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.     

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.     

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.     

Treatment of Staphylococcus aureus endocarditis using moxifloxacin

The conventional treatment of staphylococcal endocarditis requires in-patient administration, is inconvenient, and is potentially toxic. Increasing experience with well-absorbed, well-tolerated and highly active agents such as the new quinolones has prompted interest in their use as therapeutic alternatives for the treatment of such infections. We describe a case of staphylococcal endocarditis which failed to respond to conventional therapy, but where the addition of moxifloxacin, an 8-methoxyquinolone, was curative.     

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.     

Contribution of clumping factor B to pathogenesis of experimental endocarditis due to Staphylococcus aureus

Staphylococcus aureus Newman with an insertion mutation in clfB, the gene encoding clumping factor B, only marginally decreased infection rate (P>0.05) in rats with experimental endocarditis. In contrast, clfB complementation on a multicopy plasmid significantly increased infectivity (P<0.05) over the deleted mutants. Although clfB could affect endovascular infection, its importance in experimental endocarditis was limited.     

The fibrinogen- and fibronectin-binding domains of Staphylococcus aureus fibronectin-binding protein A synergistically promote endothelial invasion and experimental endocarditis

Staphylococcus aureus experimental endocarditis relies on sequential fibrinogen binding (for valve colonization) and fibronectin binding (for endothelial invasion) conferred by peptidoglycan-attached adhesins. Fibronectin-binding protein A (FnBPA) reconciles these two properties—as well as elastin binding—and promotes experimental endocarditis by itself. Here we attempted to delineate the minimal subdomain of FnBPA responsible for fibrinogen and fibronectin binding, cell invasion, and in vivo endocarditis. A large library of truncated constructs of FnBPA was expressed in Lactococcus lactis and tested in vitro and in animals. A 127-amino-acid subdomain spanning the hinge of the FnBPA fibrinogen-binding and fibronectin-binding regions appeared necessary and sufficient to confer the sum of these properties. Competition with synthetic peptides could not delineate specific fibrinogen- and fibronectin-binding sites, suggesting that dual binding arose from protein folding, irrespective of clearly defined binding domains. Moreover, coexpressing the 127-amino-acid subdomain with remote domains of FnBPA further increased fibrinogen binding by ≥10 times, confirming the importance of domain interactions for binding efficacy. In animals, fibrinogen binding (but not fibronectin binding) was significantly associated with endocarditis induction, whereas both fibrinogen binding and fibronectin binding were associated with disease severity. Moreover, fibrinogen binding also combined with fibronectin binding to synergize the invasion of cultured cell lines significantly, a feature correlating with endocarditis severity. Thus, while fibrinogen binding and fibronectin binding were believed to act sequentially in colonization and invasion, they appeared unexpectedly intertwined in terms of both functional anatomy and pathogenicity (in endocarditis). This unforeseen FnBPA subtlety might bear importance for the development of antiadhesin strategies.     

Regulation of Staphylococcus aureus type 5 capsular polysaccharides by agr and sarA in vitro and in an experimental endocarditis model

The expression of antiphagocytic polysaccharide capsules is an important pathogenetic step in establishing Staphylococcus aureus infections. Using a green fluorescent protein reporter gene (gfp) system, we examined the expression and genetic regulation of the cap5 promoter (capsular polysaccharide 5 genes) by two major global regulators of S. aureus (agr and sarA) in vitro and in a rabbit endocarditis model. In vitro, cap5 expression substantially increased during the post-exponential phase in parental, as well assarA mutant constructs. However, cap5 expression was greatly reduced in agr and agr/sarA double mutants. In the endocarditis model, the extent of cap5 expression in vegetations infected with the parental strain was substantially higher than that observed with the agr/sarA double mutants (P<0.05). Similar trends were noted in renal, but not splenic abscesses. Collectively, these data suggest that agr positively regulates cap5 expression both in vitro and in vivo, while the contribution of sarA to cap5 regulation, although modest, is readily discerned in vivo in agr minus background. In addition, the regulation ofcap5 expression by these global regulators may vary in distinct anatomic niches in vivo.     

Protective efficacy of antibodies to the Staphylococcus aureus type 5 capsular polysaccharide in a modified model of endocarditis in rats.

