Simultaneous spatiotemporal mapping of in situ pH and bacterial activity within an intact 3D microcolony structure

Abstract

Biofilms are comprised of bacterial-clusters (microcolonies) enmeshed in an extracellular matrix. Streptococcus mutans can produce exopolysaccharides (EPS)-matrix and assemble microcolonies with acidic microenvironments that can cause tooth-decay despite the surrounding neutral-pH found in oral cavity. How the matrix influences the pH and bacterial activity locally remains unclear. Here, we simultaneously analyzed in situ pH and gene expression within intact biofilms and measured the impact of damage to the surrounding EPS-matrix. The spatiotemporal changes of these properties were characterized at a single-microcolony level following incubation in neutral-pH buffer. The middle and bottom-regions as well as inner-section within the microcolony 3D structure were resistant to neutralization (vs. upper and peripheral-region), forming an acidic core. Concomitantly, we used a green fluorescent protein (GFP) reporter to monitor expression of the pH-responsive atpB (PatpB::gfp) by S. mutans within microcolonies. The atpB expression was induced in the acidic core, but sharply decreased at peripheral/upper microcolony regions, congruent with local pH microenvironment. Enzymatic digestion of the surrounding matrix resulted in nearly complete neutralization of microcolony interior and down-regulation of atpB. Altogether, our data reveal that biofilm matrix facilitates formation of an acidic core within microcolonies which in turn activates S. mutans acid-stress response, mediating both the local environment and bacterial activity in situ.

Changes in the salivary electrolytic dynamic after sucrose exposure in children with Early Childhood Caries

“…Consequently, the metabolism of intracellular and extracellular polysaccharides can promote a greater susceptibility to dental caries by prolonging the exposure of tooth surfaces to organic acids. In other words, there is an upregulation mechanism for dental demineralization and consequently a constant generation of acidic pH niches [22][23][24] . With regard to the CF group, and according to the latter-mentioned hypothesis, the increase in the salivary calcium concentration after sucrose rinse can be explained by the low basal metabolism of the biofilm in CF individuals, which is forced to increase the sucrose metabolism and ultimately shift the ionic imbalance at the interface tooth-biofilm-saliva.…”

Extracellular DNA and lipoteichoic acids interact with exopolysaccharides in the extracellular matrix of Streptococcus mutans biofilms

“…Both factors can influence the diffusion of metabolites and how antimicrobial agents reach the microorganisms. In previous studies, it was demonstrated that inside large microcolonies, acidic niches can be created that could promote tooth demineralization (Xiao et al 2012; Hwang et al 2016). The lack of distinct microcolonies could be due to the influence of S. gordonii and A. naeslundii on the construction and assembly of the 3-D structure of microcolonies, such that the EPS is spread across the biofilm and not concentrated at specific areas, appearing as a carpet-like morphology rather than the ‘peaks and valleys’ observed in single-species biofilms.…”

Glycan recognition at the saliva – oral microbiome interface

“…1). This film protects the mineralized surface of teeth from erosion by acids in the diet or acids produced by oral bacteria, the latter being a first step in the initiation of dental caries [63–65]. The lubricating effect of glycosylated mucins also protects the integrity of the tooth occlusal surfaces from attrition through antagonistic teeth and excessive wear through abrasive food components.…”