Search

Human microbiomes are predicted to assemble in a reproducible and ordered manner yet there is limited knowledge on the development of the complex bacterial communities that constitute the oral microbiome. The oral microbiome plays major roles in many oral diseases including early childhood caries (ECC), which afflicts up to 70% of children in some countries. Saliva contains oral bacteria that are indicative of the whole oral microbiome and may have the ability to reflect the dysbiosis in supragingival plaque communities that initiates the clinical manifestations of ECC. The aim of this study was to determine the assembly of the oral microbiome during the first four years of life and compare it with the clinical development of ECC. The oral microbiomes of 134 children enrolled in a birth cohort study were determined at six ages between two months and four years-of-age and their mother's oral microbiome was determined at a single time point. We identified and quantified 356 operational taxonomic units (OTUs) of bacteria in saliva by sequencing the V4 region of the bacterial 16S RNA genes. Bacterial alpha diversity increased from a mean of 31 OTUs in the saliva of infants at 1.9 months-of-age to 84 OTUs at 39 months-of-age. The oral microbiome showed a distinct shift in composition as the children matured. The microbiome data were compared with the clinical development of ECC in the cohort at 39, 48, and 60 months-of-age as determined by ICDAS-II assessment. Streptococcus mutans was the most discriminatory oral bacterial species between health and current disease, with an increased abundance in disease. Overall our study demonstrates an ordered temporal development of the oral microbiome, describes a limited core oral microbiome and indicates that saliva testing of infants may help predict ECC risk.

Glass ionomer cements (GIC) are dental restorative materials that are suitable for modification to help prevent dental plaque (biofilm) formation. The aim of this study was to determine the effects of incorporating casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) into a GIC on the colonisation and establishment of Streptococcus mutans biofilms and the effects of aqueous CPP-ACP on established S mutans biofilms. S. mutans biofilms were either established in flow cells before a single ten min exposure to 1% w/v CPP-ACP treatment or cultured in static wells or flow cells with either GIC or GIC containing 3% w/w CPP-ACP as the substratum. The biofilms were then visualised using confocal laser scanning microscopy after BacLight LIVE/DEAD staining. A significant decrease in biovolume and average thickness of S. mutans biofilms was observed in both static and flow cell assays when 3% CPP-ACP was incorporated into the GIC substratum. A single ten min treatment with aqueous 1% CPP-ACP resulted in a 58% decrease in biofilm biomass and thickness of established S. mutans biofilms grown in a flow cell. The treatment also significantly altered the structure of these biofilms compared with controls. The incorporation of 3% CPP-ACP into GIC significantly reduced S. mutans biofilm development indicating another potential anticariogenic mechanism of this material. Additionally aqueous CPP-ACP disrupted established S. mutans biofilms. The use of CPP-ACP containing GIC combined with regular CPP-ACP treatment may lower S. mutans challenge., Competing Interests: The authors have declared that no competing interests exist.

