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Endodontic Topics 2004, 9, 5–14
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Copyright r Blackwell Munksgaard
ENDODONTIC TOPICS 2004
1601-1538
Significance of bacterial
identification by molecular biology
methods
DAVID A. SPRATT
Rapid advances in molecular biology over the last 20 years have provided a bewildering array of techniques aimed at
helping us to tease apart all aspects of biology. The discipline of microbiology has gained greatly from these advances
especially with respect to detection and identification of micro-organisms. Indeed these molecular biology
techniques have changed the way we classy all life on Earth. An important part of endodontic microbiology is
detection and identification of the micro-organisms associated with initiation and progression of this polymicrobial
infection. A range of appropriate molecular techniques are reviewed in the present article and include aspects of
comparative 16S rRNA gene sequencing, polymerase chain reaction detection, strategies for identification of
unculturable bacteria, and whole community analysis. Some of these techniques are widely used in endodontic
microbiology while others are used by only a few workers. The advantages and disadvantages of all the techniques
are discussed and put into perspective.
All available surfaces in the oral cavity are colonized by
different and diverse microbial biofilms. Structures
present in the mouth but not exposed to the microflora
are usually sterile e.g. the endodontium – the pulp and
root canal system within teeth.
Endodontic infections are therefore defined as
infections of the pulp and periapical tissues. A bacterial
cause for these diseases was suggested by Miller (1) at
the end of the 19th century when he demonstrated
cocci, rods, and spirochaetes in necrotic pulps. However, because of other stronger arguments namely the
hollow tube theory, (2) a bacterial cause for these
pulpal and periapical diseases has only been attributed
since the mid 1960s when pioneering work by
Kakehashi et al. (3) demonstrated the importance of
bacteria as prerequisites to pulpal inflammation and
subsequent necrosis. Bacteria and there products gain
access to the pulp chamber in the majority of cases as a
consequence of caries. Because of significant demineralization of the enamel, cementum or dentine the pulp
can be directly exposed to insult by the biofilm
associated with the lesion. Additionally, the pulp can
be exposed by a number of other mechanisms e.g.
trauma, exposed dentinal tubules, congenital conditions, enamel lamellae and possibly anachoresis (4–7).
Before colonization bacterial products e.g. metabolic
end products, lipopolysaccharide etc can elicit an
inflammatory response from the pulp (8).
Accurate identification of micro-organisms involved
in a disease process is frequently essential not only for
effective antimicrobial therapy but also for understanding the disease initiation and progression.
Traditional microbiological
identification
Given the microbial nature of the disease, traditional or
culture-based microbiology studies were carried out by
a number of oral microbiology groups throughout the
world. Over the next two decades or so it was shown
that the bacteria species associated with these lesions
were surprisingly limited (9) given the number of taxa
potentially able to colonize and the large number of
taxa associated with periodontal lesions (see below).
This reduced diversity implies special selective pressures operating within the root canal system. While the
culture-based techniques have reported 4–12 taxa per
root canal (10, 11) when the range of taxa isolated
from root canal infections as a group as is taken into
account 20–30 genera are commonly isolated Of these
5
Spratt
the most commonly occurring species are Fusobacterium nucleatum, Streptococcus species, Porphyromonas
species, Prevotella intermedia, Peptostreptococcus species, Actimomyces species and Eubacterium species (The
genus Eubacterium is very broad and at present
undergoing significant taxonomic revision). The isolation and identification of these taxa lead to large
numbers of studies aimed at defining which taxa
were responsible for the disease, what mechanisms
they used and indeed, associating particular taxa to
different aspects of root canal infections e.g. pain,
lesion size, etc.
From early microscopy studies it was shown that
50% of the oral microbiota was unculturable (12).
Therefore, it was very possible that unculturable taxa
were present in root canal infections and were
potentially playing a role in the disease initiation or
progression or both. These unculturable taxa fall into
two broad catagories. The first are taxa that need
nutrients or other essential components that conventional sampling techniques, transport conditions or
laboratory media do not provide. This could be
sensitivity to oxygen (i.e. very strict anaerobes) or the
absolute requirement for products provided by other
taxa within the root canal (9). These taxa are therefore
broadly unknown apart from microscopy studies
although; unless distinct morphology is apparent there
is no way of knowing what proportion of the taxa
are represented in the culture dependant proportion of
the sample. The second category contains those taxa
that are known, and very often common, but for
some reason cannot be cultured, i.e. they are in a
dormant state and ‘non-culturable’ (13). The term
‘viable but not culturable’ (VBNC) was coined to
describe this state. It is thought that cells will go into
this state as a protection strategy in response to adverse
environmental conditions. It is very possible that
‘adverse’ conditions exist within root canals especially
nutrient deprivation and this may be another explanation for the limited taxa isolated for individual root
canal infections.
