Measurement and analysis of microbial fluoride resistance in dental biofilm models.

The interactions between communities of microorganisms inhabiting the dental biofilm is a major determinant of oral health. These biofilms are periodically exposed to high concentrations of fluoride, which is present in almost all oral healthcare products. The microbes resist fluoride through the action of membrane export proteins. This chapter describes the culture, growth and harvest conditions of model three-species dental biofilm comprised of cariogenic pathogens Streptococcus mutans and Candida albicans and the commensal bacterium Streptococcus gordonii. In order to examine the role of fluoride export by S. mutans in model biofilms, procedures for generating a strain of S. mutans with a genetic knockout of the fluoride exporter are described. We present a case study examining the effects of this mutant strain on the biofilm mass, acid production and mineral dissolution under exposure to low levels of fluoride. These general approaches can be applied to study the effects of any gene of interest in physiologically realistic multispecies oral biofilms.

Ancient Genomes From Bronze Age Remains Reveal Deep Diversity and Recent Adaptive Episodes for Human Oral Pathobionts.

Ancient microbial genomes can illuminate pathobiont evolution across millenia, with teeth providing a rich substrate. However, the characterization of prehistoric oral pathobiont diversity is limited. In Europe, only preagricultural genomes have been subject to phylogenetic analysis, with none compared to more recent archaeological periods. Here, we report well-preserved microbiomes from two 4,000-year-old teeth from an Irish limestone cave. These contained bacteria implicated in periodontitis, as well as Streptococcus mutans, the major cause of caries and rare in the ancient genomic record. Despite deriving from the same individual, these teeth produced divergent Tannerella forsythia genomes, indicating higher levels of strain diversity in prehistoric populations. We find evidence of microbiome dysbiosis, with a disproportionate quantity of S. mutans sequences relative to other oral streptococci. This high abundance allowed for metagenomic assembly, resulting in its first reported ancient genome. Phylogenetic analysis indicates major postmedieval population expansions for both species, highlighting the inordinate impact of recent dietary changes. In T. forsythia, this expansion is associated with the replacement of older lineages, possibly reflecting a genome-wide selective sweep. Accordingly, we see dramatic changes in T. forsythia's virulence repertoire across this period. S. mutans shows a contrasting pattern, with deeply divergent lineages persisting in modern populations. This may be due to its highly recombining nature, allowing for maintenance of diversity through selective episodes. Nonetheless, an explosion in recent coalescences and significantly shorter branch lengths separating bacteriocin-carrying strains indicate major changes in S. mutans demography and function coinciding with sugar popularization during the industrial period.

Human saliva modifies growth, biofilm architecture, and competitive behaviors of oral streptococci.

The bacteria within supragingival biofilms participate in complex exchanges with other microbes inhabiting the same niche. One example is the mutans group streptococci (Streptococcus mutans), implicated in the development of tooth decay, and other health-associated commensal streptococci species. Previously, our group transcriptomically characterized intermicrobial interactions between S. mutans and several species of oral bacteria. However, these experiments were carried out in a medium without human saliva. To better mimic their natural environment, we first evaluated how inclusion of saliva affected growth and biofilm formation of eight Streptococcus species individually and found saliva to positively benefit growth rates while negatively influencing biofilm biomass accumulation and altering spatial arrangement. These results carried over during evaluation of 29 saliva-derived isolates of various species. Surprisingly, we also found that addition of saliva increased the competitive behaviors of S. mutans in coculture competitions against commensal streptococci that led to increases in biofilm microcolony volumes. Through transcriptomically characterizing mono- and cocultures of S. mutans and Streptococcus oralis with and without saliva, we determined that each species developed a nutritional niche under mixed-species growth, with S. mutans upregulating carbohydrate uptake and utilization pathways while S. oralis upregulated genome features related to peptide uptake and glycan foraging. S. mutans also upregulated genes involved in oxidative stress tolerance, particularly manganese uptake, which we could artificially manipulate by supplementing in manganese leading to an advantage over its opponent. Our report highlights observable changes in microbial behaviors through leveraging environmental- and host-supplied resources over their competitors. IMPORTANCE: Dental caries (tooth decay) is the most prevalent disease for both children and adults nationwide. Caries are initiated from demineralization of the enamel due to organic acid production through the metabolic activity of oral bacteria growing in biofilm communities attached to the tooth's surface. Mutans group streptococci are closely associated with caries development and initiation of the cariogenic cycle, which decreases the amount of acid-sensitive, health-associated commensal bacteria while selecting for aciduric and acidogenic species that then further drives the disease process. Defining the exchanges that occur between mutans group streptococci and oral commensals in a condition that closely mimics their natural environment is of critical need toward identifying factors that can influence odontopathogen establishment, persistence, and outgrowth. The goal of our research is to develop strategies, potentially through manipulation of microbial interactions characterized here, that prevent the emergence of mutans group streptococci while keeping the protective flora intact.

