Effect of the Biofilm Age and Starvation on Acid Tolerance of Biofilm Formed by Streptococcus mutans Isolated from Caries-Active and Caries-Free Adults

Abstract

Streptococcus mutans (S. mutans) is considered a leading cause of dental caries. The capability of S. mutans to tolerate low pH is essential for its cariogenicity. Aciduricity of S. mutans is linked to its adaptation to environmental stress in oral cavity. This study aimed to investigate the effect of biofilm age and starvation condition on acid tolerance of biofilm formed by S. mutans clinical isolates. S. mutans clinical strains isolated from caries-active (SM593) and caries-free (SM18) adults and a reference strain (ATCC25175) were used for biofilm formation. (1) Both young and mature biofilms were formed and then exposed to pH 3.0 for 30 min with (acid-adapted group) or without (non-adapted group) pre-exposure to pH 5.5 for three hours. (2) The mature biofilms were cultured with phosphate-buffered saline (PBS) (starved group) or TPY (polypeptone-yeast extract) medium (non-starved group) at pH 7.0 for 24 h and then immersed in medium of pH 3.0 for 30 min. Biofilms were analyzed through viability staining and confocal laser scanning microscopy. In all three strains, mature, acid-adapted and starved biofilms showed significantly less destructive structure and more viable bacteria after acid shock than young, non-adapted and non-starved biofilms, respectively (all p < 0.05). Furthermore, in each condition, SM593 biofilm was denser, with a significantly larger number of viable bacteria than that of SM18 and ATCC25175 (all p < 0.05). Findings demonstrated that mature, acid-adapted and starvation might protect biofilms of all three S. mutans strains against acid shock. Additionally, SM593 exhibited greater aciduricity compared to SM18 and ATCC25175, which indicated that the colonization of high cariogenicity of clinical strains may lead to high caries risk in individuals.

Keywords: Starvation; Streptococcus mutans; aciduricity; biofilm formation; confocal laser scanning microscopy.

Figure 1

Confocal laser scanning microscopy (CLSM) images of Live/Dead (SYTO-9 and PI) stained young and mature biofilms treatment with acid-adapted or non-adapted. Young biofilm in non-adapted group (A1), ATCC25175; (A2), SM18; (A3), SM593; Young biofilm in acid-adapted group (B1), ATCC25175; (B2), SM18; (B3), SM593; Mature biofilm in non-adapted group (C1), ATCC25175; (C2), SM18; (C3), SM593; Mature biofilm in acid-adapted group (D1), ATCC25175; (D2), SM18; (D3), SM593 (scale bars, 5 mm; magnification 630×).

Figure 2

CLSM images of Live/Dead (SYTO-9 and PI) stained starved/non-starved biofilms after acid shock. Non-starved biofilm ((A1), ATCC25175; (A2), SM18; (A3), SM593); Starved biofilm ((B1), ATCC25175; (B2), SM18; (B3), SM593) (scale bars, 5 mm; magnification 630×).

Figure 3

Three-dimensional architecture of S. mutans biofilms under different conditions. Young biofilm in non-adapted group (A1), ATCC25175; (A2), SM18; (A3), SM593; Young biofilm in acid-adapted group (B1), ATCC25175; (B2), SM18; (B3), SM593; Mature biofilm in non-adapted group (C1), ATCC25175; (C2), SM18; (C3), SM593; Mature biofilm in acid-adapted group (D1), ATCC25175; (D2), SM18; (D3), SM593 Non-starved biofilm (E1), ATCC25175; (E2), SM18; (E3), SM593; Starved biofilm (F1), ATCC25175; (F2), SM18; (F3), SM593.