Evaluation in Vitro of the Antimicrobial Activity of Two Topical Preparations Used in the Management of Ear Infections in the Dog*

Transcription

1 Evaluation in Vitro of the Antimicrobial Activity of Two Topical Preparations Used in the Management of Ear Infections in the Dog* David H. Lloyd, PhD, B Vet Med, FRCVS, Diplomate ECVD Anne I. Lamport, BA, AIBMS Dermatology Unit Department of Small Animal Medicine and Surgery Royal Veterinary College (University of London) Hawkshead Campus North Mymms, Hertfordshire AL9 7TA, UK *This study was sponsored by Virbac, France. a Both products were from the Virbac European range. Epi-Otic NF in this study is equivalent to Epi-Otic in the United States. ABSTRACT Products designed for ear cleansing (i.e., Epi-Otic and Epi-Otic New Formula [NF] a ) were evaluated in vitro for their ability to kill clinical isolates of Staphylococcus intermedius, Pseudomonas aeruginosa, and the yeast Malassezia pachydermatis. Dilutions of the products at 1/5 and 1/25 in distilled water were inoculated with suspensions of the organisms and held at room temperature. The mixtures were sampled at intervals between 1 and 16 minutes, and numbers of viable colonyforming units of the microorganisms were determined by culture on blood agar or modified Dixon s agar (for the yeast) using the spreadplate method. At a dilution of 1/5, both products were able to eliminate S. intermedius within 1 minute. P. aeruginosa was eliminated within 2 minutes by both products. M. pachydermatis was eliminated by Epi-Otic NF and Epi-Otic within 4 and 8 minutes, respectively. Lower activity was demonstrated following dilution at 1/25. The findings support previously published data for Epi-Otic indicating antimicrobial activity both in vitro and in vivo. The findings suggest that Epi-Otic may be useful in the control of ear infections. INTRODUCTION Superficial infections of the ear canal with bacteria such as Staphylococcus intermedius and Pseudomonas aeruginosa and the yeast Malassezia pachydermatis are significant factors in otitis externa. 1 3 Often, mixed infections occur, necessitating the employment of treatments that are effective against more than one of these organisms. Selection of appropriate products depends on a knowledge not only of their antimicrobial activity but also of the processes of the diseases and the factors underlying them. 4 7 Topical therapy is an important component of the management of such conditions. Lloyd, Bond, and Lamport 8 have shown that the ear cleanser Epi-Otic (Virbac Corporation) has potent activity in vitro against otic pathogens and that this activity is also reflected by studies in vivo. The current study was designed to 43

2 Veterinary Therapeutics Vol. 1, No. 1, Winter 2000 compare the activity in vitro of Epi- Otic and Epi-Otic NF products against significant pathogens. Only the antibacterial and antifungal activity in vitro of the products as formulated has been assessed. MATERIALS AND METHODS Two randomly selected isolates from canine referral cases with otic infections of each of the following microorganisms were used: S. intermedius, P. aeruginosa, and M. pachydermatis (Table 1). They were tested against two ear cleansers, Epi- Otic and Epi-Otic NF (Virbac Corporation). These products are not marketed as antimicrobials, and there are no claims for therapeutic antibacterial or antifungal activity. The test procedures were essentially the same as those employed by Lloyd, Bond, and Lamport 8. The isolates were grown on brainheart infusion agar overnight at 37 C, or on modified Dixon s agar (for Malassezia) for 3 days at 34 C. These cultures were then suspended in Mueller Hinton broth at concentrations equivalent to colony-forming units (CFU) per ml. Twenty µl of each suspension were added to 2 ml of either a 1/5 or 1/25 dilution of the test product in sterile distilled water. The mixtures were held at room temperature (~21 C). Aliquots were then removed after 1, 2, 4, 8, 16, and 32 minutes, diluted, and spread-plated in 5% bovine blood agar or modified Dixon s agar and incubated as before. Counts of colonies growing on the media were made after 48 hours for the bacteria and 72 hours for Malassezia and expressed as CFU/mL. Control suspensions were made and tested in the same way, but with phosphatebuffered saline (0.01M, ph 7.2) instead of the test products. The control counts (~10 4 CFU/mL) remained unchanged throughout the study. TABLE 1. Identification of Canine Bacterial and Yeast Isolates and Codes Used in the Text Species Origin Isolate Code Staphylococcus intermedius Skin 37/796 S1 63/896 S2 Pseudomonas aeruginosa Ear 45/1196 P1 5/1296 P2 Malassezia pachydermatis Skin 20/1196 M1 34/1196 M2 RESULTS Staphylococcus intermedius At the 1/5 dilution, S. intermedius counts had fallen to zero in less than 1 minute with Epi-Otic and Epi-Otic NF (Table 2). At the 1/25 dilution, S. intermedius counts reached zero after 4 minutes with Epi-Otic and after 2 minutes with Epi-Otic NF. Pseudomonas aeruginosa At the 1/5 dilution, P. aeruginosa counts reached zero within 1 minute for one isolate with Epi-Otic and Epi-Otic NF; the second isolate was killed by both of these products within 2 minutes (Table 3). At 1/25 dilution, P. aeruginosa counts reached zero after 1 minute for Epi-Otic NF and after 2 minutes with Epi-Otic. Malassezia pachydermatis At the 1/5 dilution, M. pachydermatis isolates were killed within 4 minutes by Epi-Otic NF and within 8 minutes by Epi-Otic (Table 4). At the 1/25 dilution, M. pachydermatis counts were low (120 CFU/mL) or had reached zero with Epi-Otic NF at 16 minutes, but were still high with Epi-Otic (10 4 and 4240 CFU/mL). DISCUSSION The otic products tested are routinely used undiluted in the ears or on skin. It is unlikely 44

