Oral streptococci S. anginosus and S. mitis induce distinct morphological, inflammatory, and metabolic signatures in macrophages.

Oral streptococci, key players in oral biofilm formation, are implicated in oral dysbiosis and various clinical conditions, including dental caries, gingivitis, periodontal disease, and oral cancer. Specifically, Streptococcus anginosus is associated with esophageal, gastric, and pharyngeal cancers, while Streptococcus mitis is linked to oral cancer. However, no study has investigated the mechanistic links between these Streptococcus species and cancer-related inflammatory responses. As an initial step, we probed the innate immune response triggered by S. anginosus and S. mitis in RAW264.7 macrophages. These bacteria exerted time- and dose-dependent effects on macrophage morphology without affecting cell viability. Compared with untreated macrophages, macrophages infected with S. anginosus exhibited a robust proinflammatory response characterized by significantly increased levels of inflammatory cytokines and mediators, including TNF, IL-6, IL-1ß, NOS2, and COX2, accompanied by enhanced NF-κB activation. In contrast, S. mitis-infected macrophages failed to elicit a robust inflammatory response. Seahorse Xfe96 analysis revealed an increased extracellular acidification rate in macrophages infected with S. anginosus compared with S. mitis. At the 24-h time point, the presence of S. anginosus led to reduced extracellular itaconate, while S. mitis triggered increased itaconate levels, highlighting distinct metabolic profiles in macrophages during infection in contrast to aconitate decarboxylase expression observed at the 6-h time point. This initial investigation highlights how S. anginosus and S. mitis, two Gram-positive bacteria from the same genus, can prompt distinct immune responses and metabolic shifts in macrophages during infection.IMPORTANCEThe surge in head and neck cancer cases among individuals devoid of typical risk factors such as Human Papilloma Virus (HPV) infection and tobacco and alcohol use sparks an argumentative discussion around the emerging role of oral microbiota as a novel risk factor in oral squamous cell carcinoma (OSCC). While substantial research has dissected the gut microbiome's influence on physiology, the oral microbiome, notably oral streptococci, has been underappreciated during mucosal immunopathogenesis. Streptococcus anginosus, a viridans streptococci group, has been linked to abscess formation and an elevated presence in esophageal cancer and OSCC. The current study aims to probe the innate immune response to S. anginosus compared with the early colonizer Streptococcus mitis as an important first step toward understanding the impact of distinct oral Streptococcus species on the host immune response, which is an understudied determinant of OSCC development and progression.

Antimicrobial activity of probiotic Streptococcus salivarius LAB813 on in vitro cariogenic biofilms.

OBJECTIVE: To investigate the antimicrobial activity of a novel commensal strain of Streptococcus salivarius, LAB813, against Streptococcus mutans biofilms. METHODS: The inhibitory activity of LAB813 towards S. mutans was tested using mono-, dual-, and multi-species cariogenic biofilms formed on three types of orthodontic appliances (metal, ceramic, aligner). The activity of the commercially available probiotic, BLIS M18™ was used as control. RESULTS: LAB813 significantly inhibited S. mutans biofilms with cell killing approximating 99% for all materials. LAB813 showed effectiveness at inhibiting S. mutans in more complex multi-species biofilms with cell killing approximating 90% for all three materials. When comparing the killing kinetics of the probiotics, LAB813 had a faster rate of killing biofilms than M18. Experiments conducted with cell-free culture supernatant confirmed the presence of an inhibitory substance of proteinaceous nature. The addition of xylitol, a common sugar substitute used for human consumption, potentiated the inhibitory effects of LAB813 against S. mutans embedded in a more complex fungal-bacterial biofilm. CONCLUSIONS: LAB813 possesses strong antimicrobial activity, potent anti-biofilm properties, and enhanced antimicrobial activity in the presence of xylitol. The identification and characterization of strain LAB813 exhibiting antimicrobial activity towards S. mutans hold exciting promise for this novel strain to be developed as an oral probiotic for use in the prevention of dental caries.

Synthesis of a Novel Lantibiotic Using Mutacin II Biosynthesis Apparatus.

