Effect of continuous exposure to chlorhexidine digluconate on competence and genetic transformation of Streptococcus mutans

• Grant number: 24/14419-8
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: October 01, 2024
• End date: September 30, 2025
• Field of knowledge: Health Sciences - Dentistry
• Principal Investigator: Antônio Pedro Ricomini Filho
• Grantee: Luiza Bittencourt de Borba
• Host Institution: Faculdade de Odontologia de Piracicaba (FOP). Universidade Estadual de Campinas (UNICAMP). Piracicaba , SP, Brazil

Abstract

Streptococcus mutans is a naturally competent microorganism, meaning it has the ability to capture and incorporate extracellular DNA into its genome, a capability known as bacterial transformation. Competence is modulated by the expression of the sigma factor gene (sigX), which is activated by peptides such as CSP (competence stimulating peptide) and XIP (sigX inducing peptide). Sub-inhibitory concentrations of antimicrobials are stress factors that modulate these pathways, increasing or decreasing competence capability. The objective of this study will be to evaluate the effect of continuous exposure to chlorhexidine digluconate (CHX) for 1, 7, and 14 days on the activation of competence and genetic transformation of S. mutans strains. Isogenic mutants of S. mutans containing a luciferase reporter in the promoter regions of the cipB (CSP activation) and sigX (XIP activation) genes will be used. The cultures will be evaluated after 1, 7, and 14 days of continuous exposure to CHX. Culture stocks will be prepared and frozen at optical density (OD600) 0.5. The stocks will be diluted (1:10) and distributed in 96-well plates, followed by addition of 1 mM D-luciferin, 250 nM CSP and increasing CHX concentrations (0, 0.25, 0.50, 1.0 µg CHX/mL). The plates will be incubated at 37ºC for 12 h in a multidetection microplate reader, during which growth (OD600) and sigX expression (luminescence) will be measured every 30 min. Luminescence data will be normalized by OD600 values and the area under the curve (AUC) calculated. Transformation will be performed using Kanamycin resistance amplicon (aRJ02) that will be added at 150, 270 and 390 min. After incubation for 1 h the suspensions will be plated on agar plates: non-selective (TSB) and selective (TSB + 200 ¿g/ml kanamycin. Data will be analyzed by One-way ANOVA and Tukey's test (¿ = 5%).