Investigative Ophthalmology & Visual Science, Vol. 31, No. 10, October 1990
Copyright © Association for Research in Vision and Ophthalmology
CiprofloxQcin Iontophoresis for AminoglycosideResistant Pseudomonol Kerafifis
Jeffery A. Hobden,* James J. Reidy,t Richard J. O'Callaghan,* Michael 5. Insler.f and James M. Hill*t£
Studies using ciprofloxacin for the therapy of experimental aminoglycoside-resistant keratitis caused
by Pseudomonas aeruginosa were conducted using transcorneal iontophoresis as the drug-delivery
system. Corneas infected with P. aeruginosa ATCC 27853/pMG6 were treated 22 hours postinfection
with ciprofloxacin delivered by iontophoresis (0.8 mA X 10 min), mock iontophoresis (eyecup with no
current), or frequently applied topical drops. Iontophoresis of 10 mg/ml or 25 mg/ml of ciprofloxacin
significantly reduced the number of viable bacteria per cornea by more than 5 log units compared with
untreated controls (P < 0.0001). Five hours after the initiation of treatment, mock iontophoresis (10
mg/ml or 25 mg/ml) or 11 applications of topical ciprofloxicin drops (7.5 mg/ml) decreased the viable
bacteria relative to the untreated controls by 5 log units (P < 0.0001). One treatment with an eyecup
was as effective as 11 treatments with topical drops (P > 0.75). One hour after treatment with
iontophoresis or mock iontophoresis of 10 mg/ml of ciprofloxacin, aqueous humor concentrations were
83.75 ± 8.85 Mg/ml and 24.87 ± 4.0 Mg/ml (mean ± standard error of the mean), respectively. One
hour after the last of five applications of 7.5 mg/ml of ciprofloxacin (every 15 min for 1 hr) the aqueous
humor concentration was 4.2 ± 1.14 Mg/ml. These results show the value of ciprofloxacin in treating
aminoglycoside-resistant infections caused by P. aeruginosa and suggest that ciprofloxacin can be
efficiently delivered by iontophoresis. Invest Ophthalmol Vis Sci 31:1940-1944,1990
Bacterial keratitis is a potentially sight-threatening
complication associated with contact lens wear.
Pseudomonas aeruginosa is the pathogen most commonly encountered in contact lens-related bacterial
keratitis.1"3 Infections that are caused by this organism are more virulent than most other bacterial pathogens encountered in the eye. Appropriate antimicrobial treatment must be promptly instituted to
avoid significant loss of vision.
Current modes of therapy include the topical application of fortified tobramycin or gentamicin drops
every 30 min around the clock for 48-72 hr, sometimes in conjunction with daily subconjunctival injections.4 This intensive regime is disruptive to the
patient and may require hospitalization to ensure
compliance. Subconjunctival injections are painful,
and their effectiveness has been questioned.5"8
Choi and Lee9 used transcorneal iontophoresis to
deliver vancomycin into the aqueous humor and
cornea of rabbit eyes. Rootman et al10 demonstrated
From the Departments of *Microbiology, Immunology, Parasitology, and ^Pharmacology, and the |Lions Eye Research Laboratories, LSU Eye Center, Louisiana State University Medical Center
School of Medicine, New Orleans, Louisiana.
Supported in part by Public Health Service grants EY-08871,
EY-07608, and Core grant EY-02377 from the National Eye Institute, National Institutes of Health, Bethesda, Maryland.
Reprint requests: James M. Hill, LSU Eye Center, 2020 Gravier
Street, Suite B, New Orleans, LA 70112-2234.
that iontophoresis is an effective method of delivering
tobramcyin to both the cornea and anterior chamber.
Iontophoresis was also shown to be effective in delivering tobramycin to Pseudomonas-infected corneas and in treating P. aeruginosa keratitis in the
rabbit. 5 " 12 In this study we show the efficacy of iontophoretically delivered ciprofloxacin for tobramycin-resistant P. aeruginosa using a rabbit model of
keratitis.
