Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02324979 2000-10-03
WO 99/51??1 PCT/NL98/00186_
A method of interstrain differentiation of bacteria.
Summary of the invention
The subject invention lies in the field of interstrain
differentiation of bacteria. A general method has been developed with
which various types of bacteria can be differentiated into separate
individual strains. Thus in particular in the clinical setting this
method can suitably be used to determine what strain of bacterium is
present in a sample. This new method is applicable for discerning between
various strains of both Gram negative and Gram positive types of
bacteria.
Background of the invention
Previously we had disclosed a method called oligotyping for
interstrain differentiation of Mycobacterium tuberculosis strains in
W095/31569~ It was stated in this document that one of the key factors in
the control of tuberculosis is the rapid diagnosis of the disease and the
identification of the sources of infection. M. tuberculosis strain typing
has already proved to be extremely useful in outbreak investigations (6,
14, 31) and is being applied to a variety of epidemiologic questions in
numerous laboratories. Traditionally, laboratory diagnosis is done by
microscopy, culturing of the micro-organism, skin testing and X-ray
imaging. Unfortunately, these methods are often not sensitive, not
specific and are very time-consuming, due to the slow growth rate of M.
tuberculosis. Therefore, new techniques like in vitro amplification of M.
tuberculosis DNA have been developed to rapidly detect the micro-organism
in clinical specimens (14). The ability to differentiate isolates of M.
tubercutos~s by DNA techniques has revolutionarized the potential to
identify the sources of infection and to establish main routes of
transmission and risk factors for acquiring tuberculosis by infection
(1,3-10, 14, 16, 19-22, 25, 26, 2'J-33). The use of an effective universal
typing system will allow strains from different geographic areas to be
compared and the movement of individual strains to be tracked. Such data
may provide important insights and identify strains with particular
problems such as high infectivity, high virulence and/or multidrug
resistance. Analysis of large numbers of isolates may provide answers to
long-standing questions regarding the efficacy of BCG vaccination and the
CA 02324979 2000-10-03
WO 99151771 PCT/NL98/00186
2 _
frequency of reactivation versus reinfection.
The same problems identified for M. tuberculosis are inherent
in differentiation of numerous other bacteria. The problems specifically
arise for potentially epidemic pathogens and for bacteria that infect
hospitals. A more rapid and simple typing method is required. Preferably
the testing methods for various bacteria will occur in the same manner
ensuring routine use for all types of bacteria for which testing is
required. Preferably a test that can be carried out by non specialised
personnel using little laboratory space and time is sought after.
The method disclosed in W095/31569 is based on the DNA
polymorphism found at a unique chromosomal locus, the "Direct Repeat"
(DR) region, which is uniquely present in M. tuberculosis complex
bacteria. This locus was discovered by Hermans et al. {15) in M. bouts
BCG, the strain used worldwide to vaccinate against tuberculosis. The DR
region in M. bovis BCG -:onsists of Directly repeated sequences of 36 base
pairs, which are interspersed by non-repetitive DNA spacers, each 35 to
41 base pairs in length (15). The number of copies of the DR sequence in
M.bovis BCG was determined to be 49. In other strains of the M.
tuberculosis complex the number of DR elements was found to vary (15).
The vast majority of the M. tuberculosis strains contain one or more
IS6110 elements in the DR containing region of the genome.
It has been shown (12) that the genetic diversity in the DR
region is generated by differences in the DR copy number, suggesting that
homologeous recombination between DR sequences may be a major driving
force for the DR-associated DNA polymorphism (12). The high degree of DNA
polymorphism within a relatively small part of the chromosome makes this
region well-suited for a PCR-based fingerprinting technique.
Figure 1 depicts the structure of the DR region of M. bovis BCG
as determined previously by Hermans et al . and Groenen et al . ( 12 , 15 ) .
For the sake of convenience we will designate a DR plus its 3'adjacent
spacer sequence as a "Direct Variant Repeat" (DVR). Thus, the DR region
is composed of a discrete number of DVR's, each consisting of a constant
part (DR) and a variable part (the spacer).
The method disclosed in W095/31569 is based on a unique method
of fn vitro amplification of DNA sequences within the DR region and the
hybridisation of the amplified DNA with multiple, short synthetic
oligomeric DNA sequences based on the sequences of the unique spacer
DNA's within the DR region (figure 2). This differs from previous PCR
methods in the use of a set of primers with bath primers having multiple
CA 02324979 2000-10-03
WO 99/51771 PCT/NL98/00186
3 =
priming sites as opposed to having one of the primers bind to a fixed
priming site such as to a part of IS6110. Because M. tuberculosis complex
strains differ in the presence of these spacer sequences, strains can be
differentiated by the different hybridisation patterns with a set of
various spacer DNA sequences.
The method consists of in vitro amplification of nucleic acid
using amplification primers in a manner known per se in amplification
reactions such as PCR, LCR or NASBA, wherein a pair of primers is used
comprising oligonucleotide sequences sufficiently complementary to a part
of the Direct Repeat :sequence of a microorganism belonging to the M.
