Comparative Efficacy of Topical Gatifloxacin With Ciprofloxacin, Amikacin,and Clarithromycin in the Treatment of Experimental Mycobacteriumchelonae Keratitis FREE

Infectious keratitis due to nontuberculous mycobacterial species isa rare, but devastating, complication after laser in situ keratomileusis (LASIK). Mycobacterium chelonae is reported as the most common speciesisolated of the nontuberculous mycobacteria causing keratitis following LASIK.^{1 - 12} Combinationtherapy with topical amikacin and ciprofloxacin or with amikacin and clarithromycinhas been the mainstay of medical therapy.^{2 - 3 ,7 - 8} Despiteaggressive and protracted treatment, the clinical outcome of medical therapyhas been unsatisfactory in many cases. Ford et al¹³ reportedthat 55% of the cases of keratitis due to nontuberculous mycobacteria didnot respond to medical therapy alone and ultimately required a surgical procedure.

Gatifloxacin, 1 of 8-methoxy fluoroquinolones with broad-spectrum antimicrobialactivity, has been reported to be 4-fold more active than ciprofloxacin againstrapidly growing mycobacteria in vitro.¹⁴ Yet,to our knowledge, no in vivo data are available for results of treatment ofnontuberculous mycobacterial infections with gatifloxacin. Given its excellentin vitro activity, gatifloxacin can be considered as a potential candidatefor a new therapeutic strategy against nontuberculous mycobacterial keratitis.The aim of this study was to compare the efficacy of topical gatifloxacinwith ciprofloxacin, an earlier generation fluoroquinolone, and combinationtherapy with amikacin and clarithromycin in the rabbit model of M chelonae keratitis.

A stock strain of M chelonae ATCC-35752 (AmericanType Culture Collection, Rockville, Md) was selected as the test organism.This strain demonstrated via pilot laboratory testing to induce keratitiscomparable to human isolates. The suspension was diluted to 10⁷ organismsper milliliter in phosphate buffer solution. In vitro susceptibility testingdetermined that the mimimum inhibitory concentration (MIC) of amikacin was16 µg/mL (susceptible) by broth microdilution technique using NationalCommittee for Clinical Laboratory Standards' guidelines. The reports fromother separate laboratories have shown that the MIC of gatifloxacin was 0.12µg/mL or less,¹⁴ the MIC of ciprofloxacinwas 0.25 µg/mL,¹⁴ and the MIC of clarithromycinwas 0.125 µg/mL¹⁵ for this carefully selected test strain.

Topical fortified amikacin (50 mg/mL) was prepared by the Johns Hopkinsinstitutional research pharmacy from parenteral formulations according toroutine procedures. Topical clarithromycin (10 mg/mL) was prepared by theresearch pharmacy by reconstituting an oral suspension powder with sterilewater followed by dilution with artificial tears as described by Ford et al.¹³ Topical 0.3% ciprofloxacin (Ciloxan; Alcon LaboratoriesInc, Fort Worth, Tex) was used as a commercial preparation. Topical 0.3% gatifloxacinwas provided by Allergan Inc, Irvine, Calif, for the animal experiment.

Thirty-five adult male New Zealand white rabbits, weighing 3.0 to 4.0kg, were randomly divided into 5 groups. Institutional guidelines regardinganimal experimentation were followed and all animals were treated accordingto the Association of Vision Research in Ophthalmology Statement for the Useof Animals in Ophthalmic and Vision Research. Anesthesia was induced withintramuscular injection of 30 mg/kg of ketamine hydrochloride (Ketaject; PhoenixPharmaceutical Inc, St Joseph, Mo) and 5 mg/kg of xylazine hydrochloride (Xyla-ject;Phoenix Pharmaceutical Inc). Topical anesthesia was achieved with administrationof 1 drop of 0.5% proparacaine hydrochloride to the rabbit eyes. Through a30-gauge needle, a 100-µg suspension of M chelonae containing 10⁶ organisms was inoculated into the midstromaof the right cornea. Immediately after the inoculation, 0.5 mL of dexamethasonesodium phosphate (4 mg/mL) was injected subconjunctivally to each rabbit.¹⁶

