Streptococcus Oligofermentans Research Articles

Anti-caries Streptococcus spp.: A potential preventive tool for special needs patients.

Probiotics are living microorganisms that act on the host-microbiome interface to restore the microbiota's physiological homeostasis. Numerous probiotics have been marketed with inhibitory activity against Streptococcus mutans and consequently with a potential anti-caries effect, mainly of the genera Lactobacillus and Bifidobacterium, whose main disadvantage is their limited ability to settle in the oral cavity. This narrative review describes the main Streptococcus spp. with probiotic anti-Streptococcus mutans activity, whose substantivity is greater than that of Lactobacillus spp. and consequently with anti-caries potentiality. We performed a literature review in the PubMed, Science Direct and Google Scholar databases of articles published in English (without time restriction) related to caries and probiotics. The potential identified anti-caries probiotics included Streptococcus spp. A12, Streptococcus oralis (AJ3), Streptococcus oligofermentans, Streptococcus salivarius (K12, M18, JH, LAB813, 24SMB), Streptococcus spp. with arginolytic activity (S. sanguinis, S. gordonii, S. ratti, S. parasanguinis, S. intermedius, S. australis, and S. cristatus), Streptococcus rattus (JH145), Streptococcus dentisani and Streptococcus downii. The possibility of using these Streptococcus spp. as probiotics that inhibit the growth of dental plaque and the development of carious lesions represents a potential tool of particular interest for individuals with physical or intellectual disabilities that impede the routine and effective application of mechanical dental plaque removal techniques.

Elucidating the Role and Structure-Activity Relationships of the Streptococcus oligofermentans Competence-Stimulating Peptide.

Streptococcus oligofermentans is an early colonizer of the oral microbiome with documented bactericidal activity against the oral pathogen Streptococcus mutans. S. oligofermentans has been observed to possess the typical comABCDE competence regulon found within most oral streptococci; however, the competence-stimulating peptide (CSP) responsible for QS activation and the regulatory role of the competence regulon is yet to be explored. Herein, we have both confirmed the identity of the S. oligofermentans CSP and utilized a wide range of phenotypic assays to characterize its regulatory role in competence, biofilm formation, and hydrogen peroxide formation. To determine the importance of each amino acid residue in CSP/ComD binding, we performed systematic replacement of amino acid residues within the S. oligofermentans CSP and developed a luciferase-based reporter system to assess the ability of these mutated analogues to modulate the competence regulon. Additionally, we performed CD analysis on mutated CSP analogues to determine the correlation between the peptide secondary structure and QS activation. To further explore S. oligofermentans' potential as a biotherapeutic against S. mutans infection, lead QS activators and inhibitors were used in interspecies competition assays to assess the effect of QS modulation on interactions between these two species. Lastly, we have documented a lack of S. oligofermentans-induced cytotoxicity, highlighting the potential of this native flora as a biotherapeutic with minimal health risks.

RNase Z Oxidative Degradation Impedes tRNA Maturation and is Involved in Streptococcal Translation Regulation in Response to Oxidative Stress.