Unlabelled: Caseinomacropeptide (CMP), the variably phosphorylated and glycosylated forms of the bovine milk protein fragment, κ-casein(106-169), is produced during cheese production and has been shown to have a range of antibacterial bioactivities., Objectives: To characterise the biofilm disruptive component of CMP and compare its activity with the known antimicrobial agents chlorhexidine and zinc ions., Methods: Streptococcus mutans biofilms were grown in flow cells with an artificial saliva medium containing sucrose and treated with CMP and the glycosylated forms of κ-casein(106-169) (κ-casein glycopeptide, KCG). The biofilms were imaged using confocal laser scanning microscopy (CLSM) and quantified by COMSTAT software analysis. A static biofilm assay and flow cytometric analysis were used to examine the mechanism of action of chlorhexidine and a combination of KCG with the known antimicrobial agent ZnCl2 (KCG-Zn)., Results: CLSM analysis showed that S. mutans produced robust, structured biofilms with an average thickness of 7.37μm and a biovolume of 3.88μm(3)/μm(2) substratum after 16h of incubation in the flow cell system. A single application of 10mg/mL CMP that contained 2.4mg/mL KCG significantly reduced total biofilm biovolume and average biofilm thickness by 53% and 61%, respectively. This was statistically the same as a 2.4mg/mL KCG treatment that reduced the total biovolume and average thickness by 59% and 69%, respectively, suggesting the KCG was the biofilm disruptive component of CMP. Chlorhexidine treatment (0.1%) caused similar effects in the flow cell model. KCG-Zn caused significantly more disruption of the biofilms than either KCG or ZnCl2 treatment alone. In a static biofilm model chlorhexidine was shown to work by disrupting bacterial membrane integrity whilst KCG-Zn had no effect on membrane integrity., Conclusions: KCG and KCG-Zn may have potential as natural biofilm disruptive agents., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Kappacin, nonglycosylated kappa-casein(106-169), is a novel antimicrobial peptide produced from kappa-casein found in bovine milk. There are two major genetic forms of kappacin, A and B, and using synthetic peptides corresponding to the active region, kappa-casein(138-158), of these forms, we have shown that the Asp148 to Ala148 substitution is responsible for the lesser antibacterial activity of kappa-casein-B(106-169). Kappacin was shown to have membranolytic action at concentrations above 30 microM at acidic pH when tested against artificial liposomes. There was little membranolytic activity at neutral pH, which is consistent with the lack of antibacterial activity of kappacin against Streptococcus mutans at this pH. Kappacin specifically bound two zinc or calcium ions per mol, and this binding enhanced antibacterial activity at neutral pH. Nuclear magnetic resonance analysis indicated that a kappa-casein-A(138-158) synthetic peptide undergoes a conformational change in the presence of the membrane solvent trifluoroethanol and excess divalent metal ions. This change in conformation is presumably responsible for the increase in antibacterial activity of kappacin detected in the presence of excess zinc or calcium ions at neutral pH. When tested against the oral bacterial pathogen S. mutans cultured as a biofilm in a constant-depth film fermentor, a preparation of 10 g/liter kappacin and 20 mM ZnCl2 reduced bacterial viability by 3 log10 and suppressed recovery of viability. In contrast 20 mM ZnCl2 alone reduced bacterial viability by approximately 1 log10 followed by rapid recovery. In conclusion, kappacin has a membranolytic, antibacterial effect that is enhanced by the presence of divalent cations.

Caseinomacropeptide (CMP) is a heterogeneous C-terminal fragment (residues 106 to 169) of bovine milk kappa-casein composed of glycosylated and phosphorylated forms of different genetic variants. We have demonstrated that CMP has growth-inhibitory activity against the oral opportunistic pathogens Streptococcus mutans and Porphyromonas gingivalis and against Escherichia coli. CMP was fractionated using reversed-phase high-performance liquid chromatography (RP-HPLC), and each fraction was tested for activity against S. mutans in a 96-well-plate broth assay. Fractions were characterized by N-terminal sequence analysis and mass spectrometry. The active form of CMP was shown to be the nonglycosylated, phosphorylated kappa-casein (residues 106 to 169) [kappa-casein(106--169)], which we have designated kappacin. Endoproteinase Glu-C was used to hydrolyze CMP, and the generated peptides were separated using RP-HPLC and gel filtration-HPLC and then tested for activity against S. mutans. The peptide Ser(P)(149)kappa-casein-A(138--158) was the only peptide generated by endoproteinase Glu-C digestion that exhibited growth-inhibitory activity. Peptides corresponding to the sequences of the inhibitory peptide Ser(P)(149)kappa-casein-A(138--158) and its nonphosphorylated counterpart kappa-casein-A(138--158) were chemically synthesized and tested for antibacterial activity. The synthetic Ser(P)(149) kappa-casein-A(138--158) displayed growth-inhibitory activity against S. mutans (MIC, 59 microg/ml [26 microM]). The nonphosphorylated peptide, however, did not inhibit growth at the concentrations tested, indicating that phosphorylation is essential for activity.

