Effect of Treatment Duration on Pharmacokinetic/Pharmacodynamic ...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2006, p. 2919–2925 0066-4804/06/$08.00⫹0 doi:10.1128/AAC.00859-05 Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Vol. 50, No. 9

Effect of Treatment Duration on Pharmacokinetic/Pharmacodynamic Indices Correlating with Therapeutic Efficacy of Ceftazidime in Experimental Klebsiella pneumoniae Lung Infection Irma A. J. M. Bakker-Woudenberg,1* Marian T. ten Kate,1 Wil H. F. Goessens,1 and Johan W. Mouton2 Department of Medical Microbiology & Infectious Diseases, Erasmus University Medical Center Rotterdam, Rotterdam,1 and Department of Medical Microbiology and Infectious Diseases, Canisius Wilhelmina Hospital, Nijmegen,2 The Netherlands Received 7 July 2005/Returned for modification 31 October 2005/Accepted 18 June 2006

The pharmacokinetic/pharmacodynamic (PK/PD) indices that define the therapeutic effect of the betalactam ceftazidime in a rat model of Klebsiella pneumoniae lung infection were investigated in relation to treatment duration and treatment endpoint. Treatment was started 24 h after infection with dosing regimens of 3.1 up to 1,600 mg/kg of body weight/day and dosing every 6, 12, or 24 h. When animals were treated for a relatively short period of 48 h, the duration of time that unbound plasma ceftazidime levels exceeded the MIC of the antibiotic for the infecting strain was the index that best correlated with therapeutic efficacy in terms of significant bacterial killing in the infected lung (microbiological effect). The maximum effect was reached when plasma ceftazidime levels were above the MIC for 60 to 70% of the dosing interval. However, when the treatment duration was extended to a relatively long period of 18 days instead of 48 h and animal survival rate instead of microbiological efficacy was taken as the endpoint, the fAUC/MIC ratio (where AUC is the area under the concentration-time curve) was the PK/PD index that best correlated with therapeutic efficacy. The PK/PD indices that effect 50% survival of rats for the fAUC/MIC ratios were 18.0 (95% confidence interval [95% CI], 16.3 to 19.9), 20.2 (95% CI, 13.8 to 29.4), and 27.9 (95% CI, 21.3 to 36.5) for the schedules of administration of every 6, 12, and 24 h, respectively. The fAUC/MIC needed for 100% survival was >100. We conclude that the PK/PD index that best correlates with outcome is dependent on the duration of treatment and/or the parameter of outcome. The effect of long-term treatment should be studied more extensively in other models of infection. positive or gram-negative bacilli have been used. The variables included in these models are the pathogen species and the animal species, the site of infection, the choice of antimicrobial agent, the start time and the duration of treatment, the treatment endpoint, the animal host defense, and others. However, an important limitation to the majority of these studies is that the treatment duration in the experimental animal setting is relatively short and usually varies from 24 h to 48 h. A decrease in bacterial counts in infected organs is commonly used as the parameter for treatment outcome, although survival has been used as well. In addition, in most studies animals with impaired host defenses are used because an intact host immune status may interfere with the determination of the impact of PK/PD indices for therapeutic efficacy (3). It could be questioned whether similar results with respect to the PK/PD indices that determine the therapeutic outcomes obtained with antibiotics will be achieved in an experimental setup in which animals are treated for a prolonged period of time, simulating the duration of clinical treatment of a serious infection. In that respect, some studies show that the PK/PD index that determines therapeutic efficacy is not dependent on the time period of treatment. In the majority of studies a treatment duration of 24 h has been applied, and some investigators using a treatment duration of 5 days achieved similar results (29, 30). In contrast, other investigators demonstrated that the treatment duration may be relevant in this respect (J. W. Mouton, J. Leggett, and W. A. Craig, Abstr. 37th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 23, 1997). The present study was undertaken to investigate the corre-

