AU762314B2

AU762314B2 - Oligonucleotide probes for detecting enterobacteriaceae and quinolone-resistant enterobacteriaceae

Info

Publication number: AU762314B2
Application number: AU33723/99A
Authority: AU
Inventors: Fred C. Tenover; Linda M. Weigel
Original assignee: US Department of Health and Human Services
Current assignee: US Department of Health and Human Services
Priority date: 1998-04-01
Filing date: 1999-03-30
Publication date: 2003-06-19
Anticipated expiration: 2019-03-30
Also published as: EP1068355A2; WO1999050458A3; CA2324990A1; WO1999050458A2; AU3372399A

Description

WO 99/50458 PCT/US99/06963 OLIGONUCLEO)I'IDI PROBES FOR DETECTING Enterobacteriaceae AND QUINOLONE-RESISTANT Enterobacteriaceae 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 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 Enterobacteriaceae is a family of closely related, Gramnegative 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.

III. Enterobacteriaceae: Introduction and Identification. in Murray, P.

et al., Manual of Clinical Microbiology, Washington, ASM Press, 6th 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 identificatioin of these organisms include growth on selective and/or differential media followed WO 99/50458 PCTIS99/06963 2 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 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, WO 99/50458 PCT/S99/06963 3 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 Enterobacteriaceae 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, Citrobacterfreundii, 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.

Therefore, it is desirable to provide improved materials and methods for detecting and differentiating Enterobacteriaceae species and/or quinolone resistance in the clinical laboratory and research settings.

In a first aspect, the present invention provides an isolated nucleic acid probe, comprising 10 to 50 consecutive nucleotides of one of the gyrA genes of Escherichia coli (SEQ ID NO: Enterobacter aerogenes (SEQ ID NO: Enterobacter cloacae (SEQ ID NO: Klebsiella oxytoca (SEQ ID NO: Klebsiellapneumoniae (SEQ ID NO: or Providencia stuartii (SEQ ID NO: wherein the probe selectively 25 hybridizes under stringent conditions to a single gyrA gene set forth in SEQ ID NOs: 1- 8, or a complementary sequence thereof, but does not hybridize to any of the other gyrA genes set forth in SEQ ID NOs: 1-8, wherein the probe identifies an Enterobacteriaceae species as Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, or Providencia stuartii.

In a second aspect, the present invention provides an isolated nucleic acid probe, consisting essentially of 10 to 50 consecutive nucleotides of one of the gyrA genes of Citrobacterfreundii (SEQ ID NO: or Serratia marcescens (SEQ ID NO: 8), wherein the probe selectively hybridizes under stringent conditions to a single gyrA gene set forth in SEQ ID NOs: 1-8, or a complementary sequence thereof, but does not 35 hybridize to any of the other gyrA genes set forth in SEQ ID NOs: 1-8, wherein the probe identifies an Enterobacteriaceae species as Citrobacterfreundii or Serratia marcescens.

In a third aspect, the present invention provides an isolated nucleic acid probe having a nucleic acid sequence of SEQ ID NO: 17, 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.

In a fourth aspect, the present invention provides an isolated nucleic acid probe, comprising 10 to 50 consecutive nucleotides of one of the gyrA genes of Escherichia coli (SEQ ID NO: Enterobacter aerogenes (SEQ ID NO: Klebsiella oxytoca (SEQ ID NO: Klebsiellapneumoniae (SEQ ID NO: or Providencia stuartii (SEQ ID NO: wherein the probe selectively hybridizes under stringent conditions to a single gyrA gene set forth in SEQ ID NOs: 1-8, or a complementary sequence thereof, wherein a quinolone susceptible strain selectively hybridizes to the probe, and wherein a quinolone resistant strain has a one or more base pair mismatch with the probe.

In a fifth aspect, the present invention provides an isolated nucleic acid probe, consisting essentially of 10 to 50 consecutive nucleotides of one of the gyrA genes of Citrobacterfreundii (SEQ ID NO: Enterobacter cloacae (SEQ ID NO: or Serratia marcescens (SEQ ID NO: wherein the probe selectively hybridizes under stringent conditions to a single gyrA gene set forth in SEQ ID NOs: 1-8, or a complementary sequence thereof, wherein a quinolone susceptible strain selectively hybridizes to the probe, and wherein a quinolone resistant strain has a one or more base pair mismatch with the probe.

In a sixth aspect, the present invention provides an isolated nucleic acid probe :having a nucleic acid sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, S: 25 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.

In a seventh aspect, the present invention provides a method of identifying in a sample an Enterobacteriaceae species selected from the group consisting of Escherichia coli (SEQ ID NO: Citrobacterfreundii (SEQ ID NO: Enterobacter 30 aerogenes (SEQ ID NO: Enterobacter cloacae (SEQ ID NO: Klebsiella oxytoca (SEQ ID NO: Klebsiellapneumoniae (SEQ ID NO: Providencia stuartii (SEQ ID NO: and Serratia marcescens (SEQ ID NO: comprising combining the sample with the isolated nucleic acid probe according to any one of the first to third aspects, wherein the probe selectively hybridizes under stringent conditions to a single gyrA gene set forth in SEQ ID NOs: 1-8, or a complementary sequence thereof, but does not hybridize to any of the other gyrA genes set forth in SEQ ID NOs: 1-8, wherein the presence of hybridization with the single gyrA gene identifies the Enterobacteriaceae species as Escherichia coli, Citrobacterfreundii, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, Providencia stuartii, or Serratia marcescens.

In an eighth aspect, the present invention provides a method of determining in a sample the quinolone resistance of an Enterobacteriaceae species selected from the group consisting of Escherichia coli (SEQ ID NO: Citrobacterfreundii (SEQ ID NO: Enterobacter aerogenes (SEQ ID NO: Enterobacter cloacae (SEQ ID NO: Klebsiella oxytoca (SEQ ID NO: Klebsiellapneumoniae (SEQ ID NO: 6), Providencia stuartii (SEQ ID NO: and Serratia marcescens (SEQ ID NO: 8), comprising combining the sample with the isolated nucleic acid probe according to any one of the fourth to sixth aspects, wherein the probe selectively hybridizes under stringent conditions to a single gyrA gene set forth in SEQ ID NOs: 9-16, or a complementary sequence thereof, the presence of hybridization with the single gyrA gene indicating the quinolone resistance of the species, wherein a quinolone susceptible strain selectively hybridizes to the probe, and wherein a quinolone resistant strain has a one or more base pair mismatch with the probe.

