RESUMEN

PURPOSE: To determine the importance of surfactant protein D in Pseudomonas keratitis. METHODS: The surfactant D status of wild-type and surfactant D-deficient Black Swiss mice was confirmed by PCR reactions and immunoblot assay. Mouse corneas were infected with one of three strains of P. aeruginosa. At 1, 2, 3, and 6 days postinfection, eyes were scored by slit-lamp examination and bacteria per cornea quantified. RESULTS: Infected wild-type mice had slit-lamp scores on 3 and 6 days postinfection that were significantly lower than those of surfactant D-deficient mice (p

Asunto(s)

Queratitis/metabolismo , Queratitis/microbiología , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Proteína D Asociada a Surfactante Pulmonar/metabolismo , Animales , Córnea/metabolismo , Córnea/microbiología , Immunoblotting , Queratitis/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Pseudomonas aeruginosa/crecimiento & desarrollo , Proteína D Asociada a Surfactante Pulmonar/deficiencia , Factores de Tiempo

RESUMEN

PURPOSE: Pseudomonas aeruginosa PAO1 deficient in LasA protease was reported to be ocularly avirulent. However, the avirulence of this mutant could not attributed to the loss of LasA protease. The purpose of this study was to define the mechanism for such a mutant's inability to cause corneal disease. METHODS: A LasA protease--deficient mutant of P. aeruginosa PAO1 was constructed by allelic exchange. Virulence of this mutant in mouse and rabbit models of keratitis was assessed by scoring for ocular disease and quantitating viable bacteria from infected corneas. Adherence to scarified mouse corneal tissue was determined with an organ culture assay. RESULTS: In the mouse eye, the LasA protease--deficient mutant was not virulent, despite being as adherent as its parent strain. Virulence of the mutant was also significantly reduced in the rabbit eye. Complementation with lasA did not restore virulence in either model of infection. Neither the mutant nor the mutant complemented with lasA grew well in ocular tissue. An analysis of the mutant showed that it was auxotrophic for leucine. CONCLUSION: These data show that the mutant's avirulence in the eye is caused by poor growth in the ocular environment and not the loss of a functional lasA gene.

Asunto(s)

Proteínas Bacterianas , Córnea/microbiología , Úlcera de la Córnea/microbiología , Infecciones Bacterianas del Ojo/microbiología , Metaloendopeptidasas/deficiencia , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidad , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos , Pseudomonas aeruginosa/enzimología , Conejos , Virulencia

RESUMEN

PURPOSE: A mutant strain of Pseudomonas aeruginosa deficient in LasA protease (staphylolytic protease) has been described as having reduced ocular virulence, suggesting that LasA is a major virulence factor. This study was undertaken to provide further genetic analysis of the role of P. aeruginosa LasA protease in ocular infections. METHODS: LasA protease-deficient mutants of P. aeruginosa PAO1-V and ATCC 19660 were constructed by allelic replacement. Mutants and their respective wild type parent strains were evaluated for virulence and growth in the eye using mouse scarification and rabbit intrastromal injection models of keratitis. RESULTS: LasA protease-deficient mutants of both strains were as virulent as wild type strains, growing to 4 to 6 log10 CFU/cornea and causing significant ocular pathology in the mouse (P > 0.42) and rabbit (P > 0.53). CONCLUSIONS: These data show that LasA protease is not a major corneal virulence factor, suggesting that the main mechanism of corneal damage has yet to be definitively identified.

Asunto(s)

Proteínas Bacterianas , Sustancia Propia/microbiología , Úlcera de la Córnea/microbiología , Infecciones Bacterianas del Ojo/microbiología , Metaloendopeptidasas/fisiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidad , Animales , Recuento de Colonia Microbiana , Sustancia Propia/patología , Úlcera de la Córnea/patología , Infecciones Bacterianas del Ojo/patología , Femenino , Metaloendopeptidasas/deficiencia , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/aislamiento & purificación , Conejos , Virulencia

RESUMEN

PURPOSE: Alkaline protease has been associated with virulence in Pseudomonas aeruginosa corneal infections. To define the role of this enzyme in such infections, isogenic mutants of P. aeruginosa deficient in alkaline protease production were constructed. This study examines the ability of these mutants to adhere to scarified corneal tissue in vitro and to establish corneal infections in vivo. METHODS: Mutants were constructed by allelic exchange in two phenotypically different wild type strains, PAO1 (invasive) and ATCC 19660 (cytotoxic). Alkaline protease-deficient mutants were characterized by zymography and western blot analysis of bacterial culture supernatants. Allelic exchange was confirmed by PCR analysis of the disrupted aprA gene of the mutants. Adherence of wild type and mutant strains to scarified corneal epithelium was assessed by an in vitro organ culture assay, while ocular virulence of the strains was determined in vivo using a mouse scarification model of bacterial keratitis. RESULTS: Being isogenic, phenotypes of mutants were identical to their respective parents with the exception of the loss of alkaline protease production. The absence of alkaline protease did not alter corneal adherence or ocular virulence of the organisms when compared to similar wild type strains. CONCLUSIONS: These data provide evidence that alkaline protease produced by P. aeruginosa is not essential in the pathogenesis of P. aeruginosa keratitis.

