Research article
A new ophthalmic formulation containing antiseptics and dexpanthenol: In vitro antimicrobial activity and effects on corneal and conjunctival epithelial cells

https://doi.org/10.1016/j.exer.2020.108269Get rights and content

Highlights

  • Topical antibiotic overuse increases microbial resistance.

  • Therefore, the interest towards antiseptics is growing.

  • Keratosept ophthalmic solution showed a good antimicrobial activity in vitro.

  • Keratosept showed low toxicity on corneal and conjunctival epithelial cells.

Abstract

Antibiotic resistance is increasing even in ocular pathogens, therefore the interest towards antiseptics in Ophthalmology is growing. The aim of this study was to analyze the in vitro antimicrobial efficacy and the in vitro effects of an ophthalmic formulation containing hexamidine diisethionate 0.05%, polyhexamethylene biguanide (PHMB) 0.0001% disodium edetate (EDTA) 0.01%, dexpanthenol 5% and polyvinyl alcohol 1.25% (Keratosept, Bruschettini, Genova, Italy) on cultured human corneal and conjunctival cells. The in vitro antimicrobial activity was tested on Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus mitis. For each microbial strain 10 μL of a 0.5 MacFarland standardized bacterial inoculum were incubated at 25 °C with 100 μL of ophthalmic solution for up to 6 h. After different periods of time, samples were inoculated on blood agar with 5% sheep blood. Moreover, a 0.5 MacFarland bacterial inoculum was seeded in triplicate on Mueller-Hinton Agar or on Mueller-Hinton Fastidious Agar; then a cellulose disc soaked with 50 μL of ophthalmic solution was applied on the surface of agar and plates were incubated for 18 h at 37 °C, in order to evaluate the inhibition of bacterial growth around the disc. Human corneal and conjunctival epithelial cells in vitro were incubated for 5, 10 and 15 min with Keratosept or its components. The cytotoxicity was assessed through the release of cytoplasmic enzyme lactate dehydrogenase (LDH) into the medium immediately after exposure to the drugs; the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate the metabolic cell activity. Our results show that Keratosept ophthalmic solution gave an average logarithmic (log) reduction of bacterial load of 2.14 ± 0.35 within 6 h of exposure (p-value < 0.05 versus control saline solution). On agar plates, all microbial strains, excluding P. Aeruginosa, showed an inhibition zone of growth around the Keratosept-soaked discs. Keratosept and its components after 5 and 10 min did not show any cytotoxic effect on cultured corneal and conjunctival cells, and only after 15 min a significant reduction of cell viability and an increase of cytotoxicity compared to control (vehicle) was seen; dexpanthenol 5% and polyvinyl alcohol accelerated the wounding of corneal cells in vitro. In conclusion, Keratosept showed good antimicrobial activity on the tested strains; the ophthalmic solution and its components were safe and non-toxic for the corneal and conjunctival epithelial cells for 5 and 10 min at the concentrations analyzed, and dexpanthenol 5% and polyvinyl alcohol promoted the wounding of corneal cells.

Introduction

Antimicrobial resistance to antibiotics is one of the biggest public health challenges of our time and it has been continuously increasing in recent years, even in ocular pathogens (Asbell and DeCory, 2018; Bertino, 2009; Holland et al., 2014). As in all human infections, the rise of bacterial resistance can also be of concern in ophthalmology, leading to unsuccessful treatment of sight-threatening infections (Asbell and DeCory, 2018; Bertino, 2009; Holland et al., 2014).

Since the overuse of antibiotics in the treatment of ocular infectious diseases has led to the appearance of multidrug-resistant bacterial strains (Asbell and DeCory, 2018), the interest towards antiseptics is growing. In ophthalmology, the role of topical antibiotics in the prophylaxis of post-surgical endophthalmitis, especially in intravitreal injections, is under debate because their perioperative use can increase the resistance of conjunctival flora but has no proven added effectiveness in preventing endophthalmitis if combined with povidone iodine antisepsis (Grzybowski et al., 2017).

Moreover, due to their wide spectrum of activity (multi-drug resistant bacteria, methicillin-resistant staphylococcus aureus, viruses, protozoa, fungi) (Grzybowski et al., 2018; Kaehn, 2010; Maycock and Jayaswal, 2016), and the few reports on reduced microbial susceptibility which currently only concern chlorhexidine (Horner et al., 2012; Kampf, 2016), the use of antiseptics has been proposed for the treatment of viral conjunctivitis, mild bacterial conjunctivitis, or even more severe infections (such as keratitis) in developing countries, where the availability of antibiotics is often limited by costs (Grzybowski et al., 2018; Isenberg et al., 2017). Regarding Acanthamoeba keratitis, the only available treatment are the antiseptics biguanides (PHMB 0.02%, and chlorhexidine 0.02%) and diamidines (hexamidine 0.1% or propamidine 0.1%) (Carrijo-Carvalho et al., 2017; Maycock and Jayaswal, 2016).

However, antiseptics may be toxic for the ocular surface epithelia, leading to corneal epithelial defects or damage, especially at high concentrations (Shibata et al., 2014). Therefore, there is an increasing interest in topical formulations containing antiseptics with a wide antimicrobial spectrum, but with a lower tendency to damage the ocular surface. Recently, an ophthalmic solution containing antiseptics, such as hexamidine diisethionate 0.05%, PHMB 0.0001%, disodium edetate (EDTA) 0.01%, and healing agents, such as dexpanthenol (D-panthenol)5% and polyvinyl alcohol 1.25%, has been launched on the market in Italy (Keratosept, Bruschettini, Genova, Italy). The purposes of our study were to evaluate the antimicrobial activity of this product on isolates of Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus mitis, that are among the most frequent causes of ocular infections (Asbell and DeCory, 2018; Garg et al., 2017; Green et al., 2008; Han et al., 1996; Teweldemedhin et al., 2017) and to analyze in vitro the effects of this solution and of its components on cultured human corneal and conjunctival cells.

Section snippets

In vitro antimicrobial activity of the ophthalmic solution

The antimicrobial activity of the ophthalmic solution was evaluated using a modified protocol from the International Organization for Standardization- ISO 14729 guidelines (International Organization for Standardization, 2001).

Staphylococcus aureus, Methicillin-Resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus mitis (of the Viridans Group Streptococci, VGS) were used in this study. All the microbial strains were

In vitro antimicrobial activity of the ophthalmic solution

The Keratosept ophthalmic solution showed an antiseptic spectrum of activity and gave an average log reduction of 2.14 ± 0.35 within 6 h of exposure, significantly higher than the control solution (p-value < 0.05), although individual log reductions varied between microorganisms (Fig. 1). Log reductions of the fastidious species (Streptococcus pneumoniae, Streptococcus mitis and Streptococcus pyogenes) were significantly marked within 2 h of exposure to the ophthalmic solution (p-value < 0.05

Discussion

Increasing trends in antibiotic resistance in ocular infections, especially widespread fluoroquinolone non-susceptibility (Asbell and DeCory, 2018; Bertino, 2009; Holland et al., 2014), have enhanced the role of antiseptics in the prevention of infection after ocular surgery (Grzybowski et al., 2017). Nowadays, povidone-iodine (PVI) and chlorhexidine represent the elective antiseptics in ophthalmology. However, a reduced susceptibility to chlorhexidine of MRSA and other staphylococci was

Funding declaration

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of competing interest

None.

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    These authors contributed equally to this work.

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