Bacteriophage compositions and uses for treatment of escherichia coli (e. coli) colonization

Abstract

Disclosed herein are recombinant bacteriophages capable of producing Colicin M and Colicin K. Also provided are compositions comprising such recombinant bacteriophages and methods for reducing E. coli within bacterial populations, including methods of treating E. coli colonization.

Description

[0001] Attorney Docket No. L 121360 1330WO (00140)

[0002] BACTERIOPHAGE COMPOSITIONS AND USES FOR TREATMENT OF ESCHERICHIA COLI(E. COLI) COLONIZATION

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Application No. 63 / 744,617, filed January 13, 2025, the contents of which is incorporated by reference herein in its entirety.

[0005] STATEMENT REGARDING THE SEQUENCE LISTING

[0006] The instant application contains an electronic Sequence Listing which has been submitted in xml (ST.26) format via USPTO Patent Center, herein incorporated by reference in its entirety. Said xml copy named “L121360 1130WO.xml” is 29,698 bytes in size, and was created on January 12, 2026.

[0007] FIELD OF THE DISCLOSURE

[0008] The present disclosure generally relates to recombinant bacteriophages capable of producing Colicin K and Colicin M and methods for targeting E. coli. More specifically, the present disclosure relates to bacteriophage cocktails comprising recombinant bacteriophages capable of producing Colicin K and Colicin M, and methods for reducing the number of E. coli in a population of bacteria.

[0009] BACKGROUND

[0010] Phage therapy provides an approach to treatment of bacterial infections distinct from, and optionally complementary to, treatment with antibiotics. A growing interest in phage therapy stems, in part, from the rise in frequency of multi-drug-resistant bacterial infections in humans. Broad spectrum antibiotics can be useful in treatment of some multi-drug resistant bacterial infections, however, such agents kill both pathogenic and beneficial bacteria. Recent case reports of phage therapy have demonstrated clinical utility in resolving these otherwise intractable infections, including adherent invasive E. coli (AIEC) infections.

[0011] AIEC infections are associated with pathogenesis in multiple gastrointestinal disorders GI) including Crohn’s disease (CD), ulcerative colitis (UC), inflammatory bowel disease (IBD), colorectal cancer (CRC), and coeliac disease (CeD). For example, in one study, AIEC phenotype was investigated in 4,233 E. coli isolated from the ileum and colon of 14 UC and 15 CRC patients and in 38 fecal E. coli strains obtained from 17 CeD and 10 healthy children. AIEC prevalence andAttorney Docket No. L 121360 1330WO (00140)

[0012] abundance were compared with previous data from CD patients and healthy controls (Lopez-Siles etal., Front Immunol. 2022; 13:748839. doi: 10.3389 / fimmu.2022.748839). Clonality, virulence gene carriage, and phylogenetic origin were determined for the AIEC

[0013] identified. In UC, AIEC prevalence was intermediate between CD and healthy subjects (UC:

[0014] 35.7%, CD: 55.0%, H: 21.4%), and similar to CD patients with colonic disease (C-CD: 40.0%). In CRC, the prevalence was lower (6.7%) than these groups. Although none of the E. coli isolated from the CeD cohort were AIEC, E. coli strains isolated from active CeD patients showed higher invasion indices than those isolated from healthy and inactive CeD pediatric

[0015] patients. These results demonstrate the prevalence of AIEC in patients with various GI disorders.

[0016] SUMMARY

[0017] Provided herein are compositions comprising a first engineered bacteriophage and at least a second engineered bacteriophage, wherein the first bacteriophage comprises: a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; and b) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, wherein the first bacteriophage and the second bacteriophage are E. coli-iax^Qirnii. bacteriophages.

[0018] In some embodiments of the compositions, the first engineered bacteriophage comprises: a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 2; and b) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 1.

[0019] In some embodiments of the compositions, the second engineered bacteriophage comprises: a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; and b) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1.

[0020] In some embodiments of the compositions, the second engineered bacteriophage comprises: a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 2; and b) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereofAttorney Docket No. L 121360 1330WO (00140)

[0021] comprising an amino acid sequence set forth as SEQ ID NO: 1.

[0022] In some embodiments of the compositions, the composition further comprises a third engineered bacteriophage comprising: a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; and b) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1.

[0023] In some embodiments of the compositions, the composition further comprises a third engineered bacteriophage comprising: a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 2; and b) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 1.

[0024] In some embodiments of the compositions, the composition further comprises a fourth engineered bacteriophage comprising: a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; and b) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1.

[0025] In some embodiments of the compositions, the composition further comprises a fourth engineered bacteriophage comprising: a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 2; and b) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 1.

[0026] In some embodiments of the compositions, the composition further comprises a fifth engineered bacteriophage comprising: a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; and b) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1.Attorney Docket No. L 121360 1330WO (00140)

[0027] In some embodiments of the compositions, the composition further comprises a fifth engineered bacteriophage comprising: a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 2; and b) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 1.

[0028] In some embodiments of the compositions, the composition further comprises a sixth engineered bacteriophage comprising: a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; and b) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1.

[0029] In some embodiments of the compositions, the composition further comprises a sixth engineered bacteriophage comprising: a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 2; and / or b) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 1.

[0030] In some embodiments of the compositions, at least one of the engineered bacteriophage is a Tequatrovirus. In some embodiments of the compositions, at least two of the engineered bacteriophage are Tequatrovirus. In some embodiments of the compositions, the engineered bacteriophage are from at least two different genera selected from Tequatrovirus, Phapecoctavirus, Justusliebigvirus, Vequintavirus, and Krischvirus. In some embodiments of the compositions, the bacteriophage are from at least three different genera selected from Tequatrovirus, Phapecoctavirus, Justusliebigvirus, Vequintavirus, and Krischvirus.

[0031] In some embodiments of the compositions, the Colicin M polynucleotide is inserted into a structural gene site in at least one engineered bacteriophage.

[0032] In some embodiments of the compositions, the Colicin K polynucleotide is inserted into a structural gene site in at least one engineered bacteriophage.

[0033] In some embodiments of the compositions, at least one engineered bacteriophage is an obligately lytic bacteriophage.

[0034] In some embodiments of the compositions, between two and six of the engineered bacteriophage targets E. coli. In some embodiments of the compositions, the E. coli is an antibioticAttorney Docket No. L 121360 1330WO (00140)

[0035] resistant E. colt.

[0036] In some embodiments of the compositions, the first bacteriophage has a different host range than the second bacteriophage. In some embodiments of the compositions, the first bacteriophage has at least 80% sequence identity to a bacteriophage selected from p004kel36, p00cge016, p00exe327, p00mfe005, p007621e011, or p007817e005.

[0037] In some embodiments of the compositions, the second bacteriophage has at least 80% sequence identity to a bacteriophage selected from p004kel36, p00cge016, p00exe327, p00mfe005, p007621e011, or p007817e005, wherein the first and at least second bacteriophage are different.

[0038] In some embodiments of the compositions, the third bacteriophage has at least 80% sequence identity to a bacteriophage selected from p004kel36, p00cge016, p00exe327, p00mfe005, p007621e011, or p007817e005, wherein the first, second, and at least third bacteriophage are different.

[0039] In some embodiments of the compositions, the fourth bacteriophage has at least 80% sequence identity to a bacteriophage selected from p004kel36, p00cge016, p00exe327, p00mfe005, p007621e011, or p007817e005, wherein the first, second, third, and at least fourth bacteriophage are different.

[0040] In some embodiments of the compositions, the fifth bacteriophage has at least 80% sequence identity to a bacteriophage selected from p004kel36, p00cge016, p00exe327, p00mfe005, p007621e011, or p007817e005, wherein the first, second, third, fourth, and at least fifth bacteriophage are different.

[0041] In some embodiments of the compositions, the sixth bacteriophage has at least 80% sequence identity to a bacteriophage selected from p004kel36, p00cge016, p00exe327, p00mfe005, p007621e011, or p007817e005, wherein the first, second, third, fourth, fifth, and at least sixth bacteriophage are different.

[0042] In some embodiments of the compositions: a) the Tequatrovirus has at least 90% sequence identity to p004k (ATCC Accession No. PTA-127149), pOOex (ATCC Accession No. PTA-127145), p6921 (ATCC Accession No. PTA-127576), p6799 (ATCC Accession No. PTA-127577), or p69894 (ATCC Accession No. PTA- 127578); b) the Phapecoctavirus has at least 90% sequence identity to pOOcg; c) the Justusliebigvirus has at least 90% sequence identity to pOOmf; d) the Vequintavirus has at least 90% sequence identity to p007621; and e) the Krischvirus has at least 90% sequence identity to p007817; and each engineered bacteriophage comprises: a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; and b) a Colicin K polynucleotide encoding a Colicin KAttorney Docket No. L 121360 1330WO (00140)

[0043] polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1.

[0044] In some embodiments of the compositions, none of the engineered bacteriophage comprise a nucleic acid molecule encoding a CRISPR system, optionally a Type I or Type II CRISPR system, a CRISPR Cas3, a CRISPR-Cas9, a Pseudomonas aeruginosa Type 1C CRISPR.

[0045] Provided herein are compositions comprising six engineered bacteriophage, each engineered bacteriophage comprising: a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; and b) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, wherein: i) a first engineered bacteriophage is p004kel36; ii) a second engineered bacteriophage is p00cge016; iii) a third engineered bacteriophage is p00exe327; iv) a fourth engineered bacteriophage is p00mfe005; v) a fifth engineered bacteriophage is p007621e001; and vi) a sixth engineered bacteriophage is p007817e005.

[0046] In some embodiments of the above compositions, each engineered bacteriophage comprises: a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 2; and b) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 1.

[0047] In some embodiments of the above compositions, the composition comprises at least 106plaque forming units (PFU) of each engineered bacteriophage. In some embodiments of the above compositions, the composition comprises between 106and 1016plaque forming units (PFU) of each engineered bacteriophage.

[0048] Provided herein are pharmaceutical composition comprising the composition of any one of claims 1-34, and a pharmaceutically acceptable excipient. In some embodiments of the above pharmaceutical compositions, the pharmaceutical composition is formulated for oral, intravenous, or enema administration. In some embodiments of the above pharmaceutical compositions, the pharmaceutical composition is formulated for oral administration

[0049] Provided here are methods of reducing the number of E. coli within a population of bacteria comprising a plurality of E. coli, the method comprising contacting the population with any of the compositions disclosed herein, or any of the pharmaceutical compositions disclosed herein.

[0050] Provided here are methods of administering a composition comprising at least one engineered bacteriophage capable of producing at least one colicin to a subject in need thereof, theAttorney Docket No. L 121360 1330WO (00140)

[0051] method comprising administering to the subject an effective amount of any of the compositions disclosed herein, or any of the pharmaceutical compositions disclosed herein.

[0052] In some embodiments of the above methods, the effective amount: a) reduces the number of E. coli within a population of bacteria comprising a plurality of E. coli within the gastrointestinal tract of the subject; b) reduces the number of AIEC in the gastrointestinal tract of the subject; c) reduces calprotectin levels in the subject; d) reduces the level of at least inflammatory cytokine in the gastrointestinal tract of the subject; e) reduces at least one symptom associated with gastrointestinal disease in the subject; f) reduces the severity of at least one gastrointestinal disease in the subject; g) increases the relapse time in the subject; h) improves the histopathology score of at least one tissue in the subject; i) promotes at least one positive clinical response in the subject; and / or j) promotes a positive endoscopic response in the subject.

[0053] In some embodiments of the above methods, the E. coli is a pathogenic E. coli. In some embodiments of the above methods, the pathogenic E. coli is an adherent-invasive E. coli (AIEC). In some embodiments of the above methods, the gastrointestinal tract of the subject comprises the pathogenic E. coli.

[0054] In some embodiments of the above methods, the subject has been diagnosed with a gastrointestinal disease or condition selected from inflammatory bowel disease (IBD), Crohn’s disease (CD), ulcerative colitis (UC), indeterminate colitis, and gastroenteritis. In some embodiments of the above methods, the gastroenteritis is: enteroaggregative (EAEC), enterohemorrhagic (EHEC), enteroinvasive (EIEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), or diffuse adherent (DAEC).

[0055] In some embodiments of the above methods, the composition is administered systemically. In some embodiments of the above methods, the composition is administered locally. In some embodiments of the above methods, the composition or pharmaceutical composition is administered orally, intravenously, or by enema. In some embodiments of the above methods, the composition or pharmaceutical composition is administered orally.

[0056] Provided herein are engineered bacteriophages comprising a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; and b) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1.

[0057] In some embodiments of the above bacteriophages, the Colicin M polypeptide or functional fragment or variant thereof comprises an amino acid sequence set forth as SEQ ID NO: 2, and theAttorney Docket No. L 121360 1330WO (00140)

[0058] Colicin K polypeptide or functional fragment or variant thereof comprises an amino acid sequence set forth as SEQ ID NO: 1.

[0059] In some embodiments of the above bacteriophages, the engineered bacteriophage is a recombinant Tequatrovirus. In some embodiments of the above bacteriophages, the engineered bacteriophage comprises a genome nucleotide sequence having at least 90% sequence identity to the genomic nucleotide sequence of p00exe327. In some embodiments of the above bacteriophages, the engineered bacteriophage is p00exe327. In some embodiments of the above bacteriophages, the engineered bacteriophage comprises a genome nucleotide sequence having at least 90% sequence identity to the genomic nucleotide sequence of p004kel36. In some embodiments of the above bacteriophages, the engineered bacteriophage is p004kel36.

[0060] In some embodiments of the above bacteriophages, the engineered bacteriophage is a recombinant Phapecoctavirus.

[0061] In some embodiments of the above bacteriophages, the engineered bacteriophage comprises a genome nucleotide sequence having at least 90% sequence identity to the genomic nucleotide sequence of p00cge016. In some embodiments of the above bacteriophages, the engineered bacteriophage is p00cge016.

[0062] In some embodiments of the above bacteriophages, the engineered bacteriophage is a recombinant Justusliebigvirus. In some embodiments of the above bacteriophages, the engineered bacteriophage comprises a genome nucleotide sequence having at least 90% sequence identity to the genomic nucleotide sequence of p00mfe005. In some embodiments of the above bacteriophages, the engineered bacteriophage is p00mfe005.

[0063] In some embodiments of the above bacteriophages, the engineered bacteriophage is a recombinant Vequintavirus. In some embodiments of the above bacteriophages, the engineered bacteriophage comprises a genome nucleotide sequence having at least 90% sequence identity to the genomic nucleotide sequence of p007621e011. In some embodiments of the above bacteriophages, the engineered bacteriophage is p007621e011.

[0064] In some embodiments of the above bacteriophages, the engineered bacteriophage is a recombinant Krischvirus. In some embodiments of the above bacteriophages, the engineered bacteriophage comprises a genome nucleotide sequence having at least 90% sequence identity to the genomic nucleotide sequence of p007817e005. In some embodiments of the above bacteriophages, the engineered bacteriophage is p007817e005.

[0065] Provided herein are any of the above compositions, pharmaceutical compositions, or engineered bacteriophages for use in the manufacture of a medicament for the treatment of a disease.Attorney Docket No. L 121360 1330WO (00140)

[0066] Provided herein are any of the above compositions, pharmaceutical compositions, or engineered bacteriophages for use in treating a disease.

[0067] BRIEF DESCRIPTIONOF THE DRAWINGS FIG. 1 is a graph showing the host range of recombinant colicins expressed as percent of the strain panel (n=88) with AUC ratio less than 0.65 (threshold used to determine bacterial growth reduction) in response to treatment.

[0068] FIG. 2A is a graph showing the percentage of susceptible strains from subject samples from patients with Crohn’s disease, ulcerative colitis, other gastrointestinal disease, and an aggregate of all subject samples in response to treatment a combination of six recombinant bacteriophages each comprising a Colicin M polynucleotide and Colicin K polynucleotide (LBP-AIEC01). FIG. 2B is a graph showing the percentage of E. coli isolates from the subject samples and their reduction in growth in response to treatment with LBP-AIEC01.

[0069] FIG. 3A is a graph showing reduction in the number of E. coli (AIEC) by a recombinant bacteriophage comprising a Colicin M polynucleotide or a Colicin K polynucleotide compared to the parent bacteriophage and bacteria cell-only control. FIG. 3B is a graph showing reduction in the number of E. coli (AIEC) by purified Colicin M or Colicin K. FIG. 3C is a graph showing reduction in the number of E. coli (AIEC) within macrophages by Colicin M or Colicin K.

[0070] FIG. 4A shows a diagram depicting an experimental workflow to assess adherence and invasion phenotype in AIEC. FIG. 4B is a graph showing the reduction in the number of E. coli having an adherence and invasion phenotype of five AIEC strains after treatment with individual recombinant bacteriophages present within LBP-AIEC01.

[0071] FIGS. 5A-5B provide graphs demonstrating distribution of bacteriophage (bacteriophage titer) in the kidney, bladder, blood, and colon after intravenous (IV) and oral (PO) administration of a cocktail comprising three bacteriophages (i.e., LBP-EC01).

[0072] FIG. 6 is a diagram depicting experimental workflow of a study examining the impact of LBP-AIEC01 on disease severity in an AIEC / DSS-induced colitis mouse model.

[0073] FIGS. 7A-7C are graphs showing AIEC titer (FIG. 7 A), disease severity (FIG. 7B), and fecal lipocalin levels (FIG. 7C) after oral administration of AIEC alone (AIEC + Vehicle) or with LBP-AIEC01 (AIEC + Phage) in the study of FIG. 6. FIG. 7A shows AIEC titer in stool 6 hours post-administration. FIG. 7B shows disease severity at day 24 of the study. FIG. 7C shows levels of fetal lipocalin in stool at day 24 of the study.Attorney Docket No. L 121360 1330WO (00140)

[0074] DETAILED DESCRIPTION

[0075] The present disclosure now will be described more fully hereinafter. The disclosure may be embodied in many different forms and should not be construed as limited to the aspects set forth herein; rather, these aspects are provided so that this disclosure will satisfy applicable legal requirements.

[0076] Disclosed herein are compositions and methods for reducing the number of Escherichia coli within a population of bacteria. The population of bacteria can comprise a plurality of E. coli. In some instances, the plurality of E. coli comprises adherent-invasive E. coli (AIEC). The compositions and methods are particularly effective at targeting and reducing the number of adherent-invasive E. coli (AIEC) within a population of bacteria comprising a plurality of AIEC. The compositions disclosed herein can comprise at least one recombinant bacteriophage engineered to be capable of producing at least one colicin molecule. Exemplary colicin molecules are disclosed herein. For instance, the bacteriophages can be engineered to produce Colicin K and Colicin M. The bacteriophages can comprise any number of colicins disclosed herein. For instance, the bacteriophages can produce two colicins, e.g., colicin K and colicin M. Methods for generating recombinant bacteriophages capable of producing colicins are provided.

[0077] Also provided herein are compositions comprising at least one recombinant bacteriophage, e.g., a recombinant bacteriophage capable of producing colicin K and colicin M. The compositions can comprise more than one recombinant bacteriophage. For instance, compositions can comprise at least two, at least three, at least four, at least five, at least six, or more than six recombinant bacteriophages. In some instances, the composition comprises six recombinant bacteriophages. The recombinant bacteriophage can be any bacteriophage disclosed herein, e.g., any bacteriophage in Tables 1-3. In some instances, the composition comprises six bacteriophages. In such instances, the composition can comprise six bacteriophages listed in Table 3 (i.e., LBP-AIEC-01). Variants of the bacteriophages disclosed herein are also envisaged within the scope of the present application. Such variants may comprise a genome comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or greater than 99% sequence identity to any bacteriophage disclosed herein, e.g., any bacteriophage identified in Tables 1-3. The bacteriophages and variants thereof disclosed herein are lytic and capable of targeting E. coli.

