Discussion
Kerasave is a newly developed medium intended for storage of human donor corneas at 2°C–8°C for up to 14 days. This medium has the peculiarity of being supplied with an antifungal amphotericin B tablet.18
Unlike our previous study, in which only Candida spp were tested at shorter time intervals,17 18 the present study investigated nine micro-organisms incubated in Kerasave up to 14 days to simulate the eye bank common practice and cover the intended use of the device. Indeed, the release of the cornea for transplantation usually does not exceed 7 days of storage at 4°C,2 and the maximum time of use of the device indicated by the manufacturer corresponds to 14 days. Therefore, in the present study, data were not acquired beyond 14 days. All the tested micro-organisms showed significantly lower microbial concentrations in Kerasave than in the growth controls of all the tested time points, except for A. brasiliensis on day 14, which was comparable with the control. Our findings indicated that the medium effectively eliminated seven out of nine (78%) tested micro-organisms. This was accomplished despite the low storage temperature of 4°C, which is considered to be a condition resulting in poor antimicrobial efficacy.11 33
The bactericidal activity is achieved when a 3 log10 decrease is obtained.34–36 In the time-kill assay, a very high initial microbial concentration inoculum (105–106 CFU/mL) is used to allow quantification of the killing efficacy. The high inoculum concentration used in our study does not reflect a clinical situation as it is significantly higher than the bacterial concentration observed in the blood of patients with sepsis (102–103 CFU/mL)37; therefore, complete elimination of the tested micro-organism is not expected in these assays. In our study, the 3 log10 decrease indicates bactericidal activity of Kerasave for some micro-organisms. Complete microbial elimination (5–6 log10 reduction) would correspond to the performance of disinfectants and sterilisation processes, which is not expected to be achieved by Kerasave.
A log10 reduction ≥3 was achieved after 3 days of incubation in Kerasave for C. albicans, P. aeruginosa and K. pneumoniae strains, and was achieved on day 14 for S. aureus, E. faecalis and E. cloacae. The elimination of C. albicans was in line with the results of our previous study, in which C. albicans killing was assessed after 10 days of incubation.18 In this study, Kerasave was shown to be effective during the 14 days of storage. For three strains (A. brasiliensis, F. solani and B. subtilis), a low or negative log10 decrease was observed, which indicated low microbial elimination or slight microbial growth over time. For both A. brasiliensis and F. solani, which are filamentous fungi, a low killing efficacy of 2.5 µg/mL amphotericin B contained in Kerasave was expected as the minimal inhibition concentration values for amphotericin B reported in the literature were 0.03–4 µg/mL and 4–8 µg/mL for A. brasiliensis and F. solani, respectively.38–41 In a previous study, Duncan et al42 found that the supplementation of the hypothermic corneal storage medium with amphotericin B at 0.25 µg/mL effectively reduced the growth of F. solani.
Along with the lower concentration of amphotericin B, there are some other differences in the experimental conditions between this and our study, that is, a lower concentration of the inoculum (2.5×103 CFU/mL vs 105–106 CFU/mL) and a lower extent of growth reduction. Nevertheless, the authors also concluded that amphotericin B appeared to be a reasonable candidate drug for an antifungal additive to a corneal storage medium. In a recent study, Kaymar et al43 found that amphotericin B at 2.5 µg/mL was ineffective in eliminating small loads of Candida and Fusarium species (up to 103 CFU/mL). However, the goal of that study was to achieve complete elimination of fungi limited to single testing due to the scarcity of corneal tissue. Unlike our study, the growth reduction was not quantified. Thus, the differences in the experimental design might explain why the authors concluded that the eradication of fungal contamination of donor corneal tissue might require complementary approaches of antifungal supplementation of hypothermic storage media. However, based on the results of Kapur et al’s study,44 which examined the effect of the hypothermic corneal storage medium with and without tissue growth of similar species, it appears that the presence of corneal tissue does not affect viable counts.
