Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-01-16
2004-03-16
Whisenant, Ethan (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C436S094000, C536S023100, C536S024300, C536S024330
Reexamination Certificate
active
06706475
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
This invention relates in general to the field of diagnostic microbiology. In particular, the invention relates to the species-specific detection of Enterobactedaceae.
BACKGROUND OF THE INVENTION
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)).
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.
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)).
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)).
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.
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.
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
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 Enterobacieriaceae in a sample, and detection and diagnosis of Enterobacteriaceae infection in a subject.
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 stuarrii
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.
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.
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.
REFERENCES:
patent: 5645994 (1997-07-01), Huang
patent: 0 688 873 (1998-01-01), None
Bachoul et al.,Microbial Drug Resistance(1998) 4:4, pp. 271-276.
Cambau et al.,Drugs(1993) 45(Suppl. 3):15-23.
Deguchi et al.,Journal of Antimicrobial Chemotherapy(1997) 40: pp. 543-549.
Deguchi et al.,Antimicrobial Agents and Chemotherapy(1997) 41:11, pp. 2544-2546.
Dimri et al.,Nucleic Acids Research(1990) 18:151-156.
Everett et al.,Antimicrob. Agents Chemother. (1996) 40:2380-2386.
Guillemin et al.,Antimicrob. Agents Chemo. (1995) 39(9):2145-2149.
Heisig et al.,Antimicrob. Agents Chemother. (1993) 37:696-701.
Hooper,Antimicrob. Agents Chemother. (
Tenover Fred C.
Weigel Linda M.
Klarquist & Sparkman, LLP
Lu Frank Wei Min
The United States of America as represented by the Department of
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