Objectives: The proliferation of metallo-beta-lactamase-producing Pseudomonas aeruginosa represents a significant public health threat. P. aeruginosa can undergo significant phenotypic changes that can drastically impair antibiotic efficacy. This study’s objectives were (1) to quantify the time course of killing of VIM-2-producing P. aeruginosa in response to aztreonam-based therapies, and (2) to document the capacity of P. aeruginosa to undergo morphological transformations that facilitate persistence.

Methods: A well-characterized, clinical VIM-2-producing P. aeruginosa was studied in the Hollow Fiber Infection Model (HFIM) over 9 days (7 days of active antibiotic therapy, 2 days treatment withdrawal) at a 10^7.5 CFU/mL starting inoculum. HFIM treatment arms included: growth control, aztreonam, ceftazidime/avibactam, aztreonam/‌ceftazidime/‌avibactam, polymyxin B, and aztreonam/‌ceftazidime/‌avibactam/‌polymyxin B. In addition, real-time imaging studies were conducted under static conditions to determine the time course of the reversion of persister cells. 

Results: A pronounced discrepancy was observed between OD₆₂₀ and bacterial counts obtained from plating methods (hereafter referred to as ‘OD-count discrepancy’). For aztreonam monotherapy, observed counts were 0 CFU/mL by 120 h. Despite this, there was a significant OD-count discrepancy as compared to the pre-treatment 0h. Between therapy withdrawal at 168h and 216h, all arms with suppressed counts had re-grown to the system carrying capacity. Real-time imaging of the P. aeruginosa filaments after drug removal showed rapid reversion from a long, filamentous phenotype to many individual rods within 2 h.

Conclusion: Managing MBL-producing P. aeruginosa will require a multi-faceted approach, focused on maximizing killing and minimizing proliferation of resistant and persistent subpopulations, which will involve eliminating drug-induced phenotypic transformers.