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Comparative recovery of Streptococcus mutans on five isolation media, including a new simple selective medium.

For the isolation of Streptococcus mutans, several selective media have been developed, of which Mitis-Salivarius Sucrose Bacitracin agar (MSB) is the most widely used (Gold et al., 1973). Recently, the Trypticase Yeast-Extract Cystine agar medium (TYC, de Stoppelaar et al., 1967) was modified into a selective medium for S. mutans, called Trypticase Yeast-Extract Cystine Sucrose Bacitracin (TYCSB, van Palenstein Helderman et al., 1983). The aim of this study was to compare the recovery of S. mutans from clinical samples on Mitis-Salivarius agar (MS), MSB, TYC, and TYCSB. Further, a new simple selective medium for S. mutans was introduced. This medium, called TSY20B, was supposed to have the same qualities as TYCSB, but its preparation is less laborious. One hundred eighty-five plaque and saliva samples from 37 subjects were plated on MS, MSB, TYC, and TYCSB, and 285 samples from 23 subjects were plated on TYCSB and TSY20B. All plates were incubated at 37 degrees C in a 91% N2, 5% CO2, 4% H2 atmosphere for five days. The S. mutans counts on TYC and TYCSB were significantly higher than on MS or MSB by almost a factor of 10. Seventy-seven percent of the samples gave higher S. mutans counts on TYCSB than on MSB. Especially, samples with high S. mutans d/g numbers gave lower S. mutans counts on MSB. These data clearly indicate that MSB agar is inhibitory for S. mutans and should not be used. An additional advantage of TYCSB over MSB agar is the possibility of distinguishing S. mutans serotypes d/g from other serotypes.(ABSTRACT TRUNCATED AT 250 WORDS)

Agar↗

Restriction fragment length polymorphism analysis of 16S ribosomal DNA of Streptococcus and Enterococcus species of bovine origin.

Twelve bacterial species including Streptococcus uberis, S. parauberis, S. agalactiae, S. dysgalactiae, S. bovis, S. mitis, S. salivarius, S. saccharolyticus, Enterococcus faecium, E. faecalis, E. avium, and Aerococcus viridans were examined for their 16S ribosomal DNA fingerprint patterns. Oligonucleotide primers complementary to 16S rRNA genes were used to amplify by the polymerase chain reaction 16S ribosomal gene fragments from genomic DNAs. The molecular sizes of the amplified 16S ribosomal DNA (rDNA) fragments from the 12 species examined ranged from 1,400 to 1,500 bp. Restriction fragment length polymorphism analysis of 16S rDNA was performed with 11 different restriction endonucleases. All 12 species examined could be differentiated on the basis of characteristic 16S rDNA fingerprint patterns by using the restriction endonucleases HhaI, RsaI, and MspI. A scheme for the differentiation of the 12 species is presented. Eleven isolates representing 11 species were obtained from cows with intramammary infections and were examined by 16S rDNA fingerprinting. All 11 species isolated from cows were differentiated by using HhaI, RsaI, and MspI restriction endonucleases. The results of this study demonstrate the potential application of 16S rDNA fingerprinting for the identification and differentiation of bacterial species.

Animals↗

Bacteria in human mouths involved in the production and utilization of hydrogen peroxide.

