Combination medicament comprising bacteriophage and vaccine against pathogenic bacteria

Abstract

The present invention relates to a combination therapy against pathogenic bacteria in the gut, wherein a bacteriophage against the capsule of the pathogenic bacterium is combined with a vaccination against a bacterial antigen which is normally masked by the capsule.

Description

[0001] Combination medicament comprising bacteriophage and vaccine against pathogenic bacteria

[0002] Field

[0003] The present invention relates to a combination therapy against pathogenic bacteria in the gut, wherein a bacteriophage against the capsule of the pathogenic bacterium is combined with a vaccination against a bacterial antigen which is normally masked by the capsule.

[0004]

[0005] The gut microbiota is a complex ecosystem of microbes that play an important role in human health and development. While many bacterial species provide direct health benefits to the host by liberating nutrients or maintaining immunological homeostasis, some are opportunistic pathogens and can cause life-threatening disease if spread to extraintestinal sites. Certain strains of Escherichia coli (E. coli) are harmless gut commensals but have the potential to cause systemic infections as they harbor specific virulence factors. One of the most relevant of these virulence factors is the K1 polysaccharide capsule. The K1 capsule is comprised of repeating units of a-(2-8)-linked sialic acid, forming membrane-anchored poly-sialic acid (PSA). PSA is also a post-translational modification of the host neural cell adhesion molecule, making the K1 capsule a self-antigen and thus poorly immunogenic. The capsular polysaccharides are long and mask underlying surface antigens, including the LPS O-antigen and outer membrane porins. In addition, the K1 capsule enables immune evasion by inhibiting phagocytosis and by blocking the activation cascade of the complement system, promoting survival in the bloodstream. The self-antigen-nature of this capsule type limits the possibility of using vaccination to prevent intestinal colonization by E. coli K1.

[0006] Neonates are at increased risk of developing sepsis or meningitis caused by E. coli K1, due to the vulnerable intestinal architecture of the neonatal gut, with low levels of protective mucus and decreased intestinal mobility and acid secretion. These factors facilitate intestinal colonization by pathogenic facultative aerobes, such as E. coli K1, which under favorable conditions can escape the intestinal barrier and cause neonatal sepsis. The maternal gut microbiota can be a reservoir for K1 E. coli, from which it subsequently can be vertically transmitted to the neonate during or after birth. Removing the encapsulated E. coli from the maternal gastrointestinal tract prior to birth could therefore decrease the risk of neonatal sepsis and meningitis development. However, administrating antibiotics during pregnancy can have negative consequences on the microbial diversity and developing immune system of the neonate. In contrast, bacteriophages (phages) targeting E. coli K1 often use the K1 capsule as a primary phage receptor, with endosialidase domains in the tail enabling precise targeting. One of the main concerns regarding phage therapy is the rapidly emerging resistance. Here we turn this selection to our advantage by targeting an E. coli virulence factor (Kortright et al., Cell Host & Microbe, 25, 2019). We show that oral treatment with lytic K1-specific phages can be used to steer rapid within-host evolution of capsule-less E. coli in the murine gut.

[0007] Without the protective capsule, the immunogenic surface antigens of E. coli are exposed and the ability to survive outside the gastrointestinal tract is reduced. By predicting the evolutionary trajectory of E. coli K1 under phage pressure, we can couple this directed approach to an oral vaccination scheme against the capsule-less strain, where high-affinity intestinal secretory IgA (S-lgA) is elicited to trap the phage escapers via enchained growth. To further increase the intestinal clearance of E. coli K1, we introduce the probiotic niche competitor E. coli Nissle to eliminate the targeted strain via vaccine-enhanced competition. This combined approach increases colonization resistance against E. coli K1 and can be applied to pregnant dams in a murine vertical transmission model to delay gut colonization of E. coli K1 in neonatal pups and thus subsequently reducing the risk of development of neonatal sepsis.

[0008] Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to treat bacterial infections of the gut without the need for antibiotics. This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification.

[0009] Summary of the Invention

[0010] A first aspect of the invention relates to a combination medicament comprising:

[0011] a) a bacteriophage capable of killing a pathogenic bacterium expressing a specific capsule serotype (targeting a specific capsule serotype of a pathogenic bacterium); b) a vaccination against said pathogenic bacterium.

[0012] Further aspects of the invention relate to a combination medicament according to the first aspect for use in treatment of a bacterial infection.

[0013] Terms and definitions

[0014] General

[0015] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control. The terms “comprising”, “having”, “containing”, and “including”, and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open-ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of’ or “consisting of.”

[0016] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0017] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

[0018] As used herein, including in the appended claims, the singular forms “a”, “or” and “the” include plural referents unless the context clearly dictates otherwise.

[0019] "And / or" where used herein is to be taken as specific recitation of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard techniques are used for molecular, genetic, and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

[0021] Any patent document cited herein shall be deemed incorporated by reference herein in its entirety. As used herein, the term treating or treatment of any disease or disorder (e.g. gut infection) refers in one embodiment to ameliorating the disease or disorder (e.g. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. Methods for assessing treatment and / or prevention of disease are generally known in the art, unless specifically described hereinbelow.

[0022] As used herein, the term pharmaceutical composition refers to a compound of the invention, or a pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the invention is provided in a form suitable for oral administration.

[0023] As used herein, the term pharmaceutically acceptable earner includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (for example, antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington: the Science and Practice of Pharmacy, ISBN 0857110624). The invention also encompasses nanoparticles, liposomes, or cellular carriers within the meaning of pharmaceutically acceptable carrier.

[0024] Bacterial definitions

[0025] The term bacteriophage in the context of the present specification relates to virus infecting and lysing bacterial cells. The bacteriophage of the invention attaches to a saccharide of the bacterial capsule, and following this interaction the genome of the bacteriophage enters the cell. The bacteriophage of the invention is a virulent bacteriophage which kills an infected bacterium via cell lysis.

[0026] The term pathogenic bacterium in the context of the present specification relates to a harmful, non-symbiotic bacterium which is able to infect a host organism. In certain embodiments, the host organism is a human. In certain embodiments, the host organism is a livestock animal. Infection of the pathogenic bacterium takes place in the gut, but the bacterium might also be able to infect other parts of the host’s body. The goal of the invention is to eradicate the infection with the pathogenic bacterium.

[0027] The term O-antigen in the context of the present specification relates to a polysaccharide composed of multiple oligosaccharide repeating units (O units) each with two to seven residues from a broad range of common or rare sugars and their derivatives, and the O-antigen is part of the lipopolysaccharide (LPS). The O antigen is one of the most variable cell constituents, due to variation in the sugars present in the O unit and the linkages within and between O units. The diversity allows the various clones in the species to each present a surface that offers a selective advantage in its specific niche, which probably accounts for the maintenance of O antigen diversity.

[0028] The term serotype in the context of the present specification relates to a classification system for bacteria, wherein the capsular antigen and the underlying antigen underneath the capsule are considered. Almost all species of Gram-negative bacteria produce a layer of lipopolysaccharide on the outer membrane. The outermost portion of the LPS accessible to antibodies is the O antigen. As an example, E. coli isolates produce two serotype-specific surface polysaccharides: the lipopolysaccharide (LPS) O antigen and capsular polysaccharide K antigen.

[0029] The term vaccination in the context of the present specification relates to the administration of a vaccine to help the immune system develop immunity against the vaccine. In this invention, the vaccine should impart immunization against the pathogenic bacterium and the vaccine is either an attenuated version of the pathogenic bacterium or the O-antigen (or another immunogenic component) of the pathogenic bacterium. The immunogenic part of the vaccine is masked by the capsule in the wildtype pathogenic bacterium. Only after the loss of the capsule, the pathogenic bacterium will be vulnerable to the immune response caused by the vaccine.

[0030] The term inactivated capsule mutant in the context of the present specification relates to a non-pathogenic version of a usually pathogenic bacterium which carries a mutation leading to the loss of its capsule.

[0031] The term probiotic bacterium in the context of the present specification relates to a bacterium which is not harmful to the host when colonizing the gut, but rather is a physiological occupant of the host’s gut. The probiotic bacterium of the invention occupies the same nice as the pathogenic bacterium of the invention, thus, they are competitors for the same nice.

[0032] Detailed Description of the Invention

[0033] The idea of the present invention is that a pathogenic bacterium may be targeted via a bacteriophage which uses the bacterial capsule to attach before infecting the cell. The bacterium very likely evades the lysis by the bacteriophage via a mutation leading to not building the capsule anymore. That is why the invention combines the bacteriophage treatment with a vaccination targeting a bacterial antigen which is normally masked by the capsule, but exposed in the capsule-mutants. With the combination treatment, there is a much higher efficiency in treatment of the bacterial infection. This efficiency may be increased even more by co-administering a probiotic bacterium which occupies a close enough niche as the pathogenic bacterium.

[0034] A first aspect of the invention relates to a combination medicament comprising or consisting of:

[0035] a) a bacteriophage capable of killing a pathogenic bacterium expressing a specific capsule serotype (targeting a specific capsule serotype of a pathogenic bacterium); and

[0036] b) a vaccination against said pathogenic bacterium.

[0037] The bacteriophage is used synonymously with the term “bacteriophage component”. The vaccination is used synonymously with the term “vaccine component”.

[0038] The pathogenic strain is characterized by display on its surface of a bacterial wild type surface antigen.

[0039] In certain embodiments, the vaccine component comprises an inactivated, propagation incompetent vaccine strain of said bacterium. The inactivated strain displays said wild type surface displayed bacterial antigen; it induces immunity against the pathogen.

[0040] In certain embodiments, the vaccination is against an antigen underneath the capsule.

[0041] In certain embodiments, the medicament is formulated for oral administration. In certain embodiments, the medicament is a capsule comprising the vaccine, the phage, and optionally the probiotic bacterium. In certain embodiments, the medicament is an oral solution comprising the vaccine, the phage, and optionally the probiotic bacterium.

[0042] In certain embodiments, the capsule is an E. coli transporter-dependent capsular polysaccharide. In certain embodiments, the capsule is a K1 capsule.

[0043] In certain embodiments, the bacteriophage is of the order Caudovirales. In certain embodiments, the bacteriophage is of the family Autographviridae. In certain embodiments, the bacteriophage is a K1E- or a K1 F-like bacteriophage. In certain embodiments, a genome of the bacteriophage has at least (>) 85%, particularly > 90%, > 92%, > 95%, > 98%, > 99% or 100% identity to a genome of a bacteriophage of Genbank Accession number PQ476009, or PQ476011.