tubereutosts complex of microorganisms for hybridisation to a Direct
Repeat to occur and subsequently elongation of the hybridized primer to
take place, said primer being such that elongation in the amplification
reaction occurs for tine primer in the 5' Direction and for the other
primer in the 3' Direction. Due to the multiple presence of Direct
Repeats in the microorganisms to be detected the use of such primers
implies that all the spacer regions will be amplified in an efficient
manner. In particular it is not necessary for extremely long sequences to
be produced in order to obtain amplification of spacers located at a
distance from the primer. With the instant selection of the primer pairs
a heterogenous product is obtained comprising fragments all comprising
spacer region nucleic acid. Subsequently the detection of the amplified
product can occur simply by using an oligonucleotide probe directed at
one or more of the spacer regions one wishes to detect. In order to avoid
hindrance in the :amplification reactions the primers can have
oligonucleotide sequer_ces complementary to non-overlapping parts of the
Direct Repeat sequence so that when both primers hybridize to the same
Direct Repeat and undergo elongation they will not be hindered by each
other. In particular to avoid any hindrance during elongation reactions
when one primer DRa is capable of elongation in the 5' Direction and the
other primer DRb is capable of elongation in the 3' Direction the DRa is
selected such that it is complementary to a sequence of the Direct Repeat
located to the 5' side of the sequence of the Direct Repeat to which DRb
is complementary. The primer used must have an oligonucleotide sequence
capable of annealing tc~ the consensus sequence of the Direct Repeat in a
manner sufficient for amplification to occur under the circumstances of
the particular amplification reaction. A person skilled in the art of
amplification reactions will have no difficulty in determining which
length and which degree of homology is required for good amplification
CA 02324979 2000-10-03
WO 99!51771 PCT/NL98/00186
4 -
reactions to occur. The consensus sequence of the Direct Repeat of
microorganisms belonging to the M. tuberculosis complex is given in
sequence id. no. 2 and in figure 1.
In addition to what has already been disclosed in W095/31569 we
also determined the spoligotypes of M. tuberculosis strains which were
subcultured for many months both in the laboratory and in guinea pigs.
The strains selected for this purpose were those used in a previous study
on the stability of TS 6110 (2). All subcultured strains displayed the
identical spoligotype patterns compared with the primary cultures thus
indicating the pace of the molecular clock in this instance is slow
enough for use in epidemiology of the disease.
Because of the large success and simplicity of the method for
Mycobacterium tuberculosis strain differentiation and in view of problems
in strain differentiation with other microorganisms we used the Direct
Repeat consensus sequence to screen data bases with nucleic acid
sequences from other microorganisms. Unfortunately no further matches
were found. The Direct. Repeat sequence appeared to be unique for the
Mycobacterium tuberculosis as did their spacer sequences. As to date no
function had actually been attributed to the Direct Repeat sequence it
was unexpected that the sequence was universally distributed amoung other
types of microorganisms. Such would at best be expected if the sequence
had a function that was required also in other organisms.
Description of the invention
Notwithstanding the negative result after screening with the
Direct Repeat consensus sequence we considered further analysis of known
seguences by looking for a pattern in the nucleic acid sequences of other
microorganisms reminiscent of the Direct Repeat-spacer pattern in
Mycobacterium tuberculosis. Quite unexpectedly we found using a
specifically designed computer programme that such patterns existed in a
large number of other microorganisms with a broad range of genera. It
appears that the DR-like sequences are very common in prokaryotes. They
are however noticeably absent in eukaryotes. Chapter III of Bergeys
Determinative Manual of Bacteriology Ninth edition (11) provides a table
of characteristics for distinguishing prokaryotes from eukaryotes i.e.
distinguish bacterium from microoscopic eukaryotes in the shape of mold,
yeast, algae or protozoans.
All bacterial sequences analysed revealed the presence of such
CA 02324979 2000-10-03
WO 99/51771 PCT/NL98/00186
_
a sequence structure and thus the oligotyping method illustrated for
Mycobacterium tuberculosis can be applied for differentiating between all
strains of bacteria. It was totally unexpected that a consensus structure
of this type could be universally found. The Direct Repeat sequences
5 themselves are different between different genera but the general
framework of a cluster of Direct Repeat sequences, separated by a number
of non repetitive spacers is universally present in bacterial genomes.
Considering the fact that thusfar no function has been attributed to such
a region in Mycobacterium tuberculosis or in fact for any of the
sequences comprising Direct Repeat like regions in any other bacteria for
which such sequences hac- been described this is remarkable.
Bacteria can be divided into Archaebacteria and Eubacteria. The
eubacteria in turn can de distinguished into Gram-negative and Gram-
positive bacteria with cell walls and Eubacteria lacking cell walls.
Chapter IV of Bergeys determinative Manual of Bacteriology Ninth edition
(11) reveals the characteristics for each group. Over a wide range of the
subgroups in these 4 groups we have found the presence of the consensus
structure i.e. the presence of DR-like loci. The IV groups have been
subdivided by Bergey into more than 30 subgroups. We have examples in
Groups 3,4,5 and 6, Group 11, 17, 31, 32, 33~ The method according to
the invention is particularly of interest for the bacteria that are
pathogenic for humans. Group 4 comprises Gram negative bacteria. Genera
from Group 4 are Legionella (which can cause pneumonia) and Legionnaires
disease, the genus Neisseria (of which Neisseria meningitidis is well
known as causative agent of meningitis and of which Neisseria gonorrhoeae
is another example), the genus Pseudomonas (renown for hospital
infections) and the genus Bordetella (of whj.ch Bordetella pertussis is
well known as causative agent of whooping cough). In Group 5 bacteria as
defined in Bergeys Manual the Enterobacteriacae form a family of 30
genera. These bacteria form a particularly interesting group of Gram
negative bacteria that infect humans. Suitable examples of genera from
this family are Enterobacter, Escherichia, Shigella, Salmonella,
Serratia, Klebsiella and Yersinia. Other less well known pathogenic
Enterobacteriacae genera are Cedeca, Citrobacter, Kluyvera, Leclercia,
Pantoea, Proteus. Providencia and Hafnia. Other Group 5 families are
Pasteurellaceae with tha genus Haemophilus and the family Vibrionaceae
with the genus Vibrio. Haemophilus influenzae is a leading cause of
meningitis in children and also other septicemia conditions. Vibrio
cholerae is the cfiusat.ive agent of cholera, V. parahaemolyticus can cause
CA 02324979 2000-10-03
WO 99/51771 PCT/NL98/00186
6 -
food poisoning and 4. vulnificus causes highly fatal septicemia.
