Prebiotic formulations for the prevention of sepsis- and necrotizing enterocolitis-induced neurodevelopmental impairment

Abstract

Provided herein are probiotic compositions and methods for treating or preventing diseases or neurodevelopmental disorders in subjects in need thereof using the compositions. Aspects and embodiments of the present technology combine the health benefits of probiotic bacteria with the health benefits of prebiotic substances that help stimulate the exclusive growth of probiotic species, and in one aspect provide bacteria in the form of a biofilm on biocompatible microspheres.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 62 / 856,704, filed June 3, 2019, the contents of which are incorporated herein by reference. Statement of Government Support

[0002] This invention is a hygiene Grant number given by the Institute GM123482 This invention was made with Government support under the Federal Accreditation Convention. The United States Government has certain rights in this invention. Sequence Listing

[0003] This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. An ASCII copy of said listing, created on June 3, 2020, is attached hereto. Technical Field

[0004] The present disclosure relates to novel probiotic formulations and methods of use thereof for treating or preventing disease and neurodevelopmental deficiencies in infants, newborns and fetal patients. [Background technology]

[0005] Probiotics are live bacteria that confer health benefits to the host when consumed in sufficient amounts (Food and Agriculture Organization of the United Nations and World Health Organization, "Health and Nutritional Properties of Probiotics in Food Including Powdered Milk with Live Bacteria" (2001)) and are gaining support as a viable option for treating intestinal diseases (Hemarajata and Versalovic, (2013) Effects of Probiotics on Gut Microbiota: Mechanisms of Intestinal Immunomodulation and Neuromodulation, Therap Adv Gastroenterol, 6:39-51). Under the right conditions, many probiotics can effectively prevent pathogen colonization by direct (e.g., generation of antimicrobial defenses) or indirect (e.g., stimulation of host defense) mechanisms. Few probiotic species are capable of both preventing pathogen colonization and limiting excessive inflammatory responses. However, this is important because excessive colonic inflammation in response to colonic infections can lead to the development of long-lasting illnesses such as post-infectious irritable bowel syndrome. Thus, the development of probiotics that can prevent excessive immune responses to colonic pathogens while still maintaining antimicrobial immunity can prevent both short-term and long-term health effects of intestinal infections. The present disclosure provides formulations that address this unmet need and also provides related advantages. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Food and Agriculture Organization of the United Nations and World Health Organization, ``Health and Nutritional Properties of Probiotics in Food Including Powdered Milk with Live Bacteria'' (2001) [Non-Patent Document 2] Hemarajata and Versalovic, (2013) Effects of Probiotics on Gut Microbiota: Mechanisms of Intestinal Immunomodulation and Neuromodulation, Therap Adv Gastroenterol, 6:39-51 Summary of the Invention [Means for solving the problem]

[0007] overview Aspects and embodiments of the present technology combine the health benefits of probiotic bacteria with the health benefits of prebiotic substances that help stimulate the exclusive growth of probiotic species, in one aspect providing bacteria in the form of biofilms on biocompatible microspheres. The applicant has discovered that the use of biofilms on and / or within microspheres results in improved efficacy and duration of therapeutic response to alleviate neurodevelopmental deficiencies, in one aspect due to sepsis caused by various factors. It has been shown that probiotic biofilms can be grown on surfaces as a means of introducing bacteria to wound sites, where the formulations include plasters or dressings based on natural gelatin hydrocolloids to treat wounds (i.e., EP 2450062). However, there is an unmet need for lower probiotic doses and greater efficacy of probiotic bacteria and suitable formulations thereof. The compositions and methods disclosed herein are provided to address this unmet need, and to the applicant's knowledge, have not been disclosed previously.

[0008] In one aspect, a method of preventing, delaying, or treating neurodevelopmental deficiencies or promoting neurodevelopment in a subject, comprising, consisting essentially of, or consisting of administering to the subject an effective amount of a composition, wherein the composition comprises, consists essentially of, or consists of microspheres, biofilm-producing probiotic bacteria, and prebiotics, and the prebiotics comprise, consist essentially of, or consist of a nutritional supplement for the probiotic bacteria. In one aspect, the subject is suffering from, susceptible to, or has suffered from NEC, or other pathologies or conditions that have similar effects on the brain, or sepsis, and / or conditions that cause sepsis.

[0009] In another aspect, a method of preventing, reducing or delaying microglial activation in a subject is provided comprising, consisting essentially of, or consisting of administering to the subject an effective amount of a composition, wherein the composition comprises, consists essentially of, or consists of microspheres, biofilm-producing probiotic bacteria, and prebiotics, and the prebiotics comprise, consist essentially of, or consist of a nutritional supplement for the probiotic bacteria. In one aspect, the subject is a subject suffering from, susceptible to, or suffering from NEC, or other pathologies or conditions that have similar effects on the brain, or sepsis, and / or conditions that cause sepsis.

[0010] In another aspect, a method is provided for increasing expression of brain-derived neurotrophic factor (BDNF) in a subject, comprising, consisting essentially of, or consisting of administering to the subject an effective amount of a composition, wherein the composition comprises, consists essentially of, or consists of microspheres, biofilm-producing probiotic bacteria, and prebiotics, and the prebiotics comprises, consists essentially of, or consists of a nutritional supplement for the probiotic bacteria. In one aspect, the subject is a subject suffering from, susceptible to, or suffering from NEC, or other pathologies or conditions that have similar effects on the brain, or sepsis, and / or conditions that cause sepsis.

[0011] In aspects of the above methods, the subject is an infant, newborn or fetus, eg, a mammalian or human subject.

[0012] In some embodiments, the condition causing sepsis includes necrotizing enterocolitis (NEC) or other pathologies or conditions that have similar effects on the brain or neurodevelopment.

[0013] The technology also provides formulation methods that improve the efficiency and persistence of delivery of probiotic strains to the site of action.The technology specifically bypasses the rate-limiting step of biofilm formation.The technology is useful for gastrointestinal health and other aspects of the gastrointestinal tract, such as the gastrointestinal tract, where probiotic bacteria need to be colonized.

[0014] With regard to gastrointestinal health in particular and the environment in general, probiotics are a natural way to protect and restore the gut microbiota to a healthy state. Unfortunately, even under optimal conditions, probiotic bacteria (when normally delivered) are unable to colonize and persist well, so that the magnitude and duration of their beneficial effects are minimized. One of the rate-limiting steps is the ability of the introduced bacteria to form a long-term biofilm. Bacteria are more easily established and persist when they are already in the form of a biofilm (surface-attached community) rather than planktonic (free-living). The positive effects of probiotic bacteria can be enhanced by providing them in a biofilm state, which can be easily achieved by growing the bacteria with the biofilm on the surface of biocompatible and non-toxic microspheres. The biocompatible microspheres can be biodegradable polymers, non-biodegradable polymers, metals, or combinations thereof. When the surface is in the form of a microsphere, prebiotics and / or prebiofilmic substances can be added as cargo to aid in the establishment and maintenance of the biofilm of the probiotic bacteria.

[0015] In one embodiment, the biofilm-producing probiotic bacteria attach to the surface of the biocompatible microsphere before or after administration and generate a biofilm. In another embodiment, the bacteria are loaded into the core of the microsphere and then diffuse from the core to the microsphere surface. The biocompatible microspheres can be semipermeable or porous and have either a solid core or a hollow core. The biocompatible microspheres can have a hollow core that can carry prebiotics and any nutritional supplements for the probiotic bacteria as cargo, so that the bacteria gain access by diffusion from the lumen or core. The microspheres themselves can also contain prebiotics and any nutritional supplements necessary for the probiotic bacteria. The microspheres can also carry drugs, or compounds, or agents that are selective for pathogens that in one embodiment may compete with the health-inducing bacteria in the composition. In further embodiments, the microspheres can carry chemical reducing agents and / or molecules and / or surfaces that promote adsorption (into the core or surface of the microspheres) and / or molecules and / or surfaces that promote absorption (into the core or surface of the microspheres). In addition to biocompatible microspheres, biofilm-generating probiotics and prebiotics, the novel probiotic formulations can also contain prebiofilmics, which are substances that support the formation and / or persistence of biofilms, and in one embodiment, the prebiofilmics are DNA-binding polypeptides or proteins and / or DNABII polypeptides or proteins, or fragments thereof that support the formation and / or persistence of biofilms. The prebiotics are released from the hollow core and attach to the bacteria. This occurs because the surface of the microspheres is porous or semipermeable, and the prebiotics are released by diffusion or leak out of the microspheres due to the slow degradation of the microspheres and diffuse back in.The release of the prebiotics from the hollow cores can be regulated by varying the microsphere size (smaller microspheres release faster) and / or by modifying the viscosity of the prebiotics (i.e., higher viscosity releases slower).

[0016] Microspheres have an additional value in ideally providing a diffusible prebiotic cargo that can help promote colonization and survival of probiotic bacteria while limiting the attack of pathogenic bacteria. At least for the probiotic bacterium Lactobacillus reuteri, the biofilm state favors colonization of the intestine over the same bacterium in planktonic form. Furthermore, it is shown that L. reuteri introduced as a biofilm in mice has a more robust and sustained preventive effect against the pathogenesis of the enteropathogenic bacterium, Citrobacter rodentium, than its planktonic form. Based on these results, a highly integrated example is provided to generate novel formulations of probiotics that provide a greater and more long-lasting effect against dysbiosis, thereby preventing or even treating intestinal pathogenesis with a significantly reduced need for patient compliance.

[0017] In view of the above advantages, there is provided herein a composition comprising, alternatively consisting essentially of, or even further consisting of biocompatible microspheres, biofilm-producing probiotic bacteria, and prebiotics, wherein the prebiotics comprise, alternatively consisting essentially of, or even further consisting of a nutritional supplement for the probiotic bacteria. In one aspect, the composition further comprises, alternatively consisting essentially of, or even further consisting of a carrier, such as a pharma- ceutically acceptable carrier or a biocompatible scaffold.

[0018] The compositions are formulated for in vivo or ex vivo use. For in vivo use, the compositions are formulated for enteral, oral, parenteral, vaginal, nasal (inhalation), intravenous or intramuscular (injection), topical, as a suppository, as a spray (aerosol), dry application by mixing into soil, or as a solute (for mixing with an aqueous environment). In one embodiment, the compositions are formulated in a dosage form. Suitable dosage forms include, but are not limited to, enteral, parenteral, suppository, powder, liquid, capsule, chewable tablet, swallowable tablet, buccal tablet, troche, lozenge, soft chew, liquid, suspension, spray, tincture, decoction, infusion, and combinations thereof.

[0019] The present disclosure also provides a method for preparing the above-mentioned composition, comprising, alternatively consisting essentially of, or even further consisting of, the step of mixing the biocompatible microspheres with biofilm-producing probiotic bacteria, prebiotics, and in one embodiment further mixing with prebiofilmics. In a further embodiment, the method further comprises, alternatively consisting essentially of, or even further consisting of, the step of mixing an effective amount of one or more of a nutritional supplement for the probiotic bacteria, a drug active against pathogens or invertebrates, or a chemical reducing agent, and / or a molecule that promotes adsorption (into the core or onto the surface of the microsphere), and / or a molecule that promotes absorption (into the core or onto the surface of the microsphere).

[0020] The present disclosure also provides therapeutic uses of the compositions disclosed herein.

[0021] In one aspect, a method for promoting neurodevelopment or preventing, delaying or treating neurodevelopmental deficiency in an infant, fetus or newborn subject suffering from, susceptible to or suffering from necrotizing enterocolitis (NEC) or other disease states or conditions that have a similar effect on the brain, or sepsis and / or a condition that causes sepsis, is provided, comprising, consisting essentially of or consisting of administering to the subject an effective amount of a composition comprising, consisting essentially of or consisting of microspheres, biofilm-producing probiotic bacteria and prebiotics, wherein the prebiotics comprise, consist essentially of or consist of a nutritional supplement for the probiotic bacteria. The subject may be any subject, such as a mammalian or human patient, that is, has, or is susceptible to NEC or other disease states or conditions.

[0022] In some embodiments of the methods described herein, the microsphere further comprises, consists essentially of, or consists of a partial or complete biofilm coating on the exterior surface of the microsphere. In some embodiments, the microsphere comprises, consists essentially of, or consists of a material selected from the group consisting of biodegradable polymers, non-degradable polymers, and metals, and the microsphere has a diameter of about 0.5 microns to about 1000 microns.

[0023] In some embodiments of the methods described herein, the composition further comprises, consists essentially of, or consists of one or more of the following: prebiofilmics, therapeutic or therapeutic agents, chemical reducing agents, molecules that promote adsorption, and molecules that assist absorption.

[0024] In some embodiments of the methods described herein, the prebiofilmics comprises, consists essentially of, or consists of agents that support biofilm formation and persistence.

[0025] In some embodiments of the methods described herein, the prebiofilmics comprises, consists essentially of, or consists of a DNA-binding polypeptide or protein, and / or a DNABII polypeptide or protein, or equivalents of each of these.

[0026] In some embodiments of the methods described herein, the prebiotic comprises, consists essentially of, or consists of water soluble carbohydrates, inulin, oligosaccharides, oligofructose, fructooligosaccharides, galactooligosaccharides, glucose, starch, maltose, maltodextrin, polydextrose, amylose, sucrose, fructose, lactose, isomaltulose, polyols, glycerol, carbonates, thiamine, choline, histidine, trehalos, nitrogen, sodium nitrate, ammonium nitrate, phosphorus, phosphates, hydroxyapatite, potassium, potash, sulfur, homopolysaccharides, heteropolysaccharides, cellulose, chitin, vitamins, and combinations thereof.

[0027] In some embodiments of the methods described herein, the composition further comprises, consists essentially of, or consists of a pharma- ceutically acceptable carrier or a biocompatible scaffold.

[0028] One of the active ingredients of the preparation of the solvent is L.acido philus, L. crispatus, L. gasseri, L. delbrue ckii、L.salivarius、L.casei、L.paracasei、L.plantarum、L.rhamnosus、L.reuteri、L.brevis、L.b uchneri, L. fermentum, L. rhamnosus, B. adolescentis, B. angulation, B. bifidum, B. breve, B. catenulatum, B. infantis, B. lactis, B. longum, B. pseudocatenulatum, S. thermophiles, Pseudomonas fluorescens、P.protegens、P.brassicacearum、P.aeruginosa;Azospirillum.brabrasilense、A.lipferum、A.halopraeferens、A.irakense;Acetobacter diazotrophicus;Herbaspirillum seropedicae;Bacillus subtilis.Pseudomonas stutzeri、fluorescens、P.putida、P.cepacian、P.vesicularis、P.paucimobilis;Bacilluscereus、B.thuringiensis、B.sphaericus;Shewanella oneidensis;Geobacter bemidjiensis、G.metallireducens、G.sulfurreducens、G.uraniireducens、G.lovleyi;Serratia marcescens、Desulfovibrio vulgaris、D.desulfuricans、Dechloromonas aromatic、Deinococcus radiodurans、Methylibium petroleumphilum、Alcanivorax borkumensis、Archaeglobus fulgidus、Haloferax sp.、Halobacterium sp.and combinations thereof.

[0029] In some embodiments of the methods described herein, the probiotic bacteria provide one or more of: antibacterial immunity support, correction of dysbiosis, strengthening or supporting the gastrointestinal barrier, supporting or enhancing gastrointestinal motility, localized release of antibiotic compositions, antagonizing disease-associated bacterial infections, or prevent impairment in one or more of strength, coordination, righting mechanisms, auditory reflex testing, curiosity, learning ability, working memory, short-term memory, long-term memory, visual-spatial reasoning, cognition, object recognition, and serotonin.

[0030] In some embodiments of the methods described herein, the probiotic bacteria prevent pathogen colonization and / or limit excessive inflammatory responses by down-regulating cytokine and chemokine production. Methods for determining whether this is accomplished are known in the art and are briefly described herein.

[0031] In some embodiments of the methods described herein, the microspheres comprise, consist essentially of, or consist of a solid or hollow core.

[0032] In some embodiments of the methods described herein, the prebiotic is encapsulated within or comprises, consists essentially of, or consists of a hollow core of a microsphere.

[0033] In some embodiments of the methods described herein, the composition further comprises, consists essentially of, or consists of an agent, which is selective for a pathogen.

[0034] In some embodiments of the methods described herein, a complimentary agent is coated on the surface of the microsphere and / or encapsulated within the core or hollow core.

[0035] In some embodiments of the methods described herein, the microspheres comprise, consist essentially of, or consist of a metal selected from one or more of cobalt, chromium, gold, nickel, platinum, stainless steel, titanium, tantalum, nickel-titanium, alloys, and combinations thereof.

[0036] In some embodiments of the methods described herein, the microspheres are made of dextran; dextranomer; poly(lactic-co-glycolic acid), i.e., PLGA; polycaprolactone, i.e., PLC; chitosan; gelatin; DNA hydrogen; acetalated dextran; poly(lactide); poly(glycolide); poly(lactide-co-glycolide); poly(lactic acid); poly(glycolic acid); poly(lactic acid-co-glycolic acid)];Poly(lactide) / poly(ethylene glycol) copolymer;Poly(glycolide) / poly(ethylene glycol) copolymer;Poly(lactide-co-glycolide) / poly(ethylene glycol) copolymer;Poly(lactic acid) / poly(ethylene glycol) copolymer;Poly(glycolic acid) / poly(ethylene glycol) copolymer;Poly(lactic acid-co-glycolic acid) / poly(ethylene glycol) copolymer;Poly(caprolactone);Poly(caprolactone / poly(ethylene glycol) copolymer;Poly(orthoesters);Poly(phosphazenes);Poly(hydroxybutyrate);Poly(hydroxybutyrate ); poly(lactide-co-caprolactone); polycarbonate; polyesteramide; polyanhydride; poly(dioxanone); poly(alkylene alkylate); polyethylene glycol / polyorthoester copolymer; polyurethane; poly(amino acid); polyether ester; polyacetal; polycyanoacrylate; poly(oxyethylene) / poly(oxypropylene) copolymer; Sephadex® copolymer, and / or combinations thereof.

[0037] In some embodiments of the methods described herein, the microspheres comprise, consist essentially of, or consist of a non-biodegradable polymer selected from one or more of poly(ethylene vinyl acetate), poly(vinyl acetate), silicone polymers, polyurethanes, polysaccharides, such as cellulosic polymers and derivatives, acyl-substituted cellulose acetate and its derivatives, copolymers of poly(ethylene glycol) and poly(butylene terephthalate), polystyrene, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulphonated polyolefins, polyethylene oxide, and copolymers and blends thereof.

[0038] In some embodiments of the methods described herein, the microspheres are selected from the group consisting of Sephadex, Sephadex G-25, poly(lactic-co-glycolic acid) ("PLGA"); polycaprolactone ("PLC"); chitosan; gelatin; hydrogen DNA; acetalated dextran; poly(lactide); poly(glycolide); poly(lactide-co-glycolide); poly(lactic acid); poly(glycolic acid); poly(lactic acid-co-glycolic acid); poly(lactide) / poly(ethylene glycol) copolymer; poly(glycolide) / poly(ethylene glycol) copolymer; poly(lactide-co-glycolide) / poly(ethylene glycol) copolymer; poly(lactic acid / poly(ethylene glycol) copolymer; poly(glycolic acid) / poly(ethylene glycol) copolymer; poly(lactic acid-co-glycolic acid) poly(oxyethylene) / poly(ethylene glycol) copolymers; poly(caprolactone); poly(caprolactone) / poly(ethylene glycol) copolymers; poly(orthoesters); poly(phosphazenes); poly(hydroxybutyrates); poly(hydroxybutyrates); poly(lactide-co-caprolactone); polycarbonates; polyesteramides; polyanhydrides; poly(dioxanone); poly(alkylene alkylates); polyethylene glycol / polyorthoester copolymers; polyurethanes; poly(amino acids); polyetheresters; polyacetals; polycyanoacrylates; poly(oxyethylene) / poly(oxypropylene) copolymers; and combinations thereof.

[0039] In some embodiments of the methods described herein, the microspheres comprise, consist essentially of, or consist of a non-biodegradable polymer selected from one or more of poly(ethylene vinyl acetate), poly(vinyl acetate), silicone polymers, polyurethanes, polysaccharides, such as cellulosic polymers and derivatives, acyl-substituted cellulose acetate and its derivatives, copolymers of poly(ethylene glycol), poly(butylene terephthalate), polystyrene, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulfonated polyolefins, polyethylene oxide, and copolymers and blends thereof.

[0040] In some embodiments of the methods described herein, the probiotic bacteria comprises, consists essentially of, or consists of L. reuteri, and the prebiotic comprises maltose, glycerol, or histadine.

[0041] In some embodiments of the methods described herein, L. reuteri produces glucosyltransferase (GFT).

[0042] In some embodiments of the methods described herein, the microspheres comprise, consist essentially of, or consist of dextran or dextranomer.

[0043] In some embodiments of the methods described herein, the microspheres comprise, consist essentially of, or consist of dextranomer, the probiotic bacteria comprise, consist essentially of, or consist of L. reuteri, and the prebiotic comprises, consist essentially of, or consist of maltose.

[0044] In some embodiments of the methods described herein, the microspheres comprise, consist essentially of, or consist of dextranomer, the probiotic bacteria comprise, consist essentially of, or consist of L. reuteri, and the prebiotic comprises, consist essentially of, or consist of maltose.

[0045] In some embodiments of the methods described herein, the composition comprises, consists essentially of, or consists of substantially identical microspheres.

[0046] In some embodiments of the methods described herein, the composition comprises, consists essentially of, or consists of microspheres that differ from one another in composition.

[0047] In some embodiments of the methods described herein, the compositions are prepared by mixing microspheres with biofilm-producing probiotic bacteria and prebiotics, optionally in a medium that comprises, consists essentially of, or consists of a biofilm.

[0048] In some embodiments of the methods described herein, the composition comprises, consists essentially of, or consists of admixing the prebiofilmics.

[0049] In some embodiments of the methods described herein, the composition is prepared by mixing one or more of a prebiofilmic, a therapeutic or therapeutic agent, a chemical reducing agent, a molecule that promotes adsorption, and a molecule that assists absorption, and / or the prebiofilmic comprises, consists essentially of, or consists of an agent that assists in biofilm formation and persistence.

[0050] In some embodiments of the methods described herein, the prebiofilmics are DNA binding polypeptides or proteins, and / or DNABII polypeptides or proteins, or equivalents of each of these.

[0051] In some embodiments of the methods described herein, the composition is administered to the subject, or to the mother if the subject is a fetus, at a concentration of about 1×10 7 ~Approx. 1×10 9 The biofilm-producing probiotic bacteria are administered to provide 100 CFU / ml of biofilm-producing probiotic bacteria.

[0052] In some embodiments of the methods described herein, the composition is administered at about 6, 12, 18, 24, 36, 48 and 72 hours after the subject's birth.

[0053] In some embodiments of the methods described herein, the composition is administered in a single dose.

[0054] In some embodiments of the methods described herein, the composition is formulated in a dosage form selected from the group comprising, consisting essentially of, or consisting of a feeding tube, enterally, parenterally, a suppository, in a biocompatible scaffold, a powder, a liquid, a capsule, a chewable tablet, a swallowable tablet, a buccal tablet, a troche, a lozenge, a soft chew, a liquid, a suspension, a spray, a tincture, a decoction, an infusion, parenterally, and combinations thereof.

[0055] In some embodiments of the methods described herein, the compositions comprise, consist essentially of, or consist of PGLA or dextranomer biocompatible microspheres, one or more biofilm-producing probiotic bacteria including at least Lactobacillus reuteri ("L. reuteri"), and a nutritional supplement comprising, consisting essentially of, or consisting of one or more of maltose, sucrose, glycerol, or histidine in an amount to support the growth of the probiotic bacteria, and optionally the microspheres are partially or fully coated with a biofilm.

[0056] In some embodiments of the methods described herein, the microspheres have a diameter in the range of about 0.5 micrometers to 75 micrometers.

[0057] In some embodiments of the methods described herein, the release of the prebiotic is modulated by varying the microsphere size (smaller microspheres release faster) or by modifying the viscosity of the prebiotic (i.e., higher viscosity releases slower).

[0058] In some embodiments of the methods described herein, a kit is provided that comprises, consists essentially of, or consists of a composition comprising, consisting essentially of, or consisting of microspheres, biofilm-producing probiotic bacteria, and prebiotics, where the prebiotics comprise, consist essentially of, or consist of a nutritional supplement for the probiotic bacteria, and instructions for preventing, delaying, or treating neurodevelopmental deficiencies in infants susceptible to or suffering from sepsis and / or a condition that causes sepsis (i.e., necrotizing enterocolitis (NEC)).

[0059] In some embodiments of the methods described herein, preventing or promoting neurodevelopment in a subject (e.g., where the subject is on the autism spectrum), including, e.g., preventing or treating, includes, consists essentially of, or consists of partial prevention of the deficiency. Subjects were assessed for ear opening and eye opening, righting to plane, righting to air, forelimb grasp, acoustic startle, righting to plane, negative geotaxis, open field crossing, cliff aversion, Barnes maze, elevated plus maze, Jamar dynamometer, handheld dynamometry, manual muscle testing (MMT), isokinetic dynamometry, trunk stability test (TST), unilateral hip bridge endurance test (UHBE), pronation sign, Barre sign, autism, autism spectrum disorder, Romberg test, Landau reflex, particle suspension, sensory reflexes (pin pain, superficial touch, position, vibration, and charger), reflexes (biceps, triceps, brachioradial, patellar, and ankle), Moro reflex, tonic neck response, sucking reflex, palmer and planter grasp reflex. reflex, parachute reaction, somatic righting reaction (NOB), somatic righting reaction (BOB), ear opening auditory reflexreflex), static compliance, physical volume of the ear canal, contralateral reflex, ipsilateral reflex, tympanometry, Y-maze, novel object recognition task, STPI (State-Trait Personality Inventory), 5-Dimensional Curiosity Scale, Self-Curiosity Attitudes and Interests Scale, Curiosity Seeking Scale-II, State-Trait Personality Inventory (STPI), subscales of the Sensation Seeking Scale (SSS), Bayley Scales of Infant Development (BSID-III) (1-42 months), Mullen Early Learning Scale (1-68 months), Fagan Intelligence Scale for Infants (FTII) (birth-12 months), Griffith Mental Development Scale I (0-2 years), Battelle Development Scale (BDI) (birth-8 years), and Vineland Adaptation Partial prevention of failure comprising, consisting essentially of, or consisting of obtaining a score of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the score obtained by a control subject of the same age and species as the subject but not suffering from a disease or condition, e.g., sepsis, and / or a condition that causes sepsis, on one or more of any clinically recognized tests selected from the following: Promoting neurodevelopment in a subject, comprising, consisting essentially of, or consisting of the subject obtaining a score that is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the score obtained by a control subject of the same age and species as the subject but not suffering from a disease or condition, e.g., sepsis and / or a condition that causes sepsis.

[0060] In some embodiments of the disclosed methods, preventing neurodevelopmental deficiency or promoting neurodevelopment in a subject comprises, consists essentially of, or consists of a complete prevention of the deficiency or a delay in time until the subject displays one or more symptoms of the deficiency, such as impaired cognitive or social development associated with autism. The subject may display ear and eye patency, flat surface righting, air righting, forelimb grasp, acoustic startle, flat surface righting, negative geotaxis, open field crossing, cliff aversion, Barnes maze, elevated plus maze, Jamar dynamometer, manual muscle testing, manual muscle testing (MMT), isokinetic muscle testing, trunk stability test (TST), unilateral hip bridge endurance test (UHBE), pronation sign, autism, autism spectrum disorder, Barre sign, Romberg test, Landau reflex, , particle suspension, sensory reflexes (pin pain, superficial touch, position, vibration, and charger), reflexes (biceps, triceps, brachioradial, patellar, and ankle), Moro reflex, tonic neck response, sucking reflex, palmar and plantar grasp reflex, parachute response, somatic neck righting response (NOB), somatic righting response (BOB), ear opening acoustic reflex, static compliance, physical volume of the ear canal, contralateral reflex, ipsilateral reflex, tympanometry, Y-maze, novel object recognition and / or the complete prevention of failure to obtain the same score as a control subject of the same age and species not affected by a disease or condition, e.g., sepsis, and / or a condition causing sepsis (NEC), on one or more of any clinically recognized tests selected from the following: cognitive tasks, STPI (State-Trait Personality Inventory), Five-Dimensional Curiosity Scale, Self-Curiosity Attitudes and Interests Scale, Curiosity Seeking Scale-II, State-Trait Personality Inventory (STPI), subscales of the Sensation Seeking Scale (SSS), Bayley Scales of Infant Development (BSID-III) (1-42 months), Mullen Early Learning Scale (1-68 months), Fagan Scale of Intelligence for Infants (FTII) (birth-12 months), Griffith's Mental Development Scale I (0-2 years), Battelle Development Scale (BDI) (birth-8 years), and Vineland Adaptive Behavior Scales (0-18 years).

[0061] In some embodiments, scoring is performed 0-1 month, 1-6 months, 6-12 months, 12-18 months, 18-24 months, 24 months to 3 years, or greater than 3 years after administration of the pharmaceutical composition.

[0062] In some embodiments, the pharmaceutical composition is administered to a fetus of a subject in need of treatment before the fetus is born (i.e., prenatally). In some embodiments, the pharmaceutical composition is administered to a mother who is pregnant with a subject in need of treatment. In some embodiments, the pharmaceutical composition is administered to a subject having an age selected from 0-1 days, 1-6 days, 6-12 days, 12-18 days, 18-24 days, 24 days-100 days, or more than 100 days.

[0063] In some embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of PGLA or dextranomer biocompatible microspheres, one or more biofilm-producing probiotic bacteria comprising, consisting essentially of, or consisting of at least Lactobacillus reuteri ("L. reuteri"), and a nutritional supplement comprising, consisting essentially of, or consisting of one or more of maltose, sucrose, glycerol, or histidine in an amount to support the growth of the probiotic bacteria, and optionally the microspheres are partially or fully coated with a biofilm. In some embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of dextranomer biocompatible microspheres comprising, consisting essentially of, or consisting of Lactobacillus reuteri ("L. reuteri") and a nutritional supplement comprising, consisting essentially of, or consisting of at least maltose or at least maltose. In some aspects, the microsphere surface is partially or completely covered with a biofilm.

[0064] In some embodiments, the infant has an age selected from 0-1 month, 1-6 months, 6-12 months, 12-18 months, 18-24 months, 24 months-3 years, or older than 3 years. In some embodiments, the infant is a human.

