Note: Descriptions are shown in the official language in which they were submitted.
CA 02430586 2003-05-30
POPULATION PROFILING MATERIALS AND METHOIDS
FIELD OF THE INVENTION
The invention relates generally to the field of microbiology, and more
specifically to the
characterization and identification of microorganisms within a community of
microorganisms. The community of microorganisms may be isolated fre~m any
number of
sources including, but not limited to, humans, animals, plants, soil, and
other environments
known to harbor microorganisms. The invention describes a method for
identifying groups
of microorganisms within a sample of microorganisms within the community. The
invention
further discloses a method of comparing and analyzing the microorganisms using
genetic
sequences relating to specific variable regions flanked by non-variable, or
constant, regions
of nucleic acid within DNA regions of interest.
BACKGROUND
Microorganism culture-based techniques were developed that, when. combined
with
differentiation of isolates based on numerous physiological and biochemical
tests, became the
standard method for investigating a microbial community composition. A
limitation of these
methods is referred to as the great plate count anomaly (Si:aley, et al. 1985.
ArcrZU. Rev.
Microbiol. 39:321-346). That is, only a small fraction of microorganisms
present in a
population can be cultured in the laboratory, wherein the fraction may be as
low as 0.001 to
15%, depending on the community (Amann, et al. 1995. Mica°obiol. Rev.
59:143-169).
The development of recombinant DNA methods yielded a proliferation in small-
scale studies
of complex microbial communities, such as those associated with termite guts
(faster, et al.
1996. Appl. Environ. Microbiol. 62:347-352), rice paddy soil (Bai, et al.
2000. Microb.
Ecol. 39:273-281), 120-million-year-old amber (Greenblatt, et al. 1995x.
Microb. Ecol.
38:58-68), Antarctic lake ice (cordon, et al. 2000. Microb. Ecol. 39:197-202),
and leaves of
a seagrass in the northern Gulf of Eilat (Weidner, et al. 2000. Microb. Ecol.
39:22-31 ).
Molecular methods for microbial community analysis (as reviewed in: Ranjard,
et al. 2000.
Re.s. Microbiol. 151:167-177; Theron, et al. 2000. Crit. Rev. Microbial. 26:37-
57; and
Vaughan, et al. 2000. Curr. issues Ir2test. Microbiol. 1:1-12) include
denaturing gradient gel
CA 02430586 2003-05-30
electrophoresis, temperature gradient gel electrophoresis, and restriction
fragment length
polymorphism analysis. While these methods disclose rapid comparative analyses
of
populations and generate population "fingerprints," the methods do not
identify individual
organisms within the populations.
Methods that identify individual members of microbial communities are based on
PCR and
direct sequencing or cloning and sequencing of specific targets within
microbial genornes.
The most frequently used target is the 16S rRNA gene (Olsen, et al. 1986.
Arcnu. Rev.
Microbiol. 40:337-365, Pace, et al. 1986. Adv. Microb. Evol. 9:1-55). The
molecular
phylogenetic view of the microbial world is dominated by 165 rRNA sequence
relationships,
and the wealth of sequence information accumulated for 165 rRNA genes from
thousands of
organisms is stored in the Ribosomal Database Project (Maidak, et al. 1999.
Nucleic Acids
Res. 27:171-173) and is a standard tool for studying microbial communifiies.
Libraries of
total genomic DNA extracted from a community of interest can be screened for
rRNA genes,
L5 or libraries of PCR amplified rRNA genes or gene segments can be generated
and sequenced.
While studies of multiple libraries of 16s rRNA have been attempted (Leser
D.L., 2002,
Applied Environmental Microbiology 68: 673-690) this approach involves
significant
challenges which prompted the suggestion that a DNA microarray approach was
the future of
such work.
Other gene targets used in microbial identification and elucidation of
phylogenetic
relationships include rpoB (Dahllof, et al. 2000. Appl. Environ. Mtcrobic~l.
66:3376-3380),
gyrB (Kasai, et al. 1998. Genome Inform. Ser. Workshop Genorrze Inform. 9:13-
21 ), pmoA
(Bourne, et al. 2001. Appl. Environ. Microbiol. 67:3802-3809), and cpn~~0,
which encodes
the 60-kDa chaperonin found in virtually all eubacteria and the mitochondria
and chloroplasts
of eukaryotes (Sigler, et al. 1998. Arcnu. Rev. Bicchem. 67:581-608). A
molecular method is
available for the identification of microorganisms based on amplification of a
portion of the
cpn60 gene by universal, degenerate PCR primers (Gob, et al. 2000. J. Clin.
tllicrobiol.
38:3953-3959). This method has demonstrated advantages over 165 rRNA--based
methods in
that for closely related organisms, there is more phylogenetic information in
the protein-
encoding cpn60 sequence relative to the structural RNA-encoding 165 rRNA gene
(Brousseau, et al. 2001. Appl. Euviron. Mzcrobiol. 67:4828-4833).
2
CA 02430586 2003-05-30
S~TMMARY OF THE INVENTION
The invention provides, in one aspect, a method for profiling the relative
abundance of
different kinds of organisms in a population. The method involvea, generally,
the
identification of a target DNA region which is present in all organisms of
interest and within
which the exact DNA sequence varies between different kinds of organisms of
interest.
The population as a whole is sampled, and DNA is extracted, without the need
to separate
different kinds of organism before DNA extraction. The extracted DNA is then
examined to
identify what target regions it contains. These target regions are then
compared to known
target region sequences for organisms of interest to identify what organisms
of interest are
present in the population and what their relative abundance is. In many cases,
once target
regions have been identified, they will be grouped phylogenetically so that
the relative
abundance of different general types or classes of organisms can be compared.
Once a population has been profiled, this information can be used to identify
potential
concerns relating to the population or to predict further population shifts.
For example,
populations of microorganisms exist in association with the mammalian body.
Concerns
typically arise only when the ratio of different types of organism in the
population deviate
significantly from what is observed in normal, healthy subjects. Thus, thf~
invention allows
the comparison of population profiles to be obtained from subjects who may be
at risk to
those from healthy subject and (if desired) subjects having known population
profile
abnormalities of clinical or other concern.
The target DNA region can be any region present within all organisms of
interest and capable
of ready identification from a heterogeneous sample. In some instance, the
target DNA
variable region is flanked by non-variable regions which allow PCR
arr~plification of the
variable region and the production of libraries.
Target regions of particular interest can be found in cpn60 (chaperonin 60),
16s rRNA, 23s
rRNA and the 16s-23s interspacer region.
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CA 02430586 2003-05-30
In one embodiment of the invention there is provided a method, the first stage
of which is to
develop as complete an inventory or census as possible of the population of
interest based on
partial cpn60 sequences. To do this one uses the total genomic DNA from the
population as
template in PCR reactions with the universal cpn60 PCR primers H279 and H280
(see for
example US5,708,160 of Hemmingsen). These primers amplify 552,555 or 558
nucleotides
(some other very rare exceptions to these sizes) of the cpn60 gene from
genomes present.
The PCR product pool is legated into a cloning vector to create the library or
libraries.
Randomly selected clones are sequenced. The result of the sequencing is a
collection of
partial, "universal target" sequences representing population constituents.
Putative taxonomic
assignments of the sequences are made based on comparing each sequence to the
reference
database of cpn60 sequences. The frequency with which each sequence is
recovered from the
library is determined. This sequence collection of cpn60 universal targets
becomes the
jumping-off point for all downstream activities.
In the second stage of this embodiment, representatives of each unique
sequence are used to
create a phylogenetic tree and the frequencies of each sequence from each
library are applied
to the tree. The result of this is that one can identify clusters or branches
of the tree (clusters
or groups of phylogenetically related sequences) which have a ratio of
interest - could be that
the taxon of interest was many times more abundant in one of the libraries or
could be that it's
interesting because it's present in constant amounts in all libraries. Once a
target taxon has
been identified as interesting, this subset of sequences is pulled out of the
collection for
further analysis.
The third stage (after target taxon identification) is to find signature
sequences in the target
group which are in common to all members of that group and are not found in
other
population members (and preferably not in anything unrelated in the known
universe of
cpn60 sequences). These signature sequence elements can be used to design PCR
primers
which will amplify a region internal to the universal target (552-558 bp). The
size of this
PCR product will be less than 552-558 by but its particular size will depend
on the possible
location of distinguishing primers. In some. instances a product size of 100-
200 bases since
this is the preference for Taqman type real-time PCR. If SYBR green or smother
approach is
used for quantitative PCR, the product size can be less important.
4
CA 02430586 2003-05-30
The PCR products produced by the universal primers could be analysed by
techniques such
as denaturing gradient gel electrophoresis (DGGE) as has been often done ira
16s rRNA based
studies. Either instead of, or in addition to sequencing. As DGGE provides
only a
"fingerprint" and not a sequence for identification purposes, sequencing will
sometimes be
preferred.
Based on the heterogeneity of specific regions within the cpn60 gene and the
its ubiquitous
presence of the cpn60 gene mammals, a novel use of the cpn60 gene is useful in
isolating and
identifying specific families of microorganisms within a diverse community has
been
developed. Thus, this invention encompasses the design and use of primers for
the purposes
described using the cpn60 gene.
