Keratitis is an inflammation of the cornea, characterized by rapidly advancing destruction of the stroma, that can lead to complications like thinning or perforation of the cornea, but also to blindness (Dakhil et al., 2017, Lakhundi et al., 2017). It is one of the most frequent diseases causing loss of sight worldwide (Al-Mujaini et al., 2009). Epidemiology indicates more than two million cases of microbial keratitis yearly (Ung et al., 2019). Besides being a health threatening condition, it is also an economic burden (Hossain, 2021). Different bacterial species such as Pseudomonas aeruginosa and Staphylococcus aureus but also fungi such as Candida spp., Fusarium spp. and Aspergillus spp. or protozoa like Acanthamoeba spp. can cause keratitis (Kaye et al., 2010). The therapy of bacterial keratitis is very intense, especially in the early phase of acutely developed infections (Pearce et al., 2022). The emergence of multidrug resistance amongst these pathogens is a new challenge. When diagnosis and empiric therapy is deferred, half of the patients will suffer vision deterioration (Bennett et al., 1998). A delay of effective therapy due to pathogen resistance will have similar effects. Therefore, research needs to be intensified in order to find new possibilities in the treatment of bacterial keratitis and to ultimately improve the quality of life for our patients.

P. aeruginosa is a Gram-negative bacterium and an important healthcare-associated pathogen. It is responsible for many opportunistic and chronic infections, including ventilator- and cystic fibrosis-associated pneumonia. Its remarkable intrinsic and acquired resistance profile is increasingly challenging (Diggle and Whiteley, 2020, Stover et al., 2000, Strateva and Yordanov, 2009). Mechanisms including efflux pumps, antibiotic-deactivating enzymes, restricted outer membrane permeability but also horizontal gene transfers are key players in this context (Pang et al., 2019). Notably, a lower Minimal Inhibitory Concentration (MIC) of the employed antibiotic is concomitant with a better clinical outcome of Pseudomonas spp. keratitis (Kaye et al., 2010). In the United-States, most cases of keratitis are associated with contact lens use and for this reason often caused by P. aeruginosa (Konda et al., 2014, Lin et al., 2019, Schein et al., 1989).

Experimental keratitis in mice is an important tool to test new antimicrobial substances in the fight against one of the most frequent blindness causing diseases worldwide (Al-Mujaini et al., 2009, Wu et al., 2017, Zavarshani et al., 2019, Zhang et al., 2021). In this context, the corneal epithelium that protects the collagenic stroma is considered to constitute a major ocular defense against P. aeruginosa (Fleiszig and Evans, 2002, Ung and Chodosh, 2021). A better corneal penetration of tested compounds can be achieved by superficial epithelium removal prior to infection (Barequet et al., 2004, Wu et al., 2017). However, corneal self-healing during the observation period of the infection model needs to be excluded, preserving the possibility to ascribe any improvement of the clinical status to the tested medication. We will focus here on experimental P. aeruginosa keratitis in mice. Other laboratory animals like rabbits, guineapigs or even rats are also in demand (Bozkurt et al., 2021, Davis et al., 1979, Kilic et al., 2018). Marquart (Marquart, 2011) discussed the advantages and disadvantages of the use of different laboratory animals in this context. Experimental P. aeruginosa keratitis is known to be difficult to establish in mice (Urwin et al., 2020). Keratitis is, indeed, rather a human disease than a murine. Some mouse strains are more susceptible to develop a corneal inflammation than others. For instance, Th1-responder strains like C57BL/6 N suffer from IL-12 mediated corneal damage, whereas Th2-responders like BALB/c mice show an IL-18 controlled bacterial reduction with less destruction of the corneal infrastructure (Hazlett, 2005). As with respect to age, both younger (weeks dimensions) (Chojnacki et al., 2019, Kwong et al., 2007, Saraswathi and Beuerman, 2015) and older (months dimensions) (Deichelbohrer et al., 2017, Hazlett et al., 1990, Hazlett et al., 1986, Wu et al., 2017) mice have been used in experimental keratitis models. Beyond those variables, the employed technique, the selection of the bacterial strain as well as its concentration on infection are critical.

Here, we reinvestigated the impact of the age on corneal infection susceptibility in experimental designs. Serial dilutions and flow cytometry (Fluorescence Activated Cell Sorting; FACS) of eye homogenates enabled microbiological and immunological analysis, respectively. The impact of a corneal epithelium-removal procedure was also explored. We discuss here different prerequisites, including the critical role of the P. aeruginosa strain selection, to successfully establish an experimental keratitis in mice that can be used for drug testing.