Recombinant K2 bacteriophages and uses thereof

Patent metadata

Field	Value
Publication number	US-10174294-B1
Application number	US-201815908162-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 K2 bacteriophages, methods for making the same, and uses thereof. The recombinant K2 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 K2 bacteriophage nucleic acid sequence, wherein a heterologous nucleic acid sequence is inserted into the nucleic acid sequence between (a) position 25,443 and 25,444 of SEQ ID NO: 1 or (b) position 75,922 and 75,923 of SEQ ID NO: 1 wherein the heterologous nucleic acid sequence comprises 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 K2 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 K2 bacteriophage nucleic acid sequence of claim 2 , wherein the expression control sequence is an inducible promoter or a constitutive promoter.

4. The recombinant K2 bacteriophage nucleic acid sequence of claim 1 , wherein the fluorescent protein is selected from the group consisting of TagBFP, Azurite, EBFP2, mKalamal, 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 K2 bacteriophage nucleic acid sequence of claim 1 , wherein the chemiluminescent protein is β-galactosidase, horseradish peroxidase (HRP), or alkaline phosphatase.

6. The recombinant K2 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 K2 bacteriophage nucleic acid sequence of claim 6 , wherein the bioluminescent protein is nanoluciferase.

8. A recombinant K2 bacteriophage comprising the recombinant K2 bacteriophage nucleic acid sequence of claim 1 .

9. A recombinant K2 bacteriophage comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 23 and SEQ ID NO: 24.

10. The recombinant K2 bacteriophage of claim 8 , wherein the bacteriophage specifically infects E. coli strains that express K2 capsule genes.

11. A bacterial host cell comprising the recombinant K2 bacteriophage of claim 8 .

12. A vector comprising the recombinant K2 bacteriophage nucleic acid sequence of claim 1 .

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

14. The bacterial host cell of claim 13 , wherein the host cell expresses K2 capsule genes.

15. The bacterial host cell of claim 13 , wherein the host cell is a natural or non-natural host for K2 bacteriophage.

16. A kit comprising one or more coded/labeled vials that contain the recombinant K2 bacteriophage of claim 8 , instructions for use, and optionally at least one antibiotic.

17. A method for identifying at least one bacterial strain or species that expresses K2 capsule genes in a test sample obtained from a subject comprising
(a) infecting the test sample comprising bacterial cells with the recombinant K2 bacteriophage of claim 8 ; and
(b) detecting the expression of the reporter protein of the recombinant K2 bacteriophage, wherein detection of the reporter protein indicates the presence of at least one bacterial strain or species that expresses K2 capsule genes in the test sample.

18. 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 test sample comprising bacterial cells with the recombinant K2 bacteriophage.

19. 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 K2 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 K2 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 K2 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 K2 phage.

20. The method of claim 19 , wherein the antibiotic is selected from the group consisting of rifampicin, tetracycline, 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.

21. The method of claim 19 , wherein the bacterial strain or species in the test sample expresses K2 capsule genes.

22. The method of claim 19 , 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 K2 bacteriophage.

Assignees

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

Inventors

• Mcbrine Connor
• Kourepenos Georgiana
• Holder Jason

Classifications

• C12N2795/00043

  viral genome or elements thereof as genetic vector · CPC title

• C12Q1/025

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

• C12N2795/00021

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

• C12N15/74

  Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora · CPC title