Spatial and Temporal Patterns of Biocide Action against Staphylococcus epidermidis 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 data support the accepted paradigm that bacteria in biofilms are more difficult to kill than are the same microorganisms when in free aqueous suspension. Analytical techniques such as colony formation assays (plating), regrowth assays, crystal violet staining for total biomass, tetrazolium salt reduction, and ATP content measurement have been employed. These measurements provide an indication of overall efficacy, in terms of either viability reduction or biofilm removal, but do not shed light on fundamental phenomena important in the interaction of the biocide with the biofilm. In other words, there is little or no mechanistic insight that can be gleaned from these conventional testing data.The technique described in this article affords information about the time scale for penetration of biologically active concentrations of the antimicrobial agent into the biofilm interior, the failure of most of the antimicrobials to induce any release of biomass from the biofilm, the important role of hydrodynamics outside the biofilm in removing biofilm weakened by reaction with chlorine, the lack of correlation of antimicrobial molecular weight with observed penetration time, and the presence of a subpopulation of cells that are less susceptible to an antimicrobial.