[0044] In certain embodiments, said bacterium is an Enterobacteriaceae. In certain embodiments, the pathogenic bacterium is an E. coli, Salmonella spp. or Klebsiella spp. bacterium. In certain embodiments, the pathogenic bacterium is an E. coli bacterium. In certain embodiments, the pathogenic bacterium is a Salmonella spp. bacterium. In certain embodiments, the pathogenic bacterium is a Klebsiella spp. bacterium.

[0045] In certain embodiments, the vaccination comprises an inactivated capsule mutant of said pathogenic bacterium. In certain embodiments, the vaccination comprises an O-antigen (as immunogenic bacterial surface component) of said pathogenic bacterium. In certain embodiments, the O-antigen is of serotype 0-75. In certain embodiments, the O-antigen is of serotype 0-2. In certain embodiments, the vaccination is an attenuated version of the pathogen. In certain embodiments, the vaccination is the O-antigen (or another immunogenic component) of the pathogen linked to virus-like particles. In certain embodiments, the vaccination is a (standard) protein carrier, particularly tetanus toxoid or diphtheria toxoid. In certain embodiments, the vaccination is an O-antigen glycoconjugate (i.e. the O-antigen covalently linked to a protein).

[0046] In certain embodiments, the combination medicament additionally comprises a probiotic bacterium. The probiotic bacterium is used synonymously with the term “probiotic component”, (probiotic competitor able to exclude said pathogenic bacterium) In certain embodiments, the combination medicament additionally comprises a bacterium of strain E. coli Nissle. In certain embodiments, the probiotic is of the same family as the pathogenic bacterium. In certain embodiments, the probiotic is of the family Enterobacteriaceae and the pathogenic bacterium is also of the family Enterobacteriaceae.

[0047] In certain embodiments, the probiotic component comprises bacteria of a live propagation competent, or at least gut-colonization competent, avirulent strain of said pathogen, wherein said avirulent strain displays a modified variant of said surface displayed wild type bacterial antigen. The variant is capable of escaping binding by vaccine-induced immunoglobulins capable of specifically recognizing the wild-type surface antigen and colonizes ecological niches inside the patient that otherwise might be occupied by mutant variants of the pathogen that manage to escape immunity induced by the vaccine component.

[0048] A further aspect of the invention relates to a combination medicament according to the first aspect for use in treatment of urinary tract infection.

[0049] A further aspect of the invention relates to a combination medicament according to the first aspect for use in treatment of Crohn’s disease or inflammatory bowel disease.

[0050] A further aspect of the invention relates to a combination medicament according to the first aspect for use in prevention of meningitis or sepsis in a newborn, wherein said combination medicament is administered to a pregnant woman. A further aspect of the invention relates to a combination medicament according to the first aspect for use in treatment of a bacterial infection in livestock.

[0051] In certain embodiments, the combination medicament is an alternative to conventional antibiotics. Thus, any gut infection with a bacterium having a capsule may be treated with the combination medicament of the invention, instead of antibiotic treatment. K1 E. coli cells are often resistant to antibiotics, which makes sepsis or meningitis difficult to treat. The invention prevents the transmission of K1 E. coli to patients at risk, i.e. , the neonates from their mother, thus reducing the need to treat sepsis or meningitis with antibiotics. Without the treatment there is a risk of transmission of K1 E. coli to neonates.

[0052] More broadly, the invention can be used to remove unwanted capsulated bacteria from the intestinal tract of patients or livestock animals without using antibiotics.

[0053] The skilled person is aware that the combination medicament may be administered in different forms and at different time points. Thus, the bacteriophage, the vaccination and the optional probiotic bacterium can either be administered all together, or one by one.

[0054] A further aspect of the invention relates to a bacteriophage capable of killing a pathogenic bacterium expressing a specific capsule serotype for administration to a patient who has received administration of a vaccination against said pathogenic bacterium.

[0055] A further aspect of the invention relates to a vaccination against a pathogenic bacterium for administration to a patient who has received administration of a bacteriophage capable of killing said pathogenic bacterium (expressing a specific capsule serotype).

[0056] The bacteriophage and the vaccination of these further aspects are described in more detail above.

[0057] Yet another aspect of the invention relates to a probiotic pharmaceutical preparation for use in protecting a patient from infection by, or disease caused by a pathogenic strain of a bacterial pathogen. The pharmaceutical preparation comprises or consists of:

[0058] a) a bacteriophage capable of killing a pathogenic bacterium expressing a specific capsule serotype (targeting a specific capsule serotype of a pathogenic bacterium); and

[0059] b) a vaccination against said pathogenic bacterium.

[0060] Pharmaceutical Compositions, Administration / Dosage Forms and Salts

[0061] According to one aspect of the compound according to the invention, the compound according to the invention is provided as a pharmaceutical composition, pharmaceutical administration form, or pharmaceutical dosage form, said pharmaceutical composition, pharmaceutical administration form, or pharmaceutical dosage form comprising at least one of the compounds of the present invention or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent or excipient.

[0062] In certain embodiments of the invention, the compound of the present invention is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient an elegant and easily handleable product.

[0063] Certain embodiments of the invention relate to a dosage form for oral administration. In addition, the pharmaceutical compositions of the present invention can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including without limitation solutions, suspensions or emulsions).

[0064] The dosage regimen for the compounds of the present invention will vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient, and the effect desired. In certain embodiments, the compounds of the invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily.

[0065] The pharmaceutical compositions of the present invention can be subjected to conventional pharmaceutical operations such as sterilization and / or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc. They may be produced by standard processes, for instance by conventional mixing, granulating, dissolving or lyophilizing processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see for example L. Lachman et al. The Theory and Practice of Industrial Pharmacy, 4th Ed, 2013 (ISBN 8123922892).

[0066] Method of Manufacture and Method of Treatment according to the invention

[0067] The invention further encompasses, as an additional aspect, the use of a combination medicament as identified herein (bacteriophage and vaccination), for use in a method of manufacture of a medicament for the treatment or prevention of gut infection.

[0068] Similarly, the invention encompasses methods of treatment of gut infection, comprising administering to a patient in need thereof a therapeutically effective amount of a combination medicament as identified herein (bacteriophage and vaccination).

[0069] Wherever alternatives for single separable features are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein. Thus, any of the alternative embodiments for a bacteriophage may be combined with any of the alternative embodiments of a vaccination and these combinations may be combined with any probiotic bacterium mentioned herein.

[0070] The specification further encompasses the following items.

[0071] Items

[0072] 1. A combination medicament comprising:

[0073] a. a bacteriophage capable of killing a pathogenic bacterium expressing a specific capsule serotype;

[0074] b. a vaccination against said pathogenic bacterium.

[0075] 2. The combination medicament according to item 1, wherein the medicament is formulated for oral administration.

[0076] 3. The combination medicament according to any one of the preceding items, wherein said capsule is an E. coli transporter-dependent capsular polysaccharide.

[0077] 4. The combination medicament according to any one of the preceding items, wherein said capsule is a K1 capsule.

[0078] 5. The combination medicament according to any one of the preceding items, wherein the bacteriophage is of the order Caudovirales.

[0079] 6. The combination medicament according to any one of the preceding items, wherein the bacteriophage is of the family Autographviridae.

[0080] 7. The combination medicament according to any one of the preceding items, wherein said bacterium is an Enterobacteriaceae.

[0081] 8. The combination medicament according to any one of the preceding items, wherein said pathogenic bacterium is an E. coli, Salmonella spp. or Klebsiella spp. bacterium.

[0082] 9. The combination medicament according to any one of the preceding items, wherein said vaccination comprises an inactivated capsule mutant of said pathogenic bacterium.

[0083] 10. The combination medicament according to any one of the preceding items, wherein said vaccination comprises an O-antigen of said pathogenic bacterium.

[0084] 11. The combination medicament according to item 10, wherein said O-antigen is of serotype 0-75.

[0085] 12. The combination medicament according to item 10, wherein said O-antigen is of serotype O-2.

[0086] 13. The combination medicament according to any one of the preceding items, wherein the combination medicament additionally comprises a probiotic bacterium.

[0087] 14. The combination medicament according to item 13, wherein the combination medicament additionally comprises a bacterium of strain E. coli Nissle. 15. The combination medicament according to any one of items 1 to 14 for use in treatment of urinary tract infection.

[0088] 16. The combination medicament according to any one of items 1 to 14 for use in treatment of Crohn’s disease or inflammatory bowel disease.

[0089] 17. The combination medicament according to any one of items 1 to 14 for use in prevention of meningitis or sepsis in a newborn, wherein said combination medicament is administered to a pregnant woman.

[0090] 18. The combination medicament according to any one of items 1 to 14 for use in treatment of a bacterial infection in livestock.

[0091] The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

[0092] Description of the Figures

[0093] Fig. 1 shows capsule-specific phages drive attenuating through capsule loss in vitro and in the gut of colonized mice. a. Bacterial growth (n = 3 independent biological replicates) of EcK1 in presence of 100, 0NW-E1, 0NW-B1 or the K10cocktail at multiplicity of infection (M.O.I.) = 0.01 compared to growth without phage measured over 18 hours, b. Colony morphology of encapsulated and capsule-less E. coli K1 after plating on LB agar no salt plates. As indicated, capsule-less mutants exhibit a translucent colony morphology visible to the naked eye. c. Mini plaque assay (performed in 24-well plate) used to assess phage susceptibility of colonies with opaque (wild-type) and translucent (capsule-less) morphology, demonstrating that colonies with translucent morphology were resistant to phage infection d. Percent encapsulated EcK1 after 18 hours of growth in presence and absence of phages, quantified by counting colonies with translucent morphology (see Fig. 6), showing fixed capsule mutants after phage exposure, e. Survival of EcK1, EcKIAkps and an isolated capsule-less clone with mutations in neuC in human serum compared to heat-inactivated serum (n = 3 independent biological replicates), demonstrating increased susceptibility to serum killing when the capsule is lost (Paired parametric t-tests: *p < 0.05, ns = not significant), f. Survival curves of mice after subcutaneous injection of 108 CFU of EcK1 (n=39), EcKIAkps (n=35) or an evolved EcK1 with a mutation in neuC (EcK1 neuC mutant, n=36).