While microbiologists may have suspected that a
number of taxa were present and unculturable (for
whatever reason) there was very little that could be
done other than using complex media to mimic the
conditions present at the site of isolation or indeed use
co-culture strategies. At the end of the day they had to
be able to culture the taxa before they could identify or
indeed characterize them.
6
Molecular identification methods for
culture dependant techniques
With the advent of ‘molecular biology’ microbiologists
had another avenue to pursue with respect to understanding the microbiology of root canal infections.
Shortly after Kary Mullis described a polymerase chain
reaction (PCR) technique, for which he received the
Nobel Prize in 1993 (14), the flood gates opened with
respect to what was possible in the world of microbial
detection and identification. The application of PCR
and sequencing (and associated database construction
and searching software) revolutionized the detection
and identification of bacteria.
PCR is a technique, which uses a DNA polymerase
enzyme to make a huge number of copies of virtually
any given piece of DNA or gene. It facilitates a short
stretch of DNA (usually fewer than 3000 bp) to be
amplified by about a million-fold. In practical terms it
amplifies enough specific copies to be able to carry out
any number of other molecular biology applications
e.g. size determination (in bases) and its nucleotide
sequence. The particular stretch of DNA to be
amplified, called the target sequence, is identified by a
specific pair of DNA primers, oligonucleotides usually
about 20 nucleotides in length which designate the
outer limits of the amplification product.
Given that there are about 500 bacterial taxa present
in the oral cavity (15) the range and complexity of the
techniques utilized to identify this very diverse microbiota is bewildering. Molecular biology techniques
have lead to new approaches for bacterial identification.
The use of nucleotide sequence data from 16S
ribosomal RNA genes (amonge others), now makes it
possible not only to identify but to infer phylogeny for
all organisms on Earth (16). Phylogeny is defined as the
evolutionary relationships within and between taxonomic levels, particularly the patterns of lines of
descent, in a sense a family tree spanning 3.5 billion
years. Therefore within reason a single methodology
can be used to identify any bacterial isolate from any
environment. The 16S (small subunit) rRNA gene was
selected as a candidate molecule for a number of
reasons: (i) it is present in all organisms and performs
the same function, (ii) its sequence is sufficiently
conserved and contains regions of conserved, variable
and hypervariable sequence, (iii) it is of sufficient size
(ca. 1500 bases) to be relatively easily sequenced but
large enough to contain sufficient information for
Bacterial identification by molecular biology methods
identification and phylogenetic analysis. While this
technology took a few years to become popular a
drawback was how to analyze the sequence information
you had from your isolates. Until sufficient data had been
deposited in the National Centre for Biotechnology
Information (NCBI) Genbank database or specialized
ribosomal databases very few comparative identifications
could be made. This has, however, now been rectified
and the Ribosomal Database Project has over 124 000
aligned bacterial small subunit rRNA sequences deposited (as of February 2005). By visiting the RPP website
unknown 16S rRNA sequence can be easily uploaded
and compared with all the sequences on the database to
find its nearest neighbours, i.e. in most cases an
identification to the species level can be ascribed (Fig. 1).
This technology has meant that hundreds of isolates
can be identified quickly and easily. Indeed in a recent
study of 261 isolates from five infected root isolates 20
taxa were identified by comparative 16S rRNA sequence analysis at an average for 12.6 per sample (17).
Using this type of technology (a single methodology)
these isolates could have been identified with a couple
of weeks on a part time basis. This is compared with
probably some months work by an experienced
microbiologist using a large number of diverse and
complex biochemical techniques.
