidence on antimicrobial resistance (AMR) in agricultural and environmental settings. Project PIs contributed to the “Livestock Infectious Diseases in a Changing Climate” policy brief, (https://moredun.org.uk/research/diseases/livestock-infectious-diseases-in-a-changing-climate) requested by the Scottish Government, linking AMR with climate-related pressures on livestock systems. Engagement with policymakers and stakeholders was further supported through attendance at the Food Security Conference at the Royal Botanic Garden Edinburgh, which highlighted links between food security, environmental sustainability, and AMR. Project team members also participate in the launch events for the Climate Change Impacts on AMR Using a Planetary Health Framework (CLIMAR) working groups, including the Biosphere, Land System Change, Biogeochemical Flows, and Nature Working Groups, contributing scientific insight to cross-sector discussions on environmental change and AMR. Outputs from a workshop focused on updating the Scottish One Health AMR Register (SOHAR) were shared with the project, contributing to awareness of the current AMR research landscape in Scotland, and supporting the wider AMR national action plans.
Industry impact. The project supported industry and professional audiences by increasing awareness on antibiotic use and AMR. Engagement included an oral presentation and Q&A panel session at Agriscot 2025 focused on improving antibiotic use on Scottish farms, attended by farmers, veterinarians, and advisors. Project findings were also communicated through a poster presentation at the Royal Highland Show (RHS) 2025 addressing the spread of antibiotic resistance in the livestock farm environment. 
Public impact. Public-facing engagement activities improved understanding of antimicrobial resistance (AMR) as a shared animal, human, and environmental challenge. Research findings and engagement were communicated through farming-focused at national agricultural events, include Agriscot and RHS 2025. 
Academic impact. The project has delivered academic impact by generating new insights on AMR across agricultural systems. Findings were shared with the research community through poster presentations at the British Society for Antimicrobial Chemotherapy (BSAC) Winter Conference 2025 and through engagement in the Edinburgh AMR Forum workshop associated with the UKRI transdisciplinary AMR call, supporting methodological exchange and cross-disciplinary collaboration. Project team member attended an eDNA workshop held on December 2025 at the James Hutton Institute. The project has also contributed to a cross-project collaboration focused on ecosystem-scale analysis of pathogen transmission. Samples and data generated within this project, including soil, animal faecal, and biosolids samples, have been supplied to SRUC as part of an ecosystem case study developed under SRUC-B6-1.
 

O1.3. Genes from microorganisms, such as bacteria, that provide resistance to antimicrobials, can be collectively called the ‘resistome’. The first step in assessing the resistome was to validate methods for DNA extraction from the range of samples collected from both farms: soil, faecal, farm septic tank, farm water streams, and organic fertilisers. DNA extractions were successfully carried out, yielding high-quality genomic DNA (D7). To ensure robust analysis, a DNA pooling strategy was implemented, with three biological replicates per sample. This approach resulted in a total of 144 soil sample pools, 46 faecal sample pools, 36 water sample pools, 6 septic tank sample pools from Farm 1, and 6 DNA samples from each type of organic fertiliser. Resistance gene profiles of the samples were obtained using a customised qPCR array obtained from Resistomap Oy. The presence of 54 resistance genes, selected following literature review of UK AMR studies, based on their relevance to antimicrobial resistance and their association with human activity was determined. 

O3.1; O3.2. In consultation with BioSS and JHI, a pooling strategy was discussed for soil samples to analyse antimicrobials and heavy metal residues. These pooled samples will be analysed in Year 4. (D14, D15 M5). O2.8. Whole-genome sequencing of the first set of multi-drug resistant (MDR) isolates (n= 13) was completed. The results showed that these bacteria carried the presence of between zero to fourteen different resistance genes. Most of them had at least one gene encoding tetracycline resistance, a common antimicrobial drug used in animals and humans. Interestingly, one isolate contained four distinct tetracycline resistance genes, including two mosaic genes which evolved in the past 25 years following recombination of older forms of tetracycline resistance genes (O2.7, D10). Work will continue to complete analysis of the WGS data on the remaining 57 sheep isolates and compare the presence of the same genes in the same bacteria from different sources, and in different bacterial isolates. There have been no deviations from the original work-plan.

Impact

Policy: By improving understanding about the use of different fertilisation practices for grasslands and AMR transmission in farming, this work directly informs evidence-based policies to safeguard animal and human health, ensuring sustainable agricultural practices for the future. The project was presented at the Shaping the Science for Scotland’s Food Future conference (K6), which brought together 160 delegates, including Emma Harper, MSP, Co-convener of the Cross-Party Group on Rural Policy, and Arianne Burgess, MSP. The event provided valuable opportunities for engagement between scientists and policymakers throughout the day.

Industry, Public and Professional: The project was featured in the OnFARM Scottish Farming Podcast, part of a four-part series in partnership with SEFARI Gateway, Scotland’s Centre of Expertise for knowledge exchange and innovation. The episode highlighted AMR research in farming, promoting sustainable livestock management and responsible antimicrobial use. With 324 downloads, it has helped raise awareness among farmers and policymaker. The project objectives were highlights in the SEFARI blog “Understanding How Antimicrobial Resistance Spreads on Farms: The Role of Biosolids, Manure, and Slurry”. It emphasizes the project’s goal of understanding AMR transmission and promoting best practices to protect animal and human health. Since November 15, 2024, it has received 199 views from 80 active users, linking research findings to real-world agricultural and public health concerns. Engagement activities at the Royal Highland Show 2024, provided an opportunity to discuss the project aims and scope with farmers, scientists and general public (K7). Project progress was presented in the annual B6 topic level meeting (K7).

Year 2 highlights:

• Extensive sampling: Soil, animal faeces, water, and septic tank samples were collected from two research farms, adding almost 700 new samples to the project. These are being stored for detailed genetic and chemical analyses in later years.

• Tracking resistant bacteria: More than 800 E. coli strains were isolated and stored, ready for testing to assess levels of antibiotic resistance and multidrug resistance.

• Resistance findings: From sheep samples, 200 bacterial isolates were recovered, representing 53 different species. Many showed high levels of resistance, with some resistant to as many as 11 out of 12 antibiotics tested. Resistance was particularly common to tetracycline, trimethoprim, colistin sulphate, and kanamycin, though all isolates remained sensitive to chloramphenicol.

• Knowledge exchange: Results were shared with farmers and stakeholders at the Royal Highland Show and the annual B6 research meeting.

The project remains on track and is already providing vital insights into how fertiliser practices may influence the spread of AMR.

This project is investigating how different pasture fertilisation practices in Scotland affect this risk, with the aim of identifying the best farming practices to reduce the spread of resistant bacteria through the food chain.

Year 1 progress:

• Study sites and treatments: Research is taking place at two Scottish farms using different fertilisers, thermal-treated sludge pellets, animal slurry, manure, and inorganic fertiliser, alongside untreated controls. A management plan and sampling protocols were developed with BioSS.

• Sampling: Over 860 samples were collected from soils, sheep faeces, watercourses, and farm septic tanks. These will be analysed for resistance genes, heavy metals, and antibiotic residues in later years.

• Bacteria analysis: More than 1,200 E. coli strains were isolated from soils and faeces to test for resistance. In addition, resistant bacteria from sheep faeces were identified, including known species and eight potentially novel strains.

• Knowledge exchange: Findings were shared at the Royal Highland Show and at the annual B6 topic meeting.

All Year 1 objectives were achieved, keeping the project firmly on track. Ultimately, the results will help shape safer, more sustainable grassland management practices that reduce the risk of AMR spreading from farm to fork.