# Patent

## Title
Recombinant mycobacteriophages for delivery of nucleic acids of interest into mycobacteria

## Abstract

Methods are provided for detecting mycobacteria in a sample, including clinical samples. Methods are also provided for determining susceptibility of mycobacterial strains to known or potential antibiotic agents, as are kits therefor. Recombinant mycobacteriophages are also provided comprising heterologous nucleic acids of interest.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a U.S. national stage entry under 35 U.S.C. §371 of PCT International Patent Application No. PCT/US2012/61059, filed Oct. 19, 2012, which claims priority of U.S. Provisional Application No. 61/550,094, filed Oct. 21, 2011, the contents of each of which are incorporated herein by reference into the subject application.


STATEMENT OF GOVERNMENT SUPPORT
This invention was made with government support under grant numbers GM093901, CFAR AI051519 and 4R37 AI026170-23 awarded by the National Institutes of Health. The government has certain rights in the invention.


BACKGROUND OF THE INVENTION
Throughout this application various publications are referred to in square brackets by number. Full citations for these references may be found at the end of the specification. The disclosures of each of these publications, and also the disclosures of the patents and patent application publications recited herein, are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
There is an urgent need for improved assays for both diagnosis of tuberculosis (TB) and drug susceptibility testing (DST) that are accurate, rapid and inexpensive [1]. The most important advances will be assays that can be applied at the point of care in the developing world. Recent advances in nucleic acid amplification approaches (“genotypic assays”), especially the Xpert MTB/RIF assay, are important contributions to the rapid initial diagnosis of pulmonary TB. However, Xpert MTB/RIF detects only rifampicin resistance (RifR) and relies on the fact that almost all clinical RifR identified to date is due to any of three specific point mutations [2-7]. Other clinical drug resistance phenotypes have more varied genetic underpinnings (e.g., hundreds of different mutations can lead to isoniazid resistance [8]) and are therefore refractory to diagnosis via sequence-specific approaches.
In South Africa both multidrug resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB) strains are endemic; MDR-TB and XDR-TB together account for as much as 20% of all TB cases, and contribute significantly to mortality among hospitalized patients [9, 10]. Since both MDR-TB and XDR-TB are resistant to rifampicin, Xpert MTB/RIF cannot distinguish between them. Thus, because of the limitations of currently available diagnostic tests, patients with MDR-TB and XDR-TB are often put on inappropriate therapy for weeks or even months until drug susceptibility testing results become available.
Phenotypic assays, in contrast, recognize the organismal response of bacteria to antibiotics without limitation to any particular antibiotic, allele or mechanism. Culture remains the gold standard for phenotypic assays, but a classical culture identification of M. tuberculosis takes 4 to 8 weeks. Newer approaches, such as microscopic-observation drug-susceptibility (MODS), have shortened the time needed for phenotypic assays to between 1 and 2 weeks [11].
Fluorophages are a type of “reporter-phage” that inject their DNA specifically into mycobacteria [12]. Fluorescence is produced by expression of a fluorescent protein, such as Green Fluorescent Protein (GFP), gene cloned into the phage. Bacterial physiology required for growth and division is similar to that required to produce fluorescence, but the appearance of fluorescent signal after phage infection takes substantially less time than any assay requiring multiple cycles of cell growth and division. Moreover, the drugs which inhibit host gene expression likewise inhibit fluorescence. Since the proof of principle demonstration in 1993 [13], mycobacterial reporter phages have remained a potentially elegant solution to the problem of TB diagnosis. In laboratory cultures, including cultures derived from clinical isolates, reporter phages detect mycobacterial cells, and allow assays of DST in appreciably less time than culture alone [13-18]. However, existing reporter phages are unable to identify mycobacteria directly in clinical specimens, which limits their use.
The present invention address the need for improved mycobacterium phages for delivering nucleic acids of interest, including those encoding reporter proteins, into mycobacteria, and also addresses the need to identify mycobacteria directly in clinical specimens without costly equipment.
SUMMARY OF THE INVENTION
An isolated recombinant mycobacteriophage is provided comprising a vector backbone into which (a) heterologous mycobacteriophage promoter nucleic acid and (b) a heterologous nucleic acid encoding a protein of interest are integrated, and wherein (a) is upstream of (b), and wherein the vector backbone is derived from a mycobacteriophage TM4 and wherein the recombinant mycobacteriophage is conditionally propagating.
Also provided is an isolated recombinant mycobacteriophage comprising a vector into which (a) heterologous PLeft lytic promoter of mycobacteriophage L5 and (b) a heterologous nucleic acid encoding a reporter protein are integrated, and wherein (a) is upstream of (b).
The invention also provides a method of detecting a mycobacterium in a sample comprising:
a) contacting the sample with the isolated recombinant mycobacteriophage described herein under conditions permitting the mycobacteriophage to infect a mycobacterium present in the sample and the protein of interest to be expressed in the mycobacterium; and

b) detecting the protein of interest in the mycobacterium in the sample, thereby detecting the mycobacterium in the sample.

The invention further provides a method of determining if a mycobacterium is susceptible to a candidate agent comprising:
a) contacting a sample comprising the mycobacterium with the isolated recombinant mycobacteriophage described herein in (i) the absence of the candidate agent and (ii) in the presence of the candidate agent, under conditions permitting the mycobacteriophage to infect a mycobacterium present in the sample and the protein of interest to be expressed in the mycobacterium;

b) detecting the protein of interest in the mycobacterium in the sample in (i) and (ii); and

c) comparing the amount of the protein of interest detected in the presence of the candidate agent and the absence of the candidate agent,

wherein a decrease in amount of the protein of interest in the presence of the candidate agent compared to in the absence of the candidate agent indicates that the mycobacterium is susceptible to the candidate agent.

The invention additionally provides a method of identifying a candidate agent as effective in treating an infection caused by a strain of mycobacterium, the method comprising
culturing a mycobacterium with the isolated recombinant mycobacteriophage described herein in (a) the absence of the candidate agent under conditions permitting the mycobacteriophage to infect a mycobacterium present in the sample and the protein of interest to be expressed in the mycobacterium, and (b) the presence of the candidate agent under conditions permitting the mycobacteriophage to infect a mycobacterium present in the sample and the protein of interest to be expressed in the mycobacterium, and

detecting the protein of interest in the mycobacterium in the sample in (a) and in (b), and

comparing the amount of the protein of interest detected in the presence of the candidate agent and the absence of the candidate agent,

wherein a decrease in the amount of the protein of interest in the presence of the candidate agent compared to in the absence of the candidate agent indicates the agent as effective to treat an infection caused by the strain of mycobacterium.

Also provided is a method for producing recombinant mycobacteriophages comprising:
a) introducing a vector into a mycobacterium so as to form a lysogen, which vector comprises (i) a mycobacteriophage promoter nucleic acid and (ii) a heterologous nucleic acid encoding a protein of interest, and wherein (i) is upstream of (ii), and integrated into a vector backbone derived from a mycobacteriophage TM4 and is temperature-sensitive conditionally propagating;

b) maintaining the lysogen at a temperature that permits the lysogen to replicate into a plurality of lysogens; and then

c) maintaining the plurality of lysogens at a temperature that effects induction in the plurality of lysogens, so as thereby to produce recombinant mycobacteriophages comprising (i) and (ii).

