Vol. 33, No. 3INFECTION AND IMMUNITY, Sept. 1981, p. 927-9320019-9567/81/090927-06$02.00/0

Characterization of Rabbit Corneal Damage Produced bySerratia Keratitis and by a Serratia Protease

DAVID LYERLY, LARRY GRAY, AND ARNOLD KREGER*

Department ofMicrobiology and Immunology, Bowman Gray School ofMedicine of Wake ForestUniversity, Winston-Salem, North Carolina 27103

Received 17 April 1981/Accepted 5 June 1981

The structural alterations elicited in the rabbit corneal stroma by experimentalSerratia marcescens keratitis and by a highly purified serratia protease prepa-ration were compared by gross observation, biochemical analyses, and electronmicroscopic examination of the affected tissue. Acute inflammation, liquefactivenecrosis of the cornea, and descemetocele formation occurred during the devel-opment of the infection and after the intracorneal injection of submicrogramamounts of the protease. In vitro incubation of insoluble corneal stromal tissuewith the bacterium or with the protease resulted in solubilization of the stromalproteoglycan ground substance; however, specific collagenase activity was notdetected. Electron microscopic examination of corneas damaged by the bacterialinfection and by the protease revealed loss of ruthenium red staining of theproteoglycan ground substance and dispersal of ultrastructurally normal collagenfibrils. Thus, our findings indicate that the major corneal damage which occursduring serratia keratitis and after the injection of the serratia protease is causedby solubilization and loss of the ground substance of the tissue. In addition, theobservation that the major structural alterations observed during serratia keratitiscan be reproduced by the bacterial protease supports the idea that the enzyme isinvolved, at least in part, with the production of severe corneal damage by thebacterium.

Serratia marcescens is an opportunistic path-ogen capable of causing a variety of humanocular diseases, including keratoconjunctivitis,corneal abscess, purulent conjunctivitis, lacrimalduct infections, and endophthalmitis (1, 3, 7, 8,16, 20, 22-24). Bernstein and Maddox (2) pro-duced keratitis in rabbits by injecting S. mar-cescens intracorneally and reported that the ad-ministration of large numbers of bacteria (9 x107 colony-forming units) produced an infectionwhich caused liquefactive necrosis and destruc-tion of the cornea. They also noted that intra-corneal injection of a large amount (150,tg) ofthe bacterial lipopolysaccharide endotoxin elic-ited severe corneal opacification but did notcause the extensive liquefactive necrosis char-acteristically produced by the viable bacterium.In addition to the potential cornea-damagingactivity of the bacterial endotoxin, the possibleimportance of serratia extracellular proteases inthe pathogenesis of serratia keratitis was sug-gested by the observation that intracorneal in-jection ofsubmicrogram amounts of serratia pro-tease preparations elicited rapid and extensiveliquefactive necrosis of and descemetocele for-mation in the rabbit cornea (15, 17). Although

the above-cited studies suggested the possibleinvolvement of serratia endotoxin or extracellu-lar proteases or both in the pathogenesis ofserratia keratitis, additional studies were neededto characterize and compare the biochemicaland ultrastructural changes produced in corneasinfected with the bacterium with those in cor-neas treated with highly purified preparations ofthe bacterial products. In the present study, wehave investigated the possible role of serratiaextracellular proteases in experimental serratiakeratitis by characterizing and comparing thebiochemical and ultrastructural alterations oc-curring in the corneal stroma during the infec-tion with those elicited by a highly purifiedserratia protease preparation.

MATERIALS AND METHODSBacterium and bacterial enzyme. S. marcescens

strain BG was cultivated, and its extracellular metal-loprotease was purified as previously described (17).

