1 Veterans Affairs Medical Center, 500 W. Fort Street, Boise, ID 83702; 2 College of Pharmacy, Idaho State University, Pocatello, ID, USA
Received 6 August 2001; returned 4 March 2002; revised 15 April 2002; accepted 29 April 2002
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods: A one-compartment pharmacodynamic model simulated fluoroquinolone dosing regimens. Duplicate 24 h experiments were carried out in MuellerHinton broth with 3% horse blood at 1 x 108 cfu/mL. Reserpine (10 mg/L) was added to select experiments conducted with efflux-expressing strains. AME was expressed as the area under the timeconcentration kill curve (AUEC24). Strains expressing increased MIC post-timeconcentration kill curve (TCKC) were evaluated for changes in QRDR.
Results: Moxifloxacin exhibited a greater AME against all isolates. Efflux was generally associated with partial loss of AME for all fluoroquinolones, and levofloxacin retained no AME against parC-expressing S. pneumoniae. Increased fluoroquinolone MIC post-TCKC was more common with efflux expression. The addition of reserpine was associated with enhanced AME for levofloxacin and moxifloxacin, but was not associated with altered resistance selection. Isolates recovered post-TCKC from experiments involving efflux- or parC mutation-containing isolates generally exhibited a more than four-fold increase in MIC, which was associated with commonly reported substitutions in both parC and gyrA.
Conclusion: The results of this study generally indicate that resistance selection under pharmacodynamic conditions is similar to results reported with static fluoroquinolone concentrations. While moxifloxacin AME was greater than levofloxacin and sparfloxacin, the overall selection of resistant isolates did not differ.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In vitro model
TCKC experiments were conducted as in a one-compartment in vitro pharmacodyanamic model described previously.9 The model consisted of a hollow glass sealed chamber (c. 500 mL) with inflow and outflow ports. Using a peristaltic pump, antibiotic-free, phosphate-buffered MuellerHinton broth supplemented with 3% lysed horse blood (SMHB) was forced into the system at a pre-determined rate so that an equal volume of medium was displaced, resulting in the simulation of a mono-exponential pharmacokinetic process.10 Before experiments were carried out, a pre-determined inoculum of S. pneumoniae was instilled into the system, followed by a single bolus addition of either moxifloxacin, levofloxacin or sparfloxacin. Each experiment was carried out in duplicate for 24 h at 37°C.
Antibiotics and pharmacokinetics
Stock solutions of moxifloxacin (Bayer, New Haven, CT, USA), sparfloxacin (Bertek Pharmaceuticals, Sugarland, TX, USA) and levofloxacin (Ortho-McNiel, Raritan, NJ, USA) were prepared using the appropriate amounts of sterile distilled water for injection and frozen in aliquots at 70°C until they were needed for individual experiments. Reserpine (Sigma, St Louis, MO, USA) was dissolved in 12 M glacial acetic acid and diluted to a concentration of 7 mg/mL in sterile distilled water for injection. Reserpine was dissolved immediately before each TCKC experiment, and was introduced into the pharmacodynamic model as a 1 mL bolus injection. The maximal peak concentration (Cmax) of reserpine introduced to the pharmacodynamic model was c. 10 mg/L. TCKC experiments were designed to simulate serum pharmacokinetic characteristics reflective of in vivo dosage regimens.1113 Target Cmaxs were 4.5, 1.3 and 6.0 mg/L for moxifloxacin, sparfloxacin and levofloxacin, respectively. Mono-exponential half-lives (t) were 12, 16 and 6 h for moxifloxacin, sparfloxacin and levofloxacin, respectively.
The fluoroquinolone concentrations simulated in the models were confirmed utilizing a microbiological assay.14 Briefly, plates were prepared from antibiotic media 2 (Difco, Detroit, MI, USA) with the pH adjusted to c. 7.7 at 25°C, and Escherichia coli ATCC 25922 was used as the study organism at a density equivalent to a 0.5 McFarland standard. Twenty-microlitre duplicate aliquots of standard solutions prepared in SMHB, as well as samples recovered from the 1 and 8 h TCKC timepoints, were incubated at 37°C for 24 h. The levofloxacin assay was linear from 0.3 to 10.0 mg/L, whereas the sparfloxacin and moxifloxacin assays were linear from 0.3 to 5 mg/L.
