Aciduric microbial taxa including Scardovia wiggsiae and Bifidobacterium spp. in caries and caries free subjects.

Actinobacteria came into focus of being potential caries-associated pathogens and could, together with the established Streptococcus mutans and lactobacilli thus function as caries indicator species. Here we analyzed the role and diagnostic predictive value of the acidogenic-aciduric species Scardovia wiggsiae and Bifidobacterium dentium together with S. mutans, lactobacilli and bifidobacteria in biofilm of non-cavitated (n = 20) and cavitated (n = 6) caries lesions versus controls (n = 30). For the genus Bifidobacterium and for B. dentium new sets of primers were designed. Based on real-time quantitative PCR and confirmed by DNA sequencing we found a higher prevalence (61.5%) of S. wiggsiae in caries lesions than in controls (40%). However, among the controls we found three individuals with both the highest absolute and relative S. wiggsiae numbers. Testing for S. mutans revealed the same prevalence as S. wiggsiae in caries lesions (61.5%) but in controls its prevalence was only 10%. B. dentium was never found in healthy plaque but in 30.8% of clinical cases, with the highest numbers in cavitated lesions. The Bifidobacterium-genus specific PCR had less discriminative power as more control samples were positive. We calculated the relative abundances and applied receiver operating characteristic analyses. The top results of specificity (93% and 87%) and sensitivity (100% and 88%) were found when the constraint set was "Lactobacillus relative abundance ≥0.02%" and "two aciduric species with a relative abundance of each ≥0.007%". Combinatory measurement of several aciduric taxa may be useful to reveal caries activity or even to predict caries progression.

Genetic variability of mutans streptococci revealed by wide whole-genome sequencing.

BACKGROUND: Mutans streptococci are a group of bacteria significantly contributing to tooth decay. Their genetic variability is however still not well understood. RESULTS: Genomes of 6 clinical S. mutans isolates of different origins, one isolate of S. sobrinus (DSM 20742) and one isolate of S. ratti (DSM 20564) were sequenced and comparatively analyzed. Genome alignment revealed a mosaic-like structure of genome arrangement. Genes related to pathogenicity are found to have high variations among the strains, whereas genes for oxidative stress resistance are well conserved, indicating the importance of this trait in the dental biofilm community. Analysis of genome-scale metabolic networks revealed significant differences in 42 pathways. A striking dissimilarity is the unique presence of two lactate oxidases in S. sobrinus DSM 20742, probably indicating an unusual capability of this strain in producing H2O2 and expanding its ecological niche. In addition, lactate oxidases may form with other enzymes a novel energetic pathway in S. sobrinus DSM 20742 that can remedy its deficiency in citrate utilization pathway.Using 67 S. mutans genomes currently available including the strains sequenced in this study, we estimates the theoretical core genome size of S. mutans, and performed modeling of S. mutans pan-genome by applying different fitting models. An "open" pan-genome was inferred. CONCLUSIONS: The comparative genome analyses revealed diversities in the mutans streptococci group, especially with respect to the virulence related genes and metabolic pathways. The results are helpful for better understanding the evolution and adaptive mechanisms of these oral pathogen microorganisms and for combating them.

Cryptic Streptococcus mutans 5.6-kb plasmids encode a toxin-antitoxin system for plasmid stabilization.

BACKGROUND: In all Streptococcus mutans strains, 5-13% carry a 5.6-kb plasmid. Despite its frequency, little is known about its mediated functions with most of the information coming from a single study focussing on plasmid pUA140. OBJECTIVE: Here, we describe the sequence and genetic organization of two S. mutans 5.6-kb plasmids, pDC09 and pNC101. RESULTS: Based on PicoGreen dsDNA quantification and Real-Time quantitative PCR (RTQ-PCR), the plasmid copy number was found to range between 10 and 74, depending on the strain tested. In contrast to literature, we identified six instead of five open reading frames (ORFs). While the putative gene products of ORF1 (as a Rep-protein) and ORF2 (as a Mob-protein) could be confirmed as being identical to those from pUA140, the functions of ORF3 (unknown) and ORF 4 (possibly AtpE homologue) could not be further revealed. However, the product of ORF5 showed a fairly high identity (38-50%) and structural similarity (58-74%) to RelE of Streptococcus pneumoniae, Streptococcus equi, and Streptococcus downei. In addition, we identified a functionally corresponding ORF6 encoding a protein with 61-68% identity (81-86% similarity) to the S. equi and S. downei antitoxin of the RelB family. RelE and RelB together form a plasmid-encoded toxin-antitoxin (TA) system, RelBE(plas). Despite its rather limited sequence similarity with chromosomal TA systems in S. mutans (RelBE(chro), MazEF, HicBA), we found similar tertiary structures applying I-Tasser protein prediction analysis. CONCLUSION: Type II-toxins, as the plasmid-encoded RelE, are RNA endonucleases. Depending on their mRNA cleavage activity, they might 1) kill every plasmid-free progeny, thereby stabilizing plasmid transfer at the expense of the host and/or 2) help S. mutans enter a dormant state and survive unfavourable environmental conditions. Whilst a function in plasmid stabilization has been confirmed, a function in persistence under nutritional stress, tested here by inducing amino acid starvation, could not be demonstrated so far.

Dental composite materials containing carolacton inhibit biofilm growth of Streptococcus mutans.

OBJECTIVE: Caries adjacent to restorations is one of the main causes for restoration replacement. Antimicrobial substances incorporated into dental materials would potentially be able to reduce secondary caries initiation and progression. This study investigated biofilm growth of Streptococcus mutans UA159 on the surface of composite materials containing the biomolecule carolacton compared to materials containing chlorhexidine (CHX) and triclosan. METHODS: Biofilm inhibition was investigated by counting colony forming units (CFU), viability staining (Life/Dead), and real-time quantitative PCR. RESULTS: First, the antimicrobial substances were added to the cultivation medium at 2.5 µg/ml (0.0002%) and 0.25 µg/ml (0.00002%). CHX eliminated bacterial growth and biofilm formation completely. Triclosan was effective at 2.5 µg/ml, but at 0.25 µg/ml biofilm mass and viability were unchanged, yet the number of CFU increased due to disruption of cell chains and biofilm aggregates. Carolacton had a limited effect on biofilm growth and mass, but reduced viability significantly. When incorporated into composite materials carolacton (25 µg/ml resp. 0.002%, w/w) had no adverse effect on physical/mechanical properties and retained its biofilm inhibiting effect. Life/Dead staining revealed a reduction of biofilm viability of up to 64%. CFUs were reduced by 98% and qPCR demonstrated a mean inhibition of 87%. In contrast, materials containing CHX or triclosan showed an insignificant effect on biofilm formation, even at a 100 fold increased concentration (0.2%). The anti-biofilm activity of composite material containing carolacton was stable over a period of 42 days. SIGNIFICANCE: Carolacton incorporated into dental filling material has a strong biofilm-inhibiting effect on S. mutans and is therefore potentially able to prevent secondary caries formation.