a Service des Maladies Infectieuses et Tropicales, Hôpital du Bocage, BP 1542, 21034 Dijon cedex, France; b Service d'Anatomie Pathologique, Centre Hospitalier Universitaire de Dijon, France
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Abstract |
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Introduction |
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Clinical treatment failures in patients with infections caused by resistant S. pneumoniae highlight the need for evaluation of the therapeutic efficacy of antibiotics. Ketolides (HMR 3647, or telithromycin, in particular) have a broad antibacterial spectrum similar to that of erythromycin A, but with additional outstanding activity against inducible MLSB-resistant S. pneumoniae.15 This in vitro activity, together with interesting pharmacokinetic and pharmacodynamic characteristics, make HMR 3647 of potential therapeutic value, particularly in pneumonia. However, few in vivo data are available to date.
We developed a model of experimental penicillinresistant S. pneumoniae pneumonia using non-immunosuppressed animals (adult New Zealand rabbits), reproducing human pneumococcal pneumonia in immunocompetent humans, with an inoculum free of any adjuvant.16 We were also able to reproduce human serum pharmacokinetics following antibiotic administration. We used this model to evaluate the therapeutic efficacy of HMR 3647 for pneumonia caused by S. pneumoniae strains with different antibiotic susceptibilities, and compared it with treatments with amoxycillin and erythromycin.
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Materials and methods |
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Three S. pneumoniae strains isolated from blood cultures of patients with pneumonia were used (kindly provided by P. Geslin and F. Goldstein, France). The first strain (195; serotype 19) was known to be penicillin susceptible erythromycin resistant, the second strain (16089; serotype 9V) erythromycin susceptible but penicillin resistant, and the last strain (11724; serotype 19) to be resistant to both penicillin and erythromycin. Purity was confirmed throughout the study by Gram's stain and colony morphology. Working stock cultures were kept frozen at 70°C in a 15% glycerol supplemented brainheart infusion (bioMérieux Laboratories, Marcy l'Etoile, France). In order to maintain virulence, stock cultures were changed monthly using isolates from rabbits with untreated S. pneumoniae pneumonia. For all strains, MIC and MBC were determined as described previously.17 For strain 11724, MIC and MBC were also determined before and after HMR 3647 pre-exposure. Effect of HMR 3647 pre-exposure was evaluated by pre-growth (8 h at 37°C) in brainheart medium at sub-inhibitory concentrations (0.25 x MIC). The culture was then washed three times before MIC and MBC testing were performed.
Experimental inoculum
Before each experiment, several colonies of S. pneumoniae were inoculated into brainheart infusion, then subcultured on to agar plates and incubated for 24 h at 37°C in 5% CO2. Twenty-five to 30 colonies of an overnight culture were inoculated into 9 mL of brainheart infusion, incubated for 6 h at 37°C and then sub-cultured on agar plates for 18 h at 37°C in 5% CO2. Colonies from this culture were diluted in sterile saline to obtain a final concentration of 10 log10 cfu/mL. No adjuvant was used. This concentration was first determined by optical density measurement, with reference to a standard curve, and confirmed using successive dilution cultures.
Animals
Male New Zealand white rabbits (body weight: 2.73.0 kg) were obtained from Elevage Scientifique des Dombes (Romans, France). These animals were not immunosuppressed, and were virus antibody free and specific pathogen free. They were placed in individual cages and were nourished ad libitum with water and feed according to current recommendations.
Experimental pneumococcal pneumonia in rabbits
The animals were anaesthetized intramuscularly with 1.5 mL of a mixture of ketamine (50 mg/mL) and xylazine (2.75 mg/mL). A silicon catheter was introduced into the jugular vein through a lateral incision of the neck and then tunnelled subcutaneously through the interscapular area.18
Twenty-four hours later, the rabbits were anaesthetized with 0.8 mL of the ketamine + xylazine mixture iv and then by a few millilitres of propofol as required. Under view control, a silicone catheter (Sigma Medical, Nanterre, France) was introduced through the vocal cords into the trachea and into the bronchi. Freshly prepared pneumococcal inoculum (0.5 mL) was then gently flushed through the catheter. The endo-bronchial catheter was then removed and the animals were placed upright for 15 s to facilitate distal alveolar migration by gravity.
