U.S. patent application number 10/121120 was filed with the patent office on 2003-09-25 for specific and universal probes and amplification primers to rapidly detect and identify common bacterial pathogens and antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories.
Invention is credited to Bergeron, Michel G., Ouellette, Marc, Roy, Paul H..
Application Number | 20030180733 10/121120 |
Document ID | / |
Family ID | 24099022 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030180733 |
Kind Code |
A1 |
Bergeron, Michel G. ; et
al. |
September 25, 2003 |
Specific and universal probes and amplification primers to rapidly
detect and identify common bacterial pathogens and antibiotic
resistance genes from clinical specimens for routine diagnosis in
microbiology laboratories
Abstract
The present invention relates to DNA-based methods for universal
bacterial detection, for specific detection of the common bacterial
pathogens Escherichia coli, Klebsiella pneumoniae, Pseudomonas
aeruginosa, Proteus mirabilis, Streptococcus pneumoniae,
Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus
faecalis, Staphylococcus saprophyticus, Streptococcus pyogenes,
Haemophilus influenzae and Moraxella catarrhalis as well as for
specific detection of commonly encountered and clinically relevant
bacterial antibiotic resistance genes directly from clinical
specimens or, alternatively, from a bacterial colony. The above
bacterial species can account for as much as 80% of bacterial
pathogens isolated in routine microbiology laboratories. The core
of this invention consists primarily of the DNA sequences from all
species-specific genomic DNA fragments selected by hybridization
from genomic libraries or, alternatively, selected from data banks
as well as any oligonucleotide sequences derived from these
sequences which can be used as probes or amplification primers for
PCR or any other nucleic acid amplification methods. This invention
also includes DNA sequences from the selected clinically relevant
antibiotic resistance genes. With these methods, bacteria can be
detected (universal primers and/or probes) and identified
(species-specific primers and/or probes) directly from the clinical
specimens or from an isolated bacterial colony. Bacteria are
further evaluated for their putative susceptibility to antibiotics
by resistance gene detection (antibiotic resistance gene specific
primers and/or probes). Diagnostic kits for the detection of the
presence, for the bacterial identification of the above-mentioned
bacterial species and for the detection of antibiotic resistance
genes are also claimed. These kits for the rapid (one hour or less)
and accurate diagnosis of bacterial infections and antibiotic
resistance will gradually replace conventional methods currently
used in clinical microbiology laboratories for routine diagnosis.
They should provide tools to clinicians to help prescribe promptly
optimal treatments when necessary. Consequently, these tests should
contribute to saving human lives, rationalizing treatment, reducing
the development of antibiotic resistance and avoid unnecessary
hospitalizations.
Inventors: |
Bergeron, Michel G.;
(Sillery, CA) ; Ouellette, Marc; (Quebec, CA)
; Roy, Paul H.; (Loretteville, CA) |
Correspondence
Address: |
QUARLES & BRADY LLP
411 E. WISCONSIN AVENUE
SUITE 2040
MILWAUKEE
WI
53202-4497
US
|
Family ID: |
24099022 |
Appl. No.: |
10/121120 |
Filed: |
April 11, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10121120 |
Apr 11, 2002 |
|
|
|
09452599 |
Dec 1, 1999 |
|
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|
Current U.S.
Class: |
435/6.12 ;
435/6.15; 435/91.2 |
Current CPC
Class: |
C07K 14/3156 20130101;
C12Q 2600/16 20130101; C07K 14/212 20130101; C12Q 1/6895 20130101;
C07K 14/285 20130101; C07K 14/195 20130101; C07K 14/21 20130101;
C07K 14/26 20130101; C07K 14/245 20130101; C12Q 1/689 20130101;
C07K 14/31 20130101; C07K 14/315 20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
What is claimed is
1. A method using probes (fragments and/or oligonucleotides) and/or
amplification primers which are specific, ubiquitous and sensitive
for determining the presence and/or amount of nucleic acids from
bacterial species selected from the group consisting of Escherichia
coli, Klebsiells pneumoniae, Pseudomonas aeruginosa, Proteus
mirabilis, Streptococcus pneumoniae, Staphlyococcus aureus,
Staphylococcus epidermidis, Enterococcus faecalis, Staphlyococcus
saprophyticus, Streptococcus pyogenes, Haemophilus influenzae and
Moraxells catarrhalis in a any sample suspected of containing said
bacterial nucleic acid, wherein said bacterial nucleic acid or
variant or part thereof comprises a selected target region
hybridizable with said probes or primers; said method comprising
the steps of contacting said sample with said probes or primers and
detecting the presence and/or amount of hybridized probes and/or
amplified products as an indication of the presence and/or amount
of said bacterial species.
2. A method as defined in claim 1 further using probes (fragments
and/or oligonucleotides) and/or amplification primers which are
universal and sensitive for determining the presence and/or amount
of nucleic acids from any bacteria from any sample suspected of
containing said bacterial nucleic acid, wherein said bacterial
nucleic acid or variant or part thereof comprises a selected target
region hybridizable with said probes or primers; said method
comprising the steps of contacting said sample with said probes or
primers and detecting the presence and/or amount of hybridized
probes and/or amplified products as an indication of the presence
and/or amount of said any bacteria.
3. A method as defined in claim 1 further using probes (fragments
and/or oligonucleotides) and/or amplification primers which are
specific, ubiquitous and sensitive for determining the presence
and/or amount of nucleic acids from an antibiotic resistance gene
selected from the group consisting of bla.sub.tem, Bla.sub.rob,
Bla.sub.shv, aadB, aacC1, aacC2, aacC3, aacA4, mecA, vanA, vanH,
vanX, satA, aacA-aphD, vat, vga, msrA, sul and int in any sample
suspected of containing said bacterial nucleic acid, wherein said
bacterial nucleic acid or variant or part thereof comprises a
selected target region hybridizable with said probes or primers;
said method comprising the steps of contacting said sample with
said probes or primers and detecting the presence and/or amount of
hybridized probes and/or amplified products as an indication of the
presence and/or amount of said antibiotic resistance gene.
4. The method of any one of claims 1, 2 and 3 which is performed
directly on a sample obtained from human patients, animals,
environment or food.
5. The method of any one of claims 1, 2 and 3 which is performed
directly on a sample consisting of one or more bacterial
colonies.
6. The method of any one of claims 1 to 5, wherein the bacterial
nucleic acid is amplified by a method selected from the group
consisting of: a) polymerase chain reaction (PCR), b) ligase chain
reaction, c) nucleic acid sequence-based amplification, d)
self-sustained sequence replication, e) strand displacement
amplification, f) branched DNA signal amplification, g) nested PCR,
and h) multiplex PCR.
7. The method of claim 6 wherein said bacterial nucleic acid is
amplified by PCR.
8. The method of claim 7 wherein the PCR protocol is modified to
determine within one hour the presence of said bacterial nucleic
acids by performing for each amplification cycle an annealing step
of only one second at 55.degree. C. and a denaturation step of only
one second at 95.degree. C. without any elongation step.
9. A method for the detection, identification and/or quantification
of Escherichia coli directly from a test sample or from bacterial
colonies, which comprises the following steps: a) depositing and
fixing on an inert support or leaving in solution the bacterial DNA
of the sample or of a substantially homogenous population of
bacteria isolated from this sample, or inoculating said sample or
said substantially homogenous population of bacteria isolated from
this sample on an inert support, and lysing in situ said inoculated
sample or isolated bacteria to release the bacterial DNA, said
bacterial DNA being in a substantially single stranded form; b)
contacting said single stranded DNA with a probe, said probe
comprising at least one single stranded nucleic acid which
nucleotidic sequence is selected from the group consisting of SEQ
ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, a
sequence complementary thereof, a part thereof and a variant
thereof, which specifically and ubiquitously anneals with strains
or representatives of Escherichia coli, under conditions such that
the nucleic acid of said probe can selectively hybridize with said
bacterial DNA, whereby a hybridization complex is formed, said
complex being detected by labelling means, the label being present
on said probe or the label being present on a first reactive member
of said labelling means, said first reactive member reacting with a
second reactive member present on said probe; and c) detecting the
presence or the intensity of said label on said inert support or in
said solution as an indication of the presence and/or amount of
Escherichia coli in said test sample.
10. A method as defined in claim 9, wherein said probe is selected
from the group consisting of: 1) an oligonucleotide of 12-227
nucleotides in length which sequence is comprised in SEQ ID NO: 3
or a complementary sequence thereof, 2) an oligonucleotide of
12-278 nucleotides in length which sequence is comprised in SEQ ID
NO: 4 or a complementary sequence thereof, 3) an oligonucleotide of
12-1596 nucleotides in length which sequence is comprised in SEQ ID
NO: 5 or a complementary sequence thereof, 4) an oligonucleotide of
12-2703 nucleotides in length which sequence is comprised in SEQ ID
NO: 6 or a complementary sequence thereof, 5) an oligonucleotide of
12-1391 nucleotides in length which sequence is comprised in SEQ ID
NO: 7 or a complementary sequence thereof, and variants thereof
which specifically and ubiquitously anneal with strains and
representatives of Escherichia coli.
11. The method of claim 10, wherein the probe for detecting nucleic
acid sequences from Escherichia coli is selected from the group
consisting of SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID
NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53,
SEQ ID NO: 54 and a sequence complementary thereof.
12. A method for detecting the presence and/or amount of
Escherichia coli in a test sample which comprises the following
steps: a) treating said sample with an aqueous solution containing
at least one pair of oligonucleotide primers having at least 12
nucleotides in length, one of said primers being capable of
hybridizing selectively with one of the two complementary strands
of Escherichia coli DNA that contains a target sequence, and the
other of said primers being capable of hybridizing with the other
of said strands so as to form an extension product which contains
the target sequence as a template, said at least one pair of
primers being chosen from within one of the following sequences:
SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID
NO: 7; b) synthesizing an extension product of each of said primers
which extension products contain the target sequence, and
amplifying said target sequence, if any, to a detectable level; and
c) detecting the presence and/or amount of said amplified target
sequence as an indication of the presence and/or amount of
Escherichia coli in said test sample.
13. The method of claim 12, wherein said at least one pair of
primers is selected from the group consisting of: a) SEQ ID NO: 42
and SEQ ID NO: 43, b) SEQ ID NO: 46 and SEQ ID NO: 47, c) SEQ ID
NO: 55 and SEQ ID NO: 56, and d) SEQ ID NO: 131 and SEQ ID NO:
132.
14. A method for the detection, identification and/or
quantification of Klebsiella pneumoniae directly from a test sample
or from bacterial colonies, which comprises the following steps: a)
depositing and fixing on an inert support or leaving in solution
the bacterial DNA of the sample or of a substantially homogenous
population of bacteria isolated from this sample, or inoculating
said sample or said substantially homogenous population of bacteria
isolated from this sample on an inert support, and lysing in situ
said inoculated sample or isolated bacteria to release the
bacterial DNA, said bacterial DNA being in a substantially single
stranded form; b) contacting said single stranded DNA with a probe,
said probe comprising at least one single stranded nucleic acid
which nucleotidic sequence is selected from the group consisting of
SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, a sequence
complementary thereof, a part thereof and a variant thereof, which
specifically and ubiquitously anneals with strains or
representatives of Klebsiella pneumoniae, under conditions such
that the nucleic acid of said probe can selectively hybridize with
said bacterial DNA, whereby a hybridization complex is formed, said
complex being detected by labelling means, the label being present
on said probe or the label being present on a first reactive member
of said labelling means, said first reactive member reacting with a
second reactive member present on said probe; and c) detecting the
presence or the intensity of said label on said inert support or in
said solution as an indication of the presence and/or amount of
Klebsiella pneumoniae in said test sample.
15. A method as defined in claim 14, wherein said probe is selected
from the group consisting of: 1) an oligonucleotide of 12-238
nucleotides in length which sequence is comprised in SEQ ID NO: 8
or a complementary sequence thereof, 2) an oligonucleotide of
12-385 nucleotides in length which sequence is comprised in SEQ ID
NO: 9 or a complementary sequence thereof, 3) an oligonucleotide of
12-462 nucleotides in length which sequence is comprised in SEQ ID
NO: 10 or a complementary sequence thereof, 4) an oligonucleotide
of 12-730 nucleotides in length which sequence is comprised in SEQ
ID NO: 11 or a complementary sequence thereof, and variants thereof
which specifically and ubiquitously anneal with strains and
representatives of Klebsiella pneumoniae.
16. The method of claim 15, wherein the probe for detecting nucleic
acid sequences from Klebsiella pneumoniae is selected from the
group consisting of SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59,
SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID
NO: 66, SEQ ID NO: 69 and a sequence complementary thereof.
17. A method for detecting the presence and/or amount of Klebsiella
pneumoniae in a test sample which comprises the following steps: a)
treating said sample with an aqueous solution containing at least
one pair of oligonucleotide primers having at least 12 nucleotides
in length, one of said primers being capable of hybridizing
selectively with one of the two complementary strands of Klebsiella
pneumoniae DNA that contains a target sequence, and the other of
said primers being capable of hybridizing with the other of said
strands so as to form an extension product which contains the
target sequence as a template, said at least one pair of primers
being chosen from within one of the following sequences: SEQ ID NO:
8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; b) synthesizing
an extension product of each of said primers which extension
products contain the target sequence, and amplifying said target
sequence, if any, to a detectable level; and c) detecting the
presence and/or amount of said amplified target sequence as an
indication of the presence and/or amount of Klebsiella pneumoniae
in said test sample.
18. The method of claim 17, wherein said at least one pair of
primers is selected from the group consisting of: a) SEQ ID NO: 61
and SEQ ID NO: 62, b) SEQ ID NO: 67 and SEQ ID NO: 68, c) SEQ ID
NO: 135 and SEQ ID NO: 136, and d) SEQ ID NO: 137 and SEQ ID NO:
138.
19. A method for the detection, identification and/or
quantification of Proteus mirabilis directly from a test sample or
from bacterial colonies, which comprises the following steps: a)
depositing and fixing on an inert support or leaving in solution
the bacterial DNA of the sample or of a substantially homogenous
population of bacteria isolated from this sample, or inoculating
said sample or said substantially homogenous population of bacteria
isolated from this sample on an inert support, and lysing in situ
said inoculated sample or isolated bacteria to release the
bacterial DNA, said bacterial DNA being in a substantially single
stranded form; b) contacting said single stranded DNA with a probe,
said probe comprising at least one single stranded nucleic acid
which nucleotidic sequence is selected from the group consisting of
SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, a
sequence complementary thereof, a part thereof and a variant
thereof, which specifically and ubiquitously anneals with strains
or representatives of Proteus mirabilis, under conditions such that
the nucleic acid of said probe can selectively hybridize with said
bacterial DNA, whereby a hybridization complex is formed, said
complex being detected by labelling means, the label being present
on said probe or the label being present on a first reactive member
of said labelling means, said first reactive member reacting with a
second reactive member present on said probe; and c) detecting the
presence or the intensity of said label on said inert support or in
said solution as an indication of the presence and/or amount of
Proteus mirabilis in said test sample.
20. A method as defined in claim 19, wherein said probe is selected
from the group consisting of: 1) an oligonucleotide of 12-225
nucleotides in length which sequence is comprised in SEQ ID NO: 12
or a complementary sequence thereof, 2) an oligonucleotide of
12-402 nucleotides in length which sequence is comprised in SEQ ID
NO: 13 or a complementary sequence thereof, 3) an oligonucleotide
of 12-157 nucleotides in length which sequence is comprised in SEQ
ID NO: 14 or a complementary sequence thereof, 4) an
oligonucleotide of 12-1348 nucleotides in length which sequence is
comprised in SEQ ID NO: 15 or a complementary sequence thereof, and
variants thereof which specifically and ubiquitously anneal with
strains and representatives of Proteus mirabilis.
21. The method of claim 20, wherein the probe for detecting nucleic
acid sequences from Proteus mirabilis is selected from the group
consisting of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID
NO: 73, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 80, SEQ ID NO: 81,
SEQ ID NO: 82 and a sequence complementary thereof.
22. A method for detecting the presence and/or amount of Proteus
mirabilis in a test sample which comprises the following steps: a)
treating said sample with an aqueous solution containing at least
one pair of oligonucleotide primers having at least 12 nucleotides
in length, one of said primers being capable of hybridizing
selectively with one of the two complementary strands of Proteus
mirabilis DNA that contains a target sequence, and the other of
said primers being capable of hybridizing with the other of said
strands so as to form an extension product which contains the
target sequence as a template, said at least one pair of primers
being chosen from within one of the following sequences: SEQ ID NO:
12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15; b)
synthesizing an extension product of each of said primers which
extension products contain the target sequence, and amplifying said
target sequence, if any, to a detectable level; and c) detecting
the presence and/or amount of said amplified target sequence as an
indication of the presence and/or amount of Proteus mirabilis in
said test sample.
23. The method of claim 22, wherein said at least one pair of
primers is selected from the group consisting of: a) SEQ ID NO: 74
and SEQ ID NO: 75, and b) SEQ ID NO: 133 and SEQ ID NO: 134.
24. A method for the detection, identification and/or
quantification of Staphylococcus saprophyticus directly from a test
sample or from bacterial colonies, which comprises the following
steps: a) depositing and fixing on an inert support or leaving in
solution the bacterial DNA of the sample or of a substantially
homogenous population of bacteria isolated from this sample, or
inoculating said sample or said substantially homogenous population
of bacteria isolated from this sample on an inert support, and
lysing in situ said inoculated sample or isolated bacteria to
release the bacterial DNA, said bacterial DNA being in a
substantially single stranded form; b) contacting said single
stranded DNA with a probe, said probe comprising at least one
single stranded nucleic acid which nucleotidic sequence is selected
from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID
NO: 23, SEQ ID NO: 24, a sequence complementary thereof, a part
thereof and a variant thereof, which specifically and ubiquitously
anneals with strains or representatives of Staphylococcus
saprophyticus, under conditions such that the nucleic acid of said
probe can selectively hybridize with said bacterial DNA, whereby a
hybridization complex is formed, said complex being detected by
labelling means, the label being present on said probe or the label
being present on a first reactive member of said labelling means,
said first reactive member reacting with a second reactive member
present on said probe; and c) detecting the presence or the
intensity of said label on said inert support or in said solution
as an indication of the presence and/or amount of Staphylococcus
saprophyticus in said test sample.
25. A method as defined in claim 24, wherein said probe is selected
from the group consisting of: 1) an oligonucleotide of 12-172
nucleotides in length which sequence is comprised in SEQ ID NO: 21
or a complementary sequence thereof, 2) an oligonucleotide of
12-155 nucleotides in length which sequence is comprised in SEQ ID
NO: 22 or a complementary sequence thereof, 3) an oligonucleotide
of 12-145 nucleotides in length which sequence is comprised in SEQ
ID NO: 23 or a complementary sequence thereof, 4) an
oligonucleotide of 12-265 nucleotides in length which sequence is
comprised in SEQ ID NO: 24 or a complementary sequence thereof, and
variants thereof which specifically and ubiquitously anneal with
strains and representatives of Staphylococcus saprophyticus.
26. The method of claim 25, wherein the probe for detecting nucleic
acid sequences from Staphylococcus saprophyticus is selected from
the group consisting of SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO:
100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104
and a sequence complementary thereof.
27. A method for detecting the presence and/or amount of
Staphylococcus saprophyticus in a test sample which comprises the
following steps: a) treating said sample with an aqueous solution
containing at least one pair of oligonucleotide primers having at
least 12 nucleotides in length, one of said primers being capable
of hybridizing selectively with one of the two complementary
strands of Staphylococcus saprophyticus DNA that contains a target
sequence, and the other of said primers being capable of
hybridizing with the other of said strands so as to form an
extension product which contains the target sequence as a template,
said at least one pair of primers being chosen from within one of
the following sequences: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO:
23, and SEQ ID NO: 24; b) synthesizing an extension product of each
of said primers which extension products contain the target
sequence, and amplifying said target sequence, if any, to a
detectable level; and c) detecting the presence and/or amount of
said amplified target sequence as an indication of the presence
and/or amount of Staphylococcus saprophyticus in said test
sample.
28. The method of claim 27, wherein said at least one pair of
primers is selected from the group consisting of: a) SEQ ID NO: 98
and SEQ ID NO: 99, and b) SEQ ID NO: 139 and SEQ ID NO: 140.
29. A method for the detection, identification and/or
quantification of Moraxella catarrhalis directly from a test sample
or from bacterial colonies, which comprises the following steps: a)
depositing and fixing on an inert support or leaving in solution
the bacterial DNA of the sample or of a substantially homogenous
population of bacteria isolated from this sample, or inoculating
said sample or said substantially homogenous population of bacteria
isolated from this sample on an inert support, and lysing in situ
said inoculated sample or isolated bacteria to release the
bacterial DNA, said bacterial DNA being in a substantially single
stranded form; b) contacting said single stranded DNA with a probe,
said probe comprising at least one single stranded nucleic acid
which nucleotidic sequence is selected from the group consisting of
SEQ ID NO: 28, SEQ ID NO: 29, a sequence complementary thereof, a
part thereof and a variant thereof, which specifically and
ubiquitously anneals with strains or representatives of Moraxella
catarrhalis, under conditions such that the nucleic acid of said
probe can selectively hybridize with said bacterial DNA, whereby a
hybridization complex is formed, said complex being detected by
labelling means, the label being present on said probe or the label
being present on a first reactive member of said labelling means,
said first reactive member reacting with a second reactive member
present on said probe; and c) detecting the presence or the
intensity of said label on said inert support or in said solution
as an indication of the presence and/or amount of Moraxella
catarrhalis in said test sample.
30. A method as defined in claim 29, wherein said probe is selected
from the group consisting of: 1) an oligonucleotide of 12-526
nucleotides in length which sequence is comprised in SEQ ID NO: 28
or a complementary sequence thereof, 2) an oligonucleotide of
12-466 nucleotides in length which sequence is comprised in SEQ ID
NO: 29 or a complementary sequence thereof, and variants thereof
which specifically and ubiquitously anneal with strains and
representatives of Moraxella catarrhalis.
31. The method of claim 30, wherein the probe for detecting nucleic
acid sequences from Moraxella catarrhalis is selected from the
group consisting of SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110,
SEQ ID NO: 111, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ
ID NO: 117 and a sequence complementary thereof.
32. A method for detecting the presence and/or amount of Moraxella
catarrhalis in a test sample which comprises the following steps:
a) treating said sample with an aqueous solution containing at
least one pair of oligonucleotide primers having at least 12
nucleotides in length, one of said primers being capable of
hybridizing selectively with one of the two complementary strands
of Moraxella catarrhalis DNA that contains a target sequence, and
the other of said primers being capable of hybridizing with the
other of said strands so as to form an extension product which
contains the target sequence as a template, said at least one pair
of primers being chosen from within one of the following sequences:
SEQ ID NO: 28 and SEQ ID NO: 29; b) synthesizing an extension
product of each of said primers which extension products contain
the target sequence, and amplifying said target sequence, if any,
to a detectable level; and c) detecting the presence and/or amount
of said amplified target sequence as an indication of the presence
and/or amount of Moraxella catarrhalis in said test sample.
33. The method of claim 32, wherein said at least one pair of
primers is selected from the group consisting of: a) SEQ ID NO: 112
and SEQ ID NO: 113, b) SEQ ID NO: 118 and SEQ ID NO: 119, and c)
SEQ ID NO: 160 and SEQ ID NO: 119.
34. A method for the detection, identification and/or
quantification of Pseudomonas aeruginosa directly from a test
sample or from bacterial colonies, which comprises the following
steps: a) depositing and fixing on an inert support or leaving in
solution the bacterial DNA of the sample or of a substantially
homogenous population of bacteria isolated from this sample, or
inoculating said sample or said substantially homogenous population
of bacteria isolated from this sample on an inert support, and
lysing in situ said inoculated sample or isolated bacteria to
release the bacterial DNA, said bacterial DNA being in a
substantially single stranded form; b) contacting said single
stranded DNA with a probe, said probe comprising at least one
single stranded nucleic acid which nucleotidic sequence is selected
from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID
NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, a sequence complementary
thereof, a part thereof and a variant thereof, which specifically
and ubiquitously anneals with strains or representatives of
Pseudomonas aeruginosa, under conditions such that the nucleic acid
of said probe can selectively hybridize with said bacterial DNA,
whereby a hybridization complex is formed, said complex being
detected by labelling means, the label being present on said probe
or the label being present on a first reactive member of said
labelling means, said first reactive member reacting with a second
reactive member present on said probe; and c) detecting the
presence or the intensity of said label on said inert support or in
said solution as an indication of the presence and/or amount of
Pseudomonas aeruginosa in said test sample.
35. A method as defined in claim 34, wherein said probe is selected
from the group consisting of: 1) an oligonucleotide of 12-2167
nucleotides in length which sequence is comprised in SEQ ID NO: 16
or a complementary sequence thereof, 2) an oligonucleotide of
12-1872 nucleotides in length which sequence is comprised in SEQ ID
NO: 17 or a complementary sequence thereof, 3) an oligonucleotide
of 12-3451 nucleotides in length which sequence is comprised in SEQ
ID NO: 18 or a complementary sequence thereof, 4) an
oligonucleotide of 12-744 nucleotides in length which sequence is
comprised in SEQ ID NO: 19 or a complementary sequence thereof, 5)
an oligonucleotide of 12-2760 nucleotides in length which sequence
is comprised in SEQ ID NO: 20 or a complementary sequence thereof,
and variants thereof which specifically and ubiquitously anneal
with strains and representatives of Pseudomonas aeruginosa.
36. The method of claim 35, wherein the probe for detecting nucleic
acid sequences from Pseudomonas aeruginosa is selected from the
group consisting of SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89,
SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID
NO: 94, SEQ ID NO: 95 and a sequence complementary thereof.
37. A method for detecting the presence and/or amount of
Pseudomonas aeruginosa in a test sample which comprises the
following steps: a) treating said sample with an aqueous solution
containing at least one pair of oligonucleotide primers having at
least 12 nucleotides in length, one of said primers being capable
of hybridizing selectively with one of the two complementary
strands of Pseudomonas aeruginosa DNA that contains a target
sequence, and the other of said primers being capable of
hybridizing with the other of said strands so as to form an
extension product which contains the target sequence as a template,
said at least one pair of primers being chosen from within one of
the following sequences: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:
18, SEQ ID NO: 19 and SEQ ID NO: 20; b) synthesizing an extension
product of each of said primers which extension products contain
the target sequence, and amplifying said target sequence, if any,
to a detectable level; and c) detecting the presence and/or amount
of said amplified target sequence as an indication of the presence
and/or amount of Pseudomonas aeruginosa in said test sample.
38. The method of claim 37, wherein said at least one pair of
primers is selected from the group consisting of: a) SEQ ID NO: 83
and SEQ ID NO: 84, and b) SEQ ID NO: 85 and SEQ ID NO: 86.
39. A method for the detection, identification and/or
quantification of Staphylococcus epidermidis directly from a test
sample or from bacterial colonies, which comprises the following
steps: a) depositing and fixing on an inert support or leaving in
solution the bacterial DNA of the sample or of a substantially
homogenous population of bacteria isolated from this sample, or
inoculating said sample or said substantially homogenous population
of bacteria isolated from this sample on an inert support, and
lysing in situ said inoculated sample or isolated bacteria to
release the bacterial DNA, said bacterial DNA being in a
substantially single stranded form; b) contacting said single
stranded DNA with a probe, said probe comprising at least one
single stranded nucleic acid which nucleotidic sequence is selected
from the group consisting of SEQ ID NO: 36, a sequence
complementary thereof, a part thereof and a variant thereof, which
specifically and ubiquitously anneals with strains or
representatives of Staphylococcus epidermidis, under conditions
such that the nucleic acid of said probe can selectively hybridize
with said bacterial DNA, whereby a hybridization complex is formed,
said complex being detected by labelling means, the label being
present on said probe or the label being present on a first
reactive member of said labelling means, said first reactive member
reacting with a second reactive member present on said probe; and
c) detecting the presence or the intensity of said label on said
inert support or in said solution as an indication of the presence
and/or amount of Staphylococcus epidermidis in said test
sample.
40. A method as defined in claim 39, wherein said probe is selected
from the group consisting of an oligonucleotide of 12-705
nucleotides in length which sequence is comprised in SEQ ID NO: 36
and variants thereof which specifically and ubiquitously anneal
with strains and representatives of Staphylococcus epidermidis.
41. A method for detecting the presence and/or amount of
Staphylococcus epidermidis in a test sample which comprises the
following steps: a) treating said sample with an aqueous solution
containing at least one pair of oligonucleotide primers having at
least 12 nucleotides in length, one of said primers being capable
of hybridizing selectively with one of the two complementary
strands of Staphylococcus epidermidis DNA that contains a target
sequence, and the other of said primers being capable of
hybridizing with the other of said strands so as to form an
extension product which contains the target sequence as a template,
said at least one pair of primers being chosen from within the
following sequence: SEQ ID NO: 36; b) synthesizing an extension
product of each of said primers which extension products contain
the target sequence, and amplifying said target sequence, if any,
to a detectable level; and c) detecting the presence and/or amount
of said amplified target sequence as an indication of the presence
and/or amount of Staphylococcus epidermidis in said test
sample.
42. The method of claim 41, wherein said at least one pair of
primers is selected from the group consisting of: a) SEQ ID NO: 145
and SEQ ID NO: 146, and b) SEQ ID NO: 147 and SEQ ID NO: 148.
43. A method for the detection, identification and/or
quantification of Staphylococcus aureus directly from a test sample
or from bacterial colonies, which comprises the following steps: a)
depositing and fixing on an inert support or leaving in solution
the bacterial DNA of the sample or of a substantially homogenous
population of bacteria isolated from this sample, or inoculating
said sample or said substantially homogenous population of bacteria
isolated from this sample on an inert support, and lysing in situ
said inoculated sample or isolated bacteria to release the
bacterial DNA, said bacterial DNA being in a substantially single
stranded form; b) contacting said single stranded DNA with a probe,
said probe comprising at least one single stranded nucleic acid
which nucleotidic sequence is selected from the group consisting of
SEQ ID NO: 37, a sequence complementary thereof, a part thereof and
a variant thereof, which specifically and ubiquitously anneals with
strains or representatives of Staphylococcus aureus, under
conditions such that the nucleic acid of said probe can selectively
hybridize with said bacterial DNA, whereby a hybridization complex
is formed, said complex being detected by labelling means, the
label being present on said probe or the label being present on a
first reactive member of said labelling means, said first reactive
member reacting with a second reactive member present on said
probe; and c) detecting the presence or the intensity of said label
on said inert support or in said solution as an indication of the
presence and/or amount of Staphylococcus aureus in said test
sample.
44. A method as defined in claim 43, wherein said probe is selected
from the group consisting of an oligonucleotide of 12-442
nucleotides in length which sequence is comprised in SEQ ID NO: 37
and variants thereof which specifically and ubiquitously anneal
with strains and representatives of Staphylococcus aureus.
45. A method for detecting the presence and/or amount of
Staphylococcus aureus in a test sample which comprises the
following steps: a) treating said sample with an aqueous solution
containing at least one pair of oligonucleotide primers having at
least 12 nucleotides in length, one of said primers being capable
of hybridizing selectively with one of the two complementary
strands of Staphylococcus aureus DNA that contains a target
sequence, and the other of said primers being capable of
hybridizing with the other of said strands so as to form an
extension product which contains the target sequence as a template,
said at least one pair of primers being chosen from within the
following sequence: SEQ ID NO: 37; b) synthesizing an extension
product of each of said primers which extension products contain
the target sequence, and amplifying said target sequence, if any,
to a detectable level; and c) detecting the presence and/or amount
of said amplified target sequence as an indication of the presence
and/or amount of Staphylococcus aureus in said test sample.
46. The method of claim 45, wherein said at least one pair of
primers is selected from the group consisting of: a) SEQ ID NO: 149
and SEQ ID NO: 150, b) SEQ ID NO: 149 and SEQ ID NO: 151, and c)
SEQ ID NO: 152 and SEQ ID NO: 153.
47. A method for the detection, identification and/or
quantification of Haemophilus influenzae directly from a test
sample or from bacterial colonies, which comprises the following
steps: a) depositing and fixing on an inert support or leaving in
solution the bacterial DNA of the sample or of a substantially
homogenous population of bacteria isolated from this sample, or
inoculating said sample or said substantially homogenous population
of bacteria isolated from this sample on an inert support, and
lysing in situ said inoculated sample or isolated bacteria to
release the bacterial DNA, said bacterial DNA being in a
substantially single stranded form; b) contacting said single
stranded DNA with a probe, said probe comprising at least one
single stranded nucleic acid which nucleotidic sequence is selected
from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID
NO: 27, a sequence complementary thereof, a part thereof and a
variant thereof, which specifically and ubiquitously anneals with
strains or representatives of Haemophilus influenzae, under
conditions such that the nucleic acid of said probe can selectively
hybridize with said bacterial DNA, whereby a hybridization complex
is formed, said complex being detected by labelling means, the
label being present on said probe or the label being present on a
first reactive member of said labelling means, said first reactive
member reacting with a second reactive member present on said
probe; and c) detecting the presence or the intensity of said label
on said inert support or in said solution as an indication of the
presence and/or amount of Haemophilus influenzae in said test
sample.
48. A method as defined in claim 47, wherein said probe is selected
from the group consisting of: 1) an oligonucleotide of 12-845
nucleotides in length which sequence is comprised in SEQ ID NO: 25
or a complementary sequence thereof, 2) an oligonucleotide of
12-1598 nucleotides in length which sequence is comprised in SEQ ID
NO: 26 or a complementary sequence thereof, 3) an oligonucleotide
of 12-9100 nucleotides in length which sequence is comprised in SEQ
ID NO: 27 or a complementary sequence thereof, and variants thereof
which specifically and ubiquitously anneal with strains and
representatives of Haemophilus influenzae.
49. The method of claim 48, wherein the probe for detecting nucleic
acid sequences from Haemophilus influenzae is selected from the
group consisting of SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107
and a sequence complementary thereof.
50. A method for detecting the presence and/or amount of
Haemophilus influenzae in a test sample which comprises the
following steps: a) treating said sample with an aqueous solution
containing at least one pair of oligonucleotide primers having at
least 12 nucleotides in length, one of said primers being capable
of hybridizing selectively with one of the two complementary
strands of Haemophilus influenzae DNA that contains a target
sequence, and the other of said primers being capable of
hybridizing with the other of said strands so as to form an
extension product which contains the target sequence as a template,
said at least one pair of primers being chosen from within one of
the following sequences: SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID
NO: 27; b) synthesizing an extension product of each of said
primers which extension products contain the target sequence, and
amplifying said target sequence, if any, to a detectable level; and
c) detecting the presence and/or amount of said amplified target
sequence as an indication of the presence and/or amount of
Haemophilus influenzae in said test sample.
51. The method of claim 50, wherein said at least one pair of
primers comprises the following pair: SEQ ID NO: 154 and SEQ ID NO:
155.
52. A method for the detection, identification and/or
quantification of Streptococcus pneumoniae directly from a test
sample or from bacterial colonies, which comprises the following
steps: a) depositing and fixing on an inert support or leaving in
solution the bacterial DNA of the sample or of a substantially
homogenous population of bacteria isolated from this sample, or
inoculating said sample or said substantially homogenous population
of bacteria isolated from this sample on an inert support, and
lysing in situ said inoculated sample or isolated bacteria to
release the bacterial DNA, said bacterial DNA being in a
substantially single stranded form; b) contacting said single
stranded DNA with a probe, said probe comprising at least one
single stranded nucleic acid which nucleotidic sequence is selected
from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID
NO: 34, SEQ ID NO: 35, a sequence complementary thereof, a part
thereof and a variant thereof, which specifically and ubiquitously
anneals with strains or representatives of Streptococcus
pneumoniae, under conditions such that the nucleic acid of said
probe can selectively hybridize with said bacterial DNA, whereby a
hybridization complex is formed, said complex being detected by
labelling means, the label being present on said probe or the label
being present on a first reactive member of said labelling means,
said first reactive member reacting with a second reactive member
present on said probe; and c) detecting the presence or the
intensity of said label on said inert support or in said solution
as an indication of the presence and/or amount of Streptococcus
pneumoniae in said test sample.
53. A method as defined in claim 52, wherein said probe is selected
from the group consisting of: 1) an oligonucleotide of 12-631
nucleotides in length which sequence is comprised in SEQ ID NO: 30
or a complementary sequence thereof, 2) an oligonucleotide of
12-3754 nucleotides in length which sequence is comprised in SEQ ID
NO: 31 or a complementary sequence thereof, 3) an oligonucleotide
of 12-841 nucleotides in length which sequence is comprised in SEQ
ID NO: 34 or a complementary sequence thereof, 4) an
oligonucleotide of 12-4500 nucleotides in length which sequence is
comprised in SEQ ID NO: 35 or a complementary sequence thereof, and
variants thereof which specifically and ubiquitously anneal with
strains and representatives of Streptococcus pneumoniae.
54. The method of claim 53, wherein the probe for detecting nucleic
acid sequences from Streptococcus pneumoniae is selected from the
group consisting of SEQ ID NO: 120, SEQ ID NO: 121 and a sequence
complementary thereof.
55. A method for detecting the presence and/or amount of
Streptococcus pneumoniae in a test sample which comprises the
following steps: a) treating said sample with an aqueous solution
containing at least one pair of oligonucleotide primers having at
least 12 nucleotides in length, one of said primers being capable
of hybridizing selectively with one of the two complementary
strands of Streptococcus pneumoniae DNA that contains a target
sequence, and the other of said primers being capable of
hybridizing with the other of said strands so as to form an
extension product which contains the target sequence as a template,
said at least one pair of primers being chosen from within one of
the following sequences: SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:
34 and SEQ ID NO: 35; b) synthesizing an extension product of each
of said primers which extension products contain the target
sequence, and amplifying said target sequence, if any, to a
detectable level; and c) detecting the presence and/or amount of
said amplified target sequence as an indication of the presence
and/or amount of Streptococcus pneumoniae in said test sample.
56. The method of claim 55, wherein said at least one pair of
primers is selected from the group consisting of: a) SEQ ID NO: 78
and SEQ ID NO: 79, b) SEQ ID NO: 156 and SEQ ID NO: 157, and c) SEQ
ID NO: 158 and SEQ ID NO: 159.
57. A method for the detection, identification and/or
quantification of Streptococcus pyogenes directly from a test
sample or from bacterial colonies, which comprises the following
steps: a) depositing and fixing on an inert support or leaving in
solution the bacterial DNA of the sample or of a substantially
homogenous population of bacteria isolated from this sample, or
inoculating said sample or said substantially homogenous population
of bacteria isolated from this sample on an inert support, and
lysing in situ said inoculated sample or isolated bacteria to
release the bacterial DNA, said bacterial DNA being in a
substantially single stranded form; b) contacting said single
stranded DNA with a probe, said probe comprising at least one
single stranded nucleic acid which nucleotidic sequence is selected
from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, a
sequence complementary thereof, a part thereof and a variant
thereof, which specifically and ubiquitously anneals with strains
or representatives of Streptococcus pyogenes, under conditions such
that the nucleic acid of said probe can selectively hybridize with
said bacterial DNA, whereby a hybridization complex is formed, said
complex being detected by labelling means, the label being present
on said probe or the label being present on a first reactive member
of said labelling means, said first reactive member reacting with a
second reactive member present on said probe; and c) detecting the
presence or the intensity of said label on said inert support or in
said solution as an indication of the presence and/or amount of
Streptococcus pyogenes in said test sample.
58. A method as defined in claim 57, wherein said probe is selected
from the group consisting of: 1) an oligonucleotide of 12-1337
nucleotides in length which sequence is comprised in SEQ ID NO: 32
or a complementary sequence thereof, 2) an oligonucleotide of
12-1837 nucleotides in length which sequence is comprised in SEQ ID
NO: 33 or a complementary sequence thereof, and variants thereof
which specifically and ubiquitously anneal with strains and
representatives of Streptococcus pyogenes.
