Related Experiment Video
Updated: Aug 14, 2026
High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity
Published on: November 16, 2016
In vitro effects of fluoroquinolone and aminoglycoside antibiotics on human keratocytes
Andrea Leonardi1, Vincenzo Papa, Iva Fregona
1Department of Neuroscience, Ophthalmology Unit, University of Padova, Italy.
Purpose:
The purpose of this study was to assess the cytotoxic effects of the fluoquinolone ofloxacin and the aminoglycoside netilmicin on stromal human keratocytes in vitro.
Methods:
Cultured human keratocytes were exposed to various concentrations of ofloxacin or netilmicin (0.16-5.0 mg/mL). Both cell proliferation (MTT assay) and cell morphology (phase-contrast microscopy) were evaluated after 1, 4, 12, and 24 hours of incubation. Measurement of annexin V binding performed in association with the dye exclusion test using propidium iodide (PI) was also performed by FACS analysis after 4 hours of exposure.
Results:
Both antimicrobials induced dose- and time-dependent morphologic changes in keratocytes, yet the effects of netilmicin were minimal. After 24 hours of exposure, both drugs induced a dose-dependent inhibition of cell proliferation; however, ofloxacin demonstrated significantly more toxic effects than netilmicin (t test for ED50 values, P < 0.0001). Statistical differences between 2 antibiotics start at concentrations above 1.25 mg/mL (ANOVA with post-hoc test, P < 0.01). Expression of the apoptotic marker annexin V was unaffected by antibiotic exposure, whereas the uptake of the necrotic marker PI was increased by ofloxacin (5 mg/mL) but not by netilmicin (ofloxacin versus netilmicin, ANOVA, P < 0.05).
Conclusions:
Relative effects of aminoglycosides and fluoroquinolones on stromal keratocytes appear to be different: netilmicin was shown to be significantly less toxic than ofloxacin. This finding is particularly relevant in deciding the optimal antibiotic to be applied in clinical situations in which the epithelium is absent or compromised, as after photorefractive keratectomy, alkali burns, or ulcerative keratitis.
Insights
Ofloxacin is more toxic to human corneal cells than netilmicin, with significant differences observed at higher concentrations. This suggests netilmicin may be a safer choice for eye treatments when the corneal epithelium is compromised.
Area of Science:
- Ophthalmology
- Cell Biology
- Pharmacology
Background:
- Stromal human keratocytes are crucial for corneal health and wound healing.
- Antibiotic choice is critical in treating corneal conditions, especially when the epithelium is compromised.
- Fluoroquinolones and aminoglycosides are commonly used antibiotics in ophthalmology.
Purpose of the Study:
- To compare the in vitro cytotoxic effects of ofloxacin (a fluoroquinolone) and netilmicin (an aminoglycoside) on human keratocytes.
- To evaluate the impact of these antibiotics on keratocyte proliferation and morphology.
- To assess the potential for apoptosis and necrosis induced by ofloxacin and netilmicin.
Main Methods:
- Human keratocytes were exposed to varying concentrations of ofloxacin or netilmicin.
- Cell proliferation was measured using the MTT assay.
- Cell morphology was assessed via phase-contrast microscopy.
- Annexin V binding and propidium iodide uptake were analyzed by flow cytometry to detect apoptosis and necrosis.
Main Results:
- Both antibiotics caused dose- and time-dependent changes in keratocyte morphology, with ofloxacin showing more pronounced effects.
- Ofloxacin significantly inhibited cell proliferation more than netilmicin at 24 hours (P < 0.0001).
- Netilmicin demonstrated significantly lower toxicity compared to ofloxacin, particularly at concentrations above 1.25 mg/mL (P < 0.01).
- Propidium iodide uptake, indicating necrosis, increased with ofloxacin but not netilmicin (P < 0.05).
Conclusions:
- Netilmicin exhibits significantly less toxicity to human stromal keratocytes than ofloxacin in vitro.
- These findings are clinically relevant for selecting appropriate antibiotics in post-surgical or disease-compromised corneas.
- The differential toxicity highlights the importance of antibiotic selection in ophthalmic applications to minimize keratocyte damage.
