Role of Staphylococcus aureus coagulase and clumping factor in pathogenesis of experimental endocarditis.

The pathogenic role of staphylococcal coagulase and clumping factor was investigated in the rat model of endocarditis. The coagulase-producing and clumping factor-producing parent strain Staphylococcus aureus Newman and a series of mutants defective in either coagulase, clumping factor, or both were tested for their ability (i) to attach in vitro to either rat fibrinogen or platelet-fibrin clots and (ii) to produce endocarditis in rats with catheter-induced aortic vegetations. In vitro, the clumping factor-defective mutants were up to 100 times less able than the wild type strain to attach to fibrinogen and also significantly less adherent than the parents to platelet-fibrin clots. Coagulase-defective mutants, in contrast, were not altered in their in vitro adherence phenotype. The rate of in vivo infection was inoculum dependent. Clumping factor-defective mutants produced ca. 50% less endocarditis than the parent organisms when injected at inoculum sizes infecting, respectively, 40 and 80% (ID40 and ID80, respectively) of rats with the wild-type strain. This was a trend at the ID40 but was statistically significant at the ID80 (P < 0.05). Coagulase-defective bacteria were not affected in their infectivity. Complementation of a clumping factor-defective mutant with a copy of the wild-type clumping factor gene restored both its in vitro adherence and its in vivo infectivity. These results show that clumping factor plays a specific role in the pathogenesis of S. aureus endocarditis. Nevertheless, the rate of endocarditis with clumping factor-defective mutants increased with larger inocula, indicating the contribution of additional pathogenic determinants in the infective process.     

Study of Staphylococcus aureus pathogenic genes by transfer and expression in the less virulent organism Streptococcus gordonii

Because Staphylococcus aureus strains contain multiple virulence factors, studying their pathogenic role by single-gene inactivation generated equivocal results. To circumvent this problem, we have expressed specific S. aureus genes in the less virulent organism Streptococcus gordonii and tested the recombinants for a gain of function both in vitro and in vivo. Clumping factor A (ClfA) and coagulase were investigated. Both gene products were expressed functionally and with similar kinetics during growth by streptococci and staphylococci. ClfA-positive S. gordonii was more adherent to platelet-fibrin clots mimicking cardiac vegetations in vitro and more infective in rats with experimental endocarditis (P < 0.05). Moreover, deleting clfA from clfA-positive streptococcal transformants restored both the low in vitro adherence and the low in vivo infectivity of the parent. Coagulase-positive transformants, on the other hand, were neither more adherent nor more infective than the parent. Furthermore, coagulase did not increase the pathogenicity of clfA-positive streptococci when both clfA and coa genes were simultaneously expressed in an artificial minioperon in streptococci. These results definitively attribute a role for ClfA, but not coagulase, in S. aureus endovascular infections. This gain-of-function strategy might help solve the role of individual factors in the complex the S. aureus-host relationship.     

SpA, ClfA, and FnbA genetic variations lead to Staphaurex test-negative phenotypes in bovine mastitis Staphylococcus aureus isolates

Staphylococcus aureus encodes many proteins that act as virulence factors, leading to a variety of diseases, including mastitis in cows. Among these virulence factors, SpA, ClfA, ClfB, FnbA, and FnbB are important for the ability of S. aureus to adhere to and invade host cells as well as to evade host immune responses. The interaction between these S. aureus surface proteins and human immunoglobulin G and fibrinogen that are coupled to latex particles is utilized to induce latex agglutination reactions, which are used widely in diagnostic kits for confirmation of presumptive S. aureus isolates. In this study, the Staphaurex latex agglutination test was performed on a collection of confirmed bovine mastitis S. aureus isolates. Notably, 54% (43/79 isolates) of these isolates exhibited latex agglutination-negative phenotypes (Staphaurex-negative result). To gain insights into the reasons for the high frequency of Staphaurex-negative bovine mastitis S. aureus isolates, the spa, clfA, clfB, fnbA, and fnbB genes were examined. Specific genetic changes in spa, clfA, and fnbA, as well as a loss of fnbB, which may impair SpA, ClfA, FnbA, and FnbB functions in latex agglutination reactions, were detected in Staphaurex-negative S. aureus isolates. The genetic changes included a premature stop codon in the spa gene, leading to a truncated SpA protein that is unable to participate in S. aureus cell-mediated agglutination of latex particles. In addition, clfA and fnbA genetic polymorphisms were detected that were linked to ClfA and FnbA amino acid changes that may significantly reduce fibrinogen-binding activity. The genetic variations in these S. aureus isolates might also have implications for their bovine mastitis virulence capacity.     

