Mucus polymer concentration and in vivo adaptation converge to define the antibiotic response of Pseudomonas aeruginosa during chronic lung infection

ABSTRACT The airway milieu of individuals with muco-obstructive airway diseases (MADs) is defined by the accumulation of dehydrated mucus due to hyperabsorption of airway surface liquid and defective mucociliary clearance. Pathological mucus becomes progressively more viscous with age and disease severity due to the concentration and overproduction of mucin and accumulation of host-derived extracellular DNA (eDNA). Respiratory mucus of MADs provides a niche for recurrent and persistent colonization by respiratory pathogens, including Pseudomonas aeruginosa, which is responsible for the majority of morbidity and mortality in MADs. Despite high concentration inhaled antibiotic therapies and the absence of antibiotic resistance, antipseudomonal treatment failure in MADs remains a significant clinical challenge. Understanding the drivers of antibiotic tolerance is essential for developing more effective treatments that eradicate persistent infections. The complex and dynamic environment of diseased airways makes it difficult to model antibiotic efficacy in vitro. We aimed to understand how mucin and eDNA concentrations, the two dominant polymers in respiratory mucus, alter the antibiotic tolerance of P. aeruginosa. Our results demonstrate that polymer concentration and molecular weight affect P. aeruginosa survival post antibiotic challenge. Polymer-driven antibiotic tolerance was not explicitly associated with reduced antibiotic diffusion. Lastly, we established a robust and standardized in vitro model for recapitulating the ex vivo antibiotic tolerance of P. aeruginosa observed in expectorated sputum across age, underlying MAD etiology, and disease severity, which revealed the inherent variability in intrinsic antibiotic tolerance of host-evolved P. aeruginosa populations. IMPORTANCE Antibiotic treatment failure in Pseudomonas aeruginosa chronic lung infections is associated with increased morbidity and mortality, illustrating the clinical challenge of bacterial infection control. Understanding the underlying infection environment, as well as the host and bacterial factors driving antibiotic tolerance and the ability to accurately recapitulate these factors in vitro, is crucial for improving antibiotic treatment outcomes. Here, we demonstrate that increasing concentration and molecular weight of mucin and host eDNA drive increased antibiotic tolerance to tobramycin. Through systematic testing and modeling, we identified a biologically relevant in vitro condition that recapitulates antibiotic tolerance observed in ex vivo treated sputum. Ultimately, this study revealed a dominant effect of in vivo evolved bacterial populations in defining inter-subject ex vivo antibiotic tolerance and establishes a robust and translatable in vitro model for therapeutic development.


Figure S2 .
Figure S2.Mucin and eDNA do not affect growth kinetics of P. aeruginosa.(A) Growth curve of mPAO1 expressing gfp, in LB or SCFM2 with 0%, 0.5%, 1%, 2%, and 4% w/v mucin and no eDNA for 24 h.(B) WT mPAO1 growth curve in SCFM2 lacking mucin, with increasing concentrations of HMW eDNA.Fluorescence (483 / 535 nm) (A) or absorbance (600 nm) (B) measurements were taken every 15 min using a Tecan Infinite 200 PRO plate reader (Tecan Group Ltd., AG, Switzerland).Data are representative of the mean from n = 3 biological replicates in technical duplicate.(C and D) CFU of mPAO1 after 8 h of growth in SCFM2 with (C) increasing mucin or (D) increasing eDNA concentration.Statistical analysis performed by one-way ANOVA with Tukey's multiple comparison correction.ns indicates not significant (p-value > 0.05).

Figure S4 .
Figure S4.High molecular weight eDNA is necessary to impart tobramycin tolerance.(A) DNA gel electrophoresis of salmon sperm DNA from different commercial sources compared to a representative sputum from a CF subject.(B-C) % survival of mPAO1 after treatment with 300 μg/mL tobramycin in SCFM2 with (B) 0.5% or (C) 2% w/v mucin and increasing concentrations of LMW eDNA.Statistical differences were determined by one-way ANOVA.(D) Gel electrophoresis of SCFM2 containing 2% mucin and 1 mg/mL HMW DNA treated with rhDNase (3 μg/mL) for 0, 1, 2 or 3 h.(E) Effect of rhDNase (3 μg/mL) on the growth of mPAO1 in 2% mucin SCFM2 after

Figure S5 .
Figure S5.Antibiotic diffusion was not affected by polymer concentration.Diffusion rate of Texas Red conjugated tobramycin through (A) LB and SCFM2 with increasing mucin concentrations and (B) 0.5% w/v mucin SCFM2 with increasing concentrations of eDNA.Data are presented as the mean ± SD of n = 3 replicates.Statistical analysis was performed by oneway ANOVA.ns indicates not significant (p-value > 0.05).
Figure S6.PLS regression model and simulation.(A-F) Linear trend and 95% confidence intervals of the relationship between the outcome variables (tobramycin survival and

Figure S7 .
Figure S7.Antibiotic survival and complex viscosity of final media composition.(A) Tobramycin survival of mPAO1, and (B) complex viscosity of SCFM2 containing 1 mg/mL HMW eDNA and increasing % w/v mucin.Statistical differences were determined using one-way ANOVA with Tukey's multiple comparison correction.*p<0.05,**p<0.01,****p<0.0001.Data represent the mean ± SD of n ≥ 3 biological replicates.The viscosity of water is indicated by the solid blue line, and the complex viscosity at which mucus entangles and becomes gel-like (42) is indicated by the dashed gray line.+ indicates the median for each sample.

S3. P. aeruginosa exhibits mucin concentration-dependent tolerance to multiple antibiotic classes. Survival
ns Figure

In vitro mucin concentration-dependent antibiotic survival of clinical populations.
Percent survival of ex vivo treated sputum and P. aeruginosa populations recovered from each subject mock treated sputum evaluated in vitro for tobramycin tolerance in LB and SCFM2 containing 1 mg/mL HMW eDNA with increasing %w/v mucin.Hyperresistant sputum populations (MIC ≥ 80) were excluded.Data are representative of n = 3 independent replicates