Application of a high throughput Alamar blue biofilm susceptibility assay to <it>Staphylococcus aureus </it>biofilms

• Main Authors: Pettit George R (Author), Weber Christine A (Author), Pettit Robin K (Author)
• Format: Book
• Published: BMC, 2009-10-01T00:00:00Z.
• Subjects: article
• Online Access: Connect to this object online.

Summary

<p>Abstract</p> <p>Background</p> <p><it>Staphylococcus aureus </it>and <it>S. epidermidis </it>biofilms differ in structure, growth and regulation, and thus the high-throughput method of evaluating biofilm susceptibility that has been published for <it>S. epidermidis </it>cannot be applied to <it>S. aureus </it>without first evaluating the assay's reproducibility and reliability with <it>S. aureus </it>biofilms.</p> <p>Methods</p> <p><it>Staphylococcus aureus </it>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 ≤ 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 μg/ml (most >2048 μg/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)-2<it>H</it>-tetrazolium-5-carboxanilide (XTT) reduction and viable counts (CFU/ml) for <it>S. aureus </it>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.</p> <p>Results</p> <p>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).</p> <p>Conclusion</p> <p>A reliable, reproducible method for evaluating biofilm susceptibility was successfully applied to <it>S. aureus </it>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 <it>S. aureus </it>biofilm specific agents.</p>

Item Description

10.1186/1476-0711-8-28
1476-0711