[0065] In some embodiments, kits are provided that comprise, alternatively consist essentially of, or even consist of the compositions described herein and instructions for diagnostic, industrial, agricultural, or therapeutic use. In certain embodiments, for example, the following items are provided: (Item 1) A method of preventing, delaying or treating neurodevelopmental deficiency or promoting neurodevelopment in a subject suffering from, susceptible to or suffering from a neurodevelopmental deficiency, comprising the step of administering to the subject an effective amount of a composition comprising microspheres, biofilm-producing probiotic bacteria and a prebiotic, wherein the prebiotic comprises a nutritional supplement for the probiotic bacteria, and optionally the subject is suffering from NEC or other conditions having a similar effect on the brain, or sepsis, and / or a condition causing sepsis. (Item 2) A method for preventing, reducing or delaying microglial activation in a subject, comprising administering to the subject an effective amount of a composition comprising microspheres, biofilm-producing probiotic bacteria and a prebiotic, wherein the prebiotic comprises a nutritional supplement for the probiotic bacteria. (Item 3) 3. The method of claim 2, wherein microglial cell body thickness is reduced in the subject compared to a control subject. (Item 4) 3. The method of claim 1 or 2, wherein microglial dendrites are thinned in the subject compared to a control subject. (Item 5) A method for increasing expression of brain-derived neurotrophic factor (BDNF) in a subject, comprising administering to the subject an effective amount of a composition comprising microspheres, biofilm-producing probiotic bacteria, and a prebiotic, wherein the prebiotic comprises a nutritional supplement for the probiotic bacteria. (Item 6) 6. The method of claim 5, wherein the increase is an increase compared to a control subject. (Item 7) 7. The method according to item 5 or 6, wherein the increased expression of BDNF is increased expression in the hippocampus of the subject. (Item 8) A method for increasing expression of Grin2A in a subject, comprising the step of administering to the subject an effective amount of a composition comprising microspheres, biofilm-producing probiotic bacteria, and prebiotics, wherein the prebiotics comprise a nutritional supplement for the probiotic bacteria. (Item 9) 9. The method of claim 8, wherein the increase is an increase compared to a control subject. (Item 10) 10. The method according to item 8 or 9, wherein the increased expression of Grin2A is increased expression in the prefrontal cortex of the subject. (Item 11) 11. The method of any one of items 1 to 10, wherein the microspheres further comprise a partial or complete biofilm coating on the outer surface of the microspheres. (Item 12) 12. The method according to any one of items 1 to 11, wherein the bacteria and the prebiotics diffuse from the interior of the microsphere to the surface of the microsphere. (Item 13) 13. The method of any one of items 1 to 12, wherein the microsphere comprises a material selected from the group consisting of a biodegradable polymer, a non-degradable polymer, and a metal, and the diameter of the microsphere is from about 0.5 micrometers to about 1000 micrometers. (Item 14) 14. The method of any one of items 1 to 13, wherein the composition further comprises one or more of a prebiofilmic, a therapeutic or therapeutic agent, a chemical reducing agent, a molecule that promotes adsorption, and a molecule that assists absorption. (Item 15) 15. The method of claim 14, wherein the prebiofilmics comprises agents that support biofilm formation and persistence. (Item 16) 16. The method of claim 15, wherein the prebiofilmics is a DNA binding polypeptide or protein, and / or a DNABII polypeptide or protein, or an equivalent of each of these. (Item 17) 17. The method of any one of items 1 to 16, wherein the prebiotics comprise water soluble carbohydrates, inulin, oligosaccharides, oligofructose, fructooligosaccharides, galactooligosaccharides, glucose, starch, maltose, maltodextrin, polydextrose, amylose, sucrose, fructose, lactose, isomaltulose, polyols, glycerol, carbonate, thiamine, choline, histidine, trehalose, nitrogen, sodium nitrate, ammonium nitrate, phosphorus, phosphate, hydroxyapatite, potassium, potash, sulfur, homopolysaccharides, heteropolysaccharides, cellulose, chitin, vitamins, and combinations thereof. (Item 18) 18. The method according to any one of items 1 to 17, wherein the composition further comprises a pharma- ceutically acceptable carrier or a biocompatible scaffold. (Item 19) Recommended L. acidophilus, L. crispatus, L. gasseri, L. delbrueckii, L. saliva ius, L. casei, L. paracasei, L. plantarum, L. rhamnosus, L. reuteri, L. brevis, L. buchneri .fermentum, L. rhamnosus, B. adolescentis, B. angulation, B. bifidum, B. breve, B. catenulatum, B. infantis, B. lactis, B. longum, B. pseudocatenulatum, S. thermophiles, Pseudomonas fluorescens、P.protegens、P.brassicacearum、P.aeruginosa;Azospirillum.brabrasilense、A.lipferum、A.halopraeferens、A.irakense;Acetobacter diazotrophicus;Herbaspirillum seropedicae;Bacillus subtilis.Pseudomonas stutzeri、fluorescens、P.putida、P.cepacian、P.vesicularis、P.paucimobilis;Bacilluscereus、B.thuringiensis、B.sphaericus;Shewanella oneidensis;Geobacter bemidjiensis、G.metallireducens、G.sulfurreducens、G.uraniireducens、G.lovleyi;Serratia marcescens、Desulfovibrio vulgaris、D.desulfuricans、Dechloromonas aromatic、Deinococcus radiodurans、Methylibium petroleumphilum、Alcanivorax borkumensis、Archaeglobus fulgidus、Haloferax sp.、Halobacterium sp., and 1 of the strains of the sp This is a 18-year-old 18-year-old. (Item 20) 20. The method according to any one of items 1 to 19, wherein said probiotic bacteria provide one or more of the following: support of antibacterial immunity, correction of dysbiosis, strengthening or supporting the gastrointestinal barrier, supporting or enhancing gastrointestinal motility, localized release of antibiotic compositions, antagonizing disease-associated bacterial infections, or prevent impairment in one or more of strength, coordination, bounce-back mechanisms, auditory reflex testing, curiosity, learning ability, cognitive ability, social interaction, working memory, short-term memory, long-term memory, visual-spatial reasoning, cognition, object recognition and serotonin. (Item 21) 21. The method according to any one of items 1 to 20, wherein said probiotic bacteria prevent pathogen colonization and / or limit excessive inflammatory responses by downregulating cytokine and chemokine production. (Item 22) 22. The method of any one of the preceding claims, wherein the microsphere comprises a solid or hollow core. (Item 23) 23. The method of claim 22, wherein the prebiotic is encapsulated within the hollow core or constitutes the core of the microsphere. (Item 24) 24. The method of any one of the preceding claims, wherein the composition further comprises an agent, the agent being selective for a pathogen. (Item 25) 25. The method of any one of items 1 to 24, wherein a complementary agent is coated on the surface of the microsphere and / or encapsulated within the core or hollow core. (Item 26) 26. The method of any one of the preceding claims, wherein the microspheres comprise a metal selected from one or more of cobalt, chromium, gold, nickel, platinum, stainless steel, titanium, tantalum, nickel-titanium, alloys, and combinations thereof. (Item 27) The microspheres may be selected from the group consisting of dextran, dextranomer, poly(lactic-co-glycolic acid), i.e., PLGA, polycaprolactone, i.e., PLC, chitosan, gelatin, hydrogen DNA, acetalized dextran, poly(lactide), poly(glycolide), poly(lactide-co-glycolide), poly(lactic acid), poly(glycolic acid), poly(lactic acid-co-glycolic acid, poly(lactide) / poly(ethylene glycol) copolymer, poly(glycolide) / poly(ethylene glycol) copolymer, poly(lactide-co-glycolide) / poly(ethylene glycol) copolymer, poly(lactic acid) / poly(ethylene glycol) copolymer, poly(glycolic acid) / poly(ethylene glycol) copolymer, poly(lactic acid-co-glycolic acid) / poly(ethylene glycol) copolymer 27. The method of any one of items 1 to 26, comprising a biodegradable polymer selected from one or more of: poly(caprolactone); poly(caprolactone) / poly(ethylene glycol) copolymer; poly(orthoester); poly(phosphazene); poly(hydroxybutyrate); poly(hydroxybutyrate); poly(lactide-co-caprolactone); polycarbonate; polyesteramide; polyanhydride; poly(dioxanone); poly(alkylene alkylate); polyethylene glycol / polyorthoester copolymer; polyurethane; poly(amino acid); polyetherester; polyacetal; polycyanoacrylate; poly(oxyethylene) / poly(oxypropylene) copolymer; Sephadex® copolymer, and / or combinations thereof. (Item 28) 28. The method of any one of the preceding claims, wherein the microspheres comprise a non-biodegradable polymer selected from one or more of poly(ethylene vinyl acetate), poly(vinyl acetate), silicone polymers, polyurethanes, polysaccharides, such as cellulosic polymers and derivatives, acyl-substituted cellulose acetate and its derivatives, copolymers of poly(ethylene glycol) and poly(butylene terephthalate), polystyrene, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulfonated polyolefins, polyethylene oxide, and copolymers and blends thereof. (Item 29) The microspheres may be selected from the group consisting of Sephadex, Sephadex G-25, poly(lactic-co-glycolic acid) ("PLGA"); polycaprolactone ("PLC"); chitosan; gelatin; hydrogen DNA; acetalated dextran; poly(lactide); poly(glycolide); poly(lactide-co-glycolide); poly(lactic acid); poly(glycolic acid); poly(lactic acid-co-glycolic acid); poly(lactide) / poly(ethylene glycol) copolymer; poly(glycolide) / poly(ethylene glycol) copolymer; poly(lactide-co-glycolide) / poly(ethylene glycol) copolymer; poly(lactic acid / poly(ethylene glycol) copolymer; poly(glycolic acid / poly(ethylene glycol) copolymer; poly(lactic acid-co-glycol 29. The method of any one of items 1 to 28, comprising a polymer selected from: poly(oxyethylene) / poly(ethylene glycol) copolymers; poly(caprolactone); poly(caprolactone) / poly(ethylene glycol) copolymers; poly(orthoesters); poly(phosphazenes); poly(hydroxybutyrates); poly(hydroxybutyrates); poly(lactide-co-caprolactone); polycarbonates; polyesteramides; polyanhydrides; poly(dioxanone); poly(alkylene alkylates); polyethylene glycol / polyorthoester copolymers; polyurethanes; poly(amino acids); polyetheresters; polyacetals; polycyanoacrylates; poly(oxyethylene) / poly(oxypropylene) copolymers; and combinations thereof. (Item 30) 30. The method of any one of items 1 to 29, wherein the microspheres comprise a non-biodegradable polymer selected from one or more of poly(ethylene vinyl acetate), poly(vinyl acetate), silicone polymers, polyurethanes, polysaccharides, such as cellulosic polymers and derivatives, acyl-substituted cellulose acetate and its derivatives, poly(ethylene glycol), copolymers of poly(butylene terephthalate), polystyrene, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulfonated polyolefins, polyethylene oxide, and copolymers and blends thereof. (Item 31) 31. The method according to any one of the preceding claims, wherein the probiotic bacteria comprises L. reuteri and the prebiotic comprises maltose, glycerol or histidine. (Item 32) 32. The method of claim 31, wherein the L. reuteri produces glucosyltransferase (GFT). (Item 33) 33. The method of any one of the preceding claims, wherein the microspheres comprise dextran or dextranomer. (Item 34) 34. The method according to any one of the preceding claims, wherein the microspheres comprise dextranomer, the probiotic bacteria comprise L. reuteri and the prebiotic comprises maltose. (Item 35) 35. The method of claim 34, wherein the microspheres consist essentially of dextranomer, the probiotic bacteria consist essentially of L. reuteri, and the prebiotic consist essentially of maltose. (Item 36) 36. The method of any one of the preceding claims, wherein the composition comprises microspheres that are substantially identical. (Item 37) 36. The method of any one of the preceding claims, wherein the composition comprises microspheres that differ in composition from one another. (Item 38) 38. The method according to any one of the preceding claims, wherein the composition is prepared by mixing microspheres with biofilm-producing probiotic bacteria and prebiotics, optionally in a medium containing a biofilm. (Item 39) 39. The method of claim 38, wherein preparing the composition further comprises mixing prebiofilmics. (Item 40) 40. The method of any one of items 1 to 39, wherein the composition is prepared by mixing one or more of a prebiofilmic, a therapeutic or therapeutic agent, a chemical reducing agent, a molecule that promotes adsorption, and a molecule that supports absorption, and / or the prebiofilmic comprises an agent that supports biofilm formation and persistence. (Item 41) 41. The method according to item 39 or 40, wherein the prebiofilmics is a DNA binding polypeptide or protein, and / or a DNABII polypeptide or protein, or an equivalent of each of these. (Item 42) The composition is administered to the subject in an amount of about 1×10 7 ~Approx. 1×10 9 42. The method according to any one of items 1 to 41, wherein the method is administered to provide 100 CFU / ml of biofilm-producing probiotic bacteria. (Item 43) 43. The method of any one of the preceding claims, wherein the composition is administered at about 6, 12, 18, 24, 36, 48 and 72 hours after the subject's birth. (Item 44) 44. The method of any one of the preceding claims, wherein the composition is administered in a single dose. (Item 45) 45. The method of any one of items 1 to 44, wherein the composition is formulated in a dosage form selected from the group consisting of a feeding tube, enterally, a suppository, in a biocompatible scaffold, a powder, a liquid, a capsule, a chewable tablet, a swallowable tablet, a buccal tablet, a troche, a lozenge, a soft chew, a liquid, a suspension, a spray, a tincture, a decoction, an infusion, parenterally, and combinations thereof. (Item 46) 46. ​​The method of any one of items 1 to 45, wherein the composition comprises PGLA or dextranomer biocompatible microspheres, one or more biofilm-producing probiotic bacteria comprising at least Lactobacillus reuteri ("L. reuteri"), and a nutritional supplement comprising one or more of maltose, sucrose, glycerol or histidine in an amount to support the growth of the probiotic bacteria, and optionally the microspheres are partially or fully coated with a biofilm. (Item 47) 47. The method of any one of the preceding claims, wherein the microspheres have a diameter in the range of about 0.5 micrometers to 75 micrometers. (Item 48) 48. The method according to any one of the preceding claims, wherein the release of the prebiotic is regulated by varying the microsphere size (smaller microspheres release faster) or by modifying the viscosity of the prebiotic (i.e. higher viscosity releases slower). (Item 49) Preventing said neurodevelopmental deficiency or promoting neurodevelopment includes partial prevention of said deficiency, wherein said partial prevention of said deficiency is achieved by performing the following tests on the subject: ear and eye patency, flat surface righting, air righting, forelimb grasp, acoustic startle, flat surface righting, negative geotaxis, open field crossing, cliff aversion, Barnes maze, elevated plus maze, Jamar dynamometer, manual muscle testing, manual muscle testing (MMT), isokinetic muscle testing, trunk stability test (TST), unilateral hip bridge endurance test (UHB) E), pronation sign, Barre sign, Romberg test, Landau reflex, particle suspension, sensory reflexes (pin pain, superficial touch, position, vibration, and charger), reflexes (biceps, triceps, brachioradial, patellar, and ankle), Moro reflex, tonic neck response, sucking reflex, palmar and plantar grasp reflex, parachute response, somatic neck righting response (NOB), somatic body righting response (BOB), ear opening acoustic reflex, static compliance, physical volume of the ear canal, contralateral reflex, ipsilateral reflex, tympanome Tree, Y-maze, novel object recognition task, STPI (State-Trait Personality Inventory), 5-dimensional curiosity scale, Self-Curiosity Attitude Interest Scale, Curiosity Exploration Scale-II, State-Trait Personality Inventory (STPI), subscales of Sensation Seeking Scale (SSS), Bayley Scale of Infant Development (BSID-III) (1-42 months), Mullen Early Learning Scale (1-68 months), Fagan Intelligence Scale for Infants (FTII) (birth-12 weeks), Griffith Mental Development Scale I (0-2 years), Battelle Development Scale (BDI) ( 49. The method of any one of items 1 to 48, comprising obtaining a score that is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the score obtained by a control subject of the same age and species as the subject but not affected by the condition on one or more of the Vineland Adaptive Behavior Scale (birth-8 years), and the Vineland Adaptive Behavior Scale (0-18 years). (Item 50) Preventing said neurodevelopmental deficiency or promoting neurodevelopment comprises completely preventing said deficiency or delaying the time until said subject exhibits one or more symptoms of said deficiency, wherein completely preventing said deficiency comprises preventing said deficiency by detecting ear and eye patency, flat righting, air righting, forelimb grasp, acoustic startle, flat righting, negative geotaxis, open field crossing, cliff aversion, Barnes maze, elevated plus maze, Jamar dynamometer, manual muscle strength measurement, manual muscle strength testing (M MT), isokinetic strength testing, trunk stability test (TST), unilateral hip bridge endurance test (UHBE), pronation sign, Barre sign, Romberg test, Landau reflex, particle suspension, sensory reflexes (pin pain, superficial touch, position, vibration, and charger), reflexes (biceps, triceps, brachioradial, patellar, and ankle), Moro reflex, tonic neck response, sucking reflex, palmar and plantar grasp reflex, parachute response, somatotopy neck righting response (NOB), somatotopy body righting response (BOB) 49. The method of any one of items 1 to 48, comprising obtaining the same score as a control subject of the same age and species as the subject being treated, but not affected by the disorder, disease or condition, on one or more of any clinically recognized tests selected from the following: STPI (State-Trait Personality Inventory), Five-Dimensional Curiosity Scale, Self-Curiosity Attitudes and Interests Scale, Curiosity Seeking Scale-II, State-Trait Personality Inventory (STPI), subscales of the Sensation Seeking Scale (SSS), Bayley Scales of Infant Development (BSID-III) (1-42 months), Mullen Early Learning Scale (1-68 months), Fagan Scale of Infant Intelligence (FTII) (birth-12 weeks), Griffith's Mental Development Scale I (0-2 years), Battelle Developmental Scale (BDI) (birth-8 years), and Vineland Adaptive Behavior Scales (0-18 years). (Item 51) 51. The method according to item 49 or 50, wherein obtaining the score is performed 0 to 1 month, 1 to 6 months, 6 to 12 months, 12 to 18 months, 18 to 24 months, 24 months to 3 years, or more than 3 years after administration of the composition. (Item 52) 52. The method of any one of the preceding paragraphs, wherein the composition is administered to a fetus of the subject in need of treatment before the fetus is born (i.e., prenatally). (Item 53) 53. The method of any one of items 1 to 52, wherein the composition is administered to a mother pregnant with the subject in need of treatment. (Item 54) 53. The method of any one of items 1 to 52, wherein the composition is administered to the subject having an age selected from 0 to 1 days, 1 to 6 days, 6 to 12 days, 12 to 18 days, 18 to 24 days, 24 to 100 days, or more than 100 days. (Item 55) 55. The method of any one of the preceding claims, wherein the subject is an infant, fetus or newborn. (Item 56) 56. The method of any one of items 1 to 55, wherein the subject is a mammal or a human patient. (Item 57) 57. A kit comprising a composition comprising microspheres, biofilm-producing probiotic bacteria and a prebiotic, wherein said prebiotic comprises a nutritional supplement for said probiotic bacteria, and instructions for use in the method according to any one of items 1 to 56. (Item 58) A kit comprising a composition comprising microspheres, biofilm-producing probiotic bacteria and a prebiotic, wherein the prebiotic comprises a nutritional supplement for the probiotic bacteria, and instructions for increasing BDNF in a subject. (Item 59) A kit comprising a composition comprising microspheres, biofilm-producing probiotic bacteria and a prebiotic, wherein the prebiotic comprises a nutritional supplement for the probiotic bacteria, and instructions for increasing Grin2A in a subject. [Brief description of the drawings]

[0066] [Figure 1] Figures 1A and 1B illustrate that L. reuteri biofilm structural integrity depends on the presence of DNABII family proteins. Confocal microscopy images of in vitro L. reuteri biofilms stained with the LIVE / DEAD BacLight Bacterial Viability Kit (Molecular Probes). L. reuteri biofilms were grown for 24 hours at 37°C and 5% CO2, at which point the films were treated for 16 hours with a 1:50 dilution of either (Figure 1A) rabbit naive serum, (Figure 1B) rabbit anti-integration host factor polypeptide ("IHF"), or plain medium (data not shown). Anti-IHF treatment resulted in a 20% reduction in maximum height, a 35% reduction in average thickness, and a 41% reduction in biomass (data not shown).

[0067] [Diagram 2] Figure 2 illustrates that prebiotic compounds increase the mean thickness and biomass of probiotic biofilms. Addition of 10 μg / ml S.mutans HU to L.reuteri biofilms at the time of inoculation increased the mean thickness and biomass by 33% and 55%, respectively. Addition of 10 μg / ml calf thymus DNA increased the mean thickness by 44% and the biomass by 68%. Addition of 10 μg / ml HU and DNA together resulted in an increased effect compared to either alone, increasing the mean thickness by 53% and the biomass by 78%.

[0068] [Diagram 3]Figure 3 illustrates the in vivo colonization and retention of L. reuteri by a single oral dose. Mice (n=3 / condition) were administered L. reuteri by oral gavage in planktonic, planktonic+PLGA, biofilm, and biofilm+PLGA cultures. After 7 days, mice were sacrificed and L. reuteri 16S rRNA genes were PCR amplified from mouse colon. Probiotics were found in higher percentages in mice treated with biofilm cultures or cultures with PLGA present than in those treated with planktonic.

[0069] [Figure 4] Figure 4 illustrates that L. reuteri biofilms grown with PLGA microspheres and HU reduce splenic colonization by C. rodentium more effectively than biofilms and planktonic L. reuteri. Mice (n=6 / condition) were treated with a single oral gavage of L. reuteri in one of the following forms: planktonic, planktonic+PLGA+HU, biofilm, and biofilm+PLGA+HU (0.115 μg / ml PLGA, 10 μg / ml HU). After 12 hours, mice were gavaged with C. rodentium and sacrificed for necropsy 12 days after infection. Only L. reuteri biofilm+PLGA+HU showed a statistically significant reduction in C. rodentium CFU / g (P=0.0343).

[0070] [Figure 5-1] 5A and 5B show the results of a study confirming that compositions of the present disclosure are consistent with reducing inflammation and antagonizing bacterial pathogens in an animal model of NEC. [Figure 5-2] Same as above.

[0071] [Figure 6]Figures 6A-6C show L. reuteri binding to dextranomer microspheres. Confocal laser scanning microscopy (CLSM) of L. reuteri attached to DM. (Figure 6A) Water-filled DM, (Figure 6B) sucrose-filled DM, and (Figure 6C) maltose-filled DM showed that after 30 min of incubation with L. reuteri, the attachment of L. reuteri to DM was enhanced and it could incorporate biofilm-promoting cargo into the DM lumen (green: bacteria stained with SYTO 9, red: DM stained with Congo red).

[0072] [Figure 7-1] Figures 7A-7C show that microsphere composition and luminal cargo affect L. reuteri adhesion, L. reuteri adheres to DM in a GTF-dependent manner, and bacteria lacking GTF did not bind to DM. Spin column assays were performed to assess relative bacterial adhesion to microspheres. Bacteria were incubated with 5 mg of microspheres for 5 min and centrifuged at 100 × g to separate bound and unbound bacteria, and then the CFU of unattached bacteria in the flow-through fraction of the spin column was quantified. (Figure 7A) Microspheres composed of either cross-linked dextran (DM) or cross-linked cellulose (CM) were loaded with water, growth medium, or various sugars at a concentration of 1 M to determine which microsphere type supported the greatest adhesion of L. reuteri. (Figure 7B) Relative WT and ΔgtfW L. reuteri adhesion to DM showed that L. reuteri adheres to DM in a GTF-dependent manner. (FIG. 7C) Bacteria not expressing GTF were tested for microsphere adhesion as well as water- and sucrose-loaded DM. Error bars represent standard error of the mean. Statistical significance is indicated by the following: *P<0.05, **P<0.01, ***P<0.0005. [Figure 7-2] Same as above.

[0073] [Figure 8]Figure 8. Diffusion of cargo from microspheres over time. Crystal violet (CV)-loaded DM with or without glycerol (added to increase viscosity) was assayed to determine the relative rates of CV diffusion from microspheres. With 0% glycerol addition, CV diffused faster (100% diffusion after 10 hours) compared to DM that contained 40% or 80% glycerol. Applicants observed 100% diffusion from DM after 16 hours regardless of viscosity. Error bars represent standard error of the mean. Statistical significance from DM with 0% glycerol addition is indicated by the following: *P<0.05, **P<0.01, ****P<0.0001.

[0074] [Figure 9] Figure 9 shows that histamine can be produced by L. reuteri from L-histidine delivered by DM. Stationary-phase WT L. reuteri were incubated for 2 hours with either saline with or without 3% maltose or 2% glycerol, or with 4 mg / ml L-histidine with or without 3% maltose or 2% glycerol. Histamine production increased with the addition of 3% maltose to the 4 mg / ml L-histidine solution (white bars with black border) compared to 4 mg / ml L-histidine alone (black bars, and grey bars with black border). When 4 mg / ml L-histidine was provided by DM, the total level of histamine produced was significantly lower (three middle bars) compared to L-histidine provided in solution (four bars on the left), likely due to less L-histidine being readily available to the bacteria. However, when the concentration of L-histidine loaded into the DM was increased to 30 mg / ml, significantly more histamine was produced (three bars on the right), despite any caveats regarding slower access to L-histidine due to it only being available by diffusion from the DM. Error bars represent standard error of the mean. Statistical significance is indicated by the following: *P<0.05, **P<0.01.

[0075] [Figure 10] FIG. 10 shows survival in gastric acid. Viability of WT and ΔgtfW L. reuteri (107 CFU / ml) after 4 hours in synthetic gastric acid at pH 2 in the absence or presence of 5 mg DM containing water, sucrose (1M) or maltose (1M) as cargo, or 10 μl cargo alone without DM. When WT L. reuteri was attached as a biofilm on DM containing sucrose or maltose, the relative survival in acid was enhanced compared to the same volume of the same cargo delivered without DM, indicating that the biofilm phenotype contributes to better survival during exposure to low pH. ΔgtfW showed reduced resistance to acid compared to WT, regardless of the presence or absence of DM or sugar alone. Error bars represent standard error of the mean. Statistical significance is indicated by: *P<0.05, **P<0.01.

[0076] [Figure 11]Figures 11A and 11B show that delivery of L. reuteri attached to DM as a biofilm supported increased attachment to intestinal epithelial cells. (Figure 11A) L. reuteri WT and ΔgtfW attached as biofilms on DM containing either water, sucrose (1M) or maltose (1M), or to an equal volume of sugar alone (without DM), were examined for relative attachment to human colon DLD-1 cells after 60 minutes of incubation. When delivered as a biofilm on the surface of DM containing sucrose or maltose, significantly more WT attached to DLD-1 cells compared to water-filled DM or an equal volume of sugar alone. Regardless of cargo, significantly fewer ΔgtfW mutant cells attached to DLD-1 cells, indicating that GTFW proteins contribute to L. reuteri attachment. (FIG. 11B) Attachment of WT to fetal intestinal FHs 74 cells after 60 min of incubation showed that presenting L. reuteri attached on the DM surface as a biofilm with either sucrose or maltose as cargo resulted in greater attachment to intestinal cells. Error bars represent standard error of the mean. Statistical significance is indicated by the following: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0077] [Figure 12-1]Figures 12A and 12B show that increased attachment to DLD-1 colonic epithelial cells is observed when L. reuteri is delivered as a biofilm bound to DM. (Figure 12A) In vitro CLSM of DLD-1 epithelial cells (blue, DAPI), L. reuteri (green, CFSE), and DM (red, Congo red). WT L. reuteri (top four rows) compared to ΔgtfW L. reuteri (middle four rows) and no L. reuteri (bottom row). Bacteria and DM were prestained, incubated on prestained DLD-1 epithelial cells for 1 h, washed three times, and fixed for CLSM analysis. (Figure 12B) Comparison of bacterial biomass quantified by COMSTAT analysis of the green channel of CLSM images of WT and ΔgtfW L. reuteri. WT without DM (n=10) resulted in less bacterial signal compared to either WT+DM-sucrose (n=10) or WT+DM-maltose (n=10). ΔgtfW showed no difference in the relative number of bacteria attached to DLD-1 cells with or without DM. Error bars represent standard error of the mean. Statistical significance is indicated by: *P<0.05, **P<0.01. [Figure 12-2] Same as above.

[0078] [Figure 13] 13A-13F are illustrations of DM cargo loading, filtration, and addition to bacterial cultures. (Figure 13A) Dehydrated DM and desired cargo (e.g., 1 M maltose) are incubated together to allow diffusion of the solution into the DM. (Figure 13B) The DM+ solution is vortexed and pipetted into a vacuum filtration system. (Figure 13C) Excess solution is removed by vacuum filtration, leaving only the DM with absorbed cargo. (Figure 13D) The DM cargo pellet can then be removed from the vacuum filter by scraping with a sterile loop. (Figure 13E) The DM- cargo pellet is transferred to the bacterial solution, typically bacteria resuspended in saline. (Figure 13F) The final product is a solution containing bacteria+DM- cargo together, and thus this solution can be used for downstream applications (e.g., assays, oral gavage, etc.).

[0079] [Figure 14] Figures 14A and 14B are illustrations of the spin column DM adhesion assay. (Figure 14A) A mixture of bacteria + DM-cargo is incubated together on top of a spin column filter in a 1.5 or 2.0 ml microcentrifuge tube. After the desired incubation time (e.g., 5 min), the tube + column is centrifuged at ≦100×g to separate bacteria attached to the DM from those not attached. (Figure 14B) After centrifugation, non-attached cells will be present in the flow-through at the bottom of the microcentrifuge tube, while bacteria attached to the DM will remain on the surface of the filter along with the DM (the pore size of the filter is too small for the DM to pass through, but large enough for the bacterial cells). The cells present in the flow-through are counted by serial dilution plating. A bacteria-only (no DM) control is used as the baseline, and all DM experiments are subtracted from the baseline.

[0080] [Figure 15-1]Figures 15A and 15B show that sucrose induces gtfW, whereas maltose is a substrate for GTFW. (Figure 15A) A gtfW transcriptional reporter was constructed by fusing click beetle luciferase downstream of the gtfW promoter in a plasmid, which was then introduced into L. reuteri (strain LMW 501). Expression of gtfW in MRS, with or without the indicated additions, was monitored throughout growth by removing 100 μl aliquots every hour and measuring OD600nm. An additional 80 μl aliquot was removed and 20 μl of 2 mM D-luciferin was added and left to incubate at RT for 5 min, after which luminescence was detected. (Figure 15B) GTFW enzyme activity. Proteins extracted from S. mutans, L. reuteri WT, L. reuteri ΔgtfW (strain LMW 500), and E. coli carrying gtfW on an inducible plasmid (Ec) (strain LMW 502) were subjected to SDS-PAGE followed by PAS staining to examine GTFW enzyme activity. 5% sucrose or 5% maltose was used as substrate. Arrows indicate GTFW activity. [Figure 15-2] Same as above.

[0081] [Figure 16-1]Figures 16A-16D show that GTFW contributed to early biofilm formation in growth media supplemented with sucrose or maltose. L. reuteri WT and ΔgtfW were seeded in 8-well borosilicate chamber slides and incubated for 1, 3 or 6 h at 37°C, 5% CO2. At the indicated time intervals, bacteria were stained with LIVE / DEAD stain for viability, fixed, visualized by confocal microscopy (CLSM) and quantified by COMSTAT analysis of fluorescent signals. (Figure 16A) CLSM of L. reuteri biofilms at 1, 3 and 6 h showed significantly more bacteria present and increased aggregation of WT bacteria compared to the gtfW mutant (left column) in conditions with sucrose or maltose at 1 h. This was confirmed by quantification of green fluorescent signals (Figure 16B). The GTF-dependent increase in biofilms in the presence of sucrose or maltose increased after 3 h (Fig. 16A-middle column, and Fig. 16C) and further increased after 6 h (Fig. 16A-right column, and Fig. 16D). The gtfW mutant, unable to utilize maltose for biofilm formation, still benefited from sucrose in the growth medium after 1 h, which could be attributed to an increased growth rate (data not shown). Error bars represent standard error of the mean. Statistical significance is indicated by: *P<0.05, **P<0.01. [Figure 16-2] Same as above. [Figure 16-3] Same as above.

[0082] [Figure 17] Figure 17 shows that L. reuteri can produce reuterin from glycerol-loaded microspheres. L. reuteri incubated for 1 h with DM containing 0-80% glycerol as the only glycerol source in this experimental condition was measured for relative reuterin production. For comparison, the amount of reuterin produced by L. reuteri without DM in 2% glycerol solution (dotted line) was used as a control. Error bars represent standard error of the mean.

[0083] [Figure 18] Figure 18 shows that glycerol delivered by DM and the metabolites produced thereafter did not affect L. reuteri survival. Overnight cultures of WT L. reuteri were washed and resuspended in either saline or MRS medium. 5 mg of DM-water or DM-80% glycerol was then added to L. reuteri and incubated at 37°C. Every hour, aliquots were taken for subsequent serial dilutions and plating of viable CFU. After 24 hours, there was no significant difference between cultures incubated in the same medium (saline or MRS) containing either DM-water or DM-80% glycerol.

[0084] [Figure 19] Figure 19 illustrates that the maximum conversion of glycerol provided by DM to acrolein did not result in toxic levels of acrolein. The World Health Organization (WHO) recommends an intake of 7.5 μg / kg body weight or less of acrolein per day. Assuming 100% conversion of available glycerol provided by DM to acrolein by L. reuteri, a maximum of 6.24 μg of acrolein was produced by the L. reuteri and DM-glycerol dosage utilized in this study (red arrow). The dashed line (50 μg acrolein) represents <10% of the daily tolerable amount of acrolein for a 70 kg human.

[0085] [Figure 20]Figure 20 shows that L. reuteri delivered as a biofilm on DM does not inhibit adhesion to mucin. L. reuteri reporter expressing click beetle luciferase was distributed onto agar plates containing either 2% mucin + 0.8% agar or 0.8% agar, either in planktonic state or as a biofilm on the DM surface, incubated at room temperature for 1 hour, and then washed to remove unattached L. reuteri. D-luciferin (0.4 mM) was then added to the plates, and the plates were imaged for luminescence signals arising from remaining attached bacteria. To calculate the amount of bacteria attached only to mucin, the relative luminosity of the agar-only plate was subtracted from the relative luminosity of the mucin + agar plate.

[0086] [Figure 21-1] Figures 21A and 21B show that L. reuteri adhered to DM and bound to the surface of DLD-1 human colonic epithelial cells. In vitro SEM of L. reuteri and DM on a confluent monolayer of DLD-1 cells. Bacteria and DM were incubated on DLD-1 epithelial cells for 1 hour, washed three times, fixed and prepared for SEM analysis. Magnifications of 400x (Figure 21A) and 2500x (Figure 21B) showed L. reuteri attached to DM (yellow box) and several clusters of L. reuteri without DM attached to the surface of DLD-1 (white arrows). [Figure 21-2] Same as above.

