(561) Loss of Flagellar Motility Enhances Aggregation and Drives Antibiotic Tolerance in Pseudomonas aeruginosa

Background: Cystic fibrosis (CF) is characterized by accumulation of mucus within the airways and heightened presence of the immune system, with neutrophils among the most abundant cell type. Chronic bacterial infections, in which Pseudomonas aeruginosa (Pa) is the dominant pathogen, are common in people with CF. Through unknown mechanisms, chronic Pa infections exhibit high rates of antibiotic treatment failure that is not linked to in vitro antibiotic resistance. In the CF lung, Pa is known to form biofilm-like aggregates that are believed to heavily contribute to antibiotic treatment failure. Neutrophils contribute to lung pathology through degranulation and discharge of neutrophil extracellular traps (NETs), in which the serine protease, neutrophil elastase (NE), is the primary effector. During exacerbations of CF, the presence of NE in the lung dramatically increases. NE has previously been shown to degrade and downregulate Pa flagella, although it is unknown what effect NE has on Pa treatment outcomes.

Methods: We utilize a well characterized synthetic sputum medium (SSM) that recapitulates the nutritional composition of the CF lung and contains disease relevant concentrations of mucus. However, in vitro systems lack host immune factors. We performed antibiotic tolerance and persistence assays of Pa strain PAO1 in SSM containing varying neutrophil effectors found in NETs to assess the role of neutrophils on antibiotic treatment failure following high-dose tobramycin therapy. We also assess aggregation by laser scanning confocal microscopy.

Results: We show that the addition of neutrophil elastase, but not other NET effectors, significantly increases tolerance of Pa to tobramycin in SSM. We observed that aggregation also increased in the presence of neutrophil elastase. We also show that a flagellar mutant (deletion of the major flagellin subunit, fliC) of Pa exhibits significantly increased tolerance to tobramycin compared to wildtype (WT). A flagellar mutant exhibits significantly more aggregation as well. In addition, a flagellar mutant exhibited true persistence towards tobramycin, with significant survival even after 72 hours tobramycin exposure. In the absence of tobramycin, WT and ΔfliC did not exhibit any competition. However, in the presence of tobramycin, ΔfliC significantly outcompeted WT.

Conclusions: The evolutionary steps that drive loss of motility in Pa during chronic infection of the CF lung are not fully known. Given that NE is known to act on Pa flagella, and that flagellar mutants exhibit increased fitness in the presence of antibiotics, our study sheds light on the potential evolutionary pathway that drives loss of motility in chronically adapted Pa isolates. Antibiotic tolerance and persistence assays, as well as competition assays suggest that loss of flagella provides a fitness advantage in the presence of antibiotics. Additionally, increased aggregation of ΔfliC correlated with increased antibiotic tolerance, suggesting a vital role of aggregation in the development of antibiotic tolerance. In conclusion, our study highlights a previously unknown role of the immune system in driving antibiotic tolerance during chronic Pa infection. Between the neutrophilic response and antibiotic pressure, there is an evolutionary advantage for loss of flagella, leading to non-motile, high aggregating populations of Pa (Figure 1).

Acknowledgements: This work is supported by the Cystic Fibrosis Foundation grant WOLFGA19G0.

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