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Antimicrobial Agents and Chemotherapy, August 2007, p. 2848-2854, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.01376-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Dexamethasone as Adjuvant Therapy to Moxifloxacin Attenuates Valve Destruction in Experimental Aortic Valve Endocarditis Due to Staphylococcus aureus{triangledown}

Ioannis Skiadas,1,2* Angelos Pefanis,3 Apostolos Papalois,4 Aspasia Kyroudi,5 Helen Triantafyllidi,6 Thomas Tsaganos,1 and Helen Giamarellou1

Fourth Department of Internal Medicine, University of Athens School of Medicine, University General Hospital Attikon, Athens, Greece,1 Cardiology Department, Hippocration General Hospital, Athens, Greece,2 Third Department of Internal Medicine, University of Athens School of Medicine, Sotiria General Hospital for Chest Diseases, Athens, Greece,3 Experimental Research Department of ELPEN-Pharma, Athens, Greece,4 Department of Histology and Embryology, University of Athens School of Medicine, Athens, Greece,5 Second Department of Cardiology, University of Athens School of Medicine, University General Hospital Attikon, Athens, Greece6

Received 3 November 2006/ Returned for modification 12 March 2007/ Accepted 29 May 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although the beneficial effects of dexamethasone have frequently been investigated in various serious-infection settings, insufficient data on valve histology and cardiac function for infective endocarditis are available. The efficacy of moxifloxacin for the treatment of experimental aortic valve endocarditis due to methicillin-susceptible Staphylococcus aureus and the long-term effects of dexamethasone were evaluated in the current study. Sixty-eight rabbits were randomly assigned to four groups: A, B, C, and D. Group A consisted of 18 animals and functioned as a control group. Groups B and C consisted of 11 and 23 subjects, respectively, which received moxifloxacin for 5 days in a human-like pharmacokinetic simulation. Group D consisted of 16 animals that were administered moxifloxacin plus dexamethasone (0.25 mg/kg of body weight twice a day intravenously). The group B animals were sacrificed a day after the completion of treatment, and group C and D animals were sacrificed after 12 days in order to monitor any possible relapse and allow microbiological, histopathological, and echocardiographic evaluation of the long-term effects of glucocorticoids. No differences in survival, sterilization rates, or inflammatory infiltration and calcification of valve tissue were observed among the treated groups. However, the degrees of valve damage and collagenization were significantly worse, the fibroblast content was higher, and fractional shortening of the left ventricle fluctuated significantly in group C compared to group D (all groups, P < 0.05). We concluded that dexamethasone treatment for experimental S. aureus endocarditis attenuates valve destruction and preserves overall cardiac function without impeding the efficacy of moxifloxacin.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The treatment of infectious endocarditis (IE) caused by Staphylococcus aureus is a challenging problem in everyday clinical practice. S. aureus is the most common cause of IE in most of the developed world (9), is associated with mortality rates ranging from 25 to 40% (2), and is often responsible for acute infections with rapid valve destruction (20). Previous studies of S. aureus endocarditis have emphasized the difficulties in achieving both microbiological and clinical cures with the use of antibiotics alone (21). Over the past 10 years, therefore, early combined medical and surgical interventions have been favored over medical treatment alone (5, 32), and both newer antibiotics and anti-inflammatory compounds such as corticoids have been tested to alleviate these difficulties.

Moxifloxacin is a newer 8-methoxyfluoroquinolone that is more potent than former quinolones against isolates of Staphylococcus aureus and Staphylococcus epidermidis, as evidenced by its lower MICs against these pathogens (7). Moreover, it has recently been proven to be highly efficacious against staphylococcal experimental endocarditis due to methicillin-resistant S. aureus (MRSA) (8). Nonetheless, experimental data concerning the efficacy of moxifloxacin against endocarditis due to methicillin-susceptible S. aureus (MSSA) are still lacking.

In experimental models, dexamethasone has been successfully combined with antibiotics to treat staphylococcal endophthalmitis (27), septic arthritis (25), and septic nephritis (31), as well as pneumococcal (24) and Haemophilus influenzae type b (28) meningitis. There are also clinical trials and reports supporting the administration of steroids, especially dexamethasone, due to its strong anti-inflammatory properties, in combination with antibiotics for infections such as meningitis (19) and nephritis (15).

