EP1934376A2 - Dna microarray for rapid identification of candida albicans in blood cultures. - Google Patents
Dna microarray for rapid identification of candida albicans in blood cultures.Info
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- EP1934376A2 EP1934376A2 EP06806431A EP06806431A EP1934376A2 EP 1934376 A2 EP1934376 A2 EP 1934376A2 EP 06806431 A EP06806431 A EP 06806431A EP 06806431 A EP06806431 A EP 06806431A EP 1934376 A2 EP1934376 A2 EP 1934376A2
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- gene probes
- seq
- dna
- identification
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
Definitions
- the present invention provides an analytical device, especially a DNA microarray, for identification and characterisation of microorganisms in a sample or clinical specimen. Furthermore, it provides for a method for rapid identification and strain profiling of different microbial species in a sample or clinical specimen, especially in a blood culture, utilizing said analytical device.
- Isolation, identification and characterisation of bacteria and fungi from clinical specimens is a main task of microbiological routine diagnostics.
- microorganisms are ubiquitous in certain areas of the human body. For this reason isolation and identification of pathogenic bacteria from clinical material and discrimination of specific pathogens from contaminations with indigenous or environmentally encountered microorganisms is a requirement for the correct diagnosis of infectious diseases. Additionally, accurate identification of antibiotic resistance and particular virulence factors provide important information enabling the clinician to choose effective antimicrobial therapy.
- specimen types can be used for direct identification of the pathogens. These include, but are not limited to, liquor in the course of bacterial meningitis, sputum from patients with bacterial pneumonia, urine in the course of upper and lower urinary tract infections, huiate from sites of deep purulent infections (such as abscess, phlegmone, lung emphysema and septic arthritis), stool from patients with gastrointestinal tract infections, pus, swabs or wound fluid from purulent infections of the skin and wounds.
- bacteria are represented in the specimen only in minor numbers, thus, indirect identification of pathogens after culture of specimens in liquid media is employed. Important examples are enrichment cultures of food samples during outbreaks of food borne infections and blood cultures for diagnosis of bloodstream infections.
- Bacteremia is the means by which local infections spread hematogenously to distant organs. This hematogenous dissemination of bacteria is part of the pathophysiology of, e.g., meningitis and endocarditis, Pott's disease and many other forms of osteomyelitis.
- indwelling catheters are a frequent cause of bacteremia and subsequent nosocomial infections, since they provide a means by which bacteria normally found on the skin can enter the bloodstream.
- Other causes of bacteremia include dental procedures, urinary tract infections, intravenous drug use, and colorectal cancer.
- Systemic fungal infection is becoming more and more common in modern hospitals.
- the most common fungal infections are candidiasis and aspergillosis, but other systemic fungal infections such as Histoplasmosis, Blastomycosis, Coccidioidomycosis and Cryptococcosis are also of increasing relevance.
- Systemic fungal infections in hospitals are commonly seen in immune compromised patients and - like bacteremia - in patients with indewelling catheters. Due to underlying serious illnesses and possible resistance of the pathogens to antifungal agents, patients with systemic fungal infections often have poor clinical outcomes. Infections due to Candida species are the fourth most important cause of nosocomial bloodstream infection.
- Bacteremia is operationally defined as the presence of viable bacteria as evidenced by positive blood cultures. Fungemia is similarly defined as the presence of viable fungi as evidenced by positive blood cultures. When bacteremia or fungemia occurs in the presence of systemic symptoms (such as fever or chills) the condition is designated as sepsis; and in the setting of more severe disturbances of temperature, respiration, heart rate or white blood cell count, is characterised as systemic inflammatory response syndrome (SIRS).
- SIRS systemic inflammatory response syndrome
- Staphylococcus aureus Escherichia coli, Coagulase-negative staphylococci (CoNS)
- Klebsiella pneumoniae Pseudomonas aeruginosa
- Enterococcus spp. Streptococcus spp.
- Candida albicans and Enterobacter cloacae are the most frequent etiological agents of bacteremia and fungemia in Europe (Decousser, J. W. et al., J. Antimicrob. Chemother. 51: 1214-22 (2003); Lyytikainen, O. et al., Clin.
- said therapy has to be performed as empirical initial therapy (ReIIo, J. et al., Intensive Care Med. 20:94-98 (1994)), which covers the complete spectrum of relevant pathogens.
- the increase of bacterial resistance lowers the chance of success for such empirical antibiotic treatments considerably (Mylotte, J. M. and Tayara, A., Eur. Clin. Microbiol. Infect. Dis. 19: 157-163 (2000); Weinstein, M. P. et al., Clin. Infect. Dis. 24:584-602 (1997)).
- Staphylococci are the most important and frequent group of pathogens growing in blood culture, responsible for 30% to more than 50% of all bacteremia events (James, P.A. and Al-Shafi, K.M., J. Clin. Pathol. 53:231-233 (2000); Reisner, B. S. and Woods, G. L, J. Clin. Microbiol. 37:2024-2026 (1999); Velasco, E. et al., Sao Paulo Med. J. 118: 131-138 (2000)) with a mortality rate ranging from 13 to 50% (McClelland, R.S. et al., Arch. Intern. Med. 159:1244-1247 (1999); ReIIo, J.
- E. coli, Klebsiella spp., Enterobacter spp., Proteus spp., Pseudomonas aeruginosa, Streptococcus pneumoniae, beta hemolytic Streptococci and Enterococcus spp. belong to the most frequent reported pathogens causing bacteremia (Reimer, L.G. et al., Clin. Microbiol. Rev., 10:444-65 (1997); Reacher, M. H. et al., BMJ, 320:213-6 (2000); Lyytikainen, O. et al., Clin. Infect.
- microarrays were recently also used to identify viral (Wang, R.F. et al., FEMS Microbiol. Lett. 213: 175-182 (2002)) and bacterial (Bekal, S. et al., J. Clin. Microbiol. 41:2113-2125 (2003)) pathogens in environmental and clinical samples.
- microarrays for detection of bacteria and fungi are known in the art (Nakamura, M. et al., Abstracts of the general meeting of the American society for microbiology, abstract No C219 (2003); Wang, R.-F. et al., Molecular and Cellular Probes 223-224 (2004); Lehner, A. et al., FEMS Microbiol. Lett. 133-142 (2005); EP 1310569; WO 92/07096; US-B1-6,747,137).
- all these microarrays have in common the use of short oligonucleotides with a maximum length of 40 nt ("short oligonucleotides").
- short-oligonucleotide microarrays They are short-oligonucleotide microarrays. Although such short-oligonucleotide microarrays could be rapidly designed and built up they carry some intrinsic disadvantages: like all methods based on single and often short DNA sequences they show reduced reliability and sensitivity (Stears, R.L. et al., Nat. Med. 9:140-145 (2003)). To palliate the high probability of non-specific hybridisation due to the short size (20-40 bp) of the oligonucleotides it is necessary to design many partially overlapping oligonucleotides in order to confirm the presence of a gene. This consequent increase in complexity makes it extremely difficult to set up the optimal hybridisation conditions necessary for producing trustful results.
- oligonucleotide microarrays using oligonucleotides with a maximum length of 40 nt are unsuitable for routine diagnostics.
- a DNA microarray employing capture probes of more than 40 nt length amplified by PCR was described by Fitzgerald et al. (Fitzgerald, J. R. at al., Proc. Natl. Acad. Sci. USA 98(15) :8821-8826 (2001)).
- a microarray comprising 2817 complete ORFs of S. aureus strain COL was constructed, representing >90% of the S. aureus genome.
- the microarray was able to discriminate 36 S. aureus strains. However, since it was not designed for the identification of different bacterial species, it was not tested for possible cross reactions with other bacteria besides S.
- the aim of present invention is to provide a gene-segment based analytical device, especially a microarray, for species specific identification and characterisation of different microorganisms, especially different bacteria and pathogenic fungi, present in a sample or clinical specimen which does not possess the drawbacks of the short-oligonucleotide microarray as outlined above.
- Said device/microarray must allow the specific identification of the target species and should furthermore allow the differentiation (i.e. distinguish) between different target microorganisms present in the sample or clinical specimen. It must furthermore provide a high reliability and sensitivity of detection.
- the present invention provides an analytical device, which is preferably a DNA microarray, for the identification and characterisation of microorganisms in biological samples, especially of microorganisms connected with bacteremia, fungemia and sepsis.
- Species specific gene probes in this device/microarray allow the identification of different microbial species, whilst antibiotic resistance and virulence gene probes allow for the genotypic discrimination within a species.
- the device/microarray can be designed to allow species identification, virulence determination and resistance determination independently from each other or simultaneously, and furthermore said determinations can be performed for one or more different microbial species and strains with one device/microarray. Furthermore, different microbial species and strains are discriminated, even in a polymicrobial sample (specimen with more than one pathogen).
- the device/DNA microarray according to present invention thus demonstrates the feasibility of simultaneously identifying and characterising different microbial species in a sample or clinical specimen, especially in blood samples, without prior PCR amplification of target DNA or pre-identification of the pathogen. This can reduce sample processing time to a single day and less.
- the invention furthermore provides a method for rapid identification and characterisation of microorganisms, especially of bacteria, yeasts and filamentous fungi, using the device/microarray of the invention.
- the method is quick, can be automated, leads to reproducible results and allows an early choice of specific antibiotics for treatment of bacteremia, fungemia or sepsis.
- the present invention provides
- an analytical device for direct identification and characterisation of microorganisms in a sample or clinical specimen, wherein the analytical device comprises species specific gene probes which are (i) selected from DNA sequences or partial DNA sequences of the microorganisms to be identified or DNA sequences complementary or homologous thereto, and (ii) have a length of at least 100 nucleotides (nt);
- an in vitro method for identification and characterisation of microorganisms in a sample or in a clinical specimen comprising (a) isolating the total DNA from the sample or clinical specimen and labelling the DNA with a reporter molecule, preferably a fluorochrome;
- Fig. 1 DNA microarray analyses of 58 clinical isolates, reference strains and blood cultures.
- Each column shows the results of an individual hybridisation with target DNA prepared from: S. aureus ATCC 29213 (1), MW2 (2), clinical isolates (3-7), positive blood cultures (8-11); P. aeruginosa ATCC 27853 (12), clinical isolates (13-17), positive blood culture (18); E. coli ATCC 25922 (19), clinical isolates (20-25), positive blood cultures (26-27); S. epidermidis clinical isolates (28-32), positive blood cultures (33-35); clinical isolates of S. au ⁇ cula ⁇ s (36), S. capitis (37), S. haemolyticus (38), S. hominis (39), and S. warneri (40).
- Gram-negative species included a Proteus mirabilis positive blood culture (41), clinical isolates of Proteus mirabilis (42-43), Serratia marcescens (44-45), Klebsiella pneumonia (46- 48), Stenotrophomonas maltophilia (49), Acinetobacter baumannii (50), Enterobacter cloacae (51) and Enterobacter aerogenes (52); other Gram-positive species included clinical isolates of Micrococcus spp. (53), Enterococcus spp. (54), Enterococcus faecalis (55) and Streptococcus pneumoniae (56) and two positive blood cultures of S. pneumoniae (57-58).
- FIG. 2 Validation of the S. aureus microarray of example 1.11. 2 ⁇ g genomic DNA from S. aureus strain T94 were labelled either with Cy3 or Cy5, combined and hybridised as described in Example 1.11. Cy3: green signal; Cy5: red signal; double-hybridisation : yellow signal. A) Overlay of microarray scanned using Cy3 and Cy5 filter sets;
- Fig. 3 Specific identification of S. aureus from distantly related bacteria using the microarray of example 1.11. 2 ⁇ g of S. aureus DNA were co-hybridised with 2 ⁇ g of pure E. coli (A) or P. aeruginosa (B) genomic DNA. Obtained hybridisation patterns are represented as bar codes, where the 140 spotted gene segments appear subsequently and are clustered in categories (NC: negative control; PC: positive control; Antibiotic Resistance Determinants; Virulence Factors and Metabolic Functions (see Tab. 6)). Positive hybridisation is indicated by a bar while negative spots are represented by an empty area. Both assays show clear S. aureus discrimination with practically no cross hybridisation between DNA from said gram negative bacteria and S.
- FIG. 4 Specific identification of S. aureus from coagulase negative staphylococci using the microarray of example 1.11. 2 ⁇ g of S. aureus DNA were co-hybridised with 2 ⁇ g of S. epidermidis (A) or S. saprophytics (B) genomic DNA. Obtained hybridisation patterns are illustrated by scanned fluorescent picture data (A: S. aureus: green signal; S. epidermidis: red signal; B: S. aureus: red signal; S. saprophyticus: green signal) and transformed in bar codes (see legend of Fig. 3). All specific S. aureus virulence factor genes hybridised exclusively with S. aureus DNA. Yellow spots showing cross-hybridisation correspond to some shared antibiotic resistance determinants and genes associated to metabolic functions.
- Fig. 5 Specificity of the S. aureus microarray of example 1.11.
