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Experimental Therapeutics

Moxifloxacin Efficacy and Vitreous Penetration in a Rabbit Model of Staphylococcus aureus Endophthalmitis and Effect on Gene Expression of Leucotoxins and Virulence Regulator Factors

Stéphane Bronner, François Jehl, Jean-Daniel Peter, Marie-Cécile Ploy, Corinne Renault, Pierre Arvis, Henri Monteil, Gilles Prevost
Stéphane Bronner
1Institut de Bactériologie, Faculté de Médecine, Université Louis Pasteur—Hôpitaux Universitaires de Strasbourg, F-67000 Strasbourg
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François Jehl
1Institut de Bactériologie, Faculté de Médecine, Université Louis Pasteur—Hôpitaux Universitaires de Strasbourg, F-67000 Strasbourg
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Jean-Daniel Peter
1Institut de Bactériologie, Faculté de Médecine, Université Louis Pasteur—Hôpitaux Universitaires de Strasbourg, F-67000 Strasbourg
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Marie-Cécile Ploy
2Département de Microbiologie, CHU Dupuytren, F-87042 Limoges Cedex
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Corinne Renault
1Institut de Bactériologie, Faculté de Médecine, Université Louis Pasteur—Hôpitaux Universitaires de Strasbourg, F-67000 Strasbourg
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Pierre Arvis
3Bayer Pharma, F-92807 Puteaux, France
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Henri Monteil
1Institut de Bactériologie, Faculté de Médecine, Université Louis Pasteur—Hôpitaux Universitaires de Strasbourg, F-67000 Strasbourg
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Gilles Prevost
1Institut de Bactériologie, Faculté de Médecine, Université Louis Pasteur—Hôpitaux Universitaires de Strasbourg, F-67000 Strasbourg
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  • For correspondence: gilles.prevost@medecine.u-strasbg.fr
DOI: 10.1128/AAC.47.5.1621-1629.2003
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ABSTRACT

Bacterial endophthalmitis is a serious complication of ocular surgery and of eye trauma; the leading causative organisms are Staphylococcus aureus strains. Tissue damage is due both to the host inflammatory response and to toxin synthesis by bacteria. Systemic treatment remains difficult because most antibiotics show poor ocular penetration. Moxifloxacin (MXF), a novel fluoroquinolone, was evaluated for its penetration into the vitreous of normal rabbit eyes and of eyes of rabbits infected for 24 h with methicillin-susceptible and methicillin-resistant S. aureus (MSSA and MRSA) following a single intravenous administration of 5 or 20 mg/kg. MXF penetration was rapid and efficient regardless of the dose, ranging from 28 to 52%. An inflammatory state of the vitreous significantly increased penetration after the 20-mg/kg dose, with penetration reaching 52%. Concentrations determined in the vitreous cavity following a 20-mg/kg administration showed a 3.5-fold decrease of the bacterial density within 5 h for MSSA (MIC, 0.125 μg/ml) and a 1.6-fold decrease for MRSA (MIC, 4 μg/ml) strains, respectively. By using a semiquantitative reverse transcription-PCR method, the expression of luk-PV and hlgCB, but not hlgA, encoding staphylococcal leukotoxins, was detected in the vitreous without MXF treatment. A slight decrease in the expression of leucotoxins and sarA, agr, and sigB virulence regulatory factors was observed 1 h following the administration of 5 mg of MXF per kg.

Bacterial endophthalmitis is a serious complication of ocular surgery or follows a traumatic injury of the eye. The leading causative organisms are gram-positive pathogens, such as Staphylococcus epidermidis and Staphylococcus aureus (11). A systemic antibiotic treatment remains difficult because of poor ocular penetration through blood-retina barriers and the blood-aqueous humor barrier (13). Intravitreous penetration of most fluoroquinolones is generally better than that of β-lactams, aminoglycosides, or vancomycin (11, 15). Among fluoroquinolones, moxifloxacin (MXF) is active against a wide range of bacteria including gram-positive ones (9, 24, 25). In S. aureus, the primary target of MXF is the topoisoimerase IV (4). However, success in the treatment of bacterial endophthalmitis depends on both the elimination of infecting organisms and the control of the host inflammatory immune response.

Components of gram-positive bacteria such as structural components, cell wall-associated proteins, and exotoxins are able to induce an inflammatory response (18, 22, 27). Alpha-toxin was shown to be a major virulence factor responsible for corneal tissue damage (10, 30, 36). Other staphylococcal pore-forming toxins (e.g., Panton-Valentine leucocidin [PVL] and gamma-hemolysin [HlgA, HlgB, and HlgC]) are able to induce severe inflammation and intraocular damage (36, 42). Moreover, attenuation of bacteria to eliminate the production of the gamma-hemolysin (44) or several toxins by mutations in the two-component signal regulatory system agr (accessory gene regulator) (34) and in the regulatory locus sar (staphylococcal accessory regulator) (12) resulted in significant reduction of the severity of infection (6) and demonstrated that the production of toxins in situ contributes in part to the course of endophthalmitis. We recently demonstrated that the in vitro expression of staphylococcal bicomponent leucotoxins is also reduced in agr- and sar-defective strains (8).