Oral streptococci produce large quantities of organic acids as the end-products of carbohydrate fermentation. In an approach to determine if oral streptococci exhibit differential sensitivities to organic acid anions, we determined the effects of formate, lactate, and acetate on intracellular pH maintenance, glycolysis, and growth of Streptococcus mutans and Streptococcus sanguis. Growth was determined as maximum culture optical density in the presence of the organic acid anions at pH 7.1, 6.7, 6.3, and 6.1, and the effects of the anions on glycolytic activity and intracellular pH were determined at pH 7.0 and 5.0. At pH 7.1, the organic acid anions had little effect on growth of either species. At the lower pH values, all of the anions reduced the maximum culture optical density of both species in a pH- and concentration-dependent manner, with S. sanguis being more sensitive to growth inhibition than S. mutans. The organic acid anions had little or no effect on glycolytic activity of either species at pH 7.0. However, all of the organic acid anions tested reduced glycolytic activity at pH 5.0 in a concentration-dependent manner, with S. sanguis being more sensitive than S. mutans. The inhibition of glycolysis could be related to the pKa of the organic acid, with formate and lactate being more inhibitory than acetate. The organic acid anions decreased the intracellular pH of S. mutans and S. sanguis, glycolyzing at an extracellular pH of 5.0, such that the reduction in glycolytic activity caused by the organic acid anions could be directly attributed to the fall in intracellular pH. In conclusion, the production of lactic acid in plaque would not only lower pH, thereby having a disadvantageous effect on less aciduric oral streptococci, such as S. sanguis, but would also increase their sensitivity to the effects of low pH, helping S. mutans to become more dominant.

Glutamine transport in glucose-energized cells of Streptococcus mutans Ingbritt exhibited Michaelis-Menten-type kinetics with a Vmax of 13.4 nmol/mg dry weight/min and a Kt of 4.1 microM. Diffusion of glutamine into de-energized cells of S. mutans displayed similar type kinetics, with a Kt of 6.8 microM but with a markedly reduced Vmax of 53.9 pmol/mg dry weight/min. Glutamine transport in S. mutans is not proton motive force-driven, as the intracellular accumulation of glutamine by energized cells far exceeded the thermodynamic limits of the proton motive force, and the dissipation of this proton motive force by gramicidin in a high K+ medium did not decrease the intracellular glutamine concentration. Glutamine transport is therefore likely to be energized by ATP hydrolysis. The activity of the transporter was maximal between pH 6.0 and 7.0 and decreased rapidly above pH 7.0. The transport of glutamine was not competitively inhibited by asparagine, glutamate or aspartate, indicating a specific glutamine transport system. Reversed-phase high-pressure liquid chromatography of cell extracts revealed that approximately 26% of the glutamine taken into the cell was converted to glutamate within 10 min. The results are consistent with transported glutamine being converted to glutamate and ammonia by the action of an intracellular glutaminase. Glutamine therefore may be an important source of nitrogen for the cell.

The heat-stable phosphocarrier protein (HPr) of Streptococcus mutans was extracted from whole cells using sodium lauroylsarcosinate/EDTA and purified to homogeneity by a single-step, ion-exchange chromatographic procedure. The complete amino acid sequence of the protein was determined from peptides generated by trypsin, alpha-chymotrypsin, endoproteinase Glu-C, and cyanogen bromide treatment. The HPr from S. mutans contains 86 or 87 amino acyl residues, depending on removal of the N-terminal Met and the protein shows high sequence homology with HPr from other Gram-positive bacteria. The predicted tertiary structure of the S. mutans HPr, from model building by homology, is an open-faced beta-sandwich consisting of two alpha-helices and a four-stranded antiparallel beta-sheet.

