Killing of a Multispecies Biofilm Using Gram-Negative and Gram-Positive Targeted Antibiotic Released from High Purity Calcium Sulfate Beads.

Abstract

Multispecies biofilm orthopedic infections are more challenging to treat than mono-species infections. In this in-vitro study, we aimed to determine if a multispecies biofilm, consisting of Gram positive and negative species with different antibiotic susceptibilities could be treated more effectively using high purity antibiotic-loaded calcium sulfate beads (HP-ALCSB) containing vancomycin (VAN) and tobramycin (TOB) in combination than alone. Three sets of species pairs from bioluminescent strains of Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) and clinical isolates, Enterococcus faecalis (EF) and Enterobacter cloacae were screened for compatibility. PA + EF developed intermixed biofilms with similar cell concentrations and so were grown on 316L stainless steel coupons for 72 h or as 24 h agar lawn biofilms and then treated with HP-ALCSBs with single or combination antibiotics and assessed by viable count or bioluminescence and light imaging to distinguish each species. Replica plating was used to assess viability. The VAN + TOB bead significantly reduced the PA + EF biofilm CFU and reduced the concentration of surviving antibiotic tolerant variants by 50% compared to single antibiotics. The combination of Gram-negative and positive targeted antibiotics released from HP-ALCSBs may be more effective in treating multispecies biofilms than monotherapy alone.