US20050042606A9

US20050042606A9 - 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

Info

Publication number: US20050042606A9
Application number: US10/121,120
Authority: US
Inventors: Michel Bergeron; Marc Ouellette; Paul Roy
Original assignee: Bergeron Michel G.; Marc Ouellette; Roy Paul H.
Current assignee: GeneOhm Sciences Canada Inc
Priority date: 1994-09-12
Filing date: 2002-04-11
Publication date: 2005-02-24
Also published as: US20090053703A1; US20020055101A1; US20030180733A1; US20070105129A1; US7943346B2; US20070009947A1

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.

Description

BACKGROUND OF THE INVENTION

Classical Identification of Bacteria
Bacteria are classically identified by their ability to utilize different substrates as a source of carbon and nitrogen through the use of biochemical tests such as the API20E™ system. Susceptibility testing of Gram negative bacilli has progressed to microdilution tests. Although the API and the microdilution systems are cost-effective, at least two days are required to obtain preliminary results due to the necessity of two successive overnight incubations to isolate and identify the bacteria from the specimen. Some faster detection methods with sophisticated and expensive apparatus have been developed. For example, the fastest identification system, the autoSCAN-Walk-Away system™ identifies both Gram negative and Gram positive from isolated bacterial colonies in 2 hours and susceptibility patterns to antibiotics in only 7 hours. However, this system has an unacceptable margin of error, especially with bacterial species other than Enterobacteriaceae (York et al., 1992. J. Clin. Microbiol. 30:2903-2910). Nevertheless, even this fastest method requires primary isolation of the bacteria as a pure culture, a process which takes at least 18 hours if there is a pure culture or 2 to 3 days if there is a mixed culture.
Urine Specimens
A large proportion (40-50%) of specimens received in routine diagnostic microbiology laboratories for bacterial identification are urine specimens (Pezzlo, 1988, Clin. Microbiol. Rev. 1:268-280). Urinary tract infections (UTI) are extremely common and affect up to 20% of women and account for extensive morbidity and increased mortality among hospitalized patients (Johnson and Stamm, 1989; Ann. Intern. Med. 111:906-917). UTI are usually of bacterial etiology and require antimicrobial therapy. The Gram negative bacillus Escherichia coli is by far the most prevalent urinary pathogen and accounts for 50 to 60% of UTI (Pezzlo, 1988, op. cit.). The prevalence for bacterial pathogens isolated from urine specimens observed recently at the “Centre Hospitalier de 1'Université Laval (CHUL)” is given in Tables 1 and 2.
Conventional pathogen identification in urine specimens. The search for pathogens in urine specimens is so preponderant in the routine microbiology laboratory that a myriad of tests have been developed. The gold standard is still the classical semi-quantitative plate culture method in which a calibrated loop of urine is streaked on plates and incubated for 18-24 hours. Colonies are then counted to determine the total number of colony forming units (CFU) per liter of urine. A bacterial UTI is normally associated with a bacterial count of ≧10⁷CFU/L in urine. However, infections with less than 10⁷CFU/L in urine are possible, particularly in patients with a high incidence of diseases or those catheterized (Stark and Maki, 1984, N. Engl. J. Med. 311:560-564). Importantly, close to 80% of urine specimens tested are considered negative (<10⁷CFU/L; Table 3).
Accurate and rapid urine screening methods for bacterial pathogens would allow a faster identification of negative results and a more efficient clinical investigation of the patient. Several rapid identification methods (Uriscreen™, UTIscreen™, Flash Track™ DNA probes and others) were recently compared to slower standard biochemical methods which are based on culture of the bacterial pathogens. Although much faster, these rapid tests showed low sensitivities and specificities as well as a high number of false negative and false positive results (Koening et al., 1992. J. Clin. Microbiol. 30:342-345; Pezzlo et al., 1992. J. Clin. Microbiol. 30:640-684).
Urine specimens found positive by culture are further characterized using standard biochemical tests to identify the bacterial pathogen and are also tested for susceptibility to antibiotics.
Any Clinical Specimens
As with urine specimen which was used here as an example, our probes and amplification primers are also applicable to any other clinical specimens. The DNA-based tests proposed in this invention are superior to standard methods currently used for routine diagnosis in terms of rapidity and accuracy. While a high percentage of urine specimens are negative, in many other clinical specimens more than 95% of cultures are negative (Table 4). These data further support the use of universal probes to screen out the negative clinical specimens. Clinical specimens from organisms other than humans (e.g. other primates, mammals, farm animals or live stocks) may also be used.
Towards the Development of Rapid DNA-Based Diagnostic Tests
A rapid diagnostic test should have a significant impact on the management of infections. For the identification of pathogens and antibiotic resistance genes in clinical samples, DNA probe and DNA amplification technologies offer several advantages over conventional methods. There is no need for subculturing, hence the organism can be detected directly in clinical samples thereby reducing the costs and time associated with isolation of pathogens. DNA-based technologies have proven to be extremely useful for specific applications in the clinical microbiology laboratory. For example, kits for the detection of fastidious organisms based on the use of hybridization probes or DNA amplification for the direct detection of pathogens in clinical specimens are commercially available (Persing et al, 1993. Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.).
The present invention is an advantageous alternative to the conventional culture identification methods used in hospital clinical microbiology laboratories and in private clinics for routine diagnosis. Besides being much faster, DNA-based diagnostic tests are more accurate than standard biochemical tests presently used for diagnosis because the bacterial genotype (e.g. DNA level) is more stable than the bacterial phenotype (e.g. biochemical properties). The originality of this invention is that genomic DNA fragments (size of at least 100 base pairs) specific for 12 species of commonly encountered bacterial pathogens were selected from genomic libraries or from data banks. Amplification primers or oligonucleotide probes (both less than 100 nucleotides in length) which are both derived from the sequence of species-specific DNA fragments identified by hybridization from genomic libraries or from selected data bank sequences are used as a basis to develop diagnostic tests. Oligonucleotide primers and probes for the detection of commonly encountered and clinically important bacterial resistance genes are also included. For example, Annexes I and II present a list of suitable oligonucleotide probes and PCR primers which were all derived from the species-specific DNA fragments selected from genomic libraries or from data bank sequences. It is clear to the individual skilled in the art that oligonucleotide sequences appropriate for the specific detection of the above bacterial species other than those listed in Annexes 1 and 2 may be derived from the species-specific fragments or from the selected data bank sequences. For example, the oligonucleotides may be shorter or longer than the ones we have chosen and may be selected anywhere else in the identified species-specific sequences or selected data bank sequences. Alternatively, the oligonucleotides may be designed for use in amplification methods other than PCR. Consequently, the core of this invention is the identification of species-specific genomic DNA fragments from bacterial genomic DNA libraries and the selection of genomic DNA fragments from data bank sequences which are used as a source of species-specific and ubiquitous oligonucleotides. Although the selection of oligonucleotides suitable for diagnostic purposes from the sequence of the species-specific fragments or from the selected data bank sequences requires much effort it is quite possible for the individual skilled in the art to derive from our fragments or selected data bank sequences suitable oligonucleotides which are different from the ones we have selected and tested as examples (Annexes I and II).
Others have developed DNA-based tests for the detection and identification of some of the bacterial pathogens for which we have identified species-specific sequences (PCT patent application Serial No. WO 93/03186). However, their strategy was based on the amplification of the highly conserved 16S rRNA gene followed by hybridization with internal species-specific oligonucleotides. The strategy from this invention is much simpler and more rapid because it allows the direct amplification of species-specific targets using oligonucleotides derived from the species-specific bacterial genomic DNA fragments.
Since a high percentage of clinical specimens are negative, oligonucleotide primers and probes were selected from the highly conserved 16S or 23S rRNA genes to detect all bacterial pathogens possibly encountered in clinical specimens in order to determine whether a clinical specimen is infected or not. This strategy allows rapid screening out of the numerous negative clinical specimens submitted for bacteriological testing.
We are also developing other DNA-based tests, to be performed simultaneously with bacterial identification, to determine rapidly the putative bacterial susceptibility to antibiotics by targeting commonly encountered and clinically relevant bacterial resistance genes. Although the sequences from the selected antibiotic resistance genes are available and have been used to develop DNA-based tests for their detection (Ehrlich and Greenberg, 1994. PCR-based Diagnostics in Infectious Diseases, Blackwell Scientific Publications, Boston, Mass.; Persing et al, 1993. Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.), our approch is innovative as it represents major improvements over current “gold standard” diagnostic methods based on culture of the bacteria because it allows the rapid identification of the presence of a specific bacterial pathogen and evaluation of its susceptibility to antibiotics directly from the clinical specimens within one hour.
We believe that the rapid and simple diagnostic tests not based on cultivation of the bacteria that we are developing will gradually replace the slow conventional bacterial identification methods presently used in hospital clinical microbiology laboratories and in private clinics. In our opinion, these rapid DNA-based diagnostic tests for severe and common bacterial pathogens and antibiotic resistance will (i) save lives by optimizing treatment, (ii) diminish antibiotic resistance by reducing the use of broad spectrum antibiotics and (iii) decrease overall health costs by preventing or shortening hospitalizations.

SUMMARY OF THE INVENTION

In accordance with the present invention, there is provided sequence from genomic DNA fragments (size of at least 100 base pairs and all described in the sequence listing) selected either by hybridization from genomic libraries or from data banks and which are specific for the detection of commonly encountered bacterial pathogens (i.e. 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) in clinical specimens. These bacterial species are associated with approximately 90% of urinary tract infections and with a high percentage of other severe infections including septicemia, meningitis, pneumonia, intraabdominal infections, skin infections and many other severe respiratory tract infections. Overall, the above bacterial species may account for up to 80% of bacterial pathogens isolated in routine microbiology laboratories.
Synthetic oligonucleotides for hybridization (probes) or DNA amplification (primers) were derived from the above species-specific DNA fragments (ranging in sizes from 0.25 to 5.0 kilobase pairs (kbp)) or from selected data bank sequences (GenBank and EMBL). Bacterial species for which some of the oligonucleotide probes and amplification primers were derived from selected data bank sequences are Escherichia coli, Enterococcus faecalis, Streptococcus pyogenes and Pseudomonas aeruginosa. The person skilled in the art understands that the important innovation in this invention is the identification of the species-specific DNA fragments selected either from bacterial genomic libraries by hybridization or from data bank sequences. The selection of oligonucleotides from these fragments suitable for diagnostic purposes is also innovative. Specific and ubiquitous oligonucleotides different from the ones tested in the practice are considered as embodiments of the present invention.
The development of hybridization (with either fragment or oligonucleotide probes) or of DNA amplification protocols for the detection of pathogens from clinical specimens renders possible a very rapid bacterial identification. This will greatly reduce the time currently required for the identification of pathogens in the clinical laboratory since these technologies can be applied for bacterial detection and identification directly from clinical specimens with minimum pretreatment of any biological specimens to release bacterial DNA. In addition to being 100% specific, probes and amplification primers allow identification of the bacterial species directly from clinical specimens or, alternatively, from an isolated colony. DNA amplification assays have the added advantages of being faster and more sensitive than hybridization assays, since they allow rapid and exponential in vitro replication of the target segment of DNA from the bacterial genome. Universal probes and amplification primers selected from the 16S or 23S rRNA genes highly conserved among bacteria, which permit the detection of any bacterial pathogens, will serve as a procedure to screen out the numerous negative clinical specimens received in diagnostic laboratories. The use of oligonucleotide probes or primers complementary to characterized bacterial genes encoding resistance to antibiotics to identify commonly encountered and clinically important resistance genes is also under the scope of this invention.