Opposing genetic polymorphisms of two ABC transporters contribute to the variation of nukacin resistance in Streptococcus mutans.

Streptococcus mutans is a cariogenic bacterium that produces a variety of bacteriocins and retains resistance to these bacteriocins. In this study, we investigated the susceptibility of 127 S. mutans strains to nukacins produced by Staphylococcus spp., which are commensal bacteria in humans. We detected diverse susceptibilities among strains. Nineteen strains had a disrupted LctF (type I), which is responsible for nukacin susceptibility, whereas the remaining 108 strains had an intact LctF (type II) and displayed resistance to nukacins. However, the type I strains still showed resistance to nukacins to some extent. Interestingly, 18/19 (94.7%) type I strains carried a mukA-T locus, which is related to the synthesis of mutacin K8, and mukFEG, an ABC transporter. In contrast, among type II strains, only 6/108 strains (5.6%) had both the mukA-T locus and mukFEG, 19/108 strains (17.6%) carried only mukFEG, and 83/108 strains (76.9%) harbored neither mukA-T nor mukFEG. We also found that MukF had two variants: 305 amino acids (type α) and 302 amino acids (type ß). All type I strains showed a type α (MukFα), whereas most type II strains with mukFEG (22/25 strains) had a type ß (MukFß). Then, we constructed a mukFEG-deletion mutant complemented with MukFαEG or MukFßEG and found that only MukFαEG was involved in nukacin resistance. The nukacin resistance capability of type II-LctFEG was stronger than that of MukFαEG. In conclusion, we identified a novel nukacin resistance factor, MukFEG, and either LctFEG or MukFEG was active in most strains via genetic polymorphisms depending on mukA-T genes. IMPORTANCE: Streptococcus mutans is an important pathogenic bacterium not only for dental caries but also for systemic diseases. S. mutans is known to produce a variety of bacteriocins and to retain resistance these bacteriocins. In this study, two ABC transporters, LctFEG and MukFEG, were implicated in nukacin resistance and each ABC transporter has two subtypes, active and inactive. Of the two ABC transporters, only one ABC transporter was always resistant, while the other ABC transporter was inactivated by genetic mutation. Interestingly, this phenomenon was defined by the presence or absence of the mutacin K8 synthesis gene region, one of the bacteriocins of S. mutans. This suggests that the resistance acquisition is tightly controlled in each strain. This study provides important evidence that the insertion of bacteriocin synthesis genes is involved in the induction of genetic polymorphisms and suggests that bacteriocin synthesis genes may play an important role in bacterial evolution.

Suppression of Streptococcus mutans Biofilm Formation and Gene Expression by PRG Barrier Coat: A Molecular and Microscopic Study for Preventing Dental Caries.

PURPOSE: This study aimed to investigate the inhibitory effect of a PRG Barrier Coat on biofilm formation and structure by Streptococcus mutans and propose an effective method for preventing dental caries. MATERIALS AND METHODS: Streptococcus mutans MT8148 biofilms were obtained from hydroxyapatite disks with and with- out a PRG Barrier Coat. Scanning electron microscopy (SEM) was used to observe the 12- and 24-h-cultured biofilms, while reverse-transcription polymerase chain reaction (qRT-PCR) was used to quantify caries-related genes. Biofilm adhe- sion assessments were performed on glass. Statistical analysis was performed using a two-sample t-test. RESULTS: A statistically significant difference in Streptococcus mutans biofilm adhesion rate was observed between the con- trol and PRG Barrier Coat-coated samples (p < 0.01). However, there was no statistically significant difference in total bacter- ial count or biofilm volume (p > 0.05). SEM revealed that the PRG Barrier Coat inhibited biofilm formation by Streptococcus mutans. Real-time RT-PCR revealed that the material restricted the expression of genes associated with caries-related bio- film formation. However, the suppression of gtfD and dexB differed from that of other genes. CONCLUSION: PRG Barrier Coat suppressed biofilm formation by Streptococcus mutans by inhibiting the expression of in- soluble glucan synthase, which is associated with primary biofilm formation. The material also affected gene expression and altered the biofilm structure. Tooth surface-coating materials, such as PRG Barrier Coat, may improve caries preven- tion in dental practice.

