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We tested the hypothesis that the bacterial load at the infection site could impact considerably on the pharmacokinetic/pharmacodynamic (PK/PD) parameters of fluoroquinolones. Using a rat lung infection model, we measured the influence of different marbofloxacin dosage regimens on selection of resistant bacteria after infection with a low (105 CFU) or a high (109 CFU) inoculum of Klebsiella pneumoniae. For daily fractionated doses of marbofloxacin, prevention of resistance occurred for an area-under-the-concentration-time-curve (AUC)/MIC ratio of 189 h for the low inoculum, whereas for the high inoculum, resistant-subpopulation enrichment occurred for AUC/MIC ratios up to 756 h. For the high-inoculum-infected rats, the AUC/MIC ratio, Cmax/MIC ratio, and time within the mutant selection window (TMSW) were not found to be effective predictors of resistance prevention upon comparison of fractionated and single administrations. An index corresponding to the ratio of the time that the drug concentrations were above the mutant prevention concentration (MPC) over the time that the drug concentrations were within the MSW (T>MPC/TMSW) was the best predictor of the emergence of resistance: a T>MPC/TMSW ratio of 0.54 was associated with prevention of resistance for both fractionated and single administrations. These results suggest that the enrichment of resistant bacteria depends heavily on the inoculum size at the start of an antimicrobial treatment and that classical PK/PD parameters cannot adequately describe the impact of different dosage regimens on enrichment of resistant bacteria. We propose an original index, the T>MPC/TMSW ratio, which reflects the ratio of the time that the less susceptible bacterial subpopulation is killed over the time that it is selected, as a potentially powerful indicator of prevention of enrichment of resistant bacteria. This ratio is valid only if plasma concentrations achieve the MPC.
In infections such as pneumonia, the burden of microorganisms can become quite high and may frequently exceed the inverse of the frequency of mutations, leading to the development of a resistant subpopulation. This leads to the presence of a small subpopulation of resistant organisms at the time that antimicrobial therapy is initiated. Under these conditions, a drug exposure that will kill only the susceptible dominant population may allow amplification of the resistant mutant subpopulation that is present before treatment, resulting in the emergence of resistance during therapy (24, 38).
Resistance to fluoroquinolones can occur spontaneously in bacterial populations at a frequency of about 10−6 to 10−8 (6) following a stepwise process that involves mutations in the genes coding for the targets DNA gyrase and topoisomerase IV (21, 32). Therefore, we can presume that an increase in the bacterial load at the infection site could be associated with an increase in the likelihood of a resistant mutant subpopulation being present before any fluoroquinolone treatment is administered.
Until recently, optimization and individualization of antimicrobial dosing regimens have been primarily based on the pharmacokinetic/pharmacodynamic (PK/PD) indices that describe the optimal efficacy and/or the prevention of toxicity (27). However, the increasing problem of emergence of resistance under the influence of antibiotic selection pressure led to the identification of PK/PD indices that best correlate with the prevention of antimicrobial resistance (2). An approach for preventing the emergence of antimicrobial resistance has been proposed, consisting of administration of the drug at doses that produce plasma concentrations that continuously exceed the threshold for spontaneous-drug-resistant-mutant susceptibility and thereby prevent the selective amplification of any mutant subpopulation (12, 37, 38). The drug concentration capable of inhibiting the growth of the least-susceptible single-step mutant subpopulation has been called the mutant prevention concentration (MPC). In addition, it has been suggested that selective amplification of spontaneous drug-resistant mutants is more pronounced within the range of antimicrobial plasma concentrations between the MIC of the wild bacterial population and the MPC, defined as the mutant selection window (MSW) (6, 23, 36, 37). The concept of the MSW has been characterized in vitro (15, 18) and in vivo (16), and the MSW has been shown to be an association between the time a pathogen population is subjected to antimicrobial exposure within the MSW (TMSW) and the mutant enrichment. On the other hand, this concept has been challenged and debated in the literature (31, 35). More recently, publications have highlighted that the actual antimicrobial concentration within the MSW at the bottom or at the top of the MSW boundaries could also play an important role in the enrichment of a mutant subpopulation (10, 17).
