Patent Application
20100112549
1. Field of the Invention
Embodiments of the present invention relate to selective organism detection and, more particularly, to recombinant bacteriophages and the use of such recombinant bacteriophages to detect target bacteria and to determine drug(s) to which such target bacteria are resistant.
2. Description of the Related Art
The ability to identify specific types of bacteria is of great importance to health-care providers, farmers, and ultimately patients and consumers around the world. Illustrative of this fact, is that the annual worldwide bacterial in vitro diagnosis market is about (USD) $10 billion. Further, there are many different specific (but not exhaustive) illustrative examples that highlight of need for specific, sensitive, accurate, and reproducible bacterial diagnostics (products, kits, and methods), as discussed below.
For example, bovine mastitis, an infection caused by bacterial cells, results in the inflammation of the bovine breast, reduction in milk yield and a decrease in milk quality. This condition is caused by the bacteria Staphylococcus aureus and Staphylococcus agalactiae. This reduction in milk yields and quality in the western world alone cause annual financial losses estimated at $3.7 billion.
Bovine tuberculosis (Mycobacterium bovis), is another example of a bacteria that causes great financial loses worldwide. In 2005, for example, 12 of a heard of 55 cattle in a small Michigan farm tested positive for bovine tuberculosis. The farm was forced to destroy the entire herd of cattle, along with an entire herd of hogs. Tuberculosis testing in cattle requires the animal to be held for 2 days, and tests are false positive 5 percent of the time. Often entire herds have to be quarantined or destroyed. The annual worldwide financial losses are estimated at (USD) $3 billion. Moreover, M. bovis can infect humans.
Bacterial food borne diseases pose a significant threat to human health in causing ˜76 million illnesses, 325,000 hospitalizations, and 5,000 deaths in the US annually. Economic losses in the US due to insects and microbes are estimated between $5 and $17 billion annually.
For example, in 1996, juice that was contaminated with the bacteria Escherichia coli was released into the public by juice maker Odwalla which resulted in one death and 66 illnesses. The company paid a $1.5 million fine, and the recall alone cost the company $6.5 million. In 2006, an E. coli O157:H7 outbreak from contaminated Dole brand spinach originating from California resulted in 205 illnesses and 3 deaths.
Tuberculosis is a leading cause of death worldwide. One third of the world's population is infected with Mycobacterium tuberculosis, the bacterium that causes tuberculosis. Every day 25,000 people are infected and 5,000 people die from the disease. Furthermore, due primarily to poor diagnosis, multidrug resistant strains of M. tuberculosis are emerging and the reemergence of tuberculosis as a worldwide epidemic has become a real threat. The worldwide annual market for tuberculosis diagnostics is $1.8 billion.
According to leading international health organizations such as the World Health Organization (WHO), there is an essential need for quicker and more reliable diagnostics that can be feasibly implemented in developing countries that have little infrastructure and require low-cost diagnosis options. For example, India is the most afflicted country by tuberculosis, with 1.79 million new cases emerging annually and with only 46% of total infections actually detected due to poor bacterial diagnosis options available.
MRSA is a drug-resistant version of the common Staphylococcus aureus bacteria and is carried by 2.5 million people in the US. A carrier can be a healthy individual, and still be highly contagious, due to the nature of the MRSA bacterium. The bacteria are highly contagious and spread by touch. Approximately 86% of all infections occur within hospitals, and these infections carry a 20% mortality rate. This bacterium costs an average of $21,000 over the standard costs to treat, and kills approximately 19,000 people in the US annually.
There are several target areas where bacterial detection/diagnosis is advantageous including: environmental samples, plant samples, veterinary samples, food samples, livestock samples, and medical samples. These target areas/samples can be derived from many different sources including: environmental samples can be derived from sources such as water (e.g. rivers, lakes, ponds, oceans) the atmosphere, soil, mineral, as well as from surfaces and natural and synthetic materials; plant samples can be derived from sources such as live or dead natural vegetation, crops; veterinary samples can be derived from sources such as live or dead household animals, farm animals, and wild animals; food samples can be derived from sources such as any natural or synthetic food product intended for human, animal, or plant use; livestock samples can be derived from sources such as animals intended for human consumption; and medical samples can be derived from sources such as human tissue, blood, and sputum, and other bodily fluids.
A bacteriophage or phage is defined as a virus that infects bacteria. Bacteriophages have a high specificity to their corresponding host bacteria. To infect bacteria, the bacteriophage attaches to specific receptors on the surface of the bacteria. This attachment determines the host range of each bacteriophage, and normally is restricted to some genera, species, or even subspecies of bacteria. This bacteriophage specificity could provide clinicians, laboratory technicians, technicians in the field, as well as consumers, with the ability to identify (detect or diagnose) specific types of bacteria by exploiting this bacteriophage characteristic.
