Phenotypic resistance to thrombin-induced platelet microbicidal protein in vitro is correlated with enhanced virulence in experimental endocarditis due to Staphylococcus aureus.

Thrombin-induced platelet microbicidal protein (tPMP) is secreted by rabbit platelets following thrombin stimulation, and it kills common endovascular pathogens in vitro, including Staphylococcus aureus. Therefore, pathogens which exhibit tPMP resistance in vitro possess a potential survival advantage in vivo at sites of endovascular damage. We generated an isogenic S. aureus strain pair, differing in tPMP susceptibility, by transposon (Tn551) mutagenesis of a tPMP-susceptible (tPMPs) parental strain (ISP479) to derive a stably tPMP-resistant (tPMPr) strain, ISP479R. ISP479 and ISP479R were equivalent in vitro in the following phenotypes: biotyping, antiobiograms, platelet adherence and aggregation, growth kinetics, cell wall-associated protein A expression, and fibrinogen binding. Genotypic comparisons of chromosomal DNA of strains ISP479 and ISP479R following restriction endonuclease digestion revealed indistinguishable pulsed-field gel electrophoretic patterns. The genotype exhibited by strain ISP479R was linked to the tPMP-resistant phenotype, as it was transducible into the initially tPMP-susceptible parental strain, ISP479. Southern hybridization verified the presence of a single copy of Tn551 in the same chromosomal restriction site of both ISP479R and tPMPr transductants of ISP479. The correlation of in vitro tPMP susceptibility phenotypes with the ability to induce experimental endocarditis (a prototypical endovascular infection) was evaluated. Despite equivalent rates of endocarditis induction, animals infected with strain ISP479R achieved significantly higher vegetation bacterial densities over a 7-day post-challenge period than did animals infected with strain ISP479. These data suggest that tPMPr microbial strains have a selective advantage in experimental staphylococcal endocarditis. Furthermore, the major impact of tPMP resistance upon endocarditis pathogenesis appears to involve a postvalvular adherence event(s), most probably by facilitating bacterial proliferation within vegetations.     

Influence of preformed antibody on the pathogenesis of experimental Candida albicans endocarditis.

The influence of preformed antibody on the induction of experimental Candida albicans endocarditis was investigated by both in vitro and in vivo techniques. Preincubation of C. albicans with immune serum (raised in rabbits by intravenous injection of Formalin-killed yeast cells) decreased adhesion to the constituents of nonbacterial thrombotic endocarditis, e.g., fibrin plus platelets, in vitro. Two different methods, with radiolabeled or viable yeast cells, were confirmatory and demonstrated decreased adhesion of immune serum-treated C. albicans cells to 0 to 7.8% of control values (P less than 0.001). These results correlated with protection from the development of C. albicans endocarditis in the immunized rabbits. The mean (+/- standard deviation) infectious dose for 50% of the animals was 10(5.29) +/- 10(0.07) in 48 control animals versus 10(7.11) +/- 10(0.22) in 37 immunized rabbits (P less than 0.001). These studies suggest that humoral antibody may protect against C. albicans endocarditis, perhaps through inhibition of adhesion, a crucial early step in the pathogenesis of endocarditis.     

Resistance of T-cell receptor delta-chain-deficient mice to experimental Candida albicans vaginitis

Conditions consistent with tolerance or immunoregulation have been observed in experimental Candida albicans vaginal infections. The present study investigated the role of gamma/delta T cells in experimental vaginal candidiasis. Results showed that T-cell receptor delta-chain-knockout mice had significantly less vaginal fungal burden when compared to wild-type mice, suggesting an immunoregulatory role for gamma/delta T cells in Candida vaginitis.     

Detection of circulating antigen in experimental Candida albicans endocarditis by an enzyme-linked immunosorbent assay.

