WO2018183355A1 - Formulations prébiotiques - Google Patents

Formulations prébiotiques Download PDF

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Publication number
WO2018183355A1
WO2018183355A1 PCT/US2018/024604 US2018024604W WO2018183355A1 WO 2018183355 A1 WO2018183355 A1 WO 2018183355A1 US 2018024604 W US2018024604 W US 2018024604W WO 2018183355 A1 WO2018183355 A1 WO 2018183355A1
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Prior art keywords
poly
reuteri
composition
microsphere
biofilm
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PCT/US2018/024604
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English (en)
Inventor
Steven D. Goodman
Lauren O. Bakaletz
Michael Bailey
Gail BESNER
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Research Institute At Nationwide Children's Hospital
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Priority claimed from US15/649,352 external-priority patent/US10624934B2/en
Application filed by Research Institute At Nationwide Children's Hospital filed Critical Research Institute At Nationwide Children's Hospital
Publication of WO2018183355A1 publication Critical patent/WO2018183355A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin

Definitions

  • This disclosure relates to novel probiotic formulations and methods for using same for treating or preventing disease.
  • Probiotics are live microbes that when ingested in high enough quantities confer a health benefit for the host (Food and Agriculture Organization of the United Nations and World Health Organization, "Health and Nutritional Properties of Probiotics in Food
  • probiotics can effectively prevent pathogen colonization due to either direct (e.g., production of antimicrobial defenses) or indirect (e.g., stimulation of host defenses) mechanisms. Few probiotic species are able to both prevent pathogen colonization and limit excessive inflammatory responses. This is important, however, because excessive colonic inflammation in response to colonic infection can lead to the development of protracted illness, such as post-infectious irritable bowel syndrome. Thus, the development of probiotics that are able to prevent excessive immune responses to colonic pathogens, while still maintaining anti-bacterial immunity would have the ability to prevent both short-term and longer-term health effects of enteric infection. This disclosure provides formulations that address this unmet need and provides related advantages as well.
  • aspects and embodiments of this technology combine the health benefits of probiotic bacteria with prebiotic substances to help stimulate the exclusive growth of the probiotic species and, in one aspect, provide the bacteria in the form of a biofilm on a biocompatible microsphere.
  • Applicants have discovered that the use of a biofilm on the microsphere provides enhanced efficacy and duration of the therapeutic response. It has been shown that probiotic biofilms can be grown on surfaces as a means to introduce bacteria into the site of wounds, where a formulation comprising a plaster or dressing based on a hydrocolloid that is a natural gelatin to treat wounds (i.e., EP2450062).
  • a formulation comprising a plaster or dressing based on a hydrocolloid that is a natural gelatin to treat wounds (i.e., EP2450062).
  • EP2450062 a formulation comprising a plaster or dressing based on a hydrocolloid that is a natural gelatin to treat wounds
  • This technology also provides methods of formulation, which enhance the efficiency and durability of introducing probiotic strains at a site of action. It specifically bypasses the rate limiting step of biofilm formation.
  • This technology is useful for gastrointestinal gut health and any aspect where probiotic bacteria need to establish, e.g., the gastrointestinal tract, wound healing, skin, vaginal, oral, agriculture, and water purification.
  • probiotics are a natural way to protect and restore gut microbiota to a healthy state.
  • probiotic bacteria (as typically delivered) fail to establish, or sufficiently persist, minimizing the magnitude and duration of their healthful effects.
  • One of the rate limiting steps is the capacity of introduced bacteria to form a lasting biofilm.
  • bacteria are already in the form of a biofilm (a surface adhered community) as opposed to planktonic (free-living), they more readily establish and persist.
  • the positive effects of probiotic bacteria can be enhanced by providing them in a biofilm state; this can readily be accomplished by growing the bacteria on the surface of a biocompatible and nontoxic microsphere and associated with a biofilm.
  • Biocompatible microspheres can be biodegradable polymers, non-biodegradable polymers, a metal, or a combination thereof.
  • prebiotic and/or prebiofilmic substances can be added as cargo to facilitate establishment and maintenance of the probiotic bacterial biofilm.
  • the biocompatible microsphere and generates a biofilm.
  • the biocompatible microsphere is semipermeable or porous, and has either a solid or hollow core.
  • the biocompatible microsphere can carry a prebiotic and any nutritional supplementation for the probiotic bacterium as a cargo whereby the bacterium gains access via diffusion from the lumen.
  • the microsphere can also carry a drug, or a compound, or an agent, which is selective against a pathogen, that in one aspect, may compete with the health-inducing bacterium in the composition.
  • the microsphere can carry chemical reductants and/or molecules and or surfaces that promote adsorption (in the core or on the surface of the microsphere) and/or molecules and/or surfaces that promote absorption (in the core or on the surface of the microsphere).
  • a novel probiotic formulation can also contain a prebiofilmic, which is a substance that supports biofilm formation and/or durability, and in one aspect, the prebiofilmic is a DNA binding polypeptide or protein and/or a DNABII polypeptide or protein or a fragment thereof that supports biofilm formation and/or durability. The prebiotic is released from the hollow core and to adhere to the bacterium.
  • release of the prebiotic from the hollow core can be regulated by varying microsphere size (smaller microspheres release faster), and/or by altering the viscosity of the prebiotic (i.e., the higher the viscosity the slower the release).
  • Microspheres have added value in ideally providing diffusible prebiotic (nutritional supplementation specific/exclusive to probiotic bacteria) cargo that can help promote probiotic bacterial establishment and survival while limiting pathogenic bacterial challenge.
  • the biofilm state is advantageous in establishing in the gut over the same bacteria in planktonic form.
  • L. reuteri introduced into mice as biofilms are shown to have a more robust and durable prophylactic effect on the pathogenesis of the enteropathogenic bacterium, Citrobacter rodentium, than L. reuteri in its planktonic form.
  • compositions comprising, or alternatively consisting essentially of, or yet further consisting of, a biocompatible microsphere, a biofilm-generating probiotic bacterium and a prebiotic, wherein the prebiotic comprises, or alternatively consisting essentially of, or yet consisting of, a nutritional supplementation for the probiotic bacterium.
