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Antimicrobial Agents and Chemotherapy, February 2002, p. 471-477, Vol. 46, No. 2
0066-4804/01/$04.00+0     DOI: 10.1128/AAC.46.2.471-477.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Convulsant and Subconvulsant Doses of Norfloxacin in the Presence and Absence of Biphenylacetic Acid Alter Extracellular Hippocampal Glutamate but Not Gamma-Aminobutyric Acid Levels in Conscious Rats

I. Smolders,¹ C. Gousseau,² S. Marchand,² W. Couet,^2* G. Ebinger,³ and Y. Michotte¹

Department of Pharmaceutical Chemistry and Drug Analysis,¹ Department of Neurology, University Hospital, Vrije Universiteit Brussel, 1090 Brussels, Belgium,³ Equipe Emergente Médicaments et BHE, Faculté de Médecine et Pharmacie, BP 199, 86005 Poitiers Cedex, France²

Received 23 March 2001/ Returned for modification 4 September 2001/ Accepted 26 October 2001

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Fluoroquinolones are antibiotics with central excitatory side effects. These adverse effects presumably result from inhibition of -aminobutyric acid (GABA) binding to GABA_A receptors. This GABA antagonistic effect is greatly potentiated by the active metabolite of fenbufen, biphenylacetic acid (BPAA). Nevertheless, it remains questionable whether GABA receptor antagonism alone can explain the convulsant activity potentials of these antimicrobial agents. The present study was undertaken to investigate the possible effects of norfloxacin, both in the absence and in the presence of BPAA, on the extracellular hippocampal levels of GABA and glutamate, the main central inhibitory and excitatory amino acid neurotransmitters, respectively. This in vivo microdialysis approach with conscious rats allows monitoring of behavioral alterations and concomitant transmitter modulation in the hippocampus. Peroral administration of 100 mg of BPAA per kg of body weight had no effect on behavior and did not significantly alter extracellular GABA or glutamate concentrations. Intravenous perfusion of 300 mg of norfloxacin per kg did not change the rat's behavior or the concomitant neurotransmitter levels in about half of the experiments, while the remaining animals exhibited severe seizures. These norfloxacin-induced convulsions did not affect extracellular hippocampal GABA levels but were accompanied by enhanced glutamate concentrations. Half of the rats receiving both 100 mg of BPAA per kg and 50 mg of norfloxacin per kg displayed lethal seizures, while the remaining animals showed no seizure-related behavior. In the latter subgroup, again no significant alterations in extracellular GABA levels were observed, but glutamate overflow remained significantly elevated for at least 3 h. In conclusion, norfloxacin exerts convulsant activity in rats, accompanied by elevations of extracellular hippocampal glutamate levels but not GABA levels, even in the presence of BPAA.

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Fluoroquinolones (FQs) are used in the treatment of various infectious diseases because of their broad and strong antibacterial activities, especially against gram-negative bacteria. Although generally well tolerated, they may induce adverse symptoms on the central nervous system (CNS) including headache, confusion, hallucinations, anxiety, nervousness, and nightmares (3, 4, 19). Epileptic seizures have occasionally been reported, most frequently in patients with a history of epilepsy or when FQs have been coadministered with theophylline or nonsteroidal anti-inflammatory drugs such as fenbufen (4, 12, 22). It is most often admitted from the numerous studies conducted in vitro that this central excitatory effect should result from inhibition of -aminobutyric acid (GABA) binding to its receptors (1, 12, 28, 29). However, both in vitro electrophysiological and radioligand binding experiments demonstrated that FQs are only weak GABA_A receptor antagonists and that their affinities are greatly potentiated by the active metabolite of fenbufen, biphenylacetic acid (BPAA), which does not exhibit any inhibitory action per se (2, 14, 16, 21). Furthermore, other in vitro binding experiments suggested that FQs exert their convulsant effects in part by reducing central adenosine-mediated inhibition (11). They do not, however, alter the binding of glutamate-selective ligands for the ionotropic -amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate receptors, nor did they affect excitatory AMPA-, N-methyl-D-aspartate (NMDA)-, or kainate-activated currents (11, 13). More recently, a whole set of investigations has been conducted in vivo by a new approach that allows distinction between the pharmacokinetic and pharmacodynamic contributions to the convulsant activities of FQs. That approach was based upon the determination of drug concentrations within the biophase at the onset of activity (5). It was shown that both the central diffusion and the affinity for the receptors responsible for the epileptogenic activity vary considerably among FQs, from which it was concluded that these two factors must be considered for prediction of convulsant activity in vivo (7). The proconvulsant effect of BPAA on norfloxacin, selected as a representative FQ, was investigated by the same type of approach (17). The objective of the present study was to elucidate more about the mechanism of convulsant activity of norfloxacin in vivo, both in the absence and in the presence of BPAA, by intracerebral microdialysis in conscious, freely moving rats. This approach allows monitoring of the behavioral alterations and the concomitant modulation of the extracellular neurotransmitter levels. The hippocampus was selected as the area of interest because this seizure-prone region of the brain is recognized as being involved in the regulation of the brain's excitability and has been suggested as playing a pivotal role in FQ-induced convulsions (15). The neurotransmitters to be investigated are GABA and glutamate, the main inhibitory and excitatory amino acid transmitters in the CNS, respectively, that are clearly in control of hippocampal excitability and epileptic events.

