It has long been suspected that microbial biofilms harbor cells in a variety of activity states, but there have been few direct experimental visualizations of this physiological heterogeneity. Spatial patterns of DNA replication and protein synthetic activity were imaged and quantified in staphylococcal biofilms using immunofluorescent detection of pulse-labeled DNA and also an inducible green fluorescent protein (GFP) construct. Stratified patterns of DNA synthetic and protein synthetic activity were observed in all three biofilm systems to which the techniques were applied. In a colony biofilm system, the dimensions of the zone of anabolism at the air interface ranged from 16 to 38 m and corresponded with the depth of oxygen penetration measured with a microelectrode. A second zone of activity was observed along the nutrient interface of the biofilm. Much of the biofilm was anabolically inactive. Since dead cells constituted only 10% of the biofilm population, most of the inactive cells in the biofilm were still viable. Collectively, these results suggest that staphylococcal biofilms contain cells in at least four distinct states: growing aerobically, growing fermentatively, dead, and dormant. The variety of activity states represented in a biofilm may contribute to the special ecology and tolerance to antimicrobial agents of biofilms.

The dynamic antimicrobial action of chlorine, a quaternary ammonium compound, glutaraldehyde, and nisin within biofilm cell clusters of Staphylococcus epidermidis was investigated using time-lapse confocal scanning laser microscopy. The technique allowed for the simultaneous imaging of changes in biofilm structure and disruption of cellular membrane integrity through the loss of an unbound fluorophore loaded into bacterial cells prior to antimicrobial challenge. Each of the four antimicrobial agents produced distinct spatial and temporal patterns of fluorescence loss. The antimicrobial action of chlorine was localized around the periphery of biofilm cell clusters. Chlorine was the only antimicrobial agent that caused any biofilm removal. Treatment with the quaternary ammonium compound caused membrane permeabilization that started at the periphery of cell clusters, then migrated steadily inward. A secondary pattern superimposed on the penetration dynamic suggested a subpopulation of less-susceptible cells. These bacteria lost fluorescence much more slowly than the majority of the population. Nisin caused a rapid and uniform loss of green fluorescence from all parts of the biofilm without any removal of biofilm. Glutaraldehyde caused no biofilm removal and also no loss of membrane integrity. Measurements of biocide penetration and action time at the center of cell clusters yielded 46 min for 10 mg liter ؊1 chlorine, 21 min for 50 mg liter ؊1 chlorine, 25 min for the quaternary ammonium compound, and 4 min for nisin. These results underscore the distinction between biofilm removal and killing and reinforce the critical role of biocide reactivity in determining the rate of biofilm penetration.The action of a biocide or antibiotic against microorganisms in biofilms varies in time and space. There is insight to be gained into the phenomena important in this process by watching, through a microscope, the antimicrobial attack. Here we describe the application of a recently developed technique for visualizing antimicrobial action (29) to biofilms formed by Staphylococcus epidermidis. We describe distinct behaviors for the four antimicrobial agents examined, which were chlorine, glutaraldehyde, a quaternary ammonium compound (QAC), and an antimicrobial peptide, nisin.S. epidermidis, a commensal resident of the skin and an opportunistic pathogen, is a common culprit in nosocomial infections (13,15,31). In particular, this microorganism is known to form biofilms on indwelling devices such as catheters, prosthetic joints, and contact lenses. There is therefore interest in understanding the efficacy of biocides against biofilms formed by this organism in such applications as control of contamination on hospital countertops and in catheter lock solutions, skin disinfectants, and contact lens storage case disinfection.Numerous evaluations of biocide activity against S. epidermidis biofilms have been reported for such agents as chlorhexidine, hydrogen peroxide, povidone-iodine, alcohols, and chlorine (4,6,10,17,19,21,32). These ...