[0094] 4 independent experiments, shade and dotted lines represent 95% confidence intervals. Capsule-less mutants are less virulent than wild-type E. coli during sepsis, g. Experimental set-up of oral infection of LCM mice with EcK1 and subsequent oral phage treatment with the individual phages or the K1<t>cocktail (n = 6-8 per group) compared to no phage control (n = 8) with fecal sampling for 8 days post infection, h. Percent capsule-less mutants in the fecal EcK1 population, determined by translucent colony morphology, demonstrating loss of capsule after oral phage treatment, i. Fecal bacterial loads of EcK1, demonstrating no significant difference between control and phage treated groups, except on day 4 post infection (two-way ANOVA on log-normalized values with Bonferroni corrections for multiple comparisons: *p<0.05, ns = not significant), j. Fecal phage loads determined by plaque assay, p.i.; post infection, CFU; colony forming unit, PFU; plaque forming, OD; optical density, ABX; Oral antibiotics treatment, D.L.; Detection limit, <t>; phage.

[0095] Fig. 2 shows the K1<t>cocktail is not impacted by other surface molecules and can infect a range of E. coli K1 isolates, a. Bacterial growth (n = 3 independent biological replicates) of EcK1_058 in presence of K1<t>cocktail at multiplicity of infection (M.O.I.) = 0.01 compared to growth without phage measured over 24 hours, b. Flow cytometry analysis of the cultures after staining with an anti-PSA antibody. All cultures grown with phage exhibit significantly lower anti-PSA staining compared to the no phage control. Paired parametric t-test; ***p = 0.0002. c. Experimental set-up. LCM mice were orally infected with 108CFU of EcK1_058, followed by 109PFU of the K1<t>cocktail or exhausted LB (grey), d.

[0096] Fraction of the fecal EcK1_058 population with the K1 capsule present, determined by translucent colony morphology (Fig. 6), demonstrating loss of capsule after oral phage treatment (Mann-Whitney U-tests: ns = not significant, *p < 0.05). e. Fecal bacterial loads of EcK1_058 (Mann-Whitney U-tests: ns = not significant), f. Fecal phage loads determined by plaque assay. ABX; Oral antibiotics treatment, CFU; colony forming unit, PFU; plaque forming unit, p.i.; post infection, <t>; phage

[0097] Fig. 3 shows exclusion of EcK1 by EcN in mice vaccinated against capsule-less EcK1 and treated with capsule-specific phages, a. Overview of the experimental design for oral vaccination with PA-EcK1Akps or mock-vaccinated with PBS weekly for 4 weeks, followed by oral infection with EcN, EcK1 and finally the K1<t>cocktail or exhausted LB. Naive (black circles, n = 7), vaccinated (pink squares, n = 10), naive + phage treated (triangles, n = 9), vaccinated + phage treated (diamonds, n = 11). b. Fecal bacterial load of EcK1 determined by selective plating, demonstrating a reduction of EcK1 in vaccinated and phage treated mice compared to all control groups (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). If no EcK1 could be detected, the value of 104CFU / gfeces was plotted, c. Ratio of EcK1 to EcN in feces determined by selective plating, reflecting significantly lower EcK1 than EcN in the vaccinated and phage treated group compared to all other group (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Statistical analysis for b and c was performed with two-way ANOVA on log-normalized values with Bonferroni corrections for multiple comparisons, detailed plots with all comparisons can be found in Fig. 11. ABX; Oral antibiotics treatment, D.L.; Detection limit, CFU; colony forming unit, p.i.; post infection, <t>; phage.

[0098] Fig. 4 shows competitive exclusion, enhanced by vaccination and capsule-specific phages, of EcK1 from the mother’s gut protects pups from colonization after birth, a. Experimental set-up. Females were orally vaccinated with PA- EcKIAkps or mock-vaccinated with PBS weekly for 3 weeks before mating. 1 week after mating, the females received the final vaccine booster, were subsequently colonized with EcN, EcK1 and received the K1<t>cocktail or exhausted LB. Fecal bacterial loads were tracked for 7 days post infection before the pups were born approximately 12 days post infection. At day of life (D.O.L.) 10, a part of the litter was euthanized. The fecal bacterial loads in the remaining pups was tracked until D.O.L. 21 by rectal swabbing and overnight enrichment in LB. Naive (black circles, n = 16), vaccinated (squares, n = 11), naive + phage treated (triangles, n = 10), vaccinated + phage treated (diamonds, n = 21). b. Mean fecal loads of EcK1 in pregnant females, determined by selective plating (n = 6 per group), showing reduced bacterial colonization in vaccinated and phage treated mice compared to the control groups c. Mean fecal ratio of EcK1 to EcN in pregnant females, d. Fecal loads of EcK1 in pups at D.O.L. 10. Naive (n = 13), naive + phage (n = 19), vaccinated (n = 22) and vaccinated + phage treated (n = 13). When no EcK1 was detected, the value of 104CFU / gfeces is plotted. (Mann-Whitney tests: *p < 0.05, **p < 0.01, ***p < 0.001). e. Fraction of pups colonized with EcK1 at D.O.L. 10. f. Fraction of pups colonized with EcK1 at D.O.L. 11 to D.O.L. 21, illustrating that the vertical transmission of EcK1 to pups is delayed by approximately 1 week when females receive both vaccination and phage treatment. Statistical analysis for c and d was performed with two-way ANOVA on log-normalized values with Bonferroni corrections for multiple comparisons (ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001), detailed plots with all datapoints and significant comparisons can be found in Fig. 12. ABX; Oral antibiotics treatment, CFU; colony forming unit, D.O.L.; day of life, p.i.; post infection, <t>; phage. Fig. 5 shows the isolated phages can only infect E. coli expressing the K1 capsule a. Alignment of the genomes of the three capsule-specific phages to the reference phages K1E (GenBank Id: KY435490.1 (Schneider et al., Biomedical Research International, 2018, 2018)) and K1F (GenBank Id: NC_007456.1 (Scholl & Merril, Journal of Bacteriology, 187, 2005; Moller-Olsen et al., Scientific Reports, 10, 2020)). Rings show homologous sequences, with the color intensity representing the percentage of identity. Alignments and visualization were performed using BRIG. (Alikhan et al., BMC Genomics, 12, 2011) b-e. Dilutions of lysates of individual phages and the K1 cocktail were spotted on lawns of EcK1 (b), EcKIAkps (c), EcN (K5 capsule, d), E. coli K23 isolate (e, NCTC 10430). Lysis plaques can only be seen on EcK1, whereas strains lacking the capsule or containing another capsule are resistant to the isolated phages.

[0099] Fig. 6 shows capsule-less mutants exhibit translucent morphology and mutations in the capsule biosynthesis cluster, a. Capsule biosynthesis cluster with indicated mutations found after sequencing translucent colonies isolated from in vitro and in vivo experiments. b. LIPLC analysis of 1,2-diamino-4,5- methyleneoxybenzene (DMB)-labelled bacterial capsules of indicated bacterial strains. Presence of capsule is indicated by sequential peaks visible in EcK1 but absent in EcKIAkps and all isolated and constructed capsule mutants, c.

[0100] Fecal levels of the inflammation marker lipocalin-2 demonstrating no or low inflammation after EcK1 colonization, with (diamonds, n = 2) or without (grey circles, n = 5) phage treatment, comparable to uncolonized LCM mice (black triangles, n = 1). d. LIPLC analysis of DMB-labelled bacterial capsules of indicated bacterial strains, demonstrating loss of capsule in isolated translucent colonies (EcKlemrR* 1 and EcKlemrR* 2). e. Bacterial survival of EcK1, EcKIAemrR and the two natural emrR mutants in 100% human serum compared to heat-inactivated serum (n = 3 independent biological replicates), demonstrating increased susceptibility to serum killing when the capsule is lost. A.u.; arbitrary units, LCN-2; lipocalin-2, p.i.; post infection, D.L.; detection limit, LCM; low complexity microbiota, CFU; colony forming unit.

[0101] Fig. 7 shows the K1 -specific phages cannot replicate on EcK1 mutants lacking the capsule, a-f. Bacterial growth (n = 3 independent biological replicates) of EcKIAkps (a), EcKIAemrR (b), EcK1 neuC mutant (c), EcKIAneuC (d), EcK1 emrR* 1 (e) and EcK1 emrR* 2 (f) in presence of 100, 0NW-E1, 0NW-B1 or the K10cocktail at multiplicity of infection (M.O.I.) = 0.01 compared to growth without phage measured over 18 hours. No lysis (i.e. sharp reduction of the OD) was observed over time. g. Fold replication of individual phages and the KKPcocktail on EcK1 mutants lacking the capsule (EcKIAkps, EcKIAemrR, EcKIAneuC) compared to wild-type EcK1, demonstrating no replication if the capsule is not present (Unpaired t-tests: *p<0.05).

[0102] Fig. 8 shows the K1<t>cocktail can infect E. coli K1 isolates of varying O- and Flantigens. a. Fluorescence profiles after anion exchange UPLC of hydrolyzed K1 capsule where the polysialic acid is labelled with 1, 2-diamino-4, 5- methyleneoxybenzene (DMB) of indicated E. coli K1 isolates. The presence of capsule is indicated by the sequential peaks present in the EcK1 and absent in the EcKIAkps sample. One representative result per strain, b-h. Dilutions of lysates of individual phages and the K1<t>cocktail were spotted on lawns of EcK1_009 (b), EcK1_054 (c), EcK1_065 (d), EcK1_068 (e), EcK1_068Akps (f), EcK1_058 (g), EcK1_058Akps (h). Lysis plaques with the phage cocktail can be seen on all lawns of bacteria with the K1 capsule (b-e, g), whereas knocking out the capsule or spotting the phages on a E. coli isolate with a different capsule type yield no lysis plaques (f,h).

[0103] Fig. 9 shows the K cocktail can be used to drive in vitro evolution of capsule-less mutants in E. coli K1 isolates with different serological properties, a-d. Bacterial growth (n = 3 biologically independent replicates) of EcK1_009 (a), EcK1_054 (b), EcK1_065 (c), EcK1_068 (d) with K cocktail at M.O.I. = 0.01 (+K10>) compared to growth without phage (-K10) measured over 24 hours. When grown in presence of the K1<t>cocktail, all isolates demonstrate initial growth, followed by a population crash and regrowth of a phage-resistant mutant population, e. Flow cytometry analysis of the cultures after staining with an antiPSA antibody. All cultures grown with phage exhibit significantly lower anti-PSA staining compared to the no phage control. Paired parametric t-tests: ns = not significant, *p<0.05, **p<0.01. a.u.; arbitrary units, OD; optical density.

