U.S. patent application number 12/105243 was filed with the patent office on 2008-08-21 for oligonucleotide probes for detecting enterobacteriaceae and quinolone-resistant enterobacteriaceae.
This patent application is currently assigned to THE GOVERNMENT OF THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE DEPARTMENT OF. Invention is credited to Fred C. Tenover, Linda M. Weigel.
Application Number | 20080199877 12/105243 |
Document ID | / |
Family ID | 33566953 |
Filed Date | 2008-08-21 |
United States Patent
Application |
20080199877 |
Kind Code |
A1 |
Weigel; Linda M. ; et
al. |
August 21, 2008 |
OLIGONUCLEOTIDE PROBES FOR DETECTING ENTEROBACTERIACEAE AND
QUINOLONE-RESISTANT ENTEROBACTERIACEAE
Abstract
Oligonucleotide probes for detecting Enterobacteriaceae species.
Unique gyrA coding regions permit the development of probes
specific for eight different species: Escherichia coli, Citrobacter
freundii, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella
oxytoca, Klebsiella pneumoniae, Providencia stuartii and Serratia
marcescens. The invention thereby provides methods for the
species-specific identification of these Enterobacteriaceae in a
sample, and detection and diagnosis of Enterobacteriaceae infection
in a subject. Further, nucleic acids are provided for determining
quinolone-resistant status of these Enterobacteriaceae.
Inventors: |
Weigel; Linda M.; (Decatur,
GA) ; Tenover; Fred C.; (Atlanta, GA) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 S.W. SALMON STREET, SUITE 1600
PORTLAND
OR
97204
US
|
Assignee: |
THE GOVERNMENT OF THE UNITED STATES
OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE DEPARTMENT
OF
HEALTH AND HUMAN SERVICES, CENTERS FOR DISEASE CONTROL AND
PREVENTION
|
Family ID: |
33566953 |
Appl. No.: |
12/105243 |
Filed: |
April 17, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10798827 |
Mar 10, 2004 |
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12105243 |
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09647563 |
Jan 16, 2001 |
6706475 |
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PCT/US99/06963 |
Mar 30, 1999 |
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10798827 |
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60080375 |
Apr 1, 1998 |
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Current U.S.
Class: |
435/6.15 |
Current CPC
Class: |
C12Q 1/689 20130101;
C07H 21/04 20130101; Y02A 50/451 20180101; Y10T 436/143333
20150115; Y02A 50/30 20180101; C12Q 1/6827 20130101; C07H 21/02
20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
[0002] This invention was made in the Centers for Disease Control
and Prevention, an agency of the United States Government. The U.S.
Government has certain rights in this invention.
Claims
1. An isolated nucleic acid probe of 20 to 50 nucleotides in length
for identifying an Enterobacteriaceae species in a sample, wherein
the probe is at least 90% homologous to the 5' region of the
gyraseA gene of an Enterobacteriaceae species selected from the
group consisting of Citrobacter freundii (SEQ ID NO: 2),
Enterobacter aerogenes (SEQ ID NO: 3), Enterobacter cloacae (SEQ ID
NO: 4), Klebsiella oxytoca (SEQ ID NO: 5), Klebsiella pneumoniae
(SEQ ID NO: 6), Providencia stuartii (SEQ ID NO: 7), Serratia
marcescens (SEQ ID NO: 8), a complementary sequence of anone of SEQ
ID NOS: 2-8, but is not at least 90% homologous to the to the 5'
region of the gyraseA gene of. Escherichia coli (SEQ ID NO: 1) or
the complementary sequence thereof.
2. The isolated nucleic acid probe of claim 1, comprising the
nucleotide sequence set forth as one of SEQ ID NO: 18, SEQ ID NO:
19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or
SEQ ID NO: 24, or a complementary sequence thereof.
3. The isolated nucleic acid probe of claim 1, length for
identifying an Enterobacteriaceae species in a sample, wherein the
probe is at least 95% homologous to the 5' region of the gyraseA
gene of Citrobacter freundii (SEQ ID NO: 2), Enterobacter aerogenes
(SEQ ID NO: 3), Enterobacter cloacae (SEQ ID NO: 4), Klebsiella
oxytoca (SEQ ID NO: 5), Klebsiella pneumoniae (SEQ ID NO: 6),
Providencia stuartii (SEQ ID NO: 7), or Serratia marcescens (SEQ ID
NO: 8), or a complementary sequence thereof, but is not at least
95% homologous to the to the 5' region of the gyraseA gene of.
Escherichia coli (SEQ ID NO: 1) or the complementary sequence
thereof.
4. The isolated nuclear acid probe of claim 1, wherein the probe is
at least 100% homologous to the 5' region of the gyraseA gene of
Citrobacter freundii (SEQ ID NO: 2), Enterobacter aerogenes (SEQ ID
NO: 3), Enterobacter cloacae (SEQ ID NO: 4), Klebsiella oxytoca
(SEQ ID NO: 5), Klebsiella pneumoniae (SEQ ID NO: 6), Providencia
stuartii (SEQ ID NO: 7), or Serratia marcescens (SEQ ID NO: 8), or
a complementary sequence thereof, but is not at least 100%
homologous to the to the 5' region of the gyraseA gene of.
Escherichia coli (SEQ ID NO: 1) or the complementary sequence
thereof.
5. A method of identifying in a sample an Enterobacteriaceae
species selected from the group consisting of Escherichia coli,
Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae,
Klebsiella oxytoca, Klebsiella pneumoniae, Providencia stuartii and
Serratia marcescens, comprising: contacting the sample with a
nucleic acid probe of claim 1; and detecting the presence of
hybridization with a nucleic acid indicating the presence of the
respective species; thereby identifying an Enterobacteriaceae
species selected from the group consisting of Escherichia coli,
Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae,
Klebsiella oxytoca, Klebsiella pneumoniae, Providencia stuartii and
Serratia marcescens in the sample.
6. An isolated nucleic acid probe of 20 to 50 nucleotides in length
capable of determining quinolone resistance status of an
Enterobacteriaceae species selected from the group consisting of
Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae,
Klebsiella oxytoca, Klebsiella pneumoniae, Providencia stuartii and
Serratia marcescens in a sample, wherein the probe hybridizes to
the 5' region of the gyraseA gene of Citrobacter freundii (SEQ ID
NO: 2), Enterobacter aerogenes (SEQ ID NO: 3), Enterobacter cloacae
(SEQ ID NO: 4), Klebsiella oxytoca (SEQ ID NO: 5), Klebsiella
pneumoniae (SEQ ID NO: 6), Providencia stuartii (SEQ ID NO: 7), or
Serratia marcescens (SEQ ID NO: 8), or a fully complementary
sequence thereof, and wherein a nucleic acid a quinolone
susceptible strain hybridizes to the probe, and wherein a nucleic
acid a quinolone resistant strain has a one or more base pair
mismatch with the probe.
7. The probe of claim 6, comprising the nucleotide sequence set
forth as one of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID
NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33, or a
complementary sequence thereof.
8. The probe of claim 6, wherein the probe is labeled.
9. A method for determining the quinolone resistance of an
Enterobacteriaceae species selected from the group consisting of
Escherichia coli Citrobacter freundii, Enterobacter aerogenes,
Enterobacter cloacae, Klebsiella oxytoca, Klebsiella Pneumoniae,
Providencia stuartii and Serratia marcescens in a sample,
comprising contacting the sample with a nucleic acid probe, wherein
the probe selectively hybridizes to a nucleic acid sequence set
forth as one of SEQ ID NOs: 9-16, or a complementary sequence
thereof, respectively, the presence of hybridization with a nucleic
acid indicating the quinolone susceptibility of the respective
species.
10. The method of determining the quinolone resistance status of an
Enterobacteriaceae species of claim 9, comprising contacting the
sample with a nucleic acid probe, wherein the probe selectively
hybridizes to a nucleic acid of SEQ ID NO: 9, or a complementary
sequence thereof, the presence of hybridization indicating
quinolone resistance of the Escherichia coli in the sample.
11. The method of determining the quinolone resistance status of an
Enterobacteriaceae species of claim 9, wherein hybridization of the
probe to the nucleic acid sequence of SEQ ID NOs: 1-9 indicates
that the Enterobacteriaceae species is susceptible to quinolone and
a one or more base pair mismatch of the probe to the nucleic acid
sequence of one or more of SEQ ID NOs: 1-9 indicates that
Enterobacteriaceae species is resistant to quinolone.
12. The method of claim 9, wherein the probe is from about 10 to 50
nucleotides in length.
13. The method of claim 9, wherein the probe consists of the
nucleic acid sequence set forth as one of SEQ ID NOs: 25-33.
14. The method of claim 10, wherein the probe selectively
hybridizes to nucleotides 25 to 613 of SEQ ID NO: 9.
15. The method of claim 10, wherein the probe selectively
hybridizes to nucleotides 199 to 318 of SEQ ID NO:9, or a
complementary sequence thereof.
16. The method of claim 10, wherein the probe selectively
hybridizes to nucleotides 239 to 663 of SEQ ID NO: 9, or a
complementary sequence thereof.
17. The method of claim 9, wherein the probe is about 25
nucleotides in length.