Bacteria and susceptibility testing
S. pneumoniae ATCC 49619 and a clinical isolate (SP 2136) were studied in the first set of experiments. These isolates lacked alterations in the QRDR and did not phenotypically express fluoroquinolone efflux. In the second set of TCKC experiments, two additional isolates of S. pneumoniae were studied. Strain 49619 EFX/C was a derivative of ATCC 49619 that expressed an efflux phenotype and had a mutation in parC (Ser-80Tyr). This isolate was obtained by plating 104 cfu of ATCC 49619 on Trypticase soy agar (TSA) with 5% sheep blood supplemented with norfloxacin (4 x MIC). The other strain, 49619 EFX, was a mutant recovered at the 24 h time point during a TCKC experiment using levofloxacin and ATCC 49619. This strain expressed an efflux phenotype, but lacked alterations in the QRDR. Before undertaking TCKC experiments, isolates were subcultured three times consecutively in increasing volumes of SMHB to allow the organisms to attain exponential growth. The final culture (c. 1.5 L) was concentrated by centrifugation at 7000 rpm for 10 min. The pellet was resuspended in 20 mL of SMHB, and serial dilution was used to provide confirmation of the bacterial density. Depending on the isolate being studied, between 4 and 16 mL was injected into each model to provide a starting bacterial inoculum of c. 1 x 108 cfu/mL for each TCKC experiment. Growth controls (± reserpine) verified that each isolate could attain exponential growth. Susceptibility testing was performed on wild-type isolates and on all isolates recovered from the TCKC experiments after 24 h. Homogenates of a minimum of five to 10 colonies recovered from the 24 h sampling point of the TCKC were used to assess post-TCKC susceptibility. Susceptibility testing was performed using Etest according to the manufacturers specifications. Fluoroquinolone resistance post-TCKC was defined as at least a two-unit (mg/L) increase in MIC on the Etest strip as compared with the wild-type isolate.15
TCKC pharmacodynamics
At pre-determined timed intervals, samples of media were removed from the pharmacodynamic models for quantification of bacteria utilizing serial 10-fold dilution with aliquots plated on TSA supplemented with 5% sheep blood. After incubation for 1824 h at 37°C, plates containing between 30 and 300 cfu were counted. The theoretical lower limit of bacterial counting accuracy was 3.0 x 102 cfu/mL. TCKCs were constructed by plotting log10 cfu/mL versus time.
Analysis of AME was expressed as the area under the timeconcentration kill curve (AUEC24).16 The AUEC24 only incorporated timeconcentration data points that were above the lower theoretical limit of bacterial counting. To compensate for differences in initial starting inocula, AUEC24 was standardized by dividing AUEC24 by the cfu/mL at time zero.17
Efflux determination
Phenotypic expression of efflux-mediated resistance was determined by an agar dilution method:18 1 x 104 cfu of each isolate was inoculated on to duplicate plates containing serially increasing concentrations of norfloxacin or norfloxacin plus reserpine (10 mg/L). Selected experiments were conducted with increased reserpine concentrations (50 and 100 mg/L) against isolates recovered post-TCKC that exhibited high-level fluoroquinolone resistance not explained by mutations in QRDR.
Amplification of S. pneumoniae QRDRs of parC, parE, gyrA and gyrB genes
All parent isolates and all post-TCKC isolates that exhibited an increased MIC underwent genotypic analysis by PCR for changes in their QRDR. Bacterial genomic DNA was extracted using the High Pure PCR Template kit (Boehringer-Mannheim, Indianapolis, IN, USA). The QRDRs of parC, parE, gyrA and gyrB were amplified via PCR using isolated chromosomal DNA as the template. PCR primers for the QRDRs and conditions for amplification were as described by Pan et al.2 All PCR primers were synthesized by the Idaho State University Molecular Research Core Facility. To identify mutations at the level of nucleotide sequence, PCR products were electrophoresed on 2% agarose gels, the appropriate band excised from the gel and PCR product purified using the Qiagen QIAquick Gel Extraction Kit (Qiagen Inc., Valencia, CA, USA). Nucleotide sequences of PCR products were determined directly using an ABI Automated Fluorescence Sequencer and mutations were identified using the MegAlign program of DNAStar sequence analysis software.