Earlier studies have demonstrated that pure cultures of oral streptococci produce hydrogen peroxide but none has found any free peroxide in dental plaque or salivary sediment despite streptococci being major components of their mixed bacterial populations. The absence of peroxide in plaque and sediment could be due to the dominance of its destruction over its formation by bacterial constituents. To identify which of the oral bacteria might be involved in such a possibility, pure cultures of 27 different oral bacteria were surveyed (as well as dental plaque and sediment) for their peroxide-forming and peroxide-removing capabilities. Peroxide production was measured for each of the pure cultures by incubation with glucose at low and high substrate concentrations (2.8 and 28.0 mM) for 4 h and with the pH kept at 7.0 by a pH-stat. Removal of hydrogen peroxide was assessed in similar experiments where peroxide at 0, 29.4, 147.2 or 294.4 mM [0, 0.1, 0.5 and 1% (w/v)] replaced the glucose. Hydrogen peroxide formation was seen with only three of the bacteria tested, Streptococcus sanguis I and II (sanguis and oralis), and Strep. mitior (mitis biotype I); levels of hydrogen peroxide between 2.2 and 9.8 mM were produced when these micro-organisms were grown aerobically and 1.1 and 3.9 mM when grown anaerobically. Earlier reports indicate that such levels were usually sufficient to inhibit the growth of many plaque bacteria. The amounts formed were similar at the two glucose levels tested, suggesting that maximum peroxide production is reached at low glucose concentration. None of the three peroxide-producing organisms was able to utilize hydrogen peroxide but five of the other 24 tested, Neisseria sicca, Haemophilus segnis, H. parainfluenzae, Actinomyces viscosus and Staphylococcus epidermidis, could readily do so, as could the mixed bacteria in salivary sediment and dental plaque, both of which contain relatively high numbers of these peroxide-utilizing micro-organisms. The ability of the bacteria in plaque and sediment to degrade hydrogen peroxide was considerable and extremely rapid; peroxide removal and usually complete within the first 15 min of the incubation even when its initial level was as high as 294.4 mM. This almost overwhelming ability to remove peroxide was confirmed when peroxide-producing and -using cultures were mixed and when each of eight salivary sediments was incubated with glucose and with peroxide at concentrations up to 294.4 mM. In the glucose incubations, no hydrogen peroxide was observed, indicating dominance of microbial peroxide removers over hydrogen peroxide producers.(ABSTRACT TRUNCATED AT 400 WORDS)

Actinomyces viscosus↗

Assessment of head-space gas-liquid chromatography for the rapid detection of growth in blood cultures.

Blood for transfusion was inoculated with between 10(0) and 10(2) colony-forming units (CFU) per ml of each of 59 microbial isolates and added to cooked meat broth. At intervals up to 72 h incubation, the cultures were examined by conventional visual inspection and automated head-space gas-liquid chromatography (HS-GLC). Forty-six isolates including all those examined of Staphylococcus aureus, Streptococcus pyogenes, S. pneumoniae, S. faecalis, S. milleri, S. mitior, S. mitis, S. salivarius, S. sanguis, Escherichia coli, Klebsiella pneumoniae, K. oxytoca, Proteus mirabilis, Morganella morganii, Serratia sp., Enterobacter cloacae, Bacterioides fragilis, Clostridium perfringens, Candida albicans, C. krusei and Torulopsis glabrata, and three isolates of Staphylococcus epidermidis, were detected by HS-GLC. HS-GLC failed to detect the growth of eleven isolates including all those of Pseudomonas aeruginosa, Acinetobacter calcoaceticus. Haemophilus influenzae, Corynebacterium sp. and two isolates of S. epidermidis. The growth of all 59 isolates were detected by visual inspection. No significant difference was found between HS-GLC analysis and visual inspection in the speed of detection of bacterial isolates. All the yeast isolates were detected by HS-GLC after 24 h incubation, indicating that it may be possible to detect fungemias earlier by HS-GLC analysis than by other methods.

Bacteria↗

Antibiotic susceptibility of streptococci isolated from blood from neutropenic patients.

MICs were performed on 62 streptococci isolated from the blood of neutropenic patients from 1986 to 1992 using the NCCLS broth microdilution procedure. Species isolated were 43 S. mitis, 6 S.pneumoniae, 5 S.sanguis, 1 S.pyogenes, 1 beta-hemolytic streptococcus of the group G, 1 S.adjacens, 1 S.intermedius, 4 not identified isolates (2 alpha-hemolytic, 1 non-hemolytic and 1 nutritionally variant strains). 26% of all strains were resistant or intermediate to penicillin (MIC 90: 2 mg/l) and 89% to norfloxacine (MIC 90: 32 mg/l). All were susceptible to vancomycin. Among cephalosporins, all strains were susceptible to cefotaxime (MIC 90: 1 mg/l), ceftriaxone (MIC 90: 1 mg/l) and cefepime (MIC 90: 2 mg/l) while 19% were R or I to ceftazidime (MIC 90: 16 mg/l). All were susceptible to imipenem (MIC 90: 0.5 mg/l) and meropenem (MIC 90: 1 mg/l). Among the third generation cephalosporins, ceftazidime--the agent of this class of antibiotics that is most often used for the empirical therapy of febrile episodes in neutropenic patients (in combination but also in monotherapy)--has a high resistance rate as compared to the other compounds.