The concentration of amphotericin B in Kerasave represents the optimal balance between the efficacy and safety for preserving the donor cornea17 and was selected under the intended use of Kerasave, which corresponds to corneal storage rather than corneal disinfection. Although corneal disinfection as intended for disinfectants (povidone-iodine solutions) is out of Kerasave’s intended use,18 Kerasave temporarily prevented the growth of F. solani, for which a moderate 1.25 log10 decrease was observed after 14 days. In any case, we can consider Kerasave effective in decreasing or eliminating contamination from Candida spp, which is the most commonly encountered pathogen.7 Other fungi were not commonly encountered in donor corneal transplantation.23 24 45
Surprisingly, a very low log10 decrease of 0.18 was observed for B. subtilis after 14 days in Kerasave, indicating a bacteriostatic rather than bactericidal effect for this strain under the tested conditions. Data in the literature reported B. subtilis to be sensitive to gentamicin (4.0 mg/L; 0.125 µg/mL) and resistant to streptomycin45–47; the gentamicin content of Kerasave, which is significantly higher than that reported in the literature, led us to expect higher killing efficacy of B. subtilis spizizenii by Kerasave. The low killing efficacy was confirmed when we repeated the time-kill assay at 4°C and 31°C after 3 days of incubation to evaluate whether the incubation temperature could influence the killing efficacy of Kerasave (data not shown). At 31°C, a 0.6 log10 decrease was observed in Kerasave after 3 days, whereas marked growth was observed in controls (−2.8 log10 decrease; data not shown). We thus confirmed the bacteriostatic effect of Kerasave for B. subtilis under tested conditions. Moreover, we hypothesised that B. subtilis spizizenii can turn into sporulation, enabling survival under adverse conditions.48 49 However, microscopy studies to check for possible B. subtilis spizizenii sporulation were not conducted as they were beyond the scope of the present study.
With regard to growth controls, most strains maintained the initial concentration or showed growth along with incubation (A. brasiliensis, E. faecalis and E. cloacae). Previous studies15 42 compared Kerasave with other corneal storage media. Contrarily, in this study, we chose to use growth media as control samples to confirm microbial growth of the strains under tested conditions and not in comparison with other corneal storage media since the validity of the time-kill assay and evaluation of the recovery of viable micro-organisms require optimal microbial growth and absence of any factors that would interfere with it. As discussed by Tran et al,17 approaches that do not use a step to neutralise the antimicrobials present in the culture samples used for the quantification of the micro-organisms can lead to the presence of some residual amounts of drugs, which can prevent growth on culture plates.17 Kerasave contains different antimicrobials, and their interference with microbial growth is predictable. Therefore, to avoid false-negative results, RESEP was used before plating the Kerasave samples on agar plates. The treatment of Kerasave samples with RESEP was validated prior to its use in the time-kill assay using the ultra-high-performance liquid chromatography which revealed total removal of antimicrobials from Kerasave and complete recovery of viable micro-organisms was obtained in RESEP-treated or untreated samples (online supplemental tables 1 and 2).
The RESEP treatment was not performed on F. solani containing samples since growth inhibition of F. solani by Kerasave was not observed. Thus, the dilution plate technique was sufficient to obtain a reliable result in the time-kill study.
Safety study of Kerasave was out of the scope of the present study as it has been previously investigated in cells via a cytotoxicity test, biocompatibility animal studies according to International Organization for Standardization standards17 18 and in donor corneas.15 45
This study completes the data obtained in our previous work on the efficacy of the Kerasave medium, confirming the killing of the most common corneal contaminants by Kerasave under hypothermic corneal storage conditions even after a short incubation time (C. albicans, P. aeruginosa and K. pneumoniae) and preventing the microbial growth of all the other tested strains. In this study, the killing efficacy of Kerasave was directly related to the time of incubation, as observed for S. aureus, E. faecalis and E. cloacae. The killing efficacy of Kerasave could be limited in case of contaminations by micro-organisms resistant to the antimicrobials present in Kerasave (amphotericin B, streptomycin sulfate and gentamicin sulfate), and eye banks should consider that a short storage interval and storage temperature of 4°C could prevent the eradication of high contamination from donor tissues.18 Finally, the hypothermic corneal storage medium Kerasave is a promising tool for enhancing the safety of the recipient of the transplanted cornea.