Sialidase-like Enzymes Produced by Group A, B, C, G, and L Streptococci and by Streptococcus sanguis

A group of enzymes were prepared from the culture fluids of streptococci belonging to groups A, B, C, G, and L, and from a strain of Streptococcus sanguis. These streptococcal enzymes (designated St-sialidases) released a substance shown to belong to the sialic acid group from the specific substrate BSM-St, a sialomucoid prepared from bovine submaxillary gland. They were inactive on N-acetylneuramin lactose prepared from bovine colustrum and on a sialomucoid prepared from bovine submaxillary mucin, whereas these substances are susceptible to sialidases produced by group K streptococci and by Vibrio cholerae. Some of the St-sialidases were markedly activated by divalent cations, but others showed little response. The heat stability of the enzymes produced by the different strains varied. The optimal pH was between 5.5 and 6.5 with acetate buffer and was about 7 with phosphate buffer. K(m) values were determined for the St-sialidases with BSM-St as substrate.     

Characteristics of a protease of Streptococcus sanguis G9B which degrades the major salivary adhesin

An endogenous enzyme present in cell surface extracts of Streptococcus sanguis strain G9B degraded the major salivary adhesin of the organism. The enzyme showed optimal activity between 50 and 65 degrees C and was inactivated at higher temperatures. The activity at these unusually high temperatures seemed to be a consequence of release from the cell surface since intact whole G9B cells showed greater activity at 37 degrees C. The enzyme was not found in culture supernatants of G9B cells. The pH range for the enzyme was between 5 and 9. It was inhibited by iodoacetic acid, Hg2+, Cu2+, EDTA, SDS, and PMSF, but not by TLCK, TPCK, soybean trypsin inhibitor, cysteine, dithiothreitol, leupeptin, Ca2+, Mg2+ or saliva. The enzyme did not show any activity against human or rabbit IgG or human IgA. Enzyme activity was also found in S. sanguis strains Adh- (a spontaneously occurring non-adherent mutant of G9B), and M-5.     

Comparative Grouping of Mastitis Streptococci by Means of Co-Agglutination and Precipitation

Two hundred bovine mastitis streptococcal strains belonging to groups B, C, E, G and L were tested comparatively by means of the co-agglutination and precipitation technique. Identical results were obtained with the two tests in 191, or 95.5 %, of the strains. Six strains, which could not be grouped by co-agglutination, proved to belong to group B when grouped by precipitation. On the other hand, one strain which proved to belong to group G and two strains to group L when grouped by co-agglutination, could not be grouped by precipitation. Some cross-reactivity was observed between groups A and C, B and G, B and L. Only a few L strains showed marked cross-reactivity which was not easily distinguished from specific reactions. However, the cross-reactivity ought to be eliminated by dilution or adsorption. Using the precipitation test as a supplementary method, the easy and rapid co-agglutination test was found to be a suitable procedure for routine grouping of mastitis streptococci.     

Lysis and Lysogenization of Groups A, C, and G Streptococci by a Transducing Bacteriophage Induced from a Group G Streptococcus

A temperate bacteriophage, designated GT-234, was isolated from a group G Streptococcus after ultraviolet irradiation. After several single-plaque passages in a group G indicator strain, this phage formed plaques in 3 of 14 group A strains, in 3 of 15 group C strains, and in 4 of 13 group G strains-but not in some representatives of several other serogroups. After propagation in each of the sensitive strains, the progeny from each was shown to be the same phage by (i) adsorption and plaque formation in each of the other groups, (ii) lysogenization in each of the other groups, (iii) high titers on infection of each serogroup, regardless of the group of propagating strain, and (iv) neutralization of infection in each of the other groups by antiserum against the phage propagated in group G. Phage GT-234 is serologically related to virulent group A phage A25, from which it is morphologically indistinguishable. Like A25, it is a transducing phage. Other studies showed that A25, as well as a group A temperate transducing phage (AT-298), could also infect strains of group C and G. These results indicate a need for reassessment of group specificity and phage receptors among streptococci of groups A, C, and G and raise possibilities for intergroup transduction.     