Bacteremia↗

[Evaluation of a rapid method for the identification of the Enterococcus genus].

BACKGROUND: The evaluation of the reliability of a group of three tests: pyrrolidonil-arilamidase, rapid test in NaCl esculin broth and group D antistreptococci agglutination for the rapid identification of the Enterococcus genus were performed. METHODS: One hundred fifty-eight strains of streptococci: 110 strains of Enterococcus faecalis, 8 E. faecium, 3 E. avium, 16 Streptococcus intermedius, 7 S. salivarius, 4 S. bovis, 4 S. constellatus, 2 S. sanguis and 1 strain of S. equinus, S. mitis, S. uberis and S. mutans were studied. The tests used were: rapid test in NaCl- esculin broth, pyrrolidonyl-arilamidase and antigroup D streptococcus agglutination (Patho Dx Strep D/PYR; Diagnostic Product Corporation). All catalase-negative gram-positive cocci or coccibacilli which were positive in the three tests were considered to belong to the Enterococcus genus. The usefulness of Gram staining in culture in thioglycolate broth was also studied comparing it with the results obtained with solid medium. RESULTS: The results of the proposed identification scheme were evaluated calculating different parameters: sensitivity 98%, specificity 100%, positive predictive value, 100%, and negative predictive value 94%. No significant differences were observed between the results obtained with Gram staining with thioglycolate broth and those obtained with solid medium except in the case of the Streptococcus mutans strain. CONCLUSIONS: The application of a combination of tests: pyrroidonyl-arilamidase, antistreptococci group D agglutination and rapid test in NaCl-esculin broth to catalase-negative gram-positive cocci and coccobacilli allows reliable identification in a maximum of three hours of strains belonging to the Enterococcus genus.

Bacteriological Techniques↗

Caries experience, periodontal treatment needs, salivary pH, and Streptococcus mutans counts in a preadolescent Down syndrome population.

The prevalence of dental caries and periodontal treatment needs in an institutionalized population with Down syndrome was examined and the relationship between caries prevalence, salivary pH, and salivary levels of Streptococcus mutans was studied. Thirty-two children with Down syndrome, aged 8 to 13, were compared with two control groups of similar age ranges: healthy children and non-Down mentally retarded (MR) children living in the same institution as the Down syndrome population. The gender ratio mixtures in each group also matched the study sample. Caries experience as indicated by decayed, missing, and filled surfaces (DMF-S) showed significantly lower mean scores for the Down syndrome group compared with both control groups. A similar pattern was found when evaluating the mean numbers of decayed surfaces. Streptococcus mutans counts, expressed as number of colony-forming units on mitis salivarius agar plates among the Down syndrome group, were the lowest, although not statistically significant compared with the counts of the healthy children. Both groups had bacterial counts which were significantly lower than those of the MR group. Significant differences between the two institutionalized groups and the healthy group were recorded for the salivary pH levels. The periodontal treatment needs as evaluated by the Community Periodontal Index of Treatment Needs showed significant difference between the MR group versus both the healthy population and the Down syndrome group. On the whole, 84% of the Down syndrome children were cariesfree.

Adolescent↗

Clinical isolates of Streptococcus mutans serotype c with altered colony morphology due to fructan synthesis.

Streptococcus mutans MT6801 , MT6861 , and MT6879 , which form large mucoid colonies on mitis salivarius agar, were isolated from a mother and her two daughters. These isolates were identified as serotype c by immunodiffusion with serotype-specific antisera. The large colonies formed on sucrose-containing agar were found to contain water-soluble fructan . The cell-free fructosyltransferase ( FTase ) activity of the strains which formed large colonies was five to eight times higher than that of serotype c S. mutans which produced small, rough colonies typical of this serotype. Furthermore, greater quantities of fructan were synthesized from sucrose by growing cells of MT6801 when compared with MT8148 , a typical serotype c S. mutans. Glucosyltransferase and FTase could be isolated by chromatofocusing from culture supernatants of MT6801 and MT8148 . The FTase activity of both strains was eluted at pH 4.5, and glucosyltransferase was released by elution with an NaCl linear gradient. The eluted FTase activity of MT6801 was significantly higher than that of MT8148 . Strains MT6861 and MT6879 were also found to possess a similar property in terms of FTase activity. These results suggest that formation of large mucoid colonies by these strains is a consequence of high FTase activity.