Materials and Methods
Preparation of Antibiotic Solutions
A tobramycin solution was prepared by dissolving
tobramycin sulfate powder (Alcon, Fort Worth, TX)
in sterile deionized water. Ciprofloxacin solutions
were prepared by dissolving powdered ciprofloxacin
(Alcon) in a minimal volume of 1 M acetic acid
(< 0.5 ml) and adjusting the pH to 6.0 with 0.55 M
sodium hydroxide and the volume with sterile deionized water. A control solution (pH 6.0) was prepared
using equivalent volumes of acetic acid and sodium
hydroxide.
Determination of Ciprofloxacin Charge in Solution
Ciprofloxacin mobility in an electrical field was
determined by paper electrophoresis. A 10-/ul aliquot
of a ciprofloxacin solution was spotted onto a 30
1940
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No. 10
IONTOPHORESIS OF CIPROFLOXACIN / Hobden er ol
X 3-cm filter paper strip (Beckman, Belmont, CA).
Tobramycin (10 fx\) underwent electrophoresis as a
control. The electrolyte solution consisted of 0.2%
sodium chloride adjusted to pH 6.4 with 1 M sodium
hydroxide. Bromphenol blue (1 ix\ of a 0.5 mg/ml
solution; Sigma, St. Louis, MO) was used as a tracking dye. Samples underwent electrophoresis at room
temperature for 3-5 hr with a direct current of 200
Volts. Strips were then allowed to air dry. Ciprofloxacin was visualized by observing fluorescence when
illuminated with a 254-nm ultraviolet transilluminator (Ultra-Violet Products, San Gabriel, CA). Tobramycin was developed by first spraying the strip with
0.25% ninhydrin (Sigma) in acetone and then incubating at 37 °C for 1-2 hr.
In a paper electrophoresis system, bromphenol
blue, a tracking dye with a net negative charge,
moved 4.0 cm from the origin towards the anode, and
tobramycin, which has a net positive charge when in
solution, moved 5.5 cm towards the cathode. Ciprofloxacin moved 2.5 cm towards the cathode, illustrating a net positive charge.
Experimental Pseudomonal Keratitis
Treatment and care of the rabbits in this investigation were in strict compliance with the ARVO Resolution on the Use of Animals in Research. New Zealand white rabbits weighing 2-3 kg were anesthetized
with an intramuscular injection of a 1:5 mixture of
xylazine (100 mg/ml; Miles, Shawnee, KA) and ketamine hydrochloride (100 mg/ml; Bristol, Syracuse,
NY). Proparacaine hydrochloride (0.5%, Ophthaine;
Squibb, Princetown, NJ) was instilled in both eyes,
and each eye received an intrastromal injection, as
previously described,5 of 10 /zl of tryptic soy broth
containing 103 logarithmic-phase colony forming
units (CFU) of P. aeruginosa ATCC 27853/pMG6.
This strain was obtained by conjugally transferring
plasmid pMG6 from P. aeruginosa PA0303 to P.
aeruginosa ATCC 27853 as previously described.12
The number of bacteria in each inoculum and minimum inhibitory concentrations (MICs) for ciprofloxacin and tobramycin were determined retrospectively. The MICs were determined by the tube-dilution method.13
Determination of Ciprofloxacin Concentration in
Aqueous Humor
Aqueous-humor samples were collected with a
27-G needle attached to a 1-ml tuberculin syringe.
Aliquots of 20 /A of each aqueous-humor sample
were placed in triplicate on filter paper discs
(Schleicher & Schuell, Inc., Keene, NH, 740-E, 6.35
mm). The discs were then placed on 150 X 15-mm
1941
petri dishes containing 75 ml of Oxoid #2 antibiotic
agar (Oxoid, Columbia, MD) previously inoculated
with 105 CFU of Klebsiella pneumoniae ATCC
10031. The ciprofloxacin concentrations in the anterior chamber were determined by referencing the
average zone of inhibition to a standard curve for
known concentrations of ciprofloxacin ranging from
0.95-125 fig/m\. Standards were assayed with all
aqueous-humor samples to assure the precision of the
assay.