Nucleic acid hybridization and cell wall composition studies of pyogenic streptococci

Abstract DNA-rRNA and DNA-DNA hybridization studies indicate that the classical pyogenic streptococci can be divided into five homology clusters. Based on these studies the term pyogenic streptococci should be confined to the first cluster consisting of serological groups A, A-variant, C, G ('large' colony, type II) and L.
Streptococci of serological groups B and M form the second cluster. The third cluster is composed of streptococci of serological groups R and S and serological groups U, V and P are found in the fourth cluster. The fifth cluster comprises strains of Streptococcus anginosus S. intermedius, Streptococcus MG and serological groups G ('minute' colony, type I) and F (type I). Most of the test strains contain the peptidoglycan type Lys-Ala_1–3. Only streptococci of serogroups R and S reveal a directly cross-linked peptidoglycan. Rhamnose was found as characteristic component of all cell wall polysaccharides. The impact of our results on the systematics of classical pyogenic streptococci will be discussed.     

Streptococcus uberis: an Approach to its Classification

Thirty-one strains of streptococci, selected because they were considered to be or were similar to Streptococcus uberis , were examined by physiological tests and for properties of their lactate dehydrogenases (LDHs) and deoxyribonucleic acid (DNA). Twenty-nine of the 31 strains fell into two main genotypes as judged by DNA/DNA hybridization. One genotype (Group I) included the type strain of Strep. uberis and most other strains in this group were physiologically similar to it and had the same percent guanine + cytosine (%GC) in their DNA. The other genotype (Group II) contained strains which were more variable physiologically and had DNA of a lower % GC than the type strain. The European strains examined contained two distinct LDH types, one being associated with the Group I and the other with Group II strains. Three American strains examined however had LDH's which were unlike those of the European strains.     

Streptococcus faecalis sex pheromone (cAM373) also produced by Staphylococcus aureus and identification of a conjugative transposon (Tn918).

Streptococcus faecalis RC73 was found to harbor a conjugative plasmid (pAM373) which confers a mating response to a sex pheromone (cAM373) excreted by plasmid-free members of the same species. The pheromone was also detected in culture filtrates of all of 23 Staphylococcus aureus strains but in only 2 of 22 coagulase negative staphylococcus strains. Streptococcus sanguis Challis and G9B also produced the activity, but 10 other Streptococcus sanguis strains did not. The activity was also produced by Streptococcus faecium 9790. A tetracycline resistance (Tc) determinant present in S. faecalis RC73 was not associated with pAM373 but served as a useful marker in efforts to identify pAM373 among other plasmids present in the strain. Analyses of the Tc determinant showed that it was located on a conjugative transposon very similar to Tn916. Designated Tn918, the transposon could insert into pAM373 as well as into two other hemolysin plasmids. Whereas pAM373 derivatives transferred very well between strains of Streptococcus faecalis, the plasmid would not establish in Staphylococcus aureus or Streptococcus sanguis. However, a derivative of pAM373 carrying Tn918 proved to be a useful delivery vehicle for generating transposon insertions into multiple sites on the staphylococcal chromosome.     

Diversity and mobility of integrative and conjugative elements in bovine isolates of Streptococcus agalactiae, S. dysgalactiae subsp. dysgalactiae, and S. uberis

