Patent Application
20060094034
The invention relates to an array and uses thereof and particularly relates to an array for characterizing a microorganism by its virulence and antibiotic resistance, and uses thereof.
A variety of pathogenic microorganisms exist, which pose a continued health threat. An example is the bacterium Escherichia coli, which is commonly found in the environment as well as in the digestive tracts of common animal species including humans. Individual strains within Escherichia coli (E. coli) can vary in pathogenicity from innocuous to highly lethal, as evidenced by incidents of its contamination of drinking water and outbreaks of so-called hamburger disease. Pathogenic forms of Escherichia coli (E. coli) are a worldwide cause of urinary tract infections, intestinal infections as well as septicemia and nosocomial infections. It is important that medicine can intervene effectively. One of medicine's arms against the E. coli infections is the use of antibiotics. However, an increase of antibiotic resistance is observed among E. coli strains. There are well over one hundred genes known to be directly involved in determining the degree and type of antibiotic resistance of E. coli. There is currently no practical, cost-effective way to determine rapidly and simultaneously the presence or the absence of this large set of these antibiotic resistance genes within a given E. coli strain. The genetic methods like genome analysis with DNA chips provide key information for guiding antibiotic therapy. But the most important problem is that presently, no technical product is offered to rapidly and simultaneously detect many resistance genes and mutations in a single step.
The pathogenicity of a given E. coli depends on the presence or absence of virulence genes within its genome. These virulence genes are ideal targets for the determination of the pathogenicity potential of any given E. coli isolate.
For virulence, the presence of virulence genes and the pathogenic behavior (so-called pathotype) are established by various combinations of microbiological methods including bacterial culture, immunoassay, tissue culture methods, PCR and microscopic analysis of biopsy samples. The same comments about slowness and expense apply here as well.
The above methods have been used for detecting and identifying pathogenic E. coli. However, these approaches suffer from a variety of limitations, the most serious of which is related to the large variety of virulence factors distributed among the known pathotypes. Currently, there is no practical, cost-effective way to determine rapidly and simultaneously the presence or absence of this large set of these virulence genes within a given E. coli strain.
For antibiotic resistance, basic microbiology tests (disk diffusion, broth dilution, agar dilution, and gradient diffusion) are the principal approach to get the phenotype of resistance rapidly. The bacteria have to be isolated and cultured before testing. Detection of antibiotic resistance genes can be accomplished with Polymerase Chain Reaction (PCR) amplification of target DNA and amplicon confirmation by gel electrophoresis and by probe hybridization techniques. Detection of gene mutations associated with antimicrobial resistance can be possible with the use of PCR-RFLP analysis, PCR-SSCP analysis, PCR-CFLP analysis, PCR-RNA combined with RNase cleavage assay, PCR amplification combined with DNA sequencing or with microarray analysis. The majority of these assays are impossible to do in one step, so the procedures are slow, complex and expensive.
A major drawback of the basic microbiology tests is that they are slow and tests give information about the phenotype only. There are also problems with other tests used to detect antibiotic resistance genes. First, they lack sensitivity when only a few organisms are present in the sample or when inhibitors are also present. Second, different assays are required for each antimicrobial agent tested or gene tested. False-positive results may occur due to contamination of the test sample with extraneous nucleic acid or residual nucleic acid from prior samples. The general situation of the tests used to detect mutations associated with antimicrobial, resistance is that the assays are insensitive, complex, slow, costly and may require several steps. A similar situation prevails for virulence genes.
Some publications show that DNA microarrays have been used for the detection of mutation associated with antimicrobial resistance of Mycobacterium tuberculosis. There are also publications that note that microarrays have been used for the detection of two resistance genes of the non pathogenic yeast Saccharomyces cerevisiae, for the detection of one resistance gene of M. tuberculosis, but not for pathogens having a large number of antibiotic resistance and virulence genes such as E. coli strains.
The published procedures for antibiotic resistance gene analysis and for virulence gene analysis using DNA microarrays all suffer from significant drawbacks and cannot currently be considered practical or cost-effective.
It would therefore be desirable to have improved methods and materials for the detection of pathogenic microorganisms, such as bacteria (e.g. E. coli).
The invention relates to a collection of probes, e.g. in an array format, and uses thereof.
According to one aspect of the invention there is provided an apparatus for the simultaneous detection in a pathogen or in a liquid sample containing an unknown pathogen, of a plurality of antibiotic resistance and virulence genes, comprising a microarray, DNA probes e.g. synthetic oligonucleotides complementary for a plurality of currently known antibiotic resistance genes and virulence genes for a pathogen e.g. E. coli having such a plurality of known antibiotic resistance genes and virulence genes, immobilized on the microarray.
According to another aspect of the invention, a method is provided for simultaneous detection of a plurality of antibiotic resistance and virulence genes in a given liquid culture or colony of pathogen for the presence of these resistance and virulence genes comprising;
Accordingly, in one aspect, the invention provides an array comprising: a substrate and a plurality of nucleic acid probes, each of the probes being bound to the substrate at a discrete location; the plurality of probes comprising at least one probe for at least one antibiotic resistance gene of a species of a microorganism and at least another probe for at least one virulence gene of the species. In an embodiment, the array comprises at least 103 distinct nucleic acid probes. In embodiments, each of the probes are independently greater than or equal to 15, 20, 50 or 100 nucleotides in length. In an embodiment, the array comprises a subarray, wherein the subarray comprises the at least two probes at adjacent discrete locations on the substrate.
In an embodiment, the microorganism is a bacterium, in a further embodiment, of the family Enterobacteriaceae, in a further embodiment, the bacterium is E. coli.
In an embodiment, the virulence gene can be one that codes for a pathotype selected from the group consisting of: enterotoxigenic E. coli (ETEC); enteropathogenic E. coli (EPEC); enterohemorrhagic E. coli (EHEC); enteroaggregative E. coli (EAEC); enteroinvasive E. coli (EIEC); uropathogenic strains (UPEC); E. coli strains involved in neonatal meningitis (MENEC); E. coli strains involved in septicemia (SEPEC); cell-etaching E. coli (CDEC); and diffusely adherent E. coli (DAEC).