  • the composition further comprises, or alternatively consists essentially of, or yet further consisting of, a carrier, such as a pharmaceutically acceptable carrier or a biocompatible scaffold.
  • compositions are formulated for in vivo or ex vivo use.
  • the compositions are formulated for administration orally, vaginally, nasally (inhalation), intravenously or intramuscularly (injectable), topically, as a suppository, as a spray (aerosol administration), dry application by admixing in the soil, as a solute (for admixing with an aqueous environment).
  • they are formulated in a dosage form.
  • Suitable dosage forms include, but are not limited to a suppository, a powder, a liquid, a capsule, a chewable tablet, a swallowable tablet, a buccal tablet, a troche, a lozenge, a soft chew, a solution, a suspension, a spray, a tincture, a decoction, an infusion, and combinations thereof.
  • This disclosure also provides a method for preparing the above-noted composition, the method comprising, or alternatively consisting essentially of, or yet further consisting of, admixing a biocompatible microsphere with a biofilm-generating probiotic bacterium, a prebiotic, and in one aspect, further admixing a prebiofilmic.
  • the method further comprises, or alternatively consists essentially of, or yet further consists of, admixing an effective amount of one or more of: a nutritional supplement for the probiotic bacterium, a drug active against a pathogen or invertebrate, or a chemical reductant and/or molecule that promote adsorption (in the core or on the surface of the microsphere) and/or molecules that promote absorption (in the core or on the surface of the microsphere).
  • compositions as disclosed herein also provides therapeutic, industrially and agricultural use of the compositions as disclosed herein.
  • kits comprising, or alternatively consisting essentially of, or yet consisting of, a composition as described herein and instructions for use diagnostically, industrially, in agriculture or therapeutically.
  • FIGS. 1A and IB illustrate that L. reuteri biofilm structural integrity relies on the presence of DNABII family proteins Confocal microscopy images of in vitro L. reuteri biofilms stained with LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes).
  • Z. reuteri biofilms were grown for 24 hours at 37°C and 5% C02, at which time they were treated with a 1 :50 dilution of either (FIG. 1A) rabbit naive serum, (FIG. IB) rabbit anti- integration host factor polypeptide ("HtF”) , or media with nothing added (data not shown) for 16 hours.
  • Anti-IHF treatments resulted in a 20% decrease in maximum height, 35% decrease in average thickness, and 41%) decrease in biomass (data not shown).
  • FIG. 2 illustrates that prebiotic compounds increase probiotic biofilms in average thickness and biomass.
  • Addition of 10 ⁇ g/ml S. mutans HU to L. reuteri biofilm at time of seeding increased average thickness and biomass 33%, and 55%>, respectively.
  • Addition of 10 ⁇ g/ml calf thymus DNA increased average thickness 44% and biomass 68%.
  • FIG. 3 illustrates that Z. reuteri in vivo colonization and retention with a single oral administration.
  • FIG. 4 illustrates that Z. reuteri biofilm grown with PLGA microspheres and HU reduces C. rodentium spleen colonization more effectively than biofilm and planktonic Z. reuteri.
  • FIGS. 5A and 5B show the results of studies establishing that compositions of this disclosure are consistent with a reduction in inflammation and antagonization of bacterial pathogens in an animal model of NEC.
  • FIGS. 6A-6C show that Z. reuteri binds to dextranomer microspheres. Confocal laser scanning microscopy (CLSM) of Z. reuteri adhered to DMs.
  • FIG. 6A Water-filled DMs
  • FIG. 6B sucrose-filled DMs
  • FIG. 6C maltose-filled DMs after incubation with Z. reuteri for 30 minutes showed that Z. reuteri adherence to DMs can be enhanced to incorporate biofilm-promoting cargo within the DM lumen (green: bacteria stained with SYTO 9, red: DMs stained with Congo Red).
  • FIGS. 7A-7C show that microsphere composition and lumen cargo affected L. reuteri adherence, L. reuteri adhered to DMs in GTFW-dependent manner, and bacteria lacking GTF did not bind to DMs.
  • a spin column assay was performed to assess relative bacterial adherence to microspheres. Bacteria were incubated for 5 minutes with 5 mg of microspheres, centrifuged at 100 x g to separate bound and unbound bacteria, then CFU of non-adhered bacteria was quantified in the flow-through of the spin column. (FIG.
  • FIG. 7A Microspheres composed of either cross-linked dextran (DM) or cross-linked cellulose (CM) were filled with water, growth medium, or various sugars at a concentration of 1M to determine which microsphere type supported greatest adherence of L. reuteri.
  • FIG. 7B Relative WT and AgtflF L. reuteri adherence to DM showed that L. reuteri adhered to DMs in a GTF-dependent manner.
  • FIG. 7C Non-GTF expressing bacteria were similarly tested for microsphere adherence with water-loaded and sucrose-loaded DMs. Error bars represent standard error of the mean. Statistical significance is indicated by the following: * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.0005.
  • FIG. 8 Diffusion of cargo out of microspheres over time.
  • Crystal violet (CV)-loaded DMs with and without glycerol (added to increase viscosity) were assayed to determine the relative rate of CV diffusion from the microspheres.
  • CV diffused at a higher rate (100% diffusion after 10 hours) compared to DMs that contained 40% or 80% glycerol.
  • Applicant observed 100% diffusion from DMs after 16 hours regardless of viscosity. Error bars represent standard error of the mean.
  • Statistical significance from DMs with 0% added glycerol is indicated by the following: * P ⁇ 0.05, ** P ⁇ 0.01, **** P ⁇ 0.0001.
  • FIG. 9 shows that histamine can be produced by L. reuteri from L-histidine delivered via DM.
  • Stationary phase WT L. reuteri was incubated for 2 hours in either saline with and without 3% maltose or 2% glycerol, or 4 mg/ml L-histidine with and without 3% maltose or 2% glycerol.
  • Histamine production was increased with addition of 3% maltose to 4 mg/ml L-histidine solution (white bar black border) compared to just 4 mg/ml L-histidine (black bar and grey bar black border).
  • FIG. 10 shows gastric acid survival.