Studies showed that exposure of S. aureus to subinhibitory concentrations of antimicrobial agents (β-lactams and fluoroquinolones) led to activation by ciprofloxacin of the fibronectin-binding gene promoter (5) or alpha-toxin and other cell wall-associated proteins (26, 33, 37). Subinhibitory concentrations of clindamycin stimulated the sar promoters and the stress environmental regulator σB operon (23).

In this study, we evaluated the penetration of MXF into the vitreous of normal and infected nonpigmented rabbit eyes following systemic administration of two doses (5 and 20 mg/kg). Two S. aureus strains were used, the methicillin-susceptible ATCC 49775 strain (MSSA) and a methicillin-resistant strain (MRSA). Using a competitive reverse transcription-PCR (RT-PCR) method (8), we studied the expression of S. aureus genes encoding Panton-Valentine leucocidin (luk-PV) and gamma-hemolysin (hlgA and hlgCB) and virulence regulation factors (agr, sarA, and sigB) after infection and treatment.

(This work was presented in part at the 12th European Congress of Clinical Microbiology and Infectious Diseases, Milan, Italy, 24 to 27 April 2002.)

MATERIALS AND METHODS

Strains.The S. aureus strains used in this study are listed in Table 1.

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TABLE 1.

Bacterial strains and plasmids used in this study

Chemicals.MXF (molecular mass, 437.9 Da) titrated powder (batch 502714, 660434T) was kindly supplied by Bayer (Puteaux, France). Acetonitrile, tetrabutylammonium bromide, and dichloromethane (high-performance liquid chromatography [HPLC] grade) were purchased from Merck (Darmstadt, Germany).

MIC and MBC determination.The MIC of MXF were determined by microdilutions ranging from 0.008 to 256 μg/ml in brain heart infusion broth medium (Difco Becton-Dickinson, Le Pont de Claix, France). The bacterial inoculum, prepared from a 3-h culture at the exponential growth phase, was diluted to obtain 106 to 107 CFU/ml. The MBC, allowing no more than 0.01% survivors after a 24-h exposure, was determined by counting CFU in tubes with no apparent growth.

Time-kill experiments.A 600-μl volume of a 18-h culture of S. aureus ATCC 49775 was inoculated into 40 ml of the yeast-Casamino acids-pyruvate (YCP) medium (20). After 1 h of incubation at 37°C to ensure that the growth phase was obtained, 1.8 ml of bacterial culture was challenged with 200 μl of serial twofold dilutions of MXF from 32- to 0.125-fold the MIC. Bacterial counts were determined after −1, 0, 0.5, 1, 2, 4, 6 and 24 h of antibiotic exposure by plating 100 μl of 10-fold serial dilutions on 5% (vol/vol) blood agar sheep plates. The limit of detection of bacterial counting was 20 CFU/ml.

Animals.Experimental procedures were in accordance with the resolution of the Association for Research in Vision and Ophthalmology on the use of animals. Permission (no. A67482-11) to experiment on animal into a P2-classified animalery was given by the French Ministry of Agriculture and Forest.

Adult male or female New Zealand F2 rabbits weighing from 3.10 to 4.40 kg (3.80 ± 0.30 kg) were used. The animals were divided in two groups; the first group was used to determine the kinetics of MXF (5 and 20 mg/kg) in the serum and its penetration into the vitreous after intravenous injection into infected or uninfected rabbits. A subsequent group was used to semiquantify by RT-PCR the expression of S. aureus virulence factors in a model of ocular infection (44) and was further subjected to antimicrobial treatment. A 22-gauge catheter (0.9 by 25 mm; Becton-Dickinson, Le Pont de Claix, France) was inserted into a marginal ear vein to facilitate drug administration. The animals were anesthetized for about 15 min with an intravenous 10-mg/kg dose of ketamine (Ketalar; Pfizer) covering the sampling period.

Solution injection and sampling.The intravenous bolus of MXF (5 and 20 mg/kg) was administered as a 10-mg/ml solution through the marginal ear vein and was followed by 2 ml of 10-U/ml heparin (heparin Choay; Sanofi Winthrop)-0.9% (wt/vol) NaCl. All samples were collected at 0.5, 1, 2, 3, and 5 h after drug administration. Serum specimens were centrifuged at 2,000 × g for 10 min at 4°C. Serum was stored at −80°C pending analysis. For vitreous samples, a few drops of 0.4% (wt/vol) oxybuprocain chlorohydrate (Ophtadoses; Novartis Ophthalmics, Rueil Malmaison, France) was instilled into the eyes. A 23-gauge needle (0.6 by 25 mm) affixed to a low-dose insulin syringe (1 ml) was carefully inserted into the center of the vitreous cavity, and 75 μl was sampled. Samples were transferred into a 1.5-ml microcentrifuge tube and stored frozen at −80°C pending analysis. For total RNA extraction, 1 ml of vitreous was collected and transferred to a 15-ml tube containing 5 ml of RNA-Later (Ambion, Austin, Tex.). Immediately after the sampling period, the animals were euthanized with intravenous pentobarbital sodium solution (Sanofi).

