Animal Feed Components and The Impacts of Land-applying Manure on Soil Bacterial Biodiversity and Antibiotic Res

1. Animal Feed Components and The Impacts of Land-applying Manure on Soil Bacterial Biodiversity and Antibiotic Resistance Amy R. Sapkota, PhD, MPH University of Maryland College Park School of Public Health

2. Background • Animal-based food products constitute a large proportion of the U.S. diet • U.S. per capita consumption of total meats is 90.5 kg/year (199 lbs/year) (USDA, 2005) • Quality of food products is directly related to animal feeding practices • Farms and feeding practices have undergone changes • Small family farms  Concentrated animal feeding operations • Corn or grass diet  Complex mixtures • U.S. feed industry has grown to be the largest feed producer in the world • >120 million tons of feed • $25 billion industry Source: USDA, 2005. http://www.ers.usda.gov/data/

3. What is in Animal Feed and Does it Affect Human Health? • Previously, no comprehensive, peer-reviewed source existed regarding specific feed ingredients and their potential impacts on human health • Existing sources focused on one feed contaminant and one health effect

4. Methods • We reviewed the literature and FDA, USDA, and CDC databases: • To summarize existing data on current feed production practices and feed ingredients • To describe biological, chemical and other etiologic agents that have been detected in feed • To evaluate evidence whether current feeding practices may lead to adverse human health effects • To identify data gaps that prevent comprehensive assessments of human health risks associated with feed • Compiled a review article targeted to public health researchers

5. What is in animal feed? Source: Sapkota et al. 2007. EHP 115;5:663-670.

6. Etiologic Agents Detected in Feed and Potential Human Health Effects Source: Sapkota et al. 2007. EHP 115;5:663-670.

7. From Animal Feed Ingredients to Human Health

8. Take-home Messages • No comprehensive nationwide animal feed surveillance system to monitor: • The amounts and types of specific feed ingredients • The levels of contaminants in feeds and food products • Human health effects data are not appropriately linked to the minimal amount of surveillance data that do exist • Difficult to pinpoint the extent to which human health risks are associated with animal feed We need increased federal funding and surveillance from “farm to fork”

9. The Impacts of Land-applying Manure on Soil Bacterial Biodiversity and Antibiotic Resistance

10. Background • 500 million to 1 billion tons of animal manure is land-applied each year • Manure contains nutrients and contaminants, including antibiotic residues and resistant bacteria • When swine manure is land-applied, resistance genes can be transferred between swine-associated bacteria and soil bacteria, perpetuating the spread of resistance (Agerso 2005; Jensen 2002) • We hypothesized that antibiotic selective pressures resulting from the land-application of swine manure could modify soil bacterial biodiversity as well Sources: Agerso et al. 2005. Appl Env Microbiol 71:7941-7947; Jensen et al. 2002. Env Int 28:487-491.

11. Study Objectives • To use traditional culture techniques and metagenomic methods to explore antibiotic resistance and bacterial diversity in agricultural soils amended with swine manure compared to control soils

12. Methods Spectinomycin Sapkota et al. [In preparation].

13. Methods: Sample Analysis • Culture techniques: • Culturable bacteria (susceptible and resistant) were isolated from each sample and identified by PCR and sequencing • Isolates were tested for the aad(A) resistance gene and the resulting genes were sequenced • Metagenomic methods: • Total DNA was extracted from each sample • 16S rRNA genes in each sample were amplified, purified, and labeled using an in vitro transcription method • RNA was hybridized to a 16S rRNA-based taxonomic microarray • Microarray results were analyzed by principal components analysis • The aad(A) resistance gene was quantified using real-time PCR, cloned and sequenced

14. Bacterial Biodiversity in Culturable Isolates Sapkota et al. [In preparation].

15. Bacterial Biodiversity in Metagenomic DNA Samples Metagenomic microarray approach revealed more soil biodiversity, in general, and more differences between manure-amended and control soils. Sapkota et al. [In preparation].

16. Spectinomycin Resistance in Culturable Isolates The aad(A) resistance gene was only detected in isolates from manure samples and soils impacted by manure.

17. aad(A) Resistance Gene in Metagenomic DNA Samples • Similar to results observed in culturable isolates • Higher copy numbers of the aad(A) resistance gene were in soils impacted by manure • Currently, we are analyzing all aad(A) resistance gene sequences from culturable bacteria and metagenomic samples to understand diversity in the gene Samples Standards NC

18. Conclusions • Microarray results indicate that some differences exist in bacterial biodiversity between soils amended with swine manure and control soils • Spectinomycin-resistant bacteria were isolated only from manure samples and soils recovered from pig grazing fields • The aad(A) resistance gene was only detected in manure samples and soils impacted by manure • The application of swine manure to soils impacts both antibiotic resistance and bacterial biodiversity in soil

19. Funding:Center for a Livable Future, Johns Hopkins Bloomberg School of Public Health École Centrale de Lyon Colleagues:Timothy Vogel, Pascal Simonet, Elizabeth Navarro, Maude David, Jean-Michel Monier, Audra Nemir, Benoit Remenant, Saliou Fall, Sandrine Demaneche JHU Colleagues:Kellogg Schwab, Ellen Silbergeld, Robert Lawrence, Shawn McKenzie, Polly Walker, Lisa Lefferts Acknowledgements