Susceptibility of Pseudomonas Isolates From the Ears and Skin of Dogs to Enrofloxacin, Marbofloxacin, and Ciprofloxacin

Introduction

Pseudomonas spp. are ubiquitous, highly resistant, saprophytic, gram negative bacilli that are commonly isolated from dogs with chronic otitis and occasionally from cases of pyoderma.^1–3 Fluoroquinolone antibiotics have been extensively utilized and studied in Pseudomonas spp. infections of humans and animals, with enrofloxacin, marbofloxacin, and ciprofloxacin showing in vitro efficacy.^4–10 In humans, fluoroquinolone resistance emerged quickly, because a single mutation led to minimum inhibitory concentration (MIC) levels that were unreachable in serum.¹¹ In the United States, enrofloxacin is an older, third-generation fluoroquinolone; ^a marbofloxacin is a newer, third-generation fluoroquinolone;^b and ciprofloxacin is a second-generation fluoroquinolone that is currently licensed for use in humans.¹⁰ While previous studies have evaluated the susceptibility of Pseudomonas spp. to one or more fluoroquinolones, none have evaluated sensitivities of ear and skin isolates separately to enrofloxacin, marbofloxacin, and ciprofloxacin.^4–8 Additionally, none of the prior studies investigated the susceptibility of resistant Pseudomonas spp. isolates to fluoroquinolones, and many Pseudomonas spp. infections in dogs have already been treated with and may be resistant to one or more fluoroquinolones, especially in cases of chronic otitis externa.

The purposes of this study were to determine the susceptibilities of ear and skin isolates of Pseudomonas spp. to the three aforementioned fluoroquinolones, to compare susceptibility patterns between ear and skin samples, and to compare susceptibility patterns of ear isolates resistant to fluoroquinolones.

Materials and Methods

The database of a local veterinary diagnostic laboratory was searched retrospectively for canine ear and skin culture submissions received from the Animal Dermatology Clinic in San Diego, California, between April 2004 and October 2005. Submissions that grew Pseudomonas spp. from dogs diagnosed with chronic otitis externa and pyoderma were included in the analysis. Repeated cultures from the same dog were excluded from the study. No dogs had cultures included from both the ear and skin. Breed, age, gender, diseases predisposing to infection, and site cultures were recorded for each dog. Determination of previous fluoroquinolone exposure was attempted for isolates resistant to fluoroquinolones.

Samples were obtained by insertion of a sterile swab^c into the horizontal and/or vertical ear canal for otic cultures. The swab was pressed or rolled against the skin for skin cultures. Samples were delivered to the laboratory within 24 hours, and specimens were inoculated onto blood and MacConkey sorbitol agar plates and evaluated daily. Pseudomonas spp. were identified using microplate^d and biochemical testing.¹² If no growth was evident after 72 hours, plates were discarded. Sensitivity testing was performed,^e,f and isolates were classified as sensitive, intermediate, or resistant according to National Committee for Clinical Laboratory Standards (NCCLS) guidelines.¹³ For enrofloxacin, a zone of inhibition ≥20 mm was considered sensitive; 14 to 16 mm was deemed intermediate; and ≤13 mm was considered resistant. For marbofloxacin, a zone of inhibition ≥17 mm was considered sensitive; 14 to 19 mm was intermediate; and ≤13 mm was resistant. For ciprofloxacin, a zone of inhibition ≥21 mm was considered sensitive; 16 to 20 mm was intermediate; and ≤15 mm was resistant.¹³

Results

Isolates of Pseudomonas (P.) aeruginosa were cultured from the external ears (n=32) of 30 dogs. At the time of culture, dogs ranged in age from 1.0 to 15 years (mean 7.8±3.1 years). One dog was adopted as an adult, so the age was unknown. There were 17 males (14 castrated) and 13 females (12 spayed). Predisposing causes of otitis externa included atopic dermatitis (n=9), atopic dermatitis and adverse food reaction (n=1), foreign body in the horizontal ear canal (n=1), and hypothyroidism (n=1).