Of the Enterobacteriacae Shigella, Escherichia and Salmonella
are best known and difficult to differentiate. Shigella is an intestinal
pathogen of humans causing bacillary dysentery. Well known strains are S.
dysenteriae, S. flexneri, S. boydii, S. sonnei. The genus Salmonella is
well known for food poisoning. Well known Salmonella strains are S.
typhimurium, S. arizona, S. choleraesuis, S. bongori. Salmonella are also
causative agents of typhoid fever, enteric fevers, gastroenteritis and
septicemia. The genus Serratia bacteria are opportunistic pathogens for
hospitalized humans causing septicemia and urinary tract infections.
Examples are S. liquefaciens and S. marcescens. Of the Escherichia E.
coli is best known as major cause of urinary tract infections and
nosocomial infections including septicemia and meningitis. Other species
are usually associated with wound infections.
Enterobacter constitutes a problem genus of opportunistic
pathogens causing burn wound and urinary tract infections occasionally
also meningitis and septicemia. Well known species are E. cloacae, E.
sakazakii, E. aerogene~, E. agglomerans, E. gergoviae. Klebsiella are
also causative agents of bacteriemia, pneumonia, urinary tact and other
human infections in urological, neonatal, intensive care and geriatric
patients. Klebsiella pneumoniae and K, oxytoca are examples of species in
the genus.
Particularly interesting from a clinical point of view are also
the Gram positive pathogenic bacteria. The genera Streptococcus and
Staphylococcus form examples of such bacteria. Streptococcus pneumoniae,
Streptococcus pyogenas and Staphylococcus aureus are examples thereof. Of
the mentioned groups and genera the pathogenic bacteria are of interest.
These bacteria are dangerous when infecting hospitals in particular.
Due to the increasing incidence of infection differentiation of
potentially epidemiological organisms is also of interest. Such organisms
comprise Bordetella pertussis and Neisseria menigitidis the causative
organism of meningitis is of particular interest. Quite specifically
pathogenic bacteria infecting hospitals and bacteria capable of causing
epidemics are targets for the differentiation method according to the
invention.
The invention consists of a method of in vitro amplification of
nucleic acid using amplification primers in a manner known per se, in
amplification reactions such as PCR, LCR or NASBA, wherein a pair of
primers is used comprising oligonucleotide sequences sufficiently
CA 02324979 2000-10-03
WO 99/51771 PGT/NL98/00186
7 -
complementary to a part of the Direct Repeat sequence of a bacterium
other than a microorganism belonging to the M tuberculosis complex of
microorganisms for hybridisation to a Direct Repeat to occur and
subsequently elongation of the hybridised primer to take place, said
primers being such that elongation in the au:plification reaction occurs
for one primer in the 5' Direction and for the other primer in the 3'
Direction, wherein the Direct Repeat is a sequence with a length between
20-50 base pairs which occurs 5-60 times in a contiguous region of the
bacterial genome, whereby the Direct Repeat sequences are separated by
spacer sequences with a length of between 20-50 nucleotides, said spacer
sequences being non repetitive. By using the programme Patscan e.g. on
the nucleic acid data bases for microorganism genomic sequences such
motifs and thus also the identities of the various species specific
Direct Repeats and the corresponding spacer sequences can be obtained. In
the Patscan programme the Direct Repeat can be designated pi with a
length between 20-50 basepairs then search for pl 20-50 basepairs
downstream of pl. Thus this pattern in Patscan is described as
pl=(20..50)(20..50)pl{20..50)pl. The length of the sequences can be
varied as can the intermediate distance and the number of times the
Direct Repeat has to occur. A Direct Repeat can often have a length of
30-40 base pairs with a spacer length of 35-45 base pairs. Basically we
looked for a stretch of identical repeat sequences interspersed by spacer
sequences which do not necessarily share much of their sequence with the
Direct Repeat of M. tubercutosts. The patscan programme is freely
accessible at the Internet site:http://www-c.mcs.anl.gov/home/overbeek/-
PatScan/HTML/patscan.html. The programme was written by Ross Overbeek
Mathmatics and Computer Science Division Argonne National Laboratory
Building 221 Room D-236 900 S. Cass Avenue Arginne IL 60439 USA.
Most of the Repeats exhibit one or more of the following
characteristics, they end with s sequence si.railar to GAAAC i.e. exhibit
at least 3 of the nucleotides of this consensus sequence at the terminus,
preferably 4 or 5, start with CTTTG, have stretches of 3-4 identical
bases. The termini can for example be selected from GAAAC, GAAXXC GAACTC,
GXAAC, GCAAC, GAAA, GAAXC, GAAGC and AAAC. Suitable Termini are provided
in Table II.
Organisms as diverse as the Archaebacteria e.g. Methanococcus
~annasschi (Group 31), Haloferax mediterranei (Group 33). the
cyanobacteria Calotrix (Group 11), and Anabeana (Group 11), and purple
bacteria e.g. E.coli (Group 5), Mycobacterium tuberculosis {Group 21) and
CA 02324979 2000-10-03
WO 99/51771 PCTINL98/00186
8 _
Thermus thermophilus (Group 4), Archaeoglobus {Group 32) arid Thermotoga
(Group 6) were found to possess DR-like sequences upon analysis of their
genomes using the Patscan programme. In the subsequent study of
literature from which these data were derived it also became clear from
Southern blots that the Repeat sequences were also found in related
species.
