Download Porphyromonas

A Preliminary Analysis of the Microbiota of
Canine Dental Plaque
Eastman
Dental
Institute
D.R.
For Oral Health Care Sciences
www.eastman.ucl.ac.uk
1Division
1
Elliott ,
D.A.
1
Spratt ,
C.
2
Buckley ,
M-L.
2
Baillon ,
M.
1
Wilson
of Infection and Immunity, Eastman Dental Institute, University College London, United Kingdom
2Waltham Centre for Pet Nutrition, Waltham-on-the-Wolds, LE14 4RT, United Kingdom.
Introduction
Results (continued)
Results
Dental plaque is a complex multi species biofilm which has been extensively Figure 1 shows an example phylogenetic tree as used to group the isolated bacteria for each genus. Longer sequences were obtained
studied in humans owing to its importance in the aetiology of oral diseases. for selected sequences and these were used to further characterise some groups by database searches and additional phylogenetic
Relatively little work has been carried out to advance our understanding of the trees as shown in Figure 2. These data were then combined with viable counting results from primary isolations to determine the
canine oral microbiota and its relationship with canine oral health.
Intensive research attention on human dental plaque has led to numerous
suggestions and theories about the causes of oral diseases such as periodontal
disease. Most of these ideas should be equally applicable to the dental plaque of
frequency at which each group was isolated for each sample category (health or disease) as shown in Table 1.
Figure 1. Example neighbour joining phylogenetic tree for partial 16S
rRNA gene sequences (250 alignment positions) of Neisseria species
isolated from the canine oral cavity of 9 dogs.
N. dentiae
cp06.07
cp01.10
cps01.33
cp08.26
N. weaveri
cp08.01
cp09.16
cp17.05
N. flavescens
N. polysaccharea
N. elongata
understanding, the oral microbiota and ecology of the target animal must be
suitably characterised. There are many published research papers relating to the
oral microbiota of dogs, but frequently they focus on bite wounds, or animal
models of human periodontitis, while neglecting to consider its importance to the
health of the dog.
Neisseria weaveri
group
cp07.09
N. canis
cp05.07
cp06.31
cps01.16
cp01.09
cp07.07
plaque, motivated by the desire to improve oral healthcare for dogs. A culture
Neisseria canis
group
cp03.11
cp03.09
cp09.01
cp62.01
based approach was used for isolation of bacteria, coupled with 16S rRNA gene
sequencing for the identification of recovered bacterial isolates.
cp62.02
cp09.08
cp02.03
cp02.07
cps01.20
cp04.01
cp06.15
cp01.11
cp07.21
cp07.10
cp17.04
cp09.13
cp08.09
cp08.04
cp03.06
cp08.10
cp01.07
cp09.02
cp05.02
Materials and Methods
Plaque sampling was performed on dogs during routine dental treatment, after
scoring the disease status using standard indices adapted for canines. Healthy
dogs were sourced from the Waltham Centre for Pet Nutrition, and dogs with
periodontal disease were sourced from a local veterinary clinic. Plaque samples
were taken from the buccal surface of upper third premolars at the gingival margin
(healthy sites) or at the base of the periodontal pocket (diseased sites) and
Neisseria elongata
group
Neisseria canis - like
Group 1
disease
9.2%
<0.1%
A. canis - like. Possible new species*
6.1%
<0.1%
A. turicensis - like
0.7%
<0.1%
A. naeslundii / A. bowdenii*
0.9%
ND
A. hordeovulneris
0.5%
ND
A. hordeovulneris
0.4%
ND
ND
<0.1%
0.5%
ND
31.9%
77.6%
Prevotella heparinolytica
3.6%
ND
Prevotella heparinolytica
ND
18.2%
Bacteroides sp.
1.9%
ND
Porphyromonas gulae*
4.8%
27.7%
Porphyromonas canoris*
21.1%
ND
Porphyromonas cansulci
ND
1.3%
Porphyromonas endodontalis
ND
0.9%
Porphyromonas macacae
ND
29.6%
0.5%
ND
ND
ND
2.1%
3.0%
C. rectus - like. Possible new species*
1.8%
3.0%
C. Curvus
0.3%
ND
14.2%
ND
Corynebacterium sp. Likely new genus*
3.9%
ND
C. felinum*
2.4%
ND
C. jeikium - like. Likely new species*
7.5%
ND
Corynebacterium sp.
0.5%
ND
0.7%
9.6%
ND
1.1%
Actinomyces species
A. hyovaginalis - like*
Bacteroides, Porphyromonas, and
Prevotella species
Porphyromonas cangingivalis
Campylobacter species
Neisseria canis - like
Group 2
Corynebacterium species
Fusobacterium and Filifactor species
Fusobacterium alocis
the laboratory.
The samples were serially diluted and plated out onto Columbia agar base, and
anaerobe agar both containing 5 % horse blood before being incubated at 37oC
aerobically with 5 % CO2, and anaerobically respectively. After 3 - 10 days each
Figure 2. Example neighbour joining phylogenetic tree (832 alignment
positions) showing two Neisseria species which were selected for extra
sequencing.
Fusobacterium nucleatum - like. Possible new species*
0.1%
8.5%
Filifactor villosum
0.6%
ND
0.6%
<0.1%
Some isolates were selected for additional sequencing to provide a more
accurate phylogenetic classification for the group to which they belong.
Haemophilus / Actinobacillus - like. Likely new species*
0.6%
ND
H. haemoglobinophilus
ND
<0.1%
Haemophilus sp.
