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WO2008011715A1 - Identification et caractérisation de micro-organismes - Google Patents

Identification et caractérisation de micro-organismes Download PDF

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Publication number
WO2008011715A1
WO2008011715A1 PCT/CA2007/001326 CA2007001326W WO2008011715A1 WO 2008011715 A1 WO2008011715 A1 WO 2008011715A1 CA 2007001326 W CA2007001326 W CA 2007001326W WO 2008011715 A1 WO2008011715 A1 WO 2008011715A1
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WIPO (PCT)
Prior art keywords
microorganism
sample
probe
virulence
probes
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Ceased
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PCT/CA2007/001326
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English (en)
Inventor
Josée HAREL
Roland Brousseau
Gabriela Martinez
Cécile CROST
Luke Masson
Christine Maynard
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Universite de Montreal
National Research Council of Canada
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Universite de Montreal
National Research Council of Canada
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Publication of WO2008011715A1 publication Critical patent/WO2008011715A1/fr
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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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to the field of detection and analysis of microorganisms, including in particular characterization of microorganisms such as bacteria for medical or veterinary screening purposes.
  • bacterial pathogens are responsible for animal husbandry economic losses and zoonosis. For example, in Canada, financial loss resulting from porcine pleuropneumonia infections, due to the bacteria Actinobacillus pleuroneumoniae, are estimated to cost over 40 million dollars per year. Some bacterial pathogens are found in the environment, and in the digestive tracts of common animal species including humans. Monitoring of new or re-emerging pathogens is also a high concern in both animal and human public health management. Identification of new pathogens is a fastidious process that can be shortened by the use of microarray technology as demonstrated by the recent viral SARS outbreak.
  • antimicrobial resistance has increased rapidly in the last decade and has become a major public health threat worldwide. Emergence of multi-resistant bacterial strains is a serious problem in the medical field. In both developed and developing countries, bacterial resistance is increasing due to abusive use of antibiotics. Antibiotic resistance, and more particularly the development of bacteria resistant to multiple drugs, is a rapidly growing concern. Diverse factors, including patient's expectations, provider's perceptions, and use of antibiotics in agriculture, contribute to the problem. Mortality associated with bacterial infections is increasing because it may be difficult to cure a simple pathology.
  • aureus have been reported to have a decreased susceptibility to vancomycin, which has been the last remaining uniformly effective treatment.
  • Various initiatives have been introduced, such as the one organized by the World Health Organization launched in 2001, to limit bacterial resistance to antibiotics and ensure surveillance. Slow, costly, labour intensive tests for bacterial detection and analysis are often employed. These tests may include:
  • Bacterial identification often relies upon culture-based methodology and on biochemical tests. To test bacterial antibioresistance requires bacterial culturing using standard procedures like the disk diffusion method (Kirby Bauer) or minimal inhibitory concentration (MIC). Often methods for bacterial toxin detection use a combination of in vivo toxicity assays on cultured mammalian cells such as Vero or Hep-2, microscopy techniques on infected animal tissues, and various forms of immunoassays. The application of such techniques is generally limited to the detection of one particular toxin or virulence phenotype.
  • PCR polymerase chain reaction
  • the phenotypic and genetic tests alluded to above are generally slow, costly, and labour-intensive. Also, the application of such techniques may be limited to the detection of one particular toxin or virulence phenotype. Finally, the tests may not provide sufficient information for a precise epidemiologic bacterial strain profiling. Genetic profiling by PCR offers great sensitivity, good specificity and fairly good turnaround time, but suffers from shortcomings which make it unusable in routine monitoring of bacterial clinical isolates. These shortcomings include: the need of specific primers and amplification conditions for each pathogen of interest;
  • Certain exemplary embodiments provide a DNA array for medical or veterinary screening of a biological sample of human or animal origin, to screen for a presence of at least one microorganism in the sample, and to determine a species, virulence, and antibiotic resistance of each microorganism in the sample , the DNA array comprising: (a) a substrate; and
  • At least one species determination probe each comprising a nucleotide sequence characteristic of the microorganism species from which it is derived;
  • At least one virulence probe each for determining a virulence of the microorganism from which it is derived, said microorganism being detectable by the at least one species determination probe of (i); and (iii) at least one antibiotic resistance probe, each for determining an antibiotic resistance of the microorganism from which it is derived, said microorganism being detectable by the at least one species determination probe of (i).
  • a method for medical or veterinary screening of a biological sample of human or animal origin to screen for a presence of at least one microorganism in the sample, and to determine a species, virulence, and antibiotic resistance of each microorganism in the sample, said method comprising the steps of:
  • association of at least one of said sample nucleic acids with at least one species determination probe is indicative that said sample comprises a microorganism from which the nucleic acid sequence of said probe is derived;
  • association of at least one of said sample nucleic acids with at least one virulence probe is indicative of a level of virulence of a microorganism in said sample
  • association of at least one of said sample nucleic acids with at least one antibiotic resistance probe is indicative of an antibiotic resistance of at least one microorganism in said sample.
