Non Technical Summary
This project investigates how the relationship between the community of microbes in food animals respond and adapt to exposure to heavy metals and how resistance to antibiotics and resistance to heavy metals are co-occurrent.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
307	3499	1170	10%
311	3499	1040	10%
311	3499	1070	10%
311	3499	1100	10%
712	3299	1040	10%
722	3299	1170	10%
722	3499	1170	10%
722	3910	1040	10%
722	3910	1100	10%
722	3910	1170	10%

Knowledge Area
311 - Animal Diseases; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 722 - Zoonotic Diseases and Parasites Affecting Humans; 307 - Animal Management Systems;

Subject Of Investigation
3499 - Dairy cattle, general/other; 3299 - Poultry, general/other; 3910 - Cross-commodity research--multiple animal species;

Field Of Science
1040 - Molecular biology; 1070 - Ecology; 1100 - Bacteriology; 1170 - Epidemiology;

Goals / Objectives
Enhance surveillance and monitoring of antibiotic resistance and develop improved diagnostic tests. Develop and evaluate interventions (including alternatives to antibiotics) that reduce antimicrobial resistance in food production systems. Quantify animal health, public health, social, economic, and environmental impacts of antimicrobial interventions in food production systems. Determine the ecology and mechanisms involved in resistance and transmission of resistance.

Project Methods
All objectives will be attained by studies leveraging next generation sequencing technologies.Sequencing of samples from various body sites collected from food animals (cattle, poultry) as well as samples collected from animal environment (e.g. poultry litter) will be performed. Shotgun sequencing, targeted metagenomic sequencing, and a combination of targeted and untargeted methods will be applied to better understand the co-occurrence and development of antibiotic and heavy metal resistance, as well as taxonomic profile differences associated with increased resistance.Sequencing will be performed in core facilities, data analysis will be performed with open source software such as R, Qiime2 and MG-Rast, and data will be made publicly available upon publication of results.Specifically, methods for each objective are described below:(1) Enhance surveillance and monitoring of antibiotic resistance and develop improved diagnostic tests.A targeted sequencing panel is being developed and benchmarked against shotgun sequencing data collected from animals exposed to heavy-metal environmental contamination and used as ground-truth. Once the comprehensive panel is finalized, massive parallel amplicon sequencing of AMR genes of interest will be performed in samples collected from poultry houses and food samples, with the objective of identifying a large amount of AMR genes in a more comprehensive and cost-effective way when compared to shotgun sequencing or testing of bacterial isolates. Briefly, total genomic DNA will be extracted from each sample, AMR gene amplification will be carried out using the panel developed, and amplicons will be sequenced using an illumina sequencer. FASTA files will be blasted against a curated AMR genes database to determine presence/absence as well as relative abundance of various AMR genes present in the amplicon-based sequencing panel.(2)Determine the ecology and mechanisms involved in resistance and transmission of resistance.Samples from bovines present in an area that was exposed to environmental heavy metal contamination, as well as samples from a farm from the same state but that did not share the contaminated river basin were collected, had genomic DNA extracted, and sequenced in an Illumina platform using untargeted (shotgun) sequencing. Sequencing files were processed in MGrast and compared to Megares, a custom antimicrobial resistance database. Surveys and clinical examinations were administered at the time of sample collection and those data will be used to investigate the differences in resistance profile in animals exposed to heavy-metal contaminated water versus animals that were not exposed to heavy metal contamination.(3)Develop and evaluate interventions (including alternatives to antibiotics) that reduce antimicrobial resistance in food production systems.The use of intrauterine concentrated sugar solution will be evaluated as an alternative to subcutaneous use of ceftiofur in dairy cows diagnosed with clinical metritis. Briefly, animals will be blocked by parity and milk production and randomly allocated into alternative or antibiotic treatment groups. Follow up examination and sample collections will occur at days 0, 7, and 14 after diagnosis of disease. Cure rates will be calculated, and data on fertility of animals will be collected throughout the lactation to investigate potential effects of this alternative treatment in reproductive performance. At each exam date, vaginal swabs will be collected, genomic DNA extracted, and AMR genes will be assessed via amplicon-panel as described for objective 1.(4)Quantify animal health, public health, social, economic, and environmental impacts of antimicrobial interventions in food production systems.Impacts on cow fertility and milk production will be collected in a study investigating the use of an alternative treatment for clinical metritis, treatment cure rates, shifts on the microbial profile and resistome will be determined for each sampling point to determine the impact of such intervention on animal productivity and resistome profile. Sequencing methods are as described in objective 3: at each exam date, vaginal swabs will be collected, genomic DNA extracted, and AMR genes will be assessed via amplicon-panel.