Application of a high throughput Alamar blue biofilm susceptibility assay to Staphylococcus aureus biofilms.
BACKGROUND
Staphylococcus aureus and S. epidermidis biofilms differ in structure, growth and regulation, and thus the high-throughput method of evaluating biofilm susceptibility that has been published for S. epidermidis cannot be applied to S. aureus without first evaluating the assay's reproducibility and reliability with S. aureus biofilms.


METHODS
Staphylococcus aureus biofilms were treated with eleven approved antibiotics, lysostaphin, or Conflikt, exposed to the oxidation reduction indicator Alamar blue, and reduction relative to untreated controls was determined visually and spectrophotometrically. The minimum biofilm inhibitory concentration (MBIC) was defined as < or = 50% Alamar blue reduction and a purple/blue well 60 min after the addition of Alamar blue. Because all of the approved antibiotics had MBICs >128 microg/ml (most >2048 microg/ml), lysostaphin and Conflikt, with relatively low MBICs, were used to correlate Alamar blue reduction with 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction and viable counts (CFU/ml) for S. aureus ATCC 29213 and three clinical isolates. Alamar blue's stability and lack of toxicity allowed CFU/ml to be determined from the same wells as Alamar blue absorbances.


RESULTS
Overall, Alamar blue reduction had excellent correlation with XTT reduction and with CFU/ml. For ATCC 29213 and two clinical isolates treated with lysostaphin or Conflikt, Alamar blue reduction had excellent correlation with XTT reduction (r = 0.93-0.99) and with CFU/ml (r = 0.92-0.98). For one of the clinical isolates, the results were moderately correlated for Conflikt (r = 0.76, Alamar blue vs. XTT; r = 0.81, Alamar blue vs. CFU/ml) and had excellent correlation for lysostaphin (r = 0.95, Alamar blue vs. XTT; r = 0.97, Alamar blue vs. CFU/ml).


CONCLUSIONS
A reliable, reproducible method for evaluating biofilm susceptibility was successfully applied to S. aureus biofilms. The described method provides researchers with a simple, nontoxic, relatively inexpensive, high throughput measure of viability after drug treatment. A standardized biofilm Alamar blue assay should greatly increase the rate of discovery of S. aureus biofilm specific agents.
Pettit
Robin K
RK
Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287, USA. robin.pettit@asu.edu
Weber
Christine A
CA
Pettit
George R
GR
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D018441:Biofilms / Q000187:drug effects* / Q000254:growth & development*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D050296:Microbial Viability / Q000187:drug effects; D010078:Oxazines / Q000378:metabolism; D010084:Oxidation-Reduction; D015203:Reproducibility of Results; D013211:Staphylococcus aureus / Q000187:drug effects* / Q000254:growth & development*; D013223:Statistics as Topic; D013778:Tetrazolium Salts / Q000378:metabolism; D014966:Xanthenes / Q000378:metabolism
D000900:Anti-Bacterial Agents; D010078:Oxazines; D013778:Tetrazolium Salts; D014966:Xanthenes; C059087:2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazolium hydroxide; C005843:resazurin