18. The method of claim 9, wherein the method comprises the use of
a polymerase chain reaction (PCR), ligase chain reaction, or a
nucleotide array.
19. The method of claim 9, wherein the probe is labeled.
20. The method of claim 10, wherein the nucleic acid sequence set
forth as SEQ ID NO: 9 is amplified prior to combining the sample
with the nucleic acid probe.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. patent application Ser. No.
10/798,827, filed Mar. 10, 2004, which is a continuation of U.S.
patent application Ser. No. 09/647,563, filed Jan. 16, 2001, issued
as U.S. Pat. No. 6,706,475. U.S. patent application Ser. No.
09/647,563 is a .sctn. 371 U.S. national phase of PCT/US99/06963,
filed Mar. 30, 1999, which was published in English under PCT
Article 21(2), and which in turn claims the benefit of U.S.
Provisional Patent Application No. 60/080,375, filed Apr. 1, 1998.
U.S. patent application Ser. No. 10/798,827 is incorporated by
reference herein in its entirety.
TECHNICAL FIELD OF THE INVENTION
[0003] This invention relates in general to the field of diagnostic
microbiology. In particular, the invention relates to the
species-specific detection of Enterobacteriaceae.
BACKGROUND OF THE INVENTION
[0004] Enterobacteriaceae is a family of closely related,
Gram-negative organisms associated with gastrointestinal diseases
and a wide range of opportunistic infections. They are leading
causes of bacteremia and urinary tract infections and are
associated with wound infections, pneumonia, meningitis, and
various gastrointestinal disorders. (Farmer, J. J., III.
Enterobacteriaceae: Introduction and Identification. in Murray, P.
R., et al., Manual of Clinical Microbiology, Washington, D.C., ASM
Press, 6th (32): 438-449 (1998)). Many of these infections are life
threatening and are often nosocomial (hospital-acquired)
infections. (Schaberg et al., The Am. J. Med., 91:72s-75s (1991)
and CDC NNIS System Report Am. J. Infect. Control., 24:380-388
(1996)).
[0005] Conventional methods for isolation and identification of
these organisms include growth on selective and/or differential
media followed by biochemical tests of the isolated organism. Total
incubation times require 24-48 hours. Slow-growing or fastidious
strains require-extended incubation times. An additional 18-24
hours is required for susceptibility testing, usually by disk
diffusion or broth dilution. More recently, the identification of
bacteria by direct hybridization of probes to bacterial genes or by
detection of amplified genes has proven to be more time
efficient.
[0006] Quinolones are broad-spectrum antibacterial agents effective
in the treatment of a wide range of infections, particularly those
caused by Gram-negative pathogens. (Stein, Clin. Infect. Diseases,
23(Suppl 1):S19-24 (1996) and Maxwell, J. Antimicrob. Chemother.,
30:409-416 (1992)). For example, nalidixic acid is a
first-generation quinolone. Ciprofloxacin is an example of a second
generation quinolone, which is also a fluoroquinolone. Sparfloxacin
is an example of a third generation quinolone, which is also a
fluoroquinolone. As used herein, the term "quinolone" is intended
to include this entire spectrum of antibacterial agents, including
the fluoroquinolones. This class of antibiotics has many
advantages, including oral administration with therapeutic levels
attained in most tissues and body fluids, and few drawbacks. As a
result, indiscriminate use has led to the currently increasing
incidence of quinolone/fluoroquinolone resistance. Hooper, Adv.
Expmtl. Medicine and Biology, 390:49-57 (1995). Mechanisms of
resistance to quinolones include alterations in DNA gyrase and/or
topoisomerase IV and decreased intracellular accumulation of the
antibiotic due to alterations in membrane proteins. (Hooper et al.,
Antimicrob. Agents Chemother., 36:1151-1154 (1992)).
[0007] The primary target of quinolones, including the
fluoroquinolones, in Gram-negative bacteria is DNA gyrase, a type
II topoisomerase required for DNA replication and transcription.
(Cambau et al., Drugs, 45(Suppl. 3):15-23 (1993) and Deguchi et
al., J. Antimicrob. Chemother., 40:543-549 (1997)). DNA gyrase,
composed of two A subunits and two B subunits, is encoded by the
gyrA and gyrB genes. Resistance to quinolones has been shown to be
associated most frequently with alterations in gyrA. (Yoshida et
al., Antimicrob. Agents Chemother. 34:1271-1272 (1990)). These
mutations are localized at the 5' end of the gene (nucleotides
199-318 in the E. coli gene sequence) in an area designated as the
quinolone resistance-determining region, or QRDR, located near the
active site of the enzyme, Tyr-122. (Hooper, Adv. Expmtl. Medicine
and Biology, 390:49-57 (1995)).
[0008] Previous studies of fluoroquinolone-resistant strains of
Escherichia coli, Citrobacter freundii, Serratia marcescens and
Enterobacter cloacae have revealed that codons 81, 83, and 87 of
gyrA are the sites most frequently mutated in Gram-negative
organisms. (Nishino et al., FEMS Microbiology Letters, 154:409-414
(1997), and Kim et al., Antimicrob. Agents Chemother., 42:190-193
(1998)). However, the association of gyrA mutations with
fluoroquinolone resistance in Enterobacter aerogenes, Klebsiella
oxytoca, and Providencia stuartii has not been established.
[0009] Previous publications have referred to the use of gyrA
sequences to identify species within a single genus, such as
Husmann et al., J. Clin. Microbiol., 35(9):2398-2400 (1997) for
Campylobacters, and Guillemin et al., Antimicrob. Agents Chemo.,
39(9):2145-2149 (1995) for Mycobacterium. The complete gene
sequences of DNA gyrase A has previously been published for
Escherichia coli (Swanberg, et al., J. Mol. Biol., 197:729-736
(1987)) and Serratia marcescens (Kim et al., Antimicrob. Agents
Chemother., 42:190-193 (1998)). Fragments of gyrA including the
QRDR have been published for Enterobacter cloacae (Deguchi, J.
Antimicrob. Chemother. 40:543-549 (1997)) and Citobacter freundii
(Nishino et al., FEMS Microbiology Letters, 154:409-414 (1997)).
Additionally, the putative gyrA sequence for Klebsiella pneumoniae
was published (Dimri et al., Nucleic Acids Research, 18:151-156
(1990)), however, the present invention demonstrates that the most
likely organism used in that work was Klebsiella oxytoca.
[0010] The prior art has not provided enough information about
different Enterobacteriaceae to develop probes capable of
distinguishing between as many species as desirable, nor for
determining the quinolone resistance-status of the species. It
would be desirable to characterize additional gyrA genes and
mutations from quinolone-resistant Enterobacteriaceae for
species-specific identification and quinolone resistance
determination using oligonucleotide probes.
SUMMARY OF THE INVENTION
[0011] The present invention relates to oligonucleotide probes for
detecting Enterobacteriaceae species. Unique gyrA coding regions
permit the development of probes specific for identifying eight
different species: Escherichia coli, Citrobacter freundii,
Enterobacter aerogenes, Enterobacter cloacae, Klebsiella oxytoca,
Klebsiella pneumoniae, Providencia stuartii and Serratia
marcescens. The invention thereby provides methods for the
species-specific identification of these Enterobacteriaceae in a
sample, and detection and diagnosis of Enterobacteriaceae infection
in a subject.
[0012] Furthermore, the described unique DNA sequences from the 5'
end of gyrA, within or flanking the quinolone
resistance-determining region, permit the development of probes
specific for determining the quinolone-resistant status of eight
different species: Escherichia coli, Citrobacter freundii,
Enterobacter aerogenes, Enterobacter cloacae, Klebsiella oxytoca,
Klebsiella pneumoniae, Providencia stuartii and Serratia
marcescens. The invention thereby provides methods for the
species-specific identification of these quinolone-resistant
Enterobacteriaceae, and detection and diagnosis of
quinolone-resistant Enterobacteriaceae infection in a subject.
[0013] Therefore, it is an object of the invention to provide
improved materials and methods for detecting and differentiating
Enterobacteriaceae species and/or quinolone resistance in the
clinical laboratory and research settings.
[0014] These and other objects, features and advantages of the
present invention will become apparent after a review of the
following detailed description of the disclosed embodiments and the
appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIGS. 1A and 1B show the nucleic acid sequence (SEQ ID
NOS:1-8) alignments for a portion of the gyrA gene in Escherichia
coli (EC), Citrobacter freundii (CF), Enterobacter aerogenes (EA),
Enterobacter cloacae (ECL), Klebsiella oxytoca (KO), Klebsiella
pneumoniae (KP), Providencia stuartii (PS) and Serratia marcescens
(SM).
[0016] FIG. 2 shows the DNA sequence (SEQ ID NOS:9-16) similarity
of the quinolone resistance-determining region (QRDR) in
Escherichia coli, Citrobacter freundii, Enterobacter aerogenes,
Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae,
Providencia stuartii and Serratia marcescens.
[0017] FIG. 3 shows the deduced amino acid sequences (SEQ ID
NOS:36-43) of the QRDR for Escherichia coli, Citobacter freundii,
Enterobacter aerogenes, Enterobacter cloacae, Klebsiella oxytoca,
Klebsiella pneumoniae, Providencia stuartii, and Serratia
marcescens.
