Fluoroquinolone antimicrobials target bacterial enzymes that are essential for DNA replication and transcription, are rapidly bactericidal, and have a concentration-dependent mechanism of action.1 They have broad-spectrum antimicrobial activity against the gram-negative and gram-positive pathogens that most frequently affect the cornea, and have favorable adverse effect profiles.2,3 Results of in vitro and in vivo studies4-6 have made ophthalmic preparations of fluoroquinolones the medications of choice for treatment of Pseudomonas aeruginosa–associated ulcerative keratitis in a variety of species.
Although controversy exists regarding the categorization of the fluoroquinolones used for treatment of ophthalmologic conditions, they may be divided into generations on the basis of their spectrum of antimicrobial activity and chemical structure. Second- and third-generation fluoroquinolones primarily bind to either the bacterial enzyme DNA gyrase or topoisomerase IV, whereas the fourth-generation agents bind to both.1 This difference in primary drug target is believed to slow the development of resistance against fourth-generation fluoroquinolones among gram-positive bacteria because 2 mutations are generally required to establish resistance.7 Compared with earlier generations, fourth-generation fluoroquinolones have an expanded spectrum of activity against gram-positive bacteria; anaerobic bacteria; mycobacteria; and species of Chlamydia, Chlamydophila, Mycoplasma, and Ureaplasma.3,8 The gram-negative bacterial spectrum of third- and fourth-generation fluoroquinolones is preserved; however, compared with earlier generations (especially ciprofloxacin), they are less active in vitro against many gram-negative bacteria, including P aeruginosa.3,9–11 Despite this reduced in vitro activity, third- and fourthgeneration fluoroquinolones may have similar activity in vivo as a result of their improved corneal penetration properties.12–14
Pseudomonas aeruginosa is a gram-negative bacterium and an opportunistic corneal pathogen in a wide variety of species.15 It has been implicated as the causative organism in 9.4% of dogs with extraocular infections and as many as 21% of dogs with ulcerative keratitis.5,16 Pseudomonas aeruginosa has evolved a multitude of diverse virulence factors and mechanisms that permit efficient infection of ocular tissues, extensive dissolution of the corneal stroma, and rapid progression of clinical signs.17–19 Despite aggressive medical and surgical treatments, P aeruginosa–associated corneal infections often result in permanent structural ocular damage and reduced visual capacity.20,21
Rapid initiation of treatment with an appropriate antimicrobial is critical to the successful resolution of P aeruginosa–associated ulcerative keratitis; however, the high frequency of intrinsic and acquired multidrug antimicrobial resistance can make the empirical selection of medications difficult.22 Additionally, the ability of some strains of P aeruginosa to invade, survive, and multiply within corneal epithelial cells may limit the effectiveness of non–cell-permeable antimicrobials (eg, highly polar aminoglycosides).23 In a recent study,a most of the strains of P aeruginosa obtained from dogs with ulcerative keratitis were capable of invading corneal epithelial cells. Thus, antimicrobials, such as the fluoroquinolones, that achieve high intracellular concentrations may be preferable for the treatment of P aeruginosa–associated ulcerative keratitis in dogs.
The purpose of the study reported here was to determine the in vitro fluoroquinolone susceptibility of P aeruginosa isolates obtained from dogs with ulcerative keratitis. Susceptibilities of P aeruginosa isolates to second-, third-, and fourth-generation fluoroquinolones were of interest, and the evaluated medications were chosen to represent a spectrum of commercially available ophthalmic preparations of various fluoroquinolones and fluoroquinolone generations. The investigation was undertaken to determine the suitability of these antimicrobials for the initial treatment of P aeruginosa–associated ulcerative keratitis in dogs, pending the results of susceptibility testing of the infective organism.
Materials and Methods
Animals and P aeruginosa isolate collection—All protocols were performed in compliance with Cornell University institutional guidelines for research on animals, and informed consent was obtained from owners of the dogs prior to sample collection. All corneal P aeruginosa isolates from dogs with ulcerative keratitis that were evaluated at the Cornell University Hospital for Animals during the 3-year study period were included in the study.