In one report on experimental MRSA endocarditis, it was found that, immediately after the end of treatment, the severity of valve tissue damage was significantly less in the group receiving dexamethasone (26). However, the possibility that the steroids might delay valve destruction but not prevent it could not be excluded. In order to further elucidate this possibility, our study aimed to provide data from an observational period after the treatment of MSSA endocarditis, incorporating echocardiographic and histopathologic analyses as well.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial isolate. The S. aureus strain used in the present study was a clinical isolate obtained from a patient with endocarditis.

Antimicrobial susceptibility testing. The susceptibilities of the pathogen to penicillin, amoxicillin, oxacillin, and moxifloxacin (supplied by Bayer AG, Wuppertal, Germany) were determined with a manual microdilution technique in volumes of 0.1 ml, using logarithmic-growth-phase inocula of S. aureus in cation-supplemented Mueller-Hinton II broth (Becton-Dickinson, Franklin Lakes, NJ) adjusted to a final inoculum of ~0.5 x 105 CFU/ml. The concentrations of the antibiotics tested ranged from 0.007 to 512 mg/liter. Moreover, the MIC of moxifloxacin was also determined by Etest (AB Biodisk, Solna, Sweden). The presence of the mecA gene was detected by PCR.

Time-kill assays simulating the various serum concentrations during treatment were performed to evaluate the in vitro bactericidal effect of moxifloxacin. Overnight cultures of the S. aureus strain were used to prepare inocula at 5 x 105 CFU per ml and 5 x 107 CFU per ml, as the latter number of bacteria corresponds more closely to the pretreatment bacterial load in vegetations. The final antibiotic concentrations tested for both levels of inocula were 0.5 mg, 1 mg, and 8 mg of moxifloxacin per liter. To eliminate the carryover effect, specimens from all tubes, sampled at 0, 4, 6, and 24 h of incubation, were plated onto both blood agar plates (BAP) and Staphylococcus agar 110 plates (SAP) after seven dilutions, with a 1-log inoculum difference between each dilution (10–1 to 10–7). After 24 h (BAP) or 48 h (SAP) of incubation, the colonies were counted, and killing curves were then constructed to delineate bacterial survival over time.

Endocarditis model. The rabbit model of aortic valve endocarditis was used to evaluate treatment regimens (10, 26). The study received a permit from the Veterinary Directorate of the Prefecture of Athens in conformance with Greek legislation in response to EU directive 160/1991. Twenty-four hours after transcarotid placement of the polyethylene catheter across the aortic valve (day 2), the rabbits were injected through the marginal ear vein with 1 ml of saline solution containing 107 CFU of the infecting strain. Blood samples for quantitative culture (lower limit of detection, 2 CFU/ml) were taken 24 h after infection (day 3), and the rabbits were randomly assigned to one of the following treatment groups and treated for 5 days with the following regimens: no treatment (control, group A), moxifloxacin given at 20 mg/kg of body weight intravenously every 12 h (groups B and C), or moxifloxacin given at 20 mg/kg of body weight intravenously every 12 h and dexamethasone given at 0.25 mg/kg intravenously every 12 h (group D). The dose volumes for moxifloxacin and dexamethasone were 1.84 to 2.75 ml and 0.25 to 0.38 ml, respectively. The animals of group B were sacrificed 12 h after the end of the treatment (day 8) to assess the efficacy of the antibiotics, and the animals in groups C and D were sacrificed 12 days after the last dose (day 19) in order to detect possible relapses into endocarditis after the end of treatment and to monitor the physical history of the valve lesions as well as the course of cardiac function for comparison. Presumptive confirmation of the induction of endocarditis was based on positive results of blood cultures. Ultimate confirmation of bacterial endocarditis was based on macroscopic observation (vegetations and correct placement of the catheter across the aortic valve) and either bacteriological data obtained at autopsy or histopathological data obtained from observation of the aortic valves under light microscopy (presence of bacteria and evidence of inflammation).

The untreated controls, regardless of the time of death, and all treated animals sacrificed or which had died after the initiation of therapy were included in the calculation of the survival rate. Rabbits who had been treated with moxifloxacin or moxifloxacin plus dexamethasone were included in the analysis of efficacy (log10 CFU/g and sterility rate of valve tissue) if they had received three or more doses of the study drugs. The surviving animals were sacrificed by rapid intravenous injection of sodium phenobarbital (30 mg/kg). At the time of sacrifice, aortic valvular and left ventricular friable vegetations were removed aseptically for quantitative cultures, and the entire valve was excised and prepared for further histopathological evaluation. The same procedure as described above was performed whenever rabbits were found dead, with death occurring no longer than 8 h before postmortem examination.

