Time Course of Antibiotic and Antifungal Concentrations in Corneal Organ Culture
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Published on: May 12, 2020
Theo G Seiler1, Markus Tschopp, Stefan Zimmerli
1Departments of *Ophthalmology; and †Infectious Diseases, Inselspital, University of Bern, Bern, Switzerland.
Purpose:
Contamination with bacteria and/or fungi is a serious complication in organ-cultured corneas. Hence, antibiotic and antifungal agents are added to the culture medium. The concentration of different antimicrobial and antifungal additives to the media over time has so far not been investigated in detail and is the aim of this study.
Methods:
Nine human fresh corneoscleral discs were stored in corneal culture medium consisting of 2% fetal bovine serum and minimal essential medium. In addition, the culture medium contained 1200 μg/mL penicillin G, 25 μg/mL amphotericin B, 120 μg/mL streptomycin, and 100 μg/mL voriconazole. The concentration of amphotericin B used was 10 times higher than in clinical routine to facilitate its detection. The cultures were kept at 37°C for 28 days. At days 0, 7, 14, 21, and 28, samples of the culture medium were harvested for analysis of antimicrobial concentrations by liquid chromatography and electrospray ionization tandem mass spectrometry.
Results:
During corneal storage, the concentration of all antibiotics and antifungal agents declined significantly. By day 28, penicillin G was reduced to 14% of the original concentration. Amphotericin B and streptomycin retained approximately 60% of the original concentration to the end of the experiment and voriconazole maintained stable concentrations after an initial decline to approximately 80% at 7 days.
Conclusions:
Throughout the entire storage period, the concentrations of penicillin G, streptomycin, and voriconazole exceeded the minimum inhibitory concentrations of all common contaminants, obviating the need for a change of the medium for antimicrobial reasons. Based on the minimum inhibitory concentrations and our findings, the initial concentration of amphotericin B should be raised to 5 μg/mL.
Insights
Antimicrobial and antifungal agents in organ-cultured corneas decline over time. Penicillin G, streptomycin, and voriconazole remained effective, but amphotericin B may require a higher initial concentration for optimal corneal preservation.
Area of Science:
- Ophthalmology
- Microbiology
- Pharmacology
Background:
- Organ-cultured corneas are susceptible to bacterial and fungal contamination.
- Antimicrobial and antifungal agents are crucial for preventing contamination in corneal culture media.
- The stability and efficacy of these agents over time in corneal storage have not been extensively studied.
Purpose of the Study:
- To investigate the concentration changes of antimicrobial and antifungal agents in corneal culture media over a 28-day storage period.
- To assess the effectiveness of these agents in maintaining inhibitory concentrations against common contaminants.
Main Methods:
- Nine human corneoscleral discs were cultured in a medium containing penicillin G, amphotericin B, streptomycin, and voriconazole.
- Culture media samples were analyzed at multiple time points (0, 7, 14, 21, 28 days) using liquid chromatography and tandem mass spectrometry.
- Amphotericin B concentration was initially set 10 times higher than clinical routine for enhanced detection.
Main Results:
- Significant decline in the concentration of all tested antimicrobial and antifungal agents was observed over 28 days.
- Penicillin G reduced to 14% of its original concentration by day 28.
- Amphotericin B and streptomycin retained approximately 60% of their initial concentrations, while voriconazole remained stable after an initial drop to 80%.
Conclusions:
- Penicillin G, streptomycin, and voriconazole concentrations consistently exceeded minimum inhibitory concentrations, ensuring antimicrobial efficacy throughout storage.
- The initial concentration of amphotericin B should be increased to 5 μg/mL to ensure adequate antifungal protection based on its minimum inhibitory concentration and observed stability.
- No medium change was required for antimicrobial reasons during the study period.
