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(Investigative Ophthalmology and Visual Science. 2003;44:1591-1597.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.02-0656
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A New Topical Model of Staphylococcus Corneal Infection in the Mouse

Dalia O. Girgis,1 Gregory D. Sloop,2 Julian M. Reed,1 and Richard J. O’Callaghan1,3

1From the Departments of Microbiology, Immunology, and Parasitology and 2Pathology, Louisiana State University Health Sciences Center, and 3Department of Ophthalmology, LSU Eye Center, New Orleans, Louisiana.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To establish, in the scarified mouse eye, a new model of Staphylococcus aureus keratitis suitable for studies of pathogenesis and host defense mechanisms.

METHODS. Corneas of three strains of mice (BALB/c, A/J, and C57BL/6) were scarified and inoculated with S. aureus strain 8325-4. Mice underwent slit lamp examination (SLE) at 1, 3, 5, 7, and 9 days after infection and were killed. Histopathologic analyses, determination of bacterial colony-forming units (CFU), and myeloperoxidase (MPO) activity assays were performed at each time point.

RESULTS. S. aureus keratitis developed in both BALB/c and A/J strains of mice, but not in C57BL/6. The BALB/c and A/J strains demonstrated greater susceptibility to infection, as evidenced by significantly higher SLE scores and more viable bacteria per infected eye than in C57BL/6 mice at 5, 7, and 9 days after infection (P ≤ 0.0001). Histopathologic analysis and MPO assays of infected A/J mice both revealed an influx of polymorphonuclear leukocytes (PMNs). Histology demonstrated presence of leukocytes in the aqueous humor, migration of PMNs into infected tissue, corneal erosion, and edema in the eyes of infected A/J mice. Whereas infected BALB/c mice demonstrated both PMN migration and corneal edema, eyes of infected C57BL/6 mice failed to show even mild histopathologic changes.

CONCLUSIONS. These studies demonstrate the establishment of Staphylococcus keratitis in the mouse eye. This model should provide for a large range of future studies that are currently unavailable in the rabbit keratitis model, particularly those requiring a genetically altered host or specific immunologic reagents.

The bacterium Staphylococcus aureus is an opportunistic pathogen that is frequently found as normal flora on skin surfaces and is also responsible for a wide range of diseases including keratitis, endocarditis, septicemia, abscesses, and catheter-related infections.1 The most common cause of bacterial keratitis in many populations is S. aureus,2 3 4 both in normal and previously compromised corneas.3 4 5 S. aureus corneal infections occur primarily in contact lens wearers and patients with ocular defects or compromised systemic immunity.5 6 7 Staphylococcus keratitis is a serious condition with significant morbidity that can result in irreversible corneal scarring, a pathologic effect that reduces visual acuity and can lead to blindness.8 9

Whereas topical application of a large S. aureus inoculum to scarified rabbit eyes can result in inflammation, an actual infection with bacterial replication characteristic of keratitis is not produced.10 As a result, studies of corneal virulence, host defense, and the activity of specific staphylococcal proteins traditionally have been conducted in a rabbit model in which S. aureus keratitis is produced after intrastromal injection of log phase bacteria into the cornea.11

Recently, Hume et al.12 have achieved Staphylococcus keratitis with bacterial replication after topical inoculation of the rabbit eye. This model was based on the finding that S. aureus is killed in the rabbit tear film by group II phospholipase A2 (PLA2).13 PLA2 comprises a group of lipolytic enzymes that specifically release fatty acids, often arachidonic acid, from the sn-2 position of membrane phospholipids for production of essential lipid mediators.14 Such severe alterations of the membrane can activate a cell wall autolytic enzyme that directly causes cell lysis.15 The mammalian secretory (s)PLA2 is mobilized to sites of inflammation and exerts potent antibacterial activity against Gram-positive bacteria.1 The rabbit topical inoculation model of Staphylococcus keratitis in the rabbit eye involves the inhibition of PLA2 activity on bacteria by the addition of spermidine. Spermidine, a cationic molecule, binds the bacterial surface,16 thereby inhibiting subsequent digestion by PLA2 and protecting the bacteria in the tear film.13