[0018] FIGS. 4A and 4B show the alterations in GyrA amino acid
sequences and susceptibilities of quinolone resistant clinical
isolates of Escherichia coli, Citobacter freundii, Enterobacter
aerogenes, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella
pneumoniae, Providencia stuartii, and Serratia marcescens.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention provides a simple, rapid and useful
method for differentiating Enterobacteriaceae species and
determining their quinolone-resistance status. This invention
provides materials and methods to apply the species-specific probes
to isolated DNA from host samples for an in vitro diagnosis of
Enterobacteriaceae infection.
[0020] The present invention provides the nucleic acid sequences of
conserved and unique regions of the gyrA gene of the following
species of the Family Enterobacteriaceae: Escherichia coli,
Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae,
Klebsiella oxytoca, Klebsiella pneumoniae, Providencia stuartii and
Serratia marcescens. The present invention provides the nucleic
acid sequences of the quinolone resistance-determining region
(QRDR) and surrounding regions of gyrA of each species listed
above.
[0021] DNA sequence analyses revealed that gyrA is unique to each
species and highly conserved within the species. However, the gyrA
mutations resulting in amino acid substitutions which confer
quinolone resistance vary in number, type, and position depending
on the species. The invention demonstrates that these unique
sequences can be used for identification of enteric organisms
(genus and species) as well as detection of quinolone resistance
within a given species. In addition, comparisons of
Enterobacteriaceae gyrA with gyrA sequences from bacteria not
closely related to Enterobacteriaceae species suggest that gyrA
sequences are unique for all bacterial species and may be used for
identification of any species.
[0022] The invention provides unique, isolated nucleic acids
containing regions of specificity for eight different members of
the Family Enterobacteriaceae. These nucleic acids are from the
gyrA gene of the Enterobacteriaceae genome. In particular, the
invention provides isolated nucleic acids from Escherichia coli
(SEQ ID NO:1), Citrobacter freundii (SEQ ID NO:2), Enterobacter
aerogenes (SEQ ID NO:3), Enterobacter cloacae (SEQ ID NO:4),
Klebsiella oxytoca (SEQ ID NO:5), Klebsiella pneumoniae (SEQ ID
NO:6), Providencia stuartii (SEQ ID NO:7) and Serratia marcescens
(SEQ ID NO:8). These sequences can be used to identify and
distinguish the respective species of Enterobacteriaceae. FIGS. 1A
and 1B show the nucleic acids of SEQ ID NOS:1-8. The sequences
correspond to nucleotides #25-613, based on the E. coli gyrA
sequence numbers of Swanberg et al., J. Mol. Biol., 197:729-736
(1987).
[0023] The invention also provides unique, isolated nucleic acids
from the quinolone resistance-determining region of Escherichia
coli (SEQ ID NO:9), Citrobacter freundii (SEQ ID NO:10),
Enterobacter aerogenes (SEQ ID NO:11), Enterobacter cloacae (SEQ ID
NO:12), Klebsiella oxytoca (SEQ ID NO:13), Klebsiella pneumoniae
(SEQ ID NO:14), Providencia stuartii (SEQ ID NO:15) and Serratia
marcescens (SEQ ID NO:16). These sequences can be used to determine
the quinolone resistance status of each species. The QRDR nucleic
acids are shown in FIG. 2.
[0024] Furthermore, the invention provides specific examples of
isolated nucleic acid probes derived from the above nucleic acid
sequences which may be used as species-specific identifiers of
Escherichia coli (SEQ ID NO:17), Citrobacter freundii (SEQ ID
NO:18), Enterobacter aerogenes (SEQ ID NO:19), Enterobacter cloacae
(SEQ ID NO:20), Klebsiella oxytoca (SEQ ID NO:21), Klebsiella
pneumoniae (SEQ ID NO:22), Providencia stuartii (SEQ ID NO:23) and
Serratia marcescens (SEQ ID NO:24).
[0025] The invention also provides specific examples of isolated
nucleic acid probes derived from the QRDR of the above nucleic acid
sequences which may be used as determinants of quinolone resistance
for Escherichia coli (SEQ ID NOS:25 and 26), Citrobacter freundii
(SEQ ID NO:27), Enterobacter aerogenes (SEQ ID NO:28), Enterobacter
cloacae (SEQ ID NO:29), Klebsiella oxytoca (SEQ ID NO:30),
Klebsiella pneumoniae (SEQ ID NO:31), Providencia stuartii (SEQ ID
NO:32) and Serratia marcescens (SEQ ID NO:33).
[0026] Such probes can be used to selectively hybridize with
samples containing nucleic acids from species of
Enterobacteriaceae. The probes can be incorporated into
hybridization assays using polymerase chain reaction, ligase chain
reaction, or oligonucleotide arrays on chips or membranes, for
example. Additional probes can routinely be derived from the
sequences given in SEQ ID NOs:1-8, which are specific for
identifying the respective species or for determining quinolone
resistance. Therefore, the probes shown in SEQ ID NOs:17-24 and
25-33 are only provided as examples of the species-specific probes
or quinolone resistance-determining probes, respectively, that can
be derived from SEQ ID NOs:1-8.
[0027] By "isolated" is meant nucleic acid free from at least some
of the components with which it naturally occurs. By "selective" or
"selectively" is meant a sequence that does not hybridize with
other nucleic acids to prevent adequate determination of an
Enterobacteriaceae species or quinolone resistance, depending upon
the intended result. As used herein to describe nucleic acids, the
term "selectively hybridizes" excludes the occasional randomly
hybridizing nucleic acids, and thus has the same meaning as
"specifically hybridizing".
[0028] A hybridizing nucleic acid should have at least 70%
complementarity with the segment of the nucleic acid to which it
hybridizes. The selectively hybridizing nucleic acids of the
invention can have at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, and
99% complementarity with the segment of the sequence to which it
hybridizes. The exemplary probes shown in SEQ ID NOs:17-24 and
25-33 are designed to have 100% hybridization with the target
DNA.
[0029] The invention contemplates sequences, probes and primers
which selectively hybridize to the complementary, or opposite,
strand of nucleic acid as those specifically provided herein.
Specific hybridization with nucleic acid can occur with minor
modifications or substitutions in the nucleic acid, so long as
functional species-specific or quinolone resistance determining
hybridization capability is maintained. By "probe" is meant a
nucleic acid sequence that can be used as a probe or primer for
selective hybridization with complementary nucleic acid sequences
for their detection or amplification, which probe can vary in
length from about 5 to 100 nucleotides, or preferably from about 10
to 50 nucleotides, or most preferably about 25 nucleotides. The
invention provides isolated nucleic acids that selectively
hybridize with the species-specific nucleic acids under stringent
conditions. See generally, Maniatis, et al., Molecular cloning: a
laboratory manual. Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y., (1982) latest edition.
[0030] Molecular biology techniques permit the rapid detection of
hybridization, such as through confocal laser microscopy and high
density oligonucleotide arrays and chips. See, Kozal et al., Nat.
Med., 2(7): 753-759 (1996), Schummer et al., Biotech., 23:1087-1092
(1997) or Lockhart et al., Nat. Biotech. 14:1675-1680 (1996).
Another example of a detection format is the use of controlled
electric fields that permit the rapid determination of single base
mismatches, as described in Sosnowski et al., Proc. Natl. Acad.
Sci. USA, 94:1119-1123 (1997). The invention contemplates the use
of the disclosed nucleic acid sequences and probes derived
therefrom with these currently available techniques and those new
techniques discovered in the future.
[0031] If used as primers, the invention provides compositions
including at least two oligonucleotides (i.e., nucleic acids) that
hybridize with different regions of DNA so as to amplify the
desired region between the two primers. Depending on the length of
the probe or primer, the target region can range between 70%
complementary bases and full complementarity and still hybridize
under stringent conditions. For example, for the purpose of
diagnosing the presence of the Enterobacteriaceae in a clinical
sample, the degree of complementarity between the nucleic acid
(probe or primer) and the target sequence to which it hybridizes
(e.g., Enterobacteriaceae DNA from a sample) is at least enough to
distinguish hybridization with a non-target nucleic acid from other
Enterobacteriaceae. The invention provides examples of nucleic
acids having sequences unique to Enterobacteriaceae such that the
degree of complementarity required to distinguish selectively
hybridizing from nonselectively hybridizing nucleic acids under
stringent conditions can be clearly determined for each nucleic
acid.
[0032] Alternatively, the nucleic acid probes can be designed to
have homology with nucleotide sequences present in more than one
species of Enterobacteriaceae. Such a nucleic acid probe can be
used to selectively identify a group of Enterobacteriaceae species.
Additionally, the invention provides that the nucleic acids can be
used to differentiate Enterobacteriaceae species in general from
other species. Such a determination is clinically significant,
since therapies for these infections differ.
