Pseudomonas aeruginosa colonizes the sputum of most adult cystic fibrosis patients, forming difficult-to-eradicate biofilms in which bacteria are protected in their self-produced extracellular polymeric substance (EPS) matrices. EPS provide biofilms with viscoelastic properties, causing time-dependent relaxation after stress-induced deformation, according to multiple characteristic time constants. These time constants reflect different biofilm (matrix) components. Since the viscoelasticity of biofilms has been related to antimicrobial penetration but not yet bacterial killing, this study aims to relate killing of P. aeruginosa, in its biofilm mode of growth, by three antimicrobials to biofilm viscoelasticity. P. aeruginosa biofilms were grown for 18 h in a constant-depth film fermenter, with mucin-containing artificial sputum medium (ASM+), artificial sputum medium without mucin (ASM−), or Luria-Bertani (LB) broth; this yielded 100-μm-thick biofilms that differed in their amounts of matrix environmental DNA (eDNA) and polysaccharides. Low-load compression testing, followed by three-element Maxwell analyses, showed that the fastest relaxation component, associated with unbound water, was most important in LB-medium-grown biofilms. Slower components due to water with dissolved polysaccharides, insoluble polysaccharides, and eDNA were most important in the relaxation of ASM+-grown biofilms. ASM−-grown biofilms showed intermediate stress relaxation. P. aeruginosa in LB-medium-grown biofilms was killed most by exposure to tobramycin, colistin, or an antimicrobial peptide, while ASM+ provided the most protective matrix, with less water and most insoluble polysaccharides and eDNA. In conclusion, stress relaxation of P. aeruginosa biofilms grown in different media revealed differences in matrix composition that, within the constraints of the antimicrobials and growth media applied, correlated with the matrix protection offered against different antimicrobials.
Antimicrobial peptides, also known as host defense peptides, have recently emerged as a promising new category of therapeutic agents for the treatment of infectious diseases. This study evaluated the preclinical in vitro, ex vivo , and in vivo antimicrobial activity, as well as the potential to cause skin irritation, of human kininogen-derived antimicrobial peptide DPK-060 in different formulations designed for topical delivery. We found that DPK-060 formulated in acetate buffer or poloxamer gel caused a marked reduction of bacterial counts of Staphylococcus aureus in vitro (minimum microbicidal concentration <5 μg/ml). We also found that DPK-060 in poloxamer gel significantly suppressed microbial survival in an ex vivo wound infection model using pig skin and in an in vivo mouse model of surgical site infection (≥99 or ≥94% reduction in bacterial counts was achieved with 1% DPK-060 at 4 h post-treatment, respectively). Encapsulation of DPK-060 in different types of lipid nanocapsules or cubosomes did not improve the bactericidal potential of the peptide under the applied test conditions. No reduction in cell viability was observed in response to administration of DPK-060 in any of the formulations tested. In conclusion, the present study confirms that DPK-060 has the potential to be an effective and safe drug candidate for the topical treatment of microbial infections; however, adsorption of the peptide to nanocarriers failed to show any additional benefits.
Optical-coherence-tomography (OCT) is a non-destructive tool for biofilm imaging, not requiring staining, and used to measure biofilm thickness and putative comparison of biofilm structure based on signal intensity distributions in OCT-images. Quantitative comparison of biofilm signal intensities in OCT-images, is difficult due to the auto-scaling applied in OCT-instruments to ensure optimal quality of individual images. Here, we developed a method to eliminate the influence of auto-scaling in order to allow quantitative comparison of biofilm densities in different images. Auto- and re-scaled signal intensities could be qualitatively interpreted in line with biofilm characteristics for single and multi-species biofilms of different strains and species (cocci and rod-shaped organisms), demonstrating qualitative validity of auto- and re-scaling analyses. However, specific features of pseudomonas and oral multi-species biofilms were more prominently expressed after re-scaling. Quantitative validation was obtained by relating average auto- and re-scaled signal intensities across biofilm images with volumetric-bacterial-densities in biofilms, independently obtained using enumeration of bacterial numbers per unit biofilm volume. The signal intensities in auto-scaled biofilm images did not significantly relate with volumetric-bacterial-densities, whereas re-scaled intensities in images of biofilms of widely different strains and species increased linearly with independently determined volumetric-bacterial-densities in the biofilms. Herewith, the proposed re-scaling of signal intensity distributions in OCT-images significantly enhances the possibilities of biofilm imaging using OCT.
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