Note: Descriptions are shown in the official language in which they were submitted.
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PATENT
Docket No. 180.0010 0201
PRIMERS FOR USE IN DETECTING BETA-LACTAMASES
RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application
Serial No. 601502,091, filed 10 September 2003, and U.S. Provisional
Application Serial No. 60/502,885, filed 12 September, 2003, each of which is
incorporated herein by reference in its entirety.
BACKGROUND
A disturbing consequence of the use, and over-use, of ~3-lactam
antibiotics (e.g., penicillins and cephalosporins) has been the development
and
spread of ~3-lactamases. ~-lactamases are enzymes that open the ~3-lactam ring
of
penicillins, cephalosporins, and related compounds, to inactivate the
antibiotic.
The production of ~i-lactamases is an important mechanism of resistance to
~3-lactam antibiotics among Gram-negative bacteria.
Expanded-spectrum cephalosporins have been specifically designed to
resist degradation by the older broad-spectrum ~i-lactamases such as TEM-l, 2,
and SHV-1. Microbial response to the expanded-spectrum cephalosporins has
been the production of mutant forms of the older ~i-lactamases called
extended-spectrum ~3-lactamases (ESBLs). Although ESBL-producing
Enterobacteriaceae were first reported in Europe in 1983 and 1984, ESBLs have
now been found in organisms of diverse genera recovered from patients in all
continents except Antarctica. The occurrence of ESBL-producing organisms
varies widely with some types more prevalent in Europe (TEM-3), others more
prevalent in the United States (TEM-10, TEM-12 and TEM-26), while others
appear worldwide (SHV-2 and SHV-5).
Additionally, CTX-M [3-lactamases are spreading throughout North
America and have been found in a wide variety of isolates within the family
Eiaterobacteriaceae. Organisms producing CTX-M ~i-lactamases are typically
resistant to cefotaxime, have lower minimum inhibitory concentration (MIC)
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values to ceftazidime and elevated MIC values to cefepime. However, these
enzymes are capable of hydrolyzing the newer cephalosporins and aztreonam.
Further, it is of concern that the National Committee for clinical Laboratory
Standards (NCCLS) guidelines for ESBL detection in E. coli and Klebsiella spp.
include an initial screening with either cefpodoxime, cefotaxime, ceftazidime,
ceftriaxone, or aztreonam followed by a confirmation test using both
cefotaxime
and ceftazidime in combination with clavulanate (NCCLS performance
standards for antimicrobial and susceptibility testing; 12''' informational
supplement, M100-S12 (2002)), and that a practice among some clinical
laboratories is to use ceftazidime as the initial screening drug and
ceftazidime
with clavulanate as the confirmation test (Brenwald et al., J. Anti.tzzicz-ob.
C7zezzzotlzef-., 51:195-196 (2003); Dandekar et al., DiagiZ. Microbiol,
hZfect. Dis.,
49:37-39 (2004)). One study has shown, however, that about 14% of ESBL-
producing strains will not be detected if ceftazidime is used as an initial
screen,
and only about 35% _of ESBL-producing strains were reported as ESBL positive
when ceftazidime with clavulanate was the only confirmation test.
It is of further concern that genes encoding ~-lactamases are often
located on large plasmids that also contain genes for resistance to other
antibiotic classes including aminoglycosides, tetracycline, sulfonamides,
trimethoprim, and chloramphenicol. Furthermore, there is an increasing
tendency for pathogens to produce multiple ~i-lactamases. These developments,
which occur over a wide range of Gram-negative genera, represent a recent
evolutionary development in which common Gram-negative pathogens are
availing themselves of increasingly complex repertoires of antibiotic
resistance
mechanisms. Clinically, this increases the difficulty of identifying effective
therapies for infected patients.
Thus, there is a need for techniques that can quickly and accurately
identify the types of ~i-lactamases that may be present in a clinical isolate
or
sample, for example. Surveillance studies of this nature could have
significant
implications in the choice of antibiotic used in hospital settings and could
impact
the treatment of a bacterial infection.
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SUMMARY OF THE INVENTION
The present invention is directed to the use of oligonucleotide primers
specific to nucleic acids characteristic of (typically, genes encoding)
certain ~i-
lactamases. More specifically, the present invention uses primers of the
invention to identify family-specific ~i-lactamase nucleic acids (typically,
genes)
in samples, particularly, in clinical isolates of Gram-negative bacteria. Even
more specifically, the present invention provides primers to specifically
identify
groups within the CTX-M ~3-lactamase family.
In one aspect, the present invention is directed to a primer selected from
the group of: 5' - GAC GAT GTC ACT GGC TGA GC - 3' (SEQ ID NO:1);
5' - AGC CGC CGA CGC TAA TAC A - 3' (SEQ ID N0:2); 5' - GCG ACC
TGG TTA ACT ACA ATC C - 3' (SEQ ID N0:3); 5' - CGG TAG TAT TGC
CCT TAA GCC - 3' (SEQ ID N0:4); 5' - GCT GGA GAA AAG CAG CGG AG
- 3' (SEQ ID N0:5); 5' - GTA AGC TGA CGC AAC GTC TG - 3' (SEQ ID
NO:6); 5' - CGC TTT GCC ATG TGC AGC ACC - 3' (SEQ ~ NO:7); and
5' - GCT CAG TAC GAT CGA GCC - 3' (SEQ ID N0:8); and full-length
complements thereof.
In a further aspect, the present invention is directed to a primer pair
including one primer selected from the group of: 5' - GAC GAT GTC ACT
GGC TGA GC - 3' (SEQ ID NO:1); 5' - AGC CGC CGA CGC TAA TAC A - 3'
(SEQ ~ N0:2); 5' - GCG ACC TGG TTA ACT ACA ATC C - 3' (SEQ ID
N0:3); 5' - CGG TAG TAT TGC CCT TAA GCC - 3' (SEQ ID N0:4); 5' -
GCT GGA GAA AAG CAG CGG AG - 3' (SEQ ID N0:5); 5' - GTA AGC TGA
CGC AAC GTC TG - 3' (SEQ ID N0:6); 5' - CGC TTT GCC ATG TGC AGC
ACC - 3' (SEQ ID N0:7); and 5' - GCT CAG TAC GAT CGA GCC - 3' (SEQ
ID N0:8); and full-length complements thereof.