All animal experimentation was carried out according to current ethical specifications.
Experimental pneumonia examination
Experimental pneumonia was evaluated by assessing pulmonary injury levels and microbiological findings in each lobe of the lungs and the spleen. These organs were taken at different times after S. pneumoniae inoculation either after sacrifice, or after pneumonia-related death. Animals were anaesthetized with high-dose thiopental, and then killed by exsanguination after cardiac puncture. The thorax was opened, and the presence or absence of pleural effusion was noted. The lungs were then dissected free from the trachea and other structures and placed on sterile gauze for at least 5 min, to allow absorption of residual pulmonary blood. A laparotomy was then performed and the spleen removed aseptically.
For each pulmonary lobe, the macroscopic aspect was noted. Two parts of each lobe were taken, fixed in 10% neutral buffered formalin, and thereafter embedded in paraffin. Haemalumeosinsafranin staining was applied to 5 µm sections. Microscopical examination was performed by a pathologist blinded to the experimental data. Macroscopic and histopathological scores were calculated as described previously.16
Each pulmonary lobe and the spleen were weighed and homogenized in sterile water. Bacteria were counted in a sample of this crude homogenate by plating 10-fold dilutions on sheep-blood agar and incubating the plates for 2448 h at 37°C. Samples of pleural effusions were directly plated and cultured in the same conditions. Bacterial concentrations in each lobe and in spleen were determined after adjusting for weight. Threshold value was 1 log10 cfu/ mL (by using 1 mL plating volume). For each rabbit, mean pneumococcal pulmonary concentration was calculated according to each lobar bacterial concentration with lobar weight [i.e. mean concentration = (lobar concentration x lobar weight) ÷
lobar weights].
Antibiotic assay
Antibiotic concentrations in the blood were determined by a bioassay method,19 using Micrococcus luteus ATCC 9341 for amoxycillin, and Bacillus subtilis ATCC 9466 for erythromycin and HMR 3647. The growth agar was antibiotic medium no. 11 for amoxycillin and tryptic soy agar at pH 9 for erythromycin and HMR 3647 in serum (Difco Laboratories, Detroit, MI, USA). Standard curves were established with solutions of antibiotics (progression from 0.5 to 8 mg/L for amoxycillin, 1 to 10 mg/L for erythromycin and 0.25 to 3.5 mg/L for HMR 3647). The linearity of the standard curves used for disc plate bioassays were at least 0.96 (r2). Serum concentrations were derived from the standard curves with a pre-dilution step if necessary. Results were expressed as micrograms per millilitre of serum. New batches of standard samples were assayed for each experiment, and in duplicate. The within-day and the between-day coefficients of variation for replicates were <7%.
Determination of native pharmacokinetics of antibiotics in rabbits
Amoxycillin (Clamoxyl, SmithKline Beecham, Nanterre, France), erythromycin lactobionate (Erythrocine IV, Abbott, Rungis, France) and HMR 3647 (Hoechst-Marion-Roussel, Romainville, France) were reconstituted according to the manufacturer's instructions, just before each experiment. A bolus was infused iv into three or four rabbits for each antibiotic (20 mg/kg for amoxycillin, 10 mg/kg for erythromycin and 15 mg/kg for HMR 3647). Blood punctures were performed at 0, 5, 10, 15, 20, 30, 45, 60, 90 and 120 min after injection (and also 180 and 240 min after injection for HMR 3647).
