Targeting Escherichia coli cells

Abstract

The technology described herein relates to methods and compositions for targeting Escherichia coli cells, e.g., for treating or preventing infections caused by Escherichia coli cells in a human or animal subject. In one embodiment, the method comprises administering to the subject a particle mixture comprising a plurality of different transduction particles for Escherichia coli cells. In one embodiment, the method comprises administering to the subject a plurality of transduction particles encoding a nuclease for targeting the genome of B2 phylogenetic population Escherichia coli cells.

Description

Technical Field

[0001] The technology described herein relates to methods and compositions for targeting E. coli cells, such as methods and compositions for treating or preventing infections caused by E. coli cells in human or animal subjects. In one embodiment, the method comprises administering to the subject a mixture of particles comprising a plurality of different transduction particles for E. coli cells. In one embodiment, the method comprises administering to the subject a plurality of transduction particles encoding a nuclease for targeting the genome of E. coli cells of the B2 phylogroup. Background Art

[0002] E. coli infections have been identified as harmful or life-threatening in a variety of settings, such as UTI infections, transplant patients, cancer patients, and other patients who are immunocompromised or taking immunosuppressants.

[0003] Nuclease targeting of Escherichia coli, for example, by means of CRISPR / Cas system, is proposed to be delivered using transduction particles, which can target nuclease to Escherichia coli cells for chromosome type or free type cutting, thereby killing cells or reducing their growth or proliferation. Suitable transduction particles are phages or engineered particles (such as non-self-replicating transduction particles) comprising capsids, which contain nucleic acids encoding at least crRNA or gRNA (or additional Cas nucleases) for targeting. Advantageously, selective targeting can be achieved, which is impossible using conventional antibiotics such as broad-spectrum antibiotics. Such selective targeting can avoid killing beneficial species and strains in patients treated. In fact, disrupting the microbiome with broad-spectrum antibiotics is a risk factor in the preventive management of cancer patients at risk of febrile neutropenia.

[0004] Bacteriophage (phage) therapy was used before the widespread availability of antibiotics but is now experiencing renewed interest due to rising bacterial antimicrobial resistance (AMR) combined with several individual case reports of success.

[0005] Bacteriophages (phages) are a phylum of viruses that infect bacteria and are different from animal and plant viruses. Bacteriophages can have a "lytic" life cycle, a "lysogenic" life cycle that may potentially become lytic, or a "non-lytic" life cycle. Phages that replicate through a lytic cycle cause the lysis of host bacterial cells as a normal part of their life cycle. Phages that replicate through a lysogenic cycle are referred to as temperate phages and can replicate and cause the lysis of host bacteria using a lytic life cycle, or they can incorporate their DNA into host bacterial DNA and become non-infectious prophages.

[0006] The natural ability of phages to infect and kill bacteria, together with the specificity of phage-bacteria interactions, are fundamental phenomena upon which the concept of phage therapy is based. Therefore, phages possessing a lytic life cycle are suitable candidates for phage therapy.

[0007] International Patent Application No. WO 00 / 69269 discloses the use of a certain bacteriophage strain for treating infections caused by vancomycin-sensitive and -resistant strains of Enterococcus faecium, and International Patent Application No. WO 01 / 93904 discloses the use of bacteriophages, alone or in combination with other antimicrobial measures, for preventing or treating gastrointestinal diseases associated with Clostridium species.

[0008] US Patent Application No. 2002 / 0001590 discloses the use of phage therapy against multidrug-resistant bacteria, specifically methicillin-resistant Staphylococcus aureus, and International Patent Application No. WO 02 / 07742 discloses the development of bacteriophages with multiple host ranges.

[0009] The use of phage therapy for the treatment of specific bacterial infectious diseases is disclosed in, for example, US Patent Application Nos. 2002 / 0044922; 2002 / 0058027 and International Patent Application No. WO 01 / 93904.

[0010] US20160333348 describes the use of a CRISPR / Cas system using bacteriophage as a vector for delivery to host bacterial cells.

[0011] In several phylogenetic groups of Escherichia coli, it has been observed that antibiotic resistance (e.g., fluoroquinolone (FQ) resistance) and multidrug-resistant (MDR) strains are frequently found in the B2 phylogenetic group. B2 strains ST131 and ST1193 were found to be associated with antibiotic resistance. ST131 is a globally dominant multidrug-resistant clone associated with a high rate of rUTI. ST131 is a major contributor to hospital-acquired UTIs and community-acquired UTIs, as well as E. coli bloodstream infections and infections in companion animals and poultry. ST131, initially identified in 2008, is associated with the worldwide spread of the CTX-M-15 extended-spectrum β-lactamase (ESBL) resistance gene. ST131 is now strongly associated with multidrug resistance (MDR), including resistance to fluoroquinolones. Recent reports have also identified strains resistant to the last-line carbapenems. Sequence type 1193 has recently emerged as a new, virulent, and resistant lineage in fluoroquinolone-resistant E. coli.

[0012] Therefore, classical antibiotics such as FQ and broad-spectrum antibiotics are not effective enough to combat such infections.Therefore, there is a need to find alternative means to address these infections. SUMMARY OF THE INVENTION

[0014] By combining the use of selective killing with the nuclease of using the transduction particle targeting of specific type, the present invention provides the means for treating or preventing the B2 phylogeny group Escherichia coli infection of humans and animals. Granule of the present invention targets by adhering to LPS, LamB or Tsx, and surprisingly finds that this height is conducive to killing and suppressing the growth of the B2 Escherichia coli cell of many different strains (including potentially lethal ST131 and ST1193 bacterial strains). As illustrated herein, surprisingly killed more than 10 kinds of different ST131 bacterial strains (plaque formation), and killed more than 10 kinds of different ST1193 bacterial strains (plaque formation).

[0015] The present invention finds utility, for example, in treating or preventing potentially life-threatening B2 phylogenetic group E. coli infections in patients, such as immunosuppressed, cancer, transplant, and UTI patients, who are susceptible to infections caused by B2 strains (and often by multiple different B2 strains). As demonstrated in the Examples, the present invention can be used to prevent E. coli B2 phylogenetic group bacteremia (bloodstream infection caused by E. coli) in a subject.

[0016] To this end, the present invention provides:-

[0017] In the first configuration

[0018] In the first aspect:-

[0019] A composition comprising a plurality of transduction particles for use in a method of treating or preventing an infection caused by Escherichia coli cells in a human or animal subject, wherein the method comprises administering the particles to the subject, wherein

[0020] (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of an E. coli cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, wherein the nuclease is expressed in the cell and cleaves the genomic DNA of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject;

[0021] (b) the E. coli cell is a cell of E. coli phylogenetic group B2; and

[0022] (c) Each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0023] In the second aspect:-

[0024] A composition comprising a plurality of transduction particles for use in a method of treating or preventing Escherichia coli bacteremia in a human or animal subject, wherein the method comprises administering the particles to the subject, wherein

[0025] (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of a cell of the Escherichia coli, wherein the administered particle contacts the cell and introduces the nucleic acid therein, wherein the nuclease is expressed in the cell and cleaves the genomic DNA of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject;

[0026] (b) the E. coli cell is a cell of E. coli phylogenetic group B2; and

[0027] (c) Each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0028] In the second configuration

[0029] In the first aspect:-

[0030] A method for treating or preventing an infection caused by E. coli cells in a human or animal subject, the method comprising administering to the subject a plurality of transducing particles, wherein

[0031] (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of an E. coli cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, wherein the nuclease is expressed in the cell and cleaves the genomic DNA of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject;

[0032] (b) the E. coli cell is a cell of E. coli phylogenetic group B2; and

[0033] (c) Each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0034] In the second aspect:-

[0035] A method for treating or preventing an infection caused by E. coli cells in a human or animal subject, the method comprising administering to the subject a plurality of transducing particles, wherein

[0036] (a) each particle comprises a nucleic acid encoding a crRNA or a guide RNA operable with a Cas nuclease for chromosome targeting in a cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, wherein the crRNA or guide RNA is expressed and guides the Cas nuclease, wherein the nuclease cleaves the chromosome of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject;

[0037] (b) the E. coli cell is a cell of E. coli phylogenetic group B2; and

[0038] (c) Each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0039] In the third aspect:-

[0040] A method for treating or preventing Escherichia coli bacteremia in a human or animal subject, the method comprising administering to the subject a plurality of transducing particles, wherein

[0041] (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of a cell of the Escherichia coli, wherein the administered particle contacts the cell and introduces the nucleic acid therein, wherein the nuclease is expressed in the cell and cleaves the genomic DNA of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject;

[0042] (b) the E. coli cell is a cell of E. coli phylogenetic group B2; and

[0043] (c) Each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0044] In the fourth aspect:-

[0045] A method for treating or preventing Escherichia coli bacteremia in a human or animal subject, the method comprising administering to the subject a plurality of transducing particles, wherein

[0046] (a) each particle comprises a nucleic acid encoding a crRNA or a guide RNA operable with a Cas nuclease for chromosomal targeting in cells of the E. coli, wherein the administered particle contacts the cells and introduces the nucleic acid therein, wherein the crRNA or guide RNA is expressed and directs the Cas nuclease, wherein the nuclease cleaves the chromosome of the cells, thereby killing the cells or reducing the growth or proliferation of the cells in the subject;

[0047] (b) the E. coli cell is a cell of E. coli phylogenetic group B2; and

[0048] (c) Each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0049] In the third configuration

[0050] In the first aspect:-

[0051] Use of a composition comprising a plurality of transduction particles in a method for treating or preventing an infection in a human or animal subject caused by phylogenetic group B2 Escherichia coli cells, the method comprising administering the composition to the subject, wherein

[0052] (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of an E. coli cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, wherein the nuclease is expressed in the cell and cleaves the genomic DNA of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject; and

[0053] (b) Each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0054] In the second aspect:-

[0055] Use of a composition comprising a plurality of transduction particles in a method for treating or preventing an infection in a human or animal subject caused by phylogenetic group B2 Escherichia coli cells, the method comprising administering the composition to the subject, wherein

[0056] (a) each particle comprises a nucleic acid encoding a crRNA or a guide RNA operable with a Cas nuclease for chromosome targeting in a cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, wherein the crRNA or guide RNA is expressed and guides the Cas nuclease, wherein the nuclease cleaves the chromosome of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject;

[0057] (b) the E. coli cell is a cell of E. coli phylogenetic group B2; and

[0058] (c) Each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0059] In the third aspect:-

[0060] Use of a composition comprising a plurality of transducing particles in a method for treating or preventing phylogenetic group B2 Escherichia coli bacteremia in a human or animal subject, the method comprising administering the composition to the subject, wherein

[0061] (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of a cell of the E. coli, wherein the administered particle contacts the cell and introduces the nucleic acid therein, wherein the nuclease is expressed in the cell and cleaves the genomic DNA of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject; and

[0062] (b) Each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0063] In the fourth aspect:-

[0064] Use of a composition comprising a plurality of transducing particles in a method for treating or preventing phylogenetic group B2 Escherichia coli bacteremia in a human or animal subject, the method comprising administering the composition to the subject, wherein

[0065] (a) each particle comprises a nucleic acid encoding a crRNA or a guide RNA operable with a Cas nuclease for chromosomal targeting in cells of the E. coli, wherein the administered particle contacts the cells and introduces the nucleic acid therein, wherein the crRNA or guide RNA is expressed and directs the Cas nuclease, wherein the nuclease cleaves the chromosome of the cells, thereby killing the cells or reducing the growth or proliferation of the cells in the subject;

[0066] (b) the E. coli cell is a cell of E. coli phylogenetic group B2; and

[0067] (c) Each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0068] In the fourth configuration

[0069] In the first aspect:-

[0070] A composition comprising a plurality of transduction particles for use in a method of treating or preventing an infection caused by Escherichia coli cells (optionally Escherichia coli cells of the B2 phylogenetic group) in a human or animal subject, wherein the method comprises administering the particles to the subject, wherein

[0071] (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of an E. coli cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, wherein the nuclease is expressed in the cell and cleaves the genomic DNA of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject;

[0072] (b) Each particle comprised by the composition is a T-even phage capsid; optionally, a capsid of T2 phage, T2-like phage, RB69 phage or RB69-like phage.

[0073] A composition comprising a plurality of different types of transduction particles, wherein each of the particles comprises a nucleic acid and wherein the particles are capable of contacting an E. coli cell and introducing the nucleic acid therein, wherein

[0074] (a) the nucleic acid of each particle comprises a nucleotide sequence encoding a product of interest (POI), wherein the nucleic acid is capable of expressing the POI in an Escherichia coli cell;

[0075] (b) each particle comprises an adhesion moiety for recognizing and binding a cognate moiety selected from the group consisting of LPS, LamB, and Tsx (optionally selected from the group consisting of LPS and Tsx) displayed on the surface of an E. coli cell; and

[0076] (c) the plurality of different types of transduction particles comprises (i) a first type of particle comprising an LPS adhesion moiety; and (ii) a second type of particle comprising a Tsx adhesion moiety.

[0077] In the second aspect:-

[0078] A method of treating or preventing sepsis, septicemia, or diarrhea in a human or animal subject, the method comprising administering to the subject a composition of the invention, wherein the E. coli cells comprise an E. coli strain that causes sepsis, septicemia, or diarrhea in humans or animals.

[0079] In the third aspect:-

[0080] A method of treating or preventing an infection caused by E. coli cells in a human or animal subject, wherein the method comprises administering a composition of the invention to the subject, wherein the infection is treated or prevented.

[0081] In the fourth aspect:-

[0082] A method for detecting the presence of Escherichia coli (optionally Escherichia coli cells of the B2 phylogenetic group) in a sample, the method comprising

[0083] (a) contacting a sample with a composition according to this configuration; and

[0084] (b) Detecting whether E. coli cells are killed or their growth or proliferation is reduced.

[0085] In the fifth aspect:-

[0086] A method for detecting the presence of Escherichia coli (optionally Escherichia coli cells of the B2 phylogenetic group) in a sample, the method comprising

[0087] (a) contacting a sample with a composition according to this configuration; wherein the particles of the composition comprise a nucleic acid comprising or encoding a detectable label, wherein the particles contact a cell and introduce the nucleic acid therein, wherein optionally the label is expressed in the cell; and

[0088] (b) Detection of E. coli cells containing the marker.

[0089] In the sixth aspect:-

[0090] A method for modifying the genome of an E. coli cell, the method comprising contacting the cell with the configured composition, wherein a nucleic acid encoding a POI is introduced into the cell, thereby modifying the genome of the cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 The killing and growth inhibition activity of the compositions of the present invention when tested against E. coli from several different phylogenetic groups and different strains within each group (E. coli taken from clinical samples); the compositions were surprisingly highly effective in killing across many phylogenetic groups (and particularly in the B2 phylogenetic group, where multiple strains—including those known to be potentially life-threatening and associated with antibiotic resistance—were killed or their growth effectively inhibited). Six negative samples included non-E. coli and non-FQ-resistant samples.

[0092] Figure 2 : Phylogenetic division of strains used in the study into groups (Phylo-grouping), and showed particularly favorable and broad targeting of many clinical B2 phylogenetic group strains.

[0093] Figure 3: A) CRISPR-Cas driven elimination of a simplified panel of 82 E. coli clinical isolates by conjugation of CGV-EcCAS and empty vector. Conjugation efficiency was determined by spotting a dilution series of the conjugation reaction onto LB agar supplemented with antibiotics and calculated as CFU / ml. The limit of detection (LOD) for this assay was 200 CFU / mL. The experiments were performed in triplicate and are indicated by dots. CGV-EcCAS conjugation experiments indicated by arrows; empty vector conjugation indicated above the dotted line marking the LOD. B) CRISPR-armed phages during co-cultivation with a host strain susceptible to both phagesTM (CRISPR-armed phage TM (CAP) and wild-type phage fractions. CAPα15.2 increased its relative abundance compared to wild-type phage from 7% to 86% over two consecutive passages. C) CAPα20.4 outcompeted wild-type α20 by increasing its relative abundance from 10% to 68% over four consecutive passages during co-cultivation with the common target E. coli strain b230. BC) Ratios of CAP and wild-type phage during co-cultivation with a host strain (E. coli 230) susceptible to α15.2, α15, α20.4, and α20. CAPα15.2 increased its relative abundance compared to wild-type phage from 7% to 86% (G), while CAPα20.4 outcompeted wild-type α20 from 10% to 68% (H).

[0094] Figure 4 Specificity evaluation of SNIPR001 and individual CAPs against a panel of clinically relevant bacteria and E. coli strain b2480 (positive control) showed no off-target effects. Positive values ​​indicate bacterial growth (no observed killing) during the evaluated time period, while negative values ​​indicate bacterial killing after phage treatment. All values ​​are expressed as the mean and standard deviation of four biological replicates, measuring growth over a 4-hour period, measured in CFU / mL.

[0095] Figure 5: A) Unrooted phylogenetic tree of JMI strains showing a clinical panel of 382 E. coli strains covering 9 phylogenetic groups and 118 MLSTs. The plaque data reflect single plaque replicates. One strain, E. coli b4038, with long branches was truncated to 37% of its original length. The phylogenetic distance scale is indicated below the phylogenetic tree, as calculated by MASH. B) The spot assay was used to analyze the efficacy of SNIPR001 against a clinical panel of 382 E. coli strains isolated from bloodstream infections (from JMI, North Liberty, IA, USA) and an internal panel of 429 E. coli strains. The spot assay was performed as two independent experiments, with results presented as mean ± standard deviation values. The standard deviation shown is based on the difference in results between the two runs, calculated as the mean absolute deviation of the mean prevalence of a given spot type normalized for panel size. C) SNIPR001 coverage is independent of antibiotic resistance phenotype; thus, SNIPR001 targets >90% of carbapenem-resistant, extended-spectrum β-lactamase (ESBL)-producing, or multidrug-resistant (MDR) E. coli strains and 89% of fluoroquinolone-resistant E. coli strains. The numbers indicated in each green or gray bar indicate the number of bacteria susceptible or resistant to SNIPR001, respectively, for each resistance class. D) Midpoint-rooted phylogenetic tree of 72 fluoroquinolone-resistant E. coli strains isolated from fecal samples of patients with hematological cancers. 67 of the 72 strains were susceptible to at least one of the four CAPs in SNIPR001. E) Redundant distribution shows that 82% of the fluoroquinolone-resistant E. coli strains from panel D (n=72) are targeted by at least two different CAPs.

[0096] Figure 6: A) At 2x 10 12 CAP recovery in minipig excreta over one week following a single oral dose of 10 PFU of SNIPR001 (n=8, shown in green) or vehicle (n=6, shown in gray) with daily sampling. The trend line indicates the average recovered phage in PFU / g excreta, and the dots indicate individual measurements. The LOD of 33 PFU / g excreta is indicated by the dashed line. B) At 2 x 10 12Figure 2. CAP recovery in minipig excreta over one week following a single oral dose of 100 PFU of a single CAP (n=8 minipigs receiving α15.2, α20.4, or α51.5, and n=7 minipigs receiving α48.4) with daily sampling. The trend line indicates the average recovery, while the dots indicate individual measurements. Recovery is measured in PFU / g excreta. The LOD of 33 PFU / g excreta is indicated by the dashed line. C) CAP recovery in mouse excreta 8, 24, and 48 hours after initiation of three daily administrations of different doses of SNIPR001 (n=10 for low, medium, and high, indicated in green), vehicle (n=10, indicated in gray), or gentamicin (n=4, indicated in gray). Recovery is measured in PFU / g excreta, with the LOD of 371 PFU / g excreta indicated by the dashed line. D) E. coli b17 recovery in mouse feces indicates an increasing SNIPR001 effect with increasing doses. Statistical analysis was performed using a two-sided Kruskal-Wallis test for comparisons of SNIPR001-treated groups and a two-sided Mann-Whitney U test for comparisons of treated groups versus vehicle. P < 0.05 = *, 0.01 = **, 0.001 = ***, with FDR correction using Holm's method for each day. Recovery was measured in CFU / g feces, with a limit of detection of 371 CFU / g feces. Animals that began SNIPR001 treatment are indicated in green, while other animals are indicated in gray. E) E. coli b17 recovery in mouse feces 8 and 24 hours after initiation of three-daily administration of CAPs α15.2, α20.4, α48.4, or α51.5, in combination with SNIPR001, confirms the synergistic effect of CAPs. Summary of the Invention

[0097] The present invention finds application in combating harmful or life-threatening B2 E. coli infections in a variety of settings, such as UTI infections, transplant patients, cancer patients, and other patients who are immunocompromised or taking immunosuppressants.