Bacteriological evaluations. Excised vegetations were weighed, homogenized (in the presence of 1 ml of sterile 0.9% NaCl), and quantitatively cultured in duplicate onto BAP and SAP after seven dilutions, with a 1-log inoculum difference between each dilution (10–1 to 10–7). After incubation of BAP for 24 h at 35°C and SAP for 48 h at 30°C, the colonies of S. aureus growing on the agar were counted, and the results were expressed as the log10 numbers of CFU per gram of vegetation. In calculating the mean bacterial densities in the vegetations, culture-negative vegetations were considered to contain ~2 log10 CFU/g on the basis of the average weight of the vegetations.

Pharmacokinetic studies. The concentrations of total moxifloxacin in sera were determined on either day 4 or day 6 by high-performance liquid chromatography (Agilent 1100 series; Agilent Technologies, Waldbronn, Germany) (17). The protein binding of moxifloxacin in rabbit sera is 24% ± 5% (22). Blood samples of 0.5 ml were obtained at 1/2, 1, 2, 6, and 12 h after administration of the drugs had been completed. However, because of occasional technical difficulties, samples were not obtained from all animals; data from about four time points per animal were available. The limit of detection for the assay used was 0.0025 mg/liter. The linearity of the standard curves was assessed by a regression coefficient of ≥0.996. The coefficient of variation varied from 1.6% to 5.3%. The area under the serum concentration-time curve (AUC) from time zero to 12 h (AUC0->12) was calculated using the trapezoidal summation method. Antibiotic concentrations on vegetations were determined by an agar diffusion bioassay with Bacillus subtilis ATCC 6633 as the indicator organism.

Left ventricular function evaluation. All treated animals in groups C and D underwent transthoracic echocardiography at the beginning of treatment (day 3), immediately after the completion of therapy (day 8), and 12 days after the end of treatment (day 19) in order to assess the effects of the various therapeutic regimens on overall left ventricular function. Echocardiography was carried out with a 5-MHz-array sector transducer linked to an ultrasound unit (Pie Medical SC 240; Maastricht, The Netherlands). Left ventricular end-diastolic and end-systolic internal dimensions were measured in accordance with current guidelines, as previously described (35), and fractional shortening (FS) was thereafter calculated. Ideally, FS correlates with the ejection fraction, sufficiently representing the overall cardiac function (16). As our purpose was to investigate the long-term effects of steroid therapy, for statistical analysis of the echocardiographic measurements, only the animals in groups C and D that survived the experimental period and for which all sequential data until sacrifice was available were included.

Histopathology. Tissue specimens from the excised aortic valves were fixed immediately in 10% neutral buffered formalin for 24 h and embedded in paraffin. Sections (5 µm thick) were cut and stained with hematoxylin and eosin for general morphology, Giemsa for bacterial colonies, Masson-trichrome for delineation of vegetations, and Van Gieson for the assessment of collagenization. Histological findings were read by an expert pathologist (A. Kyroudi) without any information regarding previous treatment or the results of the valve cultures. The following elements were considered for histological evaluation of the valvular tissue: inflammatory cell infiltration, granulated tissue, collagenization, and calcification. Each element was graded semiquantitatively by determining the percentage of its extension within three randomly chosen fields of moderate magnification (x200): 1 for absence or occasional presence, 2 for moderate presence, or 3 for abundance. In parallel, taking into account the relative distribution of the above-mentioned elements and according to previously described methodology (26), the valvular tissue damage of each specimen was evaluated and classified into three categories: mild, moderate, and severe (with attributed score numbers 1, 2, and 3, respectively). Only data from the animals of groups C and D were included in the statistical comparisons to monitor long-term effects and compare valve damage that dated from the induction of the disease.

