In vitro evaluation of tolerance ability of cross-kingdom biofilm towards oral dynamic fluctuations.

Abstract

Objectives: The aim of this study was to investigate the resilience of Streptococcus mutans (S. mutans) and Candida albicans (C. albicans) cross-kingdom biofilms in response to environmental stresses.

Materials and methods: The growth kinetics of S. mutans, C. albicans, and their co-culture in planktonic form were assessed using a BioScreen system. Biofilms were established on 96-well plates for a duration of 48 h, after which microbial counts were determined. The biofilms were then subjected to different stress conditions, including oxidative, acid, osmotic, and heat stress, for 2 h. The survival and structural integrity of the biofilms were evaluated through colony-forming unit (CFU) counting and fluorescence microscopy, respectively. Additionally, the transcriptional levels of genes concerning matrix formation, acid tolerance, oxidative tolerance were determined by quantitative real-time PCR (RT-PCR). The data was analyzed by one-way ANOVA, and post hoc Tukey's test (α = 0.05).

Results: Co-culturing S. mutans and C. albicans resulted in an extended logarithmic growth phase compared to monocultures. Dual-species biofilm had higher microorganism counts after biofilm formation, displayed higher surviving cells, and a more complex structure after exposure to various stresses when compared to monospecies biofilm (p<0.05). The transcriptional levels of genes concerning matrix formation (gtfB, gtfC, ftf, bcr1, hwp1), acid tolerance (atpD, fabM, phr1), oxidative tolerance (nox, sodA, sod1, and trx1) were upregulated in dual-species biofilm (p<0.05).

Conclusions: Dual-species biofilm present higher tolerance to various stresses in the oral cavity. The upregulation of genes involved in matrix formation and stress tolerance may partially account for this increased resilience.