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Speciation of presumptive viridans streptococci from early onset neonatal sepsis.

Twenty isolates resembling viridans streptococci, 16 from blood and four from gastric aspirates, from 17 cases of early onset neonatal sepsis were identified by the API20 Strep, Rapid ID 32 Strep and conventional tests plus hydrolysis of methylumbelliferyl glycoside substrates. Nineteen of the isolates were identified as species of viridans streptococci and one as a Leuconostoc sp. Ten of the isolates were Streptococcus oralis, three S. mitis biotype 1, two S. mitis biotype 2 and one each of S. sanguis, S. vestibularis, S. salivarius and S. intermedius. The Rapid ID 32 Strep and conventional plus methylumbelliferyl tests gave the same species identity for 17 of the isolates. S. intermedius was identified by the Rapid ID 32 Strep as S. constellatus and S. salivarius as S. equinus, with S. salivarius at lower probability. The API20 Strep failed to identify S. vestibularis and identified S. salivarius as S. defectivus. The absence of certain critical tests, including urea hydrolysis, does not allow the API20 Strep to identify all the currently recognised species of viridans steptococci. The species distribution was unexpected and the incidence of S. oralis and other viridans streptococci in vaginal swabs from prenatal patients is being investigated further.

Bacteremia↗

Attachment of Bacteroides melaninogenicus subsp. asaccharolyticus to oral surfaces and its possible role in colonization of the mouth and of periodontal pockets.

This investigation examined the ability of cells of Bacteroides melaninogenicus subsp. asaccharolyticus 381 to adhere to surfaces that might be important for its initial colonization of the mouth and its subsequent colonization in periodontal pockets. Of 48 asaccharolytic strains of B. melaninogenicus, 47 agglutinated human erythrocytes, whereas none of 20 fermentative strains, which included reference cultures of the subspecies intermedius and melaninogenicus, were active. Electron microscopy indicated that both asaccharolytic and fermentative strains possessed pili; hence, the presence of pili did not correlate with the hemagglutinating activities of B. melaninogenicus strains. Both asaccharolytic and fermentative B. melaninogenicus strains suspended in phosphate-buffered saline adhered in high numbers to buccal epithelial cells and to the surfaces of several gram-positive bacteria tested, including Actinomyces viscosus, A. naeslundii, A. israelii, Streptococcus sanguis, and S. mitis. B. melaninogenicus subsp. asaccharolyticus 381 also attached, but in comparatively low numbers, to untreated and to saliva-treated hydroxyapatite. Addition of clarified whole saliva to suspensions of strain 381 almost completely eliminated adherence to buccal epithelial cells and to hydroxyapatite surfaces, but saliva had no detectable effect on attachment to gram-positive plaque bacteria. Both fermentative and nonfermentative strains of B. melaninogenicus also attached in high numbers to crevicular epithelial cells derived from human periodontal pockets, but normal human serum strongly inhibited attachment. Serum also inhibited attachment of strain 381 to saliva- and serum-treated hydroxyapatite, but it had little effect upon attachment to gram-positive bacteria. These observations suggested that salivary and serum components would strongly inhibit the attachment of B. melaninogenicus cells to several oral surfaces, but not to the surfaces of certain gram-positive bacteria commonly present in human dental plaque. This was confirmed by an in vivo experiment in which streptomycin-labeled cells of B. melaninogenicus 381-R were introduced into the mouths of two volunteers. After 10 min, several hundred-fold higher numbers of the organism were recovered from preformed bacterial plaque present on teeth than from clean tooth surfaces or from the buccal mucosa and tongue dorsum. High numbers of B. melaninogenicus cells were also recovered from preformed plaque after 150 min, but virtually no cells of the organism were recovered from the other surfaces studied. These data suggest that the presence of dental plaque containing Actinomyces and other gram-positive bacteria may be essential for the attachment and colonization of B. melaninogenicus cells after their initial introduction into the mouth. Similarly, the presence of subgingival plaque containing gram-positive bacteria may be necessary for its secondary colonization in periodontal pockets.

Adult↗

Streptococcus sinensis sp. nov., a novel species isolated from a patient with infective endocarditis.

