University Hospital Virgen de la Arrixaca, Murcia School of Medicine, Murcia; Cardiology and Microbiology Departments, Carretera del Palmar s/n, Murcia, Spain
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Abstract |
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Introduction |
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Parenteral administration of penicillin has long been the treatment of choice for serious pneumococcal infections, but the existence of cases of endocarditis caused by penicillin-resistant strains of S. pneumoniae presents a new challenge. Other traditional antibiotics such as vancomycin, ceftriaxone and imipenem have been proposed for treatment.1315 Others, such as trovafloxacin or quinupristin dalfopristin, have recently appeared and may constitute valid alternatives for treating these infections.1622
In previous studies using an experimental endocarditis model,23 the efficacy of cefotaxime, teicoplanin and penicillin at different doses, on two strains of S. pneumoniae with differing sensitivity to penicillin (MIC 1 and 4 mg/L) were studied. Our objective in the present study was to compare the therapeutic efficacy of penicillin, vancomycin, ceftriaxone, trovafloxacin and quinupristindalfopristin against a strain of S. pneumoniae with high-level resistance to penicillin and ceftriaxone (MIC 12 mg/L for both) in an experimental model of aortic valve endocarditis in rabbits.
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Materials and methods |
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The strain of S. pneumoniae used had originally been isolated by bronchoalveolar lavage of a male, HIV-infected patient who was admitted to hospital with a respiratory tract infection. The isolate belonged to serogroup 19.
In vitro studies
Antimicrobial susceptibility tests were performed using three different methods to determine the MIC: (i) microdilution in broth following the NCCLS criteria,24 (ii) an Etest (AB Biodisks, Solna, Sweden) according to the manufacturer's instructions and (iii) dilution in MuellerHinton agar, supplemented with 5% lysed horse blood, incubated at 35°C in an aerobic atmosphere. S. pneumoniae strain ATCC 49619 was used as the control strain.
Experimental endocarditis
All the animal studies were carried out in accordance with the Scientific Procedures Act (Animals) 1986 and the Codes of Practice for the Housing and Care of Animals Used in Scientific Procedures, 1989.
Endocarditis was induced in 145 New Zealand rabbits each weighing c. 2 kg, using Garrison & Freedman's technique25 as modified by Durack & Beeson.26 The animals were anaesthetized with 20 mg/kg of ketamine (Ketolar, Parke-Davis) through one of the marginal veins of the ear. Then, a right paratracheal cervicotomy was performed involving dissection and exteriorization of the right common carotid artery. A sterile polyethylene catheter (Centracath Vigon BP, 795440 Ecouen, France) with an internal diameter of 0.8 mm and an external diameter of 1.2 mm was introduced into the left ventricle, where it remained throughout the experiment. The catheter was considered to be correctly placed when it transmitted the heartbeat in a constant manner.
Twenty-four hours after insertion, 1 mL of saline solution containing an inoculum of c. 108 cfu S. pneumoniae (resistant to penicillin) was injected into the marginal ear vein.
Blood cultures
Immediately before antibiotic treatment was started, blood was obtained for culture by inoculating 10 mL tryptic soy broth with 0.4 mL of blood withdrawn through an ear vein. After 24 h, a preliminary macroscopic examination was carried out. The blood cultures were considered positive if the characteristic colonies were seen to be growing on the plates. Those rabbits in which no positive blood cultures were obtained were excluded from the study.
Antimicrobial treatment
The animals were divided into five study groups: 16 in the control group, 31 in the trovafloxacin group, 15 in the vancomycin group, 16 in the penicillin group, 21 in the ceftriaxone group and 20 in the quinupristindalfopristin group. Antibiotic treatment was initiated 24 h after administration of the inoculum and immediately after a blood sample had been taken for culture. The animals were treated for 5 days with the following doses of antibiotics and methods of administration: iv trovafloxacin (Trovan, Pfizer Laboratories, Madrid, Spain) 13.3 mg/kg every 12 h; iv vancomycin (Diatracin, Dista Laboratories, Madrid, Spain) 20 mg/kg every 12 h; iv ceftriaxone (Rocefalin, Roche Laboratories, Barcelona, Spain) 75 mg/kg every 24 h; im penicillin G procaine (Farmaproina, Cepa Laboratories, Madrid, Spain) 300000 U/kg every 8 h and im quinupristindalfopristin (Synercid, Rhône Poulenc Rorer Laboratories, Collegeville, PA, USA) at a dosage of 30 mg/kg every 8 h. The rabbits in the control group received no antibiotic treatment.