  • Relative survival in acid was enhanced when WT L. reuteri was adhered as a biofilm on DMs that contained sucrose or maltose compared to equivalent volumes of the same cargo delivered without DMs, which indicated that the biofilm phenotype contributed to better survival during exposure to low pH.
  • AgtflV showed decreased resistance to acid compared to the WT, regardless of the presence or absence of either DMs or sugar alone. Error bars represent standard error of the mean. Statistical significance is indicated by the following: * P ⁇ 0.05, ** P ⁇ 0.01.
  • FIGS. 11A and 11B show that delivery of L. reuteri adhered to DMs as a biofilm supported increased adherence to intestinal epithelial cells.
  • FIG. 11A L. reuteri WT and AgtflY adhered as a biofilm on DMs that contained either water, sucrose (1M), or maltose (1M), or the equivalent volume of sugar alone (without DMs), were examined for relative adherence to human colonic DLD-1 cells after incubation for 60 minutes. Significantly more WT adhered to DLD-1 cells when delivered as a biofilm on the surface of DMs that contained sucrose or maltose, compared to water-filled DMs or the equivalent volume of sugar alone.
  • FIGS. 12A and 12B show that increased adherence to DLD-1 colonic epithelial cells is observed when L. reuteri was delivered as a biofilm attached to DMs.
  • FIG. 12A In vitro CLSM of DLD-1 epithelial cells (blue, DAPI), L. reuteri (green, CFSE), and DMs (red, Congo Red). WT L. reuteri (top four rows) compared to gtfW L. reuteri (middle four rows) and no . reuteri (bottom row).
  • FIG. 12B Comparison of bacterial biomass quantified via COMSTAT analysis of the green channel of CLSM images of WT and AgtfW L. reuteri.
  • gtfW showed no difference in relative number of bacteria adhered to DLD-1 cells, regardless of the presence of DMs. Error bars represent standard error of the mean. Statistical significance is indicated by the following: * P ⁇ 0.05, ** P ⁇ 0.01.
  • FIG. 13 is an illustration of DM cargo loading, filtration, and addition to bacterial culture.
  • A Dehydrated DMs and desired cargo (e.g., 1M maltose) were incubated together to allow diffusion of solution into DMs.
  • B The DM + solution is vortexed and pipetted to a vacuum filtration system.
  • C The vacuum removes excess solution, leaving just DMs with absorbed cargo.
  • D The DM-cargo pellet can now be removed from the vacuum filter by scraping with a sterile loop.
  • E The DM-cargo pellet is transferred to a bacterial solution, typically bacteria resuspended in saline.
  • F The final product is bacteria + DM-cargo together in solution, which can then be used for downstream applications (e.g., assays, oral gavage, etc.).
  • FIG. 14 is an illustration of spin column DM adherence assays.
  • a bacteria + DM-cargo mixture is incubated together on top of a spin column filter within a 1.5 or 2.0ml microcentrifuge tube. After the desired incubation time (e.g., 5 minutes), the tube + column is centrifuged at ⁇ 100 x g to separate adhered and non-adhered bacteria to DMs.
  • B After centrifugation, non-adhered cells will be in the flow through at the bottom of the
  • microcentrifuge tube and adhered bacteria to DMs will remain on the surface of the filter with the DMs (filter pore size is too small for DM passage, but small enough for bacterial cells).
  • the cells present in the flow through are enumerated by serial dilution plating.
  • a bacteria only (no DMs) control is used as a baseline, and all DM experiments are subtracted from the baseline.
  • FIGS. 15A and 15B show that sucrose induces gtfW, but maltose is the substrate for GTFW.
  • FIG. 15A A gtfW transcriptional reporter was constructed by fusing the click beetle luciferase downstream of the gtfW promoter on a plasmid, followed by introduction into L. reuteri (strain LMW 501).
  • coli harboring gtfW on an inducible plasmid (Ec) (strain LMW 502), were subjected to SDS-PAGE followed by PAS staining to examine GTFW enzymatic activity. 5% sucrose or 5% maltose were used as substrates. The arrows indicate GTFW activity.
  • FIGS. 16A-16D show that GTFW contributed to early biofilm formation in growth medium supplemented with sucrose or maltose.
  • L. reuteri WT and AgtfW were seeded into 8- well borosilicate chamber slides and incubated for 1, 3, or 6 hours at 37°C 5% CO2.
  • the bacteria were stained for viability with LIVE/DEAD stain, fixed, visualized via confocal microscopy (CLSM), and quantified via COMSTAT analysis of the fluorescent signal.
  • CLSM confocal microscopy
  • FIG. 17 shows that . reuteri can produce reuterin from glycerol-loaded
  • L. reuteri incubated for 1 hour with DMs that contained 0-80% glycerol as the only source of glycerol in the experimental conditions were measured for relative reuterin production.
  • the amount of reuterin produced by L. reuteri without DMs in a 2% glycerol solution was used as a control (dotted line). Error bars represent standard error of the mean.
  • FIG. 18 show that glycerol delivered via DMs and any subsequently produced metabolites did not affect L. reuteri survival.
  • Overnight cultures of WT L. reuteri were washed and resuspended in either saline or MRS medium. 5mg of DM-water or DM-80% glycerol were then added to L. reuteri and incubated at 37°C. At hourly intervals the aliquots were taken for subsequent serial dilution and plating for viable CFU. After 24 hours there was no significant difference between cultures incubated in the same medium (saline or MRS) with either DM-water or DM-80% glycerol.
  • FIG. 19 illustrates maximum conversion of DM-provided glycerol to acrolein did not result in toxic levels of acrolein.
  • the World Health Organization (WHO) recommends ingestion of no more than 7.5 g kg of body weight of acrolein per day. Assuming 100% conversion of available glycerol provided via DMs into acrolein by L. reuteri, the dosage of L. reuteri and DM-glycerol utilized in this work (red arrow) resulted in a maximum of 6.24 g acrolein produced.
  • the dashed line (50 g acrolein) represents ⁇ 10% of the daily allowable amount of acrolein for a 70 kg human.
  • FIG. 20 shows that L. reuteri delivered as a biofilm on DMs does not inhibit adherence to mucin.
