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Oligonucleotide probes for detecting enterobacteriaceae and quinolone-resistant enterobacteriaceae

a technology of enterobacteriaceae and probes, applied in the field of diagnostic microbiology, can solve the problems of inability to establish i>providencia stuartii/i>, gyra mutations with fluoroquinolone resistance, and infection life-threatening,

Inactive Publication Date: 2008-08-21
THE GOVERNMENT OF THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SEC DEPT OF HEALTH & HUMAN SERVICES CENTS FOR DISEASE CONTROL & PREVENTION OFFICE OF TECH TRANSFER
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[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.

Problems solved by technology

Many of these infections are life threatening and are often nosocomial (hospital-acquired) infections.
As a result, indiscriminate use has led to the currently increasing incidence of quinolone / fluoroquinolone resistance.
However, the association of gyrA mutations with fluoroquinolone resistance in Enterobacter aerogenes, Klebsiella oxytoca, and Providencia stuartii has not been established.
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.

Method used

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  • Oligonucleotide probes for detecting enterobacteriaceae and quinolone-resistant enterobacteriaceae
  • Oligonucleotide probes for detecting enterobacteriaceae and quinolone-resistant enterobacteriaceae
  • Oligonucleotide probes for detecting enterobacteriaceae and quinolone-resistant enterobacteriaceae

Examples

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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=Ente...

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 con...

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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.

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 § 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.[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.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-specifi...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C07H21/02C07H21/04
CPCC07H21/02C07H21/04Y10T436/143333C12Q1/689C12Q1/6827Y02A50/30
Inventor WEIGEL LINDA M.TENOVER FRED C.
Owner THE GOVERNMENT OF THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SEC DEPT OF HEALTH & HUMAN SERVICES CENTS FOR DISEASE CONTROL & PREVENTION OFFICE OF TECH TRANSFER
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