Abstract
Purpose: Hyperosmolarity is one of the core mechanisms of dry eye. Its various effects on the overall dynamics of the ocular surface still need to be understood. Our previous research showed that hyperosmolarity does not affect stability of the lipid layer or the tear film. The aim of this study was to investigate the effect of hyperosmolarity on the activity of lysozyme, the most abundant antibacterial tear protein involved in ocular surface defence, in a tear like solution in-vitro against Staphylococcus aureus, a bacterial isolate in ocular infection.
Methods: An artificial tear (AT) solution with osmolarity near normal (305 mOsm/Kg) and 1.5 times salt concentration hyperosmolar (HAT) solution (478 mOsm/Kg) were prepared. Lysozyme stock solutions were prepared in AT and HAT, respectively. Overnight culture of Staphylococcus aureus was inoculated in the artificial tear solution in the presence of two lysozyme solutions at concentrations of lysozyme normally found in tears. Standard lysozyme activity assay was performed spectrophotometrically at OD450. Micrococcus luteus was used as a control for the lysozyme activity assay. After the assay, bacterial culture was diluted and plated on LB agar plates to observe viable counts.
Results: Spectrophotometric measurements showed greater decrease in optical density of the bacterial culture indicating higher lysozyme activity in HAT as compared with AT. Viable count results corroborated this by showing less number of bacterial colonies in HAT as compared with AT indicating that lysozyme was more active and killed more bacteria in HAT.
Conclusions: Hyperosmolarity has no adverse effect on the antibacterial activity of lysozyme that is normally involved in ocular surface defence. To fully understand the role of hyperosmolarity on the ocular surface defence, future research will involve studying more antimicrobial proteins and ocular pathogens in hyperosmolar conditions.
Methods: An artificial tear (AT) solution with osmolarity near normal (305 mOsm/Kg) and 1.5 times salt concentration hyperosmolar (HAT) solution (478 mOsm/Kg) were prepared. Lysozyme stock solutions were prepared in AT and HAT, respectively. Overnight culture of Staphylococcus aureus was inoculated in the artificial tear solution in the presence of two lysozyme solutions at concentrations of lysozyme normally found in tears. Standard lysozyme activity assay was performed spectrophotometrically at OD450. Micrococcus luteus was used as a control for the lysozyme activity assay. After the assay, bacterial culture was diluted and plated on LB agar plates to observe viable counts.
Results: Spectrophotometric measurements showed greater decrease in optical density of the bacterial culture indicating higher lysozyme activity in HAT as compared with AT. Viable count results corroborated this by showing less number of bacterial colonies in HAT as compared with AT indicating that lysozyme was more active and killed more bacteria in HAT.
Conclusions: Hyperosmolarity has no adverse effect on the antibacterial activity of lysozyme that is normally involved in ocular surface defence. To fully understand the role of hyperosmolarity on the ocular surface defence, future research will involve studying more antimicrobial proteins and ocular pathogens in hyperosmolar conditions.
| Original language | English |
|---|---|
| Number of pages | 2 |
| Journal | Investigative Ophthalmology & Visual Science |
| Volume | 54 |
| Issue number | 15 |
| Publication status | Published - Jun 2013 |
