Targeting E. coli cells

Abstract

The technology described herein relates to methods and compositions for targeting E. coli cells, such as for treating or preventing infection by E. coli cells in a human or animal subject. In one embodiment, the method comprises administering to the subject a particle cocktail 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 nucleases for targeting the genome of B2 strain E. coli cells.

Description

[Technical Field]

[0001] The technology described herein relates to methods and compositions for targeting E. coli cells, such as for treating or preventing infection by E. coli cells in a human or animal subject. The methods, in embodiments, include administering to the subject a particle cocktail containing a plurality of different transduction particles for E. coli cells. The methods, in embodiments, include administering to the subject a plurality of transduction particles encoding nucleases for targeting the genome of E. coli cells of the B2 phylogenetic group. [Background technology]

[0002] E. coli infections have been shown to be harmful or life-threatening in a variety of settings, including UTI infections, transplant patients, cancer patients, and other patients who are immunocompromised or receiving immunosuppressive drugs.

[0003] Nuclease targeting of E. coli, such as with the CRISPR / Cas system, has been proposed through delivery using transduction particles that can target nucleases to E. coli cells for chromosomal or episomal cleavage, thereby killing the cells or reducing their growth or proliferation. Suitable transduction particles are phages or engineered particles (such as non-self-replicating transduction particles) that contain a capsid containing nucleic acids encoding at least a crRNA or gRNA (or even a Cas nuclease) for targeting. Advantageously, selective targeting that is not possible using conventional antibiotics, such as broad-spectrum antibiotics, can be achieved. Such selective targeting can avoid the killing of beneficial species and strains in treated patients. Indeed, disruption of the microbiome by broad-spectrum antibiotics is a risk factor in the preventive management of cancer patients at risk for febrile neutropenia.

[0004] Bacteriophage (phage) therapy has been used since before antibiotics were widely available, but is now attracting renewed attention due to the rise in bacterial antimicrobial resistance (AMR) combined with reports of some successful individual cases.

[0005] Bacteriophages (phages) are a phylum of viruses that infect bacteria and are distinct from animal and plant viruses. Phages can have either a "lytic" life cycle, a "lysogenic" life cycle which may be lytic, or a "non-lytic" life cycle. Phages that replicate by the lytic cycle cause lysis of host bacterial cells as a normal part of their life cycle. Phages that replicate by the lysogenic cycle are called temperate phages and can replicate by the lytic life cycle and cause lysis of the host bacterium, or they can integrate their DNA into the host bacterial DNA and become non-infectious prophage.

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

[0007] International Patent Application No. WO 00 / 69269 discloses the use of certain phage strains to treat infections caused by vancomycin-susceptible 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 antibacterial measures, to prevent or treat gastrointestinal diseases associated with Clostridium species.

[0008] US Patent Application No. 2002 / 0001590 discloses the use of phage therapy against multidrug-resistant bacteria, particularly 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 certain bacterial infectious diseases has been disclosed, for example, in US Patent Application Nos. 2002 / 0044922; 2002 / 0058027 and International Patent Application No. WO01 / 93904.

[0010] US20160333348 describes the use of a CRISPR / Cas system delivered into a host bacterial cell using a phage as a vector.

[0011] Among several E. coli phylogroups, antibiotic resistance (e.g., fluoroquinolone (FQ) resistance) and multidrug-resistant (MDR) strains have been observed to be frequently found in the B2 phylogroup. B2 strains ST131 and ST1193 have been found to be associated with antibiotic resistance. ST131 is the globally dominant multidrug-resistant clone associated with a high rate of rUTIs. ST131 is a major cause of hospital-acquired and community-acquired UTIs, E. coli bloodstream infections, and infections in companion animals and poultry. First identified in 2008, ST131 has been linked to the global spread of CTX-M-15 extended-spectrum β-lactamase (ESBL) resistance genes. ST131 is now strongly associated with multidrug resistance (MDR), including resistance to fluoroquinolones. Recent reports have also identified strains resistant to last-line carbapenems. Sequence type 1193 has recently emerged as a new virulent and resistant lineage among fluoroquinolone-resistant E coli. Summary of the Invention [Problem to be solved by the invention]

[0012] Therefore, classical antibiotics such as FQs and broad-spectrum antibiotics are not effective enough to combat such infections, and therefore, there is a need to find alternative ways to combat these infections. [Means for solving the problem]

[0013] The present invention provides a means for treating or preventing B2 phylogenetic group E. coli infections in humans and animals by combining the use of targeted nuclease selective killing with specific types of transducing particles. The particles of the present invention target by attachment to LPS, LamB, or Tsx, which has surprisingly been found to be highly advantageous in killing and inhibiting the growth of B2 E. coli cells of many different strains, including the potentially lethal ST131 and ST1193 strains. As exemplified herein, surprisingly, more than 10 different ST131 strains were killed (plaques formed), and more than 10 different ST1193 strains were killed (plaques formed).

[0014] The present invention finds utility for treating or preventing potentially life-threatening phylogenetic group B2 E. coli infections in patients, such as immunosuppressed, cancer, transplant, and UTI patients, who are susceptible to infection with B2 strains (and often with multiple different B2 strains). As demonstrated in the Examples, the present invention is useful for preventing E. coli phylogenetic group B2 bacteremia (bloodstream infection with E. coli) in a subject. To this end, the present invention provides:

[0015] First configuration First aspect:- 1. A composition comprising a plurality of transducing particles for use in a method of treating or preventing infection with E. coli cells in a human or animal subject, the method comprising administering the particles to the subject; (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, and the nuclease is expressed within 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 cells are E. coli phylogenetic group B2 cells; (c) A composition, wherein each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0016] Second aspect:- E coli in human or animal subjects Bacteremia 1. A composition comprising a plurality of transducing particles for use in a method of treating or preventing a tumor, the method comprising administering the particles to a subject, (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of a cell of said E. coli, wherein said administered particle contacts the cell and introduces the nucleic acid therein, and the nuclease is expressed within 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 cells are E. coli phylogenetic group B2 cells; (c) A composition, wherein each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0017] Second Configuration First aspect:- 1. A method for treating or preventing infection with E. coli cells in a human or animal subject, the method comprising administering to the subject a plurality of transducing particles; (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, and the nuclease is expressed within 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 cells are E. coli phylogenetic group B2 cells; (c) The method, wherein each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0018] Second aspect:- 1. A method for treating or preventing infection with E. coli cells in a human or animal subject, the method comprising administering to the subject a plurality of transducing particles; (a) each particle contains a nucleic acid encoding a Cas nuclease and operable crRNA or guide RNA for chromosomal targeting in a cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, the crRNA or guide RNA is expressed, induces the Cas nuclease, and the nuclease cleaves the cell's chromosome, thereby killing the cell or reducing cell growth or proliferation in the subject; (b) the E. coli cells are E. coli phylogenetic group B2 cells; (c) The method, wherein each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0019] Third aspect:- 1. A method for treating or preventing E. coli bacteremia in a human or animal subject, the method comprising administering to the subject a plurality of transducing particles; (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of a cell of said E. coli, wherein said administered particle contacts the cell and introduces the nucleic acid therein, and the nuclease is expressed within 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 cells are E. coli phylogenetic group B2 cells; (c) The method, wherein each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0020] Fourth aspect:- 1. A method for treating or preventing E. coli bacteremia in a human or animal subject, the method comprising administering to the subject a plurality of transducing particles; (a) each particle comprises a nucleic acid encoding a Cas nuclease and operable crRNA or guide RNA for chromosomal targeting in a cell of the E. coli, the administered particle contacts the cell and introduces the nucleic acid therein, the crRNA or guide RNA is expressed, induces the Cas nuclease, and the nuclease cleaves the cell's chromosome, thereby killing the cell or reducing cell growth or proliferation in the subject; (b) the E. coli cells are E. coli phylogenetic group B2 cells; (c) The method, wherein each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0021] Third Configuration First aspect:- 1. Use of a composition comprising a plurality of transducing particles in a method for treating or preventing infection with phylogenetic group B2 E. coli cells in a human or animal subject, the method comprising administering the composition to the subject; (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, and the nuclease is expressed within 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) Use wherein each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0022] Second aspect:- 1. Use of a composition comprising a plurality of transducing particles in a method for treating or preventing infection with phylogenetic group B2 E. coli cells in a human or animal subject, the method comprising administering the composition to the subject; (a) each particle contains a nucleic acid encoding a Cas nuclease and operable crRNA or guide RNA for chromosomal targeting in a cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, the crRNA or guide RNA is expressed, induces the Cas nuclease, and the nuclease cleaves the cell's chromosome, thereby killing the cell or reducing cell growth or proliferation in the subject; (b) the E. coli cells are E. coli phylogenetic group B2 cells; (c) Use wherein each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0023] Third aspect:- 1. Use of a composition comprising a plurality of transducing particles in a method for treating or preventing phylogenetic group B2 E. coli bacteremia in a human or animal subject, the method comprising administering the composition to the subject; (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of a cell of said E. coli, wherein said administered particle contacts the cell and introduces the nucleic acid therein, and the nuclease is expressed within 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) Use wherein each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0024] Fourth aspect:- 1. Use of a composition comprising a plurality of transducing particles in a method for treating or preventing phylogenetic group B2 E. coli bacteremia in a human or animal subject, the method comprising administering the composition to the subject; (a) each particle comprises a nucleic acid encoding a Cas nuclease and operable crRNA or guide RNA for chromosomal targeting in a cell of the E. coli, the administered particle contacts the cell and introduces the nucleic acid therein, the crRNA or guide RNA is expressed, induces the Cas nuclease, and the nuclease cleaves the cell's chromosome, thereby killing the cell or reducing cell growth or proliferation in the subject; (b) the E. coli cells are E. coli phylogenetic group B2 cells; (c) Use wherein each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0025] Fourth Configuration First aspect:- 1. A composition comprising a plurality of transduction particles for use in a method for treating or preventing infection by E. coli cells (optionally B2 phylogenetic group E. coli cells) in a human or animal subject, the method comprising administering particles to the subject, wherein (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of an E. coli cell, the administered particles contacting the cell and introducing the nucleic acid therein, the nuclease being expressed within the cell and cleaving the genomic DNA of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject; (b) A composition, wherein each particle comprised in the composition is a capsid of a T-even phage, optionally a capsid of a T2 phage, a T2-like phage, an RB69 phage or an RB69-like phage.

[0026] A composition comprising a plurality of different types of transduction particles, each of said particles comprising a nucleic acid, said particles being capable of contacting E. coli cells and transferring the nucleic acid thereto; (a) the nucleic acid of each particle comprises a nucleotide sequence encoding a product of interest (POI), and the nucleic acid is capable of expressing the POI in E. coli cells; (b) each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB and Tsx (optionally selected from LPS and Tsx) displayed on the surface of an E. coli cell; (c) The composition, wherein the plurality of different types of transduction particles comprises (i) a first type of particle comprising an LPS attachment moiety, and (ii) a second type of particle comprising a Tsx attachment moiety.

[0027] Second aspect:- A method for 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.

[0028] Third aspect:- A method of treating or preventing an infection by E. coli cells in a human or animal subject, the method comprising administering to the subject a composition of the invention, wherein the infection is treated or prevented.

[0029] Fourth aspect:- 1. A method for detecting the presence of E. coli (optionally phylogenetic group B2 E. coli cells) in a sample, comprising: (a) contacting a sample with a composition according to the present invention; (b) detecting that the E. coli cells have been killed or their growth or proliferation has been reduced; A method comprising:

[0030] Fifth aspect:- 1. A method for detecting the presence of E. coli (optionally phylogenetic group B2 E. coli cells) in a sample, comprising: (a) contacting a sample with a composition according to the present invention, wherein particles of the composition contain a nucleic acid that includes or encodes a detectable label, and wherein the particles contact cells and introduce the nucleic acid therein, and optionally the label is expressed in the cells; (b) detecting that the E. coli cells contain the label; A method comprising:

[0031] Sixth aspect:- A method for modifying the genome of an E. coli cell, the method comprising contacting the cell with a composition of the present invention, wherein a nucleic acid encoding a POI is introduced into the cell, thereby modifying the genome of the cell. [Brief explanation of the drawings]

[0032] [Figure 1] Figure 1 shows the killing and growth inhibitory activity of compositions of the present invention when tested against E. coli of several different phylogenetic groups and different strains within each group (E. coli taken from clinical samples); the compositions were surprisingly highly effective at killing across many phylogenetic groups (particularly in the B2 phylogenetic group, where multiple strains were killed or their growth effectively inhibited, including those known to be potentially life-threatening and associated with antibiotic resistance). The six negatives included non-E. coli and non-FQ resistant samples. [Figure 2] FIG. 1 shows the phylogenetic grouping of strains used in the study and the particularly advantageous broad targeting of many clinical B2 phylogenetic group strains. [Figure 3A] A) CRISPR-Cas-driven elimination of a truncated panel of 82 E. coli clinical isolates by conjugation of CGV-EcCAS and empty vector. Conjugation efficiency was determined by spotting serial dilutions of the conjugation reaction on LB agar supplemented with antibiotics and calculated as CFU / ml. The limit of detection (LOD) of this assay was 200 CFU / ml. Experiments were performed in triplicate and are indicated by dots. CGV-EcCAS conjugation experiments are indicated by arrows; empty vector conjugations are shown above the dotted line labeled LOD. [Figure 3B-C]B) Percentage of CRISPR-armed phage™ (CAP) and WT phage during co-culture with a host strain sensitive to both phages. CAP α15.2 increases its relative abundance compared to WT phage from 7% to 86% over two serial passages. C) CAP α20.4 surpasses WT α20 by increasing its relative abundance from 10% to 68% over four serial passages during co-culture with the common target, E. coli strain b230. B-C) Ratio of CAP and WT phage during co-culture with a host strain (E. coli b230) sensitive to α15.2, α15, α20.4, and α20. CAP α15.2 increased its relative abundance from 7% to 86% compared with the WT phage (G), while CAP α20.4 surpassed WT α20 by increasing it from 10% to 68% (H). [Figure 4] Figure 1 shows specificity assessment of SNIPR001 and individual CAPs against a panel of clinically relevant bacteria and E. coli strain b2480 (positive control) showing no off-target effects. Positive values represent bacterial growth (no observed killing), while negative values indicate bacterial killing after phage treatment within the time evaluated. All values are shown as the mean of four biological replicates with standard deviation, measuring growth over a 4-hour period and measured in CFU / mL. [Figure 5A] A) Unrooted phylogenetic tree of JMI strains showing a clinical panel of 382 E. coli strains including 9 phylogenetic groups and 118 MLSTs. Plaque formation data reflect a single plaque formation replicate. One strain of E. coli b4038 with a long branch is truncated to 37% of its original length. Phylogenetic distance scale calculated by MASH is shown below the tree. [Figure 5B]B) 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. Spot assays were performed in two independent experiments, and results are shown as mean ± SD values. The standard deviation shown is based on discrepancies in results between two runs, calculated as the mean absolute deviation of the mean prevalence of a given spotting type, normalized to panel size. [Figure 5C] C) Coverage of SNIPR001 is independent of antibiotic resistance phenotype; consequently, SNIPR001 targets over 90% of carbapenem-resistant, extended-spectrum β-lactamase (ESBL)-producing, or multidrug-resistant (MDR) E. coli strains, and 89% of fluroquinolone-resistant E. coli strains. The numbers in each green or gray bar indicate the number of bacteria susceptible or resistant to SNIPR001, respectively, for each resistance category. [Figure 5D] D) Midpoint rooted phylogenetic tree of 72 fluroquinolone-resistant E. coli strains isolated from fecal samples of hematological cancer patients. 67 of the 72 strains are susceptible to at least one of the four CAPS in SNIPR001. [Figure 5E] E) Redundancy distribution showing that 82% of the fluroquinolone-resistant E. coli strains (n=72) from panel D are targeted by at least two different CAPs. [Figure 6A] A) CAP recovery in feces of minipigs after a single po administration of 2x1012 PFU of SNIPR001 (n=8, shown in green) or vehicle (n=6, shown in gray) over a 1-week period and daily sampling. The trend line shows the mean recovered phage in PFU / g feces, and the dots represent individual measurement points. The LOD of 33 PFU / g feces is indicated by the dotted line. [Figure 6B]B) CAP recovery in feces of minipigs after a single po administration of 2x10 PFU of a single CAP (n=8 minipigs received either α15.2, α20.4, or α51.5, and n=7 minipigs received α48.4) over a 1-week period and daily sampling. Trend lines indicate mean recovery, while dots represent individual measurements. Recovery was measured in PFU / g feces. The LOD of 33 PFU / g feces is indicated by the dotted line. [Figure 6C] C) CAP recovery in the feces of mice 8, 24, and 48 hours after the start of treatment with various doses of SNIPR001 (n=10 for low, medium, and high, shown in green), vehicle (n=10, shown in gray), or gentamicin (n=4, shown in gray) administered three times daily. Recovery was measured in PFU / g feces, with the LOD of 371 PFU / g feces indicated by the dotted line. [Figure 6D] D) Effect of SNIPR001 on E. coli b17 recovery in the feces of mice, which increased with increasing dose. Statistical analysis was performed using the two-tailed Kruskal-Wallis test to compare SNIPR001-treated groups and the two-tailed Mann-Whitney U test to compare vehicle and treatment groups. *P<0.05=*, 0.01=**, 0.001=***. FDR was corrected using Holm's method separately for each day. Recovery was measured as CFU / g feces, with a detection limit of 371 CFU / g feces. Animals where SNIPR001 treatment was initiated are shown in green; other animals are shown in gray. [Figure 6E] E) Recovery of E. coli b17 in the feces of mice 8 and 24 hours after the start of treatment with CAP α15.2, α20.4, α48.4 or α51.5 administered three times daily in combination with SNIPR001, confirming the synergistic effect of CAP. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention finds use 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 on immunosuppressive drugs.

[0034] Cancer treatments continue to advance, increasing survival rates for people with hematological malignancies. However, this population is immunocompromised, and chemotherapy regimens cause gastrointestinal mucositis accompanied by bone marrow suppression and increased intestinal permeability. Translocation of enteric bacteria, including E. coli, from the gastrointestinal tract is a frequent cause of bloodstream infections (BSIs). Because the mortality rate associated with BSIs can be up to 50%, antimicrobial prophylaxis is indicated for people at risk for febrile neutropenia. While there are no approved treatments for the prevention of BSIs in patients with hematological cancers, 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 supporting immunological tone and may benefit oncology treatment outcomes. Indeed, microbiome disruption by broad-spectrum antibiotics is a risk factor in the preventive management of patients at risk for febrile neutropenia. In addition to the adverse effects of fluoroquinolones, including safety warnings and precautions, resistance to fluoroquinolones is on the rise, approaching 60% in the United States.

[0035] In immunocompromised patients with hematologic malignancies at risk for developing neutropenia, E. coli accounts for 25.1-30% of all bacteremia cases, with a 90-day mortality rate of 35.8%. Furthermore, up to 65% of E. coli isolated as the causative pathogen from BSI in patients with hematologic cancers who underwent hematopoietic stem cell transplantation (HSCT) were resistant to fluoroquinolones. Therefore, novel narrow-spectrum treatments and prophylactic options are needed to prevent infections in these vulnerable patients. The present invention addresses this need.

[0036] To this end, the present invention provides compositions, methods and uses according to the above construction. Accordingly, the following description is provided by way of numbered embodiments.

[0037] 1. A composition comprising a plurality of transducing particles for use in a method of treating or preventing infection with E. coli cells in a human or animal subject, the method comprising administering the particles to the subject; (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, and the nuclease is expressed within 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 cells are E. coli phylogenetic group B2 cells; (c) A composition, wherein each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0038] Optionally, (i) each particle comprises a phage capsid containing nucleic acid; (ii) the particles of the composition target a plurality of different E. coli genes, optionally selected from essential genes and virulence genes; (iii) the composition comprises: A: Particles containing an LPS adhesive moiety and a LamB adhesive moiety. B: Particles containing an LPS adhesive moiety and a Tsx adhesive moiety, or C: Particles containing the Tsx adhesive moiety but lacking the LamB and LPS adhesive moieties Includes:

[0039] 2. A method for treating or preventing infection by E. coli cells in a human or animal subject, the method comprising administering to the subject a plurality of transducing particles, the method comprising administering to the subject the particles; (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, and the nuclease is expressed within 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 cells are E. coli phylogenetic group B2 cells; (c) The method, wherein each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0040] Optionally, (i) each particle comprises a phage capsid containing nucleic acid; (ii) the particles of the composition target a plurality of different E. coli genes, optionally selected from essential genes and virulence genes; (iii) the composition comprises: A: Particles containing an LPS adhesive moiety and a LamB adhesive moiety. B: Particles containing an LPS adhesive moiety and a Tsx adhesive moiety, or C: Particles containing the Tsx adhesive moiety but lacking the LamB and LPS adhesive moieties Includes.

[0041] Optionally, the genomic DNA is chromosomal DNA of the cell. Additionally or alternatively, the genomic DNA is plasmid DNA of the cell.

[0042] Optionally, each particle contains a nucleic acid encoding a nuclease for chromosome targeting, wherein the administered particle contacts a cell, introduces the nucleic acid therein, and the nuclease is expressed within the cell and cleaves the cell's chromosome, thereby killing the cell or reducing cell growth or proliferation in the subject.

[0043] The human may be male or female. The human may be an adult or a child. The human may be 18 years of age or older, e.g., 40, 50, 60, 70, 80 years of age or older. The human may be younger than 18 years of age, e.g., a teenager, e.g., an infant, e.g., up to 5 years of age, e.g., up to 2 years of age. The animal may be a livestock or companion animal, e.g., a dog or cat. The animal may be a bird (e.g., a poultry, e.g., 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).

[0044] The infection may be a bloodstream infection. The infection may be a hospital-acquired infection.

[0045] In one example, each adhesive moiety is a tail fiber protein. In one example, each particle comprises a phage tail fiber that includes the adhesive moiety or is fused to the adhesive moiety. In one example, each adhesive moiety is an antibody fragment, e.g., an antibody single variable domain. In one example, each adhesive moiety is a nanobody. In one example, each adhesive moiety comprises an antibody binding site capable of binding to a cognate moiety. For example, each adhesive moiety comprises an antibody single variable domain (i.e., a 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 adhesive moiety.

[0046] For example, at least two, three, or four different types of the transducing particles are administered to a subject, each type comprising one or more types of tail fibers comprising an adhesive moiety, and other particle types not comprising said one or more types of tail fiber, hi one example, each type of the plurality of tail fiber types differs from the other types by the type of adhesive moiety it comprises.

[0047] Optionally, the particles comprise adhesive moieties for binding to LPS, LamB, and Tsx. Optionally, the particles comprise adhesive moieties for binding to LamB and Tsx. Optionally, the particles comprise adhesive moieties for binding to LPS and Tsx. Optionally, the particles comprise adhesive moieties for binding to LPS and LamB.

[0048] In Gram-negative bacteria, the peptidoglycan layer is relatively thin and located inside the outer membrane, the major component of the cell wall. These two layers are connected by Braun's lipoprotein. The outer membrane is a highly structured lipid bilayer decorated with proteins, polysaccharides, and lipids, the latter two molecules forming the LPS layer. LPS is a complex consisting of three parts: lipid A, core polysaccharide, and O-polysaccharide. Lipid A generally consists of a fatty acid linked to a glucosamine phosphate disaccharide. The core polysaccharide is linked to lipid A via a ketodeoxyoctonate linker. The core polysaccharide and O-polysaccharide (O-chain or O-antigen) contain several units of sugar residues that extend beyond the outer membrane. Cells containing all three components of LPS are called smooth (S) type, while cells lacking the O-polysaccharide portion are distinguished as rough (R) type.

[0049] Optionally, the LPS is smooth or rough LPS.

[0050] For example, the particles include at least one type of particle in which the adhesive moiety is capable of binding to the O-antigen of LPS.

[0051] E. coli is a highly versatile species, and its diversity has been studied from various perspectives, emphasizing, for example, phylogenetic classification, pathotypes, and the wide range of O serotypes. The highly diverse O-antigen, the outermost part of the lipopolysaccharide component of the E. coli outer membrane, is linked to the innermost lipid A via the core region of LPS, of which five distinct structures, designated K-12, R1, R2, R3, and R4, have been characterized to date. Phylogenetic groups B2 and C strains are primarily dominated by the R1 type. Strains within phylogenetic group B2 can possess the K-12 core, for example, belonging to complex STc131, one of the major clones of extraintestinal pathogenic E. coli (ExPEC) strains.

[0052] Preferably, the LPS comprises an R1 core region. In one example, the LPS comprises an R2 core region. In one example, the LPS comprises an R3 core region. In one example, the LPS comprises an R4 core region. In one example, the LPS comprises a K-12 core region.

[0053] Optionally, LamB comprises the amino acids of SEQ ID NO: 1 or an amino acid sequence that is 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 that is 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 that is 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 that is at least 70, 80, 90, or 95% identical to SEQ ID NO: 4.

[0054] 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 E. coli nucleoside-specific channel-forming protein Tsx has a Uniprot accession number of P0A927 or is a homolog thereof. In one example, the E. coli maltose outer membrane porin (maltoporin) LamB has a Uniprot accession number of P02943 or is a homolog thereof. Homolog: A gene, nucleotide, or protein sequence related to a second gene, nucleotide, or protein sequence by descent from a common ancestral DNA or protein sequence. The term homolog may apply to the relationship between genes separated by an event of division or the relationship between genes separated by an event of gene duplication.

[0055] In one embodiment, the E coli cells comprise UPEC E coli. In one embodiment, the E coli cells comprise enteropathogenic E coli (ExPEC) cells.

[0056] 3. The composition or method of embodiment 1 or 2, respectively, wherein the method is for treating or preventing infection in a subject with an E. coli strain selected from the group consisting of ST131, ST1193, ST648, ST315, ST405, ST361, ST88 and ST453.

[0057] In a preferred embodiment, the strain is ST1193. In another preferred embodiment, the strain is ST131.

[0058] 4. The composition or method of any preceding embodiment, wherein the method is for treating or preventing infection in a subject with a plurality of different phylogenetic group B2 strains of E. coli, optionally wherein the plurality comprises E. coli ST131 cells and ST1193 cells. E. coli ST131 and / or ST1193 have been found to be pathogenic and associated with fluoroquinolone resistance. As shown in the Examples section herein, the present invention is useful for treating or preventing infections caused by such bacteria. Optionally, the plurality of bacteria includes E. coli ST131 and / or ST1193 strains. Optionally, the plurality of strains includes fluoroquinolone-resistant strains. For example, the plurality of strains includes fluoroquinolone-resistant strains of E. coli ST131 and / or ST1193. 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) have been found in the setting of UTI. In one embodiment (e.g., the subject has or is at risk for a UTI), the B2 E coli comprises one or more strains selected from 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).

[0059] 5. The composition or method of any of the preceding embodiments, wherein the subject is a transplant or cancer patient (optionally a hematological cancer patient), or the patient has or is at risk for a urinary tract infection (UTI), and optionally the transplant is a solid organ transplant or a stem cell transplant (optionally a hematopoietic cell transplant), or the transplant is a medical device transplant.

[0060] For example, the subject is a hematologic cancer patient suffering from neutropenia. For example, the subject is a hematopoietic stem cell transplant patient.

[0061] Suitable medical devices are, for example, cardiac devices (eg, ventricular assist devices such as left ventricular assist devices (LVADs)), catheters (eg, biliary catheters) or artificial prostheses (eg, joint) prostheses.

[0062] In one example, the patient has an isolated phylogroup B2 E coli infection. In one example, the E coli is isolated phylogroup B2 E coli.

[0063] For example, the subject is suffering from or 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 prostate surgery patient.

[0064] 6. The composition or method of any of the preceding embodiments, wherein the method is performed before the subject receives a transplant. For example, the transplant is a solid organ transplant or a stem cell transplant (optionally a hematopoietic cell transplant).

[0065] 7. The composition or method of any preceding embodiment, wherein the B2 E coli cells comprise a strain of E coli that causes sepsis, septicemia, or diarrhea in humans.

[0066] Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a human pathogen responsible for global epidemics of bloody diarrhea and hemolytic uremic syndrome (HUS). Conventional antimicrobial agents trigger an SOS response in EHEC, which promotes the release of potent Shiga toxins, responsible for much of the morbidity and mortality associated with EHEC infection. Cattle are the natural reservoir for EHEC, and approximately 75% of EHEC outbreaks are associated with the consumption of contaminated cattle products. EHEC causes disease in humans but is asymptomatic in adult ruminants. E. coli serotype O157:H7 (EHEC) infection is characterized by abdominal cramps and bloody diarrhea, as well as the life-threatening complication hemolytic uremic syndrome (HUS). Treatments for EHEC infection are currently needed (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 were at higher risk of developing HUS (Slutsker et al., 1998). Further studies supported the contraindication of antibiotics in EHEC infection, and children receiving antibiotic therapy for EHEC-associated hemorrhagic colitis were more likely to develop HUS (Wong et al., 2000; Zimmerhackl, 2000; Safdar et al., 2002; Tarr et al., 2005). Conventional antibiotics promote Shiga toxin production by enhancing the replication and expression of stx genes encoded within chromosomally integrated lambdoid prophage genomes. The present approach may rely on nuclease cleavage of target cell genomic DNA. Stx induction also promotes phage-mediated lysis of the EHEC cell envelope, allowing the release and spread of Shiga toxin into the environment (Karch et al., 1999; Matsushiro et al., 1999; Wagner et al., 2002). Advantageously, therefore, the present invention provides an alternative means for treating phylogroup B2 EHEC in human and animal subjects.In one example, a subject (eg, a human) has or is at risk for hemolytic uremic syndrome (HUS), for example, the subject has an E coli infection, such as an EHEC E coli infection.

[0067] 8. A composition or method according to any of the preceding embodiments for preventing hemolytic uremic syndrome (HUS), a UTI infection, sepsis, septicemia or diarrhea in a subject.

[0068] The composition or method may be for treating or preventing a bloodstream infection with pathogenic lineage group B2 E coli cells in a subject.

[0069] 9. The composition or method of any preceding embodiment, wherein each particle comprises a phage capsid containing nucleic acid, and optionally the capsid comprises capsid proteins of a T-even phage (optionally T4) or a lambda phage.

[0070] As known to those skilled in the art, transduction particles are operative to infect their cognate host cells and introduce nucleic acid therein by transduction.

[0071] 10. The composition or method of any of the preceding embodiments, wherein each particle is a phage (optionally a lytic phage) or a packaged phagemid.

[0072] 11. The composition or method of any of the preceding embodiments, wherein at least two, three, or four different types of transducing particles are administered to the subject.

[0073] For example, each particle type includes a different adhesive moiety type or collection of adhesive moiety types than other particle types.

[0074] 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.

[0075] 12. The composition or method of any of the preceding embodiments, wherein a first type of transduction particles and a second type of transduction particles are administered to a subject, wherein the first type of particles comprise a first adhesive moiety capable of recognizing and binding to a first cognate moiety selected from the group of LPS, LamB, and Tsx displayed on B2 E. coli, and the second type of particles comprise a second adhesive moiety capable of recognizing and binding to a second cognate moiety selected from said group, and wherein the first and second adhesive moieties are different from each other.

[0076] For example, the first and second adhesive moieties differ from each other by their tail fibers, and optionally the first adhesive moiety is cognate with LPS and the second moiety is cognate with LamB, or optionally the first adhesive moiety is cognate with LPS and the second moiety is cognate with Tsx, or optionally the first adhesive moiety is cognate with Tsx and the second moiety is cognate with LamB.

[0077] For example, each particle comprises a phage capsid containing the nucleic acid, and at least two, three, or four different types of transducing particles are administered to a subject, a first type of transducing particles and a second type of transducing particles are administered to a subject, the first type of particles comprising a first adhesive moiety capable of recognizing and binding to a first cognate moiety selected from the group consisting of LPS, LamB, and Tsx displayed on B2 E. coli, and the second type of particles comprising a second adhesive moiety capable of recognizing and binding to a second cognate moiety selected from the group, and the first and second adhesive moieties are different from each other.

[0078] For example, each particle comprises a phage capsid containing the nucleic acid, and at least two, three or four different types of transducing particles are administered to a subject, a first type of transducing particles and a second type of transducing particles are administered to a subject, the first type of particles comprising a first adhesive moiety capable of recognizing and binding to LPS displayed on B2 E. coli, and the second type of particles comprising a second adhesive moiety capable of recognizing and binding to LamB displayed on B2 E. coli.

[0079] For example, each particle comprises a phage capsid containing the nucleic acid, and at least two, three or four different types of transducing particles are administered to a subject, a first type of transducing particles and a second type of transducing particles are administered to a subject, the first type of particles comprising a first adhesive moiety capable of recognizing and binding to Tsx displayed on B2 E. coli, and the second type of particles comprising a second adhesive moiety capable of recognizing and binding to LamB displayed on B2 E. coli.

[0080] For example, each particle comprises a phage capsid containing the nucleic acid, and at least two, three or four different types of transducing particles are administered to a subject, a first type of transducing particles and a second type of transducing particles are administered to a subject, the first type of particles comprising a first adhesive moiety capable of recognizing and binding to LPS displayed on B2 E. coli, and the second type of particles comprising a second adhesive moiety capable of recognizing and binding to Tsx displayed on B2 E. coli.

[0081] For example, each particle comprises a phage capsid containing the nucleic acid, and at least three different types of transducing particles are administered to a subject, a first type of transducing particles, a second type of transducing particles, and a third type of transducing particles are administered to a subject, the first type of particles comprising a first adhesive moiety capable of recognizing and binding to LPS displayed on B2 E. coli, the second type of particles comprising a second adhesive moiety capable of recognizing and binding to LamB displayed on B2 E. coli, and the third type of particles comprising a second adhesive moiety capable of recognizing and binding to Tsx displayed on B2 E. coli.

[0082] For example, each particle comprises a phage capsid containing the nucleic acid, and at least four different types of transducing particles are administered to a subject, including a first type of transducing particles, a second type of transducing particles, a third type of transducing particles, and a fourth type of transducing particles, wherein the first type of particles comprise a first adhesive moiety capable of recognizing and binding to LPS displayed on B2 E. coli, the second type of particles comprise a second adhesive moiety capable of recognizing and binding to LamB displayed on B2 E. coli, the third type of particles comprise a second adhesive moiety capable of recognizing and binding to Tsx displayed on B2 E. coli, and the fourth type of particles comprise a second adhesive moiety capable of recognizing and binding to LPS displayed on B2 E. coli, and the adhesive moieties of the particles are different from each other.

[0083] For example, each particle comprises a phage capsid containing the nucleic acid, and at least four different types of transducing particles are administered to a subject, including a first type of transducing particles, a second type of transducing particles, a third type of transducing particles, and a fourth type of transducing particles, wherein the first type of particles comprise a first adhesive moiety capable of recognizing and binding to LPS displayed on B2 E. coli, the second type of particles comprise a second adhesive moiety capable of recognizing and binding to LamB displayed on B2 E. coli, the third type of particles comprise a second adhesive moiety capable of recognizing and binding to Tsx displayed on B2 E. coli, and the fourth type of particles comprise a second adhesive moiety capable of recognizing and binding to LamB displayed on B2 E. coli, and the adhesive moieties of the particles are different from each other.

[0084] For example, each particle comprises a phage capsid containing the nucleic acid, and at least four different types of transducing particles are administered to a subject, including a first type of transducing particles, a second type of transducing particles, a third type of transducing particles, and a fourth type of transducing particles, wherein the first type of particles comprise a first adhesive moiety capable of recognizing and binding to LPS displayed on B2 E. coli, the second type of particles comprise a second adhesive moiety capable of recognizing and binding to LamB displayed on B2 E. coli, the third type of particles comprise a second adhesive moiety capable of recognizing and binding to Tsx displayed on B2 E. coli, and the fourth type of particles comprise a second adhesive moiety capable of recognizing and binding to Tsx displayed on B2 E. coli, and the adhesive moieties of the particles are different from each other.

[0085] 13. The composition or method of embodiment 12, wherein the first and second cognate moieties are different from each other.

[0086] Alternatively, the first and second cognate moieties are identical. Alternatively, the first and second cognate moieties are LPS. Alternatively, the first and second cognate moieties are Tsx. Alternatively, the first and second cognate moieties are LamB.

[0087] 14. The composition or method of any of the preceding embodiments, wherein the nucleic acid of each particle comprises a nucleotide sequence (N1) encoding said nuclease, and each particle is a synthetic T-even phage (optionally a T4 phage) comprising an insertion of N1 into the genome of the phage, and the region is between the pin (protease inhibitor) gene and the iPII (intrinsic protein) gene.

[0088] Optionally, the phage comprises: (a) Coordinates (i) 1887 and 8983; (ii) 2625 and 8092; (iii) 1904 and 8113; (iv) 2668 and 7178; (v) 7844 and 11117; (vi) 8643 and 10313; (vii) 9231 and 13383; (viii) 9480 and 12224; (ix) 8454 and 17479; or (x)9067 and 16673 a synthetic T-even (e.g., T4) phage comprising a deletion of DNA from, and / or an insertion of heterologous DNA into, a region of the phage genome corresponding to the region between Coordinates are referenced to the wild-type T4 phage genome (SEQ ID NO: 5).

[0089] 15. The composition or method of any of the preceding embodiments, wherein said nuclease is an inducible nuclease, optionally a Cas, meganuclease, zinc finger nuclease or TALEN.

[0090] 16. The composition or method of any of the preceding embodiments, wherein the nuclease is a Type I, II, III, IV, V or VI nuclease, optionally Cas9 or Cas3.

[0091] 17. At least 1 x 10 7 The composition or method of any preceding embodiment, wherein particles of PFUs are administered to a subject.

[0092] In one example, 1 x 10 8 ~1×10 13 PFU of particles are administered to the subject. In one example, 1 x 10 8 ~1×10 12 PFU of particles are administered to the subject. In one example, 1 x 10 10 ~1×10 12 PFU of particles are administered to a subject.

[0093] 18. The composition or method of any of the preceding embodiments, wherein the particles are administered to the subject at an MOI (multiplicity of infection) of at least 0.01.

[0094] Optionally, the particles are administered to the subject at an MOI of 1 or less. 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.

[0095] 19. The composition or method of any of the preceding embodiments, wherein the strain or at least one of the strains is an antibiotic-resistant or MDR strain, and / or the strain or at least one of the strains is a B2-I strain.

[0096] For example, the strain or at least one strain is a strain selected from B2-I (STc131), B2-II, B2-IX, and B2-VI.

[0097] For example, at least one MDR strain is resistant to fluoroquinolones and the strain is a beta-lactamase (ESBL)-producing E coli.

[0098] 20. The composition or method of embodiment 19, wherein the antibiotic is a fluoroquinolone (optionally levofloxacin), a carbapenem, or vancomycin, and / or the E coli is a beta-lactamase (ESBL)-producing E coli.

[0099] Optionally, the antibiotic is selected from ciprofloxacin (e.g., Cipro™), gemifloxacin (e.g., Factive™), levofloxacin (e.g., Levaquin®), moxifloxacin (e.g., Avelox™), and ofloxacin.

[0100] For example, E. coli produces CTX-M-15, which is the most abundant enzyme in ESBL-producing E. coli that cause human infections.

[0101] Preferably, the antibiotic is a fluoroquinolone (FQ). For example, the FQ is levofloxacin. Levofloxacin, particularly sold under the trade name Levaquin (trademark), is an antibiotic drug. It is used to treat many bacterial infections, including acute bacterial sinusitis, pneumonia, urinary tract infections, chronic prostatitis, and some types of gastroenteritis. Levofloxacin prophylaxis is recommended to prevent gram-negative bloodstream infections (BSIs) in patients with long-term chemotherapy-induced neutropenia. However, increasing fluoroquinolone resistance may decrease the effectiveness of this approach (see, 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. (2013) found that among the patients tested, nearly one-third of hematopoietic cell transplant (HCT) recipients who were colonized with fluoroquinolone-resistant Enterobacterales (FQRE) before transplantation developed Gram-negative bloodstream infections (BSIs) while receiving levofloxacin prophylaxis, and the infections were typically caused by these colonizing strains. In contrast, levofloxacin prophylaxis was highly effective in patients who were not initially colonized with FQRE. The authors found that 23% of patients hospitalized for HCT were colonized with FQRE, with E. coli being the predominant species. Patients with hematologic malignancies receiving intensive chemotherapy, including those undergoing hematopoietic cell transplantation (HCT), frequently develop severe neutropenia and gastrointestinal mucositis, placing them at high risk for developing BSIs caused by Gram-negative Enterobacterales bacteria.Neutropenic patients often suffer severe consequences from BSIs caused by Enterobacterales, with mortality rates as high as 15–20%. Furthermore, because many fluoroquinolone-resistant Enterobacterales (FQRE) also harbor extended-spectrum β-lactamases (ESBLs), breakthrough infections that occur despite fluoroquinolone prophylaxis may be resistant to first-line antimicrobial therapy for fever and neutropenia. Finally, fluoroquinolone side effects are becoming apparent, including Clostridioides difficile infection, aortic dissection and rupture, hyperglycemia, tendinopathy, QT interval prolongation, and mental status changes. Therefore, fluoroquinolones should only be administered to patients if the clinical benefit is likely to justify these potential side effects. Therefore, although fluoroquinolones may reduce the risk of Gram-negative BSIs in many patients, patients colonized with FQRE do not benefit from fluoroquinolone prophylaxis.

[0102] The high rate of FQRE colonization and the absence of risk factors suggest that FQRE is endemic in the community. Indeed, a study of urinary isolates among 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 urinary E. coli isolates from 2017 found that one-quarter were resistant to FQRE. Furthermore, 13% to 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 isolated in this study were ST131, a common sequence type circulating worldwide, and these isolates are frequently fluoroquinolone-resistant and ESBL-producing.

[0103] Bacteremia caused by extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E), such as E. coli, is associated with inadequate empirical treatment and significant mortality in neutropenic patients (e.g., "Colonization With Levofloxacin-Resistant Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae and Risk of Bacteremia in Hematopoietic Stem Cell Transplant Recipients," Satlin et al., Clin Infect Dis. 2018 Nov 13;67(11):1720–1728. doi:10.1093 / cid / ciy363). This study found that HSCT recipients colonized with levofloxacin-resistant ESBL-E before transplantation and receiving levofloxacin prophylaxis had a high rate of bacteremia from these colonizing strains during neutropenia. This single-center study of 312 HSCT recipients found that 10% of patients were colonized with ESBL-E before their transplant. Nearly one-third of patients who were colonized with ESBL-E before transplant developed subsequent ESBL-E bacteremia during their posttransplant neutropenia, compared with less than 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.

[0104] In one example, a composition or method of the invention is for preventing the passage of lineage group B2 E coli from the gastrointestinal tract to the bloodstream of a subject, thereby preventing or reducing bacteremia in the patient.

[0105] In one example, a composition or method of the invention is for preventing the transfer of lineage group B2 E coli from the urinary tract to the bloodstream of a subject, thereby preventing or reducing bacteremia in the patient.

[0106] In one example, the E coli is contained in the digestive tract of the subject. Optionally, in these examples, the composition is administered orally to the subject.

[0107] In another example, the E. coli is in the subject's urinary tract. For example, the infection is an infection of the kidney, bladder, or urethra. Optionally, in these examples, the composition is administered to the subject's urinary tract, such as by a catheter.

[0108] 21.(i) The nuclease is Cas; (ii) each particle comprises a phage capsid containing nucleic acid; (iii) The composition or method of any preceding embodiment, wherein a first type of transduction particles and a second type of transduction particles are administered to a subject, wherein the first type of particles comprise a first adhesive moiety capable of recognizing and binding to a first cognate moiety selected from the group consisting of LPS, LamB, and Tsx displayed on B2 strain E. coli, and the second type of particles comprise a second adhesive moiety capable of recognizing and binding to a second cognate moiety selected from said group, and wherein the first and second adhesive moieties are different from each other.

[0109] 22. A method for treating or preventing infection with E. coli cells in a human or animal subject, the method comprising administering to the subject a plurality of transducing particles; (a) each particle contains a nucleic acid encoding a Cas nuclease and operable crRNA or guide RNA for chromosomal targeting in a cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, the crRNA or guide RNA is expressed, induces the Cas nuclease, and the nuclease cleaves the cell's chromosome, thereby killing the cell or reducing cell growth or proliferation in the subject; (b) the E. coli cells are E. coli phylogenetic group B2 cells; (c) The method, wherein each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0110] Each nucleic acid preferably encodes a plurality of different cRNAs containing spacer sequences that target E. coli chromosomal genes, for example, each nucleic acid preferably encodes a plurality of different cRNAs containing spacer sequences that target two, three, or four E. coli chromosomal genes selected from fimH, bolA, rpoH, lptA, and murA.

[0111] Optionally, each crRNA or guide RNA includes a spacer that targets an E. coli gene selected from the group consisting of fimH, bolA, rpoH, lptA, and murA. Optionally, each nucleic acid encodes a plurality of different cRNAs or guide RNAs, where the cRNAs or guide RNAs target at least two, three, or four (or all) of the E. coli genes selected from the group consisting of fimH, bolA, rpoH, lptA, and murA. Optionally, each nucleic acid encodes a plurality of different cRNAs or guide RNAs, where the cRNAs or guide RNAs target fimH and bolA. Optionally, each nucleic acid encodes a plurality of different cRNAs or guide RNAs, where the cRNAs or guide RNAs target rpoH and lptA. Optionally, each nucleic acid encodes a plurality of different cRNAs or guide RNAs, where the cRNAs or guide RNAs target fimH and murA. Optionally, each crRNA or guide RNA comprises a spacer sequence 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 consisting of SEQ ID NOs: 6-10.

[0112] Optionally, each nucleic acid encodes a first crRNA, a second crRNA, a third crRNA, a fourth crRNA, and a fifth cRNA, where the cRNAs are different from each other and each crRNA targets a B2 phylogenetic group E. coli gene. Optionally, each nucleic acid encodes a first crRNA, a second crRNA, a third crRNA, a fourth crRNA, and a fifth cRNA, where the cRNAs are different from each other and each crRNA is complementary to a B2 phylogenetic group E. coli gene. Optionally, each nucleic acid encodes a first crRNA, a second crRNA, a third crRNA, a fourth crRNA, and a fifth cRNA, where 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 cRNAs comprise 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 cRNAs comprise 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 cRNAs comprise 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.

[0113] 23. The method of embodiment 22, wherein the method is any one of embodiments 1 to 20, except that, optionally, the nuclease is an endogenous nuclease of the cell and is not encoded by a nucleic acid contained in the particle.