59. A method for detecting the presence and/or amount of
Streptococcus pyogenes in a test sample which comprises the
following steps: a) treating said sample with an aqueous solution
containing at least one pair of oligonucleotide primers having at
least 12 nucleotides in length, one of said primers being capable
of hybridizing selectively with one of the two complementary
strands of Streptococcus pyogenes DNA that contains a target
sequence, and the other of said primers being capable of
hybridizing with the other of said strands so as to form an
extension product which contains the target sequence as a template,
said at least one pair of primers being chosen from within one of
the following sequences: SEQ ID NO: 32 and SEQ ID NO: 33; b)
synthesizing an extension product of each of said primers which
extension products contain the target sequence, and amplifying said
target sequence, if any, to a detectable level; and c) detecting
the presence and/or amount of said amplified target sequence as an
indication of the presence and/or amount of Streptococcus pyogenes
in said test sample.
60. The method of claim 59, wherein said at least one pair of
primers is selected from the group consisting of: a) SEQ ID NO: 141
and SEQ ID NO: 142, and b) SEQ ID NO: 143 and SEQ ID NO: 144.
61. A method for the detection, identification and/or
quantification of Enterococcus faecalis directly from a test sample
or from bacterial colonies, which comprises the following steps: a)
depositing and fixing on an inert support or leaving in solution
the bacterial DNA of the sample or of a substantially homogenous
population of bacteria isolated from this sample, or inoculating
said sample or said substantially homogenous population of bacteria
isolated from this sample on an inert support, and lysing in situ
said inoculated sample or isolated bacteria to release the
bacterial DNA, said bacterial DNA being in a substantially single
stranded form; b) contacting said single stranded DNA with a probe,
said probe comprising at least one single stranded nucleic acid
which nucleotidic sequence is selected from the group consisting of
SEQ ID NO: 1, SEQ ID NO: 2, a sequence complementary thereof, a
part thereof and a variant thereof, which specifically and
ubiquitously anneals with strains or representatives of
Enterococcus faecalis, under conditions such that the nucleic acid
of said probe can selectively hybridize with said bacterial DNA,
whereby a hybridization complex is formed, said complex being
detected by labelling means, the label being present on said probe
or the label being present on a first reactive member of said
labelling means, said first reactive member reacting with a second
reactive member present on said probe; and c) detecting the
presence or the intensity of said label on said inert support or in
said solution as an indication of the presence and/or amount of
Enterococcus faecalis in said test sample.
62. A method as defined in claim 61, wherein said probe is selected
from the group consisting of: 1) an oligonucleotide of 12-1817
nucleotides in length which sequence is comprised in SEQ ID NO: 1
or a complementary sequence thereof, 2) an oligonucleotide of
12-2275 nucleotides in length which sequence is comprised in SEQ ID
NO: 2, and variants thereof which specifically and ubiquitously
anneal with strains and representatives of Enterococcus
faecalis.
63. A method for detecting the presence and/or amount of
Enterococcus faecalis in a test sample which comprises the
following steps: a) treating said sample with an aqueous solution
containing at least one pair of oligonucleotide primers having at
least 12 nucleotides in length, one of said primers being capable
of hybridizing selectively with one of the two complementary
strands of Enterococcus faecalis DNA that contains a target
sequence, and the other of said primers being capable of
hybridizing with the other of said strands so as to form an
extension product which contains the target sequence as a template,
said at least one pair of primers being chosen from within one of
the following sequences: SEQ ID NO: 1 and SEQ ID NO: 2; b)
synthesizing an extension product of each of said primers which
extension products contain the target sequence, and amplifying said
target sequence, if any, to a detectable level; and c) detecting
the presence and/or amount of said amplified target sequence as an
indication of the presence and/or amount of Enterococcus faecalis
in said test sample.
64. The method of claim 63, wherein said at least one pair of
primers is selected from the group consisting of: a) SEQ ID NO: 38
and SEQ ID NO: 39, and b) SEQ ID NO: 40 and SEQ ID NO: 41.
65. A method for the detection of the presence and/or amount of any
bacterial species directly from a test sample or from bacterial
colonies, which comprises the following steps: a) depositing and
fixing on an inert support or leaving in solution the bacterial DNA
of the sample or of a substantially homogenous population of
bacteria isolated from this sample, or inoculating said sample or
said substantially homogenous population of bacteria isolated from
this sample on an inert support, and lysing in situ said inoculated
sample or isolated bacteria to release the bacterial DNA, said
bacterial DNA being in a substantially single stranded form; b)
contacting said single stranded DNA with a universal probe which
sequence is selected from the group consisting of SEQ ID NO: 122,
SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 128, SEQ
ID NO: 129, SEQ ID NO: 130 and a sequence complementary thereof,
under conditions such that the nucleic acid of said probe can
selectively hybridize with said bacterial DNA, whereby a
hybridization complex is formed, said complex being detected by
labelling means, the label being present on said probe or the label
being present on a first reactive member of said labelling means,
said first reactive member reacting with a second reactive member
present on said probe; and c) detecting the presence or the
intensity of said label on said inert support or in said solution
as an indication of the presence and/or amount of said any
bacterial species in said test sample.
66. A method for detecting the presence and/or amount of any
bacterial species in a test sample which comprises the following
steps: a) treating said sample with an aqueous solution containing
a pair of universal primers which sequence is defined in SEQ ID NO:
126 and SEQ ID NO: 127, one of said primers being capable of
hybridizing selectively with one of the two complementary strands
of said any bacterial species DNA that contains a target sequence,
and the other of said primers being capable of hybridizing with the
other of said strands so as to form an extension product which
contains the target sequence as a template; b) synthesizing an
extension product of each of said primers which extension products
contain the target sequence, and amplifying said target sequence,
if any, to a detectable level; and c) detecting the presence and/or
amount of said amplified target sequence as an indication of the
presence and/or amount of said any bacterial species in said test
sample.
67. A method for evaluating a bacterial resistance to .beta.-lactam
antibiotics mediated by the bacterial antibiotic resistance gene
bla.sub.tem (TEM-1) directly from a test sample or from bacterial
colonies, which comprises the following steps: a) depositing and
fixing on an inert support or leaving in solution the bacterial DNA
of the sample or of a substantially homogenous population of
bacteria isolated from this sample, or inoculating said sample or
said substantially homogenous population of bacteria isolated from
this sample on an inert support, and lysing in situ said inoculated
sample or isolated bacteria to release the bacterial DNA, said
bacterial DNA being in a substantially single stranded form; b)
contacting said single stranded DNA with a probe, said probe
comprising at least one single stranded nucleic acid which
nucleotidic sequence is selected from the group consisting of SEQ
ID NO: 161, a sequence complementary thereof, a part thereof and a
variant thereof, which specifically anneals with said bacterial
antibiotic resistance gene coding for a .beta.-lactamase, under
conditions such that the nucleic acid of said probe can selectively
hybridize with said bacterial DNA, whereby a hybridization complex
is formed, said complex being detected by labelling means, the
label being present on said probe or the label being present on a
first reactive member of said labelling means, said first reactive
member reacting with a second reactive member present on said
probe; and c) detecting the presence or the intensity of said label
on said inert support or in said solution as an indication of a
bacterial resistance to .beta.-lactam antibiotics mediated by the
bacterial antibiotic resistance gene TEM-1.
68. A method as defined in claim 67, wherein said probe comprises
an oligonucleotide of at least 12 nucleotides in length which
hybridizes to SEQ ID NO: 161.
69. A method for evaluating a bacterial resistance to .beta.-lactam
antibiotics mediated by the bacterial antibiotic resistance gene
bla.sub.tem (TEM-1) in a test sample which comprises the following
steps: a) treating said sample with an aqueous solution containing
at least one pair of oligonucleotide primers having at least 12
nucleotides in length, one of said primers being capable of
hybridizing selectively with one of the two complementary strands
of said bacterial antibiotic resistance gene coding for a
.beta.-lactamase that contains a target sequence, and the other of
said primers being capable of hybridizing with the other of said
strands so as to form an extension product which contains the
target sequence as a template, said at least one pair of primers
being chosen from within the sequence defined in SEQ ID NO: 161; b)
synthesizing an extension product of each of said primers which
extension products contain the target sequence, and amplifying said
target sequence, if any, to a detectable level; and c) detecting
the presence and/or amount of said amplified target sequence as an
indication of a bacterial resistance to .beta.-lactam antibiotics
mediated by the bacterial antibiotic resistance gene TEM-1.
70. A method for evaluating a bacterial resistance to .beta.-lactam
antibiotics mediated by the bacterial antibiotic resistance gene
bla.sub.rob (ROB-1) directly from a test sample or from bacterial
colonies, which comprises the following steps: a) depositing and
fixing on an inert support or leaving in solution the bacterial DNA
of the sample or of a substantially homogenous population of
bacteria isolated from this sample, or inoculating said sample or
said substantially homogenous population of bacteria isolated from
this sample on an inert support, and lysing in situ said inoculated
sample or isolated bacteria to release the bacterial DNA, said
bacterial DNA being in a substantially single stranded form; b)
contacting said single stranded DNA with a probe, said probe
comprising at least one single stranded nucleic acid which
nucleotidic sequence is selected from the group consisting of SEQ
ID NO: 162, a sequence complementary thereof, a part thereof and a
variant thereof, which specifically anneals with said bacterial
antibiotic resistance gene coding for a .beta.-lactamase, under
conditions such that the nucleic acid of said probe can selectively
hybridize with said bacterial DNA, whereby a hybridization complex
is formed, said complex being detected by labelling means, the
label being present on said probe or the label being present on a
first reactive member of said labelling means, said first reactive
member reacting with a second reactive member present on said
probe; and c) detecting the presence or the intensity of said label
on said inert support or in said solution as an indication of a
bacterial resistance to .beta.-lactam antibiotics mediated by the
bacterial antibiotic resistance gene ROB-1.
71. A method as defined in claim 70, wherein said probe comprises
an oligonucleotide of at least 12 nucleotides in length which
hybridizes to SEQ ID NO: 162.
72. A method for evaluating a bacterial resistance to .beta.-lactam
antibiotics mediated by the bacterial antibiotic resistance gene
bla.sub.rob (ROB-1) in a test sample which comprises the following
steps: a) treating said sample with an aqueous solution containing
at least one pair of oligonucleotide primers having at least 12
nucleotides in length, one of said primers being capable of
hybridizing selectively with one of the two complementary strands
of said bacterial antibiotic resistance gene coding for a
.beta.-lactamase that contains a target sequence, and the other of
said primers being capable of hybridizing with the other of said
strands so as to form an extension product which contains the
target sequence as a template, said at least one pair of primers
being chosen from within the sequence defined in SEQ ID NO: 162; b)
synthesizing an extension product of each of said primers which
extension products contain the target sequence, and amplifying said
target sequence, if any, to a detectable level; and c) detecting
the presence and/or amount of said amplified target sequence as an
indication of a bacterial resistance to .beta.-lactam antibiotics
mediated by the bacterial antibiotic resistance gene ROB-1.
73. A method for evaluating a bacterial resistance to .beta.-lactam
antibiotics mediated by the bacterial antibiotic resistance gene
bla.sub.shv (SHV-1) directly from a test sample or from bacterial
colonies, which comprises the following steps: a) depositing and
fixing on an inert support or leaving in solution the bacterial DNA
of the sample or of a substantially homogenous population of
bacteria isolated from this sample, or inoculating said sample or
said substantially homogenous population of bacteria isolated from
this sample on an inert support, and lysing in situ said inoculated
sample or isolated bacteria to release the bacterial DNA, said
bacterial DNA being in a substantially single stranded form; b)
contacting said single stranded DNA with a probe, said probe
comprising at least one single stranded nucleic acid which
nucleotidic sequence is selected from the group consisting of SEQ
ID NO: 163, a sequence complementary thereof, a part thereof and a
variant thereof, which specifically anneals with said bacterial
antibiotic resistance gene coding for a .beta.-lactamase, under
conditions such that the nucleic acid of said probe can selectively
hybridize with said bacterial DNA, whereby a hybridization complex
is formed, said complex being detected by labelling means, the
label being present on said probe or the label being present on a
first reactive member of said labelling means, said first reactive
member reacting with a second reactive member present on said
probe; and c) detecting the presence or the intensity of said label
on said inert support or in said solution as an indication of a
bacterial resistance to .beta.-lactam antibiotics mediated by the
bacterial antibiotic resistance gene SHV-1.
74. A method as defined in claim 73, wherein said probe comprises
an oligonucleotide of at least 12 nucleotides in length which
hybridizes to SEQ ID NO: 163.
75. A method for evaluating a bacterial resistance to .beta.-lactam
antibiotics mediated by the bacterial antibiotic resistance gene
bla.sub.shv (SHV-1) in a test sample which comprises the following
steps: a) treating said sample with an aqueous solution containing
at least one pair of oligonucleotide primers having at least 12
nucleotides in length, one of said primers being capable of
hybridizing selectively with one of the two complementary strands
of said bacterial antibiotic resistance gene coding for a
.beta.-lactamase that contains a target sequence, and the other of
said primers being capable of hybridizing with the other of said
strands so as to form an extension product which contains the
target sequence as a template, said at least one pair of primers
being chosen from within the sequence defined in SEQ ID NO: 163; b)
synthesizing an extension product of each of said primers which
extension products contain the target sequence, and amplifying said
target sequence, if any, to a detectable level; and c) detecting
the presence and/or amount of said amplified target sequence as an
indication of a bacterial resistance to .beta.-lactam antibiotics
mediated by the bacterial antibiotic resistance gene SHV-1.
76. A method for evaluating a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aadB directly from a test sample or from bacterial
colonies, which comprises the following steps: a) depositing and
fixing on an inert support or leaving in solution the bacterial DNA
of the sample or of a substantially homogenous population of
bacteria isolated from this sample, or inoculating said sample or
said substantially homogenous population of bacteria isolated from
this sample on an inert support, and lysing in situ said inoculated
sample or isolated bacteria to release the bacterial DNA, said
bacterial DNA being in a substantially single stranded form; b)
contacting said single stranded DNA with a probe, said probe
comprising at least one single stranded nucleic acid which
nucleotidic sequence is selected from the group consisting of SEQ
ID NO: 164, a sequence complementary thereof, a part thereof and a
variant thereof, which specifically anneals with said bacterial
antibiotic resistance gene coding for an aminoglycoside
adenylyltransferase, under conditions such that the nucleic acid of
said probe can selectively hybridize with said bacterial DNA,
whereby a hybridization complex is formed, said complex being
detected by labelling means, the label being present on said probe
or the label being present on a first reactive member of said
labelling means, said first reactive member reacting with a second
reactive member present on said probe; and c) detecting the
presence or the intensity of said label on said inert support or in
said solution as an indication of a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aadB.
77. A method as defined in claim 76, wherein said probe comprises
an oligonucleotide of at least 12 nucleotides in length which
hybridizes to SEQ ID NO: 164.
78. A method for evaluating a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aadB in a test sample which comprises the following
steps: a) treating said sample with an aqueous solution containing
at least one pair of oligonucleotide primers having at least 12
nucleotides in length, one of said primers being capable of
hybridizing selectively with one of the two complementary strands
of said bacterial antibiotic resistance gene coding for an
aminoglycoside adenylyltransferase that contains a target sequence,
and the other of said primers being capable of hybridizing with the
other of said strands so as to form an extension product which
contains the target sequence as a template, said at least one pair
of primers being chosen from within the sequence defined in SEQ ID
NO: 164; b) synthesizing an extension product of each of said
primers which extension products contain the target sequence, and
amplifying said target sequence, if any, to a detectable level; and
c) detecting the presence and/or amount of said amplified target
sequence as an indication of a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aadB.
79. A method for evaluating a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aacC1 directly from a test sample or from bacterial
colonies, which comprises the following steps: a) depositing and
fixing on an inert support or leaving in solution the bacterial DNA
of the sample or of a substantially homogenous population of
bacteria isolated from this sample, or inoculating said sample or
said substantially homogenous population of bacteria isolated from
this sample on an inert support, and lysing in situ said inoculated
sample or isolated bacteria to release the bacterial DNA, said
bacterial DNA being in a substantially single stranded form; b)
contacting said single stranded DNA with a probe, said probe
comprising at least one single stranded nucleic acid which
nucleotidic sequence is selected from the group consisting of SEQ
ID NO: 165, a sequence complementary thereof, a part thereof and a
variant thereof, which specifically anneals with said bacterial
antibiotic resistance gene coding for an aminoglycoside
acetyltransferase, under conditions such that the nucleic acid of
said probe can selectively hybridize with said bacterial DNA,
whereby a hybridization complex is formed, said complex being
detected by labelling means, the label being present on said probe
or the label being present on a first reactive member of said
labelling means, said first reactive member reacting with a second
reactive member present on said probe; and c) detecting the
presence or the intensity of said label on said inert support or in
said solution as an indication of a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aacC1.
80. A method as defined in claim 79, wherein said probe comprises
an oligonucleotide of at least 12 nucleotides in length which
hybridizes to SEQ ID NO: 165.
81. A method for evaluating a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aaccl in a test sample which comprises the
following steps: a) treating said sample with an aqueous solution
containing at least one pair of oligonucleotide primers having at
least 12 nucleotides in length, one of said primers being capable
of hybridizing selectively with one of the two complementary
strands of said bacterial antibiotic resistance gene coding for an
aminoglycoside acetyltransferase that contains a target sequence,
and the other of said primers being capable of hybridizing with the
other of said strands so as to form an extension product which
contains the target sequence as a template, said at least one pair
of primers being chosen from within the sequence defined in SEQ ID
NO: 165; b) synthesizing an extension product of each of said
primers which extension products contain the target sequence, and
amplifying said target sequence, if any, to a detectable level; and
c) detecting the presence and/or amount of said amplified target
sequence as an indication of a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aacC1.
82. A method for evaluating a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aacC2 directly from a test sample or from bacterial
colonies, which comprises the following steps: a) depositing and
fixing on an inert support or leaving in solution the bacterial DNA
of the sample or of a substantially homogenous population of
bacteria isolated from this sample, or inoculating said sample or
said substantially homogenous population of bacteria isolated from
this sample on an inert support, and lysing in situ said inoculated
sample or isolated bacteria to release the bacterial DNA, said
bacterial DNA being in a substantially single stranded form; b)
contacting said single stranded DNA with a probe, said probe
comprising at least one single stranded nucleic acid which
nucleotidic sequence is selected from the group consisting of SEQ
ID NO: 166, a sequence complementary thereof, a part thereof and a
variant thereof, which specifically anneals with said bacterial
antibiotic resistance gene coding for an aminoglycoside
acetyltransferase, under conditions such that the nucleic acid of
said probe can selectively hybridize with said bacterial DNA,
whereby a hybridization complex is formed, said complex being
detected by labelling means, the label being present on said probe
or the label being present on a first reactive member of said
labelling means, said first reactive member reacting with a second
reactive member present on said probe; and c) detecting the
presence or the intensity of said label on said inert support or in
said solution as an indication of a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aacC2.
83. A method as defined in claim 82, wherein said probe comprises
an oligonucleotide of at least 12 nucleotides in length which
hybridizes to SEQ ID NO: 166.
84. A method for evaluating a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aacC2 in a test sample which comprises the
following steps: a) treating said sample with an aqueous solution
containing at least one pair of oligonucleotide primers having at
least 12 nucleotides in length, one of said primers being capable
of hybridizing selectively with one of the two complementary
strands of said bacterial antibiotic resistance gene coding for an
aminoglycoside acetyltransferase that contains a target sequence,
and the other of said primers being capable of hybridizing with the
other of said strands so as to form an extension product which
contains the target sequence as a template, said at least one pair
of primers being chosen from within the sequence defined in SEQ ID
NO: 166; b) synthesizing an extension product of each of said
primers which extension products contain the target sequence, and
amplifying said target sequence, if any, to a detectable level; and
c) detecting the presence and/or amount of said amplified target
sequence as an indication of a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aacC2.
85. A method for evaluating a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aacC3 directly from a test sample or from bacterial
colonies, which comprises the following steps: a) depositing and
fixing on an inert support or leaving in solution the bacterial DNA
of the sample or of a substantially homogenous population of
bacteria isolated from this sample, or inoculating said sample or
said substantially homogenous population of bacteria isolated from
this sample on an inert support, and lysing in situ said inoculated
sample or isolated bacteria to release the bacterial DNA, said
bacterial DNA being in a substantially single stranded form; b)
contacting said single stranded DNA with a probe, said probe
comprising at least one single stranded nucleic acid which
nucleotidic sequence is selected from the group consisting of SEQ
ID NO: 167, a sequence complementary thereof, a part thereof and a
variant thereof, which specifically anneals with said bacterial
antibiotic resistance gene coding for an aminoglycoside
acetyltransferase, under conditions such that the nucleic acid of
said probe can selectively hybridize with said bacterial DNA,
whereby a hybridization complex is formed, said complex being
detected by labelling means, the label being present on said probe
or the label being present on a first reactive member of said
labelling means, said first reactive member reacting with a second
reactive member present on said probe; and c) detecting the
presence or the intensity of said label on said inert support or in
said solution as an indication of a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aacC3.
86. A method as defined in claim 85, wherein said probe comprises
an oligonucleotide of at least 12 nucleotides in length which
hybridizes to SEQ ID NO: 167.
87. A method for evaluating a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aacC3 in a test sample which comprises the
following steps: a) treating said sample with an aqueous solution
containing at least one pair of oligonucleotide primers having at
least 12 nucleotides in length, one of said primers being capable
of hybridizing selectively with one of the two complementary
strands of said bacterial antibiotic resistance gene coding for an
aminoglycoside acetyltransferase that contains a target sequence,
and the other of said primers being capable of hybridizing with the
other of said strands so as to form an extension product which
contains the target sequence as a template, said at least one pair
of primers being chosen from within the sequence defined in SEQ ID
NO: 167; b) synthesizing an extension product of each of said
primers which extension products contain the target sequence, and
amplifying said target sequence, if any, to a detectable level; and
c) detecting the presence and/or amount of said amplified target
sequence as an indication of a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aacC3.
88. A method for evaluating a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aacA4 directly from a test sample or from bacterial
colonies, which comprises the following steps: a) depositing and
fixing on an inert support or leaving in solution the bacterial DNA
of the sample or of a substantially homogenous population of
bacteria isolated from this sample, or inoculating said sample or
said substantially homogenous population of bacteria isolated from
this sample on an inert support, and lysing in situ said inoculated
sample or isolated bacteria to release the bacterial DNA, said
bacterial DNA being in a substantially single stranded form; b)
contacting said single stranded DNA with a probe, said probe
comprising at least one single stranded nucleic acid which
nucleotidic sequence is selected from the group consisting of SEQ
ID NO: 168, a sequence complementary thereof, a part thereof and a
variant thereof, which specifically anneals with said bacterial
antibiotic resistance gene coding for an aminoglycoside
acetyltransferase, under conditions such that the nucleic acid of
said probe can selectively hybridize with said bacterial DNA,
whereby a hybridization complex is formed, said complex being
detected by labelling means, the label being present on said probe
or the label being present on a first reactive member of said
labelling means, said first reactive member reacting with a second
reactive member present on said probe; and c) detecting the
presence or the intensity of said label on said inert support or in
said solution as an indication of a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aacA4.
89. A method as defined in claim 88, wherein said probe comprises
an oligonucleotide of at least 12 nucleotides in length which
hybridizes to SEQ ID NO: 168.
90. A method for evaluating a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aacA4 in a test sample which comprises the
following steps: a) treating said sample with an aqueous solution
containing at least one pair of oligonucleotide primers having at
least 12 nucleotides in length, one of said primers being capable
of hybridizing selectively with one of the two complementary
strands of said bacterial antibiotic resistance gene coding for an
aminoglycoside acetyltransferase that contains a target sequence,
and the other of said primers being capable of hybridizing with the
other of said strands so as to form an extension product which
contains the target sequence as a template, said at least one pair
of primers being chosen from within the sequence defined in SEQ ID
NO: 168; b) synthesizing an extension product of each of said
primers which extension products contain the target sequence, and
amplifying said target sequence, if any, to a detectable level; and
c) detecting the presence and/or amount of said amplified target
sequence as an indication of a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aacA4.
91. A method for evaluating a bacterial resistance to .beta.-lactam
antibiotics mediated by the bacterial antibiotic resistance gene
mecA directly from a test sample or from bacterial colonies, which
comprises the following steps: a) depositing and fixing on an inert
support or leaving in solution the bacterial DNA of the sample or
of a substantially homogenous population of bacteria isolated from
this sample, or inoculating said sample or said substantially
homogenous population of bacteria isolated from this sample on an
inert support, and lysing in situ said inoculated sample or
isolated bacteria to release the bacterial DNA, said bacterial DNA
being in a substantially single stranded form; b) contacting said
single stranded DNA with a probe, said probe comprising at least
one single stranded nucleic acid which nucleotidic sequence is
selected from the group consisting of SEQ ID NO: 169, a sequence
complementary thereof, a part thereof and a variant thereof, which
specifically anneals with said bacterial antibiotic resistance gene
coding for a penicillin-binding protein, under conditions such that
the nucleic acid of said probe can selectively hybridize with said
bacterial DNA, whereby a hybridization complex is formed, said
complex being detected by labelling means, the label being present
on said probe or the label being present on a first reactive member
of said labelling means, said first reactive member reacting with a
second reactive member present on said probe; and c) detecting the
presence or the intensity of said label on said inert support or in
said solution as an indication of a bacterial resistance to
.beta.-lactam antibiotics mediated by the bacterial antibiotic
resistance gene mecA.
92. A method as defined in claim 91, wherein said probe comprises
an oligonucleotide of at least 12 nucleotides in length which
hybridizes to SEQ ID NO: 169.
93. A method for evaluating a bacterial resistance to .beta.-lactam
antibiotics mediated by the bacterial antibiotic resistance gene
mecA in a test sample which comprises the following steps: a)
treating said sample with an aqueous solution containing at least
one pair of oligonucleotide primers having at least 12 nucleotides
in length, one of said primers being capable of hybridizing
selectively with one of the two complementary strands of said
bacterial antibiotic resistance gene coding for a
penicillin-binding protein that contains a target sequence, and the
other of said primers being capable of hybridizing with the other
of said strands so as to form an extension product which contains
the target sequence as a template, said at least one pair of
primers being chosen from within the sequence defined in SEQ ID NO:
169; b) synthesizing an extension product of each of said primers
which extension products contain the target sequence, and
amplifying said target sequence, if any, to a detectable level; and
c) detecting the presence and/or amount of said amplified target
sequence as an indication of a bacterial resistance to
.beta.-lactam antibiotics mediated by the bacterial antibiotic
resistance gene mecA.
94. A method for evaluating a bacterial resistance to vancomycin
mediated by the bacterial antibiotic resistance genes vanH, vanA
and vanX directly from a test sample or from bacterial colonies,
which comprises the following steps: a) depositing and fixing on an
inert support or leaving in solution the bacterial DNA of the
sample or of a substantially homogenous population of bacteria
isolated from this sample, or inoculating said sample or said
substantially homogenous population of bacteria isolated from this
sample on an inert support, and lysing in situ said inoculated
sample or isolated bacteria to release the bacterial DNA, said
bacterial DNA being in a substantially single stranded form; b)
contacting said single stranded DNA with a probe, said probe
comprising at least one single stranded nucleic acid which
nucleotidic sequence is selected from the group consisting of SEQ
ID NO: 170, a sequence complementary thereof, a part thereof and a
variant thereof, which specifically anneals with said bacterial
antibiotic resistance genes coding for vancomycin-resistance
proteins, under conditions such that the nucleic acid of said probe
can selectively hybridize with said bacterial DNA, whereby a
hybridization complex is formed, said complex being detected by
labelling means, the label being present on said probe or the label
being present on a first reactive member of said labelling means,
said first reactive member reacting with a second reactive member
present on said probe; and c) detecting the presence or the
intensity of said label on said inert support or in said solution
as an indication of a bacterial resistance to vancomycin mediated
by the bacterial antibiotic resistance genes vanH, vanA and
vanX.
95. A method as defined in claim 94, wherein said probe comprises
an oligonucleotide of at least 12 nucleotides in length which
hybridizes to SEQ ID NO: 170.
96. A method for evaluating a bacterial resistance to vancomycin
mediated by the bacterial antibiotic resistance genes vanH, vanA
and vanX in a test sample which comprises the following steps: a)
treating said sample with an aqueous solution containing at least
one pair of oligonucleotide primers having at least 12 nucleotides
in length, one of said primers being capable of hybridizing
selectively with one of the two complementary strands of said
bacterial antibiotic resistance genes coding for
vancomycin-resistance proteins that contain a target sequence, and
the other of said primers being capable of hybridizing with the
other of said strands so as to form an extension product which
contains the target sequence as a template, said at least one pair
of primers being chosen from within the sequence defined in SEQ ID
NO: 170; b) synthesizing an extension product of each of said
primers which extension products contain the target sequence, and
amplifying said target sequence, if any, to a detectable level; and
c) detecting the presence and/or amount of said amplified target
sequence as an indication of a bacterial resistance to vancomycin
mediated by the bacterial antibiotic resistance genes vanH, vanA
and vanX.
97. A method for evaluating a bacterial resistance to streptogramin
A mediated by the bacterial antibiotic resistance gene satA
directly from a test sample or from bacterial colonies, which
comprises the following steps: a) depositing and fixing on an inert
support or leaving in solution the bacterial DNA of the sample or
of a substantially homogenous population of bacteria isolated from
this sample, or inoculating said sample or said substantially
homogenous population of bacteria isolated from this sample on an
inert support, and lysing in situ said inoculated sample or
isolated bacteria to release the bacterial DNA, said bacterial DNA
being in a substantially single stranded form; b) contacting said
single stranded DNA with a probe, said probe comprising at least
one single stranded nucleic acid which nucleotidic sequence is
selected from the group consisting of SEQ ID NO: 173, a sequence
complementary thereof, a part thereof and a variant thereof, which
specifically anneals with said bacterial antibiotic resistance gene
coding for a streptogramin A acetyltransferase, under conditions
such that the nucleic acid of said probe can selectively hybridize
with said bacterial DNA, whereby a hybridization complex is formed,
said complex being detected by labelling means, the label being
present on said probe or the label being present on a first
reactive member of said labelling means, said first reactive member
reacting with a second reactive member present on said probe; and
c) detecting the presence or the intensity of said label on said
inert support or in said solution as an indication of a bacterial
resistance to streptogramin A mediated by the bacterial antibiotic
resistance gene satA.
98. A method as defined in claim 97, wherein said probe comprises
an oligonucleotide of at least 12 nucleotides in length which
hybridizes to SEQ ID NO: 173.
99. A method for evaluating a bacterial resistance to streptogramin
A mediated by the bacterial antibiotic resistance gene satA in a
test sample which comprises the following steps: a) treating said
sample with an aqueous solution containing at least one pair of
oligonucleotide primers having at least 12 nucleotides in length,
one of said primers being capable of hybridizing selectively with
one of the two complementary strands of said bacterial antibiotic
resistance gene coding for streptogramin A acetyltransferase that
contains a target sequence, and the other of said primers being
capable of hybridizing with the other of said strands so as to form
an extension product which contains the target sequence as a
template, said at least one pair of primers being chosen from
within the sequence defined in SEQ ID NO: 173; b) synthesizing an
extension product of each of said primers which extension products
contain the target sequence, and amplifying said target sequence,
if any, to a detectable level; and c) detecting the presence and/or
amount of said amplified target sequence as an indication of a
bacterial resistance to streptogramin A mediated by the bacterial
antibiotic resistance gene satA.
100. A method for evaluating a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aacA-aphD directly from a test sample or from
bacterial colonies, which comprises the following steps: a)
depositing and fixing on an inert support or leaving in solution
the bacterial DNA of the sample or of a substantially homogenous
population of bacteria isolated from this sample, or inoculating
said sample or said substantially homogenous population of bacteria
isolated from this sample on an inert support, and lysing in situ
said inoculated sample or isolated bacteria to release the
bacterial DNA, said bacterial DNA being in a substantially single
stranded form; b) contacting said single stranded DNA with a probe,
said probe comprising at least one single stranded nucleic acid
which nucleotidic sequence is selected from the group consisting of
SEQ ID NO: 174, a sequence complementary thereof, a part thereof
and a variant thereof, which specifically anneals with said
bacterial antibiotic resistance gene coding for an aminoglycoside
acetyltransferase-phosphotra- nsferase under conditions such that
the nucleic acid of said probe can selectively hybridize with said
bacterial DNA, whereby a hybridization complex is formed, said
complex being detected by labelling means, the label being present
on said probe or the label being present on a first reactive member
of said labelling means, said first reactive member reacting with a
second reactive member present on said probe; and c) detecting the
presence or the intensity of said label on said inert support or in
said solution as an indication of a bacterial resistance to
aminoglycoside antibiotics mediated by the bacterial antibiotic
resistance gene aacA-aphD.
101. A method as defined in claim 100, wherein said probe comprises
an oligonucleotide of at least 12 nucleotides in length which
hybridizes to SEQ ID NO: 174.
102. A method for evaluating a bacterial resistance to
aminoglycoside; antibiotics mediated by the bacterial antibiotic
resistance gene aacA-aphD in a test sample which comprises the
following steps: a) treating said sample with an aqueous solution
containing at least one pair of oligonucleotide primers having at
least 12 nucleotides in length, one of said primers being capable
of hybridizing selectively with one of the two complementary
strands of said bacterial antibiotic resistance gene coding for an
aminoglycoside acetyltransferase-phosphotransferase that contains a
target sequence, and the other of said primers being capable of
hybridizing with the other of said strands so as to form an
extension product which contains the target sequence as a template,
said at least one pair of primers being chosen from within the
sequence defined in SEQ ID NO: 174; b) synthesizing an extension
product of each of said primers which extension products contain
the target sequence, and amplifying said target sequence, if any,
to a detectable level; and c) detecting the presence and/or amount
of said amplified target sequence as an indication of a bacterial
resistance to aminoglycoside antibiotics mediated by the bacterial
antibiotic resistance gene aacA-aphD.
103. A method for evaluating a bacterial resistance to
virginiamycin mediated by the bacterial antibiotic resistance gene
vat directly from a test sample or from bacterial colonies, which
comprises the following steps: a) depositing and fixing on an inert
support or leaving in solution the bacterial DNA of the sample or
of a substantially homogenous population of bacteria isolated from
this sample, or inoculating said sample or said substantially
homogenous population of bacteria isolated from this sample on an
inert support, and lysing in situ said inoculated sample or
isolated bacteria to release the bacterial DNA, said bacterial DNA
being in a substantially single stranded form; b) contacting said
single stranded DNA with a probe, said probe comprising at least
one single stranded nucleic acid which nucleotidic sequence is
selected from the group consisting of SEQ ID NO: 175, a sequence
complementary thereof, a part thereof and a variant thereof, which
specifically anneals with said bacterial antibiotic resistance gene
coding for a virginiamycin acetyltransferase, under conditions such
that the nucleic acid of said probe can selectively hybridize with
said bacterial DNA, whereby a hybridization complex is formed, said
complex being detected by labelling means, the label being present
on said probe or the label being present on a first reactive member
of said labelling means, said first reactive member reacting with a
second reactive member present on said probe; and c) detecting the
presence or the intensity of said label on said inert support or in
said solution as an indication of a bacterial resistance to
virginiamycin mediated by the bacterial antibiotic resistance gene
vat.
104. A method as defined in claim 103, wherein said probe comprises
an oligonucleotide of at least 12 nucleotides in length which
hybridizes to SEQ ID NO: 175.
105. A method for evaluating a bacterial resistance to
virginiamycin mediated by the bacterial antibiotic resistance gene
vat in a test sample which comprises the following steps: a)
treating said sample with an aqueous solution containing at least
one pair of oligonucleotide primers having at least 12 nucleotides
in length, one of said primers being capable of hybridizing
selectively with one of the two complementary strands of said
bacterial antibiotic resistance gene coding for a virginiamycin
acetyltransferase that contains a target sequence, and the other of
said primers being capable of hybridizing with the other of said
strands so as to form an extension product which contains the
target sequence as a template, said at least one pair of primers
being chosen from within the sequence defined in SEQ ID NO: 175; b)
synthesizing an extension product of each of said primers which
extension products contain the target sequence, and amplifying said
target sequence, if any, to a detectable level; and c) detecting
the presence and/or amount of said amplified target sequence as an
indication of a bacterial resistance to virginiamycin mediated by
the bacterial antibiotic resistance gene vat.
106. A method for evaluating a bacterial resistance to
virginiamycin mediated by the bacterial antibiotic resistance gene
vga directly from a test sample or from bacterial colonies, which
comprises the following steps: a) depositing and fixing on an inert
support or leaving in solution the bacterial DNA of the sample or
of a substantially homogenous population of bacteria isolated from
this sample, or inoculating said sample or said substantially
homogenous population of bacteria isolated from this sample on an
inert support, and lysing in situ said inoculated sample or
isolated bacteria to release the bacterial DNA, said bacterial DNA
being in a substantially single stranded form; b) contacting said
single stranded DNA with a probe, said probe comprising at least
one single stranded nucleic acid which nucleotidic sequence is
selected from the group consisting of SEQ ID NO: 176, a sequence
complementary thereof, a part thereof and a variant thereof, which
specifically anneals with said bacterial antibiotic resistance gene
coding for an ATP-binding protein, under conditions such that the
nucleic acid of said probe can selectively hybridize with said
bacterial DNA, whereby a hybridization complex is formed, said
complex being detected by labelling means, the label being present
on said probe or the label being present on a first reactive member
of said labelling means, said first reactive member reacting with a
second reactive member present on said probe; and c) detecting the
presence or the intensity of said label on said inert support or in
said solution as an indication of a bacterial resistance to
virginiamycin mediated by the bacterial antibiotic resistance gene
vga.
107. A method as defined in claim 106, wherein said probe comprises
an oligonucleotide of at least 12 nucleotides in length which
hybridizes to SEQ ID NO: 176.
108. A method for evaluating a bacterial resistance to
virginiamycin mediated by the bacterial antibiotic resistance gene
vga in a test sample which comprises the following steps: a)
treating said sample with an aqueous solution containing at least
one pair of oligonucleotide primers having at least 12 nucleotides
in length, one of said primers being capable of hybridizing
selectively with one of the two complementary strands of said
bacterial antibiotic resistance gene coding for an ATP-binding
protein that contains a target sequence, and the other of said
primers being capable of hybridizing with the other of said strands
so as to form an extension product which contains the target
sequence as a template, said at least one pair of primers being
chosen from within the sequence defined in SEQ ID NO: 176; b)
synthesizing an extension product of each of said primers which
extension products contain the target sequence, and amplifying said
target sequence, if any, to a detectable level; and c) detecting
the presence and/or amount of said amplified target sequence as an
indication of a bacterial resistance to virginiamycin mediated by
the bacterial antibiotic resistance gene vga.
109. A method for evaluating a bacterial resistance to erythromycin
mediated by the bacterial antibiotic resistance gene msrA directly
from a test sample or from bacterial colonies, which comprises the
following steps: a) depositing and fixing on an inert support or
leaving in solution the bacterial DNA of the sample or of a
substantially homogenous population of bacteria isolated from this
sample, or inoculating said sample or said substantially homogenous
population of bacteria isolated from this sample on an inert
support, and lysing in situ said inoculated sample or isolated
bacteria to release the bacterial DNA, said bacterial DNA being in
a substantially single stranded form; b) contacting said single
stranded DNA with a probe, said probe comprising at least one
single stranded nucleic acid which nucleotidic sequence is selected
from the group consisting of SEQ ID NO: 177, a sequence
complementary thereof, a part thereof and a variant thereof, which
specifically anneals with said bacterial antibiotic resistance gene
coding for an erythromycin resistance protein under conditions such
that the nucleic acid of said probe can selectively hybridize with
said bacterial DNA, whereby a hybridization complex is formed, said
complex being detected by labelling means, the label being present
on said probe or the label being present on a first reactive member
of said labelling means, said first reactive member reacting with a
second reactive member present on said probe; and c) detecting the
presence or the intensity of said label on said inert support or in
said solution as an indication of a bacterial resistance to
erythromycin mediated by the bacterial antibiotic resistance gene
msrA.
110. A method as defined in claim 109, wherein said probe comprises
an oligonucleotide of at least 12 nucleotides in length which
hybridizes to SEQ ID NO: 177.
