Effect on polymorphonuclear cell function of a human-specific cytotoxin, intermedilysin, expressed by Streptococcus intermedius.

Streptococcus intermedius is a member of the normal flora of the mouth but is also an opportunistic pathogen associated with purulent infections at oral and nonoral sites. Intermedilysin (ILY) has been shown to be a cytolysin capable of generating pores in the cell membrane of erythrocytes demonstrable by electron microscopy. This effect has been shown to be specific for human cells. Since polymorphonuclear cells (PMNs) are the main cell involved in innate immunity we investigated the effect of purified intermedilysin from Streptococcus intermedius on PMN function. Active ILY at a concentration of 40 ng/microl caused a significant decrease in the number of intact PMNs after 60 min. The active cytolysin, when compared with heat-inactivated ILY, did not appear to be chemotactic for the PMNs but did cause an increase in intracellular calcium, with increased cell surface CD11b expression, metabolic burst, and phagocytosis of Staphylococcus aureus. These findings may have implications for the role of ILY in deep-seated abscesses.

Mannosidase production by viridans group streptococci.

The production of mannosidase activity by all currently recognized species of human viridans group streptococci was determined using an assay in which bacterial growth was dependent on the degradation of the high-mannose-type glycans of RNase B and subsequent utilization of released mannose. RNase B is an excellent substrate for the demonstration of mannosidase activity since it is a glycoprotein with a single glycosylation site which is occupied by high-mannose-type glycoforms containing five to nine mannose residues. Mannosidase activity was produced only by some members of the mitis group (Streptococcus mitis, Streptococcus oralis, Streptococcus gordonii, Streptococcus cristatus, Streptococcus infantis, Streptococcus parasanguinis, and Streptococcus pneumoniae) and Streptococcus intermedius of the anginosus group. None of the other species within the salivarius and mutans groups or Streptococcus peroris and Streptococcus sanguinis produced mannosidase activity. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, it was demonstrated that the Man(5) glycan alone was degraded while Man(6) to Man(9), which contain terminal alpha(1-->2) mannose residues in addition to the alpha(1-->3), alpha(1-->6), and beta(1-->4) residues present in Man(5), remained intact. Investigations on mannosidase production using synthetic (4-methylumbelliferone- or p-nitrophenol-linked) alpha- or beta-mannosides as substrates indicated that there was no correlation between degradation of these substrates and degradation of the Man(5) glycan of RNase B. No species degraded these alpha-linked mannosides, while degradation of the beta-linked synthetic substrates was restricted to strains within the Streptococcus anginosus, S. gordonii, and S. intermedius species. The data generated using a native glycoprotein as the substrate demonstrate that mannosidase production within the viridans group streptococci is more widely distributed than had previously been considered.

Antimicrobial effect of a novel ozone- generating device on micro-organisms associated with primary root carious lesions in vitro.

The aims of this present study were (1) to assess the antimicrobial effect of ozone from a novel ozone-generating device (Heolozone, USA) [0.052% (v/v) in air delivered at a rate of 13.33 ml.s(-1)] on primary root carious lesions (PRCLs) and (2) to evaluate the efficacy of ozone specifically on Streptococcus mutans and Streptococcus sobrinus. In study 1, 40 soft PRCLs from freshly extracted teeth were randomly divided into two groups to test the antimicrobial effect on PRCLs from exposure to ozonated water for either 10 or 20 s. Half of a lesion was removed using a sterile excavator. Subsequently, the remaining lesion was exposed to the ozonised water for a period of either 10 or 20 s (corresponding to 0. 069 or 0.138 ml of ozone, respectively). Using paired Student t tests, a significant (p<0.001) reduction (mean +/- SE) was observed in the ozone-treated groups with either a 10-second (log(10) 3.57+/-0.37) or 20-second (log(10) 3.77+/-0.42) ozone application compared with the control groups (log(10) 5.91+/-0.15 and log(10) 6.18+/-0.21, respectively). In study 2, 40 sterile saliva-coated glass beads were randomly divided into two groups for each micro-organism. One glass bead was put into each bijou bottle with 3 ml of Todd-Hewitt broth. S. mutans and S. sobrinus were inoculated anaerobically overnight. Each glass bead was then washed with 2 ml of phosphate-buffered saline. Immediately, 10 s of ozone gas was applied to each glass bead in the test groups. There was a significant (p<0.0001) reduction (mean +/- SE) in ozone-treated samples for S. mutans (log(10) 1.01+/-0.27) and S. sobrinus (log(10) 1.09+/-0.36) compared with the control samples (log(10) 3.93+/-0.07 and log(10) 4.61+/-0.13, respectively). This treatment regime is an effective, quick, conservative and simple method to kill micro-organisms in PRCLs. Ozone gas application for a period of 10 s was also capable of reducing the numbers of S. mutans and S. sobrinus on saliva-coated glass beads in vitro.

