(563) Novel Porphyrin Targets Biofilm, Pseudomonas aeruginosa, and Staphylococcus aureus

Background: Infection and colonization with multidrug-resistant (MDR) bacteria is a major problem in cystic fibrosis (CF). Pseudomonas aeruginosa (PsA) and methicillin-resistant Staphylococcus aureus (MRSA) infection worsen lung function, decrease quality of life, and lead to hospitalization in CF, making development of novel antimicrobials a high research priority. An ideal antimicrobial would: 1) kill both Gram-positive and Gram-negative organisms, 2) deconstruct biofilm, an important bacterial defense mechanism and virulence factor in CF, 3) distribute in the lungs, and 4) allow for inhalational delivery to limit toxicity. Porphyrins are tetrapyrole macrocycles capable of binding metal ions and exhibit diverse functions in nature including oxygen transport (heme), light harvesting in plants (chlorophyll), and redox functions in cells resulting in energy production (cytochromes). We synthesized a novel zinc porphyrin molecule, ZnPor (Zn for zinc, Por for porphyrin), with activity against both Gram-positive and Gram-negative bacteria and minimal toxicity in pulmonary epithelial cells. ZnPor also deconstructs biofilms.

Methods: ZnPor’s ability to disrupt Staphylococcus aureus (SA) biofilm was assessed in vitro. We used a bioluminescent SA, which enables high throughput screening of antibiofilm activity. A single colony of an MRSA USA300 genetically engineered to stably express the Lux operon (generous gift from Dr. RD. Plaut, FDA, Bethesda, MD) was inoculated into TSB broth and grown overnight with shaking. The next day, broth containing the bacteria was diluted 1/100 and added into sterile 96-well black flat-bottomed tissue culture plates and incubated at 37^oC overnight without shaking to promote formation of static biofilms. After incubation and washing, ZnPor or TSB alone were added to wells, plates were incubated for 18 hours, and luminescence was measured using a BioTek Cytation 3 plate reader. For in vivo experiments, efficacy of ZnPor against PsA pneumonia was investigated in wildtype (WT) mice. Animals were anesthetized and infected with 5 x 10⁶ CFU PsA (strain PA01) via intratracheal route. Immediately following infection, mice were treated with ZnPor (125 µM) or PBS and monitored for signs of toxicity. At 16 h, bronchoalveolar lavage (BAL) and lungs were isolated. Bacterial titers were measured in BAL and lungs in ZnPor-treated versus untreated mice.

Results: Compared to biofilms growing in TSB alone, addition of ZnPor (50 µg/ml) resulted in significant reduction in the bioluminescence signal (598 Light Units in ZnPor-treated vs. 5206 Light Units in PBS-treated) indicating efficient inhibition of biofilm formation by ZnPor. For in vivo experiments, ZnPor treatment resulted in significantly decreased PsA infection measured in BAL compared with no treatment (mean 3.32 log (CFU/ml) for ZnPor-treated vs. mean 4.72 log (CFU/ml) for PBS-treated; p < 0.05, Mann-Whitney-U, 5 mice/group). No clinical toxicity was observed.

Conclusions: ZnPor shows activity against SA biofilm in vitro using a bioluminescent reporter model. In addition, ZnPor shows efficacy against PsA and is not toxic in an in vivo animal model of PsA pneumonia. Experiments are underway to establish anti-biofilm activity of ZnPor and antimicrobial efficacy and safety of ZnPor in vivo with the long-term goal of developing a novel antimicrobial porphyrin agent ideal in CF.

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