  • L. reuteri reporter that expressed click beetle luciferase was dispensed either planktonically or as a biofilm on the DM surface onto agar plates that contained either 2% mucin + 0.8% agar or 0.8% agar, incubated at room temperature for 1 hour, then washed to remove non-adhered L. reuteri.
  • D-luciferin (0.4 mM) was then added to the plates, and the plates were imaged for luminescent signal that originated from remaining adhered bacteria.
  • the relative luminosity of the agar-only plates was subtracted from the relative luminosity of the mucin + agar plates.
  • FIGS. 21A and 21B show that L. reuteri adhered to DMs and L. reuteri attached to the surface of DLD-1 human colonic epithelial cells.
  • FIGS. 22A and 22B show incidence and severity of NEC.
  • FIG. 22A is H&E stained intestinal tissue sections demonstrating the following grades of histologic injury: Grade 0, no visible histological villus damage; Grade 1, distal villus enterocyte detachment; Grade 2, sloughing of enterocytes to the mid villus level; Grade 3, loss of the entire villus with preservation of the crypts; and Grade 4, transmural necrosis. Grade 2 injury and above is consistent with histologic NEC. All images are 20x magnification.
  • FIG. 22B shows that rat pups were delivered prematurely, subjected to the experimental NEC protocol, and sacrificed when signs of clinical NEC developed or after 96 h. Each dot represents a single rat pup with their histologic injury score depicted. NEC incidence for each experimental group of pups is indicated. *p ⁇ 0.05.
  • FIG. 23 shows rat pup survival. The number of pups alive and free from endpoint criteria (lethargy, bloody stools, agonal breathing, cyanosis) are depicted for each
  • FIG. 24 shows intestinal permeability of rat pups subjected to experimental NEC. Intestinal permeability was determined by measuring serum levels of FITC dextran 4 h after gastric administration of FITC dextran, with greater levels of serum FITC dextran indicating greater intestinal permeability.
  • FITC fluorescein isothiocyanate. *p ⁇ 0.05.
  • FIG. 25 shows Lr Persistence in the GI tract.
  • a bioluminescent strain of Lr was generated and used to track Lr presence in the small and large intestine (as the amount of light emitted) after 48 h of the experimental NEC protocol.
  • RLU relative light units. *p ⁇ 0.05.
  • FIGS. 26A-26E show inflammatory markers. Intestinal specimens were collected and fixed in formalin. RNA was isolated and analyzed with real-time qPCR for the expression of (FIG. 26A) IL-6, (FIG. 26B) IL- ⁇ , (FIG. 26C) CCL-2, (FIG. 26D) CXCL-1, and (FIG. 26E) IL-10. Results represent the mean ⁇ SEM of 7-10 different rat pups, performed in duplicate. *p ⁇ 0.05.
  • FIG. 27 shows incidence and severity of NEC. Rat pups were delivered
  • compositions and methods include the recited elements, but do not exclude others.
  • Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination for the intended use.
  • a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like.
  • Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
  • a "biofilm” intends a thin layer or an organized community of microorganisms that at times can adhere to the surface of a structure, that may be organic or inorganic, together with the polymers, such as DNA, that they secrete and/or release.
  • the biofilms are very resistant to microbiotics and antimicrobial agents. They live on gingival tissues, teeth, and restorations, causing caries and periodontal disease, also known as periodontal plaque disease. They also cause chronic middle ear infections. Biofilms can also form on the surface of dental implants, stents, catheter lines and contact lenses. They grow on pacemakers, heart valve replacements, artificial joints and other surgical implants.
  • a "prebiotic” intends a nutritional supplement for the probiotic bacterium.
  • Prebiotics are food ingredients, for example, oligosaccharides, that are non-digestible by a subject (e.g., by a mammal such as a human), and that stimulates the growth or activity of one or more beneficial bacteria and/or inhibit the growth or activity of one or more pathogenic bacteria.
  • a prebiotic may selectively stimulate the growth and/or activity of one or a limited number of bacteria in the subject.
  • a "prebiofilmic" intends a substance that supports biofilm formation and durability, for example the prebiofilmic can be a substance that supports the extracellular matrix of the biofilm like an eDNA binding polypeptide or protein or alternatively a substrate that can be converted into a substance that facilitate adhesion, e.g., sucrose.
  • a "DNABII polypeptide or protein” intends a DNA binding protein or polypeptide that is composed of DNA-binding domains and thus have a specific or general affinity for DNA. In one aspect, they bind DNA in the minor grove.
  • Non-limiting examples of DNABII proteins are an integration host factor (IHF) protein and a histone-like protein from E. coli strain U93 (HU), examples of which are provided in the attached sequence listing and additional strains and polypeptides are provided in Table 4. Also intended are polypeptide fragments and equivalent polypeptides that have amino acid modifications that do not substantially change the biological activity of the protein or polypeptides, or active fragment thereof.
  • Active fragments can include, for example, the c-terminal half or c-terminal third of the protein or polypeptide.
  • Other DNA binding proteins that can be associated with the biofilm 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), as well as equivalent polpyeptides and active fragments thereof.
  • a "microsphere” intends a porous and/or semi-permeable biofilm-carrying and/or compound-carrying (e.g., drug-carrying) particulate or granular material within the particular size range recited.
  • a microsphere consisting of particles 50 millimeters or less in diameter, and about 1 micron or more (e.g., about 1 to about 100 or alternatively, or alternatively, about 1 to about 75 microns, or alternatively about 1 to about 50, or
  • microns alternatively about 1 to about 25, or alternatively about 1 to about 10 microns, or alternatively about 0.5 to about 200 microns, or alternatively about 0.5 to about 700 microns, or alternatively about 1 to about 600 microns, or alternatively less than about 700 microns, or alternatively less than about 600 microns, or alternatively less than 500 microns, or alternatively less than about 400 microns, or alternatively less than about 300 microns, or alternatively less than about 200 microns, or alternatively less than about 100 microns) in diameter.