Numerous studies in vitro and in animal infection models have been performed to elucidate the correlation between antimicrobial therapeutic efficacy and the pharmacokinetic/ pharmacodynamic (PK/PD) indices of antimicrobials, such as the duration of time that plasma levels remain above the MIC (T⬎MIC), the ratio of the 24-h area under the plasma concentration-time curve (AUC) to the MIC (24-h AUC/MIC ratio), and the peak concentration in plasma (Cmax) related to the MIC (Cmax/MIC ratio) (11, 13, 14, 15, 20, 24, 33, 36). The general view is that T⬎MIC is the major PK/PD index that determines the in vivo efficacies of beta-lactams, including penicillins, cephalosporins, monobactams, and carbapenems, while, on the other hand, the Cmax/MIC and 24-h AUC/MIC ratios are the important PK/PD indices that correlate with the efficacies of aminoglycosides and fluoroquinolones. These conclusions have primarily been derived from investigations with animal models of infection searching for the therapeutic rationale for antimicrobial dosing. The optimal antibiotic dosing regimens for the treatment of human infections are increasingly based on these experimental studies (11, 23) and have now been validated in a number of clinical trials (1, 2, 16, 17, 18, 19, 25, 28). A variety of animal models of infection caused by gram-

* Corresponding author. Mailing address: Department of Medical Microbiology & Infectious Diseases, Erasmus University Medical Center Rotterdam, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands. Phone: 31 10 4632175. Fax: 31 10 4633875. E-mail: i.bakker-woudenberg @erasmusmc.nl. 2919

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lation between the PK/PD indices for ceftazidime and outcome in relation to the treatment duration and treatment endpoint in an animal model of Klebsiella pneumoniae lung infection. Ceftazidime was administered at three different frequencies. In the first part of the study, a relatively short treatment duration of 48 h was applied and efficacy was assessed monoparametrically in terms of the decrease in bacterial counts from the infected tissue compared to the counts at the start of treatment (microbiological effect). In the second part of the study, the same model of infection and the same antimicrobial treatment with respect to the choice of antibiotic and dosage frequency were used to assess the predictive PK/PD indices that determine the efficacy of ceftazidime administered for a relatively long duration of 18 days, and an overall parameter for efficacy in terms of animal survival rate was used. MATERIALS AND METHODS Animals. Male specified-pathogen-free RP/AEur/RijHsd albino rats (Harlan, Horst, The Netherlands) were used in all experiments. The animals were 11 to 15 weeks old and weighed 250 to 310 g. The experimental protocols adhered to the rules laid down in the Dutch Animal Experimentation Act (1977) and the published Guidelines on the Protection of Experimental Animals by the Council of the European Community (8a). The present protocols were approved by the Institutional Animal Care and Use Committee of the Erasmus University Medical Center Rotterdam. Bacteria. K. pneumoniae ATCC 43816, capsular serotype 2, was used to infect the rats. The MIC of ceftazidime for this strain was 0.5 ␮g/ml, as determined by the tube microdilution test (8). Infection model. A left-sided pneumonia in rats was obtained as described previously (4). In brief, the rats were anesthetized with fluanisone and fentanyl citrate (Hypnorm; Janssen, Animal Health, Saunderton, United Kingdom), followed by pentobarbital (Nembutal; Sanofi Sante´ b.v., Maassluis, The Netherlands). After intubation of the left primary bronchus, a cannula was passed through the tube and the left lung was inoculated with 106 viable K. pneumoniae bacteria in the logarithmic phase of growth suspended in 20 ␮l of phosphatebuffered saline. After bacterial inoculation, the narcotic antagonist naloxon hydrochloride (Narcan; Bristol-MyersSquibb, Woerden, The Netherlands) was injected. Inoculation of the lung resulted in an acute unilateral pneumonia. The animals were killed by CO2 inhalation, a blood sample was taken, and the left lung was removed and homogenized (Polytron; Kinematica, Lucerne, Switzerland) in 5 ml of phosphate-buffered saline for 30 s at 30,000 rpm. Lung homogenate suspensions and blood were serially diluted and plated on tryptone soy agar (Oxoid, Basingstoke, United Kingdom). Animal survival was monitored daily. The animals that died from infection were dissected. Macroscopic observation of the lungs was performed, and the lungs and blood were cultured semiquantitatively. Antimicrobial treatment. Ceftazidime (Glaxo-Wellcome, Zeist, The Netherlands) was administered intramuscularly, and treatment was started 24 h after bacterial inoculation of the left lung. The duration of treatment was 48 h or 18 days. Ceftazidime doses were increased twofold. In rats treated for 48 h the doses ranged from 6.3 to 1,600 mg/kg of body weight/day, and in rats treated for 18 days the doses ranged from 3.1 to 400 mg/kg/day. The injection frequency was every 24, 12, or 6 h (q24h, q12h, and q6h, respectively). Parameters for therapeutic activity. In rats treated for 48 h therapeutic efficacy was assessed by quantification of the numbers of viable K. pneumoniae bacteria recovered from the infected left lung after dissection of the rats 72 h after bacterial inoculation (n ⫽ 3 rats per treatment group). The decrease in bacterial numbers compared to the numbers at the start of treatment (dlog10 CFU) was calculated. In rats treated for 18 days, therapeutic efficacy was assessed by determination of the survival rate for rats until day 43 after bacterial inoculation (n ⫽ 10 per treatment group). The survival of rats was monitored daily. Postmortem cultures of the left lung and blood from rats were performed to check for the presence of K. pneumoniae only. The emergence of resistance was monitored by subculture on plates containing 8 ␮g/ml ceftazidime (16 times the MIC). Pharmacokinetics. The pharmacokinetics of ceftazidime after single- and multiple-dose administration of various dosages were determined in rats with K. pneumoniae lung infection. Blood samples were obtained by puncture of the retro-orbital plexus under CO2 anesthesia at 5, 15, 30, 45, 60, 90, 120, 180, and