In an embodiment, the virulence gene encodes a polypeptide of 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. In an embodiment, the virulence gene is selected from the group consisting of afaBC3, afaE5, afaE7, afaD8, aggA, aggC, aida, bfpA, bmaE, cdt1, cdt2, cdt3, cfaI, clpG, cnf1, cnf2, cs1, cs3, cs31a, cvaC, derb122, eae, eaf, east1, ehxA, espA group I, espA group II, espA group III, espB group I, espB group II, espB group III, espC, espP, etpD, F17A, F17G, F18, F4, F41, F5, F6, fimA group I, fimA group II, fimH, mC, focG, fyuA, hlyA, hlyC, ibe10, iha, invX, ipaC, iroN, irp1, irp2, iss, iucD, iufA, katP, kfiB, kpsMTII, kpsMTIII, 17095, leoA, IngA, It, neuC, nfaE, ompA, ompT, paa, papAH, papC, papEF, papG group I, papG group II, papG group III, pai, rtbO9, rfbO101, rfbO111, rfbE 0157, rfbE O157H7, rfc O4, rtx, sfaDE, sfaA, stah, stap, stb, stx1, stx2, stxA I, stxA II, stxB I, stx B II, stxB III, tir group I, tir group II, tir group III, traT, and tsh genes. In an embodiment, the above-noted probe comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:102, or a fragment thereof, or a sequence substantially identical thereto. In the present invention, complete identity of the probes with the DNA to be detected is not essential, as partial identity or homology for detecting hybridization of the probes with the DNA to be detected can be sufficient. One skilled in the art will appreciate that by varying the hybridization conditions and the percentage of homology, same results can be achieved, depending on the selectivity or sensitivity desired for the array.
In an embodiment, the substrate is selected from the group consisting of a porous support and a support having a non-porous surface. In embodiments the support is selected from the group consisting of a slide, chip, wafer, membrane, filter and sheet. In an embodiment, the slide comprises a coating capable of enhancing nucleic acid immobilization to the slide. In an embodiment, the probes are covalently attached to the substrate.
The invention further provides a method of detecting the presence of a microorganism in a sample, the method comprising: contacting the above-mentioned array with a sample nucleic acid of the sample; and detecting association of the sample nucleic acid to a probe on the array; wherein association of the sample nucleic acid with the probe is indicative that the sample comprises a microorganism from which the nucleic acid sequence of the probe is derived. In an embodiment, the sample nucleic acid comprises a label. In an embodiment, the label is a fluorescent dye (e.g. a cyanine, a fluorescein, a rhodamine and a polymethine dye derivative). In an embodiment, the method further comprises extracting the sample nucleic acid from the sample before contacting it with the array. In an embodiment, the sample nucleic acid is not amplified by PCR prior to contacting it with the array. In an embodiment, the method further comprises digesting the sample nucleic acid with a restriction enzyme to produce fragments of the sample nucleic acid prior to contacting with the array. In an embodiment, the fragments are of an average size of about 0.2 Kb to about 12 Kb. In an embodiment, the method further comprises labeling the sample nucleic acid prior to contacting it with the array. In an embodiment, the sample nucleic acid is selected from the group consisting of DNA and RNA.
In an embodiment, the above-mentioned sample is selected from the group consisting of environmental samples, biological samples and food. In an embodiment, the environmental samples are selected from the group consisting of water, air and soil. In an embodiment, the biological samples are selected from the group consisting of blood, urine, amniotic fluid, feces, tissues, cells, cell cultures and biological secretions, excretions and discharge.
In an embodiment, the method is further for determining a pathotype and an antibiotic resistance of a species of the microorganism, wherein the probes are for a pathotype and an antibiotic resistance of the species and wherein association of the sample nucleic acid with the probes is indicative that the microorganism is of the pathotype and is resistant to the antibiotic tested.
In an embodiment, the sample is a tissue, body fluid, secretion or excretion from a subject and the method is further for diagnosing an infection by the microorganism in the subject, wherein association of the nucleic acid with the probe is indicative that the subject is infected by the microorganism.
In an embodiment, the method is for diagnosing a condition related to infection by the microorganism in the subject, wherein the probe is for a pathotype of the species and wherein association of the sample nucleic acid with the probe is indicative that the microorganism is of the pathotype and is antibiotic resistant and that the subject suffers from a condition associated with the pathotype. In an embodiment, the condition is selected from the group consisting of: diarrhea, hemorrhagic colitis, hemolytic uremic syndrome, invasive intestinal infections, dysentery, urinary tract infections, neonatal meningitis and septicemia. In an embodiment, the subject is a mammal, in a further embodiment, a human.
The invention further provides a commercial package comprising the above-mentioned array together with instructions for: (a) detecting the presence of a microorganism in a sample; (b) determining the pathotype of a microorganism in a sample; (c) determining antibiotic resistance of a microorganism in a sample; (d) diagnosing an infection by a microorganism in a subject; (e) diagnosing a condition related to infection by a microorganism, in a subject; or (f) any combination of (a) to (e).
The invention further provides a use of the above-mentioned array for: (a) detecting the presence of a microorganism in a sample; (b) determining the pathotype of a microorganism in a sample; (c) determining antibiotic resistance of a microorganism in a sample; (d) diagnosing an infection by a microorganism in a subject; (e) diagnosing a condition related to infection by a microorganism, in a subject; or (f) any combination of (a) to (e).
The invention further provides a method of producing an array for phenotyping a microorganism in a sample by its pathotype and antibiotic resistance, the method comprising: providing a plurality of nucleic acid probes, the plurality of probes comprising at least one probe for at least one antibiotic resistance gene of a species of the microorganism and at least one other probe for at least one pathotype of the species; and applying each of the probes to a different discrete location of a substrate. In an embodiment, the method further comprises the step of cross-linking by exposure of the array to ultraviolet radiation. In an embodiment, the method further comprises heating the array subsequent to the cross-linking.
The invention further provides a method of producing an array for phenotyping a microorganism in a sample by its pathotype and antibiotic resistance, the method comprising: selecting a plurality of nucleic acid probes, the plurality of probes comprising at least one probe for a first pathotype of a species of the microorganism and at least another one probe for detecting an antibiotic resistance gene of the species; and synthesizing or immobilizing each of the plurality of probes at a different discrete location of a substrate.
The invention combines the parallel processing power inherent in DNA microarrays with a very effective and robust labeling methodology, plus an optimized design of immobilized DNA probes to achieve practicality, robustness and cost effectiveness. Such a combination has not, to the inventors' knowledge, been reported in either the patent or scientific literature.
With regard to antimicrobial resistance, there are several reasons to pursue the identification of antibiotic resistance genes or mutations associated with antibiotic resistance in pathogens with DNA microarrays. First, DNA microarrays are helpful for arbitrating results which come from regular microbiology tests that are at or near the breakpoint for resistance for pathogenic species. Second, DNA microarrays can be used to detect resistance genes or mutations that result in resistance in organisms directly in clinical specimens to guide therapy early in the course of a patient's disease long before culture are positive. Third, DNA microarrays are more accurate than antibiograms for following the epidemiologic spread of a particular resistance gene in a hospital or a community setting.
The lower cost, higher reliability and increased flexibility of the new approach described herein, together with the combination of virulence and antibiotic resistance gene probes on the same array, amount to a breakthrough in usability and practicality.
The method used for fabricating microarrays (except for the material affixed to the microarrays) is substantially that described by U.S. Pat. No. 6,110,426, the disclosure of which is incorporated herein by reference.