Bovine isolates of Streptococcus agalactiae (n = 76), Streptococcus dysgalactiae subsp. dysgalactiae (n = 32), and Streptococcus uberis (n = 101) were analyzed for the presence of different integrative and conjugative elements (ICEs) and their association with macrolide, lincosamide, and tetracycline resistance. The diversity of the isolates included in this study was demonstrated by multilocus sequence typing for S. agalactiae and pulsed-field gel electrophoresis for S. dysgalactiae and S. uberis. Most of the erythromycin-resistant strains carry an ermB gene. Five strains of S. uberis that are resistant to lincomycin but susceptible to erythromycin carry the lin(B) gene, and one has both linB and lnuD genes. In contrast to S. uberis, most of the S. agalactiae and S. dysgalactiae tetracycline-resistant isolates carry a tet(M) gene. A tet(S) gene was also detected in the three species. A Tn916-related element was detected in 30 to 50% of the tetracycline-resistant strains in the three species. Tetracycline resistance was successfully transferred by conjugation to an S. agalactiae strain. Most of the isolates carry an ICE integrated in the rplL gene. In addition, half of the S. agalactiae isolates have an ICE integrated in a tRNA lysine (tRNA(Lys)) gene. Such an element is also present in 20% of the isolates of S. dysgalactiae and S. uberis. A circular form of these ICEs was detected in all of the isolates tested, indicating that these genetic elements are mobile. These ICEs could thus also be a vehicle for horizontal gene transfer between streptococci of animal and/or human origin.     

Numerical taxonomy of Streptococcus

A numerical taxonomic study of strains of Streptococcus, together with representatives of allied genera, showed 28 reasonably distinct phenons. The major areas, with their phenons, were: (a) enterococcal species group (S. faecalis, S. faecium, 'S. avium' and a proposed new species 'S. gallinarum'); (b) paraviridans species group (S. bovis, S. equinus, S. salivarius, 'S. casseliflavus', S. mutans, S. raffinolactis and an unidentified Oral Group I); (c) lactic species group (S. lactis including S. cremoris); (d) thermophilic species group (S. thermophilus); (e) viridans species group (S. mitis, S. sanguis, a proposed new species 'S. oralis' and 'S. milleri'); (f) pyogenic species group (S. agalactiae, S. pyogenes, S. equi, 'S. equisimilis' including 'S. zooepidemicus, and a cluster of Lancefield Group B strains of human origin); (g) parapyogenic species group (S. uberis, 'S. dysgalactiae', and a cluster of strains of Lancefield Groups R, S and T). Species of Aerococcus, Gemella, Leuconostoc and Pediococcus are very closely related to the streptococci.     

Regulation of glucose metabolism in oral streptococci through independent pathways of glucose 6-phosphate and glucose 1-phosphate formation

In vivo rates of glucose uptake and acid production by oral streptococci grown in glucose- or nitrogen-limited continuous culture and batch culture were compared with the glucose phosphorylation activities of harvested, decryptified cells. The strains examined contained significant phosphoenolpyruvate-phosphotransferase system (PTS) activity, measured by a glucose 6-phosphate (G6P) dehydrogenase-linked assay procedure, but this activity was insufficient to account for the in vivo glucose uptake rates. However, ATP was a superior phosphoryl donor to phosphoenolpyruvate, and unlike the PTS, phosphoryl transfer with ATP was insensitive to bacteriostatic concentrations of chlorhexidine, suggesting glucokinase-mediated G6P formation. Again, G6P formation from the PTS and glucokinase reactions was not commensurate with some of the glucose uptake rates observed, implying that other phosphorylation reactions must be occurring. Two novel reactions involving carbamyl phosphate and acetyl phosphate were identified in some of the strains. No G6P formation was detected with these potential phosphoryl donors, but in the presence of phosphoglucomutase, glucose 1-phosphate (G1P) formation was evident, which was insensitive to chlorhexidine. G1P is a precursor of glycogen, and good correlation was obtained between G1P formation activity and endogenous metabolism of washed cells measured either as a rate of acid production at a constant pH 7 or as a decrease in pH with time in the absence of titrant. A "league table" of abilities to synthesize G1P and produce acid from endogenous metabolism was compiled for oral streptococci grown in batch culture. This indicated that Streptococcus mutans Ingbritt and Streptococcus sanguis Challis were unable to form G1P or produce much acid endogenously, whereas increasing activities were obtained with Streptococcus salivarius, Streptococcus sanguis, and Streptococcus mitis. In particular, S. mitis had the highest G1P formation activities and was able to decrease the pH to less than 5 in 15 min by endogenous metabolism alone. The data are consistent with the intracellular accumulation of free glucose driven by proton motive force when PTS activities are low and the subsequent phosphorylation to either G6P for metabolism via glycolysis or G1P for glycogen biosynthesis. The accumulation of acetyl phosphate during glucose-limited growth and the availability of arginine for catabolism to carbamyl phosphate provide an explanation as to why some glucose-limited oral streptococci continue to synthesize glycogen under these conditions, which might prevail in plaque.     

