RESEARCH POSTER PRESENTATION DESIGN © 2015 www.PosterPresentations.com The goal of the research was to assess an approach to determine source and dissemination pathways for antimicrobial resistant (AMR) Escherichia coli in dairy environments. If the source of AMR bacteria on dairies and the path of transmission can be identified, then steps can be taken to limit its generation and spread into niches that would not otherwise be populated by AMR E. coli, including into the human population. The objectives of this project were to determine the intensity of sampling needed to measure diversity of isolate resistance and assess transmission dynamics within a dairy farm. Introduction Sampling This study is field-based; working with commercial dairy herds that maintain both a milking herd and rear their replacement animals on the same physical site and house at least 200 preweaned calves. For this pilot study, all isolates were from fecal samples taken from a single commercial dairy herd in central Washington. We defined 8 production niches based on housing and function: preweaned calves, weaned calves, breeding age heifers , early lactation (fresh) cows, lactating cows, non-lactating (dry) cows, lactating cows to be sold (“do not breed”=DNB), and cows in the hospital pen. Our on-farm sampling target was 9 animals per niche and 4 isolates per animal. A minimum of 3 samples were taken from each pen that housed animals. E. coli isolation We used E. coli as our model bacterium for resistance phenotypes. 1. ~ 0.10 grams of fecal sample diluted to 10 -5 in sterile saline, plated to MAC 2. Incubated 18-24 hours at 37 o C, randomly selected 8 lactose positive colonies from each plate to Columbia blood agar 3. From the blood agar, 4 oxidase test negative and indole test positive isolates were tested for susceptibility to 15 antibiotics 4. Resistance phenotypes were generated by concatenating minimum inhibitory concentration (MIC) results Data analysis For each niche, we assessed phenotypic AMR diversity based on the distribution of profiles within that niche. Biological diversity was quantified by phenotype richness (the number of AMR profiles in each niche). The relationship between sample size and diversity within each niche was modeled with rarefaction curves based on Chao1 estimates, reported with a 95% confidence interval. Materials and Methods Results Discussion In this preliminary study, we examined the resistance phenotypes of E. coli from fecal samples collected from a single commercial dairy herd. Isolates from preweaned calves had the greatest phenotypic diversity and the greatest degree of resistance. All isolates from adult animals had comparable low levels of both diversity and resistance. These results suggest that preweaned calf E.coli isolates are phenotypically distinct from the rest of the dairy, and may be a source for generation of AMR bacteria. Since this is just one sampling from one herd, these findings are far from conclusive. The rarefaction curves for every niche besides the lactating cows are approaching an asymptote, which indicates that the sample sizes were sufficiently large to capture the phenotypic diversity of the population. The sampling size may need to be increased for lactating cows to be representative of diversity in the niche. This sampling method can be used for a larger project that is currently under development to analyze source and dissemination pathways for resistance that will collect more samples across multiple dairies and time points. References Mollenkopf DF, Weeman MF, Daniels JB et al. Variable within- and between-herd diversity of CTX-M cephalosporinase-bearing Escherichia coli isolates from dairy cattle, Appl.Environ.Microbiol. 2012;78: 4552-4560. Andrews JM. Determination of minimum inhibitory concentrations, JAC. 2001;48 Suppl. S1,5-16 Watts, J. (2008). Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals: Approved standard (3rd ed.). Wayne, PA: CLSI CDC 2012, Foodborne Disease Active Surveillance Network (FoodNet): FoodNet Surveillance Report for 2011 (Final Report)., U.S. Department of Health and Human Services, CDC., Atlanta, Georgia. Roberts, C.F. (1959) A Replica Plating Technique for the isolation of Nutritionally Exacting Mutants of a FIlamentous Fungus J. gen Microbiol. 20, 540-548 Mather AE, Matthews L, Dominic JM, et al . An ecological approach to assessing the epidemiology of antimicrobial resistance in animal and human populations. Proc R Soc B. 2012; 279: 1630-1639. USDA-NIFA . 2015-68003-22998 MC Snyder; W Sischo 1 , DVM, PhD Identifying source and dissemination pathways of antimicrobial resistance on dairies 2.2 1.9 0.33 0.56 0.38 0.62 0.22 0.38 8.1 3.42 0.04 0.28 0.29 0.25 0.56 0.08 0 1 2 3 4 5 6 7 8 9 Preweaned Weaned Heifers Fresh Lactating Dry Hospital Late/DNB Production niche E. coli isolate AMR phenotype results Mean no. of phenotypes per sample Mean no. of resistances per isolate* 0 20 40 60 80 100 120 140 160 180 200 1 2 3 4 5 6 7 8 9 Expected number of phenotypes Number of samples Chao 1 Mean Chao 1 95% CI Lower Bound Chao 1 95% CI Upper Bound 0 10 20 30 40 50 60 70 80 90 1 2 3 4 5 6 7 8 9 Chao 1 Mean Chao 1 95% CI Lower Bound Chao 1 95% CI Upper Bound 0 2 4 6 8 10 12 14 16 18 1 2 3 4 5 6 7 8 9 Chao 1 Mean Chao 1 95% CI Lower Bound Chao 1 95% CI Upper Bound 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 1 2 3 4 5 6 7 8 9 Chao 1 Mean Chao 1 95% CI Lower Bound Chao 1 95% CI Upper Bound 0 50 100 150 200 250 300 1 3 5 7 9 11131517192123252729313335373941 Chao 1 Mean Chao 1 95% CI Lower Bound Chao 1 95% CI Upper Bound 0 2 4 6 8 10 12 1 2 3 4 5 6 7 8 Chao 1 Mean Chao 1 95% CI Lower Bound Chao 1 95% CI Upper Bound 0 10 20 30 40 50 60 70 80 1 2 3 4 5 6 7 8 9 10 11 12 13 Chao 1 Mean Chao 1 95% CI Lower Bound Chao 1 95% CI Upper Bound 0 0.5 1 1.5 2 2.5 1 2 3 4 5 6 7 8 9 Chao 1 Mean Chao 1 95% CI Lower Bound Chao 1 95% CI Upper Bound Preweaned rarefaction curve Weaned rarefaction curve Heifer rarefaction curve Fresh rarefaction curve Lactating rarefaction curve Late/DNB rarefaction curve Dry/Close-up rarefaction curve Hospital rarefaction curve Fig. 1: A pen of dairy cows feeding Table 1: Antibiotics tested Antibiotic Resistance concentration Ampicillin 8 μg/ml Chloramphenicol 8 μg/ml Sulfisoxazole 256 μg/ml Kanamycin 16 μg/ml Amikacin 16 μg/ml Trimethoprim/Sulfamethoxazole 2/38 μg/ml Streptomycin 32 μg/ml Tetracycline 4 μg/ml Amoxicillin/Clavulanic Acid 8/4 μg/ml Naladixic Acid 16 μg/ml Gentamicin 4 μg/ml Ceftiofur 2 μg/ml Cefotaxime 1 μg/ml Cefoxitin 8 μg/ml Chloramphenicol 8 μg/ml Ciprofloxacin 0.12 μg/ml Acknowledgments This project is supported by the USDA National Institute of Food and Agriculture Grant No. 2015-68003- 22998 The author would like to thank Dr. Bill Sischo, Lindsay Tippett, Stephanie Wright, Emily Hudson, and Russell McClanahan for their guidance and insight in this project.