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Multiple glucan-binding proteins of Streptococcus sobrinus.

Several proteins from culture supernatants of Streptococcus sobrinus were able to bind avidly to Sephadex G-75. The proteins could be partially eluted from the Sephadex by low-molecular-weight alpha-1,6 glucan or fully eluted by 4 M guanidine hydrochloride. Elution profiles were complex, yielding proteins of 16, 45, 58 to 60, 90, 135, and 145 kDa, showing that the wild-type strain possessed multiple glucan-binding proteins. Two mutants of Streptococcus sobrinus incapable of aggregation by high-molecular-weight alpha-1,6 glucan were isolated. One mutant was spontaneous, from a cell suspension to which glucan had been added, whereas the other was induced by ethyl methanesulfonate. Both mutants were devoid of a 60-kDa protein, as shown by gel electrophoresis of culture supernatants and whole cells. Amino acid analysis showed that the 58- to 60-kDa protein and the 90-kDa protein were distinct, although both were N-terminally blocked. Both mutants retained their ability to adhere to glass in the presence of sucrose and to ferment mannitol and sorbitol. Both mutants retained their glucosytransferase activities, as shown by activity gels. Western blots (immunoblots), employing antibody against a glucan-binding protein of Streptococcus mutans, failed to reveal cross-reactivity with S. sobrinus proteins. The results show that even though S. sobrinus produces several proteins capable of binding alpha-1,6 glucans, the 60-kDa protein is probably the lectin needed for glucan-dependent cellular aggregation.

Amino Acids↗

Identification and characterization of the eps (Exopolysaccharide) gene cluster from Streptococcus thermophilus Sfi6.

We report the identification and characterization of the eps gene cluster of Streptococcus thermophilus Sfi6 required for exopolysaccharide (EPS) synthesis. This report is the first genetic work concerning EPS production in a food microorganism. The EPS secreted by this strain consists of the following tetrasaccharide repeating unit:-->3)-beta-D-Galp-(1-->3)-[alpha-D-Galp-(1-->6)]-beta-D- D-Galp-(1-->3)-alpha-D-Galp-D-GalpNAc-(1-->. The genetic locus The genetic locus was identified by Tn916 mutagenesis in combination with a plate assay to identify Eps mutants. Sequence analysis of the gene region, which was obtained from subclones of a genomic library of Sfi6, revealed a 15.25-kb region encoding 15 open reading frames. EPS expression in the non-EPS-producing heterologous host, Lactococcus lactis MG1363, showed that within the 15.25-kb region, a region with a size of 14.52 kb encoding the 13 genes epsA to epsM was capable of directing EPS synthesis and secretion in this host. Homology searches of the predicted proteins in the Swiss-Prot database revealed high homology (40 to 68% identity) for epsA, B, C, D, and E and the genes involved in capsule synthesis in Streptococcus pneumoniae and Streptococcus agalactiae. Moderate to low homology (37 to 18% identity) was detected for epsB, D, F, and H and the genes involved in capsule synthesis in Staphylococcus aureus for epsC, D, and E and the genes involved in exopolysaccharide I (EPSI) synthesis in Rhizobium meliloti for epsC to epsJ and the genes involved in lipopolysaccharide synthesis in members of the Enterobacteriaceae, and finally for eps K and lipB of Neisseria meningitidis. Genes (epsJ, epsL, and epsM) for which the predicted proteins showed little or no homology with proteins in the Swiss-Prot database were shown to be involved in EPS synthesis by single-crossover gene disruption experiments.

Bacterial Proteins↗

Identification and characterization of IS1381, a new insertion sequence in Streptococcus pneumoniae.

A new insertion sequence (IS1381) was identified in the genome of Streptococcus pneumoniae R6 as an 846-bp segment containing 20-bp terminal inverted repeats and flanked by 7-bp direct repeats. The three sequenced copies of this element have two overlapping open reading frame (ORF) genes named orfA and orfB. However, significant variations between individual copies were found, suggesting that inactivating mutations have occurred in an original single ORF. Accordingly, the consensus IS1381 element derived from the comparison of the three available copies should contain a single ORF sufficient to encode a basic protein of 267 amino acids which exhibited high similarity to the putative transposases of ISL2 from Lactobacillus helveticus and of IS702 from the cyanobacterium Calothrix sp. strain PCC 7601. A minimum of five to seven copies were detected by hybridization experiments in the R6 genome. In remarkable contrast with the two previously reported pneumococcal insertion sequences, several copies of IS1381 have been detected in all of the clinical isolates tested so far. Interestingly, Streptococcus oralis NCTC 11427 (type strain), a close relative of pneumococcus, does not contain this element, but its occurrence in the type strain of Streptococcus mitis (NCTC 12261) suggests that this species has exchanged DNA with S. pneumoniae directly or through an intermediate species.