[Role and related mechanisms of LiaSR two-component system in acid tolerance and biofilm formation of Streptococcus mutans].

Objective: To investigate the role and related mechanisms of the LiaSR two-component system in acid tolerance and biofilm formation abilities of Streptococcus mutans (Sm) 593. Methods: The growth curves of various Sm strains in pH=5.5 brian heart infusion (BHI) medium were analyzed. And colony forming unit (CFU) was also performed to evaluate the acid tolerance of Sm. Laurdan probe, H+-K+adenosine triphosphate (ATP)ase activity analysis kit, proton permeability assay and real-time fluorescence quantitative PCR (RT-qPCR) were conducted to detect the acid tolerant mechanisms of LiaSR two-component system in Sm. Crystal violet staining, CFU, SYTOX probe and anthrone-sulfuric method were used to analyze the properties and structures of the Sm biofilms. RT-qPCR was conducted to detect the expression levels of underlying regulated genes. Results: The growth of mutants in acidic BHI were inhibited (P<0.05). The acid tolerance of mutants significantly decreased compared to the wild-type strain (P<0.05). In mutants, the activity of H+-ATPase (917.06±59.53 and 469.53±47.65) were elevated by 7.22-folds and 3.70-folds compared to the wild-type strain (127.00±50.71) (P<0.001, P<0.001) and the encoded gene atpD (3.39±0.21 and 1.94±0.17) were also elevated by 3.39-folds and 1.94-folds compared to the wild-type strain (1.00±0.15) (P<0.001, P=0.001). The Laurdan generalized polarization of mutants (0.18±0.04 and 0.18±0.05) increased significantly compared to the wild-type strain (0.08±0.05) (P=0.006, P=0.003) and the expression levels of fabM gene were decreased in mutants (0.52±0.11 and 0.57±0.05) by 1/2 (P=0.014, P=0.022). In liaR deletion mutant, the reduced terminal pH (4.76±0.01) can also be observed (P<0.001). The total amount of the biofilms of three Sm didn't show significant differences (P>0.05). But the number of viable bacteria of mutants' biofilms were decreased [Sm 593: (12.00±2.80)×107 CFU/ml; Sm ΔliaS: (2.95±1.13)×107 CFU/ml; Sm ΔliaR: (7.25±1.60)×107 CFU/ml] (P=0.001, P=0.024). The extracellular DNA were increased by 18.00-folds and 6.50-folds in mutants' biofilms (128.73±15.65 and 46.38±5.52) compared to the wild-type strain (7.16±3.62) (P<0.001, P=0.003). Water-soluble exopolysaccharides could be found up-regulated in liaS deletion mutant [(138.73±10.12) µg/ml] (P=0.003) along with the expression level of gtfC gene (1.65±0.39) (P=0.014). The expression level of gtfD were elevated by 47.43-folds and 16.90-folds in mutants (P<0.001, P=0.010). Conclusions: The LiaSR two-component system can promote the expression of fabM gene and increase the fluidity of Sm which contributes to acid tolerance. The LiaR can also decrease the proton permeability and restrict the entrance of H+. The LiaSR two-component system can negatively regulate the production of the extracellular matrix in Sm biofilm.

[Inhibitory effect of Streptococcus mutans antisense vicK RNA regulating the cariogenicity of oral streptococci multi-species biofilm].