[0114] 24. A composition comprising a plurality of transducing particles for use in a method for treating or preventing infection with E. coli cells in a human or animal subject according to embodiment 22 or 23, comprising: (a) each particle contains a nucleic acid encoding a Cas nuclease and operable crRNA or guide RNA for chromosomal targeting in a cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, the crRNA or guide RNA is expressed, induces the Cas nuclease, and the nuclease cleaves the cell's chromosome, thereby killing the cell or reducing cell growth or proliferation in the subject; (b) the E. coli cells are E. coli phylogenetic group B2 cells; (c) A composition, wherein each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0115] 25. A method for detecting the presence of B2 phylogenetic group E coli in a sample, the method comprising contacting a sample containing B2 phylogenetic group E coli with a composition comprising a plurality of transducing particles; (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of said E. coli cell, wherein said administered particle contacts the cell and introduces the nucleic acid therein, and the nuclease is expressed within 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) each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells; (c) the method comprises detecting that B2 phylogenetic group E coli cells have been killed or have their growth or proliferation reduced.

[0116] 26. A method for detecting the presence of B2 phylogenetic group E coli in a sample, the method comprising contacting a sample containing B2 phylogenetic group E coli with a composition comprising a plurality of transducing particles; (a) each particle comprises a nucleic acid that contains or encodes a detectable label, and the administered particles contact cells and introduce the nucleic acid therein, and optionally, the label is expressed in the cells; (b) each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells; (c) The method comprises detecting phylogenetic group B2 E coli cells containing the label.

[0117] 27. The method of embodiment 26 or 27, wherein the composition comprises the features of the composition described in any one of embodiments 1, 2 to 21, 24 and 25.

[0118] 28. The method of any one of embodiments 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.

[0119] 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), the subject is a transplant or cancer patient (optionally a blood cancer patient), or the patient has or is at risk of a urinary tract infection (UTI), and optionally the transplant is a solid transplant or a stem cell transplant (optionally a hematopoietic cell transplant).

[0120] 30. The method of any one of embodiments 26 to 30, wherein the nucleic acid of each particle comprises a nucleotide sequence (N1) that encodes the nuclease or comprises or encodes a label, and each particle is a synthetic T-even phage (optionally a T4 phage) that comprises an insertion of N1 into the genome of the phage, and the region is between the pin (protease inhibitor) gene and the iPII (internal protein) gene.

[0121] 31. At least 1 x 10 7 32. The method of any one of embodiments 26 to 31, wherein particles of PFU are contacted with the sample.

[0122] 32. The method according to any one of embodiments 26 to 32, wherein the particles are contacted with the sample at an MOI (multiplicity of infection) of at least 0.01.

[0123] Labels for detection methods are familiar to those skilled in the art. The label can be, for example, a fluorescent label, such as GFP. The sample can be blood, saliva, sputum, or a cell sample.

[0124] concept The present invention also provides the following concepts, which are well supported by Example 2. All concepts may be combined with any other features disclosed herein.

[0125] Patients with hematological malignancies frequently develop bloodstream infections due to the translocation of E. coli and other bacteria from the intestine. Antibiotic treatment to prevent these infections has detrimental effects on the microbiome and immune function and is further hindered by the rise of antibiotic resistance, particularly to fluoroquinolones. As described in Example 2, compositions of this concept target bacteria in biofilms, reduce the emergence of phage-resistant E. coli, and are able to outcompete their ancestral WT phage in coculture experiments. Compositions with broad host ranges across the E. coli phylogeny, including multidrug-resistant strains (B2 phylogenetic group), are provided. Compositions comprising different particles of this concept surprisingly reduce E. coli burden in the intestines of mice better than the individual constituent particles. Compositions, methods, and doses of this concept find utility in selectively killing E. coli, which can cause fatal infections in patients with hematological cancers, transplants, or UTIs. These compositions, methods, and doses also find utility in treating or preventing E. coli bloodstream infections.

[0126] To this end, the following is provided:

[0127] Concept A: A composition comprising a plurality of different types of transduction particles, each of said particles comprising a nucleic acid, said particles being capable of contacting E. coli cells and transferring the nucleic acid thereto; (a) the nucleic acid of each particle comprises a nucleotide sequence encoding a product of interest (POI), and the nucleic acid is capable of expressing the POI in E. coli cells; (b) each particle comprises an adhesive moiety for recognizing and binding to a cognate site selected from LPS, LamB, and Tsx (optionally selected from LPS and Tsx) displayed on the surface of an E. coli cell; (c) The composition, wherein the plurality of different types of transduction particles comprises (i) a first type of particle comprising an LPS attachment moiety, and (ii) a second type of particle comprising a Tsx attachment moiety.

[0128] Concept B 1. A composition comprising a plurality of transducing particles for use in a method of treating or preventing infection with E. coli cells in a human or animal subject, the method comprising administering the particles to the subject; (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, and the nuclease is expressed within 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 cells are E. coli phylogenetic group B2 cells; (c) A composition, wherein each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB, and Tsx (optionally selected from LPS and Tsx) displayed on the surface of a phylogenetic group B2 E. coli cell.

[0129] Concept C: 1. A composition comprising a plurality of transduction particles for use in a method for treating or preventing infection by E. coli cells (optionally B2 phylogenetic group E. coli cells) in a human or animal subject, the method comprising administering particles to the subject, wherein (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of an E. coli cell, the administered particles contacting the cell and introducing the nucleic acid therein, the nuclease being expressed within the cell and cleaving the genomic DNA of the cell, thereby killing the cell or reducing the growth or proliferation of the cell in the subject; (b) A composition, wherein each particle comprised in the composition is a capsid of a T-even phage, optionally a capsid of a T2 phage, a T2-like phage, an RB69 phage, or an RB69-like phage.

[0130] The following features are optional features that can be combined with the above concepts or any other configuration, example, embodiment or option herein.

[0131] Optionally, A: each particle of the first type comprises an LPS adhesive moiety and a LamB adhesive moiety; B: each particle of the first type comprises an LPS adhesive moiety and a Tsx adhesive moiety; or C: Each particle of the second type contains the Tsx adhesive moiety but lacks the LamB and LPS adhesive moieties. Optionally, the composition comprises particles according to A, B and C.

[0132] Optionally, each particle comprises a phage capsid containing nucleic acid. In one embodiment, the capsid comprises capsid proteins of T-even (optionally T2) or lambda phage.

[0133] Optionally, the capsid of each particle included in the composition is a T-even phage capsid, optionally a capsid of a T2 phage, a T2-like phage, an RB69 phage or an RB69-like phage.

[0134] Optionally, each particle is a phage (optionally a lytic phage) or a packaged phagemid.

[0135] Optionally, the composition comprises at least three or four different types of transducing particles, hi one embodiment, the composition has four (but not more than four) different types of transducing particles.

[0136] Optionally, the nucleic acid of each particle comprises at least one nucleotide sequence (N1) encoding the POI, and each particle is a synthetic T-even phage comprising an insertion of N1 into a modification-permissive region (MPR) of the phage genome, the MPR extending from immediately after gene 49 to gene E compared to a reference wild-type T2 phage. The nucleic acid may comprise a DNA deletion of the phage DNA in the MPR. The insertion may comprise up to 5000, 6000, 7000, or 8000 bp of DNA, and / or the deletion comprises 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 the MPR comprises at least 100 bp of DNA between gene 49 and gene E.

[0137] 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 said selected phage, which percentage may be at least 96, 97, 98, or 99%.

[0138] 12. The composition of any one of claims 8 to 11, wherein the synthetic phage genome comprises the insertion between coordinates 9000 and 21000, coordinates being nucleotide positions counting from the nucleotide immediately after gene 49 (coordinate number 1) towards gene E compared to a reference wild-type T2 phage. Optionally, the insertion is between coordinates 10300 and 19800, for example between 10359 and 19810 relative to the T2 genome. Optionally, the insertion is within a window selected from the following windows (numbers are coordinates relative to the T2 genome): 9000~21000;10000~21000, 10100~21000, 10200~21000, 10300~21000, 10400~21000, 10500~21000, 11000~21000, 15000~21000; 9000~20000;10000~20000, 10100~20000, 10200~20000, 10300~20000, 10400~20000, 10500~20000, 11000~20000, 15000~20000; 9000~19500;10000~19500, 10100~19500, 10200~19500, 10300~19500, 10400~19500, 10500~19500, 11000~19500, 15000~19500; 9000~19000;10000~19000, 10100~19000, 10200~19000, 10300~19000, 10400~19000, 10500~19000, 11000~19000, 15000~19000; 9000-18000; 10000-18000, 10100-18000, 10200-18000, 10300-18000, 10400-18000, 10500-18000, 11000-18000 and 15000-18000

[0139] Optionally, the nuclease is a dsDNA nuclease, i.e., capable of cleaving dsDNA. Optionally, the nuclease is a nickase, e.g., a Cas9 nickase.

[0140] In one embodiment, the POI is a protein. In one embodiment, the POI is an RNA, e.g., a crRNA.

[0141] Optionally, the POI: (a) a nuclease for targeting the DNA of an E. coli cell, which nuclease can be expressed within the cell to cleave the cell's DNA, thereby modifying or killing the cell; or (b) Dead Cas (dCas) for targeting the DNA of E. coli cells, where dCas can be expressed within the cells and can target the DNA of the cells, thereby modifying the cells. Optionally, when (a) applies, the nuclease is an inducible nuclease, optionally a Cas, meganuclease, zinc finger nuclease, or TALEN, or when (b) applies, the dCas is dCas9.

[0142] The nuclease can be a Type I, II, III, IV, V or VI Cas nuclease, optionally Cas9 or Cas3.

[0143] In a preferred embodiment, the nucleic acid comprises (optionally in 5' to 3' order) the cas3 gene (ygcB), and casA (ygcL, cas8e), casB (ygcK, cas11), casC (ygcJ, cas7), casD (ygcI, cas5), and casE (ygcH, cas6), and optionally a nucleotide sequence encoding a CRISPR array or a guide RNA that targets the E. coli genome.

[0144] In a preferred embodiment, the POI comprises at least one crRNA or guide RNA operable 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 a protospacer of B2 phylogenetic group E. coli cells, or E. coli cells of a strain selected from the group consisting of ST131, ST1193, ST648, ST315, ST405, ST361, ST88, and ST453.

[0145] Each nucleic acid preferably encodes a plurality of different cRNAs containing spacer sequences targeting E. coli chromosomal genes. For example, each nucleic acid preferably encodes a plurality of different cRNAs containing spacer sequences targeting two, three, or four E. coli chromosomal genes selected from fimH, bolA, rpoH, lptA, and murA. In one embodiment, a composition of the invention comprises a type of transduction particle targeting the E. coli genes bolA, rpoH, and fimH. Additionally or alternatively, a composition of the invention comprises a type of transduction particle targeting the E. coli genes lptA and murA. Optionally, the composition comprises a first type of transduction particle targeting the E. coli genes bolA, rpoH, and fimH, and a second type of transduction particle targeting the E. coli genes lptA and murA. Optionally, the first type of particle follows any other first type of particle herein that comprises an LPS-adhering moiety (e.g., comprises an LPS and a Tsx-adhering moiety). Optionally, the second type of particles follows any other first type of particles herein that include a Tsx adhesive moiety. Optionally, the second type of particles includes an LPS adhesive moiety (e.g., includes an LPS and a LamB adhesive moiety).

[0146] The protospacer sequence may be contained in a gene selected from the E. coli genes fimH, bolA, rpoH, lptA, and murA.

[0147] In one embodiment, the particles of the composition target all of the E. coli genes fimH, bolA, rpoH, lptA, and murA.

[0148] The nucleotide sequence encoding the POI may comprise a stress phase active (SPA) promoter for expression of the POI in E. coli cells. The promoter may be the E. coli bolA promoter. The promoter may comprise SEQ ID NO: 13.

[0149] In one embodiment, the E coli cells comprise a strain of E coli that causes sepsis, septicemia or diarrhea in humans or animals.

[0150] Optionally, the E coli cells are GI tract cells. Optionally, the E coli cells are in a gut microbiome. Optionally, the E coli cells are in the blood of the subject. Optionally, the E coli cells are in the urinary tract of the subject. Optionally, the E coli cells are in a microbiome selected from the microbiomes of the GI tract (e.g., stomach), blood, urinary tract, mouth, nose, eye, ear, skin, anus, or hair.

[0151] In one embodiment, the composition is for use in a method of treating or preventing infection with E coli cells in a human or animal subject, the method comprising administering the particles to the subject.

[0152] Optionally, the method is for preventing the passage of lineage group B2 E coli from the gastrointestinal or urinary tract of a subject to the bloodstream, thereby preventing or reducing bacteremia in the subject.

[0153] Optionally, the method is for preventing E. coli infection in a subject at risk of febrile neutropenia. The subject can be a cancer patient, such as a blood cancer patient. The infection can be a bloodstream infection in the subject. Thus, in one embodiment, the method is for preventing bloodstream E. coli infection in a subject at risk of febrile neutropenia, and the subject is a human cancer patient, such as a blood cancer patient.

[0154] The following is provided:- A method for treating or preventing sepsis, septicemia, or diarrhea in a human or animal subject, the method comprising administering to the subject a composition described herein (e.g., a composition of Concept A, B, or C), wherein the E coli cells comprise a strain of E coli that causes sepsis, septicemia, or diarrhea in humans or animals.

[0155] A method for treating or preventing an infection by E. coli cells in a human or animal subject, the method comprising administering to the subject a composition described herein (e.g., a composition of Concept A, B, or C), wherein the infection is treated or prevented.

[0156] The infection may be reduced or eliminated. The infection may be reduced by at least 20, 30, 40, 50, 60, 70, 80, or 90%.

[0157] The subject may be a transplant or cancer patient (optionally a blood cancer patient), or the patient is suffering from or at risk of urinary tract infection (UTI). Optionally, the transplant is a solid organ or stem cell transplant (optionally a hematopoietic cell transplant), or the transplant is a medical device transplant. The subject may be suffering from a blood cancer, for example, leukemia. The subject (e.g., a cancer patient) may be at risk of or suffer from neutropenia. Preferably, the subject is suffering from a blood cancer, for example, leukemia, and is at risk of or suffers from neutropenia.

[0158] Optionally, the method is performed before the subject undergoes a transplant or said transplant. The transplant may be a stem cell transplant, for example, if the subject is a cancer patient.

[0159] The composition or method may be for preventing hemolytic uremic syndrome (HUS), a UTI infection, sepsis, septicemia, or diarrhea in a human subject.

[0160] In a composition for use in the method, or any method described herein, at least 1 x 10 7 PFU of particles are administered to the subject. Also provided is a dose of the composition described herein, the dose being at least 1 x 10 7 For example, the dose is at least 1 x 10 PFU of the particles. 7 , 1×10 8 , 1×109 , 1×10 10 or 1×10 11 For example, the dose is 1 x 10 PFU of the particles. 7 , 2 × 10 9 or 2×10 11 For example, the dose is 1 x 10 PFU of the particles. 7 ~2×10 11 For example, the dose is 1 x 10 PFU of the particles per gram of subject body weight. 7 ~2×10 11 In the method, E coli may be reduced by at least 3 or 4 log10 CFU / g of the subject's body weight.

[0161] The dosage may be contained in a medical device or container, for example, for oral or intravenous administration. The device may be an IV device, a syringe, or may include a needle. The device or container may be sterile.

[0162] Optionally, the particles are administered to the subject at an MOI (multiplicity of infection) of at least 0.01. For example, the MOI is at least 0.1 or 1.

[0163] Preferably, 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. Optionally, the antibiotic is a fluoroquinolone (optionally levofloxacin), a carbapenem, or vancomycin, and / or the E. coli cells comprise a beta-lactamase (ESBL)-producing E. coli.

[0164] In one embodiment, (i) the composition comprises first and second types of particles according to A, B, and C; A: each particle of the first type comprises an LPS adhesive moiety and a LamB adhesive moiety; B: each particle of the first type comprises an LPS adhesive moiety and a Tsx adhesive moiety; or C: Each particle of the second type contains the Tsx attachment site but lacks the LamB and LPS attachment sites; (ii) the POI comprises an inducible nuclease for targeting the DNA of an E. coli cell, wherein the nuclease can be expressed within the cell to cleave the cell's DNA, thereby modifying or killing the cell; (iii) each particle comprises a phage capsid containing nucleic acid; (iv) The particles of the composition target a plurality of different E. coli genes, optionally selected from essential and virulence genes.

[0165] The following is provided:- 1. A method for detecting the presence of E. coli (optionally phylogenetic group B2 E. coli cells) in a sample, comprising: (a) contacting the sample with a composition described herein (e.g., according to Concept A, B, or C); (b) detecting that the E. coli cells have been killed or their growth or proliferation has been reduced; A method comprising:

[0166] 1. A method for detecting the presence of E. coli (optionally phylogenetic group B2 E. coli cells) in a sample, comprising: (a) contacting a sample with a composition described herein (e.g., according to Concept A, B, or C), wherein particles of the composition comprise a nucleic acid that includes or encodes a detectable label, and wherein the particles contact cells to introduce the nucleic acid therein, and optionally, the label is expressed within the cells; (b) detecting that the E. coli cells contain the label; A method comprising:

[0167] A method for modifying the genome of an E. coli cell, the method comprising contacting the cell with a composition 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.

[0168] When the POI is a nuclease, the nuclease is expressed within 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.

[0169] A method for treating or preventing infection with E. coli cells in a human or animal subject, comprising administering to the subject a composition described herein (e.g., according to Concept A, B, or C).

[0170] Optionally, the sample is a patient sample (e.g., a blood, urine, stool, or saliva sample), the subject is a transplant or cancer patient (optionally a blood cancer patient), or the patient has or is at risk for a urinary tract infection (UTI), and optionally the transplant is a solid transplant or a stem cell transplant (optionally a hematopoietic cell transplant).

[0171] The methods herein may, in one embodiment, be carried out in vitro. The methods herein may, in one embodiment, be carried out ex vivo.

[0172] Optionally, at least 1 × 10 7 The particles of the PFU are contacted with the sample. Optionally, the particles of the PFU are contacted with the sample.

[0173] Optionally, the particles are contacted with the sample at an MOI (multiplicity of infection) of at least 0.01.

[0174] Optionally, each particle included in the composition comprises a T-even phage capsid, optionally a T2 phage, a T2-like phage, an RB69 phage, or an RB69-like phage capsid. Each particle may comprise a Tevenvirinae phage capsid. Each particle may be a modified Tevenvirinae phage containing a genomic insertion of a nucleotide sequence encoding a POI or the nuclease (and optionally a cognate crRNA if the nuclease is a Cas).

[0175] Each particle may be a phage or a packaged phagemid. Preferably, the phage is a lytic phage. In another embodiment, the phage may be a non-lytic phage. In another embodiment, the phage may be a temperate phage.

[0176] Optionally, the phage is a modified T2 phage, T2-like phage, RB69 phage, or RB69-like phage. Preferably, the phage is a T2-like phage or RB69-like phage. Preferably, each particle is a modified first phage, and 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 percentage of identity between the phage genomes is determined by Mash analysis using kmer sizes of 17 and 1000.

[0177] Optionally, the composition comprises at least three or four different types of transduction particles, which types have different attachment moieties for recognizing and binding to cognate moieties contained in E. coli cells.

[0178] Optionally, the cell comprises phylogenetic group B2 E coli. 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.

[0179] In one embodiment, (i) the nuclease is an inducible nuclease for targeting the DNA of an E. coli cell, the nuclease being capable of being expressed within the cell to cleave the DNA of the cell, thereby killing the cell; (ii) each particle comprises a phage capsid containing nucleic acid; (iii) the particles of the composition target a plurality of different E. coli genes, optionally selected from essential genes and virulence genes; (iv) optionally, the composition comprises: A: Particles containing an LPS adhesive moiety and a LamB adhesive moiety (and lacking Tsx); B: Particles containing an LPS adhesive moiety and a Tsx adhesive moiety, or C: Particles containing the Tsx adhesive moiety but lacking the LamB and LPS adhesive moieties Includes.

[0180] The nuclease may be a Cas nuclease, and the nucleic acid encodes at least one crRNA or guide RNA operable with Cas for DNA targeting in E. coli cells. Each crRNA or guide RNA may include a spacer sequence complementary to an E. coli protospacer sequence, optionally a protospacer sequence of E. coli cells of phylogenetic group B2 or a strain selected from the group consisting of ST131, ST1193, ST648, ST315, ST405, ST361, ST88, and ST453. Each protospacer sequence may be included in a gene selected from the E. coli genes fimH, bolA, rpoH, lptA, and murA.

[0181] 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.

[0182] Expression of the POI, nuclease, or crRNA can be under the control of a stress-phase active (SPA) promoter. Optionally, the promoter is the E. coli bolA promoter or comprises SEQ ID NO: 13. Additionally or alternatively, expression of the POI, nuclease, or crRNA can be under the control of the E. coli promoter PJ23100 (SEQ ID NO: 14), e.g., a promoter having a nucleotide sequence 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.

[0183] The spacer herein may flank a direct repeat sequence in the nucleic acid contained in the particle, for example, the repeat has the sequence of SEQ ID NO: 15 (or said sequence with up to 5, 4, 3, or 2 nucleotide changes compared to SEQ ID NO: 15).

[0184] In one embodiment, expression of the POI or nuclease or crRNA may be under the control of a first promoter and expression of the adhesive moiety / adhesive moieties is under the control of a second promoter different from the first promoter.

[0185] The adhesive moiety herein is on the outer surface of the cognate particle. In one embodiment, the moiety is comprised in the tail fiber, spike or capsid of the particle, preferably the tail fiber.

[0186] The following items are provided:

[0187] item:

[0188] 1. A composition comprising a plurality of transducing particles for use in a method of treating or preventing infection with E. coli cells in a human or animal subject, the method comprising administering the particles to the subject; (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, and the nuclease is expressed within 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 cells are E. coli phylogenetic group B2 cells; (c) A composition, wherein each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0189] 2. A method for treating or preventing infection by E. coli cells in a human or animal subject, the method comprising administering to the subject a plurality of transducing particles, the method comprising administering to the subject the particles; (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, and the nuclease is expressed within 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 cells are E. coli phylogenetic group B2 cells; (c) The method, wherein each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0190] 3. The composition or method according to item 1 or 2, respectively, wherein the method is for treating or preventing infection in a subject with an E. coli strain selected from the group consisting of ST131, ST1193, ST648, ST315, ST405, ST361, ST88 and ST453.

[0191] 4. The composition or method of any preceding item, wherein the method is for treating or preventing infection in a subject with a plurality of different phylogenetic group B2 strains of E. coli, optionally wherein the plurality includes E. coli ST131 and ST1193 cells.

[0192] 5. The composition or method of any of the preceding items, wherein the subject is a transplant or cancer patient (optionally a blood cancer patient), or the patient has or is at risk for a urinary tract infection (UTI), and optionally the transplant is a solid organ transplant or a stem cell transplant (optionally a hematopoietic cell transplant), or the transplant is a medical device transplant.

[0193] 6. The composition or method of any of the preceding items, wherein the method is performed before the subject receives a transplant.

[0194] 7. The composition or method of any of the preceding items, wherein the B2 E coli cells comprise a strain of E coli that causes sepsis, septicemia, or diarrhea in humans.

[0195] 8. The composition or method of any of the preceding items for preventing hemolytic uremic syndrome (HUS), a UTI infection, sepsis, septicemia or diarrhea in a subject.

[0196] 9. The composition or method of any of the preceding items, wherein each particle comprises a phage capsid containing nucleic acid, and optionally the capsid comprises capsid proteins of a T-even phage (optionally T4) or a lambda phage.

[0197] 10. The composition or method of any of the preceding items, wherein each particle is a phage (optionally a lytic phage) or a packaged phagemid.

[0198] 11. The composition or method of any of the preceding items, wherein at least two, three, or four different types of transducing particles are administered to the subject.

[0199] 12. The composition or method of any of the preceding items, wherein a first type of transduction particles and a second type of transduction particles are administered to a subject, wherein the first type of particles comprise a first adhesive moiety capable of recognizing and binding to a first cognate moiety selected from the group consisting of LPS, LamB, and Tsx displayed on B2 E. coli, and the second type of particles comprise a second adhesive moiety capable of recognizing and binding to a second cognate moiety selected from the group, and wherein the first and second adhesive moieties are different from each other.

[0200] 13. The composition or method of item 12, wherein the first and second cognate moieties are different from each other.

[0201] 14. The composition or method of any of the preceding items, wherein the nucleic acid of each particle comprises a nucleotide sequence (N1) encoding the nuclease, and each particle is a synthetic T-even phage (optionally a T4 phage) comprising an insertion of N1 into the genome of the phage, the region being between the pin (protease inhibitor) gene and the iPII (intrinsic protein) gene.

[0202] 15. The composition or method of any of the preceding items, wherein the nuclease is an inducible nuclease, optionally a Cas, meganuclease, zinc finger nuclease or TALEN.

[0203] 16. The composition or method of any of the preceding items, wherein the nuclease is a Type I, II, III, IV, V or VI nuclease, optionally Cas9 or Cas3.

[0204] 17. At least 1 x 10 7 The composition or method of any of the preceding items, wherein particles of PFU are administered to the subject.

[0205] 18. The composition or method of any of the preceding items, wherein the particles are administered to the subject at an MOI (multiplicity of infection) of at least 0.01.

[0206] 19. The composition or method of any of the preceding items, wherein the strain or at least one of the strains is an antibiotic-resistant or MDR strain and / or the strain or at least one of the strains is a B2-I strain.

[0207] 20. The composition or method of item 19, wherein the antibiotic is a fluoroquinolone (optionally levofloxacin), a carbapenem, or vancomycin, and / or the E coli is a beta-lactamase (ESBL)-producing E coli.

[0208] 21.(i) The nuclease is Cas; (ii) each particle comprises a phage capsid containing nucleic acid; (iii) The composition or method of any preceding item, wherein a first type of transduction particles and a second type of transduction particles are administered to a subject, wherein the first type of particles comprise a first adhesive moiety capable of recognizing and binding to a first cognate moiety selected from the group consisting of LPS, LamB, and Tsx displayed on B2 strain E. coli, and the second type of particles comprise a second adhesive moiety capable of recognizing and binding to a second cognate moiety selected from the group, and wherein the first and second adhesive moieties are different from each other.

[0209] 22. A method for treating or preventing infection with E. coli cells in a human or animal subject, the method comprising administering to the subject a plurality of transducing particles; (a) each particle contains a nucleic acid encoding a Cas nuclease and operable crRNA or guide RNA for chromosomal targeting in a cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, the crRNA or guide RNA is expressed, induces the Cas nuclease, and the nuclease cleaves the cell's chromosome, thereby killing the cell or reducing cell growth or proliferation in the subject; (b) the E. coli cells are E. coli phylogenetic group B2 cells; (c) The method, wherein each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0210] 23. The method of item 22, wherein the method is described in any one of items 1 to 20, except that, optionally, the nuclease is an endogenous nuclease of the cell and is not encoded by a nucleic acid contained in the particle.

[0211] 24. A composition comprising a plurality of transducing particles for use in a method for treating or preventing infection with E. coli cells in a human or animal subject according to item 22 or 23, comprising: (a) each particle contains a nucleic acid encoding a Cas nuclease and operable crRNA or guide RNA for chromosomal targeting in a cell, wherein the administered particle contacts the cell and introduces the nucleic acid therein, the crRNA or guide RNA is expressed, induces the Cas nuclease, and the nuclease cleaves the cell's chromosome, thereby killing the cell or reducing cell growth or proliferation in the subject; (b) the E. coli cells are E. coli phylogenetic group B2 cells; (c) A composition, wherein each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells.

[0212] 25. A composition or method according to any preceding item for preventing the translocation of phylogroup B2 E coli from the gastrointestinal or urinary tract of a subject to the bloodstream, thereby preventing or reducing bacteremia in the patient.

[0213] 26. A method for detecting the presence of B2 phylogenetic group E coli in a sample, the method comprising contacting a sample containing B2 phylogenetic group E coli with a composition comprising a plurality of transducing particles; (a) each particle comprises a nucleic acid encoding a nuclease for targeting the genome of said E. coli cell, wherein said administered particle contacts the cell and introduces the nucleic acid therein, and the nuclease is expressed within 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) each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells; (c) the method comprises detecting that B2 phylogenetic group E coli cells have been killed or have their growth or proliferation reduced.

[0214] 27. A method for detecting the presence of B2 phylogenetic group E coli in a sample, the method comprising contacting a sample containing B2 phylogenetic group E coli with a composition comprising a plurality of transducing particles; (a) each particle comprises a nucleic acid that contains or encodes a detectable label, and the administered particles contact cells and introduce the nucleic acid therein, and optionally, the label is expressed in the cells; (b) each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB, and Tsx displayed on the surface of phylogenetic group B2 E. coli cells; (c) The method comprises detecting phylogenetic group B2 E coli cells containing the label.

[0215] 28. The method according to item 26 or 27, wherein the composition comprises the features of the composition according to any one of items 1, 2 to 21, 24 and 25.

[0216] 29. The method of any one of items 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.

[0217] 30. The method of any one of items 26 to 29, wherein the sample is a patient sample (e.g., a blood, urine, stool, or saliva sample), and the subject is a transplant or cancer patient (optionally a blood cancer patient), or the patient has or is at risk of a urinary tract infection (UTI), and optionally the transplant is a solid transplant or a stem cell transplant (optionally a hematopoietic cell transplant).

[0218] 31. The method according to any one of items 26 to 30, wherein the nucleic acid of each particle comprises a nucleotide sequence (N1) that encodes the nuclease or comprises or encodes a label, and each particle is a synthetic T-even phage (optionally a T4 phage) that comprises an insertion of N1 into the genome of the phage, and the region is between the pin (protease inhibitor) gene and the iPII (internal protein) gene.

[0219] 32. At least 1 × 10 7 32. The method according to any one of items 26 to 31, wherein particles of PFU are contacted with the sample.

[0220] 33. The method according to any one of items 26 to 32, wherein the particles are contacted with the sample at an MOI (multiplicity of infection) of at least 0.01.

[0221] It will be understood that the specific embodiments described herein are shown by way of illustration and not as limitations of the invention. The key features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine research, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of the present invention and encompassed by the claims. All publications and patent applications mentioned herein are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications, and all U.S. equivalent patent applications and patents, are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Reference is made to the publications mentioned herein and to equivalent publications from the United States Patent and Trademark Office (USPTO) or WIPO, the disclosures of which are incorporated herein by reference to provide disclosure that may be used in the present invention and / or to provide one or more features (e.g., of vectors) that may be included in one or more claims herein.

[0222] In the claims and / or specification, when used in conjunction with the word "comprising," the use of the phrase "a" or "an" may mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one." The use of the word "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only, or the alternatives are not mutually exclusive, although the present disclosure supports a definition that refers only to alternatives and "and / or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method employed to determine the value, or the variation that exists among study subjects.

[0223] 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.

[0224] As used herein, "or combinations thereof" or similar terms refer 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, where order is important in the particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations including repeats of one or more items or terms are expressly included, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those of skill in the art will understand that there is typically no limit to the number of items or terms in any combination, unless otherwise clear from the context.

[0225] Any part of this disclosure may be read in combination with any other part of this disclosure, unless otherwise clear from the context.

[0226] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to the compositions and / or methods, and to the steps or sequence of steps of the methods described herein, without departing from the concept, spirit, and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0227] The invention is described in more detail in the following non-limiting examples. [Example]

[0228] Surprisingly, particle compositions for targeting multiple different E. coli strains, including the B2 phylogenetic group, overview Patient samples containing a variety of different E. coli strains were challenged with a particle composition. The composition included multiple transduction particles carrying an adhesive moiety capable of binding to LPS, LamB, or Tsx present on E. coli cells. The particles contained capsids containing phage capsid proteins, which in turn contained nucleic acids encoding a CRISPR / Cas system for chromosomal targeting in E. coli. Each nucleic acid encoded multiple different cRNAs containing spacer sequences targeting E. coli chromosomal genes. The ability to kill or reduce the growth of E. coli in the samples was determined using a plaque assay, as described below. Whole-genome sequencing and genome assembly were used to assign susceptible E. coli strains to phylogenetic groups. Surprisingly, the use of such particle compositions was found to be highly effective and broadly targeted against multiple different E. coli strains (clinically relevant strains from actual patient samples). Furthermore, advantageously, numerous different strains of the B2 phylogenetic group were targeted and killed. This is significant because B2 E. coli strains often exhibit antibiotic resistance (e.g., MDR), e.g., resistance to fluoroquinolones, and cause potentially life-threatening infections in patients, such as cancer, transplant, and UTI patients. In addition to group B2, the inventors were surprisingly also able to successfully kill or inhibit the growth of multiple strains of E. coli phylogenetic groups B1, D, F, and G.

[0229] Patient sampling The tested samples (n = 71) were obtained from two prospective observational studies that enrolled adult (≥18 years) patients admitted for autologous or allogeneic hematopoietic cell transplantation (HCT) and who received levofloxacin (fluoroquinolone, FQ) prophylaxis starting the day before transplantation (day -1) (Satlin 2021 and Satlin 2018). Trimethoprim-sulfamethoxazole (TMP-SMX) was administered to allogeneic HCT recipients 2–4 days before HCT. Because antibiotic treatment does not eradicate all E. coli in patients, e.g., fluoroquinolone-resistant E. coli persist, E. coli was obtained from patients by sampling. E. coli isolates were obtained from either perianal swabs or fecal samples obtained upon admission for transplantation. Sampling timing varied from day -7 (7 days before transplantation) to day 0 (the day of transplantation).

[0230] General plaque assay: spotting for coverage This procedure describes a method for assessing the coverage of phage particle lysates against a panel of bacterial strains.

[0231] [Table 1]

[0232] Spotting was performed according to the general plaque assay described above. Bacterial strains were prepared by inoculating 5 μl of 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 in a culture tube was mixed (at 55°C) with 3 ml of pre-warmed top agar containing 5 mM CaCl2 and 5 mM MgSO4. The mixture was poured onto the top of a pre-conditioned LB plate and evenly distributed by swirling. The plate was left on the bench for 5-10 minutes to solidify. Meanwhile, the particle composition was diluted in PBS buffer to 10 μl. 0 ~10 -9The plates were diluted to 100 μL, and 5 μL of each serial dilution was spotted onto the overlay. The plates were left on a bench with the lid open for 20 minutes or until the spots were completely absorbed by the agar, and then incubated upside down at 37°C overnight.

[0233] Result evaluation: If visible plaques appeared, the result was recorded as positive, the plaques were counted, and the phage concentration was calculated as follows: number of plaques x 200 x the dilution at which plaques were observed, i.e., if 5 plaques were counted at dilution -6: 5 x 200 x 1e6 = 1e9 pfu / ml.

[0234] If no visible plaques were present but inhibition of growth was observed, the result was recorded as a zone of lysis and the lowest dilution of inhibition was noted.

[0235] If no plaques or inhibition were observed, the result was recorded as negative.

[0236] Whole genome sequencing DNA extraction was performed using the Omega Bio-tek Mag-Bind Bacterial DNA 96 Kit. Samples were eluted in 100 μL of elution buffer according to the protocol. Sequencing libraries were generated using an Illumina Nextera XT and paired-end sequencing was performed (300 cycles) on an Illumina MiSeq instrument equipped with a V2 flow cell. The average sequencing depth for all samples was 48x (range: 31-72x).

[0237] Genome assembly and phylogenetic tree reconstruction 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 kmer sizes of 17 and 1000 sketches. Neighbor-joining trees were constructed using rapidnj 2.3.2 (Simonsen et al., 2008). Final tree visualizations were generated with Interactive Tree of Life (iTOL) version 6.5.3 (Letunic et al., 2021). Phylogenetic classification of the fungus was performed in silico using the method described in "ClermonTyping: an easy-to-use and accurate in silico method for Escherichia genus strain phylotyping," Johann Beghain et al., Microb Genom. 2018 Jul;4(7):e000192, published online June 19, 2018, doi:10.1099 / mgen.0.000192, PMCID: PMC6113867, PMID: 29916797. We used the set of primer sequences described in Table S1 of that reference (available in the online version of this article).

[0238] result Surprisingly, the use of such particle compositions was found to be highly effective and broadly targeted against multiple different E. coli strains (clinically relevant strains from actual patient samples) (see Figure 1). Furthermore (see Figure 2), advantageously, numerous different strains of the B2 phylogenetic group were targeted and killed. This is significant because B2 E. coli strains often exhibit antibiotic resistance (e.g., MDR), e.g., resistance to fluoroquinolones, and cause potentially life-threatening infections in patients, including cancer, transplant, and UTI patients. More than 10 different ST131 strains were killed (forming plaques), and more than 10 different ST1193 strains were killed (forming plaques). Sequence type 1193 has recently emerged as a new pathogenic and resistant lineage among fluoroquinolone-resistant E. coli. Escherichia coli ST131 is a globally dominant multidrug-resistant clone implicated in a high proportion of rUTIs. Uropathogenic E. coli (UPEC) is a major cause of urinary tract infections (UTIs), accounting for approximately 90% of all cases. The majority of UPEC strains belong to E. coli phylogenetic groups B2 or D and are often clonal. The most commonly isolated sequence types (STs) worldwide are ST69, ST73, ST95, and ST1312. The recently emerged ST131 clone, which has spread worldwide, is a major cause of hospital-acquired and community-acquired UTIs, as well as bloodstream infections, and infections in companion animals and poultry. First identified in 2008, ST131 has been linked to the global spread of CTX-M-15 extended-spectrum β-lactamase (ESBL) resistance genes. Most ST131 strains are now strongly associated with multidrug resistance (MDR), including resistance to fluoroquinolones. Recent reports have also identified strains resistant to last-line carbapenems.

[0239] Importantly, we included clinical samples from patients who developed E. coli bacteremia (despite pretreatment with FQ / TMP-SMX). The composition was able to kill or reduce the growth of E. coli strains in these samples (further indicating the composition's potential use in preventing bacteremia in subjects). This included strains of the following Multi-Locus Sequence Typing (MLST) types (see Figure 2): ST648, ST315, ST405, ST361, ST88, ST453, ST1193, and ST131.

[0240] In addition to group B2, the inventors were also surprisingly able to successfully kill or inhibit the growth of multiple strains of E. coli phylogenetic groups B1, D, F and G.

[0241] References · Shifu Chen, Yanqing Zhou, Yaru Chen, Jia Gu; fastp: an ultra-fast all-in-one FASTQ preprocessor, Bioinformatics, Volume 34, Issue 17, 1 September 2018, Pages i884-i890, https: / / doi.org / 10.1093 / bioinformatics / bty560 · 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 · 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 · 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 · 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 · Michael J. Satlin et al, Colonization with Fluoroquinolone-resistant Enterobacterales decreases the effectiveness of fluoroquinolone prophylaxis in hematopoietic cell transplant recipients. Clinical Infectious Diseases 2021. · Michael J. Satlin et al, Colonization with Levofloxacin-resistant extended-spectrum β-lactamase-producing Enterobacteriaceae and risk of bacteremia in hematopoietic cell transplant recipients. Clinical Infectious Diseases 2018. [Example]

[0242] Phage-derived CRISPR therapy designed to reduce E. coli in patients with blood cancer summary Patients with hematological malignancies frequently develop bloodstream infections due to the translocation of E. coli and other bacteria from the intestine. Antibiotic treatment to prevent these infections has detrimental effects on the microbiome and immune function, further hindered by the rise of antibiotic resistance, particularly to fluoroquinolones. We screened a library of 162 wild-type (WT) phages and identified eight phages with broad coverage of E. coli. We engineered selected phages with novel tail fibers and CRISPR-Cas mechanisms to target clinically relevant E. coli. The engineered phages target bacteria in biofilms, reduce the emergence of phage-resistant E. coli, and outcompete their ancestral WT phages in coculture experiments. SNIPR001 contains four engineered bacteriophages with a broad host range across E. coli lineages, including multidrug-resistant strains. SNIPR001 was well tolerated in animals and reduced E. coli burden in the mouse intestine better than its constituent components, making it a novel CRISPR-Cas therapy designed to selectively target E. coli, a potentially fatal infection in patients with hematologic cancers.

[0243] Introduction Cancer treatments continue to improve, increasing survival rates for people with hematologic malignancies 1 However, chemotherapy regimens frequently used in this immunocompromised population cause myelosuppression and gastrointestinal mucositis with increased intestinal permeability. 2~4 Translocation of enteric bacteria, including E. coli, from the gastrointestinal tract is a frequent cause of bloodstream infections (BSIs). 5 .

[0244] Because the mortality rate associated with bloodstream infections caused by enteric bacteria such as E. coli is 15-20% 6 Antibiotic prophylaxis is indicated in people at risk for febrile neutropenia. 7 Although there are no approved treatments for the prevention of bloodstream infections in patients with hematologic cancers, fluoroquinolones are used off-label in the United States based on two randomized trials demonstrating a reduction in bacterial infections during neutropenia. 7、8、9 In addition to the side effects of fluoroquinolones, including safety warnings and precautions, bacterial resistance is increasing in cancer patients and is approaching 60% of E. coli bloodstream infections in the United States. 10 In immunocompromised patients with hematologic malignancies who develop chemotherapy-induced neutropenia, E. coli accounts for 25.1–30% of all bacteremia cases. 11、12 Furthermore, bloodstream infections in patients with hematopoietic cancers who have undergone hematopoietic stem cell transplantation 5 Up to 65% of E. coli isolated as causative pathogens from the study were resistant to fluoroquinolones. 13 Therefore, novel narrow-spectrum prophylactic options that also cover fluoroquinolone-resistant E. coli are needed to prevent infections in these vulnerable patients.

[0245] Bacteriophage therapy has been used since before antibiotics were widely available. 14 , with several successful individual case reports 16~18 Currently attracting renewed attention due to the rise in bacterial antimicrobial resistance in combination with 15However, few clinical trials have been conducted using wild-type (WT) phages. 19~22 Although several have been directed against E. coli, they have failed to obtain convincing results in larger randomized controlled trials due to possible incomplete coverage of target strains by the phage cocktail. 23 A recent attempt was to use Klebsiella pneumoniae strains (n=17). 24 Phages (n=41) and Vibrio strains (n=294) targeting 25 This includes a more extensive characterization of phages (n=248) targeting T3, suggesting that good coverage of target strains can be achieved by large-scale systematic screening. Synthetic biology has also been used to engineer the T3 phage tail fiber and expand the spectrum of strains that engineered phages can target. 26 Finally, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems may contribute to the effectiveness of phage killing as a complementary killing mode to their lytic activity. CRISPR-associated nuclease (Cas) and CRISPR-RNA form a complex that, in some CRISPR-Cas systems, can bind to homologous DNA target sequences and result in DNA degradation. 27、28 Because prokaryotes lack error-prone non-homologous end joining and rely solely on homologous recombination to repair DNA damage, they are prone to cell death after DNA degradation. This vulnerability has been exploited by using CRISPR-Cas as an antibacterial modality against several bacteria, including Staphylococcus aureus, E. coli, or Clostridium difficile. 29~35 .

[0246] To address the significant unmet medical need for novel prophylactic agents for patients with hematological malignancies, we report the development of SNIPR001. Our research process for designing SNIPR001 involved several steps (Figure 3). Briefly, a library of WT phages (n = 162) was tested in vitro against a phylogenetically diverse panel of E. coli strains representing the biology of the target bacterium, E. coli. WT phages with the broadest and most complementary target strain coverage were selected for further engineering. Selected WT phages were subjected to both tail fiber engineering and CRISPR-Cas arming to generate a library of CRISPR-Cas armed phages (CAPs). The CAP library was evaluated for manufacturability, in vitro stability, spectrum of efficacy, in vivo pharmacokinetics, and efficacy. A combination of four CAPs was selected to generate the development candidate SNIPR001, which is currently in clinical development (ClinicalTrials.gov ID NCT05277350).

[0247] result Wild-type lytic phages α15, α17, α20, α48, and α51 (all members of the Tevenvirinae family) were used as starting points for phage engineering to generate synthetic phage types (CRISPR-armed phages; CAPs) α15.2, α20.4, α48.4, or α51.5. The tail fiber specificities for the cognate E. coli surface ligands TSX, LPS, and LamB were as follows: α15.2 binds to LPS and Tsx α20.4 binds to LPS and LamB α48.4 binds to Tsx α51.5 binds to Tsx

[0248] A cocktail containing these phage types targeting LPS, LamB, and Tsx was generated (this cocktail is referred to herein as SNR001).

[0249] CRISPR-Cas-arming of phages to target E. coli Selected lytic phages are CRISPR-Cas-armed and CAP libraries are generated using the E. coli IE CRISPR-Cas system. 39 was engineered to target phylogenetically diverse E. coli strains. A CRISPR-Guided Vector (CGV™) was generated containing the cas3 gene (ygcB) and downstream cascade gene complexes consisting 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 distinct genes in the E. coli genome. To evaluate the killing efficiency of the CRISPR-Cas system, conjugation of CGV-EcCas to E. coli strain b52 resulted in an average of 3.5 log reduction in killing compared to the empty vector. 10 The CGV-EcCas demonstrated a reduction in CFU / mL. 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 against a simplified panel of 82 E. coli strains. Conjugate delivery of the empty vector was achieved in 75% of the isolates. For all strains delivered with CGV-EcCas, bacterial counts were reduced below the limit of detection (LOD, 200 CFU / mL), which was 1–6 log 10 This corresponds to a decrease in β-actin, highlighting the potent CRISPR-Cas-mediated killing ( Fig. 3A ).

[0250] Promoter P bolA In addition, the CRISPR-Cas system was engineered to encode a synthetic constitutively expressed E. coli promoter (P J23100 ) because targeting multiple regions has been shown to prevent the evolution of resistance. 43CRISPR 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). 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 CAP α15.2 at an equal MoI compared to WT α15. We observed increased cas3 RNA levels only during CAP α15.2 infection. We then extended this assay to all four CAPs (α15.2, α20.4, α48.4, and α51.5) and demonstrated increased cas3 transcript levels, highlighting that the CAPs expressed the CRISPR-Cas system during infection with the target strains.

[0251] To demonstrate the competitive advantage of CAPs, we performed a competition experiment in which CAPs (α20.4 and α15.2) and their WT ancestor phages were co-cultured with E. coli strain b230, which served as a target for both competing phages. They were co-cultured at an approximate initial ratio of one CAP to nine WT phages and passaged four times onto fresh target cells in liquid culture. After each passage, the relative abundance of CAP and WT phage particles was assessed. Both CAPs outcompeted their WT counterparts within four rounds, with CAP α20.4 reaching 68% after four rounds and CAP α15.2 reaching 86% after two rounds (Figure 3B-C), demonstrating improved fitness compared to the WT phages.

[0252] CAP Cocktail SNIPR001 The activity of the CAPs was tested against an E. coli panel (n=429) using a growth rate assay. To maximize our coverage, we combined the CAPs to obtain a composition (SNR001) containing phage types α15.2, α20.4, α48.4, and α51.5.