[0078] Polynucleotides encoding colicins are also provided. Polynucleotides can encode at least one colicin polypeptide or functional fragment or variant thereof. In some instances, a polynucleotide encodes at least two colicin polypeptides or functional fragments or variants thereof. Functional fragments and variants of colicin polypeptides retain biological activity of a colicin from which they are derived. Any colicin disclosed herein is envisaged as being encoded by aAttorney Docket No. L 121360 1330WO (00140)

[0079] polynucleotide that can be introduced into a bacteriophage. In some instances, a polynucleotide encodes a colicin polypeptide comprising an amino acid sequence set forth as any one of SEQ ID NOs: 1-11, or a functional fragment or variant comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to an amino acid sequence set forth as any one of SEQ ID NOs: 1-11. It would be understood that functional fragments or variants of a colicin polypeptide comprises the amino acids and / or domains within the polypeptide sequence necessary for biological activity. Polynucleotides encoding colicin polypeptides can be introduced into bacteriophages to generate engineered bacteriophages. The polynucleotides can comprise a nucleotide sequence having at least 80% sequence identity to a nucleotide sequence set forth as any one of SEQ ID NOs: 14-25. In some instances, the polynucleotides comprise at least 85%, at least 90%, at least 95%, or greater than 99% sequence identity to nucleotide sequence set forth as any one of SEQ ID NOs: 14-25. Any method for introducing polynucleotides into bacteriophages known in the art is envisaged in the present disclosure. It would be understood that the polynucleotides are stably integrated and maintained in an engineered bacteriophage. For instance, polynucleotides can be integrated into the genome of a bacteriophage. Methods for genomic integration are disclosed herein. It would be understood that bacteriophage having colicin-encoding polynucleotide(s) in the genome are capable of the methods disclosed herein. For instance, such bacteriophages such retain the ability to target and reduce the number of E. coli.

[0080] Also disclosed herein are pharmaceutical compositions comprising bacteriophages engineered to produce colicins. The pharmaceutical compositions can comprise a combination of bacteriophages, e.g., a combination of recombinant bacteriophages. Any bacteriophage, or combination of bacteriophages, disclosed herein is envisaged as being included in a pharmaceutical composition. A pharmaceutical composition can comprise at least one recombinant bacteriophage capable of producing colicin K and colicin M. In some instances, a pharmaceutical composition comprises between one and six recombinant bacteriophages capable of producing colicin C and colicin M.

[0081] The bacteriophages, and compositions comprising combinations of such bacteriophages, disclosed herein are particularly effective at reducing the number of E. coli within a population of bacteria comprising a plurality of E. coli. In some instances, the plurality of E. coli comprises a pathogenic E. coli. In some instances, the plurality of E. coli comprises AIEC. Methods for utilizing such compositions for reducing the number of E. coli within a population of bacteria are also provided. In some instances, the bacteriophages, and combinations thereof, can be utilized to reduce the number of E. coli within the gastrointestinal tract of a subject. The bacteriophages andAttorney Docket No. L 121360 1330WO (00140)

[0082] combinations of bacteriophages disclosed herein can be used to reduce the number of AIEC within a population of E. coli. AIEC are understood to drive disease progression and worse outcomes for more than a third of Crohn’s disease patients. Reducing the driving force behind inflammation and intestinal barrier disruption in such patients could cure these patients of their symptoms. Methods are provided for selectively reducing the number of E. coli within the gastrointestinal tract of a subject, such as a subject exhibiting at least one symptom of a gastrointestinal disease, e.g., Crohn’s disease. The bacteriophages and combinations of bacteriophages provided herein have been shown to reduce the adhesive and invasive phenotype associated within a population of AIEC.

[0083] Accordingly, methods are also provided for reducing the number of AIEC within the gastrointestinal tract of a subject. Such methods may reduce the amount of E. coli (e.g., AIEC) present within a stool sample of a subject having at least one symptom of Crohn’s disease and / or reduce disease severity of such a subject. In some instances, the bacteriophages and compositions disclosed herein are particularly effective at reducing the adherent-invasive capability of a plurality of AIEC within a population of bacteria. AIEC form biofilms on the gut mucosal surface and invade epithelial cells. AIEC also replicate inside of macrophages after phagocytosis. As such, also provided are methods of reducing the number of AIEC within a biofilm and / or within at least one macrophage within the gastrointestinal tract of a subject. The methods disclosed herein are effective at reducing the number of invasive AIEC capable of invading epithelial cells within the gastrointestinal tract of a subject.

[0084] I. Definitions

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0086] As used herein, “a,” “an,” or “the” can mean one or more than one. For example, “a bacteriophage” can mean a single bacteriophage or a multiplicity of bacteriophages.

[0087] As used herein, unless specifically indicated otherwise, the word “or” is used in the inclusive sense of “and / or” and not the exclusive sense of “either / or.”

[0088] The term “about” or “approximately” usually means within 5%, or more preferably within 1%, of a given value or range. When used in reference to numerical ranges, cutoffs, or specific values means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. Unless explicitly stated otherwise within the Examples or elsewhere in theAttorney Docket No. L 121360 1330WO (00140)

[0089] Specification in the context of an assay, result or embodiment, “about” means within one standard deviation per the practice in the art, or a range of up to 5%, whichever is larger.

[0090] The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.

[0091] The term “about,” when used before a list, modifies each member of the list. The term “about” is understood to encompass tolerated variation or error within the art, e.g., 2 standard deviations from the mean, or the sensitivity of the method used to take a measurement. When “about” is present before the first value of a series, it can be understood to modify each value in the series.

[0092] As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex region of “no more than 2 nucleotide base pairs” has a 2, 1, or 0 nucleotide base pairs. When “no more than” or “less than” is present before a series of numbers or a range, it is understood that each of the numbers in the series or range is modified.

[0093] In the event of a conflict between a sequence in the application and an indicated accession number or position in an accession number, the sequence in the application predominates.

[0094] It is understood that therapeutic agents, particularly biologic therapeutic agents, and engineered organisms, such as engineered bacteriophage, inherently include variations that result from the manufacturing and purification process. As used herein, an “essentially homogeneous population” e.g., an essentially homogeneous population of bacteriophage, is understood as a population in which at least 70% of the member of the population have the desired features, e.g., bacteriophage genome, desired size, e.g., molecular weight, size by chromatography method.

[0095] As used herein, “bacteriophage”, also known as “phage” is understood as a virus that infects and replicates within bacteria and archaea. Bacteriophages occur abundantly in the biosphere, with different genomes and lifestyles. Phages are classified by the International Committee on Taxonomy of Viruses (ICTV) according to morphology and nucleic acid. Bacteriophage, including engineered bacteriophage, have a number of practical uses including, but not limited to, phage therapy for treatment of bacterial colonization or infection, in the food industry to inhibit the growth of spoilage bacteria, indicators of fecal contamination in water sources, use in diagnostic tools, phage display for peptide library screening, and antimicrobial drug discovery. Although bacteriophage do not infect mammalian cells, bacteriophage are present in mammals including in the gut microbiome, both beneficially and antagonistically. Bacteriophage populations can vary with factors including disease and age. Bacteriophage have long been studied and are well characterized as discussed further below.Attorney Docket No. L 121360 1330WO (00140)

[0096] As used herein, “ . coli colonization” is understood as the persistent presence of E. coli bacteria on or in an organ of the body, optionally a human body.

[0097] As used herein, an “engineered bacteriophage” or “recombinant bacteriophage” is understood as a bacteriophage having a genome that has been manipulated by a human such that the bacteriophage does not exist in nature and, preferably, the manipulation of the bacteriophage genome results in a change in expression of at least one protein or RNA as compared to the naturally occurring bacteriophage from which it was derived or has the highest known sequence identity. A recombinant bacteriophage can comprise at least one exogenous and / or heterologous polynucleotide. For instance, a recombinant bacteriophage can comprise a colicin-encoding polynucleotide. The recombinant bacteriophages disclosed herein can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide of functional fragment or variant thereof, and a Colicin K polynucleotide encoding a Colicin K polypeptide of functional fragment or variant thereof. Accordingly, a recombinant bacteriophage of the present disclosure is capable of producing a Colicin M polypeptide or functional fragment or variant thereof, and a Colicin K polypeptide or a functional fragment or variant thereof. Such recombinant bacteriophages retain the lytic and replicative ability of the parent bacteriophages from which they are derived. In some instances, a recombinant bacteriophage is derived from a wild-type bacteriophage, e.g., a bacteriophage occurring in nature. In some instances, a recombinant bacteriophage is derived from a recombinant bacteriophage. An engineered bacteriophage can be derived from a naturally occurring isolate from a relevant source material, e.g., patient sample, soil sample, that is used as the starting material for manipulation. In certain embodiments, the engineered bacteriophage genome is manipulated to disrupt expression of a bacteriophage protein, e.g., to disrupt the lysogenic pathway of the bacteriophage. In certain embodiments, the engineered bacterial genome is manipulated to result in expression of a heterologous polypeptide. The heterologous polypeptide can be incorporated into the bacteriophage particle, e.g., as part of a capsid fusion protein. The heterologous polypeptide expresses from the bacteriophage genome may not be incorporated into the bacteriophage particle and may be released from the E. coli upon lysis of the bacteria with the bacteriophage particles without being a component of the bacteriophage particles. Manipulation of a genome can include insertion of a nucleotide sequence in the genome. Manipulation of a genome can include deletion of a nucleotide sequence in the genome. Manipulation of a genome can include mutation of a nucleotide sequence in the genome.

[0098] As used herein, a bacteriophage “targeting” a bacterial species, e.g., E. coli, is understood as able to infect the bacteria and undergo a productive lytic lifecycle. “Specifically targeting” when referring to E. coli refers to the ability of a bacteriophage to infect E. coli and undergo a productive lytic lifecycle at a higher rate than a bacteria that is not E. coli when exposed under the same orAttorney Docket No. L 121360 1330WO (00140)

[0099] similar conditions. For instance, the number of PFU produced upon exposure to E. coli can be at least 10-fold, at least 100-fold, at least 1000-fold, or greater than 1000-fold higher than upon exposure of the bacteriophage to a control bacterial species, e.g., a bacteria that is not E. coli, when exposed under the same or similar conditions. Without wishing to be bound by any theory, targetbinding proteins produced by a bacteriophage may define the ability of a bacteriophage to target a specific bacteria through specific binding to a binding protein and / or domain thereof present on a target bacteria. Target-binding proteins of a bacteriophage may be modified to enable a recombinant bacteriophage to target a certain bacteria not targeting by a parent bacteriophage from which the recombinant bacteriophage is derived.

[0100] As used herein, an “antibiotic resistant E. coli” is understood as a strain of E. coli that persists in the presence of an effective dose at least one antibiotic against a non-resistant strain. Exemplary antibiotics for which resistance has been demonstrated for E. coli include amoxicillin, cefuroxime, and cefatriaxone, ceftazidime, gentamicin, ciprofloxacin, and sulfonamides. Multidrug resistant E. coli are resistant to at least two, optionally more, antibiotics.

[0101] As used herein, “heterologous” is understood as a sequence having a different relation, relative position, or structure as compared to a naturally occurring sequence, cell, or organism. For example, a coding sequence inserted into a genome wherein the inserted coding sequence is not naturally present is a heterologous coding sequence. The inserted coding sequence can be a naturally occurring sequence from a different organism or may be a non-naturally occurring coding sequence from the same organism or a different organism. The inserted coding sequence may be inserted into the genome in an open reading frame, optionally with endogenous

[0102] regulatory sequences to promote expression of the inserted sequence. The inserted coding sequence can be inserted after a stop codon, but before the termination sequence in the genome. The inserted coding sequence may be inserted into the genome within an open reading frame to replace, completely or in part, or be inserted into the coding sequence in the open reading frame.

[0103] The polypeptide encoded by the heterologous coding sequence would be a heterologous polypeptide. A heterologous sequence can also include a sequence for a functional RNA, e.g., a guide RNA (gRNA), tRNA, or short hairpin RNA (shRNA). In certain embodiments, a heterologous sequence can be derived from within the same organism but inserted at a different relative position in the genome, preferably with a resulting change in level or timing of expression of the heterologous sequence or adjacent sequence(s).

[0104] As used herein, a “structural gene insertion site” is a site within a structural gene of a bacteriophage genome located between the promoter and the transcription terminator of a structural gene. The insertion site can be located between the stop codon and the transcription terminator of aAttorney Docket No. L 121360 1330WO (00140)

[0105] structural gene, which can encode a protein selected from major capsid protein, tail fiber, tail spike, tail sheath and head stabilization / decoration (HOC) protein. In another instance, the insertion site can be located between the promoter and start codon of a structural gene. It would be understood that insertion of a polynucleotide, e.g., polynucleotide encoding a colicin polypeptide, does not disrupt production of a structural gene product. Polynucleotides encoding colicins disclosed herein can be incorporated into a bacteriophage genome at a structural gene insertion site. Insertion of such polynucleotides into the genome of a bacteriophage does not disrupt or alter production of structural proteins or bacteriophage viability. Any location within a structural gene is envisaged for insertion of an exogenous polynucleotide does not disrupt or alter production of structural proteins or bacteriophage viability. In some instances, an exogenous polynucleotide is inserted such that the colicin coding sequence is in-frame with a structural gene coding sequence.

[0106] As used herein, “nucleic acid” and “polynucleotide" are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g, chemically synthesized) DNA or RNA and chimeras of RNA and DNA. The term polynucleotide or nucleic acid refers to a chain of nucleotides without regard to length of the chain. The term "fragment" when used to refer to a polynucleotide will be understood to mean a nucleotide sequence of reduced length relative to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of and / or consisting of a nucleotide sequence of contiguous nucleotides identical to the reference nucleic acid or nucleotide sequence. Such a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially or and / or consist of, oligonucleotides having a length ofat least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive nucleotides of a nucleic acid or nucleotide sequence according to the invention. The polynucleotides disclosed herein can encode a colicin polypeptide, e.g., Colicin K or Colicin M, or a functional fragment or variant thereof. Such polynucleotides are referred to herein as “colicinencoding polynucleotide.” In some instances, a polynucleotide encodes a Colicin M polypeptide or fragment or variant thereof (i.e., a Colicin M polynucleotide). In some instances, a polynucleotide encodes a Colicin K polypeptide or fragment or variant thereof (i.e., a Colicin K polynucleotide). Polynucleotides encoding any colicin known in the art are envisaged in the present application.

[0107] “Polypeptide” as used herein refers to a multimeric compound comprising amino acid residues that can adopt a three-dimensional conformation. Polypeptides include but are not limited to enzymes, enzyme precursor proteins, regulatory proteins, structural proteins, receptors, nucleic acid binding proteins, antibodies. Polypeptides may, but do not necessarily, comprise post-translational modifications, non-natural amino acids, prosthetic groups, and the like.Attorney Docket No. L 121360 1330WO (00140)

[0108] As used herein, “sequence identity,” “identity,” “percent identity,” and “sequence similarity” refer to a measure of the degree of similarity of two sequences based upon an alignment of the sequences that maximizes similarity between aligned amino acid residues or nucleotides, and which is a function of the number of identical or similar residues or nucleotides, the number of total residues or nucleotides, and the presence and length of gaps in the sequence alignment. A variety of algorithms and computer programs are available for determining sequence similarity using standard parameters. As used herein, sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ), and are described in, for example, Altschul et al. (1990), J. Mol. Biol. 215:403-410; Gish and States (1993), Nature Genet. 3:266-272; Madden et al. (1996), Meth. Enzymol.266: 131-141; Altschul et al.

[0109] (1997), Nucleic Acids Res. 25:3389-3402); Zhang et al. (2000), J. Comput. Biol. 7(l-2):203-14. As used herein, percent similarity of two amino acid sequences is the score based upon the following parameters for the BLASTp algorithm: word size=3; gap opening penalty=-l 1; gap extension penalty=-l; and scoring matrix=BLOSUM62. As used herein, percent similarity of two nucleic acid sequences is the score based upon the following parameters for the BLASTn algorithm: word size=l 1; gap opening penalty=-5; gap extension penalty=-2; match reward=l; and mismatch penalty=-3. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are considered to have “sequence similarity” or “similarity”. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Henikoff S and Henikoff J G. (Proc Natl Acad Sci 89:10915-9 (1992)). Identity (e.g., percent homology) can be determined using any homology comparison software, including for example, the BlastN software of the National Center of Biotechnology Information (NCBI) such as by using default parameters. When used in the context of comparing bacteriophage polynucleotides, such as genomic sequences, e.g., full-length genomeAttorney Docket No. L 121360 1330WO (00140)

[0110] sequence, percent identity can be defined by the following formula: Percent Identity = (I / La) x min (La / Lq, La / Lr), whereby: I is equal to the number of identical residues; Lais the alignment length; Lqis the query length and Lr is the reference length.

[0111] It would be understood that percent identity between two bacteriophages involves a comparison of the full length genome nucleic acid sequences of the two bacteriophages (i.e., an alignment of the entire nucleic acid sequence of the genome). Unless otherwise described, a reference to sequence identity or percent identity of a first bacteriophage (e.g., test bacteriophage) to at least a second bacteriophage (e.g., reference bacteriophage) involves comparing the degree of similarity based upon an alignment of the full genome sequences of the first and the second bacteriophage, whereby the alignment is over the full length of the genomic sequences of the bacteriophages being compared. For instance, a test bacteriophage and a reference bacteriophage have 90% sequence identity when the test bacteriophage and reference bacteriophage share 90% nucleic acid similarity across the entire genomes of the two bacteriophages.

[0112] As used herein, a first sequence is considered to be “identical” or have “100% identity” with a second sequence if an alignment of the first sequence to the second sequence shows that all of the positions of the second sequence in its entirety are matched by the first sequence. For example, the sequence AAG has 100% identity to the sequence AAGA because an alignment would give 100% identity in that there are matches, without gaps, to all three positions of the first sequence. Less than 100% identity can be calculated using routine methods. For example, ACG would have 67% identity with AAGA as two of the three positions of the first sequence are matches to the second sequence (2 / 3 = 67%). The differences between RNA and DNA (generally the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogs such as modified uridines do not contribute to differences in identity or complementarity among polynucleotides as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5 -methylcytosine, both of which have guanosine or modified guanosine as a complement). Thus, for example, the sequence 5’-AXG where X is any modified uridine, such as pseudouridine, Nl-methyl pseudouridine, or 5 -methoxyuridine, is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5’-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman- Wunsch algorithms, which are well-known in the art. One skilled in the art will understand what choice of algorithm and parameter settings are appropriate for a given pair of sequences to be aligned; for sequences of generally similar length and expected identity >50% for amino acids or >75% for nucleotides, the Needleman-Wunsch algorithm with default settings of the Needleman- Wunsch algorithm interface provided by the EBI at theAttorney Docket No. L 121360 1330WO (00140)

[0113] www.ebi.ac.uk web server is generally appropriate.

[0114] The term “fragment” refers to a portion of a polynucleotide or polypeptide sequence.

[0115] "Fragments" or "biologically active portions" include polypeptides comprising a sufficient number of contiguous amino acids, or polynucleotides comprising a sufficient number of contiguous nucleotides, to retain the biological activity. Fragments of polypeptides include those that are shorter than the full-length sequences due to the use of an alternate downstream start site. In some instances, an active cell constituent comprises a polypeptide. A biologically active portion of a cell constituent can be a polypeptide that comprises, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or more contiguous amino acid residues of any polypeptide sequence disclosed herein. In some instances, an active cell constituent comprises a polynucleotide. In such instances, a biologically active portion of a cell constituent can be a polynucleotide that comprises, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or more contiguous nucleotide sequences of a polynucleotide sequence disclosed herein. Such biologically active portions can be prepared by recombinant techniques and evaluated for activity.