  • a method for diagnosing an infection in a human or animal subject by at least one microorganism, determining a virulence of the infection, and planning antibiotic administration to treat the infection comprising the steps of:
  • association of said sample nucleic acids to probes on said DNA array wherein association of at least one of said sample nucleic acids with at least one species determination probe is indicative that said subject is infected with at least one microorganism from which the nucleic acid sequence of said at least one species determination probe is derived;
  • association of at least one of said sample nucleic acids with at least one virulence probe is indicative of a level of virulence of said at least one microorganism in said sample
  • association of at least one of said sample nucleic acids with at least one antibiotic resistance probe is indicative of an antibiotic resistance of said at least one microorganism in said sample.
  • a method for producing a DNA array for screening a biological sample for microorganisms, to simultaneously detect a species of each microorganism present, a virulence of each microorganism, and an antibiotic resistance of each microorganism comprising the steps of:
  • a) selecting a plurality of nucleic acid probes including at least one probe for detecting a species of microorganism, at least one probe for detecting a virulence of a microorganism, and at least one probe for detecting an antibiotic resistance of a microorganism; and either
  • Figure 1 is a schematic illustration of virulence gene DNA microarray.
  • Figure 2 is a schematic representation of microarray technology synthesis, detection process and analysis of the bacterial pathogens.
  • Figure 3 is a list ofcpn ⁇ O probes for identification of the bacterial pathogens.
  • Figure 4 is a list of virulence gene markers for characterization of bacterial pathogens.
  • Figure 5 is a list of antimicrobial agent resistance gene probes for the Gram-positive bacterial pathogens.
  • Figure 6 is a list of antimicrobial agent resistance gene probes for Gram-negative bacterial pathogens.
  • 'Antibiotic resistance' includes total or partial resistance to a presence of an antibiotic, compared to an equivalent organism that does not exhibit such antibiotic resistance.
  • 'Infection' refers to an infiltration of a human or non-human animal by an unwanted microbial organism. An infection may or may not cause clinically observable changes or symptoms in the human or non-human animal.
  • 'Microarray' or 'array' refers to any selection of known DNA molecules arranged in an orderly fashion upon a substrate, with each group of similar or identical DNA molecules being affixed to the substrate within one or more discreet areas or locations upon the surface of the substrate.
  • a typical method for producing such a microarray per se is described in United States Patent 6,110,416, which is incorporated herein by reference.
  • the basic concept of the use of a DNA microarray in accordance with the present invention is as following.
  • a sample possibly comprising bacteria which may come from environment, food, water, clinical sample from human or animal source is either incubated on a solid medium or in a liquid medium for culturing and multiplicating the microorganism that may be contained therein, or is used directly with PCR techniques to amplify any DNA from any microorganisms that may be present therein.
  • a detectable marker such as a fluorescent dye. If the DNA has been amplified by PCR directly, the amplified DNA is then labeled with the detectable label. The DNA labeled with the detectable label is then applied to a DNA microarray in accordance with the present invention.
  • microarrays are high density nucleic acid probe arrays, which may used for example to detect and/or monitor the expression of a large number of genes, or for detecting sequence variations, mutations and polymorphisms.
  • Microfabricated arrays of large number of oligonucleotide probes (variously described as “biological chips", “gene chips”, or “DNA chips"), allow the simultaneous nucleic acid hybridization analysis of a target DNA molecule with a very large number of oligonucleotide probes.
  • nucleic acids may include any polymer or oligomer of nucleosides or nucleotides (polynucleotides or oligonucleotides), which include pyrimidine and purine bases, preferably cytosine, thymine, and uracil, and adenine and guanine, respectively.
  • Polymers or oligomers of deoxyribonucleotides or ribonucleotides may be used, which may contain naturally occurring or modified bases, and which may contain normal internucleotide bonds or modified (e.g. peptide) bonds.
  • 'Microbe' refers to any microorganism and most preferably any prokaryotic microorganism.
  • Examples of genera of prokaryotes which are useful in accordance with the invention include, but are not limited to Staphylococcus, Pseudomonas, Escherichia, Bacillus, Salmonella, Chlamydia, Helicobacter, and Streptococcus and other prokaryotic microorganisms that are known in the art.
  • Species which can be identified by the method of the invention include, but are not limited to, S. haemolyticus, S. epidermidis, S. lugdunensis, S. hominis, E. coli, B. subtilis, Streptococcus faecalis, Bartonella henselae, B. quintana, B.
  • Bacteria represent one class of microorganisms.
  • 'Pathotype' refers to the classification of a particular strain of a microorganism by virtue of the pathogenic phenotype it may manifest when it infects a subject. A plurality of strains may thus be grouped in the same pathotype if the strains are capable of resulting in the same phenotypic manifestation (e.g.