In vitro digestion of rabbit corneal stromaltissue by bacteria and enzymes. Fourteen corneaswere excised aseptically from rabbit eyes obtainedfrom Pel-Freez Biologicals, Inc. (Rogers, Ark.), andthe epithelium, Descemet's membrane, and endothe-

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928 LYERLY, GRAY, AND KREGER

lium were removed by scraping. The stromal tissuethus obtained was cut into small pieces (about 2 by 2mm) and uniformly dispersed at 4°C in 20 ml of sterile0.02 M phosphate buffer (PB; pH 7.0) by treatment(six 1-min pulses at full power, with 1-min rest periodsbetween pulses) with a VirTis model 45 homogenizer(The VirTis Co., Inc., Gardiner, N.Y.) equipped withmidi turboshear blades. The preparation was centri-fuged (25,000 x g, 20 min, 4 °C), the supernatant fluidwas discarded, and the pellet was suspended in 5 ml ofsterile PB. Portions (1 ml) of the sterile suspension ofdispersed stromal tissue were incubated, with gentleagitation in sterile plastic tubes (17 by 100 mm), withsterile PB (100 ,ul), with a washed suspension of S.marcescens strain BG (105 colony-forming units), orwith sterile solutions (0.5 mg in 100 IlI of PB) of papain(Sigma Chemical Co., St. Louis, Mo.) activated with0.1 M cysteine hydrochloride, collagenase (Worthing-ton Biochemical Corp., Freehold, N.J.), or serratiaprotease. The tissue suspension inoculated with S.marcescens was incubated for 20 h at 37°C. The tissuesuspensions containing the various enzyme prepara-tions or the control PB were supplemented with pen-icillin G (125 yg/ml) and streptomycin sulfate (200 ytg/ml) and incubated for 6 h at 37°C. After 6 h, a secondportion of either sterile buffer (100 pl) or the variousenzyme preparations (0.5 mg) was added to the appro-priate mixtures, and the mixtures were incubated foran additional 6 h. After incubation, the sterility of thetissue suspensions incubated with the control PB andwith the various enzyme preparations was determined,and the bacteria in the tissue suspension inoculatedwith S. marcescens were quantitated by plate countson Columbia agar (BBL Microbiology Systems, Cock-eysville, Md.) supplemented with 5% (vol/vol) sheepblood. The supernatant fluids of the digestion mixtures(solubilized stromal tissue) were obtained by centrif-ugation (25,000 x g, 20 min, 4°C), one PB wash (1 ml)of the pellets was pooled with the supernatant fluids,and the solutions were stored at -80°C until analyzed.The pellets from the digestion mixtures (nonsolubil-ized stromal tissue) were suspended in 4.5-ml portionsof 4 N hydrochloric acid and incubated at 70°C for 5h to produce homogeneous suspensions. The suspen-sions were adjusted to pH 7.0 and stored at -80°Cuntil analyzed. The ability of the growing bacteria andthe protease preparation to solubilize the corneal pro-teoglycan ground substance and collagen was exam-ined by analyzing the supernatant fluids and pelletsfor glucuronic acid (4) and total hexosamines (5)(markers for ground substance) and for hydroxypro-line (25) (marker for collagen).

Production of corneal damage by bacteria andenzyme preparations. New Zealand white rabbits(ca. 2.3 kg) anesthetized with ketamine hydrochloride,ether, and topical tetracaine hydrochloride were in-jected intracorneally (30-gauge needles), in groups ofca. 16 rabbits per group, with portions (40 ,ul) of thefollowing: (i) suspensions containing various numbers(6 x 102 to 6 x 105 colony-forming units) of viable S.marcescens strain BG (right eyes in group 1); (ii)sterile 0.85% saline or heat-killed (100°C for 15 min)bacteria (control left eyes in group 1); (iii) filter-steri-lized preparations of serratia protease (250 to 2,000ng) in 0.05 M tris(hydroxymethyl)aminomethane

(Tris)-hydrochloride buffer (pH 7.5; right eyes ingroup 2); and (iv) Tris-hydrochloride buffer or heat-inactivated (100°C for 15 min) protease preparations(control left eyes in group 2).Electron microscopy. Experimental animals were

sacrificed by intravenous injection of sodium pento-barbital, their comeas were immediately excised with-out disturbing the central comeal lesions, and thetissues were processed for and examined by electronmicroscopy as previously described (14), except thatthe thin sections were stained with phosphotungsticacid (9) and examined in a Zeiss 10-A microscope withan accelerating voltage of 60 kV.