A double-antibody sandwich enzyme-linked immunosorbent assay was developed for the detection of circulating Candida albicans antigen during the course of experimental C. albicans endocarditis. The enzyme-linked immunosorbent assay was positive in 75% of rabbits with polyethylene catheter-induced experimental aortic valve C. albicans endocarditis but was negative in all controls, including catheterized animals that received intravenous Candida or catheterized but uninfected animals, and in rabbits with experimental fungal or bacterial endocarditis of other etiologies. The enzyme-linked immunosorbent assay was much more sensitive than blood culturing or fever determinations in experimental C. albicans endocarditis. This assay is more sensitive than currently available serological techniques, is highly specific, and deserves further study in the diagnosis of invasive, disseminated C. albicans infections, including endocarditis.     

Hyperproduction of alpha-toxin by Staphylococcus aureus results in paradoxically reduced virulence in experimental endocarditis: a host defense role for platelet microbicidal proteins.

Staphylococcal alpha-toxin targets several cell types which are important components of cardiac vegetations in endocarditis, including platelets, erythrocytes, and endothelial cells. We evaluated the in vivo role of Staphylococcus aureus alpha-toxin in experimental endocarditis by using isogenic strains differing in the capacity to produce functional alpha-toxin, including 8325-4 (wild-type strain), DU-1090 (a mutant strain with allelic replacement of the alpha-toxin gene [hla]), DU1090(pH35L) (a mutant strain producing a target cell-binding but nonlytic toxin), DU1090(pDU1212) (a variant of DU1090 carrying the cloned hla gene on a multicopy plasmid), and DU1090(pCL84::hla) (a variant of DU1090 with a single copy of the hla gene cloned into the chromosomal lipase locus). In vitro, wild-type alpha-toxin (from parental strain 8325-4) extensively lysed both erythrocytes and platelets. In contrast, mutant alpha-toxin [from strain DU1090(pH35L)] lysed neither cell type. Following exposure to the wild-type alpha-toxin, platelet lysates were found to contain microbicidal activity against Bacillus subtilis (but not against Micrococcus luteus), as well as against the parental and alpha-toxin variant S. aureus strains noted above. Furthermore, lysate microbicidal activity was heat stable, neutralized by polyanionic filters or compounds, and recoverable from anionic filter membranes by hypertonic saline elution. These characteristics are consistent with those of cationic platelet microbicidal proteins (PMPs). Reverse-phase high-pressure liquid chromatography and polyacrylamide gel electrophoresis confirmed the presence of three distinct PMPs (1, 2, and 3) in platelet lysates. In experimental endocarditis, the two variant staphylococcal strains producing either minimal alpha-toxin or nonlytic alpha-toxin in vitro [strains DU1090 and DU1090(pH35L), respectively] exhibited significantly lower virulence in vivo than the parental strain (decreased intravegetation staphylococcal densities). Paradoxically, the two variant staphylococcal strains producing alpha-toxin at supraparental levels in vitro [strains DU1090(p1212) and DU1090(pCL84::hla)] also exhibited significantly decreased induction rates and intravegetation staphylococcal densities in experimental endocarditis versus the parental strain. The reduced in vivo virulence of the latter variant staphylococcal strains could not be explained by differences in bacteremic clearance or initial adherence to sterile vegetations (compared to the parental strain). These findings suggest that the reduced virulence exhibited by the variant staphylococcal strains in this model was related to pathogenetic events subsequent to bacterial adherence to the damaged endocardium. Excess intravegetation secretion of alpha-toxin, leading to increased PMP release (secondary to either increased platelet secretion or lysis), may well explain the reduced virulence observed in experimental endocarditis.     

Experimental Candida albicans endocarditis: characterization of the disease and response to therapy.

Endocarditis caused by Candida albicans was induced in rabbits after insertion of a catheter across the aortic valve. The mean survival time of 34 rabbits was 26 days. Only 7% of temperature recordings taken were elevated. Candida was recovered from only 9% of blood cultures taken. Precipitating and agglutinating serum antibody was detected after 12 days of infection. Antibody titers rose progressively until death in rabbits with endocarditis, whereas titers peaked early and subsequently decreased in animals that received an intravenous injection of C. albicans without precatheterization. Three groups of rabbits were treated for 6 days with amphotericin B, 5-fluorocytosine, or the two durgs in combination. Amphotericin B alone reduced the mean titer of organisms from log10 8.79 +/- 1.46 to log 10 3.1 +/- 1.9 colony-forming units/g. 5-Fluorocytosine was less effective (mean titer after 6 days of therapy was log10 7.4 +/- 0.33 colony-forming units/g). The addition of 5-fluorocytosine to amphotericin B did not increase the rate at which Candida cells were eradicated from the vegetations. These in vivo results corrleated with the failure to demonstrate an increased rate of fungicidal activity in vitro with the two drugs.     