Adult↗

Cell surface hydrophobicity of group D and viridans streptococci isolated from patients with septicaemia.

Sixty-three strains of Group D streptococci and viridans streptococci isolated from blood cultures during a two year period were typed to the species level with conventional biochemical tests and API Strep. Streptococcus faecalis was the most common species isolated followed by S. sanguis, S. mitis and S. constellatus (S. milleri). One of the two isolates of S. faecium was a contamination. The reported increasing frequency of this organism and other Group D and viridans streptococci as well as the association of S. bovis with malignant bowel disease indicate the need for full identification of streptococcal isolates from blood cultures. Pronounced surface hydrophobicity as measured with the salt aggregation test (SAT) was expressed by 59/63 (94%) of the blood culture isolates whereas strains isolated from commercial fermentation products and strains passaged several times were hydrophilic. In the presence of human serum albumin which binds to lipoteichoic acid only one strain decreased in surface hydrophobicity. The surface hydrophobicity of two strains even slightly increased indicating that lipoteichoic acid but marginally contributes to surface hydrophobicity of streptococcal cells from these species.

Adolescent↗

Properties of a mutant of Streptococcus mutans altered in glucosyltransferase activity.

A mutant of Streptococcus mutans GS-5 has been isolated as a smooth colonial variant on mitis salivarius agar. This mutant, designated SNG-1, adheres to glass surfaces as well as the parental organism when grown in the presence of sucrose. However, in contrast to the parental organism, glucose-grown cultures of the mutant did not adhere to smooth surfaces when incubated with sucrose under nongrowing conditions. The inability of the mutant organism to adhere to glass surfaces under the latter condition was a result to markedly reduced levels of mutant cell-associated glucosyltransferase activity. In addition, the extracellular activity of the mutant was also severely depressed relative to the parental activity. The reduced levels of mutant enzyme activity appear to be a result of a mutation in a structural gene coding for glucosyltransferase activity since (i) mutant glucosyltransferase activity is much less resistant to heat inactivation compared to the parental enzymes and (ii) the migration patterns of the mutant and parental enzymes differ on polyacrylamide gels and after isoelectric focusing on polyacrylamide gels. However, the kinetic properties of the mutant enzymes are similar to those of the comparable parental activities in terms of pH and temperature optima and Km values for sucrose. The mutant enzyme responsible for soluble glucan synthesis has been purified approximately 300-fold. These results are discussed in terms of the mechanism of glucan synthesis by S. mutans.

Binding Sites↗

New method for the isolation of Streptococcus mutans and its differentiation from other oral streptococci.

A new, improved agar medium for the isolation of Streptococcus mutans, the etiological agent of dental caries, was developed. In contrast to mitis-salivarius agar, this medium not only recovers a greater number of S. mutans strains from most oral specimens but, because of its mannitol and sorbitol content, it also facilitates the differentiation of S. mutans from other oral streptococci, e.g., S. salivarius, S. mitis, and S. sanguis, which do not grow or produce scanty growth only after 10 days of incubation. The medium is easy to prepare because of its simple and unique composition, is characterized by the presence of an acid indicator, and can be utilized under aerobic and anaerobic conditions as well. The medium cannot be used to distinguish among the eight serotypes, a to g and SL-1, of S. mutans. Mannitol-utilizing bacteria such as streptococci (e.g., S. faecalis) and other microorganisms (e.g., Staphylococcus aureus) are able to grow on this medium and can be distinguished from S. mutans by their unique colony morphology.

Aerobiosis↗

Study of root caries in an artificial mouth.

The object of this project was to develop a laboratory model system to study root and enamel caries. Model plaque biofilms containing consortia of four (Streptococcus mutans, Streptococcus sobrinus, Actinomyces naeslundii, and Lactobacillus rhamnosus) or six (S mutans, S sobrinus, S mitis, A naeslundii, L rhamnosus, and Veillonella dispar) species of caries pathogens were developed in a artificial mouth. Caries lesions similar to those found in vivo were produced on enamel and dentine blocks and in intact root tissues by these consortia more effectively than biofilms of the individual species. Fluoride supplied at 5 ppm suppressed enamel caries effectively, less so dentinal lesion formation, but much more so than F incorporated into the mineralised surface of enamel and roots. This is the first direct comparison of the effect on caries of environmental F and tooth mineral F induced by a plaque-like bacterial biofilm.