The basic concept of the DNA microarray as applied to antimicrobial resistance and virulence genes detection is as following. A bacterial sample 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 microorganisms that may be present therein. When microorganisms are grown first, DNA is then extracted and labeled with 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 an antibiotic resistance and virulence gene, DNA microarray. The fluorescent DNA will stick (by hybridization) wherever a complementary probe for antibiotic resistance or virulence gene matches its DNA sequence. Since the order and position of the probes is precisely determined, the content of antibiotic resistance genes and virulence genes in the initial sample is fully determined.
The present invention provides products and methods for the detection and characterization of microorganisms, such as bacteria, (e.g. of the family Enterobacteriaceae) such as E. coli. The products and methods of the invention can be used to detect the presence of such a microorganism in a sample (e.g. a biological or environmental sample). Further, such products and methods can be used to characterize such a microorganism, e.g. determining/characterizing its pathotype (virulence) and antibiotic resistance.
Pathogenic E. coli are responsible for three main types of clinical infections (a) enteric/diarrheal disease (b) urinary tract infections and (c) sepsis/meningitis. On the basis of their distinct virulence properties and clinical symptoms of the host, pathogenic E. coli are divided into numerous categories or pathotypes. The diarrheagenic E. coli include (i) enterotoxigenic E. coli (ETEC) associated with traveller's diarrhea and porcine and bovine diarrhea, (ii) enteropathogenic E. coli (EPEC) causing diarrhea in children and animals, (iii) enterohemorrhagic E. coli (EHEC) associated with hemorrhagic colitis and hemolytic uremic syndrome in humans, (iv) enteroaggregative E. coli (EAEC) associated with persistent diarrhea in humans, and (v) enteroinvasive E. coli (EIEC) involved in invasive intestinal infections, watery diarrhea and dysentery in humans and animals (Nataro, J. P., et al. (1998) Clin Microbiol Rev. 11:142-201). Extra-intestinal infections are caused by three separate E. coli pathotypes (i) uropathogenic strains (UPEC) that cause urinary tract infections in humans, dogs and cats (Beutin, L. (1999) Vet Res. 30:285-298; Garcia, E., et al. (1988) Antonie Van Leeuwenhoek. 54:149-163; and Wilfert, C. M. (1978) Annu Rev Med. 29:129-136) (ii) strains involved in neonatal meningitis (MENEC) (Wilfert, C. M. (1978) Annu Rev Med. 29:129-136) and (iii) strains that cause septicemia in humans and animals (SEPEC) (Dozois, C. M., et al. (1997) FEMS Microbiol Lett. 152:307-312; Harel, J., et al. (1993) Vet Microbiol. 38:139-155; Martin, C., et al. (1997) Res Microbiol. 148:55-64; and Wilfert, C. M. (1978) Annu Rev Med. 29:129-136).
Numerous bioassays and molecular methods have been developed for the detection of genes involved in pathogenic E. coli virulence mechanisms. However, the sheer numbers of known virulence factors have made this a daunting task. As described herein, microarray technology offers the most rapid and practical tool to detect the presence or absence of a large set of virulence genes simultaneously within a given E. coli strain. Prior to applicants' findings herein, only a few studies have reported the use of microarrays as a diagnostic tool (Call, D. R., et al. (2001) Int J Food Microbiol. 67:71-80; Chizhikov, V., et al. (2001) Appl Environ Microbiol. 67:3258-3263; Cho, J. C., et al. (2001) Appl Environ Microbiol. 67:3677-3682; Li, J., et al. (2001) J Clin Microbiol. 39:696-704; and Murray, A. E., et al. (2001) Proc Natl Acad Sci USA. 98:9853-9858). Described herein is a new approach for detection of a large number of virulence and antibiotic resistance factors present in E. coli strains and the subsequent determination of the strain's pathotype and antibiotic resistance. As described herein, nucleic acid sequences derived from most known virulence and antibiotic factors including associated-virulence genes and antibiotic resistance genes were amplified by PCR and immobilized onto glass slides to create a virulence and antibiotic resistance DNA microarray chip. Probing this virulence/antibiotic resistance gene microarray with labeled genomic E. coli DNA, the virulence and antibiotic resistance patterns of a given strain can be assessed and its pathotype determined in a single experiment.
As a practical example in support of this invention, an E. coli virulence and antibiotic resistance factor microarray was designed and tested. It was of course recognized that applications of this microarray reach far into human health, drinking water and environmental research.
According to another aspect of the invention, a method is provided for analyzing a given liquid culture or colony of bacteria simultaneously for the presence of a number of these virulence and antibiotic resistance genes in the same experiment.
In one embodiment, an array of virulence and antibiotic resistance genes may be used by reference laboratories involved in public or veterinary health. A simplified format of the microarray focusing on a few key virulence and antibiotic resistance genes could find a broader market in routine medical or veterinary microbiological laboratory work.
Other types of virulence and antibiotic resistance genes may be represented on such an array for a variety of applications. For example, the armed forces may be interested in implementing this type technology for detection and/or identification of biological warfare agents.
The invention thus relates to products and methods which enable the parallel analysis in respect of a plurality of pathotypes of a microorganism, and possibly of various antibiotic resistance, via the use of a collection of a plurality of nucleic acid probes derived from virulence and antibiotic resistance genes of the microorganism, the collection corresponding to a plurality of pathotypes and antibiotic resistance patterns of the microorganism. In an embodiment, the plurality of pathotypes may comprise at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 pathotypes. In an embodiment, the plurality of antibiotic resistance patterns may comprise at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 antibiotic resistance genes.
Accordingly, in an aspect, the invention relates to a collection comprising a plurality of probes, the probes being derived from genetic/protein (e.g. a virulence and antibiotic resistance genes) material/information from a microorganism and correspond to a plurality of pathotypes and antibiotic resistance patterns of the microorganism. In an embodiment, the probes comprise a nucleic acid sequence derived from a microorganism or a sequence substantially identical thereto. In an embodiment, the collection can represent more than one microorganism.
“Pathotype” as used herein 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. disease symptoms) when they infect a subject. In the case of E. coli, for example, pathotypes may include those associated with intestinal and extraintestinal conditions. Such pathotypes include but are not limited to ETEC, EPEC, EHEC, EAEC, EIEC, UPEC, MENEC, SEPEC, CDEC and DAEC noted herein. As described herein, a pathotype may be identified and/or characterized using a probe based on a virulence gene associated with the pathotype, in a particular microorganism (See Table 1).
“Virulence gene” as used herein refers to a nucleic acid sequence of a microorganism, the presence and/or expression of which correlates with the pathogenicity of the microorganism. In the case of bacteria, such 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). In the case of E. coli, examples of virulence genes and classes of polypeptides encoded by such genes are described below. Virulence genes for a variety of pathogenic microorganisms are known in the art.
The term probe as used herein is intended to mean 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.