The present study was undertaken to achieve a model of S. aureus keratitis in the mouse after topical inoculation of bacteria to the scarified mouse eye. Whereas the wounded cornea model in mice has been well characterized for infective strains of Pseudomonas,17 18 19 20 such an infection using S. aureus has not been previously demonstrated. The establishment of a keratitis model in the mouse with S. aureus would provide a new means to study the interaction between Staphylococcus and its host. Determination of the role of S. aureus virulence factors in the mouse eye could provide new insights into corneal pathogenesis, particularly as related to those factors that mediate tissue damage. In addition, the new model will allow for a large range of studies that cannot be performed using the rabbit model because of the lack of immunologic reagents and genetically altered animal strains. As a result, a mouse model is anticipated to provide for advancements in understanding the effects of bacterial toxins as well as the host defense system: both innate defenses and the response to infection. These topics are of great importance because of the limited information currently available.

Therefore, the goals of this study were to define a new animal model of S. aureus keratitis by quantifying bacterial replication in the scarified mouse eye after topical Staphylococcus application and to determine the pathologic effects of this ocular infection.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Mice
Male mice (6–7 weeks of age) of the BALB/c, A/J, and C57BL/6 strains were purchased from the National Cancer Institute (Frederick, MD). All animals were maintained according to institutional guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Bacteria
S. aureus strain 8325-4 used in this study has been described for use in the rabbit intrastromal injection and topical inoculation models of keratitis.12 21 Bacteria were grown overnight in tryptic soy broth (TSB; Difco Laboratories, Detroit, MI) at 37°C and then subcultured to log phase under the same conditions.

Infection of Mice
Mice were challenged with S. aureus strain 8325-4 and anesthetized with ketamine HCl (Ketaset; Bristol Laboratories, Syracuse, NY) and xylazine (Rompum; Miles Laboratories, Shawnee, KS). A 1.5-mL volume of xylazine (100 mg/mL) was combined with 10 mL of 100 mg/mL ketamine, diluted 1:4 with saline, and injected subcutaneously at a dose of 0.1 mL/20 g body weight. Each cornea was scarified with a 30.5-gauge needle by making four parallel incisions to the corneal surface that did not penetrate beyond the superficial stroma. A 5-µL aliquot containing a 1.0 x 108-colony forming unit (CFU) suspension of bacteria was applied to each scarified cornea. For these experiments, the eyes of control mice were neither scarified nor inoculated with bacteria. BALB/c, A/J, and C57BL/6 mice were monitored by slit lamp examination (SLE) at postinfection (PI) days 1, 3, 5, 7, and 9 (n = 7 mice/group, repeated two additional times), at which times mice were killed by cervical dislocation. After sacrifice at various times after infection, eyes (n = 5/group) were used for CFU and myeloperoxidase (MPO) determinations, and remaining eyes (n = 2/group) were used for histopathologic analysis.

Quantification of Viable Bacteria
Eyes were enucleated and homogenized in 1.0 mL sterile phosphate-buffered saline (PBS) with sterile disposable tissue grinders (Kontes Scientific, Vineland, NJ). To quantify viable bacteria, a 0.1-mL aliquot of the homogenate was serially diluted 1:10 in PBS. Serial dilutions (0.1 mL/plate) were plated onto tryptic soy agar (TSA; Difco) and mannitol salt agar (MSA) plates (EM Science, Gibbstown, NJ) in triplicate and incubated at 37°C for 24 hours. Colonies were counted, and CFU per cornea were expressed as logarithmic values.