Owing to extensive metagenomic studies, we now have access to numerous sequences of novel bacteriocin-like antimicrobial peptides encoded by various cultivable and noncultivable bacteria. However, relatively rarely, we even have access to these cultivable strains to examine the potency and the targets of the predicted bacteriocins. In this study, we evaluated a heterologous biosynthetic system to produce biologically active nonnative novel lantibiotics, which are modified bacteriocins. We chose Streptococcus mutans, a dental pathogen, as the host organism because it is genetically easy to manipulate and is inherently a prolific producer of various bacteriocins. We chose the S. mutans T8 strain as the host, which produces the lantibiotic mutacin II, to express 10 selected homologs of mutacin II identified from GenBank. These lantibiotic peptides either are novel or have been studied very minimally. The core regions of the selected lantibiotic peptides were fused to the leader sequence of the mutacin II peptide and integrated into the chromosome such that the core region of the native mutacin II was replaced with the new core sequences. By this approach, using the mutacin II biosynthesis machinery, we obtained one bioactive novel lantibiotic peptide with 52% different residues compared to the mutacin II core region. This unknown lantibiotic is encoded by Streptococcus agalactiae and Streptococcus ovuberis strains. Since this peptide displays some homology with nukacin ISK-1, we named it nukacin Spp. 2. This study demonstrated that the mutacin II biosynthesis machinery can be successfully used as an efficient system for the production of biologically active novel lantibiotics. IMPORTANCE In this study, we report for the first time that Streptococcus mutans can be used as a host to produce various nonnative lantibiotics. We showed that in the T8 strain, we could produce bioactive lacticin 481 and nukacin ISK-1, both of which are homologs of mutacin II, using T8's modification and secretion apparatus. Similarly, we also synthesized a novel bioactive lantibiotic, which we named nukacin Spp. 2.

Pseudomonas aeruginosa behaviour in polymicrobial communities: The competitive and cooperative interactions conducting to the exacerbation of infections.

Pseudomonas aeruginosa is mostly associated with persistent infections and antibiotic resistance as a result of several factors, biofilms one of them. Microorganisms within the polymicrobial biofilm (PMB) reveal various transcriptional profiles and affect each other which might influence their pathogenicity and antibiotic tolerance and subsequent worsening of the biofilm infection. P. aeruginosa within PMB exhibits various behaviours toward other microorganisms, which may enhance or repress the virulence of these microbes. Microbial neighbours, in turn, may affect P. aeruginosa's virulence either positively or negatively. Such interactions among microorganisms lead to emerging persistent and antibiotic-resistant infections. This review highlights the relationship between P. aeruginosa and its microbial neighbours within the PMB in an attempt to better understand the mechanisms of polymicrobial interaction and the correlation between increased exacerbations of infection and the P. aeruginosa-microbe interaction. Researching in the literature that was carried out in vitro either in co-cultures or in the models to simulate the environment at the site of infection suggested that the interplay between P. aeruginosa and other microorganisms is one main reason for the worsening of the infection and which in turn requires a treatment approach different from that followed with P. aeruginosa mono-infection.

Methods to Study Antagonistic Activities Among Oral Bacteria.

Most bacteria in nature exist in multispecies communities known as biofilms. In the natural habitat where resources (nutrient, space, etc.) are usually limited, individual species must compete or collaborate with other neighboring species in order to perpetuate in the multispecies community. The human oral cavity is colonized by >700 microbial species known as the indigenous microbiota. This indigenous flora normally maintains an ecological balance through antagonistic as well as mutualistic interspecies interactions. However, environmental perturbation may disrupt this balance, leading to overgrowth of pathogenic species which could in turn initiate diseases such as dental caries (tooth decay) and periodontitis (gum disease). Understanding the mechanisms of diversity maintenance may help developing novel approaches to manage these "polymicrobial diseases". In this chapter, we will focus on a well-characterized form of biochemical warfare: bacteriocins produced by Streptococcus mutans, a primary dental caries pathogen, and hydrogen peroxide (H2O2) produced by several oral commensal streptococci. We will describe detailed methodologies on the competition assay, isolation, purification, and characterization of bacteriocins.

Propionate Attenuates Growth of Oral Streptococci through Enhancing Methionine Biosynthesis.