Electron microscopic visualization of the stromalproteoglycan ground substance in corneas injectedwith 0.05 M Tris-hydrochloride buffer (pH 7.5), ser-ratia protease (500 ng), or S. marcescens (6 x 104colony-forming units) was accomplished by infiltratingthe corneas (by injection) with ruthenium red-contain-ing fixative (11), excising and cutting the corneas intowedge-shaped sections, fixing with the ruthenium red-containing fixative for 2 h at 4°C, washing the tissuesections with cacodylate buffer (0.2 M, pH 7.4), andpostfixing for 3 h at room temperature in a solution ofosmium tetroxide-ruthenium red (11). Further proc-essing of the tissues for electron microscopy was doneas described in the preceding paragraph, except thatthin sections were subsequently stained with uranylacetate and lead citrate (9).

RESULTSIn vitro effect of S. marcescens and ser-

ratia protease on solubility of rabbit cor-neal stromal tissue. Neither the growing bac-teria, which reached a population density of ca.105 bacteria per ml, nor the bacterial enzymepreparation possessed significant collagenolyticactivity; however, both caused extensive solubi-lization of the stromal proteoglycans (Table 1).Collagenase and papain served as positive con-trols to demonstrate the solubilization ofstromalcollagen and proteoglycan ground substance, re-spectively.Gross corneal damage elicited by exper-

imental serratia keratitis and by the ser-ratia protease. Intracorneal injection of ca. 6x 104 bacteria caused a rapidly progressing, de-structive infectious process characterized byconjunctivitis, corneal opacification, hypopyonformation, the presence of a mucopurulent exu-date, liquefactive necrosis of the cornea, desce-metocele formation, and corneal perforation(Fig. la and b). Intracorneal injection of submi-crogram amounts of the highly purified serratiaprotease preparation caused rapid liquefactivenecrosis of the cornea and descemetocele for-mation (grossly observable within 6 h) whichclosely resembled the lesion produced by thebacterial infection (Fig. lc and d). However, theprotease usually did not elicit as extensive cor-neal opacification and mucopurulent exudate as

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TABLE 1. Effect of S. marcescens and a highlypurified serratia protease preparation on the

solubility of cornealproteoglycans (as glucuronicacid and hexosamines) and collagen (as

hydroxyproline)Amt solubilized (%)

Treatment Glucu- Hex- Hy-ronic osa- droxy-acid' minesb pro-

linecPB control 9 21 1S. marcescens 39 49 3Serratia protease 100 61 8dPapain 49 53 19dClostridium histolyticum looe 74e 99

collagenasea Determined by the method of Blumenkrantz and

Asboe-Hansen (4).b Determined by the method of Blumenkrantz and

Asboe-Hansen (5) after hydrolysis of the samples with4 N hydrochloric acid for 30 min at 1200C.

'Determined by the micromethod of Woessner (25)after sample hydrolysis with 6 N hydrochloric acid for18 h at 130°C and removal of insoluble residue bycentrifugation.

d This effect is typical of that produced by noncol-lagenolytic proteases which possess limited nonspe-cific activity against peptide bonds in the nonhelical,terminal, telopeptide regions of the collagen molecule,rather than that produced by specific collagenaseswhich cleave peptide bonds in the helical region of themolecule (13).

e The solubilization of the stromal proteoglycans bythe commercially obtained C. histolyticum collagenasepreparation was caused by contamination of the prep-aration with nonspecific clostridial proteases.

the infection. Injection of more than 1 ,ig of theprotease preparation usually resulted in comealperforation.Electron microscopic characterization of

structural alterations in corneal stromatadamaged by experimental serratia kerati-tis and by the serratia protease. Electronmicroscopic examination of comeas 3 h, 1 day,and 7 days after the injection of control prepa-rations showed no obvious structural alterationsin the extracellular matrix of the corneal stro-mata (Fig. 2a, d, and e).

