Streptococci Of Serological Group Research Articles

Septicaemia in emerald monitors ( Varanus prasinus Schlegel 1839) caused by Streptococcus agalactiae acquired from mice

The present study was performed to investigate both the identity and the source of the bacteria responsible for a fatal septicaemia observed in a group of three subadult emerald monitors ( Varanus prasinus Schlegel 1839). The emerald monitors were necropsied and examined by light microscopy, including immunohistology, and by electron microscopy. Tissue samples were additionally submitted for bacteriological, virological and parasitological examinations. The virological and parasitological results were noncontributory, whereas the bacteriological investigation resulted in the isolation of gram-positive cocci which were characterized biochemically and serologically and by molecular analysis. The death of the emerald monitors was caused by a partially leukocyte-associated septicaemic infection with streptococci of serological group B of serotype V. Phenotypically and genotypically identical group B streptococci were isolated from the intestine of subadult mice, obtained from the feed used for the monitors. The genotypical characterization included an identical DNA fingerprint of strains of both origins, indicating the epidemiological relation between the feeding mice and the infections of the monitors.

Identification of streptococci isolated from various sources by determination of <i>cfb </i>gene and other CAMP-factor genes

In the present study, the CAMP-factor (cfb) gene of streptococci of serological group B (Streptococcus agalactiae) and the CAMP-factor (cfu) gene of S. uberis could be amplified by polymerase chain reaction. A cfb specific amplicon could be observed for all 128 phenotypically CAMP-positive S. agalactiae, for the phenotypically CAMP-negative S. agalactiae strain 74-360, and for 2 S. difficile reference strains. A cfu specific amplicon could be observed for all 7 phenotypically CAMP-positive S. uberis. Four S. agalactiae strains isolated from 4 cows with mastitis appeared to be phenotypically CAMP-negative and negative in the cfb gene PCR. The CAMP-positive and CAMP-negative isolates, including both S. difficile, could be identified as S. agalactiae by amplification of a S. agalactiae specific part of the V2 region of the 16S rRNA and a species-specific part of the 16S-23S rRNA intergenic spacer region. Amplification of an internal fragment of the cfb gene with a reduced annealing temperature yielded positive reactions not only for CAMP-positive S. agalactiae, but also for phenotypically CAMP-positive S. pyogenes (n = 4), S. canis (n = 28), and S. uberis (n = 7), indicating a close relation of the CAMP genes of these 4 species. The relation could be further demonstrated by sequencing the internal fragment of the CAMP-factor (cfg) gene of S. canis and comparing the sequence with those of S. agalactiae, S. pyogenes, and S. uberis.

Properties of serological group B streptococci of dog, cat and monkey origin.

This study was designed to identify and characterize further Streptococcus agalactiae isolated during routine diagnostics from three diseased dogs and a cat, as well as from the inner organs of a monkey which died on a sepsis with beta-haemolytic streptococci. The cultures could be identified as streptococci of serological group B by cultural, biochemical and serological properties and by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified 16S ribosomal DNA. A further characterization of the isolates by serotyping and by determination of antibiotic resistances revealed a close relationship of these isolates to the human biotype of this species.

Occurrence of Extracellular Hyaluronic Acid and Hyaluronatlyase in Streptococci of Groups A, B, C, and G

Streptococci of serological groups A (GAS), B (GBS), C (GCS) and G (GGS) were examined in vitro using an optimized medium in respect of their ability to produce hyaluronic acid (HA) and hyaluronatlyase (HY). In this study, 614 GAS (including 123 streptococcal toxic shock syndrome strains, STSS), 247 GBS, 225 GCS and 143 GGS were investigated in qualitative and quantitative tests. Only 4% of GAS and 2.7% of GCS were able to express HA. In contrast to GAS, isolates of GCS showed a highly specific HA formation (to 1 g HA/g dry biomass). In all strains of GBS and GGS, not even a single isolate was positive for HA. HY expression was detectable in all four serological groups. In GAS, only 12.5% of strains were positive; the most common types being 22 and 4, whereas in GBS, GCS and GGS, 72.1%, 84% and 85.3% of isolates, respectively, could be reported as positive. The data suggest that the HA capsule only plays a secondary role in infections caused by GAS strains pathogenic for humans.

Comparative studies on streptococci of serological group G isolated from various origins.

