Dexamethasone Improves Ofloxacin Efficacy in Treating Acute Bacterial Conjunctivitis: Evidence from A Rabbit Model

Bacterial conjunctivitis represents the major part of infectious
conjunctivitis and, although it generally shows a spontaneous resolution, antibiotics
speed the elimination of bacteria from the conjunctiva thus limiting the duration of the
disease. The addition of anti-inflammatory drugs may potentiate the effectiveness of
antibiotics in eradicating bacterial conjunctivitis. Aim of the present study is to compare
the efficacy of two different eye drops based on ofloxacin without or with dexamethasone
against bacterial conjunctivitis induced by Staphylococcus aureus or Pseudomonas
aeruginosa.

for acute forms of the disease [3]. Patients developing conjunctivitis also develop an inflammatory status leading to the production of high levels of inflammatory cytokines in tears, including interleukin (IL)-6, and Transforming Growth Factor (TGF)-ß [4][5][6]. Generally, acute bacterial conjunctivitis is a self-limiting disease, with spontaneous resolution within 7-10 days after infection, although in some cases the disease may go on to become chronic [7]. Therapies using antibiotics have been shown to limit the duration of the disease and to speed the elimination of bacteria from the conjunctiva [3]. Presently, the treatments for bacterial infections of the ocular surface, including conjunctivitis, are largely empirical. Antibiotics such as fluoroquinolones because of their broad-spectrum antibacterial activity, documented safety and lack of toxicity are well suited to treat bacterial conjunctivitis [8]. Among fluoroquinolones, ofloxacin, a second-generation fluoroquinolone, is used as eye drops in the treatment of this disease, although additional fluoroquinolones of third or fourth generation with higher activity in respect to ofloxacin have been introduced in the market [9]. Combining antibiotics with additional drugs counteracting inflammatory processes associated to bacterial infection may be useful to potentiate the treatment. In this respect, corticosteroids, which are already extensively used against inflammatory ocular diseases, show the potential to treat acute conjunctivitis in combination with approved therapies, and formulations of antibiotics with dexamethasone are in clinical use or under clinical investigation [10]. In the present study, we compared the efficacy of two different eye drops based on ofloxacin without or with dexamethasone against bacterial conjunctivitis induced by S. aureus or P. aeruginosa in a rabbit model. In particular, a qualitative evaluation of the infection progression was performed on photograph images of the rabbit eyes taken at different time points after infection, while semi-quantitative assessment was made using a previously reported clinical score [11]. Whether the addition of dexamethasone may potentiate the anti-inflammatory activity of ofloxacin was also investigated by evaluating IL-6 and TGF-ß levels in tears.

Strains
S. aureus strain ATCC 6538, isolated from a human lesion, and P. aeruginosa strain ATCC 9027, isolated from outer ear infection, both ofloxacin sensitive, were obtained by ATCC (Manassas, VA, USA).
Strains were stored at -80°C in Luria Bertani (LB) broth (Fisher Scientific, Rodano, Milan, Italy) supplemented with 40% glycerol (v/v), and sub-cultured on LB agar (Fisher Scientific, Rodano, Milan, Italy) at 37°C when necessary. Bacterial pre-inoculum was prepared in 5 mL of fresh LB broth, by picking a single colony which was incubated at 37°C under agitation at 220 rpm overnight. Next day, overnight bacterial culture was diluted in fresh LB broth and then grown for 3 hours to reach the exponential phase.

Experimental Conjunctivitis
New Zealand white rabbits (n = 18) were purchased from Azienda Agricola Pampaloni (Fauglia, Italy). All animals were

Clinical Score
A semi-quantitative evaluation of the infection progression was performed by using a previously published clinical score with minor modifications [11]. Briefly, 2 parameters, hyperemia and the presence of purulence, were considered. For each parameter, a grading scale ranging from 0 to 3 (0, absence of symptom; 1, detectable; 2, intermediate; 3, maximal) was used. The sum of average scores for each eye at each time point was plotted to generate the clinical index.

