Recombinant k1-5 bacteriophages and uses thereof

Patent metadata

Field	Value
Publication number	US-2018223262-A1
Application number	US-201815893094-A
Country	US
Kind code	A1
Filing date	Feb 9, 2018
Priority date	Feb 9, 2017
Publication date	Aug 9, 2018
Grant date	—

Abstract

The present disclosure provides compositions including recombinant K1-5 bacteriophages, methods for making the same, and uses thereof. The recombinant K1-5 bacteriophages disclosed herein are useful for the identification and/or antibiotic susceptibility profiling of specific bacterial strains/species present in a sample.

First claim

1 . A recombinant K1-5 bacteriophage nucleic acid sequence, wherein the nucleic acid sequence between (a) position 1,571 and 1,690 of SEQ ID NO: 1 or (b) position 19,979 and 20,165 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, a chemiluminescent protein, or any combination thereof.

2 . The recombinant K1-5 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 K1-5 bacteriophage nucleic acid sequence of claim 2 , wherein the expression control sequence is an inducible promoter or a constitutive promoter.

4 . The recombinant K1-5 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, PAmCherry1, PATagRFP, Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, or Dronpa.

5 . The recombinant K1-5 bacteriophage nucleic acid sequence of claim 1 , wherein the chemiluminescent protein is β-galactosidase, horseradish peroxidase (HRP), or alkaline phosphatase.

6 . The recombinant K1-5 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 K1-5 bacteriophage nucleic acid sequence of claim 6 , wherein the bioluminescent protein is nanoluciferase.

8 . A recombinant K1-5 bacteriophage comprising the recombinant K1-5 bacteriophage nucleic acid sequence of claim 1 .

9 . A recombinant K1-5 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: 10 and SEQ ID NO: 11.

10 . The recombinant K1-5 bacteriophage of claim 8 , wherein the bacteriophage specifically infects E. coli strains that express either K1 or K5 capsule genes.

11 . The recombinant K1-5 bacteriophage of claim 10 , wherein the E. coli strains that express either K1 or K5 capsule genes are selected from the group consisting of ATCC #11775, ATCC #700973, ATCC #23506, ATCC #23508, and MS101.

12 . A bacterial host cell comprising the recombinant K1-5 bacteriophage of claim 8 .

13 . A vector comprising the recombinant K1-5 bacteriophage nucleic acid sequence of claim 1 .

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

15 . The bacterial host cell of claim 12 , wherein the host cell expresses either K1 or K5 capsule genes, and optionally wherein the host cell is a natural or non-natural host for K1-5 bacteriophage.

16 . The bacterial host cell of claim 14 , wherein the host cell expresses either K1 or K5 capsule genes, and optionally wherein the host cell is a natural or non-natural host for K1-5 bacteriophage.

17 . A kit comprising one or more coded/labeled vials that contain the recombinant K1-5 bacteriophage of claim 8 , instructions for use, and optionally at least one antibiotic.

18 . A method for identifying at least one bacterial strain or species that expresses K1 and/or K5 capsule genes in a test sample obtained from a subject comprising
(a) contacting the test sample comprising bacterial cells with the recombinant K1-5 bacteriophage of claim 8 ; and (b) detecting the expression of the reporter protein of the recombinant K1-5 bacteriophage, wherein detection of the reporter protein indicates the presence of at least one bacterial strain or species that expresses K1 and/or K5 capsule genes in the test sample.

19 . 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, or 90 minutes after contacting the test sample comprising bacterial cells with the recombinant K1-5 bacteriophage.

20 . 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 K1-5 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 K1-5 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 K1-5 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.

21 . The method of claim 20 , 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.

22 . The method of claim 20 , wherein the bac

Assignees

• Charles Stark Draper Laboratory Inc

Inventors

• Mcbrine Connor
• Holder Jason

Classifications

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · 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

• C07K14/005

  from viruses · CPC title

• C12N2795/10243

  viral genome or elements thereof as genetic vector · CPC title