INFECTiON AND IMMUNITY, June 1994, p. 2478-2482 Vol. 62, No. 60019-9567/94/$04.00+0Copyright © 1994, American Society for Microbiology

Corneal Virulence of Staphylococcus aureus: Roles ofAlpha-Toxin and Protein A in Pathogenesis

MICHELLE C. CALLEGAN,1 LEE S. ENGEL,' JAMES M. HILL,"2 AND RICHARD J. O'CALLAGHAN'Department of Microbiology, Immunology and Parasitology' and the Lions Eye Research Laboratories,2

Louisiana State University Medical Center, New Orleans, Louisiana 70112

Received 10 January 1994/Returned for modification 3 March 1994/Accepted 31 March 1994

Staphylococcus aureus produces a variety of proteins, including alpha-toxin and protein A, that couldcontribute to corneal tissue damage during keratitis. We examined corneal infections produced by intrastromalinjection of four S. aureus strains-three isogenic mutants, one lacking alpha-toxin (Hly- Spa'), one lackingprotein A (Hly+ Spa-), and one lacking both alpha-toxin and protein A (Hly- Spa-), and the wild type (Hly+Spa')-in a rabbit model of experimental keratitis. Rabbit corneas were injected intrastromally with 100 CFUof one of the four strains, and the eyes were examined by slit lamp biomicroscopy over a 25-h period. Cornealhomogenates were used for determination of CFU and neutrophil myeloperoxidase activity at 5-h intervals. Allstrains had the same logarithmic growth curve from 0 to 10 h postinfection, after which CFU remainedconstant at 107 CFU per cornea. By 15 h postinfection, slit lamp examination scores were significantly higherfor eyes infected with Hly+ strains than for Hly--infected eyes. At this time, distinct epithelial erosions wereseen in Hly+-infected eyes but not in Hly--infected eyes. Myeloperoxidase activity was significantly greater forHly'-infected corneas than for Hly--infected corneas at both 20 and 25 h postinfection. Spa+- andSpa--infected eyes showed no differences in slit lamp examination scores or myeloperoxidase activities. Theseresults suggest that alpha-toxin, but not protein A, is a major virulence factor in staphylococcal keratitis,mediating the destruction of corneal tissue in eyes infected with this bacterial pathogen.

Bacterial keratitis caused by Staphylococcus aureus is anincreasingly common problem, especially for individuals withcompromised corneas (20). Staphylococci are common inhab-itants of the face and anterior nares and thus have access to theeye and can infect injured ocular tissue (21). The use ofextended-wear contact lenses has been associated with staph-ylococcal keratitis (20). S. aureus keratitis progresses rapidly,and without proper and prompt treatment, it can result incorneal scarring, corneal perforation, loss of vision, and theneed for corneal transplantation.

S. aureus produces and secretes many proteins, includingcoagulase, protein A, alpha-, beta-, gamma-, and delta-toxin,and leukocidin, all ofwhich could contribute to the virulence ofthe organism. Alpha-toxin is a pore-forming hemolytic toxin(13) that causes membrane damage to many types of mamma-lian cells (1, 26). The cytolytic nature of alpha-toxin for severalcell types could be an important mechanism for cornealepithelial and stromal tissue damage during S. aureus keratitis(1, 26). Several investigators have demonstrated a major rolefor alpha-toxin in virulence of nonocular infections (3, 12, 17,23, 24).

Protein A is a cell wall-associated exoprotein that binds tothe Fc region of immunoglobulin G of most mammalianspecies (9). When administered in vivo, protein A can incite anassortment of biological effects, including anaphylaxis-typereactions (11, 15), and can activate both the classical andalternate complement pathways (8). The complement-activat-ing function of protein A suggests that protein A could inducecorneal inflammation and could be a critical factor in staphy-lococcal virulence (8). Protein A also inhibits opsonization andphagocytosis of staphylococci in vitro (10). As a result, protein

* Corresponding author. Mailing address: Louisiana State Univer-sity Medical Center, Department of Microbiology, Immunology andParasitology, 1901 Perdido St., New Orleans, LA 70112. Phone: (504)568-4072. Fax: (504) 568-2918.

A could help S. aureus avoid the host immune response andthus could contribute to the virulence of the organism.To determine the possible role of alpha-toxin and protein A

in corneal pathology, we compared the virulence of thewild-type strain with that of isogenic mutants of S. aureuslacking one or both of these specific virulence factors in anestablished rabbit model of keratitis.

