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Definitions

• This invention relates in general to the field of diagnostic microbiology.
• the invention relates to the species-specific detection of Enterobacteriaceae .
• 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.
• 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.
• Many of these infections are life threatening and are often nosocomial (hospital-acquired) infections.
• Schoaberg et al. The Am
• 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, /. Antimicrob. Chemother., 30:409-416 (1992)).
• 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.
• 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)).
• DNA gyrase a type II topoisomerase required for DNA replication and transcription.
• DNA gyrase composed of two A subunits and two B subunits, is encoded by the gyrA and gyr genes. Resistance to quinolones has been shown to be associated most frequently with alterations in gyrA.
• 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.
• 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.
• Figure 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.
• QRDR quinolone resistance-determining region
• FIG. 3 shows the deduced amino acid sequences (SEQ ID NO: 1
• 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.
• 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.
• 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.
• 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.
• 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.
• the invention provides isolated nucleic acids from Escherichia coli (SEQ ID NO: l), 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).
• Figures 1A and IB 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), 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 Figure 2.
• 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).
• 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).
• 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: l-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:l-8.
• isolated nucleic acid free from at least some of the components with which it naturally occurs.
• 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.
• selective 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 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.
• 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.
• 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. Nat I. 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.
• 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.
• the target region can range between 70% complementary bases and full complementarity and still hybridize under stringent conditions.
• the degree of complementarity between the nucleic acid (probe or primer) and the target sequence to which it hybridizes 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.
• 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, theinvention 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.
• 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).
• GYRA6 SEQ ID NO: 34
• 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.
• detection of nucleic acid hybridization can be facilitated by the use of reporter or detection moieties.
• 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.
• detectable 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.
• 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.
• 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.
• 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.
• 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
• 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 Rh ⁇ ne-Poulenc Rorer (Collegeville, PA).
• 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 S'-CGACCTTGCGAGAGAAAT-S' (SEQ ID NO:34)
• 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 ly sates. Amplifications were carried out in a Gene Amp 9600 PCR System (Perkin-Elmer, Applied Biosystems Division, Foster City, CA) in 50 ⁇ l volume containing 50 pmol of each primer, 200 ⁇ M deoxynucleoside triphosphates, 10 ul cell lysate containing approximately 100 ng template DNA, IX reaction buffer with 1.5 mM MgCl2 and 1 U native Taq polymerase (Perkin Elmer).
• 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).
• 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. 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 an ⁇ K. 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 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).
• the QRDR was identical to the sequence published by Kim et al. (ATCC 1475 ⁇ )(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 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.
• 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 (>.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)).
• high levels of resistance were found in strains with single as well as double gyrA mutations.
• pneumoniae isolates exhibited either single or double mutations involving Ser-83 and Asp-87, and ciprofloxacin MICs ranged from 1 - 16 ⁇ 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. 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 He, similar to C. freundii and E. aerogenes. MICs associated with this alteration ranged from 0.5 - 16 ⁇ g/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.
• 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 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.
• GTC 3' (SEQ ID NO:21) (307-336)
• 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.

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