Enzyme-Linked Immunosorbent Assays
Quantification of IL-6 and TNF-β protein levels was performed

Statistical Analysis
Statistical significance was evaluated with Prism 8.02

Effects of Ofloxacin on Bacterial Conjunctivitis
The effectiveness of two different ofloxacin-based eye drops, formulated without or with dexamethasone, was investigated in rabbit models of bacterial conjunctivitis induced by either S. aureus or P. aeruginosa. As shown in Figure 1, eye infection could be observed 12 hours after bacteria inoculation. Twenty-four hours after bacteria inoculation, rabbits were treated with sterile saline or the antibiotic in the absence or in the presence of dexamathasone. In

Effects of Ofloxacin on Inflammatory Cytokines
At the end of the treatment with oxofloxacin without or with dexamethasone, tears were collected and used to evaluate protein

Discussion
Whether the addition of corticosteroids to fluoroquinolones may have a beneficial role in treating bacterial conjunctivitis is a matter of debate, although formulations of antibiotics with dexamethasone are in clinical use or under clinical investigation [10]. As shown by the present findings, both S. aureus and P. aeruginosas induce an acute form of bacterial conjunctivitis in the rabbit characterized by hyperemia and purulence that spontaneously regresses. This is in line with previous findings demonstrating that almost 60% of affected people show a spontaneous regression of acute bacterial conjunctivitis within 1-2 weeks [12,13]. The conjunctiva has host defence capability mediated by goblet cells, which may counteract bacterial infections [14]. For instance, the injection of S. aureus in the rabbit conjunctiva fails to produce significant conjunctivitis due DOI: 10.26717/BJSTR.2020. 26.004289 to the activation of host defences [15]. On the other hand, compared to placebo, topical application of broad-spectrum antibiotics as eye drops improves the rate of clinical remission in patients suffering from acute bacterial conjunctivitis [16]. In particular, ofloxacin is antibiotics [18]. In addition to their antimicrobial properties, fluoroquinolones exert indeed a predominant anti-inflammatory activity [19]. For instance, besifloxacin has been shown to inhibit the production of several cytokines in human corneal epithelial cells after inflammatory damage with IL-1ß [20]. In addition, in a rat model of conjunctivitis, gatifloxacin used as ophthalmic solution significantly reduces the production of tumor necrosis factor α [21]. The present results demonstrate for the first time that the addition of 0.1% dexamethasone to 0.3% ofloxacin accelerates the resolution of conjunctiva infection indicating that corticosteroids may potentiate the antibacterial properties of fluoroquinolones.
This finding is in line with the assumption that corticosteroids are widely used in the treatment of inflammatory ocular diseases [22] thus supporting their use in the treatment of bacterial conjunctivitis.
In this respect, the American Academy of Ophthalmology guidelines on treatment of conjunctivitis include corticosteroids as a treatment option, also in association with additional drugs [23]. For instance, the association of the aminoglycoside antibiotic tobramycin with 0.1% dexamethasone may be used when a risk of bacterial ocular infection exists, while the association of the antiseptic povidoneiodine with 0.1% dexamethasone is under investigation for the treatment of inflammatory conditions associated with ocular infections, including conjunctivitis [10].
Among cytokines produced by immune resident cells and goblet cells in the conjunctiva and collected in tears, IL-6 levels are associated with several diseases of the anterior segment of the eye including conjunctivitis [5]. TGF-ß is a cytokine acting as a multifunctional growth factor that induces unfavorable inflammatory responses and tissue fibrosis and is involved in several pathologies of the eye [24]. As shown here, dexamethasone potentiates the ofloxacin-induced reduction of both IL-6 and TGF-ß, suggesting that corticosteroids potentiate the anti-inflammatory properties of fluoroquinolones. In ocular diseases, the combination between moxifloxacin and dexamethasone has been proven to be effective in reducing infection and inflammation associated with bacterial blepharitis in humans [25]. In addition, moxifloxacin added with dexamethasone prevents postoperative inflammation and infection following cataract surgery [26].
Most of all cases of bacterial conjunctivitis worldwide is represented by the acute form of the disease. To the best of our knowledge, this is the first demonstration that the addition of dexamethasone to ofloxacin potentiates the anti-inflammatory