Evaluating a polymicrobial biofilm model for structural components by co-culturing Komagataeibacter hansenii produced bacterial cellulose with Pseudomonas aeruginosa PAO1

Abstract

A polymicrobial biofilm model of Komagataeibacter hansenii and Pseudomonas aeruginosa was developed to understand whether a pre-existing matrix affects the ability of another species to build a biofilm. P. aeruginosa was inoculated onto the preformed K. hansenii biofilm consisting of a cellulose matrix. P. aeruginosa PAO1 colonized and infiltrated the K. hansenii bacterial cellulose biofilm (BC), as indicated by the presence of cells at 19 μm depth in the translucent hydrogel matrix. Bacterial cell density increased along the imaged depth of the biofilm (17-19 μm). On day 5, the average bacterial count across sections was 67 ± 4 % P. aeruginosa PAO1 and 33 ± 6 % K. hansenii. Biophysical characterization of the biofilm indicated that colonization by P. aeruginosa modified the biophysical properties of the BC matrix, which inlcuded increased density, heterogeneity, degradation temperature and thermal stability, and reduced crystallinity, swelling ability and moisture content. This further indicates colonization of the biofilm by P. aeruginosa. While eDNA fibres - a key viscoelastic component of P. aeruginosa biofilm - were present on the surface of the co-cultured biofilm on day 1, their abundance decreased over time, and by day 5, no eDNA was observed, either on the surface or within the matrix. P. aeruginosa-colonized biofilm devoid of eDNA retained its mechanical properties. The observations demonstrate that a pre-existing biofilm scaffold of K. hansenii inhibits P. aeruginosa PAO1 eDNA production and suggest that eDNA production is a response by P. aeruginosa to the viscoelastic properties of its environment.

Keywords: Biofilm; Co-culture; EPS; Extracellular DNA; Komagataeibacter hansenii; Microbial population; Pseudomonas aeruginosa PAO1; Structural integration.

Fig. 1

Scanning Electron Microscopy (SEM) images of biofilms visualized with 2K (A&B, Scale – 10 μm) and 5K (a&b, Scale – 10 μm) magnification for (A, a) BC from K. hansenii and (B, b) BC co-cultured with P. aeruginosa PAO1. Black arrow indicates EPS deposition on BC fibers and bacterial cells.

Fig. 2

SEM images of biofilms treated with NaOH at 12K (Scale – 1 μm) magnification. (a) BC control from K. hansenii monoculture (b) BC co-cultured with P. aeruginosa PAO1.

Fig. 3

XRD patterns of biofilms - BC from K. hansenii, BC co-cultured with P. aeruginosa PAO1, n = 3. (A) with NaOH and (B) without NaOH treatment.

Fig. 4

TGA and DTG thermograms of biofilms - BC from K. hansenii, BC co-cultured with P. PAO1, n = 3. TGA thermograms of the biofilms (A) with and (C) without NaOH treatment and DTG thermograms of the the biofilms (B) with and (D) without NaOH treament.

Fig. 5

FTIR-ATR spectra of BC from K. hansenii monoculture and BC co-cultured with P. aeruginosa PAO1, n = 3. (A) NaOH-treated biofilms, (B) untreated biofilms, (C) P. aeruginosa PAO1 monoculture biofilm. Gray blocks represent characteristics cellulose vibration peaks.

Fig. 6

Confocal images of biofilm. Green - YFP-Pseudomonas aeruginosa PAO1; Red - RFP-Komagataeibacter hansenii 53582; Yellow = green + red - RFP-Komagataeibacter hansenii 53582. (A) Composition and abundance of microbes in co-cultured biofilm across 19 μm-thick biofilm (day 4). (B) Monoculture P. aeruginosa PAO1 biofilm (day 5) showing eDNA at depth of 3 μm from the biofilm-medium interface. (C) Monoculture K. hansenii BC biofilm (day 5) at a depth of 3 μm from the biofilm-medium interface. (D,E,F) Co-cultured biofilms stained with SYTO 9 (green) and propidium iodide (red) a nucleic acid stain (day 5) showing infiltration of P. aeruginosa PAO1 into BC matrix at 4, 9 and 17 μm thickness, respectively. (G,H,I) Co-cultured biofilms at day 2, 3 and 5 stained with TOTO-1 (green) eDNA stain. All three images were at 3rd μm of 18 μM thick biofilm. BC co-cultured with P. aeruginosa PAO1 biofilm showing uniform distribution of bacterial cells with green eDNA fibres (day 2) and absence of eDNA at day 5 (Scale – 10 μm). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Visco-elasticity of biofilms - BC from K. hansenii, BC co-cultured with P. aeruginosa PAO1. (A) Biofilms with NaOH treatement, (B) biofilm without NaOH treatement, (C) P. aeruginosa PAO1 monoculture biofilm. Tan δ, increased storage modulus (i.e., elasticity) relative to the loss modulus (i.e., viscosity) in BC(PAO1) compared to BC control (n = 3; t statistic = 1.77, DF = 13, p = 0.099). At 0.05 confidence interval, the viscoelasticity of BC(PAO1) is not significantly different from BC control.

Fig. 8

Mechanical characterizations of co-cultured biofilms processed by press drying and freeze drying, treated with NaOH – BC control, BC co-cultured with P. aeruginosa PAO1. (A) Tensile strength of press dried films, (B) Tensile strength of freeze-dried films, (C) Tensile strength at break, inset shows low tensile strength, (D) Percentage elongation at break € Modulus of Elasticity, inset shows low elasticity (n = 8*3; Tensile strength - t statistic = 1.09, DF = 5, p = 0.32; Elongation at break - t statistic = 1.07, DF = 5, p = 0.33); Young's modulus - t statistic = 1.07, DF = 5, p = 0.35. At 0.05 confidence interval, the mechanical properties of BC(PAO1) are not significantly different from BC control.