Objective: To investigate the regulative effects of Streptococcus mutans (Sm) antisense vicK RNA (ASvicK) on the multi-species biofilm formed by three common oral streptococci (Sm, Streptococcus sanguinis, and Streptococcus gordonii) (Sm+Ss+Sg). Methods: ASvicK over-expression strain was constructed by using a recombinant plasmid, and three-species biofilm UA159+Ss+Sg and ASvicK+Ss+Sg were cultured. The phenotypes of biofilms were detected by scanning electron microscopy (SEM). Crystal violet (CV) assay was used to detect biofilm biomass. Lactate kit and anthrone-sulfuric acid colorimetric assay were used to determine the abilities of lactic acid and exopolysaccharides production, respectively. The proportions of three-species and expression levels of the cariogenic-related genes in biofilms were detected by TaqMan fluorescence quantitative PCR and real-time fluorescence quantitative PCR. A biofilm demineralization model of human enamel slabs was further constructed, and the hardness of enamel surface was detected. Results: Compared to UA159+Ss+Sg, over-expression of ASvicK could inhibit biofilm formation and lactic acid production in ASvicK+Ss+Sg biofilm significantly decreased by 78.93% (P<0.001) and 62.23% (P<0.001), respectively. With ASvicK over-expression, the amounts of water-insoluble and-soluble glucoses in ASvicK+Ss+Sg biofilm were reduced respectively by 39.13% (P<0.001) and 68.00% (P<0.001). Compared to the UA159+Ss+Sg Group, the proportion of Sm, the cariogenic bacteria, showed 33.00% reduction (P<0.01) in Sm+Ss+Sg biofilm, and the gene expressions of cariogenic-relative genes vicK/X, gtfB/C/D, and ftf significantly decreased (P<0.05). The micro-hardness value of enamel slabs after demineralization by ASvicK+Ss+Sg biofilm was significantly increased to 183.84% (P<0.001). Conclusions: ASvicK over-expression could reduce the Sm proportion and weaken the cariogenicity of oral Streptococcus biofilm, thereby possibly slowing down the progression of caries.

Mass spectrometry and split luciferase complementation assays reveal the MecA protein interactome of Streptococcus mutans.

MecA is a highly conserved adaptor protein encoded by prokaryotes from the Bacillota phylum. MecA mutants exhibit similar pleiotropic defects in a variety of organisms, although most of these phenotypes currently lack a mechanistic basis. MecA mediates ClpCP-dependent proteolysis of its substrates, but only several such substrates have been reported in the literature and there are suggestions that proteolysis-independent regulatory mechanisms may also exist. Here, we provide the first comprehensive characterization of the MecA interactome and further assess its regulatory role in Clp-dependent proteolysis. Untargeted coimmunoprecipitation assays coupled with mass spectrometry revealed that the MecA ortholog from the oral pathobiont Streptococcus mutans likely serves as a major protein interaction network hub by potentially complexing with >100 distinct protein substrates, most of which function in highly conserved metabolic pathways. The interactome results were independently verified using a newly developed prokaryotic split luciferase complementation assay (SLCA) to detect MecA protein-protein interactions in vivo. In addition, we further develop a new application of SLCA to support in vivo measurements of MecA relative protein binding affinities. SLCA results were independently verified using targeted coimmunoprecipitation assays, suggesting the general utility of this approach for prokaryotic protein-protein interaction studies. Our results indicate that MecA indeed regulates its interactome through both Clp-dependent proteolysis as well as through an as-yet undefined proteolysis-independent mechanism that may affect more than half of its protein interactome. This suggests a significant aspect of the MecA regulatory function still has yet to be discovered.IMPORTANCEDespite multiple decades of study, the regulatory mechanism and function of MecA have remained largely a mystery. The current study provides the first detailed roadmap to investigate these functions in other medically significant bacteria. Furthermore, this study developed new genetic approaches to assay prokaryotic protein-protein interactions via the split luciferase complementation assay (SLCA). SLCA technology is commonly employed in eukaryotic genetic research but has not yet been established for studies of bacterial protein-protein interactions. The SLCA protein binding affinity assay described here is a new technological advance exclusive to the current study and has not been reported elsewhere.

Production improvement of an antioxidant in cariogenic Streptococcus mutans UA140.

AIMS: This study aimed to improve the production of mutantioxidin, an antioxidant encoded by a biosynthetic gene cluster (mao) in Streptococcus mutans UA140, through a series of optimization methods. METHOD AND RESULTS: Through the construction of mao knockout strain S. mutans UA140∆mao, we identified mutantioxidin as the antioxidant encoded by mao and verified its antioxidant activity through a reactive oxygen species (ROS) tolerance assay. By optimizing the culture medium and fermentation time, 72 h of fermentation in chemically defined medium (CDM) medium was determined as the optimal fermentation conditions. Based on two promoters commonly used in Streptococcus (ldhp and xylS1p), eight promoter refactoring strains were constructed, nevertheless all showed impaired antioxidant production. In-frame deletion and complementation experiments demonstrated the positive regulatory role of mao1 and mao2, on mao. Afterward, the mao1 and mao2, overexpression strain S. mutans UA140/pDL278:: mao1mao2, were constructed, in which the production of mutantioxidin was improved significantly. CONCLUSIONS: In this study, through a combination of varied strategies such as optimization of fermentation conditions and overexpression of regulatory genes, production of mutantioxidin was increased by 10.5 times ultimately.