Also provided is a method of preparing a sputum sample for reporter phage infection comprising: a) mixing the sputum sample with diluted dithiothreitol in phosphate buffer; b) vortexing the resultant product; c) allowing the product resulting from b) to rest; d) centrifuging the product resulting from c); e) decanting the supernatant; f) mixing remainder with a diluted sodium hydroxide solution; g) vortexing the product resulting from f); h) allowing the product resulting from g) to rest; i) adding phosphate buffer saline and centrifuging the resulting product; j) re-suspending resultant pellet in a mycobacterium-supporting broth.
Additional objects of the invention will be apparent from the description which follows.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A-1D. Generation of improved fluorophage. A) Schematic representation of various fluorophages constructed in the present study. B) Comparison of fluorescence obtained from hsp60-driven mVenus fluorophage φ2GFP7 and episomal plasmid pYUB1378 expressing mVenus from same promoter. C) Flow cytometric comparison of fluorophages in M. smegmatis. D) Time lapse microscopy of mVenus expression in mycobacteria after infection with fluorophage φ2GFP10.
FIG. 2A-2B. Comparison of fluorophages in M. tuberculosis. A) Determination of exposure time to capture a fluorescent image with a similar fluorescent intensity (arbitrary level) by a Nikon Ti microscope after infection of M. tuberculosis mc26230 with the indicated fluorophages. B) Flow cytometric determination of fluorescent intensity obtained after infection of M. tuberculosis mc26230 with φ2GFP7 and φ2GFP10.
FIG. 3A-3B. Efficiency of detection of M. tuberculosis using fluorophage. M. tuberculosis mc26230 cells were infected with φ2GFP10 at different multiplicities of infection (MOI). Percent fluorescent cells was determined using flow cytometry. B) Mean Fluorescent Intensity (MFI) of M. tuberculosis mc26230 cells infected with φ2GFP10 at different MOI was determined using FlowJo analysis.
FIG. 4A-4D. Drug susceptibility test for M. tuberculosis strains using fluorophage φ2GFP10. Drug-sensitive M. tuberculosis mc26230 (A); Rifampicin-resistant M. tuberculosis mc27201 (B); Kanamycin-resistant M. tuberculosis mc27202 (C). Rifampicin- and Kanamycin-resistant M. tuberculosis mc27203 (D) were incubated with φ2GFP10 and the indicated antibiotics simultaneously, incubated for 12 hr, and then examined by microscopy and flow cytometry. Failure to fluoresce in the presence of antibiotic indicates sensitivity, and a fluorescence signal in the presence of a specific antibiotic indicates resistance to that antibiotic.
FIG. 5. Detection of M. tuberculosis in spiked sputum samples. Laboratory-cultured cells of M. tuberculosis mc26230 cells were added to sputum samples to a final concentration of 104 cells per ml. The sample was decontaminated with N-acetyl-L-cysteine (NALC) alone, or with NALC and NaOH, and then incubated with φ2GFP10 for 12 hr at 37° C. before analysis by flow cytometry.
FIG. 6A-6D. Diagnosis and DST of M. tuberculosis in primary clinical samples from active TB patients. Sputum samples were collected from TB patients and analyzed for the presence of M. tuberculosis using (A) Ziehl-Neelsen staining, or (B) φ2GFP10. The DST for (C) rifampicin and (D) kanamycin was performed on the same sample using φ2GFP10. The DST results, available within 14 hr from the time of sputum collection with φ2GFP10 showed that the patient was infected with a drug sensitive M. tuberculosis and were in corroboration with the DST results of agar proportion method available after three weeks.
FIG. 7. The vector backbone, phAE159, for the new phage was generated by the deletion of a  ̃6 kb region encompassing genes gp48-64 [29] from the temperature-sensitive mutant of TM4 known as ph101.
FIG. 8. Kinetics of accumulation of fluorescence protein in M. tuberculosis after infection with φ2GFP10. The M. tuberculosis cells were incubated with φ2GFP7. The samples were fixed at 12, 24, 36 and 48 hr with 2% paraformaldehyde and analyzed by flow cytometry. M. tuberculosis cells incubated with φ2GFP10 and fixed after 12 h were used as a positive control. All the infections were performed at a MOI of 100.
FIG. 9. Drug susceptibility test for M. tuberculosis strains using fluorophage φ2GFP10. Drug sensitive M. tuberculosis mc26230 was incubated with the indicated antibiotics. Phage φ2GFP10 was added either at the time of drug exposure or 3 h and 6 h after drug treatment and percent fluorescent population was determined for each case after 12 h of incubation at 37° C. The schematic representation of the experiment is shown at left. The percent fluorescent population was determined in each case by flow cytometry. Failure to fluoresce in the presence of antibiotic indicates sensitivity and the percent fluorescent population does not significantly decrease by longer incubation in presence of Rifampicin and Kanamycin. The six hour pre-incubation is not sufficient to determine drug sensitivity to isoniazid and ofloxacin.
FIG. 10A-10B. M. tuberculosis drug susceptibility test for ofloxacin and isoniazid using fluorophage φ2GFP10. Drug sensitive M. tuberculosis mc26230 was incubated with ofloxacin or isoniazid. Phage φ2GFP10 was added after 18 and 24 h after pre-treatment with (A) ofloxacin and (B) isoniazid. Results indicate that 18 h of pre-treatment is sufficient to determine drug susceptibility for both the antibiotics



DETAILED DESCRIPTION OF THE INVENTION
As used herein, a “heterologous” nucleic acid, with regard to its presence in a phage or vector backbone, refers to nucleic acid that is not naturally present in the phage or vector backbone, respectively.
As used herein, a “mycobacteriophage” is a phage capable of infecting one or more mycobacteria. A “recombinant” mycobacteriophage is one containing, in its genome, a heterologous nucleic acid. An “isolated” recombinant mycobacteriophage is one that is not naturally occurring.
As used herein, the term “expression,” with regard to a nucleic acid, refers to the process by which a nucleotide sequence undergoes successful transcription and, for polypeptides or proteins, translation.
As used herein, the term “promoter” refers to a minimal nucleotide sequence sufficient to direct transcription. In an embodiment of the invention described herein, the mycobacteriophage promoter controls expression of the protein of interest.
Sambrook, Molecular Cloning: A Laboratory Manual (Third Edition), Cold Spring Harbor Laboratory Press (CSH Press), 2001 (ISBN-10: 0879695773; ISBN-13: 978-0879695774), the contents of which are hereby incorporated by reference in their entirety provides an overview of molecular biology techniques that can be employed herein.
This invention provides an isolated recombinant mycobacteriophage comprising a vector backbone into which (a) heterologous mycobacteriophage promoter nucleic acid and (b) a heterologous nucleic acid encoding a protein of interest are integrated, and wherein (a) is upstream of (b), and wherein the vector backbone is derived from a mycobacteriophage TM4 and wherein the recombinant mycobacteriophage is conditionally propagating.
This invention also provides isolated recombinant mycobacteriophage comprising a vector into which (a) heterologous PLeft lytic promoter of mycobacteriophage L5 and (b) a heterologous nucleic acid encoding a reporter protein are integrated, and wherein (a) is upstream of (b). In the recombinant mycobacteriophage, the vector backbone can advantageously be derived from a mycobacteriophage TM4. In a preferred embodiment, mutations, deletions or insertions are used so as to obtain a recombinant mycobacteriophage that is conditionally propagating.
In either case, the conditional propagation property of the recombinant mycobacteriophage permits it to be introduced into specific cells without killing them, e.g. for reporting purposes. By changing the relevant condition, for example temperature, the phage can be induced to propagate, e.g. for production purposes. Novel temperature-sensitive TM4 mutants have been identified, and critical residues therefore are disclosed herein. Thus, TM4 (and derivatives thereof) are therefore useful for vector backbones for the present isolated recombinant mycobacteriophages. A recombinant phage having the property of “conditionally propagating” as used herein means that the phage replicates only under certain conditions. In preferred embodiments, the relevant condition is temperature (herein designated by “temperature-sensitive” or a grammatical equivalent thereof). In a preferred embodiment of the invention, the conditionally propagating recombinant phage is a phage which replicates at 30° C., but fails to replicate (and/or lyse its host cell) at 37° C. In an embodiment, the conditionally propagating recombinant mycobacteriophage is based on a TM4 phage, GenBank: AF068845, and in non-limiting examples is a point mutant, a deletion mutant, an insertion mutant, or a combination of point mutation(s) and deletion(s) and/or insertion(s).
As used herein, the term “vector backbone” refers to a nucleic acid molecule capable of transporting one or more other nucleic acid(s) to which it has been linked. As used herein, vector backbone “derived from TM4” is vector that comprises one or more nucleic acid sequences identical to a portion of a TM4 genome sequence but the complete sequence of which is not identical to a TM4 genome sequence. Such a vector backbone may be made, in non-limiting examples, by way of one or more of a mutation, insertion and/or deletion of a TM4 genome sequence, or by one or more subsequent mutation(s), insertion(s) and/or deletion(s) of a mutated, deleted and or inserted TM4 genome sequence. Accordingly, a vector backbone derived from a TM4 as used herein includes both directly derived from TM4, and one derived from one which in turn is derived from TM4, and so forth (e.g., in a non-limiting example, derived from phAE159). In embodiments of the isolated recombinant mycobacteriophages described herein, the vector backbone comprises a TM4 genomic sequence having a deletion in a non-essential area thereof (i.e., not essential for replication) permitting insertion of heterologous DNA. In an embodiment, there is a 5.5 kbp to 6.5 kbp deletion of non-essential DNA of the TM4 genomic sequence, and in one embodiment a 5.8 kbp deletion. This permits insertion of a large amount of heterologous nucleic acids, for example up to 10 kbp, without loss of performance. Such heterologous nucleic acids can comprise any protein of interest, as well as expression-controlling nucleic acids such as promoters and other related entities. In a specific embodiment, the vector backbone does not comprise a portion having the sequence of residues 33,877 through 39,722 of the TM4 genome. In embodiments the vector backbone does not comprise TM4 gene 49.
Various mutations of TM4 genome that contribute to temperature sensitivity/conditional propagation have been identified herein. These are used to advantageously effect conditionally propagation recombinant mycobacteriophages. In an embodiment, the isolated recombinant mycobacteriophages described herein comprise a vector backbone which does not comprise a portion having the sequence of TM4 gene 49 through the first sixteen codons of gene 64 of the TM4 genome. In an embodiment, the vector backbone of the isolated recombinant mycobacteriophages described herein does not comprise a portion having the sequence of the last twelve codons of gene 48 of the TM4 genome. In an embodiment, the vector backbone of the isolated recombinant mycobacteriophages described herein does not encode a proline at a residue equivalent to residue 220 of gene product gp48 of the TM4 genome. In an embodiment, the vector backbone encodes a serine at a residue equivalent to residue 220 of gene product gp48 of the TM4 genome. In an embodiment, the vector backbone does not encode an alanine at a residue equivalent to residue 131 of gene product gp66 of the TM4 genome. In an embodiment, the vector backbone encodes a threonine at a residue equivalent to residue 131 of gene product gp66 of the TM4 genome. In an embodiment, the vector backbone does not encode an arginine at a residue equivalent to residue 116 of gene product gp20 of the TM4 genome. In an embodiment, the vector backbone encodes a cysteine at a residue equivalent to residue 116 of gene product gp20 of the TM4 genome.
The vector phAE159 is a useful vector having a high cloning capacity and is derived from the temperature sensitive ph101 vector which in turn is derived from TM4. As such, phAE159 is useful as a vector backbone of the invention. In a preferred embodiment, the vector backbone is a phAE159 vector. In an embodiment, the phAE159 vector backbone comprises the sequence set forth in SEQ ID NO:1. In a preferred embodiment employing the phAE159 vector, the mycobacteriophage promoter nucleic acid is cloned into a PacI site of the phAE159 vector. In another embodiment, the vector backbone is a ph101 vector. Phages derived from TM4 which are useful as embodiments of the vector backbone in the present invention include, in non-limiting examples, those set forth in Genbank Accession No. JF937104 (SEQ ID NO:4); JF704106 (SEQ ID NO:5); JF704105 (SEQ ID NO:6); HM152764 (SEQ ID NO:7); and HM152767 (SEQ ID NO:8).
Insertion sites of the heterologous mycobacteriophage promoter nucleic acid, Pleft lytic promoter, and the heterologous nucleic acid encoding a protein of interest, may be within any non-essential region of the vector backbone. In a preferred embodiment the insertions are within the sequence that results from a deletion of a  ̃6 kb region encompassing genes encoding gp48-64 from the temperature-sensitive mutant of TM4 known as ph101, in the site of the deletion.
The heterologous mycobacteriophage promoter nucleic acid and heterologous nucleic acid encoding a protein of interest/reporter protein can be integrated into such vector backbones in the isolated recombinant mycobacteriophages of the invention.
In an embodiment, the mycobacteriophage promoter nucleic acid is a mycobacteriophage L5 promoter. In a preferred embodiment, the mycobacteriophage promoter nucleic acid is a PLeft lytic promoter of mycobacteriophage L5. In another embodiment, the mycobacteriophage promoter nucleic acid is a T7 promoter and the isolated recombinant phage further comprises a nucleic acid encoding a T7 polymerase. In an embodiment the L5 promoter has the following sequence:







L5 promoter sequence:


(SEQ ID NO: 3)


CGATGATAAGCGGTCAAACATGAGAATTCGCGGCCGCATAATACGACTC


 


ACTATAGGGATCTTAATTAAGGCGCCTCATGTTCTTTCCTGCGTTATCC


 


CCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCG


 


CTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGC


 


GGAAGAGCGCCCAATACGCAAACCGCCTCTCCCAGATCTGATATCGCTA


 


GAGGAAACAGCTATGACCATGATTACGCCAAGCTTGCATGCCTGCAGCT


 


AGGGCACCAATTTGCGATTAGGGCTTGACAGCCACCCGGCCAGTAGTGC


 


ATTCTTGTGTCACCGCAGCAGCAAGGCGGTAGGCGGATCCGAGAGGATC


 


GTGCCGGTGCCGGTGAAAATCCGGCGGCAAGATTCTCCGGTTTGACAGC


 


CACCCGGTTATCGGGTAAGCTGCAAGCATCACCAACTTGGACGGGAAAG


 


GGAGATCGCATATG






A “protein of interest” as used herein, includes peptides, polypeptides and proteins. In a preferred embodiment, the protein of interest is a protein. In a preferred embodiment, the protein is a reporter protein. A “reporter protein” as used herein is any detectable protein that can be encoded by a nucleic acid in a mycobacteriophage. Many reporter proteins are known in the art, and the selection of which reporter protein to use can be made on usability considerations and expected conditions. In a preferred embodiment for recombinant phages to be used in visual assays, the reporter protein is a fluorescent protein. In a further preferred embodiment, the fluorescent protein is a green or yellow fluorescent protein. In a further preferred embodiment, the fluorescent protein is green fluorescent protein derived from A. victoria. In a preferred embodiment, the yellow fluorescent protein is Venus monomeric fluorescent protein (see Nagai et al., Nat Biotechnol. 2002 January; 20(1):87-90, the content of which is hereby incorporated by reference in its entirety). In an embodiment, the Venus fluorescent protein comprises SEQ ID NO:2. In an embodiment the yellow fluorescent protein has an excitation peak of 514 nm and an emission peak is 527 nm. In other embodiments, the yellow fluorescent protein is Citrine or Ypet. Other fluorescent proteins that may be used include those described in U.S. Pat. No. 8,034,614 (the content of which is hereby incorporated by reference in its entirety).
Other fluorescent proteins that may be used as reporter proteins are set forth below in Table 1:
