  • pathotypes may include those associated with intestinal and extraintestinal conditions.
  • pathotypes include but are not limited to ETEC, EPEC, EHEC, EAEC, EIEC, UPEC, MENEC, SEPEC, CDEC and DAEC noted herein.
  • 'Probe' refers to any fragment of nucleic acid sufficient to hybridize with a target nucleic acid (generally DNA) to be detected.
  • the fragment can vary in length from 15 nucleotides up to hundreds or thousands of nucleotides. Determination of the length of the fragment is a question of the desired sensitivity, of cost and/or the specific conditions used in the assay.
  • probes may be bound to different, discrete locations of a substrate such as a microarray.
  • the length of the probes may be variable, e.g. at least 15, 20, 50, 100, 500, 1000 or 2000 nucleotides in length.
  • sample typically refers to a sample or biological origin, such as a sample derived from a liquid or solid (e.g. tissue) from a human or non-human animal.
  • a sample may also refer to a liquid or solid cell culture derived from specimens taken from a human or non-human animal.
  • 'Virulence gene' refers to a nucleic acid sequence of a microorganism, the presence and/or expression of which correlates with the pathogenicity of the microorganism.
  • virulence genes may in an embodiment comprise chromosomal genes (i.e. derived from a bacterial chromosome), or in a further embodiment comprise a non-chromosomal gene (i.e. derived from a bacterial non- chromosomal nucleic acid source, such as a plasmid).
  • Virulence genes for a variety of pathogenic microorganisms are known in the art.
  • DNA arrays also referred to herein and in the art as "DNA chips” that present significant advantages over those of the prior art.
  • DNA arrays enable analysis of a biological sample to determine (1) which microorganisms are present in the sample, (2) how virulent the microorganisms are, and (3) whether the microorganisms are resistant to a range of antibiotics, in preferred embodiments by way of a single DNA hybridization and wash cycle, without need for further diagnostic analysis, reagents, or experiments.
  • the DNA arrays of the invention permit screening and analysis of biological samples with relatively high speed, efficiency, and accuracy.
  • the invention therefore encompasses various methods involving such DNA arrays, for example for rapid diagnosis and treatment planning for either human or non-human animal patients.
  • the DNA arrays of the invention are suitable for use in a hospital or other clinical settings. Moreover, the DNA arrays may even be used for "on-farm" diagnosis and treatment planning for farm animals.
  • the DNA arrays significantly reduce the need for export of biological samples from the clinical or farm setting for extended biological analysis. This substantially reduces delays for diagnosis and treatment planning, and enables more rapid and effective infection treatment through the use of highly appropriate antibiotics specific for the infective microbial strains present. It follows that the DNA arrays of the invention, and related methods, present a significant step forwards in improving patient treatment and prognosis, as well as helping to prevent the spread of infectious disease through human and / or animal populations through rapid and effective treatment of infected individuals.
  • DNA microarray a series of DNA probes for detecting microbial species, virulence, and antibiotic resistance.
  • DNA probes include a significant number of the currently known virulence factors genes, antimicrobial agent genes, and species identification genes (e.g. cpn ⁇ O), as identifier sequences for an infection caused by a pathogenic bacteria of medical or veterinary interest.
  • the invention further provides for analysis of a given liquid culture or colony of bacteria simultaneously for the presence of all these virulence genes, the antibiotic resistance genes and the specific cpn ⁇ O in the same experiment.
  • the DNA arrays of the invention may include any probes, and may be custom- designed according predetermined parameters, with probes selected according to those parameters.
  • the DNA arrays of the invention represent the first time that probes have been grouped on the same array with the aim of achieving pathogenic organism identification, analysis of potential virulence of such organisms, and their antibiotic resistance characteristics, all in one highly parallel step.
  • the juxtaposition of such probes on the same DNA array greatly increases the usefulness of the array by simultaneously providing three substantially independent sets of very important data, useful to guide clinicians and veterinarians in the treatment of their patients.
  • Such DNA array or DNA chip products have not been described in the art to date.
  • the DNA arrays of the invention may further enable elucidation and characterization of emerging microbial strains that could present a danger to human or non-human animal health.
  • such DNA arrays and systems for their analysis may be highly transportable so that they can be taken to remote locations to screen in situ for the emergence of suspected new disease strains, for example showing novel levels or virulence or resistance to existing antibiotics.
  • the DNA arrays can also facilitate epidemiological and phylogenetic studies since the prevalence of each virulence and antibiotic resistance gene can be determined for different strains, and the phylogenetic associations elucidated between virulence pattern and serotypes of a given strain.
  • DNA array technology is compatible with the increasing number of newly recognized virulence and resistance genes since thousands of individual probes can be immobilized on one chip.
