Vet Dermatol 2019 DOI: 10.1111/vde.

12734

Characterization of the otic bacterial microbiota in dogs

with otitis externa compared to healthy individuals
Juraj Korbelik* , Ameet Singh†, Joyce Rousseau* and J. Scott Weese*
*Department of Pathobiology, Ontario Veterinary College, University of Guelph, 50 Stone Road E, Guelph, Ontario, Canada N1G 2W1
†Department of Clinical Studies, Ontario Veterinary College, University of Guelph, 50 Stone Road E, Guelph, Ontario, Canada N1G 2W1
Correspondence: Juraj Korbelik, Department of Pathobiology, Ontario Veterinary College, University of Guelph, 50 Stone Road E, Guelph, Ontario
Canada N1G 2W1. E-mail: jkorbelik@hotmail.com

Background – Otitis externa is a common multifactorial disease in dogs. The diversity of the cutaneous micro-
biota in dogs appears to decrease in diseased states. However, little is known about the microbiota of the canine
ear and how it is altered by disease.
Hypothesis/Objectives – To describe the otic bacterial microbiota in dogs with otitis externa compared to
healthy dogs.
Animals – Samples were collected from 18 dogs with clinical and cytological evidence of otitis externa, and
eight clinically normal dogs without cytological evidence of otitis externa.
Methods and materials – DNA from each sample was isolated and Illuminaâ sequencing of the V4 hypervari-
able region of the 16S rRNA gene amplicons was performed. Sequences were processed using the bioinformat-
ics software MOTHUR.
Results – Bacteria from 27 different phyla were identified. Affected ears had significantly decreased alpha diver-
sity when compared to healthy ears. Community structure and membership also differed between the two
groups. Linear discriminant analysis effect size analysis identified 153 operational taxonomic units (OTUs) that
were differentially abundant. Eleven OTUs were over-represented in the affected ears, including Staphylococcus,
Pseudomonas and Parvimonas.
Conclusions – The otic bacterial microbiota is much more complex than has been identified with previous cul-
ture-based studies; otitis externa is accompanied by broad and complex differences in the microbiota.

future, these findings may provide information on devel-

Introduction
Otitis externa is a common multifactorial disease with a
prevalence as high as 20% in dogs.1,2 Primary causes of
otitis externa include hypersensitivity and autoimmune
disorders, endocrine diseases, otic parasites, foreign bod-
ies and conformational defects.3 In dogs, a common
underlying cause of otitis externa is atopic dermatitis
(AD).4 This common inflammatory condition of the skin
that affects 10% of dogs.5 Alteration in the otic micro-
biota can complicate otitis externa.3 Different mecha-
nisms have been proposed to be the cause of alteration
in cutaneous microbiota of humans and dogs with AD. In
many cutaneous diseases, it is unclear if the condition is
caused by an alteration in the cutaneous microbiota or if
the alterations are the result of the disease itself.6
Meticillin- and multi-drug resistance are now common-
place in both veterinary and human medicine.7 Under-
standing the impact of disease states on the otic
microbiota is important to identify specific changes that
might be markers of microbiota disruption as aids for
diagnosis or for selective therapeutic repopulation. In the
oping nonantimicrobial approaches to controlling otitis
externa.
Until recently, many of the bacterial species on the skin
and in the ear canal could not be analysed using culture-
based methods, limiting our existing knowledge.8 The
development of molecular techniques to identify and
quantify microbial organisms has revolutionized our view
of the microbial world. Studies which analysed species
diversity using molecular methods have demonstrated
that the skin microbiota of dogs is much richer and more
diverse than reported previously by culture-based meth-
ods.9,10 To date, very few studies have assessed the otic
microbiota in dogs.
The aim of this study was: (i) to further evaluate the
normal bacterial microbiota of the ear canal in healthy
dogs, (ii) to investigate how the otic bacterial microbiota
of dogs with otitis externa is altered compared to clinically
normal dogs and (iii) to compare the otic microbiota of
samples within the same dog and samples between
dogs.

Methods and materials

Study population
Accepted 16 January 2019 All animals for this study were recruited through the Guelph Veteri-
Source of funding: This study was self-funded. nary Specialty Hospital following a protocol approved by the Univer-
Conflict of interest: No conflicts of interest have been declared. sity of Guelph’s Animal Care Committee. Thirty dogs that presented

