Correlation between the production of extracellular substances by type III group B streptococcal strains and virulence in a mouse model.

Twelve strains of serotype III group B streptococci (8 isolated from cases of neonatal disease, 3 isolated from asymptomatically colonized infants, and 1 laboratory reference strain) were examined for the vitro production of three potential extracellular virulence products: type-specific antigen, neuraminidase, and protease. In addition, virulence in a mouse model, expressed as 50% lethal dose, was determined for the 12 strains to determine whether a relationship existed between the production of any of the three extracellular products and virulence. Only production of extracellular type-specific antigen showed a correlation with virulence in the mouse model. The high producers of extracellular type-specific antigen were an average of 166-fold more virulent for mice than low producers of the same component. There was no correlation between virulence and either neuraminidase or protease production, nor was there a correlation between either of these two extracellular products and the levels of extracellular type-specific antigen. When levels of group B streptococci of each type (a high and low producer of extracellular type-specific antigen) in organs of infected mice were examined, comparable levels of organisms were found in the brain, spleen, and lungs of mice near death regardless of the initial inoculum. However, the high producer of extracellular type-specific antigen caused death in mice with a 2 to 3 log lower inoculum than the low producer, suggesting that these strains may be more invasive.     

Ecology and nature of immunoglobulin A1 protease-producing streptococci in the human oral cavity and pharynx.

The identity and proportional distribution of immunoglobulin A1 (IgA1) protease-producing streptococci in the oral and pharyngeal microflora were studied. A collection of 459 streptococcal strains, including reference strains of Streptococcus species, and fresh isolates from human dental plaque and buccal and pharyngeal mucosa were identified by biochemical means and were examined for IgA1 protease production. IgA1 protease production was demonstrated in some, but not all, strains of Streptococcus sanguis and Streptococcus mitior and in a group of strains of uncertain taxonomic affiliation. The property was not associated with particular biotypes within the two species. Strains of S. sanguis and S. mitior isolated from Macaca fascicularis also cleaved human IgA1. A significantly different proportion of streptococcal populations in different ecosystems produced IgA1 protease. The enzyme was released by 62.7% of streptococcal isolates from buccal mucosa in contrast to only 7.8% from pharyngeal mucosa. In samples from initial and mature dental plaque 38 to 40% of streptococcal isolates produced IgA1 protease. This difference was largely a result of the frequency by which IgA1 protease activity was present in S. mitior, the predominant streptococcal species in all samples. Among otherwise identical isolates of S. mitior, 67.8% from buccal mucosa in contrast to only 5.9% from pharyngeal mucosa produced IgA1 protease. The results indicate that IgA1 protease may confer an ecological advantage to streptococci colonizing surfaces exposed to a secretory IgA-mediated selection pressure.     

Cleavage of immunoglobulin A1, A2 and G by proteases from clinical isolates of Pasteurella multocida.

Several Pasteurella multocida strains were examined for their ability to produce extracellular enzymes that cleave immunoglobulin A and G (Ig A and Ig G) molecules. Two strains isolated from human pulmonary and genital infections produced proteases that cleaved human IgA and IgG, colostral IgA and human myeloma IgA1 and IgA2. Human IgM was not degraded by these enzymes. Examination of cleavage digests showed two main fragments with different electrophoretic mobilities. The two P. multocida strains produced a protease that cleaved IgA and IgG heavy chains outside the hinge region, and differed in this respect from the hinge-cutting proteases of other bacteria. Protease production may be a virulence mechanism for P. multocida strains.     

Properties of extracellular neuraminidase produced by group A streptococcus.

Extracellular neuraminidase production by group A streptococci was examined in 92 strains. Fourteen of these strains produced appreciable amounts of enzyme; 12 of the neuraminidase-producing strains belonged to T types 1, 4, and 12. Production of the enzyme paralleled bacterial growth in culture and was maximal in medium containing 0.2% glucose. The enzyme produced by one of these strains was partially purified by ammonium sulfate fractionation and filtration on G-200 Sephadex. Its molecular weight was estimated at 90,000. Activity was optimal at pH 5.7 and in the presence of 0.01 to 0.03 M calcium and magnesium cations. The enzyme was stable at temperatures of 4 and 37 degrees C for at least 24 h but was inactivated within 10 min at temperatures of 50 and 65 degrees C. The enzyme hydrolyzed 40% of the sialic acid in bovine submaxillary mucin, but was inactive on sialyl-lactose, porcine submaxillary mucin, oligosaccharides derived from porcine mucin, or human orosomucoid. The Km value for this enzyme with bovine submaxillary mucin as substrate was in the order of 3.6 x 10(-4) M.     