  • a DNA array for medical or veterinary screening of a biological sample of human or animal origin to screen for a presence of at least one microorganism in the sample, and to determine a species, virulence, and antibiotic resistance of each microorganism in the sample, the DNA array comprising:
  • At least one species determination probe each comprising a nucleotide sequence characteristic of the microorganism species from which it is derived;
  • At least one virulence probe each for determining a virulence of the microorganism from which it is derived, said microorganism being detectable by the at least one species determination probe of (i); and (iii) at least one antibiotic resistance probe, each for determining an antibiotic resistance of the microorganism from which it is derived, said microorganism being detectable by the at least one species determination probe of (i).
  • the substrate may comprise any material suitable for depositing thereupon, or synthesizing thereupon, oligonucleotide probes in discrete locations to establish a DNA array in accordance with the present invention.
  • Preferred substrates may include, but are not limited to, glass, resins and polymers such as nylon. Numerous other substrates are also know in the art, any of which may be used to produce the DNA arrays in accordance with the present invention.
  • the invention encompasses the use of any DNA probe or probes that fulfill the criteria specified.
  • the at least one species determination probe may comprise a plurality of species determination probes, each in a different location in the DNA array, for detection of at least one microorganism species in a single sample.
  • the at least one microorganism may comprise just one type of microorganism, with optionally multiple probes specific for that microorganism.
  • the array may include a plurality (2 or more) probes specific for a range of different microorganism species.
  • each species determination probe is derived from a or 16S-derived gene sequence. For example, cp «60-derived gene sequences are disclosed in, but not limited to, those mentioned in United States Patent 5,708,160 issued January 13, 1998, which is incorporated herein by reference.
  • the invention also encompasses the use of specific and selected nucleotide sequences for each of the at least one species determination probes. These sequences may be selected from, but are not limited to, those disclosed in Table 1 herein, also shown as SEQ ID NOS. : 1 -82 in the accompany sequence listing, or a fragment of such sequences suitable for use as a probe, or a sequence having at least 50% identity, preferably at least 70% identity, more preferably having at least 80% identity and most preferably having at least 90% identity thereto. A skilled artisan will recognize that other related DNA sequences may also be suitable for use as probes in the DNA arrays of the invention other than those specifically listed in Table 1.
  • the at least one virulence probes include, in accordance with the DNA arrays of the invention, any nucleotide sequences suitable for assessing a level or degree of virulence of any microorganism in a given biological sample.
  • a DNA array of the invention may comprise a plurality of virulence probes (i.e. at least two) each indicative of a level of virulence of at least one microorganism.
  • the virulence probes include a plurality of probes sufficient for assessing a virulence of multiple microorganisms in the sample.
  • each virulence probe may encode a polypeptide from a class of proteins selected from the group consisting of toxins, adhesion factors, secretory system proteins, capsule antigens, somatic antigens, flagellar antigens, invasins, autotransporter proteins, and aerobactin system proteins.
  • toxins adhesion factors
  • secretory system proteins capsule antigens
  • somatic antigens flagellar antigens
  • flagellar antigens flagellar antigens
  • invasins invasins
  • autotransporter proteins autotransporter proteins
  • aerobactin system proteins aerobactin system proteins
  • each of the at least one virulence probes comprises may be independently selected from, but are not limited to, those disclosed in Table 2 herein, also shown as SEQ ID NOS.: 83-144 in the accompany sequence listing, or a fragment of such sequences suitable for use as a probe, or a sequence having at least 50% identity, preferably at least 70% identity, more preferably having at least 80% identity and most preferably having at least 90% identity thereto.
  • SEQ ID NOS.: 83-144 in the accompany sequence listing, or a fragment of such sequences suitable for use as a probe, or a sequence having at least 50% identity, preferably at least 70% identity, more preferably having at least 80% identity and most preferably having at least 90% identity thereto.
  • a skilled artisan will recognize that other related DNA sequences may also be suitable for use as probes in the DNA arrays of the invention other than those specifically listed in Table 2.
  • the DNA arrays of the present invention encompass the use of any antibiotic resistance probe(s) for determining a potential for antibiotic resistance of microorganisms in a sample.
  • Such probes may include multiple probes each specific for different levels or types of antibiotic resistance for the same microorganism.
  • the antibiotic resistance probes may be relevant for a plurality (i.e. 2 or more) different species of microorganism.
  • each of the at least one antibiotic resistance probes comprises a nucleotide sequence independently selected from Tables 3 and 4 (corresponding to SEQ ID NOS.: 145- 187), or a fragment of such sequences suitable for use as a probe, or a sequence having at least 50% identity, preferably at least 70% identity, more preferably having at least 80% identity and most preferably having at least 90% identity thereto.
  • Tables 3 and 4 corresponding to SEQ ID NOS.: 145- 187
  • the DNA arrays of the invention may be designed for detection and analysis of any type of microorganism, including both eukaryotic and prokaryotic microorganisms.
