Persistence of Streptococcus pyogenes in stationary-phase cultures

In addition to causing fulminant disease, Streptococcus pyogenes may be asymptomatically carried between recurrent episodes of pharyngitis. To better understand streptococcal carriage, we characterized in vitro long-term stationary-phase survival (>4 weeks) of S. pyogenes. When grown in sugar-limited Todd-Hewitt broth, S. pyogenes cells remained culturable for more than 1 year. Both Todd-Hewitt supplemented with excess glucose and chemically defined medium allowed survival for less than 1 week. After 4 weeks of survival in sugar-limited Todd-Hewitt broth, at least 10(3) CFU per ml remained. When stained with fluorescent live-dead viability stain, there were a number of cells with intact membranes that were nonculturable. Under conditions that did not support persistence, these cells disappeared 2 weeks after loss of culturability. In persistent cultures, these may be cells that are dying during cell turnover. After more than 4 weeks in stationary phase, the culturable cells formed two alternative colony phenotypes: atypical large colonies and microcolonies. Protein expression in two independently isolated microcolony strains, from 14-week cultures, was examined by use of two-dimensional electrophoresis. The proteomes of these two strains exhibited extensive changes compared to the parental strain. While some of these changes were common to the two strains, many of the changes were unique to a single strain. Some of the common changes were in metabolic pathways, suggesting a possible alternate metabolism for the persisters. Overall, these data suggest that under certain in vitro conditions, S. pyogenes cells can persist for greater than 1 year as a dynamic population.     

Inducible and constitutive formation of fructanase in batch and continuous cultures of Streptococcus mutans

The production of extracellular beta-D-fructanase by several strains of Streptococcus mutans was studied in continuous culture. When glucose was the limiting nutrient, S. mutans K1-R and OMZ176 accumulated fructanase to maximum levels at low growth rates (dilution rate 0.05-0.10 h-1), due to the longer residence times of the bacteria in the culture vessel under these conditions. Extracellular fructanase activity was greater than has been previously reported for batch cultures. The rate of fructanase production for both S. mutans strains K1-R and OMZ176 increased with increasing growth rate when glucose was limiting. Under conditions of glucose sufficiency, the rate of fructanase production was always lower than in cultures where glucose was limiting, irrespective of the growth rate. Cultures of S. mutans Ingbritt (serotype c) grown with sorbitol- or glucose-limitation synthesized fructanase at a very low basal rate. When fructose was the limiting carbohydrate the enzyme was induced with a maximum rate of production occurring at a dilution rate of 0.40 h-1. Strains of S. mutans from other serotypes (a, d, d/g) were either not affected by changing the limiting sugar from glucose to fructose or else fructanase activity was slightly decreased in the fructose-limited medium. Fructanases from various strains of S. mutans readily hydrolysed (2----6)-beta-D-fructans, but all possessed the ability to hydrolyse (2----1)-beta-D-fructans to varying degrees.     

Natural genetic transformation of Streptococcus mutans growing in biofilms

Streptococcus mutans is a bacterium that has evolved to be dependent upon a biofilm "lifestyle" for survival and persistence in its natural ecosystem, dental plaque. We initiated this study to identify the genes involved in the development of genetic competence in S. mutans and to assay the natural genetic transformability of biofilm-grown cells. Using genomic analyses, we identified a quorum-sensing peptide pheromone signaling system similar to those previously found in other streptococci. The genetic locus of this system comprises three genes, comC, comD, and comE, that encode a precursor to the peptide competence factor, a histidine kinase, and a response regulator, respectively. We deduced the sequence of comC and its active pheromone product and chemically synthesized the corresponding 21-amino-acid competence-stimulating peptide (CSP). Addition of CSP to noncompetent cells facilitated increased transformation frequencies, with typically 1% of the total cell population transformed. To further confirm the roles of these genes in genetic competence, we inactivated them by insertion-duplication mutagenesis or allelic replacement followed by assays of transformation efficiency. We also demonstrated that biofilm-grown S. mutans cells were transformed at a rate 10- to 600-fold higher than planktonic S. mutans cells. Donor DNA included a suicide plasmid, S. mutans chromosomal DNA harboring a heterologous erythromycin resistance gene, and a replicative plasmid. The cells were optimally transformed during the formation of 8- to 16-h-old biofilms primarily consisting of microcolonies on solid surfaces. We also found that dead cells in the biofilms could act as donors of a chromosomally encoded antibiotic resistance determinant. This work demonstrated that a peptide pheromone system controls genetic competence in S. mutans and that the system functions optimally when the cells are living in actively growing biofilms.     