These features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and S* the appended claims.

Throughout this specification the word "comprise", or variations such as S "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of 25 any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the 30 field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

Brief Description of the Drawings Figures 1A and 1B show the nucleic acid sequence (SEQ ID NOS: 1-8) alignments for a portion of the gyrA gene in Escherichia coi Citrobacter freundii Enterobacter aerogenes Enterobacter cloacae (ECL), Kiebsiella oxgtoca Kiebsiella pneumoniae Providencia stuartii (PS) and Serratia marcescens (SM).

Figure 2 shows the DNA sequence (SEQ ID NOS:9-16) similarity of the quinolone resistance-determining region (QRDR) in WO 99/50458 PCT[US99/06963 Escherichia coli, Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, Providencia stuartii and Serratia marcescens.

Figure 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.

Figures 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 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.

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.

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 WO 99/50458 PCT/US99/06963 6 related to Enterobacteriaceae species suggest that gyrA sequences are unique for all bacterial species and may be used for identification of any species.

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: 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.

Figures 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).

The invention also provides unique, isolated nucleic acids from the quinolone resistance-determining region of Escherichia coli (SEQ- ID NO:9), Citrobacterfreundii (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 Figure 2.

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), Citrobacterfreundii (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).

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), Citrobacterfreundii (SEQ ID WO 99/50458 PCT/US99/06963 7 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).

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.

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".

A hybridizing nucleic acid should have at least 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.

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 WO 99/50458 PCT/US99/06963 8 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, (1982) latest edition.

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., 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.

If used as primers, the invention provides compositions including at least two oligonucleotides 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 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.

Alternatively, the nucleic acid probes can be designed to have homology with nucleotide sequences present in more than one species of WO 99/50458 PCT/US99/06963 9 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.

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.

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 resistancedetermining probe of SEQ ID NOS:25-33 is indicative of quinolone resistance in the sample.

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 WO 99/50458 PCT/US99/06963 moieties include radioactive labeling, enzyme labeling, and fluorescent labeling.

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 In this Example, the DNA sequence of the gyrA was determined for eight species ofEnterobacteriaceae. 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.

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 coli) ATCC 11775 CF Citrobacter freundii freundii) ATCC 8090 EA Enterobacter aerogenes aerogenes) ATCC 13048 ECL Enterobacter cloacae cloacae) ATCC 13047 KO Klebsiella oxytoca oxytoca) ATCC 13182 KP Klebsiella pneumoniae pneumoniae) ATCC 13883 PS Providencia stuartii stuartii) ATCC 29914 SM Serratia marcescens marcescens) ATCC 13880 WO 99/50458 PCT/US99/06963 11 Amplification of gyrA Bacterial strains and determination of antibiotic susceptibility profiles.

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.

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, CT), ofloxacin and nalidixic acid were from Sigma (St. Louis, MO) and sparfloxacin was from Rh6ne-Poulenc Rorer (Collegeville, PA).

Amplification of 5' region of gvrA.

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: GYRA6 5'-CGACCTTGCGAGAGAAAT-3' (SEQ ID NO:34) GYRA631R 5'-GTTCCATCAGCCCTTCAA-3' (SEQ ID Primer GYRA6 corresponds to nucleotides 6 to 23 and primer GYRA631R is complementary to nucleotides 610 to 631 of the E. coli gyrA sequence.

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, CA) in .tl volume containing 50 pmol of each primer, 200 gM deoxynucleoside triphosphates, 10 ul cell lysate containing approximately 100 ng template WO 99/50458 PCT/US99/06963 12 DNA, IX reaction buffer with 1.5 mM MgCl2 and 1 U native Taq polymerase (Perkin Elmer). An initial 4 minute period of denaturation at 94 0 C was followed by 30 cycles including: denaturation for 1 minute at 94*C, annealing for 30 seconds at 55C, extending for 45 seconds at 72*C, followed by a final cycle of 72*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, CA) 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, WI) analyses programs were used for the construction of DNA and amino acid sequence alignments.

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 Figures lA-1B.

The sequences provided correspond to nucleotide positions 25 to 613 of the gyrA regions for Escherichia coli.