[0033] The invention further provides methods of using the nucleic
acids to detect and identify the presence of Enterobacteriaceae, or
particular species thereof. The methods involve the steps of
obtaining a sample suspected of containing Enterobacteriaceae. The
sample, such as blood, urine, lung lavage fluids, spinal fluid,
bone marrow aspiration, vaginal mucosa, tissues, etc., may be taken
from an individual, or taken from the environment. The
Enterobacteriaceae cells in the sample can then be lysed, and the
DNA released (or made accessible) for hybridization with
oligonucleotide probes.
[0034] The DNA sample is preferably amplified prior to
hybridization using primers derived from the gyrA regions of the
Enterobacteriaceae DNA that are designed to amplify several
species. Examples of such primers are shown below as GYRA6 (SEQ ID
NO:34) and or GYRA631R (SEQ ID NO:35). Detection of and/or the
determination of quinolone resistance in the target species of
Enterobacteriaceae is achieved by hybridizing the amplified gyrA
DNA with an Enterobacteriaceae species-specific probe that
selectively hybridizes with the DNA. Detection of hybridization is
indicative of the presence of the particular species of
Enterobacteriaceae or quinolone resistance, depending upon the
probe. In the case where the species of Enterobacteriaceae is
known, for example through previous hybridization with a
species-specific identifying probe of SEQ ID NOS:17-24, the lack of
subsequent hybridization with a species-specific quinolone
resistance-determining probe of SEQ ID NOS:25-33 is indicative of
quinolone resistance in the sample.
[0035] Preferably, detection of nucleic acid hybridization can be
facilitated by the use of reporter or detection moieties. For
example, the species-specific probes can be labeled with
digoxigenin, and a universal-Enterobacteriaceae species probe can
be labeled with biotin and used in a streptavidin-coated microtiter
plate assay. Other examples of detectable moieties include
radioactive labeling, enzyme labeling, and fluorescent
labeling.
[0036] The invention further contemplates a kit containing one or
more species-specific and/or quinolone resistance-determining
probes, which can be used for the identification and/or quinolone
resistance determination of particular Enterobacteriaceae species.
Such a kit can also contain the appropriate reagents for
hybridizing the probe to the sample and detecting bound probe. The
invention may be further demonstrated by the following non-limiting
examples.
EXAMPLES
Example 1
[0037] In this Example, the DNA sequence of the gyrA was determined
for eight species of Enterobacteriaceae. Oligonucleotide primers
were designed from conserved gyrA gene sequences flanking the QRDR
and used to amplify and sequence the 5' region of gyrA from ATCC
type strains and fluoroquinolone-resistant clinical isolates. The
nucleotide and the inferred amino acid sequences were aligned and
compared.
[0038] The QRDR sequences from 60 clinical isolates with decreased
fluoroquinolone susceptibilities were analyzed for alterations
associated with fluoroquinolone resistance. The primer sequences at
the 3' and 5' ends have been removed leaving nucleotides #25-613,
based on the E. coli gyrA sequence numbers of Swanberg et al., J.
Mol. Biol., 197:729-736 (1987). The organisms, abbreviations and
ATCC type strain designation numbers are as follows.
EC=Escherichia coli (E. coli) ATCC 11775 CF=Citrobacter freundii
(C. freundii) ATCC 8090 EA=Enterobacter aerogenes (E. aerogenes)
ATCC 13048 ECL=Enterobacter cloacae (E. cloacae) ATCC 13047
KO=Klebsiella oxytoca (K. oxytoca) ATCC 13182 KP=Klebsiella
pneumoniae (K. pneumoniae) ATCC 13883 PS=Providencia stuartii (P.
stuartii) ATCC 29914 SM=Serratia marcescens (S. marcescens) ATCC
13880
Amplification of gyrA
Bacterial Strains and Determination of Antibiotic Susceptibility
Profiles.
[0039] Type strains of Enterobacteriaceae were from American Type
Culture Collection (ATCC). Fluoroquinolone resistant and
susceptible clinical isolates were selected from the Intensive Care
Antimicrobial Resistance Epidemiology (ICARE) study, collected from
39 hospitals across the U.S. between June, 1994 and April 1997
(Archibald et al., CID, 24(2):211-215 (1997)). ICARE isolates were
screened to exclude duplicate strains from the same patient.
[0040] Minimal inhibitory concentrations (MICs) were determined by
the broth microdilution method with cation-adjusted Mueller-Hinton
broth according to the methods of the National Committee for
Clinical Laboratory Standards (NCCLS M7-A4 (1997)). Ciprofloxacin
was purchased from Bayer Corporation (West Haven, Conn.), ofloxacin
and nalidixic acid were from Sigma (St. Louis, Mo.) and
sparfloxacin was from Rhone-Poulenc Rorer (Collegeville, Pa.).
Amplification of 5' Region of gyrA.
[0041] Oligonucleotide primers were designed based on homologous
regions of gyrA sequences in E. coli (Swanberg et al., J. Mol.
Biol., 1987. 197:729-736) and K. oxytoca (published by Dimri et
al., Nuc. Acids Res., 1990. 18:(1):151-156 as K. pneumonia), as
follows:
TABLE-US-00001 GYRA6 5'-CGACCTTGCGAGAGAAAT-3' (SEQ ID NO:34)
GYRA631R 5'-GTTCCATCAGCCCTTCAA-3' (SEQ ID NO:35)
[0042] Primer GYRA6 corresponds to nucleotides 6 to 23 and primer
GYRA631R is complementary to nucleotides 610 to 631 of the E. coli
gyrA sequence.
[0043] DNA fragments were amplified from chromosomal DNA in cell
lysates. Amplifications were carried out in a GeneAmp 9600 PCR
System (Perkin-Elmer, Applied Biosystems Division, Foster City,
Calif.) in 50 .mu.l volume containing 50 .mu.mol of each primer,
200 .mu.M deoxynucleoside triphosphates, 10 ul cell lysate
containing approximately 100 ng template DNA, 1.times. reaction
buffer with 1.5 mM MgCl.sub.2 and 1 U native Taq polymerase (Perkin
Elmer). An initial 4 minute period of denaturation at 94.degree. C.
was followed by 30 cycles including: denaturation for 1 minute at
94.degree. C., annealing for 30 seconds at 55.degree. C., extending
for 45 seconds at 72.degree. C., followed by a final cycle of
72.degree. C. for 10 minutes. Amplification products were
visualized by agarose gel electrophoresis and ethidium bromide
staining to determine specificity and size of gene fragments. PCR
products were purified on QIAquick spin columns (QIAGEN,
Chatsworth, Calif.) and sequenced with the ABI Prism Dye Terminator
or dRhodomine Terminator Cycle Sequencing Kit and an ABI 377
automated sequencer (Perkin Elmer). To eliminate errors due to
amplification artifacts, the forward and reverse sequences of each
QRDR were determined using products from independent PCR reactions.
The GCG (Genetics Computer Group, Madison, Wis.) analyses programs
were used for the construction of DNA and amino acid sequence
alignments.
[0044] The resultant sequences of the gyrA regions for Escherichia
coli, Citrobacter freundii, Enterobacter aerogenes, Enterobacter
cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, Providencia
stuartii and Serratia marcescens are shown below in Table 1 and in
FIGS. 1A-1B. The sequences provided correspond to nucleotide
positions 25 to 613 of the gyrA regions for Escherichia coli.
