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2020 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
Continued to collect data under Objective 1 on bacteria and environmental parameters present in the broiler house environment under raised without antibiotics broiler production system. Escherichia coli (E. coli) was isolated from the intestine of broiler chickens, broiler litter, house particulate matter/dust, feed and water during 1- 4 consecutive production cycles lasting 49 days/cycle. No airborne E. coli was detected at day 0 of flock 3, however, E. coli was measured from day 7 – 28. In contrast, coliform bacteria concentration decreased from day 14–28. Antibiotic susceptibility testing was done on a selected number of E. coli isolates with 66% of isolates susceptible to 14 antibiotics tested and 23% displaying multidrug resistance to two or more classes of antibiotics. A multiple regression test revealed that airborne E. coli was positively correlated to litter E. coli and particulate matter (2.5 microns) and negatively correlated to particulate matter (10 microns) with litter moisture having no significant effect. The study suggested that decreasing levels of particulate matter may decrease airborne bacteria concentrations. No Salmonella was detected throughout the study, but E. coli was isolated from all samples and > 600 isolates were archived in relevant cryoprotectants.
In the summer of 2019, a study was conducted to determine the poultry house environmental parameters (temperature, ammonia, humidity) that are correlated with the bacterial community residing in the broiler chicken gut or litter. Six hundred Cobb-500 broiler chicks were raised in floor pens with clean pine shavings in two houses for 49 days under environmental conditions reflecting those of commercial raised without antibiotics broiler production. Microbial community survey was performed on the meconium of day-old chicks, the ceca when the birds were 14, 28, 42 and 49-days old and on weekly litter samples. Furthermore, the effect of an 8-h feed withdrawal period prior to slaughter on the microbiome was tested. The bacterial communities in the meconium, ceca and litter were significantly different from each other throughout the study and the ceca had the highest alpha diversity. The top genera in the meconium was Enterococcus and from the ceca was Bacteroidetes. For the litter, the top genera changed from day 7 to day 49. The bacterial community in the ceca and litter were significantly different between houses (house 1 and 2) from day 28 – day 49. Temperature and morning relative humidity had a significant correlation on the beta diversity of the ceca. In addition to house temperature, relative humidity and ammonia, the pH and moisture of the litter were the important parameters correlating with the beta diversity of the litter. An 8-h feed withdrawal before slaughter had no significant correlation with the bacterial community of the ceca. The results suggested that the environmental conditions in the farmhouse significantly affected the gut microbiome of broiler chickens and bird weight.
A study was conducted at the Savannah River Site soils to examine the bacterial communities carrying metal and antibiotic resistance genes. The site 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 relative abundance, diversity, and structure of soil bacterial communities was explored using 16S rRNA gene amplicon sequencing and revealed a core microbiome consisting of eight phyla shared among 95% of the sequences. Sites with records of metal/radionuclide contamination displayed significantly lower bacterial diversity compared to the reference uncontaminated site. Metal concentrations and carbon to nitrogen ratio were the most important factors determining the microbial community structure. Metagenomic analysis indicated that among antibiotic resistance genes, multidrug and vancomycin resistance genes were most prominent in all soil samples including those from the reference site. Metal resistance genes were dominated by those associated with copper, arsenic, iron, nickel, zinc, molybdenum resistance, and multi-metal resistance. The results demonstrated that certain bacterial species are differentially affected by soil edaphic factors and heavy metal contamination, and that their presence had an important role in the dissemination and maintenance of antibiotic and metal resistance.
The horizontal transfer (conjugation and transduction) of multidrug resistance plasmids to a susceptible Salmonella Heidelberg strain was determined in vitro in the absence of antibiotics using a consortium of E. coli strains. E. coli strains were isolated from the cecal contents of broiler chicks two weeks after challenge with S. Heidelberg. Chickens were either raised on fresh pine shavings or reused litter. The E. coli strain consortium comprised of four sequence types and four phylogenetic groups. They carried multiple plasmids and antibiotic resistance genes that conferred resistance to aminoglycosides, tetracyclines, sulfonamides and ampicillin. Synthetic 2-Hexadecynoic acid and citric acid were used as inhibitors of antibiotic resistance transfer from E. coli to S. Heidelberg. E. coli strains originating from broiler chicks raised on fresh pine shavings were able to transfer resistant plasmids to S. Heidelberg in the presence or absence of 2-Hexadecynoic acid. However, antibiotic resistance transfer was abolished when sodium citrate was added. The study provided new information on the E. coli strains that can serve as donors of multidrug resistant plasmids to Salmonella and alternative to antibiotics that can limit their transfer or acquisition.
For Objective 2, a study was conducted to validate a TaqMan®-based real-time quantitative polymerase chain reaction (PCR) assays developed for targeting 39 poultry-associated genes and bacteria including 27 antibiotic resistance genes and plasmids (i.e. resistome) by investigating changes in the broiler chicken gut resistome that are correlated with Eimeria (a pathogen that causes clinical disease in broiler chickens). Male chicks of equal numbers of Cobb500 and Athens Canadian Random Bred genotypes were raised under standard husbandry management. At 14 days of age, the chicks were randomly assigned to 6 treatments groups. The treatment effects consisted of bird genotypes and three infection levels (oral gavage of sporulated Eimeria acervulina oocysts, mixed culture of sporulated E. acervulina, E. maxima and E. tenella or distilled water). At 6 days post infection, 5 birds from each treatment group were randomly sampled and their ceca were removed and used for PCR analysis. DNA was extracted from cecal contents and used in TaqMan®-based real-time qPCR assays. A principal component analysis revealed that the concentration of total bacteria, E. coli and the resistome could explain ~ 64.2 % of the total variability in the data. The observed variability was influenced by bird genotype rather than the Eimeria strain used for infection. The result confirmed that the newly developed TaqMan®-based real-time PCR assays could detect poultry-associated gut pathogens and resistome perturbed by a treatment effect. The assay was optimized for multiplex PCR and digital PCR on a Fluidgm® Biomark HD platform for high-throughput quantification and detection.
