The Canine Oral Microbiome

Abstract

Determining the bacterial composition of the canine oral microbiome is of interest for two primary reasons. First, while the human oral microbiome has been well studied using molecular techniques, the oral microbiomes of other mammals have not been studied in equal depth using culture independent methods. This study allows a comparison of the number of bacterial taxa, based on 16S rRNA-gene sequence comparison, shared between humans and dogs, two divergent mammalian species. Second, canine oral bacteria are of interest to veterinary and human medical communities for understanding their roles in health and infectious diseases. The bacteria involved are mostly unnamed and not linked by 16S rRNA-gene sequence identity to a taxonomic scheme. This manuscript describes the analysis of 5,958 16S rRNA-gene sequences from 65 clone libraries. Full length 16S rRNA reference sequences have been obtained for 353 canine bacterial taxa, which were placed in 14 bacterial phyla, 23 classes, 37 orders, 66 families, and 148 genera. Eighty percent of the taxa are currently unnamed. The bacterial taxa identified in dogs are markedly different from those of humans with only 16.4% of oral taxa are shared between dogs and humans based on a 98.5% 16S rRNA sequence similarity cutoff. This indicates that there is a large divergence in the bacteria comprising the oral microbiomes of divergent mammalian species. The historic practice of identifying animal associated bacteria based on phenotypic similarities to human bacteria is generally invalid. This report describes the diversity of the canine oral microbiome and provides a provisional 16S rRNA based taxonomic scheme for naming and identifying unnamed canine bacterial taxa.

Full text

The Canine Oral Microbiome
Floyd E. Dewhirst
1,2
*, Erin A. Klein
1
, Emily C. Thompson
1
, Jessica M. Blanton
1
, Tsute Chen
1
, Lisa Milella
3
,
Catherine M. F. Buckley
4
, Ian J. Davis
4
, Marie-Lousie Bennett
5
, Zoe V. Marshall-Jones
4
1Department of Molecular Genetics, The Forsyth Institute, Cambridge, Massachusetts, United States of America, 2Department of Oral Medicine, Infection and Immunity,
Harvard School of Dental Medicine, Boston, Massachusetts, United States of America, 3The Veterinary Dental Surgery, Byfleet, United Kingdom, 4WALTHAM Centre for
Pet Nutrition, Melton Mowbray, United Kingdom, 5Mars Pet Care Europe, Birstall, United Kingdom
Abstract
Determining the bacterial composition of the canine oral microbiome is of interest for two primary reasons. First, while the
human oral microbiome has been well studied using molecular techniques, the oral microbiomes of other mammals have
not been studied in equal depth using culture independent methods. This study allows a comparison of the number of
bacterial taxa, based on 16S rRNA-gene sequence comparison, shared between humans and dogs, two divergent
mammalian species. Second, canine oral bacteria are of interest to veterinary and human medical communities for
understanding their roles in health and infectious diseases. The bacteria involved are mostly unnamed and not linked by
16S rRNA-gene sequence identity to a taxonomic scheme. This manuscript describes the analysis of 5,958 16S rRNA-gene
sequences from 65 clone libraries. Full length 16S rRNA reference sequences have been obtained for 353 canine bacterial
taxa, which were placed in 14 bacterial phyla, 23 classes, 37 orders, 66 families, and 148 genera. Eighty percent of the taxa
are currently unnamed. The bacterial taxa identified in dogs are markedly different from those of humans with only 16.4% of
oral taxa are shared between dogs and humans based on a 98.5% 16S rRNA sequence similarity cutoff. This indicates that
there is a large divergence in the bacteria comprising the oral microbiomes of divergent mammalian species. The historic
practice of identifying animal associated bacteria based on phenotypic similarities to human bacteria is generally invalid.
This report describes the diversity of the canine oral microbiome and provides a provisional 16S rRNA based taxonomic
scheme for naming and identifying unnamed canine bacterial taxa.
Citation: Dewhirst FE, Klein EA, Thompson EC, Blanton JM, Chen T, et al. (2012) The Canine Oral Microbiome. PLoS ONE 7(4): e36067. doi:10.1371/
journal.pone.0036067
Editor: Jacques Ravel, Institute for Genome Sciences, University of Maryland School of Medicine, United States of America
Received January 27, 2012; Accepted March 30, 2012; Published April 27, 2012
Copyright: ß2012 Dewhirst et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the WALTHAM Centre for Pet Nutrition. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: fdewhirst@forsyth.org
Introduction
Bacteria of the oral cavity have been studied with great interest
since Anton van Leeuwenhoek first examined the plaque between
his teeth with his crude microscope in 1683 [1]. Using cultivable
methods, approximately 300 species from the human oral cavity
have been isolated, characterized and formally named. Studies of
the oral microbiota of other vertebrates have been less extensive.
Unfortunately, bacteria from non-human sources were often
misidentified and misclassified based on phenotypic similarity to
human microorganisms. With the advent of molecular identifica-
tion methods, primarily based on 16S rRNA sequence analysis, it
has become apparent that bacteria from different vertebrate hosts
are frequently unique, despite similar biochemical and other
phenotypic traits. While molecular methods have been valuable in
clarifying the identification and taxonomy of isolates, the greatest
strength of these methods is in the identification of the majority of
organisms which are currently uncultivated. Studies with molec-
ular methods have demonstrated that the bacterial diversity in
most environments is severely underestimated in surveys with
cultivation-based methods [2,3].
While the human oral microbiome has been surveyed using
culture-independent methods [4], the canine oral microbiome has
not. Previous canine studies were based primarily on culture-
dependant methods and sometimes sought to identify species
commonly found in human plaque [5,6,7,8].