Concentrations in serum following an iv injection of antibiotic (either amoxycillin, erythromycin or HMR 3647) could be calculated from the following equation: concentration in serum = Aet + Beßt, where t corresponded to the time elapsed since the bolus was injected. For each rabbit, a logarithmic regression of measured concentrations versus time during the elimination phase (on the basis of an open bicompartmental model) was performed by using the least squares method. Such a regression led to the determination of the ß slope and B constant of the elimination phase. The same method was used to determine the
slope and A constant of the distribution phase, with the exception that values calculated according to the elimination phase equation were withdrawn from measured concentrations.20
Human-like treatment in rabbits
To simulate human pharmacokinetics following the administration of either 1 g of amoxycillin iv,21,22 500 mg of erythromycin iv23 or 800 mg of HMR 3647 orally (data from Hoescht-Marion-Roussel Laboratories), a variable flow-rate infusion with successive levels was used. Briefly, by using the antibiotic pharmacokinetic constants in rabbits, it was possible to calculate both vascular and extra-vascular concentrations following each constant rate infusion, given any initial condition (i.e. with an empty model or not), by using the model developed by Hull.24 Indeed, intercepts A and B from the plasma concentration equation depend in part on the antibiotic dose given either in bolus or by continuous infusion. Thus, it was possible by reversing the formulae to calculate the infusion rate necessary to yield a specific plasma concentration. This method has already been successfully used in humans and animals.16,25
For each experiment, a computer-controlled pump containing an antibiotic was connected to the central venous catheter. This protected connection allowed free circulation and free food access to the rabbits. Infusion rates were controlled by programmable computer software (Softpump, World Precision Instruments, Sarasota, FL, USA). Infusion rates were modified every 30 min (HMR 3647, oral simulation) and every 5 min (amoxycillin and erythromycin, iv simulation).
To control the quality of simulations, arterial blood samples were taken during simulation, at 5, 10, 15, 20, 30, 40, 50, 60, 75, 90, 120, 150 and 180 min for iv simulation (amoxycillin and erythromycin), and at 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330 and 360 min for oral simulation (HMR 3647). Comparison between observed and expected values was made by using a correlation coefficient, and expected versus observed area under the curve (AUC) ratios.
Pharmacodynamic parameters were calculated considering the MIC (and in some cases MBC) of the studied antibiotic for each S. pneumoniae strain. Time above MIC (or MBC) was defined as the percentage of time with in vivo concentrations above the MIC (or MBC) during the study period (48 h). The average AUC24/MIC (or MBC) was calculated as the sum of the AUCs of each simulated dose during the period of experiment, divided by MIC (or MBC) and divided by the duration of the experiment (in days). The average AUC24 > MIC (or MBC) was calculated as the sum of the AUC of concentrations above MIC (or MBC) of each simulated dose during the period of the experiment, divided by the duration of the experiment (in days).
Experimental schedules
A total of 70 rabbits were used. For each strain of S. pneumoniae, 2025 rabbits were randomly assigned, just before inoculation with 0.5 mL of 10 log10 cfu/mL, to four arms: (i) control (n = 10); (ii) iv amoxycillin (n = 5); (iii) iv erythromycin (n = 5); and (iv) oral HMR 3647 (n = 5). Human-like antibiotic treatment was started 4 h after inoculation. Antibiotic concentrations in the serum and lungs were systematically assayed at time of death in treated rabbits. All treated rabbits still alive at 48 h were killed at least 10 h after the last computer-controlled antibiotic infusion, i.e. 4852 h after inoculation to avoid significant carry-over effect. In this latter group, lung antibiotic concentrations at time of death were always below the threshold of detection. Untreated rabbits still alive were also killed 48 h after inoculation. Main evaluation criteria were pneumococcal concentrations in lungs and spleen at time of death for all the rabbits. Complementary evaluation was performed taking into account the survival rate at 48 h (i.e. the number of rabbits dead from pneumonia divided by the total number of rabbits, dead and killed), and the macroscopic and histopathological scores at time of death.