Examination of vegetations
Eight hours after the last dose of the 5 day drug regimen, each animal was killed by rapid injection of 150 mg thiopental (Penthotal Sódico, Abbott Laboratories, Chicago, IL, USA) through a marginal vein in the ear. Only those animals that survived at least 24 h after bacterial challenge were included in the study. The ribcage was immediately opened and the heart was removed, the correct position of the catheter being checked at the same time. A macroscopic diagnosis of endocarditis was performed by observing the characteristic vegetations. Only those animals that survived at least 48 h after introduction of the catheter were included in the study. Those rabbits with incorrectly placed catheters or showing no vegetations were excluded.
Under aseptic conditions, the vegetations were resected from the aortic leaflets, weighed and homogenized in 0.9 mL sterile physiological saline. A series of 10-fold dilutions was then prepared in saline and 100 µL of each dilution was plated on to blood agar. Colonies were counted after 48 h incubation at 37°C in air and expressed as log10 cfu/g. Cultures were considered sterile if there were 2 log10 cfu/g. The limit of detection of the method used was 100 cfu/g and so sterile cultures were assigned a log10 value of 2. The microorganisms were identified by the characteristic appearance of the colonies.
Response to treatment
For inclusion in the analysis, blood cultures had to show the presence of S. pneumoniae before treatment, and each animal had to survive 5 days and show vegetations. The response to therapy was determined by the overall survival rate, the sterilization of blood cultures and vegetations and the number of cfu/g S. pneumoniae in each vegetation.
Twenty-six animals were excluded from further consideration in the study for the following reasons: three were from the control group (because of negative blood cultures); eight from the trovafloxacin group (five because of negative blood cultures and three deaths before 48 h); two from the vancomycin group (one because of negative blood cultures and one not showing vegetations); six from the ceftriaxone group (three negative blood cultures, one death before 48 h and two without vegetations); and six from the quinupristindalfopristin group (two negative blood cultures and four deaths before 48 h).
The cause of death was related directly to surgical intervention in five cases (three cardiac cavity perforations and two profuse bleeding from neck blood vessels) and unknown in the other four cases. Once the first 48 h had passed (24 h after infection) no mortality occurred in any of the groups, so that 119 rabbits remained for analysis.
Antibiotic serum levels
After 5 days of treatment, blood samples were taken from all the rabbits through the marginal vein in the ear. The samples were taken on two occasions: (i) 1 h after the administration of the antibiotic when it was given iv, or 1.5 h after im administration (peak value); and (ii) immediately before the administration of the next dose of the antibiotic (trough value). The drug concentrations in plasma were measured by a bioassay method using Staphylococcus aureus ATCC 25932 for penicillin, Micrococcus luteus ATCC 9341 for quinupristindalfopristin, Escherichia coli ATCC 25992 for ceftriaxone and Bacillus subtilis ATCC 6633 for trovafloxacin.27,28 Competitive enzymo-immunoanalysis with monoclonal antibodies was used to determine the concentrations of vancomycin in an ACA IV discrete clinical analyser (Dabe Behring).
Statistical analysis
The 2 test was used to determine the relationship between qualitative variables, or Fisher's exact test when the expected results were under five. Bonferroni's correction was applied when multiple comparisons were performed. For the comparison of averages between each group of treatment and the control group the MannWhitney U-test was used. The overall comparison of averages between the study groups was determined by means of the Kruskal Wallis test, with subsequent contrast analysis using Dunnett's test. The correlation between two qualitative variables was calculated using Pearson's r coefficient. Statistical significance was considered to exist when P < 0.05, except in correlation analysis, when a P < 0.01 was required.
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Results |
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The MICs for the test strain of S. pneumoniae were as follows: penicillin 12 mg/L, cefotaxime 6 mg/L, ceftriaxone 12 mg/L, erythromycin 4 mg/L, vancomycin 0.5 mg/L, trovafloxacin 0.19 mg/L and quinupristindalfopristin 0.75 mg/L.