Oral streptococci are considered as an opportunistic pathogen associated with initiation and progression of various oral diseases. However, since the currently-available treatments often accompany adverse effects, alternative strategy is demanded to control streptococci. In the current study, we investigated whether short-chain fatty acids (SCFAs), including sodium acetate (NaA), sodium propionate (NaP), and sodium butyrate (NaB), can inhibit the growth of oral streptococci. Among the tested SCFAs, NaP most potently inhibited the growth of laboratory and clinically isolated strains of Streptococcus gordonii under anaerobic culture conditions. However, the growth inhibitory effect of NaP on six different species of other oral streptococci was different depending on their culture conditions. Metabolic changes such as alteration of methionine biosynthesis can affect bacterial growth. Indeed, NaP enhanced intracellular methionine levels of oral streptococci as well as the mRNA expression level of methionine biosynthesis-related genes. Collectively, these results suggest that NaP has an inhibitory effect on the growth of oral streptococci, which might be due to alteration of methionine biosynthesis. Thus, NaP can be used an effective bacteriostatic agent for the prevention of oral infectious diseases caused by oral streptococci.

Transposon mutagenesis in oral streptococcus.

Oral streptococci are gram-positive facultative anaerobic bacteria that are normal inhabitants of the human oral cavity and play an important role in maintaining oral microecological balance and pathogenesis. Transposon mutagenesis is an effective genetic manipulation strategy for studying the function of genomic features. In order to study cariogenic related genes and crucial biological element genes of oral Streptococcus, transposon mutagenesis was widely used to identify functional genes. With the advent of next-generation sequencing (NGS) technology and the development of transposon random mutation library construction methods, transposon insertion sequencing (TIS) came into being. Benefiting from high-throughput advances in NGS, TIS was able to evaluate the fitness contribution and essentiality of genetic features in the bacterial genome. The application of transposon mutagenesis, including TIS, to oral streptococci provided a massive amount of valuable detailed linkage data between genetic fitness and genetic backgrounds, further clarify the processes of colonization, virulence, and persistence and provides a more reliable basis for investigating relationships with host ecology and disease status. This review focuses on transposon mutagenesis, including TIS, and its applicability in oral streptococci.

Infective endocarditis following invasive dental procedures: IDEA case-crossover study.

BACKGROUND: Infective endocarditis is a heart infection with a first-year mortality rate of ≈ 30%. It has long been thought that infective endocarditis is causally associated with bloodstream seeding with oral bacteria in ≈ 40-45% of cases. This theorem led guideline committees to recommend that individuals at increased risk of infective endocarditis should receive antibiotic prophylaxis before undergoing invasive dental procedures. However, to the best of our knowledge, there has never been a clinical trial to prove the efficacy of antibiotic prophylaxis and there is no good-quality evidence to link invasive dental procedures with infective endocarditis. Many contend that oral bacteria-related infective endocarditis is more likely to result from daily activities (e.g. tooth brushing, flossing and chewing), particularly in those with poor oral hygiene. OBJECTIVE: The aim of this study was to determine if there is a temporal association between invasive dental procedures and subsequent infective endocarditis, particularly in those at high risk of infective endocarditis. DESIGN: This was a self-controlled, case-crossover design study comparing the number of invasive dental procedures in the 3 months immediately before an infective endocarditis-related hospital admission with that in the preceding 12-month control period. SETTING: The study took place in the English NHS. PARTICIPANTS: All individuals admitted to hospital with infective endocarditis between 1 April 2010 and 31 March 2016 were eligible to participate. INTERVENTIONS: This was an observational study; therefore, there was no intervention. MAIN OUTCOME MEASURE: The outcome measure was the number of invasive and non-invasive dental procedures in the months before infective endocarditis-related hospital admission. DATA SOURCES: NHS Digital provided infective endocarditis-related hospital admissions data and dental procedure data were obtained from the NHS Business Services Authority. RESULTS: The incidence rate of invasive dental procedures decreased in the 3 months before infective endocarditis-related hospital admission (incidence rate ratio 1.34, 95% confidence interval 1.13 to 1.58). Further analysis showed that this was due to loss of dental procedure data in the 2-3 weeks before any infective endocarditis-related hospital admission. LIMITATIONS: We found that urgent hospital admissions were a common cause of incomplete courses of dental treatment and, because there is no requirement to record dental procedure data for incomplete courses, this resulted in a significant loss of dental procedure data in the 2-3 weeks before infective endocarditis-related hospital admissions. The data set was also reduced because of the NHS Business Services Authority's 10-year data destruction policy, reducing the power of the study. The main consequence was a loss of dental procedure data in the critical 3-month case period of the case-crossover analysis (immediately before infective endocarditis-related hospital admission), which did not occur in earlier control periods. Part of the decline in the rate of invasive dental procedures may also be the result of the onset of illness prior to infective endocarditis-related hospital admission, and part may be due to other undefined causes. CONCLUSIONS: The loss of dental procedure data in the critical case period immediately before infective endocarditis-related hospital admission makes interpretation of the data difficult and raises uncertainty over any conclusions that can be drawn from this study. FUTURE WORK: We suggest repeating this study elsewhere using data that are unafflicted by loss of dental procedure data in the critical case period. TRIAL REGISTRATION: This trial is registered as ISRCTN11684416. FUNDING: This project was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 26, No. 28. See the NIHR Journals Library website for further project information.

