Current Eye Research

Received on August 20, 1997; revised on December 3, 1997 and accepted on January 20, 1998

© 1998 Oxford University Press

Comparison of topical antibiotics for treating

Mycobacterium

chelonae

keratitis in a rabbit model

Fung-Rong Hu and I-Jong Wang

Department of Ophthalmology, National Taiwan University Hospital, Taipei, Taiwan

Abstract Introduction

Purpose.

To investigate the effect of topical amikacin (25 mg/ml), imipenem (25 mg/ml), ciprofloxacin (3 mg/ml), clarithro-mycin (20 mg/ml), amikacin combined with ciprofloxacin,amikacin combined with imipenem, and amikacin combinedwith clarithromycin on

Mycobacterium chelonae

keratitis.

Methods.

Ninety New Zealand albino rabbits were infectedwith a strain of

M. chelonae

for which minimum inhibitoryconcentration indicated

in vitro

sensitivity to the above antibi-otics. The rabbits were treated for one or two weeks. The treat-ment efficacy was judged by the size of stromal infiltrate andquantitative culture of the infected corneas.

Results.

The size of the stromal infiltrate showed no signifi-cant difference in treated eyes compared to the untreated, con-trol eyes. However, all treatments significantly reduced thenumber of organisms in treated eyes compared to untreated,control eyes (all

p

-values

,

0.05). No significant difference intreatment efficacy was found between individual treatmentgroups. In none of the cases were organisms eliminated fromthe infected eyes, even after 2 weeks of treatment.

Conclusions.

The results suggest that topical amikacin, imi-penem, ciprofloxacin and clarithromycin had some therapeuticeffect on

M. chelonae

keratitis; however, amikacin combinedwith imipenem, ciprofloxacin, and clarithromycin showed noincreased efficacy over single agent therapy during 2 weeks oftreatment. Long term treatment may be required to eradicate

M. chelonae.

Curr. Eye Res. 17: 478–482, 1998.

Key words:

antibiotics;

in vivo

efficacy; keratitis;

Mycobacte-rium chelonae

;

rabbit

Mycobacterium fortuitum

complex, composed of

M. fortuitum

and

M. chelonae

, has been known to cause corneal infectionsin humans since 1965 (1). Nontuberculous mycobacterialkeratitis was often associated with foreign body injury, surgery,contact lens wearing and corticosteroid usage (2–5). The clini-cal course is chronic and indolent, with clinical presentationsmimicking fungal keratitis or herpetic keratitis. Medical ther-apy of nontuberculous mycobacterial keratitis is often unsatis-factory because of delayed diagnosis, slow response to therapy,inadequate drug penetration, resistance to most conventionalantibiotics and the emergence of a resistant strain, especiallywith single-agent therapy (4, 6, 7). Amikacin has been the sin-gle most commonly used agent for treating nontuberculousmycobacterial keratitis, however, its efficacy is still unsatisfac-tory (4, 8). In a previous study, we found that the newly-developed antimicrobial agents, including ciprofloxacin, clari-thromycin and imipenem, have good

in vitro

activity against

M. chelonae

ocular isolates, however, no synergistic effectcould be demonstrated for combinations of an aminoglycosidewith other effective drugs (11). Because

in vitro

activity doesnot always accurately predict therapeutic efficacy

in vivo

, weconducted a study to compare the therapeutic response of ami-kacin, ciprofloxacin, clarithromycin, imipenem and amikacincombined with one effective agent for treating

M. chelonae

keratitis in a rabbit model.

Materials and methods

Fifteen strains of

M. chelonae

were isolated from cornealulcers at the Department of Ophthalmology at National TaiwanUniversity Hospital from 1989 to 1992. All of them were iden-tified by the standard bacteriological method (12) and stored inMueller-Hinton broth (Difco Laboratories, Detroit, MI) con-taining 15% glycerol at

2

70

8

C and on Lowenstein-Jensenslants. Broth microdilution susceptibility test, as described bythe National Committee for Clinical Laboratory Standards (13),was used to test the minimum inhibitory concentration (MIC)

Correspondence: Dr. Fung-Rong Hu, Department of Ophthalmology, NationalTaiwan University Hospital, 7, Chung-Shan S. Rd., Taipei, Taiwan; E-mail:fungrong@ha.mc.ntu.edu.tw