[0098] Cancer treatment continues to advance, and survival rates for patients with hematologic malignancies are gradually increasing. However, this population is immunocompromised, and chemotherapy regimens cause myelosuppression and gastrointestinal mucositis with associated increased intestinal permeability. Translocation of intestinal bacteria, including Escherichia coli, from the gastrointestinal tract is a frequent cause of bloodstream infections (BSIs). The mortality rate associated with BSIs can be as high as 50%; therefore, antimicrobial prophylaxis is used in patients at risk for febrile neutropenia. There are no approved therapies for the prevention of BSIs in patients with hematologic cancers, but fluoroquinolones are used off-label in the United States. This antibiotic prophylaxis practice contradicts the emerging paradigm that maintaining a normal microbiome is important for maintaining immune tone, potentially benefiting the outcome of oncology treatment. Indeed, perturbing the microbiome with broad-spectrum antibiotics is a risk factor in the prophylactic management of patients at risk for febrile neutropenia. In addition to the side effects of fluoroquinolones, which include safety warnings and precautions, bacterial resistance is on the rise and approaches 60% in the United States.

[0099] In immunocompromised patients with hematological malignancies at risk of developing neutropenia, E. coli is responsible for 25.1-30% of all bacteremia cases, with a 90-day mortality rate of 35.8%. In addition, up to 65% of E. coli isolated as the causative pathogen in BSIs in patients with hematological cancers undergoing hematopoietic stem cell transplantation (HSCT) are resistant to fluoroquinolones. Therefore, new narrow-spectrum treatment and prevention options are needed to prevent infection in these susceptible patients. The present invention addresses this need.

[0100] To this end, the present invention provides compositions, methods and uses according to the above configurations. Accordingly, the following description is provided with numbered embodiments.

[0101] 1. A composition comprising a plurality of transduction particles for use in a method of treating or preventing an infection caused by Escherichia coli cells in a human or animal subject, wherein the method comprises administering the particles to the subject, wherein

[0102] (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of an E. coli cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, wherein the nuclease is expressed in the cell and cleaves the genomic DNA of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject;

[0103] (b) the E. coli cell is a cell of E. coli phylogenetic group B2; and

[0104] (c) Each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0105] Optionally,

[0106] (i) Each particle comprises a phage capsid containing a nucleic acid;

[0107] (ii) particles of the composition target a plurality of different E. coli genes, optionally selected from essential genes and virulence genes; and

[0108] (iii) the composition comprises

[0109] A: Particles containing LPS-adhesive moieties and LamB-adhesive moieties;

[0110] B: particles comprising an LPS-adhesive moiety and a Tsx-adhesive moiety; or

[0111] C: Particles containing the Tsx-adherent moiety but lacking the LamB- and LPS-adherent moieties.

[0112] 2. A method for treating or preventing an infection caused by E. coli cells in a human or animal subject, the method comprising administering to the subject a plurality of transduction particles, wherein the method comprises administering the particles to the subject, wherein

[0113] (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of an E. coli cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, wherein the nuclease is expressed in the cell and cleaves the genomic DNA of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject;

[0114] (b) the E. coli cell is a cell of E. coli phylogenetic group B2; and

[0115] (c) Each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0116] Optionally,

[0117] (i) Each particle comprises a phage capsid containing a nucleic acid;

[0118] (ii) particles of the composition target a plurality of different E. coli genes, optionally selected from essential genes and virulence genes; and

[0119] (iii) the composition comprises

[0120] A: Particles containing LPS-adhesive moieties and LamB-adhesive moieties;

[0121] B: particles comprising an LPS-adhesive moiety and a Tsx-adhesive moiety; or

[0122] C: Particles containing the Tsx-adherent moiety but lacking the LamB- and LPS-adherent moieties.

[0123] Optionally, the genomic DNA is the chromosomal DNA of the cell.Additionally or alternatively, the genomic DNA is the plasmid DNA of the cell.

[0124] Optionally, each particle comprises a nucleic acid encoding a nuclease for chromosome targeting, wherein the administered particle contacts a cell and introduces the nucleic acid therein, wherein the nuclease is expressed in the cell and cleaves the cell's chromosome, thereby killing the cell or reducing cell growth or proliferation in the subject.

[0125] The human can be male or female. The human can be an adult or a child. The human can be 18 years of age or older, such as 40, 50, 60, 70, 80 years of age or older. The human can be younger than 18 years of age, such as a teenager, such as an infant, such as up to 5 years of age, such as up to 2 years of age. The animal can be a livestock or companion animal such as a dog or cat. The animal can be a bird (e.g., a poultry, such as a chicken, turkey, or duck, preferably a chicken), a cow, a sheep, a goat, or a pig (e.g., a newborn pig or a pig under 6 months of age).

[0126] The infection can be a bloodstream infection. The infection can be a hospital-acquired infection.

[0127] In one example, each adhesion moiety is a tail fiber protein. In one example, each particle comprises a phage tail fiber, which comprises the adhesion moiety or is fused to the adhesion moiety. In one example, each adhesion moiety is an antibody fragment, such as an antibody single variable domain. In one example, each adhesion moiety is a nanobody. In one example, each adhesion moiety comprises an antibody binding site that can bind to a homologous portion. For example, each adhesion moiety comprises an antibody single variable domain (i.e., dAb), such as a nanobody. In one example, each particle comprises one or more phage tail fibers or spikes, and each fiber or spike comprises the adhesion moiety.

[0128] For example, at least 2, 3, or 4 different types of transducing particles are administered to a subject, and each type comprises one or more types of tail fibers comprising an adhesion moiety, wherein the other particle types do not comprise the one or more tail fiber types. In one example, each type of the plurality of tail fiber types differs from the other types in the type of adhesion moiety it comprises.

[0129] Optionally, the particle comprises an adhesion moiety for binding LPS, LamB, and Tsx. Optionally, the particle comprises an adhesion moiety for binding LamB and Tsx. Optionally, the particle comprises an adhesion moiety for binding LPS and Tsx. Optionally, the particle comprises an adhesion moiety for binding LPS and LamB.

[0130] In Gram-negative bacteria, the peptidoglycan layer is relatively thin and located on the inner side of the outer membrane, the main component of the cell wall. The two layers are connected by Braun lipoproteins. The outer membrane is a complex structure composed of a lipid bilayer decorated with proteins, polysaccharides and lipids; the latter two molecules form the LPS layer. LPS is a complex composed of three parts: lipid A, core polysaccharide and O-polysaccharide. Generally speaking, lipid A is composed of fatty acids attached to glucosamine phosphate disaccharides. The core polysaccharide is connected to lipid A by a keto-deoxyoctanoate linker. The core polysaccharide and O-polysaccharide (O-chain or O-antigen) contain several sugar residue units extending outward to the outer membrane. Cells containing all three components of LPS are named smooth (S) type, while those lacking the O-polysaccharide part are distinguished as rough (R) type.

[0131] Optionally, the LPS is a smooth LPS or a rough LPS.

[0132] For example, the particles comprise at least one type of particle, the adhesive moiety of which is capable of binding the O-antigen of LPS.

[0133] Escherichia coli is a highly versatile species, and its diversity has been explored from various perspectives, emphasizing, for example, phylogenetic grouping, pathotypes, and the wide range of O serotypes. The highly variable O-antigen, the outermost portion of the lipopolysaccharide component of the E. coli outer membrane, is linked to the innermost lipid A by the core region of the LPS. Five different structures of the LPS core region have been characterized to date, designated K-12, R1, R2, R3, and R4. Phylogenetic groups B2 and C strains are primarily dominated by the R1 type. Strains within phylogenetic group B2 may carry the K-12 core, such as those belonging to the complex STc131, one of the major clones of extraintestinal pathogenic E. coli (ExPEC) strains.

[0134] Preferably, the LPS comprises an R1 core region. In one embodiment, the LPS comprises an R2 core region. In one embodiment, the LPS comprises an R3 core region. In one embodiment, the LPS comprises an R4 core region. In one embodiment, the LPS comprises a K-12 core region.

[0135] Optionally, LamB comprises the amino acids of SEQ ID NO: 1, or an amino acid sequence at least 70, 80, 90, or 95% identical to SEQ ID NO: 1. Optionally, Tsx comprises the amino acids of SEQ ID NO: 2, or an amino acid sequence at least 70, 80, 90, or 95% identical to SEQ ID NO: 2. Optionally, LamB is encoded by the nucleotide sequence of SEQ ID NO: 3, or an amino acid sequence at least 70, 80, 90, or 95% identical to SEQ ID NO: 3. Optionally, Tsx is encoded by the nucleotide sequence of SEQ ID NO: 4, or an amino acid sequence at least 70, 80, 90, or 95% identical to SEQ ID NO: 4.

[0136] The Escherichia coli tsx gene encodes an integral outer membrane protein (Tsx) that functions as a substrate-specific channel for deoxynucleosides and the antibiotic albicidin. In one example, the nucleoside-specific channel-forming protein Tsx of Escherichia coli has Uniprot accession number P0A927 or a homolog thereof. In one example, the maltose outer membrane porin (maltoporin) LamB of Escherichia coli has Uniprot accession number P02943 or a homolog thereof.

[0137] Homologs: A gene, nucleotide or protein sequence that is related to a second gene, nucleotide or protein sequence by inheritance from a common ancestral DNA or protein sequence. The term homolog can apply to the relationship between genes separated by an event or between genes separated by a gene duplication event.

[0138] In one embodiment, the E. coli cell comprises a UPEC E. coli. In one embodiment, the E. coli cell comprises an enteropathogenic E. coli (ExPEC) cell.

[0139] 3. The composition or method of embodiment 1 or 2, respectively, wherein the method is used to treat or prevent an infection in a subject caused by an E. coli strain selected from ST131, ST1193, ST648, ST315, ST405, ST361, ST88 and ST453.

[0140] In a preferred embodiment, the strain is ST1193. In another preferred embodiment, the strain is ST131.

[0141] 4. The composition or method of any preceding embodiment, wherein the method is for treating or preventing an infection in a subject caused by a plurality of different phylogenetic group B2 strains of Escherichia coli; optionally wherein the plurality comprises Escherichia coli ST131 and ST1193 cells.

[0142] In some embodiments, the present invention relates to a method for treating or preventing the infection of an Escherichia coli ST131 or ST1193 strain. ... E. coli strains B2-ST73 (CH24-30); B2-ST73 (CH24-103); B2-ST131 (CH40-30); B2-ST141 (CH52-5); B2-ST372 (CH103-9); B2-ST404 (CH14-27); B2-ST404 (CH14-807) and B2-ST1193 (CH14-64) were found in the UTI context. In one embodiment (e.g., wherein the subject has or is at risk of a UTI), the B2 E. coli comprises one or more strains selected from the group consisting of: B2-ST73 (CH24-30); B2-ST73 (CH24-103); B2-ST131 (CH40-30); B2-ST141 (CH52-5); B2-ST372 (CH103-9); B2-ST404 (CH14-27); B2-ST404 (CH14-807) and B2-ST1193 (CH14-64).

[0143] 5. The composition or method of any preceding embodiment, wherein the subject is a transplant or cancer patient (optionally a hematological cancer patient), or wherein the patient has or is at risk of a urinary tract infection (UTI); and optionally wherein the transplant is a solid organ or stem cell transplant (optionally a hematopoietic cell transplant), or wherein the transplant is a medical device transplant.

[0144] For example, the subject is a hematological cancer patient suffering from neutropenia. For example, the subject is a hematopoietic stem cell transplant patient.

[0145] A suitable medical device may be, for example, a cardiac device (eg, a ventricular assist device, such as a left ventricular assist device (LVAD)), a catheter (eg, a biliary catheter), or a prosthesis (eg, a joint).

[0146] In one example, the patient has a cryptic B2 phylogenetic group E. coli infection. In one example, the E. coli is a cryptic B2 phylogenetic group E. coli.

[0147] For example, the subject has or is at risk of acute bacterial sinusitis, pneumonia, urinary tract infection, chronic prostatitis, or gastroenteritis caused by B2 phylogenetic group E. coli. For example, the subject is a male human prostate surgery patient.

[0148] 6. The composition or method of any preceding embodiment, wherein the method is performed before the subject receives a transplant.

[0149] For example the transplant is a solid organ or stem cell transplant (optionally a hematopoietic cell transplant).

[0150] 7. The composition or method of any preceding embodiment, wherein the B2 E. coli cells comprise an E. coli strain that causes sepsis, septicemia, or diarrhea in humans.

[0151] Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a human pathogen responsible for outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) worldwide. Conventional antimicrobial drugs trigger the SOS response in EHEC, which promotes the release of potent Shiga toxins, which are responsible for much of the morbidity and mortality associated with EHEC infection. Cattle are the natural reservoir of EHEC, and approximately 75% of EHEC outbreaks are associated with the consumption of contaminated cattle-derived products. EHEC causes disease in humans but is asymptomatic in adult ruminants. Characteristics of E. coli serotype O157:H7 (EHEC) infection include abdominal cramps and bloody diarrhea, as well as the life-threatening complication hemolytic uremic syndrome (HUS). There is a need for treatments for EHEC infection (Goldwater and Bettelheim, 2012). The use of conventional antibiotics exacerbates Shiga toxin-mediated cytotoxicity. In an epidemiological study conducted by the Centers for Disease Control and Prevention, patients treated with antibiotics for EHEC enteritis had a higher risk of developing HUS (Slutsker et al., 1998). Additional studies support the contraindication of antibiotics in EHEC infection; children treated with antibiotic therapy for hemorrhagic colitis associated with EHEC had an increased chance of developing HUS (Wong et al., 2000; Zimmerhackl, 2000; Safdar et al., 2002; Tarr et al., 2005). Conventional antibiotics promote the production of Shiga toxins by enhancing the replication and expression of the stx gene, which is encoded within the genome of the chromosomally integrated lambdoid prophage. The methods of the present invention can rely on nuclease cleavage of the target cell genomic DNA. Stx induction also promotes phage-mediated lysis of the EHEC cell envelope, allowing the release and spread of Shiga toxins into the environment (Karch et al., 1999; Matsushiro et al., 1999; Wagner et al., 2002). Thus, advantageously, the present invention provides alternative means for treating B2 phylogenetic group EHEC in human and animal subjects. In one example, the subject (e.g., a human) suffers from or is at risk of hemolytic uremic syndrome (HUS), e.g., the subject suffers from an E. coli infection, e.g., an EHEC E. coli infection.

[0152] 8. The composition or method of any preceding embodiment, for preventing hemolytic uremic syndrome (HUS), UTI infection, sepsis, septicemia, or diarrhea in a subject.

[0153] The compositions or methods can be used to treat or prevent a bloodstream infection caused by pathogenic B2 phylogenetic group E. coli cells in a subject.

[0154] 9. The composition or method of any preceding embodiment, wherein each particle comprises a phage capsid comprising the nucleic acid; optionally wherein the capsid comprises a capsid protein of T-even (optionally T4) or lambda phage.

[0155] As is known to the skilled person, transducing particles are operable to infect their cognate host cells by transduction in order to introduce a nucleic acid therein.

[0156] 10. The composition or method of any preceding embodiment, wherein each particle is a bacteriophage (optionally a lytic phage) or a packaged phagemid.

[0157] 11. The composition or method of any preceding embodiment, wherein at least 2, 3, or 4 different types of transducing particles are administered to the subject.

[0158] For example, each particle type comprises one type of adhesion moiety or collection of adhesion moiety types that is different from particles of other types.

[0159] In one example, two different types of transduction particles are administered to a subject. In one example, three different types of transduction particles are administered to a subject. In one example, four different types of transduction particles are administered to a subject. In one example, five different types of transduction particles are administered to a subject. In one example, six different types of transduction particles are administered to a subject.

[0160] 12. The composition or method of any preceding embodiment, wherein a first type of transduction particle and a second type of transduction particle are administered to a subject, wherein the first type of particle comprises a first adhesion moiety capable of recognizing and binding to a first homology moiety selected from the group LPS, LamB, and Tsx displayed on B2 E. coli, and the second type of particle comprises a second adhesion moiety capable of recognizing and binding to a second homology moiety selected from said group, wherein the first adhesion moiety and the second adhesion moiety are different from each other.

[0161] For example, the first and second adhesion moieties differ from each other by their tail fibers, optionally wherein the first adhesion moiety is homologous to LPS and the second moiety is homologous to LamB; or optionally wherein the first adhesion moiety is homologous to LPS and the second moiety is homologous to Tsx; optionally wherein the first adhesion moiety is homologous to Tsx and the second moiety is homologous to LamB.

[0162] For example, each particle comprises a phage capsid comprising the nucleic acid; wherein at least 2, 3 or 4 different types of transduction particles are administered to a subject; and wherein a first type of transduction particle and a second type of transduction particle are administered to a subject, wherein the first type of particle comprises a first adhesion moiety capable of recognizing and binding to a first homology portion selected from the group LPS, LamB and Tsx displayed on B2 E. coli, and the second type of particle comprises a second adhesion moiety capable of recognizing and binding to a second homology portion selected from said group, wherein the first adhesion moiety and the second adhesion moiety are different from each other.

[0163] For example, each particle comprises a phage capsid comprising the nucleic acid; wherein at least 2, 3, or 4 different types of transduction particles are administered to the subject; and wherein a first type of transduction particle and a second type of transduction particle are administered to the subject, wherein the first type of particle comprises a first adhesion moiety that can recognize and bind to LPS displayed on B2 E. coli, and the second type of particle comprises a second adhesion moiety that can recognize and bind to LamB displayed on B2 E. coli.

[0164] For example, each particle comprises a phage capsid comprising the nucleic acid; wherein at least 2, 3, or 4 different types of transduction particles are administered to the subject; and wherein a first type of transduction particle and a second type of transduction particle are administered to the subject, wherein the first type of particle comprises a first adhesion moiety that can recognize and bind to Tsx displayed on B2 E. coli, and the second type of particle comprises a second adhesion moiety that can recognize and bind to LamB displayed on B2 E. coli.

[0165] For example, each particle comprises a phage capsid comprising the nucleic acid; wherein at least 2, 3, or 4 different types of transduction particles are administered to the subject; and wherein a first type of transduction particle and a second type of transduction particle are administered to the subject, wherein the first type of particle comprises a first adhesion moiety that can recognize and bind to LPS displayed on B2 E. coli, and the second type of particle comprises a second adhesion moiety that can recognize and bind to Tsx displayed on B2 E. coli.

[0166] For example, each particle comprises a phage capsid comprising the nucleic acid; wherein at least three different types of transduction particles are administered to a subject; and wherein a first type of transduction particle, a second type of transduction particle, and a third type of transduction particle are administered to a subject, wherein the first type of particle comprises a first adhesion portion that can recognize and bind to LPS displayed on B2 E. coli, the second type of particle comprises a second adhesion portion that can recognize and bind to LamB displayed on B2 E. coli, and the third type of particle comprises a second adhesion portion that can recognize and bind to Tsx displayed on B2 E. coli.

[0167] For example, each particle comprises a phage capsid comprising the nucleic acid; wherein at least four different types of transduction particles are administered to a subject; and wherein a first type of transduction particle, a second type of transduction particle, a third type of transduction particle, and a fourth type of transduction particle are administered to a subject, wherein the first type of particle comprises a first adhesion portion that can recognize and bind to LPS displayed on B2 E. coli, the second type of particle comprises a second adhesion portion that can recognize and bind to LamB displayed on B2 E. coli, the third type of particle comprises a second adhesion portion that can recognize and bind to Tsx displayed on B2 E. coli, and the fourth type of particle comprises a second adhesion portion that can recognize and bind to LPS displayed on B2 E. coli, wherein the adhesion portions of the particles are different from each other.

[0168] For example, each particle comprises a phage capsid comprising the nucleic acid; wherein at least four different types of transduction particles are administered to a subject; and wherein a first type of transduction particle, a second type of transduction particle, a third type of transduction particle, and a fourth type of transduction particle are administered to a subject, wherein the first type of particle comprises a first adhesion portion that can recognize and bind to LPS displayed on B2 E. coli, the second type of particle comprises a second adhesion portion that can recognize and bind to LamB displayed on B2 E. coli, the third type of particle comprises a second adhesion portion that can recognize and bind to Tsx displayed on B2 E. coli, and the fourth type of particle comprises a second adhesion portion that can recognize and bind to LamB displayed on B2 E. coli, wherein the adhesion portions of the particles are different from each other.

[0169] For example, each particle comprises a phage capsid comprising the nucleic acid; wherein at least four different types of transduction particles are administered to a subject; and wherein a first type of transduction particle, a second type of transduction particle, a third type of transduction particle, and a fourth type of transduction particle are administered to a subject, wherein the first type of particle comprises a first adhesion portion that can recognize and bind to LPS displayed on B2 E. coli, the second type of particle comprises a second adhesion portion that can recognize and bind to LamB displayed on B2 E. coli, the third type of particle comprises a second adhesion portion that can recognize and bind to Tsx displayed on B2 E. coli, and the fourth type of particle comprises a second adhesion portion that can recognize and bind to Tsx displayed on B2 E. coli, wherein the adhesion portions of the particles are different from each other.

[0170] 13. The composition or method of embodiment 12, wherein the first homologous portion and the second homologous portion are different from each other.

[0171] In an alternative embodiment, the first and second homologous portions are identical. In an alternative embodiment, the first and second homologous portions are LPS. In an alternative embodiment, the first and second homologous portions are Tsx. In an alternative embodiment, the first and second homologous portions are LamB.