3 TABLE 2. Viable Counts (Colony-forming Units/mL) for the Two Staphylococcus intermedius Isolates with 1/5 and 1/25 Dilutions of the Products Epi-Otic NF 5 S S S S Epi-Otic 5 S S S S TABLE 3. Viable Counts (Colony-forming Units/mL) for the Two Pseudomonas aeruginosa Isolates with 1/5 and 1/25 Dilutions of the Products Epi-Otic NF 5 P P P P Epi-Otic 5 P P P P that dilution by exudate would be greater than fivefold in vivo and thus efficacy at 1/5 suggests that they are likely to be effective in vivo. Such efficacy in vivo against otic bacteria and M. pachydermatis has been demonstrated for Epi-Otic by Lloyd, Bond, and Lamport 8 in studies of ear treatment in basset hounds. Dilution to 1/25 greatly reduced potency in vitro of the products against S. intermedius and M. pachydermatis and also revealed considerable variation in strain sensitivity between some of the isolates. However, this was not apparent at 1/5 and therefore may not affect efficacy under the conditions of normal use of the products. Both products were able to kill S. intermedius within 1 minute when diluted to 1/5. The activity that was demonstrated against P. aeruginosa was impressive for both of the products. Pseudomonas was killed within 2 minutes at a dilution of 1/5 and even at 1/25 only one isolate survived as long as 2 minutes. Pseudomonas can cause serious infections that may be difficult to treat with systemic antibiotics; therefore a topical approach is sometimes necessary. Indeed topical therapy alone or in combination with systemic therapy may sometimes be the treatment of choice both in otic 45

4 Veterinary Therapeutics Vol. 1, No. 1, Winter 2000 TABLE 4. Viable Counts (Colony-forming Units/mL) for the Two Malassezia pachydermatis Isolates with 1/5 and 1/25 Dilutions of the Products Epi-Otic NF 5 M M M1 >10000* M2 >10000 > Epi-Otic 5 M M M1 >10000 >10000 > M2 >10000 >10000 > *Confluent growth (uncountable) was estimated at >10000 CFU/mL. and cutaneous infections. 1,9 These data indicate that the topical approach is likely to be useful in the treatment and control of such infections. M. pachydermatis proved to be more resistant than the two bacterial species. At the 1/5 dilution, Epi-Otic NF was able to kill the yeast within 4 minutes; Epi-Otic was able to kill the yeast within 8 minutes. Of particular interest is the comparison of data on efficacy between the old and new formula Epi-Otic products. Reformulation appears to have increased the antimicrobial activity in vitro in every instance. It is uncertain how potent the antimicrobial activity of these products is likely to be in vivo or how long efficacy is likely to persist when they are applied to infected skin. However, Lloyd, Bond, and Lamport 8 were able to demonstrate reduction of bacterial and yeast populations in the ears of basset hounds for 24 hours after application of Epi-Otic to the ears. When using such products it is important to remember that bacterial and fungal infections of skin are seldom, if ever, primary. For this reason, it is essential, where possible, to identify and rectify underlying causative factors. CONCLUSIONS The ear cleansers evaluated in this study have potent antimicrobial activity in vitro against S. intermedius, P. aeruginosa, and M. pachydermatis. This activity in vitro, together with the previous data indicating antimicrobial activity of Epi-Otic in vivo, 8 suggests that they may be useful in the management of infections of the ear. ACKNOWLEDGMENT Adapted with permission from Lloyd DH, Lamport AI: Évaluation in vitro de l activité antimicrobienne de topiques cutanés et auriculaires chez le chien. Prat Méd Chir Anim Comp 34: , REFERENCES 1. Griffin CE: Otitis externa and otitis media, in Griffin CE, Kwochka KW, Macdonald JD (eds): Current Veterinary Dermatology: The Art and Science of Therapy. St. Louis, Mosby-Year Book, 1992, pp Scott DW, Miller WH, Griffin CE: Muller and Kirk s Small Animal Dermatology, ed 5. Philadelphia, WB Saunders, Bond R, Ferguson EA, Curtis CF, et al: Factors associated with elevated cutaneous Malassezia pachydermatis populations in dogs with pruritic skin disease. J 46

5 Sm Anim Pract 37: , Russell AD, Russell NJ: Biocides: Activity action and resistance, in Hunter PA, Darby GK, Russell NJ (eds): Fifty Years of Antimicrobials: Past Perspectives and Future Trends. Cambridge, Cambridge University Press, 1995, pp Lloyd DH, Lamport AI, Feeney C: Sensitivity to antibiotics amongst cutaneous and mucosal isolates of canine pathogenic staphylococci in the United Kingdom, Vet Dermatol 7: , Pellerin JL, Bourdeau P, Sebbag H, Person JM: Epidemosurveillance of antimicrobial compound resistance of Staphylococcus intermedius clinical isolates from canine pyoderma. Comparative Immunol, Microbiol & Infect Dis 21: , Lloyd DH, Lamport AI: Activity of chlorhexidine shampoos in vitro against Staphylococcus intermedius, Pseudomonas aeruginosa and Malassezia pachydermatis. Vet Record 144: , Lloyd DH, Bond R, Lamport AI: Antimicrobial activity in vitro and in vivo of a canine ear cleanser. Vet Record 143: , Griffin CE, Song MD: Management of otitis externa (Workshop report), in Kwochka KW, Willemse T, von Tscharner C (eds): Advanc Vet Dermatol, vol 3. Oxford, Butterworth Heinemann, 1998, pp