Differentiation of species in human beta-haemolytic group G streptococci using immunoglobulin Fc fragment receptor.

AIMS: To assess the ability of human immunoglobulin Fc fragment binding activity to differentiate human biotype large colony group G streptococci from the group G "Streptococcus milleri group". METHODS: Fifty two isolates of large colony group G streptococci and 30 group G "S milleri group" strains were tested for their ability to bind fluorescein conjugated human IgG Fc fragments after acetone fixation. Immunoblotting with peroxidase labelled human Fc fragments after resolution of bacterial polypeptides by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed for six large colony strains. RESULTS: All large colony group G streptococci showed positive Fc fragment binding whereas all "S milleri group" bacteria failed to bind Fc fragments when viewed by fluorescence microscopy. All six large colony strains showed similar immunoblot binding patterns. CONCLUSION: Immunoglobulin Fc fragment receptor content distinguishes the large colony group G streptococci from the group G "S milleri group" and mayhave a role in the rapid laboratory diagnosis of pharyngeal pathogens.     

Rapid differentiation between members of the anginosus group and Streptococcus dysgalactiae subsp. equisimilis within beta-hemolytic group C and G streptococci by PCR

Based on a pair of primers developed initially for differentiating the anginosus group from other viridans streptococci, the PCR reported here can also differentiate between members of the anginosus group and Streptococcus dysgalactiae subsp. equisimilis among beta-hemolytic group C and G streptococci. The resulting 742-bp PCR product was specific for members of the anginosus group, although a smaller, nonspecific product (361 bp) was generated from S. dysgalactiae subsp. equisimilis. Restriction digestion of the amplicon with XbaI and BsmI further differentiated Streptococcus anginosus from Streptococcus constellatus within the anginosus group.     

Grouping of beta-haemolytic streptococci by enzyme-linked immunosorbent assay

A method of grouping beta-haemolytic streptococci serologically by enzyme-linked immunosorbent assay is described. A comparison of this method with double diffusion in agar gel showed complete correlation of results when highly absorbed grouping sera were used.     

Receptor in group C and G streptococci detects albumin structures present in mammalian species.

The presence of albumin structures with the capacity to bind to a surface receptor in group C and G streptococci was studied in serum samples from 45 mammalian species representing 15 different orders, using an inhibition assay. The ability of animal sera to inhibit the uptake of radiolabeled human serum albumin by the streptococci indicated the presence of such albumin structures. Positive reactions were found in species of most orders tested, with Marsupialia as a notable exception. All Carnivora sera tested were strongly positive. In some orders such as Artiodactyla both positive and negative species were identified. Serum samples from 62 bird species representing 15 orders and from 5 fish species were also tested in the inhibition assay. None of these serum samples was capable of inhibiting the uptake of human serum albumin by streptococci. Some differences were also noted in the results obtained with group C and G streptococci from human and bovine sources, respectively, indicating the presence of two types of receptors. The present studies suggest a phylogenetic origin of albumin structures with affinity for the streptococcal receptor to a period after the divergence of Marsupialia from the other mammalian orders.     

Use of enzymes in typing group A beta-haemolytic streptococci

A series of 698 strains of group A beta-haemolytic streptococci (GAS) isolated from children with streptococcal pyoderma was tested for production of serum opacity factor (OF) and nicotinamide adenine dinucleotide glycohydrolase (NADase). OF was produced by 37% of strains and 40% produced NADase. Classification based on various combinations of OF and NADase reactions showed that 58% belonged to enzyme group el and 34% to e2. Correlation with T and M types showed the possible use of this means of classification as an epidemiological marker for GAS. The specificity of such a system in the further classification of various T types of GAS in epidemiological studies, in the light of antigenic variation among M types, is described.     

Multiplex PCR assay for rapid and accurate capsular typing of group B streptococci

We developed a simple, specific, and sensitive two-multiplex-PCR assay that enabled the detection of all known group B streptococcal (GBS) capsular polysaccharides. This test is well adapted for GBS capsular polysaccharide typing in large-scale epidemiological studies.     