- Fig. 6 Identification and characterisation of S. aureus from positive blood culture using the microarray of example 1.11. 2 ⁇ g of DNA prepared from blood culture positive for S. aureus (strain T95) was co- hybridised with 2 ⁇ g of DNA prepared from sterile blood culture or with 2 ⁇ g of pure S. aureus genomic DNA for 4 hours. Positive and negative spots are transformed in a bar code scheme (see legend of Fig. 3).
- Fig. 7 Hybridization profiles obtained in Example 2 after microarray hybridization with DNA obtained from six bacterial target strains: (A) S. aureus ATCC 29213, (B) S. epidermidis BC 1920, (C) S. pyogenes DSM 11723, (D) S. pneumoniae ATCC 49619, (E) E. faecalis UW 700700/95, (F) E. faecium VRE9182 and two non-target strains: (G) E. casseliflavus UW703/95 and (H) S. angiosus DSM 20563.. Each bar represents the fluorescent signal of one capture probe.
- Fluorescent signals of the 930 probes represent the median intensity of four spots from which the local background was substracted. Probe IDs are given in Table 8.
- Fig. 8 Specificity of the microarray for Candida albicans in Example 2.
- Fig. 9 Specificity of selected capture probes for (A) Klebsiella oxytoca, (B) K. pneumoniae, (C) Proteus vulgaris and (D) P. mirabilis does allow species discrimination.
- Fluorescence intensities refer to hybridization signals obtained for the respective probes after hybridization with DNA isolated from 44 different microbial strains (see Table 9 for strain identification).
- Fig. 10 Specificity of selected capture probes for the coagulase-negative staphylococci (A) S. epidermidis, (B) S. haemolyticus, (C) S. warneri and (D) S. saprophytics. Fluorescence intensities refer to hybridization signals obtained for the respective probes after hybridization with DNA isolated from 44 different microbial strains (see Table 9 for strain identification).
- An “analytical device” in the context of present invention is any solid support onto which DNA gene probes are attached in a way permitting hybridisation of the DNA in the sample and subsequent detection of the bound DNA.
- This includes microtiter plates coated with one or several DNA gene probes per well, glass surfaces (like, e.g., microscopic slides) with DNA spots, filter paper disks, membranes, gold electrodes and beads (particles with a diameter of from 1 nm to several ⁇ m made of glass, plastic, metal etc.) coated with DNA, etc..
- the beads may be used in a multi-chamber system, preferably in a microfluidic multi-chamber system, wherein each chamber contains a population of beads.
- Each bead has an attached DNA sequence and the whole beads population in one chamber will carry the same DNA sequence, each chamber corresponding then to a specific capture probe.
- the target DNA to be analysed flows through the multi-chamber system and will hybridize with the complementary DNA sequences attached to the beads.
- Beads could be also attached to a surface by magnetic force, i.e. paramagnetic beads coupled with DNA could be attached on the surface of the magnet and arrange in a lattice structure. Vice versa, beads made of a magnetic material could be attached to an iron surface.
- the analytical device of present application is preferably a DNA microarray, a (magnetic) bead or set of beads coated with DNA probes or a microtiter plate coated with DNA probes. More preferred it is a (magnetic) bead or set of beads coated with DNA probes or a DNA microarray. In the most preferred aspect of present invention it is a DNA microarray.
- a “DNA microarray” consists of a collection of nucleic acid sequences, preferably DNA sequences, immobilized onto a solid support, such as glass, plastic or silicon chips, in a latticed pattern (forming an "array”). Each unique sequence of said sequences forms a tiny feature on the microarray called a “spot” or “capture probe”. The size of these spots varies from one system to another, but is usually less than two hundred micrometers in diameter, thus up to tens of thousands of spots can be arrayed in a total area of a few square centimeters. DNA microarrays provide a means to detect and quantify large numbers of discrete nucleic sequences in parallel.
- the nucleic acids in the sample that is being analysed are expected to form duplexes specifically with the corresponding capture probes. Occurrence or absence of duplex formation indicate the presence or absence of said target.
- said target is commonly converted to a labelled population of nucleic acids, using reporter molecules. Hybridisation of said labelled target DNA molecules from the tested samples with complementary DNA sequences affixed in specific spots on the array can thus be detected by examination for the presence of said label on the array using a microarray scanner (M ⁇ ller, H. -J., R ⁇ der, T., "Der Experimentator: Microarrays", Spektrum Akademischer Verlag, Heidelberg (2004)).
- Gene probe or “gene probe derived from” refers to a DNA sequence present on the microarray of present invention and used as a capture probe. It is a DNA segment (see below) which is complementary to a target DNA sequence, preferably to a microbial, more preferably to a bacterial or fungal gene or gene segment.
- Said gene probe is prepared by any known method of DNA synthesis, and preferably prepared by cloning the respective PCR-amplified gene or gene segment into a plasmid/vector. The recombinant gene or gene segment is then amplified by PCR, isolated from the amplification mix, purified (preferably by ethanol-purification) and finally spotted onto the array.
- An “isolate” is a microbial, especially a fungal or bacterial strain isolated from a given specimen, wherein the isolation includes at least one in vitro propagation.
- a "clinical isolate” is an isolate from a clinical specimen.
- CoNS Coagulase-negative staphylococci
- CoNS in the context of present invention are Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugdunensis and Staphylococcus warneri, of which Staphylococcus epidermidis is especially preferred.
- isolated DNA is a DNA separated or purified from the organism it is naturally associated with or from the clinical specimen in which it occurs. This comprises biochemically or biophysically purified native DNA, recombinant DNA, chemically synthesized DNA and DNA analogues (e.g. peptide nucleic acids).
- a “DNA segment” or “gene segment” is an isolated DNA which contains or consists of a part of the native full-length sequence of a gene which is still able to hybridize to the native sequence under stringent hybridisation conditions.
- DNA DNA
- the present invention is in the following exclusively described as relating to “DNA” sequences, it is not to be construed as being limited thereto. Rather, if the term “DNA” is used in connection with the gene probes or target sequences of present invention, it includes other polynucleotides (like RNA or RNA/DNA hybrids), and DNA analogues such as PNA, phosphonate backbone DNA, artificial pentose or hexose backbone DNA which is able to hybridize with native DNA etc..
- DNA itself is the preferred polynucleotide for performance of the invention.
- the DNA sequences used as gene probes in present invention are either identical, substantially identical or homologous to the complementary native target sequences (i.e. they are "derived from” said target sequences). In the context of present invention, when a specific DNA sequence is denominated, this encompasses not only said specific sequence, but also the sequences substantially identical or homologous thereto, i.e. its substitution mutants.
- “Substantially identical” means that the DNA contains mutations of up to 10% of the total number of nt in comparison with the native DNA sequence and/or has a nucleotide identity of > 90% to the corresponding native DNA segment. Said mutations are preferably single nucleotide polymorphisms or point mutations and include the mutation of not only a single but also a few (up to 10 nt, preferably up to 5 nt) consecutive nt. "Homologous” or “homologue” refers to a DNA sequence which has a sequence identity of more than 70% of the corresponding native DNA sequence and encompasses the substantially identical DNA sequences. Preferably, the sequences used as gene probes are at least substantially identical to the corresponding native DNA sequence.
- Preferred gene probes of the present invention are the DNA sequences listed in the sequence protocol, their complementary sequences or their corresponding native DNA segment.
- the DNA sequences used as gene probes in present invention may also be deletion or addition mutants of the corresponding native DNA segments.
- the minimum length of the DNA sequences suitable as probes in present invention is 100 nt.
- the deletions take place at the 5 ' - and/or 3 ' - terminus of the native DNA segment.
- the added nucleotides may sum up to a total of 90% of the nucleotide number of the native DNA segment, if added at the 5 ' - or 3 ' -terminus of the DNA sequence.
- the additions and deletions may be of one isolated nucleotide or of 2 or more consecutive nucleotides at one or more internal site(s) of the native DNA segment.
- 0-30% nucleotides of the corresponding native DNA segment are added or deleted.
- the addition or deletion mutants used as gene probes in present invention comprise one or more segment(s) of at least 100 consecutive nt each, which are derived from one gene, and/or sequences homologous (70% homology) or complementary thereto. These segments may be embedded in or fused to other DNA sequences, which will not hybridize under stringent conditions with either human or bacterial DNA or the DNA of the target microorganism.
- Said other DNA sequences preferably have a maximum length which adds up with the length of the enclosed segment(s) to not more than the upper limit for the length of gene probes suitable for present invention.
- a "positive blood culture” is an in vitro culture started from whole blood or blood components wherein the growth of microorganisms has been detected. Said growth is indicated by a positive growth index. The detection is preferably done by monitoring CO 2 production in the blood culture.
- Direct identification of microorganisms refers to an identification method which comprises isolation of DNA from a sample or clinical specimen, but does not require an amplification of the genetic material of the microorganisms after said isolation in order to identify the microorganisms using the method of present invention.
- the isolated genetic material is labelled and applied to the DNA microarray of present invention without prior amplification, i.e. directly after isolation or after a short workup step.
- probe(s) means that a species can be identified specifically and unambiguosly using said probe or set of probes. "Differentiation” means the discrimination among distinct and different species, genera or groups of pathogens.
- a "detection method" in the context of the present invention is a method for determination of hybridisation of DNA molecules contained in a sample to the probes on the solid support of the microarray of present invention.
- This method may be any textbook method for detection of DNA hybridisation on microarrays, e.g. direct detection or labelling of target DNA with a reporter molecule and consecutive visualisation of the reporter molecule.
- Preferred detection methods are said labelling method and the direct detection by electrical biosensors or mass spectrometry (Liu, R. H. et al., Anal. Chem. 76(7): 1824-31 (2004); Stomakhin, A. A. et al., Nucleic Acids Res. 28(5):1193-8 (2000)).
- a "reporter molecule” in the context of the method of the present invention is a chemical or physical marker which allows differentiation of labelled from unlabelled DNA by physical, chemical or immunological methods.
- the labelling method includes, but is not limited to radioactive labelling (e.g. with 33 P, 32 P), fluorescent/luminescent/chromophor labelling and hapten labelling (i.e. psoralen or DIG). It is followed by an appropriate detection step necessary to determine the presence and/or quantity of the reporter molecule, namely scintillation counting (e.g. phosphoimaging); photooptic measurement (e.g. fluorescence measurement, luminescence measurement) and antibody-based detection (including colorimetric, luminescence or fluorescence detection), respectively.
- scintillation counting e.g. phosphoimaging
- photooptic measurement e.g. fluorescence measurement, luminescence measurement
- antibody-based detection including colorimetric, luminescence or fluorescence detection
- the reporter molecule is a fluorochrome/fluorophor (both terms are used as synonyms in the context of present invention) which includes but is not limited to cyanines, fluoresceins and rhodamines. More preferably, it is of the cyanine group of fluorophores. Most preferably, it is selected from the group consisting of the fluorophores Cy3, Cy5 or Alexa Fluor 647 and Alexa Fluor 546.
- the ratio of base to dye molecules (BDR) in DNA labelled with such reporter molecules is preferably less or equal to 60.
- a "target species” is a species for which species-specific capture probes are present in the microarray, allowing species identification by positive hybridisation. "Non- target species” are all other species.
- the present invention provides an analytical device, preferably a DNA microarray, and its use for rapid identification and characterisation of microorganisms in a sample or clinical specimen (embodiments (1) to (3)).
- the invention is exemplified in the following by the most preferred embodiment of the analytical device (1), namely a DNA microarray.
- the invention can, however, also be performed using any other of the analytical devices as listed above.
- DNA microarray of embodiment (1) is to be understood as "analytical device of embodiment (I)".
- the DNA microarray of embodiment (1) of the invention comprises gene specific DNA sequences as capture probes, which allow the identification of microbial species ("target species"), especially of bacterial and fungal species, and/or their further characterisation with regard to antibiotic resistance and virulence. Preferably, it allows the identification and characterisation of the target species. It is specific, applicable to the analysis of DNA isolated from blood cultures and suitable to detect resistance genes.
- the DNA microarray of embodiment (1) comprises at least 1 species specific probe per target species. In a preferred aspect of the invention, it additionally comprises one or more virulence and/or resistance gene probe(s).
- a further preferred aspect of embodiment (1) is that the DNA microarray comprises species specific probes for more than one or multiple microbial species, i.e. for a plurality of species.
- the DNA microarray of this preferred aspect of embodiment (1) allows the simultaneous detection of a plurality of microbial species in a sample without previous isolation and/or amplification of single species. It furthermore allows a one-step determination of whether certain microorganisms are present in a sample or not, even if the sample comprises a plurality of different microbial strains.
- the panel of probes can be continually extended to include sequences for additional species, variant isolates or antibiotic resistance determinants as they are characterised and available.
- the accuracy, range and discriminatory power of the gene-segment based microarray can be refined by adding or removing gene probes to the panel without significantly increasing complexity or costs.
- three important species causing bacteremia were selected to provide a proof of principle (examples 1.1-1.10).
- the range of organisms that can be identified can be easily expanded by increasing the number of gene probes on the array. For example, addition of a few probes specific for S. epidermidis and other CoNS will allow for the species identification of coagulase-negative staphylococci. Furthermore, due to a specific hybridisation pattern for each species it will also allow the identification of mixed blood cultures with more than one pathogen.
