Journal of Refractive Surgery

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Keratotomy Model of Pseudomonas Keratitis: Gentamicin Chemotherapy

E Britt Brockman, MD; Paul A Tarantino, MD; Jeffery A Hobden, MS; James M Hill, PhD; Herbert E Kaufman, MD; Michael S Insler, MD

Abstract

ABSTRACT

Background: Chemotherapy of bacterial keratitis requires frequent application of antibiotic drops. Collagen shields containing antibiotics could reduce the need for frequent antibiotic application. To determine the effect of gentamicin-containing collagen shields and gentamicin drops on Pseudomonas keratitis, a new keratotomy model of infection was employed.

Methods: Model-contact lenses (58% water content) presoaked in 1% bovine serum albumin and exposed to 10p 8 colony forming units per mL of Pseudomonas aeruginosa strain 27853, were found to reproducibly retain 5.9 (log base 10) colony-forming units. Rabbit corneas were scarified centrally with two perpendicular intersecting diamond knife cuts (5 mm × 5 mm × 0.2 mm), and bacteria-impregnated contact lenses were positioned and held in place for 24 hours with partial tarsorrhaphies. Treatment-Fourteen hours after lens removal (38 hours after infection), corneas were treated for 8 hours with collagen shields hydrated in saline (control), or shields impregnated with 800 µg gentamicin during manufacture, or one drop every 30 minutes of fortified gentamicin drops (14 mg/mL). The rabbits were killed and corneas collected for bacterial enumeration after 8 hours of treatment (46 hours after infection).

Results: Model-Slit-lamp examination and microbiologic confirmation showed uniformity of keratitis in all eyes. Treatment-Corneas treated with saline (controls) contained 6.4 (log base 10) Pseudomonas. Corneas treated with gentamicin-impregnated collagen shields (total drug = 800 µg) and fortified gentamicin drops (total drug = 21 mg) showed a reduction in viable bacteria of 2 logs and 6 logs, respectively, relative to the control.

Conclusions: In this new model of Pseudomonas keratitis, the amount of gentamicin introduced into collagen shields during manufacture effectively reduced bacterial growth in infected rabbit corneas. However, larger amounts of drug applied as fortified drops on a frequent dosing schedule were more effective by a factor of three. Treatment of keratitis with antibiotic-impregnated collagen shields may reduce the need for very frequent application of topical drops, but may be more effective with topical drop supplementation to increase the amount of drug available over the course of therapy. Refract Corneal Surg 1992;8:39-43.)

Abstract

ABSTRACT

Background: Chemotherapy of bacterial keratitis requires frequent application of antibiotic drops. Collagen shields containing antibiotics could reduce the need for frequent antibiotic application. To determine the effect of gentamicin-containing collagen shields and gentamicin drops on Pseudomonas keratitis, a new keratotomy model of infection was employed.

Methods: Model-contact lenses (58% water content) presoaked in 1% bovine serum albumin and exposed to 10p 8 colony forming units per mL of Pseudomonas aeruginosa strain 27853, were found to reproducibly retain 5.9 (log base 10) colony-forming units. Rabbit corneas were scarified centrally with two perpendicular intersecting diamond knife cuts (5 mm × 5 mm × 0.2 mm), and bacteria-impregnated contact lenses were positioned and held in place for 24 hours with partial tarsorrhaphies. Treatment-Fourteen hours after lens removal (38 hours after infection), corneas were treated for 8 hours with collagen shields hydrated in saline (control), or shields impregnated with 800 µg gentamicin during manufacture, or one drop every 30 minutes of fortified gentamicin drops (14 mg/mL). The rabbits were killed and corneas collected for bacterial enumeration after 8 hours of treatment (46 hours after infection).

Results: Model-Slit-lamp examination and microbiologic confirmation showed uniformity of keratitis in all eyes. Treatment-Corneas treated with saline (controls) contained 6.4 (log base 10) Pseudomonas. Corneas treated with gentamicin-impregnated collagen shields (total drug = 800 µg) and fortified gentamicin drops (total drug = 21 mg) showed a reduction in viable bacteria of 2 logs and 6 logs, respectively, relative to the control.

