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Research Project: Characterizing Antimicrobial Resistance in Poultry Production Environments


2018 Annual Report

1. Characterize the antimicrobial resistance (including the resistome and mobilome) of pathogens and commensals from agroecosystems and identify factors that contribute to their spread. 2. Optimize methods for detecting bacteria with antimicrobial resistance and develop tools to assess biological and environmental factors that contribute to their persistence and transmission in agroecosystems.

Goals will be accomplished by identifying environmental factors associated with the persistence of resistance in microbial populations from poultry production environments (feces, litter, water and feed) that are under different management regimes (conventional, pastured and organic). Traditional and advanced molecular analyses will be used to characterize phenotypic and genotypic properties of populations including the resistome (i.e., the collection of all resistance genes in both pathogenic and non-pathogenic bacteria) and mobilome (genetic elements associated with transfer of antibiotic resistance (AR) genes between bacterial genera and species). Data to understand the effect of environmental factors on horizontal transmission of resistance genes between populations is urgently needed to inform risk assessment models and identify targets for mitigation studies. Research conducted as part of this project will evaluate the effect of nutrients, disinfectants and antibiotic concentrations on the rate of horizontal gene transmission by plasmids to pathogens like Salmonella enterica serovar Heidelberg and Enterococcus faecalis. Separate studies will investigate molecular properties of plasmids that are mobilized from the complex poultry litter microbiota to new bacterial recipients and the effect of environment on the rate of horizontal gene transfer. Quantitative data are needed to establish functional relationships between the persistence/growth of bacteria with antimicrobial resistance and management/environmental factors. To this end, methods will be optimized for sensitive, specific detection of AR targets in complex ecosystems and studies will be conducted to quantify (1) the effect of poultry litter re-use on the development and persistence of bacteria with antibiotic resistance and (2) to evaluate the effect of residual antibiotics and manure nutrients on the native soil AR microbiota and the manure-associated AR microbiota. Functional relationships will be established to describe the ecology of antimicrobial resistance under environmentally relevant conditions and to establish the effect of these management strategies and residual antibiotics on native and litter-associated microbial flora and on AR pathogens of importance to humans.

Progress Report
An on-farm longitudinal sampling was initiated to evaluate the development, distribution and persistence of pathogens and bacteria with antimicrobial resistance in broiler production facilities. Initial studies were conducted to optimize procedures for sample collection and bacterial isolation. Methods are being used in collaborative studies to monitor targeted populations in litter used as bedding for four successive flocks across 12 sites within each of four broiler production facilities. Flocks are being followed from start-up (i.e., placing new peanut hulls as bedding) through clean out (i.e., four broiler production cycles). Litter is being sampled at the beginning of each production cycle (7 to 10 days after broiler placement) and near cycle finish (4-6 weeks). Campylobacter, Salmonella, Escherichia coli (E. coli), Staphylococcus and Enterococcus are being isolated and characterized. The popularity of farmers’ markets has increased in recent years because of an increased interest in local foods and organic produce. As part of this two-year project, we are conducting longitudinal sampling to address the microbiological quality of samples taken from four organic farms using biological soil amendments of animal origin (BSAAO) for growing squash. We are monitoring for the abundance of indicator bacteria (E. coli, coliform and Enterococci) and pathogens (Salmonella, Listeria, and Campylobacter) in BSAAO, soil, irrigation water (IW), and surface water (SW) samples collected bi-monthly during the produce growing season. In addition, squash samples are being collected on farm and at farmer’s market at harvest. Preliminary results suggest that pathogens are absent from BSAAO and therefore no pathogen contamination has been detected in soil, water or produce samples. Interestingly, produce from farmers market was found in first year sampling to have levels of indicator bacteria (E. coli, coliform and Enterococci) that were several orders of magnitude above those found in farm samples (although no on-farm produce was taken). This year we are more intensively sampling produce on-farm and at the farmers market. We are continuing characterization of isolates obtained as part of the study. This research is partially funded by a grant from the Food and Drug Administration (FDA). Completed collaborative research with United States Department of Agriculture (USDA), Animal Health and Plant Health Inspection Service (APHIS) National Animal Health Monitoring System (NAHMS) to determine the prevalence and antimicrobial resistance patterns of foodborne pathogens and commensals from fecal samples from cow-calf operations in the United States. This work is partially funded by an agreement with the USDA APHIS NAHMS. In the last year, we conducted two collaborative in vivo poultry studies to evaluate the contribution of the poultry intestinal microbiome to the resistome and mobilome of Salmonella Heidelberg. The goals of the studies were to evaluate the effect of route of exposure to S. Heidelberg on the rate of colonization of young chicks and to assess the resistome and mobilome of S. Heidelberg recovered from ceca of 14 day old chicks established on fresh or used poultry litter. The studies found that there were significant differences in the rate of colonization based on method of inoculation (higher rates in birds inoculated by oral gavage or via seeder birds than via intra cloacal inoculation). Sequencing and resistome/mobilome analysis of recovered S. Heidelberg strains is in process. Livestock manures provide important functions in field application, however, metals entering the environment through manure application (i.e., As, Cd, Cu, and Zn) may select for bacteria with genes for resistance to metals that are co-located on plasmids containing genes for antibiotic resistance (AR). A new study was initiated to evaluate the associations between residual metals, metal resistance genes and soil amendment with flue gas desulfurized gypsum (FGDG) on the occurrence, persistence and mitigation of AR in an integrated crop-livestock production system. The efficacy of remediation of metals, metal and antibiotic resistance by applying a soil amendment used to improve soil quality, FGDG, to affect both the microbiota and nutrient/metal availability. To evaluate these populations we are now developing quantitative real-time polymerase chain reaction (PCR) assays to target metal resistance genes and will also use previously designed qPCR assays to target antimicrobial resistance genes. In addition we will be applying metagenomic analyses to capture physical DNA proximity via in vivo cross-linking to enrich and fix DNA sequences that are closely associated. Soil samples (5 cm depth) will be taken from an existing continuous corn with rye & Austrian Winter Pea mix winter cover cropping system study that has been in progress for 3 years. This project is partially funded through an ARS environmental AR research grant. Recent studies have highlighted the large and diverse microbial flora present in the poultry microbiome. These populations may serve as reservoirs for the persistence and/or transmission of antibiotic resistance (AR) elements (genes, plasmids, transposons) within poultry production systems. Studies suggest that gram-positive organisms rather than gram-negatives may be important reservoirs of antimicrobial resistance in agro-ecosystems. These studies were conducted to (1) optimize methods for capturing mobile genetic elements moving between poultry litter microbiota and (2) isolate and characterize transferrable resistance elements mobilized between PL flora (donor) and Enterococcus faecalis JH2-2 (recipient) using long-read sequencing technology. Three types of poultry litter were evaluated: Conventional production (three sources), backyard litter (three sources), and organic litter (one source). Antimicrobial susceptibility testing of transconjugates suggests that between two and six resistance genes were mobilized from litter microbiota to the recipient. Work continues to annotate sequences from captured plasmids. This work was partially funded by an ARS environmental AR grant.