The primary purpose of this study was to identify major species
of bacteria present in canine oral microbiome through an
examination of subgingival plaque using culture-independent
methods. This study reports on the analysis of 5,958 16S rRNA
sequences from 65 clone libraries and provides 416 full 16S rRNA
reference sequences (.1500 base) for the 353 taxa identified. As
the vast majority of these taxa are not formally named, a
provisional taxonomic scheme is presented based on assigning
each taxon to the closest genus or higher taxa, and assigning it a
unique Canine Oral Taxon number.
Materials and Methods
Ethics Statement
Dogs were recruited in the UK from a kenneled population and
from client owned dogs presented at a specialist veterinary clinic;
informed client consent was obtained. Two studies were
performed as follows: subgingival plaque was collected from 20
dogs in the first study (10 of which were from a kenneled
population) and from 31 dogs in the second. The studies were
approved by the WALTHAM Centre for Pet Nutrition ethical
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review committee, and run under licensed authority in accordance
with the UK Animals (Scientific Procedures) Act 1986.
Plaque collection and DNA isolation
Animals were sampled under anesthesia. Each dog was given a
premedication of 0.02 mg/kg acepromazine (ACP 2 mg/ml) and
0.02 mg/kg buprenorphine (Vetergesic 0.3 mg/ml) intramuscu-
larly, then induced with 0.4 mg/kg propofol (Rapinovet 10 mg/
ml) given intravenously, and maintained on 2% inhalational
isoflurane. Initially supra-gingival and gingival margin plaque and
calculus were removed using a Gracey curette to prevent
contamination of the sub-gingival sample. A periodontal probe
was then inserted under the gingival margin and swept along the
tooth surface. Plaque from at least eight teeth was pooled. The
resulting subgingival plaque pool from each dog was suspended in
a 350 ml solution of 50 mM Tris (pH 7.6), 1 mM EDTA (pH 8.0)
and 0.5% Tween 20 and was immediately stored at 220uC prior
to DNA extraction. DNA extraction was performed using the
DNeasy Tissue Kit (Qiagen, Valencia, California) following the
manufacturer’s instructions for the isolation of genomic DNA from
Gram-positive bacteria (which also works well for Gram-negative
bacteria). For the second study DNA extraction was performed
using the Masterpure Gram Positive DNA Purification Kit
(Epicentre, USA), according to the manufacturer’s instructions
with an additional overnight lysis as follows. Plaque samples were
centrifuged at 50006g for 10 minutes and the cell pellet
resuspended in 150 ml of TE buffer (10 mM Tris-Cl and
0.5 mM EDTA, pH 9.0). Following vortexing, 1 ml Ready-Lyse
Lysozyme (Epicentre, UK) was added and the lysis mix incubated
overnight at 37uC for 18 hrs. Following the extraction, DNA was
resuspended in TE buffer.
DNA amplification
DNA samples purified from subgingival plaque of 20 dogs in
study 1 were individually amplified with ‘‘universal’’ primers F24/
Y36 (9-29F/1525-1541R) to construct 20 libraries. The sequences
of primers are given in Table S1 in the supplemental materials.
Purified DNA from the 10 of the 20 dogs was also combined into 4
pools (each pool from 2 or 3 dogs), and each pool was amplified
individually with ‘‘Bacteroidetes-selective’’, F24/F01 or ‘‘Spiro-
chaetes-selective’’, F24/M98, primers to give eight additional
libraries. In study 2, DNA samples purified from subgingival
plaque of 31 dogs were individually amplified with ‘‘universal’’
primers F24+AD35/C72 (9-27F [YM+B]/1492-1509R) to con-
struct 31 libraries. The forward primer was a combination of 4
parts of the 4-fold degenerate 9–27 ‘‘YM’’ primer F24 and one
part Bifidobacteriales primer AD35 (modified from Frank et al. [9],
to give a 5-fold degenerate primer mix for enhanced phylogenetic
coverage. Equal amount of DNA from 3 sets of ten to eleven dogs
were pooled to give 3 DNA super-pools. The three super-pools
were amplified individually with ‘‘Bacteroidetes-selective’’, F24/
F01 and ‘‘Spirochaetes-selective’’, F24/M98, primers to give six
additional libraries.
PCR was performed in thin-walled tubes with a Perkin-Elmer
9700 Thermocycler. One ml of the purified DNA template was
added to a reaction mixture (50 ml final volume) containing 20
rmole of each primer, 40 nmole of dNTPs, 2.5 units of Platinum
Taq polymerase (Invitrogen, Carlsbad, CA) in 106PCR buffer
(200 mM Tris-HCl pH 8.4, 500 mM KCl). In a hot start protocol,
samples were preheated at 94uC for 4 min followed by
amplification using the following conditions: denaturation at
94uC for 45 s, annealing at 60uC for 45 s, and elongation at
72uC for 1.5 min with an additional 1 s for each cycle. A total of
30 cycles were performed and then followed by a final elongation
step at 72uC for 15 min. The size and amount of each amplicon
was examined by electrophoresis in a 1% agarose gel. DNA was
stained with SYBR Safe DNA gel stain (Invitrogen, Carlsbad, CA)
and visualized under UV light. After checking that a strong
amplicon of the correct size was produced, a second preparative
gel was run and the full length amplicon band was cut out and
purified using a Qiagen Gel Extraction kit (Qiagen, Valencia, CA).
Cloning and Library Screening procedures
Size-purified PCR amplified DNA was cloned using a TOPO
TA Cloning Kit as previously described [4]. Approximately 90
colonies were picked for each library. Clones were amplified using
M13 forward and reverse primers and amplicon purified as
previously described [4].