In an embodiment, the above-noted collection is in the form of an array, whereby the probes are bound to different, discrete locations of a substrate. The length of the probes may be variable, e.g. at least 15, 20, 50, 100, 500, 1000 or 2000 nucleotides in length. High density nucleic acid probe arrays, also referred to as “microarrays,” may for example be used 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. In one aspect, the invention provides biological assays using such high density nucleic acid or protein probe arrays. For the purpose of such arrays, “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. A variety of methods are known for making and using microarrays, as for example disclosed in Cheung, V. G. et al. (1999) Nature Genetics Supplement, 21, 15-19; Lipshutz, R. J. et al., (1999) Nature Genetics Supplement, 21, 20-24; Bowtell, D. D. L. (1999) Nature Genetics Supplement, 21, 25-32; Singh-Gasson, S. et al. (1999) Nature Biotechnol. 17, 974-978; and, Schweitzer, B. et al. (2002) Nature Biotechnol. 20, 359-365; all of which are incorporated herein by reference. DNA chip technology is described in detail in, for instance, U.S. Pat. No. 6,045,996 to Cronin et al., U.S. Pat. No. 5,858,659 to Sapoisky et al., U.S. Pat. No. 5,843,655 to McGall et al., U.S. Pat. No. 5,837,832 to Chee et al., and U.S. Pat. No. 6,110,426 to Shalon et al., all of which are specifically incorporated herein by reference. Suitable DNA chips are available for example from Affymetrix, Inc. (Santa Clara, Calif.).
In another embodiment, a 70-mer oligonucleotide microarray was developed in order to determine simultaneously the presence or absence of a large set of virulence and antimicrobial resistance genes withinm including closely-related variants, within a given E. coli isolate. This embodiment contains oligonucleotides designed from the previous virulence midroarray, oligonucleotides specific for antimicrobial resistance genes previously characterized in various E. coli strains, and oligonucleotides specific for new putative virulence genes described in E. coli. 70-mer oligonucleotides were preferred to amplicons on the basis of earlier results obtained with amplicon-based microarrays, which found that amplicon probes had a high potential to cross-hybridize while oligonucleotide probes were more specific. Indeed, contrary to amplicon-based microarray and other molecular methods, such as membrane hybridizations, no cross-hybridization was observed between genes showing a high percentage of identity in their nucleic sequences. As an example, the absence of cross-hybridization, confirmed by PCR, between tetC and tetA genes, which show more than 75 percent of identity in their nucleic sequence, features the 70-mer oligonucleotide microarray specificity (see
Two hundred and ninety one 70-mer oligonucleotides were designed for the elaboration of the virulence and antibiotic resistance array (see Table 7). Thirty three of them correspond to 30 antimicrobial resistance genes characteristically found in E. coli strains and to the class 1 integron. Because of one false positive result obtained with the first oligonucleotide specific for class 1 integron, I have designed 2 new 70-mer oligonucleotides. These two ones, int1(2) and int1(3), were respectively specific for the conserved region (qacEdelta1) and for the integrase gene of the class 1 integron. The 258 other oligonucleotides were designed either from the previous virulence amplicon-based microarray or correspond to new putative virulence genes recently described in E. coli strains. Among them, four were specific for bacterial species (lacY-Ec for E. coli, lacY-Cf for Citrobacter freundii, Sf0315 and Sf3004 for Shigella flexnen), three were positive controls (lacZ, uldA and tnaA), and two were negative controls (gfp and Arabidopsis thaliana) (
For antimicrobial resistance genes and virulence genes from the previous virulence microarray, oligonucleotides were designed either from published PCR primers which were lengthened to 70 bases, or designed using the software program “OligoPicker”(Wang and Seed, 2003). For all of the new virulence genes or associated-virulence genes, the (public domain) “OligoPicker” software was used to design oligonucleotides. When different variants were found for a single gene, multiple alignments and phylogenetic analysis were performed to identify variant-specific probes. When 10% of divergence or more was observed between the DNA sequence of two variants, one oligonucleotide was designed for each one. Compared to the previous virulence amplicon-based microarray, this particular embodiment adds 59 oligonucleotides specific for fimbrial or afimbrial adhesins genes (30) or gene variants (29), 13 oligonucleotides specific for colicin genes and 7 oligonucleotides specific for microcins, 18 oligonucleotides specific for the different eae (intimine) gene variants, 8 oligonucleotides specific for toxins genes or gene variants, 29 oligonucleotides specific for various virulence genes or gene variants recently described in E. coli, and 6 oligonucleotides specific for putative new virulence genes.
As shown in
Validation of the oligonucleotide microarray took advantage of the availability of full genome sequences from thee references together with our large collection of characterized E. coli isolates. DNA from the three E. coli reference strains EDL933 (EHEC), CFT073 (UPEC) and MG1655 (K12), and from a collection of 20 well-characterized E. coli isolates (strains characterized with the previous virulence amplicon-based microarray or by membrane hybridizations) was hybridized to the oligonucleotide microarray. Hybridizations with these known labeled genomic DNA validated our microarray as a powerful tool for the detection of virulence and antimicrobial resistance genes in E. coli isolates. As shown in
Methods for storing, querying and analyzing microarray data have for example been disclosed in, for example, U.S. Pat. No. 6,484,183 issued to Balaban, et al. Nov. 19, 2002; and U.S. Pat. No. 6,188,783 issued to Balaban, et al. Feb. 13, 2001; Holloway, A. J. et al., (2002) Nature Genetics Supplement, 32, 481-489; each of which is incorporated herein by reference.
DNA chips 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. Preferably, 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. In the hybridization 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. In the detection step, 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. If desired, denaturing reagents such as formamide may 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 Hyb™ (Roche) buffer. In embodiments, 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. Single nucleotide mistmatches in shorter fragments (15 to 25 nucleotides in length) can be also detected if the hybridization conditions are designed accordingly. Hybridization time typically ranges from about one hour to overnight (16 to 18 hours approximately). After hybridization, 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×SSC contains 150 mM NaCl and 15 mM trisodium citrate, pH 7). In embodiments, a detergent such as sodium dodecyl sulfate [SDS; e.g. at 0.1% (w/v)] may be added to the washing buffer. Various details of hybridization conditions, some of which are described herein, are known in the art.
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. For instance, 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. If desired, 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. Most preferably, 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 32P, 33P or 35S can also be used.
When 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. In an embodiment, 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.
In embodiments, 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. In an embodiment, the nucleic acid is not amplified using polymerase chain reaction (PCR) methods prior to application to the array.
In an embodiment, 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.
In an embodiment, 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.
In embodiments, 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” as used herein 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. As the term is used herein, 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. In alternative embodiments, sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90% or 95%. As used herein, 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. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. 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. Mol. 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. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10 (or 1 or 0.1 or 0.01 or 0.001 or 0.0001), M=5, N=4, and a comparison of both strands. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments of the invention, 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.
An alternative indication that two nucleic acid sequences are substantially complementary is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, conditions. Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M NaHPO4, 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). Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO4, 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.). Generally, 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.
The above pre-existing elements were combined for the first time into a unique combination that surpasses others in terms of defining a robust, straightforward, practical and above all useable procedure. No similar work exists in the literature to the inventors' knowledge.
