Antigenic characteristics of the L forms of Streptococcus group B

Stable L-forms of group B streptococci (GBS) have been obtained and their antigenic features have been studied by the serological methods (the passive hemagglutination test, the aggregate agglutination test, the gel diffusion test), as well as by using ferritin and peroxidase labels with the subsequent electron microscopy. The use of the serological methods has made it possible to reveal the antigenic differences between the stable L-forms of GBS and their bacterial forms. Specific antigenic substances can be found in the supernatant fluid obtained after the sedimentation of the ultrasonically disintegrated cellular mass of streptococcal L-forms and bacterial cultures. The use of ferritin and peroxidase labels has revealed the specificity of GBS L-form antigen and its localization on the cytoplasmic membrane of all L-form structural elements.     

Microbial associations in chronic respiratory tract diseases

In the peripheral blood of patients with chronic bronchitis the presence of Mycobacterium pneumoniae antigens has been registered in 20.0% of cases and the presence of group A Streptococcus haemolyticus antigens, in 24.0% of cases, the transformation of streptococci into the L-form being observed in 12.0% of cases. The presence of M. pneumoniae, streptococci and their L-forms, as well as associations of these microorganisms, is characteristic of patients with chronic obstructive bronchitis, which is, probably, one of the reasons for the maintenance of bronchial obstruction. In patients with chronic pneumonia and fibrosing alveolitis the antigens of the bacterial form of streptococcus and its L-forms have been detected only in a few cases.     

Identification with the aid of various serological reactions of L forms of streptococci isolated from the live organism]

Growth inhibition, agglutination, precipitation, and passive hemagglutination tests were used for the identification of the L-forms of streptococci isolated from the organism of experimental rabbits both after the infection with the L-forms of streptococci and with the streptococci of group A. The tests were positive not only with the antiserum of homologous, but also of heterologous strains of the L-form of streptococcus, group A. The L-form cultures isolated from the experimental animals failed to differ from the laboratory strain of the L-forms of streptococcus, group A, by serological properties.     

Ultrastructure of the L forms of Streptococcus group B

The submicroscopic organization of the L-forms of beta-hemolytic streptococci of group B has been studied in the course of their cultivation. The L-forms of group B streptococci differ from those of group A streptococci by a higher growth rate. On the submicroscopic level, the activity of ATP-ase has been revealed on the internal side of the cytoplasmic membrane. Regularities in the localization of intramembranous particles sized 6-18 nm in the hydrophobic area of the membrane have been established by means of freezing-etching. With the adequate methods of fixation, the continuous three-layer structure of the cytoplasmic membrane can be determined in all elements of the L-form population.     

The role of non-type-specific protein antigens of the cell wall in Streptococcus group A in developing delayed hypersensitivity

The role of the type-nonspecific (TNS) cell-wall antigens of group A streptococci has been determined. The study has been made on guinea pigs sensitized with whole microbial cells or HCl extracts containing TNS antigens. To determine delayed hypersensitivity, the in vitro cytotoxic test on adhering lymph-node cells in the autologous system has been used. The study has shown that sensitization with group A streptococci of different types or with TNS antigens induces the development of delayed hypersensitivity to TNS antigens (or antigen), common for different types of group A streptococci, but specific for this group. HCl extracts containing TNS antigens can be recommended as the preparation for testing delayed hypersensitivity to antigens, specific for group A streptococci.     

Isolation of non-type-specific protein antigens of Streptococcus group B

Hydrochloride extracts obtained from group B streptococcal strains of different serotypes have proved to be the source of type-nonspecific protein antigens, precipitated with ethanol and studied by gel chromatography and spectrophotometric scanning in ultraviolet rays. Thus, 2 or 3 antigens, one of them found to be common for streptococci of groups A, B and G, as well as the admixture of group-specific polysaccharide, have been detected. In extracts obtained from group B streptococcal strains of different serotypes a common protein antigen, specific only for group B, has been detected. The suitability of gel chromatography with the use Toyopearl gel HW-55F for the preparative isolation of the specific fraction of protein type-nonspecific antigen with a view to the subsequent study of immune response to group B streptococci has been shown.     

