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Developing a Pseudomonas aeruginosa keratitis model in Swiss albino mice for clinical assessment
For correspondence: Dr Lalan Kumar Arya, Regional Institute of Ophthalmology, Indira Gandhi Institute of Medical Sciences, Patna 800 014, Bihar, India e-mail: lalan.mku@gmail.com
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Received: ,
Accepted: ,
Abstract
Background & objectives
Pseudomonas aeruginosa is a leading cause of bacterial keratitis, known for its rapid progression, severe corneal damage, and resistance to treatment. Existing animal models exist to study disease mechanisms and therapeutic options require specialised conditions or complex procedures. This study aimed to develop a simplified, cost-effective, and reproducible murine model of P. aeruginosa keratitis using Swiss albino mice for translational research applications.
Methods
Four female Swiss albino mice (6–8 wk old) were maintained under standard conditions. Baseline ocular evaluation was done using a handheld slit lamp. The left corneas were abraded with a sterile 26G needle and inoculated topically with 10 μL of P. aeruginosa (1.0 × 10⁶ CFU/mL, ATCC 19660). The right eyes served as uninfected controls. Clinical signs were assessed on days 1, 2, and 3 using a standardised 0–4 scoring scale. Infection was confirmed through culture, biochemical tests, and PCR targeting the exotoxin A gene. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test.
Results
All infected eyes developed progressive keratitis marked by lid swelling, corneal haze, and stromal involvement. Control eyes remained unaffected. Clinical scores increased significantly over time (P< 0.05). Culture and molecular analyses confirmed successful infection.
Interpretation & conclusions
This simplified Swiss albino mouse model effectively mimics human P. aeruginosa keratitis and enables cost-efficient study of pathogenesis and therapeutic testing. It can facilitate standardised ophthalmic infection research.
Keywords
Cornea
experimental model
infection
keratitis
Pseudomonas aeruginosa
swiss albino mouse
Bacterial keratitis is a rapidly progressing ocular infection that can result in corneal ulceration, stromal necrosis, and permanent visual impairment if not promptly diagnosed and managed. Pseudomonas aeruginosa is among the most virulent causative organisms, particularly in contact lens wearers, individuals with ocular trauma, or post-surgical eyes. It produces a wide array of virulence factors, including exotoxins, proteases, and biofilms, which contribute to its aggressive clinical course and resistance to treatment1,2. Experimental animal models have played a crucial role in deciphering the pathogenesis of P. aeruginosa keratitis and evaluating the efficacy of novel therapeutics.
Murine models, in particular, offer several advantages due to their cost-effectiveness, availability of genetically defined strains, and a well-characterised immune system that allows for targeted immunological investigations3,4. Over the past decades, several models have been developed using different mouse strains and methods of bacterial inoculation, such as corneal scratch or intrastromal injection, which have contributed valuable insights into innate immune responses, neutrophil activity, and bacterial clearance mechanisms5,6.
Recent studies have further explored cytokine regulation and inflammatory mediators, including the roles of interleukins, toll-like receptors, and programmed cell death pathways in host defence against P. aeruginosa7-9. Despite these advancements, existing models often require technical complexity, specialised mouse strains, or invasive procedures, limiting their broad utility. There remains a need for a simplified, reproducible, and ethically feasible model that can be employed in routine laboratory settings without compromising scientific rigor.
To address this gap, the present study aimed to establish a cost-effective and reproducible murine model of P. aeruginosa keratitis using Swiss albino mice. The model utilises a standardised corneal abrasion technique followed by topical inoculation of a defined bacterial dose, with subsequent clinical grading, culture confirmation, and PCR validation. This approach may provide an accessible platform for studying disease mechanisms and testing therapeutic interventions in bacterial keratitis.
Materials & Methods
Study design
This experimental animal study was designed to develop and characterize a simplified model of Pseudomonas aeruginosa keratitis. The study was conducted at the Regional Institute of Ophthalmology, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, India from October 2020 to March 2021 using four Swiss albino mice to establish and assess infection progression, as previously outlined in murine keratitis models6,10. The study was approved by the Institutional Animal Ethics Committee.
Bacterial strain and culture conditions
A standard strain of Pseudomonas aeruginosa (ATCC 19660) was used, consistent with widely utilised reference strains in keratitis models6,11. The organism was cultured overnight at 37 °C on blood agar and MacConkey agar. Identification was confirmed by Gram staining and standard biochemical tests. A single colony was transferred into Luria-Bertani (LB) broth and incubated to mid-logarithmic phase (OD₆₀₀ = 0.3), approximating 1.0 × 10⁶ CFU/mL12. To verify the bacterial concentration, a 10⁻⁴ dilution yielded 35 colonies on MacConkey agar, estimating the original culture at 3.5 × 10⁷ CFU/mL.