The present invention fully solves the problem by using synthetic oligonucleotides as gene probes. Additionally, the juxtaposition of antibiotic resistance genes and virulence genes on the same microarray greatly increases the usefulness of the Invention by simultaneously providing two independent sets of very important data.
Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Numeric ranges are inclusive of the numbers defining the range. In the claims, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to”. The following examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein.
The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
Strains and Media
E. coli strains used to produce PCR templates are listed in Table 2. E. coli isolates including characterized strains (the non-pathogenic K12-derived E. coli strain DH5α, the enterohemorrhagic strain EDL933, the uropathogenic strain J96, the enterotoxigenic strain H-10407 and the enteropathogenic strains E2348/69 and P86-1390) and uncharacterized clinical strains from bovine (B00-4830, B99-4297), avian (Av01-4156), canine (Ca01-E179) and human (H87-5406) origin were used to assess the detection thresholds and hybridization specificity of the virulence microarray. Most of the E. coli strains were obtained from the Escherichia coli laboratory collection at the Faculté de médecine vétérinaire of the Université de Montreal. E. coli strains A22, AL851, C248 were kindly provided by Carl Marrs (University of Michigan) and IA2 by J. R. Johnson (University of Minnesota) respectively. All strains were stored in Luria-Bertani broth (LB [6]) broth plus 25% (v/v) glycerol at −80° C. E. coli cultures were grown at 37° C. in LB broth for genomic DNA extraction and purification. Alternatively, the bacterial strains are kept as a culture collection at −80° C. in tryptic soy broth (TSB) medium containing 10% (v/v) glycerol. Two aliquots of each strain are simultaneously plated on tryptic soy agar (TSA) supplemented with 5% (v/v) sheep blood as a quality control (purity of the strains) and resuspended in 10 ml of LB broth. Cells are grown overnight at 37° C. An agitation of 250 rpm is required for the liquid cultures (LB broth).
Note:
Amplicons were prepared using primers noted herein and strains noted above as source of template for PCR amplification
Tables 3 and 4 list the antimicrobial resistance genes and mutations thereof tested, as well as their origin from specific control strain identified by name and accession number.
Selection and Sequence Analysis of Virulence Gene Probes
The selection included virulence genes of E. coli pathotypes involved in intestinal and extra-intestinal diseases in humans and animals (see Table 2). The primers used for probe amplification were either chosen from previous studies on virulence gene detection or designed from available gene sequences (see Table 5). One hundred three E. coli virulence genes were targeted in this study, encoding (a) toxins (heat-labile toxin LT, human heat-stable toxin STaH, porcine heat-stable toxin STaP, Shiga-toxins Stx1 and Stx2, haemolysins Hly and Ehx, East1, STb, EspA, EspB, EspC, cytolethal distending toxin Cdt, cytotoxic necrosing factor Cnf, Cva, Leo) (b) adhesion factors (Cfa, Iha, Pap, Sfa, Tir, Bfp, Eaf, Eae, Agg, Lng, Aida, Foc, Afa, Nfa, Drb, Fim, Bma, ClpG, F4, F5, F6, F17, F18, F41) (c) secretion systems (Etp) (d) capsule antigens (KfiB, KpsMTII, KpsMTIII, Neu) (e) somatic antigens (RfcO4, RfbO9, RfbO101, RfbO111, RfbEO157) (f) flagellar antigen (FliC), (g) invasins (IbeA, IpaC, InvX), (h) autotransporters (Tsh), (i) aerobactin system (lucD, TraT, lutA) and, in addition, to espP (serine-protease), katP (catalase), omp (outer membrane proteins A and T), iroN (catechol siderophore receptor), iss (serum survival gene), putative RTX family exoprotein (rtx) and paa (related attaching and effacing gene) probes. The Yersinia high-pathogenicity island (ifp1, irp2, and fyuA) present in different E. coli pathotypes and other Enterobacteriaceae was also targeted. An E. coli positive control gene, uidA, which encodes the E. coli-specific 6-glucuronidase protein and the uspA gene which encodes a uropathogenic-specific protein were added to this collection.
The selection included antibiotic resistance genes (see Table 6).
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The DNA sequence of each gene was analyzed by BLAST analysis and ClustalW alignment followed by phylogenetic analysis. When the selected gene showed sequence divergence over 10% amongst different strains, new primers were designed to amplify the probe from each phylogenetic group as was the case for espA, espB and tir genes. The new primers were selected in conserved sequence areas flanking the area of divergence in order to ensure gene discrimination at the hybridization level. Phylogenetic analysis of the attaching and effacing locus (LEE) genes espA, espB and tir permitted us to distinguish three phylogenetic groups with regard to the sequence divergence cutoff value (<10%) chosen for this study. Attaching and effacing genes from strains EDL933, E2348/69 and RDEC-1 belonging to the different phylogenetic groups have been cloned and sequenced. Genomic DNA from strains EDL933 (EHEC), E2348/69 (Human EPEC) and RDEC-1 (rabbit EPEC) were used as templates to PCR amplify the different probes espA2-espB1-tir2, espA3-espB2-tir3 and espA1-espB3-tir1 respectively. The amplified probes were sequenced to confirm their identity and printed onto the pathotype microarray as shown in
Probe Amplification, Purification and Sequencing
E coli strains were grown overnight at 37° C. in Luria-Bertani medium. A 200 μl sample of the culture was centrifuged, the pellet was washed and resuspended in 200 μl of distilled water. The suspension was boiled 10 min and centrifuged. A 5 μl aliquot of the supernatant was used as a template for PCR amplification. PCR reactions were carried out in a total volume of 100 μl containing 50 pmol of each primer, 25 pmol of dNTP, 5 μl of template, 10 μl of 10×Taq buffer (500 mM KCl, 15 mM MgCl2, 100 mM Tris-HCl, pH 9) and 2.5 U of Taq polymerase (Amersham-Pharmacia). PCR products were analyzed by electrophoresis on 1% agarose gels in TAE (40 mM Tris-acetate, 2 mM Na2EDTA), then purified with the Qiaquick™ PCR Purification Kit (Qiagen, Mississauga, Ontario) and eluted in distilled water. Since the annealing temperature of the various PCR primers ranged from 40° to 65° C. and genomric DNA from 36 E. coli strains were used as template, all the PCR amplifications were done separately. A total of 103 virulence factor probes and two positive control probes, uidA and uspA, were amplified successfully as determined by amplicon size and DNA sequence. The purity of the amplified DNA was confirmed by agarose gel electrophoresis of 50-100 ng of each amplified fragment. The size of the PCR products ranged from 117 bp (east1) to 2121 bp (katP) with an average length of 500 bp for the majority of the DNA probes (Table 1). For quality control purposes all PCR fragments were partially sequenced for gene verification (Applied Biosystem 377 DNA sequencer using the dRhodamine Terminator Cycle Sequencing Ready™ reaction Kit).