Role of Intracellular Polysaccharide in Persistence of Streptococcus mutans

Intracellular polysaccharide (IPS) is accumulated by Streptococcus mutans when the bacteria are grown in excess sugar and can contribute toward the cariogenicity of S. mutans. Here we show that inactivation of the glgA gene (SMU1536), encoding a putative glycogen synthase, prevented accumulation of IPS. IPS is important for the persistence of S. mutans grown in batch culture with excess glucose and then starved of glucose. The IPS was largely used up within 1 day of glucose starvation, and yet survival of the parental strain was extended by at least 15 days beyond that of a glgA mutant; potentially, some feature of IPS metabolism distinct from providing nutrients is important for persistence. IPS was not needed for persistence when sucrose was the carbon source or when mucin was present.Streptococcus mutans is a facultative colonizer of the human dental plaque, the microbial pellicle that covers the surface of the teeth. It is the major etiological agent of dental caries (17). Sugar metabolism is central to the behavior of S. mutans (4, 7). It can use a variety of sugars. The sugars are fermented by glycolysis with production of organic acids, particularly lactic acid (4, 7). In addition to providing energy, sucrose is used to produce extracellular polysaccharides to form the biofilm matrix that aids in the association of S. mutans with the dental plaque. Once the S. mutans biofilm becomes part of the dental plaque, the acidic by-products of sugar fermentation dissolve tooth enamel, eventually resulting in dental caries (17). The presence of sugars in the dental plaque is periodic and reflects the intake of dietary sugars. If there is excess sugar available, in addition to producing organic acids and matrix, intracellular (iodophilic) polysaccharide (IPS; glycogen) is formed.The IPS of S. mutans is a polymer of the glycogen-amylopectin type, with α-(1, 4)- and α-(1, 6)-linked glucose, and is stored as intracellular granules (10). Intracellular glycogen storage reserves in various bacterial species are synthesized from glucose-1-P via ADP-glucose (1). The synthesis involves at least three enzymes: glycogen synthase, glucose-1-phosphate pyrophosphorylase, and branching enzyme. The genes encoding these enzymes are commonly found in a glg operon, although the order of genes differs between species. In two gram-positive species, Bacillus subtilis and Bacillus stearothermophilus, the gene order is glgB-glgC-glgD-glgA-glgP (15, 29): glgA encodes glycogen synthase, glgB encodes glucan branching enzyme, and glgC and glgD encode subunits of glucose-1-phosphate pyrophosphorylase. The glgP gene encodes glycogen phosphorylase, which is unlikely to be involved in glycogen synthesis (29). Genes putatively encoding similar enzymes are present in the same order in the genome of S. mutans (29); they are thought also to form an operon.The IPS can be used as a source of carbohydrate for fermentation upon nutrient depletion (11, 13). In planktonic cultures, IPS reserves are largely consumed within 12 h of the imposition of sugar starvation (11, 13, 32). In S. mutans, IPS utilization may prolong acid production and hence the period of lowered pH of the resting (between meals) plaque, a factor that contributes to the incidence of caries. Indeed, IPS is implicated in dental caries: a mutant that synthesized elevated levels of IPS was hypercariogenic in germfree rats (27). Strains isolated from human carious lesions were nearly all stable IPS producers, whereas most strains from caries-inactive persons were variable IPS producers (13, 33).Since S. mutans deep in the dental plaque may not have access to nutrients because of competition with the bacteria at the surface of the plaque, the bacteria may need to survive longer periods of nutrient starvation. Previous studies in our laboratory showed that S. mutans can survive under sugar starvation conditions, provided that the pH remains above ∼5.5 (22). The presence of spent medium and mucin significantly prolonged survival of sugar-starved biofilms and batch cultures (22; also unpublished observations). Here we examine the role of IPS.The role of IPS (glycogen) in bacterial survival has been tested for several other bacterial species. It was found to extend survival of Aerobacter aerogenes (8) and Escherichia coli (28). Intracellular glycogen was also shown to support the survival of Streptococcus mitis during stationary-phase starvation (32). In contrast, glycogen-rich Sarcina lutea died at a higher rate during starvation than did bacteria without glycogen (2).In order to test the role of IPS in S. mutans survival, we constructed an IPS-deficient mutant by inactivating glgA (GenBank SMU.1536) (http://www.oralgen.lanl.gov/), putatively encoding the glycogen synthase. We also constructed a mutant potentially altered in IPS metabolism by inactivating the putative pullulanase structural gene, pul (SMU.1541). Pullulanases are responsible for hydrolyzing α-(1,6) linkages (and in some cases 1,4 linkages) in pullulan and in other polysaccharides (35) and may be important in determining the branching in IPS and/or affecting the catabolism of IPS. We studied the persistence of bacteria under conditions of sugar limitation and of sugar excess in both batch cultures and biofilms. We found that IPS can play a role in supporting S. mutans persistence in batch cultures but found no role for IPS in survival in biofilms.     

