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2021 Annual Report
Objectives
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.
Approach
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
This is the final report for this project which merged with project 6040-32000-012-00D, "Reduction of Foodborne Pathogens and Antimicrobial Resistance in Poultry Production Environments", refer to this project for additional information.
During the life of the project, considerable progress was made for the two objectives.
For Objective 1, live broiler chicken, in-vitro and in-situ studies were performed to identify environmental factors and stressors that contribute to the spread of antimicrobial resistance (AMR) in commensal bacteria and food-borne pathogens. Reused broiler litter was inoculated with two strains of Salmonella Heidelberg (S. Heidelberg) and the litter microbiome and S. Heidelberg strains present were characterized for 14 days. In addition, novel Bacillus subtilis strains, that reduced S. Heidelberg population in-vitro, were isolated from the litter used. The study provided novel information on the microbiome present in reused litter and provided stakeholders with the identity of the bacterial species that can be used for reducing Salmonella in poultry.
One day-old broiler chicks were inoculated with S. Heidelberg and raised on either fresh litter or reused litter. After 2 weeks, chicks on reused litter carried significantly lower levels of multidrug resistant S. Heidelberg than chicks on fresh litter. Furthermore, the bacterial species and metabolic pathways responsible for the competitive exclusion of multidrug resistant S. Heidelberg in reused litter were determined. The study revealed that reusing litter allowed for the development of a competitive litter microbiota that can exclude invading AMR food-borne pathogens like Salmonella.
Neonatal broiler chicks were challenged with an antibiotic susceptible S. Heidelberg strain using various routes of inoculation and S. Heidelberg isolates recovered from the ceca and litter were characterized. The results showed that chicks grown without antibiotics harbored AMR S. Heidelberg population 14 days after challenge and chicks challenged orally acquired AMR at a higher rate than chicks inoculated via the cloaca. Additionally, S. Heidelberg infection disturbed the microbiota of broiler chicks and the commensal Escherichia coli population was determined to be the main reservoir of transferrable antibiotic resistance genes. The acquisition of these AMR determinants from E. coli increased the fitness of S. Heidelberg when they were exposed to acidic pH in vitro. The study suggested that simply removing antibiotics from food-animal production might not be sufficient to limit the spread of AMR.
A prospective broiler cohort study was conducted to investigate the environmental drivers of Campylobacter and Salmonella persistence in the litter of a commercial broiler facility. Litter was sampled from four houses at the beginning of each production cycle and at the end of each cycle for three consecutive flocks. There was a significant effect of litter age, broiler house, grow-out period, house temperature, and litter pH and moisture on the abundance of pathogens. The recovery of Campylobacter spp. and Salmonella spp. was more likely if the litter moisture was >25% and pH was >7, and when the house temperature was <79 F. Additionally, broiler flock on fresh litter were more likely to carry pathogens compared to flock on reused litter. This research provided stakeholders with environmental management parameters to monitor or change to reduce the incidence of food-borne pathogens in live poultry.
A study to determine the relationship between particulate matter, airborne E. coli and E. coli present in the litter was conducted during a 49-day broiler chicken grow-out. No airborne E. coli was detected at day 0; however, E. coli increased from day 7 to day 28 for samples collected at bird-level heights and close to the house ceiling. Sixty-six percent of the E. coli isolates were susceptible to the 14 antibiotics tested and 23% displayed resistant to two or more classes of antibiotics. Airborne E. coli was positively correlated to particulate matter (2.5 microns) and E. coli found in litter. The study suggested that stakeholders can reduce the level of pathogenic bacteria exposure in chickens by decreasing the levels of particulate matter in the air.
A mating study was conducted to show that an antibiotic susceptible strain of Enterococcus faecalis could acquire antibiotic resistance genes from the microbiota present in broiler litter. E. faecalis recipients that acquired resistance to tetracyclines, aminoglycosides, macrolides and phenicols were characterized by whole genome sequencing. Results showed that antibiotic resistance was conferred by the horizontal transfer of antibiotic resistance genes from the broiler litter microbiota to E. faecalis. This study demonstrated that broiler litter represents an important extra-intestinal environment that may allow the transfer of antibiotic resistant genes to Enterococcus spp.
Research was conducted to evaluate how application of soil amendments, including broiler litter, flue gas desulfurization gypsum and fertilizer, affects the persistence and transport of bacterial populations and AMR genes. Pathogens and AMR genes were detected sporadically in runoff samples and 64% of the targets showed no PCR signal. Salmonella was detected in three runoff samples from soils amended with either broiler litter and gypsum or fertilizer while Campylobacter was not detected in any sample. The AMR genes detected were for sulfonamides, streptomycin, and tetracycline. The microbiota present in soils amended with broiler litter differed from soils amended with fertilizer or fertilizer and gypsum. The research showed that the application of broiler litter affected the bacterial community structure of the soil but had a negligible effect on the AMR genes present in runoff.
A two-year longitudinal study was conducted to address the microbiological quality of environmental and squash samples taken from three organic farms that use biological soil amendments of animal origin for growing squash. The amendment used by three of the four farms was from broiler litter and a fourth farm using coffee grounds served as a control. Pathogens were not detected in the amendments, soil, water, or squash samples. Squash from the control farm carried lower levels of indicator bacteria (coliforms and enterococci) than farms that used broiler litter. Antibiotic resistant Enterobacter spp, Serratia spp. and Pantoea spp. were the dominant coliforms found, while a group of novel vancomycin resistant Enterococcus spp. were the main enterococci species found. One Enterococcus strain carried mcr5 gene that conferred resistant to colistin - a last resort antibiotic. These bacterial species were higher on the outside than on the inside of squash and washing prior to sale at the farmer’s market reduced the bacterial load in squash. The result of the study emphasizes the importance of cleaning produce before consumption.
