Mary E. Marquart

Effectiveness of Ciprofloxacin, Levofloxacin, or Moxifloxacin for Treatment of Experimental Staphylococcus aureus Keratitis

ABSTRACT The purpose of this study was to quantitatively compare, in a rabbit keratitis model, the levels of effectiveness of moxifloxacin, levofloxacin, and ciprofloxacin for the treatment of Staphylococcus aureus isolates of diverse antibiotic susceptibilities. Rabbit eyes were intrastromally injected with approximately 100 CFU of methicillin-sensitive or methicillin-resistant S. aureus (MSSA or MRSA, respectively) organisms that were either sensitive or resistant to ofloxacin. One drop of moxifloxacin (0.5%), levofloxacin (0.5%), or ciprofloxacin (0.3%) was topically applied hourly from 4 to 9 (early) or 10 to 15 (late) h postinfection. At 1 h after cessation of therapy, the corneas were harvested, and the number of CFU per cornea was determined. For the ofloxacin-sensitive strains, early treatment of MSSA or MRSA with moxifloxacin, levofloxacin, or ciprofloxacin produced approximately a 5-log decrease in CFU per cornea relative to that in untreated eyes (P ≤ 0.0001). For late therapy of ofloxacin-sensitive strains, moxifloxacin, levofloxacin, and ciprofloxacin produced approximately 5-, 4-, and 2- to 3-log reductions in CFU per cornea, respectively (P ≤ 0.0001). Early treatment of the ofloxacin-resistant strains with either moxifloxacin or levofloxacin produced a ≥4-log or ≥3-log decrease, respectively, in the MSSA or MRSA strains (P ≤ 0.0001), whereas ciprofloxacin treatment produced a 1-log decrease in CFU per cornea relative to that in untreated eyes (P = 0.1540). For late treatment of ofloxacin-resistant strains, levofloxacin and ciprofloxacin failed to significantly reduce the number of CFU per cornea (P ≥ 0.3627), whereas moxifloxacin produced a significant reduction in CFU per cornea of approximately 1 log (P ≤ 0.0194). Therefore, for three of the four treatments tested, moxifloxacin demonstrated greater effectiveness than either levofloxacin or ciprofloxacin.

Properties of PASP: A Pseudomonas Protease Capable of Mediating Corneal Erosions

PURPOSE To analyze PASP in terms of its gene distribution and expression, its corneal pathologic effects, its enzymatic properties, and the protectiveness of the immune response to this protease. METHODS Twenty-five strains of P. aeruginosa were analyzed for the PASP gene and secreted protein by PCR and Western blot analysis, respectively. Active recombinant (r)PASP (10 microg/20 microL) or heat-inactivated rPASP was intrastromally injected into rabbit corneas. Pathologic changes were monitored by slit lamp examination (SLE) and histopathology. Purified rPASP was assayed for cleavage of collagens and susceptibility to TLCK. Rabbit antibody to rPASP was produced and tested for enzyme inactivation, and actively immunized rabbits were challenged by intrastromal injection of active rPASP (5 microg). RESULTS All 25 strains of P. aeruginosa analyzed were positive for the PASP gene and protein. SLE scores of eyes injected with active rPASP were significantly higher than control eyes at all postinjection times (PI; P <or= 0.004). Histopathologic studies documented the destruction of the corneal epithelial layer and portions of the corneal stroma at 9 hours PI, and polymorphonuclear (PMN) leukocyte infiltration into the cornea by 24 hours after active rPASP injection. PASP cleaved type I and IV collagens and was susceptible to TLCK inhibition. PASP was present in the cytoplasm and periplasm, but only secreted PASP was enzymatically active. A high antibody titer (ELISA titer >or= 10,000) was produced, but this antibody did not protect against active rPASP challenge. CONCLUSIONS PASP is a commonly produced Pseudomonas protease that can cleave collagens and cause corneal erosions.

Animal Models of Bacterial Keratitis

Bacterial keratitis is a disease of the cornea characterized by pain, redness, inflammation, and opacity. Common causes of this disease are Pseudomonas aeruginosa and Staphylococcus aureus. Animal models of keratitis have been used to elucidate both the bacterial factors and the host inflammatory response involved in the disease. Reviewed herein are animal models of bacterial keratitis and some of the key findings in the last several decades.