[0253] The ancestors of CAPs α15.2, α20.4, α48.4, and α51.5 are classified as the Tevenvirinae subfamily. Specifically, the ancestors of α15, α48, α20, and α51 share sequence similarity with E. coli phages T2, T4, and RB69 (as determined by Mash analysis), as shown below. In silico analysis of the SNIPR001 genome indicated that CAPs do not encode known transposase or integrase genes, suggesting that the phages are not lysogenic and therefore are not predicted to be able to insert their DNA into bacterial cells.

[0254] Similarity Matrix:

[0255] [Table 2]

[0256] This similarity was calculated, where the distance between genomes was calculated using Mash (v1.1) with a k-mer size of 21 and a sketch size of 10,000.

[0257] For a further description of Mash, see 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.

[0258] SNIPR001 does not affect other gastrointestinal-associated bacteria Ideally, phage-based therapies should not perturb non-target genera of the microbiome, so the specificity of SNIPR001 against E. coli was evaluated by investigating its effect on a panel of strains including closely related non-E. coli species and various families associated with the resident bacterial community in the gut (and E. coli as a positive control). Bacteria were cultured without CAP, with the SNIPR001 cocktail, or with individual SNIPR001CAPs (n=4). Growth in CFU / mL (DCFU / mL) was assessed over 4 hours. 4h~0h ) In parallel, E. coli b2480 was grown under the same conditions as a positive control (Figure 4). We observed no significant effect of either the SNIPR001 cocktail or SNIPR001CAP on non-E. coli strains (p>0.05, Student's t-test, FDR corrected by Holm's method), whereas E. coli growth was significantly inhibited (p<0.05, Student's t-test, FDR corrected by Holm's method). Therefore, SNIPR001 is not expected to affect the gut microbiome other than the target E. coli.

[0259] In vitro host range of SNIPR001 in clinical target populations To understand its potential effect on strains associated with hematological cancer patients, SNIPR001 coverage was tested against our internal E. coli panel (429 strains) and a set of 382 clinical E. coli strains (JMI Laboratories, North Liberty, IA, USA). These JMI strains were derived from patients with bloodstream infections admitted to hematology / oncology departments across four different regions (54 Asia-Pacific isolates, 161 Europe isolates, 26 Latin America isolates, and 141 North America isolates) between 2018 and 2020. The genotype distribution of E. coli strains in the patient population was determined using whole-genome sequencing and was found to represent nine diverse phylogenetic groups and 118 multilocus sequence types (MLSTs) (Figure 5A). Using a spotting assay, we recorded phage infectivity against the panel of JMI strains. When single plaques could not be confirmed, visible single plaques were distinguished from lysis zones. All spotting assays were performed in duplicate. We observed a total coverage of 90.4 ± 1.6% for SNIPR001 in the 382 JMI E. coli panel and 95.6 ± 0.3% for SNIPR001 in the internal E. coli panel (429 strains). Furthermore, we observed plaques in 53.1 ± 7.7% of the JMI panel strains and lysis zones in 37.3 ± 6.1% of the strains, as well as plaques in 60.5 ± 6.6% of the internal panel strains and lysis zones in 35.1 ± 6.3% of the strains (Figure 5B). SNIPR001 showed 100% coverage in the B2 phylogenetic group, which represents 53% of the JMI panel. This phylogenetic group is correlated with multidrug resistance and virulence. In addition, we observed that SNIPR001 covered 91.7% (n = 55) of strains 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 fluroquinolones such as ciprofloxacin and levofloxacin (Figure 5C).

[0260] Finally, we validated SNIPR001 against a clinical panel (n = 72) of fluroquinolone-resistant E. coli strains isolated from either fecal samples or anal swabs from patients with hematological cancers. This population represents the expected clinical target patient population being followed (SNIPR001 has been granted fast-track status by the FDA). A subset of these strains caused bloodstream infections (Figure 5D). 82% (n = 72) of E. coli strains were susceptible to at least two or more of the CAPs in SNIPR001, and 93% of strains were susceptible to the entire SNIPR001 cocktail (Figure 5E). This data demonstrates the benefit of SNIPR001 compared to individual CAPs in terms of improved efficacy spectrum.

[0261] Tolerance and gastrointestinal recovery of SNIPR001 in minipigs The tolerability and gastrointestinal recovery of SNIPR001 were evaluated in Göttingen minipigs. 12 After oral administration of PFU of SNIPR001 or vehicle, blood and feces were collected over 7 days. CAP was not recovered from plasma, indicating a lack of systemic exposure, but CAP was detected at 2 × 10 7 Peak PFU were recovered in the feces by 7 days after SNIPR001 administration (Figure 6A). Minipigs showed no clinical signs, and no significant changes were observed in hematological or biochemical parameters, particularly in any immune cell group compared to vehicle treatment, confirming that SNIPR001 was well tolerated. Similar recovery of individual CAPs was obtained (Figure 6B). In conclusion, SNIPR001 appears to be well tolerated in Göttingen minipigs with gastrointestinal recovery.

[0262] Efficacy of SNIPR001 and constitutive CAP in a mouse colonization model To evaluate the in vivo efficacy of the four selected CAPs in reducing E. coli , we performed the same method as Galtier et al. 44A mouse gastrointestinal colonization model from [1] was adapted 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 discontinued and the animals were inoculated with E. coli b17 (1 × 10 7 Mice were orally inoculated with a single dose of 1000 mg / mL (CFU). This allowed for stable colonization for 3–4 days. To assess the effectiveness of CAP against established colonization, treatment began 2 days after inoculation, and the study was terminated 4 days after inoculation, as colonization began to decline. To ensure maximum exposure to CAP, mice were treated with three doses per day, administered 8 hours apart, for a total of six doses over two days.

[0263] Mice were treated with either high, medium, or low doses (2 × 10 11 PFU, 2 × 10 9 PFU, 1 × 10 7 Mice were treated by oral gavage with 1000 PFU of SNIPR001, vehicle (negative control), or gentamicin (positive control). Fecal CAP recovery was 3 x 10 at the low dose. 7 PFU / g to 1 × 10 at high doses 10 PFU / g range, confirming successful GI transit (Figure 6C). These levels of CAP 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 24 hours after treatment (day 3). At the high dose, SNIPR001 reduced the target E. coli population by 4 log 10This resulted in a significant reduction in CFU / g (Figure 6D). Despite increased variability in bacterial recovery on day 4, likely due to clearance of colonizing strains, as noted in the vehicle group, a similar reduction was observed two days after treatment (day 4). Although the medium dose did not reach statistical significance (p<0.05, Mann-Whitney U test), there was still a numerical reduction compared to the vehicle group. We then compared the efficacy of individual CAPs with the SNIPR001 cocktail in this model. In this experiment, we observed a greater reduction in colonization of the target strain with SNIPR001 compared to either single CAP (which showed a numerical but not statistically significant reduction), highlighting the efficacy advantage of the combination (Figure 6E). We also assayed the resistance profile of randomly collected surviving bacteria and found no isolates that were resistant to the SNIPR001 cocktail. Overall, this data demonstrates the ability of SNIPR001 to reduce target E. coli in the GI tract of colonized mice.

[0264] Consideration Here, we describe the development of SNIPR001, designed to target enteric E. coli, which frequently migrates into the bloodstream and causes bloodstream infections, in neutropenic patients with hematologic cancers. Despite off-label use of fluoroquinolones, these patients continue to experience high morbidity and mortality. The use of traditional antibiotics has provided important health benefits over the past century. However, at the same time, we are now experiencing a significant rise in bacterial resistance, with an estimated 1.27 million deaths attributed to bacterial antimicrobial resistance in 2019, with E. coli being the primary pathogen. 45 Therefore, new antibiotic treatments are needed to address both the unmet medical need for antimicrobial-resistant infections in this vulnerable population. In this study, we describe the development of SNIPR001, a novel development candidate that may address these challenges.

[0265] Confirmation of SNIPR001 efficacy against a large, clinically relevant panel of strains supports the clinical potential of SNIPR001. 10 The reduction in 4950 .

[0266] SNIPR001 is an orthogonal antibacterial approach, as it has shown activity against multidrug-resistant strains. Additionally, there is emerging evidence that maintaining a normal microbiome is important for supporting immune function and may benefit cancer treatment outcomes. 51 This is also recognized in many current guidelines regarding the prophylactic management of patients at risk for febrile neutropenia. 7 In this context, in vitro studies with SNIPR001 demonstrated specificity for E. coli with no off-target effects on any of the non-E. coli strains tested, thereby indicating little detrimental impact on the microbiome. In the future, personalized combinations of narrow-spectrum antibiotics such as SNIPR001 may be used as a first-line treatment, rather than in addition to broad-spectrum antibiotics such as fluoroquinolones.

[0267] Clinical studies are currently underway in the United States to evaluate SNIPR001's ability to confirm safety and reduce E. coli in the gut without disrupting the overall gut microbiome (NCT05277350). The inventors believe that SNIPR001 represents a potentially important therapeutic advance in the field of antimicrobials 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.

[0268] [Table 3]

[0269] [Table 4]

[0270] method 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 cocktail or wastewater sample. After 6 minutes of incubation at room temperature (when infection should occur), Ca 2+ Three milliliters of prewarmed top agar containing the phage or wastewater mixture was added to the E. coli / phage mixture and immediately poured onto an LB plate. Alternatively, 10-fold dilutions of each cocktail were spotted onto lawns prepared with the isolates. After drying, the plates were incubated overnight at 37°C. Plaques were excised from each plate, resuspended in 500 μL of SM buffer, vortexed, and stored at 4°C. 10-fold dilutions were spotted onto the isolate from which the plaques were originally excised. This procedure was repeated at least three times to increase the likelihood of obtaining plaques corresponding to a single phage. Lysates were prepared from single plaques excised in previous rounds of propagation. DNA was extracted, and their genomes were sequenced.

[0271] E. coli panel and isolation procedure Three E. coli panels were included in this study: one in-house panel and two clinically relevant panels. The in-house panel consisted of 429 phylogenetically diverse E. coli strains isolated from the blood of patients with bloodstream and urinary tract infections, feces of people without known disease, animals, and the environment. The strains encompassed seven different phylogenetic groups (A, B1, B2, C, D, E, and F), 114 multilocus sequence typing (MLST) groups, serotypes (K and O), antibiotic resistance profiles, and different geographic locations of isolates.

[0272] The JMI panel consists 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 admitted to hematology-oncology departments across four different regions (54 Asia-Pacific isolates, 161 Europe isolates, 26 Latin America isolates, and 141 North America isolates) and sourced through the SENTRY Antimicrobial Surveillance Program (2018–2020), which comprises a network of over 150 medical centers in over 28 countries worldwide (https: / / www.jmilabs.com / sentry-surveillance-program).

[0273] Finally, a panel of 72 fluoroquinolone-resistant E. coli strains was isolated from either fecal samples or anal swabs of hematological cancer patients admitted for hematopoietic cell transplantation. 53、54 .

[0274] E. coli strains were grown in lysate broth (LB) at 37°C, 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 needed. The conjugation donor, E. coli JKE201, was used. 55 All media for growth of and its derivatives were supplemented with 1,6-diaminopimelic acid (DAP) (80 μg / mL) to complement their auxotrophy.

[0275] Both E. coli strain b52, used to produce α15.2, α48.4, and α51.5, and E. coli strain b2479, selected to produce α20.4, belong to phylogenetic group A. Strain E. coli b17 was used as the colonizer in the in vivo efficacy model because it is susceptible to all SNIPR001 CAPs and is part of the SNIPR Biome strain bank.

[0276] Phage screening by growth rate The in vitro susceptibility of an in-house panel of E. coli (n=429) to 162 WT phages was assessed using a growth rate assay. This assay measures bacterial metabolic activity by tracking the reduction of a tetrazolium dye to a purple compound that aggregates during bacterial growth. (Henry et al., 2012) 56 Colorimetric measurements were recorded every 15 minutes for 24 hours using an OmniLog® (Biolog, Hayward, CA, USA) adapted from. The area under the inhibition curve (iAUC) was calculated from the rate curve over the course of the experiment and defined as the ratio of the normalized AUC of the phage-treated bacterial growth curve to the bacteria-only control. Sensitivity was defined as an iAUC value ≥ 0.2.

[0277] Calculating bacterial growth inhibition using iAUC The growth inhibitory effect of SNIPR001 was determined using growth rate curves constructed using an OmniLog® instrument. To reduce technical variability in measurements between time points, a cubic smoothing spline function was applied to the data in Scala using the "umontreal.ssj.functionfit" package. To identify appropriate ρ and weight variables, all combinations of ρ and weights of 0.1 and 0.5 were applied in increments of 0.1 (i.e., 0.1, 0.2, ... 0.5). The spline with the smallest mean absolute error was selected for calculating AUC. The initial accumulated amount of fluorescent dye at the initial time points varies slightly from well to well, resulting in an artificially elevated AUC for a given well. The best smoothing squared spline was used to remove the mean signal for the first 1.5 hours before any measurable growth from all growth curves to approximate a zero-growth signal intercept. The total incremental AUC (iAUC) was calculated as the sum of Riemann midpoint sums for each time point along the smoothing squared spline. Finally, we calculated the iAUC as iAUC=1-AUC 試料 / AUC 対照 where AUC is the試料 is the AUC of the spline generated by the given bacterium and SNIPR001, while AUC 対照 represents the AUC of the spline generated for a given phage or a given bacterium without CAP, or a combination thereof. 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 occasionally results in iAUC values outside these ranges, which are considered negligible.

[0278] Host range was calculated as the proportion of the panel that had an iAUC<0.2 for each repeat. The reported standard deviation was calculated as the deviation in the number of strains with an iAUC<0.2 and then normalized to the panel size by dividing the standard deviation by the panel size.

[0279] CRISPR-Cas-armed phage for targeting E. coli Phages were CRISPR-Cas-armed using homologous recombination. We inserted the payload into the region immediately following gene 49 through gene E, compared to a reference wild-type T2 phage. Thus, the synthetic phage genome contained an insertion between coordinates 9000 and 21000, where coordinates are counted from the nucleotide immediately following gene 49 (coordinate number 1) toward gene E, compared to a reference wild-type T2 phage. Recombination was performed within bacterial cells during phage propagation. The cells carried a plasmid that served as a recombination template. The recombination template plasmid carried the sequence intended for insertion into the phage genome between approximately 200–700 bp of flanking sequences that were homologous to the phage sequence at the insertion site. For each phage, we inserted a CRISPR array targeting the endogenous E. coli type I-E CRISPR-Cas system (Genbank CP032679.1), i.e., the cas3 gene (ygcB) and downstream genes encoding the Cascade complex, casA (ygcL), casB (ygcK), casC (ygcJ), casD (ygcI), and casE (ygcH), as well as selected E. coli sequences. The cas genes from E. coli were identical for all selected CAPs. Insertion of the CRISPR-Cas system resulted in a deletion of approximately 7 kbp of phage DNA from gene 49 to gene E. The sequences of the resulting CAPs were verified by next generation sequencing (BaseClear, Leiden, The Netherlands).

[0280] Transduction of CGV in biofilms E. coli b52 cells were grown in 96-well plates and biofilms were developed on the peg lids. Each well contained 180 μL of M9 medium (Sigma, M6030) supplemented with 20 mM glucose, 2 mM MgSO, 0.1 mM CaCl, 0.1% Amicase (Sigma), and 0.1% mannitol. The wells were inoculated with 1 μL of overnight b52 culture. The peg lids were inserted, and the microtiter plates were incubated stationary at 37°C for 24 hours. The peg lids were then transferred, without washing, to a new plate with fresh medium, and the plates were incubated for an additional 24 hours. After incubation, each well received 100 μL of medium and 100 μL of CGV transducing particles (approximately 10 8 New plates with the pegs (particles) were prepared (three replicates). The biofilms grown on the pegs were rinsed three times with sterile H2O (200 μl) before being transferred to the new plates. The plates were incubated stationary at 37°C for 5 hours.

[0281] To assay the metabolic activity of cells in biofilms, the lids were rinsed three times with 200 μL of H2O and then placed in plates containing 20 μL of Alamarblue stain (ThermoFisher) and 180 μL of medium per well. The plates were incubated at 37 °C for 1.5 h and transferred to a microplate reader (Synergy H1, Biotek). Fluorescence (excitation: 560 nm; emission: 590 nm) and absorbance (600 nm) were recorded for each well.

[0282] Promoter P bolA The metabolic activity of biofilms treated with CGV carrying the cas gene was reported relative to the metabolic activity of biofilms treated with CGV not carrying the promoter transcribing the cas gene.

[0283] Plasmid and strain construction 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 The vector was cloned into a 3-spacer array targeting genes in E. coli under the control of the constitutive promoter J23100. The array contains nucleotides from the E. coli genome for each target locus separated by direct repeats (repeat sequence, SEQ ID NO: 15). Protospacer adjacent motifs (PAMs) are located adjacent to the selected target sequence in the E. coli genome.

[0284] Transformation assay Dilute (1:100) the overnight culture in fresh LB medium until it reaches mid-logarithmic phase (OD 600 The cells were grown to a pH of 1.0 (≈0.6). Cells were then prepared for electroporation by concentrating 50-fold in ice-cold MilliQ water. Cells were then electroporated with the appropriate plasmid, recovered in super optimal broth (SOB) at 37°C for 1 hour, and plated on LB plates supplemented with antibiotics.

[0285] Conjugation assay Conjugation experiments were established to evaluate the transfer and killing efficiency of CGV-EcCas using E. coli JKE201 as the donor and E. coli clinical isolates as recipients (including target and non-target E. coli strains as controls). Plasmids were conjugated into E. coli recipients by liquid mating. Briefly, overnight cultures were diluted (1:100) into fresh LB medium and the OD 600 Grow, wash, and in fresh LB until OD ≈ 0.4 600The cells were suspended to a pH of approximately 0.25. 125 μl of donor and 25 μl of recipient cell suspension were mixed at a 5:1 ratio in a 96-well microplate and incubated at 37°C for 16 hours. Conjugation efficiency was determined by plating serial dilutions of the conjugation reaction onto LB agar supplemented with antibiotics (to select for transconjugants). Specific killing efficiency was quantified by plating 90 μL of the conjugation reaction onto selective plates. The CGV-EcCas plasmid encodes resistance to kanamycin, gentamicin, and amikacin, allowing for the selection of transconjugants. Viability was calculated by counting CFU on the plates, and data were recorded as viable cell concentration (CFU / mL).

[0286] Synchronized CAP infection and cas3 expression assay Test strains from overnight LB cultures were diluted 100-fold and incubated in LB at 37°C with shaking until stationary phase, after which 10 mL aliquots were divided into 50 mL Falcon tubes. Each aliquot was then inoculated with 50 μL of high-titer lysate of the individual CAP, and incubation continued under the same conditions. In addition, a 10 mL LB volume of each CAP was also inoculated with 50 μL of CAP lysate and used for phage counting at 0 min. Aliquots were collected for total RNA extraction and phage counting at 5, 15, and 30 min after inoculation. The phage counting aliquots were syringe-filtered (0.2 μm, Sartorious, Göttingen, Germany) and subjected to plating efficiency assays. For total RNA extraction, 1 mL aliquots of each culture were centrifuged at 13.3 k × g for 15 s using a tabletop centrifuge, and the supernatant was discarded. The pellet was then immediately resuspended in cold RNA Later (Thermo Fisher Scientific, AM7020) and stored at -20°C until extraction. Total RNA was extracted using the GeneElute Total RNA Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's protocol for extracting RNA from bacteria. After the initial elution, 1 μL of Dnase I (1 U / μL) was added and incubated overnight at 37°C. The reaction was terminated by a 15-minute incubation at 70°C. RNA was repurified on a GeneElute column and eluted with 35 μL of kit elution buffer. Total RNA concentration was estimated using a NanoDrop instrument (Thermo Scientific, One / OneC), and 0.5–2 μg of RNA was added to a cDNA synthesis reaction containing SuperScript III RT enzyme (Thermo Fisher Scientific, Waltham, MA, USA) and random decamers to initiate synthesis in a 20 μL reaction volume. The cDNA reaction was diluted to 100 μL in water. Real-time PCR was performed in triplicate using 5 μL of cDNA as template, 10 μL of Power SYBR Green PCR Master mix (Thermo Fisher) and 0.2 μM of each PCR primer.PCR was performed on an AB QuantStudio5 system (Applied Biosystems, Foster City, CA, USA) using a standard two-step thermocycling protocol for Power SYBR Green PCR Master Mix with annealing / extension at 60°C. 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 for cas3 were 5'-caagtatgctaccaacggctaaag-3' (SEQ ID NO: 19) and 5'-ccaatcaaaatcaacgtcgagtga-3' (SEQ ID NO: 20). For these primer pairs, a single PCR product was confirmed by melting curve analysis. Relative transcript levels were estimated using 10-fold dilutions of purified PCR products as standards, and values were expressed as the ratio of cas3 transcripts to gapA transcripts.

[0287] Phage competition assay The lysates of the two phages were mixed at a ratio of 9:1 (WT:CAP), and the phage mixture was added to 10 ml of 2xYT medium containing 10 mM CaCl2 and 20 mM MgCl2, as well as 100 μL of overnight E. coli strain b230, which served as a target for both competing phages. After 2 hours of 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 step was repeated twice.

[0288] The phage ratio was assessed by PCR using three primers, yielding two specific products: one for WT phage and one for CAP. The PCR products were separated on a 1% agarose gel, and the DNA bands were stained with SYBRsafe and visualized and quantified using a ChemiDoc XRS+ System (Model 1708265, Biorad). The background-corrected intensity of the band corresponding to WT 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 ratio of CAP to the total phage content (WT+CAP) was determined based on a calibration curve generated by using a set of different mixtures of the two phages, and the curve was fitted to the measured band intensity ratio (WT / CAP). The estimated error of the reported values is less than 20%.

[0289] Lawn killing assay Test strains from overnight cultures in LB 9 A 100 μL aliquot of the strain adjusted to CFU / mL was added to 100 μL of 10 9 PFU / mL of either CAP α15.2 or WT α15 was mixed with 1 mL of CAP α15.2 or WT α15 in a 15 mL Falcon tube to achieve a multiplicity of infection of 1, which was then melted, mixed with 3 mL of pre-prepared top agar, and spread onto LB plates. After the lawn solidified, the plates were incubated at 37°C overnight, and the total number of surviving colonies was counted the following day for the CAP α15.2 or WT α15 group. Assays were performed as independent biological replicates, and each experiment consisted of 10 technical replicates. Statistical significance was established using both replicates using the Mann-Whitney U test.

[0290] Generalized transduction assay The transduction ability of each CAP was assessed by a generalized transduction assay. Briefly, transduction lysates were prepared by growing each CAP in E. coli MG1655 lamB::Cm. This strain was modified from WT MG1655 (Cat. No. 700926, American Type Culture Collection, Manassas, VA, USA) to carry a chloramphenicol selection marker. The experiment was performed using the well-characterized lytic T4 phage (negative control) and its transduction mutant, T4GT7. 58 After this step, the WT E. coli MG1655 strain was incubated at an OD of 0.3. 600 The cells were infected with each transduction lysate at an MOI of 0.5, 0.1, or 0.01 and plated on LB plates containing chloramphenicol. The following day, the number of transduced colonies was recorded for each CAP, control, and different MOIs. The transduction frequency was calculated by dividing the number of transductants by the titer of the transduction lysate.

[0291] Sequence analysis of CAP Individual SNIPR001 CAP sequences were analyzed for the presence of antibiotic resistance, virulence genes, and lysogeny-related genes (transposase and integrase) using databases (Table 2). Additionally, phage samples were analyzed using whole-genome sequencing, which typically yields >1000x coverage of the entire phage genome. Assemblies were constructed by downsampling the data to an average coverage of 1000x for the phage and assembled using SKESA. To detect differences between samples and detect minor mutations, raw reads were mapped back to the assembly using BWA (version 0.7.17).

[0292] [Table 5]

[0293] Phage specificity assay using liquid killing assay The killing specificity of SNIPR001 CAPs (α15.2, α20.4, α48.4, and α51.5) and SNIPR001 was assessed by biopotency assay 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).

[0294] Briefly, overnight cultures were cultured in LB broth for 10 min. 6 The CFU counts were adjusted to CFU / mL. Prior to the 4-hour incubation, SNIPR001 CAP or SNIPR001 (each CAP combined at an equal ratio) 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 the data were calculated by subtracting the initial inoculum (0 hour) from the assay endpoint CFU / mL (4 hours). 10 Expressed as CFU / mL.

[0295] [Table 6]

[0296] Spotting assay and efficiency of plating (EoP) To count the phage titer, an equal volume mixture of phage lysate or SNIPR001 CAP was serially diluted 10-fold in SM buffer or PBS, respectively. 100 μL or 300 μL of bacterial overnight culture was plated on 3 mL or 10 mL of 0.5% top agar (Ca 2+ and Mg 2+ Bacterial lawns were prepared by adding 100 μl of phage to a 100-well plate containing 100 μl of phage serotype (containing 100 μg of phage serotype) containing 100 μl of phage serotype (containing 100 μg of phage serotype), which was vortexed briefly and poured onto round or square LB plates. Five μl of serial dilutions of the test phage were then spotted onto the lawn, allowed to dry at room temperature with the lid open, and then incubated overnight at 37°C. Strains b52, b2479, and b17 were used as assay controls and were included in each round of assay.

[0297] The next day, the results were evaluated (Table 4). In this assay, susceptible strains are defined as those producing countable plaques in PFU / mL, and those strains showing no visible plaques but impaired bacterial growth (i.e., lysis zones). Coverage is defined as the percentage of the total number of susceptible strains. Images of all plates were recorded. Graphs showing the efficiency of plating results are first calculated by multiplying the titer by log 10 Transformations were performed and then standard deviations and means were calculated. The clinical panel and control strains were tested in two independent experiments.

[0298] [Table 7]

[0299] Animals and husbandry Mouse studies were conducted using female CD-1® IGS mice (approximately 6-7 weeks old upon arrival) from Charles River (Freiburg, Germany). Animals were housed in groups of 3-5 mice per cage in an air-conditioned room (temperature 20-23°C; relative humidity 30-70%) under a 12:12-h light / dark cycle (lights on 07:00-19:00). Standard pelleted food and tap water were available ad libitum. Animals were allowed to acclimate for at least 7 days before the start of experimental procedures. Thirty female Gottingen minipigs (approximately 4-7 months old upon arrival) from Ellegaard Gottingen Minipigs A / S, Denmark, were used for tolerability and kinetic studies. Animals were allowed to acclimate for at least 14 days before the start of experiments. Pigs were housed in groups of 2-3 animals and fed standard pig chow twice daily. Tap water was available ad libitum. All procedures were performed in accordance with the guidelines of the Danish Animal Experiments Inspectorate, Ministry of Environment and Food of Denmark, and in accordance with an institutional license (BioAdvice, animal license number 2015-15-0201-00540).

[0300] Mouse intestinal colonization model The mouse intestinal colonization model was developed by Galtier et al. (2016) 44 Briefly, pretreatment with streptomycin (5 g / L) in drinking water was given 3 days before inoculation with E. coli b17 to reduce the native bacterial levels. On day 0, 3 × 10 7 An inoculum of CFU of E. coli b17 was prepared from a frozen glycerol stock and administered by oral gavage to all mice at 0.25 mL.

[0301] Treatment was administered three times daily for 2 days, starting 2 days after inoculation. Immediately before each administration, the four CAPs were mixed in a 1:1:1:1 ratio to form SNIPR001 at high, medium, or low concentrations, and 2 × 10 11 , 2 × 10 9 and 1 × 10 7 Dose levels of PFU were obtained. At the time of treatment, mice received 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.

[0302] CAP Recovery and Tolerability Study Göttingen minipigs were first given a cocktail of antibiotics containing neomycin (60 mg / kg, once daily for 4 days, orally) and cefquinomere (2 mg / kg, once daily for 3 days, intramuscularly) prior to SNIPR001 or a single CAP administration to reduce the level of Gram-negative bacteria in the GI tract and thereby limit phage replication. The animals were then fasted overnight and lightly sedated, after which they were given 50 mL of 10% sodium bicarbonate orally followed by 2 × 10 in 100 mL of phage. 12 Animals were administered a single oral dose of either single CAP or the SNIPR001 cocktail at 100 PFU per day. Fecal samples were collected daily for CAP quantification by plaque assay. Additionally, blood samples were collected for hematological and blood chemistry analyses, including C-reactive protein, and plaque assay for tolerability studies. Animals were closely monitored after SNIPR001 administration, and body temperatures were recorded regularly.

[0303] Quantification of E. coli b17 and CAP in feces Fecal samples were homogenized and serially diluted in SM buffer. Triplicate 10 μl of each dilution was then spotted onto McConkey agar plates (Sigma, M7408) supplemented with streptomycin (1 mg / mL) and incubated at 37°C for 12–16 h for E. coli enumeration.

[0304] A plaque assay was 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 aliquots of each dilution were spotted onto E. coli b17 overlays and incubated at 37°C for 12–16 hours.

[0305] To quantify the presence of resistance in vivo, three colonies from fecal samples of each mouse in the medium dose group at three different time points were picked from McConkey agar plates. The colonies were incubated in LB broth at 37°C for 12-16 hours and used to create a top agar overlay on the LB agar plate. The plates were then allowed to dry on the LAF bench for 15 minutes. The SNIPR001 cocktail and four individual CAPs were added at 1x10 5 Serial dilutions were spotted from a PUF / mL stock. As a control, a top agar overlay of the colonizing strain E. coli b17 was spotted in the same manner. Plates were allowed to dry in the LAF bench with the lid on and then incubated upside down at 37°C for 12–16 hours.

[0306] Whole genome sequencing of E. coli strains from JMI Total genomic DNA was extracted and purified using the KingFisher Cell and Tissue DNA kit (Thermo Scientific, Waltham, MA, USA) on a robotic KingFisher™ Flex Magnetic Particle Processor (Thermo Scientific) workstation.

[0307] Total genomic DNA was used as input material for library construction. DNA libraries were prepared using the Nextera XT™ Library Construction Protocol and Index Kit (Illumina, San Diego, CA, USA) and sequenced on a MiSeq Sequencer (Illumina) using MiSeq Reagent Kits v3 (600 cycles).

[0308] Definition of resistance phenotypes Extended-spectrum β-lactamase (ESBL) phenotype was defined for Escherichia coli as a minimum inhibitory concentration (MIC) value of ≥2 mg / L for ceftriaxone, ceftazidime, and / or aztreonam ( https: / / clsi.org / ). Carbapenem-resistant Enterobacterales (CRE) was defined as any isolate that exhibited resistance to imipenem, doripenem, and / or meropenem with an MIC >2 mg / L ( https: / / clsi.org / ).

[0309] Assembly of whole-genome sequencing data Trimmomatic with the settings "LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36" 60 Raw sequencing reads were trimmed using SPAdes (version 0.39). Trimmed reads were then run using SPAdes with default settings. 61(version 3.14.1). Contigs shorter than 500 bp or with a sequencing depth less than 2x were removed from the final assembly.

[0310] Comparative genomics of clinical E. coli strains Multilocus sequence typing (MLST) was performed on the assembled genome of E. coli bacteria using the MLST2 database downloaded from the MLST2 repository ( https: / / bitbucket.org / genomicepidemiology / mlst_db / src / master / ) on July 1, 2021, using default settings. 62 Phylogenetic group classification was performed using Clermon Typing on the assembled E. coli genome using default settings. 63 Distance matrices for phylogenetic tree construction were generated using MASH with a k-mer size of 21 and 10,000 sketches per genome. 64 The sketches were then compared and MASH-distances were generated in a pairwise manner to create a distance matrix for the E. coli genome.

[0311] Phage synteny analysis To generate synteny plots, the wild-type sequences of the four phages included in the final cocktail and two closely related, well-known reference phages (RB69 AY303349.1 and T2 NC_054931.1) were annotated with RAST to extract predicted protein sequences. All protein sequences for each phage were re-queried against all other phage genomes using tblastn (v2.12.0) with an E-value cutoff of 1e-10. Synteny plots were then generated using a custom Python script (see data availability) using the drawSvg library (v1.9.0). The plot shows the phage genomes in order of similarity, displaying all tblastn hits as synteny blocks shaded by their protein identity. Proteins from the two reference phages were manually classified as belonging to each of the functional groups: "DNA metabolism," "structure," or "other," and color-coded accordingly.

[0312] Data Processing and Visualization Figures and key statistics were generated using R version 4.1.0. The following packages were used to generate the figures: 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 65 v. 3.0.2, systemfonts v. 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. Means and standard deviations were calculated after transforming values to the scale indicated in the given figures, e.g., log 10 If a scale is used, the mean and standard deviation are log 10 Calculate after conversion.

[0313] Data Availability The data and results generated in this study have been deposited at https: / / github.com / sniprbiome / SNIPR001_paper. The phage genome sequence has been deposited in Genbank under accession number OQ067373-76.

[0314] Code Availability All code required to create this study is available at https: / / github.com / sniprbiome / SNIPR001_paper.

[0315] References 1. Pulte, D., Jansen, L. & Brenner, H. Changes in long term survival after diagnosis with common hematologic malignancies in the early 21st century. Blood Cancer J 10, 56 (2020). 2. Riley, M. et al. Epidemiology and Burden of Mucosal Barrier Injury Laboratory-Confirmed Bloodstream Infections in Bone Marrow Transplant and Hematology-Oncology Units. Am J Infect Control 45, S38 (2017). 3. Dandoy, CE et al. Incidence, Risk Factors, and Outcomes of Patients Who Develop Mucosal Barrier Injury-Laboratory Confirmed Bloodstream Infections in the First 100 Days After Allogeneic Hematopoietic Stem Cell Transplant. Jama Netw Open 3, e1918668 (2020). 4. Velden, W. J. F. M., Herbers, A. H. E., Netea, M. G. & Blijlevens, N. M. A. Mucosal barrier injury, fever and infection in neutropenic patients with cancer: introducing the paradigm febrile mucositis. Brit J Haematol 167, 441-452 (2014). 5. Girmenia, C. et al. Incidence, Risk Factors and Outcome of Pre-engraftment Gram-Negative Bacteremia After Allogeneic and Autologous Hematopoietic Stem Cell Transplantation: An Italian Prospective Multicenter Survey. Clin Infect Dis 65, 1884-1896 (2017). 6. Klastersky, J. et al. Bacteraemia in febrile neutropenic cancer patients. Int J Antimicrob Ag 30, 51-59 (2007). 7. Taplitz, R. A. et al. Antimicrobial Prophylaxis for Adult Patients With Cancer-Related Immunosuppression: ASCO and IDSA Clinical Practice Guideline Update. J Clin Oncol 36, JCO.18.00374 (2018). 8. Cullen, M. et al. Antibacterial Prophylaxis after Chemotherapy for Solid Tumors and Lymphomas. New Engl J Med 353, 988-998 (2005). 9. Bucaneve, G. et al. Levofloxacin to Prevent Bacterial Infection in Patients with Cancer and Neutropenia. New Engl J Medicine 353, 977-987 (2005). 10. See, I., Freifeld, A. G. & Magill, S. S. Causative Organisms and Associated Antimicrobial Resistance in Healthcare-Associated, Central Line-Associated Bloodstream Infections From Oncology Settings, 2009-2012. Clin Infect Dis 62, 1203-1209 (2016). 11. Trecarichi, E. M. et al. Current epidemiology and antimicrobial resistance data for bacterial bloodstream infections in patients with hematologic malignancies: an Italian multicentre prospective survey. Clin Microbiol Infec 21, 337-343 (2015). 12. Marin, M. et al. Factors influencing mortality in neutropenic patients with haematologic malignancies or solid tumours with bloodstream infection. Clin Microbiol Infec 21, 583-590 (2015). 13. Averbuch, D. et al. Antimicrobial Resistance in Gram-Negative Rods Causing Bacteremia in Hematopoietic Stem Cell Transplant Recipients: Intercontinental Prospective Study of the Infectious Diseases Working Party of the European Bone Marrow Transplantation Group. Clin Infect Dis 65, 1819-1828 (2017). 14. Miedzybrodzki, R. et al. Bacteriophages. 1-31 (2018) doi:10.1007 / 978-3-319-40598-8_31-1. 15. Miedzybrodzki, R. et al. Bacteriophages. 921-951 (2021) doi:10.1007 / 978-3-319-41986-2_31. 16. Schooley, R. T. et al. Development and Use of Personalized Bacteriophage-Based Therapeutic Cocktails To Treat a Patient with a Disseminated Resistant Acinetobacter baumannii Infection. Antimicrob Agents Ch 61, (2017). 17. Dedrick, R. M. et al. Engineered bacteriophages for treatment of a patient with a disseminated drug resistant Mycobacterium abscessus. Nat Med 25, 730-733 (2019). 18. Jennes, S. et al. Use of bacteriophages in the treatment of colistin-only-sensitive Pseudomonas aeruginosa septicaemia in a patient with acute kidney injury-a case report. Crit Care 21, 129 (2017). 19. Jault, P. et al. Efficacy and tolerability of a cocktail of bacteriophages to treat burn wounds infected by Pseudomonas aeruginosa (PhagoBurn): a randomised, controlled, double-blind phase 1 / 2 trial. Lancet Infect Dis 19, 35-45 (2018). 20. Sarker, S. A. et al. Oral Phage Therapy of Acute Bacterial Diarrhea With Two Coliphage Preparations: A Randomized Trial in Children From Bangladesh. Ebiomedicine 4, 124-137 (2016). 21. Leitner, L. et al. Intravesical bacteriophages for treating urinary tract infections in patients undergoing transurethral resection of the prostate: a randomised, placebo-controlled, double-blind clinical trial. Lancet Infect Dis 21, 427-436 (2020). 22. Sabino, J., Hirten, R. P. & Colombel, J. Review article: bacteriophages in gastroenterology-from biology to clinical applications. Aliment Pharm Therap 51, 53-63 (2020). 23. Gorski, A., Borysowski, J. & Miedzybrodzki, R. Phage Therapy: Towards a Successful Clinical Trial. Antibiotics 9, 827 (2020). 24. Federici, S. et al. Targeted suppression of human IBD-associated gut microbiota commensals by phage consortia for treatment of intestinal inflammation. Cell 185, 2879-2898.e24 (2022). 25. Kauffman, K. M. et al. Resolving the structure of phage-bacteria interactions in the context of natural diversity. Nat Commun 13, 372 (2022). 26. Yehl, K. et al. Engineering Phage Host-Range and Suppressing Bacterial Resistance through Phage Tail Fiber Mutagenesis. Cell 179, 459-469.e9 (2019). 27. Westra, E. R. et al. CRISPR Immunity Relies on the Consecutive Binding and Degradation of Negatively Supercoiled Invader DNA by Cascade and Cas3. Mol Cell 46, 595-605 (2012). 28. Sinkunas, T. et al. Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR / Cas immune system. Embo J 30, 1335-1342 (2011). 29. Bikard, D. et al. Development of sequence-specific antimicrobials based on programmable CRISPR-Cas nucleases. Nat Biotechnol 32, 1146-1150 (2014). 30. Citorik, R. J., Mimee, M. & Lu, T. K. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nat Biotechnol 32, 1141-1145 (2014). 31. Selle, K. et al. In Vivo Targeting of Clostridioides difficile Using Phage-Delivered CRISPR-Cas3 Antimicrobials. Mbio 11, e00019-20 (2020). 32. Neil, K. et al. High-efficiency delivery of CRISPR-Cas9 by engineered probiotics enables precise microbiome editing. Mol Syst Biol 17, e10335 (2021). 33. Edgar, R. & Qimron, U. The Escherichia coli CRISPR system protects from λlysogenization, lysogens, and prophage induction. J Bacteriol 192, 6291-4 (2010). 34. Hsu, B. B. et al. In situ reprogramming of gut bacteria by oral delivery. Nat Commun 11, 5030 (2020). 35. Yosef, I., Manor, M., Kiro, R. & Qimron, U. Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria. Proc National Acad Sci 112, 7267-7272 (2015). 36. Ochman, H. & Selander, R. K. Standard reference strains of Escherichia coli from natural populations. J Bacteriol 157, 690-3 (1984). 37. Oechslin, F. Resistance Development to Bacteriophages Occurring during Bacteriophage Therapy. Viruses 10, 351 (2018). 38. Dunne, M. et al. Salmonella Phage S16 Tail Fiber Adhesin Features a Rare Polyglycine Rich Domain for Host Recognition. Structure 26, 1573-1582.e4 (2018). 39. Brouns, S. J. J. et al. Small CRISPR RNAs Guide Antiviral Defense in Prokaryotes. Science 321, 960-964 (2008). 40. Knight, D. & Girling, K. Gut flora in health and disease. Lancet 361, 1831 (2003). 41. Christensen, S. K., Mikkelsen, M., Pedersen, K. & Gerdes, K. RelE, a global inhibitor of translation, is activated during nutritional stress. Proc National Acad Sci 98, 14328-14333 (2001). 42. Vieira, H. L. A., Freire, P. & Arraiano, C. M. Effect of Escherichia coli Morphogene bolA on Biofilms. Appl Environ Microb 70, 5682-5684 (2004). 43. Uribe, R. V. et al. Bacterial resistance to CRISPR-Cas antimicrobials. Sci Rep-uk 11, 17267 (2021). 44. Galtier, M. et al. Bacteriophages to reduce gut carriage of antibiotic resistant uropathogens with low impact on microbiota composition. Environ Microbiol 18, 2237-2245 (2016). 45. Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet 399, 629-655 (2022). 46. Hyman, P. & Abedon, S. T. Bacteriophage host range and bacterial resistance. Adv Appl Microbiol 70, 217-48 (2010). 47. Lemay, M.-L., Renaud, A., Rousseau, G. & Moineau, S. Targeted Genome Editing of Virulent Phages Using CRISPR-Cas9. Bio-protocol 8, e2674 (2018). 48. Yehl, K. et al. Engineering Phage Host-Range and Suppressing Bacterial Resistance through Phage Tail Fiber Mutagenesis. Cell 179, 459-469.e9 (2019). 49. Selle, K. et al. In Vivo Targeting of Clostridioides difficile Using Phage-Delivered CRISPR-Cas3 Antimicrobials. Mbio 11, e00019-20 (2020). 50. Lam, K. N. et al. Phage-delivered CRISPR-Cas9 for strain-specific depletion and genomic deletions in the gut microbiome. Cell Reports 37, 109930-109930 (2021). 51. Lee, K. A. et al. The gut microbiome: what the oncologist ought to know. Brit J Cancer 125, 1197-1209 (2021). 52. Gencay, Y. E., Ayaz, N. D., Copuroglu, G. & Erol, I. Biocontrol of Shiga Toxigenic Escherichia coli O157:H7 in Turkish Raw Meatball by Bacteriophage. J Food Safety 36, 120-131 (2016). 53. Satlin, M. J. et al. Colonization With Fluoroquinolone-Resistant Enterobacterales Decreases the Effectiveness of Fluoroquinolone Prophylaxis in Hematopoietic Cell Transplant Recipients. Clin Infect Dis 73, 1257-1265 (2021). 54. Satlin, M. J. et al. Colonization With Levofloxacin-resistant Extended-spectrum β-Lactamase-producing Enterobacteriaceae and Risk of Bacteremia in Hematopoietic Stem Cell Transplant Recipients. Clin Infect Dis Official Publ Infect Dis Soc Am 67, 1720-1728 (2018). 55. Harms, A. et al. A bacterial toxin-antitoxin module is the origin of inter-bacterial and inter-kingdom effectors of Bartonella. Plos Genet 13, e1007077 (2017). 56. Henry, M. et al. Development of a high throughput assay for indirectly measuring phage growth using the OmniLogTM system. Bacteriophage 2, 159-167 (2014). 57. Lutz, R. & Bujard, H. Independent and Tight Regulation of Transcriptional Units in Escherichia Coli Via the LacR / O, the TetR / O and AraC / I1-I2 Regulatory Elements. Nucleic Acids Res 25, 1203-1210 (1997). 58. Young, K. K., Edlin, G. J. & Wilson, G. G. Genetic analysis of bacteriophage T4 transducing bacteriophages. J Virol 41, 345-347 (1982). 59. Gibson, S. B. et al. Constructing and Characterizing Bacteriophage Libraries for Phage Therapy of Human Infections. Front Microbiol 10, 2537 (2019). 60. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114-2120 (2014). 61. Bankevich, A. et al. SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing. J Comput Biol 19, 455-477 (2012). 62. Larsen, M. V. et al. Multilocus Sequence Typing of Total-Genome-Sequenced Bacteria. J Clin Microbiol 50, 1355-1361 (2012). 63. Beghain, J., Bridier-Nahmias, A., Nagard, H. L., Denamur, E. & Clermont, O. ClermonTyping: an easy-to-use and accurate in silico method for Escherichia genus strain phylotyping. Microb Genom 4, e000192 (2018). 64. Ondov, B. D. et al. Mash: fast genome and metagenome distance estimation using MinHash. Genome Biol 17, 132 (2016). 65. Yu, G. Using ggtree to Visualize Data on Tree-Like Structures. Curr Protoc Bioinform 69, e96 (2020). 66. Datsenko, KA & Wanner, BL One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc National Acad Sci 97, 6640-6645 (2000). 67. Hantke, K. Compilation of Escherichia coli K-12 outer membrane phage receptors - their function and some historical remarks. Fems Microbiol Lett 367, (2020).

[0316] array The amino acid sequences are written N- to C-terminally, and the DNA sequences are written 5' to 3'.