ABSTRACTWhen encountering oxidative stress, organisms selectively upregulate antioxidant genes and simultaneously suppress the translation of most other proteins. Eukaryotes employ multiple strategies to adjust translation at both the initiation and elongation stages; however, how prokaryotes modulate translation under oxidative stress remains unclear. Here, we report that upon hydrogen peroxide (H2O2) challenge, Streptococcus oligofermentans reduced translation via RNase Z (So-RNaseZ) oxidative degradation, thus hindering tRNA maturation. S. oligofermentans encodes all CCA-less tRNAs that require So-RNaseZ for 3′ end maturation. A combination of nonreducing SDS-PAGE and liquid chromatography/tandem mass spectrometry (LC/MS-MS) assays demonstrated that H2O2 oxidation induced Cys38-Cys149 disulfide linkages in recombinant So-RNaseZ protein, and serine substitution of Cys38 or Cys149 abolished these disulfide linkages. Consistently, redox Western blotting also determined intramolecular disulfide-linked So-RNaseZ in H2O2-treated S. oligofermentans cells. The disulfide-linked So-RNaseZ and monomer were both subject to proteolysis, whereas C149S mutation alleviated oxidative degradation of So-RNaseZ, suggesting that H2O2-mediated disulfide linkages substantially contributed to So-RNaseZ degradation. Accordingly, Northern blotting determined that tRNA precursor accumulation and mature tRNA species decrease in H2O2-treated S. oligofermentans. Moreover, reduced overall protein synthesis, as indicated by puromycin incorporation, and retarded growth of S. oligofermentans occurred in an H2O2 concentration-dependent manner. Overexpression of So-RNaseZ not only elevated tRNA precursor processing and protein synthesis but also partly rescued H2O2-suppressed S. oligofermentans growth. Moreover, So-RNaseZ oxidative degradation-mediated translation repression elevated S. oligofermentans survival under high H2O2 stress. Therefore, this work found that So-RNaseZ oxidative degradation-impeded tRNA maturation contributes to streptococcal translation repression and provides the oxidative stress adaptability for S. oligofermentans.IMPORTANCE Translation regulation is a common strategy used by organisms to reduce oxidative damage. Catalase-negative streptococci produce as well as tolerate high levels of H2O2. This work reports a novel translation regulation mechanism employed by Streptococcus oligofermentans in response to H2O2 challenge, in which the key tRNA endonuclease So-RNaseZ is oxidized to form Cys38-Cys149 disulfide linkages and both the disulfide-linked So-RNaseZ and monomers are subject to proteolysis; thus, tRNA maturation, protein translation, and growth are all suppressed. Notably, So-RNaseZ oxidative degradation-mediated translation repression offers oxidative adaptability to S. oligofermentans and enhances its survival against high H2O2 challenge. So-RNaseZ orthologs and H2O2-sensitive cysteines (Cys38 and Cys149) are widely distributed in Streptococcus and Lactococcus species genomes, which also encode all CCA-less tRNAs and lack catalase. Therefore, RNase Z oxidative degradation-based translation regulation could be widely employed by these lactic acid bacteria, including pathogenic streptococci, to cope with H2O2.

Metagenome association study of the gut microbiome revealed biomarkers linked to chemotherapy outcomes in locally advanced and advanced lung cancer.

BackgroundThe gut microbiome is important in the development and immunotherapy efficacy of lung cancer. However, the relationship between the intestinal flora and chemotherapy outcomes remains unclear and was investigated in this study.MethodsWe analyzed baseline stool samples from patients with locally advanced and advanced lung cancer before chemotherapy treatment, through metagenomics of the gut microbiota. The composition, diversity, function, and metabolic pathway analysis were compared among patients with different clinical outcomes.ResultsFrom 64 patients, 33 responded to treatment (responders) and 31 did not (nonresponders). Streptococcus mutans and Enterococcus casseliflavus were enriched in responders (P < 0.05), while 11 bacteria including Leuconostoc lactis and Eubacterium siraeum were enriched in nonresponders (P < 0.05) by variance analysis. Responders were associated with significantly higher Acidobacteria and Granulicella, while Streptococcus oligofermentans, Megasphaera micronuciformis, and Eubacterium siraeum were more abundant in nonresponders by Lefse analysis. Streptococcus mutans and Enterococcus casseliflavus were further identified as bacterial markers relevant to responders using unsupervised clustering, and Leuconostoc lactis and Eubacterium siraeum were related to nonresponders. The L‐glutamate degradation VIII pathway was enriched in responders (P = 0.014), and the C4 photosynthetic carbon assimilation cycle, reductive TCA cycle I, and hexitol fermentation to lactate, formate, ethanol, and acetate were enriched in nonresponders (P < 0.05). Additionally, significant associations of bacterial species with clinical phenotypes were observed by Spearman correlation analysis.ConclusionsThe specific gut microbiome of patients with lung cancer might be connected to the clinical outcomes of chemotherapy.Key pointsSignificant findings of the study Lung cancer patients with different gut microbiome compositions and microbiome metabolic pathways have different responses to chemotherapy. Microbiome species are also associated with different lung cancer clinical phenotypes. What this study adds We have identified specific gut microbiome species that can be used as relevant biomarkers for chemotherapy outcomes. This can potentially be used to guide clinical treatment decisions.

Probiotic Effects on Multispecies Biofilm Composition, Architecture, and Caries Activity In Vitro.