Intraocular infection.A 18-h culture of S. aureus ATCC 49775 or MRSA 3193 in YCP medium was diluted in 0.9% (wt/vol) NaCl. Intraocular infections were induced by injections of 280 ± 150 CFU of S. aureus ATCC 49775 or 1340 ± 394 CFU of S. aureus 3193 through the pars plana into the center of the vitreous cavity with a 30-gauge needle (0.3 by 13 mm) affixed to a low-dose insulin syringe.

After 24 h of infection, clinical evaluation of the posterior chamber prior to any sampling was performed with a direct ophthalmoscope (Heine), in accordance with the criteria of Nussenblatt et al. (35). Briefly, five increasing levels of severity were defined: 0, normal eye without vitreous haze; 1, vitreous haze allowing observation of the optic nerve and retinal vessels; 2, vitreous haze still allowing observation of major vessels and optic nerve with difficulty; 3, vitreous haze allowing observation of the optic nerve only, its boundaries being blurred; 4, vitreous haze preventing observation of the optic nerve. The clinical observation of the anterior chamber also resulted in five increasing levels of severity of the lesions as previously reported (42): 0, normal eye with no physical damage; 1, slight conjunctival hyperemia located around the site of injection; 2, presence of conjunctival hyperemia involving at least half of the surface and associated with scant discharge but without haze in the anterior chamber; 3, moderate secretions, slight blepharitis, total conjunctival hyperemia involving all the eyeball, and slight haze of the anterior chamber, still allowing observation of the iris; 4, total conjunctival hyperemia, blepharitis, edema, and secretions.

Bacterial counts in the vitreous.A 10-μl aliquot of the 50-μl vitreous sample was serially 10-fold diluted in 90 μl of sterile 0.9% (wt/vol) NaCl. A 50-μl volume of each dilution was plated on a 5% (wt/vol) sheep blood agar plate. The limit of detection of bacterial count was 2 × 102 CFU/ml.

Antibiotic assays. (i) Sample preparation.A 400-μl aliquot of serum was placed into a glass tube with 3.2 ml of dichloromethane. The resulting suspension was subjected to 10-min shake with rotation (20 rpm) prior to a 1-min manual shake. After centrifugation for 10 min, the aqueous phase was eliminated and the organic phase was placed into a new glass tube containing 200 μl of 20 mM orthophosphoric acid solution (H3PO4), pH 2.0. The suspension was shaken manually for 1 min before being centrifuged at 2,000 × g for 10 min at 10°C. The supernatant was directly injected onto the column.

The vitreous samples were briefly centrifuged before being injected onto the column. For the serum, standards were prepared with MXF concentrations of 0.5 and 5 μg/ml for rabbits receiving the 20-mg/kg dose and 0.25 and 2.5 μg/ml for the 5-mg/kg dose. For the vitreous samples, standards were prepared in 20 mM H3PO4 (pH 2.0) with an antibiotic concentration of 1 μg/ml.

(ii) Chromatographic conditions.To measure MXF concentrations, we developed a reversed-phase high-performance liquid chromatography assay (System Gold HPLC, detector model 166; Beckman-Coulter). The samples were run in a high-speed Ultrasphere C18, 75- by 4.6-mm column with 3-μm spherical beads (Beckman-Coulter, Fullerton, Calif.). The 20-μl samples were injected onto the column and monitored with UV detection at 296 nm. The mobile phases consisted of a mixture of acetonitrile (12% for serum samples, 10% for vitreous samples) and 5 mM tetrabutylammonium bromide. The pH was adjusted to 1.8 with concentrated H3PO4. The mobile-phase flow rate was settled at 1.5 ml/min.

The antibiotic concentrations were quantified by using a peak-area integration. Limits of quantification (accuracy of the determination of MXF, >80%) in the serum and in the aqueous phase were 0.02 and 0.01 μg/ml, respectively. The limit of detection, assuming that the concentration of antibiotic resulted in a signal-to-noise ratio of 3, was 0.01 μg/ml in the serum and 0.005 μg/ml in the aqueous phase. The day-to-day coefficient of variation for serum was 6.5% for a concentration of 0.05 μg/ml, 7.0% for a concentration of 0.5 μg/ml, and 8.0% for a concentration of 5 μg/ml. The within-day coefficient of variation was 7.4, 6.6, and 6.7%, respectively.

Pharmacokinetic and statistical analysis.The standard kinetic parameters were determined as described by Gibaldi and Perrier (17). The areas under the vitreous and serum curves (AUC) were determined using the trapezoidal method (Sigma Plot; SPSS Science, Chicago, III.). All results are provided as means ± standard deviations. The paired Student t test was used for statistical analysis of the pharmacokinetic data. P values of ≤0.05 were considered significant. The nonparametric test of Wilcoxon-Mann-Whitney was used for the comparison of the data obtained from clinical observations (42).

