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

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

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).

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.

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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 C_max (Table 2). The mean AUC_0.5-5h 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 AUC_0.5-5h 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 C_max 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 AUC_0.5-5h/serum AUC_0.5-5h, were 0.28 for the infected rabbits and 0.32 for the uninfected rabbits.

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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.

(ii) Following a 20-mg/kg administration (ATCC 49775 and MRSA 3193).In the group of uninfected rabbits (n = 5), the mean C_max in vitreous was 1.67 ± 0.17 μg/ml, reached 1 h after administration. The mean AUC_0.5-5h 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 AUC_0.5-5h 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 C_max 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 AUC_0.5-5h in the vitreous was 10.75 ± 1.19 μg.h/ml. The C_max in the vitreous was 2.76 ± 0.58 μg/ml within 3 h after injection of MXF. The AUC_0-5h 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 AUC_0.5-5h 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 AUC_0.5-5h in the serum for uninfected and infected rabbits. For the rabbits infected by the MRSA 3193 strain, the AUC_0.5-5h 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).

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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).

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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 = AUC_T>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.

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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 C_max 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.