In yet further aspects, the present invention is directed to a primer
selected from the group of 5' - GAC GAT GTC ACT GGC TGA GC - 3' (SEQ
m N0:1); 5' - AGC CGC CGA CGC TAA TAC A - 3' (SEQ ID N0:2); and
full-length complements thereof; a primer selected from the group of 5' - GCG
ACC TGG TTA ACT ACA ATC C - 3' (SEQ ID N0:3); 5' - CGG TAG TAT
TGC CCT TAA GCC - 3' (SEQ ID N0:4); and full-length complements thereof;
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a primer selected from the group of 5' - GCT GGA GAA AAG CAG CGG AG -
3' (SEQ ID NO:S); 5' - GTA AGC TGA CGC AAC GTC TG - 3' (SEQ ID
N0:6); and full-length complements thereof; and to a primer selected from the
group of 5' - CGC TTT GCC ATG TGC AGC ACC - 3' (SEQ >D NO:7); and 5'
- GCT CAG TAC GAT CGA GCC - 3' (SEQ )I~ N0:8); and full-length
complements thereof.
As used herein, a nucleic acid characteristic of a ~-Iactamase enzyme
includes a gene or a portion thereof. A "gene" as used herein, is a segment or
fragment of nucleic acid (e.g., a DNA molecule) involved in producing a
peptide
(e.g., a polypeptide and/or protein). A gene can include regions preceding
(upstream) and following (downstream) a coding region (i.e., regulatory
elements) as well as intervening sequences (introns, e.g., non-coding regions)
between individual coding segments (exons). The term "coding region" is used
broadly herein to mean a region capable of being transcribed to form an RNA,
the transcribed RNA can be, but need not necessarily be, further processed to
yield an mRNA.
Additionally, a method for identifying a (3-Iactamase in a clinical sample
is provided. Preferably, the clinical sample provided is characterized as Gram-
negative bacteria with resistance to (3-lactam antibiotics. In one aspect, the
method of the present invention for identifying a (3-lactamase in a clinical
sample includes, providing a pair of oligonucleotide primers specific for one
or
more groups within the CTX-M (3-lactamase family, wherein one primer of the
pair is complementary to at least a portion of the ~3-lactamase nucleic acid
in the
sense strand and the other primer of each pair is complementary to at least a
portion of the (3-lactamase nucleic acid in the antisense strand; annealing
the
primers to the ~i-lactamase nucleic acid; simultaneously extending the
annealed
primers from a 3' terminus of each primer to synthesize an extension product
that is complementary to the nucleic acid strands annealed to each primer
wherein each extension product after separation from the ~i-lactamase nucleic
acid serves as a template for the synthesis of an extension product for the
other
primer of each pair; separating the amplified products; and analyzing the
separated amplified products for a region characteristic of the (3-lactamase.
As
discussed below, the CTX-M ~i-lactamase family includes several groups of ~i-
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lactamases within the family. The present invention provides a method for
identifying one or more groups within the CTX-M ~-lactamase family.
The method, described above, employs oligonucleotide primer pairs that
are specific for one or more groups within the CTX-M family of ~-lactamases,
particularly primer pairs specific for one or more groups of CTX-M ~3-
lactamases such as the (3-lactamases of Group l: CTX-M-1, 3, 10-12, 15 (also
known as UOE-1), 22, 23, 28, 29, and 30; Group 2: CTX-M-2, 4-7, 20, and
TOHO-1, Group 3: CTX-M-8; and Group 4: CTX-M-9, 13, 14, 16-19, 21, 27,
and TOHO-2. The primers may also be specific for nucleic acid of the ~i-
lactamases of Group 5: CTX-M-25 and 26. The primer pairs may be specific for
one group within the CTX-M ~3-lactamase family or more than one group within
the CTX-M ~-lactamase family (e.g., a primer pair specific for two groups
within the CTX-M ~3-lactamase family); however, none of the primer pairs of
the
present invention are specific for all CTX-M (3-lactamase family groups (in
which case they would be only family-specific but not group-specific). That
is,
although the primer pairs of the present invention can distinguish a CTX-M ~-
lactamase from another ~-lactamase family (e.g., a TEM, SHV, or OXA family),
the primer pairs of the present invention can also distinguish between
different
groups within the CTX-M ~-lactamase family. ,
Real-time polymerase chain reaction (PCR) is recognized in the art as a
useful tool that may provide advantages over traditional PCR, as described
more
thoroughly below. Thus, in yet another aspect, the present invention is also
directed to a method for identifying a ~-lactamase in a clinical sample, the
method including:
providing a primer pair comprising one primer selected from the group of:
5' - GAC GAT GTC ACT GGC TGA GC - 3' (SEQ )D NO:1 ); 5' - AGC CGC
CGA CGC TAA TAC A - 3' (SEQ )D N0:2); 5' - GCG ACC TGG TTA ACT
ACA ATC C - 3' (SEQ >D N0:3); 5' - CGG TAG TAT TGC CCT TAA GCC -
3' (SEQ JD N0:4); 5' - GCT GGA GAA AAG CAG CGG AG - 3' (SEQ JD
N0:5); 5' - GTA AGC TGA CGC AAC GTC TG - 3' (SEQ >D N0:6); 5' - CGC
TTT GCC ATG TGC AGC ACC - 3' (SEQ ID N0:7); and 5' - GCT CAG TAC
GAT CGA GCC - 3' (SEQ )D N0:8); and full-length complements thereof; and
subjecting the primer pair to a real-time polymerase chain reaction assay.
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The present invention further provides kits useful in detecting a (3-
lactamase of interest in a clinical sample. In one aspect, the present
invention
provides a diagnostic kit for detecting a CTX-M ~-lactamase which includes
packaging, containing, separately packaged: at least one primer pair capable
of
hybridizing to a ~-lactamase nucleic acid selected from the group of members
of
Groups 1-5 of the CTX-M (3-lactamase family; a positive and negative control;
and a protocol for identification of the ~-lactamase nucleic acid of interest,
wherein the at least one primer pair is specific for one or more groups within
the
CTX-M ~-lactamase family.
In a further aspect, the present invention is directed to a diagnostic kit for
detecting a family of CTX-M ~i-lactamase which includes packaging, containing,
separately packaged: at least one primer pair capable of hybridizing to a ~3-
lactamase nucleic acid of interest; a positive and negative control; and a
protocol
for identification of the (3-lactamase nucleic acid of interest; wherein the
primers
are selected from the group of: 5' -GAC GAT GTC ACT GGC TGA GC - 3'
(SEQ ID NO:1 ); 5' -AGC CGC CGA CGC TAA TAC A - 3' (SEQ 1D N0:2); 5'
-GCG ACC TGG TTA ACT ACA ATC C - 3' (SEQ >D N0:3); 5' -CGG TAG
TAT TGC CCT TAA GCC - 3' (SEQ ID N0:4); 5' -GCT GGA GAA AAG
CAG CGG AG - 3' (SEQ ID NO:S); 5' -GTA AGC TGA CGC AAC GTC TG -
3' (SEQ III NO:6); 5' -CGC TTT GCC ATG TGC AGC ACC - 3' (SEQ )D
N0:7); and 5' -GCT CAG TAC GAT CGA GCC - 3' (SEQ ID NO:~); and full-
length complements thereof.
