Streptococcal M protein size mutants occur at high frequency within a single strain.

Streptococcal M protein, the antiphagocytic molecule on the surface of the organism, was previously found to exhibit extensive size heterogeneity between as well as within M serotypes. In this study, methods were devised to isolate M protein size mutants within a laboratory-grown culture. We were able to isolate three independent M protein deletion mutants and one additional mutant, which was derived from the first deletion mutant. We found that these deletion mutants occur at a frequency of approximately 1 in 2 X 10(3) CFUs in culture. Functional studies revealed that the deletion mutants were able to survive as well as the parental strain in human blood. They also had the determinants necessary to absorb opsonic antibodies as well as the parent. Pepsin digestion experiments localized the deletions within the N-terminal half of the M molecule, which is distal to the cell wall surface. This is the region of the molecule in which extensive sequence repeats are found. This is consistent with the suggestion that the size changes may be the result of homologous recombination between the repeat regions in the gene. These results support the idea that strains showing M protein size variation within successive clinical isolates from single patients may be derived from the initial infecting organisms, and are not the result of separate unrelated acquisitions of the same serotype. This size change may be important in the survival of the streptococcus in vivo.

Immunochemical studies on the group and type antigens of group F streptococci and the identification of a grouplike carbohydrate in a type II strain with an undesignated group antigen.

Two antigens, the group-specific carbohydrate and the Type II carbohydrate, have been isolated by cellulose column chromatography from a formamide extract of a Group F streptococcus. Chemical and immunologic analyses indicate that both antigens are free of other cellular components. Both antigens are components of the cell wall although the Type II antigen is probably more superficial than the group antigen. The Type II antigen is composed of rhamnose, glucose, galactose, and galactosamine. The Group F antigen is composed of rhamnose, glucose, galactosamine and a small percentage of glucosamine. A grouplike carbohydrate and the Type II carbohydrate have been isolated from a streptotoccal strain which lacks a serologically detectable streptococcal group antigen. This grouplike carbohydrate, which does not cross-react immunologically with Group F serum, is composed of rhamnose, galactose, and glucosamine. No chemical or immunological differences were observed between the Type II antigen isolated from the Group F strain and the Type II antigen isolated from the nongroupable strain.

The individual antigenic specificity of antibodies to streptococcal carbohydrates.

Although a single electrophoretically uniform antibody component with specificity for the group carbohydrate may comprise the bulk of the gamma-globulin in rabbits immunized with streptococcal vaccines, this is not always the case. Not infrequently, electrophoresis may reveal multiple antibody components. Nevertheless, it has been feasible by various preparative procedures to isolate from a single antiserum at least two antibody components with similar reactivity for the carbohydrate both of which are electrophoretically monodisperse. Light chains from such antibodies reveal a restricted pattern when examined by disc electrophoresis. Antibodies to streptococcal carbohydrates have been examined for their individual antigenic specificity. Goats were immunized with isolated Group C and Group A-variant antibodies raised in rabbits. Individual antigenic specificity of these antibodies was brought out by absorption of the goat anti-antiserum with Fr II of pooled normal rabbit sera. Additional absorption of the goat anti-antisera with Fr II diminished but did not eliminate the reactivity for the homologous antibody. Immunoelectrophoretic studies with papain fragments of purified streptococcal antibodies localized the specificity to the Fab fragment. Specificity was not confined to the isolated light chains of the antibody.

Immunochemical studies on the cross-reactivity between streptococcal and staphylococcal mucopeptide.

Particulate mucopeptides of Group A-variant streptococci and Staphylococcus aureus, solubilized by ultrasonic treatment, give a precipitin reaction with the sera of rabbits immunized with Group A-variant streptococci. gamma-G globulin antibodies have been recovered from these sera which react with the mucopeptides but not with the Group A-variant carbohydrate. The immunochemical basis for the cross-reactivity between the streptococcal and staphylococcal mucopeptides was investigated in detail. Three chemically different fractions have been isolated from enzymatic digests of staphylococcal mucopeptide and were employed as haptenic inhibitors of the precipitin reaction. A fraction consisting of the peptide moiety of mucopeptide was the strongest inhibitor, whereas the hexosamine-rich fraction was less effective. The third fraction, rich in glycine, was least effective. It is suggested that the immunologic cross-reactivity between streptococcal and staphylococcal mucopeptide is due to the fact that these two substances contain chemically similar tetrapeptides. The hexosamine polymer which is identical for both mucopeptides may also contribute to their cross-reactivity.

