Corneal Infection Models: Tools to Investigate the Role of Biofilms in Bacterial Keratitis

Abstract

Bacterial keratitis is a corneal infection which may cause visual impairment or even loss of the infected eye. It remains a major cause of blindness in the developing world. Staphylococcus aureus and Pseudomonas aeruginosa are common causative agents and these bacterial species are known to colonise the corneal surface as biofilm populations. Biofilms are complex bacterial communities encased in an extracellular polymeric matrix and are notoriously difficult to eradicate once established. Biofilm bacteria exhibit different phenotypic characteristics from their planktonic counterparts, including an increased resistance to antibiotics and the host immune response. Therefore, understanding the role of biofilms will be essential in the development of new ophthalmic antimicrobials. A brief overview of biofilm-specific resistance mechanisms is provided, but this is a highly multifactorial and rapidly expanding field that warrants further research. Progression in this field is dependent on the development of suitable biofilm models that acknowledge the complexity of the ocular environment. Abiotic models of biofilm formation (where biofilms are studied on non-living surfaces) currently dominate the literature, but co-culture infection models are beginning to emerge. In vitro, ex vivo and in vivo corneal infection models have now been reported which use a variety of different experimental techniques and animal models. In this review, we will discuss existing corneal infection models and their application in the study of biofilms and host-pathogen interactions at the corneal surface.

Keywords: bacterial keratitis; biofilm; cornea; ex vivo; in vitro; in vivo; infection; microbial keratitis; models.

Figure 1

The biofilm life-cycle. The biofilm life-cycle consists of three main stages: (1) Attachment: Planktonic, free-living bacteria adhere to a surface. This is mediated by a combination of physical factors (e.g., surface hydrophobicity, electrostatic interactions) and bacterial appendages (e.g., pili, flagella). (2) Biofilm maturation: Adherent bacteria proliferate to form microcolonies. Bacteria produce Extracellular Polymeric Substances (EPS), EPS matrix stabilises the bacterial network. (3) Dissemination: A subset of bacteria detach, regain some of the characteristics of planktonic bacteria and colonise distal sites.

Figure 2

Human corneal layers: (A) Schematic representation, and (B) haematoxylin and eosin staining. The cornea has six distinct layers and the outermost layer is the corneal epithelium, which is made up of 5-7 rows of tightly packed corneal epithelial cells. These cells lie on an acellular, collagenous layer named the Bowman’s layer and together the epithelium and the Bowman’s layer are essential in the protection of the underlying stromal tissue. The stroma constitutes 90% of the overall thickness of the cornea and is composed of mainly type I collagen and differentiated keratocytes. Beneath the stroma is the Pre-Descemet’s layer (also known as Dua’s layer) and the Descemet’s membrane. These collagen-rich, acellular layers separate the stromal tissue from the endothelium. The endothelium is composed of a single layer of cells, which are mainly hexagonal in shape. This layer is adjacent to the anterior chamber and constitutes the final layer of the cornea. Created with Biorender.com.

Figure 3

Scanning electron micrographs of ex vivo porcine corneas after 4 h Methicillin-Resistant Staphylococcus aureus (MRSA) infection (A), 6 h MRSA infection (B), 24 h Pseudomonas aeruginosa infection (C) and the uninfected porcine cornea (D). Arrows show MRSA adhering to corneal epithelial cells.