Construction of an arrayed CRISPRi library as a resource for essential gene function studies in Streptococcus mutans.

IMPORTANCE: The construction of arrayed mutant libraries has advanced the field of bacterial genetics by allowing researchers to more efficiently study the exact function and importance of encoded genes. In this study, we constructed an arrayed clustered regularly interspaced short palindromic repeats interference (CRISPRi) library, known as S treptococcus mutans arrayed CRISPRi (SNAP), as a resource to study >250 essential and growth-supporting genes in Streptococcus mutans. SNAP will be made available to the research community, and we anticipate that its distribution will lead to high-quality, high-throughput, and reproducible studies of essential genes.

Diverse nature of ClpX degradation motifs in Streptococcus mutans.

IMPORTANCE: Cytoplasmic Clp-related proteases play a major role in maintaining cellular proteome in bacteria. ClpX/P is one such proteolytic complex that is important for conserving protein homeostasis. In this study, we investigated the role of ClpX/P in Streptococcus mutans, an important oral pathogen. We identified several putative substrates whose cellular levels are regulated by ClpX/P in S. mutans and subsequently discovered several recognition motifs that are critical for degradation. Our study is the first comprehensive analysis of determining ClpX/P motifs in streptococci. We believe that identifying the substrates that are regulated by ClpX/P will enhance our understanding about virulence regulation in this important group of pathogens.

The inhibitory effect of ovomucoid from egg white on biofilm formation by Streptococcus mutans.

BACKGROUND: Streptococcus mutans, the main pathogen associated with tooth decay, forms cariogenic biofilms on tooth surfaces. Therefore, controlling oral biofilm helps prevent dental caries. Hen's egg is a nutrient-dense food, and egg white is a good source of protein. Ovomucoid is one of the major proteins in egg white, with a 28 kDa molecular weight. The present study aimed to investigate the inhibitory effects of ovomucoid on the biofilm formation of S. mutans by suppressing virulence factors, including bacterial adherence, cellular aggregation and exopolysaccharide (EPS) production. RESULTS: Crystal violet staining showed that biofilm formation by S. mutans was inhibited by ovomucoid at 0.25-1 mg mL-1 levels. Field emission scanning electron microscopy also confirmed this inhibition. In addition, ovomucoid reduced mature biofilm, water-insoluble EPS synthesis and the metabolic activity of bacterial cells in the biofilm. The bacterial adhesion and aggregation abilities of S. mutans were also decreased in the presence of ovomucoid. Ovomucoid downregulated the expression of comDE and vicR genes involved in the two-component signal transduction system and gtfA and ftf genes involved in EPS production. CONCLUSION: Ovomucoid has the potential for use as an anti-biofilm agent for dental caries treatment because of its inhibitory effects on the virulence factors of S. mutans. © 2023 Society of Chemical Industry.

Association of Root Biofilm Bacteriome with Root Caries Lesion Severity and Activity.

INTRODUCTION: This research aimed to assess the association of root biofilm bacteriome with root caries lesion severity and activity in institutionalised Colombian elderlies and was conducted to gather data on the root caries bacteriome in this population. METHODS: A bacteriome evaluation of biofilm samples from sound and carious root surfaces was performed. Root caries was categorised (ICDAS Root criteria) based on severity (sound surfaces, initial: non-cavitated, moderate/extensive combined: cavitated) and activity status (active and inactive). DNA was extracted and the V4 region of the 16S rRNA gene was sequenced; afterwards the classification of features was conducted employing amplicon sequence variants and taxonomic assignment via the Human Oral Microbiome Database (HOMD). Bacterial richness, diversity (Simpson's and Shannon's indices), and relative abundance estimation were assessed and compared based on root caries severity and activity status (including Sound surfaces). RESULTS: A total of 130 biofilm samples were examined: sound (n = 45) and with root caries lesions (n = 85; by severity: initial: n = 41; moderate/extensive: n = 44; by activity: active: n = 60; inactive: n = 25). Species richness was significantly lower in biofilms from moderate/extensive and active groups compared to sound sites. There was a higher relative abundance of species like Lechtotricia wadei, Capnocytophaga granulosa, Cardiobacterium valvarum, Porphyromonas pasteri - in sound sites; Dialister invisus, Streptococcus mutans, Pseudoramibacter alactolyticus and Bacteroidetes (G-5) bacterium 511 - in moderate/extensive lesions, and Fusobacterium nucleatum subsp. animalis, Prevotella denticola, Lactobacillus fermentum, Saccharibacteria (TM7) (G-5)bacterium HMT 356 - in active lesions. CONCLUSION: Root caries bacteriome exhibited differences in species proportions between the compared groups. Specifically, cavitated caries lesions and active caries lesions showed higher relative abundance of acidogenic bacteria.