Small intestinal mucosal cell proliferation and bacterial flora in the conventionalization of the germfree mouse.

The relationship between intestinal colonization and the small bowel mucosal cellular proliferation rate during conventionalization of the germfree mouse was examined. 16 mice were maintained under standard germfree conditions, and 54 others were conventionalized. Migration of the small bowel epithelial cells was followed by radioautography with administration of tritiated thymidine. Colonization was followed by qualitative and quantitative bacteriological fecal analyses. The percentages of the villi labeled (as determined by cell count) 24, 48, and 72 hr following thymidine administration showed immediate progression in the conventionalized animals from the germfree villus migration time (4 days) toward the conventional villus migration time (2 days). The epithelial migration rate of animals conventionalized for 8 days was comparable to that of conventional animals. After conventionalization, aerobic and anaerobic organisms undergo a period of extensive multiplication; however, 72 hr later the number of these microorganisms cultured in the stool decrease and are similar to those recovered from normal animals. Coliforms and streptococci are recovered in large numbers only in the first days after conventionalization, while the Bacteroides are first recovered in significant numbers on the fifth day of conventionalization. Except for smaller numbers of Bacteroides, the bacterial populations in the stools of the conventionalized animals are qualitatively and quantitatively similar by the eighth day of conventionalization to those of true conventional mice. Adaptive balance between cell proliferation and sloughing, and thus migration rate, begins immediately after conventionalization of germfree animals as bacterial populations establish themselves throughout the gastrointestinal tract, and results in a doubling of the mucosal cell turnover after 8 days. At this time both the small intestinal epithelial cell migration rate and the intestinal microflora are similar to those of conventional animals.

Intraphagocytic beta-N-acetylglucosaminidase. Properties of the enzyme and its activity on group A streptococcal carbohydrate in comparison with a soil bacillus enzyme.

The beta-N-acetylglucosaminidases of rabbit and human polymorphonuclear leukocytes and of rabbit alveolar macrophages have been studied in comparison with the beta-N-acetylglucosaminidase derived from a soil bacillus which had previously been shown to hydrolyze the group-specific polysaccharide of Group A streptococci. The phagocytic enzymes are lysosome associated and have an acid pH optimum. In contrast, the soil bacillus enzyme is an extracellular product, has a higher pH optimum, and is probaby of smaller molecular size. When tested on p-nitrophenyl-betaN-acetylglucosaminide as substrate, the K(m) of the phagocytic enzymes is slightly higher than that of the soil bacillus. However, there were extreme differences in their effect on the Group A streptococcal polysaccharide. Thus, 5 x 10(6) units of the alveolar macrophage enzyme were required to hydrolyze the available N-acetylglucosamine of 1 mg of polysaccharide in 18 hr, while 100 units of the soil bacillus enzyme were sufficient to achieve this hydrolysis. In both cases, the serological reactivity of the polysaccharide is altered with loss of Group A specificity and acquisition of a new specificity characteristic of A-variant streptococci. Possible explanations for differences in the activity of the enzymes are considered, and the role of the phagocytic enzymes in intracellular degradation of Group A streptococci is discussed.

Absence of glycerol teichoic acids in certain oral streptococci.

Glycerol teichoic acids were not detected immunochemically or chemically in phenol-water, hot saline (Rantz and Randall), or supernatant fluids of disrupted cells of Streptococcus mitis. Thus teichoic acids do not appear to be found in most Gram-positive bacteria, as has been suggested.

The polar lipids of group B Streptococci. I. Glucosylated diphosphatidylglycerol, a novel glycopholipid.

1. From group B Streptococci a novel glycophospholipid was isolated, which contained D-glucose, glycerol, acyl groups and phosphorus in a molar ratio of approx. 1:3:4:2. It was established to be 2'-O-alpha-D-glucopyranosyl-, 1',3'-bis-(1,2-diacyl-sn-glycero-3-phospho)-glycerol. 2. The structure of the deacylated core was accomplished by analyses of the breakdown products obtained on (i) strong alkaline hydrolysis, (ii) Smith-degradation, and (iii) periodate oxidation with subsequent hydrazinolysis. The four acyl groups were located by sequential degradation of the native lipid with phospholipase A2 and 98% acetic acid. 2. In group B Streptococci approximately 25% of diphosphatidylglycerol occurs in the form of its glucosylated derivative which accounts for 18% of the lipid phosphorus. The glucosylated phosphatidylglycerol analogue could not be detected. A phosphoglucolipid, however, was present, which was tentatively identified as glycerophosphodiglucosyldiacylglycerol.