We previously observed in vitro and in vivo, in neutropenic mice, that both the time that the antimicrobial concentrations are within the MSW and the bacterial inoculum size at the start of the antimicrobial treatment play an important role in the enrichment of the resistant mutant subpopulation (15, 16). The aim of the present study was to use a rat model of Klebsiella pneumoniae lung infection to investigate (i) whether different bacterial inoculum sizes at the infection site at the beginning of antimicrobial treatment could influence the selection of resistant mutants in immunocompetent animals and (ii) what PK/PD indices are best associated with the prevention of resistant-mutant enrichment after different dosage regimens of marbofloxacin, a fluoroquinolone of veterinary interest.
Klebsiella pneumoniae ATCC 43816 was used for establishment of lung infection throughout all the experiments. Marbofloxacin powder, a broad-spectrum quinolone, was kindly provided by Vetoquinol, Lure, France.
The MIC was determined in triplicate for the bacteria by a broth microdilution method according to CLSI reference methods.
The MPC was determined as described previously (6). Briefly, an overnight culture of the tested bacteria in Mueller-Hinton (MH) broth was concentrated 100 times in NaCl (0.9%) to obtain a suspension containing 1010 CFU/ml. One hundred microliters of this suspension was then plated on MH agar containing various concentrations of marbofloxacin obtained by successive twofold dilutions. The MPC was the lowest marbofloxacin concentration preventing the growth of bacterial colonies after incubation for 72 h at 37°C. Determinations were done in triplicate.
Male OFA rats (Charles River, L'arbresle, France), weighing 200 to 270 g, were used for all the studies. The rats were housed two to three per cage at room temperature with a 12-h-light/12-h-dark cycle and were acclimatized for at least 2 weeks before the beginning of the experiments. The rats had free access to food (T2014; Harlan, Gannat, France) and tap water.
All animal procedures were conducted in accordance with accepted humane standards of animal care under agreement number A 31909 for animal experimentation from the French Ministry of Agriculture.
Experimental lung infection was produced as previously described (4). In brief, rats were anesthetized with ketamine-medetomidine (Imalgene 1000 [Merial SAS, Villeurbanne, France] and Domitor [Pfizer, Paris, France]). The trachea was cannulated, and the lungs were inoculated with 0.05 ml of a saline suspension of K. pneumoniae ATCC 43816 containing 2 × 106 (group A) or 2 × 1010 (group B) CFU/ml. For each inoculum size, a control group of four rats did not receive any antimicrobial treatment and a group of three rats were sacrificed just prior to the start of therapy for baseline quantitative cultures of lung homogenates.
Marbofloxacin treatment (Marbocyl; Vetoquinol, Lure, France) was started 4 h (group A) or 24 h (group B) after the inoculation of the lungs with K. pneumoniae ATCC 43816. Marbofloxacin was administered subcutaneously at a volume of 0.3 ml. There were two modalities of treatment. The marbofloxacin doses were administered according to the following time schedule: either one single administration was used or the same total dose was fractionated into four daily administrations over 4 days. For group A, a single total marbofloxacin dose (16 mg/kg of body weight) was tested according to the time schedule. For group B, a total of three marbofloxacin doses (16, 64, and 100 mg/kg) were tested according to the time schedule. Animals were sacrificed 96 h after the first marbofloxacin administration by an intraperitoneal injection of pentobarbital sodium (Dolethal; Vetoquinol, France). The lungs were aseptically removed and homogenized in 10 ml of NaCl (0.9%). The homogenates were centrifuged at 3, 000 × g for 10 min and washed twice in 10 ml of NaCl (0.9%) to prevent drug carryover. Ten microliters of successive 10-fold dilutions of the homogenates was then plated in triplicate on an MH drug-free agar plate containing 10% activated charcoal and 10% MgSO4, and 100 μl of the homogenates was plated on MacConkey agar supplemented with 0.064 μg/ml and 0.256 μg/ml marbofloxacin. Colonies were counted after overnight incubation at 37°C. If the colonies were too small, incubation was continued for a further 24 h. The lowest level of detection was 100 CFU/lung, and bacteria were considered eradicated below this level. Ten to twelve rats were included per treatment group. The proportion of resistant bacteria was calculated as the ratio of bacterial counts observed in the presence of 0.064 or 0.256 μg/ml marbofloxacin over the total bacterial counts on plates without marbofloxacin.