[0104] Fig. 10 shows the K1 capsule allows EcK1 to escape PA-EcK1Akps vaccine-induced IgA response in the gut. a. Experimental set-up. LCM mice were orally vaccinated with PA-EcK1Akps (n = 7) or mock- vaccinated with PBS (n = 6) weekly for 4 weeks, before oral infection with 108CFU EcK1 and EcKIAkps in a 1:1 ratio, b. Fecal ratio of EcK1 over EcKIAkps normalized by the initial ratio in the inoculum, showing increased ratio of EcK1 in vaccinated mice compared to naive (Mann-Whitney tests with Holm-Sidak correction: ns = not significant, *p<0.05). c-d. Fecal load of EcK1 (c) and EcKIAkps (d), determined by selective plating, e. Intestinal IgA titers against EcK1 and EcKIAkps, determined by flow cytometry, showing increased IgA titers in vaccinated mice against EcK1 Akps compared to naive and EcK1 (Mann-Whitney tests: ns = not significant, **p<0.02, ***p<0.001). ABX; Oral antibiotics treatment, C.I.; competitive index, CFU; colony forming unit, PFU; plaque forming unit, p.i.; post infection.

[0105] Fig. 11 shows colonization resistance against EcK1 can be increased with a rationally combined strategy of phage treatment, oral vaccination, and niche competition - all measured data points, a. EcK1 Akps-specific intestinal IgA titers determined by flow cytometry, showing significantly higher IgA titers in the vaccinated groups compared to naive mice (Mann-Whitney tests: ***p<0.001, ****p<0.0001). b. Fecal ratio of EcK1 / EcN. c-d. Fecal bacterial load of EcK1 (c) and EcN (d) and determined by selective plating, e. Fecal phage titers measured by plaque assay. Phage booster shot is indicated with

[0106]

[0107] on day 3 p.i. (Mann-Whitney tests: ns = not significant), f. Percent encapsulated EcK1, quantified by translucent colony morphology (Fig. 6), showing that EcK1 only in naive mice treated with phage exhibit a capsule-less phenotype. Statistical analysis for b-d and f was performed with two-way ANOVA on log-normalized data points with Bonferroni corrections for multiple comparisons (ns = not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). MFI; median fluorescent intensity, DF; dilution factor, C.I.; competitive index, CFU; colony forming unit, PFU; plaque forming unit, p.i.; post infection, D.L.; detection limit, <t>; phage.

[0108] Fig. 12 shows fecal bacterial and phage loads in pregnant dams - all measured data points, a. Fecal ratio of EcK1 to EcN. Fecal bacterial loads of EcK1 (b) and EcN (c), determined by selective plating. Percent encapsulated EcK1, quantified by translucent colony morphology (Fig. 6), showing that EcK1 only in naive mice treated with phage exhibit a capsule-less phenotype, a-d. Statistical comparisons (performed with two-way ANOVA on log-normalized data (ns = not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). e. Fecal phage titers measured by plaque assay. (Mann-Whitney tests: ns = not significant). CFU; colony forming unit, PFU; plaque forming unit, p.i.; post infection, <t>; phage, DF; dilution factor

[0109] Fig. 13 shows UPLC analysis of translucent clones from phage treated pregnant females and their pups. a-j. UPLC analysis of hydrolyzed and DMB-labelled bacterial capsules. Presence of capsule is indicated by sequential peaks visible in EcK1 wild-type (a) but absent in EcKIAkps (b) and in all tested mutants with translucent morphology from phage-treated pregnant female 1 (c), female 2 (e) and female 3 (g), as well as their respective pups (d, f, h) and pups from female 4 (i) and female 5 (j). a.u.; arbitrary units, D.O.L.; day of life, p.i. post infection. Fig. 14 shows E. coli loads and IgA titers in pups. a. Percent encapsulated EcK1 found in pups at D.O.L. 10, showing that all pups from naive phage treated females became colonized with capsule-less EcK1, reflecting the phenotype of the bacteria transmitted from the mothers, b. Fecal EcN loads in pups at D.O.L. 10, determined by selective plating, c. Fraction of pups colonized with EcN at D.O.L.

[0110] 11 to D.O.L. 21. d. Fraction of pups with phage at D.O.L. 10 to D.O.L. 21. e-g.

[0111] EcKIAkps-specific intestinal IgA titers determined by flow cytometry from pups at D.O.L. 10 (e), and in colonized (f) and uncolonized (g) mothers at endpoint. From uncolonized mothers (g), naive mother (open grey circle) or pups (closed grey circle) show no EcKIAkps-specific intestinal IgA, whereas vaccinated mother (open square) and pups (closed square) have significantly higher titers. Without vaccination or colonization no EcKIAkps-specific IgA response is generated or transmitted to the pups. Mann- Whitney tests: ns = not significant, ***p<0.001 D.O.L.; day of life, CFU; colony forming unit, p.i.; post infection, DF; dilution factor, MFI; median fluorescent intensity, D.L.; detection limit.

[0112] Description of the Tables

[0113] Table 1 shows the sequence type (ST), O-antigen and H-antigen of six E. coli K1

[0114] isolates susceptible to the K1<t>cocktail.

[0115] Examples

[0116] Example 1: Phages can drive evolution of a capsule-less population with increased susceptibility to serum killing

[0117] Three phages requiring the K1 capsule for E. coli infection were isolated from sewage water, 100-A, NW-B1 and NW-E1 (Fig. 1a, Fig. 5a). Whole-genome sequencing revealed that the three phages belonged to the family Autographviridae, closely related to two previously characterized K1 specific phages (Moller-Olsen et al., Scientific Reports, 10, 2020; Schneider et al., Biomedical Research International, 2018, 2018) (Fig. 5a). Spotting of the individual phages or combined at 1 : 1 : 1 in a cocktail (K1 <t>cocktail) yielded plaques on lawns of EcK1 , but not on the capsule knockout EcKIAkps or on E.coli with other capsule types (EcNK5 and EcK23 , Fig. 5b-e). When EcK1 was exposed to the phages, an initial growth was followed by a reduction of the optical density (OD) due to lysis of the sensitive population (lysis phase). After this, a phage-resistant population was able to reach maximal OD (Fig. 1a). The phage- resistant clones exhibited a translucent morphology on LB agar no salt. Capsule loss was confirmed in these clones by sequencing, immuno-staining with an anti-PSA antibody and capsule labelling followed by Ultra-performance liquid chromatography (UPLC) analysis (Fig.

[0118] 1 b-d, Fig. 6a-b). The translucent colony morphology phenotype was thereafter used to quantify capsule mutants in a population by plating. Sequencing of phage-resistant clones revealed mutations in different genes within the capsule biosynthesis cluster, all leading to loss of the capsule (Fig. 6a-b, Table 2) (Vann et al., Journal of Bacteriology, 186, 2004; Annunziato et al., Journal of Bacteriology, 177, 1995). One of the resistant clones had mutations in the neuC gene (EcKIneuC*). NeuC catalyzes the formation N-acetylmannosamine (ManNAc) and UDP from UDP-GIcNAc as the first committed step in CMP-Neu5Ac biosynthesis, an essential precursor for the K1 capsule. Growth dynamics of constructed and natural mutants lacking the capsule (EcKIAkps, EcKIAneuC, EcKIneuC*) showed no lysis phase in the presence of isolated phages or the cocktail (Fig. 7a-c). Accordingly, the phages could not replicate on strains lacking the capsule (Fig. 7g). Together these results demonstrated that these phages require the K1 capsule for infection.

[0119] In line with the known functions of the K1 capsule in protecting the bacteria against the innate immune system, incubation of capsule-less EcK1 mutants (EcKIAkps, EcKIAneuC, EcK1 neuC*) in human serum revealed increased susceptibility to serum killing compared to the wildtype strain EcK1 (Fig. 1e). Accordingly, the capsule-less E. coli mutants EcKIAkps and EcKIAneuC were also less virulent in a sepsis model compared to wild-type EcK1 (Fig. 1f). This demonstrates that phage selection can be leveraged to drive virulence attenuation in pathogenic E. coli. The next question was: does this apply in a clinically relevant ecological context?

[0120] The main ecological niche of E. coli K1 is the large intestine, and invasive infections commonly arise from translocation of the patient’s own resident E. coli to extra-intestinal sites. To test the efficacy of phage-driven capsule loss in the intestinal context, antibiotic pretreated C57BL / 6J mice with low complexity microbiota (LCM) (Stecher et al., PLoS Pathogens, 6, 2010) were infected orally with 108CFU of EcK1, followed by 109PFU of the individual phages or the K1<t>cocktail (Fig. 1g). In this model, EcK1 robustly colonized the gut without causing detectable intestinal inflammation (Fig. 6c). Capsule-less mutants reached fixation at the end of the experiment in all mice except two treated with NW-B1 alone (Fig. 1h). The median bacterial loads were initially lower in phage treated groups, with significant differences in mice treated with the individual phages compared to control mice on day 4 post infection (Fig. 1i). At later time points however, there was no significant difference in fecal bacterial loads between phage-treated and control mice (Fig. 1 i), suggesting that the open niche for E. coli in these mice is rapidly filled by capsule-less mutants selected for upon phage exposure. Phage titers decayed below detectable levels by day 6 post infection in most mice (Fig. 1 j), following the trend of the increasing fraction of capsule-less EcK1, indicating that, in the absence of capsule producing preys, phages cannot be maintained in the gut. Sequencing of isolated capsule-less clones of EcK1 revealed mutations in emrR, a transcriptional regulator of the capsule biosynthesis. UPLC analysis, growth dynamic and phage replication assay revealed loss of capsule as well as abolished phage infection and the mutants were found to be susceptible to serum killing (Fig 6a, d-e, Fig. 7d-g).

[0121] 2: The K1<Pcocktail can be E. coli K1 isolates

[0122]

[0123] To investigate the generality of this approach, the individual phages and the K1<t>cocktail were spotted on lawns of other E. coli K1 isolates with varying O- and H-antigens (EcK1_009, EcK1_054, EcK1_065, EcK1_068, EcK1_058). Whereas spotting of the individual phages resulted in varying amounts of plaques formed, the K1<t>cocktail could specifically infect a range of pathogenic isolates that produce the K1 capsule without impact of the composition of other surface glycans such as the O-antigen on infectivity (Table 1, Fig 8a-h). This highlights the benefit of using a phage cocktail as opposed to individual phages. Individual bacterial strains can harbor other phage defense systems, such as CRISPR-Cas, which may be overcome by some of the phages in the cocktail. Overnight growth curve analysis in presence of the K cocktail revealed initial lysis phase and subsequent regrowth of a capsule-less population in all evaluated strains (Fig. 2a-b, Fig. 9a-e). Furthermore, we inoculated nonpretreated LCM mice with the distantly related E. coli K1 isolate EcK1_058, followed by the K1<t>cocktail (Fig. 2c). In presence of phage, the percentage of capsule-less mutants is significantly higher, confirmed by phenotypic characterization on LB agar no salt and sequencing, which revealed large deletions (36-66 kb) spanning over the capsule biosynthesis cluster (Fig. 2d, Table 2). Again, the overall bacterial load remains equal between the groups, however in this setting we observed prolonged sustainment of the phage in the gut, consistent with available prey (Fig. 2e-f).