Additionally, in yet another aspect, the present invention is also directed
to a diagnostic kit for detecting a family of CTX-M (3-lactamase using real
time polymerase chain reaction which includes packaging, containing,
separately
packaged: at least one primer pair capable of hybridizing to a ~3-lactamase
nucleic acid of interest; a positive and negative control; and a protocol for
identification of the ~-lactamase nucleic acid of interest; wherein one primer
of
the pair of primers is selected from the group of: 5' -GAC GAT GTC ACT GGC
TGA GC - 3' (SEQ >D NO:l); 5' -AGC CGC CGA CGC TAA TAC A - 3' (SEQ
)D N0:2); 5' -GCG ACC TGG TTA ACT ACA ATC C - 3' (SEQ ID N0:3); 5'
-CGG TAG TAT TGC CCT TAA GCC - 3' (SEQ JD N0:4); 5' -GCT GGA
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GAA AAG CAG CGG AG - 3' (SEQ ID NO:S); 5' -GTA AGC TGA CGC AAC
GTC TG - 3' (SEQ ID N0:6);
5' -CGC TTT GCC ATG TGC AGC ACC - 3' (SEQ ID N0:7); and 5' -GCT
CAG TAC GAT CGA GCC - 3' (SEQ ID N0:8); and full-length complements
thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1-2 are primers of the present invention.
Figure 3 is a CTX-M PCR of four primer sets representing groups of
blacTxM genes.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
The present invention is directed to the detection of nucleic acid that is
characteristic of (e.g., at least a segment of a gene that codes for) family-
specific
~3-lactamase nucleic acid in samples (e.g., clinical isolates of Gram-negative
bacteria). Specifically, the present invention is directed to the detection of
~-lactamase nucleic acid (preferably, a gene or at least a segment of a gene)
using unique primers and the polymerase chain reaction (PCR). Using the
primers and methods of the present invention, (3-lactamases belonging to the
CTX-M family (e.g., (3-lactamases of Groups 1-5 as indicated above), for
example, can be identified.
Even more specifically, the present invention provides primers to
specifically identify groups within the CTX-M (3-lactamase family.
Accordingly, using the primers and methods of the present invention, not only
can ~3-lactamases belonging to the CTX-M family be distinguished from other (3-
lactamases, but various groups within the CTX-M family (e.g., (3-lactamases of
Groups 1-5 as indicated herein) can be identified. This does not mean,
however,
that any one primer pair must be specific for only one group within the
family,
although this is preferred. Certain primers are specific for two (or more),
but not
all, groups, for example, which is still advantageous because it allows for
narrowing the group identification.
PCR amplification and sequencing of genes for use in characterizing
organisms producing CTX-M-~i-lactamases is known in the art, such as, for
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example, the molecular approach to screening ESBL-positive organisms for the
presence of CTX-M genes as described in Edelstein et al. (Antifnicr~ob.
Age~zts
Claemother., 47:3724-3732 (2003)). In this approach, consensus primers (i.e.,
"universal" primers), which recognize all known variant genes of blacTx-M
known in the art at the time, are used to generate an amplified product of 544
base pairs. Following this reaction, the specific groups of blacTx-M genes are
identified by the use of restriction fragment length polymorphism (RFLP)
analysis. The present invention provides an advantage over the above approach
in that specific primer sets are used to detect various CTX-M-~3-lactamase
genes
and/or groups of CTX-M-~-lactamase genes, negating the need to perform an
additional identification step (at least for most embodiments). That is, it
should
be understood that for most embodiments of the present invention, an
additional
identification step is not necessarily needed, but one could be used if
desired,
particularly if the primers are specific for two or more groups and there is a
desire to distinguish between these groups or to identify the specific CTX-M
~-lactamase family member.
The present invention provides amplification of a single DNA fragment
for a CTX-M-~3-lactamase gene, affording simple interpretation of results that
can be adapted for use in reference laboratories for screening multiple
isolates
for the presence of CTX-M-~i-lactamase genes. For example, to identify a (3-
lactamase of interest contained in a sample, such as CTX-M-14 (3-lactamase, a
selected primer pair of the invention (e.g., SEQ JD NO. 5 and SEQ » NO. 6 for
CTX-M-14 ~i-lactamase) is used to provide an amplified product, which,
following a gel electrophoresis assay of the amplified products, provides
identification of the ~-lactamase of interest (i.e., the CTX-M-14 ~i-
lactamase) in
a single step. That is, no additional identification steps, such as RFLP, WAVE
analysis, sequence identification (Gold Standard), or single stranded
conformational polymorphisms (SSCP), are required to identify the ~i-
lactamases
of interest, although such additional assays could be performed, if desired.
Thus, the present invention is distinguished over the methods of Edelstein et
al.
as the Edelstein et al. methods require the use of the additional RFLP
identification step and may require yet another identification step beyond the
RFLP analysis.
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Additionally, the step of analyzing the separated amplified products, as
recited in the present claims, includes a visual inspection of the gel
produced by
gel electrophoresis of the amplified products. The gel produced by the present
methods typically provides a clear, unambiguous band; thus, the primers and
methods of the present invention may be performed by personnel with lesser
training and/or experience and still provide accurate results. The methods of
Edelstein et al., however, typically do not produce as clear a product as do
the
methods of the present invention, as the results of RFLP analysis are often
difficult to interpret and do not give the single band results provided by the
primers and methods of the present invention. Thus, the methods of Edelstein
et
al. generally provide more ambiguous results, which are typically more
difficult
for an untrained person to correctly interpret.
Further, the methods of the present invention provide primer pairs
specific for one or more groups within the CTX-M (3-lactamase family, which is
defined herein to include primer pairs specific for one group (e.g., ~i-
lactamases
found in Group 1 of the CTX-M family) and also primer pairs specific for more
than one group (e.g., ~i-lactamases found in Groups 3 and 5 of the CTX-M
family); however, the primer pairs are not specific for all ~i-lactamase
family
groups. That is, the present invention provides a method for identifying in a
single step a (3-lactamase included in the CTX-M ~i-lactamase family over a ~-
lactamase included in another family of ~-lactamases (e.g., an OXA family), as
well as provides a method of distinguishing one group of the CTX-M ~-
lactamase family over another group of the CTX-M (3-lactamase family. This is
distinguished over the methods of Edelstein et al., for example, which do not
distinguish, and in particular do not distinguish in one step, between groups
within the CTX-M (3-lactamase family. Edelstein et al. provide a method
whereby universal primers are used with PCR to provide amplified products,
which may include all CTX-M ~i-lactamases from other ~3-lactamases. Further
identification is then required to determine the ~i-lactamase in the sample.