[0317] protein >LamB (SEQ ID NO: 1) MMITLRKLPLAVAVAAGVMSAQAMAVDFHGYARSGIGWTGSGGEQQCFQTTGAQSKYRLGNECETYAELKLGQEVWKEGDKSFYFDTNVAYSVAQQNDWEATDPAFREANV QGKNLIEWLPGSTIWAGKRFYQRHDVHMIDFYYWDISGPGAGLENIDVGFGKLSLAATRSSEAGGSSSFASNNIYDYTNETANDVFDVRLAQMEINPGGTLELGVDYGRANL RDNYRLVDGASKDGWLFTAEHTQSVLKGFNKFVVQYATDSMTSQGKGLSQGSGVAFDNEKFAYNINNNGHMLRILDHGAISMGDNWDMMYVGMYQDINWDNDNGTKWWTVG IRPMYKWTPIMSTVMEIGYDNVESQRTGDKNNQYKITLAQQWQAGDSIWSRPAIRVFATYAKWDEKWGYDYNGDSKVNPNYGKAVPADFNGGSFGRGDSDEWTFGAQMEIWW >Tsx (sequence number 2) MKKTLLAAGAVLALSSSFTVNAAENDKPQYLSDWWHQSVNVVGSYHTRFGPQIRNDTYLEYEAFAKKDWFDFYGYADAPVFFGGNSDAKGIWNHGSPLFMEIEPRFSIDKLTNTDLSFFGPFKEWYFANNYIYDMGRNKDGRQSTWYM GLGTDIDTGLPMSLSMNVYAKYQWQNYGAANENEWDGYRFKIKYFVPITDLWGGQLSYIGFTNFDWGSDLGDDSGNAINGIKTRTNNSIASSHILALNYDHWHYSVVARYWHDGGQWNDDAELNFGNGNFNVRSTGWGGYLVVGYNF DNA >LamB (SEQ ID NO: 3) >Tsx (Accession No. 4) ATGAAAAAAACATTACTGGCAGCCGGTGCGGTACTGGCGCTCTCTTCGTCTTTTACTGTCAACGCAGCTGAAAACGACAAACCGCAGTATCTTTCCGACTGGTGGCACCAGAGCGTTAACGTTGTCGGAAGCTATCACACCCGTTTCGGACCGCAGATCCGCAACGATACCTACCTTGAGTACGAAGCATTCGCTAAAAAAGACTGGTTCGACTTCTATGGTTATGCGGATGCGCCGGTATTCTTCGGCGGTAACTCCGATGCAAAAGGTATCTGGAACCACGGTTCTCCGCTGTTTATGGAAATCGAACCACGTTTCTCCATCGACAAGCTGACCAATACTGACCTTAGCTTCGGTCCGTTCAAAGAGTGGTACTTCGCGAACAACTACATTTACGACATGGGTCGTAATAAAGATGGTCGCCAGAGCACCTGGTACATGGGTCTGGGTACCGATATCGACACTGGCCTGCCGATGAGCCTGTCCATGAACGTCTATGCGAAATACCAGTGGCAGAACTATGGCGCAGCGAACGAAAACGAGTGGGACGGTTACCGTTTCAAAATTAAATACTTTGTGCCGATTACCGATCTGTGGGGCGGTCAGCTGAGCTACATCGGCTTCACCAACTTCGACTGGGGTTCCGATTTAGGGGATGACAGCGGTAACGCAATCAACGGTATTAAGACCCGTACTAATAACTCTATCGCTTCCAGCCATATTCTGGCTCTGAACTACGATCACTGGCACTACTCTGTCGTAGCTCGTTACTGGCACGACGGTGGTCAGTGGAACGACGATGCAGAACTGAACTTCGGCAACGGCAACTTCAACGTTCGCTCTACCGGCTGGGGTGGTTACCTGGTAGTAGGTTACAACTTCTGA