© 2019 ESVD and ACVD, Veterinary Dermatology 1

Korbelik et al.
to the Dermatology Department with clinical and cytological evidence
consistent with otitis externa were enrolled. Clinical signs consistent
with otitis externa included at least one ear with evidence of ery-
thema and discharge from the external ear canal. In order to be
included in the study, at least one ear must have had cytological evi-
dence of infectious otitis externa. This was described as having
greater than or equal to two (2+) bacteria or yeast on a semiquantita-
tive assessment scale.11 All dogs were examined by the investigator.
Cases were excluded if they had received any topical ear treatments
or cleaners within the two weeks previous to sampling, or systemic
antifungals or antibiotics within the three months previous to sam-
pling.
For the control group, ten dogs that were presented to the Guelph
Veterinary Specialty Hospital Surgery Department for elective ortho-
paedic procedures were enrolled. In order to be enrolled in the study,
the dogs must not have received any systemic antimicrobials within
the last three months nor any topical ear treatments or cleaners in
the previous two weeks. Dogs with any prior history of ear or skin
disease were excluded from the study. Each dog was examined for
evidence of otitis externa or signs of skin disease. This included ery-
thema, discharge from the ears, alopecia, excoriations, hyperpigmen-
tation of the skin or papular or pustular eruptions. Dogs with any
signs of skin or ear disease were excluded from the study. Otic cytol-
ogy was performed from both ears. Any evidence of yeast or bacteria
(1+ or greater) on cytology would lead to exclusion from the study.
Sample collection and DNA extraction
Samples were collected from affected and control dogs using a regu-
lar tipped culture swab (ESwab Regular Collection Kit, BD Diagnos-
ticsTM; Franklin Lakes, NJ, USA). Each swab was inserted into the
external ear canal up to the junction between the vertical and horizon-
tal canals. The swab was then rotated 360 degrees before being
withdrawn. Both ears were sampled separately in all dogs. Each
sample was labelled and stored at 4°C for no longer than seven days
before being transferred to a 80°C freezer until analysis. DNA was
extracted from each sample using the MoBio Power Soil DNA Extrac-
tion kit, following the manufacturer’s protocol (Qiagen Inc.; Toronto,
ON, Canada). DNA quantity and quality was assessed by spectropho-
tometry (NanoDrop, Roche; Mississauga, ON, Canada).
Amplification and sequencing
In order to assess the bacterial microbiota, PCR amplification of the
V4 hypervariable region of the 16S rRNA gene using the forward pri-
mer S-D-Bact-0564-a-S-15 and reverse primer S-D-Bact-0785-b-A-18
was performed as reported previously.12 The forward and reverse pri-
mers were designed to contain an overlapping region of the forward
and reverse Illuminaâ sequencing primers (Illuminaâ; San Diego, CA,
USA) in order to anneal them to primers containing the Illuminaâ
adaptors plus the 8 bp identifier indices.
After amplification, the PCR products were evaluated by elec-
trophoresis in 2% agarose gel and purified with the Agencourt
AMPure XP (Beckman Coulter Inc.; Mississauga, ON, Canada) by
mixing 20 lL of amplicon with 40 lL of AMPure on a 96 well plate.
After incubation for 5 min at room temperature, the beads were sep-
arated and washed twice with 80% ethanol and then eluted in 32 lL
of 10 mM Tris pH 8.5 buffer solution.
A second PCR was performed to attach dual indices and Illuminaâ
sequencing adapters using the Nextera XT Index kit (Illuminaâ). After
purification of these amplicons, the samples were quantified by spec-
trophotometry using the NanoDropâ (Roche) and normalized to a
final concentration of 2 nM. Normalization and sequencing of the
library pool was performed at the University of Guelph’s Advanced
Analysis Centre using an Illuminaâ MiSeq and 2 9 250 kit.
Data analysis
The MOTHUR package of algorithms (v1.39.5) was used for analy-
sis.13 Paired end reads were aligned. Sequences >242 or <239 bp
were removed, as well as those containing ambiguous base calls,
long runs of holopolymers (>8 bp) or those that did not align with the
correct region. Chimeras were detected using UCHIME and
2 © 2019 ESVD and ACVD, Veterinary Dermatology
removed.14 Sequences from chloroplasts, mitochondria, Archaea
and Eukaryotes were removed. Sequences were binned into OTUs
at a 3% (0.03) dissimilarity level using the nearest-neighbour
approach, and taxonomy assigned to both individual sequences and
OTUs using the RDP database.15 Subsampling was performed to nor-
malize sequence number across samples for assessment of alpha
and beta diversity to a depth of 19,451 sequences. In order to maxi-
mize sequence depth, samples that yielded relatively low sequence
numbers (<20,000 sequences) were repeated. One ear sample was
selected at random from each control dog for comparison between
affected and control ears. For dogs in which a sample from both ears
was available, one ear was assigned a value between 1 and 50 and
the other between 51 and 100. A random number generator was
used to generate a value between 1 and 100 and the sample to be
used for analysis was selected based on the resulting value.
Richness (Chao1), evenness (Shannon’s evenness) and alpha
diversity (inverse Simpson’s) indices were calculated. Comparison of
the relative abundances of taxa at different levels (phylum to species)
was performed using Wilcoxon-Mann-Whitney U-test in JMPâ
(v13.2.0, SAS Institute Inc.; Cary, NC, USA); P < 0.05 was consid-
ered significant for all comparisons. Adjustment for false discovery
rate was performed using the Benjamini–Hochberg technique16 in R
(v3.3.3, “Another Canoe”, RStudio Inc., 2017). Beta diversity was
assessed through the creation of dendrograms using both the Yue &
Clayton measure of dissimilarity (a measure of community structure,
which considers shared OTUs and their relative abundances) and the
traditional Jaccard coefficient (a measure of community member-
ship, which considers the number of shared OTUs, not their abun-
dance). Unweighted UniFrac tests were applied to evaluate the
impact of otitis externa on microbial population structure.17 Principal
coordinate analysis (PCoA) was performed based on both the Jaccard
coefficient and Yue & Clayton measure. Linear discriminant analysis
effect size (LEfSe) was used to identify OTUs that were enriched or
depleted.18
Results
From the total of 60 affected ears sampled, 31 were
removed from analysis of bacterial microbiota due to poor
DNA yield or repeated inability to obtain adequate
sequence numbers. Of the 20 control ears sampled, five
were removed from analysis of bacterial microbiota due
to repeatedly low numbers of sequences or DNA yield.
This left 29 samples from 18 affected dogs and 15 sam-
ples from eight control dogs for the analysis (Table 1). For
the purpose of comparing results between affected and
control groups, only one ear was selected at random
reducing the total number to 18 samples in affected dogs
and eight samples in control dogs. Dogs enrolled into the
affected group (those with otitis externa) ranged from
one to 10.5 years of age (median = 6.25 years) and con-
sisted of six spayed females, three intact females and
nine castrated males. Twelve pure-bred and six mixed
breed dogs were enrolled into the affected group. Four
spayed females and four castrated males ranging from
one to nine years of age (median = 5.75 years) were
enrolled into the control group. This group consisted of
five pure-bred and three mixed breed dogs.
After quality control processing, the total number of
sequences from the ears of healthy dogs was 805,196
with a median of 55,852 sequences per sample
(range = 27,113–80,634). The total number of sequences
found in affected ears was 1,706,195 with a median of
47,364 sequences per sample (range = 19,451–141,643).
Bacteria from 27 different phyla were identified. Relative
abundances were used to identify the predominant bacterial
 Otic microbiota in dogs with otitis externa

