Preserved Ocular Anti-infective Works as a Combination Agent
Research findings suggest preserved gatifloxacin kills multiple microbes faster than unpreserved moxifloxacin in treatment and surgical settings.
By Terrence P. O'Brien, M.D., Baltimore

A study comparing gatifloxacin ophthalmic solution 0.3% (Zymar) preserved with 0.005% benzalkonium chloride (BAK) against nonpreserved moxifloxacin ophthalmic solution 0.5% (Vigamox) found that gatifloxacin behaved more like a combination agent in providing more rapid kill rates than moxifloxacin.

The study, carried out at the Ocular Microbiology Laboratory of the Wilmer Eye Institute in Baltimore, examined the antimicrobial efficacy of preserved gatifloxacin and nonpreserved moxifloxacin against yeasts, mold and bacteria. Note that the two agents have similar minimal inhibitory concentrations (MICs). Their differences in efficacy must, therefore, stem from differences in formulation.

Antibacterial efficacy: methods

Our research team grew seven species of Staphylococcus aureus and one species each of Staphylococcus hominis and Staphylococcus epidermidis on blood agar plates for 18 to 24 hours at 30ºC to 35ºC. We prepared inocula suspensions in saline by harvesting and diluting growth from each plate. Aliquots of moxifloxacin, gatifloxacin and a saline control were inoculated to contain 10⁵ to 10⁶ cells per milliliter. We performed all testing in a water bath set at 37ºC. Samples of test solution were removed, neutralized and assayed for viable cells at intervals of 15, 30 and 60 minutes. We then filtered diluted samples through Pall Supor filters and washed the filters with 300-mL volumes of the control. The filters were placed onto the surfaces of tryptic soy agar plates containing neutralizers, which were then incubated for a minimum of 72 hours at 30ºC to 35ºC. We then counted the colonies and recorded the data.

Antibacterial efficacy: results

For all four genera, the concentrations of colony forming units (CFUs) of S aureus were reduced more quickly when exposed to gatifloxacin 0.3% than to moxifloxacin 0.5%. This difference in kill rates occurred at all measured time points. Similar differences in kill rates occurred when gatifloxacin and moxifloxacin were tested against strains of ocular S epidermidis, S hominis and S aureus.

At 6 hours, moxifloxacin allowed more recovery of S aureus and Escherichia coli than did gatifloxacin after the same time period. At 24 hours, moxifloxacin allowed more recovery of S aureus than did gatifloxacin after the same time period. Data during these time periods represent those cells that are not in the log phase of growth during an infection; those cells are either in the stationary phase or are otherwise not dividing or synthesizing DNA. The recovery of both species at both time points for gatifloxacin was below the level of detection for the assay.

Remember that in-vitro resistance suggested by laboratory susceptibility testing does not necessarily translate to in-vivo resistance. In at least two rabbit models of gatifloxacin-resistant S aureus, aggressive treatment with gatifloxacin was sufficient to eradicate the infection.

It appears that the formulation containing BAK was responsible for this added efficacy. When you're trying to kill bacteria, the preservative gives you an additional active agent that's lacking when you use a nonpreserved anti-infective alone. The gatifloxacin/BAK combination, gives you a two-pronged hit to the bacteria. We proved that in the laboratory setting, and I think this provides additional protective activity in the clinical and surgical setting.

The Staphylococcus species are among the most well-documented causes of ocular infections. In the United States, coagulase-negative staphylococci are responsible for about 70% of postcataract surgery endophthalmitis, followed by S aureus and viridans group streptococci. Other Gram-positive and Gram-negative microorganisms are less frequently seen causes of postoperative endophthalmitis.

Candida albicans treated with gatifloxacin had minimal recovery.	Likewise, Candida guilliermondi was sensitive to gatifloxacin.
Candida tropicalis showed negligible recovery against gatifloxacin.	Cryptococcus albidus was vulnerable to moxifloxacin at 48 hours.
Slides courtesy of Rupp, et al.

Anti-yeast efficacy: methods

Another study comparing gatifloxacin 0.3% with BAK and moxifloxacin 0.5% alone against a variety of yeast isolates in vitro offered some interesting outcomes as well.

The carriage rate of Candida species in healthy individuals may be as high as 80%. So it's not surprising that Candida species occasionally can cause mycotic exogenous endophthalmitis. Candida parapsilosis has been implicated as the cause of epidemic exogenous endophthalmitis arising from the use of contaminated irrigating solution during surgery. Aspergillus species of mold have been inculpated in cases of endophthalmitis and fungal keratitis.

We took 20 strains of yeasts and grew them at 30ºC to 35ºC for 18 to 24 hours on Sabouraud dextrose agar.

Test samples of gatifloxacin and moxifloxacin were then pooled, and equal aliquots of the test samples were incubated with each test organism at an initial concentration of 1 x 10⁵ to 1 x 10⁶ CFUs per mL of test solution. The samples were neutralized and assayed at 4, 24, 48 hours and 7, 14 and 28 days to determine the number of viable CFUs per mL of test solution.

Anti-yeast efficacy: results

At 4 hours, moxifloxacin 0.5% without preservative consistently allowed more fungal recovery than gatifloxacin 0.3% with BAK for all 20 strains of yeast. At 7 days, moxifloxacin still allowed more fungal recovery in 15 of 20 strains, while in the other five strains both agents reduced fungal recovery below the detection limit of 10 CFUs per mL. Viable cells could still be recovered from 15 of 20 yeast strains treated with moxifloxacin, whereas none of the 20 yeast strains treated with gatifloxacin with BAK for 7 days were recoverable.