Correlation between growth in antibiotic-medium and hemolytic activity of group C and G streptococci

The effect of a subminimal inhibitory concentration of penicillin on the production of bound and free hemolysins by streptococci was examined using sheep red blood cells. A marked decrease of a group C cell-free and bound activities was observed with penicillin at a concentration of 1/3 of the MIC whereas an increase was observed with those of a group G strain. Potassium ferricyanide and anti-streptolysin O (group A streptococcus) were strongly inhibitory for the free activities of both strains. The cell-bound activities were stimulated by addition of RNA during bacterial growth in control cultures and also in drug-containing media.     

Comparison of five selective media for beta-haemolytic streptococci.

Five selective media for beta-haemolytic streptococci were tested and compared with the conventional blood agar plate using 200 throat swabs from children with possible streptococcal pharyngitis. The medium described by Liebermeister and Braveny, which is based on the reduction of nutrients and enhancement of the haemolytic activity of beta-streptococci, was markedly superior to the other selective media containing inhibiting agents.     

Grouping of beta-haemolytic streptococci by agglutination.

Streptococcal grouping sera, diluted and absorbed to remove cross-reactions, were bound to staphylococci and used to group trypsinised beta-haemolytic streptococci by coagglutination. The results compared well with those obtained using the Phadebact streptococcal grouping kit. The same sera bound to staphylococci without prior dilution and absorption could be used to group enzyme extracts of haemolytic streptococci by slide agglutination, the results again comparing favourably with those of the Phadebact kit.     

Restriction endonuclease digest patterns of chromosomal DNA from group B beta-haemolytic streptococci.

Scanning densitometry and computer-assisted numerical analysis were used to examine restriction endonuclease digest patterns (RDPs) of chromosomal DNA from 26 infecting strains and 44 vaginal isolates of group B beta-haemolytic streptococci (GBS). At the 95% similarity level, HindIII RDPs of serotype Ia and III strains clustered into four and three RDP types, respectively. Nine of 10 strains from neonates with early-onset septicaemia belonged to two particular RDP types (Ia-3 and III-3). In contrast, serotype III GBS strains from meningitis cases were not characterised by particular RDP types. Associations between RDPs and certain phenotypic characteristics were also found.     

Rapid species identification of group C streptococci isolated from horses.

Two commercial systems, the API 20S (Analytab Products, Plainview, N.Y.) and the Rapid Strep (API System S.A., Montalieu-Vercieu, France), were evaluated for ease of use and accuracy in the rapid identification of group C streptococci isolated from horses. A total of 85 Streptococcus isolates were tested, including S. equi (67 isolates), S. zooepidemicus (13 isolates), and S. equisimilis (5 isolates). All S. equi and S. zooepidemicus isolates were correctly identified within 24 h by the Rapid Strep system. Specific grouping sera was necessary to distinguish between S. equisimilis and group G or L strains. The API 20S system did not provide species identification of any of these isolates. An identification of randomly selected isolates to species level was performed by conventional methods and confirmed the identification derived through the Rapid Strep system. Our results indicate that the Rapid Strep system is a valuable aid for species identification of equine isolates of group C streptococci.     

Clinical presentation and outcome of bacteraemia caused by beta-haemolytic streptococci serogroup G

There has been a considerable increase in annual cases of bacteraemia caused by beta-haemolytic streptococci serogroup G (GGS) and we therefore undertook a retrospective case review in the Danish County of Northern Jutland. During a 19-year period (1981-1999) 94 cases were identified in a regional bacteraemia register, and the annual incidence rose from 3.0 per 106 inhabitants in 1981-1987 to 7.4 in 1988-1993 and 17.2 in 1994-1999. Clinical charts were available for 92 cases. The male/female ratio was 52/40 and the median age 71 years, range 15-97 years. Twenty-seven patients had reduced functional capacity. Fifty-two cases were community-acquired. The most common site of infection was the skin followed by the lungs and the urogenital tract. Eleven patients had no apparent site of infection. Underlying disease was present in 81 patients, who shared 149 co-morbid conditions, and the overall one-month case fatality rate was 23% (95% confidence limits 15-33%). We conclude that GGS are of increasing importance as a cause of bacteraemic infections and first and foremost affect patients who are aged, debilitated, or suffer from severe or multiple underlying diseases.     

