Evaluation of ligand modified poly (N-Isopropyl acrylamide) hydrogel for etiological diagnosis of corneal infection

Abstract

Corneal ulcers, a leading cause of blindness in the developing world are treated inappropriately without prior microbiology assessment because of issues related to availability or cost of accessing these services. In this work we aimed to develop a device for identifying the presence of Gram-positive or Gram-negative bacteria or fungi that can be used by someone without the need for a microbiology laboratory. Working with branched poly (N-isopropyl acrylamide) (PNIPAM) tagged with Vancomycin, Polymyxin B, or Amphotericin B to bind Gram-positive bacteria, Gram-negative bacteria and fungi respectively, grafted onto a single hydrogel we demonstrated specific binding of the organisms. The limit of detection of the microbes by these polymers was between 10 and 4 organisms per high power field (100X) for bacteria and fungi binding polymers respectively. Using ex vivo and animal cornea infection models infected with bacteria, fungi or both we than demonstrated that the triple functionalised hydrogel could pick up all 3 organisms after being in place for 30 min. To confirm the presence of bacteria and fungi we used conventional microbiology techniques and fluorescently labelled ligands or dyes. While we need to develop an easy-to-use either a colorimetric or an imaging system to detect the fluorescent signals, this study presents for the first time a simple to use hydrogel system, which can be applied to infected eyes and specifically binds different classes of infecting agents within a short space of time. Ultimately this diagnostic system will not require trained microbiologists for its use and will be used at the point-of-care.

Keywords: Amphotericin B; Corneal infections; Ex vivo corneal infection model; Fluorescence imaging; Hydrogel; Poly N isopropyl Acrylamide (PNIPAM); Polymyxin B; Vancomycin.

Fig. 1

Schematic diagram illustrating the attachment of highly branched poly(N-isopropyl acrylamide) (PNIPAM) functionalised with ligands for binding to microbes. The microbes bind to respective ligand and then desolvation of PNIPAM segments occurs, which enhances attachment of the microbes. The immobilized microbes are then visualized by adding dyes hat are specific to the class of microbe.

Fig. 2

Schematic diagram of the overall strategy used for the evaluation of ligand modified poly (n-isopropylacrylamide) attached to a hydrogel toward developing a device for the rapid detection of bacteria and fungi from corneal ulcer cases.

Fig. 3

Clinical photographs of rabbit eyes 24 h after inoculation of microorganisms showing corneal ulcer development. Here G is control eye (G), G1 is an eye after inoculation of S. aureus, G2 is corneal ulcer by P. aeruginosa, G3 is by C. albicans, and G 4a is ulcer by mixed infection of S. aureus + C. albicans while G4b is mixed infection of P. aeruginosa and C. albicans.

Fig. 4

The effect of Amphotericin-B functionalised hydrogel on the survival of C. albicans. The histograms represent the mean optical density (at 600 nm) of C. albicans grown in the presence of PNIPAM-linked hydrogels in BHI broth for 24 h at 37 °C. The PNIPAMs had either COOH groups (non-functionalised) or Amphotericin-B at the chain ends. The positive control was a parallel culture of C. albicans without hydrogel. Results shown are the means ± SEM of 3 replicates.

Fig. 5

Binding of microorganisms to functionalised hydrogels. Histograms show the number of bacterial or fungal cells bound to the surface of respective mono functionalised hydrogels and to the triple functionalised hydrogel compared to a non-functionalised control hydrogel. The values are mean ± SEM of 8 fields of views analyzed from 3 independent experiments (Fig. 5A). Fig. 5B is the microphotograph of bacteria and fungi bound to triple hydrogel surfaces compared to the non-functionalised control hydrogels imaged using a fluorescence microscope.

Fig. 5

Binding of microorganisms to functionalised hydrogels. Histograms show the number of bacterial or fungal cells bound to the surface of respective mono functionalised hydrogels and to the triple functionalised hydrogel compared to a non-functionalised control hydrogel. The values are mean ± SEM of 8 fields of views analyzed from 3 independent experiments (Fig. 5A). Fig. 5B is the microphotograph of bacteria and fungi bound to triple hydrogel surfaces compared to the non-functionalised control hydrogels imaged using a fluorescence microscope.

Fig. 6

Results of experiments to evaluate the sensitivity of detection of the triple hydrogel as assessed by determining total ATP content and count of microorganisms attached to the hydrogel when incubated in-vitro for 1 h to increasing concentration of S. aureus, P. aeruginosa or C. albicans. Fig. 6A shows luminance values while Fig. 6B shows number of microorganisms (mean ± SD of 8 fields of view per hydrogel) from at least 3 independent experiments.

Fig. 6

Results of experiments to evaluate the sensitivity of detection of the triple hydrogel as assessed by determining total ATP content and count of microorganisms attached to the hydrogel when incubated in-vitro for 1 h to increasing concentration of S. aureus, P. aeruginosa or C. albicans. Fig. 6A shows luminance values while Fig. 6B shows number of microorganisms (mean ± SD of 8 fields of view per hydrogel) from at least 3 independent experiments.

Fig. 7

Results of experiments to determine the time required for the hydrogel to be kept on cornea for effective sampling. Fig. 7A is microphotograph of S. aureus, P. aeruginosa, and C. albicans cells adherent to the surface of the triple hydrogel at different time points. The histogram in Fig. 7B represents mean ± SD values number of microorganisms as derived from 8 fields of views per hydrogel from three independent experiments. Higher loads of organisms were observed with increased duration of application of the hydrogel on infected ex-vivo cornea infection model.

Fig. 7

Results of experiments to determine the time required for the hydrogel to be kept on cornea for effective sampling. Fig. 7A is microphotograph of S. aureus, P. aeruginosa, and C. albicans cells adherent to the surface of the triple hydrogel at different time points. The histogram in Fig. 7B represents mean ± SD values number of microorganisms as derived from 8 fields of views per hydrogel from three independent experiments. Higher loads of organisms were observed with increased duration of application of the hydrogel on infected ex-vivo cornea infection model.

Fig. 8

Microphotograph showing microorganisms bound to the triple hydrogel from single and mixed species rabbit corneal infections and corresponding microbiology results of corneal scraping. The images show visual counts of cells per field of view. Here in column A represent microphotograph of corneal scraping smear stained by Gram stain, column B represents smears stained by Calcofluor white stain, column C represents smears prepared from the growth on the culture and stained by Gram stain, and column D and E represent microphotographs of stained triple hydrogels removed aseptically from infected rabbit corneas and show bacteria and fungi bound to the hydrogel surface.

Fig. 9

Histogram representing number of S. aureus, P. aeruginosa or C. albicans per field of view attached to the triple functionalised hydrogel after application for half an hour on infected rabbit corneas. Data are mean ± SD of 8 fields of view per hydrogel.