Infective endocarditis is a life-threatening infection of the heart valves. Most people are at low risk of infective endocarditis. However, those with certain cardiac conditions are at moderate risk of infective endocarditis, and those with artificial or repaired heart valves, a history of infective endocarditis and certain congenital heart conditions are at high risk of infective endocarditis. In around 40­45% of cases, oral bacteria are the cause of infective endocarditis. For many years, those people at moderate or high risk of infective endocarditis were given antibiotics (antibiotic prophylaxis) before invasive dental procedures such as extractions to reduce the risk of infective endocarditis. There is no good-quality evidence, however, to support the effectiveness of antibiotic prophylaxis, or the link between invasive dental procedures and infective endocarditis. Many believe that the oral bacteria that cause infective endocarditis are more likely to enter the blood during daily activities (e.g. toothbrushing, flossing or chewing), particularly in those with poor oral hygiene, than on the rare occasions when invasive dental procedures are performed. The aim of this study was to link English NHS data on infective endocarditis-related hospital admissions and dental treatments to determine if infective endocarditis is more likely in the weeks immediately after an invasive dental procedure than at any other time. When we linked the data sets and plotted the occurrence of different dental treatments over the year before infective endocarditis-related hospital admission, we detected a problem in the way that dental data were recorded. Unfortunately, there was a failure to collect dental procedure data when courses of treatment were incomplete. As one of the most common reasons for not completing a course of treatment was emergency admission to hospital, this meant that the number of dental procedures recorded decreased in the weeks before any emergency hospital admission. We have attempted to correct for this, but the data loss has affected the data quality. Although the data suggest an association between invasive dental procedures and infective endocarditis in individuals at high risk of infective endocarditis, the certainty of this association has been weakened.

E-Cigarette Aerosol Exposure Favors the Growth and Colonization of Oral Streptococcus mutans Compared to Commensal Streptococci.

E-cigarettes (e-cigs) have drastically increased in popularity during the last decade, especially among teenagers. While recent studies have started to explore the effect of e-cigs in the oral cavity, little is known about their effects on the oral microbiota and how they could affect oral health and potentially lead to disease, including periodontitis and head and neck cancers. To explore the impact of e-cigs on oral bacteria, we selected members of the genus Streptococcus, which are abundant in the oral cavity. We exposed the commensals Streptococcus sanguinis and Streptococcus gordonii and the opportunistic pathogen Streptococcus mutans, best known for causing dental caries, to e-liquids and e-cig aerosols with and without nicotine and with and without menthol flavoring and measured changes in growth patterns and biofilm formation. Our results demonstrate that e-cig aerosols hindered the growth of S. sanguinis and S. gordonii, while they did not affect the growth of S. mutans. We also show that e-cig aerosols significantly increased biofilm formation by S. mutans but did not affect the biofilm formation of the two commensals. We found that S. mutans exhibits higher hydrophobicity and coaggregation abilities along with higher attachment to OKF6 cells than S. sanguinis and S. gordonii. Therefore, our data suggest that e-cig aerosols have the potential to dysregulate oral bacterial homeostasis by suppressing the growth of commensals while enhancing the biofilm formation of the opportunistic pathogen S. mutans. This study highlights the importance of understanding the consequences of e-cig aerosol exposure on selected commensals and pathogenic species. Future studies modeling more complex communities will provide more insight into how e-cig aerosols and vaping affect the oral microbiota. IMPORTANCE Our study shows that e-cigarette aerosol exposure of selected bacteria known to be residents of the oral cavity hinders the growth of two streptococcal commensals while enhancing biofilm formation, hydrophobicity, and attachment for the pathogen S. mutans. These results indicate that e-cigarette vaping could open a niche for opportunistic bacteria such as S. mutans to colonize the oral cavity and affect oral health.