MATERUILS AND METHODS

Bacterial strains. The S. aureus strains used in these studies,their sources, and the designations of specific genes and geneproducts are summarized in Table 1. Biochemical testingconfirmed the fermentation of mannitol and production ofcoagulase and deoxyribonuclease by each strain. The mutantswere derived from S. aureus 8325-4 (Hly+ Spa'), a humanisolate derived from NCTC 8325. The alpha-toxin-deficientmutant (DU1090 [Hly- Spa']) was constructed by inactivationof the structural gene for alpha-toxin (hly) by allele-replace-ment mutagenesis with hly::Emr (23). The protein A-deficientmutant (DU5723 [Hly+ Spa-]) was constructed by inactivationof the structural gene for protein A (spa) by allele-replacementmutagenesis with Aspa::ethidium bromide (EtBr)r (24). Amutant deficient in both alpha-toxin and protein A (DU5724[Hly- Spa-]) was derived by transduction of the hly::Emr locusfrom DU1090 to DU5723 by phage 485 (24).Assay for detection of alpha-toxin-producing strains. Pro-

duction of alpha-toxin was detected on Columbia blood agar(Difco Laboratories, Inc., Detroit, Mich.) containing 5.0%(vol/vol) rabbit blood. Maintenance of the hly::Emr locus wasfavored by plating strains on tryptic soy agar (Difco) containingerythromycin (100 ,ug/ml).Assay for detection of protein A production. To identify

strains producing protein A, dot immunoblots of bacterialsupernatants or bacterial suspensions were performed. Over-night cultures were centrifuged (10,000 x g for 5 min, 4°C),

2478

on June 9, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

CORNEAL VIRULENCE OF S. AUREUS EXOPROTEINS 2479

TABLE 1. Phenotypic characteristics of S. aureus strains

Strain" Phenotype" Characteristic(s)' Reference

8325-4 Hly' Spa' NCTC 8325 cured of prophages 22DUI090 Hly Spa' hly::Emr 23DU5723 Hly+ Spa- Aspa::EtBrr 24DU5724 Hly Spa- hly::Emr, .spa::EtBrr 24

a These strains were kindly supplied by Timothy J. Foster (MicrobiologyDepartment, Trinity College, Dublin, Ireland).

" Designations for pertinent gene products: Hly'. alpha-toxin produced; Hly-,alpha-toxin not produced; Spa', protein A produced; Spa -, protein A notproduced.

C Designations for antibiotic resistance genes: Emr, erythromycin resistancecoded by the site-specific mutagenized hlly gene (23): EtBrr, EtBr resistancecoded by the site-specific mutagenized spa gene (24).

and aliquots (5 RI) of cell supernatants or bacterial suspensions(5 x 105 CFU) were spotted onto nitrocellulose (Schleicher &Schuell, Keene, N.H.). Blots were developed with rabbitanti-protein A antiserum (Sigma, St. Louis, Mo.) and goatanti-rabbit immunoglobulin G-alkaline phosphatase-conju-gated antiserum (Sigma). Maintenance of the Aspa::EtBrrlocus was favored by plating strains on tryptic soy agarcontaining EtBr (8 pg/ml).

S. aureus keratitis. S. aureus strains were grown overnight intryptic soy broth (Sigma) at 37°C, subcultured 1:100 into trypticsoy broth, and grown to an optical density of 0.3 at 650 nm(approximately 108 CFU/ml). Staphylococci were diluted intryptic soy broth to 104 CFU/ml; 10 pLI of this dilutioncontaining approximately 100 CFU of logarithmic-phasestaphylococci was used for each intrastromal injection (4-6).Rabbits were treated and maintained in specific accordancewith institutional guidelines and the Association of Research inVision and Ophthalmology Resolution on the Use of Animalsin Research. New Zealand White rabbits were anesthetizedintramuscularly with a 5:1 solution of ketamine hydrochloride(Ketaset; 100 mg/ml; Bristol Laboratories, Syracuse, N.Y.) andxylazine (Rompun; 100 mg/ml; Miles Laboratories, Shawnee,Kans.). Prior to injection, proparacaine hydrochloride (Al-caine; 0.5%; Alcon Laboratories, Fort Worth, Tex.) was in-stilled into each eye. Intrastromal injection of 10 .I1 of bacterialsuspension into the corneal stroma was performed with a30-gauge needle attached to a 100-,ul syringe. The number ofviable staphylococci in the inoculum was determined retro-spectively by triplicate plating of 100-,ul aliquots onto trypticsoy agar (4-6).

