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.

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.

Role of sigmaB in the expression of Staphylococcus aureus cell wall adhesins ClfA and FnbA and contribution to infectivity in a rat model of experimental endocarditis.

Isogenic Staphylococcus aureus strains with different capacities to produce sigma(B) activity were analyzed for their ability to attach to fibrinogen- or fibronectin-coated surfaces or platelet-fibrin clots and to cause endocarditis in rats. In comparison to the sigma(B)-deficient strain, BB255, which harbors an rsbU mutation, both rsbU-complemented and sigma(B)-overproducing derivatives exhibited at least five times greater attachment to fibrinogen- and fibronectin-coated surfaces and showed increased adherence to platelet-fibrin clots. No differences in adherence were seen between BB255 and a DeltarsbUVWsigB isogen. Northern blotting analyses revealed that transcription of clfA, encoding fibrinogen-binding protein clumping factor A, and fnbA, encoding fibronectin-binding protein A, were positively influenced by sigma(B). Sigma(B) overproduction resulted in a statistically significant increase in positive spleen cultures and enhanced bacterial densities in both the aortic vegetations and spleens at 16 h postinoculation. In contrast, at 72 h postinoculation, tissues infected with the sigma(B) overproducer had lower bacterial densities than did those infected with BB255. These results suggest that although sigma(B) appears to increase the adhesion of S. aureus to various host cell-matrix proteins in vitro, it has limited effect on pathogenesis in the rat endocarditis model. Sigma(B) appears to have a transient enhancing effect on bacterial density in the early stages of infection that is lost during progression.

Toxoplasma gondii: flat-mounting of retina as a new tool for the observation of ocular infection in mice.

Ocular toxoplasmosis is the principal cause of posterior uveitis and a leading cause of blindness. Animal models are required to improve our understanding of the pathogenesis of this disease. The method currently used for the detection of retinal cysts in animals involves the observation, under a microscope, of all the sections from infected eyes. However, this method is time-consuming and lacks sensitivity. We have developed a rapid, sensitive method for observing retinal cysts in mice infected with Toxoplasma gondii. This method involves combining the flat-mounting of retina - a compromise between macroscopic observation and global analysis of this tissue - and the use of an avirulent recombinant strain of T. gondii expressing the Escherichia coli beta-galactosidase gene, visually detectable at the submacroscopic level. Single cyst unilateral infection was found in six out of 17 mice killed within 28 days of infection, whereas a bilateral infection was found in only one mouse. There was no correlation between brain cysts number and ocular infection.

Use of a human-like low-grade bacteremia model of experimental endocarditis to study the role of Staphylococcus aureus adhesins and platelet aggregation in early endocarditis.

Animal models of infective endocarditis (IE) induced by high-grade bacteremia revealed the pathogenic roles of Staphylococcus aureus surface adhesins and platelet aggregation in the infection process. In humans, however, S. aureus IE possibly occurs through repeated bouts of low-grade bacteremia from a colonized site or intravenous device. Here we used a rat model of IE induced by continuous low-grade bacteremia to explore further the contributions of S. aureus virulence factors to the initiation of IE. Rats with aortic vegetations were inoculated by continuous intravenous infusion (0.0017 ml/min over 10 h) with 10(6) CFU of Lactococcus lactis pIL253 or a recombinant L. lactis strain expressing an individual S. aureus surface protein (ClfA, FnbpA, BCD, or SdrE) conferring a particular adhesive or platelet aggregation property. Vegetation infection was assessed 24 h later. Plasma was collected at 0, 2, and 6 h postinoculation to quantify the expression of tumor necrosis factor (TNF), interleukin 1α (IL-1α), IL-1β, IL-6, and IL-10. The percentage of vegetation infection relative to that with strain pIL253 (11%) increased when binding to fibrinogen was conferred on L. lactis (ClfA strain) (52%; P = 0.007) and increased further with adhesion to fibronectin (FnbpA strain) (75%; P < 0.001). Expression of fibronectin binding alone was not sufficient to induce IE (BCD strain) (10% of infection). Platelet aggregation increased the risk of vegetation infection (SdrE strain) (30%). Conferring adhesion to fibrinogen and fibronectin favored IL-1β and IL-6 production. Our results, with a model of IE induced by low-grade bacteremia, resembling human disease, extend the essential role of fibrinogen binding in the initiation of S. aureus IE. Triggering of platelet aggregation or an inflammatory response may contribute to or promote the development of IE.

Role of the immune response induced by superantigens in the pathogenesis of microbial infections.