Amino Acid Sequence↗

Streptococcus salivarius fimbriae are composed of a glycoprotein containing a repeated motif assembled into a filamentous nondissociable structure.

Streptococcus salivarius, a gram-positive bacterium found in the human oral cavity, expresses flexible peritrichous fimbriae. In this paper, we report purification and partial characterization of S. salivarius fimbriae. Fimbriae were extracted by shearing the cell surface of hyperfimbriated mutant A37 (a spontaneous mutant of S. salivarius ATCC 25975) with glass beads. Preliminary experiments showed that S. salivarius fimbriae did not dissociate when they were incubated at 100 degrees C in the presence of sodium dodecyl sulfate. This characteristic was used to separate them from other cell surface components by successive gel filtration chromatography procedures. Fimbriae with molecular masses ranging from 20 x 10(6) to 40 x 10(6) Da were purified. Examination of purified fimbriae by electron microscopy revealed the presence of filamentous structures up to 1 microm long and 3 to 4 nm in diameter. Biochemical studies of purified fimbriae and an amino acid sequence analysis of a fimbrial internal peptide revealed that S. salivarius fimbriae were composed of a glycoprotein assembled into a filamentous structure resistant to dissociation. The internal amino acid sequence was composed of a repeated motif of two amino acids alternating with two modified residues: A/X/T-E-Q-M/phi, where X represents a modified amino acid residue and phi represents a blank cycle. Immunolocalization experiments also revealed that the fimbriae were associated with a wheat germ agglutinin-reactive carbohydrate. Immunolabeling experiments with antifimbria polyclonal antibodies showed that antigenically related fimbria-like structures were expressed in two other human oral streptococcal species, Streptococcus mitis and Streptococcus constellatus.

Amino Acids↗

Streptococcus pseudoporcinus sp. nov., a novel species isolated from the genitourinary tract of women.

Streptococcus strains from animal and human sources identified biochemically as Streptococcus porcinus were investigated by 16S rRNA gene sequencing. The nine human strains isolated between 1997 and 2005 formed a single cluster with more than 2.1% dissimilarity with S. porcinus strains from animal sources. A novel species, Streptococcus pseudoporcinus sp. nov., is proposed.

Cluster Analysis↗

Evaluation of the Rapid Strep system for the identification of clinical isolates of Streptococcus species.

A total of 247 strains of streptococci isolated from humans were tested for identification in the Rapid Strep system. The identification rates and identification levels were different for each Streptococcus species. Our data indicate that the Rapid Strep system will identify nearly all the beta-hemolytic Streptococcus species if serological procedures are used in conjunction with the rapid physiological procedures. Of the group D streptococci, 98% of the enterococci and 95% of the non-enterococci were correctly identified. Of the commonly occurring viridans species, 85% were correctly identified, but only 10% of the less frequently occurring viridans species were identified. A total of 90% of the Streptococcus pneumoniae and 60% of the Aerococcus strains were correctly identified.

Humans↗

Incidence and characterization of beta-hemolytic Streptococcus milleri and differentiation from S. pyogenes (group A), S. equisimilis (group C), and large-colony group G streptococci.

The biochemical characteristics of 172 clinical isolates of group A, C, F, or G or "nongroupable" beta-hemolytic streptococci were examined. Among these isolates, 91 were identified as beta-hemolytic strains of Streptococcus milleri. The remaining isolates included 20 Streptococcus pyogenes, 21 Streptococcus equisimilis, 37 large-colony group G streptococci, and 3 unidentified nongroupable isolates. A majority (84%) of the S. milleri strains possessed Lancefield group antigen (3 A, 27 C, 41 F, and 5 G), whereas 15 S. milleri strains (16%) were nongroupable. Serological tests did not differentiate S. milleri isolates with group A, C, or G antigen from S. pyogenes (group A), S. equisimilis (group C), or large-colony group G streptococci. Biochemical tests which were found useful for differentiation included the Voges-Proskauer test, hydrolysis of pyroglutamic acid and beta-D-glucuronide, bacitracin susceptibility, and acid production from ribose. S. milleri represented 56% of the group C, 100% of the group F, and 83% of the nongroupable beta-hemolytic streptococci isolated in our clinical laboratory, whereas the incidence of S. milleri among group A and group G streptococci was estimated to be low. The role of beta-hemolytic S. milleri as a cause of human infection remains obscured by the failure to routinely differentiate S. milleri from other beta-hemolytic streptococci.

