Role of granulocytes in the induction of an experimental endocarditis with a dextran-producing Streptococcus sanguis and its dextran-negative mutant.

The role of granulocytes in the induction of endocarditis with a dextran-producing Streptococcus sanguis and a dextran-negative mutant of this strain was studied. The number of colony-forming units of Streptococcus sanguis needed to colonize the vegetations in 50% of the rabbits (ID50) was significantly lower for the parent strain than for the dextran-negative mutant. However, in granulocytopenic rabbits the ID50s of both strains did not differ measurably. Dextran-negative streptococci were more readily cleared from the circulation than dextran-positive, but in this respect no difference was found between control and granulocytopenic rabbits, which indicates that clearance cannot account for the difference in ID50 between the two strains in the control group. At serum concentrations of 5% and lower, in-vitro granulocytes phagocytosed the dextran-negative streptococci more rapidly than the dextran-positive. The intracellular killing of the streptococci was no influenced by dextran production. This study suggests that an impaired phagocytic removal of attached bacteria from the vegetational surface can be a factor promoting the induction of endocarditis by dextran-producing streptococci.     

Role of granulocytes in the induction of an experimental endocarditis with a dextran-producing Streptococcus sanguis and its dextran-negative mutant

The role of granulocytes in the induction of endocarditis with a dextran-producing Streptococcus sanguis and a dextran-negative mutant of this strain was studied. The number of colony-forming units of Streptococcus sanguis needed to colonize the vegetations in 50% of the rabbits (ID50) was significantly lower for the parent strain than for the dextran-negative mutant. However, in granulocytopenic rabbits the ID50s of both strains did not differ measurably. Dextran-negative streptococci were more readily cleared from the circulation than dextran-positive, but in this respect no difference was found between control and granulocytopenic rabbits, which indicates that clearance cannot account for the difference in ID50 between the two strains in the control group. At serum concentrations of 5% and lower, in-vitro granulocytes phagocytosed the dextran-negative streptococci more rapidly than the dextran-positive. The intracellular killing of the streptococci was no influenced by dextran production. This study suggests that an impaired phagocytic removal of attached bacteria from the vegetational surface can be a factor promoting the induction of endocarditis by dextran-producing streptococci.     

Influence of preformed antibody on experimental Streptococcus sanguis endocarditis.

The influence of preformed, anti-whole organism antibody on the development of Streptococcus sanguis endocarditis was examined in both in vivo and in vitro systems. Antibody prevented, rather than potentiated, endocarditis in rabbits. The infectious dose in 30 control animals was 10(6.5) +/- 0.33 (mean +/- standard deviation); this increased to 10(7.71 +/- 0.05 in 36 immunized animals (P less than 0.01). No differences in bacterial clearance mechanisms were apparent between groups. Antibody also prevented the adherence of S. sanguis to the constituents of nonbacterial thrombotic endocarditis (fibrin and platelets) in vitro. When preincubated in high-titer antisera, adherence of S. sanguis was reduced compared with controls (adherence ratio mean +/- standard error of the mean, X 10(4): 174 +/- 5 versus 427 +/- 10, P less than 0.001). Preadsorption of immune sera with intact S. sanguis restored adherence to normal values, whereas preadsorption with dextran was partially effective. These studies demonstrate that preformed antibody had a protective role in vivo and suggest that a possible mechanism is blockade of adherence, a crucial early step in the pathogenesis of endocarditis.     

Effects of monocytopenia and anticoagulation in experimental Streptococcus sanguis endocarditis.

The role of blood monocytes in the attachment of streptococci to endocardial vegetations was investigated in an experimental Streptococcus sanguis endocarditis by depletion of blood monocytes with the cytostatic drug VP 16-213 alone and combined with anticoagulant treatment with warfarin sodium. The numbers of streptococci in the vegetations of control, monocytopenic, and monocytopenic/anticoagulated rabbits were comparable. In the vegetations streptococci were found mainly in areas free of phagocytic cells. It is concluded that streptococci do not have to be phagocytosed by monocytes in the circulation before being deposited on the surface of endocardial vegetations. Even the vegetations of intensively anticoagulated/monocytopenic rabbits showed colonies of streptococci embedded in polymerized fibrin and cellular material, this matrix possibly being held together by streptococcal dextran.     