Asunto(s)

Adhesión Bacteriana , Córnea/microbiología , Úlcera de la Córnea/microbiología , Infecciones Bacterianas del Ojo/microbiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidad , Serina Endopeptidasas/fisiología , Animales , Proteínas Bacterianas , Western Blotting , Úlcera de la Córnea/patología , Cartilla de ADN/química , ADN Bacteriano/análisis , Endopeptidasas/genética , Infecciones Bacterianas del Ojo/patología , Femenino , Vectores Genéticos , Ratones , Ratones Endogámicos C57BL , Mutación , Reacción en Cadena de la Polimerasa , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/enzimología , Serina Endopeptidasas/deficiencia , Virulencia

RESUMEN

PURPOSE: Pseudomonas aeruginosa proteases are thought to be important virulence factors in the pathogenesis of corneal disease. This study examined protease production from two strains of P. aeruginosa responsible for two very distinct clinical diseases: strain Paer1, isolated from a Contact Lens-induced Acute Red Eye (CLARE), and strain KEI 1025, isolated from a corneal ulcer. Strains were compared to a laboratory strain (ATCC 19660) known to produce severe keratitis in experimentally infected mice for protease production and for ocular virulence. METHODS: Protease production was examined with colorimetric assays, gelatin zymography and western blots. Elastase A activity was quantitated with a staphylolytic assay. Ocular virulence was examined using a mouse scratch model of keratitis. RESULTS: In contrast to strains KEI 1025 or ATCC 19660, Paer1 was unable to produce enzymatically active elastase A, elastase, and protease IV. All three strains produced active alkaline protease. Strains KEI 1025 and ATCC 19660 produced a fulminant keratitis in mice whereas Paer1 produced a mild transient infection. Restoration of elastase activity in Paer1 via genetic complementation did not result in a virulent phenotype. Co-infection of mouse eyes with strains Paer1 and ATCC 19660 resulted in the eventual loss of Paer1 from corneal tissue. CONCLUSIONS: These studies suggest that P. aeruginosa elastase A and/or protease IV, but not alkaline protease or elastase, contribute to the ocular virulence of this organism.

Asunto(s)

Lentes de Contacto/efectos adversos , Úlcera de la Córnea/microbiología , Exopeptidasas/metabolismo , Pseudomonas aeruginosa/enzimología , Enfermedad Aguda , Animales , Sangre , División Celular/efectos de los fármacos , Recuento de Colonia Microbiana , Úlcera de la Córnea/etiología , Medios de Cultivo/farmacología , Ojo/microbiología , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/aislamiento & purificación , Pseudomonas aeruginosa/patogenicidad , Virulencia

RESUMEN

Polymorphonuclear neutrophils (PMN) in Pseudomonas aeruginosa-infected cornea are required to clear bacteria from affected tissue, yet their persistence may contribute to irreversible tissue destruction. This study examined the role of C-X-C chemokines in PMN infiltration into P. aeruginosa-infected cornea and the contribution of these mediators to disease pathology. After P. aeruginosa challenge, corneal PMN number and macrophage inflammatory protein-2 (MIP-2) and KC levels were compared in mice that are susceptible (cornea perforates) or resistant (cornea heals) to P. aeruginosa infection. While corneal PMN myeloperoxidase activity (indicator of PMN number) was similar in both groups of mice at 1 and 3 days postinfection, by 5-7 days postinfection corneas of susceptible mice contained a significantly greater number of inflammatory cells. Corneal MIP-2, but not KC, levels correlated with persistence of PMN in the cornea of susceptible mice. To test the biological relevance of these data, resistant mice were treated systemically with rMIP-2. This treatment resulted in increased corneal PMN number and significantly exacerbated corneal disease. Conversely, administration of neutralizing MIP-2 pAb to susceptible mice reduced both PMN infiltration and corneal destruction. Collectively, these findings support an important role for MIP-2 in recruitment of PMN to P. aeruginosa-infected cornea. These data also strongly suggest that a timely down-regulation of the host inflammatory response is critical for resolution of infection.

