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JAC Advance Access originally published online on June 16, 2006
Journal of Antimicrobial Chemotherapy 2006 58(2):349-358; doi:10.1093/jac/dkl250
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

A pharmacodynamic approach to antimicrobial activity in serum and epithelial lining fluid against in vivo-selected Streptococcus pneumoniae mutants and association with clinical failure in pneumonia

Luis Alou1, María-José Giménez1, David Sevillano1, Lorenzo Aguilar1, Fabio Cafini1, Olatz Echeverría1, Emilio Pérez-Trallero2 and José Prieto1,*

1 Microbiology Department, School of Medicine, Universidad Complutense Avda. Complutense s/n, 28040 Madrid, Spain 2 Microbiology Department, Hospital Donostia, Paseo Dr Beguiristáin s/n 20014 Donostia, San Sebastián, Spain

*Corresponding author. Tel: +34-91-3941508; Fax: +34-91-3941511; E-mail: jprieto{at}med.ucm.es

Received 1 March 2006; returned 11 May 2006; revised 24 May 2006; accepted 24 May 2006


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Objectives: Emergence of resistance may be prevented by killing both the parental infecting strain and subsequent less susceptible step-mutants. The present study analyses eradication and resistance selection in Streptococcus pneumoniae with moxifloxacin, levofloxacin and azithromycin, using a parental serotype 3 clinical strain (strain A) and its correspondent step-mutant derivatives resistant to these antibiotics (B, C, D), which were selected in vivo in a patient with pneumonia.

Methods: Moxifloxacin, levofloxacin and azithromycin MICs were 1, 2 and 0.5 mg/L for the parental strain; 4, 16 and 4 mg/L for isolate B; and 4, 16 and >128 mg/L for isolates C and D, respectively. A pharmacokinetic computerized device was used to simulate serum and epithelial lining fluid (ELF) concentrations. Initial inoculum was ~108 cfu/mL. Population analysis profiles were performed using plates with increasing antimicrobial concentrations.

Results: In ELF simulations, moxifloxacin showed a bactericidal pattern against all isolates with a minority (~100 cfu/mL) of the surviving population (isolates B, C and D) growing on plates with moxifloxacin concentrations just above those in ELF. Levofloxacin and azithromycin showed a bactericidal pattern only against isolate A, with the whole population of isolates B, C and D growing on plates with levofloxacin concentrations higher (16–64 mg/L) than those in ELF and in plates with azithromycin concentrations as high as 2048 mg/L (for isolates C and D).

Conclusions: Antimicrobial activity in pulmonary tissue against possible emerging resistant mutants during pneumonia treatment may prevent failures more than the solely activity against the S. pneumoniae parental infecting strain.

Keywords: moxifloxacin , levofloxacin , azithromycin , pharmacodynamics


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Bacterial evolution towards resistance occurs in two steps: emergence and dissemination.1 Although both concepts are closely related, the specific weight of each one in Streptococcus pneumoniae resistance depends on the marker that defines it, since in the case of quinolones resistance arises within a given host due to point mutations,2 whereas in the case of ß-lactams and macrolide resistance is mostly due to acquisition of resistance genes by transformation, and de novo resistance occurs rarely in a susceptible population within a given host.2 Serotype 3, which is frequently isolated from blood and respiratory samples in adults, has been considered particularly susceptible to ß-lactams and macrolides,3 but shows low prevalence of ciprofloxacin resistance with a tendency to be clustered among macrolide non-susceptible strains,4 suggesting cross-selection of resistance. There is a high prevalence of resistance to macrolides in Spain,5 and the shift in susceptibility of pneumococcal subpopulations to higher ciprofloxacin MICs is a cause of concern,6 reaching around 5% strains with MICs ≥ 4 mg/L,5 with newer quinolones (as moxifloxacin) showing much higher activity (lower MIC90 values) than older quinolones (as ciprofloxacin, ofloxacin and levofloxacin).6,7 The remaining 95% strains with ciprofloxacin MIC ≤ 2 mg/L present 100% susceptibility to levofloxacin and moxifloxacin.6,7

