Recombinant PB1 bacteriophages and uses thereof

Patent metadata

Field	Value
Publication number	US-10174293-B1
Application number	US-201815908140-A
Country	US
Kind code	B1
Filing date	Feb 28, 2018
Priority date	Feb 28, 2018
Publication date	Jan 8, 2019
Grant date	Jan 8, 2019

Abstract

The present disclosure provides compositions including recombinant PB1 bacteriophages, methods for making the same, and uses thereof. The recombinant PB1 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 PB1 bacteriophage nucleic acid sequence, wherein the nucleic acid sequence is inserted between position 28,851 and 28,852 of SEQ ID NO: 1 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, a chemiluminescent protein, or any combination thereof.

2. The recombinant PB1 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 PB1 bacteriophage nucleic acid sequence of claim 2 , wherein the expression control sequence is an inducible promoter or a constitutive promoter.

4. The recombinant PB1 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, mKOK, 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, PAmCherryl, PATagRFP, Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, and Dronpa.

5. The recombinant PB1 bacteriophage nucleic acid sequence of claim 1 , wherein the chemiluminescent protein is β-galactosidase, horseradish peroxidase (HRP), or alkaline phosphatase.

6. The recombinant PB1 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 PB1 bacteriophage nucleic acid sequence of claim 6 , wherein the bioluminescent protein is nanoluciferase.

8. A recombinant PB1 bacteriophage comprising the recombinant PB1 bacteriophage nucleic acid sequence of claim 1 .

9. A recombinant PB1 bacteriophage comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

10. A bacterial host cell comprising the recombinant PB1 bacteriophage of claim 8 .

11. A vector comprising the recombinant PB1 bacteriophage nucleic acid sequence of claim 1 .

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

13. The bacterial host cell of claim 10 , wherein the host cell is a natural or non-natural host for PB1 bacteriophage.

14. The bacterial host cell of claim 12 , wherein the host cell is a natural or non-natural host for PB1 bacteriophage.

15. A kit comprising one or more coded/labeled vials that contain the recombinant PB1 bacteriophage of claim 8 , instructions for use, and optionally at least one antibiotic.

16. A method for identifying at least one bacterial strain or species in a test sample obtained from a subject comprising
(a) contacting the test sample comprising bacterial cells with the recombinant PB1 bacteriophage of claim 8 ; and
(b) detecting the expression of the reporter protein of the recombinant PB1 bacteriophage, wherein detection of the reporter protein indicates the presence of at least one bacterial strain or species in the test sample.

17. The method of claim 16 , 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 contacting the test sample comprising bacterial cells with the recombinant PB1 bacteriophage.

18. 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 PB1 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 PB1 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 PB1 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 PB1 bacteriophage.

19. The method of claim 18 , 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.

20. The method of claim 18 , 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 PB1 bacteriophage.

21. The method of claim 18 , wherein the test sample is blood, sputum, mucus, lavage, saliva, or a swab obtained from the subject.

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

23. A method for making the recombinant PB1 bacteriophage of claim 1 comprising (a) contacting a first PB1 bacteriophage genome comprising a single first recognition site with a first restriction enzyme in vitro under con

Assignees

• Charles Stark Draper Laboratory Inc
• Charles Stark Draper Laboratory Inc

Inventors

• Gruszka Sarah
• Dow Parker
• Holder Jason

Classifications

• C12Q1/025

  for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics (antimicrobial activity C12Q1/18) · CPC title

• C12N2795/00043

  viral genome or elements thereof as genetic vector · CPC title

• C12N2795/00021

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

• C12Q1/04

  Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor {(C12Q1/6897 takes precedence)} · CPC title