Analysis
ANOVA and Dunns test were used to compare the AME for intra- and inter-species comparisons, and P < 0.0167 was considered statistically significant for intergroup comparisons. The AME for specific fluoroquinolones with and without reserpine was compared using Students t-test, and the 2 or Fishers exact test was used to compare the likelihood of developing resistance for efflux versus non-efflux strains post-TCKC. P < 0.05 was considered statistically significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Despite initial MICs suggesting that all organisms were susceptible to all three fluoroquinolones, there were large differences in AME between moxifloxacin, sparfloxacin and levofloxacin. All three fluoroquinolones exhibited pronounced AME against isolates without QRDR changes or the presence of efflux. However, moxifloxacin, and to a lesser extent sparfloxacin, retained activity against S. pneumoniae that exhibited phenotypic fluoroquinolone efflux. Despite an MIC of 1.5 mg/L, levofloxacin retained partial AME against the isolate possessing efflux only, and almost no AME against the isolate possessing both phenotypic efflux and a parC mutation. However, the exact degree of levofloxacin AME against 49619 EFX/C was not exemplified in these TCKCs, as the organism replicated above the serial dilution capacity of most time points. Moxifloxacin retained a relatively more pronounced AME against the isolate possessing a topoisomerase IV mutation in parC.
The presence of an energy-dependent efflux transport mechanism has been reported to be present in c. 45% of fluoroquinolone-resistant S. pneumoniae.18 Pharmacokinetic simulations against isolates without QRDR changes or the presence of efflux revealed the selection of only one isolate with an increase in MIC. This isolate did not contain alterations in QRDR, but was an efflux-producing isolate. In contrast, the majority of pharmacokinetic simulations against 49619 EFX resulted in the further selection of organisms with increases in MIC and corresponding changes in parC and gyrA. These results would support the findings of Beyer et al.,3 which suggest that the probability of avoiding efflux reduces the potential for selection of further resistance.
The results seen following addition of reserpine to our TCKC experiments conducted with isolates expressing efflux indicated enhanced AME for moxifloxacin and levofloxacin, but not sparfloxacin. Theoretically, moxifloxacin AME should not have been influenced by reserpine, as moxifloxacin has been reported to be relatively unaffected by pmrA-mediated efflux due to its C-8 methoxy substitution.3 However, at least one moxifloxacin-resistant isolate exhibiting a four-fold MIC increase attributed to efflux has been described.19
In contrast to static experiments conducted with reserpine, the addition of reserpine to TCKC experiments of efflux-expressing isolates did not prevent the development of further resistance.3,20 While reserpine is freely soluble in glacial acetic acid, we cannot exclude the possibility that reserpine precipitated during TCKC because the solubility of this drug is poor in aqueous solutions. Aeschlimann et al.,21 working with NorA-expressing Staphylococcus aureus, reported difficulty in maintaining the solubility of reserpine in TCKC.
Of interest, two post-TCKC isolates from experiments conducted with moxifloxacin and levofloxacin against 49619 EFX/C exhibited high-level resistance that was not fully explained by analysis of known QRDRs. This isolate possessed an alteration in parC and expressed efflux before TCKC. The MICs of moxifloxacin and levofloxacin were 0.25 and 1.5 mg/L, respectively. Post-TCKC analysis of recovered isolates indicated moxifloxacin and levofloxacin MICs of 4 and 32 mg/L, respectively. Subsequent agar dilution experiments conducted with 10, 50 and 100 mg/L of reserpine and serial concentrations of norfloxacin revealed a two-fold decrease in MIC with the addition of 10 mg/L reserpine and a three-fold decrease in MIC with the addition of 50 mg/L reserpine. However, 50 mg/L reserpine independently inhibited the growth of both isolates, whereas 10 mg/L did not. We did not directly measure the MIC post-TCKC of moxifloxacin or levofloxacin with reserpine, but chose to maintain consistency with the agar dilution method used to assess pre-TCKC isolates for efflux expression.