Statistical methods. To compare the differences between sterile and nonsterile blood cultures and between sterile and nonsterile tissue cultures, the Fisher exact test was used. For comparison of differences between histopathological categories of severity of valvular tissue destruction, the chi-square test was used. Differences between groups in terms of histopathological scores were analyzed using the Mann-Whitney U test. The F-max index indicated that it was safe to proceed with parametric analyses for comparisons of mean bacterial burdens in either blood or tissue cultures, and the analysis of variance model was used to assess statistical significance. Finally, Kaplan-Meier curves and the log rank test were used to compare survival rates among the study groups. Two-way repeated-measures analysis of variance was utilized to test for time- and group-related differences between echocardiographic measurements of left ventricular function. A P value of ≤0.05 was considered statistically significant for all tests.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In vitro susceptibility studies. The MICs/minimum bactericidal concentrations of the antibiotics studied for the infecting strain at inocula of ~5 x 105 CFU/ml were as follows: penicillin, 2/2 mg/liter; amoxicillin, 2/4 mg/liter; oxacillin, ≤0.25/0.5 mg/liter; and moxifloxacin, 0.125/0.125 mg/liter. The strain was examined further by PCR for the presence of the mecA gene and was found to be negative. The result of the Etest for moxifloxacin was 0.064 mg/liter (sensitive).

In time-kill curves, moxifloxacin exhibited rapid bactericidal effects (>3 log10 CFU/ml difference) in vitro against S. aureus, even at the low concentration of 0.5 mg/liter.

Animal studies. (i) Survival. Of the 93 animals enrolled in the study, 12 died within less than 24 h after placement of the catheter, 8 died before randomization and the beginning of treatment, and 5 were excluded because they did not meet the inclusion criteria. Overall, 68 rabbits were evaluated for survival, while only 63 rabbits were evaluated for efficacy. The mean survival rates of all study groups are presented in Table 1. The log rank test verified that there were no statistically significant differences in the survival rates among the B, C, and D groups.


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  		TABLE 1. Survival and microbiology results of treatment of experimental aortic valve endocarditis due to S. aureus with moxifloxacin and moxifloxacin plus dexamethasonef

 
(ii) Bacteriological studies in animals. Table 1 shows the sterilization frequencies of blood and vegetations with the different therapeutic regimens.

With respect to the reductions in the bacterial burden measured in heart valve tissues (mean, log10 per gram of tissue), it is remarkable that all animals who received over three doses of moxifloxacin had sterile vegetations except for two: one from group B and the other from group C, with 3.5 and 5.6 log10 CFU/g, respectively. In comparison to the untreated animals, the animals receiving moxifloxacin alone (groups B and C) or moxifloxacin plus dexamethasone (group D) had significantly reduced CFU/g of vegetation (P < 0.001 versus the control group for both regimens). Furthermore, there were no differences among the treated groups (B, C, and D) in the microbiological parameters.

Antibiotic pharmacokinetics. The mean (± the standard deviation [SD]) concentrations of moxifloxacin (used as the single therapy; group C) in the sera 30 min (C30 min) and 12 h (C12 h) after the third dose (n = 7) were 3.73 ± 0.68 and 0.23 ± 0.02 mg/liter, respectively. The respective concentrations after the seventh dose (groups B and C; n = 19) were 4.39 ± 1.43 and 0.51 ± 0.41 mg/liter. These serum moxifloxacin concentrations resemble those observed in humans after a single 400-mg dose (33). The AUC0->12 for the third dose was 16.5 mg·h/liter, and for the seventh dose it was 22.2 mg·h/liter. A statistically significant difference in the C12 h (P = 0.018) and a tendency for the AUC0->12 and C30 min to increase after repeated doses (third and seventh) were observed (P = 0.279 and 0.094, respectively). Animals receiving moxifloxacin plus dexamethasone (group D; n = 13) had mean (± the SD) concentrations of moxifloxacin in sera of 3.33 ± 0.74 and 1.0 ± 0.17 mg/liter, 30 min and 12 h, respectively, after the seventh dose. The AUC0->12 of moxifloxacin calculated after the seventh dose in group D was 23.9 µg·h/ml. We observed that the C30 min of moxifloxacin in group D were lower and the C12 h were higher than the respective levels in the B or C groups (P = 0.010 and 0.005, respectively), and the corresponding elimination rate constants (k10) were 0.10 h–1 and 0.19 h–1 (P < 0.001). Although both differences were significant, the AUC0->12 of moxifloxacin for all treated groups (B, C, and D) did not differ (P = 0.198). Six hours, 12 h, and 12 days after the end of treatment, 2 out of 5, 4 out of 11, and all 11 vegetations, respectively, had undetectable concentrations of moxifloxacin. The mean (± the SD) levels in the three vegetations remaining at 6 h and the seven vegetations remaining at 12 h were 4.4 ± 1.6 and 3.8 ± 1.4 µg/g, respectively.