While probiotics have been tested for their anti-caries effect in vitro and also clinically, there is a lack of understanding of their effects on complex dental biofilms. We assessed two probiotics, Lactobacillus reuteri and Streptococcus oligofermentans, on a continuous-cultured model containing Streptococcus mutans, Lactobacillus rhamnosus and Actinomyces naeslundii. Cariogenic biofilms were grown on bovine enamel specimens and daily challenged with L. reuteri or S. oligofermentans whole culture (LC/SC) or cell-free supernatant (LS/SS) or medium only (negative control, NC) (n = 21/group) for 10 days. Biofilm was assessed via counting colony-forming units, quantitative polymerase chain reaction, and fluorescence in situ hybridization. Caries activity was determined by pH measurements and by assessing mineral loss (ΔZ) using transverse microradiography. Both LC and SC significantly reduced total and strain-specific cariogenic bacterial numbers (p < 0.05). ΔZ was reduced in LC (mean ± SD: 1846.67 ± 317.89) and SC (3315.87 ± 617.30) compared to NC (4681.48 ± 495.18, p < 0.05). No significant reductions in bacterial numbers and ΔZ was induced by supernatants. Biofilm architecture was not considerably affected by probiotic applications. Viable probiotics L. reuteri and S. oligofermentans, but not their culture supernatants, could reduce the caries activity of multi-species biofilms in vitro.

Environment-Specific Probiotic Supernatants Modify the Metabolic Activity and Survival of Streptococcus mutans in vitro.

A range of studies showed probiotics like Streptococcus oligofermentans and Limosilactobacillus reuteri to inhibit the cariogenic activity and survival of Streptococcus mutans, possibly via the production of substances like H2O2, reuterin, ammonia and organic acids. We aimed to assess the environment-specific mechanisms underlying this inhibition. We cultured L. reuteri and S. oligofermentans in various environments; minimal medium (MM), MM containing glucose (MM+Glu), glycerol (MM+Gly), lactic acid (MM+Lac), arginine (MM+Arg) and all four substances (MM+all) in vitro. Culture supernatants were obtained and metabolite concentrations (reuterin, ammonia, H2O2, lactate) measured. S. mutans was similarly cultivated in the above six different MM variation media, with glucose being additionally added to the MM+Gly, MM+Lac, and MM+Arg group, with (test groups) and without (control groups) the addition of the supernatants of the described probiotic cultures. Lactate production by S. mutans was measured and its survival (as colony-forming-units/mL) assessed. L. reuteri environment-specifically produced reuterin, H2O2, ammonia and lactate, as did S. oligofermentans. When cultured in S. oligofermentans supernatants, lactate production by S. mutans was significantly reduced (p < 0.01), especially in MM+Lac+Glu and MM+all, with no detectable lactate production at all (controls means ± SD: 4.46 ± 0.41 mM and 6.00 ± 0.29 mM, respectively, p < 0.001). A similar reduction in lactate production was found when S. mutans was cultured in L. reuteri supernatants (p < 0.05) for all groups except MM+Lac+Glu. Survival of S. mutans cultured in S. oligofermentans supernatants in MM+Lac+Glu and MM+all was significantly reduced by 0.6-log10 and 0.5-log10, respectively. Treatment with the supernatant of L. reuteri resulted in a reduction in the viability of S. mutans in MM+Gly+Glu and MM+all by 6.1-log10 and 7.1-log10, respectively. Probiotic effects on the metabolic activity and survival of S. mutans were environment-specific through different pathways.

Redox-Regulated Adaptation of Streptococcus oligofermentans to Hydrogen Peroxide Stress.