Staphylococcus aureus susceptibility to thrombin-induced platelet microbicidal protein is independent of platelet adherence and aggregation in vitro.

Bacterium-platelet interactions at the cardiac valve surface represent an important initial step in the induction of infective endocarditis (IE). This cell-cell interaction may play either a protagonistic role in the induction of IE via bacterial adherence to and aggregation of platelets or an antagonistic role via secretion of platelet-derived microbicidal molecules. We examined the spectrum and interrelationship of three aspects of the interaction of 20 clinical Staphylococcus aureus isolates with rabbit platelets in vitro: (i) S. aureus adherence to platelets; (ii) S. aureus-induced platelet aggregation; and (iii) S. aureus resistance to the action of thrombin-induced platelet microbicidal protein (PMP; low-molecular-weight cationic peptides contained in alpha granules). Among the 20 S. aureus isolates (11 bacteremia, 9 endocarditis), there was a heterogeneous distribution profile for each of the bacterium-platelet interaction parameters studied. For S. aureus-platelet adherence and S. aureus-induced platelet aggregation, 3 of 20 and 7 of 20 isolates tested were considered highly active for each respective parameter; 5 of 20 staphylococcal strains were deemed resistant to the bactericidal action of PMP. In addition, more endocarditis isolates (45%) were PMP resistant than strains from patients without endocarditis (19%). When analyzed concomitantly, there was a significant, positive correlation between S. aureus-platelet adherence and S. aureus-induced platelet aggregation among isolates (P = 0.003; r = 0.78). In contrast, there were no statistically significant relationships between either platelet adherence or aggregation and PMP resistance among these 20 S. aureus isolates. These data suggest that platelet adherence and aggregation are related abilities of S. aureus, while resistance to thrombin-induced PMP is an independent phenotypic characteristic and potential virulence factor.     

Candida albicans endocarditis possibly related to systemic candidiasis in a heroin addict

A case of Candida albicans endocarditis is described which developed in a heroin addict with aortic valvulopathy after an episode of cutaneous and chondrocostal candidiasis related to the use of brown heroin. To our knowledge this is the first case reported in the English literature. This complication should be suspected in all heroin addicts with this new syndrome, especially if valvulopathy is present.     

Pathogenicity of Candida albicans auxotrophic mutants in experimental infections.

Auxotrophic and prototrophic control strain pairs of Candida albicans constructed by molecular biology methodologies were evaluated for pathogenicity in a systemic mouse model. Mutants that were auxotrophic for adenine, uracil, and heme each showed a lowered level of pathogenicity relative to control strains. It can be concluded from these experiments that decreased pathogenicity in each case is due to the auxotrophic mutation, because mutant and control strains were constructed so as to differ at a single locus. These observations suggest that new therapeutic agents for Candida infections might be designed based upon the inhibition of biosynthetic pathways that, in some cases, might be absent from the host.     

Partial characterization and staphylocidal activity of thrombin-induced platelet microbicidal protein.