[0116] In general, "variants" is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a deletion and / or addition of one or more nucleotides at one or more internal sites within the native polynucleotide and / or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a "native" or “wild type” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the native amino acid sequence of the gene of interest. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode the polypeptide or the polynucleotide of interest. Generally, variants of a particular polynucleotide disclosed herein will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein. Variants of a particular polynucleotide disclosed herein (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. WhereAttorney Docket No. L 121360 1330WO (00140)

[0117] any given pair of polynucleotides disclosed herein is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

[0118] “Treatment” as used herein is understood as reducing at least one sign or symptom of the disease or condition. Reduction can include to a frequency or severity such that the sign or symptom of the disease is no longer detectable. Treatment can include administration of more than one dose of the agent. For example, in some embodiments, treatment can include administration of 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses. Treatment can include administration with other agents. Effective treatment does not require a cure or complete elimination of the disease or condition. The rate of progression or development of a disease can be compared to the progression or development of a disease in an appropriately matched control, e.g., a population control, a control from a natural history study. As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, but not limited to, reduction of frequency or severity of one or more signs or symptoms of GI disease including episodic disease events associated with one or more of inflammatory bowel disease (IBD), Crohn’s disease (CD), or ulcerative colitis (UC), or gastroenteritis, optionally gastroenteritis: enteroaggregative (EAEC), enterohemorrhagic (EHEC), enteroinvasive (EIEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), and diffuse adherent (DAEC). Treatment can include reduction of bacterial burden, particularly reduction of pathogenic bacterial burden in a subject.

[0119] As used herein, “delivering” and “administering” are used interchangeably.

[0120] “Co-administration”, as used herein, means that a plurality of substances are administered sufficiently close together in time so that the agents act together. Co-administration encompasses administering substances together in a single formulation and administering substances in separate formulations close enough in time so that the agents act together. In certain embodiments, the agents may act synergistically.

[0121] As used herein, the phrase “pharmaceutically acceptable” means that which is useful in preparing a pharmaceutical composition that is generally non-toxic and is not biologically undesirable and that are not otherwise unacceptable for pharmaceutical use. Pharmaceutically acceptable generally refers to substances that are non-pyrogenic.

[0122] As used herein, a “control” is understood as an appropriate matched sample or subject for comparison. For example, a control can be a cell population treated in the same manner as the test population except that the treatment used for the control population lacks at least one active agent,Attorney Docket No. L 121360 1330WO (00140)

[0123] e.g., an engineered bacteriophage. In certain embodiments, a control may be an internal control, e.g., a cell population or subject prior to treatment. In certain embodiments, a “control” as in a control subject is a comparator for a measurement, e.g., a diagnostic measurement of a sign or symptom of a disease. In certain embodiments, a control can be a subject sample from the same subject an earlier time point, e.g., before a treatment intervention. In certain embodiments, a control can be a measurement from a normal subject, i.e., a subject not having the disease of the treated subject, to provide a normal control, e.g., an enzyme concentration or activity in a subject sample. In certain embodiments, a normal control can be a population control, i.e., the average of subjects in the general population. In certain embodiments, a control can be an untreated subject with the same disease. In certain embodiments, a control can be a subject treated with a different therapy, e.g., the standard of care. In certain embodiments, a control can be a subject or a population of subjects from a natural history study of subjects with the disease of the subject being compared. In certain embodiments, the control is matched for certain factors to the subject being tested, e.g., age, gender. In certain embodiments, a control may be a control level for a particular lab, e.g., a clinical lab. The ability to design or select appropriate controls is within the ability of those of skill in the art. It is understood when relative values are provided, they can be considered as relative values as compared to an appropriate control.

[0124] As used herein, “purified” such as in “purified protein” or “purified nucleic acid” is understood as being substantially removed from the mixture in which it was made, e.g., a cell, a subject sample, a reaction mixture. In certain embodiments, purified is understood as at least 50% of the mixture is the purified compound, e.g., purified protein or purified nucleic acid by weight. In certain embodiments, purified is understood as at least 70% of the mixture is the purified compound by weight.

[0125] The term "antibody" as used herein refers to any molecule comprising a binding domain that binds to a target molecule. An antibody can refer to full-length immunoglobulins as well as to fragments thereof. An antibody can also refer to a receptor, or fragment or variant thereof, that retains a binding domain. Full-length immunoglobulins may be monoclonal, polyclonal, chimeric, humanized, veneered or human antibodies. An antibody can include immunoglobulin molecules including monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies, antigen binding fragments, multispecific antibodies, such as bispecific, trispecific, tetraspecific etc., dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen binding site of the required specificity. An antibody can also be comprised in part by aptamers or tag-binding moieties such as biotin / streptavidin / avidin. The antibody can be generated or derived using any methodAttorney Docket No. L 121360 1330WO (00140)

[0126] available in the art. For example, recombinant techniques for generating antibodies are well known in the art. In some embodiments, the antibody can be generated using genetic engineering techniques. In some embodiments, the antibody can be derived from click chemistry. Antibody structure and / or production method are not envisaged as limiting the antibodies disclosed herein. “Full length antibodies” are comprised of two heavy chains (HC) and two light chains (LC) inter-connected by disulfide bonds as well as multimers thereof (e.g. IgM). Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (comprised of domains CHI, hinge, CH2 and CH3). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The VH and the VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with framework regions (FR). Each VH and VL is composed of three CDRs and four FR segments, arranged from amino-to-carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Immunoglobulins can be assigned to five major classes, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (K) and lambda (1), based on the amino acid sequences of their constant domains. The antibodies described herein can include "antibody fragments", which can be portions of a full-length immunoglobulin retaining the targeting specificity of said immunoglobulin. Many but not all antibody fragments lack at least partially the constant region (Fc region) of the full-length immunoglobulin. In some embodiments, antibody fragments are produced by digestion of the full-length immunoglobulin. An antibody fragment may also be a synthetic or recombinant construct comprising parts of the immunoglobulin or immunoglobulin chains (see e.g. Holliger, P. and Hudson, J. Engineered antibody fragments and the rise of single domains. Nature Biotechnology 2005, vol. 23, no. 9, p. 1126-1136). Examples of antibody fragments include, without being limited to, include scFv, Fab, Fv, Fab', F(ab')2 fragments, dAb, VHH, nanobodies, V(NAR) or minimal recognition units. “Single chain variable fragments” or “single chain antibodies” or “scFv” are one type of antibody fragment. scFv are fusion proteins comprising the variable heavy (VH) and variable light (VL) of immunoglobulins connected by a linker. They thus lack the constant Fc region present in full-length immunoglobulins, but retain the specificity of the original immunoglobulin.

[0127] As used herein, “subject” includes primates, including human and non-human primates, mouse, and rat. In certain embodiments, the subject is a human subject. In certain embodiments, the subject is a non-human subject. In certain embodiments, the subject is a non-human subject expressing one or more human genes, e.g., a transgenic mouse. Such models are well known in theAttorney Docket No. L 121360 1330WO (00140)

[0128] art.

[0129] As used herein, the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0130] Various embodiments of this disclosure may be presented in a range format. It should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also part of this disclosure. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1-10 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 1 to 6, from 1 to 7, from 1 to 8, from 1 to 9, from 2 to 4, from 2 to 6, from 2 to 8, from 2 to 10, from 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. This applies regardless of the breadth of the range.

[0131] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between. The recitation of a numerical range for a variable is intended to convey that the present disclosure may be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable can be equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable which is inherently continuous, the variable can be equal to any real value within the numerical range, including the end-points of the range. As an example, and without limitation, a variable which is described as having values between 0 and 2 can take the values 0, 1 or 2 if the variable is inherently discrete, and can take the values 0.0, 0.1, 0.01, 0.001, or any other real values ^0 and =2 if the variable is inherently continuous.

[0132] The patent and scientific literature referred to herein establishes knowledge that is available to those of skill in the art. The issued US patents, allowed applications, published foreign applications, and references, including GenBank database sequences, which are cited herein areAttorney Docket No. L 121360 1330WO (00140)

[0133] hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference.

[0134] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference herein in their entirety.

[0135] II. Bacteriophage lifecycle and genome

[0136] The life cycle of bacteriophages tends to be either a lytic cycle or a lysogenic cycle. In addition, some phages display pseudolysogenic behaviors.

[0137] With lytic phages such as the T4 phage, bacterial cells are lysed and destroyed shortly after replication of the virion, releasing the phage progeny infect new host cells. Lytic phages are typically considered to be more suitable for phage therapy.

[0138] In contrast, the lysogenic cycle does not result in immediate lysing of the host cell. Phage able to undergo lysogeny are known as temperate phages. Their viral genome is maintained in and replicates with the host genome either integrated with the host DNA or as a plasmid. The virus remains dormant until host conditions deteriorate, e.g., depletion of nutrients, increased bacterial host population; then, the endogenous phages enter the lytic cycle, resulting in lysis of the host cell. As the lysogenic cycle allows the host cell to continue to survive and reproduce, which is typically not desirable in phage therapy. An example of a bacteriophage known to follow the lysogenic cycle and the lytic cycle is the phage lambda of E. coli.

[0139] As used herein, “obligately lytic phage” are unable to undergo the lysogenic pathway.

[0140] Obligately lytic phages occur in nature or may be engineered by disrupting expression of proteins required to complete the lysogenic lifecycle or expression of proteins that promote the

[0141] lytic lifecycle. It is understood that expression can be disrupted by engineering of coding sequences of proteins or regulatory sequences that direct the expression of proteins. Proteins required for the lysogenic lifecycle include the cl repressor gene, the ell gene, the lexA gene, and the integrase gene.

[0142] To enter a host cell, bacteriophages bind to specific receptors on the surface of bacteria, including lipopolysaccharides, teichoic acids, proteins, or even flagella. This specificity means a bacteriophage can infect only certain bacteria bearing receptors to which they can bind, which in turn, determines the phage host range. Polysaccharide-degrading enzymes are virion-associated proteins that enzymatically degrade the capsular outer layer of their hosts at the initial step of a tightly programmed phage infection process. Bacteria can become resistant to certain phage by mutation of receptors so the phage is no longer able to bind the surface of the bacteria.

[0143] Upon infection, bacterial ribosomes translate viral mRNA into protein. For RNA-based phages, RNA replicase is synthesized early in the process. Proteins modify the bacterial RNA polymeraseAttorney Docket No. L 121360 1330WO (00140)

[0144] to preferentially transcribes the viral mRNA. Host protein and nucleic acid synthesis are downregulated in favor of production of proteins encoded in the phage genome. These products go on to become part of new virions within the cell, helper proteins that contribute to the assemblage of new virions, or proteins involved in cell lysis. In the case of engineered bacteriophage, inserted coding sequences are similarly preferentially produced.

[0145] The exact mechanism of virion assembly can vary, but typically the base plates are assembled first, with the tails being built upon them afterward. The head capsids, constructed separately, spontaneously assemble with the tails. The DNA is packed efficiently within the heads. Helper proteins may act catalytically during phage morphogenesis to promote assembly.

[0146] Phages may be released via cell lysis, by extrusion, or, in a few cases, by budding. Released virions are described as free, and, unless defective, are capable of infecting a new bacterium. In contrast to virion release, phages displaying a lysogenic cycle do not kill the host and instead become longterm residents as prophages.

[0147] Bacteriophage genomes have been well characterized to identify predicted and actual open reading frames, including overlapping reading frames; regulatory sequences (e.g., promoter sequences, termination sequences), and sequences that are not predicted to or do not include an open reading frame or regulatory sequence. Those of skill in the art are readily able to identify such sequences.

[0148] Given the large number of bacteriophages, there is no single, consensus bacteriophage genome structure. However, for bacteriophage containing DNA genomes, the genes can be grouped into three general classes of early genes that are expressed first after infection that express proteins that promote expression of the phage genome and downregulate expression of host proteins; DNA replication genes; and virus structure and assembly genes sometimes referred

[0149] to as late genes. Bacteriophage genes with the highest level of expression are typically virus structure and assembly genes. Such structural genes with high levels of expression include the major capsid protein, tail fiber, tail spike, tail sheath, and head stabilization / decoration (HOC) protein.

[0150] III. Colicin peptides

[0151] Bacteriocins are ribosomal synthesized antimicrobial peptides produced by bacteria, which can kill or inhibit bacterial strains closely-related or non-related to produced bacteria, but that do not harm the bacteria that produce them (Yang et al., Front Microbiol. 2014; 5: 683. doi:

[0152] 10.3389 / fmicb.2014.00683). Bacteriocins produced by E. coli may be referred to as “colicins”.

[0153] Without being limited by theory, colicins function to kill other bacteria, such as E. coli, through variousAttorney Docket No. L 121360 1330WO (00140)

[0154] mechanisms, including targeting enzymatic activity and forming pores in target bacteria. In some embodiments, the colicins described herein function through pore formation. Colicins are present in nature to reduce environmental competitors for acquiring nutrients and living space. Colicins host ranges have been and can be characterized, and colicins having the desired host range can be selected for various purposes. As such, colicins can be selected to target pathogenic bacteria, e.g., E. coli bacteria, to reduce the population of pathogenic bacteria, while sparing non-target bacteria, e.g., beneficial bacteria.

[0155] Identification of appropriate colicins for reducing the population of undesired bacteria while sparing the population of desirable bacteria can be determined by collection of biological samples, e.g., from a subject or population of subjects, and analyzing cell viability by an optical density assay or plate-based plaquing assay, of both pathogenic and desirable bacteria.

[0156] Colicins are organized in three specific domains, an amino-terminal translocation (T) domain, which is implicated in the transfer across the outer membrane via the translocator protein; a central receptor-binding (R) domain, which is bound with a bacterial outer membrane receptor; and a carboxy-terminal cytotoxic (C) domain, which has antibacterial activity. In order to avoid poisoning by self-produced colicins, specificity immunity proteins are simultaneously produced to inactivate colicins. When a bacterial outer membrane surface has the colicins recognition receptors protein and the translocators protein system, the colicins are transported into the bacteria, killing sensitive bacterial strains. For a particular colicin, non-receptor protein bacteria are classified as resistant strains. Bacteria with a deficiency of translocator protein system are classified as tolerant strains, and those which produce immunity proteins are classified as immune strains. Resistant, tolerant, and immune strains of bacteria are not killed by corresponding colicins. There are many colicins found in succession, most of which are encoded on plasmids, while few are located in chromosomes. Atypical colicin gene cluster encodes the toxin protein, immunity protein, and lysis gene. The lysis protein, known as bacteriocin release protein (BRP), can induce the release of colicins from bacteria.

[0157] Based on the outer membrane translocation system, colicins are categorized into two groups: groups A and B. Group A colicins use the Tol protein system (Tol system) to penetrate the outer membrane of sensitive bacteria, for example: colicins El to E9, colicin A, K, N, S4, U, 28b, and DF13. Group B barteriocins use the Ton system (Ton system) to penetrate the outer membrane of sensitive bacteria, for example: colicin 5, 10, B, D, M, V, la, and lb. In the general, group A colicins are encoded on small plasmids with a lysis gene and can be released out of the bacteria, while group B colicins are encoded on large plasmids without a lysis gene. Colicins can be divided into three categories based on bactericidal mechanisms within target cells: (1) Pore-formingAttorney Docket No. L 121360 1330WO (00140)

[0158] type colicins that form pores or channels in the inner- membrane, including colicin A, B, El, la, lb, K, and N; (2) Nuclease type colicins containing DNase, 16S rRNase, and tRNase to non-specifically digest DNA and RNA of bacteria, includingcolicin E2 to E9; and (3) Peptidoglycanase type colicins that can digest the peptidoglycan precursor, leading to an inability to synthesize peptidoglycan and bacterial death, such as Colicin M.

[0159] Exemplary colicins capable of being produced by the presently disclosed bacteriophages include, but are not limited to: Colicin K, Colicin M, Colicin lb, Colicin 10, Colicin U, Colicin la, Colicin El, Colicin E2, Colicin E3, Colicin D, and Colicin B.

[0160] In some instances, the colicin is Colicin K. In some instances, the colicin is a functional fragment or variant of Colicin K. In some instances, the colicin is Colicin K or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 1. A functional fragment or a variant of Colicin K can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The colicin can be Colicin K or a functional fragment or variant thereof that is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 14. A functional fragment or variant of Colicin K can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 14. In some instances, Colicin K or a functional fragment or variant thereof comprises an amino acid sequence set forth as SEQ ID NO: 1, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 14. In some instances, the functional fragment or variant of Colicin K has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 14, or a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 14.

[0161] In some instances, the colicin is Colicin M. In some instances, the colicin is a functional fragment or variant of Colicin M. In some instances, the colicin is Colicin M or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 2. A functional fragment or a variant of Colicin M can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceAttorney Docket No. L 121360 1330WO (00140)

[0162] identity to the amino acid sequence set forth as SEQ ID NO: 2. The colicin can be Colicin M or a functional fragment or variant thereof that is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 15. A functional fragment or variant of Colicin M can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 15. In some instances, Colicin M or a functional fragment or variant thereof comprises an amino acid sequence set forth as SEQ ID NO: 2, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 15. In some instances, the functional fragment or variant of Colicin M has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 15, or a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 15. The colicin can be Colicin M or a functional fragment or variant thereof that is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 16. A functional fragment or variant of Colicin M can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 16. In some instances, Colicin M or a functional fragment or variant thereof comprises an amino acid sequence set forth as SEQ ID NO: 2, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 16. In some instances, the functional fragment or variant of Colicin M has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 16, or a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 16.

[0163] In some instances, the colicin is Colicin lb. In some instances, the colicin is a functional fragment or variant of Colicin lb. In some instances, the colicin is Colicin lb or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 3. A functional fragment or a variant of Colicin lb can have at least 80%, 81%, 82%, 83%, 84%, 85%,Attorney Docket No. L 121360 1330WO (00140)

[0164] 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 3. The colicin can be Colicin lb or a functional fragment or variant thereof that is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 17. A functional fragment or variant of Colicin lb can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 17. In some instances, Colicin lb or a functional fragment or variant thereof comprises an amino acid sequence set forth as SEQ ID NO: 3, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 17. In some instances, the functional fragment or variant of Colicin lb has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 3, and is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 17, or a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 17.

[0165] In some instances, the colicin is Colicin 10. In some instances, the colicin is a functional fragment or variant of Colicin 10. In some instances, the colicin is Colicin 10 or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 4. A functional fragment or a variant of Colicin 10 can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4. The colicin can be Colicin 10 or a functional fragment or variant thereof that is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 18. A functional fragment or variant of Colicin 10 can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 18. In some instances, Colicin 10 or a functional fragment or variant thereof comprises an amino acid sequence set forth as SEQ ID NO: 4, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 18. In some instances, the functional fragment or variant of Colicin 10 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 4, and is encoded by a polynucleotide sequence comprising aAttorney Docket No. L 121360 1330WO (00140)

[0166] nucleotide sequence set forth as SEQ ID NO: 18, or a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 18.

[0167] In some instances, the colicin is Colicin U. In some instances, the colicin is a functional fragment or variant of Colicin U. In some instances, the colicin is Colicin U or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 5. A functional fragment or a variant of Colicin U can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 5. The colicin can be Colicin U or a functional fragment or variant thereof that is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 19. A functional fragment or variant of Colicin U can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 19. In some instances, Colicin U or a functional fragment or variant thereof comprises an amino acid sequence set forth as SEQ ID NO: 5, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 19. In some instances, the functional fragment or variant of Colicin U has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 5, and is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 19, or a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 19.

[0168] In some instances, the colicin is Colicin la. In some instances, the colicin is a functional fragment or variant of Colicin la. In some instances, the colicin is Colicin la or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 6. A functional fragment or a variant of Colicin la can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 6. The colicin can be Colicin la or a functional fragment or variant thereof that is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 20. A functional fragment or variant of Colicin la can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%,Attorney Docket No. L 121360 1330WO (00140)

[0169] 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 20. In some instances, Colicin la or a functional fragment or variant thereof comprises an amino acid sequence set forth as SEQ ID NO: 6, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 20. In some instances, the functional fragment or variant of Colicin la has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 6, and is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 20, or a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 20.

[0170] In some instances, the colicin is Colicin El. In some instances, the colicin is a functional fragment or variant of Colicin El. In some instances, the colicin is Colicin El or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 7. A functional fragment or a variant of Colicin El can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 7. The colicin can be Colicin El or a functional fragment or variant thereof that is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 21. A functional fragment or variant of Colicin El can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 21. In some instances, Colicin El or a functional fragment or variant thereof comprises an amino acid sequence set forth as SEQ ID NO: 7, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 21. In some instances, the functional fragment or variant of Colicin El has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 7, and is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 21, or a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 21.