Actinomyces↗

Expression of Streptococcus mutans gtf genes in Streptococcus milleri.

The Streptococcus mutans glucosyltransferase (GTF) genes gtfB and gtfC were ligated into Escherichia coli-streptococcus shuttle plasmids and introduced into Streptococcus milleri. gtfB transformant KSB8 formed an S. mutans-like rough colony on mitis salivarius agar and expressed an extracellular GTF-I, of 158 kDa, and two cell-bound GTF-Is, of 158 and 135 kDa. gtfC transformant KSC43 formed a semirough colony on mitis salivarius agar and expressed primarily an extracellular GTF-SI, of 146 kDa, and two cell-bound GTF-SIs, of 146 and 152 kDa. The extracellular GTFs from KSB8 and KSC43 were purified and characterized. The two types of GTF also reacted specifically with monoclonal antibodies directed against each enzyme. Both enzymes synthesized significant amounts of oligosaccharides, consisting primarily of alpha-1,6-glucosidic linkages, as well as water-insoluble glucans, containing alpha-1,3-glucosidic linkages. Insoluble-glucan-synthesizing activities of both enzymes were stimulated (three- to sixfold) by the addition of dextran T10 and were inhibited in the presence of 1.5 M ammonium sulfate. The Km(s) for sucrose and the optimal pHs were also similar for both enzymes. However, when the transformants were grown in Todd-Hewitt broth supplemented with sucrose, KSC43 cells, expressing GTF-SI activity, adhered to glass surfaces in vitro, while KSB8 cells, expressing GTF-I activity, did not. These results are discussed relative to the potential role of the gtfB and gftC genes in S. mutans cariogenicity.

Ammonium Sulfate↗

Identity of viridans streptococci isolated from cases of infective endocarditis.

The oral streptococci have undergone considerable taxonomic revision in recent years but there is still little information concerning associations between the newly defined species and disease. This study examined the identities of 47 strains of oral streptococci collected from 42 confirmed cases of infective endocarditis. By means of recently described physiological schemes, the most common species identified were Streptococcus sanguis sensu stricto (31.9%), S. oralis (29.8%) and S. gordonii (12.7%). Other related species including S. mitis and "S. parasanguis" were less common. This indicates that attention should be focused on S. sanguis sensu stricto and S. oralis when considering possible pathogenic mechanisms involved in viridans streptococcal endocarditis.

Endocarditis, Bacterial↗

Effects of tetracycline on the streptococcal flora of periodontal pockets.

The effects of tetracycline on the subgingival streptococcal flora of periodontal patients were examined. Before antibiotic treatment, tetracycline-resistant isolates were obtained from 24 to 25 patients. In most patients, the proportion of the subgingival flora resistant to tetracycline increased after 2 weeks of therapy (1,000 mg of tetracycline/day) and then decreased after the cessation of treatment. Cultural conditions used for primary isolation were designed to favor the growth of facultative streptococci. Consequently, the majority (99%) of resistant isolates were identified as streptococci. Among 407 tetracycline-resistant Streptococcus isolates chosen for further classification, 9 species were identified, with S. sanguis (63%) and S. mitis (19%) predominating. There were no significant differences in the distribution of species isolated before and after treatment and after the cessation of tetracycline treatment. Plasmids were isolated from only 23 of 121 resistant streptococcal strains examined, suggesting that tetracycline resistance is not plasmid mediated in the majority of these oral streptococci.

DNA, Bacterial↗

Selective binding of blood group-reactive salivary mucins by Streptococcus mutans and other oral organisms.