The protective efficacy of antibodies to the Staphylococcus aureus type 5 capsular polysaccharide (CP5) was examined in a modified model of catheter-induced endocarditis. Rats were catheterized by surgically passing a polyethylene catheter through the right carotid artery and aortic valve into the left ventricle. The following day, the rats were injected by the intraperitoneal (i.p.) route with immunoglobulin G (IgG) purified from nonimmunized rabbits or from rabbits immunized with a conjugate vaccine composed of CP5 and CP8 linked covalently to recombinant Pseudomonas aeruginosa exotoxoid A. One day after passive immunization, the animals were challenged i.p. with one of three serotype 5 S. aureus isolates (strain Reynolds, Lowenstein, or VP) or nontypeable strain 521. Protection was evaluated by comparing quantitative cultures of blood, endocardial vegetations, and kidneys from control and immune animals. For experiments performed with S. aureus Reynolds and Lowenstein, rats given capsular antibodies (645 microg of CP5-specific IgG) showed a significantly (P < 0.05) lower prevalence of endocarditis than rats injected with nonimmune IgG. Similarly, quantitative cultures of the blood, kidneys, and aortic valve vegetations revealed that fewer S. aureus cells were recovered from rats given capsule-specific IgG than from rats administered nonimmune IgG. Rats challenged with strain VP were protected with 1.145 mg of CP5-specific IgG. Capsular antibodies did not protect against infection elicited by a nontypeable strain. These results demonstrate that capsular antibodies elicited by immunization with a polysaccharide-protein conjugate vaccine protect experimental animals against serotype 5 S. aureus infection in a modified model of endocarditis.     

Capsule-negative Staphylococcus aureus induces chronic experimental mastitis in mice

Staphylococcus aureus capsular polysaccharides (CP) have been shown to enhance staphylococcal virulence in numerous animal models of infection. Although serotype 5 CP (CP5) and CP8 predominate among S. aureus isolates from humans, most staphylococcal isolates from bovines with mastitis in Argentina are capsule negative. This study was designed to evaluate the effects of CP5 and CP8 expression on the pathogenesis of experimental murine mastitis. Lactating mice were challenged by the intramammary route with one of three isogenic S. aureus strains producing CP5, CP8, or no capsule. Significantly greater numbers of acapsular mutant cells were recovered from the infected glands 12 days after bacterial challenge compared with the encapsulated strains. Histopathological analyses revealed greater polymorphonuclear and mononuclear leukocyte infiltration and congestion in the mammary glands of mice infected with the encapsulated strains compared with the acapsular mutant, and the serotype 5 strain elicited more inflammation than the serotype 8 strain. In vitro experiments revealed that the acapsular S. aureus strain was internalized by MAC-T bovine epithelial cells in significantly greater numbers than the CP5- or CP8-producing strain. Taken together, the results suggest that S. aureus lacking a capsule was able to persist in the murine mammary gland, whereas encapsulated strains elicited more inflammation and were eliminated faster. Loss of CP5 or CP8 expression may enhance the persistence of staphylococci in the mammary glands of chronically infected hosts.     

Antibiotic treatment of Staphylococcus aureus endocarditis. A review of 119 cases

Clinical and bacteriological information of Staphylococcus aureus endocarditis was reviewed in 119 cases from all over Denmark. Overall mortality was 71%. Survival correlated with antistaphylococcal treatment, short duration from onset of infection to start of treatment, and long duration of treatment. In spite of relevant treatment, mortality was significantly lower in cases infected with penicillin-susceptible strains than when penicillin-resistant strains were isolated. There were no differences in the effect of various anti-staphylococcal treatment regimens; in particular, there were no differences in mortality with regard to beta-lactam antibiotics alone as compared to beta-lactam antibiotics in combination with aminoglycosides. However, embolic manifestations occurred more often after start of treatment with combination therapy than with beta-lactam antibiotics alone.     

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.     

Evaluation of endocarditis caused by methicillin-susceptible Staphylococcus aureus developing nonsusceptibility to daptomycin

We examined sequential methicillin-susceptible Staphylococcus aureus isolates from a patient with mitral valve endocarditis recovered during persistent bacteremia on standard therapy and relapse after treatment with daptomycin. An isolate obtained after 5 days of antimicrobial therapy, but before exposure to daptomycin, showed subtle physiological changes in response to daptomycin, with significant regrowth in the daptomycin killing assay compared to the treatment-naive strain. Once daptomycin was started, the population became more heterogeneous and tested as nonsusceptible. These organisms were examined in a simulated-vegetation in vitro pharmacodynamic model, which confirmed progressive decreases in killing with daptomycin concentrations that simulate those attained in humans with 6-mg/kg of body weight daily dosing. Early surgical intervention or combination therapy or both might have prevented the loss of daptomycin susceptibility.     

