Staphylococcus Corneal Virulence in a New Topical Model of Infection

Abstract

purpose. To develop a topical inoculation model of Staphylococcus aureus keratitis in which scarification, contact lenses, and spermidine are used to inhibit the host defenses and to investigate the role of α-toxin in this infection.

methods. An α-toxin–positive parent strain (8325-4), its isogenicα -toxin–negative mutant (DU1090), and a genetically rescued form of the mutant (DU1090/pDU1212) were bound to rabbit-specific contact lenses, treated with spermidine (50 mM), and applied to scarified rabbit corneas. Eyes were treated topically with spermidine before and after lens application. Eyes were graded for disease by slit lamp examination (SLE) every 6 hours until 24 hours PI (PI), and erosion diameters were measured. Histopathologic changes and colony forming units (CFUs) of bacteria were determined.

results. Spermidine treatment and inoculation of eyes with Staphylococcus on contact lenses resulted in significant increases in both CFUs per cornea (P = 0.0041) and SLE score (P ≤ 0.0001), compared with eyes inoculated without spermidine treatment. The CFUs in eyes infected with 8325-4, DU1090, or DU1090/pDU1212 demonstrated a similar (P ≥ 0.1959) multilog increase in CFUs over the inoculum at 24 hours PI. The α-toxin–producing strains, 8325-4 and DU1090/pDU1212, caused significantly more disease than theα -toxin–deficient mutant DU1090 at 24 hours PI (P ≤ 0.0001). Histopathology revealed bacteria in scarified regions of the corneas and, for 8325-4 and DU1090/pDU1212, extensive epithelial sloughing and severe inflammation.

conclusions. A new topical model of infection has been developed, and α-toxin is an important virulence factor in this model.

Infection of the cornea by Staphylococcus aureus often involves the spread of bacteria from the skin or lid margin to the tear film and then to the cornea. ^{1 2 3} The host defense system of the tear film, which protects the eye from such infections, has long been recognized to include lysozyme, lactoferrin, complement, and IgA antibody. ^{4 5 6 7 8} More recently, Qu and Lehrer ⁹ have demonstrated that the human eye is also protected by the action of phospholipase A₂. Recent in vitro studies from our laboratory have demonstrated that phospholipase A₂ in the rabbit eye is a potent defense against S. aureus. ¹⁰ Host defenses were so effective that topical inoculation of a scarified cornea with 10 million colony forming units (CFUs) of S. aureus adhering to a contact lens failed to cause a productive infection and resulted in efficient bacterial killing within the tear film. 

The killing of Staphylococcus on contact lenses after topical corneal application is in contrast to the extensive bacterial replication and pathologic effect of keratitis that results from the intrastromal injection of bacteria into the cornea. The injection of only 100 CFUs into the corneal stroma results in severe keratitis, an indication that the stroma does not have the host defense systems found in the tear film. Keratitis resulting from intrastromal injection is characterized by bacterial replication and severe ocular changes, including corneal edema, corneal epithelial cell destruction, and iritis, as well as migration of polymorphonuclear neutrophils (PMNs) from the eyelid to the tear film. ^{11 12 13} Genetic, immunologic, and histopathologic studies have shown that the major cause of these pathologic events is the action of α-toxin, a lytic toxin produced by S. aureus in the late log phase of growth. ^{11 14 15}  

Bacterial infection of the cornea has been hypothesized to occur in a two-step process. ¹³ Bacteria first interact with the surface of corneal epithelial cells and then penetrate into the stroma where toxins mediate severe inflammation and tissue damage. The intrastromal model of Staphylococcus keratitis has been useful for studies of chemotherapy and pathologic reactions that occur during the intrastromal stage of infection. However, the intrastromal model cannot address the events occurring earlier in the infectious process at the epithelial cell surface. To study these reactions, a model of keratitis initiated by topical inoculation is needed. 

The results of recent studies on the interaction of Staphylococcus and tears have been applied in the present study to develop a topical inoculation model of keratitis. Topical inoculation of the rabbit cornea with Staphylococcus and the incubation of bacteria in tears in vitro have been found to quickly kill large numbers of Staphylococcus. ¹⁰ In vitro studies have determined that spermidine inhibits the phospholipase activity of rabbit tears and prevents bacterial killing by tears in vitro. ¹⁰ Accordingly, in the present study spermidine treatment was used to augment bacterial survival in the tear film in an effort to produce a topical inoculation model of Staphylococcus keratitis. In addition, the importance ofα -toxin in the infectious process that occurs after topical inoculation of the rabbit cornea was tested. 