Bacteriophages experience two types of natural life cycles, or methods of viral reproduction, known as the lytic cycle and the lysogenic cycle. In the lytic cycle, host cells will be broken and suffer death after replication of the virion. In contrast, the lysogenic cycle does not result in immediate lysing of the host cell and consequential host cell death; rather, the bacteriophage genome integrates with the host DNA, or establishes itself as a plasmid, and replicates along with the organism's genome. The endogenous bacteriophage remains dormant until the host is exposed to specific conditions (e.g., stress) at which point the bacteriophage may be activated, initiating the reproductive cycle resulting in the lysis of the host cell.
There are conventional methods that exploit the specificity of the bacteriophage/bacteria interaction, which typically explore the lytic cycle of the bacteriophage. In fewer cases, these conventional methods explore the lysogenic cycle. Typically, as discussed in the reported literature, a reporter gene is incorporated into the host indiscriminately after the initial bacteriophage-host interaction takes place or a reporter molecule is fused to a phage and the amplification of this phage or the expression of the reporter molecule is detected after the recombinant bacteriophage infects its host bacteria. In some previous works, the use of bacteria-specific promoters and/or origin of replication are discussed in order to increase the specificity of the detection method. Furthermore, reporter genes are disclosed that express enzymes (e.g. luciferase, or beta galactosidase) which require indirect detection through the addition of other reporter bacteria or substrates.
Detection and analysis of bacteria from samples suffers unless steps are taken to prepare the samples for examination. This preparation can involve physical and chemical manipulations of the sample in order to improve the efficacy of a diagnosis. These steps are often taken in order to remove factors from the sample that may inhibit a detection methodology. Another predominant use of sample preparation is for concentrating the bacteria within the sample in order to improve the likelihood of detection as well as the robustness of the data that can be gathered from the bacteria in the sample.
Two specific examples where sample preparation has been addressed for clinical diagnosis of Mycobacterium tuberculosis are highlighted as follows. In the most comprehensive review of sample preparation of clinical sputum samples for detection of tuberculosis via smear microscopy to date (see Karen R Steingart, V. N., Megan Henry, Philip C Hopewell, Andrew Ramsay, Jane Cunningham, Richard Urbanczik, Mark D Perkins, and M. P. Mohamed Abdel Aziz, Sputum processing methods to improve the sensitivity of smear microscopy for tuberculosis: a systematic review. Lancet Infectious Diseases, 2006. 6: p. 664-674, which is hereby incorporated by reference herein in its entirety), the results showed that the concentration of the bacteria by centrifugation and any kind of conventional chemical processing of the sputum improved the sensitivity for detection. In another study looking specifically at processing sputum for detection of tuberculosis using a bacteriophage-based method (see D. J. Park, F. A. D., A. Meyer, and S. M. Wilson, Use of a Phage-Based Assay for Phenotypic Detection of Mycobacteria Directly from Sputum. Journal of Clinical Microbiology, 2002. 41(2): p. 680-688 which is hereby incorporated by reference herein in its entirety), both chemical and physical processes were determined to significantly improve the yield of the technique by chemically removing a factor in the sputum that inhibits bacteriophage infection and by concentrating the sample via centrifugation.
The conventional techniques for employing sample preparation require significant expertise and laboratory infrastructure for implementation. In addition, sample preparation often introduces a greater risk of contamination, to the samples hindering diagnosis, and also to clinical staff posing a higher risk for contracting disease.
Sample preparation of sputum for diagnosis of tuberculosis, for example, requires several steps of processing with chemicals and centrifugation. Several tubes are used in the process and a laboratory centrifuge is required.
Examples of sample preparation kits include the MYCOPROSAFE® sample preparation kit produced by Salburis, Inc. (Woburn, Mass.), and the FASTPlaque-Response™ tuberculosis diagnostic kit produced by Biotec Laboratories, LTD (Suffolk, England). The MYCOPROSAFE® sample preparation kit is used for processing sputum for tuberculosis diagnosis. Although this kit provides the tubes and chemicals needed for processing, it still relies on laboratory infrastructure (namely a centrifuge) for implementation. The FASTPlaque-Response™ tuberculosis diagnostic kit is used for detection and determination of rifampicin resistance of the tuberculosis bacteria. The preparation procedure is also complex and requires centrifugation.
The requirement of laboratory infrastructure and technical expertise poses a burden in any clinical setting and these requirements and associated costs make sample preparation impossible to implement in many resource-limited settings.