Antibiotics treating

Mycobacterium chelonae

keratitis

479

of various antibiotics against these 15 strains of

M. chelonae

.Among them, a strain of

M. chelonae

which was most suscep-tible to the antibiotics tested was selected for this study. TheMIC of amikacin was 8

m

g/ml, the MIC of ciprofloxacin was0.5

m

g/ml, the MIC of imipenem was 4

m

g/ml and the MIC ofclarithromycin was 0.125

m

g/ml for this strain.According to the ARVO Statement for the Use of Animals in

Ophthalmic and Vision Research, ninety New Zealand albinorabbits, weighing 1.5 to 2.5 kg each, were randomly dividedaccording to the regimen and duration of treatment into 16treatment groups and one control group. Each treatment groupconsisted of five rabbits, and the untreated control group con-sisted of ten rabbits. Sedation and anesthesia were inducedthrough an intramuscular injection of a mixture of ketaminehydrochloride (50 mg/ml, 44 mg/kg of body weight) and xyla-zine hydrochloride (20 mg/ml, 8.8 mg/kg of body weight).Unilateral intrastromal corneal inoculation of

M. chelonae

wasperformed using the same procedure as previously described(14, 15). After one drop of proparacaine hydrochloride 0.5% wasinstilled onto the corneal surface, a 30-gauge needle attached toa Hamilton syringe with 10

4

organisms (10

m

l, 10

6

organisms/ml) was inoculated into the mid-stroma of the central cornea inthe right eye of each rabbit under an operating microscope. Theinjection of air bubbles and penetration of corneas wereavoided during intrastromal injection. A subconjunctival injec-tion of dexamethasone phosphate (0.5 ml, 4 mg/ml) was givenimmediately after the inoculation in the same eye.

The rabbits were observed daily, without treatment, after inoc-ulation. If there was no evidence of infection 3 days after inocu-lation, another booster dose of subconjunctival dexamethasonewas given. Topical antibiotics were applied hourly ten timesevery day from the eighth day after inoculation or a boosterdose of subconjunctival steroid injection. Each treatment groupreceived one of the following regimens: amikacin (25 mg/ml),imipenem (25 mg/ml), ciprofloxacin (3 mg/ml), clarithromycin(20 mg/ml), 2% alcohol, amikacin (25 mg/ml) combined withciprofloxacin (3 mg/ml), amikacin (25 mg/ml) combinedwith imipenem (25 mg/ml), or amikacin (25 mg/ml) com-bined with clarithromycin (20 mg/ml). Topical amikacin andimipenem were prepared for eyedrops from intravenousinjection powder. Ciprofloxacin was obtained from commer-cially available eyedrops (Alcon Laboratories, Inc., Fort Worth,TX, USA). Topical clarithromycin was prepared by dissolvingclarithromycin in powder form in 2 ml of alcohol, and thendiluted to a concentration of 20 mg/ml. The final concentrationof alcohol is 2%. Topical 2% alcohol was used as a control forthe clarithromycin-treated group. Each application of an antibi-otic consisted of one drop (approximate 50

m

l) placed directlyon the surface of the cornea. In combined treatment groups, theeyedrop application of the two antibiotics was separated by fivemin. The duration of therapy was either 1 week or 2 weeks.There were 10 rabbits left untreated after inoculation, serving ascontrol eyes.

Rabbits were observed under a slit lamp biomicroscopedaily without treatment for 7 days after inoculation or after thebooster dose of subjunctival steroid injection. The area of themain stromal infiltrate in each eye was estimated by measur-

ing the longitudinal and transverse axes and multiplying themtogether. The conjunctival injection was recorded by using asemiquantitative grading scale (1+

5

trace; 2+

5

mild; 3+

5

moderate; and 4+

5

severe). These clinical features were eval-uated again for each eye 1 week and 2 weeks after treatment.

At the end of the therapy (i.e. on day 8 or day 15 after initia-tion of treatment), the rabbits were sacrificed by an intravenousinjection of pentobarbital sodium (100–125 mg in 2–2.5 ml)after being sedated with an intramuscular injection of chlorprom-azine (2 ml, 25 mg/ml). A corneal button was removed from eachrabbit with a 10-mm trephine under sterilized conditions. Theexcised button was placed in 1.0 ml of sterilized saline andground in a glass Tenbroek tissue grinder (Wheaton Scientific,Millville, NJ). Then 1

m

l and 10

m

l of the extracted fluid werespread uniformly onto blood agar plates using a 1

m

l and a 10

m

l standard metal loops, and were inoculated in duplicate foreach volume fluid. The plates were placed in plastic bags andincubated for 5 days at 37

8

C with 10% CO

2

. The number ofcolony forming units (CFU) per ml were determined on thefifth day. Organisms were confirmed to be

M. chelonae

by pos-itive Ziehl-Neelsen acid fast staining.

