Surfactant protein D in Pseudomonas aeruginosa keratitis.

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

Topical antibiotic therapy for the treatment of experimental Staphylococcus aureus keratitis.

A rabbit model of Staphylococcus aureus keratitis was developed to study the chemotherapeutic efficacy of ciprofloxacin, vancomycin, and cefazolin. Intrastromal injection of 100 colony forming units of log phase S. aureus ATCC strain 25923 resulted in rapid growth in the cornea, peaking at 10(7) cfu/cornea by 12 hr post-infection. Slit-lamp examination revealed that infected eyes reached 30% of maximum inflammation by 10 hr and 60% by 22 hr post-infection. Antibiotic therapy (one drop every 15 min for 5 hr) was initiated at 4 hr post-infection (experiment 1) or 10 hr post-infection (experiment 2). Another group was initiated at 10 hr post-infection and treated for 10 hr (experiment 3). In experiment 1, treatment from 4-9 hr post-infection with 0.3% ciprofloxacin drops decreased the cfu per cornea 6.1 logs, compared to placebo-treated controls (P = 0.0001), and rendered 50% of inoculated eyes sterile. Vancomycin (5.0%) and cefazolin (5.0%) each lowered the cfu per cornea 4.6 logs (P = 0.0187) but did not sterilize any eyes. In experiment 2, therapy from 10-15 hr post-infection with 0.3% ciprofloxacin reduced the cfu per cornea 0.9 logs (P = 0.0001). Vancomycin (5.0%) and cefazolin (5.0%) decreased the cfu per cornea 0.2 logs (P = 0.3973) and 0.3 logs (P = 0.1307), respectively. In experiment 3, therapy from 10-20 hr post-infection with 0.3% ciprofloxacin reduced the cfu per cornea 3.9 logs (P < 0.0001). In this keratitis model, ciprofloxacin was more effective than vancomycin or cefazolin in killing S. aureus.

Ciprofloxacin iontophoresis for aminoglycoside-resistant pseudomonal keratitis.

Studies using ciprofloxacin for the therapy of experimental aminoglycoside-resistant keratitis caused by Pseudomonas aeruginosa were conducted using transcorneal iontophoresis as the drug-delivery system. Corneas infected with P. aeruginosa ATCC 27853/pMG6 were treated 22 hours postinfection with ciprofloxacin delivered by iontophoresis (0.8 mA X 10 min), mock iontophoresis (eyecup with no current), or frequently applied topical drops. Iontophoresis of 10 mg/ml or 25 mg/ml of ciprofloxacin significantly reduced the number of viable bacteria per cornea by more than 5 log units compared with untreated controls (P less than 0.0001). Five hours after the initiation of treatment, mock iontophoresis (10 mg/ml or 25 mg/ml) or 11 applications of topical ciproflaxicin drops (7.5 mg/ml) decreased the viable bacteria relative to the untreated controls by 5 log units (P less than 0.0001). One treatment with an eyecup was as effective as 11 treatments with topical drops (P greater than 0.75). One hour after treatment with iontophoresis or mock iontophoresis of 10 mg/ml of ciprofloxacin, aqueous humor concentrations were 83.75 +/- 8.85 micrograms/ml and 24.87 +/- 4.0 micrograms/ml (mean +/- standard error of the mean), respectively. One hour after the last of five applications of 7.5 mg/ml of ciprofloxacin (every 15 min for 1 hr) the aqueous humor concentration was 4.2 +/- 1.14 micrograms/ml. These results show the value of ciprofloxacin in treating aminoglycoside-resistant infections caused by P. aeruginosa and suggest that ciprofloxacin can be efficiently delivered by iontophoresis.

Quinolones in collagen shields to treat aminoglycoside-resistant pseudomonal keratitis.

Rabbit corneas were infected with a tobramycin-resistant (minimum inhibitory concentration, 31.25 micrograms/ml) strain of Pseudomonas aeruginosa 27853 (10(3) colony-forming units) and were treated 22 hours later with collagen corneal shields hydrated in either 25 mg/ml ciprofloxacin, 40 mg/ml norfloxacin, 40 mg/ml tobramycin, or deionized water. Shields were removed at 26 hours postinfection, and 1 hour later, corneas were harvested for bacterial enumeration. Application of shields hydrated in ciprofloxacin reduced the number of viable bacteria per cornea approximately 4 log units compared with the application of shields containing tobramycin or deionized water (P less than 0.0001). Use of shields hydrated in norfloxacin reduced the number of P. aeruginosa organisms by greater than 2 log units compared with shields containing tobramycin or deionized water (P less than 0.0001). Ciprofloxacin was significantly more effective than norfloxacin in reducing the number of bacteria per cornea (P less than 0.0001). There was no significant difference in the number of bacteria recovered from corneas treated with tobramycin or deionized water (P less than 0.56).