The increase in prescription of fluoroquinolones,8 with higher rates of prescription for the older compounds,9 may limit the possibilities of therapeutic failures in community-acquired pneumonia (CAP) due to drug-resistant organisms, but may compromise the future effectiveness of this class of drugs.8 In vivo selection of resistance and subsequent therapeutic failures with levofloxacin have been described.10–12

New fluoroquinolones with high anti-pneumococcal activity may prevent emergence of resistance by killing both the parental infecting strain and the subsequent less-susceptible step-mutants.13 We explore this possibility in the present study by analysing eradication and resistance selection in S. pneumoniae with moxifloxacin, levofloxacin and azithromycin serum and epithelial lining fluid (ELF) concentrations, using a parental infecting strain and the subsequent resistant step-mutants selected in vivo in a CAP patient empirically treated with levofloxacin plus macrolide.12


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Strains

The isolates (A, B, C and D) used in the study were clinical isolates recovered along the treatment period in a patient (with allergy to penicillin) with CAP and history of chronic obstructive pulmonary disease:12 the patient was initially treated with intravenous (iv) 500 mg levofloxacin once daily for 13 days and iv 500 mg clarithromycin twice daily for 2 days; on day 14 treatment was changed to iv 200 mg ciprofloxacin twice daily plus clarithromycin iv; on day 24, due to exacerbation of the respiratory infection, the patient was treated with trimethoprim–sulfamethoxazole for 5 days; and finally, the picture resolved after 10 days of vancomycin treatment.12 Four penicillin-susceptible isolates of S. pneumoniae serotype 3 were obtained from sputum cultures on days 0, 14 and 24, and a pleural fluid culture on day 30:12 (i) the first isolate (parental strain) (A) presented a point mutation in gyrA and the MIC (mg/L) of ciprofloxacin and erythromycin were 2 and 0.5, respectively; (ii) the second isolate (B) presented point mutations in gyrA, parC and a six amino acid insertion in ribosomal protein L22 (RTAHIT) and the MIC (mg/L) of ciprofloxacin and erythromycin were 32 and 2, respectively; (iii) the third isolate (C) presented the same point mutations and resistance determinants as the second isolate plus a mutation in the sequence of the gene corresponding to the domain V of the 23S rRNA (A2058G), and the MIC (mg/L) of ciprofloxacin and erythromycin were 16 and >128, respectively; and (iv) the last isolate (D) presented all resistance determinants but RTAHIT, and the MIC of ciprofloxacin and erythromycin were same as those for the third isolate.12

Moxifloxacin, levofloxacin and azithromycin MICs were 1, 2 and 0.5 mg/L for the parental strain; 4, 16 and 4 mg/L for the second isolate; and 4, 16 and >128 mg/L for the remaining isolates, respectively.12

Antimicrobials

Moxifloxacin reference standard was supplied by Bayer, Barcelona, Spain. Levofloxacin and azithromycin laboratory reference standards were supplied by Sigma Chemical Co. (St Louis, MO, USA).

In vitro kinetic model

A previously described two-compartment dynamic model was used to expose bacteria to changing study drug concentrations avoiding the dilution of the bacterial inoculum together with the drug.14 The central compartment (spinner flask, tubing and lumina of capillaries of the dialyser of the second compartment) was filled with 480 mL of Todd–Hewitt broth (Difco laboratories, Detroit, Mich.) supplemented with 0.5% yeast extract (Difco laboratories). The extra-capillary space and the intra-dialyser circulating tubing of the second compartment (FX50 class dialyser with helixone membranes, Fresenius Medical Care S.A., Barcelona, Spain) represented the infection site. Total distribution volume was 600 mL. Both compartments were maintained at 37°C throughout the simulation process. The exponential decay of concentrations was obtained by a continuous dilution–elimination process using computerized peristaltic pumps (Masterflex, Cole-Parmer Instrument Co., Chicago, IL, USA) at the adequate rate to simulate half-lives of levofloxacin, moxifloxacin and azithromycin in serum and in ELF. Flow rates in the peristaltic pumps were synchronized throughout the simulated period using Win Lin software (Cole-Parmer Instrument Co.). Additional pumps circulated the antimicrobial-medium mixture at 50 mL/min rate between the central and peripheral compartment, and at 20 mL/min within the extra-capillary space through external tubing. A computer-controlled syringe pump (402 Dilutor Dispenser; Gilson S.A, Villiers-le-Bel, France) allowed the simulation of drug concentrations by infusion of the drug into the central compartment until the Cmax was reached.