Excitation
Emission
Molar





Maximum
Maximum
Extinction
Quantum
in vivo


Protein
(nm)
(nm)
Coefficient
Yield
Structure




















GFP (wt)
395/475
509
21,000
0.77
Monomer*


EGFP
484
507
56,000
0.60
Monomer*


Emerald
487
509
57,500
0.68
Monomer*


Superfolder GFP
485
510
83,300
0.65
Monomer*


Azami Green
492
505
55,000
0.74
Monomer


mWasabi
493
509
70,000
0.80
Monomer


TagGFP
482
505
58,200
0.59
Monomer*


TurboGFP
482
502
70,000
0.53
Dimer


AcGFP
480
505
50,000
0.55
Monomer*


ZsGreen
493
505
43,000
0.91
Tetramer


T-Sapphire
399
511
44,000
0.60
Monomer*


EBFP
383
445
29,000
0.31
Monomer*


EBFP2
383
448
32,000
0.56
Monomer*


Azurite
384
450
26,200
0.55
Monomer*


mTagBFP
399
456
52,000
0.63
Monomer


ECFP
439
476
32,500
0.40
Monomer*


mECFP
433
475
32,500
0.40
Monomer


Cerulean
433
475
43,000
0.62
Monomer*


CyPet
435
477
35,000
0.51
Monomer*


AmCyan1
458
489
44,000
0.24
Tetramer


Midori-Ishi Cyan
472
495
27,300
0.90
Dimer


TagCFP
458
480
37,000
0.57
Monomer


mTFP1 (Teal)
462
492
64,000
0.85
Monomer


EYFP
514
527
83,400
0.61
Monomer*


Topaz
514
527
94,500
0.60
Monomer*


Venus
515
528
92,200
0.57
Monomer*


mCitrine
516
529
77,000
0.76
Monomer


YPet
517
530
104,000
0.77
Monomer*


TagYFP
508
524
64,000
0.60
Monomer


PhiYFP
525
537
124,000
0.39
Monomer*


ZsYellow1
529
539
20,200
0.42
Tetramer


mBanana
540
553
6,000
0.7
Monomer


Kusabira Orange
548
559
51,600
0.60
Monomer


Kusabira Orange2
551
565
63,800
0.62
Monomer


mOrange
548
562
71,000
0.69
Monomer


mOrange2
549
565
58,000
0.60
Monomer


dTomato
554
581
69,000
0.69
Dimer


dTomato-Tandem
554
581
138,000
0.69
Monomer


TagRFP
555
584
100,000
0.48
Monomer


TagRFP-T
555
584
81,000
0.41
Monomer


DsRed
558
583
75,000
0.79
Tetramer


DsRed2
563
582
43,800
0.55
Tetramer


DsRed-Express (T1)
555
584
38,000
0.51
Tetramer


DsRed-Monomer
556
586
35,000
0.10
Monomer


mTangerine
568
585
38,000
0.30
Monomer


mRuby
558
605
112,000
0.35
Monomer


mApple
568
592
75,000
0.49
Monomer


mStrawberry
574
596
90,000
0.29
Monomer


AsRed2
576
592
56,200
0.05
Tetramer


mRFP1
584
607
50,000
0.25
Monomer


JRed
584
610
44,000
0.20
Dimer


mCherry
587
610
72,000
0.22
Monomer


HcRed1
588
618
20,000
0.015
Dimer


mRaspberry
598
625
86,000
0.15
Monomer


dKeima-Tandem
440
620
28,800
0.24
Monomer


HcRed-Tandem
590
637
160,000
0.04
Monomer


mPlum
590
649
41,000
0.10
Monomer


AQ143
595
655
90,000
0.04
Tetramer









In other embodiments, the reporter protein is a β-galactosidase encoded by a LacZ, a maltose binding protein, or a chloramphenicol acetyltransferase.
A protein of interest also includes an antigen, a peptide anti-inflammatory agent, an enzyme, a lymphokine, and a peptide antibiotic.
In an embodiment, the mycobacteriophage is a temperature-sensitive conditionally propagating mutant. In a preferred embodiment, the recombinant mycobacteriophage does not propagate in a mycobacteria at 37° C. In a preferred embodiment, the recombinant mycobacteriophage propagates in a mycobacteria at 30° C.
In an embodiment, the mycobacteria is M. smegmatis. In an embodiment, the mycobacteria is M. tuberculosis. In embodiments, the mycobacteria is M. bovis, M. bovis-BCG, M. avium, M. phlei, M. fortuitum, M. lufu, M. paratuberculosis, M. habana, M. scrofulaceum or M. intracellulare. 
Other useful elements, commonly known in the art, may also be included in the genome of the recombinant mycobacteriophages of the invention. For example, the recombinant genome may further comprise one of, more than one of, or all of (1) an antibiotic resistance gene, (2) a ColE1 sequence and (3) a λ Cos sequence upstream of (a). A preferred antibiotic resistance gene is an ampicillin resistance gene. The recombinant mycobacteriophages and vectors of the invention can optionally comprise a mycobacteriophage integration sequence. The recombinant mycobacteriophages and vectors of the invention can optionally comprise an E. coli cosmid sequence.
Methods employing the isolated recombinant mycobacteriophages of the invention are also within the scope of the invention. Methods for determining the presence of particular strains of mycobacteria in a sample, for example therapy-resistant strains, as well as determining susceptibility of a strain to a treatment, are particularly advantageous uses of the recombinant mycobacteriophages described herein.
Thus, in one aspect of the invention a method is provided of detecting a mycobacterium in a sample comprising:
a) contacting the sample with an isolated recombinant mycobacteriophage of as described herein conditions permitting the mycobacteriophage to infect a mycobacterium present in the sample and the protein of interest to be expressed in the mycobacterium; and
b) detecting the protein of interest in the mycobacterium in the sample, thereby detecting the mycobacterium in the sample.
The sample may be treated to promote infection of the mycobacteria present by the mycobacteriophage. Non-limiting suitable protocols are set forth in the specification. The protein of interest is in such methods is preferably a readily-detected reporter protein. The fluorophages made of the recombinant mycobacteriophages herein are especially useful for their high expression of visually detectable fluorescence. Thus, the presence of the targeted mycobacterium in a sample infected by the recombinant mycobacteriophages would be indicated by the presence of fluorescence, under suitable illumination, which can be readily ascertained, e.g. by standard microscopy or flow cytometry, avoiding the need for more complex and/or expensive detection strategies. Sputum samples are a preferred sample, they are easily obtained and treated. The detection of M. tuberculosis in a sputum sample is readily effected by the present method, especially if the reporter protein is a visually-detectable fluorescent protein, such as a suitable yellow fluorescent protein.
The same advantageous aspects conferred by the recombinant mycobacteriophages of the invention can be utilized to determine efficacy of treatments against mycobacteria. As such, one aspect of the invention is a method of determining if a mycobacterium is susceptible to a candidate agent comprising:
a) contacting a sample comprising the mycobacterium with an isolated recombinant mycobacteriophage as described herein in (i) the absence of the candidate agent and (ii) in the presence of the candidate agent, under conditions permitting the mycobacteriophage to infect a mycobacterium present in the sample and the protein of interest to be expressed in the mycobacterium;

b) detecting the protein of interest in the mycobacterium in the sample in (i) and (ii); and

c) comparing the amount of the protein of interest detected in the presence of the candidate agent and the absence of the candidate agent,

wherein a decrease in amount of the protein of interest in the presence of the candidate agent compared to in the absence of the candidate agent indicates that the mycobacterium is susceptible to the candidate agent.

In non-limiting embodiments, the candidate agent may be a known antibiotic or may be a suspected antibiotic, the efficacy of which is to be determined. Any other treatment method can be assayed for efficacy in the same manner.
Also within the scope of the invention is a method of identifying a candidate agent as effective in treating an infection caused by a strain of mycobacterium, the method comprising
culturing a mycobacterium with an isolated recombinant mycobacteriophage as described herein in (a) the absence of the candidate agent under conditions permitting the mycobacteriophage to infect a mycobacterium present in the sample and the protein of interest to be expressed in the mycobacterium, and (b) the presence of the candidate agent under conditions permitting the mycobacteriophage to infect a mycobacterium present in the sample and the protein of interest to be expressed in the mycobacterium, and

detecting the protein of interest in the mycobacterium in the sample in (a) and in (b), and

comparing the amount of the protein of interest detected in the presence of the candidate agent and the absence of the candidate agent,

wherein a decrease in the amount of the protein of interest in the presence of the candidate agent compared to in the absence of the candidate agent indicates the agent as effective to treat an infection caused by the strain of mycobacterium. In non-limiting embodiments, the candidate agent may be a known antibiotic or may be a suspected antibiotic, the efficacy of which is to be determined. The method may be used to identify a therapy for a mycobacterial infection otherwise resistant to treatment by other methods.

In an embodiment of the methods employing candidate agents, the candidate agent is an antibiotic. In an embodiment the candidate agent is an organic molecule of 2000 Da or less. In an embodiment the candidate agent is an organic molecule of 1500 Da or less
In an embodiment of the methods described hereinabove, the mycobacteria are Mycobacterium tuberculosis. In an embodiment of the methods, the mycobacteria are Mycobacterium bovis or Mycobacterium smegmatis. In embodiments, the mycobacteria is M. bovis-BCG, M. avium, M. phlei, M. fortuitum, M. lufu, M. paratuberculosis, M. habana, M. scrofulaceum or M. intracellulare. 
In embodiments the Mycobacterium tuberculosis is MDR-TB and XDR-TB. In embodiments the mycobacteria is resistant to, or is suspected of being resistant to kanamycin, isoniazid and/or rifampicin. In embodiments, the treatment/therapy can be an aminoglycosides (e.g., amikacin), a polypeptide (e.g., capreomycin, viomycin, enviomycin), a fluoroquinolone, (e.g., ciprofloxacin, levofloxacin, moxifloxacin), and/or a thioamide (e.g. ethionamide).
The conditions of the methods are chosen so as to reflect the conditionally propagating nature of the recombinant mycobacteriophage. In an embodiment, the conditions comprise culturing the sample with the isolated recombinant mycobacteriophage substantially at 36° C. to 38° C. In an embodiment, the conditions comprise culturing the sample with the isolated recombinant mycobacteriophage at 37° C. In an embodiment, the conditions comprise culturing the sample for infection with the mycobacteriophage at a temperature not below 31° C.
In a preferred embodiment, the methods further comprise processing the sample with a concentration of NaOH of 1% to 2% prior to culturing the mycobacterium therein for infection with the isolated recombinant mycobacteriophage.
In embodiments of the methods, suitable proteins of interest are as described hereinabove, but any protein of interest may be used. In an embodiment, the protein of interest is a fluorescent protein and the fluorescence of the fluorescent protein is detected. Preferably, the fluorescence is detected/quantified visually using a microscope or flow cytometry apparatus.
In all of the inventions described herein, the sample may be a sputum sample obtained from a mammal, including from a human. The human may be known to have tuberculosis, be at risk of tuberculosis, or be suspected as having tuberculosis.
Kits are also provided for the detecting of a mycobacterium in a sample comprising the described recombinant mycobacteriophages, packaging therefor, and written instructions for use thereof.
The recombinant mycobacteriophages of the present invention can be made in multiple ways. Background art for the concept of producing reporter mycobacteriophages is discussed in U.S. Pat. No. 6,300,061, and shuttle phasmids are discussed in U.S. Pat. No. 5,750,384, both of which patents are incorporated by reference in their entirety. With regard to the present recombinant mycobacteriophages, one method of production is described in the Experimental section hereinbelow. An additional method for producing recombinant mycobacteriophages comprises:
a) introducing a vector into a mycobacterium so as to form a lysogen, which vector comprises (i) a mycobacteriophage promoter nucleic acid and (ii) a heterologous nucleic acid encoding a protein of interest, and wherein (i) is upstream of (ii), and integrated into a vector backbone derived from a mycobacteriophage TM4 and which is temperature-sensitive conditionally propagating;