In vivo growth of S. aureus. Rabbit eyes were randomlyassigned to four groups, and each group received intrastromalinjection of one of four strains: 8325-4, DU1090, DU5723, orDU5724. The eyes were examined every 5 h postinfection byslit lamp biomicroscopy, as described below. Rabbits weresacrificed at various times postinfection (5, 10, 15, 20, and 25 h)by intravenous injection of sodium pentobarbital solution (100mg/ml; The Butler Co., Columbus, Mo.). The corneas, four ormore per time point, were harvested and assayed for viablebacteria (CFU) and myeloperoxidase (MPO) activity, as de-scribed below.

Slit lamp examination. Slit lamp examinations (TopconSL-5D; Kogaku Kikai K. K., Tokyo, Japan) were performed aspreviously described (6, 16). Briefly, inflammation was gradedfrom 0 to 4 for each of seven parameters: conjunctival injec-tion, conjunctival chemosis, iritis, fibrin in the anterior cham-ber, hypopyon in the anterior chamber, stromal infiltrate, andstromal edema. Grades for individual parameters weresummed to give a total slit lamp examination score rangingfrom 0 (normal eye) to 28 (maximally inflamed eye).

Quantification of viable S. aureus organisms per cornea.Quantification of viable staphylococci per cornea has beenpreviously described (4-6). Briefly, corneas were asepticallyremoved at the corneoscleral limbus, dissected into 8 to 10small pieces, homogenized, and centrifuged (14,000 x g, 5min). Aliquots of corneal homogenate were serially diluted insterile phosphate-buffered saline (PBS [pH 7.4]), plated ontotryptic soy agar, and incubated at 37°C for 48 h. Colonies (50or more) recovered from corneas were replica plated ontorabbit blood agar; hemolysis indicated alpha-toxin productionby individual colonies. Subcultures of individual colonies (50 ormore) were also tested by dot immunoblot to demonstrateprotein A production.MPO assay. The influx of polymorphonuclear leukocytes is

an initial inflammatory response to bacterial infection (2, 7,14). To estimate the extent of corneal inflammation, MPOactivity of infiltrated polymorphonuclear leukocytes in cornealhomogenates was assayed in a manner similar to the method ofWilliams et al. (27). Assays were performed in triplicate. Afteraddition of hexadecyltrimethylammonium bromide (Sigma) toa final concentration of 0.5%, homogenates were homogenizedagain and centrifuged to remove any remaining corneal tissue.Aliquots of homogenate (6.9 p.l) were added to 96-well micro-titer plates containing 200 pI' of potassium phosphate buffer(50 mM, pH 6.0) with o-dianisidine dihydrochloride (16.7mg/100 ml; Sigma) and hydrogen peroxide (0.0005%). Thechange in optical density with time at 450 nm was measuredevery 2 min for 10 min with a microtiter plate reader (Dyna-tech MR5000; Dynatech Laboratories, Chantilly, Va.). Calcu-lations of MPO activity were performed as described byWilliams et al. (27). One unit of MPO activity was equivalentto approximately 5 logs of polymorphonuclear leukocytes.

Statistical analysis. Statistical analysis of data was per-formed with the Statistical Analysis Systems program forpersonal computers (25). For CFU determinations, analysis ofvariance and Student's t tests between least-square means fromeach treatment group showing statistical variances were per-formed. For slit lamp examination scores and MPO activitydeterminations, nonparametric one-way analysis of variance(Kruskal-Wallis test) and Wilcoxon's test were used for com-parison among treatment groups.

RESULTS

The inocula of strains 8325-4, DU1090, DU5723, andDU5724 used to initiate experimental infections contained 106+ 26, 94 + 13, 94 + 12, and 95 + 25 CFU, respectively (mean+ standard error of the mean). The in vivo growth rates ofthese strains in terms of CFU per cornea were similar (Fig. 1).The CFU increased steadily between 0 and 10 h postinfectionand stabilized at 7.5 log,() CFU per cornea at 15 h postinfec-tion, representing corneal biphasic growth (6, 18, 19). Ran-domly selected colonies cultured from each corneal homoge-nae (50 or more per cornea) retained their wild-type ormutant phenotypes, as tested on rabbit blood agar (alpha-toxin) or by dot immunoblot (protein A).The slit lamp examination scores demonstrated markedly

greater pathology in the eyes infected with the alpha-toxin-producing (Hly5) strains of S. aureus (Fig. 2). Average slitlamp examination scores for strains 8325-4 and DU5723 (bothHly+) increased from 1.0 at 5 h to 22.0 at 25 h postinfection.Average slit lamp examination scores for strains DUI090 andDU5724 (both Hly-) increased from 1.0 at 5 h to 9.0 at 25 hpostinfection, an increase significantly less than those of thetwo Hly+ strains (P < 0.0001 [Fig. 2]).The rapidly increasing slit lamp examination scores in the