[0318] >NC_000866.4 Enterobacteria phage T4, complete genome (SEQ ID NO: 5) AATTTTCCTTATTAGGCCGCAAGGGCCTTCATAGTTTTAGCGATTTGGGAAACTTCATCATCACTTAAAG AGTTGCGATAACCGATGAAGTCGGAAACAATACGGAATTTCTTGGTAAACTCAGCAACCATTTTATCACT GTTTTTTGAAGCATTATTTGATAATACATCAAAAAGATTAGTTACTGTCCAAATGTCATGACCGATGGTA TCTTTTCCACCATTAAAATATACACCCTGTAATGAACTAACCATATTAGCGAGTCGTGTATATTCTTCAG AAACTTCATCTATACTGAAGTACTTCATCATAAAATCTAACTCAGGATACTTGATAATTTTATCAATATA TCGTTTAGCTGAACTTGAATAACCTACATACTTATCATAATCTACATCATCAAAAGCATCTACATATAAA TCACGCAAAGCTTCAAAAATACATTGGCACTGACCGAGTTCTTTTACCTTTTTCTGTAAAAGCGGACGAA TAACATAAAATTCATTAATGCCAATAAGATTAGCCATACGAATCAAAATATTCATAGATGGATGACAAAG AGATGTAGTACCATCCATAGAGAAAATATCAGAACGATGCATATACGCTACATAACCAGTAATTTCATCT GCTTCTGATGTGAGGCGTAAATAATTCCTCTTTTCCCAGCGCCCGTCTTTAATTTCAAACTTAAACGCTG TAGCAGCTTTAGGACGAGGAGCTTTACTTTTAACTACCTTTGGAATATAACTTTTTACTAAAGCTTCAAT TTCTGACAAATAATGAATGTTAACTTCATCACTTTCAAACATCGCCATAATATCAGGAAGCAAATCAATC TGCGATTCTACTTCTGGATTAATAAACAGAAGACGTTCGTTATGAATATTCAAAGTGTTATTAAATT CACTATCATCTAACGCACGTGCTAATCCACGGACAATATTAACACGATTTTTAATATTATCAATAACGAT ATTAATTTTTGTTGTATTAATACCAAACAGACGATAACTTGATGCAACGGCTGAAGTTTCATGACTTTGC TTAATGCGCTTCAGTCGAGGGTCAAGATTTACTTCATACACAACTCCCGCGTTGCATAACTTACTGTCAG GTTCAAACATGCTCTGCATCTTTTTATATGACAGATTTTTAGTCGTGAATTTGACTGAATTACTAATCAT ATAATCTCGAGCAGAATACCCCATCTTCATCAATTCACGATATGTGTGACGAGGAGATGTAGATTCTTTA AATCGTTTTACATCTTCATTAAATGCTTTCTCACTGAGTTCTTTAACTCGTTCAATAATATTTTTACGAG TGCGATCATCCAGTGAAAGAGCCTCGCGAGATGGAGCAATATCAAGTGAACCCATTGGAAACTTAATGTA ATTCACTTCATTGCGAATGCTTAGCCAGTTACGGTCTCTAATAACACCATCGATAGGATAAACAATACCA CCGTAGATAGCATATAATCCACCACGATCAGGCCAGTATCTTTCTGGATTTACACCGTAATAGTCATCAA AATCCGGAAAATAATCAATTTCGCGGTCAAGACCATTAATGATAGCCAAATCTTTGAACGGTCGCATGAT ATAAGAAACTTCATAAGCAAAGTTTCTAAAGTCTTTTTCTTCAACTGGAACTACGATTTCAATACCAGTT TTATCATCTGGACCCATTTCTTTTACGAATGTAGGTTTAATCTGTGGACCATCACCATCCATGTAAGCTA CATAACCACGAATTTCACCTTTATGATACGAAGTAATACTAAACGTATCAGTATAACTAAACGGAGATTT AGAACCTAAACCAAATCCGCCAATAAAGTCATTAGATTCAGCTTTAGAATGAACTGAAGTATGAATTATAC AACCCAGGAGAATTATCATCACCTTGAATATCAAAATCACTCATACCCCGGACCAAAATCTCGACAAACAA ATCGTGGGTCTAAACGTCCAGGAACTTGTATGATAAATTTTTCAGGATTTCCATTAAGTGCATGAGCATC AATCATGTTAGTAATCAATTCACGGACTACTGCGCGAATCTTGTTTGTATACAAATCAGATGACAGAATT TTAAATACTTTAGGAGATGCTGTGATGCTAAATGCTTTTGATTTAGAACCATTACCAAGAATTGTTTCTT TTTCAGTGGTGATAATCATAATTTCCTCATTAATTCATATTACGCTTAATAACTTCAGCAACTTCTAGTA GTTCATCTTTAGTTGCGGTGTCGGATTGAATTTTATCTCTAATATCTTTAAAGCGGGGTTTTAAATTCTTC GGCTTCTCCCATATCGAAAAAGCGTTGAATGATTCTATATTCTCGATGAACTGCTTTATCAAAAAGTTCT AAATTTACTTTATATGATTTCATTTCAATATCCTCATTTGCCCAATTAATTATACCACATCCTTGTGGTA AAGTAAACTACTGGCTCATCCATTCTTTACGAAGGTCAGCATTATCTCCCATGAGCATTTCAAAAAGCTC TTTCCAGTTCTCAGGAAGTTTAACAACATCATACTGGGGTTTTGAATCATCTCACGATATTCAGATTTT TCCAAAGAGCCAAGTCCCTTAATATAACGGATGCTATGTTTAGGTAGAGCATCTTTGGCACTCTCATATT CAGCGACTGTATAAAAACCATTCTTGTTTTTTACCGACCTGAGCGATGATTACAGGAGTTTTGACAAAGCG AATTCGTCCTTGCTCAAACAATTCTGGCCAATTACTAAAAATCCGAGCAGAGAAGGATAAATAGAACCT AATCCAATAATCTCTTAATTATGAGGTATTTCTATAGATAGCCCGAAGGCTATCCATCGTGATCTGCGTC TGTCATAATAGCGACATTCGCATAGTTCATTGAAGAGGATTTAATAGAACGACGAACATTGTTCTGCAAT TTATATTTTTTCATATCAACGCTAGAAGAATCAATTTTTACAAATTTCATTATACACCTCATAGAACTTT TCATCAGGAATCCAAACCGCGTTTAAATTCATTAAATGCTCGGCGAATAATTTTGAATTCACAGTTATAT TATTAACTGATTTCCATTTAGCAACTCCCGTTCGTTTATAATGATCGGGGTCATATTTCGTGACGTACCA TTCATATAAATTTGGTATTAATTTTACAGCCTCTGGATTTGTCGCTGATTTATTATACCATGGTTTCGCT TTTTCAAGTTCAGCTGCTTTTGCAGTAGCAGCAACAGTATTTCCAACACCAACTAATTTTTCAGTTTTAA TGCGTGGATCATTAACATGAAGACGAAAAACTTCGCCCTTTTTCATTTTTAAAGCATGTCATGCCTTTAAT TCCAGGAAGCTTAACACCTTTATTAACACCGCATCATTCCTTTGGGTTAAATGATCCTTTAATTAATAAG GCGCATTTACCCGATTTAACTACTTCTCATTCAACAACTTTATCTTTCATAACGTTTTTTGACCATTCAG ATACTGCTCTTTGATGGCTAAATTCTAGCAATTTCACTATAATTTGCACTAGAACGTAAACTATTTTTCA GTGTTTCAACATTCTATTCATCGCATATGCCATTTCACGATTACGATGAATTTTATATAGTAGAAAATAG TGCTAAAAAGTGTTCACGAAAAGTCATGTTTCACCAAATTTCGTTATCATCAGAACCTCCCATTGATCT TGGAAGGATATGATGAATTTCACCCTTAAATTTAGAAACGCGGGTTTTACCCCGCACTATTAAGTCATTA TAGATTTTTTCGTAATTCACCTACTGTTATCCATTTACCATTAATCTGTACTTCATCATTTTCATTTACG ATAATTGTATCGCCATTTAGCTCGAAAGTAAACCACTCGCCATCTTCTTTTTCTTCAAACGCTTTTTCAC CGAGAACTAGACCAGTGATTGCGCAAATATCAAATAGTTCTTTGTTTTTAAGCATATCTGCATAAGACAT ACCCCAACTGTTGAGAACTTTACCACGCAATGGATAACCACCGTGAAGTTCTTTTATCACGAACATCAATA AGATATCCGATAGCCGAATCACCCTCAGTCAAGAAAAGAGTAGTATCAGCATCTTTACCGCAAAGATTCG CTTTGATATGTTTATGAACCTTAGCTTTAGAAGCCTTTTTAGCTGCTTTAGTTTCTGCTGCTTTTTCTGC CGCCAATTTACGAGCCAAAGCAGCTTCAATAATCGGCATTAGAATTGCTTCATTATTTAGAATATCACGT GAAATCTTTTTAGCATCAAGTTGAATATGACTACGAATTTCGCCAAATGGAGAAGTCAAACGCTCTTTAG TTTGACGAATCAATCGCATGTTTTTCATATCACGAACAAACATAACGATAGTCAAACATTCTTTGACACG TGCTTTAGTCACATCAATTTTGAACTTACGTTTGATTTGTGGAAATAAGGTCTTCACAAATATCATCCATA GCGCAGTCAATGTGATGGCCACCATTCTTAGTATGAATGTTATTGACGTATGTTAATTGACGAAAACCAT CCGGTGAACGACCAACCGCAATAGAACAATTTTCTTGCTCTTGAACAATAGCATGTTCATCATACTGCCG TGCATATTTCTTAAAATTGCCCTGAACCTTTTTACCATTAAAGGTAAATTGAATATCAGGATAAACTACA GCAAGTGTCTGGAGACGATCCAGTGTAATGTCAAGATAAACTTGGGACAGCTCATTAGTTTCAAATGACA TAAAATCAGGAATGAAAGTAACACGAGTTCCTTTCCATTTTCCAGGAATATCTTCCCATGATTTATTTTC CATGCCATTTGAACAACGAACTACAATATTATTTTGACCGTCGCCAGTTTCACCGACAAACATCACAGAA AAAATGTTTGTCAAACTAGAACCAACACCGTTCATACCGCCGGTGACGCGTTCTTTATCATCACCAAAGT TACCACCTGCTTTTGGAATAGTCCATGCGGCAACAGGACCAGGAATTTCTTCACCGGTAGGTGTTTTAAC CATCGCTTGTGGAATACCGCGACCGTTATCTTCAACTGTTACTTGATTGTTTTTAATAGTAACATTAATT TTATTCGCGAATTTAAACTTAGTACGAATACCTTCATCTACTGAGTTATCGATAATTTCATCAATAAGCT TAACAAGACCAGGTACATACTGAACACTTTCCCATTTACCAAACATAAAGCGCTCATGCGTTTCATTAGC AGAAGAGCCAATGTACATGCCACTACGCTTTTTGATATGTTCAATATCGCTCAGAATTTTAATTTCATTC TTAATCATCACTTATCCTCGTTTGGTTTCGGGAATATTATACTCCGGTAATCATAAAGCTAAAGGCCCGA AGGCCTTTTATTTAAAACGAATAGTTGAATCCTTAAAGAACAGCCCAGAACATACTGTTCCTTCTACTTT CTGCCCGGTAGGTCCAATAGCACGAAATCCAGTATGCTGGAAATCATTTTCAGAGCAACCGAACCAATTA TATCCAGTGATTTCAATATTAGTAAAACCACTTGAAGACAAAACTTTGGTTGCATTAATCAGCATCAGTA CTATTAATTAAAGACACTGCTAATACTAATGCTGCAATTGAACGACTAATATATTTCATAACTACCCTTT AAGCAAGTCGTAAAATCCATTATTCCCATGCTTAGGAAGCGGAAACTAACCGAACAGCCAGCCGATGACA ATCAGGACATACACCAGTATCTCTTCCAGAAATTTTCTTGATTTTTTCGTATTCTTTTGCACAGTCTTTG GATTGACATTTATAATCATAAAGCGGCATAATTATTCCTTAAAGTAAGTAAGCTTTCAACATCTGATATAAAGA CCACGCCTGATCATTATTTCAATAGTAACTTTCATGACTGGGAATTCTGTGAAATCTTCTATTTGTTCT TGCTCTTTCTTCTCCTGCTCTTGCTCTTCAACCGCCTGATATGGATTTTCCACTTCATCAAAGAACCCAG CTTCGTTAGTAGAGAGCCAGATAAAGTTTTCGTCAAGGATATCACCGCCGGCACAACGTTTGAGTACACC TATGGATGTCATAATTTTAGTAGGACGCCCAAGATAATCAGCATCTAAAATTTTAAAAGGCTCCATACCT AAACGTCGTGCATAGATTCCGTTATCAGTATGGTCTTTAATAAAATTTTCTTGAGCTTGTTTTATTTTTAA ATTGATACCATTATTAACTTCAAATTTAATAGCCATTAATAAATTTCCTTCCCAGTAAGTTGTGCCGTCT TCAGTAATTTCACGAAATACACCATAAATTGGCTGTTTATCACCGACTTTCTCATACACATAAACAGAAG TCAAGTGAGTAAACTTGCTAGTATGTTCCTTTTGAACTACTACCAAATTTGGATCAAATAATACATCTTC AAATTCATCATTAGTGCAATTCTGAACAATTTTACGTTTCATTACAATTTCCTCATTAATTGAACAGTGG AGCGATACGTTTCAGAAGAGTATCAACACCTTTAGCGAATTTTCCATTTTATTCTCCAAGTTGTTTTCTG TATCAGTAGTTGATATTGATATAGTACCATAATCAACTACTGATGTATATAGTTTTATGAAAAAATTTAAA CTTTATGCATAGAGAGCATTGCTATAGTGTTTAATCCAACTTTCAGGAATGACTTTGTATGTTCCTAAAA ATACCACGTGTACAACTTAACACCATCTTCTACCCATTGATCGGTAATGTATCCACACATAGCGCGAGT ATAAACAACCCTTCCATCATCTTTAATAAAGTTAAATTCACAAGGAGCAATGAACTTGATAGCCTGACCG AGTTTCCACTTAAAGTCTACACCTACATGCGAAGTATCAATCGTTTCAATTCCTTTAGCAGGAACAGCTT TTAAACGCAGACTCAAGAAATTTCGCACGAACATAGCCAAACTGGGGTTTAGACTTTCCATCTTTAGG AATGATACGCACTTTTACTTCAGAATCTTCATCTTTAACACCATGCTTAAGCTGAATGCTTACAACTTCG ACCAATTTTCCTGCTGCTTTAGAACGGGATTTATCAGATACACGAGCTAATTCACCAATATTAATAATCA TAGTTATCTCTCACTTGTTAAAAGATTTTATACTCCACGGGACCATTATACTCTGGTCCCAAGAGTTTG TAAACTATTAATTCAAAATAGCTACCACTGCACTACGAGGAACTACGGAGTACTCTCCAGCATGAACTAC GTTCAGAAGTTCAACGCCATCTTCCAATCCATTGGTCAGTTACCCCAACCACCAATCGGATTCGCAAATGG ACGACGAATGTAAACTGCCTTACACAACAAATCAGTCGGGTCTTCAGGTTTTTCAATTTCTGTCAATAGA TTAAAATCTTCTTCATAGATGATGAATGATGATACCCTTCCATAATAGTTTCTAATTTCCATGTACACTG TACCAATGAGAATTCCACTATTAACACTAATGACAGTAAAAGGATATCCGCCAGTTGTACCAAGAAGTTC CCAAAATTTGCTATCAGTCGTATTCGACATTGTCTGAAAATCAATTTCAGATGGTTTTTTCATTTGATAC GCAGTATTAATTTTAATCATAATTTTCTCTTTAGTTTAAGGTAATAAAGCCTTTTAGTTCGGCATAGGAT TTACGGAACATTACTTGATGCCCGCCAATGATAACTTGGTCATCTGGTACTTCGTATACAGCAAGATAAA ATCCTTTCGAAGCTAATTCTTCACGCTCTTCTCGTGTGAACCATTTCATCATATCATATTCGCTAGCAAA AGCAAAATGATAAAGAGCTACAAACCATCCGGGAATATGATATTCTACTCCAACATAATCTTTCTTGAAC TTAGTATTAATTACGATATTAGCATTTTTAACTAATAGTTTGTCTTCGTGGCGGCACAGGAATTCTTTTAT TATCATTACTATGATGCATAAAAATTAGGTCTGTCATAACCTACATGTAATAACCACTCTTCACTCCATGA ATCTATTATACTTCTATACGGCGTTATTTGAACACAAAGATCTCGGCGTATTGTTATAGCGTCTTCATAA TTAAGAATACTAAACGATGATTCAACACGATAAATTTTCATTTTATTATCCTCAGTAGCTATGGTGTTAT AGTACCACAACTAACCGAGGAAGTAAACAACTTTTTATCGTTTTGTTGGAAGAGATAGAGGATCGCATTC TTCCTCTGATGGAGCATCTTCAAGACCCATAGCATATCGCAAAGCATACTTCATCATCAGGATGTCTTTC GCACAGTCATGAATAGAATCATGTGCAACGAATCCATCTAAAGTTCCCTTTGGAAGAGGACACGTTGTCA TATCACGAACAAGCAGAAGTGCTTCAATTCTAGTACGAATATCACGCTGATTCCAAAATTTACAAGGTTC TAACTTAAATGTATCAAGCTCATTCTCGGAAACGCCGTTAAGACGTTGAATATCGCGAATAAGATCGACT AAAATTGGAAAATCAAACGACATTCCACGGCACCAGCCTTGAGATTTCCAAGGATCGATATTATGTGCAT TGATGTAATCATTAAATTTTGCAATACCGTCGATAGTGCTTACATCTTCATCGGATGGTGCAATATTTTT TCGAGCTTCAGGAGATTGATTCTTCCACCATTCGATAGTACTTTTAGTAAAAGACGGTGTCCTTTTTGG CTTTTTAAATCAAATTTGATTTTAATGCCACGTGAAACTAATTCATCGAATGTTTCAACTACTTCTGGAT TAGGGTCAAGCAATTACAGCCAAATCAATAACCGCTGCTTTTTCACCACTTCCCATTGTTTCAAAATC TATAATAAAATCAAACATTAAATTTTCCTCGCTAAATCACGAATTTGACCTACAGTATAGTCTTGAATAT AAACTTTATTAATAGGCTCATCAATAAATTTTGCCATAGATTCAATATCTTTTTTGTATTTCTTCAAGACT GTATACTATCTTTGAAGCTTTTTCGCGAATAGTGATATTTTCAGGACCCGGATTTTCTTGAATGACAACT TTAACATTTGTCATAAGAGATTTAAACTGGTACCAACTTAATTCAATCATTAATAATCGCCTCATAAAGA TAGCTAATTTCGCCTAAAACATAATCATTGATTGTAACAGTTTTAACTTCACCGCAAAAGAATTCTAACG CAATTAAATCTCGTTCAATTTCTTCTAATTGAAGCATCAACTTACTAGATTCAATTTTTACAGTTTCACG ATTTTTGCTATAAGCTATTTCATAAATTTCGCTTACTTTATCTTGAAGAAGATAAAACTGATCTTTAGTT ATTTCCACGAATAGCTTCCTCAAATTTAATCATACATAAAACACATCATAACGACCACGGGTGACACCAA CATAAAGAAGTTGTTGAGCTAATTCAACATCTGCATAATGAATACAAGGCGTATAAATGAAAGCACGGTC TACAGACATACCCTGCGCTTTATGGAATGTTGATGCAGGAAGTGCTTTTCACTTTACTAAACTGTGATTTA GCATCCCAAAATCACTCCACGGAGCTTTTCCGCCTTTGTTCCAATTTTTATAAGTTTCTGCTGTTTTAG CTAAAAATAGGTTAAACTTATACAATTCTTCGTCAGATGAAATTATTTTAATCTTTTCACGATAATATTC ATCATCGCCATAAGTTTCTACTGTTAAATCCCAATGACGAATTAGATATTCTCCAGGAACACCACGGGCT TTAACAAACGTTGATGTATACTCTGCTTCTATAATACGAACTAATTGTCCGTTATTAAAAATAATTTCTG ACACAGGCTTCCATCAATTTTATATGTTTTAAATAATGGTTCCTGCATTACAATAATTTCACCGACAAT AAAATCTTTATCAGTTTCAAAAATCTTTTTACGAATAATGCTATTTAACTTGTCAACAGATTTATTCGTA AATGCCATTACGCGATTTTCAAACAAATCATCTAGTGATTTGACGATTGAAAAATAATTTACCATAAAAT CGCGTAAAGCGGTATCACCAGTAAATCCACGTACTCCATGCCCGTCAACAACTTTATCATAATTCCACTT ACCGTTGCGAACGTCAGTAGCTACATCAATAATAGGAGCATTACTGCGTTTAACTTCAGTGAGTTCACAC TGATAAAAATCTTTATGTGTAAAGAATGGACTGATATAAGCAGTATTTTCTCCTGGTTCACAGGTCTGA TTTGCTTATTATCCCCCTATTCCAATTATAGTACACCAAGGTGGAATAGTTGAAAGCAGAATTTTAAATAG CTTTCTATCATACATTGACACTTCGTCGCAGATTAATACTCTGCATTTGGCTAAATCAGGTACTTCTTTT TGTTCAAAAAGAACATTTTCTTCATATGTTACTGGGTTAATTTTAAGAATACTATGAATAGTACTCGCTT CTTTCCCTGATAGTTTTGAAAGAATCTTTTTAGCTGCATGTGTAGGAGCTGCTAAAATAATACCAGTTCC ACCCGTAGATATTAAAGCTTCAATGATGAACTTAGTAAGAGTAGTCTTACCGGTACCAGCAGGTCCATTA ATAGTTACATGATGTTTCTTTTCTTTAATAGCCTTCATAACAATGTTAAAGGCATTTTTCTGGCCTTCGG TCAAATCATCAAATGTCATCGTAAATTCCCTGCAATTGGTATACTAACAATACGCCCAGTATCTAAAATT CGCTGATATAATCTTTGCGTGTCTACGTCAGGCTTAACATGTTTAACTTCTATTTTATTAAACCAAAATT TACGTGGAGTCTCAACTAATCTTGGAATTCCCTTACCTAAAGCTAATCGATACTGCTCTTTAAGAGTGGT AAATACTTTATCAGCAATCTTCCATTCAAAATACAGCAGGACGATGTTCATCAAGCGGAACTGGCGCT GTAAATCCGTCTTTGTCTCGGTAAACTATCGCATATACATAAACCATATTATCCTCGGATAAGTTTAAAA ATTGAACAATTTAGCGGATATCCTCTTTTCAGTTTAAGTTTATCAATAAAGACAAATTTTGATACCGCT CTACACCTTGAATAATTTTATCACACATATCATATTGCATTTCTGCTTCTGACAACTTTTTCACAATTTT CCAATCCGAGCCTTTAAGAAGAACGTTCAATTTAACAACTTCAGCGCCTTCTTGCTATGCGAGAACCATCA ATACGTGCTTTAAGTGCTATAATTCTCAGCTTAATGTCAGAGGTCTGTTTTGATTTAGAAAGCTGAGAAA TGTGTTCAATTCGATTTTTCACCGTTTTTTCTGTATAGCTTTAATTTGATTATAAGTCTTTTTGATTTTAGC CCATTTCTTTTCATCTAAATTTAGTTTATGAACTTTTTTCGCAGATGAACGACCAATTCGCAAAGCAAAT AAATCACGCTTTTCAATCAACTCTTCTAAAGTATAATCAGAACGAAATGTATTATACTTTTTCTTTACTG CAATAACATTCCCTTTAATGTATCCAACGTTATTATCAAAACGTTCTAATGATAATTTCTCTCCTTCAAT ACGATTATCAAAAGGTTCTCCCGAGTAAGCACAAACTTTTTGATCTAAAAATGTTCTTAATGTAATTGAAG TCTAAGTTAAAATCTTTAGAACGTCTTTTTGCAGATGCCTGAGTATGCTCTAAACGACGTTTAATTTTAC GAATTTGGTTATTAGACAGCTTCATATTTTTCTCACATCTTACGGACGGTTAACTACTTATACTATAACA TTTTTACTTTAACTTGTAAACAACTTTATGAAAAATGCTTTAAAACTTTCATGGTATAATGAATCTAAGT CCTTCCATTATAGATTAAATCCTTCAAAATCAAGAGTATAGATAGTGTATGTTGAACACTTTTTATACTC ATATCTATCTGCAATTCTAAATACACTTCCAGCTGGTATCATTACTTCTTGTTCATCTGAAACTAATTCC ATATTACGATAACGATGACTATCCGGAAACTTAAAGTTTGGATTGTATTCTTTACAGCGTAGAGCTTTTA TAGCATACTCCTGGAAATTGAATACCATAGGAGCTTTGAATTCAAAAATAACTTGTGTGTTATACTCTAA ACCAGAAGCAAAATGTAGAGCTATATTTTTATCATATGAAGCTGATACGACTTTATCAAATGTAATAATA TCAATTCCTTGATTTAATACTTGTTTAGTCTCAGCTGGAACACCTCTCCAAAGAGGTTTATCGTTTGGAA CCAAACGAGATTTGATTATTTCATTTAACCAAGAATGGTCATCTGGTTTATTAGTAATACAATGAATTAA AAGTTCAATTTCAGATAAATTAAACCCTTCAGAAAGTAATTCTTCACGAATAGAAGCACGCACCGATGCA TCCATTGATTTTATTTTAAAATCTTTTAGTTGCATTACTGAGTATTTCATTCAACTACCTCAATATCATA AACTTTAAATGTTCCAAATGAATCGTGTAATTTTTCTTTTGAAATAGAAGTTATTTTATACTTTCCAATT GGAATCATCCATTCTTGTTCACGCACAATCATCATTAAGTTATCAGTACGCTCTGAATCTAATCCATCAG TATCTTCATACGTGTACTTAAACTCAGTATTAGGAGAAGAAAGTATAATATCGCTGATATGGTCAGAATA ATTAAAAGCTTTATCAGTTTTTAAACGAAGTATTGTTTCAGTGAAATATTCAGCATAAGAAAAAGAACAC GCTGTATGCAAACTAGTAGTAAATGAATCTACCCTGTTCGTTGAAAACACTTCTCCAACTTGTAAATCTT TAATGAGTTCTTTTGTCGATTTTGATATACCACGATATAATTGATAAGGCGATTTAGTTAAATGCTTTTT AATGATTTCATTTAAATGCTTATGAAGAGCTTCATTCTTTTTGGCTTCCATACATTGCCAAAGAACAGAC TGCTCAAAGTCAGTAAATTTTTCACAGACCTTTTTATACATATCATATTGAAAATCAACGCTTTCAGCTT TTATAGATAACTGTTCAACATCTGCAAGATTAATAATCATGATAGCCTCCGTATACTTCAGAAGCTATCA TATCATCGTTAGAAAGGAAAGTAAACAACTTTTTGAATTATTTTGCCCCAGGGAGCCCAAGGCGGAGGGTC AAGATGGTATGAAGCTAGTTCTTCTAGAAGAGCATCTGGGGCTTCAATTCCATAATTCTGTAATACTATA CGGTACTCTTTCTTATAATCACTAGAATCATTCTGGTTATTCGTAGAATGATTATCTTCTAACATCTCAA ATAAATCCATATTAATTCCTAGCGATAAAAACCAAATTTACGATTAGTTTCAATGATCTTTCTTTCTTCT TCGGACATTCTCCAGCGTAGTCCAACATCAAAATGAGCCCAGACCATCCTAACAAATGCATTTATATCTT TAATATCTTCAATAATGAATGTTTTACTTTTAGAAGGTTGACTCGCTAATTTAACTTTATCGTCTTCAAA CATGTCAAAAAGACCATATTCATGAGCTCTTTTATAGCCTTTAATAGTTAAAGCTTCAGAAGAAGAAAAT GGTGAATCTGTATTCTGCAAAATATCTTCAGTATGCTTTATAATTAGAATAATATTTTCTGGATATTTTC TTTCTTTTATATCTTTAATTAAAAAATCCGGATTTTCTGCTAAAGGAATAATTAATGAGCAATTTTTATC AAGTGTATTTACTGATTTACCTTCTTTAGACATAAATTCTATTGAATATAATTTTGCTACTTCAATCATG TGATTTCCTTTTGCCTACTAATGGACCGTCAGGAATTTTATTTTCCTGGATATATTTCTCATTTTCTTCC ATCATTTTACTGCCAATTTTAAGAAGCAAATCCATTGCTTCATTTGCTTTTGCTTTAGCTTCTTCTAACG TCATATCTTTGTTCATGATTTATCACCATAGATGTCTCTCATCAATTTAAGCGCTGAGCGTTCTAGTTTC TTTTCTTTCTCAGCACTAATCATTGATTTCATCCATTCTTCTGATTCATTCTGCATTTCTTTATTTGCTT GTTCAACCCAACCGTCATCAATATACATTGAGTTTGGTCTATTGAACCATTCAAGCATCTTCTTCAGAAC TTTCATTCGTTTTACCTAAAACAATAGTAGGAGCATCGTCAAATTTATGAATTTTAGCAAATTTGGATT TAAATTATTCCATAAAAGGTAATAAAATATGATAGCACCTTCGTCCGTAATTATAATTTTATTTCCT TTATCTATTTTCCAATCATATTGAATCATATGAATAGAAGGATAACGGTTTATTATTATAATATGCTT CAGCAACATCAATAGTTAGATTTAGTAAATGCTTGAATTGCCATAAATGGAGAATTTTTGTACAGTTTC AATAATTCCGATCTTTTTAAGTTTTATTTCAACATCTTCTGGAATTGGCATTGAAATAAAATCTTCTACT AGATACATATTATTGTCATATCTTTTCAATCATTACTGCTACAGTAATTGGAACATCCTTGACAAACG CCATACTAATACTATTTGATAGACATTTCAAACAAAATTGCTTCCATAATTTTCCTCAATCACAAGATGTA GATGAACAACTAGAATCACAAGAACTTCCACATGAATCACCTGCCCATACATGAACAGGAACATTAGTAT CATATGAATCAGAACTAGACTGTGTATTCTGTGTGTTAGATGTAGTAGGTGTTGACCAGCGCCCAAGG ATTTTTATAATATTCTTGGGCTTCTTCATAAGTCATAGTAACTGCTTCTACTGTTCCATCCCCCATATAA ACATATTCAACTACAGTTAAAGGAAGGTAGTCATTTGAAATAGGAACTACACCTTCCCCAGGAGTTGTAG AGAAAAAATCCGTAAAGAAACTTTTAAACCAATTAAAGATAAACATTCAAAAGCCTCTTTTGAATTCG ACTTGCTTCTCACCATAATCATATCGAATCTCTACATTAAATTCGACAGAACCATCTGCGTACATCATAA ATGAATGCACAACAACTTCTGTAGACCATGGTTGTAGTTCATATTTCTTCATTACATGTCGTGAAATGAT AATATCTAAATCTTCATTTGGTTTAATCCAACGATTTAACATAGTACTCTCCTCTATAAGATAATTCTAT TATACCATACTCATTTTGGAAAGTAAACCATTTAAATGAAAAAAGGACTCCCGAAGGAGTCCTTGAGTTA TTAACCAGTTACTTTCCACAAATCTTCATTTGCAGCAATCCATTCAGTACGTTGATTTTCTTCATATACT GTAGAATATGCTGCTTTTTCTGAAGGGAATGTCTGGTAATGAGCGCCAGAAATTTTGTGAACGTCAGAAT AAAGAGGGAAAGCTACAGAAATTTCCTTTCCTTCAATTTTCTTATTTCCATCTAATTTCTGCTCAAATGT TTTGATATTAACATAACCGCGAGTACTAGCCATGTAATTCTCCTTTATTTAAATTACATGATTATTTATA CATCTTCTTTTCTGAATAAGTAAATTAAATTCTTAAGAGCCGAACTTGTTACATCATATTTTCCTTTAAG CGCCTTTACAACCGGGCCTGTTGCTGGTTTACCTAAAGAAACCCATAACTCGTGTATTTCGCTTTTAAGT GGTTCATGCCAATGCGGTGCTTTTCTTTGCGCCCTTGAAGTTCCTTCTGAAATCTTTTTATTTCCGCCAT TCGAATAAAACTTTTTCATTACTTCAGATTGCCGAGCTTTTCTTTCTGCCCTATTTTGGGCTATACGTTG TGAATTCTTCATCCGAGTTTTTGTTTCAGGATTGTTTAATCTAAGTTTATGTTCTAATCGTTGTTGCTCA GTCCATTTTCTTCCTTCTCCACCCTGACCACCAAGGAGAAATATTAATGCAAGTATCTGGGTGTTTACGTT TTAATGCAGATATTAGCTCACGTTCAACTTCATATGATTTTTCTCTAGAACCATGGCATTTTGACCATCG TATTTTATAATTAAATCCATACTTACGATATATGTTCCATAGTATTTTACCTGAACCCGGATATTTATCA TTATATGGATTTACAATAAATGATTCATGTTTCCCAGCGTACCAAAAGACGCCTTTCGGCGTCCTAACTT TTACTATATATGTTCTATAAAAATTTTTGTTTAATATCCATTATCTTGACGTTCAAAATTATGTTTGTTCT TCAGATAATAAAGTTTAAAGATTTCTTCCGCATTCATTCCAAGGCCAACAAACATATTTAATACGAAATG AAATATATCCACAAGCTCAAATTTAATTTCGAGCTGGTCTTCGGGGGACATTTCATCAATGCGTTTTTCT TGCGCTTCAATATAACGTGCTTTCCATTTTTTCCATACAGCAGAAGCTTCTTTTTCACCACGGTGACATTT CACCAAGAGAAGTCAGAAGTTCGCGGAATTCATCATCAATACAGTCTTTTTGTTCACGCATCCAAGAAAC AACATCACCGGCAGTTTCTAATTTATCTGGATGATAGCAGTATTCGCGGACATTAGCCAAACGAATCTGT AAAAACCGCTGCATATCAAGCATAACTTGCAGCGGATCTTTTTCATCACCGAGAATATCCCAGTATTCAT TTTGAGCTTTATCAACACCTTCGATCAAATGAGCACATTCATTAAAGTGAGCCATTAGTTTTCCTTTCAA TTCATTAATAAGTTAAATAATTATATCATTTGAGTATGTAAGCAATAATTAAAAATATATACTTCATCA GTTCCATTCTTTTCTTTGGAATGATATATGTTAAAGACGTATTTTTTATTAAGATGCTTTACATTATATT TTTTAGACCATTCTTTAAGAAGAGTGTTTTCCTTTCCGTGGGTGTTCTAAAACATTCGACTGCCCAAATTT TATTCCTCTATCATTTAAAGAATCTAAAAGATTTAAAAGGTCTTTTTCTTCATCTTCTGACCAAAATTTA TTATAATCAGCAACTGTTATGAGATACGGAGGATCTACATATACAAAATCGCCGTCTAAAATTTTAACAT CTTTAAAATGCAATGAACTAAAGATTATTTTATCACAATTTTGTTTAAAGTGATTATATTGTTTTTCACT ATTTTTGTTTATAGTTCTTTTTCCAAACGGAGTAGTAAAATTTCCTTTATCGTTTATACGAATCATATTA CTAAATCCGTGAAAATGAAGAACATAAAGTAAAAGAGGATCCTAGTTTTATTATAATCTTCACGTAATT TCAAAAACTCTTCTTTTGATGTTTTTGATAGTTTGTATTGCTTTATTACTTTTAAAACGTCATCCCATGA TACATTAATAAGACGCTTATACATTTCAATAATTGGTTCTTGAATATCATTGGCCAATACAGGGCCATTA ACATTCAAAGACACTGATAAACCTCCACAAAATAAATCCACGAATCTGTTATATTTTGGAAAGTGAGATT TGAGTTCAGGTAATAATGATTGTTTATTACCTGTATACGCGATAGCTCCTAGCATTATATTCTCTCATTT ATTGCAGCAAAAATGAATTATACAATTCTTCATCATATGTATTTGATAGTAATACTAACACGTTTTGCGA TAAATATTAATTTAAAGGAGGATATATATGGTACAAAAATTAATGGCACTTGTTAATGCCATAAAAGGTA ATAAGAAACGTATAGCTTTTACTATTTCTACTATGGTAGGAATTTTACTCTGGAACTTTATTTTATCACC TGTTGCAATTGCACATGGTGTTAATATTCCAGTAGTTACTCTTGATACATTCGTAGATTTAGCATTTGCT TTAGTTGGGTTAATTTAAATCTTAGCATATTTAGATAGCCGCATTTTAGCCATTAACCCCTGGGCAATAT TATTTTTCATATATTCCATAATTTGTTCAGGGGTTGCACCTTCCTTTCTAATCATATCATTAACATCTTT TGATTTCCAGGGAGATTTATCCCAAAACATAACCCTTTCTCCTGCATCAACTAATTTAGTCATTCGTTTA ATAGTGTCAGGGTGACGAGGTTCATTATCTAAGACCCACACACGTCTATCTTTAAATGGAACAACTTCTA GGTCTAATTGACCGCCCGTAATAGCTATACCATTTTCAATAAAAAGTGAATCTATAGGTCCTTCTAGAAC ATATACATCACCATCTTTAACTCGTTCGACTCCATAGATTTTTGTTGCCTCAGGATAAGCTTCGATGGTG ATATATTTTTGAGGAGCATCTTTCTTTAATGCACGTCCTTGAAAAGACTCAGCTTTTCCATTAGCATTAT AAATTGGAATAACAAGACGAGGCTCAGAAATTTCCTTTTTGTATGTTCCCGGTGCTATGCTATTAACTAA TTTAGGCCATTCGGTTGTAAACCAAAGATATTTCCATTTATCCTTTGGAATACAACGAGCTTTTACGTAT TTTATAATTGGATGGTCTTCCGCCAGTTTATCTAATCTAACACATGACGGAAGAGATTTAATTATTTTCT TCTCGGGTTGTTTAGGAAGTTCTTTAGGTTTTTCTATTGGACGACTTTTACCTTTTTCTTTTTCTTATTTC AAAGATATACTCACGATATAAATCGGGTTCAAACTCCTTTAAATATATTCCGATTGGTGCATGATAGTTA CAGTTATAACAATGAATATTTCCTTCATTATTATCACCATAATACCATCCACCGGGCTTTATTCTGGTCGG TTTTTGAATCTCCACAAACAGGGCATCTAAACCGTAATTTAAAAGTTGAACTATTATTTACTTGTGTGAA TTTAGGTAAATGAGCTAATGCACGGTATGCAAACTCATTATCAATCCAAGGTATTGATGACATTTTTACT CTTCTTTTTTCTTTAGATTCCTCTTTTTTCTTTTTTAGGAATCTGTTCAGGACCTTTATTTACTACAGCGCC TGATGTTGTTCCAATAGAGATATTTTCAGGATTACCACCTGAATCTCCAGCGACCATATCTTCTTTGATA AATTCTTTAAATGTTTTCATATTAACCTCTATTCATAAAAGCATTAAAAATTTGGTCATCAATAGAAACA TTTACTTTAGGCTGTTTTTCAGATGGCAATTCATATCCACATATTACAATTTTATGATCAATATCAAAAT ACACAGAAGCAATATGATTAATGATATTTTCAGTAAAGTCTAAATCAACATCAATATCTTTTTGACCAAA GCCCAAAGGATAAATAATGCGAGTAATTCGATTATCTTTAACAAAGATTCCACCGACATACTCTGTGCTG CGTTTAAAGTCTACACGCCGACGAAATGAAAAATATTCAGGCTCTTTATGAGCTCGGCTCATAGGACACA ACGAATAACTAGAATAAGAGATGTCAAATCCTACGCCTTCAATATGAACTAAATTGTCATGATTAAACCA ATTATAATCATATGCCAAGTCCATTAGATTGTCATATGTGAAAAGCACCGGATTAACATCATTGGTCACA AGCATATAATTAGCAACTGCTATAATTTCATTATTTTTAACGAATACAAACCGTGATTGATGCGAATGGC CTGGACCTGCGTTTTCACGATTAATGATATAACACTGGGCACCTTTATATTTGTACGTGTCTTTACACTT GTGCATTTGATAAAGCATTATTCACCTACCACTTCAGCGATGATATTTTTGTTATTAAAGTTTTTATCGC AATACAGAACATAATTATACTGCATTACACCACCAGACTTAAGCTGTTTTTGCACTTCAGCTTTCATTTC AGGACGATCACGCTTAACGATATTCATAATATCTGCTTCAATTTGAGTTTCAACCTCAGTCTGATCCGCA GTCATAGACCATTCGCACAAATCTTTATCATAACCTGCCATAGCAGGCTGAGCAGCACAAGAAGCTAAAG CAAAAATTGTAGCAAAGATGAATTTTTTCATGATAATCTCCTCAGTAGTTTATGTTTATATAGTATCTCA ATTTCCAACAAAAGTAAACAGTTATTTTAAAATTTCTGCGTAATCACATGTTACAAACTGTTTCTCTAAC TTGACGATTTTACGAAAGTATCTTTTGCATTGGCGAATCTGCCTCTTCGTAGGGCGAACAGCAAACTTAA TAAATTCCACTCGACCAAATGGAGGGCTTTCTTCTGCTGGAATATCTAACACCAATTCCCACGTATCTGC AATAAGTGCTTTGAATTGCGTATTTTTCCTGACGTTATACGGAGTAGGTTTAAATAAAACAATATGCATA TTATCCTCGGCAATCCACTTCACATACTTTCTTGTCATCAATGAAAGCTTTAACTAATGCTTTATTAACT TCAGCATATTGAGTAGTAGCCCATTGAACGTCATCTTTCATCATTGTGATTTCTTTAGTAAACATGCTTT CATTCTTAAACCACCCCATAAAAACTACCTTTACCAATTCCATAACAATCTCCTCATTTAACCGACAAGA CTACTATACCATAGTCTTGTCAGCTTGTAAACTAAAATTTTAATTCATTCGCCAAAGCATCTAACTGAGC TCGAGTCGATTCATTTCTTTGATAGCGATTCTGCTCAGCCTGAATCTGTTGTGAACCTGCTACTTCGTTC ACTTCAGTTGGAGTAGAATCTTGTTCAATTTCTACCCATTTTTGATTTCCTTTTTGAACACCCATCAAAA ACTTATTCCACTTATTCTTATCACCATATCGTGATTTGATTTGCTTAATGAGTTGTTGTTCAGCAGCTGC TAGCTCCTCGGTTTCAATGACCGCAAGCATAAAATCGGCTGTTGCTGGAAGACCGGCAGATTCTGCAATA TCGCTCATGTTAACATCGGAAGAGTCCCAAGCTTGTTTACCAACCTGTGCTGCAGTCCAAAGAACAGTTT CGGTTTCAACAGCCAGAGCACGTAATTCCTCTGCAATAGCTTTAACAGTTGTGTAACTATTTTCTGAATA AACTCTAATGCGGCAAGATTTACAAATACCTAGATAGTCGACAATAATGATTGTTGGAACAAAATTCTTC TTGAGCTTCAATTCGTTTAAAAGTGATCGAAATGTATTAGCGTCTGCTCCACCAGTAGGATACTGTTTAA CGATTAAACGACCAAGAGTAGATTTCTCACGCCATTTTTCCATTTTTCCTTTATACTCAGCGTAAGAAAT ATGCCCATCATCAATGTCATCAAGAGAAACATCAAGCATATTAGCGTCAATACGTTTAGCACAGACTTCT TCTGCCATTTCCATGGAAATGTAAAGAACATTATGTCCGAGCTGTAAATAATCTGCCGCTAATGAACACA ATCCTAATGATTTACCAACGTTAACGCCAGCCATTAAAACGTTCAGTGTTCCAGTTTCAGCTCCGCCTTT CGTAATTTTGTTCAGAATTCTGAGTTTAAATGGAACCTTACGAGCTTTATTCATATAAGATAGCCAACGT GCTTCGTAGTCATCCATCCAATCATGACCAACGTAACTATCAAATGAAATTGATAATGCCTGGCGCATGA TGTCAGGAATAGCACCAACATCCGGCATTTTCTTATTTCGTTTTTCCGGAGGAAGCTCAGCATTAGTTTG AATTTCGATTATTTTAGACGTAGCATTAAACATCGCCCTTTGCTGAACATATTTTTCTGTTTCTTTTACT AACCAGCTGTGGTCTTCCGGAGAATCAGCCAGTTTTGAAATAAGTGTTTTTACACCAGAAATATTCTGTTT CAGTAAATGAACTATTTTCTAATGCAACATTTAACGCATTAATAGATGAAGCGCTATGGTACTCATTAAC ATGAGATTTAATTTAATTTGAATGTATTTTTAGCTGGACCACTTTCAAAAATTCTGAATCCATATATGGC CAAACTTTTGAAAAATAAGCTTGATCAAATATGAGATGAAAGAATAATTTCTACCACACTTACTCCTT AAAAGAATTTAAATTTTTTCTTTGACCTTTTATTAAATGCATCTTGCAGTTGCATTGTAATACATTTTTC TACATGAGGAGCTAACTCAGCTTTTCTTTCTTGGTCAAGAACAGCAAAGTCCATTACAACCTTTCCATCA ACCCAATCCAGTTTAGTTACATACACTATATGCGTAGAACCATCTTCTAGTTTAATGACAATCTCCTGGA TAACATTTTCCATAGCAGATTTAATTATCTTAAGAGACTCATTAAAAAAGACGTTCTTTTCTTTCTTCTTC CCCCTCCGAAGAGGGGGATTCATCGATAATTTCTAGATCTAAATCTAAATCATCTTTATTCATTAAATTC TTCCATATCACTTAGCTGTTCGAGGTCAGTTTCTAAATCAGCAGCTGATTTACTTTTACTTTCTGGAGAT TTAAATTTTTCAACCTTTGAGTTAATCAATTCATCAACTTCAGCTTCAACAATTTCATTACTATCAATAG CACCTAACTGATAAGCACGTTTAATAGCATCTCGGAATGGTTGATGCTTAAATAAAGGACCCCAGAATGT AGTGCAGTTGGTATCTTTTGCACGCCAAGATTTTTCTTCGCGAATCATCTCGCCAGTTTCTTCGTCAAGA AATTCACGAGCATACCAGCCATTTTTAGGTTTTACCACGAATCCTAATTCTAGAGCCATATCTAACAATC CAGAATAAGGATCGATACCACCGTCAAATTTAACATCAATAAAGAATTTACTTTTTTCTTTAACGGTACG AGATTTTTCTACATTTAGAACAAATTGATACCCCTGAAGATCAGAACCATCTTTAATCTGGCGTTTACCG ATAATGAATACGGTATCAGCCGAATACATCGGTCCAGTACCACCTCCCATAACTGTTTTACTAAACATTT CTTGTGTTTCGTATGTATGGTTAATAGCAATACATGGAATATTTTTAGTACTAAAATAGGGAGTTACGAT ACGAAATAAGCTTTTCATTGTTTTAGCTCTAGTCATATCACTAACAACTTTTTCATTTAAAGCATCTTCA GTTTCTTTCTTAGAAGCTAAGTTACCAAGTGAATCGATAAAAACGACTACCTTTTCGCCGCGTTCAATTG CATCCAATTGATTAACCATGTCAATACGTAATTGCTCAAGTGATTGAACCGGAGTATGAATTACTCGTTC TGGATCGACTCCCATAGACCGCAAATAAGCAGGAGTAATACCAAATTCACTATCATAAAACAAACATACT GCATCAGGATATTGACGCATGTAAGATGACACCATTGTTAATCCAAAGTTTGATTTAAATGATTTTGATG GACCTGCCAAAATTAACAGACCAGATTGCATACCACCAGTAATTTCACCAGAAAGTGCAATATTCATCAT AGGAATTTTTGTTCGAACTACATCTTTTTCATTAAAGAATTTAGATGCTGTTAATTCTGCAGTCAATTTA GAAGTAGAAGCTTTAATCAAACGAGATTTTAAATCAGACATATAATACCTTATAGTAGTGTTCTTGTTCC ACGAAATACTTCGAGCATTCTTGCATAAGATTTATTTGGAAATCCAGCTTCAATTGCTAATTTTTTAAGC TTAATAGCACCGGATTTTCCAGAGTTTTCCCATATTTCTTTAATCTTTTCAATGTTATCCCACATTATTG GGTCACGTTTATGTGATGGTTTATTTCTATTATAACCATATGGATTATTAAGCAAAGCTTCTTTTCGTTT TGCTTTAGTTTCTTCTGACTGCTTTTTGCCTAGCTGGGCTTTCCGGCAAACATCACTAAATCCAAGATGT TTTGGTTTTCCTTTTTGAGCTTTTGAAATTTTTTCTTTGGTTTCTGATGAAACTCTATAACCAATTCTTC TACCACCCTCTCCACCAATTTTTAAATTATAAGTCATAGGATCATTTACCACATCTATTGTTACTAATTC TCTTTCAGCATCACGGGCTGATTTAAAATCTTTATAAAACCCAAGAATGCTTAAATTAAAATTTTTCTTA CCATACTTTTTCTTGGCTTGTGCTAATAAAGTTCCTGATCCCATATAACCATCATTTAAATCATCGGTTG AATGAGTTCCATAGTAAATTTTATTATTAACTAAATTAGTTATAACATAAGTATAATTATATTTCTTTTC TTTATGTCTTTTCATGTCATTAATCAACTGCTATTCGATGAACTTCTCTCTTTTCTAATCAATATAACAA AAGAAGTCCCAAAAAGCAAACAGACCTAAACCGATAATAAGCAATAAAGGTCCTAACATTTATTCCACCG GTTAAAGATAAATAACTTTCTAATAATAGTTCATAATTTTTATAAATCAATAGCTTTTTTGAACGCATCT TGCCATTCGGCTTTCTTTGCACGGGTTTTATTTAAAATATCATGTTGAATAGAAAGCATCTCTTTACGCA AAACATCACTGTGTTTTAACTCATTGACTCTATCAATGAGTTCAGCACGATTATTTACATAAAAACGAGC ATCATTAATAATTCGATGTTTGGTATCAAATTCTTCGTCAATTAGCATCACTGCATCAGATGCCATTGTT TCCCAGACGCGTAAGGTAATAAAGTTGTCATTATAATTCTTGTCACCAATAATTAATGCAGCAATAGCTT GACTATTCTTTTCAGATACCATGTTCATAGGAATTTTTCCAGTGAACACCGGAGCTTTGGTCCAAGGATA TTTAGGATTTTTAAACTGTTTTTCTCGTGCATTGCCAAAAAACTCAATATTTAAACCGGTGTCAAATAAG AATTCTACCATCTTGGATTCGCGTTGACCGGACCGAAATGAACCGCCATAAATAACATCCAAAGTTTTCT TGGTAGGCTTAGATAATTGAAAATCGTTCATATGAATTTTATATTGTTCAATAGGAAAATATTCAAATTC AATAACATTATCAACTTTCTTATGCGCAGCCTTAGCAATGTCTAAATTTATACCTTGGGAAATCACTTTA ATTGGTGATTTAATTAATAGCTCTTCTTCAGTGTACAAATATGCCCATGGTCTATTTTTAACATTTGGCC AAGACTGCGAAAACGGCAAACGTATATCTGTAAATAAATAATAAATTTTACTTTTGTATTTTGCCATAAA TTTTTGCGCAGATAAAATTGCTAAATTAGGTTTACCGCCAAAAGTTAATAGAAGAATTAACAACTATC AAACGGTCATAATCATTAACATCTACTTCATCAAAAGATTTAGTGTAAACACCATTTTTAAGAGAAATAA TGTCGACATTAAGACCCATTTCAGAAATAACTTTAAAAGATAAATAGTTTCAGAAGATGGAACAGTTTT AAAATTAATAACATTATTACCCATATTAATTATAGCAATTTTCATATTATTCCTTTTATGTTAAACGATT AAGCGTATTTTCCTACATAATCTTTTTTCGAAATATGTGTTTCGCCAGTTTTCCACCAATGATCAACCAA ATAAAATGACGAGAATACACATGTAGGCTTCCAACATTCCATATAATGGAACCTGCTTTATACTGGCGA GTTGAATCACCTGCATTCAAATCAGATACTAATTTATCTAATACGTATTTTTGCCATGCATAATCATTAC GGAATCCGAAGACCACGTCATTTGAGCGCATGTTAACAACCGCATTGATTTTCTTGTCACGAATCAGGTA TTGTACTGTATTCGTGCACATGAAATCTGACATACCATCTTTATTATAGTCAAACTGCATAGATGGACGA GTATAAATCATGATACCACGTCGAGAATCAGGATTTTGACCAAGTTCAGCTAAACACATGTCATACTGAG CATAGTTATCTTCTGACCAGATAGCCCAACCATAATTCGAGTTAATTTCACCTTTAGAAGATGCTACTTG TTGCCAAATCTTCGGTGTTTCACCCGGAATATCTTTAACGAACAAGCTTTTAGATTTATACCATTCAAGT TCACGCTGAATGTATTCATCATTAAGAGCGCCAAAAATAAACGGTTCATCTGCTACAAATGATGCGCCAA TAATTTCAATAGTTTTAACACCTGTTTTATCAACTACGAAATCTTTTTCTTTTAATGCAAGCCCCAAATG AAGACGGATTTCTTCAACTGTCATAGAGTCACTAATCATTTAAACCTCAATTGATACATTCATATTTAAC TTGTAACAGTAATAAACTCCAACCTAAAATAATAGTTGGAATCATAAGAGGAACCGTTACACTATAGTAT ATACTTATTATAATCATCAAGATTAAAAGCAATGCTGCTATAATTTTGCTTTTCATTCCTTCTCTCTGAT GATAATTACCTGATTTGGCTGCGCAGACTTTTTAGTTTCACCTGCAATTGACCAAATAAATGTAATAAAC CAACCAATAATTGACCAGTTAAACAGTAAAGATGCGAAAAAGATTCCTACTGTCGATTTTGACCCACGCA TCAAGGCGATAAACCATGGAAGCATGTATATAATAATAGCCAACACGCCTGAAACTAAAACCATAAAAAT TGAACCTGCTACTAAAGTTTCCATGTTTTCCTCACTTAGGTCAAATTTTTTACACATGAATTATAAGAAT TCACTACATACTCCATCGGAGCGTTTTTACCTGTACGCCACTGGTAATTATTAGCCCAATTTGCCCAAAG CTCAGCGCAGTAGTTTTCAATTTTTTCTTCGCGTGTAATTACATCTGAATTACGATATGCTTGAGCAGAT TCATCTGGACGAATAGCTTCGTCAAAATTCGCCTGCATTTGTTCTACTGTCTGTTTTGGAGCTTCTTTAT AACACTTGACATTAGGATTATAAAATTTGCTTGAACAGTTTACAATTTTTCCTACATCAGACTGATTTAC TACCGGTCCTTGAGCTACACAACCAGCAAGACCTAATGCAATAACCAAAATAGCGATTTTCATTTCATTC TCCAAATCCGTATCAGTAGTTGATAGTTGTATAGTACCATGGAAGAACAGTCTTGTAAACAGTTTTGTGA AAAAATTTTTAGGGAATCCAAAGGGTCCAGAATCATCTCTTTTTCATAAGTATAGATTTATATTACTTGT ATGAAAGGGGACCTGGAGGTCCTAGATTTATTCTATCAGCCAAACAGGAAGTCTAACGAAGCTTTTTCT TCATAGTCCATGCCAGCCGATTCACACATACCCGCAAGCGGTTTAACAAACGATTTTTGGAACAAAGTTG AGTGGTCAATCCAAGATAGCACATCAGAACGAATTTCTTTTGGAAGTTCTGTACCCGATGGCCAAGCAAT GCACTTGTCACCAAATGGATTTCCTTCACGTAATGGAAGAACCATTACTTTATTTCCATCCAAAATTGGA GCTACACCTAAACCGCTAACAGCTCGACGATAAGTTAGCACACCACGAATATGGAACGGGCATTTAAATC CTGGCCAACCTTTATCATCATATTTCGCTATATCGTTCGCAGTTTTTTACTTCAGCAATAACTTTATAGTC AAGTTGACGATATTCTTTCTCGAAGTTCTTGTAGTATTCTTGGACAGACTCTTCACCTTCCTGAAGAATA CGACGAATACTTTCTTCGAGAGCTTCTTGCACTGCTTTTGGTGTTGAACTCTGCTGAGTTTCCATACCCA TGATTTTTAGATGCGGTTCAGCAAATCGCTTATCTTCCATATCATAAACGTTCAGAGCATAACGCTTTTT CGCTTTCCAAAATCCACCAACGCCCTTTGAACCAAGCGGAGGGCAAGAAATAGCTTCACGGTCCATATGC ATCAGATGCTCGCGGTTATTCATATAATCACATAACTCACGATATGCAACATCAATCATAGGTTCCATCT TTTTCTTACCGAACTGATTCATGAATTCAACCAAATCGTTCTGCTCTTTGAATCGGTCAAGACCAACTTT TTCAATAACTTTATCTACGCAAACATATACCGAATCAGTATCACCTGCTGCAATGAAATCTTCATCATTA GTTCCGCATACTTTATTCAGATATTCATTAATTTTACGAGCAATCCACTGAATACCGACTTGGCCGAAAA TTGTGATAGCAGTAGCATTTCGCAAATCATAGTAACGGAAATGAATATTACCAAGAGCACCATAAAGACT GTTAATGAGAATTTTACGGTTCAGCTGATTTGTATTAGCAAGTGTAGCTGCTTTTTCACATTCTTCAATC AGACTATTGAGAACAGATTCGGTGTAATTCGATAGTTCATTTAAGAAATCATCACTGAACTTAACATATC GTTCAACTTCTGGTTTAGTTGAACAAGACCCTGCGCCTTTCATAATAATCTTTTTAATAGCTTCGGCATT CATTTCTTCAGCGAACATTTTCTTTTTCCAGTCTTTACGCTGGAAAAATACTTTAGCGATTTCCTTTGGA ATGATACCTTCTTGATGTTTATCATACATCCATCCATTCGGAGAACAAGAATATTCATCACTCGGTTTAG GAGCTGTTCCTGCGATATATTCATGAATTGGATGAACTTTAAACTGACCACGAATAGTTTCAGGACTAAT GTTAACCTGGCGAATAATGCTCGGATACAGAGACGTCAAGTCAAAACTCATAATGTATCGACGTGCAATT GGTTTAGGTTCAAACACAAATGCACCCGGAAAACTCTGTTTAACGTGCGAACCTTGTTGAGGAATAACCT TATGTTCACCTTTCAATGAGTTAAAAATAATAGCATCCCAAGTTTTAATAGGACTCATTACACCAGAAAA AGGCATTTTAGCGTAATAAGACATACTTAAAACTAGATCGATAAACCCACGAATTTTATCGATTGCTTGA ACTGATTCTACGTCAATGATGTTATAACTAATGTATCGTTGATGATTAGTCTCACGAAGTTTATTAATAG GACCGTCGTATGGTAATTTACCTTTTTTGGTTTCATGTTGAGCAACTGATTCCAAAGAGAATGACGGCAA ATTAGTAAAAGCGAATTTCTTGTACAAATCTAAATAATCAAGAATAGATACGCCATCAATAGAATAAATT TTCTTTGCTACCGTACATATTTTGAATTAGTTTAGATTTTTACCCGACCGATTGGAGAGAAACGTTTCATAC TACGTTCACCCAGAATCATTTTAACACGATTCATGATATACGGAACGTCAAACCCCTCAATATTCCAACC AGTAAAAATAGCAGGTCGTTTCTGTTCCCAAAGATTGATATATTCCATGAGCATATCACGCTCATTATCG AATGGCATATAAATTACTCGGTCAAGAATTTCTTGAGGAACTTCATCACCACCTTCACAGTCAAGCTTAG CAGCTAACTTTGCATCCCATTTTGATACTGAACCGTACATTGAATTCAAAAGGTCGAAAACATAAAAACG ATCGTCAATTGAATCGTAATGAGTGATAGCATCAATTTCATATTCTGCTTTCATTGGGTCAGGAAATTTA TCACCGTAACCTCAATGTCACAGTTAGCTACACGAACAAATTTTCGGTCATAAACAATTTCTGAACCAT ATGTATCACTTATATAAGCGAGTTTAAAATCGTTCATACCGAGAGCTTCGAGACCGATGTCTTCCATTCG CTTCATCCAATCTCGAGCATCTTTCATTGATGGAAATTTTTGAGGAGCGCAGTTTTTACCATAGATGTCT TTGTATTTTGACTCTTCCTTACAATGCCTAAACATAGTTGGAAGATATTCTACTTCACGGGTACGTTCCT TTCCATTTTCATCAATATAACGTTCAACAATGTTATTTCCGACTGTTTCAATAGAGATATAAAATTCTTT CATAGATATTCCTTAGTTTATAGCCCGAGTTATTAGGCTCTTGATATATTATACTCCAAATAAGGGGCCG AAGCCCCTTGCTTAATTACCAATCGTATATTTAGGAACGAGCTTCCATTCATGTTTTTGTTTAAAAGAAA TAACTCGGAAGTTATTAGTTAAATCTTTCATAAAAGTTCTTTGACCAGGAACGATTTCAATTAGTCCCCA ATCTTCTAATAGCCATGCAATCGAATCACGACGAACTTCATCTTCTTCTGTCATTTCAACTTGACGACCA TCCATACGAAGCATTTCTTTAAAATGAACGATATAGTATAGTCCTTTTTTCTGAAGAATATGACAGGACT GATATAGAACTTTATCTTTATTATTAGCAATTCCCATACGAGTCAAAGTTTCTTTTACTTTCAGAAAATC TTCAGGTTTTTTAAGAGTAATTTCAATCATTTTACCATTCCAATGCTAGTTTTTTGAGTTGTTTCTGTTC TTTTACGTTCTTAGTCACTTCTTTCAAAAAATCATCCGTGACTAAACCTTTTAGTTCTTTTAATACTAAA GGAAGTTTTCCATTTTTAGTAAGAATTGATTTATAGTTAATTGCATCATTTGTATTAACTTGATACCGCT TAGCAAGTAACTTAATAATCAATACTTCGGTGGAATCTTCAACCAGTTTTGCCCATTTACCATATCTTTT ACCACGAGGAACTGCAGCCATTAGATAATTAAAATGAGCTTCATCACTTAAGCCTGATCCAATTAAATTC ATAGCATATACAGCTGGCATACACTCTGGAAATTGTGATAATGCATTTTCAACCATGAATTTTGAATAAT CTTTTTGAGCAATAGAGCATTTAGTTTTATTATTAATAGCTCCAATTATTTCAAAAAATTCATTTTCTGC TTTTTCTTTAAAAGAATCAGCAGCGGATTGGACAGCTGTCCAATCTTTTGAATACCAAGCAACTTGATGC TCGTTTAATTGAATATCATCTTTAAATAAGCTCATATCACTTCCACTGCATTTCGCATGCTAATTGAATG AAAAGATAAGCTAAATGCAATTCAGTATTAGCTGCAATACCATGATACTGATTATTTTCGCCGACAATTT CGTACATACGAATAATACTTTGTGGAGTTACACGTGAATAGATTTCTTCGGCAAGTTTACCCACGAACCA CGAAATATCAGCCGCATATTTTGGTGCTAAAGCTCTGAGTTGTTTAACATCTTTATTTTTGAGAGACTCA AGAACATCATCAATAGCACCACGATCGTTAGTAACCAGTGATAAAATACCAGCATCCAAAACACCTTTAG ACGAATAACTATCGAGCTCGCCAATAGTTTTACGAAATCAGGAAAATTCTTTTTAACCAAAGCTGCTAC AACTTTCATATCAGCTATAGCAATTCCTTCATGCTTGCAGATTTCAGTCAATCGACGAATCATCTGCTTC ATCATTTCAATTTTATCTTCATCAGTTGGTTGACCGAATGTAATAACTCGGCAGCGTGACTGAAGCGGTT TAATAATACCATCAATATTATTAGCAGTAATAATAATACTACAGTTTGAACTATAAGCTTCCATAAAGGA ACGAAGATGTCGCTGAGACTCTGCTAACCCTGAACGGTCAAATTCATCAATAACGATTACTTTTTGACGA CCATCAAATGAAGCGGCGCTGGCAAAATTAGTCAAAGGACCACGAACGAAATCAATTTTACAATCTGACC CATTCACAAACATCATATCAGCATTTACATCATGACATAATGCTTTTGCTACAGTTGTTTTACCTGTTCC TGGAGAAGGAGAATGAAGAATAATATGTGGAATCTTACCTTTACTTGTAATAGATTTAAAGGTTTCTTTA TCAAAAGCGGGAAGAATACATTCATCGATAGTAGATGGACGATATTTCTGTTCAAGAATGTGTTCTTTTT CATTTACAGTAATCATAATTTCCTCATTCAAGTTTTAGTGTAAATTATAAAGGCCGAAGCCCTCTATTAA AAATCGTGGGTAGAATCAGCTTCAAGAGCTACCACATAATTCGCGTGTTCACCTTCAAATTTAGCAGCAC CTTGTTTACCTTTTGCCCAAAGCAGAAGTTTATAATTTCCTGGTTGCATTTTCATATTTGCCATATTGAT AATGAAATTAAATGTATTTTCACCATCATAATCACCAAGAGTCAAAGAATATTTAACACGGGTCAGAGCA GAATCTTCTACTTTATTAAAACCGTTAATTACGATTTTACCTTCTTTTACCGTGATAGCAATTGTATCAA TTTGCAGACCACGAGATACACGCAACAGCTGTTGAAGGTCTTCAGCTTTAATTTCAGTAACAGCAGATGC TACCGGGAATGGAATTGGTTTATTAGGAGCAACTACTGTACTCGGATCGGCTGCTGGCCAAAAAATTGTT GAGCGGGCATCAGCAATTTTAATATTTCCATCTTCTGACTGGGAAATTTCTGCATCATCATTAACTAAAG ACAGAATACCGAGAAAACCGTTCAAATCGTAAATTGCTACATCAAAATCAATAACGTCAGAAATATTTGC TTCCGCATAAGTTGTACCATTAACTGCGCGAGTCATAATAAATTGACCGGATTTAAGCATAATACCAGAG TTAATAGTAGCGAAATTTTTAAGCAGAGCAGTAGTATCTTTAGACAGTTTCATGTAATTTCCTTCAATTC AAATGAGATTTAATTTTATAACTAATTTAATAAAGCAATTAACGATTAAAATCAGCCGCAATTGTTTCCG CAACAATTTGAGCAGCAACAATTAGACGTTCATCTGCATTACCGCAATAATCATCTTCAAGGCGTTCACC ACATGAAGTCATAATAAATTTAGCACCGGCGTTTAGGGATTCTGTAGTATGTTTGGCATTAGTTCAATC CATTTATTACTTACTTCACGATCGATAGCTTCATAATACGCATGACGAGCAGGGTGCAGATTTAATTTTGT TCTGAATAACTTCCATTGCGTTATCAGAAAGAGACAAAACCCATGCTCGACGAATTTTATTTTGGTTTTG TGGATTTGATTCAGAACGCACGTGTTTTGGCTGAATATCTTTTACATCAACAGTATAATTCACAGTAATT TTAGTCATAATACACCTTTAGTCATAATAATCAGTAACAGTCCAAGCTTCATTTCTATTGGACATTATTT TTGTATATTCTGCTTTAAATGCATTCCTAAGCATAGATTCAGTAACTATATGCTCTTCATTAGAAAAATT ATTTCTCAGAATATATCGTTTTATTTCAGGAATAGTTAATAGATGCTGTCCAGTTGAATATTCCATGTTT TTCCTCCATAGAGATTATACTCTAATAAATTAAAGCATAATCTCTTATAAATTAAACCATTACAGTAAAT CGACCAACTTTCTTCATTTGAAGATGCTGACCATATTCTTGCGGGTCATGGTCTTTATGCGAAATTATAA AAACGTTAGTGTTTTTCATTGAATTTATAATATTAGCTACACCTTTAATACCTTCGGCATCAAATGACCC ATCAAACACTTCATCAAGAATTAATGTACTAATACTAACACCAGATACGATAGAAGCAATATCACGCCAA GTAAATAAAAGAGCAATATCGATTCGTGCCTTTCACCTTCACTAAATGAAGCATAACTAAAATCTTCAC GACCACGGGGATTTAATTGTCTCATTAAATTCTTCACTTAATGTAAACACATAATCCGCTTCCATTATTTT AAGATAATGGTTAATCTGCTTATTAAATAATGGAATGTACTTTTTAATAATAGCACCTTTAATACCAGAA TCTTTGAGCATATCAGTCAAAATTCCTCGGTGGTATTTTTCCATACTAAATTAGTTTTTGTCTTAACAA TTTTATCAAGTTCTTCTTGAAGCAGTGCTATTTCATCAGCATGGTCAATAAACTCAGAAGATGCTTTTTC TATAGCCGCTTTAACTTTTTTAGCTTTATCTACTGCTGCGATCAGAGATTGCTTTTTATTGCGAATATCA TTTGCCAACGACTGCTGGGTTTTAAATTATCTCGGTATTCATCAACAAGAACTTTTAAATTATCACGAT GTGTTGAAAGCTGTTCAAACGAATGTGTGCATTCAGAAACTTTATCTTTAATTTTAGAAACAACTTTATC ACCGGAACTCAATTGTGACAAACAGGTTGGACATAATCCACCTTCGTGATACATATTAATGACTTTATTA TACGAGTCAATTTTTGATTTAATTAAAAATGCTTCTTGACCGATTTTATTAAATGCATCAGTCGGGTCTT CGTCCAAAACAATATTAACTAATCTTTCGTTAGCTTCTTCTATTTCCGATTTTAGCGTTCTAGCTTCTTT TGCCAAATCATCATACATATTTTGTAGACGAGTTAAGGTTGTCACCCGTTAATTTTTTCTGGCGTTCAACA TTATCATTATATATTTTAATTTGTTGGATAATACTATCTTTTTTAACATCAAGCACTTGGTTCTGCGAAT TTAATTCACGTATTAGTGCTTTATTAAGCTTATCCATTTCAGCTAATGTTCCTACCTCAAGCAGGTCTTC CACAAGCTTTCTTCGCGCAGGGGTCGACAAACCCATGAAAGGGGTATACCCTGCTGTACCAAGGACAACA ATCTGCTTGAAACTGGCATATGACATTCCGATAAGCTGTTCAAATTCTGCTTGGAAATCTTTACTGCTGG CAGATTCATTAAGACGTGTACCGTTAACGGTGATTTCGAAAACGTTTGGTTTTTGTCCTCTTTTGATATA GTACTTTTTCTCATCATATTCCATCCACAGTTCAACTAAAAGTTCTTTCTTATTTGTGCTGTTTATTAAT TGACCTTTCTTTACATCGCGAAATGGCTTACCAAAAGCCCAAATGTGATGGCTTCTAGCATAGTAGACT TACCACCGCCATTTCGTCCAGTAATAAGAGTTTTTTGAACCTTATCTAATTGAATGTCAATCCCATTTTG ACCAACTGACATTATATTTTTATATTTTACTCTATTAAGTTTAAAATTCTTCACAAAAGATTCCTTTTAA TGTATCTTTTAGACCATTCTATCATATCATCATAATCTAAAAAGTATTCATCAAAATTCAGCCATGCAAAC AACGCCTTGTGCGTTTTTGATGTGATATAAATTATTCCAACATATCTAGAATCTTCTTCGGTATAATCA ATATTTGCTATGAATTCATCATTAATGTCAAATGTCGAAAACTTCACAGTATGCATCCTTAATACAAGAT ACAGCCATATCTCGTAATGATTTAGGTGTGTCATTATCTAAAATGTTGAAGTTAAAAGATACAGCCCAGT CAGTGCAGACAGTAACCTTTTTAATACAATTATCTTCAACCTCTAACGGTTCAAACCAGTATTCAATAAT TTCTTTATGACCAATATCATCTTTTACTTCACATTTAAAATGCTGACTCATCATAACATTTTTAAATTCA TCAAAAGTCATTGTGTTGCCTCTACATATAGCTGATTTGCATATTGAATAAGTGCTTCACGGTCAGAATC AGTGATGTCTGGAATTGCATTAATATACTCTTCCATTAATGTCTGAAGCGATTGAACTTCAACTTCTTCA CTGTCATCTGACTCGACAGAGTTATCAATCTTTGACACAACTCGTAATGAATGCACAACTTTTTCTAGTT CAGATTCGAACTTCGTCAGATTTTTGTCTACTTCAGTTACTATAACACGTACTGATAGATTTGTAAAATC TTTATAGTCAATTTTTCCTTTAAATGGATAATGAATTCTACGATGCCAGGTAGTATTGTTTGGAATAAAT TCCGTTCGTTCTGTTTCTGTATCAAACATCCAGAACCCACGAGGGTCATTCTCGTCACCTGCGGTTAGTG TCCATGGTGTCCCAATATATCTGACGTTTGCAGCCTCAGAAATAGTATGGAAGTGACCAGACCACACTTC TTTATAAGTCTTAAGGAAATCGGGTTCAAGACCATGAGATTTCATTCCTTTATAAAAATAAAATCCATTC AGTCCCAGTGACCAACACAAGAAGCAGATGAAGTTTTGATATGCTCAAGAATTTCACCAGTATTTT CTTCGCACATCCAAGGAATCAAATCAATCAAACACCCGTCAAAATCTACTGTAGTAGGCTTATCATACAC TTTAACATTAGGATATTTAGCCAAAAGCTCAGTAGAAGCATTTGGATGCATTACATTTTTATAGTGGAGA