With regard to the genetic organisation the structures of the
DR-like loci in the microorganisms is rather variable {figure 3)~ In M.
tuberculosis the DR locus is large and in most isolates it is disrupted
by an insertion element. This is also the case in T. thermophilus,
however here the number of DVR's is only 11 and the DR locus is disrupted
by two insertion elements. In E. coli K12 2 PR loci are present separated
by approximately 22kb; in Anabaena the locus is of intermediate size and
interrupted by a 130 by sequence of unknown function or origin. In H.
mediterranei the DR locus is of intermediate size and not disrupted,
however there is evidence for a second DR locus on one of the mega
plasmids found in this organism. In M. jannaschii there is one locus of
intermediate size but at several other positions in the genome one or a
few other DVR's are found. In most cases the DVR's are linked to a so-
called Long Repeat (LR) element of unknown function. Also in M.
jannaschii mega plasmids are found but in contrast to H. mediterranei
they do not contain DR sequences.
Accession numbers for the sequences of various organisms for
which the DR like loci have been found are provided here. For E. coli and
Shigella M27059, M27060, U29579, 029580 and M18270. The relevant portions
of the sequences are also disclosed by Blattner for E. coli. Nakata et al
reveal in the Journal of Bacteriology (13) that downstream of the iap
region a sequence of 29 bases appears 14 times 32 or 33 base pairs apart.
Nucleotide sequences hybridizing to the 29 base pair sequence were also
detected in Shigella dysenteriae and Salmonella typhimurium.
A DR-like sequence was found in the contig 214 of S. pyogenes
Ml(ATCC 700294) of the genome sequencing project of the University of
Oklahoma. Further research into this DR-like sequence in other S.
pyogenes revealed spacer polymorphism. The DR regions of eight S.
pyogenes isolates were studied. The DR regions were isolated by PCR using
primers that were derived from the database {University of Oklahoma,
serotype M1 ATCC )00294. The sequence data is available under
http://www.genome.ou.e~du. This strain contains seven repeats and six
spacers.
CA 02324979 2000-10-03
WO 99/51771 PCT/NL98100186
9 -
Five of the isolates gave a PCR product, these were a M2
strain, a M4 strain and three M1 strains. The M4 strain contained only a
single repeat sequence that was flanked by the same sequences as the ATCC
700294. The M2 strain sequencing did not work, but the size of the PCR
fragment indicated that two repeats are present. The three M1 strains
were all the same, they contained four repeats and three spacers. The
repeats were identical to ATCC X00294, while one of the spacers was
identical to ATCC 700294 and two were different.
These studies on S. pyogenes show that the DR regions have
conserved spacers and repeat sequences.
The Salmonella genomic sequence as sequenced by the University
of Washington St Louis has also revealed the presence of DR-like
sequences. The DR exhibits high homology with the Direct Repeat of E.
coli. One of the contigs revealed 7 Repeats and 6 spacers.
A panel of rive E. coZi isolates and three ShigeZZa strains
were studied. The five E. coti isolates were selected to have an optimal
diversity, they were isolated from different species or geographic
regions. The ShigeZZa strains are considered separate (sub)species. See
Table 1. The isolates were obtained from the collection of Dr. Wim
Gaastra.
Table 1
~.
species description DRI" DRII"
E. coti 184 American isolate Southern PCR
358 human urinary tractSouthern Southern
968 , mastitis Southern PCR
1008 chicken PCR PCR
1732 human intestine Southern PCR
ShigeZta disenteriae593 Southern PCR
sonnei 595 Southern PCr
boydil 603 Southern PCR
" The DR regions were identified by Southern blot of genomic DNA and DRI
and DRII regions of E. coti K12. When PCR is indicated the DR regions
were identified by the Southern and the PCR. This PCR was done with
primers derived from the K12 sequence.
The DRI and DRII sequences that could be amplified by PCR were
CA 02324979 2000-10-03
WO 99/51771 PCT/NL98/00186
IO
cloned and sequenced. Somehow the DRI regions could not be amplified by
PCR using the primers designed on the K12 sequence, while the Southern
data demonstrate that DRI is present. Apparently, the recognitions sites
for the primers are polymorphic. The sizes of the DRII regions were found
to vary greatly between these isolates. The smallest was a single repeat
in the S. sonnet strain and the largest was a repeat cluster of at least
repeats in E. coti isolate 1008. The sequences of the repeats were
highly conserved between these isolates. The S. t~phimurium data is
obtainable from the Internet http://genome.wustl.edu/gsc/
10 bacterial/salmonella.html.
The space~ sequences almost all were unique. Approximately 40
spacers have been sequenced and only three of them were already known
from a previously sequenced DR region. This indicates a high number of
different spacer sequences in E. coZi.
15 Accession number X73453 provides the Halerofax mediterranei
sequence. The sequence can also be found in Molecular Microbiology 17 of
1995 in an article by Mojica et al. (17). The Repeat sequence has also
been found in related species.
The genomic project of the Methanococcus jannaschii reveals a
DR-like sequence as is apparent from the Bult; et al article in Science
273 of 1996 (18). The Accession number is U67459 i.a.
Accession number X87270 for Anabeana sp reveals 17 spacers and
a LTRR element. These elements also occur. in related species of
cyanobacteria such as Calotrix. The sequence data are provided by
- 25 Masepohl et al in BBA 1307 1996 (23).
Accession number AE000782 for Archaeoglobus fulgidus reveals
three DR-like Repeats with the same Repeat sequence and the this has a
slightly larger but closely related Repeat. The Repeats are present 20-30
times. The spacers are unique sequences. H.P. Klenk discloses sequence
data in Nature 390 1997 (24).
The invention also covers a method of detection of a bacterium,
said bacterium not belonging to the M. tuberculosis complex of
microorganisms said method comprising
1) amplifying nucleic acid from a sample with the amplification method
according to any of the preceding described embodiments of the
amplification method according to the invention, followed by
2) carrying out a hyuridisation test in a manner known per se, wherein
the amplification product is hybridised to an oligonucleotide probe
or a plurality of different oligonucleotide probes, each
CA 02324979 2000-10-03
WO 99/51771 PCT/NL98/00186
11
oligonucleotide being sufficiently homologous to a part of a spacer
of the Direc t Region of the bacterium to be determined for
hybridisation to occur to amplified product if such spacer nucleic
acid was present in the sample prior to amplification, said
hybridisation step optionally being carried out without prior
electrophoresis or separation of the amplified product.