ANTIMICROB. AGENTS CHEMOTHER. 240 min after intramuscular ceftazidime administration. The concentration at each time point was assessed in triplicate. Ceftazidime concentrations in plasma were determined by the standard large-plate agar diffusion assay, as described previously (7). In short, diagnostic sensitivity test agar (Oxoid) and an Escherichia coli test strain susceptible to 0.2 ␮g/ml of ceftazidime were used. Undiluted plasma samples as well as diluted plasma samples were assayed in volumes of 100 ␮l. Twofold increasing standard concentrations ranging from 0.2 to 1.6 ␮g/ml of ceftazidime were used. The lower limit of detection in this assay was 0.2 ␮g/ml ceftazidime. The correlation coefficient was 0.99, and the coefficient of variation varied from 1 to 3% over a range from 0.2 to 1.6 ␮g/ml. The level of protein binding of ceftazidime was measured by means of the ultrafiltration technique, as described previously (6). Pharmacokinetic parameters were determined by using Winnonlin (version 2.1; Pharsight Corp., Mountain View, CA) by applying a one-compartment open model with absorption. Survival analysis and PK/PD analysis. The PK/PD indices were determined for the unbound fraction of ceftazidime by using MicLab 2.32 (Medimatics, Maastricht, The Netherlands). Values were determined by simulating steadystate conditions. The pharmacokinetic parameter values used to calculate indices from doses not included in the pharmacokinetic study were obtained by interpolation. Data analysis was performed by using Graphpad Prism (version 3.0; Graphpad Software, San Diego, CA). Significant differences in survival between groups were determined by using the log rank test. Curves were compared per dose level, and if there were significant differences overall, individual curves were compared. Estimates of the 50% effective PK/PD index (EI50) and the 50% effective dose (ED50) that effected 50% survival of rats were obtained by fitting the maximum-effect model with variable slope. Statistical significance was accepted at a P value of ⱕ0.05 by using Dunn’s correction for multiple comparisons, where applicable. Statistical differences between the effects of the dosing regimens after 48 h of treatment were determined by linear regression (q6h versus q12h) or multivariate analysis (q6h versus q24h).