organisms within the ear canal. Relative abundance is the
percentage composition of an organism of a particular type
relative to the total number of organisms in that community.
The predominant bacteria identified in healthy ear canals
were from the phyla Firmicutes, Proteobacteria and Bac-
teroidetes. Within the phylum Firmicutes, the three most
common genera in healthy ears were Romboutsia, Mega-
monas and Holdemanella. Within the phylum Proteobacte-
ria, the three most common genera were Succinivibrio,
Haemophilus and Anaerobiospirillum. Prevotella, Bac-
teroides and Porphyromonas were the three most com-
mon genera within the phylum Bacteroidetes (Figure 1).
In affected ear canals, the phylum Firmicutes was the
most abundant, followed by Actinobacteria and Pro-
teobacteria. Within the phylum Firmicutes, the three
most common genera were Staphylococcus, Streptococ-
cus and Blautia. The three most common genera in the
phylum Actinobacteria were Corynebacterium, Bifidobac-
terium and Collinsella. The most common genera in the
phylum Proteobacteria were Pseudomonas, Escherichia/
Shigella and Acinetobacter (Figure 1).
After controlling for false discovery rate, the relative
abundances of 24 taxa were higher in control samples
when compared to affected samples, including the phyla
Firmicutes (P = 0.008), Bacteroidetes (P < 0.001) and the
genera Rombutsia (P < 0.001), Fusobacterium (P =
0.021) and Megamonas (P < 0.001). There were no sig-
nificant findings in the affected group (Table 2).
LEfSe analysis identified 164 OTUs that were differen-
tially abundant (P < 0.05). One hundred and fifty-three
OTUs were over-represented in healthy ears, including
the genus Rombutsia, Anaerobiospirillum, Corynebac-
terium, Megamonas and Faecalibacterium. Eleven OTUs
were over-represented in the affected ears, including
those from the genera Staphylococcus, Pseudomonas,
Parvimonas, Bacteroides and Actinomyces (Figure 2). Of
the 153 OTUs over-represented in healthy ears, 85 OTUs
(55.6%) were from the phylum Firmicutes; 58 of the 85
(68.2%) were from the order Clostridiales.
Observed richness (P = 0.004), estimated richness
(P < 0.001), evenness (P = 0.004) and diversity (P =
0.002) were significantly lower in affected ear canals
(Figure 3). Unweighted Unifrac analysis indicated that
there were significant differences in both bacterial com-
munity structure (P < 0.001) and membership (P <
0.001) between affected and control ears. These differ-
ences were depicted using PCoA plot and dendrogram
that demonstrate clearly separated clusters of samples
from healthy dogs and those with otitis externa (Fig-
ure 4).
A PCoA also was performed to compare beta diversity
of samples within the same dog and samples between
dogs. In dogs with bilateral otitis externa, the majority of
samples from the same dog were clustered closely
together. Samples from the same dog were more similar
to each other, than to samples from the ears of other
affected dogs (Figure 5). In contrast, ears from the
same healthy dog appeared to be no more similar to each
other than those of samples from other control dogs
(Figure 6).