Taxonomic study of "tufted mitior" strains of streptococci (Streptococcus sanguinis biotype 11); recognition of a new genospecies.

The taxonomic position of tufted strains of streptococci, phenotypically resembling Streptococcus mitis and previously referred to as 'tufted mitior' was investigated. By 16S rRNA sequence analysis, it was clear that the "tufted mitior" strains belonged to the mitis group of species within the genus Streptococcus. It was confirmed that these strains were taxonomically independent at the species level, sharing less than 43%, DNA-DNA similarity with all established species of the mitis group. However biochemical test data obtained, using three commercial identification kits (Rapid ID32 Strep, STREPTOGRAM, and Biolog GP-plate) together with in-house biochemical tests employing 4-MUF-linked fluorogenic substrates did not reveal sufficient differential tests with which to identify the "tufted mitior" strains unequivocally. From these data, we conclude that these "tufted mitior" strains represent a new taxon within the mitis group of the genus Streptococcus, and propose that they should be considered as a genospecies until differential phenotypic characteristics are found for their identification.

DNA-DNA reassociation studies of Streptococcus constellatus with unusual 16S rRNA sequences.

DNA-DNA reassociation studies were performed on previously described 'CI strains', which form an unusual 16S rRNA population within the 'anginosus' group of Streptococcus. The CI strains displayed reassociation values of >70% with the Streptococcus constellatus NCDO 2226T strain, with Tm values <1 degrees C, indicating phylogenetic species identity.

Distribution of the intermedilysin gene among the anginosus group streptococci and correlation between intermedilysin production and deep-seated infection with Streptococcus intermedius.

The distribution of intermedilysin, a human-specific cytolysin, among the anginosus group streptococci and the correlation of toxin production and infection by Streptococcus intermedius were investigated. PCR and Southern hybridization specific for the intermedilysin gene revealed that the toxin gene exists only in S. intermedius and no homologue to the toxin gene is distributed in S. anginosus and S. constellatus. Thus, the intermedilysin gene is useful as a marker gene of S. intermedius. Moreover, a human-specific hemolysis assay and Western blotting with intermedilysin-specific antibodies clearly demonstrated that the intermedilysin production level in isolates from deep-seated infections, such as brain and liver abscesses, is higher (6.2- to 10.2-fold, respectively) than in strains from normal habitats, such as dental plaque, or from peripheral infection sites. However, other candidate virulence factors of S. intermedius, such as chondroitin sulfate depolymerase, hyaluronidase, and sialidase activities, did not show such a clear correlation between enzymatic activity and isolation sites or disease severity. From these results, intermedilysin is likely to be the pathogenic or triggering factor of significance in inducing deep-seated infections with S. intermedius.

A study of small-colony, beta-haemolytic, Lancefield group C streptococci within the anginosus group: description of Streptococcus constellatus subsp. pharyngis subsp. nov., associated with the human throat and pharyngitis.

beta-Haemolytic, Lancefield group C streptococci within the anginosus-species group were shown by genetic and phenotypic criteria to be heterogeneous and to constitute two distinct taxa related at subspecies level to Streptococcus constellatus and Streptococcus anginosus, respectively. The first group, referred to here as DNA group 1, comprised six strains with 86-100% intragroup overall genomic DNA relatedness; five of the strains were originally isolated from the human throat and one was from an abdominal mass. They shared 61-77% DNA relatedness (delta Tm values = 1.2-1.5 degrees C) with reference strains of S. constellatus and were clearly differentiated from S. constellatus (now named Streptococcus constellatus subsp. constellatus) by the ability to produce beta-N-acetylgalactosaminidase, beta-N-acetylglucosaminidase, beta-D-fucosidase, beta-D-galactosidase and beta-D-glucosidase. The name S. constellatus subsp. pharyngis is proposed for these strains on the grounds that they are genetically and phenotypically distinct and exhibit a predeliction for the human throat, being isolated also from cases of pharyngitis. The DNA G + C content is 35-37 mol%. The type strain is MM9889aT (= NCTC 13122T). The second group (DNA group 2) was formed by five beta-haemolytic, Lancefield group C strains originally isolated from various human infections. DNA group 2 strains (81-100% intragroup DNA relatedness) shared 60-72% DNA relatedness (delta Tm values = 2.1-4.1 degrees C) with S. anginosus strains NCTC 10713T and MAS 283 but were not clearly differentiated phenotypically from S. anginosus, showed no clear pattern of clinical association, and therefore are not formally proposed as a new subspecies here.

