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Otitis externa in dogs: microbiology

and antimicrobial susceptibility
I. Tsachev

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Otitis externa in dogs: microbiology and
antimicrobial susceptibility
V. PETROV1*, G. MIHAYLOV1, I.TSACHEV1, G. ZHELEV1, P. MARUTSOV1, K. KOEV1
1
Department of Veterinary Microbiology, Infectious and Parasitic Diseases, Faculty of Veterinary Medicine, Trakia University, 6000 Stara Zagora, BULGARIA
*Corresponding author: vlado72@abv.bg
SUMMARY
he aims of the study were to determine the prevalence of bacterial/yeast
otitis externa in dogs and to analyse antimicrobial susceptibility. Among
257 swab ear samples from Bulgarian dogs with otitis externa, bacteria
and yeast species were isolated in 93.77% of cases as mono-infections
(109 cases) involving mainly coagulase positive staphylococci (60 strains),
Malassezia pachidermatis (97 strains) and Pseudomonas aeruginosa (42
strains) and more oten as poly-infections (132 cases). he other agents
scarcely identiied were β-haemolytic Streptococcus spp., Proteus mirabilis
and Escherichia coli, the 2 last species being almost exclusively found in
poly-infections. he sensitivity of isolated bacteria to antimicrobial drugs,
most commonly used treatment of otitis externa has been tested. he
high sensitivity to beta-lactams and aminoglycoside-aminocyclitols was
established in Gram positive bacteria, while Gram negative bacteria were
sensitive to aminoglycoside-aminocyclitols, polymyxin B and enroloxacin.
hese results highlight the relative high frequency of yeasts and the necessity
of coupling antimicrobial susceptibility tests to bacterial isolation.
Keywords : Otitis externa, dogs, bacterial isolation,
yeast, antimicrobial susceptibility.
Introduction
Otitis externa is an acute or chronic inlammation of the
external ear. he disease is more frequent in dogs than in cats.
Dogs with long pendulous ears are most commonly afected
[2]. Ear infections are among the ten most frequent reasons
for dogs to be presented to veterinarians and may afect up
to 20% of dogs [1, 6]. A number of predisposing factors are
identiied such as anatomical ear canal stenosis, hair in the ear
canal, pendulous ears, haired concave side of ears, increased
humidity (hinting dogs), moisture retention, washing, injury
during manipulations, foreign bodies, prolonged antibiotic
treatment, obstructive diseases (neoplasms) and systemic
conditions (immunosuppression) [17]. Other causes include
permanent factors such as bacteria (coagulase positive
Staphylococcus spp., β-haemolytic Streptococcus spp., Proteus
mirabilis, Pseudomonas aeruginosa, Escherichia coli), yeasts
(Malassezia pachidermatis, Candida albicans), as well as
RESUME
Otite externe chez le chien: analyse microbiologique et résistance aux
antibiotiques
Les objectifs de cette étude ont été de déterminer la prévalence des otites
externes bactériennes ou mycosiques chez le chien et d’établir la résistance
des germes aux antibiotiques. Parmi les 257 prélèvements auriculaires
réalisés sur des chiens atteints d’otite externe en Bulgarie, des bactéries
ou des levures ont été mises en évidence dans 93.77% des cas, sous forme
de mono-infections (109 cas) principalement dues à des staphylocoques
coagulase + (60 souches), à Malassezia pachidermatis (97 souches) et à
Pseudomonas aeruginosa (42 souches), et le plus souvent sous forme de
poly-infections (132 cas). Les autres agents plus rarement identiiés ont été