DETAILED DESCRIPTION OF THE INVENTION

Development of Species-Specific DNA Probes
DNA fragment probes were developed for the following bacterial species: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Haemophilus influenzae and Moraxella catarrhalis. (For Enterococcus faecalis and Streptococcus pyogenes, oligonucleotide sequences were exclusively derived from selected data bank sequences). These species-specific fragments were selected from bacterial genomic libraries by hybridization to DNA from a variety of Gram positive and Gram negative bacterial species (Table 5).
The chromosomal DNA from each bacterial species for which probes were seeked was isolated using standard methods. DNA was digested with a frequently cutting restriction enzyme such as Sau3AI and then ligated into the bacterial plasmid vector pGEM3Zf (Promega) linearized by appropriate restriction endonuclease digestion. Recombinant plasmids were then used to transform competent E. coli strain DH5α thereby yielding a genomic library. The plasmid content of the transformed bacterial cells was analyzed using standard methods. DNA fragments of target bacteria ranging in size from 0.25 to 5.0 kilobase pairs (kbp) were cut out from the vector by digestion of the recombinant plasmid with various restriction endonucleases. The insert was separated from the vector by agarose gel electrophoresis and purified in low melting point agarose gels. Each of the purified fragments of bacterial genomic DNA was then used as a probe for specificity tests.
For each given species, the gel-purified restriction fragments of unknown coding potential were labeled with the radioactive nucleotide α-³²P(dATP) which was incorporated into the DNA fragment by the random priming labeling reaction. Non-radioactive modified nucleotides could also be incorporated into the DNA by this method to serve as a label.
Each DNA fragment probe (i.e. a segment of bacterial genomic DNA of at least 100 bp in length cut out from clones randomly selected from the genomic library) was then tested for its specificity by hybridization to DNAs from a variety of bacterial species (Table 5). The double-stranded labeled DNA probe was heat-denatured to yield labeled single-stranded DNA which could then hybridize to any single-stranded target DNA fixed onto a solid support or in solution. The target DNAs consisted of total cellular DNA from an array of bacterial species found in clinical samples (Table 5). Each target DNA was released from the bacterial cells and denatured by conventional methods and then irreversibly fixed onto a solid support (e.g. nylon or nitrocellulose membranes) or free in solution. The fixed single-stranded target DNAs were then hybridized with the single-stranded probe. Pre-hybridization, hybridization and post-hybridization conditions were as follows: (i) Pre-hybridization; in 1 M NaCl+10% dextran sulfate+1% SDS (sodium dodecyl sulfate)+1 μg/ml salmon sperm DNA at 650° C. for 15 min. (ii) Hybridization; in fresh pre-hybridization solution containing the labeled probe at 650° C. overnight. (iii) Post-hybridization; washes twice in 3×SSC containing 1% SDS (1×SSC is 0.15M NaCl, 0.015M NaCitrate) and twice in 0.1×SSC containing 0.1% SDS; all washes were at 650° C. for 15 min. Autoradiography of washed filters allowed the detection of selectively hybridized probes. Hybridization of the probe to a specific target DNA indicated a high degree of similarity between the nucleotide sequence of these two DNAs. Species-specific DNA fragments selected from various bacterial genomic libraries ranging in size from 0.25 to 5.0 kbp were isolated for 10 common bacterial pathogens (Table 6) based on hybridization to chromosomal DNAs from a variety of bacteria performed as described above. All of the bacterial species tested (66 species listed in Table 5) were likely to be pathogens associated with common infections or potential contaminants which can be isolated from clinical specimens. A DNA fragment probe was considered specific only when it hybridized solely to the pathogen from which it was isolated. DNA fragment probes found to be specific were subsequently tested for their ubiquity (i.e. ubiquitous probes recognized most isolates of the target species) by hybridization to bacterial DNAs from approximately 10 to 80 clinical isolates of the species of interest (Table 6). The DNAs were denatured, fixed onto nylon membranes and hybridized as described above.
Sequencing of the Species-Specific Fragment Orobes
The nucleotide sequence of the totality or of a portion of the species-specific DNA fragments isolated (Table 6) was determined using the dideoxynucleotide termination sequencing method which was performed using Sequenase (USB Biochemicals) or T7 DNA polymerase (Pharmacia). These nucleotide sequences are shown in the sequence listing. Alternatively, sequences selected from data banks (GenBank and EMBL) were used as sources of oligonucleotides for diagnostic purposes for Escherichia coli, Enterococcus faecalis, Streptococcus pyogenes and Pseudomonas aeruginosa. For this strategy, an array of suitable oligonucleotide primers or probes derived from a variety of genomic DNA fragments (size of more than 100 bp) selected from data banks was tested for their specificity and ubiquity in PCR and hybridization assays as described later. It is important to note that the data bank sequences were selected based on their potential of being species-specific according to available sequence information. Only data bank sequences from which species-specific oligonucleotides could be derived are included in this invention.
Oligonucleotide probes and amplification primers derived from species-specific fragments selected from the genomic libraries or from data bank sequences were synthesized using an automated DNA synthesizer (Millipore). Prior to synthesis, all oligonucleotides (probes for hybridization and primers for DNA amplification) were evaluated for their suitability for hybridization or DNA amplification by polymerase chain reaction (PCR) by computer analysis using standard programs (e.g. Genetics Computer Group (GCG) and Oligo™ 4.0 (National Biosciences)). The potential suitability of the PCR primer pairs was also evaluated prior to the synthesis by verifying the absence of unwanted features such as long stretches of one nucleotide, a high proportion of G or C residues at the 3′ end and a 3′-terminal T residue (Persing et al, 1993. Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.).
Hybridization with Oligonucleotide Probes
In hybridization experiments, oligonucleotides (size less than 100 nucleotides) have some advantages over DNA fragment probes for the detection of bacteria such as ease of preparation in large quantities, consistency in results from batch to batch and chemical stability. Briefly, for the hybridizations, oligonucleotides were 5′ end-labeled with the radionucleotide γ³²P(ATP) using T4 polynucleotide kinase (Pharmacia). The unincorporated radionucleotide was removed by passing the labeled single-stranded oligonucleotide through a Sephadex G50 column. Alternatively, oligonucleotides were labeled with biotin, either enzymatically at their 3′ ends or incorporated directly during synthesis at their 5′ ends, or with digoxigenin. It will be appreciated by the person skilled in the art that labeling means other than the three above labels may be used.
The target DNA was denatured, fixed onto a solid support and hybridized as previously described for the DNA fragment probes. Conditions for pre-hybridization and hybridization were as described earlier. Post-hybridization washing conditions were as follows: twice in 3×SSC containing 1% SDS, twice in 2×SSC containing 1% SDS and twice in 1×SSC containing 1% SDS (all of these washes were at 65° C. for 15 min ), and a final wash in 0.1×SSC containing 1% SDS at 25° C. for 15 min. For probes labeled with radioactive labels the detection of hybrids was by autoradiography as described earlier. For non-radioactive labels detection may be calorimetric or by chemiluminescence.
The oligonucleotide probes may be derived from either strand of the duplex DNA. The probes may consist of the bases A, G, C, or T or analogs. The probes may be of any suitable length and may be selected anywhere within the species-specific genomic DNA fragments selected from the genomic libraries or from data bank sequences.
DNA Amplification
For DNA amplification by the widely used PCR (polymerase chain reaction) method, primer pairs were derived either from the sequenced species-specific DNA fragments or from data bank sequences or, alternatively, were shortened versions of oligonucleotide probes. Prior to synthesis, the potential primer pairs were analyzed by using the program oligo™ 4.0 (National Biosciences) to verify that they are likely candidates for PCR amplifications.
During DNA amplification by PCR, two oligonucleotide primers binding respectively to each strand of the denatured double-stranded target DNA from the bacterial genome are used to amplify exponentially in vitro the target DNA by successive thermal cycles allowing denaturation of the DNA, annealing of the primers and synthesis of new targets at each cycle (Persing et al, 1993. Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.). Briefly, the PCR protocols were as follows. Clinical specimens or bacterial colonies were added directly to the 50 μL PCR reaction mixtures containing 50 mM KCl, 10 mM Tris-HCl pH 8.3, 2.5 mM MgCl_2,0.4 μM of each of the two primers, 200 μM of each of the four dNTPs and 1.25 Units of Taq DNA polymerase (Perkin Elmer). PCR reactions were then subjected to thermal cycling (3 min at 95° C. followed by 30 cycles of 1 second at 95° C. and 1 second at 55° C.) using a Perkin Elmer 480™ thermal cycler and subsequently analyzed by standard ethidium bromide-stained agarose gel electrophoresis. It is clear that other methods for the detection of specific amplification products, which may be faster and more practical for routine diagnosis, may be used. Such methods may be based on the detection of fluorescence after amplification (e.g. TaqMan™ system from Perkin Elmer or Amplisensor™ from Biotronics) or liquid hybridization with an oligonucleotide probe binding to internal sequences of the specific amplification product. These novel probes can be generated from our species-specific fragment probes. Methods based on the detection of fluorescence are particularly promising for utilization in routine diagnosis as they are, very rapid and quantitative and can be automated.
To assure PCR efficiency, glycerol or dimethyl sulfoxide (DMSO) or other related solvents, can be used to increase the sensitivity of the PCR and to overcome problems associated with the amplification of target with a high GC content or with strong secondary structures. The concentration ranges for glycerol and DMSO are 5-15% (v/v) and 3-10% (v\v), respectively. For the PCR reaction mixture, the concentration ranges for the amplification primers and the MgCl₂are 0.1-1.0 μM and 1.5-3.5 mM, respectively. Modifications of the standard PCR protocol using external and nested primers (i.e. nested PCR) or using more than one primer pair (i.e. multiplex PCR) may also be used (Persing et al, 1993. Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.). For more details about the PCR protocols and amplicon detection methods see examples 7 and 8.
The person skilled in the art of DNA amplification knows the existence of other rapid amplification procedures such as ligase chain reaction (LCR), transcription-based amplification systems (TAS), self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA) and branched DNA (bDNA) (Persing et al, 1993. Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.). The scope of this invention is not limited to the use of amplification by PCR, but rather includes the use of any rapid nucleic acid amplification methods or any other procedures which may be used to increase rapidity and sensitivity of the tests. Any oligonucleotides suitable for the amplification of nucleic acid by approaches other than PCR and derived from the species-specific fragments and from selected antibiotic resistance gene sequences included in this document are also under the scope of this invention.
Specificity and Ubiquity Tests for Oligonucleotide Probes and Primers
The specificity of oligonucleotide probes, derived either from the sequenced species-specific fragments or from data bank sequences, was tested by hybridization to DNAs from the array of bacterial species listed in Table 5 as previously described. Oligonucleotides found to be specific were subsequently tested for their ubiquity by hybridization to bacterial DNAs from approximately 80 isolates of the target species as described for fragment probes. Probes were considered ubiquitous when they hybridized specifically with the DNA from at least 80% of the isolates. Results for specificity and ubiquity tests with the oligonucleotide probes are summarized in Table 6. The specificity and ubiquity of the amplification primer pairs were tested directly from cultures (see example 7) of the same bacterial strains. For specificity and ubiquity tests, PCR assays were performed directly from bacterial colonies of approximately 80 isolates of the target species. Results are summarized in Table 7. All specific and ubiquitous oligonucleotide probes and amplification primers for each of the 12 bacterial species investigated are listed in Annexes I and II, respectively. Divergence in the sequenced DNA fragments can occur and, insofar as the divergence of these sequences or a part thereof does not affect the specificity of the probes or amplification primers, variant bacterial DNA is under the scope of this invention.
Universal Bacterial Detection
In the routine microbiology laboratory a high percentage of clinical specimens sent for bacterial identification is negative (Table 4). For example, over a 2 year period, around 80% of urine specimens received by the laboratory at the “Centre Hospitalier de 1' Université Laval (CHUL)” were negative (i.e. <10⁷CFU/L) (Table 3). Testing clinical samples with universal probes or universal amplification primers to detect the presence of bacteria prior to specific identification and screen out the numerous negative specimens is thus useful as it saves costs and may rapidly orient the clinical management of the patients. Several oligonucleotides and amplification primers were therefore synthesized from highly conserved portions of bacterial 16S or 23S ribosomal RNA gene sequences available in data banks (Annexes III and IV). In hybridization tests, a pool of seven oligonucleotides (Annex I; Table 6) hybridized strongly to DNA from all bacterial species listed in Table 5. This pool of universal probes labeled with radionucleotides or with any other modified nucleotides is consequently very useful for detection of bacteria in urine samples with a sensitivity range of ≧10⁷CFU/L. These probes can also be applied for bacterial detection in other clinical samples.
Amplification primers also derived from the sequence of highly conserved ribosomal RNA genes were used as an alternative strategy for universal bacterial detection directly from clinical specimens (Annex IV; Table 7). The DNA amplification strategy was developed to increase the sensitivity and the rapidity of the test. This amplification test was ubiquitous since it specifically amplified DNA from 23 different bacterial species encountered in clinical specimens.
Well-conserved bacterial genes other than ribosomal RNA genes could also be good candidates for universal bacterial detection directly from clinical specimens. Such genes may be associated with processes essential for bacterial survival (e.g. protein synthesis, DNA synthesis, cell division or DNA repair) and could therefore be highly conserved during evolution. We are working on these candidate genes to develop new rapid tests for the universal detection of bacteria directly from clinical specimens.
Antibiotic Resistance Genes
Antimicrobial resistance complicates treatment and often leads to therapeutic failures. Furthermore, overuse of antibiotics inevitably leads to the emergence of bacterial resistance. Our goal is to provide the clinicians, within one hour, the needed information to prescribe optimal treatments. Besides the rapid identification of negative clinical specimens with DNA-based tests for universal bacterial detection and the identification of the presence of a specific pathogen in the positive specimens with DNA-based tests for specific bacterial detection, the clinicians also need timely information about the ability of the bacterial pathogen to resist antibiotic treatments. We feel that the most efficient strategy to evaluate rapidly bacterial resistance to antimicrobials is to detect directly from the clinical specimens the most common and important antibiotic resistance genes (i.e. DNA-based tests for the detection of antibiotic resitance genes). Since the sequence from the most important and common bacterial antibiotic resistance genes are available from data banks, our strategy is to use the sequence from a portion or from the entire gene to design specific oligonucleotides which will be used as a basis for the development of rapid DNA-based tests. The sequence from the bacterial antibiotic resistance genes selected on the basis of their clinical relevance (i.e. high incidence and importance) is given in the sequence listing. Table 8 summarizes some characteristics of the selected antibiotic resistance genes.

EXAMPLES

The following examples are intended to be illustrative of the various methods and compounds of the invention.

Example 1

Isolation and cloning of fragments. Genomic DNAs from Escherichia coli strain ATCC 25922, Klebsiella pneumoniae strain CK2, Pseudomonas aeruginosa strain ATCC 27853, Proteus mirabilis strain ATCC 35657, Streptococcus pneumoniae strain ATCC 27336, Staphylococcus aureus strain ATCC 25923, Staphylococcus epidermidis strain ATCC 12228, Staphylococcus saprophyticus strain ATCC 15305, Haemophilus influenzae reference strain Rd and Moraxella catarrhalis strain ATCC 53879 were prepared using standard procedures. It is understood that the bacterial genomic DNA may have been isolated from strains other than the ones mentioned above. (For Enterococcus faecalis and Streptococcus pyogenes oligonucleotide sequences were derived exclusively from data banks). Each DNA was digested with a restriction enzyme which frequently cuts DNA such as Sau3AI. The resulting DNA fragments were ligated into a plasmid vector (pGEM3Zf) to create recombinant plasmids and transformed into competent E. coli cells (DH5α). It is understood that the vectors and corresponding competent cells should not be limited to the ones herein above specifically examplified. The objective of obtaining recombinant plasmids and transformed cells is to provide an easily reproducible source of DNA fragments useful as probes. Therefore, insofar as the inserted fragments are specific and selective for the target bacterial DNA, any recombinant plasmids and corresponding transformed host cells are under the scope of this invention. The plasmid content of the transformed bacterial cells was analyzed using standard methods. DNA fragments from target bacteria ranging in size from 0.25 to 5.0 kbp were cut out from the vector by digestion of the recombinant plasmid with various restriction endonucleases. The insert was separated from the vector by agarose gel electrophoresis and purified in a low melting point agarose gel. Each of the purified fragments was then used for specificity tests.
Labeling of DNA fragment probes. The label used was α³²P(dATP), a radioactive nucleotide which can be incorporated enzymatically into a double-stranded DNA molecule. The fragment of interest is first denatured by heating at 95° C. for 5 min, then a mixture of random primers is allowed to anneal to the strands of the fragments. These primers, once annealed, provide a starting point for synthesis of DNA. DNA polymerase, usually the Klenow fragment, is provided along with the four nucleotides, one of which is radioactive. When the reaction is terminated, the mixture of new DNA molecules is once again denatured to provide radioactive single-stranded DNA molecules (i.e. the probe). As mentioned earlier, other modified nucleotides may be used to label the probes.
Specificity and ubiquity tests for the DNA fragment probes. Species-specific DNA fragments ranging in size from 0.25 to 5.0 kbp were isolated for 10 common bacterial pathogens (Table 6) based on hybridization to chromosomal DNAs from a variety of bacteria. Samples of whole cell DNA for each bacterial strain listed in Table 5 were transferred onto a nylon membrane using a dot blot apparatus, washed and denatured before being irreversibly fixed. Hybridization conditions were as described earlier. A DNA fragment probe was considered specific only when it hybridized solely to the pathogen from which it was isolated. Labeled DNA fragments hybridizing specifically only to target bacterial species (i.e. specific) were then tested for their ubiquity by hybridization to DNAs from approximately 10 to 80 isolates of the species of interest as described earlier. The conditions for pre-hybridization, hybridization and post-hybridization washes were as described earlier. After autoradiography (or other detection means appropriate for the non-radioactive label used), the specificity of each individual probe can be determined. Each probe found to be specific (i.e. hybridizing only to the DNA from the bacterial species from which it was isolated) and ubiquitous (i.e. hybridizing to most isolates of the target species) was kept for further experimentations.

Example 2

Same as example 1 except that testing of the strains is by colony hybridization. The bacterial strains were inoculated onto a nylon membrane placed on nutrient agar. The membranes were incubated at 37° C. for two hours and then bacterial lysis and DNA denaturation were carried out according to standard procedures. DNA hybridization was performed as described earlier.