Streptococcal growth and toxin production in vivo

Streptococcal growth in vivo was studied with inoculated micropore filter chambers which were implanted into the peritoneal cavities of mice. Eight of nine group A strains and one group B strain grew in vivo, achieving concentrations of 10(7) to 10(9) CFU/ml in the chambers. Experiments with the Richards strain showed that the number of viable organisms remained high at 5 and 8 weeks after infection. The use of specific inhibitors and appropriate toxin-negative strains demonstrated that both cytolytic toxins produced by group A streptococci, streptolysin S and streptolysin O, were present in the culture fluids harvested from the chambers. This finding represents the first evidence that streptolysin S is produced in vivo. The host response to streptococci growing in vivo was examined by following the increase in serum antistreptolysin O levels. The response was first detected 2 weeks after chamber implantation and appeared to be maximal after 5 weeks. In addition, the production of antibody to streptococcal cell surface antigens was demonstrated indirectly with fluorescein-labeled anti-mouse immunoglobulin G.     

Association of phenotypic and genotypic characteristics of invasive Streptococcus pyogenes isolates with clinical components of streptococcal toxic shock syndrome.

Sixty-two invasive Streptococcus pyogenes strains, including 32 strains isolated from patients with streptococcal toxic shock syndrome (STSS), were analyzed for the following phenotypic and genotypic characteristics: M-protein type, serum opacity factor production, protease production, the presence of streptococcal pyrogenic exotoxin (Spe) genes A, B, and C, and in vitro production of SpeA and SpeB. These characteristics were analyzed for possible associations with each other as well as with clinical components of STSS. M-type 1, the most commonly isolated M-type, was significantly associated with protease production. Protease activity was significantly associated with the clinical sign of soft tissue necrosis. M-type 1 and 3 strains from STSS patients were significantly associated with the clinical signs of shock and organ involvement as well as with SpeA production in vitro. Finally, the production of SpeA was significantly associated with the clinical component of shock and organ involvement as well as with rash. These data suggest that STSS does not make up a single syndrome but, rather, that the multiple STSS clinical criteria probably reflect different phenotypic characteristics of individual S. pyogenes isolates.     

Effect of urokinase on the extracellular virulence properties of Pseudomonas aeruginosa and Burkholderia cepacia

The influence of urokinase and oxygen availability on growth, siderophore, protease and lipase production in Burkholderia cepacia and non-mucoid (PA01) and mucoid (PaWH) strains of Pseudomonas aeruginosa was assessed for cells grown in batch culture under iron-restriction. Siderophore production decreased with increasing concentration of urokinase in B. cepacia independent of oxygen availability but decreased in both strains of P. aeruginosa only under oxygen-depleted conditions. Protease activity was enhanced for all three strains irrespective of oxygen content whereas lipase production increased in B. cepacia and decreased in PA01 under both sets of growth conditions and varied with oxygen availability in PaWH. The evidence presented suggests that urokinase could contribute to the pathophysiology of pulmonary infections.     