Thrombin-induced platelet microbicidal protein (PMP) is considered to play an important role in preventing an important role in preventing streptococcal endocarditis. However, the structural features and functions of PMPs have not been well characterized, and their antibacterial spectra against other common endocarditis pathogens, such as the staphylococci, are not known. Thrombin stimulation of washed rabbit platelets (10(8)/ml) yielded a PMP-rich preparation with a specific activity of approximately 25 U/mg of protein as determined by Bacillus subtilis bioassay. Twenty-eight clinical and laboratory Staphylococcus aureus isolates, exposed to a standardized PMP preparation (100 U/ml for 2 h at 37 degrees C), exhibited a Poisson-distributed heterogeneity to the bactericidal action of PMP, with approximately one-third designated as PMP resistant. Gel filtration chromatography (Sephadex G-50) identified the bioactive moiety within PMP preparations to be in the major protein elution peak; sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) presumptively identified PMP as a low-molecular-weight (MW) (8,500) protein present only in such bioactive protein peaks. Both the bioactivity of PMP preparations and the low-MW protein band were removable by specific anionic membranes (e.g., cellulose-acetate/nitrate), as well as by a variety of anionic resins, further corroborating the suspected cationic charge of PMP. In addition, both PMP bioactivity and the low-MW protein band were recoverable by 1.5 M NaCl elution of the anionic membrane filters post-PMP adsorptive removal. Adsorption of bioactive PMP preparations by highly PMP-susceptible B. subtilis (10(8) CFU/ml, 30 min) resulted in a near-complete loss of residual bioactivity; in contrast, adsorption of bioactive PMP preparations with less PMP-susceptible S. aureus strains failed to reduce bioactivity. Significant lysozyme contamination of PMP-rich preparations was ruled out by determination of differences between bioactive PMP preparations and exogenous lysozyme as regards (i) relative heat stabilities; (ii) differential bactericidal activity versus B. subtilis and Micrococcus luteus; and (iii) SDS-PAGE protein profiles. These data show that the bioactive PMP protein moiety is of low MW, is heat stable, is probably cationic (similar to leukocyte-derived defensins), and possesses potent bactericidal activity against a significant percentage of S. aureus isolates.     

Lymphocyte adhesion to Candida albicans.

Adherence of lymphocytes to the fungus is the first step in the direct lymphocyte-mediated antifungal effect against Candida albicans. In this study we identified macrophage-1 antigen (Mac-1) (CD11b/CD18, alpha(M)/beta(2)) as the lymphocyte surface structure responsible for the adhesion of activated lymphocytes to the hyphal form of the fungus. Antibodies specific for epitopes of the alpha-subunit (CD11b) and the beta(2)-subunit (CD18) of Mac-1 were shown to completely eliminate lymphocyte adhesion to C. albicans hyphae. Lymphocyte adhesion to C. albicans was also inhibited significantly by known ligands of Mac-1, including the extracellular matrix proteins laminin and fibrinogen, as well as engineered peptides containing arginine-glycine-aspartic acid sequences and the disintegrin echistatin. N-Acetyl-D-glucosamine and beta-glucan, which inhibit Mac-1-mediated adhesion to the yeast, blocked lymphocyte adhesion to hyphae. NIH 3T3 fibroblast transfectants expressing human CD11b/CD18 bound to C. albicans, and their binding was inhibited by antibodies specific for CD11b/CD18. Finally, antibodies specific for CD11b/CD18 effectively inhibited the capacity of activated lymphocytes to have an antifungal effect against hyphae. Our results clearly identify Mac-1 (CD11b/CD18) as the lymphocyte surface structure that mediates activated lymphocyte adhesion to C. albicans and the resultant antifungal effect of the lymphocytes.     

Relationship between germination of Candida albicans and increased adherence to human buccal epithelial cells.

A strong correlation was shown between germination and increased adherence of Candida albicans to human buccal epithelial cells, indicating that germination or other changes in the fungi accompanying germination were responsible for enhanced adherence. Partial inhibition of germination by cysteine resulted in a comparably lower adherence. Preferential adherence of germinated fungi occurred in competition assays with nongerminated and germinated fungi. The enhanced adherence to human mucosal cells of germinated C albicans could represent one mechanism contributing to the pathogenicity of the organism.     

Role of granulocytes in increased host resistance to Candida albicans induced by recombinant interleukin-1.

