J R Soc Med 2002;95:81-83
doi:10.1258/jrsm.95.2.81
© 2002 Royal Society of Medicine
Unculturable bacteria—the uncharacterized organisms that cause oral infections
William Wade PhD
Molecular Microbial Ecology Programme, Guy's, King's and St Thomas'
Dental Institute, King's College London, Floor 28, Guy's Tower, Guy's
Hospital, London SE1 9RT, UK
E-mail:
william.wade{at}kcl.ac.uk
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INTRODUCTION
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The first bacterial culture media were broths made either by
infusion or by
enzymatic digestion of meat from various sources.
Originally developed by
Spallanzani in the 18th century and
then refined by Pasteur in the 19th
century, these allowed the
recovery of bacteria from human disease
sites
1,2.
However, it
was quickly realized that such broths would be likely to contain
mixtures
of micro-organisms, and Robert Koch saw the need for development
of
solid culture media that would allow the physical separation
of bacterial
colonies. He first tried aseptically divided potatoes.
Material taken from
infected lesions was spread across the potato
and then incubated at body
temperature. Following incubation,
bacterial colonies were seen which could be
subcultured to further
potatoes to give pure cultures. Although successful,
Koch observed
that only a limited number of the micro-organisms present in
the
sample grew on the potato. This was probably the first recognition
of the
phenomenon of unculturability
in vitro. Nevertheless,
the success of
the technique led to the use of solidifying agents
such as gelatin and agar to
create solid media from the broths
developed by Pasteur and others. This
advance led to the golden
age of medical microbiology, in the last quarter of
the 19th
century, when many of the bacteria causing serious infections
in man
were identified. This tremendous success, however, probably
led
microbiologists to become complacent simply because so many
important
pathogenic bacteria could be cultured
in vitro in
this way.
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UNCULTURABILITY
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We are grossly ignorant of bacterial life on earth. Environmental
microbiologists
estimate that less than 2% of bacteria can be cultured in the
laboratory.
In the mouth we do rather better, with about 50% of the oral
microflora
being
culturable
3. For
other body sites, the figure is unknown
but is likely to be similar to that
found in the mouth or higher.
For example, the colonic microflora is suspected
to be predominantly
unculturable. It is therefore likely on numerical grounds
alone
that unculturable and therefore uncharacterized organisms are
responsible
for several oral and other human infections. A known instance
is
syphilis, caused by the spirochaete
Treponema pallidum, which
remains
unculturable today.
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MOLECULAR IDENTIFICATION OF BACTERIA
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It is only with the advent of molecular biology that techniques
have become
available for studying mixed bacterial communities
in their entirety, without
the biases of culture. The theoretical
basis for the development of these
methods came first from Zuckerandl
and
Pauling
4, who
suggested the use of biological macromolecules
for the elucidation of
evolutionary relationships among organisms.
This idea, which developed into
the branch of science known
as molecular phylogeny, relies on the analysis of
the DNA sequences
of genes of common ancestry, or proteins themselves, in a
range
of organisms. Mathematical techniques are used to assess the
similarity
of these sequences and to construct phylogenetic
trees which demonstrate the
evolution of the whole organisms
from which the DNA or proteins were isolated.
In practice, ‘housekeeping’
genes are used for this purpose since
they are widely distributed
among different organisms and because their
essential function
has made them relatively conserved throughout evolution,
allowing
easy alignment of the sequences. The most widely used of these
genes
to date has been the small subunit (16
S) ribosomal RNA
gene
5.
At around
1500 base pairs in length this is both long enough
to be informative and short
enough to allow easy sequencing,
particularly since the advent of automated
DNA sequencers. Phylogenetic
trees are constructed by first aligning sequences
of the same
gene from different organisms. Then the genetic distance between
pairs
of organisms in the dataset is calculated to give a matrix of
similarities.
This matrix is then further analysed by, for example, the
neighbour-joining
method in order to construct the phylogenetic tree or
dendrogram
6.
Once such a tree has been constructed, the identity of an unknown organism
can be determined by simply adding the sequence of its gene to the tree or by
running a similarity search against other sequences of the same gene in a
database. For 16S rRNA there are now several databases, containing
around 12 000 sequences in
total7,8.
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CHARACTERIZATION OF MIXED BACTERIAL COMMUNITIES
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Mixed bacterial communities can be characterized by adding polymerase
chain
reaction (PCR) and cloning steps to the procedure described
above. DNA is
first extracted directly from the biomass of the
original sample without
culture of the bacteria contained within
it. Then, the gene encoding
16
S rDNA is amplified by PCR with
primers specific for conserved
regions of the gene (i.e. regions
present in > 95% of bacterial
species
9). This will
result
in a mixture of all of the 16
S rDNA molecules from the
organisms
in the original sample. These are then cloned into a plasmid
vector,
which is used to transform an
Escherichia coli host,
thereby
establishing a library of 16
S rDNA from the sample.