Superantigens (SAgs) are microbial proteins which have potent effects on the immune system. They are presented by major histocompatibility complex (MHC) class II molecules and interact with a large number of T cells expressing specific T cell receptor V beta domains. Encounter of a SAg leads initially to the stimulation and subsequently to the clonal deletion of reactive T cells. SAgs are expressed by a wide variety of microorganisms which use them to exploit the immune system to their own advantage. Bacterial SAgs are exotoxins which are linked to several diseases in humans and animals. A classical example is the toxic shock syndrome in which the massive release of cytokines by SAg-reactive cells is thought to play a major pathogenic role. The best characterized viral SAg is encoded by mouse mammary tumour virus (MMTV) and has proved to have a major influence on the viral life cycle by dramatically increasing the efficiency of viral infection. In this paper, we review the general properties of SAgs and discuss the different types of microorganisms which produce these molecules, with a particular emphasis on the role played by the SAg-induced immune response in the course of microbial infections.

Parenteral sparfloxacin compared with ceftriaxone in treatment of experimental endocarditis due to penicillin-susceptible and -resistant streptococci.

A new, investigational, parenteral form of sparfloxacin was compared with ceftriaxone in the treatment of experimental endocarditis caused by either of three penicillin-susceptible streptococci or one penicillin-resistant streptococcus. Both drugs have prolonged half-lives in serum, allowing single daily administration to humans. Sparfloxacin had relatively low MICs (0.25 to 0.5 mg/liter) for all four organisms and was also greater than or equal to eight times more effective than the other quinolones against 21 additional streptococcal isolates recovered from patients with bacteremia. Ceftriaxone MICs were 0.032 to 0.064 mg/liter for the penicillin-susceptible strains and 2 mg/liter for the resistant isolate. Both antibiotics resulted in moderate bacterial killing in vitro. Rats with catheter-induced aortic vegetations were inoculated with 10(7) CFU of the test organisms. Antibiotic treatment was started 48 h later and lasted either 3 or 5 days. The drugs were injected at doses which mimicked the kinetics in human serum produced by one intravenous injection of 400 mg of sparfloxacin (i.e., the daily dose expected to be given to human adults) and 2 g of ceftriaxone. Both antibiotics significantly decreased the bacterial densities in the vegetations. However, sparfloxacin was slower than ceftriaxone in its ability to eradicate valvular infection caused by penicillin-susceptible bacteria. While this difference was quite marked after 3 days of therapy, it tended to vanish when treatment was prolonged to 5 days. In contrast, sparfloxacin was very effective against the penicillin-resistant isolate, an organism against which ceftriaxone therapy failed in vivo. No sparfloxacin-resistant mutant was selected during therapy. Thus, in the present experimental setting, this new, investigational, parenteral form of sparfloxacin was effective against severe infections caused by both penicillin-susceptible and penicillin-resistant streptococci.

RP 59500 prophylaxis of experimental endocarditis due to erythromycin-susceptible and -resistant isogenic pairs of viridans group streptococci.

RP 59500 is a new injectable streptogramin composed of two synergistic components (quinupristin and dalfopristin) which are active against a number of erythromycin-susceptible and -resistant gram-positive bacteria. The following experiments investigate the ability of RP 59500 to prevent experimental endocarditis due to either of two erythromycin-susceptible streptococcal isolates or their constitutively erythromycin-resistant Tn916 delta E transconjugants. RP 59500 had low MICs (0.125 to 0.5 mg/liter) for all four test organisms and was substantially bactericidal in vitro. Rats with catheter-induced aortic vegetations were given single-dose antibiotic prophylaxis 30 to 60 min before bacterial inoculation through a computerized pump system which permitted the simulation of drug kinetics for humans produced by either 7 mg of RP 59500 per kg of body weight or 1 g of vancomycin. Single-dose RP 59500 prophylaxis successfully prevented endocarditis due to both the erythromycin-susceptible parent strains and their erythromycin-resistant derivatives in rats challenged with the minimal inoculum infecting 90% of controls. In addition, RP 59500 also prevented infection in animals challenged with fivefold-larger inocula of the erythromycin-susceptible parent strains. Vancomycin successfully prevented endocarditis due to any of the four test organisms. These results underline the in vivo efficacy of RP 59500 against both erythromycin-susceptible and -resistant streptococci. Such good results against the resistant strains would not be expected with erythromycin or clindamycin, which are the standard macrolidelincosamide-streptogramin antibiotics used for endocarditis prophylaxis in humans. An oral form of RP 59500 which might advantageously replace some of the older prophylactic regimens is currently being developed.

Prophylaxis of experimental endocarditis with antiplatelet and antithrombin agents : a role for long-term prevention of infective endocarditis in humans?