In vivo bacterial protease production during Pseudomonas aeruginosa corneal infection.

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.

Aged mice fail to upregulate ICAM-1 after Pseudomonas aeruginosa corneal infection.

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.

Pseudomonas deficient in protease IV has significantly reduced corneal virulence.

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.

Pseudomonas keratitis. The role of an uncharacterized exoprotein, protease IV, in corneal virulence.

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.

Pseudomonas aeruginosa protease IV produces corneal damage and contributes to bacterial virulence.

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.

Iontophoresis of tobramycin for the treatment of experimental Pseudomonas keratitis in the rabbit.

Iontophoresis of tobramycin sulfate was employed to treat bacterial keratitis induced in rabbits by an intrastromal injection of Pseudomonas aeruginosa. Quantitation of bacterial killing was achieved by culturing corneal homogenates and counting viable bacteria per cornea after treatment. When the rabbits received two iontophoretic treatments, iontophoresis significantly reduced the number of bacterial colony-forming units, compared with the use of an eyecup without current or two subconjunctival injections of 20 mg of tobramycin sulfate, and with untreated controls. The results of iontophoresis were not significantly different from those involving treatment with topical fortified drops (40 mg/mL). A single iontophoresis treatment was compared with the use of an eyecup without current or less fortified topical drops (13.6 mg/mL), as well as with untreated controls. Iontophoresis was significantly better than either the eyecup or the topical fortified tobramycin drops in reducing viable bacteria in the corneas. Iontophoresis may be a useful adjunct in the treatment of severe corneal bacterial infections.

Treatment of experimental Pseudomonas keratitis using collagen shields containing tobramycin.

To study the effectiveness of collagen shields containing tobramycin sulfate in the treatment of Pseudomonas keratitis, rabbits were infected via an intrastromal injection of Pseudomonas aeruginosa and treated 22 hours later with either collagen corneal shields rehydrated in 4% tobramycin and applied to the cornea or 4% tobramycin drops. Bacterial killing was quantitated by culturing corneal homogenates and calculating the number of viable bacteria (colony-forming units) per cornea. Corneas receiving shields rehydrated in 4% tobramycin and applied for four hours demonstrated significantly reduced numbers of bacteria compared with untreated control corneas. The collagen shields were as effective in reducing the number of viable bacteria per cornea as 4% tobramycin drops applied every 30 minutes over a four-hour period. Over a nine-hour treatment period, the addition of four drops of 4% tobramycin to shields in situ was as effective as exchange with a new shield rehydrated in 4% tobramycin. These results suggest that collagen shields rehydrated in a water-soluble antibiotic such as tobramycin may be an effective and convenient mode of therapy for Pseudomonas keratitis.

Ciprofloxacin ointment versus ciprofloxacin drops for therapy of experimental Pseudomonas keratitis.

To compare the efficacy of ciprofloxacin hydrochloride ointment with that of ciprofloxacin drops for treatment of experimental Pseudomonas keratitis, rabbit eyes were infected by intrastromal injection of 10(3) colony-forming units (CFU) log-phase P. aeruginosa. Infected and uninfected eyes were treated with either 0.3% ciprofloxacin ointment applied hourly for 6 h or 0.3% ciprofloxacin drops applied every 15 min for 6 h. Infected eyes treated with the ointment or drop vehicle alone served as placebo controls. Ciprofloxacin ointment significantly reduced the number of viable bacteria (CFU) per cornea more than four logs compared to the placebo control (p < 0.0001). Ciprofloxacin drops significantly reduced the number of bacteria (CFU) per cornea > 7 logs as compared with placebo-treated controls (p < 0.0001). Ciprofloxacin ointment may be a useful adjunct to conventional topical drops for therapy of bacterial keratitis.

The efficacy of topical ciprofloxacin and norfloxacin in the treatment of experimental Pseudomonas keratitis.

An aminoglycoside-resistant strain of Pseudomonas aeruginosa was injected intrastromally into the corneas of rabbits, and keratitis was allowed to develop over a 22-h period. Rabbits were treated with either 0.75% ciprofloxacin, 1% norfloxacin, or 1.36% tobramycin administered topically every 15 min for 1 h and then every 30 min for the following 3 h. All therapy ceased 26 h postinoculation. Rabbits were killed 1 h after the treatment, and the number of bacteria per cornea were quantified in terms of bacterial colony-forming units. Aqueous humor specimens were obtained from rabbits receiving norfloxacin and ciprofloxacin, and bioassays were performed to determine drug concentration. Ciprofloxacin caused a 5 log reduction in the number of bacterial colony-forming units, as compared with untreated controls (p less than 0.0001); it also produced a significantly greater reduction in bacterial colony-forming units than either norfloxacin or fortified tobramycin drops (p less than 0.0001). Norfloxacin produced a 2 log reduction in bacterial colony-forming units, as compared with untreated controls (p less than 0.0001). The mean aqueous concentration of norfloxacin (7.5 micrograms/ml) was substantially less than that achieved by ciprofloxacin (30.5 micrograms/ml). We conclude that ciprofloxacin may be a useful broad spectrum, topical chemotherapeutic agent in the therapy of aminoglycoside-resistant P. aeruginosa keratitis.