Conclusions: In this new model of Pseudomonas keratitis, the amount of gentamicin introduced into collagen shields during manufacture effectively reduced bacterial growth in infected rabbit corneas. However, larger amounts of drug applied as fortified drops on a frequent dosing schedule were more effective by a factor of three. Treatment of keratitis with antibiotic-impregnated collagen shields may reduce the need for very frequent application of topical drops, but may be more effective with topical drop supplementation to increase the amount of drug available over the course of therapy. Refract Corneal Surg 1992;8:39-43.)

Both daily and extended wear soft contact lenses have been associated with an increased frequency of Pseudomonas aeruginosa infectious keratitis.1-4 The corneal epithelial barrier may be damaged by overnight wear of soft contact lenses, thus allowing pathogenic organisms such as Pseudomonas to establish an infection in the corneal stroma.5-7 If effective treatment is not instituted promptly, irreversible corneal scarring and even perforation may result.

The current therapeutic regimen in infectious keratitis includes the use of fortified topical antibiotic drops every 15 to 30 minutes for the first 48 hours to insure that bactericidal concentrations reach the site of infection.8,9 This regimen, however, is difficult for the patient and often requires hospitalization.

Collagen corneal shields (Bio-Cor, Bausch & Lomb, Clearwater, Fla) rehydrated in an antibiotic solution have been demonstrated to be an effective treatment modality for experimental Pseudomonas keratitis.10-13 In patients with corneal infections, the use of collagen shields to deliver antibiotics may reduce the need for frequent application of fortified antibiotics.10-13

In this study, we compared the effectiveness of gentamicin-impregnated collagen shields (800 µg of gentamicin), and frequent drops (14 mg/mL) in treating Pseudomonas keratitis in rabbits.

MATERIALS AND METHODS

New Zealand white rabbits (2 to 3 kg) were anesthetized by an intramuscular injection of a 1:5 mixture of xylazine hydrochloride (100 mg/mL; Miles Laboratories, Shawnee, Kan) and ketamine hydrochloride (100 mg/mL; Bristol Laboratories, Syracuse, NY). Two drops of proparacaine (0.5%) were instilled into each eye and nictitating membranectomies were performed. Two days later, rabbits were again anesthetized with intramuscular xylazine and ketamine, and the corneas anesthetized with topical proparacaine. Two perpendicular intersecting diamond knife cuts (5 mm × 5 mm × 0.2 mm) were made centrally in each cornea. Cuts were 0.20 mm deep and 5 mm in length. Contact lenses (Acuvue; Johnson & Johnson, Jacksonville, FIa) were soaked overnight in filter-sterilized 1% bovine serum albumin (BSA).14 The lenses were then exposed to 10p 8 P. aeruginosa strain 27853 for 1 hour at room temperature, rinsed with sterile PBS and then placed onto the cornea. Partial tarsorrhaphies were performed. Three additional Pseudomonas-soaked lenses were rinsed in PBS, placed into sterile PBS (3 mL), homogenized, and plated in triplicate to determine the number of Pseudomonas adhering to the lenses. Twenty-four hours later, animals were again anesthetized and the contact lenses were removed. The infections were allowed to progress for 12 additional hours. The eyes were rinsed with PBS, photographed, and clinical scores of corneal infection were assigned.

Clinical scoring divided the eye into zones I through III. Zone I consisted of an area 1 mm in all directions from the borders of the intersecting keratotomy incisions. Zone II extended from zone I to the midperiphery, and zone ?? extended from zone II to the limbus. Corneal haze in each zone was graded 1 through 4. Grade 1 was a faint, barely perceptible haze, and grade 4 was a totally opaque cornea. Grades 2 and 3 represent haze of increased intensity beyond grade 1 but less than grade 4.

Microbiologic confirmation of infection uniformity was previously performed by culturing infected corneas at 36 hours and quantifying viable bacteria. No significant difference among corneas was noted.

Three treatment groups were employed. Group 1 (3 rabbits, 6 eyes) received collagen shields hydrated in PBS. Group 2 (3 rabbits, 6 eyes) received gentamicin-impregnated collagen shields (800 µg gentamicin) hydrated in PBS. Group 3 (3 rabbits, 6 eyes) received fortified gentamicin drops (1.4%; 14 mg/ mL) every 30 minutes for 8 hours. All rabbits were kept anesthetized with urethane (1.5 gm/kg) during the treatment period and the eyes were taped shut to avoid shield displacement. Those rabbits receiving drops did not have their eyes taped shut. At the end of the 8-hour treatment period, all rabbits were killed with an overdose of pentobarbital sodium.