- a second important feature of this microarray format is the length of the DNA sequences used as gene probes. They are at least 100 nt, preferably 100-3000 nt long. In an especially preferred aspect of embodiment (1) the length of the gene probes is from 100 to 1000 nt, most preferably from 200 to 800 nt. Thus, one probe per gene is usually sufficient to produce strong signals and high specificity (Stears, R.L. et al., Nat. Med., 9:140-5 (2003)). For long probes like these, minor point mutations are likely to only slightly reduce duplex formation, which does not lead to the loss of hybridisation signals. In contrast, short oligonucleotide microarrays sometimes lack specificity and require multiple short oligonucleotides per one gene.
- the microorganisms or microbial DNA to be detected using the microarray of present invention are preferably bacteria (such as Staphylococci, Enterococci, Streptococci, E. coli, P. aeruginosa, Klebsiella spp., Proteus spp., Enterobacter spp., Acinetobacter spp. and Stenotrophomonas spp.) or fungi (such as yeasts and filamentous fungi, in particular Candida spp., Aspergillus spp., Cryptococcus spp., Malassezia spp., Trichosporin spp.), respectively bacterial or fungal DNA.
- bacteria such as Staphylococci, Enterococci, Streptococci, E. coli, P. aeruginosa, Klebsiella spp., Proteus spp., Enterobacter spp., Acinetobacter spp. and Sten
- the microarray is especially suitable for direct identification and characterisation of bacteria and C. albicans.
- the analytical device is suitable for species specific identification of one microbial strain or (preferably) a plurality of microbial strains in clinical specimens comprising microbial strains, especially bacteria and/or fungi. It furthermore allows differentiation of the target species from each other and from non-target-species contained in one sample comprising a plurality of microbial strains.
- the DNA microarray is feasible to identify and characterize any of the microorganisms, including the fungi and bacteria as defined above, known as etiological agents of fungemia, bacteremia or sepsis.
- microorganisms selected from the group consisting of S. aureus, CoNS (including Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus lugdunensis, Staphylococcus warneri, Staphylococcus saprophyticus, Staphylococcus hominis), C. albicans, Enterococcus faecalis, Enterococcus faecium, E.
- the DNA microarray is suitable for species specific identification of microorganisms selected from the group consisting of Staphylococci, E. coli and Candida sp., preferably for species specific identification of Staphylococci, especially of S. aureus. More preferably, it is suitable for species specific identification of Staphylococci and at least one of E. coli and Candida albicans.
- the DNA microarray is suitable to identify and characterize at least S. aureus, Coagulase- negative staphylococci, E. coli, Enterococcus faecalis and faecium and Candida albicans.
- the DNA microarray is in a preferred embodiment of present invention suitable for additional species specific identification or differentiation of Klebsiella pneumoniae, Klebsiella oxytoca, Streptococcus pneumoniae, Streptococcus pyogenes, Pseudomonas aeruginosa, Proteus mirabilis and/or Proteus vulgaris.
- the practicability and specificity of the DNA microarray for the identification and characterisation of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa was evaluated with clinical isolates and positive blood cultures (Examples 1.1-1.10).
- a microarray which allows identification and characterisation of S. aureus.
- the latter microarray allows the detection of every S. aureus isolate, unambiguously identifies most of important virulence genes such as tsst-1, sea, seb, eta and antibiotic resistance genes such as mecA, aacA- aphD, blaZ, ermA and specifically distinguishes S. aureus from unrelated gram negative bacteria, e.g. Escherichia coli or Pseudomonas aeruginosa, as well as from closely related CoNS (Example 1.11, Fig. 2-6).
- the microarray of (1) is suitable for diagnosis of fungemia, bacteremia or sepsis; especially for diagnosis of bacteremia, candidemia, and bacterial or Candida sepsis.
- the present invention provides a novel approach for detection of microorganisms, especially of bacteria and fungi, by microarrays: using gene-segments it allows species identification by probing a large and diverse set of species-specific genes. Such an approach is reliable since it makes possible to identify a pathogen even when some genes have been deleted from its genome.
- the selected DNA probes are at least 100 nt, preferably 200 to 800 nt long and are therefore not sensitive to single nucleotide polymorphisms or GG-content variations in the targets. Therefore, a gene segment array according to present invention is useful for indicating the presence of a gene even though the sequence may be slightly altered e.g. by point mutations (Southern, E. et al., Nat. Genet.
- the DNA microarray of embodiment (1) comprises the minimal number of species specific gene probes which is sufficient for species identification, the minimal number of virulence gene probes which is sufficient for virulence determination, and/or the minimal number of resistance gene probes which is sufficient for determination of resistance of a specific microorganism.
- the minimal number of gene probes in this aspect of the invention is: for correct species identification at least 1 species specific gene probes per target species, more preferably at least 2 different species specific gene probes per target species, even more preferably at least 10, most preferably at least 20; for virulence determination at least 1 gene probe per target species, more preferably at least 5 different gene probes, even more preferably at least 20 different gene probes, most preferably gene probes for all known virulence factors of each target species; for determination of resistance at least 1 gene probe per antibiotic class or resistance factor, more preferably at least 5 different gene probes, most preferably all known gene-coded resistance determinants in the target species.
- the DNA microarray of embodiment (1) comprises gene probes which are specific for a microbial species, bacterial/fungal species or a group of microorganisms to be identified.
- Said gene probes are preferably DNA sequences selected from three different groups, namely (a) species specific gene probes; (b) virulence gene probes; and/or (c) resistance gene probes.
- the species specific set of gene probes for each species to be identified and characterised is selected from species specific gene probes (a) for
- Staphylococcus aureus including gene probes derived from clfA, clfB, coa, lytM, NAG, sodA, sodB, epiP-bsaP, geh, hemC, hemD, hsdS, lip, menC, nuc, SAV0431, SAV0440, SAV0441, spa, ebpS, fbpA, fib, fnbB, srtA, stpC, fnbA, fern A, fmhB, fmhA;
- Escherichia coli including gene probes derived from bll69, fliCb, nfrB, yacH, ycdS, yciQ, shuA;
- Staphylococcus epidermidis including gene probes derived from ardeSEOlO ⁇ , ardeSE0107, atlE, agrB, alphSE1368, gad, glucSEll ⁇ l, icaB, mvaSSepid, nitreSE1972, nitreSE1974, nitreSE1975, oiamtSE1209, ORFISepid, ORF3bSepid, qacR, ureSE1865, ureSE1867;
- Staphylococcus haemolyticus including gene probes derived from femBShaemolyt, mvaDShaemolyt, mvaSShaemolyticus, RNApolsigm;
- Staphylococcus lugdunensis including gene probes derived from agrB2Stalugd, agrC2Stalugd, slamStalugd;
- Staphylococcus warneri including gene probes derived from msrwlStwar, nukMStwar, proDStwar, proMStwar, sigrpoStwar, tnpStwar;
- Staphylococcus saprophytics including gene probes derived from
- Candida albicans including gene probes derived from ARG56, ASL43f, BGL2,
- CCT8 CDC37, CEF3, CHSl, CHS2, CHS4, CHS5, CHTl, CHT2, CHT4, CSAl, 5triphosphatase, AAFl, ADHl, ALSl, ALS7, EDTl, ELF, ESSl, FALl, GAPl, GNAl,
- GSCl GSCl, GSLl, HISl, HTSl, HWPl, HYRl, INTIa, KRE15f, KRE6, KRE9, MIGl, MLSl,
- Enterococcus faecalis including gene probes derived from arcA, arcC, bkdA, camEl, csrA, dacA, dfr, dhoDla, ABC-eltA, agrBfs, agrCfs, dnaE, ebsA, ebsB, eep, efaR, gls24_glsB, gph, gyrAEf, metEf, mntHCb2, mob2, mvaD, mvaE, parC, pcfG, phoZ, polC, ptb, recSl, rpoN, tms, tyrDC, tyrS;
- Enterococcus faecium including gene probes derived from bglB, bglR, bglS, efmA, efmB, efmC, mreC, mreD, mvaDEfaecium, mvaEEfaecium, mvaKlEfaecium, mvaK2Efaecium, mvaSEfaecium, orf3_4Efaeciumb, orf6_7Efaecium, orf7_8Efaecium, orf9_10Efaecium;
- Klebsiella pneumonia including gene probes derived from atsA, budC, citA, citW, citX, dalK, acoA, acoB, acoC, ahlK, fimK, glfKPN2, ItrA, mdcC, mdcH, nifF, nifK, nifN, tyrP, wbbO, wzb, wzmKPN2, wztKPN2, yojH, liac; (xi ⁇ ) Klebsiella oxytoca including gene probes derived from gatY, pelX, tagH, tagK, tagT;
- Streptococcus pneumoniae including gene probes derived from caplEStrpneu, caplFStrpneu, caplGStrpneu, cap3AStrpneu, cap3BStrpneu, celAStrpneu, celBStrpneu, cglAStrpneu, cglBStrpneu, cglCStrpneu, cglDStrpneu, cinA, cpsl4EStrpneum, cpsl4FStrpneum, cpsl4GStrpneum, cpsl4HStrpneum, cpsl9aHStrpneum, cpsl9aHStrpneum, cpsl9aHStrpneum, cpsl
- Streptococcus agalactiae including gene probes derived from cpsAIStrgal, cpsBIStrgal, cpsCIStrgal, cpsDIStrgal, cpsEIStrgal, cpsGlStrgal, cpsIStragal, cpsJStragal, cpsKStragal, cpsMStragal, cpsYStragal, cylBStraga, cylEStraga, cylFStraga, cylHStraga, cyllStraga, cylJStraga, cylKStraga, 0487Straga, 0488Straga, 0493Straga, 0495Straga, 0498Straga, 0500Straga, 0502Straga, 0504Straga, folDStraga, neuAIStrgal, neuBIStrgal, neuCIStrgal, neuDIStrgal, recNStraga,
- Streptococcus pyogenes including gene probes derived from cyclStrpyog, fah_rph_hlo_Strpyog, int, int315.5, oppD, SPy0382Strpyog, SPy0390Strpyog, SpyM3_1351, vicXStrpyog;
- Streptococcus mutans including gene probes derived from 573Stprmut, 580SStprmut, 581_582SStprmut, 584SStprmut, dltAStrmut, dltBStrmut, dltCppxlStrmut, dltDStrmut, lichStrbov, lytRStprmut, lytSStprmut, pepQStrrmut, pflCStrmut, recNStprmut, ytqBStrmut;
- Proteus mirabilis including gene probes derived from atfA, atfB, atfC, ccmPrmil, cyaPrmi, flfB, flfD, flfN, flhD, floA, ftsK, gstB, hemCPrmi, hemDPrmi, hev, katA, Ipp
- Acinetobacter baumanii including gene probes derived from carO, gacS, dhbA, dhbB, sid, csuD, csuC, tnp-ACIBA, waaA-ACIBA, csuB, csuA_B / csuA, putl, por, abc, furACIBA, dec, cysl, trpE, put3, ompA-ACIBA.
- the virulence specific set of gene probes for each species to be identified and characterised is selected from virulence gene probes (b) for
- Staphylococcus aureus including gene probes derived from bsaE, bsaG, cap5h, cap5i, cap5j, cap5k, cap8H, cap ⁇ l, cap8J, cap8K, I-hld, I-hysA, I-IgGbg, EDIN, eta, etb, hglA, hglB, hglC, hla, hlb, lukF, lukS, NAG, sak, sea, seb, seel, seg, seh, sel, setl5, set6, set7, set8, sprV8, tst, I-sdrC, I-sdrD, I-sdrE;
- Escherichia coli including gene probes derived from bl202, eae, eltB, escR, escT, escU, espB, fes, fteA, hlyA, hlyB, iucA, iucB, iucC, papG, rfbE, shuA, SLTII, toxA-LTPA, VT2vaB;
- Staphylococcus epidermidis including gene probes derived from gcaD, hld_orf5, icaC, icaD, icaR, psm_betaland2, purR, spoVG, yabJ;
- Staphylococcus haemolyticus including gene probes derived from lipShaemolyt;
- Staphylococcus lugdunensis including gene probes derived from fblStalugd, slushABCStalugd
- Staphylococcus warneri including gene probes derived from gehAStwar
- Candida albicans including gene probes derived from CCNl, CDC28, CLN2,
- Enterococcus faecalis including gene probes derived from asal, aspl, cgh, cylA, cylB, cyll, cylL_cylS, cylM, ace, ef 00108, ef 00109, efOOll, efOO113, ef0012, ef0022, ef0031, ef0032, ef0040, ef0058, enlA, esa, esp, gelE, groEL, groES, rtl, sala, salb, seal, sepl, vicK, yycH, yycl, yycJ;
- Enterococcus faecium including gene probes derived from entA_entI, entD, entR, oep, sagA;
- Klebsiella pneumonia including gene probes derived from dm, aldA, hemly, pSL017, pSL020, rcsA, rmlC, rmlD, waaG, wbbD, wbbM, wbbN, wbdA, wbdC, wztKpn, yibD;
- P. aeruginosa including gene probes derived from aprA, aprE, ctx, algB, algN, algR, ExoS, fpvA, lasRa, UpA, HpH, Orfl59, Orf252, pchG, PhzA, PhzB, PLC, plcN, plcR, pvdD, pvdF, pyocinSl, pyocinSlim, pyocinS2, pys2, rbf303, rhlA, rhlB, rhlR,
- Streptococcus pneumoniae including gene probes derived from igaStrpneu, lytA, nanA, nanBStrpneu, pcpCStrpneu, ply, prtAStrpneu, pspA, SP0834Strpneu, sphtraStrpneu, wciJStrpneu, wziyStrpneu, wzxStrpneu;
- Streptococcus agalactiae including gene probes derived from CAMPfactor, 0499Straga, hylStragal, lipStragal;
- (x ⁇ /)Streptococcus pyogenes including gene probes derived from DNaseIStrpyog, fba2Strpyog, fhuAStrpyog, fhuBIStrpyog, fhuDStrpyog, fhuGStrpyog, hylA, hylP, hylp2, oppB, ropB, scpAStrpyog, sloStrpyog, smez- Strpyog, sof, speA, speB2Strpyog, speCStrpyog, speJStrpyog, srtBStrpyog, srtCStrpyog, srtEStrpyog, srtFStrpyog, srtGStrpyog, srtlStrpyog, srtKStrpyog, srtRStrpyog, srtTStrpyog, vicKStrpyog;
- Proteus mirabilis including gene probes derived from flaA, IaD, fliA, hpmA, hpmB, IpsPrmi, mrpA, mrpB, mrpC, mrpD, mrpE, mrpF, mrpG, mrpH, mrpl, mrpJ, patA, putA, uca, ureDPrmi, ureEPrmi, ureFPrmi, zapA, zapB, zapD, zapE.
