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Research Project: Characterizing Antimicrobial Resistance in Poultry Production Environments


2019 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
Completed a study to determine the limit of quantification (LOQ) and detection (LOD) of Salmonella enterica (serovars Heidelberg, Typhimurium, Enteritidis and Kentucky), Campylobacter (multiple jejuni and coli strains), E. coli (n = 4 strains) and Enterococcus (faecalis, faecium, hirae and durans) in broiler litter. These strains were inoculated together at equal concentrations (10 – 10^7 cfu/ml) into 1 g of reused litter. Brilliant Green Sulfur Agar, Campy Cefex CHROMagarTM ECC and m-Enterococcus Agar were used for enumeration of Salmonella, Campylobacter, E. coli and Enterococcus, respectively. If no colonies appeared after direct plating then an additional plate was streaked from the eluate and enriched overnight in buffered peptone broth or in Bolton’s broth for 48 h. Based on this method, the LOQ for viable and culturable Salmonella, Campylobacter, E. coli and Enterococcus in 1 g (~90 % dry weight) of reused litter was determined to be ~30, 70, 20 and 140 cfu, respectively. Their corresponding LOD was determined to be 5, 130, 6 and 2 cfu/g dry weight. The result suggests that this method is adequate for the quantification and detection of low levels of pathogens and commensals present in broiler litter. Completed an on-farm longitudinal study to evaluate the development and persistence of pathogens and bacteria with antimicrobial resistance in bedding materials used for raising three successive flock of commercial broilers. Litter (n =240) was sampled at the beginning of each production cycle (7 to 10 days after broiler placement) and near cycle finish (4-6 weeks). Campylobacter, Salmonella, E. coli, Staphylococcus and Enterococcus were quantified, isolated and tested for their susceptibility to antibiotics. Whole genome sequencing of Salmonella and E. coli isolates is completed and sequencing of Enterococcus isolates is underway. Preliminary results suggests that the reuse of the same bedding material over the grow-out of successive flocks aided in the reduction of pathogens including Salmonella and Campylobacter. Salmonella ser. Kentucky and Salmonella ser. Enteritidis were the two major serovars identified in litter and none carried antimicrobial resistance. C. jejuni was the major Campylobacter sp. identified (47/52) and they were susceptible to all drugs tested, while C. coli isolates (4/5) were resistant to tetracycline. The majority of the E. coli (35/56) and Enterococcus (78/86) isolates were resistant to at least one class of antibiotics. Further studies to characterize bacterial isolates that have been sequenced is underway. DNA extraction, water activity and nutrient/cation determination are planned on litter that was stored from this study. Completed a two-year longitudinal study in collaboration with the Food and Drug Administration (FDA) and the University of Georgia (UGA) that addresses the microbiological quality of samples taken from four organic farms using biological soil amendments of animal origin (BSAAO) for growing squash. The BSAAO used by 3 of the 4 farms are from broiler litter. Preliminary results suggest that pathogens are absent from BSAAO and therefore no pathogen contamination was detected in soil, water or produce samples. However, produce collected on-farm harbored indicator bacteria (Enterococci, E. coli and coliforms) that were several orders of magnitude higher than produce that was washed prior to selling at the farmers market. Furthermore, indicator bacteria, ampicillin resistant coliform and tetracycline resistant Enterococcus was significantly higher on the outside (skin) than on the inside of squash (pulp). The presence of indicator bacteria on produce suggests that it is important to carefully clean produce before consuming. Further studies are underway to characterize E. coli, coliforms and Enterococcus isolates recovered from squash. A study to characterize transferrable resistance genes mobilized from various reused broiler litter (donor) to Enterococcus faecalis (marked recipient) was completed. Enterococcus faecalis recipients that acquired resistance to tetracyclines, aminoglycosides, macrolides and phenicols were characterized by whole genome sequencing. Results indicate that antibiotic resistance was conferred by the transfer of mobile elements harboring resistance genes from reused broiler litter microbiota to Enterococcus faecalis. This study demonstrates that broiler litter represents an important extra-intestinal environment that may favor the transfer of antibiotic resistant mobile genes to Enterococcus. A study to evaluate the associations between residual metals, antimicrobial resistance genes and soil amendments with flue gas desulfurized gypsum (FGDG), broiler litter (BL) or fertilizer (NPK) on the occurrence, persistence and mitigation of antimicrobial resistance in an integrated crop-livestock production system was started. Amendments were applied to an existing continuous corn with rye & Austrian Winter Pea mix winter cover cropping system and soil samples were collected before and after application. A targeted gene sequencing approach that enables the detection of antibiotic resistance genes at a higher depth of coverage has been developed and validated. Furthermore, development of a digital qPCR assay that will allow the quantification of 48 genes in 48 samples at once is underway. This PCR assay will target pathogens and antibiotic/metal resistance genes associated with “poultry environments”. Both methods would be used to determine the microbiota and resistome (metal and antibiotic resistant genes) present in amendments and amended soil. So far, DNA extracted from FGDG amendments collected in 2018 was too low for molecular work. Consequently, FGDG amendments will not be analyzed moving forward. Sampling for FY’2019 was completed in April 2019 and samples have been saved appropriately. Extraction of DNA from amended soils and other amendments used has begun.