[0124] Example 3: Combination of phage therapy and oral vaccination increases colonization resistance and intestinal clearing of EcK1

[0125] Next, we set out to target the capsule-less population that evolves upon phage exposure but is not cleared from the gut. In the intestinal environment, secretory Immunoglobulin A (SlgA) elicited through oral vaccination can drive competitive exclusion of targeted strains. While the K1 capsule is poorly immunogenic, the underlying O-antigens can induce a strong SlgA response upon vaccination. To test this assumption, we prepared oral vaccine using peracetic acid (PA) to kill the bacteria while maintaining their O-antigen intact (Moor et al., Frontiers in Immunology, 7:34, 2016). As expected, we found that mice vaccinated against EcK1 Akps (PA- EcK1 Akps) produced SlgA that strongly advantage EcK1 over EcKIAkps in the gut (Fig. 10). We reasoned that combining phage against EcK1 with oral vaccination targeting EcKIAkps might suppress gut colonization by both EcK1 and capsule-less mutants. This should mitigate the risk of capsule restoration in capsule-less mutants. For example, if mutants are generated by transposon insertion in the capsule biosynthesis cluster, virulent capsulated clones could re-emerge quickly in the absence of phage pressure (Styles et al., Microbiology Spectrum, 10(3), 2022). Moreover, as LCM mice are devoid of E. coli, we also introduced the probiotic E. coli Nissle (EcN) as niche competitor to further increase intestinal clearance of the pathogen (Fig. 3a). This recapitulates the situation in humans where several E. coli strains share the intestinal niche.

[0126] Oral vaccination with PA-EcK1Akps induced production of slgA directed against EcKIAkps in the intestine (Fig. 11a). As expected, this does not affect the competition between EcK1 and E. coli Nissle (Fig. 3b, Fig. 11b). In contrast, the EcK1 load was reduced 100-fold with oral phage treatment alone (Fig. 3c, Fig. 11c). Oral vaccination and phage treatment exhibited additive effects on reducing the average EcK1 levels 1000-fold compared to the control mice (Fig. 3b). In three vaccinated and phage treated mice, no EcK1 could be detected 5 days post infection, i.e. complete clearance from the gut was achieved. There was no significant difference in the fecal loads of EcN between the treatment groups (Fig. 11 d) Correspondingly, the ratio of EcK1 to EcN was significantly reduced the last three days in the vaccine and phage treated group compared to all other groups, largely reflecting the change in EcK1 loads (Fig. 3b-c, Fig. 11b-c). Phage titers diminished over the course of the experiment (Fig. 11e). In all animals where phenotypically K1-positive EcK1 could be detected, phage titers remained at around 104PFU / gfeces. In all animals where 100% of fecal EcK1 has lost capsule, or where the entire EcK1 population was eliminated, phage titers were undetectable (Fig. 11e-f). This demonstrates that phage therapy and vaccine-induced mucosal immunity can have additive effects that results in major loss of fitness for the targeted E. coli K1 strain, in some cases allowing complete exclusion of this opportunistic pathogen from the gut.

[0127] Example 4: Combined oral vaccination and phage treatment of pregnant mice prevent vertical transmission

[0128] While vertical transmission of maternal gut microbes generally is considered beneficial for neonatal health and development, previous studies have shown that E. coli K1 colonizing the gut of mothers can be transmitted and cause sepsis in their child after birth. Therefore, we tested whether our intervention could reduce intestinal load of EcK1 in the gut of pregnant mice sufficiently to minimize transmission to neonates. Female mice were orally vaccinated against EcKIAkps or mock-vaccinated with PBS once a week for three weeks prior to mating. One week after mating, the females received the final booster of the vaccine to increase chance of IgA transmission via milk. The pregnant females were then colonized with EcN and 24 hours later with EcK1, before receiving phage treatment or exhausted LB as control (Fig. 4a). All vaccinations and colonizations were done by voluntary feeding to reduce stress during pregnancy. Daily fecal sampling from the pregnant females revealed plateauing colonization at 108CFU / gfeces in all control group and reduced EcK1 loads in the vaccinated and phage treated group, consistent with previous experiments (Fig. 4b-c, Fig. 12a-e). As the gut microbiota was not cleared with antibiotics beforehand and these mice therefore have a more complex microbiota with higher colonization resistance, we confirmed that our strategy also works at reduced colonization levels. In this setting, the fecal EcK1 bacterial loads were initially reduced by with phage treatment alone, followed by rapid re-population with the emerging capsule-less mutants (Fig. 4b-c, Fig. 12a-b,d), demonstrating the benefit of combining phage treatment with oral vaccination compared to phage treatment alone.

[0129] At day of life (D.O.L.) 10, a part of the litter was sacrificed to quantify the intestinal E. coli colonization levels in the pups. We observed transmission of EcK1 without any treatment (in 11 / 13 pups (80%)) (Fig. 4d). Vaccination alone had no effect on transmission (20 / 22 pups (90%) were colonized) (Fig. 4d), consistent with the K1 capsule blocking the O-antigen-mediated IgA binding (Fig. 10e). In the phage-treated group, 63% (12 / 19) of the pups were colonized with EcK1, however all pups in this group were colonized by capsule-less mutants, reflecting the fixation of a capsule-less population in the mothers (Fig. 4d, Fig. 12d, Fig. 13, Fig. 14a). In contrast, only 23% (3 / 13) of pups from vaccinated and phage treated mothers were colonized EcK1 and instead a larger fraction of pups was colonized by probiotic EcN compared to control groups (Fig. 4d, Fig. 14b-c). At this time, which is roughly 22 days post inoculation, the unvaccinated females also produce sufficient IgA for transfer to the pups via the milk, as can be seen in the IgA titers from the small intestinal and stomach content of the pups (Fig. 14e-g). Although long-term colonization is sufficient to produce an anti-O-antigen IgA response (Fig. 14f-g), it does not suppress E. coli transmission to the pups even in the phage-treated group, where the population is completely capsule-less and the surface antigens exposed. Therefore, we conclude that the transmitted IgA is not the main factor protecting the pups against EcK1 transmission. Instead, suppression of EcK1 gut colonization in the mother by phage mediated killing of encapsulated EcK1 and subsequent rapid exclusion of emerging phage-resistant mutants with vaccine-induced IgA and niche competition, is likely preventing the transmission of EcK1 from mothers to pups. In addition, vaccine-induced IgA is superior to naturally developed IgA in suppressing gut colonization levels as long-term colonization with a large population size is ideal for evolution of IgA-escape variants.

[0130] Example 5: Discussion

[0131] The K1 capsule is a key virulence factor in E. coli strains causing neonatal sepsis and meningitis, especially in preterm and low birth weight infants. While intra-partum administration of antibiotics has significantly reduced the burden of neonatal sepsis caused by group B Streptococcus, there are currently no successful strategies to prevent disease caused by E. coli. In addition, relying on antibiotics for treatment becomes increasingly unattractive, due to a high incidence of extended beta-lactamase resistance and long-term negative implications due to a disrupted gut microbiota. Alternatively, administration of probiotics or synbiotics to neonates has been evaluated as a preventative strategy against sepsis, where some report success and others no effect.

[0132] Here, we instead directly target the K1 capsule with phages that select for capsule-less mutants of E. coli. We were able to target a range of E. coli K1 clinical neonatal sepsis isolates in vitro and in vivo. The evolved capsule-less mutants were both more susceptible to serum killing and unable to cause disease when injected IV in mice (Fig. 1 f), confirming the reduction in virulence previously observed. Steering the evolution of attenuated bacterial populations with phages has previously been highlighted as a potential intervention to improve clinical outcome by leveraging the rapid evolution of phage resistance but has not yet been adopted clinically. As a second line of defense, we involved the host’s mucosal immune system and trapped the evolved capsule-less mutants with intestinal IgA elicited by oral vaccination against EcKIAkps (Fig. 3). The final essential part to increase colonization resistance against E. coli K1 is to introduce the niche competitor EcN, which competes with E. coli K1 for niche occupancy and further reduces the bacterial load of EcK1 and derivatives via vaccine-enhanced competition (Fig. 3). Each part of this combined approach provides an additive effect to increase colonization resistance against E. coli K1. However, there is a large variation in the final EcK1 colonization levels between the individual mice in the vaccine and phage treated group, where some mice are colonized to 107CFU / gfeces and in others no EcK1 was detected (Fig. 3c). This could potentially be explained by differences in the pace at which the competing microbiota repopulates the gut after antibiotic treatment. To improve the success rate of the combined treatments, a consortium of probiotic strains, instead of only EcN, could be used to enhance the competition against E. coli K1.

[0133] Furthermore, the strategy can be translated into a vertical transmission model where the pups are protected from E. coli K1 colonization during the first vulnerable days of life, if the mother receives both vaccination and phage treatment (Fig. 4). To our knowledge, this is the first demonstration of specific prevention of transmission of a neonatal sepsis-causing E. coli with important implications for neonatology. Phage treatment alone does not provide protection against vertical transmission during early life, however the cells transmitted were attenuated capsule-less clones unable to survive systemically, which may be sufficient to prevent disease (Fig. 14a). On the other hand, there is a risk of capsule restoration when the evolutionary pressure exerted by the phage is lifted, as the capsule can confer protection when colonizing the gut. In our setting, oral vaccination against the capsule-deficient E. coli alone did not protect from transmission of E. coli K1, in line with the K1 capsule effectively blocking IgA-O-antigen interactions (Fig. 10e). Sereme et al., on the other hand, recently proposed intraperitoneal vaccination of pregnant females with a live attenuated E. coli K1, where the transmitted antibodies protected the pups against systemic infection (Sereme et al., Nature Communications, 15, 2024). However, live attenuated vaccine is not recommended for pregnant women.