Streptococcal antagonism in oral biofilms: Streptococcus sanguinis and Streptococcus gordonii interference with Streptococcus mutans

Biofilms are polymicrobial, with diverse bacterial species competing for limited space and nutrients. Under healthy conditions, the different species in biofilms maintain an ecological balance. This balance can be disturbed by environmental factors and interspecies interactions. These perturbations can enable dominant growth of certain species, leading to disease. To model clinically relevant interspecies antagonism, we studied three well-characterized and closely related oral species, Streptococcus gordonii, Streptococcus sanguinis, and cariogenic Streptococcus mutans. S. sanguinis and S. gordonii used oxygen availability and the differential production of hydrogen peroxide (H(2)O(2)) to compete effectively against S. mutans. Interspecies antagonism was influenced by glucose with reduced production of H(2)O(2). Furthermore, aerobic conditions stimulated the competence system and the expression of the bacteriocin mutacin IV of S. mutans, as well as the H(2)O(2)-dependent release of heterologous DNA from mixed cultures of S. sanguinis and S. gordonii. These data provide new insights into ecological factors that determine the outcome of competition between pioneer colonizing oral streptococci and the survival mechanisms of S. mutans in the oral biofilm.     

Persistence of Streptococcus mutans in Stationary-Phase Batch Cultures and Biofilms

Streptococcus mutans is a member of oral plaque biofilms and is considered the major etiological agent of dental caries. We have characterized the survival of S. mutans strain UA159 in both batch cultures and biofilms. Bacteria grown in batch cultures in a chemically defined medium, FMC, containing an excess of glucose or sucrose caused the pH to decrease to 4.0 at the entry into stationary phase, and they survived for about 3 days. Survival was extended up to 11 days when the medium contained a limiting concentration of glucose or sucrose that was depleted by the time the bacteria reached stationary phase. Sugar-limited cultures maintained a pH of 7.0 throughout stationary phase. Their survival was shortened to 3 days by the addition of exogenous lactic acid at the entry into stationary phase. Sugar starvation did not lead to comparable survival in biofilms. Although the pH remained at 7.0, bacteria could no longer be cultured from biofilms 4 days after the imposition of glucose or sucrose starvation; BacLight staining results did not agree with survival results based on culturability. In both batch cultures and biofilms, survival could be extended by the addition of 0.5% mucin to the medium. Batch survival increased to an average of 26 (±8) days, and an average of 2.7 × 10⁵ CFU per chamber were still present in biofilms that were starved of sucrose for 12 days.     

In vitro activity of xanthorrhizol against Streptococcus mutans biofilms

AIMS: We determined the effect of xanthorrhizol (XTZ) purified from the rhizome of Curcuma xanthorrhiza Roxb. on the Streptococcus mutans biofilms in vitro. METHODS AND RESULTS: The biofilms of S. mutans at different phases of growth were exposed to XTZ at different concentrations (5, 10 and 50 micromol l(-1)) and for different time exposures (1, 10, 30 and 60 min). The results demonstrated that the activity of XTZ in removing S. mutans biofilm was dependent on the concentration, exposure time and the phase growth of biofilm. A concentration of 5 micromol l(-1) of XTZ completely inhibited biofilm formation by S. mutans at adherent phases of growth, whereas 50 micromol l(-1) of XTZ removed 76% of biofilm at plateau accumulated phase when exposed to S. mutans biofilm for 60 min. CONCLUSIONS: Xanthorrhizol isolated from an edible plant (C. xanthorrhiza Roxb.) shows promise as an antibacterial agent for inhibiting and removing S. mutans biofilms in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: XTZ could be used as a potential antibacterial agent against biofilm formation by S. mutans.     