Strains of Streptococcus mutans of four genetic groups and five serotypes, and strains of S. sanguis, S. mitis, S. salivarius, Actinomyces naeslundii, and A. viscosus, were found to bind blood group-reactive (BGR) mucin isolated from whole human saliva. The bacteria studied bound mucins with blood type A or B reactivity to a similar extent, suggesting that the carbohydrate moieties responsible for the A and B antigenic determinants were not involved. The organisms studied appeared to bind different fractions of BGR mucin molecules because preparations absorbed with cells of a given oral species no longer contained BGR molecules which bound to homologous organisms but still possessed BGR components which bound to varying degrees to other bacteria. Differences were even noted among S. mutans strains belonging to different genetic groups and serological types. Immunoglobulins could not be detected in the mucin preparations, and addition of anti-human immunoglobulin A (IgA), IgG, or IgM serum to reaction mixtures did not affect binding. Mucin pretreated with periodate or iodoacetate no longer bound to S. mutans H12 cells, suggesting that carbohydrate moieties and sulfhydryl groups played an essential role. Active cell metabolism was not required for BGR mucin binding; however, pretreatment of H12 cells with periodate or heat (100 degrees C for 15 min) reduced binding. Mucin labeled with [(14)C]phenyl isothiocyanate appeared to bind to S. mutans H12 cells comparably to untreated mucin; the binding also appeared to be specific because less than 15% of the labeled material became bound when incubated with an excess of streptococci. Binding of [(14)C]phenyl isothiocyanate-labeled mucin was not affected by neutral sugars tested or by preparations of c antigen, glycerol teichoic acid, dextran, or crude glucosyltransferase. However, binding was inhibited by several amines. BGR salivary mucins are present in the acquired pellicle covering teeth; the ability of bacteria to selectively bind such components suggest that they may serve as receptor molecules involved in the attachment of bacteria to teeth.

Actinomyces↗

Molecular cloning and characterization of scrB, the structural gene for the Streptococcus mutans phosphoenolpyruvate-dependent sucrose phosphotransferase system sucrose-6-phosphate hydrolase.

A DNA fragment encoding the sucrose-6-phosphate hydrolase component of the Streptococcus mutans phosphoenolpyruvate-dependent sucrose phosphotransferase system has been recovered from a plasmid-based genomic library of strain GS5. The locus, designated scrB, was found to reside within a 2.9-kilobase-pair restriction fragment present on the chimeric molecule pVA1343 (7.3 kilobase pairs). Minicell analysis of pVA1343-directed translation products revealed that the scrB product synthesized in Escherichia coli V1343 was a single peptide of Mr 57,000. This polypeptide was reactive with antiserum prepared against S. mutans intracellular invertase, which has been previously shown to have an Mr of 43,000 to 48,000. The basis of this difference in Mr was not established but may represent a posttranslational proteolytic event which occurred in S. mutans but not in recombinant V1343. Sucrose-6-phosphate hydrolase purified to homogeneity from V1343 exhibited Michaelis constants of 180 mM for sucrose and 0.08 mM for sucrose-6-phosphate. Deletion analysis of pVA1343 facilitated the assignment of a coding region for the hydrolase within the insert, as well as an orientation for the transcription of scrB. scrB-defective strains of S. mutans constructed by additive integration of an insertionally inactivated scrB locus exhibited the sucrose sensitivity characteristic of this mutant class. Similar loci were detected by DNA-DNA hybridization in additional strains of S. mutans and two strains of Streptococcus cricetus, but not in single strain representatives of S. rattus, S. sobrinus, S. sanguis I and II, S. salivarius, or S. mitis.

Animals↗

Effectiveness of ethylene oxide for sterilization of dental handpieces.

Ethylene oxide gas has been utilized as an alternative method for sterilization of dental handpieces, as it is less corrosive than steam. However, its effectiveness for sterilization of the internal components of dental handpieces has not been established. The objective of this study was to compare the effectiveness of ethylene oxide and steam for sterilization of dental handpieces. Unused handpieces and handpieces which had been exposed to clinical dental procedures ('clinical') were contaminated with Streptococcus mutans, exposed to steam or ethylene oxide, and flushed with sterile saline. Washings were plated on mitis-salivarius agar, and colonies identified and counted. No viable colonies could be established from washings from 'clinical' or 'unused' handpieces exposed to steam. However, viable colonies could be established from 'clinical' handpieces exposed to ethylene oxide. This data suggests that a substance entrapped within 'clinical' handpieces (possibly the biofilm) may protect bacteria from ethylene oxide gas, preventing adequate sterilization.

Biofilms↗