Microstructural differences between single-species and dual-species biofilms of Streptococcus mutans and Veillonella parvula, before and after exposure to chlorhexidine.

Dual-species biofilms of Streptococcus mutans and Veillonella parvula are less susceptible to antimicrobials than single-species biofilms of the same microorganisms. The microstructure of single and dual-species biofilms of S. mutans and/or V. parvula was visualized to find out whether the spatial arrangement of bacteria in biofilms is related to survival strategies against antimicrobials. Biofilms were grown in glass-bottomed 96-well microtiter plates and exposed to chlorhexidine at 48 h. Fluorescent probes were used for staining. The microstructure of biofilms was analyzed by confocal scanning laser microscopy at 48, 96, 192, and 264 h. Spatial arrangement analysis was performed using DAIME software for 48 h biofilms. A decrease in the viability and thickness in all types of biofilms was detected after chlorhexidine treatment in time. In untreated biofilms, clustering was observed. In chlorhexidine-treated single-species biofilms, bacteria were dispersed. However, the most prominent clustering was observed in chlorhexidine-treated dual-species biofilm bacteria, which had a higher survival rate compared with chlorhexidine-treated single-species biofilms. Bacteria in dual-species biofilms establish a specific spatial arrangement, forming clusters within distances below 1.2 microm as a survival strategy against antimicrobials while the same bacteria lack this defensive construction in a single-species biofilm.

Differences between single- and dual-species biofilms of Streptococcus mutans and Veillonella parvula in growth, acidogenicity and susceptibility to chlorhexidine.

Streptococcus mutans, considered a primary pathogen in dental caries, thrives in dental plaque, which is a multispecies biofilm. Metabolic interactions between S. mutans and Veillonella parvula have been suggested. In this study we developed a biofilm model to quantify single-species (S. mutans or V. parvula) and dual-species (S. mutans and V. parvula) biofilm formation, and we identified the differences between the respective biofilms in terms of growth, acid formation, and response to chlorhexidine. Polystyrene 96-well microtiter plates were used for biofilm formation. These biofilms were exposed to various chlorhexidine concentrations (0.025-0.4 mg ml(-1)) and treatment conditions. Growth of the biofilms and the effects of chlorhexidine were evaluated by viable counts. Viability of the two species in all biofilm types was similar ( approximately 10(8) colony-forming units per well) after 72 h. Lactic acid accumulation of dual-species biofilms was significantly lower at 48 and 72 h than single-species biofilms of S. mutans. Dual-species biofilms were less susceptible to chlorhexidine than single-species biofilms when a neutralization step was included. These results indicate that bacteria in dual-species biofilms have different properties from bacteria in single-species biofilms.

Effect of biofilm model, mode of growth, and strain on streptococcusmutans protein expression as determined by two-dimensional difference gel electrophoresis.

The effect of biofilm model, strain and mode of growth (biofilm or planktonic) on protein expression in Streptococcus mutans, a dental pathogen, was determined by two-dimensional difference gel electrophoresis. The bacterial strain (21-28% differentially expressed proteins) and the biofilm model (0.3-7.8% differential expression) used have a much larger effect on protein expression than the mode of growth (0.2-0.7% differential expression), something that has been ignored in biofilm studies up to now.

Development of a standard test to assess the resistance of Staphylococcus aureus biofilm cells to disinfectants.

A standardized disinfectant test for Staphylococcus aureus cells in biofilms was developed. Two disinfectants, the membrane-active compound benzalkonium chloride (BAC) and the oxidizing agent sodium hypochlorite, were used to evaluate the biofilm test. S. aureus formed biofilms on glass, stainless steel, and polystyrene in a simple system with constant nutrient flow that mimicked as closely as possible the conditions used in the current standard European disinfectant test (EN 1040). The biofilm that was formed on glass contained cell clumps and extracellular polysaccharides. The average surface coverage was 60%, and most (92%) of the biofilm cells were viable. Biofilm formation and biofilm disinfection in different experiments were reproducible. For biofilms exposed to BAC and hypochlorite the concentrations needed to achieve 4-log killing were 50 and 600 times higher, respectively, than the concentrations needed to achieve this level of killing with the European phase 1 suspension test cells. Our results show that a standardized disinfectant test for biofilm cells is a useful addition to the current standard tests.