3) detecting any hybridised products in a manner known per se.
The method can be carried out in a manner such that the
hybridisation test is carried out using a number of oligonucleotide
probes, said number comprising at least a number of oligonucleotides
probes specific for the total spectrum of bacteria it is desired to
detect. In a suitable embodiment of a method according to the invention
the oligonucleotide probe is at least seven oligonucleotides long and is
a sequence complementary to a sequence selected from any of the spacer
sequences of the Direct Repeat region of the bacterium to be determined
or is a sequence comples~entary to fragments or derivatives of said spacer
sequences, said oligonucleotide probe being capable of hybridising to
such a spacer sequence and comprising at least seven consecutive
nucleotides homologous: to such a spacer sequence and/or exhibiting at
least 60x homology, preferably exhibiting at least 80x homology with such
a spacer sequence.
Preferably the method according to the invention is carried out
to determine the presence and nature of a pathogenic bacterium selected
from the group of Gram negative bacteria of Groups 4 and 5 of Bergeys
Determinative Manual of Bacteriology ninth edition. Of particular
interest due to damage caused by such pathogens are bacteria belonging to
the families Enterobacteriaceae, Pasteurellaceae and Vibrionaceae of
Group 5, most specifically the Enterobacteriaceae. Also of interest are
the Gram positive bactaria of Group 1'j. Suitable examples of genera of
the pathogenic bacterium to be detected from the group of Gram negative
bacteria of Bergeys Determinative Manual of Bacteriology ninth edition
are Eschericchia, Shigella, Salmonella, Klebsiella, Enterobacter,
Yersinia, Serratia, Haemophilus, Vibrio, Legionella, Neisseria,
Pseudomonas and Bordetella. For the group of Gram positive bacterial
genera Staphylococcus and Streptococcus are targets for the
differentiation method.
Suitably in a method according to the invention for
differentiating the type of bacterium in a sample, said bacterium not
belonging to the M. tuberculosis complex the hybridisation pattern is
CA 02324979 2000-10-03
WO 99/51771 PCT/NL98/00186
12
compared with that obtained with a reference. Such a reference can be the
hybridisation pattern obtained with one or more known strains of the
bacterium to be determined in analogous m~inner as the strain to be
determined. Alternatively the reference is a source providing a list of
spacer sequences and sources thereof, such as a data bank. Table II
exhibits some suitable examples of sequences that occur as Direct Repeat
sequences according to the invention for the genera illustrated.
CA 02324979 2000-10-03
wo mstun ~ 3 PCT/NL98/00186
a
~
ae !
a
b
0
o~o
0 0
s
-- m '
l~ U n1
U B i
~
V .L N ~ tr
+.~a a a~ a
010 0 ~ a ~''~ g
'
1
r-i, cac~ asas c~ ~ ~ c o ~ o
a~ u~ ~ ~ i ~ a'o~
+~~ b 'o zsw ~u o ~ o a .-~
a
C: O tI1.r., 40 A V O 00 C,"it
f.~ 8 +~11 O O O O O rl rl O rl rl ClO
a!cr1 +~ ~ w .~ ~ 40
c,.. s~ .~~ s.s~ c~c~ a a a o v~ a o ra
xa x z c'~no 0 0 0 o c ~,~ c a E
t
U
U U
U U U U i
H ~ i- H H E U
i
U U U U U
d ~ d ~ '
U U U U U
U CU7 U CU'S~ CU7
C-U~ C-~~U H H H U '
N ,
U U r U ~
C' U U U U U j
U U U U
a a
A
,
a~ cz o cz
fx ~ U U U U U U U
O
O
O
w O
01
ro ~
tr d ~, p
7
F/ N
O
'N
N i3w w~01 'V .C S1
1
V ~
~1 .'3~N V t
01 ,~ ~ W Q N ~! N V
H ~ 1.~G O O ~!N ~ N O
C7 'N N <J N ~ w ,~1~" ~1.1
U fy N SJ N
r~ O ,.C 00O~ 01~ Ct ~ N
~1
W ~ ~ v1v7 ~ ~ v7
E~
O tf1
CA 02324979 2000-10-03
WO 99/51771 ~ 4 PCT/NL98/00186
.'a .~ x x
a c~ o
G.Or 4.Or3
~ v ~ ~ O 00 N _
tn O~ .-a O .-rM Ov O ~ a~0~ N
,.aU1 Lf~ ~' l~- ~O lit~ O O B
O e-I M N .-1lfl W M o g O
U M O~ L'- l~ ~ t~ Gr 40d O C',
U M v0 CO ~' ~O cd O ~ W N
GS ~ N 7G N O r1 t~ d d HO
'd vD .-~t ~-. .~i.-.
o . ~ ~ o'~no~~. a
!1 N O ~ ~7 x
.-.rl (xl L~1 O .-~-I O
U rl C1 r~-I~O O ~ ~ rl r-i
c0 v cd W LD O m-ir-1 ~ I~ cb
C. 1~ J~O O1rl B U ~ ~ 00 i~ LC1 l0
N e-1O rl ri.C 01 cd O ~ i~ to O M O
O -~ ~ ~ O rl O O .-~ M
f.~ ~ i rlO p, 01 U O M .-i ~, [~ ~
N ~ -~ .Git N O .-i r~ 'F.,'Q1
4 ,T' N U -~'fn O r7 1 rl M rl O> G)O
N tl3~' rlrl l!1cd O 'L1O r-Itf1O Ice- f~ l~ rlC1
CG d N a ~ N ~ M rl ~ cdo0fx1N fI).-1 aCM
U U
O C~7 C~7 d ~ d
d
d H ~ ~ Cd5 Cdr~
O U U ~ U
O ~ H f-U~~ C5
U d U d C5 U
C ~ ~ U U
U ~ ~ d d U Cd.7
iJ' d U d C7 U U d U
d
N U U d d U ~ U
COG d
d ~ U
w
N
N V
vi
~1 C~ N w~
V '~1 .