Hemolysis↗

Rapid detection of Streptococcus pyogenes in pediatric patient specimens by DNA probe.

A chemiluminescent DNA probe test (Group A Streptococcus Direct Test; Gen-Probe, Inc., San Diego, Calif.) for rapid, direct detection of cRNA of Streptococcus pyogenes in throat swabs was compared with conventional culture and identification techniques. Throat swabs from 277 patients suspected of having streptococcal pharyngitis were examined. By DNA probe alone, 10 specimens were positive, 51 were positive by both assays, and 8 were positive by culture alone. Thus, DNA probe sensitivity, specificity, and positive and negative predictive values were 86, 95, 84, and 96%, respectively. Including an indeterminate category, sensitivity, specificity, and positive and negative predictive values were 89, 96, 86, and 97%, respectively. After discrepancy testing, these values for the raw data improved to 90, 98, 93, and 97%, respectively. None of the 24 specimens that grew non-S. pyogenes beta-hemolytic streptococci in culture were positive by the DNA probe. Because mucoid S. pyogenes strains are more virulent than nonmucoid strains, 24 isolates were retrospectively tested with the DNA probe to ensure that both types would be detected equally well. Isolates were examined in pure cultures as well as mixed with representative normal oral flora. There was no statistical difference in detection of any of the four groups. Group A Streptococcus Direct Test is a rapid, sensitive, and specific test for S. pyogenes.

Child↗

Comparison of Gen-Probe Group A streptococcus Direct Test with culture for diagnosing streptococcal pharyngitis.

The Group A Streptococcus Direct Test (GP-ST test; Gen-Probe, Inc., San Diego, Calif.) was compared with culture for the detection of Streptococcus pyogenes from throat swabs of 767 patients with pharyngitis. Swabs were tested by the GP-ST test after inoculating a 5% sheep blood agar (SBA) plate. SBA plates were incubated at 35 degrees C in room air for 72 h. SBA plates with no evidence of beta-hemolytic colonies after 18 to 24 h of incubation were subcultured by taking a swipe across the primary inoculum from the SBA plate to an agar selective for Streptococcus spp. In a low-prevalence (11.9%) population and in comparison with the number of positive cultures detected by the 72-h single-culture method (SBA plate method), the GP-ST test had a sensitivity of 88.6%, a specificity of 97.8%, a positive predictive value of 83.9%, and a negative predictive value of 98.5%. In comparison with the growth of any colonies of S. pyogenes on the 72-h SBA plates plus a subculture onto selective blood agar, the sensitivities and specificities were as follows: 72-h SBA plate method, 96.7 and 100%, respectively; GP-ST test, 85.7 and 97.8%, respectively. The GP-ST test is an easy-to-perform, reliable test for batch screening of throat swabs for S. pyogenes.

Bacteriological Techniques↗

Species identification of members of the Streptococcus milleri group isolated from the vagina by ID 32 Strep system and differential phenotypic characteristics.

The importance of bacterial vaginosis as a risk factor in obstetric and gynecological infections has recently been recognized. The bacterial vaginosis group of organisms includes members of the Streptococcus milleri group, the identification of which has caused much confusion. We prospectively surveyed the rates of carriage of S. milleri group organisms in 397 high vaginal swabs received in our laboratory. For the identification of 99 clinical isolates and 23 control strains, we compared the results obtained by the rapid ID 32 Strep system (Analytab Products) and by a scheme utilizing six differential phenotypic characteristics (presence of beta-N-acetylglucosaminidase, alpha-glucosidase, beta-D-fucosidase, beta-galactosidase, beta-N-acetylgalactosaminidase, and beta-glucosidase) as described by Whiley et al. (R. A. Whiley, H. Fraser, J. M. Hardie, and D. Beighton, J. Clin. Microbiol. 28:1497-1501, 1990). We identified Streptococcus anginosus in 18% and Streptococcus constellatus in 0.05% of the specimens examined. Of the isolates of S. anginosus that reacted with grouping antisera, 20 of 25 belonged to Lancefield group F. The incubation conditions for bacterial cultures and for reaction mixtures affected the results of phenotypic characterization in the production of alpha-glucosidase, beta-galactosidase, and beta-glucosidase. However, by using bacterial cultures grown under hypercapnic conditions and incubating the reaction mixtures aerobically, consistent phenotypic characteristics were obtained, allowing identification similar to that obtained by the ID 32 Strep system. We therefore recommend the phenotypic scheme as an inexpensive, reliable, and convenient method for the initial identification of species of the S. milleri group.

