(539) Single Sequential Bacteriophage Therapy Decreases Pseudomonas Virulence in Cystic Fibrosis Compared to a Cocktail Approach

Background: In adults with cystic fibrosis (CF),

Pseudomonas aeruginosa

(PA) is the most common sputum pathogen and is increasingly
multidrug-resistant (MDR). To address this problem, we are using lytic bacteriophages (phages) to design personalized phage therapy. Lytic phages

are bacteria-specific viruses that self-amplify to kill bacteria. Type IV pilus (TIVP) is a virulence factor that PA uses to attach to airway epithelial cells. We are investigating mechanisms for TIVP targeting phage (TIVP-H6) to not only kill MDR PA but also reduce PA virulence by impairing TIVP function in surviving PA mutants. In addition, comparisons are made between single sequential and cocktail strategies of phage therapy.

Methods: Six patients with MDR PA were treated with TIVP-phage via FDA-approved emergency IND and IRB approval. We selected single sequential, or in a few patients cocktail, for TIVP phage therapy. Phage was delivered via nebulization daily (outpatient) or twice daily (inpatient) for 7-10 days. Sputum and spirometry were obtained pre- and post-phage therapy. Sputum was processed to measure PA density and metagenomics.
PA isolates were cultured to mid-exponential growth phase (OD₆₀₀=0.5). Pyocyanin was extracted and quantified from PA supernatant. In vitro, PA supernatant was used to stimulate airway epithelial cell (BEAS-2b) line for comparison of IL-8, IL-6 and EGFR phosphorylation via ELISA. Attachment Assay: PA was co-cultured 1-hour with CF airway epithelial cell (CFBE41o-) line and a control (16HBEo-) at air liquid interface. Adherent PA were cultured overnight for density comparison.

Results: After phage treatment, sputum PA titers decreased significantly (2.2±0.76 log reduction, p< 0.05). After phage therapy, precent predicted FEV1 (ppFEV1) increased 0%-8.9%; pre [day -30 to -1] and post [day 21 to 35] (p< 0.01). In vitro, single sequential pre-phage adhered 8.7%±SEM 1.6% compared to post-phage at 0.98%±SEM 0.35% (p< 0.01), which is consistent with the observation that PA post-TIVP-H6 decreasing TIVP. We studied PA TIVP mutant strains PA103pilA and PA103pilQ, and a similar effect was observed. PA103 isotype adhered 4.5%±SEM 0.38% compared to PA103pilA 2.6%±SEM 0.42% (and p< 0.05) and PA103pilQ 2.8%±SEM 0.95% (p=0.29). In comparison, cocktail pre-phage adhered at 3.6%±SEM 0.77% compared to post-phage at 2.7%±SEM 0.93%. Previously, we described differences between the airway epithelial inflammation between single sequential and cocktail phage therapy strategies. Using PA supernatants pre- and post-TIVP phage to stimulate airway epithelial cells we saw a similar effect in markers of in inflammation (e.g., decreased IL-6 and IL-8 production and EGFR-phosphorylation post phage therapy).

Conclusions: MDR PA is increasingly common in CF and exceedingly difficult to manage. TIVP phage therapy is a novel approach to investigate targeting a bacteria virulence factor in the human lung. We found that phage therapy decreases sputum PA titers and improves ppFEV1. Single sequential TIVP phage therapy decreased PA adherence compared to cocktail, which is consistent with our observation of the effect of TIVP phage therapy on airway epithelial inflammation. Given the interest in phage therapy, the observation that single sequential, but not cocktail, decreases virulence may influence strategies for selecting phage therapies is CF.

Acknowledgements: This work was supported by the Cystic Fibrosis Foundation [grant STANLE20D0] and the National Institutes of Health [grant 5 T32 HL007778].

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