Experimental Design
Three experiments were done. In the first experiment, nine rabbits were assigned randomly to one of
four treatment groups. Group I (2 rabbits, 4 eyes)
received iontophoresis of 25 mg/ml of ciprofloxacin
22 hr postinfection. Iontophoresis was accomplished
by placing an anode connected to a direct current
source in contact with a ciprofloxacin solution instilled in an eyecup resting upon the cornea of an
anesthetized rabbit and by attaching the cathode to a
saline-soaked gauze pad on the rabbit's ear. A current
of 0.8 mA was then passed through the solution for
10 min. Group II (2 rabbits, 4 eyes) received mock
iontophoresis of ciprofloxacin (25 mg/ml) 22 hr postinfection; ie, the same treatment as Group I, except
no current was passed through the solution. Group
III (2 rabbits, 4 eyes) received topical ciprofloxacin
drops (7.5 mg/ml) starting 22 hr postinfection, every
15 min, ending 23 hr postinfection, and then every 30
min ending 26 hr postinfection. There were 11 applications of topically administered drops over a 4-hr
period. Group IV (3 rabbits, 6 eyes) received no
treatment. All rabbits were killed 27 hr postinfection.
To ascertain any bactericidal effects of sodium acetate, the iontophoresis and mock-iontophoresis portions of the experiment were done using a control
solution (1 rabbit, 2 eyes per treatment group).
In the second experiment, rabbits were assigned to
the same four treatment groups (2 rabbits, 4 eyes/
group), as described in experiment 1. However, the
concentration of ciprofloxacin in Group I (iontophoresis) and Group II (mock iontophoresis) was decreased from 25 to 10 mg/ml. The concentration of
ciprofloxacin in Group III (7.5 mg/ml) and the dosing schedule remained unchanged. All rabbits were
killed with an overdose of sodium pentobarbital 27 hr
postinfection.
In the third experiment, rabbits were randomly assigned to similar treatment groups (2 rabbits, 4 eyes/
group), as described in experiment 2, with the exception that the animals were killed 1 hr after the initiation of treatment (23 hr postinfection). Also, rabbits
in Group III received one drop of 7.5 mg/ml ciprofloxacin every 15 min for 1 hr starting 21 hr postin-
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1942
fection. Five drops were applied over a 60-min period. To determine ciprofloxacin concentrations 23
hr postinfection, aqueous-humor samples were collected at the termination of the experiment. Drops
were terminated 1 hr before collecting aqueous
humor.
The procedure for quantitating viable bacteria per
cornea has been previously described.5 Briefly, the
corneas were excised and homogenized in sterile
phosphate-buffered saline (3 ml). An aliquot (0.5 ml)
of homogenate was serially diluted, and dilutions (0.1
ml) were plated in triplicate on tryptic soy agar plates
(TSA; Difco, Detroit, MI). All agar plates were incubated at 37°C for up to 48 hr. To determine if bacteria recovered from treated corneas retained tobramycin resistance and susceptibility to ciprofloxacin, colonies from Groups I, II, and III were subcultured
onto TSA plates, and MICs for ciprofloxacin or tobramycin were determined.
Results were analyzed using the Statistical Analysis
Systems.14 The mean number of CFU per rabbit was
used to calculate the mean number of CFU per treatment group. An analysis of variance was done on the
logarithmic number of CFU per treatment group.
After the analysis of variance, t-tests among means
were calculated. All colony counts are expressed as
base-10 logarithms.