Solute size effects on the diffusion in biofilms of Streptococcus mutans

The diffusion of poly(ethylene glycol) (PEG) (MW varying between 200 and 10,000), and of three different types of micelles was examined in Streptococcus mutans biofilms using infrared spectroscopy. PEGs were used because they show limited interactions with biological materials and their weight can be selected in order to cover a wide range of size. The study showed that a considerable fraction at the base of the biofilm was not accessible to the diffusing solute molecules and this inaccessible fraction was very dependent on the size of the diffusing molecules. In parallel, it was found that the diffusion coefficients of these solutes in the biofilms were less than those in water and this reduction was less pronounced for large macromolecules, an effect proposed to be related to their limited penetration. Triton X-100, a neutral detergent, forms micelles that behave like PEG, suggesting that the behaviour observed for neutral macromolecules can be extrapolated to neutral macroassemblies. However, the diffusion, as well as the penetration of sodium dodecylsulphate micelles (a negatively charged surfactant) and cetylpyridinium chloride micelles (positively charged), in the biofilms appeared to be significantly influenced by electrostatic interactions with biofilm components. The present findings provide useful insights associated with the molecular parameters required to efficiently penetrate bacterial biofilms. The study suggests a rationale for the limited bactericidal power of some antibiotics (the large ones). The restricted accessibility of macromolecules and macroassemblies to biofilms must be examined carefully in order to offer guidelines in the development of novel antibacterial treatments.     

Two-dimensional fluorescence difference gel electrophoretic analysis of Streptococcus mutans biofilms

Compared with traditional two-dimensional (2D) proteome analysis of Streptococcus mutans grown as a biofilm from a planktonic culture at steady state (Rathsam et al., Microbiol. 2005, 151, 1823-1837), the use of 2D fluorescence difference gel electrophoresis (DIGE) led to a 3-fold increase in the number of identified protein spots that were significantly altered in their level of expression (P < 0.050). Of the 73 identified proteins, only nine were up-regulated in biofilm grown cells. The results supported the previously surmised hypothesis that general metabolic functions were down-regulated in response to a reduction in growth rate in mature S. mutans biofilms. Up-regulation of competence proteins without any concomitant increase in stress-responsive proteins was confirmed, while the levels of glucosyltransferase C (GtfC), involved in glucan formation, O-acetylserine sulfhyrylase (cysteine synthetase A; CsyK), implicated in the formation of [Fe-S] clusters, and a hypothetical protein encoded by the open reading frame, SMu0188, were also up-regulated.     

Treatment of Streptococcus mutans biofilms with a nonthermal atmospheric plasma

AIMS: A nonthermal atmospheric plasma, designed for biomedical applications, was tested for its antimicrobial activity against biofilm cultures of a key cariogenic bacterium Streptococcus mutans. METHODS AND RESULTS: The Strep. mutans biofilms were grown with and without 0.15% sucrose. A chlorhexidine digluconate rinse (0.2%) was used as a positive antimicrobial reference. The presence of sucrose and the frequency of plasma application during growth were shown to have a significant effect on the response to treatment and antibacterial activity. CONCLUSIONS: A single plasma treatment for 1 min on biofilms cultured without sucrose caused no re-growth within the observation period. However, with either single or repeated plasma treatments of 1 min, on biofilms cultured with 0.15% sucrose, growth was only reduced. SIGNIFICANCE AND IMPACT OF THE STUDY: In summary, there may be a role for nonthermal plasma therapies in dental procedures. Sucrose and associated growth conditions may be a factor in the survival of oral biofilms after treatment.     

Antimicrobial activity of Curcuma xanthorrhiza nanoemulsions on Streptococcus mutans biofilms

Abstract

In this study, an optimal nanoemulsion formulation for Curcuma xanthorrhiza oil (Xan) was investigated using different sonication times. The antimicrobial effects of the nanoemulsion, the original emulsion, distilled water (DW), and Listerine, on Streptococcus mutans biofilms were compared. The optimum ultrasonic time, determined in terms of droplet size and stability, was found to be 10?min. Cell viability was the lowest on exposure to the nanoemulsion, and significantly different compared with exposure to DW or Listerine. The emulsion’s effect was similar to that of the nanoemulsion, but was non-uniform with a high interquartile range. Confocal microscope analysis revealed that the live/dead cell ratio in the nanoemulsion was 50% and 40% less than those in DW and Listerine, respectively. Biofilm treated with the nanoemulsion was thinner than biofilms exposed to the other treatments. Xan nanoemulsions exhibited stable and strong antimicrobial effects due to nano-sized particles, highlighting their potential use in oral health treatment.     

Activity of Nidus Vespae extract and chemical fractions against Streptococcus mutans biofilms

AIMS: To evaluate the effect of Nidus Vespae extract and chemical fractions on the viability and architecture of Streptococcus mutans biofilms. METHODS AND RESULTS: The raw material was first extracted using 95% ethanol/water. Subsequent fractions were prepared from this extract using cyclohexane/ethyl acetate, petroleum ether/ethyl acetate and chloroform/methanol. The biomass dry weight and total protein of samples treated with Nidus Vespae extract and chemical fractions were significantly less than those treated with the vehicle control (P < 0.05). Biofilms treated with Nidus Vespae also resulted in lower percentage of polysaccharide composition. The pH decrease in the biofilm matrix was retarded by Nidus Vespae compared with the vehicle control. Architecture of biofilms treated with Nidus Vespae was different than in the vehicle control and 0.05% chlorhexidine. CONCLUSIONS: Chloroform/methanol fraction was the most effective treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: The significant antibiofilm activity demonstrated by Nidus Vespae shows it to be a promising source of novel anticariogenic agents.     

