19 research outputs found
Combination treatment to improve mucociliary transport of Pseudomonas aeruginosa biofilms
People with muco-obstructive pulmonary diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) often have acute or chronic respiratory infections that are difficult to treat due in part to the accumulation of hyperconcentrated mucus within the airway. Mucus accumulation and obstruction promote chronic inflammation and infection and reduce therapeutic efficacy. Bacterial aggregates in the form of biofilms exhibit increased resistance to mechanical stressors from the immune response (e.g., phagocytosis) and chemical treatments including antibiotics. Herein, combination treatments designed to disrupt the mechanical properties of biofilms and potentiate antibiotic efficacy are investigated against mucus-grown Pseudomonas aeruginosa biofilms and optimized to 1) alter biofilm viscoelastic properties, 2) increase mucociliary transport rates, and 3) reduce bacterial viability. A disulfide bond reducing agent (tris(2-carboxyethyl)phosphine, TCEP), a surfactant (NP40), a biopolymer (hyaluronic acid, HA), a DNA degradation enzyme (DNase), and an antibiotic (tobramycin) are tested in various combinations to maximize biofilm disruption. The viscoelastic properties of biofilms are quantified with particle tracking microrheology and transport rates are quantified in a mucociliary transport device comprised of fully differentiated primary human bronchial epithelial cells. The combination of the NP40 with hyaluronic acid and tobramycin was the most effective at increasing mucociliary transport rates, decreasing the viscoelastic properties of mucus, and reducing bacterial viability. Multimechanistic targeting of biofilm infections may ultimately result in improved clinical outcomes, and the results of this study may be translated into future in vivo infection models
Mucus and mucus flake composition and abundance reflect inflammatory and infection status in cystic fibrosis
BACKGROUND: Mucus hyperconcentration in cystic fibrosis (CF) lung disease is marked by increases in both mucin and DNA concentration. Additionally, it has been shown that half of the mucins present in bronchial alveolar lavage fluid (BALF) from preschool-aged CF patients are present in as non-swellable mucus flakes. This motivates us to examine the utility of mucus flakes, as well as mucin and DNA concentrations in BALF as markers of infection and inflammation in CF airway disease. METHODS: In this study, we examined the mucin and DNA concentration, as well as mucus flake abundance, composition, and biophysical properties in BALF from three groups; healthy adult controls, and two CF cohorts, one preschool aged and the other school aged. BALFs were characterized via refractometry, PicoGreen, immunofluorescence microscopy, particle tracking microrheology, and fluorescence image tiling. RESULTS: Mucin and DNA BALF concentrations increased progressively from healthy young adult controls to preschool-aged people and school-aged people with CF. Notably, mucin concentrations were increased in bronchoalveolar lavage fluid (BALF) from preschool-aged patients with CF prior to decreased pulmonary function. Infrequent small mucus flakes were identified in normal subjects. A progressive increase in the abundance of mucus flakes in preschool and school-aged CF patients was observed. Compositionally, MUC5B dominated flakes from normal subjects, whereas an increase in MUC5AC was observed in people with CF, reflected in a reduced flaked MUC5B/MUC5AC mucin ratio. CONCLUSION: These findings suggest mucus composition and flake properties are useful markers of inflammatory and infection-based changes in CF airways
Altering the viscoelastic properties of mucus-grown Pseudomonas aeruginosa biofilms affects antibiotic susceptibility
The viscoelastic properties of biofilms are correlated with their susceptibility to mechanical and chemical stress, and the airway environment in muco-obstructive pulmonary diseases (MOPD) facilitates robust biofilm formation. Hyperconcentrated, viscoelastic mucus promotes chronic inflammation and infection, resulting in increased mucin and DNA concentrations. The viscoelastic properties of biofilms are regulated by biopolymers, including polysaccharides and DNA, and influence responses to antibiotics and phagocytosis. We hypothesize that targeted modulation of biofilm rheology will compromise structural integrity and increase antibiotic susceptibility and mucociliary transport. We evaluate biofilm rheology on the macro, micro, and nano scale as a function of treatment with a reducing agent, a biopolymer, and/or tobramycin to define the relationship between the viscoelastic properties of biofilms and susceptibility. Disruption of the biofilm architecture is associated with altered macroscopic and microscopic moduli, rapid vector permeability, increased antibiotic susceptibility, and improved mucociliary transport, suggesting that biofilm modulating therapeutics will improve the treatment of chronic respiratory infections in MOPD
Combination treatment to improve mucociliary transport of Pseudomonas aeruginosa biofilms
Representation of MCTD imaging for transport of biofilms.