[0172] 14. The composition or method of any preceding embodiment, wherein the nucleic acid of each particle comprises a nucleotide sequence (N1) encoding the nuclease, wherein each particle is a synthetic T-even phage (optionally T4 phage) comprising an insertion of N1 within the genome of the phage, wherein the region is located between the pin (protease inhibitor) gene and the iPII (internal protein) gene.

[0173] Optionally, the phage is

[0174] (a) A synthetic T-even (e.g., T4) phage comprising a DNA deletion from a genomic region of the phage and / or a heterologous DNA insertion within a genomic region of the phage corresponding to the region between the following coordinates

[0175] (i)1887 and 8983;

[0176] (ii) 2625 and 8092;

[0177] (iii) 1904 and 8113;

[0178] (iv) 2668 and 7178;

[0179] (v) 7844 and 11117;

[0180] (vi) 8643 and 10313;

[0181] (vii) 9231 and 13383;

[0182] (viii) 9480 and 12224;

[0183] (ix) 8454 and 17479; or

[0184] (x)9067 and 16673;

[0185] The coordinates are referenced to the wild-type T4 phage genome (SEQ ID NO: 5);

[0186] 15. The composition or method of any preceding embodiment, wherein the nuclease is a guided nuclease, optionally a Cas, a meganuclease, a zinc finger nuclease, or a TALEN.

[0187] 16. The composition or method of any preceding embodiment, wherein the nuclease is a Type I, II, III, IV, V, or VI nuclease, optionally Cas9 or Cas3.

[0188] 17. The composition or method of any preceding embodiment, wherein at least 1 x 10 7 Particles of PFU are administered to a subject.

[0189] In one example, 1x 10 8 to 1x 10 13 PFU of particles are administered to a subject. In one example, 1x10 8 to 1x 10 12 PFU of particles are administered to a subject. In one example, 1 x 10 10 to 1x 10 12 Particles of PFU are administered to a subject.

[0190] 18. The composition or method of any preceding embodiment, wherein the particles are administered to the subject at an MOI (multiplicity of infection) of at least 0.01.

[0191] Optionally, the particles are administered to the subject at an MOI of no more than 1. Optionally, the particles are administered to the subject at an MOI of 0.001 to 1. Optionally, the particles are administered to the subject at an MOI of 0.01 to 1. Optionally, the particles are administered to the subject at an MOI of 0.1 to 1.

[0192] 19. The composition or method of any preceding embodiment, wherein the or at least one strain is an antibiotic resistant strain or an MDR strain; and / or wherein the or at least one strain is a B2-1 strain.

[0193] For example the strain or at least one strain is a strain selected from the group consisting of B2-I (STc131), B2-II, B2-IX and B2-VI.

[0194] For example, at least one of the MDR strains is resistant to a fluoroquinolone and the strain is a β-lactamase (ESBL)-producing Escherichia coli.

[0195] 20. The composition or method of embodiment 19, wherein the antibiotic is a fluoroquinolone (optionally levofloxacin), a carbapenem, or vancomycin; and / or wherein the E. coli is a β-lactamase (ESBL)-producing E. coli.

[0196] Optionally, the antibiotic is selected from ciprofloxacin (e.g. Cipro TM ), gemifloxacin (such as Factive TM ), levofloxacin (eg Levaquin TM ), moxifloxacin (eg Avelox TM ) and ofloxacin.

[0197] For example, E. coli produces CTX-M-15, which is the most abundant enzyme in ESBL-producing E. coli that causes human infections.

[0198] Preferably, the antibiotic is a fluoroquinolone (FQ). For example, FQ is levofloxacin. TMLevofloxacin, sold as well as other levofloxacin, is an antibiotic drug. It is used to treat a variety of bacterial infections, including acute bacterial sinusitis, pneumonia, urinary tract infections, chronic prostatitis, and some types of gastroenteritis. In patients with long-term chemotherapy-induced neutropenia, levofloxacin prophylaxis is recommended to prevent Gram-negative bloodstream infections (BSIs). However, increasing fluoroquinolone resistance may reduce the effectiveness of this approach (e.g., Clin Infect Dis. 2021 Oct 5; 73(7): 1257-1265. doi: 10.1093 / cid / ciab404, “Colonization With Fluoroquinolone-Resistant Enterobacterales Decreases the Effectiveness of Fluoroquinolone Prophylaxis in Hematopoietic Cell Transplant Recipients, Michael J Satlin et al.). This study found that nearly one-third of hematopoietic cell transplant (HCT) recipients with pre-transplant fluoroquinolone-resistant Enterobacteriales (FQRE) colonization developed Gram-negative bloodstream infections (BSIs) while receiving levofloxacin prophylaxis among the patients tested, and that the infections were typically caused by their colonizing strains. In contrast, levofloxacin prophylaxis was highly effective in patients not initially colonized with FQRE. The authors found that 23% of patients admitted for HCT were colonized with FQRE, and that Escherichia coli was the dominant species. Patients with hematologic malignancies receiving intensive chemotherapy, including those undergoing hematopoietic cell transplantation (HCT), Those who develop severe neutropenia and gastrointestinal mucositis frequently are at high risk for developing bloodstream infections (BSIs) from Gram-negative enteric bacteria (Enterobacteriales). Neutropenic patients often suffer severe consequences from BSIs caused by Enterobacteriales, with mortality rates as high as 15%–20%. Furthermore, many fluoroquinolone-resistant Enterobacteriales (FQRE) also carry extended-spectrum β-lactamases (ESBLs); therefore, breakthrough infections that occur despite fluoroquinolone prophylaxis may be resistant to first-line antimicrobial therapy for fever and neutropenia. Finally, adverse effects of fluoroquinolones are becoming increasingly apparent and include Clostridioides difficile infection, aortic dissection and rupture, dysglycemia, tendinopathy, QT prolongation, and altered mental status. Therefore, fluoroquinolones should be administered to patients only if they are likely to provide clinical benefit justifying these potential adverse effects.Therefore, although fluoroquinolones may reduce the risk of Gram-negative BSI in many patients, those colonized with FQRE may not benefit from fluoroquinolone prophylaxis.

[0199] The high incidence of FQRE colonization and the lack of risk factors suggest that FQRE are prevalent in the community. Indeed, a study of urine isolates from outpatients in the United States demonstrated that 12% of E. coli isolates from young women and 29% of E. coli isolates from older women were fluoroquinolone-resistant. A surveillance study of 1,831 urine E. coli isolates from 2017 found that a quarter were FQ-resistant. Furthermore, 13%–16% of men undergoing transrectal prostate biopsy were found to be colonized with fluoroquinolone-resistant E. coli. Nearly half of the fluoroquinolone-resistant E. coli isolates in the study were ST131, a common sequence type circulating worldwide and frequently associated with fluoroquinolone resistance and ESBL producers.

[0200] Bacteremia caused by extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E), such as E. coli, is associated with inadequate empirical therapy and substantial mortality in neutropenic patients (see, e.g., "Colonization With Levofloxacin-resistant Extended-spectrum β-Lactamase-producing Enterobacteriaceae and Risk of Bacteremia in Hematopoietic Stem Cell Transplant Recipients," Satlin MJ et al., Clin Infect Dis. 2018 Nov 13;67(11):1720-1728. doi:10.1093 / cid / ciy363). The study found that HSCT recipients colonized with levofloxacin-resistant ESBL-E before transplantation and receiving levofloxacin prophylaxis had a high incidence of bacteremia from their colonizing strains during neutropenia. In this single-center study of 312 HSCT recipients, 10% of patients were found to be colonized with ESBL-E before their transplantation. Nearly one-third of patients with pretransplant ESBL-E colonization developed subsequent ESBL-E bacteremia after their transplantation while neutropenic, compared with <1% of patients who were not initially colonized with ESBL-E. Furthermore, bloodstream and gastrointestinal ESBL-E had identical MLST and PFGE profiles in all cases, suggesting that these patients developed bacteremia from their colonizing isolates.

[0201] In one example, the compositions or methods of the invention are used to prevent translocation of E. coli of phylogenetic group B2 from the gastrointestinal tract of a subject to the bloodstream, thereby preventing or reducing bacteremia in the patient.

[0202] In one example, the compositions or methods of the invention are used to prevent translocation of E. coli of phylogenetic group B2 from the urinary tract to the bloodstream of a subject, thereby preventing or reducing bacteremia in the patient.

[0203] In one example, the E. coli is contained by the gastrointestinal tract of the subject. Optionally, in these examples, the composition is administered orally to the subject.

[0204] In another example, the E. coli is contained by the subject's urinary tract. For example, the infection is a kidney, bladder, or urinary tract infection. Optionally, in these examples, the composition is administered to the subject's urinary tract, for example, via a catheter.

[0205] 21. The composition or method of any preceding embodiment, wherein

[0206] (i) The nuclease is Cas,

[0207] (ii) each particle comprises a phage capsid containing a nucleic acid; and

[0208] (iii) wherein a first type of transduction particle and a second type of transduction particle are administered to a subject, wherein the first type of particle comprises a first adhesion moiety capable of recognizing and binding to a first homology moiety selected from the group LPS, LamB and Tsx displayed on B2 strain E. coli, and the second type of particle comprises a second adhesion moiety capable of recognizing and binding to a second homology moiety selected from said group, wherein the first adhesion moiety and the second adhesion moiety are different from each other.

[0209] 22. A method for treating or preventing an infection caused by E. coli cells in a human or animal subject, the method comprising administering to the subject a plurality of transducing particles, wherein

[0210] (a) each particle comprises a nucleic acid encoding a crRNA or a guide RNA operable with a Cas nuclease for chromosome targeting in a cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, wherein the crRNA or guide RNA is expressed and guides the Cas nuclease, wherein the nuclease cleaves the chromosome of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject;

[0211] (b) the E. coli cell is a cell of E. coli phylogenetic group B2; and

[0212] (c) Each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0213] The various nucleic acids preferably encode a plurality of different cRNAs comprising a spacer sequence that targets an E. coli chromosomal gene. For example, the various nucleic acids preferably encode a plurality of different cRNAs comprising a spacer sequence that targets 2, 3 or 4 E. coli chromosomal genes selected from fimH, bolA, rpoH, lptA and murA.

[0214] Optionally, each crRNA or guide RNA comprises a spacer that targets an E. coli gene selected from the group consisting of fimH, bolA, rpoH, lptA, and murA. Optionally, various nucleic acids encode multiple different cRNAs or guide RNAs, wherein the cRNAs or guide RNAs target at least 2, 3, or 4 (or all) of the E. coli genes selected from the group consisting of fimH, bolA, rpoH, lptA, and murA. Optionally, various nucleic acids encode multiple different cRNAs or guide RNAs, wherein the cRNAs or guide RNAs target fimH and bolA. Optionally, various nucleic acids encode multiple different cRNAs or guide RNAs, wherein the cRNAs or guide RNAs target rpoH and lptA. Optionally, various nucleic acids encode multiple different cRNAs or guide RNAs, wherein the cRNAs or guide RNAs target fimH and murA. Optionally, various crRNAs or guide RNAs comprise a spacer that is complementary to an E. coli gene selected from the group consisting of fimH, bolA, rpoH, lptA, and murA. Optionally, each crRNA or guide RNA comprises a spacer sequence that is at least 80, 90 or 95% identical to a nucleotide sequence selected from the group of SEQ ID NOs: 6-10.

[0215] Optionally, the various nucleic acids encode the first crRNA, the second crRNA, the third crRNA, the fourth crRNA, and the fifth cRNA, wherein the crRNAs are different from each other and the various crRNAs target B2 phylogenetic group E. coli genes. Optionally, the various nucleic acids encode the first crRNA, the second crRNA, the third crRNA, the fourth crRNA, and the fifth cRNA, wherein the cRNAs are different from each other and the various crRNAs are complementary to B2 phylogenetic group E. coli genes. Optionally, the various nucleic acids encode the first crRNA, the second crRNA, the third crRNA, the fourth crRNA, and the fifth cRNA, wherein the cRNAs comprise SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively. Optionally, each nucleic acid encodes a first crRNA, a second crRNA, a third crRNA, a fourth crRNA, and a fifth cRNA, wherein the cRNA comprises a nucleotide sequence at least 80% identical to SEQ ID NO: 6, a nucleotide sequence at least 80% identical to SEQ ID NO: 7, a nucleotide sequence at least 80% identical to SEQ ID NO: 8, a nucleotide sequence at least 80% identical to SEQ ID NO: 9, and a nucleotide sequence at least 80% identical to SEQ ID NO: 10, respectively. Optionally, each nucleic acid encodes a first crRNA, a second crRNA, a third crRNA, a fourth crRNA, and a fifth cRNA, wherein the cRNA comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 6, a nucleotide sequence at least 90% identical to SEQ ID NO: 7, a nucleotide sequence at least 90% identical to SEQ ID NO: 8, a nucleotide sequence at least 90% identical to SEQ ID NO: 9, and a nucleotide sequence at least 90% identical to SEQ ID NO: 10, respectively. Optionally, each nucleic acid encodes a first crRNA, a second crRNA, a third crRNA, a fourth crRNA, and a fifth cRNA, wherein the cRNA comprises a nucleotide sequence at least 95, 96, 97, 98, or 99% identical to SEQ ID NO: 6, a nucleotide sequence at least 95, 96, 97, 98, or 99% identical to SEQ ID NO: 7, a nucleotide sequence at least 95, 96, 97, 98, or 99% identical to SEQ ID NO: 8, a nucleotide sequence at least 95, 96, 97, 98, or 99% identical to SEQ ID NO: 9, and a nucleotide sequence at least 95, 96, 97, 98, or 99% identical to SEQ ID NO: 10, respectively.

[0216] 23. The method of embodiment 22, wherein the method is according to any one of embodiments 1-20, optionally with the exception that the nuclease is endogenous to the cell and is not encoded by the nucleic acid contained in the particle.

[0217] 24. A composition comprising a plurality of transduction particles for use in a method of treating or preventing an infection caused by an E. coli cell in a human or animal subject according to embodiment 22 or 23, wherein

[0218] (a) each particle comprises a nucleic acid encoding a crRNA or a guide RNA operable with a Cas nuclease for chromosome targeting in a cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, wherein the crRNA or guide RNA is expressed and guides the Cas nuclease, wherein the nuclease cleaves the chromosome of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject;

[0219] (b) the E. coli cell is a cell of E. coli phylogenetic group B2; and

[0220] (c) Each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0221] 25. A method for detecting the presence of B2 phylogenetic group Escherichia coli in a sample, the method comprising contacting a sample comprising B2 phylogenetic group Escherichia coli with a composition comprising a plurality of transduction particles, wherein

[0222] (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of the E. coli cells, wherein the administered particles contact the cells and introduce the nucleic acid therein, wherein the nuclease is expressed in the cells and cleaves the genomic DNA of the cells, thereby killing the cells or reducing the growth or proliferation of the cells in the subject;

[0223] (b) each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells; and

[0224] (c) The method comprises detecting that the E. coli cells of the B2 phylogenetic group are killed or their growth or proliferation is reduced.

[0225] 26. A method for detecting the presence of B2 phylogenetic group Escherichia coli in a sample, the method comprising contacting a sample comprising B2 phylogenetic group Escherichia coli with a composition comprising a plurality of transduction particles, wherein

[0226] (a) each particle comprises a nucleic acid comprising or encoding a detectable label, wherein the administered particle contacts a cell and introduces the nucleic acid therein, wherein optionally the label is expressed in the cell;

[0227] (b) each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells; and

[0228] (c) The method comprises detecting E. coli cells of phylogenetic group B2 comprising a marker.

[0229] 27. The method of embodiment 26 or 27, wherein the composition comprises the features of the composition of any one of embodiments 1, 2-21, 24 and 25.

[0230] 28. The method according to any one of embodiments 26 to 28, wherein the E. coli comprises one or more E. coli strains selected from the group consisting of ST131, ST1193, ST648, ST315, ST405, ST361, ST88 and ST453.

[0231] 29. The method of any one of embodiments 26-29, wherein the sample is a patient sample (e.g., a blood, urine, stool, or saliva sample), wherein the subject is a transplant or cancer patient (optionally a hematological cancer patient), or wherein the patient has or is at risk for a urinary tract infection (UTI); and optionally wherein the transplant is a solid organ or stem cell transplant (optionally a hematopoietic cell transplant).

[0232] 30. The method of any one of embodiments 26-30, wherein the nucleic acid of each particle comprises a nucleotide sequence encoding the nuclease (N1), or comprises or encodes a tag, wherein each particle is a synthetic T-even phage (optionally T4 phage) comprising an N1 insertion within the genome of the phage, wherein the region is located between the pin (protease inhibitor) gene and the iPII (internal protein) gene.

[0233] 31. The method according to any one of embodiments 26 to 31, wherein at least 1 x 10 7 The particles of PFU are brought into contact with the sample.

[0234] 32. The method of any one of embodiments 26-32, wherein the particles are contacted with the sample at an MOI (multiplicity of infection) of at least 0.01.

[0235] The labels used in the detection methods are conventional for the skilled person. The label can be, for example, a fluorescent label, such as GFP. The sample can be blood, saliva, sputum or a cell sample.

[0236] concept

[0237] The present invention also provides the following concepts fully supported by Example 2. All concepts can be combined with any other features disclosed herein.

[0238] Patients with hematological malignancies frequently develop bloodstream infections due to translocation of E. coli and other bacteria from the intestine. Antibiotic treatment to prevent these infections has deleterious effects on the microbiome and immune tone and is further hampered by increased antibiotic resistance, particularly to fluoroquinolones. As described in Example 2, the composition of the concept can target bacteria in biofilms, reduce the emergence of phage-resistant E. coli, and outperform their ancestral wild-type phage in co-culture experiments. Compositions are provided with a broad host range spanning the E. coli phylogenetic tree, including multidrug-resistant strains (B2 phylogenetic group). According to the concept, compositions comprising different particles can surprisingly better reduce E. coli loads in the mouse intestine than the individual component particles. The composition, method, and dosage of the concept have been found to be effective in selectively killing E. coli that can cause life-threatening infections in patients with hematological cancers, transplants, or UTIs. The composition, method, and dosage have also been found to be effective in treating or preventing E. coli bloodstream infections.

[0239] To this end, provide:-

[0240] Concept A:

[0241] A composition comprising a plurality of different types of transduction particles, wherein each of the particles comprises a nucleic acid, and wherein the particles are capable of contacting an E. coli cell and introducing the nucleic acid therein, wherein

[0242] (a) the nucleic acid of each particle comprises a nucleotide sequence encoding a product of interest (POI), wherein the nucleic acid is capable of expressing the POI in an Escherichia coli cell;

[0243] (b) each particle comprises an adhesion moiety for recognizing and binding a cognate moiety selected from the group consisting of LPS, LamB, and Tsx (optionally selected from the group consisting of LPS and Tsx) displayed on the surface of an E. coli cell; and

[0244] (c) the plurality of different types of transduction particles comprises (i) a first type of particle comprising an LPS adhesion moiety; and (ii) a second type of particle comprising a Tsx adhesion moiety.

[0245] Concept B:

[0246] A composition comprising a plurality of transduction particles for use in a method of treating or preventing an infection caused by Escherichia coli cells in a human or animal subject, wherein the method comprises administering the particles to the subject, wherein

[0247] (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of an E. coli cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, wherein the nuclease is expressed in the cell and cleaves the genomic DNA of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject;

[0248] (b) the E. coli cell is a cell of E. coli phylogenetic group B2; and

[0249] (c) Each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB and Tsx (optionally selected from the group consisting of LPS and Tsx) displayed on the surface of phylogenetic group B2 E. coli cells.

[0250] Concept C:

[0251] A composition comprising a plurality of transduction particles for use in a method of treating or preventing an infection caused by Escherichia coli cells (optionally E. coli cells of the B2 phylogenetic group) in a human or animal subject, wherein the method comprises administering particles to the subject, wherein (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of the E. coli cells, wherein the administered particles contact the cells and introduce the nucleic acid therein, wherein the nuclease is expressed in the cells and cleaves the genomic DNA of the cells, thereby killing the cells or reducing the growth or proliferation of the cells in the subject; and

[0252] (b) Each particle comprised by the composition is a T-even phage capsid; optionally, a capsid of T2 phage, T2-like phage, RB69 phage or RB69-like phage.

[0253] The following features are optional features that may be combined with the above concepts or any other configuration, example, implementation, or option herein.

[0254] Optionally,

[0255] A: Each particle of the first type comprises an LPS-binding moiety and a LamB-binding moiety;

[0256] B: Each particle of the first type comprises an LPS adhesion moiety and a Tsx adhesion moiety; or

[0257] C: Each particle of the second type comprises a Tsx adhesion moiety but lacks LamB and LPS adhesion moieties.

[0258] Optionally, the composition comprises particles according to A, B and C.

[0259] Optionally, each particle comprises a bacteriophage capsid comprising the nucleic acid.In one embodiment, the capsid comprises the capsid protein of T-even (optionally T2) or lambda phage.

[0260] Optionally, the capsid of each particle comprised in the composition is a T-even phage capsid; optionally comprising a capsid of T2 phage, T2-like phage, RB69 phage, or RB69-like phage.