[0087] [Figure 22-1]Figures 22A and 22B show the incidence and severity of NEC. Figure 22A is an H&E stained intestinal tissue section demonstrating the following grades of histological damage: grade 0, no visible histological villus injury; grade 1, detachment of distal intestinal villus cells; grade 2, sloughing of enterocytes to the mid-villus level; grade 3, loss of all villi with preservation of crypts; and grade 4, transmural necrosis. Grade 2 injury and higher is consistent with histological NEC. All images are at 20x magnification. Figure 22B shows that rat pups were delivered prematurely, subjected to the experimental NEC protocol, and sacrificed when clinical NEC signs occurred or after 96 hours. Each point represents a single rat pup with their histological injury score indicated. The incidence of NEC for each experimental group of rat pups is shown. *p<0.05. [Figure 22-2] Same as above.

[0088] [Diagram 23] Figure 23 shows pup survival. The number of pups alive and free of endpoint criteria (lethargy, bloody stools, end-respiration, cyanosis) is plotted by experimental group at 8 hour intervals over the course of the 96 hour experimental NEC protocol.

[0089] [Figure 24] Figure 24 shows intestinal permeability in rat pups subjected to experimental NEC. Intestinal permeability was determined by measuring serum levels of FITC-dextran 4 hours after intragastric administration of FITC-dextran. Higher serum FITC-dextran levels indicate greater intestinal permeability. FITC, fluorescein isothiocyanate. *p<0.05.

[0090] [Diagram 25] Figure 25 shows Lr persistence in the GI tract. Bioluminescent strains of Lr were generated and used to track the presence of Lr (as the amount of light emitted) in the small and large intestine after 48 hours of the experimental NEC protocol. RLU, relative luminescence units. *p<0.05.

[0091] [Figure 26-1] Figures 26A-26E show inflammatory markers. Intestinal specimens were collected and fixed in formalin. RNA was isolated and analyzed by real-time qPCR for expression of (Figure 26A) IL-6, (Figure 26B) IL-1β, (Figure 26C) CCL-2, (Figure 26D) CXCL-1, and (Figure 26E) IL-10. Results represent the mean ± SEM of 7-10 different rat pups performed in duplicate. *p<0.05. [Figure 26-2] Same as above. [Figure 26-3] Same as above.

[0092] [Figure 27] Figure 27 shows the incidence and severity of NEC. Rat pups were delivered early, given a single enteral treatment as indicated, and then subjected to the experimental NEC protocol. Rat pups were sacrificed at the time of clinical NEC signs or 96 hours later, intestinal tissues were collected, and H&E sections were graded to determine the extent of intestinal injury. The incidence of NEC for each experimental group of pups is shown. The percentage of pups with grade 2, grade 3, and grade 4 damage is depicted for each treatment group. *p<0.05.

[0093] [Figure 28] Figure 28 shows the results for a unique triad of rats subjected to the Y-maze test. "Breast-fed" indicates control rats, "NEC-stress" indicates rats with NEC not administered Lactobacillus reuteri (Lr), "NEC-stress+Lr" indicates rats with NEC administered free form Lr, and "NEC-stress+Lr-DM-Maltose" indicates rats with NEC administered maltose-loaded dextranomer microspheres (DM) incubated with Lr to form biofilms.

[0094] [Figure 29]Figure 29 shows the results of the novel object recognition task. The different test groups for the rats are as indicated in the legend of Figure 28.

[0095] [Diagram 30] Figure 30 shows a neonatal rat model of experimental NEC. Neonatal rats were delivered prematurely by Caesarean section. To induce NEC, rat pups were subjected to a high calorie diet and repeated episodes of hypoxia / hypothermia for 96 hours.

[0096] [Diagram 31] Figure 31 shows the study schedule. Newborn rats were delivered prematurely by Caesarean section on E20.5. To induce NEC, rat pups were subjected to high calorie diet and repeated episodes of hypoxia / hypothermia x 96 hours. Surviving rat pups were placed with nursing mothers and subjected to daily developmental milestone testing for 23 days. Cognitive function and memory testing were performed between 4 and 8 weeks of age. At 2 months of age, rats were sacrificed and tissues were harvested.

[0097] [Diagram 32] Figures 32A-32H show developmental milestones after experimental NEC. Newborn rats were delivered prematurely by Caesarean section. To induce NEC, rat pups were subjected to high calorie diet and repeated episodes of hypoxia / hypothermia for 96 hours. Surviving rat pups were placed with nursing mothers and developmental milestones were measured daily for 23 days. (Figure 32A,B) Air righting test; (Figure 32C,D) Forelimb grasp test; (Figure 32E,F) Ear patenting; (Figure 32G,H) Acoustic startle. Error bars represent SEM and statistical significance was defined as p≦0.05 (one-way ANOVA).

[0098] [Diagram 33]Figures 33A-33D show the Y-maze and novel object test after experimental NEC. Rat pups were subjected to experimental NEC. Surviving rat pups were placed with surrogate mothers. After weaning, behavioral activity of the rats was measured. (Figure 33A, Figure 33B) Y-maze test; (Figure 33C, Figure 33D) Novel object test. Error bars represent SEM, and statistical significance was defined as p≦0.05 (one-way ANOVA).

[0099] [Diagram 34] Figures 34A-34C show the results of the Barnes maze test after experimental NEC. Rat pups were subjected to experimental NEC. Surviving rat pups were placed with surrogate mothers. After weaning, the behavioral activity of the rats was measured: (Figure 34A) Barnes maze setup; (Figure 34B) number of holes checked on the test day; and (Figure 34C) latency to find the escape hole. Error bars represent SEM and statistical significance was defined as p < 0.05 (one-way ANOVA).

[0100] [Diagram 35] Figures 35A-35C show the results of the elevated plus maze test after experimental NEC. Rat pups were subjected to experimental NEC. Surviving rat pups were placed with surrogate mothers. After weaning, behavioral activity of rats was measured. (Figure 35A) Elevated plus maze setup; (Figure 35B) Time in open lane and branch; (Figure 35C) Time in walled lane. Error bars represent SEM and statistical significance was defined as p < 0.05 (one-way ANOVA).

[0101] [Diagram 36]Figures 36A-36C show microglia in the brain 2 months after experimental NEC. Rat pups were subjected to experimental NEC and surviving pups were placed in surrogate mothers. Rats were weaned at 21-23 days of age and sacrificed at 2 months of age. Brains were collected and fixed in 4% PFA. Frozen sections were stained with Iba-1 antibody and Alexa 488-conjugated secondary antibody. At least 8 confocal pictures were taken per section using a 20x objective. (Figure 36A) Representative immunohistochemical images of the whole brain; (Figure 36B) Activated and amoeboid microglia counts / HPF; (Figure 36C) Percentage of Iba-1+ cells / HPF quantified using Image J software. Error bars represent SEM and statistical significance was defined as p≦0.05 (one-way ANOVA).

[0102] [Figure 37-1] Figures 37A-37D show neurotrophic gene expression after experimental NEC. Rat pups were subjected to experimental NEC and surviving pups were placed in surrogate mothers. Rats were sacrificed at 2 months of age, brains were collected, and the prefrontal cortex (PFC) and hippocampus were harvested. Gene expression was measured by RT-qPCR for (Figure 37A, Figure 37B) BDNF and (Figure 37C, Figure 37D) Grin2A. Relative copy numbers from independent rats are shown. Error bars represent SEM and statistical significance was defined as p≦0.05 (one-way ANOVA). [Figure 37-2] Same as above.

[0103] [Figure 38] Figure 38 shows the weight of rats after experimental NEC. Newborn rats were delivered prematurely by Caesarean section on E21. To induce NEC, rat pups were subjected to a high calorie diet and repeated episodes of hypoxia / hypothermia x 96 hours. Surviving rat pups were placed with nursing mothers and weighed daily for 21 days. Pup weights at postnatal day (DOL) 4, 10, 15 and 21 are shown. Error bars represent SEM and statistical significance was defined as p < 0.05 (one-way ANOVA).

[0104] [Figure 39] Figures 39A-39I show additional developmental milestones after experimental NEC. Newborn rats were delivered prematurely by Caesarean section on E21. To induce NEC, rat pups were subjected to high calorie diet and repeated episodes of hypoxia / hypothermia x 96 h. Surviving rat pups were placed with nursing animals and developmental milestones were measured daily for 23 days: (Figure 39A, Figure 39B) surface righting test; (Figure 39C, Figure 39D) negative geotaxis test; (Figure 39E, Figure 39F) open field test; (Figure 39G, Figure 39H) cliff aversion test; and (Figure 39I) postnatal days at eye opening. Error bars represent SEM and statistical significance was defined as p<0.05 (one-way ANOVA). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0105] It is to be understood that the invention is not limited to the particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the invention is limited only by the appended claims.

[0106] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0107] The practice of the present technology will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, within the skill of the art. For example, Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., NY); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach;Harlow and Lane eds.(1999) Antibodies, A Laboratory Manual;Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis;US Patent No. 4,683,195;Hames and Higgins eds.(1984) Nucleic Acid Hybridization;Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds.(1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir's Handbook of Experimental Immunology.

[0108] All numerical designations, including ranges, such as pH, temperature, time, concentration and molecular weight, are approximate values ​​that vary (+) or (-) by increments of 1.0 or 0.1, or alternatively by a variance of + / -15%, or alternatively by 10%, or alternatively by 5%, or alternatively by 2%, as appropriate. It will be understood, although not always expressly stated, that all numerical designations are preceded by the term "about". It will also be understood, although not always expressly stated, that the reagents described herein are merely exemplary, and that equivalents of such are known in the art.

[0109] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a bacterium" includes a plurality of bacteria, including mixtures thereof.

[0110] As used herein, the term "comprising" is intended to mean that the compositions and methods include the elements described, but do not exclude others. "Consisting essentially of" when used to define compositions and methods shall mean excluding elements other than those that are essentially significant to the combination for the intended use. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants from the isolation and purification methods, as well as pharma- ceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of" shall mean excluding trace elements of other components and more than substantial method steps for administering the compositions of the invention. Embodiments defined by each of these transition terms are within the scope of the invention.

[0111] "Biofilm" refers to a thin layer or organized community of microorganisms that can attach, sometimes together with polymers such as DNA that they secrete and / or release, to the surface of structures that can be organic or inorganic. Biofilms are highly resistant to microbiotic and antimicrobial agents. Biofilms are present on gum tissue, teeth and restorations and cause caries and periodontal disease, also known as periodontal plaque disease. Biofilms also cause chronic otitis media. Biofilms also form on dental implants, stents, catheter lines and contact lenses. Biofilms also grow on pacemakers, replacement heart valves, artificial joints and other surgical implants. The Centers for Disease Control estimate that more than 65% of nosocomial (hospital-acquired) infections are caused by biofilms. Fungal biofilms also frequently contaminate medical devices. Fungal biofilms cause chronic vaginal infections and lead to life-threatening systemic infections in those with compromised immune systems. Biofilms are also involved in numerous diseases. For example, cystic fibrosis patients have Pseudomonas infections that often result in the development of antibiotic-resistant biofilms.

[0112] "Prebiotic" refers to a nutritional supplement for probiotic bacteria. Prebiotics are food ingredients, such as oligosaccharides, that are not digestible by a subject (e.g., a mammal, such as a human), and that stimulate the growth or activity of one or more beneficial bacteria and / or inhibit the growth or activity of one or more pathogenic bacteria. Prebiotics can selectively stimulate the growth and / or activity of one or a limited number of bacteria in a subject.

[0113] By "prebiofilmics" is intended a substance that supports the formation and persistence of a biofilm, for example, a prebiofilmic can be a substance that supports the extracellular matrix of a biofilm, such as an eDNA-binding polypeptide or protein, or alternatively, a substance that can be converted to a substance that promotes attachment, such as sucrose.

[0114] "DNABII polypeptide or protein" refers to a DNA-binding protein or polypeptide that is composed of a DNA-binding domain and thus has a specific or general affinity for DNA. In one embodiment, they bind in the minor groove of DNA. Non-limiting examples of DNABII proteins are the integration host factor (IHF) protein and the histone-like protein from E. coli U93 strain (HU), examples of which are provided in the attached sequence listing, and further strains and polypeptides are provided in Table 5. Polypeptide fragments and equivalent polypeptides with amino acid modifications that do not substantially change the biological activity of the protein or polypeptide or its active fragments are also contemplated. An active fragment may, for example, include the c-terminal half or the c-terminal one-third of the protein or polypeptide. Other DNA-binding proteins that may associate with biofilms include DPS (Genbank Accession No. CAA49169), H-NS (Genbank Accession No. CAA47740), Hfq (Genbank Accession No. ACE63256), CbpA (Genbank Accession No. BAA03950) and CbpB (Genbank Accession No. NP_418813), and equivalent polypeptides and active fragments thereof.

[0115] "Microsphere" means a porous and / or semi-permeable biofilm-bearing and / or compound-bearing (eg, drug-bearing) particle or granular material within the particular size range stated. As used herein, a microsphere is comprised of particles having a diameter of 50 millimeters or less and a diameter of about 1 micrometer or greater (e.g., about 1 to about 100, or alternatively, about 1 to about 75 micrometers, or alternatively about 1 to about 50, or alternatively about 1 to about 25, or alternatively about 1 to about 10 micrometers, or alternatively about 0.5 to about 200 micrometers, or alternatively about 0.5 to about 700 micrometers, or alternatively about 1 to about 600 micrometers, or alternatively less than about 700 micrometers, or alternatively less than about 600 micrometers, or alternatively less than 500 micrometers, or alternatively less than about 400 micrometers, or alternatively less than about 300 micrometers, or alternatively less than about 200 micrometers, or alternatively less than about 100 micrometers). Non-limiting examples of such include hollow microspheres that are porous and / or semipermeable and in some embodiments contain pharmaceutical or drug substances, microcapsules (in which excipients form an envelope or shell that surrounds and contains a cargo such as a drug, a chemical reducing agent, or an adsorbing or absorbing molecule), and microparticles, which are used as a generic term for any particle in the stated size range, whether spherical or not, as these terms are typically used in the art. Table 6 provides non-limiting examples of commercially available microspheres and their properties.

[0116] "In one embodiment, aubject" as used herein refers to a subject having sepsis or a sepsis-causing condition (i.e., NEC), exposed to a condition that develops sepsis or a sepsis-causing condition, or at risk of sepsis or a sepsis-causing condition, and receiving treatment with a composition according to the present invention. The subject may be a subject suffering from, susceptible to, or suffering from necrotizing enterocolitis. The subject may be an infant, fetus, or newborn. In one embodiment, the subject is an infant subject of a certain age selected from 0-1 day, 1-6 days, 6-12 days, 12-18 days, 18-24 days, 24 days-100 days, or more than 100 days.

[0117] "Control subject" refers to either 1) a subject suffering from, susceptible to, or afflicted with a disease or condition, e.g., sepsis, or a condition that causes the same sepsis as in infant subjects (i.e., NEC), or autism, or an autism spectrum disorder), but (btu) who has not been administered the composition, and who is of the same age and species as the subject being treated, or 2) a subject of the same age and species as the subject being treated, but not suffering from the disease or condition.

[0118] "Biodegradable polymer" means a polymer that is biocompatible and can be broken down in vivo by bodily processes into products that are readily processable by the body and therefore should not accumulate in the body.

[0119] "Biocompatible" means that the components of the delivery system do not cause tissue injury or injury to the human biological system. To impart biocompatibility, polymers and excipients that have a history of safe use in humans or have GRAS (Generally Recognized As Safe) status are preferentially used. Biocompatibility means that the components and excipients used in the composition are ultimately "absorbed" or eliminated by the body without adverse effects on the body. For a composition to be considered biocompatible and non-toxic, it must not cause toxicity to cells. Similarly, the term "bioresorbable" refers to microspheres made of materials that are bioresorbed over time in vivo to avoid long-term accumulation of materials in the patient's body. Biocompatible nanoparticles are absorbed into the body over a period of less than two years, preferably less than one year, and even more preferably less than six months. The rate of bioresorption is related to the size of the particle, the materials used, and other factors well recognized by those skilled in the art. Mixtures of bioresorbable, biocompatible materials can be used to form the microspheres used in the present invention.

[0120] "Integration host factors" or "IHF" proteins are bacterial proteins used by bacteriophages to integrate their DNA into host bacteria. They are DNA-binding proteins that function in genetic recombination and in transcriptional and translational regulation. They also bind to extracellular microbial DNA. Genes encoding IHF protein subunits in E. coli are himA (Genbank Accession No. POA6X7.1) and himD (POA6Y1.1) genes. Non-limiting examples of such are provided in the attached sequence listing and set forth in Table 5.

[0121] "HU" or "histone-like protein from E. coli strain U93" refers to a class of heterodimeric proteins typically associated with E. coli. HU proteins are known to bind to DNA junctions. Related proteins have been isolated from other microorganisms. The complete amino acid sequence of E. coli HU was reported by Laine et al. (1980) Eur. J. Biochem. 103(3):447-481. Antibodies against HU proteins are commercially available from Abcam. Non-limiting examples of such are provided in the attached sequence listing.

[0122] The terms "protein", "peptide" and "polypeptide" are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits may be linked by peptide bonds. In alternative embodiments, the subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide contains at least two amino acids, and there is no limit to the maximum number of amino acids that may make up a protein's or peptide's sequence. As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acid, including glycine and both D and L optical isomers, amino acid analogs and peptidomimetics.

[0123] "C-terminal polypeptide" refers to the C-terminal half or C-terminal third of a polypeptide. By way of example, for a polypeptide containing 90 amino acids, the C-terminal polypeptide includes amino acids 46 through 90 or amino acids 60 through 90. In another embodiment, the term refers to the C-terminal 20 amino acids from the carboxy terminus.

[0124] "N-terminal polypeptide" refers to the n-terminal half of a polypeptide. By way of example, for a polypeptide containing 90 amino acids, the c-terminal polypeptide includes amino acids 1 through 45. In another embodiment, the term refers to the c-terminal 20 amino acids from the amino terminus.

[0125] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure, if present, can be imparted prior to or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

[0126] A polynucleotide is composed of a specific sequence of the four nucleotide bases adenine (A), cytosine (C), guanine (G), and thymine (T), with uracil (U) replacing thymine if the polynucleotide is RNA. Thus, the term "polynucleotide sequence" is an alphabetical representation of a polynucleotide molecule. This alphabetical representation can be entered into a database of a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.

[0127] The terms "isolated" or "recombinant" as used herein with respect to nucleic acids, such as DNA or RNA, refer to molecules separated from other DNA or RNA, respectively, present in the natural source of the polymer as well as polypeptides. The term "isolated or recombinant nucleic acid" is intended to include nucleic acid fragments that do not occur naturally as fragments and are not found in nature. The term "isolated" is also used herein to refer to polynucleotides, polypeptides, antibodies and proteins that are isolated from other cellular proteins and is intended to encompass both purified and recombinant polynucleotides. In other embodiments, the term "isolated or recombinant" means separated from cellular and other components with which it is normally associated in nature in a cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof. For example, an isolated cell is one that is separated from tissues or cells of a different phenotype or genotype. An isolated polynucleotide is separated from the contiguous 3' and 5' nucleotides with which it is normally associated in its native or natural environment, e.g., on a chromosome. As will be apparent to one of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragment(s) thereof does not require "isolation" in order to distinguish it from its naturally occurring counterpart.

[0128] Glucotransferase is an enzyme that establishes glycosidic bonds. Non-limiting examples of GTF protein sequences are available in DSM 20016. gtfW ABQ83597.1 is provided in DSM 17938 gtfA WP _003671465. See also Walter et al. (2008) Microbiology 154(Pt 1):72-80.

[0129] Unless expressly stated and unless otherwise intended, when the present invention relates to a polypeptide, protein, polynucleotide or antibody, it is to be presumed that equivalents or biological equivalents of such are intended within the scope of the present invention. As used herein, the term "biological equivalent thereof" is intended to be synonymous with "equivalent thereof" when the reference to a reference protein, antibody, polypeptide, polynucleotide or nucleic acid is intended to have a minimal degree of homology and still maintain the desired structure or functionality. Unless otherwise stated herein, any nucleic acid, polynucleotide, polypeptide or protein referred to herein is intended to include its equivalent. For example, equivalent means a homology or identity percentage of at least about 70%, or alternatively 80%, across the protein or a particular fragment thereof, and alternatively at least about 85%, or alternatively at least about 90%, or alternatively at least about 95%, or alternatively 98%, and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid.

[0130] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned, a percentage of bases (or amino acids) are the same comparing the two sequences. Alignment and homology or percent sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST using default parameters. Specifically, the preferred programs are BLASTN and BLASTP, using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expectation=10; matrix=BLOSUM62; description=50 sequences; sort order=high score; database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0131] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for purposes of comparison. If a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A "non-related" or "non-homologous" sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present invention.

[0132] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in eukaryotic cells.

[0133] The term "encodes," as the term is applied to a polynucleotide, refers to a polynucleotide that is said to "encode" a polypeptide when, in its natural state, or when manipulated by methods well known to those of skill in the art, it can be transcribed and / or translated to produce mRNA for the polypeptide and / or fragments thereof. The antisense strand is the complementary strand of such a nucleic acid, from which a coding sequence can be deduced.

[0134] A "subject" or "patient" of diagnosis or treatment is a cell or an animal, e.g., a mammal or a human. Non-human animals are those that are undergoing diagnosis or treatment, are the subject of infectious diseases or animal models, e.g., monkeys, murines, e.g., rats, mice, chinchillas, canines, e.g., dogs, lagomorphs, e.g., rabbits, farm animals, sport animals, and pets.

[0135] As used herein, the terms "treat", "treatment" and the like are used herein to mean obtaining a desired pharmacological and / or physiological effect. This effect may be prophylactic, in terms of preventing, completely or partially, the disorder or its signs or symptoms, and / or may be therapeutic, in terms of curing, partially or completely, the disorder and / or adverse effects resulting from the disorder.

[0136] "Preventing" means preventing a disorder or effect in vitro or in vivo in a system or subject predisposed to the disorder or effect. An example of such would be preventing the formation of a biofilm in a system infected with a microorganism known to produce biofilms or alternatively prevent gastrointestinal disorders by supporting the intestinal health of the patient.

[0137] The term "culturing" refers to the propagation of cells or organisms in vitro on or in various types of media. It is understood that the progeny of cells grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell. "Expanded" refers to any proliferation or division of cells.

[0138] "Pharmaceutically acceptable carrier" refers to any diluent, excipient or carrier that can be used in the composition of the present invention. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in the field. They are preferably selected with respect to the intended form of administration, i.e. oral tablets, capsules, elixirs, syrups, etc., and in accordance with conventional pharmaceutical practice.

[0139] "Biocompatible scaffold" refers to a scaffold or matrix that is capable of supporting biofilm growth upon administration to a subject. In other embodiments, the biocompatible scaffold is a precursor to an implantable device that is capable of performing its intended function, has a desired degree of incorporation into the host body, and does not cause undesirable local or systemic effects in the host. Biocompatible scaffolds are described in U.S. Patent Nos. 6,638,369 and 8,815,276. In one aspect, the microspheres described herein are biocompatible scaffolds.

[0140] "Administration" refers to the delivery of a substance to a subject, such as an animal or human. Administration can be continuous or intermittent throughout the course of treatment in one dose. Methods for determining the most effective means and dosage of administration are known to those skilled in the art and vary with the composition used for treatment, the purpose of the treatment, and the age, health, or sex of the subject being treated. Single or multiple administrations can be made, with the dosage level and pattern selected by the attending physician or, in the case of pets and animals, by the attending veterinarian. Suitable dosage formulations and methods of administration of agents are known in the art. Routes of administration can also be determined, and methods for determining the most effective route of administration are known to those skilled in the art and vary with the composition used for treatment, the purpose of the treatment, the health or disease stage of the subject being treated, and the target cells or tissues. Non-limiting examples of routes of administration include enterally, orally, vaginally, nasally (inhalation), injection, topical application, and by suppository.

[0141] The term "effective amount" refers to an amount sufficient to achieve a beneficial or desired result or effect. For therapeutic or prophylactic applications, the effective amount depends on the type and severity of the condition in the issue, as well as the characteristics of the individual subject, such as general health, age, sex, weight, and tolerance to the pharmaceutical composition. For therapeutic compositions, in some embodiments, the effective amount is an amount sufficient to generate a defense response against pathogens, or alternatively, to support a healthy body condition. In some embodiments, this amount is an amount sufficient to accomplish one or more of the following: 1) eliminate pathogens; 2) restore healthy microflora; 3) modulate the immune system; 4) maintain metabolism and metabolic pathways; 5) reduce toxic compounds in the environment (toxic compounds in water, soil, air, and compounds such as heavy metals (e.g., chromium, arsenic, mercury, radioactive actinides, uranium, plutonium, thorium, polycyclic aromatic hydrocarbons (PAHs), petroleum hydrocarbons, crude oil, refined oil, herbicide contaminants, or pesticide contaminants); and 6) modify biofilms.

[0142] "Physical strength" refers to the ability of a subject to perform physical work, also known in the art as muscle force production. "Coordination" refers to the organization of different components of a complex body or activity so that they can work together effectively. Physical strength and / or coordination in infants can be measured by clinical tests known to those skilled in the art, such as Jamar dynamometer, manual muscle test, manual muscle test (MMT), isokinetic muscle test, trunk stability test (TST), unilateral hip bridge endurance test (UHBE), pronation sign, Barre sign, Romberg test, Landau reflex, particle suspension, sensory reflex (pin pain, superficial touch, position, vibration, and charger), reflex (biceps, triceps, brachioradial, patellar, and ankle), Moro reflex, tonic neck response, sucking reflex, palmar and plantar grasp reflex, and parachute response.

[0143] The "righting mechanism" or righting reflex, also known as labyrinthine righting reflex, is a reflex that corrects the orientation of the body when the body is positioned out of its normal upright position. The righting mechanism can be measured clinically using one or more recognized techniques known to those skilled in the art, such as the cervical righting reaction (NOB) or the bodily righting reaction (BOB).

[0144] "Acoustic reflex" refers to the reflex of the ear upon the introduction of a stimulus. Acoustic reflex can be measured by clinical techniques known to those skilled in the art, such as the ear opening acoustic reflex, static compliance, the physical volume of the ear canal, the contralateral reflex, the ipsilateral reflex, and tympanometry.

[0145] "Curiosity" refers to the desire to learn or explore something, often something new.Curiosity can be assessed using methods known in the art, such as the Y-maze, novel object recognition task, STPI (state-trait personality scale), 5-dimensional curiosity scale, self-curiosity attitude interest scale, curiosity exploration scale-II, state-trait personality scale (STPI), and subscales of sensation seeking scale (SSS).

[0146] "Learning ability" refers to the ability to grasp; understand and benefit from experience. "Working memory" refers to the part of short-term memory that is involved in immediate conscious perception and language processing. "Short-term memory" refers to the ability to hold small amounts of information in mind for a short period of time in a useful, readily available state without manipulation. "Long-term memory" is the stage of the Atkinson-Shiffrin memory model where information knowledge is permanently retained. Long-term memory is defined in contrast to short-term and working memory, which last only about 18-30 seconds. Long-term memory is usually classified into explicit (declarative), as well as episodic, semantic, autobiographical, and implicit (procedural) memory. "Cognition" is the mental activity and process of acquiring and understanding knowledge through thought, experience, and the senses, and encompasses many aspects of intellectual functions and processes, such as attention, knowledge formation, memory and working memory, judgment and evaluation, reasoning and calculation, problem solving and decision making, and the comprehension and production of language. "Visuospatial reasoning" refers to the ability to understand, infer, and retain spatial relationships between objects and spaces. "Object recognition" refers to the ability to remember an object that was previously shown, then removed from view, and then shown again. Any of the traits defined in this paragraph can be measured using clinical scales or tests known to those skilled in the art for measuring cognitive abilities in infants, such as the Bayley Scales of Infant Development (BSID-III) (1-42 months), the Mullen Early Learning Scale (1-68 months), the Fagan Scales of Intelligence for Infants (FTII) (birth-12 months), the Griffith Mental Development Scale I (0-2 years), the Battelle Development Scales (BDI) (birth-8 years), and the Vineland Adaptive Behavior Scales (0-18 years).

[0147] "Infant" as used herein may refer to a very young human or animal, such as 0-1 month, 1-6 months, 6-12 months, 12-18 months, 18-24 months, 24 months-3 years, or older than 3 years. Infants refer not only to humans, but also to animals such as monkeys, bovines, porcines, murines, rats, avian animals, and reptilian animals.

[0148] "Sepsis," as used herein, refers to a life-threatening condition that occurs when the body's response to an infection causes injury to its tissues and organs. This initial stage is followed by suppression of the immune system. Sepsis can be diagnosed using tools known to those skilled in the art, including but not limited to blood cultures, urine cultures, other cultures, blood tests, lumbar punctures, and x-rays.

[0149] The glutamate [NMDA] receptor subunit epsilon-1 is a protein that in humans is encoded by the GRIN2A gene, or "Grin2A" as used herein.

[0150] "Brain-derived neurotrophic factor" (BDNF) refers to a protein that in humans is encoded by the BDNF gene. BDNF acts on certain neurons in the central and peripheral nervous systems to help support the survival of existing neurons and promote the growth and differentiation of new neurons and synapses. Changes in the expression of this protein can be measured, for example, by reverse transcription polymerase chain reaction (RT-PCR), Western blot, or mass spectrometry.

[0151] "Activated microglia" refers to an inflammatory response mediated by resident immune cells of the CNS, which usually respond to neuronal damage and remove damaged cells by phagocytosis. Microglial activation is a hallmark of brain pathology. Activated microglia can be identified by imaging techniques, including but not limited to positron emission tomography (PET), such as those described in J Neuroimmune Pharmacol. 2009 June; 4(2): 227-243, the disclosure of which is incorporated herein by reference.

[0152] "Neurodevelopmental deficiency," as used herein, refers to neurodevelopmental disorders of growth and development of the brain and / or central nervous system. A narrower use of the term refers to disorders of brain function that affect emotions, learning abilities, self-control, and memory as individuals develop and grow. Neurodevelopmental disorders within the scope of this definition include, but are not limited to: Intellectual Disability (ID) or Intellectual Developmental Disability (IDD), formerly known as mental retardation; Specific learning disabilities, such as dyslexia or dyscalculia; Autism spectrum disorders, such as Asperger's syndrome or autistic disorder; Movement disorders, including developmental coordination disorder and stereotypic movement disorder; Tic disorders, including Tourette's syndrome; Traumatic brain injury (including congenital injuries, such as those that cause cerebral palsy); Communication, speech, and language disorders; Genetic disorders, such as fragile X syndrome, Down's syndrome, attention deficit hyperactivity disorder, schizophrenia, schizotypal disorder, hypogonadotropic hypogonadism syndrome. Behavioral disorders, including conduct disorder. Attention-deficit hyperactivity disorder.Neurodevelopmental deficiencies may be detected by tests described herein or known to those of skill in the art, such as ear and eye opening, plane righting, air righting, forelimb grasp, acoustic startle, plane righting, negative geotaxis, open field crossing, cliff aversion, Barnes maze, elevated plus maze, Jamar dynamometer, manual muscle testing, manual muscle testing (MMT), isokinetic muscle testing, trunk stability test (TST), unilateral hip bridge endurance test (UHBE), pronation sign, Barre sign, Romberg test, Landau reflex, particle suspension, sensory reflexes (pin pain, superficial touch, position, vibration, and charger), reflexes (biceps, triceps, brachioradial, patellar, and ankle), Moro reflex, tonic neck response, sucking reflex, palmar and plantar grasp reflex, parachute response, somatic neck righting response (NOB), and / or ventral flexion reflex (STR). ), somatic righting response (BOB), ear opening acoustic reflex, static compliance, physical volume of the ear canal, contralateral reflex, ipsilateral reflex, tympanometry, Y-maze, novel object recognition task, STPI (State-Trait Personality Inventory), 5-Dimensional Curiosity Scale, Self-Curiosity Attitude Interest Scale, Curiosity Seeking Scale-II, State-Trait Personality Inventory (STPI), subscales of the Sensation Seeking Scale (SSS), Bayley Scales of Infant Development (BSID-III) (1-42 months), Mullen Early Learning Scale (1-68 months), Fagan Intelligence Scale for Infants (FTII) (birth-12 weeks), Griffith Mental Development Scale I (0-2 years), Battelle Development Scale (BDI) (birth-8 years), and Vineland Adaptive Behavior Scales (0-18 years).

[0153] "Infection" as used herein may refer to a bacterial infection, a viral infection, a fungal infection or a protozoan infection.

