(511) Loss of pilus-mediated twitching motility in early Pseudomonas aeruginosa infections in cystic fibrosis patients

Background: Up to 40% of patients fail eradication therapy with inhaled tobramycin (TOB), which is commonly used in new-onset
Pseudomonas aeruginosa (PA) infections to prevent progression to chronic infection in Cystic Fibrosis (CF) patients. Our group and others have previously found that PA isolates from patients who failed eradication therapy were associated with a non-twitching (loss of pilus-mediated motility) phenotype, and demonstrated resistance to in vitro neutrophil (Nφ)-mediated opsonophagocytosis and intracellular bacterial killing (OPK). Furthermore, the loss of twitching phenotype was a significant predictor of resistance to in vitro OPK among PA clinical isolates. In this study, we asked whether PA isolates that exhibit impaired in vitro OPK also persist in vivo, and whether restoring pilus-mediated motility is sufficient to restore the isolates’ susceptibility to in vitro OPK and in vivo bacterial clearance.

Methods: From a cohort of CF children with new-onset PA infections (SickKids, Toronto), we used PA clinical isolates collected immediately prior to TOB. We selected representative Eradicated isolates (N=2) from patients who successfully eradicated PA, and Persistent isolates (N=2) from those who failed PA eradication. We first tested these PA isolates for in vitro OPK, ROS production and degranulation (CD63 surface expression) using primary murine bone marrow-derived Nφs. In vivo bacterial clearance was compared in a murine pulmonary infection model. In a larger subset of Persistent and Eradicated isolates (N=15), we performed targeted comparative genome analysis of genes required for twitching motility to identify mutations potentially responsible for loss of twitching motility. Genetic complementation of pilA was done by chromosomal insertion of an inducible pBAD-pilA construct into non-twitching clinical PA isolates to restore twitching motility.

Results: Compared to Eradicated isolates, Persistent isolates elicited lower ex vivo OPK and CD63 expression in primary mouse Nφs. Mice infected with Eradicated isolates 513 and 558 completely cleared their infection by 96h.p.i, while none of the mice infected with Persistent isolates 565 and 505 did (100% vs. 0% clearance, P< 0.0001). Comparative genome analysis revealed that pilA was absent in 565, but a 369 bp truncated pilA found in 14 out of 15 isolates did not cause complete loss of twitching motility. We also restored twitching motility and susceptibility to Nφ-mediated OPK in non-twitching Persistent isolates 565 and 578 by genetically complementing pilA. Studies to determine whether restoration pilus-mediated motility improves in vivo clearance is underway.

Conclusions: Persistent PA isolates from patients who failed eradication therapy show resistance to Nφ-mediated antibacterial functions and bacterial persistence in vivo. Loss of pilus-mediated motility alters Nφ-PA interaction and contributes to impaired bacterial clearance of these Persistent PA isolates.

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