The cloned genes
can then be sequenced individually and submitted
for identification to the
databases mentioned above, typically
via the World Wide Web. In this way, the
composition of the
microflora in the sample can be determined. If this
procedure
is complemented with traditional culture then the 16
S rRNA
genes
sequenced from the culturable organisms can be ‘subtracted’
from
the total microflora displayed by the molecular analysis, to
reveal the
unculturable portion. This technique, or a variation
of it, was first used to
describe the microflora of environmental
sites such as hot springs and deep
seawater
10,11.
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MOLECULAR ANALYSIS OF THE ORAL MICROFLORA
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Molecular analysis of the oral microflora to date has focused
on the
bacteria associated with dento-alveolar abscesses and
periodontitis. Dymock
et al.
12
performed molecular analysis
of pus aspirated with three dento-alveolar
abscesses and found
five groups of unculturable, and therefore previously
uncharacterized,
organisms that were predominant in the samples. Two of these
represented
totally novel eubacterial lineages. In addition, it was found
that
the numbers of two culturable species,
Fusobacterium nucleatum and
Porphyromonas endodontalis, were grossly underestimated
in the
samples. These observations suggest that some cultivable
species include
uncultivable biotypes.
A substantial number of novel taxa have also been identified in molecular
studies of
gingivitis13 and
periodontitis14. It
is noteworthy that the majority of novel lineages isolated from oral
infections are concentrated in two divisions of the phylogenetic
tree—the Cytophagas and the low G+C Gram-positive bacteria.
The studies of the oral microflora described above have all made use of
universal PCR primers for the initial 16S rDNA amplification, to
maximize the recovery of organisms from the sample. The technique can also be
adapted to use group-specific primers to specifically seek unculturable and
uncharacterized organisms within specific regions of the phylogenetic tree.
Spratt et
al.15 designed
primers for the oral asaccharolytic Eubacterium branch of the low G+C
Gram-positives and identified two groups of novel organisms within this branch
from a single advanced periodontitis sample. Similar studies are now underway
to investigate the microflora associated with endodontic infections, dentinal
caries, and the early stages of plaque development.
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WHY ARE SOME BACTERIA UNCULTURABLE?
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As discussed above, it has been known for over a hundred years
that
in-vitro culture conditions may not allow the growth of
all bacteria
in a sample. Some of the possible reasons are that
a required nutrient is not
present in the culture medium, that
the culture medium itself is toxic, or
that other bacteria in
the sample produce substances inhibitory to the target
organism.
In addition, we know that bacteria can depend on each other
for
growth. Oral bacteria, in particular, have evolved over
millions of years in a
mixed community in a biofilm. Over this
time, some may have acquired mutations
in essential synthetic
pathways but are able to obtain the substances required
from
other bacteria in the biofilm. Of course, a bacterium dependent
on
another will not be able to grow independently
in vitro.
One example
of this is
Bacteroides forsythus, a Gram-negative
anaerobe implicated
in periodontitis, which has an absolute
requirement for N-acetyl muramic
acid
16, one of the
essential
components of peptidoglycan. This organism grows very poorly
in pure
culture but grows well either in co-culture with other
organisms or in media
supplemented with N-acetyl muramic acid.
Another reason for non-culturability in vitro may be the
disruption of bacterial cytokine networks. Bacterial
cytokines17 are
thought to be the mediators of bacterial—bacterial signalling and may be
particularly important in coordinating the growth of component organisms in
bacterial biofilms such as dental plaque. Mukamolova et
al.18 have
reported the existence of a resuscitation-promoting factor (RpF) in
Micrococcus luteus which stimulates the growth of other Gram-positive
bacteria at picomolar concentrations. It is possible then that bacterial
growth is controlled by networks of such cytokines, which may be responsible
for the shifts in plaque composition in response to environmental factors.
Obviously, the separation of bacteria on solid media would disrupt such
networks and may explain why some organisms are unculturable.
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CONCLUSIONS
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The PCR-cloning-sequencing technique described in this review
now allows
the essentially complete description of complex bacterial
communities.
Originally developed for the dissection of environmental
ecosystems, these are
now being applied to the human microflora,
especially the flora associated
with oral infections. These
studies have confirmed that the estimates of
bacterial unculturability
made from combined microscopic/cultural studies are
essentially
correct and that 50% of the oral flora is unculturable. This
group
is highly likely to include novel pathogens, so we may
well be entering a new
golden era of microbiology when associations
between specific organisms and
infectious diseases can be assigned
with greater certainty than ever before.
We are also becoming
increasingly aware of the extent to which pathogenic
bacteria
in mixed infections communicate with each other and their mammalian
host.
The normal flora organisms in man have evolved as a biofilm
and may be
dependent on each other for nutrition, working together
in consortia to cause
disease in susceptible hosts.
In-vitro investigation of such
organisms should take into account this
interdependence.
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TOP
INTRODUCTION
UNCULTURABILITY
