Figure 5: Cephalexin resistance accumulation assay.

(a) Schematic representation of the assay, starting from a shared pre-culture that was divided into two pre-treatments, by adding no antibiotics (−) to one and 0.56 μg ml⁻¹ cephalexin (+) (LEX) to the other. After 2 h of pre-treatment, both cultures were exposed to 1.2-μg ml⁻¹ cephalexin for 4 h, followed by competence induction with 100 ng ml⁻¹ competence-stimulating peptide (CSP). DNA encoding pbp2x G601V was provided, allowing for genetic resistance accumulation, and cells that successfully incorporated this resistance factor were selected on plates containing 2.6 μg ml⁻¹ cephalexin. (b) Development of the viable cell count of the control (−) and the pre-treated (+) culture in the course of the assay; dotted bars indicate the number of CFUs after mutagenesis on plates containing 2.6 μg ml⁻¹ cephalexin; average and s.e.m. of duplicates are shown; asterisks indicate significance (P<0.01, t-test). (c) Plate reader assay sets in duplicates of S. pneumoniae D-PEP22 pbp2x G601V cells treated with a concentration series of cephalexin, measuring cell density (closed symbols) and gene expression (symbols without outline). Experiments were replicated at least three times.