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<p>Our long-term goal is to develop solutions for mitigating antibiotic resistance (AR) in farm animals. Much of the research to date on selection/acquisition of antimicrobial resistance in food animal production has focused on feedlots. In our preliminary studies, we have found high levels of ARMs in cow/calf operations here in Florida, suggesting that the pathways involved in acquisition of ARMs are more complex - and begin earlier in the production cycle - than had been anticipated. On the basis of our findings, our central hypothesis is that current management practices for grass-fed cattle create conditions conducive to the acquisition and development of antibiotic resistance in the gastrointestinal tract. Development of rational mitigation strategies requires a comprehensive understanding of when, where, and how resistance was acquired, and how resistance genes and ARMs move among animals. However, little data is available on factors contributing to the acquisition of ARMs in agricultural systems. Therefore, understanding of AR occurrence from earlier in the food animal production cycle is essential to develop mitigation strategies. This integrated research and extension project focusing on identifying associated risk factors and origin for the presence of such resistance in farm animals to develop methods for reducing ARMs in farm animals. Conventional and new extension approaches will be used to better educate stakeholders and the public to reduce ARMs in cattle, enhancing the sustainability of US agriculture and global food safety and security.The specific objectives of the proposal are as follows:</p><p><ol><li> Understand AR occurrence in cow/calf operations to identify risk factors raising AR in cattle that will explain how ARMs are developed and prevalent in earlier stage of production. This will be applied in the development of a farm management model to mitigate ARMs. Obj.1A: Evaluate prevalence of antibiotic resistant microorganisms in cow/calf operations. Obj. 1B: Identify associated risk factors for the presence of such resistance, including antibiotic use, environmental factors, soil, water, feeds, and animal management practices.</li><li> Identify mechanisms of antimicrobial resistance, especially Extended-Spectrum β-Lactamases (ESBLs), to understand how and where antibiotic resistance acquired in cattle that can be applied in the development of a cattle management model to mitigate ARMs. Obj. 2A: Speciate ESBL producing microorganisms in cattle. Obj. 2B: Identify resistance genes and understand mechanisms of acquisition of ESBLs in cattle.</li><li> Determine the points where ARMs are introduced into cattle identify critical control points that will provide the basis for development of intervention strategies for antimicrobial resistance and use this information to develop a dynamic ARM transmission model. Obj. 3A: Trace animals from birth to slaughter to understand ARM occurrence, prevalence, and transmission in cow/calf operations. Obj. 3B: Identify animal factors that affect the prevalence of ARMs in cattle, including genetic and physiological factors. Obj. 3C: Develop a mathematical model that will predict ARM occurrence in cattle.</li><li> Develop and deliver a comprehensive extension/education program on best farm and cattle management practices to mitigate AR on farms. Obj. 4A: Develop best AR mitigation strategies on the basis of outcomes of this proposed research that can be applied in cow/calf operations. Obj. 4B: Transfer mitigation strategies to food animal producers and scientists by publications, training, and education through scientific journals, personal contact, and web-based resources including eXtension system. Obj. 4C: Involve undergraduate, graduate, and pre-veterinary students in best cattle management practices and research activities to reduce ARMs to stimulate interest towards graduate school and food animal oriented careers.</li></ol></p>
<p>Aim #1: This aim is an integrated project combining research and extension components. The goal of the research component in Aim #1 is to understand AR occurrence in cow/calf operations in Florida to identify risk factors raising AR in cattle that will explain how ARMs are developed and prevalent in earlier stage of production and will be applied in development of farm management model to mitigate ARMs.Obj.1A: Evaluate prevalence of antibiotic resistant microorganisms in cow/calf operations to confirm our findings that ESBL-producing microorganisms are ubiquitous in the cattle consuming forage-based diets, we will expand our studies through investigation of farms in north Florida. The north Florida region will be used because small and medium-sized farms, which feed grass to cattle, can be easily identified for sampling compared to other regions. In addition, we found this region has different soil composition and vulnerability to ground water pollution, suggesting this region may give us unique insights to predict risk factors that raise the prevalence and transmission of ESBL-producing microorganisms1A.1. Sampling strategy in cattle operation Cattle operations in north Florida will be recruited for this project.1A. 2. Prevalence of ARMs in cow/calf operations and collection of isolates To measure ESBL prevalence in small/medium size farms in grass-fed cattle, we will analyze samples obtaining from RAJ, soil, water, and feeds.1A. 3. Statistical analyses For the prevalence analysis, we will model the probability of a positive swab sample as a function of county, farm size category, and cow/calf status using a generalized linear mixed model with a logit link, as follows:logit(pijk )=α+βLargeij + ηCowijk +ui, where i indexes county, j indexes farm, and k indicates swab, Largeij indicates whether farm ij is a large farm or not, Cowijk indicates whether swab ijk is from a cow or a calf, and ui is a random effect indicating correlation of prevalences within a county. For the risk factor analysis, we will incorporate covariates into the above model and test two-sided statistical significance at the α = 0.05 level.1A. 4. Metagenomic analysisTo understand if animal microflora affects on the prevalence of ARMs in cattle, we will conduct culture independent metagenomic analysis. We will select about 100 cattle (50 cows and 50 calves) and RAJ swab samples will be used for this analysis.1A. 4. 