Amplification of over 2,000 unique cpn60 sequence isolates from within a
diverse microbial
population allows specific primers characteristic of the cpn60 homologues
expressed within
each population to be engineered. The present invention thus relates generally
to the methods
for designing the present population-, or taxa-specific primers. The
specifically engineered
primers may be useful in accurately determining the number, types, and
identities of specific
populations of microorganisms present within an environment. The disclosed
identification
method allows for rapid determination of causative agents as, for example, in
disease,
malnutrition, and infection. Kits using these primers may be designed and used
for the rapid
identification of certain microorganisms.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts in bar graph format the species population found in C, B and W
libraries and
depicts in bar graph format the number of clones recovered from the C, B and W
libraries.
FIG. 2 illustrates the phylogenetic relationship between 136 sequences
originating from the C
library using a phylogenetic tree generated by phylogenetic analysis using a
maximum
likelihood distance calculation followed by the neighbor-joining method.
5
CA 02430586 2003-05-30
PIG. 3 illustrates the phylogenetic relationship of 171 unique nucleotide
sequences derived
from the B library using a phylogenetic tree generated by phylogenetic
analysis using a
maximum likelihood distance calculation followed by the neighbor joining
method.
FIG. 4 illustrates the phylogenetic relationship between 110 sequences
originating from the
W library using a phylogenetic tree generated by phylogenetic analysis using a
maximum
likelihood distance calculation followed by the neighbor-joining method.
FIG. 5 illustrates the phylogenetic relationship between 316 sequences derived
from the C, B
and W libraries using a phylogenetic tree generated by phylogenteic analysis
using a
maximum likelihood distance calculation followed by the neighbor-joining
method.
FIG. 6 lists the unique nucleic acid sequences identified in microorganisms in
human vaginal
samples and listed in the BV-1 and BV-2 libraries.
FIG. 7 depicts a comparison of frequencies of recovery of clones corresponding
to various
taxonomic groups (genera) from human vaginal flora libraries BV-1 and BV-2
depicted in
bar graph format.
FIG. 8 illustrates the phylogenetic analysis of CFB group sequences recovered
from human
vaginal flora libraries.
FIG. 9 lists the unique nucleic acid sequences identified in microorganisms in
crucifer
maggot (Delia redicum ) midget samples.
FIG. 10 depicts an experimental scheme for the creation of cpn60 sequence
libraries from the
midget microflora of Delia radicum.
FIG. 11 illustrates the peptide sequence distance tree for sequences derived
from Delia
redicum midget flora cpn60 library and selected cpn60 reference sequences.
6
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FIG. 12 A. frequency distribution of unique nucleotide sequences recovered
from the
combined pig faeces cpn60 libraries. B. Taxonomic breakdown of total library
contents.
Assignment to a taxonomic graup was based on comparisons of clone sequences to
a
database of cpn60 reference sequences.
FIG.13 phylogenetic relationships of 280 unique Cpn60 peptide sequences
translated from
398 unique nucleotide sequences. Distance calculations were made using the
Dayhoff PAM
matrix and the dendrogram was produced by neighbour joining. The scalebar
represents 0.1
substitutions per site. Branches are coloured according to the assigned
taxonomic group of
the sequences (red, CFB group; green, Proteobacteria gamma; blue,
bacillus./clostridium
group; black, other).
FIG. 14 phylogenetic relationships of 12 clone peptide sequences assigned to
the CFB
group, including the two most abundant cloned sequences (represented by 001
f12 and
002 a03). The tree is a consensus of 100 neighbour joined trees. Distance
calculations were
made using the Dayhoff PAM matrix and branch lengths were imposed on tl'ae
consensus tree
using fitch. Nodes with bootstrap values >50% are indicated with white dots.
Reference
sequences used in the tree are Flavobacterium hydatis (GenBank accession
AAK32145),
Flavobacteriacm ferrugineum (AAK32146), Bergeyella zoohelcum (ATCC43767),
Chryseobacterium meningosepticum (ATCC13253), Chryseohacterium gleam
(ATCC35910), Bacteroides forsythus (CAB43992), Bacteroides vulgatus
(ATCC8482),
Bacteroides uniformis (ATCC8492), Bacteroides ovatus (ATCC8483), Prevotella
bivia
(ATCC29303), Prevotella intermedia (ATCC25611 ), Rhodotlzermus marinas (strain
ITI 376,
AAD37976), and Chlorobium tepidccm (derived from contig 3499, TIGR unfinished
genome
database).
7
CA 02430586 2003-05-30
FIG. 15 cummulative frequency distribution plots for the pig faeces library
(solid line), a
population of individual species from 172 different eubacterial and eukaryotic
genera,
a single taxonomic subclass (77 species from 34 genera of Proteobacteria
gamma) and a
single genus, Lactobacillus. Plots were generated from DNA identity matrices
derived from
CLUSTALw multiple sequence alignments using GeneDoc.
FIG. 16 A. frequency distributions of unique nucleotide sequences recovered
from clone
libraries P56, P40, B40 and B56. B. Taxonomic composition of libraries 840,
856, P40 and
P56.
FIG. 17 taxonomic composition of groups of library clones pooled by PCR
annealing
temperature used in library construction (left panel) or genomic DNA template
extraction
method (right panel).
IS
8
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DETAILED DESCRIPTION
The present invention discloses a method for identifying a microorganism or
characterizing
populations or groups of organism through amplification of variab:fe regions
of a
polynucleotide sequence using PCR or related molecular approaches. The
generated
amplieon can be utilized to identify and distinguish unique microorganisms at
the species
level.
The development of high-throughput technologies for genomics applications
presents an
opportunity to conduct large-scale, even comprehensive, studies of complex
microbial
communities. It is now possible to conduct a study of the application of
genomics technology
and the molecular diagnostic method disclosed herein to cataloging the
diversity in a
microbial community. Results confirm the potential for this method in larger
studies of
microbial communities and the establishment of universal targets for studying
the
phylogenetic relationships of microorganisms in complex communities.
One potentially useful application of these new methodologies concerns the use
of antibiotics
in mammalian feed stocks. Modern North American pig rearing practices involve
the
inclusion of subclinical doses of antibiotics in feed to control populations
of pathogenic
bacteria. Pigs undergoing weaning and the coincident shift from a
predominantly Gram-
negative to a predominantly Gram-positive flora are particularly vulnerable to
pathogens such
as Escherichia coli and Clostridium ~erfringens. With increasing pressures to
eliminate the
use of antibiotics to control intestinal diseases in pigs comes an increasing
interest in
understanding the role of normal intestinal flora in mammalian health and in
utilizing the
potential prebiotic properties of feed ingredients.
Genomics-inspired technologies such as robotic colony picking, template
preparation, and
sequencing and automated data assembly and analysis can be employed to produce
potentially comprehensive profiles of important microbial communities. The
libraries of
sequence data produced will be tools for developing methods to quantitate
organisms within a
population, for the detection of pathogens or specific organisms of interest,
to monitor
9
CA 02430586 2003-05-30
changes in populations over time or treatment, and for creating specific
probes for techniques,
such as fluorescence in situ hybridization.
The potential extension of these new technologies to identifying Unique
microorganisms in
human subjects is an opportunity to develop novel diagnostic kits. Rapid and
accurate
identification of microorganisms within mammals allows rapid and more
effective treatment
of mammalian subjects. The savings in time potentially yields new life saving
measures
through more efficient targeting of microorganisms using selective anti-
microbial agents. In
many cases the methods disclosed herein will be useful in examining
populations resident in
a range of animals, including mammals, birds and fish.
The present invention discloses oligonucleotide primers far identifying unique
microorganisms within a large community of microorganisms by amplifying; a
polynucleotide
sequence having a variable region. Examples of polynucleotide sequences of
interest
include: cpn60, 16s rRNA, 23s rRNA and the 16s-23s interspacer region. This is
accomplished by using primers having a sequence that is substantially
homolagous to
sequences of non-variable regions flanking the variable regions of the
polynucleotide region
of interest. Oligonucleotide primers described herein may be isolated (pure),
may be double
or single stranded, may consist of I3NA, RNA, or any other nucleic acid
molecule
substantially homologous to the regions specified such that the primers may
hybridize to the
target region under stringent conditions and be used in the current invention.
The length of
the primers of the present invention may depend on many factors including;,
but not limited
to, an identity of a host organism or environment, amount of material
available, techniques
used, and other unforeseen variables. The primers should be able to hybridize
to the locus to
be amplified under conditions that allows accurate synthesis of nucleic acid,
thus requiring
the primers to be substantially complimentary to each strand of the non-
variable regions
flanking the variable regions within the genomic locus to be amplified.
As used herein, the term "organism" includes insects and microorganisms and
includes
protozoans, gram-positive bacteria, gram-negative bacteria, yeasts, fungi,
insects, and any
other organism whose genome encodes for the chaperonin-60 protein o~r its
equivalent.
Nucleic acid from the organism is prepared using any number of techniquE;s
used to isolate
nucleic acid. The non-variable regions flanking the variable regions within
the genomic
CA 02430586 2003-05-30
locus to be amplified are allowed to anneal to the primers under conditions
which allow
efficient and accurate hybridization. Various techniques may be used to
encourage maximum
diversity in the selection of targets during the annealing process, including,
but not limited to,
splitting the target DNA into equivalent lots to be processed in parallel at
different annealing
temperatures.
Target DNA (or a portion thereof) is amplified using any number of known DNA
amplification techniques such as the addition of agents that catalyze
reproduction of DNA
such as E. cop DNA polymerise I, T4 DNA polymerise, Taq. and Vent polymerises,
reverse
transcriptase, and other known enzymes.. Products may be purified using a
variety of known
isolation methods. These products may be sequenced to identify the unique
organisms from
which the original variable sequence originated. The sequencing can be
accomplished a
number of ways, including, ligating the amplified variable regions containing
flanking ends
into a vector for the purpose of transfection into reproducing bacterial hosts
(such as E. cola).
