Safety and efficacy of McCarey-Kaufman medium supplemented with colistin (polymyxin E) and amphotericin B in inhibiting the multidrug-resistant Pseudomonas aeruginosa using an ex vivo donor corneal infection model

Abstract

Purpose: This study aimed to evaluate the efficacy and safety of McCarey-Kaufman (MK) medium supplemented with colistin and amphotericin B in inhibiting the growth of multidrug-resistant Pseudomonas (P.) aeruginosa , using an ex vivo experimental model with human donor corneas.

Methods: Cadaveric human corneas deemed unsuitable for corneal transplantation were obtained, and MK media were supplemented with colistin and amphotericin B. Multidrug-resistant P. aeruginosa was cultured and used to infect the human donor corneas ex vivo . Infected corneas were placed in the MK media with additional antibiotics (colistin and amphotericin B) and the standard MK media, which served as the control arm for comparison. Corneal opacity due to infiltration and quantitative analysis of colony-forming units (CFUs) were assessed. The viability of the corneal endothelium was assessed using trypan blue staining.

Results: Corneas incubated in MK media supplemented with additional antibiotics showed less corneal opacification compared with those in standard MK media at both 48- and 96-hour (hr) time points. Quantitative analysis revealed a lower bacterial load and a significant reduction in CFU in the corneas incubated in MK media with additional antibiotics compared with the control group. At 48 hrs, there was 84% ( P value = 0.024) reduction in bacterial load, and at 96 hr, a 53% ( P value = 0.016) reduction was observed in comparison with those placed in standard MK media. The trypan blue staining tests revealed that the extent of endothelial cell loss in corneas incubated in supplemented MK media was comparable to the ones in standard MK media.

Conclusion: The addition of colistin and amphotericin B to MK media demonstrated efficacy in inhibiting the growth of multidrug-resistant P. aeruginosa in an ex vivo cornea infection model. The supplemented media had no detrimental effect on the corneal endothelium. The findings suggest that supplementing the MK media with these broad-spectrum antimicrobial agents may help mitigate the risk of postoperative donor-related infection in the recipients by reducing and containing the load of microbial contamination in donor corneas.

Figure 1

Representative image illustrating the absence of corneal contamination after overnight incubation in DMEM F12 + FBS media

Figure 2

Representative image showing a secure seal between the metal rings and the corneas

Figure 3

Illustration of corneal opacification due to infiltration, followed by an infection at 48 and 96 hr. It is evident from the image that the corneas incubated in supplemented MK were comparatively more transparent than the corneas incubated in standard MK media at both 48 and 96 hr

Figure 4

(a) Representative image of CFUs plated at the 48-hr timepoint. The image shows a significant decrease in the colony count in the supplemented MK medium plate in comparison with the standard MK medium plate (n = 6); (b) Graph showing the CFUs recovered from standard MK and supplemented MK at the 48-hr time point

Figure 5

(a) Representative image of CFUs plated at the 96-hr timepoint. The image shows a significant decrease in the colony count in the supplemented MK medium plate in comparison with the standard MK medium plate (n = 6); (b) Graph showing the CFUs recovered from standard MK and supplemented MK at the 96-hr time point

Figure 6

(a) Representative image depicting the area of endothelial cell loss on day 1 and day 4 in corneas incubated in standard MK media and supplemented MK media. It is observed that corneas incubated in supplemented MK media showed comparatively less endothelial cell loss on day 4 than the ones placed in standard MK media (n = 6); (b) representative figure showing the portion of stained and unstained regions for calculating the percentage of cell loss in ImageJ software. *Red color denotes the area of endothelial cell loss (stained).*Green color denotes the area of viable endothelial cells (unstained); (c) graph illustrates the percentage of endothelial cell loss. It is evident from the data that the endothelial cell loss was significantly lower in the corneas incubated in supplemented MK media (P value = <0.0001)