Left ventricle function. Fourteen animals in group C and 10 animals in group D survived the experiment and underwent serial echocardiographic evaluations (Fig. 1). At the induction of endocarditis, the left ventricular function of the animals in group C was more deteriorated than that of the animals in group D, but the difference was not statistically significant. In the course of the first days, there was an increase in FS, as measured on day 8 (Fig. 1), reflecting the establishment of aortic regurgitation, which is characterized by false, augmented FS, a common finding among many researchers (35). In the same way, for both groups C and D at follow-up (third evaluation, almost 3 weeks after the onset of disease), there was deterioration of the left ventricular function. This final evaluation (Fig. 2) represents the decisive hemodynamic effects of aortic valvulopathy at stabilization, after the healing of endocarditis, as all the evaluated animals had sterile blood and sterile vegetations at autopsy. Compared separately at each time point of evaluation (days 3, 8, and 19), the indices of cardiac function for the two groups did not differ significantly (P = 0.13, 0.08, and 0.33, respectively). However, the group treated with moxifloxacin plus dexamethasone had worse FS at the beginning and, in the long term follow-up, had a more-preserved cardiac function than the animals treated only with moxifloxacin who had a reverse course (Fig. 1). Accordingly, multiple comparisons of the repeated measurements within each group demonstrated significant fluctuation of FS over time in group C (P < 0.001), attributable to the momentous divergent rise and fall of the FS, whereas the animals in group D showed no significant wavering in their consecutive measurements of systolic cardiac function (P = 0.219).


Figure 1
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  		FIG. 1. Echocardiography. The animals treated with the addition of dexamethasone (group D; broken line) showed remarkable stability in their cardiac functions (multiple comparisons of the sequential measurements of FS within group D yielded no significance; P = 0.219). On the other hand, the FS of group C (straight line) animals had a momentous divergent rise and fall (multiple comparisons of the sequential measurements of FS within this group differed significantly; P < 0.001).

 

Figure 2
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  		FIG. 2. Representative echocardiographic evaluation at end of the follow-up period for two animals that received either moxifloxacin (A) or dexamethasone plus moxifloxacin (B). The animal that received only the antibiotic (A) exhibited a more-dilated left ventricle (2.0 versus 1.55 mm), worse FS (30% versus 37%), and more tachycardia than the animal that received dexamethasone in addition to moxifloxacin (B).

 
Histopathology. Histopathological examination allowing for the assessment and semiquantitative grading of the degree of aortic valve damage verified the presence of classical endocarditis lesions. The aortic valves of all the animals included revealed various degrees of ulceration of the endothelial surfaces and formation of infected thrombotic vegetations. The vegetations from all the animals consisted of acellular fibrin, platelets, and a matrix colonized by bacteria. Various densities of the bacterial colonies were seen only in the vegetations. Inflammatory infiltration was observed at the peripheries of the vegetations, particularly in the marginal area of each valve.

The valves of animals receiving only antibiotics presented greater deformation, due to extensive collagenization and a higher tendency for calcified lesions. Inflammatory infiltrations were also occasionally observed (Fig. 3A). In contrast, the valves of the animals treated additionally with dexamethasone were characterized by young granulomatous tissue with few collagen fibers and invariable inflammatory cell infiltration. Calcification tended to be rare (Fig. 3B). On the basis of the above, the fundamental finding was the substantially lesser degree of damage observed in the group treated with moxifloxacin plus dexamethasone (group D) in comparison to that in the counterpart group C (P = 0.012) (Table 2). The overall median histopathologic scores and the distribution of the various degrees of valve damage for each evaluated parameter as determined in the two test-of-cure groups (C and D) are indicated in detail in Tables 2 and 3, respectively. In particular, collagenization was significantly lower in group D (P < 0.001). Furthermore, there was less calcification, and inflammatory infiltration tended to be greater in this group, findings that are in agreement with those of previous studies (10, 26). However, these differences in inflammatory infiltration and calcification yielded no statistical significance (P = 0.274 and 0.296, respectively) (Table 2). Therefore, the animals in group C presented with much more deformed and fibrotic valves, still without any concurrent differences in inflammatory infiltration and calcification compared to the animals in group D. Finally, there was an interesting observation that on average the endothelia of dexamethasone-treated animals appeared less coherent than that of the animals on single-antibiotic therapy, but this was not quantified.


