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<P>The major goal of this project is to address mitigation strategies for antimicrobial resistant (AMR) bacteria in cattle operations by investigating novel identification and typing methods for these microorganisms. Specifically, Matrix-Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS) will be evaluated and developed for this purpose using presumptive cephalosporin and fluoroquinolone resistant E. coli, Enterococcus, Staphylococcus spp. isolates collected from cattle facilities and mammalian and avian wildlife from cattle facilities' surroundings. <br>Objective 1: Identification of isolates from livestock facilities using MALDI-TOF MS. Approximately 3,000 presumptive AMR E. coli, Enterococcus, Staphylococcus spp. isolates will be identified via MALDI-TOF MS using formic acid/ethanol extractions (suitable for genus, and usually species identifications) for sample preparation. <br>Objective 2: Development and optimization of sample preparation and data analysis strategies to enable subtyping by MALDI-TOF MS. A variety of sample preparation procedures (solvent systems, detergent extractions, enzymatic treatments, sample fractionation, and multiple MALDI matrices) will be evaluated using panels of well characterized bacteria from environmental, human, and food sources, with specific focus on E. coli, Enterococcus, Staphylococcus spp. <br>Objective 3: Using optimized protocols in Objective 2, we will perform subtyping analysis on the confirmed target AMR bacteria and generate novel mass spectral reference libraries. The optimal sample preparation and data analysis procedures determined in Specific Aim 2 will be applied to isolates identified in Specific Aim 1 as E. coli, Enterococcus, Staphylococcus spp. Representative panels of the confirmed isolates will also be subjected to pulsed-field gel electrophoresis (PFGE) and antimicrobial sensitivity testing for comparative evaluations of MALDI-TOF MS typing efficacy. The discriminatory mass spectral data, determined through extensive bioinformatic interpretations and by indirect comparisons to PFGE/antimicrobial sensitivity profiles, will be used to build reference mass spectral libraries. </P>
<P>NON-TECHNICAL SUMMARY: The dissemination of antimicrobial resistant (AMR) bacteria by wildlife to animal feeding operations (AFOs), can jeopardize food safety, and consequently, adversely impact production. The prevalence of AMR bacteria in cattle AFOs may be effectively reduced by interventions which limit contact of wildlife with livestock. Thus, it is imperative to characterize and monitor the transfer of AMR bacteria between wildlife and AFOs, ultimately leading to improved interventions that target the specific problem wildlife vector(s). As a result, methods for rapid and reliable identification and subtyping of AMR bacteria are urgently needed to assess the transmission of AMR bacteria found in wildlife to AFOs. In this application we propose to develop methods using Matrix-Assisted Laser Desorption Ionization Time-of- Flight mass spectrometry (MALDI-TOF MS) for rapid and specific identification and typing of AMR Escherichia coli, Staphylococcus, Enterococcus spp. from agricultural and wildlife sources. Microbial identification using this technology is based on the comparison of mass spectral patterns obtained directly from cells or cell extracts against reference library spectra using a pattern recognition algorithm. This technology is currently available in the form of commercial platforms, and a considerable amount of studies have been generated demonstrating its utility for identification and typing of human clinical strains (at the species level), though it is not yet officially approved for use in diagnostics applications in the U.S. There is, however, a lack of knowledge regarding the use of MALDI-TOF MS for identification/typing of bacterial isolates from environmental, animal, and food sources, and few reference spectra for these isolates are represented in the currently available mass spectral libraries. The proposed study will address these issues by: 1) Applying MALDI-TOF MS to biotype approximately 3,000 AMR isolates from cattle AFO- related (fecal, feed, and water) and wildlife sources, 2) Optimizing sample preparation procedures for MALDI-TOF MS identification and typing of AMR isolates, 3) Generation of comparative reference mass spectral libraries of AMR isolates to enable future diagnostic applications. </P>
<P>APPROACH: This project will not be performed on vertebrate animals, but it will exclusively employ bacterial strains previously isolated in a collaborative study with The Ohio State University, USDA-APHIS NWRC, and Colorado State University. The bacterial strains were isolated in concordance with animal welfare and care regulations using the USDA-APHIS NWRC protocols QA-1945 and QA-1986 for which Institutional Animal Care and Use Committee (IACUC) approval was obtained. We will optimize existing sample preparation procedures for MALDI-TOF MS and develop new methods for AMR bacteria that will specifically enrich for compounds capable of distinguishing not only between bacterial genera and species, but also subspecies and populations. MALDI-TOF MS will be performed using the Bruker Ultraflex II TOF/TOF (Bruker Daltonik) pre-calibrated with bacterial test standard (Bruker Daltonik). Empirical evaluations to determine the optimal MALDI-TOF MS instrument settings will be determined for each sample preparation procedure tested. Mass spectral analyses for bacterial identifications will be performed using the Bruker MALDI Biotyper Realtime Classification software (Ver. 3.1) (Bruker Daltonik) and corresponding reference database. Closely related and genetically similar bacteria will be suntyped by discerning unique phenotypic traits discernible through optimized MALDI-TOF MS sample preparation procedures. Principal Component Analysis will be performed to reduce the dimensionality of the multivariate spectral data sets and employed to visualize the homo/heterogenicity between isolates spectra, which equate to similarities/differences in taxonomy and/or phenotype. These data will then be converted into multivariate 3D scatter plots, dendrograms (hierarchical clustering), or heat maps (correlational clustering) for easy visualization and interpretation of phenotypic and taxonomic properties. The project will reach its target audiences through scientific publications and presentations as well as meetings with industry representatives. </P>