- the resistance specific set of gene probes is selected from resistance gene probes (c) derived from genes coding for
- beta-lactams resistance including gene probes derived from blaIMP-7, mecISepid, blaOXA-10, blaB, ampC, 1-biaR, blaOXA-32, bla-CTX-M-22, pbp2aStrpneu, blaSHV-1, biaOXA-2, blaRShaemolyt, blaIMP-7, I-mecR, blaOXY, dacCStrpyog, mecA, blaIShaemolyt, b Ia vim, pbp2b, pbp2primeSepid, pbp2x, pbp3Saureuc, pbp4, pbp ⁇ Efaecium, pbpC, I-mecI, pbpla, I-blal, blaTEM-106, blaOXY-KLOX, ftsWEF, cumA, blaPER-1, bla_FOX-3, blaA
- aminoglycosides resistance including gene probes derived from aacA_aphDStwar, aacCl, aacC2, strB, aadA, aadB, aadD, aacA4, strA, aph-A3, aacCl, aacA4, aacA-aphD, I-spc, aphA3; aacA4ENCL, aac(6p)-lb7;
- multiple target resistance including gene probes derived from acrB, mexB, I- qacA, sull, sul, cadBStalugd, mexA, acrR, emeA, acrA, rtn, abcXStrpmut, qacEdeltal, elkT-abcA, I-cadA, albA, wzm, msrCb, nov, wzt, wbbl, norA23, mexR, arr2, mreA, I-cadC, uvrA, AdeR-ACIBA, adeA-ACIBA, adeB-ACIBA, adeC-ACIBA, AdeS-ACIBA;
- (ix) fungicides resistance especially C. albicans fungicide resistance, including gene probes derived from CRD2, CDRl, MET3, FET3, FTR2, MDR1-7, ERGIl, SEC20.
- the resistance specific set of gene probes is selected from resistance gene probes (c) derived from genes coding for
- beta-lactams resistance including gene probes derived from bla-CTX-M-22, blaSHV-1, blaTEM-106, mecA, blaZ;
- aminoglycosides resistance including gene probes derived from aacCl, aacC2, aadA, aadB, aadD, aacA4, aph-A3, aacCl, aacA4, aacA-aphD, aphA3;
- tetracyclines resistance including gene probes derived from tetAJ, tetL, tetM
- glycopeptides resistance including gene probes derived from vanA, vanB, vanC-2.
- the most relevant resistance gene probes are probes derived from and specific for mecA. This is due to the fact that mecA is common to all Staphylococci including S. aureus and CoNS. Since the same resistance phenotype is determined by many different genotypes, it is preferred to use a plurality of resistance gene probes for unambiguous and comprehensive prediction of antibiotic resistance. The largest available set of resistance probes is most preferred.
- the microarray may contain a set of gene probes which serve as controls.
- a set of control gene probes is selected from group (d) consisting of control gene probes coding for
- negative controls namely DNA sequences which will not hybridise with human DNA or bacterial, fungal or the microbial target DNA under the hybridisation conditions of the method of present invention, including gene probes derived neither from fungal, bacterial or target microbial nor from human genes, preferably gene probes derived from plant genes, more preferably from Arabidopsis thaliana or Glycine max genes;
- positive controls including segments of ribosomal DNA from bacterial target species, preferably 16S DNA, and segments of conserved human genes;
- positive controls specific for DNA added to the sample (“spiked DNA") namely DNA sequences which will not hybridise with human DNA or the fungal, bacterial or microbial target DNA under the hybridisation conditions of the method of present invention, including gene probes derived neither from fungal, bacterial or target microbial nor from human genes, preferably gene probes derived from mouse or amoeba genes, most preferably from Mus musculus or Dictyostelium discoideum genes
- control gene probes are necessary to a) detect non-specific hybridisation; b) optimise hybridisation conditions and image acquisition and analysis; c) provide positive controls for the quality of probe preparation, hybridisation and detection; and/or d) control technical aspects of the entire detection procedure including labelling, hybridisation and detection steps.
- the microarray contains DNA sequences selected from the group consisting of the SEQ ID NOs: 1-918 and 2842-2908, complementary sequences thereto, addition mutants, deletion mutants, substitution mutants and homologues thereof as gene probes.
- the gene probes of group (a) are selected from SEQ ID NO: 1-99, 142-152, 174-199, 209-214, 216-219, 222-229, 231-291, 308-342, 377-393, 399- 431, 449-490, 523-591, 606-639, 645-656, 687-701, 706-749, 776-781, 2843- 2863, 2902 and 2903 (compare Tab. 1).
- the gene probes of group (b) are selected from SEQ ID NO: 100-141, 153-173, 200-208, 215, 220-221, 230, 292- 307, 343-376, 394-398, 432-448, 491-522, 592-605, 640-644, 657-686, 702-705, 750-775 and 782-784 (compare Tab. 1).
- the gene probes of group (c) are selected from SEQ ID NO:785-918, 2864-2875, 2888 and 2907-2908, preferably from SEQ ID NO: 785-909, 2864-2875, 2888 and 2907-2908 (compare Tab. 1).
- the gene probes of group (d) are selected from SEQ ID NO:919-947, preferably from SEQ ID NO:919-925 and 944-947, more preferably from SEQ ID NO: 919 and 921 (compare Tab. 1).
- Tab. 1 Preferred gene probes for species identification, virulence determination and resistance determination of microorganisms
- the DNA microarray of (1) is preferably suitable for
- identification of Staphylococcus aureus and comprises one or more or all gene probes selected from SEQ ID NO:3-6, 31, 40, 50, 51, 58, 59, 63, 64, 66-69, 71, 74, 76, 77, 79, 2902 and 2903, preferably at least one of the gene probes represented by SEQ ID NO:71, 68, 4 and 69; and/or
- identification of Escherichia coli and comprises one or more or all gene probes selected from SEQ ID NO: 142, 144, 145, 148, 150-152, 160, 161 and 170, preferably at least one of the gene probes represented by SEQ ID NO: 145, 160, 161 and 170; and/or
- (III) identification of Staphylococcus epidermidis and comprises gene probes selected from SEQ ID NO: 174, 175, 177, 178, 180-182, 185-193, 198 and 199, preferably at least one of the gene probes represented by SEQ ID NO: 177, 178 and 190; and/or
- identification of Staphylococcus haemolyticus and comprises one or more or all gene probes selected from SEQ ID NO: 211, 213 and 214, preferably at least one of the gene probes represented by SEQ ID NO:211 and 214; and/or
- (V) identification of Staphylococcus lugdunensis and comprises one or more or all gene probes selected from SEQ ID NO:216, 217 and 219-221, preferably at least one of the gene probes represented by SEQ ID NO:216, 219, 220 and 221; and/or
- (X) identification of Enterococcus faecalis and comprises one or more or all gene probes selected from SEQ ID NO: 308-310 and 312-342, preferably at least one of the gene probes represented by SEQ ID NO:308, 310 and 314; and/or
- identification of Pseudomonas aeruginosa comprises one or more or all gene probes selected from SEQ ID NO:470-485, 487-493 and 505, preferably at least one of the gene probes represented by SEQ ID NO:471, 474, 488 and 505; and/or
- (XVII) identification of Streptococcus pyogenes and comprises one or more or all gene probes selected from SEQ ID NO:645-648, 652, 655, 656, 658 and 660, preferably at least one of the gene probes represented by SEQ ID NO:645, 658 and 660; and/or
- (XVIII) identification of Streptococcus mutans and comprises one or more or all gene probes selected from SEQ ID NO:687-701, preferably at least one of the gene probes represented by SEQ ID NO:687, 691 and 692; and/or (XIX) identification of Proteus mirabilis and comprises one or more or all gene probes selected from SEQ ID NO:706-710, 712-742 and 744-749, preferably at least one of the gene probes represented by SEQ ID NO:721, 725 and 735; and/or
- (XX) identification of Proteus vulgaris and comprises one or more or all gene probes selected from SEQ ID NO:776-778 and 780-781, preferably at least one of the gene probes represented by SEQ ID NO:776, 777 and 781; and/or
- the DNA microarray of embodiment (1) is suitable for species specific identification of at least S. aureus and preferably comprises gene probes selected from SEQ ID NO:3-6, 31, 40, 50, 51, 58, 59, 63, 64, 66-69, 71, 74, 76, 77, 79, 2902 and 2903, more preferably from SEQ ID NO:4, 68, 69 and 71, even more preferably comprises at least SEQ ID NO:71.
- the DNA microarray is suitable for species specific identification of at least S. aureus, E. coli, CoNS, Enterococcus sp., and/or Candida sp., and preferably comprises gene probes selected from a) SEQ ID NO:4, 68, 69 and 71, preferably SEQ ID NO: 71 for identification of S. aureus) b) SEQ ID NO: 145, 160, 161 and 170, preferably SEQ ID NO: 145 for identification of E. coli] c) SEQ ID NO: 177, 178 and 190, preferably SEQ ID NO: 178 for identification of S.
- These microorganisms are the prevalent microorganisms in clinical samples and/or are of the highest diagnostic relevance.
- the probes listed under (a) to (h) are the most reliable probes for identification of said microorganisms.
- a DNA microarray comprising one, several or all of said four probes is suitable for species specific detection or differentiation of (i) S. aureus if it comprises SEQ ID NO: 71;
- Candida albicans if it comprises SEQ ID NO: 249. This set of four probes thus forms an especially preferred set of probes for embodiment (1).
- Sets (B), (C) and (D) are preferred, set (D) is especially preferred.
- the DNA microarray of embodiment (1) may be suitable for additional species specific identification or differentiation of one or more of Klebsiella pneumoniae, Klebsiella oxytoca, Streptococcus pneumoniae, Streptococcus pyogenes, Pseudomonas aeruginosa, Proteus mirabilis and Proteus vulgaris.
- the DNA microarray of (1) is suitable for
- (I) virulence determination of Staphylococcus aureus and comprises one or more or all of the gene probes of group (b) selected from SEQ ID NO: 100-141; and/or
- V virulence determination of Staphylococcus lugdunensis and comprises one or more or all of the gene probes of group (b) selected from SEQ ID NO:220-221; and/or
- VI virulence determination of Staphylococcus warneri and comprises the gene probe of group (b) represented by SEQ ID NO: 230; and/or
- VI virulence determination of Candida albicans and comprises one or more or all of the gene probes of group (b) selected from SEQ ID NO:292-307; and/or
- (X) virulence determination of Klebsiella pneumonia and comprises one or more or all of the gene probes of group (b) selected from SEQ ID NO:432-448; and/or
- (XIII) virulence determination of Streptococcus pneumoniae and comprises one or more or all of the gene probes of group (b) selected from SEQ ID NO:592-605; and/or
- (XIV) virulence determination of Streptococcus agalactiae and comprises one or more or all of the gene probes of group (b) selected from SEQ ID NO:640-644; and/or
- (XV) virulence determination of Streptococcus pyogenes and comprises one or more or all of the gene probes of group (b) selected from SEQ ID NO:657-686; and/or
- XVI virulence determination of Streptococcus mutans and comprises one or more or all of the gene probes of group (b) selected from SEQ ID NO:702-705; and/or (XVII) virulence determination of Proteus mirabilis and comprises one or more or all of the gene probes of group (b) selected from SEQ ID NO:750-775; and/or
- the DNA microarray of (1) is suitable for antibiotic resistance determination of (I) Staphylococcus aureus, (II) Escherichia coli, (III) Staphylococcus epidermidis, (IV) Staphylococcus haemolyticus, (V) Staphylococcus lugdunensis, (VI) Staphylococcus warneri, (VIII) Enterococcus faecalis, (IX) Enterococcus faecium, (X) Klebsiella pneumonia, (XI) Klebsiella oxytoca, (XII) Pseudomonas aeruginosa, (XIII) Streptococcus pneumoniae, (XIV) Streptococcus agalact
- the microarray of (1) is suitable for identification and characterisation, i.e. virulence and/or resistance determination, of the target microorganism and comprises one or more or all of the gene probes of group (a) and additionally one or more or all of the gene probes of group (b) and group (c) for each organism as listed above.
