Multiparameter Assessments To Determine the Effects of Sugars and Antimicrobials on a Polymicrobial Oral Biofilm

Abstract

Clinical studies indicate relationships between dental plaque, a naturally formed biofilm, and oral diseases. The crucial role of nonmicrobial biofilm constituents in maintaining biofilm structure and biofilm-specific attributes, such as resistance to shear and viscoelasticity, is increasingly recognized. Concurrent analyses of the diverse nonmicrobial biofilm components for multiparameter assessments formed the focus of this investigation. Comparable numbers of Actinomyces viscosus, Streptococcus sanguinis, Streptococcus mutans, Neisseria subflava, and Actinobacillus actinomycetemcomitans cells were seeded into multiple wells of 96-well polystyrene plates for biofilm formation. Quantitative fluorescence and confocal laser scanning microscopy (CLSM) examined the influences of dietary sugars, incubation conditions, ingredients in oral hygiene formulations, and antibiotics on biofilm components. Biofilm extracellular polymeric substances (EPS) were examined with an optimized mixture of fluorescent lectins, with biofilm proteins, lipids, and nucleic acids detected with specific fluorescent stains. Anaerobic incubation of biofilms resulted in significantly more biofilm EPS and extractable carbohydrates than those formed under aerobic conditions (P < 0.05). Sucrose significantly enhanced biofilm EPS in comparison to fructose, galactose, glucose, and lactose (P < 0.05). CLSM demonstrated thicker biofilms under sucrose-replete conditions, along with significant increases in biofilm EPS, proteins, lipids, and nucleic acids, than under conditions of sucrose deficiency (P < 0.05). Agents in oral hygiene formulations (chlorhexidine, ethanol, and sodium lauryl sulfate), a mucolytic agent (N-acetyl-L-cysteine), and antibiotics with different modes of action (amoxicillin, doxycycline, erythromycin, metronidazole, and vancomycin) inhibited biofilm components (P < 0.05). Multiparameter analysis indicated a dose-dependent inhibition of biofilm EPS and protein by chlorhexidine and sodium lauryl sulfate, along with distinctive inhibitory patterns for subinhibitory concentrations of antibiotics. Collectively, these results highlight multiparameter assessments as a broad platform for simultaneous assessment of diverse biofilm components.

Influence of a model human defensive peroxidase system on oral streptococcal antagonism

“…In contrast, BHI supplemented with SCN 2 did not decrease in biomass, indicating that bLPO alone is sufficient to exert an effect on the biofilm-forming abilities of oral streptococci. The terminal cell density of all species (determined spectrophotometrically) was not influenced by bLPO, SCN 2 or bLPO+SCN 2 (versus the BHI control) under the tested conditions, suggesting that the reduced biomass was not a consequence of decreased growth (data not shown).Earlier reports have shown that S. mutans is better able to withstand shear stress (Yang et al, 2006) and antimicrobials (Kreth et al, 2008b) when grown in the presence of sucrose. This has been attributed to the production of extracellular glucan polymers.…”

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

“…This result is in agreement with a report by Sissons et al (28), who reported that 10% ethanol had almost no inhibitory effect on microcosm dental plaque. Similarly, Yang et al (36) found that ethanol concentrations below 20% did not demonstrate significant inhibition against a polymicrobial oral biofilm.…”

Antimicrobial Penetration and Efficacy in an In Vitro Oral Biofilm Model

“…Very few studies have addressed the issue of active penetration into dental plaque or oral biofilm (14,18,34,38). There are also few studies that reported measurements of oral biofilm removal by chemical treatments (7,41).…”