Jeffery A. Hobden

Macrophage Inflammatory Protein-2 Is a Mediator of Polymorphonuclear Neutrophil Influx in Ocular Bacterial Infection

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.

The role of pneumolysin in ocular infections with Streptococcus pneumoniae

Pneumolysin, a cytolytic protein produced by Streptococcus pneumoniae, has many properties which suggest it may be an important virulence factor in pneumococcal ocular infections. To directly test this possibility, we have constructed pneumolysin-negative strains of S. pneumoniae and compared their virulence with that of the wild type in a rabbit model of intracorneal infection. A pneumolysin-negative strain produced by chemical mutagenesis (probably a point mutant) was found to be no less virulent than the parent strain. However, a strain bearing a deletion in the pneumolysin gene showed greatly reduced virulence. This strain produced less pathology while showing significantly higher bacterial counts. These results suggest that a property of the pneumolysin molecule other than its cytolytic (hemolytic) activity may be involved in its pathogenic mechanism of action. This property may be the ability to activate complement, known to be a function of pneumolysin, which results in influx of PMNs, reducing the bacterial counts but also producing tissue damage.

Pseudomonas aeruginosa Proteases and Corneal Virulence

Pseudomonas aeruginosa is a common cause of corneal infections, particularly among users of soft contact lenses. Previous studies with chemically induced mutants deficient in alkaline protease (AP) or elastase (LasB) suggested that these proteases contributed to the rapid liquifactive stromal necrosis characteristic of P. aeruginosa corneal infections. Because these mutants might harbor other chromosomal changes that could affect virulence, the role of these proteases in the pathogenesis of corneal disease (as well as a second elastase, LasA protease) was reexamined by constructing isogenic mutants deficient only in these enzymes. Allelic exchange was used to construct mutants of P. aeruginosa PAO1-V deficient in AP (PAO1-V AP[ - ]), LasB and LasA protease (PDO801 LasB[ - ]), or all three proteases (PDO801 TM). These mutants were then evaluated for virulence using mouse scratch and rabbit intrastromal injection models of corneal disease. Loss of AP significantly increased disease scores in the rabbit (P < 0.030) but not the mouse (P > 0.060) model of infection. Loss of both elastases had no effect on ocular virulence in either animal model of corneal disease (P > 0.100). The loss of all three proteases significantly decreased disease scores in the rabbit (P < 0.035), but not in the mouse (P > 0.110). Taken together, these data suggest that AP, LasB, and LasA protease are not essential for initiating or maintaining a corneal infection. Furthermore, AP appears to be an important mediator of pathology depending on the location of the organism within the cornea and whether or not concomitant elastolytic activity is present.

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

&NA; 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 < 0.0001); it also produced a significantly greater reduction in bacterial colony‐forming units than either norfloxacin or fortified tobramycin drops (p < 0.0001). Norfloxacin produced a 2 log reduction in bacterial colony‐forming units, as compared with untreated controls (p < 0.0001). The mean aqueous concentration of norfloxacin (7.5 μg/ml) was substantially less than that achieved by ciprofloxacin (30.5 μg/ml). We conclude that ciprofloxacin may be a useful broad spectrum, topical chemotherapeutic agent in the therapy of aminoglycoside‐resistant P. aeruginosa keratitis.

EFFECTIVENESS OF SPECIFIC ANTIBIOTIC/STEROID COMBINATIONS FOR THERAPY OF EXPERIMENTAL PSEUDOMONAS AERUGINOSA KERATITIS

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)

Pseudomonas aeruginosa keratitis in leukopenic rabbits

To study the role of the host inflammatory response in Pseudomonas aeruginosa keratitis, rabbits were made leukopenic with intravenous injections of cyclophosphamide and dexamethasone. Twenty-four hr later, keratitis was initiated in all rabbits with an intrastromal injection of 1,000 log phase P. aeruginosa strain 27853. Slit lamp examination of eyes showed that leukopenic rabbits had significantly less (P < 0.0001) ocular pathology at 16, 22, and 27 hr postinfection. The number of viable bacteria recovered from corneas of leukopenic rabbits was the same as the number recovered from nonleukopenic rabbits (P = 0.95). These results suggest that the host inflammatory response significantly contributes to the overall ocular pathology associated with P. aeruginosa keratitis, but does not influence the survival of the infecting organism in the cornea at the height of the infection.

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.

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.

Iontophoretic application of tobramycin to uninfected and Pseudomonas aeruginosa-infected rabbit corneas.

Pseudomonas aeruginosa keratitis was induced in rabbits to study the effects of corneal infection on the delivery of tobramycin by iontophoresis. Some rabbits were treated by use of an eye cup with no current as a control for iontophoresis, and others were treated with fortified drops (1.36%) delivered topically for comparison with results of earlier studies. One hour after treatment with tobramycin, the concentration of drug in the infected corneas was compared with that achieved in mock-infected and uninfected eyes. Iontophoresis of 25 mg of tobramycin per ml at 0.8 mA for 10 min delivered significantly more drug (P = 0.0001) to corneal tissue than did drops or use of an eye cup without current in P. aeruginosa-infected eyes mock-infected eyes, or uninfected eyes. Tobramycin concentrations in the infected corneas (605.9 micrograms/g) were not significantly different (P = 0.815) from the concentrations in mock-infected eyes (641.4 micrograms/g), but were lower (P = 0.007) than those obtained by iontophoresis in uninfected corneas (853.6 micrograms/g). Use of an eye cup without current delivered tobramycin equally to infected, mock-infected, and normal eyes, i.e., 176.5, 171.0, and 163.1 micrograms/g, respectively (P greater than 0.709). Tobramycin delivered by use of fortified drops delivered topically was detectable in mock-infected corneas (20 micrograms/g) and P. aeruginosa-infected corneas (6.0 micrograms/g). These results suggest that iontophoresis has value as an ocular drug delivery system and that an eye cup could also be useful in a therapeutic regimen for ocular infections.

Ciprofloxacin and prednisolone therapy for experimental Pseudomonas keratitis.

Rabbit corneas were injected intrastromally with Pseudomonas aeruginosa. Sixteen hours after injection, the rabbits were divided randomly into four treatment groups (3 rabbits/6 eyes per group: 1, ciprofloxacin and prednisolone; 2, ciprofloxacin only; 3, prednisolone only; 4, untreated. Ocular signs of inflammation were graded in a masked fashion by slit lamp examination before injection and 16 and 27 hours after injection. Slit lamp examination scores were significantly lower in eyes receiving ciprofloxacin and prednisolone or prednisolone alone, compared with scores in untreated eyes. Slit lamp examination scores were not significantly lower in eyes receiving ciprofloxacin alone, compared with untreated controls. The numbers of viable bacteria in the corneas treated with ciprofloxacin and in the corneas treated with ciprofloxacin and prednisolone were similar and were significantly less (P less than 0.0001) than those in untreated corneas, indicating that the presence of the steroid did not interfere with the bactericidal action of ciprofloxacin.