These hosts may be plated and colonies isolated for the purpose of growing
large quantities
of the variable region carried by the plasmid within the host cells. The
plismids may be
isolated allowing sequencing of the variable region and comparison of these
sequences.
The aforementioned techniques are examples by which one of ordinary skill in
the art are
able to identify and compare sequences of unique members of a diverse
population within a
large community of microorganisms. ~ne of ordinary skill in the art in light,
of the disclosure
herein may be able to design other methods for isolating and amplifying the
isolated variable
regions for sequencing that would be equivalent to the method described
heroin.
The method of the invention can be readily applied to a range of DNA regions
so long as (a)
the DNA region is present in all the organisms of interest; (b) the DNA region
has non-
variable regions flanking a variable region so as to allow selective
amplification of the
variable region; and, (c) the DNA region is phylogenetically informative
(allowing
association of the DNA region with a particular species, genus or variety o:f
organism). For
example, the DNA sequence encoding 16S r RNA is known for a wide range of
species, as
are "universal primers" useful to amplify a variable region in 16S r RNA
suitable to allow
phylogenetic identification of the source organism.
CA 02430586 2003-05-30
In some instances, the variable region will preferably be between about 2.5-
1000 bases. In
some cases 50-500 bases will be desired. In some cases 50-200 bases will be
desired.
The invention described herein further discloses a method for designing
primers specific for
unique target taxa found within communities of microorganisms. Upon comparison
of the
sequences to a suitable database (such as the cph~0 database ("epnl~B") where
the target is in
cpn60), the nearest neighbors of the identified and sequenced clones may be
determined. The
nearest neighbors may be considered in a group for the purpose of designing a
new template
specific for the target taxa. These new templates may be used as starting
materials for
detecting the presence or absence of the unique taxa group within various
samples.
Useful cpn60 primers are disclosed in US 5,989,821 and US 5,7089,160. Also
useful are
H729: 5'-CGC CAG GGT TTT CCC AGT CAC GAC GAI III GCI G(3I GAY GGI ACI
ACIAC-3'
H730: 5'-AGC GGA TAA CAA TTT CAC ACA GGA YK IYK ITC IC'C RAA ICC IGG
IGC YTT -3'
165 rRNA is useful as a target. Primers of particular interest for use with
16~s rRNA are 5'-
TCCTACGGGAGGCAGCAGT -3' and GGACTACCAGGG'TATCTAATCCTGTT -3'
(universal primers for amplification of what is vaauably called the V3 region
of the I6s gene),
and 5'-GAGTTTGATCCTGGCTCAG -3' and 5'-GWATTACCGCGGCKGCTG-3'(universal
16s rRNA primers which amplify the regions variably called Vl and V6,
encompassing from
nucleotides I 1-536 on the E. coli sequence).
23S rRNA is also useful as a potential target. Primers of interest include 5'-
GCGATTTCYGAAYGGGGRAACCC-3' and 5'-TTCGCCTTTCCCTCAC'.GGTACT -3'
The 16s-23s interspacer region is also a useful target. Primers of interest
include 16UN IX
(5'-GGT GAA GTC GTA ACA AG-3') and 23UN II (5'-TGC CAA GGC A7f C CAC C-3')
In some instances the target sequence is a chaperonin sequence. In some cases
it is a type 1
chaperonin sequence. Non-limiting examples of cpn60 sequences can bf: found
with the
12
CA 02430586 2003-05-30
following accession numbers: AF338228, AF274871, AF406639, AY263150, AY263147m
AY123725 (all from the GenBank nucleotide database).
As used herein, a "cpn60 like" sequence is an oligonucleotide sequence having
at least 40%
homology to (a) the E.coli groEL sequence (in prokayotes) or (b) the cadida
albicans hsp60 in
eukasyotes) and capable of encoding a protein which, when expressed under
suitable
conditions, has a biological function similar to that of epn60, groEL or
hsp60. In some
instances, homology of at least 50%, 75%, 80%, or 90% will be preferred. For
the purposes
of this document, "a biological function similar to that of cpn60, groEL or
hsp60" means a
role in the folding of proteins andJor the assembly of protein subunits in
complexes. This
role does not normally include forming a part of the final structure. Based on
the disclosure
herein, one skilled in the art could readily screen a protein to determine if
it had this function.
As used herein, the term "cpn60 variant" refers to a cpn60-like sequence which
is at least
70% homologous to the most highly expressed cpn60, groEL or hsp60 gene found
in the
organism being examined. As herein the term "DNA region" refers to a
continuous
deoxyoligonucleotide sequence found within a genome of an organism (including
nuclear,
mitochondrial, chlosoplast, and plasmid DNAs). While the term DNA region may
refer to a
gene or a part of a gene, it may also refer to a regulatory or structural DIVA
region, intron
DNA, and/or unexpressed genes or pseudo genes.
As used herein, when a DNA region or gene is said to be "represented in a
species" the
means that the species has an oligonucleotide sequence having a high degree:
of homology the
DNA region and the oligonucleotide sequence performs substantially the same
role as the
DNA region or gene. In some instances the oligonucleotide sequence will be at
least about
40% homologous to the DNA region. In some cases the homology will be at least
about
50%, 75%, 90% or 95%. Complete sequence identity is not required.
For the purposes of phylogenetic analysis, it is reasonable to assign an
identified sequence
with the taxonomic classification from which the most closely related known
sequence is
derived. In many cases sequences having 97% homogy are considered to derive
from the
same species.
13
CA 02430586 2003-05-30
The invention is further explained by the following examples. The following
examples are
intended as illustration only and not intended to represent a limitation of
the present
invention. There exist numerous additional applications known to be possible
by those
skilled in the art. Additionally, the techniques mentioned below are intended
as
representations of examples of techniques which may be used to achieve the
results
enumerated herein. One of ordinary skill in the art would see that alternative
and equally
successful techniques may be used to achieve the same goals of each step of
the procedure
discussed above.
It will be apparent to those skilled in the art, in light of the disclosure
herein, that the
methods, primers, and libraries of the invention are useful in screening for,
predicting,
diagnosing, and developing strategies for treating a wide range of human and
animal
conditions. Of particular interest are conditions thought to be associated
with abnormal
"flora" (abnormal populations of organisms). These populations may be abnormal
due to the
presence, absence, or abnormal relative abundance of one or more species of
organism.
Some specific examples of disorders thought to be associated with "abnorrr~al"
flora include:
Inflammatory Bowel Diseases in humans (Chrohn's, colitis, etc.), necrotic
enteritis in
chickens (linked to Clostridium overgrowth and C. perfringens in particular)
dental or oral
health issues such as gum disease and tooth decay, as well as cardiac
conditions having an
oral infection connection. Intestinal flora profiles are also related to
livestock performance,
since the flora affects nutrient absorption, digestion, etc. . The microbial
flora on the surface
of plant tissue is thought to play a role in disease resistance and plant
health.
Applications outside the realm of human, animal or plant medicine are also
contemplated.
For example, it is frequently useful to know the profile of organisms in an
environment. By
way of non-limiting example, quantities of nitrogen-fixing microorganisms in
agricultural
soils, toxin-degrading microorganisms in the soil at sites of contamination
(bioremediation).
Additionally, industrial processes depend on certain flora - for example, pulp
mill in-mill
streams contain microorganisms vital to proper flocculation.
l4
CA 02430586 2003-05-30
Example 1
Example lA - Pig Faecal Flora
Pig feces is a tractable microbial community, rich in microbial life
(estima.ted to exceed 10"
organisms per g of feces, Finegold, et al. 1974. Am. J. Cli~2. Nutr. 27:1456-
1469, and
Moore, et al. 1974. Appl. Microbiol. 27:961-979), for which there is a wealth
of descriptive
literature. However, the invention described herein may be applied to any
number of various
sources of microorganism communities as would be apparent by one of skill in
the art.
Wheat and barley are the primary feed ingredients for some pigs, while corn is
the major
ingredient of feed in other parts of North America. Wheat and barley have
higher levels of
non-starch polysaccharides than corn and could have an effect on the
composition of gut
microflora in mammals eating these diets. Total genomic; DNA from the deal
contents
samples from pigs fed antibiotic-free diets containing either corn, wheai; or
barley as the
primary energy source is used to create cpn60 sequence libraries in order to
investigate the
effects of corn, wheat or barley-based diets on pig intestinal microflora. A
phylogenetic
analysis of the resulting sequence data is used as the basis for designing
primer-probe sets to
target specific taxonomic groups within the populations for quantitative PCR.
A real-time
quantitative PCR approach can be taken to validate the representation of the
targeted
sequences in the cloned libraries and to develop molecular tools for the
monitoring of
taxonomic groups of interest within the pig ileum populations. In an
independent study using
the same digesta samples, bacterial populations can be enumerated using
selective agar.
Pigs and feces collection.
Fecal samples were obtained from the recta of 6-week-old pigs (n = 5) housed
in a
commercial swine facility (Prairie Swine Centre Inc., Saskatoon, Saskatchewan,
Canada). A
medicated (chlortetracycline at 308 mg/kg, sulfamethazine at 308 mg/kg, and
penicillin at
154 mg/kg) wheat and soybean meal-based diet formulated to meet nutrient
requirements was
fed from 21 days of age (weaning). Fecal samples were pooled (a total of
approximately 2 g)
and stored at -20° C until genomic DNA was extracted.
Genomic DNA extraction.