Example 3

Same as example 1 except that bacteria were detected directly from clinical samples. Any biological samples were loaded directly onto a dot blot apparatus and cells were lysed in situ for bacterial detection. Blood samples should be heparizined in order to avoid coagulation interfering with their convenient loading on a dot blot apparatus.

Example 4

Nucleotide sequencina of DNA fragments. The nucleotide sequence of the totality or a portion of each fragment found to be specific and ubiquitous (Example 1) was determined using the dideoxynucleotide termination sequencing method (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA. 74:5463-5467). These DNA sequences are shown in the sequence listing. Oligonucleotide probes and amplification primers were selected from these nucleotide sequences, or alternatively, from selected data banks sequences and were then synthesized on an automated Biosearch synthesizer (Millipore™) using phosphoramidite chemistry.
Labeling of oliaonucleotides. Each oligonucleotide was 5′ end-labeled with γ³²P-ATP by the T4 polynucleotide kinase (Pharmacia) as described earlier. The label could also be non-radioactive.
Specificity test for oligonucleotide probes. All labeled oligonucleotide probes were tested for their specificity by hybridization to DNAs from a variety of Gram positive and Gram negative bacterial species as described earlier. Species-specific probes were those hybridizing only to DNA from the bacterial species from which it was isolated. Oligonucleotide probes found to be specific were submitted to ubiquity tests as follows.
Ubiquity test for oligonucleotide probes. Specific oligonucleotide probes were then used in ubiquity tests with approximately 80 strains of the target species. Chromosomal DNAs from the isolates were transferred onto nylon membranes and hybridized with labeled oligonucleotide probes as described for specificity tests. The batteries of approximately 80 isolates constructed for each target species contain reference ATCC strains as well as a variety of clinical isolates obtained from various sources. Ubiquitous probes were those hybridizing to at least 80% of DNAs from the battery of clinical isolates of the target species. Examples of specific and ubiquitous oligonucleotide probes are listed in Annex 1.

Example 5

Same as example 4 except that a pool of specific oligonucleotide probes is used for bacterial identification (i) to increase sensitivity and assure 100% ubiquity or (ii) to identify simultaneously more than one bacterial species. Bacterial identification could be done from isolated colonies or directly from clinical specimens.

Example 6

PCR amplification. The technique of PCR was used to increase sensitivity and rapidity of the tests. The PCR primers used were often shorter derivatives of the extensive sets of oligonucleotides previously developed for hybridization assays (Table 6). The sets of primers were tested in PCR assays performed directly from a bacterial colony or from a bacterial suspension (see Example 7) to determine their specificity and ubiquity (Table 7). Examples of specific and ubiquitous PCR primer pairs are listed in annex II.
Specificity and ubiquity tests for amplification primers. The specificity of all selected PCR primer pairs was tested against the battery of Gram negative and Gram positive bacteria used to test the oligonucleotide probes (Table 5). Primer pairs found specific for each species were then tested for their ubiquity to ensure that each set of primers could amplify at least 80% of DNAs from a battery of approximately 80 isolates of the target species. The batteries of isolates constructed for each species contain reference ATCC strains and various clinical isolates representative of the clinical diversity for each species.
Standard precautions to avoid false positive PCR results should be taken. Methods to inactivate PCR amplification products such as the inactivation by uracil-N-glycosylase may be used to control PCR carryover.

Example 7

Amplification directly from a bacterial colony or suspension. PCR assays were performed either directly from a bacterial colony or from a bacterial suspension, the latter being adjusted to a standard McFarland 0.5 (corresponds to 1.5 ×10⁸bacteria/mL). In the case of direct amplification from a colony, a portion of the colony was transferred directly to a 50 μL PCR reaction mixture (containing 50 mM KCl, 10 mM Tris pH 8.3, 2.5 mM MgCl_2,0.4 μM of each of the two primers, 200 μM of each of the four dNTPs and 1.25 Unit of Taq DNA polymerase (Perkin Elmer)) using a plastic rod. For the bacterial suspension, 4 μL of the cell suspension was added to 46 μL of the same PCR reaction mixture. For both strategies, the reaction mixture was overlaid with 50 μL of mineral oil and PCR amplifications were carried out using an initial denaturation step of 3 min. at 95° C. followed by 30 cycles consisting of a 1 second denaturation step at 95° C. and of a 1 second annealing step at 55° C. in a Perkin Elmer 480™ thermal cycler. PCR amplification products were then analyzed by standard agarose gel (2%) electrophoresis. Amplification products were visualized in agarose gels containing 2.5 μg/mL of ethidium bromide under UV at 254 nm. The entire PCR assay can be completed in approximately one hour.
Alternatively, amplification from bacterial cultures was performed as described above but using a “hot start” protocol. In that case, an initial reaction mixture containing the target DNA, primers and dNTPs was heated at 85° C. prior to the addition of the other components of the PCR reaction mixture. The final concentration of all reagents was as described above. Subsequently, the PCR reactions were submitted to thermal cycling and analysis as described above.

Example 8

Amplification directly from clinical specimens. For amplification from urine specimens, 4 μL of undiluted or diluted (1:10) urine was added directly to 46 μL of the above PCR reaction mixture and amplified as described earlier.
To improve bacterial cell lysis and eliminate the PCR inhibitory effects of clinical specimens, samples were routinely diluted in lysis buffer containing detergent(s). Subsequently, the lysate was added directly to the PCR reaction mixture. Heat treatments of the lysates, prior to DNA amplification, using the thermocycler or a microwave oven could also be performed to increase the efficiency of cell lysis.
Our strategy is to develop rapid and simple protocols to eliminate PCR inhibitory effects of clinical specimens and lyse bacterial cells to perform DNA amplification directly from a variety of biological samples. PCR has the advantage of being compatible with crude DNA preparations. For example, blood, cerebrospinal fluid and sera may be used directly in PCR assays after a brief heat treatment. We intend to use such rapid and simple strategies to develop fast protocols for DNA amplification from a variety of clinical specimens.

Example 9

Detection of antibiotic resistance genes. The presence of specific antibiotic resistance genes which are frequently encountered and clinically relevant is identified using the PCR amplification or hybridization protocols described in previous sections. Specific oligonucleotides used as a basis for the DNA-based tests are selected from the antibiotic resistance gene sequences. These tests can be performed either directly from clinical specimens or from a bacterial colony and should complement diagnostic tests for specific bacterial identification.

Example 10

Same as examples 7 and 8 except that assays were performed by multiplex PCR (i.e. using several pairs of primers in a single PCR reaction) to (i) reach an ubiquity of 100% for the specific target pathogen or (ii) to detect simultaneously several species of bacterial pathogens.
For example, the detection of Escherichia coli requires three pairs of PCR primers to assure a ubiquity of 100%. Therefore, a multiplex PCR assay (using the “hot-start” protocol (Example 7)) with those three primer pairs was developed. This strategy was also used for the other bacterial pathogens for which more than one primer pair was required to reach an ubiquity of 100%.
Multiplex PCR assays could also be used to (i) detect simultaneously several bacterial species or, alternatively, (ii) to simultaneously identify the bacterial pathogen and detect specific antibiotic resistance genes either directly from a clinical specimen or from a bacterial colony.
For these applications, amplicon detection methods should be adapted to differentiate the various amplicons produced. Standard agarose gel electrophoresis could be used because it discriminates the amplicons based on their sizes. Another useful strategy for this purpose would be detection using a variety of fluorochromes emitting at different wavelengths which are each coupled with a specific oligonucleotide linked to a fluorescence quencher which is degraded during amplification to release the fluorochrome (e.g. TaqMan™, Perkin Elmer).

Example 11

Detection of amplification Products. The person skilled in the art will appreciate that alternatives other than standard agarose gel electrophoresis (Example 7) may be used for the revelation of amplification products. Such methods may be based on the detection of fluorescence after amplification (e.g. Amplisensor™, Biotronics; TaqMan™) or other labels such as biotin (SHARP Signal™ system, Digene Diagnostics). These methods are quantitative and easily automated. One of the amplification primers or an internal oligonucleotide probe specific to the amplicon(s) derived from the species-specific fragment probes is coupled with the fluorochrome or with any other label. Methods based on the detection of fluorescence are particularly suitable for diagnostic tests since they are rapid and flexible as fluorochromes emitting different wavelengths are available (Perkin Elmer).

Example 12

Species-specific, universal and antibiotic resistance gene amplification primers can be used in other rapid amplification procedures such as the ligase chain reaction (LCR), transcription-based amplification systems (TAS), self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA) and branched DNA (bDNA) or any other methods to increase the sensitivity of the test. Amplifications can be performed from an isolated bacterial colony or directly from clinical specimens. The scope of this invention is therefore not limited to the use of PCR but rather includes the use of any procedures to specifically identify bacterial DNA and which may be used to increase rapidity and sensitivity of the tests.

Example 13

A test kit would contain sets of probes specific for each bacterium as well as a set of universal probes. The kit is provided in the form of test components, consisting of the set of universal probes labeled with non-radioactive labels as well as labeled specific probes for the detection of each bacterium of interest in specific clinical samples. The kit will also include test reagents necessary to perform the pre-hybridization, hybridization, washing steps and hybrid detection. Finally, test components for the detection of known antibiotic resistance genes (or derivatives therefrom) will be included. Of course, the kit will include standard samples to be used as negative and positive controls for each hybridization test.
Components to be included in the kits will be adapted to each specimen type and to detect pathogens commonly encountered in that type of specimen. Reagents for the universal detection of bacteria will also be included. Based on the sites of infection, the following kits for the specific detection of pathogens may be developed:

- A kit for the universal detection of bacterial pathogens from most clinical specimens which contains sets of probes specific for highly conserved regions of the bacterial genomes.
- A kit for the detection of bacterial pathogens retrieved from urine samples, which contains eight specific test components (sets of probes for the detection of Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus saprophyticus, Staphylococcus aureus and Staphylococcus epidermidis).
- A kit for the detection of respiratory pathogens which contains seven specific test components (sets of probes for detecting Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus pyogenes and Staphylococcus aureus).
- A kit for the detection of pathogens retrieved from blood samples, which contains eleven specific test components (sets of probes for the detection of Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes and Staphylococcus epidermidis).
- A kit for the detection of pathogens causing meningitis, which contains four specific test components (sets of probes for the detection of Haemophilus influenzae, Streptococcus pneumoniae, Escherichia coli and Pseudomonas aeruginosa).
- A kit for the detection of clinically important antibiotic resistance genes which contains sets of probes for the specific detection of at least one of the 19 following genes associated with bacterial resistance : bla_tem, bla_rob, bla_shv, aadB, aacC1, aacC2, aacC3, aacA4, mecA, vanA, vanH, vanX, satA, aacA-aphD, vat, vga, msrA, sul and int.
- Other kits adapted for the detection of pathogens from skin, abdominal wound or any other clinically relevant kits will be developed.

Example 14

Same as example 13 except that the test kits contain all reagents and controls to perform DNA amplification assays. Diagnostic kits will be adapted for amplification by PCR (or other amplification methods) performed directly either from clinical specimens or from a bacterial colony. Components required for universal bacterial detection, bacterial identification and antibiotic resistance genes detection will be included.
Amplification assays could be performed either in tubes or in microtitration plates having multiple wells. For assays in plates, the wells will be coated with the specific amplification primers and control DNAs and the detection of amplification products will be automated. Reagents and amplification primers for universal bacterial detection will be included in kits for tests performed directly from clinical specimens. Components required for bacterial identification and antibiotic resistance gene detection will be included in kits for testing directly from colonies as well as in kits for testing directly from clinical specimens.
The kits will be adapted for use with each type of specimen as described in example 13 for hybridization-based diagnostic kits.

Example 15

It is understood that the use of the probes and amplification primers described in this invention for bacterial detection and identification is not limited to clinical microbiology applications. In fact, we feel that other sectors could also benefit from these new technologies. For example, these tests could be used by industries for quality control of food, water, pharmaceutical products or other products requiring microbiological control. These tests could also be applied to detect and identify bacteria in biological samples from organisms other than humans (e.g. other primates, mammals, farm animals and live stocks). These diagnostic tools could also be very useful for research purposes including clinical trials and epidemiological studies.

TABLE 1


Distribution of urinary isolates from positive urine samples
(≧10⁷ CFU/L) at the Centre Hospitalier
de l′Université Laval (CHUL) for the
1992-1994 period.

% of isolates

	Nov 92	April 93	July 93	Jan 94
Organisms	n = 267^a	n = 265	n = 238	n = 281

Escherichia coli	53.2	51.7	53.8	54.1
Enterococcus faecalis	13.8	12.4	11.7	11.4
Klebsiella pneumoniae	6.4	6.4	5.5	5.3
Staphylococcus epidermidis	7.1	7.9	3.0	6.4
Proteus mirabilis	2.6	3.4	3.8	2.5
Pseudomonas aeruginosa	3.7	3.0	5.0	2.9
Staphylococcus saprophyticus	3.0	1.9	5.4	1.4
Others^b	10.2	13.3	11.8	16.0

^an = total number of isolates for the indicated month.
^bSee table 2.

TABLE 2


Distribution of uncommon^aurinary isolates from positive urine
samples ( ≧10⁷CFU/L) at the Centre Hospitalier de
I′Université Laval (CHUL) for the 1992-1994 period.

% of isolates

Organisms^a	Nov 92	April 93	July 93	Jan 94

Staphylococcus aureus	0.4	1.1	1.3	1.4
Staphylococcus spp.	2.2	4.9	1.7	6.0
Micrococcus spp.	0.0	0.0	0.4	0.7
Enterococcus faecium	0.4	0.4	1.3	1.4
Citrobacter spp.	1.4	0.8	0.4	0.7
Enterobacter spp.	1.5	1.1	1.3	1.4
Klebsiella oxytoca	1.1	1.5	2.5	1.8
Serratia spp.	0.8	0.0	0.5	0.0
Proteus spp.	0.4	0.4	0.0	1.1
Morganella and Providencia	0.4	0.8	0.4	0.0
Hafnia alvei	0.8	0.0	0.0	0.0
NFB^b(Stenotrophomonas,	0.0	0.4	1.3	1.1
Acinetobacter)
Candida spp.	0.8	1.9	0.7	0.4

^aUncommon urinary isolates are those identified as “Others” in Table 1.
^bNFB: non fermentative bacilli.

TABLE 3


Distribution of positive^a(bacterial count ≧10⁷CFU/L)
and negative (bacterial count <10⁷CFU/L) urine specimens tested
at the Centre Hospitalier de l′Université Laval (CHUL) for
the 1992-1994 period.

Number of isolates (%)

Specimens	Nov 92	April 93	July 93	Jan 94

received:	1383(100)	1338(100)	1139(100)	1345(100)
positive:	267(19.3)	265(19.8)	238(20.9)	281(20.9)
negative:	1116(80.7)	1073(80.2)	901(79.1)	1064(79.1)

^aBased on standard diagnostic methods, the minimal number of bacterial pathogens in urine samples to indicate an urinary tract infection is normally 10⁷CFU/L.

TABLE 4


Distribution of positive and negative clinical specimens tested
in the Microbiology Laboratory of the CHUL.