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Chemicals and reagents. Norfloxacin·HCl was prepared from commercially available norfloxacin (Sigma, St. Louis, Mo.) as described before (5). The norfloxacin·HCl solution (76.6 mg/ml) for intravenous (i.v.) administration was dissolved in 5% glucose (VUB Hospital, Brussels, Belgium). A suspension of BPAA (Sigma) for oral (p.o.) administration was prepared in 0.5% carboxymethyl cellulose (CMC; Cooperation Pharmaceutique Française, Melun, France). Rats that received sham i.v. injections or sham p.o. administrations were given adequate amounts of the respective vehicle solutions. GABA and L-glutamate standards were supplied by Sigma. All other chemicals were analytical reagent grade or better and were supplied by Merck (Darmstadt, Germany). Aqueous solutions were made with purified water (Seralpur pro 90 CN; Belgolabo, Overijse, Belgium) and filtered through a 0.2-µm-pore-size membrane filter. The perfusion fluid for microdialysis, so-called modified Ringer's solution, contained 147 mM NaCl, 2.3 mM CaCl₂, and 4 mM KCl.

Animals and surgery. The protocols used in the present study are in accordance with national rules on experiments with animals and were approved by the Ethics Committee on Animal Experiments of the Faculty of Medicine and Pharmacy of the Free University of Brussels, V.U.B., Brussels, Belgium. Male albino Wistar rats (weight, between 270 and 300 g) were kept under standard laboratory conditions (room temperature, 22 ± 1°C; a cycle of 12 h of light and 12 h of dark with lights on at 7 a.m.; and food and water ad libitum). The rat subjected to surgery was anesthetized with a mixture of ketamine and diazepam (50:5 mg/kg). A lateral incision was made on the ventral surface of the neck, and the left jugular vein was exposed by using blunt dissection. The vessel was tied at the anterior end and a hole was punctured posterior to it. A polyurethane catheter (inside diameter, 0.58 mm; outside diameter, 0.98 mm; Plastimed Laboratories, Saint-Leu-La-Foret, France) was inserted into the hole and was pushed about 2.5 cm toward the heart. This catheter was secured to the vessel with a suture tied around it, was cleared with a saline solution of heparin (100 IU/ml), and was plugged to maintain patency. The catheter was brought through a subcutaneous tunnel and externalized between the two shoulder blades. Following the placement of the i.v. catheter, the animal was mounted on a stereotaxic frame to implant a guide for intracerebral microdialysis. A vertical incision was made in the skin to expose the skull. An intracranial guide (CMA/Microdialysis, Stockholm, Sweden) was implanted in the dorsal hippocampus and was fixed with dental cement. Coordinates toward the bregma were L +4.6, A -5.6, V +4.6 (18). Rats received an intraperitoneal injection of ketoprofen (4 mg/kg of body weight) to provide postoperative analgesia.

Microdialysis. Immediately after surgery, the guide cannula obturator was replaced by a CMA/12 microdialysis probe (membrane length, 3 mm; CMA/Microdialysis). The probe was continuously perfused with modified Ringer's solution at a flow rate of 2 µl/min (CMA/100 microdialysis pump; CMA/Microdialysis). The animal was allowed to recover from surgery overnight in its experimental microdialysis cage. During the experiment, on the next day, dialysates were collected every 20 min from the hippocampus of the freely moving rat. For the first six collection periods, samples were collected under baseline conditions, i.e., before any drug or vehicle was administered. During the next collection period, the animals received BPAA p.o. or a sham p.o. administration of the 0.5% CMC vehicle (volumes were always adapted to the rat's individual weight). One hour later, i.e., during collection period 10, the rats received an appropriate dose of norfloxacin as a short i.v. infusion of 1 min or a sham i.v. infusion of the 5% glucose solution. Dialysates were sampled for another 13 sampling intervals. Rats were divided into four treatment groups. The treatments are summarized in Table 1.

