title: Biofilm Antimicrobial Susceptibility Increases With Antimicrobial Exposure Time.
abstract: 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.
authors.affiliation: Banner - University Medical Center Phoenix, 1320 N 10th Street, Phoenix, AZ, 85006, USA. pcastan6@gmail.com.|Center for Interventional Biomaterials, Arizona State University, Tempe, AZ, USA. pcastan6@gmail.com.
authors.forename: Paulo
authors.identifier: http://orcid.org/0000-0003-0653-4380
authors.initials: P
authors.lastname: Castaneda
authors.affiliation: Banner - University Medical Center Phoenix, 1320 N 10th Street, Phoenix, AZ, 85006, USA.|College of Medicine-Phoenix, University of Arizona, Phoenix, AZ, USA.|Sonoran Biosciences, Chandler, AZ, USA.|Center for Interventional Biomaterials, Arizona State University, Tempe, AZ, USA.
authors.forename: Alex
authors.initials: A
authors.lastname: McLaren
authors.affiliation: Center for Interventional Biomaterials, Arizona State University, Tempe, AZ, USA.
authors.forename: Gamuchirai
authors.initials: G
authors.lastname: Tavaziva
authors.affiliation: Sonoran Biosciences, Chandler, AZ, USA.|Center for Interventional Biomaterials, Arizona State University, Tempe, AZ, USA.
authors.forename: Derek
authors.initials: D
authors.lastname: Overstreet
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D018441:Biofilms / Q000187:drug effects* / Q000254:growth & development; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000187:drug effects* / Q000254:growth & development; D055624:Methicillin-Resistant Staphylococcus aureus / Q000187:drug effects / Q000254:growth & development; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000254:growth & development; D013210:Staphylococcus / Q000187:drug effects* / Q000254:growth & development; D013212:Staphylococcus epidermidis / Q000187:drug effects / Q000254:growth & development; D013997:Time Factors; D014031:Tobramycin / Q000494:pharmacology*; D014640:Vancomycin / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D014640:Vancomycin; D014031:Tobramycin