Selection of phage-resistent clones of streptococcus group A and their properties]

In studying the effects induced by virulent phage CAI in the sensitive cultures of streptococcus, group A, a possibility was shown of selection of phage-resistant clones with the altered enzymatic activity. These clones lost their capacity to produce proteinase and retained residual lipoproteinase activity. This evidence correlates with literature data indicating that phage-resistant streptococci served as good producers of M-protein--the main virulence factor. Infection of the culture producing streptokinase with phage CAI with a definite infection multiplicity led to an increase of the enzyme activity in the culture fluid. This process was accompanied by selection of the resistant strains characterized by greater streptokinase production and greater enzyme stability. As suggested, the latter could result from the absence of proteolytic activity in the phage-resistant clone.     

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.     

A mutant of group A streptococcus resistant to high levels of kanamycin exhibits characteristics of penicillin tolerance in vitro

Abstract In studies of penicillin tolerance in group A streptococci, we observed that a mutant of group A streptococcus isolated for high-level resistance to kanamycin exhibited several characteristics of penicillin tolerance: significant disparity between the penicillin MIC and MBC, delayed killing by penicillin concentrations of 16 times the MIC, and survival in areas of superinhibitory penicillin concentrations when strains were transferred from a penicillin-gradient plate to a penicillin-free replicate plate. In contrast, the parent strain of group A streptococcus and its mutant isolated for high-level resistance to streptomycin were nontolerant for penicillin. Moreover, a clinical isolate of group A streptococcus possessing high-level resistance to kanamycin was also found to be tolerant to penicillin. These findings suggest that genetic mechanisms responsible for high-level resistance to kanamycin may be related with the expression of penicillin tolerance of group A streptococci in vitro.     

Effect of filtrates from transformable and nontransformable streptococci on the transformation of streptococci

Perry, Dennis (Northwestern University Medical School, Chicago, Ill.), and Hutton D. Slade. Effects of filtrates from transformable and nontransformable streptococci on the transformation of streptococci. J. Bacteriol. 91:2216-2222. 1966.-The nature of the transformation competence factor from a group H streptococcus was investigated. The activity of competence factor reached a maximum at the time that optimal competence was attained, the maxima of both occurring in the early log phase of growth. The decrease in competence factor was much more gradual than the decrease in number of competent cells. No inhibitor, however, was detected as being responsible for the decrease in either competent cells or competence factor activity. Efforts to induce transformation in other serological groups of streptococci with the use of group H competence factor were unsuccessful. The development of competence in group H when grown in the presence of nontransformable group A strains resulted in a significant increase in the number of transformants. Culture filtrates from early log phase group A cells also caused an increase in the number of transformants from the group H strain. The addition of 10(-4)m ethylenediaminetetraacetic acid to group A (or group H) culture filtrates caused significant increases in the number of transformants. These results thus indicate that group A streptococci, although nontransformable, produce low levels of "competence factor." Late culture filtrates from the group H streptococcus and several strains of group A streptococci possessed deoxyribonuclease-like activity which inhibited the transformation of the group H strain. This activity in the A filtrates, however, was not prevented by group A anti-deoxyribonuclease sera. Instead, these sera also inhibited transformation. Evidence indicates that the lack of transformation of group A streptococci is due to factors other than the production of deoxyribonuclease.     

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.     

Effect of Culture Medium Composition and pH on the Production of M Protein and Proteinase by Group A Streptococci