The
present invention, advantageously, provides the ability to identify in a
clinical
sample, in a single step, the presence of one group over another group of the
CTX-M (3-lactamase family.
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The methods of the present invention involve the use of the polymerise
chain reaction sequence amplification method (PCR) using novel primers. U.S.
Patent No. 4,683,195 (Mullis et al.) describes a process for amplifying,
detecting, and/or cloning nucleic acid. Preferably, this amplification method
relates to the treatment of a sample containing nucleic acid (typically, DNA)
of
interest from bacteria, particularly Gram-negative bacteria, with a molar
excess
of an oligonucleotide primer pair, heating the sample containing the nucleic
acid
of interest to yield two single-stranded complementary nucleic acid strands,
adding the primer pair to the sample containing the nucleic acid strands,
allowing each primer to anneal to a particular strand under appropriate
temperature conditions that permit hybridization, providing a molar excess of
nucleotide triphosphates and polymerise to extend each primer to form a
complementary extension product that can be employed in amplification of a
desired nucleic acid, detecting the amplified nucleic acid, and analyzing the
amplified nucleic acid for a size specific amplicon (as indicated below)
characteristic of the specific ~i-lactamase of interest. This process of
heating,
annealing, and synthesizing is repeated many times, and with each cycle the
desired nucleic acid increases in abundance. Within a short period of time, it
is
possible to obtain a specific nucleic acid, e.g., a DNA molecule, that can be
readily purified and identified.
The oligonucleotide primer pair, which may include at least one primer
selected from the group of SEQ ID NO. 1 to SEQ )D NO. 8, includes one primer
that is substantially complementary to at least a portion of a sense strand of
the
nucleic acid and one primer that is substantially complementary to at least a
portion of an antisense strand of the nucleic acid. The process of forming
extension products preferably involves simultaneously extending the annealed
primers from a 3' terminus of each primer to synthesize an extension product
that is complementary to the nucleic acid strands annealed to each primer
wherein each extension product after separation from the [3-lactamase nucleic
acid serves as a template for the synthesis of an extension product for the
other
primer of each pair. The amplified products are preferably detected by size
fractionization using gel electrophoresis. Variations of the method are
described in U.S. Patent No. 4,683,194 (Saiki et al.). The polymerise chain
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reaction sequence amplification method is also described by Saiki et al.,
Sciei2ce, 230, 1350-1354 (1985) and Scharf et al., Scie~zce, 324, 163-166
(1986).
An "oligonucleotide," as used herein, refers to a polymeric form of
nucleotides of any length, either ribonucleotides or deoxyribonucleotides. The
term oligonucleotide refers particularly to the primary structure, and thus
includes double and single-stranded DNA molecules and double and single-
stranded RNA molecules.
A "primer," as used herein, is an oligonucleotide that is complementary
to at least a portion of nucleic acid of interest and, after hybridization to
the
nucleic acid, may serve as a starting-point for the polymerase chain reaction.
The terms "primer" or "oligonucleotide primer," as used herein, further refer
to
a primer, having a nucleotide sequence that possesses a high degree of nucleic
acid sequence similarity to at least a portion of the nucleic acid of
interest.
"High degree" of sequence similarity refers to a primer that typically has at
least
about 80% nucleic acid sequence similarity, and preferably about 90% nucleic
acid sequence similarity. Sequence similarity may be determined, for example,
using sequence techniques such as GCG FastA (Genetics Computer Group,
Madison, Wisconsin), MacVector 4.5 (I~odaklIBI software package) or other
suitable sequencing programs or methods known in the art.
The terms "complement" and "complementary" as used herein, refer to a
nucleic acid that is capable of hybridizing to a specified nucleic acid
molecule
under stringent hybridization conditions. Stringent hybridization conditions
include, for example, temperatures from about 50°C to about
65°C, and
magnesium (Mg) concentrations from about 1.5 millimolar (mM) to about 2.0
mM. Thus, a specified DNA molecule is typically "complementary" to a
nucleic acid if hybridization occurs between the specified DNA molecule and
the nucleic acid. "Complementary," further refers to the capacity of purine
and
pyrimidine nucleotides to associate through hydrogen bonding in double
stranded nucleic acid molecules. The following base pairs are complementary:
guanine and cytosine; adenine and thymine; and adenine and uracil.
As used herein, the terms "amplified molecule," "amplified fragment,"
and "amplicon" refer to a nucleic acid molecule (typically, DNA) that is a
copy
of at least a portion of the nucleic acid and its complementary sequence. The
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copies correspond in nucleotide sequence to the original molecule and its
complementary sequence. The amplicon can be detected and analyzed by a wide
variety of methods. These include, for example, gel electrophoresis, single
strand conformational polymorphism (SSCP), restriction fragment length
polymorphism (RFLP), capillary zone electrophoresis (CZE), and the like.
Preferably, using methods and primers of the present invention, the amplicon
can be detected, and hence, the type of (3-lactamase identified, using gel
electrophoresis and appropriately sized markers, according to techniques known
to one of skill in the art.
The primers are oligonucleotides, either synthetic or naturally occurring,
capable of acting as a point of initiating synthesis of a product
complementary
to the region of the DNA molecule containing the ~i-lactamase of interest. The
primer includes nucleotides capable of hybridizing under stringent conditions
to
at least a portion of at least one strand of a nucleic acid molecule of a (3-
lactamase selected from the group of CTX-M-l, CTX-M-2, CTX-M-3, CTX-
M-4, CTX-M-5, CTX-M-6, CTX-M-7, CTX-M-8, CTX-M-9, CTX-M-10,
CTX-M-11, CTX-M-12, CTX-M-13, CTX-M-14, CTX-M-15 (also known as
UOE-1), CTX-M-16, CTX-M-17, CTX-M-18, CTX-M-19, CTX-M-20, CTX-
M-21, CTX-M-22, CTX-M-23, CTX-M-25, CTX-M-26, CTX-M-27, CTX-M-
28, CTX-M-29, CTX-M-30, TOHO-1, TOHO-2, or any combination of these ~-
lactamases.
Preferably, the primers of the present invention typically have at least
about 15 nucleotides, preferably at least about 18 nucleotides, and more
preferably at least about 20 nucleotides. Typically, the primers have no more
than about 30 nucleotides, preferably no more than about 28 nucleotides, more
preferably no more than about 26 nucleotides, even more preferably no more
than about 24 nucleotides, and still more preferably no more than about 22
nucleotides. The primers are chosen such that they preferably produce a primed
product of at least about 300 base pairs and preferably no greater than about
600
base pairs, more preferably no greater than about 500 base pairs.
