Endotracheal Tube Mucus for the Growth and Analysis of Pseudomonas aeruginosa Biofilms

Introduction: Muco-obstructive pulmonary diseases (MOPD) including cystic fibrosis (CF) are characterized by the accumulation of hyperconcentrated mucus within the airways that promotes chronic inflammation and infection.1 With the implementation of highly effective modulator therapies (HEMTs), the availability of CF sputum has decreased drastically, limiting the biophysical and biochemical analyses that can be performed on physiologically relevant mucus specimens. Further Pseudomonas aeruginosa infection continues to persist despite HEMT usage. Endotracheal tube (ETT) mucus has previously been shown to be compositionally and rheologically similar to human bronchial epithelial (HBE) mucus that is often used for CF research.2 Herein, the utilization of ETT mucus for the growth and analysis of Pseudomonas aeruginosa biofilms is described in comparison to HBE mucus to serve as a physiologically relevant growth model for infection research in CF.

Methods: Endotracheal tube mucus was collected from the UNC surgical unit post-op as previously described.2 Mucus samples from a minimum of 10 patients were pooled, corrected for tonicity, and diluted to 4% total solids and characterized with SEC-MALS, macrorheology, and microrheology. Mucus from HBE cultures was prepared at 4% solids for comparison. P. aeruginosa biofilms were grown in HBE and ETT mucus as previously described,3 treated with tobramycin, and characterized for changes in viscoelastic moduli and viability. Mucociliary transport rates in coordinated ciliated cultures were quantified via fluorescence video microscopy.

Results: Mucin complexes in ETT were an order of magnitude larger than those in HBE mucus. Biofilms grown in ETT and HBE mucus were composed of ~1010 CFU/mL and were similar susceptible to tobramycin with a 5-log reduction in viability after treatment at 1 mg/mL. Biofilms grown in ETT mucus were mechanically robust and exhibited complex viscosity values 4x greater than those grown in HBE mucus. Transport of ETT mucus was faster than HBE mucus at 4% solids. While biofilm transport rates were slower than those of mucus, biofilms grown in ETT mucus likewise transported more rapidly than those grown in HBE mucus.

Conclusions: Endotracheal tube mucus represents an attractive and physiologically relevant growth model for biofilm infection in MOPD such as CF. Similar to HBE mucus, the rheological properties can be tuned via adjusting concentration. Uniquely, ETT mucus contains a higher non-mucin protein content than HBE mucus which may contribute to altered rheological behavior of biofilms grown therein. As ETT mucus is directly collected from human subjects, this mucus may serve as a more relevant growth environment model of P. aeruginosa biofilms. Both mucus types support the formation of mechanically robust biofilms with similar bacterial loads and antibiotic susceptibility, but viscoelastic properties and mucociliary transport rates vary between mucus types. This phenomenon may be due in part to the disparity in mucin complex size between ETT and HBE mucus.