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TABLE 1. Treatment summary

Chromatographic assays. Chromatographic conditions and precolumn derivatization procedures for the amino acids have previously been described in detail (24). For GABA determinations, isocratic, reversed-phase, microbore liquid chromatography (LC) with amperometric detection was used. Precolumn derivatization was performed with o-phthalaldehyde-tert-butylthiol and iodoacetamide. Glutamate determinations were carried out after precolumn derivatization with o-phthalaldehyde-ß-mercaptoethanol by gradient, reversed-phase, microbore LC with fluorescence detection.

Statistical analysis. All results presented in the figures are expressed as the mean ± standard error of the mean (SEM) amino acid concentrations (micromolar). These dialysate concentrations were not corrected for the recovery across the dialysis membrane. Recovery of glutamate and GABA varies between 10 and 15%. The levels for the first six dialysate collection periods of 20 min each were averaged, and the average was taken as the mean baseline level for that experiment. All other time points correspond to successive collection periods of 20 min. Statistical analysis of alterations of the amino acid concentrations with time compared to the baseline levels within one experimental group was performed by analysis of variance (ANOVA) for repeated measurements and Fisher's protected least-significant difference (PLSD) post hoc tests ( = 0.05).

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Sham p.o. administration, sham i.v. infusion group (n = 4). The basal hippocampal dialysate concentrations were 0.031 ± 0.008 µM for GABA (Fig. 1a)and 0.383 ± 0.138 µM for glutamate (Fig. 1b). During the collection of the samples for determination of baseline levels, the animals were usually very quiet or even somnolent. The sham p.o. administration of the CMC vehicle variably distressed the animals, after which they soon regained their normal, quiet behavior. The sham i.v. infusion of the glucose solution did not induce any behavioral alterations. The sham p.o. administration and the sham i.v. infusion did not significantly change the extracellular GABA (Fig. 1a) (P = 0.74) or glutamate (Fig. 1b) (P = 0.52) levels. Nevertheless, in one animal in this control group, we noticed a fourfold increase in extracellular glutamate levels during the collection period in which the rat received the vehicle p.o., resulting in a large variation for that particular time point (Fig. 1b).

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FIG. 1. Sham experiments and lack of effects of BPAA (100 mg/kg) alone. The extracellular (EC) levels (mean ± SEM) of GABA (a) and glutamate (b) sampled from the rat hippocampus by in vivo microdialysis are indicated. The first six dialysates were sampled under basal conditions. These stable 120-min dialysate concentrations were pooled and taken as the mean baseline level for that experimental group. Each of the remaining time points represents a subsequent 20-min collection period. After 140 min (collection period 7), the rats received a sham p.o. administration of 0.5% CMC vehicle (sham p.o. administration, sham i.v. infusion group; n = 4) or a p.o. administration of 100 mg of BPAA per kg (p.o. BPAA administration, sham i.v. infusion group; n = 6), indicated by the first arrow, labeled p.o. One hour later, during collection period 10, all rats received a sham i.v. infusion of the 5% glucose solution, indicated by the second arrow, labeled i.v. Statistical analysis was done by one-way ANOVA for repeated measures and Fisher's PLSD post hoc tests; the corresponding P values are denoted.

p.o. BPAA administration, sham i.v. infusion group (n = 6). Baseline hippocampal dialysate concentrations were 0.027 ± 0.013 µM for GABA (Fig. 1a) and 0.391 ± 0.115 µM for glutamate (Fig. 1b). During the collection of the samples for determination of baseline levels, the animals were usually very quiet. The p.o. administration of 100 mg of BPAA per kg briefly distressed the rats, after which they immediately displayed their normal behavior. The sham i.v. infusion of the glucose solution 1 h later did not induce any behavioral changes. The p.o. administration of 100 mg of BPAA per kg and the sham i.v. infusion did not significantly alter extracellular GABA (Fig. 1a) (P = 0.52) or glutamate (Fig. 1b) (P = 0.84) concentrations.