- the array comprises preferably at least the core gene probes designated in example 1.7, more preferably all the sequences listed in Tab. 2 and/or Tab. 6. Even more preferred, it consists of said sequences.
- the gene probes were considered as most preferable if they were i) known previously to be species-specific, ii) bioinformatically selected to have the least chance to hybridise with nontarget genes and iii) empirically proven to be specific in a series of experiments (see Examples).
- the DNA microarray of (1) comprises the following gene probes, even more preferably consists of the following gene probes: (I) When the DNA microarray is suitable for identification and characterisation of Staphylococcus aureus, it comprises
- the DNA microarray When the DNA microarray is suitable for identification and characterisation of Escherichia coli, it comprises (a) the gene probes represented by SEQ ID NO: 142, 144, 145, 148, 150-152, 160, 161 and 170; and at least one of
- the DNA microarray When the DNA microarray is suitable for identification and characterisation of Staphylococcus warneri, it comprises (a) the gene probes represented by SEQ ID NO:224-228 and 230; and at least one of
- Staphylococcus saprophyiticus it comprises
- Staphylococcus horn in is, it comprises
- the DNA microarray When the DNA microarray is suitable for identification and characterisation of Streptococcus agalactiae, it comprises (a) the gene probes represented by SEQ ID NO:606-639; and at least one of
- the DNA microarray When the DNA microarray is suitable for identification and characterisation of Proteus mirabilis, it comprises (a) the gene probes represented by SEQ ID NO:706-710, 712-742 and 744-749; and at least one of
- Acinetobacter baumanii it comprises
- the DNA microarray which is a preferred aspect of embodiment (1) can be fabricated using textbook methods for microarray production, including printing with fine-pointed pins onto the solid support, photolithography using pre-made masks or dynamic micromirror devices, ink-jet printing or electrochemistry on microelectrode arrays (M ⁇ ller, H. -J., R ⁇ der, T., "Der Experimentator: Microarrays, Spektrum Akademischer Verlag, Heidelberg (2004)).
- Preferred fabrication methods are printing methods spotting the gene probes onto the solid surface of the microarray.
- the attachment of the spotted DNA to the surface is achieved by covalent or non-covalent binding, preferably by non-covalent binding, more preferably by electrostatic interaction (ionic binding), most preferably by ionic binding of the DNA to amino groups present on the surface of the solid support.
- Any amino-functionalized microarray support can be used, but gamma aminopropyl silane (GAPSTM) coated slides, especially UltraGAPSTM coated glass slides, are preferred in present invention.
- the amount of DNA per spot printed onto the array is from 0.1 to 15.0 ng, preferably from 0.1 to 0.2 ng.
- the present invention also pertains to a method for fabrication of a microarray of embodiment (1), which method comprises spotting the gene probes listed above to an appropriate solid support.
- the sample of embodiments (1) to (4) may be any sample containing microorganisms, including food samples, environmental samples and clinical specimens.
- a sample which is a clinical specimen is preferred.
- the sample or clinical specimen of embodiments (1) to (4) is preferably selected from the group consisting of whole blood, serum, urine, saliva, liquor, sputum, yakate, stool, pus, swabs, wound fluid and positive blood cultures, more preferably is whole blood or a positive blood culture, most preferably is a positive blood culture. If blood culture is used as DNA source, 0.5 ml positive blood culture is sufficient for identification and characterisation of the microorganisms and bacteria present without prior amplification of the target DNA.
- microarray of present application is (i) a robust diagnostic tool, detecting all tested bacterial reference strains and clinical isolates;
- the DNA microarray of embodiment (1) is especially suitable for diagnosis of
- bacteremia fungemia or sepsis
- the device preferably comprises probes for species specific identification of at least S. aureus, E. coli, CoNS, Enterococcus sp., and Candida sp.;
- the device preferably comprises probes for species specific identification of at least Candida sp., S. aureus and P. aeruginosa; and/or (iii) urinary tract infections, wherein the device preferably comprises probes for species specific identification of at least E. coli, Enterococci sp., Candida sp. and Proteus sp..
- microarray hybridisation allows for reliable prediction of oxacillin, penicillin, erythromycin, tobramycin and gentamicin resistance in a single assay.
- microarray hybridisation it is furthermore possible to discriminate multi-resistant and multi-susceptible MRSA (strain MW2).
- Multi-susceptible MRSA have been shown to be susceptible to tobramycin and erythromycin (Polyzou, A. et al., J. Antimicrob. Chemother. 48:231-4 (2001); Pournaras, S. et al., J. Clin. Microbiol. 39:779-81 (2001)).
- simultaneous comprehensive resistance genotyping for oxacillin, macrolide and aminoglycoside resistance genes preferably mecA, aadD, aacA-aphD, ermA,B,C and msrSA
- microarray hybridisation allows the rapid discrimination of multi-resistant or multi-susceptible strains and in consequence other therapeutic options with e.g. macrolides and may reduce reliance on vancomycin (Polyzou, A. et al., J. Antimicrob. Chemother. 48:231-4 (2001); Pournaras, S. et al., J. Clin. Microbiol. 39:779-81 (2001)).
- One preferred aspect of embodiment (1) is a DNA microarray for the identification and characterisation of the three important bacteremia causing species Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa in a sample, preferably in blood culture.
- the microarray allows simultaneous species identification and detection of important virulence and antibiotic resistance genes in a single assay.
- this array consists of 2-20 species specific gene probes, 1-20 virulence gene probes and 1-20 resistance gene probes of at least 100 nt length, more preferably of 200-800 nt length.
- One especially preferred embodiment is an array comprising or consisting of the gene probes listed in Tab. 2.
- the probes may be amplified from recombinant plasmids or synthesized by any other method know in the art. These probes represent genes encoding house-keeping proteins, virulence factors and antibiotic resistance determinants. Evaluation with 42 clinical isolates, 3 reference strains and 13 positive blood cultures revealed that this DNA microarray is highly specific in identifying S. aureus, E. coli and P. aeruginosa strains and in discriminating them from closely related Gram-positive and Gram- negative bacterial strains also known to be etiological agents of bacteremia. In Example 1.6 and 1.7, this array was successful in identifying all tested 27 E. coli, P. aeruginosa and S.
- One further preferred aspect of embodiment (1) of the invention is a DNA microarray for the identification and characterisation of S.
- the DNA microarray is - in the context of embodiment (2) - preferably used for in vitro differentiation of a plurality of different microbial strains contained in one sample and/or for species-specific identification of one or more microbial strain(s) contained in a mixture of a plurality of microorganisms.
- the DNA microarray of embodiment (1) is advantageous for this kind of use, as it allows the simultaneous determination of the presence or absence in the analysed sample of all those microbial strains for which the device comprises species specific probes.
- the array is also suitable for identification and determination of single or of a selection of microbial strains in a mixture of strains, especially in a clinical sample containg additional component, without prior isolation of the target strain.
- the method of embodiment (3) comprises - after isolating the total DNA (including non-microbial DNA) from a sample - the steps of immediate labelling and microarray-based detection of this isolated DNA with or without, preferably without, further DNA amplification steps after the DNA isolation. It is one advantage of the method (3) that it can be performed without said further DNA amplification steps, i.e. the isolated DNA is labelled and applied to the microarray without prior amplification.
- a DNA preparation protocol employing sonication for simultaneous cell disruption and target DNA fragmentation is the method of choice to increase the sensitivity of the microarray, in particular towards low-copy number and/or plasmid encoded genes which may be underrepresented in the target DNA.
- the method of embodiment (3) is preferably a method for diagnosis of bacteremia, fungemia or sepsis.
- the sample or clinical specimen used in embodiment (3) is preferably blood or derived from blood, more preferably is a blood culture. Most preferably, the clinical specimen is a positive blood culture.
- 100 pg of purified genomic microbial DNA may be sufficient (lower detection limit), but preferably at least 1 ng of said DNA should be present in the sample.
- at least 10 ng, preferably at least 20 ng, more preferably at least 1 ⁇ g of purified genomic microbial DNA or at least 1 ⁇ g, preferably at least 2 ⁇ g of DNA extracted from blood culture are required.
- 500 ⁇ l of positive blood culture yield enough DNA for several hybridisations.
- the DNA isolated in step (a) is labelled and applied to the analytical device without prior amplification, preferably is labelled by random priming.
- the DNA isolated in step (a) is fragmented before the labelling reaction. Both aspects simplify and speed up the analysis in comparison to convention methods.
- the ratio of microbial DNA to total DNA isolated from said sample or clinical specimen is less than or equal to 100 %, preferably is from 1% to 99%, more preferably from 30 to 60%.
- the labelling reaction of the method of embodiment (3) may be any DNA labelling reaction known in the art. However, chemical labelling reactions consisting of chemical attachment of a reporter molecule to the sample DNA and labelling by integration of labelled nucleotides into the sample DNA are preferred.
- the reporter molecules are fluorophores, more preferably are of the cyanine group of fluorophores.
- the DNA is labelled with Cy3, Cy5 and/or Alexa Fluor 647 and Alexa Fluor 546.
- the ratio of bases to dye molecules (BDR) is preferably less or equal to 60.
- the detection of the reporter molecule in the method of embodiment (3) of the invention is preferably done by using a suitable detection system for the bound reporter molecule. This detection system is preferably based on visualization of the reporter molecule, more preferably on fluorescence detection. Furthermore, the detection is preferably done by a microarray scanner or microarray reader.
- the DNA microarray can be substituted by any other solid support onto which DNA gene probes are attached in a way permitting hybridisation of the DNA in the sample and subsequent detection of the bound DNA.
- the beads are preferably used in a multi-chamber system, more preferably in a microfluidic multi-chamber system, wherein each chamber contains a population of beads.
- Each bead has an attached DNA sequence and the whole beads population in one chamber will carry the same DNA sequence, each chamber corresponding then to a specific capture probe.
- the target DNA to be analysed flows through the multi-chamber system and will hybridize with the complementary DNA sequences attached to the beads.
- Beads could be also attached to a surface by magnetic force, i.e. paramagnetic beads coupled with DNA could be attached on the surface of the magnet and arrange in a lattice structure. Complimentary, beads made of a magnetic material could be attached to an iron surface.
- the use of the DNA coated beads or of a DNA microarray of embodiment (1) is preferred.
- the use of a DNA array is especially preferred.
- the analytical device is a DNA microarray.
- the detection is preferably performed using a DNA microarray reader.
- the analytical device is a DNA coated bead or a set of DNA coated beads (plurality of DNA coated beads).
- the application and/or detection step is preferably performed in a microfluidic device.
- the kit of embodiment (4) of the invention may additionally comprise reagents for the labelling reactions of embodiment (3) and/or reagents necessary for the hybridisation step of the method of embodiment (3).
- Example 1.1 Materials and Methods Reference strains, clinical isolates and culture conditions: Bacterial reference strains were obtained from the American Type Culture Collection (ATCC, Manassas, Va.), the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) or the network on antimicrobial resistance in Staphylococcus aureus (NARSA, Herndon, Virginia). Clinical isolates were obtained from the inventors ' clinical routine microbiology laboratory.
- ATCC American Type Culture Collection
- DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen
- NARSA Herndon, Virginia
- Clinical isolates were obtained from the inventors ' clinical routine microbiology laboratory.
- Staphylococcus aureus ATCC 25923, NRS123 alias MW2, 5 clinical isolates
- Staphylococcus epidermidis 5 clinical isolates
- Staphylococcus capitis clinical isolate
- Staphylococcus haemolyticus clinical isolate
- Staphylococcus hominis clinical isolate
- Staphylococcus warneri clinical isolate
- Staphylococcus auriculahs (clinical isolate)
- Micrococcus spp Micrococcus spp.
- Bacterial strains and clinical isolates were grown over night at 37 0 C with constant shaking in 5 ml Luria-Bertani (LB) broth or tryptic soy broth (TSB, 30 g/l, Merck) containing 3 g/l yeast extract. Enterococci and streptococci were grown in 10 ml TSB plus yeast without agitation under 5% CO 2 . Overnight cultures were harvested at 2,560 g for 10 min. After discarding the supernatant the pellet was washed in 1 ml TE (10 mM Tris-HCI, pH 7.5 and 1 mM EDTA) and recovered by centrifugation at 17,900 g for 10 min. Cell pellets were used for DNA preparation.
