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

Abstract

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 stressinduced 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.

Clinical Characteristics of Biofilms in Patients with Chronic Rhinosinusitis: A Prospective Case–Control Study

“…The penetration of antibiotics into biofilm is significantly reduced, and bacteria in the biofilm are exposed to the drug in gradually increasing doses. This gradual exposure can result in stress-induced metabolic or transcriptional changes in the bacteria and increased resistance to antibiotic therapy [30]. Some authors have reported multiple microbes in biofilms, and different biofilm species are associated with differences in disease severity and postoperative results [31,32].…”

Lipid and polymer nanoparticles for drug delivery to bacterial biofilms

“…In studies using fluorescent dyes, diffusion was found to be the main mode of transport inside biofilm clusters. [115][116][117][118][119] Two important parameters in this respect are the particle size and the surface characteristics of the nanoparticles. Good penetration into biofilms is essential to deliver the antimicrobial agent to all bacterial cells inside the biofilm.…”

The role of small-colony variants in failure to diagnose and treat biofilm infections in orthopedics

“…Jefferson et al (2005) showed that the antibiotic vancomycin can fully penetrate the biofilm but the diffusion rate in a biofilm is significantly reduced as compared to in water. This suggests that whereas planktonic bacteria are quickly exposed to a high concentration of antibiotics, bacteria in the deeper layers of the biofilm are exposed to a gradually increasing antibiotic concentration.…”