Microbial Ecology of Soils Treated with Animal Manure

<p>The project consists of three specific aims. Methods are listed under each specific aim.</p><p>Specific Aim 1: Determine the effect of long-term manure amendment on microbial communities in soil. Total DNA will be extracted from soil treated with different animal manure. A fragment of 16S rRNA gene would be amplified by PCR. Resulting amplicons will be primer-tagged and sequence analyzed using Illumina MiSeq technology resulting in ~300bp paired-end fragments. Reads would be assembled into OTUs and taxonomies assigned. Principal Component analyses would be performed to ascertain differences and trends among samples.</p><p>Specific Aim 2: Determine the presence and abundance of antibiotic-resistant organisms in soil treated with different manure and determine the survivability of such organisms over time. Soil would be suspended in PBS media and plated on agar plates containing different antibiotics. Bacteria that grow on these plates would be pooled. DNA from the pooled samples would be extracted, end-repaired, and cloned into a Fosmid vector. Fosmid clones would be grown in media supplemented with antibiotics and resulting clones would be sequence analyzed to identify them. A second approach would utilize a battery of ~75 PCR assays developed against known antibiotic resistant genes.</p><p>Specific Aim 3: Determine the presence, abundance and type of antibiotic residues in soil. Soil samples will be homogenized and filtered. Filtrate would be used to impregnate 20mm filter disks and air-dried. These disks would be used in antibiotic sensitivity assays on plates containing either E. coli, Staphylococcus aureus, or Proteus mirabilis. Soil would also be tested using a panel of commercial ELISA tests designed to detect Bacitracin, Carbadox, Chlortetracycline, Lincomysin, Penicillin, and Monensin.</p>