Comparative Efficacy of Topical Gatifloxacin with Ciprofloxacin, Amikacin, and Clarithromycin in the Treatment of Experimental Mycobacterium Chelonae Keratitis

LABORATORY SCIENCES Comparative Efficacy of Topical Gatifloxacin With Ciprofloxacin, Amikacin, and Clarithromycin in the Treatment of Experimental Mycobacterium chelonae Keratitis

Joon-Young Hyon, MD; Myung-Jin Joo, MD; Stacey Hose; Debasish Sinha, PhD; James D. Dick, PhD; Terrence P. O’Brien, MD

Objective: To determine the comparative efficacy of topi- more significantly than the controls that were treated with cal gatifloxacin with ciprofloxacin, fortified amikacin, and a topical balanced salt solution (both PϽ.001). Therapy clarithromycin against Mycobacterium chelonae keratitis with 0.3% gatifloxacin was more effective than 0.3% cipro- in an animal model. floxacin alone (PϽ.001) and demonstrated synergy by enhancing the efficacy of the combination of fortified ami- Methods: Experimental M chelonae keratitis was in- kacin (50 mg/mL) and clarithromycin (10 mg/mL) duced via intrastromal inoculation in a rabbit model. (PϽ.001). Neither 0.3% ciprofloxacin nor the combina- Thirty-five rabbits were randomly divided into 5 groups tion of fortified amikacin (50 mg/mL) and clarithromy- and each group was treated hourly for 12 hours with topi- cin (10 mg/mL) demonstrated a significant difference in cal 0.9% balanced salt solution, 0.3% gatifloxacin, 0.3% activity against mycobacteria compared with the topical ciprofloxacin hydrochloride, a combination of topical for- balanced salt solution. tified amikacin sulfate (50 mg/mL) and clarithromycin (10 mg/mL), or a triple combination of topical 0.3% gati- Conclusion: These results suggest that topical 0.3% gatifloxacin, fortified amikacin sulfate (50 mg/mL), and cla- floxacin ophthalmic solution can be a new initial treat- rithromycin (10 mg/mL). Antibacterial efficacy of each ment agent against M chelonae keratitis. regimen was determined by quantitative bacteriologic analysis. Clinical Relevance: Topical gatifloxacin 0.3% may provide an initial alternative in thereapy of M chelonae Results: Treatment with 0.3% gatifloxacin or the triple keratitis. combination of 0.3% gatifloxacin, topical fortified amikacin sulfate (50 mg/mL), and clarithromycin (10 mg/ mL) reduced the number of mycobacterial organisms Arch Ophthalmol. 2004;122:1166-1169

NFECTIOUS KERATITIS DUE TO NON- crobial activity, has been reported to be tuberculous mycobacterial spe- 4-fold more active than ciprofloxacin cies is a rare, but devastating, against rapidly growing mycobacteria in complication after laser in situ vitro.14 Yet, to our knowledge, no in vivo keratomileusis (LASIK). Myco- data are available for results of treatment bacteriumI chelonae is reported as the most of nontuberculous mycobacterial infec- common species isolated of the nontuber- tions with gatifloxacin. Given its excel- culous mycobacteria causing keratitis fol- lent in vitro activity, gatifloxacin can be lowing LASIK.1-12 Combination therapy considered as a potential candidate for a with topical amikacin and ciprofloxacin or new therapeutic strategy against nontu- From the Ocular Microbiology with amikacin and clarithromycin has been berculous mycobacterial keratitis. The aim and Immunology Laboratory, the mainstay of medical therapy.2,3,7,8 De- of this study was to compare the efficacy Wilmer Eye Institute spite aggressive and protracted treat- of topical gatifloxacin with ciprofloxa- (Drs Hyon, Joo, Sinha, and ment, the clinical outcome of medical cin, an earlier generation fluoroquino- O’Brien and Ms Hose), therapy has been unsatisfactory in many lone, and combination therapy with ami- Department of Pathology cases. Ford et al13 reported that 55% of the kacin and clarithromycin in the rabbit (Dr Dick), The Johns Hopkins cases of keratitis due to nontuberculous model of M chelonae keratitis. University School of Medicine, Baltimore, Md. Dr O’Brien is a mycobacteria did not respond to medical nonsalaried ad hoc consultant therapy alone and ultimately required a METHODS for Alcon Laboratories Inc, surgical procedure. Allergan Inc, Pharmacia Corp, Gatifloxacin, 1 of 8-methoxy fluoro- A stock strain of M chelonae ATCC-35752 and Santen Inc. quinolones with broad-spectrum antimi- (American Type Culture Collection, Rock-