Effect of immunization on susceptibility to experimental Streptococcus mutans and Streptococcus sanguis endocarditis.

It has been asserted that humoral immunity is an important potentiating factor in pathogenesis of infective endocarditis, in that prior immunization to certain bacteria may predispose the host to endocarditis caused by those organisms. If so, possible future vaccination of humans with streptococcal antigens for the prevention of dental caries might increase the susceptibility of the population to streptococcal endocarditis. To examine this hypothesis further, we immunized rabbits with killed Streptococcus sanguis or Streptococcus mutans. After complement-fixing antibody had developed, the rabbits were tested for susceptibility to experimental infective endocarditis. Rabbits with high titers of complement-fixing antibody to the infecting organism developed streptococcal endocarditis less often (13%) than animals with lower titers (69%; P less than 0.0002). These findings do not support the hypothesis that pre-immunization predisposes to infective endocarditis and lend no credence to the concept that vaccination of human subjects against dental caries might increase their susceptibility to streptococcal endocarditis. On the contrary, the results of these experiments indicate that specific antibody can confer relative immunity to infective endocarditis.     

Role of phagocytosis in activation of the coagulation system in Streptococcus sanguis endocarditis.

The formation of vegetations consisting of fibrin, cellular elements, humoral factors, and bacteria is the central event in the pathogenesis of bacterial endocarditis. Fibrin formation occurs on the vegetation, the coagulation system being activated locally via the expression of tissue factor (TF) on fibrin-adherent monocytes. This study was performed to assess the importance of phagocytosis of fibrin-adherent Streptococcus sanguis in the stimulation of TF expression on fibrin-adherent monocytes, as well as a role for "frustrated" phagocytosis. With the latter process, these cells are unable to remove bacteria from the fibrin surface but nonetheless might be activated to generate TF. We found that serum was not required for the stimulation of TF expression by fibrin-adherent monocytes in the presence of S. sanguis in an in vitro model for bacterial endocarditis. The bacterial adhesin dextran did not influence the TF activity (TFA) of fibrin-adherent monocytes: TFA was the same after stimulation with a dextran-positive streptococcus as with its dextran-negative mutant. Furthermore, dextran did not influence the TFA of endocardial vegetations, which was the same for vegetations isolated from rabbits infected either with dextran-positive S. sanguis or its dextran-negative mutant. These results do not support the hypothesis that in bacterial endocarditis (frustrated) phagocytosis significantly contributes to TF expression on vegetation-adherent monocytes. Fibronectin, however, although not influencing the fibrin binding of the streptococci, did enhance the TFA of monocytes in a concentration-dependent manner. We conclude that although streptococci do enhance expression of TFA on monocytes, phagocytosis and bacterial adhesins do not play a major role in this process. Stimulation of monocyte TFA may be more dependent on interactions between monocytes and the vegetational surface via fibronectin receptors, such as VLA 4 and VLA 5 (very late antigens 4 and 5).     

Role of granulocytes and monocytes in experimental Staphylococcus epidermidis endocarditis.

The role of granulocytes and monocytes during the induction and course of Staphylococcus epidermidis endocarditis was investigated by the selective depletion of monocytes with the drug VP16-213 and of both granulocytes and monocytes with nitrogen mustard. The induction of endocarditis was influenced only by the depletion of monocytes: the 50% infective dose differed significantly, being 3.4 X 10(5) CFU in control rabbits and 3.4 X 10(4) CFU in the monocyte-depleted rabbits, whereas no significant differences were found between the latter and those depleted of both granulocytes and monocytes. Also, control rabbits injected with 10(6) or 10(7) CFU had a significantly higher incidence of sterile vegetations than did rabbits selectively depleted of granulocytes or monocytes. Compared with baseline values, mean monocyte numbers at the time of bacterial inoculation were significantly increased in control rabbits whose vegetations remained sterile, whereas this effect was not seen in rabbits whose vegetations became infected. The course of the endocarditis appeared to be significantly influenced by both granulocytes and monocytes. Comparison showed that a decrease of the same numbers of these cells per microliter of blood was accompanied for the monocytes by an approximately fourfold higher increase of the number of staphylococci in the vegetations. The correlation between the number of granulocytes and of monocytes on the one hand and the number of staphylococci in the vegetations on the other was not substantially influenced by the duration of the disease or the number of staphylococci injected to induce the endocarditis. The number injected proved to be significantly correlated with the number of staphylococci in the vegetations. In rabbits with numbers of CFU per gram of vegetation exceeding 10(7), blood cultures were usually positive. This finding applied rarely to control rabbits, but generally to drug-treated rabbits. In the latter animals a significant correlation between the number of staphylococci in the vegetations and in the circulation was found. We conclude that only monocytes have a measurable effect on the induction of Staphylococcus epidermidis endocarditis but during its course both granulocytes and monocytes keep the endocardial infection in check.     

