Journal: Nature Communications
Article Title: Microscale insights into pneumococcal antibiotic mutant selection windows
Figure Lengend Snippet: 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.
Article Snippet: Microtitre plate reader experiments were performed using a TECAN infinite pro 200 plate reader (Tecan Group) by measuring every 10 min with the following protocol: 5 s shaking, OD (595 nm) measurement with 25 flashes, luminescence measurement with an integration time of 1 s. Plate reader assays proved to represent a straightforward method for analysing growth and gene expression profiles of pneumococci in a high-throughput manner.
Techniques: Mutagenesis, Expressing, Sampling, Sequencing