Implications of Antimicrobial Combinations in Complex Wound Biofilms Containing Fungi

Abstract

Diabetic foot ulcer treatment currently focuses on targeting bacterial biofilms, while dismissing fungi. To investigate this, we used an in vitro biofilm model containing bacteria and fungi, reflective of the wound environment, to test the impact of antimicrobials. Here we showed that while monotreatment approaches influenced biofilm composition, this had no discernible effect on overall quantity. Only by combining bacterium- and fungus-specific antibiotics were we able to decrease the biofilm bioburden, irrespective of composition.

Keywords: Candida; antimicrobial; biofilm; chronic wound; polymicrobial.

FIG 1

Increased microbial complexity of biofilms leads to reduced susceptibility to antimicrobial agents. Biofilm (sessile) MIC values were calculated using the alamarBlue viability test. All tests were carried out in quadruplicates on three separate occasions as described in the text. Monoculture (S. aureus [Sa]) biofilms were compared to coculture biofilms (S. aureus plus C. albicans [Sa + Ca] or S. aureus plus P. aeruginosa [Sa + Pa]) and triadic biofilms (S. aureus plus C. albicans plus P. aeruginosa [Sa + Ca + Pa]). Data were analyzed using a two-way analysis of variance (ANOVA) with Tukey's multiple-comparison test to compare each mono- or coculture at each antimicrobial (ciprofloxacin) concentration (in micrograms per milliliter).

FIG 2

Triadic biofilm composition is influenced by antimicrobial treatment. The antibiotics flucloxacillin (FLX) and ciprofloxacin (CIPX) and the antifungal fluconazole (FLC) were used to treat the biofilms, either alone or in combination as described in the text. Biofilm percentage composition is shown in the bar graphs (A), while absolute numbers of viable colony-forming equivalents (CFE) present are shown below (B). Treated biofilms were compared to untreated controls using a two-tailed unpaired t test (*, P < 0.05). Pa/PA, P. aeruginosa; Sa/SA, S. aureus; Ca/CA, C. albicans.

FIG 3

Triple antimicrobial treatment elicits the largest impact on biofilms. The heat map shows a fold change increase in viable CFE (red), decrease (blue), or no change (white) after treatment with flucloxacillin (FLX), ciprofloxacin (CIPX), and fluconazole (FLC), either alone or in combination. Hierarchical clustering analysis (left) shows that the total bioburden is closely related to C. albicans (CA), suggesting this is the component of the biofilm that is integral to infection. SA, S. aureus; PA, P. aeruginosa.