Impacts of sarA and agr in Staphylococcus aureus strain Newman on fibronectin-binding protein A gene expression and fibronectin adherence capacity in vitro and in experimental infective endocarditis

We investigated the impacts of sarA and agr on fnbA expression and fibronectin-binding capacity in Staphylococcus aureus in vitro and in experimental endocarditis. Although sarA up-regulated and agr down-regulated both fnbA expression and fibronectin binding in vitro and in vivo, fnbA expression was positively regulated in the absence of both global regulators. Thus, additional regulatory loci contribute to fnbA regulation and fibronectin-binding capacities in S. aureus.     

Antibodies to capsular polysaccharides are not protective against experimental Staphylococcus aureus endocarditis.

The protective efficacy of antibodies to the Staphylococcus aureus capsular polysaccharide was examined in a rat model of catheter-induced endocarditis. Capsular antibodies were induced either by active immunization with killed S. aureus or by passive immunization with hyperimmune rabbit antiserum to S. aureus. Control rats were injected with phosphate-buffered saline or passively immunized with normal rabbit serum or rabbit antiserum to a nonencapsulated strain. Animals with indwelling catheters were challenged intravenously with 5 x 10(4) to 4 x 10(6) CFU of the homologous S. aureus strain (capsular serotype 5 strain Reynolds or serotype 1 strain SA1 mucoid). Both immunized and control rats developed S. aureus endocarditis. The numbers of S. aureus cells recovered from the blood and aortic valve vegetations of immunized rats were similar to those of control rats, indicating that capsule-specific antibodies were not protective. To determine whether the presence of an indwelling catheter interfered with antibody-mediated protection against S. aureus endocarditis, catheters were removed 2 h after insertion in additional groups of rats. An inoculum of 10(8) CFU of strain Reynolds was needed to provoke endocarditis in rats catheterized for 2 h, compared with 5 x 10(4) CFU for rats with indwelling catheters. Passively transferred capsular antibodies were not protective since both immunized and nonimmunized animals developed endocarditis, and quantitative cultures of blood and valvular vegetations revealed no differences between immunized and control animals. The findings of this study indicate that antibodies to the capsular polysaccharide are not protective in the rat model of experimental S. aureus endocarditis.     

Role of the sar locus of Staphylococcus aureus in induction of endocarditis in rabbits.

A regulatory locus on the Staphylococcus aureus chromosome, designated sar, is involved in the expression of cell wall proteins, some of which are potentially important in the pathogenesis of endocarditis. For instance, mutant 11D2 (sar::Tn917LTV1) was found to bind substantially less to matrix proteins (i.e., fibrinogen and fibronectin) than parent strain DB. Remarkably, these two strains did not differ in other phenotypes considered important in the initiation of endocarditis (e.g., binding to platelets and resistance to platelet-derived microbicidal proteins). The isogenic pair were compared for pathogenicity in a rabbit endocarditis model. There were significant differences in infectivity rates between the two strains (71 and 88% for DB versus 17 and 42% for mutant 11D2 at inocula of 10(3) and 10(4) CFU, respectively). In early adherence studies, parent DB adhered substantially better than the mutant to valvular vegetations at an inoculum of 10(6) CFU (P = 0.05). Southern blot analysis of colonies indicated that the location of the Tn917LTV1 insert in mutant 11D2 remained stable after animal passage. In vitro adherence assays revealed that mutant 11D2 was less adherent to cultured human endothelium than parent DB. These studies suggest that the sar locus is involved in the initial adherence of S. aureus to the fibrin-platelet-endothelium matrix on damaged valvular endothelium.     

Reduced adherence to traumatized rat heart valves by a low-fibronectin-binding mutant of Staphylococcus aureus.

Isogenic strains of Staphylococcus aureus, differing in fibronectin binding, were constructed for studies of the contribution of fibronectin binding to the in vivo pathogenesis of staphylococcal disease. Mutagenesis of S. aureus 879R4S was accomplished by mating with Enterococcus faecalis FA378 that carried the transposon Tn918. Four low-fibronectin-binding mutants were identified that bound 24- to 35-fold less fibronectin than the parent strain did. A spectinomycin-resistant strain, R4SSp, was transduced by a bacteriophage (80 alpha) lysate propagated on a low-binding mutant of 879R4S to produce R4SSp/1536, which bound less fibronectin, contained a single copy of the transposon, and grew on spectinomycin-containing medium. Using a rat model of endocarditis, we determined the distribution of S. aureus R4SSp and its transductant in normal and cardiac catheterized rats. Cultures of heart tissue showed that catheterized rats challenged with the fibronectin-binding parent strain had over 250-fold more organisms in the left heart than did rats challenged with the low-binding transductant. The ability to bind fibronectin had no effect on the number of S. aureus cells cultured from other tissues. These data suggest that the ability of S. aureus to bind fibronectin is an important factor in establishing adherence to damaged heart valves in vivo.     