Total RNA extraction.Vitreous samples were centrifuged at 4°C in RNA-Later for 10 min at 5,000 × g. RNA was isolated using the FastRNA kit, Blue (Q-BIOgene, Illkirch-Graffenstaden, France), after an orbital stirring centrifugation of twice 25 seconds, with a speed settled to 6.5 in the FastPrep FP120 instrument (Q-BIOgene), as previously described (8).

RT-PCR.To semiquantify the expression of bicomponent leucotoxin-encoding genes and virulence regulatory factors agr, sar, and sigB, competitive RT-PCR procedures were used as previously described (8), and the results are expressed as numbers of specific mRNA copies per CFU. Briefly, total RNA preparation was treated by using the DNA-free kit (Ambion), as specified by the manufacturer, to ensure complete removal of DNA. Then cDNA synthesis was performed with a specific antisense 3′ primer, using Moloney murine leukemia virus M-MuLV reverse transcriptase (Perkin-Elmer, Foster City, Calif.).

A competitive PCR was then performed with a constant amount of cDNA coamplified with a dilution of the corresponding plasmid competitor (Table 1), ranging from 100 ng to 0.01 fg. The PCR amplifications were performed with Taq DNA polymerase (Invitrogen, Life Technologies) in a thermocycler (Perkin-Elmer model 9700). The primers used for the RT and amplification of the leucotoxin mRNAs, luk-PV, hlgA, hlgCB, and the PCR conditions were described previously (8). The hlgC and hlgB genes (hlgCB in the text) are cotranscribed (8).

After cDNA synthesis of the sigB, sarA, and agr transcripts and before the amplification step, the RNA-DNA heteroduplex was denatured for 3.5 min at 94°C. Amplification was achieved by 35 cycles of denaturation for 1.5 min at 92°C, annealing for 1.5 min at 55°C for sarA and 1 and 0.5 min at 60°C for sigB and agr, respectively, and polymerization at 72°C (1.5 min for sarA and sigB, 1 min for agr). Amplification ended with 8 min of incubation at 72°C. The primers used for RT (the second listed for each gene) and amplification were 5′-GAGTTGTTATCAATGGTCACTTAT-3′ and 5′-GCTTCAGTGATTCATTTATTTAC-3′ for sarA (SAU46541, nucleotides [nt] 896 to 1218), 5′-CTAATTGAGTCATTGGCATATAAAT-3′ and 5′-TGACTTAAACCGATACGCTCACCT-3′ for sigB (Y09929, nt 2810 to 3393), and 5′-GGTCTCATAATGATGATTAACTCA-3′ and 5′-AAGGAAGGAGTGATTTCAATGGCA-3′ for RNAIII (agr X52543, nt 1014 to 1504).

Some authors reported the use of RT-PCR of gyrA expression as an internal standard (19). However, this gene was constantly expressed at 1 mRNA/1,000 CFU, which corresponds to the detection limit of our test, which was influenced in part by the necessary dilution of total RNA due to the presence of leukocyte RNAs in the infected vitreous and the necessary dilution of cDNA used in PCR (1/200 of total RNA preparation). These constraints made the use of an internal standard inefficient. Nevertheless, the test was previously evaluated for its reproducibility and sensitivity (8).

RESULTS

In vitro susceptibility.The MIC and MBC of MXF were 0.125 and 0.250 μg/ml, respectively, for S. aureus strain ATCC 49775 and 4 and 32 μg/ml, respectively, for the MRSA strain 3193.

Time-kill experiments.Figure 1 shows the time-kill curves of MXF for strain S. aureus ATCC 49775 cultured in YCP medium. The time-kill rates were dependent on concentrations, and the bacterial counts decreased faster for high concentrations (>2× MIC). The bactericidal reduction was 0.8 log unit after a 6-h exposure at 2× MIC (0.25 μg/ml), corresponding to the MBC. A 1-log-unit regrowth was obtained between 6 and 24 h of exposure at 1× MIC (0.125 μg/ml). No bactericidal activity was obtained with MXF concentrations lower than the MIC.

FIG. 1.
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FIG. 1.

In vitro time-kill curves for S. aureus ATCC 49775 exposed to different concentrations of MXF (1:8 to 32 MIC).

Intraocular infection.Experimental infections with viable bacteria were initiated by injection of 250 ± 150 CFU of S. aureus ATCC 49775 and 1,340 ± 394 CFU of S. aureus 3193.

At 24 h after infection with the ATCC 49775 MSSA strain, clinical modifications in both the posterior and anterior eye chambers of rabbits were recorded. In the posterior chamber, only the optic nerve could be still observed (level 3 by Nussenblatt's criteria). The anterior chamber showed 75 to 100% conjunctival hyperemia around the cornea, moderate secretions and slight blepharitis, and moderate haze where details of the iris remained observable.

After infection with MRSA strain 3193 (24 h), the optic nerve was not observable (level 4 by Nussenblatt's criteria). In the anterior chamber, the haze did not allow iris details to be seen; conjunctival hyperemia, blepharitis, edema, and secretions were more acute even though the bacterial counts remained similar to those for the ATCC 49775 infections. These differences from the results of infection with ATCC 49775 after 24 h were significant (P < 0.005) according to the Wilcoxon-Mann-Whitney test (42).