[0154] "Sepsis-causing condition," as used herein, may refer to any condition in a subject or in the mother (maternal) that causes or results in sepsis, such as, for example, necrotizing enterocolitis (NEC), Escherichia coli (E coli) in the maternal amniotic fluid, Listeria, Streptococcus, Neisseria meningitidis, Salmonella, Haemophilus influenzae type b, herpes simplex virus, cytomegalovirus (CMV), Candida, bacterial infection of the maternal amniotic fluid, premature birth, ruptured maternal amniotic membrane, fungal infection, viral infection, respiratory syncytial virus (RSV), protozoan infection, lung infection, brain infection, urinary tract infection, skin infection, urinary catheterization of an infant subject, intravenous placement of an infusion line in an infant subject, inflammation and / or immune dysregulation in an infant subject, and infections resulting from surgery on an infant subject.

[0155] "Cell body thickness," as used herein, refers to the thickness of the cell body as measured, for example, by transmission electron microscopy, transmission through dye microscopy, liquid displacement, Coulter method, absorption, fluorescence, light obscuration, or confocal scanning.

[0156] For in vitro or ex vivo applications, in some embodiments, the effective amount depends on the size and nature of the application in question.The effective amount also depends on the nature and sensitivity of the in vitro target and method used.The person skilled in the art can determine the effective amount based on these and other considerations.The effective amount may include one or more administrations of the composition depending on the embodiment.

[0157] The agents and compositions, e.g., the active ingredients in a pharmaceutical composition, can be used in the manufacture of medicaments and can be used to treat humans and other animals by administration in accordance with conventional procedures.

[0158] The agent or composition of the present invention can be administered by any suitable route of administration for therapy. It is understood that the preferred route will vary depending on the condition and age of the recipient and the disease being treated.

[0159] Necrotizing enterocolitis ("NEC") is a medical condition seen primarily in premature infants in which parts of the intestine die (tissue death). Necrotizing enterocolitis occurs after birth (i.e., not in stillborn infants) and is the second most common cause of death. Seven percent of all neonatal intensive care unit admissions are NEC-related. The mortality rate is 12%. Methods for carrying out the present disclosure Microsphere Composition

[0160] The present disclosure provides a composition comprising, consisting essentially of, or consisting of microspheres, biofilm-producing probiotic bacteria, and prebiotics, where the prebiotics comprise, consist essentially of, or consist of a nutritional supplement for the probiotic bacteria. In one embodiment, the composition further comprises, consists essentially of, or consists of one or more of the following: biofilm, prebiofilmics, a coating on the surface of the microsphere, a therapeutic or therapeutic agent, a chemical reducing agent, a molecule that promotes adsorption, and a molecule that assists absorption. The microsphere comprises, consists essentially of, or consists of a solid core, a hollow core, and in one embodiment, the microsphere encapsulates the prebiotic within the hollow core. The microsphere can be biocompatible and / or semipermeable. In one embodiment, the microsphere comprises a biofilm layer or coating on the outer surface of the microsphere. Microsphere Components

[0161] In one embodiment, the biocompatible microsphere comprises, consists essentially of, or consists of a material selected from the group consisting of biodegradable polymers, non-degradable polymers, and metals, and the diameter of the microsphere is from about 0.5 micrometers to about 1000 micrometers. Additional preferred ranges are described herein and incorporated by reference. The microsphere can be porous and / or semi-permeable.

[0162] Non-limiting examples of biodegradable polymers include dextran; dextranomer; poly(lactic-co-glycolic acid), i.e., PLGA; polycaprolactone, i.e., PLC; chitosan; gelatin; hydrogen DNA; acetalized dextran; poly(lactide); poly(glycolide); poly(lactide-co-glycolide); poly(lactic acid); poly(glycolic acid); poly(lactic acid-co-glycolic acid); poly(lactide) / poly(ethylene glycol) copolymer; poly(glycolide) / poly(ethylene glycol) copolymer; poly(lactide-co-glycolide) / poly(ethylene glycol) copolymer; poly(lactic acid) / poly(ethylene glycol) copolymer; poly(glycolic acid) / poly(ethylene glycol) copolymer; poly(lactic acid-co-glycolic acid) / poly(ethylene glycol) copolymers; poly(caprolactone); poly(caprolactone) / poly(ethylene glycol) copolymers; poly(orthoesters); poly(phosphazenes); poly(hydroxybutyrates); poly(hydroxybutyrates); poly(lactide-co-caprolactone); polycarbonates; polyesteramides; polyanhydrides; poly(dioxanone); poly(alkylene alkylates); polyethylene glycol / polyorthoester copolymers; polyurethanes; poly(amino acids); polyetheresters; polyacetals; polycyanoacrylates; poly(oxyethylene) / poly(oxypropylene) copolymers; Sephadex® copolymers, and / or combinations thereof.

[0163] Non-limiting examples of non-biodegradable polymers are selected from one or more of poly(ethylene vinyl acetate), poly(vinyl acetate), silicone polymers, polyurethanes, polysaccharides, such as cellulosic polymers and derivatives, acyl-substituted cellulose acetate and its derivatives, copolymers of poly(ethylene glycol) and poly(butylene terephthalate), polystyrene, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulfonated polyolefins, polyethylene oxide, and copolymers and blends thereof.

[0164] Non-limiting examples of polymers that comprise, consist essentially of, or consist of microspheres include Sephadex, Sephadex G-25, poly(lactic-co-glycolic acid) ("PLGA"); polycaprolactone ("PLC"); chitosan; gelatin; DNA hydrogen; acetalated dextran; poly(lactide); poly(glycolide); poly(lactide-co-glycolide); poly(lactic acid); poly(glycolic acid); poly(lactic acid-co-glycolic acid); poly(lactide) / poly(ethylene glycol) copolymer; poly(glycolide) / poly(ethylene glycol) copolymer; poly(lactide-co-glycolide) / poly(ethylene glycol) copolymer; poly(lactic acid / poly(ethylene glycol) copolymer; poly(glycolic acid / poly poly(ethylene glycol) copolymers; poly(lactic acid-co-glycolic acid) / poly(ethylene glycol) copolymers; poly(caprolactone); poly(caprolactone) / poly(ethylene glycol) copolymers; poly(orthoesters); poly(phosphazenes); poly(hydroxybutyrates); poly(hydroxybutyrates); poly(lactide-co-caprolactone); polycarbonates; polyesteramides; polyanhydrides; poly(dioxanone); poly(alkylene alkylates); polyethylene glycol / polyorthoester copolymers; polyurethanes; poly(amino acids); polyetheresters; polyacetals; polycyanoacrylates; poly(oxyethylene) / poly(oxypropylene) copolymers; and combinations thereof.

[0165] Non-limiting examples of metals include cobalt, chromium, gold, nickel, platinum, stainless steel, titanium, tantalum, nickel-titanium, alloys, and combinations thereof. Prebiotics

[0166] Non-limiting examples of prebiotics of the composition include one or more of water soluble carbohydrates, inulin, oligosaccharides, oligofructose, fructooligosaccharides, galactooligosaccharides, glucose, starch, maltose, maltodextrin, polydextrose, amylose, sucrose, fructose, lactose, isomaltulose, polyols, glycerol, carbonate, thiamine, choline, histidine, trehalose, nitrogen, sodium nitrate, ammonium nitrate, phosphorus, phosphate, hydroxyapatite, potassium, potash, sulfur, homopolysaccharides, heteropolysaccharides, cellulose, chitin, vitamins, and combinations thereof.

[0167] In another embodiment, the prebiotic is selected from one or more of trehalose, nitrogen as in sodium nitrate, ammonium nitrate, phosphorus as in phosphates such as hydroxyapatite, potassium as in potash, sulfur, oligosaccharides, homopolysaccharides, heteropolysaccharides, cellulose, chitin, glucose, fructose, sucrose, maltose, starch, polydextrose, amylose, glycerol, carbonates, and combinations thereof.

[0168] In still further embodiments, the prebiotic of the composition comprises, consists essentially of, or consists of one or more of a vitamin mixture for stimulating microbial growth, nitrogen as in sodium nitrate, ammonium nitrate, etc., phosphorus as in phosphates such as hydroxyapatite, potassium as in potash, sulfur, oligosaccharides, homopolysaccharides, heteropolysaccharides, cellulose, chitin, glucose, fructose, sucrose, maltose, starch, polydextrose, amylose, glycerol, carbonates, and combinations thereof. Probiotic bacteria

[0169] In one embodiment, the probiotic bacteria are selected to provide one or more of antibacterial immune support, enhancing or supporting the health of a subject, enhancing or supporting the gastrointestinal barrier, or antagonizing disease-associated bacterial infection. In another embodiment, the probiotic bacteria are selected to prevent pathogen colonization and / or limit and / or eliminate pathogens and / or limit excessive inflammatory responses by downregulating cytokine and chemokine production.

[0170] The main strains of the fermentation plants are L. acidophilus, L. crispatus, L. gasseri, L. delbrueckii, and L. sali varius, L. casei, L. paracasei, L. plantarum, L. rhamnosus, L. reuteri, L. brevis, and L. buchneri L. fermentum, L. rhamnosus, B. adolescentis, B. angulation, B. bifidum, B. breve, B. catenulatum, B. infantis, B. lactis, B. longum, B. pseudocatenulatum, S. thermophiles, Pseudomonas fluorescens、P.protegens、P.brassicacearum、P.aeruginosa;Azospirillum.brabrasilense、A.lipferum、A.halopraeferens、A.irakense;Acetobacter diazotrophicus;Herbaspirillum seropedicae;Bacillus subtilis.Pseudomonas stutzeri、fluorescens、P.putida、P.cepacian、P.vesicularis、P.paucimobilis;Bacilluscereus、B.thuringiensis、B.sphaericus;Shewanella oneidensis;Geobacter bemidjiensis、G.metallireducens、G.sulfurreducens、G.uraniireducens、G.lovleyi;Serratia marcescens、Desulfovibrio vulgaris、D.desulfuricans、Dechloromonas aromatic、Deinococcus radiodurans、Methylibium petroleumphilum、Alcanivorax borkumensis、Archaeglobus fulgidus、Haloferax sp.、Halobacterium 1 of the strains of sp.

[0171] In another embodiment, the probiotic is L. reuteri, which produces the GTF protein or contains the GTFW gene (ATCC 23272). Prebiofilmics

[0172] In other embodiments, the prebiofilmics comprises, consists essentially of, or consists of agents that support the formation and persistence of biofilms, non-limiting examples of which include DNA-binding polypeptides or proteins, and / or DNABII polypeptides or proteins, or equivalents of each of these, optionally polypeptides comprising, consisting essentially of, or consisting of one or more of the attached sequence listing, or biologically active fragments or equivalents of each of these, alone or in combination. Complementary drugs

[0173] The microspheres and compositions containing the microspheres can further comprise an agent, which is selective for pathogens that may compete with the probiotic organisms. The complementary agent can be present in the core of the microsphere in the composition containing the microsphere, on the surface. Non-limiting examples of such include chemical reducing agents; molecules and / or surfaces that promote adsorption (into the core or on the surface of the microsphere); molecules and / or surfaces that promote absorption (into the core or on the surface of the microsphere). In one embodiment, the chemical reducing agent and the molecules and / or surfaces that promote absorption are coated on the surface of the microsphere. Biofilm layer

[0174] In one embodiment, the microsphere composition further comprises a biofilm layer on the exterior surface of the microparticle. This layer can be about 0.5 micrometers to about 1 millimeter deep, for example, in the range between about 1 micrometer to about 500 micrometers, about 1 micrometer to about 250 micrometers, about 1 micrometer to about 200 micrometers, about 1 micrometer to about 100 micrometers, about 1 micrometer to about 50 micrometers, about 1 micrometer to about 40 micrometers, about 1 micrometer to about 30 micrometers, about 2 micrometers to about 100 micrometers, about 2 micrometers to about 50 micrometers, about 2 micrometers to about 40 micrometers, about 2 micrometers to about 30 micrometers, about 3 micrometers to about 100 micrometers, about 3 micrometers to about 50 micrometers, about 3 micrometers to about 40 micrometers, about 3 micrometers to about 30 micrometers, about 5 micrometers to about 100 micrometers, about 5 micrometers to about 50 micrometers, about 5 micrometers to about 40 micrometers, and about 5 micrometers to about 30 micrometers. composition

[0175] The present disclosure also provides one or more microsphere compositions described herein in combination with a carrier, such as a pharma- ceutically acceptable carrier or a biocompatible scaffold.Non-limiting examples of pharma-ceutically acceptable carriers include diluents, excipients or carriers that can be used in the compositions of the present disclosure.Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of vegetable saturated fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

[0176] Non-limiting examples of biocompatible scaffolds include scaffolds or matrices that have the ability to support biofilm growth upon administration to a subject or environment to be treated.

[0177] In one embodiment, the composition comprises multiple microspheres that are the same or different from one another, e.g., same or different diameters, same or different microsphere components, same or different probiotics, same or different complementary agents, same or different prebiofilmics, and hollow and / or solid cores.

[0178] The composition can be formulated into a dosage form of biofilm-producing probiotic bacteria, e.g., or a microsphere composition for end use in an effective amount, e.g., about 1×10 5 ~1×10 11 CFU / ml, or alternatively approximately 1 × 10 5 ~Approx. 1×10 10 CFU / ml, or approximately 1 x 10 5 ~Approx. 1×10 9CFU / ml, or approximately 1 x 10 6 ~Approx. 1×10 11 CFU / ml, or approximately 1 x 10 6 ~Approx. 1×10 9 CFU / ml, or approximately 1 x 10 7 ~Approx. 1×10 11 CFU / ml, or approximately 1 x 10 7 ~Approx. 1×10 10 CFU / ml, or approximately 1 x 10 7 ~Approx. 1×10 9 CFU / ml, or approximately 1 x 10 8 CFU / ml can be provided.

[0179] The compositions may be formulated or processed for ease of administration, storage and application, for example, they may be frozen, lyophilized, suspended (suspension formulations) or powdered, and may be processed as suppositories, tablets, liquids, suspensions, pills, capsules, sustained release formulations. Application and Use

[0180] A method of preventing, delaying or treating neurodevelopmental deficiency or promoting neurodevelopment in a subject suffering from, susceptible to or suffering from neurodevelopmental deficiency is provided, comprising administering to the subject an effective amount of a composition comprising microspheres, biofilm-producing probiotic bacteria and a prebiotic, wherein the prebiotic comprises a nutritional supplement for the probiotic bacteria. In one aspect, the subject suffers from NEC or other conditions with similar effects on the brain, or sepsis and / or a condition that causes sepsis.

[0181] Also provided is a method of preventing, reducing or delaying microglial activation in a subject, comprising administering to the subject an effective amount of a composition comprising microspheres, biofilm-producing probiotic bacteria and prebiotics, wherein the prebiotics comprise a nutritional supplement for the probiotic bacteria. In one aspect, the subject suffers from NEC or other pathologies with similar effects on the brain, or sepsis, and / or conditions that cause sepsis. In one aspect, the thickness of the microglial cell body is reduced in the treated subject compared to a control subject. In another aspect, the microglial dendrites are thinned in the subject compared to a control subject.

[0182] Further provided is a method of increasing expression of brain-derived neurotrophic factor (BDNF) in a subject, comprising administering to the subject an effective amount of a composition comprising microspheres, biofilm-producing probiotic bacteria, and a prebiotic, wherein the prebiotic comprises a nutritional supplement for the probiotic bacteria. In one aspect, the subject is suffering from NEC or other pathologies with similar effects on the brain, or sepsis, and / or a condition that causes sepsis.

[0183] In the above method, the increase is an increase compared to a control subject. In one embodiment, the increased expression of BDNF is increased expression in the hippocampus of the subject.

[0184] Further provided is a method for increasing the expression of Grin2A in a subject, comprising administering to the subject an effective amount of a composition comprising microspheres, biofilm-producing probiotic bacteria, and prebiotics, wherein the prebiotics comprises a nutritional supplement for the probiotic bacteria. In one embodiment, the subject suffers from NEC or other pathologies that have a similar effect on the brain, or sepsis, and / or a condition that causes sepsis. In one embodiment, the increase is compared to a control subject. In another embodiment, the increased expression of Grin2A is increased in the prefrontal cortex of the treated subject.

[0185] In embodiments of the disclosed methods, the microspheres further comprise a partial or complete biofilm coating on the exterior surface of the microsphere. In further embodiments, the bacteria and / or prebiotics diffuse from the interior of the microsphere to the surface of the microsphere.

[0186] Generally, the compositions of the present disclosure are used in therapeutic applications, and the components of the composition and the carriers and additional agents are selected for the designated use.

[0187] In one embodiment, the composition provides one or more of antimicrobial immune support, strengthening or supporting the gastrointestinal barrier, or antagonizing disease-associated bacterial infections. In another embodiment, the composition prevents pathogen colonization and / or limits excessive inflammatory responses by downregulating cytokine and chemokine production.

[0188] In one aspect, the compositions are useful for treating mammals, such as humans, monkeys, murines, e.g., rats, mice, chinchillas, canines, e.g., dogs, lagomorphs, e.g., rabbits, farm animals, sport animals and pets.

[0189] Indications and uses vary depending on the environment. The compositions can be used for the treatment or prevention of diseases, such as psychological disorders, such as depression or anxiety; the treatment or prevention of any of the chronic and / or recurrent diseases caused by intestinal infectious diseases, infection-induced colitis, traveler's diarrhea, inflammatory bowel disease (IBD), colitis, diarrheal diseases, vaginitis, wounds, burns, psoriasis, dermatitis, dental caries, periodontitis, sinusitis, or pathogenic bacteria replacing healthy bacteria or necrotizing enterocolitis (NEC) (to support antibacterial immunity, strengthen or support the gastrointestinal barrier, and modify or support the intestinal flora due to dysbiosis (and even in the absence of disease)); the treatment or prevention of diseases or disorders involving intestinal dysmotility (to enhance or support gastrointestinal motility or antagonize disease-associated bacterial infections); the treatment or prevention of vaginitis, colitis or traveler's diarrhea, peritonitis, postoperative ileus, irritable bowel syndrome (IBS), pseudo-obstruction, and / or constipation. In one embodiment, treatment does not include prophylaxis.

[0190] Thus, in one aspect, the present disclosure provides a method for treating or preventing a disease or disorder as provided herein that is suitably treated by a biofilm in a subject in need thereof, the method comprising, consisting essentially of, or consisting of administering to the subject an effective amount of a composition as disclosed herein having ingredients selected for a particular treatment. Non-limiting examples of diseases include psychological disorders such as depression or anxiety, intestinal infectious diseases, autism, autism spectrum disorder, infection-induced colitis, traveler's diarrhea, inflammatory bowel disease (IBD), colitis, diarrheal diseases, vaginitis, wounds, burns, psoriasis, dermatitis, dental caries, periodontitis, sinusitis, or any chronic and / or recurrent disease caused by pathogenic bacteria replacing healthy bacteria or necrotizing enterocolitis (NEC), as well as diseases or disorders involving intestinal dysmotility (enhancing or supporting gastrointestinal motility or antagonizing disease-associated bacterial infections), to support antibacterial immunity, strengthen or support the gastrointestinal barrier, modify or support the gut flora due to dysbiosis (and even in the absence of disease), vaginitis, colitis or traveler's diarrhea, peritonitis, post-operative ileus, irritable bowel syndrome (IBS), pseudo-obstruction, and / or constipation. In addition, the compositions are useful for promoting health and / or maintaining intestinal homeostasis.

[0191] The present disclosure also provides a method for delivering probiotics to a subject, the method comprising, consisting essentially of, or consisting of administering the probiotics by administering a dose of a composition disclosed herein.

[0192] The dosage and components of the composition will vary depending on the subject and the purpose of treatment. In one embodiment, the composition comprises about 1×10 7 ~Approx. 1×10 9 The composition can be formulated into a dosage form, e.g., or an effective amount of the microsphere composition for the end use, e.g., about 1×10 5~1×10 11 CFU / ml, or alternatively, approximately 1 × 10 5 ~Approx. 1×10 10 CFU / ml, or approximately 1 x 10 5 ~Approx. 1×10 9 CFU / ml, or approximately 1 x 10 6 ~Approx. 1×10 11 CFU / ml, or approximately 1 x 10 6 ~Approx. 1×10 9 CFU / ml, or approximately 1 x 10 7 ~Approx. 1×10 11 CFU / ml, or approximately 1 x 10 7 ~Approx. 1×10 10 CFU / ml, or approximately 1 x 10 7 ~Approx. 1×10 9 CFU / ml, or approximately 1 x 10 8 CFU / ml can be provided.

[0193] The compositions can be administered at about 6, 12, 18, 24, 36, 48 and 72 hour intervals, or can be administered in a single dose.

[0194] The compositions can be administered orally, vaginally, topically, by inhalation, intravenously, intramuscularly, or by suppository. The compositions can be administered in any suitable formulation.

[0195] The amounts and components of the composition vary depending on the purpose of treatment and whether it is a single dose or multiple doses. In one embodiment, the composition is administered in a dose of about 1×10 7 ~Approx. 1×10 9 The composition can be formulated into a dosage form, e.g., or an effective amount of the microsphere composition for the end use, e.g., about 1×10 5 ~1×10 11 CFU / ml, or alternatively, approximately 1 × 10 5 ~Approx. 1×10 10 CFU / ml, or approximately 1 x 10 5 ~Approx. 1×10 9 CFU / ml, or approximately 1 x 106 ~Approx. 1×10 11 CFU / ml, or approximately 1 x 10 6 ~Approx. 1×10 9 CFU / ml, or approximately 1 x 10 7 ~Approx. 1×10 11 CFU / ml, or approximately 1 x 10 7 ~Approx. 1×10 10 CFU / ml, or approximately 1 x 10 7 ~Approx. 1×10 9 CFU / ml, or approximately 1 x 10 8 CFU / ml can be provided. The composition can be delivered in multiple doses, for example, every 6, 12, 18, 24, 36, 48 and 72 hours, or can be administered in a single dose.

[0196] In the methods of the present disclosure, the composition administered can include one or more microsphere compositions described herein in combination with a carrier, such as a pharma- ceutically acceptable carrier or a biocompatible scaffold.Non-limiting examples of pharma-ceutically acceptable carriers include diluents, excipients, or carriers that can be used in the compositions of the present disclosure.

[0197] In a further aspect, the microsphere further comprises, consists essentially of, or consists of a biofilm layer partially or completely surrounding the microsphere.

[0198] In one embodiment, the composition comprises multiple microspheres that are the same or different from one another, e.g., same or different diameters, same or different microsphere components, same or different probiotics, same or different complementary agents, same or different prebiofilmics, same or different biofilm layers, and hollow and / or solid cores.

[0199] The composition can be formulated into a dosage form of biofilm-producing probiotic bacteria, e.g., or a microsphere composition for end use in an effective amount, e.g., about 1×105 ~1×10 11 CFU / ml, or alternatively, approximately 1 × 10 5 ~Approx. 1×10 10 CFU / ml, or approximately 1 x 10 5 ~Approx. 1×10 9 CFU / ml, or approximately 1 x 10 6 ~Approx. 1×10 11 CFU / ml, or approximately 1 x 10 6 ~Approx. 1×10 9 CFU / ml, or approximately 1 x 10 7 ~Approx. 1×10 11 CFU / ml, or approximately 1 x 10 7 ~Approx. 1×10 10 CFU / ml, or approximately 1 x 10 7 ~Approx. 1×10 9 CFU / ml, or approximately 1 x 10 8 CFU / ml can be provided. Medicinal, nutritional or therapeutic uses

[0200] The present disclosure provides a composition for nutritional or pharmaceutical use, comprising, essentially consisting of, or consisting of microspheres, biofilm-producing probiotic bacteria, and prebiotics, wherein the prebiotics comprise, essentially consisting of, or consisting of a nutritional supplement for the probiotic bacteria.In one embodiment, the composition further comprises, essentially consisting of, or consisting of one or more of the following: prebiotics, biofilm layer, therapeutic agent, or therapeutic agent.The microsphere comprises, essentially consisting of, or consists of a solid core, a hollow core, and in one embodiment, the microsphere encapsulates the prebiotics in the hollow core.

[0201] In one embodiment, the microsphere comprises, consists essentially of, or consists of a material selected from the group consisting of a biodegradable polymer, a non-degradable polymer, or a metal, and the microsphere has a diameter of about 0.5 micrometers to about 1000 micrometers, or alternatively about 0.5 micrometers to about 100 micrometers, or alternatively less than 100 micrometers.

[0202] Non-limiting examples of biodegradable polymers for pharmaceutical use include dextran, dextranomer, poly(lactic-co-glycolic acid) or PLGA, polycaprolactone or PLC, chitosan, gelatin, hydrogen DNA, acetalized dextran, poly(lactide), poly(glycolide), poly(lactide-co-glycolide), poly(lactic acid), poly(glycolic acid), poly(lactic acid-co-glycolic acid), poly(lactide) / poly(ethylene glycol) copolymer, poly(glycolide) / poly(ethylene glycol) copolymer, poly(lactide-co-glycolide) / poly(ethylene glycol) copolymer, poly(lactic acid) / poly(ethylene glycol) copolymer, poly(glycolic acid) / poly(ethylene glycol) copolymer, poly(lactic acid-co-glycolic acid), poly(lactic acid-co-glycolic acid), poly(lactide ... In some embodiments, the poly(ethylene glycol) / poly(ethylene glycol) copolymers may be selected from one or more of poly(ethylene glycol) / poly(ethylene glycol) copolymers, poly(caprolactone), poly(caprolactone) / poly(ethylene glycol) copolymers, poly(orthoesters), poly(phosphazenes), poly(hydroxybutyrates), poly(hydroxybutyrates), poly(lactide-co-caprolactone), polycarbonates, polyesteramides, polyanhydrides, poly(dioxanone), poly(alkylene alkylates), polyethylene glycol / polyorthoester copolymers, polyurethanes, poly(amino acids), polyetheresters, polyacetals, polycyanoacrylates, poly(oxyethylene) / poly(oxypropylene) copolymers, Sephadex® copolymers, and / or combinations thereof.

[0203] Non-limiting examples of non-biodegradable polymers for pharmaceutical use are selected from one or more of poly(ethylene vinyl acetate), poly(vinyl acetate), silicone polymers, polyurethanes, polysaccharides, such as cellulosic polymers and derivatives, acyl-substituted cellulose acetate and its derivatives, copolymers of poly(ethylene glycol) and poly(butylene terephthalate), polystyrene, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulfonated polyolefins, polyethylene oxide, and copolymers and blends thereof.

[0204] Non-limiting examples of metals include cobalt, chromium, gold, nickel, platinum, stainless steel, titanium, tantalum, nickel-titanium, alloys, and combinations thereof.

[0205] Non-limiting examples of prebiotics of compositions for pharmaceutical use include, consist essentially of, or consist of one or more of water soluble carbohydrates, inulin, oligofructose, fructooligosaccharides, galactooligosaccharides, glucose, maltose, maltodextrin, polydextrose, sucrose, fructose, lactose, isomaltulose, polyols, glycerol, thiamine, choline, histidine, and combinations thereof.

[0206] Non-limiting examples of probiotic bacteria are one or more of L. acidophilus, L. crispatus, L. gasseri, L. delbrueckii, L. salivarius, L. casei, L. paracasei, L. plantarum, L. rhamnosus, L. reuteri, L. brevis, L. buchneri, L. fermentum, L. rhamnosus, B. adolescentis, B. angulation, B. bifidum, B. breve, B. catenulatum, B. infantis, B. lactis, B. longum, B. pseudocatenulatum, S. thermophiles, and combinations thereof.

[0207] Non-limiting examples of prebiofilmics include, consist essentially of, or consist of agents that support the formation and persistence of biofilms, including DNA-binding polypeptides or proteins, and / or DNABII polypeptides or proteins, or equivalents of each of these, optionally polypeptides comprising, consisting essentially of, or consisting of one or more of the attached sequence listings, or biologically active fragments or equivalents of each of these, alone or in combination.

[0208] The microspheres and compositions containing the microspheres can further comprise an agent that is selective for pathogens that may compete with the probiotic organisms. The complementary agent can be present within the core of the microspheres in the compositions containing the microspheres, on the surface.

[0209] In a further aspect, the microsphere further comprises, consists essentially of, or consists of a biofilm layer partially or completely surrounding the microsphere.

[0210] Compositions for pharmaceutical use can be provided as compositions comprising, consisting essentially of, or consisting of one or more microsphere compositions described herein in combination with a carrier, e.g., a pharma- ceutically acceptable carrier or a biocompatible scaffold.

[0211] In one embodiment, the composition comprises multiple microspheres that are the same or different from one another, e.g., same or different diameters, same or different microsphere components, same or different biofilm layers, same or different probiotics, same or different complementary agents, same or different prebiofilmics, and hollow and / or solid cores.

[0212] The composition can be formulated into a dosage form of biofilm-producing probiotic bacteria, e.g., or a microsphere composition for end use in an effective amount, e.g., about 1×10 5 ~1×10 11 CFU / ml, or alternatively, approximately 1 × 10 5 ~Approx. 1×10 10 CFU / ml, or approximately 1 x 10 5 ~Approx. 1×10 9 CFU / ml, or approximately 1 x 10 6 ~Approx. 1×10 11 CFU / ml, or approximately 1 x 10 6 ~Approx. 1×10 9 CFU / ml, or approximately 1 x 10 7 ~Approx. 1×10 11 CFU / ml, or approximately 1 x 10 7 ~Approx. 1×10 10 CFU / ml, or approximately 1 x 10 7 ~Approx. 1×10 9 CFU / ml, or approximately 1 x 10 8 CFU / ml can be provided.

[0213] The compositions may be formulated or processed for ease of administration, storage and application, for example, they may be frozen, lyophilized, suspended (suspension formulations) or powdered, and may be processed as suppositories, tablets, liquids, suspensions, pills, capsules, sustained release formulations.

[0214] The compositions are useful for treating mammals, such as humans, monkeys, murines, eg, rats, mice, chinchillas, canines, eg, dogs, lagomorphs, eg, rabbits, farm animals, sport animals and pets.

[0215] The compositions can be used for the treatment or prevention of diseases, such as psychological disorders, such as depression or anxiety; the treatment or prevention of any of the chronic and / or recurrent diseases caused by intestinal infectious diseases, infection-induced colitis, traveler's diarrhea, inflammatory bowel disease (IBD), colitis, diarrheal diseases, vaginitis, wounds, burns, psoriasis, dermatitis, dental caries, periodontitis, sinusitis, or pathogenic bacteria replacing healthy bacteria or necrotizing enterocolitis (NEC) (to support antibacterial immunity, strengthen or support the gastrointestinal barrier, modify or support the intestinal flora due to dysbiosis (and even in the absence of disease)); the treatment or prevention of diseases or disorders involving intestinal dysmotility (to enhance or support gastrointestinal motility or antagonize disease-associated bacterial infections); the treatment or prevention of vaginitis, colitis or traveler's diarrhea, peritonitis, postoperative ileus, irritable bowel syndrome (IBS), pseudo-obstruction, and / or constipation.

[0216] Thus, in one aspect, the present disclosure provides a method for treating or preventing a disease that is preferably treated by healthy bacteria and / or biofilms in a subject in need thereof, the method comprising, consisting essentially of, or consisting of administering to the subject an effective amount of a composition disclosed herein having ingredients selected for a particular treatment. Non-limiting examples of diseases include those identified above (and incorporated herein by reference) and include one or more of psychological disorders such as depression or anxiety, intestinal infectious diseases, infection-induced colitis, traveler's diarrhea, inflammatory bowel disease (IBD), colitis, diarrheal diseases, vaginitis, wounds, burns, psoriasis, dermatitis, dental caries, periodontitis, sinusitis, or any chronic and / or recurrent disease caused by pathogenic bacteria replacing healthy bacteria or necrotizing enterocolitis (NEC) (to support antibacterial immunity, strengthen or support the gastrointestinal barrier or antagonize against disease-associated bacterial infections), vaginitis, colitis or traveler's diarrhea, peritonitis, post-operative ileus, irritable bowel syndrome, pseudo-obstruction, constipation.

[0217] Thus, the present disclosure provides a method for delivering a probiotic formulation to a subject in need thereof, e.g., a subject suffering from a disease or condition disclosed herein, by administering to the subject an effective amount of a suitable disease-related or health-promoting composition disclosed herein. The composition may be administered by any suitable method of administration, e.g., orally, vaginally, by inhalation, by injection, topically, or by suppository.