	No. of	% of	% of
Clinical	samples	positive	negative
specimens^a	tested	specimens	specimens

Urine	17,981	19.4	80.6
Haemoculture/marrow	10,010	6.9	93.1
Sputum	1,266	68.4	31.6
Superficial pus	1,136	72.3	27.7
Cerebrospinal fluid	553	1.0	99.0
Synovial fluid-articular	523	2.7	97.3
Bronch./Trach./Amyg./Throat	502	56.6	43.4
Deep pus	473	56.8	43.2
Ears	289	47.1	52.9
Pleural and pericardial fluid	132	1.0	99.0
Peritonial fluid	101	28.6	71.4

^aSpecimens tested from February 1994 to January 1995.

TABLE 5


Bacterial species (66) used for testing the specificity of DNA fragment
probes, oligonucleotide probes and PCR primers.

	Number of		Number of
Bacterial species	strains	Bacterial species	strains
Gram negative:	tested	Gram positive:	tested

Proteus mirabilis	5	Streptococcus pneumoniae	7
Klebsiella pneumoniae	5	Streptococcus salivarius	2
Pseudomonas aeruginosa	5	Streptococcus viridans	2
Escherichia coli	5	Streptococcus pyogenes	2
Moraxella catarrhalis	5	Staphylococcus aureus	2
Proteus vulgaris	2	Staphylococcus epidermidis	2
Morganella morganii	2	Staphylococcus saprophyticus	5
Enterobacter cloacae	2	Micrococcus species	2
Providencia stuartii	1	Corynebacterium species	2
Providencia species	1	Streptococcus groupe B	2
Enterobacter agglomerans	2	Staphylococcus simulans	2
Providencia rettgeri	2	Staphylococcus ludgunensis	1
Neisseria mucosa	1	Staphylococcus capitis	2
Providencia alcalifaciens	1	Staphylococcus haemolyticus	2
Providencia rustigianii	1	Staphylococcus hominis	2
Burkholderia cepacia	2	Enterococcus faecalis	2
Enterobacter aerogenes	2	Enterococcus faecium	1
Stenotrophomonas maltophilia	2	Staphylococcus warneri	1
Pseudomonas fluorescens	1	Enterococcus durans	1
Comamonas acidovorans	2	Streptococcus bovis	1
Pseudomonas putida	2	Diphteroids	2
Haemophilus influenzae	5	Lactobacillus acidophilus	1
Haemophilus parainfluenzae	2
Bordetella pertussis	2
Haemophilus parahaemolyticus	2
Haemophilus haemolyticus	2
Haemophilus aegyptius	1
Kingella indologenes	1
Moraxella atlantae	1
Neisseria caviae	1
Neisseria subflava	1
Moraxella urethralis	1
Shigella sonnei	1
Shigella flexneri	1
Klebsiella oxytoca	2
Serratia marcescens	2
Salmonella typhimurium	1
Yersinia enterocolitica	1
Acinetobacter calcoaceticus	1
Acinetobacter lwoffi	1
Haftnia alvei	2
Citrobacter diversus	1
Citrobacter freundii	1
Salmonella species	1

TABLE 6


Species-specific DNA fragment and oligonucleotide probes for hybridization.

Number of fragment probes^b

Number of oligonucleotide probes

Organisms^a	Tested	Specific	Ubiquitous^c	Synthesized	Specific	Ubiquitous^c

E. coli ^d	—	—	—	20	12	9^f
E. coli	14	2	2^e	—	—	—
K. pneumoniae ^d	—	—	—	15	1	1
K. pneumoniae	33	3	3	18	12	8
P. mirabilis ^d	—	—	—	3	3	2
P. mirabilis	14	3	3^e	15	8	7
P. aeruginosa ^d	—	—	—	26	13	9
P. aeruginosa	6	2	2^e	6	0	0
S. saprophyticus	7	4	4	20	9	7
H. influenzae ^d	—	—	—	16	2	2
H. influenzae	1	1	1	20	1	1
S. pneumoniae ^d	—	—	—	6	1	1
S. pneumoniae	19	2	2	4	1	1
M. catarrhalis	2	2	2	9	8	8
S. epidermidis	62	1	1	—	—	—
S. aureus	30	1	1	—	—	—
Universal probes^d	—	—	—	7	—	7^g

^aNo DNA fragment or oligonucleotide probes were tested for E. faecalis and S. pyogenes.
^bSizes of DNA fragments range from 0.25 to 5.0 kbp.
^cA specific probe was considered ubiquitous when at least 80% of isolates of the target species (approximately 80 isolates) were recognized by each specific probe. When 2 or more probes are combined, 100% of the isolates are recognized.
^dThese sequences were selected from data banks.
^eUbiquity tested with approximately 10 isolates of the target species.
^fA majority of probes (8/9) do not discriminate E. coli and Shigella spp.
^gUbiquity tests with a pool of the 7 probes detected all 66 bacterial species listed in Table 5.

TABLE 7


PCR amplification for bacterial pathogens commonly encountered in
urine, sputum, blood, cerebrospinal fluid and other specimens.

Primer pair^a

Amplicon

DNA amplification

Organism	# (SEQ ID NO)	size (bp)	Ubiquity^b	from colonies^c	from specimens^d

E. coli	l^e(55-56)	107	75/80	+	+
	2^e(46-47)	297	77/80	+	+
	3 (42-43)	102	78/80	+	+
	4 (131-132)	134	73/80	+	+
	1 + 3 + 4	—	80/80	+	+
E.faecalis	1^e(38-39)	200	71/80	+	+
	2^e(40-41)	121	79/80	+	+
	1 + 2	—	80/80	+	+
K. pneumoniae	1 (67-68)	198	76/80	+	+
	2 (61-62)	143	67/80	+	+
	3^h(135-136)	148	78/80	+	N.T.ⁱ
	4 (137-138)	116	69/80	+	N.T.
	1 + 2 + 3	—	80/80	+	N.T.
P. mirabilis	1 (74-75)	167	73/80	+	N.T.
	2 (133-134)	123	80/80	+	N.T.
P. aeruginosa	1^e(83-84)	139	79/80	+	N.T.
	2^e(85-86)	223	80/80	+	N.T.
S. saprophyticus	1 (98-99)	126	79/80	+	+
	2 (139-140)	190	80/80	+	N.T.
M. catarrhalis	1 (112-113)	157	79/80	+	N.T.
	2 (118-119)	118	80/80	+	N.T.
	3 (160-119)	137	80/80	+	N.T.
H. influenzae	1^e(154-155)	217	80/80	+	N.T.
S. pneumoniae	1^e(156-157)	134	80/80	+	N.T.
	2^e(158-159)	197	74/80	+	N.T.
	3 (78-79)	175	67/80	+	N.T.
S. epidermidis	1 (147-148)	175	80/80	+	N.T.
	2 (145-146)	125	80/80	+	N.T.
S. aureus	1 (152-153)	108	80/80	+	N.T.
	2 (149-150)	151	80/80	+	N.T.
	3 (149-151)	176	80/80	+	N.T.
S. pyogenes ^f	1^e(141-142)	213	80/80	+	N.T.
	2^e(143-144)	157	24/24	+	N.T.
Universal	1^e(126-127)	241	194/195^g	+	+

See notes on next page

a All primer pairs are specific in PCR assays since no amplification was observed with DNA from 66 different species of both Gram positive and Gram negative bacteria other than the species of interest (Table 5).
b The ubiquity was normally tested on 80 strains of the species of interest. All retained primer pairs amplified at least 90% of the isolates. When combinations of primers were used, an ubiquity of 100% was reached.
c For all primer pairs and multiplex combinations, PCR amplifications directly performed from a bacterial colony were 100% species-specific.
d PCR assays performed directly from urine specimens.
e Primer pairs derived from data bank sequences. Primer pairs with no “e” are derived from our species-specific fragments.
f For S. pyogenes, primer pair #1 is specific for Group A Streptococci (GAS). Primer pair #2 is specific for the GAS-producing exotoxin A gene (SpeA).
g Ubiquity tested on 195 isolates from 23 species representative of bacterial pathogens commonly encountered in clinical specimens.
h Optimizations are in progress to eliminate non-specific amplification observed with some bacterial species other than the target species.

N.T.: not tested.

TABLE 8


Selected antibiotic resistance genes for diagnostic purposes.

Genes	Antibiotics	Bacteria^a	SEQ ID NO

(bla_tem) TEM-1	β-lactams	Enterobacteriaceae,	161
		Pseudomonadaceae,
		Haemophilus, Neisseria
(bla_rob) ROB-1	β-lactams	Haemophilus, Pasteurella	162
(bla_shv) SHV-1	β-lactams	Klebsiella and other	163
		Enterobacteriaceae
aadB, aacC1, aacC2,	Aminoglycosides	Enterobacteriaceae,	164, 165, 166
aacC3, aacA4		Pseudomonadaceae	167, 168
mecA	β-lactams	Staphylococci	169
vanH, vanA, vanX	Vancomycin	Enterococci	170
satA	Macrolides	Enterococci	173
aacA-aphD	Aminoglycosides	Enterococci, Staphylococci	174
vat	Macrolides	Staphylococci	175
vga	Macrolides	Staphylococci	176
msrA	Erythromycin	Staphylococci	177
Int and Sul	β-lactams, trimethoprim,	Enterobacteriaceae,	171, 172
conserved sequences	aminoglycosides, antiseptic,	Pseudomonadaceae
	chloramphenicol

^aBacteria having high incidence for the specified antibiotic resistance genes. The presence in other bacteria is not excluded.



Annex I: Specific and ubiquitous oligonucleotides probes
for hybridization

Originating DNA fragment

SEQ ID NO	Nucleotide Sequence	SEQ ID NO	Nucleotide position

Bacterial species: Escherichia coli

44	5′-CAC CCG CTT GCG TGG CAA GCT GCC C	5^a	213-237

45	5′-CGT TTG TGG ATT CCA GTT CCA TCC G	5^a	489-513

48	5′-TGA AGC ACT GGC CGA AAT GCT GCG T	6^a	759-783

49	5′-GAT GTA CAG GAT TCG TTG AAG GCT T	6^a	898-922

50	5′-TAG CGA AGG CGT AGC AGA AAC TAA C	7^a	1264-1288

51	5′-GCA ACC CGA ACT CAA CGC CGG ATT T	7^a	1227-1251

52	5′-ATA CAC AAG GGT CGC ATC TGC GGC C	7^a	1313-1337

53	5′-TGC GTA TGC ATT GCA GAC CTT GTG GC	7^a	111-136

54	5′-GCT TTC ACT GGA TAT CGC GCT TGG G	7^a	373-397

Bacterial species: Proteus mirabilis

70^b	5′-TGG TTC ACT GAC TTT GCG ATG TTT C	12	23-47

71	5′-TCG AGG ATG GCA TGC ACT AGA AAA T	12	53-77

72^b	5′-CGC TGA TTA GGT TTC GCT AAA ATC TTA TTA	12	80-109

73	5′-TTG ATC CTC ATT TTA TTA ATC ACA TGA CCA	12	174-203

^aSequences from data banks
^bThese sequences are from the opposite DNA strand of the sequences given in the Sequence listing



Annex I: Specific and ubiquitous oligonucleotides probes
for hybridization

Originating DNA fragment

SEQ ID NO	Nucleotide Sequence	SEQ ID NO	Nucleotide position

Bacterial species: Proteus mirabilis

76	5′-CCG CCT TTA GCA TTA ATT GGT GTT TAT AGT	13	246-275

77	5′-CCT ATT GCA GAT ACC TTA AAT GTC TTG GGC	13	291-320

80^b	5′-TTG AGT GAT GAT TTC ACT GAC TCC C	14	18-42

81	5′-GTG AGA CAG TGA TGG TGA GGA CAC A	15^a	1185-1209

82	5′-TGG TTG TCA TGC TGT TTG TGT GAA AAT	15^a	1224-1230

Bacterial species: Klebsiella pneumoniae

57	5′-GTG GTG TCG TTC AGG GGT TTC AC	8	45-67

58	5′-GCG ATA TTC ACA CCC TAC GCA GCC A	9	161-185

59^b	5′-GTC GAA AAT GCC GGA AGA GGT ATA CG	9	203-228

60^b	5′-ACT GAG CTG CAG ACC GGT AAA ACT CA	9	233-258

63^b	5′-CGT GAT GGA TAT TCT TAA CGA AGG GC	10	250-275

64^b	5′-ACC AAA CTG TTG AGC CGC CTG GA	10	201-223

65	5′-GTG ATC GCC CCT CAT CTG CTA CT	10	77-99

66	5′-CGC CCT TCG TTA AGA ATA TCC ATC AC	10	249-274

69	5′-CAG GAA GAT GCT GCA CCG GTT GTT G	11^a	296-320

^aSequences from data banks
^bThese sequences are from the opposite DNA strand of the sequences given in the Sequence listing



Annex I: Specific and ubiquitous oligonucleotides probes
for hybridization

Originating DNA fragment

SEQ ID NO	Nucleotide Sequence	SEQ ID NO	Nucleotide position

Bacterial species: Pseudomonas aeruginosa

87	5′-AAT GCG GCT GTA CCT CGG CGC TGG T	18^a	2985-3009

88	5′-GGC GGA GGG CCA GTT GCA CCT GCC A	18^a	2929-2953

89	5′-AGC CCT GCT CCT CGG CAG CCT CTG C	18^a	2821-2845

90	5′-TGG CTT TTG CAA CCG CGT TCA GGT T	18^a	1079-1103

91	5′-GCG CCC GCG AGG GCA TGC TTC GAT G	19^a	705-729

92	5′-ACC TGG GCG CCA ACT ACA AGT TCT A	19^a	668-692

93	5′-GGC TAC GCT GCC GGG CTG CAG GCC G	19^a	505-529

94	5′-CCG ATC TAG ACC ATC GAG ATG GGC G	20^a	1211-1235

95	5′-GAG CGC GGC TAT GTG TTC GTC GGC T	20^a	2111-2135

Bacterial species: Streptococcus pneumoniae

120	5′-TCT GTG CTA GAG ACT GCC CCA TTT C	30	423-447

121	5′-CGA TGT CTT GAT TGA GCA GGG TTA T	31^a	1198-1222

^aSequences from data banks
^bThese sequences are from the opposite DNA strand of the sequences given in the Sequence listing