[0261] Optionally, each particle is a bacteriophage (optionally a lytic phage) or a packaged phagemid.

[0262] Optionally, the composition comprises at least 3 or 4 different types of transducing particles. In one embodiment, the composition has 4 (but not more than 4) different types of transducing particles.

[0263] Optionally, the nucleic acid of each particle comprises at least one nucleotide sequence (N1) encoding the POI, wherein each particle is a synthetic T-even phage comprising an N1 insertion within a modification permissive region (MPR) of the phage genome, wherein the MPR is immediately after gene 49 and toward gene E when compared to a reference wild-type T2 phage. The nucleic acid may comprise a DNA deletion of phage DNA in the MPR. The insertion may comprise up to 5000, 6000, 7000, or 8000 bp of DNA and / or the deletion may comprise up to 5000, 6000, 7000, or 8000 bp of DNA. The MPR may comprise contiguous DNA between gene 49 and gene E, wherein the contiguous DNA is at least 1000 bp in length; or wherein the MPR comprises at least 100 bp of DNA between gene 49 and gene E.

[0264] Optionally, the T-even phage herein is a phage selected from T2, T4 or T6 phage, or comprises a genome that is at least 95% identical to the genome of the selected phage. The percentage may be at least 96, 97, 98 or 99%.

[0265] The composition of any one of claims 8 to 11, wherein the synthetic phage genome comprises the insertion between coordinates 9000 and 21000, wherein the coordinates are nucleotide positions counted from the nucleotide immediately following gene 49 (coordinate number 1) towards gene E when compared to a reference wild-type T2 phage. Optionally, the insertion is between coordinates 10300 and 19800, for example, between 10359 and 19810 for the reference T2 genome.

[0266] Optionally, the insertion is in a window selected from the following windows (numbers are coordinates with reference to the T2 genome): -9000-21000; 10000-21000, 10100-21000, 10200-21000, 10300-21000, 10400-21000, 10500-21000, 11000-21000, 15000-21000;

[0267] 9000-20000; 10000-20000, 10100-20000, 10200-20000, 10300-20000, 10400-20000, 10500-20000, 11000-20000, 15000-20000;

[0268] 9000-19500; 10000-19500, 10100-19500, 10200-19500, 10300-19500, 10400-19500, 10500-19500, 11000-19500, 15000-19500;

[0269] 9000-19000; 10000-19000, 10100-19000, 10200-19000, 10300-19000, 10400-19000, 10500-19000, 11000-19000, 15000-19000;

[0270] 9000-18000; 10000-18000, 10100-18000, 10200-18000, 10300-18000, 10400-18000, 10500-18000, 11000-18000 and 15000-18000.

[0271] Optionally, the nuclease is a dsDNA nuclease, i.e. capable of cleaving dsDNA. Optionally, the nuclease is a nickase, such as a Cas9 nickase.

[0272] In one embodiment, the POI is a protein. In one embodiment, the POI is an RNA, such as a crRNA.

[0273] Optionally, the POI contains

[0274] (a) a nuclease for targeting DNA of an Escherichia coli cell, wherein the nuclease can be expressed in the cell and used to cut the DNA of the cell, thereby modifying or killing the cell; or

[0275] (b) Dead Cas (dCas) for targeting DNA of E. coli cells, wherein the dCas can be expressed in the cells and targeted to the DNA of the cells, thereby modifying the cells.

[0276] Optionally, when (a) is applicable, the nuclease is a guide nuclease, optionally a Cas, a meganuclease, a zinc finger nuclease or a TALEN; or when (b) is applicable, the dCas is dCas9.

[0277] The nuclease can be a type I, II, III, IV, V or VI Cas nuclease, optionally Cas9 or Cas3.

[0278] In a preferred embodiment, the nucleic acid comprises (optionally in 5' to 3' order) a cas3 gene (ygcB) and a cognate Cascade gene complex comprising casA (ygcL, cas8e), casB (ygcK, cas11), casC (ygcJ, cas7), casD (ygcI, cas5) and casE (ygcH, cas6), and optionally a CRISPR array or nucleotide sequence encoding a guide RNA that targets the E. coli genome.

[0279] In a preferred embodiment, the POI comprises at least one crRNA or guide RNA that can be used with Cas for DNA targeting in E. coli cells. Optionally, each crRNA or guide RNA comprises a spacer sequence complementary to an E. coli protospacer sequence; optionally, the protospacer region of an E. coli cell of the B2 phylogenetic group or a strain selected from the groups ST131, ST1193, ST648, ST315, ST405, ST361, ST88, and ST453.

[0280] Various nucleic acids preferably encode multiple different cRNAs comprising spacer sequences, and the spacer sequences target E. coli chromosomal genes. For example, various nucleic acids preferably encode multiple different cRNAs comprising spacer sequences, and the spacer sequences target 2, 3 or 4 E. coli chromosomal genes selected from fimH, bolA, rpoH, lptA and murA. In one embodiment, the composition of the present invention comprises a type of transduction particle targeting E. coli genes bolA, rpoH and fimH. Additionally or alternatively, the composition of the present invention comprises a type of transduction particle targeting E. coli genes lptA and murA. Optionally, the composition comprises a first type of transduction particle targeting E. coli genes bolA, rpoH and fimH; and a second type of transduction particle targeting E. coli genes lptA and murA. Optionally, the first type of particle is according to any other first type of particle herein, which comprises an LPS adhesion portion (e.g., comprising an LPS and Tsx adhesion portion). Optionally, the second type of particle is according to any other first type of particle herein, which comprises a Tsx adhesion portion. Optionally, the second type of particle comprises an LPS attachment moiety (eg comprises an LPS and a LamB attachment moiety).

[0281] The protospacer sequence may be comprised in a gene selected from the group consisting of the E. coli genes fimH, bolA, rpoH, lptA and murA.

[0282] In one embodiment, the particles of the composition target all of the E. coli genes fimH, bolA, rpoH, lptA, and murA.

[0283] The nucleotide sequence encoding the POI may comprise a stress-phase active (SPA) promoter for expressing the POI in E. coli cells. The promoter may be the E. coli bolA promoter. The promoter may comprise SEQ ID NO: 13.

[0284] In one embodiment, the E. coli cells comprise an E. coli strain that causes sepsis, septicemia, or diarrhea in humans or animals.

[0285] Optionally, the E. coli cells are gastrointestinal cells. Optionally, the E. coli cells are contained in the intestinal microbiome. Optionally, the E. coli cells are contained in the blood of the subject. Optionally, the E. coli cells are contained in the urinary tract of the subject. Optionally, the E. coli cells are contained in a microbiome selected from the gastrointestinal tract (e.g., stomach), blood, urinary tract, mouth, nose, eye, ear, skin, anus, or hair microbiome.

[0286] In one embodiment, the composition is for use in a method of treating or preventing an infection caused by E. coli cells in a human or animal subject, wherein the method comprises administering the particles to the subject.

[0287] Optionally, the method is used to prevent translocation of B2 phylogroup E. coli from the gastrointestinal tract or urinary tract of a subject to the subject's bloodstream, thereby preventing or reducing bacteremia in the subject.

[0288] Optionally, the method is used to prevent an E. coli infection in a subject at risk for febrile neutropenia. The subject can be a cancer patient, such as a hematological cancer patient. The infection can be a bloodstream infection in the subject. Thus, in one embodiment, the method is used to prevent an E. coli infection in a subject at risk for febrile neutropenia, wherein the subject is a human cancer patient, such as a hematological cancer patient.

[0289] supply:-

[0290] A method of treating or preventing sepsis, septicemia or diarrhoea in a human or animal subject, the method comprising administering to the subject a composition as described herein (e.g. a composition of Concept A, B or C), wherein the E. coli cells comprise an E. coli strain that causes sepsis, septicemia or diarrhoea in humans or animals.

[0291] A method of treating or preventing an infection caused by Escherichia coli cells in a human or animal subject, wherein the method comprises administering a composition as described herein (e.g. a composition of Concept A, B or C) to the subject, wherein the infection is treated or prevented.

[0292] Infection may be reduced or eliminated. Infection may be reduced by at least 20, 30, 40, 50, 601 70, 80 or 90%.

[0293] The subject may be a transplant or cancer patient (optionally a hematological cancer patient), or wherein the patient is suffering from a urinary tract infection (UTI) or is at risk of a urinary tract infection (UTI). Optionally, the transplant is a solid organ or stem cell transplant (optionally a hematopoietic cell transplant) or wherein the transplant is a transplant of a medical device. The subject may be suffering from a hematological cancer, such as a leukemia. The subject (e.g., a cancer patient) may be at risk of neutropenia or may be suffering from neutropenia. Preferably, the subject is suffering from a hematological cancer, such as a leukemia, and is at risk of neutropenia or is suffering from neutropenia.

[0294] Optionally, the method is performed before the subject receives a transplant or said transplant.The transplant may be a stem cell transplant, for example when the subject is a cancer patient.

[0295] The composition or method can be used to prevent hemolytic uremic syndrome (HUS), UTI infection, sepsis, septicemia, or diarrhea in a human subject.

[0296] In the composition for use in the method or any method described herein, at least 1 x 10 7 PFU of particles are administered to a subject. Also provided is a dose of the composition described herein, wherein the dose is at least 1 x 10 7 PFU of the particles. For example, a dose of at least 1 x 10 7 , 1x 10 8 , 1x 10 9 , 1x 10 10 or 1x 10 11 PFU of the particles. For example, a dose of 1 x 10 7 , 2x 10 9 or 2x 10 11 PFU of the particles. For example, a dose of 1 x 10 7 Up to 2x 10 11 PFU of the particles. For example, a dose of 1 x 10 7 Up to 2x10 11 In the method, the E. coli may be reduced by at least 3 or 4 log10 CFU / g body weight of the subject.

[0297] The dose can be contained in a medical device or container, for example, for oral or intravenous administration. The device can be an intravenous device, a syringe, or contain an injection needle. The device or container can be sterile.

[0298] Optionally, the particles are administered to the subject at an MOI (multiplicity of infection) of at least 0.01, for example an MOI of at least 0.1 or 1.

[0299] Preferably, the E. coli cells comprise at least one strain that is an antibiotic-resistant or MDR strain; and / or at least one B2-1 strain. Optionally, the antibiotic is a fluoroquinolone (optionally levofloxacin), a carbapenem, or vancomycin; and / or wherein the E. coli cells comprise E. coli that produces β-lactamase (ESBL).

[0300] In one embodiment,

[0301] (i) The composition comprises the first and second types of particles according to A, B and C

[0302] A: Each particle of the first type comprises an LPS-binding moiety and a LamB-binding moiety;

[0303] B: Each particle of the first type comprises an LPS adhesion moiety and a Tsx adhesion moiety; or

[0304] C: Each particle of the second type contains the Tsx adhesion moiety but lacks the LamB and LPS adhesion moieties;

[0305] (ii) the POI comprises a nuclease that is directed to the DNA of an E. coli cell, wherein the nuclease is capable of being expressed in the cell and used to cleave the DNA of the cell, thereby modifying or killing the cell;

[0306] (iii) each particle comprises a phage capsid containing a nucleic acid; and

[0307] (iv) The particles of the composition target a plurality of different E. coli genes, optionally selected from essential genes and virulence genes.

[0308] supply:-

[0309] A method for detecting the presence of Escherichia coli (optionally Escherichia coli cells of the B2 phylogenetic group) in a sample, the method comprising

[0310] (a) contacting a sample with a composition as described herein (e.g. according to concept A, B or C); and

[0311] (b) Detecting whether E. coli cells are killed or their growth or proliferation is reduced.

[0312] A method for detecting the presence of Escherichia coli (optionally Escherichia coli cells of the B2 phylogenetic group) in a sample, the method comprising

[0313] (a) contacting a sample with a composition as described herein (e.g. according to concept A, B or C); wherein the particles of the composition comprise a nucleic acid comprising or encoding a detectable label, wherein the particles contact a cell and introduce the nucleic acid therein, wherein optionally the label is expressed in the cell; and

[0314] (b) Detection of E. coli cells containing the marker.

[0315] A method for modifying the genome of an E. coli cell, the method comprising contacting the cell with a composition as described herein (e.g. according to concept A, B or C), wherein a nucleic acid encoding a POI is introduced into the cell, thereby modifying the genome of the cell.

[0316] When the POI is a nuclease, the nuclease is expressed in the cell and cleaves the cell's genomic DNA, thereby killing the cell or reducing the growth or proliferation of the cell in the subject.

[0317] A method for treating or preventing an infection caused by E. coli cells in a human or animal subject, the method comprising administering to the subject a composition as described herein (eg according to concept A, B or C).

[0318] Optionally, the sample is a patient sample (e.g., a blood, urine, stool, or saliva sample), wherein the subject is a transplant or cancer patient (optionally a hematological cancer patient), or wherein the patient has or is at risk of a urinary tract infection (UTI); and optionally wherein the transplant is a solid organ or stem cell transplant (optionally a hematopoietic cell transplant).

[0319] In one embodiment, the methods herein can be performed in vitro. In one embodiment, the methods herein can be performed ex vivo.

[0320] Optionally, make at least 1 x 10 7 Contacting the Particles of PFUs with the Sample Optionally, the particles of PFUs described above are contacted with the sample.

[0321] Optionally, the particles are contacted with the sample at an MOI (multiplicity of infection) of at least 0.01.

[0322] Optionally, each particle contained in the composition comprises a T-even phage capsid; optionally comprising a capsid of T2 phage, T2-like phage, RB69 phage, or RB69-like phage. Each particle may comprise a capsid of a Tevenvirinae phage. Each particle may be a modified Tevenvirinae phage comprising a genomic insertion of a nucleotide sequence encoding a POI or the nuclease (and optionally comprising a cognate crRNA when the nuclease is Cas).

[0323] Each particle can be a bacteriophage or a packaged phagemid. Preferably, the bacteriophage is a lytic phage. In another embodiment, the bacteriophage can be a non-lytic phage. In another embodiment, the phage can be a temperate phage.

[0324] Optionally, the phage is a modified T2 phage, a T2-like phage, an RB69 phage, or an RB69-like phage. Preferably, the phage is a T2-like phage or an RB69-like phage. Preferably, each particle is a modified first phage, wherein the genome of the first phage is at least 95, 96, 97, 98, or 99% identical (by nucleotide sequence identity) to the genome of wild-type T2 or RB69. Preferably, the percentage is at least 95%. Preferably, the percent identity between phage genomes is determined by Mash analysis using kmer sizes of 17 and 1000.

[0325] Optionally, the composition comprises at least 3 or 4 different types of transduction particles, wherein the types have different adhesion moieties for recognizing and binding to homologous moieties contained in E. coli cells.

[0326] Optionally, the cell comprises Escherichia coli of phylogenetic group B2. The cell is preferably a pathogenic cell. The cell is preferably pathogenic to the subject. The cell may mediate a disease or condition in the subject.

[0327] In one embodiment,

[0328] (i) the nuclease is a guided nuclease for targeting DNA of Escherichia coli cells, wherein the nuclease can be expressed in the cells and used to cut the DNA of the cells, thereby killing the cells;

[0329] (ii) each particle comprises a phage capsid containing a nucleic acid; and

[0330] (iii) particles of the composition target a plurality of different E. coli genes, optionally selected from essential genes and virulence genes; and

[0331] (iv) optionally the composition comprises

[0332] A: Particles containing both the LPS-adhesive moiety and the LamB-adhesive moiety (and lacking Tsx);

[0333] B: particles comprising an LPS-adhesive moiety and a Tsx-adhesive moiety; or

[0334] C: Particles containing the Tsx-adherent moiety but lacking the LamB- and LPS-adherent moieties.

[0335] The nuclease may be a Cas nuclease, and the nucleic acid encodes at least one crRNA or guide RNA that can be used with Cas for DNA targeting in E. coli cells. The various crRNAs or guide RNAs may comprise spacer sequences complementary to: E. coli protospacer sequences; optionally, the protospacer regions of E. coli cells of the B2 phylogenetic group or strains selected from groups ST131, ST1193, ST648, ST315, ST405, ST361, ST88, and ST453. Each protospacer sequence may be contained in a gene selected from the E. coli genes fimH, bolA, rpoH, lptA, and murA.

[0336] Optionally, the particles of the composition target at least one virulence gene and at least one essential gene. Optionally, the particles of the composition target at least three genes selected from virulence genes and essential genes. Optionally, the particles of the composition target all of the E. coli genes fimH, bolA, rpoH, lptA, and murA.

[0337] The expression of the POI or nuclease or crRNA can be under the control of a stress-activated (SPA) promoter. Optionally, the promoter is the E. coli bolA promoter or comprises SEQ ID NO: 13. Additionally or alternatively, the expression of the POI or nuclease or crRNA can be under the control of the E. coli promoter PJ23100 (SEQ ID NO: 14), for example, a promoter having a nucleotide sequence that is at least 80, 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 14. For example, the promoter has the nucleotide sequence of SEQ ID NO: 16.

[0338] The spacer herein may be adjacent to a direct repeat sequence in the nucleic acid contained in the particle, for example a repeat of the sequence having SEQ ID NO: 15 (or said sequence having up to 5, 4, 3 or 2 nucleotide changes compared to SEQ ID NO: 15).

[0339] In one embodiment, expression of the POI or nuclease or crRNA can be under the control of a first promoter, and expression of the adhesion moiety / adhesion moieties is under the control of a second promoter different from the first promoter.

[0340] The adhesion moiety herein is located on the outer surface of the homologous particle. In one embodiment, the moiety is comprised in the tail fiber, spike or capsid of the particle, preferably the tail fiber.

[0341] Provide the following paragraphs.

[0342] paragraph:

[0343] 1. A composition comprising a plurality of transduction particles for use in a method of treating or preventing an infection caused by Escherichia coli cells in a human or animal subject, wherein the method comprises administering the particles to the subject, wherein

[0344] (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of an E. coli cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, wherein the nuclease is expressed in the cell and cleaves the genomic DNA of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject;

[0345] (b) the E. coli cell is a cell of E. coli phylogenetic group B2; and

[0346] (c) Each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0347] 2. A method for treating or preventing an infection caused by E. coli cells in a human or animal subject, the method comprising administering to the subject a plurality of transduction particles, wherein the method comprises administering the particles to the subject, wherein

[0348] (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of an E. coli cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, wherein the nuclease is expressed in the cell and cleaves the genomic DNA of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject;

[0349] (b) the E. coli cell is a cell of E. coli phylogenetic group B2; and

[0350] (c) Each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0351] 3. The composition or method of paragraph 1 or 2, respectively, wherein the method is used to treat or prevent an infection in a subject caused by an Escherichia coli strain selected from the group consisting of ST131, ST1193, ST648, ST315, ST405, ST361, ST88 and ST453.

[0352] 4. The composition or method of any preceding paragraph, wherein the method is used to treat or prevent an infection in a subject caused by a plurality of different phylogenetic group B2 E. coli strains; optionally wherein the plurality comprises E. coli ST131 and ST1193 cells.

[0353] 5. The composition or method of any preceding paragraph, wherein the subject is a transplant or cancer patient (optionally a hematological cancer patient), or wherein the patient has or is at risk of a urinary tract infection (UTI); and optionally wherein the transplant is a solid organ or stem cell transplant (optionally a hematopoietic cell transplant), or wherein the transplant is a medical device transplant.

[0354] 6. The composition or method of any preceding paragraph, wherein the method is performed before the subject receives a transplant.

[0355] 7. The composition or method of any preceding paragraph, wherein the B2 E. coli cells comprise an E. coli strain that causes sepsis, septicemia, or diarrhea in humans.

[0356] 8. The composition or method of any preceding paragraph, for preventing hemolytic uremic syndrome (HUS), UTI infection, sepsis, septicemia, or diarrhea in a subject.

[0357] 9. The composition or method of any preceding paragraph, wherein each particle comprises a phage capsid comprising the nucleic acid; optionally wherein the capsid comprises a capsid protein of T-even (optionally T4) or lambda phage.

[0358] 10. The composition or method of any preceding paragraph, wherein each particle is a bacteriophage (optionally a lytic phage) or a packaged phagemid.

[0359] 11. The composition or method of any preceding paragraph, wherein at least 2, 3, or 4 different types of transducing particles are administered to the subject.

[0360] 12. The composition or method of any preceding paragraph, wherein a first type of transduction particle and a second type of transduction particle are administered to a subject, wherein the first type of particle comprises a first adhesion moiety capable of recognizing and binding to a first homology moiety selected from the group LPS, LamB, and Tsx displayed on B2 E. coli, and the second type of particle comprises a second adhesion moiety capable of recognizing and binding to a second homology moiety selected from said group, wherein the first adhesion moiety and the second adhesion moiety are different from each other.

[0361] 13. The composition or method of paragraph 12, wherein the first and second homologous portions are different from each other.

[0362] 14. The composition or method of any preceding paragraph, wherein the nucleic acid of each particle comprises a nucleotide sequence (N1) encoding the nuclease, wherein each particle is a synthetic T-even phage (optionally a T4 phage) comprising an N1 insertion within the genome of the phage, wherein the region is located between the pin (protease inhibitor) gene and the iPII (internal protein) gene.