Asunto(s)

Infecciones Bacterianas del Ojo/inmunología , Infecciones Bacterianas del Ojo/patología , Monocinas/fisiología , Infiltración Neutrófila/inmunología , Animales , Quimiocina CXCL2 , Recuento de Colonia Microbiana , Susceptibilidad a Enfermedades , Infecciones Bacterianas del Ojo/metabolismo , Sueros Inmunes/administración & dosificación , Inmunidad Innata , Inyecciones Intraperitoneales , Queratitis/inmunología , Queratitis/metabolismo , Queratitis/patología , Recuento de Leucocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Monocinas/genética , Monocinas/inmunología , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/citología , Pseudomonas aeruginosa/inmunología , ARN Mensajero/metabolismo , Proteínas Recombinantes/administración & dosificación

RESUMEN

In this study, the role of intercellular adhesion molecule 1 (ICAM-1) in the pathogenesis of Pseudomonas aeruginosa keratitis was examined by using inbred ICAM-1-deficient knockout mice. These mice had significantly less (P

Asunto(s)

Molécula 1 de Adhesión Intercelular/fisiología , Queratitis/microbiología , Infecciones por Pseudomonas/etiología , Animales , Ojo/inmunología , Ojo/patología , Queratitis/inmunología , Queratitis/patología , Ratones , Ratones Noqueados , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/patología

RESUMEN

Pseudomonas aeruginosa is the most common pathogen among contact lens-associated infections. This study investigated the response of the murine cornea to infection with an ocular strain of P. aeruginosa isolated from a subject with an inflammatory adverse response to contact lens wear termed CLARE. Although this bacterium was isolated in confluency (greater than 2000 cfu lens-1) from the lens at the time of the inflammatory episode, no infection of the cornea subsequently developed. Male C57BL/6J mice (20 per strain) had their corneas scratched with a 26 gauge needle (3 parallel 1.0 mm wounds in the left eye only). The incisions were centered over the pupillary axis and penetrated the epithelial cell basal lamina and into the superficial stroma. The CLARE strain was found to persist (viable bacteria could be cultured from corneal homogenates) up to 8 hr, as did the virulent control strain ATCC 19660. At 24 hr, only ATCC 19660 could be cultured, indicating an inability of the strain isolated from CLARE, Paer1, to persist in the eye consistent with the human inflammatory episode. Histological examination of the mouse tissue showed further differences between infection by the two strains. Infection with ATCC 19660 resulted in tissue necrosis and a large population of polymorphonuclear leukocytes (PMNs) recruited to the wound site. In contrast, during infection with the CLARE strain, PMN recruitment was reduced and temporally delayed. The CLARE strain grew as well as ATCC 19660 in vitro but produced less protease activity, in particular less elastase. The decreased PMN response and decreased protease production by the CLARE strain may have been responsible for the lack of ocular damage and apparent healing of the wound. P. aeruginosa strains are considered to be invasive or cytotoxic to corneal tissue, however this strain may represent a third inflammatory type consistent with its differing pathology.

Asunto(s)

Conjuntivitis Bacteriana/microbiología , Lentes de Contacto de Uso Prolongado/efectos adversos , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/patogenicidad , Animales , Conjuntivitis Bacteriana/inmunología , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Activación Neutrófila , Virulencia

RESUMEN

PURPOSE: To determine whether environmental factors or bacterial viability affect the binding of two strains of P. aeruginosa to mouse cornea. METHODS: Scarified corneas were placed in organ culture and inoculated with P. aeruginosa cell suspensions containing either ATCC 19660 or PAO1 bacterial strains classed as cytotoxic or invasive, respectively. Eyes were incubated in vitro for 1 h after bacterial application at different pH or temperature conditions or in PBS containing various divalent cations. The adhesion of heat-killed or formalin-fixed bacteria was tested similarly. Scanning electron microscopy (scanning EM) was used to quantitate adherent bacteria. RESULTS: P. aeruginosa ATCC 19660 showed an increase in binding at pH 8.0, favored higher temperatures and required both calcium and magnesium for optimum binding. Adherence of PAO1 was enhanced at pH 6.5 and decreased at pH 8.0. This strain favored binding at lower temperatures and did not require either divalent cation for optimum binding. In addition, the presence of magnesium ions resulted in reduced binding for this strain. Both strains exhibited less binding ability after formalin fixation or heat killing. CONCLUSION: Environmental factors and bacterial viability are important factors which influence the ability of both cytotoxic and invasive strains of P. aeruginosa to bind to the scarified cornea.

Asunto(s)

Adhesión Bacteriana/fisiología , Cationes Bivalentes/farmacología , Córnea/microbiología , Pseudomonas aeruginosa/fisiología , Temperatura , Cicatrización de Heridas , Animales , Adhesión Bacteriana/efectos de los fármacos , Calcio/farmacología , Córnea/efectos de los fármacos , Lesiones de la Cornea , Concentración de Iones de Hidrógeno , Magnesio/farmacología , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Rastreo , Técnicas de Cultivo de Órganos