Leucine transport in glucose-energized cells of Streptococcus mutans exhibited Michaelis-Menten-type kinetics at low extracellular concentrations, with a K1 of 15.3 microM and a Vmax of 6.1 nmol/mg dry weight/min. At high extracellular leucine concentrations, the transmembrane diffusion of leucine was not saturable, indicating that passive diffusion becomes a significant mechanism of leucine transmembrane movement under these conditions. The proton motive force (PMF) was measured in glucose-energized cells of S. mutans and was found to have a maximum value of 126 mV at an extracellular pH (pH0) of 5.0; this decreased to 45 mV at pH0 8.0. The intracellular accumulation of leucine was significantly correlated with the magnitude of the PMF. The addition of excess isoleucine or valine caused a marked decrease in the leucine transport rate. Maximal rates of leucine transport occurred at pH0 6.0, and the rate of leucine transport was independent of the growth medium. The results suggest that there is a PMF-driven, branched-chain amino acid carrier in S. mutans with a proton: substrate stoichiometry of 1.

The intracellular pH (pHi) optimum for glycolysis in Streptococcus mutans Ingbritt was determined to be 7.0 by use of the ionophore gramicidin for manipulation of pHi. Glycolytic activity decreased to zero as the pHi was lowered from 7.0 to 5.0. In contrast, glycolysis had an extracellular pH (pHo) optimum of 6.0 with a much broader profile. The relative insensitivity of glycolysis to the lowering of pHo was attributed to the ability of S. mutans to maintain a transmembrane pH gradient (delta pH, inside more alkaline) at low pHo. At a pHo of 5.0, glycolyzing cells of S. mutans maintained a delta pH of 1.37 +/- 0.09 units. The maintenance of this delta pH was dependent on the concentration of potassium ions in the extracellular medium. Potassium was rapidly taken up by glycolyzing cells of S. mutans at a rate of 70 nmol/mg dry weight/min. This uptake was dependent on the presence of both ATP and a proton motive-force (delta p). The addition of N-N'-dicyclohexylcarbodiimide (DCCD) to glycolyzing cells of S. mutans caused a partial collapse of the delta pH. Growth of S. mutants at pHo 5.5 in continuous culture resulted in the maintenance of a delta pH larger than that produced by cells grown at pH 7.0. These results suggest the presence of a proton-translocating F1Fo-ATPase in S. mutans whose activity is regulated by the intracellular pH and transmembrane electrical potential (delta psi). The production of an artificial delta p of 124 mV across the cell membrane of S. mutans did not result in proton movement through the F1Fo-ATPase coupled to ATP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

The transmembrane movement of radiolabeled, nonmetabolizable glucose analogs in Streptococcus mutants Ingbritt was studied under conditions of differing transmembrane electrochemical potentials (delta psi) and pH gradients (delta pH). The delta pH and delta psi were determined from the transmembrane equilibration of radiolabeled benzoate and tetraphenylphosphonium ions, respectively. Growth conditions of S. mutants Ingbritt were chosen so that the cells had a low apparent phosphoenolpyruvate (PEP)-dependent glucose:phosphotransferase activity. Cells energized under different conditions produced transmembrane proton potentials ranging from -49 to -103 mV but did not accumulate 6-deoxyglucose intracellularly. An artificial transmembrane proton potential was generated in deenergized cells by creating a delta psi with a valinomycin-induced K+ diffusion potential and a delta pH by rapid acidification of the medium. Artificial transmembrane proton potentials up to -83 mV, although producing proton influx, could not accumulate 6-deoxyglucose in deenergized cells or 2-deoxyglucose or thiomethylgalactoside in deenergized, PEP-depleted cells. The transmembrane diffusion of glucose in PEP-depleted, KF-treated cells did not exhibit saturation kinetics or competitive inhibition by 6-deoxyglucose or 2-deoxyglucose, indicating that diffusion was not facilitated by a membrane carrier. As proton-linked membrane carriers have been shown to facilitate diffusion in the absence of a transmembrane proton potential, the results therefore are not consistent with a proton-linked glucose carrier in S. mutans Ingbritt. This together with the lack of proton-linked transport of the glucose analogs suggests that glucose transmembrane movement in S. mutans Ingbritt is not linked to the transmembrane proton potential.