Methods

Bacteria

S. aureus strain 8325-4 is an α-toxin–producing strain analyzed previously in the rabbit intrastromal injection keratitis model. ^{11 16} S. aureus strain DU1090 is an α-toxin mutant in which the α-toxin gene has been inactivated by allele replacement. ^{16 17} S. aureus strain DU1090/pDU1212 is the α-toxin–negative mutant that contains the plasmid pDU1212 that codes for α-toxin and restores α-toxin production to wild-type levels. ^{17 18} DU1090 and DU1090/pDU1212 have previously been analyzed in the rabbit intrastromal injection keratitis model. ^{11 16}  

Animals

New Zealand White rabbits (2.0–3.0 kg) were maintained in strict accordance with the institutional guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All rabbits were anesthetized by subcutaneous injection of a 1:5 mixture of xylazine (100 mg/ml; Rompum; Miles Laboratories, Shawnee, KS) and ketamine hydrochloride (100 mg/ml; Ketaset; Bristol Laboratories, Syracuse, NY). Proparacaine hydrochloride (0.5% Alcaine; Alcon Laboratories, Fort Worth, TX) was topically applied to each eye before scarification. 

Contact Lenses and Bacterial Adhesion

Bacteria were grown overnight in tryptic soy broth (TSB; Difco Laboratories, Detroit, MI) and subcultured 1:100 in TSB to approximately 10⁸ CFU/ml (optical density at 650 nm = 0.285; midlog phase). Contact lenses (61.4% polymacon, 38.6% water) designed for the rabbit eye with a base curve of 7.5 and diameter of 14.4 mm (Gelflex Laboratories, Perth, Australia) were incubated for 1 hour at room temperature in a 1.5-ml suspension of midlog-phase bacteria diluted to 10⁵ CFU/ml. After 30 minutes, 1 ml 50 mM spermidine (Sigma-Aldrich, St. Louis, MO) was added to each lens already soaking in bacterial culture. Contact lenses (n ≥ 4) used to determine the number of bacteria bound were selected at random, rinsed in sterile PBS, and assayed by homogenizing each in sterile PBS (3.0 ml), diluting the homogenate in PBS, and inoculating onto tryptic soy agar (TSA; Difco). Control lenses for 8325-4, DU1090, and DU1090/pDU1212 each contained a similar number of adherent bacteria per contact lens (3.95 ± 0.10 log CFU, 3.94 ± 0.07 log CFU, and 4.15 ± 0.05 log CFU, respectively; P ≥ 0.0801). 

Animal Model

Sixty and 30 minutes before and at the time of application of contact lenses, 40 μl spermidine (50 mM), dissolved in distilled water and filter sterilized, was applied to each rabbit eye. Scarifications, sufficient to penetrate the corneal stroma, were performed on rabbit corneas using a 22-gauge needle making three parallel scratches (approximately 8 mm long) in the center of each cornea. Contact lenses with adherent bacteria were then placed on each rabbit eye (n ≥ 5 per strain), underneath the nictitating membrane and remained for the duration of the experiment. After lens insertion, rabbit eyes were again treated with 40 μl spermidine (50 mM) at 30, 60, and 90 minutes postinfection (PI). 

Slit Lamp Examinations

Rabbit eyes, with lenses in place, were examined for changes by slit lamp examination (SLE) every 6 hours until 24 hours PI. SLE of rabbit eyes was performed by biomicroscope (Topcon; Koaku Kikai KK, Tokyo, Japan) by two masked observers. Each of seven ocular parameters (injection, chemosis, corneal infiltrate, corneal edema, fibrin in the anterior chamber, hypopyon formation, and iritis) was graded on a scale of 0 (none) to 4 (severe), as previously described. ¹⁹ The parameter grades were totaled to produce a single SLE score, ranging from 0 (normal eye) to a theoretical maximum of 28. Corneal erosions were detected with fluorescein (Fluor-I-Strip AT; Wyeth-Ayerst Laboratories Inc., Philadelphia, PA) and erosion diameters measured. Average diameters were expressed in millimeters. 

Bacterial Quantification

Rabbits were killed at 24 hours PI by an injection of pentobarbital sodium (100 mg/ml; The Butler Company, Columbus, OH). Corneas (n ≥ 5 per strain) were aseptically removed and cut in half. One half was used for histopathology, and the other half was cultured to enumerate viable bacteria. The number of viable S. aureus per cornea was determined by culturing corneal homogenates in triplicate, as previously described. ^{11 16} Contact lenses were also collected, homogenized, and plated on TSA at 24 hours PI. CFUs were expressed as base 10 logarithms. Bacteria recovered from infected eyes were tested for hemolysin production by inoculating individual colonies onto rabbit blood agar plates (PML Microbiologicals, Wilsonville, OR). 