RESUMEN

PURPOSE: A Pseudomonas mutant deficient in protease IV has significantly reduced virulence in experimental keratitis. In the present study, the corneal toxicity of purified protease IV and its ability to augment the virulence of protease-IV-deficient bacteria were analyzed. METHODS: The toxicity of purified protease IV was determined by intrastromally injecting the exoenzyme (20-200 ng) into the cornea. The effects of protease IV on the corneal virulence of the protease-IV-deficient strain, PA103-29::Tn9, were determined by injecting eyes with 1000 CFU of log phase bacteria plus either 200 ng active purified protease IV or 200 ng heat-inactivated protease IV. Changes in ocular disease, determined by slit-lamp examination, were measured at 3, 16, 22, and 27 hours after infection. Colony-forming units per cornea were quantified at 27 hours after infection. RESULTS: Purified protease IV at doses from 50 to 200 ng induced epithelial defects within 3 hours of injection. Injection of 20 ng active protease IV or heat-inactivated protease IV (200 ng) had no effect on ocular tissue. Corneal virulence of the protease-IV-deficient strain was augmented by intrastromal injection with purified protease IV but not with heat-inactivated protease IV (P < or = 0.0001). Neither active nor heat-inactivated protease IV altered the growth of bacteria in the cornea (6 log units; P = 0.81). CONCLUSIONS: The important role of protease IV in corneal virulence was demonstrated by direct toxicity and by its ability to significantly augment the virulence of protease-IV-deficient Pseudomonas.

Asunto(s)

Córnea/efectos de los fármacos , Úlcera de la Córnea/microbiología , Infecciones Bacterianas del Ojo/microbiología , Péptido Hidrolasas/farmacología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidad , Animales , Recuento de Colonia Microbiana , Córnea/microbiología , Córnea/patología , Úlcera de la Córnea/inducido químicamente , Úlcera de la Córnea/patología , Infecciones Bacterianas del Ojo/inducido químicamente , Infecciones Bacterianas del Ojo/patología , Péptido Hidrolasas/aislamiento & purificación , Infecciones por Pseudomonas/inducido químicamente , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/enzimología , Conejos , Virulencia

RESUMEN

Corneal clarity in young adult Swiss (HSD:ICR) mice is restored after Pseudomonas aeruginosa infection. Previous data showed that this response involves a rapid up-regulation of constitutive intercellular cell adhesion molecule-1 (ICAM-1) and migration of inflammatory cells into the cornea. In contrast, in aged mice, there is no up-regulation of corneal ICAM-1, inflammatory cell infiltration into the cornea is delayed, and the cornea perforates. Therefore, the aim of this study was to test whether specific cytokines which up-regulate ICAM-1 expression differ in young and aged mice. Corneas of young (6- to 8-week-old) and aged (1- to 2-year-old) mice were scarified and inoculated with P. aeruginosa. The eyes were graded for pathologic changes (score 0 to +4); at 6, 12, 24, and 48 h postinfection (p.i.), six mice from each age group were sacrificed. Three corneas from each respective group were excised for quantitation of interleukin-1beta (IL-1beta), tumor necrosis factor alpha, and gamma interferon (IFN-gamma) by enzyme-linked immunosorbent assay. The remaining three corneas from each age group were harvested for quantitation of viable bacteria by direct plate count determination and for infiltrating polymorphonuclear leukocytes (PMNs) by a myeloperoxidase (MPO) assay. Compared to those of young mice, the corneas of infected aged mice had less IL-1beta at 6 h p.i. (P < or = 0.04) and less IFN-gamma at 12 to 48 h p.i. (P < or = 0.05). Also, compared to those of young mice, corneas of aged mice had fewer PMNs (P < or = 0.008) by the MPO assay at 6 h p.i. and more viable bacteria (P < or = 0.01) per cornea by plate count determination at 24 h p.i. These data suggest that the lack of up-regulation of ocular ICAM-1 in aged mice may reflect a reduction in both IL-1beta and IFN-gamma levels in the infected cornea. Consequently, a sufficient number of PMNs and other inflammatory cells fail to rapidly migrate into the infected corneas of aged mice, the bacterial load is initially greater than that in young mice, and the cornea perforates.

Asunto(s)

Envejecimiento/fisiología , Interferón gamma/deficiencia , Interleucina-1/deficiencia , Queratitis/etiología , Infecciones por Pseudomonas/etiología , Factor de Necrosis Tumoral alfa/deficiencia , Animales , Córnea/química , Córnea/patología , Femenino , Técnicas In Vitro , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-1/análisis , Ratones , Ratones Endogámicos ICR , Neutrófilos/citología , Peroxidasa/análisis , Factor de Necrosis Tumoral alfa/análisis , Regulación hacia Arriba