The Mann-Whitney U test with two tail

p

-values was used inthis study to compare the mean infiltrate areas and the treat-ment efficacy between treatment and control groups. A

p

-value

,

0.05 was recognized as statistically significant.

Results

M. chelonae

keratitis developed by day 3 after inoculation in87 rabbits. The other three rabbits developed keratitis afterreceiving a booster dose of a subconjunctival steroid injection.Seven rabbits died during this period; the others lived well andcould see with the fellow eye. Typical keratitis developed asdense mid-stromal infiltrates with an epithelial defect and pro-fuse yellowish-white discharges. The eyelids were almoststuck together by the profuse, sticky discharges during the firstweek. No corneal perforation was found. Conjunctival injec-tion appeared with corneal ulcer and persisted during the wholecourse (Table 1). Following treatment, the discharge graduallydisappeared, but stromal infiltration persisted (Table 1). Themean stromal infiltrate area did not change significantly in sizeafter the two-week course of therapy in either single-agent orcombination-therapy groups.

Table 2 showed the number of residual colonies after oneweek of topical antibiotics therapy. All of these antibioticscould suppress the proliferation of organisms in rabbit corneas.The number of organisms was significantly reduced in single-agent or combined therapy groups when compared with thecontrol group (

p

,

0.05). Similar results were observed after 2weeks of treatment (Table 3). There was no significant differ-ence among the various treatment groups, except that clarithro-mycin was less effective compared to amikacin and ciprofloxa-cin in reducing the number of organisms after one week oftreatment. Compared with the 2% alcohol-treated group, theclarithromycin-treated group showed no difference in cultureresults after one and two weeks of treatment. Combinationtherapy did not show any improved efficacy over single agent

480

F.-R. Hu and I-J. Wang

therapy. Nevertheless, none of the therapies could eradicate theorganisms in rabbit corneas even after two weeks of treatment.

Discussion

Most reported cases of nontuberculous mycobacterial keratitishave been caused by

M. fortuitum

-complex organisms, whichare composed of

M. fortuitum

and

M. chelonae

(3). Nontuber-culous mycobacterial keratitis is difficult to treat, because theorganism is generally resistant to conventional antituberculous

agents such as isoniazid, rifampin, streptomycin, ethambutol,and para-aminosalicylate (4, 16). Amikacin has been the singlemost commonly used agent for nontuberculous mycobacterialkeratitis; however, its efficacy remains unsatisfactory, even atconcentrations up to 100 mg/ml (8, 15). Highly concentratedtopical amikacin was poorly tolerated and is toxic to cornealand conjunctival epithelium (17, 18); therefore, the search forother effective agents against nontuberculous mycobacterialkeratitis is worth investigating.

Recently, many new agents were found to be effective for

M.fortuitum

complex

in vitro

, including ciprofloxacin, clarithromy-cin, and imipenem (11, 19, 20). According to reports, the MICsof ciprofloxacin against

M. chelonae

ranged from 0.12 to 512

m

g/ml (9, 10, 19, 20). Topical application of ciprofloxacin couldyield an aqueous humor concentration of 4.82

±

2.15

m

g/ml inthe presence of an intact epithelium, and a concentration of12.9

±

4.39

m

g/ml after the debridement of the corneal epithe-lium (21). Although this achievable drug concentration ishigher than the MICs for most of the

M. chelonae

strain re-ported in the literature, the therapeutic response of ciprofloxa-cin to

M. chelonae

keratitis is difficult to predict (22, 23). Lin

et al.

(15) conducted a study to compare the efficacy of topicalciprofloxacin for treating

M. fortuitum

and

M. chelonae

keratitisin the rabbits. They found that ciprofloxacin is effective againstboth organisms, but it is less effective against

M. chelonae

than

M. fortuitum

. Although ciprofloxacin can significantly reducethe number of organisms in treated eyes, there was no clinicaleffect observed for

M. chelonae

keratitis. In this study, we alsofound that the number of organisms is significantly reduced af-ter ciprofloxacin treatment, as compared to the control group.However, the stromal infiltrate area did not change significantlyafter 2 weeks of ciprofloxacin treatment, even when infectedwith a rather sensitive strain of

M. chelonae

(MIC

5

0.5

m

g/ml).

Table 1.

