We request NIH support to investigate the use of functionalized gold nanoclusters as an adjuvant therapy for chronic suppurative otitis media (CSOM). CSOM is characterized by a chronically discharging infected middle ear that is most frequently caused by Pseudomonas aeruginosa (PA) and is a leading cause of permanent hearing loss. In CSOM, PA exists in bacterial communities known as biofilms. Fluoroquinolone eardrops are the primary method of treatment due to its non-ototoxicity. However, this treatment is ineffective in fully eradicating the infection. This is a result of the inability of fluoroquinolone to target a subpopulation of metabolically inactive bacteria known as persister cells within the biofilms. Persister cells repopulate the biofilm niche after therapy is discontinued, causing a relapse of CSOM. The end result is multiple rounds of surgery and a lifelong struggle with this disease. There is a large unmet medical need to develop new medical therapies aimed at persister cells in biofilms of CSOM. Our lab has recently created and validated a novel PA CSOM animal model with bioluminescent strains of PA allowing real time tracking of disease. It mimics the human condition by persisting beyond six months, is recalcitrant to topical fluoroquinolone therapy, and leads to hair cell death (i.e. hearing loss) over time. Using our unique model of PA CSOM, we are now able to test novel therapeutics for CSOM. Consequently, we have created an anionic hydrophilically functionalized gold nanocluster covalently conjugated with cell penetrating peptide (AuNC@CPP) as an adjuvant for fluoroquinolones. We have shown, in vitro, that co-administration of the AuNC@CPP with ofloxacin completely eliminates persister cells in both stationary phase and biofilms of PA. The exceptional killing ability of AuNC@CPP plus ofloxacin opens up the possibility to develop the first therapeutic that specifically targets PA CSOM. We theorize that our AuNC@CPP combined with ofloxacin therapy can eradicate PA CSOM in vivo. Because our preliminary data shows that AuNC@CPP does not increase intracellular ofloxacin concentration, we hypothesize that AuNC@CPP acts as both an enhancer of reactive oxygen species (ROS)-mediated ofloxacin lethality and inhibits bacterial DNA repair. We propose to determine the mechanism(s) behind how AuNC@CPP potentiates ofloxacin lethality of persister cells by measuring intracellular ROS production and evaluating RecA inhibition followed by in vivo studies for efficacy and ototoxicity in comparison to ofloxacin alone. The proposed work will develop the first adjuvant therapy that specifically targets recalcitrant CSOM and lays the foundation for future research of this treatment in other chronic biofilm infections.
Chronic suppurative otitis media (CSOM) is a significant world health problem affecting more than 330 million people worldwide that has no effective therapy and is the most common cause of permanent hearing loss among children in the developing world. Fluoroquinolone eardrops are the primary method of treatment but are ineffective in fully eradicating the infection, of which Pseudomonas aeruginosa (PA) is a leading culprit, due to the inability of the drug to target the metabolically inactive persister cells within the biofilm. In this research project, we aim to evaluate the mechanisms, efficacy, and ototoxicity of a novel, functionalized gold nanocluster covalently conjugated with cell penetrating peptide (AuNC@CPP) as an adjuvant for fluoroquinolone treatment of CSOM, which in preliminary in vitro studies has shown a remarkable ability to eliminate persister cells when co-administered with ofloxacin.