[0171] In some instances, the colicin is Colicin E2. In some instances, the colicin is a functionalAttorney Docket No. L 121360 1330WO (00140)

[0172] fragment or variant of Colicin E2. In some instances, the colicin is Colicin E2 or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 8. A functional fragment or a variant of Colicin E2 can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 8. The colicin can be Colicin E2 or a functional fragment or variant thereof that is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 22. A functional fragment or variant of Colicin E2 can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 22. In some instances, Colicin E2 or a functional fragment or variant thereof comprises an amino acid sequence set forth as SEQ ID NO: 8, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 22. In some instances, the functional fragment or variant of Colicin E2 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 8, and is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 22, or a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 22.

[0173] In some instances, the colicin is Colicin E3. In some instances, the colicin is a functional fragment or variant of Colicin E3. In some instances, the colicin is Colicin E3 or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 9. A functional fragment or a variant of Colicin E3 can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 9. The colicin can be Colicin E3 or a functional fragment or variant thereof that is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 23. A functional fragment or variant of Colicin E3 can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 23. In some instances, Colicin E3 or a functional fragment or variant thereof comprises an amino acid sequence set forth as SEQ ID NO: 9, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 23. In some instances, the functional fragment orAttorney Docket No. L 121360 1330WO (00140)

[0174] variant of Colicin E3 has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 9, and is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 23, or a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 23.

[0175] In some instances, the colicin is Colicin D. In some instances, the colicin is a functional fragment or variant of Colicin D. In some instances, the colicin is Colicin D or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 10. A functional fragment or a variant of Colicin D can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 10. The colicin can be Colicin D or a functional fragment or variant thereof that is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 24. A functional fragment or variant of Colicin D can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 24. In some instances, Colicin D or a functional fragment or variant thereof comprises an amino acid sequence set forth as SEQ ID NO: 10, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 24. In some instances, the functional fragment or variant of Colicin D has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 10, and is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 24, or a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 24.

[0176] In some instances, the colicin is Colicin B. In some instances, the colicin is a functional fragment or variant of Colicin B. In some instances, the colicin is Colicin B or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 11. A functional fragment or a variant of Colicin B can have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 11. The colicin can be Colicin B or aAttorney Docket No. L 121360 1330WO (00140)

[0177] functional fragment or variant thereof that is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 25. A functional fragment or variant of Colicin B can be encoded by a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 25. In some instances, Colicin B or a functional fragment or variant thereof comprises an amino acid sequence set forth as SEQ ID NO: 11, is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 25. In some instances, the functional fragment or variant of Colicin B has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 11, and is encoded by a polynucleotide sequence comprising a nucleotide sequence set forth as SEQ ID NO: 25, or a polynucleotide sequence comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 25.

[0178] Provided herein are polynucleotides comprising a nucleotide sequence encoding a colicin, or a functional fragment or variant thereof. Such polynucleotides can be incorporated into a bacteriophage. For instance, such polynucleotide can be incorporated into the genome of a bacteriophage disclosed herein. The coding sequences can be codon optimized for expression in a host organism. For instance, the coding sequences can be codon optimized for expression in bacteria. In some instances, the coding sequences are codon optimized for expression in bacteriophage.

[0179] Colicins produced by the bacteriophages disclosed herein can comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence set forth as any one of SEQ ID NOs: 1-11. In some instances, a colicin produced by a bacteriophage disclosed herein comprises an amino acid sequence set forth as any one of SEQ ID NOs: 1-11. Colicins produced by the bacteriophages disclosed herein can comprise an amino acid sequence encoded by a polynucleotide comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence set forth as any one of SEQ ID NOs: 14-25. In some instances, a colicin produced by a bacteriophage disclosed herein comprises an amino acid sequence encoded by a polynucleotide comprising a nucleotide sequence set forth as any one of SEQ ID NOs: 14-25.

[0180] It is understood that multiple colicin homologs or orthologs exist among bacterial speciesAttorney Docket No. L 121360 1330WO (00140)

[0181] having various nucleotide and amino acid sequences. Such sequences can be readily identified using sequence databases such as the NCBI database. Coding sequences can be changed without changing the encoded sequence for purposes of codon optimization, or to facilitate cloning or expression. As provided herein, a colicin polypeptide retains similar activity to the colicin sequence provided herein. For example, a colicin having

[0182] less than 100% identity to a colicin provided herein has at least 90% activity as compared to a colicin having a sequence disclosed herein as compared by an assay provided herein, e.g., an optical density assay or a plaque forming assay as provided herein.

[0183] IV. Other antimicrobial agents and peptides

[0184] In some embodiments, a bacteriophage disclosed herein (e.g., bacteriophage capable of producing colicin K and colicin M) expresses at least one antimicrobial agent or peptide disclosed herein. In some embodiments, a bacteriophage disclosed herein is further genetically modified to express an antibacterial peptide, or a functional fragment of an antibacterial peptide. In some embodiments, the bacteriophage comprises a nucleic acid that encodes a peptide that prevents bacteriophage degradation or a peptide that assists in breaking down or degrading biofilm matrix. In some embodiments, the bacteriophage expresses at least two antimicrobial agents or peptides disclosed herein.

[0185] In some embodiments, a bacteriophage described herein comprises a nucleic acid that encodes a peptide that prevents bacteriophage degradation or enables escape of the bacteriophage from the host defenses. In some embodiments, a bacteriophage disclosed herein comprises a nucleic acid sequence that encodes an enzybiotic where the protein product of the nucleic acid sequence targets bacteriophage resistant bacteria. In some embodiments, the peptide comprises Ipi (e.g., a sequence at least 80% identical to SEQ ID NO: 63). The antimicrobial agent or peptide is encoded by a nucleotide sequence comprising at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 61.

[0186] In some embodiments, the bacteriophage comprises nucleic acids which encode enzymes which assist in breaking down or degrading biofilm matrix. In some embodiments, a bacteriophage disclosed herein comprises nucleic acids encoding Dispersin D aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,Attorney Docket No. L 121360 1330WO (00140)

[0187] polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase or lyase. In some embodiments, the enzyme is selected from the group consisting of cellulases, such as glycosyl hydroxylase family of cellulases, such as glycosyl hydroxylase 5 family of enzymes also called cellulase A; poly glucosamine (PGA) depolymerases; and colonic acid depolymerases, such as 1,4-L-fucodise hydrolase, colanic acid, depolymerazing alginase, DNase I, or

[0188] combinations thereof. In some embodiments, a bacteriophage disclosed herein secretes an enzyme disclosed herein. In some embodiments, the peptide disrupts quorum sensing and biofilm formation. In some embodiments, the peptide increases the sensitivity of a bacterial cell to an antibiotic. In some embodiments, the enzyme comprises DNAse I (e.g., a sequence at least 80% identical to SEQ ID NO: 62). The antimicrobial agent or peptide can be encoded by a nucleotide sequence comprising at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 60.

[0189] In some embodiments, a bacteriophage disclosed herein is capable of producing an antimicrobial agent or peptide. In some embodiments, the antimicrobial agent or peptide comprises PLNC8 a. In some embodiments, the antimicrobial agent or peptide comprises PLNC8p. In some embodiments, the antimicrobial agent or peptide comprises LytM. In some embodiments, the antimicrobial agent or peptide comprises an anti-restriction modification enzyme.

[0190] In some embodiments, a bacteriophage is capable of producing an antimicrobial peptide. In some embodiments, a bacteriophage is capable of producing an antibiotic such as ampicillin, penicillin, penicillin derivatives, cephalosporins, monobactams, carbapenems, ofloxacin, ciproflaxacin, levofloxacin, gatifloxacin, norfloxacin, lomefloxacin, trovafloxacin, moxifloxacin, sparfloxacin, gemifloxacin, pazufloxacin. Any antibiotic disclosed herein can be produced by a bacteriophage. In some embodiments, a bacteriophage comprises a nucleic acid sequence encoding an antibacterial peptide and / or is capable of producing an antibacterial peptide or a peptide that aids or enhances killing of a target bacteria. The target bacteria can be any bacteria disclosed herein, e.g., E. coli or AEIC. A bacteriophage can comprise a nucleic acid sequence encoding a peptide and / or is capable of producing a peptide, such as an antibacterial peptide.

[0191] In certain embodiments, the compositions and engineered bacteriophages provided herein do not comprise a nucleic acid molecule encoding an antibacterial peptide that is not a colicin peptide.

[0192] In certain embodiments, the compositions and engineered bacteriophages provided herein do not comprise a nucleic acid molecule encoding CRISPR system (i.e., a CRISPR enzyme and guide RNA), a CRISPR enzyme, or a CRISPR guide RNA. In certain embodiments, the compositions and engineered bacteriophages do not comprise a nucleic acid molecule encoding at least one of a Type I or Type II CRISPR sy sterna, CRISPR-Cas3, CRISPR-Cas9, or a PseudomonasAttorney Docket No. L 121360 1330WO (00140)

[0193] aeruginosa Type I C.

[0194] V. Polynucleotides

[0195] Disclosed herein are polynucleotides encoding antimicrobial agents, including, for instance, peptides, such as antimicrobial peptides. Also disclosed are polynucleotides encoding at least one colicin polypeptide, or fragment, or variant thereof (i.e., colicin-encoding polynucleotides). Any colicin polypeptide disclosed herein can be encoded by the polynucleotide. For instance, a polynucleotide can comprise a nucleotide sequence encoding a colicin having an amino acid sequence set forth as any one of SEQ ID NOs: 1-11, or a functional fragment or variant thereof comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence set forth as any one of SEQ ID NOs: 1-11. In some instances, a polynucleotide comprises a nucleotide sequence encoding a colicin having an amino acid sequence set forth as any one of SEQ ID NOs: 1-11. Such polynucleotides can encode more than one colicin, or fragment, or variant thereof. In such instances, any combination of colicins disclosed herein (e.g., colicins having an amino acid sequence set forth as any one of SEQ ID NOs: 1-11, or functional fragments or variants thereof) can be encoded by the polynucleotide. Polynucleotides can comprise more than one nucleotide sequence encoding more than one colicin. For instance, a polynucleotide may comprise a first nucleotide sequence encoding a first colicin, or fragment, or variant thereof, and a second nucleotide sequence encoding a second colicin, or fragment, or variant thereof. In such instances, the first colicin and the second colicin can be different colicins. Different colicins have amino acid sequences having less than 95% sequence identity, e.g., less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, or less than 10% sequence identity.

[0196] In some instances, a polynucleotide comprises a nucleotide sequence encoding a colicin comprising an amino acid sequence set forth as SEQ ID NO: 1 (i.e., Colicin K), or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. Such a polynucleotide is referred to herein as a ’’Colicin K polynucleotide.” A polynucleotide sequence can comprise a nucleotide sequence set forth as SEQ ID NO: 14. In some instances, a polynucleotide sequence can comprise a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence setAttorney Docket No. L 121360 1330WO (00140)

[0197] forth as SEQ ID NO: 14.

[0198] In some instances, a polynucleotide comprises a nucleotide sequence encoding a colicin comprising an amino acid sequence set forth as SEQ ID NO: 2 (i.e., Colicin M), or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2. . Such a polynucleotide is referred to herein as a ’’Colicin M polynucleotide.” A polynucleotide sequence can comprise a nucleotide sequence set forth as SEQ ID NO: 15 or 16. In some instances, a polynucleotide sequence can comprise a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 15 or 16.

[0199] In some instances, a polynucleotide can comprise a first nucleotide sequence encoding a first colicin comprising an amino acid sequence set forth as SEQ ID NO: 1 (i.e., Colicin K), or a functional fragment or variant thereof comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1, and a second nucleotide sequence encoding a second colicin comprising an amino acid sequence set forth as SEQ ID NO: 2 (i.e., Colicin M), or a functional fragment or variant thereof comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2. In such instances, a polynucleotide can comprise a first nucleotide sequence encoding a first colicin comprising an amino acid sequence set forth as SEQ ID NO: 1 (i.e., Colicin K), and a second nucleotide sequence encoding a second colicin comprising an amino acid sequence set forth as SEQ ID NO: 2. A polynucleotide sequence can comprise a first nucleotide sequence set forth as SEQ ID NO: 14, and a second nucleotide sequence set forth as SEQ ID NO: 15. A polynucleotide sequence can comprise a first nucleotide sequence set forth as SEQ ID NO: 14, and a second nucleotide sequence set forth as SEQ ID NO: 16. In some instances, a polynucleotide sequence can comprise a first nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 14, and a second nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 15. In some instances, a polynucleotide sequence can comprise a first nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,Attorney Docket No. L 121360 1330WO (00140)

[0200] 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 14, and a second nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth as SEQ ID NO: 16.

[0201] In some instances, a polynucleotide comprises a nucleotide sequence encoding a colicin having an amino acid sequence set forth as any one of SEQ ID NOs: 1-11.

[0202] In some instances, a polynucleotide comprises a nucleotide sequence encoding a colicin comprising an amino acid sequence set forth as SEQ ID NO: 3, or a functional fragment or variant thereof comprising an amino sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence set forth as SEQ ID NO: 3. In such instances, the polynucleotide can comprise a nucleotide sequence set forth as SEQ ID NO: 17, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 17.

[0203] In some instances, a polynucleotide comprises a nucleotide sequence encoding a colicin comprising an amino acid sequence set forth as SEQ ID NO: 4, or a functional fragment or variant thereof comprising an amino sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence set forth as SEQ ID NO: 4. In such instances, the polynucleotide can comprise a nucleotide sequence set forth as SEQ ID NO: 18, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 18.

[0204] In some instances, a polynucleotide comprises a nucleotide sequence encoding a colicin comprising an amino acid sequence set forth as SEQ ID NO: 5, or a functional fragment or variant thereof comprising an amino sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence set forth as SEQ ID NO: 5. In such instances, the polynucleotide can comprise a nucleotide sequence set forth as SEQ ID NO: 19, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 19.

[0205] In some instances, a polynucleotide comprises a nucleotide sequence encoding a colicinAttorney Docket No. L 121360 1330WO (00140)

[0206] comprising an amino acid sequence set forth as SEQ ID NO: 6, or a functional fragment or variant thereof comprising an amino sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence set forth as SEQ ID NO: 6. In such instances, the polynucleotide can comprise a nucleotide sequence set forth as SEQ ID NO: 20, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 20.

[0207] In some instances, a polynucleotide comprises a nucleotide sequence encoding a colicin comprising an amino acid sequence set forth as SEQ ID NO: 6, or a functional fragment or variant thereof comprising an amino sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence set forth as SEQ ID NO: 6. In such instances, the polynucleotide can comprise a nucleotide sequence set forth as SEQ ID NO: 20, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 20.

[0208] In some instances, a polynucleotide comprises a nucleotide sequence encoding a colicin comprising an amino acid sequence set forth as SEQ ID NO: 7, or a functional fragment or variant thereof comprising an amino sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence set forth as SEQ ID NO: 7. In such instances, the polynucleotide can comprise a nucleotide sequence set forth as SEQ ID NO: 21, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 21.

[0209] In some instances, a polynucleotide comprises a nucleotide sequence encoding a colicin comprising an amino acid sequence set forth as SEQ ID NO: 8, or a functional fragment or variant thereof comprising an amino sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence set forth as SEQ ID NO: 8. In such instances, the polynucleotide can comprise a nucleotide sequence set forth as SEQ ID NO: 22, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence set forth as SEQ IDAttorney Docket No. L 121360 1330WO (00140)

[0210] NO: 22.

[0211] In some instances, a polynucleotide comprises a nucleotide sequence encoding a colicin comprising an amino acid sequence set forth as SEQ ID NO: 9, or a functional fragment or variant thereof comprising an amino sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence set forth as SEQ ID NO: 9. In such instances, the polynucleotide can comprise a nucleotide sequence set forth as SEQ ID NO: 23, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 23.

[0212] In some instances, a polynucleotide comprises a nucleotide sequence encoding a colicin comprising an amino acid sequence set forth as SEQ ID NO: 10, or a functional fragment or variant thereof comprising an amino sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence set forth as SEQ ID NO: 10. In such instances, the polynucleotide can comprise a nucleotide sequence set forth as SEQ ID NO: 24, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 24.

[0213] In some instances, a polynucleotide comprises a nucleotide sequence encoding a colicin comprising an amino acid sequence set forth as SEQ ID NO: 11, or a functional fragment or variant thereof comprising an amino sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence set forth as SEQ ID NO: 11. In such instances, the polynucleotide can comprise a nucleotide sequence set forth as SEQ ID NO: 25, or a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 25.

[0214] Colicin-encoding polynucleotides can be introduced into a bacteriophage, e.g., any bacteriophage disclosed herein, to generate a recombinant bacteriophage capable of producing a colicin molecule. A recombinant bacteriophage comprising a colicin-encoding polynucleotide retains the ability to target E. coli. In particular, such a recombinant bacteriophage retains its lytic activity. Recombinant bacteriophages capable of producing colicins retain their replicative ability. Colicin-encoding polynucleotides can be introduced into a bacteriophage using any method forAttorney Docket No. L 121360 1330WO (00140)

[0215] introducing heterologous polynucleotides into bacteriophages known in the art. Methods for generating recombinant bacteriophages, including, for instance, introducing exogenous nucleic acids, e.g., polynucleotides disclosed herein, are readily understood and established in the art. Any method known in the art for is envisaged for production of recombinant bacteriophages comprising exogenous and / or heterologous nucleic acids. The colicin-encoding polynucleotides can be introduced into a bacteriophage as exogenous nucleic acid. The colicin-encoding polynucleotides can be heterologous (i.e., colicins not naturally encoded by wild-type bacteriophage in nature).

[0216] Methods for manipulating bacteriophages, including introducing nucleic acids into bacteriophages to generate engineered bacteriophages, as well as assays and for analysis thereof, are well established and widely described in the scientific literature (see, e.g., Kutter et al., Bacteriophages: Biology and Applications, CRC Press, 2005; Calendar, The Bacteriophages, 2nd Ed., Oxford University Press, 2006). Such methods include, but are not limited to, homologous recombination, in vitro packaging of modified genomes, phagemid systems, and CRISPR-Cas mediated genome editing. Any suitable means known to those skilled in the art can be employed to introduce exogenous nucleic acid sequences into bacteriophage genomes, including chemical, biochemical, physical, or molecular biology techniques, without limitation. The choice of method may depend on the bacteriophage type, desired modification, and experimental conditions.

[0217] Exemplary methods for introducing nucleic acids, e.g., exogenous and / or heterologous nucleic acids, into bacteriophages are described in WO2021092254, W02021092210, WO2022098916, and WO2022098899, the contents of which are incorporated herein by reference in their entirety. WO2023215798, the contents of which are incorporated herein by reference in their entirety, describes introduction of colicin-encoding nucleic acids into bacteriophages. Any method known in the art for introducing nucleic acids into bacteriophages is envisaged within the scope of the present application for generated the disclosed engineered bacteriophages.

[0218] The colicin-encoding polynucleotides disclosed herein can be optimized for stable expression in the bacteriophage genome. This includes considerations such as optimizing the insertion site, modifying secondary structures, removing DNA modification sites, and codon optimization to match the bacteriophage genome codon usage. The polynucleotides can be retained through multiple generations of bacteriophage passaging. In some instances, the polynucleotides are retained through at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, or more than 30 passages. Methods for assessing the presence of nucleotide sequences within a bacteriophage, e.g., a bacteriophage genome are readilyAttorney Docket No. L 121360 1330WO (00140)

[0219] understood in the art. Any method can be utilized to confirm the presence of polynucleotides.

[0220] Additional modifications to nucleic acids including for example, codon optimization and removal of restriction enzyme motifs or DNA methylation sites can be used to improve the stability and expression of the inserted nucleic acid within the bacteriophage.