Ocular kinetics of MXF and intravitreal penetrations. (i) Following a 5-mg/kg administration (ATCC 49775).In the vitreous, the variations in MXF concentrations obtained between 1 and 3 h following administration were very weak and the concentrations remained close to the Cmax (Table 2). The mean AUC0.5-5h was 5.40 ± 3.26 μg.h/ml in the serum for uninfected rabbits (n = 5) and 1.73 ± 0.37 μg.h/ml in the vitreous (Fig. 2A). The mean vitreous peak level was 0.45 ± 0.18 μg/ml within 3 h following drug administration. For the infected rabbits (n = 6), the mean AUC0.5-5h was 8.55 ± 3.72 μg.h/ml in the serum and 2.38 ± 0.74 μg.h/ml in the vitreous (Fig. 2B). The Cmax in the vitreous was 0.68 ± 0.28 μg/ml at 2 h following administration. The ratio between the concentrations in the vitreous and in the serum increased during the time following administration, to reach 94.3% at 5 h. Penetration ratios, determined by vitreous AUC0.5-5h/serum AUC0.5-5h, were 0.28 for the infected rabbits and 0.32 for the uninfected rabbits.

FIG. 2.
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FIG. 2.

Mean concentrations of MXF in the serum and vitreous following a single intravenous administration of 5 mg/kg (A and B) or 20 mg/kg (C and D). (A and C) Rabbits with uninfected eyes. (B and D) Rabbits with eyes infected by S. aureus ATCC 49775. Error bars indicate standard deviation.

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TABLE 2.

MXF concentrations in serum and vitreous following intravenous administration of 5 or 20 mg/kg and kinetic parameters

(ii) Following a 20-mg/kg administration (ATCC 49775 and MRSA 3193).In the group of uninfected rabbits (n = 5), the mean Cmax in vitreous was 1.67 ± 0.17 μg/ml, reached 1 h after administration. The mean AUC0.5-5h was 19.43 ± 11.16 μg.h/ml in the serum and 6.64 ± 0.76 μg.h/ml in the vitreous (Fig. 2C). The penetration ratio was 0.34.

In the subsequent group (n = 8) with eyes infected by the ATCC 49775 strain, the mean AUC0.5-5h was 19.62 ± 10.99 μg.h/ml in the serum and 9.12 ± 1.81 μg.h/ml in the vitreous (Fig. 2D). The Cmax in the vitreous was 2.50 ± 0.67 μg/ml within 2 h after injection. The penetration ratio was 0.46.

For the rabbits infected by the MRSA 3193 strain (n = 6), the mean AUC0.5-5h in the vitreous was 10.75 ± 1.19 μg.h/ml. The Cmax in the vitreous was 2.76 ± 0.58 μg/ml within 3 h after injection of MXF. The AUC0-5h in the serum was 20.52 ± 6.31 μg/ml, which led to a penetration ratio of 0.52.

Comparison of MXF penetration into the vitreous of uninfected and infected rabbits.In the uninfected rabbits, the MXF penetration into the vitreous was high and did not vary much with the dose. For the 20-mg/kg dose, the ratios of MXF penetration into the vitreous were 0.34 in the uninfected group and 0.46 in the group infected by S. aureus ATCC 49775. For the 20-mg/kg dose, the penetration of MXF, compared with the AUC0.5-5h in the vitreous of all rabbits in each group, was significantly better (P < 0.02) in the infected eyes than in the uninfected eyes. In the same way, no significant differences were found between the AUC0.5-5h in the serum for uninfected and infected rabbits. For the rabbits infected by the MRSA 3193 strain, the AUC0.5-5h in the vitreous was also significantly increased (P < 0.001) compared to the uninfected rabbits. However, no significant difference in the vitreous penetration was found between the uninfected and infected rabbits (ATCC 49775) given the 5-mg/kg dose (P > 0.1).

Bactericidal activity of MXF against S. aureus ATCC 49755 in eyes.Concurrently with the kinetics of MXF penetration, viable bacteria were counted in the vitreous after 24 h of infection and before the administration of MXF (T = 0 h), and at 0.5, 1, 2, 3, and 5 h. After 24 h of infection, the bacterial counts were 6.42 ± 0.49 log10 CFU/ml before the administration of the 5-mg/kg dose and 5.20 ± 0.25 log10 CFU/ml before the administration of the 20-mg/kg dose. A 3.5-log-unit decrease of viable bacteria was obtained with the 20-mg/kg dose within 5 h (Fig. 3B), and a 1.5-log decrease was obtained with the 5-mg/kg dose in the same period (Fig. 3A).

FIG. 3.
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FIG. 3.

(A) Mean bacterial count of S. aureus ATCC 49775 in the vitreous after administration of a 5-mg/kg dose of MXF. (B) Mean bacterial count of S. aureus ATCC 49775 in vitreous after administration of a 20-mg/kg dose of MXF. Error bars indicate standard deviation.