VOL. 62? 1994

on June 9, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

2480 CALLEGAN ET AL.

u-0 5 10 15 20 25

Hours PostinfectionFIG. 1. Growth of S. aureus strains in the rabbit cornea. The

number of CFU per cornea (mean ± standard error) was determinedat various times postinfection following intrastromal injection of 100CFU per cornea.

eyes infected with the Hly+ strains were indicative of progres-sive corneal damage. From 5 to 10 h postinfection, conjunctivalinjection and chemosis increased and small punctate infiltratesappeared in the cornea at the injection site. By 15 h postinfec-

25* 8325-4 (Hly+ Spa+)A DU1090 (Hly- Spa+)

20- 0 DU5723 (Hly+ Spa-)DU5724 (Hly- Spa-)

CZ

15-

; 10

CZ

0

0 5 10 15 20 25

Hours PostinfectionFIG. 2. Slit lamp examination scores (mean standard error) of

rabbit corneas infected with 100 CFU of one of four strains of S.aureus.

FIG. 3. Rabbit cornea infected with an alpha-toxin-producing(Hly) strain of S. aureus, photographed at 20 h postinfection.

tion, Hly+-infected eyes had marked epithelial defects (i.e.,sloughing of the epithelium within a defined border) at the siteof injection. Chemosis and iritis ranged from moderate tosevere, and fibrin and leukocytes were present in the anteriorchamber. By 20 h postinfection, conjunctival injection andchemosis were severe. Epithelial defects at this stage werelarge (greater than 5 mm in diameter) and filled with pus (Fig.3). Fibrin accumulation in the anterior chamber ranged frommoderate to severe. Desmetocele formation was seen in 50%of Hly+-infected corneas by 25 h postinfection.

In contrast, eyes infected with Hly- strains showed lesscorneal pathology (Fig. 4). Hly--infected eyes developed mildto moderate amounts of conjunctival chemosis, injection, andiritis, even when infections were allowed to progress to 25 h.Punctate infiltrates were visible at the injection site by 10 hpostinfection and began to coalesce by 25 h postinfection.Distinct epithelial defects were absent; the corneal epitheliumand stroma at the injection site remained essentially intact.Fibrin accumulation in the anterior chamber ranged from traceto mild. Desmetoceles did not form.

Corneal inflammation resulting from infiltrating polymor-phonuclear leukocytes, as measured by MPO activity, in-creased rapidly after 15 h postinfection in Hly+-infected eyesbut not in Hly--infected eyes (Fig. 5). The differences weresignificant at both 20 and 25 h postinfection (P < 0.0014).

Cd

a)Eo

0

u

C4C)

._

-

toCD

8

6-,

4--

2 i

* 8325-4 (Hly+ Spa+)0-8-

6--

24

* DU1090 (Hly- Spa+)

8

6--

4-/

2o DU5723 (Hly+ Spa-)

8-

6-A

4-

2| DU5724 (Hly- Spa-)

INFEcr. IMMUN.

on June 9, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

CORNEAL VIRULENCE OF S. AUREUS EXOPROTEINS 2481

15 --* 83254 (Hly+ Spa+)A DU1090 (Hly- Spa+)C DU5723 (Hly+ Spa-)e DU5724 (Hly- Spa-)

1o-0

*_

Ct._

X0

1-45 ,~~~~~~~~~,/-

/

0 5 10 15 20 25Hours Postinfection

FIG. 4. Rabbit cornea infected with an alpha-toxin-deficient(Hly ) strain of S. aureus, photographed at 20 h postinfection.

DISCUSSIONS. aureus produces a number of proteins that could contrib-

ute to corneal virulence. Mutants of S. aureus lacking theability to produce various exoproteins have not been studiedpreviously with any ocular models of infection. Our studies ofcorneal virulence in the rabbit keratitis model were performedon the basis of strains that had identical growth rates in thecornea and differed only in the genetic loci stated.