A bacterium was isolated from the blood culture of a patient with infective endocarditis. The cells were facultative anaerobic, nonsporulating, gram-positive cocci arranged in chains. The bacterium grows on sheep blood agar as alpha-hemolytic, gray colonies of 0.5 to 1 mm in diameter after 24 h of incubation at 37 degrees C in ambient air. Growth also occurs in 10 or 40% bile and on bile esculin agar but not in 6% NaCl. No enhancement of growth is observed in 5% CO(2). It is nongroupable with Lancefield groups A, B, C, D, F, or G antisera and is resistant to optochin and bacitracin. The organism is aflagellated and is nonmotile at both 25 and 37 degrees C. It is Voges-Proskauer test positive. It produces leucine arylamidase and beta-glucosidase but not catalase, urease, lysine decarboxylase, or ornithine decarboxylase. It hydrolyzes esculin and arginine. It utilizes glucose, lactose, salicin, sucrose, pullulan, trehalose, cellobiose, hemicellulase, mannose, maltose, and starch. 16S rRNA gene sequencing showed that there were 3.6, 3.7, 4.3, 4.7, and 5.9% differences between the 16S rRNA gene sequence of the bacterium and those of Streptococcus gordonii, Streptococcus intermedius, Streptococcus constellatus, Streptococcus sanguis, and Streptococcus anginosus, respectively. The G+C content of it (mean plus minus standard deviation) was 53.0% plus minus 2.9%. Based on phylogenetic affiliation, it belongs to the mitis or anginosus group of Streptococcus. For these reasons a new species, Streptococcus sinensis sp. nov., is proposed, for which HKU4 is the type strain. Further studies should be performed to ascertain the potential of this bacterium to become an emerging cause of infective endocarditis.

Adult↗

Effect of acidic pH on expression of surface-associated proteins of Streptococcus oralis.

Streptococcus oralis, a member of the mitis group of oral streptococci, is implicated in the pathogenesis of infective endocarditis and is the predominant aciduric non-mutans-group streptococcus in dental plaque. We undertook to identify the most abundant surface-associated proteins of S. oralis and to investigate changes in protein expression when the organism was grown under acidic culture conditions. Surface-associated proteins were extracted from cells grown in batch culture, separated by two-dimensional gel electrophoresis, excised, digested with trypsin, and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Putative functions were assigned by homology to a translated genomic database of Streptococcus pneumoniae. A total of 27 proteins were identified; these included a lipoprotein, a ribosome recycling factor, and the glycolytic enzymes phosphoglycerate kinase, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and enolase. The most abundant protein, phosphocarrier protein HPr, was present as three isoforms. Neither lactate dehydrogenase nor pyruvate oxidase, dominant intracellular proteins, were present among the proteins on the gels, demonstrating that proteins in the surface-associated pool did not arise as a result of cell lysis. Eleven of the proteins identified were differentially expressed when cells were grown at pH 5.2 versus pH 7.0, and these included superoxide dismutase, a homologue of dipeptidase V from Lactococcus lactis, and the protein translation elongation factors G, Tu, and Ts. This study has extended the range of streptococcal proteins known to be expressed at the cell surface. Further investigations are required to ascertain their functions at this extracellular location and determine how their expression is influenced by other environmental conditions.

Bacterial Proteins↗

Streptococcus oligofermentans sp. nov., a novel oral isolate from caries-free humans.

Five streptococcal strains were isolated from dental plaque and saliva of caries-free humans. The cells were gram-positive, non-spore-forming, non-motile cocci that were arranged in short chains. The strains were catalase-negative, facultatively anaerobic and produced lactic acid exclusively from glucose fermentation. Biochemical analysis that used both conventional methods and the commercial API 20 Strep system showed that the five strains fermented only a few kinds of sugar. The mean DNA G + C content of the five novel strains was 39.5 +/- 0.8 mol%. Phylogenetic analysis based on 16S rDNA sequence homology indicated that the new isolates represented a novel member of the mitis group of the genus Streptococcus, related most closely to the recently described species Streptococcus sinensis. DNA-DNA relatedness between novel strain LMG 21535T and type strains of phylogenetically related species of oral streptococci was 7.1-16.4%. Therefore a novel Streptococcus species, Streptococcus oligofermentans sp. nov., is proposed. The type strain is LMG 21535T=AS 1.3089T.

Base Composition↗

Identification of viridans streptococci associated with bacteraemia in neutropenic cancer patients.

Twenty-three viridans streptococcal isolates from pyrexial neutropenic patients with various malignant diseases were studied in a comprehensive identification scheme. Fourteen isolates were identified as Streptococcus oralis, five as S. mitis and two as S. salivarius but the remaining two could not be identified reliably. The virulence mechanisms associated with the ability of these species to survive and grow in vivo require further investigation but may involve the production of specific glycosidase and proteolytic enzyme activities.

Bacteremia↗

Competition for glucose between Candida albicans and oral bacteria grown in mixed culture in a chemostat.