[0363] 15. The composition or method of any preceding paragraph, wherein the nuclease is a guide nuclease, optionally a Cas, a meganuclease, a zinc finger nuclease, or a TALEN.

[0364] 16. The composition or method of any preceding paragraph, wherein the nuclease is a Type I, II, III, IV, V, or VI nuclease, optionally Cas9 or Cas3.

[0365] 17. The composition or method of any preceding paragraph, wherein at least 1 x 10 7 Particles of PFU are administered to a subject.

[0366] 18. The composition or method of any preceding paragraph, wherein the particles are administered to the subject at an MOI (multiplicity of infection) of at least 0.01.

[0367] 19. The composition or method of any preceding paragraph, wherein the or at least one strain is an antibiotic-resistant or MDR strain; and / or wherein the or at least one strain is a B2-1 strain.

[0368] 20. The composition or method of paragraph 19, wherein the antibiotic is a fluoroquinolone (optionally levofloxacin), a carbapenem, or vancomycin; and / or wherein the E. coli is a β-lactamase (ESBL)-producing E. coli.

[0369] 21. The composition or method of any preceding paragraph, wherein

[0370] (i) The nuclease is Cas,

[0371] (ii) each particle comprises a phage capsid containing a nucleic acid; and

[0372] (iii) wherein a first type of transduction particle and a second type of transduction particle are administered to a subject, wherein the first type of particle comprises a first adhesion moiety capable of recognizing and binding to a first homology moiety selected from the group LPS, LamB and Tsx displayed on B2 strain E. coli, and the second type of particle comprises a second adhesion moiety capable of recognizing and binding to a second homology moiety selected from said group, wherein the first and second adhesion moieties are different from each other.

[0373] 22. A method for treating or preventing an infection caused by E. coli cells in a human or animal subject, the method comprising administering to the subject a plurality of transducing particles, wherein

[0374] (a) each particle comprises a nucleic acid encoding a crRNA or a guide RNA operable with a Cas nuclease for chromosome targeting in a cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, wherein the crRNA or guide RNA is expressed and guides the Cas nuclease, wherein the nuclease cleaves the chromosome of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject;

[0375] (b) the E. coli cell is a cell of E. coli phylogenetic group B2; and

[0376] (c) Each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0377] 23. The method of paragraph 22, wherein the method is according to any of paragraphs 1-20, optionally except that the nuclease is endogenous to the cell and is not encoded by a nucleic acid contained in the particle.

[0378] 24. A composition comprising a plurality of transduction particles for use in a method of treating or preventing an infection caused by an E. coli cell in a human or animal subject according to paragraph 22 or 23, wherein

[0379] (a) each particle comprises a nucleic acid encoding a crRNA or a guide RNA operable with a Cas nuclease for chromosome targeting in a cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, wherein the crRNA or guide RNA is expressed and guides the Cas nuclease, wherein the nuclease cleaves the chromosome of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject;

[0380] (b) the E. coli cell is a cell of E. coli phylogenetic group B2; and

[0381] (c) Each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0382] 25. The composition or method of any preceding paragraph, for preventing translocation of phylogenetic group B2 Escherichia coli from the gastrointestinal tract or urinary tract of a subject to the subject's bloodstream, thereby preventing or reducing bacteremia in the patient.

[0383] 26. A method for detecting the presence of B2 phylogenetic group Escherichia coli in a sample, the method comprising contacting a sample comprising B2 phylogenetic group Escherichia coli with a composition comprising a plurality of transduction particles, wherein

[0384] (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of the E. coli cells, wherein the administered particles contact the cells and introduce the nucleic acid therein, wherein the nuclease is expressed in the cells and cleaves the genomic DNA of the cells, thereby killing the cells or reducing the growth or proliferation of the cells in the subject;

[0385] (b) each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells; and

[0386] (c) The method comprises detecting that the E. coli cells of the B2 phylogenetic group are killed or their growth or proliferation is reduced.

[0387] 27. A method for detecting the presence of B2 phylogenetic group Escherichia coli in a sample, the method comprising contacting a sample comprising B2 phylogenetic group Escherichia coli with a composition comprising a plurality of transduction particles, wherein

[0388] (a) each particle comprises a nucleic acid comprising or encoding a detectable label, wherein the administered particle contacts a cell and introduces the nucleic acid therein, wherein optionally the label is expressed in the cell;

[0389] (b) each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells; and

[0390] (c) The method comprises detecting E. coli cells of phylogenetic group B2 comprising a marker.

[0391] 28. The method of paragraph 26 or 27, wherein the composition comprises the features of the composition of any one of paragraphs 1, 2-21, 24 and 25.

[0392] 29. The method of any one of paragraphs 26-28, wherein the E. coli comprises one or more E. coli strains selected from the group consisting of ST131, ST1193, ST648, ST315, ST405, ST361, ST88 and ST453.

[0393] 30. The method of any one of paragraphs 26-29, wherein the sample is a patient sample (e.g., a blood, urine, stool, or saliva sample), wherein the subject is a transplant or cancer patient (optionally a hematological cancer patient), or wherein the patient has or is at risk of a urinary tract infection (UTI); and optionally wherein the transplant is a solid organ or stem cell transplant (optionally a hematopoietic cell transplant).

[0394] 31. The method of any of paragraphs 26-30, wherein the nucleic acid of each particle comprises a nucleotide sequence encoding the nuclease (N1), or comprises or encodes a marker, wherein each particle is a synthetic T-even phage (optionally T4 phage) comprising an N1 insertion within the genome of the phage, wherein the region is located between the pin (protease inhibitor) gene and the iPII (internal protein) gene.

[0395] 32. The method of any one of paragraphs 26 to 31, wherein at least 1 x 10 7 The particles of PFU are brought into contact with the sample.

[0396] 33. The method of any of paragraphs 26-32, wherein the particles are contacted with the sample at an MOI (multiplicity of infection) of at least 0.01.

[0397] It should be understood that the specific embodiments described herein are shown by way of illustration and are not intended to limit the present invention. Without departing from the scope of the present invention, the main features of the present invention can be used in various embodiments. Those skilled in the art will recognize or use numerous equivalents that can identify the specific procedures described herein without exceeding conventional research. Such equivalents are considered to be within the scope of the present invention and are covered by the claims. All publications and patent applications mentioned in the specification indicate the technical level of those skilled in the art to which the present invention belongs. All publications and patent applications and all U.S. equivalent patent applications and patents are incorporated by reference to the extent that individual publications or patent applications each clearly and individually indicate that they are incorporated by reference. With reference to the publications mentioned herein and the equivalent publications of the U.S. Patent and Trademark Office (USPTO) or WIPO, its disclosure is incorporated by reference herein for providing the disclosure that can be used for the present invention and / or to provide one or more features (such as the feature of the carrier) that may be included in one or more claims herein.

[0398] When used in conjunction with the term "comprising" in the claims and / or the specification, the use of the word "a" or "an" may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one". Unless expressly indicated to refer only to alternatives or the alternatives are mutually exclusive, the use of the term "or" in the claims is used to mean "and / or", although the present disclosure supports definitions referring only to alternatives and "and / or". Throughout this application, the term "about" is used to indicate that a value includes the inherent error variation of the device, the method used to determine the value, or the variation that exists among the study subjects.

[0399] As used in this specification and claims, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0400] As used herein, the term "or combinations thereof" or the like refers to all permutations and combinations of the items listed before the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in the particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, specifically included are combinations containing repetitions of one or more items or terms, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and the like. A skilled artisan will understand that there is typically no limit to the number of items or terms in any combination, unless otherwise apparent from the context.

[0401] Any part of this disclosure may be read in combination with any other part of this disclosure, unless otherwise apparent from the context.

[0402] All compositions and / or methods disclosed herein and claimed for protection can be prepared and performed without undue experimentation in view of the present disclosure. Although the compositions and methods of the present invention are described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the compositions and / or methods described herein and the steps or sequence of steps of the methods without departing from the concept, spirit and scope of the present invention. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope and concept of the present invention as defined by the appended claims.

[0403] The present invention is described in more detail in the following non-limiting examples. DETAILED DESCRIPTION

[0404] Example

[0405] Example 1: Particles for surprisingly targeting multiple different E. coli strains including the B2 phylogenetic group Granular composition

[0406] Overview

[0407] Patient samples containing various different E. coli strains are challenged with a particle composition. The composition comprises a plurality of transduction particles carrying an adhesion portion that can bind to LPS, LamB or Tsx present on the E. coli cells. The particles comprise a capsid containing a phage capsid protein, wherein the capsid contains a nucleic acid encoding a CRISPR / Cas system for chromosome targeting in E. coli. Each nucleic acid encodes a plurality of different cRNAs comprising a spacer sequence that targets an E. coli chromosomal gene. The ability to kill or reduce the growth of E. coli in a sample is determined using a plaque assay as described below. Whole genome sequencing and genome assembly are used to assign susceptible E. coli strains to phylogenetic groups. It is surprising to see that the use of such particle compositions can very effectively and widely target a plurality of different E. coli strains (these strains are clinically relevant to actual patient samples). In addition, advantageously, a large number of different B2 phylogenetic group strains are targeted and killed. This is significant because B2 E. coli strains often display antibiotic resistance (e.g., MDR), such as resistance to fluoroquinolones, which causes potentially life-threatening infections in patients, such as cancer, transplant, and UTI patients. In addition to the B2 group, surprisingly, we were also able to successfully kill or inhibit the growth of multiple strains of E. coli phylogenetic groups B1, D, F, and G.

[0408] Patient sampling

[0409] The samples tested (n=71) were obtained from recruited adults ( > Two prospective observational studies were conducted in patients (aged 18 years and older) who were admitted for autologous or allogeneic hematopoietic cell transplantation (HCT) and received levofloxacin (fluoroquinolone, FQ) prophylaxis starting on the day before transplantation (day -1) (Satlin 2021 and Satlin 2018). Trimethoprim-sulfamethoxazole (TMP-SMX) was administered to allogeneic HCT recipients 2 to 4 days before HCT. Antibiotic treatment cannot eradicate all E. coli in patients; for example, fluoroquinolone-resistant E. coli will persist, and therefore E. coli from patients was sampled. E. coli isolates were derived from anal swabs or fecal samples obtained at the time of admission for transplantation. The timing of sampling varied from day -7 (7 days before transplantation) to day 0 (the day of transplantation).

[0410] General plaque assay: spot assay for coverage

[0411] This procedure describes a method for assessing the coverage of a panel of bacterial strains by phage particle lysates.

[0412]

[0413]

[0414] The plaque assay was performed according to the general plaque assay above. The bacterial strain was prepared by inoculating 5 μl of the frozen stock into 250 μl of LB broth in a 96-well plate. The plate was incubated overnight at 37°C and 250 rpm. The next day, 100 ml of the overnight strain was mixed with 3 ml of preheated top agar (at 55°C) containing 5 mM CaCl2 and 5 mM MgSO4 in a culture tube. The mixture was poured onto the top of the pre-adapted LB plate and evenly distributed by rotation. The plate was placed on the laboratory bench for 5-10 minutes to solidify. At the same time, the particle composition was prepared from 10 0 Dilute to 10 -9 , and 5 μL of each serial dilution was spotted on top of the overlay. The plate was left uncovered on the bench for 20 minutes or until the spots were completely absorbed by the agar, and then incubated upside down at 37°C overnight.

[0415] Result evaluation:

[0416] If visible plaques appear, the result is recorded as positive, the plaques are counted, and the phage concentration is calculated. Calculation of phage concentration: number of plaques x 200 x the dilution in which the plaques were observed. ie: if 5 plaques are counted for dilution -6: 5 x 200 x 1e6 = 1e9 pfu / ml

[0417] If no visible plaques were present, but inhibition of growth was observed, the result was recorded as zone of lysis, and the lowest dilution of inhibition was noted.

[0418] If no plaques or inhibition were observed, the result was recorded as negative.

[0419] Whole-genome sequencing

[0420] DNA extraction was performed using the Omega Bio-tek, Mag-Bind Bacterial DNA 96 kit. The protocol was followed and samples were eluted in 100 μL of elution buffer.

[0421] Sequencing libraries were generated using an Illumina Nextera XT and paired-end sequenced on an Illumina MiSeq instrument with a V2 flow cell (300 cycles). The average sequencing depth for all samples was 48x (range: 31-72x).

[0422] Genome assembly and phylogenetic tree reconstruction

[0423] Raw data were trimmed for adapter sequences and low-quality bases using fastp 0.22.0 (Chen et al. 2018). Genomes were assembled using SKESA 2.4.0 (Souvorov et al. 2018). Phylogenetic groups for each sample were determined using EzClermont 0.7.0 (https: / / github.com / nickp60 / EzClermont). Genomic distances were estimated using Mash 1.1 (Ondov et al. 2016) with a kmer size of 17 and 1000 sketches. Neighbor-joining phylogenetic trees were constructed using rapidnj 2.3.2 (Simonsen et al. 2008). Final phylogenetic tree visualizations were generated in Interactive Tree of Life (iTOL) version 6.5.3 (Letunic et al. 2021). Phylotyping of strains was performed in silico using the method published in Microb Genom. 2018 Jul;4(7):e000192, published online June 19, 2018. doi:10.1099 / mgen.0.000192, PMCID:PMC6113867, PMID:29916797, “Clermon Typing: an easy-to-use and accurate in silico method for Escherichia genus strain phylotyping”, Johann Beghain et al. A set of primer sequences described in Table S1 of the reference (available in the online version of this article) was used.

[0424] result

[0425] It was surprising to see that the use of such particle compositions was able to very effectively and broadly target multiple different E. coli strains that were clinically relevant to actual patient samples, see Figure 1 In addition (see Figure 2), advantageously, targets and kills a large number of different B2 phylogenetic group strains. This is significant because B2 E. coli strains often display antibiotic resistance (e.g., MDR), such as resistance to fluoroquinolones, which causes potentially life-threatening infections in patients, such as cancer, transplant, and UTI patients. Kills (plaque forming) more than 10 different ST131 strains, and kills (plaque forming) more than 10 different ST1193 strains. Sequence type 1193 has recently emerged as a new, virulent, and resistant lineage among fluoroquinolone-resistant E. coli. E. coli ST131 is the world's dominant multidrug-resistant clone associated with a high incidence of rUTI. Uropathogenic E. coli (UPEC) is the leading cause of urinary tract infections (UTIs), responsible for ~90% of all cases. Most UPEC strains belong to Escherichia coli phylogenetic group B2 or D and are often clonal, with the most common sequence types (STs) isolated worldwide being ST69, ST73, ST95, and ST1312. The recently emerged and globally disseminated ST131 clone is a major contributor to hospital-acquired UTIs and community-acquired UTIs, as well as bloodstream infections and infections in companion animals and poultry. ST131, originally identified in 2008, is associated with the worldwide spread of the CTX-M-15 extended-spectrum beta-lactamase (ESBL) resistance gene. Most ST131 strains are currently closely associated with multidrug resistance (MDR), including resistance to fluoroquinolones. Recent reports have also identified strains resistant to the last-line carbapenems.

[0426] Of note, we included clinical samples from patients who went on to develop E. coli bacteremia despite pre-treatment with FQ / TMP-SMX. The composition could kill or reduce the growth of the E. coli strains in these samples (and this suggests the potential for the composition to prevent bacteremia in the subject). This included strains of the following Multi-Locus Sequence Typing (MLST) types (see Figure 2 ): ST648, ST315, ST405, ST361, ST88, ST453, ST1193 and ST131.

[0427] In addition to group B2, surprisingly we were also able to successfully kill or inhibit the growth of multiple strains of E. coli phylogenetic groups Bl, D, F and G.

[0428] References

[0429] ●Shifu Chen, Yanqing Zhou, Yaru Chen, Jia Gu; fastp: an ultra-fast all-in-one FASTQ preprocessor, Bioinformatics, Volume 34, Issue 17, September 1, 2018, Pages i884–i890, https: / / doi.org / 10.1093 / bioinformatics / bty560

[0430] ●Alexandre Souvorov, Richa Agarwala and David J. Lipman. SKESA: strategic k-mer extension for scrupulous assemblies. Genome Biology 2018 19:153. doi.org / 10.1186 / s13059-018-1540-z

[0431] ●Ondov, B.D., Treangen, T.J., Melsted, P., et al. Mash: fast genome and metagenome distance estimation using MinHash. Genome Biol 17, 132 (2016). https: / / doi.org / 10.1186 / s13059-016-0997-x

[0432] ●Martin Simonsen, Thomas Mailund and Christian N.S. Pedersen. Rapid Neighbour Joining. Proceedings of the 8th Workshop in Algorithms in Bioinformatics (WABI), LNBI 5251, 113-122, Springer Verlag, October 2008. doi:10.1007 / 978-3-540-87361-7_10

[0433] ●Letunic I and Bork P (2021) Nucleic Acids Res doi:10.1093 / nar / gkab301 Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation

[0434] ●Michael J. Satlin et al., Colonization with Fluoroquinolone-resistant Enterobacterales decreases the effectiveness of fluoroquinolone prophylaxisin hematopoietic cell transplant recipients. Clinical Infectious Diseases 2021.

[0435] ●Michael J.Satlin et al., Colonization with Levofloxacin-resistantextended-spectrumβ-lactamase-producing Enterobacteriaceae and risk ofbacteremia in hematopoietic cell transplant recipients. Clinical InfectiousDiseases 2018.

[0436] Example 2: Phage-derived CRISPR therapeutics designed to reduce E. coli in patients with hematological cancers

[0437] summary

[0438] Patients with hematological malignancies frequently develop bloodstream infections due to Escherichia coli and other bacteria translocation from the intestine. Antibiotic treatment to prevent these infections has a deleterious effect on the microbiome and immune tone, and is further hindered by increasing antibiotic resistance, particularly antibiotic resistance to fluoroquinolones. We screened a library of 162 wild-type (WT) phages and identified 8 phages with broad coverage against Escherichia coli. The selected phages were engineered with novel tail fibers and a CRISPR-Cas mechanism targeting clinically relevant Escherichia coli. Engineered phages target bacteria in biofilms, reduce the appearance of phage-tolerant Escherichia coli, and outperform their ancestral wild-type phages in co-culture experiments. SNIPR001 comprises four engineered bacterial phages with a broad host range across the Escherichia coli system, including multidrug-resistant strains. SNIPR001 is well tolerated in animals and better reduces the E. coli load in the mouse intestine than its constituent components. SNIPR001 represents a novel CRISPR-Cas therapeutic designed to selectively target Escherichia coli, which can cause fatal infections in patients with hematological cancers.

[0439] introduction

[0440] Cancer treatments are improving, and survival rates are increasing for people with hematologic malignancies. 1 However, chemotherapy regimens frequently used in this immunocompromised population cause myelosuppression and gastrointestinal mucositis with associated increased intestinal permeability. 2-4 Translocation of enteric bacteria, including Escherichia coli, from the gastrointestinal tract is a frequent cause of bloodstream infections. 5 .

[0441] Bloodstream infections caused by enteric bacteria such as E. coli are associated with a mortality rate of 15-20%. 6 Therefore, antimicrobial prophylaxis should be used in people at risk for febrile neutropenia. 7 There are no approved therapies for preventing bloodstream infections in patients with hematologic cancers, but two randomized trials demonstrated that they reduced bacterial infections during neutropenia. 7,8,9 , approved for off-label use in the U.S. In addition to the side effects of fluoroquinolones, which include safety warnings and precautions, bacterial resistance is on the rise in cancer patients and accounts for nearly 60% of E. coli bloodstream infections in the U.S. 10 In immunocompromised patients with hematological malignancies who develop chemotherapy-induced neutropenia, E. coli is responsible for 25.1–30% of all bacteremia cases. 11,12 In addition, in patients with hematological cancers undergoing hematopoietic stem cell transplantation, 5 As many as 65% of Escherichia coli isolated as the causative pathogen in the 13 Therefore, new narrow-spectrum prophylaxis options that also cover fluoroquinolone-resistant E. coli are needed to prevent infection in these susceptible patients.

[0442] Bacterial phage therapy was used before the widespread availability of antibiotics 14 However, due to the rise of bacterial antimicrobial resistance and the reports of several successful individual cases 16-18 , now with renewed interest 15 Despite this, few clinical trials with wild-type (WT) phage have been conducted. 19-22 , and although several trials have targeted E. coli, these have not been able to generate convincing results in larger randomized controlled trials, possibly due to incomplete coverage of the target strains by the phage cocktails. 23 The most recent effort involved targeting Klebsiella pneumoniae strains (n=17) 24Phages targeting Vibrio (n=41) and Vibrio strains (n=294) 25 More extensive characterization of phages (n=248) suggests that better coverage of target strains can be achieved with large-scale systematic screening. Synthetic biology was also used to engineer T3 phage tail fibers, expanding the spectrum of strains targeted by engineered phages. 26 Finally, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system can enhance efficacy by acting as a complementary killing mode to the lytic activity of bacteriophages. CRISPR-associated nucleases (Cas) and CRISPR-RNA form complexes that, in some CRISPR-Cas systems, can bind to cognate DNA target sequences and cause DNA degradation. 27,28 Since prokaryotes lack error-prone non-homologous end joining and rely solely on homologous recombination to repair DNA damage, they are prone to cell death following DNA degradation. This vulnerability is exploited by using CRISPR-Cas as an antibacterial modality for several bacteria, including Staphylococcus aureus, Escherichia coli, or Clostridium difficile. 29–35 .