TABLE 1 Gvrase 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 TGACGTAATC GGTAAATACC ATCCCCATGG TGACTCGGCG GTTIATGACA CGATCGTCCG TATGGCGCAG CCATTCTCGC TGCGTTACAT GCTGGTAGAC GGTCAGGGTA ACTTCGGTTC CATCGACGGC GACTCTGCGG CGGCAATGCG TTATACGGAA ATCCGTCTGG CGAAAATGC CCATGAACTG ATGGCTGATC TCGAAAAAGA GACGGTCGAT TTCGTTGATA ACTATGACGG TACGGAAAAA ATTCCGGACG TCATGCCAAC CAAAATTCCT AACCTGCTGG TGAACGGTTC TTCCGGTATC WO 99/50458 PTU9/66 PCTIUS99/06963 13 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 ATGGCTGACC TGGAAAAAGA AACGGTTGAT TTCGTCGATA ACTACGACGG CACCGAACAA ATTCCTGACG TCATGCCGAC CAAAATTCCT AACCTGCTGG TGAACGGTTC GTCCGGTATC GCGGTAGGTA TGGCGACCAA CATFTCCGCCG CACAACCTGA CTGAAGTGAT CAACGGCTGT CTGGCATATA TTGACGATGA AGACATCAGC A (SEQ ID NO:2) Enterobacter aero genes 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 TGCGTTfATAT GCTGGTCGAT GGCCAGGGTA ACTTTGGTTC TGTCGATGGC GACTCCGCTG CAGCGATGCG TTATACGGAA ATCCGTATGT CGAAGATCGC TCATGAGCTG ATGGCCGATC TCGAAAAAGA GACGGTTGAT TITCGTCGACA ACTATGACGG CACGGAGAAA ATCCCTGACG TCATGCCGAC AAAAATCCCT AACCTGCTGG TGAACGGTf C TTCCGGTATC GCCGTAGGTA TGGCGACCAA CAYJ7CCGCCG 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 WO 99/50458 PCT/US99/06963 14 TGGAATAAAG CCTACAAAAA ATCTGCCCGT GTCGTFTGGTG ACGTAATCGG TAAATACCAT CCCCATGGTG ATTCCGCGGT GTACGACACC ATCGTTCGTA TGGCGCAGCC TTTCTCGCTG CGTTACATGC TGGTAGATGG TCAGGGTAAC TTTGGTTCTA TCGACGGCGA CTCCGCCGCG GCAATGCGTT ATACGGAAAT CCGTCTGGCG AAAATFFGCCC ATGAGCTGAT. GGCCGACCTG GAAAAAGAGA CGGTFTGATTT CGTTGATAAC TACGATGGCA CGGAAAAAAT TCCTGACGTC ATGCCAACGA AGATCCCTAA CCTGCTGGTG AACGGTTCGT CCGGTATCGC CGTAGGGATG GCGACCAACA TTCCGCCGCA CAACATCACC GAAGTGATCA ACGGCTGCCT GGCCTATATC GACGATGAAG ACATCAGCA (SEQ ID NO:4) Kiebsiella oxytoca ACACCGGT CAACATTGAG GAAGAGCTGA AGAGCTCCTA TCTGGATTAT GCGATGTCGG TCATTGTTGG CCGTGCGCTG CCGGATGTCC GAGATGGCCT GAAGCCGGTA CACCGTCGCG TACTATACGC CATGAACGTA 'TTGGGCAATG ACTGGAACAA AGCCTATAAA AAATCTGCCC GTGTCGTGGG TGACGTCATC GGTAAATACC ACCCTCATGG TGATACTGCC GTATACGACA CCATTGTACG TATGGCGCAG CCATTCTCCC TGCGTTACAT GCTGGTAGAT GGCCAGGGTA ACTrGGTrC GGTCGACGGG GACTCCGCCG CAGCGATGCG TTATACGGAA ATCCGTATGT CGAAGATCGC CCATGAACTG ATGGCCGACC TCGAAAAAGA GACGGTGGAT TTCGTCGATA ACTATGACGG CACGGAGAAA ATCCCTGACG TTrATGCCGAC CAAAATCCCG AACCTGCTAG TCAACGGTTC GTCCGGTATC GCGGTAGGTA TGGCGACTAA TATTfCCGCCG CACAACCTGA CCGAAGTGAT CAACGGCTGT CTGGCCTACG TTGAAAACGA AGACATCAGC A (SEQ ID Kiebsiella pneumoniae ACACCGGT CAACATTGAG GAAGAGCTTA AGAACTCTTA TCTGGATTAT GCGATGTCGG TCATTG'ITGG CCGTGCGCTG CCGGATGTCC GAGATGGCCT GAAGCCGGTA CACCGTCGCG TACTITACGC CATGAACGTA TFTGGGCAATG ACTGGAACAA AGCCTATAAA AAATCAGCCC GTGTCGTTGG TGACGTAATC GGTAAATACC ACCCGCACGG CGACTCCGCG GTATACGACA CCATCGTGCG TATGGCGCAG CCGTTCTCGC TGCGTIACAT GCTGGTGGAC GGGCAGGGTA AC1TTGGT-rC CATCGACGGC GACTCCGCCG CGGCGATGCG TTATACCGAA ATTCGTCTGG CGAAAATCGC TCATGAGCTG ATGGCCGATC TTGAAAAAGA GACGGTCGAT TTCGTCGACA ACTATGACGG TACGGAGCGT AT-rCCGGACG TCATGCCGAC CAAAATTCCT AACCTGCTGG TGAACGGCGC CTCCGGGATC GCCGTAGGGA TGGCCACCAA CATACCGCCA CATAACCTGA CGGAAGTGAT WO 99/50458 PTU9/66 PCT/US99/06963 TAACGGCTGT CTGGCGTATG TTGACGATGA AGACATCAGC A (SEQ ID NO:6) Providencia stuarti ACACCGGT CAATATCGAA GAAGAACTCA AAAGTTCGTA TTTGGATTAT GCGATGTCCG TTATTGTCGG GCGCGCGCTT CCAGATG'ITC GAGATGGACT GAAGCCAGTACACCGCAGAG TACTGTTTGC GATGAATGTA TTGGGAAATG ATTGGAATAA ACCCTATAAA AAATCTGCCC GTATAGTCGG GGACG2ITATC GGTAAATACC ATCCACATGG TGATAGCGCT G3TTATGAGA CAATCGTF7CG TCTTGCTCAG CCTTTTTCTA TGCGTTATAT GCTGGTAGAT GGTCAGGGGA ACTTTGGTTC AGTTGACGGA GATTCCGCAG CTGCAATGCG TTATACGGAA ATCCGTATGG CGAAAATTfGC CCATGAAATG TTAGCGGATC TTGAAAAAGA GACCGTTGAT TTCGTCCCAA ACTATGATGG TACAGAGCAA ATCCCTGAAG TTATGCCTAC GAAAATCCCT AACCTATTGG TTAATGGTTC GTCAGGTATT GCTG'ITGGGA TGGCAACGAA CATTCCTCCA CACAACCTAG GGGAAGTGAT CAGCGGTFIGC C'1TGCTrfATA TAGATGATGA AGATATJ'AGC A (SEQ ID NO:7) Serratia marcescens ACACCGGT AAACATCGAA GAGGAGTTGA AAAACTCGTA TCTGGACTAT GCGATGTCCG TTATTGTCGG ACGTGCCCTG CCAGATGYITC GTGATGGACT GAAGCCGGTT CACCGCCGCG TTCTGTACGC GATGAGCGTA TTGGGTAACG ACTGGAATAA ACCATACAAG AAATCGGCCC GTGTCGTCGG GGACGTGATC GGTAAATATC ACCCGCACGG TGACAGCGCG GTITTACGACA CTATCGTGCG TATGGCTCAG CCGTI'TTCAC TGCGCTACAT GCTGGTGGAC GGTCAGGGTA ACTFTCGGTTC CGTCGACGGC GACTCCGCGG CGGCGATGCG TATACCGAA GTGCGCATGT CCAAGATTGC TCACGAACTG TTGGCGGATC TGGAAAAAGA AACCGTCGAC TTCGTGCCTA ACTATGATGG CACCGAGCAG ATCCCGGCCG TCATGCCGAC CAAGATCCCG AACCTGCTGG TCAACGGCTC GTCGGGCATC GCCGTGGGCA TGGCTACCAA TATI'CCGCCG CACAACCTGG CGGAAGTCGT CAACGGCTGC CTGGCCTATA TCGACGATGA AAACATCAGC A (SEQ ID NO:8) The QRDR sequences- from positions 199 to 318 (relative to E.

cc ii) are shown below in Table 2.

