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.

Keywords: antibiotic tolerance; antibiotic-loaded bone cement; biofilm; periprosthetic joint infection; persister; pseudomonas; staphylococcus.

Figure 1

Killing of lawn biofilms of P. aeruginosa (PA-Xen41) and S. aureus (SAP231) with vancomycin and tobramycin loaded beads at the center. Zone of biofilm killing (ZOB-K) was monitored every day for seven days, and antibiotic-resistant phenotypes were observed in PA-Xen41 beginning at day four.

Figure 2

Different zones were developed after placement of vancomycin and tobramycin loaded beads on lawn biofilms of (A) PA-Xen41 and (B) SA-SAP231. Zone of biofilm killing (ZOB-K), zone of biofilm resistance (ZOB-R), and zone of biofilm lawn (ZOB-L) were evident over time after antibiotic bead placement. Images in insets show vancomycin and tobramycin treated day five lawn biofilms of Xen41 (A) and day six lawn biofilms of SAP231 (B).

Figure 3

Effect of bead placement on killing of PA-Xen41 (A,B) and SA-SAP231 (C,D) biofilms treated with vancomycin and tobramycin, where (A,C) are original plates and (B,D) are replica plates of original petri-plates at day seven.

Figure 4

Killing of lawn biofilms of (A) PA-Xen41 and (B) SA-SAP231 treated with vancomycin and tobramycin. Dotted horizontal lines in the graphs represents edges of the Petri dishes and vertical lines represents days of complete lawn biofilm killing.

Figure 5

(A) Influence of bead number on clearing of lawn biofilms of PA-Xen41 after 24 h of treatment with antibiotic-loaded calcium sulfate beads (ALCSB) containing vancomycin and tobramycin. Image analysis (Image J) of beads in clusters showing profile plot across cleared zone (mm); (B) Influence of bead number on total area and area cleared per bead of lawn biofilms of PA-Xen41 (i) and SA-SAP231 (ii) after 24 h of treatment with beads containing vancomycin and tobramycin. Distance cleared from edge of bead cluster was relatively independent of the number of beads. More beads cleared a greater area; however, the area cleared per bead was dramatically reduced when more beads were clustered together.