Results
The base-10 log numbers of viable bacteria per
cornea after treatment with iontophoresis or mock
iontophoresis of 25 mg/ml of ciprofloxacin or with
topical ciprofloxacin drops (7.5 mg/ml) are shown in
Table 1. There was no significant difference in the log
number of bacteria remaining after treatment with
iontophoresis of the placebo (7.16 ± 0.24; mean
Table 1. Viable bacteria per cornea 27 hours
postinfection after treatment with ciprofloxacin
Group
Treatment
Conditions
I
Iontophoresis
0.8 mA X 10 min
25 mg/ml
22 hr postinfection
10 min
25 mg/ml
22 hr postinfection
7.5 mg/ml
Every 15 min for
first hr, then
every 30 min for
II
III
Eye cup
Drops
CFU*
0.25 ±0.17
Table 2. Viable bacteria per cornea 27 hours
postinfection after treatment with ciprofloxacin
Group
Treatment
Conditions
I
Iontophoresis
0.8 mA X 10 min
10 mg/ml
22 hr postinfection
10 min
10 mg/ml
22 hr postinfection
7.5 mg/ml
Every 15 min for
first hr, then
every 30 min for
II
Eye cup
III
Drops
IV
None
None
—
•Log base 10±SEM.
t Significantly different than group I (P <, 0.008).
X Not significantly different than group II (P = 0.80).
0.57 ±0.17
1.71 ±0.17f
1.63 ± 0.17ft
^
—
7.06 ± 0.17f
* Log base 10±SEM.
t Significantly different than group I (P <, 0.0001).
X Not significantly different than group II (P = 0.75).
± standard error of the mean) or mock iontophoresis
of the placebo (7.31 ± 0.24) relative to untreated
controls (7.01 ± 0.24; P > 0.65).
The base-10 log numbers of viable bacteria per
cornea after treatment with iontophoresis or mock
iontophoresis of 10 mg/ml of ciprofloxacin or with
topical ciprofloxacin drops (7.5 mg/ml) are shown in
Table 2. Iontophoresis was significantly more effective than mock iontophoresis or topically applied
drops (P < 0.0001). Treatment with an eyecup was
equally as effective as treatment with 11 doses of topical drops in reducing the number of bacteria per cornea (P = 0.75).
The results of experiment 3 are shown in Table 3.
At 23 hr postinfection, iontophoresis of 10 mg/ml of
ciprofloxacin reduced the number of viable bacteria
per cornea greater than 6 log units compared with
untreated controls (P < 0.0001).
The ciprofloxacin concentration in the aqueous
humor 1 hr after the last application (five applications) of 7.5 mg/ml of ciprofloxacin was 4.20 ±1.14
Table 3. Viable bacteria per cornea 23 hours
postinfection after treatment with ciprofloxacin
Group
Treatment
Conditions
I
Iontophoresis
0.8 mA X 10 min
10 mg/ml
22 hr postinfection
10 min
10 mg/ml
22 hr postinfection
7.5 mg/ml
Every 15 min for
first hr
0.36 ± 0.29
—
6.91 ±0.24f
1.18±0.17f
II
Eye cup
1.11 ± 0.17ft
6.23 ± 0.14t
CFU*
3hr
III
Drops
IV
None
3hr
IV
Vol. 31
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Ocrober 1990
* Log base 10 ± SEM.
t Significantly different than group I (P ^ 0.0001).
X Not significantly different than group I (P = 0.08).
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CFU*
1.11 ±0.29*
2.27 ± 0.29f
No. 10
IONTOPHORESIS OF CIPROFLOXACIN / Hobden er ol
jig/ml. The eyecup produced an aqueous-humor
concentration 1 hr after treatment of 24.85 ± 4.0
/ug/ml. Iontophoresis produced an aqueous-humor
concentration 1 hr after treatment of 83.75 ± 8.85. Of
50 colonies tested after isolation from infected corneas, all retained their susceptibility to ciprofloxacin
(MIC = 0.4 Mg/ml) and their resistance to tobramycin
(MIC = 31.25 /zg/ml).
Corneas receiving iontophoresis or mock iontophoresis (eyecup with no current) of the ciprofloxacin
solutions were edematous immediately after treatment. Corneal edema was also seen in eyes treated
with iontophoresis or mock iontophoresis of the control solution. The edema resolved within 20 min after
completion of treatment.