~1 N ~ ~ ~f'1
B F U
d V V O N
M O .r ti t3 fi O tf 'I~
O O C V .4 O~
N C1 E B t1 i~.~0.0, Cl d B CS
N p1 Cl N N ,~ .x ~ rte'
E.,c. .~ .~ x ~ b cs o, d x ~
u~ E. ~ N ~ U x ~C ~ +~ ~c
O ~ O
.-r .-i N
CA 02324979 2000-10-03
WO 99/51771 PCTINL98/00186
Not only the above methods fall within the scope of the
invention but also specifically selected primer pairs for carrying out
such a method. A pair of primers according to the invention is a pair
wherein both primers comprise oligonucleotide sequences of at least '7
5 oligonucleotides and are sufficiently complementary to a part of the
Direct Repeat sequence of the microorganism E. cola for hybridisation to
occur and subsequently ~alongation of the hybridised primer to take place,
said primers being such that elongation in the amplification reaction
occurs for one primer in the 5' Direction and for the other primer in the
10 3' Direction and wherein sufficiently complementary means said
oligonucleotide sequence comprises at least seven consecutive nucleotides
homologous to such a Direct Repeat sequence and/or exhibits at least 60x
homology, preferably at least 80x homology, most preferably more than 90x
homology with the corresponding part of the Direct Repeat sequence.
15 Suitable Direct Repeat sequences are provided in Table II. In particular
such a primer pair can comprise one primer DRa capable of elongation in
the 5' Direction and the other primer DRb capable of elongation in the 3'
Direction with DRa being complementary to a sequence of the Direct Repeat
located to the 5' side of the sequence of the Direct Repeat to which DRb
is complementary, the Direct Repeat being present in the Direct Region of
E. cola. Another suitable pair comprises primers with oligonucleotide
sequences of at least ~ oligonucleotides and are sufficiently
complementary to a part of the Direct Repeat sequence of the
microorganism S. typhimurium for hybridisation to occur and subsequently
elongation of the hybridised primer to take place, said primers being
such that elongation in the amplification reaction occurs for one primer
in the 5' Direction. and for the other primer in the 3' Direction and
wherein sufficiently complementary means said oligonucleotide sequence
comprises at least seven consecutive nucleotides homologous to such a
Direct Repeat sequence in particular the Sequence provided in Table II
and/or exhibits at least 60x homology, preferably at least 80x homology,
most preferably more th:~n 90x homology with the corresponding part of the
Direct Repeat sequence. In particular such a pair comprises one primer
DRa capable of elongation in the 5' Direction and the other primer DRb
capable of elongation in the 3' Direction with DRa being complementary to
a sequence of the DirFct Repeat located to the 5' side of the sequence of
the Direct Repeat to which DRb is complementary, the Direct Repeat being
present in the Direct Region of S. typhimurium.
Kits for carrying out a differentiation method according to any
CA 02324979 2000-10-03
WO 99/51771 PCT/NL98/00186
i6
of the described embodiments also fall within the scope of the invention.
Such kits comprise a primer pair according to any of the described
embodiments and optionally an oligonucleotide probe or a carrier, said
carrier comprising at least 1 oligonucleotide probe specific for a spacer
region of a bacterium to be determined said bacterium not belonging to M
tuberculosis complex, preferably the oligonucleotide probe as defined,
being an oligonucleotide probe of at least 10 nucleotides, preferably
more than 12 nucleotides, in particular comprising between 12 to 40
nucleotides, said probe being sufficiently homologous to any of the
spacer sequences or to fragments or derivatives of such spacer sequences
to hybridise to such a spacer sequence, said oligonucleotide probe
comprising at least 10 consecutive nucleotides homologous to such a
spacer sequence and/or exhibiting at least 60x homology, preferably
exhibiting at least 80': homology, most preferably exhibiting more than
90x homology with the corresponding part of the spacer sequence. Suitably
a kit according to the invention comprises a data carrier with required
reference patterns of the bacterial strain to be determined.
REFERENCES
1. Beck-Sague, C., S.W. Dooley, M. D. Hutton, J. Otten, A.
Breeden, J.T. Crawford, A.E. Pitchenik, C. Woodley, C.
Cauthen,
W.R. Jarvis. 1992 Hospital outbreak of multidrug-resistant
Mycobacterium tubercutosts infections. JAMA. 268: 1280-1286.
2. Van Soolingen, D., P.W.M. Hermans, P.E.W. de Haas, D.
R. Soll,
and J.D.A. van Embden. 1991. The occurrence and stability
of
insertion sequences in lKycobactertum tubercutosts complex
strains; evaluation of IS-dependent DNA polymorphism as
a tool
in the epidemiology of tuberculosis. J. Clin. Microbiol.
29:2578-2586.
3. CDC. 1992. Transmission of multidrug-resistant tuberculosis
among immunocompromised persons in a correctional system.
New
York 1991. MMWR 41: 507-509.
4. CDC. 1991. Nosoconial transmission of multidrug resistant
tuberculosis among HIV-infected persons, Florida and New
York,
1988-1991. MMwR 40: 585-591.
5. Cave, M.D., K,D. Eisenach, P.F. McDermott, J.H. Bates,
and J.T.
CA 02324979 2000-10-03
WO 99/51771 PGT/NL98/00186
17
Crawford. 1991. IS61I0: Conservation of sequence in the
Mycobacterium tuberculosis complex and its utilization in DNA
fingerprinting. Mol. Cell. Probes 5:73-80
6. Daley, C.L. P.M. Small, G.F. Schecter, G.K. Schoolnik,
R.A.