Sample collection—Corneal swab specimens for microbial culture were collected during initial evaluation of the dogs. Sterile polyester-tipped swabs, moistened with sterile saline (0.9% NaCl) solution, were brushed against ulcerated regions of the cornea and immediately inserted into transport medium.b Samples were delivered to the laboratory for processing within 1 hour of collection.
Bacteria identification—Standard microbiologic culturing methods were used. Direct cultures on solid media (trypticase soy agar with 5% sheep blood, chocolate agar, Levine eosin methylene blue agar, and Columbia colistin-nalidixic acid agar) and in enrichment broth (brain-heart infusion broth) were performed for each sample. Direct cultures and enrichment subcultures were incubated at 37°C in 6% CO2 and examined after 24 and 48 hours. All microorganism identifications were performed by use of an automated system.c Biochemical identifications were supplemented with conventional tube biochemicals (cytochrome oxidase, Pseudomonas agar F, and Pseudomonas agar P) to confirm identifications when necessary.
Fluoroquinolone susceptibility determinations— Pseudomonas aeruginosa isolates were tested in vitro for susceptibility to ciprofloxacin, ofloxacin, norfloxacin, lomefloxacin, levofloxacin, gatifloxacin, and moxifloxacin via the disk diffusion method. All susceptibility determinations were performed by the same technician (LMH) at the Cayuga Medical Center, Ithaca, NY. A standardized inoculum of each isolate (1.5 × 108 CFUs/mL) was prepared and inoculated onto Mueller-Hinton agar plates. Filter paper disks,d each impregnated with one of the antimicrobial agents, were applied to the agar surface and incubated for 18 to 24 hours at 35°C in an ambient-air incubator. The disk content of each drug used was as follows: ciprofloxacin, 5 μg; ofloxacin, 5 μg; norfloxacin, 10 μg; lomefloxacin, 10 μg; levofloxacin, 5 μg; gatifloxacin, 5 μg; and moxifloxacin, 5 μg. Following incubation, the diameters of the inhibition zones were measured to the nearest millimeter. The size of the zone of inhibition was used to generate a qualitative report of susceptible, intermediate, or resistant designation for each isolate in accordance with CLSI guidelines.24 No interpretive values were available for the interaction of moxifloxacin and P aeruginosa, so values for Enterobacteriaceae were used. Quality-control organisms (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and P aeruginosa ATCC 27853) were tested on each day of susceptibility determinations according to recommendations of the CLSI.24
Statistical analysis—Isolates were designated as susceptible, intermediate, or resistant to the various antimicrobials. Susceptibility was defined as the proportion of isolates scored as susceptible among all isolates evaluated for a particular drug or generation. Only 1 isolate for 1 drug was scored as intermediate; it was included in the resistant category in the analyses. The percentage of susceptible isolates was compared among individual fluoroquinolones and among fluoroquinolone generations by use of the Fisher exact test. For the latter comparisons, the susceptibility results of isolates for all drugs within a generation were combined to determine the susceptibility of isolates for that generation and compared across the generations. Statistical significance was defined by a value of P ≤ 0.05 for all comparisons. Because none of the comparisons were significant at the P ≤ 0.05 level, no corrections were necessary for multiple comparisons.
Results
Pseudomonas aeruginosa was isolated from 27 dogs with ulcerative keratitis during the 3-year study period. None of these dogs had received topical or systemic fluoroquinolone treatment prior to referral. Twenty-four of the 27 isolates were susceptible to all fluoroquinolones evaluated, and susceptibility percentages ranged from 88.9% to 100% for individual antimicrobials (Table 1). The percentage of susceptible isolates was highest (100% [27/27 isolates]) for ciprofloxacin and levofloxacin and lowest (88.9% [24/27 isolates]) for moxifloxacin. Among the individual fluoroquinolones or fluoroquinolone generations, there were no significant differences in the percentage of isolates that was designated as susceptible. Qualitycontrol–organism results were within reference ranges on each day of testing.