Preexposure to a low concentration of H2O2 significantly increases the survivability of catalase-negative streptococci in the presence of a higher concentration of H2O2 However, the mechanisms of this adaptation remain unknown. Here, using a redox proteomics assay, we identified 57 and 35 cysteine-oxidized proteins in Streptococcus oligofermentans bacteria that were anaerobically cultured and then pulsed with 40 μM H2O2 and that were statically grown in a 40-ml culture, respectively. The oxidized proteins included the peroxide-responsive repressor PerR, the manganese uptake repressor MntR, thioredoxin system proteins Trx and Tpx, and most glycolytic proteins. Cysteine oxidations of these proteins were verified through redox Western blotting, immunoprecipitation, and liquid chromatography-tandem mass spectrometry assays. In particular, Zn2+-coordinated Cys139 and Cys142 mutations eliminated the H2O2 oxidation of PerR, and inductively coupled plasma mass spectrometry detected significantly decreased amounts of Zn2+ in H2O2-treated PerR, demonstrating that cysteine oxidation results in Zn2+ loss. An electrophoretic mobility shift assay (EMSA) determined that the DNA binding of Mn2+-bound PerR protein (PerR:Zn,Mn) was abolished by H2O2 treatment but was restored by dithiothreitol reduction, verifying that H2O2 inactivates streptococcal PerR:Zn,Mn through cysteine oxidation, analogous to the findings for MntR. Quantitative PCR and EMSA demonstrated that tpx, mntA, mntR, and dpr belonged to the PerR regulons but that only dpr was directly regulated by PerR; mntA was also controlled by MntR. Deletion of mntR significantly reduced the low-H2O2-concentration-induced adaptation of S. oligofermentans to a higher H2O2 concentration, while the absence of PerR completely abolished the self-protection. Therefore, a low H2O2 concentration resulted in the cysteine-reversible oxidations of PerR and MntR to derepress their regulons, which function in cellular metal and redox homeostasis and which endow streptococci with the antioxidative capability. This work reveals a novel Cys redox-based H2O2 defense strategy employed by catalase-negative streptococci in Mn2+-rich cellular environments.IMPORTANCE The catalase-negative streptococci produce as well as tolerate high levels of H2O2 This work reports the molecular mechanisms of low-H2O2-concentration-induced adaptation to higher H2O2 stress in a Streptococcus species, in which the peroxide-responsive repressor PerR and its redox regulons play the major role. Distinct from the Bacillus subtilis PerR, which is inactivated by H2O2 through histidine oxidation by the Fe2+-triggered Fenton reaction, the streptococcal PerR is inactivated by H2O2 oxidation of the structural Zn2+ binding cysteine residues and thus derepresses the expression of genes defending against oxidative stress. The reversible cysteine oxidation could provide flexibility for PerR regulation in streptococci, and the mechanism might be widely used by lactic acid bacteria, including pathogenic streptococci, containing high levels of cellular manganese, in coping with oxidative stress. The adaptation mechanism could also be applied in oral hygiene by facilitating the fitness and adaptability of the oral commensal streptococci to suppress the pathogens.

Influence of environmental factors on the two-species biofilm formed by Streptococcus oligofermentans and Streptococcus mutans

Objective: To study the influence of environmental factors on the two-species biofilm formed by the combinations of Streptococcus oligofermentans (So) with Streptococcus mutans (Sm) and Streptococcus sanguinis (Ss) with Sm so as to evaluate the role of So in maintaining the microecological balance of the oral cavity. Methods: Single-and two-species biofilms were grown on saliva-coated surfaces (glass tube and 96-well plate). Colony-counting method and safranin staining method were used to measure the biofilms formed under various oxygen conditions (aerobic and anaerobic), sucrose conditions (0%, 1% and 5% sucrose concentrations) and pH conditions (5.5, 6.0, 6.5, 7.0, 7.5 and 8.0). Results: Comparing the numbers of Sm in two co-cultures under various conditions, Sm counts in So+Sm group [(7.70±2.46)×10(8) CFU/ml] were significantly lower than those in Ss+Sm group [(9.00±1.13)×10(8) CFU/ml] in aerobic environment (P<0.05). Sm counts in So+Sm group [(2.80±0.52)×10(8) CFU/ml] were also significantly lower than those in the Ss+Sm group [(4.00±1.25)×10(8) CFU/ml] in anaerobic environment (P<0.05). The Sm counts in So+Sm group [(8.90±0.82)×10(8) CFU/ml] were significantly higher than those in Ss+Sm group [(7.50±1.73)×10(8) CFU/ml] in 0% sucrose environment (P<0.05). The Sm counts in So+Sm group [(5.70±2.94)×10(8) CFU/ml] were significantly lower than those in Ss+Sm group [(10.30±3.21) ×10(8) CFU/ml] in 1% sucrose environment (P<0.05). The Sm counts in So+Sm group [(6.10±1.71)×10(8) CFU/ml] were also significantly lower than those in Ss+Sm group [(7.40±1.20)×10(8) CFU/ml] in 5% sucrose environment (P<0.05). The Sm counts in So+Sm group [(3.50±1.50)×10(8) CFU/ml] were significantly lower than those in Ss+Sm group [(10.70±2.80)×10(8) CFU/ml] in pH7.0 environment (P<0.05). Comparing the formation of biofilm after 24 h cultivation, the Sm counts in So+Sm group were significantly lower than those in Ss+Sm group both in aerobic and anaerobic environments (P<0.05). The Sm counts in So+Sm group were significantly higher than those in Ss+Sm group in 0% sucrose environment (P<0.05). The Sm counts in So+Sm group were significantly lower than those in Ss+Sm group in 1% and 5% sucrose and pH 7.0 environments (P<0.05). Both So and Ss had no inhibitory effect on Sm in pH5.5 and pH8.0 environments. Conclusions: In the in vitro two-species co-culture systems, So showed stronger inhibitory effects than Ss on Sm and its inhibitory ability might influenced by various environmental factors.

