Identification and antimicrobial susceptibility patterns of Staphylococcus spp. isolated from canine chronic otitis externa

Identificação e susceptibilidade a antimicrobianos de Staphylococcus spp. isolados de cães com otite externa crônica

a01v53n2

Identification and antimicrobial susceptibility patterns of Staphylococcus spp. isolated from canine chronic otitis externa

[Identificação e susceptibilidade a antimicrobianos de Staphylococcus spp. isolados de cães com otite externa crônica]

N. Silva

Escola de Veterinária da UFMG

Caixa Postal 567

30123-970 – Belo Horizonte, MG

Recebido para publicação, após modificações, em 8 de novembro de 2000.

E-mail: [e-mail]

ABSTRACT

Swab samples obtained from 96 dogs with chronic otitis externa were cultured for the isolation of Staphylococcus species. Of 57 staphylococcal strains, 41 (72%) were coagulase-negative (CNS). The identification of staphylococci strains was made by standard procedures for the routine identification of staphylococci in clinical practice. S. sciuri was the most frequent species isolated (22.8%) from chronic otitis externa in dogs followed by S. intermedius (12.3%), S. auricularis (10.5%) and S. aureus (8.8%). Three (5.2%) CNS strains could not be identified. Bacterial isolates were susceptible to enrofloxacin, gentamicin, cephalothin, chloramphenicol and neomycin. Resistance was most common to penicillin G, oxacillin and ampicillin.

Keywords: Dog, otitis externa, staphylococci, antimicrobial

RESUMO

Foram utilizadas 96 amostras de material obtidas de cães com otite externa crônica para isolamento de Staphylococcus spp. Foram identificadas 57 amostras de estafilococos por meio de um sistema simples e prático desenvolvido para identificação de Staphylococcus spp. (SIS) a partir de material obtido de amostras clínicas. Entre os estafilococos, cerca de 41 (72%) foram amostras de Staphylococcus coagulase-negativo (SCN). S. sciuri foi o microrganismo mais envolvido com as otites crônicas em cães (22.8%), seguido pelo S. intermedius (12.3%), S. auricularis (10.5%) e S. aureus (8.8%). Três amostras de SCN (5.2%) não foram identificadas pelo SIS. Testes de susceptibilidade a antimicrobianos revelaram que enrofloxacina, gentamicina, cefalotina, cloranfenicol e neomicina foram os mais efetivos frente aos microrganismos isolados. Resistência foi apresentada frente a penicilina G, oxacilina e ampicilina.

Palavras-chave: Cão, otite externa, Staphylococcus, antimicrobiano

INTRODUCTION

Otitis externa is considered the most common disease of the ear canal in the dog, characterized by an acute or chronic inflammation of the epithelium of the external auditory meatus, sometimes involving the pinna (Acosta et al., 1992; Rosychuk, 1994). Although the microbial flora of the ear of dogs has been studied and some authors tried to correlate the signs of the disease with the aetiological agent (Kowalski, 1988), it is generally accepted that the laboratory identification of the agent is necessary.

The different roles played by pathogenic and saprophytic microorganisms implicated in otitis externa are unclear. In dogs, S. intermedius was one of the major coagulase-positive (CPS) species (Cox et al., 1988). S. aureus is also usually associated with pyodermas as a result of the high frequency of nasal carriage (Medleau et al., 1986). On the other hand, there are few reports about the significance of coagulase-negative staphylococci (CNS) in canine otitis and skin lesions (Acosta et al., 1992).

The purpose of the present study was to identify species of Staphylococcus isolated from dogs with chronic otitis externa and to determine their antimicrobial susceptibility.

MATERIALS AND METHODS

Samples were obtained from 96 adults dogs with chronic otitis externa referred to the Veterinary Hospital of Federal University of Minas Gerais, from 1995 to 1999. The ear canals were swabbed with sterile cotton-tipped applicators. The swabs were streaked on 5% (v/w) sheep blood agar and inoculated into tubes containing brain heart infusion broth (BHI-Difco) and incubated for 24-48h at 37°C. Presumptive identification of isolates as coagulase-positive or coagulase-negative Staphylococcus was based on colony morphology, Gram stain, catalase and coagulase activity using rabbit plasma. The bacteria were stored in glycerol 50% (v/v) at -80°C until use.

Before final identification, the stock cultures were subcultured twice on 5% (v/w) sheep blood agar at 37°C for 24-48h to ensure purity prior to use. The isolates were biotyped by a simple staphylococci identification system (SIS) developed in the laboratory, where microwell plastic trays were used. For acid production, carbohydrate solutions were prepared at 1% (w/v) final concentration in bacto purple broth base medium², supplemented with either maltose, D-trehalose, D-mannitol, D-xylose, D-cellobiose, sucrose, D-mannose, D-ribose, raffinose, lactose and fructose. Solutions for acetoin production, arginine dihydrolization, urea hydrolysis, nitrate reduction and ONPG were prepared as described by Quinn et al. (1994). Each medium (200ml) was poured into a separate microwell. Bacteria to be tested were suspended in 2.0ml of BHI Broth, adjusted to a McFarland 2.0 standard, and 20ml of the bacterial suspension, corresponding to 6 ´ 10⁶ CUF/ml (colony unit forming), were inoculated into each well using a multichannel pipette (Jencons Ltd. USA). Wells for arginine dihydrolase and urease tests were covered with sterile mineral oil. The microwells were covered with sterile tape and incubated in a moist chamber for 24-48 h at 37°C in an aerobic atmosphere.

