Spatial and temporal patterns of antimicrobial action against Staphylococcus Epidermidis biofilms

Abstract

This study investigated the spatio-temporal patterns of antimicrobial action against Staphylococcus epidermidis planktonic and biofilm bacteria. Bacteria were stained with a fluorogenic esterase substrate, Calcein-AM, which allowed for the visualization of cells that possessed intact cell membranes. Four different antimicrobial agents were tested for their effect upon cell viability as associated with membrane integrity. The four biocides were Barquat®, glutaraldehyde, chlorine, and nisin. Planktonic bacteria were analyzed with flow cytometry, observing fluorescence loss during 1 h antimicrobial treatment. Treatment with Barquat resulted in initial fluorescence loss, which increased during the treatment period to levels which were present prior to the introduction of biocide, along with a decrease in cell density. Treatments with glutaraldehyde and chlorine resulted in increased average fluorescence intensity for the cell population, accompanied by decreased cell density for chlorine and increased cell density for glutaraldehyde. Nisin treatment resulted in a decrease in CAM fluorescence with an increase in cell density. Viable cell plate counts showed average log reductions in CFU/mL of 3.61, 3.83, 4.12, 4.26, and 4.67 for Barquat, glutaraldehyde, high and low concentrations of chlorine, and nisin treatments, respectively. There was no apparent correlation between plate counts and flow cytometry data. Biofilm bacteria were analyzed with time-lapse confocal scanning laser microscopy, observing fluorescence loss during biocide treatment. Biofilms treated with Barquat lost an average of 91.5% of their initial fluorescence, and clusters decreased in areal coverage by 9%. Fluorescence loss during Barquat treatment suggested the presence of a tolerant subpopulation of bacteria in the interior regions of the biofilm. Glutaraldehyde treatment reduced the average fluorescence by 16%, and cluster area did not change. There was CTC staining after glutaraldehyde treatment only. The high and low concentrations of chlorine treatment showed averages of 100% and 79% reductions in CAM staining, with liquefaction of biomass causing erosion events which reduced areal coverage by 90% and 43%, respectively. Nisin treatment reduced CAM staining by an average of 100%, while shrinking the cluster area by 8%. Corner biofilms showed qualitative differences during treatment than isolated clusters. Mathematically-predicted biocide diffusion times were much faster than experimentally observed fluorescence loss in biofilms.