Sensitivity to marbofloxacin was undetermined for two isolates. Of the 30 ear isolates for which all three fluoroquinolones were evaluated, 21 (70%) had identical sensitivity patterns. Fifteen (50%) were susceptible to all three fluoroquinolones, and six (20%) were resistant to all three antibiotics [Table 1]. Ear isolates were significantly less susceptible to enrofloxacin than to ciprofloxacin (P=0.021). No significant differences were seen in susceptibility of ear isolates to enrofloxacin when compared to marbofloxacin or when marbofloxacin was compared to ciprofloxacin.

Fifteen ear isolates were resistant to at least one of the three fluoroquinolones [Table 2]. These resistant ear isolates were significantly less susceptible to enrofloxacin, as compared to ciprofloxacin (P<0.001) and marbofloxacin (P=0.014). No significant difference was seen in susceptibility of resistant isolates to marbofloxacin when compared to ciprofloxacin. Previous fluoroquinolone exposure was known for 10 dogs with fluoroquinolone-resistant isolates, with eight previously exposed to topical enrofloxacin, seven exposed to oral enrofloxacin, and two exposed to oral marbofloxacin. Ear isolates resistant to both marbofloxacin and ciprofloxacin were found despite no previous exposure to these antibiotics.

Pseudomonas aeruginosa (n=17), unidentified Pseudomonas spp. (n=3), and P. fluorescens (n=1) were cultured from the skin of 18 dogs. At the time of skin culture, the dogs ranged in age from 1 to 14 years (mean 7.4±3.2 years). There were 11 males (10 castrated) and seven spayed females. Infections from which isolates were cultured included superficial pyoderma (n=5), deep pyoderma (n=3), pododermatitis (n=3), perivulvar dermatitis (n=3), lip fold pyoderma (n=2), neck fold pyoderma (n=1), and tail fold pyoderma (n=1). Predisposing diseases identified in the dogs included atopic dermatitis (n=7), generalized demodicosis (n=2), hepatocutaneous syndrome (n=1), and atopic dermatitis with adverse food reaction (n=1).

Sensitivity to ciprofloxacin was undetermined for one isolate. Of the 20 skin isolates for which all three fluoroquinolones were evaluated, 17 (85%) had identical sensitivity patterns, with 14 (70%) susceptible to all three fluoroquinolones and three (15%) resistant to all three antibiotics [Table 1]. Six samples showed fluoroquinolone resistance, but the subset was considered too small for further analysis. There were no significant differences in susceptibility patterns between any of the fluoroquinolones.

When the susceptibilities of ear isolates were compared to susceptibilities of skin isolates for each fluoroquinolone, the ear isolates were significantly less susceptible to enrofloxacin (P=0.034) than were the skin isolates. No significant differences were seen between the ear and skin samples in their susceptibilities to marbofloxacin or ciprofloxacin.

Discussion

Geographical variations in fluoroquinolone susceptibility have been documented in humans, possibly from drug availability and usage.^14,15 Susceptibility of ear isolates to enrofloxacin in the study reported here (46.9%) was higher than that found by Cole et al. in both middle ear (35%) and horizontal ear canal isolates (12.5%) from dogs with otitis media.¹⁶ Susceptibility was lower, however, than that found in prior studies (51% to 71%) of dogs with chronic otitis externa.^4–6

In the current study, susceptibility of the ear isolates to marbofloxacin (66.7%) was the lowest reported from any region to date, and it was noticeably lower than prior reported susceptibilities (91.3% to 93.4%).^4,5 Similarly, susceptibility of ear isolates to ciprofloxacin (75.0%) was also the lowest reported to date, with prior reports ranging from 78.3% to 93.4%.^4,5,7 Interestingly, a recent study of marbofloxacin susceptibility of Pseudomonas from otitis and dermatological infections in Europe from 1994 to 2001 reported no increase in resistance over time, and Peterson et al. concluded there was no significant change in ciprofloxacin susceptibility from 1992 to 1997 in isolates from dogs with otitis and pyoderma.^7,8 The lower susceptibility to marbofloxacin and ciprofloxacin documented in the current study suggests that susceptibility of Pseudomonas spp. to these latter two fluoroquinolones must be closely monitored, especially as they become more commonly prescribed by veterinarians.