There are a number of advantages and indeed
disadvantages associated with the comparative sequencing technique. On the plus side the single protocol is
easily learned has relatively high through put and a
good level of identification can be expected. On the
down side is, however, that microbiologists and
molecular biologists with very little experience can
perform these techniques. The potential upshot of this
is the mis-identification of isolates. While the technique
is very straightforward the interpretation of the data
Prepare DNA from
a pure culture
Agar plate
Identification
Closest match on
database in
seconds
PCR Amplify
16S rRNA gene
30 cycles of:
Denaturing
Annealing
Extension
Analyse base
sequence
online
BLAST or RDP
produced is not as easy as it first looks. For example new
un-named isolates can be forced into species ‘pigeon
holes’ or indeed isolates from very closely related
groups can be misidentified, i.e. mitis group streptococci. Additionally in point (iii) above mention is made
of the size of the gene being sufficient to contain
enough information for identification and phylogenetic
analysis. While this is true (in most cases) it is common
practice not to sequence all 1500 bases, since this
would take a number of sequencing runs. Commonly a
single sequence is used ranging form 300–700 bases
and identity is conferred on this basis. In most cases this
may be acceptable (depending on what conclusions are
drawn or claims made) but care must be used in
interpreting the data without information on the
complimentary sequence (the other DNA strand) or
more complete sequence.
While the 16S rRNA revolution has undoubtedly
been a quantum leap for microbiology it has not been as
good as originally hoped. Some bacterial groups are
very closely related and the sequence information
within the gene is not sufficient to resolve these taxa
with any certainty. Difficult groups to resolve which are
relevant in endodonotic infections include: mitis group
streptococci (S. mitis, S. oralis, S. sangiunis, and S.
gordonii), Actinomyces spp, (A. naeslundii, A. israelii,
A. meyeri, A. odontolyticus, A. viscosus, A. gerencseriae
and A. radicidentis), coagulase-negative staphylococci
(S. epidermidis, S. warneri, S. lentus etc) and Veillionella
spp (V. parvula, V. atypica and V. dispar).
In light of this other candidate genes have been
proposed and used for comparative sequence analysis
studies. Not all of these follow the ‘rules’ above but are
often useful once 16S rRNA or indeed biochemical
characterization has identified the isolate as a ‘difficult
group’. Manganese dependant superoxide dismutase
Check for
“good” PCR
product
Clean
Product
Gel electrophoresis
Analyse DNA
sequence
Capillary or gel
electrophoresis
Sequence
PCR product
Fig. 1. Flow diagram showing the steps involved in bacterial identification using a 16S rRNA sequencing approach.
7
Spratt
(sodA) is one such gene that has been successfully used to
identify the oral streptococci, including the mitis group
(18) and the coagulase-negative staphylococci (19). A
number of other genes have been used to identify
coagulase-negative staphylococci but not commonly
within oral microbiology these are the hsp60 and rpoB.
Molecular identification methods for
culture independant techniques
The advent of PCR not only led to gene sequencing
and identification of culturable taxa. It also provided a
technique to circumvent the whole cultivation aspect of
species detection and identification. In its simplest form
this developed on the premise that given that bacteria
can be identified by differences in certain DNA
sequences (16S rRNA gene). It should then be
possible, using specific PCR primers, to identify a
particular species from any given sample whether that
be from a root canal, a periodontal pocket, carious
plaque or indeed saliva (Fig. 2).
This has led to a large body of literature pertaining to
the prevalence of specific taxa or groups in root canal
infections (and is not reviewed here). This technique is
relatively straightforward once the PCR parameters and
the specificities have been ascertained and high throughput approaches can deal with hundreds of samples per day.
Indeed, multiplex approaches allow more than one target
to be detected in each PCR reaction (Fig. 3). Multiple
primer sets can be used for at least three separate taxa (20).
As you might expect there are some problems with
this approach. The main problem is specificity for
example, if a positive result occurs for a sample how do
you know that it is actually an amplified product for the
target taxon rather than a similar one or indeed
something completely different (but happens to share
sequence homology)? The specificity testing only takes
into account the strains used in the test and this is
usually no more than 10–30. Given the potential of any
of up to 500 taxa being present, 50% of which are
unculturable it is not hard to see where false positives
can arise. As long as this is appreciated by the researches
than there are some remedies especially if 16S rRNA
genes are the target. The simplest of which is to
randomly select a proportion of the amplification
products (10–20%) and subject them to comparative
sequence analysis, i.e. identify them.
A further consideration is the detection limit of the
particular PCR technique (this may vary with user,
reagents and equipment). It must always be borne in
mind that to fail to amplify a product does not mean the
target template was not present in the sample – it means
that it was not there in sufficient quantity to be amplified.