Molecular aspects of immunoglobulin A1 degradation by oral streptococci

Using a panel of 143 strains classified according to a novel taxonomic system for oral viridans-type streptococci, we reexamined the ability of oral streptococci to attack human immunoglobulin A1 (IgA1) molecules with IgA1 protease or glycosidases. IgA1 protease production was an exclusive property of all strains belonging to Streptococcus sanguis and Streptococcus oralis (previously S. mitior) and of some strains of Streptococcus mitis biovar 1. These are all dominant initiators of dental plaque formation. Degradation of the carbohydrate moiety of IgA1 molecules accompanied IgA1 protease activity in S. oralis and protease-producing strains of S. mitis biovar 1. Neuraminidase and beta-galactosidase were identified as extracellular enzymes in organisms of these taxa. By examination with enzyme-neutralizing antisera, four distinct IgA1 proteases were detected in S. sanguis biovars 1 to 3, S. sanguis biovar 4, S. oralis, and strains of S. mitis, respectively. The cleavage of IgA1 molecules by streptococcal IgA proteases was found to be influenced by their state of glycosylation. Treatment of IgA1 with bacterial (including streptococcal) neuraminidase increased susceptibility to protease, suggesting a cooperative activity of streptococcal IgA1 protease and neuraminidase. In contrast, a decrease in susceptibility was observed after extensive deglycosylation of the hinge region with endo-alpha-N acetylgalactosaminidase. The effector functions of IgA antibodies depend on the carbohydrate-containing Fc portion. Hence, the observation that oral streptococci may cleave not only the alpha 1 chains but also the carbohydrate moiety of IgA1 molecules suggests that the ability to evade secretory immune mechanisms may contribute to the successful establishment of these bacteria in the oral cavity.     

The gene encoding a new mitogenic factor in a Streptococcus pyogenes strain is distributed only in group A streptococci.

We recently cloned a gene encoding a new mitogenic factor (MF) from Streptococcus pyogenes NY-5. In the present study, we determined the distribution of this MF gene (mf) by PCR based upon its sequence. Of 371 streptococcal group A strains isolated from clinical specimens, 370 (99.7%) were positive for mf. The strain that was negative for the MF gene was also negative for the streptolysin O gene (slo). Some streptococcal strains belonging to groups C and G were negative for mf but positive for slo. Group B strains were negative for both. Furthermore, we examined the presence of mf in 54 strains belonging to 28 families and found mf only in group A streptococci. These results indicate that mf is distributed specifically in group A streptococci and the presence of mf in clinical samples strongly suggests infection with group A streptococci.     

Coaggregation of Streptococcus sanguis and other streptococci with Candida albicans.

Thirteen strains of viridans group streptococci and two strains of other streptococci were tested for coaggregation with Candida albicans. Streptococcus sanguis strains generally exhibited low levels of adherence to 28 degrees C-grown exponential-phase yeast cells, but starvation of yeast cells for glucose at 37 degrees C (or at 28 degrees C) increased their coaggregating activity with these streptococci by at least tenfold. This was a property common to four C. albicans strains tested, two of which were able to form mycelia (6406 and MEN) and two of which were not (MM2002 and CA2). The expression of the coaggregation adhesin during yeast cell starvation was inhibited by addition of trichodermin or amphotericin B. The strains of S. sanguis, Streptococcus gordonii, and Streptococcus oralis tested for coaggregating activity encompassed a diverse range of physiological and morphological types, yet all exhibited saturable coaggregation with starved C. albicans cells. There was no correlation of cell surface hydrophobicity, of either yeast or streptococcal cells, with their abilities to coaggregate. Strains of Streptococcus anginosus also coaggregated with starved yeast cells; Streptococcus salivarius and Streptococcus pyogenes coaggregated to a lesser degree with C. albicans, and the coaggregation with S. pyogenes was not promoted by yeast cell starvation; Streptococcus mutans and Enterococcus faecalis did not coaggregate with yeast. The coaggregation reactions of S. sanguis and S. gordonii with C. albicans were inhibited by EDTA and by heat or protease treatment of the yeast cells and were not reversible by the addition of lactose or other simple sugars. These observations extend the range of intergeneric coaggregations that are known to occur between oral microbes and suggest that coaggregations of C. albicans with viridans group streptococci may be important for colonization of oral surfaces by the yeast.     

Extracellular neuraminidase production by clinical isolates of group B streptococci from infected neonates.

A total of 73 clinical isolates of group B streptococci obtained from diseased infants in 23 states and Puerto Rico were examined for extracellular neuraminidase production. The association of elevated levels of neuraminidase with serotype III isolates was evident in a broad geographical distribution.     