DNA isolation and PCR amplification
Genomic DNA was isolated from colonies using the Qiagen DNA extraction kit (Hilden, Germany) with tissue lysis protocol as per manufacturer instructions. Strain identity was confirmed via PCR targeting the Exotoxin A gene, as described in previous study10. Primers ETA1 (5′-GAC AAC GCC CTC AGC ATC ACC AGC-3′) and ETA2 (5′-CGC TGG CCC ATT CGC TCC AGC GCT-3′) were used, yielding a 367 bp amplicon, consistent with molecular diagnostics methods13.
Animals and housing conditions
Four Swiss albino mice (6–8 wk old) were obtained from a CPCSEA-registered vendor and housed under a 12-h light/dark cycle with controlled temperature and ad libitum access to food and water, in accordance with established guidelines14. All animal procedures complied with the NIH Guide for the Care and Use of Laboratory Animals.
Anaesthesia and corneal scarification
Mice were anesthetised intraperitoneally with a combination of ketamine (100 mg/mL) and xylazine (10 mg/mL) mixed in a 1:4 ratio, administered at a dose of 0.1 mL per 20 g of body weight15. A central 1 mm corneal epithelial defect was then created using a sterile 26-gauge needle, ensuring the underlying stroma remained intact11. The right eye was left untreated to serve as a non-infected control.
Infection protocol
A 10 μL inoculum of P. aeruginosa (∼1.0 × 10⁶ CFU) was applied topically to the abraded left cornea under anaesthesia, following a previously described method6. The unscarified right eye remained untreated and served as an internal control.
Clinical observation and grading
Eyes were examined on days 1, 2, and 3 using a handheld slit lamp and surgical microscope, based on a standardised 0–4 scoring system15,16: 0 = clear; 1 = mild opacity; 2 = dense partial opacity; 3 = full corneal opacity; 4 = perforation or phthisis. Fluorescein staining and cobalt blue light were used to evaluate epithelial defects. Images were documented microscopically.
Statistical analysis
Clinical scores were analysed using GraphPad Prism v10.2.0. Results were presented as mean±standard deviation (SD). Group comparisons were performed using one-way ANOVA followed by Tukey’s post hoc test. A P value < 0.05 was considered statistically significant, consistent with previously described analytical protocols6.
Results
An experimental model of P .aeruginosa keratitis was successfully established in Swiss albino mice following controlled corneal epithelial abrasion and topical inoculation with a standardised suspension of P. aeruginosa (ATCC 19660) on day 1. As illustrated in figure A show the naive (uninfected) eyes of four Swiss albino mice, while (Figure B) depict the induction of infection through central corneal epithelial abrasion (∼1 mm in diameter) using a sterile 26-gauge needle, followed by topical application of 10 μL of P. aeruginosa suspension (∼1.0 × 10⁶ CFU/mL) to the left eye. The right eyes were left unscarified and uninoculated to serve as internal controls and remained clinically unaffected throughout the observation period.
- Ocular Examination and Induction of Pseudomonas aeruginosa Keratitis. (A) Naïve (uninfected) mouse eyes. (B) Corneal epithelial abrasion (∼1 mm) made with a 26-gauge needle, followed by topical inoculation with Pseudomonas aeruginosa (Day 1). (C–D) Day 1 post-infection: fluorescein-stained images under a surgical microscope and cobalt blue filter. (E–F) Day 2 post-infection: corresponding fluorescein and cobalt blue images showing progression of infection. Progressive changes include lid edema, conjunctival hyperaemia, enlarged epithelial defects, and stromal edema.
Establishment of experimental keratitis and day 1 post infection clinical findings
On day 1 post-infection, the inoculated eyes of all four mice demonstrated initial signs of keratitis including lid swelling, conjunctival hyperaemia, and mild-to-moderate corneal haze (Figure C), fluorescein staining revealed epithelial defects, which were further highlighted under a cobalt blue filter (Figure D) confirming early inflammatory changes. The right (control) eyes showed no pathological changes.
Clinical progression of corneal lesions
Corneal disease severity was evaluated using a standardised 0–4 scoring system based on opacity and structural involvement. On day 1 post infection, all infected eyes exhibited slight-to-moderate corneal opacity (mean score: 1.25 ± 0.50), mainly localised to the central cornea. By day 2 post infection, lesion severity worsened, with increased lid oedema and dense corneal opacities partially obscuring the visual axis (mean score: 2.50 ± 0.58). On day 2 post-infection (Figure E and F), clinical examination revealed significant progression to full-thickness corneal opacification, stromal edema, and early descemetocele formation (mean score: 3.25 ± 0.50), indicating advanced inflammation and tissue damage (Table). During anaesthesia for day 3 assessment, one mouse died, likely due to procedure induced hyperthermia. This incident underscores the importance of thermal regulation and monitoring during repeated anaesthesia in small animal models.