As a rule of thumb the lower detection limit of PCR
(single round) is about 1000 cells of target (20). Further
modifications of PCR can take the detection limit down
to about 10 cells this is termed nested PCR. Essentially,
following the first PCR reaction another one is
performed with a different set of primers using the
product from the first PCR as a template. This technique,
while being a very sensitive, is prone to contamination
and false positive reactions are very common.
Simply increasing the detection limit does not really
answer any further questions like for example, which
taxa are there in high proportions and which are there
as very small proportions? Information on proportions
may provide clues as to which taxa are important in
disease initiation and progression bearing in mind that
Patient samples
C
1
2
3
4
5
6
7
8
9
10
C
Fig. 2. Diagrammatic representation of the visualisation of polymerase chain reaction products from 10 subjects where
taxon ‘A’ was targeted with a specific primer set. It shows a band present in subject samples 1, 2, 4, 5, and 10. This
indicates that taxon ‘A’ was detected in these five subjects and not in subjects 3, 6, 7, 8, or 9. C denotes a control sample of
target taxon alone.
8
Bacterial identification by molecular biology methods
Patient samples
C
1
2
3
4
5
6
7
8
9
10
C
D
E
F
Fig. 3. Diagrammatic representation of the visualisation of multiplex polymerase chain reaction (PCR). Amplification
products from 10 subjects where three specific taxa ‘D’, ‘E’, and ‘F’ were targeted in one PCR reaction. All three taxa are
only detected in subject 2. None of the target taxa were detected in subjects 6, 7, and 9.
DNA from 5 root
canal samples
1 2 3 4 5
A
B
1 2 3 4 5
a
b
DNA from
5 taxa
c
a
b
Wash and
detect
c
d
d
e
e
Fig. 4. Diagram of Checkerboard DND–DNA hybridization showing; A – vertical lanes containing sample DNA
and horizontal lanes with DNA from five known taxa. B –
following hybridization, washing and detection the
presence or absence of the various taxa in the patient
samples can be ascertained. Taxon b is present in all
samples, taxon c is not detected in any sample, taxon a is
detection in samples 2 and 5, etc.
in a polymicrobial infection the key taxa may be
different for each stage.
A further development of PCR has meant that not
only could specific taxa be detected but they could be
quantified as well. This technique, real-time PCR or
quantitative PCR, uses fluorescence to detect PCR
products as they accumulate. Theoretically, there is a
quantitative relationship between the amount of
starting material and the PCR product at any cycle
(21). Therefore using PCR primers from above (or
modifications of these) specific bacterial taxa can be
detected and quantified. Indeed if a global 16S rRNA
gene PCR primer (theoretically amplifies all 16S rRNA
genes from all bacterial taxa) is used as well (in a
separate reaction) the total number of bacteria present
in the sample can be ascertained. Therefore an
estimation of the proportion (as a function of
the whole microbiota) of a target taxon can be made.
The technique obviously suffers from similar drawbacks mentioned above but as long as these are carefully
controlled for and considered, quantitative PCR will
provide crucial information pertaining to the progression and nature of root canal infections. A quantitative PCR approach has been used to study the
microbiology of carious dentine (22) and showed a
greater bacterial load by quantitative PCR than culturing methods and quantified a number of important taxa
(Micromonas micros, Porphyromonas endodontalis and
P. gingivalis).
Molecular techniques for bacterial detection and
identification are not restricted to PCR alone and a
notable alternative technique is checkerboard DNA–
DNA hybridization. This technique involves deposition
of bacterial DNA from clinical samples (root canal, plaque
etc) in parallel (vertical) lines on a nylon membrane.
Digoxigenin-labelled whole genomic DNA probes are
run at right angles to the samples (horizontal). Following
washing the bound probe is detected and quantified (Fig.
4). This method was pioneered and extensively used by
Sigmund Socransky in Boston, MA, USA (23–26). The
technique utilizes whole genomic DNA for 40 bacterial
taxa and 28 patient samples per membrane this makes it a
very high throughput technique and thousands of samples
can be analyzed very quickly generating huge amounts of
data regarding the detection rates of the forty taxa in each
sample. The technique is semi-quantitative and standards
containing known numbers of cells are used (105 and
106). A potential drawback is however the unknown cross
reactivity with unknown taxa present in the sample.
Additionally the technique can only provide information
on known culturable taxa and while very valuable does not
address the unculturable proportion of any sample. The
technique has been used to a limited extent in endodontic
microbiology studies (for details see (27–30)).