RESUMEN

PURPOSE: The role of protease IV in the pathogenesis of Pseudomonas aeruginosa keratitis was investigated by comparing a mutant strain completely deficient in protease IV activity with its protease IV activity-producing parent. METHODS: A protease IV-deficient Pseudomonas strain PA103-29::Tn9 was generated by mutagenesis of strain PA103-29, which produces protease IV, through transposon insertion. Protease IV activity was determined by a casein agar assay, zymography, and cleavage of the chromogenic substrate, Chromozym PL. Corneal virulence was evaluated by slit lamp examination and bacterial cultures in both a rabbit intrastromal model and a mouse topical model of keratitis. RESULTS: The protease IV-deficient strain PA103-29::Tn9 had significantly reduced corneal virulence relative to its parent strain PA103-29 in both a rabbit intrastromal model and a mouse topical model of infection. In the rabbit model, ocular damage (slit lamp examination score) mediated by the parent strain was severe at 32 hours after infection, whereas damage mediated by the mutant was minimal at both 32 and 55 hours after infection. This difference in virulence was not a result of differences in growth in vivo, because both strains grew equally. In the mouse model, eyes inoculated with the protease IV-producing parent strain had significant corneal damage as early as 24 hours after infection, whereas the protease IV-deficient mutant strain produced no significant corneal damage during 6 days of infection. CONCLUSIONS: The ability to produce active protease IV was the determining factor in the severity of corneal virulence. Protease IV appears to mediate corneal virulence and should be considered as a target in the development of medications designed to minimize corneal damage during Pseudomonas keratitis.

Asunto(s)

Péptido Hidrolasas/deficiencia , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/patogenicidad , Animales , Recuento de Colonia Microbiana , Córnea/microbiología , Enfermedades de la Córnea/patología , Sustancia Propia/microbiología , Femenino , Ratones , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/genética , Conejos , Especificidad de la Especie

RESUMEN

The goal of this study was to test whether bacterial exoproducts, such as elastase or alkaline protease contribute to the initial binding of Pseudomonas aeruginosa to mouse corneal epithelium. Each protease, purified from P. aeruginosa, when applied exogenously at concentrations of either 25 or 50 ng ml-1, elevated binding of Pseudomonas to mouse cornea in organ culture. Polyclonal antibodies against bacterial alkaline protease, but not elastase, interfered with bacterial binding and reduced significantly the number of organisms bound to cornea in an organ culture binding inhibition assay. Zymographic analysis of conditioned media from additional organ culture experiments showed that the P. aeruginosa strain used, which is highly virulent in cornea in vivo, secretes detectable levels of alkaline protease, but not elastase in vitro and that secretion was enhanced if the corneal epithelium was wounded. Lastly, how alkaline protease enhanced bacterial binding to the corneal epithelium of the organ cultured eye was examined. Data from this study suggest that exposure of lipase-sensitivity epithelial receptors represents at least one mechanism.

Asunto(s)

Adhesión Bacteriana , Córnea/microbiología , Endopeptidasas/fisiología , Elastasa Pancreática/fisiología , Pseudomonas aeruginosa/enzimología , Animales , Anticuerpos Antibacterianos/farmacología , Córnea/efectos de los fármacos , Lesiones de la Cornea , Medios de Cultivo Condicionados/análisis , Endopeptidasas/metabolismo , Femenino , Técnicas In Vitro , Lipasa/farmacología , Ratones , Elastasa Pancreática/antagonistas & inhibidores , Elastasa Pancreática/metabolismo , Inhibidores de Proteasas/farmacología

RESUMEN

A polyclonal antibody (pAb) against gangliotetraosylceramide (asialo GM1), a glycolipid to which bacterial pili and LPS bind, and a mAb against a 66 kDa pilus-binding protein purified from adult mouse corneal epithelium were used to determine if antibodies against host receptors for bacterial adhesins could inhibit bacterial binding to wounded corneal epithelium and protect ocularly challenged mice from corneal perforation when topically applied. Bacteria were mixed with anti-66 kDa mAb, a mixture of anti-asialo GM1 pAb and anti-66 kDa mAb, an irrelevant control mAb (anti-human histocompatibility Ag HLA-DR5) or PBS prior to application to scarified corneas in organ culture. The combination of the two antibodies or the anti-66 kDa mAb alone was effective in reducing bacterial adherence compared with either PBS or the antibody control. To determine if these antibodies were protective in vivo, corneas of C57BL/6J mice were scarified and inoculated with Pseudomonas aeruginosa. Eyes were treated topically with anti-asialo GM1 pAb, anti-66 kDa mAb, a mixture of the two or control mouse serum. More serum-treated corneas perforated compared to corneas from any other group (P < or = 0.005) by 30 days postinfection. Treatment with a combination of the two antibodies resulted in significantly less corneal pathology 30 days p.i. when compared to any other treatment (P < or = 0.005). These data provide evidence that antibodies against host corneal receptors significantly inhibit bacterial binding in vitro and when applied topically in vivo, lessen the severity of ocular disease characteristic of P. aeruginosa keratitis.