Annex I: Specific and ubiquitous oligonucleotides probes
for hybridization

Originating DNA fragment

SEQ ID NO	Nucleotide Sequence	SEQ ID NO	Nucleotide position

Bacterial species: Staphylococcus saprophyticus

96	5′-CGT TTT TAC CCT TAC CTT TTC GTA CTA CC	21	45-73

97^b	5′-TCA GGC AGA GGT AGT ACG AAA AGG TAA GGG	21	53-82

100	5′-CAC CAA GTT TGA CAC GTG AAG ATT CAT	22	89-115

101^b	5′-ATG AGT GAA GCG GAG TCA GAT TAT GTG CAG	23	105-134

102	5′-CGC TCA TTA CGT ACA GTG ACA ATC G	24	20-44

103	5′-CTG GTT AGC TTG ACT CTT AAC AAT CTT GTC	24	61-90

104^b	5′-GAC GCG ATT GTC ACT GTA CGT AAT GAG CGA	24	19-48

Bacterial species: Moraxella catarrhalis

108	5′-GCC CCA AAA CAA TGA AAC ATA TGG T-3′	28	81-105

109	5′-CTG CAG ATT TTG GAA TCA TAT CGC C-3′	28	126-130

110	5′-TGG TTT GAC CAG TAT TTA ACG CCA T-3′	28	165-189

111	5′-CAA CGG CAC CTG ATG TAC CTT GTA C-3′	28	232-256

114	5′-TTA CAA CCT GCA CCA CAA GTC ATC A-3′	29	97-121

115	5′-GTA CAA ACA AGC CGT CAG CGA CTT A-3′	29	139-163

116	5′-CAA TCT GCG TGT GTG CGT TCA CT-3′	29	178-200

117	5′-GCT ACT TTG TCA GCT TTA GCC ATT CA-3′	29	287-312

^aSequences from data banks
^bThese sequences are from the opposite DNA strand of the sequences given in the Sequence listing



Annex I:
Specific and ubiquitous oligonucleotides probes
for hybridization

Originating DNA fragment

		SEQ ID	Nucleotide
SEQ ID NO	Nucleotide sequence	NO	position

Bacterial species:	Haemophilus influenzae
105^b	5′-GCG TCA GAA AAA GTA GGC GAA ATG AAA G	25	138-165

106^b	5′-AGC GGC TCT ATC TTG TAA TGA CAC A	26^a	770-794

107^b	5′-GAA ACG TGA ACT CCC CTC TAT ATA A	27^a	5184-5208

	Universal probes^c
122^b	5′-ATC CCA CCT TAG GCG GCT GGC TCC A	—	—

123	5′-ACG TCA AGT CAT CAT GGC CCT TAC GAG TAG G	—	—

124^b	5′-GTG TGA CGG GCG GTG TGT ACA AGG C	—	—

125^b	5′-GAG TTG CAG ACT CCA ATC CGG ACT ACG A	—	—

128^b	5′-CCC TAT ACA TCA CCT TGC GGT TTA GCA GAG AG	—	—

129	5′-GGG GGG ACC ATC CTC CAA GGC TAA ATA C	—	—

130^b	5′-CGT CCA CTT TCG TGT TTG CAG AGT GCT GTG TT	—	—

^aSequences from data banks
^bThese sequences are from the opposite DNA strand of the sequences given in the Sequence listing
^cUniversal probes were derived from 16S or 23S ribosomal RNA gene sequences not included in the Sequence listing



Annex II: Specific and ubiquitous primers for DNA amplification

Originating DNA fragment

SEQ ID NO	Nucleotide Sequence	SEQ ID NO	Nucleotide position

Bacterial species: Escherichia coli

42	5′-GCT TTC CAG CGT CAT ATT G	4	177-195

43^b	5′-GAT CTC GAC AAA ATG GTG A	4	260-278

46	5′-TCA CCC GCT TGC GTG GC	5^a	212-228

47^b	5′-GGA ACT GGA ATC CAC AAA C	5^a	490-508

55	5′-GCA ACC CGA ACT CAA CGC C	7^a	1227-1245

56^b	5′-GCA GAT GCG ACC CTT GTG T	7^a	1315-1333

131	5′-CAG GAG TAC GGT GAT TTT TA	3	60-79

132^b	5′-ATT TCT GGT TTG GTC ATA CA	3	174-193

Bacterial species: Enterococcus faecalis

38	5′-GCA ATA CAG GGA AAA ATG TC	1^a	69-88

39^b	5′-CTT CAT CAA ACA ATT AAC TC	1^a	249-268

40	5′-GAA CAG AAG AAG CCA AAA AA	2^a	569-588

41^b	5′-GCA ATC CCA AAT AAT ACG GT	2^a	670-689

^aSequences from data banks
^bThese sequences are from the opposite DNA strand of the sequences given in the Sequence listing



Annex II: Specific and ubiquitous primers for DNA amplification

Originating DNA fragment

SEQ ID NO	Nucleotide Sequence	SEQ ID NO	Nucleotide position

Bacterial species: Klebsiella pneumoniae

61	5′-GAC AGT CAG TTC GTC AGC C	9	37-55

62^b	5′-CGT AGG GTG TGA ATA TCG C	9	161-179

67	5′-TCG CCC CTC ATC TGC TAC T	10	81-99

68^b	5′-GAT CGT GAT GGA TAT TCT T	10	260-278

135	5′-GCA GCG TGG TGT CGT TCA	8	40-57

136^b	5′-AGC TGG CAA CGG CTG GTC	8	170-187

137	5′-ATT CAC ACC CTA CGC AGC CA	9	166-185

138^b	5′-ATC CGG CAG CAT CTC TTT GT	9	262-281

Bacterial species: Proteus mirabilis

74	5′-GAA ACA TCG CAA AGT CAG T	12	23-41

75^b	5′-ATA AAA TGA GGA TCA AGT TC	12	170-189

133	5′-CGG GAG TCA GTG AAA TCA TC	14	17-36

134^b	5′-CTA AAA TCG CCA CAC CTC TT	14	120-139

^aSequences from data banks
^bThese sequences are from the opposite DNA strand of the sequences given in the Sequence listing



Annex II: Specific and ubiquitous primers for DNA amplification

Originating DNA fragment

SEQ ID NO	Nucleotide Sequence	SEQ ID NO	Nucleotide position

Bacterial species: Staphylococcus saprophyticus

98	5′-CGT TTT TAC CCT TAC CTT TTC GTA CT	21	45-70

99^b	5′-ATC GAT CAT CAC ATT CCA TTT GTT TTT A	21	143-170

139	5′-CTG GTT AGC TTG ACT CTT AAC AAT C	24	61-85

140^b	5′-TCT TAA CGA TAG AAT GGA GCA ACT G	24	226-250

Bacterial species: Pseudomonas aeruginosa

83	5′-CGA GCG GGT GGT GTT CAT C	16^a	554-572

84^b	5′-CAA GTC GTG GTG GGA GGG A	16^a	674-692

85	5′-TCG CTG TTC ATC AAG ACC C	17^a	1423-1441

86^b	5′-CCG AGA ACC AGA CTT CAT C	17^a	1627-1645

Bacterial species: Moraxella catarrhalis

112	5′-GGC ACC TGA TGT ACC TTG	28	235-252

113^b	5′-AAC AGC TCA CAC GCA TT	28	375-391

118	5′-TGT TTT GAG CTT TTT ATT TTT TGA	29	41-64

119^b	5′-CGC TGA CGG CTT GTT TGT ACC A	29	137-158

160	5′-GCT CAA ATC AGG GTC AGC	29	22-39

119^b	5′-CGC TGA CGG CTT GTT TGT ACG A	29	137-158

^aSequences from data banks
^bThese sequences are from the opposite DNA strand of the sequences given in the Sequence listing



Annex II: Specific and ubiquitous primers for DNA amplification

Originating DNA fragment

SEQ ID NO	Nucleotide Sequence	SEQ ID NO	Nucleotide position

Bacterial species: Staphylococcus epidermidis

145	5′-ATC AAA AAG TTG GCG AAC CTT TTC A	36	21-45

146^b	5′-CAA AAG AGC GTG GAG AAA AGT ATC A	36	121-145

147	5′-TCT CTT TTA ATT TCA TCT TCA ATT CCA TAG	36	448-477

148^b	5′-AAA CAC AAT TAC AGT CTG GTT ATC CAT ATC	36	593-622

Bacterial species: Staphylococcus aureus

149^b	5′-CTT CAT TTT ACG GTG ACT TCT TAG AAG ATT	37	409-438

150	5′-TCA ACT GTA GCT TCT TTA TCC ATA CGT TGA	37	288-317

149^b	5′-CTT CAT TTT ACG GTG ACT TCT TAG AAG ATT	37	409-438

151	5′-ATA TTT TAG CTT TTC AGT TTC TAT ATC AAC	37	263-292

152	5′-AAT CTT TGT CGG TAC ACG ATA TTC TTC ACG	37	5-34

153^b	5′-CGT AAT GAG ATT TCA GTA GAT AAT ACA ACA	37	83-112

^aSequences from data banks
^bThese sequences are from the opposite DNA strand of the sequences given in the Sequence listing



Annex II: Specific and ubiquitous primers for DNA amplification

Originating DNA fragment

SEQ ID NO	Nucleotide Sequence	SEQ ID NO	Nucleotide position

Bacterial species: Haemophilus influenzae

154	5′-TTT AAC GAT CCT TTT ACT CCT TTT G	27^a	5074-5098

155^b	5′-ACT GCT GTT GTA AAG AGG TTA AAA T	27^a	5266-5290

Bacterial species: Streptococcus pneumoniae

78	5′-AGT AAA ATG AAA TAA GAA CAG GAC AG	34	164-189

79^b	5′-AAA ACA GGA TAG GAG AAC GGG AAA A	34	314-338

156	5′-ATT TGG TGA CGG GTG ACT TT	31^a	1401-1420

157^b	5′-GCT GAG GAT TTG TTC TTC TT	31^a	1515-1534

158	5′-GAG CGG TTT CTA TGA TTG TA	35^a	1342-1361

159^b	5′-ATC TTT CCT TTC TTG TTC TT	35^a	1519-1538

Bacterial species: Streptococcus pyogenes

141	5′-TGA AAA TTC TTG TAA CAG GC	32^a	286-305

142^b	5′-GGC CAC CAG CTT GCC CAA TA	32^a	479-498

143	5′-ATA TTT TCT TTA TGA GGG TG	33^a	966-985

144^b	5′-ATC CTT AAA TAA AGT TGC CA	33^a	1103-1122

^aSequences from data banks
^bThese sequences are from the opposite DNA strand of the sequences given in the Sequence listing



Annex II: Specific and ubiquitous primers for DNA amplification

Originating DNA fragment

SEQ ID NO	Nucleotide Sequence	SEQ ID NO	Nucleotide position

Universal primers^c

126	5′-GGA GGA AGG TGG GGA TGA CG	—	—

127^b	5′-ATG GTG TGA CGG GCG GTG TG	—	—

^aSequences from data banks
^bThese sequences are from the opposite DNA strand of the sequences given in the Sequence listing
^cUniversal primers were derived from the 16S ribosomal RNA gene sequence not included in the Sequence listing



Annex III
Selection of Universal Probes by Alignment of the
Sequences of Bacterial 16S and 23S Ribosomal RNA Genes.

Reverse strand of SEQ ID NO: 122	TGGAGCC AGCCGCCTA GGTGGGAT

	1461 1510
Streptococcus salivarius	TGAGGTAACC TTTTGGAGCC AGCCGCCTAA GGTGGGATAG ATGANNGGGG

Proteus vulgaris	TAGCTTAACC TTCGGGAGGG CGCTTACCAC TTTGTGATTC ATGACTGGGG

Pseudomonas aeruginosa	TAGTCTAACC GCAAGGGGGA CGGTTACCAC GGAGTGATTC ATGACTGGGG

Neisseria gonorrhoeae	TAGGGTAACC GCAAGGAGTC CGCTTACCAC GGTATGCTTC ATGACTGGGG

Streptococcus lactis	TTGCCTAACC GCAAGGAGGG CGCTTCCTAA GGTAAGACCG ATGACNNGGG



Annex III. Selection of universal probes by alignment of the
sequences of bacterial 165 and 23S ribosomal RNA genes.

SEQ ID NO: 123	ACGTCAAGTC ATCATGGC CCTTACGAGT AGG

	1251 1300
Haemophilus influenzae	GGTNGGGATG ACGTCAAGTC ..ATCATGGC CCTTACGAGT AGGGCTACAC

Neisseria gonorrhoeae	GGTGGGGATG ACGTCAAGTC ..CTCATGGC CCTTATGACC AGGGCTTCAC

Pseudomonas cepacia	GGTNGGGATG ACGTCAAGTC ..CTCATGGC CCTTATGGGT AGGGCTTCAC

Serratia marcescens	GGTGGGGATG ACGTCAAGTC ..ATCATGGC CCTTACGAGT AGGGCTACAC

Escherichia coli	GGTGGGGATG ACGTCAAGTC ..ATCATGGC CCTTACGACC AGGGCTACAC

Proteus vulgaris	GGTGGGGATG ACGTTAAGTC GTATCATGGC CCTTACGAGT AGGGCTACAC

Pseudomonas aeruginosa	GGTGGGGATG ACGTCAAGTC ..ATCATGGC CCTTACGGCN AGGGCTACAC

Clostridium perfringens	GGTGGGGATG ACGTNNAATC ..ATCATGCC CNTTATGTGT AGGGCTACAC

Mycoplasma hominis	GGTGGGGATG ACGTCAAATC ..ATCATGCC TCTTACGAGT GGGGCCACAC

Helicobacter pylori	GGTGGGGACG ACGTCAAGTC ..ATCATGGC CCTTACGCCT AGGGCTACAC

Mycoplasma pneumoniae	GGAAGGGATG ACGTCAAATC ..ATCATGCC CCTTATGTCT AGGGCTGCAA



Annex III. Selection of universal probes by alignment of the
sequences of bacterial 16S and 23S ribosomal RNA genes.