Corneal virulence of LasA protease--deficient Pseudomonas aeruginosa PAO1.

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.

A Pseudomonas aeruginosa strain isolated from a contact lens-induced acute red eye (CLARE) is protease-deficient.

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.

Environmental factors influence P. aeruginosa binding to the wounded mouse cornea.

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.

Alkaline protease-deficient mutants of Pseudomonas aeruginosa are virulent in the eye.

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.

Methicillin-resistant Staphylococcus aureus keratitis in the rabbit: therapy with ciprofloxacin, vancomycin and cefazolin.

To determine the efficacy of a fluoroquinolone antibiotic in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) keratitis, topical administration of 0.3% ciprofloxacin was compared with topical 5.0% vancomycin or 5.0% cefazolin in experimental infections in the rabbit eye. The infections were established by intrastromal injection of 100 colony forming units (CFU) of MRSA, which resulted in greater than 10(6) CFU per cornea by 12 hr postinfection. Chemotherapy (one drop every 15 min) was given from 4-9, 10-15, or 10-20 hr postinfection. Early therapy (4-9 hr postinfection) with ciprofloxacin rendered all eyes free of bacteria; ciprofloxacin was significantly more effective than vancomycin or cefazolin. When treatment was initiated 6 hr later (10-15 hr postinfection), no corneas became free of bacteria, but ciprofloxacin was again more effective than vancomycin or cefazolin. Bacterial killing by ciprofloxacin after treatment from 10-20 hr postinfection was also significantly greater than that of vancomycin. Overall, the results show that ciprofloxacin is effective in killing methicillin-resistant staphylococcus aureus, and is most effective when applied during the very early stages of infection.

Confirmation of the role of pneumolysin in ocular infections with Streptococcus pneumoniae.

In earlier experiments on the role of the cytolytic toxin pneumolysin in ocular infections with pneumococcus, we found that a strain carrying a deletion in the gene encoding pneumolysin was considerably less virulent than wild type when tested in an intracorneal model of keratitis in the rabbit. To confirm this result, we have constructed a strain in which pneumolysin activity was restored by transformation of the deleted strain with a plasmid bearing the complete pneumolysin gene. Hemolytic titers of pneumolysin indicated that only one copy of the plasmid per bacterium expresses the pneumolysin gene in this strain. The virulence of this strain was compared with that of wild type and deleted strains transformed with the vector lacking the pneumolysin gene. Slit lamp examination (SLE) scores for eyes infected with the restored strain were similar to those for eyes infected with wild type and significantly greater than those for the pneumolysin-deleted strain. Molecular analysis of bacteria recovered from infected corneas showed that the vector plasmid was retained; however, in most isolates of the restored strain, the plasmid underwent an excision and lost the pneumolysin gene. The cloned gene apparently persisted long enough to induce the pathologic changes, and the results confirm the importance of pneumolysin as a virulence factor in ocular infections.

Prednisolone acetate or prednisolone phosphate concurrently administered with ciprofloxacin for the therapy of experimental Pseudomonas aeruginosa keratitis.

This study was conducted to determine the therapeutic efficacy of 3.0 mg/ml ciprofloxacin administered concurrently with one of two salts of prednisolone for the treatment of experimental pseudomonal keratitis. Rabbit corneas were injected intrastromally with Pseudomonas aeruginosa ATCC strain 27853. Sixteen hr after injection, rabbits were randomly divided into four treatment groups (3 rabbits, 6 eyes per group): 1) ciprofloxacin plus prednisolone acetate; 2) ciprofloxacin plus prednisolone phosphate; 3) ciprofloxacin only; 4) untreated. Signs of inflammation were graded in a masked fashion by slit lamp examination (SLE) and by estimating polymorphonuclear leukocyte (PMN) numbers in corneas 27 hr after injection. SLE scores and PMN numbers were significantly lower (P < 0.02) in eyes receiving either salt of prednisolone plus ciprofloxacin compared to the untreated controls. In contrast, SLE scores and PMN numbers were not significantly different in eyes treated with ciprofloxacin alone, compared to untreated controls (P > 0.13). No viable bacteria were recovered from any eye treated with ciprofloxacin (groups 1, 2, and 3). Ciprofloxacin concentrations in the aqueous humor of eyes in groups 1, 2, and 3 were greater than 15-fold higher than the MIC for P. aeruginosa 27853. These results suggest that either salt of prednisolone, when combined with ciprofloxacin, reduces ocular inflammation without affecting the antimicrobial efficacy of the antibiotic.