b) maintaining the lysogen at a temperature that permits the lysogen to replicate into a plurality of lysogens; and then

c) maintaining the plurality of lysogens at a temperature that effects induction in the plurality of lysogens, so as thereby to produce recombinant mycobacteriophages comprising (i) and (ii).

In embodiments of this method of production the vector backbone derived from a mycobacteriophage TM4 comprises a mutation and/or a deletion relative to TM4, in one of, more than one of, or all of (i) a gp48 gene, (ii) a gp66 gene and (iii) a gp20 gene of TM4, which mutation(s) and/or a deletion(s) confer temperature-sensitivity of phage propagation. In embodiments, the vector backbone derived from a mycobacteriophage TM4 comprises a mutation and/or a deletion relative to TM4 that confers temperature-sensitivity of repression, and mutation and/or a deletion relative to TM4 that confers conditional-sensitivity of a lysis function thereof. In an embodiment, the method further comprises recovering the recombinant mycobacteriophages produced by a means available in the art. One such way is to purify the recombinant mycobacteriophages on a CsCl gradient. In an embodiment of the methods of production, the plurality of lysogens is induced by subjecting the plurality to a temperature that releases repression and/or permits lysis. In an embodiment, the mycobacterium of the method of production is M. smegmatis. In an embodiment of the methods of production, the temperature-sensitivity results in replication of the mycobacteriophages in the mycobacterium at 30° C. but not at 37° C.
In an embodiment of the methods, the vector backbone of the isolated recombinant mycobacteriophage does not comprise TM4 gene 49. In an embodiment, the vector backbone comprises a TM4 genomic sequence having a 5.5 kbp to 6.5 kbp deletion of non-essential DNA thereof. In an embodiment, the vector backbone does not comprise a portion having the sequence of residues 33,877 through 39,722 of the TM4 genome. In an embodiment, the vector backbone:
(i) does not comprise a portion having the sequence of gene 49 through the first sixteen codons of gene 64 of the TM4 genome;
(ii) does not comprise a portion having the sequence of the last twelve codons of gene 48 of the TM4 genome;
(iii) does not encode a proline at a residue equivalent to residue 220 of gene product gp48 of the TM4 genome;
(iv) encodes a serine at a residue equivalent to residue 220 of gene product gp48 of the TM4 genome;
(v) does not encode an alanine at a residue equivalent to residue 131 of gene product gp66 of the TM4 genome;
(vi) encodes a threonine at a residue equivalent to residue 131 of gene product gp66 of the TM4 genome;
(vii) does not encode an arginine at a residue equivalent to residue 116 of gene product gp20 of the TM4 genome; and/or
(viii) encodes a cysteine at a residue equivalent to residue 116 of gene product gp20 of the TM4 genome.
In an embodiment, the vector backbone comprises the sequence set forth in Genbank Accession No. JF937104; JF704106; JF704105; HM152764; or HM152767. These backbones confer preferred temperature sensitivities/conditional propagation. In embodiments, the vector backbone is a phAE159 vector or a ph101 vector.
In the methods described, unless otherwise indicated, any mycobacteriophage promoter known in the art may be employed. In a preferred embodiment, the mycobacteriophage promoter nucleic acid is a PLeft lytic promoter of mycobacteriophage L5.
Treatment of samples for analysis with fluorophages is known in the art (e.g. see [13]) and can be used with the methods described herein. However, a novel and preferred method for treating sputum samples for infection by the isolated recombinant mycobacteriophages of the invention is provided by this invention comprising: a) mixing the sputum sample with diluted dithiothreitol in phosphate buffer; b) vortexing the resultant product; c) allowing the product resulting from b) to rest; d) centrifuging the product resulting from c); e) decanting the supernatant; f) mixing remainder with a diluted sodium hydroxide solution; g) vortexing the product resulting from f); h) allowing the product resulting from g) to rest; i) adding phosphate buffer saline and centrifuging the resulting product; j) re-suspending resultant pellet in a mycobacterium-supporting broth.
In embodiments, the sputum sample is mixed with 30-70% diluted dithiothreitol. In a preferred embodiment, the sputum sample is mixed with 35-60% diluted dithiothreitol. In a most preferred embodiment, the sputum sample is mixed with 50% diluted dithiothreitol. In a further preferred embodiment, the sputum sample is mixed with an equal volume of the diluted dithiothreitol.
In embodiments, in step b) the product is vortexed at 2000-6000 rpm. In a preferred embodiment, the product is vortexed at 3500-5000 rpm. In a most preferred embodiment, the product is vortexed at 4000 rpm. In embodiments, in step b) the product is vortexed for 1-25 mins. In a preferred embodiment, the product is vortexed for 5-15 mins. In a most preferred embodiment, the product is vortexed for about 10 mins or for 10 mins.
In an embodiment, in step f) the remainder is mixed with 0.4-2.0% NaOH. In a preferred embodiment, the remainder is mixed with 0.8-1.6% NaOH. In a most preferred embodiment, the remainder is mixed with 1% NaOH.
In embodiments, in step g) the product is vortexed at 2000-6000 rpm. In a preferred embodiment, the product is vortexed at 3500-5000 rpm. In a most preferred embodiment, the product is vortexed at 4000 rpm.
In embodiments, in step g) the product is vortexed for 10-30 mins. In a preferred embodiment, the product is vortexed for 15-25 mins. In a most preferred, embodiment the product is vortexed for about 20 mins or for 20 mins.
In one preferred embodiment, the method comprises: a) mixing the sputum sample with an equal volume of 50% diluted dithiothreitol in phosphate buffer; b) vortexing the resultant product; c) allowing the product resulting from b) to rest; d) centrifuging the product resulting from c) at 4000 RPM for about 10 minutes; e) decanting the supernatant; f) mixing remainder with 1% sodium hydroxide solution; g) vortexing the product resulting from f); h) allowing the product resulting from g) to rest; i) adding phosphate buffer saline and centrifuging the resultant at 4000 RPM for about 20 minutes; j) re-suspending resultant pellet in a suitable mycobacterium-supporting broth; k) adding reporter phage and incubating therewith at 37° C. One suitable broth well known in the art is Middlebrook 7H9 Broth (for example, available from BD, Franklin Lakes, N.J.).
In the inventions described herein, the mycobacteria may be M. tuberculosis, M. smegmatis, M. bovis, M. bovis-BCG, or any other known mycobacteria, including those described hereinabove.
The methods disclosed herein involving subjects can be used with any mammalian subject. Preferably, the mammal is a human.
All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
This invention will be better understood from the Experimental Details, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.
EXPERIMENTAL RESULTS
Introduction
To date, the key limitations of reporter phages have been (a) their relatively poor reporter signals, which require a relatively long exposure time to discern fluorescent mycobacteria from cells displaying autofluorescence, and (b) the incompatibility of common sputum processing methods with phage infection. Herein a new unexpectedly high-intensity fluorophage is disclosed for rapid and inexpensive point-of-care diagnostic tool and as a tool for drug susceptibility testing of M. tuberculosis in clinical sputum samples. This high-intensity fluorophage for the first time allows direct optics-to-human microscopic observation of phage-encoded fluorescence of M. tuberculosis cells in sputum samples. The fluorophage permitted M. tuberculosis diagnosis made at 12 hr and drug susceptibility testing results were complete within 36 hr of sputum collection from TB patients.
Materials and Methods
Generation of high-intensity fluorophage: Vector pYUB1378 was generated by cloning the mVenus gene downstream of hsp60 promoter in pMV261 [19] (mVenus obtainable from IKEN Brain Science Institute, Saitama, JAPAN-Atsushi Miyawaki). (Venus is disclosed in Nagai T et al., Nat. Biotechnol. 2002 January; 20(1):87-90, A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications). Vector pYUB1228 was generated for reporter phage construction by introducing a lambda cos site and a unique PacI site at the BspH1 site in pEXP 1-DEST (Invitrogen). The hsp60-mVenus cassette was cloned from pYUB1378 into pYUB1228 at BglII-NaeI sites to generate pYUB1167. Vector pYUB1361 encoding mVenus gene from T7 promoter was generated by replacing the hsp60 promoter with T7 promoter using BglII and NdeI sites. Plasmid pYUB1383 was generated by cloning T7 polymerase gene [20, 21] upstream of pYUB1361 at BglII site. The L5 promoter [22] was cloned in the forward orientation upstream of the mVenus gene between the BglII and NdeI site in plasmid pYUB1167 to generate pYUB1391. The plasmids pYUB1167, pYUB1361, pYUB1383 and pYUB1391 were cloned individually into phAE159 at a unique PacI site to obtain phasmids [23]. M. smegmatis mc2155 [24] was used to generate the reporter phage using the method described previously [13]. The phasmids harboring pYUB1167, PYUB1168, pYUB1361, pYUB1383, and pYUB1391 resulted in fluorophages φ2GFP7, φ2GFP8, φ2GFP9, and φ2GFP10, respectively (FIG. 1A). Individual plaques were picked and propagated at 30° C. to obtain high-titer fluorophages [23].