TCGTGATTTCCTACAATAGTGTGTAATGTAATTCCAGCATCATCAAGCGTTTGAACTATTTCACGGGCAA ACTCCATAGTTTTATGTGTGATCGCTTTTCGCACATCAAAAATATCACCGTATTGAATCCAGGTAGTAAT TCCATTTTTCTTAGAATATTCTATCGCTTGCTTAATTCCATCAATTTGAATACCGCGAATCCACTCATCA TCAGCTTTAACGCCTAAATGCCAATCACCTAAATTTAAAATTTTCATATATCAAGAACCGTCATTGAAAT GCAAAATAAAATTATTGAAATAAATCCATCTGGAGTGCTAAAGAACCCAATCCAACATGCTCTAGTGAAT AGATAAAATGCAAGAAAAAGTATCACATATCCAAGAAATATCATTATATCAAACTCCGTATAAAGCTAAA GGGCCGAAGCCCTTTATTTTGTAATAATGTCAAACTGTTCTTTAAAGCAGAAGCTTGAATCTTGATGCTG ATACAAAAATTCATATGCTTTTTCTCGCTCACGGTCATAAAGAGCTCGGTCAGATGACAGTTCTTTAATA CGTTCAAATGTTGATTCCATATCATTTTCATCAAACCAAATGATACCGCTATCATGCGAGGTCAAAGGAG TATTATCAACACGGAATTTTAAATTTTCGCCAGTAGATTTCCAAAATACCGGAATTGTTCCACATGCACC AAGCTCGAGATGAGTATATTCGAGTGAGCGTTGTAAGTATTTCTGGTTAAGTTTACTCAACTGATATCCA AAGCCAGATTTACTCATTCGTTCAAGCATTTCACTATTAATATAACAATCTAGGATTTGTGCCGGTTGAT TCGGCGCGAGATTCATTTTATCAATCTCACGATTACCGTAATATTCATACGGAATACCTTTTTCCTTAAT TGCAATAAAAGCAGGGGAACGTTCCAGACCTTCCATTACAGTGGATTTACCAGCAGGTTTTAAGAATTTT TCATGAAAATCAAACATCTGGTAAAAACCTTTCCATGTAGTCGTACGACCAATCCAACGGTTGATATTCA TGTTAATTTCAGAAACATCTTTCCAATAAGTTGACCGAACCTTCACAATATCCATAGGAGGCTGAAAATT ATATACTGTCGGTGCTTCTTCAATATCATCAAACAGAGAAACAGTTTCTGGATACCATTCTTTCATCAGA ACTTTATTAAAATCACCATTATCAGAATGGCTAAAAATAACATCAGCTCGACGAACAGTTTCTTCTAATC CCAAATTTCGACGCAAAGAAAGAACAGAATGATCATGCTGATAAACTACAACACGAATAGAAGGTTTAAT ATTATCTAAAAGTTTTTTATAGTTATTAATCGTAGCTTCTTGAACGGAAGTAGCAGGAACAGAATTAATA ATTAGAATATCACAATCATTTACTAGCTTAAGTGCTTTATCGTATTCTTTAGCTAAAATAACTGGAATTG AAAATGATTTGTGGTCATGAGAACTTGTACGAGTAAATGATTTATCTTTAGCATAAACCAAAGTTACTTC ATGACCATTTTTAATAAACCAATCACGTTGCTCGAGTGAGAATTTTGTTACACCACAACCTTCAAGACCT CGAGCCATAAAAATGCAAATACGCATAGTTTTCCTCTTTTCATTTAATAAATCATGTAAATAATATTTTA TTTTCTATAAACGCTACGAATAGGCCCAAACATATCCATAAGCAATTTTTTGCTTTTTCCAGATATGCACT TTTTAATATTAGACATGCATCCTTTAACGGATACAGCTGCGTCTTTAATTCTAGGAAATACTCGTAATAA TTTTCCAGTAGTATCATATTGAAATACTGGCACATTTCTTTTCTGAGTTATCCCACGTTTAGATTTAGAC ATTTTTGTTTAGTTGCATTAGACATCCTAGAGGGCTTTCCTATGTTATCAGAATAATAATATTTCCATT GAAATCCTCCAGCGGTTTTCCTTTTACCATCTACACATTGTTTAATTGAAGTTGAACAGCTATATGACAT ATCTTCTGCAGCATCTGTAATACATCTATATTTGCGAATAAAATTTCCATTTAAATCATATTGATAAATT GGTTTTCCAGCATTTCGTCTAGCGTTAGACATGTATTTTTTATTATCATCATCATCTTTCCAGAACTCTTTCA TGCTTTCCGATATTGAAGAAGATACAGCTGTTTTAATCCATCCATACATTTTATTATTTAGTCTTGTTCC GTCAGAACTATAACACATCATACGAATAGCTAAAGCCAATTTAGGAAGTCTATAAATTTTAAATAATAAT AAATGCGCGGTAAAATGTTCTTCTGGTGTCAAAAGAACTAAATTAGTTTTATCATCTGTACCACCCATAC ATCTAGGAATTATATGATGTGTTTCAGTATAGTATGTCAAAAGACTTTTATCATTGCCTCTGTTTAGTCC TTTTTCGATCAGTAAATTATATATGTTTAAATAATTCATTTTAGTTTATTTTACCAAAAAATTTATAAAG CAATATAGGAGCCGAAGCTCCCTATCCACATAATACGCCATACAGAGGCTCGTTAGAACTTTTAAATTTTA TGCGCTTATATGTTATAGTTCCTTCTGCTTTAGCTTTATCATGAGCCTCTTTAAAGCGTTCCATCATTTC CTCTCTAGAGGAACGAATTTTATTATAATCTATTTCAGAAGTCGGATGTTCATCTTTCACAGTTGCCACC ATTTTTGACTTGATAAGAATCAACCCACACTTTCATATTAGGGTCTGCTCTTACAATAGGAGGATTAAC TTCTTTGATTGAACTATCACGGTATTCTTTTTCAGAAATTTTCACGCAAAGATGACCATTCAAAGATTCA GTAAATCCTGCATTAATTTTAAGACGCTTGAATTTTACGTGTGTAAGCATCAATCATATCCTCAATCTGC GATCTAGTAGTCTTCCAAAGAATACTGATGAGTTCATCGTTATATGGCTGTTTTAGAATATCCCGACTTT TCTTGATAGCATATTCGTATTGAGCAAAATTATTATTTTTCAGGCTGCGATCTGAGCGTGCTTATAAAGACG ATTCAGTTCGCGTTTGTTTTTAGACAATAACTTATTTGCTTTTCTTTCTGCTTCAAGACGGTTCTTTTCT TCAATAGAAGAAATAAGCTTTTCCACTTCATCATTAATTTCGGGTTTATCAGTCATATTATTTCTCTAAT ATAAAATAAAAATCATCATCTGTTAAATGATACCGATAGTTTAATTCTACACCATTAGATTTAAAAGCGG TATCATACGGATTTTCTGGATCAATATCAATGTCAAGAGCTAAAACTTCCCTGAGATACATTTTAAGTAA ATAGGGAATAGCTTCAACTTCAGGTATTTCTTCCAAGAATCCGGAGAGGTTAATCGTTAGCCCTCATATAA AAATCCAAACTAGGAGAATCATCTACAACACTTTTCTTTTCAGCCCCCGGTGTTCTATAGGTTGATTCT TCGTAATGCGTCATTTTATCATAGATGTCTTGAATAAAAGTTTCATCTACTAACGCAACCATATCGTCGT CACGGCTGTCATAGACATTGTGAACGAAGTAACTATATTTCTTTGCAACTTCCTTACGTTCTTTTTAAT ACGTTGGACGAATGCATTAAAACAAGCTTGAGTTATATACGCATGTGGGTTTTTATATTTCGTTTCATCA AAATTGTGAAGCCCCTTAATAGAAGCTTCTATACCATCTGCAATCATTTCTTGTTTCCAAGACTGGGTGT ATCCTGAAAAGTGAAACGTTTAGATAAGCCTTCTGCAATAAGCATAATGGCTAATCCGATAGTATCATT CTGACGAACTACTTTATTTGGGTCTTTATTATTTGCTAATTCTGTTTTCCAATCAATAATAGCTTGTAAA AGCTCTTATTGTTTACGTAATTATATTTAGGCTTAGTTTCTGACATTTTCACCCTCTTAGCTCAATTCAT AGATCTATTATATCATAATATTTGAAGACCTATCTTAAAGCATAGAGGATGAATCAATTTCAAGCACTTC ATCAGATTAGCCGCTCCAAGAGCTGCATCTAGTGAATCAAATTGGTCAACATATTCAATTAATTCGCCGT AATTAGCGTATAACCACCATTGGCTAAATTCATACTCAAGGATGAATCCATTTCCTTCAATTTGAGTTAA ACCAATGCCATTTGTATTTACTTCATACCCAGCGAGACGTAAATCGTTAATAAGAGCTTCGTTCATAATT ATACCTTAGTAATTTTCAGGTCTGCAAATTTTTTCTTGCGTTGATTTTTCATGCGACGAATAGTTTTATC GGAAATTTCATGCTTTTGATAAGCTTTAGATTCTACACCAAAAGCTTTAACATCAAATTCTGACAAGATA TATTGAACCAACAATTCACGGACAGTATTGCGTCCAATCTTCTGATCATTCTGTTTCATCGTTTGATGAA GCTCTTTTTCCCATTTATCCAAAATTTGAGGAGTTACAATATCGCCTTTTTCTAGCAAAGAAACTACTTT ATCATATGCGTAAGAATTGATGGCGTTTTTAATTTGAATAGTCATACATTATCCTCAATTACGTTAAAAT TTTATTATCCAAAAAGGCCGAAGCCCTTAGGCTAAACTTTTTGGCACCCTTCCAGCCTTCGTACATCATT GCGACTGACAATGACAAAGCTCCTTCACATGCTGCATACTTATTATTCCAGAACCAATTTAGAAAAACTT CATCCTCAATACCGTTGTGTTTCATGTTCTGGAAAAATTTGGCGCGTTCTTCAGCAATTCGCTGCGATAA TTTAGATTCAGGATTCATTTAAATTTCCCAATTGCCATTTTCATCAATAAATTTAATCCAGTCATTTACT GACCACTTGGTCGTATCGCCTTTTGGAGTAACATTTAAAGTGTATAGCCCTTGCTTAAAAAGCATGCGTT TGATATTCATATTTTCCTCAGCTGTAACGATAACACTCGTTTGATTTACGTTTAGCAACTCGTTGAGAAG TATTATAATCAAAATCATCATCAATGTAAACTGATTTTTTCAACTTTCTTACTTCACCGCGTAATTGACG ATTTAACTCATCTTTAACTTCTGAATCAATACCTTTCATTCTACGCCATTTATCTGAGCGAAAAATGTTT TCAACCATATCTTTATGACTTACCCCATCAGGAGCTTTACGCTTTCCGAAATAGTCATAATCACGCACTT TTAAGTCTTTACGACGATACGTTTTACCCATGGAGTTTAATTTCCTTAGCAACTGAACTAAATACAGCAC GATCACAAATCATACGTTTATGTAACTTGAGTATAAGATAGTAAACATCAAAACCATTTACATAAGTAAC ACGAACAACATTACCATTCACGAGATAATACTGTCCCTTTTTAATCTCTTTATCAACCACAACCATATCA ATTCCTCAAAGGTAATTCATATGTTAATAATACCACGGTTTGAACTTGTTGTAAACAACTTTGTGAAAAA TATTTTAGGGAATGATAAGAAAGGAACGATAGCTTAGAATGGTAATATACAGAATGTGAGAAAGAAAGGC CCAGAGGGCCCGTCTTAATCTTCTATGATATCTCTATCATATCCAAGTGAAATGAGAGTTTCTTTGAAGT GTTTAATGTTCTTTTGTCTAGAATCATTAATGAAAATGACTGGATAACGAATGTTAAGAGATGTGAATCC AGCGCGTTTAGCAAGAGATACAATCAGCGGACGATCATACTCAATCTTACCATTATTTGTAAGAACTTTA TAGAAAGTAAAAGGAGCATTGAGCTCCTTTAGAAGTTTTGTAACTGATTGACATCCAGGACAACGACCTA CTTCATCTGGAATTCCATAGACTTCAATCTTATTTTGTTTCACAACTATTCCTTACTAAGAGCAGCATTC AGCTTGTTAGTAATTTTATCCAGACGCTCATTGAATTCACTAGAAGATAAGCCCTTTTCTGGAGAAATCA AACTAATCACGAAAAATATAGCAATAAAAGGAATAAGGAAAATAGCTCCAACTGCCATAAACAGAAAGAA TGTTACAGTTGTAAGAAAATCAGCTAAACCTTTACGAAATTTATACATTTACATTTACCTTTAATTGATT AACCAAGCATTGATAAGCACTAAACTATACTGCGAATAAAATTCTGGACCAAAATGAAAATCATATCATT TATAGTATCCATAATGTAATTCAATTTAATCATGTTTCCACACCCCATCGGTATTTGACCAAAGTCGCTG ATTATCTGATCCTCGCCACAGCTTTTTGGTCGGAAGATTTTTCTCATACTTCCCATCAATAATAACATCA ACATATTTAAGCATTTCTAGTTGTTTAATATCTTCAAACTTATATCCTGTCCACAACCAGATATCTTTCT CCGGAAATCTTGCTTTAACCCAAGAAACTAAATTTGAAATCTCTTCTCGGTTCTGTGGATAAAGTGGGTC ACCGCCGGTTAAGGTAAGGCCTTGGATATACGATTTGCTTAAATGGGACGCAAGTTCTTTAACGGTATTC ATAGTGAATAACTGTCCGTTACGAGCATTCCAAGTACTACGATTATAACAACCTTCGCATTTATGCAAAC ATCCAGTGACGAAGAGAACGACCCTACATCCAGGGCCGTTAACGAAATCGCATGGATAAATTCTATCATA ATTCATCTTTATATTTCCAAGTATATCCCTTATGAATAGATTGAATTCCTTTGCAACATTTATAAACAGC AGAATGAAAAAATCCTGCATTTCTTATTTCAGTTGCTCCAGTTAGTTCTATTGTATTTCCGTCTGGTGAA TAGCCAATAACCGTTTTGGTGTTAAAGCGATTTCTAGAACTATTTTTGATATGCTCTTTATGATTAGTAG ACAAAGTTCTTCCTGTTAGCCCATCAGATATCCTTTTTCCAAAATCATCAGGAAGAGTTACTTTAGACCT AGCTATGCTCATTAGCTTCTTAGTTTTATCGCTTCTTTTTGCGCCACGCTGATTTTCAAATTTCTTAGCT TTTGCATACGCGTATTGTGAAGAAGTAACTTTTCTATGCTTACTATTACTCATTATCCAATATGCATCAT ACATCTTTCCACCATATATTTTAGCCAATAAATGGTGAGCAGTATAGTGGGCCTTATAAGTTAAAAATAC TAAGTTGTCTAAATCATCTGAACCACCCATACTTCTTGGAATTATATGATGAAGTTCATACCCGGCTTTA GTTTGACGGGGCTTAGATGGGTGTTCAGCAGAATTAACTAGATTTGAATAGATTCTGTCATAATTCATTG GTGCTTAACCCTATGCATGATTTCTTTATTTTTACCGAGATTAAATCCGCGTTCGTTCGGATTTCCCAAA TAACCACATGTTCTTCTTATGGTATTCATCTTTTTAGGATCAGTTTCTCCACAAATAGAACAAACAATAC CGTTTTCAGTAGGAGTCATTTCATGGGTACTTCCACATGTAAAACATTTATCTACTGGCATATTAACACC AAAATAATCTAAATGCTGTGCAGCATAATCCCACACGGCCTCAAGACCTTTTAAGTTATTTTTCATATCA GGAAGTTCAACATAAGAAATGTGACCACCTGTCGCAATGAAATGATATGGCGCTTCACGAGAAATCTTTT CAAACGGAGTAATATTTTCTTCTACTGAAACATGGAAACTGTTAGTGACCAGCCTTTATCGGTAACATC TTTTACGCTTCCATATTTTTCTGTATCGAGTTTACAGAAGCGATAACACAGGTTTTCAGCAGGAGTCGAA TATAAACTAAAAGCAAATCCGGTTCTTTCAGTCCACTGTTTAAGATGAGCATTCATTTTAGTTAAAATTT CTCGTCCAATATCACGACCGACAAGAATATTCAATTCGTGAATACCAATGTATCCTAAAGACACTGAACT TCTACCGTTTTTAAATAACTCAATTATGTCGTCATCAGGTTTAAGACGAACCCCGAATGCACCTTCTTGG TAAAGAATAGGAGCAACAGTAGCTTTAACTCCTTTTAAGGAACTAATTCTACACATCAAAGCTTCAAAAC ATAAATCCATTCGTTCATTAAATAGCTCAACAAATTTCTGTTCATTGAACTGTGTTCCAATATAAGAATC TAACGCGATGCGAGGAAGATTCAGTGTTACAACACCAAGATTATTGCGTCCATCAAGAATTTCATTGCCA GTCGAATCTTTCCATACGCTCAAGAAACTACGGCAACCCATCGGAGAAACAGGAACAGATGAACCAGTGA TAGCTTTATTGTTCTTAGCTGAAATAATATCAGGATACATCCTTTTGCTTGCGCACTCTAGAGCAAGCTG TTTAATATCATAGTTCGGATCGTCTTTATAAAGATTAACACCTTCTTCAACGAACATAACAAGCTTAGGG AAAATAGGAGTTATCCCATCACGACCAAGACCTTTAATACGATTTTTCAGAATTGCTTTCTGAATCATTC GTTCAGTCCAGTCAGTTCCCGTACCAAATGTAATTGTTACAAAAGGAGTCTGTCCGTTTGAACTGAATAA CGTGTTCACTTCATCAATGTGTTCAGTAGAGTTCGCTACTTCTCTACCCGTTTATATGAAACAATGTAAT TTGGATAATTGTCATCTAAACACAAATCTTTAATACGACTTGGGTGGAGTTTTTAAATCTTTAGCAGCATC CACAAATGATTTATAGATATTGTCATTTATTTTAACATATTCTGGGACGGGATGAATTGTTATTTTTATA TCTATAACATTTGGATGATTACTAACCTGTTCTTCAGAGCATTTAAGAAATTTTGCAGCTGATTTAAAAC TTCTAAATTTATTTCCGGATTTTAAAGCTATAGAAACAGTCTTTTTAGCCGTTCTACTACCAATATTATT TTTAACATGAGCTTCACTTCTACTATTATTTTTATAATGCTCAATCAATTTCTCTCGTATCTTATCTTTA TGTTTTAAAGATAAGATTTTACCTTTATGGGCATTACTAAGTTTTTGCTTATGTTCTCTGAATCCGGAT ATTTGTTAAATTTATATCCACCTATAGATTTATTAAGAATAAATTCGTTATTAAAATATTTCCTAATAAG CATTTCTTCATGTTTAAGGGCCGATTCATAAGAATCAAAAACTTGAAGAATTATCCACTTAGCTTTATAA TCTTTAAGCTTTTCTTTAACAAGCTTAGATGACGAATTGTATTCTTTCCAATTTGTATCTTTACCATATA TAGTTTTGAATTTTTTAAAACCTATATAAAAAGACTTATCAGGAAATCTTACCATATAGGTAAATGCTAC AGAATTGGCAATTTTTAAGCTTTTTCTTAATTTCCATTTCATCGTTTCACCTCGTATTCATATTTATACG AGGATAAACAGCTGCATGTCACCATGCAGTTCAGACTATATCTTCAACTCTTAGAGTTGTCTGCCGTTTC GGGTCGCTTGACCCTACTCCCTTACATTCATCAGGGATAGTCGTTGGGCATTTACAGCTACTGCTGATTT AGCAACGGATTGTCTCAGTGAGAGTTTCCCGTTTTAGGCAGATTTTACATGAGCTTGACTTACGTTAACT CATAAGCTTGGAATGCATCGTATACGTCTTTTTCTGTTTTAGATTGAGCATAATTCAACGCATCAGCGAT TTGCCATTTTTCTGCATCCTCAATATGTTTTGCATAGGTGCGTTTAACATAAGGAGAAAGTACTTTATCT ACATTCGCAAAAGTCGTTCCGCCGTATTGGTGAGAAGCAACTTGCGCAGTAATTTGTGCCATAATTGCAG TAGCAACTCCAATTGATTTAGGAGTTTCAATCTGCGCATTACCAAGCTTAAATCCGTTTTCAAGCATTCC TTTTAAATCTACTAAACAGCAATTAGTAAATGGAAGAGCAGGGGAATAATCAATATCATGCACGTGAATA ATTCCGCTTTCATGCGCTTTCATAATAAGAACGGGACCATATTTTTGGCAATGTGTTTAGACACAATAC CAGCCATAAGGTCCCGTTGAGTTGGAAAAACACGAGAATCTTTATTAGCATTCTCGTTAAAAGGTCTTT ATTAGTTTTATGAATTAATCCTTCAATTTCTTTTTCAATTGTCATTTTAAACTCTTTCTAAGCTGCTTCT TGAATGAAGCTATTAATTGTGTTTTGGTGTCAGATTCATTATATTCAAATCCTCTTTGAAGCATCTCGGC CATCATTTCCTCTTTTCCTAAACGAGAAAATTCCTTTGATTTATCTCCAACAAAGTTAGGGTGAATATTA TTTTGGGTGTAATCGGATTTTAAATAAGTAAGTAAATTTTCTAACCATTCAAGATAATCAACACCTTGTC CCTTTAAGCCAGAACGATTAAATTTATGCTTCATTTGACCCTTCTGCAGCATTGCATAGATTACAAAGCAA TCCACGCACCTTTCCTGCTTTTGGTCCATTTAATTCATGGTCATGGTCGAGGTGATTAGCTTGAACATCA GGATTTAGTTCTCGTTGGCAAATTAAGCATTTACCGTTTTGTGCATCATAAAATTTCTGTTTTTCTTCTT TGTATAATTTGCCAGTCAATAACATAATAAACCCTTACCTTAAATAGATAAGGGTATTTATTATTTTCAA GTATTGTAAAACATTTGATGCAATCGCTTATATTGCTGAATCATTCGGTCAGAAAAAGAAATTTGAGTTT CAAGCCATTCAATGTACTCTGCGGCAGCTTGCATTAAATTTCCTTCATAGCCATCGTTATTTTCTTGTGC AGCTAATTTAGCTAATGCGTATGAAATACGTTCACCTTGAAAATCGGCTTTAGGCTTCTGAACAACTTGA TTAGTTCGCTCTACAACTTCTTCAATTTCGCCATTTTCTACTGATTCAGTATTCCACAAACACCAATACG TAATTGGCTTATCGTAGATGTTAATAATCTTTCCATCAGAAAGTTCAATTTCAATAATTCCAGTGTCAGG CTCTATATCATCTTCACATTCTTTTACAAGTTCACGAACTTTAAAGACAGTACCTGCACTAAGTTCCGGC CAGTAATTACACAGCCCTGTATCAGCACGAATTAATTCTAAACCATTTATCTACTGTAATCATGTCCCATC TCCATATCAATTAAGTCATTTATCGTTGGTTCATTATACACCGTTTTCTTCATCAGTGTAAACCGGTTCTT CCGGCTCTGGCTCTACAGTTCCCATCTAGCCGCCCACCAAGGTTTTATAGCGTAATCCATTCTCGTACT GTCTGAGTTACTACACGTTCTCGAAGCTCAACCAGTTCAACAGTAGGAACTGCATAATACACAGTCAGAAT GGTAAGAACCTGAGCGAGATTCATTTACAGCCACATGTACATTATGTTTTGGACTAAAAATAAACACACTG ACGATATTGGCATTTACCATCTTGCACCCAGTCTTCTTATTTCGATAGGTTTAAGATAGTCGGAAAAATCG AAATCATAGTTTTCCGAAAATGCATCATGGTCTTCAATGATTTCGCTCAGAACTGCATCAACATCTAATT TATTTTCAATATTCATTTTTCACACCACCAACTGCTTACTTCAAAATCGGGTTCGCCATGTTCCCAGAAC CAGGGGCCGATAGGAATCCACTTACCTTCATCGGTGACGTCATATTCTTCGGATTTAAGCCCAACGTATC GCCATTCGACTTCATAAGCGGTACCACCGATTACAACTGTGTTCTCGCCATACGGGTTAGACACAACAAA GTCTATACCTTCTGCTTTAGCTAACCAAATCATGTATTCATAGAGTTTATATTCCAGGTCGCCTTCTGTT CCATCGCCTAGAAAAATAATTTGTTCATTTAATTCTATCATTTAAAGTATTCCCGCAATTGGTCAAATCC ACCAATATGACTTCCATCAGGAGCAAATACCTGAGGCATTGTTAAGCCGATTTGAGTATCACGACCTAGT TTAGTCAGAAGCTCAGCAATTTTCTCATCATCAAAAACACCTTTTTCCGGCATAATGTTGATAAATTCAA ACGGCTGTTTCTTCACAGTCAAAAGACGTTTTGCATTATCGCAATACACACATTTATGAATGTTGCTATC ATAACCATATACTTTAAACATATTATTCCTTAATTCCTAGTACTTGTTTAAAAGTCTCGTCGTAATCAAG ACTTTGGCCCGTTTGTTCTTTATGCTTGTATATAATATCACTTACTTCTGATAGCATATTTTTATATGAA CGAGTTAAAGCAGATTTAAGCACGCTGTATCTATCAGGAAATTTACCGTGTTCATTATAATAAGCTATTG CTAATTCACGGACTGCCTTTTCAGCAGTCTCCATATATTCTTTACGCTTTGTCATTTTCTTCTCGGTCAA ATCGGGTTTACAATGGCGACATTTATAACGAAGATTATTAGTCTGCCAATGGACCAATTGCACCTGTTC AGTTCCACATTCAGGGCAATTAGGAACGTTTTTAGAAGCCCTTTCGCGACGTTCAACCATGACCATTACA GAATCCCAATTAACAGGAGGGCTATAATCATCACAACCATAAATCTTTCCACGCATTTCCAGATCGTCTT CTTCACCAGCCATAATAATTTTAATTAGACTAGAATTACTTGAAGCTGCAATGTCTTCTAATAGACGCTT TTTCATTTCAATACCTCAATAGCATTACGTAAACCATTTGCTTTGGCGGTTAAATCCTTAAGAACTTTAG TATGCTCTTCAATCTGTTTTTCAACTTCAATCAAACGGGTACTGAGATATTCGCGTTCTTCTTTCATAGT ATCATTCCCATGATTGGGCTTTGGCGTAGGTTTAAATTTATTAGGGTCCTTTAACTTAATAACAACTTTA GTTTCAAACAGTGAAAGACCATAATTCGTATTATCATCAAAGGATTCAACTACGTCCATTTTAGACAATA ACGTACGAAGTTTATAAGTCAAAGAACGAATCGTTTTATTATGTTTATTAGCTTCACATGGTTTCATATA TGGAAGATTTTTATATATTTCATGCGGAGCACAAACAAATATTAACAATTCAAATGGTCCATTCTGAGAT GATGGAATGAATTTAGCCGTAACCCAATCTTTTACATTTACATTAATAGTACGATCATCGCCATGACAAA ACAGTCTACGAGAATGCTTAAAAAGCATATTCACATTATTATTAAATTCGTGTGAAAACTCACTGGCAAA TTTTTCTTTACTATCTTTAGAAAACCATTCTTCTAAATAAGTTCTTTTCACGTCGTTAATAATATTAAAT GTAACAACATTATCATTTGATACATTAGTCTCAATGTAGCTTGGATACATAAATTTTTTAACATTATTAA CAGCTATCACAGAAGATAGTAATCGGGAACGTTTGAACCACTCGAGCAAAGAACCAAGAGAATGTGAATA CGCATTTTGTGTATCTTTACAAATATGGTTTTCCCATCCAATATAGTCTAAAAATCACGAAGAATATTA TTGATTACTTCTCTATGTCCCTTTCTGATAATCACGATGTTTAGTAATAAGACTGTCAAAATAATCAAATAT AATGTTTACGAGTTTTCATGTTCTTCTCACTTGGTTAATGATTTATACTCCGAGCCATCCTTGGCTTTAA ATTTTACTTAATTAACTGCAAAGCTTGTTCTAGACGATCAAGACGATTAACGGATTCTTCCAAATCTTT TTAGCCTGCTCATATTCTTTCTGCGCTTTATTAGAAATTTCTAGAACTTCTTTATAAGCTTTTTCTAGTG CAATAACTTCTGGACGAATTTCTATCGGTTCAGGTGAATCATCAAGACATTCCATTAGTTCCTCAAGGGT AGTTTCTTCTTTAGGAGTATTCACAATTTCATCACATTTTTGTTGGTAAATTTCTTTACTAGTAGGTGAA TAAGCACATTTCACTTCACGAAGGCTAATTACGAGTTTATATCCTTCCCAACCGCATATATTCTTAAAAG GATATGTATGAATATTTTCTACTGTTTCCATACAAAGTAATGCGGTCTCTAATTGACGAGTTATAGTGCT AGCAATTGAGAAAAATTTATTAATATTCTTTTGGTTAGTTTCTGGTGTAAAAAATCCATAATTCACAAAA ATAGATACTTTATTTTTATCAAGTTCATATTCTTTTATGATAATCATATCAGAAGCCAAAGGATGAATTT GACGATAATCGCCATAACATAATTTAGAAGCTGATTTTAGAATTTGCTTCTTGAAAAGTCTGAAATTACT AATCCAACGACGAGTAAAAATATTCTCAGGGTCTTCTTTATTATTGAGATGATAAGAATTAACATCACCG AACCAATTATATCCTACTACATCTTTAGTTCGTTTAATTTCTTTCCCAAAATTACCAAGAATATCCCGAT TAACCAAATATGAAAGAAGACGAGACTCTTTAAAGGTTTTCTTGATAACATCTTTATCTACACGGCTAGA AATTTTACGAATTTCATGAATAAATTTAGTAGAAGAAACAATACTTGCATCAACACTATTGAGCCACTGA TTGATAATAGAAAGAACGCTATTTTGGCCAAACATCGGTATAGCTTTATCATCAATAACGCTATTGAATG ATTTGATATATTCGTTACGAGTCATATTAATCTCCTCAGTAGAAAGTAAGAACATTATACCACATCCTTG TGGCAAAGTAAACTAGTTCAGTGCATTTAGTGCATTGTTCAGTTTAGAACGTTGCTTTGTCAGATTTTGA ACCCTTGACTGAGCTTGTTCTAACGCCTTTTCAGCTTCTAGCACTTCATTGGTCGCTTTAACTAATTCAG CATCTACTGCCTTAAGAGACTTCTCAATCGCATCCGCGTGCCATTTTTCAACAGGTTTAAGACTTGGATT TTCAACAGGAAGAAAATTCACTTTATTGAATTTCCATGCATCTTTATTACCTGAGCTATACATCCAAAAT TTAGGATCGTTTGATAAGTAATTAGATAAATTTAAGTTATCTTGCACTTCTTGCTTTTTCTCTTGTGTGA CTTCGTCCTTTCTCAAAAATTCATATTTAAATGAAACGATCATATCAAGTTCATATGATGTTGTATCTAA CTTAAATCGTTCAACGAGGTAAAATCTTAGATTGAACAGCAGCAACAACACATCCATATACTTAAATGCT TCTGTCAACTGCGATTTAAGGCATTCGCAAATTGAAAGAGAATTTTTTGTATTAGGTTTAAAGCTAATTC GTGCAGTTCTATTATTTTCTTTTAACGGTCTTACTTCCATCTGAAGAGTATAACCATCAAATTTCAGATT CTTTAAGTTAATGTCAGAGCCTTTTAATCGACTAGCAGTAGACAAAATTTGCTTTAATTGTCTACGGAAC AGAGCAATAAATCTCCATTCAATACGATAATGATTTGGATTGAAAAATCCAGATGATAAATCGACCTTAC TTTGACCAACGCCTTGAACAAATAGAGGATTTTTATAATCAATAGTTTTACAGAAATCGTGAGCTGTTC ACGAGCAGTCATTTGAGTAATATACCCTGCTTTACTAAAATTACGCACCCATTCACTGCTATTCGTATAC TTAAAGTGATGAATAACACGATTAACTTTATCAGGGTCTAAATTATTTTCACACAAAAATGTCATAACAT CACGAGTATAGCTGGCATTACGAACCATATCTTCAATTTGAGAACGAGTTTTCATGGTGTTCCTTAAGAT TTAAGTAAATCAACAATTTTAATTAACTTTTCACGCTCAGATTTAGCTTTACTACTCAATCCAGATAGTC TGAAAATTTCATCATCATATTGTTGAATAGAAAATATCAGCTCTTCAATTTGCTTATTAAAATAATCAATT TGTTCAGAATGTTTTTCGTTACTACGAACTGGTACAGGTTTTGTAGGCAATTTAGTTGAACTGGATTCAT TCGGCGATAAATTAAAATGCAATTTGAACCAATCGGGCATTTAGCACCAGATGAATATTCTAACGTTCC AGCTTCTTTTAAAACTTCACAAGCTAAACAGAGATGATGCCCCATATTAACATAATCAGTAGAGCGAGCT CTGATGTAATAATCATCTCCACGAGTGCTAATTTAAAGCATTTAAGGTCTTCTGTATTATTAGTTTTAA AAATCATTTGTTTATCTAGACCTTTAGCCAATCGAGCACCTAATGCTAATAATCGTCGTTGATTTTCCCA CAAATCTGCGATCATTTGGTCAAATGATAATTTCGGCATTAGCCGACCATAAAGGTCATATCCTTTATTA TAATTGCGAAGGATTTCACTCGCATTAATACGAGACAACGCTCCAATTTTATTAAGGGCTTTCATAATAC GATTAGATAAACTCATTCCAGACCCGTTTGTTCGCTGTAAATGTTTTTCTAATCCAAATCCAATATCAAC TTTAAATTTATCTAAAATTTCAGCATGAACATCTCTGTCAATAACATTCAAATCCAAAGTTGGGTTAAAT CTATGAAAAAATTTATCTGGCTCTCCACGACGTAAACAGTCCATTCATTCATCATTTTTTTATTAACTA AAGATTTAATTACTGCGTTGACATTATTAATTACTACTGACATATTTTCCTCACTCAATTTTACCAATTA CGCGGAATAAGATTGAACAGACTATATAAGTACCACATATAGATTGAACTAATGCCATTCCAAAGAACCA AACAATATTATCAAACCATGTCTGTTTTACGTCGAAAGGACGTAAACTTACAGTAATATTATCACCTTTT TCTATTGAAGAATACGTCTCTGGGGAAATATATTCACTAAATCTATAACCGTCTTTTGAGTTCATATACAG CAATAAACGATAAACTAGACCCCTTTCCTTGAGTTCCTGTAAGGGTATTAACTACAGTAACATCATAATC TTTATAATGCATATAATCATTAATTGCGTAATAACCATATGCAATTACTATACATAAACAACATATCAAT AAATTCAATCTTTTAATTATCAACTGTTTCATAATAATCTCAATTAAAAGGGCTTAGAACCATTATACCA TCCTTGGTATAAAGCGGTTATGCGAGTACCGTATTTAACCGTTCTTCAAACTTCCGAAGAGTGTTCTGGC GTTCAGCTCTTTGCTTTTTGTAAGTTTCAATACGCTCTGAAATGAGAGTGTATCGTTCATTTACTGATTC TTTCATAAAATCAGGAATTTCTCGAGAAGCTTTAATCTCGTCAAATTTATCAATAACAGCTTGCTCTTCA GCAATTAAGTTATCATACATCAAAATATCTTTCTTGATGAACTCAATATCTTCTTGAGTTACACGAGATA ATTTAGATGCTTTATCCTTTTTGTACTGTTCGTTAGTATCACGAGACCAGTGTAATGTACGATTTTTATT CGTATTCTTGTAAATTTCTACAATACCAATCTCATCGATAATAACGATCCAATTCCAACGGGATTTGTAA ATTGTCCTCCATTAACAGTGATTTCACCCTCCGATTGAGATGTCATTAAAGAACTTGCTTTGTGCTTCTG ATTTAAATTTACCATCGTTGTAATTTACCAGGTTGAAAATATCTTTAGCGTTCATTTTGTGTTCCTCCGT AGTTGATAGTTGTATAGTACCACAGAGGAACGGTCTTGTAAACAACTAAAAGAAACTTCTTTCACAATTT TTTCCACTGAACCAAGCGCTCACTGCTTTCTTAGTTTCAGGAGCAGTGTTATCCATAAACCATTCAAAGG CAGCCTTTTTTATGATTCTGGAGGGCTTCTCGGGCTTTAATCTGCTCACGGTCTATTAACACTAACATATG AGCCTTTCTTGTCACCAGGGGCTTCTTATGATTTTTTGAATACTCCCAATCATTTGTCCATCGCATCGTT GTTGCGAATTGAAATACAGCTTCTTTAATCTTAGTTTCCGTAAATTTCACGAGCCTTTGAGTATAACATCA TTACCTCCATTTACCAGTTTAATTCTAGTCATCTTTTTGATGGCAGTCCATATAATCTATTTCTGAACTG CCTTTTTGTCTTAGAAGGCCTCTTATGAATTTATTTCAGAAGAGTAACCCGTAGCGATTTCTTCCCAACC GTTTTTGTCGGTCATAATAAAGTCAGCAAGATAAAGTGCAGTACGCAGTGAAACATTACGTAAACGGTTA ACATTGACTTTCATCCATGATAATGCTTTATAAGTTTCTTCATCAGAAAGACCACGCTTTTGCATCATGT CGGTTGAAAGAATAACATCTTCAACCCTGACCATAATTTCTTCATTAGTGTGAACACCCAAATCCAAATA AACTGAGCGGGACACTAATGCTTGTAAATGTGGAGCAAGTTTAGTACCACGGTCTAATTCGCGGTCAATG TCAACGTTTGTGATAAAAACAATTGTTCCTTTAAATTCGAGCTCACGCTCAATGCCTTTTTCTTCTAAGT AAGAAGATGCAGTGCTCCAGCAGACTTTACGGGTCTCTCCAGTGTCCAGAGCAGCTTTCAGAAGATTAAG AATGTCCATATCAGAGAAAACATCCACATCATCAATCAAAAGGACAGAATTCTCTTCACGATTATTCCAA AGCTGTTCATAAAGACCGATACCAGAGATTTTACCGTTAATGCTTTTATACTCAATGTATCCAATATCAT TTGCTTTATTCAAAGCTTTATCTAAAGAATATGTTTTACCAATACCCGCCGCACCAGAGATAATTAATGA ACGAATATTTCCGTTAATAATACCATTCGTCATCATTCCCATAACATTAAATCTTTTATTAATGCGGGTT TTCATATCTTCATATGATTCTTTAACTTCTTCAACTTTTACACCATCATATGAAATGTCTGATTTGTAAA CCCAAACACCGCGACGTTTACCGTCAATTTCAACAAAAACTTTACCATCTCCTTGTGCATCTACCGGAGC ATTATCAGGGAACCATTCACCTAAGAGCTCAAAAGTTCCAGAGATTTCTTTACCGAAGTACATACCCTTA TTGATAGTTACAGTTTTCATTTTATTCTCCAAATCCGTATCAGTTGATAGTTGTATAGTACCATAAAGCT TTATGCTTGTAAACCGTTTTGTGAAAAAATTTTGAAATAAAAAAGGGAGCCCGAAGGCTCCCTATCATTT ATAATAACTTCGATGGTTTTCAAGATAAACCCTCTCAAGGAAGTCATCCCAGAAACTCATGTCTACTTTT TGCTGCATACCGTTCTTAGAAGCTTCAGTAGATGCTGCTTCTACTTGATCGACCACATCTTCCAAAAACT CTTGAACGGTTTTAAATGGATGCTTACCCAACTTCACGTCGAGAATAAATGGAGCATCTTGGAGTGGATA AACCAAGTCACCAGTTTTGTAAATTTCCAATAGTTGAAGTCCACCACGACAAGCATGGCTCAGAGCTTTC CAGTCAATGCCTTCATTGGCTTCGGCCTTACGAGCACGTTCGCCGTATTCAGCATCTAATTTGTTCAGTG ACTGCTTAAGCTCAATAAGAGAAAGCGTTGTCTGATATTTACGACCCAACACTGTGTAAAACGTTTGTGG GCCTGTTTTCTCATGATTATGGAACACCCATTCACAGAATTCGTTTTCTGGAAGACGATGCTTAATATCT TCAACTTTAGTACGACGCTGCTTAATGGAACCATCTTCTTGGTAATCAACCCATTGCTCAGGGATTTGAT TAACTACTTTCAATACATCGCGTAATGCAGCCAAACGAGAACCCTTGACGCCGTATTTAGAAGCTTGCTT GCGGACATATCCTAAATATGATTTCATGTTAGTCGTATAAAAACGAGAACGGTTGTCTTGAATAAACTTC CAMACATCAGGCAAATCGGATTTAACCACTAGTTCAGGTGGAGTGTGAAGCATATCCAATGCTACAGGTT CACCATCTGCTGCTAATTTAAAGAAATACTTAAGACTATACAATTCGTGGTCAATATTATCTTTAGTGTT TTTAGATGATGTGTTGTTGGTATTTTTACTCATGTGCTCTTTGACGTTTCCAATAAGAATATCGCGAGCA GGAGGAACAAAGATTTCTTTAAAATCTACATCAGATTCTGGAGTAGAAGTTCCATAAAGATGACTACCAA AATAAGACTTAACTACAGTTTTCATTATTAGACCTTTCATAATCTTCATTAAATTGTACAATCAATCGAT GATAATTAGATTTTGGATATCCTAATTTACTTATATAAGTTCCGAATGACCCACGTTTAGGTCTATTTAA TTTAATCCATAACTTATAAAGTAAGTCATAGTCTTGCCAATGTTTACCAGTTCTTTGTCTAATTTTAGAT GAATTTGATTGTTTCTTTTTAGCTTCAAAATTATTCAAAGATTTTTTGACTGCAGCAGAGTGTTTTTCTT TAACTCCCGGTCGTTTAAAAGCAGCTTTCACCCCTTTAGATATTTTTTCTTTAACTTCCGGTTTGTTTTG TGCTTCTAACTGGGATTCTGAAATTTTAGCCCTTATTTCAGGAAGACTTAATGTTATCGAACGTTTTTCT CTGTATTCATTAGACTTCCACATTTCTTTCATTGTATTAGAATGTATTTTTGAAAATTTTTCTCTAACTA ACTGATATCCTCTTGAAGTTAATTTGAGATTTCTTCCTAAAGAATCTTCTCCAAAATTATAAAATGACCA CCATGCATAAATTAATCCAGGCGAATTGTAATGAATTTTAGCTAAAAGCCAATGGGCTATAAAATGCTCT CTAGCTGTTAATAAAACTAGATTATCAGAATCATCATTACCACCATACAAGATGGAATAATATGATGCT TTTCCGTATAAAAATTTAATTTAGATTTATCTAATTTTTCTGGTTTTCCTTTCTTAATTAAATTATTATA TACTTTAGTATAATTCATTGGTTCTTCGTCTCATTTAATTTTGCTTTGCATTTATAACACATGTCTGTTC CACTTGAAAATAAACATATTTCCTCACTTTGAAATCATAGTTGGAATAACAGAATCAAGATAAGTCTTTAG TGCAATAGCTTCCTCTTTCTTTAATGTAATGATATGTGATCGAAAATCATCAATTTGACGAATAGATACT ACATCTCCCTCTTCATAGCATTTTGAAACATTTAAAATAGTTTCATCATTCTGATTACAGGAGTTAGTAA TAATAGCATTACATTTTTTAAACCATTTTAAATTATGTTTTCTTTTAGTAGAATCATAAAAATATTTAAT GTTAGTTATAAAATTATCCCAATTATTAATTGATAAGCACATTGACTCGCTTTTTAATATTAAATCCTGGG CATGAAGAATAAAAATGAATTTTATGCTCATCATTAATGCTTACAATTTTATCAGTGTAAGCATATTCAA TTTGGGTTAAACGAACAATTTCGCTAGGCGTAAAATATAACATGTCATCTTCTTGCGTCAAACGATACAT GTTATTTACTTTTTCTATAGCCAATTCACCAAAAAGTGGACTAACTTCTAATACTAGTCGTTTCGCCTCG CTCATCATTACATACTCCTCTGAATCATATTAATAATGTTATTCACCAGATTATAAGTAAACATTGGGTA ATTATATTGAATTCACATTATACAAACAAAACTTTCATTCTCTTCTCCTTGGCAGTTGAATTATA ACTATACCATAATCTTGTCAACTTGTAAACCATTAAATGACGTTTTCGATAATTTTGAAGCTTTGTAT GAGCATCAACCATGATTTTCATTTCTTCCTTGGAAAAGCTGTGCCTCTTTCCCGAGAAGAACATTCATAG TCAGAAACTGCATTAGCATATTCTTCAATTAGCTTCATTAAAAACATCTGTTTTTCAGTTTTCATTATTC CACCTAATCATTTCAAGATATTGAACTAACTTAGCTTTGGATTTATCCAAATCCCTTTTAGCTGCTTCTA TACCGTCGTATGAATATCCTTCACAATGCTCAACTGCTAATTGATATGAATCTATTTCAATATTCATGCGC TAATTTAATGATTTTCAAACTGTTCGCGTGTTAGCATACTAACTCTCGTATTATGATCGATAATTT CATCAAGAAACATATCTAACGCTTCTACAGCATTATTAACTTTAGCTTCAAATTCTTCAATACCTGATAA GGCAAGTTAATGCGTTCCTCATCTGCTTTACGAATTAAAGCTACCAATTCCTTAATTTTATCCGCTGTGT TCGATACTAATCATTATTCCACCATATGAAAGAGAATATTGCACACGCCATGTGAGTTGCAACTTC ATCACATATTAACGTTTCTTAAGAAGTTCTACAAGTTCTTCACTAGTAACTTCATCCATGTCGACG AAAAAATCACCATTAATGATGACGTAGATATTTCCTTCTTGATTGAGTGCTTCAATTTTCATGATGTTCT CCTCTTTATCCGATGGTTGTATAGTACCACAGCTCAAACGGAAAGTAAACCGGTAAAATGAAAAAAAGTC TCCCGAAGGAGACTAATGTTATTCGAGGGAAAGAAGATACTTACTCTGGTAAAACATTCCAGTAATATCA TCTATCGTGCTTTGGATGGCTGGAGGCATTTCTTTATAAATGCTGTTAGATTGGTCTAGTATGCGATCAA TCATTTTAATTGTGTCGGTAGGAAGTTTACTGGCATCTGGAATTGAAGGCGTGTATTTTCGACCAGAATA CCCCAAATATTGCTCACCAATTTATCAATCAAATCTGGCAACTCGGAAAAAAAATAAATCGTATGCTTG TGTCTAGCATAACTTTTAGTTTCAAAATGTGCAGAATGAAAATAAGCTTGTGCAGCCATTAATAAACCTA AGTATTCATCTGCCTTTGAAGGTTTTCCACTTTGTGAAAAGTCGCTGAATTTCATTCAGTCTCCAATTTA ATGTTCATAATTCTAGCGTATGATTGTGCCATCTCCGCGCCTCGCTCTATACATTCAAAATCAGAAGAGC ACGGGTCATTTTTATAGGTCGTTCTCATAAAACTATAGAATTGTTCAGAGAGATTCTACGCTTTTATTTTC AAAAAGCATATAAACGTGCCTAATACCAGATTCCATAAATTTATCAAAATGAGGATCGACATTCGCTTCA ATCGATGGAGATAAACAAATGACAATCCTAGCATGGCAAAGGTGCTGTTGCTTTTAAGGCCATAAAGG CCTCCTATCATTTTTGTCCTGTATTTACTTTGTGCCGATTGCACGGCCTTAACTTTATCAAGGTATTTTTC AAAATTTCGCAATCTAGTATAGTCTGCCGGAGATTGGTTGAGTGATACTTCTCGACGCAAAGCTGAAATG ATATTTCCAACTTCCCTACGAATTTCATCTAATTGAAGAACAGTAAGATTGCGAAGTTGCTTTTCAGTTA ATTGTAGCATATATACCCCTTTAGTTAGATAAACCTATTTATAACTTTTGCACTAACCGAGCTTTTTAGT TAATTCATTCCAATGTTTTCTACACAAAGAAACATAAATTTCATCACCAATACAAATTTGATTACCTTCT TTAACTGGTGTTCCATCTTCCATTAATCGAGCTGTCATAATCGCTTTTTTACCACAATGACAAACTGCTT TTAGTTCAATAAGTTTATCTGCAATCGCTAAAAGTTCTTTAGAACCTTCAAATAATTTTCCAGCGAAATC AGTCCTTAGCCCATAAGCCATAACAGGAACATTATATGTATCAACAATTCGGCTCAATTGATGCACCTGT TCAGTTTTTAAAAACTGAGCTTCATCTACAAATACGCAATGAATATCTTTTTGTGCTTCAGCCCATTTAT AGAACTCGAAAATATCCATATCATCTGTAATAATATTCGCTTCCTGCTTAATTCCAATGCGAGAAACGAC TTCACAGACAGAATCGCGAGTATCAATAGCAGGCTTAAGAACTAATACACTCATTCCACGTTCTTTATAA TTATGTGCAGCAATCAAAAGAGAAGCAGATTTTCCAGCATTCATTGCTGCATAAGTAAAAATTAAACTCG CCATCTTAGTCCTTAGTTAAATTTTCTAAATATGTTTCTAAATCATTTTCAGCTTTATCGATAGATTTTA CTAATTCGTAATATGTTTCGGCATCTCCATATTCAGAAGATATTTCAAAAGACAAATCCTTTTCTAAACT AATAATTTCACCAACTAAAAATAATATTTCGTTCTTTTGTTCGCGAGTAATCATAAGGAATTTATATAAT CAATGAGTTCTTGTTCTTTATTATCGAATTCTTTAGAAAGTTCTTCGTACTCGTTTGCGCTAAAAGGACC GCATTCATTACAAACTTTTTCCAATTCACTATTTTTATCCATAACTTCGTGGATAAGAGAAAAGAGTGTG TCTTTTTGTTCTTTGCTTAAACTCATAACCATGTCACCTTTTAAGCAGTATTCTTCTACATGCTGTTTACG ACCTTTCTTATCAATAAAGGTATATTCAACGAATGTTCCAATGTAGTCTTTATCTACATCATGTGGACTA TTAATTGGACATTTAGTGCGACAAATGCGTTCCCATTGACGAATAATTACTGCCTTATTCTTTGGGTCAT ATGGATGTGGATAATGTATATTCATAATAATGGTTCCCAATCAACAATCACAATTTCTAATTTAGAGGAA TATGTATCTAAAATCCCCTCAATAATATCCCAGTTCCCTTTACCTATGCCTGCACCAATCCTAGGCATAT AGATTGTAGGTTTAATCAGTTTATTTTCACCAAACTCATTTAATTCTAACATACAATTCATTAAAGCGGA ATACTCAAAATTTGGCCCTGGTTGAAATTGAGTATAAAGATTGAAGCAGTAAGCTTTATGAGTCCTAAAG TATTTTTCATAGACTGAGTAAGAACCGAGTTTAGTTACATCACCCCATTCAGTCTGTAATTTATCAGCTT CCAAAATTTTAGGGAAAGCTTTGGTTAATTGACCCGCTACGCCTGAACCCATAGTATGAAAACAATTACA TCCATGTGCAATATTTTTACCTTCAGCGAAAAGGGCGACAATATCGCCCTTGATATATTTTACAATCATC TAGTACTCAATCCTCGATTATAAGAATCTACCAAACGGTCAACCATTGAATGACAAGCGGCTTTATCTTT CTCCTCCGCAACTGAACATTCTAAGGTATTCCACTTTTTAGCATTCGTTTTAACAATGTATCGTTTTTG TATCTGCTTGATTTATCTCTTTCTCCGTCTTTATATGCATATATTAATTTCTGTGCAAATTCAGCTTGGC ATGCCTTATTTTTCCCACAATAATCTGCCGCAGTGCGGTTTACATATTCTCTAATTTCAGTATATGATGT ATCTGCTGACGCAGAAGCAGAATTGAAATTAATCCTATACATAAAACCAAAATTTTAGTCATTTACTAT TTCCAAAAGTTTATTATTTTTAAGGTAATTAGCCTTTTCTAGGACTTCAGAAGCATATTTAGAACCTGCT TTAACATTCCATCCCGAATTATAAAGAGGATATTGCTTTTCTTATATCGCCCTTATGTATATTTAACCAAT AAGAAAGTTCAATGTACGCCCAGGAAGCTGAATTGGATCGTTTATTCAACATTCTTTTTATTTCAGCATC GGTCATATTATAACCAAGTTCCTTAACTCTTGCTCGCATAGTAGGCAAATAATTTTGGAACATTCCGTAG GCGTGATGCTTTGGTTTAGATTTTAAATTAACTCCGCCAGAGCTTTCTTGCCATAAAATGGCAGCCATTA TATGACCTAATCCGCTCTTGTGGATATTTTTGTGTGTTTTATATTTTCCATCCTTAGAAAATTGTTCCCC GAATTGATACGCGTAACGCATGTTATCGAGTTGGACATTACTGAAAGTATGCTCGGAGCTATGTGCCATC ATTGAAATGGCCAATAGACCAGCGAGTAGTGCTTTTCTCATGCTTACCTCATTGAGTTTTAATTACTGCT TTAGAAGCCTTTCCTGGTAAACGACGACTGTTGATAATTGCCATCCTACATTGAAGTGACGGGTCTTTGA ACTTCTCGTTAGGTTTACAAACTGTAAATCCAAGCCAAAGATTTCCATCTGTGATTTCTAAACGTCCAGG ACGATATTCAACCCCATCAATAAAATCCTCGTCAATGTCAGGACGCGGAGGCATACTCAGGAATTCATTA ACTTCTAAAACATGGTCTTTTATTTTATGGAATAATTCAAAAACGTATGTCTCATCAATCTCCCGTTGAA TTGCGCGATCAAGAAGATGTTGAGAATATTTTAGATGAAACGATGAGACTCCTGCTGCTTTTGATGCCTC ACGAATCTCATTGTTAATTTGACGAAACTCCGACTCAAAGTGACGACGAAGCTTATTTCGACGGATAAAA ACTTCTGTATTGATAGTCATGTTATTCTCTCTTAACTGATAGAAAAATTATACCACAGTCAAGAGGAAA AGTAAACAGTTATTCTTTAAATTCAATTATTCATAGACTTTGAAACTTCGGCACGAACCTCATGT AGATTTTTGAGCTGTTCAAGACGCTGCGTATAGTAAGCAATTTCATCTTCTTCGAGACAGTCCTGCGAAT CTTCTTTAAGATAACGTGCATAGTCCTGGAAAGCGTTACGGACTACTTCCTGGAAGTCATCAAGACTTTG AATTTTCTTAGGAGCAACAGATACACGACGAGGGGCAGTATAATACTCATAACCAAACCCTGCGCTTAAT TGAGCCATTAGTATTTTTCCTCTGGTTGGAACACTGCACGACAAGCCCACATACTGGCTTCTTTGAGTTT CGTTTTAGCAATAGTTAACTGATCGAGACTTTCAGCATAATTCTTCGCGAATTCACAGTCTTCGCAATTA TCTAGTGCTTCCCAGAATTCATCATATAAAGCATCAAAGATAAGTCCTAAACGAACTTCAGCGTCTTTAA TAGCATTTACTTTACCGATTTTCTCTTCAGTATGTGGTTTATAACCCTTAATATCTTCAATCATATTTGA CTTCCTCACCAGTACATAATACGTATTCAACTAAACGAATAGGTTCATGAATGCCATAGCCTTGAACAGA AATTTCTGTCGTAGGATAAATTCCATCAATATCACCCATATTCCACGCTTCATTAAATTGCTGTTCGCCT GAGTTACTAAACCACTCGCGAAAGCATTTAGCACATCTTCAGAACCTTCAATAATTATCTTTGCCATTAC AAACTTTCAGTAAAGGTACGAGCGATAACGTCGCGCTGCTGTTCCGGAGTCAGAGAGTTAAAGCGAACTG CATAACCGGATACACGGATGGTCAGCTGCGGATATTTTTCCGGATGCTTAACTGCATCTTCCAGAGTTTC ATGACGCAGAACGTTAACGTTCAGGTGTTGACCACCTTCAATTTTAACTGTAGGTTGTGGCTCAATTTCA ATTTCACGGGCATGCAAACCATAGAAAATTTCTGGGTCTACAAAAAGGTCCTCTTTAAGGTTTTAGAGA CAATAATTCGTGCTTGAATACCATCTTCAAAATAAATAGTACCTTTATGTGTGCCTTCAAGAATTTGATA TGCTTTCATATAAACCTCAATTAGAAAATAAATTTATCCAAGATTGTTCTTTAATTAAAATGGCTCAGA ATCATATGCCATTAAACTTTGCGTAATTAATCCTTTAAAAGGTCCATCAATAAAATTCCATGGTAAATAT GGAATTTTATTCATTAGCCGTGCATTAGGAGCAGTGCACAAAACTCTGCATCCTTTGAATACGCCTTTTT GTAATTTGTATTGCTTGGGATAAAATTCGCTCAAAATGTTATTTTTTGCCAAAATTTCAAAATGATTCAC CAATTTATTTTTAATAGTTTTTGGCGAAAAATAAAGATATTCGAAAAGCTGAGTGTCTGTCATCATTGCA TTCCGATTACGAAAAACTGTGGACGAGTAATACCACCAATGCAACATTTACTATTACAGCAGTAGTGTAC GGTGTCAATATGGACACTATAAATCTTATCCATATCAGGAGATTTGACAGGCTCATCAATTATATACAAA ATTCGCGAAAGCTTTAAACCTCTGAACTTGCTTCCTTTATTACCAATAAAACTGCGTACAGAATCAGTAA ATAAACGAAAACGTATATCATCATTAGAATAACGCGAAAATTCCCTTTTTGATGTTATTTGCAGAAATTTT AGCATAAGCTGAAGTATTAGAAAGAACAATAACTGTTCCGCCATCATACAACCAATTAGCAGCAAAGTTA GTCACAGCAATTGATTTACCGGATTGACGTCCACCATCTAGTCGAAGTGTACAATACTGTTTAAGTAAGT CTTCAAATGGCGGGATATATTCGTTTTTACAAATTTCTTCTACTCTAGCATCAGAATGGTGTGTAAAAGC ATTCATCAGGGATAGATAAGGACCAGTTAAAAATGTTCTCATTTCTTCTCTATAAGCTCTATAAGTTTGG GCCATTCCGTGGCACATGAATTGTCCATTTCTGTATTTACCCATTACCGCGCTTGGGCTCGACCTTATTA CAGGTTGGCGGGAATCCCTCATATAATCATGAGGTCCAGGTTGTTCCCTTATGCATAAATCGCCTTACCG TAGTATTTGTACCAAGTAGGACGTTGTGCAATTTTTTCATCTAAACGAGCTTGTGATATAGCAATAGAAG CTTCATGGGGAATATAATCACCACGGAATTCCTGAGGAATATCACTAATATCCTGGACTGTAGTATCCTT GATATTAAAACCACGTTTTAAACATTCAGCTATAAGCTCAATTTGACGTTTACGTAAGAACTCGAGCTTA TCGTAAAAGAATGTAACATGACCTGCGCCAAGGATAAAAGTAGGACTGATTTTAAAATCACGAACACGTT TACCGTTAGCAACATGCTTACGAACTGCACCAAAAACACGCGGCAATTCACGATATTCAGCCATTAAGTG TTGGTCAGCCAATTCAGATACTAAAGTAAGGTTGATACGAGTCATTTTAGTGTTCTCCTGTAGTTGATAG GTCTATAGTATCATACCTACAGGAGATGTAAACTGTTATTTATCTTTAATTGCTTTAGCTGCTTCGATAG CCGCTTGCTGAAGGTCATCCATAGACATGCCGAACTTAGAAGCAAAGTTATCAATTTTCTTTTCGACGGC ATTCAAAGGCTTGGCCTGTTTACCTTCATTAGCGCCAGGAAGAGCGAGAATACGCTCTCTGTCAATATAA AGGCTTACAAGTTTCTTACGGTCTTTATCGGGCAAATCATGAAATGAATGGGCCTTTTTATTTACAGCGG CTTCTAATTTACCTGCGCCCACTCGCGCTTCGGCAATAAATTCTTGATATGTTTTCATATGTTTCCTTTA AATGTAAATATTTTTATTATTCTATCCTAGAATTGTGATAATATATTCACAATTCTAGGAGTTGTAAACT GCTTTTATTTAAGCGTCCCAAGTATAAGCTTTATTAAGAATTACCACGGGCTGCATTAGCAACGGCGTAA GCGTACTGAATATTAGCGTCTTTAAACTTACCTTTAGAGGTATCTATTTCTGCCTTAAAGCCGCCTTTAG TCATAACATCGGCAAATTCTTTACGGAAAGCCATTGCATCAAGACCTTTCCATGATGATTTATGCTTGGC AAATTCAAGACCTGCGAAGTTGACAGCTTTAGCCAATTTATTATCAATGACCCATTTACCGGCTTTAGGC ACAAACTTCGGGCCTTTCTGTTTAGAGAACAGTTTTAAATTCCAGCGGCGAAGGTCTGCATCAGCTTTAA CAAATTCTGAGTCTAAGTTACTAGCAACAACATCTTCAATATGAGCAAATTTAAGTCCGTCAACTTCAAT ATTTAAATCGGTACGCCAGCGAAGTCCTTCCCAAGCGAAGGCTTTAAAGTCGGAAGCCTTTGTAGCTAAG TACCGTTCAATAGGAGCTGTTTTAGGGTCAAAGCCGTTTCCTGATCGGTAGGTCCACTCATCTTTGTTAA TGCCTTTGGCCTTACTACAGAAGCTTCGGCAATAAAATTCTTGATATGTTTTCATATGTTTCCTTTAAAT ATTTTAATTAGTAATTGTCTATTCAAGTAATTGTGAATATACTATCACAATTTCAAGAGAAAGTAAACAG CTTTATAGATTTTTATACGCGTCCCAAGTGCCAGTTCTAAACGTTGTAATGACTCGTTTTGCGCGATTAG GTGTTTGATTATACCATATACTTTTAGCTAAGTTAACTGCTGCTTCATCCCAGCGTTTTTGTTGAAGCAT ACGTAAAGAGTTAGTAAATCCTGCCACACCGGTTTCTCCCATTTGGAAAACCATATTAATCAATGCACAG CGACGAACCGCATCAAGAGAATCATAAACCGGTTTTAATTTAGCATTTCTCAGAATTCCGCGAACAGCAG CATCAACATCCTGATTAAAGAGTTTTTCAGCCTCATCTTTTGTAATTACACCATTGCAATTACGCCCAAT AGCTTTATCTAATTCAGATTTAGCAGCATTAAGTGATGGACTTTTTGTAAGCAATGACCGATGCAATA GTGTAATAGCCTTCTGTGTCTTTATAGATTTTAAGTCTAAGACGTTCATCTATACGTAACATTTCAAATA TATTCATAATACCTCCTAAGTATTTATAGAAGGTATTTATAAAAATTAAAAGAGGTTGTCATTATTCGGT AAAGTGAAGGACCCATCACATATTGCCACTGAGTACGAGGAATAAGAGCAAAAGCGTCCATCTCTGGAAT CATAACGCCATCTTTATTTTCAAAATAAGACTCGCAACGGCAATTTCTGAACATCTCATGCTCTACTGGA ATCGTGTAATAAAATAACTGTAGGTCTTTATTACTAGAATATTTAAATACACCTAGGTCTTCTAGAAGGT CTGGATTATAATTGCTAAAACCAGTCTCTTCTAAACATTCTCTTCGTGCTGCATCTAATGCGCTTAAATC AGAATTTTCTACACGGCCCTTTGGAATATCCCAACGATGTGCCATCATTCCAGTCTTACGAGAACCAGTA ACCCGACCCATAAATAAATCTTTATCTTCTGTCATAAAGATAATACCAGCTGATAATGTTTTCATTTTAA TTTCCTGCATTCAGTGATAAAGTTATTTAAATTTTGAGCATATTTCTTTTCATCAAAAATCTTTTGCTGT CTGCGTAACCGCCATGGCATTTCAATGAACATACGCCATATCCCTAGATAATACCGCTGCTGTAAAAATA TTAACAAGTATAGTTAAAAGAATCCAATCGCCTATTCTGTCCATTGGATTTTTATAAAAAAGTAAAATAC GAATGATGATATAGGAAGACTAATGATATACCACAGAAGAACCTTCTTATCTGTGAACCAATCAGCATTC GTTAACTTAGCGCGACCATTTTGAATACACACGAATTTATCATCTGTTACAGTAAATGGCTTAGCTGCTT GATATCCCATTCTAAACTCCCTAATTAATCGTTTCTTTGTATCTTCGGAACAACCATTCCAATCAACTCT ATCAACTGGAATGCCATCATCCCCATCATCTAAATCATACCAGCGAGTTTTTAAAATCATTTAATTTTCC TGCAATCAATCACAAACTCTTTCATTGATTCATTTTCAATATAAGACATGTAGCTATTATATTCTTTTAA TTGTATTTTGTAATCCTTTTTTCTTTGCCAATTTATTTTAAAATTATCATAATGAAAATATAAAATGATA CCAAAGAATGAAAACAATGAAATAATTTTAGTATAACAAAGCTCGTCCCAACTTCTATTATATCTACTG TACCACTGATTTTTAAAATAAAACAGTCAATTAATAGTCCAATAAGACTACCTGTAAGAGCTGCAGCCAA CGCCACAGCAAAAATTAAATGACTCAGAAAACGAATATTTGACTTTATTTAGCTTTGGCTTTTGCATC GTGATTCCTTAACAAATTTCATAATTTCATTAAATTCATACTCAGCAAGTTTAAGCTGGGTGTTCCTTTTT AATCTTTTTGCACTGGGCTTTCCAATCACGTACGCGTTTACGATAATGTCTTCCTTGATACCAGTATCCT ATCCAATTTACGGGTACTAATAAAAATGGAACTACCAATGGAAGAGTTAGCATTAACATAATTATTACGC CAGAATCAATATCAGTCATAACATCTAAAACACCTCCAATAATCAATAGAATTACAAATGATATAGCTAT CACAGGACCTATTAATACATCAGTAGAAATTATCTGGCGCTTTAATTCATACTTCAAAGGTTTACTTGGA AGGTATAGTGATGGCTTTGACATATTCTCTACATTCCTTAACAAATTTTTTCTAGTAATAAATCACTTTCA AAATTAGGATTTTCCACTAATTTATCAAAAGATCATCAACAATATTCAAGATATTTCTTTTACTAAGAA TACGTTTATTTTCATGCTTCGTTTCAGAATCAACTATAAGAGTAAAGAAATTTTCTTTCCCTGAAATTT TACCGTAGTATCAATATAAAATAAATTTGACTTTTGTAAATTACGTTTAAACCATGCGTCACTTAAACTA TAAACACCGAGATAATCAAAATCGTCGTTTAAATAACAAACTGACCATTCAGGAAGAATGAAATCAGTAA ATTCAACATCAAAATCACATGTCAATGAATGAATTGATTCAATACTGTTAATAAGTATTCCAGGACGTAT TAAAGACTTTTTACCTCTGGAAAATCTTCCAGAAAGACTTTCATCAGTTTCATATGAAGAAACCCCAATAA TAATTACGTCCTTTTGCCATATGTTTAAGAGCATTTAGTAATTGGTCTGGAACATCAACGTGTCTTTGGA ACTCTTCAAACATTGAATTGAAATCACTTTGCATTTTCATTCCTATTTACTCCAAGTAATAGGGGCCGAA GCCCCTTATCATTATTTCAGAGAATTAATATATTCCTGAACATCGGCAGAGGTAGTTTCAACCCCAGAAA TATTACCATTAAAGGTTTCAACTCGAGCAAGAGTATCTTCAATATCAACCTTAGTCAGTGCTGCAATTTC AACTACATCATCAGCAGTACTAATTCCAAGGGCATTTGCTGCACGAGTTTCACGGATATATTCCAATTTA ACTGCAAGTTCTTGGCGAGCATCATCTAACTCAACTACTTTCTTGGCGATTTCAATTCGCATTTCAGCAT AACCATCAGCTTTAGTAGTCAGCTGTTCAGCTGTTCGACGATATAGCAAGCCGAGTTTAGCATGCATTGT TACATCTTGACCTTCGGAAAGAAGCTTGCGAATTTCACGCTCTTTTGATTCGGCCTGTTTATTCTTTTCA ACAATAAGTTCACGAATACGTTTTTCTTCATTAATAGATTTAACAGAAGCAGTTTTTAGGTCTTTAATTT TATCAAGTAGTTTTGCTGCTGCAGCAGTATACTGTTCTTCAACAGATAGATTTTTAGCCATTGCAGAACC AAGTTTAGTGGCGAATAAACTCAACAATTTTCTTCAGTGTGTTCATAGTATTTCCTTAGGTTGGTATAATT AGATAATATAATATCACGTTTCTAATAGATTGTAAACTTATTCTTCGTCTAGCTCGTCGATAAAGGCGTT GATGGCCTCGATAATGGCATCATTGATAGCCAATAAAATAAAATCATCGTCAGTACCTTTAGAAGATTCT AAAGCATTGATATATGCTTGGTTGACGAGTTCCCAAGCCTTTTTAAAATAAGGAGCTTCATCATCAGGAC AAATATCCCGCACGCCTTCAAAGATACGTTTGGCATAATCTAACACCCATTGTACAGGCATGTTTTGAGA ACGTTCGTTAAACTCTTTAAAGTCCTTAGATTCAAAAAGCTCTTCAGGATAATTATTTCTATTACAAAAA GCTTTACTAAAGTTACGTTTCATAATGTTTTCCTCATTTGTATAGGCTCATAATATCTCAATCATSAGCC TATGTAAACTTATTTCATATTATTGAAATATTCTTCTGCGATTTCGTCGTTATCATGGTAAACTTTAGAA GACAGTTTAACATAACTTTCAGCAGTGAACATGTTAATCACAACCTTTACAGTATACCACTGACCGTCTT CATTACCCATTACTGCGTAAGTTTCAAACATCGGATGGTCAGGACCGATAACTTTAATATCATTCACCGT ACGACCGAAATCTTCTGAAACACATTTCATAAAGAAGTTGAAAAGTTCACCGTAATTATCCATTTTATTC TCCAAGTTATTTTCTGTATCAGTAGTTGATAGTTGTATAGTACCATGGAAGAACAAGGATGTAAACAGTT TTGTGAAAAAATTTTTAAAAAGTTTTAGGGAATTCTAGGGCGGAGAGGGGCAATTAAAAGATAGGATAAT ATATTATAAAGGGTATAAACTAAATGATGCCTAGAGAGGTCTGGAAAGGCTTAGATACCAAAAAGCCCCA ACCTTTCGGTCGGGGCTAACCGTTGCGGCAACCTTGTCGGGGTTCCACCTGCCAAGGCAAGTGTTTGTAC GAAACGCCGGGATTCGAACCCGGTTATTAAGTAGTTGACGCTACTCAATATTTTTAAAAGGCCATATCTC AACCATATCCGAACGTTCCGTCAAAAACGCTACTCGGCTTACGGCAAAGATATTTCCTCGAATCGATAAT TTGGTGCGCCGTTTCTGCTGTGATGTAAGAGGGCATCAATAAACGCAAAGATTATTAACGCAATTCCTTA CTCAGGGAACCATCAGTCCGACGACTTACCGGTAGCGACCCGGTTTCTCATTTGGTATCCCGCCCTGGGA TCGAACCAGGACCGCAAACTTAGAAGGATCGTATGCTATCCATTACACCAGCGGGACGTAATTTAAAATT TCATTTTTCGACCTTTAAACCATCCTTCTGGAATTGGGTCAGTTTTCTTAATACGTTTAGAAACTTTTTC ATCTAATGAATGAATCCACATCATACCGAATTGGGAATTCTTTTCACCTTTCTGGTGATTATTTTGGCG TGAGATTCTTTCATTTTATTAATAGTTTCAGGAGTATGATGCTTATTTAGAAATCTGCTATTATTTAAAA ATTTTCCCTGTATTCAGGAGTTGACCACAAACGTTTAAATACATTTGAACCAATTTTACGATATTTTTC TTGAAGTAAAATATCATTTTCAAAACGTGACTTAAACGATTTAGCTCCTTTTAAGCTAGCATCTTTCTTC TGGTTTAGCATTCCAGGAATATTTACATGATCCCATCCACCTTCACCGCCAAGTTTTAAATTATACACAT CTGGTCTATTTAAAAACTCTTCTGTGACAATATTTTTCTCGGCTTCAAGCATAGATTCTTTATCGTCAAA ATACTCTAATATTTCTTTAGAAAAATTTTCTATACCATATTTATCTTGGGCTCTTTTTAATAATTTACCA GAACCCATATATCCATCATCTAAATTTTCGGTAGAATGCACACCAATATAAATTTTATTATTAATTTTAT TTGTTATTTTATAAGTGTAATAGAACATAAATATCTCCTATTTCTAAGAGTATTTATGTTCTCAAAATAT GACCCAGACCAGATTTGAACTGGTAACCTTTCCCTTATGAGGGGACTGCTGCTAACCATTGAGCTACAGG GCCTTGGTGCTGATTGACGGAATCGAACCGCCGACATCCTCATTACAAGTGAGGTGCTCTACCTACTGAG CTAAATCAGCAAAATTACCGGAGGCGATAGGATTTGAACCTATGAGTCGCCGGAGCGACTGCCGGTTTTCA AGACCGGTGCATTAAACCACTCTGCCACGCCTCCAGTCTCCATACAAGGATTTGAACCTTGGACCTCCTG ATCCCAAATCAGGCGCTCTACCAAACTGAGCTACACGGAGTAAATTAAATTGGAGCGGATAATGAGAATC GAACTCACATCATCAGATTGGAAGTCTGAGGTAATACCATTATACGATATCCGCAAATTTGGTGCGAGAA GTGGGACTCGAACCCACAAGGAAATCATTCCGCAGCATTTTAAGTGCTGTGCCTTTACCAATTTGACCAT