There is a need for quicker and more reliable bacterial diagnostics (products, kits, and methods) that can be feasibly implemented, and that go beyond the bacteriophage-host binding infecting event. This need includes bacterial diagnostics that are more specific, sensitive, accurate, and reproducible, as compared to conventional bacterial diagnostics. There is also a need to further exploit the intrinsic high specificity of bacteriophages to their corresponding host bacteria.
In addition, there is also a need for a sample preparation apparatus that is low in cost, easy to use, and does not require technical expertise or any additional laboratory infrastructure to perform.
It is therefore an object and advantage of the present invention to provide less costly, more efficient, more specific, faster, more accessible, and better adaptable processes and apparatuses for selective organism (e.g., bacterial) detection than provided by currently available technology.
It is also an object and advantage of the present invention to provide bacterial diagnostics (products, kits, devices, and methods) that further exploit the intrinsic high specificity of bacteriophages to their corresponding host bacteria, as compared with conventional bacterial diagnostics.
In accordance with the foregoing objects and advantages, an embodiment of the present invention provides recombinant bacteriophages, a method for constructing and producing such recombinant bacteriophages, and use of such recombinant bacteriophages for detecting target bacteria and/or for determining drug(s)/antibiotics to which the target bacteria is resistant.
In accordance with an embodiment of the present invention, products, kits, and methods that are capable of detecting specific types of bacteria, for example, by probing for the presence of specific nucleic acid sequences and/or genes within (that is characteristic of) a targeted viable bacterium rather than merely through the bacteriophage/host binding/infecting event, are provided. Generic examples of such products/methods may include those based on: bacterial culture; bacterial staining and microscopy; enzyme-Linked ImmunoSorbent Assay (ELISA); polymerase chain reaction (PCR); and other bacteriophage-based methods.
It is further object and advantage of the present invention to provide the capability to probe other specific nucleic acid sequences in order to detect for characteristics that, for example, give rise to a bacterium's drug resistance. Thus, the detection of a specific bacterial nucleic acid sequence can be made dependent upon the expression and detection of the reporter gene(s) used.
In accordance with an embodiment with the present invention, a method to detect specific nucleic acid sequences in a target (i.e. viable bacteria) in a sample comprises the following steps that may occur alone or in combination, as appropriate: (a) the bacterium in the sample is exposed to infection by genetically engineered bacteriophages, which have had their lytic cycle repressed or deleted. Reporter gene(s) are incorporated in the genome of the bacteriophage. This reporter gene(s) is placed downstream of a promoter and flanked by nucleic acid sequences homologous to a target nucleic acid sequence to be detected in the bacteria; (b) the infected bacteria express the reporter gene(s) only if the target nucleic acid sequence(s) or gene(s) is/are present in the bacteria, and homologous recombination with gene replacement occurs; and (c) the reporter gene(s) may then be detected directly or indirectly.
In accordance with an embodiment with the present invention, in vitro diagnostic kits and devices for detecting target bacterial organisms are provided.
In accordance with an embodiment with the present invention, sample preparation apparatuses for preparing samples for detection and analysis using bacteriophage-based techniques, that are low in cost, easy to use, and do not require technical expertise or any additional laboratory infrastructure to perform, are provided.
The present invention will be more fully understood and appreciated by reading the following Detailed Description in conjunction with the accompanying drawings, in which:
a is a graphical illustration showing a bacteriophage probing construct comprising a primary reporter gene (RG), which is placed downstream of a promoter (P) and flanked by nucleic acid sequences (5′ HT; 3′ HT) homologous to a specific target nucleic acid sequence (Target) to be detected within a target bacterium, according to an embodiment of the present invention.
b is a graphical illustration showing a gene replacement event from a double crossover event between the homologous nucleic acid sequences (5′ HT; 3′ HT) flanking the reporter gene (RG) in the bacteriophage as shown in
Embodiments of the invention will be more fully understood and appreciated by reading the following Detailed Description in conjunction with the accompanying drawings, wherein like reference numerals refer to like components.
The technology underlying the embodiments of the present invention herein is based on the intrinsic high specificity of bacteriophages (i.e. viruses that infect bacteria) to their corresponding host bacteria, as discussed supra.
In accordance with an embodiment of the present invention, the process of employing phages capable of being detected via a reporter molecule that is fused to the phage, benefits from a production strategy that produces a phage that lacks the expressed reporter molecule, but contains the reporter molecule DNA. After infection, however, the phage progeny incorporate the expressed reporter molecule. Small molecules (i.e. smaller than 40 KDa), such as small proteins, peptides, epitopes, and oligomers with desired characteristics that allow for their detection (e.g. high binding affinity, immunogenicity, chemical reactivity, conductivity, electrochemical activity, etc) may be used as reporter molecules, although larger molecules with the desired characteristics may also be used if desired.