TABLE-US-00002 TABLE 1 Gyrase A 5' Region Sequences Escherichia
coli ACACCGGT CAACATTGAG GAAGAGCTGA AGAGCTCCTA TCTGGATTAT
GCGATGTCGG TCATTGTTGG CCGTGCGCTG CCAGATGTCC GAGATGGCCT GAAGCCGGTA
CACCGTCGCG TACTTTACGC CATGAACGTA CTAGGCAATG ACTGGAACAA AGCCTATAAA
AAATCTGCCC GTGTCGTTGG TGACGTAATG GGTAAATACC ATCCCCATGG TGACTCGGCG
GTTTATGACA CGATCGTCCG TATGGCGCAG CCATTCTCGC TGCGTTACAT GCTGGTAGAC
GGTCAGGGTA ACTTCGGTTC CATCGACGGC GACTCTGCGG CGGCAATGCG TTATACGGAA
ATCCGTCTGG CGAAAATTGC CCATGAACTG ATGGCTGATC TCGAAAAAGA GACGGTCGAT
TTCGTTGATA ACTATGACGG TACGGAAAAA ATTCCGGACG TCATGCCAAC CAAAATTCCT
AACCTGCTGG TGAACGGTTC TTCCGGTATC GCCGTAGGTA TGGCAACCAA CATCCCGCCG
CACAACCTGA CGGAAGTCAT CAACGGTTGT CTGGCGTATA TCGATGATGA AGACATCAGC A
(SEQ ID NO:1) Citrobacter freundii ACACCGGT CAACATTGAG GAAGAGCTGA
AGAGCTCCTA TCTGGATTAT GCGATGTCGG TCATTGTTGG CCGTGCGCTG CCAGACGTCC
GAGATGGCCT GAAGCCGGTT CACCGTCGCG TACTTTACGC CATGAACGTA TTGGGCAACG
ACTGGAATAA AGCCTATAAA AAATCTGCCC GTGTCGTTGG TGACGTAATC GGTAAATACC
ACCCTCATGG TGATACCGCC GTTTACGACA CCATTGTTCG TATGGCGCAG CCATTCTCCT
TGCGTTACAT GCTGGTAGAT GGTCAGGGTA ACTTTGGTTC TGTCGATGGC GACTCCGCAG
CGGCGATGCG TTATACGGAA ATCCGTATGT CGAAAATCGC CCATGAGCTG ATGGCTGACG
TGGAAAAAGA AACGGTTGAT TTCGTCGATA ACTACGACGG CACCGAACAA ATTCCTGACG
TCATGCCGAC CAAAATTCCT AACCTGCTGG TGAACGGTTC GTCCGGTATC GCGGTAGGTA
TGGCGACCAA CATTCCGCCG CACAACCTGA CTGAAGTGAT CAACGGCTGT CTGGCATATA
TTGACGATGA AGACATCAGC A (SEQ ID NO:2) Enterobacter aerogenes
ACACGGGT CAACATTGAG GAAGAGCTGA AAAGCTCGTA TCTGGATTAT GCGATGTCGG
TCATTGTTGG CCGTGCGCTG CCGGATGTCC GAGATGGCCT GAAGCCGGTA CACCGTCGCG
TACTATACGC CATGAACGTA TTGGGCAATG ACTGGAACAA AGCCTATAAA AAATCAGCCC
GTGTCGTTGG CGACGTAATC GGTAAATACC ACCCGCATGG TGATACCGCC GTTTATGACA
CCATCGTACG TATGGCGCAG CCGTTCTCCT TGCGTTATAT GCTGGTCGAT GGCCAGGGTA
ACTTTGGTTC TGTCGATGGC GACTCCGCTG CAGCGATGCG TTATACGGAA ATCCGTATGT
CGAAGATCGC TCATGAGCTG ATGGCCGATC TCGAAAAAGA GACGGTTGAT TTCGTCGACA
ACTATGACGG CACGGAGAAA ATCCCTGACG TCATGCCGAC AAAAATCCCT AACCTGCTGG
TGAACGGTTC TTCCGGTATC GCCGTAGGTA TGGCGACCAA CATTCCGCCG CATAACCTGA
CGGAAGTTAT CAACGGCTGC CTGGCATACG TTGATAACGA AGACATCAGC A (SEQ ID
NO:3) Enterobacter cloacae ACACCGGTTA ACATCGAGGA AGAGCTGAAG
AGCTCCTATC TGGACTATGC GATGTCGGTC ATTGTTGGCC GTGCGCTGCC GGACGTCCGC
GATGGCCTGA AGCCGGTACA CCGTCGCGTA CTATACGCCA TGAACGTATT GGGCAATGAC
TGGAATAAAG CCTACAAAAA ATCTGCCCGT GTCGTTGGTG ACGTAATCGG TAAATACCAT
CCCCATGGTG ATTCCGCGGT GTACGACACC ATCGTTCGTA TGGCGCAGCC TTTCTCGCTG
CGTTACATGC TGGTAGATGG TCAGGGTAAC TTTGGTTCTA TCGACGGCGA CTCCGCCGCG
GCAATGCGTT ATACGGAAAT CCGTCTGGCG AAAATTGCCC ATGAGCTGAT GGCCGACCTG
GAAAAAGAGA CGGTTGATTT CGTTGATAAC TACGATGGCA CGGAAAAAAT TCCTGACGTC
ATGCCAACGA AGATCCCTAA CCTGCTGGTG AACGGTTCGT CCGGTATCGC CGTAGGGATG
GCGACCAACA TTCCGCCGCA CAACATCACC GAAGTGATCA ACGGCTGCCT GGCCTATATC
GACGATGAAG ACATCAGCA (SEQ ID NO:4) Klebsiella oxytoca ACACCGGT
CAACATTGAG GAAGAGCTGA AGAGCTCCTA TCTGGATTAT GCGATGTCGG TCATTGTTGG
CCGTGCGCTG CCGGATGTCC GAGATGGCCT GAAGCCGGTA CACCGTCGCG TACTATACGC
CATGAACGTA TTGGGCAATG ACTGGAACAA AGCCTATAAA AAATCTGCCC GTGTCGTGGG
TGACGTCATC GGTAAATACC ACCCTCATGG TGATACTGCC GTATAGGACA CCATTGTACG
TATGGCGCAG CCATTCTCCC TGCGTTACAT GCTGGTAGAT GGCCAGGGTA ACTTTGGTTC
GGTCGACGGC GACTCCGCCG CAGCGATGCG TTATACGGAA ATCCGTATGT CGAAGATCGC
CCATGAACTG ATGGCCGACC TCGAAAAAGA GACGGTGGAT TTCGTCGATA ACTATGACGG
CACGGAGAAA ATCCCTGACG TTATGCCGAC CAAAATCCCG AACCTGCTAG TCAACGGTTC
GTCCGGTATC GCGGTAGGTA TGGCGACTAA TATTCCGCCG CACAACCTGA CCGAAGTGAT
CAACGGCTGT CTGGCCTACG TTGAAAACGA AGACATCAGC A (SEQ ID NO:5)
Klebsiella pneumoniae ACACCGGT CAACATTGAG GAAGAGCTTA AGAACTCTTA
TCTGGATTAT GCGATGTCGG TCATTGTTGG CCGTGCGCTG CCGGATGTCC GAGATGGCCT
GAAGCCGGTA CACCGTCGCG TACTTTACGC CATGAACGTA TTGGGCAATG ACTGGAACAA
AGCCTATAAA AAATCAGCCC GTGTCGTTGG TGACGTAATC GGTAAATACC ACCCGCACGG
CGACTCCGCG GTATACGACA CCATCGTGCG TATGGCGCAG CCGTTCTCGC TGCGTTACAT
GCTGGTGGAC GGCCAGGGTA ACTTTGGTTC CATCGACGGC GACTCCGCCG CGGCGATGCG
TTATACCGAA ATTCGTCTGG CGAAAATCGC TCATGAGCTG ATGGCCGATC TTGAAAAAGA
GACGGTCGAT TTCGTCGACA ACTATGACGG TACGGAGCGT ATTCCGGACG TCATGCCGAC
CAAAATTCCT AACCTGCTGG TGAACGGCGC CTCCGGGATC GCCGTAGGGA TGGCCACCAA
CATACCGCCA CATAACGTGA CGGAAGTGAT TAACGGCTGT CTGGCGTATG TTGACGATGA
AGACATCAGC A (SEQ ID NO:6) Providencia stuartii ACACCGGT CAATATCGAA
GAAGAACTCA AAAGTTCGTA TTTGGATTAT GCGATGTCCG TTATTGTCGG GCGCGCGCTT
CCAGATGTTC GAGATGGACT GAAGCCAGTACACCGCAGAG TACTGTTTGC GATGAATGTA
TTGGGAAATG ATTGGAATAA ACCCTATAAA AAATCTGCCC GTATAGTCGG GGACGTTATC
GGTAAATACC ATCCACATGG TGATAGCGCT GTTTATGAGA CAATCGTTCG TCTTGCTCAG
CCTTTTTCTA TGCGTTATAT GCTGGTAGAT GGTCAGGGGA ACTTTGGTTC AGTTGACGGA
GATTCCGCAG CTGCAATGCG TTATACGGAA ATCCGTATGG CGAAAATTGC CCATGAAATG
TTAGCGGATC TTGAAAAAGA GACCGTTGAT TTCGTCCCAA ACTATGATGG TACAGAGCAA
ATCCCTGAAG TTATGCCTAC GAAAATCCCT AACCTATTGG TTAATGGTTC GTCAGGTATT
GCTGTTGGGA TGGCAACGAA CATTCCTCCA CACAACCTAG GGGAAGTGAT CAGCGGTTGC
CTTGCTTATA TAGATGATGA AGATATTAGC A (SEQ ID NO:7) Serratia
marcescens ACACCGGT AAACATCGAA GACGAGTTGA AAAACTCGTA TCTGGACTAT
GCGATGTCCG TTATTGTCGG ACGTGCCCTG CCAGATGTTC GTGATGGACT GAAGCCGGTT
CACCGCCGCG TTCTGTACGC GATGAGCGTA TTGGGTAACG ACTGGAATAA ACCATACAAG
AAATCGGCCC GTGTCGTCGG GGACGTGATC GGTAAATATC ACCCGCACGG TGACAGCGCG
GTTTACGACA CTATCGTGCG TATGGCTCAG CCGTTTTCAC TGCGCTACAT GCTGGTGGAC
GGTCAGGGTA ACTTCGGTTC CGTCGACGGC GACTCCGCGG CGGCGATGCG TTATACCGAA
GTGCGCATGT CCAAGATTGC TCACGAACTG TTGGCGGATC TGGAAAAAGA AACCGTCGAC
TTCGTGCCTA ACTATGATGG CACCGAGCAG ATCCCGGCCG TCATGCCGAC CAAGATCCCG
AACCTGCTGG TCAACGGCTC GTCGGGCATC GCCGTGGGCA TGGCTACCAA TATTCCGCCG
CACAACCTGG CGGAAGTCGT CAACGGCTGC CTGGCCTATA TCGACGATGA AAACATCAGC A
(SEQ ID NO:8)
[0045] The QRDR sequences from positions 199 to 318 (relative to E.
coli) are shown below in Table 2.