TCTCGCGCTGGGAATAAAGGACTCGAACCTTTGCATCTAGCAGTCAAAGTGCTATGCCTTACCAACTTGG CTAATTCCCAATTATTAACAAAGGCTCTCTAACAAGAACCCTTGATGATAGAGGGTATTAATCAGTGCGG TATGAGTTAATAATAACAAATAATTCTTAAAGCATATTTACCATTTATGATGATACGTATTTACGATACA TTCAAGACCCAAAGGATTCTTGAAAATATCATATTCAAGAGGACCTTTTTCTGTTTCAATAAAGAAATCA AAATTTACTGTATTAAATTTACGGTCTTCCTTTACTAATTTAACTTGAGAAGATGAACGATCAATGTAAA CCTTTTCAACTTCAAAACACGTTAAAATGCCATAATCATCAATCAAGGCTTTAGCTGCTTCTTGATCATA TTTATATCCATTTTCAACGGATGATACTTTCGCATAAAGAATCATCATCAACCTCTATCAACAATAGCAT GAGTATGGGCATTTACGATTTGCCACCAGTCGAAACGATTGGAACCATAATCTGGTTTATTTTCATTTTC TTTAATGATATCACGCAGTTTATCTTCTGTTTCAGCATACGCAATTAAATCATCATATCCACCACAAGGA TAATAATTATCACCTGCGAATAAAAGAAAATTTACCTTAGATGGATTTACGTAATAATGGTCTTTAGGAT ATTTAGTTCCTCTCCAATCAGTTACTTCAACATAACGGTAAGAAAATCCATTTTTACTTTCAATCCAACT CCACGCTTCAAAAGGAGTATTAAAAACTTTATCAGGTATTAAATTACCTTCAAAATGAGAAGGATTTGCA TAATCCCCGGCATAAACATAATATTCGTTAATACTCATTTATTCACCTTTAGAAATTTTATCCATAACGA TAGCAATTAAACCAATTAAAAATGCTACTACAAGTGAAAACACATTTTCTGCTGTAGTTAATAATCCGCA TATAAATCCAACAAACATTGAAAAACTAAAAGCAGAAGCAGAAATTGCAATAGCAACATTTCGAATTAAT TCACAGCGTTTCATTTTATTCTCCTCAGTAGTTGATAGGGTAATAGTATCACAGCTAAAACCCTATGTAA ACAACTTTGTGAAATATTTATTACAAAAGATTTTTAGCAATAATCTTGAGATGTGCCGCAGAAATGTGTT TAGCTTTAAACAACGCAGTTTCTTCAGCAGGAGAGATAACGATTGTAGCACCATCCTTTTTAGCAGACCA CCCATCACCTAGGTAAACAGTACCTTTGATTTCTTCGCCATCAACCAGACTAATCATTGGTTTACCTTCT CGTCCTTTATTTGCTTTAATAACTTCAGAAGTAAGAGTAGCTTCGGTAATGGTAGAAACCGGGGTAGTTG TAGAAGTAAATTCTTTAAATGTTTTCATTTTTATTTTCCTAATTAATTTTGATGAGGTAATAGTATCACT ACCTCATCAGTATGTAAACAACTTTGTGAAATTATTTTAAATCATCTGCCCAATCGAGTTTAAGAGGCTC TTTGTATTCACGATCTAATACGACCGGAATTTGTACATCACCGCTAAATGATAAGGGCCCAACATTATAA GACAATGTTATAATGCGGTGTGTAATCATCAAAATCATGTGTAGCACCTAGTGCCCGCGCATACATGTGTC GACAGCGCAGATATTCAGAATCTAGCACAAGTACAAGAGTCGATCCATCTTGTGTTTCCATACTTCTAA ATGTCCAGAAGAAGCTACTTCAAAACTTCCACTCGATGGAACATATGGAACATTTACTCTTGAATAACAT ATAGTCGAATGAATTTTTTCTCTAGGAACTGGATTAGGAACACGTAAAGAGCGCTGAAGTTCTTCCAGCG CATCAAGTGTTAATTCTGAAACTTAGCTGCTACATAAAGACCCGTTGAAAAGTCTTTAAATTCCATCAT TCTTCATCTTTTGCTTCATCTGCAGATTCAGCAGTAAGATTTTTGACAGCTTCAACGATTTCTTCAACTT TGATAGTATCGCCAGTGATACCTACTGCACGAGCAATTTCAGCCAAAGTTCCTTGCAGATTTTGGATTC TTCCATCAGACGAGCAGCTTGATCCTGCGTATCAAGAATGCGAGATTTCAGAGTTACGATTTCAGCAGAC AGTTTTTGTTCAACAGTTTGTTCAGACATTATAGTACCTTTAGTGTATTTTTAATTTTTAGAAAAAGTTC TTCAAGAGAACCATCGTTTGTAATTACTAAATCGCCATCACGAATTGGCAATCCAGCTTCTGTAATATGT GTATCATTGGATTTTTGACCAGGACGAACTACATGAATTACTGTAGCACCCATCGCCCTAGCCGCATCCA TTTCATGATCTTGACGGGTATCAGGAACGATAATAATCATAACCTGAGTTAATTTATCAAGATATC TAAAGCAAATTTTACCCAGTACATGCGGTCGAAGTTATTAACAATCAAATCCGTACCTAGGGCTTGC ATCAGACGACGGACTGACCATTGATCTTCAATATTATTTATAACGTCAGTAATCTTATTAAATGCTACGA AATTAACTGATTCTTTTCCTTCGTCATCAAAAACAAACACCTTTAATTGGGCTTTTACCATTAAGATA ACAAAATGCTTGTTCCATAATCGTGATTACTTCTAATTTAGTCAGATTTAAATTAGTCTCACGATCATAG TCAATTCCTTCAAACTCTTTACGAGTTAAGCAAGGATAGTCAGTGTTTGCTGCAAATACTCCCCATGCAT AAGCCAATGCATCCTTAATAGGACCAGCAAGTTGGTATTTAACTGCAGAATATTGCTCATGATAAAATC AGCAGTAGTATCTTTTCCACTACGCTTTACACCGCTTAAAAAGATTAGTTTCATGTGTTTCTCCTCAAAT TTAATTAAGATTAACACACAAACTGAAGCATTAAACTTCTGCTATAATTTTACCATCTTTTTCTACT TGAAAATAGGTGTAAGGAATTGTTGCAGTACATACTAAAGCCGGGTCTGAATCTTCCGTGTAGCTAAATT CTACTTCAGATAGGTCAGAAACCCAAGGCTTATAAAAATTTATTGACATCACGATTTCAGTTTTGCTATT ATCTAAGATGTAAAGCGTAATGTACTCAGGACCTGTTTTTTGGGCAGTATTTTCACCTGTAAGATAGTTG CTAGTTCCTAGCATCCATTCATACATTCCTATCCACGACTTAAGTTCTTCATCAACTATAAATCTCACAA TGAGTGGATCATACTCAAATGTAACACCTGGACGTTGTGCTCGGCCCAGTCCAAACGGCCCAGTCACGGT ATCAGTAACAGGTATTCTAATTCCTGGAATAGGAACTGACTGAGCATTTAAAGTAAAAGCAGATGTAGTA TTACTATGTGGTATTGATACTACAAAGTTAGTTGTATTTGCTTGGTTAAAAATTTGTTGCAGAGCTTGCG ACATATATTCCTCATAATGCTTTATAACTGTTGGTGGTATAATGGGTCTAAGTCCCTTCCATTCAATTCC ATTTAGAACAAACAACAGAAAAGAATGGAAGATAATAGAATTAGATATTTGACCAGACTTTGTTTGCAGA GAAACGTTTTCCTTTTGAAACGAACTGCTGAAGTGGCATCAACACAACGTTCGCCCAGTCTTTCGGGGCG ATTTCAACAAGGCTACCCATAATATTACCAGGTATATATGCCTTAATCATTTGGTCTGCACCCCTAAATC CTTTCACTTGACTCCAATCAATTTTTAATTTCGTTTTATTAGTAATAGTAGGTGTATTTGCATATTGCTT TAAAAGCTCTTCTAGGAATTGCTGACGAGCTTTAGGTGGAATATAGTGCAAGTTTAATCCGTACATTAAA TTATGCTTACCTAAACCAAGGTAAATTATCAAAGGAAATTTATCCCAGTAAGGAAGAGTTTCCTTGTGTT TAGCATCATAAGCAAAAGCATATATTCGTCCCGGCTGCGGGCGAACAACTTTATGTCCTTTTACTTGCTT AATAGTTTCAGCAAACCACTTTCTGGTTTTATTATTAATTGCTGCGCCTTCATTACGAATTTTATTCACGC AATGTTTGTCTGAATGAATTTATCATAAGCAGTTGTCTTTCTTGCTTATTGAGTTTATTCATTGGTTTTG ATTCAAGTTTTTGAATCTTTTCAGCCGTTTTAATTCCTGAAGCATATTTTGACATTGCTGAAGTAAACGT AGAGTATTTGATTCCTCTTTCTTCAGCAAATTGCTTTCCTGTCATTCCTTTTGCTTTGGCCTTTTTATAT TCAAGACCTATCTGAATCCATTTCTTTTCGTTTAATGATTGCTTAACCTTTGGAACTTGGGGAGTGCTT CATTAATTATTTGAAAAATAGCCATTATGCCCCCTTAAAGCCAAGAGCTCGTAATCCATCTTCTGTTAGA ATTCTAAATTTTATTCCACGCTTTTCAGCTAAAGATTGTGCTGCTTTCCATTTGTCAGTGTTCACAGACC AGGTATAAATTTCATTCATAAATCTTTTCTTCGCTGCGGTCGTTAGATGTGCTGGTTTAACTGGTGGTTG TGTTTCTTTTTTAGGTTTTATTTCAATAAAAAAATTCTTGTCCAGAAGAATCTTTCATCCAAATATCCATG AAGTATCTACGTTTTTTCCCTTCTGCATTACAAAATAAGGAATTACTGCTGTTTCACTACCCCATGCAA TAATTTCTGGATTTTTATCTAACCATTCAAAAAAGAATTTTTCCCAATTTGATCTATACGTAATTTTTTT AGGGTCACCTCTATACTTTGATATATTTTTAGGAACCCATTTTCCAGAATATGCCATTGGATTCTCCTTA TAAATAGATAATATATTTATAAACAGGAGGGCCCATGCTCTTTACATTTTTTGATCCGATTGAATATGCG GCCAAAACGGTGAATAAAAACGCGCCGACTATTCCTATGACAGATATTTTTAGAAACTATAAAGACTATT TTAAACGCGCTCTTGCGGGATACCGCTTACGTACTTATTATATTAAAGGTTCACCACGCCCGGAAGAATT AGCAAATGCTATATATGGAAATCCACAGCTGTATTGGGTTTTATTGATGTGTAATGATAATTATGACCCG TATTATGGATGGATTACTTCGCAAGAAGCTGCTTATCAAGCATCTATACAAAAATACAAAAACGTAGGTG GAGACCAAATAGTATATCATGTGAATGAGAACGGTGAAAAATTTTATAATTTAATATCATACGATGATAA TCCATATGTTTGGTATGATAAAGGCGATAAAGCTAGAAAATATCCTCAATATGAAGGAGCGCTTGCTGCG GTCGATACGTATGAAGCTGCTGTTCTTGAAAATGAAAAACTTCGTCAAATAAAAATAATAGCAAAATCAG ACATCAATTCATTTATGAACGACCTTATACGTATAATGGAGAAATCTTATGGAAATGATAAGTAATAACC TTAATTGGTTTGTCGGTGTTGTTGAAGATAGAATGGACCCATTAAAATTAGGTCGTGTTCGTGTTCGTGT GGTTGGTCTGCATCCACCTCAAAGAGCACAAGGTGATGTAATGGGTATTCCAACTGAAAAATTACCATGG ATGTCAGTTATTCAACCTATAACTTCTGCAGCAATGTCTGGAATTGGAGGTTCTGTTACTGGACCAGTAG AAGGAACTAGAGTTTATGGTCATTTTTTAGACAAATGGAAAACTAATGGAATTGTCCTTGGCACGTATGG TGGAATAGTTCGCGAAAAACCGAATAGACTTGAAGGATTTTCTGACCCAACTGGGCAGTATCCTAGACGT TTAGGAAATGATACTAACGTACTAAACCAAGGTGGAGAAGTAGGATATGATTCGTCTTCTAACGTTATCC AAGATAGTAACTTAGACACCGCAATAAATCCCGATGATAGACCGCTATCAGAGATTCCGACCGATGATAA TCCAAATATGTCAATGGCTGAAATGCTTCGCCGTGATGAAGGATTAAGATTAAAAGTTTATTGGGATACC GAAGGATATCCGACAATTGGTATTGGTCATCTTATCATGAAGCAGCCAGTTCGTGATATGGCTCAAATTA ATAAAGTTTTATCAAAACAAGTTGGTCGTGAAATTACAGGAAATCCAGGTTCTATTACAATGGAAGAGGC GACGACTTTATTTGAGCGTGATTTGGCTGATATGCAACGGGACATTAAATCACATTCTAAAGTAGGACCA GTCTGGCAAGCTGTCAACCGTTCTCGTCAAATGGCGTTAGAAAATATGGCATTTCAGATGGGTGTTGGTG GTGTAGCTAAATTTAACACAATGTTAACTGCTATGTTAGCAGGAGATTGGGAAAAAGCGTATAAAGCCGG TCGTGATTCATTGTGGTATCAACAAAAAAAGGCCGTGCATCCCGTGTTACCATGATTATTCTTTACGGGG AATTTGGAATCATATGGTGTTGAAGTGAAACCCCAGCTAGGTCTCTATCAGCAATGGCTGCTACTGTAG CTAAATCTTCTGACCCTGCTGACCCTCCTATTCCAAATGACTCGAGAATTTTATTCAAAGAACCAGTTTC TTCATATAAAGGTGAATATCCTTATGTGCATACAATGGAAACTGAAAGCGGACATATTCAGGAATTTGAT GATACCCCTGGGCAAGAACGATATAGATTAGTTCATCCAACTGGAACTTATGAAGAAGTATCACCATCAG GAAGAAGAACAAGAAAAACTGTTGATAATTTGTATGATATAACCAATGCTGATGGTAATTTTTTGGTAGC CGGTGATAAAAAAGACTAACGTCGGTGGTTCAGAAATTTATTATAACATGGATAATCGTTTACATCAAATC GATGGAAGCAATACATTTGTACGTGGAGACGAAACGAAAACTGTTGAAGGTAATGGAACTATCCTAG TTAAAGGTAATGTTACTATTATAGTTGAAGGTAATGCTGACATTCAGTTAAAGGAGATGCTACCACTTT AGTTGAAGGAAATCAAACTAAACAGTAAATGGAAATCTTTCTTGGAAAGTTGCCGGGACAGTTGATTGG GATGTCGGTGGTGATTGGACAGAAAAATGGCATCTATGAGTTCTATTTCATCTGGTCAATACACAATTG ATGGATCGAGGATTGACATTGGCTAATATACTTCCAATGAGCGCTGATTTAGGAGAATCCATGGAAGGTT CTTCTATCGACGTCACCTTTACCGCTCAATTAGAAACAGGTGAAACGTTAGTATCTATAAATATAACTAG TTACGAAGAAACTCCTGGGGTTTTAGTAGAAGAAAATCGCTTATATGGAACATATGAATCTGTATTTGGT TTCGGAAATGACGCGTTGAAATATCGTTTAGGCGATGAATTTAAAACTGCTGCTTCATGGGAAGAACTTC CTACTGATTCTGATACTCAGTTGTATTTGTGGAAAGCTCCTCAAAACCTCCAGAAGACATTCACTTACGA AGTAACATTAATATATGACTACCAAGAACAAAGTGAATCTGGGGGTTCTGGCAGTAATTCTAGGTCATCT TCTGATACTACTGAACCGACAGATCCTCCTGCTCCAGTAAGAAAAACTCTAGTTAAAAATTATACTAAAA CTATAGTTGGAAATTGGAGTCGTTGGGCTAATAAACTGAGAAAATATGCCTATGCAAGACCATAAATATT TTTATTTGTATTCAATAACTAATAAAACAACAGAAAAAATTTATGTAGGCGTCCACAAAACTTCAAATTT GGATGATGGGTATATGGGTTCTGGCGTTGCCATTAAAAATGCCATTAAAAAAATATGGCATAGATAATTTT TATAAGCATATTATAAAAATTCTTTGAATCTGAAAAAGCTATGTATGACGCAGAGGCAGAAATAGTCACAG AGGAATTTGTTAAATCTAAGAAAACTTATAATATGAAACTAGGCGGTATCGGTGGCTTCCCAAAACATAA CACAGCGGGTGCTAAAATGGATTTTACGGTAAATCTCATTCGCGTGAAACTAGATTGAAAATTAGCATT AAATCGTCTAGAAAAAGAGGGCCTAGAGGGCTAGAGGTAAAACTCTGAAGATGTGTGGCGCCAATAACCC AAGGTATGGCAAAATAGCCCCTAATGCTAAATCTGTTATTATCAACGGCGTTTTATATAAGGTATTAAA ATCGCAGCTAAAGCTCTTAATATAAATTATAGTACCTTAAAGGGGCGAGTTAAAGCGGGGTATTATAAAT GTCAGGATTAAGTTATGATAAGTGTGTTACTGCTGGCCATGAAGCGTGGCCTCCAACAGTTGTGAATGCT ACACAAAGTAAAGTATTCACTGGAGGAATTGCTGTTCTCGTAGCAGGCGATCCAATTACAGAACATACAG AAATTAAAAAGCCGTATGAAACACATGGCGGAGTGACACAACCTAGAACTTCTAAGGTATATGTCACTGG AAAGAAAGCTGTTCAAATGGCTGATCCAATATCATGCGGTGATACTGTGGCTCAGGCATCATCTAAAGTA TTCATTAAATAGGATTTAATTGGCAAATACCCCTGTAAATTATCAATTAACAAGAACAGCAAATGCTAT TCCCGAGATATTCGTCGGGGGTACATTTGCTGAAATAAAACAAAACCTCATTGAATGGCTTAATGGCCAA AATGAATTTTTGGATTATGATTTTGAAGGCTCAAGATTAAACGTTCTGTGTGACCTTTTAGCTTATAATA CATTATACATTCAGCAGTTTGGTAATGCTGCTGTGTATGAAAGCTTTATGCGTACTGCTAACTTACGAAG TTCAGTTGTTCAAGCTGCACAAGATAACGGATATTTACCTACTTCAAAATCCGCTGCGCAGACCGAATT ATGTTAACATGCACTGACGCATTGAATAGGAATTACATTACTATTCCTCGCGGAACTCGCTTTTTAGCAT ATGCAAAAGATACTTCTGTTAATCCATATAACTTCGTTTCTAGGGAAGACGTTATTGCTATTCGTGATAA AAATAACCAATATTTTCCGCGTTTAAAATTGGCCCAGGGACGTATAGTAAGAACTGAAATCATTTATGAT AAATTAACACCTATTATCATTTATGATAAAAATATTGATAGAAACCAGGTTAAATTATACGTTGATGGAG CGGAATGGATTAACTGGACGAGAAAGTCAATGGTTCATGCTGGTTCAACATCAACGATTTACTATATGCG TGAAACTATTGATGGAAACACTGAATTCTATTTTGGTGAAGGTGAAATTTCTGTTAATGCTTCTGAAGGA GCTTTGACCGCTAATTATATCGGAGGTCTTAAACCTACTCAGAACTCTACGATTGTTATTGAGTACATTA GTACTAATGGTGCTGACGCGAACGGAGCAGTCGGATTTTCATACGCAGATACATTAACAAATATAACTGT CATCAATATTAATGAAAATCCAAACGATGATCCAGATTTTGTTGGGGCAGATGGAGGCGGTGATCCAGAA GATATTGAGCGTATTCGCGAATTGGGTACTATTAAACGCGAAACCCAACAACGATGCGTAACTGCGACTG ACTATGATACATTCGTTTCAGAGAGATTTGGTTCTATTATTCAAGCTGTTCAGACTTTCACTGATTCTAC TAAACCTGGGTATGCATTTATTGCTGCTAAACCTAAATCAGGATTGTATTTAACTACCGTACAGCGTGAA GATATTAAAAATTATCTCAAAGACTATAATTTAGCTCCTATTACGCCATCAATTATTCTCCTAATTATTC TTTTTATTAAAGACTAATTTAAAAGTCACATATGCTTTAAATAAACTGCAAGAATCCGAACAGTGGCTTGA AGGTCAAATAATTGATAAAATAGATCGCTTATTATACCGAAGATGTAGAAATTTTTAACTCGTCTTTCGCT AAATCTAAGATGTTGACATATGTAGATGATGCAGATCATTCTGTCATGGTTCATCAGCGACTATTCAAA TGGTTCGTGAAGTCACAAAACTTCTATAAAACGCCTGAAGCGGGTATTAAATACAATAATCAAATAAAGA TCGTTCTATGGAATCTAATACGTTTTCATTTAATTCTGGACGAAAGGTTGTAAATCCTGATACTGGTTTA GAAGAAGATGTATTATATGACGTTCGTATAGTATCAACAGACCGAGATTCTAAAGGAATTGGTAAAGTTA TTATTGGTCCATTTGCTTCTGGCGATGTTACAGAAATGAAAACATTCAGCCTATACAGGCAACGATTT TAACAAATTAGCAAATTCTGATGGACGCGACAAATACTATGTTATCGGTGAAATAAATTATCCAGCTGAT GTGATTTATTGGAATATCGCTAAAATTTAATTTAACATCTGAAAAAATTGAAGTTCAGACCATTGAATTAT ATTCTGACCCAACCGATGATGTTATCTTTACTCGCGATGGTTCACTGATTGTATTGAAAATGACTTACG TCCACAATACTTAACTATCGATTTGGAGCCTATATCCACAATGACAGTAAAAGCACCTTCAGTCACTAGTC TCAGAATTTCCAAGTTATCCGCAAATCAGGTGCAAGTACGCTGGGATGACGTTGGTGCTAATTTCTACTA TTTTGTAGAAATCGCTGAGACAAAACAAACTCGGGGGAAAATCTCCCGAGTAATCAATATCGTTGGATT AATTTAGGATATACAGCAAATAATAGTTTCTTTTTTGATGATGCTGATCCATTAACAACATACATTATTA GAGTAGCCACAGCTGCGCAAGATTTTGAGCAGTCTGATTGGATTTATACCGAAGAGTTTGAAACTTTTGC TACAAATGCTTATACATTTCAAAACATGATTGAAATGCAATTAGCCAATAAATTCATTCAGGAAAAATTT ACTCTTAATAATTCTGATTATGTTAATTTTAATAATGATACTATAATGGCTGCATTGATGAATGAATCAT TCCAATTCAGCCCATCGTATGTTGATGTTTCATCAATAAGTAATTTTATTATTGGTGAAAATGAGTATCA TGAAATACAAGGTTCTATTCAGCAAGTATGTAAGGATATTAACCGAGTTTATTTGATGGAATCAGAAGGA ATTCTATATCTTTTTGAGCGCTATCAACCTGTAGTTAAAGTATCCAATGATAAAGGACAAACCTGGAAAG CTGTAAAGCTCTTCAATGACCGTGTAGGATATCCTTTATCTAAGACAGTATATTACCAATCTGCGAACAC AACATACGTCTAGGATACGACAAGATTTTCTATGGCCGCAAATCTACTGATGTTAGATGGTCAGCCGAT GATGTCAGATTTAGTTCTCAGGATATAACATTTGCTAAACTTGGCGACCAATTACATCTAGGATTTGATG TAGAAATTTTTTGCCACTTACGCGACTTTACCAAGCGAATGTATACCGCATTGCAGAAGCTATTACTTGCAC CGATGATTACATTTACGTTGTCGCCAGAGACAAAGTTAGATACATAAAAACGAGTAATGCACTTATAGAT TTTGATCCATTATCTCCAACATATTCGGAAAGACTTTTTGAACCTGATACCATGACTATAACCGGAAATC CTAAAGCAGTATGCTATAAAATGGATTCTATCTGTGATAAAGTTTTTGCTCTTATTATTGGTGAAGTTGA AACATTAAATGCTAATCCTAGAACATCAAAAATAATTGATTCCGCTGATAAAGGAATATATGTTTTAAAT CATGACGAAAAAACATGGAAGAAGGTTTTTGGTAATACCGAAGAAGAAAGAAGACGTATTCAACCCGGAT ATGCGAATATGTCAACTGACGGTAAATTAGTTTCTCTGTCTTCGAGTAATTTTAAATTTTTAAGTGATAA TGTTGTTAATGACCCTGAAACTGCAGCAAAATATCAGTTAATTGGCGCTGTTAAATATGAATTTCCTCGT GAATGGTTAGCTGATAAGCATTATCATATGATGGCATTTATAGCGGATGAAACATCTGATTGGGAGACTT TTACTCCTCAACCAATGAAATACTACGCAGAACCATTCTTTAACTGGTCTAAAAAATCTAACACACGTTG TTGGATAAACAACTCTGATAGAGCTGTGGTAGTTTATGCTGATTTAAAATACACTAAAGTTATAGAAAAT ATTCCGGAAACATCACCAGATAGATTAGTTCATGAATACTGGGATGATGGTGATTGCACTATAGTAATGC CAAATGTCAAATTCACTGGATTTAAAAAATACGCATCAGGAATGCTTTTCTATAAAGCCTCCGGTGAAAT AATTTCTTACTATGATTTTAACTATCGTGTGAGAGATACAGTAGAAATTATTTGGAAGCCAACTGAAGTA TTTTTAAAAGCATTTTTACAAAACCAAGAGCATGAGACTCCTTGGTCACCAGAAGAAGAGCGTGGATTAG CTGACCCTGATTTAAGACCATTAATTGGCACAATGATGCCTGATTCTTATTGTTACAGGATTCGAATTT TGAGGCATTTTGCGAAGCATATATTCAGTATCTTTCTGATGGATATGGAACTCAATACAATAATTTACGA AATTTAATTCGTAACCAATATCCACGAGAAGAGCACGCATGGGAATATTTGTGGTCAGAGATATATAAAAA GAAACATTTATTTAAATGCTGATAAACGCGATGCTGTTGCGAGATTCTTTGAATCACGTAGCTATGATTT TTATTCTACTAAAGGAATTGAAGCATCATACAAGTTTCTTTTTAAAGTTCTTTATAATGAAGAAGTTGAA ATTGAAATTGAATCTGGGGCTGGTACTGAATATGATATAATCGTTCAATCTGATTCTTTGACTGAAGATT TAGTAGGACAAACGATTTATACGGCAACAGGAAGATGTAATGTTACTTATATAGAAAGAAGCTATTCTAA TGGTAAATTGCAATGGACCGTAACTATTCATAATCTTTTGGGACGATTAATTGCTGGTCAAGAAGTTAAA GCAGAAAGACTCCCTAGTTTTGAAGGCGAAAATTATTCGTGGGGTTAAAGGAAAGGATTTGCTTCAAAACA ATATAGACTATATTAATAGAAGTAGATCATACTATGTAATGAAAATTAAATCCAATTTACCTTCTTCCCG CTGGAAATCTGACGTTATTCGTTTTGTTCATCCAGTAGGATTTGGATTTATAGCAATTACCCTTTTAACA ATGTTTATTAATGTTGGTTTAACTCTTAAACATACAGAGACTATAATTAATAAATACAAAAACTATAAAT GGGATTCTGGATTGCCTACTGAATATGCCGACAGAATAGCTAAATTAACTCCAACCGGTGAAATTGAGCA TGATTCAGTAACAGGCGAAGCAATTTATGAGCCTGGCCCAATGGCTGGTGTAAAATATCCTCTTCCTGAT GACTATAATGCTGAAAATAATAATTCAATATTTCAAGGTCAATTGCCGTCTGAACGACGTAAATTAATGA GTCCTTTATTTGATGCATCTGGAACAACATTTGCGCAATTTAGAGATTTAGTTAATAAACGTCTAAAAGA TAATATAGGAAATCCAAGAGACCCTGAAAATCCAACACAGGTTAAAATAGATGAATGATTCAAGTGTTAT CTATCGTGCGATAGTTACTTCAAAATTTAGAACAGAAAAAATGTTGAATTTTTATAATTCAATTGGAAGT GGTCCGGATAAAAACACTATCTTTATCACATTTGGAAGATCAGAACCGTGGTCATCAAATGAAAATGAGG TGGGCTTTGCCCCACCTTATCCAACCGATTCTGTATTAGGCGTAACTGACATGTGGACGCATATGATGGG AACAGTAAAAGTTCTTCCATCAATGCTTGATGCTGTTATTCCTCGCAGAGATTGGGGAGATACTAGATAT CCGGATCCATACACATTTAGAATTAACGATATTGTAGTGTGTAACTCAGCTCCTTACAACGCTACTGAAT CAGGCGCTGGTTGGTTAGTGTATCGTTGTTTAGATGTTCCTGATACCGGAATGTGTTCAATAGCATCTTT AACTGATAAAGATGAATGCCTTAAGTTAGGTGGAAAATGGACTCCTTCTGCTAGGTCAATGACTCCGCCT GAAGGTCGAGGAGATGCTGAAGGAACAATTGAACCCGGAGACGGGTATGTGTGGGAATATCTATTTGAGA TTCCGCCTGATGTATCTATAAATAGATGCACGAATGAATATATCGTGGTTCCTTGGCCTGAGGAATTAAA AGAAGACCCGACTAGATGGGGATATGAAGATAATCTCACTTGGCAACAAGATGATTTTGGATTAATTTAC CGTGTTAAGGCAAATACTATCCGTTTTAAAGCATATTTAGATTCAGTTTATTTTCCTGAAGCTGCATTAC CAGGAAATAAAGGATTTAGACAAATATCAATAATCACGAATCCTCTTGAAGCTAAAGCTCATCCAAATGA CCCAAACGTTAAAGCTGAAAAGGATTATTATGACCCAGAAGATTTAATGAGGCATTCGGGTGAAATGATT TATATGGAAAATAGGCCACCTATTATTATGGCAATGGATCAAACAGAAGAAATCAATATTCTGTTTACAT TTTAAATTAAGGGAGCCCATGGGCTCCCTTTTTCTTTATAAATACTATAAACTCATAAGGAAACCGCTAT GTTCATTCAAGAACCAAAGAAATTGATTGATACCGGCGAAATTGGTAACGCTTCTACTGGTGATATCTTA TTCGACGGTGGTAATAAAATTAATAGTGATTTTAACGCAATTTATAATGCGTTTGGCGATCAGCGTAAAA TGGCAGTAGCAAATGGCACTGGAGCAGATGGTCAAATTATCCATGCTACTGGATATTATCAAACAACTC TATTACAGAGTACGCAACTCCAGTGAAAGTTGGCACTAGACATGATATTGATACCTCTACTGTAGGTGTT AAAGTTATCATTGAAAGAGGCGAACTCGGCGATTGTGTTGAATTCATTAACTCTAATGGATCAATATCAG TTACTAATCCTTTGACAATTCAAGCTATTGATTCAATTAAAGGTGTTTCAGGTAATTTAGTAGTAACTAG CCCATATAGTAAAGTTACTTTACGCTGTATTTCATCTGATAATTCTACGTCGGTTTGGAATTATTCTATT GAAAGTATGTTTGGACAAAGGAATCACCAGCTGAAGGTACATGGAATATTTCTACATCTGGATCAGTTG ACATTCCATTATTTCATCGTACTGAATACAATATGGCTAAATTGCTAGTTACGTGCCAATCGGTAGATGG AAGAAAAATTAAAACAGCAGAAATAAATATTCTTGTGGATACTGTTAATTCAGAGGTAATTTCTTCTGAA TATGCTGTCATGCGAGTTGGGAATGAAACCGAAGAAGACGAAATCGCTAATATTGCATTTAGTATTAAAG AAAATTATGTAACGGCGACTATAAGTTCTTCAACTGTCGGTATGAGAGCAGCAGTTAAAGTTATCGCTAC GCAGAAAATCGGGGTGGCTCAATAATGAAACAAAATATTAATATCGGTAATGTTGTAGATGATGGTACCG GTGACTACCTGCGTAAAGGTGGTATAAAAATAAATGAAAACTTTGATGAGCTTTATTATGAACTCGGTGA TGGTGATGTTCCATATTCAGCCGGTGCCTGGAAAACTTATAATGCTTCATCAGGACAAACATTAACAGCA GAATGGGGGAAATCATACGCTATTAATACATCTTCTGGAAGAGTGACTATAAATCTTCCAAAGGGTACAG TTAATGATTACAACAAGGTAATTAGAGCTAGAGACGTATTTGCTACATGGAACGTCAACCCAGTTACACT AGTAGCTGCTTCCGGCGATACGATTAAAGGGTCTGCAGTACCAGTTGAAATTAATGTTCGATTCAGCGAT TTAGAACTAGTGTATTGTGCCCCAGGACGTTGGGAATATGTCAAAAATAAACAAATTGACAAAATTACCA GTTCAGACATTAGTAATGTAGCTCGCAAAGAATTTTTAGTTGAAGTTCAAGGACAAACAGACTTTTTAGA TGTTTTCCGTGGAACTAGTTATAATGTAAATAACATCAGAGTAAAACATCGTGGTAACGAATTGTATTAC GGCGATGTGTTTAGCGAAAACAGCGATTTTGGCTCTCCAGGCGAAAATGAAGGAGAACTGGTTCCTCTTG ATGGATTTAACATTCGATTAAGACAGCCTTGTAATATTGGTGACACTGTTCAAATTGAAACATTTATGGA TGGTGTATCACAGTGGAGAAGTTCATATACAAGACGTCAAATTAGATTGTTAGATTCAAAATTAACGTCA AAAACTTCTTTAGAAGGAAGCATTTACGTTACTGATTTATCAACAATGAAATCAATTCCATTTTCTGCTT TTGGATTAATTCCAGGAGAACCTATTAATCCTAACTCTCTTGAGGTTCGTTTTAACGGGATTTTACAGGA ATTGGCTGGCACAGTTGGAATGCCATTATTTCATTGTGTTGGTGCCGATTCAGACGATGAAGTAGAATGC TCTGTTTTAGGTGGAACTTGGGAACAATCTCATACCGATTATTCAGTTGAAACTGATGAAAACGGCATAC CAGAAATTTTACATTTCGATAGCGTATTTGAGCATGGTGACATTATCAATATCACCTGGTTTAATAATGA TTTGGGTACATTATTAACAAAAGATGAGATTATTGATGAAACTGATAATCTCTATGTATCGCAAGGACCT GGAGTAGATATTTCTGGTGATGTAAATTTAACAGACTTCGATAAAATTGGTTGGCCAAATGTAGAAGCAG TTCAATCTTATCAACGCGCATTTAATGCTGTTTCAAATATCTTTGATACGATTTATCCTATTGGAACTAT ATATGAAAACGCTGTTAATCCAAATAACCCTGTTACATATATGGGATTCGGCTCATGGAAATTATTTGGG CAAGGAAAAGTTTTAGTTGGATGGAATGAAGATATTTCGGACCCTAACTTTGCTCTAAATAACAACGATT TAGATTCGGGTGGAAATCCTTCACATACCGCAGGTGGAACAGGTGGTTCTACTTCTGTTACATTGGAAAA TGCTAATCTTCCTGCAACTGAAACAGATGAAGAAGTTCTAATAGTTGATGAAAATGGATCAGTCATTGTT GGTGGGTGTCAATACGATCCAGATGAATCCGGTCCAATTTACACTAAATACCGTGAAGCTAAAGCATCTA CTAACTCTACTCACACTCCGCCAACATCAATAACTAACATTCAACCATATATTACAGTTTATCGTTGGAT AAGGATTGCATAATGAGTTTACTTAATAATAAAGCGGGAGTTATTTCCCGCTTAGCCGATTTTCTTGGTT TTAGACCTAAAACTGGCGACATTGATGTAATGAATCGTCAATCAGTCGGGTCAGTGACAATATCTCAATT AGCGAAAGGATTTTATGAACCAAACATAGAATCAGCTATTAATGACGTTCATAATTTTTCTATAAAAGAC GTTGGCACAATTATTACTAATAAAACTGGTGTTTCTCCTGAGGGTGTTTCTCAAACTGATTATTGGGCAT TTTCTGGAACTGTAACAGACGATTCTCTTCCTCCGGGTTCTCCTATTACGGTATTAGTATTTGGTCTTCC AGTTTCAGCAACAACTGGAATGACGGCAATTGAGTTTGTTGCAAAAGTTCGCGTTGCACTACAAGAAGCT ATTGCGTCATTTACTGCTATCAATTCATATAAAGACCATCCAACTGATGGTAGTAAATTAGAAGTTACTT ATTTAGATAATCAAAAACATGTATTAAGCACATATTCTACATATGGAATAACTATTTCCCAAGAAATTAT ATCTGAGTCTAAGCCTGGCTATGGTACATGGAATTTATTGGGCGCACAAACTGTAACTTTAGATAATCAG CAGACTCCTACAGTATTTTATCATTTTGAGAGAACAGCATGAGTAATAATACATATCAACACGTTTCTAA TGAATCTCGTTATGTAAAATTTGATCCTACCGATACGAATTTTCCACCGGAGATTACTGATGTTCACGCT GCTATAGCAGCCATTTCTCCTGCTGGAGTAAATGGAGTTCCTGATGCATCGTCAACAACAAAGGGAATTC TATTTATTCCCACTGAACAGGAAGTTATAGATGGAACTAATAATACCAAAGCAGTTACACCAGCAACGTT GGCAACAAGATTATCTTATCCAAATGCAACTGAAACTGTTTACGGATTAACAAGATATTCAACCAATGAT GAAGCCATTGCCGGAGTTAATAATGAATCTTCTATAACTCCAGCTAAATTTACTGTCGCCCTTAATAATG CGTTTGAAACGCGAGTTTCAACTGAATCCTCAAATGGTGTTATTAAAATTTCATCTCTACCGCAAGCATT AGCTGGTGCAGATGATACTACTGCAATGACTCCATTAAAAACACAGCAGTTAGCTATTAAATTAATTGCG CAAATTGCTCCTTCTGAAACCACAGCTACCGAATCGGACCAAGGTGTTGTTCAATTAGCAACAGTAGCGC AGGTTCGTCAGGGAACTTTAAGAGAAGGCTATGCAATTTCTCCTTATACGTTTATGAATTCATCTTCTAC TGAAGAATATAAAGGCGTAATTAAATTAGGAACACAATCAGAAGTTAACTCGAATAATGCTTCTGTTGCG GTTACTGGCGCAACTCTTAATGGTCGTGGTTCTACGACGTCAATGAGAGGCGTAGTTAAATTAACTACAA CCGCCGGTTCACAGAGTGGAGGCGATGCTTCATCAGCCTTAGCTTGGAATGCTGACGTTATCCAGCAAAG AGGTGGTCAAATTATCTATGGAACACTCCGCATTGAAGACACATTTACAATAGCTAATGGTGGAGCAAAT ATTACGGGTACCGTCAGAATGACTGGCGGTTATATTCAAGGTAACCGCATCGTAACACAAAATGAAATTG ATAGAACTATTCCTGTCGGAGCTATTATGATGTGGGCCGCTGATAGTCTTCCTAGTGATGCTTGGCGCTT CTGCCATGGTGGAACTGTTTCAGCGTCAGATTGTCCATTATATGCTTCTAGAATTGGAACAAGATATGGC GGAAACCCATCAAATCCTGGATTGCCTGACATGCGTGGTCTTTTGTTCGTGGTTCTGGTCGTGGTTTCTC ACTTAACAAATCCAAATGTTAATGGTAATGACCAATTTGGTAAACCTAGATTAGGTGTAGGTTGTACCGG TGGATATGTTGGTGAAGTACAGATACAACAGATGTCTTATCATAAACATGCTGGTGGATTTGGTGAGCAT GATGATCTGGGGGCATTCGGTAATACCCGTAGATCAAATTTTGTTGGTACACGTAAAGGACTTGACTGGG ATAACCGTTCATACTTCACCAATGACGGATATGAAATTGACCCAGAATCACAACGAAATTCCAAATATAC ATTAAATCGTCCTGAATTAATTGGAAATGAAACACGTCCATGGAAACTTTCTTTAAACTACATAATTAAG GTAAAAGAATGACAGATATTGTACTGAATGACTTACCATTCGTTGACGGCCCTCCTGCAGAGGGCCAGAG CCGCATTTCCTGGATTAAAAACGGCGAAGAAATATTAGGAGCTGACACACAGTATGGAAGTGAAGGCTCA ATGAATAGAACCTACGGTTTCTGTACTAAGAAATGTTGAAGTTCTTGATAAAAACATTGGAATACTTAAAAA CATCTTTAGAAACCGCAAATAGTGATATTAAAACAATTCAGGGCATCTTAGATGTATCTGGTGATATTGA AGCTTTGGCCCAAATAGGTATCAATAAAGGATATTTCTGACCTCAAAACGCTAACCAGTGAACACACA GAAATATTAAATGGAACTAATAATACGGTTGACAGTATTCTTGCCGATATTGGTCCATTTAACGCCGAGG CCAACTCTGTATACAGAACGATCAGAAATGATTTACTGTGGATAAAGCGTGAACTTGGACAATACACTGG TCAAGATATTAATGGTCTTCCTGTTGTAGGAAATCCTAGTAGTGGAATGAAGCATCGCATTATTAATAAT ACTGATGTCATCACTTCGCAGGGAATACGTTTAAGCGAATTAGAAACAAAATTTATTGAATCTGATGTAG GTTCTTTGACCATTGAAGTTGGTAATCTTCGTGAAGAGCTTGGACCGAAACCACCATCATTTTCACAGAA CGTTTATAGTCGTTTAAATGAAATTGACACTAAACAGACAACAGTTGAGTCTGACATTAGTGCTATTAAG ACCTCAATAGGATATCCAGGAAATAATTCGATTATCACGAGTGTTAATACAAACACTGATAATATTGCAT CTATTAATTTAGAGCTAAATCAAAGTGGAGGTATTAAACAGCGTTTAACCGTTTATTGAAACTTCCATTGG TTCAGATGATATTCCTTCGAGTATTAAAGGTCAAATCAAAGATAATACAACTTCAATCGAATCTCTAAAT GGAATCGTCGGTGAAAACACTTCATCTGGCTTAAGAGCGAATGTTTCATGGTTAAACCAAATTGTTGGAA CTGATTCTAGCGGTGGACAACCTTCTCCTCCTGGGTCTCTTTAAACCGAGTTTCTACAATTGAAACTTC TGTTTCAGGCTTGAATAACGCTGTTCAAAACCTACAAGTAGAGATTGGTAATAACAGCGCAGGAATTAAAA GGGCAAGTTGTAGCGTTAAATACTTTAGTAAATGGAACTAATCCAAACGGTTCAACTGTTGAAGAGCGCG GATTAACCAATTCAATAAAAGCTAACGAAACTAACATTGCATCAGTTACACAAGAAGTGAATACAGCTAA AGGCAATATATCTTCTTTTACAAGGTGATGTTCAAGCTCTCCAAGAAGCCGGTTATATTCCTGAAGCTCCA AGAGATGGGCAAGCTTACGTTCGTAAAGATGGCGAATGGGTATTCCTTTCTACCTTTTTATCACCAGCAT AACATGGGGCCGCAAGGCCCCCAAAGGATTTTAAATGTCAGGATATAATCCTCAGAATCCAAAGGAACTCA AAGATGTCATTCTAAGACGTTTAGGGGCTCCAATTATTAATGTTGAGTTAACACCCGATCAAATTTACGA TTGTATCCAGCGTGCCCTAGATTATACGGTGAATACCATTTTGATGGACTCAATAAAGGTTTTCATGTT TTTTATGTAGGGGATGATGAAGAAGGTACAAGACCGGAGTCTTCGATTTAAGAGGTTCTAACGTATTTG CAGTAACCGCATTTTACGCACAAATATTGGGTCAATAACATCTATGGATGGAAACGCTACATACCATCCGTG GTTTACTGACTTTCTTTTAGGAATGGCTGGTATTAATGGCGGAATGGGAACGTCTTGTAATAGATTTTAT GGACCAAATGCCTTTGGAGCTGATTTAGGATATTTTACCCACAGCTTACCAGTTATATGGGAATGATGCAAG ATATGCTCTCTCCTATTCCAGACTTTTGGTTTAATTCAGCAAATGAACAGCTCAAAGTCCATGGGAAACTT CCAAAAATATGATTTAATTATCGTAGAAAGCTGGACTAAATCATACATTGATACAAACAAAATGGTTGGA AATACAGTAGGATATGGAACAGTCGGTCCACAAGATAGCTGGTCATTATCTGAACGATATAATAACCCAG ACCACAATTTAGTAGGTCGTGTTGTCGGCCAAGATCCGAATGTTAAACAGGGTGCTTATAATAATCGTTG GGTGAAAGACTATGCAACAGCTTTAGCTAAAGAATTGAACGGTCAAATTTTAGCACGCCACCAAGGTATG ATGCTTCCGGGCGGTGTTACAATTGATGGGCAGCGCTTAATATAGAAGAAGCCAGATTAGAAAAAGAAGCAC TGCGCGAAGAATTACTTTACTTGATCCTCCATTTGGAATTTTGGTAGGTTAATATGGCTACTTATGATA AAAATCTTTTTGCTAAATTGGGAAAACCGCACAGGTTATTCTCAGACCAATGAAACTGAAATATTAAATCC TTATGTAAATTTCCAATCATTATAAAAAACAGCCAAATATTAGCTGATGTATTAGTAGCTGAAAGCATTCAA ATGCGAGGTGTAGAATGCTATTATGTTCCAAGAGAGTATGTTTCCCCTGATTTGATATTCGGCGAAGACT TAAAAAATAAATTTACTAAAGCTTGGAAATTTGCTGCATATTTAAATTCATTTGAAGGATATGAAGGAGC TAAATCGTTCTTTAGTAACTTTGGTATGCAAGTACAAGACGAAGTGACTTTATCTTAACCCAAATTTTA TTTAAGCATCAAGTTAACGGAAAAGAACCCAAGGAAGGTGATTTGATATATTTTCCTATGGATAACAGCT TATTTGAAATTAACTGGGTTGAACCATATGATCCATTTTCAATTAGGCCAAAACGCTATTCGTAAAAT TACGGCAGGTAAATTCATTTTATTCTGGAGAAGAAATTAATCCAGTTCTACAGAAAAATGAAGGAATTAAC ATTCCAGAATTTAGTGAATTAGAATTAATGCTGTTCGCAATCTTAACGGTATTCATGACATTAATATTG ATCAGTATGCTGAAGTAGATCAAATTAATTCTGAAGCTAAAGAATACGTTGAACCTATGTTGTTGTCAA TAACAGAGGCAAATCTTTCGAATCTAGCCCATTTGACAATGATTTCATGGATTAATAAATATTAACT AATTAAAGCCCGGATTAGGAGAAGTCATGTTTGGTTATTTTTATAATTCGTCTTTTAGACGATATGCTAC CTTGATGGGCGATTTGTTTTCAAATATCCAAATCAAACGTCAGTTAGAATGGTGATAAGTTTATACGT GTTCCTATTACGTATGCATCAAAGGAACACTTTGATGAAATTGAATAAATGGACATCAATAAATTCAC AAGAAGATGTAGCTAAAGTTGAAACTATTCTACCTCGTATAAATTTACATTTAGTTGATTTTAGCTATAA TGCTCCATTTAAAACAAACATTTAATCAGAATTTACTGCAAAAAGGTGCAACTTCTGTAGTATCGCAG TATAATCCATCTCCTATTAAAATGATTTATGAATTGAGTATCTTTACTCGCTATGAAGATGATATGTTTC AAATAGTTGAACAGATTCTTCCATATTTTCAACCTCATTTTAATACAACTATGTACGAGCAGTTTGGAAA TGATATTCCATTTAAAAGGGATATTAAAATTGTACTGATGTCTGCTGCTATAGACGAAGCTATAGATGGG GATAATTTATCTCGTCGTAGAATTGAATGGTCATTAACATTTGAAGTAAATGGATGGATGTATCCTCCAG TAGATGATGCAGAAGGATTAATTCGTACTACTTATACAGATTTTCACGCCAATACAAGAGATTTGCCTGA CGGCGAAGGTGTTTTTGAATCTGTCGATAGCGAAGTTGTTCCTCGAGATATTGACCCAGAAGACTGGGAT GGAACAGTAAAACAAACTTTCACTAGTAATGTAAATAGACCAACACCGCCAGAACCTCCTGGCCCAAGAA CATAGAGGTTATTATGGAAGGTCTTGATATAAACAAACTTTTAGATATTTCTGACCTCCCCGGAATTGAC GGGGAGGAAATCAAAGTGTATGAACCTCTGCAATTAGTAGAAGTTAAAAGCAATCCACAAAACCGTACTC CAGACTTAGAAGATGATTATGGAGTAGTTCGTCGAAATATGCATTTTCAGCAACAAATGCTAATGGACGC TGCCAAGATTTTTCTTGAGACAGCAAAGAATGCTGATTCTCCTCGTCACATGGAAGTATTTGCAACTCTT ATGGGGCAAATGACTACGACGAACAGAGAAATACTGAAGCTTCATAAAGATATGAAAGACATTACATCTG AGCAGGTTGGCACCAAAGGCGCTGTTCCTACAGGTCAAATGAATATTCAGAATGCGACAGTATTCATGGG TTCACCAACAGAATTAATGGACGAAATTGGTGATGCTTACGAGGCTCAAGAAGCTCGTGAGAAGGTGATA AATGGAACAACCGATTAATGTATTAAATGATTTCCATCCGTTAAATGAAGCTGGAAAAATTTTAATAAAA CACCCAAGCTTAGCGGAAAGAAAAGATGAAGATGGAATTCATTGGATAAAATCTCAGTGGGATGGAAAAT GGTATCCTGAAAAATTCAGTGATTACCTTCGTCTACACAAAATAGTAAAAATTCCAAACAACTCTGATAA GCCTGAATTATTTCAAACTTATAAAGATAAGAATAATAAAAGATCTCGGTATATGGGTCTTCCTAACTTG AAACGAGCTAATATTAAAACACAATGGACTCGTGAAATGGTTGAGGAATGGAAAAAATGCCGAGATGATA TTGTTTATTTTGCAGAAACATACTGTGCCATTACTCATATTGACTATGGTGTCATAAAGGTTCAATTACG TGACTATCAGCGTGATATGCTCAAAATAATGTCATCTAAACGTATGACTGTTTGTAATCTATCGCGCCAG CTCGGTAAAACCACCGTAGTAGCTATTTTCCTTGCACACTTTGTATGTTTTAACAAAGATAAAGCTGTAG GTATTCTTGCACACAAAGGCTCAATGTCTGCGGAAGTTTTAGACCGTACTAAGCAAGCAATTGAACTGCT TCCTGACTTTTTACAACCAGGAATTGTTGAATGGAATAAGGGTTCAATTGAACTAGATAATGGTTCTTCA ATTGGCGCTTATGCTTCCTCTCCTGACGCAGTTCGTGGTAACTCGTTCGCAATGATTTACATTGACGAAT GTGCGTTTATTCCAAACTTCCATGATTCCTGGCTTGCTATTCAACCAGTAATTTCATCTGGTCGTCGTTC GAAAATTATTATTACTACGACTCCTAATGGATTAAATCATTTTTATGATATTTGGACTGCTGCTGTCGAA GGTAAATCTGGATTTGAACCATATACTGCTATTTGGAATTCAGTTAAAGAACGTCTTTATAACGATGAAG ATATTTTTGACGATGGATGGCAATGGAGCATACAAACCATTAATGGTTCTTCATTAGCTCAATTCCGTCA AGAACATACTGCAGCGTTTGAAGGGACTTCTGGTACATTAATTTCAGGAATGAAATTAGCTGTTATGGAT TTTATTGAAGTAACACCAGATGATCATGGTTTTCACCAATTTAAAACCTGAACCAGATAGAAAATATA TTGCAACTCTAGATTGCTCAGAAGGTCGTGGGCAAGATTACCACGCTTTGCATATTATTGATGTTACTGA TGATGTGTGGGAACAGGTTGGTGTTTTGCATTCAAACACTATTTCTCATTTAATTCTACCTGACATCGTT ATGCGTTATTTAGTAGAATACAATGAATGCCCAGTTTATATTGAATTAAATAGTACTGGTGTGTCAGTTG CAAAATCGCTTTATATGGATTTAGAATACGAAGGTGTTATCTGCGATTCATATACTGATTTAGGAATGAA ACAAACTAAACGCACGAAAGCAGTAGGATGTTCCACGCTAAAAGACCTTATTGAAAGATAAGCTTATT ATTCATCACCGAGCGACTATTCAAGAATTTAGAACGTTTAGTGAAAAAGGCGTGTCTTGGGCGGCTGAAG AAGGTTATCATGACGATTTAGTAATGTCTTTAGTGATTTTTGGATGGTTATCAACGCAGTCAAAATTTAT TGATTATGCGGATAAAGATGACATGCGATTAGCATCTGAAGTATTTTTCAAGAGGCTTCAGGATATGAGC GACGACTACGCGCCAGTTATATTTGTGGATTCGGTTCATTCTGCTGAGTATGTTCCAGTATCTCATGGTA TGTCAATGGTATAAATATATTAAAGCATATTAAAGAGGATTAAAAATGACTTTATTATCTCCGGGCATTG AGCTCAAAGAAACTACGGTTCAAAGCACCGTAGTTAATAACTCTACTGGTACAGCAGCTTTGGCCGGTAA ATTCCAGTGGGGTCCTGCTTTTCAGATTAAACAGGTTACAAATGAAGTAGATTTAGTTAATACTTTTGGT CAACCAACCGCTGAAACTGCTGACTATTTTATGTTCTGCGATGAATTTCTTGCAGTACGGAAATGACTTAC GAGTAGTTCGTGCTGTTGATAGAGATACCGCTAAAAACTCATCGCCAATTGCTGGTAATATTGATTACAC AATTTCTACCCCAGGTAGTAACTATGCGGTTGGAGATAAAATCACGGTCAAATATGTTTCAGATGATATT GAAACTGAAGGTAAAATTACTGAAGTAGACGCAGATGGAAAAATTAAGAAAATTAATATTCCTACTGGCA AAAATTACGCTAAAGCGAAAGAAGTCGGTGAATATCCAACACTAGGTTCTAACTGGACTGCGGAAATTTC TTCATCTTCCTCTGGTTTAGCTGCAGTAATAACTCTTGGAAAAATTATTACTGATTCTGGTATTTTATTA GCTGAAATTGAAATGCTGAAGCTGCTATGACAGCGGTTGACTTTCAAGCAAATCTTAAAAAATATGGAA TTCCAGGAGTAGTAGCGCTTTATCCAGGCGAATTAGGCGATAAAATTGAAATTGAAATCGTATCTAAAGC TGACTATGCAAAAGGAGCTTCTGCATTACTCCCAATTTATCCAGGTGGTGGTACTCGTGCATCTACTGCT AAAGCAGTGTTTGGATATGGACCGCAAACTGATTCACAGTACGCTATTATAGTTCGTCGTAATGATGCTA TTGTTCAAAGCGTTGTTCTTTCAACTAAGCGTGGTGAAAAAGATATTTACGATAGTAACATCTATATCGA TGACTTTTTCGCAAAAGGTGGTTCAGAATATATTTTTGCAACTGCACAAAACTGGCCAGAAGGCTTCTCT GGAATTTTAACTCTGTCTGGTGGATTATCATCAAATGCTGAAGTAACAGCAGGAGATTTGATGGAAGCTT GGGACTTCTTTGCTGACCGTGAATCTGTTGACGTTCAACTGTTTATTGCAGGTTCTTGTGCCGGTGAATC TTTAGAAACAGCATCTACTGTCCAAACACGTCGTTTCAATTGGGGATGCTCGCCAAGATTGCTTAGTA TTGTGCTCTCCTCCGCGTGAAACTGTAGTTGGAATTCCTGTAACCCGTGCTGTTGATAACCTAGTCAATT GGAGAACTGCGGCAGGTTCATACACTGATAATAACTTTAATATCAGTTCAACCTATGCAGCAATTGATGG TAACCATAAGTATCAGTATGACAAATATAATGATGTGAATCGTTGGGTTCCATTAGCAGCTGATATTGCT GGTTTATGCGCAAGAACTGATAACGTATCTCAGACTTGGATGTCTCCAGCTGGTTATAATCGTGGTCAGA TTCTTAACGTTATTAAACTTGCTATTGAAACTCGCCAGGCTCAGCGCGACCGTTTATACCAAGAAGCTAT CAACCCGGTAACCGGTACAGGTGGTGATGGTTACGTATTGTATGGTGATAAAACAGCTACTTCTGTTCCT TCTCCATTTGATCGTATAACGTTCGTCGTCTGTTTAATATGTTGAAAACGAATATCGGACGTAGTTCAA AATATCGTTTGTTCGAATTAAACAACGCGTTTACTCGTTCATCATTCCGCACAGAAACTGCCCAGTACTT ACAAGGGAATAAAGCTCTCGGTGGAATTTATGAATATCGTGTAGTTTGCGATACAACAAATAACACTCCG TCAGTAATTGATAGAAATGAGTTTGTTGCAACATTCTACATCCAACCGGCTAGAAGCATTAACTACATTA CCTTAAACTTCGTAGCAACTGCTACTGGTGCAGATTTCGATGAGTTAACTGGTCTTGCTGGTTAATACGG TGCATTCTAAAGGCCTGTTTCGGCAGGCCATATAAATACACTATATCCTTAATTCTTTAATTCTATATGC CCTAGGTTAAACATAGGGATATAAATACTACAGAGGCTAATATGTTTGTAGATGATGTAACACGAGCGTT TGAATCTGGTGATTTTGCTCGACCTAACTTATTCCAAGTAGAAATTTCTTATCTTGGACAAAATTTTACG TTCCAATGTAAAGCTACTGCTCTACCAGCTGGTATTGTAGAAAAAATTCCAGTCGGATTTATGAACCGTA AAATTAACGTAGCAGGCGATCGTACATTCGATGACTGGACTGTTACAGTAATGAACGATGAAGCTCATGA TGCTCGTCAGAAGTTCGTTGATTGGCAAAGCATTGCTGCGGGGCAAGGAAACGAAATTACTGGTGGAAAA CCTGCAGAGTATAAAGAGGCGCTATCGTTCGTCAATATGCTCGTGACGCTAAAACAGTAACAAAAGAAA TTGAAAATTAAAGGTCTGTGGCCTACTAACGTGGGTGAACTTCAATTAGATTGGGATTCAAACAATGAAAT CCAAACTTTTGAAGTAACTCTTGCTCTCGATTATTGGGAATAAAATGAATGGGGAGAAATCCCCATCCTG CTTAAAGCAGAGAAGTCCATTATAAATATAACTATAATTCCCATTTGGAGAATACAATGAAATTTAATGT ATTAAGTTTGTTTGCTCCATGGGCTAAAATGGACGAACGAAATTTTAAAGACCAAGAAAAAGAAGATCTT GTTTCCATTACAGCCCCAAAGCTTGATGATGGAGCAAGAGAATTTGAAGTAAGCTCGAATGAAGCTGCTT CTCCTTATAATGCTGCATTCCAAACAATTTTTGGTTCATATGAACCAGGAATGAAAACTACTCGTGAGCT TATTGATACATATCGTAATCTCATGAATAACTATGAAGTAGATAATGCAGTTTCAGAATCGTTTCAGAT GCTATCGTCTATGAAGATGATACTGAAGTCGTAGCGTTAAATTTGGATAAATCTAAATTTAGCCCAAA TTAAAAATATGATGTTAGATGAATTTAGTGATGTATTAAATCATCTATCGTTTCAACGAAAAGGTTCTGA TCATTTTAGACGTTGGTATGTTGATTCAAGAATTTTCTTTCATAAAATCATTGATCCAAAACGTCCAAAA GAAGGCATAAAAGAATTACGTAGATTAGACCCTCGCCAAGTTCAGTATGTTCGTGAAATTATAACAGAAA CTGAAGCTGGCACAAAAATAGTTAAAGGTTACAAAGAATATTTTATATATGATACTGCCCATGAGTCATA TGCATGTGATGGTAGAATGTATGAAGCTGGCACAAAAATAAAAATTCCTAAAGCTGCCGTCGTTTATGCC CATTCTGGATTAGTCGATTGTTGCGGTAAAAATATCATCGGGTATTTGCATCGTGCTGTTAAACCTGCTA ACCAATTAAAATTATTAGAAGATGCTGTAGTCATTTATCGCATTACTCGTGCTCCTGACCGTGTTTG GTATGTAGACACAGGTAATATGCCTGCTCGTAAAGCTGCTGAGCACATGCAACATGTTATGAACACGATG AAAAACCGTGTAGTATATGATGCATCAACAGGTAAAATAAAAAATCAACAGCATAATATGTCTATGACCG AAGACTATTGGTTGCAGCGCCGTGATGGTAAAGCTGTGACAGAAGTTGATACTCTTCCTGGTGCTGATAA TACTGGCAATATGGAAGATATTCGTTGGTTTAGACAAGCTCTTTATATGGCATTACGTGTTCCTCTTTCA CGCATTCCGCAAGACCAACAAGGCGGTGTGATGTTTGATTCTGGAACTAGCATTACACGTGATGAATTAA CGTTTGCTAAATTTATTCGTGAGTTACAGCACAAGTTTGAAGAAGTTTTCCTAGATCCGCTTAACAACAAA TCTTTTGCTTAAAGGTATAATCACAGAAGATGAGTGGAATGATGAAATAAATAATATTAAGATAGAATTT CATCGGGATAGCTACTTTGCTGAGCTCAAAGAAGCAGAAATTTTGGAACGAAGAATTAATATGCTAACCA TGGCAGAACCATTTATTGGTAAATATATTTCTCACAGAACTGCTATGAAAGACATTTTGCAGATGACTGA TGAAGAAATAGAACAAGAAGCCAAGCAAATTGAAGAAGAGTCTAAAGAGGCTCGTTTCCAAGACCCCGAC CAAGAACAAGAGGATTTTTAATGGAAGGTTTAATTGAAGCTATTAAATCAAACGACCTCGTAGCCGCTCG TAAATTATTTGCTGAAGCCATGGCTGCAAGAACGATTGATTTAATTAAAGAAGAAAAATCGCTATCGCT CGCAATTTCTTAATCGAAGGTGAAGAACCTGAAGACGAGGATGAAGATGAAGATGACGAAGATAGTGATG ATAAAGACGACAAAAAAGACGAAGACTCTGACGAAGACGAGGATGATGAATAATGCTTCTGATCCCTGAA ACTCATGAATTAGTTCTCGAGAATGTCGAAGCACTTATTCCTGAAGCACAGGGTCGCTTTGACGAATTGT CTTCTGCTTTAAATAAAGACGATATAAATACAATTGTCGAGAATATGCTTGATGATGAAACTGATTTAGC GGTTGCATTAGCTTCTATTAATGAAAATATGCCGTTAAATGAATTCATCGTTAAACATGTTTCTCGCCCGT GGGTGAATTACTCGCACTAAAGACCGCAAAACGCGTGAACGAAATGCATTTCAAACCACTGGGCTGTCTA AAGCAAAACGTAGACAAATTGCTCGTAAAGCTACCAAAACGAAGATTGCCAATCCAGCAGGTCAATCTCG TGCTCAGCGTAAGCGTAAAAAAGCTCTTAAACGCCGTAAAGCATTAGGATTAAGCTAATGAATGAACCCC AATTACTAATTGAAACTTGGGGTCAACCTGGCGAAATTATTGATGGCGTACCAATGCTTGAATCTCATGA TGGAAAAGACTTAGGTTTAAAACCGGGTTTATACATCGAAGGAATATTCATGCAAGCGGAAGTCGTCAAT AGAAATAAACGTCTTTATCCAAAAACGTATATTAGAAAAGCGGTAAAAGACTATATTAATGAGCAAGTTT TAACTAAACAAGCTCTCGGAGAATTAAATCATCCTCCACGCGCTAATGTTGACCCGATGCAAGCCGCTAT CATTATAGAAGATATGTGGTGGAAAGGAAATGACGTATACGGACGAGCTCGTGTTATTGAAGGTGACCAT GGTCCTGGAGATAAATTAGCAGCTAATATTCGTGCCGGATGGATTCCAGGAGTTTCTTCTCGTGGATTAG GTTCATTGACTGACACAAATGAAGGTTATCGTATCGTAAACGAAGGATTCAAATTAACTGTAGGTGTTGA TGCAGTATGGGGTCCAAGTGCTCCAGATGCATGGGTAACTCCTAAGGAAATTACCGAATCACAGAGACGGCG GAAGCCGATACAAGTGCCGATGACGCCTATATGGCTCTCGCAGAGGCCATGAAAAAAGCGTTATAAATAT TATTATCTAAACAACAGGACTACAAAATGCTTAAAGAACAACTGATTGCCGAAGCGCAGAAAATTGATGC TTCCGTTGCTCTTGATAGTATTTTCGAATCAGTTAATATTTCTCCGGAAGCAAAAGAAACTTTCGGCACT GTATTCGAAGCTACCGTCAAGCAGCACGCCGTTAAATTAGCTGAATCTCATATCGCTAAAATTGCTGAAA AAGCAGAAGAAGAAGTAGAAAAAAATAAAGAAGAAGCCGAAGAAAAAGCTGAGAAGAAAATCGCTGAGCA AGCTTCTAAATTCATTGACCATCTTGCAAAAGAATGGCTCGCTGAAAATAAATTAGCAGTTGATAAAGGC ATCAAAGCCGAACTGTTTGAATCCATGCTTGGTGGATTAAAAGAGCTCTTTGTTGAACACAACGTTGTTG TTCCAGAAGAATCAGTTGATGTTGTAGCTGAAATGGAAGAAGAGCTGCAAGAACATAAAGAAGAATCGCC TCGTCTGTTCGAAGAACTGAATATGCGCGACGCATATATCAATTATGTGCAGCGTGAAGTGGCATTGAGC GAAAGTACTAAAGATCTGACTGAGTCTCAAAAAGAAAAAGTCTCTGCTCTGGTCGAAGGTATGGATTATT CAGATGCATTCTCAAGTAAATTGAGTGCAATCGTAGAAATGGTGAAGAAATCTAATAAAGATGAAAGCAC TATTACTGAGAGTATAAATACTCCTGATACTGAAGCAGCCGGACTGAATTTCGTCACTGAAGCTGTAGAA GATAAAGCTGCACAGGGTGCAGAAGATATTGTAAGTGTATATGCGAAAGTCGCATCTCGTTTCTAATTTT AAAGGTTAACACAAATGACTATCAAAACTAAAGCTGAACTTTTGAACAAATGGAAGCCATTACTGGAAGG TGAAGGTTTACCGGAAATTGCTAATAGCAAACAAGCGATTATCGCTAAAATCTTTGAAAACCAGGAAAAA GATTTCCAGACAGCTCCGGAATATAAAGACGAAAAAATTGCTCAGGCATTCGGTTCTTTCTTAACAGAAG CTGAAATCGGTGGTGACCACGGTTACAATGCTACCAACATCGCTGCAGGTCAGACTTCTGGCGCAGTAAC TCAGATTGGCCCAGCTGTTATGGGTATGGTACGTCGTGCTATTCCTAACCTGATTGCTTTCGATATTTGT GGTGTTCAGCCGATGAACAGCCCGACTGGCCAGGTATTCGCACTGCGCGCAGTATATGGTAAAGACCCAG TGGCTGCCGGTGCTAAAGAAGCATTCCACCCAATGTATGGTCCAGATGCAATGTTCTCTGGTCAGGGTGC TGCTAAGAAATTCCCAGCTCTGGCTGCTAGCACACAAACCACAGTAGGTGATATCTATACTCACTTCTTC CAGGAAACTGGTACTGTATATCTGCAAGCTTCTGTTCAAGTAACAATCGATGCTGGTGCGACTGATGCTG CTAAATTAGATGCTGAAATTAAGAAACAAATGGAAGCTGGTGCACTGGTAGAAATCGCTGAAGGTATGGC TACTTCTATCGCTGAACTCCAGGAAGGTTTCAATGGTTCTACCGATAACCCATGGAATGAAATGGGCTTC CGTATCGATAAGCAAGTTATCGAAGCTAAATCTCGTCAGCTGAAAGCTGCTTACTCTATTGAATTAGCAC AAGACCTCCGCGCTGTTCACGGTATGGATGCTGATGCTGAACTGTCTGGTATTCTGGCTACAGAAATTAT GCTGGAAATCAACCGTGAAGTTGTTGATTGGATTAACTACTCAGCTCAGGTTGGTAAATCTGGTATGACC CTGACTCCGGGTTCTAAAGCTGGTGTATTTGACTTCCAGGACCCAATTGATATTCGTGGTGCTCGCTGGG CGGGTGAATCCTTTAAAGCTCTGTTGTTCCAGATTGACAAAGAAGCAGTTGAAATTGCTCGTCAGACCGG TCGTGGTGAAGGTAACTTCATTATCGCTTCCCGTAACGTAGTTAACGTTTTGGCTTCAGTTGATACCGGC ATTTCTTATGCTGCACAGGGTCTGGCTACCGGCTTTAGCACTGATACTACCAAGTCAGTATTTGCTGGTG TTCTGGGTGGTAAATACCGCGTATATATCGACCAGTATGCTAAACAGGATTATTTCACTGTAGGTTATAA AGGTCCGAACGAAATGGATGCTGGTATTTACTATGCTCCATATGTAGCTCTGACTCCGCTGCGTGGTTCC GATCCGAAGAACTTCCAACCGGTAATGGGATTCAAAACTCGTTACGGTATCGGTATCAACCCATTTGCAG AATCCGCTGCTCAGGCTCCGGCTTCTCGCATCCAGAGCGGTATGCCTTCTATTCTGAATAGCCTTGGTAA AAACGCTTACTTTAGACGTGTATATGTTAAAGGTATCTAATCTCTAACGATAGAAACACAATTTTAGGGA ACCTTCGGGTTCCCTTTTTTCTATTTTATACGATAGCAATCAGGCATATCATCCGCATTTATCCAATTGC GAATAGTTTTAGGACTAACTTTAAAATGCTCCGCTGCGTAATCAGGATTATCAAATTTAACGCCCTTTAT ACATATTGGAATAAATTTTTTAATACCACCAAGTTTTTCAGAAATAGCTTTACGATGTGAAATCGATATA GGTTTGTTTTTTCGTGGATGAACATGTGTTTTATAATATTCATTGCGCCCTTTAACTCGCTTCGCAATAG TTTCATCAGATTGCTTAACGCCTGTTTTTGCCTTTGATATTTTTCGTTTAGCTTCCACAGTCATTCCTTC TTTTGTTCGTATTGATAACATATTACGATATGAAGGATCTTGCAAATGAACTATAACTGGATTTCCTCTG CCACCAATAGCAGCATTATAGGTATCAGTTCTCATAACGAATTCCTCATTAACTAGTAAAGCTTCCATTT TATACATCTCCTCAGATGAGGAGAAAGAATAAAGAATTTCTTTTTTAAAGTTATGAATACCATATTTTTT GATGGATTTTTTGATGTTTACGCCAGAACCCATATAACCATCGTTTTCGTCAAGAGTAGCATGAGCTCCG ATGTAAATTTTTCCATTGATGATATTAGTAATTTGATATATTAAATATTTCATTTTAAACATCACTCCGT TTGTATATGATTATAATATCATATTACTTTGGTCTTGTAAATAACTTTATAAATAGTATTATATTTCAAC AAGGAAAATACAATGGCTAAAATCAACGAACTTCTGCGCGAATCAACCACAACGAATAGCAACTCAATCG GTCGCCCAAATCTCGTTGCTTTGACTCGCGCTACCACTAAATTAATATATTCTGACATTGTAGCAACGCA AAGAACTAATCAACCTGTTGCTGCTTTTTATGGTATCAAATACCTTAACCCAGACAACGAATTTACATTT AAAACTGGTGCTACTTACGCTGGCGAAGCTGGATATGTAGACCGAGAACAAATCACAGAATTAACAGAAG AGTCTAAATTAACTCTCAATAAAGGCGATTTATTCAAATATAATAATATCGTTTATAAAGTATTAGAAGA TACTCCATTTGCTACTATCGAAGAAAGTGATTTAGAATTAGCTCTTCAGATTGCAATCGTTCTTTTAAAG GTTCGTCTATTTTCTGACGCAGCGTCAACAAGCAAATTTGAAAGCTCTGATAGTGAAATTGCGGATGCTA GATTCCAGATTAATAAATGGCAAACTGCAGTTAAATCTCGTAAACTTAAAACTGGCATCACAGTTGAATT AGCGCAAGATTTAGAAGCAAATGGATTCGATGCTCCTAATTTCTTGGAAGATTTGCTTGCAACTGAAATG GCAGATGAAATCAATAAAGACATTCTGCAGTCTTTGATTACAGTGTCAAAACGCTATAAAGTTACAGGAA TTACTGATAGTGGATTCATCGATTTGAGTTATGCATCTGCTCCTGAAGCTGGTCGTTCATTATACCGAAT GGTATGTGAAATGGTTTCGCATATCCAAAAAGAATCAACTTATACAGCAACGTTCTGTGTTGCTTCAGCT CGTGCCGCTGCGATTCTTGCTGCATCAGGCTGGTTAAAACATAAACCAGAAGATGACAAATATCTTTCAC AAATGCCTACGGGTTCTTAGCTAATGGTTTACCGCTTTATTGCGATACTAACAGCCCATTAGATTATGT AATCGTTGGCGTAGTAGAAAATATCGGTGAAAAAGAAATTGTTGGATCAATTTTCTATGCTCCGTATACA GAAGGTCTCGACTTAGATGACCCTGAACATGTAGGTGCATTTAAAGTTGTTGTTGATCCAGAAAGCTTAC AACCATCTATCGGTTTATTAGTTAGATATGCTTTATCAGCAAATCCTTATACTGTAGCAAAAGATGAAAA AGAAGCAAGAATAATTGACGGTGGAGACATGGATAAAATGGCAGGTCGTTCAGATTTGTCTGTTTTATTA GGTGTTAAGCTACCAAAAATTATCATTGATGAATAAAACAAAGGGACCTTTCGGTCCCTTTTTATTTAAC TTACCAACTCAATCCAAGCTGGACGAAGTACATCTTGTACCATTTTAACTAATTCCTTTTTA...