9
Spratt
Detection and identification of
unculturable taxa using molecular
methods
To determine the unculturable microbiota in a sample,
culture and PCR approaches are often used in a
subtractive technique. A given root canal sample is
processed routinely by culture – that is to culture on a
range of media (selective and non-selective) in both
aerobic and anaerobic atmospheres. Isolates are identified by 16S rRNA gene sequence analysis (as above).
Additionally an aliquot can be analyzed using a PCRcloning approach. DNA is isolated and purified from
this aliquot. Using a similar PCR technique used for
bacterial identification (see above), 16S rRNA genes
are amplified. This will produce a mixed product i.e.
instead of amplifying DNA from a pure culture (one
taxon) this amplifies DNA from all the taxa present in
the sample. Because of this the PCR product cannot be
simply sequenced since, sequencing a number of
different 16S rRNA genes from different bacteria at
the same time will produce nonsense sequence data
Amplicon
A C B
from taxon B
C
A
B
B
A
B A B
C
D C C
B
C
C
A
which cannot be analyzed. Therefore the different 16S
rRNA genes present need to be separated. This is done
by cloning the PCR products (see Fig. 5). Once the
PCR products are singularized (each one separated into
a plasmid and transformed into a host cell) they can be
sequenced and identified as for culturable taxa (above).
At this point there are two lists of bacteria identified
from the sample; a culture dependent list and a culture
independent list. Those taxa present on the culture
independent list but not on the culture dependent list
are therefore counted as ‘unculturable’ (Fig. 6).
The taxa determined as unculturable maybe either new
unknown taxa or indeed well known and usually
culturable taxa (possibly in a VBNC state). To understand
the prevalence of these newly detected taxa in the infection
specific PCR primers can be obtained or designed for
straightforward PCR detection assays as detailed above.
The subtractive PCR cloning approach is very powerful
but is very time consuming and expensive to perform on
large numbers of samples. It can however provide detailed
information on the richness of the microbiota at any given
site and provide targets for further studies.
C
Insert
B
C
A
D
A
Plasmid
B
B
Mixed PCR product
Ligate products into a Plasmid
Universal primers used to amplify 16S
rRNA genes from a 4-membered
community (A-D)
Plasmid contains insert and gene
encoding antibiotic resistance amongst
other things
A
Cells can now be
replicated and
stored.
Insert can be
sequenced and
identified
Plasmid
E. coli cell
B
Transformed colony
Each colony consists of cells with
one plasmid containing one 16S
rRNA gene
Cells can grow only if the plasmid is present
since it contains resistance to the antibiotic
used
Transform E. coli cells with vector
One plasmid per cell.
Culture on antibiotic containing media
Fig. 5. Diagramatic representation of the polymerase chain reaction (PCR) cloning process used to singularise mixed
PCR products.
10
Bacterial identification by molecular biology methods
Root canal Sample
Paper point or pus aspirate
Culture (+/− enrichment)
on agar media
Isolate and purify DNA
directly from clinical sample
Non-selective or selective
Aerobic & Anaerobic
PCR amplify 16S rRNA
gene and sequence See Figure 1
Randomly Select
30− 50 isolates
Singularise by TA cloning
Characterise
Colony morphology
Gram morphology
Catalase test
Oxidase test
Randomly Select 30− 50 clones
containing 16S rDNA insert
PCR amplify 16S rRNA
gene and sequence
Amplify 16S rRNA
gene and sequence
See Figure 1
See Figure 1
Compare
Identify
Any taxon present as a
clone and not as an
isolate is considered
unculturable
Identify
Fig. 6. Strategy defining the unculturable microbiota in a root canal sample.
The main perceived drawback of this technique is a
concern that the universal primers used in the PCR are
not as universal as once hoped for example, selective
amplification of templates with a low GC content (31,
32). Additionally previous studies have also reported
that all of the steps involved in the production of a gene
library may have some biases (33–36). Therefore,
although it is often boasted that this technique negates
the biases inherent in culture it is less frequently
mentioned that it might have a number of biases itself!
A number of studies have been carried out with root
canal samples (37–39, 17) and pus from alveolar
abscesses (40, 41). In most of these studies novel taxa
were detected and described. Indeed, in a culture and
cloning study similar to that described in Fig. 6
Munson et al. (17) detected 65 taxa from only five
root canal samples, 27 of which were novel.