Reverse of the probe SEQ ID NO: 124	GCCTTGTACA CACCGCCCGT CACAC

	1451 1490
Escherichia coli	ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG

Neisseria gonorrhoeae	ACGTTCCCNG NNCTTGTACA CACCGCCCGT CACACCATGG

Pseudomonas cepacia	ACGTTCCCGG GTCTTGTACA CACNGCCCGT CACACCATGG

Serratia marcescens	ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG

Proteus vulgaris	ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG

Haemophilus influenzae	ACGTTCCCGG GCNTTGTACA CACCGCCCGT CACACCATGG

Pseudomonas aeruginosa	ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG

Clostridium perfringens	ACGTTCCCNG GTCTTGTACA CACCGCNCGT CACACCATGA

Mycoplasma hominis	ACGTTCTCGG GTCTTGTACA CACCGCCCGT CACACCATGG

Helicobacter pylori	ACGTTCCCGG GTCTTGTACT CACCGCCCGT CACACCATGG

Mycoplasma pneumoniae	ACGTTCTCGG GTCTTGTACA CACCGCCCGT CAAACTATGA

Reverse strand of SEQ ID NO 125:	TCG TAGTCCGGAT TGGAGTCTGC AACTC

	1361 1400
Escherichia coli	AAGTGCGTCG TAGTCCGGAT TGGAGTCTGC AACTCGACTC

Neisseria gonorrhoeae	AAACCGATCG TAGTCCGGAT TGCACTCTGC AACTCGAGTG

Pseudomonas cepacia	AAACCGATCG TAGTCCGGAT TGCACTCTGC AACTCGAGTG

Serratia marcescens	AAGTATGTCG TAGTCCGGAT TGGAGTCTGC AACTCGACTC

Proteus vulgaris	AAGTCTGTCG TAGTCCGGAT TGGAGTCTGC AACTCGACTC

Haemophilus influenzae	AAGTACGTCT AAGTCCGGAT TGGAGTCTGC AACTCGACTC

Pseudomonas aeruginosa	AAACCGATCG TAGTCCGGAT CGCAGTCTGC AACTCGACTG

Clostridium perfringens	AAACCAGTCT CAGTTCGGAT TGTAGGCTGA AACTCGCCTA

Mycoplasma hominis	AAGCCGATCT CAGTTCGGAT TGGAGTCTGC AATTCGACTC

Helicobacter pylori	ACACC..TCT CAGTTCGGAT TGTAGGCTGC AACTCGCCTG

Mycloplasma pneumoniae	AAGTTGGTCT CAGTTCGGAT TGAGGGCTGC AATTCGTCCT

Reverse strand of SEQ ID NO: 128	CT CTCTGCTAAA CCGCAAGGTG ATGTATAGGG

	1991 2040
Lactobacillus lactis	AAACACAGCT CTCTGCTAAA CCGCAAGGTG ATGTATAGGG GGTGACGCCT

Escherichia coli	AAACACAGCA CTGTGCAAAC ACGAAAGTGG ACGTATACGG TGTGACGCCT

Pseudomonas aeruginosa	AAACACAGCA CTCTGCAAAC ACGAAAGTGG ACGTATAGGG TGTGACGCCT

Pseudomonas cepacia	AAACACAGCA CTCTGCAAAC ACGAAAGTGG ACGTATAGGG TGTGACGCCT

Bacillus stearothermophilus	AAACACAGGT CTCTGCGAAG TCGTAAGGCG ACGTATAGGG GCTGACACCT

Micrococcus luteus	AAACACAGGT CCATGCGAAG TCGTAAGACG ATGTATATGG ACTGACTCCT

SEQ ID NO: 129	GGGGGGACC ATCCTCCAAG GCTAAATAC

	481 530
Escherichia coli	TGTCTGAATA TGGGGGGACC ATCCTCCAAG GCTAAATACT CCTGACTGAC

Pseudomonas aeruginosa	TGTCTGAACA TGGGGGGACC ATCCTCCAAG GCTAAATACT ACTGACTGAC

Pseudomonas cepacia	TGTCTGAAGA TGGGGGGACC ATCCTCCAAG GCTAAATACT CGTGATCGAC

Lactobacillus lactis	AGTTTGAATC CGGGAGGACC ATCTCCCAAC CCTAAATACT CCTTAGTGAC

Micrococcus luteus	CGTGTGAATC TGCCAGGACC ACCTGGTAAG CCTGAATACT ACCTGTTGAC

Reverse strand of SEQ ID NO: 130	AACACAGCA CTCTGCAAAC ACGAAAGTGG ACG

	1981 2030
Pseudomonas aeruginosa	TGTTTATTAA AAACACAGCA CTCTGCAAAC ACGAAAGTGG ACGTATAGGG

Escherichia coli	TGTTTATTAA AAACACAGCA CTGTGCAAAC ACGAAAGTGG ACGTATACGG

Pseudomonas cepacia	TGTTTAATAA AAACACAGCA CTCTGCAAAC ACGAAAGTGG ACGTATAGGG

Bacillus stearothermophilus	TGTTTATCAA AAACACAGGT CTCTGCGAAG TCGTAAGGCG ACGTATAGGG

Lactobacillus lactis	TGTTTATCAA AAACACAGCT CTCTGCTAAA CCACAAGGTG ATGTATAGGG

Micrococcus luteus	TGTTTATCAA AAACACAGGT CCATGCGAAG TCGTAAGACG ATGTATATGG



Annex IV. Selection of the universal PCR primers by alignment of the bacterial
16S ribosomal RNA gene

SEQ ID NO: 126	GGAGGAA GGTGGGGATG ACG

Reverse strand of SEQ ID NO: 127	CA CACCGCCCGT CACACCAT

	1241 1270......1461 1490
Escherichia coli	ACTGGAGGAA GGTGGGGATG ACGTCAAGTC......GCCTTGTACA CACCGCCCGT CACACCATGG

Neisseria gonorrhoeae	GCCGGAGGAA GGTGGGGATG ACGTCAAGTC......NNCTTGTACA CACCGCCCGT CACACCATGG

Pseudomonas cepacia	ACCGGAGGAA GGTNGGGATG ACGTCAAGTC......GTCTTGTACA CACNGCCCGT CACACCATGG

Seratia marcescens	ACTGGAGGAA GGTGGGGATG ACGTCAAGTC......GCCTTGTACA CACCGCCCGT CACACCATGG

Proteus vulgaris	ACCGGAGGAA GGTGGGGATG ACGTTAAGTC......GCCTTGTACA CACCGCCCGT CACACCATGG

Haemophilus influenzae	ACTGGAGGAA GGTNGGGATG ACGTCAAGTC......GCNTTGTACA CACCGCCCGT CACACCATGG

Legionella pneumophila	ACCGGAGGAA GGCGGGGATG ACGTCAAGTC......GCCTTGTACA CACCGCCCGT CACACCATGG

Pseudomonas aeruginos	ACCGGAGGAA GGTGGGGATG ACGTCAAGTC......GCCTTGTACA CACCGCCCGT CACACCATGG

Clostridium perfringens	CCAGGAGGAA GGTGGGGATG ACGTNNAATC......GTCTTGTACA CACCGCNCGT CACACCATGA

Mycoplasma hominis	CTGGGAGGAA GGTGGGGATG ACGTCAAATC......GTCTTGTACA CACCGCCCGT CACACCATGG

Helicobacter pylori	GGAGGAGGAA GGTGGGGACG ACGTCAAGTC......GTCTTGTACT CACCGCCCGT CACACCATGG

Mycoplasma pneumoniae	ATTGGAGGAA GGAAGGGATG ACGTCAAATC......GTCTTGTACA CACCGCCCGT CAAACTATGA

Claims

1. A method using probes (fragments and/or oligonucleotides) and/or amplification primers which are specific, ubiquitous and sensitive for determining the presence and/or amount of nucleic acids from bacterial species selected from the group consisting of Escherichia coli, Klebsiells pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus pneumoniae, Staphlyococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Staphlyococcus saprophyticus, Streptococcus pyogenes, Haemophilus influenzae and Moraxells catarrhalis in a any sample suspected of containing said bacterial nucleic acid, wherein said bacterial nucleic acid or variant or part thereof comprises a selected target region hybridizable with said probes or primers; said method comprising the steps of contacting said sample with said probes or primers and detecting the presence and/or amount of hybridized probes and/or amplified products as an indication of the presence and/or amount of said bacterial species.

2. A method as defined in claim 1 further using probes (fragments and/or oligonucleotides) and/or amplification primers which are universal and sensitive for determining the presence and/or amount of nucleic acids from any bacteria from any sample suspected of containing said bacterial nucleic acid, wherein said bacterial nucleic acid or variant or part thereof comprises a selected target region hybridizable with said probes or primers; said method comprising the steps of contacting said sample with said probes or primers and detecting the presence and/or amount of hybridized probes and/or amplified products as an indication of the presence and/or amount of said any bacteria.

3. A method as defined in claim 1 further using probes (fragments and/or oligonucleotides) and/or amplification primers which are specific, ubiquitous and sensitive for determining the presence and/or amount of nucleic acids from an antibiotic resistance gene 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 in any sample suspected of containing said bacterial nucleic acid, wherein said bacterial nucleic acid or variant or part thereof comprises a selected target region hybridizable with said probes or primers; said method comprising the steps of contacting said sample with said probes or primers and detecting the presence and/or amount of hybridized probes and/or amplified products as an indication of the presence and/or amount of said antibiotic resistance gene.

4. The method of any one of claims 1, 2 and 3 which is performed directly on a sample obtained from human patients, animals, environment or food.

5. The method of any one of claims 1, 2 and 3 which is performed directly on a sample consisting of one or more bacterial colonies.

6. The method of any one of claims 1 to 5, wherein the bacterial nucleic acid is amplified by 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.

7. The method of claim 6 wherein said bacterial nucleic acid is amplified by PCR.

8. The method of claim 7 wherein the PCR protocol is modified to determine within one hour the presence of said bacterial nucleic acids by performing for each amplification cycle an annealing step of only one second at 55° C. and a denaturation step of only one second at 95° C. without any elongation step.

9. A method for the detection, identification and/or quantification of Escherichia coli directly from a test sample or from bacterial colonies, which comprises the following steps:

a) depositing and fixing on an inert support or leaving in solution the bacterial DNA of the sample or of a substantially homogenous population of bacteria isolated from this sample, or

inoculating said sample or said substantially homogenous population of bacteria isolated from this sample on an inert support, and lysing in situ said inoculated sample or isolated bacteria to release the bacterial DNA,

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, a sequence complementary thereof, a part thereof and a variant thereof, which specifically and ubiquitously anneals with strains or representatives of Escherichia coli, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of the presence and/or amount of Escherichia coli in said test sample.

10. A method as defined in claim 9, wherein said probe is selected from the group consisting of:

1) an oligonucleotide of 12-227 nucleotides in length which sequence is comprised in SEQ ID NO: 3 or a complementary sequence thereof,

2) an oligonucleotide of 12-278 nucleotides in length which sequence is comprised in SEQ ID NO: 4 or a complementary sequence thereof,

3) an oligonucleotide of 12-1596 nucleotides in length which sequence is comprised in SEQ ID NO: 5 or a complementary sequence thereof,

4) an oligonucleotide of 12-2703 nucleotides in length which sequence is comprised in SEQ ID NO: 6 or a complementary sequence thereof,

5) an oligonucleotide of 12-1391 nucleotides in length which sequence is comprised in SEQ ID NO: 7 or a complementary sequence thereof, and

variants thereof which specifically and ubiquitously anneal with strains and representatives of Escherichia coli.

11. The method of claim 10, wherein the probe for detecting nucleic acid sequences from Escherichia coli is selected from the group consisting of SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54 and a sequence complementary thereof.

12. A method for detecting the presence and/or amount of Escherichia coli in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Escherichia coli DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within one of the following sequences: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7;

b) synthesizing an extension product of each of said primers which extension products contain the target sequence, and amplifying said target sequence, if any, to a detectable level; and

c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of Escherichia coli in said test sample.

13. The method of claim 12, wherein said at least one pair of primers is selected from the group consisting of:

a) SEQ ID NO: 42 and SEQ ID NO: 43,

b) SEQ ID NO: 46 and SEQ ID NO: 47,

c) SEQ ID NO: 55 and SEQ ID NO: 56, and

d) SEQ ID NO: 131 and SEQ ID NO: 132.

14. A method for the detection, identification and/or quantification of Klebsiella pneumoniae directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, a sequence complementary thereof, a part thereof and a variant thereof, which specifically and ubiquitously anneals with strains or representatives of Klebsiella pneumoniae, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of the presence and/or amount of Klebsiella pneumoniae in said test sample.

15. A method as defined in claim 14, wherein said probe is selected from the group consisting of:

1) an oligonucleotide of 12-238 nucleotides in length which sequence is comprised in SEQ ID NO: 8 or a complementary sequence thereof,

2) an oligonucleotide of 12-385 nucleotides in length which sequence is comprised in SEQ ID NO: 9 or a complementary sequence thereof,

3) an oligonucleotide of 12-462 nucleotides in length which sequence is comprised in SEQ ID NO: 10 or a complementary sequence thereof,

4) an oligonucleotide of 12-730 nucleotides in length which sequence is comprised in SEQ ID NO: 11 or a complementary sequence thereof, and

variants thereof which specifically and ubiquitously anneal with strains and representatives of Klebsiella pneumoniae.

16. The method of claim 15, wherein the probe for detecting nucleic acid sequences from Klebsiella pneumoniae is selected from the group consisting of SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 69 and a sequence complementary thereof.

17. A method for detecting the presence and/or amount of Klebsiella pneumoniae in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Klebsiella pneumoniae DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within one of the following sequences: SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11;

c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of Klebsiella pneumoniae in said test sample.

18. The method of claim 17, wherein said at least one pair of primers is selected from the group consisting of:

a) SEQ ID NO: 61 and SEQ ID NO: 62,

b) SEQ ID NO: 67 and SEQ ID NO: 68,

c) SEQ ID NO: 135 and SEQ ID NO: 136, and

d) SEQ ID NO: 137 and SEQ ID NO: 138.

19. A method for the detection, identification and/or quantification of Proteus mirabilis directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, a sequence complementary thereof, a part thereof and a variant thereof, which specifically and ubiquitously anneals with strains or representatives of Proteus mirabilis, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of the presence and/or amount of Proteus mirabilis in said test sample.

20. A method as defined in claim 19, wherein said probe is selected from the group consisting of:

1) an oligonucleotide of 12-225 nucleotides in length which sequence is comprised in SEQ ID NO: 12 or a complementary sequence thereof,

2) an oligonucleotide of 12-402 nucleotides in length which sequence is comprised in SEQ ID NO: 13 or a complementary sequence thereof,

3) an oligonucleotide of 12-157 nucleotides in length which sequence is comprised in SEQ ID NO: 14 or a complementary sequence thereof,

4) an oligonucleotide of 12-1348 nucleotides in length which sequence is comprised in SEQ ID NO: 15 or a complementary sequence thereof, and

variants thereof which specifically and ubiquitously anneal with strains and representatives of Proteus mirabilis.

21. The method of claim 20, wherein the probe for detecting nucleic acid sequences from Proteus mirabilis is selected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82 and a sequence complementary thereof.

22. A method for detecting the presence and/or amount of Proteus mirabilis in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Proteus mirabilis DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within one of the following sequences: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15;

c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of Proteus mirabilis in said test sample.

23. The method of claim 22, wherein said at least one pair of primers is selected from the group consisting of:

a) SEQ ID NO: 74 and SEQ ID NO: 75, and

b) SEQ ID NO: 133 and SEQ ID NO: 134.

24. A method for the detection, identification and/or quantification of Staphylococcus saprophyticus directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, a sequence complementary thereof, a part thereof and a variant thereof, which specifically and ubiquitously anneals with strains or representatives of Staphylococcus saprophyticus, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of the presence and/or amount of Staphylococcus saprophyticus in said test sample.

25. A method as defined in claim 24, wherein said probe is selected from the group consisting of:

1) an oligonucleotide of 12-172 nucleotides in length which sequence is comprised in SEQ ID NO: 21 or a complementary sequence thereof,

2) an oligonucleotide of 12-155 nucleotides in length which sequence is comprised in SEQ ID NO: 22 or a complementary sequence thereof,

3) an oligonucleotide of 12-145 nucleotides in length which sequence is comprised in SEQ ID NO: 23 or a complementary sequence thereof,

4) an oligonucleotide of 12-265 nucleotides in length which sequence is comprised in SEQ ID NO: 24 or a complementary sequence thereof, and

variants thereof which specifically and ubiquitously anneal with strains and representatives of Staphylococcus saprophyticus.

26. The method of claim 25, wherein the probe for detecting nucleic acid sequences from Staphylococcus saprophyticus is selected from the group consisting of SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104 and a sequence complementary thereof.