In drug-free experiments, flow rates were adjusted to 0.55 mL/min.

Kinetic simulations

Pharmacokinetic profiles in serum and ELF after oral moxifloxacin 400 mg once daily, levofloxacin 500 mg once daily and azithromycin 500 mg once daily were simulated over 24 h. The target pharmacokinetic parameters, based on mean values reported in humans, were Cmax = 5.7 mg/L and t1/2 = 7 h for levofloxacin15–18 and Cmax = 3.0 mg/L and t1/2 = 12.5 h for moxifloxacin.19–22 Because serum concentrations of azithromycin decline in a polyphasic manner, and as its relatively short serum half-life between 8 and 24 h after dosing indicates its initial rapid tissue distribution, the target Cmax and t1/2 parameters were directly obtained from human serum concentration-versus-time curves.19,23 Experimentally, the profile was divided into two quasi-linear portions with apparent half-lives of 1.71 h (Ke; 0.4 h–1) from 2.5 to 5 h and of 10.5 h (Ke; 0.06 h–1) from 5 to 24 h representing further distribution and elimination. Final target pharmacokinetic parameters were Cmax = 0.4 mg/L and t1/2 = 10.5 h.

Human ELF concentrations of moxifloxacin and levofloxacin were simulated using previously described pharmacokinetic data of these drugs in the bronchopulmonary region.19,24 Levofloxacin half-lives from 0 to 12 h and from 12 to 24 h were 7 and 10 h, respectively. The moxifloxacin half-life was 20 h. The target peak and trough concentrations were 15.2 mg/L (at 4 h) and 2.94 mg/L for levofloxacin and 11.6 mg/L (at 4 h) and 5.7 mg/L for moxifloxacin, respectively.19,24 This approximation was recently employed24 using a one-compartment system. The ELF profile of azithromycin was also simulated using this approximation. Target half-life and peak and trough concentrations were 20 h, 2.18 mg/L (at 8 h) and 1.01 mg/L, respectively.19,25

Measurement of antibacterial effect

Bacterial suspensions in Todd–Hewitt broth supplemented with 0.5% yeast extract, from an overnight culture on Mueller–Hinton agar supplemented with 5% sheep blood, were allowed to grow to a density of 7.5 x 107 to 1 x 108 cfu/mL, as measured by a UV- spectrophotometer (Hitachi U-1100). Of this inoculum 60 mL was introduced into the peripheral compartment (~4.5–6 x 109 cfu in 60 mL). Samples (0.5 mL) from the peripheral compartment were collected at 0, 1, 2, 3, 4, 6, 8, 10, 12 and 24 h and were serially diluted in 0.9% sodium chloride. At least four dilutions of each sample were spread onto Mueller–Hinton agar supplemented with 5% sheep blood and incubated at 37°C, and colonies were counted after 24 h. The limit of detection was 50 cfu/mL, and each experiment was performed in triplicate.

Antibacterial effects of study drugs were calculated by determining log10 changes in viable counts over 24 h with respect to time 0. Emax was defined as the maximum difference in log10 cfu/mL with respect to time 0. The area under the bacterial-kill curve (AUBC0–24; log10 cfu/mL x h) was calculated using the log linear trapezoidal rule for the period 0–24 h.

Pharmacokinetic analysis

For the measurement of simulated antimicrobial concentrations, additional aliquots (0.5 mL) were taken at 0, 1, 1.5, 2, 2.5, 4, 6, 8, 10, 12 and 24 h, and stored at –50°C. Concentrations were determined by bioassay using Escherichia coli NCTC 10418 for levofloxacin and moxifloxacin26 and Micrococcus luteus ATCC 9341 for azithromycin.27 Plates were inoculated with an even lawn of indicator organism and incubated for 18–24 h at 37°C. Standards were prepared in Todd–Hewitt broth supplemented with 0.5% yeast extract, with a concentration range of 0.06–4 mg/L for moxifloxacin and levofloxacin and of 0.06–0.5 mg/L for azithromycin. Lower limits of detection were 0.06, 0.03 and 0.06 mg/L for levofloxacin, moxifloxacin and azithromycin, respectively. Intra-day coefficients of variation were 3.1%, 3.2% and 4.2% while inter-day coefficients of variation were 5.7%, 4.0% and 6.0% for levofloxacin (0.75 mg/L), moxifloxacin (0.75 mg/L) and azithromycin (0.1 mg/L), respectively.