Structural alterations in the extracellular ma-trix of comeal stromata infected with S. marces-cens were studied 1 day and 7 days after theinjection of ca. 6 x 104 bacteria. Only smallnumbers of infiltrating polymorphonuclear leu-kocytes (PMNL) were observed in the comealstroma at 1 day postinfection, thus suggestingthat the bulk of the structural alterations ob-served at that time were not attributable tosubstances released from the PMNL. Stromalalterations observed 1 day postinfection con-

sisted ofthe dispersal of ultrastructurally normalcollagen fibrils (Fig. 2b) and the loss of ruthe-nium red-staining of the proteoglycan groundsubstance, especially in areas adjacent to bacte-ria (Fig. 2f and g). In addition, electron-densebeads indicative of degraded proteoglycanground substance (21) were visible on many ofthe collagen fibrils (Fig. 2g). Corneal stromataexamined late in the infection (7 days postinfec-tion) showed structural changes which werequalitatively similar to, but quantitatively moreextensive than, those seen at 1 day postinfection.In addition to the dispersed collagen fibrils andthe loss of proteoglycan ground substance, nu-merous deposits of fibrin and necrotic PMNLwere observed.The structural changes elicited in the extra-

cellular matrix of the corneal stromata by theserratia protease were identical to those ob-served during the bacterial infection (Fig. 2c andh), except that the enzyme preparation elicitedless PMNL infiltration of the stromata than theinfection.Stromata damaged by the experimental infec-

tion or by the serratia protease did not showtactoid formation, which is thought to be at firstview evidence of collagen breakdown and faultyfibrillar accretion (19) or of fragmented collagenfibrils, both of which are produced by intracor-neal injection of clostridial collagenase or con-centrated PMNL lysosomal preparations (10,19).

DISCUSSIONThe extensive grossly observable liquefactive

necrosis and descemetocele formation which oc-curs in rabbit corneas during serratia keratitisand after the intracorneal injection of a highlypurified serratia protease preparation suggeststhat the major cause of the observed cornealdestruction is the alteration or loss of the majorstructural component of the cornea, i.e., theextracellular stromal matrix composed of colla-gen and proteoglycan ground substance. Theobservations described in this study indicatethat both viable S. marcescens and the serratiaprotease preparation severely damage the rabbitcornea by causing the solubilization and loss ofthe corneal proteoglycan ground substance. Theproteoglycan is believed to maintain the orderand interfibrillar attachments of the corneal col-lagen fibrils (18, 21); thus, its loss could result indispersal of undegraded collagen fibrils, weak-ening of the comea, and subsequent desceme-tocele formation and corneal perforation by theanterior chamber pressure.The strong similarity between the tissue dam-

age produced by the infectious process and that

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930 LYERLY, GRAY, AND KREGER

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FIG. 1. Liquefactive necrosis and descemetocele formation produced in rabbit corneas by S. marcescenskeratitis and by a highly purified serratia protease preparation. (a) and (b) 3 and 7 days, respectively, afterthe intracorneal injection of ca. 6 x 104 bacteria. Injection of heat-killed bacteria produced only mildconjunctivitis and corneal opacification, which resolved by 3 to 4 days postinjection. (c) and (d) 6 and 16 h,respectively, after the intracorneal injection of ca. 500 ng of serratia protease preparation. Injection of thediluents or heat-inactivated enzyme produced immediate opacification at the site of injection; however, thecornea appeared normal by 3 to 4 h postinjection.

caused by the purified bacterial enzyme supportsthe idea that the ability of the bacterium to elicitcorneal destruction is dependent, at least in part,on the in vivo production by the bacterium ofcornea-damaging proteases. However, our obser-vations should not be construed as suggestingthat the bacterium may not synthesize other

cornea-damaging toxins or enzymes capable ofplaying a role in the pathogenesis of serratiakeratitis. One difference we noted between thein vivo response elicited by the bacterium andthat elicited by the protease was that moremucopurulent exudate and more PMNL infiltra-tion of the comneal stroma was produced by the