1. Dynamics between horizontal gene transfer and acquired antibiotic resistance in S. Heidelberg following in vitro incubation in chicken ceca. Salmonella enterica serovar Heidelberg is a clinically important serovar, linked to food-borne illness and among the top 5 serovars isolated from poultry in the USA and Canada. Acquisition of new genetic material from microbial flora in the gastrointestinal tract of food animals, including broilers, may contribute to increased fitness of pathogens like S. Heidelberg and may increase their level of antibiotic tolerance. Therefore, it is critical to gain a better understanding on the dynamic interactions that occur between important pathogens and the commensals present in the animal gut and other agroecosystems. ARS researchers in Athens, Georgia, used commercial broiler ceca as a model to investigate antibiotic resistance genes that can be transferred in vitro from ceca flora to Salmonella enterica serovar Heidelberg (S. Heidelberg). They showed that the native flora in the chicken ceca were capable of transferring a plasmid producing beta-lactamase to an important food-borne pathogen S. Heidelberg. In vitro, this plasmid was transferrable between E. coli and S. Heidelberg strains, but transfer was unsuccessful between S. Heidelberg strains. An in-depth genetic characterization of transferred plasmids suggests that they share significant homology with bacteriophages. This study contributes to our understanding of the dynamics between an important food-borne pathogen and the chicken gut microbiome.

2. Reused litter decreases the transfer frequency of multidrug resistant plasmids from the broiler gut microbiota to Salmonella Heidelberg. Horizontal gene transfer plays an important role in the spread of antimicrobial resistance and virulence genes to food-borne bacterial pathogens. The notion that these genes are horizontally transferred from commensal bacteria residing in the animal gut or environment to food-borne pathogens is becoming more recognized. Likewise, plasmid-mediated antibiotic resistance gene transfer is thought to occur between intestinal commensal bacteria and putative pathogens like Salmonella. ARS researchers in Athens, Georgia, completed an in vivo broiler study evaluating the interactions between broiler gut microbiome, litter age and antimicrobial resistance acquisition in Salmonella Heidelberg. They showed that litter reuse resulted in a lower transfer of multidrug resistant plasmids (transfer frequency = 3.2 %) from the broiler gut microbiome to inoculated S. Heidelberg strain than broilers raised on fresh litter (transfer frequency = 22.5%) after 2 weeks of grow-out. This study supports the notion that consecutive reuse of litter promotes an “unfavorable” environment for E. coli and Salmonella carrying multidrug resistant plasmids, therefore limiting their potential for transfer.

Review Publications
Oladeinde, A., Cook, K.L., Orlek, A., Zock, G.S., Herrington, K., Plumblee Lawrence, J.R., Hall, M.C., Cox Jr, N.A. 2018. Hotspot mutations and ColE1 plasmids contribute to the fitness of Salmonella Heidelberg in poultry litter. PLoS One.
Oladeinde, A., Lipp, E., Chen, C., Murihead, R., Glenn, T., Cook, K.L., Molina, M. 2018. Transcriptome changes of Escherichia coli, Enterococcus faecalis and Escherichia coli O157:H7 laboratory strains in response to photo-degraded DOM. Frontiers in Microbiology.
Tyson, G., Bodeis-Jones, S., Caidi, H., Cook, K.L., Dessai, U., Haro, J., Mccullough, A., Meng, J., Morales, C., Plumblee Lawrence, J.R., Tillman, G., Winslow, A., Miller, R. 2018. Proposed epidemiological cut-off values for ceftriaxone, cefepime, and colistin in Salmonella. Foodborne Pathogens and Disease.
Smalla, K., Cook, K.L., Djordjevic, S., Klumper, U., Gillings, M. 2018. Environmental dimension of antibiotic resistance: Assessment of basic science gaps. FEMS Microbiology Ecology.
Netthisinghe, A., Cook, K.L., Gilfillen, R., Woosley, P., Sistani, K.R. 2018. Managing beef backgrounding residual soil contaminants by alum and biochar amendments. Journal of Environmental Quality.