WO 99/50458 WO 9950458PCTIUS99/06963 16 TABLE 2 Ouinglone Resistan c e-Determining Region Sequences Escherichia ccli GCCCG TGTCGTTGGT GACGTAATCG GTAAATACCA TCCCCATGGT GACTCGGCGG TTTATGACAC GATCGTCCGT ATGGCGCAGC CATTCTCGCT GCGTFIACATG CTGGTAGACG GTCAG (SEQ ID NO:9) Citrobacterfr-eundii GCCCG TGTCGTTGGT GACGTAATCG GTAAATACCA GCCTCATGGT GATACCGCCG TTTACGACAC CATTGTTCGT ATGGCGCAGC CATTCTCCTT GCGTTACATG CTGGTAGATG GTCAG (SEQ ID NO: Enterobacter aero genes G C -CCGTGTCGTT GGCGACGTAA TCGGTAAATA CCACCCGCAT GGTGATACCG CCGTTTATGA CACCATCGTA CGTATGGCGC AGCCGTTCTC CTTGCGTf AT ATGCTGGTCG ATGGCCAG (SEQ ED NO: 11) Enterobacter cloacae GC CCGTGTCGTT GGTGACGTAA TCGGTAAATA CCATCCCCAT GGTGATTCCG CGGTGTACGA CACCATCGTT CGTATGGCGC AGCCTTTCTC GCTGCGTTAC ATGCTGGTAG ATGGTCAG (SEQ ID NO: 12) Kiebsiella oxytoca GCCCGTGTC GTGGGTGACG TCATCGGTAA ATACCACCCT CATGGTGATA CTGCCGTATA CGACACCATT GTACGTATGG CGCAGCCATT CTCCCTGCGT TACATGGTGG TAGATGGCCA G (SEQ ID NO: 13) Kiebsiella pneumoniae GC CCGTGTCGTT GGTGACGTAA TCGGTAAATA CCACCCGCAC GGCGACTCCG CGGTATACGA CACCATCGTG CGTATGGCGC AGCCGFFCTC GCTGCGT7AC ATGCTGGTGG ACGGCCAG (SEQ ID NO: 14) Providencia stuartii GCCCGTATAG TCGGGGACGT TATCGGTAAA TACCATCCAC ATGGTGATAG CGCTGTTTAT GAGACAATCG TTCGTCTTGC TCAGCCTTTT TCTATGCGTT ATATGCTGOT AGATGGTCAG (SEQ ID NO: WO 99/50458 PCT/US99/06963 17 Serratia marcescens GCCCGTGTC GTCGGGGACG TGATCGGTAA ATATCACCCG CACGGTGACA GCGCGGTTTA CGACACTATC GTGCGTATGG CTCAGCCGTT TTCACTGCGC TACATGCTGG TGGACGGTCA G (SEQ ID NO: 16) 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).

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. Eighty-seven of 120 nucleotides 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 andK. pneumoniae and K. oxytoca shared 89.3 identity in this region, less similarity than between several of the different genera.

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 Mol. Biol. 197:729-736 (1997)) and 4 nucleotide differences were detected at positions 255 (C 267 (T 273 (C and 300 (T C).

When the QRDR sequence from theK. pneumoniae type strain was compared with thegyrA gene sequence from K. pneumoniae strain published by Dimri and Das (Nucleic Acids Research, 18:151-156 (1990)), differences were detected in 15 of 120 nucleotides. Of these 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 (M5al). When the M5al gyrA sequence was compared with that of theK. 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 WO 99/50458 PCTIUS99/06963 18 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 M5al strain most likely was from a strain of K. oxytoca and not K. pneumoniae.

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.

The deduced amino acid sequences of the QRDR were highly conserved (Fig. 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.

After determining the DNA sequence of the QRDR from the quinolone-susceptible type strains, the 5' region of gyrA in ciprofloxacinresistant 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 (>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 WO 99/50458 PCT/US99/06963 19 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)).

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 ltg/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 J.g/ml ciprofloxacin.

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.

K. pneumoniae isolates exhibited either single or double mutations involving Ser-83 and Asp-87, and ciprofloxacin MICs ranged from 1 16 p.g/ml. Again, double mutations were not required for highlevel resistance and no specific mutation (Ser-83 to Phe or Tyr) was associated with low or high levels of fluoroquinolone resistance.

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 pg/ml ciprofloxacin.

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 WO 99/50458 PCT/US99/06963 second position resulting in Ser-to-Ile mutations. MICs ranged from 2 to 16 ptg/ml ciprofloxacin.

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)).

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 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.

WO 99/50458 PCT/US99/06963 21 TABLE 3 Oligonucleotide probes for identification of Enterobacteriaceae E. coli 5' ACT TTA CGC CAT GAA CGT ACT AGG C 3' (SEQ ID NO:17) (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 TCG GTC 3' (SEQ ID NO:21) (307-336) K. pneumoniae 5' GTG CGT ATG GCG CAG CCG TTC TCG CTG 3' (SEQ ID NO:22) (268-294) P. stuartii 5' CGT CTT GCT CAG CCT TTT TCT ATG C 3' (SEQ ID NO:23) (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 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 WO 99/50458 PTU9/66 PCT/US99/06963 22 will be detected as one or more basepair mismatch with the susceptible strain sequence.

TABLE 4 Oligonucleotide probes for guinolone resistance in Enterobacteriaceae E. ccli 5' ATO GTG ACT CGG CGO TTT ATG ACA C 3' (SEQ ID OR 5'ATG GTG ACT COG CGG TCT ATG ACA C 3' (SEQ ID N026) C. freundii 5' ATG GTG ATA CCG CCG TTT ACG ACA C 3' (SEQ ID NO:27) E. aerogenes 5' ATG OTO ATA CCO CCG T7FJ ATG ACA C 3' (SEQ ID NO:28) E. cloacae 5' ATG GTG ATT CCG CGG TOT ACG ACA C 3' (SEQ ID NO:29) K. oxytoca (SEQ ID 5' ATG GTG ATA CTO CCG TAT ACO ACA C 3' K. pneumoniae 5'ACG GCG ACT CCG COG TAT ACG ACA C 3' (SEQ ID NO:3 1) P. stuartii 5' ATG OTO ATA OCO CTG TTIT ATG AGA C 3' (SEQ ID NO:32) S. marcescens 5'AG OTG ACA GCG COG FJ7T ACG ACA C 3' (SEQ ID NO:33) EDITORIAL NOTE APPLICATION NUMBER 33723/99 The following Sequence Listing pages 1 to 8 are part of the description. The claims pages follow on pages "23" to "29".