  • the probes may be designed for the detection of prokaryotic microorganisms such as bacteria.
  • bacteria may include, but are not limited to, those listed by the Centres for Disease Control and Prevention at http://www.cdc.gov/ncidod/dbmd/diseaseinfo/ .
  • the DNA array may be organized into subarrays in a manner well known in the art, and described herein for example with reference to Figure 1.
  • the array may comprise at least two subarrays each containing at least two identical probes at adjacent discrete locations on said substrate. In this way, false positive or false negative annealing to any given probe may be identified by way of the presence of two identical probes at adjacent positions on the array.
  • a method for medical or veterinary screening of a biological sample of human or animal origin to screen for a presence of at least one microorganism in the sample, and to determine a species, virulence, and antibiotic resistance of each microorganism in the sample, said method comprising the steps of: a) contacting any DNA array of the invention with sample nucleic acids of said sample; and b) detecting association of said sample nucleic acids to probes on said DNA array; wherein association of at least one of said sample nucleic acids with at least one species determination probe is indicative that said sample comprises a microorganism from which the nucleic acid sequence of said probe is derived; wherein association of at least one of said sample nucleic acids with at least one virulence probe is indicative of a level of virulence of a microorganism in said sample; and wherein association of at least one of said sample nucleic acids with at least one antibiotic resistance probe is indicative of an antibiotic resistance of at least one microorganism in said
  • the DNA arrays of the invention permit simultaneous identification and characterization (e.g. virulence and antibiotic resistance characteristics) of such microorganisms from a sample.
  • the contacting step may involve any means for bringing the DNA of the sample into contact with the DNA array, and the detecting step may involve any means known in the art for determining specific hybridization.
  • Such method steps would be known to the skilled artisan, and additional guidance in the regard is provided herein, and by way of references cited herein, which are incorporated by reference.
  • a preparatory step or steps may be required to obtain or substantially isolate useful DNAs from the biological sample, so as to bring them into a form or condition suitable for the contacting step.
  • a preparatory step or steps may be required to obtain or substantially isolate useful DNAs from the biological sample, so as to bring them into a form or condition suitable for the contacting step.
  • DNAs may need to be extracted and purified from the sample, and many techniques are known in the art to achieve this, including commercially available DNA extraction kits such as those available from the companies Promega or Qiagen.
  • the DNAs, once isolated, may also be subjected to further processing such as, for example, PCR amplification and / or restriction endonuclease digestion to obtain a more appropriate DNAs suitable for the contacting step.
  • DNAs in the sample (or processed as described) may be further modified or tagged, for example with fluorescent tags, in accordance with known techniques for DNA array analysis.
  • the processing of the sample to obtain DNAs from the sample may depend in part upon the nature of the sample.
  • biological samples may be selected from, but not limited to, the group consisting of blood, urine, amniotic fluid, feces, tissue, cells, biological secretions, excretions, discharge, body fluid, or a human or animal patient-derived cell culture. Therefore, such biological samples may be liquid, solid, gelatinous, soft solid, viscous, slurry-like etc. depending upon their contents.
  • the methods of the invention, and the use of corresponding DNA arrays include the option of additional steps or processing of the biological sample so that DNAs may be obtained that are useful for application to a DNA array.
  • the methods of the invention may further comprise the step of: c) tabulating results for probes for each species, virulence, and antibiotic resistance, based upon intensity of the association detected upon the DNA array. Further, the methods may further comprise the step of: d) processing information derived from step b), and / or tabulated results from step c) if present, to generate a profile for said sample indicative of microorganism(s) present in said sample, together with an indication of a virulence and an antibiotic resistance of each of said microorganism(s).
  • processing of raw data obtained from analysis of the DNA array may permit elucidation of the nature of the microorganisms in the sample, such that each is provided with a detailed profile with regard to its species, virulence, and antibiotic resistance.
  • at least step b), and optionally steps c) and d) if present may be conducted by an automated, computer-controlled, DNA array reader, and / or a computer associated therewith.
  • hybridization and optionally washing of the sample DNAs with a DNA array of the invention may permit automated output of a detailed sample profile in a rapidly obtained, and concise report, for review by a skilled artisan or technician, detailing the species and virulence / antibiotic resistance properties of all microorganisms present.
  • exemplary embodiments of the methods of the invention include a method for diagnosing an infection in a human or animal subject by at least one microorganism, determining a virulence of the infection, and planning antibiotic administration to treat the infection, said method comprising the steps of:
  • association of at least one of said sample nucleic acids with at least one species determination probe is indicative that said subject is infected with at least one microorganism from which the nucleic acid sequence of said at least one species determination probe is derived;
  • association of at least one of said sample nucleic acids with at least one virulence probe is indicative of a level of virulence of said at least one microorganism in said sample
  • association of at least one of said sample nucleic acids with at least one antibiotic resistance probe is indicative of an antibiotic resistance of said at least one microorganism in said sample.
