Figure 4: Rifampicin resistance mutation assay.

(a,b) S. pneumoniae D-PEP22 populations seeded in 44 microtitre plate wells at an inoculation density of OD 0.001 in the presence of 0.038 μg ml⁻¹ RIF (a) and at an inoculation density of OD 0.02 in the presence of 0.38 μg ml⁻¹ RIF (b), followed over time by measuring cell density (OD595) and gene expression (RLU; insets). Note that results are shown on linear scales, which more clearly display the emergence of resistant populations. (c,d) Number of resistant cells (CFUs ml⁻¹) after plating pooled samples of 44 microtitre plate wells (75 μl sampling volume per well per time point) originating from 0.038 μg ml⁻¹ rifampicin (c) and 0.38 μg ml⁻¹ rifampicin (d) treatments (duplicate assays of the ones shown in a,b based on the same pre-culture) directly into 0, 0.38, and 3.8 μg ml⁻¹ RIF, respectively; average and s.e.m. of duplicates are shown. (e) Sequencing of rpoB of 12 isolates originating from spontaneously resistant populations during 0.038-μg ml⁻¹ RIF treatment, with the relevant RpoB amino-acid sequence (bold) together with the codons inside the dashed frame and their position in italics above; below, point mutations, the increase in resistance they generate and their frequency of occurrence are indicated.