Figure 3
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  		FIG. 3. Histological findings for aortic valve tissue in areas proximal to vegetations (Ve). The tissues were stained with hematoxylin and eosin. (A) Tissue from animal receiving only moxifloxacin (group C), with scattered inflammatory cells (arrowhead) and collagen-rich fibrotic stroma (arrows). (B) Tissue from animal treated with dexamethazone plus moxifloxacin (group D), depicting inflammatory infiltrations (arrowhead) and granulated tissue (arrows).

 

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  		TABLE 2. Histopathology results depicting median scoresa for tissue valve damage, inflammatory cell infiltrations, collagenization, fibroblast content, and calcification for the test-of-cure groups

 

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  		TABLE 3. Distribution of the degrees of valve damage (mild, moderate, and severe) among the test-of-cure groups

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Fluoroquinolone antibacterials are treatment options of increasing importance for bacterial infections. In the present study, moxifloxacin in particular, being very active in vitro against S. aureus and in vivo, especially against soft tissue infections, exhibited its excellent in vivo activity with experimental endocarditis due to MSSA. S. aureus was selected as the pathogen, as such virulent strains lead rapidly to extensive valve destruction, with subsequent high mortality and morbidity rates, and are increasingly incriminated in both in community-acquired and hospital-acquired cardiac infections (9, 11). Nonetheless, moxifloxacin was so efficacious that it sterilized all but two of the treated animals, with no relapse, even when dexamethasone was coadministered on top of the antibiotics. Impressively, moxifloxacin proved to be rapidly bactericidal in vegetations, even sterilizing animals that died prematurely and received only three doses of the regimen. These ideal results are consistent with the advantageous pharmacodynamic properties of moxifloxacin, exhibiting maximum concentration of the drug in serum/MIC and AUC0->24/MIC ratios of 27.6 and 347.8, respectively, values with predicted importance for the successful treatment of endocarditis with fluoroquinolones, as proposed by Andes and Craig (1). Taken together, the results of this study, increasing experience in humans, and advantageous pharmacokinetic/pharmacodynamic and safety profiles (13) indicate that moxifloxacin is a useful treatment alternative for staphylococcal endocarditis.

In recent years, immunomodulatory treatments of infectious diseases have attracted the attention of researchers, increasing their expectations for additional benefits apart from that of the antibiotics. After the first approach by Francioli and Freedman (10) to the role of dexamethasone in endocarditis a quarter of a century ago, where mortality was not affected by dexamethasone in animals with streptococcal aortic valve endocarditis, it was only recently found that the addition of dexamethasone to vancomycin in the treatment of experimental aortic valve endocarditis due to MRSA is beneficial (26). However, the latter study had limitations: posttreatment discontinuation of the follow-up observation period and absence of data such as echocardiographic criteria on cardiac function.

The present study was undertaken to overcome these limitations. Our study verified significantly less tissue damage in the aortic valves of animals treated with dexamethasone added to moxifloxacin and gave further evidence that this finding persists long term (Table 3). More specifically, histopathology showed that valve tissue damage long after the disease was cured was more pronounced in group C, while in parallel, echocardiography revealed that cardiac function fluctuated significantly, as typically seen in patients with severe acute aortic regurgitation, again only in group C (Fig. 1).

The precise mechanisms leading to this beneficial effect are not fully elucidated. Accumulated knowledge attributes the beneficial effect of dexamethasone to its potential to reduce inflammatory mediators (3). By blocking several inflammatory pathways, dexamethasone alleviates the pathogenic immunological interaction between the intruder and the host and leads to less tissue damage. Furthermore, in the inflammatory scenery, leukocytes are classically presumed to be major coactors. Many forms of beneficial interactions between dexamethasone and leukocytes, promoting less valve destruction (26) as observed in the present study, have been described for bacterial endocarditis. There are in vitro studies which also support the finding that dexamethasone promotes phagocytosis by human monocytes, not only of S. aureus, but of other macromolecules as well, and thereby may contribute to tissue repair after immune-mediated tissue damage or infection (29, 30).