16S rRNA Sequencing
Purified DNA was sequenced using an ABI prism cycle-
sequencing kit (BigDyeHTerminator Cycle Sequencing kit) on
an ABI 3100 Genetic Analyser (Applied Biosystems, Foster City,
CA). The sequencing primers, Table S1 in supplementary
materials, were used in a quarter-dye chemistry following the
manufacturer’s instructions.
16S rRNA data analysis
Approximately 500 bases of sequence were determined using
primer Y31 (519–533R) to allow preliminary identification of
clones. If the clone sequence appeared novel (differing by more
than 7 bases from previously identified canine oral reference
sequences), a full sequence of approximately 1,500 bases was
obtained using 6 to 8 sequencing primers for full double strand
coverage (Table S1). The sequencing primers used over the course
of the two studies evolved. Primers in Table S1 which failed to
produce readable sequence for multiple taxa due to mismatches
are labeled ‘‘limited’’ and were not used in subsequent studies.
Primers which proved successful empirically and by alignment
with human and canine oral reference sequences are labeled
‘‘general’’. Full 16S rRNA sequences were assembled from the
ABI electropherogram files using Sequencher (Gene Codes
Corporation, Ann Arbor, Michigan). Programs for data entry,
editing, sequence alignment, secondary structure comparison,
similarity matrix generation, and phylogenetic tree construction
were written by F.E. Dewhirst [10]. Consensus neighbor-joining
trees [11] were constructed from our aligned sequences using
MEGA 4 [12]. The similarity matrices were corrected for multiple
base changes at single positions by the method of Jukes and Cantor
[13]. Comparisons with missing data were eliminated pairwise.
The consensus trees were based on 1,000 bootstrap resamplings.
Sequences were checked for the possibility of being chimeric
using a custom program [4] which checked the phylogenetic
distance between the best BLAST match of the ends of each
sequence with the canine reference set excluding self matches.
Sequences whose ends diverged .5% were examined using
Mallard [14] and heuristically for sequence consistency with
phylogenetic neighbors in our overall sequence alignment sorted
phylogenetically.
Nucleotide Sequences
The full 16S rRNA sequences for 416 clones representing 353
canine oral taxa were deposited in GenBank and received
accession numbers JN713151–JN713566. The accession numbers
are also included for each phylotype in Figs. 1, 2, 3, and 4. The
partial 16S RNA sequences (the 59-end ,500 bases) of 5,959
clones were deposited in GenBank as JQ294075–JQ300033.
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Results and Discussion
Oral samples for 16S rDNA clone library construction came
from a wide variety of dog breeds. The breed and age of each dog
for each library is given in Table S2 of supplementary materials.
The breeds examined include large (Saint Bernard) and small
(Papillion) breeds, and those with long (Australian Collie) and short
(Shih Tzu) snouts and ranged in age from 3 to 8 years old. While
the breeds examined in this study are originally from geograph-
ically diverse locations, the dogs sampled are from a limited area of
the United Kingdom. Thus, future studies employing samples
from dogs living in different countries could well find additional
canine microbial diversity. Because the 51 dogs examined came
from 25 breeds, there was no attempt to compare microbiomes
between breeds as the number of dogs/breed were too low.
Cloning studies
A total of 6,025 clones were examined from 65 libraries of
approximately 90 clones per library. Sixty-seven clones which had
sequences shorter than 350 trimmed bases or which were found to
be chimeric were excluded for a total of 5,958 validated clones
used for analyses. The validated clones from the first cloning
library were initially grouped into provisional phylotypes based on
their 500 base partial sequences. A full sequence was then
determined for a representative of each phylotype. The phylotypes
were given arbitrary Canine Oral Taxon numbers (COT-001
through COT-399) in the order they were identified and the full
length sequences used as a reference set against which subsequent
clones were examined by BLASTN analysis. In this study, a
phylotype or COT is defined as a set of one or more 16S rRNA
sequences with greater than 98.5% full sequence similarity (23 or
fewer base differences for a 1530 base sequence). This phylotype
definition was chosen because the 16S rRNA sequence divergence
for most strains of named oral species examined is less than 1.5%
and inter-species divergence is usually greater than 1.5%. As
subsequent clone libraries were screened, any clone with a partial
500-base sequence not matching a reference set sequence by at
least 98% (7 base mismatches) was fully sequenced and added as a
new reference sequence and given a COT number. Thus all 5,958
partial clone sequences match a reference sequence at a similarity
of greater than 98%. Some taxa have two or more reference
sequences because members of a taxon can differ by up to 23 base
differences and appear ,98% similar in their first 500 bases. A
total of 353 phylotypes were identified. Seventy of these phylotypes
(19.8%) were identified as named species based on greater than
98.5% sequence similarity to a type strains in BLASTN searches of
GenBank [15] and Greengenes [16]. The remaining 284
phylotypes (80.2%) represent currently unnamed taxa. As this
study made no attempt to cultivate members of the canine
microbiome, we are not in a position to address what percent of
the unnamed taxa are cultivable or as yet uncultivated as has been
done for human taxa [4].
Taxonomy
Each canine taxon was placed in a phylum or candidate division
based initially on BLASTN results against the Human Oral
Microbiome Database (HOMD) [4], GenBank databases Refer-
ence RNA sequences (refseq_rna) and RNA and Nucleotide
collection (nr/nt) [15], and using tools at Greengenes [16]. The
Greengenes site was particularly useful for classifying and placing
sequences from the rare phyla or candidate divisions Chlorobi,
Chloroflexi, GN02 and WPS-2. The 16S rRNA sequences of all
canine taxa were placed in an aligned database (hand-aligned
based on secondary structure) and analyzed extensively by tree
construction anchored to named reference sequences. As was
previously done for the human oral microbiome [4], a provisional
six level taxonomy was created consistent with the 16S rRNA tree
structure. The full taxonomy is presented in Table S3 in
supplementary materials. The 353 canine bacterial phylotypes
were placed in 14 bacterial Phyla, 23 Classes, 37 Orders, 66
Families, and 148 Genera. The number of taxa and clones in each
phylum or candidate division are shown in Table 1.