These experiments indicate that the high-level resistance post-TCKC might be due in part to hyperproduction of efflux; however, the full effect of inhibition of efflux with reserpine remains unknown. Hyperproduction of efflux without mutation in QRDR has rarely been shown to be responsible for high-level resistance to ofloxacin.8 An alternative possibility is that topoisomerase II mutations or other mutations outside of the sequenced QRDR resulted in phenotypic changes in MIC.22
Finally, the majority of mutations reported in our study involved Ser-80Tyr for parC, and Ser-83
Phe, Ser-83
Tyr or Glu-87
Lys for gyrA, all of which have been reported as relatively frequent mutations in fluoroquinolone-resistant S. pneumoniae.6 The results of our investigation tend to suggest that selection of specific mutational changes recovered from clinical infections, as well as those selected under static fluoroquinolone concentrations, parallel those selected by dynamic conditions utilizing in vitro pharmacodynamic models.18
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Pan, X. S., Ambler, J., Mehtar, S. & Fisher, L. M. (1996). Involvement of topoisomerase IV and DNA gyrase as ciprofloxacin targets in S. pneumoniae. Antimicrobial Agents and Chemotherapy 40, 23216.[Abstract]
3
.
Beyer, R., Pestova, E., Millichap, J. J., Stosor, V., Noskin, G. A. & Peterson, L. R. (2000). A convenient assay for estimating the possible involvement of efflux of fluoroquinolones by Streptococcus pneumoniae and Staphylococcus aureus: evidence for diminished moxifloxacin, sparfloxacin, and trovafloxacin efflux. Antimicrobial Agents and Chemotherapy 44, 798801.
4 . Gootz, T. D., Zaniewski, R., Haskell, S., Schmieder, B., Tankovic, J., Girard, D. et al. (1996). Activity of the new fluoroquinolone trovafloxacin against DNA gyrase and topoisomerase IV mutants of S. pneumoniae selected in vitro. Antimicrobial Agents and Chemotherapy 40, 26917.[Abstract]
5
.
Drugeon, H. B., Juvin, M. E. & Bryskier, A. (1999). Relative potential of fluoroquinolone-resistant S. pneumoniae strains by levofloxacin: comparison with ciprofloxacin, sparfloxacin and ofloxacin. Journal of Antimicrobial Chemotherapy, 43, Suppl. C, 559.
6
.
Jones, M. E., Sahm, D. F., Martin, N., Scheuring, S., Heisig, P., Thornsberry, C. et al. (2000). Prevalance of gyrA, gyrB, parC, and parE mutations in clinical isolates of S. pneumoniae with decreased susceptibilities to different fluoroquinolones and originating from Worldwide Surveillance Studies during the 19971998 Respiratory Season. Antimicrobial Agents and Chemotherapy 44, 4626.
7 . Tankovic, J., Perichon, B., Duval, J. & Courvallin, P. (1996). Contributions of mutations in gyrA and parC genes to fluoroquinolone resistance of mutants of S. pneumoniae obtained in vivo and in vitro. Antimicrobial Agents and Chemotherapy 40, 250510.[Abstract]
8
.
Broskey, J., Coleman, K., Gwynn, M. N., McCloskey, L., Traini, C., Voelker, L. et al. (2000). Efflux and target mutations as quinolone resistance mechanisms in clinical isolates of S. pneumoniae. Journal of Antimicrobial Chemotherapy 45, Suppl. S1, 959.