The polar lipids of group B Streptococci. II. Composition and positional distribution of fatty acids.

1. The polar lipids from group B Streptococci have been isolated. Unless overlapping fractions are worked up on each step molecular species can be lost resulting in a fatty acid composition different from the original one. 2. The lipids were shown to be 1(3),2-diacyl-3(1)-O-alpha-D-glucopyranosyl-sn-glycerol, 1(3),2-diacyl-3(1)-O-[alpha--D-glucopyranosyl-(1,2)-O-alpha-D-glucopyranosyl]-sn-glycerol, 1,2-diacyl-sn-glycero-3-phospho-1'-sn-glycerol, lysylphosphatidylglycerol and 1'-3'-bis(1,2-diacyl-sn-glycero-3-phospho-glycerol. The sterochemical configuration of the phospholipids was achieved by combinations of chemical degradations. 3. The fatty acid composition of group B Streptococci lipids is qualitatively the same as in other Streptococci, but differs by a high content of stearic acid and by a low degree of unsaturation and cyclopropanization. 4. The forementioned polar lipids as well as the glucosyldiphsphatidylglycerol,, which is also found in this organism, showed a very similar composition and the same non-radom positional distribution of fatty acids. Apart from short chain fatty acids the positioning differentiates between saturated and unsaturated fatty acids: short chain and unsaturated fatty acids are accumulated at Position 2, saturated acids are preferentially linked to Position 1. 5. The uniform fatty acid make-up in all polar lipids favours the hypothesis that their diacylglycerol portions are derived from a common phosphatidic acid precursor with negligible postsynthetic rearrangements of the constituent fatty acids.

Molecular surface characterization of oral streptococci by Fourier transform infrared spectroscopy.

In order to characterize the molecular composition of oral streptococci, infrared transmission spectroscopy on freeze-dried cells dissolved in KBr was used. All infrared spectra show similar absorption bands for the strains studied with the most important absorption bands located at 2930 cm-1 (CH), 1653 cm-1 (AmI), 1541 cm-1 (AmII) and two bands at 1236 cm-1 and 1082 cm-1, which were assigned to phosphate and sugar groups. However, calculation of absorption band ratios normalized with respect to the integrated intensity of the CH stretching region around 2930 cm-1, show significant differences between the strains. Both Streptococcus mitis strains possess high AmI/CH and AmII/CH absorption band ratios compared to the other strains. Streptococcus salivarius HBC12, a mutant strain devoid of all proteinaceous surface appendages, shows significantly lower AmI/CH and AmII/CH band ratios with respect to its parent strain S. salivarius HB. Two positive relationships could be established both between the AmII/CH absorption band ratio and the N/C elemental surface concentration ratio of the strains previously, determined from X-ray photoelectron spectroscopy (XPS) and also between AmI/CH and the fraction of carbon atoms at the surface involved in amide bonds, determined by XPS as well. From this comparison, it is concluded that transmission infrared spectroscopy can be employed as a technique to study the molecular surface composition of freeze-dried microorganisms.

On the relationship between glycerophosphoglycolipids and lipoteichoic acids in Gram-positive bacteria. II. Structures of glycerophosphoglycolipids.

1. Eight glycerophosphoglycolipids were isolated from six Gram-positive bacteria. Besides sn-glycero-1-phospho-beta-gentiobiosyldiacylglycerol (i) and sn-glycero-1-phospho-alpha-kojibiosyldiacylglycerol (ii), three novel structures have been established: 1,2-di-O-acyl-3-O-[6-(sn-glycero-1-phospho)-alpha-D-glucopyranosyl-(1 leads to 2)-(6-O-acyl-alpha-D-glucopyranosyl)]glycerol (iii), 1,2-di-O-acyl-3-O-[6-(sn-glycero-1-phospho)-beta-D-glucopyranosyl-(1 leads to 6)-alpha-D-galactopyranosyl-(1 leads to 2)-alpha-D-glucopyranosyl]glycerol (iv), and 1,2-di-O-acyl-3-O-[6-(sn-glycero-1-phospho)-beta-D-glucopyranosyl-(1 leads to 6)-alpha-D-galactopyranosyl-(1 leads to 2)-(6-O-acyl-alpha-D-glucopyranosyl)]glycerol (v). 2. Compound i was isolated from Bacillus licheniformis, Bacillus subtilis and Staphylococcus aureus, compound ii from a group B Streptococcus, compounds ii and iii from Streptococcus lactis, compounds iv and v from Lactobacillus casei. Lactobacillus plantarum contained besides compounds iv and v a glycerophosphate derivative of 1,2-di-O-acyl-3-O-[alpha-D-galactopyranosyl (1 leads to 2)-alpha-D-glucopyranosyl]glycerol. 3. Identical structural features of the described glycerophosphoglycolipids and the corresponding lipoteichoic acids are discussed.