Shown in Figs 1, 2, 3, and 4 are consensus neighbor-joining
trees based on the aligned full 16S rRNA sequences for the 353
canine taxa. Each taxon header includes name (genus and species),
Canine Oral Taxon number (COT), clone designation, GenBank
accession number, and number of clones identified for each taxon
out of a total of 5,958. The 51 taxa with 30 or more clones are
shown in bold as major taxa. Those 58 taxa marked with a filled
circle are taxa shared with humans, as defined by the canine
reference sequences sharing .98.5% similarity with reference
sequences in the Human Oral Microbiome Database by BLASTN
comparison (www.homd.org). Where a taxon is ,90% similar to a
named genus, it is designated using the family, or most specific
higher taxa name, [G-1] sp. where ‘‘[G-1]’’ indicates it belongs to
a novel genus. Family level grouping in the Clostridia (Figs 1 & 2)
include the widely recognized classification of Collins et al. [17].
Thus, Clostridium viride is written ‘Clostridium’ IV viride to indicate it is
not in the genus Clostridium sensu stricto but rather is a member of
Collins Cluster IV.
Firmicutes and Tenericutes
The majority of taxa in the Firmicutes are shown in Fig. 1 in the
cluster marked by encircled ‘‘1’’. The Firmicutes families
Peptostreptococcaceae and Lachnospiraceae are shown in Fig. 2. The
phylum Tenericutes, previously the class Mollicutes within the
Firmicutes [18], is marked with an encircled ‘‘3’’ in Fig. 1. The
Firmicutes class Erysipelotrichi, marked with an encircled ‘‘11’’,
branches within the ‘‘phylum’’ Tenericutes, demonstrating
phylogenetic inconsistencies created by elevating class level
branches within the Firmicutes to phylum level. One hundred
sixty-two taxa were identified as members of the phylum
Firmicutes.
Clostridia
The dominant class within the Firmicutes is Clostridia,
containing 138 taxa. The Clostridia clade is shown in Fig. 1,
marked encircled ‘‘4’’, and all taxa in Fig. 2. The cluster of 10
taxa, marked encircled ‘‘6’’ in Fig. 1, fall into unnamed genera in
Collins Clusters III and IV, except for one taxa falling in the genus
Faecalibacterium. Sixteen taxa fall into two family level Clusters with
Figure 1. Consensus neighbor-joining tree for canine oral tax in phyla Actinobacteria, Firmicutes and Tenericutes. The name of each
taxon is followed by Canine Oral Taxon number, reference clone designation, GenBank accession number, and the number of clones out of 5958 that
were identified as this taxon. Taxa marked with a filled circle are also found in the human oral cavity. Taxa for which there were 30 or more clones are
shown in bold. The tree was constructed with MEGA 4 using the Jukes and Cantor correction neighbor-joining distance matrix. Comparisons with
missing data were eliminated pairwise. The numbers to the left of the branches indicate the percent of time the clade was recovered out of 1,000
bootstrap resamplings. Only bootstrap percentages greater than 50 are shown. Roman numerals following a genus name indicate Collins’ Clostridia
cluster numbers [17] The scale bar shows 5% sequence divergence. The encircled numbers mark clades discussed in the text.
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no named members, marked encircled ‘‘7’’ and ‘‘8’’, for novel
families F-2 and F-1 respectively. Three taxa fall in the family
Peptococcaceae, marked encircled ‘‘9’’, related to the human
associated species Peptococcus niger. The family Veillonellaceae,
previously Acidaminococcaceae, is marked encircled ‘‘10’’. We chose
not to follow the suggestion of Marchandin et. al. [19], to elevate
this family to a class as we believe it is taxonomically unjustified.
The Veillonellaceae cluster contains members of the genera Dialister,
Anaeroglobus, Phascolarctobacterium, Schwartzia, Selenomonas, and an
unnamed genus. Nine of these taxa are also found in humans.
Shown in Fig. 2 are those Clostridia taxa falling in Collins Clusters
XI, XIII, and XIVa, with the first two clusters constituting the
family Peptostreptococceae and the last cluster the family Lachnospir-
aceae. These two families contain the majority of the Clostridia taxa
in both dogs and humans. In Collins Cluster XI, the cluster of taxa
marked encircled ‘‘1’’ contains 18 taxa. Most are in 7 unnamed
genera which may be unique to dogs. This cluster contains some
named taxa shared with humans such as ‘Eubacterium’ XI infirmum,
Mogibacterium timidum and M. diversum, and ‘Eubacterium’ XI nodatum.
The cluster marked encircled ‘‘2’’ contains 11 taxa in 3 unnamed
genera distantly related to Fusibacter paucivorans. The cluster marked
encircled ‘‘3’’ contains five Filifactor species, including F. alocis and
F. villosus, and two taxa related to human associated species
‘Eubacterium’ XI yurii. The cluster marked encircled ‘‘4’’ contains 16
taxa in the genus Peptostreptococcus sensu stricto, Proteocatella, and an
unnamed genus distantly related to ‘Clostridium’ XI sticklandii. The
validly named reference bacterium Proteocatella sphensci [20] was
initially called ‘Frigovirgula patagoniensis’ in GenBank (AF450134)
and the name ‘Frigovirgula’ unfortunately persists causing minor
confusion. Within Collins Cluster XIII, clusters marked encircled
‘‘5’’, ‘‘6’’, & ‘‘7’’, are 11 taxa in the genera Helcococcus, Parvimonas,
Tissierella, Peptoniphilus, and three unnamed genera. Five taxa,
including P. micra, are shared with humans. Seventeen canine taxa
fall in the Lachnospiraceae [21], Collins Cluster XIVa, with major
subclusters marked encircled ‘‘8’’ and ‘‘9’’. The subclusters contain
taxa in the genera Blautia, Butyrivibrio, Catonella, Shuttleworthia,as
well as 7 unnamed genera. Two taxa, including S. satelles, are
shared with humans.