TABLE-US-00003 TABLE 2 Quinolone Resistance-Determining Region
Sequences Escherichia coli GCCCG TGTCGTTGGT GACGTAATCG GTAAATACCA
TCCCCATGGT GACTCGGCGG TTTATGACAC GATCGTCCGT ATGGCGCAGC CATTCTCGCT
GCGTTACATG CTGGTAGACG GTCAG (SEQ ID NO:9) Citrobacter freundii
GCCCG TGTCGTTGGT GACGTAATCG GTAAATACCA CCCTCATGGT GATACCGCCG
TTTACGACAC CATTGTTCGT ATGGCGCAGC CATTCTCCTT GCGTTACATG CTGGTAGATG
GTCAG (SEQ ID NO:10) Enterobacter aerogenes GC CCGTGTCGTT
GGCGACGTAA TCGGTAAATA CCACCCGCAT GGTGATACCG CCGTTTATGA CACCATCGTA
CGTATGGCGC AGCCGTTCTC CTTGCGTTAT ATGCTGGTCG ATGGCCAG (SEQ ID NO:11)
Enterobacter cloacae GC CCGTGTCGTT GGTGACGTAA TCGGTAAATA CCATCCCCAT
GGTGATTCCG CGGTGTACGA CACCATCGTT CGTATGGCGC AGCCTTTCTC GCTGCGTTAC
ATGCTGGTAG ATGGTCAG (SEQ ID NO:12) Klebsiella oxytoca GCCCGTGTC
GTGGGTGACG TCATCGGTAA ATACCACCCT CATGGTGATA CTGCCGTATA CGACACCATT
GTACGTATGG CGCAGCCATT CTCCCTGCGT TACATGCTGG TAGATGGCCA G (SEQ ID
NO:13) Klebsiella pneumoniae GC CCGTGTCGTT GGTGACGTAA TCGGTAAATA
CCACCCGCAC GGCGACTCCG CGGTATACGA CACCATCGTG CGTATGGCGC AGCCGTTCTC
GCTGCGTTAC ATGCTGGTGG ACGGCCAG (SEQ ID NO:14) Providencia stuartii
GCCCGTATAG TCGGGGACGT TATCGGTAAA TACCATCCAC ATGGTGATAG CGCTGTTTAT
GAGACAATCG TTCGTCTTGC TCAGCCTTTT TCTATGCGTT ATATGCTGGT AGATGGTCAG
(SEQ ID NO:15) Serratia marcescens GCCCGTGTC GTCGGGGACG TGATCGGTAA
ATATCACCCG CACGGTGACA GCGCGGTTTA CGACACTATC GTGCGTATGG CTCAGCCGTT
TTCACTGCGC TACATGCTGG TGGACGGTCA G (SEQ ID NO:16)
[0046] Oligonucleotide primers GYRA6 and GYRA631R successfully
amplified the expected 626 bp DNA fragment from Escherichia coli,
Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae,
Klebsiella oxytoca, Klebsiella pneumoniae, Providencia stuartii and
Serratia marcescens (FIGS. 1A-1B). In additional experiments,
amplification with GYRA6 and GYRA631 produced the expected GYRA
fragment from S. typhimurium (data not shown).
[0047] The PCR products were sequenced and the 120 bp regions of
gyrA known as the QRDR were analyzed. Alignment of the QRDR DNA
sequences of the type strains revealed numerous nucleotide
substitutions when compared with the E. coli sequence (FIG. 2).
Eighty-seven of 120 nucleotides (72.5%) were conserved. Similarity
to the E. coli sequence varied from 93.3% for E. cloacae to 80.8%
for P. stuartii (FIGS. 4A-4B). Significant diversity was noted when
the gyrA QRDR sequences of two species from one genus were aligned.
E. aerogenes and E. cloacae shared 90.5% identity and K. pneumoniae
and K. oxytoca shared 89.3% identity in this region, less
similarity than between several of the different genera.
[0048] The gyrA QRDR sequence of the E. coli type strain (ATCC
11775) was compared with the E. coli K12 gyrA sequence published by
Swanberg and Wang (J. Mol. Biol. 197:729-736 (1997)) and 4
nucleotide differences were detected at positions 255 (C->T),
267 (T->C), 273 (C->T), and 300 (T->C).
[0049] When the QRDR sequence from the K. pneumoniae type strain
was compared with the gyrA gene sequence from K. pneumoniae strain
M5a1 published by Dimri and Das (Nucleic Acids Research, 18:151-156
(1990)), differences were detected in 15 of 120 nucleotides. Of
these 15 nucleotides, only one resulted in an amino acid change. At
nucleotide position 247 a T to A change altered the deduced amino
acid from Ser-83 (ATCC type strain) to Thr (M5a1). When the M5a1
gyrA sequence was compared with that of the K. oxytoca type strain,
only 4 nucleotide differences were detected. In addition, Ser was
consistently found at position 83 in the
fluoroquinolone-susceptible strains of K. pneumoniae and Thr was
consistently found at this position in the K. oxytoca strains
(FIGS. 4A and 4B). These data indicate that the Dimri and Das
sequence of the M5a1 strain most likely was from a strain of K.
oxytoca and not K. pneumoniae.
[0050] In the sequence from the S. marcescens type strain (ATCC
13880), the QRDR was identical to the sequence published by Kim et
al. (ATCC 14756) (Antimicrob. Agents Chemother., 42:190-193
(1998)). One nucleotide difference was found in the flanking region
(nt 321, T to C) with no change in amino acid sequence (data not
shown). The C. freundii QRDR sequence was identical to that of
Nishino et al. (FEMS Microbiology Letters, 154:409-414 (1997)),
however, an additional 393 nucleotides are presented herein.
[0051] The deduced amino acid sequences of the QRDR were highly
conserved (FIG. 3). E. cloacae, K. pneumoniae and S. marcescens
shared identical amino acid sequences with E. coli. In C. freundii,
E. aerogenes and K. oxytoca, one conservative substitution, Ser-83
to Thr was found. Only P. stuartii exhibited more than one amino
acid substitution in this region. In this organism two conservative
changes were detected, Val-69 to Ile and Asp-87 to Glu. In
addition, the Leu-92 and Met-98 positions were reversed when
compared with the amino acid sequences of other members of the
Enterobacteriaceae family included in this study. The Glu at
position 87 is typical for gyrA in Gram-positive organisms
(Tankovic et al., Antimicrob. Agents Chemother., 40:2505-2510
(1996)), but has not previously been described for a Gram-negative
organism.
[0052] After determining the DNA sequence of the QRDR from the
quinolone-susceptible type strains, the 5' region of gyrA in
ciprofloxacin-resistant and -susceptible clinical isolates was
amplified, sequenced, and analyzed for mutations leading to amino
acid changes associated with fluoroquinolone resistance (FIGS. 4A
and 4B). Comparisons of the fluoroquinolone-susceptible type strain
and the resistant clinical isolates of E. coli revealed single
mutations in codon 83 in gyrA associated with low levels of
resistance and double mutations (codons 83 and 87) with high levels
of resistance (.gtoreq.16 ug/ml ciprofloxacin) as previously
described (Vila et al., Antimicrob. Agents Chemother., 38:2477-2479
(1994) and Heisig et al., Antimicrob. Agents Chemother., 37:696-701
(1993)). However, in all other species in this study, high levels
of resistance were found in strains with single as well as double
gyrA mutations. MICs varied significantly among strains with the
same mutation, confirming that factors other than gyrA are involved
in determining the level of resistance to fluoroquinolones (Everett
et al., Antimicrob. Agents Chemother., 40:2380-2386 (1996) and
Piddock, Drugs, 49 (Suppl):29-35 (1995)).
[0053] All clinical isolates of C. freundii with reduced
susceptibility to fluoroquinolones were found to have Thr-83 to Ile
mutations, resulting from C-to-T substitutions at nucleotide
position 248. Two isolates also displayed alterations of Asp-87 to
Gly. However, as noted for isolate C. freundii 9023 (FIGS. 4A and
4B), the presence of a double mutation was not required for
high-level resistance (MICs of 16 .mu.g/ml ciprofloxacin). The
nucleotide substitutions in codon 83 of E. aerogenes gyrA (Thr-83
to Ile) were identical to those of C. freundii. No double mutations
were detected in gyrA from 7 strains of E. aerogenes with reduced
levels of susceptibility to fluoroquinolones. However, MICs of
isolates with the single mutation ranged from 2-16 .mu.g/ml
ciprofloxacin.
[0054] Clinical isolates of E. cloacae exhibited numerous
substitutions resulting in Ser-83 changes to Phe, Tyr, or Ile with
no single amino acid change associated with either low level or
high level resistance. There was no alteration of Ser-83 in the
clinical isolate E. cloacae 1524 which had a marginal decrease in
susceptibility to the fluoroquinolones. However, Asp-87 was changed
to Asn. This alteration, found as part of a double mutation in E.
cloacae 1224, may contribute to high-level resistance if additional
changes occur in the QRDR of E. cloacae 1524.