The competition for glucose as a growth-limiting substrate between Candida albicans and a mixed community of oral bacteria was investigated. A chemostat was operated under glucose-limiting and glucose excess conditions at a dilution rate of 0.05/h. A mixed population of oral bacteria was established and after a steady state had been reached the chemostat was inoculated with C. albicans. Seven bacterial species Streptococcus sanguis, S. sobrinus, S. mitis, Lactobacillus casei, Veillonella dispar, Eubacterium saburreum and Fusobacterium nucleatum - were able to establish stable populations under glucose-limiting conditions. The yeast was unable to grow with the bacteria under glucose limitation. Only three bacterial species, S. sobrinus, L. casei and E. saburreum, became established under glucose-excess conditions. C. albicans was also able to become established in the glucose-excess chemostat and could grow and maintain a steady state in a mixed culture with these organisms. L. casei, S. mitis and S. sobrinus had faster glucose consumption rates than C. albicans. All the bacteria, except for E nucleatum, had maximum specific growth rates higher than C. albicans. The results suggest that glucose may act as a growth-limiting substrate for C. albicans in the establishment and growth of the yeast in a mixed community of oral bacteria.

Bacteria↗

Application and evaluation of the interlaboratory reproducibility of tRNA intergenic length polymorphism analysis (tDNA-PCR) for identification of Streptococcus species.

The discriminatory power, speed, and interlaboratory reproducibility of tRNA intergenic length polymorphism analysis (tDNA-PCR) combined with capillary electrophoresis was evaluated for the identification of streptococci. This method was carried out in three different laboratories under highly standardized conditions for 54 strains belonging to 18 different species. It was concluded that interlaboratory reproducibility of tDNA fingerprints produced by means of capillary electrophoresis was sufficiently high to permit the exchange between different laboratories and the construction of common libraries which can be consulted for comparison with fingerprints obtained independently in separate laboratories. In a second step, 17 other species were included in the study and examined in one of the participating laboratories. All Streptococcus species studied, except S. mitis, S. oralis, S. parasanguinis, S. pneumoniae, S. thermophilus, and S. vestibularis, showed distinguishable tDNA fingerprints. A database of well-characterized strains was constructed to enable computer-aided identification of unknown streptococcal isolates.

DNA Fingerprinting↗

Viridans streptococci in infective endocarditis: species distribution and susceptibility to antibiotics.

A method for the speciation of viridans streptococci (non-groupable) is described. The major identification criteria are based on the reactions to a series of biochemical tests, including acid production in lactose, inulin, raffinose, mannitol and sorbitol, hydrolysis of arginine, esculin and Na hippurate, and production of polysaccharides in 5% sucrose media. A total of 450 strains was isolated from blood cultures, 183 of which were from confirmed cases of subacute endocarditis. The latter were identified as follows (%): Streptococcus sanguis I (25.7), S. mitis (19.7), S. sanguis II (19.7), S. mutans (17.5), S. milleri (12), S. morbillorium (3.2) and S. salivarius (2.2). Susceptibility to antibiotics was studied for 129 of these strains: 68% were susceptible to all drugs tested, 20% were resistant only to tetracycline, 4% only to penicillin (MIC = 0.5-4 micrograms ml-1) and 8% were multiply resistant (tetracycline, macrolides and related drugs, chloramphenicol, penicillin, high-level resistance to kanamycin and/or streptomycin [MIC = 1000-80.00 micrograms ml-1].

Anti-Bacterial Agents↗

Evaluation of the capacity of oral streptococci to produce hydrogen peroxide.

The capacity of 11 strains of oral streptococcal species (Streptococcus sanguis, S. oralis, S. mitis and S. sobrinus) to produce hydrogen peroxide (H2O2) was studied in vitro. Detection of this property in solid media, particularly with trypticase soy agar-benzidine-peroxidase, was more sensitive than in liquid media. The addition of carbohydrates (arabinose, xylose, mannose, sorbose and lactose), sorbitol and saccharine to buffered trypticase soy broth increased H2O2 production in S. oralis NCTC 11427, although the concentrations obtained with some substrates (glucose, galactose, mannitol and xylitol) were lower than those obtained in controls. In S. sanguis NCTC 7863, H2O2 production was detected only with galactose, sorbitol, lactose and saccharin.

Carbohydrate Metabolism↗

Changing agglutination activities of salivary immunoglobulin A preparations against oral streptococci.

