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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 37oC, 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 Sischo1, DVM, PhD

Identifying source and dissemination pathways of antimicrobial resistance on dairies

2.2 1.9

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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*

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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.