Slit Lamp Examination
The ocular disease was evaluated both macroscopically and microscopically using a slit lamp biomicroscope (Topcon Biomicroscope SL-5D; Kogaku Kikai KK, Tokyo, Japan) up to PI day 9. Observations of S. aureus-infected mouse eyes were graded with a modification of the scale described by Hazlett et al.22 : 0, clear and normal; +1, readily detectable opacity; +2, dense opacity or opacity partially covering the entire corneal surface over pupil; +3, dense opacity covering entire corneal surface over pupil; +4, moderate to dense opacity covering entire corneal surface with corneal erosion. Corneal erosions were detected with fluorescein (Fluor-I-Strip AT; Wyeth-Ayerst Laboratories Inc., Philadelphia, PA).

Histopathology
Eyes from control and infected BALB/c, A/J, and C57BL/6 mice were enucleated at PI days 1, 3, 5, 7, and 9. Whole eyes of uninfected and infected mice were immediately fixed in 10% neutral buffered formalin (EK Industries, Joliet, IL). After fixation, eyes were bisected and processed as previously described.23 Briefly, fixed tissue was dehydrated in a series of ethanol baths of increasing concentration. Once dehydrated, sections were held in xylene. The dehydrated tissue was embedded in paraffin and cut into sections of 5 µm. Sections were then rehydrated and stained with hematoxylin and eosin.

MPO Activity Assay
To assess PMN activity in the mouse eye, we quantified the amount of MPO in eye homogenates of uninfected and infected BALB/c, A/J, and C57BL/6 mice, as previously described.24 25 26 Briefly, hexadecyltrimethylammonium bromide (CTAB; Sigma, St. Louis, MO) was added to each sample at a final concentration of 0.5% and an MPO microtiter assay, based on an o-dianisidine-based colorimetric reaction, was used. Reactions were incubated at room temperature, and the change in optical density at 450 nm was determined every 2 minutes for 12 minutes. The units of MPO were calculated as described previously for the microtiter plate assay.25 One unit of MPO activity has been reported to be equivalent to approximately 2 x 105 PMNs.26 All assays were performed in triplicate.

As an additional control, the MPO activity in uninfected scarified mouse eyes (n = 3/group) of each strain was measured. Scarification did not significantly affect the level of MPO in uninfected eyes (P ≥ 0.61).

Statistical Analysis
The mean ± SEM of SLE scores, CFU per homogenate, and units of MPO was determined on computer (Statistical Analysis Systems software; SAS, Inc., Cary, NC). Statistical analyses were performed with a one-way nested analysis of variance on each group. Protected t-tests were then determined between least-square means derived from each variance analysis on each group. P ≤ 0.05 was considered significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
After the inoculation of scarified eyes of all three strains of mice, the eyes of BALB/c and A/J mice, but not C57BL/6, demonstrated gross pathologic signs of infection and significant increases in SLE scores (Fig. 1) . The SLE scores of A/J infected mice increased steadily from PI days 3 to 9 and were significantly higher than BALB/c and C57BL/6 mice at PI days 5, 7, and 9 (Fig. 1 , P ≤ 0.0001). An increase in SLE scores of BALB/c mice was demonstrated at PI days 3 and 5, but remained constant thereafter through PI day 9 (Fig. 1) . The C57BL/6 mice showed no disease as evident by SLE analysis throughout the course of infection (Fig. 1) .



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  		FIGURE 1. Slit lamp examination (SLE) of ocular disease in infected BALB/c, A/J, and C57BL/6 mice. Corneas were scarified and topically inoculated with S. aureus (500,000 CFU). Ocular disease was graded, and mean SLE scores were calculated by summation of the scores for each group (n = 5/group) of mice divided by the total number of mice graded at each time point. Data are expressed as the mean ± SEM.

 
The observed pathologic changes in BALB/c and A/J mice included severe iritis, corneal infiltrate, and corneal erosions. At PI day 1, no pathologic changes were observed in either strain of mice, and corneas were clear (grade 0). At PI day 3, slight or no corneal opacity (0 to +1) was observed in infected BALB/c mice compared with slight corneal opacity (+1) in A/J mice. By PI day 5, some corneal opacity (+1 to +2) was observed in BALB/c mice (Figs. 2A 2B 2C) , and dense corneal opacity (+2 to +3) was observed in infected A/J mice (Figs. 2D 2E 2F) . Whereas disease in BALB/c mice did not progress beyond +2, even up to PI day 9 (Figs. 3A 3B 3C) , dense opacities that, in nearly all cases, covered the entire corneal surface in addition to corneal erosions (+4) were observed in A/J mice by PI day 9 (Figs. 3D 3E 3F) .