111. A method for evaluating a bacterial resistance to erythromycin
mediated by the bacterial antibiotic resistance gene msrA in a test
sample which comprises the following steps: a) treating said sample
with an aqueous solution containing at least one pair of
oligonucleotide primers having at least 12 nucleotides in length,
one of said primers being capable of hybridizing selectively with
one of the two complementary strands of said bacterial antibiotic
resistance gene coding for an erythromycin resistance protein that
contains a target sequence, and the other of said primers being
capable of hybridizing with the other of said strands so as to form
an extension product which contains the target sequence as a
template, said at least one pair of primers being chosen from
within the sequence defined in SEQ ID NO: 177; b) synthesizing an
extension product of each of said primers which extension products
contain the target sequence, and amplifying said target sequence,
if any, to a detectable level; and c) detecting the presence and/or
amount of said amplified target sequence as an indication of a
bacterial resistance to erythromycin mediated by the bacterial
antibiotic resistance gene msrA.
112. A method for evaluating potential bacterial resistance to
.beta.-lactams, aminoglycosides, chloramphenicol and/or
trimethoprim mediated by the bacterial antibiotic resistance gene
int directly from a test sample or from bacterial colonies, which
comprises the following steps: a) depositing and fixing on an inert
support or leaving in solution the bacterial DNA of the sample or
of a substantially homogenous population of bacteria isolated from
this sample, or inoculating said sample or said substantially
homogenous population of bacteria isolated from this sample on an
inert support, and lysing in situ said inoculated sample or
isolated bacteria to release the bacterial DNA, said bacterial DNA
being in a substantially single stranded form; b) contacting said
single stranded DNA with a probe, said probe comprising at least
one single stranded nucleic acid which nucleotidic sequence is
selected from the group consisting of SEQ ID NO: 171, a sequence
complementary thereof, a part thereof and a variant thereof, which
specifically anneals with said bacterial antibiotic resistance gene
coding for an integrase, under conditions such that the nucleic
acid of said probe can selectively hybridize with said bacterial
DNA, whereby a hybridization complex is formed, said complex being
detected by labelling means, the label being present on said probe
or the label being present on a first reactive member of said
labelling means, said first reactive member reacting with a second
reactive member present on said probe; and c) detecting the
presence or the intensity of said label on said inert support or in
said solution as an indication of potential bacterial resistance to
.beta.-lactams, aminoglycosides, chloramphenicol and/or
trimethoprim mediated by the bacterial antibiotic resistance gene
int.
113. A method as defined in claim 112, wherein said probe comprises
an oligonucleotide of at least 12 nucleotides in length which
hybridizes to SEQ ID NO: 171.
114. A method for evaluating potential bacterial resistance to
.beta.-lactams, aminoglycosides, chloramphenicol and/or
trimethoprim mediated by the bacterial antibiotic resistance gene
int in a test sample which comprises the following steps: a)
treating said sample with an aqueous solution containing at least
one pair of oligonucleotide primers having at least 12 nucleotides
in length, one of said primers being capable of hybridizing
selectively with one of the two complementary strands of said
bacterial antibiotic resistance gene coding for an integrase that
contains a target sequence, and the other of said primers being
capable of hybridizing with the other of said strands so as to form
an extension product which contains the target sequence as a
template, said at least one pair of primers being chosen from
within the sequence defined in SEQ ID NO: 171; b) synthesizing an
extension product of each of said primers which extension products
contain the target sequence, and amplifying said target sequence,
if any, to a detectable level; and c) detecting the presence and/or
amount of said amplified target sequence as an indication of
potential bacterial resistance to .beta.-lactams, aminoglycosides,
chloramphenicol and/or trimethoprim mediated by the bacterial
antibiotic resistance gene int.
115. A method for evaluating potential bacterial resistance to
.beta.-lactams, aminoglycosides, chloramphenicol and/or
trimethoprim mediated by the bacterial antibiotic resistance gene
sul directly from a test sample or from bacterial colonies, which
comprises the following steps: a) depositing and fixing on an inert
support or leaving in solution the bacterial DNA of the sample or
of a substantially homogenous population of bacteria isolated from
this sample, or inoculating said sample or said substantially
homogenous population of bacteria isolated from this sample on an
inert support, and lysing in situ said inoculated sample or
isolated bacteria to release the bacterial DNA, said bacterial DNA
being in a substantially single stranded form; b) contacting said
single stranded DNA with a probe, said probe comprising at least
one single stranded nucleic acid which nucleotidic sequence is
selected from the group consisting of SEQ ID NO: 172, a sequence
complementary thereof, a part thereof and a variant thereof, which
specifically anneals with said bacterial antibiotic resistance gene
coding for a sulfonamide resistance protein under conditions such
that the nucleic acid of said probe can selectively hybridize with
said bacterial DNA, whereby a hybridization complex is formed, said
complex being detected by labelling means, the label being present
on said probe or the label being present on a first reactive member
of said labelling means, said first reactive member reacting with a
second reactive member present on said probe; and c) detecting the
presence or the intensity of said label on said inert support or in
said solution as an indication of potential bacterial resistance to
.beta.-lactams, aminoglycosides, chloramphenicol and/or
trimethoprim mediated by the bacterial antibiotic resistance gene
sul.
116. A method as defined in claim 115, wherein said probe comprises
an oligonucleotide of at least 12 nucleotides in length which
hybridizes to SEQ ID NO: 172.
117. A method for evaluating potential bacterial resistance to
.beta.-lactams, aminoglycosides, chloramphenicol and/or
trimethoprim mediated by the bacterial antibiotic resistance gene
sul in a test sample which comprises the following steps: a)
treating said sample with an aqueous solution containing at least
one pair of oligonucleotide primers having at least 12 nucleotides
in length, one of said primers being capable of hybridizing
selectively with one of the two complementary strands of said
bacterial antibiotic resistance gene coding for a sulfonamide
resistance protein that contains a target sequence, and the other
of said primers being capable of hybridizing with the other of said
strands so as to form an extension product which contains the
target sequence as a template, said at least one pair of primers
being chosen from within the sequence defined in SEQ ID NO: 172; b)
synthesizing an extension product of each of said primers which
extension products contain the target sequence, and amplifying said
target sequence, if any, to a detectable level; and c) detecting
the presence and/or amount of said amplified target sequence as an
indication of potential bacterial resistance to .beta.-lactams,
aminoglycosides, chloramphenicol and/or trimethoprim mediated by
the bacterial antibiotic resistance gene sul.
118. A nucleic acid having the nucleotide sequence of any one of
SEQ ID NOs: 1 to 37, SEQ ID NOs: 161 to 177, a part thereof and
variants thereof which, when in single stranded form, ubiquitously
and specifically hybridize with a target bacterial DNA as a probe
or as a primer.
119. An oligonucleotide having a nucleotidic sequence of any one of
SEQ ID NOs: 38 to 160.
120. A recombinant plasmid comprising a nucleic acid as defined in
claim 118.
121. A recombinant host which has been transformed by a recombinant
plasmid according to claim 120.
122. A recombinant host according to claim 121 wherein said host is
Escherichia coli.
123. A diagnostic kit for the detection and/or quantification of
the nucleic acids of any combination of the bacterial species
defined in any one of claims 9, 14, 19, 24, 29, 34, 39, 43, 47, 52,
57 and 61, comprising any combination of probes defined
therein.
124. A diagnostic kit for the detection and/or quantification of
the nucleic acids of any combination of the bacterial species
defined in any one of claims 10, 11, 15, 16, 20, 21, 25, 26, 30,
31, 35, 36, 40, 44, 48, 49, 53, 54, 58, 62 and 65, comprising any
combination of oligonucleotide probes defined therein.
125. A diagnostic kit for the detection and/or quantification of
the nucleic acids of any combination of the bacterial species
defined in any one of claims 12, 13, 17, 18, 22, 23, 27, 28, 32,
33, 37, 38, 41, 42, 45, 46, 50, 51, 55, 56, 59, 60, 63, 64 and 66
comprising any combination of primers defined therein.
126. A diagnostic kit for the detection and/or quantification of
the nucleic acids of any combination of the bacterial resistance
genes defined in any one of claims 67, 70, 73, 76, 79, 82, 85, 88,
91, 94, 97, 100, 103, 106 and 109 comprising any combination of
probes defined therein.
127. A diagnostic kit for the detection and/or quantification of
the nucleic acids of any combination of the bacterial resistance
genes defined in any one of claims 68, 71, 74, 77, 80, 83, 86, 89,
92, 95, 98, 101, 104, 107 and 110 comprising any combination of
oligonucleotide probes defined therein.
128. A diagnostic kit for the detection and/or quantification of
the nucleic acids of any combination of the bacterial resistance
genes defined in any one of claims 69, 72, 75, 78, 81, 84, 87, 90,
93, 96, 99, 102, 105, 108 and 111 comprising any combination of
primers defined therein.
129. A diagnostic kit for the simultaneous detection and
quantification of nucleic acids of any combination of the bacterial
species defined in claim 123, comprising any combination of the
bacterial probes defined therein and any combination of the probes
to the antibiotic resistance genes defined in any one of SEQ ID
NOs: 161 to 177 in whole or in part.
130. A diagnostic kit for the simultaneous detection and
quantification of nucleic acids of any combination of the bacterial
species defined in claim 124, comprising any combination of the
bacterial oligonucleotide probes defined therein and any
combination of oligonucleotide probes that hybridize to the
antibiotic resistance genes defined in any one of SEQ ID NOs: 161
to 177.
131. A diagnostic kit for the simultaneous detection and
quantification of nucleic acids of any combination of the bacterial
species defined in claim 125, comprising any combination of the
primers defined therein and any combination of primers that anneal
to the antibiotic resistance genes defined in any one of SEQ ID
NOs: 161 to 177.
Description
BACKGROUND OF THE INVENTION
[0001] Classical Identification of Bacteria
[0002] Bacteria are classically identified by their ability to
utilize different substrates as a source of carbon and nitrogen
through the use of biochemical tests such as the API20E.TM. system.
Susceptibility testing of Gram negative bacilli has progressed to
microdilution tests. Although the API and the microdilution systems
are cost-effective, at least two days are required to obtain
preliminary results due to the necessity of two successive
overnight incubations to isolate and identify the bacteria from the
specimen. Some faster detection methods with sophisticated and
expensive apparatus have been developed. For example, the fastest
identification system, the autoSCAN-Walk-Away system.TM. identifies
both Gram negative and Gram positive from isolated bacterial
colonies in 2 hours and susceptibility patterns to antibiotics in
only 7 hours. However, this system has an unacceptable margin of
error, especially with bacterial species other than
Enterobacteriaceae (York et al., 1992. J. Clin. Microbiol.
30:2903-2910). Nevertheless, even this fastest method requires
primary isolation of the bacteria as a pure culture, a process
which takes at least 18 hours if there is a pure culture or 2 to 3
days if there is a mixed culture.
[0003] Urine Specimens
[0004] A large proportion (40-50%) of specimens received in routine
diagnostic microbiology laboratories for bacterial identification
are urine specimens (Pezzlo, 1988, Clin. Microbiol. Rev.
1:268-280). Urinary tract infections (UTI) are extremely common and
affect up to 20% of women and account for extensive morbidity and
increased mortality among hospitalized patients (Johnson and Stamm,
1989; Ann. Intern. Med. 111:906-917). UTI are usually of bacterial
etiology and require antimicrobial therapy. The Gram negative
bacillus Escherichia coli is by far the most prevalent urinary
pathogen and accounts for 50 to 60% of UTI (Pezzlo, 1988, op.
cit.). The prevalence for bacterial pathogens isolated from urine
specimens observed recently at the "Centre Hospitalier de
1'Universit Laval (CHUL)" is given in Tables 1 and 2.
[0005] Conventional pathogen identification in urine specimens. The
search for pathogens in urine specimens is so preponderant in the
routine microbiology laboratory that a myriad of tests have been
developed. The gold standard is still the classical
semi-quantitative plate culture method in which a calibrated loop
of urine is streaked on plates and incubated for 18-24 hours.
Colonies are then counted to determine the total number of colony
forming units (CFU) per liter of urine. A bacterial UTI is normally
associated with a bacterial count of .gtoreq.10.sup.7 CFU/L in
urine. However, infections with less than 10.sup.7 CFU/L in urine
are possible, particularly in patients with a high incidence of
diseases or those catheterized (Stark and Maki, 1984, N. Engl. J.
Med. 311:560-564). Importantly, close to 80% of urine specimens
tested are considered negative (<10.sup.7 CFU/L; Table 3).
[0006] Accurate and rapid urine screening methods for bacterial
pathogens would allow a faster identification of negative results
and a more efficient clinical investigation of the patient. Several
rapid identification methods (Uriscreen.TM., UTIscreen.TM., Flash
Track.TM. DNA probes and others) were recently compared to slower
standard biochemical methods which are based on culture of the
bacterial pathogens. Although much faster, these rapid tests showed
low sensitivities and specificities as well as a high number of
false negative and false positive results (Koening et al., 1992. J.
Clin. Microbiol. 30:342-345; Pezzlo et al., 1992. J. Clin.
Microbiol. 30:640-684).
[0007] Urine specimens found positive by culture are further
characterized using standard biochemical tests to identify the
bacterial pathogen and are also tested for susceptibility to
antibiotics.
[0008] Any Clinical Specimens
[0009] As with urine specimen which was used here as an example,
our probes and amplification primers are also applicable to any
other clinical specimens. The DNA-based tests proposed in this
invention are superior to standard methods currently used for
routine diagnosis in terms of rapidity and accuracy. While a high
percentage of urine specimens are negative, in many other clinical
specimens more than 95% of cultures are negative (Table 4). These
data further support the use of universal probes to screen out the
negative clinical specimens. Clinical specimens from organisms
other than humans (e.g. other primates, mammals, farm animals or
live stocks) may also be used.
[0010] Towards the Development of Rapid DNA-Based Diagnostic
Tests
[0011] A rapid diagnostic test should have a significant impact on
the management of infections. For the identification of pathogens
and antibiotic resistance genes in clinical samples, DNA probe and
DNA amplification technologies offer several advantages over
conventional methods. There is no need for subculturing, hence the
organism can be detected directly in clinical samples thereby
reducing the costs and time associated with isolation of pathogens.
DNA-based technologies have proven to be extremely useful for
specific applications in the clinical microbiology laboratory. For
example, kits for the detection of fastidious organisms based on
the use of hybridization probes or DNA amplification for the direct
detection of pathogens in clinical specimens are commercially
available (Persing et al, 1993. Diagnostic Molecular Microbiology:
Principles and Applications, American Society for Microbiology,
Washington, D.C.).
[0012] The present invention is an advantageous alternative to the
conventional culture identification methods used in hospital
clinical microbiology laboratories and in private clinics for
routine diagnosis. Besides being much faster, DNA-based diagnostic
tests are more accurate than standard biochemical tests presently
used for diagnosis because the bacterial genotype (e.g. DNA level)
is more stable than the bacterial phenotype (e.g. biochemical
properties). The originality of this invention is that genomic DNA
fragments (size of at least 100 base pairs) specific for 12 species
of commonly encountered bacterial pathogens were selected from
genomic libraries or from data banks. Amplification primers or
oligonucleotide probes (both less than 100 nucleotides in length)
which are both derived from the sequence of species-specific DNA
fragments identified by hybridization from genomic libraries or
from selected data bank sequences are used as a basis to develop
diagnostic tests. Oligonucleotide primers and probes for the
detection of commonly encountered and clinically important
bacterial resistance genes are also included. For example, Annexes
I and II present a list of suitable oligonucleotide probes and PCR
primers which were all derived from the species-specific DNA
fragments selected from genomic libraries or from data bank
sequences. It is clear to the individual skilled in the art that
oligonucleotide sequences appropriate for the specific detection of
the above bacterial species other than those listed in Annexes 1
and 2 may be derived from the species-specific fragments or from
the selected data bank sequences. For example, the oligonucleotides
may be shorter or longer than the ones we have chosen and may be
selected anywhere else in the identified species-specific sequences
or selected data bank sequences. Alternatively, the
oligonucleotides may be designed for use in amplification methods
other than PCR. Consequently, the core of this invention is the
identification of species-specific genomic DNA fragments from
bacterial genomic DNA libraries and the selection of genomic DNA
fragments from data bank sequences which are used as a source of
species-specific and ubiquitous oligonucleotides. Although the
selection of oligonucleotides suitable for diagnostic purposes from
the sequence of the species-specific fragments or from the selected
data bank sequences requires much effort it is quite possible for
the individual skilled in the art to derive from our fragments or
selected data bank sequences suitable oligonucleotides which are
different from the ones we have selected and tested as examples
(Annexes I and II).
[0013] Others have developed DNA-based tests for the detection and
identification of some of the bacterial pathogens for which we have
identified species-specific sequences (PCT patent application
Serial No. WO 93/03186). However, their strategy was based on the
amplification of the highly conserved 16S rRNA gene followed by
hybridization with internal species-specific oligonucleotides. The
strategy from this invention is much simpler and more rapid because
it allows the direct amplification of species-specific targets
using oligonucleotides derived from the species-specific bacterial
genomic DNA fragments.
[0014] Since a high percentage of clinical specimens are negative,
oligonucleotide primers and probes were selected from the highly
conserved 16S or 23S rRNA genes to detect all bacterial pathogens
possibly encountered in clinical specimens in order to determine
whether a clinical specimen is infected or not. This strategy
allows rapid screening out of the numerous negative clinical
specimens submitted for bacteriological testing.
[0015] We are also developing other DNA-based tests, to be
performed simultaneously with bacterial identification, to
determine rapidly the putative bacterial susceptibility to
antibiotics by targeting commonly encountered and clinically
relevant bacterial resistance genes. Although the sequences from
the selected antibiotic resistance genes are available and have
been used to develop DNA-based tests for their detection (Ehrlich
and Greenberg, 1994. PCR-based Diagnostics in Infectious Diseases,
Blackwell Scientific Publications, Boston, Mass.; Persing et al,
1993. Diagnostic Molecular Microbiology: Principles and
Applications, American Society for Microbiology, Washington, D.C.),
our approch is innovative as it represents major improvements over
current "gold standard" diagnostic methods based on culture of the
bacteria because it allows the rapid identification of the presence
of a specific bacterial pathogen and evaluation of its
susceptibility to antibiotics directly from the clinical specimens
within one hour.
[0016] We believe that the rapid and simple diagnostic tests not
based on cultivation of the bacteria that we are developing will
gradually replace the slow conventional bacterial identification
methods presently used in hospital clinical microbiology
laboratories and in private clinics. In our opinion, these rapid
DNA-based diagnostic tests for severe and common bacterial
pathogens and antibiotic resistance will (i) save lives by
optimizing treatment, (ii) diminish antibiotic resistance by
reducing the use of broad spectrum antibiotics and (iii) decrease
overall health costs by preventing or shortening
hospitalizations.
SUMMARY OF THE INVENTION
[0017] In accordance with the present invention, there is provided
sequence from genomic DNA fragments (size of at least 100 base
pairs and all described in the sequence listing) selected either by
hybridization from genomic libraries or from data banks and which
are specific for the detection of commonly encountered bacterial
pathogens (i.e. Escherichia coli, Klebsiella pneumoniae,
Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus
pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis,
Enterococcus faecalis, Staphylococcus saprophyticus, Streptococcus
pyogenes, Haemophilus influenzae and Moraxella catarrhalis) in
clinical specimens. These bacterial species are associated with
approximately 90% of urinary tract infections and with a high
percentage of other severe infections including septicemia,
meningitis, pneumonia, intraabdominal infections, skin infections
and many other severe respiratory tract infections. Overall, the
above bacterial species may account for up to 80% of bacterial
pathogens isolated in routine microbiology laboratories.
[0018] Synthetic oligonucleotides for hybridization (probes) or DNA
amplification (primers) were derived from the above
species-specific DNA fragments (ranging in sizes from 0.25 to 5.0
kilobase pairs (kbp)) or from selected data bank sequences (GenBank
and EMBL). Bacterial species for which some of the oligonucleotide
probes and amplification primers were derived from selected data
bank sequences are Escherichia coli, Enterococcus faecalis,
Streptococcus pyogenes and Pseudomonas aeruginosa. The person
skilled in the art understands that the important innovation in
this invention is the identification of the species-specific DNA
fragments selected either from bacterial genomic libraries by
hybridization or from data bank sequences. The selection of
oligonucleotides from these fragments suitable for diagnostic
purposes is also innovative. Specific and ubiquitous
oligonucleotides different from the ones tested in the practice are
considered as embodiments of the present invention.
[0019] The development of hybridization (with either fragment or
oligonucleotide probes) or of DNA amplification protocols for the
detection of pathogens from clinical specimens renders possible a
very rapid bacterial identification. This will greatly reduce the
time currently required for the identification of pathogens in the
clinical laboratory since these technologies can be applied for
bacterial detection and identification directly from clinical
specimens with minimum pretreatment of any biological specimens to
release bacterial DNA. In addition to being 100% specific, probes
and amplification primers allow identification of the bacterial
species directly from clinical specimens or, alternatively, from an
isolated colony. DNA amplification assays have the added advantages
of being faster and more sensitive than hybridization assays, since
they allow rapid and exponential in vitro replication of the target
segment of DNA from the bacterial genome. Universal probes and
amplification primers selected from the 16S or 23S rRNA genes
highly conserved among bacteria, which permit the detection of any
bacterial pathogens, will serve as a procedure to screen out the
numerous negative clinical specimens received in diagnostic
laboratories. The use of oligonucleotide probes or primers
complementary to characterized bacterial genes encoding resistance
to antibiotics to identify commonly encountered and clinically
important resistance genes is also under the scope of this
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0020] Development of Species-Specific DNA Probes
[0021] DNA fragment probes were developed for the following
bacterial species: Escherichia coli, Klebsiella pneumoniae,
Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus
pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis,
Staphylococcus saprophyticus, Haemophilus influenzae and Moraxella
catarrhalis. (For Enterococcus faecalis and Streptococcus pyogenes,
oligonucleotide sequences were exclusively derived from selected
data bank sequences). These species-specific fragments were
selected from bacterial genomic libraries by hybridization to DNA
from a variety of Gram positive and Gram negative bacterial species
(Table 5).
[0022] The chromosomal DNA from each bacterial species for which
probes were seeked was isolated using standard methods. DNA was
digested with a frequently cutting restriction enzyme such as
Sau3AI and then ligated into the bacterial plasmid vector pGEM3Zf
(Promega) linearized by appropriate restriction endonuclease
digestion. Recombinant plasmids were then used to transform
competent E. coli strain DH5.alpha. thereby yielding a genomic
library. The plasmid content of the transformed bacterial cells was
analyzed using standard methods. DNA fragments of target bacteria
ranging in size from 0.25 to 5.0 kilobase pairs (kbp) were cut out
from the vector by digestion of the recombinant plasmid with
various restriction endonucleases. The insert was separated from
the vector by agarose gel electrophoresis and purified in low
melting point agarose gels. Each of the purified fragments of
bacterial genomic DNA was then used as a probe for specificity
tests.
[0023] For each given species, the gel-purified restriction
fragments of unknown coding potential were labeled with the
radioactive nucleotide .alpha.-.sup.32P(dATP) which was
incorporated into the DNA fragment by the random priming labeling
reaction. Non-radioactive modified nucleotides could also be
incorporated into the DNA by this method to serve as a label.
[0024] Each DNA fragment probe (i.e. a segment of bacterial genomic
DNA of at least 100 bp in length cut out from clones randomly
selected from the genomic library) was then tested for its
specificity by hybridization to DNAs from a variety of bacterial
species (Table 5). The double-stranded labeled DNA probe was
heat-denatured to yield labeled single-stranded DNA which could
then hybridize to any single-stranded target DNA fixed onto a solid
support or in solution. The target DNAs consisted of total cellular
DNA from an array of bacterial species found in clinical samples
(Table 5). Each target DNA was released from the bacterial cells
and denatured by conventional methods and then irreversibly fixed
onto a solid support (e.g. nylon or nitrocellulose membranes) or
free in solution. The fixed single-stranded target DNAs were then
hybridized with the single-stranded probe. Pre-hybridization,
hybridization and post-hybridization conditions were as follows:
(i) Pre-hybridization; in 1 M NaCl+10% dextran sulfate+1% SDS
(sodium dodecyl sulfate)+1 .mu.g/ml salmon sperm DNA at 650.degree.
C. for 15 min. (ii) Hybridization; in fresh pre-hybridization
solution containing the labeled probe at 650.degree. C. overnight.
(iii) Post-hybridization; washes twice in 3.times.SSC containing 1%
SDS (1.times.SSC is 0.15M NaCl, 0.015M NaCitrate) and twice in
0.1.times.SSC containing 0.1% SDS; all washes were at 650.degree.
C. for 15 min. Autoradiography of washed filters allowed the
detection of selectively hybridized probes. Hybridization of the
probe to a specific target DNA indicated a high degree of
similarity between the nucleotide sequence of these two DNAs.
Species-specific DNA fragments selected from various bacterial
genomic libraries ranging in size from 0.25 to 5.0 kbp were
isolated for 10 common bacterial pathogens (Table 6) based on
hybridization to chromosomal DNAs from a variety of bacteria
performed as described above. All of the bacterial species tested
(66 species listed in Table 5) were likely to be pathogens
associated with common infections or potential contaminants which
can be isolated from clinical specimens. A DNA fragment probe was
considered specific only when it hybridized solely to the pathogen
from which it was isolated. DNA fragment probes found to be
specific were subsequently tested for their ubiquity (i.e.
ubiquitous probes recognized most isolates of the target species)
by hybridization to bacterial DNAs from approximately 10 to 80
clinical isolates of the species of interest (Table 6). The DNAs
were denatured, fixed onto nylon membranes and hybridized as
described above.
[0025] Sequencing of the Species-Specific Fragment Orobes
[0026] The nucleotide sequence of the totality or of a portion of
the species-specific DNA fragments isolated (Table 6) was
determined using the dideoxynucleotide termination sequencing
method which was performed using Sequenase (USB Biochemicals) or T7
DNA polymerase (Pharmacia). These nucleotide sequences are shown in
the sequence listing. Alternatively, sequences selected from data
banks (GenBank and EMBL) were used as sources of oligonucleotides
for diagnostic purposes for Escherichia coli, Enterococcus
faecalis, Streptococcus pyogenes and Pseudomonas aeruginosa. For
this strategy, an array of suitable oligonucleotide primers or
probes derived from a variety of genomic DNA fragments (size of
more than 100 bp) selected from data banks was tested for their
specificity and ubiquity in PCR and hybridization assays as
described later. It is important to note that the data bank
sequences were selected based on their potential of being
species-specific according to available sequence information. Only
data bank sequences from which species-specific oligonucleotides
could be derived are included in this invention.
[0027] Oligonucleotide probes and amplification primers derived
from species-specific fragments selected from the genomic libraries
or from data bank sequences were synthesized using an automated DNA
synthesizer (Millipore). Prior to synthesis, all oligonucleotides
(probes for hybridization and primers for DNA amplification) were
evaluated for their suitability for hybridization or DNA
amplification by polymerase chain reaction (PCR) by computer
analysis using standard programs (e.g. Genetics Computer Group
(GCG) and Oligo.TM. 4.0 (National Biosciences)). The potential
suitability of the PCR primer pairs was also evaluated prior to the
synthesis by verifying the absence of unwanted features such as
long stretches of one nucleotide, a high proportion of G or C
residues at the 3' end and a 3'-terminal T residue (Persing et al,
1993. Diagnostic Molecular Microbiology: Principles and
Applications, American Society for Microbiology, Washington,
D.C.).
[0028] Hybridization with Oligonucleotide Probes
[0029] In hybridization experiments, oligonucleotides (size less
than 100 nucleotides) have some advantages over DNA fragment probes
for the detection of bacteria such as ease of preparation in large
quantities, consistency in results from batch to batch and chemical
stability. Briefly, for the hybridizations, oligonucleotides were
5' end-labeled with the radionucleotide .gamma..sup.32P(ATP) using
T4 polynucleotide kinase (Pharmacia). The unincorporated
radionucleotide was removed by passing the labeled single-stranded
oligonucleotide through a Sephadex G50 column. Alternatively,
oligonucleotides were labeled with biotin, either enzymatically at
their 3' ends or incorporated directly during synthesis at their 5'
ends, or with digoxigenin. It will be appreciated by the person
skilled in the art that labeling means other than the three above
labels may be used.
[0030] The target DNA was denatured, fixed onto a solid support and
hybridized as previously described for the DNA fragment probes.
Conditions for pre-hybridization and hybridization were as
described earlier. Post-hybridization washing conditions were as
follows: twice in 3.times.SSC containing 1% SDS, twice in
2.times.SSC containing 1% SDS and twice in 1.times.SSC containing
1% SDS (all of these washes were at 65.degree. C. for 15 min ), and
a final wash in 0.1.times.SSC containing 1% SDS at 25.degree. C.
for 15 min. For probes labeled with radioactive labels the
detection of hybrids was by autoradiography as described earlier.
For non-radioactive labels detection may be calorimetric or by
chemiluminescence.
[0031] The oligonucleotide probes may be derived from either strand
of the duplex DNA. The probes may consist of the bases A, G, C, or
T or analogs. The probes may be of any suitable length and may be
selected anywhere within the species-specific genomic DNA fragments
selected from the genomic libraries or from data bank
sequences.
[0032] DNA Amplification
[0033] For DNA amplification by the widely used PCR (polymerase
chain reaction) method, primer pairs were derived either from the
sequenced species-specific DNA fragments or from data bank
sequences or, alternatively, were shortened versions of
oligonucleotide probes. Prior to synthesis, the potential primer
pairs were analyzed by using the program oligo.TM. 4.0 (National
Biosciences) to verify that they are likely candidates for PCR
amplifications.
[0034] During DNA amplification by PCR, two oligonucleotide primers
binding respectively to each strand of the denatured
double-stranded target DNA from the bacterial genome are used to
amplify exponentially in vitro the target DNA by successive thermal
cycles allowing denaturation of the DNA, annealing of the primers
and synthesis of new targets at each cycle (Persing et al, 1993.
Diagnostic Molecular Microbiology: Principles and Applications,
American Society for Microbiology, Washington, D.C.). Briefly, the
PCR protocols were as follows. Clinical specimens or bacterial
colonies were added directly to the 50 .mu.L PCR reaction mixtures
containing 50 mM KCl, 10 mM Tris-HCl pH 8.3, 2.5 mM MgCl.sub.2, 0.4
.mu.M of each of the two primers, 200 .mu.M of each of the four
dNTPs and 1.25 Units of Taq DNA polymerase (Perkin Elmer). PCR
reactions were then subjected to thermal cycling (3 min at
95.degree. C. followed by 30 cycles of 1 second at 95.degree. C.
and 1 second at 55.degree. C.) using a Perkin Elmer 480.TM. thermal
cycler and subsequently analyzed by standard ethidium
bromide-stained agarose gel electrophoresis. It is clear that other
methods for the detection of specific amplification products, which
may be faster and more practical for routine diagnosis, may be
used. Such methods may be based on the detection of fluorescence
after amplification (e.g. TaqMan.TM. system from Perkin Elmer or
Amplisensor.TM. from Biotronics) or liquid hybridization with an
oligonucleotide probe binding to internal sequences of the specific
amplification product. These novel probes can be generated from our
species-specific fragment probes. Methods based on the detection of
fluorescence are particularly promising for utilization in routine
diagnosis as they are, very rapid and quantitative and can be
automated.
[0035] To assure PCR efficiency, glycerol or dimethyl sulfoxide
(DMSO) or other related solvents, can be used to increase the
sensitivity of the PCR and to overcome problems associated with the
amplification of target with a high GC content or with strong
secondary structures. The concentration ranges for glycerol and
DMSO are 5-15% (v/v) and 3-10% (v.backslash.v), respectively. For
the PCR reaction mixture, the concentration ranges for the
amplification primers and the MgCl.sub.2 are 0.1-1.0 .mu.M and
1.5-3.5 mM, respectively. Modifications of the standard PCR
protocol using external and nested primers (i.e. nested PCR) or
using more than one primer pair (i.e. multiplex PCR) may also be
used (Persing et al, 1993. Diagnostic Molecular Microbiology:
Principles and Applications, American Society for Microbiology,
Washington, D.C.). For more details about the PCR protocols and
amplicon detection methods see examples 7 and 8.
[0036] The person skilled in the art of DNA amplification knows the
existence of other rapid amplification procedures such as ligase
chain reaction (LCR), transcription-based amplification systems
(TAS), self-sustained sequence replication (3SR), nucleic acid
sequence-based amplification (NASBA), strand displacement
amplification (SDA) and branched DNA (bDNA) (Persing et al, 1993.
Diagnostic Molecular Microbiology: Principles and Applications,
American Society for Microbiology, Washington, D.C.). The scope of
this invention is not limited to the use of amplification by PCR,
but rather includes the use of any rapid nucleic acid amplification
methods or any other procedures which may be used to increase
rapidity and sensitivity of the tests. Any oligonucleotides
suitable for the amplification of nucleic acid by approaches other
than PCR and derived from the species-specific fragments and from
selected antibiotic resistance gene sequences included in this
document are also under the scope of this invention.
[0037] Specificity and Ubiquity Tests for Oligonucleotide Probes
and Primers
[0038] The specificity of oligonucleotide probes, derived either
from the sequenced species-specific fragments or from data bank
sequences, was tested by hybridization to DNAs from the array of
bacterial species listed in Table 5 as previously described.
Oligonucleotides found to be specific were subsequently tested for
their ubiquity by hybridization to bacterial DNAs from
approximately 80 isolates of the target species as described for
fragment probes. Probes were considered ubiquitous when they
hybridized specifically with the DNA from at least 80% of the
isolates. Results for specificity and ubiquity tests with the
oligonucleotide probes are summarized in Table 6. The specificity
and ubiquity of the amplification primer pairs were tested directly
from cultures (see example 7) of the same bacterial strains. For
specificity and ubiquity tests, PCR assays were performed directly
from bacterial colonies of approximately 80 isolates of the target
species. Results are summarized in Table 7. All specific and
ubiquitous oligonucleotide probes and amplification primers for
each of the 12 bacterial species investigated are listed in Annexes
I and II, respectively. Divergence in the sequenced DNA fragments
can occur and, insofar as the divergence of these sequences or a
part thereof does not affect the specificity of the probes or
amplification primers, variant bacterial DNA is under the scope of
this invention.
[0039] Universal Bacterial Detection
[0040] In the routine microbiology laboratory a high percentage of
clinical specimens sent for bacterial identification is negative
(Table 4). For example, over a 2 year period, around 80% of urine
specimens received by the laboratory at the "Centre Hospitalier de
1' Universit Laval (CHUL)" were negative (i.e. <10.sup.7 CFU/L)
(Table 3). Testing clinical samples with universal probes or
universal amplification primers to detect the presence of bacteria
prior to specific identification and screen out the numerous
negative specimens is thus useful as it saves costs and may rapidly
orient the clinical management of the patients. Several
oligonucleotides and amplification primers were therefore
synthesized from highly conserved portions of bacterial 16S or 23S
ribosomal RNA gene sequences available in data banks (Annexes III
and IV). In hybridization tests, a pool of seven oligonucleotides
(Annex I; Table 6) hybridized strongly to DNA from all bacterial
species listed in Table 5. This pool of universal probes labeled
with radionucleotides or with any other modified nucleotides is
consequently very useful for detection of bacteria in urine samples
with a sensitivity range of .gtoreq.10.sup.7 CFU/L. These probes
can also be applied for bacterial detection in other clinical
samples.
[0041] Amplification primers also derived from the sequence of
highly conserved ribosomal RNA genes were used as an alternative
strategy for universal bacterial detection directly from clinical
specimens (Annex IV; Table 7). The DNA amplification strategy was
developed to increase the sensitivity and the rapidity of the test.
This amplification test was ubiquitous since it specifically
amplified DNA from 23 different bacterial species encountered in
clinical specimens.
[0042] Well-conserved bacterial genes other than ribosomal RNA
genes could also be good candidates for universal bacterial
detection directly from clinical specimens. Such genes may be
associated with processes essential for bacterial survival (e.g.
protein synthesis, DNA synthesis, cell division or DNA repair) and
could therefore be highly conserved during evolution. We are
working on these candidate genes to develop new rapid tests for the
universal detection of bacteria directly from clinical
specimens.
[0043] Antibiotic Resistance Genes
[0044] Antimicrobial resistance complicates treatment and often
leads to therapeutic failures. Furthermore, overuse of antibiotics
inevitably leads to the emergence of bacterial resistance. Our goal
is to provide the clinicians, within one hour, the needed
information to prescribe optimal treatments. Besides the rapid
identification of negative clinical specimens with DNA-based tests
for universal bacterial detection and the identification of the
presence of a specific pathogen in the positive specimens with
DNA-based tests for specific bacterial detection, the clinicians
also need timely information about the ability of the bacterial
pathogen to resist antibiotic treatments. We feel that the most
efficient strategy to evaluate rapidly bacterial resistance to
antimicrobials is to detect directly from the clinical specimens
the most common and important antibiotic resistance genes (i.e.
DNA-based tests for the detection of antibiotic resitance genes).
Since the sequence from the most important and common bacterial
antibiotic resistance genes are available from data banks, our
strategy is to use the sequence from a portion or from the entire
gene to design specific oligonucleotides which will be used as a
basis for the development of rapid DNA-based tests. The sequence
from the bacterial antibiotic resistance genes selected on the
basis of their clinical relevance (i.e. high incidence and
importance) is given in the sequence listing. Table 8 summarizes
some characteristics of the selected antibiotic resistance
genes.
EXAMPLES
[0045] The following examples are intended to be illustrative of
the various methods and compounds of the invention.
Example 1
[0046] Isolation and cloning of fragments. Genomic DNAs from
Escherichia coli strain ATCC 25922, Klebsiella pneumoniae strain
CK2, Pseudomonas aeruginosa strain ATCC 27853, Proteus mirabilis
strain ATCC 35657, Streptococcus pneumoniae strain ATCC 27336,
Staphylococcus aureus strain ATCC 25923, Staphylococcus epidermidis
strain ATCC 12228, Staphylococcus saprophyticus strain ATCC 15305,
Haemophilus influenzae reference strain Rd and Moraxella
catarrhalis strain ATCC 53879 were prepared using standard
procedures. It is understood that the bacterial genomic DNA may
have been isolated from strains other than the ones mentioned
above. (For Enterococcus faecalis and Streptococcus pyogenes
oligonucleotide sequences were derived exclusively from data
banks). Each DNA was digested with a restriction enzyme which
frequently cuts DNA such as Sau3AI. The resulting DNA fragments
were ligated into a plasmid vector (pGEM3Zf) to create recombinant
plasmids and transformed into competent E. coli cells (DH5.alpha.).
It is understood that the vectors and corresponding competent cells
should not be limited to the ones herein above specifically
examplified. The objective of obtaining recombinant plasmids and
transformed cells is to provide an easily reproducible source of
DNA fragments useful as probes. Therefore, insofar as the inserted
fragments are specific and selective for the target bacterial DNA,
any recombinant plasmids and corresponding transformed host cells
are under the scope of this invention. The plasmid content of the
transformed bacterial cells was analyzed using standard methods.
DNA fragments from target bacteria ranging in size from 0.25 to 5.0
kbp were cut out from the vector by digestion of the recombinant
plasmid with various restriction endonucleases. The insert was
separated from the vector by agarose gel electrophoresis and
purified in a low melting point agarose gel. Each of the purified
fragments was then used for specificity tests.
[0047] Labeling of DNA fragment probes. The label used was
.alpha..sup.32P(dATP), a radioactive nucleotide which can be
incorporated enzymatically into a double-stranded DNA molecule. The
fragment of interest is first denatured by heating at 95.degree. C.
for 5 min, then a mixture of random primers is allowed to anneal to
the strands of the fragments. These primers, once annealed, provide
a starting point for synthesis of DNA. DNA polymerase, usually the
Klenow fragment, is provided along with the four nucleotides, one
of which is radioactive. When the reaction is terminated, the
mixture of new DNA molecules is once again denatured to provide
radioactive single-stranded DNA molecules (i.e. the probe). As
mentioned earlier, other modified nucleotides may be used to label
the probes.
[0048] Specificity and ubiquity tests for the DNA fragment probes.
Species-specific DNA fragments ranging in size from 0.25 to 5.0 kbp
were isolated for 10 common bacterial pathogens (Table 6) based on
hybridization to chromosomal DNAs from a variety of bacteria.