Summary of local findings in rabbits with

M. chelonae

keratitis

Stromal infiltrate (mm

2

) Conjunctival injection*

Group

Beforetreatment

(mean

±

SD)

1 week oftreatment

(mean

±

SD)

2 weeks of treatment

(mean

±

SD)

Beforetreatment(mean)

1 week oftreatment(mean)

2 weeks oftreatment(mean)

Control 7.43

±

0.04 6.00

±

0.50 5.5

±

0.79 3.75 3.30 2.98Amikacin 7.46

±

0.22 6.46

±

0.22 4.95

±

0.44 3.56 2.20 2.00Ciprofloxacin 7.53

±

0.15 6.48

±

0.10 5.14

±

0.42 3.56 2.25 1.60Clarithromycin 7.56

±

0.36 6.24

±

0.61 4.78

±

0.28 3.6 2.40 2.252% Alcohol 7.34

±

0.36 6.30

±

0.16 4.62

±

0.65 3.6 2.40 2.44Imipenem 7.12

±

0.49 5.92

±

0.79 4.56

±

0.58 3.6 2.25 2.25Amikacin +

ciprofloxacin 7.36

±

0.56 5.80

±

1.14 4.46

±

0.40 3.6 2.60 2.25Amikacin +

imipenem 7.24

±

0.73 5.54

±

0.92 4.40

±

0.70 3.6 2.25 2.25Amikacin +

clarithromycin

7.30

±

0.74

6.80

±

0.65

4.55 ± 0.26 3.75 2.40 2.40

*Conjunctival injection was scored using an ordinal grading scale: trace 5 1; mild 5 2; moderate 5 3; severe 5 4. These numbers indicate the persistence of theconjunctival injection, but no statistical interpretations are applied to them.

Table 2. Quantitative cultures of M. chelonae in treated anduntreated groups after one week of treatment

Culture result(mean ± SE)

Treatment groups (CFU 3 103/ml) p

Untreated eyes (n 5 3) 183.3 ± 44.1Amikacin-treated (n 5 5) 2.0 ± 0.9 0.0245*Ciprofloxacin-treated (n 5 4) 3.75 ± 1.69 0.0339*Clarithromycin-treated (n 5 5) 1.70 ± 6.3 0.0100*2% Alcohol-treated (n 5 5) 15.8 ± 4.6 0.0253*Imipenem-treated (n 5 5) 7.6 ± 4.9 0.0219*Amikacin- + ciprofloxacin-treated

(n 5 5) 24.0 ± 17.1 0.0245*Amikacin- + imipenem-treated

(n 5 5) 7.4 ± 1.9 0.0245*Amikacin- + clarithromycin-

treated (n 5 4) 11.3 ± 4.0 0.0339*

*p-value < 0.05 when compared with untreated group by Mann-Whitney U test.

Antibiotics treating Mycobacterium chelonae keratitis 481

Clarithromycin is a new acid-stable macrolide with good invitro activity against M. chelonae (24, 25). Topical clarithro-mycin has been shown to have good tissue penetration; thehighest mean corneal concentration was 160 mg/ml in rabbitsreceiving 20 mg/ml of clarithromycin (26). Topical clarithro-mycin has been shown to be useful against M. fortuitum kerati-tis in a rabbit model (17), but no report regarding the in vivoefficacy of clarithromycin against M. chelonae keratitis isavailable in the literature. Our results showed that the numbersof organisms left in the corneas of the clarithromycin-treatedgroup were more than those in the ciprofloxacin- and amika-cin-treated groups. This result suggested that clarithromycinmight be effective in vitro, but was not as effective as amikacinor ciprofloxacin in vivo against M. chelonae keratitis. The pos-sible cause of this discrepancy may be due to the poor solubil-ity of clarithromycin in a solvent, and its being a bacteriostaticagent instead of a bactericidal agent. In this study, we used 2%alcohol to dissolve clarithromycin because of its poor solubil-ity in distilled water. We also tried unsuccessfully to use 5%glucose water to dissolve it. Because 2% alcohol was used todissolve clarithromycin, there was the possibility that the bac-tericidal effect came from the alcohol instead of the clarithro-mycin. The number of organisms left in the corneas of the 2%alcohol-treated group was not different from that of the cla-rithromycin-treated group. These results suggest that althoughclarithromycin has good in vitro activity against M. chelonae,the poor solubility of clarithromycin might limit its use in clin-ical situations unless this problem is solved.