  • the invention therefore further provides for methods of clinical relevance, by permitting a clinician or veterinarian with a simple and rapid means to screen one or more human or non-human animal patients for potentially a wide range of microorganisms, and simultaneously obtain details of the virulence and antibiotic resistance of those microorganisms, by way of a single DNA array.
  • Such methods involving the use of the DNA arrays of the invention, offer unprecedented efficient and substantially comprehensive mechanisms of patient analysis, permitting rapid screening, and rapid and more effective treatment administration.
  • Such methods of the invention may involve, and under some circumstances benefit from, initial processing of the biological sample to generate a more suitable extract of DNAs for presentation to a DNA array.
  • DNAs may also be processed for example by PCR and / or restriction endonuclease or other digestion.
  • Such methods may also involve any type of biological sample, including but not limited to: blood, urine, amniotic fluid, feces, tissue, cells, biological secretions, excretions, discharge, body fluid, or a human or animal patient-derived cell culture.
  • the methods may further comprise a step of: c) tabulating results for probes for each species, virulence, and antibiotic resistance, based upon intensity of the association detected upon the DNA array.
  • the methods may further comprise: d) processing information derived from step b), and / or tabulated results from step c) if present, to generate a diagnosis and treatment plan for said subject suitable to at least partially eradicate microorganism(s) present in said subject.
  • at least step b), and optionally steps c) and d) if present are conducted by an automated, computer-controlled, DNA array reader, and / or a computer associated therewith.
  • selected methods of the invention may permit automated (or at least substantially automated) analysis of a biological sample, to achieve an automatically generated patient / infection diagnosis, optionally together with a proposed treatment plan or regime based upon information deduced from the DNA arrays with regard to virulence and antibiotic resistance of the microorganisms present.
  • the invention provides a method for producing a DNA array for screening a biological sample for microorganisms, to simultaneously detect a species of each microorganism present, a virulence of each microorganism, and an antibiotic resistance of each microorganism, the method comprising the steps of: a) selecting a plurality of nucleic acid probes, including at least one probe for detecting a species of microorganism, at least one probe for detecting a virulence of a microorganism, and at least one probe for detecting an antibiotic resistance of a microorganism; and either b) applying each probe onto a different, discrete location of a substrate; or c) synthesizing each probe at a different, discrete location upon a substrate. Any methods may be used to apply probes upon a substrate of a DNA array, or to synthesize oligonucleotide probes upon the substrate, that are known in the art, and described for example in references cited herein.
  • the DNA microarrays or chips of the invention may generally include a solid substrate or support, and an array of oligonucleotide probes immobilized on the substrate.
  • the substrate can be, for example, silicon or glass, and can have the thickness of a glass microscope slide or a glass cover slip. Substrates that are transparent to light are useful when the method of performing an assay on the chip involves optical detection. Suitable substrates include a slide, chip, wafer, membrane, filter, sheet and bead.
  • the substrate can be porous or have a non-porous surface.
  • oligonucleotides are arrayed on the substrate in addressable rows and columns.
  • a "subarray” may thus be designed which comprises a particular grouping of probes at a particular area of the array, the probes immobilized at adjacent locations or within a defined region of the array.
  • a hybridization assay is performed to determine whether a target DNA molecule has a sequence that is complementary to one or more of the probes immobilized on the substrate. Because hybridization between two nucleic acids is a function of their sequences, analysis of the pattern of hybridization provides information about the sequence of the target molecule. DNA chips are useful for discriminating variants that may differ in sequence by as few as one or a few nucleotides.
  • Hybridization assays on the DNA chip involve a hybridization step and a detection step.
  • a hybridization mixture containing the labeled target nucleic acid sequence is brought into contact with the probes of the array and incubated at a temperature and for a time appropriate to allow hybridization between the target and any complementary probes.
  • the array may optionally be washed with a wash mixture which does not contain the target (e.g. hybridization buffer) to remove unbound target molecules, leaving only bound target molecules.
  • the probes to which the target has hybridized are identified. Since the nucleotide sequence of the probes at each feature is known, identifying the locations at which target has bound provides information about the particular sequences of these probes.
  • Hybridization may be carried out under various conditions depending on the circumstances and the level of stringency desired. Such factors shall depend on the specificity and degree of differentiation between target sequences for any given analysis. For example, to distinguish target sequences which differ by only one or a few nucleotides, conditions of higher stringency are generally desirable. Stringency may be controlled by factors such as the content of hybridization and wash solutions, the temperature of hybridization and wash steps, the number and duration of hybridization and wash steps, and any combinations thereof. In embodiments, the hybridization may be conducted at temperatures ranging from about 4° C. up to about 80° C, depending on the length of the probes, their G+C content and the degree of divergence to be detected.