CA 02430586 2003-05-30
Two methods of genomic extraction were used. In a modification of the
benzylchloride
extraction method (Zhu et al. 1993. Nucleic Acids Rer. 21:5279-5280),
approximately 0.8 g
of feces was thawed and dispersed in 5 ml of benzylchloride extraction buffer
(I00 mM Tris-
HCI, pH 9.0, 40 mM EDTA). To 500 ul of the suspension was added 100 ~1 of 10%
sodium
dodecyl sulfate (SDS) and 300 ql of benzyl chloride. 'lChe remaining 4.5 ml of
fecal
suspension was reserved at -20°C. The sample was mixed by vortexing and
incubated at
50°C for 30 min, with vortexing at 5-min intervals. 300 ~l of~ 3 M
sodium <~cetate (pH 5) was
added and the sample was mixed by inversion and incubated on ice for 15 min,
followed by
centrifugation at maximum speed in a microcentrifuge at 4°C for 15 min
to separate the
aqueous and organic phases. The supernatant was transferred to a clean tube
and nucleic
acids were precipitated by the addition of 400 ~1 of isopropanol followed by
centrifugation at
top speed in a microcentrifuge for 10 min at 4°C. The pellet was washed
in cold 70%
ethanol, dried, and resuspended in 100 ~l of TE (100 rnM Tris-HCI, pH 8, I mM
EDTA).
Approximately 0.8 g of feces was dispersed in 5 ml of 25% sucrose-40 mIVI
Tris, pH 8. To
500 ~1 of the suspension, 100 ~l of lysozyme (10 mg/ml in 25 mM Tris, pH 8)
was added,
and the sample was incubated at 4°C for 10 min, followed by the
addition of 100 ~l EDTA
(0.5 M, pH 8) and incubation at 4°C for 10 min. I ml of lysis buffer
(62.5 mM EDTA, 50
mM Tris, pH 8, 1 % (vol/vol) Triton X-100) was added and the sample wa<,;
incubated at 4°C
for 15 min with periodic mixing. The lysate was extracted twice with 25:24:1
(vol/vol/vol)
phenol-chloroform-isoamyl alcohol and nucleic acids were precipitated by the
addition of 85
~1 of 3 M sodium acetate and 850 ~,1 of isopropanol, followed by
centrifugation at maximum
speed in a microcentrifuge for 10 min at 4°C. Pellet was washed once
with 70% ethanol, air
dried, and resuspended in 100 ~1 of TE.
An alternative genomic DNA extraction method which can be useful is based on
the Phenol:
Chloroform: Isoamyl (P:C:1 ) alcohol method. 200mg wet weight material, 50mg
bacterial
pellet or 200u1 of turbid bacterial suspension is used as starting material.
(Extraction buffer
Tris EDTA 100 mM Tris - HCL pH 9.0 (1.21 g in 100m1 ddH~O) and 40 rnM EDTA
(1.49 g
in the same 100m1 ddH20). Store at RT with steril filtration. (TE buffer c;an
also be used.)
10% SDS lOg in 100m1 ddH20 (heat to dissolve) pH 7.2, sterile filtration. 3M
\Sodium
Acetate pH 5.0 (24.618 CZN3Na0~) dissolve in 60 - 80 ml ddH2~A heat to
dissolve. Ilse
conc. HCL to pH. Make to 100 ml. Protinase K 20 mg/ml_keep stock frozen.
Phenol:
16
CA 02430586 2003-05-30
Chloroform: Isoamyl Alcohol (25:24:1) Sigma P-2069 Saturated with lOMM Tris,
pH 8.0,
1mM EDTA.)
Procedure: Place material into head beating tube. Bead beating tubes may be
commercial or
our own (bead volume will vary on material being extracted). Add to tu>=>e
30u1 10% SDS,
3u1 protinae K and TE buffer up to 600u1 total volume. TE buffer volume will
vary
depending on tube and sample weight. Vortex well and incubate for 1-2 hourse@
37°C. To
tube add equal volume of P:C:I solution and place on ice. Bead beat using
FastPrep unit
(PBI) 20 seconds x 10 setting 5. Place tubes on ice minimum of 1 time during
beating to
reduce over heating. Add 1/10'c' the volume sodium acetate and invert 2 or 3
times. Place
tubes on ice 10 minutes. Centrifuge tubes 3-5 minutes at 14-,000rpm. Remove
top phase to
new tube. Approximately 600 - 800u1. Add equal volume isopropyl alcohol or
more if there
is room in the tube. Invert a few times. Centrifuge tubes 3-5 minutes at
14,000 rpm remove
liquid. Add lml of 70% alcohol to the tube to wash DNA pellet. Don't mix or
invert the
tube. Centrifuge tubes 3-5 minutes at 14,000rpm and aspirate off the alcohol
very carefully
as not to disrupt the pellet. Let the tubes air dry for 20 minui:es approx.
Acid desired volume
of TE buffer of ddHzO. To help DNA pellet dissolve you can heat tubes in water
bath
@60°C or let them set at room temperature over night. Store dissolved
DNA extractions at -
20°C. Specs or agarose gels may be run to determine concentration and
DNA profile of
extractions, if desired.
PCR and cloning of PCR products.
Genomic DNA extracted from feces ( 1 ul of either benzylchloride or phenol-
chloroform-
extracted DNA) was used as the template in PCR reactions (PCRs). The PCR
primers used
were H279, 5'-GAI III GCI GGI GA(C/T) GGI ACI ACI AC-3' (SEQ ID NO 1 ), and
H280,
5'-(C/T)(G/T)I (C/T)(G/T)I TCI CC(A/G) AAI CCI GGI GC(C/T) TT-3' (SEQ ID NO
2).
Inosine (I) was used to reduce the degeneracy of the sequences (Ohtsuka, et
al. I 985. J. Eiol.
Chefn. 260:2605-2608). Primers were designed to amplify the region between
codons 92 and
277 based on the Escherichaa coli groEL sequence (GenBank accession number
X07850,
groEL is the E. coli homologue of cpn60). The PCRs contained 50 mM KCl, 10 mM
Tris-
HCl (pH 8.3), 1.5 mM MgCl2, 250 ~M each of the four deoxy:nucleoside
triphosphates, 2 U of
Taq DNA polymerase, and 0.5 ~g (50 pmoI) of each primer.
17
CA 02430586 2003-05-30
PCRs were performed on a Stratagene RobocyclerTM hermocycler according to the
following
parameters: 3 min at 95°C, 40 cycles of 1 min at 95°C, 1 min at
40°C, 1 miin at 72°C, and 10
min at 72°C. PCRs included a negative control reaction containing no
template DNA to
ensure that no contaminating template was present in the reactions. An
additional set of
PCRs were done as described except that the annealing temperature was
56°C. The resulting
four PCR products were agarose gel purified and ligated into vector pCR2.1-
TOPO with the
TOPO T-A cloning kit (Invitrogen), and transformed Escherichia coli was plated
on Luria-
Bertani agar (LB) containing ampicillin and 5-bromo-4-chloro-3-indolyl-13-D-
galactopyranoside (X-Gal). The resulting libraries were named according to the
template
extraction method and PCR annealing temperature used in their production: B56
and B40
(benzylchloride template amplified with an annealing temperature of
56°C or 40°C,
respectively); P56 and P40 (phenol-chloroform template amplified with an
annealing
temperature of 56°C or 40°C, respectively). Colonies (576 white
colonies from each library)
were picked and used to inoculate 96-well plates containing 100 ~,l of LB with
ampicillin (50
~g/ml) per well. Culture plates were incubated overnight in humidified
containers at 37°C
with shaking. Glycerol (100 ~ul of 30% glycerol in LB) was added to each well,
and plates
were sealed and stored at -80°C.
Plasmid DNA isolation and DNA sequencing.
Plasmid DNA for sequencing template was isolated either by the Qiagen
R.F?.A.L. Prep 96TM
plasmid kit according to the manufacturer's protocol or by a solid-phase
reversible
immobilization method modified from an earlier published procedure (Hawkins,
et al. 1994.
Nucleic Acids Res. 22:4543-4544) for use on an integrated automation platform
(ELMS; see
for example http://bioinfo.pbi.nrc.ca/robotics). For the robotic plasmid
preparation,
recombinant clones were cultured in 1.2 ml of Terrific Broth in deep-well (2
ml) 96-channel
microtiter plates, pelleted by centrifugation, and lysed by an alkaline-SDS
procedure. Lysates
were made up to 10% polyethylene glycol 8000 and 0.5 M NaCI prior to the
addition of 200
~g of COOH-derivatized paramagnetic beads (SeradynTM ). The bead slurry
mixture was
incubated with shaking for 5 min, and the beads were subsequently fractionated
over
permanent magnets, washed in 50% ethanol, dried, and resuspended in double-
distilled HZO.
Plasmid concentration was estimated by resolving plasmid preps on 1 % agarose
gels.
18
CA 02430586 2003-05-30
High-throughput DNA sequencing reactions were conducted in 384-well microtiter
plate
format, by using 100 to 300 ng of template DNA in combination with 5'-
biotinylated T7 and
M13RP sequencing primers, in a 1/3 volume Big DyeTM sequence reaction (PE
BiosystemsTM
). Reactions were assembled by the robotic system described above and
thermocycled
according to the supplier's recommended protocol. Sequence extension reaction
products
were purified by addition of 10 ug of streptavidin-paramagnetic beads (M-280;
Dynal
CorporationTM ), followed by fractionation over permanent: magnets.