FIG. 2. Ultrastructural corneal alterations produced by experimental S. marcescens keratitis and by theserratia protease. Line markers on all micrographs represent 0.1 ttm. (a) Normal structure and orderedarrangement ofcollagen fibrils (longitudinal section) in control cornea. (b) Longitudinal section ofextensivelydispersed but ultrastructurally normal collagen fibrils observed 1 day after the intracorneal injection of ca.6 x 104 bacteria. (c) Longitudinal section ofextensively dispersed but ultrastructurally normal collagen fibrilsobserved 6 h after the intracorneal injection of ca. 500 ng of serratia protease. (d) and (e) Normal stainingpattern of corneal proteoglycan ground substance by the ruthenium red technique. Note the electron-dense,ruthenium red-stained ground substance surrounding and coating the coUagen fibrils. (d) Cross section. (e)Longitudinal section. (f) and (g) Cross section and longitudinal section, respectively, demonstrating thereduction of ruthenium red staining of the proteoglycan ground substance 1 day after the intracornealinjection of ca. 6 x 104 bacteria. The glycocalyx ofa bacterium in the field of (f) is also stained with rutheniumred, thus confirming the penetration of the stain into the tissue. Electron-dense beads indicative of degradedproteoglycan ground substance (21) are visible on many of the ultrastructurally normal collagen fibrils. (h)Longitudinal section demonstrating the reduction of ruthenium red staining of the proteoglycan groundsubstance 6 h after the intracorneal injection of ca. 500 ng of serratia protease.

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932 LYERLY, GRAY, AND KREGER

infection than by the enzyme. This observationsuggests that chemotactic factors, which are notpresent in the purified protease preparation, areproduced by the bacterium during the develop-ment of the infectious process. In this regard,intracorneal injection of the lipopolysaccharideendotoxin of the bacterium was reported (2) toelicit severe corneal edema and opacificationand intense PMNL infiltration of the cornealstroma. However, the substance was not foundto cause the extensive liquefactive necrosis anddescemetocele formation characteristically pro-duced by the bacterium and the bacterial pro-tease. For further evaluating the possible impor-tance of the bacterial protease in the pathogen-esis of serratia keratitis, studies are currently inprogress in our laboratory to determine whetheractive immunization against the purified pro-tease can reduce or prevent the damage pro-duced after intracorneal challenge with the bac-terium and whether the protease can be detectedin situ in corneas experimentally infected withS. marcescens.Although the data presented in this paper

indicate that the observed solubilization and lossof the stromal proteoglycan ground substanceresult from direct degradation by the serratiaprotease, the ground substance also may be de-graded in vivo by host-derived or endogenousenzymes. For example, the presence of largenumbers of necrotic PMNL in the tissue duringthe later stages of the infectious disease processand after injection of the bacterial protease sug-gests the possibility that enzymes released fromthe necrotic cells may enhance the damage elic-ited by the bacterial protease. Brown et al. (6)reported that PMNL lysosomes possess an en-zyme or enzymes capable of degrading cornealproteoglycan at neutral pH in vitro.

ACKNOWLEDGMENTSThis investigation was supported by Public Health Service

grant EY-01104 from the National Eye Institute.

LITERATURE CITED1. Atlee, W. E., R. P. Burns, and M. Oden. 1970. Serratia

marcescens keratoconjunctivitis. Am. J. Ophthalmol.70:31-33.

2. Bernstein, H. N., and Y. T. Maddox. 1973. Cornealpathogenicity of Serratia marcescens in the rabbit.Trans. Am. Acad. Ophthalmol. Otolaryngol. 77:432-440.

3. Bigger, J. R., G. Meltzer, A. Mandell, and R. M.Burde. 1971. Serratia marcescens endophthalmitis.Am. J. Ophthalmol. 72:1102-1105.

