Specific and universal probes and amplification primers to rapidly detect and identify common bacterial pathogens and antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories

Abstract

The present invention relates to DNA-based methods for universal bacterial detection, for specific detection of the common bacterial pathogens Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Staphylococcus saprophyticus, Streptococcus pyogenes, Haemophilus influenzae and Moraxella catarrhalis as well as for specific detection of commonly encountered and clinically relevant bacterial antibiotic resistance genes directly from clinical specimens or, alternatively, from a bacterial colony. The above bacterial species can account for as much as 80% of bacterial pathogens isolated in routine microbiology laboratories. The core of this invention consists primarily of the DNA sequences from all species-specific genomic DNA fragments selected by hybridization from genomic libraries or, alternatively, selected from data banks as well as any oligonucleotide sequences derived from these sequences which can be used as probes or amplification primers for PCR or any other nucleic acid amplification methods. This invention also includes DNA sequences from the selected clinically relevant antibiotic resistance genes. With these methods, bacteria can be detected (universal primers and/or probes) and identified (species-specific primers and/or probes) directly from the clinical specimens or from an isolated bacterial colony. Bacteria are further evaluated for their putative susceptibility to antibiotics by resistance gene detection (antibiotic resistance gene specific primers and/or probes). Diagnostic kits for the detection of the presence, for the bacterial identification of the above-mentioned bacterial species and for the detection of antibiotic resistance genes are also claimed. These kits for the rapid (one hour or less) and accurate diagnosis of bacterial infections and antibiotic resistance will gradually replace conventional methods currently used in clinical microbiology laboratories for routine diagnosis. They should provide tools to clinicians to help prescribe promptly optimal treatments when necessary. Consequently, these tests should contribute to saving human lives, rationalizing treatment, reducing the development of antibiotic resistance and avoid unnecessary hospitalizations.

1 . A method to detect and identify the presence of a at least three target bacterial species in a sample, from amongst a plurality of possible bacterial species comprising, 
 contacting the sample with a set of amplification primers comprising a plurality of different amplification primer pairs, wherein said plurality of primer pairs comprises a first, second and third primer pair directed against target DNA from a first, second and third target bacterial species, respectively, wherein    (i) said first, second and third primer pairs hybridize solely to target DNA of said first, second and third target bacterial species, respectively, and are ubiquitous to at least 80% to said first, second and third target bacterial species, respectively, and;    (ii) each of said first, second and third primer pairs is derived from a DNA fragment that also hybridizes solely to said first, second and third target bacterial species, respectively, and is ubiquitous to at least about 80% of said first, second and third target bacterial species, respectively;    (iii) the primers are all chosen to allow amplification, under a single amplification protocol,    allowing amplification to proceed under said single amplification protocol; and    detecting the presence or amount of amplified product(s) from said first, second and third primer pairs as an indication of the presence of the first, second, or third target bacterial species in the sample, respectively.    
     
     
         2 . The method of  claim 1 , wherein at least one of said plurality of primer pairs has a ubiquity of less than 100% for DNA of one of said target bacterial species, and wherein said set of amplification primers further comprises at least one additional pair of amplification primers for said target bacterial species  
     
     
         3 . The method of  claim 1 , further comprising 
 contacting said sample with at least one universal amplification primer, wherein said at least one universal primer is chosen to allow amplification under said single amplification protocol, and    allowing amplification to proceed under said single amplification protocol.    
     
     
         4 . The method of  claim 1 , further comprising 
 contacting said sample with at least one antibiotic resistance gene amplification primer pair that hybridizes to a target antibiotic resistance gene, wherein said at least one antibiotic resistance gene primer pair is chosen to allow amplification under said single amplification protocol, and    allowing amplification to proceed under said single amplification protocol.    
     
     
         5 . The method of  claim 1 , wherein at least one target bacterial species is selected from the group consisting of  Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Staphylococcus saprophyticus, Streptococcus pyogenes, Haemophilus influenzae, Moraxella catarrhalis  and group A  Streptococci  bearing exotoxin A gene speA.  
     
     
         6 . The method of  claim 4 , wherein said antibiotic resistance gene is selected from the group consisting of bla tem , bla rob , bla shv , aadb, aacC1, aacC2, aacC3, aacA4, mecA, vanA, vanH, vanx, satA, aacA-aphD, vat, vga, msrA, sul, and int.  
     
     
         7 . The method of  claim 1 , wherein said DNA fragment(s) is or are selected from: 
 SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and a complementary sequence thereof, for determining the presence or amount of  Escherichia coli;      SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and a complementary sequence thereof, for determining the presence or amount of  Klebsiella pneumoniae;      SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and a complementary sequence thereof, for determining the presence or amount of  Pseudomonas aeruginosa;      SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and complementary sequence thereof, for determining the presence or amount of  Proteus mirabilis;      SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 35 and complementary sequence thereof, for determining the presence or amount of  Streptococcus pneumoniae;      SEQ ID NO: 37 and a complementary sequence thereof, for determining the presence or amount of  Staphylococcus aureus;      SEQ ID NO: 36 and a complementary sequence thereof, for determining the presence or amount of  Staphylococcus epidermidis;      SEQ ID NO: 1, SEQ ID NO: 2 and a complementary sequence thereof, for determining the presence or amount of  Enterococcus faecalis;      SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and a complementary sequence thereof, for determining the presence or amount of  Staphylococcus saprophyticus;      SEQ ID NO: 33, and a complementary sequence thereof, for determining the presence or amount of a bacterial species bearing exotoxin A gene speA;    SEQ ID NO: 32 and a complementary sequence thereof, for determining the presence or amount of  Streptococcus pyogenes;      SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and a complementary sequence thereof, for determining the presence or amount of  Haemophilus influenzae,  and    SEQ ID NO: 28, SEQ ID NO: 29 and a complementary sequence thereof, for determining the presence or amount of  Moraxella catarrhalis.      
     