Bacilli
The second most dominant class within the Firmicutes is the
Bacilli with 18 taxa. The Bacilli clade is marked with an encircled
‘‘5’’ in Fig. 1. All taxa can be placed in the following genera:
Abiotrophia, Aerococcus, Enterococcus, Gemella, Globicatella, Granulicatella,
Jeotgalicoccus, Lactobacillus and Streptococcus. While three streptococ-
cal species are shared with humans, streptococci appear to
represent a minor genus in dog. This is not surprising as simple
carbohydrates and sugars are not normally a major constituent of
the canine diet and canine saliva has a pH of approximately 8.0
(WALTHAM, unpublished data 2011) which may be hostile to
members of this aciduric genus.
Erysipelotrichi
Five taxa in this Firmicutes class, marked encircled ‘‘11’’ in
Fig. 1, were identified. None were sufficiently close to reference
species to place them in the genera Erysipelothrix or Bulleidia.
Novel Firmicutes Class
Firmicutes [G-1] sp. COT-309 appears to be a member of a
novel deeply branching linage marked encircled ‘‘12’’ in Fig. 1.
The closest named species had only 80% sequence similarity,
however, a clone from the microbiome of fiber adherent species
from rumen fluid was 93% similar (EU844484) supporting this
canine taxa as a member of a mammal host associated lineage.
Tenericutes
Six members of this phylum were identified and are marked
encircled ‘‘3’’ in Fig. 1, but excluding the Class Erysipelotrichi
discussed above. In this tree, the ‘‘phylum’’ does not branch as a
monophyletic entity. Mycoplasma canis and an Ureaplasma parvum-
related taxon can be placed in named genera, but four additional
taxa fall into unnamed genera.
Actinobacteria
Twelve Actinobacteria were identified and are marked encircled
‘‘2’’ in Fig. 1. Taxa in the genera Actinomyces, Leucobacter,
Pseudoclavibacter, Propionibacterium, were identified as well as a deeply
branching taxa Actinobacteria [G-1] sp. COT-376. None of these
canine oral taxa are shared with humans. In study 1 using the
standard 9–27F and 1525–1541R primers, only one Actinobac-
teria clone was recovered. Because the 1525–1541R primer has
been reported to discriminate against Actinobacteria [22], we
switched to the 1492–1505R primer in hopes of obtaining less
biased coverage in our second study. Eleven clones were obtained
with the revised ‘‘universal’’ primers and eight additional clones by
fortuitous mispriming using the ‘‘Bacteroidetes-selective’’ primer
set. It appears that no truly ‘‘universal’’ 16S rRNA primers exist
and studies of diversity benefit from the use of multiple primer sets.
Actinomyces sp. COT-083 fell in the genus Actinomyces, and is 97%
similar to Actinomyces coleocanis, a species isolated from the vagina of
a dog [23].
Proteobacteria
Fifty-two phylotypes were identified from the phylum Proteo-
bacteria, and are marked with an encircled ‘‘1’’ in Fig. 3. The five
classes are marked with Greek letters. The 22 Betaproteobacteria taxa
include 11 from the mammalian host associated genera Neisseria,
Eikenella and Conchiformibius. Whether the taxa associated with
other genera in the Betaproteobacteria are truly part of the
endogenous oral microbiome, or are transient common environ-
mental bacteria remains to be determined. The 18 Gammaproteo-
bacteria taxa include the host associated genera Cardiobacterium,
Moraxella and species in the families Pasteurellaceae and Enterbacter-
iaceae. Taxa in the genera Luteimonas and Stenotrophomonas may be
transient common environmental bacteria. One deeply branching
Alphaproteobacteria taxon, distantly related to named species (81%
similarity), was identified. The five Epsilonproteobacteria and six
Deltaproteobacteria taxa are related to well-known mammalian host
associated genera except for Chondromyces, which is generally
associated with soil or decaying organic matter.
Spirochaetes
Thirty-seven phylotypes from the phylum Spirochaetes were
identified and are marked by encircled ‘‘2’’ in Fig. 3. Thirty-four
taxa are members of the genus Treponema, marked encircled ‘‘5’’,
Figure 2. Consensus neighbor-joining tree for class
Clostridia
, families
Peptostrepto-coccaceae
and
Lachnospiraceae
.Labeling and
methods used are as described in Fig. 1.
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including the named species T. amylovorum, T. denticola, T.
maltophilum, T. medium, T. parvum, T. socranskii, and T. vincentii
which are also found in the human oral cavity. A total of 14 canine
Treponema spp. are shared with humans. Unlike previous studies of
the human oral cavity [4], three taxa outside the genus Treponema
were identified and marked encircled ‘‘3’’ and ‘‘4’’. Spirochaeta sp.
COT-379 is most closely related to Spirochaeta coccoides
(NR_042260; not shown) and Spirochaeta sp. Buddy. These two
species are not helical cells, typical of spirochetes, but rather have
a coccoid morphology. Spirochaeta sp. COT-314 is 92% similar to a
strain isolated from the marine bristle worm Alvinella pompejana
(AJ431240; not shown) and Spirochaeta isovalerica.Spirochaetes [G1]
sp. COT-373 is a deeply-branching taxa with 93% similarity to a
clone sequence from the termite gut, EF453883. Thus it appears
that the diversity of spirochetes in the mammalian oral cavity may
be broader than just the genus Treponema. The vast majority of the
spirochete clones came from the 7 libraries produced using
‘‘spirochete-selective’’ primers (Table 1), which demonstrates the
utility of using selective primers.