Table 1. Signalment, cytological results and concurrent medications of the affected (n = 18) and healthy dogs (n = 8) in which otic samples were
sequenced
Case # Health status Sex Breed Age (years) Ear Cytology Concurrent medication
A1 Affected FI Miniature pinscher 9 Left 4+ rods & cocci None
A2 Affected MN Jack Russell terrier 10.5 Left 4+ yeast Oclacitinib
A3 Affected FS Fox terrier 8 Right 4+ rods & cocci None
A4 Affected MN Mixed breed 6 Right 4+ cocci & rods None
A5 Affected MN German shepherd dog 1 Left 4+ rods & cocci Oclacitinib
A6 Affected FS Maltese terrier 6.5 Right 4+ cocci Oclacitinib
A7 Affected FS Chihuahua 7.5 Left 4+ yeast None
A8 Affected MN French bulldog 1.5 Right 2+ yeast None
A9 Affected FS Boxer 1 Right 3+ yeast Oclacitinib
A10 Affected FI Mixed breed 1 Left 4+ yeast None
A11 Affected MN Spino 2 Right 4+ rods, Prednisone
Italiano 3+ cocci,
1+ yeast
A12 Affected MN Mixed breed 9 Left 4+ rods & cocci Prednisone
A13 Affected FS Mixed breed 9 Right 4+ rods, 2+ cocci Prednisone
A14 Affected MN Mixed breed 5 Left 4+ rods & cocci Meloxicam
A15 Affected FI Mixed breed 2.5 Right 4+ yeast None
A16 Affected MN German shepherd dog 6 Left 3+ cocci, 4+ yeast None
A17 Affected MN Mastiff 7.5 Right 4+ cocci Prednisolone/trimeprazine tartate
A18 Affected FS Cocker 7 Right 4+ rods & cocci None
spaniel
C1 Healthy FS Mixed breed 8 Left NSF None
C2 Healthy FS Dachshund 9 Right NSF None
C3 Healthy MN Bernese mountain dog 5.5 Left NSF Meloxicam
C4 Healthy MN Mixed breed 6 Left NSF Meloxicam
C5 Healthy FS Mixed breed 4 Right NSF Meloxicam
C6 Healthy MN Pug 6 Right NSF None
C7 Healthy FS Cane corso 2.5 Left NSF None
C8 Healthy MN Havanese 4 Right NSF None
FI female intact, FS female spayed, MN male neutered, NSF no significant findings.

© 2019 ESVD and ACVD, Veterinary Dermatology 3

Korbelik et al.

samples from healthy dogs. Beta diversity also was

Discussion
This study identified a much richer bacterial community
than had been identified previously using culture-based
methods,19,20 as was expected based on the advantages
of sequence-based methods. Comparison of the micro-
biota of healthy dogs versus those with otitis externa
identified various interesting differences. Bacterial alpha
diversity was reduced in samples from the ear canal of
dogs affected by otitis externa when compared to
altered in affected ears when compared to healthy con-
trols. This is consistent with previous studies in dogs and
humans, which have shown decreased cutaneous bacte-
rial diversity in samples from atopic individuals when
compared to healthy controls.9,10,21,22 These changes
indicate that there were significant differences not only in
the overall membership of the population, but also the
distribution of those members within the microbiota.
Thus, the changes present go well beyond simple

Figure 1. Relative abundance of the most common bacterial taxa isolated from the ear canal of healthy dogs (C1 to C8) and those affected with
otitis externa (A1 to A18).

Table 2. Relative abundance of taxa that were significantly different in the aural microbiota of dogs with otitis externa (affected, n = 18) or healthy
controls (control, n = 8)
Taxonomic level Taxon Control median Affected median P-value Adjusted P-value
Phylum Firmicutes 71.4 30.7 0.008 0.023
Phylum Bacteroidetes 4.97 0.12 <0.001 0.001
Genus Romboutsia 26.9 0.018 <0.001 0.001
Genus Fusobacterium 0.75 0.026 0.021 0.049
Genus Megamonas 9.12 0.006 <0.001 0.001
Genus Faecalibacterium 5.98 0.026 <0.001 0.001
Genus Catenibacterium 5.7 0.004 <0.001 0.001
Genus Holdemanella 6.05 0.035 <0.001 0.001
Genus Parvimonas 0.353 0 <0.001 0.001
Genus Lactobacillus 2.4 0.047 <0.001 0.001
Genus Bacteroides 1.77 0.026 0.001 0.003
Genus Bifidobacterium 1.41 0.194 0.005 0.015
Genus Parasutterella 0.189 0 <0.001 0.001
Genus Clostridium s.s. 1.64 0.031 <0.001 0.001
Genus Prevotella 2.62 0.01 <0.001 0.001
Genus Haemophilus 1.63 0.003 <0.001 0.001
Genus Succinivibrio 2.08 0.008 <0.001 0.001
Genus Turicibacter 1.6 0.004 <0.001 0.001
Genus Porphyromonas 0.142 0.005 0.003 0.009
Genus Neisseria 0.102 0 0.012 0.03
Genus Anaerobiospirillum 1.07 0.006 <0.001 0.001
Genus Phascolarctobacterium 0.301 0.021 0.009 0.026
Genus Blautia 0.68 0.054 <0.001 0.001
Genus Actinomyces 0.177 0.008 0.008 0.023
Wilcoxon-Mann-Whitney U-tests were used to compare groups. P-values were adjusted using the Benjamini–Hochberg technique.