des streptocoques β-hémolytiques, Proteus mirabilis et Escherichia coli, les
2 dernières espèces étant presqu’exclusivement retrouvées dans des cas de
poly-infections. La sensibilité des isolats aux diférents antimicrobiens les
plus couramment utilisés dans le traitement des otites externes du chien
a été évaluée  : les bactéries Gram + ont montré une forte sensibilité aux
β-lactames et aux aminoglycosides / aminocyclitols alors que les bactéries
Gram – ont surtout été sensibles aux aminoglycosides / aminocyclitols, à
la polymyxine B et à l’enroloxacine. Ces résultats soulignent la fréquence
relativement élevée des levures et la nécessité de déterminer la résistance aux
antibiotiques des germes en parallèle de leur isolement.
Mots-clés: Otite externe, chien, isolation bactérienne,
levures, résistance aux antibiotiques.
progressing pathological alterations (hyperplasia, oedema,
ibrosis  [17]. he normal ear canal microlora consists
of coagulase-positive, coagulase-negative Staphylococcus
spp., β-haemolytic Streptococcus spp., Pr. mirabilis, E. coli,
Ps. aeruginosa etc. he last agent is isolated in only 1% of
healthy dogs and in up to 20% of dogs with chronic otitis
externa [7, 18]. In healthy feline ears, Pasteurella multocida
could be also isolated. Apart these bacterial agents, M.
pachidermatis, Microsporum canis and Otodectes cynoti could
be also present without clinical signs of inlammation [5]. It
should be noticed that bacteria and yeasts are opportunists
and not primary pathogens causing solely otitis externa,
but they found a favourable medium for growth provided
that another cause is present [17, 18]. he successful
treatment of the disease requires appropriate medications
including anti-inlammatory drugs, antifungal agents and
chemotherapeutics in relation to the sensitivity of the
aetiological agents [7, 9].
Revue Méd. Vét., 2013, 164, 1, 18-22
BACTERIAL OTITIS EXTERNA IN DOGS
he purpose of the present study was to determine the
microbial aetiological agents of canine otitis externa, their
prevalence and behaviour to antibacterial drugs that are
most commonly used for treatment of this disease.
Material and methods
ANIMALS AND SAMPLES
he study included 193 dogs from both genders, from
diferent breeds and from 8 months to 14 years old, all
with clinical signs speciic for otitis externa. Patients were
presented with unilateral or bilateral dropping of ears, head
shaking, pruritus, pain when palpated, erythema and swelling
of ear skin or of ear canal with increased amount of cerumen.
A total of 257 samples of ear secretion were collected using
sterile cotton swabs. he study was performed between
January 2007 and December 2011.
MICROBIOLOGICAL TESTS
Samples were cultivated on blood agar containing 5%
sheep blood (Bul-bio Base, National Institute of Parasitic and
Infectious Diseases, Soia) and on McConkey agar (Difco).
Cultures were incubated aerobically for 24-48 hours at 37°C.
Mycological tests included aerobic cultivation of the samples
on Sabouraud dextrose agar (supplemented with 0.4 g/L
chloramphenicol and 0.5 g/L cyclohexamide) for 2-7 days at
37°C. Ater incubation, isolates were identiied according to
conventional microbiological methods [16].
he behaviour of isolates to antimicrobial drugs was
tested by the disk difusion method and the results were
interpreted by the three-score system of Bauer et al. [3],
as per requirements of NCCLS [14]. Disks loaded with
antimicrobial drugs: amoxicillin/clavulanic acid (20/10 µg),
