Fig 1.
Scanning electron micrographs of biofilms for evaluating morphological changes throughout their development.
Representative micrographs are shown for biofilms composed of P. aeruginosa and S. aureus as mono-species and dual-species biofilms. All biofilms were cultivated for 24, 48, and 72 h prior to evaluation. Scale bar = 5 μm.
Fig 2.
Quantification of colony forming units of adherent biofilm bacteria.
(A) Mono-species biofilms of P. aeruginosa and S. aureus as well as dual-species biofilms of both species were cultivated for 24, 48 and 72 h. After each time point, the total number of viable, adherent bacteria was determined. (B) For dual-species biofilms, their bacterial composition was evaluated after 24, 48 and 72 h of co-cultivation. *p < 0.05, **p < 0.01, and ***p < 0.001.
Fig 3.
Raman spectra and false color images of different biofilms.
Raman scans of the three different biofilms were acquired on their surfaces after 24, 48 and 72 h. (A) Representative Raman peaks are highlighted in the mean biomass spectra of the samples. (B) False color images revealed the distribution of biomass (shown in light and dark green for P. aeruginosa, yellow and orange for S. aureus and light and dark blue for the dual-species biofilm) and fluorescent regions (shown in pink) on the fiber network (shown in grey). Scale bar = 5 μm.
Fig 4.
Susceptibility testing of the biofilms to the antibiotic gentamicin.
(A) P. aeruginosa and S. aureus, cultivated as mono-species and dual-species biofilms for 24, 48 or 72 h, were treated with a 10 μg/mL gentamicin solution for 24 h. Subsequently, CFUs were quantified and compared to the untreated control. (B) For dual-species biofilms, the ratio of both species was determined after the treatment with gentamicin.
Fig 5.
Raman analysis of the penetration ability of gentamicin into the biofilms.
Fixed mono- and dual-species biofilms were incubated with a 100 mg/mL gentamicin solution. For every Raman spectrum, the area under the curve (AUC) of a distinct gentamicin peak was determined and the ratio between the gentamicin applied and the AUC of the gentamicin in the biofilm was calculated.
Fig 6.
Scratch assays for evaluating the virulence of biofilm components regarding their effect on wound healing.
A confluent monolayer of human skin keratinocytes were wounded and then treated with biofilm-conditioned media from mono-species biofilms of P. aeruginosa and S. aureus as well as dual-species biofilms. Concentrations of 75%, 50%, and 25% were applied. Further, (A) acquired images, from which representatives are shown, were used for calculating the (B) wound closure relative to the initial wound area.
Fig 7.
Analysis of the host cells’ pro-inflammatory response and viability following treatment with cell-free biofilm components.
(A) The supernatant of scratch assays was used to evaluate the release of the cytokine IL-6 by an ELISA assay. Results are expressed as fold changes related to the untreated, wounded control. The dashed line indicates a fold change of 1. (B) Keratinocytes were treated with biofilm-conditioned media (BCM) from P. aeruginosa and S. aureus mono-species and dual-species biofilms in concentrations of 75%, 50%, and 25% to further determine the cell viability after BCM treatment by an MTT assay. The results were calculated in relation to the untreated control. *p < 0.05, **p < 0.01, and ***p < 0.001.