Infection of Mycobacterial strains with fluorophages: M. tuberculosis mc26230 was used as the drug-sensitive strain [25]. Drug-resistant mutants of M. tuberculosis mc26230 were isolated by selecting cells on 7H10 plates in the presence of various drugs at 5 times the MIC value [26, 27]. M. tuberculosis mc27201, mc27202 and mc27203 are the strains resistant to rifampicin, kanamycin, or both rifampicin and kanamycin respectively. Mycobacterial cells were grown to an OD600 nm of 0.8-1.0 in 7H9+OADC+0.05% Tween. Before infection, the cells were washed twice with MP buffer and re-suspended in 7H9+OADC to an OD600 nm of 1.0. 200 μl of fluorophage was added to 5 μl of cells. The samples were incubated for 12 hr at 37° C. The cells were visualized on a fluorescence microscope (Nikon Ti) using a DIC filter for bright field and a FITC filter for green fluorescence. Fluorescent bacilli after incubation with phage indicate the presence of mycobacteria. To determine relative fluorescence and the percentage of cells infected as a function of the multiplicity of infection (MOI), M. tuberculosis mc26230 cells were infected as described above with the appropriate phage dilution to obtain an MOI of 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, and 0.00001. After infection, cells were acquired using flow cytometry and the data was analyzed using FlowJo 7.6.1.
When performing drug susceptibility testing (DST), antibiotics were added simultaneously with phage or pre-incubated as noted in the respective experiments. Antibiotics used for DST were kanamycin (4 μg/ml), rifampicin (2 μg/ml), isoniazid (0.4 μg/ml) and ofloxacin (10 μg/ml). 200 μl of fluorophage (MOI=100) was added to 5 μl of cells. After phage addition samples were incubated for 12 hr at 37° C. and either analyzed on FACS caliber to quantitate fluorescence per cell and to determine the percentage of fluorescent cells or on a fluorescence microscope. Some samples were fixed in 2% paraformaldehyde before assay and this did not appreciably change fluorescence. Absence of fluorescence in the presence of antibiotic and phage indicated a drug-sensitive sample. Fluorescence in the presence of antibiotic and phage indicated that the sample was resistant to that particular drug.
M. tuberculosis detection and DST in sputum samples: Anonymous, de-identified sputum samples were collected from smear-positive TB patients in Durban, South Africa prior to initiation of treatment. Samples were treated using a standard protocol, except that the concentration of NaOH was reduced to 0.625%. Samples were re-suspended in 500 μl of 7H9+OADC. 100 μl of sample was used for Ziehl Neelsen staining and 100 μl of sample was incubated with 500 μl of phage in the presence or absence of antibiotic. After incubation for 12 hr at 37° C., the samples were fixed in 2% paraformaldehyde for 3 hr. The samples were then centrifuged, re-suspended in 10 μl of MP buffer, and analyzed by fluorescence microscopy.
Results
Development of high-intensity fluorophage: The first generation fluorophages provided an important proof-of-principle for the use of phage-mediated delivery of fluorescent reporter genes as a diagnostic tool for detecting M. tuberculosis and assessing drug susceptibilities [17, 28]. However, they were of limited fluorescence and could not be used with clinical samples. To address the main shortfall of that approach, which is poor signal to background ratio, two complementary strategies were investigated, although it was not clear whether they would be successful: 1) a new the vector backbone was engineered to increase cloning capacity, and 2) promoters originating from bacteriophages were used to enhance the expression of reporter genes. The new vector backbone, phAE159, was generated by the deletion of a  ̃6 kb region encompassing genes gp48-64 [29] from the temperature-sensitive mutant of TM4 known as ph101 (FIG. 7, [30]). Similar to parent ph101 and phAE87 [31], shuttle phasmid phAE159 propagates as a phage at 30° C., but at 37° C. will inject its DNA without propagating. The phAE159 can accommodate  ̃10 Kb of recombinant DNA without compromising its ability to form phage particles. First, the monomeric fluorescent protein mVenus [32] was cloned under the hsp60 promoter [19] to generate φ2GFP7 (FIG. 1A). However, the fluorescence intensity obtained from φ2GFP7 was similar to previous reporter phage phAE87::hsp60-EGFP [28] and was at least 10-fold lower than cells harboring the multi-copy plasmid pYUB1378 (FIG. 1B).
The enhanced cloning capacity of phAE159 was used to evaluate various chromosomal promoters from M. tuberculosis and M. smegmatis for their potential improvement of reporter expression. No promoters were found that increased the reporter signal to a level appreciably higher than hsp60 promoter in the phage (data not shown). It was investigated whether phage promoters might be better suited for high expression from mycobacteriophages, since phages have evolved to express large quantities of their structural and lytic proteins in short periods of time. First tested was the late promoter of E. coli T7 phage, which has been extensively used on episomal plasmids for the overexpression of recombinant proteins in E. coli [33, 34]. Fluorophage φ2GFP8 was generated by cloning the T7 phage promoter upstream of mVenus (FIG. 1A). The T7 promoter functions only with its cognate RNA polymerase [21, 33], therefore no fluorescence signal was observed when φ2GFP8 infected M. smegmatis strain (mc2155) lacking T7 polymerase. In contrast, infection of a M. smegmatis strain expressing T7 polymerase (mc24517) with φ2GFP8 resulted in a high level of fluorescence. Fluorescent cells were detected as early as 2 hr after infection. The population of fluorescent cells increased with time, and more than 90% of the cells were fluorescent after 12 hr of infection with φ2GFP8. Following the above results, the T7 polymerase gene was cloned upstream of the T7 promoter in φ2GFP8 to generate φ2GFP9. φ2GFP9 overcame the requirement of a strain to endogenously express T7 polymerase and strong fluorescent signal was obtained when φ2GFP9 infected the wild type M. smegmatis strain (mc2155) (FIG. 1C). Based on these results another reporter phage, φ2GFP10, was generated using PLeft lytic promoter of mycobacteriophage L5 [35]. This strong phage promoter requires only the mycobacterial host RNA polymerase. The comparison of these two phage promoters with hsp60 promoter shows that the fluorescence obtained from φ2GFP10 was almost 100-fold greater than that from cells infected with φ2GFP7 and was marginally better than φ2GFP9 under similar conditions (FIG. 1C). Fluorescent cells were observed after 1 hr of infection with φ2GFP10 and the fluorescence signal increased over time. More than 90% of the cells were fluorescent after 4 and one half hr and resulted in the saturation of the fluorescent detector (FIG. 1D).
Comparison of fluorophages in M. tuberculosis: The attenuated M. tuberculosis strain mc26230 [25] was infected with the reporter phages described in the previous section. Individual bacilli were fluorescent in all cases, but displayed a wide range of fluorescence intensity. M. tuberculosis cells infected with φ2GFP10 required an average exposure time of 15-20 ms to record an image, and φ2GFP9 required an average time of 100-300 ms. Significantly, both φ2GFP9- and φ2GFP10-infected cells could be visualized easily through the microscope eye-piece. By contrast, mc26230 cells infected with φ2GFP7 were not identifiable with optics alone (eyes on the microscope eyepiece with no intervening camera and image processing), and required an average exposure time of 4-5 sec to boost the signal of cells to a similar level (FIG. 2A). Flow cytometry data paralleled microscopy results and showed that mc26230 infected with φ2GFP10 fluoresced at approximately a 100-fold greater intensity than mc26230 infected with φ2GFP7 (FIG. 2B). The fluorescence intensity did not increase significantly after prolonged incubation of φ2GFP7 with mc26230 and significant differences were also observed after 48 hr of φ2GFP7 and φ2GFP10 infected M. tuberculosis mc26230 cells (FIG. 8). Overall, the PLeft promoter of L5 mycobacteriophage in φ2GFP10 was found to drive the strongest reporter expression from the phage.
Next, the relationship of MOI to signal was determined. The proportion of fluorescent cells in a given sample increased with increasing MOI of φ2GFP10 (FIG. 3A). At MOIs of 1, 10, and 100, approximately 38%, 72%, and 95.5% of the cells, respectively, were fluorescent (FIG. 3A). The Mean Fluorescent Intensity (MFI) signal per cell also increased as a function of MOI (FIG. 3B). The increase in this ratio implies that a single mycobacterial cell can be incited to express product from more than one reporter phage. M. smegmatis or M. tuberculosis cells infected with other fluorophages φ2GFP7, φ2GFP9) also resulted in increased MFI with increased MOI (data not shown) suggesting that the new vector backbone supports multiple infections from the same phage. Based on the results, an MOI of 100 was used in subsequent experiments.
Phenotypic drug susceptibility testing using fluorophage φ2GFP10. Reporter phage have previously been shown to allow the assay of drug sensitivity in laboratory cultures. The reporter signal is unchanged by antibiotic in a genetically resistant strain but in a sensitive strain the reporter signal is greatly attenuated by the antibiotic [18]. Drug-susceptible M. tuberculosis mc26230 was incubated with antibiotics, and phage φ2GFP10 was added either simultaneously (i.e., at time t=0), or after 3 hr or 6 hr preincubation with antibiotic. The proportion of fluorescent population was determined in each case 12 hr after φ2GFP10 addition. In the absence of drug treatment, approximately 90% of M. tuberculosis cells were positive for fluorescence after incubation with φ2GFP10 by both flow cytometry and microscopy (FIG. 4A). When rifampicin or kanamycin were added to cells along with φ2GFP10 (time t=0), fluorescence of the M. tuberculosis cells diminished by approximately 85%, as analyzed by flow cytometry. The remaining 10-15% of cells were just above the cut-off level on the FACS plot and were not detected by fluorescence microscopy (FIG. 4A). Longer pre-incubation with these drugs (t=3 hr and t=6 hr) did not significantly alter the results indicating that both the drug and the phage can be added simultaneously at the time of assay (FIG. 9). In contrast, isoniazid-inhibition of fluorescence was sensitive to the time of addition. When isoniazid was added simultaneously with the phage (t=0), fluorescent cells were detected by both flow cytometry and microscopy, although MFI was lower than the untreated control (FIG. 4A). Isoniazid pre-treatment for 18 hr prior to phage addition increased the difference between treated and non-treated samples. Ofloxacin behaved similarly to isoniazid, in that an 18 hr pretreatment with antibiotic increased the signal differences in DST (FIG. 10).