4 © 2019 ESVD and ACVD, Veterinary Dermatology


Figure 2. Operational taxonomic units (OTUs) identified by linear discriminant analysis effect size analysis as differentially abundant (P < 0.05) in
the aural microbiota of dogs affected with otitis externa (n = 18) and healthy control dogs (n = 8).
Only OTUs with a linear discriminant analysis (LDA) >3.4 are depicted.
changes in one or a few individual components of the
microbiota.
Bacteria from 27 different phyla were identified in the
ear canal of normal dogs. This is higher than a previous
study that used next generation sequencing to assess
the microbiota of healthy dogs and dogs with AD but
without clinical signs of otitis externa.22 In that study, the
most common phyla identified in normal ear canals were
Proteobacteria, Actinobacteria, Firmicutes and Bac-
teroidetes.22 Although our study also identified these
same four phyla as the most common, the order was
altered, with the phylum Firmicutes being the most com-
mon, followed by Proteobacteria, Bacteroidetes and then
Actinobacteria. In both studies, the order of the most
common phyla found in affected ears was the same: Fir-
micutes was the most common, followed by Actinobac-
teria and Proteobacteria.22 Although a preliminary study
found that the microbial community did not appear to be
influenced by individual factors and environment in dogs,9
several other studies indicate that these factors do affect
cutaneous microbiota.23,24 This may explain any differ-
ence in findings between our two studies.22 Additionally,
this study employed primers which encoded the V1–V3
region of the 16s rRNA gene, utilized a different DNA
extraction kit and assigned taxonomy using a different
© 2019 ESVD and ACVD, Veterinary Dermatology
Otic microbiota in dogs with otitis externa
database than our study.22 These dissimilarities in
methodology could also account for differences between
our studies. Standard approaches, including primer
choices, sequence platforms and bioinformatics meth-
ods, are not yet defined and there is the potential for
some degree of variation based on methodology. It is
expected that these differences would be rather subtle,
but side-by-side comparison of different methods is lack-
ing. The V4 region was chosen here because it allows for
nearly full overlap of the sequence and primers, minimiz-
ing sequencing errors and OTU inflation.25
A comparison of relative abundance between affected
and control ear canals identified that bacteria from the
genus Porphyromonas were increased in samples from
normal ears when compared to those affected with otitis
externa. Another study also found that the genus Por-
phyromonas was preferentially decreased on the pinna
and axillae of dogs with AD.10 Bacteria from the genus
Porphyromonas are anaerobic, Gram-negative bacilli.26
Some species have been implicated in respiratory,
mucosal, dental and skin infections in both humans and
animals.26
The LEfSe analysis identified that the genus Escheri-
chia was preferentially increased in healthy ears while the
genus Staphylococcus was increased in affected ear
5
Korbelik et al.

Figure 3. Alpha diversity measurements in dogs with otitis externa (affected, n = 18) and healthy dogs (control, n = 8).
(a) Number of species observed; (b) estimated richness (Chao1); (c) estimated diversity (inverse Simpson’s diversity index); and (d) estimated
evenness (Shannon’s evenness index).

Figure 4. (a) Dendrogram depicting the Yue & Clayton measure of dissimilarity in the aural microbiota of dogs with otitis externa (red) and healthy
controls (blue).
(b) Principal coordinate analysis plot was generated by JMPâ using distance calculation performed in MOTHUR using the Jaccard coefficient met-
ric. Clear coverage differences are depicted between affected samples (red, n = 18) and control samples (blue, n = 8). Ellipsoid coverage is 50%.

canals. Another study, which assessed the microbiota of mentioned previously, this study targeted the V1–V3
healthy dogs and dogs with AD but without clinical signs region making directed comparison between studies diffi-
of otitis externa, had similar findings.22 It was suggested cult.22 Our findings suggest that a reduction in the genera
that these findings could indicate a dysbiosis of the ear Porphyromonas and Escherichia may be an important
canal and that bacteria from the genus Escherichia may indicator for the presence of a bacterial dysbiosis due to a
serve a protective role in the canine ear canal.22 As disease state.
6 © 2019 ESVD and ACVD, Veterinary Dermatology