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©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 ville, Md) was selected as the test organism. This strain demonstrated via pilot laboratory testing to induce keratitis com- Quantitative Mycobacterial Culture Results in Experimental parable to human isolates. The suspension was diluted to 107 Mycobacterium chelonae Keratitis* organisms per milliliter in phosphate buffer solution. In vitro susceptibility testing determined that the mimimum inhibi- Bacterial Load, P †tory concentration (MIC) of amikacin was 16 µg/mL (suscep- Treatment Group CFUs؋10−3 Values tible) by broth microdilution technique using National Com- 0.9% BBS (n = 7) 5383 (4263) NA mittee for Clinical Laboratory Standards’ guidelines. The reports 0.3% Gatifloxacin (n = 7) 3.89 (9.65) Ͻ.001 from other separate laboratories have shown that the MIC of 0.3% Ciprofloxacin hydrochloride (n = 7) 1156.44 (926.95) .21 gatifloxacin was 0.12 µg/mL or less,14 the MIC of ciprofloxa- Amikacin sulfate (50 mg/mL) and 1505.45 (1413.02) .51 cin was 0.25 µg/mL,14 and the MIC of clarithromycin was 0.125 clarithromycin (10 mg/mL) (n = 7) µg/mL15 for this carefully selected test strain. Amikacin sulfate (50 mg/mL), clarithromycin 1.11 (1.10) Ͻ.001 Topical fortified amikacin (50 mg/mL) was prepared by (10 mg/mL), and 0.3% gatifloxacin (n = 7) the Johns Hopkins institutional research pharmacy from par- enteral formulations according to routine procedures. Topical Abbreviations: BSS, topical balanced salt solution; CFUs, colony-forming clarithromycin (10 mg/mL) was prepared by the research phar- units; NA, not applicable. *Data are given as mean (SD). macy by reconstituting an oral suspension powder with sterile †The P value was determined using the 1-way analysis of variance test with water followed by dilution with artificial tears as described by multiple comparisons compared with the BSS treatment group using Ford et al.13 Topical 0.3% ciprofloxacin (Ciloxan; Alcon Labo- logarithm-transformed data. ratories Inc, Fort Worth, Tex) was used as a commercial prepa- ration. Topical 0.3% gatifloxacin was provided by Allergan Inc, Irvine, Calif, for the animal experiment. RESULTS Thirty-five adult male New Zealand white rabbits, weigh- ing 3.0 to 4.0 kg, were randomly divided into 5 groups. Insti- By 4 days mycobacterial keratitis developed after inocu- tutional guidelines regarding animal experimentation were fol- lation in all 35 rabbits. Corneal infiltrates appeared as fo- lowed and all animals were treated according to the Association cal, fluffy white deposits with fine radiating projections, of Vision Research in Ophthalmology Statement for the Use of similar to observed and reported clinical signs in culture- Animals in Ophthalmic and Vision Research. Anesthesia was proven cases of mycobacterial keratitis in humans. induced with intramuscular injection of 30 mg/kg of ket- The quantitative culture results are summarized in amine hydrochloride (Ketaject; Phoenix Pharmaceutical Inc, St Joseph, Mo) and 5 mg/kg of xylazine hydrochloride (Xyla- the Table. The 0.3% gatifloxacin and triple combina- ject; Phoenix Pharmaceutical Inc). Topical anesthesia was tion therapy with 0.3% gatifloxacin, 50 mg/mL of forti- achieved with administration of 1 drop of 0.5% proparacaine fied amikacin sulfate, and 10 mg/mL of clarithromycin hydrochloride to the rabbit eyes. Through a 30-gauge needle, significantly reduced the number of M chelonae com- a 100-µg suspension of M