Some of the advantages of an embodiment of the present invention when compared with the conventional bacterial detection methods and products include: (i) homologous recombination with gene replacement adds additional specificity and functionality to the detection system of an embodiment of the present invention by allowing the detection of specific sequences in the genomic DNA of the target bacterium; (ii) the ability to produce a recombinant bacteriophage without the presence of any of the reporter molecule(s) used in the detection system, but possessing in the bacteriophage DNA sequence(s) coding for one or more of the reporter molecule(s) controlled by different conditional promoter(s). The presence of the different conditional promoters each individually controlled adds more sensitivity to the present system. No reporter molecule will be present prior to the infection of the target bacterium by the recombinant bacteriophage, minimizing false positive results. In addition to the increased sensitivity of the present method, the use of reporter molecules controlled by different conditional promoters allows a rational control of the reporter molecules expression by the presence or absence of the promoter's inducers or repressors at different steps in the detection protocol. This rational control incorporates the ability to detect the susceptibility of the infected target bacterium to different conditions and/or treatments, such as the presence of drugs like antibiotics or bactericides and/or to physical treatments like temperature into the system.
Advantages of the invention are illustrated by the following Examples. However, the particular materials and amounts thereof recited in these examples, as well as other conditions and details, are to be interpreted to apply broadly in the art and should not be construed to unduly restrict or limit the invention in any way.
This Example describes the construction of a recombinant bacteriophage, in accordance with an embodiment of the present invention. The construction of a recombinant bacteriophage comprises the modification of the bacteriophage's genome. The bacteriophage's genome (e.g. Mycobacterium species bacteriophages: L5, D29, TM4, Bxb1, DS6A, Barnyard, Bxz1, Bxz2, Che8, Che9c, Che9d, Cjw1, Corndog, Omega, Che12, Bethlehem, and U2; Staphylococcus aureus bacteriophages: P1, P14, CDC 47, 42E, CDC 52, CDC 52A, CDC 79, CDC 53, and UC 18; Enterococcus faecalis bacteriophages: VD13, 42, phiEF24C, PlyV12, and phiFC1; Clostridium difficile bacteriophages: phiC2, phiCD119, PhiC5, PhiC6, PhiC8, C2, and CD630) is modified such that one or more bacteriophage's gene(s) is/are controlled by conditional promoter(s) (e.g. heat shock promoters, where 42° C. is the restrictive temperature and the permissive temperature can be, for example, room temperature to 37° C. Conditional promoters may also be associated with growth phase, stage of infection, and growth conditions, such as presence or absence of a nutrient. One example of a promoter repressed by the presence of a chemical agent (repressor) is the xylR PxylA promoter system where PxylA is repressed by xylose in Staphylococcus aureus. In contrast, the Pcad promoter system is induced by the presence of cadmium in Staphylococcus) and can be fused to genes that code for Detectable Reporter Molecules, such as peptide, protein, DNA, and/or RNA. Furthermore, these molecules can be designed to generate a detectable electrical, chemical, or optical signal and/or to exhibit a specific affinity to other molecules that can be used to capture the reporter molecules. In addition, the reporter molecules can be naturally occurring molecules such as fluorescent proteins and antibodies in the case of proteins or ribozymes and copies of the host or phage DNA or RNA in the case of nucleotides. In addition, the reporter molecule can be synthetically designed such as small peptides or oligomers designed to generate specific electrical, chemical, or optical signals and/or designed to exhibit affinity to other molecules as well as fragments of antibodies. Oligomer-derived reporter molecules could be designed to confer specific affinity to other molecules simply based on affinity to complementary nucleotide sequences and/or through designing oligomers to generate aptamers with specific affinity to a variety of molecules and other materials). Exogenous DNA or RNA fragments coding for Detectable Reporter Molecule(s), is/are incorporated into a bacteriophage's DNA or RNA and are autonomously controlled by conditional promoter(s).