Fractionated beads
were resuspended in 12 ~I of 50% deionized formamide and treated at
95°C for ~ min prior to
immobilization and transfer of up to 12 ~ul of the reaction product-containing
supernatant to a
fresh 384-well microtiter plate. Completed reactions were sealed and stored at
-80°C prior to
resolution on a PE-3700 capillary sequencing device
Sequence data assembly and analysis.
All sequence data assembly, analysis, and storage were done by software
available from the
Canadian Bioinformatics Resource (http://www.cbr.nrc.ca). (Note however that
other
methods and tools of analysis are known in the art and could readily be
applied, in light of the
disclosure herein). Raw sequencing data were assembled into contigs (sets of
overlapping
segments of DNA) for each template by Pregap4 (version I .1 TM ) and Gap4
(version 4.6TM )
in the StadenTM software package (release 2000.0; J. Bonfield, K. Beal, M.
Betts, M. Jordan,
and R. Staden, 2000). Contig nucleotide and peptide sequences were compared to
a database
of approximately 1,000 cpn60 sequences by BlastP and BlastN. Sequence data,
template
information, and Blast results were deposited in a MySQL database for data
storage and
further analysis. Sequence manipulations, such as format changes and amino
acid
translations, were done by GCG (Wisconsin package, versiion 10.1 for IJnix).
Sequence
alignments were done with ClustalW rM and viewed with GeneDocTM .
Phylogenetic analysis was performed by programs in the PHYL1PT~~ software
package.
Specifically, alignments were sampled for bootstrap analysis by SeqbootT~~,
distances were
calculated with the PAM option of ProtdistTM (for peptide sequences) or the
maximum-
likelihood option of DnadistTM. Dendrograms were constructed from distance
data by using
neighbor-joining by neighbor. Consensus trees were calculated by ConsenseT'~',
and branch
19
CA 02430586 2003-05-30
lengths were superimposed on consensus trees by FitchTM. Completed trees were
viewed by
TreeViewTM and manipulated for presentation with Microsoft Powerl>ointT~~.
Unique
nucleotide sequences (encoding 280 unique peptide sequences) were deposited in
GenBank as
a phylogenetic study and assigned accession numbers AF436893 to AF437290.
These
sequences are incorporated into this application by reference.
The present invention is suited to use as a kit for the purpose of identifying
unique strains of
microorganisms within a diverse and large community of microorganisms. Such a
kit may
comprise a carrier means being compartmentalized to receive in close
confinement one or
more container means such as vials, W bes, and the like, each of the container
means
comprising one of the separate elements to be used in the method. For example,
one of the
container means may comprise means for amplifying target DNA including the
necessary
enzymes) and oligonucleotide primers for amplifying the target DNA from the
sample
obtained from the host. The oligonucleotide primers include primers having a
sequence
similar or identical to SEQ ID NO. 3 through SEQ ID NO. 5 and SEQ ID NO. 7
through SEQ
ID NO. 9 or primer sequences substantially complimentary thereto.
Cpn60 gene sequences amplified from piglet faeces total DNA.
To provide a mixed DNA template representing a complex microbial community,
total DNA
was extracted from piglet faeces. From this template, a region of the cpn60
gene sequence
was amplified using universal, degenerate primers. Four independently
amplified DNA
products were produced by application of two methods for DNA extraction
combined with
two annealing temperatures for PCR, 40°C and 56°C. The amplified
products were cloned
independently to produce four libraries. high quality sequence data were
obtained for 1125
clones that were randomly selected from the four libraries (278 from 840, 332
from 856, 293
from P40 and 222 from P56). Disregarding the flanking degenerate primer
sequences, the
cloned cpn60 gene region was either 552, 555 or 558 nucleotides in length
(184, 185 or 186
codons, respectively).
CA 02430586 2003-05-30
Pairwise comparisons of the I 125 sequences using CLUSTALw revealed the
presence of 398
unique nucleotide sequences (encoding 280 unique peptide sequences). These
were
deposited in GenBank as a phylogenetic study and assigned the accession
numbers
AF436893-AF437290. Figure 12A shows the number of timers each unique
nucleotide
sequence was recovered from the total library. A few sequences were recovered
frequently,
one sequence 148 times; whereas, 307 sequences were recovered only once. Only
l0
sequences were recovered more than 20 times. Pairwise comparisions among the
398 unique
sequences ranged from 47-99% nucleotide sequence identity.
Phylo~enetic analysis of cpn60 sequence data
Each DNA and peptide sequence was compared to a database of cpn60 sequences
using
BLAST. The database is a curated and growing collection of approximately 1100
eubacterial
and eukaryotic cpn60 sequences harvested from public databases or generatf~d
in the
laboratories of a network of collaborating researchers. The nearest database
neighbours of
the most frequently recovered library sequences are shown in Table L. The
estimated
taxonomic breakdown of the total library contents, based on nearest neighbour
taxonomy, is
illustrated in Figure 12B and Table 7. The largest taxonomic group,
represented by 55% of
the total library clones and 54% of the unique nucleotide sequences, was the
cytophaga-
flexibacter-bacteroides (CFB) group. The bacillus/clostridium subgroup of Gram-
positive
bacteria represented 36% of the total library clones and 42% of the unique
nucleotide
sequences and gamma-class Proteobacteria accounted for 8% of the total clones
and 3% of
the unique nucleotide sequences. The group labeled "others" in Figure 12B
consisted of
clones whose nearest database neighbours were in the spirochete, chlamydiales
or
Proteobacteria beta families (see Table 7 for details). Sequence length was
strictly correlated
with taxonomic assignment. That is, all clones with nearest neighbours in the
CFB group had
21
CA 02430586 2003-05-30
lengths of 558 by (186 codons) whereas all the clones with nearest neighbours
in the
Proteobacteria gamma group and bacillus/clostridium group are 555 by (185
codons) and 552
by (184 codons) respectively. These are identical to the lengths observed for
database
reference sequences from each of these groups.
The most abundant sequence in the library (recovered 148 times), represented
by clone
002 a03, is 88% identical at the amino acid level (78% nucleotide identity) to
Prevotella
intermedia ATCC25611. Other sequences recovered at least 4 times from the
library are
identified in Table 6 along with their nearest database neighbours. In three
cases, library
clones shared 100% DNA sequence identity with database reference strains:
Lactobacillass
amylovorus ATCC33620, Lactobacillces acidophilus T13 and Enterococcus ~asini
ss-1501.
Another clone shared 100% amino acid sequence identity and 98% nucleotide
sequence
identity with Pediococeus peatocaceus ATCC43200. Overall, the level of
sequence identity
between each of the 398 unique library sequences and its nearest database
neighbour ranged
from 56-100% DNA identity (51-100% peptide identity, 71-100% peptide
similarity) with
only 2 clones having less than 60% peptide identity to their nearest database
neighbour.
Table 2 shows the overall composition of recovered sequences in terms of their
nearest
database neighbours.
Inferred phylogenetic relationships amongst unique library sequences are
illrastrated in Figure
13. The 280 unique peptide sequences translated from the 398 unique nucleotide
sequences
were subjected to a multiple sequence alignment using CLUSTALw. P~airwise
distances
between the aligned sequences were calculated using the protdist program
within PHYLIP
(PAM matrix) and the tree was generated by neighbour-joinin g. Branches were
colour-coded
according to the taxonomic group of the nearest database neighbour of each
clone sequence.
22
CA 02430586 2003-05-30
Overall, the phylogenetic relationships outlined in the tree in Figure 2
reflect the initial
taxonomic estimates made based on the BLAST results.
Following the gross phylogenetic analysis presented in Figurl3, groups of
cloned sequences
from each of the represented taxonomic categories were selected for detailed
phylogenetic
tree construction, incorporating reference sequences from the cpn60 database.
Using this
technique, clone sequences were tentatively identified to the level of
taxonomic subclass,
family or genus. An example of this analysis, including ten cl'~.one sequences
with nearest
database neighbours in the CFB group and reference sequences from the genera
Chlorobium,
Rhodothermus, Flavobacterium, Bergeyella, Chryseobacterium, Bacteroides and
Weeksella
is show in Figure 14.
Genetic Diversity of Sampled Microorganisms
The cumulative frequency distribution (CFD) was plotted for the DNA sequence
identity
scores from all pairwise comparisons of library clone sequences (Figure 15).
To produce
plots for comparison to this CFD plot of our experimental population, three
other populations
of cpn60 sequences were synthesized by selecting sequences from our datab;~se
of cpsi60
reference sequences. The first of these populations consisted of individual
species from 172
different genera (including both prokaryotes and eukaryotes) represented in
t:he database. A
second population was constructed by pooling cpsz60 universal target sequences
from 77
species (34 genera) of Proteobacteria gamma. The third population consists of
37 species
from a single genus, Lactobacillus. The experimental, pig faeces library
population, while
less diverse than the population of l 72 genera, was more diverse than the
genus
Lactobacillass, or the Proteobacteria gamma taxon, with approximately half of
the pairwise
comparisons within the library having DNA identities of 60% or less.
23
CA 02430586 2003-05-30
Sequence accuracy and microheterogeneity
Clusters of nearly identical clone sequences (98-99% nucleotide identity)
ranging in size
from 2 to 20 sequences (191 total sequences) were further analysed to
determine the nature of
the differences between the sequences. Multiple alignments of these groups of
sequences
showed that a disproportionate (p<0.001) number of the differences within the
alignments
were synonymous changes, occurring in the third position of codons.
Examination of a total
of 320 differences revealed that 61 were in the first position of codons, 63
were in the second
position and 196 were in the third position. Almost all third position
differences (191/196)
were synonymous changes in terms of their effects on the encoded peptide
sequence. No in-
frame stop codons were observed in any of the 1125 clone sequences detemlined.