Histopathology

One half of each cornea harvested at 24 hours PI was subjected to histopathologic analyses, as previously described. ¹⁵ Briefly, corneas were fixed immediately in 10% formalin (EK Industries, Joliet, IL). A tissue processor (Hypercenter XP Processor; Shandon, Pittsburgh, PA) was used to prepare the corneal tissue as follows: immersion overnight in 10% zinc formalin, dehydration in alcohol (70%, 80%, 95%, and three changes of absolute alcohol), and immersion in xylene three times to clear the tissue. Corneal tissues were embedded in paraffin. The resultant paraffin blocks were then cut into 4-μm-thick sections with a rotary microtome and stained with hematoxylin and eosin for pathologic examination. 

Statistical Analysis

Data were analyzed by computer (Statistical Analysis System; SAS, Cary, NC). ²⁰ For CFU determination, analysis of variance and Student’s t-tests between least-squares means from each group showing statistical variances were performed. For SLE scores, nonparametric one-way analysis of variance (Kruskal-Wallis test) and the Wilcoxon test were used for comparison among groups. P ≤ 0.05 was considered significant. 

Results

Previous studies from this laboratory have determined that rabbit tears kill S. aureus in vitro, and spermidine protects the bacteria from this killing action. ¹⁰ Therefore, experiments were conducted to determine the ability of spermidine (50 mM) to weaken host defenses and protect S. aureus 8325-4 on contact lenses applied to scarified rabbit corneas. Application of lenses without the use of spermidine resulted in no significant change in the number of bacteria on the lens from the time of inoculation (10⁴ CFU per lens) through 24 hours (P = 0.9355). However, similar inoculations augmented by the use of spermidine resulted in more than a 2-log increase in CFUs per lens by 24 hours PI (P = 0.0027; Table 1 ). More important, spermidine treatment resulted in an increase in the number of bacteria in the cornea, an increase that was significantly greater than the count in the inoculum (P = 0.0002). Corneas of inoculated eyes not treated with spermidine did not show a significant increase in CFUs over the inoculum (P = 0.4129). The bacteria on spermidine-treated lenses caused an infection that increased the SLE score significantly over that produced by bacteria on lenses not treated with spermidine (P ≤ 0.0001; Table 1 ). 

Infection of the rabbit cornea by S. aureus 8325-4 adherent to contact lenses treated with spermidine was confirmed by histopathologic analysis (Figs. 1A 1B) . The corneas at 24 hours after inoculation showed destruction of the corneal epithelium and bacterial penetration into the stroma. An association of bacteria and neutrophils was seen within the corneal fissures created by scarification (Fig. 1C) . Thus, rabbit corneas topically inoculated with S. aureus adherent to contact lenses and treated with spermidine resulted in keratitis, as measured by bacterial replication, inflammation, and pathologic changes. 

To determine the role of α-toxin in the topical model of infection, the replication and virulence of the parent 8325-4 strain was compared with that of the α-toxin–deficient mutant, DU1090, and its rescued strain, DU1090/pDU1212. The CFUs in the cornea of the parent,α -toxin–deficient, and rescued strains were not significantly different at 24 hours PI (6.59 ± 0.13, 6.65 ± 0.16, 6.12 ± 0.25 CFU/ml, respectively; P ≥ 0.1959). Each colony recovered from eyes infected with the parent or rescue strain retained the hemolytic activity of an α-toxin–producing strain, whereas those colonies recovered from eyes inoculated with the mutant strain did not have the hemolytic activity characteristic ofα -toxin. The SLE scores in eyes infected with the rescued strain were comparable to those of eyes infected with the parent strain (P ≥ 0.1123) and scores with both strains were higher than those of the α-toxin–deficient mutant at all times tested from 6 to 24 hours PI (P ≤ 0.0081; Fig. 2 ). Eyes inoculated with the parent or the rescued strains showed severe ocular inflammation, including conjunctival injection, chemosis, corneal infiltration, corneal edema, hypopyon formation, fibrin accumulation in the anterior chamber, and iritis. The changes in the eyes infected with the α-toxin–deficient mutant were trace to mild and were limited to conjunctival injection, chemosis, corneal infiltrate, and iritis. Corneas infected with the parent or rescued strain had severe epithelial erosions, whereas each cornea infected with the α-toxin–negative mutant had an intact corneal epithelium (parent, 9.56 ± 0.87 mm; rescue, 8.40 ± 0.87 mm; mutant, 0.0 ± 0.0 mm; P ≤ 0.0001). Inflammatory changes in eyes infected with each strain, as described earlier, were visible grossly (Fig. 3) . 