| Comparison | Mean Score ± SD (group 1) | Mean Score ± SD (group 2) | P value |
|---|---|---|---|
| Day 1 vs. Day 2 | 2.1 ± 0.3 | 3.5 ± 0.4 | < 0.05 |
| Day 1 vs. Day 3 | 2.1 ± 0.3 | 3.7 ± 0.5 | < 0.01 |
| Day 2 vs. Day 3 | 3.5 ± 0.4 | 3.7 ± 0.5 | > 0.05 |
SD, standard deviation
Quantitative assessment of disease severity
Statistical analysis of the clinical grading scores revealed a significant and consistent progression of keratitis over time. One-way ANOVA followed by Tukey’s post hoc test showed that the increase in clinical scores from day 1 to day 2 (P< 0.05), and from day 1 to day 3 (P< 0.01), was statistically significant, validating the reliability of the disease model.
These results collectively confirm successful establishment of infection, progressive corneal pathology, and clinical reliability of the model for studying bacterial keratitis and evaluating potential therapeutic interventions.
Microbiological and molecular confirmation of infection
The presence of P. aeruginosa in the infected eyes was confirmed by culture, biochemical tests, and PCR. Corneal swabs from infected eyes showed consistent bacterial growth on blood agar (with beta-haemolysis) and MacConkey agar (non-lactose fermenting colonies with green pigment). The isolates were oxidase-positive, motile, and produced the characteristic blue-green pigment pyocyanin. For molecular confirmation, genomic DNA was extracted from infected corneal tissues. PCR amplification targeting the P. aeruginosa-specific exotoxin1 gene yielded a distinct band of 367 bp, confirming the P. aeruginosa infection. No amplification was observed in samples from the control eyes.
Discussion
In this study, we successfully established a simplified and reproducible murine model of P. aeruginosa keratitis using Swiss albino mice, with the goal of simulating key pathological features of human bacterial keratitis. This model was developed using a consistent corneal scratch technique followed by inoculation with a defined concentration of P. aeruginosa, leading to rapid disease progression within 24-72 h. Clinical scoring, microbial culture, and PCR-based molecular confirmation validated the onset and progression of keratitis, ensuring the reliability of the model.
Our model is particularly innovative in its simplicity, cost-effectiveness, and translational relevance. Unlike rabbit models which require intensive care, surgical precision, and higher maintenance costs17, this murine model is easy to establish and maintain, making it accessible to resource-constrained laboratories. Swiss albino mice were chosen due to their anatomical and immunological similarities to humans, enabling reproducible results and a meaningful extrapolation of findings3,4. This model addresses previous inconsistencies noted in literature, such as variations in corneal injury techniques and bacterial inoculum delivery that often impair reproducibility5,6.
Clinically, the model closely mimics human P. aeruginosa keratitis, characterised by intense corneal infiltration, oedema, ulceration, and progressive opacification. These features make it suitable for testing novel antimicrobial agents, especially against multi-drug resistant P. aeruginosa strains18,19. Moreover, the model can be extended to study immunopathogenesis, including cytokine profiling and immune cell infiltration, which are central to the host’s inflammatory response to infection1,2. It may also serve as a preliminary screening tool prior to more complex or expensive preclinical systems.
Our findings align with earlier studies that used C57BL/6 or BALB/c mice to investigate keratitis induced by various pathogens, including Staphylococcus aureus, Fusarium solani, and Acanthamoeba spp20,21. However, our use of Swiss albino mice offers an alternative model that is genetically homogeneous and well-suited to histopathological and therapeutic evaluation. This is particularly important given the growing emphasis on cost-effective research approaches under ethical mandates such as the 3Rs—reduction, refinement, and replacement22.
Recent studies have improved mouse models of P. aeruginosa keratitis. One study found that using the appropriate age of mice and the correct bacterial dose led to more consistent infections7. Another study demonstrated that the new antibiotic cefiderocol was effective in treating drug-resistant infections in these models23. A further study discovered that a protein called IL-24 increased inflammation and bacterial load in infected eyes8. These findings support the continued relevance of murine models in advancing translational keratitis research.
Despite the promising outcomes, the study has some limitations. The small sample size (n=4) restricts the statistical power and generalisability of therapeutic findings. While sufficient for a proof-of-concept, larger cohorts will be needed in future studies to validate reproducibility and detect subtle immunological differences. Only a single inoculum concentration was tested, and dose–response experiments are planned in subsequent phases. Additionally, the current study did not include histopathological analysis or cytokine profiling, although these are integral components of future work to further characterize host-pathogen dynamics and disease mechanisms.
Overall, this model serves as a valuable platform for exploring bacterial keratitis, especially for preclinical evaluation of antimicrobial therapies and immunomodulatory strategies. The simplicity, reliability, and ethical compliance of the model make it highly adaptable for both basic and translational research. With further refinement, this murine system can significantly contribute to the development of new therapeutic interventions for vision-threatening corneal infections.