1. ColE1 plasmids contribute to the fitness of Salmonella Heidelberg in poultry litter. Salmonella enterica subsp. enterica serovar Heidelberg (S. Heidelberg) is a clinically-important serovar, linked to food-borne illness, and commonly isolated from poultry. Investigations of a large, multistate outbreak in the USA in 2011 identified poultry litter (PL) as an important extra-intestinal environment that may have selected for specific S. Heidelberg strains. In this study, ARS researchers at Athens, Georgia, performed a series of controlled laboratory experiments which assessed the evolution of two S. Heidelberg strains (SH-2813 and SH-116) in PL previously used to raise 3 flocks of broiler chickens. Whole genome sequencing was performed on 86 isolates recovered after 0, 1, 7 and 14 days of evolution in PL. Only strains carrying an IncX1 (37kb), 2 ColE1 (4 and 6kb) and 1 ColpVC (2kb) plasmids survived more than 7 days in PL. Competition experiments showed that acquisition of these plasmids increased fitness and was associated with an increased copy number of IncX1 and ColE1 plasmids.

2. Characterization of environmental Escherichia coli (E. coli) for use as surrogates for manure-borne pathogens. Non-pathogenic bacterial strains are used in place of foodborne pathogens to assess control measures for pathogen mitigation. However, generic laboratory strains may not possess properties important to pathogen persistence in non-enteric environments. ARS researchers at Athens, Georgia, assessed 63 environmental E. coli strains from poultry, swine, dairy and surface waters for genotypic (phylotype, virulence, adhesion and biofilm genes) and phenotypic (biofilm formation, growth rate, lettuce adhesion and cell surface properties) properties similar to those of the produce associated foodborne pathogens E. coli O157:H7 and Salmonella enterica serotype Typhimurium (S. Typhimurium). Selected environmental E. coli isolates were non-pathogenic serotypes that lacked virulence genes and exhibited cell surface characteristics (zeta potential, hydrophobicity and exopolysaccharide composition) that were within 15% of values for S. Typhimurium. Survival of environmental E. coli (from poultry litter or surface water), E. coli O157:H7, Salmonella and a generic E. coli quality control (QC) strain on lettuce plants and in soils (sandy loam and silt loam) with or without poultry litter amendments was compared. Results suggest that the environment may provide a valuable resource for selection of superior bacterial surrogates for some foodborne pathogens.

Review Publications
Bandy, A., Cook, K.L., Fryar, A., Polk, J. 2018. Use of molecular markers to compare Escherichia coli transport to traditional groundwater tracers in epikarst. Journal of Environmental Quality. 47:88-95. doi:10.2134/jeq2017.10.0406.
Durso, L.M., Cook, K.L. 2018. Antibiotic resistance in agroecosystems: A One Health perspective. EcoHealth. 14:1-6.
Bolster, C.H., Brooks, J.P., Cook, K.L. 2018. Effect of manure application rate and rainfall timing on the leaching of antibiotic-resistant bacteria and their associated genes. Water, Air, and Soil Pollution.
Cox Jr, N.A., Cosby, D.E., Thippareddi, H., Ritz, C.W., Berrang, M.E., Jackson, J.S., Mize, S.C., Kumar, S., Howard, A.K., Rincon, A.M., Ukidwe, M., Landrum, M., Frye, J.G., Plumblee Lawrence, J.R., Hiott, L.M., Jackson, C.R., Hinton Jr, A., Cook, K.L. 2018. Incidence, species and antimicrobial resistance of naturally occurring Campylobacter isolates from quail carcasses sampled in a commercial processing facility. Journal of Food Safety. 2018:38:e12438.
Cook, K.L., Givan, E., Mayton, H., Parekh, R.R., Taylor, R., Walker, S. 2017. Using the agricultural environment to select better surrogates for foodborne pathogens associated with fresh produce. International Journal of Food Microbiology. 262/80-88. doi: 10.1016/j.ijfoodmicro.2017.09.017.