As seen in
Section (II) Shows different possible constructions that could be used, each coding for one or more Detectable Reporter Molecule (DeRM) (e.g. MBP, GST, HP thioredoxin, V5 epitope, GB1, poly-Pro-Phe-Tyr, and 6×HisTag) downstream of Autonomous Promoters (P) with desired characteristics (e.g. constitutive, conditional, etc). Each construction may be flanked with sequences homologous with specific target genes in a bacterium of interest (e.g. Enterococcus faecalis vanA and vanC1, associated with Vancomycin resistance; Staphylococcus aureus mecA, associated with methicillin resistance; Mycobacterium tuberculosis katG, gyrA, gyrB, and inhA associated with Isoniazid resistance, and pstB, Rv1258c, Rv1410c, and other efflux pumps that may be associated with one or more of the following antibiotics, rifampicin, isoniazid, ethambutol, and streptomycin; Clostridium difficile gyrA, gyrB, and efflux pump genes which are associated with resistance to Fluoroquinolones on this microorganism). Such flanking sequences are incorporated in the bacteriophage constructions in the case where gene-specific detection in the bacterium is desired. At the extremities of each construction, different restriction sites are shown. The examples shown in section (II), as well as the protocols used to insert one or more of these constructions into the bacteriophage genome shown in (I) can be achieved through conventional molecular cloning techniques and protocols as described by Sambrook et al., 1989. J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning. In: (2nd ed.), A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), which is hereby incorporated by reference herein in its entirety. A diagram representing a final product, with the various Detectable Reporter Molecule (II) constructions integrated into the bacteriophage genome (I) is shown in (III).
This Example describes the production of recombinant bacteriophages, in accordance with an embodiment of the present invention. Recombinant host bacteria are used to produce the bacteriophage. The recombinant host bacteria contain exogenous DNA coding for the wild type gene(s) with respect to the constructed recombinant bacteriophages described in Example 1, although without any of the described modifications and/or fusions. The recombinant bacteriophage is allowed to infect this recombinant host bacterium under conditions that neither allows the expression of the modified gene(s) in the constructed recombinant bacteriophage's genome described supra, nor the additional Detectable Reporter Molecules(s). Functional bacteriophages are then produced using the unmodified wild type proteins present in the recombinant phage genome and those in the recombinant host bacteria, without producing exogenous reporter molecule(s). This production of recombinant bacteriophages is further illustrated in
Turning to
As shown in
With the expression of all the DeRM repressed, in case some DeRM were fused to any structural protein (SP) of the recombinant bacteriophage, the plasmid in the recombinant bacterium expresses the wild type structural protein without the fused DeRM. This structural protein expressed from the plasmid present into the bacteria and without any fusion is then assembled with the other bacteriophage protein(s) expressed from genes introduced by the bacteriophage. The result is a progeny bacteriophage absent of any Detectable Report Molecule(s).
The resultant recombinant bacteriophage that is produced can be used, for example, in a kit as pursuant to the methodology described in Example 3, infra. The resultant recombinant bacteriophage contains in its genome all of the necessary information to produce the different DeRM(s), but do not have any expressed detectable reporter molecule, and is not fused to any expressed structural protein.
This Example describes how the recombinant bacteriophages produced in Example 2 are used to detect the presence of target bacteria in a sample. The detection of target bacteria in a sample occurs through the expression of Detection Reporter Molecules, as described in Example 2.
The recombinant bacteriophages produced in Example 2 can be used to infect target bacterial cells that are present within a sample. The infected target bacterial cells (i.e. lysogens) are kept in a condition such that the exogenous Detection Reporter Molecules (described in Example 2) is expressed (e.g. presence of an inducer or absence of a repressor), thereby providing a means for identifying the target bacteria by detecting the expressed Detection Reporter Molecules.
Turning to
This Example describes a method of determining if specific target bacteria are resistant to a specific drug. In brief, the lysogens formed by the infection of the target bacteria with the recombinant bacteriophage (as described in Example 3), are exposed to a drug. After a pre-determined period of time, the expression of the Detectable reporter Molecule (as described in Example 2) is activated (e.g., by the addition of an inducer molecule or inactivation of a repressor), thus producing the Detectable Reporter Molecule. More than one Detectable Reporter Molecule can be present and controlled by different promoters, allowing the detection of bacteria's resistance to more than one drug. In this manner, if a drug does not affect the target bacteria, Detectable Reporter Molecules will be expressed. Detection of these Detectable Reporter Molecules thus allows for the determination of resistance by the target bacteria to specific drugs.
As illustrated in
After infection of the target bacteria that was present in the sample by the recombinant bacteriophage and production of DeRM1 after the recombinant bacteriophage entered its lysogenic cycle (as discussed in Example 3-(B)), the target bacteria are exposed to a drug to be tested. If the bacteria are sensitive to the drug (not resistant), they will be killed (become metabolically inactive) by the drug (D). If the bacteria are resistant to the drug being tested, they will survive (metabolically active).
After a pre-determined time following the drug treatment step (long enough for the drug to kill any sensitive bacteria), a molecule (Inducer) is added to the media (F). This inducer will act on the promoter controlling the second reporter molecule (DeRM2), inducing its production in the bacteria that survived the addition of the drug. Detection of DeRM2 indicates that the remaining viable target bacteria are resistant to the drug tested.