Expression and diversity of the sialic acid-binding adhesin and its homologs associated with oral streptococcal infection.

Streptococcus gordonii, one of the early colonizers of oral biofilms, is involved in the development of dental caries, periodontal disease, and infective endocarditis. The Hsa adhesin of S. gordonii DL1 has the ability to bind strongly to the terminal sialic acid groups of host glycoproteins via the binding region, nonrepetitive region 2 (NR2), which is important for the pathogenicity of S. gordonii DL1. Low similarity with the NR2 of Hsa homologs among other streptococcal species has been reported. However, the reports have been limited to certain strains. This study attempted to assess frequency of the expression on the bacterial cell surface and to analyze the diversity of Hsa homologs among different wild strains of oral streptococci. We isolated 186 wild-type strains of oral streptococci from healthy volunteers and analyzed their hemagglutinating (HA) activity on human erythrocytes and their Hsa homologs and NR2 homologous regions by dot immunoblotting using anti-Hsa and anti-NR2 antisera, respectively. We found 30 strains reacted with anti-NR2 antiserum (NR2 positive) and determined the sequence of the NR2 regions. Many strains with high HA activity were also NR2 positive, suggesting that the NR2 region may be associated with HA activity. Among the NR2-positive strains, four different amino acid sequence patterns were observed, demonstrating diversity in the NR2 region. Notably, S. gordonii strains frequently possessed Hsa homologs and NR2-like antigens compared with other streptococci. It is speculated that the possessing frequency of Hsa homologs and the amino acid sequence of NR2 region may vary among streptococcal species.

Effect of Oral Streptococci Expressing Pneumococcus-like Cross-Reactive Capsule Types on World Health Organization Recommended Pneumococcal Carriage Detection Procedure.

BACKGROUND: Carriage studies are fundamental to assessing the effects of pneumococcal vaccines. Because a large proportion of oral streptococci carry homologues of pneumococcal genes, non-culture-based detection and serotyping of upper respiratory tract (URT) samples can be problematic. In the current study, we investigated whether culture-free molecular methods could differentiate pneumococci from oral streptococci carried by adults in the URT. METHODS: Paired nasopharyngeal (NP) and oropharyngeal (OP) samples were collected from 100 older adults twice a month for 1 year. Extracts from the combined NP + OP samples (n = 2400) were subjected to lytA real-time polymerase chain reaction (PCR). Positive samples were subjected to pure culture isolation, followed by species confirmation using multiple approaches. Multibead assays and whole-genome sequencing were used for serotyping. RESULTS: In 20 of 301 combined NP + OP extracts with positive lytA PCR results, probable pneumococcus-like colonies grew, based on colony morphology and biochemical tests. Multiple approaches confirmed that 4 isolates were Streptococcus pneumoniae, 3 were Streptococcus pseudopneumoniae, 12 were Streptococcus mitis, and 1 were Streptococcus oralis. Eight nonpneumococcal strains carried pneumococcus-like cps loci (approximate size, 18-25 kb) that showed >70% nucleotide identity with their pneumococcal counterparts. While investigating the antigenic profile, we found that some S. mitis strains (P066 and P107) reacted with both serotype-specific polyclonal (type 39 and FS17b) and monoclonal (Hyp10AG1 and Hyp17FM1) antisera, whereas some strains (P063 and P074) reacted only with polyclonal antisera (type 5 and FS35a). CONCLUSION: The extensive capsular overlap suggests that pneumococcal vaccines could reduce carriage of oral streptococci expressing cross-reactive capsules. Furthermore, direct use of culture-free PCR-based methods in URT samples has limited usefulness for carriage studies.

Transcriptomic Responses to Coaggregation between Streptococcus gordonii and Streptococcus oralis.