On the relationship between glycerophosphoglycolipids and lipoteichoic acids in Gram-positive bacteria. I. The occurrence of phosphoglycolipids.

1. Gram-positive bacteria out of the families of Streptococcaceae, Lactobacillaceae, Micrococcaceae and Bacillaceae were investigated with respect to the occurrence and the concentration of phosphoglycolipids. 2. Phosphatidylglycolipids occur exclusively in group D Streptococci and in Streptococcus hemolyticus D-58. Phosphatidyl-alpha-kojibiosyldiacylglycerol, the prevalent species, accounts for up to 28% of the polar lipids. The related glycerophospho-phosphatidyl-alpha-kojibiosyldiacylglycerol is restricted to Streptococcus faecalis. 3. Glycerophosphoglycolipids, usually minor components, comprise thirteen compounds most of which have so far not been described. Except Micrococcus lysodeikticus all examined bacteria contained one or more glycerophosphoglycolipids. Their occurrence parallels, therefore, that of lipoteichoic acids, which supports the hypothesis of a metabolic relationship between these two membrane components.

Nuclear magnetic resonance spectra of lipoteichoic acid.

Lipoteichoic acid acids with a range of chemical compositions have been studied using 1H; 13C- and 31P-nuclear magnetic resonance. Proton spectroscopy provided a rapid method for demonstrating whether alanine in a sample is covalently bound to the polyglycerophosphate chains and for monitoring hydrolysis of alanine. The nature of sugar substituents can be determined, with some limitations, from the 13C spectra, and the proportions of glycerol residues substituted by alanine and sugar can be measured. The 31P spectra of lipoteichoic acid provided information about both the degree of substitution and the distribution of the substituent along the polyglycerophosphate chain, except when the substituent was galactose. The polyglycerophosphate chains were shown to undergo rapid internal rotation and no evidence for tertiary structure was found either in the presence or absence of magnesium ions. Magnesium ions exchange rapidly between the bound and free state and the binding constant to lipoteichoic acid of 64 M-1 is typical for monophosphates in aqueous solution. There was no evidence that alanine substitution affects the binding constant for magnesium ions.

Screening for bacterial Fc-receptor activity on nitrocellulose membranes.

Fc receptors released from staphylococci and streptococci could be readily detected by direct cultivation of the bacteria on nitrocellulose membranes or by microfiltration of their culture supernatants. The nitrocellulose membranes used in both assays were subsequently treated with human immunoglobulin (Ig) G. The reactions of the Fc receptor with IgG could be demonstrated by use of the respective peroxidase-labelled antibodies against IgG. The resulting color formation, which indicated Fc-receptor activity of the bacterial culture, closely corresponded to the 125I-labelled IgG-binding reactivity.

New receptor for human plasminogen on gram positive cocci.

180 bacterial strains representing 17 different species of gram positive cocci were tested for the ability to interact with human plasminogen. Receptors for plasminogen could be detected on 23/24 strains of S. pyogenes, 15/15 strains of S. equisimilis, 14/16 strains of human group G streptococci and 14/14 strains of S. pneumoniae. Eight of nineteen strains representing five species of alpha-hemolytic streptococci were also positive. S. equisimilis demonstrated the highest uptake with a median value of 58 per cent (20%-67%). On the other hand, all strains of S. agalactiae, the majority of S. faecalis and all S. aureus, S. epidermidis and S. saprophyticus strains tested were negative. The concentration of unlabelled plasminogen causing a 50 per cent reduction of bound tracer was between 50 and 150 mM. These estimates of the dissociation constant confirmed the specific nature of the interaction. Binding of plasminogen could be blocked by addition of plasmin-aprotinin complex, suggesting that plasminogen and plasmin bind to the same receptor. Binding was also blocked by the plasminogen fragment kringle 1-3, but not by miniplasminogen, a fragment containing kringle 5 and the B-chain region. As streptokinase interacts mainly with the B-chain of plasmin it is clear that the bacterial receptor for plasminogen is not identical to streptokinase.