Use of the Streptosec test for grouping beta-haemolytic streptococci.

The Streptosec test, which embodies the coagglutination principle for grouping beta-haemolytic streptococci, was used against 72 streptococci previously grouped by precipitin methods. Only two of the 72 strains failed to react. The test is easy to manipulate, represents a considerable saving in time and effort, and produces results with an acceptable degree of accuracy.     

Extraction and characterization of IgG Fc receptors from group C and group G streptococci

Immunoglobulin G (IgG) Fc receptors (FcRs) were extracted by proteolytic digestion of four strains each of group C and group G streptococci. The solubilized proteins were analyzed in Western blots and multiple IgG-binding bands were obtained. The banding patterns of some of the strains were very similar, but this property was independent of which streptococcal group the strains belonged to. Highly purified FcRs were prepared from one group C and one group G strain. The 13 N-terminal amino acids were determined, and found to be identical, whereas comparison with the sequence of staphylococcal protein A did not reveal any homology. The isolated streptococcal FcRs also appeared closely related antigenically and functionally. Thus, both molecules were capable of inhibiting each others binding to immobilized IgG, and the radiolabelled group G FcR was completely inhibited from binding to IgG by an antibody to the group C FcR. Finally, in a direct binding assay both proteins were capable of reacting to a similar degree with a wide variety of IgGs, thereby demonstrating the great potential of streptococcal FcRs as tools for binding and detection of IgG antibodies.     

Demonstration of specific binding sites for human serum albumin in group C and G streptococci.

A total of 297 bacterial strains belonging to 27 species was tested for quantitative uptake of radiolabeled human serum albumin. Specific binding sites with high affinity for human serum albumin were found exclusively in group C and G streptococci. The albumin binding was found to be a time-dependent, saturable, and displaceable process which obeyed simple kinetic equations. Scatchard analysis revealed that human serum albumin bound to a homogeneous population of receptors with an affinity in the order ot 10(7) liters/mol and that the average bacterial cell carried more than 80,000 binding sites. The albumin receptor is a heat-stable component susceptible to proteolytic digestion. It has a surface localization separate from the receptors for immunolgobulin G, fibrinogen, aggregated beta 2-microglobulin, and haptoglobin. In individual strains, albumin reactivity was also detected independently of these other types of interactions with human proteins.     

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.     

groESL sequence determination,phylogenetic analysis,and species differentiation for viridans group streptococci

The full-length sequences of the groESL genes (also known as cpn10/60) of Streptococcus anginosus, Streptococcus constellatus, Streptococcus gordonii, and Streptococcus sanguis and the near full-length sequence of the groESL genes of Streptococcus intermedius, Streptococcus bovis, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, and Streptococcus salivarius were determined. The lengths of the groES genes from the 10 species listed above ranged from 282 to 288 bp, and the full-length sequences of groEL determined for 4 species (S. anginosus, S. constellatus, S. gordonii, and S. sanguis) revealed that each was 1,623 bp. The intergenic region (spacer) between the groES and groEL genes varies in size (15 to 111 bp) and sequence between species. The variation of the groES sequences among the species tested was greater (62.1 to 95.1% nucleotide sequence identities) than that of the groEL sequences (77.2 to 95.2% nucleotide sequence identities). Phylogenetic analysis of the groES and groEL genes yielded evolutionary trees similar to the tree constructed by use of the 16S rRNA gene. The intraspecies variation of the spacer was minimal for clinical isolates of some species. The groESL sequence data provide an additional parameter for identification of viridans group streptococcal species.