Contribution of anti-inflammatory and anti-virulence effects of azithromycin in the treatment of experimental Staphylococcus aureus keratitis

Azithromycin at concentrations of 0.01, 0.1, and 1% along with its vehicle was prepared for this study by Senju Pharmaceutical Co., Ltd. (Osaka, Japan). These formulations all contained DuraSite®, which is a proprietary polymeric mucoadhesive delivery system, prepared by Senju Pharmaceutical Co., Ltd. An ophthalmic corticosteroid, betamethasone sodium phosphate 0.1% (Rinderon® ophthalmic, otic, and nasal solution 0.1%), was purchased from Shionogi & Co., Ltd. (Osaka, Japan). Saline (Otsuka normal saline) was purchased from Otsuka Pharmaceutical Factory, Inc. (Tokushima, Japan).

Ten milligrams of azithromycin dihydrate, which was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) for in vitro assays, was dissolved in 1 mL of ethanol. That solution was then filtered and diluted with bacterial growth medium. Preserved rabbit blood was purchased from Kojin Bio Co., Ltd. (Tokyo, Japan).

Seventy-three male Japanese white rabbits weighing 1.3–1.9 kg were supplied by Kitayama Labes Co., Ltd. (Nagano, Japan). All procedures were performed in accordance with the statement of the Association for Research in Vision and Ophthalmology and the guidelines for animal experimentation of Senju Pharmaceutical Co., Ltd., and the protocol was approved by the Institutional Animal Care and Use Committee (approval nos. 20,140,903–01, and 20,160,310–01).

Staphylococcus aureus ATCC 25923 supplied by the American Type Culture Collection (Manassas, VA) was used in this study. The minimal inhibitory concentrations of AZM was reported to be 2 μg/mL for this strain, and it was defined as AZM-susceptible based on the Clinical Laboratory Standards Institute breakpoint (M100-S18) [16]. Furthermore, it has been reported that this strain can induce keratitis in rabbits [17, 18]. Staphylococcus aureus was grown at 32 °C on soybean casein digest (SCD) agar (Nihon Pharmaceutical Co., Ltd., Tokyo, Japan) or tryptic soy broth (TSB) (Becton, Dickinson and Company, Franklin Lakes, NJ).

Forty-one rabbits were anesthetized by intramuscular injection of a 3:1 mixture of 50 mg/mL ketamine (Ketalar® for intramuscular injection 500 mg; Daiichi-Sankyo Propharma Co., Ltd., Tokyo, Japan) and 20 mg/mL xylazine (Celactar® 2% injection; Bayer Yakuhin, Ltd., Tokyo, Japan). Staphylococcus aureus cells were suspended with saline. The viable bacteria were quantified using the agar dilution plate method (4.7 × 10⁶ colony-forming units (CFU) per milliliter). A 30 μL of bacterial suspension was injected into the corneal stroma of right eye. The left eye was not used for throughout the experiment. To avoid that the shortage of the number of viable bacteria limited the assessable dose-response range, the number was set higher than those of the previous reports. Oxybuprocaine hydrochloride ophthalmic solution (Benoxil® ophthalmic solution 0.4%; Santen Pharmaceutical Co., Ltd., Osaka, Japan) was topically applied prior to inoculation. Slit-lamp examination (SLE) was performed at 5, 12, 24, 36, and 48 h after inoculation, and the clinical severity of ocular discharge, hypopyon, and infection signs in the cornea, conjunctiva, nictitating membrane, and iris were scored according to a previously reported standard scale of ocular infection [19]. A masked observer performed all SLE scoring. Eleven rabbits were excluded due to leakage of inoculated suspension from corneal stroma immediately after administration. Thirty rabbits that were successfully inoculation were selected and randomly divided into four groups (n = 7–8). Azithromycin at concentrations of 0.01, 0.1, and 1% or its vehicle was instilled into the right eye (50 μL/eye, twice daily for 2 days). The topical treatment was started 5 h post inoculation when animals presented sign of ocular infection. The rabbits were euthanized by an intravenous overdose of sodium pentobarbital after SLE at 48 h. The corneas were removed, finely minced, serially diluted with sterile saline, and plated onto mannitol salt agar plates for quantification of viable bacteria in the cornea.

Bacterial cells were grown in TSB with or without (control group) AZM at concentrations of 0.008–8 μg/mL for approximately 16 h under agitation. After measuring the optical density at 660 nm to evaluate the growth of bacterial cells in the culture solution, bacterial culture supernatant was collected by centrifugation at 1610×g for 2 min and the residual bacterial cells were removed using a 0.22-μm filter. The bacterial supernatant was incubated with equal volume of 1% rabbit red blood cells suspended in saline for 10 min at 37 °C. The reaction mixture was centrifuged at 1000×g for 10 min to remove the intact red blood cells, and the supernatant was transferred into a 96-well microplate. Hemolysis was detected by measuring the absorbance at 440 nm as an indicator of hemoglobin release into the supernatant. The absorbance of the control group was defined as 100% and the bacterial number and the hemolysis rate of each group were calculated.

Inoculation of S. aureus into the corneal stroma in rabbits induced clinical signs mainly in the cornea, conjunctiva, nictitating membrane, and iris. In the vehicle group, the SLE score was increased at 5 h after inoculation and reached the maximum at 36 h after inoculation (Fig. 1, Table 1). Azithromycin decreased the SLE scores in a dose-dependent manner. The SLE score in the 0.01 and 0.1% AZM groups reached the maximum at 24 and 36 h, respectively, and they were significantly lower than that of the vehicle group at 36 h after inoculation (p < 0.05; Table 1). The SLE score of the 1% AZM group reached the maximum at 36 h after inoculation, and the scores were significantly lower than those of the vehicle group from 12 to 48 h after inoculation. Treatment with 1% AZM significantly decreased the number of viable bacteria per cornea compared with that of the vehicle (p < 0.05), although the numbers of viable bacteria per cornea in the 0.01 and 0.1% AZM groups were similar to that of the vehicle group (Table 2).

Administration of SaS induced the clinical signs hyperemia and edema in the conjunctiva, redness and edema in the nictitating membrane, hyperemia in the iris, and ocular discharge (Fig. 2). In the saline group, the SLE score was increased at 3 h after induction and reached the maximum at 12 h after induction (Fig. 3, Table 3). The SLE scores of the AZM groups were lower than those of the saline group, and treatment with 1% AZM significantly reduced the SLE score at 12 h (p = 0.0113). The SLE score of the 0.1% betamethasone sodium phosphate group was significantly lower than that of the saline group at 6 and 12 h (p = 0.0085 and 0.0037, respectively). The SLE score of the 1% AZM group at 12 h was similar to that of the 0.1% betamethasone sodium phosphate group.

Bacterial growth was significantly inhibited by AZM at or above 0.5 μg/mL concentration (Fig. 4). Hemolytic activity was significantly inhibited by AZM at or above 0.125 μg/mL concentration.