ampicillin/cloxacillin (6.69/18.31 µg), gentamicin (10 µg),
tobramycin (10 µg), amikacin (30 µg), enroloxacin (5 µg),
chloramphenicol (30 µg), doxycycline (30 µg), lincomycin/
spectinomycin (9 / 100 µg), and polymyxin B (10 µg) were
used.
Results
he microbiological tests conducted on 257 samples
showed that only 16 did not contain pathogenic
microorganisms. From the other 241 samples, 388 microbial
agents were isolated. heir distribution by species is shown on
igure 1. Coagulase-positive staphylococci were the prominent
species, representing 43.6% of the microbial agents identiied
and were found in more than 70% (169/241) of dogs with
bacterial otitis externa. Malassezia pachidermatis represented
25% of the identiied germs and was isolated in 40% of
patients whereas Pseudomonas aeruginosa (17%), Proteus
mirabilis, Escherichia coli and β-haemolytic streptococci
were more rarely observed and were isolated in 17%, 11%
Revue Méd. Vét., 2013, 164, 1, 18-22
19
Figure 1 : Number and distribution of microbial isolates (n = 388) from
dog ear secretion samples positive for microbiological agents (n = 241).
and 10% of dogs with bacterial otitis externa respectively.
In addition, Pseudomonas aeruginosa and Proteus mirabilis
were isolated only from animals with chronic otitis or
from dogs that failed to respond to previous antibiotic
treatment. As already mentioned, the other bacterial species
(β-haemolytic streptococci and E. coli) rarely caused otitis
externa independently, but were rather seen in association
with staphylococci or Pseudomonas aeruginosa.
Among the 241 positive samples for microbiological tests,
mono-infections were observed only in 109 samples (45.2%).
Coagulase-positive staphylococci were most commonly
identiied when a single organism was isolated from otitis
patients corresponding to 60 cases (25% of all diseased dogs)
followed by M. pachidermatis in 31 cases (13%). he other
bacterial species (β-haemolytic streptococci, E. coli and
Pseudomonas aeruginosa) rarely caused mono-infections,
only in 11, 2 and 5 cases, respectively. By contrast, Proteus
mirabilis was not detected as a sole agent of canine otitis.
Two and multiple microbial species were isolated from
132 samples: 119 samples with 2 microbial species, 11 with
3 germs and 2 with 4 pathogens (Table I). he most frequent
microbial species involved in poly-infections were again
Coagulase positive Staphylococcus spp (109/132, i.e. 82.58%),
Malassezia pachidermatis (50.00%) and Pseudomonas
aeruginosa (28.03%). However, among the species scarcely
isolated in bacterial otitis externa, Proteus mirabilis and E.
coli were oten associated with other germs in 100% and
infections with the b-haemolytic Streptococcus spp. and of
92.86% of positive samples whereas the proportions of mono-
poly-infections involving them remained roughly similar
(44% and 56%, respectively). Animals with 3 or 4 microbial
agents simultaneously isolated exhibited chronic otitis.
he behaviour of bacterial isolates to tested antibacterial
drugs is summarized in Table II. he analysis of strains
behaviour towards antibiotics showed preserved high
sensitivity to β-lactam combinations amoxicillin/
clavulanic acid and ampicillin/cloxacillin in staphylococci
and streptococci. Similar behaviour was exhibited by
Gram + bacteria towards aminoglycoside-aminocyclitols
gentamicin, amikacin and tobramycin, with percentage
of sensitive strains between 74% and 91%. With regard
20 PETROV (V.) AND COLLABORATORS