Quantitative comparison of fluoroquinolone therapies of experimental Gram-negative bacterial keratitis

Purpose. To determine the effectiveness of topically applied fluoroquinolones for experimental Pseudomonas or Serratia keratitis. Methods. Bacteria were injected intrastromally (10 3 colony forming units [CFU]). From 16 to 22 hours post-infection (PI), a single topical drop of moxifloxacin (Vigamox ®, 0.545%), levofloxacin (Quixin™, 0.5%), ofloxacin (Ocuflox ®, 0.3%) or ciprofloxacin (Ciloxan ®, 0.3%) was applied every 30 minutes. At 23 hours PI, corneas were cultured quantitatively. Results. For Pseudomonas keratitis, untreated eyes contained 7 log CFU/cornea and antibiotic-treated eyes demonstrated a = 5-log reduction in CFU/cornea (p = 0.0001). Moxifloxacin, levofloxacin, or ciprofloxacin therapies were not significantly different from each other (p = 0.67). For Serratia keratitis, untreated eyes contained 7 log CFU/cornea whereas treated eyes had a = 2-log reduction (p = 0.0001). Moxifloxacin therapy proved most effective (p = 0.001). Conclusions. Overall, moxifloxacinwas the most effective of the four fluoroquinolones in reducing CFU/cornea in the rabbit model of Gram-negative keratitis.

Corneal Virulence of Pseudomonas aeruginosa Elastase B and Alkaline Protease Produced by Pseudomonas putida

Purpose: To measure the specific virulence contributions of two Pseudomonas aeruginosa proteases, elastase B and alkaline protease, when expressed separately by Pseudomonas putida in a rabbit model of bacterial keratitis. Methods: P. putida KT2440 was transformed with plasmids that enabled the extracellular production of either elastase or alkaline protease. Protease expression was confirmed by zymography and immunoblotting. P. putida expressing elastase, alkaline protease, or vector alone was injected intrastromally (103 colony forming units [CFU]) into rabbit corneas (n = 6). Infected eyes were graded by slit-lamp examination (SLE) at 20, 24, 28, and 32 hr postinfection (PI). Rabbits were sacrificed at 33 hr PI, and the log CFU (±SEM) per cornea was determined. Results: SLE scores for eyes infected with P. putida producing elastase were significantly higher than those infected with vector alone at all time points (p ≤ 0.008). SLE scores for eyes infected with P. putida producing alkaline protease were not significantly higher than the control (p ≥ 0.1), but small erosions formed in 33% of corneas. At both 24 and 28 hr PI, the SLE scores for corneas infected with P. putida producing elastase were significantly higher than those infected with P. putida producing alkaline protease (p ≤ 0.002). Conclusions: Elastase production by P. putida caused significant increases in SLE scores whereas expression of alkaline protease caused limited corneal erosions. This suggests that the production of elastase during P. aeruginosa keratitis enhances ocular pathology, whereas alkaline protease production contributes to limited corneal erosion.

The Cholesterol-Dependent Cytolysin Pneumolysin from Streptococcus pneumoniae Binds to Lipid Raft Microdomains in Human Corneal Epithelial Cells

Streptococcus pneumoniae (pneumococcus) is an opportunistic bacterial pathogen responsible for causing several human diseases including pneumonia, meningitis, and otitis media. Pneumococcus is also a major cause of human ocular infections and is commonly isolated in cases of bacterial keratitis, an infection of the cornea. The ocular pathology that occurs during pneumococcal keratitis is partly due to the actions of pneumolysin (Ply), a cholesterol-dependent cytolysin produced by pneumococcus. The lytic mechanism of Ply is a three step process beginning with surface binding to cholesterol. Multiple Ply monomers then oligomerize to form a prepore. The prepore then undergoes a conformational change that creates a large pore in the host cell membrane, resulting in cell lysis. We engineered a collection of single amino acid substitution mutants at residues (A370, A406, W433, and L460) that are crucial to the progression of the lytic mechanism and determined the effects that these mutations had on lytic function. Both PlyWT and the mutant Ply molecules (PlyA370G, PlyA370E, PlyA406G, PlyA406E, PlyW433G, PlyW433E, PlyW433F, PlyL460G, and PlyL460E) were able to bind to the surface of human corneal epithelial cells (HCECs) with similar efficiency. Additionally, PlyWT localized to cholesterol-rich microdomains on the HCEC surface, however, only one mutant (PlyA370G) was able to duplicate this behavior. Four of the 9 mutant Ply molecules (PlyA370E, PlyW433G, PlyW433E, and PlyL460E) were deficient in oligomer formation. Lastly, all of the mutant Ply molecules, except PlyA370G, exhibited significantly impaired lytic activity on HCECs. The other 8 mutants all experienced a reduction in lytic activity, but 4 of the 8 retained the ability to oligomerize. A thorough understanding of the molecular interactions that occur between Ply and the target cell, could lead to targeted treatments aimed to reduce the pathology observed during pneumococcal keratitis.

Infectious keratitis: secreted bacterial proteins that mediate corneal damage.

Ocular bacterial infections are universally treated with antibiotics, which can eliminate the organism but cannot reverse the damage caused by bacterial products already present. The three very common causes of bacterial keratitis—Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae—all produce proteins that directly or indirectly cause damage to the cornea that can result in reduced vision despite antibiotic treatment. Most, but not all, of these proteins are secreted toxins and enzymes that mediate host cell death, degradation of stromal collagen, cleavage of host cell surface molecules, or induction of a damaging inflammatory response. Studies of these bacterial pathogens have determined the proteins of interest that could be targets for future therapeutic options for decreasing corneal damage.