The carbon catabolite repressor CcpA mediates optimal competence development in Streptococcus oligofermentans through post-transcriptional regulation.

Natural transformation increases the genetic diversity of bacteria, but is costly and must be strictly controlled. We previously found that deletion of ccpA, a key regulator of carbon catabolite repression (CCR), reduced transformation efficiency of Streptococcus oligofermentans, the current work further investigated the regulatory mechanisms of CcpA. The competence operon comCDE is subjected to basal and autoregulatory transcription. A luciferase reporter detected a transcriptional readthrough (TRT) from the upstream tRNAArg into the comCDE operon, which was induced by L -arginine. Insertion of the Escherichia coli T1T2 terminator downstream of tRNAArg abolished TRT, and reduced the basal comCDE transcription by 77% and also the transformation efficiency. Deletion of ccpA increased tRNAArg TRT and tRNAArg -comCDE polycistronic transcript by twofold. An in vitro transcription assay determined that CcpA promoted the transcription termination of tRNAArg TRT, and RNA EMSA and SPR assays detected equal binding affinity of CcpA to both the RNA and DNA of tRNAArg . These results indicate that CcpA controls the basal comCDE transcription by post-transcriptional actions. Overexpression of comDE or its phospho-mimicking mutant comDED58E reduced transformation efficiency, indicating that excessive ComE impairs competence development. CCR-regulated competence was further confirmed by higher tRNAArg TRT but lower transformation efficiency in galactose than in glucose.

A Streptococcus aquaporin acts as peroxiporin for efflux of cellular hydrogen peroxide and alleviation of oxidative stress

Aquaporins (AQPs) are transmembrane proteins widely distributed in various organisms, and they facilitate bidirectional diffusion of water and uncharged solutes. The catalase-negative bacterium Streptococcus oligofermentans produces the highest H2O2 levels reported to date, which has to be exported to avoid oxidative stress. Here, we report that a S. oligofermentans aquaporin functions as a peroxiporin facilitating bidirectional transmembrane H2O2 transport. Knockout of this aquaporin homolog, So-AqpA, reduced H2O2 export by ∼50% and increased endogenous H2O2 retention, as indicated by the cellular H2O2 reporter HyPer. Heterologous expression of So-aqpA accelerated exogenous H2O2 influx into Saccharomyces cerevisiae and Escherichia coli cells, indicating that So-AqpA acts as an H2O2-transferring aquaporin. Alanine substitution revealed Phe-40 as a key residue for So-AqpA-mediated H2O2 transport. Northern blotting, qPCR, and luciferase reporter assays disclosed that H2O2 induces a >10-fold expression of So-aqpA Super-resolution imaging showed that H2O2 treatment increases So-AqpA protein molecules per cell by 1.6- to 3-fold. Inactivation of two redox-regulatory transcriptional repressors, PerR and MntR, reduced H2O2-induced So-aqpA expression to 1.8- and 4-fold, respectively. Electrophoretic mobility shift assays determined that MntR, but not PerR, binds to the So-aqpA promoter, indicating that MntR directly regulates H2O2-induced So-aqpA expression. Importantly, So-aqpA deletion decreased oxic growth and intraspecies competition and diminished the competitive advantages of S. oligofermentans over the caries pathogen Streptococcus mutans Of note, So-aqpA orthologs with the functionally important Phe-40 are present in all streptococci. Our work has uncovered an intrinsic, H2O2-inducible bacterial peroxiporin that has a key physiological role in H2O2 detoxification in S. oligofermentans.

Factors Influencing the Competition between Streptococcus oligofermentans and Streptococcus mutans in Dual-Species Biofilms