[0055] K. pneumoniae isolates exhibited either single or double
mutations involving Ser-83 and Asp-87, and ciprofloxacin MICs
ranged from 1-16 .mu.g/ml. Again, double mutations were not
required for high-level resistance and no specific mutation (Ser-83
to Phe or Tyr) was associated with low or high levels of
fluoroquinolone resistance.
[0056] K. oxytoca mutations were confined to the Thr-83 codon and
were consistent C-to-T substitutions in the second position
resulting in amino acid change to Ile, similar to C. freundii and
E. aerogenes. MICs associated with this alteration ranged from
0.5-16 .mu.g/ml ciprofloxacin.
[0057] Changes in the QRDR of P. stuartii gyrA were also confined
to codon 83, however, the nucleotide substitutions varied. The
single nucleotide substitutions included A-to-C at the first
position or C-to-G at the third position, both resulting in
Ser-to-Arg mutations, or G-to-T in the second position resulting in
Ser-to-Ile mutations. MICs ranged from 2 to 16 .mu.g/ml
ciprofloxacin.
[0058] S. marcescens displayed the greatest diversity in mutations
with Gly-81, Ser-83, or Asp-87 involved. No double mutations were
detected in the QRDR of gyrA from 6 fluoroquinolone-resistant
clinical isolates. An unusual mutation of Gly-81 to Cys was found
in two isolates. However, this mutation has been described in E.
coli (Yoshida et al., Antimicrob. Agents Chemother., 34:1271-1272
(1990)).
[0059] The data in this Example provides for the first time enough
comparative nucleic acid sequence data for the gyrA gene to enable
one to prepare probes that will selectively hybridize to target
nucleic acid to identify the species and/or quinolone resistance of
Escherichia coli, Citrobacter freundii, Enterobacter aerogenes,
Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae,
Providencia stuartii and Serratia marcescens.
Example 2
Development of Probes
Identification of Enterobacteriaceae Species
[0060] Oligonucleotide probes can be selected for species-specific
identification of Enterobacteriaceae in or near the QRDR of gyrA.
The region which includes the codons most often associated with
fluoroquinolone resistance (nucleotides 239-263) was not used for
the reason that if identification were based on one or more
nucleotide changes, the changes associated with resistance would
interfere with identification. Each probe for identification was
selected for maximum difference, and it is recognized that a
smaller region within some probes could be used, based on single
base changes. However, most of the probes have at least two
nucleotide differences compared with the same region in other
strains. When there were variations, other than those associated
with resistance, within the susceptible and/or the resistance
strains for any given species, the position of the probe was
shifted to a region which was completely conserved for all strains
sequenced. For this reason, the probes were in the region 5' of the
QRDR.
TABLE-US-00004 TABLE 3 Oligonucleotide probes for identification of
Enterobacteriaceae E. coli 5' ACT TTA CGC CAT GAA CGT ACT AGG (SEQ
ID NO:17) C 3' (144-168) C. freundii 5' TGG GCA ACG ACT GGA ATA AAG
CC 3' (SEQ ID NO:18) (164-186) E. aerogenes 5' TTA TAT GCT GGT CGA
TGG CCA G 3' (SEQ ID NO:19) (297-323) E. cloacae 5' GCC GGA CGT CCG
CGA TGG CCT 3' (SEQ ID NO:20) (102-122) K. oxytoca 5' GTA GAT GGC
CAG GGT AAC TTT GGT (SEQ ID NO:21) TCG GTC 3' (307-336) K.
pneumoniae 5' GTG CGT ATG GCG CAG CCG TTC TCG (SEQ ID NO:22) CTG 3'
(268-294) P. stuartii 5' CGT CTT GCT CAG CCT TTT TCT ATG (SEQ ID
NO:23) C 3' (271-295) S. marcescens 5' GGA ATA AAC CAT ACA AGA AA
3' (SEQ ID NO:24) (176-195) Note: Numbers in parentheses refer to
base positions in E. coli sequence
Fluoroquinolone Resistance Probes
[0061] Simultaneous identification of the species and mutations
leading to resistance can be determined by using one of the above
oligonucleotide probes in combination with the resistance probes
set forth below. All oligonucleotide probes shown in Table 4 for
quinolone resistance span the region containing the amino acid
codons most frequently associated with resistance (nucleotides
239-263). Susceptible strains will hybridize to the resistance
probe for that species and resistance will be detected as one or
more basepair mismatch with the susceptible strain sequence.
TABLE-US-00005 TABLE 4 Oligonucleotide probes for quinolone
resistance in Enterobacteriaceae E. coli 5' ATG GTG ACT CGG CGG TTT
ATG ACA (SEQ ID NO:25) C 3' OR 5' ATG GTG ACT CGG CGG TCT ATG ACA
(SEQ ID NO:26) C 3' C. freundii 5' ATG GTG ATA CCG CCG TTT ACG ACA
(SEQ ID NO:27) C 3' E. aerogenes 5' ATG GTG ATA CCG CCG TTT ATG ACA
(SEQ ID NO:28) C 3' E. cloacae 5' ATG GTG ATT CCG CGG TGT ACG ACA
(SEQ ID NO:29) C 3' K. oxytoca 5' ATG GTG ATA CTG CCG TAT ACG ACA
(SEQ ID NO:30) C 3' K. pneumoniae 5' ACG GCG ACT CCG CGG TAT ACG
ACA (SEQ ID NO:31) C 3' P. stuartii 5' ATG GTG ATA GCG CTG TTT ATG
AGA (SEQ ID NO:32) C 3' S. marcescens 5' ACG GTG ACA GCG CGG TTT
ACG ACA (SEQ ID NO:33) C 3'
Sequence CWU 1
1
351589DNAEscherichia coli 1acaccggtca acattgagga agagctgaag
agctcctatc tggattatgc gatgtcggtc 60attgttggcc gtgcgctgcc agatgtccga
gatggcctga agccggtaca ccgtcgcgta 120ctttacgcca tgaacgtact
aggcaatgac tggaacaaag cctataaaaa atctgcccgt 180gtcgttggtg
acgtaatcgg taaataccat ccccatggtg actcggcggt ttatgacacg
240atcgtccgta tggcgcagcc attctcgctg cgttacatgc tggtagacgg
tcagggtaac 300ttcggttcca tcgacggcga ctctgcggcg gcaatgcgtt
atacggaaat ccgtctggcg 360aaaattgccc atgaactgat ggctgatctc
gaaaaagaga cggtcgattt cgttgataac 420tatgacggta cggaaaaaat
tccggacgtc atgccaacca aaattcctaa cctgctggtg 480aacggttctt
ccggtatcgc cgtaggtatg gcaaccaaca tcccgccgca caacctgacg
540gaagtcatca acggttgtct ggcgtatatc gatgatgaag acatcagca
5892589DNACitrobacter freundii 2acaccggtca acattgagga agagctgaag
agctcctatc tggattatgc gatgtcggtc 60attgttggcc gtgcgctgcc agacgtccga
gatggcctga agccggttca ccgtcgcgta 120ctttacgcca tgaacgtatt
gggcaacgac tggaataaag cctataaaaa atctgcccgt 180gtcgttggtg
acgtaatcgg taaataccac cctcatggtg ataccgccgt ttacgacacc
240attgttcgta tggcgcagcc attctccttg cgttacatgc tggtagatgg
tcagggtaac 300tttggttctg tcgatggcga ctccgcagcg gcgatgcgtt
atacggaaat ccgtatgtcg 360aaaatcgccc atgagctgat ggctgacctg
gaaaaagaaa cggttgattt cgtcgataac 420tacgacggca ccgaacaaat
tcctgacgtc atgccgacca aaattcctaa cctgctggtg 480aacggttcgt
ccggtatcgc ggtaggtatg gcgaccaaca ttccgccgca caacctgact
540gaagtgatca acggctgtct ggcatatatt gacgatgaag acatcagca
5893589DNAEnterobacter aerogenes 3acacgggtca acattgagga agagctgaaa
agctcgtatc tggattatgc gatgtcggtc 60attgttggcc gtgcgctgcc ggatgtccga