Antibodies, Bacterial↗

Species belonging to the "Streptococcus milleri" group: antimicrobial susceptibility and comparative prevalence in significant clinical specimens.

The association between the three species belonging to the "Streptococcus milleri" group and different sites of isolation was examined for 73 successive strains recovered from clinically significant, purulent infections. Susceptibility testing was performed on 64 of these strains. The present study supports the association of particular species with different clinical sources. Susceptibility data suggest that emerging penicillin resistance among Streptococcus anginosus and Streptococcus intermedius isolates may represent a potential clinical problem in the therapeutic management of infections caused by these species.

Anti-Bacterial Agents↗

Septicemia caused by Streptococcus canis in a human.

We describe a case of septicemia due to Streptococcus canis in a 77-year-old man. The organism was presumably transmitted from a domestic animal. Ulcers of the lower limbs were the likely portals of entry. The differentiation between Streptococcus canis and Streptococcus dysgalactiae was based on biochemical properties and DNA macrorestriction analysis by pulsed-field gel electrophoresis.

Aged↗

Use of resequencing oligonucleotide microarrays for identification of Streptococcus pyogenes and associated antibiotic resistance determinants.

Group A streptococci (GAS) are responsible for a wide variety of human infections associated with considerable morbidity and mortality. Ever since the first systematic effort by Lancefield to group Streptococcus species by M protein variants, the detection and characterization of Streptococcus by different methods have been an evolving process. The ideal assay for GAS identification not only would provide quick and accurate diagnostic results but also would reveal antibiotic resistance patterns and genotype information, aiding not only in treatment but in epidemiologic assessment as well. The oligonucleotide microarray is a promising new technology which could potentially address this need. In this study, we evaluated the usefulness of oligonucleotide resequencing microarrays for identifying GAS and its associated antibiotic resistance markers. We demonstrated an assay platform that combines the use of resequencing DNA microarrays with either random nucleic acid amplification or multiplex PCR for GAS detection. When detecting Streptococcus pyogenes from coded clinical samples, this approach demonstrated an excellent concordance with a more established culture method. To this end, we showed the potential of resequencing microarrays for efficient and accurate detection of GAS and its associated antibiotic resistance markers with the benefit of sequencing information from microarray analysis.

Anti-Bacterial Agents↗

Identification of 43 Streptococcus species by pyrosequencing analysis of the rnpB gene.

Pyrosequencing technology was evaluated for identification of species within the Streptococcus genus. Two variable regions in the rnpB gene, which encodes the RNA subunit of endonuclease P, were sequenced in two reactions. Of 43 species, all could be identified to the species level except strains of the species pairs Streptococcus anginosus/S. constellatus and S. infantis/S. peroris. A total of 113 blood culture isolates were identified by pyrosequencing analysis of partial rnpB sequences. All but eight isolates could be unambiguously assigned to a specific species when the first 30 nucleotides of the two regions were compared to an rnpB database comprising 107 streptococcal strains. Principal coordinate analysis of sequence variation of strains from viridans group streptococci resulted in species-specific clusters for the mitis and the salivarius groups but not for the anginosus group. The identification capacity of pyrosequencing was compared to the biochemical test systems VITEK 2 and Rapid ID 32 Strep. The concordance between pyrosequencing and VITEK 2 was 75%, and for Rapid ID 32 Strep the corresponding figure was 77%. Isolates with discrepant identifications in the three methods were subjected to entire rnpB DNA sequence analysis that confirmed the identifications by pyrosequencing. In conclusion, pyrosequencing analysis of the rnpB gene can reliably identify Streptococcus species with high resolution.

Bacterial Typing Techniques↗

Rapidly fatal necrotising fasciitis caused by Streptococcus pyogenes.

AIMS: To describe the morbid anatomical and bacteriological features in a series of five cases of rapidly fatal Streptococcus pyogenes necrotising fasciitis. METHODS: Post mortem and bacteriological examinations were made of five patients dying within 48 hours from rapidly fatal necrotising fasciitis. RESULTS: All five cases died rapidly from a toxic Streptococcus toxin syndrome as a result of developing necrotising fasciitis following trivial injury. CONCLUSIONS: Necrotising fasciitis caused by Streptococcus pyogenes infection can be rapidly fatal. This is probably the result of a toxic shock syndrome. Rapid, early diagnosis and swift and probably empirical treatment is required to avoid a fatal outcome.