Early proper flock distribution is a proxy used by broiler chicken producers to predict if a flock will achieve expected health, welfare and food-safety milestones at the end of a production cycle. However, this process is manually done and therefore prone to human error. A machine vision-based method was developed to automatically count the number and distribution of chickens in an experimental broiler house. The method recognizes birds’ image distribution on pen floor by virtually dividing the floor into drinking, feeding, and rest/exercise zones. As the broiler chickens grew, images were collected from each individual day and analyzed separately to avoid the bias caused by change of body weight/size over days. Results showed that the identification accuracy of birds’ distribution in the drinking and feeding zones was 0.94 and 0.95, respectively. The study provided the basis/proof-of-concept for developing an automatic system to monitor flock health in a commercial production system.
A study on the effect of litter age on Salmonella fitness was initiated using Salmonella Heidelberg and Salmonella Enteritidis as Salmonella-models. The isolates were used in a series of in vitro inoculation studies with fresh pine shavings (0 flock of broiler chickens) or “reused litter” of different ages (previously used for growing 1 and 3 flock of broiler chickens). Concentration of antibiotic susceptible and multidrug resistance Salmonella was monitored for 21 days after inoculation. In addition, antibiotic susceptibility and whole genome sequencing was performed on selected number of isolates. The population of multidrug resistant S. Heidelberg surviving in fresh litter was higher (68%) in fresh pine shavings compared to 1-flock old reused litter (53%). For S. Enteritidis, multidrug resistant population was only 1% in reused litter and 6% in fresh pine shavings. Preliminary result suggested that reused litter had a greater potential of reducing antibiotic resistant Salmonella than fresh pine shavings. This study was part of a collaboration with Agriculture and Agri-Food Canada and Canada is responsible for bacterial genome sequencing.
Accomplishments
1. Reused litter microbiome has benefits. Reusing litter to raise consecutive flocks of broiler chickens is a widespread and well accepted practice in the United States but frowned upon in Europe and Canada. However, what constitutes a beneficial or deleterious litter microbiome remains unknown or suggestive. Pre-harvest interventions, that can reduce the population of multidrug resistant Salmonella and pathogens in litter, will have a better potential to limit their transfer to the chicken gut. ARS researchers in Athens, Georgia, the University of Georgia and Colorado State University, inoculated reused broiler litter with two strains of Salmonella Heidelberg (S. Heidelberg) and characterized the litter microbiome and S. Heidelberg strains present for 14 days. In addition, they isolated novel Bacillus subtilis strains from litter that can reduce S. Heidelberg populations in vitro. The study provided novel information on the microbiome present in reused litter and the bacterial species with potential for development into “Litterbiotics”. The publication of the study resulted in press releases by the American Society for Agronomy and poultry-focused magazines for its importance to the poultry industry.
2. Reads2Resistome: A high throughput bioinformatic tool for bacterial genome analysis. There is no doubt that next-generation sequencing has significantly improved our understanding of the bacterial genome architecture. However, there are still multiple bioinformatic hurdles to maneuver before a bacterial culture becomes an informative annotated genome. This usually entails using several stand-alone tools that makes the process cumbersome and prone to specialist/human error. ARS researchers in Athens, Georgia and Colorado State University, developed Reads2Resistome, a bioinformatic tool that streamlines this arduous effort. Reads2Resistome allows 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 and collection of all the antibiotic resistance genes, virulence genes, and other resistance elements within the chromosome, plasmids or bacteriophage. The pipeline is executable on both Mac and Linux operating systems and is well-suited for institutions and organizations which maintain, or have access to, a high-performance cluster for the analysis of "big data”. Reads2Resistome is the first pipeline to our knowledge that performs both genome assembly and in-depth genome characterization. It has been made publicly available on GitHub and accessible to USDA researchers via SCINet.
Review Publications
Bucher, M., Zwirzitz, B., Oladeinde, A.A., Cook, K.L., Plymel, C., Zock, G., Aggrey, S., Ritz, C., Looft, T.P., Lipp, E., Agga, G.E., Sistani, K.R. 2020. Reused poultry litter microbiome with competitive exclusion potential against Salmonella Heidelberg. Journal of Environmental Quality. 49(4):869-881. https://doi.org/10.1002/jeq2.20081.
Guo, Y., Chai, L., Aggrey, S., Oladeinde, A.A., Johnson, J., Zock, G. 2020. A machine vision-based method for monitoring broiler chicken floor distribution. Sensors. http://doi.org/10.3390/s20113179.
Thomas, J., Oladeinde, A.A., Kierant, T., Finger, J., Vasquez, N., Cartee, J., Beasley, J., Seaman, J., Mcarthur, V., Rhodes, O., Glenn, T. 2020. Co-occurrence of antibiotic, biocide, and heavy metal resistance genes in bacteria from metal and radionuclide contaminated soils at the Savannah River Site. Microbial Biotechnology. https://doi.org/10.1111/1751-7915.13578.
Cox Jr, N.A., Oladeinde, A.A., Cook, K.L., Zock, G.S., Berrang, M.E., Ritz, C.W., Hinton Jr, A. 2020. Research Note: Evaluation of several inoculation procedures for colonization of day-old broiler chicks with Salmonella Heidelberg. Poultry Science. 99(3):1615-1617. https://doi.org/10.1016/j.psj.2019.10.020.