gatggcctga agccggtaca ccgtcgcgta 120ctatacgcca tgaacgtatt
gggcaatgac tggaacaaag cctataaaaa atcagcccgt 180gtcgttggcg
acgtaatcgg taaataccac ccgcatggtg ataccgccgt ttatgacacc
240atcgtacgta tggcgcagcc gttctccttg cgttatatgc tggtcgatgg
ccagggtaac 300tttggttctg tcgatggcga ctccgctgca gcgatgcgtt
atacggaaat ccgtatgtcg 360aagatcgctc atgagctgat ggccgatctc
gaaaaagaga cggttgattt cgtcgacaac 420tatgacggca cggagaaaat
ccctgacgtc atgccgacaa aaatccctaa cctgctggtg 480aacggttctt
ccggtatcgc cgtaggtatg gcgaccaaca ttccgccgca taacctgacg
540gaagttatca acggctgcct ggcatacgtt gataacgaag acatcagca
5894589DNAEnterobacter cloacae 4acaccggtta acatcgagga agagctgaag
agctcctatc tggactatgc gatgtcggtc 60attgttggcc gtgcgctgcc ggacgtccgc
gatggcctga agccggtaca ccgtcgcgta 120ctatacgcca tgaacgtatt
gggcaatgac tggaataaag cctacaaaaa atctgcccgt 180gtcgttggtg
acgtaatcgg taaataccat ccccatggtg attccgcggt gtacgacacc
240atcgttcgta tggcgcagcc tttctcgctg cgttacatgc tggtagatgg
tcagggtaac 300tttggttcta tcgacggcga ctccgccgcg gcaatgcgtt
atacggaaat ccgtctggcg 360aaaattgccc atgagctgat ggccgacctg
gaaaaagaga cggttgattt cgttgataac 420tacgatggca cggaaaaaat
tcctgacgtc atgccaacga agatccctaa cctgctggtg 480aacggttcgt
ccggtatcgc cgtagggatg gcgaccaaca ttccgccgca caacatcacc
540gaagtgatca acggctgcct ggcctatatc gacgatgaag acatcagca
5895589DNAKlebsiella oxytoca 5acaccggtca acattgagga agagctgaag
agctcctatc tggattatgc gatgtcggtc 60attgttggcc gtgcgctgcc ggatgtccga
gatggcctga agccggtaca ccgtcgcgta 120ctatacgcca tgaacgtatt
gggcaatgac tggaacaaag cctataaaaa atctgcccgt 180gtcgtgggtg
acgtcatcgg taaataccac cctcatggtg atactgccgt atacgacacc
240attgtacgta tggcgcagcc attctccctg cgttacatgc tggtagatgg
ccagggtaac 300tttggttcgg tcgacggcga ctccgccgca gcgatgcgtt
atacggaaat ccgtatgtcg 360aagatcgccc atgaactgat ggccgacctc
gaaaaagaga cggtggattt cgtcgataac 420tatgacggca cggagaaaat
ccctgacgtt atgccgacca aaatcccgaa cctgctagtc 480aacggttcgt
ccggtatcgc ggtaggtatg gcgactaata ttccgccgca caacctgacc
540gaagtgatca acggctgtct ggcctacgtt gaaaacgaag acatcagca
5896589DNAKlebsiella pneumoniae 6acaccggtca acattgagga agagcttaag
aactcttatc tggattatgc gatgtcggtc 60attgttggcc gtgcgctgcc ggatgtccga
gatggcctga agccggtaca ccgtcgcgta 120ctttacgcca tgaacgtatt
gggcaatgac tggaacaaag cctataaaaa atcagcccgt 180gtcgttggtg
acgtaatcgg taaataccac ccgcacggcg actccgcggt atacgacacc
240atcgtgcgta tggcgcagcc gttctcgctg cgttacatgc tggtggacgg
ccagggtaac 300tttggttcca tcgacggcga ctccgccgcg gcgatgcgtt
ataccgaaat tcgtctggcg 360aaaatcgctc atgagctgat ggccgatctt
gaaaaagaga cggtcgattt cgtcgacaac 420tatgacggta cggagcgtat
tccggacgtc atgccgacca aaattcctaa cctgctggtg 480aacggcgcct
ccgggatcgc cgtagggatg gccaccaaca taccgccaca taacctgacg
540gaagtgatta acggctgtct ggcgtatgtt gacgatgaag acatcagca
5897589DNAProvidencia stuartii 7acaccggtca atatcgaaga agaactcaaa
agttcgtatt tggattatgc gatgtccgtt 60attgtcgggc gcgcgcttcc agatgttcga
gatggactga agccagtaca ccgcagagta 120ctgtttgcga tgaatgtatt
gggaaatgat tggaataaac cctataaaaa atctgcccgt 180atagtcgggg
acgttatcgg taaataccat ccacatggtg atagcgctgt ttatgagaca
240atcgttcgtc ttgctcagcc tttttctatg cgttatatgc tggtagatgg
tcaggggaac 300tttggttcag ttgacggaga ttccgcagct gcaatgcgtt
atacggaaat ccgtatggcg 360aaaattgccc atgaaatgtt agcggatctt
gaaaaagaga ccgttgattt cgtcccaaac 420tatgatggta cagagcaaat
ccctgaagtt atgcctacga aaatccctaa cctattggtt 480aatggttcgt
caggtattgc tgttgggatg gcaacgaaca ttcctccaca caacctaggg
540gaagtgatca gcggttgcct tgcttatata gatgatgaag atattagca
5898589DNASerratia marcescens 8acaccggtaa acatcgaaga cgagttgaaa
aactcgtatc tggactatgc gatgtccgtt 60attgtcggac gtgccctgcc agatgttcgt
gatggactga agccggttca ccgccgcgtt 120ctgtacgcga tgagcgtatt
gggtaacgac tggaataaac catacaagaa atcggcccgt 180gtcgtcgggg
acgtgatcgg taaatatcac ccgcacggtg acagcgcggt ttacgacact
240atcgtgcgta tggctcagcc gttttcactg cgctacatgc tggtggacgg
tcagggtaac 300ttcggttccg tcgacggcga ctccgcggcg gcgatgcgtt
ataccgaagt gcgcatgtcc 360aagattgctc acgaactgtt ggcggatctg
gaaaaagaaa ccgtcgactt cgtgcctaac 420tatgatggca ccgagcagat
cccggccgtc atgccgacca agatcccgaa cctgctggtc 480aacggctcgt
cgggcatcgc cgtgggcatg gctaccaata ttccgccgca caacctggcg
540gaagtcgtca acggctgcct ggcctatatc gacgatgaaa acatcagca
5899120DNAEscherichia coli 9gcccgtgtcg ttggtgacgt aatcggtaaa
taccatcccc atggtgactc ggcggtttat 60gacacgatcg tccgtatggc gcagccattc
tcgctgcgtt acatgctggt agacggtcag 12010120DNACitrobacter freundii
10gcccgtgtcg ttggtgacgt aatcggtaaa taccaccctc atggtgatac cgccgtttac
60gacaccattg ttcgtatggc gcagccattc tccttgcgtt acatgctggt agatggtcag
12011120DNAEnterobacter aerogenes 11gcccgtgtcg ttggcgacgt
aatcggtaaa taccacccgc atggtgatac cgccgtttat 60gacaccatcg tacgtatggc
gcagccgttc tccttgcgtt atatgctggt cgatggccag 12012120DNAEnterobacter
cloacae 12gcccgtgtcg ttggtgacgt aatcggtaaa taccatcccc atggtgattc
cgcggtgtac 60gacaccatcg ttcgtatggc gcagcctttc tcgctgcgtt acatgctggt
agatggtcag 12013120DNAKlebsiella oxytoca 13gcccgtgtcg tgggtgacgt
catcggtaaa taccaccctc atggtgatac tgccgtatac 60gacaccattg tacgtatggc
gcagccattc tccctgcgtt acatgctggt agatggccag 12014120DNAKlebsiella
pneumoniae 14gcccgtgtcg ttggtgacgt aatcggtaaa taccacccgc acggcgactc
cgcggtatac 60gacaccatcg tgcgtatggc gcagccgttc tcgctgcgtt acatgctggt
ggacggccag 12015120DNAProvidencia stuartii 15gcccgtatag tcggggacgt
tatcggtaaa taccatccac atggtgatag cgctgtttat 60gagacaatcg ttcgtcttgc
tcagcctttt tctatgcgtt atatgctggt agatggtcag 12016120DNASerratia
marcescens 16gcccgtgtcg tcggggacgt gatcggtaaa tatcacccgc acggtgacag
cgcggtttac 60gacactatcg tgcgtatggc tcagccgttt tcactgcgct acatgctggt
ggacggtcag 1201725DNAEscherichia coli 17actttacgcc atgaacgtac taggc
251823DNACitrobacter freundii 18tgggcaacga ctggaataaa gcc
231922DNAEnterobacter aerogenes 19ttatatgctg gtcgatggcc ag
222021DNAEnterobacter cloacae 20gccggacgtc cgcgatggcc t
212130DNAKlebsiella oxytoca 21gtagatggcc agggtaactt tggttcggtc
302227DNAKlebsiella pneumoniae 22gtgcgtatgg cgcagccgtt ctcgctg
272325DNAProvidencia stuartii 23cgtcttgctc agcctttttc tatgc
252420DNASerratia marcescens 24ggaataaacc atacaagaaa
202525DNAEscherichia coli 25atggtgactc ggcggtttat gacac
252625DNAEscherichia coli 26atggtgactc ggcggtctat gacac
252725DNACitrobacter freundii 27atggtgatac cgccgtttac gacac
252825DNAEnterobacter aerogenes 28atggtgatac cgccgtttat gacac
252925DNAEnterobacter cloacae 29atggtgattc cgcggtgtac gacac
253025DNAKlebsiella oxytoca 30atggtgatac tgccgtatac gacac
253125DNAKlebsiella pneumoniae 31acggcgactc cgcggtatac gacac
253225DNAProvidencia stuartii 32atggtgatag cgctgtttat gagac
253325DNASerratia marcescens 33acggtgacag cgcggtttac gacac
253418DNAEnterobacter sp. 34cgaccttgcg agagaaat
183518DNAEnterobacter sp. 35gttccatcag cccttcaa 18
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