The procedure for quantifying viable bacteria per cornea has been described previously.15 Briefly, corneas were rinsed, excised, and homogenized in 3 ml of sterile PBS. An aliquot (0.5 mL) of homogenate was serially diluted and 0.1 mL of each dilution was plated in triplicate on tryptic soy agar plates. Bacterial counts were performed by a masked observer after 24 hrs of incubation at 37°C.

Results were analyzed with a Statistical Analysis Systems program.16 An analysis of variance was performed on the logarithm of the number of colonies per cornea. Where an analysis of variance showed a significant difference, t-tests between least square means from each treatment group were performed. Colony counts per cornea are expressed in base 10 logarithms ± SEM.

RESULTS

The contact lenses (Acuvue®) that were applied to the cornea after 1 hour of exposure to 10p 8 Pseudomonas aeruginosa contained 5.86 ± 0.07 Gog base 10) CFU. Slit-lamp examinations and photographs of each cornea were performed at 0 and 39 hours postinfection (PI). Prior to incision, all corneas were normal. At 39 hours PI, all eyes showed a purulent exudate, severe conjunctivitis, and a fulminant ulcerative keratitis centered on the linear incisions. Ten corneas showed keratitis extending to the midperiphery with penetration into the midstroma (zone II), and seven corneas demonstrated keratitis within 1 mm of the keratotomy incisions (zone I). One cornea was noted to have lost the contact lens and was excluded from the study. The eyes were then randomly assigned to three treatment groups, insuring that each treatment group had approximately equal representation with corneas demonstrating infection in zones I and II. We have previously shown in unpublished data, however, that the corneas at 36 hours irrespective of clinical zones have similar bacterial counts and are not statistically different.

Following 8 hours of treatment (47 hour PI), bacterial counts were determined for all corneas (Figure). Gentamicin-impregnated collagen shields applied to corneas for 8 hours reduced the number of CFUs in corneas by approximately 2 logs compared with corneas receiving shields soaked in only saline (P < .0001). Gentamicin drops (1.4%) applied to corneas every 30 minutes for 8 hours reduced the number of CFUs by 6 logs compared with corneas receiving shields soaked in only saline (P < .0001). In this experiment, antibiotic-soaked collagen shields were less effective than frequent antibiotic drops in reducing the number of CFUs in the cornea (P < .0001).

DISCUSSION

To achieve a therapeutic effect, a sufficient concentration of antibiotic must reach the microorganisms at the site of infection. Contact time, lipid solubility, and concentration are important factors in determining penetration of a compound from the surface of the eye into the corneal stroma.17 Recently, the corneal collagen shield has been introduced as a promising drug delivery system.18

The collagen shield is a thin membrane made of porcine or bovine scleral collagen that conforms to the shape of the eye and dissolves over time. Shields, hydrated in antibiotic solution or made with antibiotic incorporated into the collagen matrix (currently manufactured and under investigation by Bausch & Lomb, Clearwater, FIa), act to prolong the contact time of the drug with the corneal surface. This improves drug penetration into the cornea and anterior chamber.19 It has been shown in an intrastromal model of Pseudomonas keratitis in the rabbit that a collagen shield hydrated in 4.0% tobramycin was as effective as 4.0% tobramycin drops in reducing the number of colony-forming units of bacteria in the cornea.12 The drops were applied every 30 minutes for 4 hours while only one shield per eye was used.

Several models of Pseudomonas keratitis have been developed previously and employed to evaluate antibiotics and drug delivery systems.20,26 Previous animal models of Pseudomonas keratitis have induced keratitis by introducing epithelial defects,20 trephining into the stroma,21 injecting microorganisms into the cornea,12,13,22,23 or scratching the cornea.24-25 Others have induced keratitis without direct manipulation of the cornea by inducing hypoxic states with tarsorrhaphies prior to the placement of Pseudomonas-contairang contact lenses.26-27

These models are not ideal because they either do not mimic the type of corneal trauma associated with human keratitis or lack precision in the delivery of a known amount of bacteria to a reproducible area of injury. Our model more fully addresses the former, but is at best just as good as the intrastromal model in the exactness of bacterial delivery.