Histopathologic analysis of eyes infected with either of theα -toxin–producing strains demonstrated the loss of the corneal epithelium and the accumulation of PMN in the tear film within the fissures created by scarification (Figs. 4A 4C) . In contrast, eyes infected with the α-toxin–negative mutant retained the corneal epithelium and demonstrated few PMNs in the tear film associated with the cornea (Fig. 4B) . 

Discussion

This investigation has shown that Staphylococcus adherent to rabbit-specific contact lenses, in conjunction with spermidine treatment, can cause keratitis when applied to scarified rabbit corneas. Such topical inoculations result in bacterial replication on lenses and in the cornea, inflammation, and pathologic changes typically associated with keratitis. The inflammatory and pathologic changes produced include destruction of the corneal epithelium, influx of PMNs into the tear film and corneal stroma, chemosis and injection within the conjunctiva, edema of the cornea, hypopyon formation, fibrin accumulation in the anterior chamber, and iritis. 

Previous studies have shown that Staphylococcus on contact lenses designed for human use are killed in the rabbit eye and that phospholipase A₂ has a critical role in this potent defense reaction in the host. ¹⁰ Thus, the uses of the lenses specifically designed for the rabbit eye and spermidine treatment were important factors in the development of keratitis after this inoculation procedure. Rabbit-specific contact lenses, unlike human-specific lenses, conform to the curvature of the rabbit cornea assuring a closer interaction with the cornea. Staphylococcus replication after topical inoculation is apparently fostered in part by the barrier effect of the contact lenses, which limits tear film flow into fissures created during scarification. In addition, bacterial replication is aided by the inhibition of the bactericidal action of phospholipase A₂ as a result of multiple applications of spermidine. The lenses may also protect bacteria from some of the PMNs that reach the tear film from the overlying eyelid or limbus. ¹²  

The severe inflammatory and pathologic changes associated with this new topical model of Staphylococcus keratitis are dependent on the production of α-toxin during infection. Corneal epithelial destruction and severe ocular inflammatory responses were present in eyes infected with the toxin-producing strains, yet absent in eyes inoculated with the α-toxin–deficient mutant. We have previously demonstrated that α-toxin causes extensive pathologic damage in an intrastromal injection model of Staphylococcus keratitis. ^{11 15 16} α-Toxin, however, is not essential for the growth of bacteria within the cornea in the intrastromal model, ¹¹ and α-toxin was not essential for growth of bacteria in the cornea after topical inoculation. 

The topical model of keratitis is expected to be useful in the analysis of the early stages of infection. One key question that could be addressed in this model is the mechanism by which Staphylococcus enters the corneal stroma. Johnson et al. ²¹ found that S. aureus adhered to rabbit corneal epithelial cells in vitro; however, this finding was not demonstrated in vivo. One explanation could be that bacteria replicate in corneal fissures and some are able to enter the stroma. The fibrous collagen bundles of the stroma are cut during scarification, and the spaces created between bundles could provide a route for the bacteria to diffuse from the fissures to the stroma. The penetration of theα -toxin–deficient mutant into the stroma suggests that the toxic and inflammatory effects of α-toxin are not needed for bacterial invasion of the stroma. 

The topical model could also be useful for determining the ability of antibody to protect against corneal infection. Antibody toα -toxin has been found to protect the cornea from tissue damage and inflammation after intrastromal injection. ¹⁴ However, antibody to α-toxin does not interfere with Staphylococcus replication in the cornea. It is possible that antibody, particularly IgA class antibody in the tear film, specific for a Staphylococcus antigen other than α-toxin can prevent Staphylococcus corneal infection. The topical model could provide evidence of such protection and a determination of the immunogen as well as the immunization procedure that best induces a protective immune state. 

This study has shown that Staphylococcus can infect the scarified rabbit cornea, provided the phospholipase A₂ defense of the tear film is compromised. The model should allow analysis of the early bacterial infectious process and of the ability of innate as well as acquired host defenses (antibodies) to protect against keratitis. 

²  Present affiliation: Co-operative Research Centre for Eye Research and Technology, University of New South Wales, Sydney, New South Wales, Australia.

 Supported by National Institutes of Health Grant RO1 EY10974.

 Submitted for publication April 23, 2001; revised July 2, 2001; accepted August 1, 2001.

 Commercial relationships policy: N.

