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
). 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
) 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
, 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
). 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
) and in vivo (16
). However, we did not observe in our experiment such a relationship between TMSW
and R-8×MIC subpopulation enrichment (Fig. , panels A1 and A2), which is in agreement with a previous in vitro study with Staphylococcus aureus
), 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
). 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. ) 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
ratio (Fig. , panels C1 and C2, and Fig. , 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
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
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 ). 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).