chelonae containing 106 organisms pared with the control of 0.9% balanced salt solution (both was inoculated into the midstroma of the right cornea. Imme- PϽ.001). Gatifloxacin therapy demonstrated greater an- diately after the inoculation, 0.5 mL of dexamethasone so- timycobacterial activity than ciprofloxacin therapy dium phosphate (4 mg/mL) was injected subconjunctivally to Ͻ 16 (P .001). Triple combination therapy with gatifloxa- each rabbit. cin, amikacin, and clarithromycin resulted in a more fa- After the intrastromal inoculation, infection was allowed vorable result in reduction of bacterial colonies than to proceed for 5 days before initiation of antibiotic therapy. From the fifth day after the inoculation, topical antibiotics were ap- monotherapy with 0.3% gatifloxacin, but there was no plied to the affected right eyes hourly for 12 hours and each group statistical significance. There was no significant differ- was treated with one of the following regimens: (1) 3 mg/mL of ence observed between control eyes and eyes treated with gatifloxacin; (2) 3 mg/mL of ciprofloxacin; (3) a combination ciprofloxacin or with combination therapy using 50 of 50 mg/mL of fortified amikacin and 10 mg/mL of clarithro- mg/mL of amikacin sulfate and 10 mg/mL of clarithromycin; (4) a triple combination of topical 3 mg/mL of gati- mycin (Figure). floxacin, 50 mg/mL of fortified amikacin and 10 mg/mL of clarithromycin; or (5) 0.9% balanced salt solution as a control. The rabbits were randomly allocated to each treatment COMMENT group prior to intrastromal inoculation. Nontuberculous mycobacterial keratitis represents a per- One hour after the final instillation of the antibiotic drops, the animals were killed with an overdose of barbiturate (Beutha- sistent challenge in diagnosis and treatment because of nasia D-Special; Schering-Plough Animal Health Corp, Union, NJ) its indolent clinical course and poor susceptibility to con- and uniform corneal buttons were excised with a sterile 8.5-mm ventional antibacterial therapy. Most patients having non- trephine (Saber Medical Inc, Westchester, Pa). Corneal buttons tuberculous mycobacterial keratitis report a history of an- were ground in 1-mL sterile phosphate buffer solution using a tecedent trauma or prior surgery such as penetrating disposable tissue homogenizer (The Kendall Co, Mansfield, Mass). keratoplasty, radial keratotomy, or refractive surgery. The Then serial dilutions were prepared in sterile phosphate buffer proper diagnosis and treatment is often delayed and the solution. Twenty microliters from each dilution was plated on use of exogenous corticosteroid may suppress local im- blood agar plates and incubated at 37°C with 5% carbon diox- munity and prolong the clinical course. Biofilm of the ide. Quantitative mycobacteriological analysis of viable M chelo- mycobacterium may play a role in evading host defense nae colonies was conducted on the sixth day of incubation. mechanism and promoting resistance to conventional dis- For quantitative analysis, data were transformed to loga- 17 rithmic values, and 1-way analysis of variance (ANOVA) with infection. Recently, there have been increasing con- multiple comparisons was used to compare the efficacy of each cerns regarding mycobacteria as opportunistic patho- regimen against the M chelonae keratitis. PϽ.05 was consid- gens associated with LASIK. Review of the ophthalmic ered statistically significant. literatures discloses 31 reported cases of post-LASIK non-

