Mucus polymer concentration and
in vivo
adaptation converge to define the antibiotic response of
Pseudomonas aeruginosa
during chronic lung infection
Authors: Greenwald Matthew A. , Meinig Suzanne L. , Plott Lucas M. , Roca Cristian , Higgs Matthew G. , Vitko Nicholas P. , Markovetz Matthew R. , Rouillard Kaitlyn R. , Carpenter Jerome , Kesimer Mehmet , Hill David B. , Schisler Jonathan C. , Wolfgang Matthew C. 
Published: April 2024

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.