The streptococcal cultures used in the present study were isolated from dogs, bovines and humans and could be classified into Lancefield's serological group G. Most of the group G streptococci grew in fluid media as granular sediment with clear supernatant and formed compact colonies in soft agar. The majority of the group G streptococci from dogs and bovines displayed CAMP-like synergistic haemolytic activities on sheep blood agar, fermented lactose and salicin and produced the enzyme alpha-D-galactosidase. The group G streptococci from humans mainly fermented trehalose and produced the enzyme beta-D-glucuronidase. In addition, some of the group G streptococci reacted with type antigen X and R and two cultures with M6 specific antiserum. A positive opacity factor reaction could be observed with few group G streptococci isolated from dogs and bovines, but not with those from humans. In binding studies with 125I-labelled plasma proteins most of the cultures interacted with 125I-immunoglobulin G and 125I-albumin. Binding of 125I-IgG was more pronounced among group G streptococci isolated from humans. The determination of antibiotic susceptibility revealed that most of the group G streptococci were susceptible to bacitracin, cefoxitin, clindamycin, erythromycin, gentamicin, penicillin and sulfamethoxazole-trimethoprim. Some of the cultures were resistant to minocycline, neomycin and tetracycline. All this data clearly distinguished group G streptococci isolated from animals and humans and could additionally be used for individual characterization of this microorganism. This might be useful in epidemiological aspects and contribute to understanding infections caused by these bacteria.

Adherence of haemagglutinating streptococci of serological group B to alveolar macrophages of rabbits.

In this study, selected haemagglutination-positive and haemagglutination-negative streptococci of serological group B were investigated in HeLa-cell- and alveolar-macrophage adherence tests. The cultures, isolated from bovine milk samples, had been previously serotyped and further characterized. All haemagglutination-positive group B streptococci adhered in high numbers to HeLa cells and alveolar-macrophages of rabbits. Haemagglutination-negative group B streptococci showed no comparable adherence. The adherence to alveolar-macrophages of rabbits was further characterized. This adherence appeared to be time- and temperature-dependent and could be inhibited by heat or proteolytic treatment of the bacteria. After opsonization of the bacteria, an adherence to alveolar-macrophages could also be observed for haemagglutination-negative group B streptococci, indicating a separate adherence mechanism. The group B streptococcal adherence might represent an important prerequisite for invasion and possibly intracellular survival of this bacterial species.

Phase variation in streptococci of serological group B

Encapsulation is thought to be a critical virulence factor in streptococci of serological group B. In the present study two encapsulated low-density variants could be separated from their unencapsulated original strains by Percoll gradient centrifugation. The original strains had been isolated from human endocarditis and bovine mastitis. Type antigen preparations of the encapsulated human and bovine group B streptococcus reacted with type III- and type IV-specific antiserum, respectively. No comparable reactions could be observed with their unencapsulated parent strains. In contrast to the original strains, the encapsulated variants grew with uniform turbidity in fluid medium and formed diffuse colonies in soft agar. The original strains grew as granular sediment and formed compact colonies in soft agar. In addition, the original strains appeared to have a more hydrophobic surface and showed significantly greater adherence to epithelial cells. In contrast to the nonencapsulated parent strains, the encapsulated variants were less phagocytosed by polymorphonuclear leukocytes. These findings may help our understanding of the pathogenic importance of phase variants in infections with this bacterial organism.

Relationship between group B streptococcal serotypes and cell surface hydrophobicity.

Cell surface hydrophobicities of streptococci of serological group B were determined by the adherence of the bacteria to hexadecane droplets. A significant adherence to hexadecane was observed with the group B streptococcal type reference strains Ib, V, Ic, R and X, but not with those of serotype Ia, II, III and IV. Cultivation of the bacteria in microcapsule-inducing media reduced the hexadecane adherence properties. The adherence to hexadecane was not related to fibrinogen binding properties of the cultures. Screening a large number of group B streptococci isolated from humans and bovines revealed that those with polysaccharide type antigen alone were generally hydrophilic, those with protein antigen alone or with protein antigen in combination with polysaccharide antigen were mostly hydrophobic. Cultivation of the bacteria under microaerobic conditions or after a single mouse passage enhanced microcapsule production and correspondingly reduced the hexadecane adherence values. Treatment of the bacteria by guanidinium chloride or by neuraminidase enhanced the hexadecane adherence. The hydrophobic component on group B streptococcal surface appeared to be partly inactivated by heat or proteolytic treatment of the bacteria.

Antibiotic Resistance Patterns and Pigment Production of Streptococci of Serological Group B Isolated from Bovines and Humans

All 127 streptococci of serological group B isolated from bovines and humans were resistant to bacitracin (0.04 U) and gentamicin and susceptible to penicillin. Of the bovine isolates examined 10%, 8%, 22%, 2% and 30% were resistant to bacitracin (10 U), cefoxitin, clindamycin, erythromycin and tetracycline respectively. Of the human isolates 52%, 21%, 38%, 3% and 65% were resistant to bacitracin (10 U), cefoxitin, clindamycin, erythromycin and tetracycline respectively. Most of the bovine strains were susceptible, most of the human strains were resistant to bacitracin and tetracycline. A relation of tetracycline resistance to group B streptococcal microcapsule was proposed. Pigmentation studies revealed generally a weak pigmentation of bovine and a strong pigmentation of human group B streptococci. Antibiotic resistance patterns together with the ability to form a yellow to orange-red pigment might be used for characterization of individual cultures of group B streptococci.