RESULTS K. pneumoniae lung infection. At 24 h after bacterial inoculation of the left lung with 106 K. pneumoniae bacteria, the bacterial numbers in the left lung had increased approximately 103-fold to 9.8 log10 CFU (range, 9.1 to 9.9 log10 CFU). At 72 h after bacterial inoculation, the bacterial numbers in untreated rats had increased ⱖ104-fold. The infection developed progressively. Untreated rats developed septicemia and pleuritis and eventually died from day 3 after bacterial inoculation. All untreated rats died by day 8. Pharmacokinetics. The pharmacokinetic characteristics of ceftazidime were determined after administration of single doses of 12.5, 25, 100, and 800 mg/kg in infected rats at 24 h after bacterial inoculation of the lung. Figure 1A shows the linear relationship between log dose (in mg/kg) and log AUC, including the 95% confidence interval (CI). To determine whether the AUCs differed significantly after 18 days of therapy and whether ceftazidime showed unexpectedly high levels of accumulation, the pharmacokinetic profile of ceftazidime was determined in a group of rats after 18 days of therapy of 25 mg/kg/day q6h. Although the AUC was slightly higher, the value obtained fell within the 95% CI of the dose-AUC regression line. Likewise, Fig. 1B shows the relationship between the elimination rate constant and dose, including the rate constant obtained after 18 days of treatment. The volume of distribution was, however, significantly lower after 18 days of treatment (Fig. 1C). The protein binding of ceftazidime was 11%. Therapeutic efficacy (microbiological outcome) of ceftazidime after 48 h of treatment. Treatment with ceftazidime, which was started at 24 h after bacterial inoculation, resulted in a decrease in the bacterial counts in the left lung to a maximum of 3 log10 CFU compared to the counts in the untreated con-

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THERAPEUTIC EFFICACY OF CEFTAZIDIME

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FIG. 1. Relationship between dose and various pharmacokinetic parameters of ceftazidime. Closed symbols, results obtained after administration of a single dose to infected rats; open symbols, results obtained after 18 days of treatment of infected rats; k10, elimination rate constant; Vd, volume of distribution.

trols at 72 h. Figure 2A shows that ceftazidime administration four times daily was more effective than twice-daily administration (P ⬍ 0.01) and once-daily administration (P ⬍ 0.01) in achieving a significant reduction in the bacterial counts. These results indicate that the efficacy of ceftazidime is dependent on the dosing regimen and is more effective when the same daily dose is administered in divided doses rather than as one dose. Therapeutic efficacy of ceftazidime during 18 days of treatment. The rate of survival of rats was assessed for a period of 43 days after bacterial inoculation. All dead animals showed macroscopic lesions in the infected lung, and semiquantitative cultures exhibited high bacterial loads in infected lung and blood. As shown in Fig. 2B, treatment with ceftazidime administered at various dosing frequencies resulted in a significant increase in the rat survival rate in a dose-dependent manner. There was no significant difference between the q6h, q12h, and q24h dosing regimens. One hundred percent survival of the rats was obtained at doses that were well tolerated. Sigmoidal dose-response curves were observed and were similar for all treatment schedules. These results indicate that, in contrast to the results obtained after 48 h of treatment, the efficacy of ceftazidime after 18 days of treatment is dependent on the total daily dose rather than the dosing regimen and is equally effective when the same daily dose is administered in divided doses or as one dose. Whereas the dose-response curves were similar for the three regimens, the product-moment survival curves showed some