 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.

 

Table 1.

 

Effect of Spermidine Treatment on the Replication of S. aureus 8325-4 Inoculated into Rabbit Eyes on Rabbit-Specific Contact Lenses

Group	Spermidine Treatment*	Cornea (Log CFU, † )	Lens (Log CFU, ‡ )	SLE Score, §
1	None*	4.56 ± 1.28	3.87 ± 1.75	10.13 ± 2.32
2	Before and After Inoculation*	6.59 ± 0.13	6.74 ± 0.22	15.52 ± 0.29

*   S. aureus 8325-4 was applied to rabbit-specific contact lenses which, after rinsing, contained 3.95 ± 0.10 log CFU per lens. One set of lenses with adherent Staphylococcus was soaked only in saline and applied to scarified corneas (n = 4) without any treatment. The remaining lenses were soaked in spermidine (50 mM) and applied to scarified rabbit corneas (n = 13) that were treated with spermidine at 60 and 30 minutes before inoculation and at 30, 60, and 90 minutes after applying the lenses.

^†  The log number CFU per cornea was determined at 24 hours PI. Data are expressed as the mean ± SEM. The CFU per cornea in group 1 was significantly lower than in group 2 (P = 0.0041).

^‡  The log number CFU of the contact lens was determined at 24 hours PI. Data are expressed as the mean ± SEM. The CFU per lens for group 1 was significantly lower than in group 2 (P = 0.0040).

^§  The SLE score was determined at 24 hours PI. Data are the mean ± SEM. The SLE score in group 1 was significantly lower than in group 2 (P ≤ 0.0001).

Figure 1.

 

Histopathologic evidence of S. aureus 8325-4 replication in the rabbit eye after topical inoculation and spermidine treatment. Contact lenses designed for rabbit eyes with adherent S. aureus 8325-4 (10⁴ CFU per lens) were applied to scarified rabbit eyes. Eyes and contact lenses were treated with spermidine as described in the text. Corneas were harvested at 24 hours after inoculation. (A) A scratch extending into the outer third of the cornea and staphylococci associated with the denuded corneal stroma (arrow). An inflammatory exudate is present in the scratch fissure. (B) Extension of cocci (arrow) into the corneal stroma. (C) A fibrinopurulent exudate within the fissure created by a scratch. Staphylococci are present within PMNs (arrows). Original magnification, (A) ×25; (B, C)× 250.

Figure 2.

 

SLE scores during S. aureus keratitis after topical inoculation with strains differing in α-toxin production. Eyes topically infected with contact lenses containing 10⁴ CFU of strain 8325-4, DU1090, or DU1090/pDU1212 were analyzed by SLE every 6 hours PI. Open bars: The parent strain 8325-4; filled bars: the α-toxin–negative strain DU1090; striped bars: the genetically rescued strain DU1090/pDU1212. Data are expressed as average SLE score ± SEM.

Figure 3.

 

Rabbit eyes inoculated topically with S. aureus strains differing in α-toxin production. Rabbit eyes topically infected with either of the α-toxin–producing strains (8325-4 or DU1090/pDU1212) or the α-toxin–negative strain (DU1090) were photographed at 24 hours PI. (A) Rabbit eye infected with anα -toxin–producing strain. Severe ocular pathologic changes could be seen that are typical of infection caused by either of theα -toxin–producing strains tested. (B) Rabbit eye infected with the α-toxin–negative mutant. Trace to mild ocular pathologic changes were evident in this rabbit eye infected with theα -toxin–negative strain.

Figure 4.

 

Histopathology of rabbit corneas infected with S. aureus strains differing in α-toxin production. Corneas topically infected with strain 8325-4, DU1090, or DU1090/pDU1212 were harvested at 24 hours PI and prepared for histopathologic examination as described in the text. (A) Cross section of a cornea infected with strain 8325-4. Severe infiltration by PMNs within the scratch site (filled arrow), a denuded corneal stroma (arrowhead), and PMNs within the corneal stroma (open arrow) were visible. (B) Cornea infected with strain DU1090. The corneal epithelium remained intact (filled arrow) and minimal PMN infiltration was seen within the scratch site (open arrow). (C) Cornea infected with strain DU1090/pDU1212. Severe infiltration of PMNs into the scratch fissure (filled arrow), PMNs within the corneal stroma (open arrow), and a denuded corneal stroma (arrowhead) were visible. Original magnification, ×50.

The authors thank Kiana Nelson for technical assistance, Brett Thibodeaux for help with the figure preparations, and Mary Marquart and Megan Austin for critical review of the manuscript.