Imipenem, a new beta-lactamase-resistant carbapenem anti-biotic, has been shown to have good in vitro antimycobacterialactivity (27). Imipenem was used successfully in the mono-therapy of a case of M. chelonae lung disease (28). In ourstudy, we found that imipenem is as effective as amikacin and

ciprofloxacin for treating M. chelonae keratitis. However, imi-penem discolored gradually and turned a brown color within24 to 48 h after dissolving into an aqueous solution. We wererequired to prepare a new solution every day, which makeslong-term topical usage of imipenem very expensive andimpractical. Due to the lack of information regarding the sta-bility and half-life of imipenem for topical use, the potentialapplicability of this agent for clinical ophthalmic usage isuncertain. Further studies of the ocular pharmacokinetics andpharmacodynamics of imipenem are necessary to establish theactual roles that imipenem could play in the long-term treat-ment of M. chelonae keratitis.

Multiple-drug therapy was preferred for most mycobacterialinfections because of frequent development of clinical resis-tance during single agent therapy (7). Matoba et al. (20) haveshown that the combination of amikacin with imipenem orciprofloxacin led to results ranging from antagonistic to addi-tive effects. We have found that amikacin combined with cipro-floxacin, clarithromycin and imipenem showed an antagonisticin vitro effect (11). Helm et al. (17) found no evidence of re-duced or improved clinical efficacy of the combination of ami-kacin and vancomycin in experimental M. fortuitum keratitis.In our study, we found that amikacin combined with cipro-floxacin, imipenem or clarithromycin showed no improved ef-ficacy for treating M. chelonae keratitis compared to singleagent therapy. However, M. chelonae is difficult to completelyeradicate, even after 2 weeks of treatment, in contrast to thecomplete eradication within several h of gram-negative bacilliin a similar rabbit model (21). Because prolonged medicaltreatment for M. chelonae keratitis is necessary, combinedtherapy should still be considered, to decrease the risk of devel-oping antibiotic resistance over time. Further study to investi-gate other antibiotic combinations that show a synergisticeffect could produce better therapeutic results in treating M.chelonae keratitis.

Corticosteroid usage has been implicated as a contributingfactor in the development of severe and prolonged M. fortuitumkeratitis in humans (1, 2). Paschal et al. demonstrated thatinoculation of M. fortuitum alone in rabbit corneas results in aself-limited keratitis of short duration; however, concomitantsubconjunctival steroid injection at the time of mycobacterialinoculation could induce an indolent, progressive keratitis (14).In the steroid-treated eyes, an acute inflammatory reaction wasseen at weeks 1, 2, and 4 after inoculation, but granulomatousinflammation was seen only at week 4. Although the number oforganisms remaining in the rabbit corneas decreased spontane-ously three weeks after inoculation in the steroid-treated eyes,corneal infiltrates persisted until week 4 when granulomatousinflammation appeared (14). In this study, we induced M. che-lonae keratitis in the rabbits with the same method as describedby Paschal et al. We found that the number of organismsremaining in the cornea decreased spontaneously three weeksafter inoculation in the untreated eyes. Nevertheless, the stro-mal infiltrates and conjunctival injection did not change signif-icantly in either treated or untreated eyes until 4 weeks afterinoculation. The keratitis resolved progressively thereafter,even without treatment. These results suggest that granuloma-

Table 3. Quantitative cultures of M. chelonae in treated anduntreated groups after two weeks of treatment

Culture result (mean ± SE)

Treatment Groups (CFU 3 103/ml) p

Untreated eyes (n 5 5) 54.6 ± 16.7Amikacin-treated (n 5 4) 11.3 ± 5.2 0.0139*Ciprofloxacin-treated (n 5 5) 0.6 ± 0.4 0.0082*Clarithromycin-treated (n 5 4) 7.5 ± 4.8 0.0139*2% Alcohol-treated (n 5 5) 0.2 ± 0.2 0.0071*Imipenem-treated (n 5 5) 9.0 ± 5.8 0.0278*Amikacin- + ciprofloxacin-treated

(n 5 5) 4.2 ± 1.4 0.0163*Amikacin- + imipenem-treated

(n 5 5) 3.0 ± 1.2 0.0163*Amikacin- + clarithromycin-

treated (n 5 4) 0.3 ± 0.3 0.0127*

*p-value < 0.05 when compared with untreated group by Mann-WhitneyU test.

482 F.-R. Hu and I-J. Wang

tous inflammation plays an important role in limiting thespread of organisms and leading to resolution of disease.Although there are probably some differences in host defensebetween rabbits and human, corticosteroid therapy, which willsuppress granulomatous inflammation, should be avoided inthe treatment of M. chelonae keratitis.

Acknowledgments

This study was supported by grant NSC 83-0412-B002-005from the National Science Council, Taiwan.

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