A novel selective medium Sucrose-Bacitracin agar 10 for accurate isolation and identification of Streptococcus mutans

Abstract

Background: To develop and evaluate Sucrose-Bacitracin agar with 10% sucrose (SB-10), a novel selective medium designed to enhance the isolation and identification of Streptococcus mutans, a key cariogenic pathogen in dental caries.

Methods: SB-10 was compared to Mitis Salivarius Bacitracin agar with 20% sucrose (MSB-20) for its ability to support S. mutans growth, promote distinctive colony morphology, and differentiate it from other oral bacteria. Sugar particle formation was assessed using wild-type and mutant strains of S. mutans lacking glucosyltransferases B and C (GtfB and GtfC). Liquid chromatography was performed to characterize the sugar particles around colonies. The specificity and clinical utility of SB-10 were further assessed by culturing clinical saliva samples and confirming S. mutans colonies via 16S rRNA sequencing.

Results: SB-10 supported robust S. mutans growth with larger colonies and distinctive sugar particle formation compared to MSB-20. GtfB and GtfC were essential for sugar particle deposition around colonies. SB-10 demonstrated high specificity by suppressing the growth of non-target oral bacteria and accurately isolating S. mutans from clinical saliva samples, with all colonies confirmed as S. mutans via 16S rRNA sequencing.

Conclusions: SB-10 is a novel selective medium that enhances the isolation and identification of S. mutans, characterized by unique sugar particles deposition and colony morphology.

Keywords: Streptococcus mutans; Dental caries; Glucosyltransferases; Selective medium; Sucrose-Bacitracin agar.

Fig. 1

CFU counts of S. mutans on BHIA under aerobic and anaerobic conditions. The colony-forming units (CFU) of S. mutans were quantified after incubation on BHI plates under aerobic and anaerobic conditions. “ND” indicates not detected

Fig. 2

Effects of sucrose concentration on CFU counts and colony morphology of S. mutans. a CFU of S. mutans on BHIA, SB and MSB media with varying sucrose concentrations. b Colony diameters of S. mutans on BHIA, SB and MSB media with varying sucrose concentrations. Data are presented as mean ± standard deviation from three independent biological replicates, with 10 colonies per replicate. c Representative microscopic image (20 × magnification) showing colony morphology of S. mutans grown on SB-10 medium. Contrast was modified to highlight morphological features. The red dashed circle indicates the typical sugar particle deposition surrounding bacterial colonies. Statistical significance is indicated as ***P < 0.001, and ****P < 0.0001, and “NS” indicates not significant

Fig. 3

Effects of glucosyltransferases on CFU counts and colony morphology of S. mutans. a CFU of S. mutans UA159, UA159 ΔgtfB, and UA159 ΔgtfB/C grown on SB-10, and MSB-20 media. b Colony diameters of S. mutans UA159, UA159 ΔgtfB, and UA159 ΔgtfB/C on SB-10, and MSB-20 media. Data are presented as mean ± standard deviation from three independent biological replicates, with 10 colonies per replicate. c Morphological characteristics of UA159 ΔgtfB on SB-10. d Morphological characteristics of UA159 ΔgtfB/C colonies on SB-10. Contrast was modified to highlight morphological features. Statistical significance is indicated as ****P < 0.0001 and “NS” indicates not significant

Fig. 4

Growth and morphological characteristics of common oral bacteria. a CFU of S. mutans, S. epidermidis, E. faecalis, S. sobrinus, S. oralis, S. salivarius, S. gordonii, and S. sanguinis on BHI, SB-10, and MSB-20 media. b  Colony diameters of S. mutans, S. epidermidis, E. faecalis, S. sobrinus, S. oralis, S. salivarius, S. gordonii, and S. sanguinis on BHI, SB-10, and MSB-20 media. Data are presented as mean ± standard deviation from three independent biological replicates, with 10 colonies per replicate. c Morphological characteristics of S. mutans on SB-10 media under oblique light. d Morphological characteristics of S. sobrinus colony on SB-10 media under oblique light. The contrast was modified to highlight morphological features. Statistical significance is indicated as ****P < 0.0001, “NS” indicates not significant, and “ND” indicates not detected

Fig. 5

Morphological comparison of S. mutans colonies from clinical saliva samples incubated on SB-10 and MSB-20 agar media. a Representative image of S. mutans colonies from clinical saliva samples incubated on SB-10 agar medium for 72 h under anaerobic conditions. The inset shows a microscopic image at 20 × magnification, with the red dashed circle indicating the characteristic sugar particle deposition around the colony edges. b Representative image of S. mutans colonies from clinical saliva samples incubated on MSB-20 agar medium for 72 h under anaerobic conditions. The inset shows a microscopic image at 20 × magnification, with no noticeable sugar particle deposition observed