Bactericidal activity of MXF against S. aureus MRSA 3193 in eyes.After 24 h of infection, the mean concentration of bacteria in the vitreous was 6.36 ± 1.20 log10 CFU/ml before the administration of the 20-mg/kg dose of MXF. At 5 h after administration, the mean concentration of bacteria was 4.71 ± 2.06 log10 CFU/ml, corresponding to a 1.6-log decrease (Fig. 4).

FIG. 4.
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FIG. 4.

Mean bacterial count of MRSA 3193 in the vitreous after administration of a 20-mg/kg dose of MXF. Error bars indicate standard deviation.

Pharmacodynamics.MXF, like other fluoroquinolones, has a concentration-dependent bactericidal activity, and the area above the inhibitory curve in the serum (AUIC = AUCT>MIC/MIC) is therefore the predictive parameter of antibiotic efficiency (7). For S. aureus ATCC 49775 (MFX MIC = 0.125 μg/ml), the AUIC in the serum determined from 0.5 to 5 h was 152.48 μgml−1h−1 at the 20-mg/kg dose and 63.92 μgml−1h−1 at the 5-mg/kg dose. When determined in the vitreous, the AUIC reached 68.48 and 14.56 μgml−1h−1, respectively.

Semiquantification of pvl, hlgA, hlgCB, sarA, agr, and sigB gene expression by RT-PCR.A number of genes related to the virulence of S. aureus were checked for their expression by using a competitive RT-PCR test. In this approach, samples were considered only when obtained at a time when the MXF concentration was critical but when bacteria were still not affected by a strong bactericidal effect. Thus, a 5-mg/kg dose of MXF was chosen and the vitreous was sampled 1 h after MXF injection.

(i) Without antibiotic administration.A group of four untreated rabbits was used as control. The mean volume of vitreous collected at 24 + 1 h of infection was 0.92 ± 0.04 ml. The mean bacterial count for the ATCC 49775 strain was 6.32 ± 0.17 log10 CFU/ml. Results were expressed as the number of specific mRNA molecules with respect to the number of CFU measured in the vitreous cavity. The expression of hlgA was under the limit of detection (<1 mRNA/1,000 CFU), whatever the animal considered. The expression of luk-PV mRNA, resulting from the cotranscribed lukF-PV and lukS-PV genes that encode the PVL (8), was 22.2 ± 19.1 mRNA/100 CFU (Fig. 5). Concomitantly, the expression of hlgCB was 4 ± 2 mRNA/100 CFU. To evaluate the potential influence of the regulatory factors, the expression of sarA, agr (RNAIII), and sigB was semiquantified. The number sarA mRNAs was 290 ± 200 mRNA/100 CFU, while RT-PCR of the RNAIII showed that less than 5 ± 4 mRNA/1,000 CFU remained. The expression of the transcriptional factor sigB was assessed at 40 ± 20 mRNA/100 CFU.

FIG. 5.
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FIG. 5.

Influence of MXF on the expression of luk-PV, hlgCB, sarA, agr (RNAIII), and sigB by S. aureus ATCC 49775 in a rabbit endophthalmitis model, 1 h after intravenous administration of 5 mg/kg.

(ii) Following administration of 5 mg/kg.A mean volume of 0.88 ± 0.15 ml of vitreous (n = 4) was collected 1 h after the administration of MXF (5 mg/kg) and before the Cmax was reached. This elapsed time was chosen to detect the potential effect of MXF on virulence factor expression within the first period of antibiotic and bacterial contact. At 1 h, the mean concentration of MXF was 0.496 ± 0.134 μg/ml, corresponding to 4× the MIC of S. aureus ATCC 49775 strain, and 2× the MBC, and the mean bacterial count was 6.21 ± 0.49 log10 CFU/ml. Thus, comparison between the bacterial density in the vitreous before administration and after 1 h indicated no significant bactericidal activity. The concomitant expression of luk-PV determined by semiquantitative RT-PCR decreased to 3 ± 2 mRNA/100 CFU. The expression for hlgCB and sarA was 6 ± 4 mRNA/1,000 CFU and 13 ± 6 mRNA/100 CFU, respectively. The expressions of hlgA, sigB, and RNAIII were less than 1 mRNA/1,000 CFU.

(iii) Following administration of 20 mg/kg.A mean volume of 0.95 ± 0.05 ml (n = 4) was collected 1 h after the administration of MXF and corresponded to a bacterial density of 4.5 ± 1.3 log10 CFU/ml. At this time, the mean concentration of MXF determined by HPLC was 2.04 ± 0.17 μg/ml. The expressions of hlgA, sigB, sarA, and RNAIII were lower than the limit of detection of the RT-PCR test (<1 mRNA/1,000 CFU). The expressions of hlgCB and luk-PV were detected only in one assay, with 5 and 15 mRNA/1,000 CFU, respectively. This may be due to the low bacterial densities that resulted in the significant bactericidal activity in the vitreous cavities after the administration of 20 mg of MXF per ml and to the necessary dilutions for the RT-PCR test.