Determination of antibodies to Streptococcus group A polysaccharide by an immunodiffusion method in human sera in various pathological processes of streptococcal etiology

A method for the assay of antibodies to the specific antigenic determinant of group A streptococcal polysaccharide (A-polysaccharide) in human sera was developed. The sera were tested in the precipitation test in agar gel with different doses of A-polysaccharide. The presence of a high level of the above-mentioned antibodies is indicative of infection caused by group A streptococcus, but not streptococci of other groups or by the L-forms of streptococci. In 87.5% of patients with primary rheumatism a high level of antibodies to the specific antigenic determinant of A-polysaccharide was detected during the first day of the disease, which confirms most convincingly the etiological role of group A streptococcus in rheumatism. Considerable differences in the level of antibodies to A-polysaccharide in the active and non-active phases of rheumatism have been established, which makes it possible to use the presence of a high level of these antibodies as an indicator of the rheumatic process activity. A considerable percentage of sera with a high level of antibodies to A-polysaccharide was also detected in erysipelas and acute glomerulonephritis patients.     

Localization of group A streptococcal nontype-specific antigens and their occurrence among streptococci of different groups]

It was shown by immunodiffusion methods that nontypespecific antigens revealed in the HCl extracts of streptococcus, group A, were localized in the cell wall. In B, E, H, K, L, M, P, S, T streptococci groups there was revealed only one, and in C and G streptococci groups--two antigens identical to the HTC antigens of streptococci, group A. Besides, an antigen, which was apparently specific specific for group A streptococcus only, was detected. The data obtained should be taken into consideration in the elaboration of improved method of grouping and typing group A streptococcus.     

Role of hydrophobic surface proteins in mediating adherence of group B streptococci to epithelial cells.

Determination of the cell-surface hydrophobicity of group B streptococci by hydrophobic interaction chromatography on phenyl-Sepharose revealed that human and bovine group B streptococcal isolates with protein surface antigens, either alone or in combination with polysaccharide antigens, were mainly hydrophobic, whereas those with polysaccharide antigens alone were mainly hydrophilic. Removal of capsular neuraminic acid enhanced, and pronase treatment reduced, surface hydrophobicity. The hydrophobic surface proteins, solubilized by mutanolysin treatment of the bacteria and isolated by hydrophobic interaction chromatography, appeared in SDS-PAGE as numerous protein bands. Staphylococcal carrier cells loaded with antibodies produced against hydrophobic surface proteins agglutinated specifically with hydrophobic group B streptococci. No agglutination reaction was observed with hydrophilic cultures. Hydrophobic group B streptococci adhered to buccal epithelial cells in significantly higher numbers than did hydrophilic cultures. The adherence of group B streptococci to epithelial cells was inhibited in the presence of isolated hydrophobic proteins and in the presence of specific antibodies produced against hydrophobic proteins. The results of this study demonstrate a close relation between the occurrence of type-specific antigens, surface hydrophobicity and the adherence of group B streptococci to epithelial cells.     

The complex serologic diagnosis of typhoid fever

A parallel serological study of the blood sera of typhoid patients has been made by the methods of countercurrent immunoelectrophoresis and the indirect hemagglutination test with a view to establish the presence of soluble typhoid antigens and their corresponding antibodies. As shown in this study, the occurrence of Salmonella typhi O- and Vi-antigens is essentially higher than the content of specific antibodies in diagnostically significant titers.     

Electron microscopic study of the interrelation of the L forms of beta-hemolytic Streptococcus group A with mouse peritoneal macrophages

Interrelations between the L-forms of group A beta-hemolytic streptococci and mouse peritoneal macrophages have been studied by electron microscopy. The macrophages have been shown to actively phagocytize L-form cells in great amounts. Most of the phagocytized L-forms are destroyed and become nonviable, but a few of them survive as elementary bodies within 48 hours.     