Statistics
Results were expressed as the mean or percentage ± S.D. Proportions were analysed as quantitative values by using angular transformation. After verification of the homogeneity of the variances using Hartley's table, continuous variables were analysed with one-way analysis of variance. In case of a significant test, post hoc analyses comparing results between two arms were conducted by using Scheffé's test. To compare relationships between quantitative values, the correlation coefficient (r) and r2 values were calculated by the linear regression model. For all the tests, a P value < 0.05 was considered significant.
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Results |
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MICs and MBCs of antibiotics for the different strains of S. pneumoniae are summarized in Table I. HMR 3647 MBC was very close to MIC for strains 195 and 16089, with a MBC/MIC ratio of 2. By contrast, the MBC/MIC ratio was much higher for strain 11724 (MBC/MIC = 32). Only for the erythromycin-resistant S. pneumoniae strain (195), pre-exposure to HMR 3647 induced a four-fold increase of HMR 3647 MBC whereas no modification of MIC was observed. Lastly, the inoculum size did not appear to significantly modify either HMR 3647 MIC or MBC for the three S. pneumoniae strains studied.
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Amoxycillin, erythromycin and HMR 3647 concentrations in serum following an iv bolus in rabbits fitted into a bicompartmental model. Pharmacokinetic constants are summarized in Table II. Simulations of human pharmacokinetics following iv amoxycillin 1 g, iv erythromycin 500 mg and oral HMR 3647 800 mg administration are shown in the Figure
(ac). The correlation coefficients between concentrations obtained by simulation and the expected human values were 0.998 for amoxycillin, 0.979 for erythromycin and 0.993 for HMR 3647. The ratios between observed AUC and human expected AUC were 1.03 for amoxycillin, 1.45 for erythromycin and 0.90 for HMR 3647.
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Untreated rabbits.
As expected, 4 h after inoculation, rabbits had a crude bacteraemic pneumonia, with pneumococcal concentrations reaching 5 log10 cfu/g in lungs and 3 log10 cfu/g in spleen. Histopathological and macroscopic scores were both close to 10 (non inoculated = 0) at this time.
Forty-eight hours after inoculation, a significant pneumonia was observed with all strains of S. pneumoniae in all untreated rabbits. Mean pneumococcal concentrations in lung and spleen were 5.3 ± 1.7 and 4.0 ± 2.1 log10 cfu/g, respectively, whereas mean macroscopic and mean histopathological scores were 14.7 ± 8.0 and 11.9 ± 5.8, respectively. No significant differences were observed between S. pneumoniae strains for lung bacterial concentrations and lung lesions. However, there was a significant difference between strains for spleen pneumococcal concentration (P = 0.05), with significantly lower concentrations in rabbits infected with strain 11724 than in those infected with strain 195 (P = 0.05, Scheffé's test). At the same time, mortality was lower in rabbits inoculated with strain 11724 than in those inoculated with strains 195 (0 versus 40%, P = 0.07) and 16089 (0 versus 40%, P = 0.07).
Treated rabbits.
The efficacy of human simulated treatment of experimental pneumococcal pneumonia in rabbits is summarized in Table III.
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Discussion |
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Antibiotic treatment was started 4 h after inoculation at a time when pulmonary consolidation with high pneumococcal concentrations was present, and before the first untreated pneumonia-related deaths occurred. Lung lesions observed 4 h after inoculation ensured that antibiotic treatment was a therapeutic treatment and did not simply act as prophylaxis. Antibiotic therapy reproduced in rabbits the human serum pharmacokinetics of the antibiotics. To ascertain the quality of HMR 3647 simulation, all serum samples were assayed not only by the plate bioassay method, repeated twice, but also by HPLC with a very good correlation between these two methods (data not shown). Results obtained after simulation were very close to the expected (human) concentrations. Treatment duration allowed an evaluation 48 h after inoculation, without any carry-over effect, at a time when bacterial concentrations are high in untreated rabbits. We can therefore argue that such an experimental model allowed a good evaluation in vivo of the efficacy of antibiotic treatments in pneumonia.