     
         8 . The method of  claim 1 , further comprising performing said method directly on a sample obtained from human patients, animals, environment or food.  
     
     
         9 . The method of  claim 1 , further comprising performing said method directly on a sample consisting of one or more bacterial colonies.  
     
     
         10 . The method of  claim 1 , wherein said amplification step comprises a method selected from the group consisting of: 
 (a) polymerase chain reaction (PCR),    (b) ligase chain reaction,    (c) nucleic acid sequence-based amplification,    (d) self-sustained sequence replication,    (e) strand displacement amplification,    (f) branched DNA signal amplification,    (g) nested PCR, and    (h) multiplex PCR.    
     
     
         11 . The method of  claim 10 , wherein said amplification step comprises PCR.  
     
     
         12 . The method of  claim 11 , wherein said PCR comprises an annealing step of only one second at about 55° C. and a denaturation step of only one second at about 95° C. without any time specifically allocated to an elongation step.  
     
     
         13 . The method of  claim 1 , wherein said amplification and detection comprises: 
 (a) contacting said sample with an aqueous solution comprising said first, second and third oligonucleotide primers pairs,    wherein each primer is at least twelve nucleotides in length,    wherein each primer pair comprises a first and a second oligonucleotide primer,    wherein said first primers of said first, second, and third primer pairs hybridize solely with one of the two complementary strands of said first, second, or third target bacterial species DNA, respectively, comprising of any one of said sequences for determining the presence or amount of said bacterial species, that contain a target sequence, and    wherein said second primer of said primer pairs hybridizes solely with the other strands of said target DNA of said first second, or third target bacterial species such that each primer pair can form an extension product which contains the target sequence as a template;    (b) synthesizing an extension product of each of said primers which extension product contains the target sequence, and amplifying said target sequence, if any, to a detectable level; and    (c) detecting the presence or amount of said amplified target sequence as an indication of the presence or amount of said target bacterial species in said test sample.    
     
     
         14 . The method of  claim 13 , wherein step (c) further comprises hybridizing said amplified target sequence with a probe.  
     
     
         15 . The method of  claim 1 , wherein said plurality of different amplification primer pairs comprises at least one amplification primer pair selected from the group consisting of: 
 SEQ ID NO: 112 and SEQ ID NO: 113,    SEQ ID NO: 118 and SEQ ID NO: 119, and    SEQ ID NO: 160 and SEQ ID NO: 119 for the detection of  Moraxella catarrhalis;      SEQ ID NO: 83 and SEQ ID NO: 84, and    SEQ ID NO: 85 and SEQ ID NO: 86; for the detection of  Pseudomonas aeruginosa;      SEQ ID NO: 145 and SEQ ID NO: 146, and    SEQ ID NO: 147 and SEQ ID NO: 148, for the detection of  Staphylococcus epidermidis;      SEQ ID NO: 149 and SEQ ID NO: 150,    SEQ ID NO: 149 and SEQ ID NO: 151, and    SEQ ID NO: 152 and SEQ ID NO: 153, for the detection of  Staphylococcus aureus;      SEQ ID NO: 78 and SEQ ID NO: 79,    SEQ ID NO: 156 and SEQ ID NO: 157, and    SEQ ID NO: 158 and SEQ ID NO: 159; for the detection of  Streptococcus pneumoniae;      SEQ ID NO: 143 and SEQ ID NO: 144, for the detection of a bacterial species bearing exotoxin A gene speA,    SEQ ID NO: 141 and 142, for the detection of  Streptococcus pyogenes,      SEQ ID NO: 38 and SEQ ID NO: 39, and    SEQ ID NO: 40 and SEQ ID NO: 41, for the detection of  Enterococcus faecalis,      SEQ ID NO: 61 and SEQ ID NO: 62,    SEQ ID NO: 67 and SEQ ID NO: 68,    SEQ ID NO: 135 and SEQ ID NO: 136, and    SEQ ID NO: 137 and SEQ ID NO: 138, for the detection of  Klebsiella pneumoniae;      SEQ ID NO: 74 and SEQ ID NO: 75, and    SEQ ID NO: 133 and SEQ ID NO: 134, for the detection of  Proteus mirabilis;      SEQ ID NO: 98 and SEQ ID NO: 99, and    SEQ ID NO: 139 and SEQ ID NO: 140, for the detection of  Staphylococcus saprophyticus;  or    SEQ ID NO: 154 and SEQ ID NO: 155, for the detection of  Haemophilus influenzae.