Financial support & sponsorship
The study received funding support from Science and Engineering Research Board, Government of India and Regional Institute of Ophthalmology, Indira Gandhi Institute of Medical Sciences, Patna, India (Grant Number- SERB/F/3631).
Conflicts of Interest
None.
Use of Artificial Intelligence (AI)-Assisted Technology for manuscript preparation
The authors confirm that there was no use of AI-assisted technology for assisting in the writing of the manuscript and no images were manipulated using AI.
References
- Pathogenesis of contact lens-associated microbial keratitis. Optom Vis Sci. 2010;87:225-32.
- [CrossRef] [PubMed] [Google Scholar]
- The type III secretion system of Pseudomonas aeruginosa: Infection by injection. Nat Rev Microbiol. 2009;7:654-65.
- [CrossRef] [PubMed] [PubMed Central] [Google Scholar]
- Distribution and phenotype of dendritic cells and resident tissue macrophages in the mouse eye. Invest Ophthalmol Vis Sci. 1999;40:621-32.
- [Google Scholar]
- Granulocytes in innate immunity: Anti-microbial strategies and inflammatory responses. Microbiol Spectr. 2019;7
- [Google Scholar]
- Pseudomonas aeruginosa corneal infection: studies on the role of neutrophils and macrophages. Invest Ophthalmol Vis Sci. 2007;48:4150-58.
- [Google Scholar]
- TLR4 and TLR5 regulate innate immunity and tissue damage following infection with Pseudomonas aeruginosa. J Immunol. 2010;185:2086-94.
- [Google Scholar]
- Establishment of an optimized mouse model of Pseudomonas aeruginosa keratitis for evaluating therapeutic interventions. Ocul Surf. 2023;28:47-54.
- [Google Scholar]
- IL-24 contributes to corneal inflammation and bacterial burden in Pseudomonas aeruginosa keratitis. Exp Eye Res. 2024;230:109550.
- [Google Scholar]
- Programmed cell death pathways regulate bacterial keratitis in diabetic mice. Mol Vis. 2024;30:110-20.
- [Google Scholar]
- Development of a mouse model of Pseudomonas aeruginosa keratitis. Exp Eye Res. 2019;181:232-39.
- [CrossRef] [PubMed] [Google Scholar]
- Evidence for recurrent corneal infection with Pseudomonas aeruginosa in mice. Curr Eye Res. 2007;32:391-98.
- [Google Scholar]
- Growth phase analysis and CFU correlation of Pseudomonas aeruginosa in LB broth. J Microbiol Methods. 2009;78:163-5.
- [Google Scholar]
- Rapid identification of Pseudomonas aeruginosa from ocular isolates by PCR using exotoxin A-specific primers. Mol Cell Probes. 2000;14:199-204.
- [CrossRef] [PubMed] [Google Scholar]
- Immunomodulatory and therapeutic effects of Ocimum sanctum Linn. on experimental animal model of chronic bacterial prostatitis. Int Immunopharmacol. 2011;11:1967-73.
- [CrossRef] [PubMed] [Google Scholar]
- Development of a murine model of Pseudomonas aeruginosa keratitis. Exp Eye Res. 2014;127:43-48.
- [Google Scholar]
- Mutants in genes related to stress responses show altered virulence in Pseudomonas aeruginosa keratitis models. Infect Immun. 2010;78:1549-56.
- [Google Scholar]
- Bacterial adaptation during chronic respiratory infections. Ocul Immunol Inflamm. 2021;29:1043-50.
- [Google Scholar]
- Extended-spectrum beta-lactamases: A clinical update. Clin Microbiol Rev. 2005;18:657-86.
- [CrossRef] [PubMed] [PubMed Central] [Google Scholar]
- Emerging therapies against infections with Pseudomonas aeruginosa. F1000 Res. 2019;8:Rev-1371.
- [Google Scholar]
- MIP-2 (CXCL2) is critical for PMN infiltration and pathogen clearance in bacterial keratitis. Curr Eye Res. 1998;17:963-70.
- [Google Scholar]
- TLR4 is required for host resistance in Pseudomonas aeruginosa keratitis. Invest Ophthalmol Vis Sci. 2006;47:4910-6.
- [CrossRef] [PubMed] [Google Scholar]
- The ethics of animal research. EMBO Reports. 2007;8:526-30.
- [CrossRef] [PubMed] [PubMed Central] [Google Scholar]
- Cefiderocol is an effective topical monotherapy for experimental extensively drug-resistant Pseudomonas aeruginosa keratitis. Ophthalmol Sci. 2023;4:100452.
- [CrossRef] [PubMed] [PubMed Central] [Google Scholar]