DISCUSSION

Previous studies showed that MXF efficiently penetrates into tissues (32, 45), and cerebrospinal fluid (CSF) (38). It also has an excellent penetration into the human aqueous humor, reaching levels of 2.33 ± 0.85 μg/ml 10 h after a single oral 400-mg dose (16). Our study revealed a good and rapid penetration of MXF into the vitreous of rabbits after a single intravenous administration of 5 or 20 mg/kg. The penetration ratios of MXF into the uninfected rabbit vitreous were close to those of ofloxacin (30%) (39). These results are in accordance with the relationship between lipophilicity, low molecular weight, and the ability of fluoroquinolones to cross the blood-ocular barrier and to penetrate into the vitreous, as shown by Liu et al. (28). Other parameters are known to influence ocular penetration: protein binding in serum and the molecular weight of the antibiotic. The protein binding of MXF in the rabbit serum was 24% (38) versus 34% for ofloxacin. As previously shown (1), ocular inflammation increased the penetration of fluoroquinolones and other drugs into the vitreous. In this study, we demonstrated a significant increase of MXF penetration into the vitreous infected by S. aureus ATCC 49775 for the 20-mg/kg dose (P < 0.02) but not for the 5-mg/kg dose (P > 0.1). However, for both these doses, the Cmax in the infected eyes was 1.5-fold higher than in the uninfected eyes. In the uninfected groups, the AUC0.5-5h in the serum and in the vitreous was fourfold higher for the 20-mg/kg dose than for the 5-mg/kg dose. However, for the infected rabbits, the AUC0.5-5h in the vitreous was fourfold higher for the 20-mg/kg dose than for the 5-mg/kg dose, whereas for the serum AUC0.5-5h, this difference was only twofold. Consequently, the penetration ratio for the 5-mg/kg dose (0.28) was probably underestimated. The intensity of inflammation, which may be linked to the importance of haze due to the presence of leukocytes, may influence the ocular penetration of MXF. This was suggested by the increased penetration of MXF (AUC0.5-5h = 10.75 ± 1.19 μg.h/ml) in the case of infection with the MRSA 3193 strain compared to that of infection with ATCC 49775 (AUC0.5-5h = 9.12 ± 1.81 μg · h/ml). Effectively, infection with the MRSA strain generated a significantly greater haze of the vitreous. Previous studies suggested the existence of a partial active efflux system of quinolones across the retinal cell barrier and across the blood-brain barriers (28, 38). In the vitreous, the MXF concentrations were relatively steady from 1 h to 5 h following administration. At 24 h, the elimination of MXF from the vitreous was important, and the concentrations were highly reduced. Østergaard et al. determined a higher Cmax in infected rabbit CSF than in uninfected CSF for the 10-mg/kg dose but not for the 20- or 40-mg/kg doses (38).

The MIC and MBC for S. aureus ATCC 49775 were 0.125 and 0.25 μg/ml, respectively. For the 20-mg/kg dose, a 3.5-log-unit decrease of bacterial counts (ATCC 49775) was observed in the vitreous after a short exposure (5 h) to MXF. Additional administrations of MXF should be required to eradicate bacteria to the vitreous. For the MRSA 3193 strain, a single treatment with MXF reached concentrations in the vitreous that were not effective for a significant bactericidal activity.

The recommended management of bacterial endophthalmitis includes direct injection of antibiotics into the vitreous. However, the high sensitivity to drug administration of the retinal cells directly adjacent to the vitreous restricts some direct intraocular administrations (11). When fluoroquinolones were used at therapeutic doses, administered systemically and topically, retinal toxicity did not occur (43). The most common antibiotics used in direct administration for endophthalmitis treatment are vancomycin, amikacin, and ceftazidime (11). Systemic administration of antibiotics with efficient intraocular penetration, e.g., fluoroquinolones, is be preferable and may reduce the doses of directly administrated antibiotics required. Fluoroquinolones have become widely used as antibacterial agents in the treatment of ocular infections, with topical, intravitreal, and systemic routes of administration being used (11).

The penetration of MXF into the human vitreous should be good, at least as good as the penetration into uninfected human aqueous humor (16). However, the protein binding of MXF in serum is higher in humans (54%) than in rabbits (24%) and may slightly reduce its ocular penetration. For ciprofloxacin, penetration into the human eye, which has more retinal vessels than does the rabbit eye, was demonstrated to be higher than penetration into the rabbit vitreous (31). Elimination of MXF is more rapid in rabbits than in humans because of the increased metabolism. This results in an elimination half-life of MXF in human serum that is 5- to 10-fold longer than that in rabbit serum (12 to 15 h versus 1 to 2 h) (38). Following administration of a 20-mg/kg dose in rabbits, the AUCs in the serum were close to that in humans given the usual doses (7 mg/kg). Consequently, kinetic studies of penetration into the vitreous in humans must be performed with conventional doses (7 mg/kg orally or intravenously over 60 min), since the Cmax in serum is lower but the half-life in serum is longer in humans, resulting in close AUCs.