Genomic DNA Extraction and Labeling
Cells, collected by centrifuging 5 ml of an overnight culture at 12,000 rpm, were washed with 4 ml of solution 1 (0.5 M NaCl, 0.01 M EDTA pH 8), resuspended in 1.2 ml of buffer 2 (solution 1 containing 1 mg/ml of lysozyme), then incubated at room temperature for 30 min. After proteinase K and SDS additions, a two hours incubation at 37° C. and a phenol-chloroform extraction, total DNA was precipitated by adding one volume of isopropanol. The harvested pellet was washed with one volume of 70% (v/v) ethanol, dried then resuspended in 100 μl of Tris-EDTA buffer. When desired, a volume of 5 ul of RNAse (10 mg/mL) was added to remove any trace of unwanted RNA in the suspension.
Before labeling, total DNA was reduced in size by restriction enzyme digestion (New England BioLabs, Mississauga, Ontario) and following digestion, the enzymes removed by phenol-chloroform extraction. Cy 3 dye was covalently attached to DNA using a commercial chemical labeling method (Mirus' Label IT™, PANVERA) with the extent of labeling depending primarily on the ratio of reagent to DNA and the reaction time. These parameters were varied to generate labeled DNA of different intensity. Two μg of the digested DNA were chemically labeled using 4 μl of Label IT™reagent, 3 μl of 10× Mirus™ labeling buffer A and distilled water in a 30 μl total volume. The reactions were carried out at 37° C. for 3 h. Labeled DNA was then separated from free dye by washing four times with water and centrifugation through Microcon™ YM-30 filters (Millipore, Bedford, USA). The amount of incorporated fluorescent cyanine dye was quantified by scanning the probe from 200 nm to 700 nm and subsequently inputting the data into the % incorporation calculator found at http://www. Dangloss.com/seidel/Protocols/Dercent inc.html. This method is based on the calculation of the ratio of μg of incorporated fluorescence: μg of labeled DNA. Alternatively, genomic E. coli DNA is fluorescently labeled with a simple random-priming protocol based on invitrogen's Bioprime DNA Labeling kit. The kit is used as a source of random octamers, reaction buffer, and high concentration klenow (40 U/pl). The dNTP mix provided in the kit, which contains biotin-labeled dCTP, is replaced by 1.2 mM dATP, 1.2 mM dGTP, 1.2 mM dTTP and 0.6 mM dCTP in 10 mM Tris pH 8.0 and 1 mM EDTA. In addition, 2 μl of Cy5-dCTP 1 mM from NEN were used to fluorescently label the DNA. The labeled samples are then purified on QIAquick™ columns according to the manufacturer's protocol after adding 2.5 μl 3 M NaOAcetate pH 5.2 to lower the pH of the solution. The microarrays are pre-hybridized for 1 hour at hybridization temperature with DIG buffer (Roche) and 10% (v/v) salmon sperm DNA (10 mg/ml), washed for 10 minutes in water and dried with gaseous; nitrogen 500 ng of labeled DNA, dried and resuspended in 6 μl of DIG buffer with salmon sperm DNA was used for the hybridization which is performed at 47° C. under a 11 mm×11 mm coverslip. Three stringency washes are performed after the hybridization: 1×SSC-0.2% (w/v) SDS at 42° C., 0.1×SSC-0.2% (w/v) SDS at 37° C. and 0.1×SSC at 37° C. The slide is dried with gaseous nitrogen and scanned.
Optimization of Microarray Detection Threshold Using a Prototype Microarray
A prototype chip was constructed and used to assess parameters, namely fragment length and extent of fluorescent labeling of the target (test) DNA, to optimize the spot detection threshold of the microarray. DNA amplicons from 34 E. coli virulence genes including the following EHEC virulence gene probes: espP, EHEC-hlyA, stx1, stx2, stxc, stxaII, paa and eae were generated by PCR amplification and printed in triplicate. The probe lengths ranged from 125 bp (east1) to 1280 bp (irp1). A HindIII/EcoRI digestion was used to generate large fragments (average size ˜6 Kb) and Sau3A/AluI digestion to produce smaller DNA fragments (average size ˜0.2 Kb) from E. coli O157:H7 strain STJ348 genomic DNA. The restricted DNAs were labeled and used as the target for hybridization with the prototype microarray. In the present experiments, the strongest hybridization signal was obtained by using larger fragments labeled at an optimal Cy3 rate in the range of 7.5 to 12.5. An estimate of the microarray's sensitivity was calculated by the following equation as described by De Boer and Beumer (De Boer, E., et al. (1999) Int J Food Microbiol. 50:119-130):
Sensitivity (%)=(number of true positive spots (p)/p+number of false negative spots)×100.
Construction of the E. coli Pathotype Microarray
Virulence factor probes were grouped by pathotype with the resulting array being composed of eight subarrays each corresponding to well characterized E. coli categories (
Some virulence genes, such as fimA, fimH, irp1, irp2, iss, fyuA, ompA, east1, iha, fliC, tsh and ompT are shared by several E. coli pathotypes, and are thus indicative of subsets of pathotypes rather than specific to any one pathotype in particular. Finally a positive control, the uidA gene probe as well as a negative control composed of 50% (v/v) DMSO solution were added. An estimate of the specificity of the virulence microarray was calculated by the following equation (De Boer, E., et al. (1999) Int J Food Microbol. 50:119-130):
Specificity (%)=(number of true negative spots(n)/n+number of false positive spots)×100.
Printing and Processing of the Microarrays
Two μg of each DNA amplicon were lyophilized in a speed-vacuum and resuspended in filtered (0.22 μm) 50% (v/v) DMSO. The concentration of amplified products was adjusted to 200 ng/μl and 10 μl of each DNA amplicon were transferred to a 384-well microplate and stored at −20° C. until the printing step. DNA was then spotted onto CMT-GAPS™ slides (Corning Co., Corning, N.Y.) using a VIRTEK ChipWriter™ with Telechem SMP3™ microspotting pins. Each DNA probe was printed in triplicate on the microarray. After printing, the arrays were subjected to ultraviolet crosslinking at 1200 μJoules (U.V. Stratalinker™1800, STRATAGEN) followed by heating at 80° C. for four hours. Slides were then stored in the dark at room temperature until use.
Microarray Hybridization and Analysis
Microarrays were prehybridized at 42° C. for one hour under a 22×22 mm coverslip (SIGMA) in 20 μl of pre-warmed solution A (DIG Easy Hyb™ buffer, Roche, containing 10 μg of tRNA and 10 μg of denatured salmon sperm DNA). After the coverslip was removed by dipping the slide in 0.1×SSC (1×SSC contained 150 mM NaCl and 15 mM trisodium citrate, pH 7), the array was rinsed briefly in water and dried by centrifugation at room temperature in 50 ml conical tubes for five min at 800 rpm. Fluorescently-labeled DNA was chemically denatured as described by the manufacturer and added to 20 μl of a fresh solution of pre-warmed solution A. Hybridization was carried out overnight at 42° C. as recommended by the manufacturer. After hybridization, the coverslip was then removed in 0.1×SSC and the microarray washed three times in pre-warmed 0.1×SSC/0.1% (w/v) SDS solution and once in 0.1×SSC for 10 min at 50° C. After drying by centrifugation (800 rpm, five min, room temperature), the array was analyzed using a fluorescent scanner (Canberra-Packard, Mississauga, Ontario). The slides were scanned at a resolution of 5 μm at 85% laser power and the fluorescence quantified after background subtraction using QuantArray™ software (Canberra-Packard). All hybridization experiments were replicated between two to five times per genome.