Surfaces coated with the naturally-occurring polysaccharide chitosan (partially deacetylated poly N-acetyl glucosamine) resisted biofilm formation by bacteria and yeast. Reductions in biofilm viable cell numbers ranging from 95% to 99.9997% were demonstrated for Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Candida albicans on chitosan-coated surfaces over a 54-h experiment in comparison to controls. For instance, chitosan-coated surfaces reduced S. epidermidis surface-associated growth more than 5.5 (10)log units (99.9997%) compared to a control surface. As a comparison, coatings containing a combination of the antibiotics minocycline and rifampin reduced S. epidermidis growth by 3.9 (10)log units (99.99%) and coatings containing the antiseptic chlorhexidine did not significantly reduce S. epidermidis surface associated growth as compared to controls. The chitosan effects were confirmed with microscopy. Using time-lapse fluorescence microscopy and fluorescent-dye-loaded S. epidermidis, the permeabilization of these cells was observed as they alighted on chitosan-coated surfaces. This suggests chitosan disrupts cell membranes as microbes settle on the surface. Chitosan offers a flexible, biocompatible platform for designing coatings to protect surfaces from infection.

Fluorescently tagged daptomycin accessed the interior of Staphylococcus epidermidis biofilm cell clusters within minutes. The diffusion coefficient of daptomycin in the biofilm was 28% of its value in pure water. Daptomycin activity against staphylococci embedded in biofilms is unlikely to be limited by penetration of the antibiotic into the biofilm.Infections associated with microbial biofilms that form on implanted medical devices are notoriously difficult to resolve with antimicrobial chemotherapy (9). One of the obvious and oft-mentioned explanations for the failure of antibiotic treatment is that the antibiotic may not penetrate the biofilm. If the time required for an antibiotic to penetrate the biofilm is long compared to the treatment duration, then slow penetration is a plausible explanation for the observed antibiotic tolerance of the biofilm. On the other hand, if the time required for an antibiotic to penetrate the biofilm is short compared to the treatment time, then biofilm penetration is probably not the limiting step. In this latter case, biological explanations for biofilm tolerance should be sought.There are few direct experimental visualizations of antibiotic penetration into bacterial biofilms. Stone et al. imaged the rapid delivery of tetracycline into thin Escherichia coli biofilms (10). This drug penetrated biofilms that were approximately 15-m thick within 10 min. Jefferson et al. demonstrated that vancomycin partially permeated a Staphylococcus aureus biofilm during 1 h of exposure to the drug under static conditions (4).The purpose of the work reported in this article was to determine the time course of diffusive penetration of daptomycin into large, dense clusters of staphylococcal biofilm.Biofilms of S. epidermidis strain RP62A (ATCC 35984) were grown in a flow cell at 37°C with continuous flow of 1/10 strength tryptic soy broth (7). Time-lapse confocal scanning laser microscopy was then used to analyze diffusive penetration of a fluorescent solute as described previously (7). Daptomycin was fluorescently tagged by adding aqueous NaHCO 3 and either Bodipy or rhodamine to daptomycin dissolved in dimethylformamide. Fluorescently labeled daptomycin was dissolved in a phosphate buffer containing 2 mM Mg 2ϩ to a final concentration of 40 g ml Ϫ1 and pumped through the capillary biofilm reactor at a flow rate of 1 ml min Ϫ1 . Diffusion measurements were obtained inside isolated biofilm clusters attached to the ceiling of the glass capillary, at a focal plane approximately 10 m from the glass surface. Diffusion experiments were conducted at an ambient temperature of approximately 23°C.S. epidermidis formed dense, heterogeneous biofilms in glass capillary tube reactors during the 20-h growth period. Thick biofilm formed in the corners of the flow cell and also occasionally as clusters in the middle of the tube walls. Diffusion experiments were conducted on the relatively isolated clusters. The radial dimension of cell clusters (R) ranged from approximately 100 to 200 m. The overall mean radiu...

Nanomedicine directed at diagnosis and treatment of infections can benefit from innovations that have substantially increased the variety of available multifunctional nanoplatforms. Here, we targeted a spherical, icosahedral viral nanoplatform to a pathogenic, biofilm-forming bacterium, Staphylococcus aureus. Density of binding mediated through specific protein-ligand interactions exceeded the density expected for a planar, hexagonally close-packed array. A multifunctionalized viral protein cage was used to load imaging agents (fluorophore and MRI contrast agent) onto cells. The fluorescence-imaging capability allowed for direct observation of penetration of the nanoplatform into an S. aureus biofilm. These results demonstrate that multifunctional nanoplatforms based on protein cage architectures have significant potential as tools for both diagnosis and targeted treatment of recalcitrant bacterial infections.