differences (Fig. 3). The survival curves were similar for all dose levels, resulting in less than 100% survival. However, the dose level that resulted in 0% survival (3.1 mg/kg/day) showed significantly earlier death during the q6h dosing regimen (log rank test). PK/PD analysis. A PK/PD analysis was performed to more closely study concentration-effect relationships. Figure 4A and B shows the relationship between fT⬎MIC and dlog CFU and between fAUC/MIC and dlog CFU in the left lung after 48 h of therapy, respectively. From the results in Fig. 4, it can be concluded that fT⬎MIC correlated the best with the therapeutic effect. It is noteworthy, however, that the relationship does not follow a classic sigmoidal curve relationship. Although the PK/PD index value for a static effect is 18% and the maximum effect seems to be reached at approximately 30% fT⬎MIC, increasing fT⬎MIC values result in a better effect until about 60 to 70% fT⬎MIC. Because of this, the fit of a maximum-effect model with variable slope did result in R2 values that were too low to be meaningful. The relationship between fAUC/MIC and dlog CFU in the left lung was significantly different between the q6h, q12h, and q24h dosing regimens. Figure 4C and D shows that the PK/PD index that best correlated with rat survival after 18 days of treatment was the fAUC/MIC. This indicates that the efficacy of treatment was independent of the dosing schedule, although the EI50 for fAUC/MIC was slightly higher for the q24h regimens than for the q12h and q6h regimens. This is further substantiated by the significant difference between the effect curves relating mor-

FIG. 2. Effect of increasing doses of ceftazidime on outcome in rats with K. pneumoniae lung infection. (A) Decrease in bacterial numbers in the left lung after 48 h of treatment compared to that at start of treatment (dlog CFU; n ⫽ 3 per group); (B) 43-day survival of rats after 18 days of treatment (n ⫽ 10 per group).

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ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. EI50s and ED50s of three different ceftazidime dosing regimens, based on rat survival on day 43 Dosing frequency

% fT⬎MIC

fAUC/MIC

ED50

q6h q12h q24h

24.5 (23.8–25.2)a 15.0 (13.5–16.5) 10.1 (8.2–12.0)

18.0 (16.3–19.9) 20.2 (13.8–29.4) 27.9 (21.3–36.5)

15.7 (13.8–17.8) 14.6 (9.9–21.4) 15.6 (11.6–20.3)

a

Values in parentheses are 95% CIs.

DISCUSSION FIG. 3. Survival curves for rats with K. pneumoniae lung infection treated for 18 days with 12.5 mg/kg/day (the ED50) and 3.1 mg/kg/day ceftazidime; the numbers in the legends indicate the doses. The shapes of the curves are similar and not significantly different.

tality with fT⬎MIC. Table 1 shows the EI50s for fT⬎MIC and fAUC/MIC, as well as the ED50s, including the 95% CIs. The model fits were very good, with R2 being ⬎0.96 for all nine curves. The relationship between fCmax/MIC and the PK/PD index was also determined, but this relationship was not as obvious as that for the fAUC/MIC ratio (results not shown). Emergence of resistant strains. A sample of the left lung homogenate from every nonsurviving rat was plated on medium containing 8 ␮g/ml ceftazidime to determine whether ceftazidime-resistant strains had emerged during therapy. Resistant strains were never found. In addition, only K. pneumoniae was present in the infected lungs of rats that had died.

