Biofilm Antimicrobial Susceptibility Increases With Antimicrobial Exposure Time

Background: The antimicrobial concentration required to kill all the bacteria in a biofilm, known as the minimum biofilm eradication concentration (MBEC), is typically determined in vitro by exposing the biofilm to serial concentrations of antimicrobials for 24 hours or less. Local delivery is expected to cause high local levels for longer than 24 hours. It is unknown if longer antimicrobial exposures require the same concentration to eradicate bacteria in biofilm. Questions/purposes Does MBEC change with increased antimicrobial exposure time?

Methods: Biofilms were grown for 24 hours using five pathogens (methicillin-sensitive Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa) and then exposed to four antimicrobials regimens: tobramycin, vancomycin, and tobramycin combined with vancomycin in 3:1 and 1:1 ratios by weight in concentrations of 62.5, 125, 250, 500, 1000, 2000, 4000, and 8000 μg/mL for three durations, 1, 3, and 5 days, in triplicate. MBEC was measured as the lowest concentration that killed all bacteria in the biofilm determined by 21-day subculture.

Results: MBEC was lower when antimicrobial exposure time was longer. For the staphylococcus species, the MBEC was lower when exposure time was 5 days than 1 day in 11 of 12 antimicrobial/microorganism pairs. The MBEC range for these 11 pairs on Day 1 was 4000 to > 8000 μg/mL and on Day 5 was < 250 to 8000 μg/mL. MBEC for tobramycin/P. aeruginosa was 2000 μg/mL on Day 1 and ≤ 250 μg/mL on Day 5, and for E. coli, 125 μg/mL on Day 1 and ≤ 62.5 on Day 5.

Conclusions: Although antimicrobial susceptibility was lower for longer exposure times in the microorganisms we studied, confirmation is required for other pathogens. Clinical Relevance One-day MBEC assays may overestimate the local antimicrobial levels needed to kill organisms in biofilm if local levels are sustained at MBEC or above for longer than 24 hours. Future studies are needed to confirm that antimicrobial levels achieved clinically from local delivery are above the MBEC at relevant time points and to confirm that MBEC for in vitro microorganisms accurately represents MBEC of in vivo organisms in an clinical infection.