Direct bacterial damage and the release of cytokines at the inflammatory site are responsible for the tissue damage associated with bacterial infections. Leuckotoxins are composed of class S (LukS-PV, HlgA, and HlgC) and class F (LukF-PV and HlgB) components, which act synergistically. The intravitreal injection into the rabbit eye with the hypothetical combinations of HlgA + LukF-PV and HlgA + HlgB led to a highest inflammatory intensity among all combinations of classes S and F (42). At the same dose, alpha-toxin was less inflammatory. Strinkingly, in vivo, only the expressions of luk-PV and hlgCB were detected by RT-PCR in this model. These expressions occurred at bacterial densities 2 or 3 log units lower than in culture media (8). As in vitro, hlgA expression was lower than for other leukotoxins and was not significant in the vitreous cavity. For regulatory factors in vivo, the expression of RNAIII was dramatically reduced. This was in accordance with other studies that showed an inactivation of RNAIII in vivo (19). SarA expression levels were close to the in vitro expression in the YCP medium, where bacterial densities were at least 2 log units greater. SarA regulates the transcription of virulence factors in two ways, by up-regulating the agr locus (agr-dependent pathway) or by an agr-independent pathway. In the latter way, SarA is able to bind promoters of the target virulence genes and then up-regulates these transcriptions (14). The environmental conditions in the vitreous cavity probably modulate the expression of the virulence-associated regulatory factors and consequently the expression of toxins. In vivo, the influence of agr (RNAIII) on toxin expression remains unclear, and other regulatory circuits (e.g., sae) may act rather than those observed in vitro (19). The presence of working concentrations of the agr autoinducible peptide in vivo remains questionable. Leukotoxins expression by S. aureus ATCC 49775 was slightly reduced in the vitreous in the presence of 4× the MIC of MXF compared with that in the untreated rabbit. The MXF activity on these expressions may be beneficial and may contribute to a reduction of intraocular damage by toxins. Similarly, the expression of the virulence regulatory factors seemed to be reduced following MXF administration, which may contribute to the attenuation of the virulence. It is reasonable to think that expression of other toxins, which are regulated by these factors, was also reduced by MXF. No significant expression of leukotoxins or regulatory factors was detected in the vitreous 1 h after the administration of the 20-mg/kg dose, either because of the low bacterial density or because of a potential reduction of the expression. Worlitzsch et al. (46) reported a decrease of total hemolytic activity by S. aureus in infected rat pouches treated with MXF.

Unlike the β-lactams, quinolones do not directly affect cell wall synthesis and thus reduce the release of proinflammatory cell wall products during antibiotic killing. A possible immunomodulatory effect of MXF was previously reported in a rabbit model of meningititis (41). Effectively, MXF has an immunomodulatory effect through its capacity to inhibit the secretion of interleukin-1α and tumor necrosis factor alpha by human monocytes (2). Tumor necrosis factor alpha activates NF-κB and provokes an intraocular inflammatory reaction when injected intravitreally (11).

In conclusion, with an efficient and rapid intraocular penetration, an immunomodulatory effect, and a decrease of the toxin expression in S. aureus, systemic MXF might be of interest as a therapeutic agent in cases of bacterial endophthalmitis. Moreover, MXF has better antibacterial activity than most of the other fluoroquinolones against bacteria potentially involved in endophthalmitis (29). The concentrations reached in the vitreous are above the MIC for 90% of the populations these organisms (16). Additional investigations of the possible use of MFX in bacterial endophthalmitis is warranted.

ACKNOWLEDGMENTS

We thank Bayer Pharma France for financial support. This work was also supported by grant EA 3432 from the French “Direction de la Recherche et des Etudes Doctorales” (DRED).

FOOTNOTES

    • Received 20 May 2002.
    • Returned for modification 17 November 2002.
    • Accepted 4 February 2003.
  • Copyright © 2003 American Society for Microbiology

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Moxifloxacin Efficacy and Vitreous Penetration in a Rabbit Model of Staphylococcus aureus Endophthalmitis and Effect on Gene Expression of Leucotoxins and Virulence Regulator Factors
Stéphane Bronner, François Jehl, Jean-Daniel Peter, Marie-Cécile Ploy, Corinne Renault, Pierre Arvis, Henri Monteil, Gilles Prevost
Antimicrobial Agents and Chemotherapy May 2003, 47 (5) 1621-1629; DOI: 10.1128/AAC.47.5.1621-1629.2003

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Moxifloxacin Efficacy and Vitreous Penetration in a Rabbit Model of Staphylococcus aureus Endophthalmitis and Effect on Gene Expression of Leucotoxins and Virulence Regulator Factors
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Moxifloxacin Efficacy and Vitreous Penetration in a Rabbit Model of Staphylococcus aureus Endophthalmitis and Effect on Gene Expression of Leucotoxins and Virulence Regulator Factors
Stéphane Bronner, François Jehl, Jean-Daniel Peter, Marie-Cécile Ploy, Corinne Renault, Pierre Arvis, Henri Monteil, Gilles Prevost
Antimicrobial Agents and Chemotherapy May 2003, 47 (5) 1621-1629; DOI: 10.1128/AAC.47.5.1621-1629.2003
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  • Article
    • ABSTRACT
    • MATERIALS AND METHODS
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KEYWORDS

anti-infective agents
Aza Compounds
endophthalmitis
Exotoxins
fluoroquinolones
quinolines
staphylococcal infections

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