  • Non-limiting examples of such include hollow microspheres that are porous and/or semi-permeable, and can, in some aspects, contain a pharmaceutical or a drug, microcapsules, (in which the excipient forms a skin or shell that surrounds and contains a cargo, such as a drug, a chemical reductant, or absorptive or adsorptive molecules), and microparticles, which are used as a generic term for any particles in the recited size range, whether spherical or not, as those terms are typically used in the art.
  • Table 6 provides non-limiting examples of microspheres that are commercially available and their characteristics.
  • a “biodegradable polymer” intends polymers that are biocompatible and can degrade in vivo by bodily processes to products that are readily disposable by the body and should not accumulate in the body.
  • biocompatible it is meant that the components of the delivery system will not cause tissue injury or injury to the human biological system.
  • polymers and excipients that have had history of safe use in humans or with GRAS
  • biocompatibility it is meant that the ingredients and excipients used in the composition will ultimately be “bioabsorbed” or cleared by the body with no adverse effects to the body.
  • biocompatible and be regarded as non-toxic, it must not cause toxicity to cells.
  • bioabsorbable refers to microspheres made from materials which undergo
  • bioabsorption in vivo over a period of time such that long term accumulation of the material in the patient is avoided.
  • the biocompatible nanoparticle is bioabsorbed over a period of less than 2 years, preferably less than 1 year and even more preferably less than 6 months.
  • the rate of bioabsorption is related to the size of the particle, the material used, and other factors well recognized by the skilled artisan.
  • a mixture of bioabsorbable, biocompatible materials can be used to form the microspheres used in this invention.
  • IHF protein is a bacterial protein that is used by bacteriophages to incorporate their DNA into the host bacteria. These are DNA binding proteins that function in genetic recombination as well as in transcription and translational regulation. They also bind extracellular microbial DNA. The genes that encode the IHF protein subunits in E. coli are himA (Genbank accession No. : POA6X7.1) and himD
  • 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 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 to the HU protein are commercially available from Abeam. Non-limiting examples of such are provided in the attached sequence listing.
  • protein 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 another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc.
  • a protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
  • a "c-terminal polypeptide” intends the c-terminal half or c-terminal third of a polypeptide.
  • the c-terminal polypeptide would comprise amino acids 46 through 90 or amino acids 60 through 90.
  • the term intends the c-terminal 20 amino acids from the carboxy terminus.
  • a "n-terminal polypeptide” intends the n-terminal half of a polypeptide.
  • the c-terminal polypeptide would comprise amino acids 1 through 45.
  • the term intends the c-terminal 20 amino acids from the amino terminus.
  • 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 may perform any function, known or unknown.
  • polynucleotides a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), 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.
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • the term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA.
  • A adenine
  • C cytosine
  • G guanine
  • T thymine
  • U uracil
  • polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.
  • isolated or recombinant refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule as well as polypeptides.
  • isolated or recombinant nucleic acid is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • isolated is also used herein to refer to polynucleotides, polypeptides, antibodies and proteins that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
  • the term "isolated or recombinant” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature.
  • an isolated cell is a cell that is separated from tissue or cells of dissimilar phenotype or genotype.
  • An isolated polynucleotide is separated from the 3 ' and 5' contiguous nucleotides with which it is normally associated in its native or natural environment, e.g., on the chromosome.
  • a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof does not require "isolation" to distinguish it from its naturally occurring counterpart.
  • Glucotransferases are enzymes that establish glycosidic linkages.
  • a non-limiting example of a sequence of the GTF protein is available at DSM 20016.
  • gtfW ABQ83597.1 is provided at DSM 17938 gtfA WP _003671465. See also, Walter et al. (2008) Microbiology 154(Pt l):72-80.
  • biological equivalent thereof is intended to be synonymous with "equivalent thereof when referring to a reference protein, antibody, polypeptide, polynucleotide or nucleic acid, intends those having minimal homology while still maintaining desired structure or functionality. Unless specifically recited herein, it is contemplated that any nucleic acid, polynucleotide, polypeptide or protein mentioned herein also includes equivalents thereof.
  • an equivalent intends at least about 70%, or alternatively 80 % homology or identity and alternatively, at least about 85 %, or alternatively at least about 90 %, or alternatively at least about 95 %, or alternatively 98 % percent homology or identity across the protein or a particular fragment thereof, and exhibits substantially equivalent biological activity to the reference protein, polypeptide or nucleic acid.
  • a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
  • the alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1.
  • default parameters are used for alignment.
  • a preferred alignment program is BLAST, using default parameters.
  • Homology refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or “non-homologous" sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present invention.
  • expression refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in an eukaryotic cell.
  • encode refers to a polynucleotide which is said to "encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof.
  • the antisense strand is the
  • a "subject" or “patient” of diagnosis or treatment is a cell or an animal such as a mammal or a human.
  • Non-human animals subject to diagnosis or treatment and are those subject to infections or animal models, for example, simians, murines, such as, rats, mice, chinchilla, canine, such as dogs, leporids, such as rabbits, livestock, sport animals and pets
  • the terms “treating,” “treatment” and the like are used herein to mean obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disorder or sign or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disorder and/or adverse effect attributable to the disorder.
  • To "prevent” intends to prevent a disorder or effect in vitro or in vivo in a system or subject that is predisposed to the disorder or effect. Examples of such is preventing the formation of a biofilm in a system that is infected with a microorganism known to produce one or alternatively, prevent a gastrointestinal disorder by supporting a healthy state of the patient' s gut.
  • the term “culturing” refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell. By “expanded” is meant any proliferation or division of cells.
  • “Pharmaceutically acceptable carriers” refers to any diluents, excipients or carriers that may be used in the compositions of the 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, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium
  • Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. They are preferably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like and consistent with conventional pharmaceutical practices.
  • a “biocompatible scaffold” refers to a scaffold or matrix for with the ability to support biofilm proliferation upon administration to a subject.
  • a biocompatible scaffold is a precursor to an implantable device which has the ability to perform its intended function, with the desired degree of incorporation in the host, without eliciting an undesirable local or systemic effects in the host.
  • Biocompatible scaffolds are described in U.S. Patent Nos. 6,638,369 and 8,815,276.
  • the microsphere as described herein is a biocompatible scaffold.