- LB Luria-Bertani
- TSB tryptic soy broth
- Blood cultures Aerobic and anaerobic blood culture bottles (BACTEC ® , Becton Dickinson, Heidelberg, Germany) were inoculated with blood from patients with suspected sepsis and placed in a BACTEC ® 9240 blood culture system (Becton Dickinson), a continuous-reading, automated, and computed blood culture system that detects the growth of microorganisms by monitoring CO 2 production. Incubation was performed according to the manufacturer's recommendations. Bottles with a positive growth index were removed from the incubator, and aliquots of 1 ml of the blood culture suspensions were taken aseptically with a needle syringe.
- BACTEC ® 9240 blood culture system Becton Dickinson
- the organisms grown on agar plates were characterised and tested for susceptibility using a VITEK-2 system (bioMerieux, Inc., N ⁇ rtingen, Germany), Etest strips (AB BIODISK, Solna, Sweden) or disk diffusion tests following the method recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (Standards, N.C.f.C.L., Approved standard M2-4a, Villanova, PA (1990)).
- VITEK-2 system bioMerieux, Inc., N ⁇ rtingen, Germany
- Etest strips AB BIODISK, Solna, Sweden
- disk diffusion tests following the method recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (Standards, N.C.f.C.L., Approved standard M2-4a, Villanova, PA (1990)).
- DNA was prepared from 13 blood cultures positive for S. aureus (4), S. epidermidis (3), S. pneumoniae (2), P. aeruginosa (1), E. coli (2) and P. mirabilis (1).
- Total cellular DNA was extracted and purified either by using the First-DNA All- tissue kit (GEN-IAL GmbH, Troisdorf, Germany) following the instructions of the supplier or by enzymatic lysis followed by phenol/chloroform extraction. For the latter protocol, cell pellets were resuspended in 500 ⁇ l lysis buffer (20 mM Tris-HCI, pH 8.0, 2 mM EDTA, pH 8.0, and 1.2% Triton ® -X-100) and lysozyme (Sigma, Taufkirchen, Germany) was added to reach a final concentration of 0.8 mg/ml.
- lysis buffer (20 mM Tris-HCI, pH 8.0, 2 mM EDTA, pH 8.0, and 1.2% Triton ® -X-100
- lysostaphin Sigma was added to a final concentration of 0.2 mg/ml to promote staphylococcal lysis or mutanolysin (0.5 U/ ⁇ l; Sigma) was added to lyse Streptococci and Enterococci. After incubation at 37°C for one hour, cell lysates were treated with Proteinase K (1 mg/ml; Sigma) for 1 hour at 55°C and then with RNase A (0.2 mg/ml; Qiagen, Hilden, Germany) for 1 hour at 37°C. The volume was increased by the addition of 200 ⁇ l TE and the salt concentration was adjusted to 0.7 M by addition of 5 M IMaCI.
- CTAB cetyltrimethylammonium bromide
- Total DNA from commercially available reference strains, clinical isolates and blood cultures was labelled by a non-enzymatic chemical labelling method using the Label It Cy3/Cy5 kits (Mirus, Madison, USA) or the ULYSIS Alexa Fluor 467 Nucleic Acid Labelling Kit (Molecular Probes; Eugene, USA).
- each target DNA was spiked with three gene segments (1 ⁇ l each, 30 ng/ ⁇ l) amplified by PCR from selected recombinant plasmids to serve as internal positive controls.
- Cy3/Cy5 kit 5 ⁇ g of high molecular weight DNA (>20 kb) were mixed with 7.5 ⁇ l reagent in a total volume of 50 ⁇ l and incubated for 2 hours at 37°C according to the recommendations by the supplier. After adjusting the volume to 200 ⁇ l with H 2 O and adding 0.1 volume of 5 M NaCI, unbound label was removed by precipitation with 2 volumes of ice-cold absolute ethanol for at least 30 min at -20 0 C. The labelled DNA was recovered by centrifugation at 17,900 g for 30 min. The pellet was washed with 70% ethanol and resuspended in 70 ⁇ l TE.
- This ratio and the amount of recovered labelled DNA was determined by measuring the absorbance of the nucleic acids at 260 nm and the absorbance of the dye at its absorbance maximum using a lambda40 UV- spectrophotometer (PerkinElmer) and plastic disposable cuvettes for the range from 220 nm to 1,600 nm (UVette; Eppendorf, Hamburg, Germany).
- Example 1.4 Microarray construction Cloned PCR-products were used to generate probes for the DNA microarray. All together 120 gene segments representing virulence genes, antibiotic resistant determinants and species specific metabolic and structural genes from S. aureus (40), E. coli (31) and P. aeruginosa (49) were represented on the microarray (Tab. 2). Tab. 2: Gene probes with SEQ ID NOs, function, gi numbers and primer sequences. E. coli gene probes (1-31), P. aeruginosa gene probes (32-80), S. aureus gene probes (81-120).
- S. aureus, E. coli and P. aeruginosa genes were selected from the literature and databases, and compared by BLAST analysis to all other sequences available in the NCBI database. Primers were designed to amplify gene segments of 200-810 bp length and devoid of apparent homology with genes of other bacterial species and Homo sapiens. Gene segments were amplified by using the puReTaq Ready-To-Go PCR beads (Amersham Biosciences, Freiburg, Germany) and cloned into the pDrive Cloning Vector (Qiagen, Hilden, Germany) according to the recommendations of the suppliers and transformed into competent Escherichia coli (XL-1-Blue) cells using the calcium chloride protocol (Sambrook, J., Russel D. W., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY (2001)).
- Example 1.5 Hybridisation and scanning All experiments described represent dual co-hybridisations of two different target DNA samples labelled respectively with Cy3, Cy5 or Alexa647. After removal of unbound label, Cy3 and Cy5/Alexa647 labelled DNAs were pooled and mixed with 10 ⁇ g of Salmon Sperm DNA and 50 ⁇ g of poly-A-DNA. The mixture was frozen in liquid nitrogen and lyophilised in the dark.
- the target DNA Prior to hybridisation the target DNA was reconstituted in 33 ⁇ l H 2 O and 55 ⁇ l 2x hybridisation solution (Memorec Biotec GmbH, Cologne, Germany) and chemically denatured with 11 ⁇ l denaturation buffer Dl (Mirus) and neutralized with 11 ⁇ l buffer Nl (Mirus) according the instructions of the supplier.
- Hybridisation was automatically performed with a TECAN Hybridisation Station (HS400, TECAN, Salzburg, Austria). The arrays were prewashed at 60 0 C for 1 min with 0.2% SDS and 4x SSC and prehybridised in 120 ⁇ l denatured prehybridisation buffer (Memorec) for 30 min at 6O 0 C at mild agitation.
- hybridisation was performed at 60 0 C for 18 hours at mild agitation.
- the arrays were washed at 50 0 C in primary wash buffer (Memorec) - five cycles of 1 min wash time and 30 s soak time - and in secondary wash buffer (Memorec) - five cycles of 20 s wash time and 30 s soak time -, and finally dried at 30 0 C with N 2 (2.7 bar) for 3 min.
- Hybridised arrays were scanned with a Scan Array 5000 laser scanner (PerkinElmer). Laser light of wavelengths at 532 and 635 nm was used to excite Cy3 dye and Cy5/Alexa647 dye, respectively. Fluorescent images were analysed by the ImaGene software (BioDiscovery, El Segundo, CA, USA).
- DNA-chip The specificity of the DNA-chip was validated firstly (compare Example 1.1) with 45 well characterised clinical isolates and reference strains of the three target species as well as other related bacteria and secondly (compare Example 1.2) with 13 blood cultures from sepsis patients.
- Example 1.7A Detection and discrimination of E. coli All DNA samples from 9 E. coli strains hybridised always with seven E. coli gene probes (envZ, fes (1) and (2), nfrB, yacti, yagX, ycdS) (Fig. IA, columns 19 to 27); in the following these genes are designated as core genes. With 14 E. coli gene probes variable hybridisation was observed including the antibiotic resistance gene probes bla-TEMlO ⁇ , sul, strB and aacC2. Such a variable hybridisation profile is expected for antibiotic resistance genes since acquired resistance to antimicrobials is strain specific. For 11 E.
- coli virulence gene probes (eae, eltB, escR, escT, escU, espB, hlyA, hlyB, SLTII 1 toxA-LTPA, VT2vaB) no hybridisation signals were detected with any of the tested E. coli isolates and blood cultures. Since these virulence genes are known to be specific for particular E. coli pathotypes (Bekal, S. et al., J. Clin. Microbiol., 41 :2113-25 (2003)), it was not surprising that they were not present in the tested strains.
- the eae, esc and esp genes for example are encoded on a chromosomal pathogenicity island, which is typical for enteropathogenic E.
- AE attaching and effacing
- the alpha-hemolysin (hly) operon is encoded on a large plasmid of enterohemorrhagic E. coli strains (Schmidt, H. et al., Infect. Immun. 63: 1055-61 (1995)).
- Example 1.7B Detection and discrimination of Pseudomonas aeruginosa
- DNA samples obtained from P. aeruginosa uniformly hybridised with 32 out of 49 P. aeruginosa specific gene segments including the mexA gene probe (core genes). Variable hybridisation was observed with 17 probes allowing for discrimination of individual P. aeruginosa isolates (Fig. IB, columns 12 to 18).
- Example 1.7C Detection and discrimination of S. aureus
- Hybridisation to the core gene probes permitted the identification of S. aureus, while hybridisation to antibiotic resistance gene probes allowed for discrimination of strains.
- Example 1.7D Discrimination of E. coli, P. aeruginosa and S. aureus from related bacterial species
- the Micrococcus spp. isolate showed no hybridisation with the DNA-chip (column 53). Streptococci (column 56 to 58) and enterococci (columns 54 and 55) showed hybridisation with the staphylococcal 16S RNA gene probe and once with the staphylococcal aph-A3 aminoglycoside resistance gene probe (Enterococcus spp.) (Fig. 1C). Out of 12 strains of seven Gram-negative species (columns 41 to 52), two hybridised with the S. aureus 16S rRNA gene probe (Klebsiella pneumoniae and Proteus mirabilis, Fig.
- Tab. 3 Microarray hybridisation signals obtained with different target DNA preparations of Pseudomonas aeruginosa isolates.
- Example 1.9 Detection and characterisation of pathogens in blood cultures
- DNA prepared from blood cultures comprises a mixture of human and bacterial DNA
- the resulting hybridisation signals obtained with DNA from 1 ml positive blood culture allowed a clear and unambiguous characterisation of S. aureus, E. coli and P. aeruginosa present in 13 tested blood specimens (Fig. 1).
- positive BACTEC ® cultures were identified by microarray hybridisation as multi-resistant MRSA (Fig. 1C, column 8), penicillin-resistant S. aureus (column 9 and 11), multi-susceptible S. aureus (column 10), E. coli (Fig. IA, columns 26 and 27), P.
- aeruginosa (Fig. IB, columnl ⁇ ), and discriminated from oxacillin resistant Staphylococcus epidermidis (columns 33-35), Proteus mirabilis (column 43) and Streptococcus pneumoniae (columns 57 and 58).
- Example 1.10 Correlation between susceptibility testing and microarray hybridisation of selected antibiotic resistance genes
- S. aureus For 11 Staphylococcus aureus strains and blood cultures, susceptibility results determined by the VITEK2 system, Etest strips and disk diffusion tests were compared with the results of the microarray hybridisation assay for the simultaneous detection of antibiotic resistance genes (Tab. 4). The presence or absence of resistance genes as indicated by microarray hybridisation was confirmed by PCR with gene specific primers (results not shown). Tab. 4: Correlation between phenotypic and genotypic antibiotic resistance for 11 S. aureus isolates and blood cultures.
- Phenotypic resistance to penicillin correlated 100% with the hybridisation of the mecA gene (Table 4b), between resistance to erythromycin and hybridisation to the erythromycin resistance genes ermA, ermC or msrSA (Tab. 4c) and between resistance to tobramycin and hybridisation to the aadD gene (Tab. 4d).
- E. coli and other Gram negative bacteria The prototype microarray harboured only four E. coli and one P. aeruginosa resistance gene probes which do not yet allow a comprehensive prediction of antibiotic resistances. Nevertheless, hybridisation with the E. coli resistance gene probe blaTEM106 was observed in one P. mirabilis and four E. coli strains and correlated with phenotypic ampicillin resistance for all five strains (Tab. 5).
- Tab. 5 Correlation between ampicillin/penicillin resistance, gentamicin/tobramycin resistance and streptomycin resistance and hybridisation with the resistance gene probes blaTEM-106, aacC2, aph-A3 and strB, respectively.
