Direct Visualization of Spatial and Temporal Patterns of Antimicrobial Action within Model Oral Biofilms

Abstract

A microscopic method for noninvasively visualizing the action of an antimicrobial agent inside a biofilm was developed and applied to describe spatial and temporal patterns of mouthrinse activity on model oral biofilms. Three species biofilms of Streptococcus oralis, Streptococcus gordonii, and Actinomyces naeslundii were grown in glass capillary flow cells. Bacterial cells were stained with the fluorogenic esterase substrate Calcien AM (CAM). Loss of green fluorescence upon exposure to an antimicrobial formulation was subsequently imaged by time-lapse confocal laser scanning microscopy. When an antimicrobial mouthrinse containing chlorhexidine digluconate was administered, a gradual loss of green fluorescence was observed that began at the periphery of cell clusters where they adjoined the flowing bulk fluid and progressed inward over a time period of several minutes. Image analysis was performed to quantify a penetration velocity of 4 m/min. An enzyme-based antimicrobial formulation led to a gradual, continually slowing loss of fluorescence in a pattern that was qualitatively different from the behavior observed with chlorhexidine. Ethanol at 11.6% had little effect on the biofilm. None of these treatments resulted in the removal of biomass from the biofilm. Most methods to measure or visualize antimicrobial action in biofilms are destructive. Spatial information is important because biofilms are known for their structural and physiological heterogeneity. The CAM staining technique has the potential to provide information about the rate of antimicrobial penetration, the presence of tolerant subpopulations, and the extent of biomass removal effected by a treatment.Direct time-lapse microscopic observation has proven to be an extremely powerful tool over the last decade in developing insight into the complex, spatially structured behaviors of microbial biofilms. This approach has revealed, for example, fluid flow patterns in the channels and interstices of heterogeneous biofilm structures (31), dynamic biomass movement and viscoelastic flow (17,30), the contribution of cell migration to the formation of mushroom-like structures (18), transient patterns of gene expression (25, 37), the time course of diffusive penetration of solutes into biofilms (15,24), and rapid motility of cells trapped in hollow biofilm cell clusters (14, 34).There have been few applications of time-lapse microscopy to investigate the action of antimicrobial agents against biofilms (4, 11, 16). Most of the techniques used to assess the action of antimicrobial agents on biofilms involve sacrificial sampling followed by plating or endpoint staining. By these methods it is difficult to gain insight into spatiotemporal patterns because it is not possible to observe a single spot in time.We have developed a microscopic method for noninvasively visualizing the action of an antimicrobial agent inside a biofilm. The first step in this method is to load bacterial cells with a fluorescent dye by incubating them with a fluorogenic esterase substrate (2...

Real-Time Microsensor Measurement of Local Metabolic Activities in Ex Vivo Dental Biofilms Exposed to Sucrose and Treated with Chlorhexidine

“…However, in deep layers of oral biofilms not all bacteria were reached (49). Direct visualization by fluorescence microscopy of the CHX effect was described by Takenaka et al (44) for a three-species oral biofilm grown in vitro. Cell damage started from the periphery of bacterial aggregates and slowly continued into the depths.…”

“…Electron microscopic examinations showed that CHX binds to and damages bacterial cell membranes and leads to structural changes and leakage of cytoplasm (3). Furthermore, contraction of in vitro grown biofilms after CHX exposure was shown in addition to cell damage (19,44), which could cause changes in the diffusion behavior by changes to extracellular polymeric slime density. However, in deep layers of oral biofilms not all bacteria were reached (49).…”

Correlative Time-Resolved Fluorescence Microscopy To Assess Antibiotic Diffusion-Reaction in Biofilms

“…Fluorescence microscopy represents an essential and noninvasive method to assess antimicrobial dynamics and reactivity in biofilms, contributing to our understanding of what makes bacteria resistant in these biostructures (3,5,8,29). In this respect, confocal laser scanning microscopy (CLSM) has been shown to be a powerful tool to analyze antimicrobial actions indirectly by a time course (hour-to-day scale) visualization of live and dead cells through the biofilm structure (4,17,29,34).…”

Adverse Influences of Antimicrobial Strategy against Mature Oral Biofilm

“…We performed a direct time-lapse microscopic observation throughout continuous exposure of commercial mouthrinses to an oral biofilm model [10]. Consequently, no removal of biomass was observed in control, ethanol (EtOH), 0.12% chlorhexidine gluconate (CHG), and Biotene, which contains lysozyme, lactoferrin, lactoperoxidase, glucose oxidase, and potassium thiocyanate, even after 20 min exposure.…”