Ceragenin-mediated disruption of Pseudomonas aeruginosa biofilms.

BackgroundMicrobial biofilms, as a hallmark of cystic fibrosis (CF) lung disease and other chronic infections, remain a desirable target for antimicrobial therapy. These biopolymer-based viscoelastic structures protect pathogenic organisms from immune responses and antibiotics. Consequently, treatments directed at disrupting biofilms represent a promising strategy for combating biofilm-associated infections. In CF patients, the viscoelasticity of biofilms is determined mainly by their polymicrobial nature and species-specific traits, such as Pseudomonas aeruginosa filamentous (Pf) bacteriophages. Therefore, we examined the impact of microbicidal ceragenins (CSAs) supported by mucolytic agents-DNase I and poly-aspartic acid (pASP), on the viability and viscoelasticity of mono- and bispecies biofilms formed by Pf-positive and Pf-negative P. aeruginosa strains co-cultured with Staphylococcus aureus or Candida albicans.MethodsThe in vitro antimicrobial activity of ceragenins against P. aeruginosa in mono- and dual-species cultures was assessed by determining minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC). Inhibition of P. aeruginosa mono- and dual-species biofilms formation by ceragenins alone and in combination with DNase I or poly-aspartic acid (pASP) was estimated by the crystal violet assay. Additionally, the viability of the biofilms was measured by colony-forming unit (CFU) counting. Finally, the biofilms' viscoelastic properties characterized by shear storage (G') and loss moduli (G"), were analyzed with a rotational rheometer.ResultsOur results demonstrated that ceragenin CSA-13 inhibits biofilm formation and increases its fluidity regardless of the Pf-profile and species composition; however, the Pf-positive biofilms are characterized by elevated viscosity and elasticity parameters.ConclusionDue to its microbicidal and viscoelasticity-modifying properties, CSA-13 displays therapeutic potential in biofilm-associated infections, especially when combined with mucolytic agents

The relative mass of polymicrobial biofilms formed by Pf-negative <i>and</i> Pf-positive <i>P</i>. <i>aeruginosa</i> isolates with <i>S</i>. <i>aureus</i> Xen30 (SA, red bars), and <i>C</i>. <i>albicans</i> 1408 (CA, grey bars) strains in dual-species mixtures.

Recorded values were compared to the anti-biofilm activity of CSA-13 alone (A). Biofilm mass was measured in the presence of DNase I (B), pASP (C), and Pf1 bacteriophage (D) at concentrations from 0.5 to 5 μg/mL, 1 to 200 μg/mL and 0.01 to 0.5 mg/mL, respectively and co-administrated with CSA-13 at doses of 5 (E-G) or 20 μg/mL (H-J). Results are presented as mean ± SD of three repetitions. * indicates statistical significance when compared to dual-species mixtures of biofilm with Pf-negative P. aeruginosa isolates.</p

Phyloproteomic relatedness of Pf-negative (blue color) and Pf-positive (green color) <i>P</i>. <i>aeruginosa</i> strains estimated based on MALDI-TOF mass spectra fingerprints.

Analysis was performed using ‘taxonomy module’ in Saramis v4.12 Vitek MS-Plus RUO software. (TIF)</p

Fig 1 -

(A) Minimal inhibitory concentrations and minimal bactericidal concentrations (MIC/MBC; μg/mL) of CSA-13, CSA-44, and CSA-131 against five Pf-negative P. aeruginosa strains (black bars) and five Pf-positive P. aeruginosa strains (blue bars). (B) Inhibitory concentration at 50%-level (IC50) was recorded for tested ceragenins (CSA-13, CSA-44, and CSA-131) against five Pf-negative P. aeruginosa strains (black bars) and Pf-positive P. aeruginosa strains blue bars). The IC50 values were calculated by interpolation of the dose-response curve when biofilm formation in the presence of tested agents was measured. Results are presented as mean ± SD from 5 strains of three repetitions. Results were not statistically significant. (C) Relative mass of biofilm formed by five strains of Pf-negative P. aeruginosa (black bars) and Pf-positive P. aeruginosa (blue bars) in the presence of CSA-13 at doses of 5 and 20 μg/mL. Results are presented as mean ± SD from 5 strains of three repetitions. * indicates statistical significance when compared to Pf-negative P. aeruginosa isolates.</p

Rheological measurements of mono-species biofilm formed by <i>P</i>. <i>aeruginosa</i> (PA), <i>S</i>. <i>aureus</i> (SA), and <i>C</i>. <i>albicans</i> (CA) strains.

Shear storage modulus as a function of shear strain amplitude (A), phase shift as a function of shear strain amplitude (B), storage modulus as a function of oscillation frequency (C), and dynamic viscosity (D) were measured. (TIF)</p

The antibiotic susceptibility profiles of <i>P</i>. <i>aeruginosa</i> strains.

The antibiotic susceptibility profiles of P. aeruginosa strains.</p

S3 Fig -

Viability of biofilms formed by P. aeruginosa (PA), S. aureus (SA), and C. albicans (CA) strains in mono-species biofilm treated with DNase I (B), pASP (C) and Pf1 bacteriophage (D) at concentrations of 5 μg/mL, 10 μg/mL and 0.1 mg/mL, respectively and co-administrated with CSA-13 at dose of 20 μg/mL (E-G). Recorded values were compared to the anti-biofilm activity of CSA-13 alone (A). Green bars indicate percentage of bacteria grown. Results are presented as mean ± SD from 5 strains with three repetitions. * indicates statistical significance when compared to Pf-negative isolates. (TIF)</p

Minimal inhibitory concentration (MIC; μg/mL) and minimal bactericidal/fungicidal concentrations (MBC/MFC; μg/mL) of CSA-13 against Pf-negative <i>and</i> Pf-positive <i>P</i>. <i>aeruginosa</i> isolates, <i>S</i>. <i>aureus</i> Xen30 and <i>C</i>. <i>albicans</i> 1408 both in single-species culture or polymicrobial conditions.

Minimal inhibitory concentration (MIC; μg/mL) and minimal bactericidal/fungicidal concentrations (MBC/MFC; μg/mL) of CSA-13 against Pf-negative and Pf-positive P. aeruginosa isolates, S. aureus Xen30 and C. albicans 1408 both in single-species culture or polymicrobial conditions.</p