Asunto(s)

Anticuerpos Antibacterianos/farmacología , Lesiones Oculares Penetrantes/inmunología , Lesiones Oculares Penetrantes/prevención & control , Gangliósido G(M1)/inmunología , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/fisiología , Receptores Inmunológicos/inmunología , Animales , Unión Competitiva/inmunología , Electroforesis en Gel Bidimensional , Epitelio/inmunología , Femenino , Fimbrias Bacterianas/química , Ratones , Ratones Endogámicos C57BL

RESUMEN

PURPOSE: The role of exoproteins in the pathogenesis of Pseudomonas aeruginosa keratitis was investigated in three animal models by assessing the relationship between corneal virulence and the activities of exotoxin A, elastase, alkaline protease, and an uncharacterized protease, protease IV. METHODS: The four Pseudomonal strains tested included a prototype strain (ATCC 27853) producing exotoxin A, elastase, and alkaline protease; a parent strain (PA103) producing only exotoxin A and protease IV; a mutant (PA103-29) producing only protease IV; and a mutant (PA103-AP1) producing exotoxin A and having only approximately 5% of the protease IV activity of its parent. Corneal virulence was evaluated in the mouse scratch, rabbit scratch, and rabbit intrastromal models in terms of clinical signs (slit lamp examination, slit lamp examination), and viable bacteria. RESULTS: Protease IV, the only protease produced by PA103 and PA103-29, was found to produce a unique band on zymograms (120 kDa) and to react distinctively with a synthetic substrate. Evidence for the role of protease IV in corneal virulence included two findings: PA103-29,which produced protease IV but not the other exoproteins, caused infections that were as severe as those caused by the prototype strain (ATCC 27853) in all three models (P>0.24); and PA103-AP1, the strain deficient in 95% of the parent protease IV activity, mediated infections characterized by slit lamp examination scores significantly lower than those of infections caused by the parent (PA103) or the prototype strain (ATCC 27853) in the rabbit and mouse scratch models (P<0.02). CONCLUSIONS: Protease IV was found to be a novel Pseudomonas protease contributing to corneal virulence in rabbits and mice when infections were initiated at the corneal surface. Furthermore, production of protease IV in low quantities was sufficient for virulence when the topical stages of keratitis were bypassed by an intrastromal injection of Pseudomonas.

Asunto(s)

ADP Ribosa Transferasas , Toxinas Bacterianas , Córnea/microbiología , Exotoxinas/fisiología , Infecciones Bacterianas del Ojo/etiología , Queratitis/microbiología , Péptido Hidrolasas/fisiología , Infecciones por Pseudomonas/etiología , Pseudomonas aeruginosa/patogenicidad , Factores de Virulencia , Animales , Recuento de Colonia Microbiana , Infecciones Bacterianas del Ojo/fisiopatología , Femenino , Queratitis/fisiopatología , Ratones , Infecciones por Pseudomonas/fisiopatología , Pseudomonas aeruginosa/enzimología , Conejos , Serina Endopeptidasas/fisiología , Virulencia , Exotoxina A de Pseudomonas aeruginosa

RESUMEN

PURPOSE: To establish if active pseudomonal proteases are present in vivo during corneal infection with Pseudomonas aeruginosa and to determine if the mouse strains used in these and previous studies have the ability to mount a nonocular antibody response to the purified proteases because antibodies to the bacterial proteases were not detected previously during in vivo ocular infection. METHODS: At certain times after corneal infection with P. aeruginosa, corneas were harvested and supernatants from the corneal homogenates were analyzed for proteolytic activity by zymography and immunoreactivity by immunoblotting. The efficiency of the extraction procedures used in these studies was determined by incubating uninfected corneal homogenates with the purified proteases. The resultant supernatants were analyzed for alkaline protease and elastase activity. Additionally, mice were immunized intraperitoneally with the purified proteases with and without adjuvant to determine if the animals could mount a nonocular antibody response. RESULTS: Corneas infected with P. aeruginosa demonstrated the presence of alkaline protease, but not elastase, by the two methods examined. The kinetics of the in vivo alkaline protease response closely parallels previously reported bacterial clearance studies in that peak alkaline protease activity was detected in corneal tissue when peak bacterial numbers also were observed in the eye, and it was absent when the eyes were sterile or nearly sterile. In addition, C57BL/6J mice were capable of mounting a nonocular antibody response to microgram quantities of both proteases only in the presence of adjuvant. CONCLUSIONS: In the model described, enzymatically active alkaline protease, but not elastase, was demonstrated in corneal tissues during in vivo infection. Concentrations of these proteases were much lower than those required to stimulate an antibody response.

