Synergy of AcpP PPMO and Piperacillin/Tazobactam in the Breakdown of Pseudomonas aeruginosa PA01 Biofilms

INTRODUCTION: Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium and one of the most common causes of hospital-acquired infection, especially in immunocompromised patients. It is particularly pathogenic because of its ability to form biofilm, an extra-cellular matrix that makes it more resistant to host defenses and antibiotic therapies. Combination therapies have proven to be more effective at clearing biofilms because they target different processes or cell populations, but concerns about toxicity and antibiotic resistance have led to the exploration of alternative therapies that target biofilm formation at a genetic level. One such alternative is the use of peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), which are sequence-specific antisense oligomers that target the mRNA of bacterial genes and prevent translation of particular proteins. HYPOTHESIS: PPMOs targeting the essential gene AcpP will act synergistically with the antibiotic Piperacillin/Tazobactam (Pip/Tazo) in the breakdown of Pseudomonas aeruginosa PA01 biofilms in vitro. METHODS: To test synergy between the PPMO and antibiotic, PA01 biofilms were grown in filtered Mueller Hinton broth II (MHII) on minimum biofilm eradication concentration (MBEC) plates. MBEC plates are 96-well plates that have pegs attached to the lid to provide additional surface area for biofilm growth. After 24 hours of growth, the biofilm-covered pegs were switched to another 96-well plate containing fresh media with different combinations of antibiotic and PPMO in each well. Three such doses were administered every 8 hours, and at 48 hours of total growth, the biofilm remaining on the pegs was analyzed by one of four methods: 1) crystal violet assay, 2) resazurin assay, 3) CFU count, and 4) confocal microscopy. RESULTS: Crystal violet and resazurin assays demonstrated that Pip/Tazo and AcpP PPMO were potentially synergistic in biofilm breakdown for Pip/Tazo 0.5-0.0625 ug/mL and AcpP 5-0.625 uM. For several combinations, CFU measurements yielded 2-log or 3-log reduction in CFU compared to the control and synergistic effects on biofilm breakdown, particularly in cases where the antibiotic alone had no effect. While confocal microscopy demonstrated a decrease in viable bacteria with antibiotic treatment and PPMO treatment, the most significant eradication of biofilm occurred with combined treatment. CONCLUSION: The synergy demonstrated in vitro between AcpP PPMO and Pip/Tazo in the breakdown of P. aeruginosa PA01 biofilms is promising for in vivo studies as well, since PPMOs have the potential to increase biofilm sensitivity to lower doses of traditional antibiotics.