[0221] Colicin-encoding polynucleotides can be incorporated into the genome of a bacteriophage. Genomic insertion sites are described elsewhere herein. In some instances, a nucleotide sequence encoding a colicin is incorporated into a genome between the stop codon and the transcription terminator of a gene, such as a structural gene. Polynucleotides may be incorporated into non-essential genes or coding sequences thereof or within lysogenic genes, or coding sequences thereof. Any such insertion may serve to enhance lytic activity of an engineered bacteriophage, compared to a bacteriophage lacking insertion of a polynucleotide. In other instances, colicin-encoding polynucleotides can be maintained within the bacteriophage extra-chromosomally. Engineered bacteriophages comprising colicin-encoding polynucleotides incorporated into the genome retain the ability to target E. coli. In particular, such recombinant bacteriophages retain lytic activity. Recombinant bacteriophages having colicin-encoding polynucleotides incorporated into the genome retain their replicative ability.

[0222] Any polynucleotide disclosed herein can be introduced into a bacteriophage. For instance, any polynucleotide can be incorporated into the genome of a bacteriophage. It would be readily understood that more than one polynucleotide disclosed herein encoding at least one colicin can be integrated into the genome of a bacteriophage. In some instances, a polynucleotide comprising a nucleotide sequence encoding a colicin having an amino acid sequence set forth as any one of SEQ ID NOs: 1-11, or a functional fragment or variant thereof comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence set forth as any one of SEQ ID NOs: 1-11 is incorporated into the genome of a bacteriophage. In some instances, more than one polynucleotide disclosed herein (e.g., encoding a colicin having an amino acid sequence set forth as any one of SEQ ID NOs: 1-11, or a functional fragment or variant thereof) is incorporated into the genome of a bacteriophage. In such instances, two polynucleotides encoding two colicins can be incorporated into the genome of a bacteriophage. In such instances, a single polynucleotide can encode two colicins. In some instances, a single polynucleotide encodes more than two colicins.

[0223] Polynucleotide sequences are operatively associated with a variety of promoters, terminators and other regulatory elements for expression in various organisms or cells. In some embodiments, the nucleic acid sequence further comprises a leader sequence. In someAttorney Docket No. L 121360 1330WO (00140)

[0224] embodiments, the nucleic acid sequence further comprises a promoter sequence. In some embodiments, at least one promoter and / or terminator is operably linked to the nucleic acid encoding the colicin. Any promoter useful with this disclosure is used and includes, for example, promoters functional with the organism of interest as well as constitutive, inducible, developmental regulated, tissue-specific / preferred- promoters. A regulatory element as used herein is endogenous or heterologous. In some embodiments, an endogenous regulatory element derived from the subject organism is inserted into a genetic context in which it does not naturally occur (e.g. a different position in the genome than as found in nature), thereby producing a recombinant or non-native nucleic acid.

[0225] In some embodiments, expression of the nucleic acid sequence is constitutive, inducible, temporally regulated, developmentally regulated, or chemically regulated. In some embodiments, the expression of the nucleic acid sequence is made constitutive, inducible, temporally regulated, developmentally regulated, or chemically regulated by operatively linking the nucleic acid sequence to a promoter functional in an organism of interest. In some embodiments, repression is made reversible by operatively linking the nucleic acid sequence to an inducible promoter that is functional in an organism of interest. The choice of promoter disclosed herein varies depending on the quantitative, temporal and spatial requirements for expression, and also depending on the host cell to be transformed or expressed from.

[0226] Exemplary promoters for use with the methods, bacteriophages and compositions disclosed herein include promoters that are functional in bacteria. For example, L-arabinose inducible (araBAD, PBAD) promoter, any lac promoter, L-rhamnose inducible (rhaPBAD) promoter, T7 RNA polymerase promoter, trc promoter, tac promoter, lambda bacteriophage promoter (pLpL-9G-50), anhydrotetracycline-inducible (tetA) promoter, trp, Ipp, phoA, recA, proU, cst-1, cadA, nar, Ipp-lac, cspA, 11-lac operator, T3-lac operator, T4 gene 32, T5-lac operator, nprM- lac operator, Vhb, Protein A, corynebacterial-E. coli like promoters, thr, horn, diphtheria toxin promoter, sig A, sig B, nusG, SoxS, katb, a-amylase (Pamy), Ptms, P43 (comprised of two overlapping RNA polymerase G factor recognition sites, oA, GB), Ptms, P43, rplK-rplA, ferredoxin promoter, and / or xylose promoter. In some embodiments, the promoter is a BBa_J23102 promoter. In some embodiments, the promoter works in a broad range of bacteria, such as BBa_J23104, BBa_J23109, Bba_J23102. In some embodiments the promoter is derived from the target bacterium, such as endogenous colicin promoter, pl 6, plpp, or ptat. In some embodiments, the promoter is a bacteriophage promoter, such as the promoter for gpl05 or gp245.

[0227] In some embodiments, the promoter comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQAttorney Docket No. L 121360 1330WO (00140)

[0228] ID NOs: 28-30. In some instances, the promoter comprises at least or about 95% sequence identity to any one of SEQ ID NOS: 28-30. In some instances, the promoter comprises at least or about 97% sequence identity to any one of SEQ ID NOS: 28-30. In some instances, the promoter comprises at least or about 99% sequence identity to any one of SEQ ID NOS: 28-30. In some instances, the promoter comprises 100% sequence identity to any one of SEQ ID NOS: 28-30. In some instances, the promoter comprises at least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more than 50 nucleotides of any one of SEQ ID NOS: 28-30. In some instances, the promoter comprises at least a portion having at least or about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, or more than 215 nucleotides of any one of SEQ ID NOS: 28-30. In some embodiments, the promoter comprises 10-300, 20-300, 30-300, 40-300, 50-300, 60-300, 70-300, 80-300, 90-300, 100-300, 110-300, 120-300, 130-300, MO-SOO, 150-300, 160-300, 170-300, 180-300, 190-300, 210-300, 220-300, 230-300, 240-300, 250-300, 260-300, 270-300, 280-300, 290-300 nucleotides with sequence identity to any one of SEQ ID NOS: 28-30.

[0229] In some embodiments, inducible promoters are used. In some embodiments, chemical-regulated promoters are used to modulate the expression of a gene in an organism through the application of an exogenous chemical regulator. The use of chemically regulated promoters enables RNAs and / or the polypeptides encoded by the nucleic acid sequence to be synthesized only when, for example, an organism is treated with the inducing chemicals. In some embodiments where a chemical-inducible promoter is used, the application of a chemical induces gene expression. In some embodiments wherein a chemical-repressible promoter is used, the application of the chemical represses gene expression. In some embodiments, the promoter is a light-inducible promoter, where application of specific wavelengths of light induces gene expression. In some embodiments, a promoter is a light- repressible promoter, where application of specific wavelengths of light represses gene expression.

[0230] A polynucleotide can comprise an expression cassette. In some embodiments, expression cassettes are designed to express a nucleotide sequence disclosed herein. In some embodiments, the nucleic acid sequence is an expression cassette encoding a colicin. The expression cassette can be chimeric, for instance, whereby at least one component is heterologous with respect to at least one other components. An expression cassette can be naturally occurring, but obtained in a recombinant form useful for heterologous expression. In some instances, an expression cassette includes a transcriptional and / or translational termination region (i.e. termination region). Such elements areAttorney Docket No. L 121360 1330WO (00140)

[0231] understood to be functional in the host organism. In some embodiments, termination regions are responsible for the termination of transcription beyond the heterologous nucleic acid sequence of interest and for correct mRNA polyadenylation. In some embodiments, the termination region is native to the transcriptional initiation region, is native to the operably linked nucleic acid sequence of interest, is native to the host cell, or is derived from another source (i.e., foreign or heterologous to the promoter, to the nucleic acid sequence of interest, to the host, or any combination thereof). In some embodiments, terminators are operably linked to the nucleic acid sequence disclosed herein. An expression cassette can include a nucleotide sequence encoding a selectable marker. In some embodiments, the nucleotide sequence encodes either a selectable or a screenable marker, depending on whether the marker confers a trait that is selected for by chemical means, such as by using a selective agent (e.g. an antibiotic), or on whether the marker is simply a trait that one identifies through observation or testing, such as by screening (e.g., fluorescence)

[0232] Regulation of expression of heterologous sequences can be regulated by heterologous regulatory sequences. For example, constructs may include a transcriptional activator sequence. Regulatory sequences should be selected for expression of the heterologous coding sequence with consideration of the bacteriophage lifecycle. For example, strong bacterial promoters in the absence of a bacteriophage infection may not be strong promoters in the context of a bacteriophage infection.

[0233] The nucleotide sequences disclosed herein can be optimized for stable expression in a host organism, such as a bacteriophage. For instance, a nucleotide sequence encoding a colicin can be sequence optimized for stable and / or efficient expression when integrated into a bacteriophage genome. In some embodiments, the insert is stable through at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 generations of passaging. In some embodiments, the nucleic acid sequence is optimized by optimizing the insertion site, modifying secondary structures, modifying DNA modification sites, modifying restriction enzyme motifs, codon optimization, GC% optimization, or a combination thereof. In some embodiments, the insertion site of the nucleic acid sequence is optimized. In some embodiments, the nucleic acid sequence is modified to remove secondary structures.

[0234] In certain embodiments, a bacteriophage comprises a nucleic acid insert modified from an exogenous nucleic acid described herein, wherein the nucleic acid comprises a first plurality of codons encoding for a first protein, and the nucleic acid insert comprises a second plurality of codons encoding for a second protein, wherein the first protein and the second protein have at least 90% amino acid sequence identity, and wherein at least 50% of the second plurality of codons are high frequency codons in the bacteriophage genome. In certain aspects, described herein is a method of inserting an exogenous sequence comprising a plurality of codons encoding a firstAttorney Docket No. L 121360 1330WO (00140)

[0235] protein into a bacteriophage, the method comprising substituting one or more of the plurality of codons with a codon native to the bacteriophage to generate a nucleic acid insert encoding a second protein, wherein the first protein and the second protein have at least 90% amino acid sequence identity. In certain aspects, described herein is a nucleic acid insert modified from an exogenous nucleic acid, wherein the nucleic acid comprises a first plurality of codons encoding for a first protein, and the nucleic acid insert comprises a second plurality of codons encoding for a second protein, wherein the first protein and the second protein have at least 90% amino acid sequence identity, and wherein at least 50% of the second plurality of codons are high frequency codons in the bacteriophage genome. In some embodiments, the first protein and the second protein have at least 95%, 97.5%, 99% or 99.5% sequence identity. In some embodiments, at least 50%< 60%, 70%, 80%, 90% or more than 90% of the second plurality of codons are high frequency codons in the bacteriophage genome. In some embodiments, the second plurality of codons match the profile of codons in the bacteriophage genome.

[0236] In some embodiments, the nucleic acid sequence is modified to remove DNA modification sites. In some embodiments, the DNA modification sites comprise DNA methylation sites.

[0237] In some embodiments, the nucleic acid sequence is modified to remove restriction enzyme motifs. In some embodiments, the nucleic acid sequence is modified to remove restriction enzyme motifs for a restriction enzyme derived from a bacterial species described herein. In some embodiments, the nucleic acid insert does not comprise, or comprises fewer than 10 sites recognized by a bacterial enzyme.

[0238] In some embodiments, the nucleic acid sequence is codon optimized for expression in any species of interest. Codon optimization involves modification of a nucleotide sequence for codon usage bias using species-specific codon usage tables. The codon usage tables are generated based on a sequence analysis of the most highly expressed genes for the species of interest. When the nucleotide sequences are to be expressed in the nucleus, the codon usage tables are generated based on a sequence analysis of highly expressed nuclear genes for the species of interest. The modifications of the nucleotide sequences are determined by comparing the species-specific codon usage table with the codons present in the native polynucleotide sequences. Codon optimization of a nucleotide sequence results in a nucleotide sequence having less than 100% identity (e.g., 50%, 60%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and the like) to the native nucleotide sequence but which still encodes a polypeptide having the same function as that encoded by the original nucleotide sequence. In some embodiments, the nucleic acid sequences of this disclosure are codon optimized for expression in the organism / species of interest.Attorney Docket No. L 121360 1330WO (00140)

[0239] In some embodiments, the nucleic acid sequence is modified to optimize the percent GC content. In some embodiments, the percent GC content is modified so that at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% 90% or more than 90% of the nucleotides comprises guanine or cytosine. In some embodiments, the percent GC content is modified so that no more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% 90% or more than 90% of the nucleotides comprises guanine or cytosine.

[0240] In some embodiments, the exogenous nucleic acid is a bacterial nucleic acid. In some embodiments, the nucleic acid and the bacterial nucleic acid have less than 100%, 95%, 90%, 80%, 70%, 60%, or 50% sequence identity. In some embodiments, the bacterial nucleic acid comprises a nucleotide sequence encoding an antimicrobial agent and / or peptide, such as a colicin.

[0241] In some embodiments, a heterologous polynucleotide sequence is inserted into a bacteriophage genome. Insertion of a polynucleotide sequence into a bacteriophage genome preserves the lytic activity of the bacteriophage. Any location of a bacteriophage genome is envisaged for insertion of a polynucleotide sequence. For instance, the polynucleotide sequence can be inserted at a transcription terminator site at the end of an operon. In some embodiments, the nucleic acid sequence is inserted into the bacteriophage genome as a replacement for one or more non-essential genes, or portions thereof. In such instances, the integrated polynucleotide sequence replaces such non-essential genes, or portions thereof, which are removed from the genome. In such instances, a polynucleotide sequence replaces one or more lysogenic genes. Non-essential genes refer to at least one gene that is not required to be present or functional (e.g., encode a functional gene product) for the survival of the bacteriophage. Non-essential genes are not required in a recombinant bacteriophage to practice any of the methods disclosed herein (e.g., reducing the number of E. coli in a population of bacteria). The replacement of non-essential and / or lysogenic genes does not affect the lytic activity of the bacteriophage. That is, a recombinant bacteriophage retains lytic activity. In some instances, the non-essential gene is a gene that is non-essential for the induction and / or maintenance of lytic cycle. In some instances, the replacement of non-essential and / or lysogenic genes preserves lytic activity of the bacteriophage. In some instances, the replacement of non-essential and / or lysogenic genes enhances lytic activity of the bacteriophage. In some instances, the replacement of non-essential and / or lysogenic genes renders a lysogenic bacteriophage lytic.

[0242] In some embodiments, the nucleic acid sequence is introduced into the bacteriophage genome at a first location while one or more non-essential and / or lysogenic genes are separately removed and / or inactivated from the bacteriophage genome at a separate location. In some embodiments, the nucleic acid sequence is introduced into the bacteriophage at a first locationAttorney Docket No. L 121360 1330WO (00140)

[0243] while one or more non-essential and / or lysogenic genes are separately removed and / or inactivated from the bacteriophage genome at multiple separate locations. In some embodiments, the removal and / or inactivation of one or more non-essential and / or lysogenic genes does not affect the lytic activity of the bacteriophage. In some embodiments, the removal and / or inactivation of one or more non-essential and / or lysogenic genes preserves the lytic activity of the bacteriophage. In some embodiments, the removal of one or more non-essential and / or lysogenic genes renders a lysogenic bacteriophage into a lytic bacteriophage.

[0244] In some embodiments, the bacteriophage is a temperate bacteriophage which has been rendered lytic by any of the aforementioned means. In some embodiments, a temperate bacteriophage is rendered lytic by the removal, replacement, or inactivation of one or more lysogenic genes. In some embodiments, the lytic activity of the bacteriophage is due to the removal, replacement, or inactivation of at least one lysogeny gene. In some embodiments, the lysogenic gene plays a role in the maintenance of lysogenic cycle in the bacteriophage. In some embodiments, the lysogenic gene plays a role in establishing the lysogenic cycle in the bacteriophage. In some embodiments, the lysogenic gene plays a role in both establishing the lysogenic cycle and in the maintenance of the lysogenic cycle in the bacteriophage. In some embodiments, the lysogenic gene is a repressor gene. In some embodiments, the lysogenic gene is cl repressor gene. In some embodiments, the lysogenic gene is an activator gene. In some embodiments, the lysogenic gene is ell gene. In some embodiments, the lysogenic gene is lexA gene. In some embodiments, the lysogenic gene is int (integrase) gene. In some embodiments, two or more lysogeny genes are removed, replaced, or inactivated to cause arrest of a bacteriophage lysogeny cycle and / or induction of a lytic cycle. In some embodiments, a temperate bacteriophage is rendered lytic by the insertion of one or more lytic genes. In some embodiments, a temperate bacteriophage is rendered lytic by the insertion of one or more genes that contribute to the induction of a lytic cycle. In some embodiments, a temperate bacteriophage is rendered lytic by altering the expression of one or more genes that contribute to the induction of a lytic cycle. In some embodiments, a temperate bacteriophage phenotypically changes from a lysogenic bacteriophage to a lytic bacteriophage. In some embodiments, a temperate bacteriophage is rendered lytic by environmental alterations. In some embodiments, environmental alterations include, but are not limited to, alterations in temperature, pH, or nutrients, exposure to antibiotics, hydrogen peroxide, foreign DNA, or DNA damaging agents, presence of organic carbon, and presence of heavy metal (e.g. in the form of chromium (VI). In some embodiments, a temperate bacteriophage that is rendered lytic is prevented from reverting to lysogenic state.

[0245] In some embodiments, the replacement, removal, inactivation, or any combination thereof,Attorney Docket No. L 121360 1330WO (00140)

[0246] of one or more non-essential and / or lysogenic genes is achieved by chemical, biochemical, and / or any suitable method. In some embodiments, the insertion of one or more lytic genes is achieved by any suitable chemical, biochemical, and / or physical method by homologous recombination.

[0247] The nucleotide sequence can be codon optimized for expression in any species of interest. It would be readily understood by one having ordinary skill in the art that codon optimization may improve gene expression efficiency in a particular host, e.g., a bacteriophage. Without wishing to be bound by any theory, codon optimization may improve expression efficiency by aligning a nucleotide sequence with a host’s preferred codons, balancing translation speed, avoiding rare codons, and / or managing mRNA stability and structure. Codon optimization involves modification of a nucleotide sequence for codon usage bias using, for example, species-specific codon usage tables. The codon usage tables are generated based on a sequence analysis of the most highly expressed genes for the species of interest. When the nucleotide sequences are to be expressed in the nucleus, the codon usage tables are generated based on a sequence analysis of highly expressed nuclear genes for the species of interest. The modifications of the nucleotide sequences are determined by comparing the species-specific codon usage table with the codons present in the native polynucleotide sequences. Codon optimization of a nucleotide sequence results in a nucleotide sequence having less than 100% identity (e.g., 50%, 60%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,) to the native nucleotide sequence but which still encodes a polypeptide having the same function as that encoded by the original nucleotide sequence. In some embodiments, the nucleic acid sequences of this disclosure are codon optimized for expression in the organism / species of interest.

[0248] It would be understood that introduction of heterologous polynucleotides into bacteriophages may impact recognition of a bacteriophage by a host immune response and that minimizing immune activation or pro-inflammatory response to a recombinant bacteriophage would be beneficial. As such, heterologous polynucleotides may be modified prior to incorporation into a bacteriophage such that immune recognition to such sequences and bacteriophages is minimized. For instance, heterologous polynucleotides may be sequence optimized to generate variants exhibiting reduced innate immune recognition or increased genome stability. The mammalian innate immune system relies on evolutionarily conserved pattern-recognition mechanisms that enable host cells to detect and respond to foreign nucleic acids. Among the molecular features that serve as signatures of microbial DNA, unmethylated cytosine-phosphate-guanine (CpG) dinucleotides are particularly significant. In vertebrates, CpG motifs are relatively rare and frequently methylated; therefore, their abundance and unmethylated state in microbialAttorney Docket No. L 121360 1330WO (00140)

[0249] genomes make them potent indicators of non-self DNA. CpG motifs act as microbe-associated molecular patterns (MAMPs) recognized primarily by Toll-like receptor 9 (TLR9) within the endosomal compartments of immune cells, B cells, and a range of epithelial cell types. Upon binding CpG-containing DNA, TLR9 undergoes a conformational change that initiates MyD88-dependent signaling cascades that activate NF-KB, promote the expression of

[0250] pro-inflammatory cytokines, and stimulate type I interferon responses. Historically, much of the focus on CpG-mediated immune recognition has come from studies of bacterial and eukaryotic viral DNA. However, it has been shown that the same principles are relevant to bacteriophages, whose genomes vary widely in CpG content and organization. In particular, bacteriophage genomes with higher CpG frequencies are more likely to activate TLR9, while CpG-poor phage genomes may evade or dampen innate immune detection (see Kharrat L. et al. Large-Scale Genomic Analysis of CpG-Mediated Immunogenicity in Bacteriophages and a Novel Predictive Risk Index. bioRxiv, 16 May 2025, the contents of which are incorporated herein by reference in their entirety). Importantly, CpG abundance alone does not seem to fully predict immunogenic potential, as the spatial arrangement of CpG motifs, e.g., the inter-CpG distance, appears to play a crucial role. In particular, closely spaced motifs, especially those separated by fewer than 30 nucleotides, facilitate TLR9 homodimer formation and enhance receptor activation. It would be understood that polynucleotide variants disclosed herein may contain CpG motifs that are spatially arranged in order to reduce host immune recognition and activation. CpG motifs may be separated by at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, or at least 50 nucleotides. In some instances, CpG motifs are separated by at least 20 nucleotides. In some instances, CpG motifs are separated by at least 25 nucleotides. In some instances, CpG motifs are separated by at least 30 nucleotides. In some instances, CpG motifs are separated by at least 35 nucleotides. In some instances, CpG motifs are separated by at least 40 nucleotides. In some instances, CpG motifs are separated by at least 45 nucleotides. In some instances, CpG motifs are separated by at least 50 nucleotides.