To identify known virulence genes and consequently, the pathotype of the E. coli strain being examined, genomic DNA from several previously characterized E. coli strains was labeled and hybridized to the pathotype microarray. The K12-derived E. coli strain DH5α was included as a nonpathogenic control. Interestingly, E. coli DH5α produced a fluorescent hybridization signal with the uidA, fimA1, fimA2, fimH, ompA, ompT, traT, fliC and iss probes (
Since the genomic sequence of E coli O157:H7 strain EDL933 is available on GENBANK (NC—002655), this strain represented a good choice to assess the detection threshold and hybridization specificity of the E. coli virulence factors on the microarray. After hybridizing the pathotype microarray with Cy3-labeled genomic DNA from E. coli O157:H7, the scanned image (
The UPEC strain J96 (O4:K6) is a prototype E. coli strain from which various extraintestinal E. coli virulence factors have been cloned and characterized. This strain possesses two copies of the gene clusters encoding P (pap-encoded) and P-related (prs-encoded) fimbriae, produces FIC (focG), contains two hly gene clusters encoding hemolysin and produces cytotoxic necrosing factor type 1 (cnf1). E. coli strain J96 DNA was labeled and hybridized to the pathotype microarray. The scanned array resulted in a UPEC pathotype hybridization pattern (
An enterotoxin-producing strain of E. coli isolated from a case of cholera-like diarrhea, E. coli strain H-10407, was used as a control strain to assess the ability of the microarray to identify the ETEC pathotype (
To further validate the pathotype chip, virulence gene detection was assessed by hybridization with genomic DNA from five clinical E. coli strains isolated from human (H87-5406) and animal (Av01-4156, B004830, Ca01-E179, B99-4297) sources. Genomic DNAs from these strains were fragmented and Cy3-labeled and the microarray hybridization patterns obtained were compared with PCR amplification results.
The virulence gene pattern obtained after microarray hybridization analysis with Cy3-labeled E. coli genomic DNA of avian-origin (Av01-456) showed the presence of the extra-intestinal E. coli virulence genes (iucD, iroN, traT, iut4) and genes present in our K12 strain (fimA1, fimA2, fimH, iss, ompA, and ompt) (
When the pathotype microarray was hybridized with genomic DNA from strain B004830 isolated from bovine ileum, genes encoding ETEC fimbriae F5 and heat stable toxin StaP were detected (
The virulence pattern obtained after microarray hybridization analysis with Cy3-labeled human-origin E. coli genomic DNA H87-5406 strain was very complex and did not fall within a single pathotype category. The hybridization pattern revealed the presence of espP, iss, rtx, fimA1, formA2, fimH, ompA, and ompT genes as well as Shiga-toxin gene, stx1, detected in the enterohemorragic pathotype (
The virulence patterns of two other isolates, the pulmonary isolated strain Ca01-E179 and the bovine strain B994297 (used elsewhere in this study) were clearly identified as UPEC pathotype and Shiga-toxin positive E. coli respectively. The presence of all the pathotype-specific virulence factors that were positively identified by the microarray data for the above animal and human isolates, was further confirmed by PCR amplification of each positive signal.
Given the importance of the stx gene family, amplicons sbcA1 and stxA2 specific for the A subunits of the stc1 and stx2 family (Table 5) were designed, in addition to using the published amplicons stx1 and stx2 (Table 2) which overlap the A and B subunits of the genes. Sequence similarity is of the order of 57% between the published stx1 and stx2 amplicons; similarity between the stxA1 and stxA2 amplicons designed herein is slightly higher, at 61%. As shown in
To further explore the potential of microarrays to distinguish gene variants within homologous gene families, primers used for cnf1 and cnf2 probe amplification were derived from studies on the detection of cnf variant genes by PCR amplification. The resulting amplicons have 85% sequence similarity. Hybridization results obtained with genomic DNA from cnf-positive strains H87-5406 and Ca01-E1799 (
Since the DNA microarray showed initial promise in discriminating between the known gene variants of stx and cnf, a more defined group of genes were selected in order to test the ability of the pathotype microarray to differentiate between different phylogenetic groups of genes with a sequence divergence cutoff value of >10%. The DNA sequence similarity values of espA, espB and tir probes from the three different groups are summarized in
A prototype of microarray for testing antibiotic resistance has been constructed.
Other results in the form of a comparison between two multiresistant Escherichia coli enterotoxigenic strains (ETEC 329 and ETEC 399) are shown in
The present invention also allow to discriminate a single base pair mutation.
In accordance with the present invention, there is provided together several known methods optimized to achieve the various steps described above. The key elements are i) the use of synthetic oligonucleotides as DNA probes (see Table 7 below for examples)—these are superior to generally used PCR amplicons in terms of ease of manufacture and purification, but require optimized DNA labeling and hybridization conditions in order to generate sufficient signal. The optimized DNA labeling procedures are described in Bekal et al. (Bekal, S., et al., Journal of Clinical Microbiology, 2003. 41 (5): p. 2113-2125), the disclosure of which is incorporated herein by reference; ii) the use of a bias-free, combined DNA amplification and labeling method to save time, reduce costs and, greatly improve sample processing and robustness of the procedure. Amplification is based upon commercial kits, which is generally known in the art; and iii) the use of shortened hybridization time under carefully controlled conditions to save time. Hybridization time has been shortened from overnight (18 h) to four hours, with partial results available after one hour, in one embodiment of the invention.
The studies described herein entailed designing a DNA microarray containing 103 gene probes distributed into eight subarrays corresponding to various E. coli pathotypes. To evaluate the microarray regarding the specificity of the amplified virulence factor gene fragments, genomic DNAs from different E. coli strains were labeled and hybridized to the virulence factor microarray. To this end, applicants developed a simple protocol for probe and target preparation, labeling and hybridization. The use of PCR amplification for probe generation, and fragmented genomic DNA as labeled target allowed the detection of all known virulence factors within characterized E. coli strains. Direct chemical labeling of genomic DNA with a single fluorescent dye (Cy3) facilitated the work.