[0218] Successful treatment can be determined by monitoring relief of disease symptoms, cytokine production, and / or by assaying for the presence of probiotic cultures in the subject. Nutritional supplements

[0219] The compositions disclosed herein are also useful as nutritional supplements for promoting overall health and well-being, and for maintaining intestinal health and / or homeostasis.Therefore, in one aspect, the present disclosure also provides a method for promoting health and / or maintaining intestinal homeostasis in a subject in need thereof, comprising or consisting essentially of or consisting of administering to the subject an effective amount of the compositions described herein, and optionally, the surface of the microsphere is porous and / or semi-permeable, and prebiotics are released by diffusion or leak out of the microsphere and diffuse due to the slow degradation of the microsphere.By determining whether intestinal discomfort is reduced or alleviated, those skilled in the art can determine whether gut homeostasis as well as better overall health is achieved. Diarrhea and GI Disorders

[0220] Diarrhea affects approximately 4 billion people annually worldwide and is responsible for over 2 million deaths. Among the most important bacterial causes of diarrheal disease in infants and young children are the adherent and evolving (A / E) pathogens, which upon colonization induce diarrhea associated with increased inflammatory cytokines and structural changes to the colony tissue. This acute infection has long-term effects on intestinal health, and infection with A / E pathogens and exaggerated inflammatory responses are known risk factors for the development of post-infectious irritable bowel syndrome.

[0221] Probiotics are a natural way to protect and restore the gut microbiota to a healthy state and have been shown to promote health distal to the site of colonization. See Mackos et al. (2013) Infection and Immunity 81, No. 9 (3253-3263). Unfortunately, even under optimal conditions, probiotic bacteria are unable to establish or persist sufficiently, thereby minimizing the magnitude and duration of their health benefits. One of the rate-limiting steps is the ability of the introduced bacteria to form long-term biofilms. Bacteria are more easily established and persist when they are already in the form of a biofilm (surface-attached community) rather than planktonic (free-living). The positive effects of probiotic bacteria can be enhanced by providing them in a biofilm state, which can be easily achieved by growing the bacteria on the surface of biocompatible, non-toxic microspheres. The biocompatible microspheres can be biodegradable polymers, non-biodegradable polymers, metals, or combinations of these. When the surface is in the form of a microsphere, prebiotic and / or prebiofilmic substances can be added as cargo to aid in the establishment and maintenance of probiotic bacterial biofilms.

[0222] Microspheres have an additional value in that they ideally provide a diffusible prebiotic cargo that can help promote colonization and survival of probiotic bacteria while limiting the attack of pathogenic bacteria. At least for the probiotic bacterium Lactobacillus reuteri, the biofilm state favors colonization of the mouse intestine over the same bacterium in planktonic form.

[0223] Furthermore, L. reuteri introduced into mice as a biofilm has a more robust and sustained preventive effect against the pathogenesis of the enteropathogenic bacterium, Citrobacter rodentium, than its planktonic form. Based on these results, three examples of highly integrated probiotics have been developed that provide greater and longer-lasting effects against dysbiosis, thereby preventing or even treating intestinal pathogenesis with a significantly reduced need for patient compliance.

[0224] Biofilm-producing probiotic bacteria attach to the surface of biocompatible microspheres and produce biofilms. Biocompatible microspheres have either solid or hollow cores. When biocompatible microspheres have hollow cores, they can carry prebiotics and any nutritional supplements for probiotic bacteria as cargo. In one aspect, for microspheres with hollow cores, the surface of the sphere can be semi-permeable to allow the cargo to diffuse to the associated bacteria at high local concentration, or impermeable but slowly degrade to allow the release of contents. Prebiotics can be encapsulated in the hollow core. Microspheres can also carry drugs or compounds or agents that are selective for the growth or proliferation of pathogens. In addition to biocompatible microspheres, biofilm-generating probiotics and prebiotics, the novel probiotic formulation can also contain prebiofilmics, which are substances that support the formation and persistence of biofilms, and specifically, prebiofilmics are DNA-binding polypeptides or proteins, and / or DNABII polypeptides or proteins, fragments and / or equivalents thereof.Non-limiting examples of such are provided in the attached sequence listing.One or more drugs, compounds or agents, as well as one or more prebiofilmics, can be present in a single microsphere.

[0225] Prebiotics can support the growth of any probiotic bacteria, including biofilm-producing bacteria.Prebiotics are usually water-soluble carbohydrates, such as one or more of inulin, oligofructose, fructooligosaccharides, galactooligosaccharides, glucose, maltose, maltodextrin, polydextrose, sucrose, fructose, lactose, isomaltulose, polyols, and glycerol.Combinations of various prebiotics can be used to support the growth of probiotics.

[0226] Probiotics are any type of microorganism that has health benefits. Probiotics are also commonly consumed as live active cultures, for example in yogurt, soy yogurt, as part of fermented foods, or as dietary supplements. Probiotics can also be used as suppositories. Some non-exhaustive examples of probiotics are L.acidophilus, L.crispatus, L.gasseri, L.delbrueckii group, L.salivarius, L.casei, L.paracasei, L.plantarum, L.rhamnosus, L.reuteri, L.brevis, L.buchneri, L.fermentum, L.rhamnosus, B.adolescentis, B.angulation, B.bifidum, B.breve, B.catenulatum, B.infantis, B.lactis, B.longum, B.pseudocatenulatum, and S.thermophiles. In one embodiment, the probiotic is L. reuteri expressing GTF proteins. All strains of L. reuteri have at least one GTF protein, but they may differ between strains, for example, in DSM 20016, the GTF is GTFW, which uses maltose as its only substrate, while in DSM 17938, the GTF is GTFA, which uses sucrose as its only substrate.

[0227] Probiotics support antibacterial immunity by preventing pathogen colonization and / or limiting excessive inflammatory responses. Without being bound by theory, probiotics down-regulate cytokine and chemokine production.

[0228] The biocompatible microspheres can be one or more of biodegradable polymers, non-biodegradable polymers, metals or mixtures thereof. Biodegradable polymers include dextran, dextranomers, poly(lactic-co-glycolic acid), i.e. PLGA, polycaprolactone, i.e. PLC, chitosan, gelatin, DNA hydrogen, acetalized dextran, poly(lactide), poly(glycolide), poly(lactide-co-glycolide), poly(lactic acid), poly(glycolic acid), poly(lactic acid-co-glycolic acid), poly(lactide) / poly(ethylene glycol) copolymers, poly(glycolide) / poly(ethylene glycol) copolymers, poly(lactide-co-glycolide) / poly(ethylene glycol) copolymers, poly(lactic acid) / poly(ethylene glycol) copolymers, poly(glycolic acid) / poly(ethylene glycol) copolymers, poly(lactic acid-co-glycolic acid) / poly(ethylene glycol) copolymers. The polymer may be selected from, but is not limited to, polymers; poly(caprolactone); poly(caprolactone) / poly(ethylene glycol) copolymers; poly(orthoesters); poly(phosphazenes); poly(hydroxybutyrates); poly(hydroxybutyrates); poly(lactide-co-caprolactone); polycarbonates; polyesteramides; polyanhydrides; poly(dioxanone); poly(alkylene alkylates); polyethylene glycol / polyorthoester copolymers; polyurethanes; poly(amino acids); polyetheresters; polyacetals; polycyanoacrylates; poly(oxyethylene) / poly(oxypropylene) copolymers; Sephadex® copolymers (made from dextran crosslinked with epichlorohydrin, commercially available from Sigma-Aldrich and described in Koo and Wankat (1988) Korean Biochem. J. 21(1)), and / or combinations thereof.The non-biodegradable polymers can be selected from, but are not limited to, poly(ethylene vinyl acetate), poly(vinyl acetate), silicone polymers, polyurethanes, polysaccharides, such as cellulosic polymers and derivatives, acyl-substituted cellulose acetate and its derivatives, copolymers of poly(ethylene glycol) and poly(butylene terephthalate), polystyrene, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulfonated polyolefins, polyethylene oxide, and copolymers and blends thereof. The metals can be selected from, but are not limited to, cobalt, chromium, gold, nickel, platinum, stainless steel, titanium, tantalum, nickel-titanium, and alloys and combinations thereof.

[0229] Microspheres are selected, identified, and characterized to promote the durability and robustness of probiotic biofilms. Probiotic biofilms formed on poly(lactic-co-glycolic acid) (PLGA), a biodegradable (and FDA approved) surface, have been shown to result in biofilms that are superior in preventing pathogen translocation through the epithelial barrier. Other FDA approved or Generally Regarded as Safe (GRAS) materials that can be used to generate surfaces for growing biofilms are also investigated. Results are prioritized using biological efficacy and persistence in animal models and shelf life as fundamental criteria. Finally, to further improve the efficacy of probiotic film introduction and maintenance, prebiotic substances are provided to the probiotic biofilm surface by diffusible cargo within the microspheres.

[0230] In a further aspect, the microspheres are partially or completely coated with a biofilm layer. This layer can be about 0.5 micrometers to about 1 millimeter deep, for example, in the range between about 1 micrometer to about 500 micrometers, about 1 micrometer to about 250 micrometers, about 1 micrometer to about 200 micrometers, about 1 micrometer to about 100 micrometers, about 1 micrometer to about 50 micrometers, about 1 micrometer to about 40 micrometers, about 1 micrometer to about 30 micrometers, about 2 micrometers to about 100 micrometers, about 2 micrometers to about 50 micrometers, about 2 micrometers to about 40 micrometers, about 2 micrometers to about 30 micrometers, about 3 micrometers to about 100 micrometers, about 3 micrometers to about 50 micrometers, about 3 micrometers to about 40 micrometers, about 3 micrometers to about 30 micrometers, about 5 micrometers to about 100 micrometers, about 5 micrometers to about 50 micrometers, about 5 micrometers to about 40 micrometers, and about 5 micrometers to about 30 micrometers. Compositions for the treatment of diarrhea and other GI related disorders

[0231] The present disclosure provides a composition comprising, alternatively consisting essentially of, or even further consisting of, biocompatible microspheres, biofilm-producing probiotic bacteria, and prebiotics, where the prebiotics comprises, alternatively consisting essentially of, or even further consisting of a nutritional food source or supplement for the culture and / or growth of the probiotic bacteria. The composition can further comprise a prebiofilmic. The prebiofilmic may specifically comprise, consist essentially of, or consist of a substance that supports the formation and persistence of biofilms, and the prebiofilmic may be a DNA-binding polypeptide or protein, and / or a DNABII polypeptide or protein. In one embodiment, the composition is frozen, e.g., flash-frozen. In another embodiment, the composition is lyophilized or dried in powder form. In a further embodiment, the composition is formulated as a suppository or in an ingestible form (e.g., a tablet) for administration. The composition may further comprise a mixture of the above-mentioned microspheres, e.g., a mixture containing two or more probiotic bacteria, and / or two or prebiofilmics and / or two or more nutritional supplements and / or supplements to support the cultivation and / or growth of the probiotic bacteria.

[0232] In some embodiments, the prebiotic comprises, consists essentially of, or consists of water-soluble carbohydrates selected from, but not limited to, one or more of inulin, oligofructose, fructooligosaccharides, galactooligosaccharides, glucose, maltose, maltodextrin, polydextrose, sucrose, fructose, lactose, isomaltulose, polyols, glycerol, and combinations thereof. In one aspect, the composition further comprises, consists essentially of, or consists of a solid or liquid carrier, such as a pharma- ceutically acceptable carrier.

[0233] As will be apparent to those skilled in the art, the prebiotics and prebiofilmics are selected in each composition to specifically support the growth of the probiotic bacteria. By way of example only, when the probiotic bacteria comprises, consists essentially of, or consists of L.reuteri, the composition comprises, consists essentially of, or consists of an effective amount of sucrose, glycerol, and optionally a HU polypeptide or protein to support the growth and maintenance of the probiotic when administered to a subject or patient. Non-limiting examples of prebiofilmic compositions include, but are not limited to, one or more of the polypeptides provided in the attached sequence listing, c-terminal fragments thereof, or n-terminal fragments thereof, or additional strains and polypeptides and fragments thereof, such as full-length or c-terminal or n-terminal fragments of those provided in Table 5, and equivalents of each of these. Additional nutritional supplements for the support of other probiotic bacteria can be found in Bergey's Manual of Determinative Bacteriology, 9 th Ed. Holt et al., Williams Wilkins (1994).

[0234] Non-limiting examples of probiotic bacteria for use in the composition include, but are not limited to, one or more of L.acidophilus, L.crispatus, L.gasseri, L.delbrueckii group, L.salivarius, L.casei, L.paracasei, L.plantarum, L.rhamnosus, L.reuteri, L.brevis, L.buchneri, L.fermentum, L.rhamnosus, B.adolescentis, B.angulation, B.bifidum, B.breve, B.catenulatum, B.infantis, B.lactis, B.longum, B.pseudocatenulatum, S.thermophiles, or combinations thereof.As will be clear to those skilled in the art, one or more bacteria can be combined in a single composition.In some embodiments, the probiotic bacteria is Lactobacillus reuteri, which in a further aspect expresses GTF protein. In other aspects, Lactobacillus reuteri expresses GTFA proteins. The bacteria are available from commercial sources, such as the American Type Culture Collection (ATCC). In one aspect, the one or more probiotic bacteria in the composition support antibacterial immunity. In other aspects, the one or more probiotic bacteria in the composition prevent pathogen colonization and / or limit excessive inflammatory responses by downregulating cytokine and chemokine production. In some embodiments, the composition further comprises, consists essentially of, or consists of an agent, which is selective for a pathogen, such as a competing pathogen.

[0235] Biocompatible microspheres comprise, consist essentially of, or consist of one or more of biodegradable polymers, non-biodegradable polymers, metals, or combinations thereof. In some embodiments, the microspheres comprise, consist essentially of, or consist of a solid core. In some embodiments, the microspheres comprise, consist essentially of, or consist of a hollow core. In some embodiments, the prebiotics are encapsulated within the hollow core of the microsphere and can be released in high concentrations only relative to the attached probiotics, either due to the semipermeable nature of the microsphere surface or due to the gradual degradation of the microsphere.

[0236] In one aspect, the present disclosure provides a composition comprising, alternatively consisting essentially of, or even further consisting of PGLA biocompatible microspheres, one or more biofilm-producing probiotic bacteria, and a nutritional supplement comprising, consisting essentially of, or consisting of one or more of sucrose or glycerol in an amount that supports the growth of the probiotic bacteria. The biofilm-producing probiotic bacteria may optionally include Lactobacillus reuteri ("L.reuteri"), which can express GTF proteins. The composition may further comprise, alternatively consisting essentially of, or even further consisting of an effective amount of an IHF or HU polypeptide or protein. The composition may further comprise a pharmaceutically acceptable carrier or a biocompatible scaffold, and is optionally formulated as a suppository.

[0237] The size of the microspheres can range from about 0.5 micrometers to about 100 micrometers. In certain embodiments, the microspheres are less than about 100 micrometers in diameter. In other embodiments, the microspheres are less than about 50 micrometers, or less than about 40 micrometers, or less than about 30 micrometers, or less than about 20 micrometers, or less than about 10 micrometers, or less than about 5 micrometers, or less than about 3 micrometers to 0.5 micrometers in diameter. In further embodiments, the microspheres are about 0.5 micrometers to about 90 micrometers, or to about 80 microns, or to about 70 micrometers, or to about 60 micrometers, or to about 50 micrometers, or to about 40 micrometers, or to about 30 micrometers, or to about 20 micrometers, or to about 10 micrometers, or to about 5 micrometers, or to about 3 micrometers, or to about 2 micrometers, or to about 1 micrometer, or to about 2 micrometers, or to about 1 micrometer, or to about 5 micrometers, or to about 3 micrometers, or to about 2 micrometers, or to about 1 micrometer. Alternatively, the diameter is from about 1 to about 100, or alternatively from about 1 to about 75, or alternatively from about 1 to about 50, or alternatively from about 1 to about 25, or alternatively from about 1 to about 15, or alternatively from about 1 to about 10 micrometers in diameter.

[0238] In some embodiments, the microspheres are biodegradable polymers, non-limiting examples of which include dextran, dextranomer; poly(lactic-co-glycolic acid) ("PLGA"); polycaprolactone ("PLC"); chitosan; gelatin; hydrogen DNA; acetalated dextran; poly(lactide); poly(glycolide); poly(lactide-co-glycolide); poly(lactic acid); poly(glycolic acid); poly(lactic acid-co-glycolic acid); poly(lactide) / poly(ethylene glycol) copolymer; poly(glycolide) / poly(ethylene glycol) copolymer; poly(lactide-co-glycolide) / poly(ethylene glycol) copolymer; poly(lactic acid) / poly(ethylene glycol) copolymer; poly(glycolic acid / poly (ethylene glycol) copolymers; poly(lactic acid-co-glycolic acid) / poly(ethylene glycol) copolymers; poly(caprolactone); poly(caprolactone) / poly(ethylene glycol) copolymers; poly(orthoesters); poly(phosphazenes); poly(hydroxybutyrates); poly(hydroxybutyrates); poly(lactide-co-caprolactone); polycarbonates; polyesteramides; polyanhydrides; poly(dioxanone); poly(alkylene alkylates); polyethylene glycol / polyorthoester copolymers; polyurethanes; poly(amino acids); polyetheresters; polyacetals; polycyanoacrylates; poly(oxyethylene) / poly(oxypropylene) copolymers; and combinations thereof. In some embodiments, the biodegradable polymer is poly(lactic acid-co-glycolic acid), i.e., PLGA.

[0239] In some embodiments, the microsphere comprises, consists essentially of, or consists of a non-biodegradable polymer.Non-limiting examples of non-biodegradable polymers include, but are not limited to, one or more of poly(ethylene vinyl acetate), poly(vinyl acetate), silicone polymers, polyurethanes, polysaccharides, such as cellulose-based polymers and cellulose derivatives, acyl-substituted cellulose acetate and its derivatives, copolymers of poly(ethylene glycol) and poly(butylene terephthalate), polystyrene, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chlorosulfonated polyolefins, polyethylene oxide, and copolymers and blends thereof.

[0240] In some embodiments, the microspheres comprise, consist essentially of, or consist of a metal, which may be selected from, but is not limited to, one or more of cobalt, chromium, gold, nickel, platinum, stainless steel, titanium, tantalum, nickel-titanium, and alloys and combinations thereof. Pharmaceutical Compositions

[0241] The composition can be formulated as a frozen composition, e.g., flash frozen, dried or lyophilized for storage and / or transportation. In addition, the composition can be administered alone or in combination with a carrier, e.g., a pharma- ceutically acceptable carrier or a biocompatible scaffold. The composition of the present invention can be administered in a conventional manner rectally as a suppository, enterally, parenterally, by injection, e.g., intravenously, subcutaneously or intramuscularly. Additional formulations suitable for other administration methods include oral formulations. Oral formulations include such commonly utilized excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suppositories, suspensions, tablets, pills, capsules, sustained release formulations, or powders and contain about 10% to about 95%, preferably about 25% to about 70%, of the active ingredient.

[0242] Typically, the composition is administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective against the disease or condition being treated. The amount administered will depend on the subject being treated. The exact amount of the composition administered will depend on the judgment of the practitioner. The preferred initial and booster administration regimen is variable, but is typically an initial administration, followed by subsequent administrations.

[0243] In many cases, it will be desirable to have multiple administrations of the composition for about, at most about, or at least about 3, 4, 5, 6, 7, 8, 9, 10 or more days. Administration will usually range from 2 days to 12 weeks apart, more usually 1 to 2 weeks apart. Periodic booster administrations at 0.5 to 5 year intervals, usually 2 year intervals, are desirable to maintain the status of the immune system.

[0244] In some embodiments, additional pharmaceutical compositions are administered to the subject to support or enhance the compositions described herein. Different aspects of the present invention include administering an effective amount of the composition to the subject. In addition, such compositions can be administered in combination with immune system modifiers. Such compositions are generally dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0245] The phrase "pharmacologically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to animals or humans. As used herein, "pharmacologically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except in the case where any conventional media or agent is incompatible with the active ingredient, its use in immunogenic and therapeutic compositions is contemplated.

[0246] The carrier can be a solvent or dispersion medium, for example, containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid poly(ethylene glycol), etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating agent, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of undesirable microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars and sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents delaying absorption, for example, aluminum monostearate and gelatin, in the composition.

[0247] The effective amount of the therapeutic composition is determined based on the intended purpose. The term "unit dose" or "dosage" refers to physically discrete units suitable for use in subjects, each containing a predetermined amount of the composition calculated to produce the desired response discussed above in connection with its administration, i.e., appropriate route and regimen. The amount administered depends on both the number of treatments and the unit dose, but also on the desired outcome and / or protection. The exact amount of the composition also depends on the judgment of the practitioner and is peculiar to each individual. Factors that affect the dosage include the physical and clinical condition of the subject, the route of administration, the intended purpose of the treatment (alleviation of symptoms versus cure), and the potency, stability, and toxicity of the particular composition. Once formulated, the solution is administered in a manner compatible with the dosage formulation and in such amount as will be therapeutically or prophylactically effective. The formulation is easily administered in a variety of dosage forms, such as the above-mentioned types of injectable solutions. Process for preparing the composition

[0248] The present disclosure also provides a method for preparing the compositions described herein, which comprises, alternatively consists essentially of, or even further consists of, mixing, contacting or culturing biocompatible microspheres with biofilm-producing probiotic bacteria and prebiotics.In one embodiment, the method further comprises, consists essentially of, or consists of adding or mixing prebiofilmics that support the formation and growth of biofilms by bacteria.Non-limiting examples of such biofilmics include one or more of DNA-binding polypeptides or proteins and / or DNABII polypeptides or proteins.In a further embodiment, the microspheres are contacted with biofilms or placed in a culture that supports the growth of biofilms on the surface of the microspheres.Additional components as disclosed herein can be further mixed with the microspheres, etc. Treatments for Colon and GI Health

[0249] Diarrheal disease is a major cause of morbidity and mortality worldwide, resulting in approximately 15% of childhood deaths. Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are the two most common bacterial causes of pediatric diarrhea. The mechanisms by which these pathogens cause diarrhea are not yet fully understood but are initiated once the pathogens colonize the intestinal epithelium (Nataro and Kaper (1998) Diarrheagenic Escherichia Coli, Clin Microbiol Rev. 11:142-201).

[0250] A closely related pathogen, Citrobacter rodentium, is a murine pathogen that is widely used to model human EPEC and EHEC infections because mice are relatively resistant to both EPEC and EHEC. In mice, C. rodentium produces colonic pathogens that are nearly indistinguishable from those produced by EPEC and EHEC in humans (Borenshtein, M. et al. (2008) Utility of the Citrobacter rodentium Infection Model in Laboratory Mice, Curr Opin Gastroenterol, 24:32-7; Luperchio and Schauer (2001) Molecular Pathogenesis of Citrobacter rodentium and Transmissible Murine Colonic Hyperplasia, Microbes Infect, 3:333-40; Mundy, TT et al. (2005) Citrobacter rodentium of Mice and Man, Cell Microbiol 7:1697-706). This may be expected since C. rodentium possesses a homolog of the enterocyte effacement locus (LEE) pathogenicity island carried by EPEC and EHEC that encodes effector proteins required for the development of attachment and retraction (A / E) lesions. These lesions are accompanied by colonic hyperplasia and pathological colitis characterized by epithelial defects and leukocyte infiltration (Luperchio and Schauer (2001) Molecular Pathogenesis of Citrobacter rodentium and Transmissible Murine Colonic Hyperplasia, Microbes Infect. 3:333-40).

[0251] The intestinal epithelium provides a formidable barrier against enteric pathogens. To cause disease, enteric pathogens must adhere to or penetrate / invade host epithelial cells. Thus, interaction with epithelial cells is the first step in the virulence of all enteric pathogens, and this step can be studied by the use of A / E pathogens to assess colonic colonization and resulting pathology.

[0252] Colonization of the colon by A / E pathogens depends on the composition of the gut microbiota. Induction of dysbiosis (disruption of the native population of beneficial bacteria) within the colonic microbiota by administering antibiotics (Wlodarska, B. et al. (2011) Antibiotic Treatment Alters the Colonic Mucus Layer and Predisposes the Host to Exacerbated Citrobacter rodentium-Induced Colitis, Infect Immun, 79:1536-45) or by inducing an inflammatory response (Lupp, ML et al. (2007) Host-Mediated Inflammation Disrupts the Intestinal Microbiota and Promotes the Overgrowth of Enterobacteriaceae, Cell Host Microbe, 2:119-29) has been shown to greatly enhance pathogen colonization.

[0253] Colonic dysbiosis may further exacerbate the inflammatory response to colonic pathogens (Wlodarska, B. et al. (2011) Antibiotic Treatment Alters the Colonic Mucus Layer and Predisposes the Host to Exacerbated Citrobacter rodentium-Induced Colitis, Infect Immun. 79:1536-45), but even in the absence of pathogen challenge, dysbiosis may contribute to the pathogenesis of inflammatory bowel disease (Machiels et al. (2013) A Decrease of the Butyrate-Producing Species Roseburia hominis and Faecalibacterium prausnitzii Defines Dysbiosis in Patients with Ulcerative Colitis, Gut (First published online September 10, 2013);Morgan et al. (2012) Dysfunction of the Intestinal Microbiome in Inflammatory Bowel Disease and Treatment, Genome Biol., 13:R79) or irritable bowel syndrome (Carroll et al. (2012) Alterations in Composition and Diversity of the Intestinal Microbiota in Patients with Diarrhea-Pdominant Irritable Bowel Syndrome, Neurogastroenterol Motil. 24:521-30, e248;Chassard, M. et al. (2012) Functional Dysbiosis within the Gut Microbiota of Patients with Constipated-Irritable Bowel Syndrome, Aliment Pharmacol Ther.Genetically susceptible individuals may propagate inflammatory responses, as evidenced by findings of dysbiosis in patients with HIV-1 infection (35:828-38).

[0254] In some embodiments, a method for treating or preventing disease in a subject is provided, comprising, consisting essentially of, or consisting of administering an effective amount of a composition as described above to a subject in need of such treatment. As used herein, "subject" refers to an animal (e.g., murine, bovine, canine, feline, equine, or simian) or human. Non-limiting diseases to be treated include, but are not limited to, any of the diseases and disorders listed above (and incorporated herein by reference), such as psychological disorders, e.g., depression or anxiety, intestinal infectious disease, infection-induced colitis, traveler's diarrhea, inflammatory bowel disease (IBD), colitis, diarrheal disease, vaginitis, wounds, burns, psoriasis, dermatitis, dental caries, periodontitis, sinusitis, or chronic and / or recurrent diseases caused by pathogenic bacteria replacing healthy bacteria or necrotizing enterocolitis (NEC). In addition, the composition can be administered to support antibacterial immunity, strengthen or support gastrointestinal barrier, or antagonize disease-related bacterial infection. In some embodiments, the disease is vaginitis. In some embodiments, the disease is colitis or traveler's diarrhea. As will be understood by those skilled in the art, the composition is specifically selected for the disease to be treated. In some embodiments, the composition further comprises, consists essentially of, or consists of prebiofilmic. In some embodiments, the prebiofilmic comprises, consists essentially of, or consists of DNA binding polypeptide or protein, and / or DNABII polypeptide or protein, such as IHF or HU, fragments thereof, and / or equivalents of each of these. In some embodiments, the composition is administered as a suppository.

[0255] In some embodiments, the composition of the method comprises about 1×10 7 ~Approx. 1×10 9 The composition is administered to provide 100 CFU / ml of biofilm-producing probiotic bacteria. In some embodiments, the composition is administered at about 6, 12, 18, 24, 36, 48 and 72 hours. In some embodiments, the composition is administered in a single dose.

[0256] In some embodiments, a method of administering probiotics is provided, comprising, consisting essentially of, or consisting of administering a dose of a composition as described above comprising, or alternatively consisting essentially of, or even consisting of, biocompatible microspheres, biofilm-producing probiotic bacteria, prebiotics and prebiofilmics to a subject in need of such treatment. In some embodiments, the composition of the method comprises about 1×10 7 ~Approx. 1×10 9 The composition is administered to provide 100 CFU / ml of biofilm-producing probiotic bacteria. In some embodiments, the composition is administered at about 6, 12, 18, 24, 36, 48 and 72 hours. In some embodiments, the composition is administered in a single dose. kit

[0257] Also provided is a kit comprising a composition comprising microspheres, biofilm-producing probiotic bacteria, and a prebiotic, where the prebiotic comprises a nutritional supplement for the probiotic bacteria, and instructions for use in a method described herein, e.g., a method for promoting neurodevelopment or treating or preventing a neurodevelopmental disorder in a subject in need thereof.

[0258] Further provided is a kit comprising a composition comprising microspheres, biofilm-producing probiotic bacteria and a prebiotic, wherein the prebiotic comprises a nutritional supplement for the probiotic bacteria, and instructions for increasing BDNF in a subject.

[0259] Still further provided is a kit comprising a composition comprising microspheres, biofilm-producing probiotic bacteria and a prebiotic, wherein the prebiotic comprises a nutritional supplement for the probiotic bacteria, and instructions for increasing Grin2A in a subject. Therapeutic methods for the treatment of NEC-induced neurodevelopmental defects

[0260] Necrotizing enterocolitis (NEC) is a serious medical condition that primarily affects low birth weight infants, with an overall mortality rate after surgical treatment as high as 30% and reaching 50% in very low birth weight infants. Currently, there is no known cure for the disease. More than half of survivors of NEC subsequently exhibit some degree of cognitive impairment and neurodevelopmental deficits.

[0261] Provided is a method of preventing, delaying, or treating neurodevelopmental deficiency in an infant subject suffering from, susceptible to, or suffering from necrotizing enterocolitis (NEC), comprising, or alternatively consisting essentially of, or even further consisting of, administering to the subject an effective amount of any of the pharmaceutical compositions disclosed herein. In one aspect, the method comprises, or consists essentially of, treatment. In another aspect, the method comprises, or consists essentially of, or even further consists of prevention. In one aspect, the administration is intrauterine administration. In another aspect, the administration is postnatal administration. In a further aspect, the administration is enteral administration.

[0262] In some embodiments, the neurodevelopmental deficiency comprises, consists essentially of, or consists of deficiencies in one or more of: physical strength, coordination, interpersonal interaction, resilience mechanisms, auditory reflex testing, curiosity, learning ability, working memory, short-term memory, long-term memory, visuospatial reasoning, cognition, object recognition, and serotonin.

[0263] In some embodiments, preventing neurodevelopmental deficiency includes, consists essentially of, or consists of partial prevention of the deficiency.Partial prevention of the deficiency includes, consists essentially of, or consists of partial prevention of the deficiency, in which the subject is able to perform a variety of tests, including Jamar dynamometer, manual muscle testing, manual muscle testing (MMT), isokinetic dynamometry, trunk stability test (TST), unilateral hip bridge endurance test (UHBE), pronation sign, Barre sign, Romberg test, Landau reflex, particle suspension, sensory reflexes (pin pain, superficial touch, position, vibration, and charger), reflexes (biceps, triceps, brachioradial, patellar, and ankle), Moro reflex, Tonic neck response, sucking reflex, palmar and plantar grasp reflex, parachute response, somatic neck righting (NOB), somatic righting (BOB), ear opening acoustic reflex, static compliance, ear canal physical volume, contralateral reflex, ipsilateral reflex, tympanometry, Y-maze, novel object recognition task, State-Trait Personality Inventory (STPI), Five-Dimensional Curiosity Scale, Self-Curiosity Attitudes and Interests Scale, Curiosity Seeking Scale-II, State-Trait Personality Inventory (STPI), subscales of the Sensation Seeking Scale (SSS), Bayley Scales of Infant Development (BSID-III) (1-42 months), Mullen Early Learning Scale (1-68 months), Fagan Intelligence Scale for Infants (FTII) (birth-12 months), Griffith Mental Development Scale I (0-2 years), Battelle Development Scale (BDI) (birth-8 years), and Vineland Adaptive Behavior Scale (0-18 years). In some embodiments, the method may include obtaining a score that is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the score obtained by a control subject of the same age and species who is not afflicted with NEC on one or more of any clinically recognized tests selected from the group consisting of a subject having a pulmonary function, ...