WO 99/50458 PTU9166 PCT/US99/06963 SEQUENCE LISTING <110> The Government of the United States of America <120> Oligonucleotide Probes for Detecting Enterobacteriaceae and Quinolone-Resistant Enterbacteriaceae <130> 03063-0430wp <140> <141> <150> 60/080375 <151> 1998-04-01 <160> <170> Patentln Ver. <210> 1 <211> 589 <212> DNA <213> Escherichia coli <400> 1 acaccggtca attgttggcc ctttacgcca gtcgttggtg atcgtccgta ttcggttcca aaaattgccc tatgacggta aacggttctt gaagtcatca acattgagga gtgcgctgcc tgaacgtact acgtaatcgg tggcgcagcc tcgacggcga atgaactgat cggaaaaaat ccggtatcgc acggttgtct agagctgaag agatgtccga aggcaatgac taaataccat attctcgctg ctctgcggcg ggctgatctc tccggacgtc cgtaggtatg ggcgtatatc agctcctatc gatggcctga tggaacaaag ccccatggtg cgttacatgc gcaatgcgtt gaaaaagaga atgccaacca gcaaccaaca gatgatgaag tggattatgc agccggtaca cctataaaaa actcggcggt tggtagacgg atacggaaat cggtcgattt aaattcctaa tcccgccgca acatcagca gatgtcggtc ccgtcgcgta atctgcccgt ttatgacacg tcagggtaac ccgtctggcg cgttgataac cctgctggtg caacctgacg <210> 2 <211> 589 <212> DNA <213> Citrobacter freundii <400> 2 acaccggtca attgttggcc ctttacgcca gtcgttggtg attgttcgta tttggttctg acattgagga gtgcgctgc tgaacgtatt acgtaatcgg tggcgcagcc tcgatggcga agagctgaag a ga cgt ccg a gggcaacgac taaataccac attctccttg ctccgcagcg agctcctatc gatggcctga tggaataaag cctcatggtg cgttacatgc gcgatgcgtt tggattatgc agccggttca cctataaaaa ataccgccgt tggtagatgg atacggaaat gatgtcggtc ccgtcgcgta atctgcccgt ttacgacacc tcagggtaac ccgtatgtcg WO 99/50458 WO 9950458PCT/US99/06963 aaaatcgccc atgagctgat ggctgacctg tacgacggca ccgaacaaat tcctgacgtc aacggttcgt ccggtatcgc ggtaggtatg gaagtgatca acggctgtct ggcatatatt <210> 3 <211> 589 <212> DNA <213> Enterobacter aerogenes gaaaaagaaa cggttgatit cgtcqataac atqccgacca aaattcctaa cctgctggtg gcgaccaaca ttccgccgca caacctgact gacgatgaag acatcagca <400> 3 acacgggtca attgttggcc ctatacgcca gtcgttggcg atcgtacgta tttggttctg aagatcgctc iatgacggca aacggttctt gaagttatca acattgagga gtgcgctgcc tgaacgtatt acgtaatcgg tggcgcagcc tcgatggcga atgagctgat cggagaaaat ccggtatcgc acggctgcct agagctgaaa ggatgtccga gggcaatgac taaataccac gttctccttg ctccgctgca ggccgatctc ccctgacgtc cgtaggtatg ggcatacgtt agctcgtatc ga tggcctga tggaacaaag ccgcatggtg cgttatatgc gcgatgcgtt gaaaaagaga atgccgacaa gcgaccaaca gataacgaag tggattatgc agccggtaca cctataaaaa ataccgccgt tggtcgatgg atacggaaat cggttgattt aaaiccctaa ttccgccgca acatcagca gatgtcggtc ccgtcgcgta atcagcccgt ttatgacacc ccagggtaac ccgtatgtcg cgtcgacaac cctgctggtg taacctgacg <210> 4 <211> 589 <212> DNA <213> Enterobacter cloacae <400> 4 acaccggtta attgttggcc ctatacgcca gtcgttggtg atcgttcgta tttggttcta aaaattgccc tacgatggca aacggttcgi gaagtgatca acatcgagga gtgcgctgcc tgaacgtatt acgtaatcgg tggcgcagcc tcgacggcga atgagctgat cggaaaaaat ccggtatcgc acggctgcct agagctgaag ggacgtccgc gggcaatgac taaataccat tttctcgctg ctccgccgcg ggccgacctg tcctgacgtc cgtagggatg agctcctatc gatggcctga tggaataaag ccccatggtg cgttacatgc gcaatgcgtt gaaaaagaga atgccaacga gcgaccaaca tggactatgc agccggtaca cctacaaaaa attccgcggt tggtagatgg atacggaaat cggttgattt agatccctaa ttccgccgca gatgtcggtc ccgtcgcgta atctgcccgt gtacgacacc tcagggtaac ccgtctggcg cgttgataac cctgctggtg caacatcacc 120 180 240 300 360 420 480 540 ggcctatatc gacgatgaag acatcagca <210> <211> 589 <212> DNA <213> Kiebsiella oxytoca <400> acaccggtca acatigagga agagctgaag agctcctatc tggattatgc gatgtcggtc attgttggcc gtgcgctgcc ggatgtccga gatggcctga agccggtaca ccgtcgcgia 120 ctatacgcca tgaacgtatt gggcaatgac tggaacaaag cctataaaaa atctgcccgt 180 WO 99/50458 WO 9950458PCT/US99/06963 gtcgtgggtq .ittqtacgta tttggttcg aagatcgccc tatgacggca aacggttcg~t gaagtgatca acgtca tcgg tqgcqcaqcc tcgacggcga atgaactgat cqgagaaaat ccggtatcgc acggctgtct taaataccac attctcccig ctccgccgca ggccgacctc ccctgacgtt ggtaggtatg ggcctacgtt cc t Citqq tq Cqttacatqc gcgatgcqt t gaaaaagaga atgccqacca gcgactaata gaaaacgaag ,Iat C t C tqgtagatgg a tacqgaaat cggtggattt aaatcccgaa ttccgccgca acatcagca c a c ga ca cc Ccagggtaac ccgtatgtcg cgtcgataac cctgctagtc caacctgacc <210> 6 <211> 589 <212> DNA <213> Kiebsiella pneumoniae <400> 6 acaccggtca attgttggcc ctttacgcca gtcgttggtg atcgtgcgta tttggttcca aaaatcgctc tatgacggta aacggcgcct gaagtgatta acattgagga gtgcgctgcc tgaacgtatt acgtaatcgq tggcgcagcc tcgacggcga atgagctgat cggagcgtat ccgggatcgc acggctgtct agagcttaag ggatgtccga gggcaatgac taaataccac gttctcgctg ctccgccgcg ggccgatctt tccggacgtc cgtagggatg ggcgtatgtt aactcttatc gatggcctga tggaacaaag ccgcacggcg cgttacatgc gcgatgcgtt gaaaaagaga atgccgacca gccaccaaca gacgatgaag tggattatgc a gc cggtac a cctataaaaa actccgcggt tggtggacqg ataccgaaat cggtcgattt aaattcctaa taccgccaca acatcagca gatgtcggtc ccgtcqgta atcagcccgjt atacgacacc ccagggtaac tcgtctggcg cgtcgacaac cctgctggtg taacctgacg <210> 7 <211> 589 <212> DNA <213> Providencia stuartii <400> 7 acaccggtca attgtcgggc ctgtttgcga atagtcgggg atcgttcgtc tttggttcag aaaattgcc tatgatggta aatggttcgt atatcgaaga gczgcgcttcc tgaatgtatt acgttatcgg ttgctcagcc ttgacggaga atgaaatgtt cagagcaaat caggtattgc aqaactcaaa agatgttcga gggaaatgat taaataccat tttttctatg ttccgcagct agcggatctt ccctgaagtt tgttgggatg agttcgtatt gatggactga tggaa taaac ccacatggtg cgttatatgc gcaatgcgtt gaaaaagaga atgcctacga gcaacgaaca tggattatgc agccagtaca cctataaaaa atagcgctgt tqgtagatgg atacggaaat ccgttgattt aaatccctaa ttcctccaca qatgtccgtt ccgcagagta atctgCCCgt ttatgagaca tcaggggaac ccgtatggcg cgtcccaaac cctattgqtt caacctaggg gaagtgatca gcggttgcct tgcttatata gatgatgaag atattagca <210> 8 <211> 589 <212> DNA <213> Serratia inarcescens <400> 8 WO 99/50458 PCTIUS99/06963 acaccggtaa attgtcggac ctgtacgcga gtcgtcgggg atcgtgcgta ttcggttCcg aagattgctC tatgatggca aacggctcgt gaagtcgtca acatcgaaga gtgccctgCC tgagcgtatt acgtgatcgg tggctcagcc tcgacggcga acgaactgtt ccgagcagat cgggcatcgC acggctgcct cgagttgaaa agatgttcgt gggtaacgac taaatatcac gttttcactg ctccgcggcg ggcggatctg cccggccgtc cgtgggcatg ggcctatatc aactcgtatc gatggactga tggaataaac ccgcacggtg cgctacatgc gcgatgcgtt gaaaaagaaa atgccgacca gctaccaata gacgatgaaa tggactatgc agccggitca catacaagaa acagcgcggt tggtggacgg ataccgaagt ccgtcgactt agatcccgaa ttccgccgca acatcagca gatgtccgtt ccgccgcgtt atcggcccgt ttacgacact tcagggtaac gcgcatgtcc cgtgcctaac cctgctggtC caacctggcg <210> 9 <211> 120 <212> DNA <213> Escherichia coli <400> 9 gcccgtgtcg ttggtgacgt aatcggtaaa taccatcccc atggtgactc ggcggtttat gacacgatcg tccgtatggc gcagccattc tcgctgcgtt acatgctggt agacggtcag 120 <210> <211> 120 (212> DNA (213> Citrobacter freundii (400> gcccgtgtcg ttggtgacgt aatcggtaaa taccaccctc atggtgatac cgccgtttac gacaccattg ttcgtatggc gcagccattc tccttgcgtt acatgctggt agatggtcaq 120 (210> 11 (211> 120 (212> DNA (213> Enterobacter aerogenes <400> 11 gcccgtgtcg ttggcqacgt aatcggtaaa taccacccgc atggtgatac cgccgtttat gacaccatcg tacgtatggc gcagccgttc tccttgcgtt atatgctggt cgatggccag 120 <210> 12 '211> 120 <212> DNA (213> Enterobacter cloacae (400> 12 gcccgtgtcg ttggtgacqt aatcggtaaa taccatcccc atggtgattc cgcggtgtac gacaccatcg ttcgtatggc gcaqcctttc tcgctgcgtt acatgctggt agatggtcag 120 (210> 13 WO 99/50458 PTU9/66 PCT/US99/06963 <211> 120 <212> DNA <213> Kiebsiella oxytoca <400> 13 gcccgtgtcg tgggtgacgt catcggtaaa taccaccctc atggtgatac tgccgtatac gacaccattg tacgtatggc gcagccattc tccctgcgtt acatgctggt agatggccag 120 <210> 14 <211> 120 <212> DNA <213> Kiebsiella pneumoriiae <400> 14 gcccgtgtcg ttggtgacgt aatcggtaaa taccacccgc acggcgactc cgcggtatac gacaccatcg tgcgtatggc gcagccgttc tcgctgcgtt acatgctggt ggacggccag 120 <210> <211> 120 <212> DNA <213> Providencia stuartii <400> gcccgtatag tcggggacgt tatcggtaaa taccatccac atggtgatag cgctgtttat gagacaatcg ttcgtcttgc tcagcctttt tctatgcgtt atatgctggt agatggtcag 120 <210> 16 <211> 120 <212> DNA <213> Serratia marcescens <400> 16 gcccgtgtcg tcggggacgt gatcggtaaa tatcacccgc acggtgacag cgcggtttac gacactateg tgcgtatggc tcagccgttt tcactgcgct acatgctggt ggacggtcag 120 <210> 17 <211> <212> DNA <213> Escherichia coli <400> 17 actttacgcc atgaacgtac taggc <210> 18 <211> 23 <212> DNA <213> Citrobacter freundii WO 99/50458 WO 9950458PCTIUS99/06963 <400> 18 tgggcaacga ctggaataaa gcc 23 <210> 19 <211> 22 <212> DNA <213> Enterobacter aerogenes <400> 19 ttatatgctg gtcgatggcc ag 22 <210> <211> 21 <212> DNA <213> Enterobacter cloacae <400> gccggacgtc cgcgatggcc t 21 <210> 21 <211> <212> DNA <213> Kiebsiella oxytoca <400> 21 gtagatggcc agggtaactt tggttcggtc <210> 22 <211> 27 <212> DNA <213> Kiebsiella pneumoniae <400> 22 gtgcgtatgg cgcagccgtt ctcgctg 27 <210> 23 <211> <212> DNA <213> Providencia stuartii <400> 23 cgtcttgctc agcctttttc tatgc <210> 24 <211> <212> DNA <213> Serratia inarcescens WO 99/50458 WO 9950458PCT/US99/06963 <400> 24 ggaataaacc atacaagaaa <210> <211> (212> DNA <213> Escherichia coli <400> atggtgactc ggcggtttat gacac <210> 26 <211> <212> DNA <213> Escherichia coli <400> 26 atggtgactc ggcggtctat gacac (210> 27 <211> <212> DNA <213> Citrobacter freundii (400> 27 atggtgatac cgccgtttac gacac <210> 28 <211> <212> DNA <213> Enterobacter aerogenes <400> 28 atggtgatac cgccgtttat gacac <210> 29 <211> <212> DNA <213> Enterobacter cloacae <400> 29 atggtgatte cgcggtgtac gacac <210> <211> <212> DNA <213> Kiebsiella oxytoca WO 99/50458 WO 9950458PCTIUS99/06963 <400> atggtgatac tgccgtatac gacac <210> 31 <211> <212> DNA <213> Kiebsiella pneumoniae <400> 31 acggcgactc cgcggtatac gacac <210> 32 <211> <212> DNA <213> Providencia stuartii <400> 32 atggtgatag cgctgtttat gagac <210> 33 <211> <212> DNA <213> Serratia marcescens <400> 33 acggtgacag cgcggtttac gacaC <210> 34 <211> 18 <212> DNA <213> Enterobacter sp.