Sham p.o. administration, i.v. norfloxacin infusion group (n = 9). Basal hippocampal dialysate concentrations were 0.025 ± 0.005 µM for GABA and 0.498 ± 0.185 µM for glutamate. During the collection of the samples for determination of baseline levels, the rats remained very quiet or even somnolent. The sham p.o. administration briefly distressed the animals, after which they soon regained their normal, quiet behavior. After the i.v. infusion of 300 mg of norfloxacin per kg, four of the nine rats did not display any further behavioral alterations, and they remained quiet until the end of the experiment. In this subgroup of animals (the no convulsions group), we observed no significant alterations in extracellular GABA (Fig. 2a)(P = 0.11) or glutamate (Fig. 2b) (P = 0.47) levels. Five of the nine animals exhibited severe epileptic convulsions starting approximately 20 min after the start of the i.v. perfusion with 300 mg of norfloxacin per kg. These seizures persisted for about 3 to 4 h, after which the rats recovered and behaved normally for the next 24 h. The norfloxacin-induced convulsions did not significantly alter extracellular hippocampal GABA levels (Fig. 2a) (P = 0.31) but were accompanied by significantly enhanced extracellular hippocampal glutamate concentrations between 1 and 2 h after the start of the i.v. perfusion with norfloxacin and toward the end of the convulsive episode (Fig. 2b) (P = 0.05).

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FIG. 2. Subconvulsant and convulsant effects of norfloxacin in the absence of BPAA. The extracellular (EC) levels (mean ± SEM) of GABA (a) and glutamate (b) sampled from the rat hippocampus by in vivo microdialysis are indicated. Data are presented as described in the legend to Fig. 1. After 140 min (collection period 7), all rats (n = 9) received a sham p.o. administration of the CMC vehicle, indicated by the first arrow, labeled p.o. One hour later (collection period 10), all animals received an i.v. perfusion of 300 mg of norfloxacin per kg, as indicated by the second arrow, labeled i.v. The animals were subdivided into rats displaying no behavioral alterations after the i.v. norfloxacin perfusion (no convulsions; n = 4) and rats exhibiting norfloxacin-induced convulsions (n = 5). Statistical analysis was done by one-way ANOVA for repeated measures and Fisher's PLSD post hoc tests; the corresponding P values are denoted, and asterisks mark values significantly different from the corresponding baseline levels.

p.o. BPAA administration, i.v. norfloxacin infusion group (n = 7). Baseline hippocampal dialysate levels were 0.035 ± 0.008 µM for GABA and 0.557 ± 0.075 µM for glutamate. During the collection of the samples for baseline concentration determination, the rats were usually quiet. The p.o. administration of 100 mg of BPAA per kg variably distressed the animals, after which they soon displayed their normal behavior. After the additional i.v. infusion of 50 mg of norfloxacin per kg, four of the seven rats did not show any behavioral alterations and remained quiet until the end of the experiment. A significant increase in extracellular glutamate levels was observed in this subgroup during the collection period in which the rats received BPAA p.o. (Fig. 3b).During the next collection period, however, these glutamate concentrations returned to baseline levels. Nevertheless, immediately after the beginning of the concomitant perfusion of norfloxacin, the extracellular glutamate overflow remained significantly elevated for at least 3 h (Fig. 3b) (P = 0.01). No significant alterations in extracellular GABA concentrations were observed in the same subgroup of animals not exhibiting convulsions (Fig. 3a) (P = 0.75). Three of the seven animals receiving both 100 mg of BPAA per kg and 50 mg of norfloxacin per kg 1 h later exhibited very severe seizures starting approximately 20 min after the beginning of the norfloxacin perfusion. These rats displayed frequent and violent convulsive episodes resembling status epilepticus and died of exhaustion and asphyxia within 40 min. Therefore, no additional experiments were performed to extend the number of experimental animals in this population receiving both BPAA and norfloxacin. In the small number of dialysates that were collected from the latter subgroup, no significant changes were observed in extracellular GABA levels (Fig. 3a) (P = 0.61). Although glutamate concentrations seemed to increase in these rats, as in those not exhibiting seizures (Fig. 3b), the changes were not statistically significant (P = 0.43), probably due to the high interindividual variability and small number of rats (n = 3).