[0134] In conclusion, we have successfully developed a strategy to increase colonization resistance against pathogenic E. coli with high clinical relevance, with some examples of complete clearance from the gut. Furthermore, the strategy can be implemented to protect pups from E. coli K1 colonization during early life, likely due to suppression of colonization in the pregnant mother and consequently reduced probability of transmission during birth. While E. coli K1 is the most prevalent Gram-negative species causing neonatal sepsis, there are several other clinically relevant pathogens with non-immunogenic properties against which the same strategy could be applied. For example, E. coli K5 or Klebsiella species are clinically important pathogens that harbor self-mimicking capsules and could be potential future targets for phagevaccine combined treatments.

[0135] Example 6: Materials and Methods

[0136] Culture conditions

[0137] The E. coli K1 isolates originate from the Swiss Child Sepsis Cohort (Agyeman, P. K. A. etal., Lancet Child Adolesc Health., 1 (2): 124-133, 2017). Bacteria were cultivated in Luria-Bertani (LB) media at 37°C with shaking at 180 or 200 rpm (Infors HT Multitron Standard) unless otherwise stated. Media was supplemented with appropriate antibiotics (100 pg / ml ampicillin (AppliChem; 50 pg / ml apramycin (Merck); 6 pg / ml chloramphenicol (AppliChem); 50 pg / ml streptomycin (AppliChem)).

[0138] All dilutions were prepared in phosphate buffer saline (PBS).

[0139] Phage isolation from sewage water

[0140] Inlet sewage water from Emmenbrucke LU and Basel BS was filtered (0.45 pM), concentrated by adding ZnCh to a final concentration of 40 mM and pelleted by centrifugation. The pellet was resuspended in 1 ml SM buffer (100 mM NaCI, 8 mM MgSO4, 50 mM Tris-CI pH 7.5) and 150 respectively 600 pl of it was mixed with 250 pl EcK1 overnight culture, incubated still at room temperature for 15 minutes and mixed with 10 ml warm top agar (0.5% agar, 1 mM MgSCL, 1 mM CaCh) and poured onto LB agar square plates (12 cm x 12 cm, Greiner). After drying, plates were incubated at 37°C overnight. Morphologically different plaques were transferred into 1 ml LB. Dilutions of phage lysates were spotted onto a lawn of EcK1. This process was repeated three times to purify the obtained phages. Phage stocks were prepared by adding phages to 10 ml LB with EcK1 grown to OD6000.1 at M.O.I. 0.1 and incubating shaking at 37°C overnight. Simultaneously 1000 PFU of phage was added to 10 ml warm top agar with 100 pl overnight culture of EcK1, poured onto an LB agar plate and incubated at 37°C overnight. Using a 50 ml conical plastic tube (TPP, Switzerland), the lysis zones in the top agar were scraped off and mixed with the overnight culture containing phage. The mix was pelleted and the supernatant filter sterilized.

[0141] Plaque assay

[0142] The samples containing phages were sterilized using 0.22 pm filter for in vitro samples. Fecal samples were sterilized by adding 20 pL of chloroform to 200 pL of sample, vortexed for 15 seconds and centrifuged for 3 minutes at 14000 rpm. 10 ml warm top agar was mixed with 200 pl liquid culture of the reporter strain and poured on LB agar plates (1.5% agar). 10 or 5 pl of different dilutions of phage samples were spotted onto the bacterial lawn and the plates were incubated overnight at 37°C.

[0143] Construction of E. coli mutants

[0144] Mutants were constructed by lambda red recombination (Yu, D. etal., Proc Natl Acad Sci U S A., 97(11):5978-5983, 2000). Electrocom petent cells were prepared by inoculating 2 ml LB no salt supplemented with appropriate antibiotics with the parental strain and incubated overnight at 37°C or 30°C if containing temperature-sensitive plasmid such as pKD46 (Datsenko, K. A. et al., Proc Natl Acad Sci U SA, 97(12): 6640-6645, 2000). The overnight culture was diluted 1:100 in fresh LB no salt supplemented with appropriate antibiotics and incubated at 37°C. At OD600 = 0.3, EDTA was added to a final concentration of 5 pM and further grown until OD600 reached 1. If the parental strain contained a temperature-sensitive plasmid such as pKD46, the day culture was additionally supplemented with 0.2% L-arabinose and grown at 30°C. The cells were pelleted and washed twice with ice-cold sterile water.

[0145] Phage replication assay

[0146] 20 pl of E. coli cultures grown in LB supplemented with 100 pg / ml ampicillin (AppliChem) overnight at 37°C 200 rpm was diluted in 2 ml of LB in a 50 ml conical plastic tube (TPP) and incubated 2 hours at 37°C and 200 rpm. 103PFU phage was added to each tube and further incubated for 4 hours. 1 ml was pelleted and either filter sterilized (0.22 pm filter) or treated with 20 pl chloroform. Phage titers were determined with plaque assay.

[0147] Growth curves

[0148] E. coli overnight cultures were diluted 1:100 in 200 pl LB in a transparent 96 well plate (TPP) and infected with phage at M.O.I. 0.01. The plate was incubated with lid overnight at 37°C with orbital shaking in Infinite M Plex (Tecan) or Synergy H4 (Biotek) platereader with continuous OD600 measurements every 10 minutes. UPLC quantification of sialic acid

[0149] 1 ml E. coli overnight culture was pelleted, washed once with 1 ml MilliQ H2O and resuspended in 40 pl 20 mM trifluoroacetic acid (TFA). 10 pl cell sample was mixed with 1,2-diamino-4,5-methyleneoxybenzene (DMB) labelling solution (10 mM DMB, 500 mM beta-mercaptoethanol, 20 mM sodium dithionite and 20 mM TFA) and incubated 2-3 hours at 45°C. To avoid lactonization of sialic acid, each sample was supplemented with 20 pl 140 mM NaOH and further incubated at 45°C for 30 minutes. 200 pl of 20 mM Tris (pH 7.5) was added to the samples and they were centrifuged at 20’000xg for 20 minutes. 80 pl of the supernatant was injected on a DNAPac (ThermoFisher Scientific) column and separated in a gradient of NaCI (0-1 M) in 20 mM Tris pH 7.5 with a flow rate of 1.2 ml / min. The samples were run at 35°C. Fluorescence intensity was measured after excitation at 373 nm and emission at 448 nm. The chromatograms were analysed in Chromeleon 7.2 (Thermo Fisher Scientific).

[0150] Serum killing assay

[0151] Heat- inactivated serum was produced by inactivation at 56°C for 30 minutes. Three biologically independent cultures in exponential phase were diluted to 2*105CFU / ml in 200 pl of active or heat-inactivated human serum (H4522, Merck) and incubated at 37°C for 1 hour. At time point 0, 30 minutes and 60 minutes, a 20 pl sample was taken and dilutions were plated on LB agar for assessing bacterial viability.

[0152] Ethical statement

[0153] All mouse colonization experiments were performed in accordance with Swiss Federal regulations approved by the Commission for Animal Experimentation of the Kanton Zurich (licenses 120 / 2019 and 066 / 2021; Kantonales Veterinaramt Zurich, Switzerland, 33580; Kantonales Veterinararmt Basel, Switzerland). Low complexity microbiota (LCM) (Stecher et al., 2010 ibid) C57BL / 6J mice were used in all experiments. Mice were bred and housed in individually ventilated cages with a 12 hours light / dark cycle in the ETH Phenomics Center (EPIC, RCHCI), ETH Zurich or in ISOcages at WRO Basel and were fed a standard chow diet. All mice included in experiments were 4 weeks or older and objectively healthy as determined by routine health checks. Wherever possible an equal number of males and females was used in each group.

[0154] The mouse sepsis experimental protocol (APAFIS#4948) was approved by the French Ministry of Research and by the Ethical Committee for Animal Experiments, CEEA-121, Comite d’ethique Paris-Nord. Housing conditions for the mice were in agreement with the French law, with dark / light cycle, and constant ambient temperature (21°C + / -2°C) and humidity (50% + / - 10%). The mice were inoculated in a blind experiment by the zootechnician who ignores the status of the strain. Mouse work

[0155] In all mouse colonization experiments, EcK1 with an apramycin cassette inserted in glm (McKenzie, G. J. etal., BMC Microbiol., 6:39, 2006) EcK1 Tn7: aac3_VI for selection and EcN transformed with pBAD24 (Guzman, L.M. et al., J Bacteriol, 177, 1995) to allow antibiotic pretreatment with ampicillin, were used. At endpoint, mice were euthanized with CO2 and cardiac heart puncture. Blood from the heart was collected in serum tubes (Sarstedt) and centrifuged at 10000 xg for 5 minutes to obtain serum, heat inactivated at 56°C for 30 minutes and stored at -20°C until further analysis. Intestinal lavage was collected in a 2 ml Eppendorf tube by flushing the small intestine with 1 ml PBS using a cannula. Bacterial and fecal particles were cleared from the intestinal lavage by centrifugation for 10 minutes at 16’000 xg and stored at -20°C until further analysis.

[0156] Mouse model of sepsis

[0157] The virulence of E. coli K1 and derivative strains was assessed using a mouse model of sepsis as previously described (Johnson, J. R. et al, Journal of Infectious Diseases, 194, 2006). Briefly, 4 week old (14-16 g) female OF1 mice from Charles River (L’Arbresle, France) were inoculated with 108E. coli cells subcutaneously in the neck and monitored for six days. The bacteria were cultivated overnight in LB then washed and re-suspended in 200 pl of physiological water before inoculation.

[0158] Time to death was recorded during the following 7 days. Mice surviving more than 7 days were considered cured and sacrificed (Picard et al., Infection and Immunity, Q7, 1999). In each experiment, the E. coli CFT073 strain was used as a positive control killing all the inoculated mice whereas the E. coli K-12 MG1655 strain was used as a negative control for which all the inoculated mice survive (Johnson, J. R. et al, 2006 ibid).

[0159] Sequencing

[0160] Bacterial DNA was purified using Quick-DNA Miniprep Plus Kit (Zymo) and Oxford Nanopore MinlON long-read sequencing was performed in-house at the Biozentrum, using the MinlON Flow Cells R10 and the Rapid Barcoding Kit 24 V14 (Oxford Nanopore). Basecalling was performed with Guppy (Oxford Nanopore). Reads were mapped on the reference genome using BWA-MEM (Li, arXiv preprint, 1303.3997, 2013) and variants were called using LoFreq (Wilm et al., Nucleic Acids Research, 40, 2012).