Antimicrobial concentrations were analysed by a non-compartmental approach using WinNonlin Professional program (Pharsight, Mountainview, CA, USA). Cmax and Tmax were obtained directly from observed data. The area under the concentration-time curve from 0 to 24 h (AUC0–24) was calculated by the trapezoidal rule.

Measurement of emergence of resistance

Drug resistance was assessed using population analysis profiles (PAP)28 at time 0 (pre-exposure) and 24 h after exposure. Samples were plated onto antibiotic-free agar and onto agar containing moxifloxacin, levofloxacin or azithromycin in their respective simulations, at concentrations 1x, 2x, 4x, 8x and 16x MIC in order to quantify the resistant subpopulation. The highest azithromycin concentration that could be used in the medium was 2048 mg/L. Resistance was measured as differences in bacterial counts on plates containing antibiotic between 0 and 24 h. The AUC for the PAP (AUC-PAP) was calculated using the log-linear trapezoidal rule at time 0 and at 24 h.

Statistical analysis

Inter-strain comparisons of reductions in log10 cfu/mL at 24 h and differences between AUC-PAP at time 0 and 24 h after exposure were performed by the ANOVA.


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Pharmacokinetics

Figure 1 shows experimental and target concentrations of study drugs in serum and ELF. Experimental Tmax (h), Cmax (mg/L) and AUC0–24 (h x mg/L) in serum were 0.9 ± 0.2, 3.0 ± 0.1 and 39.6 ± 1.0, respectively, for moxifloxacin; 1.6 ± 0.2, 5.8 ± 0.2 and 53.0 ± 2.1, respectively, for levofloxacin; and 2.5 ± 0.0, 0.4 ± 0.0 and 2.7 ± 0.2, respectively, for azithromycin. Experimental Tmax (h), Cmax (mg/L) and AUC0–24 (h x mg/L) in ELF were 4.0 ± 0.0, 12.0 ± 0.1 and 201.0 ± 9.0, respectively, for moxifloxacin; 4.0 ± 0.0, 15.5 ± 0.6 and 188.0 ± 6.4, respectively, for levofloxacin; and 7.5 ± 1.0, 2.3 ± 0.1 and 40.0 ± 2.0, respectively, for azithromycin. Serum half-lives (h) were 12.6 ± 0.3, 6.7 ± 0.2 and 11.1 ± 0.7 for moxifloxacin, levofloxacin and azithromycin, respectively.


Figure 1
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  		Figure 1. Experimental and target concentrations in serum and ELF of moxifloxacin, levofloxacin and azithromycin.

 
Pharmacodynamics

In serum, moxifloxacin Cmax/MIC, AUC0–24/MIC and t > MIC (% dosing interval) were 3.0, 39.6 and 88.0 for isolate A, and 0.2, 9.9 and 0 for isolates B, C and D. Levofloxacin Cmax/MIC, AUC0–24/MIC and t > MIC were 2.9, 26.5 and 43.8 for isolate A, and 0.4, 3.3 and 0 for isolates B, C and D. Azithromycin Cmax/MIC, AUC0–24/MIC and t > MIC were 0.8, 5.5 and 0 for isolate A; 0.1, 0.7 and 0 for isolate B; and 0, 0 and 0 for isolates C and D.

In ELF, moxifloxacin Cmax/MIC, AUC0–24/MIC and t > MIC were 12.1, 201.0 and 100 for isolate A, and 0.8, 50.3 and 100 for isolates B, C and D. Levofloxacin Cmax/MIC, AUC0–24/MIC and t > MIC were 7.7, 94.0 and 100 for isolate A, and 1.0, 11.8 and 100 for isolates B, C and D. Azithromycin Cmax/MIC, AUC0–24/MIC and t > MIC were 4.6, 80.0 and 100 for isolate A; 0.5, 10.0 and 0 for isolate B; and 0, 0 and 0 for isolates C and D.