Samples of whole cell DNA for each bacterial strain listed in Table
5 were transferred onto a nylon membrane using a dot blot
apparatus, washed and denatured before being irreversibly fixed.
Hybridization conditions were as described earlier. A DNA fragment
probe was considered specific only when it hybridized solely to the
pathogen from which it was isolated. Labeled DNA fragments
hybridizing specifically only to target bacterial species (i.e.
specific) were then tested for their ubiquity by hybridization to
DNAs from approximately 10 to 80 isolates of the species of
interest as described earlier. The conditions for
pre-hybridization, hybridization and post-hybridization washes were
as described earlier. After autoradiography (or other detection
means appropriate for the non-radioactive label used), the
specificity of each individual probe can be determined. Each probe
found to be specific (i.e. hybridizing only to the DNA from the
bacterial species from which it was isolated) and ubiquitous (i.e.
hybridizing to most isolates of the target species) was kept for
further experimentations.
Example 2
[0049] Same as example 1 except that testing of the strains is by
colony hybridization. The bacterial strains were inoculated onto a
nylon membrane placed on nutrient agar. The membranes were
incubated at 37.degree. C. for two hours and then bacterial lysis
and DNA denaturation were carried out according to standard
procedures. DNA hybridization was performed as described
earlier.
Example 3
[0050] Same as example 1 except that bacteria were detected
directly from clinical samples. Any biological samples were loaded
directly onto a dot blot apparatus and cells were lysed in situ for
bacterial detection. Blood samples should be heparizined in order
to avoid coagulation interfering with their convenient loading on a
dot blot apparatus.
Example 4
[0051] Nucleotide sequencina of DNA fragments. The nucleotide
sequence of the totality or a portion of each fragment found to be
specific and ubiquitous (Example 1) was determined using the
dideoxynucleotide termination sequencing method (Sanger et al.,
1977, Proc. Natl. Acad. Sci. USA. 74:5463-5467). These DNA
sequences are shown in the sequence listing. Oligonucleotide probes
and amplification primers were selected from these nucleotide
sequences, or alternatively, from selected data banks sequences and
were then synthesized on an automated Biosearch synthesizer
(Millipore.TM.) using phosphoramidite chemistry.
[0052] Labeling of oliaonucleotides. Each oligonucleotide was 5'
end-labeled with .gamma..sup.32P-ATP by the T4 polynucleotide
kinase (Pharmacia) as described earlier. The label could also be
non-radioactive.
[0053] Specificity test for oligonucleotide probes. All labeled
oligonucleotide probes were tested for their specificity by
hybridization to DNAs from a variety of Gram positive and Gram
negative bacterial species as described earlier. Species-specific
probes were those hybridizing only to DNA from the bacterial
species from which it was isolated. Oligonucleotide probes found to
be specific were submitted to ubiquity tests as follows.
[0054] Ubiquity test for oligonucleotide probes. Specific
oligonucleotide probes were then used in ubiquity tests with
approximately 80 strains of the target species. Chromosomal DNAs
from the isolates were transferred onto nylon membranes and
hybridized with labeled oligonucleotide probes as described for
specificity tests. The batteries of approximately 80 isolates
constructed for each target species contain reference ATCC strains
as well as a variety of clinical isolates obtained from various
sources. Ubiquitous probes were those hybridizing to at least 80%
of DNAs from the battery of clinical isolates of the target
species. Examples of specific and ubiquitous oligonucleotide probes
are listed in Annex 1.
Example 5
[0055] Same as example 4 except that a pool of specific
oligonucleotide probes is used for bacterial identification (i) to
increase sensitivity and assure 100% ubiquity or (ii) to identify
simultaneously more than one bacterial species. Bacterial
identification could be done from isolated colonies or directly
from clinical specimens.
Example 6
[0056] PCR amplification. The technique of PCR was used to increase
sensitivity and rapidity of the tests. The PCR primers used were
often shorter derivatives of the extensive sets of oligonucleotides
previously developed for hybridization assays (Table 6). The sets
of primers were tested in PCR assays performed directly from a
bacterial colony or from a bacterial suspension (see Example 7) to
determine their specificity and ubiquity (Table 7). Examples of
specific and ubiquitous PCR primer pairs are listed in annex
II.
[0057] Specificity and ubiquity tests for amplification primers.
The specificity of all selected PCR primer pairs was tested against
the battery of Gram negative and Gram positive bacteria used to
test the oligonucleotide probes (Table 5). Primer pairs found
specific for each species were then tested for their ubiquity to
ensure that each set of primers could amplify at least 80% of DNAs
from a battery of approximately 80 isolates of the target species.
The batteries of isolates constructed for each species contain
reference ATCC strains and various clinical isolates representative
of the clinical diversity for each species.
[0058] Standard precautions to avoid false positive PCR results
should be taken. Methods to inactivate PCR amplification products
such as the inactivation by uracil-N-glycosylase may be used to
control PCR carryover.
Example 7
[0059] Amplification directly from a bacterial colony or
suspension. PCR assays were performed either directly from a
bacterial colony or from a bacterial suspension, the latter being
adjusted to a standard McFarland 0.5 (corresponds to 1.5
.times.10.sup.8 bacteria/mL). In the case of direct amplification
from a colony, a portion of the colony was transferred directly to
a 50 .mu.L PCR reaction mixture (containing 50 mM KCl, 10 mM Tris
pH 8.3, 2.5 mM MgCl.sub.2, 0.4 .mu.M of each of the two primers,
200 .mu.M of each of the four dNTPs and 1.25 Unit of Taq DNA
polymerase (Perkin Elmer)) using a plastic rod. For the bacterial
suspension, 4 .mu.L of the cell suspension was added to 46 .mu.L of
the same PCR reaction mixture. For both strategies, the reaction
mixture was overlaid with 50 .mu.L of mineral oil and PCR
amplifications were carried out using an initial denaturation step
of 3 min. at 95.degree. C. followed by 30 cycles consisting of a 1
second denaturation step at 95.degree. C. and of a 1 second
annealing step at 55.degree. C. in a Perkin Elmer 480.TM. thermal
cycler. PCR amplification products were then analyzed by standard
agarose gel (2%) electrophoresis. Amplification products were
visualized in agarose gels containing 2.5 .mu.g/mL of ethidium
bromide under UV at 254 nm. The entire PCR assay can be completed
in approximately one hour.
[0060] Alternatively, amplification from bacterial cultures was
performed as described above but using a "hot start" protocol. In
that case, an initial reaction mixture containing the target DNA,
primers and dNTPs was heated at 85.degree. C. prior to the addition
of the other components of the PCR reaction mixture. The final
concentration of all reagents was as described above. Subsequently,
the PCR reactions were submitted to thermal cycling and analysis as
described above.
Example 8
[0061] Amplification directly from clinical specimens. For
amplification from urine specimens, 4 .mu.L of undiluted or diluted
(1:10) urine was added directly to 46 .mu.L of the above PCR
reaction mixture and amplified as described earlier.
[0062] To improve bacterial cell lysis and eliminate the PCR
inhibitory effects of clinical specimens, samples were routinely
diluted in lysis buffer containing detergent(s). Subsequently, the
lysate was added directly to the PCR reaction mixture. Heat
treatments of the lysates, prior to DNA amplification, using the
thermocycler or a microwave oven could also be performed to
increase the efficiency of cell lysis.
[0063] Our strategy is to develop rapid and simple protocols to
eliminate PCR inhibitory effects of clinical specimens and lyse
bacterial cells to perform DNA amplification directly from a
variety of biological samples. PCR has the advantage of being
compatible with crude DNA preparations. For example, blood,
cerebrospinal fluid and sera may be used directly in PCR assays
after a brief heat treatment. We intend to use such rapid and
simple strategies to develop fast protocols for DNA amplification
from a variety of clinical specimens.
Example 9
[0064] Detection of antibiotic resistance genes. The presence of
specific antibiotic resistance genes which are frequently
encountered and clinically relevant is identified using the PCR
amplification or hybridization protocols described in previous
sections. Specific oligonucleotides used as a basis for the
DNA-based tests are selected from the antibiotic resistance gene
sequences. These tests can be performed either directly from
clinical specimens or from a bacterial colony and should complement
diagnostic tests for specific bacterial identification.
Example 10
[0065] Same as examples 7 and 8 except that assays were performed
by multiplex PCR (i.e. using several pairs of primers in a single
PCR reaction) to (i) reach an ubiquity of 100% for the specific
target pathogen or (ii) to detect simultaneously several species of
bacterial pathogens.
[0066] For example, the detection of Escherichia coli requires
three pairs of PCR primers to assure a ubiquity of 100%. Therefore,
a multiplex PCR assay (using the "hot-start" protocol (Example 7))
with those three primer pairs was developed. This strategy was also
used for the other bacterial pathogens for which more than one
primer pair was required to reach an ubiquity of 100%.
[0067] Multiplex PCR assays could also be used to (i) detect
simultaneously several bacterial species or, alternatively, (ii) to
simultaneously identify the bacterial pathogen and detect specific
antibiotic resistance genes either directly from a clinical
specimen or from a bacterial colony.
[0068] For these applications, amplicon detection methods should be
adapted to differentiate the various amplicons produced. Standard
agarose gel electrophoresis could be used because it discriminates
the amplicons based on their sizes. Another useful strategy for
this purpose would be detection using a variety of fluorochromes
emitting at different wavelengths which are each coupled with a
specific oligonucleotide linked to a fluorescence quencher which is
degraded during amplification to release the fluorochrome (e.g.
TaqMan.TM., Perkin Elmer).
Example 11
[0069] Detection of amplification Products. The person skilled in
the art will appreciate that alternatives other than standard
agarose gel electrophoresis (Example 7) may be used for the
revelation of amplification products. Such methods may be based on
the detection of fluorescence after amplification (e.g.
Amplisensor.TM., Biotronics; TaqMan.TM.) or other labels such as
biotin (SHARP Signal.TM. system, Digene Diagnostics). These methods
are quantitative and easily automated. One of the amplification
primers or an internal oligonucleotide probe specific to the
amplicon(s) derived from the species-specific fragment probes is
coupled with the fluorochrome or with any other label. Methods
based on the detection of fluorescence are particularly suitable
for diagnostic tests since they are rapid and flexible as
fluorochromes emitting different wavelengths are available (Perkin
Elmer).
Example 12
[0070] Species-specific, universal and antibiotic resistance gene
amplification primers can be used in other rapid amplification
procedures such as the ligase chain reaction (LCR),
transcription-based amplification systems (TAS), self-sustained
sequence replication (3SR), nucleic acid sequence-based
amplification (NASBA), strand displacement amplification (SDA) and
branched DNA (bDNA) or any other methods to increase the
sensitivity of the test. Amplifications can be performed from an
isolated bacterial colony or directly from clinical specimens. The
scope of this invention is therefore not limited to the use of PCR
but rather includes the use of any procedures to specifically
identify bacterial DNA and which may be used to increase rapidity
and sensitivity of the tests.
Example 13
[0071] A test kit would contain sets of probes specific for each
bacterium as well as a set of universal probes. The kit is provided
in the form of test components, consisting of the set of universal
probes labeled with non-radioactive labels as well as labeled
specific probes for the detection of each bacterium of interest in
specific clinical samples. The kit will also include test reagents
necessary to perform the pre-hybridization, hybridization, washing
steps and hybrid detection. Finally, test components for the
detection of known antibiotic resistance genes (or derivatives
therefrom) will be included. Of course, the kit will include
standard samples to be used as negative and positive controls for
each hybridization test.
[0072] Components to be included in the kits will be adapted to
each specimen type and to detect pathogens commonly encountered in
that type of specimen. Reagents for the universal detection of
bacteria will also be included. Based on the sites of infection,
the following kits for the specific detection of pathogens may be
developed:
[0073] A kit for the universal detection of bacterial pathogens
from most clinical specimens which contains sets of probes specific
for highly conserved regions of the bacterial genomes.
[0074] A kit for the detection of bacterial pathogens retrieved
from urine samples, which contains eight specific test components
(sets of probes for the detection of Escherichia coli, Enterococcus
faecalis, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas
aeruginosa, Staphylococcus saprophyticus, Staphylococcus aureus and
Staphylococcus epidermidis).
[0075] A kit for the detection of respiratory pathogens which
contains seven specific test components (sets of probes for
detecting Streptococcus pneumoniae, Moraxella catarrhalis,
Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas
aeruginosa, Streptococcus pyogenes and Staphylococcus aureus).
[0076] A kit for the detection of pathogens retrieved from blood
samples, which contains eleven specific test components (sets of
probes for the detection of Streptococcus pneumoniae, Moraxella
catarrhalis, Haemophilus influenzae, Proteus mirabilis, Klebsiella
pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Enterococcus
faecalis, Staphylococcus aureus, Streptococcus pyogenes and
Staphylococcus epidermidis).
[0077] A kit for the detection of pathogens causing meningitis,
which contains four specific test components (sets of probes for
the detection of Haemophilus influenzae, Streptococcus pneumoniae,
Escherichia coli and Pseudomonas aeruginosa).
[0078] A kit for the detection of clinically important antibiotic
resistance genes which contains sets of probes for the specific
detection of at least one of the 19 following genes associated with
bacterial resistance : bla.sub.tem, bla.sub.rob, bla.sub.shv, aadB,
aacC1, aacC2, aacC3, aacA4, mecA, vanA, vanH, vanX, satA,
aacA-aphD, vat, vga, msrA, sul and int.
[0079] Other kits adapted for the detection of pathogens from skin,
abdominal wound or any other clinically relevant kits will be
developed.
Example 14
[0080] Same as example 13 except that the test kits contain all
reagents and controls to perform DNA amplification assays.
Diagnostic kits will be adapted for amplification by PCR (or other
amplification methods) performed directly either from clinical
specimens or from a bacterial colony. Components required for
universal bacterial detection, bacterial identification and
antibiotic resistance genes detection will be included.
[0081] Amplification assays could be performed either in tubes or
in microtitration plates having multiple wells. For assays in
plates, the wells will be coated with the specific amplification
primers and control DNAs and the detection of amplification
products will be automated. Reagents and amplification primers for
universal bacterial detection will be included in kits for tests
performed directly from clinical specimens. Components required for
bacterial identification and antibiotic resistance gene detection
will be included in kits for testing directly from colonies as well
as in kits for testing directly from clinical specimens.
[0082] The kits will be adapted for use with each type of specimen
as described in example 13 for hybridization-based diagnostic
kits.
Example 15
[0083] It is understood that the use of the probes and
amplification primers described in this invention for bacterial
detection and identification is not limited to clinical
microbiology applications. In fact, we feel that other sectors
could also benefit from these new technologies. For example, these
tests could be used by industries for quality control of food,
water, pharmaceutical products or other products requiring
microbiological control. These tests could also be applied to
detect and identify bacteria in biological samples from organisms
other than humans (e.g. other primates, mammals, farm animals and
live stocks). These diagnostic tools could also be very useful for
research purposes including clinical trials and epidemiological
studies.
1TABLE 1 Distribution of urinary isolates from positive urine
samples (.gtoreq.10.sup.7 CFU/L) at the Centre Hospitalier de
l'Universit Laval (CHUL) for the 1992-1994 period. % of isolates
Nov 92 April 93 July 93 Jan 94 Organisms n = 267.sup.a n = 265 n =
238 n = 281 Escherichia coli 53.2 51.7 53.8 54.1 Enterococcus
faecalis 13.8 12.4 11.7 11.4 Klebsiella pneumoniae 6.4 6.4 5.5 5.3
Staphylococcus epidermidis 7.1 7.9 3.0 6.4 Proteus mirabilis 2.6
3.4 3.8 2.5 Pseudomonas aeruginosa 3.7 3.0 5.0 2.9 Staphylococcus
saprophyticus 3.0 1.9 5.4 1.4 Others.sup.b 10.2 13.3 11.8 16.0
.sup.an = total number of isolates for the indicated month.
.sup.bSee table 2.
[0084]
2TABLE 2 Distribution of uncommon.sup.a urinary isolates from
positive urine samples ( .gtoreq.10.sup.7 CFU/L) at the Centre
Hospitalier de I'Universit Laval (CHUL) for the 1992-1994 period. %
of isolates Organisms.sup.a Nov 92 April 93 July 93 Jan 94
Staphylococcus aureus 0.4 1.1 1.3 1.4 Staphylococcus spp. 2.2 4.9
1.7 6.0 Micrococcus spp. 0.0 0.0 0.4 0.7 Enterococcus faecium 0.4
0.4 1.3 1.4 Citrobacter spp. 1.4 0.8 0.4 0.7 Enterobacter spp. 1.5
1.1 1.3 1.4 Klebsiella oxytoca 1.1 1.5 2.5 1.8 Serratia spp. 0.8
0.0 0.5 0.0 Proteus spp. 0.4 0.4 0.0 1.1 Morganella and Providencia
0.4 0.8 0.4 0.0 Hafnia alvei 0.8 0.0 0.0 0.0 NFB.sup.b
(Stenotrophomonas, 0.0 0.4 1.3 1.1 Acinetobacter) Candida spp. 0.8
1.9 0.7 0.4 .sup.aUncommon urinary isolates are those identified as
"Others" in Table 1. .sup.bNFB: non fermentative bacilli.
[0085]
3TABLE 3 Distribution of positive.sup.a (bacterial count
.gtoreq.10.sup.7 CFU/L) and negative (bacterial count <10.sup.7
CFU/L) urine specimens tested at the Centre Hospitalier de
l'Universit Laval (CHUL) for the 1992-1994 period. Number of
isolates (%) Specimens Nov 92 April 93 July 93 Jan 94 received:
1383(100) 1338(100) 1139(100) 1345(100) positive: 267(19.3)
265(19.8) 238(20.9) 281(20.9) negative: 1116(80.7) 1073(80.2)
901(79.1) 1064(79.1) .sup.aBased on standard diagnostic methods,
the minimal number of bacterial pathogens in urine samples to
indicate an urinary tract infection is normally 10.sup.7 CFU/L.
[0086]
4TABLE 4 Distribution of positive and negative clinical specimens
tested in the Microbiology Laboratory of the CHUL. No. of % of % of
Clinical samples positive negative specimens.sup.a tested specimens
specimens Urine 17,981 19.4 80.6 Haemoculture/marrow 10,010 6.9
93.1 Sputum 1,266 68.4 31.6 Superficial pus 1,136 72.3 27.7
Cerebrospinal fluid 553 1.0 99.0 Synovial fluid-articular 523 2.7
97.3 Bronch./Trach./Amyg./Throat 502 56.6 43.4 Deep pus 473 56.8
43.2 Ears 289 47.1 52.9 Pleural and pericardial fluid 132 1.0 99.0
Peritonial fluid 101 28.6 71.4 .sup.aSpecimens tested from February
1994 to January 1995.
[0087]
5TABLE 5 Bacterial species (66) used for testing the specificity of
DNA fragment probes, oligonucleotide probes and PCR primers. Number
of Number of Bacterial species strains Bacterial species strains
Gram negative: tested Gram positive: tested Proteus mirabilis 5
Streptococcus pneumoniae 7 Klebsiella pneumoniae 5 Streptococcus
salivarius 2 Pseudomonas aeruginosa 5 Streptococcus viridans 2
Escherichia coli 5 Streptococcus pyogenes 2 Moraxella catarrhalis 5
Staphylococcus aureus 2 Proteus vulgaris 2 Staphylococcus
epidermidis 2 Morganella morganii 2 Staphylococcus saprophyticus 5
Enterobacter cloacae 2 Micrococcus species 2 Providencia stuartii 1
Corynebacterium species 2 Providencia species 1 Streptococcus
groupe B 2 Enterobacter agglomerans 2 Staphylococcus simulans 2
Providencia rettgeri 2 Staphylococcus ludgunensis 1 Neisseria
mucosa 1 Staphylococcus capitis 2 Providencia alcalifaciens 1
Staphylococcus haemolyticus 2 Providencia rustigianii 1
Staphylococcus hominis 2 Burkholderia cepacia 2 Enterococcus
faecalis 2 Enterobacter aerogenes 2 Enterococcus faecium 1
Stenotrophomonas maltophilia 2 Staphylococcus warneri 1 Pseudomonas
fluorescens 1 Enterococcus durans 1 Comamonas acidovorans 2
Streptococcus bovis 1 Pseudomonas putida 2 Diphteroids 2
Haemophilus influenzae 5 Lactobacillus acidophilus 1 Haemophilus
parainfluenzae 2 Bordetella pertussis 2 Haemophilus
parahaemolyticus 2 Haemophilus haemolyticus 2 Haemophilus aegyptius
1 Kingella indologenes 1 Moraxella atlantae 1 Neisseria caviae 1
Neisseria subflava 1 Moraxella urethralis 1 Shigella sonnei 1
Shigella flexneri 1 Klebsiella oxytoca 2 Serratia marcescens 2
Salmonella typhimurium 1 Yersinia enterocolitica 1 Acinetobacter
calcoaceticus 1 Acinetobacter lwoffi 1 Haftnia alvei 2 Citrobacter
diversus 1 Citrobacter freundii 1 Salmonella species 1
[0088]
6TABLE 6 Species-specific DNA fragment and oligonucleotide probes
for hybridization. Number of fragment probes.sup.b Number of
oligonucleotide probes Organisms.sup.a Tested Specific
Ubiquitous.sup.c Synthesized Specific Ubiquitous.sup.c E.
coli.sup.d -- -- -- 20 12 9.sup.f E. coli 14 2 2.sup.e -- -- -- K.
pneumoniae.sup.d -- -- -- 15 1 1 K. pneumoniae 33 3 3 18 12 8 P.
mirabilis.sup.d -- -- -- 3 3 2 P. mirabilis 14 3 3.sup.e 15 8 7 P.
aeruginosa.sup.d -- -- -- 26 13 9 P. aeruginosa 6 2 2.sup.e 6 0 0
S. saprophyticus 7 4 4 20 9 7 H. influenzae.sup.d -- -- -- 16 2 2
H. influenzae 1 1 1 20 1 1 S. pneumoniae.sup.d -- -- -- 6 1 1 S.
pneumoniae 19 2 2 4 1 1 M. catarrhalis 2 2 2 9 8 8 S. epidermidis
62 1 1 -- -- -- S. aureus 30 1 1 -- -- -- Universal probes.sup.d --
-- -- 7 -- .sup. 7.sup.g .sup.aNo DNA fragment or oligonucleotide
probes were tested for E. faecalis and S. pyogenes. .sup.bSizes of
DNA fragments range from 0.25 to 5.0 kbp. .sup.cA specific probe
was considered ubiquitous when at least 80% of isolates of the
target species (approximately 80 isolates) were recognized by each
specific probe. When 2 or more probes are combined, 100% of the
isolates are recognized. .sup.dThese sequences were selected from
data banks. .sup.eUbiquity tested with approximately 10 isolates of
the target species. .sup.fA majority of probes (8/9) do not
discriminate E. coli and Shigella spp. .sup.gUbiquity tests with a
pool of the 7 probes detected all 66 bacterial species listed in
Table 5.
[0089]
7TABLE 7 PCR amplification for bacterial pathogens commonly
encountered in urine, sputum, blood, cerebrospinal fluid and other
specimens. Primer pair.sup.a Amplicon DNA amplification Organism #
(SEQ ID NO) size (bp) Ubiquity.sup.b from colonies.sup.c from
specimens.sup.d E. coli l.sup.e (55-56) 107 75/80 + + 2.sup.e
(46-47) 297 77/80 + + 3 (42-43) 102 78/80 + + 4 (131-132) 134 73/80
+ + 1 + 3 + 4 -- 80/80 + + E.faecalis 1.sup.e (38-39) 200 71/80 + +
2.sup.e (40-41) 121 79/80 + + 1 + 2 -- 80/80 + + K. pneumoniae 1
(67-68) 198 76/80 + + 2 (61-62) 143 67/80 + + 3.sup.h (135-136) 148
78/80 + N.T..sup.i 4 (137-138) 116 69/80 + N.T. 1 + 2 + 3 -- 80/80
+ N.T. P. mirabilis 1 (74-75) 167 73/80 + N.T. 2 (133-134) 123
80/80 + N.T. P. aeruginosa 1.sup.e (83-84) 139 79/80 + N.T. 2.sup.e
(85-86) 223 80/80 + N.T. S. saprophyticus 1 (98-99) 126 79/80 + + 2
(139-140) 190 80/80 + N.T. M. catarrhalis 1 (112-113) 157 79/80 +
N.T. 2 (118-119) 118 80/80 + N.T. 3 (160-119) 137 80/80 + N.T. H.
influenzae 1.sup.e (154-155) 217 80/80 + N.T. S. pneumoniae 1.sup.e
(156-157) 134 80/80 + N.T. 2.sup.e (158-159) 197 74/80 + N.T. 3
(78-79) 175 67/80 + N.T. S. epidermidis 1 (147-148) 175 80/80 +
N.T. 2 (145-146) 125 80/80 + N.T. S. aureus 1 (152-153) 108 80/80 +
N.T. 2 (149-150) 151 80/80 + N.T. 3 (149-151) 176 80/80 + N.T. S.
pyogenes.sup.f 1.sup.e (141-142) 213 80/80 + N.T. 2.sup.e (143-144)
157 24/24 + N.T. Universal 1.sup.e (126-127) 241 .sup.
194/195.sup.g + + See notes on next page
[0090] a All primer pairs are specific in PCR assays since no
amplification was observed with DNA from 66 different species of
both Gram positive and Gram negative bacteria other than the
species of interest (Table 5).
[0091] b The ubiquity was normally tested on 80 strains of the
species of interest. All retained primer pairs amplified at least
90% of the isolates. When combinations of primers were used, an
ubiquity of 100% was reached.
[0092] c For all primer pairs and multiplex combinations, PCR
amplifications directly performed from a bacterial colony were 100%
species-specific.
[0093] d PCR assays performed directly from urine specimens.
[0094] e Primer pairs derived from data bank sequences. Primer
pairs with no "e" are derived from our species-specific
fragments.
[0095] f For S. pyogenes, primer pair #1 is specific for Group A
Streptococci (GAS). Primer pair #2 is specific for the
GAS-producing exotoxin A gene (SpeA).
[0096] g Ubiquity tested on 195 isolates from 23 species
representative of bacterial pathogens commonly encountered in
clinical specimens.
[0097] h Optimizations are in progress to eliminate non-specific
amplification observed with some bacterial species other than the
target species.
[0098] N.T.: not tested.
8TABLE 8 Selected antibiotic resistance genes for diagnostic
purposes. Genes Antibiotics Bacteria.sup.a SEQ ID NO (bla.sub.tem)
TEM-1 .beta.-lactams Enterobacteriaceae, 161 Pseudomonadaceae,
Haemophilus, Neisseria (bla.sub.rob) ROB-1 .beta.-lactams
Haemophilus, Pasteurella 162 (bla.sub.shv) SHV-1 .beta.-lactams
Klebsiella and other 163 Enterobacteriaceae aadB, aacC1, aacC2,
Aminoglycosides Enterobacteriaceae, 164, 165, 166 aacC3, aacA4
Pseudomonadaceae 167, 168 mecA .beta.-lactams Staphylococci 169
vanH, vanA, vanX Vancomycin Enterococci 170 satA Macrolides
Enterococci 173 aacA-aphD Aminoglycosides Enterococci,
Staphylococci 174 vat Macrolides Staphylococci 175 vga Macrolides
Staphylococci 176 msrA Erythromycin Staphylococci 177 Int and Sul
.beta.-lactams, trimethoprim, Enterobacteriaceae, 171, 172
conserved sequences aminoglycosides, antiseptic, Pseudomonadaceae
chloramphenicol .sup.aBacteria having high incidence for the
specified antibiotic resistance genes. The presence in other
bacteria is not excluded.
[0099]
9 Annex I: Specific and ubiquitous oligonucleotides probes for
hybridization Originating DNA fragment SEQ ID NO Nucleotide
Sequence SEQ ID NO Nucleotide position Bacterial species:
Escherichia coli 44 5'-CAC CCG CTT GCG TGG CAA GCT GCC C 5.sup.a
213-237 45 5'-CGT TTG TGG ATT CCA GTT CCA TCC G 5.sup.a 489-513 48
5'-TGA AGC ACT GGC CGA AAT GCT GCG T 6.sup.a 759-783 49 5'-GAT GTA
CAG GAT TCG TTG AAG GCT T 6.sup.a 898-922 50 5'-TAG CGA AGG CGT AGC
AGA AAC TAA C 7.sup.a 1264-1288 51 5'-GCA ACC CGA ACT CAA CGC CGG
ATT T 7.sup.a 1227-1251 52 5'-ATA CAC AAG GGT CGC ATC TGC GGC C