Asunto(s)

Infecciones Bacterianas del Ojo/enzimología , Queratitis/enzimología , Infecciones por Pseudomonas/enzimología , Pseudomonas aeruginosa/enzimología , Serina Endopeptidasas/biosíntesis , Animales , Anticuerpos Antibacterianos/análisis , Córnea/enzimología , Infecciones Bacterianas del Ojo/inmunología , Inmunización , Immunoblotting , Queratitis/microbiología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Elastasa Pancreática/biosíntesis , Elastasa Pancreática/inmunología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Conejos , Serina Endopeptidasas/inmunología

RESUMEN

PURPOSE: In young Swiss (HSD:ICR) outbred mice, corneal clarity is restored after Pseudomonas aeruginosa ocular infection, whereas disease in aged outbred mice progresses to corneal perforation. This study was conducted to elucidate further the mechanism responsible for this age-related disparity in disease response. METHODS: Corneas of young (6 to 8 weeks of age) and aged (1.5 to 2 years) female mice were scarified and inoculated with 1.0 x 10(8) colony-forming units of P. aeruginosa ATCC 19660. Eyes were scored for corneal pathology (0 to +4) at 6, 12, 24, 48, 72, 96, and 120 hours after infection. At each time point, six mice were killed from each age group, and both eyes were enucleated. Eyes (three infected, three uninfected) were embedded in OCT compound, frozen in liquid nitrogen, sectioned on a cryostat, and stained for ICAM-1 and LFA-1 immunoreactivity. The remaining six eyes (three infected, three uninfected) were embedded in eponaraldite resin, thick sectioned, and stained for light microscopic histopathologic examination. RESULTS: Immunostaining of slight to moderate intensity for ICAM-1 was seen on conjunctival fibroblasts, stromal keratocytes, corneal epithelium, and endothelium and conjunctival blood vessel endothelium of uninfected contralateral eyes in both age groups. In response to P. aeruginosa infection, only young animals were capable of upregulating ICAM-1 (as evidenced by an increase in the intensity of immunostaining) on these cells when compared to aged mice. Conversely, the intensity of immunostaining for LFA-1, a ligand for ICAM-1 on infiltrating leukocytes, was similar despite animal age. On gross observation, corneal pathology was more severe in young mice 24 to 96 hours after infection. Histopathologically, in contrast to young mice, eyes of aged animals 24 to 48 hours after infection had significantly fewer inflammatory cells, such as polymorphonuclear leukocytes (PMNs), infiltrating the corneal stroma and adhering to the endothelium near wound sites. CONCLUSION: These data suggest that the disparate response to ocular P. aeruginosa infection in young versus aged mice is due, at least in part, to the inability of aged animals to upregulate ICAM-1 above constitutively expressed levels. Consequently, the migration of inflammatory cells (PMNs) into infected corneas of aged mice is delayed, perhaps facilitating bacterial growth and contributing to a poor prognosis.

Asunto(s)

Envejecimiento/fisiología , Úlcera de la Córnea/metabolismo , Infecciones Bacterianas del Ojo/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Infecciones por Pseudomonas/metabolismo , Regulación hacia Arriba/fisiología , Animales , Anticuerpos Monoclonales , Córnea/metabolismo , Úlcera de la Córnea/microbiología , Úlcera de la Córnea/patología , Infecciones Bacterianas del Ojo/patología , Femenino , Técnicas para Inmunoenzimas , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Ratones , Infecciones por Pseudomonas/patología

RESUMEN

Our previous work has demonstrated the importance of pneumolysin in the virulence of S. pneumoniae in a rabbit intracorneal model. This was accomplished by showing that deletion of the gene encoding pneumolysin resulted in reduced virulence, whereas restoration of the wild-type gene resulted in restoration of the virulent phenotype. To assess the importance of a particular domain in the pneumolysin molecule, we have now constructed a strain which produces a pneumolysin molecule which is hemolytic but which bears a site-specific mutation in the domain known to be associated with the complement-activating properties of this molecule. Comparison of the virulence of this strain with that of a strain bearing the wild-type gene showed statistically significantly lower total slit lamp examination (SLE) scores at 12, 18, 24, and 36 h (particularly with respect to fibrin formation), but no difference at 48 h. Determination of colony forming units (CFU) in eyes infected with the two strains showed approximately 10(6) bacteria per cornea until 36 h. Between 36 and 48 h, the bacteria were almost completely cleared with very few bacteria recoverable at the later time point. The loss of virulence observed with this mutation in the complement-activation domain of pneumolysin, though less than that observed with the gene deletion mutant, suggests that complement activation by pneumolysin has a significant role in the pathology observed in this model of corneal infection.