Two satellite groups (groups C and D) of male OFA rats (Charles River, L'arbresle, France) weighing 250 to 270 g were catheterized in the femoral vein. After 3 days, the rats' lungs were inoculated with 0.05 ml of an inoculum of 2 × 106 CFU/ml (group C) or 2 × 1010 CFU/ml (group D) of K. pneumoniae ATCC 43816. Four hours after the inoculation, group C was given a single subcutaneous dose of marbofloxacin. For group D, marbofloxacin administration was 24 h after the K. pneumoniae ATCC 43816 inoculation. The doses were 4 and 16 mg/kg for group C and 4, 16, and 100 mg/kg for group D. Blood samples (200 μl) were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 h after dosing. Two to four rats were included per treatment group. After each serial blood sample, a volume of physiological saline equivalent to the collected blood volume was administered. Blood samples were centrifuged at 7,000 × g for 10 min at 4°C, and plasma was stored at −20°C until the assay. A high-performance-liquid-chromatography method with fluorescence detection (λexcitation, 295 nm; λemission, 500 nm) (Agilent 1100) was adapted from the method of Schneider et al. (30) to determine the marbofloxacin concentrations in the rat plasma. Briefly, marbofloxacin was obtained by liquid-liquid extraction: 0.1 ml plasma was added to 1 ml of dichloromethane and mixed for 10 s. A 0.2-ml volume of a methanol (2% HCl)-H2O mixture (90:10) was added to the organic layer, and 100 μl of the supernatant was injected into a C18e (LiChrospher [5 μm 125 by 4 mm]; Merck) column and with a phosphoric acid (0.01 M)-triethylamine (0.004 M) (pH 2)-acetonitrile gradient elution. The calibration curve of marbofloxacin was established over the concentration range from 20 to 500 ng/ml with a linear regression model. The plasma samples were diluted to ensure that the concentrations were within the range of the calibration curve. The accuracy varied from 104.1 to 107.5%, and the intraday and interday precision levels were lower than 6.2% and 8.2%, respectively. The limit of quantification was 20 ng/ml.
Pooled pharmacokinetic data were analyzed using software dedicated to population pharmacokinetic analysis (MONOLIX) (22), an approach that is consistent with properly analyzing sparse data as described by Burtin et al. (7). Estimations were performed using MONOLIX version 2.1 with the stochastic approximation version of the expectation maximization algorithm. Marbofloxacin pharmacokinetic parameters were obtained with a two-compartment model with extravascular input and without lag time. These parameters were then used to calculate PK/PD indices for each dosing regimen. The derived PK/PD indices selected were the ratio of the area under the plasma concentration-time curve (AUC) over the MIC (AUC/MIC), the ratio of the peak of plasma concentration over the MIC (Cmax/MIC), the time that the concentrations are above the MPC (T>MPC), and the time that the concentrations are within the MSW (TMSW). The AUC/MIC ratio was calculated using the AUC at steady state over 24 h for the fractionated administrations as defined by Mouton et al. (28) and using the AUC from 0 h to infinity for the single administrations as proposed by Toutain et al. (33). TMSW and T>MPC were calculated in the same way, over 24 h for the fractionated administrations and over 96 h for the single administrations. As the level of plasma protein binding of marbofloxacin is lower than 10% in the rat (14), the PK/PD indices were determined from total plasma concentrations.
The sigmoid inhibitory Emax models describing the relationships between PK/PD indices and the proportion of resistant bacteria for the high-level inoculum 96 h after the start of marbofloxacin treatment were delineated with WinNonlin version 5.2 (Pharsight Corporation, Mountain View, CA).