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FIG. 3. Subconvulsant and convulsant effects of norfloxacin in the presence of BPAA. The extracellular (EC) levels (mean ± SEM) of GABA (a) and glutamate (b) sampled from the rat hippocampus by in vivo microdialysis are indicated. Data are presented as described in the legend to Fig. 1. After 140 min, all rats (n = 7) received a p.o. administration of 100 mg of BPAA per kg, indicated by the first arrow, labeled p.o. One hour later (collection period 10), all animals received an i.v. perfusion of 50 mg of norfloxacin per kg, indicated by the second arrow, labeled i.v. The animals were subdivided into rats showing no behavioral alterations after the i.v. norfloxacin perfusion (no convulsions; n = 4) and rats exhibiting lethal convulsions (n = 3). Statistical analysis was done by one-way ANOVA for repeated measures and Fisher's PLSD post hoc tests; the corresponding P values are denoted, and asterisks mark values significantly different from the corresponding baseline levels.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present in vivo study investigated for the first time the possible effects of the i.v. administration of norfloxacin, with and without BPAA, on the extracellular hippocampal GABA and glutamate levels in conscious rats sampled by microdialysis. Norfloxacin was selected as a usual (17) and as an appropriate representative FQ for this study. In vitro binding experiments have demonstrated that norfloxacin possesses a much greater affinity for GABA_A receptors than most FQs, especially in the absence of BPAA (1). It is known that BPAA itself shows no binding affinity for GABA_A receptors (2) and has no effect on GABA-activated currents (12). We observed earlier that even at oral doses as high as 300 mg/kg, BPAA alone does not induce seizures in rats (17). Accordingly, neither behavioral changes nor alterations in GABA and glutamate overflows in the hippocampus were observed in the animals treated with BPAA (100 mg/kg p.o.) and sham i.v. infusions in the present experiments.

We have conducted several studies in the past with i.v. infusions of norfloxacin in rats until the onset of maximal seizures (5, 6, 7, 17). The dose of norfloxacin that induced convulsant activity varied with the infusion rate and therefore the duration (5) but was, on average, on the order of 2 mmol/kg, which is close to 600 mg/kg. However, under the latter conditions, norfloxacin-induced convulsions were lethal on most occasions, precluding investigations by microdialysis. By reducing the norfloxacin dose to 300 mg/kg, administered as a short i.v. infusion, we were able to observe in the present study severe but not lethal convulsions in about 50% of the rats. Dose adjustments in rats treated with both BPAA and norfloxacin were more challenging because two drugs were involved. We have shown earlier that the proconvulsant effect of BPAA tended toward a maximum when the administered dose increased, and in practice, an oral dose of 50 mg/kg would produce a proconvulsant effect close to the maximum (17). A dose of 100 mg/kg was therefore selected in order to avoid any absence of effects due to an insufficient dose of BPAA. However, under these conditions, a dose of 300 mg of norfloxacin per kg would produce lethal convulsions in every animal. Therefore, the dose of norfloxacin in the presence of BPAA also had to be adjusted. Consistent with the fact that BPAA increases the convulsant effect of norfloxacin by about sixfold (17), it was decided to reduce the norfloxacin dose sixfold (from 300 to 50 mg/kg) in rats pretreated with BPAA. This adjustment turned out to be appropriate since in this group, too, about 50% of the rats exhibited severe seizures, allowing comparisons between animals treated with norfloxacin alone and animals treated with norfloxacin in the presence of BPAA.

The first finding of the present study worth mentioning was that norfloxacin alone or norfloxacin in the presence of BPAA had no effect on the extracellular hippocampal GABA levels. The convulsant activity of FQs is generally attributed to their pharmacological antagonism with endogenous GABA at the GABA_A receptor sites (1, 12, 28, 29). Despite the rich presence of GABA_A receptors and GABAergic interneurons in the hippocampal formation, one should not expect GABA_A receptor ligands acting at the postsynaptic level to directly alter extracellular GABA concentrations. Indeed, different doses (1 to 100 µM) of the prototypical GABA_A receptor antagonist bicuculline, perfused via a microdialysis probe into the hippocampus, were unable to alter the extracellular GABA levels (20). One may argue that microdialysis with 20-min collection intervals may not be able to capture transient changes in neurotransmitter levels. It should, however, be mentioned that, using a similar experimental microdialysis approach in the pilocarpine rat model for intractable partial seizures, we observed that both the extracellular hippocampal GABA and the glutamate levels initially decreased during the pilocarpine perfusion and then significantly increased during the subsequent pilocarpine-induced convulsions (25, 26). Thus, the fact that norfloxacin did not alter the hippocampal GABA overflow certainly does not exclude the possibility that this antibiotic did bind to the hippocampal GABA_A receptors.