Genetic exchange between oral streptococci during mixed growth

To determine whether oral streptococci might exchange genetic information in the oral cavity, paired transformable strains of Streptococcus mutans, Streptococcus sanguis and Streptococcus milleri were growth together. Chromosomal and plasmid-borne antibiotic resistance markers could be readily transferred from S. mutans GS-5 to S. milleri NCTC 10707 or S. sanguis Challis during mixed growth. However, no exchange from the latter two organisms to strain GS-5 could be detected under these conditions. The transfer of genetic information from S. sanguis to S. milleri was also observed.     

The effect of a novel anti-adherent compound on the adherence of oral streptococci to hydroxyapatite

The effect of the copolymer M239, 144 with and without chlorhexidine on the adherence of oral streptococci to saliva-coated hydroxyapatite was investigated. At 1% w/v M239, 144 reduced the adherence of Streptococcus sanguis NCTC 7863 by 94%. It had a moderate effect on the adherence of other Strep. sanguis strains and a Streptococcus gordonii strain but had no effect on the adherence of Streptococcus oralis or Streptococcus mutans. Chlorhexidine did not influence the anti-adhesive properties of 1% w/v M239, 144.     

Competition between oral Streptococcus species in the chemostat under alternating conditions of glucose limitation and excess

Abstract Oral Streptococcus species experience carbohydrate limitation interrupted by periods of substrate excess following food intake by the host. To investigate the competitiveness of various streptococcal species under fluctuating carbohydrate supply, 2-membered chemostat cultures were run.
Under continuous limitation of glucose or sucrose, all 6 Streptococcus mutans test strains were outcompeted by Streptococcus sanguis P4A7 or Streptococcus milleri B448. This indicated that S. mutans had a lower affinity for glucose and sucrose than S. sanguis and S. milleri .
Mixed cultures were then subjected to hourly pulses with glucose. Under these conditions S. mutans Ny344 competed successfully with S. milleri B448, but still lost the competition against S. sanguis P4A7. The streptococci responded to pulses by taking up glucose at the maximum rate almost instantaneously. S. sanguis P4A7 had the highest rate of glucose uptake while the q _max value of S. mutans Ny344 was higher than that of S. milleri B448. This suggested a causal relationship between q _max and competitiveness.     

Pulsed-Field Gel Electrophoresis Is More Discriminating Than Multilocus Enzyme Electrophoresis and Random Amplified Polymorphic DNA Analysis for Typing Pyogenic Streptococci

The SmaI restriction endonuclease digestion patterns of chromosomal DNAs from 99 pyogenic streptococci belonging to Lancefield group A (41 Streptococcus pyogenes), group C (seven S. dysgalactiae, 11 \QS. equisimilis\W, three S. equi, eight S. zooepidemicus) and group G (25 human group G Streptococcus, four S. canis) were analyzed by pulsed-field gel electrophoresis (PFGE), and the results were compared with those previously obtained by multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA analysis (RAPD). PFGE revealed 93 distinct types among the 99 strains, and no patterns were common to strains of different species. The discriminatory power of PFGE was greater than that of MLEE and RAPD for groups A and G streptococci. The polymorphism among group C streptococci was similar with the three techniques. PFGE is, therefore, the most efficacious method for epidemiological typing of pyogenic streptococci. Received: 31 July 1996 / Accepted: 17 August 1996     

Prevalence of IgA receptors in clinical isolates of Streptococcus pyogenes and Streptococcus agalactiae; Serologic distinction between the receptors by blocking antibodies

