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Antimicrobial Agents and Chemotherapy, June 2005, p. 2467-2473, Vol. 49, No. 6
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.6.2467-2473.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Use of Confocal Microscopy To Analyze the Rate of Vancomycin Penetration through Staphylococcus aureus Biofilms

Kimberly K. Jefferson,1* Donald A. Goldmann,2 and Gerald B. Pier1

The Channing Laboratory, Brigham and Women's Hospital, Harvard Medical School, 181 Longwood Avenue, Boston, Massachusetts 02115,1 Children's Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, Massachusetts 021152

Received 28 July 2004/ Returned for modification 11 October 2004/ Accepted 30 January 2005

When bacteria assume the biofilm mode of growth, they can tolerate levels of antimicrobial agents 10 to 1,000 times higher than the MICs of genetically equivalent planktonic bacteria. The properties of biofilms that give rise to antibiotic resistance are only partially understood. Inhibition of antibiotic penetration into the biofilm may play a role, but this has not been proven directly. In this report, penetration of the glycopeptide antibiotic vancomycin into viable Staphylococcus aureus biofilms was analyzed by confocal scanning laser microscopy using a fluorescently labeled derivative of the drug. We found that while vancomycin bound to free-floating bacteria in water within 5 min, it took more than 1 h to bind to cells within the deepest layers of a biofilm. These results indicate that the antibiotic is transported through the depth of the biofilm but that the rate is significantly reduced with respect to its transport through flowing water. This suggests that, whereas planktonic bacteria were rapidly exposed to a full bolus of vancomycin, the bacteria in the deeper layers of the biofilm were exposed to a gradually increasing dose of the drug due to its reduced rate of penetration. This gradual exposure may allow the biofilm bacteria to undergo stress-induced metabolic or transcriptional changes that increase resistance to the antibiotic. We also investigated the role of poly-N-acetylglucosamine, an important component of the S. aureus biofilm matrix, and found that its production was not involved in the observed decrease in the rate of vancomycin penetration.

* Corresponding author. Mailing address: Channing Laboratory, 181 Longwood Avenue, Boston, MA 02115. Phone: (617) 525-2680. Fax: (617) 731-1541. E-mail: kjefferson{at}rics.bwh.harvard.edu.

Antimicrobial Agents and Chemotherapy, June 2005, p. 2467-2473, Vol. 49, No. 6
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.6.2467-2473.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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