Fluoroquinolone susceptibility determinations for 27 Pseudomonas aeruginosa isolates obtained from 27 dogs with ulcerative keratitis.
| Fluoroquinolone | No. of isolates | ||
|---|---|---|---|
| Susceptible | Intermediate | Resistant | |
| Second generation | |||
| Ciprofloxacin | 27 | 0 | 0 |
| Lomefloxacin | 26 | 0 | 1 |
| Norfloxacin | 26 | 1 | 0 |
| Ofloxacin | 25 | 0 | 2 |
| Third generation | |||
| Levofloxacin | 27 | 0 | 0 |
| Fourth generation | |||
| Gatifloxacin | 26 | 0 | 1 |
| Moxifloxacin | 24 | 0 | 3 |
Discussion
By use of the CLSI standards, the in vivo efficacy of antimicrobials can be predicted from the results of in vitro susceptibility testing of microorganisms. These standards are based on anticipated serum drug concentrations that are safely achievable after administration of a given medication. Currently, no separate standards for ocular tissue concentrations are available to guide topical ophthalmic antimicrobial treatment choices. The serum standards may be used to interpret susceptibility of microorganisms to drugs in preparations for topical ocular use; however, it is generally anticipated that corneal tissue concentrations of antimicrobials would be equal to or greater than serum concentrations.25 Consequently, resistance of an organism to a drug in vitro may occasionally be overcome by high corneal antimicrobial concentrations, thereby potentially resulting in an underestimation of the percentage of isolates susceptible to a given drug in vivo.26 Nevertheless, results of in vitro testing of bacterial isolates for susceptibility to fluoroquinolones are known to have predictive value for the therapeutic response rate among humans with bacterial keratitis.27
The emergence of fluoroquinolone-resistant strains of P aeruginosa in human and veterinary medicine has increased in the last few years.28–31 The primary mechanisms of P aeruginosa resistance to fluoroquinolones are modifications in the target enzymes by point mutations in genes for subunits of DNA gyrase and topoisomerase IV, decreased bacterial permeability to the antimicrobial as a result of decreased amounts of outer membrane porin proteins, and overexpression of efflux pumps that actively reduce intracellular antimicrobial concentrations.32,33 Many fluoroquinolone-resistant strains of P aeruginosa have cross-resistance to structurally unrelated antimicrobials because of the broad substrate specificity of the efflux pumps.34,35
Among ocular bacterial isolates, resistance to fluoroquinolones was first detected in gram-positive bacteria isolated from the eyes of humans, partially prompting the development of the fourth-generation fluoroquinolones for ophthalmic use.36,37 Since that time, an increasing prevalence of ocular fluoroquinolone-resistant P aeruginosa isolates in humans has also been described, including strains that are resistant to all currently available generations of fluoroquinolones.38–43 Limited information is available regarding fluoroquinolone resistance among ocular isolates obtained from dogs. However, in a recent study,5 7% of P aeruginosa isolates obtained from dogs with bacterial keratitis were resistant in vitro to ciprofloxacin and no pattern of increased resistance was detected over a 10-year period. Presently, it is unclear whether this information can be extrapolated to other canine populations, as dramatically different susceptibility rates have been reported for isolates obtained from humans in different geographic regions.41,44,45 In the present study, the prevalence of fluoroquinolone resistance was low among the canine ocular P aeruginosa isolates evaluated.
In veterinary medicine, ciprofloxacin is often the sole fluoroquinolone evaluated in susceptibility testing; from those results, assumptions regarding other fluoroquinolone susceptibilities are made.28 It is known that ciprofloxacin does not adequately assess the in vitro susceptibility of P aeruginosa to many other fluoroquinolones, and there is no apparent correlation of the susceptibility of canine P aeruginosa isolates to ciprofloxacin with susceptibilities to other fluoroquinolones used in veterinary medicine.28,32 These results suggest that caution should be exercised in extrapolating the results of susceptibility testing for 1 fluoroquinolone to another because some isolates could be susceptible to 1 drug but resistant to others.32 This may be of particular importance for the extrapolation of susceptibility testing results for ciprofloxacin to later generations of fluoroquinolones, which may have comparatively less antimicrobial activity against P aeruginosa.3,9,10,28,32
Case selection bias may have been introduced into the present study by the increasingly widespread use of ophthalmic fluoroquinolone preparations (almost exclusively ciprofloxacin) by the hospital's veterinarian referral base. This practice could have favored selection of dogs with ulcerative keratitis associated with more resistant strains of P aeruginosa for inclusion in the study because the canine patient population at the Cornell University Hospital for Animals is largely based on referrals. If this did occur, it could be anticipated that an even higher proportion of isolates would have been susceptible to the evaluated fluoroquinolones because dogs with ulcerative keratitis associated with fluoroquinolone-susceptible P aeruginosa strains that were receiving these medications would be more likely to have negative culture results.