Increased fecal ethanol and enriched ethanol-producing gut bacteria Limosilactobacillus fermentum, Enterocloster bolteae, Mediterraneibacter gnavus and Streptococcus mutans in nonalcoholic steatohepatitis.

Background: Non-alcoholic steatohepatitis (NASH) has become a major public health issue as one of the leading causes of liver disease and transplantation worldwide. The instrumental role of the gut microbiota is emerging but still under investigation. Endogenous ethanol (EtOH) production by gut bacteria and yeasts is an emerging putative mechanism. Microbial metagenomics and culture studies targeting enterobacteria or yeasts have been reported, but no culturomics studies have been conducted so far. Aim: To assess fecal EtOH and other biochemical parameters, characterize NASH-associated dysbiosis and identify EtOH-producing gut microbes associated with the disease, fecal samples from 41 NASH patients and 24 controls were analyzed. High-performance liquid chromatography (HPLC) was used for EtOH, glucose, total proteins, triglyceride and total cholesterol. Viable bacteria were assessed with microbial culturomics. Microbial genetic material was assessed using 16S metagenomics targeting the hypervariable V3V4 region. Results: Fecal EtOH and glucose was elevated in the stools of NASH patients (p < 0.05) but not triglyceride, total cholesterol or proteins. In culturomics, EtOH-producing Enterocloster bolteae and Limosilactobacillus fermentum were enriched in NASH. V3V4 16S rRNA amplicon sequencing confirmed the enrichment in EtOH-producing bacteria including L. fermentum, Mediterraneibacter gnavus and Streptococcus mutans, species previously associated with NASH and other dysbiosis-associated diseases. Strikingly, E. bolteae was identified only by culturomics. The well-known Lacticaseibacillus casei was identified in controls but never isolated in patients with NASH (p < 0.05). Conclusion: Elevated fecal EtOH and glucose is a feature of NASH. Several different EtOH-producing gut bacteria may play an instrumental role in the disease. Culturomics and metagenomics, two complementary methods, will be critical to identify EtOH-producing bacteria for future diagnostic markers and therapeutic targets for NASH. Suppression of EtOH-producing gut microbes and L. casei administration are options to be tested in NASH treatment.

Transcriptome Analysis of Streptococcus mutans Quorum Sensing-Mediated Persisters Reveals an Enrichment in Genes Related to Stress Defense Mechanisms.

Persisters are a small fraction of growth-arrested phenotypic variants that can survive lethal concentrations of antibiotics but are able to resume growth once antibiotics are stopped. Their formation can be a stochastic process or one triggered by environmental cues. In the human pathogen Streptococcus mutans, the canonical peptide-based quorum-sensing system is an inducible DNA repair system that is pivotal for bacterial survival. Previous work has shown that the CSP-signaling peptide is a stress-signaling alarmone that promotes the formation of stress-induced persisters. In this study, we exposed S. mutans to the CSP pheromone to mimic DNA damage conditions and isolated the antibiotic persisters by treating the cultures with ofloxacin. A transcriptome analysis was then performed to evaluate the differential gene expression between the normal stationary-phase cells and the persisters. RNA sequencing revealed that triggered persistence was associated with the upregulation of genes related to several stress defense mechanisms, notably, multidrug efflux pumps, the arginine deaminase pathway, and the Opu/Opc system. In addition, we showed that inactivation of the VicK kinase of the YycFG essential two-component regulatory system abolished the formation of triggered persisters via the CSP pheromone. These data contribute to the understanding of the triggered persistence phenotype and may suggest new therapeutic strategies for treating persistent streptococcal infections.