Characterization of Haptoglobin-Binding Properties of Streptococci of Serological Group G

Two group G streptococcal cultures (G 10187, G 11122) with surface antigen T4 possess surface receptors for human haptoglobin (Hp). G 10187 additionally interacted with immunoglobulin G and albumin, G 11122 with fibrinogen and fibronectin. Binding of 125I-Hp 2-1 was time-dependent, saturable, reversible in the presence of unlabelled Hp and could be inhibited by unlabelled human-Hp 2-1, -Hp 2-2, -Hp 1-1, Hp-hemoglobin complexes and by Hp preparations from pigs, horses and rabbits. The Hp binding sites could be destroyed by heat treatment (95 degrees C) and by proteolytic treatment of the bacteria. Hp binding sites were solubilized from group G streptococcal surface by heat treatment of the bacteria at acid pH and subsequently isolated by affinity chromatography on Hp 2-1 sepharose. SDS-PAGE and Western blotting of the Hp binding proteins revealed numerous protein bands with 125I-Hp 2-1 binding activity. Specific antibodies against G streptococcal binding proteins prepared in chickens inhibited binding of 125I-Hp to group G and group A streptococci, but not to Actinomyces pyogenes.

Distribution of B Streptococcal Type Antigens Among Streptococci of Serological Groups B, G and L

Serotyping of streptococci of serological group B isolated from humans revealed mainly the type patterns Ia/c and III/R. The protein antigen c components were c alpha beta, c alpha and less frequently, c beta. Extracts of two group B streptococcal cultures reacted with specific antiserum against type candidate 7271. Bovine group B streptococci mainly expressed the type antigens IV/X and NT/X. Protein antigen c occurred as c alpha beta, c alpha and less frequently as c beta component. Among the non group B streptococcal cultures used in this study, only those of serological groups G and L reacted with specific antisera against protein antigens R and X.

Interaction of Type II IgG Fc-Receptors from Streptococci of Serological Group A with Murine IgG

A group A streptococcal strain demonstrated binding activities for 125I-murine IgG3 and 125I-human IgG. This 125I-murine IgG3 binding could be inhibited by unlabelled equine IgG but not by IgG from cattle, chickens and dogs, indicating binding properties of IgG Fc-receptors of type II. In contrast to binding sites of Streptococcus dysgalactiae (serogroup C) carrying type III IgG Fc-receptors, the binding sites of the group A streptococcus appeared to be antigenically different, extremely sensitive to trypsin and did not show any cross reactions with human albumin. The group A and group C streptococcal binding sites could be solubilized by lysozyme treatment of the bacteria and subsequently isolated by affinity chromatography on human IgG-Sepharose. Further analysis of the group A and group C streptococcal binding proteins by SDS-PAGE and Western blotting revealed numerous, almost identical protein bands with binding activities for 125I-murine IgG3.

Further characterization of haptoglobin binding to streptococci of serological group A

Certain group A streptococci with surface antigen T 4 possess surface receptors for human haptoglobin (Hp). Binding of 125I Hp 2-1 to two representative group A streptococcal cultures could be inhibited by unlabelled Hp 2-1, Hp 2-2 and Hp 1-1 but not by the alpha 1, alpha 2 or beta chains of Hp. Hp complexes formed with equine hemoglobin and asialo-Hp also reduced 125I-Hp 2-1 binding to group A streptococci. Hp binding proteins could be solubilized from streptococcal surface by hot acid treatment of the bacteria and purified by subsequent affinity chromatography on human Hp 2-1 sepharose. The isolated Hp binding proteins specifically inhibited 125I-Hp 2-1 binding to group A streptococci and retained their 125I-Hp 2-1 binding activity in a dot binding assay on nitrocellulose membranes. SDS-PAGE and protein blots of Hp binding proteins developed with 125I-labeled Hp 2-1 revealed numerous high molecular weight proteins with 125I-Hp 2-1 binding activity.

Interactions of streptococci with human colostral immunoglobulin A

Streptococci of serological groups A, B, C, G and L were examined for interactions with human colostral IgA. Of 28 A-streptococcal cultures, 12 bound IgA with a mean of 38.7%. The other streptococci had little or no IgA-binding activities. Of the 12 IgA-binding A-streptococcal cultures, 8 contained the M-protein M 4 and 2 the M-protein M 60. The specific binding sites for IgA were heat-sensitive (60 min, 80°C) and susceptible to trypsin and pronase.

