Table 3.
Quantitative Reverse-Transcriptase Polymerase Chain Reaction Analysis of spoIIR and spoIIID Expression Following Drug Addition at T2
| Time | Control (no drugs), Mean (±SD) | Fidaxomicin, Mean (±SD) | OP-1118, Mean (±SD) | Vancomycin, Mean (±SD) |
|---|---|---|---|---|
| Strain CD196: expression ratio spoIIR/16s rRNA | ||||
| T0 | 1 (0.59–1.68) | … | … | … |
| T2 | 1.11 (0.66–2.77) | … | … | … |
| T4 | 2.4 (1.92–3.00) | 0.50 (0.26–0.95) | 0.28 (0.22–0.37) | 1.74 (1.03–2.95) |
| Strain CD196: expression ratio spoIIID/16s rRNA | ||||
| T0 | 1 (0.58–1.71) | … | … | … |
| T2 | 7.03 (1.93–25.62) | … | … | … |
| T4 | 25.75 (16.61–41.02) | 2.51 (0.81–7.77) | 1.03 (0.72–1.49) | 25.99 (13.90–48.60) |
Clostridium difficile strain CD196 was grown in 3% bacto-tryptose, 2% yeast extract, and 0.1% thioglycolate, pH 7.4 medium to the second hour (T2) after the onset of the stationary phase (T0). At that time, the culture was divided into aliquots that received no drug, fidaxomicin (0.25 µg/mL), OP-1118 (2.5 µg/mL), or vancomycin (2.5 µg/mL), as indicated. RNA was harvested from untreated or drug-treated cells 2 hours later (ie, at T4), complementary DNA was synthesized, and quantitative polymerase chain reaction was performed using gene-specific primers for spoIIR or spoIIID. Results were normalized to an internal control gene (16s rRNA) and are presented as the ratio of each transcript level relative to wild-type and no-drug controls. Mean and standard deviation from the mean values reflect results from at least 2 biological replicates.
Abbreviation: SD, standard deviation.