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  		FIGURE 2. Photomicrographs at PI day 5 of ocular disease in BALB/c and A/J mice infected with S. aureus. (A–C) Infected BALB/c mice (n = 5/group) demonstrated mild (A, +1) to dense opacities (B, C, +2) partially covering the corneal surface. (D–F) Infected A/J mice (n = 5/group) demonstrated partial dense opacity (D, +2) and dense opacity completely covering the entire corneal surface (E, F, +3).

 


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  		FIGURE 3. Photomicrographs at PI day 9 of ocular disease in BALB/c and A/J mice infected with S. aureus. (A–C) Infected BALB/c mice (n = 5/group) demonstrated mild (A, +1) to dense opacities (B, C, +2), partially covering the corneal surface. (D–F) Infected A/J mice (n = 5/group) demonstrated dense opacities covering the entire corneal surface in addition to corneal erosions (+4).

 
To further characterize the course of infection of BALB/c, A/J, and C57BL/6 mice, we quantified the number of viable S. aureus (CFU) recovered from infected mouse eyes at PI days 1, 3, 5, 7, and 9. The number of CFU recovered from infected eyes of A/J mice was found to be significantly higher than that in BALB/c mice at PI days 1 and 3 (Fig. 4 , P = 0.0007 and P = 0.0331, respectively). The bacterial loads detected in BALB/c and A/J mice at PI days 5, 7, or 9 were equivalent to each other, yet significantly higher than that of C57BL/6 infected mice (Fig. 4 , P ≤ 0.0001). The CFU per eye in C57BL/6 infected mice was equivalent to that of A/J mice at PI day 3, then decreased rapidly to near sterility from PI days 5 to 9 (Fig. 4) .



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  		FIGURE 4. Quantification of viable S. aureus in infected mouse eyes. CFU per infected mouse eye (BALB/c, A/J, and C57BL/6 strains, n = 5/group) after topical inoculation of S. aureus (500,000 CFU). Data are expressed as the mean ± SEM.

 
PMN infiltration into normal and infected mouse eyes was determined by an MPO assay at various PI times. Uninfected control eyes had minimal MPO activity (Fig. 5) . At PI day 1, the MPO activity in infected A/J eye homogenates was approximately two times higher than that in infected BALB/c and C57BL/6 tissues (Fig. 5 , P ≤ 0.0001). At PI day 3, no significant differences in the number of PMNs in infected eyes of all three strains were detected. On PI days 5, 7, and 9, the MPO activities of infected BALB/c and A/J mice were equivalent and both significantly higher than that in C57BL/6 mice (Fig. 5 , P ≤ 0.006).



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  		FIGURE 5. Effects of keratitis on MPO activity in infected mouse eyes. MPO activity in homogenates collected from infected BALB/c, A/J, and C57BL/6 mice (n = 5/group) at each time point during infection. One unit of MPO activity was equivalent to approximately 2 x 105 PMNs. Data are expressed as the mean ± SEM.

 
All three strains of mice underwent histopathologic analysis from PI days 1 to 9. However, shown herein are results from PI days 5 and 9, demonstrating the full extent of pathologic changes observed in BALB/c and A/J infected eyes. In contrast to BALB/c and A/J infected mice, histopathologic analysis of eyes of infected C57BL/6 mice on PI days 5 and 9 failed to demonstrate even mild pathologic changes associated with infection.

Histopathologic analysis of the infected BALB/c mice demonstrated congestion of the vascularized tissue of the anterior chamber (Fig. 6A) and severe edema (Fig. 6B) at PI day 5. These changes persisted until PI day 9 (data not shown). In addition, migration of neutrophils into the aqueous humor was observed by PI day 9 (Fig. 6C) .