We concluded that gatifloxacin 0.3% with BAK kills a variety of potentially pathogenic yeasts to a greater extent than does unpreserved moxifloxacin 0.5%.

In all 20 strains, gatifloxacin produced a lower CFU per mL count within 4 hours than did moxifloxacin. The earlier time points bear greater relevance than the later ones for clinical and surgical applications.

This added benefit can reduce the potential introduction of yeasts to the eye and surrounding structures during surgery and during patient use for more extended durations.

Antifungal efficacy: methods

In a study comparing the antimicrobial preservative efficacy of gatifloxacin 0.3% preserved with BAK and unpreserved moxifloxacin 0.5% against a variety of molds, gatifloxacin killed all strains of filamentous fungi tested at room temperature to a greater extent than did the unpreserved moxifloxacin.

We took 12 fungal strains and grew them on potato dextrose agar for 1 week at room temperature. We inoculated equal aliquots of gatifloxacin 0.3% and moxifloxacin 0.5% with two samples of each test organism, one per sample, containing approximately 1 x 10⁵ to 1 x 10⁶ CFUs per mL of test solution. We filtered mycelia out of these challenge suspensions and determined the numbers of viable CFUs per mL of test solution at 24 and 48 hours, and 7, 14 and 28 days.

Antifungal efficacy: results

The moxifloxacin 0.5% solution allowed more fungal recovery than did the gatifloxacin 0.3% solution for all 12 fungal strains at all time points through day 7.

In all 12 fungal species, the gatifloxacin solution resulted in growth reductions after 24 and 48 hours that were at least 2-log (99%) greater than those produced by the moxifloxacin solution.

Eight species treated with moxifloxacin 0.5% remained at levels of at least 10⁵ CFU per mL at 24 hours; eight strains treated with gatifloxacin 0.3% were reduced to no recovery.

By 28 days, all species treated with gatifloxacin 0.3% were reduced to no recovery, while five strains treated with moxifloxacin 0.5% remained at levels of at least 3 x 10² CFU per mL.

A separate study lends credence

The Antimicrobial Preservative Efficacy Test performed by Rupp and colleagues (Microbiology Department, Allergan) used gatifloxacin and moxifloxacin against a particularly virulent mold, Aspergillus niger, at room temperature. Gatifloxacin produced a reduction in growth after 7, 14 and 28 days that was approximately 2-log (99%) greater than that of moxifloxacin.

The lower limit of detection for A niger was reached only with gatifloxacin. After 24 and 48 hours, gatifloxacin produced reductions in growth that were approximately 2-log (99.0%) to 3-log (99.9%) greater than those of unpreserved moxifloxacin. This antifungal benefit is likely to prevent ocular infection from fungal contamination of an ophthalmic medication bottle.

Important implications

The findings from these studies indicate that:

• Preserved gatifloxacin kills Staphylococcus faster than nonpreserved moxifloxacin.
• Gatifloxacin's faster anti-infective protection at body temperature compared with moxifloxacin may have important implications in a surgical setting.
• In-vivo use of gatifloxacin may suffice to overcome in-vitro bacterial resistance to gatifloxacin.

Gatifloxacin's antimold benefit at room temperature carries potential for preventing ocular infection caused by fungal contamination from a medication bottle. The findings in yeast, mold and bacteria may have important implications in surgical and treatment settings. But ultimately, the findings of more rapid bacterial killing are the most exciting and clinically relevant.

Dr. O'Brien is an internationally recognized expert in refractive surgery, anterior segment and eye infections. He practices at the External Diseases and Cornea service at Johnson Hopkins Wilmer Eye Institute.

References

Riddell IJ, McNeil SA, Johnson TM, Bradley SF, Kazanjian PH, Kauffman CA. Endogenous Aspergillus endophthalmitis: report of 3 cases and review of the literature. Medicine (Baltimore). 2002;81:311-320.

Sridhar MS, Gopinathan U, Rao GN. Fungal keratitis associated with vernal keratoconjunctivitis. Cornea. 2003;22:80-81.

Callegan MC, Ramirez R, Kane ST, et al. Antibacterial activity of the 4th-generation fluoroquinolones gatifloxacin and moxifloxacin against ocular pathogens. Adv Ther 2003;20(5):246-252.

Callegan MC, Engelbert M, Parke DW 2nd, Jett BD, Gilmore MS. Bacterial endophthalmitis: epidemiology, therapeutics, and bacterium-host interactions. Clin Microbiol Rev. 2002;15:111-124.

Mah FS, Romanowski EG, Yates KA, Kowalski RP, Gordon YJ. The successful treatment of gatifloxacin-resistant Staphylococcus aureus keratitis with gatifloxacin (Zymar) in a NZW rabbit model. OMIG. 2003.

Tungsiripat T, Sarayba MA, Kaufman MB, Sweet PM, Taban M, Carpenter TR, McDonnell PJ. Fluoroquinolone therapy in multiple-drug resistant staphylococcal keratitis after lamellar keratectomy in a rabbit model. Am J Ophthalmol 2003;136:76-81.