Quite relevant, as it utilizes a considerable number of common mediators, is the healing process, consisting of three phases: i.e., inflammatory, proliferative, and remodeling. Though the process is continuous and the phases overlap, it has been suggested that the essential phase is the inflammatory one, characterized by increased vascular permeability, chemotaxis of the cells into the wound milieu, and local release of cytokines and growth factors (34). In experimental models, corticoids managed to reduce inflammation, which in turn affected cell migration, proliferation, and angiogenesis, promoting delayed wound healing (14). This is in agreement with the findings of the present study, where fibroblast content was less in group D. Moreover, as corticosteroids inhibit collagen synthesis both in wounded tissues and in fibroblast cell cultures (23), collagenization and calcification were less commonly seen in group D (Table 2). In agreement with the above, in the present study the valve bulk of animals in group C demonstrated increased fibrosis and extensive collagenization, confirming that normal flexible substrate tissue was replaced with fibrotic tissue. The induced plethora of the collagenous scar could be attributed to a more rapid healing process and justifies the more deformed and dysfunctional valvular apparatus of these animals.

In an effort to enhance the robustness of the findings of the present study, echocardiography was incorporated to assess the status of cardiac function. In aortic endocarditis, left ventricular function is influenced by the extent of valvular destruction, volume overload (35), and overall myocardial dysfunction (12). Both treatments in these groups (C and D) were of similar volumes, as all animals received the same dose of moxifloxacin, and the total volume of dexamethasone infused in 5 days in group D was 2.5 to 3.8 ml, which is hemodynamically indifferent. Moreover, as both treatments resulted in the same rapid bacteriological cure, it is obvious that if any differences in the cardiac function were to occur in the course of the disease, it should be attributed to a more-severe valvulopathy. Consecutive echocardiographic assessments pointed out that only the animals receiving the antibiotic (group C) revealed a more-pronounced pseudonormalization of the FS immediately after the end of treatment and exhibited a significantly steeper decline of the systolic function at the follow-up period (Fig. 1 and 2) compared to the nonsignificant changes of FS throughout the experimental period in group D. These observations imply that only the animals in group C had biphasic changes of acute aortic regurgitation (4, 18). On the other hand, the almost unchanged FS observed in group D is indicative of hemodynamic stability as well. Hence, coadministration of dexamethasone is advantageous, as there is general consensus that decompensated heart failure and acute valve destruction represent the two major criteria for valve replacement and the most sensitive indices of mortality for patients with infective endocarditis (2).

The issues of dosage and duration of the glucocorticoid therapy, however, are intriguing, taking into account in particular the fact that prolonged or high-dose steroid therapy has multiple side effects. Further research, both on newly tested drugs that dissociate the beneficial and detrimental effects of glucocorticoids (e.g., GRalpha selective nonsteroidal antagonists) (6) and on the relative timing, duration, and intensity of the anti-inflammatory scheme, is required.

In short, in the current study moxifloxacin proved to be highly effective against MSSA endocarditis. Furthermore, in accordance with our prior experimental data (26), the coadministration of dexamethasone demonstrated beneficial long-term actions against staphylococcal endocarditis, as assessed with histopathologic and cardiac functional parameters. These beneficial effects are of key relevance to the severity and prognosis of the disease and the need for surgical intervention. Therefore, the results of the current study contribute further to the understanding of the immunological aspects of infective endocarditis, providing an auspicious perspective for the successful conservative treatment of S. aureus endocarditis.


   ACKNOWLEDGMENTS
 
We thank Z. Chrysouli and I. Galani for assistance with the microbiological procedures, M. Liontos for assistance with the microphotographs, and A. Galanos for his help in statistical analysis.


   FOOTNOTES
 
* Corresponding author. Mailing address: Cardiology Department, Hippocration General Hospital, 114 Vas. Sofias Avenue, 11527 Athens, Greece. Phone: 0030-2107483770. Fax: 0030-2107754676. E-mail: iskiadas{at}med.uoa.gr Back

{triangledown} Published ahead of print on 11 June 2007. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Antimicrobial Agents and Chemotherapy, August 2007, p. 2848-2854, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.01376-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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