Previous studies have shown that Streptococcus oligofermentans inhibits the growth of cariogenic Streptococcus mutans in biofilms in vitro and is considered a probiotic candidate for caries prevention. This study aimed to examine the effects of various environmental factors on the competition between S. oligofermentans and S. mutans in a dual-species biofilm model. Single or dual S. oligofermentans and S. mutans biofilms were grown in a 96-well active attachment model for 48 h. Several growth conditions were examined in the model, namely: S. oligofermentans was inoculated 24 h before S. mutans or vice versa; the growth medium was supplemented with 0.2% sucrose or 0.4% glucose; biofilms were grown under a constantly neutral pH or pH-cycling condition, which included 8 h of neutral pH and 16 h of pH 5.5. The 48-h biofilms were examined for viable cell counts and lactic acid and hydrogen peroxide production ability. When S. oligofermentans was inoculated first, it clearly inhibited the growth of S. mutans and reduced the biofilm lactic acid production by up to 8-fold through hydrogen peroxide production, independently of sugar supply and pH conditions. When S. mutans was inoculated first, the level of inhibition by S. oligofermentans varied depending on the sugar supply and pH conditions. Thus, the inhibition efficacy of S. oligofermentans against S. mutans in dual-species biofilms is influenced by environmental factors. This study provides practical information on how to maximize the efficacy of S. oligofermentans.

Re-evaluation of the taxonomy of the Mitis group of the genus Streptococcus based on whole genome phylogenetic analyses, and proposed reclassification of Streptococcus dentisani as Streptococcus oralis subsp. dentisani comb. nov., Streptococcus tigurinus as Streptococcus oralis subsp. tigurinus comb. nov., and Streptococcus oligofermentans as a later synonym of Streptococcus cristatus.

The Mitis group of the genus Streptococcus currently comprises 20 species with validly published names, including the pathogen S. pneumoniae. They have been the subject of much taxonomic confusion, due to phenotypic overlap and genetic heterogeneity, which has hampered a full appreciation of their clinical significance. The purpose of this study was to critically re-examine the taxonomy of the Mitis group using 195 publicly available genomes, including designated type strains for phylogenetic analyses based on core genomes, multilocus sequences and 16S rRNA gene sequences, combined with estimates of average nucleotide identity (ANI) and in silico and in vitro analyses of specific phenotypic characteristics. Our core genomic phylogenetic analyses revealed distinct clades that, to some extent, and from the clustering of type strains represent known species. However, many of the genomes have been incorrectly identified adding to the current confusion. Furthermore, our data show that 16S rRNA gene sequences and ANI are unsuitable for identifying and circumscribing new species of the Mitis group of the genus Streptococci. Based on the clustering patterns resulting from core genome phylogenetic analysis, we conclude that S. oligofermentans is a later synonym of S. cristatus. The recently described strains of the species Streptococcus dentisani includes one previously referred to as 'S. mitis biovar 2'. Together with S. oralis, S. dentisani and S. tigurinus form subclusters within a coherent phylogenetic clade. We propose that the species S. oralis consists of three subspecies: S. oralis subsp. oralis subsp. nov., S. oralis subsp. tigurinus comb. nov., and S. oralis subsp. dentisani comb. nov.

Native valve endocarditis caused by Streptococcus oligofermentans: a case report

Introduction: Viridans group streptococci (VGS), which are commensal flora in the oral cavity, can act as opportunistic pathogens causing serious infections such as infective endocarditis (IE). Species identification of these organisms is difficult because of variability in phenotypic traits among strains of the same species. Case presentation: We report a case of endocarditis in a 64-year-old male patient caused by Streptococcus oligofermentans, which was identified by 16S rRNA gene sequencing. S. oligofermentans is a commensal of the oral cavity and rarely causes disease. This is the second report of IE caused by S. oligofermentans. Conclusion: Molecular methods are necessary to ascertain the identity of VGS strains, particularly those that rarely cause human disease.

Streptococcus oligofermentans Inhibits Streptococcus mutans in Biofilms at Both Neutral pH and Cariogenic Conditions.

Homeostasis of oral microbiota can be maintained through microbial interactions. Previous studies showed that Streptococcus oligofermentans, a non-mutans streptococci frequently isolated from caries-free subjects, inhibited the cariogenic Streptococcus mutans by the production of hydrogen peroxide (HP). Since pH is a critical factor in caries formation, we aimed to study the influence of pH on the competition between S. oligofermentans and S. mutans in biofilms. To this end, S. mutans and S. oligofermentans were inoculated alone or mixed at 1:1 ratio in buffered biofilm medium in a 96-well active attachment model. The single- and dual-species biofilms were grown under either constantly neutral pH or pH-cycling conditions. The latter includes two cycles of 8 h neutral pH and 16 h pH 5.5, used to mimic cariogenic condition. The 48 h biofilms were analysed for the viable cell counts, lactate and HP production. The last two measurements were carried out after incubating the 48 h biofilms in buffers supplemented with 1% glucose (pH 7.0) for 4 h. The results showed that S. oligofermentans inhibited the growth of S. mutans in dual-species biofilms under both tested pH conditions. The lactic acid production of dual-species biofilms was significantly lower than that of single-species S. mutans biofilms. Moreover, dual-species and single-species S. oligofermentans biofilms grown under pH-cycling conditions (with a 16 h low pH period) produced a significantly higher amount of HP than those grown under constantly neutral pH. In conclusion, S. oligofermentans inhibited S. mutans in biofilms not only under neutral pH, but also under pH-cycling conditions, likely through HP production. S. oligofermentans may be a compelling probiotic candidate against caries.