Role of granulocytes and monocytes in experimental Escherichia coli endocarditis.

The role of granulocytes and monocytes during the induction and course of Escherichia coli endocarditis was investigated in rabbits by selectively depleting monocytes from the circulation with the drug VP16-213 and granulocytes and monocytes with nitrogen mustard. For induction, the number of E. coli needed to infect the vegetations in 50% of the rabbits was significantly lower in rabbits with combined granulocytopenia and monocytopenia than in those with selective monocytopenia or in control rabbits, whereas the number of E. coli needed to infect 50% of the rabbits did not differ between the latter two. During the course of the endocarditis in endocardial vegetations, the numbers of CFU per gram of vegetation were significantly higher in the rabbits with combined granulocytopenia and monocytopenia than in the monocytopenic and control rabbits but did not differ between the latter two. The numbers of granulocytes in the circulation and the numbers of CFU per gram of vegetation showed a significant negative correlation that was not measurably influenced by the duration of the disease but was dependent on the number of E. coli injected for the induction of endocarditis. Granulocytes were found to be most effective at the lowest numbers of bacteria injected. In the circulation, too, the numbers of CFU per milliliter were significantly higher in rabbits with combined granulocytopenia and monocytopenia than in those with selective monocytopenia and control rabbits, and there was a significant negative correlation between the numbers of granulocytes and CFU per milliliter of blood. From these findings we conclude that granulocytes play a protective role during the induction and course of E. coli endocarditis in rabbits, whereas no role is demonstrable for monocytes.     

The platelet interactivity phenotype of Streptococcus sanguis influences the course of experimental endocarditis.

A strain of Streptococcus sanguis that induced rabbit platelets to aggregate in vitro (Agg+ phenotype) was hypothesized to be a more virulent pathogen than an Agg- strain in experimental endocarditis in rabbits. A left ventricular catheter was implanted, and then an Agg+ or Agg- strain was inoculated intravenously. Vegetations formed on the aortic semilunar valves but were unaffected by the duration of implantation of the catheter. Vegetations enlarged by accumulating platelets and their mass increased directly with the duration of endocarditis. Inoculation of the Agg+ strain consistently caused endocarditis with significantly larger vegetations, a more severe clinical course (including febrile episodes, hematological changes, and signs of myocardial ischemia), more gross lesions in major organs, and greater mortality than inoculation with the Agg- strain, saline, or the Agg+ strain pretreated with monospecific rabbit immunoglobulin G or Fab fragments against its platelet aggregation-associated protein (PAAP; class II). In experimental endocarditis, PAAP expressed by Agg+ S. sanguis appeared to be an important virulence factor.     

Experimental Streptococcus sanguis endocarditis: immune complexes and renal involvement.

An increase of agglutinins and circulating immune complexes (CIC) was observed in the sera of rabbits after the induction of bacterial endocarditis (BE) by intravenous injection of live streptococci into animals with a non-bacterial thrombotic endocarditis. Rabbits immunized for 12 days with heat-killed streptococci developed higher levels of agglutinins and CIC than did those unimmunized during BE. The levels remained elevated in the immunized rabbits after the induction of streptococcal endocarditis. In rabbits with very high levels of CIC complement activation occurred in vivo, possibly via the classical pathway. Kidneys from both unimmunized and immunized rabbits with streptococcal endocarditis contained bacteria and also showed inflammatory reactions but only the renal tissue of immunized rabbits with endocarditis showed a little mesangial deposition of immunoglobulin. The findings in this study indicate that in rabbits, renal inflammation due to BE is initiated more often by septic emboli than by immune complex deposition, at least during the first 12 days after infection.     