Bacteroidetes
Forty-three phylotypes were identified as members of the
phylum Bacteroidetes, marked by encircled ‘‘1’’ in Fig. 4. Eleven
named genera include: Porphyromonas, Tannerella, Proteiniphilum,
Paludibacter, Bacteroides, Prevotella, Odoribacter, Bergeyella, Cloacibacter-
ium, Capnocytophaga and Sporocytophaga, and 5 unnamed deeply
branching genera what are not anchored to named taxa. The use
of ‘‘Bacteroidetes-selective’’ primers with DNA from 7 super-pools
produced 420 clones in the Bacteroidetes phylum and increased
the depth and diversity of taxa identified over that produced from
‘‘universal’’ primers (Table 1).
There are naming issues for a number of species in the
Bacteroidetes phylum. Porphyromonas gingivicanis and Porphyromonas
crevioricanis were properly named and validly published by
Hirasawa & Takada in 1994 [24]. Unfortunately, no 16S rRNA
sequences for the type strains of these species were deposited by
anyone for 12 years (see DQ677835 & DQ67736) and for 14 years
by the authors (see AB430828 & AB430829). While these
sequences were unavailable, Collins et al. named Porphyromonas
cansulci [25] and deposited its 16S rRNA sequence in GenBank as
entry X76260. ‘‘Porphyromonas canis’’ was invalidly named by
Sakamoto & Benno in 1999 as GenBank entry AB034799. From
the 16S rRNA sequences, we now know that P. cansulci is a
synonym for P. crevioricanis, and that ‘‘P. canis’’ is an invalid
synonym for P. gingivicanis.Odoribacter denticanis was named and
validly published by Hardham et al. [26], but was challenged by
Euzeby in comments in the List of Prokaryotic Names with
Standing in Nomenclature (http://www.bacterio.cict.fr/) for not
having a type strain available. This appears to be rectified as the
type strain is now available from three national collections. This
species was also previously referred to as ‘‘Wernerella denticanis’’ and
‘‘Porphyromonas denticanis’’. Bacteroides sp. COT-183 has been called
‘‘Bacteroides denticanum’’ by Elliott (see DQ156993) and ‘‘Bacteroides
denticanoris’’ by Hardham et al. (see AY54431) in GenBank and
patent filings, but never validly described in any publication.
Chlorobi
Two phylotypes from the phylum Chlorobi, marked with
encircled ‘‘2’’ in Fig. 4, were identified. The original cultivable
members of the phylum Chlorobi, previously called Green Sulfur
Bacteria or Chlorobia, are phototropic organisms [27]. Cultivation
independent molecular methods have identified members from
diverse environments. Recently a non-photosynthetic member of
the phylum, Ignavibacterium album, has been described [28].
Sequences in GenBank with greater than 84% similarity to canine
Chlorobi phylotypes COT-046 & COT-312 have been recovered
from manure drainage, penguin dropping sediment, hydrothermal
worm mucus, and from an anaerobic digester. A sequence with
99% similarity to COT-046 has been recovered from the oral
cavity of a cat (unpublished observation), supporting the
association of this taxa with the oral cavity of mammals. Nine
clones from skin swabs of the volar forearms of four human
subjects (based on subject identification number in GenBank
entries) have a sequence similarity of 99% to canine Chlorobi taxa
(for example HM278300 and HM330153 to COT-046). These
four human subjects appear to have had the volar surface of their
arms licked by dogs prior to sampling as their clone libraries
include 23 to 51 canine oral taxa.
Fusobacteria
Ten taxa from the phylum Fusobacteria, marked encircled ‘‘3’’
in Fig. 4, were identified, including the genera Fusobacterium,
Streptobacillus, and Leptotrichia. The Fusobacteria spp. includes four
taxa that overlap the human F. nucleatum cluster. Streptobacillus sp.
COT-370 is closely related to the rat bite fever organism S.
moniliformis. It was suggested previously that dogs may be colonized
with S. moniliformis by eating rats [29], but the current study
suggests that dogs may be naturally colonized with a distinct, but
closely related species. It is notable that clones from this phylum
were not present in the 10 libraries made by PCR with standard
9–27F and 1525–1541R primers, but were present (110 clones) in
21 libraries using an extend specificity 9–27F and 1492–1509R
primers (see methods and Table S1).
GN02
Four taxa from the as-yet-uncultured GN02 candidate division,
marked encircled ‘‘4’’ in Fig. 4 were identified. GN02 is one of 15
candidate divisions proposed in a study of the Guerrero Negro
hypersaline microbial mat [30]. The canine phylotypes were
originally placed in this division using BLASTN searches of the
Greengenes database. In the past year, related taxa from human
mouth and skin have started to appear in GenBank as human
microbiome data have been submitted (for example FJ976283 &
HM249743).
SR1
Three taxa from the as-yet-uncultured SR1 candidate division,
marked encircled ‘‘5’’ in Fig. 4 were identified. The SR1 division
was named for clones identified in a study of sediment with
microbial streamers from the Sulphur River in Parkers Cave,
Kentucky [31]. The SR1 division was previously part of candidate
division OP11, so older references to a closely related taxa from
the human oral cavity referred the human taxon as OP11 clone
X112 [32]. The human phylotype, now designated SR1 sp. HOT-
345, has been identified in multiple clone libraries [4].