The MIC of marbofloxacin was 0.032 μg/ml for Klebsiella pneumoniae (ATCC43816), and the MPC was 0.512 μg/ml. The term R-2×MIC in the present paper represents bacteria growing on 0.064 μg/ml marbofloxacin (2× MIC), and the term R-8×MIC represents bacteria growing on 0.256 μg/ml marbofloxacin (8× MIC).
Low inocula (2 × 106 CFU/ml) never contained bacteria resistant to 0.064 or 0.256 μg/ml of marbofloxacin. High inocula (2 × 1010 CFU/ml) contained between 2.1 and 5.0 log10 CFU/ml of bacteria resistant to 0.064 μg/ml marbofloxacin and 1.1 and 2.2 log10 CFU/ml of bacteria resistant to 0.256 μg/ml marbofloxacin.
At the start of the marbofloxacin treatment, the bacterial counts in the lungs were 5.4 ± 0.1 log10 CFU for group A (4 h after infection) and 10 ± 0.4 log10 CFU for group B (24 h after infection). For group A at 4 h after the inoculation, we never found any bacteria resistant to 0.064 or 0.256 μg/ml marbofloxacin. For group B at 24 h after the inoculation, there was between 3.2 and 3.4 log10 CFU of bacteria resistant to 0.064 μg/ml marbofloxacin (R-2×MIC) and 2.75 to 3.06 log10 CFU of bacteria resistant to 0.256 μg/ml (R-8×MIC) marbofloxacin in the lungs. At the beginning of the marbofloxacin treatment, taking into account both total bacterial counts and resistant-subpopulation counts (R-2×MIC and R-8×MIC), we calculated that the initial numbers of both resistant subpopulations for the high inoculum were about 10−7 to 10−8.
For the low inoculum challenge (group A), the bacterial counts in the lungs of untreated animals were 4.2 to 11.2 log10 CFU 4 days after the infection. This large range of bacterial load in the lungs might be due to the immunocompetent status of the animals and reflect the interindividual variability in the evolution of this infection. However, this did not affect our study, since the antimicrobial treatment started 4 h after the K. pneumoniae ATCC 43816 inoculation and, at this time, the bacterial load was rather homogeneous (see below).
For the high inoculum challenge (group B), all untreated animals were dead 72 h after infection.
The total bacterial population 96 h after the start of marbofloxacin treatment is reported for each inoculum size and each marbofloxacin dosing regimen in Table Table1.1. For the low inoculum, the marbofloxacin dose of 16 mg/kg, fractionated or not, led to a significant decrease in K. pneumoniae ATCC 43816 in the lungs. For the high inoculum, the three marbofloxacin doses, fractionated or not, reduced the bacterial population by at least 2 log, but no difference was detected between marbofloxacin dosing regimens. With the lower marbofloxacin dose (16 mg/kg) given as a single administration, 4 out of 10 rats died between 72 and 96 h after marbofloxacin, whereas for the fractionated administration, all the animals survived. With the higher marbofloxacin dose (100 mg/kg) given in a single administration, 3 out of 12 rats died during the first 24 h after marbofloxacin administration. The death of animals in group B treated with 100 mg/kg in a single administration occurred earlier during the experiment than that of animals in group B treated with 16 mg/kg of marbofloxacin in a single administration or untreated animals (24 h versus 72 to 96 h, respectively). Despite the lack of observed adverse effects, such as convulsions, the three deaths were probably due to marbofloxacin toxicity in ill animals.
The percentages of animals with R-2×MIC and R-8×MIC bacteria in their lungs 96 h after the start of marbofloxacin treatment are shown for each inoculum size and each dosing regimen in Table Table1.1. In rats infected with the low inoculum and treated with 16 mg/kg of marbofloxacin, neither R-2×MIC nor R-8×MIC bacteria were detected at the end of the experiment. In contrast, rats infected with the high inoculum had both R-2×MIC and R-8×MIC bacteria in their lungs. The frequency of rats harboring these two K. pneumoniae ATCC 43816 subpopulations was negatively associated with the marbofloxacin doses. Moreover, for the same total marbofloxacin dose, fractionated administration seemed to be associated with a lower number of resistant subpopulations than single-dose administration.