<400> 34 cgaccttgcg agagaaat 18 <210> <211> 18 <212> DNA <213> Enterobacter sp.

<400> gttccatcag cccttcaa 18

Claims

1. An isolated nucleic acid probe, comprising 10 to 50 consecutive nucleotides of one of the gyrA genes of Escherichia coli (SEQ ID NO: Enterobacter aerogenes (SEQ ID NO: Enterobacter cloacae (SEQ ID NO: Klebsiella oxytoca (SEQ ID NO: Klebsiella pneumoniae (SEQ ID NO: or Providencia stuartii (SEQ ID NO: wherein the probe selectively hybridizes under stringent conditions to a single gyrA gene set forth in SEQ ID NOs: 1-8, or a complementary sequence thereof, but does not hybridize to any of the other gyrA genes set forth in SEQ ID NOs: 1-8, wherein the probe identifies an Enterobacteriaceae species as Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, or Providencia stuartii.

2. The isolated nucleic acid probe of claim 1, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Escherichia coli (SEQ ID NO: or a complementary sequence thereof.

3. The isolated nucleic acid probe of claim 1, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Enterobacter aerogenes (SEQ ID NO: or a complementary sequence thereof

4. The isolated nucleic acid probe of claim 1, wherein the probe selectively .hybridizes under stringent conditions to the gyrA gene of Enterobacter cloacae (SEQ ID NO: or a complementary sequence thereof.

5. The isolated nucleic acid probe of claim 1, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Klebsiella oxytoca (SEQ ID NO: or a complementary sequence thereof

6. The isolated nucleic acid probe of claim 1, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Klebsiella pneumoniae (SEQ ID NO: or a complementary sequence thereof.

7. The isolated nucleic acid probe of claim 1, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Providencia stuartii (SEQ ID NO: or a complementary sequence thereof. 24

8. An isolated nucleic acid probe, consisting essentially of 10 to 50 consecutive nucleotides of one of the gyrA genes of Citrobacter freundii (SEQ ID NO: or Serratia marcescens (SEQ ID NO: wherein the probe selectively hybridizes under stringent conditions to a single gyrA gene set forth in SEQ ID NOs: 1-8, or a complementary sequence thereof, but does not hybridize to any of the other gyrA genes set forth in SEQ ID NOs: 1-8, wherein the probe identifies an Enterobacteriaceae species as Citrobacterfreundii or Serratia marcescens.

9. The isolated nucleic acid probe of claim 8, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Citrobacterfreundii (SEQ ID NO: or a complementary sequence thereof.

The isolated nucleic acid probe of claim 8, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Serratia marcescens (SEQ ID NO: or a complementary sequence thereof.

11. An isolated nucleic acid probe having a nucleic acid sequence of SEQ ID NO: 17, 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

12. An isolated nucleic acid probe, comprising 10 to 50 consecutive nucleotides of one of the gyrA genes of Escherichia coli (SEQ ID NO: Enterobacter aerogenes (SEQ ID NO: Klebsiella oxytoca (SEQ ID NO: Klebsiella pneumoniae (SEQ ID NO: or Providencia stuartii (SEQ ID NO: wherein the probe selectively hybridizes under stringent conditions to a single gyrA gene set forth in SEQ ID NOs: 1- 8, or a complementary sequence thereof, wherein a quinolone susceptible strain selectively hybridizes to the probe, and wherein a quinolone resistant strain has a one or more base pair mismatch with the probe. 30

13. The isolated nucleic acid probe of claim 12, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Escherichia coli (SEQ ID NO: or a complementary sequence thereof.

14. The isolated nucleic acid probe of claim 12, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Enterobacter aerogenes (SEQ ID NO: or a complementary sequence thereof.

The isolated nucleic acid probe of claim 12, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Klebsiella oxytoca (SEQ ID NO: or a complementary sequence thereof.

16. The isolated nucleic acid probe of claim 12, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Klebsiella pneumoniae (SEQ ID NO: or a complementary sequence thereof.

17. The isolated nucleic acid probe of claim 12, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene ofProvidencia stuartii (SEQ ID NO: or a complementary sequence thereof.