Abstract Group A and B streptococci ( Streptococcus pyogenes and Streptococcus agalactiae ) are the only known bacterial pathogens expressing IgA Fc-receptors. However, the IgA binding proteins of the two species have been found genetically unrelated. In the present investigation the binding of human IgA among clinical isolates of group A and group B streptococci was studied and the respective IgA-binding epitopes were compared serologically. Surface binding of radiolabelled, monoclonal human IgA1 occurred in 38% of 115 unselected group A streptococcal isolates. Comparing four predominant T-types, IgA-binding was found in 77% and 85%, respectively, of types T4 and T28 strains but only in 5% and 25%, respectively, of T1 and T12 strains. In group B streptococci, 70% of 58 type Ib strains but only 2% of 399 strains of other serotypes bound IgA. Using rabbit immune sera raised to the two streptococcal species it was found that strains exhibiting IgA Fc-receptors often induced antibodies blocking the binding of IgA to bacteria. Furthermore, the blocking shown by an individual serum was restricted to the streptococcal group used for immunization showing that also the IgA-binding epitopes in group A and B streptococci are conformationally distinct. Though infections with serotypes often binding IgA, compared to other types, are not known to differ, it is assumed that the non-immune binding of IgA might favour mucosal colonization of the organisms.     

The hydrophobicity of 'viridans' streptococci isolated from the human mouth

The hydrophobicity of human oral streptococci was measured with the hexadecane assay modified by the incorporation of polyethylene glycol 6000. Large variability in the hydrophobicity between cultures of some strains grown on different occasions was observed whereas other strains were less variable. The variation in hydrophobicity was significantly reduced by growing the cells in continuous culture in a chemostat under glucose-limiting conditions. The Streptococcus mutans strains used all had low hydrophobicity and the mean hydrophobicity of this species was significantly lower ( P < 0.05) than the mean hydrophobicity of Strep. salivarius, Strep. sanguis Type I and Strep. sanguis Type II strains. This finding supports the view that hydrophobicity is a contributing factor in the adhesion of viridans streptococci to oral surfaces.     

The hydrophobicity of 'viridans' streptococci isolated from the human mouth

The hydrophobicity of human oral streptococci was measured with the hexadecane assay modified by the incorporation of polyethylene glycol 6000. Large variability in the hydrophobicity between cultures of some strains grown on different occasions was observed whereas other strains were less variable. The variation in hydrophobicity was significantly reduced by growing the cells in continuous culture in a chemostat under glucose-limiting conditions. The Streptococcus mutans strains used all had low hydrophobicity and the mean hydrophobicity of this species was significantly lower (P less than 0.05) than the mean hydrophobicity of Strep. salivarius, Strep. sanguis Type I and Strep. sanguis Type II strains. This finding supports the view that hydrophobicity is a contributing factor in the adhesion of viridans streptococci to oral surfaces.     

Genetic and biochemical characterization of the F-ATPase operon from Streptococcus sanguis 10904

Oral streptococci utilize an F-ATPase to regulate cytoplasmic pH. Previous studies have shown that this enzyme is a principal determinant of aciduricity in the oral streptococcal species Streptococcus sanguis and Streptococcus mutans. Differences in the pH optima of the respective ATPases appears to be the main reason that S. mutans is more tolerant of low pH values than S. sanguis and hence pathogenic. We have recently reported the genetic arrangement for the S. mutans operon. For purposes of comparative structural biology we have also investigated the F-ATPase from S. sanguis. Here, we report the genetic characterization and expression in Escherichia coli of the S. sanguis ATPase operon. Sequence analysis showed a gene order of atpEBFHAGDC and that a large intergenic space existed upstream of the structural genes. Activity data demonstrate that ATPase activity is induced under acidic conditions in both S. sanguis and S. mutans; however, it is not induced to the same extent in the nonpathogenic S. sanguis. Expression studies with an atpD deletion strain of E. coli showed that S. sanguis-E. coli hybrid enzymes were able to degrade ATP but were not sufficiently functional to permit growth on succinate minimal media. Hybrid enzymes were found to be relatively insensitive to inhibition by dicyclohexylcarbodiimide, indicating loss of productive coupling between the membrane and catalytic subunits.