Ocular reactivation phenotype of HSV-1 strain F(MP)E, a corticosteroid-sensitive strain

Purpose. Elucidate the ocular reactivation of HSV-1 strain F(MP)E. Methods. Rabbit corneas were infected with HSV-1 strains McKrae and F(MP)E. Latency was established and rabbits were treated with epinephrine iontophoresis or corticosteroid injection (immunosuppression). Cultured tear films were used to determine the presence of infectious virus. Eyes of immunosuppressed rabbits were also examined by slit lamp biomicroscopy. Trigeminal ganglia were co-cultured on indicator cells at time of sacrifice for detection of virus. Quantitative real-time PCR was used to detect the number of viral genome copies in the trigeminal ganglia. Results. Acute infections by strains McKrae and F(MP)E were the same. Ocular reactivation of strain F(MP)E by epinephrine iontophoresis was significantly reduced compared to McKrae (P = 0.0001), but was not significantly different from McKrae after corticosteroid injection (P > 0.29). Slit lamp examination following corticosteroid injection showed a correlation of recurrent herpetic lesions with infectious virus in tear film swabs for both McKrae and F(MP)E. Trigeminal ganglia from rabbits latently infected with each strain and each method of induced reactivation resulted in infectious virus (P = 0.69). Genome copies for both strains were present and were highly variable. Conclusions. HSV-1 strain F(MP)E has low reactivation following epinephrine iontophoresis compared to McKrae, but has high reactivation like McKrae in response to corticosteroids. The difference in reactivation following epinephrine iontophoresis is not due to a difference in the establishment of latency.

The Streptococcus pneumoniae Capsule Is Required for Full Virulence in Pneumococcal Endophthalmitis

PURPOSE To determine whether Streptococcus pneumoniae capsule was necessary for pathogenesis of pneumococcal endophthalmitis. METHODS An isogenic capsule-deficient strain was created using homologous recombination. New Zealand White rabbits were injected intravitreously with 10(2) colony-forming units (CFU) of the parent strain or the capsule mutant. Slit lamp examination (SLE), electroretinography, and myeloperoxidase activity were performed 24 and 48 hours postinfection (PI). Serial dilutions of vitreous were plated to quantitate CFU, eyes were extracted for histology, and host cytokine mRNA expression was determined. RESULTS Eyes infected with the parent strain had significantly higher SLE scores than eyes infected with the capsule-deficient strain 24 and 48 hours PI (P < 0.001). CFU recovered from eyes infected with the capsule mutant were significantly fewer than CFU recovered from eyes infected with the parent strain 24 and 48 hours PI (P < 0.001). The parent strain caused a significantly greater decrease in retinal function and more retinal destruction than the mutant strain 48 hours PI (P = 0.026). Vitreal IL-1β, IL-6, and TNF-α were upregulated by both the parent and mutant strain 12 hours PI. By 48 hours PI, there was significantly more neutrophil infiltration in the vitreous infected with the parent strain. CONCLUSIONS Endophthalmitis caused by the encapsulated strain is more damaging to retinal function and structural integrity. These findings indicate that capsule is an important virulence factor of S. pneumoniae endophthalmitis, in contrast to keratitis, suggesting that the anatomic host site in pneumococcal ocular infections is important.

Efficacy of Besifloxacin in an Early Treatment Model of Methicillin-Resistant Staphylococcus Aureus Keratitis

PURPOSE To determine the effectiveness of topically applied besifloxacin, gatifloxacin, and moxifloxacin for the early treatment of experimental methicillin-resistant Staphylococcus aureus (MRSA) keratitis. METHODS Ten hours post-MRSA infection, rabbit eyes were treated topically with 19 doses of phosphate-buffered saline (PBS), besifloxacin, gatifloxacin, or moxifloxacin. Slit-lamp examinations were performed before and after the inoculation. Corneas were harvested for bacterial quantitation and minimal inhibitory concentrations (MICs) were determined. RESULTS All 3 fluoroquinolones significantly lowered the clinical severity of the infection as compared to treatment with PBS (P < 0.05). However, the mean log(10) colony-forming unit (CFU) recovered from besifloxacin-treated corneas was significantly lower than all other treatment groups (P < 0.01). CFU recovered from corneas treated with moxifloxacin and PBS showed no significant difference (P = 0.12). Corneas treated with gatifloxacin had a significantly lower log(10) CFU recovered as compared to PBS-treated corneas (P < 0.01). The MICs for gatifloxacin and moxifloxacin were 8 microg/mL, whereas the MIC for besifloxacin was 1 microg/mL. CONCLUSIONS All 3 fluoroquinolones significantly lowered the clinical severity of the infection. Besifloxacin had an 8-fold lower MIC for MRSA than gatifloxacin and moxifloxacin, and was significantly more effective than gatifloxacin and moxifloxacin in reducing the number of MRSA in the rabbit cornea.