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  		FIGURE 6. Corneal histopathology at PI days 5 and 9 in BALB/c mice infected with S. aureus. (A) Ciliary body of BALB/c mouse at 5 days PI, showing congestion of vasculature (arrow). (B) Severe corneal edema at PI day 5 in a BALB/c mouse. (C) Ciliary body of BALB/c mouse at PI day 9. PMNs ready to migrate into the aqueous humor are demonstrated (arrows). Original magnification: (A, B) x400; (C) x1000.

 
Histopathologic analysis of infected A/J eyes at PI day 5 demonstrated adhesion of neutrophils to the corneal endothelium (Fig. 7A) , a protein-rich exudate in the anterior chamber (Fig. 7A) , and loss of intracellular and cell-basement membrane attachments in the corneal epithelium, resulting in focal loss of epithelial cells (Fig. 7B) . By PI day 9, the eyes of infected A/J mice showed numerous neutrophils adhering to the endothelium (Fig. 7C) , migration of neutrophils across the endothelium into the corneal stroma (Fig. 7C) , regional destruction of the endothelium (Fig. 7C) , and neutrophils migrating across Descemet’s membrane into epithelium with formation of microabscesses (Fig. 7C) .



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  		FIGURE 7. Corneal histopathology at PI days 5 and 9 in A/J mice infected with S. aureus. (A) A/J cornea at PI day 5. Adhesion of neutrophils to Descemet’s membrane. The aqueous humor contains numerous PMNs (open arrow) and a protein-rich exudate (filled arrow). (B) Loss of intracellular and cell-basement membrane attachments (arrows) in an A/J cornea at PI day 5. (C) Adhesion of PMNs to Descemet’s membrane and microabscess (open arrow) in epithelium of A/J cornea at PI day 9. Focal separation of endothelium from Descemet’s membrane is observed (filled arrow). Original magnification, x400.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that the scarified corneas of certain strains of mice are susceptible to infection by S. aureus. Infected A/J mice demonstrated a significantly greater susceptibility to infection than did infected BALB/c mice, as evidenced by higher initial bacterial loads and more severe ocular disease. Unlike either A/J or BALB/c mice, the C57BL/6 mice were highly resistant to infection and showed no pathologic effects of infection.

The pathologic effects of Staphylococcus keratitis in susceptible mice differed markedly from that observed in rabbits and humans. The human or rabbit eye infected with Staphylococcus demonstrates an intense infiltration of leukocytes into the tear film accompanied by a mucopurulent discharge,5 27 whereas in the infected mouse eye relatively few leukocytes appeared in the tear film with no discharge observed. In the infected mouse eye, leukocytes accumulated appreciably only in the aqueous humor, leading to PMN invasion of the cornea through its posterior surface. Whereas gross signs of infection appear within 20 hours in the infected rabbit eye, such signs require 3 to 5 days to appear in the infected mouse eye.

This study also demonstrated a marked difference between Staphylococcus and Pseudomonas relative to their ocular infection of various mouse strains. The eyes of C57BL/6 mice inoculated with a large Staphylococcus inoculum (500,000 CFU) did not show any signs of ocular infection, and most inoculated eyes of C57BL/6 mice were sterile by PI day 9. In contrast, the eyes of A/J mice infected with Staphylococcus had maximal CFU loads of nearly 4 log, with nearly 2 log CFU remaining in the heavily inflamed eyes at PI days 7 to 9. Thus, there was a multilog difference in maximal Staphylococcus loads in the eyes of the susceptible mouse strains compared with the resistant C57BL/6 mouse eyes. These observations are in contrast to Pseudomonas infection of mice in which mouse strains defined as susceptible or resistant each exhibit extensive Pseudomonas replication and considerable pathologic changes.28 29 30 31 However, strains of mice resistant to Pseudomonas infection, unlike susceptible ones, demonstrate corneal healing and are able to restore corneal clarity within a few days to a few weeks.28 31 32 33 34 As a result, the nearly complete resistance to Staphylococcus infection observed in C57BL/6 mice is unique.

