Abstract

Biofilm formation of ESKAPE pathogens creates a major problem in healthcare-associated infections, and nutrient supply significantly contributes to biofilm structure and bacterial survival. An understanding of how various nutritional environments influence polymicrobial biofilm dynamics is important for the development of targeted antimicrobials. In the present work, biofilm formation of ESKAPE pathogens was tested under three different nutritional environments—nutrient broth with no supplementation (NB; minimal), nutrient broth with supplementation of 2% glucose + 2% sucrose (NB+G+S; moderate), and Brain Heart Infusion supplemented with 2% glucose + 2% sucrose (BHI-G+S; Rich). Furthermore, we studied the effect of bacterial mixtures on biofilm features in each medium. Standard ATCC strains of Staphylococcus aureus (S), Enterococcus faecalis (E), Klebsiella pneumoniae (K), and Pseudomonas aeruginosa (P) were grown alone or in combination as mono-, dual-, tri-, and tetra-species. Biofilm formation was determined by crystal violet spectrophotometric assay (n = nine biological replicates, with three technical replicates each). LIVE/DEAD staining and confocal laser scanning microscopy (CLSM) were used for samples with absorbance values > 0.5, including 3-D architectural analysis of viability using ImageJ software. Biofilm height was quantified by using the ZEN depth code tool. Statistical analysis included one-way ANOVA with Tukey’s post hoc test (p < 0.05), and unpaired t-tests were performed when applicable to compare variables. Biofilm formation was drastically increased in rich medium compared to moderate and minimal NB. The tetra-species cocktail SKEP reached the highest biofilm dry weight in BHI+G+S and greater elevation/viability compared to NB+G+S, whereas P. aeruginosa monospecies exhibited an opposite trend (greater elevation in NB+G+S than BHI+G+S). Interestingly, the E. faecalis and P. aeruginosa group exhibited strong biofilm characteristics in a range of nutritional environments. Confocal analysis showed structurally distinct biofilms: dense, vertical biofilms in BHI+G+S and sparse, horizontal patterns in NB+G+S. Nutrient composition significantly impacts polymicrobial biofilm formation, with nutrient-rich conditions (BHI+G+S) supporting robust viable three-dimensional structures, particularly within complex bacterial communities. These observations are of potential significance with respect to biofilm formation in hospital settings, such as in wounds, and may be used in strategies against the targeted intervention of polymicrobial infections.

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