After the intrastromal inoculation, infection was allowed to proceedfor 5 days before initiation of antibiotic therapy. From the fifth day afterthe inoculation, topical antibiotics were applied to the affected right eyeshourly for 12 hours and each group was treated with one of the following regimens:(1) 3 mg/mL of gatifloxacin; (2) 3 mg/mL of ciprofloxacin; (3) a combinationof 50 mg/mL of fortified amikacin and 10 mg/mL of clarithromycin; (4) a triplecombination of topical 3 mg/mL of gatifloxacin, 50 mg/mL of fortified amikacinand 10 mg/mL of clarithromycin; or (5) 0.9% balanced salt solution as a control.The rabbits were randomly allocated to each treatment group prior to intrastromalinoculation.

One hour after the final instillation of the antibiotic drops, the animalswere killed with an overdose of barbiturate (Beuthanasia D-Special; Schering-PloughAnimal Health Corp, Union, NJ) and uniform corneal buttons were excised witha sterile 8.5-mm trephine (Saber Medical Inc, Westchester, Pa). Corneal buttonswere ground in 1-mL sterile phosphate buffer solution using a disposable tissuehomogenizer (The Kendall Co, Mansfield, Mass). Then serial dilutions wereprepared in sterile phosphate buffer solution. Twenty microliters from eachdilution was plated on blood agar plates and incubated at 37°C with 5%carbon dioxide. Quantitative mycobacteriological analysis of viable M chelonae colonies was conducted on the sixth day of incubation.

For quantitative analysis, data were transformed to logarithmic values,and 1-way analysis of variance (ANOVA) with multiple comparisons was usedto compare the efficacy of each regimen against the M chelonae keratitis. P<.05 was considered statisticallysignificant.

By 4 days mycobacterial keratitis developed after inoculation in all35 rabbits. Corneal infiltrates appeared as focal, fluffy white deposits withfine radiating projections, similar to observed and reported clinical signsin culture-proven cases of mycobacterial keratitis in humans.

The quantitative culture results are summarized in Table 1. The 0.3% gatifloxacin and triple combination therapy with0.3% gatifloxacin, 50 mg/mL of fortified amikacin sulfate, and 10 mg/mL ofclarithromycin significantly reduced the number of M chelonae compared with the control of 0.9% balanced salt solution (both P<.001). Gatifloxacin therapy demonstrated greater antimycobacterialactivity than ciprofloxacin therapy (P<.001).Triple combination therapy with gatifloxacin, amikacin, and clarithromycinresulted in a more favorable result in reduction of bacterial colonies thanmonotherapy with 0.3% gatifloxacin, but there was no statistical significance.There was no significant difference observed between control eyes and eyestreated with ciprofloxacin or with combination therapy using 50 mg/mL of amikacinsulfate and 10 mg/mL of clarithromycin (Figure1).

Nontuberculous mycobacterial keratitis represents a persistent challengein diagnosis and treatment because of its indolent clinical course and poorsusceptibility to conventional antibacterial therapy. Most patients havingnontuberculous mycobacterial keratitis report a history of antecedent traumaor prior surgery such as penetrating keratoplasty, radial keratotomy, or refractivesurgery. The proper diagnosis and treatment is often delayed and the use ofexogenous corticosteroid may suppress local immunity and prolong the clinicalcourse. Biofilm of the mycobacterium may play a role in evading host defensemechanism and promoting resistance to conventional disinfection.¹⁷ Recently,there have been increasing concerns regarding mycobacteria as opportunisticpathogens associated with LASIK. Review of the ophthalmic literatures discloses31 reported cases of post-LASIK nontuberculous mycobacterial keratitis sinceReviglio et al¹ first reported it in 1998. Mycobacterium chelonae was the most common pathogen identifiedamong these cases.

The initial choice for treatment of M chelonae keratitishas been topical amikacin combined with ciprofloxacin or clarithromycin. Clarithromycinis a macrolide antibiotic with excellent in vitro activity against M chelonae,¹⁵ and topical clarithromycinhas been reported to penetrate through intact epithelium.¹⁸ Amikacinalso has been the drug of choice based on in vitro activity, animal studies,and clinical experiences.¹⁷ Adjunctive surgicaltreatment is required in many cases of M chelonae keratitisfollowing LASIK. Among 17 cases of M chelonae keratitisfollowing LASIK reported in the literature, 10 (59%) of 17 patients eventuallyunderwent removal of the flap or therapeutic penetrating keratoplasty to controlthe infection.