Immunoglobulin A (IgA) was prepared from samples of parotid saliva collected from two individuals over a 150-day period. Standardized solutions of the IgA were tested for agglutinating antibody activity against strains of streptococcus salivarius, S. sanguis, S. mitis, and S. mutans that had been isolated from a homologous individual on day 30 and preserved in the lyophilized state. The agglutinating activity of the standardized IgA preparations against these strains was found to change over time, whereas the agglutination patterns of IgA prepared from saliva samples collected 3 days apart were generally similar. Differences in agglutination titers were also observed between IgA samples prepared from saliva collected on day 0 and day 150. The data indicate that the oral cavity may harbor streptococci of different antigenic composition representing the same species at a given time and that parotid IgA agglutinating antibody against these serotypes changes over time.

Adult↗

Antibacterial effectiveness of dentin bonding systems.

This study examined the antibacterial activities of several commercially available glass ionomer cements, dentin bonding systems and luting agents by employing both agar plate diffusion (APD) and growth inhibition (GI) methods. Amalgam and resin composites were also tested as control materials. In both methods (APD and GI), cylindrical specimens were used. Four bacteria strains were tested: Streptococcus mutans, S. salivarius, S. mitis and S. sanguis. These studies were performed using standardized innoculums with selective media, and the assayed materials were directly applied on the assay cultures and plates. The results of agar plate assay were in accordance with the results of growth inhibition method. The glass ionomer cements showed marked antibacterial activity. On the contrary, amalgam, composites, luting agents and dentinal bonding systems did not affect bacterial growth. The sensitivity of the growth method showed that all the strains were inhibited in the same way by each inhibitory material. The data suggest that the use of glass ionomer cements as cavity liners/bases may reduce the consequences of microleakage due to its antibacterial properties.

Analysis of Variance↗

Antimicrobial properties of topical anesthetic liquids containing lidocaine or benzocaine.

Six species of microorganisms commonly found within the oral cavity were exposed for either one minute or two hours to 5% lidocaine liquid topical anesthetic and benzocaine liquid topical anesthetic. Mixtures of microorganisms and anesthetics were diluted and plated onto a brain heart infusion medium. Reduction in cell viability was 73-100% after exposure to the anesthetic agents when compared with the saline/buffer controls. A significant reduction (p < .005) in cell growth by Streptococcus mutans, S. sanguis, S. mitis, S. salivarius, Actinomyces viscosus, and Candida albicans was associated with a one-minute and two-hour exposure to lidocaine, benzocaine, 5% lidocaine, and the benzocaine vehicle control. Five percent lidocaine reduced growth of the test orgainisms more than benzocaine in one-minute exposures to S. mutans, A. viscosus and S. salivarius and with a two-hour exposure to S. salivarius.Five percent lidocaine was bacteriocidal or fungicidal to all microorganisms for both time periods whereas, benzocaine liquid topical anesthetic was predominately bacteriostatic or fungistatic after one-minute exposures and bacteriocidal or fungicidal after two hours.The results indicated that two dental liquid topical anesthetics containing lidocaine or benzocaine possessed considerable antimicrobial activity to selected oral microorganisms. The exclusive use of a topical liquid anesthetic may be an adequate means to render the oral mucosa aseptic before injection of a local anesthetic.

Actinomyces↗

Influence of a fluoride mouthrinse on mutans streptococci in schoolchildren.

This study aimed to determine whether the long-term use of a fluoride mouthrinse affects the salivary levels of mutans streptococci. Two hundred and fifteen schoolchildren (aged 9-10 years) participated. One hundred and forty-nine of these children had used a fluoride mouthrinse since 5 years of age at nursery school, and the remaining 66 children had not. DFT (decayed and filled teeth) was recorded, and the salivary levels of Streptococcus mutans and Streptococcus sobrinus were measured using mitis salivarius bacitracin agar. The group that had used a fluoride mouthrinse had a significantly lower prevalence of both S. mutans and S. sobrinus (p = 0.038) and a significantly lower DFT score (p < 0.001) than the other group. Using logistic regression analysis including caries experience at baseline as a dependent variable, the odds ratio of carrying S. mutans alone was 8.0 (p = 0.066) and that of carrying both S. mutans and S. sobrinus was 16.5 (p = 0.022) in the group that had not used the fluoride mouthrinse. Children carrying both S. mutans and S. sobrinus had a higher caries incidence in 1 year than the others, with odds ratios of 5.73 (p = 0.067) in the group with a fluoride mouthrinse and 3.47 (p = 0.035) in the group without it. These results show that the long-term use of a fluoride mouthrinse is associated with reduced salivary levels of mutans streptococci and this bacterial reduction may partly contribute to the suppression of dental caries in children using a long-term fluoride mouthrinse.

Cariostatic Agents↗

Microbial complexes in subgingival plaque.