Colistin efficacy in an experimental model of Acinetobacter baumannii endocarditis

The in-vivo activity of colistin was evaluated in an experimental rabbit model of Acinetobacter baumannii endocarditis with a strain susceptible to colistin and intermediate to imipenem. Compared to a control group, colistin was effective (p < 0.05) in bacterial clearance from blood and in the sterilisation of blood cultures, but was not effective in clearing A. baumannii from vegetations. Thus, although colistin may be effective in treating bacteraemia caused by susceptible strains of A. baumannii, it may not be a suitable treatment for endocarditis, perhaps because of poor penetration into vegetations and a low C(max)/MIC ratio in tissue.     

Experimental endocarditis in the rat secondary to septic arthritis induced by Staphylococcus aureus

Objective: To develop a modified model for experimental infective endocarditis (IE) in the rat. The goal was to induce a primary infectious focus in the temporomandibular joint (TMJ) of a rat. Hematogenous translocation of the bacteria to the traumatized aortic valve was desired.
Methods: Catheterization of the right carotid artery through the aortic valve was performed 7 days after induction of arthritis, which was done by intra-articular injection of glucocorticosteroid (triamcinolone acetonide, 1 mg) and intravenous challenge with 10⁷ CFU Staphylococcus aureus .
Results: TMJ arthritis could be induced by intra-articular triamcinolone acetonide followed by intravenous bacterial challenge. Joints not given glucocorticosteroid were not affected. Only rats with arthritis developed IE subsequent to catheterization as a result of bacteremia generated from the arthritis.
Conclusions: The present model may serve as a complement to the conventional method for induction of IE, in which a high intravenous challenge has to be given. In the present model, IE was instead the result of a continuous low level of bacteremia from an infectious focus in the TMJ. This model mimics the natural development of IE in patients, and may assist as a setting for prophylactic and therapeutic trials.     

Changing Italian nosocomial-community trends and heteroresistance in Staphylococcus aureus from bacteremia and endocarditis

Bloodstream infections due to Staphylococcus aureus (BSI) are serious infections both in hospitals and in the community, possibly leading to infective endocarditis (IE). The use of glycopeptides has been recently challenged by various forms of low-level resistance. This study evaluated the distribution of MSSA and MRSA isolates from BSI and IE in 4 Italian hospitals, their antibiotic susceptibility—focusing on the emergence of hVISA—and genotypic relationships. Our results demonstrate that the epidemiology of MRSA is changing versus different STs possessing features between community-acquired (CA)- and hospital-acquired (HA)-MRSA groups; furthermore, different MSSA isolated from BSI and IE were found, with the same backgrounds of the Italian CA-MRSA. The hVISA phenotype was very frequent (19.5%) and occurred more frequently in isolates from IE and in both the MSSA and MRSA strains. As expected, hVISA were detected in MRSA with vancomycin minimum inhibitory concentrations (MICs) of 1–2?mg/l, frequently associated with the major SCCmec I and II nosocomial clones; this phenotype was also detected in some MSSA strains. The few cases of MR-hVISA infections evaluated in our study demonstrated that 5 out of 9 patients (55%) receiving a glycopeptide, died. Future studies are required to validate these findings in terms of clinical impact.     

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.     

Bacterial concentration correlations in experimental endocarditis caused by Staphylococcus epidermidis.

Using 13 strains of Staphylococcus epidermidis to produce catheter-induced experimental endocarditis in rats, we found that bacterial concentrations in blood cultures obtained at the time of sacrifice correlated significantly with the number of organisms per gram of endocardial vegetation (P less than 0.001) and the total number of organisms per vegetation (P less than 0.001). Furthermore, blood culture concentrations correlated with vegetation weights (P less than 0.001) and sizes of infecting inocula (P less than 0.0001). Mean bacterial concentrations in vegetations more than doubled as bacterial concentrations in blood rose from less than 10 to greater than 100 CFU/ml. Mean values for vegetation weights, total organisms per vegetation, and sizes of infecting inocula were also reflected by the intensity of bacteremia. Moreover, intracardiac catheters were more likely colonized as bacterial concentrations in blood cultures increased, with all catheters culture positive in the 25 animals that exhibited high-grade bacteremia (greater than or equal to 100 CFU/ml). Slime production by the bacteria did not influence the above-mentioned correlations. These data indicate that the blood concentration of bacteria reflects the microbiologic status of infected vegetations in experimental infective endocarditis.