Also, 29 of
the 38 sequences in Table 6 (sequences occurring at least 4 times) were
recc>vered from at
least two of the four libraries.
DNA extraction methods and PCR conditions used affect organisms sannpled
To assess the effects of library construction parameters on library contents,
sequence data
were grouped by library of origin and clone frequencies and taxonomic
distributions were
analyzed for each of the 4 data sets (Figure 16A). The B40, B56, P40 and P56
libraries
contained 156, 125, 112 and 91 different sequences respectively. 'The
frequency distributions
of unique sequences varied markedly between libraries. While the most
prevalent clone in
the P56 and P40 libraries was recovered approximately 70 times from each
library
(accounting for 25-30% of clones sequenced) and the most prevalent clone in
the B56 library
was recovered 49 times (15% of clones), the most abundant clone in the B40
library was
recovered only 23 times (8% of clones). Figure 5B shows the taxonomic
composition of each
of the four libraries. The relative proportions of each taxon clearly varied
between the four
24
CA 02430586 2003-05-30
libraries, with the largest proportion of Proteobacteria gamma-like clones
occurring in the
B56 library while this taxon was completely absent from the 1P40 library.
Data were also grouped for analysis according to the PCR annealing temperature
or genomic
DNA extraction method used in library construction. Figure 6 shows the
taxonomic
composition of clones produced with a PCR annealing temperature of 40°C
versus 56°C and
DNA template prepared by the benzylchloride versus phenol:chloroform
exl:raction methods.
While all 4 taxonomic subclasses (CFB group, bacillus/clostridium group,
Proteobacteria
gamma and others) were detected in each group, the relative proportions of
each taxon
present varied with library construction conditions. Fox example, the highest
proportion of
Proteobacteria gamma class clones were produced with a PCR annealing
temperature of 56°C
and a benzylchloride-extracted template (see also figure l6A),.
Example 1B-Pig Diet Flora
Wheat and barley are the primary feed ingredients for some pigs, while corn is
the major
ingredient of feed in other parts of North America. Wheat and barley have
higher levels of
non-starch polysaccharides than com and could have an effect on the
composition of gut
rnicroflora in mammals eating these diets. Total genomic DNA from the deal
contents
samples from pigs fed antibiotic-free diets containing either corn, wheat or
barley as the
primary energy source is used to create cpn60 sequence libraries in order t:o
investigate the
effects of corn, wheat or barley-based diets on pig intestinal microflora. A
phylogenetic
analysis of the resulting sequence data is used as the basis for designing
primer-probe sets to
target specific taxonomic groups within the populations for quantitative PCR.
A real-time
quantitative PCR approach can be taken to validate the representation of the
targeted
sequences in the cloned libraries and to develop molecular tools for the
monitoring of
taxonomic groups of interest within the pig ileum populations. In an
independent study using
the same digests samples, bacterial populations can be enumerated using
selective agar.
CA 02430586 2003-05-30
Forty-five pigs (35 days of age) were fed diets containing corn (yellow dent),
wheat (Laura)
or barley (Brier) as the primary source of energy for 3 weeks. Pig diets were
formulated to
contain similar digestible energy and 3.15 g of digestible lysine per Mcal
digestible energy.
These diets did not contain any antibiotics. Pig body weight and feed intalke
were measured.
At the end of the experiment, pigs were euthanized by COZ asphyxiation and
exsanguination
and their intestinal tracts removed. Samples of digestive contents (digests)
were collected
aseptically from the mid-ileum (75% of the distance between the duodenum and
the ileo-
caecal junction) and caecum. The numbers of total aerobes, total
anaerobe°s, Enterobacteria,
Lactobacillus spp., Clostridium spp. and Streptococcus spp. present in the
digests samples
were enumerated as described previously (Estrada et al. 2001. Can.J.Anim.~>ci.
81:141-748).
Total genomic DNA was isolated from 200 mg of deal digests using a combination
of
phenol:chloroform extraction and bead-beating. A pool of DNA from the 15
mammals in
each diet group was created, resulting in 3 templates for PCR amplification.
Each DNA pool
was used as a template in 4 PCR reactions that differed only in the annealing
temperature
used. Previous studies indicated that amplifying samples over a range of
annealing
temperatures (42-56°C) increases the diversity of the resulting pool of
PC°.R products. The
PCR products produced in each set of 4 reactions were pooled, agarose gel
purified and
ligated into vector pCR2.l-TOPO. Ligation mixtures were used to transform E.
coli Top-10.
The 3 resulting libraries (C (corn), B (barley) and W (wheat)) were plated on
LB/ampicillin/X-gal and 1248 white colonies were picked from each library.
Colonies were
cultured in 96-well plates (13 plates per library) and stored as glycerol
stocks at -80°C until
sequencing template preparation. Plasmid purification, quantification and
sequencing
reaction assembly, thermocycling and resolution were done according to the
methods
previously described (Hill, et al. 2002. Appl. Environ. Microbiol. 68:3055-
3066).
The total numbers of clones successfully sequenced (succ:ess is defined as a
complete
sequence of the full "universal target" with no sequence ambiguities) are
summarized in
Table L. Over 900 sequences were obtained for each of the C, B and W
libraries. The
"universal target" refers to the sequence amplified from any cpn60 gene with
the primers
H279 and H280. In reference to E. coli, the universal target encompasses
nucleotides 247-
854 (including primer landing sites) or 274-828 (excluding degenerate prirr~er
landing sites).
26
CA 02430586 2003-05-30
In general, the universal target (excluding degenerate primer Landing sites)
is 552, 555 or 558
by in length and the length varies in a consistent way with the taxonomy of
the subject, e.g.
gamma class proteobacteria have 555 nt, low-GC gram positives are all 552 and
Bacteroides
ssp. are all 558.
Table 1. Number of sequences generated for each library
Library Number of sequencesNumber of unique
Sequences
C 909 136
B 930 171
W 915 110
C+B+W 2751 316
Following assembly and editing of the sequences, each sequence was compared to
cpnDB
using FASTA (for nucleotide sequences) or BLASTp (for peptide sequences). The
nearest
database neighbor for each clone was recorded. A graphical summary of the
contents of each
library based on the genus or taxonomic group of the nearest database neighbor
for each
clone is shown in FIG. l . In general, all three libraries were dominated by
Lactobacillus-like
sequences, which constituted 84% of the C library, 92% of the B library and
90% of the W
library.
Most of these Lactobacillus-like sequences were 95-100% identical to
Lactobacillus
amylovorc~s ATCC33620. In fact, sequences 90-100% identical to L. amylovorous
ATCC33620 account for 1399 of the 2751 clones sequenced. Clostridium-like
sequences
were more abundant in the C library (12% of clones sequenced) compared to the
B library
(2% of clones) or the W library (8%). Another difference between the libraries
was the
relative prevalence of Streptococcus-like sequences in the B library (6% of
clones) compared
to the C and W libraries ('1% of each). These Streptococcus-like clones
included sequences
identical to Streptococcus orisratti, S. thermophilus and S. alactolyticus.
27
CA 02430586 2003-05-30
Phylogenetic analyses are shown in FIG. 2, FIG. 3 and FIG. 4. The contents of
the three
libraries were compared to determine the degree of overlap in sequences
recovered. Table 2
summarizes the overlaps determined. Most of the B-specific sequences were
found within
the Lactobacillus-like and Streptococcus-like sequence groups while most of
the C-specific
sequences were found in the Clostridiufn-like groups. W-specific sequences
were more
evenly distributed across all sequence groups.
Table 2. Summary of library overlap.
Library Numher of sequences
C only 76
B only 127
W only 51
C and B 10
C and W 22
B and W 8
C and B 22
and W
TOTAL, 316
A phylogenetic analysis of the 316 unique nucleotide sequences found in the
pooled C, B and
W libraries is shown in FIG. 5. 'This analysis was used to identify clusters
of closely related
sequences within the pooled libraries and to calculate the numbers of clones
in each group
recovered from each library. This calculation resulted in a "C:B:W" ratio for
each sequence
group. For example, group C 1, which includes a closely related group of
Clostridium-Like
sequences, has a C:B:W ratio of 88:18:69, indicating that sequences falling
into this group
were recovered 88 times from the C library, 18 times from the B library and 69
times from
the W library.
The relative proportions of Lactobacillus, Clostridium, Streptococcus and
Proteobacteria-
gamma-like sequences are consistent with those observed in the culture-based
assessment of
the ileum populations where lactobacilli were found to be present at 10g cfu/g
starting
28
CA 02430586 2003-05-30
material, clostridia at 10~ cfu/g, streptococci at 10~ cfu/g and
enterobacteria (Proteobacteria
gamma) at 105 to 106efu/g depending on the library.
In the enumeration study, the barley diet was associated with decreased
numbers of
enterobacteria while the barley and wheat diets were associated with an
increase in the
number of lactobacilli. These patterns were not observed in the library
sequencing study.
Numbers of Lcactobacillus-like sequences recovered from each library were
approximately
equal and too few enterobacterial (gamma proteobacteria) sequences were
recovered to draw
any conclusions about diet-associated effects on this taxon. Sequence data
indicated a
relative decrease in the frequency of Clostridicem-like and bacillales-like
sequences and a
corresponding increase in Streptococcus-like sequences associated with the
barley diet.
Comparisons of culture data and sequence data are naturally problematic since
the criteria
used to assign organisms or sequences to a given taxon are different.