This example describes the use of a lateral flow device as a specific example of detecting target bacteria and/or drug resistant target bacteria in a biological sample. Lateral flow devices should be understood by those skilled in the art, and need not be described in great detail herein. Briefly, reporter molecules can be designed for use in a lateral flow device where the reporter molecules would exhibit affinity to dye particles for visualization on the lateral flow membrane, as well as affinity to other molecules immobilized on the lateral flow membrane, for localization on the lateral flow membrane. A solution containing such reporter molecules can then be applied to the lateral flow device where they may be conjugated to dye particles and then become localized by the immobilized molecules on the lateral flow membrane. A visual signal is generated by the localized accumulation of conjugated dye particles.
Turning to
An alternative embodiment of the invention will now be described with reference to the following Examples. This alternative embodiment provides a method to detect specific nucleic acid sequences within viable bacteria. This alternative embodiment also provides genetically modified bacteriophages.
In this detection method, target bacteria present in a sample (e.g., blood, sputum, urine, food, water, soil, etc.) are specifically infected by a genetically modified bacteriophage that has had its lytic cycle repressed or deleted, and that contains at least one reporter gene. The primary reporter gene is placed downstream of a promoter which may be conditional or constitutive and is flanked by nucleic acid sequences that are homologous to specific target nucleic acid sequences present within a bacterium forming a probing construct. Additional probing constructs may be included that may contain different reporter genes and can probe for different target nucleic acid sequences. In the presence of the target nucleic acid sequence(s), a gene replacement event takes place through a double crossover event between the homologous nucleic acid sequences of the bacteriophage and the target sequences present in the bacteria. This double crossover event results in the replacement of the target nucleic acid sequence present in the bacteria with the reporter gene included in the reporter construct. This will activate the reporter genes, which will be expressed by the bacteria making these expressed molecules available for detection and identification. Additionally, the probing construct may be regulated by a conditional promoter that may restrict expression of the reporter molecule in the presence of an inhibitor or in the absence of an inducer.
Pursuant to this method of an alternative embodiment of the present invention, a specific organism (e.g., specific target bacteria) can be detected by probing for nucleic acid sequences and/or genes that are characteristic of the organism; for example, when detecting the bacterium Escherichia coli, the signature nucleic acid sequence present in 16S rDNA fragments can be used as the flanking sequence.
Reporter genes introduced by an engineered bacteriophage will only be expressed if specific target nucleic acid sequence(s) within the specific host bacterium is/are present. In this manner a specific organism can be detected via probing for nucleic acid sequence(s) and/or gene(s) that is characteristic and specific to the organism, and not simply through the bacteriophage-host infection event. Since many bacteriophages may be specific for several different types of bacteria within a single genus, the use of this method is highly desirable when one specific type of bacteria within the genus needs to be identified. In other words, this bacteriophage detection method of an alternative embodiment of the present invention comprises another layer of increased specificity with respect to the identification of specific target bacteria within a sample.
Furthermore, pursuant to this method of an alternative embodiment of the present invention, other specific nucleic acid sequence(s) and/or gene(s) may be probed in order to detect genotypes (bacterial characteristics) that would indicate or give rise to a bacterium's drug resistance, or the ability to produce enzymes or other functional protein that would produce a phenotype of interest (such as the ability to grow on specific conditions, metabolize different substrates, or produce different metabolic products and/or proteins).
For example, the nucleic acid sequence of a Multidrug Efflux Pump can be used as the flanking sequence when probing for multidrug resistance in Pseudomonas aeruginosa. Thus, in order to diagnose an organism's drug resistance, the disclosed technique does not require the addition of the drug for selective purposes. In another example the nucleic acid sequence of a specific enzyme that allows the use of a specific substrate by the cell can be used as a flanking sequence. In this case the gene replacement event could act to simply delete this phenotype and analysis of the inability of the cells to metabolize the specific substrate could allow the determination of the presence of a targeted cell without the need for generating an exogenous reporting signal. In some cases, the target cells capability of producing an enzyme may result in the formation of toxic compounds, thus the detection of the genes in a target cell that code for such enzyme would result in a means of detecting a target cell's capability of producing a specific toxin. In another example, another phenotype of interest is the nucleic acid sequence that directly codes for the production of a specific toxin (e.g. enterotoxin A, B, or C of Staphylococcus aureus). In this case, toxin-coding sequences would constitute the flanking sequence when probing for the ability of bacteria species to produce specific toxins.
