In vitro susceptibility of established biofilms composed of a clinical wound isolate of Pseudomonas aeruginosa treated with lactoferrin and xylitol

Abstract

The medical impact of bacterial biofilms has increased with the recognition of biofilms as a major contributor to chronic wounds such as diabetic foot ulcers, venous leg ulcers and pressure ulcers. Traditional methods of treatment have proven ineffective, therefore this article presents in vitro evidence to support the use of novel antimicrobials in the treatment of Pseudomonas aeruginosa biofilm. An in vitro biofilm model with a clinical isolate of P. aeruginosa was subjected to treatment with either lactoferrin or xylitol alone or in combination. Combined lactoferrin and xylitol treatment disrupted the structure of the P. aeruginosa biofilm and resulted in a >2log reduction in viability. In situ analysis indicated that while xylitol treatment appeared to disrupt the biofilm structure, lactoferrin treatment resulted in a greater than two-fold increase in the number of permeabilised bacterial cells. The findings presented here indicated that combined treatment with lactoferrin and xylitol significantly decreases the viability of established P. aeruginosa biofilms in vitro and that the antimicrobial mechanism of this treatment includes both biofilm structural disruption and permeablisation of bacterial membranes.

Fig. 1.

Penetration of lactoferrin into established Pseudomonas aeruginosa biofilm. Left panel, transmission light microscopy imaging through a single plane indicates the microcolony structure of biofilms. Right panel, epifluorescent microscopy imaging of a single plane through the microcolonies indicates that Alexa Fluor®-labelled lactoferrin can penetrate into the interior of the biofilm within 1 h.

Fig. 2.

Altered macrostructure in lactoferrin- and xylitol-treated Pseudomonas aeruginosa biofilms. 4′,6-Diamidino-2-phenylindole (DAPI)-stained biofilms are imaged directly on the coupon subsurface. Images indicate structural changes in samples treated either with control medium, 5% (w/v) xylitol, 2% (w/v) lactoferrin or 5% (w/v) xylitol and 2% (w/v) lactoferrin.

Fig. 3.

Synergistic activity of lactoferrin and xylitol treatment in reducing the viability of Pseudomonas aeruginosa bacteria. Data are presented as log reduction relative to the control, calculated from plate counts of bacteria recovered from treated biofilms. Statistically significant differences are indicated.

Fig. 4.

Live/dead imaging of treated Pseudomonas aeruginosa biofilms in situ. Epifluorescent imaging of BacLight™ LIVE/DEAD®-stained biofilms indicates cells with intact membranes (green) versus cells with permeabilised membranes (red). Samples include control, 5% (w/v) xylitol-treated cells, 2% (w/v) lactoferrin-treated cells and 5% (w/v) xylitol + 2% (w/v) lactoferrin-treated cells.

Fig. 5.

Reduction in the ratio of live cells relative to dead cells in Pseudomonas aeruginosa biofilms following treatment with lactoferrin or xylitol alone or in combination. Live versus dead cells were detected by BacLight™ LIVE/DEAD® staining and epifluorescent microscopy quantification. Statistically significant differences between ratios are indicated.

Fig. 6.

Reduction in the number of intact biofilm-grown Pseudomonas aeruginosa bacterial cells following treatment with lactoferrin or xylitol alone or in combination. Intact cells were detected and quantified by SYTO® 9 staining and epifluorescent microscopy quantification. Samples were normalised to the control. Statistically significant difference is indicated.

Fig. 7.

Increase in the number of permeabilised cells in established Pseudomonas aeruginosa biofilms following treatment with lactoferrin or xylitol alone or in combination. Permeabilised cells were detected by propidium iodide staining and were quantified by epifluorescent microscopy. Quantified samples were normalised to the control sample.