The fluorescence pattern of the rifampicin-resistant strain mc27201 after infection with φ2GFP10 was similar in the presence and absence of rifampicin. Rifampicin resistance did not change the result of treatment with kanamycin (FIG. 4B). Conversely, for kanamycin-resistant M. tuberculosis, mc27202 fluorescence decreased in the presence of rifampicin, but not in the presence of kanamycin (FIG. 4C). Fluorescence results consistently agreed with CFU plating (data not shown). M. tuberculosis mc27203 is doubly-resistant to kanamycin and rifampicin and its fluorescence intensity did not change in the presence of either antibiotic (FIG. 4D). These results show that φ2GFP10 distinguishes M. tuberculosis strains that are resistant to a single drug from those strains that are resistant to multiple drugs.
Detection of M. tuberculosis cells in spiked sputum samples with φ2GFP10. The high signal per cell encouraged us to proceed with reconstruction experiments in which defined laboratory grown log phase mycobacteria were spiked into human sputum. A pool of sputum from healthy volunteers was spiked with 104 M. tuberculosis cells per ml, processed with NALC and NaOH, and incubated for 12 hr with φ2GFP10. A fluorescent signal was readily observed if and only if M. tuberculosis cells were present. Processing with NALC alone or with NALC with 0.5% NaOH did not decrease the reporter signal (FIG. 5). However, fluorescence intensity/cell decreased 10-fold when samples were processed with a higher concentration of NaOH (1-2%) prior to phage incubation (FIG. 5). Treatment with 0.5% NaOH was sufficient to eliminate outgrowth of non-mycobacterial species during the assay period (data not shown). These data show that with φ2GFP10, 90% of M. tuberculosis cells were detected in the spiked sputum samples (FIG. 5).
Detection of M. tuberculosis cells in clinical samples: Considering the differences in metabolism, as reflected in the transcriptome of laboratory-grown M. tuberculosis compared to the expression thought to occur in patient sputum samples [36], it was not certain that φ2GFP10 would be able to detect bacilli in clinical specimens. Clinical sputums also have variable amounts of background fluorescence which make further demands on signal strength. Sputum samples with smear-positive TB were obtained from patients in Durban, South Africa. These were processed using the procedure detailed above in the spiking experiments, and incubated with fluorophage for 12 hr at 37° C. Fluorescent bacilli were readily detected by fluorescence microscopy (FIG. 6). The total time from sputum collection to detection was approximately 14 hr.
DST of the clinical M. tuberculosis was determined for kanamycin and rifampicin in parallel as shown in FIG. 6. Results obtained after 14 hr by fluorophage indicated that the strains were sensitive to both of the drugs and were in 100% agreement with the results of DST determined using the agar proportion method, which were available only after 3 weeks.
Treatment of Clinical Specimen: An improved protocol for preparing decontaminating and liquefying clinical sputum specimens prior to phage infection has been determined. This non-limiting protocol describes the steps for processing the suspected or known Tuberculosis (TB) clinical sputum sample in order to allow for reporter phage infection and direct visualization of Mycobacterium tuberculosis. The advanced protocol allows sufficient liquefaction for processing in a clinical flow cytometer and decontaminates the specimen without inhibiting reporter phage infection.
1. Add equal volume 50% diluted Dithiothreitol in phosphate buffer (Sputuolysin) to sputum sample;
2. Vortex×30 secs.-1 minute;
3. If sample still mucoid, add another equal volume of 50% diluted Dithiothreitol in phosphate buffer;
4. Let sit for 10 minutes;
5. Centrifuge at 4000 RPM×10 minutes;
6. Decant supernatant;
7. Add 5 ml 1% sodium hydroxide solution;
8. Vortex×30 secs.;
9. Let sit for 15 minutes;
10. Add PBS to make up volume to 10 ml;
11. Centrifuge at 4000 RPM×20 minutes;
12. Re-suspend pellet in 250 μl 7H9;
13. Transfer 100 microliters to microcentrifuge tube;
14. Add 200 microliters of reporter phage, e.g. fluorophage;
15. Place in 37° C. incubator overnight
16. Conduct analysis of sample to detect Mycobacterium tuberculosis using, for example, either fluorescent microscopy or flow cytometry.
Discussion
A high-intensity fluorophage, φ2GFP10 with a mycobacteriophage L5 promoter driving a GFP derivative, yielded 100-fold more per-cell signal than previously described GFP reporter phage [28]; it also allowed real-time visualization using standard fluorescence microscopy of individual M. tuberculosis bacilli in primary clinical sputum samples. It is speculated that the phage promoter works so well because phages have been selected during evolution to achieve the maximum level of gene expression in the shortest amount of time. For example, mycobacteriophage D29 yields 120 new phages from a single infected cell of M. tuberculosis in 3 hr; the doubling time of M. tuberculosis itself is approximately 24 hr [37]. The new reporter phage vector, based on mycobacteriophage TM4, is also deleted for TM4 gp49 a gene that may inhibit bacteriophage superinfection [38]. Deletion of gp49 might be responsible for the enhanced the per cell signal at greater MOI (FIG. 3). Unlike all previous reporter phage φ2GFP10 illuminated M. tuberculosis directly from clinical sputum samples without prior culture. The immediate assay of clinical samples has not been possible with any previous reporter phage because the background fluorescence was too large compared to the weaker signal from the reporter phage.
DST by φ2GFP10 has advantages over both nucleic acid tests, such as MTB/RIF, and culture assays. Nucleic acid tests are dependent upon the foreknowledge of a specified candidate sequence leading to drug resistance. DST by culture is not limited to known genotypes but does requires 4-8 weeks for classical culture and 1-2 weeks for MODS [39]. The reporter phage φ2GFP10 yielded DST results from primary clinical samples within 36 hr. Rifampicin and kanamycin (which block transcription and translation, respectively) inhibited reporter gene expression in sensitive cells when drug and phage were added concurrently at time zero and gave antibiotic susceptibility results in 12 hr. The strongest differential signal for isoniazid and ofloxacin (which block mycolic acid biosynthesis and DNA replication, respectively) required pretreatment of bacilli for 18 hr prior to phage addition and the DST results were obtained 36 hr after drug addition to the bacteria. Isoniazid and ofloxacin disrupt mycolic acid metabolism and DNA replication respectively. Since φ2GFP10 assesses the transcription and translation ability of the cells, we reason that the inhibition of transcription and translation are later and secondary effects of these drugs. All four drugs displayed similar kinetics of killing, as measured by CFU, and in all cases a drop in CFU was observed only after 48 hr of antibiotic treatment. Thus φ2GFP10 detects the inhibition of essential metabolic processes prior to the inhibition becoming irreversible. Thus φ2GFP10 gives an extremely rapid but absolutely predictive DST.
Two results with φ2GFP10 imply distinct metabolic subpopulations within log phase cultures of M. tuberculosis: 1) When isoniazid and phage were added together at time zero, the average fluorescence measured at 12 hr decreased by 64%, yet a subpopulation of cells retained maximal fluorescence (FIG. 4). 2) In experiments performed to determine the optimum MOI of infection the whole population shifted to a higher MFI with increasing MOI, yet the population spread of dynamic range of fluorescence intensity per cell, i.e. the variance around the MFI, did not change with increasing MOI (FIG. 3A: compare MOI 10 versus 100).
According to the persister model a metabolically less active subpopulation survives transient antibiotic exposure [40]. An alternative possibility is that a rapidly metabolizing subpopulation survives e.g. by inducing a drug efflux pump [41]. Further work will be required to learn if persisters are enriched in one or the other metabolic subpopulations identified by φ2GFP10 and to distinguish these alternative mechanisms for persistence in M. tuberculosis. 
The fluorescence microscope and appropriate filters needed for DST with φ2GFP10 are already included in the recommended diagnostic equipment for laboratories in resource-poor settings. Reagents are inexpensive and could be renewed locally by growing fluorophage stocks. Fluorophages have potential as a point-of-care test for resource poor-settings, since M. tuberculosis diagnosis can be available within as little as 12 hr and complete DST completed within 36 hr.
To the best of applicants' knowledge, φ2GFP10 offers the most rapid assessment of metabolic inhibition by antimicrobials against M. tuberculosis. The disappearance of acid-fast bacteria in sputum, loss of CFU, and diminution of mycobacterial DNA detected through PCR are all expected to occur weeks or months after metabolic inhibition. The applications of φ2GFP10 extend beyond initial diagnosis. Treatment efficacy is currently followed by sputum conversion from positive to negative and is typically noted at two months. φ2GFP10 allows a much earlier indication of treatment efficacy or failure. Shortening the time to determine if initial treatment is working can improve the efficiency of follow up and of EBA (Early Bactericidal Activity) trials.
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## Claims