L forms of Streptococcus in the cultures of blood from patients with rheumatic fever in the active and inactive phases

It was shown that L-forms of streptococci were constantly isolated by means of the method used by the authors from patients with rheumatism, irrespective of the activity and the course of the disease. The data obtained pointed to the presence of septicemia caused by the L-forms of streptococci in patients with rheumatism both at the active and inactive phase. In this connection it should be noted that modern clinical criteria of the activity of rheumatic process are unreliable.     

An ELISA for the detection of Bacillus subtilis L-form bacteria confirms their symbiosis in strawberry

AIMS: To develop an ELISA for the detection of antigens derived from stable Bacillus subtilis L-form bacteria and to detect these in plants injected with L-form bacteria. METHODS AND RESULTS: A sandwich ELISA was developed and its specificity was investigated using L-forms and cell-walled forms of B. subtilis, different Bacillus species and a range of bacteria isolated from glasshouse-grown strawberry plants. The detection limits of the ELISA were approximately 10(3) viable cells ml(-1) for L-forms compared with 10(7) viable cells ml(-1) for cell-walled forms. Results showed that L-forms survived and moved within strawberry tissues injected with L-form bacteria. CONCLUSION: An ELISA that selectively detects B. subtilis L-form bacteria was developed and shown to confirm the presence of L-forms in plants. SIGNIFICANCE AND IMPACT OF THE STUDY: This will be a valuable rapid method to further studies on L-form plant interactions.     

The suppression of delayed hypersensitivity to BCG antigens in BALB/c mice during the production of antibodies to the rhamnose determinants of Streptococcus group A polysaccharide]

The data obtained for the first time in our studies indicate that the production of antibodies to group A streptococcal polysaccharide (A-PS), one of the cross-reacting streptococcal antigens, may suppress delayed hypersensitivity (DH) to microbial antigens. The existence of sharply pronounced correlation between the suppression of DH and the presence of antibodies to the rhamnose area of A-PS in the blood of BALB/c mice immunized with the pepsin-treated culture of group A streptococci has been shown. The suppression of DH is absent in the immunized animals of the same group whose blood contains antibodies to the determinant, specific for A-PS. As revealed in this study, the effect of the suppression of antigen-specific cytotoxicity linked with DH to BCG antigens can be reproduced by mixing lymph node cells taken from these two groups of the animals. The data thus obtained are possibly linked with the activation of nonspecific T suppressors in the production of antibodies to the rhamnose determinants of A-PS in the animals immunized with streptococci. The mechanism of the newly discovered phenomenon is discussed.     

Monoclonal antibodies against the common polysaccharide ofStreptococcus agalactiœ

A monoclonal antibody giving a dominant reaction with the group-specific polysaccharide of streptococcus group B in an ELISA test has been developed. The purified polysaccharide exhibited a high positivity with reference anti B streptococcal antiserum in the ELISA test. Cross-tests of antibodies with other groups of streptococci provided a minimum cross-reaction only in the case of G streptococci. Monoclonal antibodies were prepared usingStreptococcus agalactiœ S 589 MT strain isolated from a case of bovine mastitis which does not express Ia, Ib, II, III, IV and V type antigens, nor C, R and X protein antigens.     

