Recombinant B11 bacteriophages and uses thereof

Patent metadata

Field	Value
Publication number	US-10329539-B1
Application number	US-201815908197-A
Country	US
Kind code	B1
Filing date	Feb 28, 2018
Priority date	Feb 28, 2018
Publication date	Jun 25, 2019
Grant date	Jun 25, 2019

Abstract

The present disclosure provides compositions including recombinant B11 bacteriophages, methods for making the same, and uses thereof. The recombinant B11 bacteriophages disclosed herein are useful for the identification and/or antibiotic susceptibility profiling of specific bacterial strains/species present in a sample.

First claim

The invention claimed is:

1. A recombinant B11 bacteriophage nucleic acid sequence, wherein the nucleic acid sequence between position 65,939 and position 65,940 of SEQ ID NO: 1 is replaced with a heterologous nucleic acid sequence comprising an open reading frame that encodes a reporter protein, wherein the reporter protein is a bioluminescent protein, a fluorescent protein, or a chemiluminescent protein.

2. The recombinant B11 bacteriophage nucleic acid sequence of claim 1 , wherein the open reading frame of the heterologous nucleic acid sequence is operably linked to an expression control sequence that is capable of directing expression of the reporter protein.

3. The recombinant B11 bacteriophage nucleic acid sequence of claim 2 , wherein the expression control sequence is an inducible promoter or a constitutive promoter.

4. The recombinant B11 bacteriophage nucleic acid sequence of claim 1 , wherein the fluorescent protein is selected from the group consisting of TagBFP, Azurite, EBFP2, mKalama1, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi-Cyan, TagCFP, mTFP1, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira-Orange, mKOκ, mKO2, mOrange, mOrange2, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP, mKeima Red, LSS-mKate1, LSS-mKate2, PA-GFP, PAmCherry1, PATagRFP, Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, or Dronpa.

5. The recombinant B11 bacteriophage nucleic acid sequence of claim 1 , wherein the chemiluminescent protein is β-galactosidase, horseradish peroxidase (HRP), or alkaline phosphatase.

6. The recombinant B11 bacteriophage nucleic acid sequence of claim 1 , wherein the bioluminescent protein is Aequorin, firefly luciferase, Renilla luciferase, red luciferase, luxAB, or nanoluciferase.

7. The recombinant B11 bacteriophage nucleic acid sequence of claim 6 , wherein the bioluminescent protein is nanoluciferase.

8. A recombinant B11 bacteriophage comprising the recombinant B11 bacteriophage nucleic acid sequence of claim 1 .

9. A bacterial host cell comprising the recombinant B11 bacteriophage of claim 8 .

10. A vector comprising the recombinant B11 bacteriophage nucleic acid sequence of claim 1 .

11. A bacterial host cell comprising the vector of claim 10 .

12. The bacterial host cell of claim 9 , wherein the host cell is a natural or non-natural host for B11 bacteriophage.

13. The bacterial host cell of claim 11 , wherein the host cell is a natural or non-natural host for B11 bacteriophage.

14. A kit comprising one or more coded/labeled vials that contain the recombinant B11 bacteriophage of claim 8 , instructions for use, and optionally at least one antibiotic.

15. A method for identifying at least one bacterial strain or species in a test sample obtained from a subject comprising
(a) infecting the test sample comprising bacterial cells with the recombinant B11 bacteriophage of claim 8 ; and
(b) detecting the expression of the reporter protein of the recombinant B11 bacteriophage, wherein detection of the reporter protein indicates the presence of at least one bacterial strain or species in the test sample.

16. The method of claim 15 , wherein the expression of the reporter protein is measured in about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 120 minutes after infecting the test sample comprising bacterial cells with the recombinant B11 bacteriophage.

17. A method for determining the antibiotic susceptibility of a bacterial strain or species in a test sample obtained from a subject comprising
(a) infecting a plurality of test samples comprising bacterial cells with the recombinant B11 bacteriophage of claim 8 and an antibiotic, wherein the plurality of test samples is derived from the subject;
(b) detecting the expression of the reporter protein of the recombinant B11 bacteriophage in the plurality of test samples; and
(c) determining that the antibiotic is effective in inhibiting the bacterial strain or species in a test sample when the reporter protein expression levels of the recombinant B11 phage infected bacterial cells in the test sample are reduced relative to that observed in an untreated control sample comprising bacterial cells, wherein the untreated control sample is derived from the subject and is infected with the recombinant B11 bacteriophage.

18. The method of claim 17 , wherein the antibiotic is selected from the group consisting of rifampicin, tetracycline, levofloxacin, ampicillin, penicillin G, methicillin, oxacillin, amoxicillin, cefadroxil, ceforanid, cefotaxime, ceftriaxone, doxycycline, minocycline, amikacin, gentamicin, levofloxacin, kanamycin, neomycin, streptomycin, tobramycin, azithromycin, clarithromycin, erythromycin, ciprofloxacin, lomefloxacin, norfloxacin, chloramphenicol, clindamycin, cycloserine, isoniazid, rifampin, teicoplanin, quinupristin/dalfopristin, linezolid, pristinamycin, ceftobiprole, ceftaroline, dalbavancin, daptomycin, mupirocin, oritavancin, tedizolid, telavancin, tigecycline, ceftazidime, cefepime, piperacillin, ticarcillin, virginiamycin, netilmicin, paromomycin, spectinomycin, geldanamycin, herbimycin, rifaximin, loracarbef, ertapenem, doripenem, imipenem/cilastatin, meropenem, cefazolin, cefalotin, cephalexin, cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefpodoxime, ceftibuten, ceftizoxime, lincomycin, dirithromycin, roxithromycin, troleandomycin, telithromycin, spiramycin, aztreonam, furazolidone, nitrofurantoin, posizolid, radezolid, torezolid, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, penicillin V, temocillin, bacitracin, colistin, polymyxin B, enoxacin, gatifloxacin, gemifloxacin, moxifloxacin, nalidixic acid, ofloxacin, trovafloxacin, grepafloxacin, sparfloxacin, temafloxacin, mafenide, sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimethoxine, sulfamethizole, sulfamethoxazole, sulfanilimide, sulfasalazine, sulfisoxazole, trimethoprim-sulfamethoxazole (Co-trimoxazole) (TMP-SMX), sulfonamidochrysoidine, demeclocycline, oxytetracycline, clofazimine, dapsone, capreomycin, ethambutol, ethionamide, pyrazinamide, rifabutin, rifapentine, arsphenamine, fosfomycin, fusidic acid, metronidazole, platensimycin, thiamphenicol, tinidazole, trimethoprim(Bs) and vancomycin.

19. The method of claim 17 , wherein the expression of the reporter protein is measured in about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 120 minutes after infecting the plurality of test samples comprising bacterial cells with the recombinant B11 bacteriophage.

20. The method of claim 15 , wherein the test sample is blood, sputum, mucus, lavage, saliva, or a swab obtained from the subject.

21. The method of claim 20 , wherein the subject is human.

Assignees

• Charles Stark Draper Laboratory Inc

Inventors

• Mcbrine Connor
• Kourepenos Georgiana
• Dow Parker
• Holder Jason

Classifications

• C12N2795/10043

  viral genome or elements thereof as genetic vector · CPC title

• C12N2795/10031

  Uses of virus other than therapeutic or vaccine, e.g. disinfectant · CPC title

• C12N7/00 Primary

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• C12N2795/10021

  Viruses as such, e.g. new isolates, mutants or their genomic sequences · CPC title

• C12N15/1086

  Preparation or screening of expression libraries, e.g. reporter assays · CPC title