When the susceptibilities of the ear isolates were compared for each of the three fluoroquinolones in this study, isolates were significantly less susceptible to enrofloxacin than to ciprofloxacin. This resistance to enrofloxacin may have developed because it is approved and widely used in both oral and topical formulations in dogs. In addition to the commercially available formulation of enrofloxacin, offlabel otic preparations are also produced using injectable enrofloxacin.⁹ Conversely, otic and systemic uses of ciprofloxacin are not approved for dogs. No commercial otic preparations containing marbofloxacin are available in the United States, and although oral marbofloxacin is now available, it has not been on the market as long as enrofloxacin.¹⁰ Results of the study suggest that marbofloxacin and ciprofloxacin may be better antibiotic selections to treat fluoroquinolone-resistant P. aeruginosa than enrofloxacin (at least in vitro).

Fluoroquinolone-resistant isolates in this study were resistant to one, two, or all three fluoroquinolones. Potential mechanisms of resistance include mutation in the gyrA subunit of DNA gyrase (the enzyme targeted by fluoroquinolones) and the presence of substrate-specific multidrug efflux pumps, of which a single pump can have differing fluoroquinolone preference.^11,17 Pseudomonas spp. utilizing the MexAB-Opr efflux pump may eliminate enrofloxacin more readily than marbofloxacin and ciprofloxacin, because enrofloxacin is more lipophilic.¹⁸ Whether this mechanism played a role in the fluoroquinolone resistance found in the current study was undetermined.

When comparing ear isolates to skin isolates for each fluoroquinolone, ear isolates were significantly less susceptible to enrofloxacin. This difference in susceptibility may have arisen because otitis externa is treated with enrofloxacin more frequently than is pyoderma; however, the reason for locational differences in susceptibilities has not been elucidated. These differences in susceptibility between body locations were similar to findings in a prior study where otic isolates of P. aeruginosa were significantly less susceptible to gentamicin and amikacin as compared to skin isolates.⁷

No statistically significant differences in susceptibility were found between fluoroquinolones for the skin isolates, possibly because skin isolates are treated less frequently with fluoroquinolones as compared to cases of chronic otitis externa. Similar to the findings for ear isolates, the susceptibility of skin isolates to ciprofloxacin was lower in this study (80%) than the susceptibility found previously (95%).⁷

Limitations of the current study included its retrospective nature, which made determination of fluoroquinolone exposure for all dogs unattainable. Susceptibility data also were acquired by the disc diffusion method, which gives a conservative interpretation of sensitivity as compared to MIC testing.⁶

It is important to note that the Pseudomonas spp. isolated in this study were from cases of chronic otitis externa and pyoderma examined at a referral hospital. Acute cases of Pseudomonas otitis or pyoderma in general practice may be more susceptible to these same fluoroquinolones. Additionally, in vitro susceptibility does not always equate to in vivo activity. Because P. aeruginosa isolated from chronic otitis externa may have variable susceptibilities, fluoroquinolone selection should be based on culture and sensitivity testing that includes enrofloxacin, marbofloxacin, and ciprofloxacin. Also, products approved for this use in dogs should be used whenever possible.^19,20 Pseudomonas spp. are also opportunistic invaders, so every effort must be made to concurrently diagnose and treat underlying diseases and to correct secondary pathological changes of the ear and skin, in order to minimize antibiotic usage and potentially lower resistance rates.

Conclusion

Pseudomonas aeruginosa isolates from dogs with chronic otitis externa were significantly more susceptible to ciprofloxacin than enrofloxacin, and fluoroquinolone resistant ear isolates were significantly more susceptible to both marbofloxacin and ciprofloxacin than enrofloxacin. Pseudomonas spp. isolated from the skin of dogs with pyoderma showed no significant differences in fluoroquinolone susceptibility. Additional prospective studies evaluating prior topical and systemic antibiotic exposure are needed to further characterize and monitor fluoroquinolone resistance in dogs with Pseudomonas spp. infections.

Acknowledgments

Thanks to Edward Cogger, PhD, Professor Emeritus, California State Polytechnic University in Pomona, California, for providing statistical analysis.