- AMP ampicillin
- GEN gentamicin
- STR streptomycin
- TOB tobramycin
- i intermediate
- E. coli gene probes S. aureus gene probes
- Reference strains and clinical isolates The following bacteria were purchased from the American Type Culture Collection (ATCC, Manassas, Va.) or the Deutsche Sammlung f ⁇ r Mikroorganismen und Zellkulturen (DMSZ, Braunschweig, Germany) and were used for evaluation of the specificity of the microarray: Staphylococcus aureus (ATCC 29213), Staphylococcus epidermidis (ATCC 12228; ATCC 18610) Staphylococcus saprophytics (ATCC 14953), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853).
- Staphylococcus aureus ATCC 29213
- Staphylococcus epidermidis ATCC 12228; ATCC 18610
- Staphylococcus saprophytics ATCC 14953
- Escherichia coli ATCC 25922
- Pseudomonas aeruginosa ATCC 27853
- Bacterial cultures Bacterial strains and clinical isolates were plated either onto sheep blood or onto Mueller-Hinton agar from 50% glycerol stocks. One colony was then picked and transferred to 5 ml Luria-Bertani (LB) broth and cultured overnight at 37°C.
- Blood cultures Aerobic blood culture bottles (BACTEC ® Plus aerobic, Becton Dickinson, Heidelberg, Germany) were inoculated with 100 CFU of S. aureus after adding 10 ml blood from healthy volunteers.
- a BACTEC ® 9240 blood culture system (Becton Dickinson) - a continuous reading, automated, and computed system detecting the growth of microorganisms by monitoring CO 2 production - was used for incubation according to the manufacturer's recommendations. Bottles with a positive growth index were removed from the incubator, and an aliquot of 1 ml of the blood culture suspension was taken aseptically with a needle syringe. The aliquot was equally divided, with one part for subculture on agar plates and CFU determination, and one part for DNA isolation.
- samples were collected from ten clinical positive blood culture specimens cultivated under the same conditions as described above. Six of them were positive for different S. aureus strains and four for other bacterial species (Staphylococcus epidermidis, Streptococcus mitis, E. coli and Klebsiella oxytoca). Blood culture aliquots of 500 ⁇ l were used for DNA preparation.
- Tab. 6 Selected S. aureus genes, selected segments (SEQ ID NO) and primers used for segment amplification (SEQ ID NO)
- each selected gene was compared to all other gene sequences available in the NCBI database using the BLAST algorithm. From that comparison, regions (ranging from 104 to 1434 bp) devoid of apparent homology with genes of other bacterial species and Homo sapiens were defined and amplified by PCR using specifically designed primers (see Tab. 6). A mixture of the total DNA from three different S. aureus reference strains and 100 clinical isolates was used as template for amplification of S. aureus gene segments, increasing therefore the chances to amplify more seldom occurring virulence and antibiotic resistance genes.
- PCR products were cloned into the plasmid pCR 2.1-Topo Vector (Invitrogen, Karlruhe, Germany) which were used to transform competent Escherichia coli (XL-1-Blue) cells using the Calcium Chloride protocol (Seidman, CE. et al., in: Ausubel, F. M. (ed.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (2000)).
- Recombinant plasmids containing selected gene segments were screened by restriction analysis and verified by sequencing.
- the plasmid library constructed was used for re-amplification and production of the bulk DNA (10 ⁇ g at a concentration of 1 ⁇ M) from each clone necessary for printing the microchips.
- a Microgrid II spotter BioRobotics, Cambridge, UK
- CMT-GAPSTM coated glass slides Corning Incorporated, Corning, USA
- Bacterial cultures Overnight cultures (5 ml) were harvested at 2,56Og for 10 minutes. After discarding the supernatant the pellet was washed in ImI TE (10 mM Tris-HCI, pH 7.5 - 1 mM EDTA) and recovered by centrifugation at 17,900 g for 2 min.
- ImI TE 10 mM Tris-HCI, pH 7.5 - 1 mM EDTA
- Blood cultures One ml of blood culture was mixed with 1 ml 0.1% Triton ® -X-100 and kept at room temperature for 5 min in order to disrupt blood human cells and resolve bacterial clumps. Bacterial cells were then harvested at 17,900 g for 10 min. Pellets were washed in 1 ml TE and recovered as described above.
- Pellets of harvested cells were resuspended in 500 ⁇ l lysis buffer (20 mM Tris-HCI, pH 8.0 - 2 mM EDTA, pH 8.0 - 1.2% Triton ® -X-100).
- lysis buffer 20 mM Tris-HCI, pH 8.0 - 2 mM EDTA, pH 8.0 - 1.2% Triton ® -X-100.
- lysozyme and lysostaphin Sigma, Taufkirchen, Germany
- Genomic DNA in the aqueous phase was sonified (3 x 10 s at 12% amplitude with 20 s breaks between pulses) in a Digital Sonifier (Branson, Schwaebisch Gmuend, Germany) to obtain fragments of around 1 kb, then precipitated with one volume of isopropanol and pelleted by centrifugation for 30 min at 4°C in a microcentrifuge at 17,900 g. The pellets were washed in 70% ethanol and resuspended in 50-100 ⁇ l TE (10 mM Tris-HCI, pH 7.5 - 1 mM EDTA). This DNA preparation was used when a high yield (hundreds of ⁇ g) was necessary, for example to prepare samples for several hybridisations experiments.
- a second protocol using DNeasy Tissue Kit (QIAGEN, Hilden, Germany) adapted to bacterial cells and allowing DNA preparation in two hours, was also used when fast preparation was the priority.
- the bacterial pellet was resuspended in 1 ml ddH 2 O and the cell suspension frozen in liquid N 2 for 1 minute and then placed in a 60° C thermo-block for 2 minutes. Such a treatment was repeated once and bacteria were centrifuged again for 5 minutes at 14,000g.
- the resulting pellet was resuspended in 180 ⁇ l lysis buffer (20 mM Tris-HCI, pH 8.0 - 2 mM EDTA, pH 8.0 - 1.2% Triton-X-100).
- lysostaphin 0.2mg/ml
- buffer AL for gram positive bacteria
- buffer ATL for gram negative
- 25 ⁇ l of the Proteinase K solution delivered with the kit were added and incubated at 70 0 C for 30 minutes.
- 200 ⁇ l of 100% ethanol were added and the suspension transferred to a DNeasy Mini Column placed into a collection tube.
- the column was centrifuged at 6,000 g for 1 minute, washed first with 500 ⁇ l of buffer AWl, centrifuged at 6,000 g for 1 minute, washed then with 500 ⁇ l of buffer AW2, and centrifuged at 14,000 g for 3 minutes. The column was then placed in a 1.5 ml tube and centrifuged once more at 14,000 g for 1 minute. DNA was eluted with 130 ⁇ l of buffer AE. After one minute the column was centrifuged at 6,00Og for 1 minute. The eluate was re- loaded in the column and centrifuged again under the same conditions in order to increase the DNA yield.
- slides were dried by a 2 min centrifugation step (1000 g) and read in a Scan Array 5000 (Perkin Elmer, Boston, USA) using emission filters for Cy3 and Cy5 in two separate channels. Fluorescence intensities as hybridisation indicators were then analyzed by the software ImaGene (BioBiscovery, Marina Del Rey, USA). Spots were found and segmented in order to select areas of recognizable signals for analysis. Intensity of fluorescence of each spot was measured, signal to local background ratios were calculated, spot morphology and deviation from expected spot position were considered. Cut off values for those parameters were empirically determined in pilot experiments and used to tag spots either as positive or as negative.
- S. aureus DNA samples in decreasing amounts were labelled and hybridised in order to determine the minimum amount of DNA producing the expected hybridisation pattern for a certain strain.
- expected patterns were defined as those produced by the hybridisation of 2 ⁇ g of DNA. From 2 ⁇ g to 50 ng no significant differences in the hybridisation pattern were observed with no false negative spots. Detection of 20 ng DNA was still satisfying with only 5% of false negative and false positive. However, 5 ng of labelled DNA yielded weak signals with almost 95% of false negative spots (data not shown).
- the limit of sensitivity of the S. aureus microarray was then considered as being 20 ng DNA which corresponds approximately to 7 x 10 6 S. aureus CFU (S. aureus genome 2.5 x 10 6 bp. 2.8 fg DNA per cell).
- S. aureus microchip The specificity of the S. aureus microchip was demonstrated by six independently performed co-hybridisation experiments. Visual examination of pictures showing results of co-hybridisation of S. aureus DNA with Pseudomonas aeruginosa or
- Escherichia coli DNA revealed no cross-hybridisation between S. aureus selected gene segments and DNA probes from those Gram negative bacteria (data not shown). Transcribing these data in a bar code showing positive or negative spots (Fig. 3A and B) confirmed that only the S. aureus DNA sample hybridised with spotted probes.
- S. aureus probes cross-hybridised with S. epidermidis and S. saprophytics DNA samples. This is not surprising as these species are phylogenetically closely related.
- genes coding for S. aureus specific proteins as nuclease (nuc), clumping factors A and B (clfA and B) 1 protein A ⁇ spa), V8 serine protease (sprV8) and alpha and beta hemolysins (hla and hlb) exclusively hybridised with S. aureus DNA. The presence/absence of such genes allowed unambiguous discrimination between S. aureus and CoNS.
- S. aureus microarray was tested as a tool for strain profiling.
- a distinctive hybridisation pattern could be established for reference strains and 10 selected clinical isolates. For instance when DNA from clinical isolates TlOO and T103 were labelled with Cy5 and Cy3, respectively, and co-hybridised, both isolates were identified as S. aureus, since both contained species-specific genes as e.g. clumping factor A and B (Fig. 5A).
- T103 contains the genes encoding enterotoxines A ⁇ eta) and B (etb) while in TlOO the gene encoding enterotoxin C ⁇ etc) is present.
- the presence or absence of these genes was confirmed by PCR assays (Fig. 5B) and the antibiotic resistance was verified by classical antibiograms (Sahm, D. & Washington, J. A. (1991). Antibacterial susceptibility tests: dilution methods. In: Manual of Clinical Microbiology (Balows, A., Ed.), pp. 1105-16. American Society for Microbiology, Washington DC, USA) (data not shown).
- S. aureus microarray detects the bacterium growing in blood culture, i.e. after the BACTEC ® signals bacterial growth. Blood culture bottles were spiked with 100 CFU of S. aureus. After the automated culturing system indicated bacterial growth, 1 ml was withdrawn for DNA extraction.
- DNA samples prepared from sterile blood culture show no crosshybridisation with spotted S. aureus probes.
- a 2 ⁇ g DNA sample derived from blood culture containing S. aureus cells revealed a hybridisation pattern almost completely identical to a DNA sample isolated from an overnight LB culture inoculated with a S. aureus colony (Fig. 6B).
- Example 2.1 Materials and Methods Reference strains, clinical isolates and culture conditions: Bacterial reference strains were obtained from the American Type Culture Collection (ATCC, Manassas, Va.), the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany), the Collection Institute Pasteur (CIP, Paris, France) or the network on antimicrobial resistance in Staphylococcus aureus (NARSA, Herndon, Virginia). Klebsiella pneumoniae serotype 03 and serotype 08 were provided by E. M. Nielsen (Department of Bacteriology, Mycology and Parasitology, Statens Serum Institut, Copenhagen, Denmark). Clinical isolates were obtained from the inventors ' clinical routine microbiology laboratory.
- Acinetobacter baumannii (DSM 30008, 1 clinical isolate), Pseudomonas aeruginosa (ATCC27853), Escherichia coil (ATCC 25922, CIP 105893, 81.88, 74.14 and 3 clinical isolates), Klebsiella oxytoca (DSM 4798, 1 clinical isolate), Klebsiella pneumoniae (DSM 681, serotype 03 strain 390 and serotype 08 strain 889), Proteus mirabilis (DSM 788, 2 clinical isolates), Proteus vulgaris (DSM 2140), Candida albicans (ATCC 10231), Enterococcus casseliflavus (clinical isolate), Enterococcus faecalis (ATCC 29212, 1 clinical isolate), Enterococcus faecium (clinical isolate), Enterococcus gallinarum (clinical isolate), Streptococcus
- Bacterial and fungal reference strains and clinical isolates were grown over night at 37 0 C with constant shaking in 5 ml Luria-Bertani (LB) broth or tryptic soy broth (TSB, 30 g/l, Merck) containing 3 g/l yeast extract. Enterococci and streptococci were grown in 10 ml TSB plus yeast without agitation under 5% CO 2 . Overnight cultures were harvested at 2,560 g for 10 min. After discarding the supernatant the pellet was washed in 1 ml TE (10 mM Tris-HCI, pH 7.5 and 1 mM EDTA) and recovered by centrifugation at 17,900 g for 10 min. Cell pellets were used for DNA preparation.
- LB Luria-Bertani
- TSB tryptic soy broth
- DNA was prepared from the strains listed in Example 2.1.
- Total cellular DNA was extracted and purified by using the Bacterial Genomic DNS Purification Kit (Edge BioSystems, Gaithersburg, USA). Cell pellets were resuspended in 200 ⁇ l lysis buffer (20 mM Tris-HCI, pH 7.5, 50 mM NaCI and 10 mM EDTA, pH 8.0) and lysozyme (Sigma, Taufkirchen, Germany) was added to reach a final concentration of 7.5 mg/ml.