Bacterial / yeast associations Number and frequency (%)

2 species
Malassezia pachidermatis 43 (17.84%)
Pseudomonas aeruginosa 21 (8.71%)
Coagulase positive
Proteus mirabilis 15 (6.22%)
Staphylococcus spp
Escherichia coli 10 (4.15%)
β-haemolytic Streptococcus spp. 10 (4.15%)

Escherichia coli 6 (2.49%)

Pseudomonas aeruginosa
Malassezia pachidermatis 5 (2.08%)
β-haemolytic Streptococcus spp. 3 (1.24%)

Escherichia coli Malassezia pachidermatis 6 (2.49%)

3-4 species
Proteus mirabilis / Malassezia pachidermatis 9 (3.73%)
Coagulase positive Staphylococ-
cus spp β-haemolytic Streptococcus spp. / Pseudomonas aeruginosa
1 (0.41%)
/ Escherichia coli

Proteus mirabilis / Malassezia pachidermatis 2 (0.83%)

Escherichia coli Proteus mirabilis / Malassezia pachidermatis / Pseudomo-
1 (0.41%)
nas aeruginosa

Table I : Distribution of microbial agents by species involved in co-infections (132 cases) in dogs with bacterial otitis externa cases (n = 241)

Staphylococcus
spp. Streptococcus spp. Pseudomonas
Proteus mirabilis Escherichia coli
Antibiotics (coagulase (β-haemolytic) aeruginosa
(n = 27) (n = 28)
positive) (n = 25) (n = 42)
(n = 169)
Amoxicillin-clavulanic acid 5% 5% 100% 30% 32%
Ampicillin/cloxacillin 19% 9% 100% 100% 100%
Gentamicin 20% 9% 2% 28% 18%
Tobramycin 26% 23% 20% 0% 5%
Amikacin 15% 21% 0% 0% 0%
Enroloxacin 32% 100% 38% 22% 14%
Chloramphenicol 40% 0% 100% 74% 0%
Doxycycline 49% 26% 100% 81% 22%
Lincomycin/Spectinomycin 59% 40% 40% 55% 0%
Polymyxin B 66% 100% 0% 74% 0%

Table II : Antibiotic resistance (%) of microbial agents isolated from dogs with bacterial otitis externa.