In this study, gatifloxacin therapy exerted a significant antimycobacterialactivity and was more active than ciprofloxacin against M chelonae keratitis. Ciprofloxacin therapy has been found to havegood in vitro activity against M chelonae¹⁹ but to be less effective against M chelonae keratitis than against Mycobacterium fortuitum keratitis in vivo.²⁰ In one seriesof nontuberculous mycobacterial keratitis, M chelonae isolateswere generally sensitive to amikacin and clarithromycin treatment but resistantto ciprofloxacin treatment.¹³ Fluoroquinolneshave 2 targets on chromosomal DNA, which are DNA gyrase (type II topoisomerase)and topoisomerase IV. Microbial resistance develops when mutations occur inthe target topoisomerase enzyme. The C-8 methoxy side chain of gatifloxacinmay prevent bacteria from developing resistance by requiring 2 topoisomerasemutations for resistance to develop.²¹ Brown-Elliottet al¹⁴ reported that 97% of M chelonae isolates were susceptible or intermediately susceptibleto gatifloxacin at an MIC of 4 µg/mL or less.

This study has shown that topical 0.3% gatifloxacin enhances the efficacyof topical fortified amikacin sulfate (50 mg/mL) and clarithromycin (10 mg/mL)when administered in triple combination therapy (P<.01).The efficacy of triple combination therapy with gatifloxacin, amikacin, andclarithromycin was superior to that of monotherapy with gatifloxacin in absolutecolony-forming unit counts, but statistical significance was not demonstrated.

Ciprofloxacin and combination therapy with fortified amikacin and clarithromycinfailed to show statistically significant antimycobacterial activity despitegood in vitro activity. This observed result may be attributed to the useof corticosteroid therapy in our animal model. In 1 series of mycobacterialkeratitis, 80% of the eyes with organisms that were sensitive but did notrespond to topical therapy were shown to have had a corticosteroid administered.¹³ Corneal epithelial penetration of each antibioticalso might affect the resulting antimycobacterial activity in this study.In the animal model of this study, mycobacterial keratitis was induced byintrastromal injection of organisms that spared the intact epithelial barrier.Although topical clarithromycin has been shown to achieve therapeutic levelsin corneal tissue in a rabbit model,¹⁸ topicalamikacin has been reported to have poor corneal penetration.²²

Although gatifloxacin and triple combination therapy with gatifloxacin,fortified amikacin, and clarithromycin have displayed significant antimycobacterialactivity by reducing colony-forming unit counts in this study, cultures remainedpositive for M chelonae in every cornea from 35 rabbitsafter frequent administration of antibiotic therapy. This result advocatesfor a more prolonged course of medical therapy and consideration of surgicaldebridement to debulk organisms in the treatment of M chelonae keratitis.

To our knowledge, this is the first reported in vivo activity of gatifloxacinagainst M chelonae and the results confirmed thetherapeutic efficacy of topical 0.3% gatifloxacin against M chelonae keratitis in an animal model. The current experiment wasdesigned not to directly simulate clinical situations but to compare the invivo efficacy of various treatment regimens, including different generationsof fluoroquinolones, monotherapy, and combination therapy, in treating M chelonae keratitis. Thus, the experimental keratitiswas treated during a short period in the early stage of the disease, whichis unlikely to happen in clinical settings. Our results suggest, however,that gatifloxacin can be a valuable new initial therapeutic agent in the treatmentof M chelonae keratitis. Treating clinicians shouldconsider that multiple combination agent therapy will still be beneficialand surgical debridement may be necessary especially in post-LASIK mycobacterialkeratitis to successfully eradicate these challenging pathogens.

Correspondence: Terrence P. O'Brien, MD, Ocular Microbiology andImmunology Laboratory, Wilmer Eye Institute, 600 N Wolfe St, Baltimore, MD21287-9121 (tobrien@jhmi.edu).

Submitted for publication April 30; final revision received November17, 2003; accepted March 26, 2004.

This study was supported by the BK21 project for Medicine, Dentistry,and Pharmacy, (Dr Hyon).