The effect of human recombinant interleukin-1 alpha (IL-1 alpha) on a systemic candidal infection in mice under various conditions of immunosuppression was investigated. In normal mice and in mice pretreated with cyclophosphamide, hydrocortisone acetate, or sublethal total body irradiation, the outgrowth of Candida albicans in the kidney was significantly reduced by the administration of a single intraperitoneal dose of 80 ng of IL-1 (P less than 0.05). In mice treated with either cyclophosphamide or irradiation, IL-1 also significantly reduced the outgrowth of C. albicans in the spleen. The protective effect of IL-1 was present when given 24 h before injection of C. albicans but also when IL-1 was given simultaneously with or 6 h after injection of C. albicans in cyclophosphamide-treated mice. The effect of IL-1 was independent of the presence or recruitment of granulocytes, since IL-1 inhibited the outgrowth of C. albicans in the kidneys and spleens of mice that were rendered severely granulocytopenic (less than 50 granulocytes per mm3) throughout the duration of the infection by either repeated injections of cyclophosphamide or sublethal total body irradiation. These results indicate that the enhancement of host resistance by IL-1 is not due solely to increased granulocytopoiesis or chemotaxis of granulocytes but strongly suggest that other mechanisms play a role in the protective effect of IL-1 against systemic infections.     

Influence of platelets and platelet microbicidal protein susceptibility on the fate of Staphylococcus aureus in an in vitro model of infective endocarditis

Several lines of evidence indicate that platelets protect against endovascular infections such as infective endocarditis (IE). It is highly likely that a principal mechanism of this platelet host defense role is the release of platelet microbicidal proteins (PMPs) in response to agonists generated at sites of endovascular infection. We studied the ability of platelets to limit the colonization and proliferation of Staphylococcus aureus in an in vitro model of IE. Three isogenic S. aureus strains, differing in their in vitro susceptibility to thrombin-induced platelet microbicidal protein-1 (tPMP), were used: ISP479C (parental strain; highly susceptible to tPMP [tPMP(s)]); ISP479R (transposon mutant derived from ISP479; tPMP resistant [tPMP(r)]); or 757-5 (tPMP(r) transductant of the ISP479R genotype in the ISP479 parental background). Time-kill assays and in vitro IE models were used to examine the temporal relationship between thrombin-induced platelet activation and S. aureus killing. In time-kill studies, early platelet activation (30 min prior to bacterial exposure) correlated with a significant bactericidal effect against tPMP(s) ISP479C (r(2) > 0.90, P < 0.02) but not against tPMP(r) strains, ISP479R or 757-5. In the IE model, thrombin activation significantly inhibited proliferation of ISP479C within simulated vegetations compared to strains ISP479R or 757-5 (P < 0.05). The latter differences were observed despite there being no detectable differences among the three S. aureus strains in initial colonization of simulated vegetations. Collectively, these data indicate that platelets limit intravegetation proliferation of tPMP(s) but not tPMP(r) S. aureus. These findings underscore the likelihood that platelets play an important antimicrobial host defense role in preventing and/or limiting endovascular infections due to tPMP(s) pathogens.     

Multiple experimental designs to evaluate the role of T-cell-mediated immunity against experimental vaginal Candida albicans infection.

Studies to date suggest a limited protective role for Candida-specific Th1-type cell-mediated immunity (CMI) against Candida albicans vaginitis, despite protection against other mucosal Candida infections. Recent evidence suggests this may be due to immunoregulatory mechanisms that inhibit a more profound CMI response against C. albicans vaginal infections. The present study was designed to conduct an evaluation of the protective role of CMI against experimental C. albicans vaginitis using multiple approaches, including the use of T-cell-immunodeficient (SCID, Nude) and knockout (CD4) mice and several immunization designs in immunocompetent mice. Results showed, with few exceptions, that most T-cell-immunodeficient or knockout mice had a vaginal fungal burden similar to that of wild-type strains throughout the observation period. In addition, no correlation was observed between vaginal T-helper and proinflammatory cytokines and fungal burden, suggesting a generalized state of immunoregulation. Evaluation of the effects of various immunization designs that included different Candida antigens, routes of delivery and strains of mice yielded no protection against vaginal candidiasis. These studies provide further evidence of a lack of a protective role of T cells against C. albicans vaginitis, and continue to support the concept of immunoregulation against vaginal CMI responses.     

Pathogenicity of morphological and auxotrophic mutants of Candida albicans in experimental infections.