Figure 5. Principal coordinate analysis plot of samples from affected
ears (n = 11) generated by JMPâ using distance calculation per-
formed in MOTHUR using the Jaccard coefficient metric. Samples
from the same dog are indicated using the same colour.
Figure 6. Principal coordinate analysis plot of samples from control
ears (n = 7) generated by JMPâ using distance calculation performed
in MOTHUR using the Jaccard coefficient metric. Samples from the
same dog are indicated using the same colour.
Bacterial from the genera Staphylococcus and Pseu-
domonas are commonly associated with otitis externa in
dogs.19,20 These two genera were identified by LEfSe
analysis to be over-represented in affected ears. Two
studies also found that the genus Staphylococcus was
increased on the skin and ears of dogs with AD.10,22 As a
cross-sectional study, we could not discern whether pro-
liferation of these genera, or the depletion of normal bac-
terial flora and subsequent replacement with these
genera, was the driving force for the development of oti-
tis externa. Otitis externa caused by Pseudomonas aerug-
inosa has been reported to have an incidence of 11–
© 2019 ESVD and ACVD, Veterinary Dermatology
Otic microbiota in dogs with otitis externa
13%.27 Pseudomonas aeruginosa is both intrinsically
resistant to many antibiotics and can rapidly acquire fur-
ther resistance through injudicious use of antibiotics.28
Pseudomonas aeruginosa also has been implicated in the
formation of biofilms, which is an important virulence fac-
tor that helps facilitate resistant bacterial infections within
the ear canal and hamper the elimination of infections by
both the immune system and antimicrobials.29
Interestingly, the genera Parvimonas and Fusobac-
terium were increased in dogs with otitis externa. Parvi-
monas micra, the only species in this genus,30 is able to
form biofilms and has been implicated in causing chronic
periodontitis in both humans and dogs.31,32 In conjunction
with other micro-organisms (including Fusobacterium
spp.), P. micra allows for the production of biofilms with
proteolytic activity, which causes progressive tissue
destruction and inflammation in canine periodontal dis-
ease.31 It is possible that P. micra has the same effects
in chronic otitis externa and as a strict anaerobe has been
overlooked in culture-based studies, although further
study would be needed to confirm this hypothesis.
The role of otitis externa caused by Corynebacterium
spp. has been disputed due to its presence as part of the
normal microbiota on the external ear canal and because
the genus has always been reported in combination with
other micro-organisms in cases of otitis externa.33 This
study found that the genus Corynebacterium was
increased in healthy ears. Analysis of relative abundance
did not find that this genus was significantly increased in
dogs with otitis externa. This would support the hypothe-
sis that this bacterium is not pathogenic and requires the
presence of other bacteria in order to proliferate.33
This study found that bacterial species diversity, richness
and evenness were significantly reduced in samples from
affected ears when compared to healthy ears. This is similar
to findings of a previous study, which also found decreased
bacterial richness and alpha diversity on the skin and ears of
dogs affected by AD.9,10 One study, which assessed the
differences in alpha diversity of the otic microbiota between
normal dogs and atopic dogs, did not find a significant differ-
ence between the two groups.22 However, samples from
atopic dogs in this study were collected from individuals
that did not show evidence of otitis externa.22
In addition to the decrease in alpha diversity, there was
a clear decrease in bacterial beta diversity in dogs with
otitis externa. Significant alterations in both community
membership and structure were present within the
microbiota of healthy dogs and those with otitis externa.
These findings were similar to a previous study that
assessed the differences between the otic microbiota of
healthy dogs and those with AD (but without evidence of
otitis externa).22 Therefore, the changes noted go beyond
simple alterations in individual components of the bacte-
rial microbiota but affected its entire composition.
In dogs, interindividual variability of the skin microbiome
is less than intraindividual variability when assessing vari-
ous body sites.9 PCoA plots to compare the bacterial com-
munity and membership structure between ear canals in
healthy dogs indicated that samples from within the same
dog were not more similar to each other, than to samples
from ears of other dogs. However, PCoA plots of dogs with
bilateral otitis externa indicated that samples from within
7
Korbelik et al.
the same dog were more similar to each other, than to
samples from the ears of other affected dogs. A decrease
in diversity and proliferation of a few bacterial genera in
affected ears could cause samples from the same dog to
become more uniform and thus cluster closer together.
A limitation of this study is that several of the dogs
enrolled in the affected group were receiving imm-
unomodulatory therapy at the time of sampling. One
study determined that treatment with ciclosporin and cor-
ticosteroids does not significantly impact the cutaneous
microbiota.34 It is unknown whether treatment with
oclacitinib, immunotherapy or antihistamines could alter
the cutaneous microbiota. Additionally, we had difficulty
obtaining adequate DNA and sequence yields from sam-
ples, resulting in a large proportion of samples being
removed from analysis. Because a larger proportion of
samples were removed from the affected group (51.7%)
than the control group (25%), we hypothesize that secre-
tions in affected ears may produce a large number of PCR
inhibitors, which interfere with DNA extraction. Low bac-
terial DNA burdens in control samples may be responsible
for poor DNA and sequence yields. This led to the analy-
sis of samples from only a subset of the enrolled popula-
tion and thus it is unclear whether this could have
introduced bias into this study. Additional cycles during
DNA amplification may have improved DNA yield but this
was avoided as it could have also introduced bias result-
ing in less meaningful results.
Next Generation Sequencing has revealed that the bac-
terial microbiota is much more complex than identified
previously using culture-based studies. This study identi-
fied the composition of the bacterial microbiota of the
healthy canine ear canal and how it is altered in otitis
externa. Reduced bacterial richness and diversity is pre-
sent in ears affected with otitis externa indicating that a
dysbiosis is present. This study did not discriminate
between different underlying causes of otitis externa and
further study is needed to investigate whether alterations
in the microbiota are present in otitis externa caused by
different primary disease states.
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Re sume 
Contexte – L’otite externe est une maladie multifactorielle fre quente chez le chien. La diversite  du micro-
biote cutane  chez le chien semble diminuer dans les e tats pathologiques. Cependant, on sait peu de chose
sur le microbiote de l’oreille canine et comment il est alte  re
 en fonction des maladies.
Hypothe ses/Objectifs – De crire le microbiote bacte rien auriculaire des chiens avec otite externe compare 
aux chiens sains.
Sujets – Des e chantillons ont e  te
 pre
leves de 18 chiens avec des signes cliniques et cytologiques d’otite
externe et huit chiens cliniquement sains sans signe cytologique d’otite externe.
Mate riel et me thode – L’ADN de chaque e chantillon a e  te
 isole
 et un sequencßage a e  te
 re
alise par Illu-
minaâ pour la re gion hypervariable V24 et les amplicons du ge ne 16S ARNr. Les se quences ont e  te