[0443] To address the important unmet medical need for new preventive agents for patients with hematological malignancies, we report the development of SNIPR001. Our research process for designing SNIPR001 includes several steps (Figure 3). In short, a library of wild-type phages (n=162) was tested in vitro on a panel of phylogenetically diverse E. coli strains representing the biology of the target bacterium E. coli. The wild-type phages with the broadest and most complementary target strain coverage were selected for further engineering. The selected wild-type phages were subjected to both tail fiber engineering and CRISPR-Cas arming to create CRISPR-Cas armed phages ( C RISPR-Cas- A The CAP library was evaluated for manufacturability, in vitro stability, efficacy profile, in vivo pharmacokinetics, and efficacy. A combination of four CAPs was selected to create the development candidate SNIPR001, which has now entered clinical development (ClinicalTrials.gov ID NCT05277350).

[0444] result

[0445] Wild-type lytic phages α15, α17, α20, α48, and α51 (all members of the T-even virus subfamily) were the starting point for phage engineering to generate synthetic phage types (CRISPR-armed phages; CAPs) α15.2, α20.4, α48.4, or α51.5. Tail fiber specificities for the cognate E. coli surface ligands TSX, LPS, and LamB were as follows:

[0446] α15.2 binds LPS and Tsx

[0447] α20.4 binds LPS and LamB

[0448] α48.4 binds to Tsx

[0449] α51.5 binds to Tsx

[0450] A cocktail containing these phage types targeting LPS, LamB, and Tsx was prepared (the cocktail is referred to herein as SNR001).

[0451] Bacteriophages armed with CRISPR-Cas to target E. coli

[0452] To arm selected lytic phages with CRISPR-Cas and generate CAP libraries, E. coli 39 The IE type CRISPR-Cas system was engineered to target phylogenetically diverse Escherichia coli strains. A CRISPR-guided vector (CGV TM ), which contains the cas3 gene (ygcB) and the downstream cascade gene complex composed of casA (ygcL, cas8e), casB (ygcK, cas11), casC (ygcJ, cas7), casD (ygcI, cas5) and casE (ygcH, cas6), as well as a CRISPR array (CGV-EcCas) targeting multiple different genes of the Escherichia coli genome. In order to evaluate the killing efficiency of the CRISPR-Cas system, CGV-EcCas was conjugated to Escherichia coli strain b52, showing a 3.5 log reduction compared to the empty vector. 10 The average reduction in CFU / mL was 2.3%. As expected, no effect was observed after conjugating CGV-EcCas to non-target E. coli strains. The killing efficiency of CGV-EcCas was further evaluated on a simplified experimental panel of 82 E. coli strains. Conjugated delivery of the empty vector was completed in 75% of the isolates. For all strains in which CGV-EcCas was delivered, bacterial counts were reduced to below the limit of detection (LOD, 200 CFU / mL), corresponding to 1-6 log 10 , emphasizing the potent CRISPR-Cas-mediated killing ( Figure 3A ).

[0453] Except for promoter P bolA In addition, the CRISPR-Cas system was engineered to express from a synthetic, constitutively expressed E. coli promoter (P J23100 ) expression. CRISPR arrays were designed to target multiple virulence genes (spacers 1, 2, and 3) or essential genes (spacers 4 and 5) (SEQ ID NOs: 6-10), as targeting multiple regions has been shown to prevent the evolution of resistance. 43 To confirm CRISPR-Cas activity in CAPs, we used RT-qPCR to measure cas3 transcripts in samples obtained 5, 15, and 30 minutes after synchronous infection with CAPα15.2 at an equal MoI compared to wild-type α15, and observed elevated levels of cas3 RNA only after infection with CAPα15.2. Next, we extended this assay to all four CAPs (α15.2, α20.4, α48.4, α51.5) and confirmed elevated levels of cas3 transcripts, emphasizing that CAPs express the CRISPR-Cas system during infection of the target strain.

[0454] To demonstrate the competitive superiority of CAPs, we performed competition experiments in which CAPs (α20.4 and α15.2) and their wild-type progenitor phages were co-cultured with E. coli strain b230, which served as the target of the two competing phages. Approximately the initial ratio of 1 CAP to 9 wild-type phages was co-cultured on fresh target cells in liquid culture and passaged four times. After each passage, the relative abundance of CAP and wild-type phage particles was assessed. Both CAPs outperformed their wild-type counterparts within four rounds; CAP α20.4 reached 68% after four rounds, while CAP α15.2 reached 86% after two rounds (Figures 3B-C), demonstrating improved fitness compared to the wild-type phages.

[0455] CAP mixture SNIPR001

[0456] The activity of CAPs was tested against an E. coli panel (n=429) using a growth kinetics assay.To maximize our coverage, we combined CAPs to produce a composition (SNR001) containing phage types α15.2, α20.4, α48.4, and α51.5.

[0457] The ancestors of CAP α15.2, α20.4, α48.4, and α51.5 are classified under the subfamily Tevenvirinae. Specifically, ancestors α15, α48, α20, and α51 have sequence similarity with E. coli phages T2, T4, and RB69, as shown below (as determined by Mash analysis). Computer analysis of the genome of SNIPR001 showed that CAP does not encode known transposase or integrase genes, indicating that the phage is not temperate and is therefore predicted to be unable to insert its DNA into bacterial cells.

[0458] Similarity Matrix:

[0459]

[0460] This similarity was calculated using Mash (v1.1) with a k-mer size of 21 and a sketch size of 10 000 to calculate distances between genomes.

[0461] For further discussion of Mash, see Ondov, BD, Treangen, TJ, Melsted, P. et al., “Mash: fast genome and metagenome distance estimation using MinHash”, Genome Biol 17, 132 (2016). https: / / doi.org / 10.1186 / s13059-016-0997-x

[0462] SNIPR001 does not affect other gut-associated bacteria

[0463] Ideally, phage-based therapies should not disrupt non-targeted genera of the microbiome, so the specificity of SNIPR001 against E. coli was assessed by investigating its effects on a panel of strains that included non-E. coli species that are relatives of E. coli, as well as a range of families associated with commensal bacterial communities in the gut (as well as E. coli as a positive control). Bacteria were cultured without CAP, with a mixture of SNIPR001, or with individual SNIPR001 CAPs (n=4). Growth in CFU / mL (DCFU / mL) was assessed over a 4-hour period. 4h-0h In parallel, E. coli b2480 was grown under the same conditions as a positive control ( Figure 4We did not observe a significant effect of the SNIPR001 cocktail or any of the SNIPR001 CAPs on non-E. coli strains (p > 0.05, Student's t-test, FDR corrected by Holm's method), whereas the growth of E. coli was significantly inhibited (p < 0.05, Student's t-test, FDR corrected by Holm's method). Therefore, SNIPR001 is not expected to affect target bacteria other than E. coli of the gut microbiome.

[0464] In vitro host range of SNIPR001 in clinical target populations

[0465] In order to understand the potential effects in strains associated with hematological cancer patients, the coverage of SNIPR001 was tested for our internal E. coli experimental group (429 strains) and 382 clinical E. coli strain groups (JMI Laboratories, North Liberty, IA, USA). These JMI strains are derived from patients with bloodstream infections hospitalized in hematological oncology departments across four different regions from 2018 to 2020 (54 isolates in the Asia-Pacific region, 161 isolates in Europe, 26 isolates in Latin America, and 141 isolates in North America). The genotype distribution of the E. coli strains in the patient population was determined using whole genome sequencing, and it was found that the genotype distribution was diverse, representing 9 phylogenetic groups and 118 multilocus sequence types (MLST) ( Figure 5A ). Using the spot assay, we documented phage infectivity against the panel of JMI strains. In cases where individual plaques could not be verified, visible individual plaques were distinguished from lysis zones. All spot assays were run in duplicate. We observed 90.4 ± 1.6% total coverage of SNIPR001 across the 382 JMI E. coli panel and 95.6 ± 0.3% total coverage of SNIPR001 on the internal E. coli panel (429 strains). In addition, we observed plaques in 53.1 ± 7.7% of the JMI panel strains and lysis zones in 37.3 ± 6.1% of the JMI panel strains, and similarly plaques in 60.5 ± 6.6% of the internal panel strains and lysis zones in 35.1 ± 6.3% of the internal panel strains ( Figure 5B ). SNIPR001 showed 100% coverage in the B2 phylogenetic group, representing 53% of the JMI panel. Furthermore, we observed that SNIPR001 covered 91.7% (n=55) of the The strains were classified as multidrug-resistant, 100% (n=5) of carbapenem-resistant strains, 92.2% (n=95) of extended-spectrum β-lactamase-producing strains and 88.9% (n=176) of strains resistant to fluoroquinolones such as ciprofloxacin and levofloxacin Strains ( Figure 5C ).

[0466] Finally, we validated SNIPR001 on a clinical panel (n=72) of fluoroquinolone-resistant E. coli strains isolated from fecal samples or perianal swabs from patients with hematological cancers. This population represents the intended clinical target patient population being pursued (SNIPR001 was designated accelerated approval status by the FDA). A subset of these strains developed bloodstream infections ( Figure 5D ). 82% of E. coli strains (n=72) were susceptible to at least two or more CAPs in SNIPR001, and 93% of the strains were susceptible to the entire SNIPR001 mixture ( Figure 5E ). These data confirm that SNIPR001 interacts with individual CAPs. Than regarding the benefits of improving efficacy spectrum.

[0467] Tolerability and gastrointestinal recovery of SNIPR001 in miniature pigs

[0468] The tolerability and gastrointestinal recovery of SNIPR001 were evaluated in Göttingen minipigs. 12 Following oral administration of PFU of SNIPR001 or vehicle, blood and feces were sampled for 7 days. No CAP was recovered from plasma, indicating no systemic exposure, whereas CAP was recovered in feces up to 7 days after SNIPR001 administration, with a peak of 2 x 10 7 PFU( Figure 6A ). Minipigs showed no clinical signs and no significant changes in hematological or biochemical parameters were observed compared to vehicle treatment, specifically, no changes were seen in any immune cells, supporting that SNIPR001 was well tolerated. Similar recoveries were obtained with individual CAPs ( Figure 6B ). In conclusion, SNIPR001 seems to It was well tolerated in Göttingen minipigs with gastrointestinal recycling.

[0469] Efficacy of SNIPR001 and constitutive CAP in a mouse colonization model

[0470] To evaluate the in vivo efficacy of the four selected CAPs in reducing E. coli, we adapted the method from Galtier et al. 44 The mouse intestinal colonization model was used for E. coli strain b17. Streptomycin was administered for 3 days to reduce Gram-negative bacteria from the mouse gastrointestinal tract, after which streptomycin administration was stopped and the animals were inoculated with E. coli b17 (1×10 7The mice were orally inoculated once with 100 CFU (1000 IU / mL) of CAP. This allowed for 3 to 4 days of stable colonization. The goal was to evaluate the efficacy of CAP on established colonization, with treatment starting 2 days after inoculation and the study terminated on day 4 after inoculation as colonization began to decline. To ensure maximum exposure to CAP, mice were treated with 3 daily doses administered 8 hours apart, for a total of 6 doses over 2 days.

[0471] High, medium or low doses (2 x 10 11 PFU, 2x 10 9 PFU, 1x10 7 Mice were treated with SNIPR001 (PFU), vehicle (negative control), or gentamicin (positive control). CAP recovery in feces ranged from 3 x 10 7 PFU / g to 1x 10 in high doses 10 PFU / g, which confirmed successful gastrointestinal passage ( Figure 6C After 24 hours of treatment (day 3), the levels of these CAPs were associated with a significant (p < 0.05, Mann-Whitney U test, FDR corrected) dose-dependent reduction in the target E. coli population compared to vehicle-treated mice. At the high dose, SNIPR001 caused a 4 log 10 CFU / g reduction ( Figure 6D ). Although there was an increase in variability in bacterial recovery on day 4, likely due to clearance of the colonizing strain as illustrated in the vehicle group, a similar reduction was observed after 2 days of treatment (day 4). Although the middle dose did not reach statistical significance (p < 0.05, Mann-Whitney U test), there was a numerical reduction compared to the vehicle group. Subsequently, the efficacy of individual CAPs was compared to the SNIPR001 mixture in this model. In this experiment, a greater reduction in colonization of the target strain was observed with SNIPR001 compared to any of the single CAPs (which showed a numerical but not statistically significant reduction), emphasizing the benefit from the efficacy of the combination ( Figure 6E We also determined the resistance profiles of randomly sampled surviving bacteria and found no isolates resistant to the SNIPR001 cocktail. In short, This data demonstrates the ability of SNIPR001 to reduce target E. coli in the gastrointestinal tract of colonized mice.

[0472] discuss

[0473] Here we describe the development of SNIPR001, designed to target enteric Escherichia coli, which frequently translocates in the bloodstream to cause bloodstream infections in neutropenic patients with hematological cancers. Although fluoroquinolones are being approved for off-label use, these patients continue to have high morbidity and mortality. Over the past century, the use of traditional antibiotics has produced important health benefits. However, in parallel, we are now experiencing significant development of bacterial resistance, and the number of deaths attributable to bacterial antimicrobial resistance in 2019 was estimated to be 1.27 million, with E. coli being the main pathogen. 45 Therefore, new antibiotic modalities are needed to address the unmet medical needs of antimicrobial-resistant infections in this vulnerable population. In this study, we describe the development of SNIPR001, a novel development candidate with the potential to address these challenges.

[0474] The demonstration of SNIPR001's efficacy in a large and clinically relevant panel of strains supports the clinical potential of SNIPR001. The 4 log 10 The reduction in E. coli was compared with previous studies 4950 definite improvement.

[0475] SNIPR001 represents an orthogonal antibacterial approach as it demonstrates activity against multidrug-resistant strains. Furthermore, there is growing evidence that maintaining a normal microbiome is important for maintaining immune tone and potentially beneficial for cancer treatment outcomes. 51 , and this is also recognized in the latest guidelines for the prophylactic management of patients at risk for febrile neutropenia 7 In this context, in vitro studies with SNIPR001 demonstrated specificity against E. coli without off-target effects against any non-E. coli strains tested, resulting in fewer deleterious effects on the microbiome. In the future, personalized combinations of narrow-spectrum antibiotics such as SNIPR001 could be used first-line, rather than as an add-on to broad-spectrum antibiotics such as fluoroquinolones.

[0476] A clinical study evaluating SNIPR001's ability to ascertain safety and its ability to reduce E. coli in the gut without perturbing the overall gut microbiome is currently underway in the United States (NCT05277350). We believe SNIPR001 exemplifies a potentially significant therapeutic advance in the field of antimicrobial agents for high-risk patient populations and may serve as a blueprint for narrow-spectrum therapies against other life-threatening antimicrobial-resistant pathogens in high-risk patient populations.

[0477] Table 1a: Overview of the four CAPs that comprise SNIPR001. E. coli genes targeted by the five individual spacers The genes and sequences are listed in Table 1b.

[0478] α15.2 contains spacer regions 1, 2, and 3

[0479] α20.4 contains spacer regions 4 and 5

[0480] α48.4 contains spacer regions 4 and 5

[0481] α51.5 contains spacer regions 4 and 5

[0482] Table 1b: E. coli genes targeted by the five individual spacers contained in the SNR001 CAP, and the spacer sequences used in the CAP.

[0483]

[0484]

[0485] method

[0486] Phage isolation was performed using a panel of E. coli strains. Briefly, 100 μL of an overnight culture of each E. coli strain was mixed with 100 μL of each phage mix or wastewater sample. After a 6-minute incubation at room temperature (during which infection should have occurred), 3 mL of a Ca-containing 2+ coli / phage or wastewater mixture and immediately poured onto LB plates. Alternatively, 10-fold dilutions of each mixture were spotted onto a lawn prepared with the isolated strain. After drying, the plates were incubated at 37°C overnight. Plaques were picked from each plate and resuspended in 500 μL of SM buffer, vortexed and stored at 4°C. The ten-fold dilutions were spotted onto the isolated strain from which the plaques were originally picked. In order to increase the probability of obtaining plaques corresponding to a single phage, the procedure was repeated at least three times. Lysates were prepared from the single plaques picked in the previous round of propagation, DNA was extracted and their genomes were sequenced.

[0487] Escherichia coli experimental panel and isolation procedure

[0488] Three E. coli panels, one internal panel, and two clinically relevant panels were included in this study. The internal panel consisted of 429 phylogenetically diverse E. coli strains isolated from the blood of patients with bloodstream and urinary tract infections, from feces of humans without known illnesses, and from animals and the environment. The strains covered seven different phylogenetic groups (A, B1, B2, C, D, E, F), 114 multilocus sequence typing (MLST) groups, serotypes (K and O), antibiotic resistance profiles, and diverse geographic locations of isolation.

[0489] The JMI panel consisted of a clinical collection of 382 E. coli strains obtained from JMI Laboratories (North Liberty, IA, USA). These strains were isolated from patients with bloodstream infections hospitalized in hematology and oncology departments across four different regions (54 isolates in the Asia-Pacific region, 161 isolates in Europe, 26 isolates in Latin America, and 141 isolates in North America) and were derived from the SENTRY Antimicrobial Surveillance Program (2018-2020), a network of more than 150 medical centers in more than 28 countries worldwide (https: / / www.jmilabs.com / sentry-surveillance-program).

[0490] Finally, a panel of 72 fluoroquinolone-resistant Escherichia coli strains was isolated from fecal samples or perianal swabs of patients with hematologic cancers hospitalized for hematopoietic cell transplantation. 53,54 .

[0491] E. coli strains were cultured in lysogeny broth (LB) at 37°C at 250 rpm in liquid medium or on agar plates containing 1.5% (w / v) agar. Cultures were supplemented with ampicillin (100 μg / mL), kanamycin (50 μg / mL), gentamicin (15 μg / mL), or amikacin (50 μg / mL) as necessary. 55 All media for the growth of β-lactamase and its derivatives were supplemented with 1,6-diaminopimelate (DAP) (80 μg / mL) to compensate for their auxotrophic deficiencies.

[0492] E. coli strain b52 used to produce α15.2, α48.4, and α51.5, and E. coli strain b2479 selected to produce α20.4 both belong to phylogenetic group A. Strain E. coli bl7 was used as the colonizing strain in the in vivo efficacy model because it is susceptible to all SNIPR001 CAPs and is part of the SNIPR Biome strain library.

[0493] Phage screening by growth kinetics

[0494] The in vitro susceptibility of an in-house E. coli panel (n=429) to 162 wild-type phages was evaluated using a growth kinetics assay. The assay measures the metabolic activity of bacteria by following the reduction of a tetrazolium dye to a purple compound that accumulates during bacterial growth. (Biolog, Hayward, CA, USA)—Adapted from Henry et al., 2012 56 Colorimetric readings were recorded every 15 minutes over a 24-hour period. The inhibition area under the curve (iAUC) was calculated from the kinetic curves over the course of the experiment and was defined as the ratio between the growth curve of the phage-treated bacteria and the normalized AUC of the bacteria-only control. Susceptibility was defined as an iAUC value ≥ 0.2.

[0495] Calculation of bacterial growth inhibition using iAUC

[0496] Use The growth kinetics curve constructed by the device was used to determine the growth inhibitory effect of SNIPR001. In order to limit the technical variability of the measurements between time points, a cubic smoothing spline function was applied to the data in Scala using the "umontreal.ssj.functionfit" package. In order to identify appropriate ρ and weight variables, ρ and each combination of weights 0.1 and 0.5 were applied in 0.1 increments (i.e., 0.1, 0.2, ... 0.5). The spline with the lowest mean absolute error was selected for AUC calculation. The initial cumulative amount of fluorescent dye at the initial time point was slightly different between the wells, resulting in artificial expansion of the AUC of some wells. Using the best smoothed square spline, the average signal of the first 1.5 hours was removed from all growth curves before any measurable growth to approximate the zero growth signal intercept. The total incremental AUC (iAUC) was calculated as the sum of the Riemann midpoint sums at each time point along the smoothed square spline. Finally, we calculated iAUC as iAUC=1-AUC 样品 AUC 对照 , where AUC 样品 is the AUC of the spline created by the given bacteria and SNIPR001, and AUC 对照 iAUC refers to the AUC of a sample created with a given bacterium without a given phage or CAP, or a combination of these. Thus, iAUC values ​​typically lie between 0 and 1, with 0 indicating no growth inhibition and 1 indicating complete growth inhibition. Some biological and technical noise does occasionally result in iAUC values ​​outside these limits, but this is considered negligible.