[0161] Murine single infection with E. coli and phage

[0162] Optionally, the mice were orally treated with 20 mg ampicillin (AppliChem) by oral gavage 24 hours before colonization. E. coli was grown in 2 ml LB supplemented with 100 pg / ml ampicillin for EcK1 , and no supplement for EcK1_058 overnight at 37°C, 200 rpm, then diluted 1 :20 in 2 ml fresh LB and cultivated for another 4 hours. The day culture was pelleted and washed twice with sterile PBS. The mice were orally infected with 108CFU E. coli by voluntary feeding on Zwieback (Migros) and after 30 minutes with 109PFU phage on Zwieback. Feces were collected daily and homogenized in 1 ml PBS by bead beating (3 mm steel or 2x 2 mm glass ball, 25 Hz for 3 min in a TissueLyser (Qiagen)). Bacteria were quantified by selective plating on LB agar or LB agar without salt supplemented with appropriate antibiotics. Phage titer was determined by plaque assay. At endpoint, bacterial load in cecum and feces was determined as described above.

[0163] Murine competition assay

[0164] The mice were orally treated with 20 mg ampicillin (AppliChem) by oral gavage 24 hours before colonization. EcK1 and EcKIA ps (EcK1 kps:.cat) were grown overnight in 2 ml LB supplemented with 100 pg / ml ampicillin at 37°C, 200 rpm and then diluted 1:20 in 2 ml fresh LB and cultivated for another 4 hours. The day cultures were pelleted and washed twice with sterile PBS. The cultures were mixed 1:1 and 108CFU of the mixture was fed to the mice by voluntary feeding on Zwieback (Migros). The initial ratio was estimated by selective plating. Feces were collected and treated as described above. Bacteria were quantified by selective plating on LB agar or LB agar without salt supplemented with appropriate antibiotics. At endpoint, bacterial load in cecum and feces was determined as described above.

[0165] Vaccinations

[0166] Peracetic acid-killed (PA-killed) vaccine of EcKIA ps was prepared as described (Moor et al., 2016 ibid). In short, EcKIA pswas grown overnight in 2 L LB media at37°C, 180 rpm shaking. The following day, the culture was pelleted at 10’000xg for 10 minutes and the pellet was washed once with 50 ml PBS before incubating the cells with 0.4% peracetic acid (Sigma-Aldrich) for 1 hour. The PA-killed cells were then washed once with 50 ml 10X PBS followed by three washes with 1X PBS before finally resuspending it to a total volume of maximum 15 ml. 101°-1011cells were then orally fed to the mice by pipetting the solution on Zwieback (Migros). Vaccinations were carried out once a week for 4 weeks. Mock-vaccinations were carried out by pipetting 100 pl PBS on Zwieback.

[0167] Murine colonization with niche competitor and EcK1 and phage treatment

[0168] Mice were orally pre-treated with 20 mg ampicillin by oral gavage 24 hours before oral infection with EcN followed by EcK1 infection 24 hours later. Bacterial strains were cultivated overnight in 2 ml LB at 37°C, 200 rpm. Day cultures were prepared by diluting the overnight culture 1:20 in 2 ml fresh LB and cultivating further for 4 hours at 37°C 200 rpm. 1 ml cells were pelleted and washed twice with 1 ml sterile PBS. 108CFU bacteria was fed to the mice by voluntary feeding on Zwieback (Migros, Switzerland). 30 minutes after infection with EcK1, 109PFU K1 <t>cocktail or 100 l PBS was fed to the mice on Zwieback (Migros).

[0169] Feces were collected daily and homogenized in 1 ml PBS by bead beating (3 mm steel or 2x 2 mm glass ball, 25 Hz for 3 min in a TissueLyser (Qiagen)). Bacteria were quantified by selective plating on LB agar with 50 pg / ml streptomycin and LB agar without salt supplemented with 50 pg / ml apramycin. Phage titer was determined by plaque assay. At endpoint, intestinal lavage was collected and bacterial load in cecum and feces was determined as described above.

[0170] Vertical transmission

[0171] 5-6 weeks old female LCM mice were orally vaccinated with PA-killed EcK1 Mps vaccine by voluntary feeding on Zwieback weekly for four weeks. One male was introduced in a cage of two females after three weeks of vaccination (age of females: 7-8 weeks) and removed two weeks later. One day after the last vaccination, 108CFU EcN was prepared as described above and orally fed to the mice on Zwieback followed by 108CFU EcK1 24 hours later. 30 minutes after EcK1 infection, 109PFU K1<t>cocktail was fed to the mice on Zwieback.

[0172] Feces were sampled daily for 9 days and processed as described above. Approximately 12 days post infection, pups were born. 10 days post birth, a part of the litter was euthanized by decapitation. Blood was collected in serum tubes (Sarstedt), centrifuged at 10000 xg for 5 min to obtain serum, heat inactivated at 56°C for 30 minutes and stored at -20°C until further analysis. Feces and cecum content were collected and processed as described above. The stomach and small intestine were collected and homogenized in 1 ml sterile PBS by bead beating (3 mm steel ball, 25 Hz for 3 min).

[0173] From 10-21 days post birth, feces from females were collected daily and processed as described above. The pups were sampled by rectal swabbing using a sterile pipette tip which was placed in 1 ml LB and enriched overnight at 37°C 180 rpm. The overnight culture was diluted and plated selectively on LB agar with 50 pg / ml streptomycin and LB no salt with 50 pg / ml apramycin for qualitative identification of bacterial strains. Detection of phages was done with plaque assay as previously described. At endpoint, mice were euthanized with CO2 and cardiac heart puncture. Blood from the heart was collected in serum tubes and processed as described above. Intestinal lavage was collected by flushing the small intestine with 1 ml PBS using a cannula. Feces and cecum content was collected, and process as described above. Mesenteric lymph nodes were homogenized in 1 ml PBS by bead beating (3 mm steel ball, 25 Hz for 3 min) and bacteria quantified by selective plating on LB agar and LB agar no salt. Analysis of antibody titers by bacterial flow cytometry

[0174] The specific antibody titers in mouse intestinal lavage were measured by flow cytometry as previously described (Moor et al., Nature Protocols, 11(8), 2016). In short, intestinal lavage and blood were collected as described above. Bacterial targets were grown overnight in filtered LB (0.22 pm), then pelleted 3 minutes at 7000 xg. The pellet was washed twice with 0.22 pm-filtered PBS / 0.02% azide / 1% BSA. After thawing, the intestinal lavages were centrifuged at 16’000 xg for 10 minutes and the supernatant was used to make serial dilutions in 96-well plates (TPP). 105washed cells were added to each well and incubated for 1 hour on ice, followed by three washes with 150 pl 0.22 pm-filtered PBS / 0.02% azide / 1% BSA by centrifugation at 7000x g for 5 minutes. Pellets were resuspended in 25 pl of 0.22 pm-filtered PBS containing monoclonal Brilliant Violet 421 Rat Anti-Mouse IgA (BD Bioscience, 2 pg / ml, 743293, AB_2741405) for detection of intestinal IgA. After 1 hour of incubation, cells were washed as described above but with 150 pl filtered PBS (0.22 pm) and resuspended in 100 pl PBS. Flow cytometry acquisition was performed on a Beckman Coulter Cytoflex S using SSC and FSC parameters in logarithmic scale. Data was analysed with FlowJo (Treestar). After gating around bacteria, median fluorescent intensity (MFI) was extracted. Log MFI was plotted against log dilution factor and four parameter logistic curves were fitted using Prism 9 / 10 (GraphPad). From these curve fits, specific titers were calculated as the dilution factor giving an above-background signal (typically MFI = 1000).

[0175] Analysis of fecal Lipocalin-2 levels by ELISA

[0176] Lipocalin-2 (LCN-2) levels in feces were detected using the Mouse Lipocalin-2 / NGAL DuoSet kit (R&D Systems). Feces were homogenized as described above and centrifuged 5 minutes at 16’000 xg. LCN-2 levels were analyzed in undiluted and 1:5 and 1:10 dilutions. 96-well ELISA plates (ThermoFisher) were coated with 100 pl capture antibody (4 pg / ml) and incubated at 4°C overnight. The plates were washed 3 times with PBS / 0.05% Tween20 and blocked with 300 pl PBS / 1%BSA for 3 hours at RT. The plates were washed 3 times with PBS / 0.05% Tween20 and 100 pl the dilutions of the feces and the LCN-2 standard was added and incubated overnight at 4°C. The next day, the plates were washed 6 times with PBS / 0.05% Tween20 before adding 100 pl of the detection antibody (500 ng / ml). The plates were incubated for 1 hour at RT, washed 6 times with PBS / 0.05% Tween20 and 100 pl Streptavidin-HRP was added. After incubation for 1 hour at RT protected from sunlight, the plates were washed 6 times with PBS / 0.05% Tween20. 100 pl of 1:1 mix of TMB and H2O2 was added, and the plates were incubated until desired signal was reached (20 minutes) at RT before adding 50 pl 1M H2SO4 to stop the reaction. The optical density at 450 nm was measured in Tecan microplate reader. Cited references:

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[0178] Agyeman PKA, Schlapbach LJ, Giannoni E, et al. Epidemiology of blood culture-proven bacterial sepsis in children in Switzerland: a population-based cohort study. Lancet Child Adolesc Health. 2017; 1 (2): 124- 133. doi: 10.1016 / S2352-4642(17)30010-X

[0179] Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL. An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U SA.

[0180] 2000;97(11):5978-5983. doi: 10.1073 / pnas.100127597

[0181] Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U SA. 2000;97(12):6640-6645.

[0182] doi: 10.1073 / pnas.120163297

[0183] McKenzie GJ, Craig NL. Fast, easy and efficient: site-specific insertion of transgenes into enterobacterial chromosomes using Tn7 without need for selection of the insertion event. BMC Microbiol. 2006;6:39. Published 2006 Apr 28. doi: 10.1186 / 1471-2180-6-39 Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol.

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[0185] Johnson JR, Clermont O, Menard M, Kuskowski MA, Picard B, Denamur E. Experimental mouse lethality of Escherichia coli isolates, in relation to accessory traits, phylogenetic group, and ecological source. J Infect Dis. 2006; 194(8): 1141-1150. doi: 10.1086 / 507305

[0186] Picard B, Garcia JS, Gouriou S, et al. The link between phylogeny and virulence in Escherichia coli extraintestinal infection. Infect Immun. 1999;67(2):546-553.