The second important finding of the present study was the demonstration for the first time that doses of norfloxacin that induce convulsant or subconvulsant activity increase extracellular hippocampal glutamate concentrations both in the presence and in the absence of BPAA. Transient increases in extracellular glutamate levels were also occasionally observed during the collection period corresponding to p.o. administration, both in the sham p.o. administration, sham i.v. infusion group and the p.o. BPAA administration, i.v. norfloxacin infusion group. These short-lasting effects were probably due to the handling and distress of these particular animals and cannot be considered drug-related effects since they appeared both in the sham-treated and in the treated groups. Noticeably, similar observations have been reported for glutamate sampled from the prefrontal cortex, ventral tegmental area, and locus ceruleus (27). It is known that FQs neither alter the binding of glutamate-selective ligands for the ionotropic glutamate receptors nor interfere with the AMPA-, NMDA-, and kainate-activated currents (11, 13). Several quinolones (ciprofloxacin, lomefloxacin, ofloxacin) were, however, found to increase in a dose-dependent manner the amplitude of electrically evoked field potentials in the CA1 region of rat hippocampal slices in vitro (9), which confirmed that FQs indeed increase the brain's excitability. Some FQs (nalidixic acid, oxolinic acid) were reported to lower the threshold for electroshock-induced seizures (30). Moreover, glutamate receptor ligands, such as noncompetitive NMDA receptor blockers, agonists at the NMDA-glycine site, and some AMPA-kainate receptor antagonists, were able to reduce epileptiform activity in in vitro slice preparations and exerted anticonvulsant effects in mouse epilepsy models (8, 10, 30). The observed enhanced extracellular glutamate levels might be essential for FQ-induced seizure generation, spread, and maintenance and for the successive glutamate receptor-mediated processes. The present findings are in accordance with the hypothesis that excessive activation of excitatory amino acid receptors might occur secondarily to or concomitantly with the impairment of the GABA_A receptor-mediated neurotransmission caused by the FQs (8) and strongly suggest that glutamate plays a key role in the CNS excitability-related side effects induced by these antimicrobial agents.

Taking the present effects on extracellular GABA and glutamate concentrations in rat hippocampus into account, we hypothesize that norfloxacin alone or as an intermolecular complex with BPAA (2) presumably inhibits postsynaptic GABA_A receptors, which then leads to a disinhibition of the hippocampal glutamatergic neurons. This does not exclude a possible involvement of adenosine A₁ receptors (11). This hypothesis can be reconciled with previously published studies. Indeed, there is a large body of evidence that FQs, with or without BPAA, pharmacologically antagonize GABA_A receptors (1, 12, 28, 29). Furthermore, GABA is known to exert a tonic inhibitory control on hippocampal glutamatergic circuits (20, 23). A sufficient decrease in GABA_A receptor-mediated inhibition can therefore result secondarily in augmented hippocampal excitability. This is reflected in our experiments by enhanced extracellular hippocampal glutamate levels during the norfloxacin-induced seizures and norfloxacin's proconvulsant activity in the presence of BPAA. This might then also explain why some excitatory amino acid receptor antagonists were shown to possess anticonvulsant activity against FQ-induced seizures.

In conclusion, norfloxacin exerts convulsant activity in rats. This activity is accompanied by elevations of extracellular hippocampal glutamate but not GABA levels, even in the presence of BPAA.

 
I. Smolders is a postdoctoral research fellow of the Fund for Scientific Research Flanders (FWO-Vlaanderen, Brussels, Belgium). We are most obliged to the FWO-Vlaanderen, the Vrije Universiteit Brussel, and the Koningin Elisabeth Stichting for financial support.

We appreciate the excellent technical assistance provided by R. Berckmans, G. De Smet, and C. De Rijck.

 
* Corresponding author. Mailing address: Laboratoire de Pharmacie Galénique et de Biopharmacie, Faculté de Médecine et Pharmacie, Université de Poitiers, 34 Rue du Jardin des Plantes, BP 199, 86005 Poitiers Cedex, France. Phone: 33-5-49-45-43-79. Fax: 33-5-49-45-43-78. E-mail: william.couet{at}univ-poitiers.fr.

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Antimicrobial Agents and Chemotherapy, February 2002, p. 471-477, Vol. 46, No. 2
0066-4804/01/$04.00+0     DOI: 10.1128/AAC.46.2.471-477.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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