Claims

1. A composition comprising a plurality of different types of transduction particles, each of said particles comprising a nucleic acid, said particles being capable of contacting E. coli cells and transferring said nucleic acid thereto; (a) the nucleic acid of each particle comprises a nucleotide sequence encoding a product of interest (POI), and the nucleic acid is capable of expressing the POI in an E. coli cell; (b) each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS and Tsx displayed on the surface of an E. coli cell; (c) The composition, wherein 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. A: each particle of said first type comprises an LPS adhesive moiety and a LamB adhesive moiety; B: each particle of said first type comprises an LPS adhesive moiety and a Tsx adhesive moiety; or C: The composition of claim 1, wherein each particle of the second type comprises a Tsx adhesive moiety but lacks a LamB and LPS adhesive moiety.

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

4. 4. The composition or method of claim 1, wherein each particle comprises a phage capsid containing the nucleic acid.

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

6. 6. The composition of any one of claims 1 to 5, wherein each particle is a phage (optionally a lytic phage) or a packaged phagemid.

7. 7. The composition of claim 1, wherein the composition comprises at least three or four different types of transduction particles.

8. 8. The composition of any one of claims 1 to 7, wherein the nucleic acid of each particle comprises at least one nucleotide sequence (N1) encoding the POI, and each particle is a synthetic T-even phage comprising an insertion of N1 into a modification-permissive region (MPR) of the genome of the phage, the MPR extending from immediately after gene 49 to gene E compared to a reference wild-type T2 phage.

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

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

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

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

13. The POI is (a) a nuclease for targeting the DNA of an E. coli cell, which can be expressed within the cell to cleave the DNA of the cell, thereby modifying or killing the cell; or (b) a dead Cas (dCas) for targeting the DNA of an E. coli cell, wherein the dCas can be expressed within the cell and target the DNA of the cell, thereby modifying the cell.

13. The composition of any one of claims 1 to 12, comprising:

14. The composition of claim 13 (a), wherein the nuclease is an inducible 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. 15. The composition of claim 13 or 14, wherein the nuclease is a Type I, II, III, IV, V or VI Cas nuclease, optionally Cas9 or Cas3.

16. 16. The composition of any one of claims 1 to 15, wherein the POI comprises at least one crRNA or guide RNA operable with Cas for DNA targeting in E. coli cells.

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

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

19. 20. 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. 20. The composition of any one of claims 1 to 19, wherein the nucleotide sequence encoding the POI comprises a stress phase active (SPA) promoter for expression of the POI in an E. coli cell, optionally wherein the promoter is an E. coli bolA promoter or comprises SEQ ID NO:

13.

21. 21. The composition of any one of claims 1 to 20, wherein the E coli cells comprise a strain of E coli that causes sepsis, septicemia, or diarrhea in humans or animals.

22. 22. The composition of any one of claims 1 to 21, wherein the composition is for use in a method of treating or preventing infection with E. coli cells in a human or animal subject, the method comprising administering the particles to the subject.

23. 23. A method of treating or preventing sepsis, septicemia or diarrhea in a human or animal subject, said method comprising administering to said subject a composition of any one of claims 1 to 22, wherein said E coli cells comprise a strain of E coli that causes sepsis, septicemia or diarrhea in humans or animals.

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

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

26. 26. The composition or method of claim 25, wherein the method is performed before the subject receives a transplant.

27. 27. A composition or method according to any one of claims 1 to 26 for preventing hemolytic uremic syndrome (HUS), UTI infection, sepsis, septicemia or diarrhea in a human subject.

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

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

01.

30. 30. The composition, method or dose of any one of claims 1 to 29, wherein the E coli cells comprise at least one strain that is antibiotic resistant or an MDR strain, and / or at least one B2-I strain.

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

32. (i) the composition comprises first and second types of particles according to claim 2A, claim 2B, and claim 2C; (ii) the POI comprises an inducible nuclease for targeting the DNA of an E. coli cell, wherein the nuclease can be expressed within the cell to cleave the DNA of the cell, thereby modifying or killing the cell; (iii) each particle comprises a phage capsid containing said nucleic acid; (iv) The composition, method or dose of any one of claims 1 to 31, wherein the particles of the composition target a plurality of different E coli genes, optionally selected from essential genes and virulence genes.

33. 1. A method for detecting the presence of E. coli (optionally phylogenetic group B2 E. coli cells) in a sample, comprising: (a) contacting the sample with the composition of any one of claims 1 to 32; (b) detecting that the E. coli cells have been killed or their growth or proliferation has been reduced; A method comprising:

34. 1. A method for detecting the presence of E. coli (optionally phylogenetic group B2 E. coli cells) in a sample, comprising: (a) contacting the sample with the composition of any one of claims 1 to 32, wherein particles of the composition comprise or comprise a nucleic acid encoding a detectable label, and wherein the particles contact cells and introduce the nucleic acid therein, and optionally the label is expressed in the cells; (b) detecting that the E. coli contains the label; A method comprising:

35. 33. A method for modifying the genome of an E. coli cell, the method comprising contacting the cell with the composition of any one of claims 1 to 32, wherein a nucleic acid encoding a POI is introduced into the cell, thereby modifying the genome of the cell.

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

37. At least 1 x 10 ７ 37. The method of any one of claims 33 to 36, wherein particles of PFUs contact the sample.

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

01.

39. 1. A composition comprising a plurality of transducing particles for use in a method of treating or preventing infection with E. coli cells in a human or animal subject, said method comprising administering said particles to said subject; (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, and the nuclease is expressed within the cell and cleaves the genomic DNA of the cell, thereby killing or reducing the growth or proliferation of the cell in the subject; (b) the E. coli cell is an E. coli lineage B2 cell; (c) each particle comprises an adhesive moiety for recognizing and binding to a cognate moiety selected from LPS, LamB, and Tsx displayed on the surface of said phylogenetic group B2 E. coli cells.

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

41. 41. The composition of claim 39 or 40, wherein each particle is a phage (optionally a lytic phage) or a packaged phagemid.

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

43. 43. The composition of any one of claims 39 to 42, wherein the composition comprises at least three or four different types of transduction particles, the types having different attachment sites for recognizing and binding to cognate sites contained in E. coli (optionally B2 lineage group E. coli) cells.

44. (i) the nuclease is an inducible nuclease for targeting the DNA of an E. coli cell, wherein the nuclease can be expressed within the cell to cleave the DNA of the cell, thereby killing the cell; (ii) each particle comprises a phage capsid containing said nucleic acid; (iii) the particles of the composition target a plurality of different E. coli genes, optionally selected from essential genes and virulence genes; (iv) optionally, the composition comprises: A: Particles containing an LPS adhesive moiety and a LamB adhesive moiety; B: Particles containing an LPS adhesive moiety and a Tsx adhesive moiety, or C:: Particles containing the Tsx adhesive moiety but lacking the LamB and LPS adhesive moieties 44. The composition of any one of claims 39 to 43, comprising:

45. 44. 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 operable with the Cas to target DNA in an E. coli cell.

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

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

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

49. 49. The composition of any one of claims 39 to 48, wherein the encoding nucleotide sequence comprises a stress phase active (SPA) promoter for expression of the nuclease in an E. coli cell, and optionally 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. 50. The composition of any one of claims 39 to 49, wherein the E coli cells comprise a strain of E coli that causes sepsis, septicemia, or diarrhea in humans or animals.

51. 51. The composition of any one of claims 39 to 50, wherein the composition is for use in a method of treating or preventing infection by E coli cells in a human or animal subject, the method comprising administering the particles to the subject.

52. 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 of any one of claims 39 to 51, wherein the E coli cells comprise a strain of E coli that causes sepsis, septicemia, or diarrhea in humans or animals.

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

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

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

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

57. At least 1 x 10 ７ 57. The composition or method of any one of claims 52 to 56, wherein PFU of particles are administered to the subject, or the dose is at least 1 x 10 ７ 28. A dose of the composition of any one of claims 1 to 22 and 25 to 27, wherein the dose is of the particles in PFU.

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

01.

59. 59. The composition, method or dose of any one of claims 39 to 58, wherein the E coli cells comprise at least one strain that is antibiotic resistant or an MDR strain, and / or at least one B2-I strain.

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

Images