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6.00

5.00

4.00

3.00 Bacterial Load, log CFUs ∗ 2.00

∗ 1.00

0.00 0.9% BSS 0.3% Gatifloxacin 0.3% Ciprofloxacin Fortified Amikacin 0.3% Gatifloxacin, Fortified Hydrochloride Sulfate (50 mg/mL) and Amikacin Sulfate (50 mg/mL), Clarithromycin (10 mg/mL) and Clarithromycin (10 mg/mL)

Treatment Group

Results of treatment groups of 35 adult male New Zealand rabbits with Mycobacterium chelonae keratitis. Asterisks indicate PϽ.05; BSS, topical balanced salt solution; and CFUs, colony-forming units.

tuberculous mycobacterial keratitis since Reviglio et al1 sistance to develop.21 Brown-Elliott et al14 reported that first reported it in 1998. Mycobacterium chelonae was the 97% of M chelonae isolates were susceptible or interme- most common pathogen identified among these cases. diately susceptible to gatifloxacin at an MIC of 4 µg/mL The initial choice for treatment of M chelonae kera- or less. titis has been topical amikacin combined with cipro- This study has shown that topical 0.3% gatifloxa- floxacin or clarithromycin. Clarithromycin is a macro- cin enhances the efficacy of topical fortified amikacin sul- lide antibiotic with excellent in vitro activity against fate (50 mg/mL) and clarithromycin (10 mg/mL) when M chelonae,15 and topical clarithromycin has been re- administered in triple combination therapy (PϽ.01). The ported to penetrate through intact epithelium.18 Amika- efficacy of triple combination therapy with gatifloxacin, cin also has been the drug of choice based on in vitro ac- amikacin, and clarithromycin was superior to that of tivity, animal studies, and clinical experiences.17 monotherapy with gatifloxacin in absolute colony- Adjunctive surgical treatment is required in many cases forming unit counts, but statistical significance was not of M chelonae keratitis following LASIK. Among 17 cases demonstrated. of M chelonae keratitis following LASIK reported in the Ciprofloxacin and combination therapy with forti- literature, 10 (59%) of 17 patients eventually under- fied amikacin and clarithromycin failed to show statis- went removal of the flap or therapeutic penetrating kera- tically significant antimycobacterial activity despite good toplasty to control the infection. in vitro activity. This observed result may be attributed In this study, gatifloxacin therapy exerted a signifi- to the use of corticosteroid therapy in our animal model. cant antimycobacterial activity and was more active than In 1 series of mycobacterial keratitis, 80% of the eyes with ciprofloxacin against M chelonae keratitis. Ciprofloxa- organisms that were sensitive but did not respond to topi- cin therapy has been found to have good in vitro activ- cal therapy were shown to have had a corticosteroid ad- ity against M chelonae19 but to be less effective against M ministered.13 Corneal epithelial penetration of each an- chelonae keratitis than against Mycobacterium fortuitum tibiotic also might affect the resulting antimycobacterial keratitis in vivo.20 In one series of nontuberculous my- activity in this study. In the animal model of this study, cobacterial keratitis, M chelonae isolates were generally mycobacterial keratitis was induced by intrastromal in- sensitive to amikacin and clarithromycin treatment but jection of organisms that spared the intact epithelial bar- resistant to ciprofloxacin treatment.13 Fluoroquinolnes rier. Although topical clarithromycin has been shown to have 2 targets on chromosomal DNA, which are DNA gy- achieve therapeutic levels in corneal tissue in a rabbit rase (type II topoisomerase) and topoisomerase IV. Mi- model,18 topical amikacin has been reported to have poor crobial resistance develops when mutations occur in the corneal penetration.22 target topoisomerase enzyme. The C-8 methoxy side chain Although gatifloxacin and triple combination therapy of gatifloxacin may prevent bacteria from developing re- with gatifloxacin, fortified amikacin, and clarithromy- sistance by requiring 2 topoisomerase mutations for re- cin have displayed significant antimycobacterial activ-