1. An isolated recombinant mycobacteriophage comprising a vector backbone into which (a) a heterologous mycobacteriophage promoter nucleic acid and (b) a heterologous nucleic acid encoding a protein of interest are integrated, and wherein (a) is upstream of (b) and operatively linked thereto, and wherein the vector backbone is derived from a mycobacteriophage TM4 and comprises a TM4 genomic sequence having a 5.5 kbp to 6.5 kbp deletion of non-essential DNA thereof, and wherein the recombinant mycobacteriophage is conditionally propagating.

2. The isolated recombinant mycobacteriophage of claim 1 wherein the heterologous mycobacteriophage promoter nucleic acid is a heterologous PLeft lytic promoter of mycobacteriophage L5.

3. The isolated recombinant mycobacteriophage of claim 1, wherein the vector backbone does not comprise TM4 gene 49.

4. The isolated recombinant mycobacteriophage of claim 1, wherein the vector backbone derived from a mycobacteriophage TM4 does not comprise a portion having the sequence of residues 33,877 through 39,722 of the TM4 genome.

5. The isolated recombinant mycobacteriophage of claim 1, wherein the vector backbone comprises the nucleic acid sequence set forth in Genbank Accession No. JF937104 (SEQ ID NO:4); JF704106 (SEQ ID NO:5); JF704105 (SEQ ID NO:6); HM152764 (SEQ ID NO:7); or HM152767 (SEQ ID NO:8).

6. The isolated recombinant mycobacteriophage of claim 1, wherein the mycobacteriophage is a temperature-sensitive conditionally propagating mutant.

7. The isolated recombinant mycobacteriophage of claim 1, wherein the recombinant mycobacteriophage does not propagate in a mycobacteria at 37° C.

8. The isolated recombinant mycobacteriophage of claim 1, wherein the recombinant mycobacteriophage propagates in a mycobacteria at 30° C.

9. A method of detecting a mycobacterium in a sample comprising:
a) acting the sample with the isolated recombinant mycobacteriophage of claim 1 under conditions permitting the mycobacteriophage to infect a mycobacterium present in the sample and the protein of interest to be expressed in the mycobacterium; and
b) detecting the protein of interest in the mycobacterium in the sample, thereby detecting the mycobacterium in the sample.

10. A method of determining if a mycobacterium is susceptible to a candidate agent comprising:
a) contacting a sample comprising the mycobacterium with the isolated recombinant mycobacteriophage of claim 1 in (i) the absence of the candidate agent and (ii) in the presence of the candidate agent, under conditions permitting the mycobacteriophage to infect a mycobacterium present in the sample and the protein of interest to be expressed in the mycobacterium;
b) detecting the protein of interest in the mycobacterium in the sample in (i) and (ii); and
c) comparing the amount of the protein of interest detected in the presence of the candidate agent and the absence of the candidate agent,

wherein a decrease in amount of the protein of interest in the presence of the candidate agent compared to in the absence of the candidate agent indicates that the mycobacterium is susceptible to the candidate agent.

11. A method of identifying a candidate agent as effective in treating an infection caused by a strain of mycobacterium, the method comprising:
culturing a mycobacterium with the isolated recombinant mycobacteriophage of claim 1 in (a) the absence of the candidate agent under conditions permitting the mycobacteriophage to infect a mycobacterium present in the sample and the protein of interest to be expressed in the mycobacterium, and (b) the presence of the candidate agent under conditions permitting the mycobacteriophage to infect a mycobacterium present in the sample and the protein of interest to be expressed in the mycobacterium, and
detecting the protein of interest in the mycobacterium in the sample in (a) and in (b), and comparing the amount of the protein of interest detected in the presence of the candidate agent and the absence of the candidate agent,
wherein a decrease in the amount of the protein of interest in the presence of the candidate agent compared to in the absence of the candidate agent indicates the agent as effective to treat an infection caused by the strain of mycobacterium.

12. The method of claim 10, wherein the candidate agent is an antibiotic.

13. The method of claim 9, wherein the mycobacteria is a Mycobacterium tuberculosis.

14. A kit for the detecting of a mycobacterium in a sample comprising the mycobacteriophage of claim 1, packaging therefor and written instructions for use thereof.