  • administering intends the delivery of a substance to a subject such as an animal or human. Administration can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, as well as the age, health or gender of the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician or in the case of pets and animals, treating vetrenanan. Suitable dosage formulations and methods of administering the agents are known in the art.
  • Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated and the target cell or tissue.
  • route of administration include oral administration, vaginal, nasal administration (inhalation), injection, topical application and by suppository.
  • the term "effective amount" refers to a quantity sufficient to achieve a beneficial or desired result or effect.
  • the effective amount will depend on the type and severity of the condition at issue and the characteristics of the individual subject, such as general health, age, sex, body weight, and tolerance to pharmaceutical compositions.
  • the effective amount is the amount sufficient to result in a protective response against a pathogen or alternatively to support a healthy state of being.
  • the amount is sufficient to accomplish one or more of 1) clear pathogen; 2) restore healthy microbiota; 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 (PAH), petroleum hydrocarbon, crude oil, refined oil, herbicide contamination or pesticide contamination); and 6) remediate a biofilm).
  • heavy metals e.g., chromium, arsenic, mercury, radioactive actinides, uranium, plutonium, thorium, polycyclic aromatic hydrocarbons (PAH), petroleum hydrocarbon, crude oil, refined oil, herbicide contamination or pesticide contamination
  • the effective amount will depend on the size and nature of the application in question. It will also depend on the nature and sensitivity of the in vitro target and the methods in use. The skilled artisan will be able to determine the effective amount based on these and other considerations.
  • the effective amount may comprise one or more administrations of a composition depending on the embodiment.
  • the agents and compositions can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures, such as an active ingredient in pharmaceutical compositions.
  • An agent or composition of the present invention can be administered for therapy by any suitable route of administration. It will also be appreciated that the preferred route will vary with the condition and age of the recipient and the disease being treated.
  • Necrotizing enterocolitis is a medical condition primarily seen in premature infants where portions of the bowel undergo necrosis (tissue death). It occurs postnatally (i.e., is not seen in stillborn infants) and is the second most common cause of mortality. 7% of all neonatal intensive care unit admissions are NEC related. The mortality rate is 12%.
  • compositions comprising a microsphere, a biofilm- generating probiotic bacterium and a prebiotic, wherein the prebiotic comprises a nutritional supplementation for the probiotic bacterium.
  • the composition further comprises one or more of: a biofilm, a prebiofilmic, coating on the surface of the microsphere a therapeutic drug or agent, a chemical reductant, a molecule that promotes adsorption, a molecule that supports absorption.
  • the microsphere comprises a solid core, a hollow core, wherein in one aspect, the microsphere encapsulates the prebiotic within the hollow core.
  • the microsphere can be biocompatible and/or semi-permeable.
  • the microsphere comprise a biofilm layer or coating on the external surface of the microsphere.
  • the biocompatible microsphere comprises a material selected from the group of: a biodegradable polymer, a non-degradable polymer, a metal, and wherein the diameter of the microsphere is from about 0.5 microns to about 1000 microns. Additional preferred ranges are described herein and incorporated herein by reference.
  • microspheres can be porous and/or semi-permeable.
  • biodegradable polymers are selected from one or more of: dextran; dextranomer; poly(lactic-co-glycolic acid) or PLGA; polycaprolactone or PLC; Chitosan; Gelatin; DNA hydrogen; acetalated dextran; poly(lactide); poly(glycolide);
  • Non-limiting examples of non-biodegradable polymers are selected from one or more of poly(ethylene vinyl acetate), poly(vinyl acetate), silicone polymers, polyur ethanes, polysaccharides such as a cellulosic polymers and cellulose derivatives, acyl substituted cellulose acetates and derivatives thereof, copolymers of poly(ethylene glycol) and poly(butylene terephthalate), polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chorosulphonated polyolefins, polyethylene oxide, and copolymers and blends thereof.
  • Non-limiting examples of polymers comprising the microsphere are selected from one or more of: Sephadex, Sephadex G-25, poly(lactic-co-glycolic acid)(" PLGA”), polycaprolactone ("PLC”), chitosan; gelatin, DNA hydrogen; acetalated dextran,
  • Non-limiting examples of metals include cobalt, chromium, gold, nickel, platinum, stainless steel, titanium, tantalum, nickel-titanium, an alloy, and combinations thereof.
  • Non-limiting examples of the prebiotic of the composition comprise one or more of: a water-soluble carbohydrate, inulin, oligosaccharides, oligofructose, fructo-oligosaccharide, galacto-oligosaccharide, glucose, starch, maltose, maltodextrins, polydextrose, amylose, sucrose, fructose, lactose, isomaltulose, polyols, glycerol, carbonate, thiamine, choline, histidine, trehalos, nitrogen, sodium nitrate, ammonium nitrate, phosphorus, phosphate salts, hydroxyapatite, potassium, potash, sulfur, homopolysaccharide, heteropolysaccharide, cellulose, chitin, vitamins, and combination thereof.
  • the prebiotic is selected from one or more of trehalose; nitrogen such as in sodium nitrate, ammonium nitrate, phosphorus such in phosphate salts like hydroxyapatite, potassium such as in potash, sulfur, oligosaccharide, homopolysaccharide, heteropolysaccharide, cellulose, chitin, glucose, fructose, sucrose, maltose, starch, polydextrose, amylose, glycerol, carbonate, and combinations thereof.
  • nitrogen such as in sodium nitrate, ammonium nitrate, phosphorus such in phosphate salts like hydroxyapatite
  • potassium such as in potash, sulfur, oligosaccharide, homopolysaccharide, heteropolysaccharide, cellulose, chitin, glucose, fructose, sucrose, maltose, starch, polydextrose, amylose, glycerol, carbonate, and combinations thereof.
  • the prebiotic of the composition comprises one or more of vitamin mixtures to stimulate microbial growth, nitrogen such as in sodium nitrate, ammonium nitrate, phosphorus such in phosphate salts like hydroxyapatite, potassium such as in potash, sulfur, oligosaccharide, homopolysaccharide, heteropolysaccharide cellulose, chitin; glucose, fructose, sucrose, maltose, starch, polydextrose, amylose, glycerol, carbonate, and combinations thereof.