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.     

Induction of experimental endocarditis by continuous low-grade bacteremia mimicking spontaneous bacteremia in humans

Transient high-grade bacteremia following invasive procedures carries a risk of infective endocarditis (IE). This is supported by experimental endocarditis. On the other hand, case-control studies showed that IE could be caused by cumulative exposure to low-grade bacteremia occurring during daily activities. However, no experimental demonstration of this latter possibility exists. This study investigated the infectivity in animals of continuous low-grade bacteremia compared to that of brief high-grade bacteremia. Rats with aortic vegetations were inoculated with Streptococcus intermedius, Streptococcus gordonii or Staphylococcus aureus (strains Newman and P8). Animals were challenged with 10(3) to 10(6) CFU. Identical bacterial numbers were given by bolus (1 ml in 1 min) or continuous infusion (0.0017 ml/min over 10 h). Bacteremia was 50 to 1,000 times greater after bolus than during continuous inoculation. Streptococcal bolus inoculation of 10(5) CFU infected 63 to 100% vegetations compared to 30 to 71% infection after continuous infusion (P > 0.05). When increasing the inoculum to 10(6) CFU, bolus inoculation infected 100% vegetations and continuous infusion 70 to 100% (P > 0.05). S. aureus bolus injection of 10(3) CFU infected 46 to 57% valves. This was similar to the 53 to 57% infection rates produced by continuous infusion (P > 0.05). Inoculation of 10(4) CFU of S. aureus infected 80 to 100% vegetations after bolus and 60 to 75% after continuous infusion (P > 0.05). These results show that high-level bacteremia is not required to induce experimental endocarditis and support the hypothesis that cumulative exposure to low-grade bacteremia represents a genuine risk of IE in humans.     

Influence of Staphylococcus aureus antibody on experimental endocarditis in rabbits.

To evaluate the potential protective benefit of antibody to whole cells of Staphylococcal aureus for the prevention of endocarditis, the rabbit endocarditis model was used. Methicillin-sensitive (17A) and methicillin-resistant (173) S. aureus strains were evaluated in rabbits with or without indwelling intracardiac catheters. All immunized rabbits developed significant homologous agglutinating antibody titers (the mean reciprocal titers were 15,300 to strain 17A and 1,150 to strain 173). After challenge, virtually no significant differences were observed between immunized and unimmunized animals with respect to (i) incidence of endocarditis, (ii) concentration of bacteria in infected vegetations, (iii) incidence of metastatic renal abscesses, or (iv) concentrations of bacteria in infected kidneys. The clearance of homologous S. aureus strains from blood cultures was similar for immunized and unimmunized animals at 10 to 90 min after intravenous challenge. In vivo adherence of homologous S. aureus strains to aortic valves and vegetations was similar in immunized and unimmunized animals when evaluated at 30 and 90 min postchallenge. Even without catheterization, the incidence of bacteremia and renal abscesses was the same in immunized and unimmunized rabbits. Whole-cell-induced S. aureus antibody did not prevent or modify any stage in the development of endocarditis in rabbits.     

Detection by PCR of adhesins genes and slime production in clinical Staphylococcus aureus

The presence of the ica loci and adhesins genes in clinical Staphylococcus aureus strains were considered important factors of virulence. In this study, 46 strains of Staphylococcus aureus were isolated from auricular infection, and were investigated for slime production using Congo Red Agar method (CRA). In order to detect the adhesins genes (ica A, ica D, fnb A, cna, Clf A) Polymerase Chain Reaction was used. Qualitative biofilm production of S. aureus using CRA plates revealed that 56.5% of strains were slime producers. In addition 78.26% of strains were ica A and ica D positive. While the fnbA gene was present in 76.1% of isolated strains. Furthermore, 56.5% of strains have the cna gene and 30.4% were clfA positives. Overall this study confirms the presence of fnb A and ica A/ica D genes in the majority of studies S. aureus strains isolated from Staphylococcal sepsis. ((c) 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).     

Adsorption of fibronectin onto polymethylmethacrylate and promotion of Staphylococcus aureus adherence