Cell-cell adhesion between oral bacteria plays a key role in the development of polymicrobial communities such as dental plaque. Oral streptococci such as Streptococcus gordonii and Streptococcus oralis are important early colonizers of dental plaque and bind to a wide range of different oral microorganisms, forming multispecies clumps or "coaggregates." S. gordonii actively responds to coaggregation by regulating gene expression. To further understand these responses, we assessed gene regulation in S. gordonii and S. oralis following coaggregation in 25% human saliva. Coaggregates were formed by mixing, and after 30 min, RNA was extracted for dual transcriptome sequencing (RNA-Seq) analysis. In S. oralis, 18 genes (6 upregulated and 12 downregulated) were regulated by coaggregation. Significantly downregulated genes encoded functions such as amino acid and antibiotic biosynthesis, ribosome, and central carbon metabolism. In total, 28 genes were differentially regulated in Streptococcus gordonii (25 upregulated and 3 downregulated). Many genes associated with transporters and a two-component (NisK/SpaK) regulatory system were upregulated following coaggregation. Our comparative analyses of S. gordonii-S. oralis with different previously published S. gordonii pairings (S. gordonii-Fusobacterium nucleatum and S. gordonii-Veillonella parvula) suggest that the gene regulation is specific to each pairing, and responses do not appear to be conserved. This ability to distinguish between neighboring bacteria may be important for S. gordonii to adapt appropriately during the development of complex biofilms such as dental plaque. IMPORTANCE Dental plaque is responsible for two of the most prevalent diseases in humans, dental caries and periodontitis. Controlling the formation of dental plaque and preventing the transition from oral health to disease requires a detailed understanding of microbial colonization and biofilm development. Streptococci are among the most common colonizers of dental plaque. This study identifies key genes that are regulated when oral streptococci bind to one another, as they do in the early stages of dental plaque formation. We show that specific genes are regulated in two different oral streptococci following the formation of mixed-species aggregates. The specific responses of S. gordonii to coaggregation with S. oralis are different from those to coaggregation with other oral bacteria. Targeting the key genes that are upregulated during interspecies interactions may be a powerful approach to control the development of biofilm and maintain oral health.

Deacidification of Cranberry Juice Reduces Its Antibacterial Properties against Oral Streptococci but Preserves Barrier Function and Attenuates the Inflammatory Response of Oral Epithelial Cells.

Cranberry (Vaccinium macrocarpon) may be a potent natural adjuvant for the prevention of oral diseases due to its anti-adherence, anti-cariogenic, and anti-inflammatory properties. However, the high titrable acidity of cranberry juice (CJ) has been reported to cause gastrointestinal discomfort, leading consumers to restrict their intake of this beverage. Electrodialysis with a bipolar membrane (EDBM) can reduce the organic acid content of CJ while retaining the flavonoids associated with potential health benefits. This study aimed to assess how the deacidification of CJ by EDBM impacts the antibacterial properties of the beverage against cariogenic (Streptococcus mutans, Streptococcus sobrinus) and commensal (Streptococcus gordonii, Streptococcus oralis, Streptococcus salivarius) streptococci, and how it affects oral epithelial barrier function and inflammatory response in an in vitro model. The removal of organic acids from CJ (deacidification rate ≥42%) reduced the bactericidal activity of the beverage against planktonic S. mutans and S. gordonii after a 15-min exposure, whereas only the viability of S. gordonii was significantly impacted by CJ deacidification rate when the bacteria were embedded in a biofilm. Moreover, conditioning saliva-coated hydroxyapatite with undiluted CJ samples significantly lowered the adherence of S. mutans, S. sobrinus, and S. oralis. With respect to epithelial barrier function, exposure to CJ deacidified at a rate of ≥19% maintained the integrity of a keratinocyte monolayer over the course of 24 h compared to raw CJ, as assessed by the determination of transepithelial electrical resistance (TER) and fluorescein isothiocyanate-conjugated dextran paracellular transport. These results can be in part attributed to the inability of the deacidified CJ to disrupt two tight junction proteins, zonula occludens-1 and occludin, following exposure, unlike raw CJ. Deacidification of CJ impacted the secretion of IL-6, but not of IL-8, by oral epithelial cells. In conclusion, deacidification of CJ appears to provide benefits with respect to the maintenance of oral health.

Proteomic response in Streptococcus gordonii DL1 biofilm cells during attachment to salivary MUC5B.

BACKGROUND: Salivary mucin MUC5B seems to promote biodiversity in dental biofilms, and thereby oral health, for example, by inducing synergistic 'mucolytic' activities in a variety of microbial species that need to cooperate for the release of nutrients from the complex glycoprotein. Knowledge of how early colonizers interact with host salivary proteins is integral to better understand the maturation of putatively harmful oral biofilms and could provide key insights into biofilm physiology. METHODS: The early oral colonizer Streptococcus gordonii DL1 was grown planktonically and in biofilm flow cell systems with uncoated, MUC5B or low-density salivary protein (LDP) coated surfaces. Bacterial cell proteins were extracted and analyzed using a quantitative mass spectrometry-based workflow, and differentially expressed proteins were identified. RESULTS AND CONCLUSIONS: Overall, the proteomic profiles of S. gordonii DL1 were similar across conditions. Six novel biofilm cell proteins and three planktonic proteins absent in all biofilm cultures were identified. These differences may provide insights into mechanisms for adaptation to biofilm growth in this species. Salivary MUC5B also elicited specific responses in the biofilm cell proteome. These regulations may represent mechanisms by which this mucin could promote colonization of the commensal S. gordonii in oral biofilms.