Optionally, a primer used in accordance with the present invention
includes a label constituent. The label constituent can be selected from the
group of an isotopic label, a fluorescent label, a polypeptide label, and a
dye
12
CA 02538270 2006-03-07
WO 2005/024045 PCT/US2004/029695
release compound. The label constituent is typically incorporated in the
primer
by including a nucleotide having the label attached thereto. Isotopic labels
preferably include those compounds that are beta, gamma, or alpha emitters,
more preferably isotopic labels are selected from the group of 3'-p, 3sS,
and'ZSI.
Fluorescent labels are typically dye compounds that emit visible radiation in
passing from a higher to a lower electronic state, typically in which the time
interval between adsorption and emission of energy is relatively short,
generally
on the order of about 10-$ to about 10-3 second. Suitable fluorescent
compounds
that can be utilized include fluorescien and rhodamine, for example. Suitable
polypeptide labels that can be utilized in accordance with the present
invention
include antigens (e.g., biotin, digoxigenin, and the like) and enzymes (e.g.,
horse
radish peroxidase). A dye release compound typically includes
chemiluminescent systems defined as the emission of absorbed energy
(typically as light) due to a chemical reaction of the components of the
system,
including oxyluminescence in which light is produced by chemical reactions
involving oxygen.
While PCR, through the use of repetitive multiplication of template
molecules, is a sensitive and extremely useful analytical tool, such
repetitive
multiplication also provides a drawback in that small differences in the
template
can result in significant differences in the amount of product. One answer to
this concern is the use of "real-time" PCR. Thus, in addition to the use of
PCR
described above (i.e., "traditional" PCR), primers, methods, and kits of the
present invention may also be useful in carrying out real-time PCR.
Real-time PCR monitors the fluorescence emitted during the PCR
reaction as an indicator of amplicon production during the PCR cycle (i.e., in
real-time) to provide data collected in the exponential phase of the reaction.
Traditional PCR, on the other hand, provides data on the amount of amplified
product only at the end-point of the reaction.
Real-time PCR is based on the detection and quantitation of a fluorescent
reporter or intercalation of a florescent dye, such as SYBR"Green into the
amplified PCR product. The signal increases in direct proportion to the amount
of PCR product in a reaction and, by recording the amount of fluorescence
emission at each cycle, the PCR reaction may be monitored during the
13
CA 02538270 2006-03-07
WO 2005/024045 PCT/US2004/029695
exponential phase. Additionally, real-time PCR substantially eliminates the
need for post-PCR processing of PCR products, thus increasing throughput and
decreasing the possibility of carryover contamination. Further, compared to
traditional PCR, real-time PCR provides a wider dynamic range of an assay
(i.e., determination of how much the target concentration can vary and still
be
quantified), and, thus, improved accuracy in quantitation of the PCR product.
Real-time PCR may also be used for applications that would be less effective
with traditional PCR (Dorak, Real-Time PCR, available on the World Wide
Web at http//dorakmt.tripod.com/genetics/realtime.html, 2004).
Real-time PCR methods typically use primers that are shorter than those
used in traditional PCR methods; however, smaller amplicons are generally
provided, typically at least about 150 base pairs, more preferably at least
about
100 base pairs, and preferably no greater than about 200 base pairs.
Amplification of shorter products aids in the advantage provided by real-time
PCR, as the longer the product, the longer time it takes for amplification,
thus
the "real-time" analysis will likely be jeopardized.
Real-time PCR may be used with primers and methods of the present
invention wherein the primer pair used includes one primer selected from the
primers of SEQ ID No. 1 to SEQ ID NO. 8, and another primer, resulting in an
amplicon having, typically, no fewer than about 100 base pairs and no more
than about 250 base pairs. Additionally, the present invention provides a kit
for
use with real-time PCR, the kit including a primer pair wherein one primer of
the primer pair is selected from the group of SEQ ID NO. 1 to SEQ m NO. 8.
Preferred examples of primers of the present invention specific for
certain (3-lactamases are as follows, wherein "F" in the designations of the
primers refers to a 5' upstream primer and "R" refers to a 3' downstream
primer.
For those ~i-lactamases that have more than one upstream primer and more than
one downstream primer listed below as preferred primers, various combinations
can be used. Typically, hybridization conditions utilizing primers of the
invention include, for example, a hybridization temperature of at least about
50°
C and no greater than about 62° C, and a Mg concentration of at least
about 1.5
mM (millimolar) and no greater than about 2.0 mM. Although lower
14
CA 02538270 2006-03-07
WO 2005/024045 PCT/US2004/029695
temperatures and higher concentrations of Mg can be employed, this may result
in decreased primer specificity.
The following primers are specific for nucleic acid characteristic of the
CTX-M-l, 3, 10-12, 15 (UOE-1), 22, 23, 28, 29 and 30 ~i-lactamase enzymes.
Primer Name: CTXM1-F3
Primer Sequence: 5'-GAC GAT GTC ACT GGC TGA GC - 3' (SEQ ID NO:l)
Primer Name: CTXMl-R2
Primer Sequence: 5' - AGC CGC CGA CGC TAA TAC A - 3' (SEQ >D N0:2)
Employing a primer pair containing the primer sequences of SEQ lD
NO:1 and SEQ ID N0:2 to a sample containing a CTX-M-l, 3, 10-12, 15
(UOE-1), 22, 23, 28, 29 and/or 30 ~i-lactamase (a Group 1 CTX-M (3-lactamase),
a size-specific amplicon of 499 base pairs will typically be obtained.
The following primers are specific for nucleic acid characteristic of the
CTX-M-2, 4, 5, 6, 7, and 20 ~i-lactamase enzymes, as well as TORO-1 ~-
lactamase.
Primer Name: TORO 1-2F
Primer Sequence: 5' -GCG ACC TGG TTA ACT ACA ATC C - 3' (SEQ >D
N0:3)
Primer Name: TOHO 1-1R
Primer Sequence: 5' -CGG TAG TAT TGC CCT TAA GCC -.3' (SEQ >D
N0:4)
Employing a primer pair containing the primer sequences of SEQ ID
NO:3 and SEQ >D N0:4 to a sample containing a CTX-M-2, 4, 5, 6, 7, andlor
20 ~-lactamase, and/or a TORO-1 ~3-lactamase (a Group 2 CTX-M ~-
lactamase), a size-specific amplicon of 351 base pairs will typically be
obtained.