PCR-Based methods for genotyping viridans group streptococci.

Standard repetitive extragenic palindromic (REP)-PCR, enterobacterial repetitive intergenic consensus-PCR, and Salmonella enteritidis repetitive element-PCR methods for bacterial strain typing were performed with DNA extracted by boiling members of each of the currently recognized species of human viridans group streptococci. Each of the methods was reproducible. The unique isolates (n = 72) from 15 species of viridans group streptococci were readily distinguishable, with no two isolates showing greater than 90% per cent similarity. The majority of strains exhibited much less than 90% similarity. Isolates identical by REP-PCR were also identical by the other two methods. These PCR-based typing methods, although they do not permit determination of the species of the isolates, are simple to perform and are suitable for clinical and ecological investigations of viridans group streptococci.

Current classification of the oral streptococci.

The classification of the oral streptococci has long remained a difficult area of streptococcal taxonomy. This article reviews the current classification of these bacteria into four species groups, and each group is described in detail. The often confusing changes that have taken place in the classification, identification and nomenclature of the member species are reviewed against a historical background of gradually improving techniques and approaches, leading towards a natural classification based primarily on genotypic evidence. Identification schemes currently in use employing biochemical tests are also reviewed, together with alternative molecular approaches.

Occurrence of lipoteichoic acid in oral streptococci.

The heterogeneous bacterial group known as oral streptococci was screened for the presence of cellular polyglycerolphosphate-containing lipoteichoic acid. This compound was detected in phenol extracts of lyophilized cells by an immunoassay in which polyglycerolphosphate-specific monoclonal antibody was used. Polyglycerolphosphate-containing lipoteichoic acid occurred in all 86 strains of oral streptococci examined except the Streptococcus mitis and Streptococcus oralis strains. This confirms the findings of Rosan (B. Rosan, Science 201:918-920, 1978) and Hamada et al. (S. Hamada, J. Mizuno, S. Kotani, and M. Torii, FEMS Microbiol. Lett. 8:93-96, 1980), is consistent with the results of the taxonomic study of oral streptococci performed by Kilian et al. (M. Kilian, L. Mikkelsen, and J. Henrichsen, Int. J. Syst. Bacteriol. 39:471-484, 1989), who emended the descriptions of Streptococcus sanguis, S. oralis, and S. mitis, and reflects the phylogenetic relationship among S. mitis, S. oralis, and Streptococcus pneumoniae.

Intermedilysin, a novel cytotoxin specific for human cells secreted by Streptococcus intermedius UNS46 isolated from a human liver abscess.

A novel cytotoxin (intermedilysin) specific for human cells was identified as a cytolytic factor of Streptococcus intermedius UNS46 isolated from a human liver abscess. Intermedilysin caused human cell death with membrane blebs. Intermedilysin was purified from UNS46 culture medium by means of gel filtration and hydrophobic chromatography. The purified toxin was resolved into major and minor bands of 54 and 53 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These proteins reacted with an antibody against intermedilysin. Five internal peptide fragments of intermedilysin were sequenced and found to have 42 to 71% homology with the thiol-activated cytotoxin pneumolysin. However, the action of intermedilysin differed from that of thiol-activated cytotoxins, especially in terms of a lack of activation by dithiothreitol and resistance to treatments with N-ethylmaleimide and 5,5'-dithio-bis-(2-nitrobenzoic acid), although cholesterol inhibited the toxin activity. Intermedilysin was potently hemolytic on human erythrocytes but was 100-fold less effective on chimpanzee and cynomolgus monkey erythrocytes. Intermedilysin was not hemolytic in nine other animal species tested. Since human erythrocytes treated with trypsin were far less sensitive to intermedilysin than were the intact cells, a cell membrane protein(s) may participate in the intermedilysin action. These data demonstrated that intermedilysin is distinguishable from all known bacterial cytolysins.

Interactions between Eikenella corrodens and 'Streptococcus milleri-group' organisms: possible mechanisms of pathogenicity in mixed infections.

