Complete Killing of Agar Lawn Biofilms by Systematic Spacing of Antibiotic-Loaded Calcium Sulfate Beads

Abstract

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.

Ultrastructure imaging of Pseudomonas aeruginosa lawn biofilms and eradication of the tobramycin-resistant variants under in vitro electroceutical treatment

“…To expose the lawn biofilms to a combination of antibiotic and electroceutical treatments, a modified Kirby-Bauer method was used 13 . In this method, blank paper disks (6 mm diameter, Remel Blank Paper Disk, Thermo Fisher Scientific) loaded with 100 µg tobramycin (TOB), delivered in a 10 µL drop and allowed to dry, were used.…”

“…Treatment of antibiotic-resistant variants in PA-Xen41 lawn biofilms. TOB loaded paper disks were placed over the 24 h grown PA-Xen41 lawn biofilm at t = 0 h, which was incubated at 37 °C and 5% CO 2 until t = 72 h. The antibiotic-resistant phenotypes emerge in the cleared lawn at t = 72 h in IVIS imaging 13 . After emergence of the antibiotic-resistant phenotypes, electroceutical treatment was applied for an additional 24 h (i.e., from t = 72 h to t = 96 h).…”

“…After the electroceutical treatment was stopped at t = 24 h, the culture plates continued to be incubated until t = 72 h. IVIS images were recorded every 24 h to observe the metabolic activity maps of PA-Xen41 lawn biofilms in response to the treatment conditions. The extended incubation period was used since the antibiotic-resistant variants of Pseudomonas aeruginosa often have a delayed lag phase or slow growth phenotype 13 .…”

“…However, the use of electroceuticals under different treatment conditions and the impact of these electrochemical treatments on the biofilm ultrastructure, which is defined as the detailed structure of bacterial cells including sub-cellular structures and the structure of the cell envelope, is not well understood. Furthermore, it is unknown if the treatment efficacy of electrochemically generated bactericidal species extends to the antibiotic-resistant variants that have been shown to emerge after standard antibiotic treatment of biofilms 13 .…”

“…Despite growing evidence for ROS and RCS being the anti-bacterial agents in the electrochemical treatment of biofilms, the effect of these bactericidal species on biofilm ultrastructure at any of the electrical poles i.e., the anode or the cathode is unknown. Furthermore, there are no systematic reports with a reliable investigation of the treatment efficacy of electrochemically generated bactericidal species at different treatment conditions, including when the antibiotic-resistant variants emerge from a lawn biofilm after the application of a standard antibiotic treatment 13 .…”