The pooled pharmacokinetic data obtained with different marbofloxacin doses were successfully analyzed using the same compartmental model, indicating dose proportionality of marbofloxacin kinetics within both the low- and the high-inoculum groups. This proportionality enabled PK/PD indices to be calculated for the different marbofloxacin doses. Moreover, no significant difference in marbofloxacin pharmacokinetic parameters was observed between the low- and high-inoculum groups receiving the same doses (data not shown). The predicted concentrations versus the time profiles of marbofloxacin for the different dosage regimens are given in Fig. Fig.11.
The PK/PD indices for the different marbofloxacin dosage regimens are shown in Table Table2.2. The proportions of resistant K. pneumoniae ATCC 43816 subpopulations observed after the different dosage regimens in rats infected with the high inoculum are shown in Fig. Fig.2.2. In all animals carrying resistant bacteria, the proportions of both R-2×MIC and R-8×MIC bacteria were higher than 10−6, indicating an enrichment in comparison with the level for the initial inoculated K. pneumoniae ATCC 43816 population (<10−6).
For the lowest dose of 16 mg/kg, the AUC/MIC ratios were 4 × 189 and 1 × 756 h for the fractionated and single-dose administrations, respectively. These ratios were associated with enrichment of R-2×MIC and R-8×MIC subpopulations in animals infected with the high inoculum, whereas there was no enrichment for animals infected with the low inoculum (Table (Table1).1). In animals infected with the high inoculum, AUC/MIC ratios of 4 × 756 and 1 × 3,026 h for the fractionated and single 64-mg/kg administrations, respectively, were also associated with R-2×MIC and R-8×MIC subpopulations, whereas ratios of 4 × 1,182 and 1 × 4,728 h, corresponding to the 100-mg/kg total dose, were associated with the absence of detection of an R-8×MIC subpopulation (Tables (Tables11 and and22).
In animals infected with the low inoculum (group A), Cmax/MIC ratios of 4 × 54 and 1 × 217 for fractionated and single 16-mg/kg administrations, respectively, were associated with the absence of R-2×MIC or R-8×MIC subpopulation enrichment (Tables (Tables11 and and2).2). In animals infected with the high inoculum (group B), the higher the Cmax/MIC ratio was, the less the R-2×MIC and R-8×MIC subpopulation enrichment occurred for each dosing regimen (Tables (Tables11 and and2).2). The Cmax/MIC ratios that were associated with the absence of R-8×MIC bacteria enrichment were 4 × 339 and 1 × 1,357 for the fractionated and single 100-mg/kg administrations, respectively. The 4 × 339 ratio value for the fractionated administration was also associated with a lower frequency of R-2×MIC subpopulation enrichment (Tables (Tables11 and and22).