The Genus Streptococcus – Part II: New Species and Pathogens in the “Miscellaneous” Streptococci and “Viridans” Streptococci Groups

In the second part of this two-part series on an update on the streptococci, new species and emergent human pathogens in the “viridans streptococci” and the “miscellaneous streptococci” groups are discussed. Among the “miscellaneous streptococci,” the most important organism in human infectious diseases is Streptococcus suis. This organism is primarily an agent of disease in swine and other animals and has now emerged as a significant human pathogen, causing bacteremia and meningitis, particularly among populations in Asia and the Far East. While S. suis capsular serotype 2 is the most frequently isolated agent, infections caused by other serotypes are increasingly being reported. In addition, disease caused by S. suis is now being described more frequently in North America in individuals with significant occupational exposure to swine. Among the viridans streptococci, several new species in the mitis-sanguinis group have been described as human pathogens, including Streptococcus pseudopneumoniae, Streptococcus oligofermentans, and Streptococcus tigurinus. Other newly reported viridans group species either comprise part of the human oral microbiome or have been isolated from animals. This article presents information on these bacterial agents, including characteristics for the recognition and identification of the more important clinical isolates in these streptococcal groups.

Phylogenomic and MALDI-TOF MS analysis of Streptococcus sinensis HKU4T reveals a distinct phylogenetic clade in the genus Streptococcus.

Streptococcus sinensis is a recently discovered human pathogen isolated from blood cultures of patients with infective endocarditis. Its phylogenetic position, as well as those of its closely related species, remains inconclusive when single genes were used for phylogenetic analysis. For example, S. sinensis branched out from members of the anginosus, mitis, and sanguinis groups in the 16S ribosomal RNA gene phylogenetic tree, but it was clustered with members of the anginosus and sanguinis groups when groEL gene sequences used for analysis. In this study, we sequenced the draft genome of S. sinensis and used a polyphasic approach, including concatenated genes, whole genomes, and matrix-assisted laser desorption ionization-time of flight mass spectrometry to analyze the phylogeny of S. sinensis. The size of the S. sinensis draft genome is 2.06 Mb, with GC content of 42.2%. Phylogenetic analysis using 50 concatenated genes or whole genomes revealed that S. sinensis formed a distinct cluster with Streptococcus oligofermentans and Streptococcus cristatus, and these three streptococci were clustered with the “sanguinis group.” As for phylogenetic analysis using hierarchical cluster analysis of the mass spectra of streptococci, S. sinensis also formed a distinct cluster with S. oligofermentans and S. cristatus, but these three streptococci were clustered with the “mitis group.” On the basis of the findings, we propose a novel group, named “sinensis group,” to include S. sinensis, S. oligofermentans, and S. cristatus, in the Streptococcus genus. Our study also illustrates the power of phylogenomic analyses for resolving ambiguities in bacterial taxonomy.

PerR-Regulated Manganese Ion Uptake Contributes to Oxidative Stress Defense in an Oral Streptococcus

