Effect of Chlorhexidine on biofilm formation and detachment of bacteria isolated from dental plaque

Objectives: The objective of the present study was to understand the mechanism of dental plaque biofilm development using cultivable taxa and to determine the effect of chlorhexidine in removal of biofilms.
Methods: Dental plaque samples were collected from the patients visiting the private clinic located in Lahore and was processed for the isolation and characterization of different bacteria. The biofilm formation potential of the isolated strains was studied using various techniques such as their ability to produce slime was analyzed by culturing on congo red medium. Their adhesion potential was determined on the basis of hydrophobicity by bacterial adhesion to hydrocarbon-BATH test and salt aggregation test. The role of extracellular polymeric substance in adherence of microorganisms to one another and to diverse surfaces was determined by its extraction and analyses through Fourier transform infrared spectroscopy (FTIR) with the aim to identify the functional groups present in the EPS. The isolated bacterial strains were further studied for their resistance to commonly used anti-plaque agent (chlorhexidine) used in mouthwashes and other oral health formulations.
Results: A total of five morphologically distinct strains were characterized biochemically and genetically. The 16S rRNA ribotyping showed them to be Neisseria flavescens, Streptococcus salivarius, Erwinia pyrifoliae, and Pseudomonas stutzeri. The two strains Streptococcus salivarius and Erwinia pyrifoliae were found to produce slime layers. All the five isolated strains were hydrophobic in the range of slightly hydrophobic to moderately hydrophobic. All these strains formed biofilm on both the surfaces i.e. glass and polystyrene. The EPS characterization results showed that EPS matrix was made up of different biomolecules such as carbohydrates and proteins as confirmed by FT-IR analysis, showing the major presence of carboxylic, hydroxylic and amino groups. All the isolated strains showed tolerance to 125 µgml^-1 of chlorhexidine.

Conclusions: The biomolecular characterization of EPS could be exploited to develop effective antiseptics to disrupt and penetrate the complex biofilms.