Protease production by Streptococcus sanguis associated with subacute bacterial endocarditis.

A viridans streptococcus (Streptococcus sanguis biotype II) isolated from the blood of a patient with subacute bacterial endocarditis was examined for protease production. In broth culture, extracellular proteolytic enzymes were not produced by this organism until after the early exponential phase of growth, with maximal protease production occurring during the stationary phase. Four distinct proteases were isolated and purified from the supernatant fluids of stationary-phase cultures, employing a combination of ion-exchange column chromatography, gel filtration column chromatography, and polyacrylamide gel electrophoresis. All four proteases could be eluted from a diethylaminoethyl cellulose column at a sodium chloride gradient concentration of 0.25 M but were separable by gel filtration chromatography on a Sephadex G-100 column. They varied in molecular weights as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis from approximately 13,000 to 230,000. All four proteases had pH optima of between 8.0 and 9.0, and two of the proteases were active against casein, human serum albumin, and gelatin but were not active against elastin and collagen. The remaining two proteases were able to degrade only casein and gelatin. These results show that S. sanguis is able to excrete maximal levels of potentially destructive enzymes when the organisms are not actively multiplying. This finding may explain some of the damage caused in heart tissue by these organisms during subacute bacterial endocarditis.     

Phenotypic characterization of Streptococcus sanguis virulence factors associated with bacterial endocarditis.

Certain strains of Streptococcus sanguis adhere (Adh+) selectively to human platelets and, in plasma, induce them to aggregate (Agg+) into in vitro thrombi. In this study, we examined 18 recent endocarditis and dental plaque isolates of microorganisms that were biotyped as S. sanguis for coexpression of platelet interactivity phenotypes with another possible virulence factor in bacterial endocarditis, dextran synthesis. Detectable production of extracellular glucosyltransferase ranged from 0.2 to 66 mU/mg of culture fluid for 10 representative strains tested. Production of extracellular or cell-associated glucosyltransferase, fructosyltransferase, and soluble or insoluble dextrans was not necessarily coexpressed with platelet interactivity phenotypes, since the levels of production of soluble and insoluble dextrans varied among representative Adh+ Agg+ and Adh- Agg- strains. Analysis of a second panel of 38 fresh dental plaque isolates showed that S. sanguis distributes in a reproducible manner into the possible phenotype groups. Strains with different platelet interactivity phenotypes were distinguished with a panel of four murine monoclonal antibodies (MAbs) raised against Adh+ Agg+ strain 133-79 and screened to rule out artifactual reactions with antigenic components in culture media. The MAbs reacted selectively with Adh+ Agg+ strains in a direct-binding, whole-cell, enzyme-linked immunosorbent assay and also inhibited their interactions with platelets. Analysis of minimal tryptic digests of many strains, including variants that failed to bind the MAbs, suggested that some noninteractivity phenotypes possess cryptic surface determinants. Since the ability to adhere to platelets and induce them to aggregate is relatively stable, these traits may be useful in a phenotyping scheme for these Lancefield nontypeable streptococci.     

Role of sialic acid in saliva-induced aggregation of Streptococcus sanguis.

The ability of saliva to induce aggregation of Streptococcus sanguis was destroyed by treating the saliva with protease or neuraminidase. Loss of aggregating activity could be correlated with the appearance of free sialic acid. Clarified saliva contains an endogenous neuraminidase that modifies aggregating activity. Aggregation was inhibited by mixed ganglioside preparations but less effectively by acid-hydrolyzed gangliosides. The aggregating activity of S. sanguis was not related to the rhamnose or phosphorous content of the cell wall or to antigen a, b, c, d, or e.     

Recombination-deficient Streptococcus sanguis.

A UV-sensitive derivative was obtained from Streptococcus sanguis Challis. The organism could be transformed with a number of small streptococcal plasmids at frequencies equal to, or 1 logarithm below, the transformation frequencies for the parent organism. However, transformation with chromosomal DNA was greatly impaired in the UV-sensitive derivative.     

Neuraminidase production by a Streptococcus sanguis strain associated with subacute bacterial endocarditis.