Discussion
The use of aminoglycoside antibiotics such as tobramycin for the treatment of eye infections caused
by P. aeruginosa is well established.15 However, when
tobramycin is used as the primary agent in these infections, emergence of resistant strains can occur.16
Our results show the value of ciprofloxacin in treating
a tobramycin-resistant strain of P. aeruginosa.
Fluoroquinolones such as ciprofloxacin, enoxacin,
and norfloxucin have excellent in vitro activity
against various ocular bacterial pathogens.17 Topical
fluoroquinolone therapy for experimental tobramycin-sensitive P. aeruginosa-induced corneal ulcers in
the rabbit have been described by Sugar et al18 and
Darrell et al.19 Sugar et al18 reported a 3-4 log unit
reduction in bacterial counts in corneas treated with
10 mg/ml of enoxacin drops delivered topically on an
hourly basis for 24 hr. Although no quantitative cultures were performed, Darrell et al19 reported negative bacterial cultures after P. aeruginosa-infected
corneas were treated with a 10 mg/ml suspension of
norfloxacin applied topically four times a day for 4
days. More recently, O'Brien et al20 were unable to
recover aminoglycoside-resistant P. aeruginosa from
rabbit corneas treated with 3 mg/ml of ciprofloxacin
drops applied every 30 min for 12 hr.
In our study we report the successful treatment of
P. aeruginosa-induced keratitis in the rabbit with
iontophoresis of ciprofloxacin. In contrast to the previously described studies in which repeated topical
applications of fluoroquinolones were used,18'20 we
were able to achieve a dramatic reduction in viable
bacteria per cornea with a single ionotrophoretic application of ciprofloxacin. One 10-min iontophoresis
of 10 mg/ml or 25 mg/ml of ciprofloxacin almost
sterilized the cornea (< ten bacteria per cornea). Also,
one 10-min application of an eyecup containing a
solution of 10 or 25 mg/ml ciprofloxacin induced a
1943
5-log unit reduction compared with untreated controls and was as effective as 11 applications of 7.5
mg/ml of ciprofloxacin topical drops. One hour after
a single iontophoretic treatment, concentrations of
ciprofloxacin in the aqueous humor were over 20
times higher than drops applied every 15 min. Initial
aqueous-humor concentrations of ciprofloxacin
achieved after a single application with an eyecup
were over six times greater than levels achieved with
frequent topical drops. Thus, iontophoresis and the
eyecup represent a more efficient means of delivering
ciprofloxacin to the aqueous humor compared with
frequently applied topical drops. If given frequently
enough and over a longer period of time, topical
drops of 7.5 mg/ml of ciprofloxacin might be as efficacious as iontophoresis or the eyecup in reducing
corneal P. aeruginosa.
The transient edema of the cornea seen after treatment with iontophoresis or mock iontophoresis of
ciprofloxacin was also seen in eyes treated with iontophoresis or mock iontophoresis of the control solution, implying that edema was not associated with
ciprofloxacin but rather with the solution used. The
ciprofloxacin solution and the control solution contained sodium acetate at an acidic pH (6.0). Ciprofloxacin iontophoresis and mock iontophoresis were
associated with transient edema that resolved within
approximately 20 min.
Iontophoresis of tobramycin has previously been
shown to be effective in experimental pseudomonal
keratitis. 5 " 12 Our results show that iontophoresis of
ciprofloxacin is an efficient treatment of pseudomonal keratitis. Further studies are needed to establish appropriate antibiotic concentrations that balance antimicrobial efficacy against corneal toxicity.
Key words: iontophoresis, ciprofloxacin, pseudomonal keratitis, rabbit, cornea
Acknowledgments
The authors thank Dr. Hilary W. Thompson for his help
with the statistical analyses and Ms. Ada Rivera for typing
the manuscript. None of the authors have any commercial,
proprietary, or financial interest in any of the companies or
products described in this study.
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