Our keratotomy model employs a reproducible corneal injury introduced with a diamond knife. Then, a known quantity of bacteria is placed over the site of the keratotomy in a precise manner with the Pseudomonas-contaimng contact lenses. The quantity delivered to the cornea, however, may not be as precise as the intrastromal injection of a known quantity of bacteria. Finally, tarsorrhaphies are employed for 24 hours while the Pseudomonas-containing contact lenses are in place. This induces a moderate hypoxic state, thus further injuring corneal epithelium and aiding infection,26'27 as well as holding the contact lens in place. Of note, hypoxia may have been induced during the treatment phase in those eyes which were taped (those with collagen shields) and may have influenced the amount of epithelial damage in those eyes versus eyes which received drops only and were not taped. The hypoxic condition may impair aminoglycoside uptake, as well as reduce bacterial growth and associated susceptibility to antibiotic action.

Figure: Efficacy of gentamicin-impregnated shields (800 µg gentamicin) relative to gentamicin drops (1.4% every 30 minutes) and control (shield hydrated in PBS). Treatment modalities were performed (for 8 hours) on eyes previously keratotomized and infected (for 38 hours) with a known quantity of Pseudomonas.

Figure: Efficacy of gentamicin-impregnated shields (800 µg gentamicin) relative to gentamicin drops (1.4% every 30 minutes) and control (shield hydrated in PBS). Treatment modalities were performed (for 8 hours) on eyes previously keratotomized and infected (for 38 hours) with a known quantity of Pseudomonas.

The ability of a contact lens to bind bacteria in a quantitative fashion is well documented.14,28-30 The bacterial binding of the contact lens is enhanced by presoaking the contact lens in bovine serum albumin (1%). Butrus et al14 have previously shown that presoaking contact lenses in BSA increased the adherence of Pseudomonas aeruginosa to the contact lens by over 300%.

Epithelial damage seems to be a prerequisite for Pseudomonas aeruginosa adherence to the cornea 24,31-35 -phe depth of injury (0.2 mm) in our study exposes corneal stroma to the Pseudomonas bacteria. Pseudomonas aeruginosa has been reported to be more adherent to corneal stroma than to corneal epithelium,20 although Stern et al36 found the opposite to be true. In our model, the adherence was sufficient to produce a fulminating keratitis in all corneas.

This study demonstrates that gentamicinimpregnated collagen shields produced a significant reduction in bacteria, yet this therapy was less effective than 1.4% gentamicin drops administered every 30 minutes. The calculated total quantity of gentamicin contained in a collagen shield was 800 µg. This was less than V20 of the total quantity (21 mg) applied to the cornea using 1.4% drops every 30 minutes for 8 hours. This suggests that gentamicin therapy using impregnated shields should be supplemented, perhaps by changing the shield at least once during the period of treatment or by applying gentamicin drops to the shield.

Differences between drops and shields in efficacy for Pseudomonas keratitis were not observed in studies by Hobden et al.12 They used an intrastromal injection model of Pseudomonas keratitis and showed equal effectiveness of collagen shields hydrated with 4% tobramycin and frequently applied 4% tobramycin drops. The intrastromal model differs from this keratotomy model in that the intrastromal model has only a small or no corneal defect, while the keratectomy model has both large corneal epithelial and stromal defects. These substantial corneal defects may allow increased penetration of drops into the corneal stroma. The combination of increased penetration and 20-fold excess of drug used contribute to the efficacy of the drops relative to the collagen shield.

Sawusch et al37 found collagen shields plus frequent drops (3 mg/mL every 30 minutes) to be more efficacious than frequent drops alone over a 12-hour period. Thus, both groups received the same total amount of drug, and the collagen shield appeared to enhance the effect of the drops. In our model, one group received 21 mg of drug in frequent drops and the other group received 800 µg of drug in a collagen shield with no drops. The shields were effective, but the frequent drops were more effective. However, due to the differences in the amounts of drug applied, the results of the two studies cannot be directly compared.

Future experiments should address the question of what frequency of antibiotic drops applied to collagen shields is as effective in decreasing bacterial counts as fortified antibiotic drops every 30 minutes. Clinically, this finding would have relevance in nursing care and/or home compliance if drops placed over collagen shields were as efficacious even when applied on a far less frequent schedule.

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10.3928/1081-597X-19920101-11

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