- lysostaphin Sigma was added to a final concentration of 0.2 mg/ml to promote Staphylococcal lysis or mutanolysin (0.5 U/ ⁇ l; Sigma) was added to lyse Streptococci and Enterococci. After incubation at 37 0 C for one hour, 400 ⁇ l Sphaeroblast buffer were added and DNA was extracted following the instructions of the supplier.
- Candida albicans DNA was extracted using the MasterPure Yeast DNA purification kit (Epicentre Biotechnologies, Madison USA) following the instructions of the manufacturer.
- high molecular weight DNA Prior to labelling, high molecular weight DNA (>12 kb) was fragmented by sonication for 30 sec at an amplitude of 80% (energy input 1500 kJ) using an ultrasonic homogenizer (Sonoplus HD 3080, Bandelin, Berlin, Germany) equipped with a BR30 booster cup for high-intensive irradiation of small and sensitive sample volumes.
- the size of the fragmented DNA 500-8000 bp was checked by 1.5% agarose gel electrophoresis.
- each target DNA was spiked with three gene segments (1 ⁇ l each, 30 ng/ ⁇ l) amplified by PCR from selected recombinant plasmids to serve as internal positive controls. After 2 hours incubation at 37°C, the reaction was interrupted by adding 5 ⁇ l of 0.5 M EDTA and unbound label was removed using the QIAquick Purification Kit (QIAGEN, Hilden, Germany). The purified labelled DNA was eluted in 80 ⁇ l TE and the relative labelling efficiency of a reaction was evaluated by calculating the approximate ratio of bases to dye molecules (acceptable labelling ratios for nucleic acid were ⁇ 60).
- This ratio and the amount of recovered labelled DNA was determined by measuring the absorbance of the nucleic acids at 260 nm and the absorbance of the dye at its absorbance maximum using a lambda40 UV- spectrophotometer (PerkinElmer) and plastic disposable cuvettes for the range from 220 nm to 1,600 nm (UVette; Eppendorf, Hamburg, Germany).
- Example 2.4 Microarray construction
- probes were represented on the microarray (Tab. 7). They comprised probes for virulence genes, species specific metabolic and structural genes from Candida albicans (86), Acinetobacter baumannii (21), Enterobacter cloacae (11), Escherichia coli (31), Enterococcus faecalis (69), E. faecium (23), Klebsiella oxytoca (21), K. pneumoniae (50), P. aeruginosa (53), Proteus mirabilis (70), P.
- the arrays were prewashed at 42 0 C for 1 min with 5x SSC and prehybridized in 110 ⁇ l denatured prehybridization buffer (30% formamide, 0,1% SDS, 5xSSC, 10mg/ml BSA) for 30 min at 42 0 C at mild agitation. After injection of 110 ⁇ l labelled DNA, hybridization was performed at 60 0 C for 18 hours at medium agitation.
- the arrays were washed at 42 0 C in wash buffer I (Ix SSC, 0.1% SDS) - three cycles of 30 sec wash time and 2 min soak time -, in wash buffer II (O.lx SSC, 0.1% SDS) - five cycles of 30 sec wash time and 2 min soak time - and wash buffer III (O.lx SSC) - four cycles of 30 sec wash time and 2 min soak time - and finally dried at 3O 0 C with N 2 (2.7 bar) for 3 min.
- Hybridized arrays were scanned with GenPix Personal Axon 4100A laser scanner (Axon Instruments, Union City, CA, USA). Laser light of wavelengths at 532 and 635 nm was used to excite Cy3 dye and Cy5 dye, respectively.
- Fluorescent images were analyzed by the GenePix Pro 6.0 and Acuity 4.0 software (Axon Instruments). For each feature (gene probe) the median pixel intensity of wavelength 635 nm or 532 nm, respectively, was determined and the median background of the respective wavelength subtracted (F635 Median - B635 and F532 Median - B532, respectively).
- a microarray comprising a set of 930 gene probes of 200 to 800 bp length was developed (Tab. 7).
- the clinically most relevant sepsis causing pathogens were represented on the microarray by gene probes specific for the genera and species E. coli (31), Staphylococcus aureus (69) and coagulase negative staphylococci (58), P. aeruginosa (53), Streptococcus spp. (185), Enterococcus spp. (92), Proteus spp. (79), Klebsiella spp. (71), Enterobacter spp.
- the array contained 131 bacterial resistance gene probes.
- a set of 29 control probes was included. Different 16S rRNA gene probes (18) served as positive hybridization controls for bacterial DNA.
- the gene probe rbcL_l_2 (segment of the rubisco gene of Hordeum vulgaris) was prelabelled with Cy3 and Cy5 and spotted onto each subarray for visualisation of the array orientation.
- Tab. 8 Microorganism strains used for microarray validation. Non-target species are Nos 21, 25, 27 and 30.
- Example 2.7 Specificity of hybridization profiles for fungi DNA of the fungus Candida albicans hybridized specifically with the Candida gene probes (Probe Nos. 157-242) including Candida resistance probes but not with bacterial 16 rRNA or species specific probes (Fig. 8, panel A). The specificity of two selected Candida probes is demonstrated in Fig. 8 panel B, the probes ALSl and ASL43f hybridized only with DNA obtained from C. albicans and not with any DNA obtained from the 43 bacterial strains.
- Example 2.8 Specificity of hybridization profiles for Gram-negative bacteria Strains of the genus Klebsiella showed specific hybridization with the Klebsiella gene probes (Probe Nos. 399-469). For this genus cross hybridization with lower intensity of the fluorescent signals was observed with some E. coli and P. aeruginosa probes (Nos. 275-306 and 470-522, respectively). This is also the case for bacterial strains of the genus Proteus, which show major hybridization with the Proteus gene probes allowing unambiguous identification (Probe Nos. 523-601). Vice versa, P. aeruginosa and E.
- the E. coli reference strain CIP 105893 and the clinical isolate U10164-2 show nearly identical hybridization profiles, demonstrating the high reproducibility of the assay.
- Strains of the non-fermenting Gram-negative bacterium A. baumannii were readily identified based on their microarray hybridization profile showing specific hybridization to the A. baumannii gene probes (Nos. 243-263). The specificity of selected species specific probes is shown in Figure 9.
- the A. baumannnii probe csuA hybridized only with labelled DNA preparations derived from A.
- the P. aeruginosa probe PhzA showed hybridization signals with a high intensity >60000 (Median fluorescence - background) only with DNA of the P. aeruginosa reference strain but with no other pathogen, demonstrating that although some P. aeruginosa probes (eg. aprA) show cross-hybridization with other Gram-negative species, unambiguous identification is feasible. Equally specific results were obtained with the E. coli probe shuA, which showed significant hybridization signals > 40000 only with DNA of the seven E. coli reference strains and clinical isolates. The closely related species K.
- the P. mirabilis probe hpmB was highly specific for the three P. mirabilis strains and isolates, while probe enzZPrvu was specific for P. vulgaris.
- Example 2.9 Specificity of hybridization profiles for Gram-positive bacteria of the genus Enterococcus
- the microarray assay was highly specific in the identification of Gram-positive target species.
- Clinical isolates of the species E. faecalis and E. faecium could be identified and discriminated unambiguously by their hybridization profiles (Probe Nos. 307-375 and 376-398, respectively) (Fig. 7, panels E and F).
- the vancomycin resistant non-target strain E. casseliflavus (Fig. 7, panel G) showed hybridization to the bacterial 16S rRNA probes, the antibiotic resistance gene probes vanC-2 (vancomycin resistance), arr2 (Rifampin resistance) and tetM (tetracycline resistance) and the S.
- aureus probes gyrA DNA gyrase subunit A
- rpoB RNA polymerase B subunit
- sstC iron transport protein
- Example 2.10 Specificity of hybridization profiles for Gram-positive bacteria of the genus Streptococcus
- Microarray hybridization assays performed with streptococcal DNA obtained from reference strains of S. pneumoniae, S. pyogenes, S. mutans and S. agalactae revealed species specific hybridization profiles and an excellent identification and discrimination of these target organisms (Fig. 7).
- the species S. dysgalactiae and S. bovis (S. viridans group) are each represented by a single gene probe on the array (fasCAXStrdysg and lichStrbov, respectively). These probes however exhibited specific hybridization to the target DNA only, and in this way permitted identification of the two species. Additionally both species showed hybridization with the 16S rRNA gene probes and pbp2b (penicillin binding protein of S. pneumoniae).
- S. dysgalactiae DNA hybridized with the probes dacCStrpyog and murEStrpyog and S. bovis DNA with gyrA, rpoB and sstC as E. casseliflavus.
- the non-target species S. gordonii and S. angiosus were readily discriminated by their hybridization profiles from other streptococci, S. gordonii showed hybridization to the 16S rRNA genes only, S. angiosus DNA hybridized additionally to gyrB and rpoB (Fig. 7 H).
- Example 2.11 Specificity of hybridization profiles for Gram-positive bacteria of the genus Staphylococcus
- Hybridization assays performed with S. aureus strains and S. epidermidis DNA produced very specific hybridization profiles with little cross hybridization (Fig. 7 AB).
- the specificity of selected probes for coagulase-negative staphylococci is shown in Fig. 10.
- S. saprophytics , S. haemolyticus, S. lugdnunensis, S. warneri and S. hominis produced hybridization profiles distinct of those from S. aureus and S. epidermidis.
- the following species specific probes were detected: RNAposigmSsapro_l and _2 for S. saprophytics, RNApolisigm and mvaDShaemolyt for S.
- S. hominis no probe proved to be species specific.
- the S. hominis derived probe ydhK cross hybridized with DNA of S. hominis, S. epidermidis and S. haemolyticus.
- certain probe patterns seem to be species specific for S. hominis and may allow identification and discrimination from S. haemolyticus and other CoNS (eg. hybridization of ydhK, tnpStwar and sin and absence of mvaDShaemolyt and RNApolsigm).
- VITEK2 Susceptibility results determined by the VITEK2 system were compared to the results of the microarray hybridization assay for the simultaneous detection of antibiotic resistance genes.
- ESBL extended spectrum ⁇ -lactamases AMP, Ampicillin; ASU, Ampicillin/Sublactam; MEZ, Mezlocillin; PRL, Piperacillin; KZ, Cefazolin; CXM, Cefuroxim; IMP, Imipenem; i, intermediary resistance
- Fluorescence signals >10000 were considered positive. c Fluorescence ⁇ 10000; most fluorescence signals were ⁇ 30000 for the hybridization assay with P. vulgaris DMS 2140
- Tab. 11 Phenotypic and qenotvpic resistance of Enterococcus strains.
- VAN vancomycin
- DA clindamycin
- E erythromycin
- QD quinupristin/dalfopristin (streptogramins)
- STR streptomycin
- TET tetracycline
- i intermediary resistance.
- the tested streptococci showed phenotypic susceptibility to all tested antiobitics.
- PEN penicillin
- OXA oxacillin
- DA clindamycin
- E erythromycin
- TOB tobramycin
- GEN gentamicin
- i intermediary resistance.
- Relative low fluorescence intensity Median fluorescence - background ⁇ 18.000).
- Example 2.14 Strain discrimination and detection of virulence genes in S. aureus Virulence gene probes, showing varying fluorescence intensities after hybridization with DNA of four different S. aureus strains are listed in Table 13.
- aureus serotypes 5 and 8 account for «25% and 50%, respectively, of isolates recovered from humans. Moreover, these two serotypes, carrying the genes cap5 and cap8, are prevalent among isolates from clinical Infections as well as from commensal sources.
- the cap5 gene was detected in the ATCC 29213 strain and the clinical MRSA isolate C5010, while cap8 was detected in the clinical isolate P2116 and the community-aquired MRSA strain MW2 (Table 13).
- the latter strain hybridized to many virulence gene probes including the leukocidin gene probes lukF and lukS and the enterotoxln gene probes sea, sec, seh and sel.
- This microarray gene profile is in perfect concordance with genome sequence of this fully sequenced strain, which produces the Panton-Valentine leukocidin (PVL), encoded by lukF and lukS. Panton-Valentine leukocidin forms non-specific pores in leukocyte plasma membranes, which result in increased permeability and eventual host cell lysis. While strain MW2 does not harbor the gene seg encoding enterotoxin G, this gene was detected in the ATCC strain and the clinical MRSA isolate C5010, which both also showed hybridization with sea (Enterotoxin A). In contrast, the clinical isolate P2116 showed no or only minor hybridization with these virulence probes. From these results it can be concluded that microarray hybridization patterns allow the discrimination of different S. aureus strains as well as the detection of clinically relevant virulence determinants.
- PVL Panton-Valentine leukocidin
- Example 2.15 Strain discrimination and detection of virulence genes in E. coli Virulence gene probes, showing varying fluorescence intensities after hybridization with DNA of seven different E. coli strains are listed in Table 14. Tab. 14: Hybridization of E. coli virulence gene probes: -, Median fluorescence ⁇ 10000; +, Median fluorescence >10000 -20000; ++, Median fluorescence >20000-50000; + + + + , Median fluorescence ⁇ 50000.
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