1. DNA sample extraction and sequencing.Fecal samples (0.1g) will be used to extract DNA sample, and 16S rRNA amplicons will be sequenced using a Illumina MiSeq instruments and reagents (47).1A. 4. 2. Sequencing data analysis and Identification of BacteriaThe sequencing processing will filter out short sequences <200bp, quality score <Q25, sequences with ambiguous base calls, and sequences with homopolymer >6bp.1A. 4. 3. Statistical Analyses for metagenomic analyses Rarefaction curves and bacterial diversity (Chao1 and Shannon) will be evaluated using the R software (http://www.r-project.org). Hierarchical clustering of microbial species will be generated by a heat map with Euclidean distance using the R. The dissimilarity of bacterial communities between samples will be analyzed by principal coordinate analysis (PCoA) of weighted UniFrac distances (http://bmf.colorado.edu/unifrac). Mean values will be plotted in the relative abundance of bacteria and data will be compared with Student's t-test using the GraphPad Prism InStat 3.1. P values ≤0.05 will be considered as statistically significant.Obj. 1B: Identify associated risk factors for the presence of such resistance, including antibiotic use, environmental factors, soil, water, feeds, and animal management practices We will accomplish the goal of objective 1B by collecting farm-specific data to assess possible risk factors for ARM carriage. We will prepare questionnaires to ask farmers and farm employees to identify high risk factors, such as antibiotic use for the therapeutic or subtherapeutic purposes, types of antibiotics if used, age of animals, types of feed, questions related to animal husbandry, source of drinking water for animals, animal migration among farms etc. (during the 1st farm visit for sampling).</p><p>Aim #2: The goal of this aim is to identify mechanisms of antimicrobial resistance, especially Extended-Spectrum β-Lactamases (ESBLs), to understand how and where ESBLs are acquired in cattle. This information will be applied in the development of a cattle management model to mitigate ARMs.Obj. 2A: Speciate ESBL producing microorganisms in cattle 2A. 1. Phenotypic screening & speciationThe goal of this objective is to speciate ESBL producing microorganisms in cattle. We want a collection that includes both clinically important isolates and species that may serve as donors for resistance genes.2A. 2. Identification of resistance genes by amplifying known ESBL genes Isolates will be screened for specific ESBL-producing genes using a multiplex PCR as previously described. In brief, this method uses 9 different primer sets (blaTEM, blaSHV, blaCTX-M, blaCMY, blaOXA-1, blaOXA-2, blaACC-like, blaVEB, and blaDHA).Obj. 2B: Identify resistance genes and understand mechanisms of acquisition of ESBLs in cattle. The goal of objective 2B is to identify specific antimicrobial resistance genes and mechanisms of gene transfer among strains. All cefotaxime-resistant isolates selected from MacConkey plates (from cattle, soil, and water), as described above, will be screened.2B. 1. Functional metagenomics to identify unknown cefotaxime resistance genes Identification of resistance genes will be conducted using functional metagenomic selections, which are suited to characterize resistance because they identify any gene sufficient to confer resistance to bacteria.2A. 2. Whole genome sequencing for strain relatedness/genetic epidemiology To examine strain relatedness and to shed light on the possible transmission route of the isolated strains, we will conduct whole-genome sequencing of cefotaxime resistant strains. Next-generation sequencing will be conducted using Illumina Miseq sequcer using 250 bp paired end reads. </p><p>Aim #3: The goal of this aim is to determine the points where ARMs are introduced into cattle to identify critical control points that will provide the basis for the development of intervention strategies for antimicrobial resistance. In addition, a dynamic ARM transmission model will be developed using outcomes of this proposed research Obj. 3A: Trace animals from birth to slaughter to understand ARM occurrence, prevalence, and transmission in cow/calf operations Obj. 3B: Identify animal factors that affect the prevalence of ARMs in cattle, including genetic and physiological factors. In this research we will study the animal from birth to slaughter to find potential ARM introduction points. This will help to identify critical control points that will provide the basis for development of intervention strategies for antimicrobial resistance. In addition, we will evaluate the GI tract microflora change using metagenomic analysis as animals grow to identify if microflora affect of the prevalence of ARMs in cattle. </p><p>Aim #4: The goal of this aim is to develop and deliver a comprehensive extension/education program on best farm and cattle management practices to mitigate AR on farms.Obj. 4A: Develop best AR mitigation strategies based on the outcomes of the proposed research.Obj. 4B: Transfer of mitigation strategies to food animal producers and scientists.</p><p>