Adult↗

Purification of proteins similar to HPr and enzyme I from the oral bacterium Streptococcus salivarius. Biochemical and immunochemical properties.

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is made of several proteins. Two of them are designated general proteins because they are required for the transport and phosphorylation of all sugars of the PTS. These two proteins are found in the soluble fraction of cellular extracts and are termed HPr and enzyme I (EI). We reported in this work the purification and the characterization of these two proteins from Streptococcus salivarius ATCC 25975. HPr was purified by DEAE-cellulose chromatography, molecular sieving on Ultrogel AcA44, and carboxymethylcellulose chromatography. Sodium dodecyl sulfate electrophoresis in the presence of urea revealed a single band with a molecular weight of 6700. The protein contained no tryptophan and had a pI of 4.8. The purification scheme of EI was as follows: DEAE-cellulose chromatography, hydroxylapatite chromatography, DEAE-Sephadex A-50 chromatography, preparative electrophoresis, and molecular sieving on Ultrogel AcA34. The five-step purification for EI produced a 199-fold purified preparation with a specific activity of 530 mumol of HPr phosphorylated per minute per milligram of protein at 37 degrees C. The fraction obtained after filtration on Ultrogel AcA34 gave one band (68 000) on sodium dodecyl sulfate - polyacrylamide gel electrophoresis. The molecular weight of the native enzyme determined by gel filtration at 4 degrees C was 135 000, suggesting that it was a dimer. Enzyme I had a pI of 4.2, a pH optimum of 6.7, a Km for HPr of about 27 microM, a Km for phosphoenolpyruvate of 0.48 mM, and kinetics that were consistent with a Ping-Pong mechanism. Evidence had been obtained which indicated that S. salivarius enzyme I was antigenically very similar to enzyme I from various strains of Streptococcus mutans, but not to the enzyme from Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis, and Escherichia coli.

Amino Acids↗

Production of mutacin-like substances by Streptococcus mutans.

Production of inhibitory substances by strains of the Streptococcus mutans group is well documented, but the nature of the substances implied is often unknown. Of nine laboratory strains known to produce inhibitory substances, the optimal conditions for producing inhibition zones on solid media were found to vary between strains but good production was generally obtained on all-purpose media with Tween 80 at 37 degrees C after 2-4 days of aerobic incubation. Streptococcus sanguis Ny101 was found to be more sensitive than Streptococcus rattus LG-1 to all inhibitory substances produced by the S. mutans strains tested. While all strains showed some inhibition, only six showed inhibition after neutralization; arginine incorporated in agar at 0.75% completely eliminated all inhibition zones. However 1% arginine in the overlays did not affect the production of inhibition zones by strains of S. mutans C67-1, Ny257, Ny266, and T8. These strains were shown to elaborate (in a reproducible fashion) inhibitory substances which were not organic acids. Inhibitory activity was never obtained in liquid preparations, except for strains Ny257 and T8 where it was found to be very unstable.

Aerobiosis↗

[Isolation of group A beta-hemolytic Streptococcus in children with perianal dermatitis].

INTRODUCTION: Perianal dermatitis due to group A beta-hemolytic streptococcus is a rare diagnosis in pediatrics. Clinical manifestations include erythema, edema, and perianal pruritus associated with vulvovaginitis or balanitis, painful defecation, constipation, anal fissures, purulent discharge, and/or rectal bleeding. MATERIAL AND METHODS: A descriptive, observational, prospective study was conducted between May, 2004 and April, 2005. In children presenting to the emergency room with erythema, edema, and perianal pruritus, a rapid streptococcal antigen test and bacterial cultures of their perianal secretions were obtained. Patients with a positive result in the rapid test were treated with oral phenoxymethylpenicillin (40-50 mg/kg/day) for 10 days, after which clinical and bacteriological evaluations were conducted. RESULTS: Of the 24 patients presenting with erythema, edema, and perianal pruritus, 19 patients (12 boys and 7 girls), aged between 6 months and 4 years (median age: 30 months), tested positive for group A beta-hemolytic streptococcus. Nine patients also presented with constipation, 5 with anal fissures, 4 with rectal bleeding, 2 with vulvovaginitis, and 1 with streptococcal pharyngoamygdalitis. Outcome was favorable in 85 % of the patients, and perianal isolation of group A beta-hemolytic streptococcus was negative after treatment in 95 %. CONCLUSIONS: In patients presenting with erythema, edema, and perianal pruritus, perianal streptococcal dermatitis should be ruled out. The rapid test for streptococcal antigen detection is a quick and reliable method for this diagnosis. The antibiotic of choice is oral penicillin.

Anus Diseases↗