7.sup.a 1313-1337 53 5'-TGC GTA TGC ATT GCA GAC CTT GTG GC 7.sup.a
111-136 54 5'-GCT TTC ACT GGA TAT CGC GCT TGG G 7.sup.a 373-397
Bacterial species: Proteus mirabilis 70.sup.b 5'-TGG TTC ACT GAC
TTT GCG ATG TTT C 12 23-47 71 5'-TCG AGG ATG GCA TGC ACT AGA AAA T
12 53-77 72.sup.b 5'-CGC TGA TTA GGT TTC GCT AAA ATC TTA TTA 12
80-109 73 5'-TTG ATC CTC ATT TTA TTA ATC ACA TGA CCA 12 174-203
.sup.aSequences from data banks .sup.bThese sequences are from the
opposite DNA strand of the sequences given in the Sequence
listing
[0100]
10 Annex I: Specific and ubiquitous oligonucleotides probes for
hybridization Originating DNA fragment SEQ ID NO Nucleotide
Sequence SEQ ID NO Nucleotide position Bacterial species: Proteus
mirabilis 76 5'-CCG CCT TTA GCA TTA ATT GGT GTT TAT AGT 13 246-275
77 5'-CCT ATT GCA GAT ACC TTA AAT GTC TTG GGC 13 291-320 80.sup.b
5'-TTG AGT GAT GAT TTC ACT GAC TCC C 14 18-42 81 5'-GTG AGA CAG TGA
TGG TGA GGA CAC A 15.sup.a 1185-1209 82 5'-TGG TTG TCA TGC TGT TTG
TGT GAA AAT 15.sup.a 1224-1230 Bacterial species: Klebsiella
pneumoniae 57 5'-GTG GTG TCG TTC AGG GGT TTC AC 8 45-67 58 5'-GCG
ATA TTC ACA CCC TAC GCA GCC A 9 161-185 59.sup.b 5'-GTC GAA AAT GCC
GGA AGA GGT ATA CG 9 203-228 60.sup.b 5'-ACT GAG CTG CAG ACC GGT
AAA ACT CA 9 233-258 63.sup.b 5'-CGT GAT GGA TAT TCT TAA CGA AGG GC
10 250-275 64.sup.b 5'-ACC AAA CTG TTG AGC CGC CTG GA 10 201-223 65
5'-GTG ATC GCC CCT CAT CTG CTA CT 10 77-99 66 5'-CGC CCT TCG TTA
AGA ATA TCC ATC AC 10 249-274 69 5'-CAG GAA GAT GCT GCA CCG GTT GTT
G 11.sup.a 296-320 .sup.aSequences from data banks .sup.bThese
sequences are from the opposite DNA strand of the sequences given
in the Sequence listing
[0101]
11 Annex I: Specific and ubiquitous oligonucleotides probes for
hybridization Originating DNA fragment SEQ ID NO Nucleotide
Sequence SEQ ID NO Nucleotide position Bacterial species:
Pseudomonas aeruginosa 87 5'-AAT GCG GCT GTA CCT CGG CGC TGG T
18.sup.a 2985-3009 88 5'-GGC GGA GGG CCA GTT GCA CCT GCC A 18.sup.a
2929-2953 89 5'-AGC CCT GCT CCT CGG CAG CCT CTG C 18.sup.a
2821-2845 90 5'-TGG CTT TTG CAA CCG CGT TCA GGT T 18.sup.a
1079-1103 91 5'-GCG CCC GCG AGG GCA TGC TTC GAT G 19.sup.a 705-729
92 5'-ACC TGG GCG CCA ACT ACA AGT TCT A 19.sup.a 668-692 93 5'-GGC
TAC GCT GCC GGG CTG CAG GCC G 19.sup.a 505-529 94 5'-CCG ATC TAG
ACC ATC GAG ATG GGC G 20.sup.a 1211-1235 95 5'-GAG CGC GGC TAT GTG
TTC GTC GGC T 20.sup.a 2111-2135 Bacterial species: Streptococcus
pneumoniae 120 5'-TCT GTG CTA GAG ACT GCC CCA TTT C 30 423-447 121
5'-CGA TGT CTT GAT TGA GCA GGG TTA T 31.sup.a 1198-1222
.sup.aSequences from data banks .sup.bThese sequences are from the
opposite DNA strand of the sequences given in the Sequence
listing
[0102]
12 Annex I: Specific and ubiquitous oligonucleotides probes for
hybridization Originating DNA fragment SEQ ID NO Nucleotide
Sequence SEQ ID NO Nucleotide position Bacterial species:
Staphylococcus saprophyticus 96 5'-CGT TTT TAC CCT TAC CTT TTC GTA
CTA CC 21 45-73 97.sup.b 5'-TCA GGC AGA GGT AGT ACG AAA AGG TAA GGG
21 53-82 100 5'-CAC CAA GTT TGA CAC GTG AAG ATT CAT 22 89-115
101.sup.b 5'-ATG AGT GAA GCG GAG TCA GAT TAT GTG CAG 23 105-134 102
5'-CGC TCA TTA CGT ACA GTG ACA ATC G 24 20-44 103 5'-CTG GTT AGC
TTG ACT CTT AAC AAT CTT GTC 24 61-90 104.sup.b 5'-GAC GCG ATT GTC
ACT GTA CGT AAT GAG CGA 24 19-48 Bacterial species: Moraxella
catarrhalis 108 5'-GCC CCA AAA CAA TGA AAC ATA TGG T-3' 28 81-105
109 5'-CTG CAG ATT TTG GAA TCA TAT CGC C-3' 28 126-130 110 5'-TGG
TTT GAC CAG TAT TTA ACG CCA T-3' 28 165-189 111 5'-CAA CGG CAC CTG
ATG TAC CTT GTA C-3' 28 232-256 114 5'-TTA CAA CCT GCA CCA CAA GTC
ATC A-3' 29 97-121 115 5'-GTA CAA ACA AGC CGT CAG CGA CTT A-3' 29
139-163 116 5'-CAA TCT GCG TGT GTG CGT TCA CT-3' 29 178-200 117
5'-GCT ACT TTG TCA GCT TTA GCC ATT CA-3' 29 287-312 .sup.aSequences
from data banks .sup.bThese sequences are from the opposite DNA
strand of the sequences given in the Sequence listing
[0103]
13 Annex I: Specific and ubiquitous oligonucleotides probes for
hybridization Originating DNA fragment SEQ ID Nucleotide SEQ ID NO
Nucleotide sequence NO position Bacterial species: Haemophilus
influenzae 105.sup.b 5'-GCG TCA GAA AAA GTA GGC GAA ATG AAA G 25
138-165 106.sup.b 5'-AGC GGC TCT ATC TTG TAA TGA CAC A 26.sup.a
770-794 107.sup.b 5'-GAA ACG TGA ACT CCC CTC TAT ATA A 27.sup.a
5184-5208 Universal probes.sup.c 122.sup.b 5'-ATC CCA CCT TAG GCG
GCT GGC TCC A -- -- 123 5'-ACG TCA AGT CAT CAT GGC CCT TAC GAG TAG
G -- -- 124.sup.b 5'-GTG TGA CGG GCG GTG TGT ACA AGG C -- --
125.sup.b 5'-GAG TTG CAG ACT CCA ATC CGG ACT ACG A -- -- 128.sup.b
5'-CCC TAT ACA TCA CCT TGC GGT TTA GCA GAG AG -- -- 129 5'-GGG GGG
ACC ATC CTC CAA GGC TAA ATA C -- -- 130.sup.b 5'-CGT CCA CTT TCG
TGT TTG CAG AGT GCT GTG TT -- -- .sup.aSequences from data banks
.sup.bThese sequences are from the opposite DNA strand of the
sequences given in the Sequence listing .sup.cUniversal probes were
derived from 16S or 23S ribosomal RNA gene sequences not included
in the Sequence listing
[0104]
14 Annex II: Specific and ubiquitous primers for DNA amplification
Originating DNA fragment SEQ ID NO Nucleotide Sequence SEQ ID NO
Nucleotide position Bacterial species: Escherichia coli 42 5'-GCT
TTC CAG CGT CAT ATT G 4 177-195 43.sup.b 5'-GAT CTC GAC AAA ATG GTG
A 4 260-278 46 5'-TCA CCC GCT TGC GTG GC 5.sup.a 212-228 47.sup.b
5'-GGA ACT GGA ATC CAC AAA C 5.sup.a 490-508 55 5'-GCA ACC CGA ACT
CAA CGC C 7.sup.a 1227-1245 56.sup.b 5'-GCA GAT GCG ACC CTT GTG T
7.sup.a 1315-1333 131 5'-CAG GAG TAC GGT GAT TTT TA 3 60-79
132.sup.b 5'-ATT TCT GGT TTG GTC ATA CA 3 174-193 Bacterial
species: Enterococcus faecalis 38 5'-GCA ATA CAG GGA AAA ATG TC
1.sup.a 69-88 39.sup.b 5'-CTT CAT CAA ACA ATT AAC TC 1.sup.a
249-268 40 5'-GAA CAG AAG AAG CCA AAA AA 2.sup.a 569-588 41.sup.b
5'-GCA ATC CCA AAT AAT ACG GT 2.sup.a 670-689 .sup.aSequences from
data banks .sup.b These sequences are from the opposite DNA strand
of the sequences given in the Sequence listing
[0105]
15 Annex II: Specific and ubiquitous primers for DNA amplification
Originating DNA fragment SEQ ID NO Nucleotide Sequence SEQ ID NO
Nucleotide position Bacterial species: Klebsiella pneumoniae 61
5'-GAC AGT CAG TTC GTC AGC C 9 37-55 62.sup.b 5'-CGT AGG GTG TGA
ATA TCG C 9 161-179 67 5'-TCG CCC CTC ATC TGC TAC T 10 81-99
68.sup.b 5'-GAT CGT GAT GGA TAT TCT T 10 260-278 135 5'-GCA GCG TGG
TGT CGT TCA 8 40-57 136.sup.b 5'-AGC TGG CAA CGG CTG GTC 8 170-187
137 5'-ATT CAC ACC CTA CGC AGC CA 9 166-185 138.sup.b 5'-ATC CGG
CAG CAT CTC TTT GT 9 262-281 Bacterial species: Proteus mirabilis
74 5'-GAA ACA TCG CAA AGT CAG T 12 23-41 75.sup.b 5'-ATA AAA TGA
GGA TCA AGT TC 12 170-189 133 5'-CGG GAG TCA GTG AAA TCA TC 14
17-36 134.sup.b 5'-CTA AAA TCG CCA CAC CTC TT 14 120-139
.sup.aSequences from data banks .sup.bThese sequences are from the
opposite DNA strand of the sequences given in the Sequence
listing
[0106]
16 Annex II: Specific and ubiquitous primers for DNA amplification
Originating DNA fragment SEQ ID NO Nucleotide Sequence SEQ ID NO
Nucleotide position Bacterial species: Staphylococcus saprophyticus
98 5'-CGT TTT TAC CCT TAC CTT TTC GTA CT 21 45-70 99.sup.b 5'-ATC
GAT CAT CAC ATT CCA TTT GTT TTT A 21 143-170 139 5'-CTG GTT AGC TTG
ACT CTT AAC AAT C 24 61-85 140.sup.b 5'-TCT TAA CGA TAG AAT GGA GCA
ACT G 24 226-250 Bacterial species: Pseudomonas aeruginosa 83
5'-CGA GCG GGT GGT GTT CAT C 16.sup.a 554-572 84.sup.b 5'-CAA GTC
GTG GTG GGA GGG A 16.sup.a 674-692 85 5'-TCG CTG TTC ATC AAG ACC C
17.sup.a 1423-1441 86.sup.b 5'-CCG AGA ACC AGA CTT CAT C 17.sup.a
1627-1645 Bacterial species: Moraxella catarrhalis 112 5'-GGC ACC
TGA TGT ACC TTG 28 235-252 113.sup.b 5'-AAC AGC TCA CAC GCA TT 28
375-391 118 5'-TGT TTT GAG CTT TTT ATT TTT TGA 29 41-64 119.sup.b
5'-CGC TGA CGG CTT GTT TGT ACC A 29 137-158 160 5'-GCT CAA ATC AGG
GTC AGC 29 22-39 119.sup.b 5'-CGC TGA CGG CTT GTT TGT ACG A 29
137-158 .sup.aSequences from data banks .sup.bThese sequences are
from the opposite DNA strand of the sequences given in the Sequence
listing
[0107]
17 Annex II: Specific and ubiquitous primers for DNA amplification
Originating DNA fragment SEQ ID NO Nucleotide Sequence SEQ ID NO
Nucleotide position Bacterial species: Staphylococcus epidermidis
145 5'-ATC AAA AAG TTG GCG AAC CTT TTC A 36 21-45 146.sup.b 5'-CAA
AAG AGC GTG GAG AAA AGT ATC A 36 121-145 147 5'-TCT CTT TTA ATT TCA
TCT TCA ATT CCA TAG 36 448-477 148.sup.b 5'-AAA CAC AAT TAC AGT CTG
GTT ATC CAT ATC 36 593-622 Bacterial species: Staphylococcus aureus
149.sup.b 5'-CTT CAT TTT ACG GTG ACT TCT TAG AAG ATT 37 409-438 150
5'-TCA ACT GTA GCT TCT TTA TCC ATA CGT TGA 37 288-317 149.sup.b
5'-CTT CAT TTT ACG GTG ACT TCT TAG AAG ATT 37 409-438 151 5'-ATA
TTT TAG CTT TTC AGT TTC TAT ATC AAC 37 263-292 152 5'-AAT CTT TGT
CGG TAC ACG ATA TTC TTC ACG 37 5-34 153.sup.b 5'-CGT AAT GAG ATT
TCA GTA GAT AAT ACA ACA 37 83-112 .sup.aSequences from data banks
.sup.bThese sequences are from the opposite DNA strand of the
sequences given in the Sequence listing
[0108]
18 Annex II: Specific and ubiquitous primers for DNA amplification
Originating DNA fragment SEQ ID NO Nucleotide Sequence SEQ ID NO
Nucleotide position Bacterial species: Haemophilus influenzae 154
5'-TTT AAC GAT CCT TTT ACT CCT TTT G 27.sup.a 5074-5098 155.sup.b
5'-ACT GCT GTT GTA AAG AGG TTA AAA T 27.sup.a 5266-5290 Bacterial
species: Streptococcus pneumoniae 78 5'-AGT AAA ATG AAA TAA GAA CAG
GAC AG 34 164-189 79.sup.b 5'-AAA ACA GGA TAG GAG AAC GGG AAA A 34
314-338 156 5'-ATT TGG TGA CGG GTG ACT TT 31.sup.a 1401-1420
157.sup.b 5'-GCT GAG GAT TTG TTC TTC TT 31.sup.a 1515-1534 158
5'-GAG CGG TTT CTA TGA TTG TA 35.sup.a 1342-1361 159.sup.b 5'-ATC
TTT CCT TTC TTG TTC TT 35.sup.a 1519-1538 Bacterial species:
Streptococcus pyogenes 141 5'-TGA AAA TTC TTG TAA CAG GC 32.sup.a
286-305 142.sup.b 5'-GGC CAC CAG CTT GCC CAA TA 32.sup.a 479-498
143 5'-ATA TTT TCT TTA TGA GGG TG 33.sup.a 966-985 144.sup.b 5'-ATC
CTT AAA TAA AGT TGC CA 33.sup.a 1103-1122 .sup.aSequences from data
banks .sup.bThese sequences are from the opposite DNA strand of the
sequences given in the Sequence listing
[0109]
19 Annex II: Specific and ubiquitous primers for DNA amplification
Originating DNA fragment SEQ ID NO Nucleotide Sequence SEQ ID NO
Nucleotide position Universal primers.sup.c 126 5'-GGA GGA AGG TGG
GGA TGA CG -- -- 127.sup.b 5'-ATG GTG TGA CGG GCG GTG TG -- --
.sup.a Sequences from data banks .sup.b These sequences are from
the opposite DNA strand of the sequences given in the Sequence
listing .sup.c Universal primers were derived from the 16S
ribosomal RNA gene sequence not included in the Sequence
listing
[0110]
20 Annex III Selection of Universal Probes by Alignment of the
Sequences of Bacterial 16S and 23S Ribosomal RNA Genes. Reverse
strand of SEQ ID NO: 122 TGGAGCC AGCCGCCTA GGTGGGAT 1461 1510
Streptococcus salivarius TGAGGTAACC TTTTGGAGCC AGCCGCCTAA
GGTGGGATAG ATGANNGGGG Proteus vulgaris TAGCTTAACC TTCGGGAGGG
CGCTTACCAC TTTGTGATTC ATGACTGGGG Pseudomonas aeruginosa TAGTCTAACC
GCAAGGGGGA CGGTTACCAC GGAGTGATTC ATGACTGGGG Neisseria gonorrhoeae
TAGGGTAACC GCAAGGAGTC CGCTTACCAC GGTATGCTTC ATGACTGGGG
Streptococcus lactis TTGCCTAACC GCAAGGAGGG CGCTTCCTAA GGTAAGACCG
ATGACNNGGG
[0111]
21 Annex III. Selection of universal probes by alignment of the
sequences of bacterial 165 and 23S ribosomal RNA genes. SEQ ID NO:
123 ACGTCAAGTC ATCATGGC CCTTACGAGT AGG 1251 1300 Haemophilus
influenzae GGTNGGGATG ACGTCAAGTC ..ATCATGGC CCTTACGAGT AGGGCTACAC
Neisseria gonorrhoeae GGTGGGGATG ACGTCAAGTC ..CTCATGGC CCTTATGACC
AGGGCTTCAC Pseudomonas cepacia GGTNGGGATG ACGTCAAGTC ..CTCATGGC
CCTTATGGGT AGGGCTTCAC Serratia marcescens GGTGGGGATG ACGTCAAGTC
..ATCATGGC CCTTACGAGT AGGGCTACAC Escherichia coli GGTGGGGATG
ACGTCAAGTC ..ATCATGGC CCTTACGACC AGGGCTACAC Proteus vulgaris
GGTGGGGATG ACGTTAAGTC GTATCATGGC CCTTACGAGT AGGGCTACAC Pseudomonas
aeruginosa GGTGGGGATG ACGTCAAGTC ..ATCATGGC CCTTACGGCN AGGGCTACAC
Clostridium perfringens GGTGGGGATG ACGTNNAATC ..ATCATGCC CNTTATGTGT
AGGGCTACAC Mycoplasma hominis GGTGGGGATG ACGTCAAATC ..ATCATGCC
TCTTACGAGT GGGGCCACAC Helicobacter pylori GGTGGGGACG ACGTCAAGTC
..ATCATGGC CCTTACGCCT AGGGCTACAC Mycoplasma pneumoniae GGAAGGGATG
ACGTCAAATC ..ATCATGCC CCTTATGTCT AGGGCTGCAA
[0112]
22 Annex III. Selection of universal probes by alignment of the
sequences of bacterial 16S and 23S ribosomal RNA genes. Reverse of
the probe SEQ ID NO: 124 GCCTTGTACA CACCGCCCGT CACAC 1451 1490
Escherichia coli ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG
Neisseria gonorrhoeae ACGTTCCCNG NNCTTGTACA CACCGCCCGT CACACCATGG
Pseudomonas cepacia ACGTTCCCGG GTCTTGTACA CACNGCCCGT CACACCATGG
Serratia marcescens ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG
Proteus vulgaris ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG
Haemophilus influenzae ACGTTCCCGG GCNTTGTACA CACCGCCCGT CACACCATGG
Pseudomonas aeruginosa ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG
Clostridium perfringens ACGTTCCCNG GTCTTGTACA CACCGCNCGT CACACCATGA
Mycoplasma hominis ACGTTCTCGG GTCTTGTACA CACCGCCCGT CACACCATGG
Helicobacter pylori ACGTTCCCGG GTCTTGTACT CACCGCCCGT CACACCATGG
Mycoplasma pneumoniae ACGTTCTCGG GTCTTGTACA CACCGCCCGT
CAAACTATGA
[0113]
23 Annex III. Selection of universal probes by alignment of the
sequences of bacterial 16S and 23S ribosomal RNA genes. Reverse
strand of SEQ ID NO 125: TCG TAGTCCGGAT TGGAGTCTGC AACTC 1361 1400
Escherichia coli AAGTGCGTCG TAGTCCGGAT TGGAGTCTGC AACTCGACTC
Neisseria gonorrhoeae AAACCGATCG TAGTCCGGAT TGCACTCTGC AACTCGAGTG
Pseudomonas cepacia AAACCGATCG TAGTCCGGAT TGCACTCTGC AACTCGAGTG
Serratia marcescens AAGTATGTCG TAGTCCGGAT TGGAGTCTGC AACTCGACTC
Proteus vulgaris AAGTCTGTCG TAGTCCGGAT TGGAGTCTGC AACTCGACTC
Haemophilus influenzae AAGTACGTCT AAGTCCGGAT TGGAGTCTGC AACTCGACTC
Pseudomonas aeruginosa AAACCGATCG TAGTCCGGAT CGCAGTCTGC AACTCGACTG
Clostridium perfringens AAACCAGTCT CAGTTCGGAT TGTAGGCTGA AACTCGCCTA
Mycoplasma hominis AAGCCGATCT CAGTTCGGAT TGGAGTCTGC AATTCGACTC
Helicobacter pylori ACACC..TCT CAGTTCGGAT TGTAGGCTGC AACTCGCCTG
Mycloplasma pneumoniae AAGTTGGTCT CAGTTCGGAT TGAGGGCTGC
AATTCGTCCT
[0114]
24 Annex III. Selection of universal probes by alignment of the
sequences of bacterial 16S and 23S ribosomal RNA genes. Reverse
strand of SEQ ID NO: 128 CT CTCTGCTAAA CCGCAAGGTG ATGTATAGGG 1991
2040 Lactobacillus lactis AAACACAGCT CTCTGCTAAA CCGCAAGGTG
ATGTATAGGG GGTGACGCCT Escherichia coli AAACACAGCA CTGTGCAAAC
ACGAAAGTGG ACGTATACGG TGTGACGCCT Pseudomonas aeruginosa AAACACAGCA
CTCTGCAAAC ACGAAAGTGG ACGTATAGGG TGTGACGCCT Pseudomonas cepacia
AAACACAGCA CTCTGCAAAC ACGAAAGTGG ACGTATAGGG TGTGACGCCT Bacillus
stearothermophilus AAACACAGGT CTCTGCGAAG TCGTAAGGCG ACGTATAGGG
GCTGACACCT Micrococcus luteus AAACACAGGT CCATGCGAAG TCGTAAGACG
ATGTATATGG ACTGACTCCT SEQ ID NO: 129 GGGGGGACC ATCCTCCAAG GCTAAATAC
481 530 Escherichia coli TGTCTGAATA TGGGGGGACC ATCCTCCAAG
GCTAAATACT CCTGACTGAC Pseudomonas aeruginosa TGTCTGAACA TGGGGGGACC
ATCCTCCAAG GCTAAATACT ACTGACTGAC Pseudomonas cepacia TGTCTGAAGA
TGGGGGGACC ATCCTCCAAG GCTAAATACT CGTGATCGAC Lactobacillus lactis
AGTTTGAATC CGGGAGGACC ATCTCCCAAC CCTAAATACT CCTTAGTGAC Micrococcus
luteus CGTGTGAATC TGCCAGGACC ACCTGGTAAG CCTGAATACT ACCTGTTGAC
[0115]
25 Annex III. Selection of universal probes by alignment of the
sequences of bacterial 16S and 23S ribosomal RNA genes. Reverse
strand of SEQ ID NO: 130 AACACAGCA CTCTGCAAAC ACGAAAGTGG ACG 1981
2030 Pseudomonas aeruginosa TGTTTATTAA AAACACAGCA CTCTGCAAAC
ACGAAAGTGG ACGTATAGGG Escherichia coli TGTTTATTAA AAACACAGCA
CTGTGCAAAC ACGAAAGTGG ACGTATACGG Pseudomonas cepacia TGTTTAATAA
AAACACAGCA CTCTGCAAAC ACGAAAGTGG ACGTATAGGG Bacillus
stearothermophilus TGTTTATCAA AAACACAGGT CTCTGCGAAG TCGTAAGGCG
ACGTATAGGG Lactobacillus lactis TGTTTATCAA AAACACAGCT CTCTGCTAAA
CCACAAGGTG ATGTATAGGG Micrococcus luteus TGTTTATCAA AAACACAGGT
CCATGCGAAG TCGTAAGACG ATGTATATGG
[0116]
26 Annex IV. Selection of the universal PCR primers by alignment of
the bacterial 16S ribosomal RNA gene SEQ ID NO: 126 GGAGGAA
GGTGGGGATG ACG Reverse strand of SEQ ID NO: 127 CA CACCGCCCGT
CACACCAT 1241 1270......1461 1490 Escherichia coli ACTGGAGGAA
GGTGGGGATG ACGTCAAGTC......GCCTTGTACA CACCGCCCGT CACACCATGG
Neisseria gonorrhoeae GCCGGAGGAA GGTGGGGATG
ACGTCAAGTC......NNCTTGTACA CACCGCCCGT CACACCATGG Pseudomonas
cepacia ACCGGAGGAA GGTNGGGATG ACGTCAAGTC......GTCTTGTACA CACNGCCCGT
CACACCATGG Seratia marcescens ACTGGAGGAA GGTGGGGATG
ACGTCAAGTC......GCCTTGTACA CACCGCCCGT CACACCATGG Proteus vulgaris
ACCGGAGGAA GGTGGGGATG ACGTTAAGTC......GCCTTGTACA CACCGCCCGT
CACACCATGG Haemophilus influenzae ACTGGAGGAA GGTNGGGATG
ACGTCAAGTC......GCNTTGTACA CACCGCCCGT CACACCATGG Legionella
pneumophila ACCGGAGGAA GGCGGGGATG ACGTCAAGTC......GCCTTGTACA
CACCGCCCGT CACACCATGG Pseudomonas aeruginos ACCGGAGGAA GGTGGGGATG
ACGTCAAGTC......GCCTTGTACA CACCGCCCGT CACACCATGG Clostridium
perfringens CCAGGAGGAA GGTGGGGATG ACGTNNAATC......GTCTTGTAC- A
CACCGCNCGT CACACCATGA Mycoplasma hominis CTGGGAGGAA GGTGGGGATG
ACGTCAAATC......GTCTTGTACA CACCGCCCGT CACACCATGG Helicobacter
pylori GGAGGAGGAA GGTGGGGACG ACGTCAAGTC......GTCTTGTACT CACCGCCCGT
CACACCATGG Mycoplasma pneumoniae ATTGGAGGAA GGAAGGGATG
ACGTCAAATC......GTCTTGTACA CACCGCCCGT CAAACTATGA
[0117]
Sequence CWU 1
1
177 1 1817 DNA Enterococcus faecalis 1 acagtaaaaa agttgttaac
gaatgaattt gttaacaact tttttgctat ggtattgagt 60 tatgaggggc
aatacaggga aaaatgtcgg ctgattaagg aatttagata gtgccggtta 120
gtagttgtct ataatgaaaa tagcaacaaa tatttacgca gggaaagggg cggtcgttta
180 acgggaaaaa ttagggagga taaagcaata cttttgttgg gaaaagaaat
aaaaggaaac 240 tggggaagga gttaattgtt tgatgaaggg aaataaaatt
ttatacattt taggtacagg 300 catctttgtt ggaagttcat gtctattttc
ttcacttttt gtagccgcag aagaacaagt 360 ttattcagaa agtgaagttt
caacagtttt atcgaagttg gaaaaggagg caatttctga 420 ggcagctgct
gaacaatata cggttgtaga tcgaaaagaa gacgcgtggg ggatgaagca 480
tcttaagtta gaaaagcaaa cggaaggcgt tactgttgat tcagataatg tgattattca
540 tttagataaa aacggtgcag taacaagtgt tacaggaaat ccagttgatc
aagttgtgaa 600 aattcaatcg gttgatgcaa tcggtgaaga aggagttaaa
aaaattgttg cttctgataa 660 tccagaaact aaagatcttg tctttttagc
tattgacaaa cgtgtaaata atgaagggca 720 attattttat aaagtcagag
taacttcttc accaactggt gaccccgtat cattggttta 780 taaagtgaac
gctacagatg gaacaattat ggaaaaacaa gatttaacgg aacatgtcgg 840
tagtgaagta acgttaaaaa actcttttca agtaacgttt aatgtaccag ttgaaaaaag
900 caatacggga attgctttac acggaacgga taacacaggg gtttaccatg
cagtagttga 960 tggcaaaaat aattattcta ttattcaagc gccatcacta
gcgacattaa atcagaatgc 1020 tattgacgcc tatacgcatg gaaaatttgt
gaaaacatat tatgaagatc atttccaacg 1080 acacagtatt gatgatcgag
ggatgcccat cttgtcagtt gttgatgaac aacatccaga 1140 tgcttatgac
aatgcttttt gggatggaaa agcaatgcgt tatggtgaaa caagtacacc 1200
aacaggaaaa acgtatgctt cctctttaga tgtagttggt catgaaatga cacatggtgt
1260 gacggaacat actgccggtt tagaatattt aggacaatca ggtgccttga
atgaatctta 1320 ttctgatttg atgggttata ttatttcggg tgcatctaat
ccagaaattg gtgcggatac 1380 tcagagtgtt gaccgaaaaa caggtattcg
aaatttacaa acgccaagta aacacggaca 1440 accagaaacc atggctcaat
acgacgatcg agcacggtat aaaggaacgc cttattatga 1500 tcaaggcggt
gttcattata acagtggaat tattaatcgg attggttaca ccattatcca 1560
gaacttaggc attgaaaaag cacagactat tttctacagc tcgttagtaa attacttaac
1620 acctaaagca caattcagtg atgctcgtga tgcgatgctt gctgctgcaa
aagttcaata 1680 tggcgatgaa gcagcttcag tggtgtcagc agcctttaac
tctgctggaa tcggagctaa 1740 agaagacatt caggtaaacc aaccaagtga
atctgttctg gtcaatgaat gaaaaaaatt 1800 ccccaattaa ataaaaa 1817 2
2275 DNA Enterococcus faecalis 2 ggtaccaaag aaaaaaacga acgccacaac
caacagcctc taaagcaaca cctgcttctg 60 aaattgaggg agatttagca
aatgtcaatg agattctttt ggttcacgat gatcgtgtcg 120 ggtcagcaac
gatgggaatg aaagtcttag aagaaatttt agataaagag aaaatttcaa 180
tgccgattcg aaaaattaat attaatgaat taactcaaca aacacaggct ttaattgtca
240 caaaagctga actaacggaa caagcacgta aaaaagcacc gaaagcgaca
cacttatcag 300 taaaaagtta tggttaatcc ccaaaaatat gaaacagtgg
gtttcgctct taaaagaaag 360 tgcctagaga ggaagaaaac aatggaaaat
cttacgaata tttcaattga attaaatcaa 420 cagtttaata caaaagaaga
agctattcgc ttttccggcc agaaactagt cgaggcaggc 480 tgtgttgagc
ccgcttatat cgaagcaatg attgaaagag accaattgct atctgcccat 540
atggggaatt ttattgccat tcctcatgga acagaagaag ccaaaaaatt agtgaaaaaa
600 tcaggaatct gtgtagtgca agtcccagag ggcgttaatt ttggcaccga
agaagatgaa 660 aaaattgcta ccgtattatt tgggattgcc ggagtcggtg
aagaacattt gcaattagtc 720 caacaaattg cactttattg tagtgatatg
gataacgtgg tgcaacttgc cgatgcatta 780 agtaaagaag aaataacaga
aaatttagcc attgcttaaa ggagagaata agaatgaacg 840 cagtacattt
tggagcagga aatattggac gcggctttat tggcgaaatt ttagctaaaa 900
cgggtttcat attaccgttt gtggatgtta atggaaacca tcatcaagcg ttaaaagaac
960 gtaaaagtta tacaattgaa ttggccgatg cctcacatca acaaattaac
gttgaaaatg 1020 tgaccgggtt aaataacatg acagaaccag aaaaagtagt
agaagcaatt gcggaagccg 1080 atttagtcac gacggcaatt ggtcctaata
ttttaccaag aattgctgaa ttaattgctc 1140 aaggaattga tgcacgtgcc
gaagcaaatt gtcaaaacgg cccgctggat attatcgctt 1200 gtgaaaatat
gattggtggt tcaacctttt tagcagaaga agtggccata atatttgaaa 1260
aacccagctt atctgaacaa tggattggtt ttcctgatgc ggcagttgat cggattgttc
1320 cattacaaaa acataaagat ccactttttg ttcaagttga gcctttttgt
gaatgggtca 1380 ttgatgatac caaccgaaaa gccaaagaga ttcagttaga
aggcgtcatt acttgtcgat 1440 tagagccgta tattgaacga aaattattta
gtgtaaccag tggccatgct acagttgcct 1500 atacaggggc gttgttaggc
tatcaaacca ttgacgaagc gatgcaggac gccttagtgg 1560 tagcgcaact
caaatcagtt ttgcaggaaa ccggtaaact tttagtggcc aaatggaatt 1620
ttgatgaaca agaacatgca gcctatattg aaaaaattat caaccgtttc caaaataaat
1680 atatttcaga tgctattaca cgtgtagcac ggacaccaat cagaaaatta
ggtgcgcaag 1740 aacggtttat tcgaccaatc cgtgaattac aggaacgcaa
tctagtgtcg gccgcattta 1800 tagcaatgat tggtattgtc tttaattatc
atgatccaga agatgaacaa agccgtcaat 1860 tacaggaaat gcttgaccaa
gaaagtgttg atacagtgga tcgctgaagt aacgggcatt 1920 gaagatccag
aaacggttaa aaatattaaa caaaacgtag aactgctatg cgcgaccaca 1980
agtagcataa ttaacaaaat ccttctacca agatacttca catttcttaa ttaaagaaaa
2040 aacaaccgcg cctcacctga gccgaccccc aaaagttaga cctagaaatc
taacttttgg 2100 aggttttttt gtatggcaaa atacagtttt gaaatttaaa
cttaaacttg ttcatgacta 2160 cttatatggt caaggaggtc taaggtttct
cgcaaagaag tatgggttta aagatagtct 2220 caaataagca aatggataaa
tgcctataaa gaacttggtg aagaaggggg gatcc 2275 3 227 DNA Escherichia
coli 3 gatccgccat gggttgtttt ccgattgagg attttataga tggtttctgg
cgacctgcac 60 aggagtacgg tgatttttaa ttattgcaat tgcacaagag
tcagttctcc cccaaagaca 120 gcaccggtat caatataatg caggttgcca
atatccacgc gatggcgcaa aggtgtatga 180 ccaaaccaga aatgatcggc
cacctgcatc gccagttcgc gagtcgg 227 4 278 DNA Escherichia coli 4
gatctaaatc aaattaattg gttaaagata accacagcgg ggccgacata aactctgaca
60 agaagttaac aaccatataa cctgcacagg acgcgaacat gtcttctcat
ccgtatgtca 120 cccagcaaaa taccccgctg gcggacgaca ccactctgat
gtccactacc gatctcgctt 180 tccagcgtca tattggggcg cgctacgttg
gggcgtgggc gtaattggtc aatcaggcgc 240 ggggtcagcg gataaacatt
caccattttg tcgagatc 278 5 1596 DNA Escherichia coli 5 atggctgaca
ttctgctgct cgataatatc gactctttta cgtacaacct ggcagatcag 60
ttgcgcagca atgggcataa cgtggtgatt taccgcaacc atataccggc gcaaacctta
120 attgaacgct tggcgaccat gagtaatccg gtgctgatgc tttctcctgg
ccccggtgtg 180 ccgagcgaag ccggttgtat gccggaactc ctcacccgct
tgcgtggcaa gctgcccatt 240 attggcattt gcctcggaca tcaggcgatt
gtcgaagctt acgggggcta tgtcggtcag 300 gcgggcgaaa ttctccacgg
taaagcctcc agcattgaac atgacggtca ggcgatgttt 360 gccggattaa
caaacccgct gccggtggcg cgttatcact cgctggttgg cagtaacatt 420
ccggccggtt taaccatcaa cgcccatttt aatggcatgg tgatggcagt acgtcacgat
480 gcggatcgcg tttgtggatt ccagttccat ccggaatcca ttctcaccac
ccagggcgct 540 cgcctgctgg aacaaacgct ggcctgggcg cagcataaac
tagagccagc caacacgctg 600 caaccgattc tggaaaaact gtatcaggcg
cagacgctta gccaacaaga aagccaccag 660 ctgttttcag cggtggtgcg
tggcgagctg aagccggaac aactggcggc ggcgctggtg 720 agcatgaaaa
ttcgcggtga gcacccgaac gagatcgccg gggcagcaac cgcgctactg 780
gaaaacgcag cgccgttccc gcgcccggat tatctgtttg ctgatatcgt cggtactggc
840 ggtgacggca gcaacagtat caatatttct accgccagtg cgtttgtcgc
cgcggcctgt 900 gggctgaaag tggcgaaaca cggcaaccgt agcgtctcca
gtaaatctgg ttcgtccgat 960 ctgctggcgg cgttcggtat taatcttgat
atgaacgccg ataaatcgcg ccaggcgctg 1020 gatgagttag gtgtatgttt
cctctttgcg ccgaagtatc acaccggatt ccgccacgcg 1080 atgccggttc
gccagcaact gaaaacccgc accctgttca atgtgctggg gccattgatt 1140
aacccggcgc atccgccgct ggcgttaatt ggtgtttata gtccggaact ggtgctgccg
1200 attgccgaaa ccttgcgcgt gctggggtat caacgcgcgg cggtggtgca
cagcggcggg 1260 atggatgaag tttcattaca cgcgccgaca atcgttgccg
aactgcatga cggcgaaatt 1320 aaaagctatc agctcaccgc agaagacttt
ggcctgacac cctaccacca ggagcaactg 1380 gcaggcggaa caccggaaga
aaaccgtgac attttaacac gtttgttaca aggtaaaggc 1440 gacgccgccc
atgaagcagc cgtcgctgcg aacgtcgcca tgttaatgcg cctgcatggc 1500
catgaagatc tgcaagccaa tgcgcaaacc gttcttgagg tactgcgcag tggttccgct
1560 tacgacagag tcaccgcact ggcggcacga gggtaa 1596 6 2703 DNA
Escherichia coli 6 gacgacttag ttttgacgga atcagcatag ttaatcactt
cactgtggaa aatgaggaaa 60 tattattttt tttgcgcttc gtaattaatg
gttataaggt cggccagaaa cctttctaat 120 gcaagcgatg acgttttttt
atgtgtctga atttgcactg tgtcacaatt ccaaatcttt 180 attaacaact
cacctaaaac gacgctgatc cagcgtgaat actggtttcc cttatgttca 240
tcagattcat ttaagcaagg gtttcttctt cattcctgat gaaagtgcca tctaaaaaga
300 tgatcttaat aaatctatta agaatgagat ggagcacact ggatatttta
cttatgaaac 360 tgtttcactc ctttacttaa tttatagagt taccttccgc
tttttgaaaa tacgcaacgg 420 ccattttttg cacttagata cagattttct
gcgctgtatt gcattgattt gatgctaatc 480 ctgtggtttg cactagcttt
aagtggttga gatcacattt ccttgctcat ccccgcaact 540 cctccctgcc
taatcccccg caggatgagg aaggtcaaca tcgagcctgg caaactagcg 600
ataacgttgt gttgaaaatc taagaaaagt ggaactccta tgtcacaacc tatttttaac
660 gataagcaat ttcaggaagc gctttcacgt cagtggcagc gttatggctt
aaattctgcg 720 gctgaaatga ctcctcgcca gtggtggcta gcagtgagtg
aagcactggc cgaaatgctg 780 cgtgctcagc cattcgccaa gccggtggcg
aatcagcgac atgttaacta catctcaatg 840 gagtttttga ttggtcgcct
gacgggcaac aacctgttga atctcggctg gtatcaggat 900 gtacaggatt
cgttgaaggc ttatgacatc aatctgacgg acctgctgga agaagagatc 960
gacccggcgc tgggtaacgg tggtctggga cgtctggcgg cgtgcttcct cgactcaatg
1020 gcaactgtcg gtcagtctgc gacgggttac ggtctgaact atcaatatgg
tttgttccgc 1080 cagtcttttg tcgatggcaa acaggttgaa gcgccggatg
actggcatcg cagtaactac 1140 ccgtggttcc gccacaacga agcactggat
gtgcaggtag ggattggcgg taaagtgacg 1200 aaagacggac gctgggagcc
ggagtttacc attaccggtc aagcgtggga tctccccgtt 1260 gtcggctatc
gtaatggcgt ggcgcagccg ctgcgtctgt ggcaggcgac gcacgcgcat 1320
ccgtttgatc tgactaaatt taacgacggt gatttcttgc gtgccgaaca gcagggcatc
1380 aatgcggaaa aactgaccaa agttctctat ccaaacgaca accatactgc
cggtaaaaag 1440 ctgcgcctga tgcagcaata cttccagtgt gcctgttcgg
tagcggatat tttgcgtcgc 1500 catcatctgg cggggcgtga actgcacgaa
ctggcggatt actaagttat tcagctgaac 1560 gatacccacc caactatcgc
gattccagaa ctgctgcgcg tgctgatcga tgagcaccag 1620 atgagctggg
atgacgcttg ggccattacc agcaaaactt tcgcttacac caaccatacc 1680
ctgatgccag aagcgctgga acgctgggat gtgaaactgg tgaaaggctt actgccgcgc
1740 cacatgcaga ttattaacga aattaatact cgctttaaaa cgctggtaga
gaaaacctgg 1800 ccgggcgatg aaaaagtgtg ggccaaactg gcggtggtgc
acgacaaaca agtgcatatg 1860 gcgaacctgt gtgtggttgg cggtttcgcg
gtgaacggtg ttgcggcgct gcactcggat 1920 ctggtggtga aagatctgtt
cccggaatat caccagctat ggccgaacaa attccataac 1980 gtcaccaacg
gtattacccc acgtcgctgg atcaaacagt gcaacccggc actggcggct 2040
ctgttggata aatcactgca aaaagagtgg gctaacgatc tcgatcagct gatcaatctg
2100 gttaaattgg ctgatgatgc gaaattccgt cagctttatc gcgtgatcaa
gcaggcgaat 2160 aaagtccgtc tggcggagtt tgtgaaagtt cgtaccggta
ttgacatcaa tccacaggcg 2220 attttcgata ttcagatcaa acgtttgcac
gagtacaaac gccagcacct gaatctgctg 2280 cgtattctgg cgttgtacaa
agaaattcgt gaaaacccgc aggctgatcg cgtaccgcgc 2340 gtcttcctct
tcggcgcgaa agcggcaccg ggctactacc tggctaagaa tattatcttt 2400
gcgatcaaca aagtggctga cgtgatcaac aacgatccgc tggttggcga taagttgaag
2460 gtggtgttcc tgccggatta ttgcgtttcg gcggcggaaa aactgatccc
ggcggcggat 2520 atctccgaac aaatttcgac tgcaggtaaa gaagcttccg
gtaccggcaa tatgaaactg 2580 gcgctcaatg gtgcgcttac tgtcggtacg
ctggatgggg cgaacgttga aatcgccgag 2640 aaagtcggtg aagaaaatat
ctttattttt ggtcatacgg tcaaacaagt gaaggcaatc 2700 gac 2703 7 1391
DNA Escherichia coli 7 agagaagcct gtcggcaccg tctggtttgc ttttgccact
gcccgcggtg aaggcattac 60 ccggcgggat gcttcagcgg cgaccgtgat
gcggtgcgtc gtcaggctac tgcgtatgca 120 ttgcagacct tgtggcaaca