[0497] Host range was calculated as the fraction of panels with iAUC < 0.2 for each replicate. The reported standard deviation was calculated as the deviation in the number of strains with iAUC < 0.2 and then normalized for panel size by dividing the standard deviation by the panel size.

[0498] CRISPR-Cas armed bacteriophages to target Escherichia coli

[0499] Phage is armed with CRISPR-Cas by using homologous recombination. When compared with the reference wild-type T2 phage, we inserted the payload into the region immediately after gene 49 and towards gene E. Thus, the synthetic phage genome contains an insertion between coordinates 9000 and 21000, wherein when compared with the reference wild-type T2 phage, the coordinates are nucleotide positions counted from the nucleotide immediately after gene 49 (coordinate number 1) towards gene E. Recombination is carried out in bacterial cells during phage propagation. The cells carry a plasmid that serves as a recombination template. The recombination template plasmid carries a sequence whose goal is to insert into the phage genome between 200-700bp of sequence flanks homologous to the phage sequence at the insertion site. For each phage, we inserted the endogenous IE type CRISPR-Cas system of E. coli (Genbank CP032679.1), namely the cas3 gene (ygcB) and the downstream genes encoding the Cascade complex, casA (ygcL), casB (ygcK), casC (ygcJ), casD (ygcI), and casE (ygcH), as well as a CRISPR array targeting selected E. coli sequences. For all selected CAPs, the cas genes derived from E. coli were the same. The insertion of the CRISPR-Cas system caused a ~7 kbp deletion of phage DNA in gene 49–gene E. The sequence of the resulting CAP was verified by NGS (BaseClear, Leiden, The Netherlands).

[0500] CGV transduction in biofilms

[0501] Escherichia coli b52 cells were grown in 96-well plates and biofilms were allowed to develop on peg lids. Each well contained 180 μL of M9 culture medium (Sigma, M6030), which was supplemented with 20 mM glucose, 2 mM MgSO 4 , 0.1 mM CaCl 2 , 0.1% casein acid hydrolysate (Amicase) (Sigma) and 0.1% mannitol. The wells were inoculated with 1 μL of overnight b52 culture. The peg lid was inserted and the microtiter plate was left to incubate at 37° C. for 24 hours. Next, the peg lid was transferred to a new plate with fresh culture medium without washing, and the plate was incubated for an additional 24 hours. After incubation, a 100 μL culture medium and 100 μL of CGV conductive particles (˜10 8 The biofilms grown on the nails were rinsed three times in sterile H2O (200 μl) and then transferred to new plates. The plates were incubated at 37°C for 5 hours.

[0502] To determine the metabolic activity of cells in the biofilm, the coverslips were rinsed three times in sterile HO (200 μL) and then placed in plates containing 20 μL of Alamarblue stain (ThermoFisher) and 180 μL of culture medium in each well. The plates were incubated at 37°C for 1.5 hours and moved to a microplate reader (Synergy H1, Biotek). Fluorescence (excitation: 560 nm; emission 590 nm) and absorbance (600 nm) were recorded for each well.

[0503] Compared to the metabolic activity of biofilms treated with CGV without a promoter for transcription of cas genes, we report that biofilms treated with CGV carrying a promoter P bolA Metabolic activity of CGV-treated biofilms.

[0504] Plasmid and strain construction

[0505] Plasmids were constructed by InFusion HD cloning using PCR-generated DNA fragments. To construct CGV-EcCas, the cas3 and cascade genes from E. coli were amplified and cloned into the ColE1-type plasmid pZE21. 57 In addition, a three-spacer array targeting genes in E. coli is included in the vector under the control of the constitutive promoter J23100. The array contains nucleotides / target loci from the E. coli genome separated by direct repeats (repeat sequence, SEQ ID NO: 15). The protospacer adjacent motif (PAM) is positioned adjacent to the selected target sequence in the E. coli genome.

[0506] Transformation assay

[0507] Overnight cultures were diluted (1:100) in fresh LB medium and grown to mid-exponential phase (OD 600 ≈0.6). Subsequently, cells were prepared for electroporation and concentrated 50-fold in ice-cold MilliQ water. Cells were then electroporated with the appropriate plasmid, allowed to recover in SOB (super optimal broth) at 37°C for 1 hour, and plated onto LB plates supplemented with antibiotics.

[0508] Conjugation assay

[0509] Using E. coli JKE201 as donor and E. coli clinical isolates as recipients (including target and non-target E. coli strains as controls), a conjugation experiment was established to evaluate the transfer and killing efficiency of CGV-EcCas. The plasmid was conjugated into the E. coli recipient by liquid conjugation. Briefly, the overnight culture was diluted (1:100) in fresh LB medium and grown to an OD of600 ≈0.4, washed and suspended in fresh LB to OD 600 ≈0.25. 125 μl of donor and 25 μl of recipient cell suspension were mixed for 5: 1 conjugation in 96-well microplates and incubated at 37 ° C for 16 hours. Conjugation efficiency was determined by plating a dilution series of the conjugation reaction onto LB agar supplemented with antibiotics (to select transconjugants). Specific killing efficiency was quantified by plating 90 μL of the conjugation reaction onto a selection plate. The CGV-EcCas plasmid encodes kanamycin, gentamicin, and amikacin resistance to enable selection of transconjugants. Viability was calculated by counting the CFU on the plate, and the data were recorded as viable cell concentration (CFU / mL).

[0510] Synchronous CAP infection and Cas3 expression assay

[0511] The overnight culture of the test strain in LB is diluted 100 times and incubated to the stationary phase at 37 ℃ with shaking in LB, and 10-mL aliquots are divided into 50-mL falcon tubes subsequently. Each aliquot is then inoculated with the high titer lysate of the individual CAP of 50 μ L, and incubation is continued under the same conditions. In addition, the simulation 10mL LB volume of each CAP is also inoculated with the CAP lysate of 50 μ L, and is used for 0 minute phage counting. After inoculation, 5 minutes, 15 minutes and 30 minutes, aliquots are collected and used for total RNA extraction and phage counting. The phage counting aliquot is syringe filtered (0.2 μ m, Sartorious, The total RNA was extracted using a 1 mL aliquot of the culture medium at 13.3 k × g for 15 seconds and the supernatant was discarded. The pellet was then immediately resuspended in a cold RNA Later (Thermo Fischer Scientific, AM7020) and stored at -20°C until extraction. Following the manufacturer's protocol for extracting RNA from bacteria, total RNA was extracted using a GeneElute total RNA kit (Sigma-Aldrich, St. Louis, MO, USA). After the first elution, 1 μL of DNase I (1 U / μL) was added and incubated overnight at 37°C. The reaction was terminated by incubation at 70°C for 15 minutes. The RNA was repurified on a GeneElute column and eluted in 35 μL of the kit elution buffer. Total RNA concentration is estimated on NanoDrop instrument (Thermo Scientific, One / OneC), and 0.5 to 2 μ g of RNA is added to cDNA synthetic reaction, which contains SuperScriptIII RT enzyme (ThermoFisher, Waltham, MA, USA) and random decamer to initiate synthesis in 20-μ L reaction volume. The cDNA reaction is diluted to 100 μ L in water. Using 5 μ L of the cDNA as template, 10 μ L of Power SYBR Green PCR Master mix (Thermo Fisher) and the various PCR primers of 0.2 μ M, real-time PCR is carried out in triplicate. Use the standard two-step thermal cycling scheme for Power SYBR Green PCR Master Mix with 60 ℃ of annealing / extensions, PCR is carried out on AB QuantStudio5 system (Applied Biosystems, Foster City, CA, USA). The forward and reverse primers for gapA (reference gene) were 5′-cgctaacttcgacaaatatgctggc-3′ (SEQ ID NO: 17) and 5′-aggacgggatgatgttctgggaa-3′ (SEQ ID NO: 18), and the forward and reverse primers for cas3 were 5′-caagtatgctaccaacggctaaag-3′ (SEQ ID NO: 19) and 5′-ccaatcaaaatcaacgtcgagtga-3′ (SEQ ID NO: 20). Single PCR products from these primer pairs were confirmed by melting curve analysis.Relative levels of transcripts were estimated using 10-fold dilutions of purified PCR products as standards, and values ​​are expressed as the ratio of cas3 / gapA transcripts.

[0512] Phage competition assay

[0513] The lysates of the two phages were mixed at a 9:1 (WT:CAP) ratio, and the phage mixture was added to 10 ml of 2xYT medium containing 10 mM CaCl and 20 mM MgCl and 100 μL of overnight E. coli strain b230, which served as the target of the two competing phages. After a 2-hour incubation in a 37°C shaking incubator, the culture was centrifuged and 1 μL of the supernatant was added to a new b230 culture. The same procedure was repeated twice.

[0514] The ratio of phage was assessed by PCR performed with three primers to obtain two specific products, one for wild-type phage and one for CAP. PCR products were separated on 1% agarose gels, and DNA bands were stained with SYBRsafe and visualized and quantified by the ChemiDoc XRS+ system (model 1708265, Biorad). The background-corrected intensity of the band corresponding to the wild-type phage was divided by the intensity of the band corresponding to CAP in the same lane to obtain the ratio of the two band intensities (WT / CAP). The CAP score compared to the total phage content (WT+CAP) was determined based on a calibration curve made by using a set of different mixtures of two phages and fitting the curve to the measured band intensity ratios (WT / CAP). The estimated error of the reported values ​​is less than 20%.

[0515] Fungal lawn killing assay

[0516] Adjust the overnight culture of the test strain in LB to 10 9 CFU / mL. Mix 100 μL aliquot of the CFU / mL adjusted strain with 100 μL of 10 9 PFU / mL was mixed to achieve a multiplicity of infection of 1 for CAPα15.2 or wild-type α15 in a 15 mL falcon tube, mixed with 3 mL of molten and pre-tempered top agar, and plated onto LB plates. After the lawn solidified, the plates were incubated overnight at 37°C, and the total number of surviving colonies was counted the next day for the CAPα15.2 or wild-type α15 groups. The assay was performed as independent biological replicates, with each experiment containing ten technical replicates. Statistical significance was established using two replicates using the Mann-Whitney U test.

[0517] Universal transduction assay

[0518] The transduction capacity of various CAPs was evaluated by a universal transduction assay. Briefly, transduction lysates were prepared by propagating various CAPs on E. coli MG1655 lamB::Cm. This strain was modified from wild-type MG1655 (Cat. No. 700926, American Type Culture Collection, Manassas, VA, USA) to carry the chloramphenicol selection marker. The experiments were performed with a well-characterized lytic T4 phage (negative control) and its transduction mutant T4GT7. 58 After this step, the cells were transduced with various lysates at an OD of 0.3 at MoIs of 0.5, 0.1, and 0.01. 600 Wild-type E. coli MG1655 strain was infected and plated on LB plates containing chloramphenicol. The next day, the number of transduced colonies was recorded for each CAP, control, and different MOIs. The transduction frequency was calculated as the number of transduced colonies divided by the titer of the transduced lysate.

[0519] Sequence analysis of CAP

[0520] Using the database (Table 2), the sequence of individual SNIPR001 CAP was analyzed for the presence of antibiotic resistance, virulence genes and lysogeny-related genes (transposase and integrase). In addition, phage samples were analyzed using whole genome sequencing. This typically results in a coverage of >1000x for the entire phage genome. Assembly was performed by downsampling the data to 1000x average coverage of the phage and assembling using SKESA. In order to detect differences between samples and to detect non-majority mutations, the original reads were mapped back to the assembly using BWA (version 0.7.17).

[0521] Table 2: List of databases used to analyze SNIPR001 CAP sequences

[0522]

[0523] Phage specificity determination using liquid killing assay

[0524] SNIPR001 CAP (α15.2, α20.4, α48.4, and α51.5) and SNIPR001 killing specificity were evaluated via biopotency assays against a panel of human-relevant aerobic (n=6) and anaerobic (n=3) bacterial strains. E. coli strain b2480 was included as a positive control for phage-mediated killing (Table 3).

[0525] Briefly, overnight cultures were adjusted to 10 6CFU / mL. Before incubation for 4 hours, SNIPR001 CAP or SNIPR001 (wherein various CAPs were combined in equal proportions) was added at an MOI of 1. Untreated bacteria were cultured in parallel as a control for bacterial growth. CFU counts were recorded at 0 and 4 hours after phage treatment, and data were expressed as Δlog by subtracting the initial inoculum (0 hour) from the assay endpoint CFU / mL (4 hours). 10 CFU / mL.

[0526] Table 3: Panel of bacterial strains tested via biopotency assay (aerobic: n=6, anaerobic: n=3, Aerobic / Anaerobic: n=1), Gram type classification, growth conditions and source / ID are shown

[0527]

[0528]

[0529] Spotting determination and Efficiency of Spotting (EoP)

[0530] To count phage titers, phage lysates or equal volume mixtures of SNIPR001 CAP were serially diluted 10-fold in SM buffer or PBS, respectively. 100 μL or 300 μL of overnight bacterial culture was added to 3 mL or 10 mL of 0.5% top agar (containing Ca 2+ and Mg 2+ ), vortexed briefly, and poured onto round or square LB plates to prepare a bacterial lawn. 5 μl of a dilution series of the test phage was then spotted onto the lawn and allowed to dry uncovered at room temperature before incubation at 37°C overnight. Strains b52, b2479, and b17 served as controls and were included in each round of the assay.

[0531] The following day, the results were evaluated (Table 4). In this assay, susceptible strains were defined as strains that produced plaques that could be counted in PFU / mL and strains that had no visible plaques but showed impaired bacterial growth (i.e., zones of lysis). Coverage defined the percentage of the total number of susceptible strains. Images of all plates were recorded. Graphs illustrating plaque coverage results were first log titer. 10 The transformations were performed and then the standard deviation and mean values ​​were subsequently calculated.The clinical panel and control strains were tested in two independent experiments.

[0532] Table 4. Criteria used to evaluate the results of the spot assay and to follow the standard definition of strain susceptibility. 46,59

[0533]

[0534] Animals and husbandry

[0535] Mouse studies used females from Charles River (Freiburg, Germany). IGS mice (approximately 6-7 weeks of age upon arrival) were used. Animals were housed in groups of 3 to 5 mice per cage in a climate-controlled room (temperature 20–23°C; relative humidity 30–70%) under a 12:12 h light-dark cycle (lighting on 07:00–19:00). Standard pelleted chow and tap water were available ad libitum. Animals were allowed at least 7 days of acclimatization before the start of the experimental procedures. 30 female Gottingen miniature pigs (approximately 4-7 months old when arriving) from minipigs A / S, Denmark were used for tolerance and kinetic studies. Before the experiment began, animals were allowed to have an adaptation period of at least 14 days. Pigs were grouped in 2 to 3 animals and given a standard pig diet twice a day, and tap water was available at will. All procedures were carried out in accordance with the guidelines of the Danish Animal Experiments Inspectorate from the Ministry of Environment and Food of Denmark and in accordance with institutional license (BioAdvice, Animal License No. 2015-15-0201-00540).

[0536] Mouse intestinal colonization model

[0537] The mouse intestinal colonization model was adapted from Galtier et al. (2016) 44 Briefly, 3 days before inoculation with E. coli b17, pre-treatment with streptomycin (5 g / L) in drinking water was given to reduce the level of natural bacteria. On day 0, 3 x 10 7 coli b17 inoculum of CFU and administered to all mice by oral gavage at 0.25 mL.

[0538] Treatment was administered three times daily for two days starting two days after inoculation. Just before each administration, the four CAPs were mixed in a ratio of 1:1:1:1 to form high, medium or low concentrations of SNIPR001 to give 2 x 10 11 , 2x 10 9 and 1x 10 7 Dose Levels of PFU. At the time of treatment, mice were administered 0.1 mL of 10% sodium bicarbonate by oral gavage, followed by oral administration of 0.3 mL of SNIPR001, saline (vehicle), or 43.5 mg / kg gentamicin.

[0539] CAP recovery and tolerance studies

[0540] Before SNIPR001 or single CAP administration, Göttingen minipigs were first given a mixture of antibiotics including neomycin (60 mg / kg, orally, once daily for 4 days) and cefquinome (2 mg / kg, intramuscularly once daily for 3 days) to reduce the level of Gram-negative bacteria in the gastrointestinal tract and thus limit phage replication. The animals were then fasted overnight and lightly sedated, and then, after oral administration of 50 mL of 10% sodium bicarbonate, 2 x 10 in 100 mL were added. 12 PFU were orally administered once with a single CAP or SNIPR001 mixture. Fecal samples were collected daily for CAP quantification by plaque assay. In addition, for tolerance studies, blood samples were collected for hematology and blood chemistry analysis, including C-reactive protein and plaque assay. Animals were closely monitored after SNIPR001 administration, and their body temperatures were recorded regularly.

[0541] Quantification of Escherichia coli b17 and CAP in feces

[0542] The fecal samples were homogenized and serially diluted in SM buffer. 10 μl of each dilution in triplicate was then spotted onto McConkey agar plates (Sigma, M7408) supplemented with streptomycin (1 mg / mL) and incubated at 37°C for 12 to 16 hours for E. coli counts.

[0543] Plaque assays were performed to enumerate CAP in fecal samples. Briefly, homogenized samples were centrifuged at 10,000 g for 10 minutes, and the supernatant was serially diluted. Triplicate 10 μl of each dilution was spotted onto an E. coli b17 overlay and incubated at 37°C for 12 to 16 hours.

[0544] To quantify the presence of resistant bacteria in vivo, three colonies from each mouse fecal sample in the medium dose group were selected from MacConkey agar plates at three different time points. The colonies were incubated in LB broth at 37°C for 12 to 16 hours and used to prepare a top agar overlay on the LB agar plate. The plate was then dried on a LAF bench for 15 minutes. The SNIPR001 mixture and the four individual CAPs were used as a culture medium from 1 x 10 5 A dilution series of the PUF / mL stock solution was spotted. As a control, a top agar overlay of the colonizing strain E. coli b17 was spotted in the same manner. The plates were left uncovered on a LAF bench to air dry and then incubated upside down at 37°C for 12 to 16 hours.

[0545] Whole genome sequencing of Escherichia coli strains from JMI

[0546] In the robot KingFisher TM Flex magnetic particle processor (Kingfisher TM Total genomic DNA was extracted and purified using the KingFisher Cell and Tissue DNA Kit (Thermo Scientific, Waltham, MA, USA) in a Flex Magnetic Particle Processor (Thermo Scientific) workstation.

[0547] Total genomic DNA was used as input for library construction. DNA libraries were prepared using Nextera XT TM The library construction protocol and indexing kit (Illumina, San Diego, CA, USA) were prepared and sequenced (600 cycles) on a MiSeq sequencer (Illumina) using the MiSeq reagent kit v3.

[0548] Resistance phenotype definition

[0549] Extended-spectrum β-lactamase (ESBL) phenotype against Escherichia coli was defined as a minimum inhibitory concentration (MIC) value of ceftriaxone, ceftazidime, and / or aztreonam ≥ 2 mg / L ( https: / / clsi.org / ).

[0550] Carbapenem-resistant Enterobacteriales (CRE) were defined as any isolate displaying imipenem, doripenem, and / or meropenem resistance with an MIC > 2 mg / L (https: / / clsi.org / ).

[0551] Assembly of whole-genome sequencing data

[0552] Using Trimmomatic 60 (Version 0.39) Trim raw sequencing reads with the settings "LEADING:3TRAILING:3SLIDINGWINDOW:4:15MINLEN:36". Use SPAdes 61 Trimmed reads were assembled using the default settings of the ELISA (version 3.14.1). Contigs shorter than 500 bp or with a sequencing depth lower than 2x were removed from the final assembly.

[0553] Comparative genomics approaches for clinical Escherichia coli strains

[0554] Using MLST2 62The assembled genome of Escherichia coli was subjected to multilocus sequence typing (MLST) using default settings, where the MLST database was downloaded from the MLST2 database (https: / / bitbucket.org / genomicepidemiology / mlst_db / src / master / ) on July 1, 2021. Clermon Typing was used. 63 The assembled E. coli genome was classified into phylogenetic groups using default settings using MASH with a k-mer size of 21 and 10,000 drafts / genome. 64 A distance matrix was generated for phylogenetic tree construction. The sketches were then compared in a pairwise manner to create the MASH distance to create a distance matrix for the E. coli genome.

[0555] Phage synteny analysis

[0556] In order to generate synteny graph, the wild-type sequence of the four kinds of phages included in the final mixture of RAST annotation was added to two closely related and well-known reference phages (RB69 AY303349.1 and T2NC_054931.1) to extract the protein sequence of prediction. Use tblastn (v2.12.0) to query all protein sequences of each phage for all other phage genomes, with an E value intercept of 1e-10. Then use custom Python script (see data availability) to generate synteny graph using drawSvg library (v 1.9.0). Described figure shows phage genome in similarity order and all tblastn hits are presented as the synteny block of adding shade by their protein identity. The protein manual classification of two reference phages is as belonging to each functional group " DNA metabolism ", " structure " or " other ", and is colored accordingly.