to enroloxacin, chloramphenicol and polymyxin B, the
sensitivity of staphylococci and streptococci was diferent.
All streptococcal isolates were resistant to enroloxacin while
68% of staphylococci were sensitive. he opposite was true
for chloramphenicol: all streptococci were sensitive whereas
40% of staphylococci were resistant to this antibiotic. Some
staphylococci (34%) were sensitive to polymyxin B, but all
streptococci were resistant. he sensitivity to tetracyclines as
well as to the combination lincomycin/spectinomycin was
higher for streptococci than for staphylococcal isolates.
Gram - bacteria showed a very diferent behaviour to
beta-lactam combinations. All Ps. aeruginosa, Pr. mirabilis
and E. coli strains were resistant to ampicillin/cloxacillin. At
the same time, more than 2/3 of Proteus and E. coli isolates
were sensitive to amoxicillin/clavulanic acid, whereas all
Pseudomonas isolates were resistant to this combination. he
resistance pattern to aminoglycosides was similar to that of
Gram + bacteria although the percentage of sensitive strains
was usually higher (ranging from 72% to 100%). he majority
of Gram - bacteria was highly sensitive to enroloxacin (from
62% to 86%). All Pseudomonas and the majority of Pr.
mirabilis strains were resistant to chloramphenicol (74%) and
tetracyclines (81%), while E. coli strains were highly sensitive.
Ps. aeruginosa and Pr. mirabilis isolates were oten resistant
to lincomycin/spectinomycin (40% and 55% respectively)
Revue Méd. Vét., 2013, 164, 1, 18-22
BACTERIAL OTITIS EXTERNA IN DOGS
whereas all E. coli strains were sensitive. All Pseudomonas
and E. coli isolates were sensitive to the polypeptide antibiotic
polymyxin B while the sensitivity of Proteus spp. strains was
however low (< 20%).
Discussion
In this study, 43.56% of all microbial isolates belonged to
the group of coagulase-positive staphylococci, among which
Staphylococcus intermedius (recently called Staphylococcus
pseudointermedius) and Staphylococcus aureus are already
known as canine pathogens [7]. A survey of microbial agents
of canine otitis externa in Bulgaria was performed in the
period 1993-1995 by BORISSOV et al. [4] that demonstrated
the presence of coagulase-positive staphylococci in 54% of
all dogs with otitis. In this investigation, coagulase-positive
staphylococci were present both in mono- or co-infections
in 70% of dogs with otitis externa. his prevalence is higher
than that reported by BORISSOV et al. [4] and similar to
that found by HARIHARAN et al. [8], HARVEY et al. [9],
LILENBAUM et al. [11], LYSKOVA et al. [12], and OLIVERA
et al. [15]. ZAMANKHAN et al. [21] observed that 73.8%
of isolates in their study belonged to Staphylococcus spp.
vs. only 43.56% in ours. he authors have investigated the
bacterial agents of otitis in a warmer climate (Iran) and
this is a probably cause for the higher prevalence observed.
Our data however are considerably diferent from those of
BORNAND [5] who isolated S. intermedius in only 23% of
canine otitis cases.
he incidence of Gram - bacteria in the present study
was not very diferent from data reported in Bulgaria [4]:
Ps. aeruginosa isolates were 9% of all strains vs. almost 11%
in this study. he study of BORISSOV et al. [4] did not
present data about the involvement of yeasts, but our data
corresponded to those of BORNAND [5] in a similar setting
in Switzerland, where M. pachidermatis was isolated from
56% of dogs with otitis (40% in our study). With regard to
this species, the present results were closer to those obtained
by LYSKOVA et al. [12] in the Czech Republic. Prevalences
of Ps. aeruginosa, Pr. mirabilis and E. coli observed in the
present study are similar to those reported by KROGH et al.
[10], LYSKOVA et al. [12] and OLIVERA et al. [15]. he last
research group report also data about the incidence of co-
infections and microbial species involved. heir results are
similar to ours in that mixed infections occurred in more
than half of diseased dogs. he combination Staphylococcus
intermedius + Malassezia pachidermatis was the most
commonly seen.
he sensitivity of staphylococci isolated from dogs
with otitis externa in the present study coincide with data
of LILENBAUM et al. [11] with respect to ampicillin and
gentamicin, except that the proportions of resistant strains to
tetracyclines observed here was elevated (almost 50% vs. 7% in
their study). he results in the present study on staphylococci
resistance to amoxicillin/clavulanic acid, gentamicin,
amikacin and enroloxacin are also highly comparable to
Revue Méd. Vét., 2013, 164, 1, 18-22
21
those of VANNI et al. [19] who has conducted a very detailed
investigation on the resistance of Staphylococcus intermedius
and Staphylococcus schleiferi in Italy.
he general opinion that Ps. aeruginosa is the most
resistant microbial agent involved in the aetiology of canine
otitis externa was also conirmed in the present study. Similar
data are published by HARIHARAN et al. [8], demonstrating
38% resistance to enroloxacin, 99% to chloramphenicol,
98% to doxycycline but also a rather higher resistance to
aminoglycoside-aminocyclitols gentamicin (15%) and
amikacin (11%), contrary to present results (2% and 0%).
Nevertheless, ZAMANKHAN et al. [21] have obtained 100%
resistance of Ps. aeruginosa strains to amoxicillin/clavulanic
acid and lincomycin/spectinomycin combinations and 10%
to gentamicin and 0% to amikacin, which are closer to our
observations. hey have also observed that 20% of isolates
were sensitive to oxytetracycline and 30% to chloramphenicol
whereas all isolates in the present study were resistance to
these drugs. Others [13, 20] reported a higher resistance
to aminoglycosides and luoroquinolones, consequently to
the frequent use of medications containing gentamicin and
enroloxacin and recommended to replace them with other
drugs from the same class as tobramycin and marboloxacin.
With regard to Pr. mirabilis and E. coli isolates, the current
results were highly similar to those of HARIHARAN et al.
[8] and ZAMANKHAN et al. [21].
As a conclusion, it could be assumed that the commonest
bacterial agents isolated from canine otitis externa were
coagulase-positive staphylococci. It should be however
remembered that yeasts are also frequently involved in
the aetiology of ear canal inlammations and that in many
instances, clinical cases were caused by co-infections.
Microbiological tests are always recommended for isolation
and identiication of microbial agents and for determination
of their sensitivity to chemotherapeutics used for treatment
of otitis externa in dogs.
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