Bindings of plasma proteins to streptococci of serological group L with special reference to their immunoglobulin G Fc-receptor activity

Of 33 streptococcal cultures belonging to serological group L, all bound human immunoglobulin (Ig) G, fibrinogen, and fibronectin; 32 bound bovine IgG; 31 bound alpha 2-macroglobulin; 5 bound albumin; and none bound either haptoglobin or IgA. The binding sites for IgG could be isolated from the L streptococci by trypsinization and purified by affinity chromatography on human IgG-Sepharose. The purified Fc receptors reacted with IgG subclasses 1, 2, 3, 4 of humans, 1 and 2 of bovines, ovines, and caprines as well as a, b, c, and T of equines. They had a molecular mass of approximately 49,000 Da. Thus, the Fc receptors from L streptococci corresponded to type III Fc receptors of Streptococcus dysgalactiae.

Streptococcal group B type antigen X in group L streptococci.

Extracts from 19 of 29 group L streptococcal cultures reacted distinctly with antiserum against group B streptococcal type antigen X in coagglutination and immunodiffusion tests. The X antigen corresponded to those obtained by streptococci of serological groups B and G.

Isolation and properties of a novel IgG-binding protein from streptococci of serological group U.

A nonimmune binding of immunoglobulin (Ig) G has been detected in streptococci of group U. The group U Fc-binding site differed from the five previously known types of staphylococcal and streptococcal Fc-binding sites by its strong affinity for murine IgG, with dissociation constants in nanomolar range for rat and mouse IgG, as well as for mouse IgG subclasses 1, 2a, 2b and 3. It also differed from other binding sites by the high sensitivity towards trypsin. The Fc-binding protein could be solubilized from the streptococci of group U with papain and purified by gel filtration on sephacryl S-200 and by subsequent affinity chromatography on human IgG-Sepharose. The purified binding protein was homogeneous on SDS-polyacrylamide gel electrophoresis and had a molecular weight of approximately 58,000 daltons. It retained its binding activities for murine IgG subclasses as revealed by western blotting. Coupled to CNBr-activated sepharose, the purified Fc-binding protein could be effectively used for the isolation of murine IgG subclasses by affinity chromatography.

β-D-galactosidase activity in streptococci of serological group B

Group B streptococci isolated from humans differed significantly in beta-D-galactosidase-activity from those of bovine mastitis. This could be demonstrated in a relatively simple and rapid test using a fluorogenic 4-methylumbelliferyl-beta-D-galactoside conjugate. Only 10 (12%) of 82 group B streptococcal cultures from human produced beta-D-galactosidase. On the other hand, 74 (96%) of 77 "bovine" cultures formed this enzyme. Thus, beta-D-galactosidase activity could be used as an additional marker for the differentiation between group B streptococci of human and bovine origin.

Interactions of plasma proteins with group A, B, C and G streptococci

Streptococci of serological groups A, B, C and G displayed different binding activities for plasma proteins. Most of the streptococci studied, except those of group B, bound immunoglobulin G. All streptococci reacted with fibrinogen and, except those of group B, with fibronectin. The majority of streptococci, but none of group B, had an affinity for α2-macroglobulin. Albumin was bound by all cultures of group G and a few of group C. Haptoglobulin interacted with only 1 group A culture. None of the streptococci bound transferrin. The specificity of binding sites for 125I-labelled plasma proteins was revealed in a series of inhibition experiments with the unlabelled proteins. The binding sites on streptococci of group G showed different sensitivities to trypsin and pepsin. Reactivities for immunoglobulin G, however, remained unaffected after treatments of the streptococci with trypsin. Exposure to heat (30 min, 80°C) partially inactivated binding activities for the plasma proteins. Sodium dodecyl-sulphate and acetylimidazole strongly reduced binding of albumin and to a lesser extent that of α 2-macroglobulin. They had no or little effect on the interaction with the other plasma proteins. Dioxane decreased almost all binding activities. Ethanol partially diminished the binding of immunoglobulin G, fibrinogen, fibronectin and α 2-macroglobulin. Treatments of group G streptococci with guanidine, urea, formamide or methanol-HCl did not affect their plasma protein binding activities.

Taxonomic Studies on Streptococci of Serological Groups C, G and L and Possibly Related Taxa

Summary The taxonomic relationships among selected species of ‘pyogenic’ and ‘viridans’ streptococci were examined using DNA-DNA hybridization, DNA base composition and biochemical tests. The results of the present study indicate that Streptococcus dysgalactiae, S. equisimilis and streptococci of Lancefield serological groups C, G and L are a single species. Strains of S. equi and “S. zooepidemicus” were found to be closely related on the basis of DNA-DNA hybridizations. It is suggested that “S. zooepidemicus” be reclassified as S. equi subsp. zooepidemicus nom. rev., comb. nov. and an emended description of the species S. dysgalactiae is given.