Table I shows the MICs of the different antibiotics for S. pneumoniae and the average peak and trough serum levels of each antibiotic. The values of quinupristin dalfopristin and the trough values of trovafloxacin were <4 and 0.5 mg/dL, respectively, these being the minimum values detectable by the technique used.
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Table II shows the weight of the vegetations, the log10 of the cfu/g of vegetation and the percentage of sterile vegetations in all the groups.
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S. pneumoniae were observed in the vegetations of all the rabbits of the control group, meaning that there was no spontaneous healing. The numbers sterilized in all the groups attained statistical significance in comparison with the control group (P < 0.001). Between the groups themselves there were only significant differences between the vancomycin and trovafloxacin group (P < 0.05).
All the antimicrobials were to a greater or lesser extent effective in significantly reducing the bacterial load of the vegetations. The average of the log10 cfu/g of vegetations in the control group was the highest of all the groups, followed by those observed in the trovafloxacin, ceftriaxone, penicillin, quinupristindalfopristin and vancomycin groups. The average cfu/g in the trovafloxacin group was only significantly different from the average values observed in the control and vancomycin groups. The vancomycin group revealed the greatest decrease in average cfu/g (6.69 points below the value of the control group), although the differences were only significant between it and the control and trovafloxacin groups; the differences were on the limit of significance with respect to the ceftriaxone group. The differences observed in the penicillin and quinupristin dalfopristin groups were only significant when compared with the control group.
A comparative analysis between the penicillin and control groups revealed no significant differences between the weight of vegetations in the respective groups, although there were significant differences (P < 0.001) with regard to the bacterial load and numbers of vegetations sterilized.
A similar comparative analysis between the quinupristin dalfopristin and control groups revealed no significant differences between the weight of the vegetations, although, once again, the differences were significant (P < 0.001) when the bacterial load and numbers sterilized (P < 0.001) were compared.
When the trovafloxacin and the control groups were compared, significant differences were seen to exist between the respective weights of the vegetations (P < 0.05), the bacterial load (P < 0.001) and numbers sterilized (P < 0.001).
The average weight of the sterile vegetations was 77.91 ± 61.6 mg, and that of their non-sterile counterparts 124.4 ± 154 mg; statistical analysis showed that significant differences existed between both groups (P < 0.05), and when a comparative analysis between the weight of the vegetations and their bacterial load was performed, the result was a correlation of 0.295 at P < 0.001. Thus, it may be stated that there is a significant correlation between the weight of the vegetations and their bacterial load.
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Discussion |
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To date, the only study of an experimental model of penicillin-resistant S. pneumoniae is that published by Ferández Guerrero et al.23 These authors used two different strains of pneumococci with different sensitivities to penicillin (MIC 1 and 4 mg/L), and studied the effectiveness of penicillin, cefotaxime and teicoplanin. Their observations suggest that the animals treated with doses of penicillin G procaine that achieved serum levels near the MIC for pneumococci, showed a significant reduction in log10 cfu/g of vegetation compared with the control group, although only 20% of the animals showed sterile vegetations. When the serum levels of penicillin were three to four times the MIC, there was a greater reduction in log10 cfu/g of vegetation, and 88% of the animals showed sterile vegetations. Only the doses of penicillin that maintained serum concentrations above the MIC between doses provided constant sterilization of the cardiac vegetations. In terms of antimicrobial efficacy, cefotaxime and teicoplanin induced a similar response to that produced by high doses of penicillin. There are no studies in the literature comparing vancomycin, ceftriaxone, trovafloxacin and quinupristindalfopristin with penicillin.