The following primers are specific for nucleic acid characteristic of the
CTX-M-9, 13, 14, 16, 17, 18, 19, 21, and 27 ~-lactamase enzymes, as well as
TOHO-2 ~i-lactamase.
Primer Name: CTXM 914F
Primer Sequence: 5' -GCT GGA GAA AAG CAG CGG AG - 3' (SEQ ID
N0:5)
CA 02538270 2006-03-07
WO 2005/024045 PCT/US2004/029695
Primer Name: CTXM914R
Primer Sequence: 5' -GTA AGC TGA CGC AAC GTC TG - 3' (SEQ ID N0:6)
Employing a primer pair containing the primer sequences of SEQ >D
N0:5 and SEQ JD N0:6 to a sample containing a CTX-M-9, 13, 14, 16, 17, 18,
19, 21, and/or 27 ~i-lactamase, and/or a TORO-2 ~3-lactamase (a Group 4 CTX-
M ~i-lactamase), a size-specific amplicon of 474 base pairs will typically be
obtained.
The following primers are specific for nucleic acid characteristic of the
CTX-M-8, 25, and 26 (3-lactamase enzymes.
Primer Name: CTXM825F
Primer Sequence: 5' -CGC TTT GCC ATG TGC AGC ACC - 3' (SEQ >D
N0:7)
Primer Name: CTXM825R
Primer Sequence: 5' -GCT CAG TAC GAT CGA GCC - 3' (SEQ >D N0:8)
Employing a primer pair containing the primer sequences of SEQ )D
N0:7 and SEQ ID N0:8 to a sample containing a CTX-M-8, 25, and/or 26 ~i-
lactamase (a Group 3 or Group 5 CTX-M (3-lactamase), a size-specific amplicon
of 307 base pairs will typically be obtained.
Various other primers, or variations of the primers described above, can
also be prepared and used according to methods of the present invention. For
example, alternative primers can be designed based on targeted ~3-lactamase
genes known or suspected to contain regions possessing high G/C content (i.e.,
the percentage of guanine and cytosine residues). As used herein, a "high G/C
content" in a target nucleic acid, typically includes regions having a
percentage
of guanine and cytosine residues of about 60% to about 90%. Thus, changes in
a prepared primer will alter, for example, the hybridization or annealing
temperatures of the primer, the size of the primer employed, and the sequence
of
the specific resistance gene or nucleic acid to be identified. Therefore,
manipulation of the G/C content, e.g., increasing or decreasing, of a primer
or
primer pair may be beneficial in increasing detection sensitivity in the
method.
16
CA 02538270 2006-03-07
WO 2005/024045 PCT/US2004/029695
Oligonucleotides of the invention can readily be synthesized by
techniques known in the art (see, for example, Crea et al., Proc. Natl. Acad.
Sci.
(U.S.A.) 75:5765 (1978)).
Once the primers are designed, their specificity can be tested using the
following method. Depending on the target nucleic acid of clinical interest, a
nucleic acid is isolated from a bacterial control strain known to express or
contain the resistance gene. This control strain, as used herein, refers to a
"positive control" nucleic acid (typically, DNA). Additionally, a "negative
control" nucleic acid (typically, DNA) can be isolated from one or more
bacterial strains known to express a resistance gene other than the target
gene of
interest. Using the polymerase chain reaction, the designed primers are
employed in a detection method, as described above, and used in the positive
and negative control samples and in at least one test sample suspected of
containing the resistance gene of interest. The positive and negative controls
provide an effective and qualitative (or grossly quantitative) means by which
to
establish the presence or the absence of the gene of interest of test clinical
samples. It should be recognized that with a small percentage of primer pairs,
possible cross-reactivity with other ~3-lactamase genes might be observed.
However, the size and/or intensity of any cross-reactive amplified product
will
be considerably different and can therefore be readily evaluated and dismissed
as a negative result.
The invention also relates to kits for identifying a family-specific ~3-
lactamase enzyme by PCR analysis. bits of the invention typically include one
or more primer pairs specific for a ~i-lactamase of interest, one or more
positive
controls, one or more negative controls, and protocol for identification of
the ~i-
lactamase of interest using polymerase chain reaction.
Primer pairs useful in kits of the present invention include those selected
from the group of SEQ ID NO. 1 to SEQ ID NO. 8. Additionally, kits useful
with real-time PCR methods include primer pairs wherein one primer of the pair
is selected from the group of SEQ ID No. 1 to SEQ m NO. 8. A negative
control includes a nucleic acid (typically, DNA) molecule encoding a resistant
~i-lactamase gene other than the ~3-lactamase gene of interest. The negative
control nucleic acid may be a naked nucleic acid (typically, DNA) molecule or
17
CA 02538270 2006-03-07
WO 2005/024045 PCT/US2004/029695
inserted into a bacterial cell. Preferably, the negative control nucleic acid
is
double-stranded; however, a single-stranded nucleic acid may be employed. A
positive control includes a nucleic acid (typically, DNA) that encodes a ~i-
lactamase gene from the family of ~-lactamase genes of interest. The positive
control nucleic acid may be a naked nucleic acid molecule or inserted into a
bacterial cell, for example. Preferably, the positive control nucleic acid is
double-stranded, however, a single-stranded nucleic acid may be employed.
Typically, the nucleic acid is obtained from a bacterial lysate.
Accordingly, the present invention provides a kit for characterizing and
identifying a family-specific (3-lactamase gene that would have general
applicability. Preferably, the kit includes a polymerase (typically, DNA
polymerase) enzyme, such as Taq polymerase, and the like. A kit of the
invention also preferably includes at least one primer pair that is specific
for a
~i-lactamase. A buffer system compatible with the polymerase enzyme is also
included and are well known in the art. Optionally, the at least one primer
pair
may contain a label constituent, a fluorescent label, a polypeptide label, and
a
dye release compound. The kit may further contain at least one internal sample
control, in addition to one or more further means required for PCR analysis,
such as a reaction vessel. If required, a nucleic acid from the bacterial
sample
can be isolated and then subjected to PCR analysis using the provided primer
set
of the invention.
In another embodiment, family-specific (3-lactamase genes in clinical
samples, particularly clinical samples containing Gram-negative bacteria, can
be
detected by the primers described herein in a "microchip" detection method. In
a microchip detection method, nucleic acid, e.g., genes, of multiple (3-
lactamases
in clinical samples can be detected with a minimal requirement for human
intervention. Techniques borrowed from the microelectronics industry are
particularly suitable to these ends. For example, micromachining and
photolithographic procedures are capable of producing multiple parallel
microscopic scale components on a single chip substrate. Materials can be mass
produced and reproducibility is exceptional. The microscopic sizes minimize
material requirements. Thus, human manipulations can be minimized by
18
CA 02538270 2006-03-07
WO 2005/024045 PCT/US2004/029695
designing a microchip type surface capable of immobilizing a plurality of
primers of the invention on the microchip surface.