The genetically modified bacteriophage may contain genes that will increase the host's metabolism, for example, by the expression of proteins which will increase the bacteria's uptake of nutrients from the media. Further, the genetically modified bacteriophages may contain genes that will improve the expression of the reporter genes, for example, by the repression of genes involved in the secondary metabolism in the host cell, or by the expression of proteins directly involved in the protein synthesis machinery in the host cell. These “helper” genes will be activated and expressed as soon as the bacteriophage genomic material naturally establishes into the host bacteria's genome. The genetically modified bacteriophage may also contain genes that will increase the frequency of homologous recombination in the host bacteria's genome by the expression of RecA protein, for example. The genetically modified bacteriophage may contain a lytic cycle on its genome, but it can be conditionally controlled and/or repressed through the use of conditional promoters. Nucleic acid molecules that are introduced by the phage may be controlled by a variety of promoters that can be host specific and/or derived from the phage or other sources. For example, appropriate promoters may be chosen from a source that will confer over-expression of the phage-introduced nucleic acids.
An embodiment of the present invention provides a plurality of genetically engineered bacteriophages, each of which may specifically infect a different target bacterium, from different genera, species, or subspecies. A plurality of genetically modified bacteriophages can have different reporter genes that may be used together, and the presence of one or more target bacteria and/or one or more type of nucleic acid sequence and/or genes could be identified.
In accordance with an embodiment of the present invention, the reporter gene may be expressed by producing a fluorescent protein, for example, a green fluorescent protein, which remains inside of the host and can be detected through its optical excitation and resulting optical emission visually or via a light detection apparatus. Additionally, the reporter gene may be expressed and secreted by the host bacteria, which can then react with a component in the medium or within the host cell, or which catalyzes a reaction in the medium to give a detectable signal. The reporter gene may be expressed and secreted by the host bacteria and can then be sensed by a device or a device surface in order to be detected by electrical means through the device. The reporter genes used on the same bacteriophage may be a plurality of the reporter genes referred to above, and the product of the reporter genes used on the same bacteriophage could be differentiated (e.g., different colors of fluorescent protein).
This Example describes a method to detect specific nucleic acid sequences in a target (i.e. viable bacteria) by the use of genetically modified bacteriophages that have had their lytic cycle repressed or deleted, and that contain at least one reporter gene present in its DNA sequence (as described supra).
Turning to
Turning to
Accordingly, a specific organism (e.g., a specific bacteria) may be detected by probing for specific nucleic acid sequence(s) and/or gene(s) that is characteristic to the organism, and not simply through the bacteriophage-host infection event. Similarly, other specific nucleic acid sequence(s) and/or gene(s) may be probed in order to detect for characteristics that, for example, give rise to a bacterium's drug resistance.
In accordance with a further embodiment of the present invention, an additional application of the engineered (generically modified) bacteriophages relates to a detection apparatus or device. According to this embodiment, engineered bacteriophages may be located and immobilized (e.g., on a micron scale) on a surface (or substrate) in a manner that can be multiplexed resulting in localization of the reporter signals for the development of a multi-target bacterial sensing and analysis device. This embodiment provides a device with high specificity, lower limits of detection, and shorter turnaround times, all of which have benefits relating to potentially higher sensitivity and superior efficacy over conventional and/or existing technologies.
The genetically engineered bacteriophages may be held to a substrate/matrix, and may be localized within areas of any dimension that may be genetically modified to hold the target bacteria even after its infection, functioning analogously to an antibody. The substrate/matrix may include an antibody and/or aptamer, which specifically recognize, bind and hold to the target bacteria and/or any of its surface components. The antibody may be a polyclonal antibody or a monoclonal antibody and/or fragments thereof.
Different genetically modified bacteriophages could be held in a different and predetermined localized area of any dimension on a solid substrate. Each of the different bacteriophages may also be modified to hold the target bacteria on the substrate even after its infection, functioning analogously as an antibody.
Development of the genetically engineered bacteriophage, as discussed supra, can be accomplished via conventional genetic engineering, microbiological, and phage display techniques, tools, and reagents. Genetic engineering methods can be used to design the bacteriophage genome, microbiological methods can be used to produce as well as characterize the activity of the engineered phage and also the employed antibodies, and phage display methods can be used to control and optimize phage-host interactions as well as phage-surface interactions for their immobilization.
Development of immobilized phages can be facilitated by the design of the genetically engineered bacteriophage and accomplished via standard surface chemistry and microfabrication techniques—For example, through depositing drops of engineered bacteriophages suspended in a liquid medium onto a surface modified to contain primary amine-reactive N-hydroxysuccinimide (NHS) esters that would react with primary amines available on the engineered bacteriophages forming a covalent amide bond between the phage and the substrate.