The influence of nutritional and cultural factors on CAMP factor production by group B streptococci

Twelve group B streptococci were examined for their capacity to produce CAMP factor in liquid culture media in which the carbohydrate type and concentration and/or the buffering capacity of the media were altered. The effects of these variations of media content on CAMP factor production were determined and related to effects on growth yield and pH changes during growth. For 9 of the 12 strains tested higher CAMP factor titres were obtained in maltose broth than in glucose broth, and accompanying changes in pH were greater and more rapid in glucose containing media. It is suggested that the lesser pH changes occurring during growth in maltose broth may be the mechanism whereby higher CAMP titres were obtained with these strains. The influence of carbohydrate type was not observed with the remaining 3 strains tested, but maximum CAMP factor yields were obtained in the presence of maltose and non-inhibitory concentrations of buffer. CAMP factor production for all strains of GBS may be increased by the addition of maltose and buffer to standard media.     

Stimulation of complement component C3 synthesis in macrophagelike cell lines by group B streptococci.

Complement levels and complement activation are key determinants in streptococcus-induced inflammatory responses. Activation of macrophage functions, such as complement synthesis, by group B streptococci (GBS) was examined as a possible component of GBS-induced chronic inflammation. Using an enzyme-linked immunosorbent assay, secreted C3 from mouse macrophagelike cell lines (PU5-1.8 and J774A.1) was monitored after cultivation with GBS. Whole, heat-killed GBS (1 to 10 CFU per macrophage) of both type Ia and III strains induced 25 to 300% increases in secreted C3 in both cell lines after a 24-h cultivation. GBS-treated cell lines exhibited increases in secreted lysozyme (10%) and in cellular protein (25 to 50%). Inhibition of macrophage phagocytosis by cytochalasin B inhibited GBS stimulation of C3. Purified cell walls of GBS type III strain 603-79 (1 to 10 micrograms/ml) also enhanced C3 synthesis. Local enhancement of macrophage C3 production by ingested streptococci or by persistent cell wall antigens may serve to promote chronic inflammatory responses.     

In vitro method to differentiate isolates of type III Streptococcus agalactiae from symptomatic and asymptomatic patients

Streptococcus agalactiae (group B streptococci) isolates from infected infants have been demonstrated to have three- to fourfold or higher levels of cell-associated lipoteichoic acid than isolates from asymptomatically colonized infants, suggesting a role for this cell surface polymer in the relative virulence of these organisms. The present study indicates that symptomatic isolates of type III group B streptococci can be readily differentiated from asymptomatic strains by their response to various levels of phosphate in a chemically defined medium (FMC). Both classes of isolates had the same doubling time (TD of 30 to 35 min) in FMC containing 65 mM sodium phosphate. However, levels of phosphate greater than 125 mM distinguished the two classes of strains. Asymptomatic strains pregrown in 65 mM phosphate to the stationary phase rapidly initiated growth at elevated phosphate levels, while symptomatic strains initiated growth only after a prolonged incubation period (greater than 400 min). These results suggest that the physiological growth response of clinical isolates of group B streptococci to phosphate can serve as a diagnostic aid in screening potentially virulent strains in pregnant women and newborn infants.     

Association of elevated levels of extracellular neuraminidase with clinical isolates of type III group B streptococci.

The level of total extracellular neuraminidase produced by 74 clinical isolates of group B streptococci isolated from diseased or asymptomatically colonized infants was assayed. Extracellular neuraminidase was obtained from concentrated filtrates of exponentially growing cultures of group B streptococci grown in a chemically defined medium (FMC) containing supplemental protein. The total activity of extracellular enzyme produced by these clinical isolates ranged from less than 10 to 360 nmol of sialic acid released per min per mg of cell dry weight. Strains were arbitrarily classified as either nonproducers (less than 10 nmol/min per mg of cell dry weight), low producers (greater than 10 to less than or equal 140 nmol/min per mg of cell dry weight), or high producers (greater than 140 to 360 nmol/min per mg of cell dry weight). Type III isolates from diseased infants were significantly more often classified as high producers than strains of group B streptococci of other serotypes from diseased infants (P less than 0.001). Furthermore, the serotype III strains isolated from neonatal infections were more often high producers than those of the same serotype from asymptomatically colonized infants (P less than 0.025). These results suggest that the ability to produce elevated levels of neuraminidase may be related to the frequent association of type III strains with disease among neonates.     