[0264] In some embodiments, preventing a neurodevelopmental deficiency comprises, consists essentially of, or consists of a complete prevention of the deficiency or a delay in time until a subject exhibits one or more symptoms of the deficiency. Complete prevention of failure will be achieved if the subject undergoes the following tests: Jamar dynamometer, manual muscle testing, manual muscle testing (MMT), isokinetic dynamometry, trunk stability test (TST), unilateral hip bridge endurance test (UHBE), pronation sign, Barre sign, Romberg test, Landau reflex, particle suspension, sensory reflexes (pin pain, superficial touch, position, vibration, and charger), reflexes (biceps, triceps, brachioradial, patellar, and ankle), Moro reflex, tonic neck response, sucking reflex, palmar and plantar grasp reflex, parachute response, somatic neck righting (NOB), somatic righting (BOB), ear opening acoustic reflex, static compliance, physical volume of the ear canal, contralateral reflex, ipsilateral reflex, tympanometry, Y-maze, novel object recognition task. The subject may include obtaining the same score as a control subject of the same age and species not affected by NEC on one or more of any clinically recognized tests selected from the following: STPI (State-Trait Personality Inventory), Five-Dimensional Curiosity Scale, Self-Curiosity Attitudes and Interests Scale, Curiosity Seeking Scale-II, State-Trait Personality Inventory (STPI), subscales of the Sensation Seeking Scale (SSS), Bayley Scales of Infant Development (BSID-III) (1-42 months), Mullen Early Learning Scales (1-68 months), Fagan Scale of Intelligence for Infants (FTII) (birth-12 months), Griffith's Mental Development Scale I (0-2 years), Battelle Development Scale (BDI) (birth-8 years), and Vineland Adaptive Behavior Scales (0-18 years).

[0265] In some embodiments, scoring is performed 0-1 month, 1-6 months, 6-12 months, 12-18 months, 18-24 months, 24 months to 3 years, or greater than 3 years after administration of the pharmaceutical composition.

[0266] In some embodiments, administration is selected from enteral administration, oral administration, nasal administration, sublingual administration, buccal administration, rectal administration, vaginal administration, intravenous administration, intraarterial administration, intradermal administration, intraperitoneal administration, intrathecal administration, intramuscular administration, epidural administration, intracerebral administration, intraventricular administration, transdermal administration, or any combination thereof.

[0267] In some embodiments, the pharmaceutical composition is administered to a fetus of a subject in need of treatment before the fetus is born (i.e., prenatally). In some embodiments, the pharmaceutical composition is administered to a mother who is pregnant with a subject in need of treatment. In some embodiments, the pharmaceutical composition is administered to a subject having an age selected from 0-1 days, 1-6 days, 6-12 days, 12-18 days, 18-24 days, 24 days to 100 days, or more than 100 days.

[0268] In some embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of PGLA or dextranomer biocompatible microspheres, one or more biofilm-producing probiotic bacteria comprising, consisting essentially of, or consisting of at least Lactobacillus reuteri ("L. reuteri"), and a nutritional supplement comprising, consisting essentially of, or consisting of one or more of maltose, sucrose, glycerol, or histidine in an amount to support the growth of the probiotic bacteria, and optionally the microspheres are partially or fully coated with a biofilm. In some embodiments, the pharmaceutical composition comprises, consists essentially of, or consists of dextranomer biocompatible microspheres comprising, consisting essentially of, or consisting of Lactobacillus reuteri ("L. reuteri") and at least maltose or a nutritional supplement comprising, consisting essentially of, or consisting of at least maltose. In some aspects, the microsphere surface is partially or completely covered with a biofilm.

[0269] In some embodiments, the microspheres have a diameter in the range of about 0.5 micrometers to 75 micrometers.

[0270] In some embodiments, the infant has an age or age selected from 0-1 month, 1-6 months, 6-12 months, 12-18 months, 18-24 months, 24 months-3 years, or older than 3 years. In some embodiments, the infant is a human.

[0271] In some embodiments, the composition of the method comprises about 1×10 7 ~Approx. 1×10 9The composition is administered to provide 100 CFU / ml of biofilm-producing probiotic bacteria. In some embodiments, the composition is administered at about 6, 12, 18, 24, 36, 48 and 72 hours. In some embodiments, the composition is administered in a single dose.

[0272] In some embodiments, a method of administering probiotics is provided, comprising, consisting essentially of, or consisting of administering a dose of a composition comprising, or alternatively consisting essentially of, or even consisting of biocompatible microspheres, biofilm-producing probiotic bacteria, prebiotics and prebiofilmics, as described above, to a subject in need of such treatment. In some embodiments, the composition of the method comprises about 1×10 7 ~Approx. 1×10 9 The composition is administered to provide 100 CFU / ml of biofilm-producing probiotic bacteria. In some embodiments, the composition is administered at about 6, 12, 18, 24, 36, 48 and 72 hours. In some embodiments, the composition is administered in a single dose. EXAMPLES

[0273] Experimental Example Example 1 To determine whether L. reuteri in a biofilm state is superior to planktonic bacteria for colonizing the mouse intestine, L. reuteri was introduced by oral gavage, but a single dose of L. reuteri was administered rather than repeated daily gavage, which is typically required for the retention of planktonic bacteria and for beneficial effects. Lactobacillus reuteri was grown in biofilm cultures or in biofilms grown on poly(lactic-co-glycolic acid) microspheres, such as PLGA, or other FDA approved biodegradable microspheres (which are hydrolyzed to lactic and glycolic acids) ranging from 20 to 300 μm in diameter (Beer, et al., (1998) Poly (Lactic-Glycolic) Acid Copolymer Encapsulation of Recombinant Adenovirus Reduces Immunogenicity in Vivo, Gene Ther, 5: 740-6; Kumari, et al., (2010) Biodegradable Polymeric Nanoparticles Based Drug Delivery Systems, Colloids Surf B Biointerfaces, 75:1-18).

[0274] Similar preparations of planktonic bacteria were prepared, but with the addition of PLGA microspheres and prebiofilmics immediately prior to gavage. As shown in Figure 3, the number of mice with L. reuteri detected in the mouse colon after 7 days was increased when introduced as a biofilm versus planktonic cells. The presence of PLGA also increased the number of mice positive for L. reuteri, whether the bacteria were in planktonic or biofilm form, compared to conditions in which PLGA was absent. This may indicate that L. reuteri can initiate binding to PLGA, a precursor step to biofilm formation, even during this brief interaction (<30 min). In the stomach, the only condition in which all mice retained L. reuteri was biofilm-grown cells with the addition of PLGA. It is therefore clear that growth of L. reuteri in biofilms in the presence of PLGA enhances colonization and persistence in the stomach and colon compared to planktonic cells. Example 2 In vitro activity of Lactobacillus reuteri against C. rodentium

[0275] To determine whether L. reuteri has the ability to better compete with C. rodentium in vitro as either a biofilm or planktonic state, a competition assay was developed. Here, C. rodentium biofilms were performed in glass chamber slides (LB medium, 24 h, 37 °C, 5% CO2). L. reuteri (108 colony forming units (CFU)) was then added as treatments to a medium compatible with both organisms, either as planktonic or in one of three biofilm forms (biofilm, PLGA biofilm, PLGA+HU biofilm; preparations as in Figure 3). After 16 h, the biofilm contents of the chamber slides were removed and aliquots were plated on a medium selective for L. reuteri (MRS) and a medium selective for C. rodentium (LB). C. rodentium treated with L. reuteri biofilms showed a more than 2-fold reduction in CFU / ml compared to untreated ones, regardless of the state of L. reuteri introduced (Table 1). More interestingly, although all L. reuteri grew during the 16-h challenge, L. reuteri introduced in biofilm form gave rise to >10-fold more CFU than when added in planktonic form. [Table 1]

[0276] In the reverse experiment, L. reuteri biofilm was first introduced, followed by planktonic and biofilm forms of C. rodentium (10 8In contrast to previous experiments, L. reuteri was still able to grow to a >10-fold increase in CFU regardless of the presence of C. rodentium (<2-fold difference between conditions), but C. rodentium did not grow during the 16-h challenge and, in fact, CFU was reduced when introduced in planktonic form. These in vitro results indicate that C. rodentium biofilms can be effectively challenged with L. reuteri and that L. reuteri persists better when introduced in biofilm form than planktonic cells. Furthermore, preformed L. reuteri biofilms create a poor environment for challenge with planktonic C. rodentium to lead to establishment. Example 3 Lactobacillus reuteri against C. rodentium in vivo

[0277] To determine whether L. reuteri is better able to compete with C. rodentium in vivo as either a biofilm or planktonic state, we utilized a version of a published competition assay (Mackos, et al., (2013) Probiotic Lactobacillus reuteri Attenuates the Stressor-Enhanced Severity of Citrobacter Rodentium Infection, Infect Immun, 81:3253-63). Briefly, L. reuteri was introduced by oral gavage into mice as described above (planktonic vs. biofilm L. reuteri in vivo). After 12 hours, an equal number of planktonic C. rodentium was also added by gavage. After 12 hours, all mice were sacrificed for necropsy. Unlike published studies showing that C. rodentium penetration of brush border epithelium and dissemination to the spleen can be prevented by daily administration of planktonic L. reuteri (Mackos, et al., (2013) Probiotic Lactobacillus reuteri Attenuates the Stressor-Enhanced Severity of Citrobacter rodentium Infection, Infect Immun, 81:3253-63), there was a statistically significant 10-fold reduction in C. rodentium penetration to the spleen in prebiofilmic-treated L. reuteri biofilms using a single dose (Figure 4). This result is consistent with the magnitude and robustness of the more persistent phenotype that prebiofilmic-treated probiotic biofilms have in vivo. Example 4 Characterization of biofilm durability and robustness for probiotic therapy

[0278] This example provided strong evidence that probiotics in a biofilm state result in a formulation that is superior to bacteria grown in a planktonic state. This example also provides an example of how to prepare these biofilms, including the frequency of administration. In addition, this example explores the properties of the biofilm itself to begin to determine why this state outperforms planktonic bacteria. Finally, this example explores the shelf life of the preparation prior to embodiment in a human host. Together, this example identifies and characterizes the conditions and components for the preparation of probiotic biofilms. Example 4.1: Effect of growth phase

[0279] We show that L. reuteri forms robust biofilms in vitro and that 24-h biofilms of L. reuteri are sufficient to colonize the mouse intestine. This example varies the age of the biofilms to determine the optimal age for biofilm colonization.

[0280] In vivo L. reuteri biofilms. When exposed to a surface, L. reuteri begins to attach almost immediately. After 6 hours, sufficient biomass has been produced to be both visible and to begin to form classical biofilm structures (e.g., mushrooms, Abee and Kuipers, (2011) Understanding Microbial Behavior within and Outside the Host to Improve Food Functionality and Safety, Curr Opin Biotechnol, 22:133-5). L. reuteri biofilms grown (as described above) on PGLA microspheres with HU and calf thymus DNA are isolated at approximately 6, 12, 18, 24, 36, 48 and 72 hours, normalized to CFU (108), and introduced by gavage into mice (9 per time point from triplicate experiments). Each mouse is assessed daily for 12 days by enumerating total lactobacillus levels in fecal samples (cultured on MRS agar).

[0281] In addition, this example uses real-time PCR to evaluate the 16S rRNA gene sequence copy number for the genus Lactobacillus (including some species of Weisella, Pediococcus and Leuconostoc due to difficulties with primer specificity) and especially for L. reuteri. 16S rRNA gene copy number is determined using real-time PCR in feces every day for 12 days after oral inoculation, and in colon, cecum, small intestine (including ileum, jejunum and duodenum) and stomach (including forestomach) on days 1, 3, 6 and 12. Sham mice with and without planktonic cells serve as controls. A significant increase in L. reuteri levels in mice treated with biofilm-grown L. reuteri compared to mice treated with sham or planktonic cells is an indication of persistence and robustness. Example 4.2: Effect of growth conditions

[0282] One set of growth conditions has been used so far: static culture in MRS medium at 37° C. (Jones, and Versalovic, (2009) Probiotic Lactobacillus reuteri Biofilms Produce Antimicrobial and Anti-Inflammatory Factors, BMC Microbiol, 9:35). Although not an exhaustive list, the present examples vary the medium, prebiofilmics as well as pH and aerobicity.

[0283] Various growth conditions in vitro. In this example, other media, such as LB, THYE (THB containing yeast extract), and mTSB (modified tryptic soy agar broth), are used to grow biofilms instead of MRS, with L. reuteri growing to different extents in each. In addition, this example changes the starting pH to about 5.5, 6, 6.5, or 7, since L. reuteri growth is favored under more acidic conditions. L. reuteri can be grown microaerophilically under 5% CO2, but stressful time conditions favor biofilm growth (Flemming, and Wingender, (2010) The Biofilm Matrix, Nat Rev Microbiol, 8:623-33). Here, L. reuteri biofilms are also grown in air or in the absence of oxygen (anaerobic chamber). Finally, this example alters the prebiofilmics of HU (approximately 0.1, 1, 10, 100 μg / ml) and calf thymus DNA (approximately 0.1, 1, 10, 100 μg / ml). All biofilms described above are assessed by CSLM with LIVE / DEAD® staining in triplicate for height, average thickness and biomass as indicators of robust growth.

[0284] Different growth conditions in vivo. The optimal conditions for biofilm growth are compared against both the initial standard conditions as well as the conditions that produce the poorest biofilms (controls). Biofilms are introduced by oral gavage to 9 mice (from triplicate experiments) per test under the conditions optimized in Example 4.1. Sham mice with and without planktonic cells serve as controls. L. reuteri levels are assessed as in Example 4.1 at 1, 3, 6 and 12 days after challenge. Example 4.3: Effect of bacterial administration

[0285] Dosing of L. reuteri; frequency and size. Determine the RHe frequency and / or size of dosing that improves the persistence and robustness of L. reuteri introduction. Grow L. reuteri biofilms for 24 hours (or age conditions as determined in Examples 4.1 and 4.2) on PLGA microspheres supplemented with HU and calf thymus DNA. Various doses (10 7 , 10 8 and 10 9 A matrix of L. reuteri biofilms is introduced into mice by oral gavage, with a range of L. reuteri CFU (CFU) and frequency (single dose, or daily doses up to 3 days), resulting in 9 different conditions. L. reuteri levels are assessed in vivo 1, 3, 6 and 12 days after gavage as outlined in Example 4.1. Nine mice (from triplicate experiments) are used per condition at each time point. Sham mice with and without planktonic cells serve as controls. Example 4.4: Testing of Dispersed Biofilm Bacteria

[0286] Testing Dispersed L. reuteri from Biofilms. Dispersed bacteria in the mouse gut are investigated for their durability and robustness. L. reuteri biofilms can be dispersed by antisera against DNABII family members (e.g., E. coli IHF). Here, this example tests the bacteria released (dispersed) due to anti-IHF treatment. 24-hour L. reuteri biofilms grown in chamber slides (no PLGA, HU or DNA added to aid dispersion) are treated with anti-IHF. Peak dispersion occurs approximately 8-12 hours after treatment (Goodman, et al. (2011) Biofilms Can Be Dispersed by Focusing the Immune System on a Common Family of Bacterial Nucleoid-Associated Proteins, Mucosal Immunol, 4:625-37), and conditioned medium containing dispersed L. reuteri after 12 hours of antibody treatment is introduced into mice by oral gavage. Assess L. reuteri levels in vivo at days 1, 3, 6 and 12 post-challenge as outlined in Example 4.1. Use nine mice (from triplicate experiments) per time point, with similar numbers for the control using planktonic bacteria and the optimized biofilm bacteria (Examples 4.1-4.3).

[0287] Biofilms prove to be superior for colonization, survival and persistence of probiotic bacteria in the intestine. This is true not only for the biofilm itself, which has better characteristics than planktonic bacteria, but also for the bacteria dispersed from the biofilm. In effect, biofilms act as dispersed bacteria generators. In fact, physiological differences (e.g. in terms of antibiotic susceptibility) have been observed in dispersed bacteria compared to laboratory-grown planktonic bacteria. Example 4.5: Shelf Life

[0288] For ease of implementation and use, the L. reuteri preparation needs to be in a sufficiently stable form.

[0289] Freezing. L. reuteri biofilms were flash frozen with no reduction in CFU and minimum inhibitory concentration, or MIC (>2 mg / ml ampicillin; MIC for planktonic L. reuteri <4 μg / ml). This suggests that L. reuteri retains at least one characteristic of its biofilm state: a high MIC. Optimized L. reuteri biofilms (Examples 4.1-4.3) are investigated for ambient air freezing to -20°C and -80°C with or without glycerol (cryoprotectant; see also Example 2), and flash freezing to -80°C (storage tubes containing freshly prepared bacterial suspensions are placed in dry ice-ethanol).

[0290] The medium is first removed, and the resulting biofilm is scraped, processed and frozen. The bacteria are stored at these temperatures for one day, one week or one month, then thawed at ambient temperature and used for introduction into mice by oral gavage. Nine mice from triplicate experiments are used, with similar numbers for the control using planktonic bacteria and the optimized biofilm bacteria (Examples 4.1-4.3). Each mouse is evaluated as in Example 4.1.

[0291] Drying. Optimized L. reuteri biofilms (Examples 4.1-4.3) are investigated by freeze-drying followed by freezing using the optimization technique in Example 4.5. The dried bacteria are stored at room temperature for about one day, one week or one month, then rehydrated with the original biofilm volume of sterile distilled water at ambient room temperature and used for introduction into mice by gavage. Nine mice from triplicate experiments are used, with similar numbers for the control using planktonic bacteria and the optimized biofilm bacteria (Examples 4.1-4.3). Each mouse is evaluated as in Example 4.1.

[0292] Finally, L. reuteri strain (ATCC23272) is utilized. Additional strains of L. reuteri (e.g., 100-23, ATCCPTA6475, ATCC55730 strains) are also investigated to evaluate strain differences. As additional controls, commercially available L. reuteri strains (Fleet® Pedia-Lax™ Probiotic Yums™ approximately 100,000,000 CFU / tablet, L. reuteri Protectis® DSM 17938 and Gerber® Soothe Colic Drop approximately 100,000,000 CFU / serving (5 drops, approximately 200ul), L. reuteri Protectis® DSM 17938) are investigated. This example shows that by dissolving each product in water and using them directly in in vitro competition experiments with C. rodentium, each product shows comparable activity to a strain of L. reuteri in planktonic form. Example 5 Identification and characterization of biodegradable surfaces and prebiotic materials to promote durability and robustness of probiotic biofilms

[0293] Other types of microspheres and specific cargoes that can either promote probiotic growth or inhibit pathogens are explored. Example 5.1: Testing of empty microspheres

[0294] In vitro binding of empty microspheres, DNA, gelatin, polylactic acid, poly-α-caprolactone, chitosan and acetalized dextran is investigated in this example.

[0295] Although we utilize PLGA microspheres as a surface for growing the biofilm, there are other FDA approved or GRAS biodegradable microspheres that may prove advantageous for the purpose. Five additional types of microspheres have been investigated, as shown in Table 2 (Chellat, F. et al.(2000) In Vitro and in Vivo Biocompatibility of Chitosan-Xanthan Polyionic Complex, J Biomed Mater Res., 51:107-16;Costa, D. et al.(2012) Swelling Behavior of a New Biocompatible Plasmid DNA Hydrogel, Colloids Surf B Biointerfaces 92:106-12;Kauffman et al.(2012) Synthesis and Characterization of Acetalated Dextran Polymer and Microparticles with Ethanol as a Degradation Product, ACS Appl Mater Interfaces, 4:4149-55;Kumari et al.(2010) Biodegradable Polymeric Nanoparticles Based Drug Delivery Systems, Colloids Surf B Biointerfaces, 75:1-18;Sinha et al. (2004) Poly-Epsilon-Caprolactone Microspheres and Nanospheres: An Overview, Int J Pharm. 278:1-23; Topuz and Okay (2009) Formation of Hydrogels by Simultaneous Denaturation and Cross-Linking of DNA, Biomacromolecules 10:2652-61).Therefore, since DNA is the basis of the EPS of biofilms, DNA can be used as the microsphere material.

[0296] This example is an example of the in vitro and in vivo optimization strategy from Example 4. Microspheres from materials in Table 2 and repeat Examples 4.1-4.5 are constructed. Microspheres that cannot support robust biofilm growth in vitro using height, thickness and biomass as the initial metrics are no longer considered. Similarly, microsphere types that are substantially unable to exceed the metrics in vivo against planktonic bacteria are no longer considered. Shelf life with or without bacteria, stability at low pH (gastric conditions) are also contemplated. [Table 2] Example 5.2: Testing prebiotic nutrients and additives as cargo to favor probiotics

[0297] It is known that the cargo of PLGA diffuses slowly or not at all compared to the hydrolysis rate of the microspheres (Fredenberg, et al. (2011) The Mechanisms of Drug Release in Poly(Lactic-Co-Glycolic Acid)-Based Drug Delivery Systems--a Review, Int J Pharm, 415:34-52). Here, we synthesized microspheres with prebiotic cargo and evaluated their ability to support the growth of L. reuteri in vitro and in vivo in a mouse model.

[0298] This example investigates nutrients in vitro. As a first test, cargoes are loaded (encapsulated inside the microspheres) into PLGA microspheres during their synthesis. These cargoes include, but are not limited to, inulin, fructooligosaccharides and galactooligosaccharides, as they support lactic acid bacteria growth. In addition, microspheres are made with MRS medium and / or glycerol, the former being restrictive for gram-negative bacteria, some of which are pathogens, and the latter glycerol, as it stimulates reuterin production, an antimicrobial molecule thought to give L. reuteri an advantage over competing bacteria. L. reuteri biofilm growth on these microspheres is performed under the conditions found in Example 4 (or Example 5.1 for variant microspheres), and height, thickness and biomass are determined by CSLM.

[0299] This example tests prebiofilmics in vitro. As in Example 4.2, the ability of prebiofilmics (HU and DNA) as cargo in PLGA microspheres (and the microsphere types from Example 5.1) to support biofilm growth in vitro was tested. In each case, biofilms are grown under the conditions seen in Example 4 using microspheres synthesized in the presence of HU and / or DNA (for encapsulation inside the microspheres) and determined by CSLM for height, thickness and biomass.

[0300] This example tests combinations of prebiotics and prebiofilmics in vitro, where a matrix of combinations of two probiotic cargos and two prebiofilmic cargos (all 16 combinations of two, all 4 combinations of three, and all four single combinations equaling a total of 21 combinations) is created to find a suitable prebiotic or prebiofilmic. In each case, biofilms are grown under the conditions found in Example 1 using PLGA microspheres (and microsphere types from Example 5.1) synthesized in the presence of the cargo, and height, thickness, and biomass are determined by CSLM.

[0301] This example tests the optimized components in vivo. The conditions from Example 5.2 that produce biofilms are used for the in vivo experiments. The four most promising conditions for PLGA microsphere cargo (or the two most promising PLGA and the two most promising other types of microspheres from Example 5.1) are tested in nine mice each from triplicate experiments. Each mouse is evaluated as in Example 4.1 at 1, 3, 6 and 12 days after L. reuteri challenge. Sham mice (without bacteria) and planktonic bacteria serve as controls. Example 5.3: Prebiotic nutrients that thwart pathogens

[0302] Microspheres containing various probiotic cargoes are investigated to determine whether they support pathogen biofilm growth. Microspheres containing the prebiotics are contacted with the pathogen (i.e., C. rodentium strain DBS120(pCRP1::Tn5)) as well as the probiotics.

[0303] This example tests pathogen-hindering nutrients in vitro. Biofilms are grown in vitro using the same prebiotic and prebiofilmic substances from Example 5.2 as cargo. C. rodentium is grown in LB medium and used to seed biofilms with PLGA and the aforementioned cargo. Biofilms are determined for height, thickness and biomass by CSLM in comparison to empty PLGA microspheres.

[0304] This example tests pathogen-hindering nutrients in vivo. Considering the results from the in vitro biofilm data in Example 5.3, we investigate four cargos for C. rodentium biofilm growth and their use in vivo in a mouse model. Nine mice (from triplicate experiments) for each condition per time point are used, with planktonic C. rodentium as control and sham (no bacteria). Fecal C. rodentium levels are determined by cultures on all days after oral C. rodentium administration. On days 1, 6, 12 and 24 after oral C. rodentium administration, colons are removed and cut longitudinally to assess inflammatory cytokines (e.g., TNF-α), inflammatory mediators (e.g., inducible nitric oxide synthase (iNOS)), and chemokines (e.g., CCL2) in half of the colon by real-time RT-PCR. In the remaining half of the tissue, immunohistochemistry is used to evaluate leukocyte infiltration into the colon (e.g., F4 / 80+ macrophages; myeloperoxidase (MPO)+ polymorphonuclear cells). Although the aforementioned immune components are necessary for protective immunity against C. rodentium, when overproduced, they can result in tissue-damaging colitis. Therefore, the remaining half of the tissue is used to evaluate colonic pathology by H&E staining.

[0305] As mentioned above, microsphere biofilm preparations can contain different types of microspheres and different cargoes. It is the applicant's belief that biofilms (regardless of surface) are superior to planktonic bacteria in terms of seeding probiotic colonization in vivo.

[0306] Non-limiting examples of cargo include, but are not limited to, specific effectors of innate immunity that reduce inflammation, which is part of the process leading to dysbiosis. For example, the microspheres can contain conditioned medium from L. reuteri, since L. reuteri produces such substances. Similarly, other bacteria are within the scope of the present disclosure (e.g., C. rodentium and L. reuteri) for pathologies that generally result from dysbiosis. Example 6 Characterization of the ability of L. reuteri to restrict or displace the enteropathogenic bacterium C. rodentium in the mouse intestine.

[0307] The previous example identified and characterized a means to create L. reuteri biofilms with good durability and robustness in the mouse gut, and also investigated how these conditions may affect mouse enteropathogenic C. rodentium. This example investigated L. reuteri biofilm preparations to determine whether they could reduce the effects of C. rodentium or even eliminate to some extent introduced or existing pathogens. Example 6.1: Testing optimized L. reuteri biofilm growth conditions upon C. rodentium challenge; production of L. reuteri

[0308] In vitro challenge of L. reuteri with C. rodentium. This example systematically determines which conditions improve L. reuteri protection against C. rodentium challenge. As shown in Table 3, this example involves growing L. reuteri in biofilms (approximately 12, 24, and 48 hour biofilms to reflect different ages) and then incubating the biofilms with various amounts of planktonic C. rodentium (10 7 , 10 8 and 10 9 Systematically perform in vitro experiments in which L. reuteri biofilm growth conditions from Example 4.2 (e.g., for prebiofilmics, since the medium for challenge should at least support the growth of both species) as well as 2.1, 2.2 and 2.3 are investigated. Evaluate the mixed biofilms by CSLM and by plate counting on selective media after 12 or 24 hours of treatment to determine which species composition and number predominate under each condition. Controls include each species without the other under each condition (e.g., addition of C. rodentium added to PLGA microspheres without L. reuteri in each chamber slide). All experiments are performed in triplicate. [Table 3]

[0309] In vivo challenge of L. reuteri with C. rodentium. Prioritize L. reuteri biofilm preparations for introduction into animals based on maximum retention or dominance of L. reuteri observed. In addition, prepare L. reuteri based on any success obtained from Examples 4.1, 4.4 and 4.5. In general, introduce L. reuteri biofilm 12 hours before oral challenge with C. rodentium. Triplicate experiments are performed with a final sample size of 9 mice per condition and time point, and are evaluated at 1, 6, 12 and 24 days after challenge (peak C. rodentium infection occurs at about day 12). Fecal C. rodentium levels are evaluated, and pathogen-induced colitis is evaluated as in Example 5.3. L. reuteri levels are also evaluated as in Example 4.1. In all cases, controls include C. rodentium without L. reuteri, and C. rodentium challenge plus planktonic L. reuteri. Example 6.2: Testing challenge conditions

[0310] Frequency and timing of L. reuteri administration following challenge with C. rodentium in vivo. This example tests how administration of L. reuteri affects its ability to act as a prophylaxis against C. rodentium challenge. This example prioritizes the top three L. reuteri administration conditions to reflect the most robust and durable results from Example 4.3. This example then uses these conditions to challenge these L. reuteri-treated mice with C. rodentium (approximately 12, 24 or 36 hours after the final L. reuteri treatment). Nine mice (from triplicate experiments) are used per condition and time point. Vehicle mice infected with C. rodentium and single planktonic L. reuteri serve as controls. C. rodentium levels and pathogen-induced colitis are assessed as in Example 5.3 at 1, 6, 12 and 24 days after challenge, and L. reuteri levels are assessed as in Example 4.1. Example 6.3: Testing therapeutic probiotic challenge after pathogenic treatment based on the results in Examples 6.1 and 6.2

[0311] In Examples 6.1 and 6.2, conditions were optimized for the use of L. reuteri as a prophylaxis against pathology caused by C. rodentium, where C. rodentium was introduced before L. reuteri to determine the effect of L. reuteri challenge on existing C. rodentium pathogenesis.

[0312] Attack of C. rodentium by L. reuteri biofilms in vitro. This example shows that L. reuteri biofilms attacked C. rodentium biofilms more effectively than planktonic L. reuteri. Here, we use L. reuteri in biofilm form under conditions that mimic the three conditions from Example 6.1. Briefly, C. rodentium biofilms (12, 24 or 36 h) were incubated with L. reuteri biofilms (10 7 , 10 8 and 10 9 CFU) are challenged against the C. rodentium biofilms. After challenge with L. reuteri, the mixed biofilms are assessed 12 or 24 hours later by CSLM or by plate counts on selective media to determine which species composition and numbers predominated under each condition, respectively. Controls include each species without the other under each condition (e.g., addition of L. reuteri to chamber slides with no pre-existing C. rodentium). All experiments are performed in triplicate.

[0313] Challenge of C. rodentium with L. reuteri biofilm in vivo. Here, this example determines whether L. reuteri biofilm can attack a prior C. rodentium infection in a mouse model. Three different C. rodentium conditions (single gavage 12, 24 or 36 hours) were investigated before challenge with L. reuteri. Four L. reuteri biofilm conditions, including administration (Example 6.2), are used to challenge C. rodentium. At least two of these conditions are taken from Example 6.1. Nine mice from triplicate experiments are used to test each of these 12 conditions. Pathogen-induced colitis is assessed as in Example 5.3 and L. reuteri levels are assessed as in Example 4.1.

[0314] Here, this example determines how effective L. reuteri introduced in biofilm form is as a prophylaxis against C. rodentium attack and as a treatment for existing C. rodentium infection. So far, L. reuteri has not been able to eliminate pathogens such as C. rodentium under these conditions, so it is particularly important to find conditions where probiotics can prevent or even cure enteropathogenic infections. The results here provide a rationale for future probiotic approaches.

[0315] Finally, in vitro assays will be performed with other pathogens as a precursor to future in vivo experiments. Pathogens included in the in vitro investigations are enteric pathogens with different modes of infection, including invasive pathogens (e.g., Salmonella enterica subspecies Typhimurium and Shigella flexneri), additional A / E pathogens (e.g., enterohaemorrhagic E. coli O157:H7; and enterotoxigenic E. coli), and toxigenic pathogens (e.g., Vibrio cholera and enterotoxigenic E. coli), and the rate-limiting step in these experiments will be to find co-culture conditions that sufficiently mimic the in vivo situation. Example 7 Statistical analysis and sample size determination

[0316] The majority of experiments involve multiple parameters and groups. Therefore, two, three or four-way analysis of variance (ANOVA) is mainly used. As an example of statistical approach, in Example 4.1, between-subject ANOVA is used with probiotic (i.e., probiotic vs. vehicle control), condition (i.e., planktonic vs. biofilm), and incubation time (i.e., 6, 12, 18, 24 or 36 hours) as between-subject variables. Since different mouse groups are collected on days 1, 3, 6 and 12 after oral inoculation, collection date is also used as between-subject variable.

[0317] First, significant four-way interactions are interpreted using post-hoc independent samples t-tests with a modified Bonferroni correction factor applied for multiple comparisons. Three-way and two-way interactions are then interpreted by post-hoc tests, followed by main effects. This general approach is followed for both in vitro and in vivo experiments.

[0318] Due to the inherent variability of in vivo experiments, a significant amount of time is spent determining the sample size required to identify statistically significant differences between groups. A power analysis performed with six different groups (preliminary sample size of 6), population mean of 3.95, and population variance of 0.75 using preliminary data investigating C. rodentium levels after L. reuteri administration showed that a sample size of n=9 per condition per time point was required to obtain statistical significance at α=0.05 while maintaining a power of 0.8. Thus, all animal studies involve a sample size of 9 per treatment and time point. This is accomplished by combining data from triplicate experiments, each containing n=3 mice per treatment and time point.

[0319] Probiotics are widely used for their health benefits of promoting digestion, but few actually prevent pathogen colonization and reduce inflammatory responses. The effectiveness of probiotic bacteria can be significantly improved by the way they are introduced into the host, especially by growing them in the form of a biofilm. Data suggest that in vivo colonization by probiotic L. reuteri is greatly enhanced when grown as a biofilm compared to planktonic cells. In addition, colonization was also increased when L. reuteri was grown in the presence of a biodegradable surface (PLGA). This indicates that conditions have been optimized, which allows for a significant improvement in L. reuteri establishment in the host body.

