FIG 3.

CLR increased lytA gene transcription and LytA release into culture supernatant. (A to C) MRSP NU4471 was incubated in the presence or absence of 5 μg/ml CLR or ERY until it reached the stationary phase of growth (OD₆₀₀, 0.55). (A) LytA protein levels in cell-free culture supernatants were determined by Western blotting. (B) The relative intensities of the bands were quantitatively analyzed. (C) Pneumococcal eDNA in the culture supernatant was quantified using real-time PCR. Supernatant from S. pneumoniae wild-type strain D39 and the lytA-isogenic mutant (ΔlytA) were used as controls. (D) Real-time PCR was performed to quantify the transcription levels of lytA in MRSP treated with 5 μg/ml CLR or ERY for 2 h. The relative quantity of the gene was normalized to the relative quantity of 16S rRNA. In panels B to D, the data represent the mean ± SD of quadruplicate experiments and were evaluated using one-way analysis of variance with Tukey’s multiple-comparison test. *, Significant difference between the indicated groups at P < 0.05. CLR, clarithromycin; Ctrl, control; eDNA, extracellular DNA; ERY, erythromycin; SD, standard deviation.