9 . Zabinski, R. A., Vance-Bryan, K., Krinke, A. J., Walker, K. J., Moody, J. A. & Rotschafer, J. C. (1993). Evaluation of activity of temafloxacin against Bacteroides fragilis by an in vitro pharmacodynamic system. Antimicrobial Agents and Chemotherapy 37, 24548.[Abstract]
10 . Walker, K. J., Larsson, A. J., Zabinski, R. A. & Rotschafer, J. C. (1994). Evaluation of antimicrobial activities of clarithromycin and 14-hydroxyclarithromycin against three strains of Haemophilus influenzae by using an in vitro model. Antimicrobial Agents and Chemotherapy 38, 20037.[Abstract]
11
.
Stass, H., Dalhoff, A., Kubitza, D. & Schuly, U. (1998). Pharmacokinetics, safety, and tolerability of ascending single doses of moxifloxacin, a new 8-methoxy quinolone, administered to healthy subjects. Antimicrobial Agents and Chemotherapy 42, 20605.
12 . Goa, K. L., Bryson, H. M. & Markham, A. (1997). Sparfloxacin: a review of its antibacterial activity, pharmacokinetic properties, clinical efficacy, and tolerability in lower respiratory tract infections. Drugs 53, 70025.[ISI][Medline]
13 . North, D. S., Fish, D. N. & Redington, J. J. (1998). Levofloxacin, a second generation fluoroquinolone. Pharmacotherapy 18, 91535.[ISI][Medline]
14 . Edberg, S. C. (1986). The measurement of antibiotics in human body fluids: techniques and significance. In Antibiotics in Laboratory Medicine, 2nd edn (Lorian V., Ed.), pp. 381463. Williams & Wilkins, Baltimore, MD, USA.
15 . Madaras-Kelly, K. J. & Demasters, T. (2000). In vitro characterization of fluoroquinolone concentration/MIC antimicrobial activity while simulating clinical pharmacokinetics of levofloxacin, ofloxacin, and ciprofloxacin against multiple drug resistant isolates of S. pneumoniae. Diagnostic Microbiology and Infectious Disease 37, 25360.[ISI][Medline]
16 . Madaras-Kelly, K. J., Ostergaard, B., Hovde, L. B. & Rotschafer, J. C. (1996). Twenty-four hour area under the concentrationtime curve/MIC ratio as a generic predictor of fluoroquinolone antimicrobial effect by using three strains of Pseudomonas aeruginosa and an in vitro pharmacodynamic model. Antimicrobial Agents and Chemotherapy 40, 62732.[Abstract]
17 . Madaras-Kelly, K. J., Larsson, A. J. & Rotschafer, J. C. (1996). A pharmacodynamic evaluation of ciprofloxacin and ofloxacin against two strains of P. aeruginosa. Journal of Antimicrobial Chemotherapy 37, 70310.[Abstract]
18
.
Brenwald, N. P., Gill, M. J. & Wise, R. (1998). Prevalence of a putative efflux mechanism among fluoroquinolone resistant clinical isolates of S. pneumoniae. Antimicrobial Agents and Chemotherapy 42, 20325.
19
.
Markham, P. N. (1999). Inhibition of the emergence of ciprofloxacin resistance in Streptococcus pneumoniae by the multi-drug efflux inhibitor reserpine. Antimicrobial Agents and Chemotherapy 43, 9889.
20 . Davies, T. A., Pankuch, G. A., Jacobs, M. R. & Appelbaum, P. C. (2000). Activity of BMS 284756 compared with other quinolones against quinolone resistant pneumococci. In Program and Abstracts of the Fortieth Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 2000. Abstract 1057, p. 175. American Society for Microbiology, Washington, DC, USA.
21 . Aeschlimann, J. R., Dresser, L., Kaatz, G. W. & Rybak, M. J. (1999). Effects of NorA inhibitors on in vitro antibacterial activities and post-antibiotic effects of levofloxacin, ciprofloxacin, and norfloxacin in genetically related strains of Staphylococcus aureus. Journal of Antimicrobial Pharmacotherapy 43, 33540.
22
.
Weigel, L. M., Anderson, G. J., Facklam, R. R. & Tenover, F. C. (2001). Genetic analysis of mutations contributing to fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae. Antimicrobial Agents and Chemotherapy 45, 351723.