[0187] doi: 10.1128 / IAI .67.2.546-553.1999

[0188] Li H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv: 1303.3997 [q-bio.GN], 2013. Available from: https: / / arxiv.org / abs / 1303.3997

[0189] Wilm A, Aw PP, Bertrand D, et al. LoFreq: a sequence-quality aware, ultra-sensitive variant caller for uncovering cell-population heterogeneity from high-throughput sequencing datasets. Nucleic Acids Res. 2012;40(22):11189-11201. doi:10.1093 / nar / gks918 Schneider G, Szentes N, Horvath M, et al. Kinetics of Targeted Phage Rescue in a Mouse Model of Systemic Escherichia coli K1. Biomed Res Int. 2018;2018:7569645. Published 2018 Jul 11. doi: 10.1155 / 2018 / 7569645 Scholl D, Merril C. The genome of bacteriophage K1F, a T7-like phage that has acquired the ability to replicate on K1 strains of Escherichia coli. J Bacterial. 2005;187(24):8499-8503. doi: 10.1128 / JB.187.24.8499-8503.2005

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[0195] Moor K, Wotzka SY, Toska A, Diard M, Hapfelmeier S, Slack E. Peracetic Acid Treatment Generates Potent Inactivated Oral Vaccines from a Broad Range of Culturable Bacterial Species. Front Immunol. 2016;7:34. Published 2016 Feb 11. doi:10.3389 / fimmu.2016.00034 Styles KM, Locke RK, Cowley LA, Brown AT, Sagona AP. Transposable Element Insertions into the Escherichia coli Polysialic Acid Gene Cluster Result in Resistance to the K1 F Bacteriophage. Microbiol Spectr. 2022;10(3):e0211221. doi:10.1128 / spectrum.02112-21 Sereme Y, Schrimp C, Faury H, et al. A live attenuated vaccine to prevent severe neonatal Escherichia coli K1 infections. Nat Common. 2024;15(1):3021. Published 2024 Apr 8. doi: 10.1038 / S41467-024-46775-x

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[0198] All scientific publications and patent documents cited in the present specification are incorporated by reference herein.

[0199] Table 1: Serotypes

[0200] K1 Isolate ST O-antigen H-antigen

[0201] Ec K1_009 428 0:107

[0202]

[0203] Ec K1_054 95 0:1 H:7 Ec K1_065 567 0:18 H:7

[0204] Ec K1_068 95 0:18 H:7

[0205] Ec K1_058 95 0:2 H:4

[0206] Ec K1 1193 0:75 H:5

[0207]

[0208] Table 2: Bacterial strains used in this study

[0209] Collection Name in Genotype Resistance Reference

[0210] ID figure / text ESETH_C73 E. coli LU 100 AmpR EcK1ESETH_C70 E. coli LU009

[0211] ESETH_C60 E. coli LU054

[0212] ESETH_C72 E. coli LU065

[0213] ESETH_C61 E. coli LU068

[0214] ESETH_C58 E. coli CH058

[0215] MDBZ1109 E. coli LU 100 kps ..cat AmpR, CmR This work EcK1Akps LLETH_242 E. coli LU068 kps:.cat CmR This work EcK1-068Akps E. coli CH058

[0216] LLETH_238 KanR This work EcK1-058Akps kps.'.aphT

[0217] E. coli LU 100 neuC AmpR, 11 bp deletion A620- MDBZ1346 This work EcK1neuC mutant mutant 630

[0218] MDBZ1727 E. coli LU 100 emrR.. cat AmpR, CmR This work EcK1AemrR LLETH_257 E. coli LU100 neuC.. cat AmpR, CmR This work EcK1AneuC E. coli LU 100 emrR AmpR, insertion sequence at

[0219] MDBZ1646 This work EcK1emrR* 1 mutant 1 487

[0220] E. coli LU 100 emrR

[0221] MDBZ1647 AmpR, T35P This work EcK1emrR*2 mutant 2

[0222] AmpR, 15 bp deletion A361- MDBZ1877 E. coli LU100 T1_100A This work EcK1neuB*

[0223] 375 in neuB

[0224] E. coli LU 100

[0225] MDBZ1878 AmpR, neuD mutation G108A This work EcK1neuD* T1_NWE1

[0226] E. coli LU 100 AmpR, 15 bp deletion A361- MDBZ1879 This work EcK1neuB* T1_NWB1 375 in neuB

[0227] MDBZ0548 E. coli Nissle pBAD24 SmR, AmpR This work EcN

[0228]

[0229] E. coli K23 reference from NCTC

[0230] ESETH_B99 E. coli K23 EC

[0231] NCTC 10430K23

[0232] E. coli LU 100

[0233] MDBZ1163 AmpR, AprR This work Ec

[0234] Tn7: ,aac3_VIK1

[0235] E. coli CH058

[0236] LLETH_246 AprR This work

[0237] Tn7: ,aac3_VI

[0238] E. coli LU 100 kps-. :frt

[0239] MDBZ1409 AmpR, CmR This work EcK1Ekps PXG10-SF

[0240] E. coli LU 100

[0241] LLETH_252 AmpR, KanR This work Ec pM965_kanRK1

[0242] E. coli LU 100 T1 D.O.L. AmpR, 1'587.711 kb deletion Female 1 D.O.L. LLETH_278 This work

[0243] 21 D42 2321472-3909183 21 (~D33 p.i.) E. coli LU100 T1 D9 p.i. AmpR, 779 bp insertion in

[0244] LLETH_277 This work Female 1 D9 p.i.

[0245] D42 neuD at pos 3887364

[0246] Pup 1 from E. coli LU 100 T1 D.O.L. AmpR, 1'587.711 kb deletion

[0247] LLETH_275 This work female 1 D.O.L.

[0248] 21 Pup 1 D42 2321472-3909183

[0249] 21

[0250] Pup 2 from E. coli LU 100 T1 D.O.L.

[0251] LLETH_276 AmpR This work female 1 D.O.L.

[0252] 21 Pup 2 D42

[0253] 21

[0254] E. coli LU 100 T1 D.O.L. AmpR, 57.528 kb deletion Female 2 colony LLETH_271 This work

[0255] 10 D497 3867971-3925499 (kps genes) 1 D.O.L. 10 E. coli LU 100 T2 D.O.L. Female 2 colony LLETH_272 AmpR This work

[0256] 10 D497 2 D.O.L. 10 Pup 1 from E. coli LU 100 T1 D.O.L. AmpR, 38.209 kb deletion

[0257] LLETH_283 This work female 2 D.O.L.

[0258] 10 Pup 1 from D497 between 3867971-3906179

[0259] 10

[0260] E. coli LU 100 T1 D.O.L. AmpR, 111 bp deletion in Female 3 colony LLETH_273 This work

[0261] 10 D498 neuC A321-432 1 D.O.L. 10 E. coli LU 100 T2 D.O.L. Female 3 colony LLETH_274 AmpR This work

[0262] 10 D498 2 D.O.L. 10 Pup 1 from E. coli LU 100 T1 D.O.L. AmpR, 38.209 kb deletion

[0263] LLETH_284 This work female 3 D.O.L.

[0264] 10 Pup 1 from D498 between 3867971-3906179

[0265] 10

[0266] Pup 2 from E. coli LU 100 T1 D.O.L.

[0267] LLETH_285 AmpR This work female 3 D.O.L.

[0268] 10 Pup 2 from D498

[0269] 10

[0270] Pup 3 from E. coli LU 100 T1 D.O.L.

[0271] LLETH_286 AmpR This work female 3 D.O.L.

[0272] 10 Pup 3 from D498

[0273] 10

[0274]

[0275] AmpR, A->G mutation at Pup 1 from E. co / / LU 100 T1 D.O.L.

[0276] LLETH_279 3504029 (12 bp upstream of This work female 4 D.O.L.

[0277] 10 Pup 1 from E25

[0278] emrR) 10

[0279] Pup 3 from E. co / / LU 100 T1 D.O.L.

[0280] LLETH_280 AmpR This work female 4 D.O.L.

[0281] 10 Pup 3 from E25

[0282] 10

[0283] Pup 2 from E. co / / LU 100 T1 D.O.L.

[0284] LLETH_281 AmpR This work female 5 D.O.L.

[0285] 10 Pup 2 from E26

[0286] 10

[0287] Pup 4 from E. co / / LU 100 T1 D.O.L.

[0288] LLETH_282 AmpR This work female 5 D.O.L.

[0289] 10 Pup 4 from E26

[0290] 10

[0291] E. coli CH_058 66.128 kb deletion between

[0292] LLETH_313 This work

[0293] translucent 1 2870609-2936724

[0294] E. coli CH_058 36.255 kb deletion between

[0295] LLETH_314 This work

[0296] translucent 2 2870545-2906722

[0297]

Claims

Claims1. A combination medicament comprising:a. a bacteriophage capable of killing a pathogenic bacterium expressing a specific capsule serotype;b. a vaccination against said pathogenic bacterium.

2. The combination medicament according to claim 1, wherein the medicament is formulated for oral administration.

3. The combination medicament according to any one of the preceding claims, wherein said capsule is an E. coli transporter-dependent capsular polysaccharide.

4. The combination medicament according to any one of the preceding claims, wherein said capsule is a K1 capsule.

5. The combination medicament according to any one of the preceding claims, wherein the bacteriophage is of the order Caudovirales, particularly wherein the bacteriophage is of the family Autographviridae.

6. The combination medicament according to any one of the preceding claims, wherein said bacterium is an Enterobacteriaceae.

7. The combination medicament according to any one of the preceding claims, wherein said pathogenic bacterium is an E. coli, Salmonella spp. or Klebsiella spp. bacterium.

8. The combination medicament according to any one of the preceding claims, wherein said vaccination comprises an inactivated capsule mutant of said pathogenic bacterium.

9. The combination medicament according to any one of the preceding claims, wherein said vaccination comprises an O-antigen of said pathogenic bacterium, particularly wherein said O-antigen is of serotype 0-75 or of serotype 0-2.

10. The combination medicament according to any one of the preceding claims, wherein the combination medicament additionally comprises a probiotic bacterium.

11. The combination medicament according to claim 10, wherein the combination medicament additionally comprises a bacterium of strain E. coli Nissle.

12. The combination medicament according to any one of claims 1 to 11 for use in treatment of urinary tract infection.

13. The combination medicament according to any one of claims 1 to 11 for use in treatment of Crohn’s disease or inflammatory bowel disease.

14. The combination medicament according to any one of claims 1 to 11 for use in prevention of meningitis or sepsis in a newborn, wherein said combination medicament is administered to a pregnant woman.

15. The combination medicament according to any one of claims 1 to 11 for use in treatment of a bacterial infection in livestock.

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