Reservoirs and Within-Host Dynamics of Antibiotic Resistance Associated with Small-Scale Poultry Production in Northwest Ecuador Open Access

Braykov, Nikolay Petrov (2015)

Permanent URL: https://etd.library.emory.edu/concern/etds/bc386j86f?locale=en
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Abstract

Small-scale poultry production that includes non-therapeutic use of antibiotics is becoming increasingly common in developing countries, contributing to an environmental reservoir of antibiotic-resistant (AR) bacteria and posing a threat to human health. We conducted a multifactorial experiment in a poultry farming facility to assess the effect of non-therapeutic antimicrobial use and high stocking density on the within-host dynamics of resistant and susceptible Escherichia coli in broiler chickens. To validate conclusions from the experiment, we used data from an observational study in 17 remote communities in Northwest Ecuador, which allowed to assess the potential for transmission of AR between different ecological reservoirs, including 'production birds' (broiler chickens and laying hens raised for sale), coop surfaces and soil, household soil and drinking water. In both studies we found high levels of resistance associated with production birds, and evidence suggesting that AR strains associated with poultry production have a source external to our study system. The effect of management practices on the ecology of AR in E. coli was limited.


Table of Contents

Acknowledgements................................................................................................................................................................ 6

Chapter I: Environmental Reservoirs of Antibiotic Resistance in 17 Village Communities - an Observational Study 1

CHAPTER SUMMARY.................................................................................................................................................... 1

INTRODUCTION................................................................................................................................................................. 3

METHODS............................................................................................................................................................................... 5

Study area........................................................................................................................................................................ 5

Ethnography.................................................................................................................................................................. 5

Household surveys................................................................................................................................................... 6

Poultry feed samples.............................................................................................................................................. 6

Sample collection & laboratory analysis................................................................................................ 7

Poultry samples........................................................................................................................................................... 7

Environmental samples....................................................................................................................................... 8

Sample processing.................................................................................................................................................... 9

Statistical analysis.................................................................................................................................................. 11

RESULTS............................................................................................................................................................................... 14

Quantitative characterization of poultry production............................................................. 14

Qualitative characterization of poultry production................................................................. 14

Chemical analysis of commercial poultry feed............................................................................ 16

Sample characterization................................................................................................................................... 16

Custom susceptibility breakpoints......................................................................................................... 18

Antibiotic resistance in poultry samples........................................................................................... 21

Antibiotic resistance in environmental samples........................................................................ 28

Cluster analysis of resistance patterns across all sample sources............................. 30

DISCUSSION....................................................................................................................................................................... 32

Study limitations and avenues for future research................................................................ 36

Chapter II: Impact of Antibiotic Exposure and Stocking Density on the Genotype and Resistance Phenotype of E. Coli Shed by Broiler Chickens - a Factorial Experiment................................................................................................................................................................................ 39

CHAPTER SUMMARY................................................................................................................................................. 39

INTRODUCTION.............................................................................................................................................................. 41

METHODS............................................................................................................................................................................ 45

Study design............................................................................................................................................................... 45

Sample processing................................................................................................................................................. 46

Antibiotic susceptibility testing.................................................................................................................. 46

Genotyping................................................................................................................................................................... 47

Statistical analysis.................................................................................................................................................. 47

RESULTS............................................................................................................................................................................... 50

Sample characteristics....................................................................................................................................... 50

Prevalence of resistance over time and treatment group, by flock.......................... 51

Prevalence of resistance over time and stocking density, by flock........................... 55

Effects of pre-colonization with integrase 1 on pre-slaughter resistance.......... 58

Phenotypic and genotypic dynamics over time, by trial..................................................... 60

Diversity dynamics by treatment and cage density............................................................... 64

Diversity dynamics by presence of resistance genes............................................................ 65

Genotypic dissimilariy between experimental trials on different days................ 67

Genotypic dissimilariy between sampling days.......................................................................... 67

Genotypic dissimilariy between treatment groups on different days................... 67