atttctacaa aacacttgat actgtatgag catacagtat 180 aattgcttca
acagaacata ttgactatcc ggtattaccc ggcatgacag gagtaaaaat 240
ggctatcgac gaaaacaaac agaaagcgtt ggcggcagca ctgggccaga ttgagaaaca
300 atttggtaaa ggctccatca tgcgcctggg tgaagaccgt tccatggatg
tggaaaccat 360 ctctaccggt tcgctttcac tggatatcgc gcttggggca
ggtggtctgc cgatgggccg 420 tatcgtcgaa atctacggac cggaatcttc
cggtaaaacc acgctgacgc tgcaggtgat 480 cgccgcagcg cagcgtgaag
gtaaaacctg tgcgtttatc gatgctgaac acgcgctgga 540 cccaatctac
gcacgtaaac tgggcgtcga tatcgacaac ctgctgtgct cccagccgga 600
caccggcgag caggcactgg aaatctgtga cgccctggcg cgttctggcg cagtagacgt
660 tatcgtcgtt gactccgtgg cggcactgac gccgaaagcg gaaatcgaag
gcgaaatcgg 720 cgactctcac atgggccttg cggcacgtat gatgagccag
gcgatgcgta agctggcggg 780 taacctgaag cagtccaaca cgctgctgat
cttcatcaac cagatccgta tgaaaattgg 840 tgtgatgttc ggtaacccgg
aaaccactac cggtggtaac gcgctgaaat tctacgcctc 900 tgttcgtctc
gacatccgtc gtatcggcgc ggtgaaagag ggcgaaaacg tggtgggtag 960
cgaaacccgc gtgaaagtgg tgaagaacaa aatcgctgcg ccgtttaaac aggctgaatt
1020 ccagatcctc tacggcgaag gtatcaactt ctacggcgaa ctggttgacc
tgggcgtaaa 1080 agagaagctg atcgagaaag caggcgcgtg gtacagctac
aaaggtgaga agatcggtca 1140 gggtaaagcg aatgcgactg cctggctgaa
agataacccg gaaaccgcga aagagatcga 1200 gaagaaagta cgtgagttgc
tgctgagcaa cccgaactca acgccggatt tctctgtaga 1260 tgatagcgaa
ggcgtagcag aaactaacga agatttttaa tcgtcttgtt tgatacacaa 1320
gggtcgcatc tgcggccctt ttgctttttt aagttgtaag gatatgccat gacagaatca
1380 acatcccgtc g 1391 8 238 DNA Klebsiella pneumoniae 8 tcgccaggaa
ggcggcattc ggctgggtca gagtgacctg cagcgtggtg tcgttcagcg 60
ctttcacccc caacgtctcg ggtccctttt gcccgagggc aatctcgcgg gcgttggcga
120 tatgcatatt gccagggtag ctcgcgtagg gggaggctgt tgccggcgag
accagccgtt 180 gccagctcca gacgatatcc tgcgctgtaa tggccgtgcc
gtcagaccag gtcagacc 238 9 385 DNA Klebsiella pneumoniae 9
cagcgtaatg cgccgcggca taacggcgcc actatcgaca gtcagttcgt cagcctgcag
60 cctgggctga atctgggacc atggcgcctg ccgaactaca gcacctatag
ccacagcgat 120 aacaacagcc gctgggagtc ggtttactcc tatcttgccc
gcgatattca caccctacgc 180 agccagctgg tggtcggtaa tacgtatacc
tcttccggca ttttcgacag tttgagtttt 240 accggtctgc agctcagttc
gacaaagaga tgctgccgga tagcctgcat gctttgcgcc 300 gacgattcga
gggatcgcgc gcaccaccgc ggaggtctcg gtttatcaga atggttacag 360
catttataaa accaccgtcg ctacc 385 10 462 DNA Klebsiella pneumoniae 10
ctctatattc aggacgaaca tatctggacc tctggcgggg tcagttccgg ctttgatcgc
60 cctgcacccg cagcgggtga tcgcccctca tctgctactg cggcgctgca
acaggcgacg 120 atcgatgacg ttattcctgg ccagcaaaca gcagaccaat
taaggtctga tagtggctct 180 cttcctccgg cgcgcgacgg tccaggcggc
tcaacagttt ggtgcatagc gctttgcggt 240 tgagatgacg cccttcgtta
agaatatcca tcacgatctc cgtccatgga gagtagcgtt 300 tattccagaa
tagggttttt caggatctca tggatctgcg cctgcttatc gctattttgt 360
aaccagatcg cataaagtgg acgggataac gtagcgctgt ccatgaccgt atgtaaccca
420 tgcttctctt tcgcccagcg agcaggtagc caacagcagc cg 462 11 730 DNA
Klebsiella pneumoniae 11 gctgaccgct aaactgggtt acccgatcac
tgacgatctg gacatctaca cccgtctggg 60 cggcatggtt tggcgcgctg
actccaaagg caactacgct tcaaccggcg tttcccgtag 120 cgaacacgac
actggcgttt ccccagtatt tgctggcggc gtagagtggg ctgttactcg 180
tgacatcgct acccgtctgg aataccagtg ggttaacaac atcggcgacg cgggcactgt
240 gggtacccgt cctgataacg gcatgctgag cctgggcgtt tcctaccgct
tcggtcagga 300 agatgctgca ccggttgttg ctccggctcc ggctccggct
ccggaagtgg ctaccaagca 360 cttcaccctg aagtctgacg ttctgttcaa
cttcaacaaa gctaccctga aaccggaagg 420 tcagcaggct ctggatcagc
tgtacactca gctgagcaac atggatccga aagacggttc 480 cgctgttgtt
ctgggctaca ccgaccgcat cggttccgaa gcttacaacc agcagctgtc 540
tgagaaacgt gctcagtccg ttgttgacta cctggttgct aaaggcatcc cggctggcaa
600 aatctccgct cgcggcatgg gtgaatccaa cccggttact ggcaacacct
gtgacaacgt 660 gaaagctcgc gctgccctga tcgattgcct ggctccggat
cgtcgtgtag agatcgaagt 720 taaaggtatc 730 12 225 DNA Proteus
mirabilis 12 cgctactgtt taaatctcat ttgaaacatc gcaaagtcag tgaaccacat
attcgaggat 60 ggcatgcact agaaaatatt aataagattt tagcgaaacc
taatcagcgc aatatcgctt 120 aattatttta ggtatgttct cttctatcct
acagtcacga ggcagtgtcg aacttgatcc 180 tcattttatt aatcacatga
ccaatggtat aagcgtcgtc acata 225 13 402 DNA Proteus mirabilis 13
acattttaaa taggaagcca cctgataaca tccccgcagt tggatcatca gatttatagc
60 ggcatttggt atccgctaga taaaagcagt ccaacgatcc cgccaattgt
tagatgaaat 120 tggactattc tttttatttg ctccgcttta tcacagtggt
tttcgctttg ccgcccctgt 180 gcgccaacag ctaagaacac gcacgctctt
taatgtgtta ggcccattaa ttaatccagc 240 gcgttccgcc tttagcatta
attggtgttt atagtcctga attattaatg cctattgcag 300 ataccttaaa
tgtcttgggc tacaaacgtg cggcagtggt ccatagtggt ggaatggatg 360
aagtgtcatt acatgctccc acacaagtgg ctgagttaca ca 402 14 157 DNA
Proteus mirabilis 14 ctgaaacgca tttatgcggg agtcagtgaa atcatcactc
aattttcacc cgatgtattt 60 tctgttgaac aagtctttat ggcaaaaaat
gcagactcag cattaaaatt aggccaagca 120 agaggtgtgg cgattttagc
ggcagtcaat aatgatc 157 15 1348 DNA Proteus mirabilis 15 tttctcttta
aaatcaattc ttaaagaaat tattaataat taacttgata ctgtatgatt 60
atacagtata atgagtttca acaagcaaaa tcatatacgt tttaatggta gtgacccatc
120 tttatgcttc actgcccaga gggagataac atggctattg atgaaaacaa
acaaaaagca 180 ttggccgcag cacttggtca aattgaaaag caatttggta
aaggttctat catgcgtctg 240 ggcgaagacc gttccatgaa cgtagaaact
atctctacag gatctttatc attagacgtt 300 gctttaggtg caggtggatt
gccacgtggc cgtattgttg aaatctatgg ccctgaatct 360 tctggtaaaa
caaccttgac tctacaagtt attgcctctg ctcagcgtga aggaaaaatt 420
tgtgcattta ttgatgctga acatgcatta gacccaattt atgctcaaaa gctaggtgtc
480 gatatcgata atctactctg ctctcaacct gacacaggtg aacaagctct
ggaaatttgt 540 gatgcattat ctcgctctgg tgcggtcgat gttattgtcg
tggactccgt ggcagcatta 600 acaccaaaag ctgaaattga aggtgaaatt
ggtgattcac acgttggttt agccgcacgt 660 atgatgagcc aagctatgcg
taaactagcg ggtaacctta aaaactctaa tacactgctg 720 attttcatta
accaaattcg tatgaaaatc ggtgttatgt ttggtaaccc agaaaccacg 780
accggtggta atgcgcttaa attctatgct tctgttcgtt tagacattcg tcgcattggc
840 tctgtcaaaa atggtgatga agtcattggt agtgagactc gcgttaaagt
tgttaaaaat 900 aaagtggctg caccgtttaa acaagctgaa ttccaaatta
tgtacggtga aggtattaat 960 acctatggcg aactgattga tttaggtgtt
aaacataagt tagtagagaa agcaggtgct 1020 tggtatagct acaatggcga
aaaaattggt caaggtaaag ctaacgcaac caattactta 1080 aaagaacatc
ctgaaatgta caatgagtta aacactaaat tgcgtgaaat gttgttaaat 1140
catgctggtg aattcacaag tgctgcggat tttgcaggtg aagagtcaga cagtgatgct
1200 gacgacacaa aagagtaatt agctggttgt catgctgttt gtgtgaaaat
agaccttaaa 1260 tcattggcta ttatcacgac agcatcccat agaataactt
gtttgtataa attttattca 1320 gatggcaaag gaagccttaa aaaagctt
1348 16 2167 DNA Pseudomonas aeruginosa 16 ggtaccgctg gccgagcatc
tgctcgatca ccaccagccg ggcgacggga actgcacgat 60 ctacctggcg
agcctggagc acgagcgggt tcgcttcgta cggcgctgag cgacagtcac 120
aggagaggaa acggatggga tcgcaccagg agcggccgct gatcggcctg ctgttctccg
180 aaaccggcgt caccgccgat atcgagcgct cgcacgcgta tggcgcattg
ctcgcggtcg 240 agcaactgaa ccgcgagggc ggcgtcggcg gtcgcccgat
cgaaacgctg tcccaggacc 300 ccggcggcga cccggaccgc tatcggctgt
gcgccgagga cttcattcgc aaccgggggg 360 tacggttcct cgtgggctgc
tacatgtcgc acacgcgcaa ggcggtgatg ccggtggtcg 420 agcgcgccga
cgcgctgctc tgctacccga ccccctacga gggcttcgag tattcgccga 480
acatcgtcta cggcggtccg gcgccgaacc agaacagtgc gccgctggcg gcgtacctga
540 ttcgccacta cggcgagcgg gtggtgttca tcggctcgga ctacatctat
ccgcgggaaa 600 gcaaccatgt gatgcgccac ctgtatcgcc agcacggcgg
cacggtgctc gaggaaatct 660 acattccgct gtatccctcc gacgacgact
tgcagcgcgc cgtcgagcgc atctaccagg 720 cgcgcgccga cgtggtcttc
tccaccgtgg tgggcaccgg caccgccgag ctgtatcgcg 780 ccatcgcccg
tcgctacggc gacggcaggc ggccgccgat cgccagcctg accaccagcg 840
aggcggaggt ggcgaagatg gagagtgacg tggcagaggg gcaggtggtg gtcgcgcctt
900 acttctccag catcgatacg cccgccagcc gggccttcgt ccaggcctgc
catggtttct 960 tcccggagaa cgcgaccatc accgcctggg ccgaggcggc
ctactggcag accttgttgc 1020 tcggccgcgc cgcgcaggcc gcaggcaact
ggcgggtgga agacgtgcag cggcacctgt 1080 acgacatcga catcgacgcg
ccacaggggc cggtccgggt ggagcgccag aacaaccaca 1140 gccgcctgtc
ttcgcgcatc gcggaaatcg atgcgcgcgg cgtgttccag gtccgctggc 1200
agtcgcccga accgattcgc cccgaccctt atgtcgtcgt gcataacctc gacgactggt
1260 ccgccagcat gggcggggga ccgctcccat gagcgccaac tcgctgctcg
gcagcctgcg 1320 cgagttgcag gtgctggtcc tcaacccgcc gggggaggtc
agcgacgccc tggtcttgca 1380 gctgatccgc atcggttgtt cggtgcgcca
gtgctggccg ccgccggaag ccttcgacgt 1440 gccggtggac gtggtcttca
ccagcatttt ccagaatggc caccacgacg agatcgctgc 1500 gctgctcgcc
gccgggactc cgcgcactac cctggtggcg ctggtggagt acgaaagccc 1560
cgcggtgctc tcgcagatca tcgagctgga gtgccacggc gtgatcaccc agccgctcga
1620 tgcccaccgg gtgctgcctg tgctggtatc ggcgcggcgc atcagcgagg
aaatggcgaa 1680 gctgaagcag aagaccgagc agctccagga ccgcatcgcc
ggccaggccc ggatcaacca 1740 ggccaaggtg ttgctgatgc agcgccatgg
ctgggacgag cgcgaggcgc accagcacct 1800 gtcgcgggaa gcgatgaagc
ggcgcgagcc gatcctgaag atcgctcagg agttgctggg 1860 aaacgagccg
tccgcctgag cgatccgggc cgaccagaac aataacaaga ggggtatcgt 1920
catcatgctg ggactggttc tgctgtacgt tggcgcggtg ctgtttctca atgccgtctg
1980 gttgctgggc aagatcagcg gtcgggaggt ggcggtgatc aacttcctgg
tcggcgtgct 2040 gagcgcctgc gtcgcgttct acctgatctt ttccgcagca
gccgggcagg gctcgctgaa 2100 ggccggagcg ctgaccctgc tattcgcttt
tacctatctg tgggtggccg ccaaccagtt 2160 cctcgag 2167 17 1872 DNA
Pseudomonas aeruginosa 17 gaattcccgg gagttcccga cgcagccacc
cccaaaacac tgctaaggga gcgcctcgca 60 gggctcctga ggagatagac
catgccattt ggcaagccac tggtgggcac cttgctcgcc 120 tcgctgacgc
tgctgggcct ggccaccgct cacgccaagg acgacatgaa agccgccgag 180
caataccagg gtgccgcttc cgccgtcgat cccgctcacg tggtgcgcac caacggcgct
240 cccgacatga gtgaaagcga gttcaacgag gccaagcaga tctacttcca
acgctgcgcc 300 ggttgccacg gcgtcctgcg caagggcgcc accggcaagc
cgctgacccc ggacatcacc 360 cagcaacgcg gccagcaata cctggaagcg
ctgatcacct acggcacccc gctgggcatg 420 ccgaactggg gcagctccgg
cgagctgagc aaggaacaga tcaccctgat ggccaagtac 480 atccagcaca
ccccgccgca accgccggag tggggcatgc cggagatgcg cgaatcgtgg 540
aaggtgctgg tgaagccgga ggaccggccg aagaaacagc tcaacgacct cgacctgccc
600 aacctgttct cggtgaccct gcgcgacgcc gggcagatcg ccctggtcga
cggcgacagc 660 aaaaagatcg tcaaggtcat cgataccggc tatgccgtgc
atatctcgcg gatgtccgct 720 tccggccgct acctgctggt gatcggccgc
gacgcgcgga tcgacatgat cgacctgtgg 780 gccaaggagc cgaccaaggt
cgccgagatc aagatcggca tcgaggcgcg ctcggtggaa 840 agctccaagt
tcaagggcta cgaggaccgc tacaccatcg ccggcgccta ctggccgccg 900
cagttcgcga tcatggacgg cgagaccctg gaaccgaagc agatcgtctc cacccgcggc
960 atgaccgtag acacccagac ctaccacccg gaaccgcgcg tggcggcgat
catcgcctcc 1020 cacgagcacc ccgagttcat cgtcaacgtg aaggagaccg
gcaaggtcct gctggtcaac 1080 tacaaggata tcgacaacct caccgtcacc
agcatcggtg cggcgccgtt cctccacgac 1140 ggcggctggg acagcagcca
ccgctacttc atgaccgccg ccaacaactc caacaaggtt 1200 gccgtgatcg
actccaagga ccgtcgcctg tcggccctgg tcgacgtcgg caagaccccg 1260
cacccggggc gtggcgccaa cttcgtgcat cccaagtacg gcccggtgtg gagcaccagc
1320 cacctgggcg acggcagcat ctcgctgatc ggcaccgatc cgaagaacca
tccgcagtac 1380 gcctggaaga aagtcgccga actacagggc cagggcggcg
gctcgctgtt catcaagacc 1440 catccgaagt cctcgcacct ctacgtcgac
accaccttca accccgacgc caggatcagc 1500 cagagcgtcg cggtgttcga
cctgaagaac ctcgacgcca agtaccaggt gctgccgatc 1560 gccgaatggg
ccgatctcgg cgaaggcgcc aagcgggtgg tgcagcccga gtacaacaag 1620
cgcggcgatg aagtctggtt ctcggtgtgg aacggcaaga acgacagctc cgcgctggtg
1680 gtggtggacg acaagaccct gaagctcaag gccgtggtca aggacccgcg
gctgatcacc 1740 ccgaccggta agttcaacgt ctacaacacc cagcacgacg
tgtactgaga cccgcgtgcg 1800 gggcacgccc cgcacgctcc cccctacgag
gaaccgtgat gaaaccgtac gcactgcttt 1860 cgctgctcgc ca 1872 18 3451
DNA Pseudomonas aeruginosa 18 tcgagacggg aagccactct ctacgagaag
acagaagccc ctcacagagg cctctgtcta 60 cgcctactaa agctcggctt
attcatatgt atttatattc tttcaataga tcactcagcg 120 ctattttaag
ttcaccctct gtaagttcac ctgggcgctc tttctttcct tcggtaaagc 180
tgtcggccag accaaacatt aaactcaagc atctcccaag cgatgcatca tcttgggcca
240 gcatccctga atcgcgcgtc ggacctccaa gtcttaaaaa attcttcgct
gaaggttttc 300 ccatcaatcg atgaggctaa tagcttcttt gcaatatcta
tcatttccat gctcacctta 360 aagcacctca tttttcatgt aaaaattgta
ttgatccgtg ccagactcaa tcctccaccc 420 agaaacaaac atcccatcct
ctccaatgat aacaacaata ttagtcctgg cattgtaatg 480 tacttttgag
tttacttcgg agtggtaagt ccctttttct acggttgcag gatcagcaag 540
gtgctcaaga attttatccc taaactctgc aagcgttcca ttgttggcgc ttttttcacc
600 cagcccaaaa tcatatttgt ggctatcaaa ttttttctgt agttgcctcc
gtgtgaagat 660 accactatca agaggactac tgagcattac ataaacaggt
ttgactccag aatccgccgg 720 gaaaatcacg atcagatcgt ttaggtccag
tagcattccc ggataggact ccgggccggt 780 cttcaacggt gtgagggccg
ctccctcata taccggcacc ggcttcggta tgaccggagt 840 ggtactcgaa
gggttctggt ttcctggagg actcgccggc gtccaagtca ggatcagtgg 900
cggcgcttct gcgaccgtag agggaaccgt aacctcgtac agtcctgttg cggcgttata
960 ggccccatcc ggaccggaac gctttcggaa cgctcacacc atcggtctga
ccaccgaaag 1020 gtcgtcgtgt tgcctcgcgc ctcgttggtc aggcgcatcg
gcagatcgac ggtaccgctg 1080 gcttttgcaa ccgcgttcag gtttacgctt
gggggaagcc ccaatttagc ggcatccatg 1140 cccagggcgt aacgaacgct
atcgggcgtt tggtcctgcc attgctcggc agtccgggag 1200 agtaggtcag
actggcaagc cacggccatc accgaggtgc tgaagccagg accgccagga 1260
cggcaatcgc atcggagatc gcttgagcaa gggatgcggc gcctgtgcga cctggatcag
1320 accccgctgc ggcggtggcg cacccgctgc cattggctgg catggcataa
gtattggcag 1380 ccctgatcgc cgcttgacga gcgatttcct tgcgccttgc
cgtttcggcg ttcagcttgt 1440 ccagccgtgc ttgcaggctg gcgatttcat
ccactaggta ggacatcggc gttgtaggtt 1500 gccttttgtt tctccagtgc
attgggtgcc ttggcaatca aggcattgtt tgcagtctgc 1560 aattcttctt
attgcgatcg cctgcgtaag gagttgagta gcgcgttcaa gccactgctc 1620
tggcgttgga ttggtcagtt gaggcaaagc attcccagcc tggtcaagct cggactgcac
1680 ttttttctcg acatttgcct tcctggcctt gtagtccgcc tccacctcag
cagcggctcg 1740 ctgggcttct gcttccaatg accgggcttt attctccagc
tcttgagacg tttgtttcaa 1800 gatagcgatt tgcgccttat agatatcggc
gctgtacgct ttggccagct cactcatatg 1860 gcgatccagg aactctccat
agaattttcg gctggccagc aactgactct ggtacatcga 1920 ctctgacttc
tgaggaaagt ctgaagccgt ataaagattg gccgggcgat cctcaatgac 1980
ctttagcgat tttgctttgg catccatgag tgcatcaacg atactctttt catcgcggat
2040 gtcattggca ctgaccgctt tacctggcaa ccccgcttca ctcttgagtt
catcaacctc 2100 cttcagggtt tcatttttca ggtttttctt gagttctgaa
tgggacttat caagcgtact 2160 tcttagcttc ctgtactcct gcattccagt
accgacatac ggacttggtc ctggtgggac 2220 aaatggtgga gtaccgtagc
ttgatcgagc aggaatatac tggattatgt cacgcccacc 2280 accctgcaca
tgtgtaataa ccatcgaacc aggttcgtaa tcattgacag ccatagatcg 2340
cccctacatt aatttgaaag tgtaatgtat tgagcgactc ccacctagag aaccctctcc
2400 cagtcaataa gccccaatgc atcggcaata cactgcaatc aacttcaata
tcccgtgttt 2460 agatgatcca gaaggtgcgc tctctcgcct cttataatcg
cgcctgcgtc aaacggtcat 2520 ttccttaacg cacacctcat ctaccccggc
cagtcacgga agccgcatac cttcggttca 2580 ttaacgaact cccactttca
aaattcatcc atgccgcccc ttcgcgagct tccggacaaa 2640 gccacgctga
ttgcgagccc agcgtttttg attgcaagcc gctgcagctg gtcaggccgt 2700
ttccgcaacg cttgaagtcc tggccgatat accggcaggg ccagccatcg ttcgacgaat
2760 aaagccacct cagccatgat gccctttcca tccccagcgg aaccccgaca
tggacgccaa 2820 agccctgctc ctcggcagcc tctgcctggc cgccccattc
gccgacgcgg cgacgctcga 2880 caatgctctc tccgcctgcc tcgccgcccg
gctcggtgca ccgcacacgg cggagggcca 2940 gttgcacctg ccactcaccc
ttgaggcccg gcgctccacc ggcgaatgcg gctgtacctc 3000 ggcgctggtg
cgatatcggc tgctggccag gggcgccagc gccgacagcc tcgtgcttca 3060
agagggctgc tcgatagtcg ccaggacacg ccgcgcacgc tgaccctggc ggcggacgcc
3120 ggcttggcga gcggccgcga actggtcgtc accctgggtt gtcaggcgcc
tgactgacag 3180 gccgggctgc caccaccagg ccgagatgga cgccctgcat
gtatcctccg atcggcaagc 3240 ctcccgttcg cacattcacc actctgcaat
ccagttcata aatcccataa aagccctctt 3300 ccgctccccg ccagcctccc
cgcatcccgc accctagacg ccccgccgct ctccgccggc 3360 tcgcccgaca
agaaaaacca accgctcgat cagcctcatc cttcacccat cacaggagcc 3420
atcgcgatgc acctgatacc ccattggatc c 3451 19 744 DNA Pseudomonas
aeruginosa 19 gggttcagca agcgttcagg ggcggttcag taccctgtcc
gtactctgca agccgtgaac 60 gacacgactc tcgcagaacg gagaaacacc
atgaaagcac tcaagactct cttcatcgcc 120 accgccctgc tgggttccgc
cgccggcgtc caggccgccg acaacttcgt cggcctgacc 180 tggggcgaga
ccagcaacaa catccagaaa tccaagtcgc tgaaccgcaa cctgaacagc 240
ccgaacctcg acaaggtgat cgacaacacc ggcacctggg gcatccgcgc cggccagcag
300 ttcgagcagg gccgctacta cgcgacctac gagaacatct ccgacaccag
cagcggcaac 360 aagctgcgcc agcagaacct gctcggcagc tacgacgcct
tcctgccgat cggcgacaac 420 aacaccaagc tgttcggcgg tgccaccctc
ggcctggtca agctggaaca ggacggcaag 480 ggcttcaagc gcgacagcga
tgtcggctac gctgccgggc tgcaggccgg tatcctgcag 540 gagctgagca
agaatgcctc gatcgaaggc ggctatcgtt acctgcgcac caacgccagc 600
accgagatga ccccgcatgg cggcaacaag ctgggctccc tggacctgca cagcagctcg
660 caattctacc tgggcgccaa ctacaagttc taaatgaccg cgcagcgccc
gcgagggcat 720 gcttcgatgg ccgggccgga aggt 744 20 2760 DNA
Pseudomonas aeruginosa 20 ctgcagctgg tcaggccgtt tccgcaacgc
ttgaagtcct ggccgatata ccggcagggc 60 cagccatcgt tcgacgaata
aagccacctc agccatgatg ccctttccat ccccagcgga 120 accccgacat
ggacgccaaa gccctgctcc tcggcagcct ctgcctggcc gccccattcg 180
ccgacgcggc gacgctcgac aatgctctct ccgcctgcct cgccgcccgg ctcggtgcac
240 cgcacacggc ggagggccag ttgcacctgc cactcaccct tgaggcccgg
cgctccaccg 300 gcgaatgcgg ctgtacctcg gcgctggtgc gatatcggct
gctggccagg ggcgccagcg 360 ccgacagcct cgtgcttcaa gagggctgct
cgatagtcgc caggacacgc cgcgcacgct 420 gaccctggcg gcggacgccg
gcttggcgag cggccgcgaa ctggtcgtca ccctgggttg 480 tcaggcgcct
gactgacagg ccgggctgcc accaccaggc cgagatggac gccctgcatg 540
tatcctccga tcggcaagcc tcccgttcgc acattcacca ctctgcaatc cagttcataa
600 atcccataaa agccctcttc cgctccccgc cagcctcccc gcatcccgca
ccctagacgc 660 cccgccgctc tccgccggct cgcccgacaa gaaaaaccaa
ccgctcgatc agcctcatcc 720 ttcacccatc acaggagcca tcgcgatgca
cctgataccc cattggatcc ccctggtcgc 780 cagcctcggc ctgctcgccg
gcggctcgtc cgcgtccgcc gccgaggaag ccttcgacct 840 ctggaacgaa
tgcgccaaag cctgcgtgct cgacctcaag gacggcgtgc gttccagccg 900
catgagcgtc gacccggcca tcgccgacac caacggccag ggcgtgctgc actactccat
960 ggtcctggag ggcggcaacg acgcgctcaa gctggccatc gacaacgccc
tcagcatcac 1020 cagcgacggc ctgaccatcc gcctcgaagg cggcgtcgag
ccgaacaagc cggtgcgcta 1080 cagctacacg cgccaggcgc gcggcagttg
gtcgctgaac tggctggtac cgatcggcca 1140 cgagaagccc tcgaacatca
aggtgttcat ccacgaactg aacgccggca accagctcag 1200 ccacatgtcg
ccgatctaca ccatcgagat gggcgacgag ttgctggcga agctggcgcg 1260
cgatgccacc ttcttcgtca gggcgcacga gagcaacgag atgcagccga cgctcgccat
1320 cagccatgcc ggggtcagcg tggtcatggc ccagacccag ccgcgccggg
aaaagcgctg 1380 gagcgaatgg gccagcggca aggtgttgtg cctgctcgac
ccgctggacg gggtctacaa 1440 ctacctcgcc cagcaacgct gcaacctcga
cgatacctgg gaaggcaaga tctaccgggt 1500 gctcgccggc aacccggcga
agcatgacct ggacatcaaa cccacggtca tcagtcatcg 1560 cctgcacttt
cccgagggcg gcagcctggc cgcgctgacc gcgcaccagg cttgccacct 1620
gccgctggag actttcaccc gtcatcgcca gccgcgcggc tgggaacaac tggagcagtg
1680 cggctatccg gtgcagcggc tggtcgccct ctacctggcg gcgcggctgt
cgtggaacca 1740 ggtcgaccag gtgatccgca acgccctggc cagccccggc
agcggcggcg acctgggcga 1800 agcgatccgc gagcagccgg agcaggcccg
tctggccctg accctggccg ccgccgagag 1860 cgagcgcttc gtccggcagg
gcaccggcaa cgacgaggcc ggcgcggcca acgccgacgt 1920 ggtgagcctg
acctgcccgg tcgccgccgg tgaatgcgcg ggcccggcgg acagcggcga 1980
cgccctgctg gagcgcaact atcccactgg cgcggagttc ctcggcgacg gcggcgacgt
2040 cagcttcagc acccgcggca cgcagaactg gacggtggag cggctgctcc
aggcgcaccg 2100 ccaactggag gagcgcggct atgtgttcgt cggctaccac
ggcaccttcc tcgaagcggc 2160 gcaaagcatc gtcttcggcg gggtgcgcgc
gcgcagccag gacctcgacg cgatctggcg 2220 cggtttctat atcgccggcg
atccggcgct ggcctacggc tacgcccagg accaggaacc 2280 cgacgcacgc
ggccggatcc gcaacggtgc cctgctgcgg gtctatgtgc cgcgctcgag 2340
cctgccgggc ttctaccgca ccagcctgac cctggccgcg ccggaggcgg cgggcgaggt
2400 cgaacggctg atcggccatc cgctgccgct gcgcctggac gccatcaccg
gccccgagga 2460 ggaaggcggg cgcctggaga ccattctcgg ctggccgctg
gccgagcgca ccgtggtgat 2520 tccctcggcg atccccaccg acccgcgcaa
cgtcggcggc gacctcgacc cgtccagcat 2580 ccccgacaag gaacaggcga
tcagcgccct gccggactac gccagccagc ccggcaaacc 2640 gccgcgcgag
gacctgaagt aactgccgcg accggccggc tcccttcgca ggagccggcc 2700
ttctcggggc ctggccatac atcaggtttt cctgatgcca gcccaatcga atatgaattc
2760 21 172 DNA Staphylococcus saprophyticus 21 ttgatgaaat
gcatcgatta ataaattttc atgtacgatt aaaacgtttt tacccttacc 60
ttttcgtact acctctgcct gaagttgacc acctttaaag tgattcgttg aaatccatta
120 tgctcattat taatacgatc tataaaaaca aatggaatgt gatgatcgat ga 172
22 155 DNA Staphylococcus saprophyticus 22 gttccattga ctctgtatca
cctgttgtaa cgaacatcca tatgtcctga aactccaacc 60 acaggtttga
ccacttccaa tttcagacca ccaagtttga cacgtgaaga ttcatcttct 120
aatatttcgg aattaatatc atattattta aatag 155 23 145 DNA
Staphylococcus saprophyticus 23 acatagaaaa actcaaaaga tttacttttt
tcaaatggaa aataagggta cacacgatat 60 ttcccgtcat cttcagttac
cggtacaaca tcctctttat taacctgcac ataatctgac 120 tccgcttcac
tcatcaaact actaa 145 24 266 DNA Staphylococcus saprophyticus 24
tttcactgga attacatttc gctcattacg tacagtgaca atcgcgtcag atagtttctt
60 ctggttagct tgactcttaa caatcttgtc taaattttgt ttaattcttt
gattcgtact 120 agaaatttta cttctaattc cttgtaattc ataacttgca
ttatcatata aatcataagt 180 atcacatttt tgatgaatac tttgatataa
atctgacaat acaggcagtt gctccattct 240 atcgttaaga atagggtaat taatag
266 25 845 DNA Haemophilus influenzae 25 tgttaaattt ctttaacagg
gattttgtta tttaaattaa acctattatt ttgtcgcttc 60 tttcactgca
tctactgctt gagttgcttt ttctgaaacc gcctctttca tttcacttgc 120
tttttctgat gctgcttctt tcatttcgcc tactttttct gacgctgctt ctgttgctga
180 tttaattact tctttcgcat cttccacttt ctctgctact ttatttttca
cgtctgtaga 240 aagctgctgt gctttttcct ttacttcagt cattgtatta
gctgcagcat cttttgtttc 300 tgatgcgact gatgctacag tttgcttcgt
atcctcaact ttttgttttg cttcttgctt 360 atcaaaacaa cctgtcacga
ctaaagctga acctaaaacc aatgctaatg ttaatttttt 420 cattattttc
tccatagaat aatttgattg ttacaaagcc ctattacttt gatgcagttt 480
agtttacggg aattttcata aaaagaaaaa cagtaatagt aaaactttac ctttctttaa
540 aaagattact ttataaaaaa acatctaaga tattgatttt taatagatta
taaaaaacca 600 ataaaaattt tattttttgt aaaaaaaaag aatagtttat
tttaaataaa ttacaggaga 660 tgcttgatgc atcaatattt ctgatttatt
accatcccat aataattgag caatagttgc 720 aggataaaat gatattggat
ttcgttttcc atacagttca gcaacaattt ctcccactaa 780 gggcaaatgg
gaaacaatta atacagattt aacgccctcg tcttttagca cttctaaata 840 atcaa
845 26 1598 DNA Haemophilus influenzae 26 gaatagagtt gcactcaata
gattcgggct ttataattgc ccagattttt atttataaca 60 aagggttcca
aatgaaaaaa tttaatcaat ctctattagc aactgcaatg ttgttggctg 120
caggtggtgc aaatgcggca gcgtttcaat tggcggaagt ttctacttca ggtcttggtc
180 gtgcctatgc gggtgaagcg gcgattgcag ataatgcttc tgtcgtggca
actaacccag 240 ctttgatgag tttatttaaa acggcacagt tttccacagg
tggcgtttat attgattcta 300 gaattaatat gaatggtgat gtaacttctt
atgctcagat aataacaaat cagattggaa 360 tgaaagcaat aaaggacggc
tcagcttcac agcgtaatgt tgttcccggt gcttttgtgc 420 caaatcttta
tttcgttgcg ccagtgaatg ataaattcgc gctgggtgct ggaatgaatg 480
tcaatttcgg tctaaaaagt gaatatgacg atagttatga tgctggtgta tttggtggaa
540 aaactgactt gagtgctatc aacttaaatt taagtggtgc ttatcgagta
acagaaggtt 600 tgagcctagg tttaggggta aatgcggttt atgctaaagc
ccaagttgaa cggaatgctg 660 gtcttattgc ggatagtgtt aaggataacc
aaataacaag cgcactctca acacagcaag 720 aaccattcag agatcttaag
aagtatttgc cctctaagga caaatctgtt gtgtcattac 780 aagatagagc
cgcttggggc tttggctgga atgcaggtgt aatgtatcaa tttaatgaag 840
ctaacagaat tggtttagcc tatcattcta aagtggacat tgattttgct gaccgcactg
900 ctactagttt agaagcaaat gtcatcaaag aaggtaaaaa aggtaattta
acctttacat 960 tgccagatta cttagaactt tctggtttcc atcaattaac
tgacaaactt gcagtgcatt 1020 atagttataa atatacccat tggagtcgtt
taacaaaatt acatgccagc ttcgaagatg 1080 gtaaaaaagc ttttgataaa
gaattacaat acagtaataa ctctcgtgtt gcattagggg 1140 caagttataa
tctttatgaa aaattgacct tacgtgcggg tattgcttac gatcaagcgg 1200
catctcgtca tcaccgtagt gctgcaattc cagataccga tcgcacttgg tatagtttag
1260 gtgcaaccta taaattcacg ccgaatttat ctgttgatct tggctatgct
tacttaaaag 1320 gcaaaaaagt tcactttaaa gaagtaaaaa caataggtga
caaacgtaca ttgacattga 1380 atacaactgc aaattatact tctcaagcac
acgcaaatct ttacggtttg aatttaaatt 1440 atagtttcta atccgttaaa
aaatttagca taataaagca caattccaca ctaagtgtgc 1500 ttttctttta
taaaacaagg cgaaaaatga ccgcacttta ttacacttat tacccctcgc 1560
cagtcggacg gcttttgatt ttatctgacg gcgaaaca 1598 27 9100 DNA
Haemophilus influenzae 27 gtcaaaaatt gcgtgcattc tagcgaaaaa
atgggctttt gggaactgtg ggatttattt 60
aaaatcttag aaaatcttac cgcactttta agctataaag tgcggtgaaa tttagtggcg
120 tttataatgg agaattactc tggtgtaatc cattcgactg tccagcttcc
agtaccttct 180 ggaactaatg tttttgtgag ataaggcaaa atttctttca
tttgggtttc taatgtccaa 240 ggtggattaa ttaccaccat accgctcgca
gtcattcctc gttgatcgct atctgggcga 300 acggcgagtt caatttttag
aatttttcta attcccgttg cttctaaacc cttaaaaata 360 cgtttagttt
gttggcgtaa tacaacagga taccaaatcg cataagtgcc agtggcaaaa 420
cgtttatagc cctcttcaat ggctttaaca acgagatcat aatcatcttt taattcataa
480 ggcggatcga tgagtactaa gcctcggcgt tcttttggcg gaagcgttgc
tttgacttgt 540 tgaaagccat tgtcacattt tacggtgaca tttttgtcgt
cgctaaaatt attgcgaaga 600 attggataat cgctaggatg aagctcggtc
aatagtgcgc gatcttgtga gcgcaacaat 660 tccgcggcaa ttaatggaga
acccgcgtaa taacgtagtt ctttgccacc ataattgagt 720 tttttgatca
tttttacata acgagcaata tcttcgggta aatctgtttg atcccacagg 780
cgtccaatac cttctttata ttcccccgtt ttttctgatt catttgagga taaacgataa
840 cgccccacac cagagtgcgt atccaaataa aaaaagcctt tttctttgag
tttaagattt 900 tccaaaatga gcattaaaac aatatgtttc aagacatcgg
catgattgcc agcgtgaaat 960 gagtgatgat aactcagcat aatatattcc
ttatatattc cttatttgtt taataacgaa 1020 ggcgagccaa ttgactcgcc
cgattacaca ctaaagtgcg gtcattttta gaagagttct 1080 tgtggttgcg
tcgctggcgt attgccttca ttatttaagc gttgctgtaa ctcagtagga 1140
acataataac cacgctcttg catttccgaa agataggtac gtgtcggttc tgttcccgca
1200 ataaaatatt ctttgcgccc accgtttgga gaaagcaaac ctgtcaaagt
atcaatgttt 1260 ttttccacaa tttttggcgg tagcgacaat ttacgttctg
gcttatcact caaagccgtt 1320 ttcatataag tgatccaagc aggcattgct
gtttttgctc ctgcttctcc acgcccaagt 1380 actcgtttgt tatcatcaaa
cccgacataa gttgtggtta ctaagtttgc accaaatccc 1440 gcataccaag
ccacttttga actgttggta gtacctgttt taccgcctat atcgctacgt 1500
ttaatgcttt gtgcaatacg ccagctggtg cctttccagt ctaaaccttg ttcgccataa
1560 attgccgtat ttaaggcact acgaatgaga aaagcaagtt cgccactaat
gacacgtggc 1620 gcatattcta ttttcgacga agcatttttt gcagcagcca
ttaaatcaat cgcatcttct 1680 ttaagtgcgg tcatatttga ttgtaattct
ggcagttcag gcacagtttc aggttgttga 1740 tctaattctt cgccattggt
gctgtcatct gttggtttta aggcattctc gcctaaagga 1800 atattggcaa
agccgttgat tttgtctttg gtttcgccat aaattacagg tatatcatta 1860
cattcaatgc aagcaatttt agggtttgca ataaataagt ctttacccgt gttatcttga
1920 attttttcaa tgatataagg ttcaatgagg aagccaccat tatcaaacac
cgcataagct 1980 cgcgccattt ctaatggtgt gaaagaggct gcgccaagtg
ctaaggcttc actggcaaaa 2040 tattgatcac gtttaaaacc aaaacgttgt
aaaaattctg ctgtgaaatc aatacctgcc 2100 gtttggatag cacgaatagc
aattatattt ttggattgac ctaatcctac gcgtaaacgc 2160 atcgggccat
cataacgatc aggcgagttt ttcggttgcc acattttttg tcccggtttt 2220
tgaatagaaa tcgggctgtc ttgtaatacg cttgaaagtg ttaagccttt ttctaatgct
2280 gccgcgtaaa taaatggttt gatagaagaa cccacttgaa ctaaagactg
tgtggctcga 2340 ttgaatttac tttgttcata gctaaagcca ccgaccactg
cttcaatcgc accattatct 2400 gaattaagag aaactaatgc tgaatttgct
gcgggaattt gtcctaattg ccattcccca 2460 ttagcacgct gatgaatcca
aatttgctcg ccgactttca caggattgct tctgcctgtc 2520 caacgcattg
cattggttga taaggtcatt ttttccccag aagcgagcaa tatatcagca 2580
ccgcctttta caattccaat cactgccgca ggaataaatg gctctgaatc aggtagtttg
2640 cgtagaaaac cgacaatgcg atcattgtcc caagcggctt catttttttg
ccataatggc 2700 gcgccaccgc gataaccgtg acgcatatcg taatcaatca
agttattacg cacagctttt 2760 tgggcttcag cttggtcttt tgaaagtaca
gtggtaaata ctttataacc actggtgtaa 2820 gcattttctt cgccaaaacg
acgcaccatt tcttgacgca ccatttcagt gacataatcg 2880 gctcgaaatt
caaattttgc gccgtgatag ctcgccacaa tcggctcttt caatgcagca 2940
tcatattctt ctttgctgat gtatttttca tctaacatac ggcttagcac cacattgcgg
3000 cgttcttctg aacgttttaa agaataaagc gggttcattg ttgaaggtgc
tttaggtaaa 3060 ccagcaataa tcgccatttc cgataaggtc aattcattca
atgatttacc gaaataggtt 3120 tgtgctgccg ctgcaacacc ataagaacga
tagcctaaaa agattttgtt taaataaagc 3180 tctaatattt cttgtttgtt
gagagtattt tcgatttcta ccgcaagcac ggcttcacga 3240 gctttacgaa
taatggtttt ttctgaggtt aagaaaaagt tacgcgctaa ttgttgagta 3300
atcgtacttg cgccttgtga tgcaccgcca ttactcactg cgacaaacaa tgcacgggca
3360 atgccgatag ggtctaatcc gtgatgatcg taaaaacgac tgtcttccgt
cgctaaaaat 3420 gcgtcaatta agcgttgtgg cacatcggct aatttcactg
gaatacggcg ttgctcaccc 3480 acttcgccaa ttaatttacc gtcagccgta
taaatctgca ttggttgctg taattcaacg 3540 gtttttaatg tttctactga
gggcaattca gattttaagt ggaaatacaa cattccgcct 3600 gctactaaac
ctaaaataca taaagttaat agggtgttta atattaattt tgcgatccgc 3660
atcgtaaaat tctcgcttcg ttaatgaata ttcttgtcaa gagacctatg atttggctgt
3720 taagtataaa agattcagcc tttaaagaat aggaaagaat atgcaattct
ccctgaaaaa 3780 ttaccgcact ttacaaatcg gcattcatcg taagcagagt