18. An isolated nucleic acid probe, consisting essentially of 10 to 50 consecutive nucleotides of one of the gyrA genes of Citrobacter freundii (SEQ ID NO: 2), Enterobacter cloacae (SEQ ID NO: or Serratia marcescens (SEQ ID NO: 8), wherein the probe selectively hybridizes under stringent conditions to a single gyrA gene set forth in SEQ ID NOs: 1-8, or a complementary sequence thereof, wherein a quinolone susceptible strain selectively hybridizes to the probe, and wherein a quinolone resistant strain has a one or more base pair mismatch with the probe.

19. The isolated nucleic acid probe of claim 18, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Citrobacterfreundii (SEQ ID NO: or a complementary sequence thereof.

20. The isolated nucleic acid probe of claim 18, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Enterobacter cloacae (SEQ ID NO: or a complementary sequence thereof.

21. The isolated nucleic acid probe of claim 18, wherein the probe selectively 30 hybridizes under stringent conditions to the gyrA gene of Serratia marcescens (SEQ ID NO: or a complementary sequence thereof. *e

22. An isolated nucleic acid probe having a nucleic acid sequence of SEQ ID NO: SEQ ID NO: 26, 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.

23. A method of identifying in a sample an Enterobacteriaceae species selected from the group consisting of Escherichia coli (SEQ ID NO: Citrobacter freundii (SEQ ID NO: Enterobacter aerogenes (SEQ ID NO: Enterobacter cloacae (SEQ ID NO: Klebsiella oxytoca (SEQ ID NO: Klebsiella pneumoniae (SEQ ID NO: Providencia stuartii (SEQ ID NO: and Serratia marcescens (SEQ ID NO: 8), comprising combining the sample with the isolated nucleic acid probe according to any one of claims 1 to 11, wherein the probe selectively hybridizes under stringent conditions to a single gyrA gene set forth in SEQ ID NOs: 1-8, or a complementary sequence thereof, but does not hybridize to any of the other gyrA genes set forth in SEQ ID NOs: 1-8, wherein the presence of hybridization with the single gyrA gene identifies the Enterobacteriaceae species as Escherichia coli, Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, Providencia stuartii, or Serratia marcescens.

24. The method of claim 23, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Escherichia coli (SEQ ID NO: or a complementary sequence thereof, the presence of hybridization identifying Escherichia coli in the sample. 20

25. The method of claim 23, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Citrobacter freundii (SEQ ID NO: or a complementary sequence thereof, the presence of hybridization identifying Citrobacter freundii in the sample.

26. The method of claim 23, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Enterobacter aerogenes (SEQ ID NO: or a complementary sequence thereof, the presence of hybridization identifying Enterobacter aerogenes in the sample.

27. The method of claim 23, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Enterobacter cloacae (SEQ ID NO: or a *complementary sequence thereof, the presence of hybridization identifying Enterobacter cloacae in the sample.

28. The method of claim 23, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Klebsiella oxytoca (SEQ ID NO: or a complementary sequence thereof, the presence of hybridization identifying Klebsiella oxytoca in the sample.

29. The method of claim 23, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Klebsiella pneumoniae (SEQ ID NO: or a complementary sequence thereof, the presence of hybridization identifying Klebsiella pneumoniae in the sample.

The method of claim 23, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Providencia stuartii (SEQ ID NO: or a complementary sequence thereof, the presence of hybridization identifying Providencia stuartii in the sample.

31. The method of claim 23, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Serratia marcescens (SEQ ID NO: or a complementary sequence thereof, the presence of hybridization identifying Serratia marcescens in the sample.

32. A method of determining in a sample the quinolone resistance of an Enterobacteriaceae species selected from the group consisting of Escherichia coli (SEQ ID NO: Citrobacterfreundii (SEQ ID NO: Enterobacter aerogenes (SEQ ID NO: Enterobacter cloacae (SEQ ID NO: Klebsiella oxytoca (SEQ ID NO: Klebsiella pneumoniae (SEQ ID NO: Providencia stuartii (SEQ ID NO: and Serratia marcescens (SEQ ID NO: comprising combining the sample with the isolated nucleic acid probe according to any one of claims 12 to 22, wherein the probe "selectively hybridizes under stringent conditions to a single gyrA gene set forth in SEQ ID NOs: 9-16, or a complementary sequence thereof, the presence of hybridization with the single gyrA gene indicating the quinolone resistance of the species, wherein a quinolone susceptible strain selectively hybridizes to the probe, and wherein a quinolone resistant strain has a one or more base pair mismatch with the probe. 28

33. The method of claim 32, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Escherichia coli (SEQ ID NO: or a complementary sequence thereof, the presence of hybridization indicating the quinolone resistance of the Escherichia coli in the sample.

34. The method of claim 32, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Citrobacter freundii (SEQ ID NO: 10), or a complementary sequence thereof, the presence of hybridization indicating the quinolone resistance of the Citrobacterfreundii in the sample.

The method of claim 32, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene ofEnterobacter aerogenes (SEQ ID NO: 11), or a complementary sequence thereof, the presence of hybridization indicating the quinolone resistance of the Enterobacter aerogenes in the sample.

36. The method of claim 32, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Enterobacter cloacae (SEQ ID NO: 12), or a complementary sequence thereof, the presence of hybridization indicating the quinolone resistance of the Enterobacter cloacae in the sample.

37. The method of claim 32, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Klebsiella oxytoca (SEQ ID NO: 13), or a complementary sequence thereof, the presence of hybridization indicating the quinolone resistance of the Klebsiella oxytoca in the sample.

38. The method of claim 32, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Klebsiella pneumoniae (SEQ ID NO: 14), or a complementary sequence thereof, the presence of hybridization indicating the S: quinolone resistance of the Klebsiellapneumoniae in the sample.

39. The method of claim 32, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Providencia stuartii (SEQ ID NO: 15), or a complementary sequence thereof, the presence of hybridization indicating the quinolone resistance of the Providencia stuartii in the sample. The method of claim 32, wherein the probe selectively hybridizes under stringent conditions to the gyrA gene of Serratia marcescens (SEQ ID NO: 16), or a complementary sequence thereof, the presence of hybridization indicating the quinolone resistance of the Serratia marcescens in the sample. Dated this twentieth day of March 2003 The Government of the United States of America, represented by The Secretary of the Department of Health and Human Services Patent Attorneys for the Applicant: F B RICE CO *°ee :ee *ee .e eeo