ND
<0.1%
not determined
7.3%
4.3%
no sequence available
4.0%
2.3%
not determined - ambiguous or few rel's
3.3%
1.9%
11.0%
1.7%
0.9%
ND
N. Canis – like, group 1
ND
1.7%
N. canis – like, group 2*
9.7%
ND
N. Weaveri
0.5%
<0.1%
23.0%
3.6%
C. gingivalis - like*
0.6%
ND
Weeksella zoohelcum*
1.3%
ND
G. palaticanis*
5.8%
ND
Leptotrichia - like. Likely new species.*
0.7%
ND
Peptostreptococcus sp.
0.0%
3.6%
M. caviae
<0.1%
ND
P. stomatis / P. dagmatis*
4.6%
0.0%
Cardiobacterium sp.
3.2%
ND
Ultramicrobacterium sp.*
S. suis
4.9%
1.9%
ND
0.0%
7.91E+04
2.12E+07
N. denitrificans
distinguishable colony morphotype was isolated and subjected to comparative
N. animalis
sequence analysis of the 16S rRNA gene.
N. canis*
The 16S rRNA gene was amplified by PCR using the primers 27F and 1492R.
cp04.01
DNA sequencing was performed with an ABI 310 Genetic Analyser using the 357F
cp05.07
primer for all isolates, and also using the 27F and 530F primers for extended
Sequences were identified by searching public databases, and
N. canis
grouped according to bacterial genus. Phylogenetic trees were then generated for
N.macaca
each group using ClustalX to align sequences, and the PHYLIP DNADist and
Neighbour programs to generate trees. Sequences of related bacteria from public
N. flavescens
databases were also included in the trees, which were used to group the isolated
N.gonorrhoea
bacteria into phylotypes. The distance separating known species on each tree
was used as a guide for the assignment of phylotypes so that they should
approximately reflect distinct species.
Actinobacillus and Haemophilus
species
Neisseria species
N. elongata
sequencing.
health
Por. catoniae - like. Likely new species*
0.1
immediately transferred into reduced transport fluid before being transported to
Isolate identity
A. europaeus
cp08.11
cp08.12
The aim of this study was to investigate the bacterial composition of canine dental
Table 1. Summary of bacterial species isolated
from dental plaque of healthy dogs (n=3) and
dogs with periodontal disease (n=2).
Species identities are based on closest clustering
sequences and BLAST searches on public
databases.
cp62.05
cps01.28
dogs and other animals, but to make the most of these advances in
0.01
University College London
N.polysacc
Key for Figures 1 & 2
Isolate numbers from this study shown in black
Isolates from this study chosen for additional
sequencing shown in blue
Sequences obtained from public databases for
comparison shown in red
Others
Total viable count per ml
ND = Not Detected
* Indicates species identities marked which were determined using longer
sequences of approximately 900 bases.
Other species identities determined using sequences of approximately 300 bases.
From Table 1 it can be seen that the bacterial composition of dental plaque was
different between samples from healthy and diseased sites. In particular,
Actinomyces, Corynebacterium, and Neisseria species were abundant at healthy
sites (9.2 %, 14.2 %, 11.0 %), while they were rare or not detected at diseased
sites.
The disease associated microbiota was, however, dominated by
Porphyromonas, Prevotella, and Fusobacterium species, which together
comprised 87.2 % of the biota. It is also evident that within genera, the
predominant species was often different depending upon the disease status of the
sample; for example Porphyromonas gulae and Porphyromonas macacae were
isolated at high frequency from disease associated samples and low frequency
from health associated samples, while the inverse was true of Porphyromonas
canoris which was only detected in healthy sites.
Discussion
Grouping bacteria using phylogenetic trees based on 16S rRNA gene sequences
allowed isolates from different samples to be compared and the relatedness of
new species and clades to be assessed.
A broad range of bacterial species belonging to at least 17 genera were isolated
from the dental plaque of 5 dogs. These genera are typical of those found in
human dental plaque, including Actinomyces, Porphyromonas, Fusobacterium,
Neisseria, and Streptococcus. Identification of the bacteria to species level,
however, revealed that the vast majority were not species normally found in the
human oral cavity. In addition the proportions of certain genera in the canine
plaque did not match those typically found in human plaque; for example
Streptococcus species, which are common in human plaque (e.g. S. sanguis),
were detected in only one sample, comprising less than 2 % of the total healthy
microbiota, and again the species was one not normally found in the human biota
(S. suis).
The greatest difference between health and disease samples was detected in the
Bacteroides, Porphyromonas, and Prevotella species, which were observed to
more than double in frequency from 32 % of the biota in health to 78 % in
disease. This result is in agreement with the literature which has repeatedly
demonstrated an association of these genera with periodontal disease in both
humans and dogs.
However, Porphyromonas gingivalis, which is widely
considered the most important periodontal pathogen in humans, was not detected
at all, though it is frequently reported in canine studies. This result suggests that
P. gingivalis may not be a normal member of the canine oral microbiota as has
been previously reported, but may have been reported in the literature either due
to mis-identification, or because the reports were made before species now
recognised as distinct were separated.
Conclusions
• The dental plaque biofilm of dogs has a distinct composition of bacterial
species compared to human dental plaque, though it shares close similarities
at the genus level.
• Continuing with this approach, and also using molecular methods to detect
culture resistant bacteria could help to accurately identify the bacteria
associated with oral health and disease in dogs and other animals, thus
allowing oral healthcare measures to be more accurately targeted.
• Comparison of the periodontal pathogens found in different animals could
provide a valuable insight into the aetiology of periodontal diseases.
Acknowledgements
This work was funded by the Biotechnology and Biological Sciences Research
Council, and the Waltham Centre for Pet Nutrition.