The effects of pH, yeast extract, and neopeptone on the production of extracellular proteinase and M protein by group A streptococci were studied with a type 1 strain capable of producing both M protein and proteinase. The strain DS 2036-66 grew moderately well in a semisynthetic broth. M protein was produced without adding peptides to the medium. When added to a medium with 1% glucose, yeast extract (0.1%) was found to stimulate both growth and proteinase formation. Limiting the glucose to 0.25% prevented a drop in pH below 6.7 and prevented proteinase formation. Although less growth occurred with limited glucose, M protein of high specific activity was produced with an actual increase in acid-extractable M protein during the stationary phase of growth. When the medium was buffered at pH 7.85 with tris(hydroxymethyl)aminomethane buffer, 0.5% neopeptone prevented proteinase formation. This was true even in the presence of 1% glucose and 0.1% yeast extract, which resulted in a fall in pH to about 4.8 by 48 hr. Growth was greater than in Todd Hewitt broth, but the specific activity of M protein was considerably less than that found in the medium with glucose limited to 0.25%. Neopeptone was found to have little direct action on crude streptococcal proteinase. Instead, the evidence suggested that neopeptone somehow prevents proteinase elaboration. Yeast extract, on the other hand, appears to stimulate proteinase elaboration. To prevent proteinase formation, neopeptone must be added early, during the logarithmic phase of growth or at the start. In contrast, when yeast extract was added as late as 24 hr, it resulted in the elaboration of extracellular proteinase and in the decline of M protein. When 38 M nontypable strains from the diagnostic laboratory were tested for proteinase activity under conditions similar to those used in the diagnostic laboratory, only six produced much proteinase.     

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.     

Interaction of group A streptococci with a culture of HEp-2 cells]

An initial stage of the interaction of the virulent (matt) and avirulent (glossy) strains of group A streptococcus with the human epithelial tissue (Hep-2) was studied. M+ and M- variants of three strains of group A streptococcus belonging to serological types 2 and 4 possessed different biological activity against the Hep-2 epithelial cells in the monolayer. M+-variant actively affected the great majority of the cells of the monolayer, multiplied of their surface and formed microcolonies. M--variant affected only an insignificant number of cells and failed to multiply on them. In difference from M+-streptococci, the activity of M+-variants is explained by their capacity to adhere to the surface of the animal cells irreversibly. This process started immediately and terminated by 1 1/2 hours of the microbial incubation in vitro with the Hep-2 monolayer. Trypsin treatment of M+-streptococci sharply diminished their capacity to adhesion, this apparently being the result of M-protein digestion. The data presented point to the important role played by M-protein in the phenomenon of the streptococci adhesion to the epithelial cells.     

Enrichment Procedure for Use with the Membrane Filter for the Isolation and Enumeration of Fecal Streptococci in Water

By the use of PYC enrichment medium, the recovery of fecal streptococci from river water has been increased more than twofold over that of M-Enterococcus Agar. Of the isolates tested, 94.6% could be classified as enterococci or enterococcus biotypes. This method seems to yield a larger number of strains which would not normally be revealed. Serological typing of atypical streptococcus strains isolated indicates that the majority of these “biotypes” can be placed in the enterococcus group.     

Role for a secreted cysteine proteinase in the establishment of host tissue tropism by group A streptococci

Primary infection of the human host by group A streptococci (GAS) most often involves either the epidermis of the skin or the oropharyngeal mucosa. A humanized in vivo model for impetigo was used to investigate the basis for host tissue tropism among GAS. Disruption of the speB gene (encoding for a secreted cysteine proteinase) led to a loss of virulence for two impetigo-derived strains (M-types 33 and 53), as evidenced by a diminution in tissue damage and a lack of reproductive growth. The level of cysteine proteinase activity in overnight cultures was associated with the extent of gross pathological changes induced by strains displaying varied degrees of virulence in the impetigo model. Moreover, high levels of secreted cysteine proteinase activity correlated with a genetic marker for preferred tissue site of infection at the skin (emm pattern D). The addition of exogenous SpeB to a speB mutant (emm pattern D) or to an avirulent throat-like strain (emm pattern A) led to increased bacterial reproduction at the skin. The data provide both experimental and epidemiological evidence for a critical role of a secreted bacterial protease in promoting host tissue-specific infection.     

Streptococcal L forms and phage. A clinical-epidemiologic study

This study showed that streptococcal L forms could not be isolated from children who were either carriers of group A streptococci or had disease due to this pathogen. It was possible to induce L colony formation in 15 strains of group A. Streptococcal bacteriophages were demonstrated in 20% of group A streptococci isolated from school children who were carriers, but did not have clinical evidence of streptococcal disease, and from 44.9% of children whose physicians considered they had acute streptococcal upper respiratory infections. Lysogeny (bacteriophage) was demonstrated more frequently during 1969-70 when carrier rates were high and from children who had manifest streptococcal disease, suggesting a possible positive relationship between lysogeny, high carrier rates, and infection in the children studied. Lysogeny and erythrogenic toxin production by group A streptococci occurred simultaneously in approximately half of the strains of group A streptococci tested, suggesting that lysogeny is not a sine qua non for erythrogenic toxin production.     

