Figure 1.
Structure of the ceragenin CSA-13.
Table 1.
Streptococcal strains used in the present work, and associated CSA-13 MIC values.
Figure 2.
Effect of CSA-13 on streptococcal species.
Exponentially growing cultures were incubated in C+Y to an A550 of about 0.2. CSA-13 was then added to aliquots of the cultures and incubation continued without shaking at 37°C. (a) S. pneumoniae R6. (b) S. pneumoniae Spain23F-1. (c) Streptococcus sp. strain 11923/96. (d) S. pseudopneumoniae. (e) S. gordonii. (f) S. mitis. (g) S. sanguinis. (h) S. mutans. (i) S. agalactiae. (j) S. pyogenes. Solid circles represent untreated, control cultures. CSA-13 was added (open symbols) at the concentrations: triangles, 1 µg/ml; circles, 10 µg/ml; squares, 25 µg/ml. (k) Lytic effect of CSA-13 (10 µg/ml; open symbols) added to a culture of the pneumococcal strain R6 at the times indicated by the arrows. (l) Bactericidal effect of CSA-13 on different streptococcal species (initial A550 corresponding to 1–2×108 CFU/ml). Grey, dashed and blackened bars correspond, respectively, to bacterial survival after 0, 2 h, and 6 h incubation in the presence (+) or absence (–) of the ceragenin (10 µg/ml). Standard error bars are shown.
Figure 3.
Fluorescence microscopy images of S. pneumoniae R6 cells either untreated (panels a–c) or treated with CSA-13 (100 µg/ml; 3 h) (panels d–f) and stained with the BacLight bacterial viability kit.
Live and non-viable bacteria fluoresce green (b, e) and red (c, f) respectively. Panels a and d are phase contrast micrographs. The arrows indicate cells treated with CSA-13 and lacking any cytoplasmic content. Panel d (enlarged inset) shows four apparently empty cocci. Some cellular material appears to be undergoing release from two bacteria (arrowheads).
Figure 4.
Growth and lysis curves and survival of S. oralis (a, b) and S. oralis (pLSE5) expressing LytA (c, d) treated with CSA-13.
Cells were grown in C+Y at 37°C to an A550 of about 0.2. CSA-13 was then added to an aliquot of the culture and incubation continued without shaking at 37°C. Solid circles represent untreated control cultures. CSA-13 was added (open symbols) at 2.5 µg/ml (triangles) or 10 µg/ml (circles). For panels b and d, grey, dashed and blackened bars correspond, respectively, to bacteria survival after 0, 2 h, and 6 h incubation in the presence (+) or absence (–) of the ceragenin (10 µg/ml). Standard error bars are shown.
Figure 5.
Triggering of the LytA autolysin by CSA-13.
(a) An exponentially growing of S. pneumoniae strain P103 (lytA::aphIII) was incubated in C+Y medium with (open symbols) or without (solid symbols) pure LytA enzyme (10 µg/ml) for 30 min at 37°C. The culture was then diluted to an A550 of 0.2 and divided into two portions. CSA-13 was added at 5 µg/ml (diamonds) to one while the other was left untreated (circles). Incubation was continued at 37°C. Survival of the culture treated only with CSA-13 was determined by plating at different incubation times (dotted line). (b) Effect of CSA-13 on the activity of cell wall hydrolase LytA using radioactively labeled pneumococcal cell walls as substrate. Data represent the percentage activity of LytA in the absence of CSA-13 and are the means of three independent experiments. (c) CD spectra of LytA in the far-UV region in the absence and presence of CSA-13 (50 µg/ml) and/or 48 mM choline chloride (cho).
Figure 6.
Disaggregation of biofilms in the presence of CSA-13.
S. pneumoniae R6 (a) and P103 (lytA::aphIII) (b) were inoculated into 200 µl of C medium in the wells of a microtiter plate (4.5×106 CFU/ml). After biofilm development (6 h at 34°C), CSA-13 was added at different concentrations, and incubation allowed to proceed for 1 h at 34°C before staining with crystal violet to quantify biofilm formation (open bars). Percentage viability (solid lines) after treatment with CSA-13 was determined by plating on blood agar plates. Standard error bars are shown. Asterisks indicate a P value<0.05.
Figure 7.
Confocal laser scanning microscopy image of the viability of biofilm-grown S. pneumoniae R6 in the presence of CSA-13.
Pneumococcal strains (4.5×104 CFU) were grown on glass-bottom dishes (WillCo-dish) in 2 ml of C medium for 12 h at 34°C. The pneumococcal biofilms were rinsed with C medium to remove non-adherent bacteria, and then incubated with CSA-13 at 5 µg/ml (b) or 25 µg/ml (c) for 90 min at 34°C. Panel a shows untreated control biofilm. Cells in the biofilms were stained with the BacLight bacterial viability kit to reveal living (green) and dead (red) bacteria.
Table 2.
Hemolytic activity of CSA-13 on sheep and human blood.