4. Blumenkrantz, N., and G. Asboe-Hansen. 1973. Newmethod for quantitative determination of uronic acids.

Anal. Biochem. 54:484-489.5. Blumenkrantz, N., and G. Asboe-Hansen. 1976. An

assay for total hexosamine and a differential assay forglucosamine and galactosamine. Clin. Biochem. 9:269-274.

6. Brown, S. I., C. W. Hook, and M. P. Tragakis. 1972.Presence, significance, and inhibition of lysosomal pro-teoglycanases. Invest. Ophthalmol. 11:149-152.

7. Cooper, R. L., and L. J. Constable. 1977. Infectivekeratitis in soft contact lens wearers. Br. J. Ophthalmol.61:250-254.

8. Davis, J. T., E. Foltz, and W. S. Blakemore. 1970.Serratia marcescens. A pathogen of increasing clinicalimportance. J. Am. Med. Assoc. 214:2190-2192.

9. Dawes, C. J. 1971. Biological techniques in electron mi-croscopy. Barnes and Noble Books, New York.

10. Gray, L. D., and A. S. Kreger. 1975. Rabbit cornealdamage produced by Pseudomonas aeruginosa infec-tion. Infect. Immun. 12:419-432.

11. Hayat, M. A. 1972. Basic electron microscopy techniques,p. 85. Van Nostrand Reinhold Co., New York.

12. Huber, J. D., F. Parker, and G. F. Odland. 1968. Abasic fuchsin and alkalinized methylene blue rapid stainfor epoxy-embedded tissue. Stain Technol. 43:83-87.

13. Kessler, E., H. E. Kennah, and S. L. Brown. 1977.Pseudomonas protease. Purification, partial character-ization, and its effect on collagen, proteoglycan, andrabbit corneas. Invest. Ophthahmol. Visual Sci. 16:488-497.

14. Kreger, A. S., and L. D. Gray. 1978. Purification ofPseudomonas aeruginosa proteases and microscopiccharacterization of pseudomonal protease-induced rab-bit corneal damage. Infect. Immun. 19:630-648.

15. Kreger, A. S., and O. K. Griffin. 1975. Cornea-damagingproteases of Serratia marcescens. Invest. Ophthalmol.14:190-198.

16. Lazachek, G. W., G. L. Boyle, A. L. Schwartz, and I.H. Leopold. 1971. Serratia marcescens, an ocular path-ogen. Arch. Ophthalmol. 86:599-603.

17. Lyerly, D., and A. Kreger. 1979. Purification and char-acterization of a Serratia marcescens metalloprotease.Infect. Immun. 24:411-421.

18. Maurice, D. M. 1969. The cornea and sclera, p. 489-600.In H. Davson (ed.), The eye, vol. 1. Academic Press,Inc., New York.

19. Rowsey, J. J., R. M. Nisbet, J. L. Swedo, and L.Katona. 1976. Corneal collagenolytic activity in rabbitpolymorphonuclear leukocytes. J. Ultrastruct. Res. 57:10-21.

20. Salceda, S. R., J. Lapuz, and R. Vizeonde. 1973. Ser-ratia marcescens endophthalmitis. Arch. Ophthalmol.89:163-166.

21. Smith, J. W., and J. Frame. 1969. Observations on thecollagen and proteinpolysaccharide complex of rabbitcorneal stroma. J. Cell Sci. 4:421-436.

22. Stenderup, A., 0. Faergeman, and M. Ingerslev. 1966.Serratia marcescens infections in premature infants.Acta Pathol. Microbiol. Scand. 68:157-160.

23. Tokunga, T., T. Fujino, K. Yamamoto, and A. Ideda.1969. A case of serpiginous corneal ulcer caused bySerratia ofEnterobacteriaceae. Folia Ophthalmol. Jpn.20:862-868.

24. Von Graevenitz, A., and S. J. Rubin. 1980. The genusSerratia. CRC Press, West Palm Beach, Fla.

25. Woessner, J. F., Jr. 1961. The determination of hydrox-yproline in tissue and protein samples containing smallproportions of this imino acid. Arch. Biochem. Biophys.93:440-447.

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