realise
es a l’aide du logiciel MOTHUR.
Re sultats – Les bacte ries de 27 phyla diffe rents ont e te
 identifie es. Les oreilles atteintes avaient une diver-
site alpha significativement plus basse compare e aux oreilles saines. La structure communautaire et
l’appartenance diffe raient aussi entre les deux groupes. Une analyse discriminante line aire de l’effet taille a
identifie 153 OTUs (operational taxonomic units) qui e taient diffe rentiellement abondant. Onze OTUs
taient surrepre
e sentes dans les oreilles atteintes, dont Staphylococcus, Pseudomonas et Parvimonas.
Conclusions – Le microbiote bacte rien auriculaire est bien plus complexe que ce qui avait e  te
 mis en evi-
dence par les e tudes anterieures base es sur les cultures; l’otite externe est accompagne e par des diffe ren-
ces larges et complexes du microbiote.

RESUMEN
Introduccio  n – la otitis externa es una enfermedad multifactorial comu n en los perros. La diversidad de la
microbiota cutanea en perros parece disminuir en estados de enfermedad. Sin embargo, poco se sabe
sobre la microbiota del oıdo canino y co mo se altera por enfermedad.
Hipo tesis/objetivos – describir la microbiota bacteriana o tica en perros con otitis externa en comparacio n
con perros sanos.
Animales – se recogieron muestras de 18 perros con pruebas clınicas y citolo gicas indicativas de otitis
externa y ocho perros clınicamente normales sin pruebas citolo gicas de otitis externa.
Me todos y materiales – se aislo  el DNA de cada muestra y se realizo  la secuenciacio
n con Illuminaâ de la
n hipervariable V4 de los amplicones del gen 16S rRNA. Las secuencias fueron procesadas utilizando
regio
el software de bioinformatica MOTHUR.
Resultados – Se identificaron bacterias de 27 filos diferentes. Las orejas afectadas tuvieron una dismi-
nucion significativa de la diversidad alfa en comparacio n con las orejas sanas. La estructura de la comuni-
dad y las especies tambie n diferıan entre los dos grupos. El an
alisis discriminante y el an
alisis del efecto en
el taman~o identificaron 153 unidades taxono micas operativas (OTU) que eran diferencialmente abundan-
tes. Once OTUs estaban sobre representadas en los oıdos afectados, incluyendo Staphylococcus, Pseudo-
monas y Parvimonas.
Conclusiones – la microbiota bacteriana o tica es mucho m as compleja de lo que se ha identificado en estu-
dios previos basados en cultivos; la otitis externa se acompan ~a de amplias y complejas diferencias en la
microbiota.

Zusammenfassung
Hintergrund – Die Otitis externa ist eine h€
aufige multifaktorielle Erkrankung von Hunden. Die Diversit€at
der kutanen Mikrobioma bei Hunden scheint in Krankheitszust€ anden abzunehmen. Es ist jedoch wenig
€ber das Mikrobiom des Hundeohres und wie es sich im Krankheitszustand ver€
bekannt u andert.
Hypothese/Ziele – Eine Beschreibung der otischen bakteriellen Mikrobioma bei Hunden mit Otitis externa
im Vergleich zu gesunden Hunden.Tiere – Es wurden Proben von 18 Hunden mit klinischer und zytologi-
scher Evidenz einer Otitis externa genommen sowie von acht klinisch normalen Hunden ohne zytologi-
scher Evidenz einer Otitis externa.
Methoden und Materialien – Es wurde DNA von jeder Probe isoliert und Illuminaâ Sequenzierung der V4
hypervariablen Region des 16S rRNA Gen Amplikons durchgefu €hrt. Die Sequenzen wurden mittels Bioin-
formatics Software MOTHUR verarbeitet.
Ergebnisse – Bakterien von 27 unterschiedlichen Phyla wurden identifiziert. Die betroffenen Ohren zeigten
eine signifikant verminderte Alpha Diversit€ at im Vergleich zu gesunden Ohren. Die Struktur der

© 2019 ESVD and ACVD, Veterinary Dermatology 9

Korbelik et al.