Acid tolerance of Lactobacillus spp. on root carious lesions: A complex and multifaceted response.

Lactobacillus spp. are acidogenic and aciduric bacteria and are among the main cariogenic microorganisms associated with the carious process. OBJECTIVE: This study aimed to identify genes involved in the acid-tolerance of Lactobacillus spp. and potential functions attributed to these genes within the metatranscriptome of sound root surfaces and carious root surfaces. DESIGN: Genomic libraries were built from mRNA isolated from the biofilm samples (10 from sound root and 9 from carious root using Illumina HiSeq 2500). Reads generated by RNA-seq were mapped against 162 oral microbial genomes and genes potentially related to acid tolerance were manually extracted from the Lactobacillus spp. genomes using L. paracasei ATCC 344 as reference genome. The R package DESeq2 was used to calculate the level of differential gene expression between those two clinical conditions. RESULTS: Fifteen Lactobacillus spp. genomes were identified and a total of 653 acid tolerance genes were overexpressed in carious root surfaces. Multiple functions, as translation, ribosomal structure and biogenesis, transport of nucleotides and amino acids, are involved in Lactobacillus spp. acid tolerance. Species-specific functions also seem to be related to the fitness of Lactobacillus spp. in acidified environments such as that of the cariogenic biofilm associated with carious root lesions. CONCLUSIONS: The response of Lactobacillus spp. to an acidic environment is complex and multifaceted. This finding suggests several possible avenues for further research into the adaptive mechanisms of these bacteria.

Small regulatory RNAs are mediators of the Streptococcus mutans SloR regulon.

Dental caries is among the most prevalent chronic diseases worldwide. Streptococcus mutans, the chief causative agent of caries, uses a 25-kDa manganese-dependent SloR protein to coordinate the uptake of essential manganese with the transcription of its virulence attributes. Small non-coding RNAs (sRNAs) can either enhance or repress gene expression, and reports in the literature ascribe an emerging role for sRNAs in the environmental stress response. Herein, we focused our attention on 18-50 nt sRNAs as mediators of the S. mutans SloR and manganese regulons. Specifically, the results of RNA sequencing revealed 19 sRNAs in S. mutans, which were differentially transcribed in the SloR-proficient UA159 and SloR-deficient GMS584 strains, and 10 sRNAs that were differentially expressed in UA159 cells grown in the presence of low vs high manganese. We describe SmsR1532 and SmsR1785 as SloR- and manganese-responsive sRNAs that are processed from large transcripts and that bind SloR directly in their promoter regions. The predicted targets of these sRNAs include regulators of metal ion transport, growth management via a toxin-antitoxin operon, and oxidative stress tolerance. These findings support a role for sRNAs in coordinating intracellular metal ion homeostasis with virulence gene control in an important oral cariogen. IMPORTANCE Small regulatory RNAs (sRNAs) are critical mediators of environmental signaling, particularly in bacterial cells under stress, but their role in Streptococcus mutans is poorly understood. S. mutans, the principal causative agent of dental caries, uses a 25-kDa manganese-dependent protein, called SloR, to coordinate the regulated uptake of essential metal ions with the transcription of its virulence genes. In the present study, we identified and characterized sRNAs that are both SloR and manganese responsive. Taken together, this research can elucidate the details of regulatory networks that engage sRNAs in an important oral pathogen and that can enable the development of an effective anti-caries therapeutic.

The Effect of Sub-MIC of Salvadora persica on Growth and Virulence Gene Expressions in Streptococcus mutans.

Dental caries is a multifactorial infectious chronic disease caused by particular bacteria and their virulence products that causes demineralization and progressive deterioration of the dental enamel. Many studies have proven miswak to have a critical antibacterial impact, particularly on cariogenic bacteria and periodontal pathogens, in the oral cavity. This study aimed to investigate the effect of different concentrations of Salvadora persica plant extract on growth and virulence gene expressions at mRNA levels in S. mutans. A total of 191 clinical samples from tooth swabs were collected, and sub-cultured on specific medium agar identified using biochemical and molecular approaches. MIC for the extract was determined and a bacterial growth curve was made to determine the growth phases and the optimum time for adding the extract at different concentrations. RT-qPCR technique was performed, and the REST-2009 software program was used for data analysis. Out of 191 swabs from the tooth 31 isolates were identified using several biochemical and molecular tests. Several S. mutans biofilm-related virulence genes and their Ct values were produced from RT-PCR under the effect of low and high doses of Meswak concentrations. Ct values and reaction efficiency were produced in RT-qPCR by Rotorgen3000, data then were analysed by REST-2009 software. Five isolates were selected to examine the effect of the extract on the mRNA levels using qPCR after growing them with both doses of the extract for about 30hrs. Levels of virulence gene mRNA were regulated differentially in cultures with added both extract doses. The isolates produced significantly lower virulence gene mRNA levels in cultures grown with both plant extract doses. The results produced in this study here provide new insights regarding several virulence gene expressions in S. mutans at the molecular levels when grown under different concentrations of Salvadora persica plant extract.