  • denaturing reagents such as formamide may be used to decrease the hybridization temperature at which perfect matches will dissociate.
  • Commonly used conditions involve the use of buffers containing about 30% to about 50% formamide at temperatures ranging from about 20° C. to about 50° C.
  • An example of such a partially denaturing buffer which is commercially available is the DIG Easy HybTM (Roche) buffer.
  • un-labelled nucleic acids such as transfer RNA (tRNA) and salmon sperm DNA may be added to the hybridization buffers to reduce background noise. Under certain conditions, a divergence of 15% over long fragments (greater than 50 bases) can be reliably detected.
  • Hybridization time typically ranges from about one hour to overnight (16 to 18 hours approximately).
  • microarrays are typically washed one to five times in buffered salt solutions such as saline-sodium citrate, abbreviated SSC, for periods of time and at salt concentrations and temperature appropriate for a particular objective.
  • a representative procedure may for example comprise three washes in pre-warmed (50° C.) 0.1 *SSC (1 xSSC contains 150 mM NaCl and 15 mM trisodium citrate, pH 7).
  • a detergent such as sodium dodecyl sulfate [SDS; e.g. at 0.1% (w/v)] may be added to the washing buffer.
  • SDS sodium dodecyl sulfate
  • Hybridization may be performed under absolute or differential formats.
  • the former refers to hybridization of nucleic acids from one sample to an array, and the detection of the nucleic acids thus hybridized.
  • the differential hybridization format refers to the application of two samples, labeled with different labels (e.g. Cy3 and Cy5 fluorophores), to the array. In this case differences and similarities between the two samples may be assessed.
  • Many steps in the use of the DNA chip can be automated through use of commercially available automated fluid handling systems.
  • the chip can be manipulated by a robotic device which has been programmed to set appropriate reaction conditions, such as temperature, add reagents to the chip, incubate the chip for an appropriate time, remove unreacted material, wash the chip substrate, add reaction substrates as appropriate and perform detection assays.
  • appropriate reaction conditions such as temperature, add reagents to the chip, incubate the chip for an appropriate time, remove unreacted material, wash the chip substrate, add reaction substrates as appropriate and perform detection assays.
  • the chip can be appropriately packaged for use in an automated chip reader.
  • the target polynucleotide whose sequence is to be determined is usually labeled at one or more nucleotides with a detectable label (e.g. detectable by spectroscopic, photochemical, biochemical, chemical, bioelectronic, immunochemical, electrical or optical means).
  • the detectable label may be, for instance, a luminescent label.
  • Useful luminescent labels include fluorescent labels, chemi-luminescent labels, bio-luminescent labels, and colorimetric labels, among others.
  • the label is a fluorescent label such as a cyanine, a fluorescein, a rhodamine, a polymethine dye derivative, a phosphor, and so forth.
  • Suitable fluorescent labels are described in for example Haugland, Richard P., 2002 (Handbook of Fluorescent Probes and Research Products, ninth edition, Molecular. Probes).
  • the label may be a light scattering label, such as a metal colloid of gold, selenium or titanium oxide. Radioactive labels such as 32 P, 33 P or 35 S can also be used.
  • the target strand is prepared in single-stranded form, the sense of the strand should be complementary to that of the probes on the chip.
  • the target is fragmented before application to the chip to reduce or eliminate the formation of secondary structures in the target. Fragmentation may be effected by mechanical, chemical or enzymatic means. The average size of target segments following fragmentation is usually larger than the size of probe on the chip.
  • the target or sample nucleic acid may be extracted from a sample or otherwise enriched prior to application to or contacting with the array.
  • Samples may amplified by suitable methods, such as by culturing a sample in suitable media (e.g. Luria-Bertani media) under suitable culture conditions to effect growth of microorganisms in the sample. Extraction may be performed using methods known in the art, including various treatments such as lysis (e.g. using lysozyme), heating, detergent (e.g. SDS) treatment, solvent (e.g. phenol-chloroform) extraction, and precipitation/resuspension.
  • the nucleic acid is not amplified using polymerase chain reaction (PCR) methods prior to application to the array.
  • PCR polymerase chain reaction
  • the probes may be provided, for example as a suitable solution, and applied to different, discrete regions of the substrate. Such methods are sometimes referred to as "printing” or “pinning", by virtue of the types of apparatus and methods used to apply the probe samples to the substrate. Suitable methods are described in for example U.S. Pat. No. 6,110,426 to Shalon et al.
  • the probe samples may be prepared by a variety of methods, including but not limited to oligonucleotide synthesis, as a PCR product using specific primers, or as a fragment obtained by restriction endonuclease digestion of a nucleic acid sample. Interaction/binding of the probe to the substrate may be enforced by non-covalent interactions and covalent attachment, for example via charge-mediated interactions as well as attachment to the substrate via specific reactive groups, crosslinking and/or heating.
  • the arrays may be produced by, for example, spatially directed oligonucleotide synthesis.