Comparison of API Rapid Strep, Baxter MicroScan Rapid Pos ID Panel, BBL Minitek Differential Identification System, IDS RapID STR System, and Vitek GPI to conventional biochemical tests for identification of viridans streptococci.

Viridans group streptococci (36 stock strains and 167 single patient blood culture isolates) were assessed using API Rapid Strep, Baxter MicroScan Rapid Pos ID Panel, BBL Minitek Differential Identification System, IDS RapID STR System, and Vitek GPI methods. Identification data obtained with these systems were compared with those indicated by conventional biochemical procedures. API, Baxter MicroScan, BBL, IDS, and Vitek corresponded with conventional biochemical identification in 74%, 66%, 65%, 50%, and 61% of the isolates, respectively; using recommended supplemental tests, agreement was augmented in 9%, 11%, 20%, 11%, and 21% of the isolates, respectively. Disagreement with conventional biochemical methods occurred in 14%, 17%, 14%, 32%, and 10% of the commercial techniques, respectively; no identification was possible in 2%, 5%, fewer than 1%, 6%, and 8% of specimens, respectively. BBL, API, and Baxter MicroScan systems provided the most reliable rapid identification, although supplemental testing often was required. Until a higher percentage of correct identification data can be obtained without supplemental procedures, conventional biochemical techniques will remain the methods of choice for identification of viridans streptococci.

Structural determination of glucans from Streptococcus mutans JC-2 (dental caries bacterium) by carbon-13 nuclear magnetic resonance.

The 13C NMR spectra of glucans from Streptococcus mutans JC-2 show that those glucans have alpha-(1,3) and alpha-(1,6) linkages.

Determination of viridans streptococci surface lipoteichoic acid by enzyme linked immune sorbent assay.

An enzyme linked immune sorbent assay (ELISA) was developed to measure bacterial surface lipoteichoic acid (LTA). Numerous strains of oral streptococci belonging to the 'viridans' group were examined on three separate occasions. The results show that, under these cultural conditions, oral streptococci do not normally express LTA on the cell surface. Occasionally strains produced amounts of LTA detectable using the ELISA but this was not a reproducible phenomenon.

Identification of viridans streptococci by pyrolysis-gas chromatography.

An isothermal method of pyrolysis-gas chromatography (Py-GC) was used for the identification of viridans streptococci. Pyrograms from 104 reference strains were subjected to a discriminant analysis to produce classification coefficients for the identification of 74 test organisms. Five groups representing recognised species were discriminated but Streptococcus milleri strains could not be distinguished from S. sanguis. If S. milleri and S. sanguis are regarded as a single pyrogroup, only three strains out of 74 were incorrectly identified by Py-GC. A multidimensional scaling analysis of the Py-GC data produced a similar species grouping, but this statistical method was less satisfactory for pyrogram data than discriminant analysis. While Py-GC was moderately successful for the identification of viridans streptococci, this study indicated that the technique has limited use in diagnostic medical microbiology because it is time-consuming and lacks flexibility.

Differentiation of Streptococcus sanguis and S. mitior by whole-cell rhamnose content and possession of arginine dihydrolase.

Whole-cell rhamnose concentrations were measured in 48 strains of streptococci resembling Streptococcus sanguis and S. mitior. Physiological characteristics were tested by the API-20/Strep system, and it was found that "typical" S. sanguis (arginine positive, aesculin positive) contained significant amounts of rhamnose, while "typical" S. mitior (arginine negative, aesculin negative) contained very low or undetectable amounts of rhamnose. Both groups contained dextran-positive and dextran-negative strains. Organisms that were more difficult to speciate (those giving positive results in the arginine or the aesculin test, but not in both) could also be divided into a rhamnose-positive and a rhamnose-negative group; with one exception, all of the rhamnose-positive strains gave a positive result with arginine in the API-20/Strep test. There were several discrepancies between the results of conventional tests for arginine and aesculin hydrolysis and those of the corresponding API test. The results of conventional tests for arginine hydrolysis did not correlate closely with rhamnose content, and conventional tests for aesculin hydrolysis were less sensitive than API tests. With the API-20/Strep system, S. sanguis can almost always be distinguished from S. mitior by its ability to hydrolyse arginine.