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76
Opinion
genera at a presence of over 1% in both enamel and dentin
lesions. However, each lesion appears to harbor a different
combination of bacteria. There was only one case in which
the lesion was dominated by a single bacterial genus
(marked as sample CA085, tooth 47 in the figure). This
exceptional instance involved Lactobacillus, which was
found to represent 99% of the RNA-based population in
a hidden dentin cavity (55% of the reads had maximal
similarity to Lactobacillus gasseri, but three other species
of lactobacilli were present). However, all of the other
samples showed diverse bacterial compositions that varied
dramatically between individuals, even within the same
carious lesion type (Figure 1). The estimated bacterial
diversity was lowest for enamel carious lesions, with an
average of 177 species-level phylotypes per sample, and
highest for open dentin cavities that were exposed to the
oral cavity, which had an average of 251 species per
sample. This suggests that open dentin cavities have an
input of microorganisms from saliva, even if the biofilm
formed on top of the lesion is eliminated from the sample,
as was the case here. Hidden dentin cavities, which have
almost no contact with the oral cavity except for a minimal
lesion through the enamel, had an estimated number of
species-level phylotypes of 201 [20]. The observation that
multiple species are detected by their RNA in carious
lesions unequivocally demonstrates that they are alive
and supports the concept that consortia formed by multiple
microorganisms act collectively to initiate and expand the
cavity. It has been shown that several oral species can act
synergistically to increase their pathogenic effect [21]. In a
fascinating example of metabolite cross-feeding, Streptococcus gordonii was shown to increase the virulence of
Aggregatibacter actinomycetemcomitans in an animal abscess model [22]. Mixed cultures of S. mutans and Veillonella alcalescens were also found to produce higher acid
levels than biofilms containing only one of these species
[23]. In addition, Veillonella may favor acid-producing bacteria in caries through nitrate reduction [24], given that low
concentrations of nitrite killed several cariogenic organisms. Finally, it must be remembered that not only bacterial
interactions but also bacterialfungal associations can be
vital for promoting virulence in disease-associated consortia
Opinion
TRENDS in Microbiology
Figure I. Bacterial composition of different oral samples from the same individual. Each ring corresponds to DNA extracted from drooling saliva, dental plaque from
sound surfaces, and an enamel carious lesion, as well as from RNA from the same lesion (inner circle). Font size is related to the proportion of each taxonomic group in
the sample.
Opinion
100%
Key:
90%
Other
Veillonella
Streptococcus
Selenomonas
80%
Schlegelella
Rothia
Pseudoramibacter
70%
Prevotella
Porphyromonas
60%
Neisseria
Leptotrichia
Lactobacillus
50%
Kingella
Granulicatella
Gemella
40%
Fusobacterium
Eubacterium
30%
Corynebacterium
Capnocytophaga
20%
Atopobium
Ac!nomyces
10%
Abiotrophia
CA
0
CA 21.3
02 4W
CA 1.3 S
0 6
CA 21.4 WS
0 6
CA 22.1 WS
0 3
CA 22.4 WS
02 6W
CA 2.4 S
0 7
CA 78.3 WS
0 6
CA 78.3 WS
0 7
CA 78.4 WS
07 5W
CA 8.4 S
07 6
CA 8.4 WS
08 7W
CA 8.4 S
0 7
CA 89.4 WS
1 5
CA 01.1 WS
10 2W
1. S
23
W
CA
S
07
CA 7.3
0 8
CA 77.4 DD
0 8
CA 79.1 DD
0 5
CA 79.2 DD
0 1
CA 82.3 DD
08 4D
8. D
46
DD
CA
07
CA 8.3
0 7
CA 78.4 H
08 7H
CA 1.4
0 8
CA 83.2 H
0 7
CA 85.4 H
08 7H
6.
28
H
0%
Samples
TRENDS in Microbiology
Figure 1. Metatranscriptomic profile of caries lesions. RNA was extracted from initial, white-spot enamel caries (WS), the deep layer from open dentin cavities (DD), and
hidden dentin cavities (H). After cDNA construction, bacterial taxonomy was obtained by Titanium-plus pyrosequencing of 800 bp PCR products encompassing
hypervariable regions V1 through V4 and using the Ribosomal Database Project (RDP) classifier. Over 4000 reads per sample were obtained, giving information about the
active microbial community. The two numbers to the right of the patient code indicate the tooth from where the sample was obtained. Drawn from data in [20].
in predicting risk of oral diseases and could have potential diagnostic value.
Thus, the change in paradigm in the etiology of tooth
decay must be translated to appropriate therapies
[35]. Given the polymicrobial nature of dental caries, we
predict that diagnostic and preventive strategies directed
toward specific bacterial species will not be universally
effective. These include diagnostic kits of caries risk assessment, which are traditionally focused on culture
counts of mutans streptococci and lactobacilli [4] whereas
the data show that the former are found at extremely low
proportions even within cavities and the latter are found
only in several dentin cavities and not in initial, enamel
caries lesions (Figure 1), confirming that this species is not
involved in the initiation of caries [36]. Regarding preventive or therapeutic approaches, passive immunization
strategies such as the use of synthetic antibodies directed
against S. mutans-specific antigens [37] are predicted to be
inefficient given the multispecies nature of caries initiation. Active immunization strategies (i.e., a caries vaccine)
are being explored mainly against mutans streptococci
antigens [6,38] and even the multispecies approach that
79
Opinion
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TRENDS in Microbiology
Figure 2. The bacterial composition of caries is tissue dependent. A sample from an initial, white-spot enamel caries (WS) and a hidden dentin lesion were taken from the
same tooth (the second molar from the fourth quadrant). The lack of contact between the two lesions was assessed radiographically. The active, RNA-based bacterial
taxonomy was obtained by pyrosequencing of long PCR products encompassing hypervariable regions V1 through V4. Drawn from data in [20].
Opinion
Type of disease
Infectious
Dysbiosis
Present in health
No (invasive)
Yes (commensal)
Virulent behavior
Pathogenic
Pathobiont
Immune response
Active
Basal
Treatment
Antimicrobial
Restore balance
Opinion
19 Duran-Pinedo, A.E. et al. (2014) Community-wide transcriptome of the
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20 Simon-Soro, A. et al. (2014) Metatranscriptomics reveals active
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