Single Treatment of Mature 3D Single-, Dual- and Poly-Species Biofilms Using a Combination Therapy of Phage or Phage-Hetero-Cocktails and Ciprofloxacin

Background/Objectives: Biofilms are a form of defense that enables bacteria to withstand antibiotic pressure and demonstrate antibiotic resistance. It is crucial to develop anti-biofilm strategies in order to combat chronic and persistent multidrug-resistant (MDR) infections. Methods: In this study, we developed 3D biofilms of single-, dual-, and poly-species MDR ESKAPE components, including the pathogens P. aeruginosa S. aureus and K. pneumoniae, in CF Mu3Gel. We evaluated the efficacy of using a phage, a di-hetero phage cocktail or a poly-hetero phage cocktail in combination with ciprofloxacin to eliminate mature biofilm biomass after 72 h or one week in a single treatment. Results: The phage components mostly exhibited synergistic behavior when combined with ciprofloxacin and with each other in di- and poly-hetero-cocktails. The reduction in 72-h dual- and poly-species biofilms was one log higher than that of one-week biofilms treated with the phage–antibiotic combination. The greatest reductions were observed in the 72-h single-species biofilm with combination therapy, at 1.4–3.0 log. Reductions of 2.16 and 1.6 log were observed in the dual-species P. aeruginosa and S. aureus biofilm and the poly-species biofilm, respectively. Conclusions: This study examined how a single application of phages or phage cocktails, either alone or in combination with ciprofloxacin, impacted established biofilm models, and how this affected the proportion of microcolonies of different species within each model. These insights will facilitate the development of strategies for multiple follow-up treatments, as well as the reordering of phages, phage cocktails, and combinations with antibiotics, to improve outcomes. The 3D biofilm models developed here could be used to screen phages or phage cocktails either on their own or alongside other therapies. This would facilitate the application of in vitro findings to real physiological settings.