Interactions between the 'Streptococcus milleri-group' organisms (SMG; S. intermedius, S. constellatus and S. anginosus) and Eikenella corrodens were investigated. Coaggregation reactions occurred frequently between S. anginosus (83% of strain combinations) or S. constellatus (87%) and E. corrodens isolates, but were infrequent between S. intermedius and E. corrodens (28%). No enhancement of enzyme activities against lipid, phosphate, peptide and saccharide substrates tested were detected with combinations of species in comparison to the species alone. Exponential growth of S. constellatus and S. intermedius, in mixed culture with E. corrodens, occurred within 6h post inoculation, in comparison to 25h without E. corrodens. No growth stimulation of S. anginosus was observed. It is concluded that both coaggregation and growth stimulation occur between E. corrodens and SMG isolates, and may be important mechanisms in the establishment of mixed infections involving these bacteria.

Genetic relatedness within and between serotypes of Streptococcus pneumoniae from the United Kingdom: analysis of multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, and antimicrobial resistance patterns.

A collection of 54 isolates of invasive Streptococcus pneumoniae of serotypes 3 and 14 and serogroups 6, 9, 19, and 23 was investigated. Multilocus enzyme electrophoresis and pulsed-field gel electrophoresis suggested that two clones were represented in the collection, one of serotype 14 isolates, most of which were resistant to erythromycin, and one of serotype 9V isolates, in which resistance to penicillin (MIC, 1 microgram/ml), cefotaxime, and co-trimoxazole was common. Among other isolates there were only a limited correlation between genetic relatedness measured by multilocus enzyme electrophoresis and expression of the same capsule type. However, isolates with highly related pulsed-field gel electrophoresis patterns always shared the same serotype and highly related allele profiles. Calculation of the index of association suggests a freely recombining population structure with epidemic spread of successful clones.

Comparative efficacy of oral doses of clindamycin and erythromycin in the prevention of bacteraemia.

Two antibiotics, clindamycin and erythromycin, were compared in a double-blind trial to test their efficacy in the prevention of post-dental extraction bacteraemia with streptococci in a group of 40 healthy patients. Tolerance to the oral doses was tested by questionnaire. Levels of drug in the serum were estimated using a microbiological assay. An in-vitro blood culture system was used as an analogy of the persistence of a bacteraemia in the presence of high levels of antibiotic. Isolates of streptococci were identified to species level. Minimum inhibitory concentrations of clindamycin and of erythromycin for each isolate were estimated. Results showed satisfactory levels of antibiotics in the blood for activity against oral streptococci. Clindamycin was slightly more effective than erythromycin in the prevention of post-extraction streptococcal bacteraemia but that efficacy was only 45%. Clindamycin as a single oral dose of 600 mg was well tolerated by patients compared with erythromycin 1.5 g.

Heterogeneity among 16S-23S rRNA intergenic spacers of species within the 'Streptococcus milleri group'.

The 16S-23S rRNA intergenic spacer has been suggested as a suitable region of the bacterial genome from which to derive useful taxonomic information, particularly with regard to identification at the species level. To investigate this approach as an aid to the identification of the three species comprising the 'Streptococcus milleri group' (SMG), the spacers of isolates of Streptococcus intermedius, Streptococcus anginosus and Streptococcus constellatus were amplified by PCR and length polymorphisms determined by agarose gel electrophoresis. Phenotypically atypical isolates which had been identified presumptively as belonging to these three species were also included. Spacers from two representatives of each spacer length found within the three SMG species were sequenced. 16S-23S rRNA intergenic spacer length polymorphisms allowed discrimination between S. anginosus (350 bp or 450 bp amplification product) and S. constellatus (380 bp amplification product), species that are difficult to differentiate phenotypically. S. intermedius (330 bp or 450 bp amplification product) and S. anginosus (350 bp or 450 bp amplification product) were not reliably distinguished by this method but are phenotypically distinct. Sequencing data demonstrated that the spacers had a central region of highly variable length flanked by conserved regions which included a single tRNA(Ala) gene. Polymorphism in the length of the 16S-23S spacer determined by PCR provides a rapid and useful adjunct to strain identification for S. anginosus and S. constellatus, which are not readily differentiated phenotypically.

Effects of different acidulants on growth of 'Streptococcus milleri group' strains isolated from various sites of the human body.

Growth of human clinical isolates of Streptococcus constellatus, Strep. intermedius and Strep. anginosus in HCl-, acetate and lactate acidified media was investigated. Under aerobic conditions, Strep. constellatus and Strep. anginosus were significantly more tolerant to all the acidulants than was Strep. intermedius. Under anaerobic conditions, Strep. anginosus and Strep. intermedius were significantly more tolerant to acetic acid (pH < 4.5) than Strep. constellatus.