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.

甚至在眼中的病原体中，抗生素抗性也在增加，因此对眼科防腐剂的兴趣也在增长。这项研究的目的是分析含有六甲双异硫氰酸酯0.05％，聚六亚甲基双胍（PHMB）0.0001％乙二胺四乙酸二钠（EDTA）0.01％，右芬太酚5％和聚乙烯醇1.25的眼用制剂的体外抗菌功效和体外作用％（Keratosept，Bruschettini，热那亚，意大利）在培养的人角膜和结膜细胞上。测试了体外对金黄色葡萄球菌，耐甲氧西林金黄色葡萄球菌（MRSA），铜绿假单胞菌，肺炎链球菌，化脓性链球菌和链球菌炎。对于每种微生物菌株，将10μL的0.5 MacFarland标准化细菌接种物与100μL的眼用溶液在25°C下孵育6小时。在不同的时间段之后，将样品用5％羊血接种在血琼脂上。此外，将0.5 MacFarland细菌接种物一式三份接种在Mueller-Hinton琼脂或Mueller-Hinton耐久琼脂上；然后将浸有50μL眼药水的纤维素圆盘涂在琼脂表面上，并将平板在37°C下孵育18 h，以评估对圆盘周围细菌生长的抑制作用。将人角膜和结膜上皮细胞在体外与角膜塑剂或其成分一起孵育5、10和15分钟。通过暴露于药物后立即将细胞质酶乳酸脱氢酶（LDH）释放到培养基中来评估细胞毒性。进行3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物（MTT）测定以评估代谢细胞活性。我们的结果表明，在暴露6小时内，Keratosept眼药水平均对数（log）减少了2.14±0.35的细菌载量（相对于对照盐水溶液，p值<0.05）。在琼脂平板上，所有微生物菌株，不包括 在暴露6小时内达到35（相对于对照盐溶液，p值<0.05）。在琼脂平板上，所有微生物菌株，不包括 在暴露6小时内达到35（相对于对照盐溶液，p值<0.05）。在琼脂平板上，所有微生物菌株，不包括铜绿假单胞菌，表明围绕Keratosept浸泡圆盘生长的抑制区域。在5和10分钟后，角蛋白原及其成分对培养的角膜和结膜细胞未显示任何细胞毒性作用，仅在15分钟后，与对照（载体）相比，细胞活力显着降低，细胞毒性增加；5％的泛醇和聚乙烯醇在体外加速了角膜细胞的损伤。总之，Keratosept对测试的菌株显示出良好的抗菌活性。在所分析的浓度下，该眼用溶液及其成分对角膜和结膜上皮细胞在5和10分钟内是安全且无毒的，而5％的泛醇和聚乙烯醇可促进角膜细胞的损伤。