McAdam, W.R. Jacobs, Jr., and P.C. Hopewell. 1992. An
outbreak
of tuberculosis with accelerated progression among persons
infected with the human immunodeficiency virus: an analysis
using restriction fragment length polymorphisms. N. Engl.
J.
Med. 326:231-235.
7. Dooley, S.W., M.E. Villarino, M. Lawrence, et al. 1992.
Nosocomial transmission of tuberculosis in a hospital
unit for
HIV-infected patients. JAMA. 267:2632-2634.
8. Dwyer, B., K. Jackson, K. Raios, A. Sievers, E. Wilshire
and B.
Ross. 1993. DNA Restriction fragment analysis to define
an
extended cluster of tuberculosis in homeless men and their
associates. J. Inf. Diseases, L67: 490-494.
9. Edlin, B.R., J.I. Tokars, M.H. Grieco, J.T. Crawford,
J.
Williams, E.M. Sordillo, K.R. Ong, J.O. Kilburn, S.W..
iMoley,
K.G. Castro, W.R. Jarvis and S.D, Holmberg. 1992. An outbreak
of multidrug-resistent tuberculosis among hospitalized
patients
with the acquired immunodeficiency syndrome. N. Eng. J.
Med.
326:1514-1521.
10. Fischl, M.A, R.B. Uttamchandani, G.L. Daikoe, et al. 1992.
An
outbreak of. tuberculosis caused by multiple-drug resistant
tubercle bacilli among patients with HIV infection. Ann.
Intern. Med. 117:177-183.
il. Bergeys Determinative Manual of Bacteriology Ninth Edition
12. Groenen, P.M.A., A.E. van Bunschoten, D. van Soolingen,
and
J.D.A, van Embden. 1993 Nature of DNA polymorphism in
the
direct repeat cluster of Mycobacterium tuberculosis;
Application for strain differentiation by a novel method.
Mol.
Microbiol. (1993) 10 (5) 1057-1065.
13. Nakata et al Journal of Bacteriology June 1989 p3553-3556
14. Hermans, P.W.M., D. van Soolingen, J.W. Dale, A.R. Schuitema,
R.A. McAdam, D. Catty, and J.D.A. van Embden. l9go. Insertion
element IS986 from Mycobacterium tubercuZosts: a useful
tool
for diagnosis and epidemiology of tuberculosis. J. Clin.
Microbiol. 28:2051-205$.
15. Hermans, P.IJ.M., D. van Soolingen, E.M. Bik, P.E.W. de
Haas,
CA 02324979 2000-10-03
WO 99/51771 ' PCTINL98/00186
18
J.W. Dale, and J.D.A. van Embden. 1991. The insertion element
IS987 from M. bovis BCG is located in a hot spot integration
region for insertion elements in M. tubercutosis complex
strains. Infect. Immun. 59:2695-2'705.
16. Hermans, P.W.M., D. van Soolingen, and J.D.A. van Embden.
1992.
Characterization of a major polymorphic tandem repeat
in
Mycobacterium tubercutosis and its potential use in the
epidemiology of Mycobacterium kansasii and Micobacterium
gordonae. J. Bacteriol. 1'74:4157-4165.
17. Mo~ica et al., Molecular Microbiology 17, 1995
18. 8ult et al., Science 273, 1996
19. Mazurek, G.H., M.D. Cave, K.D. Eisenach, R.J. Wallace
JR, J.H.
Bates, and J.T. Crawford. 1991. Chromosomal DNA fingerprint
patterns produced with IS6110 as strain specific markers
for
' epidemiologic study of tuberculosis. J. Clin. Microbiol.
29:2030-2033.
20. McAdam, R.A., P.W.M. Hermans, D. van Soolingen, Z.F. Zafnuddin,
D. Catty, J.D.A. van Embden, and J.W. Dale. 1990.
Characterization of a Mycobacterium tuberculosis insertion
sequence belonging to the IS3 family. Mol. Microbiol.
4:1607-
1613.
21. Mendiola, M.V., C,. Martin, I. Otal, and B. Gicquel. 1992.
Analysis of regions responsible fur IS6110 RFLP in a single
Mycobacterium tuberculosis strain. Res. Microbiol. 143:767-772.
22. Otal, I., C. Martin, V. Vincent-Levy-Frebault, D. Thierry,
and
B. Gicquel. 1991. Restriction fragment length polymorphism
analysis using IS6110 as an epidemiological marker in
tuberculosis. J. Clin. Microbiol. 29:1252-1254.
23. Masepohl et al., kBBA 1307 1996
24. Klenk, H.P.,Nature 390, 1997
25. Palittapongarnpim, P.S., S. Rienthong, and W. Panbangred:
1993.
Comparison of restriction fragment length polymorphism
of M.
tuberculosis isolated from cerebrospinal fluid and sputum:
a
preliminary report. Tubercle and lung disease 1993 74.
204-
.207.
26. Ross, C., K. Raios, K. Jackson, and B. Dwyer. 1992. Molecular
cloning of a hightly repeated element from Mycobacterium
tuberculosis and its use as an epidemiological tool. J. Clin.
Microbiol. 30:942-946.
CA 02324979 2000-10-03
WO 99/51771 PCT/NL98/00186
19
27. Small, P.M., R.W. Schafer, P.C. Hapewell, S.P. Singh,
M.J.
Murphy, E. Desmond, M.F. Sierra, and G.K. Schoolnik. 1993
Exogenous reinfection with multidrug-resistant M. tubercutosfs
in patients with advanced HIV infection. N. Eng. J. Med.
328:1137-1144.
28. Thierry, D., M.D. Cave, K.D. Eisenach, J.T. Crawford,
J.H.