[0320] Unexpectedly and surprisingly, the applicant demonstrated both in vitro and in vivo that treatment of L. reuteri as a biofilm in the presence of PLGA prior to challenge with the bacterial pathogen C. rodentium caused a significant reduction in the number of C. rodentium compared to planktonic L. reuteri treatment. These data reveal that probiotics can be better established when provided as a biofilm, indicating that the way in which the bacteria are introduced can greatly reflect disease outcome. Example 8

[0321] Probiotic microorganisms have also been shown to reduce anxiety and depression in otherwise healthy humans and laboratory animals. A combination of Lactobacillus helviticus and B. longum administered daily for 30 days was shown to reduce anxiety and depression in healthy human volunteers and in healthy rats (Messoudi et al. (2011) Beneficial psychological effects of a probiotic formulation (Lactobacillus helveticus R0052 and Bifidobacterium longum R0175) in healthy human volunteers. Br J Nutri, 105:755-764).

[0322] This study will test whether L. reuteri is superior in reducing the sickness, anxiety-like and depression-like behaviors induced by infectious colitis, using the same experimental design as the study described above evaluating the effect on infectious colitis itself, with minor modifications. The main difference is that we will also evaluate the animals' behavior, as well as circulating cytokines, circulating hormones and neuronal activation in the brain.

[0323] To determine whether prophylactic L. reuteri can prevent illness, such as anxiety-like and depression-like behavior, induced by C. rodentium, prophylactic treatment with L. reuteri biofilm is evaluated to determine whether C. rodentium prevents illness, anxiety-like and depression-like behavior induced by the bacteria. Preparations of L. reuteri biofilm that prove to be superior in in vitro assays are administered to mice by oral gavage 12 hours prior to oral challenge with C. rodentium. Triplicate experiments are performed with evaluation at 1, 6, 12 and 24 days post-challenge (peak C. rodentium infection occurs at approximately day 12) for a final sample size of 9 mice per condition and time point. At each time point, the animals' behavior is evaluated for locomotor activity (e.g., in the open field test), anxiety-like behavior (e.g., in the light-dark preference test and the elevated plus maze), depression-like behavior (e.g., in the tail suspension test and the Porsolt forced swim task), and sickness behavior (e.g., using the sucrose preference test). Serum cytokines related to emotion and sickness behavior (e.g., IL-1α / β and IL-6) are evaluated on each day. Circulating corticosterone levels are also evaluated. Neuronal activation in the brain, particularly in the hypothalamic paraventricular nucleus, is evaluated using c-Fos immunoreactivity.

[0324] It is also evaluated whether L. reuteri can be used as a therapeutic agent to treat sickness, anxiety-like and depression-like behavior induced by C. rodentium. For example, a composition is tested to determine whether treatment of established C. rodentium infection reduces sickness, anxiety-like and depression-like behavior. Preparations of L. reuteri that prove to be superior in in vitro assays are administered to mice by oral gavage 12, 24 and / or 36 hours after oral challenge with C. rodentium. At 1, 6, 12 and 24 days after C. rodentium challenge, the behavior of animals is evaluated for locomotor activity (e.g., in the open field test), anxiety-like behavior (e.g., in the light-dark preference test and the elevated plus maze), depression-like behavior (e.g., in the tail suspension test and the Porsolt forced swim task), and sickness behavior (e.g., using the sucrose preference test). Circulating cytokines (e.g., IL-1α / β and IL-6) related to emotion and sickness behavior are evaluated each day. Circulating corticosterone levels are also assessed.Neuronal activation in the brain, specifically in the paraventricular nucleus of the hypothalamus, is assessed using c-Fos immunoreactivity.

[0325] These examples allow conditions to be modified to create more robust and long-lasting probiotics, and once established, allow these conditions to be tested in in vivo models that may ultimately represent treatments for bacterial infections and human diseases. Example 9 NEC

[0326] Probiotic administration can be beneficial in preventing NEC. However, probiotics must be administered daily to achieve beneficial effects. Applicants herein describe a novel probiotic delivery system that grows probiotics as a biofilm on the surface of prebiotic-loaded biocompatible microspheres, thereby enabling enhanced and more sustained efficacy after just one treatment.

[0327] After delivery by Caesarean section, neonatal rats were subjected to experimental NEC [hypoxia / hypothermia / hypertonic diet (stress)]. On day 1, pups were randomized to receive a single enteral administration of: (1) vehicle only (100 μL sterile water) (N=32); (2) 1 × 10 9 CFU / mL of Lactobacillus reuteri (N = 9); (3) prebiotic-loaded biocompatible microspheres (N = 12); or (4) 1 × 10 9 CFU / mL of L. reuteri (N=33). Control pups were not stressed (N=10). Pups were sacrificed when clinical signs of NEC occurred or up to 96 hours after birth. The incidence and severity of NEC was measured using a validated histological NEC injury grading system, with grade 2 or greater injury considered equivalent to NEC.

[0328] As depicted graphically in Figure 5A, 69% of stressed, untreated rat pups developed NEC. Compared to stressed, untreated rat pups, 67% of L. reuteri-treated rat pups (p=0.329), 50% of prebiotic-loaded microsphere-treated rat pups (p=0.364), and 33% of L. reuteri-treated rat pups combined with prebiotic-loaded microspheres (p=0.003) developed NEC. No unstressed rat pups developed NEC. As depicted in Figure 5B, administration of both L. reuteri and prebiotic-loaded microspheres was sufficient to induce sufficient numbers of L. reuteri (>10 8 ) and prebiotic microspheres (≥5 mg) were required.

[0329] A single dose of Lactobacillus biofilm combined with prebiotic-loaded biocompatible microspheres is an effective treatment to reduce the incidence of NEC. Without being bound by theory, the compositions disclosed herein are prophylactic in their use in subjects in need of such treatment. Example 10 Desiccation tolerance assay

[0330] Another advantage of Applicant's invention is the improved long-term survival of probiotic bacteria. A desiccation tolerance assay was used to test the stability and viability of bacteria combined with microspheres. The assay can generally be performed by performing the following steps: To grow the bacterial culture, 1 ml to a1.5 ml of culture is transferred to a microcentrifuge tube (one tube per condition per time period to be tested). Approximately 10 μl of hydrated microspheres, trehalose, or nothing is added to the tube. The tube is incubated for 30 minutes and then the cells are pelleted by centrifugation. The supernatant is removed and the pellet is washed twice with sterile saline. All liquid is then removed from the pellet. The uncapped tube is placed on top of a dry light in a closed container and the container is placed in a 40° C. incubator. After 7 days, the tube is removed, rehydrated, and the pellet is suspended in 1 ml of growth medium for 5 minutes. Serial dilutions and plating for surviving colony forming units are then performed. Finally, rehydration and plating is repeated at 30 and 90 days.

[0331] After 90 days of incubation at 40°C and on the powerful desiccant DriLite, P. fluorescens and the proprietary Azospirillum sp. were placed and then rehydrated and tested for viability. P. fluorescens without microspheres shows a complete loss of colony forming units (CFU) after only one week in these conditions, whereas incubation with cellulose microspheres shows a loss of 10 colony forming units (CFU) after 90 days in these conditions. 5Azospirillum sp. shows a significant loss of CFU after 30 days and a complete loss after 90 days when grown without microsphere formulation, but when stored with microspheres under harsh conditions, even after 90 days, there are only 10 6 CFU / ml of Azospirillum sp. are viable. Example 11 Acid resistance protocol (48-well plate)

[0332] To form a biofilm that enhances survival at low pH, microspheres loaded with L. reuteri growth medium are utilized to provide a surface that leaches buffer nutrients to the bacteria. Bacterial cells with microspheres show a greater than 2 log increase in viable colony forming units compared to cells without microspheres after 4 hours in gastric acid at pH 2. Furthermore, L. reuteri with microspheres shows increased attachment to mouse colonic cells, addressing the problem of poor colonization and sustainability of orally administered bacteria. In summary, the novel microsphere formulation not only increases survival at low pH but also contributes to the colonization of beneficial bacteria in the gut, thus making L. reuteri a more efficient probiotic.

[0333] Acid tolerance protocol assays such as those used to generate the above information can generally be performed by performing the following steps: First, grow 5 ml cultures overnight at 37° C. (5% CO2 or anaerobically), then dilute the cultures 1:2500 in fresh medium. Transfer 500 ml per condition into a 48-well plate for each period to be tested. Transfer approximately 10 ul of hydrated microspheres to the wells or transfer nothing to the wells. Then incubate overnight at 37° C., 5% CO2 (or anaerobically) for 20 hours. At the 20 hour time point, remove the spent medium from the biofilm and replace with stomach acid at pH 2. At the 2 and 4 hour time points, remove the acid from the biofilm and suspend the cells in growth medium by pipette mixing. Finally, serially dilute and plate the cells. Example 12 Cell attachment assay

[0334] To form a biofilm that enhances survival at low pH, microspheres loaded with L. reuteri growth medium are utilized to provide a surface that leaches buffer nutrients to the bacteria. Bacterial cells with microspheres show a greater than 2 log increase in viable colony-forming units compared to cells without microspheres after 4 hours in gastric acid at pH 2. Furthermore, L. reuteri with microspheres shows increased attachment to mouse colonic cells, addressing the problem of poor colonization and survival of orally administered bacteria. These results indicate that the novel microsphere formulation not only increases survival at low pH, but also contributes to the colonization of beneficial bacteria in the gut, thus making L. reuteri a more efficient probiotic.

[0335] Cell attachment assays such as those used to generate the above information can generally be performed by carrying out the following steps: First, a mammalian cell culture is grown and incubated for approximately 10 6 Dilute to cells / ml. Transfer 500ul of the diluted mammalian cell line into a 48-well plate. Then grow to confluence (times vary but at least 16 hours) and allow the bacterial culture to grow overnight. Then transfer 500ul of the bacterial culture into 1.5ml microcentrifuge tubes (one microcentrifuge tube per condition per time period). Pellet the bacterial cells by centrifugation and wash the pellet 2-3 times to remove all growth medium. Resuspend the pelleted bacteria in cell line culture medium. Add cell line culture medium hydrated microspheres, MRS hydrated microspheres, or nothing to the suspended bacteria.

[0336] Growth medium is removed from confluent mammalian cell culture wells. Bacterial condition with cell line growth medium is aspirated into mammalian cell culture wells. Incubate at 37°C, 5% CO2. After 1 hour, spent medium supernatant is removed from each well and cells are washed twice with sterile PBS to remove non-attached bacteria. 500ul of trypsin is added to each well to dislodge attached mammalian cells from the plastic and incubate at 37°C for 5-10 minutes. The liquid in each well is mixed thoroughly to resuspend the mammalian cells. Serial dilutions and plating are then performed to calculate the number of bacteria that remained attached to the mammalian cells. At 4 and 8 hours, spent medium supernatant is removed from each well and cells are washed twice with sterile PBS to remove non-attached bacteria. 500ul of trypsin is added to each well to dislodge attached mammalian cells from the plastic and incubate at 37°C for 5-10 minutes. The liquid in each well is mixed thoroughly to resuspend the mammalian cells. Serial dilutions and plating are then performed to calculate the number of bacteria that remained attached to the mammalian cells. (Example 13) Increased Probiotic Potential

[0337] In one embodiment, the probiotic formulation contains the L. reuteri extracellular glucosyltransferase (GTF) protein, which catalyzes the formation of glucose exopolysaccharide (glucan) from its only known substrate maltose in the strain of L. reuteri used in this study (containing GTFW encoded by gtfW, DSM 20016) (Leemhuis et al., 2013; Bai et al., 2015). By way of background, GTF proteins usually have a glucan-binding domain that recognizes the exopolysaccharide it produces (Monchois et al., 1999; Kralj et al., 2004). GTF proteins, their substrates and the resulting glucan products are highly strain specific in L. reuteri, with some characterized as producing dextran (predominantly α-1,6 linkages), mutan (predominantly α-1,3 linkages) or the aptly named reuteran (predominantly α-1,4 linkages) (Kralj et al., 2002; Kralj et al., 2004). Cell aggregation, biofilm formation and intestinal colonization are directly linked to the activity of GTFAs in L. reuteri strain TMW1.106, and inactivation of gtfA significantly reduces the ability of L. reuteri to aggregate in vivo, form biofilms and colonize the GI tract (Walter et al., 2008).

[0338] The applicant's novel approach involves the selection of dextranomer microspheres [macroporous microspheres commercially available for size-exclusion chromatography (Porath and Flodin, 1959)] as a biocompatible surface to exploit the inherent ability of the GTFW of L. reuteri to bind to this cross-linked dextran (Tieking et al., 2005; Schwab et al., 2007; Walter et al., 2008). The GTFW-dependent binding of L. reuteri to DM results in: 1, selectivity of binding to DM and, as a consequence of the good binding of L. reuteri, to colonic epithelial cells; 2, protection against low pH; and 3, the ability of L. reuteri to acquire the luminal contents of DM at a concentration high enough to enhance the probiotic effect of L. reuteri. Example 13.1: Strains and culture conditions

[0339] The strains, plasmids and oligonucleotides used are listed in Table 4. Lactobacillus reuteri (ATCC 23272) and Lactobacillus rhamnosus GG (ATCC 53103) were grown in MRS (de man, Rogosa, Sharpe) medium (De Man et al., 1960) (BD, Franklin Lakes, NJ) for 16 h at 37°C and 5% CO2. Salmonella typhi (strain JSG698) and Citrobacter rodentium (ATCC 51459) were grown in lysogeny broth (LB, #63) at 37°C and 5% CO2. Clostridium difficile (strain R20291) was grown in degassed brain heart infusion (BHI) medium (BD, Franklin Lakes, NJ) at 37°C in an anaerobic chamber (Thermo Forma Scientific, 1025 Anaerobic System, Hampton, NJ) in which an atmosphere of 5% H2, 85% N2, and 10% CO2 was established. DLD-1 (ATCC CCL-221) human colon cells were grown at 37°C and 5% CO2 in RPMI medium supplemented with 10% fetal bovine serum. FHs 74 Int (ATCC CCL-241) human fetal small intestinal cells were grown at 37°C and 5% CO2 in Hybri-Care medium (ATCC 46-X) ​​supplemented with 30 ng / ml epidermal growth factor (EGF) and 10% fetal bovine serum. A gtfW deletion strain (LMW500) was constructed by inserting a chloramphenicol resistance cassette (Venereau et al.) into the gtfW open reading frame by allelic exchange as previously described (Mashburn-Warren et al., 2012). Briefly, 1 kb fragments upstream and downstream of gtfW were amplified by PCR using oligos oSG1082-1083 and oSG1084-1085 and then cloned into pFED760 (Mashburn-Warren et al., 2012) using the NotI / SalI and SalI / XhoI restriction sites, respectively.Oligos LMW34-35 were used to amplify the cat cassette from pEVP3 (Mashburn-Warren et al., 2012) and then cloned into pFED760 containing the upstream and downstream fragments of gtfW using the SalI restriction site. The resulting gtfW knockout construct plasmid (pWAR500) was then introduced into L. reuteri ATCC 23272 by electroporation. 5 ml cultures were grown at 37°C with 5% CO2 in MRS to an OD of approximately 1.0. 600nm L. reuteri electrocompetent cells were prepared by growing L. reuteri at 37 °C for 2 h. The cells were then pelleted and resuspended twice in 10 ml of sterile cold 0.5 M sucrose and 10% glycerol, followed by a final resuspension in 100 μl of sterile cold 0.5 M sucrose and 10% glycerol. To this resuspension, 1 μg of pWAR500 was added and the cell / DNA mixture was placed in an ice-cold 2 mm electroporation cuvette (BioRad, Hercules, CA). The cells were electroporated at 2500 V, 25 μF and 400 Ω using a BioRad Gene Pulser Xcell (BioRad, Hercules, CA). Immediately after electroporation, the cells were resuspended in 1 mL of MRS and incubated at 30 °C for 2 h, then serially diluted and plated on MRS agar containing 5 μg / ml chloramphenicol and incubated at 30 °C. Mutants were selected and confirmed as previously described ( Chang et al., 2011 ). [Table 4-1] [Table 4-2] [Table 4-3] CmR: chloramphenicol resistance; ErmR: erythromycin resistance; AmpR: ampicillin resistance *Bold sequences indicate restriction enzyme sequences.

[0340] gtfW promoter (PgtfW To estimate transcription from P, we amplified 350 bp of the promoter region upstream of the gtfW start codon (including the natural ribosome binding site) by PCR using oligos oSG1102-1103. gtfW A -CBluc reporter plasmid was constructed. The resulting DNA fragment was inserted into pJC156 using the XhoI / SalI restriction sites. The click beetle luciferase (CBluc) gene was amplified from Streptococcus mutans ldhCBGSm strain (Merritt et al., 2016) using oligos oSG1067-1068 and inserted downstream of the gtfW promoter region of pJC156 using the SalI / NotI restriction sites. The resulting reporter plasmid pWAR501 was transformed into L. reuteri 23272 as described above to generate the reporter strain LMW501.

[0341] An E. coli gtfW overexpression strain (LMW 502) was generated by amplifying the L. reuteri gtfW open reading frame (including the stop codon) using primers oSG1120-1126. The resulting DNA fragment was inserted into pTXB1 (New England BioLabs, Ipswich, MA) using NheI / SapI restriction sites. The resulting plasmid, pWAR502, was then transformed into the E. coli expression strain ER2566 (New England BioLabs, Ipswich, MA), selected on Luria-Bertani agar containing 100 μg / ml ampicillin, and confirmed by DNA sequencing. This strain allows overexpression of untagged GTFW protein.

[0342] To generate L. reuteri strains constitutively expressing click beetle luciferase, a reporter plasmid was constructed by amplifying 250 bp of the promoter region (including the natural ribosome binding site) upstream of the elongation factor Tu (EF-Tu) start codon by PCR using oligos oSG1069-1070. The resulting DNA fragment was inserted into pJC156 using XhoI / SalI restriction sites. The click beetle luciferase (CBluc) gene was amplified from S. mutans ldhCBGSm strain (Merritt et al., 2016) using oligos oSG1067-1068 and inserted downstream of the EF-Tu promoter region in pJC156 using SalI / NotI restriction sites. The resulting reporter plasmid, pWAR503, was transformed into L. reuteri 23272 as described above to generate LMW503. Example 13.2: Preparation and application of microspheres

[0343] Anhydrous dextranomer microspheres (DM; Sephadex® G-25 Superfine) were purchased from GE Healthcare Life Sciences (Pittsburgh, PA). Anhydrous cellulose microspheres (CM; Cellulobeads D50) were obtained from Kobo Products, Inc. (South Plainfield, NJ). Anhydrous microspheres were hydrated at 50 mg / ml in growth medium or water and then autoclaved for 20 minutes. For conditions using microspheres containing maltose, sucrose, fructose or only glucose, the autoclaved microspheres in water were removed from solution with a vacuum filtration apparatus and approximately 50 mg was collected by a sterile loop into 1 ml of a sterile filtered 1 M solution of sugar (see FIG. 13). The microsphere mixture was then vortexed vigorously and incubated at room temperature for 24 hours to reach equilibrium.

[0344] For L. reuteri application, microspheres loaded with water, 1M maltose, 1M sucrose, 1M glucose or 1M fructose were removed from solution with a vacuum filtration apparatus and collected with a 10 μl sterile loop. Approximately 5 mg of hydrated microspheres were then removed from 1 ml of 2×10 microspheres from overnight cultures that had been previously pelleted by centrifugation at 3220×g for 10 min. 9 CFU of L. reuteri were added, washed twice with 0.9% sterile saline, and resuspended in 1 ml of sterile saline. For experiments involving eukaryotic cell lines, 2 × 10 9 CFU of bacteria were resuspended in 1 ml of RPMI rather than saline. For experiments without microspheres but with an equal volume of cargo, 10 μl of cargo was added to 1 ml of bacteria in either sterile saline or RPMI. For all experiments, mixtures of bacteria and microspheres were incubated together at room temperature for 30 min (unless otherwise stated) to promote bacterial attachment to and biofilm formation on the microsphere surface and were then used in the assay. Example 13.3: Microsphere adhesion assay

[0345] L. reuteri cultures were grown and prepared as described above and incubated with microspheres loaded with either water, 1 M maltose, 1 M sucrose, 1 M fructose or 1 M glucose. To investigate bacterial adhesion to the microspheres, 300 μl of bacteria (approximately 2 × 10 9 CFU-containing 5 mg of microspheres (from an overnight culture) were combined and incubated in a Micro Bio-Spin column (BioRad, Hercules, CA) for 5 min (see FIG. 14). The column was then centrifuged (100×g) for 1 min. The flow-through fraction was serially diluted and plated to calculate the total number of non-adherent bacteria, and this value was subtracted from the total number of starting bacteria to obtain the total number of adherent bacteria. A control preparation consisting of bacteria without microspheres was used for all experiments. Example 13.4: Reporter Assay

[0346] The reporter strain LMW501 was grown at 37°C with 5% CO2 in MRS or in MRS containing 3% glucose, sucrose, fructose or maltose, and the optical density (OD) of the cultures was monitored throughout growth using an Epoch Microplate Spectrophotometer (BioTek Instruments Inc., Winooski, VT). 600nm At the indicated time points, 80 μl aliquots of bacterial culture were mixed with 20 μl of 2 mM D-luciferin in 0.1 M citrate buffer, pH 6.0, and placed in Falcon white flat-bottom 96-well plates (Becton, Dickinson Labware, Franklin Lakes, NJ), after which luminescence was detected using a Veritas Microplate Luminometer (Turner BioSystems Inc., Sunnyvale, CA). Example 13.5: GTF Enzyme Assay

[0347] S. mutans was cultured in Todd Hewitt Broth at 37°C with 5% CO2 in early log phase (OD 600nm L. reuteri WT and ΔgtfW mutants were grown at 37°C with 5% CO in MRS until late log phase (OD ~ 0.3) for optimal gtf expression. 600nm The E. coli gtfW overexpressing strain was grown in Luria-Bertani broth at 37 °C with shaking (200 rpm) until mid-log phase (OD approx. 1.0). 600nm 0.4), after which 1 mM IPTG was added to induce gtfW expression and then grown for an additional 2 h with shaking at 37°C. Whole cells of S. mutans, L. reuteri WT, L. reuteri ΔgtfW and E. coli gtfW overexpressing strains were assayed for GTF activity as previously described ( Bai et al., 2015 ) using periodic acid-Schiff staining of SDS-PAGE gels. Example 13.6: Cargo Diffusion Assay

[0348] Cargo diffusion rates from the microspheres were determined by tracking the low molecular weight dye, crystal violet (407.979 g / mol) (Fisher Scientific, Hampton, NJ). Microspheres were loaded with a 0.1% solution of crystal violet by incubating 20 mg of microspheres overnight in 1 ml of 0.1% crystal violet solution with or without the addition of glycerol (40% or 80% v / v) to increase viscosity and thereby slow the diffusion rate. After 16 hours, excess crystal violet solution was removed from the microspheres using a vacuum filtration apparatus as described above. The crystal violet-loaded microspheres were then placed in 1 ml of water, an aliquot of the water was removed, and the diffusion of crystal violet into the solution was monitored by OD using an Epoch Microplate Spectrophotometer (BioTek, Winooski, VT). 590nm The cells were analyzed at hourly intervals for 16 h at RT. Percent diffusion was calculated using an equivalent amount of crystal violet in microspheres (10 μl) in water as a control corresponding to 100% cargo diffusion. Example 13.7: Reuterin Assay

[0349] Production of reuterin by L. reuteri was measured by a quantitative colorimetric assay (Cadieux et al., 2008). This assay does not distinguish between similar aldehyde products, so measurements included 3-HPA as well as any possible derivatives, such as acrolein and 3-HPA hydrate. L. reuteri was grown overnight in MRS as above and 2 × 10 91 ml aliquots of CFU were pelleted at 3220×g for 10 min, washed twice with sterile saline and resuspended in either 1 ml of sterile saline or 1 ml of sterile saline containing 2% v / v glycerol. DM containing 0%, 2%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% glycerol were prepared as above for the other cargos and added to the resuspended L. reuteri in saline (hence the only source of glycerol available for reuterin production was from the microsphere cargo) for 1 h at 37° C. Cells were then pelleted again and the reuterin-containing supernatant removed, filtered through a 0.45 μm filter and assayed for reuterin as described in Cadieux et al., 2008 without modification. A standard curve using known concentrations of reuterin was used to extrapolate bacterially produced reuterin concentrations from the DM-glycerol and 2% v / v glycerol control experimental conditions. Example 13.8: L. reuteri survival in DM-80% glycerol

[0350] Overnight cultures of WT L. reuteri were aliquoted into microcentrifuge tubes, centrifuged, washed twice with sterile saline, and resuspended in either 1 ml saline or 1 ml MRS medium. 5 mg of either DM-water or DM-80% glycerol was then added to the tubes and incubated at 37° C. Every hour, the tubes were mixed thoroughly and aliquots were taken for subsequent serial dilutions and plating of viable CFU of bacteria. Example 13.9: Histamine Assay

[0351] Histamine production from L-histidine by L. reuteri was measured by ELISA (Enzo Life Sciences, Inc., Farmingdale, NY). L. reuteri was grown overnight in MRS as described above, and 2 × 10 9One ml aliquots of CFU were pelleted at 3220×g for 10 min, washed twice with sterile saline, and resuspended in one of the following conditions: sterile saline, saline containing 3% maltose, saline containing 2% v / v glycerol, 4 mg / ml L-histidine (Sigma-Aldrich, St. Louis, MO), 4 mg / ml L-histidine containing 3% maltose, or 4 mg / mL L-histidine containing 2% v / v glycerol. Five mg of DM containing either 4 mg / ml or 30 mg / ml L-histidine was added to media lacking L-histidine, and thus the only source of L-histidine for L. reuteri was L-histidine as cargo diffusing from the DM. Each condition was then incubated at 37° C. for 2 hours, after which time the contents were pelleted and the supernatant removed for quantification of histamine by histamine ELISA kit (Enzo Life Sciences, Inc., Farmingdale, NY) following the manufacturer's instructions without modification. All conditions were performed in at least triplicate. Example 13.10: pH Viability Assay

[0352] Bacteria were exposed to a synthetic gastric acid equivalent to determine survival at pH 2. The gastric acid equivalent is a modified version of synthetic gastric fluid (Cotter et al., 2001) consisting of 0.1 M HCl, 0.1 M NaCl and 0.01 M KCl, with the pH adjusted to 2 using 0.1 M NaOH. For the assay, 1 ml of 2 × 10 9CFU of L. reuteri were pelleted at 3220×g for 10 min, washed twice with sterile saline, and resuspended in 1 ml of 0.9% sterile saline. Cells were incubated with approximately 5 mg of loaded or unloaded microspheres as described above for 30 min, and the bacteria-microsphere mixture was added directly to gastric acid equivalents at a 1:100 dilution. Aliquots of the inoculum acid solution were mixed, serially diluted, and plated at hourly time points over a 4-h period to determine the number of viable bacteria. Bacteria without microspheres were used as a control. Example 13.11: Adhesion to Intestinal Epithelial Cells

[0353] DLD-1 colonocytes and FHs 74 small intestinal cells were cultured as described above. When the adherent epithelial cells reached confluence, the growth medium was removed, the cells were washed twice with sterile phosphate-buffered saline (PBS), and trypsin-EDTA (0.25%) was added for 10 min at 37°C to dislodge the cells from the culture flask surface. Total epithelial cells were counted using a hemocytometer (Hausser Scientific, Horsham, PA). The cells were then diluted to 5 × 10 5 The cells were diluted to a concentration of 1000 x 10 cells / ml and 1 ml was seeded per well into 24-well plates and incubated at 37°C, 5% CO. After growth for either 48 hours (for DLD-1 cells) or 120 hours (for FHs 74 cells), spent medium was removed and 2 x 10 cells were added. 9The CFU of L. reuteri alone, L. reuteri and 5 mg water-filled DM, L. reuteri and 5 mg sucrose-filled DM, or L. reuteri and 5 mg maltose-filled DM were replaced with 1 ml of RPMI or Hybri-Care medium. After 1 h of incubation, the spent medium was removed and the wells were washed three times with 1 ml of sterile PBS to remove non-adherent bacteria. The remaining epithelial cells, which were adherent bacteria, were then trypsinized as above, serially diluted, and plated on solid MRS medium for total adherent bacterial enumeration. For confocal microscopy experiments on DLD-1, Nunc Lab-Tek 8-well borosilicate chamber slides (Fisher Scientific, Hampton, NJ) were used instead of 24-well plates. Prior to DLD-1 seeding, chamber slides were treated with collagen to improve cell attachment using the following protocol: a mixture of 100 μl 7.5% BSA (Sigma-Aldrich, St. Louis, MO), 50 μl 3.79 mg / ml collagen (Millipore, Temecula, CA), 100 μl 1 mg / ml rat fibronectin (Biomedical Technologies, Stoughton, MA), and 9.75 ml PBS was prepared and 200 μl of this solution was added per chamber slide well. After incubation for 1 hour at 37°C, the solution was removed from the wells and epithelial cells were seeded and grown as described above. Example 13.12: Mucin adhesion assay

[0354] Mucin agar plates were made using porcine gastric mucin (Sigma-Aldrich, St. Louis, MO). The mucin agar plates contained 2% mucin and 0.8% agar to mimic the consistency of the mucus layer found in vivo (Macfarlane et al., 2005; Van den Abbeele et al., 2009). To assess the ability of L. reuteri to bind to mucin, 2 × 10 L. reuteri cells were cultured either in suspension or bound to 5 mg DM-water, DM-sucrose, or DM-maltose containing a plasmid encoding the expression of click beetle luciferase enzyme. 9 CFU of L. reuteri were incubated at room temperature on both stationary mucin agar and agar without mucin. After 60 min, unattached L. reuteri were removed by washing the plates twice with sterile saline. The luciferase substrate D-luciferin (Sigma-Aldrich, St. Louis, MO) was then added to the plates at a concentration of 0.4 mM to visualize the remaining attached bacteria. The relative light intensity resulting from the bacteria on the plates was measured using a FluorChem E system (ProteinSimple, San Jose, CA) with a 20 min exposure setting. To estimate the number of bacteria bound to mucin in the plates (and without any possible background binding to the agar in the plates), the amount of luminescence signal from the agar-only plates was subtracted from the mucin agar plates. Example 13.13: Confocal Microscopy

[0355] All confocal laser scanning microscopy (CLSM) was performed using a Zeiss LSM 510 confocal microscope (Ziess AG, Oberkochen, Germany). For fluorescent staining, dextranomer and cellulose microspheres were prestained with Congo Red (Fisher Scientific, Hampton, NJ) prior to incubation with cargo (e.g., sucrose) and experiments with bacteria. L. reuteri was stained with SYTO 9 (Life Technologies, Carlsbad, CA). Differential fluorescence visualization was performed using the following settings: Congo Red excitation 554 nm / emission 568 nm, and SYTO 9 excitation 490 nm / emission 525 nm. Samples were fixed using a custom biofilm fixative (Devaraj et al., 2015) containing 1.5% paraformaldehyde, 0.025% glutaraldehyde, 4.0% acetic acid, and 0.1 M phosphate buffer, pH 7.4. All microscopy was performed on samples in Nunc Lab-Tek 8-well borosilicate chamber slides (Fisher Scientific, Hampton, NJ). For CLSM experiments with DLD-1 epithelial cells, DLD-1 was stained with 4',6-diamidino-2-phenylindole (DAPI, Life Technologies, Carlsbad, CA) and L. reuteri was stained with carboxyfluorescein succinimidyl ester (CFSE, Life Technologies, Carlsbad, CA). Images were analyzed and figures were generated from CLSM images using AxioVision software (Ziess AG, Oberkochen, Germany) and ICY (de Chaumont et al., 2012). Bacterial biomass in CLSM images was quantified using COMSTAT (Heydorn et al., 2000) software.