Prevalence, diversity and transferability of the Tn916-Tn1545 family ICE in oral streptococci.

Background: The Tn916-Tn1545 family of Integrative Conjugative Elements (ICE) are mobile genetic elements (MGEs) that play a role in the spread of antibiotic resistance genes. The Tn916 harbors the tetracycline resistance gene tet(M) and it has been reported in various bacterial species. The increase in the levels of tetracycline resistance among oral streptococci is of great concern primarily due to the abundance of these species in the oral cavity and their ability to act as reservoirs for antibiotic resistance genes.Methods: In the current study, we screened 100 Norwegian clinical oral streptococcal isolates for the presence and diversity of the Tn916-Tn1545 family. In addition, we investigated the transferability the elements, and the associated transfer frequencies.Results: We observed that 21 isolates harboured the Tn916-Tn1545 family and that two of these elements were the novel Tn6815 and Tn6816. The most prevalent member of the Tn916 -Tn1545 family observed in the Norwegian clinical oral streptococcal isolates was the wild type Tn916.Conclusion: The detection of other members of this family of ICE and varying transfer frequencies suggests high versatility of the Tn916 element in oral streptococci in Norway.

Evaluating the intrinsic capacity of oral bacteria to produce hydrogen peroxide (H2O2) in liquid cultures: Interference by bacterial growth media.

This work highlights the issue of interference by growth media when measuring bacterial H2O2 production. H2O2 was shown to be stable in phosphate buffered saline (PBS) but not in growth media. The protocol used for evaluating the intrinsic capacity of oral streptococci to produce H2O2 was shown to be reliable.

Spectrum and resistance determinants of oral streptococci clinical isolates.

The profiles of oral streptococci sensitivity to antibacterial drugs may reflect information about the presence of macroorganism resistance determinants. The aim of the work was to isolate the spectrum of oral streptococci from the microbiota of the oral cavity of patients and to determine their sensitivity to a wide range of antibiotics. A total of 342 microbial streptococcal isolates were isolated from saliva samples and a periodontal pocket and tested for antibiotic sensitivity. Species identification of streptococci was carried out using biochemical API test systems. Evaluation of antibiotic resistance was performed using E-tests. Real-time PCR was used to identify the presence of tetracycline and macrolide resistance genes. The study identified six types of oral streptococci: S. oralis, S. salivarius, S. mitis, S. sanguinis, S. anginosus and S. mutans. All streptococci were sensitive to linezolid and meropenem. The proportion of penicillin-resistant streptococci in the subgroup S. oralis / mitis / mutans was 47,8% versus 23,5% in the subgroup S. salivarius / sanguinis / anginosus (p = 0.020). Significant levels of resistance were revealed to macrolides (erythromycin) - 47,9%, tetracyclines (tetracycline) - 44,4% and quinolones (ofloxacin) - 41%. Multiple drug resistance (MDR) was detected in 31,9% of oral streptococcal isolates, a combination of erythromycin, tetracycline and ofloxacin resistance was prevalent in 79 isolates (23,1%). The most common genotypes of macrolides and tetracycline resistant oral streptococci (in 127 streptococcal isolates with combined resistance) were ermB-mefE + and tetM + tetQ-, respectively. Thus, S. oralis / mitis / mutans group streptococci predominated in the structure of antibiotic-resistant oral streptococci, including MDR. So, being in one of the most densely populated biotopes of a macroorganism, oral streptococci can mediate the transfer of resistance determinants to more pathogenic and clinically significant microorganisms, which requires careful monitoring of their level of susceptibility to antimicrobial agents.

Expression of an Extracellular Protein (SMU.63) Is Regulated by SprV in Streptococcus mutans.