Bactericidal activity

Colony counts (log10 cfu/mL) at time 0 and time 24 h in drug-free simulations (controls) were 7.9 ± 0.2 and 8.4 ± 0.2 for isolate A, 7.8 ± 0.2 and 8.4 ± 0.4 for isolate B, 7.8 ± 0.2 and 8.3 ± 0.3 for isolate C, and 7.8 ± 0.2 and 8.4 ± 0.3 for isolate D.

Table 1 shows {Delta}24 h, Emax and AUBC0–24 values for antimicrobial simulations. Figure 2 shows antibacterial activity over time of serum and ELF simulated concentrations of moxifloxacin, levofloxacin and azithromycin against the parental isolate and successive selected mutants.


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  		Table 1. Antibacterial activity: MICs (mg/L), changes in colony counts at 24 h ({Delta}24 h; log10 cfu/mL), and maximal effect (Emax; log10 cfu/mL) and area under bactericidal curves 0–24 h (AUBC0–24; h x log10 cfu/mL) of moxifloxacin (MXF), levofloxacin (LVX) and azithromycin (AZM) in serum and ELF (negative values mean regrowth)

 

Figure 2
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  		Figure 2. Antibacterial activity (log10 cfu/mL over time) of serum concentrations (left-hand side) and ELF concentrations (right-hand side) of moxifloxacin, levofloxacin and azithromycin against the parental isolate. A, open circles; and successive selected mutants: B, open squares; C, filled triangles; and D, asterisks. The broken line represents the limit of detection.

 
With serum concentrations, moxifloxacin was the only antimicrobial that eradicated (drove colony counts under the detection limit) the parental isolate (isolate A), with high Emax values and a very low AUBC0–24 (Table 1). Significant (P < 0.001) differences were found between final colony counts of this isolate and isolates B, C and D. In the case of levofloxacin although a high Emax value was obtained, regrowth occurred without significant differences in final colony counts between isolate A and isolates B, C and D. With azithromycin, although no eradication was obtained, a high Emax value was achieved (with low AUBC0–24) and significant differences in final colony counts (P < 0.001) were found between isolate A and isolates B, C and D.

With ELF concentrations, no significant differences were found in final colony counts between the four isolates with moxifloxacin, with high Emax values and low AUBC0–24. For levofloxacin significant differences were found (P < 0.001) between isolate A and the remaining three isolates with high Emax values and low AUBC0–24 for isolate A, but very low Emax values and high AUBC0–24 for isolates B, C and D. Both quinolones eradicated isolate A. In the case of azithromycin similar behaviour was found with strains A and B, with significant (P < 0.001) differences between them and isolates C and D. High Emax value was obtained with isolates A and B, but very low Emax with high AUBC0–24 with the other two strains (Table 1).

Population analysis profiles (PAP)

Tables 2–4 show PAP and AUC-PAP for the four isolates. When studying changes in subpopulation distribution (growth on plates with increasing concentrations) produced by study drugs by comparing the AUC-PAP pre-exposure with those obtained after 24 h serum and ELF simulations, moxifloxacin significantly (P < 0.01) decreased AUC-PAP of isolate A in serum simulations (without changes in isolates B, C and D) and AUC-PAP of isolates A, B and C in ELF simulations. Levofloxacin significantly (P < 0.001) increased AUC-PAP of isolate A in serum simulations (without changes for the other three isolates) and significantly (P < 0.001) decreased AUC-PAP of isolate A in ELF simulations, with increases in the AUC-PAP of the other three isolates. Azithromycin significantly (P < 0.001) increased AUC-PAP for isolate A in serum simulations (without changes for the other three isolates) and significantly (P < 0.001) decreased AUC-PAP of isolates A and B but not of C and D in ELF simulations.