tattttgatt ttgtgtggtt 3840 tgatgatctc gaacagccac aaagttatca
aatctttgtt aatgatcgtt attttaaaaa 3900 tcgtttttta caacagctaa
aaacacaata tcaagggaaa acctttcctt tgcagtttgt 3960 agcaagcatt
cccgcccact taacttggtc gaaagtatta atgttgccac aagtgttaaa 4020
tgcgcaagaa tgtcatcaac aatgtaaatt tgtgattgaa aaagagctgc ctattttttt
4080 agaagaattg tggtttgatt atcgttctac cccgttaaag caaggttttc
gattagaggt 4140 tactgcaatt cgtaaaagta gcgctcaaac ttatttgcaa
gattttcagc catttaatat 4200 taatatattg gatgttgcgt caaatgctgt
tttgcgtgca tttcaatatc tgttgaatga 4260 acaagtgcgg tcagaaaata
ccttattttt atttcaagaa gatgactatt gcttggcgat 4320 ttgtgaaaga
tctcagcaat cacaaatttt acaatctcac gaaaatttga ccgcacttta 4380
tgaacaattt accgaacgtt ttgaaggaca acttgaacaa gtttttgttt atcaaattcc
4440 ctcaagtcat acaccattac ccgaaaactg gcagcgagta gaaacagaac
tcccttttat 4500 tgcgctgggc aacgcgctat ggcaaaaaga tttacatcaa
caaaaagtgg gtggttaaat 4560 gtcgatgaat ttattgcctt ggcgtactta
tcaacatcaa aagcgtttac gtcgtttagc 4620 tttttatatc gctttattta
tcttgcttgc tattaattta atgttggctt ttagcaattt 4680 gattgaacaa
cagaaacaaa atttgcaggc acagcaaaag tcgtttgaac aacttaatca 4740
acagcttcat aaaactacca tgcaaattga tcagttacgc attgcggtga aagttggtga
4800 agttttgaca tctattccca acgagcaagt aaaaaagagt ttacaacagc
taagtgaatt 4860 accttttcaa caaggagaac tgaataaatt taaacaagat
gccaataact taagcttgga 4920 aggtaacgcg caagatcaaa cagaatttga
actgattcat caatttttaa agaaacattt 4980 tcccaatgtg aaattaagtc
aggttcaacc tgaacaagat acattgtttt ttcactttga 5040 tgtggaacaa
ggggcggaaa aatgaaagct ttttttaacg atccttttac tccttttgga 5100
aaatggctaa gtcagccttt ttatgtgcac ggtttaacct ttttattgct attaagtgcg
5160 gtgatttttc gccccgtttt agattatata gaggggagtt cacgtttcca
tgaaattgaa 5220 aatgagttag cggtgaaacg ttcagaattg ttgcatcaac
agaaaatttt aacctcttta 5280 caacagcagt cggaaagtcg aaaactttct
ccagaactgg ctgcacaaat tattcctttg 5340 aataaacaaa ttcaacgttt
agctgcgcgt aacggtttat ctcagcattt acgttgggaa 5400 atggggcaaa
agcctatttt gcatttacag cttacaggtc attttgaaaa aacgaagaca 5460
tttttatccg cacttttggc taattcgtca cagctttctg taagtcggtt gcaatttatg
5520 aaacccgaag acggcccatt gcaaaccgag atcatttttc agctagataa
ggaaacaaaa 5580 tgaaacattg gtttttcctg attatattat tttttatgaa
ttgcagttgg ggacaagatc 5640 ctttcgataa aacacagcgt aaccgttctc
agtttgataa cgcacaaaca gtaatggagc 5700 aaacagaaat aatttcctca
gatgtgccta ataatctatg cggagcggat gaaaatcgcc 5760 aagcggctga
aattcctttg aacgctttaa aattggtggg ggtagtgatt tctaaagata 5820
aagcctttgc cttgttgcaa gatcaaggtt tgcaagttta cagcgtttta gagggcgttg
5880 atgtggctca agagggctat attgtagaaa aaatcaacca aaacaatgtt
caatttatgc 5940 gtaagctagg agagcaatgt gatagtagtg aatggaaaaa
attaagtttt taaaggaaga 6000 ttatgaagaa atatttttta aagtgcggtt
attttttagt atgtttttgt ttgccattaa 6060 tcgtttttgc taatcctaaa
acagataacg aacgtttttt tattcgttta tcgcaagcac 6120 ctttagctca
aacactggag caattagctt ttcaacaaga tgtgaattta gtgattggag 6180
atatattgga aaacaagatc tctttgaaat taaacaatat tgatatgcca cgtttgctac
6240 aaataatcgc aaaaagtaag catcttactt tgaataaaga tgatgggatt
tattatttaa 6300 acggcagtca atctggcaaa ggtcaggttg caggaaatct
tacgacaaat gaaccgcact 6360 tagtgagtca cacggtaaaa ctccattttg
ctaaagcttc tgaattaatg aaatccttaa 6420 caacaggaag tggctctttg
ctttctcccg ctgggagcat tacctttgat gatcgcagta 6480 atttgctggt
tattcaggat gaacctcgtt ctgtgcaaaa tatcaaaaaa ctgattgctg 6540
aaatggataa gcctattgaa cagatcgcta ttgaagcgcg aattgtgaca attacggatg
6600 agagtttgaa agaacttggc gttcggtggg ggatttttaa tccaactgaa
aatgcaagac 6660 gagttgcggg cagccttaca ggcaatagct ttgaaaatat
tgcggataat cttaatgtaa 6720 attttgcgac aacgacgaca cctgctggct
ctatagcatt acaagtcgcc aaaattaatg 6780 ggcgattgct tgatttagaa
ttgagtgcgt tggagcgtga aaataatgta gaaattattg 6840 caagccctcg
cttactcact accaataaga aaagtgcgag cattaaacag gggacagaaa 6900
ttccttacat cgtgagtaat actcgtaacg atacgcaatc tgtggaattt cgtgaggcgg
6960 tgcttggttt ggaagtgacg ccacatattt ctaaagataa caatatctta
cttgatttat 7020 tggtaagtca aaattcccct ggttctcgtg tcgcttatgg
acaaaatgag gtggtttcta 7080 ttgataaaca agaaattaat actcaggttt
ttgccaaaga tggggaaacc attgtgcttg 7140 gcggcgtatt tcacgataca
atcacgaaaa gcgaagataa agtgccattg cttggcgata 7200 tacccgttat
taaacgatta tttagcaaag aaagtgaacg acatcaaaaa cgtgagctag 7260
tgattttcgt cacgccacat attttaaaag caggagaaaa cgttagaggc gttgaaacaa
7320 aaaagtgagg gtaaaaaata actttttaaa tgatgaattt ttttaatttt
cgctgtatcc 7380 actgtcgtgg caatcttcat atcgcaaaaa atgggttatg
ttcaggttgc caaaaacaaa 7440 ttaaatcttt tccttattgc ggtcattgtg
gttcggaatt gcaatattat gcgcagcatt 7500 gtgggaattg tcttaaacaa
gaaccaagtt gggataagat ggtcattatt gggcattata 7560 ttgaacctct
ttcgatattg attcagcgtt ttaaatttca aaatcaattt tggattgacc 7620
gcactttagc tcggctttta tatcttgcgg tacgtgatgc taaacgaacg catcaactta
7680 aattgccaga ggcaatcatt ccagtgcctt tatatcattt tcgtcagtgg
cgacggggtt 7740 ataatcaggc agatttatta tctcagcaat taagtcgttg
gctggatatt cctaatttga 7800 acaatatcgt aaagcgtgtg aaacacacct
atactcaacg tggtttgagt gcaaaagatc 7860 gtcgtcagaa tttaaaaaat
gccttttctc ttgctgtttc gaaaaatgaa tttccttatc 7920 gtcgtgttgc
gttggtggat gatgtgatta ctactggttc tacactcaat gaaatctcaa 7980
aattgttgcg aaaattaggt gtggaggaga ttcaagtgtg ggggctggca cgagcttaat
8040 ataaagcact ggaaaaaaaa gcgcgataag cgtattattc ccgatacttt
ctctcaagta 8100 tttaggacat aattatggaa caagcaaccc agcaaatcgc
tatttctgat gccgcacaag 8160 cgcattttcg aaaactttta gacacccaag
aagaaggaac gcatattcgt attttcgcgg 8220 ttaatcctgg tacgcctaat
gcggaatgtg gcgtatctta ttgccccccg aatgccgtgg 8280 aagaaagcga
tattgaaatg aaatataata ctttttctgc atttattgat gaagtgagtt 8340
tgcctttctt agaagaagca gaaattgatt atgttaccga agagcttggt gcgcaactga
8400 ccttaaaagc accgaatgcc aaaatgcgta aggtggctga tgatgcgcca
ttgattgaac 8460 gtgttgaata tgtaattcaa actcaaatta acccacagct
tgcaaatcac ggtggacgta 8520 taaccttaat tgaaattact gaagatggtt
acgcagtttt acaatttggt ggtggctgta 8580 acggttgttc aatggtggat
gttacgttaa aagatggggt agaaaaacaa cttgttagct 8640 tattcccgaa
tgaattaaaa ggtgcaaaag atataactga gcatcaacgt ggcgaacatt 8700
cttattatta gtgagttata aaagaagatt tataatgacc gcacttttga aagtgcggtt
8760 atttttatgg agaaaaaatg aaaatacttc aacaagatga ttttggttat
tggttgctta 8820 cacaaggttc taatctgtat ttagtgaata atgaattgcc
ttttggtatc gctaaagata 8880 ttgatttgga aggattgcag gcaatgcaaa
ttggggaatg gaaaaattat ccgttgtggc 8940 ttgtggctga gcaagaaagt
gatgaacgag aatatgtgag tttgagtaac ttgctttcac 9000 tgccagagga
tgaattccat atattaagcc gaggtgtgga aattaatcat tttctgaaaa 9060
cccataaatt ctgtggaaag tgcggtcata aaacacaaca 9100 28 525 DNA
Moraxella catarrhalis 28 aaaaatcgac tgccgtcatt ttcaaccacc
acatagctca tattcgcaag ccaatgtatt 60 gaccgttggg aataataaca
gccccaaaac aatgaaacat atggtgatga gccaaacata 120 ctttcctgca
gattttggaa tcatatcgcc atcagcacca gtatggtttg accagtattt 180
aacgccatag acatgtgtaa aaaaattaaa taacggtgca agcatgagac caacggcacc
240 tgatgtacct tgtacgatga cctcacctgc tgtggcaacc ataccaagtc
cattgcctgt 300 gatatttttg cgaaaagaca aacttaccac acagaccaag
ccgatgattg agatgacaaa 360 ataaaaccaa tccaaatgcg tgtgagctgt
tgtggtccaa aatccagtaa atagtgcaat 420 aaatccgcaa acaaaccaaa
gtagcaccca gcttgttgtc caatcttttt taccaaagcc 480 tgtgatgtta
tctaaaatat caattttcat cagattttcc ctaat 525 29 466 DNA Moraxella
catarrhalis 29 taatgataac cagtcaagca agctcaaatc agggtcagcc
tgttttgagc tttttatttt 60 ttgatcatca tgcttaagat tcactctgcc
atttttttac aacctgcacc acaagtcatc 120 atcgcatttg caaaaatggt
acaaacaagc cgtcagcgac ttaaacaaaa aaaggctcaa 180 tctgcgtgtg
tgcgttcact tttacaaatc accatgcacc gctttgacat tgttggtgaa 240
tttcatgacc atgcacaccc ttattatatt aactcaaata aaatacgcta ctttgtcagc
300 tttagccatt cagataatca agtcgctctc atcatcagct taacaccttg
tgccattgac 360 atagaagtta acgatattaa atacagtgtg gttgaacgat
actttcatcc caatgaaatt 420 tatctactta ctcaatttag ctctactgat
aggcaacagc ttatta 466 30 631 DNA Streptococcus pneumoniae 30
gatctttgat tttcattgag tattactctc tcttgtcact tctttctatt ttaccataaa
60 gtccagcctt tgaagaactt ttactagaag acaaggggct tctgtctcta
tttgccatct 120 taggcatcaa aaaagagggg tcatccctct ttacgaattc
aatgctacta gggtatccaa 180 atactggttg ttgatgactg ccaaaatata
ggtatctgct ttcaagaggt catctggtcc 240 aaattcaaca tccaatgggg
aattttcctg ctctcggaaa cccaaaatat tcagattgta 300 tttgccacgg
aggtctaatt tacttcagac tttgacctgc ccaagactga ggaattttca 360
tctccacgat agacacattt ttatccaact gaaagacatc aacactatta tgaaaagaat
420 ggtctgtgct agagactgcc ccatttcata ctctggcgag ataaccgagt
cagctccaat 480 cttttctagc actttcttag cggtctgact tttgacctta
gcaataacag tcggtacccc 540 caaactctta cagtgcataa ccgcaagcac
actcgactcc agattttcac ctgtcgcgac 600 tacaacggta tcgcaggtat
caatccctgc t 631 31 3754 DNA Streptococcus pneumoniae 31 ccaatatttt
ggtcagcata gtgttctttt tcagtggtaa cagcttgcaa tacttgagca 60
gaaatggcag atttatcaag gaaaaagtta acgtaaggtc ctgttgcgac aactttttca
120 aaggcttggc tgttcatttt ttcagccagt tcagccgcaa tcatttgtgg
tgctttacgt 180 tcgacttttg caagagaaaa agcagggaaa gcaatgtctc
ccatttctga gtttttaggg 240 gtttccagta actttaaaat agcctcttgg
tccaggctat caatgatgct agataattcg 300 ctagcaatca attcttttgt
attcattaag agctcctttt tggacttttc tactatttta 360 tcacaatttt
aaagaaagaa gaaaaaattt ttgaaatctc ctgttttttt ggtataatat 420
ggttataaat atagttataa atatagttat aaatatgcac gcaagaggat tttatgagaa
480 aaagagatcg tcatcagtta ataaaaaaaa tgattactga ggagaaatta
agtacacaaa 540 aagaaattca agatcggttg gaggcgcaca atgtttgtgt
gacgcagaca accttgtctc 600 gtgatttgcg cgaaatcggc ttgaccaagg
tcaagaaaaa tgatatggtg tattatgtac 660 tagtaaatga gacagaaaag
attgatttgg tggaattttt gtctcatcat ttagaaggtg 720 ttgcaagagc
agagtttacc ttggtgcttc ataccaaatt gggagaagcc tctgttttgg 780
caaatattgt agatgtaaac aaggatgaat ggattttagg aacagttgct ggtgccaata
840 ccttattggt tatttgtcga gatcagcacg ttgccaaact catggaagat
cgtttgctag 900 atttgatgaa agataagtaa ggtcttggga gttgctctca
agacttattt ttgaaaagga 960 gagacagaaa atggcgatag aaaagctatc
acccggcatg caacagtatg tggatattaa 1020 aaagcaatat ccagatgctt
ttttgctctt tcggatgggt gatttttatg aattatttta 1080 tgaggatgcg
gtcaatgctg cgcagattct ggaaatttcc ttaacgagtc gcaacaagaa 1140
tgccgacaat ccgatcccta tggcgggtgt tccctatcat tctgcccaac agtatatcga
1200 tgtcttgatt gagcagggtt ataaggtggc tatcgcagag cagatggaag
atcctaaaca 1260 agcagttggg gttgttaaac gagaggttgt tcaggtcatt
acgccaggga cagtggtcga 1320 tagcagtaag ccggacagtc agaataattt
tttggtttcc atagaccgcg aaggcaatca 1380 atttggccta gcttatatgg
atttggtgac gggtgacttt tatgtgacag gtcttttgga 1440 tttcacgctg
gtttgtgggg aaatccgtaa cctcaaggct cgagaagtgg tgttgggtta 1500
tgacttgtct gaggaagaag aacaaatcct cagccgccag atgaatctgg tactctctta
1560 tgaaaaagaa agctttgaag accttcattt attggatttg cgattggcaa
cggtggagca 1620 aacggcatct agtaagctgc tccagtatgt tcatcggact
cagatgaggg aattgaacca 1680 cctcaaacct gttatccgct acgaaattaa
ggatttcttg cagatggatt atgcgaccaa 1740 ggctagtctg gatttggttg
agaatgctcg ctcaggtaag aaacaaggca gtcttttctg 1800 gcttttggat
gaaaccaaaa cggctatggg gatgcgtctc ttgcgttctt ggattcatcg 1860
ccccttgatt gataaggaac gaatcgtcca acgtcaagaa gtagtgcagg tctttctcga
1920 ccatttcttt gagcgtagtg acttgacaga cagtctcaag ggtgtttatg
acattgagcg 1980 cttggctagt cgtgtttctt ttggcaaaac caatccaaag
gatctcttgc agttggcgac 2040 taccttgtct agtgtgccac ggattcgtgc
gattttagaa gggatggagc aacctactct 2100 agcctatctc atcgcacaac
tggatgcaat ccctgagttg gagagtttga ttagcgcagc 2160 gattgctcct
gaagctcctc atgtgattac agatggggga attatccgga ctggatttga 2220
tgagacttta gacaagtatc gttgcgttct cagagaaggg actagctgga ttgctgagat
2280 tgaggctaag gagcgagaaa actctggtat cagcacgctc aagattgact
acaataaaaa 2340 ggatggctac tattttcatg tgaccaattc gcaactggga
aatgtgccag cccacttttt 2400 ccgcaaggcg acgctgaaaa actcagaacg
ctttggaacc gaagaattag cccgtatcga 2460 gggagatatg cttgaggcgc
gtgagaagtc agccaacctc gaatacgaaa tatttatgcg 2520 cattcgtgaa
gaggtcggca agtacatcca gcgtttacaa gctctagccc aaggaattgc 2580
gacggttgat gtcttacaga gtctggcggt tgtggctgaa acccagcatt tgattcgacc
2640 tgagtttggt gacgattcac aaattgatat ccggaaaggg cgccatgctg
tcgttgaaaa 2700 ggttatgggg gctcagacct atattccaaa tacgattcag
atggcagaag ataccagtat 2760 tcaattggtt acagggccaa acatgagtgg
gaagtctacc tatatgcgtc agttagccat 2820 gacggcggtt atggcccagc
tgggttccta tgttcctgct gaaagcgccc atttaccgat 2880 ttttgatgcg
atttttaccc gtatcggagc agcagatgac ttggtttcgg gtcagtcaac 2940
ctttatggtg gagatgatgg aggccaataa tgccatttcg catgcgacca agaactctct
3000 cattctcttt gatgaattgg gacgtggaac tgcaacttat gacgggatgg
ctcttgctca 3060 gtccatcatc gaatatatcc atgagcacat cggagctaag
accctctttg cgacccacta 3120 ccatgagttg actagtctgg agtctagttt
acaacacttg gtcaatgtcc acgtggcaac 3180 tttggagcag gatgggcagg
tcaccttcct tcacaagatt gaaccgggac cagctgataa 3240 atcctacggt
atccatgttg ccaagattgc tggcttgcca gcagaccttt tagcaagggc 3300
ggataagatt ttgactcagc tagagaatca aggaacagag agtcctcctc ccatgagaca
3360 aactagtgct gtcactgaac agatttcact ctttgatagg gcagaagagc
atcctatcct 3420 agcagaatta gctaaactgg atgtgtataa tatgacacct
atgcaggtta tgaatgtctt 3480 agtagagtta aaacagaaac tataaaacca
agactcacta gttaatctag ctgtatcaag 3540 gagacttctt tgacaattct
ccactttttt gctagaataa catcacacaa acagaatgaa 3600 aagggctgac
gcattgtcgc tcccttttgt ctatttttta aggagaaagt atgctgattc 3660
agaaaataaa aacctacaag tggcaggccc tgcttcgctc ctgatgacag gcttgatggt
3720 tgctagttca cttctgcaac cgcgttatct gcag 3754 32 1337 DNA
Streptococcus pyogenes 32 aacaaaataa aagaacttac ctattttcca
tccaaaatgt ttagcaatca tcatctgcaa 60 ggcaacgtat tgcatggcat
tgatgtgatg agcaactaat atgtcattag aacgttgcgt 120 caaactagca
tctaaataaa gatcgaaatg cagttatcaa aaatgcaagc tcctatcggc 180
ccttgtttta attattactc acattgcctt aatgtattta cttgcttatt attaactttt
240 ttgctaagtt agtagcgtca gttattcatt gaaaggacat tattatgaaa
attcttgtaa 300 caggctttga tccctttggc ggcgaagcta ttaatcctgc
ccttgaagct atcaagaaat 360
tgccagcaac cattcatgga gcagaaatca aatgtattga agttccaacg gtttttcaaa
420 aatctgccga tgtgctccag cagcatatcg aaagctttca acctgatgca
gtcctttgta 480 ttgggcaagc tggtggccgg actggactaa cgccagaacg
cgttgccatt aatcaagacg 540 atgctcgcat tcctgataac gaagggaatc
agcctattga tacacctatt cgtgcagatg 600 gtaaagcagc ttatttttca
accttgccaa tcaaagcgat ggttgctgcc attcatcagg 660 ctgggcttcc
tgcttctgtt tctaatacag ctggtacctt tgtttgcaat catttgatgt 720
atcaagccct ttacttagtg gataaatatt gtccaaatgc caaagctggg tttatgcata
780 ttccctttat gatggaacag gttgttgata aacctaatac agctgccatg
aacctcgatg 840 atattacaag aggaattgag gctgctattt ttgccattgt
cgatttcaaa gatcgttccg 900 atttaaaacg tgtagggggc gctactcact
gactgtgacg ctactaaacc tattttaaaa 960 aaacagagat atgaactaac
tctgtttttt ttgtgctaaa aatgaaagac ctagggaaac 1020 ttttcatcgg
tctttctcaa ttgtcatctt aatctaatac tacttctaac atcagcgggt 1080
atagtttgcc agtaattaag aaacgttgtt gatctaaatg agcaatccca ttcaaaacat
1140 taaggtcagg gtaatgggac ttatcaagat ttaaggcttt taacaaagga
ctaatatcat 1200 aggtggctac cacctttcca gaatcaggtt ggagtttgac
aatagtattg gtttgccaaa 1260 tattggcata gagataacca tctacatact
ctaattcgtt aagcattgag atagggacac 1320 tttctatagc aactagt 1337 33
1837 DNA Streptococcus pyogenes 33 tcatgtttga cagcttatca tcgataagct
tacttttcga atcaggtcta tccttgaaac 60 aggtgcaaca tagattaggg
catggagatt taccagacaa ctatgaacgt atatactcac 120 atcacgcaat
cggcaattga tgacattgga actaaattca atcaatttgt tactaacaag 180
caactagatt gacaactaat tctcaacaaa cgttaattta acaacattca agtaactccc
240 accagctcca tcaatgctta ccgtaagtaa tcataactta ctaaaacctt
gttacatcaa 300 ggttttttct ttttgtcttg ttcatgagtt accataactt
tctatattat tgacaactaa 360 attgacaact cttcaattat ttttctgtct
actcaaagtt ttcttcattt gatatagtct 420 aattccacca tcacttcttc
cactctctct accgtcacaa cttcatcatc tctcactttt 480 tcgtgtggta
acacataatc aaatatcttt ccgtttttac gcactatcgc tactgtgtca 540
cctaaaatat accccttatc aatcgcttct ttaaactcat ctatatataa catatttcat
600 cctcctacct atctattcgt aaaaagataa aaataactat tgtttttttt
gttattttat 660 aataaaatta ttaatataag ttaatgtttt ttaaaaatat
acaattttat tctatttata 720 gttagctatt ttttcattgt tagtaatatt
ggtgaattgt aataaccttt ttaaatctag 780 aggagaaccc agatataaaa
tggaggaata ttaatggaaa acaataaaaa agtattgaag 840 aaaatggtat
tttttgtttt agtgacattt cttggactaa caatctcgca agaggtattt 900
gctcaacaag accccgatcc aagccaactt cacagatcta gtttagttaa aaaccttcaa
960 aatatatatt ttctttatga gggtgaccct gttactcacg agaatgtgaa
atctgttgat 1020 caacttttat ctcacgattt aatatataat gtttcagggc
caaattatga taaattaaaa 1080 actgaactta agaaccaaga gatggcaact
ttatttaagg ataaaaacgt tgatatttat 1140 ggtgtagaat attaccatct
ctgttattta tgtgaaaatg cagaaaggag tgcatgtatc 1200 tacggagggg
taacaaatca tgaagggaat catttagaaa ttcctaaaaa gatagtcgtt 1260
aaagtatcaa tcgatggtat ccaaagccta tcatttgata ttgaaacaaa taaaaaaatg
1320 gtaactgctc aagaattaga ctataaagtt agaaaatatc ttacagataa
taagcaacta 1380 tatactaatg gaccttctaa atatgaaact ggatatataa
agttcatacc taagaataaa 1440 gaaagttttt ggtttgattt tttccctgaa
ccagaattta ctcaatctaa atatcttatg 1500 atatataaag ataatgaaac
gcttgactca aacacaagcc aaattgaagt ctacctaaca 1560 accaagtaac
tttttgcttt tggcaacctt acctactgct ggatttagaa attttattgc 1620
aattctttta ttaatgtaaa aaccgctcat ttgatgagcg gttttgtctt atctaaagga
1680 gctttacctc ctaatgctgc aaaattttaa atgttggatt tttgtatttg
tctattgtat 1740 ttgatgggta atcccatttt tcgacagaca tcgtcgtgcc
acctctaaca ccaaaatcat 1800 agacaggagc ttgtagctta gcaactattt tatcgtc
1837 34 841 DNA Streptococcus pneumoniae 34 gatcaatatg tccaagaaac
cacatgttcc taagacaaga gctaacagac tggccgtcaa 60 taatagtatt
gttctttttt tcatcattac tccttaacta gtgtttaact gattaattag 120
ccagtaaata gtttatcttt atttacacta tctgttaaga tatagtaaaa tgaaataaga
180 acaggacagt caaatcgatt tctaacaatg ttttagaagt agaggtatac
tattctaatt 240 tcaatctact atattttgca cattttcata aaaaaaatga
gaactagaac tcacattctg 300 ctctcatttt tcgttttccc gttctcctat
cctgttttta ggagttagaa aatgctgcta 360 cctttactta ctctccttta
ataaagccaa tagtttttca gcttctgcca taatagtatt 420 gttgtcctgg
gtgccaaata gtaaattatt ttttaatcct gtgagagtct ctttggcatt 480
ggacttgata attggattct ggatttttcc aagtaaatct tcagcctctc tcagttttct
540 taacctttca gtctcgacct gaggttcttc tgattcctct ggtgattctt
ctggtgattc 600 ttcttctggt tcctctgttg gttttggaga ctctggtttc
tcgctttgcg gtttctcttc 660 tcgaggggtt tcttcctcag gtttttctgt
ctgaggtttc tcctcgtttg gtttttccgt 720 ttgattggta tcagcttgac
catttttgtt tctttgaaca tggtcgctag cgttaccaaa 780 accattatct
gaatgcgacg ttcgtttgga tgttcgacat agtacttgac agtcgccaaa 840 a 841 35
4500 DNA Streptococcus pneumoniae 35 gatcaggaca gtcaaatcga
tttctaacaa tgttttagaa gtagatgtgt actattctag 60 tttcaatcta
ttatatttat agaatttttt gttgctagat ttgtcaaatt gcttaaaata 120
atttttttca gaaagcaaaa gccgatacct atcgagtagg gtagttcttg ctatcgtcag
180 gcttgtctgt aggtgttaac acttttcaaa aatctcttca aacaacgtca
gctttgcctt 240 gccgtatata tgttactgac ttcgtcagtt ctatctgcca
cctcaaaacg gtgttttgag 300 ctgacttcgt cagttctatc cacaacctca
aaacagtgtt ttgagctgac ttcgtcagtt 360 ctatccacaa cctcaaaaca
gtgttttgag ctgactttgt cagtcttatc tacaacctca 420 aaacagtgtt
ttgagcatca tgcggctagc ttcttagttt gctctttgat tttcattgag 480
tataaaaaca gatgagtttc tgttttcttt ttatggacta taaatgttca gctgaaacta
540 ctttcaagga cattattata taaaagaatt ttttgaaact aaaatctact
atattacact 600 atattgaaag cgttttaaaa atgaggtata ataaatttac
taacacttat aaaaagtgat 660 agaatctatc tttatgtata tttaaagata
gattgctgta aaaatagtag tagctatgcg 720 aaataacaga tagagagaag
ggattgaagc ttagaaaagg ggaataatat gatatttaag 780 gcattcaaga
caaaaaagca gagaaaaaga caagttgaac tacttttgac agtttttttc 840
gacagttttc tgattgattt atttcttcac ttatttggga ttgtcccctt taagctggat
900 aagattctga ttgtgagctt gattatattt cccattattt ctacaagtat
ttatgcttat 960 gaaaagctat ttgaaaaagt gttcgataag gattgagcag
gaagtatggt gtaaatagca 1020 taagctgatg tccatcattt gcttataaag
agatatttta gtttaattgc agcggtgtcc 1080 tggtagataa actagattgg
caggagtctg attggagaaa ggagagggga aatttggcac 1140 caatttgaga
tagtttgttt agttcatttt tgtcatttaa atgaactgta gtaaaagaaa 1200
gttaataaaa gacaaactaa gtgcattttc tggaataaat gtcttatttc agaaatcggg
1260 atatagatat agagaggaac agtatgaatc ggagtgttca agaacgtaag
tgtcgttata 1320 gcattaggaa actatcggta ggagcggttt ctatgattgt
aggagcagtg gtatttggaa 1380 cgtctcctgt tttagctcaa gaaggggcaa
gtgagcaacc tctggcaaat gaaactcaac 1440 tttcggggga gagctcaacc
ctaactgata cagaaaagag ccagccttct tcagagactg 1500 aactttctgg
caataagcaa gaacaagaaa ggaaagataa gcaagaagaa aaaattccaa 1560
gagattacta tgcacgagat ttggaaaatg tcgaaacagt gatagaaaaa gaagatgttg
1620 aaaccaatgc ttcaaatggt cagagagttg atttatcaag tgaactagat
aaactaaaga 1680 aacttgaaaa cgcaacagtt cacatggagt ttaagccaga
tgccaaggcc ccagcattct 1740 ataatctctt ttctgtgtca agtgctacta
aaaaagatga gtacttcact atggcagttt 1800 acaataatac tgctactcta
gaggggcgtg gttcggatgg gaaacagttt tacaataatt 1860 acaacgatgc
acccttaaaa gttaaaccag gtcagtggaa ttctgtgact ttcacagttg 1920
aaaaaccgac agcagaacta cctaaaggcc gagtgcgcct ctacgtaaac ggggtattat
1980 ctcgaacaag tctgagatct ggcaatttca ttaaagatat gccagatgta
acgcatgtgc 2040 aaatcggagc aaccaagcgt gccaacaata cggtttgggg
gtcaaatcta cagattcgga 2100 atctcactgt gtataatcgt gctttaacac
cagaagaggt acaaaaacgt agtcaacttt 2160 ttaaacgctc agatttagaa
aaaaaactac ctgaaggagc ggctttaaca gagaaaacgg 2220 acatattcga
aagcgggcgt aacggtaaac caaataaaga tggaatcaag agttatcgta 2280
ttccagcact tctcaagaca gataaaggaa ctttgatcgc aggtgcagat gaacgccgtc
2340 tccattcgag tgactggggt gatatcggta tggtcatcag acgtagtgaa
gataatggta 2400 aaacttgggg tgaccgagta accattacca acttacgtga
caatccaaaa gcttctgacc 2460 catcgatcgg ttcaccagtg aatatcgata
tggtgttggt tcaagatcct gaaaccaaac 2520 gaatcttttc tatctatgac
atgttcccag aagggaaggg aatctttgga atgtcttcac 2580 aaaaagaaga
agcctacaaa aaaatcgatg gaaaaaccta tcaaatcctc tatcgtgaag 2640
gagaaaaggg agcttatacc attcgagaaa atggtactgt ctatacacca gatggtaagg
2700 cgacagacta tcgcgttgtt gtagatcctg ttaaaccagc ctatagcgac
aagggggatc 2760 tatacaaggg taaccaatta ctaggcaata tctacttcac
aacaaacaaa acttctccat 2820 ttagaattgc caaggatagc tatctatgga
tgtcctacag tgatgacgac gggaagacat 2880 ggtcagcgcc tcaagatatt
actccgatgg tcaaagccga ttggatgaaa ttcttgggtg 2940 taggtcctgg
aacaggaatt gtacttcgga atgggcctca caagggacgg attttgatac 3000
cggtttatac gactaataat gtatctcact taaatggctc gcaatcttct cgtatcatct
3060 attcagatga tcatggaaaa acttggcatg ctggagaagc ggtcaacgat
aaccgtcagg 3120 tagacggtca aaagatccac tcttctacga tgaacaatag
acgtgcgcaa aatacagaat 3180 caacggtggt acaactaaac aatggagatg
ttaaactctt tatgcgtggt ttgactggag 3240 atcttcaggt tgctacaagt
aaagacggag gagtgacttg ggagaaggat atcaaacgtt 3300 atccacaggt
taaagatgtc tatgttcaaa tgtctgctat ccatacgatg cacgaaggaa 3360
aagaatacat catcctcagt aatgcaggtg gaccgaaacg tgaaaatggg atggtccact
3420 tggcacgtgt cgaagaaaat ggtgagttga cttggctcaa acacaatcca
attcaaaaag 3480 gagagtttgc ctataattcg ctccaagaat taggaaatgg
ggagtatggc atcttgtatg 3540 aacatactga aaaaggacaa aatgcctata
ccctatcatt tagaaaattt aattgggact 3600 ttttgagcaa agatctgatt
tctcctaccg aagcgaaagt gaagcgaact agagagatgg 3660 gcaaaggagt
tattggcttg gagttcgact cagaagtatt ggtcaacaag gctccaaccc 3720
ttcaattggc aaatggtaaa acagcacgct tcatgaccca gtatgataca aaaaccctcc
3780 tatttacagt ggattcagag gatatgggtc aaaaagttac aggtttggca
gaaggtgcaa 3840 ttgaaagtat gcataattta ccagtctctg tggcgggcac
taagctttcg aatggaatga 3900 acggaagtga agctgctgtt catgaagtgc
cagaatacac aggcccatta gggacatccg 3960 gcgaagagcc agctccaaca
gtcgagaagc cagaatacac aggcccacta gggacatccg 4020 gcgaagagcc
agccccgaca gtcgagaagc cagaatacac aggcccacta gggacagctg 4080
gtgaagaagc agctccaaca gtcgagaagc cagaatttac agggggagtt aatggtacag
4140 agccagctgt tcatgaaatc gcagagtata agggatctga ttcgcttgta
actcttacta 4200 caaaagaaga ttatacttac aaagctcctc ttgctcagca
ggcacttcct gaaacaggaa 4260 acaaggagag tgacctccta gcttcactag
gactaacagc tttcttcctt ggtctgttta 4320 cgctagggaa aaagagagaa
caataagaga agaattctaa acatttgatt ttgtaaaaat 4380 agaaggagat
agcaggtttt caagcctgct atcttttttt gatgacattc aggctgatac 4440
gaaatcataa gaggtctgaa actactttca gagtagtctg ttctataaaa tatagtagat
4500 36 705 DNA Staphylococcus epidermidis 36 gatccaagct tatcgatatc
atcaaaaagt tggcgaacct tttcaaattt tggttcaaat 60 tcttgagatg
tatagaattc aaaatattta ccatttgcat agtctgattg ctcaaagtct 120
tgatactttt ctccacgctc ttttgcaatt tccattgaac gttcgatgga ataatagttc
180 ataatcataa agaatatatt agcaaagtct tttgcttctt cagattcata
gccaatttta 240 tttttagcta gataaccatg taagttcatt actcctagtc
caacagaatg tagttcacta 300 ttcgcttttt ttacacctgg tgcattttga
atatttgctt catcacttac aactgtaaga 360 gcatccatac ctgtgaacac
agaatctctg aatttacctg attccataac attcactata 420 ttcaatgagc
ctaagttaca tgaaatatct cttttaattt catcttcaat tccatagtcg 480
ttaattactg atgtctcttg taattggaaa atttcagtac ataaattact cattttaatt
540 tgcccaatat ttgaattcgc atgtactttg tttgcattat ctttaaacat
aagatatgga 600 taaccagact gtaattgtgt ttgtgcaatc atatttaaca
tttcacgtgc gtcttttttc 660 tttttatcga tttcgaaccc ggggtaccga
attcctcgag tctag 705 37 442 DNA Staphylococcus aureus 37 gatcaatctt
tgtcggtaca cgatattctt cacgactaaa taaacgctca ttcgcgattt 60
tataaatgaa tgttgataac aatgttgtat tatctactga aatctcatta cgttgcatcg
120 gaaacattgt gttctgtatg taaaagccgt cttgataatc tttagtagta
ccgaagctgg 180 tcatacgaga gttatatttt ccagccaaaa cgatattttt
ataatcatta cgtgaaaaag 240 gtttcccttc attatcacac aaatatttta
gcttttcagt ttctatatca actgtagctt 300 ctttatccat acgttgaata
attgtacgat tctgacgcac catcttttgc acacctttaa 360 tgttatttgt
tttaaaagca tgaataagtt tttcaacaca acgatgtgaa tcttctaaga 420
agtcaccgta aaatgaagga tc 442 38 20 DNA Enterococcus faecalis 38
gcaatacagg gaaaaatgtc 20 39 20 DNA Enterococcus faecalis 39
cttcatcaaa caattaactc 20 40 20 DNA Enterococcus faecalis 40
gaacagaaga agccaaaaaa 20 41 20 DNA Enterococcus faecalis 41
gcaatcccaa ataatacggt 20 42 19 DNA Escherichia coli 42 gctttccagc
gtcatattg 19 43 19 DNA Escherichia coli 43 gatctcgaca aaatggtga 19
44 25 DNA Escherichia coli 44 cacccgcttg cgtggcaagc tgccc 25 45 25
DNA Escherichia coli 45 cgtttgtgga ttccagttcc atccg 25 46 17 DNA
Escherichia coli 46 tcacccgctt gcgtggc 17 47 19 DNA Escherichia
coli 47 ggaactggaa tccacaaac 19 48 25 DNA Escherichia coli 48
tgaagcactg gccgaaatgc tgcgt 25 49 25 DNA Escherichia coli 49
gatgtacagg attcgttgaa ggctt 25 50 25 DNA Escherichia coli 50
tagcgaaggc gtagcagaaa ctaac 25 51 25 DNA Escherichia coli 51
gcaacccgaa ctcaacgccg gattt 25 52 25 DNA Escherichia coli 52
atacacaagg gtcgcatctg cggcc 25 53 26 DNA Escherichia coli 53
tgcgtatgca ttgcagacct tgtggc 26 54 25 DNA Escherichia coli 54
gctttcactg gatatcgcgc ttggg 25 55 19 DNA Escherichia coli 55
gcaacccgaa ctcaacgcc 19 56 19 DNA Escherichia coli 56 gcagatgcga
cccttgtgt 19 57 23 DNA Klebsiella pneumoniae 57 gtggtgtcgt
tcagcgcttt cac 23 58 25 DNA Klebsiella pneumoniae 58 gcgatattca
caccctacgc agcca 25 59 26 DNA Klebsiella pneumoniae 59 gtcgaaaatg
ccggaagagg tatacg 26 60 26 DNA Klebsiella pneumoniae 60 actgagctgc
agaccggtaa aactca 26 61 19 DNA Klebsiella pneumoniae 61 gacagtcagt
tcgtcagcc 19 62 19 DNA Klebsiella pneumoniae 62 cgtagggtgt
gaatatcgc 19 63 26 DNA Klebsiella pneumoniae 63 cgtgatggat
attcttaacg aagggc 26 64 23 DNA Klebsiella pneumoniae 64 accaaactgt
tgagccgcct gga 23 65 23 DNA Klebsiella pneumoniae 65 gtgatcgccc
ctcatctgct act 23 66 26 DNA Klebsiella pneumoniae 66 cgcccttcgt
taagaatatc catcac 26 67 19 DNA Klebsiella pneumoniae 67 tcgcccctca
tctgctact 19 68 19 DNA Klebsiella pneumoniae 68 gatcgtgatg
gatattctt 19 69 25 DNA Klebsiella pneumoniae 69 caggaagatg
ctgcaccggt tgttg 25 70 25 DNA Proteus mirabilis 70 tggttcactg
actttgcgat gtttc 25 71 25 DNA Proteus mirabilis 71 tcgaggatgg
catgcactag aaaat 25 72 30 DNA Proteus mirabilis 72 cgctgattag
gtttcgctaa aatcttatta 30 73 30 DNA Proteus mirabilis 73 ttgatcctca
ttttattaat cacatgacca 30 74 19 DNA Proteus mirabilis 74 gaaacatcgc
aaagtcagt 19 75 20 DNA Proteus mirabilis 75 ataaaatgag gatcaagttc
20 76 30 DNA Proteus mirabilis 76 ccgcctttag cattaattgg tgtttatagt
30 77 30 DNA Proteus mirabilis 77 cctattgcag ataccttaaa tgtcttgggc
30 78 26 DNA Streptococcus pneumoniae 78 agtaaaatga aataagaaca
ggacag 26 79 25 DNA Streptococcus pneumoniae 79 aaaacaggat
aggagaacgg gaaaa 25 80 25 DNA Proteus mirabilis 80 ttgagtgatg
atttcactga ctccc 25 81 25 DNA Proteus mirabilis 81 gtcagacagt
gatgctgacg acaca 25 82 27 DNA Proteus mirabilis 82 tggttgtcat
gctgtttgtg tgaaaat 27 83 19 DNA Pseudomonas aeruginosa 83
cgagcgggtg gtgttcatc 19 84 19 DNA Pseudomonas aeruginosa 84
caagtcgtcg tcggaggga 19 85 19 DNA Pseudomonas aeruginosa 85
tcgctgttca tcaagaccc 19 86 19 DNA Pseudomonas aeruginosa 86
ccgagaacca gacttcatc 19 87 25 DNA Pseudomonas aeruginosa 87
aatgcggctg tacctcggcg ctggt 25 88 25 DNA Pseudomonas aeruginosa 88
ggcggagggc cagttgcacc tgcca 25 89 25 DNA Pseudomonas aeruginosa 89
agccctgctc ctcggcagcc tctgc 25 90 25 DNA Pseudomonas aeruginosa 90
tggcttttgc aaccgcgttc aggtt 25 91 25 DNA Pseudomonas aeruginosa 91
gcgcccgcga gggcatgctt cgatg 25 92 25 DNA Pseudomonas aeruginosa 92
acctgggcgc caactacaag ttcta 25 93 25 DNA Pseudomonas aeruginosa 93
ggctacgctg ccgggctgca ggccg 25 94 25 DNA Pseudomonas aeruginosa 94
ccgatctaca ccatcgagat gggcg 25 95 25 DNA Pseudomonas aeruginosa 95
gagcgcggct atgtgttcgt cggct 25 96 29 DNA Staphylococcus