An unexpected finding in this study was that the absence of PLA2 in one mouse strain did not fully correlate with susceptibility to Staphylococcus corneal infection after topical inoculation (Table 1) . PLA2 has been shown to be a major host defense molecule in the rabbit tear film12 13 35 and to be active in the human tear film.36 Inhibition of the bactericidal effects of PLA2 in the rabbit tear film allowed the topical inoculation with Staphylococcus to result in extensive bacterial replication and pathologic changes typical of keratitis.12 Despite this strong evidence of the critical role of PLA2 in host defense, the absence of this protective molecule in C57BL/6 mice37 38 did not result in their susceptibility to infection. The nonspecific host defense system of the murine tear film can involve more than PLA2 activity and an unknown mechanism is quite active in the C57BL/6 strain. Overall, there is a correlation between PLA2 and susceptibility to S. aureus keratitis, but the enzyme is supported by additional host defense mechanisms(s) in some animals (Table 1) .


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  		TABLE 1. Susceptibility to S. aureus Keratitis Associated with PLA2 Activity

 
In contrast to these unexpected results with C57BL/6 mice, the A/J strain, which is deficient in PLA2 activity,37 38 was found to be the strain most susceptible to Staphylococcus keratitis compared with BALB/c mice, which express active PLA2.37 38 The bacterial loads achieved in infected eyes of A/J mice were significantly higher than those of BALB/c mice. Similarly, infected A/J mice demonstrated an initial influx of PMN that was two times higher than that observed with infected BALB/c mice. As a result, the more severe ocular disease observed in A/J mice correlated with the absence of PLA2 activity and was most likely attributable to the presence of a greater number of Staphylococcus and PMN in the infected tissue.

The accumulation of leukocytes at the posterior corneal surface in the aqueous humor without an equivalent leukocyte response in the tear film suggests that the release and activity of chemotactic factors is more limited in the mouse tear film. In the rabbit, the PMN infiltration of the tear film was found to involve the inflammatory response of the overlying eyelid, implying that the chemotactic factors originating in the infected rabbit cornea stimulated this eyelid response.27 The absence of an equivalent response in the mouse tear film could imply that the chemotactic factors did not travel from the infected cornea to the overlying eyelid. Anatomic or biochemical barriers to the successful diffusion of the inflammatory signal, a lack of appropriate receptors for the inflammatory signal, or an inability of leukocytes to respond could explain the failure of the mouse tear film to demonstrate discharge indicative of significant inflammatory changes.

Although apoptosis has been documented in the rabbit model of S. aureus keratitis,23 the mechanisms responsible for pathogenesis in the mouse eye have not yet been analyzed. However, preliminary data indicate that apoptosis is induced in corneal epithelial cells of infected A/J mice (data not shown).

This study introduces a model of Staphylococcus infection that can be used to study the innate host defenses of the eye, the mechanisms of host response to infection, and the action of bacterial toxins in the induction of pathologic processes. Several aspects of the mouse infection appear unique, especially with regard to the potent host defense distinct from PLA2 in C57BL/6 mice. Analysis of the differences between mouse strains as well as differences observed among mice, rabbits, and humans could provide new biological information on Staphylococcus keratitis.


   Acknowledgements
 
The authors thank Brett Thibodeaux and Joseph Dajcs for technical assistance with this research.


   Footnotes
 
Supported by National Eye Institute Grant R01 EY10974 with additional support from National Eye Institute Core Grant EY02377.

Submitted for publication July 2, 2002; revised September 27 and October 25, 2002; accepted November 4, 2002.

Disclosure: D.O. Girgis, None; G.D. Sloop, None; J.M. Reed, None; R.J. O’Callaghan, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Richard J. O’Callaghan, The Department of Microbiology, Immunology and Parasitology, LSU Health Sciences Center, 1901 Perdido Street, New Orleans, LA 70112; rocall{at}lsuhsc.edu.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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