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©2004 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 ity by reducing colony-forming unit counts in this study, after laser in situ keratomileusis successfully treated with medical therapy and cultures remained positive for M chelonae in every cor- flap removal. Am J Ophthalmol. 2000;129:382-384. 3. Gelender H, Carter HL, Bowman B, et al. Mycobacterium keratitis after laser in nea from 35 rabbits after frequent administration of an- situ keratomileusis. J Refract Surg. 2000;16:191-195. tibiotic therapy. This result advocates for a more pro- 4. Suresh PS, Campbell I, Herzig S, et al. Mycobacterium keratitis following hyper- longed course of medical therapy and consideration of opic laser in situ keratomileusis. Can J Ophthalmol. 2001;36:272-274. surgical debridement to debulk organisms in the treat- 5. Garg P, Bansal AK, Sharma S, Vemuganti GK. Bilateral infectious keratitis after laser in situ keratomileusis: a case report and review of the literature. Ophthal- ment of M chelonae keratitis. mology. 2001;108:121-125. To our knowledge, this is the first reported in vivo 6. Kouyoumdjian GA, Forstot SL, Durairaj VD. Infectious keratitis after laser refrac- activity of gatifloxacin against M chelonae and the results tive surgery. Ophthalmology. 2001;108:1266-1268. confirmed the therapeutic efficacy of topical 0.3% gati- 7. Solomon A, Karp CL, Miller D, et al. Mycobacterium interface keratitis after laser floxacin against M chelonae keratitis in an animal model. in situ keratomileusis. Ophthalmology. 2001;108:2201-2208. 8. Chandra NS, Torres MF, Winthrop KL, et al. Cluster of Mycobacterium chelonae The current experiment was designed not to directly simu- keratitis cases following laser in-situ keratomileusis. Am J Ophthalmol. 2001; late clinical situations but to compare the in vivo efficacy 132:819-830. of various treatment regimens, including different gen- 9. Giaconi J, Pham R, Ta CN. Bilateral Mycobacterium abscessus keratitis after la- erations of fluoroquinolones, monotherapy, and combi- ser in situ keratomileusis. J Cataract Refract Surg. 2002;28:887-890. 10. Seo KY, Lee JB, Lee K, et al. Non-tuberculous mycobacterial keratitis at the in- nation therapy, in treating M chelonae keratitis. Thus, the terface after laser in situ keratomileusis. J Refract Surg. 2002;18:81-85. experimental keratitis was treated during a short period 11. Fulcher SF, Fader RC, Rosa RH Jr, et al. Delayed-onset mycobacterial keratitis in the early stage of the disease, which is unlikely to hap- after LASIK. Cornea. 2002;21:546-554. pen in clinical settings. Our results suggest, however, that 12. Pache M, Schipper I, Flammer J, et al. Unilateral fungal and mycobacterial kera- gatifloxacin can be a valuable new initial therapeutic agent titis after simultaneous laser in situ keratomileusis. Cornea. 2003;22:72-75. 13. Ford JG, Huang AJ, Pflugfelder SC, et al. Nontuberculous mycobacterial kerati- in the treatment of M chelonae keratitis. Treating clini- tis in South Florida. Ophthalmology. 1998;105:1652-1658. cians should consider that multiple combination agent 14. Brown-Elliott BA, Wallace RJ Jr, Crist CJ, et al. Comparison of in vitro activities therapy will still be beneficial and surgical debridement of gatifloxacin and ciprofloxacin against four taxa of rapidly growing mycobac- may be necessary especially in post-LASIK mycobacte- teria. Antimicrob Agents Chemother. 2002;46:3283-3285. 15. Brown BA, Wallace RJ Jr, Onyi GO, et al. Activities of four macrolides, including rial keratitis to successfully eradicate these challenging clarithromycin, against Mycobacterium fortuitum, Mycobacterium chelonae, and pathogens. 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