Abstract
The present study aimed at investigating the influence of norspermidine on the formation of dual-species biofilms composed of Streptococcus mutans (S. mutans) and Streptococcus sanguinis (S. sanguinis). Crystal violet assay was conducted to assess the formation of single-species biofilms of S. mutans and S. sanguinis, and the growth curve was carefully observed to monitor the growth of these two species of bacteria. Fluorescence in situ hybridization (FISH) and MTT array were used to analyze the composition and metabolic activity of the dual-species biofilms, respectively. Extracellular polysaccharides (EPS)/bacteria staining, anthrone method, and scanning electron microscopy (SEM) imaging were conducted to study the synthesis of EPS by dual-species biofilms. Lactic acid assay and pH were measured to detect dual-species biofilm acid production. We found that norspermidine had different effects on S. mutans and S. sanguinis including their growth and biofilm formation. Norspermidine regulated the composition of the dual-species biofilms, decreased the ratio of S. mutans in dual-species biofilms, and reduced the metabolic activity, EPS synthesis, and acid production of dual-species biofilms. Norspermidine regulated dual-species biofilms in an ecological way, suggesting that it may be a potent reagent for controlling dental biofilms and managing dental caries.
Highlights
Yan Sun,1 Yihuai Pan,1 Yu Sun,1 Mingyun Li,2 Shengbin Huang,3 Wei Qiu,2 Huanxin Tu,1 and Keke Zhang 4
Biofilm Formation of S. mutans and S. sanguinis Showed Differing Sensitivities to Norspermidine. e crystal violet staining results showed that S. mutans and S. sanguinis could barely form biofilms in the presence of 7.0 mM and 14.0 mM norspermidine (Figure 1(a)). e quantitative results of crystal violet assay showed a similar trend, with norspermidine leading to 20.4% and 9.8% reductions in optical density (OD) value of S. mutans compared with the control (Figure 1(b))
In S. sanguinis, there were no significant differences in OD value in the presence of 7.0 mM, 3.5 mM, and 1.8 mM norspermidine when compared with the control (p > 0.05; Figure 1(c))
Summary
Additional studies revealed that they compete with each other by producing hydrogen peroxide (S. sanguinis) and mutacin (S. mutans) [8] This relationship between the two strains could be an artifact of more complicated interactions in the dental biofilm [9]. In 2012, Ilana Kolodkin-Gal et al [11] found that Bacillus subtilis (B. subtilis) produces norspermidine to induce biofilm disassembly during the biofilm life cycle [11] This biofilm disassembly factor could inhibit the biofilm formation. Some of the results reported by Ilana Kolodkin-Gal et al were challenged by a subsequent research in which B. subtilis was lacking the biosynthetic pathway for norspermidine, and the concentration of norspermidine required to inhibit B. subtilis biofilm formation was much higher [11]. Norspermidine displayed the ability to inhibit and disperse biofilms in multidrug-resistant clinical isolates related to persistent infections in wounds to extremities [15]
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