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  		Table 2. Population analysis profiles (PAP) (log10 cfu/mL) and area under the curve of the population profiles (AUC-PAP) (log10 cfu/mL x MIC) for the four isolates used in the study prior to and after moxifloxacin (MOX) exposure in serum and ELF

 
When integrating bactericidal activity and PAP results in serum simulations, the mean decrease at 24 h of ≥4 log10 cfu/mL obtained against isolate A with moxifloxacin and azithromycin, with negligible changes for isolates B, C and D or for isolate A with levofloxacin (Table 1), is related to pre- and post-exposure population profiles. Pre- and post-exposure PAPs were similar for strains B, C and D regardless the antimicrobial agent (Tables 2–4). Isolate A showed post-exposure growth on plates, with 4–8x MIC in the case of levofloxacin, 4x MIC in the case of azithromycin and absence of growth in the case of moxifloxacin due to its high bactericidal activity (5.6 log decrease).

With ELF simulated concentrations, a mean decrease at 24 h of ≥4.6 log10 cfu/mL was obtained with moxifloxacin against isolates C and D (Table 1), with colony counts near the detection limit in plates containing moxifloxacin concentrations equal to 2x and 4x MIC (Table 2). Against these isolates, the mean decrease with levofloxacin was ≤0.3 log10 cfu/mL (Table 1) with colony counts 10-fold higher than those with moxifloxacin on plates with concentrations equal to 2x MIC and near the detection limit in plates with 4x MIC (Table 3). In the case of azithromycin, the mean decrease against isolates C and D was ≤0.3 log10 cfu/mL (Table 1) but colony counts on plates with concentrations of 2048 mg/L were similar to pre-exposure colony counts (≥107 cfu/mL) (Table 4).


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  		Table 3. Population analysis profiles (PAP) (log10 cfu/mL) and area under the curve of the population profiles (AUC-PAP) (log10 cfu/mL x MIC) for the four isolates used in the study prior to and after levofloxacin (LVX) exposure in serum and ELF

 

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  		Table 4. Population analysis profiles (PAP) (log10 cfu/mL) and area under the curve of the population profiles (AUC-PAP) (log10 cfu/mL x MIC) for the four isolates used in the study prior to and after azithromycin (AZM) exposure in serum and ELF

 
In ELF, moxifloxacin showed a bactericidal pattern against all isolates with a minority (~100 cfu/mL) of the survival population (isolates B, C and D) growing on plates with moxifloxacin concentrations higher than those obtained in ELF. Levofloxacin and azithromycin showed a bactericidal pattern only against isolate A, with the whole population of isolates B, C and D growing on plates with levofloxacin concentrations higher (16–64 mg/L) than those obtained in ELF and on plates with azithromycin concentrations as high as 2048 mg/L (for isolates C and D).


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Quinolones are recommended for the treatment of respiratory tract infections due to the penicillin/macrolide resistance prevalence in S. pneumoniae, or in case of ß-lactam allergy. The increasing use of quinolones in the community has been related to reduced susceptibility in S. pneumoniae.29 Within a given host, quinolone resistance arises after point mutations,2 and the subsequent selection of the resistant subpopulations by antibiotic treatment can drive to therapeutic failures. Because of the low spontaneous mutation frequency, this is more likely to occur in respiratory tissues than in blood, since bacteraemia, when it occurs, is of a very low order of magnitude. Most sputum cultures in patients with chronic bronchitis yield S. pneumoniae in inter-exacerbation periods30 and the same pneumococcus can persist in the bronchial secretions for long time periods.11

In the present study we report the in vitro pharmacodynamic activity of three oral once-daily drugs against the parental strain and three successive resistant mutants isolated after iv quinolone and macrolide treatment of a CAP episode in a patient with chronic bronchitis. The study was performed simulating serum and ELF concentrations of levofloxacin (which shows bioequivalence between oral and iv formulations),16 azithromycin (which exhibited similar in vitro susceptibility to clarithromycin against the four isolates)12 and moxifloxacin (a quinolone with an intrinsic activity higher than levofloxacin) in order to comparatively study their potential in eradicating and preventing resistance emergence.