Our results show that amoxycillin is effective not only for the penicillin-susceptible strain but also for strain 16089, despite its penicillin MIC of 4 mg/L. Erythromycin was effective against this latter strain, probably because it was a macrolide efflux strain with low MIC. HMR 3647 was effective for both strain 16089 and strain 195, which is consistent with in vitro data both for penicillin-resistant2630 and erythromycin-resistant strains, even though ketolides have been shown to be less active against constitutive MLSB-resistant strains.2632
HMR 3647 was shown not to be effective for the very highly erythromycin-resistant strain 11724 despite a low MIC (0.02 mg/L). There is no clear explanation for this, although it is possible that it may have an immunosuppressant effect, as observed with some macrolides33 or other ketolides.34 In our model, we failed in previous experiments to show any difference in pulmonary injuries between treated versus untreated rabbits (whatever the treatment and its microbiological efficacy), suggesting that inflammatory phenomena evolve in an autonomous fashion, without strict correlation with residual bacterial inoculum.16 Here, it is of particular interest that the pulmonary injury scores (macroscopic and histopathological) in this experiment were more often lower in rabbits treated with HMR 3647 than in those untreated or treated with other antibiotics. However, the differences observed were never statistically significant, and it could not be explained why such an immunodepression would promote bacterial pneumonia induced by strain 11724 and not those induced by other strains.
Microbiological characteristics suggest an alternative explanation. Some strains with inducible erythromycin resistance may express high-level methylase activity which inhibits in vivo the HMR 3647 efficacy, as already observed in vitro for another ketolide (HMR 3004).31 In contrast to erythromycin, HMR 3004 has been shown to be a weak inducer of methylase production, but some strains may express methylase in the absence of inducer.31 In our study, it could be speculated that after the first dose of HMR 3647, in some lung areas with high inoculum concentrations, some persisting S. pneumoniae 11724 modified their phenotype and became less susceptible to HMR 3647. However, in vitro data showed that neither pre-exposure to HMR 3647 nor use of 107 cfu/mL inoculum resulted in increasing HMR 3647 MIC and MBC for this strain. Moreover, we failed to find in vivo mutants by culturing of strains 11724 isolated from lungs after a 48 h HMR 3647 treatment in a broth supplemented with 0.02 mg/L of HMR 3647 (data not shown). It is improbable that inducible resistance could explain the HMR 3647 failure in pneumonia induced by strain 11724.
Pharmacodynamic data were useful in predicting antibiotic efficacy for both amoxycillin and erythromycin. Indeed, for these two antibiotics, time above MIC must be 33% to allow therapeutic efficacy, whereas treatment failed when time above MIC was <25%. For HMR 3647, pharmacodynamic parameters including MIC were equivalent for all of these strains, and were not useful in explaining the difference in efficacy observed for HMR 3647. It was of particular interest to study pharmacodynamic data integrating MBC. It appears that time above MBC < 25% was predictive of treatment failure, whereas it was predictive of therapeutic success when >33%, not only for amoxycillin or erythromycin, but also for HMR 3647. Pharmacodynamic considerations may therefore be contributive to explaining the treatment failure observed with strain 11724.
In conclusion, it appears that in some cases, pharmacodynamic data using MBC may be useful in predicting treatment success or failure, although more experimental data are necessary to confirm this hypothesis. Overall, it appears that HMR 3647 may be useful in the treatment of infections of the upper and lower respiratory tracts, even against penicillin-resistant and erythromycin-resistant S. pneumoniae strains.
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Acknowledgments |
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Notes |
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References |
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Received 22 May 2000; returned 16 August 2000; revised 18 September 2000; accepted 25 September 2000