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Targeted Disruption of the Extracellular Polymeric Network of Pseudomonas aeruginosa Biofilms by Alginate Oligosaccharides

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Targeted Disruption of the Extracellular Polymeric Network of Pseudomonas aeruginosa Biofilms by Alginate Oligosaccharides

Lydia C Powell et al. NPJ Biofilms Microbiomes.

Abstract

Acquisition of a mucoid phenotype by Pseudomonas sp. in the lungs of cystic fibrosis (CF) patients, with subsequent over-production of extracellular polymeric substance (EPS), plays an important role in mediating the persistence of multi-drug resistant (MDR) infections. The ability of a low molecular weight (Mn = 3200 g mol-1) alginate oligomer (OligoG CF-5/20) to modify biofilm structure of mucoid Pseudomonas aeruginosa (NH57388A) was studied in vitro using scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) with Texas Red (TxRd®)-labelled OligoG and EPS histochemical staining. Structural changes in treated biofilms were quantified using COMSTAT image-analysis software of CLSM z-stack images, and nanoparticle diffusion. Interactions between the oligomers, Ca2+ and DNA were studied using molecular dynamics (MD) simulations, Fourier transform infrared spectroscopy (FTIR) and isothermal titration calorimetry (ITC). Imaging demonstrated that OligoG treatment (≥0.5%) inhibited biofilm formation, revealing a significant reduction in both biomass and biofilm height (P < 0.05). TxRd®-labelled oligomers readily diffused into established (24 h) biofilms. OligoG treatment (≥2%) induced alterations in the EPS of established biofilms; significantly reducing the structural quantities of EPS polysaccharides, and extracellular (e)DNA (P < 0.05) with a corresponding increase in nanoparticle diffusion (P < 0.05) and antibiotic efficacy against established biofilms. ITC demonstrated an absence of rapid complex formation between DNA and OligoG and confirmed the interactions of OligoG with Ca2+ evident in FTIR and MD modelling. The ability of OligoG to diffuse into biofilms, potentiate antibiotic activity, disrupt DNA-Ca2+-DNA bridges and biofilm EPS matrix highlights its potential for the treatment of biofilm-related infections.

Conflict of interest statement

D.W.T. has a consultancy relationship and has, with K.E.H., received research funding from AlgiPharma AS. P.D.R. is a director/owner of AlgiPharma AS. The remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Effect of OligoG on inhibition of mucoid biofilm formation: Imaging and quantification of P. aeruginosa (NH57388A) biofilms grown for 24 h at 37 °C in MH broth ± OligoG (0.5%, 2% & 6%). a SEM imaging of biofilms (Scale bar, 10 µm). b CLSM 3D imaging (aerial view) of LIVE/DEAD® staining of biofilms (Scale bar, 20 µm). c COMSTAT image analysis of the corresponding biofilm CLSM z-stack images. *P < 0.05 significance was determined by comparison to the untreated control. Error bars represent the standard deviation of the data set (n = 3)
Fig. 2
Fig. 2
Effect of OligoG on disruption of mucoid established biofilms: CLSM 3D imaging (side view) with LIVE/DEAD® staining of P. aeruginosa (NH57388A) biofilms grown for 24 h at 37 °C in MH broth followed by 4 or 24 h OligoG treatment (0.5, 2 & 6%) (Scale bar, 15 µm) with COMSTAT image analysis of the corresponding biofilm CLSM z-stack images. Treatment times a 4 h. b 24 h. *P < 0.05 significance was determined by comparison to the untreated control. Error bars represent the standard deviation of the data set (n = 3)
Fig. 3
Fig. 3
Effect of OligoG on the EPS components of mucoid biofilms. 3D CLSM imaging of P. aeruginosa (NH57388A) biofilms stained with ConA (EPS polysaccharides, red) and TOTO-1 (eDNA, green; scale bar, 8 µm) in a biofilm formation assay, where biofilms are grown for 24 h in MH broth ± OligoG (0.5%, 2% & 6%) and b the biofilm disruption model where biofilms grown for 24 h in MH broth, followed by 24 h treatment of OligoG (0.5%, 2% & 6%). Corresponding mean fluorescence intensities (arbitrary units ×106) achieved from CLSM 3D imaging of the c biofilm formation assay and the d biofilm disruption assay. *P < 0.05 significance was determined by comparison to the untreated control. Error bars represent the standard deviation of the data set (n = 3)
Fig. 4
Fig. 4
Transwell® biofilm diffusion studies. a Schematic diagram of Transwell® device showing particle diffusion through the biofilm and microporous membrane. Boxplots of mean fluorescence intensity (arbitrary units) in the biofilm Transwell® assay after 4 h OligoG treatment (n = 5), where fluorescence was measured at b 1 h and c 2 h after fluosphere addition. *P < 0.05 significance was determined by comparison to the untreated control. Error bars represent the standard deviation of the data set (n = 3)
Fig. 5
Fig. 5
Molecular dynamics (MD) simulations of calcium, DNA and OligoG interactions. MD simulations at early and late binding of DNA double strand (15 bp) in the presence of Ca2+ (green spheres) at a 1 ns and b 58 ns. G-oligomer (DPn 16) was added to the simulations where the binding affinity was assessed at c 0 ns and d 50 ns
Fig. 6
Fig. 6
FTIR and ITC analysis of interactions of OligoG with DNA and Ca2+. FTIR absorbance spectra of the molecular interaction of OligoG with calcium and DNA a from 1800 to 900 cm−1 of pure OligoG (blue), OligoG + 5 mM Ca2+ (green), OligoG + 10 mM DNA + 5 mM Ca2+ (grey), and OligoG + 10 mM DNA (red) and b from 1200–900 cm−1 (colours as in A). Isothermic calorimetric titrations of 101 mM OligoG into 1 mM fish sperm (FS) DNA containing c 1 mM CaCl2 or d 5 mM CaCl2 (and corresponding reference experiments); buffer into buffer (grey), buffer into DNA (red), OligoG into buffer (blue), OligoG into FS DNA (green). Concentrations of OligoG and FS DNA are in terms of monomeric units and base pairs, respectively. Error bars present the standard deviations of the data set (n = 3)

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