[0251] Nucleotide sequences surrounding CpG motifs may also contribute to immune recognition. Certain tetramer contexts, such as ACGT, CCGT, and TCGT, are associated with enhanced TLR9 activation, whereas motifs embedded within GCGC, GCGG, or CCGC tetramers can suppress or weaken receptor engagement (see Kharrat L. et al. Large-Scale Genomic Analysis of CpG-Mediated Immunogenicity in Bacteriophages and a Novel Predictive Risk Index. bioRxiv, 16 MayAttorney Docket No. L 121360 1330WO (00140)

[0252] 2025). As such, it would be understood that multiple aspects related to CpG content within polynucleotides, e.g., CpG quantity, spacing, and surrounding nucleotides, impacts recognition of bacteriophage DNA by the host immune system.

[0253] Polynucleotides disclosed herein (e.g., polynucleotides comprising a nucleotide sequence encoding a colicin) may be optimized to reduce immune activation in a host. In some instances, CpG quantity, spacing, and / or surrounding nucleotides may be modified in the presently disclosed polynucleotide variants, such that compared to the unmodified polynucleotide sequence. In some instances, CpG quantity may be reduced as described elsewhere herein. In some instances, CpG spacing may be increased as described elsewhere herein. In some instances, the nucleotide sequences surrounding at least one CpG motif may be sequences that suppress or weaken immune activation e.g., reduce TLR9 activation.

[0254] One having ordinary skill in the art would readily understand how to modify CpG motifs and / or surrounding nucleotides to reduce immune activation. Any method for predicting and / or reducing immune activation known in the art can be utilized. For instance, Kharrat L et al describe a Bacteriophage Risk Index (BRI) that quantifies phage immunogenic potential based on underlying CpG-associated factors, wherein high BRI scores, corresponding to CpG-rich and immunostimulatory genomic patterns, induce broad and robust pro-inflammatory and antiviral responses in human epithelial cells and lower CpG-mediated risk profiles trigger more limited immune activation. As such, one having ordinary skill in the art would readily be able to predict immunostimulatory patterns of polynucleotides using such methods known in the art and generate polynucleotide variants that when incorporated into bacteriophages, such as those disclosed herein, generate bacteriophages having reduced immune activation. In some instances, the polynucleotide variants, when incorporated into a bacteriophage, reduce immune activation, e.g., host inflammation in at least one tissue, such as the gastrointestinal tract, of a subject. For instance, bacteriophages may exhibit reduce pro-inflammatory response in a subject. A pro-inflammatory response may be reduced in any tissue of a subject. For example, a bacteriophage may exhibit a reduce pro-inflammatory response in the gastrointestinal tract of a subject. It would be understood that immune activation is reduced compared to a similar bacteriophage, e.g., same bacteriophage strain, having a polynucleotide sequence that is not modified to reduce immune activation.

[0255] For instance, a heterologous polynucleotide may be optimized to generate a variant polynucleotide having decreased CpG content. CpG content can be decreased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. One having ordinary skill in the art would readilyAttorney Docket No. L 121360 1330WO (00140)

[0256] understand that synonymous codon substitutions could be utilized to minimize CpG content in heterologous polynucleotides. Polynucleotide variants are capable of encoding and producing functional gene products. For instance, polynucleotide variants disclosed herein can produce functional colicins when incorporated into bacteriophages. In some instances, bacteriophages can comprise polynucleotide variants having minimized CpG content that retain capability of producing colicins, such as Colicin M and / or Colicin K.

[0257] Polynucleotides and / or expression cassettes disclosed herein can be expressed transiently and / or stably incorporated into the genome of a host organism. Polynucleotides and / or expression cassettes can be introduced into a cell by any method known in the art. Exemplary methods of transformation include transformation via electroporation of competent cells, passive uptake by competent cells, chemical transformation of competent cells, as well as any other electrical, chemical, physical (mechanical) and / or biological mechanism that results in the introduction of nucleic acid into a cell, including any combination thereof. In some embodiments, transformation of a cell comprises nuclear transformation. In some embodiments, transformation of a cell comprises plasmid transformation and conjugation.

[0258] When more than one nucleic acid sequence is introduced, the nucleotide sequences can be assembled as part of a single nucleic acid construct, or as separate nucleic acid constructs, and can be located on the same or different nucleic acid constructs. In some embodiments, nucleotide sequences are introduced into the cell of interest in a single transformation event, or in separate transformation events. One having ordinary skill in the art would readily understand how to engineer recombinant bacteriophages with polynucleotide disclosed herein. For instance, it would be fully within the skill of an ordinarily skilled artisan to generate a recombinant bacteriophage having a polynucleotide sequence comprising a nucleotide sequence encoding a colicin integrated into the genome. Any bacteriophage disclosed herein can be utilized to generate a recombinant bacteriophage, e.g., capable of producing at least one colicin. Any method for introducing polynucleotides into a bacteriophage can be utilized.

[0259] VI. Wild-type bacteriophages

[0260] The host range of bacteriophage varies, both in the identity and the breadth of the hosts. As with colicins, bacteriophage can be selected to have a broad or narrow host range. As such, colicins can be selected to target pathogenic bacteria, e.g., E. coli bacteria, to reduce the population of pathogenic bacteria, while sparing non-target, e.g., beneficial bacteria.

[0261] Identification of appropriate bacteriophage for reducing the population of target bacteria while sparing the population of non-target bacteria can be determined by collection of biological samples,Attorney Docket No. L 121360 1330WO (00140)

[0262] e.g., from a subject or population of subjects, and analyzing cell viability by an optical density assay or plate-based plaquing assay.

[0263] Provided herein are recombinant bacteriophages that are particularly useful for expression of heterologous antimicrobial proteins, e.g., colicin proteins, e.g., colicin M and K. It would be understood that a polynucleotide comprising a nucleotide sequence encoding a heterologous protein can be inserted anywhere in the bacteriophage genome such that the lytic lifecycle of the bacteriophage is retained. Nucleotide sequences that are integrated into the bacteriophage genome are expressed at a sufficiently high level for practicing the uses and methods disclosed herein (e.g., reducing the number of E. coli in a population of bacteria). Relative levels of expression can be determined using conventional methods known in the art for examining gene expression in bacteriophages, for example, using a luminescent reporter gene assay.

[0264] In some instances, a nucleotide sequence encoding an antimicrobial agent, such as a colicin, is integrated at a site in a bacteriophage genome located after the stop codon of a structural gene and before its termination sequence (i.e., between the stop codon and termination sequence).

[0265] Exemplary structural genes can include major capsid protein, tail fiber protein, tail spike protein, tail sheath protein, and head stabilization / decoration (HOC) protein. Typically, heterologous sequences are not inserted into perceived operons or overlapping genes within the genome.

[0266] Identification of such sites in a bacteriophage genome is well within the ability of those of skill in the art. Upon integration of a heterologous nucleotide sequence, bacteriophage viability is retained. Such bacteriophages retain the ability to target E. coli and proliferate.

[0267] The bacteriophage constructs provided herein can be based on any bacteriophage that infects E. coli. Exemplary genera of such bacteriophage include Justusliebigvirus, Tequatrovirus, Felixounavirus, Mosigvirus, Phapecoctavirus, Tequintavirus, Peduovirus, Warwickvirus, Gaprivervirus, Krischvirus, Dhillonvirus, Tunavirus, Gequatrovirus, Mydovirus, Bonnellvirus, Enterogokushovirus, Comdogvirus, Drulisvirus, Inovirus, Przondovirus, Yonseivirus, Seunavirus, Asteriusvirus, Deseoctovirus, Caminolopintovirus, Saltrevirus, Skarprettervirus, Hungariovirus, Vidquintavirus, Derbicusvirus, Vectrevirus, Vequintavirus, Kagunavirus, Nonagvirus, Gamaleyavirus, Kuravirus, Guelphvirus, Christensenvirus, Loudonvirus, ogunavirus, Kayfunavirus, Agtrevirus, Hanrivervirus, Seuratvirus, Shuimuvirus, Swiduovirus,

[0268] Lederbergvirus, Cuauhtlivirus, Rosemountvirus, Xuanwuvirus, Lietduovirus, and Ripduovirus. In certain embodiments, the recombinant bacteriophage comprises a genera including Tequatrovirus, Phapecoctavirus, Justusliebingvirus, Vequintavirus, or Krischvirus. It is understood that the recombinant bacteriophages provided herein comprise a heterologous nucleotide sequence. Such nucleotide sequence can be integrated into the genome. As such, the recombinant bacteriophage canAttorney Docket No. L 121360 1330WO (00140)

[0269] comprise a Justusliebigvirus, Tequatrovirus, Felixounavirus, Mosigvirus, Phapecoctavirus, Tequintavirus, Peduovirus, Warwickvirus, Gaprivervirus, Krischvirus, Dhillonvirus, Tunavirus, Gequatrovirus, Mydovirus, Bonnellvirus, Enterogokushovirus, Corndogvirus, Drulisvirus, Inovirus, Przondovirus, Yonseivirus, Seunavirus, Asteriusvirus, Deseoctovirus, Caminolopintovirus, Saltrevirus, Skarprettervirus, Hungariovirus, Vidquintavirus, Derbicusvirus, Vectrevirus, Vequintavirus, Kagunavirus, Nonagvirus, Gamaleyavirus, Kuravirus, Guelphvirus, Christensenvirus, Loudonvirus, ogunavirus, Kayfunavirus, Agtrevirus, Hanrivervirus, Seuratvirus, Shuimuvirus, Swiduovirus, Lederbergvirus, Cuauhtlivirus, Rosemountvirus, Xuanwuvirus, Lietduovirus, or Ripduovirus, wherein said bacteriophage comprises a heterologous polynucleotide comprising a nucleotide sequence encoding a colicin.

[0270] Thus, the recombinant bacteriophages provided herein can comprise at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity sequence identity to a wild-type bacteriophage. In such instances, the recombinant bacteriophage can comprise at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity sequence identity to a Justusliebigvirus, Tequatrovirus, Felixounavirus, Mosigvirus, Phapecoctavirus, Tequintavirus, Peduovirus, Warwickvirus, Gaprivervirus, Krischvirus, Dhillonvirus, Tunavirus, Gequatrovirus, Mydovirus, Bonnellvirus, Enterogokushovirus, Comdogvirus, Drulisvirus, Inovirus, Przondovirus, Yonseivirus, Seunavirus, Asteriusvirus, Deseoctovirus, Caminolopintovirus, Saltrevirus, Skarprettervirus, Hungariovirus, Vidquintavirus, Derbicusvirus, Vectrevirus, Vequintavirus, Kagunavirus, Nonagvirus, Gamaleyavirus, Kuravirus, Guelphvirus, Christensenvirus, Loudonvirus, ogunavirus, Kayfunavirus, Agtrevirus, Hanrivervirus, Seuratvirus, Shuimuvirus, Swiduovirus, Lederbergvirus, Cuauhtlivirus, Rosemountvirus, Xuanwuvirus, Lietduovirus, or Ripduovirus.

[0271] A recombinant bacteriophage disclosed herein can comprise at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity sequence identity to a wild-type bacteriophage that targets E. coli. A recombinant bacteriophage retains the ability to target E. coli. In some instances, a recombinant bacteriophage has been modified to expand E. coli targeting (e.g., increased number of E. coli strains or increased protein targets).Attorney Docket No. L 121360 1330WO (00140)

[0272] In some instances, the recombinant bacteriophage comprises at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to a Tequatrovirus. In such instances, the recombinant bacteriophage can comprise at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to a Tequatrovirus. In such instances, the recombinant Tequatrovirus retains the ability to target Escherichia spp. In some instances, the bacteriophage is a recombinant Tequatrovirus. In some instances, the recombinant Tequatrovirus is capable of producing Colicin K. In such instances, the recombinant Tequatrovirus can comprise at least one polynucleotide sequence comprising a nucleotide sequence encoding a Colicin K. In some instances, the bacteriophage is a recombinant Tequatrovirus capable of producing Colicin M. In such instances, the recombinant Tequatrovirus can comprise at least one polynucleotide sequence comprising a nucleotide sequence encoding a Colicin M. In some instances, the bacteriophage is a recombinant Tequatrovirus capable of producing Colicin K and Colicin M. In such instances, the recombinant Tequatrovirus can comprise a first polynucleotide sequence comprising a nucleotide sequence encoding Colicin M, and a second polynucleotide sequence comprising a nucleotide sequence encoding Colicin K.

[0273] In some instances, the recombinant bacteriophage comprises at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to a Phapecoctavirus. In such instances, the recombinant bacteriophage can comprise at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to a Phapecoctavirus. In such instances, the recombinant Phapecoctavirus retains the ability to target Escherichia spp. In some instances, the bacteriophage is a recombinant Phapecoctavirus. In some instances, the recombinant Phapecoctavirus is capable of producing Colicin K. In such instances, the recombinant Phapecoctavirus can comprise at least one polynucleotide sequence comprising a nucleotide sequence encoding a Colicin K. In some instances, the bacteriophage is a recombinant Phapecoctavirus capable of producing Colicin M. In such instances, the recombinant Phapecoctavirus can comprise at least one polynucleotide sequence comprising a nucleotideAttorney Docket No. L 121360 1330WO (00140)

[0274] sequence encoding a Colicin M. In some instances, the bacteriophage is a recombinant Phapecoctavirus capable of producing Colicin K and Colicin M. In such instances, the recombinant Phapecoctavirus can comprise a first polynucleotide sequence comprising a nucleotide sequence encoding Colicin M, and a second polynucleotide sequence comprising a nucleotide sequence encoding Colicin K.

[0275] In some instances, the recombinant bacteriophage comprises at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to a Justusliebingvirus. In such instances, the recombinant bacteriophage can comprise at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to a Justusliebingvirus. In such instances, the recombinant Justusliebingvirus retains the ability to target Escherichia spp. In some instances, the bacteriophage is a recombinant Justusliebingvirus. In some instances, the recombinant Justusliebingvirus is capable of producing Colicin K. In such instances, the recombinant Justusliebingvirus can comprise at least one polynucleotide sequence comprising a nucleotide sequence encoding a Colicin K. In some instances, the bacteriophage is a recombinant Justusliebingvirus capable of producing Colicin M. In such instances, the recombinant Justusliebingvirus can comprise at least one polynucleotide sequence comprising a nucleotide sequence encoding a Colicin M. In some instances, the bacteriophage is a recombinant Justusliebingvirus capable of producing Colicin K and Colicin M. In such instances, the recombinant Justusliebingvirus can comprise a first polynucleotide sequence comprising a nucleotide sequence encoding Colicin M, and a second polynucleotide sequence comprising a nucleotide sequence encoding Colicin K.

[0276] In some instances, the recombinant bacteriophage comprises at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to a Vequintavirus. In such instances, the recombinant bacteriophage can comprise at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to a Vequintavirus. In such instances, the recombinant Vequintavirus retains the ability to target Escherichia spp. In someAttorney Docket No. L 121360 1330WO (00140)

[0277] instances, the bacteriophage is a recombinant Vequintavirus. In some instances, the recombinant Vequintavirus is capable of producing Colicin K. In such instances, the recombinant Vequintavirus can comprise at least one polynucleotide sequence comprising a nucleotide sequence encoding a Colicin K. In some instances, the bacteriophage is a recombinant Vequintavirus capable of producing Colicin M. In such instances, the recombinant Vequintavirus can comprise at least one polynucleotide sequence comprising a nucleotide sequence encoding a Colicin M. In some instances, the bacteriophage is a recombinant Vequintavirus capable of producing Colicin K and Colicin M. In such instances, the recombinant Vequintavirus can comprise a first polynucleotide sequence comprising a nucleotide sequence encoding Colicin M, and a second polynucleotide sequence comprising a nucleotide sequence encoding Colicin K.

[0278] In some instances, the recombinant bacteriophage comprises at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to a Krischvirus. In such instances, the recombinant bacteriophage can comprise at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to a Krischvirus. In such instances, the recombinant Krischvirus retains the ability to target Escherichia spp. In some instances, the bacteriophage is a recombinant Krischvirus. In some instances, the recombinant Krischvirus is capable of producing Colicin K. In such instances, the recombinant Krischvirus can comprise at least one polynucleotide sequence comprising a nucleotide sequence encoding a Colicin K. In some instances, the bacteriophage is a recombinant Krischvirus capable of producing Colicin M. In such instances, the recombinant Krischvirus can comprise at least one polynucleotide sequence comprising a nucleotide sequence encoding a Colicin M. In some instances, the bacteriophage is a recombinant Krischvirus capable of producing Colicin K and Colicin M. In such instances, the recombinant Krischvirus can comprise a first polynucleotide sequence comprising a nucleotide sequence encoding Colicin M, and a second polynucleotide sequence comprising a nucleotide sequence encoding Colicin K.

[0279] Exemplary Tequatrovirus wild type genomes for insertion of a heterologous sequence include the sequence of p004k (ATCC Accession No. PTA-127149), pOOex (ATCC Accession No. PTA-127145), p6921 (ATCC Accession No. PTA-127576), p6977 (ATCC Accession No. PTA-127577), and p6984 (ATCC Accession No. PTA-127578).

[0280] Exemplary Phapecoctavirus wild type genomes for insertion of a heterologous sequenceAttorney Docket No. L 121360 1330WO (00140)

[0281] include the sequence of pOOcg and pOOgc.

[0282] Exemplary Justusliebingvirus wild type genomes for insertion of a heterologous sequence include the sequence of pOOmf, and strains can be confirmed based on the taxonomic characterization provided, for example at the website found at

[0283] www.ncbi.nlm. nih.gov / Taxonomy / Browser / wwwtax.cgi?mode=Undef&id=2948775&lvl=3&kee p=l&srchmode=l&unlock and img.jgi.doe.gov / cgi-bin / m / main.cgi?section=TaxonDetail&page=taxonDetail&taxon_oid=2974713075.

[0284] Exemplary Vequintavirus wild type genomes for insertion of a heterologous sequence include the sequence of p007621, and Genome assembly ViralProj248531 (NCBI RefSeq assembly GCF 000919935.1) and Genome assembly ViralProj30613 (NCBI RefSeq assembly GCF_000875465.1).

[0285] Exemplary Krischvirus wild type genomes for insertion of a heterologous sequence include the sequence of p007817, and strains can be confirmed based on the taxonomic characterization provided, for example at the websites found at

[0286] www.ncbi.nlm. nih.gov / Taxonomy / Browser / wwwtax.cgi?name=Krischvirus and www.ncbi.nlm.nih.gov / Taxonomy / Browser / wwwtax.cgi?mode=Info&id=1913651&lvl=3&lin=f &keep= 1 & srchmode= 1 &unlock.