TM7
Seven canine phylotypes were identified as members of the
candidate division TM7, which is marked with an encircled ‘‘6’’ in
Figure 3. Consensus neighbor-joining tree for phyla Proteobacteria and Spirochaetes. Labeling and methods used are as described in
Fig. 1. The Greek letters mark the respective Proteobacteria classes.
doi:10.1371/journal.pone.0036067.g003
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Fig. 4. The phylum TM7 is a major lineage of Bacteria with no
known pure-culture representatives [33]. TM7 organisms have
been recognized in 16S rRNA cloning studies of many habitats,
including soils, fresh ground water, seawater, and mammalian
clinical samples [33]. They have been recovered from the human
oral cavity [4,32,34], the human distal esophagus [35], and mouse
feces [36].









Figure 4. Consensus neighbor-joining tree for phyla Bacteroidetes, Fusobacteria, Chlorobi, Chloroflexi, Synergistetes and
candidate divisions TM7, SR1, GN02 and WPS-2. Labeling and methods used are as described in Fig. 1.
doi:10.1371/journal.pone.0036067.g004
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WPS-2
The candidate division WPS-2, marked with encircled ‘‘7’’ in
Fig. 4, is known from only 39 environmental clones in Greengenes
otu_4420, mainly from soils. The WPS-2 division was one of two
named for clones identified in a study of Wittenberg polluted soil,
Germany [37]. WPS-2 sp. COT-220 is closest to GenBank entry
DQ520181, and is the 40
th
member of this rarely observed
candidate division marked encircled ‘‘7’’ in Fig. 4. As this taxon
was detected as a single clone, and no related clones have been
identified from human or other mammalian sources, it remains to
be determined if this taxon is part of the endogenous canine oral
microflora, or an environmental transient.
Chloroflexi
A single phylotype of the Chloroflexi phylum was identified and
is marked with encircled ‘‘8’’ in Fig. 4. The Chloroflexi phylum,
previously called green non-sulfur bacteria, has many cultivated
species [38], and several were named subsequent to the description
in Bergey’s Manual of Systematic Bacteriology [39]. The canine
Chloroflexi sp. COT-306 is 96% similar to human oral taxon
Chloroflexi sp. HOT-439 and 86% similarity to named species
Anaerolinea thermophila [40] in the class Anaerolineae [41].
Synergistetes
The phylum Synergistetes is known mainly from clone
sequences, but contains about a dozen cultivated species including
Synergistes jonesii, a rumen bacterium that degrades toxic pyridine-
diols [42] and Pyramidobacter piscolens, a species from the human oral
cavity [43]. Organisms from the Synergistetes phylum have
previously been mistakenly included in the phylum Firmicutes or
placed in the phylum Deferribacteres (a sister phylum of
Synergistetes and Flexistipes) [32]. As marked by an encircled
‘‘9’’ in Fig. 4, 13 canine phylotypes were identified. Six canine
phylotypes match previously identified human phylotypes at
.98.5% similarity [4].
Primer biases
The number of clones identified in each phylum for libraries
generated with two different ‘‘universal’’ primer pairs, a
‘‘Spirochaetes-selective’’ pair, and a ‘‘Bacteroidetes-selective’’ pair
are shown in Table S1. A marked difference in the diversity
recovered in clone libraries using different initial PCR primers is
apparent. In study 1, the commonly used ‘‘universal’’ 9–27 YM
forward (F24) and 1525–1541 reverse (Y36) primers produced
more than one clone only for the four common phyla Firmicutes,
Proteobacteria, Bacteroides, and Spirochaetes. In the second
study, using expanded coverage 9–27 forward primers (F24/
AD35) [9] and the ‘‘universal’’ 1492–1509 reverse primer (C72),
clones from 12 phyla/candidate divisions were recovered. Of
particular note is the recovery of Fusobacteria taxa only with the
second set of ‘‘universal’’ primers and recovery of significantly
more Actinobacteria clones with the second primer set. PCR with
the ‘‘Spirochaetes-selective’’ reverse primer M98 (1483–1501)
yielded expected results: organisms from the Spirochaetes and
Synergistetes phyla. Bacteria in these two taxa have ‘‘GG’’ at
position 1484-5 whereas most other bacteria have ‘‘CT’’. The
‘‘Bacteroidaetes-selective’’ reverse primer F01 (1487–1505) selects
for organisms with a ‘‘CT’’ at position 1490-1 whereas most non
Bacteroidaetes have other bases at these positions. While the F24/
F01 primer set yielded mostly clones from the Bacteroidetes
phylum, clones for 12 phyla/candidate divisions were recovered.
The recovery of Chlorobi clones was expected based on perfect
primer sequence match; the recovery of TM7 and SR1, which
have a one base mismatch ‘‘TT’’, is also expected; but recovery of
other taxa, such as Firmicutes, Proteobacteria and Fusobacteria, is
somewhat unexpected as they have 2 base mismatches. While the
‘‘Spirochaetes–selective’’ primers are truly selective, the ‘‘Bacter-
Table 1. Bacterial phyla identified in canine subgingival plaque.