Controlled localization in microscopic dimensions may be achieved through using tools such as the commercially available BioForce Nanosciences NanoArrayer System. This could also be accomplished through microfabrication techniques similar to those outlined in: Bhatnagar, P; Strickland, A D; Kim, I, et al. Integrated reactive ion etching to pattern cross-linked hydrophilic polymer structures for protein immobilization Applied Physics Letters, 90(14):144107, 2007, which is hereby incorporated by reference herein in its entirety.
Characterization of a device for detecting the presence of a specific nucleic acid sequence in a viable bacterial organism employing a fluorescent reporter protein as described supra, can be achieved using fluorescent microscopes.
An exemplary detection device, as described in the following Example, may be implemented, for example, as a tuberculosis in vitro diagnostic tool. The design benefits from the advantages of the disclosed technology (i.e., low limit of detection, short turnaround time, and high specificity) and is, in addition, lower in cost and simple to use.
This Example describes an embodiment of a device and diagnosis method of the present invention for detecting the presence of a specific nucleic acid sequence in a viable bacterial organism (pursuant to the method as described on Example 6).
Turning to
The bacterial in vitro diagnosis is performed as shown in
In accordance with an alternative embodiment of the diagnostic device, the main sensing platform (similar to the strip B1 described in
In accordance with an embodiment with the present invention, other exemplary embodiments of the sensing platform are provided. For example, the sensing platform can be designed to be used with a sample of interest through a modified preparatory device (similar to the illustrative container described in
In accordance with an embodiment with the present invention, sample preparation apparatuses for preparing samples for detection and analysis using bacteriophage-based techniques, that are low in cost, easy to use, and do not require technical expertise or any additional laboratory infrastructure to perform, are provided.
An exemplary autonomous bacterial sample preparation device may incorporate one or more compartments that may be separated by filters with pore sizes ranging from millimeters to sub-micrometers, one or more chemical agents that may be physically separated into the different compartments, and mechanisms for transferring fluid between the different compartments. The exemplary bacterial sample preparation device may further include an outlet port that may be sealed and which may contain a filter membrane of sub-micrometer pore sizes that retains bacteria within the device but allows liquid and small molecules to exit the device.
This exemplary bacterial sample preparation device will have the capability to:
(a) Homogenize said sample
(b) Neutralize and/or separate contaminants within said sample
(c) Capture bacteria within said sample
(d) Concentrate said bacteria
This exemplary bacterial sample preparation device can be designed to process, without limitation thereto, environmental samples, plant samples, veterinary samples, food samples, livestock samples, and medical samples. Such samples may further include soil samples, water samples, vegetable samples, meat samples, blood samples, urine samples, tissue biopsy samples, mucus samples, fecal samples, and sputum samples.
This sample preparation device will include the capability to stain target bacteria within the device with a dye incorporated within the sample preparation device. In another aspect, this exemplary bacterial sample preparation device can be designed to incorporate a solution of recombinant bacteriophages specific for target bacteria, such as the recombinant bacteriophages described herein, and will designed to allow the infection of the bacteria by the bacteriophages within the device and to allow production of reporter molecules in the case where the recombinant bacteriophages described herein are used.
This exemplary device can additionally allow for the lysis, extraction, and separation of macromolecules that may include proteins, peptides, oligomers, DNA, RNA, and lipids from within bacteria processed with the device.
This exemplary device may also integrate a lateral flow device designed to detect reporter molecules generated from the infection of target bacteria within the device by recombinant bacteriophages described herein and/or other macromolecules that may be released from lysis of the target bacteria. This lateral flow device could be integrated into the sample preparation device such that the molecules released from the target bacteria are applied to the lateral flow device upon breakage of the seal on the outlet of the sample preparation device.
Three additional exemplary embodiments of the apparatus for preparing samples for detection and analysis using bacteriophage-based techniques are described herein. These embodiments allow for chemical processing and concentration of bacteria in a sputum sample.
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A sample preparation procedure for preparing samples for detection and analysis using bacteriophage-based techniques, and using the sample preparation apparatus as set forth in
While the invention is susceptible to various modifications, and alternative forms, specific examples thereof have been shown in the drawings and are herein described in detail. It should be understood, however, that the invention is not to be limited to the particular forms or methods disclosed, but to the contrary, the invention is to cover all modifications, equivalents and alternatives falling within the spirit and scope of the appended claims.
This application claims priority to U.S. Provisional Patent Application Ser. No. 60/912,553, filed Apr. 18, 2007, the entirety of which is hereby incorporated by reference herein.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US08/60836 | 4/18/2008 | WO | 00 | 10/12/2009 |
| Number | Date | Country | |
|---|---|---|---|
| 60912553 | Apr 2007 | US |