Although there is no substitute for laboratory assessment of the antimicrobial susceptibility profile of an infective organism, the results of the present study have suggested that administration of the evaluated fluoroquinolones for empirical antimicrobial treatment of confirmed or suspected P aeruginosa–associated ulcerative keratitis in dogs has a high probability of success. The increased usage of the fluoroquinolones, however, has been correlated with increased resistance of P aeruginosa to these medications; thus, judicious and appropriate treatment of severe infections with confirmed or suspected susceptible organisms is recommended.46 To counter the development of resistance to fluoroquinolones in ophthalmic preparations, a dosing strategy that achieves a high drug concentration at the site of infection for only as long as necessary to eradicate the bacteria should be applied. Long-term, insufficient, or tapering dosing regimens may contribute to the development of resistance and should be avoided.47,48
ABBREVIATIONS
| CLSI | Clinical and Laboratory Standards Institute |
| ATCC | American Type Culture Collection |
Ledbetter EC, Kern TJ, Riis RC, et al. Pathogenic phenotype and genotype of canine ocular Pseudomonas aeruginosa isolates (abstr), in Proceedings. 37th Annu Meet Am Coll Vet Ophthalmol 2006;42.
BBL Port-A-Cul tube, Becton, Dickinson & Co, Sparks, Md.
Sensititre, Trek Diagnostic Systems Inc, Cleveland, Ohio.
BBL Sensi-Disc antimicrobial susceptibility test discs, Becton, Dickinson & Co, Sparks, Md.
References
- 2
McGee DH, Holt WF, Kastner PR, et al. Safety of moxifloxacin as shown in animal and in vitro studies. Surv Ophthalmol 2005;50:S46–S54.
- 3
Kowalski RP, Dhaliwal DK, Karenchak LM, et al. Gatifloxacin and moxifloxacin: an in vitro susceptibility comparison to levofloxacin, ciprofloxacin, and ofloxacin using bacterial keratitis isolates. Am J Ophthalmol 2003;136:500–505.
- 4
Guzek JP, Chacko D, Kettering JD, et al. Comparison of topical ciprofloxacin to conventional antibiotic therapy in the treatment of experimental Pseudomonas aeruginosa keratitis. Cornea 1994;13:500–504.
- 5↑
Tolar EL, Hendrix DV, Rohrbach BW, et al. Evaluation of clinical characteristics and bacterial isolates in dogs with bacterial keratitis: 97 cases (1993–2003). J Am Vet Med Assoc 2006;228:80–85.
- 6
Sauer P, Andrew SE, Lassaline M, et al. Changes in antibiotic resistance in equine bacterial ulcerative keratitis (1991–2000): 65 horses. Vet Ophthalmol 2003;6:309–313.
- 7↑
Ince D, Hooper DC. Mechanisms and frequency of resistance to gatifloxacin in comparison to AM-1121 and ciprofloxacin in Staphylococcus aureus. Antimicrob Agents Chemother 2001;45:2755–2764.
- 8
Fung-Tomc J, Minassian B, Kolek B, et al. In vitro antibacterial spectrum of a new broad-spectrum 8-methoxy fluoroquinolone, gatifloxacin. J Antimicrob Chemother 2000;24:437–446.
- 9
Saravolatz LD, Leggett J. Gatifloxacin, gemifloxacin, and moxifloxacin: the role of 3 newer fluoroquinolones. Clin Infect Dis 2003;37:1210–1215.
- 10
Blondeau JM, Laskowski R, Bjarnason J, et al. Comparative in vitro activity of gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin and trovafloxacin against 4151 gram-negative and gram-positive organisms. Int J Antimicrob Agents 2000;14:45–50.
- 11
Kowalski RP, Pandya AN, Karenchak LM, et al. An in vitro resistance study of levofloxacin, ciprofloxacin, and ofloxacin using keratitis isolates of Staphylococcus aureus and Pseudomonas aeruginosa. Ophthalmology 2001;108:1826–1829.