Clones
Phyla Phylotypes Universal 1525 R
a
Universal 1492 R
b
Selective Spiro
c
Selective Bact
d
Total
Firmicutes 162 1,148 1,379 0 213 2,740
Proteobacteria 52 224 569 0 68 861
Bacteroidetes 43 213 516 0 420 1,149
Spirochaetes 37 17 22 366 4 409
Synergistetes 13 1 5 511 9 526
Actinobacteria 12 1 11 0 8 20
Fusobacteria 10 0 112 0 58 170
TM7 70 701320
Tenericutes 6 0 7 0 3 10
GN02 4 0 5 0 6 11
SR1 30 001313
Chlorobi 2 1 12 0 13 26
Chloroflexi 1 0 2 0 0 2
WPS-2 1 1 0 0 0 1
Total 353 1,606 2,647 877 828 5,958
a
Clones from libraries made using 9–27F (F24) and 1525–1541R (Y36) primers.
b
Clones from libraries made using expanded coverage 9–27F (F24/AE35) and 1492–1509R (C72) primers.
c
Clones from libraries made using ‘‘Spirochaetes-selective’’ F24/M98 primer pair.
d
Clones from libraries made using ‘‘Bacteroidetes-selective’’ F24/F01 primer pair.
doi:10.1371/journal.pone.0036067.t001
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oidetes-selective’’ primers produced clones from 12 of 14 phyla
and appear to be useful in recovering a number of rare phyla/
candidate divisions. The recovery of taxa from diverse phyla was
clearly aided by using multiple primer sets for PCR of DNA prior
to library construction. Because this study used taxa selective
primers (as all studies ultimately do) to construct libraries, it is
impossible to say anything valid about relative abundance of
canine oral species from the abundance of clones recovered.
Taxa abundance
The rank abundance of clones for each canine oral taxon is
presented in Table S4 in supplemental materials. Because a variety
of primers with various biases were used for library construction,
the clone abundance data reflect only clone numbers found in
these libraries and cannot be used to validly infer the underlying
population structure. With the caveat noted, the most prevalent
taxa, Porphyromonas gingivicanis COT-022, constituted 5.3% of the
clones. Clones from 28 taxa were recovered at level of greater than
one percent. The 89 singleton clones were present as 0.017% of
5,958 clones identified. Of the 50 most common taxa, it is striking
that 40, or 80%, are unnamed. The taxon rank abundance profile
for this canine study is very similar to that previously found for the
human oral microbiome [4]. In the human study of about 35,000
clones, it was estimated that the number of taxa necessary to
identify 90%, 95% and 98% of the clones was 259, 423 and 655
taxa respectively. Assuming the canine and human oral cavities
contain about equal microbial diversity and similar rank
abundance profiles, 353 canine taxa should allow identification
of about 93% of clones in a study of similar size. This estimate is
approximate, but suggests that 353 taxa capture a significant
portion of the microbiome. While the current study provides good
initial coverage of the canine oral microbiome, the oral samples
examined were limited to the subgingival sites. Further studies
sampling other oral habitats such as teeth, tongue, cheek, hard and
soft palates, and tonsils will no doubt expand the number of canine
taxa to approach the more than 1,000 currently defined for the
human oral microbiome [4]. One goal of the current study was to
obtain essentially full length 16S rDNA reference sequences, which
are required for recognition and placement of previously
unrecognized rare taxa members such as those in candidate
divisions GN02 and WPS-2. Future studies using next generation
sequencing methods will no doubt sequence more deeply,
producing tens to hundreds of thousands of short sequences.
Studies require tradeoffs between sequence length (full length
better for phylogenetic studies), and sequence number (higher
numbers better for determining community composition).
Comparison of reference 16S rDNA sequences from the canine
oral cavity with those of the human oral cavity reveals that only
16.4% of the taxa are shared by BLASTN analysis at a threshold
of 98.5% sequence similarity (see taxa marked with filled circle in
Figs. 1, 2, 3, and 4). This indicates that there is a large divergence
in the oral microbiomes of divergent mammalian species. Of the
83.6% of taxa that differ, the differences are not only at the species
level, but also at genus through phylum levels. It is apparent from
the results presented here, however, that the majority of oral
bacteria from divergent mammalian species are unique and the
practice of naming mammalian (or even more distantly related
animal) isolates after the most phenotypically similar species from
humans is likely to be shown invalid by using molecular tools.
The Canine Oral Microbiome
The provisional taxonomic scheme presented in supplementary
materials Table S3, and the linked 16S rRNA reference sequences,
provide the most comprehensive resource to date for identifying
and referencing both the named and the 80% as yet unnamed
canine oral taxa. This sequence based identification resource
should facilitate future molecular studies of canine health and
disease as well as the zoonotic potential of canine oral microbes in
human and veterinary infectious diseases. The taxonomic scheme
presented here currently includes only those taxa for which clones
were identified in this study. It is anticipated future efforts will
expand this taxonomy and reference sequence set to include all
named canine-associated species, and isolates of novel taxa, for
which full length 16S rRNA sequences exist.
Conclusions
The results of this study provide the groundwork for describing
the diversity of taxa present in the canine oral cavity. The
provisional scheme of giving each taxon a canine oral taxon
number and placing it in a phylogenetic context should facilitate
future studies of the canine oral microbiome and its role in canine
health and disease. The canine oral microbiome is widely
divergent from that of human, hence these results will also help
in the interpretation of human microbiome studies where canine
oral bacteria appear to be present in large numbers in certain
human skin samples and in veterinary and human medical studies
where previously unnamed canine taxa are recovered from clinical
samples.
Supporting Information
Table S1 PCR and sequencing primers.
(DOCX)
Table S2 16S rDNA clones libraries.
(XLSX)
Table S3 Canine taxonomy.
(XLS)
Table S4 Rank abundance of clones in canine oral taxa.
(XLS)
Acknowledgments
We thank Sara Barbuto for her assistance in DNA sequencing and Liang
Yang for depositing sequences in GenBank.
Author Contributions
Conceived and designed the experiments: FED CMFB IJD MLB ZVMJ.
Performed the experiments: EAK ECT JMB LM. Analyzed the data: FED
TC IJD ZVMJ. Contributed reagents/materials/analysis tools: TC. Wrote
the paper: FED IJD ZVMJ.
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