Novobiocin resistance was determined by a disk-diffusion assay using Muller-Hinton Agar¹ plates with 6mg of novobiocin per disk. The interpretation of the biochemical test was made following Quinn et al. (1994) and the identification of Staphylococcus strains according to Schleifer (1986).

Susceptibility tests using agar disk-diffusion tests were done as described by Sahm & Washington (1992) and 10 antimicrobial agents (Sensibiodisc – CECON-Brazil) as penicillin G (10UI), ampicillin (10mg), oxacillin (5mg), cephalotin (30mg), chloramphenicol (30mg), erythromycin (15mg), gentamicin (10mg), trimethoprim-sulfamethoxazole (25mg), neomycin (30mg), and enrofloxacin (10mg) were used.

RESULTS

A total of 57 staphylococcal strains were isolated and identified from ear canals of dogs with chronic otitis externa. Of these, 16 were coagulase-positive, and 41 were coagulase-negative. Using the staphylococci identification system (SIS), seven of the coagulase-positive were identified as S. intermedius, five as S. aureus and four as S. hyicus. Of 41 coagulase-negative staphylococci, 13 isolates were S. sciuri, six were S. auricularis, and five were S. xylosus.

Three strains (5.2%), classified as coagulase-negative staphylococci, could not be identified through the biochemical reaction patterns as seen on Table 1.

The results of the antimicrobial susceptibility tests for coagulase-positive and coagulase-negative staphylococci are given in Table 2. Bacteria isolates were susceptible to enrofloxacin, gentamicin, cephalothin, chloramphenicol and neomycin. Resistance was most common to penicillin G, oxacillin and ampicillin. Among the coagulase-negative staphylococci isolates, S. sciuri was susceptible to enrofloxacin (100%), gentamicin (92 %), cephalothin (85%) and neomycin (85%). S. intermedius and S. aureus isolates presented the same susceptibility pattern.

DISCUSSION

Inflammatory disease of the external ear is commonly encountered in dogs. Despite advances in therapeutic approach, refractory cases of the otitis externa remain common. In view of this fact, the identification of microorganisms involved in this pathology and the determination of their antimicrobial susceptibility rates are important tools in canine practice. Results from 57 staphylococcal isolates showed S. intermedius as the major coagulase-positive staphylococci (CPS) associated with chronic otitis externa such as described by some authors (Cox et al., 1988; Acosta et al., 1992; Cole et al., 1998). Despite CPS are considered to be the main bacterial agents in canine otitis (Blanco et al., 1996; Cole et al., 1998), in the present study, CNS were the most common bacteria (72.0%) isolated and among them, S. sciuri was the most frequently found (22.8%). CNS has a questionable pathogenicity, rarely considered in canine otitis (Acosta et al., 1992). However they are considered as potential pathogens since they were isolated in canine deep pyoderma (Medleau et al., 1986) and are the most frequent reported as bloodstream pathogens in hospitals (Kloss & Bannerman, 1994) or reservoirs and transmitters of antibiotic resistance genes in the hospital environment (Rupp & Archer, 1994). There are some reports on the presence of CNS in other animal species causing otitis and they have been isolated from skin surface of clinically normal cats (Medleau & Blue, 1988; Lilenbaum et al., 1998).

The genus Staphylococcus includes nearly 40 different species and identification of all species is uncommon in routine practice (Kloss & Bannerman, 1994). These results were possible by the use of SIS a simple and inexpensive identification system, which permitted the identification of 95% of staphylococcal strains from clinical otitis in dogs referred to the Veterinary Hospital. Similar results were described for identification of staphylococci from clinical lesions, using another simplified identification systems (Piccolomini et al., 1992; Monsen et al., 1998). According to these authors, 4% of clinically isolated CNS were not identified due to inconclusive biochemical tests.

In canine practice, the topical application of antibiotics in otitis externa is especially effective, because with this procedure it is possible to reach higher antibiotic concentrations (Guedeja-Marrón et al., 1998). Systemic therapy could be also employed as an alternative approach. For treatment, the possible increased resistance of staphylococci isolated from dogs with otitis externa should be taken into account. In the present study, antibiotics as enrofloxacin, gentamicin, chloramphenicol, cephalothin and neomycin are indicated for treatment of clinical cases as demonstrated by antimicrobial susceptibility tests. Similar suggestions were given by others (Medleau et al., 1986; Cole et al., 1998; Guedeja-Marrón et al., 1998). Some years ago, Magalhães et al. (1985) reported different antibiotic susceptibility patterns presented by Staphylococcus species from otitis of dogs referred to the Veterinary Hospital of UFMG. This fact support the idea that the increasing resistance of staphylococci to different antibiotics are given by its indiscriminate use in clinical practice causing the appearance of bacterial resistance. All the species were obtained from dogs with chronic otitis and previous history of empiric antibiotic treatments.