Asunto(s)

Córnea/microbiología , Infecciones Bacterianas del Ojo/microbiología , Queratitis/microbiología , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/patogenicidad , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas , Secuencia de Bases , Recuento de Colonia Microbiana , Activación de Complemento/genética , ADN Bacteriano/genética , Infecciones Bacterianas del Ojo/patología , Regulación Bacteriana de la Expresión Génica/genética , Queratitis/patología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Infecciones Neumocócicas/patología , Conejos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/crecimiento & desarrollo , Estreptolisinas/química , Estreptolisinas/genética , Virulencia

RESUMEN

Ciprofloxacin and prednisolone, but not an aminoglycoside and dexamethasone, were previously found to be effective in killing bacteria and reducing inflammation for the treatment of Pseudomonas keratitis. We investigated the therapeutic effectiveness of tobramycin/prednisolone and ciprofloxacin/dexamethasone in a rabbit model of experimental keratitis to increase our understanding of the effectiveness of antibiotic/steroid combinations. To our knowledge, this is the first analysis of the effectiveness of a combination of ciprofloxacin and dexamethasone for experimental keratitis. Two experiments were conducted. In the first experiment, 36 rabbits were divided into six groups: 1) untreated; 2) prednisolone acetate, 1.0%; 3) prednisolone phosphate, 1.0%; 4) tobramycin, 1.36%; 5) tobramycin plus prednisolone acetate; 6) tobramycin plus prednisolone phosphate. In the second experiment, 23 rabbits were divided into four groups: 1) untreated; 2) ciprofloxacin, 0.3%, plus dexamethasone alcohol, 0.1%; 3) ciprofloxacin; 4) dexamethasone alcohol. Topical antibiotic and/or steroid was given for 10 h, from 16 to 26 h postinfection, one drop every 15 min for the first hour and then every 30 min for the remaining 9 h. At 27 h postinfection, eyes were evaluated by slit lamp examination (SLE) and assayed for the presence of bacteria in terms of colony forming units (CFU) per cornea. Both prednisolone acetate and prednisolone phosphate reduced ocular inflammation (as determined by SLE), compared with no treatment (P < or = 0.036); the phosphate was more effective (P = 0.005). Tobramycin alone and in combination with prednisolone also significantly reduced SLE, compared with no treatment (P < or = 0.006). The bactericidal activity of tobramycin was not affected by either steroid formulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Antibacterianos/uso terapéutico , Infecciones Bacterianas del Ojo/tratamiento farmacológico , Glucocorticoides/uso terapéutico , Queratitis/tratamiento farmacológico , Infecciones por Pseudomonas/tratamiento farmacológico , Animales , Antibacterianos/administración & dosificación , Ciprofloxacina/administración & dosificación , Ciprofloxacina/uso terapéutico , Recuento de Colonia Microbiana , Córnea/efectos de los fármacos , Córnea/microbiología , Dexametasona/administración & dosificación , Dexametasona/uso terapéutico , Modelos Animales de Enfermedad , Quimioterapia Combinada , Infecciones Bacterianas del Ojo/patología , Glucocorticoides/administración & dosificación , Queratitis/microbiología , Queratitis/patología , Soluciones Oftálmicas , Prednisolona/administración & dosificación , Prednisolona/uso terapéutico , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/aislamiento & purificación , Conejos , Tobramicina/administración & dosificación , Tobramicina/uso terapéutico

RESUMEN

This study was conducted to determine whether the age of the host influences the pathogenesis and therapeutic outcome of drug-treated Pseudomonas aeruginosa keratitis. Young (3- to 5-month-old) and old (1.5- to 3-year-old) rabbits were intrastromally infected with P. aeruginosa ATCC 27853. Sixteen hours later, rabbits in both age subpopulations were divided into three groups and treated topically as follows: group 1, phosphate-buffered saline; group 2, 0.3% ciprofloxacin; and group 3, 0.3% ciprofloxacin, 1.0% prednisolone, and 0.03% flurbiprofen. Drops were given every 15 min for 1 h and then every 30 min for 9 h. At 27 h postinfection, ocular pathology was graded with a slit lamp examination (SLE) scoring system. Aqueous humor was collected for ciprofloxacin quantitation, and corneas were harvested for bacterial enumeration and estimation of polymorphonuclear leukocytes. Young rabbits had more severe inflammation and pathology than old rabbits. At 27 h postinfection, SLE scores and polymorphonuclear leukocyte numbers were significantly higher for young rabbits than old rabbits (P < 0.02), regardless of treatment. Prednisolone and flurbiprofen significantly reduced SLE scores in both age groups (P < 0.03) without affecting the antimicrobial efficacy of ciprofloxacin.

Asunto(s)

Envejecimiento/fisiología , Ciprofloxacina/uso terapéutico , Flurbiprofeno/uso terapéutico , Queratitis/tratamiento farmacológico , Prednisolona/uso terapéutico , Infecciones por Pseudomonas/tratamiento farmacológico , Animales , Humor Acuoso/metabolismo , Bioensayo , Ciprofloxacina/farmacocinética , Ciprofloxacina/farmacología , Córnea/microbiología , Córnea/patología , Quimioterapia Combinada , Queratitis/patología , Recuento de Leucocitos , Neutrófilos/efectos de los fármacos , Estimulación Luminosa , Infecciones por Pseudomonas/patología , Conejos , Resultado del Tratamiento