Bates, B. Gecquel, and J.L. Guesdon. 1990. IS610, an IS-like
element of M. tuberculosis complex. Nucleic Acids Res.
18:188.
29. Van Embden, J.D.A., D. van Soolingen, P.M. Small, and
P.W.M.
Hermans. 1992. Genetic markers for the epidemiology of
tuberculosis. Res. Microbiol. 143: 385-391
30. Van Embden, J.D.A., M.D. Cave, J.T. Crawford, J.W. Dale,
K.D.
Eisenach, B. Gicquel, P.W.M. Hermans, C. Martin, R. McAdam,
T.M. Shinnick, and P.M. Small. 1993 Strain identification
of
Mycobacterium tubercutosis by DNA fingerprinting;
Recommendations for a standardized Methodology J. Clin.
Microbiol. 31:406-409.
31. Van Soolingen, D., P.W.M. Hermans, P.E.W, de Haas, and
J.D.A.
van Embd~en. 1992. Insertion element IS1081-associated
Restriction Fragment Length Polymarphism in Mycobacterium
tuberculosis Complex species: a reliable tool for recognizing
tlycobactertum bouts BCG. J. Clin. Microbiol. 30:1772-1777.
32. Van Soolingen, D., P.E.W. de Haas, P.W.M. Hermans, P.M.A.
Groenen and J.D.A. van Fmbden. 1993 Comparison of various
repetitive DNA elements as genetic markers for strain
differentiation and epidemiology of Mycobacterium tuberculosis.
J. Clin. Microbiol. 31:1987-1995.
33 Yuen L.K., B.C. Ross, K.M. Jackson, and B. Dwyer. 1992.
Characterization of Mycobacterium tuberculosis strains
from
Vietnamese patients by southern blot hybridization. J.
Clin.
Micro. 31: 1615-1618.
34. Kamerbeek J., L.M. Schouls, A. Kolk, M. van Agterveld,
D. van
Soolingen, S. Kui~per, J.E. Bunschoten, H. Molhuizen,
R. Shaw,
M. Goyal and J.D.A. van Embden 1997. Simultaneous detection
and strain differentiation of Mycobacterium tubercutosts
for
diagnosis and epidemiology. J. Clin. Microbiol. 35: 907-gi4.
CA 02324979 2000-10-03
WO 99/51771 PG"T/NL98/00186
DESCRIPTION OF THE FIG;3RES
Figure 1 depicts the structure of the DR region of M. bovis BCG as
determined previously by Hermans et al. and Groenen et al. (12, 15). For
5 the sake of convenience we will designate a DR plus its 3'adjacent spacer
sequence as a "Direct Variant Repeat" (DVR). Thus, the DR region is
composed of a discrete number of DVR's, each consisting of a constant
part (DR) and a variable part (the spacer).
10 Figure 2 depicts multiple, short synthetic oligomeric DNA sequences based
on the sequences of the unique spacer DNA's within the DR region.
Figure 3 shows the genetic organisation of the structures of the DR-like
loci in various bacterial species.
15 ~ depicts the transcription direction of open reading frame (ORF)
For M. tuberculosis: MTCY 168'7.26, 2~ and 30C are unknown genes/proteins.
For E. coli: iap gene function is alkaline phosphatase isozyme
20 conversion. ORF f94, f305, YGCE and f223 are unknown genes/proteins.
For S. pyogenes: ORF1 and 2 are unknown genes/proteins.
For T, thermophilus: ORFC and D are unknown genes/proteins and ORF lA and
1B are possibly transposases of IS elements 1000 and 1000A.
For Anabaena: No ORFs were annotated in the flanking sequences. The 130
by insert is of unknown origin.
For Haloferax mediterranei: ORF21 is an unknown gene/protein. Probably
another repeat cluster a_s also present on the megaplasmid pHM500.
For Methanococcus jannaschii: Comprises about 10 repeat clusters, the
largest one of which comprises 25 repeats. All repeat clusters are
coupled to a Long Repeat ( LR ) segment of 425bp . There are 18 LR' s , some
of which contain only one repeat. Smaller LR segments are also present,
SLR. In one case, a cluster contains 5 repeats without LR (see ref. 18)
For M. thermoautrophicum: Two repeat clusters SRI and SRII flanked by
CA 02324979 2000-10-03
WO 99151771 PG"f/NL98/00186
21
LRI, LRII. LRI and LRII are almost identical and are homologues of the LR
segment of M. jannaschii. SRI and SRII are separated by 500 kb in the
genome.
For Thermatoga maritima:CelA gene encodes cellulase: endo-1,4-beta-
glucanase (EC 3.2.1.4) and CelB is also a cellulase exhibiting 58x
identity with celA.
Far Archaeoglobus fulgi.dus: The SRIA and SRIB repeat clusters have the
same Repeat Sequence and the SRII Repeat Sequence is also clearly
homologous. The SR clusters are separated by about 400bp. SRIB and SRII
are located near tRNA genes. SRIA lies adjacent to an unknown ORF3.
Figure 4
Hybridization Patterns of 17 E. coli isolates. Thirty four different
spacer oligonucleotides were covalently linked to a membrane and PCR
amplified DNA of E. coli was hybridized as described (Kamerbeek et al.
1997). except that the primers used to amplify the DR locus were specific
for the DR sequence from E. coli. Note the polymorphism observed in E.
coli due to the strain-dependent presence or absence of spacer DNA.
Figure 5
Hybridization Patterns of 4 Salmonella typhimurium isolates. Six
different spacer oligonucleotides were covalently linked to a membrane
and PCR amplified Salmonella DNA was hybridized as described (Kamerbeek
et al 1997 ) , except that the primers used to amplify the DR locus were
specific for the DR 7.ocus of E. coli. Note the polymorphism observed in
Salmonella due to the strain-dependent presence or absence of spacer DNA.
