Date of Award
Master of Science
The conjugative transposon Tn916 contains the tetM determinant that confers resistance to the antibiotic tetracycline. Previous studies in this lab have shown that nearly 35% of fecal enterococci from swine manure contain transferable Tn916-like gene sequences. The aim of this research is to develop the methodology that enables monitoring of Tn916-like elements following manure application to soil. To accomplish this goal, soil DNA extraction and polymerase chain reaction (PCR) efficiency were evaluated based on the minimum sensitivity of detection of signal from (i) pure culture Enterococcus faecalis CG110 DNA (ii) soil seeded with pure culture E. faecalis CG110 DNA and (ii) soil seeded with varying concentrations of E. faecalis CG110 cells. Cell lysis was investigated and found to be efficient based on obtaining a minimum detection sensitivity of 1.7 x 103 copies of Tn916 from both E. faecalis cell and DNA spiked soil. In addition, using a series of purification procedures, 1.7 x 103 copies of Tn916 were detected in the presence of total DNA extracted from 10 g of soil. To determine if Tn916-like elements, found in fecal enterococci and indigenous to swine manure, become established in native (non-sterile) soil bacterial populations at a rate greater than that in sterile soil, swine lot effluent was added to sterile and non-sterile soil microcosms and levels of Tn916-like elements were monitored weekly for 42 days by using PCR. These results in combination with weekly plate counts on media containing 40 [mu]g/ml tetracycline, suggest that levels of Tn916 in effluent soil microcosms declined at a rate similar to that of tetracycline resistant populations.
Jay Howard King
King, Jay Howard, "PCR detection of Tn916-like elements in agricultural soil after the application of swine waste" (2003). Retrospective Theses and Dissertations. 19456.