Protective properties of certain external proteins of group B streptococci

On the basis of genes, which control synthesis of externally localized proteins of group B streptococci (bac and scaAB), recombinant polypeptides P6 and ScaAB were obtained. Data on protective activity of these polypeptides during experimental infection of immunized mice as well as in opsonophagocytic test on cultivated peritoneal macrophages are presented. It has been shown that protective effect of specific antibodies to P6 was dependent from intensity of immune response. Titer of specific IgG to P6 equal 1:25000 was protective for mice during challenge with LD50. During sublethal challenge level of humoral immunity determined both rate of microorganism elimination and degree of decrease of concentration of streptococci in the spleen. Recombinant polypeptide ScaAB also had marked protective activity and protective titer ScaAB-specific IgG was significantly lower compared with the first polypeptide (1:1600). It has been established that both types of antibodies have opsonizing activity against different strains of group B streptococci. Opsonizing properties of antibodies to P6 were restricted to Bac protein-producing streptococci whereas specificity of antibodies to ScaAB was not restricted by type and group borders. Opsonization of both group B and group A streptococci was revealed. It has been established that protective efficacy mediated by antibodies was dependent not only from their opsonizing characteristics but also from availability of protein antigens, which under certain conditions can be shielded by capsular polysaccharide. It has been assumed that vaccine preparation developed on the basis of polypeptides P6 and ScaAB is promising for further research.     

伤寒杆菌和甲型副伤寒杆菌的稳定L型外膜蛋白和染色体DNA酶切研究

非高渗培养基传代培养的伤寒杆菌和甲型副伤寒杆菌的稳定L型丧失了主要外膜蛋白、特异性表面抗原和染色体DNA部分片段,保留了沙门氏菌共同的内部抗原和形成L型独特的表面抗原。提示伤寒杆菌和甲型副伤寒杆菌的稳定L型多种特性的丧失同其基因或主要外膜蛋白的缺失有关。     

In vitro binding ofBifidobacterium bifidum DSM 20082 to mucosal glycoproteins and hemagglutinating activity

Adhesive properties ofBifidobacterium bifidum strain DSM 20082 were studied by the hemagglutination test and an enzyme-linked immunosorbent assay (ELISA).B. bifidum caused agglutination of human A, B, and O erythrocytes and rabbit erythrocytes, but the interactions were not specific of blood group antigens. The hemagglutination was inhibited by porcine gastric mucin and rat intestinal and colonic mucin.B. bidifum was shown to adhere to different immobilized mucosal glycoproteins and to glycophorin A, a specific erythrocyte membrane glycoprotein. The data obtained with many glycosylated components indicated thatB. bifidum receptors involved in the hemagglutination test were not the same as those that adhere to mucus glycoproteins. The results suggest that the mucosal preparations contain receptors for specific bacterial adhesins, but their structures remain to be determined.     

Cross-reacting antigens of human thymus myoid cells and stable L-forms of group A streptococci

Indirect immunofluorescence has shown a similarity between the antigen components of group A streptococcus L-forms and human thymus myoid cells. An analogous antigen (or antigens) is present in the cytoplasmic membrane of human myocardial cell fibers. The depletion of antiserum to the streptococcal L-forms both by the culture of L-forms grown in meat or casein media and by the homogenate of the cardiac muscle leads to the inhibition of immunofluorescence. The depletion of serum by the homogenate of other tissues (liver) or by L-form culture does not virtually affect the immunofluorescence intensity. According to the authors' opinion, the similarity of antigens of group A streptococcus L-forms to the antigenic components of organ tissues is likely to be responsible for long-term persistence of the microorganisms under consideration and to favour, in some cases, the occurrence of autoantibodies. The latter circumstance might lead to pathological changes in organs containing cross-reacting antigens.     

Reversion to the Streptococcal State of Enterococcal Protoplasts, Spheroplasts, and L-Forms

A method is described for predetermining whether lysozyme-damaged enterococci grow as either the parent strain or as L-forms. Organisms treated with lysozyme grew as L-forms on media solidified with low concentrations of agar, or the damaged cells grew as streptococci on media solidified with high concentrations of gelatin. After induction, some of the L-forms reverted to the parent strain, but most did not during three routine subcultures. Continued spontaneous reversion occurred through approximately 30 subcultures after induction. However, subsequent progeny did not revert, even when subjected to conditions such as the gelatin medium which strongly favors growth in the streptococcal phase.