Genotypic dissimilariy between stocking density groups on different days.... 67

Genotypic dissimilarity in a multivariable model...................................................................... 68

DISCUSSION....................................................................................................................................................................... 69

References................................................................................................................................................................................ 75

Supplementary material................................................................................................................................................ 89

LIST OF EXHIBITS

Table 1: Counts of E. coli isolates, by sample type collected from 17 villages in Esmeraldas Province, Ecuador, 2010-2013. Production birds include broilers and laying hens.................................................................................................................................................. 18

Table 2: Official and custom susceptibility breakpoints. Custom breakpoints used to derive categorical resistance profiles for all N=3860 isolates used in the analysis............................................................................................................................................................................... 20

Table 3: Percentage of E. coli isolates resistant to a panel of 12 antibiotics, by sample type 23

Table 4: Number of chickens, samples and isolates in each flock.................................................. 51

Table 5: Multivariable analysis of dissimilarity (Adonis) model................................................... 68

Figure 1: Kernel density estimates of inhibition zones and categorical interpretation of susceptibility tests of E. coli isolates from production (red) and household (blue) bird samples;.................................................................................................................................. 24

Figure 2: Categorical resistance of E. coli isolates from production birds (broilers and laying hens), by age of bird 26

Figure 3: Antibiotic resistance in E. coli isolates from production birds (broilers and laying hens) of age <2 weeks by purchase location27

Figure 4: Kernel density estimates of inhibition zone profiles and categorical interpretation of susceptibility tests for E. coli isolates from household and coop surfaces, and production birds (broilers and laying hens)..................................................... 29

Figure 5: Hierarchical clustering by sample type and location........................................................ 31

Figure 6: proportion of resistant isolates over time, by trial............................................................ 53

Figure 7: proportion of resistant isolates over time and across treatment groups, by trial 54

Figure 8: proportion of resistant isolates over time and across stocking density groups, by trial 56

Figure 9: forest plots of univariate associations of phenotypic resistance and carriage of resistance genes 59

Figure 10: proportion of resistant isolates over time and across stocking density groups, by trial 61

Figure 11: Dynamics of genotypic diversity within treatment groups...................................... 64

Figure 12: Dynamics of genotypic diversity within stocking density groups....................... 65

Figure 13: Dynamics of genotypic diversity, by presence of resistance genes..................... 66

Supplementary Exhibits

Supplementary table 1: Chemical analysis of feed samples of the most commonly sold brand of feed (Nutril) purchased at a veterinary store in the large regional center.......................................................................................................................................................... 89

Supplementary table 2: oligonucleotide primers used in the amplification of resistance genes 98

Supplementary figure 1: Monthly census of production birds (broilers and laying hens) in 15 study sites 90

Supplementary figure 2: Histograms of inhibition zones for production bird E. coli isolates and the fitted two-component mixture models91

Supplementary figure 3: Kernel density estimates of inhibition zone profiles and categorical interpretation of susceptibility tests for E. coli isolates from household drinking water samples, by farming history of household......................... 94

Supplementary figure 4: Kernel density estimates of inhibition zone profiles and categorical interpretation of susceptibility tests for E. coli isolates from household soil samples, by farming history of household..................................................... 95

Supplementary figure 5: Kernel density estimates of inhibition zone profiles and categorical interpretation of susceptibility tests for E. coli isolates from household drinking water samples, by intensity of farming in the year prior to sampling96

Supplementary figure 6: Kernel density estimates of inhibition zone profiles and categorical interpretation of susceptibility tests for E. coli isolates from household soil samples, by intensity of farming in the year prior to sampling... 97

Supplementary figure 7: forest plots of bivariate models for the proportion of resistant isolates over time and across stocking density groups, by trial............................................................................................................................................................................................................ 99

Supplementary figure 8: forest plots of bivariate models for the proportion of resistant isolates over time and across treatment groups, by trial100

Supplementary figure 9: proportion of resistant isolates over time and across stocking density groups, by trial 101


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