  • Methods for spatially directed oligonucleotide synthesis include, without limitation, light-directed oligonucleotide synthesis, microlithography, application by ink jet, microchannel deposition to specific locations and sequestration with physical barriers. In general these methods involve generating active sites, usually by removing protective groups; and coupling to the active site a nucleotide which, itself, optionally has a protected active site if further nucleotide coupling is desired.
  • the probes can be bound to the substrate through a suitable linker group. Such groups may provide additional exposure to the probe.
  • Such linkers are adapted to comprise a terminal portion capable of interacting or reacting with the substrate or groups attached thereto, and another terminal portion adapted to bind/attach to the probe molecule.
  • Samples of interest e.g. samples suspected of comprising a microorganism, for analysis using the products and methods of the invention include for example environmental samples, biological samples and food.
  • Environmental sample refers to any medium, material or surface of interest (e.g. water, air, soil).
  • Biological sample as used herein refers to a sample obtained from an organism, including tissue, cells or fluid. Biological excretions and secretions (e.g. feces, urine, discharge) are also included within this definition.
  • Such biological samples may be derived from a patient, such as an animal (e.g. vertebrate animal, humans, domestic animals, veterinary animals and animals typically used in research models). Biological samples may further include various biological cultures and solutions.
  • the probes utilized herein may in embodiments comprise a nucleotide sequence identical to a nucleic acid derived from a microorganism or substantially identical, homologous or orthologous to such a nucleic acid.
  • "Homology” and “homologous” refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing each position in the aligned sequences.
  • a degree of homology between nucleic acid or between amino acid sequences is a function of the number of identical or matching nucleotides or amino acids at positions shared by the sequences.
  • a nucleic acid sequence is "homologous" to another sequence if the two sequences are substantially identical and the functional activity of the sequences is conserved (as used herein, the term 'homologous' does not infer evolutionary relatedness as orthologous does).
  • Two nucleic acid sequences are considered “substantially identical” if, when optimally aligned (with gaps permitted), they share at least about 50% sequence similarity or identity, or if the sequences share defined functional motifs.
  • sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90% or 95%.
  • a given percentage of homology between sequences denotes the degree of sequence identity in optimally aligned sequences.
  • An "unrelated" or “non-homologous” sequence shares less than 40% identity, though preferably less than about 25% identity, with a sequence of interest.
  • Substantially complementary nucleic acids are nucleic acids in which the "complement" of one molecule is substantially identical to the other molecule.
  • Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv Appl Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J MoI Biol 48:443, the search for similarity method of Pearson and Lipman, 1988, Pr oc Natl Acad Sci USA 85: 2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis., U.S.A.).
  • Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al, 1990, J MoI Biol 215:403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (through the internet at http://www.ncbi.nim.nih.gov/).
  • the BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold.
  • Initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the BLAST program may use as defaults a word length (WV) of II, the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad.
  • nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO 4 , 7% (w/v) sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C, and washing in 0.2 ⁇ SSC/0.1% (w/v) SDS at 42° C. (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3).
  • hybridization to filter- bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO 4 , 7% (w/v) SDS, 1 mM EDTA at 65 *C, and washing in 0.1 ⁇ SSC/0.1% (w/v) SDS at 68° C. (see Ausubel, et al. (eds), 1989, supra).
  • Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, N. Y.).
  • stringent conditions are selected to be about 5° C. lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
  • Table 1 List of cpn60 probes for identification of the bacterial pathogens SEQ Histophilus AAT TGA ACA GGT TGG TAC AAT TTC CGC Histophilu Histophilus NZ AAB 429-
  • Table 3 List of antimicrobial agent gene probes for the Gram-positive bacterial pathogens.
  • Table 4 List of antimicrobial agent resistance gene probes for the Gram-negative bacterial pathogens.

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Abstract

On utilise des réseaux d'ADN pour détecter la présence (ou l'absence) d'acides nucléiques dans des échantillons. Les réseaux d'ADN de l'invention permettent le criblage d'échantillons biologiques d'origine humaine ou animale pour y détecter la présence d'au moins un micro-organisme et en déterminer l'espèce, la virulence, la résistance aux antibiotiques. L'invention porte également sur des méthodes de criblage d'échantillons, sur des méthodes de diagnostic de patients et d'établissement de traitements anti-infectieux.
PCT/CA2007/001326 2006-07-26 2007-07-26 Identification et caractérisation de micro-organismes Ceased WO2008011715A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016051177A3 (fr) * 2014-10-01 2016-05-19 Arcis Biotechnology Holdings Limited Procédés et kits
EP3305917A1 (fr) * 2014-10-01 2018-04-11 Arcis Biotechnology Holdings Limited Procédés et kits
US10711264B2 (en) 2014-10-01 2020-07-14 Arcis Biotechnology Holdings Limited Methods and kits
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