Triclosan inhibition of membrane enzymes and glycolysis of Streptococcus mutans in suspensions and biofilms

Triclosan was found to be a potent inhibitor of the F(H+)-ATPase of the oral pathogen Streptococcus mutans and to increase proton permeabilities of intact cells. Moreover, it acted additively with weak-acid transmembrane proton carriers, such as fluoride or sorbate, to sensitize glycolysis to acid inhibition. Even at neutral pH, triclosan could inhibit glycolysis more directly as an irreversible inhibitor of the glycolytic enzymes pyruvate kinase, lactic dehydro genase, aldolase, and the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Cell glycolysis in suspensions or biofilms was inhibited in a pH-dependent manner by triclosan at a concentration of about 0.1 mmol/L at pH 7, approximately the lethal concentration for S. mutans cells in suspensions. Cells in intact biofilms were almost as sensitive to triclosan inhibition of glycolysis as were cells in suspensions but were more resistant to killing. Targets for irreversible inhibition of glycolysis included the PTS and cytoplasmic enzymes, specifically pyruvate kinase, lactic dehydrogenase, and to a lesser extent, aldolase. General conclusions are that triclosan is a multi-target inhibitor for mutans streptococci, which lack a triclosan-sensitive FabI enoyl-ACP reductase, and that inhibition of glycolysis in dental plaque biofilms, in which triclosan is retained after initial or repeated exposure, would reduce cariogenicity.     

Effect of carbohydrates on fructosyltransferase expression and distribution in Streptococcus mutans GS-5 biofilms

Streptococcus mutans produces a fructosyltransferase (FTF) enzyme, which synthesizes fructan polymers from sucrose. Fructans contribute to the virulence of the biofilm by acting as binding sites for S. mutans adhesion and as extracellular nutrition reservoir for the oral bacteria. Antibodies raised against a recombinant S. mutans FTF were used to test the effect of glucose, fructose, and sucrose on FTF expression in S. mutans GS-5 biofilms. Biofilms formed in the presence of fructose and glucose showed a higher ratio of FTF compared to biofilms formed in the presence of sucrose. Confocal laser scanning microscopy images of S. mutans biofilms indicated a carbohydrate-dependent FTF distribution. The layer adjacent to the surface and those at the liquid interface displayed high amounts cell-free FTF with limited amount of bacteria while the in-between layers demonstrated both cell-free FTF and cells expressing cell-surface FTF. Biofilm of S. mutans grown on hydroxyapatite surfaces expressed several FTF bands with molecular masses of 160, 125, 120, 100, and 50 kDa, as detected by using FTF specific antibodies. The results show that FTF expression and distribution in S. mutans GS-5 biofilms is carbohydrate regulated.     

Characterization of Pseudomonas aeruginosa fatty acid profiles in biofilms and batch planktonic cultures

The fatty acid composition of Pseudomonas aeruginosa PAO1 was compared between biofilm and batch planktonic cultures. Strain PAO1 biofilms were able to maintain a consistent fatty acid profile for up to 6 days, whereas strain PAO1 batch planktonic cultures showed a gradual loss of cis-monounsaturated fatty acids over 4 days. Biofilms exhibited a greater proportion of hydroxy fatty acids but a lower proportion of both cyclopropane fatty acids and saturated fatty acids (SAFAs). SAFAs with >=16 carbons, in particular, decreased in biofilms when compared with that in batch planktonic cultures. A reduced proportion of SAFAs and a decline in overall fatty acid chain length indicate more fluidic biophysical properties for cell membranes of P. aeruginosa in biofilms. Separating the biofilms into 2 partitions and comparing their fatty acid compositions revealed additional trends that were not observed in the whole biofilm: the shear-nonremovable layer consistently showed greater proportions of hydroxy fatty acid than the bulk liquid + shear-removable portion of the biofilm. The shear-nonremovable portion demonstrated a relatively immediate decline in the proportion of monounsaturated fatty acids between days 2 and 4; which was offset by an increase in the proportion of cyclopropane fatty acids, specifically 19:0cyc(11,12). Simultaneously, the shear-removable portion of the biofilm showed an increase in the proportion of trans-monounsaturated fatty acids and cyclopropane fatty acids.