MCT occurs counterclockwise with nā„3 videos per quadrant per biofilm. Figure created with biorender.com.</p
HBE mucus rheology and transport as a function of concentration and treatment conditions.
A) Mucus complex viscosity distribution with % solids concentration. Data is representative of the distribution of every tracked particle complex viscosity. B) Mucus ensemble complex viscosity as a function of % solids Ā± treatment with 10 mM TCEP. Data is presented as the mean Ā± standard deviation of complex viscosity values for nā„3 separately prepared and evaluated mucus samples. Statistical significance was determined using single factor ANOVA with post hoc Tukey analysis. C) MCT rates of mucus as a function of % solids Ā± treatment with 10 mM TCEP. Data is presented as the mean Ā± standard deviation of MCT rates for nā„6 videos of mucus transport. Statistical significance was determined using single factor ANOVA with post hoc Tukey Kramer analysis. D) Power law relationship between mucus complex viscosity values and measured MCT rates in racetrack cultures.</p
Characterization of cilia beat frequency and mucociliary transport in racetrack cultures.
A) Average cilia beat frequency for each quadrant of two separate cultures. B) Overall average cilia beat frequency from five cultures. C) Mean MCT rates of beads in PBS for five separately prepared and evaluated cultures. Data is representative of the average and standard deviation of ā„3 individual measurements.</p
Matrix of biofilm treatment combinations.
People with muco-obstructive pulmonary diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) often have acute or chronic respiratory infections that are difficult to treat due in part to the accumulation of hyperconcentrated mucus within the airway. Mucus accumulation and obstruction promote chronic inflammation and infection and reduce therapeutic efficacy. Bacterial aggregates in the form of biofilms exhibit increased resistance to mechanical stressors from the immune response (e.g., phagocytosis) and chemical treatments including antibiotics. Herein, combination treatments designed to disrupt the mechanical properties of biofilms and potentiate antibiotic efficacy are investigated against mucus-grown Pseudomonas aeruginosa biofilms and optimized to 1) alter biofilm viscoelastic properties, 2) increase mucociliary transport rates, and 3) reduce bacterial viability. A disulfide bond reducing agent (tris(2-carboxyethyl)phosphine, TCEP), a surfactant (NP40), a biopolymer (hyaluronic acid, HA), a DNA degradation enzyme (DNase), and an antibiotic (tobramycin) are tested in various combinations to maximize biofilm disruption. The viscoelastic properties of biofilms are quantified with particle tracking microrheology and transport rates are quantified in a mucociliary transport device comprised of fully differentiated primary human bronchial epithelial cells. The combination of the NP40 with hyaluronic acid and tobramycin was the most effective at increasing mucociliary transport rates, decreasing the viscoelastic properties of mucus, and reducing bacterial viability. Multimechanistic targeting of biofilm infections may ultimately result in improved clinical outcomes, and the results of this study may be translated into future in vivo infection models.</div
This file contains supporting table and supporting figures.
This file contains supporting table and supporting figures.</p
Triple combination treatment of PAO1 biofilms.
A) Biofilm complex viscosity distribution as a function of treatment conditions. Data is representative of every tracked particle complex viscosity for three separately prepared and evaluated specimens. B) Biofilm microscopic moduli as a function of treatment. The elastic modulus (Gā, solid) and the viscous modulus (Gā, dashed) are shown in green for the solid-like biofilm component and in blue for the more watery component as determined via Gaussian mixture modeling. C) Viability of PAO1 biofilms as a function of triple combination treatment. Significance was determined using single factor ANOVA with post hoc Tukey analysis. D) Mean MCT rates of biofilms as a function of triple combination treatment. Data is presented as the mean Ā± standard deviation of tracked particles for nā„6 videos. Significance was determined using single factor ANOVA with post hoc Tukey Kramer analysis.</p