[0287] Exemplary Mosigvirus wild type genomes for insertion of a heterologous sequence include the sequence of pOOcO (ATCC Accession No. PTA-127143).

[0288] Exemplary Myovinidae wild type genomes for insertion of a heterologous sequence include the sequence of pOOke (ATCC Accession No. PTA-127148).

[0289] In some embodiments, the bacteriophage comprises a bacteriophage listed in Table 1, or a bacteriophage having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to a bacteriophage listed in Table 1. For instance, a bacteriophage can comprise at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to a bacteriophage of Table 1.

[0290] Table 1. Exemplary wild-type bacteriophages.Attorney Docket No. L 121360 1330WO (00140)

[0291]

[0292] An engineered bacteriophage can comprise a genome sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to p004k (i.e., the engineered bacteriophage comprises a polynucleotide sequence, e.g., a full-length genomic sequence, having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to p004k). In such instances, the engineered bacteriophage can comprise a genome sequence comprising at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to p004k. In some instances, the engineered bacteriophage can comprise about 97%, about 98%, or about 99% sequence identity to p004k. In such instances, the engineered bacteriophage can comprise about 98% sequence identity to p004k. In some instances, the engineered bacteriophage can comprise a genome sequence comprising at least 97%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, at least 98%, at least 98.1%, atAttorney Docket No. L 121360 1330WO (00140)

[0293] least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to p004k. In such instances, the engineered bacteriophage retains the ability to target Escherichia spp. The engineered bacteriophage retains lytic activity. The engineered bacteriophage can retain replicative ability. In some instances, the bacteriophage is an engineered p004k. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p004k is capable of producing Colicin K polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise at least one Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p004k is capable of producing Colicin M polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise at least one Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p004k is capable of producing Colicin K polypeptide or a functional fragment or variant thereof, and Colicin M polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof. The engineered bacteriophage can have at least one colicin-encoding polynucleotide comprising a nucleotide sequence encoding at least one colicin or functional fragment or variant thereof. In one instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p004k comprises a Colicin M polynucleotide encoding a Colicin M polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin M polypeptide. In another instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p004k comprises a Colicin K polynucleotide encoding a Colicin K polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin K polypeptide. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p004k comprises more than one colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding moreAttorney Docket No. L 121360 1330WO (00140)

[0294] than one colicin. In one instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p004k comprises a first colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding a first colicin or functional fragment or variant thereof, and a second colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding a second colicin or functional fragment or variant thereof. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p004k can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin M polypeptide, and a Colicin K polynucleotide encoding a Colicin K polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin K polypeptide. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p004k can comprise a Colicin M polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 2 (i.e., Colicin M), or a functional fragment or variant thereof comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide comprising a nucleotide sequence encoding an amino acid sequence set forth as SEQ ID NO: 1 (i.e., Colicin K), or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 85% sequence identity with p004k and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 85% sequence identity with p004k can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. TheAttorney Docket No. L 121360 1330WO (00140)

[0295] engineered bacteriophage comprising at least 85% sequence identity with p004k can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 90% (i.e., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more than 99%) sequence identity with p004k and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 90% sequence identity with p004k can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 90% sequence identity with p004k can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 95% (i.e., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more than 99%) sequence identity with p004k and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 95% sequence identity with p004k (e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%) can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ IDAttorney Docket No. L 121360 1330WO (00140)

[0296] NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 95% sequence identity with p004k can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In such instances, the engineered bacteriophage having at least 95% sequence identity with p004k can comprise a Colicin M polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage having at least 95% sequence identity with p004k comprises a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, or a variant thereof comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 15 or 16, or a variant thereof comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 15 or 16. In such instances, the engineered bacteriophage having at least 95% sequence identity with p004k can comprise a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 15. In certain embodiments, a bacteriophage with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty. In another instance, the engineered bacteriophage having at least 95% sequence identity with p004k can comprise a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 16. In certain embodiments, a bacteriophage with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty. The engineered bacteriophage comprising at least 97% sequence identity with p004k (e.g., at least 97%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, at least 98%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%) can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an aminoAttorney Docket No. L 121360 1330WO (00140)

[0297] acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 99% sequence identity with p004k can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In such instances, the engineered bacteriophage having at least 99% sequence identity with p004k can comprise a Colicin M polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage having at least 99% sequence identity with p004k comprises a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, or a variant thereof comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 15 or 16, or a variant thereof comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 15 or 16. In such instances, the engineered bacteriophage having at least 99% sequence identity with p004k can comprise a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 15. In certain embodiments, a bacteriophage with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty. In another instance, the engineered bacteriophage having at least 99% sequence identity with p004k can comprise a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 16. In certain embodiments, a bacteriophage with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0298] The engineered bacteriophage comprising about 98% sequence identity with p004k can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequenceAttorney Docket No. L 121360 1330WO (00140)

[0299] having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising about 98% sequence identity with p004k can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In such instances, the engineered bacteriophage having about 98% sequence identity with p004k can comprise a Colicin M polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage having about 98% sequence identity with p004k comprises a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, or a variant thereof comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 15 or 16, or a variant thereof comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 15 or 16. In such instances, the engineered bacteriophage having about 98% sequence identity with p004k can comprise a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 15. In certain embodiments, a bacteriophage with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty. In another instance, the engineered bacteriophage having about 98% sequence identity with p004k can comprise a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 16. In certain embodiments, a bacteriophage with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0300] An engineered bacteriophage can comprise a genome sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to pOOcO (i.e., the engineered bacteriophage comprises a polynucleotide sequence, e.g., a full-length genomic sequence, having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, orAttorney Docket No. L 121360 1330WO (00140)

[0301] greater than 99% sequence identity to pOOcO). In such instances, the engineered bacteriophage can comprise a genome sequence comprising at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to a pOOcO. In such instances, the engineered bacteriophage retains the ability to target Escherichia spp. The engineered bacteriophage retains lytic activity. The engineered bacteriophage can retain replicative ability. In some instances, the bacteriophage is an engineered pOOcO. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOcO is capable of producing Colicin K polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise at least one Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOcO is capable of producing Colicin M polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise at least one Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOcO is capable of producing Colicin K polypeptide or a functional fragment or variant thereof, and Colicin M polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof. The engineered bacteriophage can have at least one colicin-encoding polynucleotide comprising a nucleotide sequence encoding at least one colicin or functional fragment or variant thereof. In one instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOcO comprises a Colicin M polynucleotide encoding a Colicin M polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin M polypeptide. In another instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOcO comprises a Colicin K polynucleotide encoding a Colicin K polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin K polypeptide. In some instances, the engineered bacteriophage having at leastAttorney Docket No. L 121360 1330WO (00140)

[0302] 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOcO comprises more than one colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding more than one colicin. In one instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOcO comprises a first colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding a first colicin or functional fragment or variant thereof, and a second colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding a second colicin or functional fragment or variant thereof. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOcO can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin M polypeptide, and a Colicin K polynucleotide encoding a Colicin K polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin K polypeptide. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOcO can comprise a Colicin M polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 2 (i.e., Colicin M), or a functional fragment or variant thereof comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide comprising a nucleotide sequence encoding an amino acid sequence set forth as SEQ ID NO: 1 (i.e., Colicin K), or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 85% sequence identity with pOOcO and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 85% sequence identity with pOOcO can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a ColicinAttorney Docket No. L 121360 1330WO (00140)

[0303] K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 85% sequence identity with pOOcO can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 90% (i.e., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more than 99%) sequence identity with pOOcO and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 90% sequence identity with pOOcO can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 90% sequence identity with pOOcO can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 95% (i.e., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more than 99%) sequence identity with pOOcO and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 95% sequence identity with pOOcO can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a ColicinAttorney Docket No. L 121360 1330WO (00140)

[0304] K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 95% sequence identity with pOOcO can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOcO can comprise a Colicin M polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOcO comprises a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, or a variant thereof comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 15 or 16, or a variant thereof comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 15 or 16. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOcO can comprise a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 15. In certain embodiments, a bacteriophage with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty. In another instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOcO can comprise a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 16. In certain embodiments, a bacteriophage with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0305] An engineered bacteriophage can comprise a genome sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to pOOex (i.e., the engineered bacteriophage comprises a polynucleotide sequence, e.g., a full-length genomicAttorney Docket No. L 121360 1330WO (00140)

[0306] sequence, having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to pOOex). In such instances, the engineered bacteriophage can comprise a genome sequence comprising at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to a pOOex. In some instances, the engineered bacteriophage can comprise a genome sequence comprising at least 95%, at least 95.1%, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, at least 96%, at least 96.1%, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, or at least 97% sequence identity to pOOex. In such instances, the engineered bacteriophage retains the ability to target Escherichia spp. The engineered bacteriophage retains lytic activity. The engineered bacteriophage can retain replicative ability. In some instances, the bacteriophage is an engineered pOOex. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOex is capable of producing Colicin K polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise at least one Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOex is capable of producing Colicin M polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise at least one Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOex is capable of producing Colicin K polypeptide or a functional fragment or variant thereof, and Colicin M polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof. The engineered bacteriophage can have at least one colicin-encoding polynucleotide comprising a nucleotide sequence encoding at least one colicin or functional fragment or variant thereof. In one instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOex comprises a Colicin M polynucleotide encoding a Colicin M polypeptide, or aAttorney Docket No. L 121360 1330WO (00140)

[0307] functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin M polypeptide. In another instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOex comprises a Colicin K polynucleotide encoding a Colicin K polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin K polypeptide. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOex comprises more than one colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding more than one colicin. In one instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOex comprises a first colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding a first colicin or functional fragment or variant thereof, and a second colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding a second colicin or functional fragment or variant thereof. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOex can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin M polypeptide, and a Colicin K polynucleotide encoding a Colicin K polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin K polypeptide. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOex can comprise a Colicin M polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 2 (i.e., Colicin M), or a functional fragment or variant thereof comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide comprising a nucleotide sequence encoding an amino acid sequence set forth as SEQ ID NO: 1 (i.e., Colicin K), or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 85% sequence identity with pOOex and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragmentAttorney Docket No. L 121360 1330WO (00140)

[0308] or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 85% sequence identity with pOOex can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 85% sequence identity with pOOex can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 90% (i.e., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more than 99%) sequence identity with pOOex and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 90% sequence identity with pOOex can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 90% sequence identity with pOOex can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 95% (i.e., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more than 99%) sequence identity with pOOex and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide orAttorney Docket No. L 121360 1330WO (00140)

[0309] a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 95% sequence identity with pOOex can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 95% sequence identity with pOOex can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOex can comprise a Colicin M polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOex comprises a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, or a variant thereof comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 15 or 16, or a variant thereof comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 15 or 16. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOex can comprise a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 15. In certain embodiments, a bacteriophage with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty. In another instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOex can comprise a Colicin K polynucleotide comprising a nucleotide sequence set forth asAttorney Docket No. L 121360 1330WO (00140)

[0310] SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 16. In certain embodiments, a bacteriophage with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0311] An engineered bacteriophage can comprise a genome sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to pOOjc (i.e., the engineered bacteriophage comprises a polynucleotide sequence, e.g., a full-length genomic sequence, having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to pOOjc). In such instances, the engineered bacteriophage can comprise a genome sequence comprising at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to a pOOjc. In such instances, the engineered bacteriophage retains the ability to target Escherichia spp. The engineered bacteriophage retains lytic activity. The engineered bacteriophage can retain replicative ability. In some instances, the bacteriophage is an engineered pOOjc. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOjc is capable of producing Colicin K polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise at least one Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOjc is capable of producing Colicin M polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise at least one Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOjc is capable of producing Colicin K polypeptide or a functional fragment or variant thereof, and Colicin M polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof. The engineered bacteriophage can have at least one colicin-encodingAttorney Docket No. L 121360 1330WO (00140)

[0312] polynucleotide comprising a nucleotide sequence encoding at least one colicin or functional fragment or variant thereof. In one instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOjc comprises a Colicin M polynucleotide encoding a Colicin M polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin M polypeptide. In another instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOjc comprises a Colicin K polynucleotide encoding a Colicin K polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin K polypeptide. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOjc comprises more than one colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding more than one colicin. In one instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOjc comprises a first colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding a first colicin or functional fragment or variant thereof, and a second colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding a second colicin or functional fragment or variant thereof. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOjc can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin M polypeptide, and a Colicin K polynucleotide encoding a Colicin K polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin K polypeptide. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOjc can comprise a Colicin M polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 2 (i.e., Colicin M), or a functional fragment or variant thereof comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide comprising a nucleotide sequence encoding an amino acid sequence set forth as SEQ ID NO: 1 (i.e., Colicin K), or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,Attorney Docket No. L 121360 1330WO (00140)

[0313] 97%, 98%, or 99% sequence identity to an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 85% sequence identity with pOOjc and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 85% sequence identity with pOOjc can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 85% sequence identity with pOOjc can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 90% (i.e., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more than 99%) sequence identity with pOOjc and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 90% sequence identity with pOOjc can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 90% sequence identity with pOOjc can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth asAttorney Docket No. L 121360 1330WO (00140)

[0314] SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 95% (i.e., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more than 99%) sequence identity with pOOjc and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 95% sequence identity with pOOjc can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 95% sequence identity with pOOjc can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOjc can comprise a Colicin M polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOjc comprises a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, or a variant thereof comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 15 or 16, or a variant thereof comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 15 or 16. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOjc can comprise a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 15. In certain embodiments, a bacteriophage with these characteristics is depositedAttorney Docket No. L 121360 1330WO (00140)

[0315] under Accession No. X under the terms of the Budapest Treaty. In another instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOjc can comprise a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 16. In certain embodiments, a bacteriophage with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0316] An engineered bacteriophage can comprise a genome sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to pOOke (i.e., the engineered bacteriophage comprises a polynucleotide sequence, e.g., a full-length genomic sequence, having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to pOOke). In such instances, the engineered bacteriophage can comprise a genome sequence comprising at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to a pOOke. In such instances, the engineered bacteriophage retains the ability to target Escherichia spp. The engineered bacteriophage retains lytic activity. The engineered bacteriophage can retain replicative ability. In some instances, the bacteriophage is an engineered pOOke. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOke is capable of producing Colicin K polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise at least one Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOke is capable of producing Colicin M polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise at least one Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOke is capable of producing Colicin K polypeptide or a functional fragment or variant thereof, and Colicin M polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage canAttorney Docket No. L 121360 1330WO (00140)

[0317] comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof. The engineered bacteriophage can have at least one colicin-encoding polynucleotide comprising a nucleotide sequence encoding at least one colicin or functional fragment or variant thereof. In one instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOke comprises a Colicin M polynucleotide encoding a Colicin M polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin M polypeptide. In another instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOke comprises a Colicin K polynucleotide encoding a Colicin K polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin K polypeptide. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOke comprises more than one colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding more than one colicin. In one instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOke comprises a first colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding a first colicin or functional fragment or variant thereof, and a second colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding a second colicin or functional fragment or variant thereof. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOke can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin M polypeptide, and a Colicin K polynucleotide encoding a Colicin K polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin K polypeptide. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOke can comprise a Colicin M polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 2 (i.e., Colicin M), or a functional fragment or variant thereof comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence set forth as SEQ ID NO: 2, and aAttorney Docket No. L 121360 1330WO (00140)

[0318] Colicin K polynucleotide comprising a nucleotide sequence encoding an amino acid sequence set forth as SEQ ID NO: 1 (i.e., Colicin K), or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 85% sequence identity with pOOke and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 85% sequence identity with pOOke can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 85% sequence identity with pOOke can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 90% (i.e., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more than 99%) sequence identity with pOOke and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 90% sequence identity with pOOke can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 90%Attorney Docket No. L 121360 1330WO (00140)

[0319] sequence identity with pOOke can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 95% (i.e., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more than 99%) sequence identity with pOOke and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 95% sequence identity with pOOke can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 95% sequence identity with pOOke can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOke can comprise a Colicin M polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOke comprises a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, or a variant thereof comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 15 or 16, or a variant thereof comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 15 or 16. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identityAttorney Docket No. L 121360 1330WO (00140)

[0320] with pOOke can comprise a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 15. In certain embodiments, a bacteriophage with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty. In another instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with pOOke can comprise a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 16. In certain embodiments, a bacteriophage with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0321] An engineered bacteriophage can comprise a genome sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to p5516 (i.e., the engineered bacteriophage comprises a polynucleotide sequence, e.g., a full-length genomic sequence, having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to p5516). In such instances, the engineered bacteriophage can comprise a genome sequence comprising at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to a p5516. In such instances, the engineered bacteriophage retains the ability to target Escherichia spp. The engineered bacteriophage retains lytic activity. The engineered bacteriophage can retain replicative ability. In some instances, the bacteriophage is an engineered p5516. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p5516 is capable of producing Colicin K polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise at least one Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p5516 is capable of producing Colicin M polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise at least one Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof. In some instances, the engineered bacteriophage having at least 80%, atAttorney Docket No. L 121360 1330WO (00140)

[0322] least 85%, at least 90%, or at least 95% sequence identity with p5516 is capable of producing Colicin K polypeptide or a functional fragment or variant thereof, and Colicin M polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof. The engineered bacteriophage can have at least one colicin-encoding polynucleotide comprising a nucleotide sequence encoding at least one colicin or functional fragment or variant thereof. In one instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p5516 comprises a Colicin M polynucleotide encoding a Colicin M polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin M polypeptide. In another instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p5516 comprises a Colicin K polynucleotide encoding a Colicin K polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin K polypeptide. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p5516 comprises more than one colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding more than one colicin. In one instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p5516 comprises a first colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding a first colicin or functional fragment or variant thereof, and a second colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding a second colicin or functional fragment or variant thereof. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p5516 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin M polypeptide, and a Colicin K polynucleotide encoding a Colicin K polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin K polypeptide. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p5516 can comprise a Colicin M polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 2 (i.e., ColicinAttorney Docket No. L 121360 1330WO (00140)

[0323] M), or a functional fragment or variant thereof comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide comprising a nucleotide sequence encoding an amino acid sequence set forth as SEQ ID NO: 1 (i.e., Colicin K), or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 85% sequence identity with p5516 and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 85% sequence identity with p5516 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 85% sequence identity with p5516 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 90% (i.e., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more than 99%) sequence identity with p5516 and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 90% sequence identity with p5516 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a ColicinAttorney Docket No. L 121360 1330WO (00140)

[0324] K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 90% sequence identity with p5516 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 95% (i.e., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more than 99%) sequence identity with p5516 and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 95% sequence identity with p5516 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 95% sequence identity with p5516 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p5516 can comprise a Colicin M polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p5516 comprises a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, or a variant thereof comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 15 or 16, or a variant thereof comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,Attorney Docket No. L 121360 1330WO (00140)

[0325] 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 15 or 16. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p5516 can comprise a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 15. In certain embodiments, a bacteriophage with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty. In another instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p5516 can comprise a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 16. In certain embodiments, a bacteriophage with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0326] An engineered bacteriophage can comprise a genome sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to p6921 (i.e., the engineered bacteriophage comprises a polynucleotide sequence, e.g., a full-length genomic sequence, having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to p6921). In such instances, the engineered bacteriophage can comprise a genome sequence comprising at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to a p6921. In such instances, the engineered bacteriophage retains the ability to target Escherichia spp. The engineered bacteriophage retains lytic activity. The engineered bacteriophage can retain replicative ability. In some instances, the bacteriophage is an engineered p6921. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6921 is capable of producing Colicin K polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise at least one Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6921 is capable of producing Colicin M polypeptide or aAttorney Docket No. L 121360 1330WO (00140)