Groups CL, B1 and S1 had C:B:W ratios of 88:18:69, 16:0:7 and 1:48:4,
respectively. These
groups were chosen as targets for the design of group-specific PCR primers and
probes for
real-time PCR. Using the SignatureOligoTM software (LifeIntel, Inc.TM), PCR
primers were
designed which would amplify sequences from the target group, but not an,y
other sequences
in the C, B or W libraries or any sequences in epnDB. Primer and probe
sequences and
predicted product sizes are outlined in Table 3. The specificity of eaclh
primer set was
verified by testing them against a range of templates including clones derived
from the target
group as well as neighboring groups. While the signature OligoTM software was
employed in
this example, it will be readily understood that a variety of tools and
approaches to primer
design are available to be used to design such primers. In designing PCR
primers for this
purpose, it is preferable to examine all the target sequences to identify
"signature sequences"
sequence elements common to the target group members but not found in non-
target
sequences. At least one of the PCR primers should preferably bind target
sequences but not
non-target sequences. Both primers preferably bind target sequences. Primers
are preferably
about 15-25 nucleotides in length. Primer pairs are preferably chosen to have
similar
predicted melting temperatures. Thus, in light of the disclosure herein, one
skilled in the art
could readily design and use suitable PCR primers.
29
CA 02430586 2003-05-30
Table 3. Primers and probes for real-time PCR of sequence groups B L, C 1 and
S l .
Target Primer sequences Probe sequenceProduct
size
B 1 forward 5'-TGCAGGAGCAAATCCAATGAT-3' NA 173
(SEQ ID NO 3)
reverse 5'-GCATGGCTTCGGCAATTAAA-3'
(SEQ ID NO 4)
C1 forward 5'-GCTGTTGATGTAGCAGT'TGA-3' 5- 155
(SEQ ID NO 5) TGTTGCT'GCGG
GCATGAACC-3'
(SEQ ID NO
6)
reverse 5'-ATAACCCCTTCGTTTCCTAC-3'
(SEQ ID NO 7)
Sl forward 5'-AACTTGACGTGGTTGAAGGG-3' NA 172
(SEQ ID NO 8)
reverse 5'-GTTTTCAAGACTTCTTCAAGCAA-3'
(SEQ ID NO 9)
Results of SYBR-green PCR using Ci primers and the C, B and W genomic DNA
templates
are shown in Table 4.
CA 02430586 2003-05-30
Table 4. SYBR-green results for B1, C1 and S1 primers on genomic DNA from
ileal contents of
pigs on corn (C), barley (B) or wheat (W) diets.
Template DNA Ct(expt. 1) Ct(expt. 2) Mean Ct Number of
clones recovered
C 24.2 25.5 24.8 16
B NDz ND2 NDz 0
W 30.3 31.9 31.1 7
C1 Primers
C 20.8 20.7 20.75 88
B 24.2 23.8 24.0 18
W 23.2 22.9 23.05 69
S1 Primers
C 30.8 30.9 30.8 1
B 27.6 28.5 28.0 48
W 28.6 29.4 29.0 4
'C, equals threshold cycle. Lower values indicate higher copy number in test
sample
2Not detectable
Example 2
Vaginal swab samples were obtained from two individual volunteers. Using
methods similar
to those described in Example l, total genomic DNA was isolated from the
samples and
subjected to universal cp;a260 primer PCR to amplify partial cpn60 gene
sequences. Libraries
of partial cpn60 sequences were created by ligating the PCR products into
cloning vectors.
Ninety-six clones were randomly chosen from each library (:BV 1 and BV2).
Sequencing of
the two libraries yielded 84 and 74 complete, unambiguous sequences from BVl
and BV2
libraries respectively. Unique sequences within sack library were identified
(see FIG. 6) and
frequencies of each of the sequences were calculated. Each unique sequence was
compared
to a reference database of cpn60 sequence data and putative identifications
were made (Table
5). Frequencies of various taxonomic groups were compared across the two
libraries (FIG. 7)
and detailed phylogenetic analysis of library constituents was performed to
solidify their
sequence-similarity-based identification (FIG. 8 for examplo). Results of the
analysis and
inter-library comparison clearly demonstrate significant differences in the
flora of the two
31
CA 02430586 2003-05-30
individuals. Library BVl is typical of healthy vaginal flora, while the
contents of library
BV2 are indicative of bacterial vaginosis. (Note that "CFB" is an abbreviation
for
Chlorobium-Flexibacter-Bacteroids, also known as Bacteroidetes/chlorobi group
with respect
to the sequences, the sequences of BV-010 and BVl-087 are the same, and the
sequences of
BV-020 and BVl-050 are the same. Both sequences are shown in the Figure.
Bacterial vaginosis ("BV") occurs where there is an overgrowth of undesirable
vagina flora
(and particularly undesirable bacteria). In healthy subjects vagina flora is
predominately
lactobacillus spp. and similar. Significant levels of Gardnerella vaginalis
Cp.va inalis) are an
indicator of BV. In some instances, G.va-inalis (and closely related bacteria)
levels of at
least 10% will be a cause for concern. In other instances, the level will be
at least 20%, 30%,
40% or 50%.
Profiles of vaginal bacterial populations may be determined and used as
indicators of risk for
conditions such as BV. Thus, there is provided a method o-f assessing a
subject's risk for
developing a vaginal-related condition comprising profiling vaginal bacteria
and assessing
the level of G. va rg'nalis and closely related bacteria.
Example 3.
Using methods similar to those described in Example l, midguts were dissected
from 50
Delia radicurrc (crucifer root maggots) and total genomic DNA was isolated
from these
tissues. Universal cpn60 primer PCR was performed using the total genomic DNA
as
template. Libraries of partial cpn60 sequences were created by ligating the
PCR products
into cloning vectors. The experimental scheme for the creation of cpn60
sequence libraries
from the midgut microflora of Delica radicum is depicted in FIG. 10. Thirty-
six randomly
selected clones were sequenced (see FIG. 9). Sequences were pooled according
to identities
and representatives of each unique sequence were compared to the reference'
cpn60 sequence
database for identification. Phylogenetic analysis of the library sequence
data was also
performed (FIG. 11 ).
Example 4
In one embodiment of the invention, it is a 3-step method. The first step. is
the universal
primer PCR and creation of libraries of cpn60 clones for sequencing. The
second step is the
clustering of sequence data based on phylogenetic analysis, calculation of
c:lane frequencies
32
CA 02430586 2003-05-30
corresponding to each sequence cluster and the creation of the frequency ratio
(like the
C:B:W ratio in the pig ileum example). The third stage is the use of the
information in step 2
for the rational design of taxon or cluster-specific primer sets for
quantitative PC12. Step two
is essential and is something has not appeared in the literature yet.
Biopsy samples are obtained from 2 individuals - one with "x" disease and one
normal.
cpn60 sequence libraries are produced for both as described above. The:
sequence data is
collected (1000 per library). After clustering the data and calculating the
frequency with
which sequences in each sequence cluster occur, it is determined that the
frequency ratio for
Bacteroides-like sequences is 50:635 for "x":normal. This points out that the
Bacteroides-
like sequence cluster would be an excellent target for further investigation
with respect to
"x". The sequences in that cluster are used to design primers which will
specifically amplify
these sequences and none of the others in the library (or in the know universe
of cpn60
sequences). Thus there is provideda powerful tool for doing in vivo studies
where one
quantitatively screen large numbers of such biopsies for this sequence cluster
and
demonstrate a link between the pathology and this taxon. Thus, there is
provided a means of
identifying taxons with potential links to pathologies.
33
CA 02430586 2003-05-30
Tab le,oi'. Putative identities
and frequencies of
sequences recovered
from clone
libraries
BVl
and
BV2.