A RAPD-PCR genotyping assay which correlates with serotypes of group B streptococci

AIMS: The purpose of this study was to determine if DNA polymorphisms generated by RAPD-PCR could be used to characterize Group B streptococci (GBS) for epidemiological purposes. METHODS AND RESULTS: 30 unrelated, previously serotyped strains were analysed by RAPD-PCR using two 10-mer primers (5' TGCGAGAGTC 3' and 5' AGAGGGCACA 3'). Both primers generated DNA electropherotype patterns which, on analysis, clustered the isolates within their respective serotypes. A blind test of a further 3 field isolates also defined these strains within their subsequently determined serotypes. The detection of DNA polymorphisms between isolates within a serotype confirmed previous reports of the heterogenous nature of individual GBS serotypes. CONCLUSIONS: The RAPD-PCR is a potentially useful assay for the rapid characterization of neonatal infections associated with group B streptococci. The method appears to be more discriminatory than conventional serological assays. SIGNIFICANCE AND IMPACT OF THE STUDY: The RAPD-PCR assay is faster, more convenient and easier to perform than alternative DNA analytical procedures such as Pulsfield Gel Electrophoresis. We were able to reproduce the same results following re-testing of all isolates some 12 months later which suggests that the assay may be robust enough for use in routine epidemiological investigations.     

Fluorescent-Antibody Techniques for the Identification of Group D Streptococci: Direct Staining Method

Fluorescent-antibody (FA) techniques were employed in an attempt to develop a rapid test for the identification of group D streptococci. Fresh isolates were obtained from sewege and feces of sheep, cattle, horses, rabbits, chickens, geese, and rats. Identification to species were made by the conventional physiological, biochemical, and serological tests. Both whole and disrupted cells of representative strains of each species were used for the preparation of the group D streptococcus vaccine. Globulin fractions of individual and pooled antisera were labeled with fluorescein isothiocyanate, and the resulting conjugates were tested with homologous and heterologous antigens. The specificity of the conjugates and staining was assessed by adsorption and inhibition tests utilizing controls with homologous and heterologous antigens. Employing the direct staining method and individual and pooled conjugates, it was possible to obtain 84 and 85% positive FA reactions, respectively, with group D streptococcal strains. Trypsinization of the smears prior to staining eliminated all FA cross-reactions observed with non-group D streptococci and staphylococci. These findings suggest that the direct staining method will be of value in the rapid identification of group D streptococci.     

Proteinase inhibition, immunoglobulin-binding proteins and a novel antimicrobial principle

Recent work has demonstrated that a tripeptide derivative mimicking the active proteinase-binding site of cystatin C, a human cysteine proteinase inhibitor, can block growth of group A streptococci and replication of herpes simplex virus (HSV). In the case of HSV, intact cystatin C was also found to inhibit replication of the virus. Many streptococcal strains and HSV-infected cells produce immunoglobulin (Ig)-binding proteins, and a possible connection between such proteins and proteolytic activity was indicated by the finding that bacterial Ig-binding proteins also show affinity for proteinase inhibitors. The significance of these various observations is not clear, but available data suggest that proteinases play a role in vital microbial functions (e.g. viral replication) and may be utilized as targets for antimicrobial agents. The results discussed here also indicate that peptide derivatives based on the structure of proteinase inhibitors occurring in nature could be used as such agents.     

Detection of streptococcal mutants presumed to be defective in sugar catabolism.

The tetrazolium method for detection of bacterial mutants defective in sugar catabolism was modified for use with streptococci. The critical factors were (i) the concentration of tetrazolium, which must be titrated to determine the optimum concentration for each species or even strain, and (ii) anaerobic incubation of tetrazolium-containing agar plates. When used with standard mutagenesis protocols, this method yielded lactose-negative mutants of nine streptococcal strains representing six species. A collection of lactose-negative mutants of streptococcus, sanguis Challis was characterized and contained phospho-beta-galactosidase, lactose phosphotransferase, and general phosphotransferase mutants.     