Since the fluorescent assay used herein was based on direct detection (single Cy dye) rather than differential hybridization (multiple dyes), optimization of the signal detection threshold was performed. It was determined that the signal intensity, apart from DNA homology and DNA labeling efficiency, depended on (i) immobilized amplicon size (ii) gene copy number in target genomic DNA and (iii) size of the labeled target DNA. Within the large range of probe sizes (117 bp and 2121 bp) tested, hybridization signal intensity could be affected by probe length when using homologous DNA. Quality control analysis of the printed microarray using terminal transferase showed heterogeneity in the spotted amplicons. Using two strains with known genomes (K12 and EDL933), the level of accuracy (sensitivity and specificity) of the current virulence/antibiotic resistance chip as outlined in the Examples herein can be estimated. The average sensitivity or accuracy in discriminating among the different virulence or antibiotic resistance genes approached 97%.
Gene location is another factor to consider when designing gene detection microarrays. After hybridization with genomic DNA from E. coli O157:H7 strain EDL933, it was found strong hybridization signals to etpD, ehxA, L7590, katP and espP. Since these genes are located on the pO157 plasmid (Accession number AF074613), the stronger signal can be attributed to a higher copy number or gene dose. Moreover, many virulence genes are located on mobile elements like plasmids, phages, or transposons and are encoded by foreign DNA acquired via horizontal gene transfer and inserted in the genome. These pathogenicity islands (PAIs) are highly unstable and are constantly shuttled between strains. However, in addition to their total horizontal transfer or deletion, several studies suggested that PAIs are subject to continuous modifications in their virulence factor composition. In earlier work, the detection of a single PAI gene reflected the presumed presence of all the additional virulence genes encoded by the PAI but due to the potential for genetic rearrangements described above, this assumption is risky. Microarray technology represents an excellent tool to circumvent this PAI plasticity and identify genetic rearrangements by gene deletion or insertion on PAI clusters.
Recent investigations of E. coli virulence have revealed new information regarding the prevalence of virulence genes within a specific E. coli pathotype. For example the cytolethal-distending factor (cdt) was first described as virulence factor associated with EPEC E. coli and other diarrhea-associated pathotypes. Later, this gene was detected in strains involved in extraintestinal infections in humans and dogs. More recently, cdt and the urinary tract infection-associated gene (omp T) have been found to be as or more prevalent than traditional neonatal bacterial meningitis NBM-associated traits, such as ibeA, sfaS, and K1 capsule. The usefulness of the virulence microarray concept for exploring the global virulence pattern of strains and the potential detection of unexpected virulence genes was revealed by total genomic hybridizations with uncharacterized clinical strains. The rtx probe (encoding a putative RTX family exoprotein, accession number AE005229) located on the O157:H7 chromosome was amplified using genomic DNA from strain EDL933. Blast analysis did not reveal significant similarities with any available sequences. Analysis of the hybridization patterns of the extraintestinal strain Av014156 and strain H87-5406 revealed a strong signal with the rtx probe indicating the presence of a gene homologous to the rtx probe (
The potential for possessing different combinations or sets of virulence genes within a given E. coli strain could lead to the emergence of new pathotypes. Consistent with this hypothesis, it was found that in the clinical strain H87-5406, a combination of virulence factors from different pathotypes was observed. Moreover, microarray hybridization permitted detection of the Shiga-toxin gene stx1 associated with EHEC strains in addition to virulence genes involved in extra-intestinal infections (cdt2, cdt3, afaD8, bmaE, iucD, iroN, traT, iutA). Starcic et al. (Starcic, M., et al. (2002) Vet Microbiol. 85:361-77) recently reported a case of a “bifunctional” E. coli strain isolated from dogs with diarrhea. When analyzed, only a few strains were positive for heat stable toxin (ST) and none of them produced diarrhea-associated fimbriae K88 or K99 in contrast with previous studies. However, most of these strains were positive for cytonecrosing toxin (cnf1) as well as P-fimbriae and hemolysin (hly) that are involved in extra-intestinal infections in humans and animals. It was thus concluded that hemolytic E. coli isolated from dogs with diarrhea have characteristics of both uropathogenic and necrotoxigenic strains.
Another example illustrating the ability of the virulence microarray to provide a more thorough analysis of virulence genes and consequently the detection of potentially new pathotypes is further supported by the present study in which the ETEC pathotype of the bovine clinical strain B00-4830 was confirmed. In addition to the presence of the ETEC-associated virulence genes encoding StaP and F5 revealed in the hybridization pattern, the etpD gene, described by Schmidt et al. (Schmidt, H., et al. (1997) FEMS Microbiol Lett. 148:265-72) as an EHEC type 11 secretion pathway, was unexpectedly found to be present. In their study, Schmidt et al. (supra), reported that the etp gene cluster was detected in all 30 of the EHEC strains tested by hybridization (using the 11.9 Kb etp cluster from EDL933 as a probe) and by PCR using etpD-specific primers. However, none of the other E. coli pathotypes tested (EPEC, EAEC, EIEC, and ETEC) were positive for the etp gene cluster. As our results are contrary to this study, we assayed for the presence of the etpD gene in strain B00-4830 by PCR using the reverse primer described by Schmidt et al (supra) and a forward one designed in our study. Amplification of the expected 509 bp fragment was consistent with the microarray results confirming that etpD gene can be found in ETEC strains.
Another unexpected finding of the study described herein was the prevalence of fimH and ompT genes that have been epidemiologically associated with extraintestinal infections. BLAST analysis of ompT and fimH genes indicated the presence of both genes in E. coli K12 strain MG1655 and in enterohemorrhagic E. coli O157:H7 strain EDL933 and strain RIMD 0509952. In addition, the hybridization results herein revealed the presence of the formH gene in all strains tested in this study, including non-pathogenic E. coli, EPEC, ETEC and UPEC strains. The ompT gene was less prevalent but present in the Shiga-toxin producing strain H87-5406. It was also found in another Shiga-toxin producing strain B99-4297 as well as in the EPEC strains P86-1390 and E2348/69. The use of these genes as indicators of the UPEC pathotype should be reconsidered.
The studies described herein thus demonstrate that DNA microarray technology can be a valuable tool for pathotype and antibiotic resistance identification and assessing the virulence potential and the antibiotic resistance of E. coli strains including the emergence of new pathotypes or new resistances. The DNA chip design described herein should facilitate epidemiological and phylogenetic studies since the prevalence of each virulence and antibiotic resistance gene can be determined for different and strains and the phylogenefic associations elucidated between virulence pattern and serotypes of a given strain. In addition, unlike traditional hybridization formats, microchip 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.
The DNA labeling methodology, hybridization and pathotype/antibiotic resistance assessment described herein is both rapid and sensitive. The applications of such microarrays extend broadly from the medical field to drinking water, food quality control and environmental research, and can easily be expanded to virulence and antibiotic resistance gene detection in a variety of microorganisms.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.
This application is a continuation-in-part application of application Ser. No. 10/425,821 filed Apr. 30, 2003, still pending and also claims priority on U.S. provisional application Ser. 60/753,850 filed May 25, 2004, still pending, the entire content of both prior application being hereby incorporated in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10425821 | Apr 2003 | US |
| Child | 11136524 | May 2005 | US |