Metal homeostasis plays a critical role in antioxidative stress. Streptococcus oligofermentans, an oral commensal facultative anaerobe lacking catalase activity, produces and tolerates abundant H2O2, whereas Dpr (an Fe(2+)-chelating protein)-dependent H2O2 protection does not confer such high tolerance. Here, we report that inactivation of perR, a peroxide-responsive repressor that regulates zinc and iron homeostasis in Gram-positive bacteria, increased the survival of H2O2-pulsed S. oligofermentans 32-fold and elevated cellular manganese 4.5-fold. perR complementation recovered the wild-type phenotype. When grown in 0.1 to 0.25 mM MnCl2, S. oligofermentans increased survival after H2O2 stress 2.5- to 23-fold, and even greater survival was found for the perR mutant, indicating that PerR is involved in Mn(2+)-mediated H2O2 resistance in S. oligofermentans. Mutation of mntA could not be obtained in brain heart infusion (BHI) broth (containing ~0.4 μM Mn(2+)) unless it was supplemented with ≥2.5 μM MnCl2 and caused 82 to 95% reduction of the cellular Mn(2+) level, while mntABC overexpression increased cellular Mn(2+) 2.1- to 4.5-fold. Thus, MntABC was identified as a high-affinity Mn(2+) transporter in S. oligofermentans. mntA mutation reduced the survival of H2O2-pulsed S. oligofermentans 5.7-fold, while mntABC overexpression enhanced H2O2-challenged survival 12-fold, indicating that MntABC-mediated Mn(2+) uptake is pivotal to antioxidative stress in S. oligofermentans. perR mutation or H2O2 pulsing upregulated mntABC, while H2O2-induced upregulation diminished in the perR mutant. This suggests that perR represses mntABC expression but H2O2 can release the suppression. In conclusion, this work demonstrates that PerR regulates manganese homeostasis in S. oligofermentans, which is critical to H2O2 stress defenses and may be distributed across all oral streptococci lacking catalase.

Influence of pH on inhibition of Streptococcus mutans by Streptococcus oligofermentans

Streptococcus oligofermentans is a novel strain of oral streptococcus that can specifically inhibit the growth of Streptococcus mutans. The aims of this study were to assess the growth of S.oligofermentans and the ability of S.oligofermentans to inhibit growth of Streptococcus mutans at different pH values. Growth inhibition was investigated in vitro using an interspecies competition assay. The 4-aminoantipyine method was used to measure the initial production rate and the total yield of hydrogen peroxide in S.oligofermentans. S.oligofermentans grew best at pH 7.0 and showed the most pronounced inhibitory effect when it was inoculated earlier than S.mutans. In terms of the total yield and the initial production rate of hydrogen peroxide by S.oligofermentans, the effects of the different culture pH values were as follows: pH 7.0>6.5>6.0>7.5>5.5=8.0 (i.e. there was no significant difference between pH 5.5 and pH 8.0). Environmental pH and the sequence of inoculation significantly affected the ability of S.oligofermentans to inhibit the growth of S.mutans. The degree of inhibition may be attributed to the amount of hydrogen peroxide produced.

Complete Genome Sequence of an Oral Commensal, Streptococcus oligofermentans Strain AS 1.3089

Streptococcus oligofermentans, an oral commensal, inhibits the growth of the dental caries pathogen Streptococcus mutans by producing large amounts of hydrogen peroxide. Therefore, it can be a potential probiotic for oral health. Here we report the complete genome sequence of S. oligofermentans strain AS 1.3089.

Role of Operon aaoSo-mutT in Antioxidant Defense in Streptococcus oligofermentans

Previously, we have found that an insertional inactivation of aaoSo, a gene encoding L-amino acid oxidase (LAAO), causes marked repression of the growth of Streptococcus oligofermentans. Here, we found that aaoSo and mutT, a homolog of pyrophosphohydrolase gene of Escherichia coli, constituted an operon. Deletion of either gene did not impair the growth of S. oligofermentans, but double deletion of both aaoSo and mutT was lethal. Quantitative PCR showed that the transcript abundance of mutT was reduced for 13-fold in the aaoSo insertional mutant, indicating that gene polarity derived from the inactivation of aaoSo attenuated the expression of mutT. Enzymatic assays were conducted to determine the biochemical functions of LAAO and MutT of S. oligofermentans. The results indicated that LAAO functioned as an aminoacetone oxidase [47.75 nmol H2O2 (min·mg protein)–1]; and MutT showed the pyrophosphohydrolase activity, which removed mutagens such as 8-oxo-dGTP. Like paraquat, aaoSo mutations increased the expression of SOD, and addition of aminoacetone (final concentration, 5 mM) decreased the mutant’s growth by 11%, indicating that the aaoSo mutants are under ROS stress. HPLC did reveal elevated levels of cytoplasmic aminoacetone in both the deletion and insertional gene mutants of aaoSo. Electron spin resonance spectroscopy showed increased hydroxyl radicals in both types of aaoSo mutant. This demonstrated that inactivation of aaoSo caused the elevation of the prooxidant aminoacetone, resulting the cellular ROS stress. Our study indicates that the presence of both LAAO and MutT can prevent endogenous metabolites-generated ROS and mutagens. In this way, we were able to determine the role of the aaoSo-mutT operon in antioxidant defense in S. oligofermentans.