The properties of an extracellular neuraminidase produced by a Streptococcus sanguis strain (isolated from a confirmed case of subacute bacterial endocarditis) during growth in a defined medium was examined in this investigation. This enzyme, isolated from concentrated culture supernatants of S. sanguis biotype II, was active against human alpha-1 acid glycoprotein, N-acetylneuramin lactose, bovine submaxillary mucin, and fetuin. Neuraminidase production paralleled bacterial growth in defined medium and was maximal in the early stationary phase of growth but decreased dramatically, probably owing to protease production, during the late stationary phase. The enzyme was purified to near homogeneity by a combination of salt fractionation, ion-exchanged chromatography on DEAE-Sephacel, and gel filtration on Sephadex G-200. These procedures yielded an enzyme preparation that possessed a specific activity of 174.4 mumol of sialic acid released per min per mg of protein against human alpha-1 acid glycoprotein. The Km value for this enzyme with human alpha-1 acid glycoprotein as substrate was 2.5 X 10(-3) M, and the enzyme possessed a pH optimum of 6.5. The S. sanguis neuraminidase had a molecular weight of approximately 85,000 as estimated by gel filtration and approximately 90,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was stable at temperatures of 4 and 37 degrees C for 3 h, but approximately 50% of the enzymatic activity was lost within 30 min at 50 degrees C, with 100% of the enzymatic activity being destroyed within 10 min at temperatures of greater than or equal to 65 degrees C.     

Antigens of Streptococcus sanguis

An antigenic analysis of the alpha-hemolytic streptococci isolated from dental plaque was performed by use of antisera against a strain of Streptococcus sanguis (M-5) which was isolated from dental plaque. Immunoelectrophoretic and Ouchterlony tests of Rantz and Randall extracts of 45 strains gave positive reactions with the M-5 antisera. These strains represented 60% of the strains tested. The number of antigens which could be identified in these extracts varied from one to five and were designated a to e. The a antigen was found in 36 of the strains tested, including reference strains of S. sanguis and the group H streptococci. The strains reacting with the M-5 antisera were divided into two majors types: type I consisted of 23 strains in which the a antigen was found alone or with one or more of the c, d, and e antigens; type II consisted of 13 strains in which both the a and b antigens were found with or without one or more of the c, d, and e antigens. The remaining strains contained, either singly or in combination, the b, c, d, and e antigens but not the a antigen. Biochemical tests of representatives of each serotype and reference strains indicated that strains reacting with M-5 antisera were S. sanguis. These findings suggest that S. sanguis strains share common physiological and serological properties.     

Role of sialic acid in the kinetics of Streptococcus sanguis adhesion to artificial pellicle.

Evaluation of the kinetics of adhesion of Streptococcus sanguis 10556 to saliva-coated hydroxylapatite revealed that sialic acid played a role in the formation of a stable cell-substratum complex. In a previous paper (M. M. Cowan, K. G. Taylor, and R. J. Doyle, J. Dent. Res. 65:1278-1283, 1986) the adhesion was found to take place in two distinct stages: a reversible equilibrium, probably governed by long-range forces, followed by a transition to higher-affinity binding. In the present study, artificial pellicle was treated with neuraminidase, and kinetic adsorption and desorption experiments with S. sanguis were conducted. The depletion of sialic acid from pellicle decreased the initial adsorption rate constant only slightly. The rate constant describing the initial desorption was unaffected. However, no transition to the second (high-affinity) association occurred. While S. sanguis desorption from control pellicles exhibited two sequential rates, with the second rate being approximately 10 times slower than the first, all desorption from sialo-deficient pellicles occurred at one rate that was equivalent to the initial rate constant for control desorption. The cells did not reach an equilibrium with the sialo-deficient pellicle, even after 6 h. Competing sialic acid did not decrease the rate or extent of adsorption, but desorption occurred to a greater extent when cells had adsorbed in the presence of sialic acid. These data suggest that sialic acid plays little role in the initial association of cell and pellicle but that it is necessary for the transition to high-affinity binding and the concomitant decreased propensity to desorb.     

Immunoelectron microscopic identification and localization of Streptococcus sanguis with peroxidase-labeled antibody: localization of Streptococcus sanguis in intact dental plaque.