Analysis of the virulence of isogenic mutants suggests thatprotein A does not appear to be a major virulence factor forstaphylococcal keratitis in this model. On the basis of MPOactivities and slit lamp examination scores (Fig. 2 and 5),differences in corneal pathology could not be attributed to amutation at the spa locus. In nonocular models, studiesexamining the virulence of mutants lacking protein A haveyielded conflicting results. Spa- mutants were less virulentthan Spa' strains in both mouse peritonitis and subcutaneouslesion models (24). Other studies found no difference invirulence between Spa' and Spa- strains in a mouse mastitismodel (17).

In this keratitis model, alpha-toxin is an important virulencefactor and could be responsible for much of the cornealdamage occurring during progressive S. aureus keratitis. Ocu-lar inflammation, assessed by slit lamp examination and MPOactivity (Fig. 2 and 5), showed the striking difference in ocularpathology between Hly+ and Hly- strains. A functional hlylocus seems to be necessary for rapid epithelial damage,

FIG. 5. MPO activity (mean + standard error) from infiltratingpolymorphonuclear leukocytes in rabbit corneas at various timespostinfection following intrastromal injection of 100 CFU of isogenicstrains of S. aureus per cornea.

stromal invasion, and ulceration. These ocular findings relatewell to studies with nonocular models, showing that Hly+strains are significantly more virulent than Hly- strains (3, 12,17, 23, 24). Further studies are needed to determine themechanisms by which alpha-toxin contributes to corneal dam-age.

ACKNOWLEDGMENTS

We thank Timothy J. Foster for the S. aureus strains used in theseexperiments. We also thank Timothy J. Foster, Mary Kay Johnson, andJeffery Hobden for reading the manuscript and acknowledge Mark A.Cumella, Bryan M. Hemard, Nataskia 0. Lampe, and Patrick M.O'Callaghan for excellent technical assistance.

This work was supported in part by Public Health Service grantsEY08871 and EY02377 from the National Eye Institute, NationalInstitutes of Health, Bethesda, Md.

REFERENCES1. Bhakdi, S., and J. Tranum-Jensen. 1987. Damage to mammalian

cells by proteins that form transmembrane channels. Rev. Physiol.Biochem. Pharmacol. 107:147-223.

2. Bradley, P. P., D. A. Priebat, R. D. Christensen, and G. Rothstein.1982. Measurement of cutaneous inflammation: estimation ofneutrophil content with an enzyme marker. J. Invest. Dermatol.78:206-209.

3. Bramley, A. J., A. H. Patel, M. O'Reilly, R. Foster, and T. J. Foster.1989. Roles of alpha-toxin and beta-toxin in virulence of Staphy-lococcus aureus for the mouse mammary gland. Infect. Immun.57:2489-2494.

4. Callegan, M. C., L. S. Engel, J. M. Hill, and R. J. O'Callaghan.1994. Ciprofloxacin versus tobramycin for the treatment of staph-ylococcal keratitis. Invest. Ophthalmol. Vis. Sci. 35:1033-1037.

5. Callegan, M. C., J. M. Hill, M. S. Insler, J. A. Hobden, and R. J.O'Callaghan. 1992. Methicillin-resistant Staphylococcus aureuskeratitis in the rabbit: therapy with ciprofloxacin, vancomycin andcefazolin. Curr. Eye Res. 11:1111-1119.

6. Callegan, M. C., J. A. Hobden, J. M. Hill, M. S. Insler, and R. J.O'Callaghan. 1992. Topical antibiotic therapy for the treatment ofexperimental Staphylococcus aureus keratitis. Invest. Ophthalmol.Vis. Sci. 33:3017-3023.

7. Chusid, M. J., and S. D. Davis. 1985. Polymorphonuclear leuko-cyte kinetics in experimentally induced keratitis. Arch. Ophthal-mol. 103:270-274.

8. Espersen, F. 1985. Complement activation by clumping factor andprotein A from Staphylococcus aureus strain E 2371. Acta Pathol.

I

s. -A

k

:.i

VOL. 62, 1994

I ik,"11

/

on June 9, 2020 by guesthttp://iai.asm

.org/D

ownloaded from

2482 CALLEGAN ET AL.

Microbiol. Immunol. Scand. 93:59-64.9. Forsgren, A., and J. Sjoquist. 1966. "Protein A" from Staphylo-

coccus aureus. J. Immunol. 97:822-827.10. Gemmell, C. G., R. Tree, A. Patel, M. O'Reilly, and T. J. Foster.