BACKGROUND: Infective endocarditis (IE) mostly occurs after spontaneous low-grade bacteremia. Thus, IE cannot be prevented by circumstantial antibiotic prophylaxis. Platelet activation following bacterial-fibrinogen interaction or thrombin-mediated fibrinogen-fibrin polymerization is a critical step in vegetation formation. We tested the efficacy of antiplatelet and antithrombin to prevent experimental IE. METHODS: A rat model of experimental IE following prolonged low-grade bacteremia mimicking smoldering bacteremia in humans was used. Prophylaxis with antiplatelets (aspirin, ticlopidine [alone or in combination], eptifibatide, or abciximab) or anticoagulants (antithrombin dabigatran etexilate or anti-vitamin K acenocoumarol) was started 2 days before inoculation with Streptococcus gordonii or Staphylococcus aureus. Valve infection was assessed 24 hours later. RESULTS: Aspirin plus ticlopidine, as well as abciximab, protected 45%-88% of animals against S. gordonii and S. aureus IE (P < .05). Dabigatran etexilate protected 75% of rats against IE due to S. aureus (P < .005) but failed to protect against S. gordonii (<30% protection). Acenocoumarol was ineffective. CONCLUSIONS: Antiplatelet and direct antithrombin agents may be useful in the prophylaxis of IE in humans. In particular, the potential dual benefit of dabigatran etexilate might be reconsidered for patients with prosthetic valves, who require life-long anticoagulation and in whom S. aureus IE is associated with high mortality.

Vancomycin-intermediate Staphylococcus aureus selected during vancomycin therapy of experimental endocarditis are not detected by culture-based diagnostic procedures and persist after treatment arrest.

OBJECTIVES: Laboratory detection of vancomycin-intermediate Staphylococcus aureus (VISA) and their heterogeneous VISA (hVISA) precursors is difficult. Thus, it is possible that vancomycin failures against supposedly vancomycin-susceptible S. aureus are due to undiagnosed VISA or hVISA. We tested this hypothesis in experimental endocarditis.¦METHODS: Rats with aortic valve infection due to the vancomycin-susceptible (MIC 2 mg/L), methicillin-resistant S. aureus M1V2 were treated for 2 days with doses of vancomycin that mimicked the pharmacokinetics seen in humans following intravenous administration of 1 g of the drug every 12 h. Half of the treated animals were killed 8 h after treatment arrest and half 3 days thereafter. Population analyses were done directly on vegetation homogenates or after one subculture in drug-free medium to mimic standard diagnostic procedures.¦RESULTS: Vancomycin cured 14 of 26 animals (54%; P<0.05 versus controls) after 2 days of treatment. When vegetation homogenates were plated directly on vancomycin-containing plates, 6 of 13 rats killed 8 h after treatment arrest had positive cultures, 1 of which harboured hVISA. Likewise, 6 of 13 rats killed 3 days thereafter had positive valve cultures, 5 of which harboured hVISA. However, one subculture of vegetations in drug-free broth was enough to revert all the hVISA phenotypes to the susceptible pattern of the parent. Thus, vancomycin selected for hVISA during therapy of experimental endocarditis due to vancomycin-susceptible S. aureus. These hVISA were associated with vancomycin failure. The hVISA phenotype persisted in vivo, even after vancomycin arrest, but was missed in vitro after a single passage of the vegetation homogenate on drug-free medium.¦CONCLUSIONS: hVISA might escape detection in clinical samples if they are subcultured before susceptibility tests.

Effects of immunosuppressive drugs on HIV infection: implications for solid-organ transplantation.

With the advent of highly active antiretroviral therapy (HAART), HIV infection has become a chronic disease. Various end-stage organ failures have now become common co-morbidities and are primary causes of mortality in HIV-infected patients. Solid-organ transplantation therefore has been proposed to these patients, as HIV infection is not anymore considered an absolute contraindication. The initial results of organ transplantation in HIV-infected patients are encouraging with no differences in patient and graft survival compared with non-HIV-infected patients. The use of immunosuppressive drug therapy in HIV-infected patients has so far not shown major detrimental effects, and some drugs in combination with HAART have even demonstrated possible beneficial effects for specific HIV settings. Nevertheless, organ transplantation in HIV-infected patients remains a complex intervention, and more studies will be required to clarify open questions such as long-term effects of drug interactions between antiretroviral and immunosuppressive drugs, outcome of recurrent HCV infection in HIV-infected patients, incidence of graft rejection, or long-term graft and patient survival. In this article, we first review the immunological pathogenesis of HIV infection and the rationale for using immunosuppression combined with HAART. We then discuss the most recent results of solid-organ transplantation in HIV-infected patients.

Antibiotic treatment of experimental endocarditis due to methicillin-resistant Staphylococcus epidermidis.