  • the probiotic bacterium is selected to provide one or more of supporting anti -bacterial immunity, enhancing or supporting a healthy state in the subject,t enhancing or supporting the gastrointestinal barrier, or antagonizing disease-related bacterial infections.
  • the probiotic bacterium is seleted to prevent pathogen colonization and/or limit and/or clear the pathogen, and /or limit excessive inflammatory responses by down-regulating cytokine and chemokine production.
  • Non-limiting examples of the probiotic bacterium is one or more of L. acidophilus, L. crispatus, L. gasseri, group L. delbrueckii, L. salivarius, L. casei, L. paracasei, L.
  • the probiotic is L. reuteri that produces GTF protein or containing the GTFW gene (ATCC 23272).
  • the prebiofllmic comprises an agent that supports biofilm formation and durability, non-limiting examples of such include a DNA binding polypeptide or protein and/or a DNABII polypeptide or protein or an equivalent of each thereof, optionally, a polypeptide comprising one or more of the attached sequence listing, or a biologically active fragment or equivalent of each thereof, alone or in combination.
  • the microspheres and compositions containing the microspheres can further an agent, wherein the agent is selective against a pathogen that may compete with the probiotic organism.
  • the complimentary agents can be in the core, on the surface of the microsphere in in the composition containing the microspheres.
  • Non-limiting examples of such include chemical reductants; molecules and/or surfaces that promote adsorption (in core or on surface of microsphere); molecules and/or surfaces that promote absorption (in core or on surface of microsphere).
  • the chemical reductants and molecules and/or surfaces that promote absorption are coated on the surface of the microsphere.
  • the microsphere compositions further comprise a biofilm layer on the external surface of the microparticle.
  • the layer can be from about about 0.5 micron to about 1 millimiter in depth, and ranges in between, e.g., about 1 micron to about 500 microns, about 1 micron to about 250 microns, about 1 micron to about 200 microns, about 1 micron to about 100 microns, about 1 micron to about 50 microns, about 1 micron to about 40 microns, about 1 micron to about 30 microns, about 2 micron to about 100 microns, about 2 microns to about 50 microns, about 2 microns to about 40 microns, about 2 microns to about 30 microns, about 3 microns to about 100 microns, about 3 microns to about 50 microns, about 3 microns to about 40 microns, about 3 microns to about 30 microns, about 5 microns to about 100 microns, about 5 microns to about 50 micron microns,
  • This disclosure also provides one or a plurality of microsphere compositions as described herein in combination with a carrier, e.g., a pharmaceutically acceptable carrier or a biocompatible scaffold.
  • a carrier e.g., a pharmaceutically acceptable carrier or a biocompatible scaffold.
  • pharmaceutically acceptable carriers include diluents, excipients or carriers that may be used in the compositions of the 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 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, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, polyethylene glycol and wool fat.
  • 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
  • Non-limiting examples of biocompatible scaffolds include a scaffold or matrix for with the ability to support biofilm proliferation upon administration to a subject or an environment to be treated.
  • compositions comprise a plurality of microspheres that are the same or different from each other, e.g, the same or different diameters, the same or different microsphere components, the same or different probiotics, the same or different
  • compositions can be formulated into dosage forms of the biofilm-generative probiotic bacterium, e.g., or provide from an effective amount of the microsphere composition for the end use, e.g., from about 1 X 10 5 to 1 X 10 11 CFU/ml, or alternatively from about 1 X 10 5 to about 1 X 10 10 CFU/ml, or about 1 X 10 5 to about 1 X 10 9 CFU/ml, or about 1 X 10 6 to about 1 X 10 11 CFU/ml, or about 1 X 10 6 to about 1 X 10 9 CFU/ml, or about 1 X 10 7 to about 1 X 10 u CFU/ml, or about 1 X 10 7 to about 1 X 10 10 CFU/ml, or about 1 X 10 7 to about 1 X 10 9 CFU/ml, or about 1 X 10 8 CFU/ml.
  • compositions can be formulated or processed for ease of administration, storage and application, e.g., frozen, lyophilized, suspended (suspension formulation) or powdered; and processed as a suppository, tablet, solution, suspensions, pills, capsules, sustained release formulation.
  • compositions of this disclosure find use in therapeutic, agricultural and industrial microbial support, biofilm support, and/or or biofilm remediation, and the components of the compositions and the carriers and additional agents are selected for the specified use.
  • the compositions provide one or more of supporting anti-bacterial immunity, enhancing or supporting the gastrointestinal barrier, or antagonizing disease- related bacterial infections. In another aspect, the compositions prevents pathogen colonization and/or limits excessive inflammatory responses by down-regulating cytokine and chemokine production.
  • compositions are useful for the treatment of a mammal such as a human; simians, murines, such as, rats, mice, chinchilla, canine, such as dogs, leporids, such as rabbits, livestock, sport animals and pets.
  • a mammal such as a human
  • simians, murines such as, rats, mice, chinchilla
  • canine such as dogs
  • leporids such as rabbits, livestock, sport animals and pets.
  • they are useful to treat agricultural crops such as corn, wheat, soybeans, and potatoes; domestic garden plants such as tomatoes, peppers, spinach, and beans.
  • they are useful for the treatment of contaminated water or soil, machinery and manmade structures.
  • compositions can be used in the treatment or prevention of disease, e.g., psychological disorders, such as depression or anxiety, enteric infectious disease, infection-induced colitis, traveler's diarrhea, inflammatory bowel disease (IBD), colitis, diarrheal illness, vaginosis, wound, burns, psoriasis, dermatitis, tooth decay, periodontitis, sinusitis, or any of chronic and/or recurrent disease that is caused by pathogenic bacteria displacing healthy bacteria or nectrotizing enterocolitis (NEC), o support anti-bacterial immunity, enhancing or supporting the gastrointestinal barrier, correcting or supporting dysbiotic gut flora (and even in the absence of diseases), disease or disorders involving intestinal dysmobility, enhancing or supporting the gastrointestinal mobility, or antagonizing disease-related bacterial infection; vaginosis; colitis or traveler' s diarrhea, peritonitis, post-operative ileus, irritable bowel syndrome (IBS
  • this disclosure provides method for treating or preventing a disease or disorder suitably treated by a biofilm in a subject in need thereof is provided herein.
