FIG 5.

qRT-PCR analysis of tcdA expression following treatment with surotomycin, metronidazole, or vancomycin at 8× MIC. Strain UK1 was grown in CaCl₂-supplemented TY medium for ∼12 h as described above, at which time the culture was split and drugs were added at 8× MIC. At 2, 4, 8, and 24 h after addition of drugs, cell samples were harvested, RNA was extracted, and cDNA was synthesized as described in Materials and Methods. cDNA corresponding to tcdA mRNA was quantified by real-time PCR. Reactions were performed in triplicate using cDNA synthesized from each of a minimum of three biological replicates, and results are presented as the means and SEM of the data obtained. Results were calculated using the threshold cycle (2^−ΔΔCT) method, in which the amount of target mRNA was normalized to that of an internal control transcript (rpoC).