The dosage regimen had an important impact on TMSW. Marbofloxacin plasma concentrations were within the MSW 28, 40, and 41% of the time (over 96 h) for single administrations and 50, 67, and 60% of the time (over 24 h) for fractionated administrations for the 16-, 64-, and 100-mg/kg marbofloxacin doses, respectively (Table (Table2).2). The TMSW was lower for the 100-mg/kg dose than for the 64-mg/kg dose as calculated over 24 h, and the T>MPC was higher for the 100-mg/kg dose than for the 64-mg/kg dose (40% versus 33%, respectively) (Table (Table2).2). The relationships between the time indices and the proportions of R-2×MIC and R-8×MIC bacteria observed after treatment with the different marbofloxacin dosage regimens for animals infected with the high inoculum are presented in Fig. Fig.2.2. No clear relationship existed between TMSW and R-2×MIC and R-8×MIC subpopulation enrichments (Fig. (Fig.2,2, panels A1 and A2). For T>MPC, a relationship between T>MPC and the R-2×MIC or R-8×MIC subpopulation enrichment was found only when each dosage regimen was considered separately (fractionated or single) (Fig. (Fig.2,2, panels B1 and B2). In fact, there was a relationship between R-2×MIC or R-8×MIC subpopulation enrichment and the T>MPC/TMSW ratio (Fig. (Fig.2,2, panels C1 and C2). Irrespective of the dosage regimen, the closer the T>MPC/TMSW ratio came up to 1, the more the R-2×MIC or R-8×MIC subpopulation enrichment was limited. These observations were confirmed by fitting the percentages of rats with resistant bacteria in the lungs versus TMSW, T>MPC, and T>MPC/TMSW by using a sigmoid Emax model. Indeed, the best fit was obtained for the percentages of rats with resistant bacteria in the lungs versus T>MPC/TMSW, with R2 values of 0.99 and 0.97 for R-2×MIC and R-8×MIC, respectively (Fig. (Fig.3,3, panels B1 and B2), whereas the R2 values were 0.87 and 0.93 for T>MPC with a misfit at the end of the curve (Fig. (Fig.3,3, panels B1 and B2) and 0.54 and 0.60 for TMSW (data not shown).
The aim of this study was to investigate the influence of bacterial load at the start of a quinolone treatment on resistant-subpopulation enrichment and the ability of PK/PD parameters (AUC/MIC, Cmax/MIC, T>MPC, and TMSW) to predict resistant-subpopulation enrichment in a lung infection model in immunocompetent rats.
In order to fully characterize the model of K. pneumoniae ATCC 43816 lung infection in immunocompetent rats, we evaluated the pharmacokinetics of marbofloxacin in infected animals. Indeed, bacterial infections have been shown to alter the pharmacokinetics of drugs (19), including fluoroquinolone antimicrobials, such as marbofloxacin (20, 29). Moreover, a similar investigation by our group in an Escherichia coli thigh infection model with neutropenic mice indicated that marbofloxacin pharmacokinetics was altered by infection. There were considerable differences in marbofloxacin exposure between animals infected by a low and a high bacterial inoculum, leading to a large difference in the PK/PD parameters for the same marbofloxacin dose (16). In the present study, we did not observe any difference in the pharmacokinetic parameters between animals infected with the low (group A) and the high (group B) K. pneumoniae ATCC 43816 inoculum. The characteristics of these two infectious models (lungs versus thigh and K. pneumoniae ATCC 43816 versus E. coli) or/and a possible difference in the level of inflammation might explain these discrepancies.
Previous in vitro studies of E. coli, Staphylococcus aureus, and Pseudomonas aeruginosa (15, 26) and an in vivo study of E. coli in immunocompromised mice (16) showed an impact of inoculum size on the enrichment of resistant mutants. The present study confirms these results and clearly shows this impact in immunocompetent animals. Indeed, with an early antimicrobial treatment on an initial small bacterial population at the infection site (group A), we never observed an R-2×MIC or R-8×MIC subpopulation enrichment. In addition, the total bacterial load decreased dramatically. However, with a delayed start of the antimicrobial treatment on an initial large bacterial population (group B), we observed a limited decrease in the total bacterial population, accompanied by enrichments of R-2×MIC and R-8×MIC subpopulations, depending on the marbofloxacin dosing regimen. Moreover, for the smaller marbofloxacin dose (16 mg/kg) administered as a single administration, 4 out of 10 rats died between 72 and 96 h after the start of marbofloxacin treatment. These deaths were attributed to the infection, based upon observed clinical signs of infection in these rats. However, the three deaths observed with the highest single marbofloxacin dose (100 mg/kg) were unlikely to have been due to infection, because they occurred in the first 48 h after the inoculation, while in nontreated animals, death always occurred after 72 h. These early deaths could be attributed to the marbofloxacin toxicity in ill animals.