27. A method for detecting the presence and/or amount of Staphylococcus saprophyticus in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Staphylococcus saprophyticus DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within one of the following sequences: SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24;

c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of Staphylococcus saprophyticus in said test sample.

28. The method of claim 27, wherein said at least one pair of primers is selected from the group consisting of:

a) SEQ ID NO: 98 and SEQ ID NO: 99, and

b) SEQ ID NO: 139 and SEQ ID NO: 140.

29. A method for the detection, identification and/or quantification of Moraxella catarrhalis directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 29, a sequence complementary thereof, a part thereof and a variant thereof, which specifically and ubiquitously anneals with strains or representatives of Moraxella catarrhalis, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of the presence and/or amount of Moraxella catarrhalis in said test sample.

30. A method as defined in claim 29, wherein said probe is selected from the group consisting of:

1) an oligonucleotide of 12-526 nucleotides in length which sequence is comprised in SEQ ID NO: 28 or a complementary sequence thereof,

2) an oligonucleotide of 12-466 nucleotides in length which sequence is comprised in SEQ ID NO: 29 or a complementary sequence thereof, and

variants thereof which specifically and ubiquitously anneal with strains and representatives of Moraxella catarrhalis.

31. The method of claim 30, wherein the probe for detecting nucleic acid sequences from Moraxella catarrhalis is selected from the group consisting of SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117 and a sequence complementary thereof.

32. A method for detecting the presence and/or amount of Moraxella catarrhalis in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Moraxella catarrhalis DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within one of the following sequences: SEQ ID NO: 28 and SEQ ID NO: 29;

c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of Moraxella catarrhalis in said test sample.

33. The method of claim 32, wherein said at least one pair of primers is selected from the group consisting of:

a) SEQ ID NO: 112 and SEQ ID NO: 113,

b) SEQ ID NO: 118 and SEQ ID NO: 119, and

c) SEQ ID NO: 160 and SEQ ID NO: 119.

34. A method for the detection, identification and/or quantification of Pseudomonas aeruginosa directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, a sequence complementary thereof, a part thereof and a variant thereof, which specifically and ubiquitously anneals with strains or representatives of Pseudomonas aeruginosa, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of the presence and/or amount of Pseudomonas aeruginosa in said test sample.

35. A method as defined in claim 34, wherein said probe is selected from the group consisting of:

1) an oligonucleotide of 12-2167 nucleotides in length which sequence is comprised in SEQ ID NO: 16 or a complementary sequence thereof,

2) an oligonucleotide of 12-1872 nucleotides in length which sequence is comprised in SEQ ID NO: 17 or a complementary sequence thereof,

3) an oligonucleotide of 12-3451 nucleotides in length which sequence is comprised in SEQ ID NO: 18 or a complementary sequence thereof,

4) an oligonucleotide of 12-744 nucleotides in length which sequence is comprised in SEQ ID NO: 19 or a complementary sequence thereof,

5) an oligonucleotide of 12-2760 nucleotides in length which sequence is comprised in SEQ ID NO: 20 or a complementary sequence thereof, and

variants thereof which specifically and ubiquitously anneal with strains and representatives of Pseudomonas aeruginosa.

36. The method of claim 35, wherein the probe for detecting nucleic acid sequences from Pseudomonas aeruginosa is selected from the group consisting of SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95 and a sequence complementary thereof.

37. A method for detecting the presence and/or amount of Pseudomonas aeruginosa in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Pseudomonas aeruginosa DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within one of the following sequences: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20;

c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of Pseudomonas aeruginosa in said test sample.

38. The method of claim 37, wherein said at least one pair of primers is selected from the group consisting of:

a) SEQ ID NO: 83 and SEQ ID NO: 84, and

b) SEQ ID NO: 85 and SEQ ID NO: 86.

39. A method for the detection, identification and/or quantification of Staphylococcus epidermidis directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 36, a sequence complementary thereof, a part thereof and a variant thereof, which specifically and ubiquitously anneals with strains or representatives of Staphylococcus epidermidis, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of the presence and/or amount of Staphylococcus epidermidis in said test sample.

40. A method as defined in claim 39, wherein said probe is selected from the group consisting of an oligonucleotide of 12-705 nucleotides in length which sequence is comprised in SEQ ID NO: 36 and variants thereof which specifically and ubiquitously anneal with strains and representatives of Staphylococcus epidermidis.

41. A method for detecting the presence and/or amount of Staphylococcus epidermidis in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Staphylococcus epidermidis DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the following sequence: SEQ ID NO: 36;

c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of Staphylococcus epidermidis in said test sample.

42. The method of claim 41, wherein said at least one pair of primers is selected from the group consisting of:

a) SEQ ID NO: 145 and SEQ ID NO: 146, and

b) SEQ ID NO: 147 and SEQ ID NO: 148.

43. A method for the detection, identification and/or quantification of Staphylococcus aureus directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 37, a sequence complementary thereof, a part thereof and a variant thereof, which specifically and ubiquitously anneals with strains or representatives of Staphylococcus aureus, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of the presence and/or amount of Staphylococcus aureus in said test sample.

44. A method as defined in claim 43, wherein said probe is selected from the group consisting of an oligonucleotide of 12-442 nucleotides in length which sequence is comprised in SEQ ID NO: 37 and variants thereof which specifically and ubiquitously anneal with strains and representatives of Staphylococcus aureus.

45. A method for detecting the presence and/or amount of Staphylococcus aureus in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Staphylococcus aureus DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the following sequence: SEQ ID NO: 37;

c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of Staphylococcus aureus in said test sample.

46. The method of claim 45, wherein said at least one pair of primers is selected from the group consisting of:

a) SEQ ID NO: 149 and SEQ ID NO: 150,

b) SEQ ID NO: 149 and SEQ ID NO: 151, and

c) SEQ ID NO: 152 and SEQ ID NO: 153.

47. A method for the detection, identification and/or quantification of Haemophilus influenzae directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, a sequence complementary thereof, a part thereof and a variant thereof, which specifically and ubiquitously anneals with strains or representatives of Haemophilus influenzae, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of the presence and/or amount of Haemophilus influenzae in said test sample.

48. A method as defined in claim 47, wherein said probe is selected from the group consisting of:

1) an oligonucleotide of 12-845 nucleotides in length which sequence is comprised in SEQ ID NO: 25 or a complementary sequence thereof,

2) an oligonucleotide of 12-1598 nucleotides in length which sequence is comprised in SEQ ID NO: 26 or a complementary sequence thereof,

3) an oligonucleotide of 12-9100 nucleotides in length which sequence is comprised in SEQ ID NO: 27 or a complementary sequence thereof, and

variants thereof which specifically and ubiquitously anneal with strains and representatives of Haemophilus influenzae.

49. The method of claim 48, wherein the probe for detecting nucleic acid sequences from Haemophilus influenzae is selected from the group consisting of SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107 and a sequence complementary thereof.

50. A method for detecting the presence and/or amount of Haemophilus influenzae in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Haemophilus influenzae DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within one of the following sequences: SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27;

c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of Haemophilus influenzae in said test sample.

51. The method of claim 50, wherein said at least one pair of primers comprises the following pair: SEQ ID NO: 154 and SEQ ID NO: 155.

52. A method for the detection, identification and/or quantification of Streptococcus pneumoniae directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 35, a sequence complementary thereof, a part thereof and a variant thereof, which specifically and ubiquitously anneals with strains or representatives of Streptococcus pneumoniae, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of the presence and/or amount of Streptococcus pneumoniae in said test sample.

53. A method as defined in claim 52, wherein said probe is selected from the group consisting of:

1) an oligonucleotide of 12-631 nucleotides in length which sequence is comprised in SEQ ID NO: 30 or a complementary sequence thereof,

2) an oligonucleotide of 12-3754 nucleotides in length which sequence is comprised in SEQ ID NO: 31 or a complementary sequence thereof,

3) an oligonucleotide of 12-841 nucleotides in length which sequence is comprised in SEQ ID NO: 34 or a complementary sequence thereof,

4) an oligonucleotide of 12-4500 nucleotides in length which sequence is comprised in SEQ ID NO: 35 or a complementary sequence thereof, and

variants thereof which specifically and ubiquitously anneal with strains and representatives of Streptococcus pneumoniae.

54. The method of claim 53, wherein the probe for detecting nucleic acid sequences from Streptococcus pneumoniae is selected from the group consisting of SEQ ID NO: 120, SEQ ID NO: 121 and a sequence complementary thereof.

55. A method for detecting the presence and/or amount of Streptococcus pneumoniae in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Streptococcus pneumoniae DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within one of the following sequences: SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 34 and SEQ ID NO: 35;

c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of Streptococcus pneumoniae in said test sample.

56. The method of claim 55, wherein said at least one pair of primers is selected from the group consisting of:

a) SEQ ID NO: 78 and SEQ ID NO: 79,

b) SEQ ID NO: 156 and SEQ ID NO: 157, and

c) SEQ ID NO: 158 and SEQ ID NO: 159.

57. A method for the detection, identification and/or quantification of Streptococcus pyogenes directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, a sequence complementary thereof, a part thereof and a variant thereof, which specifically and ubiquitously anneals with strains or representatives of Streptococcus pyogenes, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of the presence and/or amount of Streptococcus pyogenes in said test sample.

58. A method as defined in claim 57, wherein said probe is selected from the group consisting of:

1) an oligonucleotide of 12-1337 nucleotides in length which sequence is comprised in SEQ ID NO: 32 or a complementary sequence thereof,

2) an oligonucleotide of 12-1837 nucleotides in length which sequence is comprised in SEQ ID NO: 33 or a complementary sequence thereof, and

variants thereof which specifically and ubiquitously anneal with strains and representatives of Streptococcus pyogenes.

59. A method for detecting the presence and/or amount of Streptococcus pyogenes in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Streptococcus pyogenes DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within one of the following sequences: SEQ ID NO: 32 and SEQ ID NO: 33;

c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of Streptococcus pyogenes in said test sample.

60. The method of claim 59, wherein said at least one pair of primers is selected from the group consisting of:

a) SEQ ID NO: 141 and SEQ ID NO: 142, and

b) SEQ ID NO: 143 and SEQ ID NO: 144.

61. A method for the detection, identification and/or quantification of Enterococcus faecalis directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, a sequence complementary thereof, a part thereof and a variant thereof, which specifically and ubiquitously anneals with strains or representatives of Enterococcus faecalis, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of the presence and/or amount of Enterococcus faecalis in said test sample.

62. A method as defined in claim 61, wherein said probe is selected from the group consisting of:

1) an oligonucleotide of 12-1817 nucleotides in length which sequence is comprised in SEQ ID NO: 1 or a complementary sequence thereof,

2) an oligonucleotide of 12-2275 nucleotides in length which sequence is comprised in SEQ ID NO: 2, and

variants thereof which specifically and ubiquitously anneal with strains and representatives of Enterococcus faecalis.

63. A method for detecting the presence and/or amount of Enterococcus faecalis in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Enterococcus faecalis DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within one of the following sequences: SEQ ID NO: 1 and SEQ ID NO: 2;

c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of Enterococcus faecalis in said test sample.

64. The method of claim 63, wherein said at least one pair of primers is selected from the group consisting of:

a) SEQ ID NO: 38 and SEQ ID NO: 39, and

b) SEQ ID NO: 40 and SEQ ID NO: 41.

65. A method for the detection of the presence and/or amount of any bacterial species directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a universal probe which sequence is selected from the group consisting of SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130 and a sequence complementary thereof, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of the presence and/or amount of said any bacterial species in said test sample.

66. A method for detecting the presence and/or amount of any bacterial species in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing a pair of universal primers which sequence is defined in SEQ ID NO: 126 and SEQ ID NO: 127, one of said primers being capable of hybridizing selectively with one of the two complementary strands of said any bacterial species DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template;

c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of said any bacterial species in said test sample.

67. A method for evaluating a bacterial resistance to β-lactam antibiotics mediated by the bacterial antibiotic resistance gene bla_tem(TEM-1) directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 161, a sequence complementary thereof, a part thereof and a variant thereof, which specifically anneals with said bacterial antibiotic resistance gene coding for a β-lactamase, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of a bacterial resistance to β-lactam antibiotics mediated by the bacterial antibiotic resistance gene TEM-1.

68. A method as defined in claim 67, wherein said probe comprises an oligonucleotide of at least 12 nucleotides in length which hybridizes to SEQ ID NO: 161.

69. A method for evaluating a bacterial resistance to β-lactam antibiotics mediated by the bacterial antibiotic resistance gene bla_tem(TEM-1) in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of said bacterial antibiotic resistance gene coding for a β-lactamase that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the sequence defined in SEQ ID NO: 161;

c) detecting the presence and/or amount of said amplified target sequence as an indication of a bacterial resistance to β-lactam antibiotics mediated by the bacterial antibiotic resistance gene TEM-1.

70. A method for evaluating a bacterial resistance to β-lactam antibiotics mediated by the bacterial antibiotic resistance gene bla_rob(ROB-1) directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 162, a sequence complementary thereof, a part thereof and a variant thereof, which specifically anneals with said bacterial antibiotic resistance gene coding for a β-lactamase, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of a bacterial resistance to β-lactam antibiotics mediated by the bacterial antibiotic resistance gene ROB-1.

71. A method as defined in claim 70, wherein said probe comprises an oligonucleotide of at least 12 nucleotides in length which hybridizes to SEQ ID NO: 162.

72. A method for evaluating a bacterial resistance to β-lactam antibiotics mediated by the bacterial antibiotic resistance gene bla_rob(ROB-1) in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of said bacterial antibiotic resistance gene coding for a β-lactamase that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the sequence defined in SEQ ID NO: 162;

c) detecting the presence and/or amount of said amplified target sequence as an indication of a bacterial resistance to β-lactam antibiotics mediated by the bacterial antibiotic resistance gene ROB-1.

73. A method for evaluating a bacterial resistance to β-lactam antibiotics mediated by the bacterial antibiotic resistance gene bla_shv(SHV-1) directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 163, a sequence complementary thereof, a part thereof and a variant thereof, which specifically anneals with said bacterial antibiotic resistance gene coding for a β-lactamase, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of a bacterial resistance to β-lactam antibiotics mediated by the bacterial antibiotic resistance gene SHV-1.

74. A method as defined in claim 73, wherein said probe comprises an oligonucleotide of at least 12 nucleotides in length which hybridizes to SEQ ID NO: 163.

75. A method for evaluating a bacterial resistance to β-lactam antibiotics mediated by the bacterial antibiotic resistance gene bla_shv(SHV-1) in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of said bacterial antibiotic resistance gene coding for a β-lactamase that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the sequence defined in SEQ ID NO: 163;

c) detecting the presence and/or amount of said amplified target sequence as an indication of a bacterial resistance to β-lactam antibiotics mediated by the bacterial antibiotic resistance gene SHV-1.

76. A method for evaluating a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aadB directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 164, a sequence complementary thereof, a part thereof and a variant thereof, which specifically anneals with said bacterial antibiotic resistance gene coding for an aminoglycoside adenylyltransferase, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aadB.