The present investigation shows that the PK/PD indices important to outcome (bacterial killing versus rat survival) of the beta-lactam ceftazidime in an experimental K. pneumoniae lung infection in rats depend on the experimental setup of the study. The experimental approach with a relatively short treatment duration and the microbiological effect as a parameter for the outcome yielded results different from those obtained by use of an experimental approach with a relatively long treatment duration and the animal survival rate as the overall parameter for efficacy. When the animals were treated for 48 h only, lower doses given more frequently, which resulted in substantial periods of time that plasma antibiotic levels exceeded the MIC, appeared to be more advantageous than higher doses given less frequently for effective treatment. The PK/PD index that correlated with the microbiological effect was fT⬎MIC. In contrast, when animals were treated for 18 days, the PK/PD index that correlated with the animal survival

FIG. 4. Relationship between fT⬎MIC or 24-h fAUC/MIC ratio of ceftazidime and outcome in rats with K. pneumoniae lung infection. (A and B) decrease in bacterial numbers in the left lung after 48 h of treatment compared to the numbers at the start of treatment (dlog CFU; n ⫽ 3 per group); (C and D) 43-day survival of rats after 18 days of treatment (n ⫽ 10 per group).

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rate as the parameter for overall cure was the fAUC/MIC ratio and not fT⬎MIC. The primary purpose of the 48-h treatment studies was to confirm that T⬎MIC is the PK/PD index value that correlates with the outcome in our infection model during short-term treatment and in that respect did not differ from other models of infection. The dosing regimens were specifically chosen for that purpose rather than for determination of the effect of the dosing regimens at every possible dose level. The drawback of this approach is that there are no datum points for all dose levels at the three dosing intervals studied, and conclusions need to be drawn for the whole dosing range and can be drawn only for the whole dosing range. This approach did show that the experimental results obtained at the end of the 48-h treatment period are in agreement with the observations of other investigators who used a short-term treatment duration with beta-lactams and the microbiological effect as the parameter for the treatment outcome (11, 12, 13, 15, 20, 30, 31, 33, 36). In these studies, fT⬎MIC was the PK/PD index that primarily correlated with efficacy; and a significant difference in effect between the q6h, q12h, and q24h dosing regimens was observed. This is usually explained by the fact that beta-lactams exhibit time-dependent killing which is not strongly concentration dependent. Maximum bacterial killing occurs at concentrations three to four times the MIC, with further increases in the drug concentrations having little effect (26, 34). Besides the microbiological effect, an increase in the animal survival rate has also been used as the endpoint for therapeutic efficacy in various infection models (5, 15, 29, 30). In our own studies, using the same experimental pneumonia model used in the present study, we found that the continuous infusion of ceftazidime is more efficacious than treatment at 6-h intervals (29, 30). These data, together with data from other studies that used a variety of infection models, show that the PK/PD index required for overall cure (animal survival) and microbiological effect (decrease in bacterial counts) is fairly consistent and is T⬎MIC (10). However, the duration of treatment was relatively short in those studies and was never longer than 4 days. We found that the maximum decrease in bacterial numbers in the infected lung tissue was related to the time that the ceftazidime concentrations in plasma remained above the MIC for 60% to 70% of the dosing interval. Other investigators also demonstrated in animal models of infection caused by members of the family Enterobacteriaceae that the levels of several broad-spectrum cephalosporins in plasma should be maintained above the MIC for 60% to 70% of the dosing interval for maximum killing of bacteria (10), and values in this range have been used to determine the optimal dosing regimens and breakpoints for this drug (32) and other cephalosporins (P. G. Ambrose, S. M. Bhavnani, R. N. Jones, W. A. Craig, and M. N. Dudley, Abstr. 44th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 138, 2004). Other animal studies suggest that the percent fT⬎MIC required for efficacy was higher for cephalosporins than for the penicillins (15). The differences were attributed to differences in the rates of killing. Most experimental studies were performed with the neutropenic murine thigh and pneumonitis models (3, 9, 15, 21, 22, 33, 35). The conclusions with respect to the PK/PD index for determination of microbiological efficacy are relatively similar for various