The ability of PK/PD indices such as AUC/MIC, Cmax/MIC, T>MPC, and TMSW to predict resistant-subpopulation enrichment has already been studied. Previous studies suggested that AUC/MIC was the PK/PD index that best correlated with efficacy of fluoroquinolones in both neutropenic and nonneutropenic murine thigh and lung infection models (3, 5, 13, 34). In our study, for the low inoculum (group A) with the lowest marbofloxacin dose (16 mg/kg), the AUC/MIC ratio of 4 × 189 or 1 × 756 h for fractionated or single-dose administration, respectively, was associated with the prevention of the emergence of any resistant subpopulation after 96 h of treatment. For the high inoculum (group B), the AUC/MIC ratios required to prevent R-8×MIC subpopulation enrichment were higher than for the low inoculum, i.e., 4 × 1,182 and 1 × 4,728 h for fractionated and single administrations, respectively. However, considering the MPC, which is linked to the MIC of the R-8×MIC bacteria, to calculate the corresponding PK/PD indices, it appears that the AUC/MPC ratio which was associated with the prevention of the enrichment of an R-8×MIC subpopulation is now 4 × 74 or 1 × 295 h for fractionated or single administration, respectively. These AUC/MPC values are of the same order of magnitude as the AUC/MIC ratios required to prevent an R-8×MIC subpopulation enrichment with a low initial inoculum. In other words, and as previously suggested (38), to be predictive, the AUC/MIC ratio should take into account the MIC of the subpopulation having the highest MIC and not the MIC of the dominant population. Moreover, the differences observed between R-8×MIC enrichments for fractionated and single-dose administrations indicated that, for the same total exposure (over 96 h), the effectiveness of the fractionated dosage regimen is greater than that of the single-dose administration; this suggest that enrichment of resistant subpopulations is codependent on both the total exposure and the time that the concentrations are above some critical level (see below).
The time the fluoroquinolone concentrations were within the MSW has previously been shown to be associated with a promotion of resistant bacterial-subpopulation enrichment in vitro (1, 9, 10, 15) and in vivo (16). However, we did not observe in our experiment such a relationship between TMSW and R-8×MIC subpopulation enrichment (Fig. (Fig.2,2, panels A1 and A2), which is in agreement with a previous in vitro study with Staphylococcus aureus (8), although it is important to note that the target of mutation resistance differs between gram-positive and gram-negative bacteria (11). Recently, it was suggested that the apparent inability of TMSW to predict resistant-mutant enrichment may be explained by the confounding influence of the actual antimicrobial concentrations at the edges of the selection window (10, 17). In other words, for a given TMSW, situations are not equivalent when time outside the MSW is under the MIC or above the MPC. In the present study, for the fractionated 16-mg/kg dose, the half-life of marbofloxacin of 2.41 h (Fig. (Fig.1)1) led plasma concentrations to decay below the MSW, whereas for the two other doses (64 and 100 mg/kg), most of the exposure to marbofloxacin was at the top of the MSW. This could explain the absence of a relationship between TMSW and R-8×MIC enrichment. For the single-dose administration, the pharmacokinetic profiles showed that for the two highest doses (64 and 100 mg/kg), marbofloxacin concentrations reside for almost the same time within the MSW, while the residence time within the MSW was shorter for the lower dose (16 mg/kg). There was thus no relationship between the TMSW and R-8×MIC subpopulation enrichment. In contrast, there were relationships between both R-2×MIC and R-8×MIC subpopulation enrichments and the T>MPC/TMSW ratio (Fig. (Fig.2,2, panels C1 and C2, and Fig. Fig.3,3, panels B1 and B2). With this ratio, it is now possible to discriminate between situations characterized by the same TMSW, but with different levels of antimicrobial concentrations within this MSW, and the higher this ratio is, the lower the risk of R-8×MIC subpopulation enrichment is. The ability of this new index to predict enrichment of resistant mutants is due to the fact that it consists of two terms reflecting two antagonistic and sequential processes: T>MPC, which reflects the time during which the antibiotic eliminates all pathogens, including resistant mutants, and TMSW, which reflects the time during which the antibiotic produces selection of resistant-mutant subpopulations. It is noteworthy that this ratio allows an unbiased comparison of fractionated and single-dose administrations, given that for fractionated