Pseudomonas aeruginosa is an opportunistic bacterial pathogen and is known to produce biofilms. We have previously shown the emergence of colony variants in the presence of tobramycin-loaded calcium sulfate beads. In this study, we characterized the variant colonies, which survived the antibiotic treatment and identified three distinct phenotypes - classically resistant colonies, viable but non-culturable colonies (VBNC), and phoenix colonies. Phoenix colonies, described here for the first time, grow out of the zone of clearance of antibiotic loaded beads from lawn biofilms while there are still very high concentrations of antibiotic present, suggesting an antibiotic resistant phenotype. However, upon sub-culturing these isolates, phoenix colonies return to wild-type levels of antibiotic susceptibility. Compared with wild-type, phoenix colonies are morphologically similar aside from a deficiency in green pigmentation. Phoenix colonies do not recapitulate the phenotype of any previously described mechanisms of resistance, tolerance or persistence, and, thus, form a novel group with their own phenotype. Growth under anaerobic conditions suggests that an alternative metabolism could lead to the formation of phoenix colonies. These findings suggest that phoenix colonies could emerge in response to antibiotic therapies and lead to recurrent or persistent infections particularly within biofilms where microaerobic or anaerobic environments are present.

Background: Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) are the major causative agents of acute and chronic infections. Antibiotic-loaded calcium sulfate beads (ALCSB) are used in the management of musculoskeletal infections such as periprosthetic joint infections (PJI). Methods: To determine whether the number and spatial distribution of ALCSB are important factors to totally eradicate biofilms, ALCSBs containing vancomycin and tobramycin were placed on 24 h agar lawn biofilms as a single bead in the center, or as 16 beads placed as four clusters of four, a ring around the edge and as a group in the center or 19 beads evenly across the plate. Bioluminescence was used to assess spatial metabolic activity in real time. Replica plating was used to assess viability. Results: For both strains antibiotics released from the beads completely killed biofilm bacteria in a zone immediately adjacent to each bead. However, for PA extended incubation revealed the emergence of resistant colony phenotypes between the zone of eradication and the background lawn. The rate of biofilm clearing was greater when the beads were distributed evenly over the plate. Conclusions: Both number and distribution pattern of ALCSB are important to ensure adequate coverage of antibiotics required to eradicate biofilms.

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