[0356] For in vitro biofilm assays, overnight cultures of WT and ΔgtfW L. reuteri were added to freshly prepared MRS growth medium to an OD of 0.01. 600nmand incubated at 37 °C, 5% CO for 2.5 h at an OD of 0.65. 600nm The bacteria were incubated until reaching a viability of 100%, diluted 1:2500 in either MRS, MRS + 3% sucrose, or MRS + 3% maltose, plated into 8-well borosilicate chamber slides, and incubated for 1, 3, or 6 hours at 37°C and 5% CO2. At the indicated time intervals, bacteria were stained with LIVE / DEAD stain for viability, fixed, visualized by confocal microscopy, and quantified by COMSTAT analysis of the fluorescent signal. Example 13.14: Scanning Electron Microscopy

[0357] All scanning electron microscopy (SEM) was performed using a Hitachi S-4800 field emission SEM (Hitachi High-Technologies Corporation, Tokyo, Japan). Samples were prepared as described under "Adhesion to Colonic Cells" except that DLD-1 human colonic epithelial cells were grown on 15 mm diameter thermanox coverslips (Electron Microscopy Sciences, Hatfield, PA) placed in the wells of a 12-well plate. DLD-1 cell and attached bacterial samples were fixed overnight at 4°C in a solution of 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2). The specimens were washed with double-distilled water, stained with a 1% solution of osmium tetroxide (Sigma-Aldrich, St. Louis, MO) in 0.1 M phosphate buffer (pH 7.2) for 1 h, washed for 5 min, stained with a 1% solution of thiocarbohydrazide (Sigma-Aldrich, St. Louis, MO), washed for 5 min, and further stained with 1% osmium tetroxide for 30 min. The samples were then dehydrated using a graded series of ethanol: 25% ethanol for 15 min, 50% ethanol for 15 min, 70% ethanol for 30 min, 95% ethanol for 15 min (twice), 100% ethanol (twice), a 1:1 mixture of 100% ethanol and 100% hexamethyldisilazane (HMDS, Sigma-Aldrich, St. Louis, MO) for 100 min, 100% HMDS for 15 min, and finally immersed in 100% HMDS, which was left to air dry overnight. The coverslips of the dehydrated samples were then mounted onto a 15 mm diameter metal SEM stage (Electron Microscopy Sciences, Hatfield, PA) using colloidal silver (Electron Microscopy Sciences, Hatfield, PA). The outer edge where the stage and coverslip meet was then coated with a thin layer of colloidal silver and left to dry overnight. The samples were sputter coated with gold and palladium for 2 min at 25 mA using an Emitech K550X sputter coater (Quorum Technologies Ltd., Laughton, United Kingdom). Example 13.15: Statistical Analysis

[0358] All experiments were performed a minimum of three times, and statistical analysis was performed by Student's t test using GraphPad Prism software (GraphPad Software, Inc., La Jolla, Calif.), with P values ​​less than 0.05 accepted as significant. result Example 13.16: Maltose or sucrose in the lumen of DM enhanced L. reuteri adhesion to DM in a GTF-dependent manner

[0359] Dextranomer microspheres (DM) were stained with Congo Red, L. reuteri with SYTO 9, and binding was investigated by confocal laser scanning microscopy (CLSM) to determine whether the probiotic bacteria could attach to the biocompatible surface and induce biofilm formation. As shown in Figures 6A-6C, bacterial aggregates were associated with the surface of a large number of DMs, indicating that L. reuteri could attach to the DM surface within the allotted time. Since DM is a cross-linked glucan similar to the natural reuteran produced by L. reuteri, without being bound by theory, applicants hypothesized that an increase in GTFW (to enhance binding to DM) or the production of glucan to stimulate aggregation and biofilm formation would promote the adherent state of L. reuteri. To this end, we compared the attachment of L. reuteri to DMs containing either sucrose (an inducer of gtfW expression, but not a substrate for GTFW; see Figures 15A and 15B) or maltose (the only substrate for GTFW) as luminal cargo. As shown in Figures 6B and 6C, higher numbers of L. reuteri attached to DMs with either sugar as cargo compared to DMs containing only water in the lumen (Figure 6A).

[0360] To further explore L. reuteri's ability to bind DM, other DM luminal compounds were tested, based on the theory that these materials should not affect GTFW protein-mediated binding and therefore are unlikely to support increased attachment to DM. The monosaccharide subunits of maltose and sucrose (e.g., glucose for maltose, glucose and fructose for sucrose) were chosen for this assay because the GTF enzymes cannot utilize them to catalyze glucan polymers. Interestingly, fructose was shown to induce gtfW expression at a similar rate as sucrose (glucose did not), but did not result in the increased binding to DM seen with sucrose (Figure 15A, Figure 7A).

[0361] To determine whether this GTFW-dependent binding is specific to the glycosyl bonds of DM, we compared L. reuteri binding to CM (cellulose microspheres), which are composed of polymers of glucose with α-linkages, whereas CM has β-linkages between glucose units (Updegraff, 1969; Kralj et al., 2002). As shown in Figure 7A, only about 10% of L. reuteri attached to CM, regardless of luminal contents. Taken together, the data in Figures 7A-7C show that L. reuteri did not bind to CM, that binding to DM was GTFW-dependent, and further, that inclusion of maltose or sucrose significantly enhanced L. reuteri binding to DM. We hypothesized that the predicted glucan-binding domain of GTFW is an essential component of L. reuteri's ability to attach to DM. To further test whether attachment to DM is GTF-dependent, a mutant strain of L. reuteri (LMW500) was generated with a chloramphenicol resistance gene inserted in place of the gtfW gene. As shown in Figure 7B, the ΔgtfW strain was unable to bind DM as effectively as the wild type (WT) in spin column assays, regardless of the cargo in the DM lumen. To further demonstrate the difference between WT and ΔgtfW, we investigated biofilm formation in medium supplemented with sucrose or maltose on glass chamber slides (Figures 16A-16D). After 1 h of incubation, the WT had more bacteria present and significantly more bacterial aggregation when sucrose or maltose was added to the growth medium (Figures 16A and 16B). After 3 and 6 hours with medium supplemented with sucrose or maltose, the WT displayed significantly more robust biofilms with higher biomass than the gtfW mutant under all conditions, and significantly more cells were present when sucrose or maltose was present in the growth medium (Figures 16A, 16C, and 16D).

[0362] Next, we tested whether bacteria that do not express similar GTFs lack the adhesion phenotype shown in Figures 7A and 7B. To investigate this, we performed the DM adhesion assays disclosed herein with another probiotic bacterium and three enteric pathogens that L. reuteri may encounter in the gastrointestinal tract: Lactobacillus rhamnosus GG, a Gram-positive bacterium commonly found in the urogenital system and commercially sold as a probiotic; Salmonella typhi, a Gram-negative bacterium that causes typhoid fever in humans; Citrobacter rodentium, a Gram-negative bacterium that causes colitis in rodents; and Clostridium difficile, a Gram-positive spore-forming bacterium that can cause severe colitis and recurrent infections in humans. As shown in Figure 7C, all of the non-GTF-expressing bacteria showed little adhesion to DMs, regardless of the cargo present in the DM lumen. Example 13.17: Diffusion of cargo from DM

[0363] The initial binding of bacteria to the DM is essential for the microsphere composition. However, equally important is the ability to co-deliver beneficial luminal cargo required by the attached bacteria as the DM passes through the gastrointestinal tract. Targeted delivery of maltose (or any other beneficial compound) directly to the probiotic bacteria by diffusion from the DM over time is a desirable feature of this system. However, this method of delivery of cargo is by diffusion through the porous surface of the microsphere and not its degradation, as occurs, for example, in poly(lactic-co-glycolic) acid (PLGA) microspheres (Danhier et al., 2012), so the rate of diffusion depends on the size of the microsphere, the mass of the solute, and the viscosity of the diluent.

[0364] The DM was loaded with crystal violet, a low molecular weight stain (407.979 g / mol), and the rate of diffusion of the dye out of the DM was tested with and without varying the viscosity of the solute within the DM lumen. As shown in FIG. 8, crystal violet diffused out of the DM lumen with a half-life of approximately 6 hours. When the viscosity was increased by the addition of 40% glycerol, the release half-life was extended to approximately 8 hours. At 80% glycerol, the crystal violet release half-life was further enhanced to 12 hours. By 16 hours, >95% of all crystal violet had been released under all conditions tested. Example 13.18: L. reuteri produced reuterin from glycerol-loaded microspheres

[0365] An important feature of L. reuteri's function as a probiotic bacterium is its ability to compete with pathogenic bacteria within the host, potentially through the production of antimicrobial agents, such as extracellular reuterin (Cleusix et al., 2007; Spinler et al., 2008). Due to limited availability of glycerol, sufficient reuterin production may be limited by suboptimal endogenous concentrations of glycerol in the GI tract. To obviate the need to provide high glycerol levels to meet the optimal requirements of L. reuteri, applicants provide a targeted delivery of glycerol directly to the bacteria bound to the surface of DM. To test this in vitro, a colorimetric assay for reuterin production (Cadieux et al., 2008) was utilized. As shown in Figure 17, DM loaded with glycerol at concentrations ranging from 10 to 80% was able to induce reuterin production. Compared to the 2% glycerol solution control, DM loaded with 80% glycerol produced an average of 53% more reuterin at 1 hour (average concentration of reuterin produced: 2% glycerol = 40 mM, DM-80% glycerol = 61 mM). To determine whether 80% glycerol, or the reuterin / downstream metabolites resulting from glycerol fermentation produced by L. reuteri, were toxic to L. reuteri, we compared the colony forming units (CFU) of L. reuteri incubated with either DM-water or DM-80% glycerol in either sterile saline or MRS growth medium every hour. As shown in Figure 18, there was no loss of CFU when L. reuteri was incubated in MRS, regardless of the DM cargo. Incubation of L. reuteri in saline resulted in a steady loss of viable CFU over time, but there was no difference in viability over time between DM-water and DM-80% glycerol, suggesting that the loss of CFU was not due to any potentially toxic compounds from glycerol fermentation, such as reuterin or acrolein (Figure 18).Since acrolein is known to be particularly toxic to humans and is a by-product of reuterin production, we next calculated the maximum possible amount of acrolein that could be produced from the dosage of L. reuteri and the volume of glycerol produced by DM, assuming a 1:1 conversion of all available glycerol to acrolein. As shown in Figure 19, the amount of acrolein that could possibly be produced by this formulation is about 6 μg (for reference, the World Health Organization recommends less than 7.5 μg / kg body weight per day) (Gomes et al., 2002). From these results and the data presented in Figure 8, we propose that glycerol-loaded DM has two beneficial effects in vivo: delaying the release of beneficial cargo and providing a substrate for reuterin production. Example 13.19: L. reuteri produced histamine from L-histidine loaded microspheres

[0366] Histamine produced by L. reuteri has previously been shown to inhibit proinflammatory cytokines such as TNF via H2 receptors and to reduce colitis in animal models (Thomas et al., 2012; Gao et al., 2015). The microsphere formulation described herein provides a unique method of delivery of the histamine precursor substrate L-histidine to L. reuteri. To test this in vitro, DM was loaded with 30 mg / ml and 4 mg / ml L-histidine and the amount of histamine produced by the bacteria was measured when the only source of L-histidine was by diffusion from the DM. As shown in Figure 9, DM-L-histidine (4 mg / ml) produced histamine levels that were only slightly lower than those produced when bacteria were incubated in 4 mg / ml L-histidine without DM. When the DM was loaded with higher concentrations of L-histidine, the amount of histamine produced was 6-7 times higher than the lower concentration of 4 mg / ml, which is consistent with DM-L-histidine (30 mg / ml), which provides about 7 times more L-histidine than DM-L-histidine (4 mg / ml) (Figure 9). In addition, we also tested whether other cargo-related DM cargo substrates, such as maltose and glycerol, negatively affected histamine production. The addition of glycerol did not result in a reduction in histamine production, whether L-histidine was in solution or provided by DM (Figure 9). With respect to the addition of maltose, histamine production actually increased when L-histidine was provided in solution, but was statistically unchanged when L-histidine was provided by DM (Figure 9). Example 13.20: Microspheres loaded with sucrose or maltose improved survival of L. reuteri at low pH

[0367] Orally ingested probiotics face significant pH changes upon reaching the stomach, where pH values ​​are as low as 1.5 when the stomach is empty (Dressman et al., 1990). Enhancing the ability to deliver maximum numbers of viable L. reuteri to the colon is crucial for its sustainability and efficacy as a probiotic. L. reuteri bound to the surface of DM in the form of a biofilm should have extended survival upon exposure to acid, and the DM loaded with sucrose or maltose would result in even longer survival in a GTFW-dependent manner. As shown in Figure 10, less than 0.1% of WT L. reuteri without DM survived in synthetic gastric acid after 4 hours at pH 2, resulting in a loss of nearly 3 logs of viable probiotics. The addition of water-filled DM did not significantly alter the survival of WT L. reuteri in gastric acid, whereas nearly 1 log more survived gastric acid stress when either DM-sucrose or DM-maltose was delivered with WT (Figure 10). To demonstrate that this protective effect was dependent on the microspheres and not on the cargo in the DM lumen, L. reuteri was incubated with the same amount of diffusible cargo without DM. Survival in acid in the presence of cargo alone was not different from L. reuteri alone (Figure 10), strongly indicating that the bacterial biofilm delivery system on DM is important for the observed protective effect.

[0368] To investigate whether this phenotype was GTFW-dependent, we tested survival in synthetic gastric acid using the L. reuteri ΔgtfW strain and found that the beneficial effects of DM-sucrose and DM-maltose were lost (Figure 10). Interestingly, the mutant also showed a lack of survival in acid without DM compared to the WT, indicating that the role of GTFW in cell aggregation and biofilm formation (Figures 16A-16D) may significantly contribute to survival in synthetic gastric acid. Example 13.21: Microspheres promote L. reuteri adhesion to human intestinal epithelial cells

[0369] This example investigated how DM, DM luminal cargo, and the product of the gtfW gene affect the relative adhesion of L. reuteri when delivered as planktonic cells or as a biofilm on DM to human intestinal cell lines DLD-1 (adult human colonic epithelial cells) and FHs 74 Int (3-4 weeks pregnant, small intestinal epithelial cells) in vitro. As shown in Figure 11A, after 1 hour of incubation on DLD-1 cells, significantly more WT L. reuteri (without DM) attached to colonocytes compared to ΔgtfW with or without DM, indicating that GTFW contributes to host cell attachment. When L. reuteri attached to DM containing sucrose or maltose was added to colonocytes, the relative adhesion of WT L. reuteri to colonocytes increased 4.7-fold for DM containing sucrose and 5.2-fold for DM containing maltose (Figure 11A). Although overall fewer WT L. reuteri adhered to FHs 74 cells than to DLD-1 cells, delivery of bacteria with either DM-sucrose or DM-maltose resulted in 1.8-fold (DM-sucrose) or 2.7-fold (DM-maltose) more adherent cells compared to WT bacteria without DM ( Fig. 11B ).

[0370] To further demonstrate that maltose and sucrose DM luminal cargos improve the relative adhesion of L. reuteri to epithelial cells in vitro, we used CSLM to analyze the adhesion of WT and ΔgtfW L. reuteri after 1 hour incubation on DLD-1 cells (Figures 12A and 12B). Similar to the CFU data presented in Figures 11A and 11B, delivery of WT L. reuteri as a biofilm on maltose or sucrose-loaded DM supported adhesion to DLD-1 cells better than that delivered with water-loaded DM or without DM, both by visual inspection (Figure 12A) and when analyzed by quantification of bacterial biomass using COMSTAT analysis of CSLM images (Figure 12B). The observed adhesion was significantly reduced in the ΔgtfW mutant compared to the wild type, consistent with the measured CFU (Figure 11A).

[0371] Finally, we tested the effect of the ability of DM-adherent WT L. reuteri to bind to mucin. Although probiotic binding to cells appears to play a role in colonization, the healthy GI tract has a mucus layer on the apical surface of epithelial cells, the main component of which is mucin (Turner, 2009). Indeed, healthy commensals are found primarily within this layer, so it is preferable that the formulation maintain its enhanced probiotic effect in the presence of mucin. As mucin adhesion is not GTF-dependent, but rather controlled by specific mucin-binding proteins (Miyoshi et al., 2006; Lukic et al., 2012), we predicted that binding to DM would have no effect on L. reuteri's ability to adhere to mucin. As shown in Figure 20, after 60 min of incubation on mucin agar plates, there is no significant difference in the relative adhesion of WT L. reuteri to mucin when delivered as a planktonic bacterial suspension or as a biofilm attached to DM. Example 13.22: Observations

[0372] A single dose of L. reuteri delivered as a biofilm attached to DM has been shown to reduce the incidence of necrotizing enterocolitis (NEC) by 50% in a pup rat model (Olson et al., 2016). Example 13 shows that L. reuteri bound to DM with the appropriate luminal cargo promoted significantly increased survival at low pH and supported increased attachment to human epithelial cells in vitro. Importantly, L. reuteri and DM are considered "generally recognized as safe" by the FDA (Grasser et al.). Indeed, DMs are used in medicines that remain in the body for long periods of time (years) without adverse effects (Hoy, 2012), such as Debrisan®, a scar-forming wound dressing (Jacobsson et al., 1976), Deflux®, a bulking gel used to treat vesicoureteral reflux (VUR) in children (Stenberg and Lackgren, 1995), and Solesta™, a bulking gel injected submucosally into the anal canal to treat fecal incontinence (Hoy, 2012). The results described herein indicate a small subset of potentially beneficial cargoes that can be placed in the DM lumen for utilization by L. reuteri and for many applications where the luminal cargo precursors can be tailored to the desired L. reuteri-produced effect (e.g., reuterin and histamine). Furthermore, this formulation eliminates the need for recombinant versions of probiotics, an approach that is not currently approved by the FDA (Venugopalan et al., 2010).

[0373] An interesting feature of our novel formulation is that it can deliver beneficial compounds directly to probiotic bacteria that are attached to the DM surface as a biofilm (Figure 22B). Combining beneficial compounds (prebiotics) with beneficial bacteria to stimulate growth is a well-established concept in probiotic research and commercial applications (Collins and Gibson, 1999;de Vrese and Schrezenmeir, 2008). There is significant evidence that probiotic-prebiotic synergy increases the total population of probiotic bacteria (de Vrese and Schrezenmeir, 2008;van Zanten et al., 2014), as well as promoting the effective treatment of diseases such as inflammatory bowel disease (Geier et al., 2007) and necrotizing enterocolitis (Asmerom et al., 2015). However, a major drawback of traditional prebiotics is that they are usually limited to carbohydrates that the host cannot digest or absorb to ensure sufficient availability to the probiotic bacteria in the intestine. The compositions disclosed herein effectively solve this problem, since the probiotic bacteria L. reuteri are now delivered (1) in association with the DM to which they are attached in greater numbers, (2) in the form of a biofilm that confers resistance to clearance, (3) with a cargo of nutrients that promotes bacterial growth, (4) with a cargo that promotes the production of antimicrobial reuterin or histamine, (5) in a form that is resistant to acid-mediated killing, thus promoting improved survival when passing through the acidic stomach, and (6) in a manner that appears to better support attachment to intestinal epithelial cells, thus potentially promoting persistence in the intestine. With regard to L. reuteri-induced release of substances that are potentially beneficial to the host, reuterin has been suggested to inhibit competition by other intestinal flora, and histamine has been shown to have an anti-inflammatory effect.Secondary metabolites produced from glycerol metabolism to generate reuterin (e.g., acrolein) and histamine can result in high levels of adverse effects, but the maximum amounts produced in our formulations are <1% and <40% of what is considered problematic in humans for acrolein (Figure 19) and histamine, respectively (Maintz and Novak, 2007; Thomas et al., 2012; Engels et al., 2016).

[0374] The use of maltose as cargo is particularly valuable for several reasons; maltose is a substrate for the glucosyltransferase (GTFW) of this L. reuteri strain (Leemhuis et al., 2013; Bai et al., 2015), which induces L. reuteri to aggregate in a GTF-dependent manner (Walter et al., 2008), causing L. reuteri to grow significantly faster and to higher densities (CFU / ml). In this experiment, we show that both maltose and sucrose have a favorable effect on L. reuteri adhesion to microspheres, promote L. reuteri adhesion to human intestinal epithelial cells, and improve bacterial survival in gastric acid (Figures 6A-6C, 7A-7C, 11A and 11B, 12A and 12B, and 13). The S. mutans and L. reuteri GTF proteins are highly similar in sequence and structure. Sucrose is the only substrate for S. mutans and most L. reuteri GTF proteins (Tieking et al., 2005; Walter et al., 2008), and it has previously been shown that sucrose rapidly aggregates L. reuteri cultures in a GTF-dependent manner (Walter et al., 2008). The favorable effect of sucrose in inducing GTFW-dependent adhesion may be due to the ability of GTFW (through the glucan-binding domain) to act as an adhesin to DM, and to induce gtfW expression (Figure 15A). Indeed, the inability of sucrose to affect L. reuteri adhesion to CM (cross-linked glucan with variant glycosidic bonds) supports this view. Sucrose-dependent biofilm formation has previously been linked to a two-component regulatory system in the rodent strain of L. reuteri 100-23 ( Frese et al., 2011 ; Su and Ganzle, 2014 ), but the genes required for this phenomenon appear to be absent in the human-derived strain of L. reuteri (23272 / DSM 20016) used in this study.Since sucrose is the preferred carbon source for L. reuteri used in this study through its sucrose phosphorylase-mediated metabolism (Ganzle and Follador, 2012), it was not surprising that sucrose had a positive effect on biofilm formation and increased attachment to DM, which may be due to the increased doubling time of L. reuteri in the presence of sucrose. The inability of glucose (a carbon source, but not a gtfW inducer or GTFW substrate) and fructose (an inducer of gtfW, but not a carbon source (Figures S15A and S15B) or a substrate for GTFW) to enhance attachment to DM suggests that understanding bacterial physiology will be essential to select beneficial luminal cargoes.

[0375] Delivery of L. reuteri as a biofilm on the surface of DM containing beneficial cargo can improve many parameters important for L. reuteri survival and sustainability in the host. The resulting increased availability of probiotic bacteria in the host, along with more viable bacteria available after low pH challenge and support for increased adhesion to intestinal epithelial cells, should potentially result in increased beneficial effects. Furthermore, targeted nutrients and substances can be delivered directly to bacteria attached to the DM surface, which has far-reaching implications for the types of compounds that can be co-delivered with orally ingested L. reuteri that have previously been limited to carbohydrates that the host cannot digest. Example 14 Protection from necrotizing enterocolitis in rats

[0376] To determine NEC incidence and survival, rat pups were delivered early, given a single intestinal Lr treatment, and subjected to experimental NEC (high calorie diet / hypoxia / hypothermia). Pups were sacrificed 96 hours after delivery or when clinical NEC occurred. Tissues were collected for histological evaluation and measurement of inflammatory markers. Intestinal mucosal barrier integrity was assessed by serum levels of enterally administered FITC-dextran. Bioluminescent strains of Lr were constructed to assess persistence in the GI tract. GtfW deletion strains of Lr were generated to evaluate the role of biofilm formation. Lr attached to sucrose- or maltose-loaded DM significantly reduced experimental NEC compared to Lr attached to unloaded DM. Lr attached to sucrose- or maltose-loaded DM improved survival, reduced intestinal permeability, and reduced intestinal inflammation.

[0377] Neonatal rat model of experimental NEC. All animal studies were performed in compliance with protocol #AR15-00012 approved by the IACUC of The Research Institute at Nationwide Children's Hospital. Sprague-Dawley rat pups of gestational age 20.5 days were delivered by cesarean section under CO2 anesthesia from timed-pregnant dams (Envigo, Indianapolis, IN). Immediately after delivery, pups were randomly assigned to experimental groups receiving a single enteral Lr or a control treatment by gavage. The rat pups were then subjected to our well-established experimental NEC model ( Dressman et al., 1990 ; Freire et al., 2016 ), which is a modified version of the stress protocol to induce NEC first introduced by Barlow et al. in 1974 ( Freire et al., 2011 ; Freise et al., 2011 ). Briefly, rat pups were subjected to repeated episodes of: 1) a hypertonic, high-calorie formula diet by oral gavage five times daily of 15 g Similac 60 / 40 (Abbott Nutrition, Columbus, Ohio) in 75 mL Esbilac (Pet-Ag, New Hampshire, IL) providing a combined total of 836.8 kJ / kg / day; 2) three hypoxic and hypothermic episodes daily (placement in a N2 gas chamber calibrated to FiO2 < 1.5% for 90 seconds immediately followed by placement in a 4°C environment for 10 minutes); and 3) gavage of 2 mg / kg lipopolysaccharide (LPS, Sigma-Aldrich, St. Louis, MO) on the first day of life. Between each of these episodes, rat pups were housed in a 35°C incubator. Breast-fed control rat pups were placed with their surrogate dams immediately after delivery by Caesarean section and were not exposed to experimental stress.

[0378] Preparation and administration of Lr biofilms. Human fecal-derived Lactobacillus reuteri 23272 was purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in de Man, Rogosa and Sharpe (MRS) broth (Fisher Scientific, Pittsburg, PA). 35 For suspension Lr administration, Lr were pelleted, resuspended in sterile 0.9% saline, and administered to neonatal rats at 2 × 10 8 Lr was administered at a dose of 100 CFU / pup (a dose consistent with other published studies) (Gao et al., 2015). For Lr administered in its biofilm state, Lr was introduced into DM prior to administration as previously described (Eaton et al., 2011). Briefly, sterile dry DM (Sephadex G-25 Superfine, GE Healthcare Bio-Sciences, Pittsburgh, PA) was hydrated to 50 mg / mL with water and then autoclaved for 20 min. For treatment groups containing sucrose or maltose, DM was removed from the solution and collected in 1 mL of sterile 1 M sugar solution. The solution was vortexed and incubated at RT for 24 h. DM was then removed from the solution using a vacuum filter, aseptically scraped, and collected by a sterile loop into a tube containing resuspended bacteria. Lr was incubated with DM for 30 min at room temperature to encourage binding and biofilm formation. 2 × 10 8 The pups were gavaged with 100 μL of bacteria-DM solution resulting in a final dose of CFU / pup.

[0379] Incidence and severity of experimental NEC. Immediately after delivery, rat pups were randomly assigned to one of seven experimental groups receiving one of the following treatments by gavage: 1) 100 μL sterile water (vehicle control) (n=49); 2) DM-Sucr (n=44); 3) 2 × 10 8 CFU Lr (n = 46); 4) 2 x 10 8 CFU Lr+DM (n=43); 5) 2×10 8CFU Lr+DM-Sucr(n=50); or 6)2×10 8 CFU Lr+DM-Malt (n=47). An additional group of pups were returned to surrogate dams to serve as non-stressed breast-fed controls. After receiving the single treatment dose, the pups were subjected to the experimental NEC protocol described previously. Pups were sacrificed when signs of NEC occurred (bloody stools, severe abdominal distension, lethargy, respiratory distress, cyanosis). All remaining pups were sacrificed 96 hours after delivery. Upon sacrifice, intestinal tissues were collected and fixed in 10% formalin for 24 hours. Fixed tissues were embedded in paraffin and then hematoxylin and eosin (H&E) stained cross-sections were prepared. Two independent observers graded each section in a blinded fashion using the histological damage grading scale originally established by Caplan et al. (Dressman et al., 1990; Freire et al., 2016; Geier et al., 2007). Histological damage was classified as follows: grade 0, no visible histological villus injury; grade 1, detachment of distal intestinal villus cells; grade 2, sloughing of enterocytes to the mid-villus level; grade 3, loss of all villi and preservation of crypts; and grade 4, transmural necrosis (Figure 2A). Experimental NEC was defined as an injury score of grade 2 or higher.

[0380] Intestinal permeability. Immediately after delivery, rat pups were randomly assigned to one of the following: 1) 100 μL sterile water (vehicle control) (n=20); 2) 2 × 10 8 CFU Lr(n=20);3)2×10 8 CFU Lr+DM-Sucr(18); or 4) 2 × 10 8CFU Lr+DM-Malt (n=15). An additional group of pups was returned to surrogate dams to serve as non-stressed breast-fed controls. The pups were then subjected to the experimental NEC protocol for 48 hours, at which point each pup received 1500 mg / kg of fluorescein isothiocyanate (FITC)-labeled dextran (FD70, MW 70,000) (Sigma-Aldrich Inc., St. Louis, MO) suspended in sterile PBS by oral gavage. Four hours later, the pups were sacrificed and serum was collected in BD Microtainer SST tubes (Becton, Dickinson and Company, Franklin Lakes, NJ). Serum was extracted and fluorescence was measured on a fluorescence plate reader (SpectraMax M2, Molecular Devices, Sunnyvale, CA) using a 492 / 518 nm filter set. A standard curve generated from 1:2 serial dilutions of known FD70 concentrations was then used to extrapolate the plasma concentration of FD70 for each rat pup.

[0381] After delivery by Caesarean section, rat pups were randomized to receive one of two enteral treatments: 1) 2 × 10 8 CFU Lr-Luc (n = 6); or 2) 2 × 10 8 CFU Lr-Luc+DM-Sucr (n=6). The rat pups were then subjected to experimental NEC. After 48 hours, all rat pups were sacrificed and their GI tracts were collected. The contents of the small intestine and cecum / colon were isolated and homogenized by bead crushing with 0.7 mm beads for 2.5 minutes. 2 mM D-luciferin (luciferase substrate) was added to each tissue homogenate, and bioluminescence was measured using a Xenogen IVIS Spectrum imaging system (PerkinElmer,Inc, Waltham, MA).

[0382] Statistical analysis. All data are expressed as mean ± SEM. One-way ANOVA followed by pairwise comparisons was performed using GraphPad Prism 7 (La Jolla, CA) and SAS 9.4 software (SAS Institute, Inc., Cary, NC). For animal survival, a log-rank test was performed. For RNA expression, outliers were identified using the IQR method. Statistical significance was defined as p ≤ 0.05. result

[0383] Effect of Lr biofilm formation on NEC incidence and severity. Nearly two-thirds of untreated rat pups subjected to the experimental protocol developed NEC (Figure 22B). The incidence of NEC was statistically unchanged for rat pups treated with DM-Sucr alone (p=0.343) or planktonic Lr alone (p=0.334). In contrast, there was a significant reduction in NEC incidence (p<0.001) in rat pups treated with Lr+DM compared to untreated rat pups. Finally, a single dose of Lr+DM-Sucr or Lr-DM-Malt resulted in a further reduction in NEC...

Claims

1. 1. A composition for use in a method of treating neurodevelopmental deficiency in a subject suffering from or suffering from Necrotizing Enterocolitis (NEC), the composition comprising microspheres containing biofilm-producing L. reuteri bacteria and a prebiotic, the bacteria forming a biofilm that partially or completely coats the surface of the microspheres, the prebiotic comprising a nutritional supplement for the L. reuteri bacteria, the method comprising administering to the subject an effective amount of the composition.

2. The composition for use according to claim 1, wherein administration of the effective amount of the composition prevents, reduces or delays microglial activation in the subject.

3. The composition for use according to claim 1, wherein administration of the effective amount of the composition increases expression of brain-derived neurotrophic factor (BDNF) in the subject.

4. The composition for use according to claim 3, wherein the increase is an increase compared to a control subject, or the increased expression of BDNF is increased expression within the hippocampus of the subject.

5. The composition for use according to claim 1 , wherein administration of the effective amount of the composition increases expression of Grin2A in the subject.

6. The composition for use according to claim 5, wherein the increase is an increase compared to a control subject and / or the increased expression of Grin2A is increased expression within the prefrontal cortex of the subject.

7. 7. The composition for use according to any one of claims 1 to 6, wherein the microsphere further comprises a partial or complete biofilm coating on the outer surface of the microsphere.

8. 8. The composition for use according to any one of claims 1 to 7, wherein the prebiotic comprises maltose, glycerol or histidine, and optionally the L. reuteri produces glucosyltransferase (GTF).

9. 9. A composition for use according to any one of claims 1 to 8, wherein the microspheres comprise dextranomer and the prebiotic comprises maltose.

10. 10. A composition for use according to any one of claims 1 to 9, wherein the composition is prepared by mixing microspheres with biofilm-producing probiotic bacteria and prebiotics, optionally in a medium containing a biofilm.

11. 11. A composition for use according to any one of claims 1 to 10, wherein the composition comprises PGLA or dextranomer biocompatible microspheres and a nutritional supplement comprising one or more of maltose, sucrose, glycerol or histidine in an amount to support the growth of the probiotic bacteria, and optionally the microspheres are entirely coated with a biofilm.

12. 12. A composition for use according to any one of claims 1 to 11, wherein the release of the prebiotic is regulated by varying the microsphere size or by modifying the viscosity of the prebiotic.

13. 13. The composition for use according to any one of claims 1 to 12, wherein the subject has or has had Bell stage 2 or 3 NEC, or the NEC has been treated with surgery.

14. The composition of any one of claims 1 to 13, wherein the bacteria form a biofilm that completely coats the outer surface of the microspheres.

15. The composition of any one of claims 1 to 14, wherein the bacteria and the prebiotics diffuse from the interior of the microsphere to the surface of the microsphere.

16. 1. A composition for use in a method of preventing or delaying neurodevelopmental failure in a subject suffering from or having suffered from Bell's stage 2 or 3 necrotizing enterocolitis (NEC), or having said NEC treated surgically, said composition comprising microspheres containing biofilm-producing L. reuteri bacteria and a prebiotic, said bacteria forming a biofilm that partially or completely coats the surface of said microspheres, said prebiotic comprising a nutritional supplement for said L. reuteri bacteria, said method comprising administering to said subject an effective amount of said composition.

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