The relative pathogenicities of yeast and mycelial forms of Candida albicans were determined after intravenous injection of the two forms into mice. Yeast and mycelial forms of C. albicans CMI45348 were prepared in chemostat culture. Both morphological forms were pathogenic, but the histology of kidney sections always showed a mixture of the yeast and mycelial elements of the organism. Similarly, infection of mice with prototrophic strains produced a mixture of morphological forms at the site of infection. The yeast (CA2) and mycelial (hOG301) morphological mutants of C. albicans were pathogenic, and sections from the kidneys of the infected mice showed that the mutants retained their original morphological forms. These data indicate that both the yeast and mycelial forms of C. albicans can adhere, invade, and proliferate in an infected host. Auxotrophic diploid mutants were nonpathogenic. However, construction of a prototrophic tetraploid strain from two auxotrophs restored the pathogenicity of the organism.     

Comparison between methods for serotyping of Candida albicans produces discrepancies in results.

Serotyping of 101 clinical isolates of Candida albicans was done with two sets of Hasenclever original anti-Candida typing sera (HSN 1 and 2) and Iatron Candida Check factor 6 typing serum (IF6). The results of these two methods were compared with slide agglutination reactions of yeast with monoclonal antibody H9. Agglutination reactions with this antibody have been previously shown to correlate with serotype. Results indicate the following correlations: between HSN 1 and HSN 2 serotyping, 93% (kappa = 0.85; 95% confidence interval [CI], 0.70 to 0.99); between IF6 and HSN 1, 60% (kappa = 0.39, 95% CI, 0.19 to 0.58); and between IF6 and HSN 2, 74% (kappa = 0.77; 95% CI, 0.64 to 0.90). Results with HSN 1 and 2 antisera correlated with H9 reactivity at 85 and 89% (kappa = 0.88; 95% CI, 0.75 to 1.00; and kappa = 0.85; CI, 0.70 to 0.99, respectively), while agreement between IF6 and H9 reactivities was less than or equal to 64% (kappa less than or equal to 0.43; 95% CI, 0.14 to 0.60). Autoagglutination of yeast during IF6 serotyping occurred with 21 of the 101 (20.8%) yeast strains. In every case, these yeast strains were serotyped by the HSN methods without autoagglutination and were uniformly type B. This study implies that it may not be possible to make valid comparisons between studies which compare serotype prevalence unless the same methods are used to serotype the yeast. The practicality and utility of serotyping in epidemiological studies are discussed, as are some of the problems associated with the available methods.     

Expression of Candida albicans SAP1 and SAP2 in experimental vaginitis.

Several strains of Candida albicans were compared for their ability to cause vaginal infection in a rat model, and their vaginopathic potentials were correlated with the expression of two aspartyl proteinases genes (SAP1 and SAP2) and adherence in vivo to the vaginal epithelium. Dot blot reactions and Northern blot analysis with RNA extracted from the vaginal fluid of rats infected with the highly vaginopathic strains H12 and 10261 demonstrated the expression of both SAP1 and SAP2 during the first week of infection. In contrast, neither gene was expressed during infection by a nonvaginopathic strain (N), even though the organism could be recovered during the first 24 h postinfection. A moderately vaginopathic strain (P) also expressed both genes, but the level of SAP1 mRNA appeared to decrease prior to that of SAP2. Neither gene was expressed, even by the highly vaginopathic strains, after the first week of infection, concomitant with a decrease in the number of organisms recovered from the vaginas. Analysis of in vivo adherence showed that the nonvaginopathic strain (N) adhered to vaginal epithelial cells less readily than the highly vaginopathic strain (H12) and moderately vaginopathic strain (P). Thus, in addition to its inability to express SAP1 and SAP2 in vivo, the nonvaginopathic strain does not colonize host cells to the same extent as the other strains tested. Our results demonstrate the early in vivo expression of two aspartyl proteinase gene during candidal vaginitis and suggest its association with the establishment of a vaginal infection.     

Biotyping of Candida albicans: results of an international collaborative survey.

An agar plate system for biotyping isolates of Candida albicans was evaluated in four laboratories for 18 coded yeast isolates, each tested in triplicate on duplicate series of agar plates. The results showed that the biotyping system gave excellent intralaboratory reproducibility. However, because the concordance of data among laboratories was poor, the method must be regarded as suitable only for research applications and not for routine use.