Recent data suggest that fibronectin may favor Staphylococcus aureus infection by promoting attachment to either injured tissues or implanted foreign bodies. We studied the quantitative adsorption of fibronectin onto polymethylmethacrylate (PMMA) cover slips by using a 125I-labeled preparation of the purified plasma glycoprotein. Fibronectin in buffer solutions showed a high affinity to PMMA coverslips. Adherence of S. aureus Wood 46 was studied on PMMA pre-exposed to fibronectin, using an assay specifically adapted to the cover slip model. Whereas S. aureus adherence in an albumin-containing buffer was less than or equal to 10(3) CFU on control uncoated cover slips, adherence in the same medium increased up to maximum of 7.7 X 10(4) CFU on cover slips preincubated in a solution of fibronectin (125-micrograms/ml). At intermediate fibronectin concentrations, bacterial adherence was a linear function of both the quantity in solution and of the quantity adsorbed on the PMMA cover slips. The presence of human serum proteins, as represented by a fibronectin-depleted pool, essentially prevented adsorption of radiolabeled fibronectin on PMMA and subsequent bacterial adherence on the cover slips. Precoating of PMMA with denatured collagen resulted in increased fibronectin adsorption on PMMA, even in the presence of serum proteins, and S. aureus adherence was optimal on such surfaces. Collagen may therefore play a role as a cofactor contributing to S. aureus adherence onto fibronectin-coated substrata or foreign bodies.     

Platelet microbicidal protein alone and in combination with antibiotics reduces Staphylococcus aureus adherence to platelets in vitro.

Bacterial adherence to platelets on the cardiac valve surface is believed to be critical in the induction of infective endocarditis. Recent studies have confirmed that thrombin-activated platelets secrete platelet microbicidal protein (PMP), which can both kill and exert nonlethal antiadherence effects against endovascular pathogens. In the present study, we quantified the influence of antibiotic and/or PMP exposures on in vitro platelet adherence of two Staphylococcus aureus strains, identical by DNA restriction and cell wall protein profiles, that differed in their susceptibility to PMP-induced killing (PMPs or PMPr, respectively). Adherence assays were performed by flow cytometry in the presence of sublethal PMP concentrations (1 to 2.5 micrograms/ml) alone or in combination with ampicillin (AMP) alone, sulbactam (SUL) alone, or AMP plus SUL (AMP-SUL), at levels achievable in serum. Exposure of the PMPs and PMPr S. aureus strains to antibiotics (for 2 h at 37 degrees C) prior to flow cytometry resulted in no substantive changes in the percent adherence to platelets compared with that for S. aureus cells not exposed to antibiotics, except for modestly increased adherence of both PMPs and PMPr cells exposed to AMP-SUL (18.5 and 15.8% increases, respectively). Addition of PMP to antibiotic-S. aureus mixtures (final 30 min) caused a significant decrease in S. aureus adherence to platelets, for both the PMPs and PMPr S. aureus strains, compared with antibiotic exposure alone (e.g., reduction in platelet adherence from 57.9 +/- 8.2% to 12.2 +/- 3.6% for PMPs cells exposed to AMP-SUL and PMP [P = 0.01]). Moreover, addition of PMP following exposure of the PMPs and PMPr strains to AMP-SUL reversed the enhanced bacterium-platelet adherence observed with such antibiotic exposures alone (P < or = 0.005). These data demonstrate that PMP exerts a potent antiplatelet adherence effect which is independent of its microbicidal capacity, rendering S. aureus cells less adherent to platelets in the presence or absence of antibiotics. Reduction of microbial adherence to platelets by PMP alone or with antibiotics provides further insight into the mechanism(s) that may be involved in host defense and antibiotic prophylaxis of infective endocarditis and other endovascular infections.     

Reconsideration of the role of fibronectin binding in endocarditis caused by Staphylococcus aureus.

The adherence characteristics in vivo and virulence of two isogenic strains of Staphylococcus aureus differing in fibronectin binding were compared in a rat model of catheter-induced infective endocarditis. No differences were found between the two strains. The results strongly point to the multifactorial nature of bacterial adherence to damaged heart valves and suggest that other binding functions can compensate for the lack of fibronectin binding in S. aureus.     

Role of fibronectin-binding proteins A and B in in vitro cellular infections and in vivo septic infections by Staphylococcus aureus

Fibronectin-binding protein A (FnBPA) and FnBPB are important adhesins for Staphylococcus aureus infection. We constructed fnbA and/or fnbB mutant strains from S. aureus SH1000, which possesses intact rsbU, and studied the role of these adhesins in in vitro and in vivo infections. In intravenous infection, all fnb mutants caused a remarkable reduction in the colonization rate in kidneys and the mortality rate of mice. fnbB mutant caused a more severe decrease in body weight than that caused by fnbA mutant. Serum levels of interleukin-6 and nuclear factor κB (NF-κB) activation in spleen cells were remarkably reduced in fnbA or fnbA fnbB mutant infections; however, there was no significant reduction in fnbB mutant infections. In in vitro cellular infection, FnBPA was shown to be indispensable for adhesion to and internalization by nonprofessional phagocytic cells upon ingestion by inflammatory macrophages and NF-κB activation. However, both FnBPs were required for efficient cellular responses. The results showed that FnBPA is more important for in vitro and in vivo infections; however, cooperation between FnBPA and FnBPB is indispensable for the induction of severe infection resulting in septic death.     

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.