Sialidase activity of the "Streptococcus milleri group" and other viridans group streptococci

Viridans group streptococci were examined for the production of sialidase (neuraminidase) activity, using the fluorescent substrate 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid in a simple and rapid (15-min) assay. Sialidase was produced by all strains of Streptococcus oralis and S. intermedius and by a majority of S. mitis strains. S. mutans, S. sobrinus, S. gordonii, S. sanguis, S. vestibularis, S. salivarius, S. anginosus, S. constellatus, "S. parasanguis," and the "tufted fibril group" were uniformly negative. Sialidase production may be a useful characteristic to assist in the identification of viridans group streptococci.     

A survey of IgA protease production among clinical isolates of Proteeae

A collection of 100 strains of Proteeae, in which all species within the tribe were represented, was examined for IgA protease production. The strains were isolated from various clinical specimens from sick and healthy persons in several countries. IgA protease-producing strains were not found amongst species of Providencia and Morganella but were common in Proteus spp. All the strains of P. mirabilis and P. penneri and many of the strains of P. vulgaris examined produced an EDTA-sensitive protease that cleaved the IgA heavy chain outside the hinge region. The proteus enzyme was different in this respect from the EDTA-sensitive, hinge-cutting proteases of other bacteria. The ability to produce IgA protease was unrelated to the O antigenicity, biotype or bacteriocin type of the strain. IgA protease production may be an important virulence mechanism for Proteus strains.     

The M protein is dispensable for maturation of streptococcal cysteine protease SpeB

The streptococcal pyrogenic exotoxin B (SpeB) is an important virulence factor of group A streptococci (GAS) with cysteine protease activity. Maturation of SpeB to a proteolytically active form was suggested to be dependent on cell-wall-anchored M1 protein, the major surface protein of GAS (M. Collin and A. Olsen, Mol. Microbiol. 36:1306-1318, 2000). Collin and Olsen showed that mutant GAS strains expressing truncated M protein secrete a conformationally different form of unprocessed SpeB with no proteolytic activity. Alternatively, we hypothesized that a truncated M protein may interfere with processing of this secreted protease, and therefore we tested cysteine protease activity in genetically defined mutant strains that express either no M protein or membrane-anchored M protein with an in-frame deletion of the AB repeat region. Measurements of SpeB activity by cleavage of a substrate n-benzoyl-Pro-Phe-Arg-p-nitroanilide hydrochloride showed that the proteolytic activities in culture supernatants of both mutants were similar to those from the wild-type strain. In addition, Western blot analysis of culture supernatants showed that SpeB expression and processing to a mature form was unaffected by either deletion mutation. Therefore, we conclude that M protein is not required for maturation of the streptococcal cysteine protease SpeB.     

Source of group B streptococci in the female genital tract.

Swabs were taken from the posterior fornix, perineum, and anorectum of 135 patients on three occasions during their pregnancy. Multiple isolates of beta-haemolytic streptococci of group B were obtained from 24 women, in 21 of whom the strains were examined by a highly discriminative serotyping and phage typing method. In 18 of these patients their own isolates were indistinguishable but different from those of other women with multiple isolates. Women yielding group B streptococci from the posterior fornix usually carried an indistinguishable strain in the anorectum.     

New phenotypic typing scheme for group B streptococci.

AIMS: To develop a new typing system for group B streptococci based on 35S-methionine-labelled protein profiles of bacterial proteins. METHODS: 377 clinical isolates of group B streptococci were examined by incorporation of 35S-methionine into bacterial proteins under strict anaerobic conditions. After sodium dodecylsulphate-polyacrylamide gel electrophoresis, autoradiography was performed. The patterns produced were visually analysed and categorised into clusters of organisms based on the pattern of band production between 32-46 kilodaltons. RESULTS: 294 of the typed strains classified into seven different groups designated a-g. 32 strains failed to incorporate 35S-methionine sufficiently to be grouped and 11 strains did not fall into one of the seven identified groups. Typability, reproducibility, and discrimination of the system was evident. CONCLUSIONS: This typing system may help to distinguish between colonising and invasive strains of the organism.