Catalase produced by Candida albicans protects Streptococcus mutans from H2O2 stress-one more piece in the cross-kingdom synergism puzzle.

Co-infection with Streptococcus mutans and Candida albicans is associated with dental caries, and their co-cultivation results in enhanced biofilm matrix production that contributes to increased virulence and caries risk. Moreover, the catalase-negative S. mutans demonstrates increased oxidative stress tolerance when co-cultivated in biofilms with C. albicans, a catalase-producing yeast. Here, we sought to obtain mechanistic insights into the increased H2O2 tolerance of S. mutans when co-cultivated with clinical isolates of Candida glabrata, Candida tropicalis, and C. albicans. Additionally, the C. albicans SC5314 laboratory strain, its catalase mutant (SC5314Δcat1), and S. mutans UA159 and its glucosyltransferase B/C mutant (UA159ΔgtfB/C) were grown as single- and dual-species biofilms. Time-kill assays revealed that upon acute H2O2 challenge, the survival rates of S. mutans in dual-species biofilms with the clinical isolates and C. albicans SC5314 were greater than when paired with SC5314Δcat1 or as a single-species biofilm. Importantly, this protection was independent of glucan production through S. mutans GtfB/C. Transwell assays and treatment with H2O2-pre-stimulated C. albicans SC5314 supernatant revealed that this protection is contact-dependent. Biofilm stability assays with sublethal H2O2 or peroxigenic Streptococcus A12 challenge resulted in biomass reduction of single-species S. mutans UA159 and dual-species with SC5314Δcat1 biofilms compared to UA159 biofilms co-cultured with C. albicans SC5314. S. mutans oxidative stress genes were upregulated in single-species biofilms when exposed to H2O2, but not when S. mutans was co-cultivated with C. albicans SC5314. Here, we uncovered a novel, contact-dependent, synergistic interaction in which the catalase of C. albicans protects S. mutans against H2O2. IMPORTANCE It is well established that co-infection with the gram-positive caries-associated bacterium Streptococcus mutans and the yeast pathobiont Candida albicans results in aggressive forms of caries in humans and animal models. Together, these microorganisms form robust biofilms through enhanced production of extracellular polysaccharide matrix. Further, co-habitation in a biofilm community appears to enhance these microbes' tolerance to environmental stressors. Here, we show that catalase produced by C. albicans protects S. mutans from H2O2 stress in a biofilm matrix-independent manner. Our findings uncovered a novel synergistic trait between these two microorganisms that could be further exploited for dental caries prevention and control.

Mutanase from Trichoderma harzianum inductively Produced by Mutan: Short-Term Treatment to Degrade Mature Streptococcus mutans Biofilm.

This study aimed to evaluate the disruptive effect of fungal mutanase against cariogenic biofilm after short-term treatment. For that, mature Streptococcus mutans biofilms (n = 9) were exposed to active or inactivated enzymes produced by Trichoderma harzianum for 1 min, two times per day. Biofilms were analyzed by amount of matrix water-insoluble polysaccharides, bacterial viability, acidogenicity, and morphology by scanning electron microscopy (SEM). The group treated with active enzymes (AE) had a significantly lower amount of insoluble polysaccharides (893.30 ± 293.69) when compared to the negative control group (NaCl, 2192.59 ± 361.96), yet no significant difference was found when comparing to the positive control group (CHX, 436.82 ± 151.07). Also, there was no significant effect on bacteria metabolism and viability (P-value < 0.05). Data generated by the quantitative analysis were confirmed through scanning electron microscopy images. Thus, fungal mutanase degraded the biofilm after a short-term treatment without interfering with bacterial viability and metabolism. Such findings offer insight to the development of routine oral care products containing this input.