Oligonucleotide probes for detecting enterobacteriaceae and quinolone-resistant enterobacteriaceae

Abstract

Oligonucleotide probes for detecting Enterobacteriaceae species. Unique gyrA coding regions permit the development of probes specific for eight different species: Escherichia coli, Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, Providencia stuartii and Serratia marcescens . The invention thereby provides methods for the species-specific identification of these Enterobacteriaceae in a sample, and detection and diagnosis of Enterobacteriaceae infection in a subject. Further, nucleic acids are provided for determining quinolone-resistant status of these Enterobacteriaceae.

1 . An isolated nucleic acid probe of 20 to 50 nucleotides in length for identifying an Enterobacteriaceae species in a sample, wherein the probe is at least 90% homologous to the 5′ region of the gyraseA gene of an Enterobacteriaceae species selected from the group consisting of  Citrobacter freundii  (SEQ ID NO: 2),  Enterobacter aerogenes  (SEQ ID NO: 3),  Enterobacter cloacae  (SEQ ID NO: 4),  Klebsiella oxytoca  (SEQ ID NO: 5),  Klebsiella pneumoniae  (SEQ ID NO: 6),  Providencia stuartii  (SEQ ID NO: 7),  Serratia marcescens  (SEQ ID NO: 8), a complementary sequence of anone of SEQ ID NOS: 2-8, but is not at least 90% homologous to the to the 5′ region of the gyraseA gene of.  Escherichia coli  (SEQ ID NO: 1) or the complementary sequence thereof. 
     
     
         2 . The isolated nucleic acid probe of  claim 1 , comprising the nucleotide sequence set forth as one of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24, or a complementary sequence thereof. 
     
     
         3 . The isolated nucleic acid probe of  claim 1 , length for identifying an Enterobacteriaceae species in a sample, wherein the probe is at least 95% homologous to the 5′ region of the gyraseA gene of  Citrobacter freundii  (SEQ ID NO: 2),  Enterobacter aerogenes  (SEQ ID NO: 3),  Enterobacter cloacae  (SEQ ID NO: 4),  Klebsiella oxytoca  (SEQ ID NO: 5),  Klebsiella pneumoniae  (SEQ ID NO: 6),  Providencia stuartii  (SEQ ID NO: 7), or  Serratia marcescens  (SEQ ID NO: 8), or a complementary sequence thereof, but is not at least 95% homologous to the to the 5′ region of the gyraseA gene of.  Escherichia coli  (SEQ ID NO: 1) or the complementary sequence thereof. 
     
     
         4 . The isolated nuclear acid probe of  claim 1 , wherein the probe is at least 100% homologous to the 5′ region of the gyraseA gene of  Citrobacter freundii  (SEQ ID NO: 2),  Enterobacter aerogenes  (SEQ ID NO: 3),  Enterobacter cloacae  (SEQ ID NO: 4),  Klebsiella oxytoca  (SEQ ID NO: 5),  Klebsiella pneumoniae  (SEQ ID NO: 6),  Providencia stuartii  (SEQ ID NO: 7), or  Serratia marcescens  (SEQ ID NO: 8), or a complementary sequence thereof, but is not at least 100% homologous to the to the 5′ region of the gyraseA gene of.  Escherichia coli  (SEQ ID NO: 1) or the complementary sequence thereof. 
     
     
         5 . A method of identifying in a sample an Enterobacteriaceae species selected from the group consisting of  Escherichia coli, Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, Providencia stuartii  and  Serratia marcescens , comprising:
 contacting the sample with a nucleic acid probe of  claim 1 ; and   detecting the presence of hybridization with a nucleic acid indicating the presence of the respective species;   thereby identifying an Enterobacteriaceae species selected from the group consisting of  Escherichia coli, Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, Providencia stuartii  and  Serratia marcescens  in the sample.   
     
     
         6 . An isolated nucleic acid probe of 20 to 50 nucleotides in length capable of determining quinolone resistance status of an Enterobacteriaceae species selected from the group consisting of  Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella pneumoniae, Providencia stuartii  and  Serratia marcescens  in a sample, wherein the probe hybridizes to the 5′ region of the gyraseA gene of  Citrobacter freundii  (SEQ ID NO: 2),  Enterobacter aerogenes  (SEQ ID NO: 3),  Enterobacter cloacae  (SEQ ID NO: 4),  Klebsiella oxytoca  (SEQ ID NO: 5),  Klebsiella pneumoniae  (SEQ ID NO: 6),  Providencia stuartii  (SEQ ID NO: 7), or  Serratia marcescens  (SEQ ID NO: 8), or a fully complementary sequence thereof, and wherein a nucleic acid a quinolone susceptible strain hybridizes to the probe, and wherein a nucleic acid a quinolone resistant strain has a one or more base pair mismatch with the probe. 
     
     
         7 . The probe of  claim 6 , comprising the nucleotide sequence set forth as one of SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO: 33, or a complementary sequence thereof. 
     
     
         8 . The probe of  claim 6 , wherein the probe is labeled. 
     
     
         9 . A method for determining the quinolone resistance of an Enterobacteriaceae species selected from the group consisting of  Escherichia coli Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, Klebsiella oxytoca, Klebsiella Pneumoniae, Providencia stuartii  and  Serratia marcescens  in a sample, comprising
 contacting the sample with a nucleic acid probe, wherein the probe selectively hybridizes to a nucleic acid sequence set forth as one of SEQ ID NOs: 9-16, or a complementary sequence thereof, respectively, the presence of hybridization with a nucleic acid indicating the quinolone susceptibility of the respective species.   
     
     
         10 . The method of determining the quinolone resistance status of an Enterobacteriaceae species of  claim 9 , comprising contacting the sample with a nucleic acid probe, wherein the probe selectively hybridizes to a nucleic acid of SEQ ID NO: 9, or a complementary sequence thereof, the presence of hybridization indicating quinolone resistance of the  Escherichia coli  in the sample. 
     
     
         11 . The method of determining the quinolone resistance status of an Enterobacteriaceae species of  claim 9 , wherein hybridization of the probe to the nucleic acid sequence of SEQ ID NOs: 1-9 indicates that the Enterobacteriaceae species is susceptible to quinolone and a one or more base pair mismatch of the probe to the nucleic acid sequence of one or more of SEQ ID NOs: 1-9 indicates that Enterobacteriaceae species is resistant to quinolone. 
     
     
         12 . The method of  claim 9 , wherein the probe is from about 10 to 50 nucleotides in length. 
     
     
         13 . The method of  claim 9 , wherein the probe consists of the nucleic acid sequence set forth as one of SEQ ID NOs: 25-33. 
     
     
         14 . The method of  claim 10 , wherein the probe selectively hybridizes to nucleotides 25 to 613 of SEQ ID NO: 9. 
     
     
         15 . The method of  claim 10 , wherein the probe selectively hybridizes to nucleotides 199 to 318 of SEQ ID NO:9, or a complementary sequence thereof. 
     
     
         16 . The method of  claim 10 , wherein the probe selectively hybridizes to nucleotides 239 to 663 of SEQ ID NO: 9, or a complementary sequence thereof. 
     
     
         17 . The method of  claim 9 , wherein the probe is about 25 nucleotides in length. 
     
     
         18 . The method of  claim 9 , wherein the method comprises the use of a polymerase chain reaction (PCR), ligase chain reaction, or a nucleotide array. 
     
     
         19 . The method of  claim 9 , wherein the probe is labeled. 
     
     
         20 . The method of  claim 10 , wherein the nucleic acid sequence set forth as SEQ ID NO: 9 is amplified prior to combining the sample with the nucleic acid probe.