77. A method as defined in claim 76, wherein said probe comprises an oligonucleotide of at least 12 nucleotides in length which hybridizes to SEQ ID NO: 164.

78. A method for evaluating a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aadB in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of said bacterial antibiotic resistance gene coding for an aminoglycoside adenylyltransferase that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the sequence defined in SEQ ID NO: 164;

c) detecting the presence and/or amount of said amplified target sequence as an indication of a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aadB.

79. A method for evaluating a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacC1 directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 165, a sequence complementary thereof, a part thereof and a variant thereof, which specifically anneals with said bacterial antibiotic resistance gene coding for an aminoglycoside acetyltransferase, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacC1.

80. A method as defined in claim 79, wherein said probe comprises an oligonucleotide of at least 12 nucleotides in length which hybridizes to SEQ ID NO: 165.

81. A method for evaluating a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aaccl in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of said bacterial antibiotic resistance gene coding for an aminoglycoside acetyltransferase that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the sequence defined in SEQ ID NO: 165;

c) detecting the presence and/or amount of said amplified target sequence as an indication of a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacC1.

82. A method for evaluating a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacC2 directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 166, a sequence complementary thereof, a part thereof and a variant thereof, which specifically anneals with said bacterial antibiotic resistance gene coding for an aminoglycoside acetyltransferase, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacC2.

83. A method as defined in claim 82, wherein said probe comprises an oligonucleotide of at least 12 nucleotides in length which hybridizes to SEQ ID NO: 166.

84. A method for evaluating a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacC2 in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of said bacterial antibiotic resistance gene coding for an aminoglycoside acetyltransferase that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the sequence defined in SEQ ID NO: 166;

c) detecting the presence and/or amount of said amplified target sequence as an indication of a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacC2.

85. A method for evaluating a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacC3 directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 167, a sequence complementary thereof, a part thereof and a variant thereof, which specifically anneals with said bacterial antibiotic resistance gene coding for an aminoglycoside acetyltransferase, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacC3.

86. A method as defined in claim 85, wherein said probe comprises an oligonucleotide of at least 12 nucleotides in length which hybridizes to SEQ ID NO: 167.

87. A method for evaluating a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacC3 in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of said bacterial antibiotic resistance gene coding for an aminoglycoside acetyltransferase that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the sequence defined in SEQ ID NO: 167;

c) detecting the presence and/or amount of said amplified target sequence as an indication of a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacC3.

88. A method for evaluating a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacA4 directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 168, a sequence complementary thereof, a part thereof and a variant thereof, which specifically anneals with said bacterial antibiotic resistance gene coding for an aminoglycoside acetyltransferase, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacA4.

89. A method as defined in claim 88, wherein said probe comprises an oligonucleotide of at least 12 nucleotides in length which hybridizes to SEQ ID NO: 168.

90. A method for evaluating a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacA4 in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of said bacterial antibiotic resistance gene coding for an aminoglycoside acetyltransferase that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the sequence defined in SEQ ID NO: 168;

c) detecting the presence and/or amount of said amplified target sequence as an indication of a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacA4.

91. A method for evaluating a bacterial resistance to β-lactam antibiotics mediated by the bacterial antibiotic resistance gene mecA directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 169, a sequence complementary thereof, a part thereof and a variant thereof, which specifically anneals with said bacterial antibiotic resistance gene coding for a penicillin-binding protein, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of a bacterial resistance to β-lactam antibiotics mediated by the bacterial antibiotic resistance gene mecA.

92. A method as defined in claim 91, wherein said probe comprises an oligonucleotide of at least 12 nucleotides in length which hybridizes to SEQ ID NO: 169.

93. A method for evaluating a bacterial resistance to β-lactam antibiotics mediated by the bacterial antibiotic resistance gene mecA in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of said bacterial antibiotic resistance gene coding for a penicillin-binding protein that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the sequence defined in SEQ ID NO: 169;

c) detecting the presence and/or amount of said amplified target sequence as an indication of a bacterial resistance to β-lactam antibiotics mediated by the bacterial antibiotic resistance gene mecA.

94. A method for evaluating a bacterial resistance to vancomycin mediated by the bacterial antibiotic resistance genes vanH, vanA and vanX directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 170, a sequence complementary thereof, a part thereof and a variant thereof, which specifically anneals with said bacterial antibiotic resistance genes coding for vancomycin-resistance proteins, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of a bacterial resistance to vancomycin mediated by the bacterial antibiotic resistance genes vanH, vanA and vanX.

95. A method as defined in claim 94, wherein said probe comprises an oligonucleotide of at least 12 nucleotides in length which hybridizes to SEQ ID NO: 170.

96. A method for evaluating a bacterial resistance to vancomycin mediated by the bacterial antibiotic resistance genes vanH, vanA and vanX in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of said bacterial antibiotic resistance genes coding for vancomycin-resistance proteins that contain a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the sequence defined in SEQ ID NO: 170;

c) detecting the presence and/or amount of said amplified target sequence as an indication of a bacterial resistance to vancomycin mediated by the bacterial antibiotic resistance genes vanH, vanA and vanX.

97. A method for evaluating a bacterial resistance to streptogramin A mediated by the bacterial antibiotic resistance gene satA directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 173, a sequence complementary thereof, a part thereof and a variant thereof, which specifically anneals with said bacterial antibiotic resistance gene coding for a streptogramin A acetyltransferase, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of a bacterial resistance to streptogramin A mediated by the bacterial antibiotic resistance gene satA.

98. A method as defined in claim 97, wherein said probe comprises an oligonucleotide of at least 12 nucleotides in length which hybridizes to SEQ ID NO: 173.

99. A method for evaluating a bacterial resistance to streptogramin A mediated by the bacterial antibiotic resistance gene satA in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of said bacterial antibiotic resistance gene coding for streptogramin A acetyltransferase that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the sequence defined in SEQ ID NO: 173;

c) detecting the presence and/or amount of said amplified target sequence as an indication of a bacterial resistance to streptogramin A mediated by the bacterial antibiotic resistance gene satA.

100. A method for evaluating a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacA-aphD directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 174, a sequence complementary thereof, a part thereof and a variant thereof, which specifically anneals with said bacterial antibiotic resistance gene coding for an aminoglycoside acetyltransferase-phosphotransferase under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacA-aphD.

101. A method as defined in claim 100, wherein said probe comprises an oligonucleotide of at least 12 nucleotides in length which hybridizes to SEQ ID NO: 174.

102. A method for evaluating a bacterial resistance to aminoglycoside; antibiotics mediated by the bacterial antibiotic resistance gene aacA-aphD in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of said bacterial antibiotic resistance gene coding for an aminoglycoside acetyltransferase-phosphotransferase that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the sequence defined in SEQ ID NO: 174;

c) detecting the presence and/or amount of said amplified target sequence as an indication of a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacA-aphD.

103. A method for evaluating a bacterial resistance to virginiamycin mediated by the bacterial antibiotic resistance gene vat directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 175, a sequence complementary thereof, a part thereof and a variant thereof, which specifically anneals with said bacterial antibiotic resistance gene coding for a virginiamycin acetyltransferase, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of a bacterial resistance to virginiamycin mediated by the bacterial antibiotic resistance gene vat.

104. A method as defined in claim 103, wherein said probe comprises an oligonucleotide of at least 12 nucleotides in length which hybridizes to SEQ ID NO: 175.

105. A method for evaluating a bacterial resistance to virginiamycin mediated by the bacterial antibiotic resistance gene vat in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of said bacterial antibiotic resistance gene coding for a virginiamycin acetyltransferase that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the sequence defined in SEQ ID NO: 175;

c) detecting the presence and/or amount of said amplified target sequence as an indication of a bacterial resistance to virginiamycin mediated by the bacterial antibiotic resistance gene vat.

106. A method for evaluating a bacterial resistance to virginiamycin mediated by the bacterial antibiotic resistance gene vga directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 176, a sequence complementary thereof, a part thereof and a variant thereof, which specifically anneals with said bacterial antibiotic resistance gene coding for an ATP-binding protein, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of a bacterial resistance to virginiamycin mediated by the bacterial antibiotic resistance gene vga.

107. A method as defined in claim 106, wherein said probe comprises an oligonucleotide of at least 12 nucleotides in length which hybridizes to SEQ ID NO: 176.

108. A method for evaluating a bacterial resistance to virginiamycin mediated by the bacterial antibiotic resistance gene vga in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of said bacterial antibiotic resistance gene coding for an ATP-binding protein that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the sequence defined in SEQ ID NO: 176;

c) detecting the presence and/or amount of said amplified target sequence as an indication of a bacterial resistance to virginiamycin mediated by the bacterial antibiotic resistance gene vga.

109. A method for evaluating a bacterial resistance to erythromycin mediated by the bacterial antibiotic resistance gene msrA directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 177, a sequence complementary thereof, a part thereof and a variant thereof, which specifically anneals with said bacterial antibiotic resistance gene coding for an erythromycin resistance protein under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of a bacterial resistance to erythromycin mediated by the bacterial antibiotic resistance gene msrA.

110. A method as defined in claim 109, wherein said probe comprises an oligonucleotide of at least 12 nucleotides in length which hybridizes to SEQ ID NO: 177.

111. A method for evaluating a bacterial resistance to erythromycin mediated by the bacterial antibiotic resistance gene msrA in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of said bacterial antibiotic resistance gene coding for an erythromycin resistance protein that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the sequence defined in SEQ ID NO: 177;

c) detecting the presence and/or amount of said amplified target sequence as an indication of a bacterial resistance to erythromycin mediated by the bacterial antibiotic resistance gene msrA.

112. A method for evaluating potential bacterial resistance to β-lactams, aminoglycosides, chloramphenicol and/or trimethoprim mediated by the bacterial antibiotic resistance gene int directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 171, a sequence complementary thereof, a part thereof and a variant thereof, which specifically anneals with said bacterial antibiotic resistance gene coding for an integrase, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of potential bacterial resistance to β-lactams, aminoglycosides, chloramphenicol and/or trimethoprim mediated by the bacterial antibiotic resistance gene int.

113. A method as defined in claim 112, wherein said probe comprises an oligonucleotide of at least 12 nucleotides in length which hybridizes to SEQ ID NO: 171.

114. A method for evaluating potential bacterial resistance to β-lactams, aminoglycosides, chloramphenicol and/or trimethoprim mediated by the bacterial antibiotic resistance gene int in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of said bacterial antibiotic resistance gene coding for an integrase that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the sequence defined in SEQ ID NO: 171;

c) detecting the presence and/or amount of said amplified target sequence as an indication of potential bacterial resistance to β-lactams, aminoglycosides, chloramphenicol and/or trimethoprim mediated by the bacterial antibiotic resistance gene int.

115. A method for evaluating potential bacterial resistance to β-lactams, aminoglycosides, chloramphenicol and/or trimethoprim mediated by the bacterial antibiotic resistance gene sul directly from a test sample or from bacterial colonies, which comprises the following steps:

said bacterial DNA being in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 172, a sequence complementary thereof, a part thereof and a variant thereof, which specifically anneals with said bacterial antibiotic resistance gene coding for a sulfonamide resistance protein under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and

c) detecting the presence or the intensity of said label on said inert support or in said solution as an indication of potential bacterial resistance to β-lactams, aminoglycosides, chloramphenicol and/or trimethoprim mediated by the bacterial antibiotic resistance gene sul.

116. A method as defined in claim 115, wherein said probe comprises an oligonucleotide of at least 12 nucleotides in length which hybridizes to SEQ ID NO: 172.

117. A method for evaluating potential bacterial resistance to β-lactams, aminoglycosides, chloramphenicol and/or trimethoprim mediated by the bacterial antibiotic resistance gene sul in a test sample which comprises the following steps:

a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of said bacterial antibiotic resistance gene coding for a sulfonamide resistance protein that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the sequence defined in SEQ ID NO: 172;

c) detecting the presence and/or amount of said amplified target sequence as an indication of potential bacterial resistance to β-lactams, aminoglycosides, chloramphenicol and/or trimethoprim mediated by the bacterial antibiotic resistance gene sul.

118. A nucleic acid having the nucleotide sequence of any one of SEQ ID NOs: 1 to 37, SEQ ID NOs: 161 to 177, a part thereof and variants thereof which, when in single stranded form, ubiquitously and specifically hybridize with a target bacterial DNA as a probe or as a primer.

119. An oligonucleotide having a nucleotidic sequence of any one of SEQ ID NOs: 38 to 160.

120. A recombinant plasmid comprising a nucleic acid as defined in claim 118.

121. A recombinant host which has been transformed by a recombinant plasmid according to claim 120.

122. A recombinant host according to claim 121 wherein said host is Escherichia coli.

123. A diagnostic kit for the detection and/or quantification of the nucleic acids of any combination of the bacterial species defined in any one of claims 9, 14, 19, 24, 29, 34, 39, 43, 47, 52, 57 and 61, comprising any combination of probes defined therein.

124. A diagnostic kit for the detection and/or quantification of the nucleic acids of any combination of the bacterial species defined in any one of claims 10, 11, 15, 16, 20, 21, 25, 26, 30, 31, 35, 36, 40, 44, 48, 49, 53, 54, 58, 62 and 65, comprising any combination of oligonucleotide probes defined therein.

125. A diagnostic kit for the detection and/or quantification of the nucleic acids of any combination of the bacterial species defined in any one of claims 12, 13, 17, 18, 22, 23, 27, 28, 32, 33, 37, 38, 41, 42, 45, 46, 50, 51, 55, 56, 59, 60, 63, 64 and 66 comprising any combination of primers defined therein.

126. A diagnostic kit for the detection and/or quantification of the nucleic acids of any combination of the bacterial resistance genes defined in any one of claims 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106 and 109 comprising any combination of probes defined therein.

127. A diagnostic kit for the detection and/or quantification of the nucleic acids of any combination of the bacterial resistance genes defined in any one of claims 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107 and 110 comprising any combination of oligonucleotide probes defined therein.

128. A diagnostic kit for the detection and/or quantification of the nucleic acids of any combination of the bacterial resistance genes defined in any one of claims 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108 and 111 comprising any combination of primers defined therein.

129. A diagnostic kit for the simultaneous detection and quantification of nucleic acids of any combination of the bacterial species defined in claim 123, comprising any combination of the bacterial probes defined therein and any combination of the probes to the antibiotic resistance genes defined in any one of SEQ ID NOs: 161 to 177 in whole or in part.

130. A diagnostic kit for the simultaneous detection and quantification of nucleic acids of any combination of the bacterial species defined in claim 124, comprising any combination of the bacterial oligonucleotide probes defined therein and any combination of oligonucleotide probes that hybridize to the antibiotic resistance genes defined in any one of SEQ ID NOs: 161 to 177.

131. A diagnostic kit for the simultaneous detection and quantification of nucleic acids of any combination of the bacterial species defined in claim 125, comprising any combination of the primers defined therein and any combination of primers that anneal to the antibiotic resistance genes defined in any one of SEQ ID NOs: 161 to 177.