Thus, an object of the present invention is to provide a parallel
screening method wherein multiple serial reactions are automatically performed
individually within one reaction well for each of the plurality of nucleic
acid
strands to be detected in the plural parallel sample wells. These serial
reactions
are performed in a simultaneous run within each of the multiple parallel lanes
of
the device. "Parallel" as used herein means wells identical in function.
"Simultaneous" means within one preprogrammed run. The multiple reactions
automatically performed within the same apparatus minimize sample
manipulation and labor.
Thus, the present invention provides multiple reaction wells, the reaction
wells being reaction chambers, on a microchip, each reaction well containing
an
individualized array to be used for detecting a ~3-lactamase gene uniquely
specified by the substrates provided, the reaction conditions and the sequence
of
reactions in that well. The chip can thus be used as a method for identifying
~i-
lactamase genes in clinical samples.
Objects and advantages of this invention are further illustrated by the
following example, but the particular materials and amounts thereof recited in
these examples, as well as other conditions and details, should not be
construed
to unduly limit this invention.
Example 1
CTX-M Type-Specific Identification in Enterobacteriaceae Using CTX-M
Family-Specific Primers
Methods:
Twenty two isolates representing E. coli (Ec), Citrobacter frem2dii (Cf)
and C. koseri (Ck) were studied. All of these strains had MICs of greater than
(>) 32 micrograms per milliliter (mg/ml) of cefotaxime, >16 mg/ml of cefepime,
and ranged between 1 to ~ mg/ml for ceftazidime. Dendrogram analysis of
CTX-M genes deposited in GenBank was performed, and based on these
analyses, known CTX-M-genes were divided into four families. Based on these
19
CA 02538270 2006-03-07
WO 2005/024045 PCT/US2004/029695
families, four sets of family-specific primers were designed. Specificity of
the
primers was tested on all 22 of the isolates.
Polymerase Chain Reaction (PCR) amplifications were carried out under
conditions as indicated in Fig. 2, which shows diagnostic primer sets and PCR
conditions, with hybridization temperature indicated in degrees Centigrade and
1.5 millimolar (mM) MgCl2 concentration. Selected PCR products were then
sequenced to verify family specificity.
Results:
DNA from all strains was tested with every primer set. Seventy-seven
percent (17/22) of the isolates were identified as carrying CTX-M genes. Of
these, 68% (15/22) were E. coli, and 5% (1122) were Citrobacter fi-eurzdii. No
CTX-M genes were identified in the C. koseri isolates. Of the positive CTX-M
E. coli isolates, 60% (9/15) were CTX-M-14-like and 40% (6/15) were CTX-M-
1-like. The Citrobacter fremZdai isolate carried a CTX-M-1-like gene.
Sequencing of selected amplicons verified that the amplicons belonged to the -
CTX-M-14 or CTX-M-1 group of CTX-M genes. No~amplification products
were observed for the other two family-specific primer sets.
Conclusions:
Susceptibility profiles alone cannot predict CTX-M producing
organisms. Twenty-three percent of isolates with similar MIC profiles were
negative for CTX-M-like genes. Accurate surveillance of CTX-M producers
will require susceptibility profiles combined with diagnostic PCR using family-
specific primer sets.
Example 2
Identification of (3-lactamase Genes in Clinical Strains producing Extended-
Spectrum ~i-lactamases Using CTX-M Family-Specific Primers
Methods:
A total of 175 isolates representing E. coli (n=168), K. pneuffioraiae (n=7)
were studied. Minimum inhibitory concentrations (MICs) to the following drugs
CA 02538270 2006-03-07
WO 2005/024045 PCT/US2004/029695
were determined by Vitek (Vitek AMS; bioMerieux Vitek 5 Systems Inc.,
Hazelwood, MO); piperacillin (PIP), piperacillin/tazobactam (TZP),
cefpodoxime (CPD), cefotaxime (CTX), ceftazidime (CAZ). The quality
control strains used for this study were E. coli ATCC 25922, E. coli 35218,
Pseudornozzas aez°ugizzosa ATCC 27853, Staphylococcus aureus ATCC
29213,
and K. pJZeuzzzoniae ATCC 700603. Throughout this study, results were
interpreted using National Committee for Clinical Laboratory Standards
(NCCLS) criteria for broth dilution.
The presence of ESBLs was evaluated in both the control strains and the
recent clinical isolates. Screening was performed with Vitek (Vitek AMS;
bioMerieux Vitek Systems, Inc., Hazelwood, MO) using 1 ~.g/ml CPD, CAZ,
and CTX. Screening and disk confirmation tests using CTX (30 ~.g) and CAZ
(30 ~.g) disks in combination with 10 ~,g clavulanate (CLA) were performed and
interpreted using NCCLS criteria for ESBL screening and disk confirmation
tests. Disks for ESBL confirmation tests were obtained from Oxoid Inc.
(Nepean, Ontario, Canada). K. pzzeumoiziae ATCC 700603 and E. coli ATCC
25922 were used as positive and negative controls, respectively.
Polymerase Chain Reaction (PCR) amplifications were carried out on a
Thermal Cycler 9600 instrument (Applied Biosystems, Norwalk, CT) as
indicated in Example 1, with the primers, the size of the expected
amplification
product, and annealing temperatures used for the PCR as listed in Table 1.
Magnesium chloride concentrations were 1.5 mM for all PCR reactions.
21
CA 02538270 2006-03-07
WO 2005/024045 PCT/US2004/029695
Table 1- Control Strains producing well-characterized ~3-lactamases
CTX-M [3-
Strain Organism ~i-lactamase lactamase
family Group
CF2 E. cloacae CTX-M-1 I
Rio-4 Proteus mirabilis CTX-M-2 II
VER-1 E. cloacae CTX-M-3 I
Cfr2525/96 Citrobacter freundiiCTX-M-3 I
Eco3553/98 E. coli CTX-M-15 I
34 SalnzozZella typlzimuriunzCTX-M-5 II
Rio-3 E. aerogerzes CTX-M-8 III
785D E. coli CTX-M-9 IV
EC97/38582 E. coli CTX-M-10 I
EC984167 E. coli CTX-M-14 IV
CF1 E. coli CTX-M-14 IV
Rio-6 E. coli CTX-M-16 IV
BM4493 K. pueunzoiziae CTX-M-17 IV
ILT-2 K. pneunzozziae CTX-M-18 IV
IL,T-3 K. p~zeunzoz2iae CTX-M-19 IV
CTl~ E. coli TOHO-1 II
S 1 * E. coli SHV-2 -
S2~ E. coli SHV-7 -
T 1 ~ E. coli TEM-3 -
T4* E. coli TEM-10 -
T5r E. coli TEM-50 -
*These strains are
part of an unpublished
isogenic panel.