~ ana
clonenearest reference identityputative taxonomy frequency
sequence
bv1-095Gardnerella vaginalis99.275Bacteria;Gram +;Actinobacteria48
ATCC14018
bv1-075Gardnerella vaginalis99.094Bacteria;Gram +;Actinobacteria6
ATCC14018
bv1-087Prevotella intermedia84.74 Bacteria;Other Prokaryotes;CFB4
ATCC25611 group
bv1-099Gardnerella vaginalis98.913Bacteria;Gram +;Actinobacteria3
ATCC14018
bv1-093Gardnerella vaginalis99.094Bacferia;Gram ~-;Actinobacteria3
ATCC14018
bv1-090Enterococcus raffinosus72.232Bacteria;Gram j-;Bacilfusiclosfidium2
0286-86 group
bv1-069Gardnerella vaginalis98.913Bacteria;Gram +;Actinobacteria2
ATCC14018
bv1-027Gardnerella vaginalis98.913Bacteria;Gram +;Actinobacteria2
ATCC14018
bv1-080Lactobacillus acidophilus85.326Bacteria;Gram +;BacillusJdostridiuvn2
(AP4 92.0f) group
bv1-050Prevotella intermedia77.738Bacteria;Other Prokaryotes;CFB2
ATCC25611 group
bv1-072Bacillus mycoides 72.414Bacteria;Gram +;Bacillus/clostridium1
CECT 4128 group
bv1-033Bacillus mycoides 72.595Bacteria;Gram +;BacillusJclostridium1
CECT 4128 group
bv1-012Sacteroides forsythus69.3 Bacteria;Other Prokaryotes;CFB1
ATCC43037 group
bv1-082Clostridium acetobutylicum76.757Bacteria;Gram +;Bacilluslclostridium1
ATCC824 group
bv1-015Clostridium acetobutylicum76.937Bacteria;Gram +;Bacilluslclostridium1
ATCC824 group
bv1-096Gardnerella vaginalis98.37 Bacteria;Gram +;Actinobacteria1
ATCC14018
bv1-067Gardnerella vaginalis99.271Bacteria;Gram +;Actinobacteria1
ATCC14018
bv1-061Gardnerella vaginalis98.732Bacteria;Gram +;Actinobacteria1
ATCC14018
bv1-045Gardnerella vaginalis98.007Bacteria;Gram +;Actinobacteria1
ATCC14018
bv1-004Gardnerella vaginalis99.094Bacteria;Gram +;Actinobacteria1
ATCC14018
bv1-044Lactobacillus acidophilus84.964Bacteria;Gram +;Bacilluslclostridiurn1
(API 92.0%) group
bv1-077Prevotella intermedia84.56 Bacteria;Other Prokaryotes;CFB1
ATCC25611 group
bv1-043Rhodothermus marinus 70.9098acteria;Other Prokaryotes;CFB1
ITf 376 group
bv2-089Lactobacillus acidophilus100 Bacteria;Gram +;Bacillus/clostridiurn50
T-13 group
bv2-081Lactobacillus acidophifus99.819Bacteria;Gram +;Baciliuslciostridiurn9
T-13 group
bv2-086Bacillus mycoides 72.414Bacteria;Gram +;Bacillus/clostridiunn4
CECT 4128 group
bv2-085Clostridium thermocellum66.788Bacteria;Gram +;Bacilluslclostridium3
ncib10682 group
bv2-037Prevotella intermedia77.778Bacteria;Other Prokaryotes;CFB2
ATCC25611 group
bv2-057Clostridium thermocellum66.788Bacteria;Gram +;Bacilluslclostridium1
ncib10682 group
bv2-032Homo sapiens mitochondrion94.054Eukarya;AnimaIs;Mammals 1
bv2-068Lactobacillus acidophilus93.116Bacteria;Gram +;Bacillus/ciostridium1
T-13 group
bv2-054Lactobacillus acidophilus99.819Bacteria;Gram +;Bacilluslclostridium1
T-13 group
bv2-023Lactobacillus acidophilus99.819Bacteria;Gram +;Bacilluslclostridium1
T-13 group
bv2-041Lactobacillus fermentum86.232Bacteria;Gram +;Bacifluslclostridium1
(API 93.7%) group
CA 02430586 2003-05-30
l
Table: Nearest cpn60 database neighbours ofi sequences recovered firom library
at least four times out
of 1125 clones.
cpn60 database neighbour GenBank Taxonomic group
name accession iaennryt;>~mnantylsaennry
002 Prevotella intermedia AF440234CFB group 88(94) 78 148*
a03 ATCC25611
001 PrevotellabiviaATCC29303AF440233CFBgroup 88192) 73 98*
f12
002_a11Anaerobiospirillum succiniciproducensAF441383Proteobacteria86(94) 80
65*
ATCC700195 gamma
OOi Prevotella bivia ATCC29303AF440233CFB group 88(92) 72 38*
c11
001 Bacillus halodurans AP001508Bacillus/clostridium64(83) 64 26*
e03 group
001 Clostridium thermocellum268137Bacillus/clostridium71(85) 69 25*
a02 ncib10682 group
001_g07Clostridium thermocellum268137Bacillus/clostridium72(86) 70 23*
ncib10682 group
005_a04Prevotella bivia ATCC29303AF440233CFB group 88(92) 73 22*
002 Bacteroides ovatus ATCC8483AF440236CFB group 97(97) 83 2i*
c12
002_b08Clostridium diilicile79-685AF080547Bacilluslclostridium73(88) 63
19*
group
003_b04Clostridium thermocellum268137Bacillus/clostridium72(86) 70 17*
ncib10682 group
001 Chryseobacterium gleum AF440235CFB group 68(84) 67 15*
a05 ATCC35910
002_g03Thermoanaerobacterbrockii056021Bacilluslclosfridium75(89) 68 15*
Rt8.G4 group
002_e03Clostridium thermocellum268137Bacilluslclostridium71 (85) 70 14*
ncib10682 group
005_b04Prevotella bivia ATCC29303AF440233CFB group 88(92) 73 13*
002 Bacteroides ovatus ATCC8483AF440236CFB group 96(96) 80 11*
e11
003 Anaerobiospirilfum succiniciproducensAF441383Proteobacteria83(91 ) 79
i 1
a04 ATCC700195 gamma
005_c01Clostridium thermocellum268137Bacillus/clostridium74(88) 71 8*
ncib10682 group
005_e05Clostridium thermocellum268137Bacillus/clostridium72(88) 72 8*
ncib10682 group
001 Closfridium perfringens X62914Bacillus/clostridium71 (88) 66 7*
h02 group
003_f04Prevotella bivia ATCC29303AF440233CFB group 88(93) 74 7*
008_h10Clostridiumdifficile79-685AF090547Bacillus/clostridiumgroup73(89) 64
7*
002 Prevotella bivia ATCC29303AF080547CFB group 87(93) 77 6*
c10
001 Lactobacillus amylovorousATCC33620 Bacillus/clostridium100(100) 100
5
h09 group
001 Bacillus halodurans AP001508Bacillus/clostridium64(83) 64 5*
h11 group
003_b12Pediococcus pentosaceus Bacillus/clostridium95(96) 84 5*
ATCC43200 group
003_d12Clostridium thermocellum268137Bacillus/clostridium72(86) 70 5
ncib10682 group
005_e02Prevotella intermedia AF440234CFB group 89(93) 82 5
ATCC25611
005_e06Clostridium thermocellum268137Bacillus/clostridium72(36) 70 5
ncib10682 group
006 Clostridium perfringens X62914Bacilluslclostridium64(84) 64 5*
b07 group
006-f02Clostridium thermocellum268137Bacillus/clostridium78(89) 71 5*
ncib10682 group
001 Clostridium thermocellum268137Bacillus/clostridium74(!38) 71 4*
e12 ncib10682 group
002 Prevotella infermedia AF440234CFB group 88(94) 78 4
d06 ATCC25611
002_d12Clostridiumthermocellumncib10682268137Bacillus/clostridiumgroup71(136)
68 4
002_g10Prevotella intermedia AF440234CFB group 88(E34) 78 4*
ATCC25611
O11 Lactobacillus acidophilus Bacilluslclostridium100(100) 100 4*
c01 T-13 group
014_a12Clostridium perfringens X62914Bacillus/closfridium72(87) 70 4
group
018 Lactococcus garvieae AF245674Bacilluslclostridium66(86) 63 4
b06 ATCC43921 group
*recovered
from
at
least
two
libraries.
Hill et al. - High throughput methods for microbial community analysis page 27
CA 02430586 2003-05-30
'Table q2Summary of all library clones classified by nearest cpn60 database
neighlbours.
Nearest cprr60 DatabaseTaxonomic GenBankNumber % % peptide
Neighbour group of DINA
Number
accessionuniqueof identityidentity(similarity)
sequencesclones
Sacteroides forsythusCFB group AJ00651621 25 69-7576(86)-88(92)
ATCC43037
Bacteroides ovatus AF44023629 68 66-t3369(81 )-97(97)
ATCC8483
Bacteroides vulgatus AF44023810 10 63-13570(82)-94(95)
ATCC8482
Chryseobacterium AF4402353 17 67-E3868(84)-73(82)
gleum ATCC35910
Prevotella bivia AF44023387 274 72-7984(89)-89(94)
ATCC29303
Prevotella infermedia AF44023444 210 72-8272(84)-93(96)
ATCC25611
Prevotella nigrescens AF44138219 20 68-8167(83)-89(93)
ATCC33563
Bacillus coagulans BacilluslclostridiumAF4413795 6 68-7072(87)-74(86)
CECT 12 group
Bacillus firmus AF4413801 2 66 65(82)
CECT 14
Bacillushalodurans AP00150811 42 63-8764(84)-71(86)
Bacillus psychrophilus AF4413818 9 66-7072(86)-73(88)
CECT 4073
Bacillus sp. MS A80284523 3 68-Ei972(87)
Clostridium acetobutylicum M745722 3 66-6865(84)-73(87)
Clostridiumdifficile79-685 AF08054714 42 61-E.564(78)-74(89)
Clostridium perfringens X6291412 28 63-7063(83)-73(88)
Clostridium fhermocellum 26813788 210 65-7567(83)-82(91)
ncib10682
Enterococcus asini AF2456717 11 59-10071 (85)-100(100)
ss-1501
Globicatella sanguinis AF4413841 1 62 63(83)
ATCC51173
Lactobacillus acidaphilus 2 5 99-1(X199(99)-100(100)
T-13
LactobacillusamylovorousATCC33620 3 9 95-1()097(99)-
100(100)
Lactobacillus jensenii 1 1 64 66(83)
ATCC25258
Lactococcus garvieae AF2456741 4 63 66(86)
ATCC43921
Pediococcus pentosaceus 5 10 84-9895(96)-100(100)
ATCC43200
ThermoanaerobacterbrockiiRt8.G4 0560212 16 68 75(89)
Anaerobiospirillum ProteobacteriaAF44138312 88 79-8083(91)-86(94)
succiniciproducens gamma
ATCC700195
BurkhoJderia vietnamiensisProteobacteriaAF1049081 1 82 87(93)
DSM 11319 beta
Chlamydia muridarumchlamydiales NP i 2 55 50(68)
296764
8orrelia burgdorferispirochetes NC_0013183 5 62-6765(82)-67(83)
Treponema pallidum AE0011881 1 64 71 (89)
AL 398
Hill et al. - High throughput methods for microbial community analysis page 28