In Streptococcus mutans, SprV (SMU.2137) is a pleiotropic regulator that differentially regulates genes related to competence, mutacin production, biofilm formation, and the stress tolerance response, along with some other pathways. In this study, we established a link between SprV and an ∼67-kDa protein in the culture supernatant of strain UA159 that was later confirmed as SMU.63 by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. We discovered that SprV downregulates the transcription and translation of SMU.63. We found that the seven amino acids from the C-terminal region of SprV were also crucial for the expression of SMU.63. Deletion of smu.63 led to increased sucrose-independent biofilm formation and competence. The sprV deletion also increased biofilm formation although this could be partially attributed to the downregulation of smu.63 In an smu.63 sprV double mutant, a synergistic effect was observed in biofilm formation in contrast to effects on competence development. We found that low or excess magnesium ion repressed sprV transcription that, in turn, affected the expression of smu.63 As expected, a magnesium ion-dependent effect of competence and biofilm formation was observed in the UA159 strain. We also replicated the results of SMU.63 expression and competence in S. mutans GS5 that encodes both SprV and SMU.63 homologs and found that the GS5 strain behaves similarly to the UA159 strain, indicating that SprV's effect is strain independent.IMPORTANCE We previously identified a pleiotropic regulator, SprV, in Streptococcus mutans This regulator appears to be highly conserved among streptococci. Here, we showed that SprV regulates the expression of a secreted protein encoded by SMU.63 in S. mutans SMU.63 has been known to impact biofilm formation and genetic competence, two important characteristics that help in colonization of the organism. SMU.63 is also unique since it is known to form amyloid fiber. We found that SprV regulates the expression of SMU.63 at both the transcriptional and translational levels. We also found that the expression of SprV is regulated by magnesium ion concentration. Interestingly, both low and high magnesium ion concentrations affected biofilm formation and genetic competence. Since SMU.63 is also highly conserved among streptococci, we hypothesized that SprV will have a similar effect on its expression.

Evaluating the relationship between acidogenicity and acid tolerance for oral streptococci from children with or without a history of caries.

Background: Dental caries etiology is attributed to a dysbiotic imbalance within the plaque microbiome leading to a dominance of strong acidogens. Some studies that investigate the link between acidogens and caries quantify the recovery of acid tolerant strains on acid agar as a measure of acidogenic potential. This methodology assumes that acidogenic potential and acid tolerance are directly related. Aim: The validity of that assumption was investigated by statistically evaluating that relationship using streptococci recovered from children with or without a history of dental caries. Methods: Thirty streptococcal isolates were isolated from each of 13 subjects. Acidogenicity was quantified by measuring the terminal pH after overnight growth in Brain Heart Infusion (BHI) and Chemically Defined Medium (CDM). Acid tolerance was quantified by measuring the lowest pH acid agar displaying growth. Results: A significant difference in acidogenicity in CDM between levels of acid tolerance was found, but no significant difference in acidogenicity in BHI was noted. Moreover, there were no significant interactions between acid tolerance and caries history on acidogenicity measures in either medium. Conclusion: An ability to grow on acid agar below pH 5.0 is best aligned with strong acidogenicity and best able to distinguish between subjects with differing caries histories.

Rapid Isolation and Purification of Secreted Bacteriocins from Streptococcus mutans and Other Lactic Acid Bacteria.

Bacteriocins are small ribosomally synthesized antimicrobial peptides produced by some microorganisms including lactic acid bacteria (LAB), a group of Gram-positive bacteria (cocci, rods) expressing high tolerance for low pH. Bacteriocins kill bacteria rapidly and are biologically active at very low concentrations. Bacteriocins produced by LAB are primarily active against closely related bacterial species. Many bacteriocins have been investigated with respect to their potential use in promoting human, plant, and animal health, and as food biopreservatives. Bacteriocins produced by LAB are particularly interesting since several LAB have been granted GRAS (Generally Recognized as Safe) status. Because it is not always possible to extract active bacteriocins secreted from cells grown in liquid medium, we developed a simple and inexpensive peptide extraction procedure using a semi-solid nutrient-rich agar medium. We hereby present a detailed procedure that leads to the rapid extraction of secreted bioactive bacteriocin peptides from the oral species Streptococcus mutans, a prolific bacteriocin-producing species, and its potential application for bacteriocin extraction from other LAB (e.g., Streptococcus, Lactococcus, Enterococcus). We also present a simple method for the detection of bacteriocin activity from the purified extracellular peptide extract.