- 12
Thibodeaux BA, Dajcs JJ, Caballero AR, et al. Quantitative comparison of fluoroquinolone therapies of experimental gram-negative bacterial keratitis. Curr Eye Res 2004;28:337–342.
- 13
Jensen H, Zerouala C, Carrier M, et al. Comparison of ophthalmic gatifloxacin 0.3% and ciprofloxacin 0.3% in healing of corneal ulcers associated with Pseudomonas aeruginosa-induced ulcerative keratitis in rabbits. J Ocul Pharmacol Ther 2005;21:36–43.
- 14
Robertson SM, Curtis MA, Schlech BA, et al. Ocular pharmaco-kinetics of moxifloxacin after topical treatment in animals and humans. Surv Ophthalmol 2005;50 (suppl 1):S32–S45.
- 15↑
Kreger AS. Pathogenesis of Pseudomonas aeruginosa ocular diseases. Rev Infect Dis 1983;5 (suppl 5):931–935.
- 16
Gerding PA Jr, McLaughlin SA, Troop MW. Pathogenic bacteria and fungi associated with external ocular disease in dogs: 131 cases (1981–1986). J Am Vet Med Assoc 1988;193:242–244.
- 17
Fleiszig SM, Evans DJ. The pathogenesis of bacterial keratitis: studies with Pseudomonas aeruginosa. Clin Exp Optom 2002;85:271–278.
- 18
Wyman M, Swanson C, Kowalski JJ, et al. Experimental Pseudomonas aeruginosa ulcerative keratitis model in the dog. Am J Vet Res 1983;44:1135–1140.
- 19
Van Horn DL, Davis SD, Hyndiuk RA, et al. Experimental Pseudomonas keratitis in the rabbit: bacteriologic, clinical, and microscopic observations. Invest Ophthalmol Vis Sci 1981;20:213–221.
- 20
Sweeney CR, Irby NL. Topical treatment of Pseudomonas sp–infected corneal ulcers in horses: 70 cases (1977–1994). J Am Vet Med Assoc 1996;209:954–957.
- 21
Kroll P, Busse H. A case of total keratomalacia due to soft contact lens wearing. Ann Ophthalmol 1980;12:1374–1376.
- 22↑
Bonomo RA, Szabo D. Mechanisms of multidrug resistance in Acinetobacter species and Pseudomonas aeruginosa. Clin Infect Dis 2006;43:S49–S56.
- 23↑
Lee EJ, Truong TN, Mendoza MN, et al. A comparison of invasive and cytotoxic Pseudomonas aeruginosa strain-induced corneal disease responses to therapeutics. Curr Eye Res 2003;27:289–299.
- 24↑
National Committee for Clinical Laboratory Standards. In:Performance standards for antimicrobial disk susceptibility tests; approved standard M2-A. 9th ed. Wayne, Pa: Clinical and Laboratory Standards Institute, 2006;1–35.
- 25↑
Kowalski RP, Yates KA, Romanowski EG, et al. An ophthalmologist's guide to understanding antibiotic susceptibility and minimum inhibitory concentration data. Ophthalmology 2005;112:1987e1–1987e6.
- 26↑
Romanowski EG, Mah FS, Yates KA, et al. The successful treatment of gatifloxacin-resistant Staphylococcus aureus keratitis with Zymar (gatifloxacin 0.3%) in a NZW rabbit model. Am J Ophthalmol 2005;139:867–877.
- 27↑
Wilhelmus KR, Abshire RL, Schlech BA. Influence of fluoroquinolone susceptibility on the therapeutic response of fluoroquinolone-treated bacterial keratitis. Arch Ophthalmol 2003;121:1229–1233.
- 28↑
Riddle C, Lemons CL, Papich MG, et al. Evaluation of ciprofloxacin as a representative of veterinary fluoroquinolones in susceptibility testing. J Clin Microbiol 2000;38:1636–1637.
- 29
Martin Barrasa JL, LupiolaGomez P, Gonzalez Lama Z, et al. Antibacterial susceptibility patterns of Pseudomonas strains isolated from chronic canine otitis externa. J Vet Med B Infect Dis Vet Public Health 2000;47:191–197.