It has been recognized for some time that bacterial species exist in complexes in subgingival plaque. The purpose of the present investigation was to attempt to define such communities using data from large numbers of plaque samples and different clustering and ordination techniques. Subgingival plaque samples were taken from the mesial aspect of each tooth in 185 subjects (mean age 51 +/- 16 years) with (n = 160) or without (n = 25) periodontitis. The presence and levels of 40 subgingival taxa were determined in 13,261 plaque samples using whole genomic DNA probes and checkerboard DNA-DNA hybridization. Clinical assessments were made at 6 sites per tooth at each visit. Similarities between pairs of species were computed using phi coefficients and species clustered using an averaged unweighted linkage sort. Community ordination was performed using principal components analysis and correspondence analysis. 5 major complexes were consistently observed using any of the analytical methods. One complex consisted of the tightly related group: Bacteroides forsythus, Porphyromonas gingivalis and Treponema denticola. The 2nd complex consisted of a tightly related core group including members of the Fusobacterium nucleatum/periodonticum subspecies, Prevotella intermedia, Prevotella nigrescens and Peptostreptococcus micros. Species associated with this group included: Eubacterium nodatum, Campylobacter rectus, Campylobacter showae, Streptococcus constellatus and Campylobacter gracilis. The 3rd complex consisted of Streptococcus sanguis, S. oralis, S. mitis, S. gordonii and S. intermedius. The 4th complex was comprised of 3 Capnocytophaga species, Campylobacter concisus, Eikenella corrodens and Actinobacillus actinomycetemcomitans serotype a. The 5th complex consisted of Veillonella parvula and Actinomyces odontolyticus. A. actinomycetemcomitans serotype b, Selenomonas noxia and Actinomyces naeslundii genospecies 2 (A. viscosus) were outliers with little relation to each other and the 5 major complexes. The 1st complex related strikingly to clinical measures of periodontal disease particularly pocket depth and bleeding on probing.

Adult↗

Plaque pH and microflora of dental plaque on sound and carious root surfaces.

Given the hypothesis that root caries is the result of acid formation by acidogenic micro-organisms, the present study was performed to relate sucrose-induced pH response of dental plaque on root surfaces to the microbial composition of the overlying plaque. Seventeen caries-active elderly Chinese with poor oral hygiene and with both sound and carious root surfaces were examined. Plaque pH was measured before and up to one hour after a controlled sucrose mouthrinse. Plaque samples for microbiologic analyses were collected from 2 sound and 2 or 3 carious pH-measurement sites in each subject. The prevalence of the following micro-organisms was assessed as % of total viable counts on Brucella agar: Prevotella intermedia, Prevotella melaninogenica, Fusobacterium nucleatum, Campylobacter rectus, Capnocytophaga spp., Actinomyces viscosus, Actinomyces naeslundi, Streptococcus spp., S. sanguis, S. mitis, S. mutans, S. sobrinus, Lactobacillus spp., and Candida spp. There was no difference in plaque pH response on sound and carious root surfaces. The plaque pH response was more pronounced in the maxilla than in the mandible for both sound and carious sites. There was no difference in microbial composition of dental plaque on sound and carious root surfaces. The pH response to sucrose was the same regardless of the presence or absence of mutans streptococci. Our results thus do not readily support the traditional concept of caries formation.

Aged↗

Composition of the oral streptococcal flora in healthy children.

OBJECTIVES: To identify the predominant streptococcal species in the mouths of healthy children and to investigate the composition of the oral streptococcal flora over a period of 4 months. PATIENTS AND METHODS: The subjects were 33 fit, healthy schoolchildren aged between 5 and 16 years. These children were part of a large study and were the matched controls for a group of subjects undergoing bone marrow transplantation. The oral flora was sampled using an oral rinse technique on two separate occasions 4 months apart. The outcome measures were the number of each streptococcal species per millilitre of oral rinse; the isolation frequency of each species; the proportion of each species as a percentage of both the total streptococcal count and the total anaerobic count. RESULTS: The predominant species were Streptococcus salivarius, S. oralis and S. mitis. There was no significant variation in the composition of the oral streptococcal flora over the 4 month period. CONCLUSIONS: The oral rinse technique provides a reliable method of sampling the streptococcal flora of children.

Adolescent↗

The effect of adsorption on the acid production of caries and noncaries-producing streptococci.

Eighteen different Streptococcus strains including S mutans, sanguis, mitis, salivarius, bovis, and 2F2, were assayed for their relative adsorption to synthetic hydroxyapatite and for quantities of lactic acid produced while in the adsorbed state. The results indicated that all S mutans and some S sanguis have an enhanced metabolic activity while adsorbed to hydroxapatite as compared to control suspensions without hydroxyapatite.

Adsorption↗