It is interesting to note that penicillin administered at the doses used in our study is able to produce serum concentrations two or three times greater than the MIC and represents an effective antimicrobial for the treatment of this type of endocarditis. It was placed third as regards its capacity to decrease the cfu/g of vegetation and sterilized 81.2% of the vegetations. The serum concentrations obtained with this antibiotic were higher than those obtained by Fernández Guerrero et al.23 using the same dose. These differences may be explained partly by the fact that the above authors took their blood samples to measure penicillin levels 1 h after the im administration of the drug, whereas we took them 1.5 h afterwards. Penicillin trough levels were below the MIC, which would explain why fewer than 100% of the vegetations were sterilized, since only doses that can provide serum concentrations above the MIC on a continuous basis can have a continuous sterilization effect.23 It is known that patients with pneumococcal meningitis caused by strains highly resistant to penicillin do not respond to treatment with penicillin or ampicillin because neither reaches sufficiently high concentrations in the cerebrospinal fluid to eliminate these resistant strains.29,30 However, penicillin can reach high concentrations in the region of cardiac vegetations, which explains why it may still be effective in the treatment of endocarditis
Quinupristindalfopristin was shown to be an effective antibiotic in this model of endocarditis, with no significant differences being seen between it and vancomycin and penicillin. After vancomycin, it was the most effective antimicrobial in reducing the cfu/g of vegetation, sterilizing 85% of the aortic vegetations. This streptogramin has the advantage of not being affected by the resistance of the pneumococci to penicillin and it has demonstrated its value as an alternative antibiotic in the treatment of infections caused by penicillin-resistant S. pneumoniae.19,20,31 The method used to measure the serum concentrations allowed us to determine only that they were less than 4 mg/L. In previous experimental endocarditis studies in the rabbit,21 using the same dose per kg and the same time intervals and methods of administration, peak values of 3.0 ± 0.8 mg/L were obtained, which are similar to our findings, and three to four times above the MIC. Quinupristindalfopristin has consistently demonstrated a postantibiotic effect against Gram-positive bacteria, and the duration of this effect appears to be dependent on drug concentration rather than on the length of exposure.32,33 This effect should be borne in mind when evaluating the results, to ascertain the drug's effect on the decrease in the log10 cfu/g of vegetation.
Trovafloxacin was chosen for this study because it is the most active of all the new fluoroquinolones against S. pneumoniae and because it shows no cross-resistance with penicillin.16,34,35 It has already produced good results in the case of S. pneumoniae penicillin-resistant pneumonia and meningitis.17,18 In our study, serum peak levels were between 14 and 32 times higher than the MIC and trough levels were similar. Trovafloxacin was shown to be effective in reducing the bacterial load and in rendering vegetations sterile (51.6%), although it was significantly less effective than vancomycin in reducing both parameters. This study was made before the use of the drug in Europe was provisionally suspended due to the existence of important side effects and the death of some patients that may have been related to its use.
Ceftriaxone was included in the study because of the increased resistance of S. pneumoniae to cephalosporins and because it is frequently recommended for this type of infection.3,13 High serum concentrations can be obtained with this drug.15 The peak serum concentrations are 2225 times above the MIC, and the trough values slightly above the MIC. In a comparison between ceftriaxone and vancomycin, there was a tendency for the latter to be more effective than the former only in reducing the bacterial load. Ceftriaxone was able to sterilize 61.9% of the vegetations and was more effective than trovafloxacin only in reducing the cfu/g of vegetation. Given that ceftriaxone is very highly bound to serum proteins, high concentrations in serum would not reflect high concentrations of free drug. In addition, the concentrations of this antibiotic found in vegetations vary between 18 and 47% of serum concentrations. Taken together these observations might partly explain the lesser effect of ceftriaxone with respect to penicillin, observed in the present study.36
Vancomycin sterilized all the animals, which confirms its usefulness for the control of infections caused by resistant S. pneumoniae,1315 as this microorganism continues to be very susceptible to vancomycin, with an MIC of <0.5 mg/L.9 Serum concentrations remained above the MIC throughout the 24 h, with peak values 6884 times higher than the MIC. Comparative analysis with penicillin and quinupristindalfopristin did not reach statistical significance, and we were unable to conclude whether one drug was more effective than the other.
There was a close relationship between the weight of the vegetations and their bacterial load. Thus, a correlation exists between their size and the therapeutic effectiveness of agents used, as judged by the cfu/g and the rate of sterilization of the vegetations.
Subsequent research should evaluate the combined use of the different antibiotics used in this study with, for example, aminoglycosides.
Although we used the infectious endocarditis model in rabbits due to its simplicity, reproducibility, relatively low cost, the possibility of using large numbers of animals and because of its histological similarity to human endocarditis, there are differences between human endocarditis and that produced experimentally in rabbits, so great care should be taken before extrapolating these results to humans.
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Notes |
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References |
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Received 1 June 2000; returned 2 October 2000; revised 23 November 2000; accepted 15 January 2001