The Savannah River Site (SRS) soils was examined for bacterial communities carrying metal and antibiotic resistance genes. The SRS served as the Department of Energy production and refinement facility for nuclear materials from 1950 - 1980, thus carries widespread contaminants including heavy metals, metalloids, and radionuclides. The results showed that strong relationships exist between the bacterial community in soils at the SRS, and the presence of chronic heavy metal and radionuclide contamination. The study supports the notion that heavy metals play a key role in the dissemination and maintenance of antibiotic resistance genes.
For Objective 2, new methods were developed and validated to accurately quantify, recover and characterize food-borne pathogens and antimicrobial resistance determinants in poultry production environments. A study was conducted to determine the limit of quantification (LOQ) and detection (LOD) of Salmonella, Campylobacter, E. coli and Enterococcus in litter. Multiple serovars/species or strains of each bacterium were inoculated together into litter at varying concentrations and the LOQ, LOD and recovery efficiency of the method used was calculated. The method performed poorly for Campylobacter, while Salmonella and Enterococcus had the best LOQ and LOD, respectively. Campylobacter had the lowest recovery, while Salmonella had the highest recovery. This study provided a template that poultry researchers can use when developing newer methods or for validation of current methods used for pathogen detection in litter.
TaqMan®-based real-time quantitative PCR assays were developed to measure the abundance of 39 poultry-associated genes and bacteria including 27 AMR genes and plasmids. The assays were validated by investigating changes in the abundance of each gene between chicks infected with Eimeria compared to uninfected chicks. The result confirmed that the newly developed qPCR assays can be used for the accurate quantification/detection of poultry gut pathogens and AMR genes.
Reads2Resistome, a bioinformatic tool, was developed to allow users with experience using Linux basic commands to analyze bacterial genomes sequenced using either short and/or long read sequencing technologies. Reads2Resistome takes sequence reads as input and performs assembly, annotation, and genome characterization with the goal of producing an accurate and comprehensive description of the bacterial genome. Reads2Resistome is publicly available on GitHub and accessible to USDA scientists via SCINet.
Accomplishments
1. Proper broiler litter management during downtime is critical to control food-borne pathogens. What constitutes a responsibly managed broiler litter remains subjective and is commonly judged by the moisture level of the litter and the amount of ammonia emitted from it. Consequently, litter treatments that can aid in lower litter moisture and pH are likely to be adopted and encouraged. ARS researchers in Athens, Georgia, Ames, Iowa, and colleagues from the University of Georgia at Athens, Institute of Food Safety, Univ. of Veterinary Medicine, Vienna, Austria, and Khon Kaen University at Khon Kaen, Thailand, determined the changes in bacterial populations present in broiler litter during downtime (the period after a flock was removed and before new broiler chicks were placed) and 24 h before and after the application of a sodium bisulfate-based amendment. The abundance and type of bacterial species present in litter was stable for 18 days after broiler chickens were removed, however the application of sodium bisulfate affected the microbiome in the top layer of litter topdressed with fresh pine shavings. Escherichia coli decreased by >90% during downtime, but an increase was observed for topdressed litter after sodium bisulfate was applied. The study provides stakeholders with new information on the minimum period of downtime needed to achieve a significant reduction in enteric bacteria and highlights the negative effect topdressing may have on litter microbiota.
2. Poultry is not a major reservoir of extended-spectrum beta-lactamase E. coli in the United States. Antimicrobial resistance associated with genes, known as plasmid-encoded extended-spectrum ß-lactamase (ESBL), that confers resistance to third generation cephalosporin antibiotics is increasing worldwide. However, data on the population of ESBL producing E. coli in different animal sources and their antimicrobial characteristics are limited. ARS researchers in Athens, Georgia, and Riverside, California, investigated the potential reservoirs of ESBL-encoded genes in E. coli isolated from swine, beef, dairy, and poultry collected from different regions of the United States using whole-genome sequencing. Of the 300 E. coli isolates, a total of nine isolates were confirmed as ESBL producers by double-disk synergy testing and multidrug resistant to at least three antimicrobial drug classes. A significantly higher numbers of ESBL-E. coli were detected in swine and dairy manure than from any other animal sources, suggesting that these may be the primary animal sources for ESBL producing E. coli.
Review Publications
Guo, Y., Aggrey, S.E., Oladeinde, A.A., Johnson, J., Zock, G.S., Chai, L. 2021. A machine vision-based method optimized for restoring broiler chicken images occluded by feeding and drinking equipment. Animals. 11(1):123. https://doi.org/10.3390/ani11010123.
Ibekwe, A.M., Durso, L.M., Ducey, T.F., Oladeinde, A., Jackson, C.R., Frye, J.G., Dungan, R.S., Moorman, T.B., Brooks, J.P., Obayiuwana, A., Karathia, H., Fanelli, B., Hasan, N.A. 2021. Diversity of plasmids and genes encoding resistance to extended-spectrum ß-Lactamase in Escherichia coli from different animal sources. Microorganisms. 9(5). Article 1057. https://doi.org/10.3390/microorganisms9051057.
Johnson, J., Zwirzitz, B., Oladeinde, A.A., Milfort, M.C., Looft, T.P., Chai, L., Zock, G.S., Sommers, M., Tunim, S., Aggrey, S.E. 2021. Succession patterns of the bacterial community in poultry litter after bird removal and sodium bisulfate application. Journal of Environmental Quality. 50(4):923-933. https://doi.org/10.1002/jeq2.20248.