administration, the ratios are found to be the same when calculated over 24 h and over the total duration of the treatment (96 h). Cutoff values for T>MPC/TMSW associated with the prevention of resistant-subpopulation enrichment need to be known; as shown in the present experiment, the ratio is influenced by the likelihood of having resistant mutants already present at the start of antimicrobial treatment. With the low inoculum (group A), T>MPC/TMSW values of 0.31 and 0.30 for fractionated and single-dose administrations, respectively, were required to prevent mutant resistant enrichment. In contrast, for the high inoculum (group B), the corresponding values were 0.67 and 0.54, respectively. From our results, it appears that the most desirable situation for carrying out an antimicrobial therapy that does not simultaneously promote antimicrobial resistance is one in which the inoculum level at the initiation of the treatment is low or null (metaphylaxis or prophylaxis). In a curative setting, characterized by a greater likelihood of the antimicrobial facing a high bacterial load, our results suggest that the best strategy would be to immediately achieve plasma concentrations above the MPC. Besides such intuitive findings, the present study offers the first evidence regarding the rational PK/PD approach for selecting the antimicrobial dosage regimens adapted to either preventive or curative settings. In addition, as shown by our results, the T>MPC needs to be long enough to produce an early reduction of the inoculum load for the situation to be the same as that with an initial low-inoculum load. In the present experiment, it appears that with the single marbofloxacin dose (mimicking the so-called one-shot treatment that is usually recommended in veterinary medicine), the T>MPC is not long enough to achieve this goal. However, it is important to note that in our study, the tested doses always achieved concentrations above the MPC for fractionated administration but the same total fluoroquinolone dose which achieves a concentration above the MPC for a single but not for a fractionated administration would be better in a single than in a fractionated administration for mutant prevention. Second, the terminal half-life of marbofloxacin in domestic animal species may be much longer (up to 13 h) than in rats (2.41 h in our study), and a single-dose administration in large animal species may give a longer T>MPC than one in rats.
In addition to R-8×MIC subpopulation enrichment, we also studied the R-2×MIC enrichment which could be attributed to efflux pump overexpression in bacteria (25). In some animals, we observed an enrichment of the R-2×MIC subpopulation without simultaneous R-8×MIC subpopulation enrichment. For the two highest marbofloxacin doses (64 and 100 mg/kg), the R-2×MIC subpopulation enrichment was greater for the single administration than for the fractionated one (Table (Table1).1). This could in turn explain the higher percentage of animals harboring R-8×MIC bacteria in their lungs with a single rather than a fractionated administration of marbofloxacin at 64 mg/kg. Indeed, if the R-2×MIC subpopulation enrichment is promoted, the ability of these bacteria to survive antimicrobial concentrations closer to the MIC favors the possibility of generating a new and more resistant subpopulation (R-8×MIC bacteria), as has been suggested by Louie et al. (25). Thus, despite the same impact on the total bacterial population, in our model the single antimicrobial administration seems to be less beneficial than the fractionated administration in preventing resistant-mutant enrichment.
In conclusion, our results show that the bacterial load at the start of antimicrobial treatment plays a critical role in the pattern of selection of resistant K. pneumoniae ATCC 43816 mutants. A low initial bacterial load limits resistant-mutant enrichment due to the lower likelihood of having a resistant mutant subpopulation already present at the beginning of the treatment. With a high bacterial load at the start of treatment, we have shown the ability of the T>MPC/TMSW ratio to define the relevant features of antibiotic exposure to prevent mutant enrichment. This PK/PD index might contribute to the rational selection of more-adapted antimicrobial dosage regimens. Nevertheless, further investigations with different infectious and animal models are needed to confirm what we observed with our trials and to quantify the cutoff value for T>MPC/TMSW in target patients (humans and domestic animal species).
We thank Jacqueline Manceau for doing the analytical assays and Pamela Louapre, Stephane Marteau, and Jean-Guy Rolland for technical support.
Published ahead of print on 8 September 2009.