In conclusion, these findings suggest that CNS plays an important role in the pathogenicity of canine chronic otitis externa. The test scheme has a value for identification of staphylococcal species and should be performed, following by antimicrobial susceptibility tests.

ACKNOWLEDGEMENTS

The authors are grateful to Dr. Jacques Robert Nicoli for his helpful suggestions and his revision of the English version of this manuscript.

• ACOSTA, B., REAL, F., FERRER, O. et al. Otitis recidivantes caninas: evaluación del control laboratorial. Med. Vet., v.9, p.369-374, 1992.
• BLANCO, J.L., GUEDEJA-MARRÓN, J., HONTECILLAS, R. et al. Microbiological diagnoses of chronic otitis externa in the dogs. J. Vet. Med. B, v.43, p.475-482, 1996.
• COLE, L.K., KWOCHKA, K.W., KOWLASKI, J.J. et al. A. Microbial flora and antimicrobial susceptibility patterns of isolates pathogens from the horizontal ear canal and middle ear in dogs with otitis media. J. Am. Vet. Med. Assoc., v.212, p.534-538, 1998.
• COX, H.U., HOSKINS, J.D., NEWMAN, S.S. et al. Temporal study of staphylococcal species on healthy dogs. Am. J. Vet. Res., v.49, p.747-751, 1988.
• GUEDEJA-MARRÓN, J., BLANCO, J.L., RUPEREZ, C. et al. Susceptibility of bacterial isolates from chronic canine otitis externa to twenty antibiotics. J. Vet. Med. B, v.45, p.507-512, 1998.
• KLOSS, W.E., BANNERMAN, T.L. Update on clinical significance of coagulase-negative staphylococci. Clin. Microbiol. Rev., v.7, p.117-140, 1994.
• KOWALSKI, J.J. The microbial environment of the ear canal in health and disease. Vet. Clin. North Amer.: Small Anim. Prac., v.18, p.743-754, 1988.
• LILENBAUM, W., NUNES, E.L.C., AZEREDO, M.A.I. Prevalence and antimicrobial susceptibility of staphylococci isolated from skin surface of clinically normal cats. Letters Appl. Microbiol., v.27, p.224-228, 1998.
• MAGALHÃES, M.J., SILVA, N., MARQUES Jr., A.P. Otite externa em cães atendidos no Hospital Veterinário da UFMG. Etiologia, freqüência e sensibilidade antibiótica. Arq. Bras. Med. Vet. Zootec., v.37, p.333-341, 1985.
• MEDLEAU, L., BLUE, J. Frequency and microbial susceptibility of Staphylococcus spp. isolated from feline skin lesions. J. Am. Vet. Med. Assoc., v.193, p.1080-1081, 1988.
• MEDLEAU, L., BROWN, R.E., MILLER, W.H. Frequency and antimicrobial susceptibility of Staphylococcus species isolated from canine pyodermas. Am. J. Vet. Res., v.47, p.229-231, 1986.
• MONSEN, T., RÖNNMARK, M., OLOFSSON, C. et al. An inexpensive and realible method for routine identification of staphylococcal species. Eur. J. Clin. Microbiol. Infect. Dis., v.17, p.327-335, 1998.
• PICCOLOMINI, R., CATAMO, G., PICCIANI, C. et al. Evaluation of Staf-System 18 R for identification of Staphylococcal clinical isolates to the species level. J. Clin. Microbiol., v.32, p.649-653, 1992.
• QUINN, P.J., CARTER, M.E., MARKEY, B. et al. Clinical veterinary microbiology, London: Wolfe, 1994. 648 p.
• ROSYCHUK, R.A.W. Management of otitis externa in the dog. Vet. Clin. North Amer.: Small Anim. Prac., v.24, p.921-952, 1994.
• RUPP, M.E., ARCHER, G.L. Coagulase-negative staphylococci: pathogens associated with medical progress. Clin. Infec. Dis., v.19, p.231-243, 1994.
• SAHM, D.F., WASHINGTON, J.A. Antimicrobial susceptibility tests: dilution methods. In: Ballows A. (Ed) Manual of clinical microbiology, Washington: American Society for Microbiology, 1992, p.1105-1125..
• SCHLEIFER, K.H. Gram-positive cocci. Family I. Micrococcaceae In: SNEATH, P.H.A., MAIR, N.S., SHARPE, M.E., (Eds) Bergey's manual of determinative bacteriology, 9.ed.,. Baltimore: The Williams & Wilkins Co. 1986, p.999-1032.

Publication Dates

• Publication in this collection
  05 June 2002
• Date of issue
  Apr 2001

History

• Received
  08 Nov 2000