Eye Clinic, Department of Neurosciences, Psychology, Pharmacology and Child Health (NEUROFARBA), University of Florence, Largo Brambilla 3, 50134, Florence, Italy.
AOU Città della Salute e della Scienza di Torino, Microbiology and Virology Unit, Turin, Italy.
Exp Eye Res. 2020 Dec;201:108269. doi: 10.1016/j.exer.2020.108269. Epub 2020 Sep 24.
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.
抗生素耐药性甚至在眼部病原体中也在增加，因此眼科领域对防腐剂的兴趣日益浓厚。本研究的目的是分析含有己二胺二异噻唑啉 0.05%、聚六亚甲基双胍（PHMB）0.0001%乙二胺四乙酸二钠（EDTA）0.01%、5%泛醇和 1.25%聚乙烯醇（Keratosept，Bruschettini，Genova，意大利）的眼用制剂对培养的人角膜和结膜细胞的体外抗菌功效和体外作用。体外抗菌活性测试针对金黄色葡萄球菌、耐甲氧西林金黄色葡萄球菌（MRSA）、铜绿假单胞菌、肺炎链球菌、化脓链球菌和酿脓链球菌。对于每种微生物菌株，将 10 μL 的 0.5 MacFarland 标准化细菌接种物与 100 μL 眼科溶液在 25°C 下孵育长达 6 小时。经过不同的时间后，将样品接种到含 5%绵羊血的血琼脂上。此外，将 0.5 MacFarland 细菌接种物在 Mueller-Hinton 琼脂或 Mueller-Hinton 苛养琼脂上以三复孔接种；然后将浸透 50 μL 眼科溶液的纤维素圆盘应用于琼脂表面，并在 37°C 下孵育 18 小时，以评估圆盘周围细菌生长的抑制情况。体外培养的人角膜和结膜上皮细胞用 Keratosept 或其成分孵育 5、10 和 15 分钟。细胞毒性通过药物暴露后立即向培养基中释放细胞质酶乳酸脱氢酶（LDH）来评估；通过 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴盐（MTT）测定法评估代谢细胞活性。我们的结果表明，Keratosept 眼用溶液在暴露 6 小时内使细菌负荷的平均对数（log）减少了 2.14±0.35（与对照生理盐水相比，p 值<0.05）。在琼脂平板上，除铜绿假单胞菌外，所有测试菌株周围的生长均显示出抑菌区。Keratosept 及其成分在 5 和 10 分钟后对培养的角膜和结膜细胞没有任何细胞毒性作用，仅在 15 分钟后，与对照（载体）相比，细胞活力明显降低，细胞毒性增加；5%泛醇和聚乙烯醇加速了体外角膜细胞的损伤。总之，Keratosept 对测试菌株表现出良好的抗菌活性；眼用溶液及其成分在分析浓度下对角膜和结膜上皮细胞在 5 和 10 分钟时是安全且无毒的，而 5%泛醇和聚乙烯醇促进了角膜细胞的损伤。
