Determination of Antibiotic Resistance in E. coli Isolated from Mid-Michigan Streams

Large livestock facilities account for the majority of antibiotic use in this country.  Studies have shown that prolific and continual use of antibiotics in these facilities has led to increased resistance in many types of bacteria.  Past studies from Mid-Michigan streams have identified antibiotic resistance in fecal coliform bacteria, including E. coli.  Correlating resistance with nutrient loading data may help identify the proliferating source of resistant strains, when sampling at the livestock facilities themselves is impossible.  This study attempted to identify and characterize the presence and possible sources of antibiotic resistant fecal coliform bacteria in surface water around large livestock facilities by testing for and isolating resistant strains and assessing associated nutrient loading at sampling sites.  Sediment and pore water samples were collected from streams both upstream and downstream from large livestock facilities and areas of known negative impacts.  Pore water extracted from the samples was plated on a media, selective for gram-negative fecal coliform bacteria.  Several plates were dosed with different concentrations of Tetracycline Hydrochloride and resistant bacteria were isolated.  Tetracycline Hydrochloride is an antibiotic widely used in livestock, but rarely used in the human population.  Resistant bacteria were analyzed using EStrips, which assess the Minimal Inhibitory Concentration (MIC) of multiple antibiotics.  PCR and gel electrophoresis were used on resistant isolates to test for the presence of the Tetracycline Hydrochloride resistance Tet(W) gene.  Results show fecal coliform bacteria from the Mid-Michigan stream have significant resistance to multiple antibiotics including Tetracycline Hydrochloride, Erythromycin, and Streptomycin.  The Tet(W) gene was identified which demonstrates that resistance is most likely mutated from livestock facilities that commonly use Tetracycline Hydrochloride.  Corroboration of these results with high nutrient loading and Biochemical Oxygen Demand downstream from these facilities, strongly suggests that large livestock operations in the region are the source of these mutations.  Additionally, these results show the importance of connecting geochemical and biological techniques to assess environmental impacts of large livestock facilities.