saprophyticus 96 cgtttttacc cttacctttt cgtactacc 29 97 30 DNA
Staphylococcus saprophyticus 97 tcaggcagag gtagtacgaa aaggtaaggg 30
98 26 DNA Staphylococcus saprophyticus 98 cgtttttacc cttacctttt
cgtact 26 99 28 DNA Staphylococcus saprophyticus 99 atcgatcatc
acattccatt tgttttta 28 100 27 DNA Staphylococcus saprophyticus 100
caccaagttt gacacgtgaa gattcat 27 101 30 DNA Staphylococcus
saprophyticus 101 atgagtgaag cggagtcaga ttatgtgcag 30 102 25 DNA
Staphylococcus saprophyticus 102 cgctcattac gtacagtgac aatcg 25 103
30 DNA Staphylococcus saprophyticus 103 ctggttagct tgactcttaa
caatcttgtc 30 104 30 DNA Staphylococcus saprophyticus 104
gacgcgattg tcactgtacg taatgagcga 30 105 28 DNA Haemophilus
influenzae 105 gcgtcagaaa aagtaggcga aatgaaag 28 106 25 DNA
Haemophilus influenzae 106 agcggctcta tcttgtaatg acaca 25 107 25
DNA Haemophilus influenzae 107 gaaacgtgaa ctcccctcta tataa 25 108
25 DNA Moraxella catarrhalis 108 gccccaaaac aatgaaacat atggt 25 109
25 DNA Moraxella catarrhalis 109 ctgcagattt tggaatcata tcgcc 25 110
25 DNA Moraxella catarrhalis 110 tggtttgacc agtatttaac gccat 25 111
25 DNA Moraxella catarrhalis 111 caacggcacc tgatgtacct tgtac 25 112
18 DNA Moraxella catarrhalis 112 ggcacctgat gtaccttg 18 113 17 DNA
Moraxella catarrhalis 113 aacagctcac acgcatt 17 114 25 DNA
Moraxella catarrhalis 114 ttacaacctg caccacaagt catca 25 115 25 DNA
Moraxella catarrhalis 115 gtacaaacaa gccgtcagcg actta 25 116 23 DNA
Moraxella catarrhalis 116 caatctgcgt gtgtgcgttc act 23 117 26 DNA
Moraxella catarrhalis 117 gctactttgt cagctttagc cattca 26 118 24
DNA Moraxella catarrhalis 118 tgttttgagc tttttatttt ttga 24 119 22
DNA Moraxella catarrhalis 119 cgctgacggc ttgtttgtac ca 22 120 25
DNA Streptococcus pneumoniae 120 tctgtgctag agactgcccc atttc 25 121
25 DNA Streptococcus pneumoniae 121 cgatgtcttg attgagcagg gttat 25
122 25 DNA Artificial Sequence Description of Artificial Sequence
Oligonucleotide 122 atcccacctt aggcggctgg ctcca 25 123 31 DNA
Artificial Sequence Description of Artificial Sequence
Oligonucleotide 123 acgtcaagtc atcatggccc ttacgagtag g 31 124 25
DNA Artificial Sequence Description of Artificial Sequence
Oligonucleotide 124 gtgtgacggg cggtgtgtac aaggc 25 125 28 DNA
Artificial Sequence Description of Artificial Sequence
Oligonucleotide 125 gagttgcaga ctccaatccg gactacga 28 126 20 DNA
Artificial Sequence Description of Artificial Sequence
Oligonucleotide 126 ggaggaaggt ggggatgacg 20 127 20 DNA Artificial
Sequence Description of Artificial Sequence Oligonucleotide 127
atggtgtgac gggcggtgtg 20 128 32 DNA Artificial Sequence Description
of Artificial Sequence Oligonucleotide 128 ccctatacat caccttgcgg
tttagcagag ag 32 129 28 DNA Artificial Sequence Description of
Artificial Sequence Oligonucleotide 129 ggggggacca tcctccaagg
ctaaatac 28 130 32 DNA Artificial Sequence Description of
Artificial Sequence Oligonucleotide 130 cgtccacttt cgtgtttgca
gagtgctgtg tt 32 131 20 DNA Escherichia coli 131 caggagtacg
gtgattttta 20 132 20 DNA Escherichia coli 132 atttctggtt tggtcataca
20 133 20 DNA Proteus mirabilis 133 cgggagtcag tgaaatcatc 20 134 20
DNA Proteus mirabilis 134 ctaaaatcgc cacacctctt 20 135 18 DNA
Klebsiella pneumoniae 135 gcagcgtggt gtcgttca 18 136 18 DNA
Klebsiella pneumoniae 136 agctggcaac ggctggtc 18 137 20 DNA
Klebsiella pneumoniae 137 attcacaccc tacgcagcca 20 138 20 DNA
Klebsiella pneumoniae 138 atccggcagc atctctttgt 20 139 25 DNA
Staphylococcus saprophyticus 139 ctggttagct tgactcttaa caatc 25 140
25 DNA Staphylococcus saprophyticus 140 tcttaacgat agaatggagc aactg
25 141 20 DNA Streptococcus pyogenes 141 tgaaaattct tgtaacaggc 20
142 20 DNA Streptococcus pyogenes 142 ggccaccagc ttgcccaata 20 143
20 DNA Streptococcus pyogenes 143 atattttctt tatgagggtg 20 144 20
DNA Streptococcus pyogenes 144 atccttaaat aaagttgcca 20 145 25 DNA
Staphylococcus epidermidis 145 atcaaaaagt tggcgaacct tttca 25 146
25 DNA Staphylococcus epidermidis 146 caaaagagcg tggagaaaag tatca
25 147 30 DNA Staphylococcus epidermidis 147 tctcttttaa tttcatcttc
aattccatag 30 148 30 DNA Staphylococcus epidermidis 148 aaacacaatt
acagtctggt tatccatatc 30 149 30 DNA Staphylococcus aureus 149
cttcatttta cggtgacttc ttagaagatt 30 150 30 DNA Staphylococcus
aureus 150 tcaactgtag cttctttatc catacgttga 30 151 30 DNA
Staphylococcus aureus 151 atattttagc ttttcagttt ctatatcaac 30 152
30 DNA Staphylococcus aureus 152 aatctttgtc ggtacacgat attcttcacg
30 153 30 DNA Staphylococcus aureus 153 cgtaatgaga tttcagtaga
taatacaaca 30 154 25 DNA Haemophilus influenzae 154 tttaacgatc
cttttactcc ttttg 25 155 25 DNA Haemophilus influenzae 155
actgctgttg taaagaggtt aaaat 25 156 20 DNA Streptococcus pneumoniae
156 atttggtgac gggtgacttt 20 157 20 DNA Streptococcus pneumoniae
157 gctgaggatt tgttcttctt 20 158 20 DNA Streptococcus pneumoniae
158 gagcggtttc tatgattgta 20 159 20 DNA Streptococcus pneumoniae
159 atctttcctt tcttgttctt 20 160 18 DNA Moraxella catarrhalis 160
gctcaaatca gggtcagc 18 161 861 DNA Escherichia coli 161 atgagtattc
aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 60
gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
120 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
ttttcgcccc 180 gaagaacgtt ttccaatgat gagcactttt aaagttctgc
tatgtggcgc ggtattatcc 240 cgtgttgacg ccgggcaaga gcaactcggt
cgccgcatac actattctca gaatgacttg 300 gttgagtact caccagtcac
agaaaagcat cttacggatg gcatgacagt aagagaatta 360 tgcagtgctg
ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 420
ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
480 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
caccacgatg 540 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg
gcgaactact tactctagct 600 tcccggcaac aattaataga ctggatggag
gcggataaag ttgcaggacc acttctgcgc 660 tcggcccttc cggctggctg
gtttattgct gataaatctg gagccggtga gcgtgggtct 720 cgcggtatca
ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 780
acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
840 tcactgatta agcattggta a 861 162 918 DNA Pasteurella haemolytica
162 atgttaaata agttaaaaat cggcacatta ttattgctga cattaacggc
ttgttcgccc 60 aattctgttc attcggtaac gtctaatccg cagcctgcta
gtgcgcctgt gcaacaatca 120 gccacacaag ccacctttca acagactttg
gcgaatttgg aacagcagta tcaagcccga 180 attggcgttt atgtatggga
tacagaaacg ggacattctt tgtcttatcg tgcagatgaa 240 cgctttgctt
atgcgtccac tttcaaggcg ttgttggctg gggcggtgtt gcaatcgctg 300
cctgaaaaag atttaaatcg taccatttca tatagccaaa aagatttggt tagttattct
360 cccgaaaccc aaaaatacgt tggcaaaggc atgacgattg cccaattatg
tgaagcagcc 420 gtgcggttta gcgacaacag cgcgaccaat ttgctgctca
aagaattggg tggcgtggaa 480 caatatcaac gtattttgcg acaattaggc
gataacgtaa cccataccaa tcggctagaa 540 cccgatttaa atcaagccaa
acccaacgat attcgtgata cgagtacacc caaacaaatg 600 gcgatgaatt
taaatgcgta tttattgggc aacacattaa ccgaatcgca aaaaacgatt 660
ttgtggaatt ggttggacaa taacgcaaca ggcaatccat tgattcgcgc tgctacgcca
720 acatcgtgga aagtgtacga taaaagcggg gcgggtaaat atggtgtacg
caatgatatt 780 gcggtggttc gcataccaaa tcgcaaaccg attgtgatgg
caatcatgag tacgcaattt 840 accgaagaag ccaaattcaa caataaatta
gtagaagatg cagcaaagca agtatttcat 900 actttacagc tcaactaa 918 163
864 DNA Klebsiella pneumoniae 163 atgcgttata ttcgcctgtg tattatctcc
ctgttagcca ccctgccgct ggcggtacac 60 gccagcccgc agccgcttga
gcaaattaaa ctaagcgaaa gccagctgtc gggccgcgta 120 ggcatgatag
aaatggatct ggccagcggc cgcacgctga ccgcctggcg cgccgatgaa 180
cgctttccca tgatgagcac ctttaaagta gtgctctgcg gcgcagtgct ggcgcgggtg
240 gatgccggtg acgaacagct ggagcgaaag atccactatc gccagcagga
tctggtggac 300 tactcgccgg tcagcgaaaa acaccttgcc gacgcaatga
cggtcggcga actctgcgcc 360 gccgccatta ccatgagcga taacagcgcc
gccaatctgc tactggccac cgtcggcggc 420 cccgcaggat tgactgcctt
tttgcgccag atcggcgaca acgtcacccg ccttgaccgc 480 tgggaaacgg
aactgaatga ggcgcttccc ggcgacgccc gcgacaccac taccccggcc 540
agcatggccg cgaccctgcg caacgttggc ctgaccagcc agcgtctgag cgcccgttcg
600 caacggcagc tgctgcagtg gatggtggac gatcgggtcg ccggaccgtt
gatccgctcc 660 gtgctgccgg cgggctggtt tatcgccgat aagaccggag
ctggcgagcg gggtgcgcgc 720 gggattgtcg ccctgcttgg cccgaataac
aaagcagagc gcattgtggt gatttatctg 780 cgggataccc cggcgagcat
ggccgagcga aatcagcaaa tcgccgggat cggcaaggcg 840 ctgtacgagc
actggcaacg ctaa 864 164 534 DNA Klebsiella pneumoniae 164
atggacacaa cgcaggtcac attgatacac aaaattctag ctgcggcaga tgagcgaaat
60 ctgccgctct ggatcggtgg gggctgggcg atcgatgcac ggctagggcg
tgtaacacgc 120 aagcacgatg atattgatct gacgtttccc ggcgagaggc
gcggcgagct cgaggcaata 180 gttgaaatgc tcggcgggcg cgtcatggag
gagttggact atggattctt agcggagatc 240 ggggatgagt tacttgactg
cgaacctgct tggtgggcag acgaagcgta tgaaatcgcg 300 gaggctccgc
agggctcgtg cccagaggcg gctgagggcg tcatcgccgg gcggccagtc 360
cgttgtaaca gctgggaggc gatcatctgg gattactttt actatgccga tgaagtacca
420 ccagtggact ggcctacaaa gcacatagag tcctacaggc tcgcatgcac
ctcactcggg 480 gcggaaaagg ttgaggtctt gcgtgccgct ttcaggtcgc
gatatgcggc ctaa 534 165 465 DNA Unknown Organism Description of
Unknown Organism Enterobacteriaceae 165 atgggcatca ttcgcacatg
taggctcggc cctgaccaag tcaaatccat gcgggctgct 60 cttgatcttt
tcggtcgtga gttcggagac gtagccacct actcccaaca tcagccggac 120
tccgattacc tcgggaactt gctccgtagt aagacattca tcgcgcttgc tgccttcgac
180 caagaagcgg ttgttggcgc tctcgcggct tacgttctgc ccaggtttga
gcagccgcgt 240 agtgagatct atatctatga tctcgcagtc tccggcgagc
accggaggca gggcattgcc 300 accgcgctca tcaatctcct caagcatgag
gccaacgcgc ttggtgctta tgtgatctac 360 gtgcaagcag attacggtga
cgatcccgca gtggctctct atacaaagtt gggcatacgg 420 gaagaagtga
tgcactttga tatcgaccca agtaccgcca cctaa 465 166 861 DNA Escherichia
coli 166 atgcatacgc ggaaggcaat aacggaggcg cttcaaaaac tcggagtcca
aaccggtgac 60 ctattgatgg tgcatgcctc acttaaagcg attggtccgg
tcgaaggagg agcggagacg 120 gtcgttgccg cgttacgctc cgcggttggg
ccgactggca ctgtgatggg atacgcatcg 180 tgggaccgat caccctacga
ggagactcgt aatggcgctc ggttggatga caaaacccgc 240 cgtacctggc
cgccgttcga tcccgcaacg gccgggactt accgtgggtt cggcctgctg 300
aatcagtttc tggttcaagc ccccggcgcg cggcgcagcg cgcaccccga tgcatcgatg
360 gtcgcggttg gtccactggc tgaaacgctg acggagcctc acaagctcgg
tcacgccttg 420 ggggaagggt cgcccgtcga gcggttcgtt cgccttggcg
ggaaggccct gctgttgggt 480 gcgccgctaa actccgttac cgcattgcac
tacgccgagg cggttgccga tatccccaac 540 aaacggcggg tgacgtatga
gatgccgatg cttggaagca acggcgaagt cgcctggaaa 600 acggcatcgg
attacgattc aaacggcatt ctcgattgct ttgctatcga aggaaagccg 660
gatgcggtcg aaactatagc aaatgcttac gtgaagctcg gtcgccatcg agaaggtgtc
720 gtgggctttg ctcagtgcta cctgttcgac gcgcaggaca tcgtgacgtt
cggcgtcacc 780 tatcttgaga agcatttcgg aaccactccg atcgtgccag
cacacgaagt cgccgagtgc 840 tcttgcgagc cttcaggtta g 861 167 816 DNA
Pseudomonas aeruginosa 167 atgaccgatt tgaatatccc gcatacacac
gcgcaccttg tagacgcatt tcaggcgctc 60 ggcatccgcg cggggcaggc
gctcatgctg cacgcatccg ttaaagcagt gggcgcggtg 120 atgggcggcc
ccaatgtgat cttgcaggcg ctcatggatg cgctcacgcc cgacggcacg 180
ctgatgatgt atgcgggatg gcaagacatc cccgacttta tcgactcgct gccggacgcg
240 ctcaaggccg tgtatcttga gcagcaccca ccctttgacc ccgccaccgc
ccgcgccgtg 300 cgcgaaaaca gcgtgctagc ggaatttttg cgcacatggc
cgtgcgtgca tcgcagcgca 360 aaccccgaag cctctatggt ggcggtaggc
aggcaggccg ctttgctgac cgctaatcac 420 gcgctggatt atggctacgg
agtcgagtcg ccgctggcta aactggtggc aatagaagga 480 tacgtgctga
tgcttggcgc gccgctggat accatcacac tgctgcacca cgcggaatat 540
ctggccaaga tgcgccacaa gaacgtggtc cgctacccgt gcccgattct gcgggacggg
600 cgcaaagtgt gggtgaccgt tgaggactat gacaccggtg atccgcacga
cgattatagt 660 tttgagcaaa tcgcgcgcga ttatgtggcg cagggcggcg
gcacacgcgg caaagtcggt 720 gatgcggatg cttacctgtt cgccgcgcag
gacctcacac ggtttgcggt gcagtggctt 780 gaatcacggt tcggtgactc
agcgtcatac ggatag 816 168 498 DNA Pseudomonas aeruginosa 168
atgctctatg agtggctaaa tcgatctcat atcgtcgagt ggtggggcgg agaagaagca
60 cgcccgacac ttgctgacgt acaggaacag tacttgccaa gcgttttagc
gcaagagtcc 120 gtcactccat acattgcaat gctgaatgga gagccgattg
ggtatgccca gtcgtacgtt 180 gctcttggaa gcggggacgg atggtgggaa
gaagaaaccg atccaggagt acgcggaata 240 gaccagttac tggcgaatgc
atcacaactg ggcaaaggct tgggaaccaa gctggttcga 300 gctctggttg
agttgctgtt caatgatccc gaggtcacca agatccaaac ggacccgtcg 360
ccgagcaact tgcgagcgat ccgatgctac gagaaagcgg ggtttgagag gcaaggtacc
420 gtaaccaccc cagatggtcc agccgtgtac atggttcaaa cacgccaggc
attcgagcga 480 acacgcagtg atgcctaa 498 169 2007 DNA Staphylococcus
aureus 169 atgaaaaaga taaaaattgt tccacttatt ttaatagttg tagttgtcgg
gtttggtata 60 tatttttatg cttcaaaaga taaagaaatt aataatacta
ttgatgcaat tgaagataaa 120 aatttcaaac aagtttataa agatagcagt
tatatttcta aaagcgataa tggtgaagta 180 gaaatgactg aacgtccgat
aaaaatatat aatagtttag gcgttaaaga tataaacatt 240 caggatcgta
aaataaaaaa agtatctaaa aataaaaaac gagtagatgc tcaatataaa 300
attaaaacaa actacggtaa cattgatcgc aacgttcaat ttaattttgt taaagaagat
360 ggtatgtgga agttagattg ggatcatagc gtcattattc caggaatgca
gaaagaccaa 420 agcatacata ttgaaaattt aaaatcagaa cgtggtaaaa
ttttagaccg aaacaatgtg 480 gaattggcca atacaggaac acatatgaga
ttaggcatcg ttccaaagaa tgtatctaaa 540 aaagattata aagcaatcgc
taaagaacta agtatttctg aagactatat caacaacaaa 600 tggatcaaaa
ttgggtacaa gatgatacct tcgttccact ttaaaaccgt taaaaaaatg 660
gatgaatatt taagtgattt cgcaaaaaaa tttcatctta caactaatga aacagaaagt
720 cgtaactatc ctctagaaaa agcgacttca catctattag gttatgttgg
tcccattaac 780 tctgaagaat taaaacaaaa agaatataaa ggctataaag
atgatgcagt tattggtaaa 840 aagggactcg aaaaacttta cgataaaaag
ctccaacatg aagatggcta tcgtgtcaca 900 atcgttgacg ataatagcaa
tacaatcgca catacattaa tagagaaaaa gaaaaaagat 960 ggcaaagata
ttcaactaac tattgatgct aaagttcaaa agagtattta taacaacatg 1020
aaaaatgatt atggctcagg tactgctatc caccctcaaa caggtgaatt attagcactt
1080 gtaagcacac cttcatatga cgtctatcca tttatgtatg gcatgagtaa
cgaagaatat 1140 aataaattaa ccgaagataa aaaagaacct ctgctcaaca
agttccagat tacaacttca 1200 ccaggttcaa ctcaaaaaat attaacagca
atgattgggt taaataacaa aacattagac 1260 gataaaacaa gttataaaat
cgatggtaaa ggttggcaaa aagataaatc ttggggtggt 1320 tacaacgtta
caagatatga agtggtaaat ggtaatatcg acttaaaaca agcaatagaa 1380
tcatcagata acattttctt tgctagagta gcactcgaat taggcagtaa gaaatttgaa
1440 aaaggcatga aaaaactagg tgttggtgaa gatataccaa gtgattatcc
attttataat 1500 gctcaaattt caaacaaaaa tttagataat gaaatattat
tagctgattc aggttacgga 1560 caaggtgaaa tactgattaa cccagtacag
atcctttcaa tctatagcgc attagaaaat 1620 aatggcaata ttaacgcacc
tcacttatta aaagacacga aaaacaaagt ttggaagaaa 1680 aatattattt
ccaaagaaaa tatcaatcta ttaaatgatg gtatgcaaca agtcgtaaat 1740
aaaacacata aagaagatat ttatagatct tatgcaaact taattggcaa atccggtact
1800 gcagaactca aaatgaaaca aggagaaagt ggcagacaaa ttgggtggtt
tatatcatat 1860 gataaagata atccaaacat gatgatggct attaatgtta
aagatgtaca agataaagga 1920 atggctagct acaatgccaa aatctcaggt
aaagtgtatg atgagctata tgagaacggt 1980 aataaaaaat acgatataga tgaataa
2007 170 2607 DNA Enterococcus faecium 170 atgaataaca tcggcattac
tgtttatgga tgtgagcagg atgaggcaga tgcattccat 60 gctctttcgc
ctcgctttgg cgttatggca acgataatta acgccaacgt gtcggaatcc 120
aacgccaaat ccgcgccttt caatcaatgt atcagtgtgg gacataaatc agagatttcc
180 gcctctattc ttcttgcgct gaagagagcc ggtgtgaaat atatttctac
ccgaagcatc 240 ggctgcaatc atatagatac aactgctgct aagagaatgg
gcatcactgt cgacaatgtg 300
gcgtactcgc cggatagcgt tgccgattat actatgatgc taattcttat ggcagtacgc
360 aacgtaaaat cgattgtgcg ctctgtggaa aaacatgatt tcaggttgga
cagcgaccgt 420 ggcaaggtac tcagcgacat gacagttggt gtggtgggaa
cgggccagat aggcaaagcg 480 gttattgagc ggctgcgagg atttggatgt
aaagtgttgg cttatagtcg cagccgaagt 540 atagaggtaa actatgtacc
gtttgatgag ttgctgcaaa atagcgatat cgttacgctt 600 catgtgccgc
tcaatacgga tacgcactat attatcagcc acgaacaaat acagagaatg 660
aagcaaggag catttcttat caatactggg cgcggtccac ttgtagatac ctatgagttg
720 gttaaagcat tagaaaacgg gaaactgggc ggtgccgcat tggatgtatt
ggaaggagag 780 gaagagtttt tctactctga ttgcacccaa aaaccaattg
ataatcaatt tttacttaaa 840 cttcaaagaa tgcctaacgt gataatcaca
ccgcatacgg cctattatac cgagcaagcg 900 ttgcgtgata ccgttgaaaa
aaccattaaa aactgtttgg attttgaaag gagacaggag 960 catgaataga
ataaaagttg caatactgtt tgggggttgc tcagaggagc atgacgtatc 1020
ggtaaaatct gcaatagaga tagccgctaa cattaataaa gaaaaatacg agccgttata
1080 cattggaatt acgaaatctg gtgtatggaa aatgtgcgaa aaaccttgcg
cggaatggga 1140 aaacgacaat tgctattcag ctgtactctc gccggataaa
aaaatgcacg gattacttgt 1200 taaaaagaac catgaatatg aaatcaacca
tgttgatgta gcattttcag ctttgcatgg 1260 caagtcaggt gaagatggat
ccatacaagg tctgtttgaa ttgtccggta tcccttttgt 1320 aggctgcgat
attcaaagct cagcaatttg tatggacaaa tcgttgacat acatcgttgc 1380
gaaaaatgct gggatagcta ctcccgcctt ttgggttatt aataaagatg ataggccggt
1440 ggcagctacg tttacctatc ctgtttttgt taagccggcg cgttcaggct
catccttcgg 1500 tgtgaaaaaa gtcaatagcg cggacgaatt ggactacgca
attgaatcgg caagacaata 1560 tgacagcaaa atcttaattg agcaggctgt
ttcgggctgt gaggtcggtt gtgcggtatt 1620 gggaaacagt gccgcgttag
ttgttggcga ggtggaccaa atcaggctgc agtacggaat 1680 ctttcgtatt
catcaggaag tcgagccgga aaaaggctct gaaaacgcag ttataaccgt 1740
tcccgcagac ctttcagcag aggagcgagg acggatacag gaaacggcaa aaaaaatata
1800 taaagcgctc ggctgtagag gtctagcccg tgtggatatg tttttacaag
ataacggccg 1860 cattgtactg aacgaagtca atactctgcc cggtttcacg
tcatacagtc gttatccccg 1920 tatgatggcc gctgcaggta ttgcacttcc
cgaactgatt gaccgcttga tcgtattagc 1980 gttaaagggg tgataagcat
ggaaatagga tttacttttt tagatgaaat agtacacggt 2040 gttcgttggg
acgctaaata tgccacttgg gataatttca ccggaaaacc ggttgacggt 2100
tatgaagtaa atcgcattgt agggacatac gagttggctg aatcgctttt gaaggcaaaa
2160 gaactggctg ctacccaagg gtacggattg cttctatggg acggttaccg
tcctaagcgt 2220 gctgtaaact gttttatgca atgggctgca cagccggaaa
ataacctgac aaaggaaagt 2280 tattatccca atattgaccg aactgagatg
atttcaaaag gatacgtggc ttcaaaatca 2340 agccatagcc gcggcagtgc
cattgatctt acgctttatc gattagacac gggtgagctt 2400 gtaccaatgg
ggagccgatt tgattttatg gatgaacgct ctcatcatgc ggcaaatgga 2460
atatcatgca atgaagcgca aaatcgcaga cgtttgcgct ccatcatgga aaacagtggg
2520 tttgaagcat atagcctcga atggtggcac tatgtattaa gagacgaacc
ataccccaat 2580 agctattttg atttccccgt taaataa 2607 171 1288 DNA
Pseudomonas aeruginosa 171 ggatccatca ggcaacgacg ggctgctgcc
ggccatcagc ggacgcaggg aggactttcc 60 gcaaccggcc gttcgatgcg
gcaccgatgg ccttcgcgca ggggtagtga atccgccagg 120 attgacttgc
gctgccctac ctctcactag tgaggggcgg cagcgcatca agcggtgagc 180
gcactccggc accgccaact ttcagcacat gcgtgtaaat catcgtcgta gagacgtcgg
240 aatggccgag cagatcctgc acggttcgaa tgtcgtaacc gctgcggagc
aaggccgtcg 300 cgaacgagtg gcggagggtg tgcggtgtgg cgggcttcgt
gatgcctgct tgttctacgg 360 cacgtttgaa ggcgcgctga aaggtctggt
catacatgtg atggcgacgc acgacaccgc 420 tccgtggatc ggtcgaatgc
gtgtgctgcg caaaaaccca gaaccacggc caggaatgcc 480 cggcgcgcgg
atacttccgc tcaagggcgt cgggaagcgc aacgccgctg cggccctcgg 540
cctggtcctt cagccaccat gcccgtgcac gcgacagctg ctcgcgcagg ctgggtgcca
600 agctctcggg taacatcaag gcccgatcct tggagccctt gccctcccgc
acgatgatcg 660 tgccgtgatc gaaatccaga tccttgaccc gcagttgcaa
accctcactg atccgcatgc 720 ccgttccata cagaagctgg gcgaacaaac
gatgctcgcc ttccagaaaa ccgaggatgc 780 gaaccacttc atccggggtc
agcaccaccg gcaagcgccg cgacggccga ggtcttccga 840 tctcctgaag
ccagggcaga tccgtgcaca gcaccttgcc gtagaagaac agcaaggccg 900
ccaatgcctg acgatgcgtg gagaccgaaa ccttgcgctc gttcgccagc caggacagaa
960 atgcctcgac ttcgctgctg cccaaggttg ccgggtgacg cacaccgtgg
aaacggatga 1020 aggcacgaac ccagtggaca taagcctgtt cggttcgtaa
gctgtaatgc aagtagcgta 1080 tgcgctcacg caactggtcc agaaccttga
ccgaacgcag cggtggtaac ggcgcagtgg 1140 cggttttcat ggcttgttat
gactgttttt ttgtacagtc tatgcctcgg gcatccaagc 1200 agcaagcgcg
ttacgccgtg ggtcgatgtt tgatgttatg gagcagcaac gatgttacgc 1260
agcagggcag tcgccctaaa acaaagtt 1288 172 1650 DNA Pseudomonas
aeruginosa 172 gttagatgca ctaagcacat aattgctcac agccaaacta
tcaggtcaag tctgctttta 60 ttatttttaa gcgtgcataa taagccctac
acaaattggg agatatatca tgaaaggctg 120 gctttttctt gttatcgcaa
tagttggcga agtaatcgca acatccgcat taaaatctag 180 cgagggcttt
actaagcttg ccccttccgc cgttgtcata atcggttatg gcatcgcatt 240
ttattttctt tctctggttc tgaaatccat ccctgtcggt gttgcttatg cagtctggtc
300 gggactcggc gtcgtcataa ttacagccat tgcctggttg cttcatgggc
aaaagcttga 360 tgcgtggggc tttgtaggta tggggctcat aattgctgcc
tttttgctcg cccgatcccc 420 atcgtggaag tcgctgcgga ggccgacgcc
atggtgacgg tgttcggcat tctgaatctc 480 accgaggact ccttcttcga
tgagagccgg cggctagacc ccgccggcgc tgtcaccgcg 540 gcgatcgaaa
tgctgcgagt cggatcagac gtcgtggatg tcggaccggc cgccagccat 600
ccggacgcga ggcctgtatc gccggccgat gagatcagac gtattgcgcc gctcttagac
660 gccctgtccg atcagatgca ccgtgtttca atcgacagct tccaaccgga
aacccagcgc 720 tatgcgctca agcgcggcgt gggctacctg aacgatatcc
aaggatttcc tgaccctgcg 780 ctctatcccg atattgctga ggcggactgc
aggctggtgg ttatgcactc agcgcagcgg 840 gatggcatcg ccacccgcac
cggtcacctt cgacccgaag acgcgctcga cgagattgtg 900 cggttcttcg
aggcgcgggt ttccgccttg cgacggagcg gggtcgctgc cgaccggctc 960
atcctcgatc cggggatggg atttttcttg agccccgcac cggaaacatc gctgcacgtg
1020 ctgtcgaacc ttcaaaagct gaagtcggcg ttggggcttc cgctattggt
ctcggtgtcg 1080 cggaaatcct tcttgggcgc caccgttggc cttcctgtaa
aggatctggg tccagcgagc 1140 cttgcggcgg aacttcacgc gatcggcaat
ggcgctgact acgtccgcac ccacgcgcct 1200 ggagatctgc gaagcgcaat
caccttctcg gaaaccctcg cgaaatttcg cagtcgcgac 1260 gccagagacc
gagggttaga tcatgcctag cattcacctt ccggccgccc gctagcggac 1320
cctggtcagg ttccgcgaag gtgggcgcag acatgctggg ctcgtcagga tcaaactgca
1380 ctatgaggcg gcggttcata ccgcgccagg ggagcgaatg gacagcgagg
agcctccgaa 1440 cgttcgggtc gcctgctcgg gtgatatcga cgaggttgtg
cggctgatgc acgacgctgc 1500 ggcgtggatg tccgccaagg gaacgcccgc
ctgggacgtc gcgcggatcg accggacatt 1560 cgcggagacc ttcgtcctga
gatccgagct cctagtcgcg agttgcagcg acggcatcgt 1620 cggctgttgc
accttgtcgg ccgaggatcc 1650 173 630 DNA Enterococcus faecium 173
atgggtccga atcctatgaa aatgtatcct atagaaggaa acaaatcagt acaatttatc
60 aaacctattt tagaaaaatt agaaaatgtt gaggttggag aatactcata
ttatgattct 120 aagaatggag aaacttttga taagcaaatt ttatatcatt
atccaatctt aaacgataag 180 ttaaaaatag gtaaattttg ctcaatagga
ccaggtgtaa ctattattat gaatggagca 240 aatcatagaa tggatggctc
aacatatcca tttaatttat ttggtaatgg atgggagaaa 300 catatgccaa
aattagatca actacctatt aagggggata caataatagg taatgatgta 360
tggataggaa aagatgttgt aattatgcca ggagtaaaaa tcggggatgg tgcaatagta
420 gctgctaatt ctgttgttgt aaaagatata gcgccataca tgttagctgg
aggaaatcct 480 gctaacgaaa taaaacaaag atttgatcaa gatacaataa
atcagctgct tgatataaaa 540 tggtggaatt ggccaataga cattattaat
gagaatatag ataaaattct tgataatagc 600 atcattagag aagtcatatg
gaaaaaatga 630 174 1440 DNA Enterococcus faecalis 174 atgaatatag
ttgaaaatga aatatgtata agaactttaa tagatgatga ttttcctttg 60
atgttaaaat ggttaactga tgaaagagta ttagaatttt atggtggtag agataaaaaa
120 tatacattag aatcattaaa aaaacattat acagagcctt gggaagatga
agtttttaga 180 gtaattattg aatataacaa tgttcctatt ggatatggac
aaatatataa aatgtatgat 240 gagttatata ctgattatca ttatccaaaa
actgatgaga tagtctatgg tatggatcaa 300 tttataggag agccaaatta
ttggagtaaa ggaattggta caagatatat taaattgatt 360 tttgaatttt
tgaaaaaaga aagaaatgct aatgcagtta ttttagaccc tcataaaaat 420
aatccaagag caataagggc ataccaaaaa tctggtttta gaattattga agatttgcca
480 gaacatgaat tacacgaggg caaaaaagaa gattgttatt taatggaata
tagatatgat 540 gataatgcca caaatgttaa ggcaatgaaa tatttaattg
agcattactt tgataatttc 600 aaagtagata gtattgaaat aatcggtagt
ggttatgata gtgtggcata tttagttaat 660 aatgaataca tttttaaaac
aaaatttagt actaataaga aaaaaggtta tgcaaaagaa 720 aaagcaatat
ataatttttt aaatacaaat ttagaaacta atgtaaaaat tcctaatatt 780
gaatattcgt atattagtga tgaattatct atactaggtt ataaagaaat taaaggaact
840 tttttaacac cagaaattta ttctactatg tcagaagaag aacaaaattt
gttaaaacga 900 gatattgcca gttttttaag acaaatgcac ggtttagatt
atacagatat tagtgaatgt 960 actattgata ataaacaaaa tgtattagaa
gagtatatat tgttgcgtga aactatttat 1020 aatgatttaa ctgatataga
aaaagattat atagaaagtt ttatggaaag actaaatgca 1080 acaacagttt
ttgagggtaa aaagtgttta tgccataatg attttagttg taatcatcta 1140
ttgttagatg gcaataatag attaactgga ataattgatt ttggagattc tggaattata
1200 gatgaatatt gtgattttat atacttactt gaagatagtg aagaagaaat
aggaacaaat 1260 tttggagaag atatattaag aatgtatgga aatatagata
ttgagaaagc aaaagaatat 1320 caagatatag ttgaagaata ttatcctatt
gaaactattg tttatggaat taaaaatatt 1380 aaacaggaat ttatcgaaaa
tggtagaaaa gaaatttata aaaggactta taaagattga 1440 175 660 DNA
Staphylococcus aureus 175 ttgaatttaa acaatgacca tggacctgat
cccgaaaata ttttaccgat aaaagggaat 60 cggaatcttc aatttataaa
acctactata acgaacgaaa acattttggt gggggaatat 120 tcttattatg
atagtaagcg aggagaatcc tttgaagatc aagtcttata tcattatgaa 180
gtgattggag ataagttgat tataggaaga ttttgttcaa ttggtcccgg aacaacattt
240 attatgaatg gtgcaaacca tcggatggat ggatcaacat atccttttca
tctattcagg 300 atgggttggg agaagtatat gccttcctta aaagatcttc
ccttgaaagg ggacattgaa 360 attggaaatg atgtatggat aggtagagat
gtaaccatta tgcctggggt gaaaattggg 420 gacggggcaa tcattgctgc
agaagctgtt gtcacaaaga atgttgctcc ctattctatt 480 gtcggtggaa
atcccttaaa atttataaga aaaaggtttt ctgatggagt tatcgaagaa 540
tggttagctt tacaatggtg gaatttagat atgaaaatta ttaatgaaaa tcttcccttc
600 ataataaatg gagatatcga aatgctgaag agaaaaagaa aacttctaga
tgacacttga 660 176 1569 DNA Staphylococcus aureus 176 atgaaaataa
tgttagaggg acttaatata aaacattatg ttcaagatcg tttattgttg 60
aacataaatc gcctaaagat ttatcagaat gatcgtattg gtttaattgg taaaaatgga
120 agtggaaaaa caacgttact tcacatatta tataaaaaaa ttgtgcctga
agaaggtatt 180 gtaaaacaat tttcacattg tgaacttatt cctcaattga
agctcataga atcaactaaa 240 agtggtggtg aagtaacacg aaactatatt
cggcaagcgc ttgataaaaa tccagaactg 300 ctattagcag atgaaccaac
aactaactta gataataact atatagaaaa attagaacag 360 gatttaaaaa
attggcatgg agcatttatt atagtttcac atgatcgcgc ttttttagat 420
aacttgtgta ctactatatg ggaaattgac gagggaagaa taactgaata taaggggaat
480 tatagtaact atgttgaaca aaaagaatta gaaagacatc gagaagaatt
agaatatgaa 540 aaatatgaaa aagaaaagaa acgattggaa aaagctataa
atataaaaga acagaaagct 600 caacgagcaa ctaaaaaacc gaaaaactta
agtttatctg aaggcaaaat aaaaggagca 660 aagccatact ttgcaggtaa
gcaaaagaag ttacgaaaaa ctgtaaaatc tctagaaacc 720 agactagaaa
aacttgaaag cgtcgaaaag agaaacgaac ttcctccact taaaatggat 780
ttagtgaact tagaaagtgt aaaaaataga actataatac gtggtgaaga tgtctcgggt
840 acaattgaag gacgggtatt gtggaaagca aaaagtttta gtattcgcgg
aggagacaag 900 atggcaatta tcggatctaa tggtacagga aagacaacgt
ttattaaaaa aattgtgcat 960 gggaatcctg gtatttcatt atcgccatct
gtcaaaatcg gttattttag ccaaaaaata 1020 gatacattag aattagataa
gagcatttta gaaaatgttc aatcttcttc acaacaaaat 1080 gaaactctta
ttcgaactat tctagctaga atgcattttt ttagagatga tgtttataaa 1140
ccaataagtg tcttaagtgg tggagagcga gttaaagtag cactaactaa agtattctta
1200 agtgaagtta atacgttggt actagatgaa ccaacaaact ttcttgatat
ggaagctata 1260 gaggcgtttg aatctttgtt aaaggaatat aatggcagta
taatctttgt atctcacgat 1320 cgtaaattta tcgaaaaagt agccactcga
ataatgacaa ttgataataa agaaataaaa 1380 atatttgatg gcacatatga
acaatttaaa caagctgaaa agccaacaag gaatattaaa 1440 gaagataaaa
aacttttact tgagacaaaa attacagaag tactcagtcg attgagtatt 1500
gaaccttcgg aagaattaga acaagagttt caaaacttaa taaatgaaaa aagaaatttg
1560 gataaataa 1569 177 1467 DNA Staphylococcus epidermidis 177
atggaacaat atacaattaa atttaaccaa atcaatcata aattgacaga tttacgatca
60 cttaacatcg atcatcttta tgcttaccaa tttgaaaaaa tagcacttat
tgggggtaat 120 ggtactggta aaaccacatt actaaatatg attgctcaaa
aaacaaaacc agaatctgga 180 acagttgaaa cgaatggcga aattcaatat
tttgaacagc ttaacatgga tgtggaaaat 240 gattttaaca cgttagacgg
tagtttaatg agtgaactcc atatacctat gcatacaacc 300 gacagtatga
gtggtggtga aaaagcaaaa tataaattac gtaatgtcat atcaaattat 360
agtccgatat tacttttaga tgaacctaca aatcacttgg ataaaattgg taaagattat
420 ctgaataata ttttaaaata ttactatggt actttaatta tagtaagtca
cgatagagca 480 cttatagacc aaattgctga cacaatttgg gatatacaag
aagatggcac aataagagtg 540 tttaaaggta attacacaca gtatcaaaat
caatatgaac aagaacagtt agaacaacaa 600 cgtaaatatg aacagtatat
aagtgaaaaa caaagattgt cccaagccag taaagctaaa 660 cgaaatcaag
cgcaacaaat ggcacaagca tcatcaaaac aaaaaaataa aagtatagca 720
ccagatcgtt taagtgcatc aaaagaaaaa ggcacggttg agaaggctgc tcaaaaacaa
780 gctaagcata ttgaaaaaag aatggaacat ttggaagaag ttgaaaaacc
acaaagttat 840 catgaattca attttccaca aaataaaatt tatgatatcc
ataataatta tccaatcatt 900 gcacaaaatc taacattggt taaaggaagt
caaaaactgc taacacaagt acgattccaa 960 ataccatatg gcaaaaatat
agcgctcgta ggtgcaaatg gtgtaggtaa gacaacttta 1020 cttgaagcta
tttaccacca aatagaggga attgattgtt ctcctaaagt gcaaatggca 1080
tactatcgtc aacttgctta tgaagacatg cgtgacgttt cattattgca atatttaatg
1140 gatgaaacgg attcatcaga atcattcagt agagctattt taaataactt
gggtttaaat 1200 gaagcacttg agcgttcttg taatgttttg agtggtgggg
aaagaacgaa attatcgtta 1260 gcagtattat tttcaacgaa agcgaatatg
ttaattttgg atgaaccaac taatttttta 1320 gatattaaaa cattagaagc
attagaaatg tttatgaata aatatcctgg aatcattttg 1380 tttacatcac
atgatacaag gtttgttaaa catgtatcag ataaaaaatg ggaattaaca 1440
ggacaatcta ttcatgatat aacttaa 1467
* * * * *