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A pair-matched longitudinal study conducted on three dairy farms in the U.S. High-Plains explored the temporal effects of two-dose ceftiofur crystalline-free acid (CCFA) treatment for metritis on third-generation cephalosporin (3GC) resistance among enteric E. coli in Holstein-Friesian cows. The current 13-day slaughter withholding period does not account for rising populations of third-generation cephalosporin (3GC) resistant bacteria in feces of animals following CCFA treatment. A total of 124 matched-pairs of cows were enrolled in the study. Cows diagnosed with postpartum metritis received the product twice at the labeled dose of 6.6 mg/kg subcutaneously at the base of alternating ears. Untreated cows–absent clinical metritis–were matched on lactation number and calving date. Feces were collected per rectum on days 0 (baseline), 6, 16, 28, and 56. Environmental samples, from watering troughs as well as surface manure from fresh-cow, hospital, maternity, and milking pens, and from the compost pile were collected prior to the animal sample collection period. Historical data on metritis rates and CCFA use were compiled from herd records. On day 0, cows exhibited an overall mean difference of over 4 log10 colony forming units (CFU) comparing 3GC resistant E. coli to the general E. coli population. At the first eligible slaughter date, the difference declined to 3.31 log10 CFU among cows in the CCFA group (P<0.01 compared to control cows). Such differences were no longer observed between the treated and control groups by day 28. Results suggest a 13-day withholding period following the final treatment is insufficient to allow levels of 3GC resistant E. coli to return to baseline. This effect varied by farm and was dependent upon the starting level of resistance. A farm-specific extended slaughter-withholding period could reduce the microbial risk to food products at slaughter. [ABSTRACT FROM AUTHOR]