90株链球菌血清学及生化特征分群鉴定的研究

本文报告90株链球菌血清学和生化特征分群鉴定结果。研究结果表明,90株链球菌用血清学分群,属A群的63株,B群3株,C群12株,D群10株,G群2株。采用生化鉴定,对链球菌属的确认有参考价值。对分群鉴定无鉴别意义。我国过去沿用的非血清学推测性分群法,只适用于B、D群和绝大部分A群菌的鉴定,对C群和G群菌无鉴别意义。因此,在链球菌分群鉴定时,要采用血清学方法,对非血清学推测性分群需进一步完善,以提高其鉴定的准确性。     

Detection of streptococcal mutants presumed to be defective in sugar catabolism

The tetrazolium method for detection of bacterial mutants defective in sugar catabolism was modified for use with streptococci. The critical factors were (i) the concentration of tetrazolium, which must be titrated to determine the optimum concentration for each species or even strain, and (ii) anaerobic incubation of tetrazolium-containing agar plates. When used with standard mutagenesis protocols, this method yielded lactose-negative mutants of nine streptococcal strains representing six species. A collection of lactose-negative mutants of streptococcus, sanguis Challis was characterized and contained phospho-beta-galactosidase, lactose phosphotransferase, and general phosphotransferase mutants.     

Intracellular form of streptococcal proteinase: A clue to a novel mechanism of secretion

The intracellular form of streptococcal proteinase has been isolated and compared with its extracellular form. As shown by double-immunodiffusion studies and radiosequence analysis, the intracellular proteinase was identical to that of the extracellular proteinase. However, the unusual mixed disulfide, protein-S-SR, shown to be present in the extracellular proteinase, was missing in the intracellular proteinase. Protease activity is dependent upon the free sulfhydryl group of the proteinase. Thus, the intracellular proteinase was enzymatically active, while the extracellular proteinase requires activation by exposure to a reducing agent. Because this appears to be the only difference between the intracellular and extracellular protease, it is proposed that the modification of the protein-SH to form protein-S-SR is a process that is intimately related to the mechanism of secretion of the proteinase into the culture fluid by streptococci.     

Immunological cross reactions between polysaccharides of Streptococci groups A and L]

Antibodies on sepharose immunosorbents containing A-polysaccharide-sepharose or synthetic beta-N-acetylglucosamine, were isolated from the sera of rabbits immunized with streptococci, group A, by means of affinity chromatography. Antibodies obtained from some sera with both immunosorbents reacted with streptococcus, group A and L polysaccharides. Partial identity of these polysaccharides was revealed by the immunodiffusion test. Absorption of antibodies with polysaccharides, group A and L, showed their different specificities. These antibodies could apparently be directed against the end parts of molecules of streptococcus, group A polysaccharide.     

Role of group A streptococcal IgG Fc-receptor in induction of anti-IgG by immunization in the rabbit

Abstract Previous work has demonstrated that streptococcal IgG Fc-receptors (FcR) may trigger production of anti-IgG after immunization of rabbits with group A streptococci. This effect seemed dependent on in vitro binding of IgG, derived from the growth medium, to the vaccine strains. In the experiments presented here, IgG was eluted from streptococcal strains to be used for immunization of rabbits by 1 M KSCN and washing, a treatment which did not affect the capacity of the strains to bind newly added IgG. Using two IgG FcR-positive group A streptococcal strains (M-types 1 and 22) for intravenous immunization, anti-IgG was found in the sera of 26 out of 28 rabbits, examined 8 weeks after immunization. In contrast, anti-IgG was not induced in 16 rabbits receiving either group A, type T27 or group B, type Ia streptococci both of which lack surface FcR activity. Finally, immunization with purified streptococcal IgG FcR (0.35 mg, given subcutaneously combined with Freund's complete adjuvant and two weeks later intraconjunctivally without adjuvant) also induced anti-IgG. In all rabbits, anti-human rather than anti-rabbit IgG was detected. It is proposed that in vivo interaction between the bacterial FcR and rabbit IgG, resulting in conformation changes in IgG, is a prerequisite for the induction of anti-IgG. Thus, streptococcal triggering of anti-IgG, ascribable to IgG Fc-receptor activity and not requiring presence of foreign IgG, has been demonstrated in the rabbit.