*Article* **Thermal Shock and Ciprofloxacin Act Orthogonally on** *Pseudomonas aeruginosa* **Biofilms**

**Haydar Aljaafari 1,2, Yuejia Gu <sup>1</sup> , Hannah Chicchelly <sup>1</sup> and Eric Nuxoll 1,\***


**Abstract:** Bacterial biofilm infections are a major liability of medical implants, due to their resistance to both antibiotics and host immune response. Thermal shock can kill established biofilms, and some evidence suggests antibiotics may enhance this efficacy, despite having an insufficient effect themselves. The nature of this interaction is unclear, however, complicating efforts to integrate thermal shock into implant infection treatment. This study aimed to determine whether these treatments were truly synergistic or simply orthogonal (i.e., independent). *Pseudomonas aeruginosa* biofilms of different architectures and stationary-phase population density were subjected to various thermal shocks, antibiotic exposures, or combinations thereof, and examined either immediately after treatment or after subsequent reincubation. Population decreases from the combination treatment matched the product of the decreases of individual treatments, indicating their orthogonality. However, reincubation showed binary behavior, where biofilms with an immediate population decrease beyond a critical factor (~10<sup>4</sup> ) died off completely during reincubation, while biofilms with a smaller immediate decrease regrew. This critical factor was independent of the initial population density and the combination of treatments that achieved the immediate decrease. While antibiotics do not appear to enhance thermal shock directly, their contribution to achieving a critical population decrease for biofilm elimination can make the treatments appear strongly synergistic, strongly decreasing the intensity of thermal shock needed.

**Keywords:** biofilms; prosthesis-related infections; heat shock; ciprofloxacin; antibacterial agents