- 30
Polk RE, Johnson CK, McClish D, et al. Predicting hospital rates of fluoroquinolone-resistant Pseudomonas aeruginosa from fluoroquinolone use in US hospitals and their surrounding communities. Clin Infect Dis 2004;39:497–503.
- 31
Fridkin SK, Hill HA, Volkova NV, et al. Temporal changes in prevalence of antimicrobial resistance in 23 US hospitals. Emerg Infect Dis 2002;8:697–701.
- 32↑
Tejedor MT, Martin JL, Navia M, et al. Mechanisms of fluoroquinolone resistance in Pseudomonas aeruginosa isolates from canine infections. Vet Microbiol 2003;94:295–301.
- 33
Hooper DC. Emerging mechanisms of fluoroquinolone resistance. Emerg Infect Dis 2001;7:337–341.
- 34
Kriengkauykiat J, Porter E, Lomovskaya O, et al. Use of an efflux pump inhibitor to determine the prevalence of efflux pump-mediated fluoroquinolone resistance and multidrug resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 2005;49:565–570.
- 35
Thomas LE, Couetdic G, Clermont O, et al. In vivo selection of a target/efflux double mutant of Pseudomonas aeruginosa by ciprofloxacin therapy. J Antimicrob Chemother 2001;48:553–555.
- 36
Alexandrakis G, Alfonso EC, Miller D. Shifting trends in bacterial keratitis in South Florida and emerging resistance to fluoroquinolones. Ophthalmology 2000;107:1497–1502.
- 37
Goldstein MH, Kowalski RP, Gordon YJ. Emerging fluoroquinolone resistance in bacterial keratitis: a 5-year review. Ophthalmology 1999;106:1313–1318.
- 38
Garg P, Sharma S, Rao GN. Ciprofloxacin-resistant Pseudomonas keratitis. Ophthalmology 1999;106:1319–1323.
- 39
Rhee MK, Kowalski RP, Romanowski EG, et al. A laboratory evaluation of antibiotic therapy for ciprofloxacin-resistant Pseudomonas aeruginosa. Am J Ophthalmol 2004;138:226–230.
- 40
Moshirfar J, Mirzaizn G, Feiz V, et al. Fourth-generation fluoroquinolone-resistant bacterial keratitis after refractive surgery. J Cataract Refract Surg 2006;32:515–518.
- 41
Chaudry NA, Flynn HW, Murray TG, et al. Emerging ciprofloxacin-resistant Pseudomonas aeruginosa. Am J Ophthalmol 1999;128:509–510.
- 42
Epstein SP, Bottone EJ, Asbell PA. Susceptibility testing of clinical isolates of Pseudomonas aeruginosa to levofloxacin, moxifloxacin, and gatifloxacin as a guide to treating Pseudomonas ocular infection. Eye Contact Lens 2006;32:240–244.
- 43
Song A, McCulley TJ, Lam BL, et al. Pseudomonas aeruginosa in vitro corneal isolate sensitivity to ofloxacin, ciprofloxacin, and trovafloxacin: a comparative study. Am J Ophthalmol 2001;131:795–796.
- 44
Kunimoto DY, Sharma S, Garg P, et al. In vitro susceptibility of bacterial keratitis pathogens to ciprofloxacin: emerging resistance. Ophthalmology 1999;106:80–85.
- 45
Lomholt JA, Kilian M. Ciprofloxacin susceptibility of Pseudomonas aeruginosa isolates from keratitis. Br J Ophthalmol 2003;87:1238–1240.
- 46↑
Hsu DI, Okamoto MP, Murthy R, et al. Fluoroquinolone-resistant Pseudomonas aeruginosa: risk factors for acquisition and impact on outcomes. J Antimicrob Chemother 2005;55:535–541.
- 47
Burgess DS. Use of pharmokinetics and pharmacodynamics to optimize antimicrobial treatment of Pseudomonas aeruginosa infections. Clin Infect Dis 2005;40:S99–S104.
- 48
Nakano M, Yasuda M, Yokoi S, et al. In vivo selection of Pseudomonas aeruginosa with decreased susceptibilities to fluoroquinolones during fluoroquinolone treatment of urinary tract infection. Urology 2001;58:125–128.