[0327] functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise at least one Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6921 is capable of producing Colicin K polypeptide or a functional fragment or variant thereof, and Colicin M polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof. The engineered bacteriophage can have at least one colicin-encoding polynucleotide comprising a nucleotide sequence encoding at least one colicin or functional fragment or variant thereof. In one instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6921 comprises a Colicin M polynucleotide encoding a Colicin M polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin M polypeptide. In another instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6921 comprises a Colicin K polynucleotide encoding a Colicin K polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin K polypeptide. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6921 comprises more than one colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding more than one colicin. In one instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6921 comprises a first colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding a first colicin or functional fragment or variant thereof, and a second colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding a second colicin or functional fragment or variant thereof. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6921 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin M polypeptide, and a Colicin K polynucleotide encoding a Colicin K polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceAttorney Docket No. L 121360 1330WO (00140)

[0328] identity to a Colicin K polypeptide. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6921 can comprise a Colicin M polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 2 (i.e., Colicin M), or a functional fragment or variant thereof comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide comprising a nucleotide sequence encoding an amino acid sequence set forth as SEQ ID NO: 1 (i.e., Colicin K), or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 85% sequence identity with p6921 and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 85% sequence identity with p6921 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 85% sequence identity with p6921 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 90% (i.e., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more than 99%) sequence identity with p6921 and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 90%Attorney Docket No. L 121360 1330WO (00140)

[0329] sequence identity with p6921 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 90% sequence identity with p6921 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 95% (i.e., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more than 99%) sequence identity with p6921 and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 95% sequence identity with p6921 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 95% sequence identity with p6921 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6921 can comprise a Colicin M polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6921 comprises a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, or a variant thereof comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,Attorney Docket No. L 121360 1330WO (00140)

[0330] 98%, or 99% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 15 or 16, or a variant thereof comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 15 or 16. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6921 can comprise a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 15. In certain embodiments, a bacteriophage with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty. In another instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6921 can comprise a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 16. In certain embodiments, a bacteriophage with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0331] An engineered bacteriophage can comprise a genome sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to p6977 (i.e., the engineered bacteriophage comprises a polynucleotide sequence, e.g., a full-length genomic sequence, having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to p6977). In such instances, the engineered bacteriophage can comprise a genome sequence comprising at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to a p6977. In such instances, the engineered bacteriophage retains the ability to target Escherichia spp. The engineered bacteriophage retains lytic activity. The engineered bacteriophage can retain replicative ability. In some instances, the bacteriophage is an engineered p6977. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6977 is capable of producing Colicin K polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise at least one Colicin KAttorney Docket No. L 121360 1330WO (00140)

[0332] polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6977 is capable of producing Colicin M polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise at least one Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6977 is capable of producing Colicin K polypeptide or a functional fragment or variant thereof, and Colicin M polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof. The engineered bacteriophage can have at least one colicin-encoding polynucleotide comprising a nucleotide sequence encoding at least one colicin or functional fragment or variant thereof. In one instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6977 comprises a Colicin M polynucleotide encoding a Colicin M polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin M polypeptide. In another instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6977 comprises a Colicin K polynucleotide encoding a Colicin K polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin K polypeptide. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6977 comprises more than one colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding more than one colicin. In one instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6977 comprises a first colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding a first colicin or functional fragment or variant thereof, and a second colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding a second colicin or functional fragment or variant thereof. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6977 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceAttorney Docket No. L 121360 1330WO (00140)

[0333] identity to a Colicin M polypeptide, and a Colicin K polynucleotide encoding a Colicin K polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin K polypeptide. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6977 can comprise a Colicin M polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 2 (i.e., Colicin M), or a functional fragment or variant thereof comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide comprising a nucleotide sequence encoding an amino acid sequence set forth as SEQ ID NO: 1 (i.e., Colicin K), or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 85% sequence identity with p6977 and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 85% sequence identity with p6977 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 85% sequence identity with p6977 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 90% (i.e., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more than 99%) sequence identity with p6977 and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a ColicinAttorney Docket No. L 121360 1330WO (00140)

[0334] K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 90% sequence identity with p6977 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 90% sequence identity with p6977 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage comprises at least 95% (i.e., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more than 99%) sequence identity with p6977 and comprises a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 95% sequence identity with p6977 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1. The engineered bacteriophage comprising at least 95% sequence identity with p6977 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding a Colicin K polypeptide comprising an amino acid sequence set forth as SEQ ID NO: 1. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6977 can comprise a Colicin M polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 2, and a Colicin K polynucleotide encoding an amino acid sequence set forth as SEQ ID NO: 1. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequenceAttorney Docket No. L 121360 1330WO (00140)

[0335] identity with p6977 comprises a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, or a variant thereof comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 15 or 16, or a variant thereof comprising a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence set forth as SEQ ID NO: 15 or 16. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6977 can comprise a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 15. In certain embodiments, a bacteriophage with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty. In another instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6977 can comprise a Colicin K polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 14, and a Colicin M polynucleotide comprising a nucleotide sequence set forth as SEQ ID NO: 16. In certain embodiments, a bacteriophage with these characteristics is deposited under Accession No. X under the terms of the Budapest Treaty.

[0336] An engineered bacteriophage can comprise a genome sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to p6984 (i.e., the engineered bacteriophage comprises a polynucleotide sequence, e.g., a full-length genomic sequence, having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99% sequence identity to p6984). In such instances, the engineered bacteriophage can comprise a genome sequence comprising at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to a p6984. In such instances, the engineered bacteriophage retains the ability to target Escherichia spp. The engineered bacteriophage retains lytic activity. The engineered bacteriophage can retain replicative ability. In some instances, the bacteriophage is an engineered p6984. In some instances, the engineeredAttorney Docket No. L 121360 1330WO (00140)

[0337] bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6984 is capable of producing Colicin K polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise at least one Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6984 is capable of producing Colicin M polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise at least one Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6984 is capable of producing Colicin K polypeptide or a functional fragment or variant thereof, and Colicin M polypeptide or a functional fragment or variant thereof. In such instances, the engineered bacteriophage can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof, and a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof. The engineered bacteriophage can have at least one colicin-encoding polynucleotide comprising a nucleotide sequence encoding at least one colicin or functional fragment or variant thereof. In one instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6984 comprises a Colicin M polynucleotide encoding a Colicin M polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin M polypeptide. In another instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6984 comprises a Colicin K polynucleotide encoding a Colicin K polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin K polypeptide. In some instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6984 comprises more than one colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding more than one colicin. In one instance, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with p6984 comprises a first colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding a first colicin or functional fragment or variant thereof, and a second colicin-encoding polynucleotide sequence comprising a nucleotide sequence encoding a second colicin or functional fragment or variant thereof. In such instances, the engineered bacteriophage having at least 80%, at least 85%, at least 90%, or at leastAttorney Docket No. L 121360 1330WO (00140)

[0338] 95% sequence identity with p6984 can comprise a Colicin M polynucleotide encoding a Colicin M polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a Colicin M polypeptide, and a Colicin K polynucleotide encoding a Colicin K polypeptide, or a functional fragment or variant thereof having at least 80%, 81%, 82%, 8...

Claims

Attorney Docket No. L121360 1330WO (00140)What is claimed is:

1. A composition comprising a first engineered bacteriophage and at least a second engineered bacteriophage, wherein the first bacteriophage comprises:a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; andb) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1,wherein the first bacteriophage and the second bacteriophage are E. co / z-targeting bacteriophages.

2. The composition of claim 1, wherein the first engineered bacteriophage comprises:a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 2; andb) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 1.

3. The composition of claim 1 or 2, wherein the second engineered bacteriophage comprises:a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; andb) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1.

4. The composition of any one of claims 1-3, wherein the second engineered bacteriophage comprises:a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 2; andAttorney Docket No. L121360 1330WO (00140)b) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 1.

5. The composition of any one of claims 1-4, wherein the composition further comprises a third engineered bacteriophage comprising:a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; andb) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1.

6. The composition of any one of claims 1-4, wherein the composition further comprises a third engineered bacteriophage comprising:a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 2; andb) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 1.

7. The composition of claim 5 or 6, wherein the composition further comprises a fourth engineered bacteriophage comprising:a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; andb) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1.

8. The composition of claim 5 or 6, wherein the composition further comprises a fourth engineered bacteriophage comprising:a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragmentAttorney Docket No. L121360 1330WO (00140)or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 2; andb) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 1.

9. The composition of claim 7 or 8, wherein the composition further comprises a fifth engineered bacteriophage comprising:a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; andb) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1.

10. The composition of claim 7 or 8, wherein the composition further comprises a fifth engineered bacteriophage comprising:a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 2; andb) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 1.

11. The composition of claim 9 or 10, wherein the composition further comprises a sixth engineered bacteriophage comprising:a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; andb) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1.

12. The composition of claim 9 or 10, wherein the composition further comprises a sixth engineered bacteriophage comprising:Attorney Docket No. L121360 1330WO (00140)a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 2; and / orb) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 1.

13. The composition of any one of claims 1-12, wherein at least one of the engineered bacteriophage is a Tequatrovirus.

14. The composition of any of claims 1-13, wherein at least two of the engineered bacteriophage are Tequatrovirus.

15. The composition of any one of claims 1-14, wherein the engineered bacteriophage are from at least two different genera selected from Tequatrovirus, Phapecoctavirus, Justusliebigvirus, Vequintavirus, and Krischvirus.

16. The composition of any one of claims 5-15, wherein the bacteriophage are from at least three different genera selected from Tequatrovirus, Phapecoctavirus, Justusliebigvirus, Vequintavirus, and Krischvirus.

17. The composition of any one of claims 1-16, wherein the Colicin M polynucleotide is inserted into a structural gene site in at least one engineered bacteriophage.

18. The composition of any one of claims 1-17, wherein the Colicin K polynucleotide is inserted into a structural gene site in at least one engineered bacteriophage.

19. The composition of any one of claims 1-18, wherein at least one engineered bacteriophage is an obligately lytic bacteriophage.

20. The composition of any one of claims 5-19, wherein between two and six of the engineered bacteriophage targets E. coli.Attorney Docket No. L121360 1330WO (00140)21. The composition of any one of claims 1-20, wherein the E. coli is an antibiotic resistant E. coli.

22. The composition of any one of claims 1-21, wherein the first bacteriophage has a different host range than the second bacteriophage.

23. The composition of any one of claims 1-22, wherein the first bacteriophage has at least 80% sequence identity to a bacteriophage selected from p00exe327, p004kel36, p00cge016, p00mfe005, p007621e011, or p007817e005.

24. The composition of any one of claims 1-23, wherein the second bacteriophage has at least 80% sequence identity to a bacteriophage selected from p004kel36, p00cge016, p00mfe005, p007621e011, p007817e005, or p00exe327, wherein the first and at least second bacteriophage are different.

25. The composition of any one of claims 1-24, wherein the third bacteriophage has at least 80% sequence identity to a bacteriophage selected from p00cge016, p00mfe005, p007621e011, p007817e005, p00exe327, or p004kel36, wherein the first, second, and at least third bacteriophage are different.

26. The composition of any one of claims 1-25, wherein the fourth bacteriophage has at least 80% sequence identity to a bacteriophage selected from p00mfe005, p007621e011, p007817e005, p00exe327, p004kel36, or p00cge016, wherein the first, second, third, and at least fourth bacteriophage are different.

27. The composition of any one of claims 1-26, wherein the fifth bacteriophage has at least 80% sequence identity to a bacteriophage selected from p007621e011, p007817e005, p004kel36, p00cge016, p00exe327, or p00mfe005, wherein the first, second, third, fourth, and at least fifth bacteriophage are different.

28. The composition of any one of claims 1-27, wherein the sixth bacteriophage has at leastAttorney Docket No. L121360 1330WO (00140)80% sequence identity to a bacteriophage selected from p007817e005, p004kel36, p00cge016, p00exe327, p00mfe005, or p007621e011, wherein the first, second, third, fourth, fifth, and at least sixth bacteriophage are different.

29. The composition of any one of claims 15-28, wherein:a) the Tequatrovirus has at least 90% sequence identity to pOOex (ATCC Accession No. PTA-127145), p004k (ATCC Accession No. PTA-127149), p6921 (ATCC Accession No. PTA-127576), p6799 (ATCC Accession No. PTA- 127577), or p69894 (ATCC Accession No. PTA-127578);b) the Phapecoctavirus has at least 90% sequence identity to pOOcg;c) the Justusliebigvirus has at least 90% sequence identity to pOOmf;d) the Vequintavirus has at least 90% sequence identity to p007621; ande) the Krischvirus has at least 90% sequence identity to p007817; andwherein each engineered bacteriophage comprises:a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; andb) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1.

30. The composition of any one of claims 1-29, wherein none of the engineered bacteriophage comprise a nucleic acid molecule encoding a CRISPR system, optionally a Type I or Type II CRISPR system, a CRISPR Cas3, a CRISPR-Cas9, a Pseudomonas aeruginosa Type 1C CRISPR.

31. A composition comprising six engineered bacteriophage, each engineered bacteriophage comprising:a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; andAttorney Docket No. L121360 1330WO (00140)b) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1,wherein:i) a first engineered bacteriophage is p00exe327;ii) a second engineered bacteriophage is p004kel36;iii) a third engineered bacteriophage is p00cge016;iv) a fourth engineered bacteriophage is p00mfe005;v) a fifth engineered bacteriophage is p007621e001; andvi) a sixth engineered bacteriophage is p007817e005.

32. The composition of claim 31, wherein each engineered bacteriophage comprises:a) a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 2; andb) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence set forth as SEQ ID NO: 1.

33. The composition of any one of claims 1-32, wherein the composition comprises at least 106plaque forming units (PFU) of each engineered bacteriophage.

34. The composition of claim 33, wherein the composition comprises between 106and 1016plaque forming units (PFU) of each engineered bacteriophage.

35. A pharmaceutical composition comprising the composition of any one of claims 1-34, and a pharmaceutically acceptable excipient.

36. The pharmaceutical composition of claim 35, wherein the pharmaceutical composition is formulated for oral, intravenous, or enema administration.

37. The pharmaceutical composition of claim 35 or 36, wherein the pharmaceutical composition is formulated for oral administrationAttorney Docket No. L121360 1330WO (00140)38. A method of reducing the number of E. coli within a population of bacteria comprising a plurality of E. coli, the method comprising contacting the population with the composition of any one of claims 1-34, or the pharmaceutical composition of any one of claims 35-37.

39. A method of administering a composition comprising at least one engineered bacteriophage capable of producing at least one colicin to a subject in need thereof, the method comprising administering to the subject an effective amount of the composition of any one of claims 1-34, or the pharmaceutical composition of any one of claims 35-37.

40. The method of claim 39, wherein the effective amount:a) reduces the number of E. coli within a population of bacteria comprising a plurality of E. coli within the gastrointestinal tract of the subject;b) reduces the number of AIEC in the gastrointestinal tract of the subject;c) reduces calprotectin levels in the subject;d) reduces the level of at least inflammatory cytokine in the gastrointestinal tract of the subject;e) reduces at least one symptom associated with gastrointestinal disease in the subject; f) reduces the severity of at least one gastrointestinal disease in the subject;g) increases the relapse time in the subject;h) improves the histopathology score of at least one tissue in the subject;i) promotes at least one positive clinical response in the subject; and / orj) promotes a positive endoscopic response in the subject.

41. The method of any one of claims 38-40, wherein the E. coli is a pathogenic E. coli.

42. The method of any one of claim 41, wherein the pathogenic E. coli is an adherent-invasive E. coli (AIEC).

43. The method of claim 41 or 42, wherein the gastrointestinal tract of the subject comprises the pathogenic E. coli.Attorney Docket No. L121360 1330WO (00140)44. The method of any one of claims 39-43, wherein the subject has been diagnosed with a gastrointestinal disease or condition selected from inflammatory bowel disease (IBD), Crohn’s disease (CD), ulcerative colitis (UC), indeterminate colitis, and gastroenteritis.

45. The method of claim 44, wherein the gastroenteritis is: enteroaggregative (EAEC), enterohemorrhagic (EHEC), enteroinvasive (EIEC), enteropathogenic (EPEC), enterotoxigenic (ETEC), or diffuse adherent (DAEC).

46. The method of any one of claims 39-45, wherein the composition is administered systemically.

47. The method of any one of claims 39-45, wherein the composition is administered locally.

48. The method of any one of claims 39-47, wherein the composition or pharmaceutical composition is administered orally, intravenously, or by enema.

49. The method of any one of claims 39-48, wherein the composition or pharmaceutical composition is administered orally.

50. An engineered bacteriophage comprising a Colicin M polynucleotide encoding a Colicin M polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 2; and b) a Colicin K polynucleotide encoding a Colicin K polypeptide or a functional fragment or variant thereof comprising an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth as SEQ ID NO: 1.

51. The engineered bacteriophage of claim 50, wherein the Colicin M polypeptide or functional fragment or variant thereof comprises an amino acid sequence set forth as SEQ ID NO: 2, and the Colicin K polypeptide or functional fragment or variant thereof comprises an amino acid sequence set forth as SEQ ID NO: 1.Attorney Docket No. L121360 1330WO (00140)52. The engineered bacteriophage of claim 50 or 51, wherein the engineered bacteriophage is a recombinant Tequatrovirus.

53. The engineered bacteriophage of any one of claims 50-52, wherein the engineered bacteriophage comprises a genome nucleotide sequence having at least 90% sequence identity to the genomic nucleotide sequence of p00exe327.

54. The engineered bacteriophage of any one of claims 50-53, wherein the engineered bacteriophage is p00exe327.

55. The engineered bacteriophage of any one of claims 50-52, wherein the engineered bacteriophage comprises a genome nucleotide sequence having at least 90% sequence identity to the genomic nucleotide sequence of p004kel36.

56. The engineered bacteriophage of any one of claims 50-52 or 55, wherein the engineered bacteriophage is p004kel36.

57. The engineered bacteriophage of claim 50 or 51, wherein the engineered bacteriophage is a recombinant Phapecoctavirus.

58. The engineered bacteriophage of any one of claims 50, 51, or 57, wherein the engineered bacteriophage comprises a genome nucleotide sequence having at least 90% sequence identity to the genomic nucleotide sequence of p00cge016.

59. The engineered bacteriophage of any one of claims 50, 51, 57, or 58, wherein the engineered bacteriophage is p00cge016.

60. The engineered bacteriophage of claim 50 or 51, wherein the engineered bacteriophage is a recombinant Justusliebigvirus.Attorney Docket No. L121360 1330WO (00140)61. The engineered bacteriophage of any one of claims 50, 51, or 60, wherein the engineered bacteriophage comprises a genome nucleotide sequence having at least 90% sequence identity to the genomic nucleotide sequence of p00mfe005.

62. The engineered bacteriophage of any one of claims 50, 51, 60, or 61, wherein the engineered bacteriophage is p00mfe005.

63. The engineered bacteriophage of claim 50 or 51, wherein the engineered bacteriophage is a recombinant Vequintavirus.

64. The engineered bacteriophage of any one of claims 50, 51, or 63, wherein the engineered bacteriophage comprises a genome nucleotide sequence having at least 90% sequence identity to the genomic nucleotide sequence of p007621e011.

65. The engineered bacteriophage of any one of claims 50, 51, 63, or 64, wherein the engineered bacteriophage is p007621e011.

66. The engineered bacteriophage of claim 50 or 51, wherein the engineered bacteriophage is a recombinant Krischvirus.

67. The engineered bacteriophage of any one of claims 50, 51, or 66, wherein the engineered bacteriophage comprises a genome nucleotide sequence having at least 90% sequence identity to the genomic nucleotide sequence of p007817e005.

68. The engineered bacteriophage of any one of claims 50, 51, 66, or 67, wherein the engineered bacteriophage is p007817e005.

69. The composition of any one of claims 1-34, the pharmaceutical composition of any one of claims 35-37, or the engineered bacteriophage of any one of claims 50-68 for use in the manufacture of a medicament for the treatment of a disease.Attorney Docket No. L121360 1330WO (00140)70. The composition of any one of claims 1-34, the pharmaceutical composition of any one of claims 35-37, or the engineered bacteriophage of any one of claims 50-68 for use in treating a disease.

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