Results:
The designed primers
were tested for
specificity in
separate PCR
reactions using
DNA template prepared
from control strains
known to produce
22
CA 02538270 2006-03-07
WO 2005/024045 PCT/US2004/029695
specific CTX-M ~-lactamases or strains producing ESBLs other than CTX-M [3-
lactamases (Tables 1 and 2, Fig 3). PCR amplification of DNA template
prepared from strains CF2, VER-l, Cfr2525/96, Eco3553/98, and EC97/38582,
resulted in a single amplified product of 499 base pairs (bp) when CTX-M
group I primers were used. No amplified product was identified with this
primer
set when DNA template from the rest of the control strains in Table 1 were
used
during PCR amplification. Group II primers amplified a single 351 by fragment
when DNA template was prepared from control strains Rio-4, 34, and CT1. All
other control strain DNA templates resulted in no amplification product for
this
primer set.
23
CA 02538270 2006-03-07
WO 2005/024045 PCT/US2004/029695
a.
' ' ' ' ' ' ~ + ~ + + + +
bn
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.
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~',, a, + ' + + + ~ ~ m
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+ + + + + + + + + + + + +
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CA 02538270 2006-03-07
WO 2005/024045 PCT/US2004/029695
.-,
1 I I I I 1
v
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! I 1 1 1 1 J t
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+ + + + + + '~. ~ V
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-~,-1- ~- ~- r +
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-j--j--j-~- c
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CA 02538270 2006-03-07
WO 2005/024045 PCT/US2004/029695
Group IV primers amplified a 474 by product from DNA prepared from
strains 785D, EC984167, CF1, Rio-6, BM4493, IL,T-2, and ILT-3. This primer
set was also very specific resulting in no amplification of DNA when template
was prepared from the other control strains. Using the group III primer set
resulted in amplification of DNA prepared from only one strain, Rio-3, which
produced CTX-M-8. An isolate producing CTX-M-25 was requested but not
obtained. Therefore, we were unable to examine the ability of the Group III
primer set to amplify blacTx-M-zs. A representative gel indicating the
specificity
of the primer pairs is shown in Fig. 2. These data indicate a high level of
specificity for these group-specific primer pairs.
All the ESBL-producing E. coli and Klebsiella spp. were examined by
PCR for the presence of blacTx-M genes. Of the 168 E. coli isolated during the
study period, 24 ( 14 %) were positive for blacTx-M genes from the CTX-M-I
group, indicating CTX-M-1-like ~i-lactamases. Ninety-three (55%) were
positive for blacTx-M genes from the CTX-M-IV group indicating CTX-M-14-
like ~i-lactamases and the remaining 51 (31 %) were negative for blacTx-m
genes
(Table 4). Of the 7 K. pneumoniae isolated during the study period, 2 (29%)
were positive for blacTx-M genes from the CTX-M-IV group and the remaining 5
(71 %) were negative for blacTx-M genes (Table 3).
26
CA 02538270 2006-03-07
WO 2005/024045 PCT/US2004/029695
r
H
U
v
N
N 'b
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b'~ ~ ~. fi g ~' ~a
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v' O ~ ~ > ~ j > re c C a ~
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N
CA 02538270 2006-03-07
WO 2005/024045 PCT/US2004/029695
Example 3
Population-based Surveillance for ESBL-producing E. coli Infections
Methods:
A total of 157 isolates were collected from a population-based
surveillance of ESBL-producing E.coli infections. The E. coli was isolated by
standard techniques, and susceptibilities to antimicrobial agents were
determined using Vitek AMS (bioMe'rieux Vitek Systems). The presence of an
ESBL was established on the basis of NCCLS guidelines. All strains with an
MIC of cefpodoxime of >_8 ~.g/mL were subjected to the NCCLS disk
confirmation tests by cefotaxime (CTX; 30 ~,g) and ceftazidime (CAS; 30 ~,g)
disks in combination with 10 ~g of clavulanate (CLA). The results were
interpreted using NCCLS criteria.
PCR amplification for CTX-M (3-lactamase genes was performed using a
Thermal Cycler 9600 instrument (Applied Biosystems, Norwalk, CT) with
standard PCR conditions, as described in Example 1 using primer pairs for
CTX-M Groups 1-5 (3-lactamases, set forth as SEQ ID NO. 1 through SEQ ID
NO. 8 indicated above and in Fig. 1.
All analyses were performed using Stata statistical software, version 8.0
(StataCorp, College Station, TX). Differences in proportions among categorical
data were assessed using Fisher's exact test. Incidence rates were calculated
by
using regional demographic data as the denominator and compared with Poisson
counts. Category-specific risks were calculated and reported as relative risk
(RR) with 95% CIs, as described in Laupland et al., J. hZfect. Dis., l ~7:1452-
1459 (2003). A multivariable logistic regression model was developed to assess
factors associated with the presence of blacTx-M genes. Variables in the
initial
model included all of those significant to in P < 0.1 univariate analysis,
age, sex,
and community/nosocomial onset. Backward stepwise variable elimination was
performed to derive the final model. Calibration and discrimination were
assessed using the Hosmer-Lemeshow goodness-of fit test and the area under
the receiver operator curve (ROC), respectively. Results were reported as ORs
and 95% CIs.
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CA 02538270 2006-03-07
WO 2005/024045 PCT/US2004/029695
Results:
All ESBL-producing E. coli were examined by PCR for the presence of
blacTx-M genes. Twenty-three (15%) of 157 were positive for bla~TX-M genes
from the CTX-M-I subgroup (referred to as "CTX-M-1-like ~3-lactamases), 87
(55%) were positive for blacTx_n,, genes from CTX-M-III subgroup (CTX-M-
14-like (3-lactamases), and the remaining 47 (30%) were negative for,,~UcTx-M
genes (CTX-M negative).
The complete disclosures of the patents, patent documents, and
publications cited herein are incorporated by reference in their entirety as
if each
were individually incorporated. Various modifications and alterations to this
invention will become apparent to those skilled in the art without departing
from
the scope and spirit of this invention. It should be understood that this
invention is not intended to be unduly limited by the illustrative embodiments
and examples set forth herein and that such examples and embodiments are
presented by way of example only with the scope of the invention intended to
be
limited only by the claims set forth herein as follows.
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