[0557] Data processing and visualization

[0558] Figures and key statistics were generated using R version 4.1.0. The following packages were used for figure generation: RcolorBrewer v.1.1-2, ape v.5.5, ggsignif v.0.6.2, ggpubr v.0.4.0, matrixStats 0.59, reshape2 v.1.4.4, ggimage v.0.3.0, here v.1.0.1, purr v.0.3.4, ggtree 65v.3.0.2, systemfontsv.1.0.2, Cairo v.1.5-12.2, cowplot v.1.1.1, reaxxl v.1.3.1, and ggplot2 v.3.3.3. The mean and standard deviation are calculated after converting the values ​​to the scale displayed on a given plot, e.g. when using log 10 When the scale is set, the mean and standard deviation are in log 10 Calculation is performed after conversion.

[0559] Data availability

[0560] The data and results generated during this study are deposited in https: / / github.com / sniprbiome / SNIPR001_paper The phage genome sequence was deposited in Genbank under accession number OQ067373-76

[0561] Code availability

[0562] All code required to produce this study is available at https: / / github.com / sniprbiome / SNIPR001_ paper get.

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[0631] sequence

[0632] Amino acid sequences are written in N-terminal to C-terminal direction, and DNA sequences are written in 5' to 3' direction.

[0633] protein

[0634] >LamB (SEQ ID NO: 1)

[0635] MMITLRKLPLAVAVAAGVMSAQAMAVDFHGYARSGIGWTGSGGEQQCFQTTGAQSKYRLGNECETYAELKLGQEVWKEGDKSFYFDTNVAYSVAQQNDWEATDPAFREANVQGKNLIEWLPGSTIWAGKRFYQRHDVHMIDFYYWDISGPGAGLENIDVGFGKLSLAATRSSEAGGSSSFASNNIYDYTNETANDVFDVRLAQMEINPGGTLELGVDYGRANLRDNYRLVDGASKDGWLFTAEHTQSVLKGFNKFVVQYATDSMTSQGKGLSQGSGVAFDNEKFAYNINNNGHMLRILDHGAISMGDNWDMMYVGMYQDINWDNDNGTKWWTVGIRPMYKWTPIMSTVMEIGYDNVESQRTGDKNNQYKITLAQQWQAGDSIWSRPAIRVFATYAKWDEKWGYDYNGDSKVNPNYGKAVPADFNGGSFGRGDSDEWTFGAQMEIWW

[0636] >Tsx(SEQ ID NO:2)

[0637] MKKTLLAAGAVLALSSSFTVNAAENDKPQYLSDWWHQSVNVVGSYHTRFGPQIRNDTYLEYEAFAKKDWFDFYGYADAPVFFGGNSDAKGIWNHGSPLFMEIEPRFSIDKLTNTDLSFGPFKEWYFANNYIYDMGRNKDGRQSTWYMGLGTDIDTGLPMSLSMNVYAKYQWQNYGAANENEWDGYRFKIKYFVPITDLWGGQLSYIGFTNFDWGSDLGDDSGNAINGIKTRTNNSIASSHILALNYDHWHYSVVARYWHDGGQWNDDAELNFGNGNFNVRSTGWGGYLVVGYNF

[0638] DNA

[0639] >LamB(SEQ ID NO:3)

[0640]

[0641] >Tsx(SEQ ID NO:4)

[0642] ATGAAAAAAACATTACTGGCAGCCGGTGCGGTACTGGCGCTCTCTTCGTCTTTTACTGTCAACGCAGCTGAAAACGACAAACCGCAGTATCTTTCCGACTGGTGGCACCAGAGCGTTAACGTTGTCGGAAGCTATCACACCCGTTTCGGACCGCAGATCCGCAACGATACCTACCTTGAGTACGAAGCATTCGCTAAAAAAGACTGGTTCGACTTCTATGGTTATGCGGATGCGCCGGTATTCTTCGGCGGTAACTCCGATGCAAAAGGTATCTGGAACCACGGTTCTCCGCTGTTTATGGAAATCGAACCACGTTTCTCCATCGACAAGCTGACCAATACTGACCTTAGCTTCGGTCCGTTCAAAGAGTGGTACTTCGCGAACAACTACATTTACGACATGGGTCGTAATAAAGATGGTCGCCAGAGCACCTGGTACATGGGTCTGGGTACCGATATCGACACTGGCCTGCCGATGAGCCTGTCCATGAACGTCTATGCGAAATACCAGTGGCAGAACTATGGCGCAGCGAACGAAAACGAGTGGGACGGTTACCGTTTCAAAATTAAATACTTTGTGCCGATTACCGATCTGTGGGGCGGTCAGCTGAGCTACATCGGCTTCACCAACTTCGACTGGGGTTCCGATTTAGGGGATGACAGCGGTAACGCAATCAACGGTATTAAGACCCGTACTAATAACTCTATCGCTTCCAGCCATATTCTGGCTCTGAACTACGATCACTGGCACTACTCTGTCGTAGCTCGTTACTGGCACGACGGTGGTCAGTGGAACGACGATGCAGAACTGAACTTCGGCAACGGCAACTTCAACGTTCGCTCTACCGGCTGGGGTGGTTACCTGGTAGTAGGTTACAACTTCTGA

[0643] >NC_000866.4 Enterobacteriaceae phage T4, complete genome (SEQ ID NO: 5)

[0644]

[0645] Spacer targeting E. coli fimH (SEQ ID NO: 6)

[0646] CGAATGACCAGGCATTTACCGACCAGCCCATC

[0647] Spacer targeting E. coli bolA (SEQ ID NO: 7)

[0648] AGTGGGAAGGGTTGCAGGACACCGTCTTTGCC

[0649] Targeting spacer region of E. coli rpoH (SEQ ID NO: 8)

[0650] CCGATGTTACCTTCCTGAATCAAATCCGCCTG

[0651] Spacer targeting E. coli lptA (SEQ ID NO: 9)

[0652] TGATTGACGGCTACGGTAAACCGGCAACGTTC

[0653] Spacer targeting E. coli murA (SEQ ID NO: 10)

[0654] GCTGTTAACGTACGTACCGCGCCGCATCCGGC

[0655] Escherichia phage T2 DNA, complete sequence (NCBI Reference Sequence: NC_054931.1) (SEQ ID NO: 11)

[0656]

[0657] Enterobacteriaceae phage RB69, complete genome (GenBank: AY303349.1) (SEQ ID NO: 12)

[0658]

[0659] bolA promoter sequence (SEQ ID NO: 13)

[0660] AACCTAAATATTTGTTGTTAAGCTGCAATGGAAACGGTAAAAGCGGCTAGTATTTAAAGGGATGGATGACATCTCAGCGTTGTCG

[0661] P J23100 Promoter sequence (SEQ ID NO: 14)

[0662] TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGC

[0663] Repeat sequence (SEQ ID NO: 15)

[0664] CGGTTTATCCCCGCTGGCGCGGGGAACTC

[0665] Promoter sequence (SEQ ID NO: 16)

[0666] TTGACGCGTAGCTCAGAGGTAGGTATAATGCTAGAAC

Claims

1. A composition comprising a plurality of different types of transduction particles, wherein each of the particles comprises a nucleic acid, and wherein the particles are capable of contacting an E. coli cell and introducing the nucleic acid therein, wherein (a) the nucleic acid of each particle comprises a nucleotide sequence encoding a product of interest (POI), wherein the nucleic acid is capable of expressing the POI in an E. coli cell; (b) each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from LPS and Tsx displayed on the surface of an E. coli cell; and (c) the plurality of different types of transduction particles comprises (i) a first type of particle comprising an LPS adhesion moiety; and (ii) a second type of particle comprising a Tsx adhesion moiety.

2. The composition according to claim 1, wherein A: Each particle of the first type comprises an LPS adhesion moiety and a LamB adhesion moiety; B: each particle of the first type comprises an LPS adhesion moiety and a Tsx adhesion moiety; or C: Each particle of the second type comprises the Tsx adhesion moiety but lacks the LamB and LPS adhesion moieties.

3. The composition of claim 2, wherein the composition comprises particles according to claims 2A, 2B and 2C.

4. A composition or method according to any preceding claim, wherein each particle comprises a bacteriophage capsid comprising a nucleic acid.

5. The composition according to claim 4, wherein the capsid of each particle contained in the composition is a T-even phage capsid; optionally a T2 phage, a T2-like phage, a RB69 phage or a RB69-like phage capsid.

6. A composition according to any preceding claim, wherein each particle is a bacteriophage (optionally a lytic phage) or a packaged phagemid.

7. A composition according to any preceding claim, wherein the composition comprises at least 3 or 4 different types of transducing particles.

8. A composition according to any preceding claim, wherein the nucleic acid of each particle comprises at least one nucleotide sequence (N1) encoding the POI, wherein each particle is a synthetic T-even phage comprising an insertion of N1 within a modification-permitting region (MPR) of the genome of the phage, wherein the MPR is immediately after gene 49 and towards gene E when compared to a reference wild-type T2 phage.

9. The composition of claim 8, wherein the nucleic acid comprises a DNA deletion of a bacteriophage DNA in an MPR.

10. The composition of claim 8 or 9, wherein the insertion comprises at most 8000 bp of DNA and / or the deletion comprises at most 8000 bp of DNA.

11. The composition of any one of claims 8-10, wherein the MPR comprises continuous DNA between gene 49 and gene E, wherein the length of the continuous DNA is at least 1000 bp; or wherein the MPR comprises at least 100 bp of DNA between gene 49 and gene E.

12. The composition of any one of claims 8-11, wherein the synthetic phage genome comprises the insertion between coordinates 9000 and 21000, wherein the coordinates are nucleotide positions counted from the nucleotide immediately following gene 49 (coordinate number 1) toward gene E when compared to a reference wild-type T2 phage.

13. A composition according to any preceding claim, wherein the POI comprises (a) a nuclease for targeting DNA of an E. coli cell, wherein the nuclease can be expressed in the cell and used to cleave the DNA of the cell, thereby modifying or killing the cell; or (b) Dead Cas (dCas) for targeting DNA of E. coli cells, wherein the dCas can be expressed in the cells and targeted to the DNA of the cells, thereby modifying the cells.

14. The composition of claim 13(a), wherein the nuclease is a guide nuclease, optionally a Cas, a meganuclease, a zinc finger nuclease, or a TALEN; or the composition of claim 13(b), wherein the dCas is dCas9.

15. A composition according to claim 13 or 14, wherein the nuclease is a type I, II, III, IV, V or VI Cas nuclease, optionally Cas9 or Cas3.

16. A composition according to any preceding claim, wherein the POI comprises at least one crRNA or guide RNA operable with Cas for DNA targeting in E. coli cells.

17. A composition according to claim 16, wherein each cRNA or guide RNA comprises a spacer sequence complementary to an E. coli protospacer sequence; optionally comprising a protospacer region of an E. coli cell of the B2 phylogenetic group or a strain selected from the groups ST131, ST1193, ST648, ST315, ST405, ST361, ST88 and ST453.

18. The composition of claim 17, wherein the protospacer sequence is contained in a gene selected from the group consisting of E. coli genes fimH, bolA, rpoH, lptA and murA.

19. The composition of claim 18, wherein the particles of the composition target all of the E. coli genes fimH, bolA, rpoH, lptA, and murA.

20. A composition according to any preceding claim, wherein the nucleotide sequence encoding the POI comprises a stress phase activated (SPA) promoter for expressing the POI in E. coli cells; optionally wherein the promoter is the E. coli bolA promoter or comprises SEQ ID NO:

13.

21. A composition according to any preceding claim, wherein the E. coli cells comprise an E. coli strain that causes sepsis, septicemia or diarrhoea in humans or animals.

22. A composition according to any preceding claim, wherein the composition is for use in a method of treating or preventing an infection caused by E. coli cells in a human or animal subject, wherein the method comprises administering the particles to the subject.

23. A method of treating or preventing sepsis, septicemia or diarrhea in a human or animal subject, the method comprising administering to the subject a composition according to any preceding claim, wherein the E. coli cells comprise an E. coli strain that causes sepsis, septicemia or diarrhea in humans or animals.

24. A method of treating or preventing an infection caused by E. coli cells in a human or animal subject, wherein the method comprises administering to the subject a composition according to any one of claims 1-21, wherein the infection is treated or prevented.

25. The composition or method of any one of claims 22-24, wherein the subject is a transplant or cancer patient (optionally a hematological cancer patient), or wherein the patient has or is at risk of a urinary tract infection (UTI); and optionally wherein the transplant is a solid organ or stem cell transplant (optionally a hematopoietic cell transplant), or wherein the transplant is a transplant of a medical device.

26. The composition or method of claim 25, wherein the method is performed prior to the subject receiving a transplant.

27. A composition or method according to any preceding claim for use in preventing hemolytic uremic syndrome (HUS), UTI infection, sepsis, septicemia or diarrhea in a human subject.

28. The composition or method according to any one of claims 22-27, wherein at least 1 x 10 7 PFU of particles are administered to a subject; or a dose of the composition according to any one of claims 1-22 and 25-27, wherein the dose is at least 1 x 10 7 The dose of the particles is in PFU.

29. The composition or method of any one of claims 22-28, wherein the particles are administered to the subject at an MOI (multiplicity of infection) of at least 0.

01.

30. A composition, method or dosage according to any preceding claim, wherein the E. coli cells comprise at least one strain that is an antibiotic-resistant or MDR strain; and / or at least one B2-I strain.

31. The composition, method or dose of claim 30, wherein the antibiotic is a fluoroquinolone (optionally levofloxacin), a carbapenem or vancomycin; and / or wherein the E. coli cells comprise β-lactamase (ESBL) producing E. coli.

32. A composition, method or dosage according to any preceding claim, wherein (i) the composition comprises first and second types of particles according to claims 2A, 2B and 2C; (ii) the POI comprises a nuclease for targeting the DNA of an E. coli cell, wherein the nuclease can be expressed in the cell and used to cleave the DNA of the cell, thereby modifying or killing the cell; (iii) each particle comprises a phage capsid containing a nucleic acid; and (iv) The particles of the composition target a plurality of different E. coli genes, optionally selected from essential genes and virulence genes.

33. A method for detecting the presence of Escherichia coli (optionally Escherichia coli cells of phylogenetic group B2) in a sample, the method comprising (a) contacting a sample with a composition according to any preceding claim; and (b) Detecting whether E. coli cells are killed or their growth or proliferation is reduced.

34. A method for detecting the presence of Escherichia coli (optionally Escherichia coli cells of phylogenetic group B2) in a sample, the method comprising (a) contacting a sample with a composition according to any preceding claim; wherein the particles of the composition comprise a nucleic acid comprising or encoding a detectable label, wherein the particles contact a cell and introduce the nucleic acid therein, wherein optionally the label is expressed in the cell; and (b) detecting E. coli cells comprising the marker.

35. A method for modifying the genome of an E. coli cell, the method comprising contacting the cell with a composition according to any preceding claim, wherein a nucleic acid encoding a POI is introduced into the cell, thereby modifying the genome of the cell.

36. The method of any one of claims 33-35, wherein the sample is a patient sample (e.g., a blood, urine, stool, or saliva sample), wherein the subject is a transplant or cancer patient (optionally a hematological cancer patient), or wherein the patient has or is at risk of a urinary tract infection (UTI); and optionally wherein the transplant is a solid organ or stem cell transplant (optionally a hematopoietic cell transplant).

37. The method according to any one of claims 33 to 36, wherein at least 1 x 10 7 PFU of particles are contacted with the sample.

38. The method of any one of claims 33-37, wherein the particles are contacted with the sample at an MOI (multiplicity of infection) of at least 0.

01.

39. A composition comprising a plurality of transduction particles for use in a method of treating or preventing an infection caused by E. coli cells in a human or animal subject, wherein the method comprises administering the particles to the subject, wherein (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of an E. coli cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, wherein the nuclease is expressed in the cell and cleaves the genomic DNA of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject; (b) the E. coli cell is a cell of E. coli phylogenetic group B2; and (c) Each particle comprises an adhesion moiety for recognizing and binding a homologous moiety selected from the group consisting of LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

40. The composition of claim 39, wherein each particle contained in the composition comprises a T-even phage capsid; optionally a T2 phage, a T2-like phage, a RB69 phage, or a RB69-like phage capsid.

41. A composition according to claim 39 or 40, wherein each particle is a bacteriophage (optionally a lytic phage) or a packaged phagemid.

42. A composition comprising a plurality of transduction particles for use in a method of treating or preventing an infection caused by an E. coli cell (optionally an E. coli cell of the B2 phylogenetic group) in a human or animal subject, wherein the method comprises administering the particles to the subject, wherein (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of an E. coli cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, wherein the nuclease is expressed in the cell and cleaves the genomic DNA of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject; and (b) Each particle contained in the composition is a T-even phage capsid; optionally, a capsid of T2 phage, T2-like phage, RB69 phage or RB69-like phage.

43. The composition of any one of claims 39-42, wherein the composition comprises at least 3 or 4 different types of transduction particles, wherein the types have different adhesion moieties for recognizing and binding to homologous moieties contained in E. coli (optionally B2 phylogenetic group E. coli) cells.

44. A composition according to any one of claims 39-43, wherein (i) the nuclease is a guide nuclease for targeting DNA of Escherichia coli cells, wherein the nuclease can be expressed in the cells and used to cut the DNA of the cells, thereby killing the cells; (ii) Each particle contains a phage capsid containing nucleic acid ； and (iii) the particles of the composition target a plurality of different E. coli genes, the E. coli genes optionally being selected from essential genes and virulence genes; and (iv) Optionally, the composition comprises A: Particles containing LPS adhesion moiety and LamB adhesion moiety; B: particles comprising an LPS adhesion moiety and a Tsx adhesion moiety; or C: Particles containing the Tsx-adherent moiety but lacking the LamB- and LPS-adherent moieties.

45. The composition of any one of claims 39-43, wherein the nuclease is a Cas nuclease and the nucleic acid encodes at least one crRNA or guide RNA that can operate with Cas for DNA targeting in E. coli cells.

46. ​​A composition according to claim 45, wherein each cRNA or guide RNA comprises a spacer sequence complementary to an E. coli protospacer sequence; optionally comprising a protospacer region of an E. coli cell of the B2 phylogenetic group or a strain selected from the groups ST131, ST1193, ST648, ST315, ST405, ST361, ST88 and ST453.

47. The composition of claim 46, wherein the protospacer sequence is contained in a gene selected from the group consisting of the E. coli genes fimH, bolA, rpoH, lptA and murA.

48. The composition of claim 47, wherein the particles of the composition target all of the E. coli genes fimH, bolA, rpoH, lptA, and murA.

49. A composition according to any one of claims 39-48, wherein the encoded nucleotide sequence comprises a stress phase activated (SPA) promoter for expressing the nuclease in E. coli cells; optionally wherein the promoter is the E. coli bolA promoter or comprises SEQ ID NO: 13 (or a promoter sequence that is at least 80, 90, 95, 96, 97, 98 or 99% identical to SEQ ID NO: 13).

50. The composition of any one of claims 39-49, wherein the E. coli cells comprise an E. coli strain that causes sepsis, septicemia, or diarrhea in humans or animals.

51. A composition according to any preceding claim, wherein the composition is for use in a method of treating or preventing an infection caused by E. coli cells in a human or animal subject, wherein the method comprises administering the particles to the subject.

52. A method of treating or preventing sepsis, septicemia or diarrhea in a human or animal subject, the method comprising administering to the subject a composition according to any one of claims 39-51, wherein the E. coli cells comprise an E. coli strain that causes sepsis, septicemia or diarrhea in humans or animals.

53. A method of treating or preventing an infection caused by E. coli cells in a human or animal subject, wherein the method comprises administering to the subject a composition according to any one of claims 39-51, wherein the infection is treated or prevented.

54. The composition or method of any one of claims 51-53, wherein the subject is a transplant or cancer patient (optionally a hematological cancer patient), or wherein the patient has or is at risk of a urinary tract infection (UTI); and optionally wherein the transplant is a solid organ or stem cell transplant (optionally a hematopoietic cell transplant), or wherein the transplant is a transplant of a medical device.

55. The composition or method of claim 54, wherein the method is performed before the subject receives a transplant.

56. The composition or method of any one of claims 39-55, for preventing hemolytic uremic syndrome (HUS), UTI infection, sepsis, septicemia, or diarrhea in a human subject.

57. The composition or method according to any one of claims 52-56, wherein at least 1 x 10 7 PFU of particles are administered to a subject; or a dose of the composition according to any one of claims 1-22 and 25-27, wherein the dose is at least 1 x 10 7 The dose of the particles is in PFU.

58. The composition or method of any one of claims 52-57, wherein the particles are administered to a subject at an MOI (multiplicity of infection) of at least 0.

01.

59. according to any one of claims 39-58 compositions, method or dosage, wherein said Escherichia coli cells comprise at least one bacterial strain that is antibiotic resistance or MDR bacterial strain; And / or at least one B2-1 bacterial strain.

60. The composition, method or dose of claim 59, wherein the antibiotic is a fluoroquinolone (optionally levofloxacin), a carbapenem or vancomycin; and / or wherein the E. coli cells comprise β-lactamase (ESBL) producing E. coli.

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