Not all quinolones need the same AUC0–24/MIC ratio to maximize antimicrobial killing and prevent the emergence of resistance.24 With respect to antimicrobial killing, it has been proposed that ratios of AUC0–24/MIC of 15:1 to >100:1 exhibited the best in vivo correlation of quinolone activity against pneumococci.31 In the present study bactericidal activity (>3 log10 reduction) was obtained with AUC0–24/MIC values around 40 or higher for moxifloxacin (as occurred in ELF simulations against the four isolates and against isolate A in serum). With levofloxacin and azithromycin, AUC0–24/MIC values of around 80–90 were able to eradicate isolate A in ELF. In a previous animal model moxifloxacin needed AUC0–24/MIC values lower than those of levofloxacin to obtain in vivo efficacy.32 It has been postulated that new fluoroquinolones with high anti-pneumococcal activity may prevent emergence of resistance by killing both the parental infecting strain and the subsequent less susceptible step-mutants.13 Since in the patient from whom isolates were obtained failure was due to development of resistance, and both moxifloxacin and levofloxacin were able to eradicate the parental strain with ELF concentrations, it seems that prevention of emergence of resistance is based on a higher degree of capability of killing of the subsequent less susceptible mutants. In this sense, and against all isolates, only moxifloxacin was able to achieve bactericidal activity in ELF (with an Emax of >5 log10), and after 24 h no differences were found in final colony counts between the four isolates (similar AUBC0–24).

In relation to resistant subpopulations after antimicrobial exposure, the study drugs exhibited different capabilities to change pre-exposure intra-strain detectable subpopulation distribution. In ELF simulations all drugs decreased the most resistant pre-exposure subpopulations (those growing in plates with 2x MIC concentrations) of isolate A to negligible values, as shown by the significant decrease in AUC-PAP. Problems arise with the successive resistant mutants (isolates B, C and D). Against isolates B and C only moxifloxacin produced a significant AUC-PAP decrease in ELF simulations. Both quinolones showed a small subpopulation (but 10-fold higher for levofloxacin) that grew on plates containing 2 x MIC concentrations (8 mg/L for moxifloxacin and 32 mg/L for levofloxacin), but only moxifloxacin was able to eradicate the majority of the pre-exposure population. It should be considered that the limit of detection in the present study was 50 cfu/mL, a value lower than in other studies.33 This may have conditioned the subpopulation detection because in the case of moxifloxacin colony counts on plates with concentrations of 2x and 4x MIC were near the detection limit. By pharmacodynamic criteria, moxifloxacin offers an advantage over the other antibiotics tested due to reduced selection of first-step mutants in ELF. Azithromycin was not able to eradicate pre-exposure bacterial counts, with all the population growing on plates with very high azithromycin concentrations.

As mentioned, mutations are more likely to occur in respiratory tissues than bronchi. Thus it is important to use antimicrobials with a lower capability of selection of first-step mutants. Although moxifloxacin selected a resistant subpopulation (detectable when using methods with very low limits of detection), the very small size of this population suggests that it could be removed by ELF immunodefences plus moxifloxacin at the time of approaching the epithelia. If not, these resistant subpopulations have the potential to penetrate into the blood stream where even moxifloxacin may not be able to eradicate them as shown in Figure 2.

Prevention of resistance seems to depend more on the eradication of possible emerging mutants than on the eradication of the parental susceptible strain. In the present study, moxifloxacin concentrations in ELF eradicated both the parental strain and its emerging mutants. This fact together with the bactericidal activity of moxifloxacin ELF concentrations against multi-drug resistant S. pneumoniae34 may provide advantages over previous quinolones and macrolides in preventing failures in an environment of high penicillin/erythromycin resistance prevalence, moreover when first-step mutants are not detected by MIC values and may have been selected by previous antibiotic treatment. The goal of antibacterial therapy should consider not only eradicating the dominant bacterial population but also achieving an exposure preventing, as much as possible, the growth of resistant subpopulations, that is preventing resistance.35


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None to declare.


   Acknowledgements
 
We thank F. Ros for his critical review of the manuscript. This study was supported in part by an unrestricted grant from Bayer, S.A., Barcelona, Spain.


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1 Courvalin P. (2005) Antimicrobial drug resistance: prediction is very difficult, especially about the future. Emerg Infect Dis 11:1503–6.[Web of Science][Medline]

2 Nuermberger EL and Bishai WR. (2004) Antibiotic resistance in Streptococcus pneumoniae: what does the future hold? Clin Infect Dis 38:Suppl 4, S363–71.[CrossRef][Medline]

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