Streptococcus sanguis has been localized ultrastructurally within intact dental plaque by means of an indirect technique which utilizes horseradish peroxidase-labeled antibody. The technique allows for complete diffusion of the reagents to all portions of the plaque specimens. Control procedures can be carried out on serial sections of plaque with a bacterial composition similar to that of the experimental specimen. The 30-mum-thick sections can be examined in the light microscope to localize areas specifically labeled with peroxidase prior to cutting ultra-thin sections for electron microscopy. This study demonstrated that specific bacteria can be localized within intact dental plaque. The results also indicated that S. sanguis grows in dental plaque as columnar shaped microcolonies perpendicular to the tooth surfaces. Growth appears to be by cell division rather than deposition of new cells at the surfaces. Despite their relatively good structural preservation, the cells in the deeper (older) layers of plaque appear to have lost some of their antigenic activity in comparison to the cells near the surface.     

Characterization of salivary alpha-amylase binding to Streptococcus sanguis.

The purpose of this study was to identify the major salivary components which interact with oral bacteria and to determine the mechanism(s) responsible for their binding to the bacterial surface. Strains of Streptococcus sanguis, Streptococcus mitis, Streptococcus mutans, and Actinomyces viscosus were incubated for 2 h in freshly collected human submandibular-sublingual saliva (HSMSL) or parotid saliva (HPS), and bound salivary components were eluted with 2% sodium dodecyl sulfate. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer, alpha-amylase (EC 3.2.1.1) was the prominent salivary component eluted from S. sanguis. Studies with 125I-labeled HSMSL or 125I-labeled HPS also demonstrated a component with an electrophoretic mobility identical to that of alpha-amylase which bound to S. sanguis. Purified alpha-amylase from human parotid saliva was radiolabeled and found to bind to strains of S. sanguis genotypes 1 and 3 and S. mitis genotype 2, but not to strains of other species of oral bacteria. Binding of [125I]alpha-amylase to streptococci was saturable, calcium independent, and inhibitable by excess unlabeled alpha-amylases from a variety of sources, but not by secretory immunoglobulin A and the proline-rich glycoprotein from HPS. Reduced and alkylated alpha-amylase lost enzymatic and bacterial binding activities. Binding was inhibited by incubation with maltotriose, maltooligosaccharides, limit dextrins, and starch.     

Role of monocytes in experimental Staphylococcus aureus endocarditis

In the pathogenesis of bacterial endocarditis (BE), the clotting system plays a cardinal role in the formation and maintenance of the endocardial vegetations. The extrinsic pathway is involved in the activation of the coagulation pathway with tissue factor (TF) as the key protein. Staphylococcus aureus is a frequently isolated bacterium from patients with BE. We therefore investigated whether S. aureus can induce TF activity (TFA) on fibrin-adherent monocytes, used as an in vitro model of BE. We also assessed in vivo in rabbits with catheter induced vegetations, the effect of S. aureus infection on vegetational TFA. In vitro experiments showed that adherent S. aureus induced TFA on fibrin-adherent monocytes which was optimal at a bacterium/monocyte ratio of 1 to 1. Monocyte damage occurred when this ratio exceeded 4 to 1 (visually) or 6 to 1 (propidium iodide influx) Consequently, TFA decreased. In vivo S. aureus led to very high bacterial numbers in the vegetations and a significant increase of their weight. However, TFA of infected vegetations was the same as of sterile ones. This may be due to the high bacteria to monocyte ratio as well as bacterium-induced monocyte damage. Teicoplanin treatment of infected rabbits reduced bacterial numbers in the blood and in the vegetations. Two-day treatment resulted in an increase of vegetational TFA, but after four-day treatment vegetational TFA dropped, most probably due to a suboptimal bacterium/monocyte ratio. S. aureus endocarditis in etoposide (Vepesid)-treated rabbits, leading to a selective monocytopenia, caused a rapid death of the animals. In these rabbits no vegetations were found at all. We conclude that, like Streptococcus sanguis and Staphylococcus epidermidis, S. aureus is able to induce TFA in fibrin-adherent blood monocytes. In addition, monocytes have a protective effect during the course of S. aureus endocarditis.