Effectiveness of antimicrobial peptide immobilization for preventing perioperative cornea implant-associated bacterial infection

Abstract

Titanium (Ti) is a promising candidate biomaterial for an artificial corneal skirt. Antimicrobial peptide (AMP) immobilization may improve the bactericidal effect of the Ti substrate. In this study, we tested the bactericidal efficacy of a functionalized Ti surface in a rabbit keratitis model. A corneal stromal pocket was created by a femtosecond laser. The Ti films were then inserted into the pocket, and Staphylococcus aureus or Pseudomonas aeruginosa was inoculated into the pocket above the implant films. The corneas with Ti-AMP implants were compared with the corneas implanted with unprotected Ti by slit lamp observation and anterior segment optical coherence tomography (AS-OCT). Inflammatory responses were evaluated by bacterium counting, hematoxylin-eosin staining, and immunostaining. There was a lower incidence and a lesser extent of infection on rabbit corneas with Ti-AMP implants than on those with unprotected Ti implants. The bactericidal effect of AMP against S. aureus was comparable to that of postoperative prophylactic antibiotic treatment; hence, SESB2V AMP bound to the Ti implant provided functional activity in vivo, but its efficacy was greater against S. aureus than against P. aeruginosa. This work suggests that SESB2V AMP can be successfully functionalized in a rabbit keratitis model to prevent perioperative corneal infection.

FIG 1

Design of the rabbit study.

FIG 2

In vitro bactericidal effect and stability of SESB2V AMP. (A) MICs of the full-length and truncated SESB2V peptides. (B) Representative images from the Live/Dead bactericidal assay. Green, live bacteria; red, dead bacteria. The live/dead ratios were determined and graphed. *, P < 0.05 (n = 6, Student's t test). (C) Fluorescence images of Ti surface after immobilization with SESB2V-FITC peptide in phosphate-buffered saline.

FIG 3

In vivo bactericidal effect of SESB2V AMP against S. aureus. (A) Slit lamp examination of the rabbit eyes before and after implantation and bacterial inoculation. (B) AS-OCT scanning of the rabbit eyes before and after implantation and bacterial inoculation. (C) Measurement of corneal thickness. Only corneal thickness anterior to the implant was measured at pre- and postinoculation day 1 and day 2. *, P < 0.05 (one-way ANOVA) between the pristine Ti and Ti-AMP groups at postinoculation day 1; **, P < 0.05 (one-way ANOVA) between the pristine Ti and Ti-AMP groups at postinoculation day 2.

FIG 4

Bacteria counts of S. aureus and P. aeruginosa cells. Results are presented as log₁₀ CFU/cornea ± standard error of the mean (SEM). * and **, P < 0.05 (n = 3 in each group; one-way ANOVA).

FIG 5

Hematoxylin-eosin (H&E) pictures of rabbit corneas after inoculation with S. aureus. (A) Normal rabbit cornea. (B to D) Rabbit cornea with pristine Ti implantation and bacterial infection: corneal epithelium (B), central corneal stroma (C), and corneal stroma surrounding the implant pocket (D). (E to G) Rabbit cornea with Ti-AMP implantation and bacterial infection: corneal epithelium (E), central corneal stroma (F), and corneal stroma surrounding the implant pocket (G).

FIG 6

Immunostaining pictures of rabbit corneas after infection with S. aureus. Green, inflammatory cell markers; blue, DAPI cell nucleus counterstaining. (A to C) CD11b staining: normal rabbit cornea (A), cornea with pristine Ti implant (B), and cornea with Ti-AMP implant (C). (D to F) F4/80 staining: normal rabbit cornea (D), cornea with pristine Ti implant (E), and cornea with Ti-AMP implant (F). (G to I) MMP9 staining: normal rabbit cornea (G), cornea with pristine Ti implant (H), and cornea with Ti-AMP implant (I).

FIG 7

Antibiotic treatment of rabbit corneas after inoculation with S. aureus and P. aeruginosa. (A and C) Scoring of slit lamp examinations after inoculation of S. aureus (A) and P. aeruginosa (C). (B and D) Bacterium counting of corneal tissue after inoculation of S. aureus (B) and P. aeruginosa (D). *, P < 0.05 (one-way ANOVA).