1991. Susceptibility to opsonophagocytosis of protein A, ox-haemo-lysin and 1-toxin deficient mutants of S. aureus isolated byallele-replacement. Zentralbl. Bakteriol. 21(Suppl.):273-277.

11. Gustafson, G. T., G. Stalenheim, A. Forsgren, and J. Sjoquist.1968. "Protein A" from Staphylococcus aureus. IV. Production ofanaphylaxis-like cutaneous and systemic reactions in non-immu-nized guinea-pigs. J. Immunol. 100:530-534.

12. Haraldsson, I., and P. Jonsson. 1984. Histopathology and patho-genesis of mouse mastitis induced with Staphylococcus aureusmutants. J. Comp. Pathol. 94:183-196.

13. Harshman, S., P. Boquet, E. Duflot, J. E. Alouf, C. Montecucco,and E. Papini. 1989. Staphylococcal a-toxin: a study of membranepenetration and pore formation. J. Biol. Chem. 264:14978-14984.

14. Hazlett, L. D., M. Zucker, and R. S. Berk. 1992. Distribution andkinetics of the inflammatory cell response to ocular challenge withPseudomonas aeruginosa in susceptible versus resistant mice. Oph-thalmic Res. 24:32-39.

15. Heczko, P. B., A. Grov, and P. Oeding. 1973. In vivo reactions ofstaphylococcal antigens. Acta Pathol. Microbiol. Scand. Sect. B81:731-740.

16. Johnson, M. K., J. A. Hobden, M. Hagenah, R. J. O'Callaghan,J. M. Hill, and S. Chen. 1990. The role of pneumolysin in ocularinfections with Streptococcus pneumoniae. Curr. Eye Res. 9:1107-1114.

17. Jonsson, P., M. Lindberg, I. Haraldsson, and T. Wadstrom. 1985.Virulence of Staphylococcus aureus in a mouse mastitis model:studies of alpha hemolysin, coagulase, and protein A as possible

virulence determinants with protoplast fusion and gene cloning.Infect. Immun. 49:765-769.

18. Kupferman, A., and M. Leibowitz. 1976. Quantitation of bacterialinfection and antibiotic effect in the cornea. Arch. Ophthalmol.94:1981-1984.

19. Kupferman, A., and M. Leibowitz. 1977. Topical antibiotic therapyof staphylococcal keratitis. Arch. Ophthalmol. 95:1634-1637.

20. Limberg, M. B. 1991. A review of bacterial keratitis and bacterialconjunctivitis. Am. J. Ophthalmol. 112(Suppl.):2S-9S.

21. Mannis, M. J. 1988. Bacterial conjunctivitis, p. 189-200. In H. E.Kaufman, B. A. Barron, M. B. McDonald, and S. R. Waltman(ed.), The cornea. Churchill Livingstone, New York.

22. Novick, R. P. 1963. Properties of a cryptic high-frequency trans-ducing phage in Staphylococcus aureus. Virology 33:155-166.

23. O'Reilly, M., J. C. S. DeAzavedo, S. Kennedy, and T. J. Foster.1985. Inactivation of the alpha-haemolysin gene of Staphylococcusaureus 8325-4 by site-directed mutagenesis and studies on theexpression of its haemolysins. Microb. Pathog. 1:125-138.

24. Patel, A. H., P. Nowlan, E. D. Weavers, and T. Foster. 1987.Virulence of protein A-deficient and alpha-toxin-deficient mutantsof Staphylococcus aureus isolated by allele replacement. Infect.Immun. 55:3103-3110.

25. SAS Institute, Inc. 1985. Statistical analysis systems users guide,statistics, version 5 edition. SAS Institute, Cary, N.C.

26. Thelestam, M., and L. Blomqvist. 1988. Staphylococcal alphatoxin-recent advances. Toxicon 26:51-65.

27. Williams, R. N., C. A. Paterson, K. E. Eakins, and P. Bhattach-erjee. 1982. Quantification of ocular inflammation: evaluation ofpolymorphonuclear leucocyte infiltration by measuring myeloper-oxidase activity. Curr. Eye Res. 2:465-470.

INFEC'F. IMMUN.

on June 9, 2020 by guesthttp://iai.asm

.org/D

ownloaded from