Gemeinschaft und Zugeho €rigkeit unterschied sich ebenfalls zwischen den zwei Gruppen. Die
Groۧenanalyse der linearen Diskriminanzanalyse identifizierte 153 funktionierende taxonomische Einheiten
(OTUs), die unterschiedlich reichlich vorhanden waren. Elf OTUs waren u €berrepr€
asentiert in den betroffe-
nen Ohren, inklusive Staphylococcus, Pseudomonas und Parvimonas.
Schlussfolgerungen – Das otische bakterielle Mikrobiom ist wesentlich komplexer als bisher mittels Kul-
tur-basierter Studien herausgefunden werden konnte; die Otitis externa wird von breiten und komplexen
Unterschieden im Mikrobiom begleitet.

要約
背景 – 外耳炎は犬の一般的な多因子疾患である。犬の皮膚細菌叢の多様性は病的状態で減少するようで
ある。しかし、犬の耳細菌叢および耳細菌叢が病気によってどのように変化するかについてはほとんど
わかっていない。
仮説/目的 – 本研究の目的は、健常犬と比較した外耳炎犬の耳細菌叢を記述することである。
被験動物 – 外耳炎の臨床的および細胞学的証拠のある18頭の犬、および外耳炎の細胞学的証拠のない8頭
の臨床的に健常な犬からサンプルを採取した。
材料と方法 – 各サンプルからDNAを分離し、16S rRNA遺伝子アンプリコンのV4超可変領域のIllumina®シ
ーケンスを実施した。バイオインフォマティクスソフトウェアMOTHURを用いて塩基配列を処理した。
結果 – 27の異なる系統の細菌を同定した。罹患耳は、健常耳と比較した場合、a多様性を有意に減少させ
た。群集構造およびメンバーシップも2グループ間で異なった。線形判別分析効果量解析は、差別的に豊
富であった153の運用分類単位(OTU)を識別した。 Staphylococcus、Pseudomonas、Parvimonasなど、
11OTUが罹患耳に過剰発現していた。
結論 – 耳細菌叢は、既存の培養ベースの研究で確認されたものよりもはるかに複雑であ理、さらに、外
耳炎は細菌叢において広く複雑な差異を伴っている。

摘要
背景 – 犬外耳炎是常见的多因素疾病。犬皮肤微生物群的多样性似乎在患病状态下降低。然而,人们对犬耳
道微生物群及其如何被疾病改变知之甚少。
假设/目标 – 描述与健康犬相比,外耳炎患犬的耳道细菌菌群。
动物 – 从18只临床和细胞学都证明患有外耳炎的犬,以及8只临床和细胞学证明无外耳炎正常犬的耳道采集
样品。
方法和材料 – 分离每个样品的DNA,进行16S rRNA基因V4高变区的扩增,并送Illumina®公司测序。使用
生物信息学软件MOTHUR处理序列。
结果 – 共鉴定出27种不同门的细菌。与健康耳道相比,患病耳道的a多样性明显降低。两组之间的细菌种群
结构和种类也不同。线性判别分析的效应量分析确定了丰度差异的153个可操作分类单位(OTU)。患病耳道
中有11个OTU过多,包括葡萄球菌、假单胞菌和细单胞菌等。
结论 – 耳道细菌菌群比以往培养研究所鉴定的要复杂得多; 外耳炎常伴随广泛而复杂的微生物群差异。

Resumo
Contexto – A otite externa e  uma doencßa multifatorial comum em c~ aes. A diversidade da microbiota
cut^anea parece sofrer reducß~ao durante estados patolo gicos. Entretanto, pouco se sabe sobre a microbiota
da orelha canina e as suas alteracßo~es causadas por enfermidades.
Hipo  tese/Objetivos – Descrever a microbiota bacteriana o tica em c~ aes com otite externa comparada a
c~aes saud aveis.
Animais – Coletou-se amostras de 18 c~aes com evide ^ncia clınica e citolo
gica de otite externa, e oito c~
aes
clinicamente normais sem evide ^ncia citolo
gica de otite externa.
Me todos e materiais – O DNA de cada amostra foi isolado e realizou-se o sequenciamento da regi~ ao
hipervariavel V4 dos amplicons do gene 16S rRNA. As seque ^ncias foram processadas utilizando o software
de bioinformatica MOTHUR.
Resultados – Bacte rias de 27 filos diferentes foram identificadas. As orelhas afetadas apresentaram alfa
diversidade significativamente reduzida comparado a orelhas normais. Houve tambe m diferencßa entre os
grupos na estrutura da comunidade e no parentesco. A an alise do efeito da an alise discriminante linear
identificou 153 unidades taxono ^micas operacionais (operational taxonomic units, OTUs) que foram diferen-
temente abundantes. Onze OTUs estavam super-representadas nas orelhas afetadas, incluindo Staphylo-
coccus, Pseudomonas e Parvimonas.
Concluso ~ es – A microbiota bacteriana otica e
 muito mais complexa do que havia sido identificado anterior-
mente nos estudos baseados em cultura; a otite externa est a acompanhada de diferencßas amplas e com-
plexas na microbiota.

10 © 2019 ESVD and ACVD, Veterinary Dermatology