Whole community analysis
Rather than trying to dissect the microbial nature of root
canal infections (or indeed any polymicrobial infection)
an alternative approach can be taken. This involves
defining the community and its characteristics as a whole
from a root canal and comparing these characteristics
with other root canals. While the above techniques can
be considered as community analysis the only one, which
really counts with respect to ‘whole’ is the culture/PCRcloning approach and this is impractical for detailed
comparisons between a number of samples.
The precise nature of these techniques is still being
developed for oral microbiology but a good example is
denaturing gradient gel electrophoresis (DGGE).
DGGE is a PCR based technique with a difference;
rather than using the sequence of bases in the amplified
11
Spratt
Patient samples
1
2
3
4
5
6
7
8
9
10
Fig. 7. Diagrammatic representation of the visualization of a DGGE gel. Directly amplified 16S rRNA genes from root
canal samples from 10 subjects showing different banding patterns (fingerprints). Lane 2 and 3 show complex patterns
indicating a high species richness while lanes 4 and 6 show a less complex pattern indicating low species richness.
product for identification (i.e. sequencing a 16S rRNA
gene) DGGE separates DNA fragments according to
their sequence information (42). The basis of this
technique is that DNA fragments of the same size but
with differing base-pair sequence can be separated (43).
This separation by DGGE relies on the electrophoretic
mobility of partially denatured DNA molecules in a
polyacrylamide gel, which is encumbered in comparison
with the completely helical form of the molecule (43).
A banding pattern is formed based on the number of
taxa present in the sample (Fig. 7). If 16S rRNAgene is
used as the target for the PCR then these bands can be
cut out from the gel and sequenced to provide an
identity for the particular band.
Since a PCR amplification step is used then the biases
previously mentioned may be operating, however, the
first step towards overcoming these is acknowledging
their existence. A further problem is the interpretation
of the data at a community fingerprint level. While
cutting out bands and sequencing them gives valuable
information it is also time consuming and costly. What
is needed is a way to compare banding patterns within
and between gels (samples) such that a particular
pattern or specific bands in a pattern is indicative of
certain clinical parameter. A number of techniques have
been used but none have been broadly adopted perhaps
because the most applicable one has not been developed to date.
DGGE has been applied in environmental microbiology (44–46) and in the analysis of microbial communities
in the human body (47–50) Recently DGGE has also
been applied to analyse the bacterial diversity of human
subgingival plaque (51, 52) as well as laboratory-grown
dental plaque microcosms (53). Siqueira et al. (54) have
12
successfully used this technique for root canal samples
and found differences in banding pattern between
symptomatic and asymptomatic infections assigning a
mean of seven taxa to asymptomatic endodontic infections and 12 taxa to symptomatic infections.
Polyphasic approaches
The molecular biology approaches described above
have given endodontic microbiologists a range of
powerful tools to understand the complex nature of
root canal infections. There is now a simple technique
to identify isolates, in most cases, to species level and
sometimes beyond. High throughput techniques have
been developed to detect specific taxa in large numbers
of samples. Even those taxa, which we have previously
termed ‘unculturable’ are now being detected and
characterized. Indeed, the concept of ‘community’ is
also being explored.
However, given the large number and variety of
molecular techniques available for the detection,
quantification and identification of micro-organisms
from root canal infections one might be forgiven for
thinking that traditional culture is redundant and
destined for microbiology history textbooks – this is
however far from the case. As I hope I have demonstrated here there are a large number of molecular
biology techniques used to detect and identify bacteria
but none of them is without flaw. A major draw back
with most of these techniques is that, because of there
very nature as culture independent techniques, they do
not provide access to the whole genome. This has major
implications if the molecular detection technique of
choice shows a very strong correlation with an as yet
Bacterial identification by molecular biology methods
unculturable taxon. In the past using culture dependent techniques the isolate in question can be subjected
to a battery of biochemical tests to ascertain what
virulence factors it has. On the basis of this information
further biochemical and molecular biology techniques
are used to characterize the nature of these factors with
respect to their role in disease initiation and progression. A culture independent technique to proved whole
cells or whole genomes is therefore eagerly awaited.
These techniques (culture dependant and culture
independent) are not exclusive to each other and
should be used together by endodontic microbiologists
in an informed polyphasic manner to understand the
complex nature of root canal infections.
13.
14.
15.
16.
17.
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