The natural history and treatment of experimental endocarditis due to heterogeneous and homogeneous methicillin-resistant Staphylococcus epidermidis was investigated. Amoxicillin/clavulanate or vancomycin were administered for 3 days via a computerized pump to mimic human drug kinetics in animals. After challenge with the minimum inoculum producing 90% of infections (ID90), bacteria in the vegetations grew logarithmically for 16 h. Then, bacterial densities stabilized (at approximately 10(8) cfu/g) and growth rates sharply declined. Both regimens cured &gt; or = 60% of endocarditis (due to heterogeneous or homogeneous bacteria) when started 12-16 h after infection, although the bacterial densities in the vegetations had increased by 20 times in between. In contrast, treatment started after 24 h failed in most animals, while bacterial densities had not increased any more. Thus, while both regimens were equivalent, the therapeutic outcome was best predicted by growth rates in the vegetations, not by bacterial densities. These observations highlight the importance of phenotypic tolerance developing in vivo.

Treatment of experimental endocarditis due to erythromycin-susceptible or -resistant methicillin-resistant Staphylococcus aureus with RP 59500.

RP 59500 is a new injectable streptogramin composed of two synergistic components (quinupristin and dalfopristin) which are active against erythromycin-susceptible and -resistant gram-positive pathogens. The present experiments compared the therapeutic efficacy of RP 59500 with that of vancomycin against experimental endocarditis due to either of two erythromycin-susceptible or two constitutively erythromycin-resistant isolates of methicillin-resistant Staphylococcus aureus. RP 59500 had low MICs for the four test organisms as well as for 24 additional isolates (the MIC at which 90% of the isolates were inhibited was &lt; 1 mg/liter) which were mostly inducibly (47%) or constitutively (39%) erythromycin resistant. Aortic endocarditis in rats was produced with catheter-induced vegetations. Three-day therapy was initiated 12 h after infection, and the drugs were delivered via a computerized pump, which permitted the mimicking of the drug kinetics produced in human serum by twice-daily intravenous injections of 7 mg of RP 59500 per kg of body weight or 1 g of vancomycin. Both antibiotics reduced vegetation bacterial titers to below detection levels in ca. 70% of animals infected with the erythromycin-susceptible isolates (P &lt; 0.05 compared with titers in controls). Vancomycin was also effective against the constitutively resistant strains, but RP 59500 failed against these isolates. Further experiments proved that RP 59500 failures were related to the very short life span of dalfopristin in serum (&lt; or = 2 h, compared with &gt; or = 6 h for quinupristin), since successful treatment was restored by artificially prolonging the dalfopristin levels for 6 h. Thus, RP 59500 is a promising alternative to vancomycin against methicillin-resistant S. aureus infections, provided that pharmacokinetic parameters are adjusted to afford prolonged levels of both of its constituents in serum. This observation is also relevant to humans, in whom the life span of dalfopristin in serum is also shorter than that of quinupristin.

Efficacy of levofloxacin in the treatment of experimental endocarditis caused by viridans group streptococci.

Levofloxacin was investigated against viridans group streptococci in vitro and in rats with experimental aortic endocarditis. The MIC(90)s of levofloxacin and ciprofloxacin for 20 independent isolates of such bacteria were 1 and 8 mg/L, respectively. Rats were infected with two types of organism: either fully susceptible to levofloxacin MIC < or = 0.5 mg/L) or borderline susceptible (MIC 1-2 mg/L). Fully levofloxacin-susceptible bacteria comprised one penicillin-susceptible (MIC 0.004 mg/L) Streptococcus gordonii, and one penicillin-tolerant as well as one intermediate penicillin-resistant (MIC 0.125 mg/L) isogenic strains. Borderline levofloxacin-susceptible bacteria comprised one penicillin-susceptible Streptococcus sanguis and one highly penicillin-resistant Streptococcus mitis (MIC 2 mg/L). Rats were treated for 5 days with drug dosages simulating the following treatments in humans: (i) levofloxacin 500 mg orally once a day (q24 h), (ii) levofloxacin 500 mg orally twice a day (q12 h), (iii) levofloxacin 1 g orally q24 h, (iv) ciprofloxacin 750 mg orally q12 h, and (v) ceftriaxone 2 g iv q24 h. Levofloxacin was equivalent or superior to ceftriaxone, and was successful in treating experimental endocarditis irrespective of penicillin resistance. Nevertheless, standard levofloxacin treatment equivalent to 500 mg q24 h in human was less effective than twice daily 500 mg or once daily 1 g doses against borderline-susceptible organisms. Ciprofloxacin, used as a negative control, was ineffective and selected for resistant isolates. This underlines the importance of MIC determinations when treating severe streptococcal infection with quinolones. In the case of borderline-susceptible pathogens, total daily doses of 1 g of levofloxacin should be considered.