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sites of infection, various pathogens, and various drugs within the same class, provided that free drug levels are used (3), although a few differences have been observed (15, 33). The present study shows that the fT⬎MIC is not the major PK/PD index that correlated with efficacy in terms of overall cure after a long treatment duration. In contrast, the AUC/ MIC ratio, or the daily dose, was the index that best correlated with the animal survival rate. It is clear that the antimicrobial treatment duration and the actual treatment endpoint used may be important determinants for the interpretation of the experimental data and their relevance to clinical practice. Experimental studies investigating the impact of PK/PD indices during short-term treatment on bacterial killing in the infected tissues provide relevant knowledge with respect to the first day(s) of treatment. At the same time, studies on the effect of long-term treatment on maximum animal survival are relevant for better understanding of the impact of PK/PD indices in infections requiring treatment for a relatively long duration. The role of beta-lactams in this respect is important, as relatively long treatment of serious infections caused by gram-negative bacteria with beta-lactams is common (15). Remarkably, the fAUC/MIC needed for 100% animal survival was about 100, a value that correlates with clinical efficacy for other classes of drugs as well, in particular, the quinolones (18). The explanations for our findings are not easy to find. Although the pharmacokinetic parameters are slightly different after 18 days of therapy, these do not explain the results. The volume of distribution after 18 days of therapy was lower, probably because the animals had cleared the infection and the fluid shift to the lungs after the early days of infection had cleared and resulted in a lower peak concentration. The elimination rate, however, was not very different; and the trough levels were comparable to those found in the initial phase of infection. There is no reason to assume that the pharmacokinetics in the three treatment groups differ in such a way as to compensate for the dosing schedule, and this would also be highly unlikely. This argument applies to changes in pharmacokinetics during the first few days of infection as well. Although it is highly likely that these changes do occur during various stages of infection, it is not very likely that these changes compensate for the findings in such a way that they explain our observations. In a similar fashion, the survival of K. pneumoniae bacteria intracellularly and their regrowth or outgrowth after the end of therapy in a dose-dependent manner also cannot explain our results. All animals, with three exceptions, either died or survived during the 18-day treatment period and not after. Of note here are the results of the survival curves. At the dose level that resulted in 0% survival, animals died significantly earlier in the q6h regimen group, indicating a difference in treatment effect that may help explain the differences found. One possible explanation may be the target of the drug. The main reason that beta-lactams show a killing pattern that is relatively concentration independent is that the overall effect is observed only when the majority of the penicillin binding proteins (PBPs) are saturated (27). It is only at between 90% and 100% of saturation that killing is observed, and this is one of the explanations for the steep concentration-effect curve of the beta-lactams. If, over time, the number of PBPs would de-

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crease substantially, this could result in a more concentrationdependent killing pattern for the beta-lactam. Alternatively, it could be speculated that there are other targets or different targets that are not important during the initial stage of treatment or that at least do not drive the therapeutic outcome, while in later stages of exposure these do become important. It is often observed, in both time-kill curve studies and here, that not all bacteria are killed after 24 or 48 h of exposure, even at the higher concentrations/doses. These “persisters” could consist of a subpopulation that does have these different pharmacodynamic characteristics and that over time could drive the therapeutic outcome. This effect would be even more enhanced if these bacteria reside in third or “deep” compartments where the initial concentrations are relatively low and steady state may be reached faster, depending on the initial peak concentration. One such compartment could be intracellular, thus allowing the prolonged survival of intracellular bacteria. Because we used a nonneutropenic model, this effect may have been larger than that in neutropenic animals. A concentration-dependent transport mechanism in and out of the cell could also play a role there. These explanations would have to be investigated in an alternative experimental setting. We conclude that the relationship between antibiotic PK/PD indices and outcome is dependent on the experimental setup. The PK/PD index that determines bacterial killing after 48 h of treatment is different from the PK/PD index that determines rat survival after 18 days if treatment in this nonneutropenic rat model. The present findings should be extended and validated with other models of infection. The results of this study indicate that investigation of different experimental approaches is relevant in the search for the therapeutic rationale for antimicrobial dosing in severe infections and may have important consequences for dosing in the severely ill patient, especially over a prolonged period of administration.

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