  • the method comprises administering to the subject an effective amount of the composition as disclosed herein, having the components selected for the particular therapy.
  • Non-limiting examples of diseases include psychological disorders, such as depression or anxiety, enteric infectious disease, infection-induced colitis, traveler's diarrhea, inflammatory bowel disease (IBD), colitis, diarrheal illness, vaginosis, wound, burns, psoriasis, dermatitis, tooth decay, periodontitis, sinusitis, or any of chronic and/or recurrent disease that is caused by pathogenic bacteria displacing healthy bacteria or nectrotizing enterocolitis (NEC), and to support anti-bacterial immunity, enhancing or supporting the gastrointestinal barrier, correcting or supporting dysbiotic gut flora (and even in the absence of diseases), disease or disorders involving intestinal dysmobility, enhancing or supporting the gastrointestinal mobility, or antagonizing disease-related bacterial infection; vaginosis; colitis or traveler' s diarrhea, peritonitis, post-operative ileus, irritable bowel syndrome (IBS), intestinal pseudoobstruction, and/or constipation. Additionally, the compositions are useful to promote health and
  • This disclosure also provides a method for delivering a probiotic to a subject comprising administering a dose of a composition as disclosed herein to the subject, thereby administering the probiotic.
  • the dosage and components of the composition will vary with the subject and purpose of the therapy.
  • the composition is administered to provide from about 1 x 10 7 to about 1 x 10 9 CFU/ml of the biofilm-generating probiotic bacterium.
  • the compositions can be formulated into dosage forms, e.g., or provide from an effective amount of the microsphere composition for the end use, e.g., from about 1 X 10 5 to 1 X 10 11 CFU/ml, or alternatively from about 1 X 10 5 to about 1 X 10 10 CFU/ml, or about 1 X 10 5 to about 1 X 10 9 CFU/ml, or about 1 X 10 6 to about 1 X 10 11 CFU/ml, or about 1 X 10 6 to about 1 X 10 9 CFU/ml, or about 1 X 10 7 to about 1 X 10 11 CFU/ml, or about 1 X10 7 to about 1 X 10 10 CFU/ml, or about 1 X 10 7 to about 1 X 10 7
  • compositions can be administered at about 6, 12, 18, 24, 36, 48, and 72 hours, or can be administered in a single dose.
  • compositions can be administered orally, vaginally, topically, by inhalation, intravenously, intramuscularly, or by suppository. They can be administered in any suitable formulation.
  • the composition are useful for the treatment of desiccation, nutrient starvation, nutrient depletion, bacterial pathogen infection, invertebrate antagonism, pollution; severe weather, physical stress, hypoxia, soil acidification.
  • this disclosure also provides methods for treating a plant, by administering to the plant directly or in its environment, a composition as disclosed herein.
  • the dosage and components of the composition will vary with the plant and purpose of the treatment.
  • the composition is administered to provide from about 1 x 10 7 to about 1 x 10 9 CFU/ml of the biofilm-generating probiotic bacterium.
  • compositions can be formulated into dosage forms, e.g., or provide from an effective amount of the microsphere composition for the end use, e.g., from about 1 X 10 5 to 1 X 10 11 CFU/ml, or alternatively from about 1 X 10 5 to about 1 X 10 10 CFU/ml, or about 1 X 10 5 to about 1 X 10 9 CFU/ml, or about 1 X 10 6 to about 1 X 10 11 CFU/ml, or about 1 X 10 6 to about 1 X 10 9 CFU/ml, or about 1 X 10 7 to about 1 X 10 11 CFU/ml, or about 1 X10 7 to about 1 X 10 10 CFU/ml, or about 1 X 10 7 to about 1 X 10 9 CFU/ml, or about 1 X 10 8 CFU/ml.
  • compositions can be administered at about 6, 12, 18, 24, 36, 48, and 72 hours, or can be administered in a single dose.
  • the composition is administered by spraying the plant or by irrigating the plant or admixing the composition with water applying to the plant or its environment. It can by sprayed onto the plant or the soil surrounding the plant, applied dry into the soil surface surrounding the plant, adding the compositions to the irrigation or watering system, or mixing the composition with the soil prior to seeding.
  • this disclosure also provides methods to deliver a composition and/or treat or prevent a disease or condition, and/or treat an environment (soil, plant, water, or surface) by contacting the surface or delivering an effective amount of the composition as disclosed herein.
  • hydrocarbons such as crude and refined oil, herbicide or pesticide contamination.
  • the compositions can be formulated or processed for ease of administration, storage and application, e.g., frozen, lyophilized, suspended (suspension formulation) or powdered, and processed for use in industrial applications, e.g., for the treatment of contaminated water or soil, machinery, and manmade structures, e.g., bioreactor, biopile, bio-venting, land-farming, filter surface, permeable reactive barrier, in situ administration via wet or dry application to water or soil.
  • compositions will vary with the purpose of the treatment.
  • the composition is administered to provide from about 1 x 10 7 to about 1 x 10 9 CFU/ml of the biofilm-generating probiotic bacterium.
  • the compositions can be formulated into dosage forms, e.g., or provide from an effective amount of the
  • microsphere composition for the end use, e.g., from about 1 X 10 5 to 1 X 10 11 CFU/ml, or alternatively from about 1 X 10 5 to about 1 X 10 10 CFU/ml, or about 1 X 10 5 to about 1 X 10 9 CFU/ml, or about 1 X 10 6 to about 1 X 10 11 CFU/ml, or about 1 X 10 6 to about 1 X 10 9 CFU/ml, or about 1 X 10 7 to about 1 X 10 11 CFU/ml, or about 1 X10 7 to about 1 X 10 10 CFU/ml, or about 1 X 10 7 to about 1 X 10 9 CFU/ml, or about 1 X 10 8 CFU/ml.
  • the compositions can be delivered in multiple doses, e.g., every 6, 12, 18, 24, 36, 48, and 72 hours, or can be administered in a single dose.
  • this disclosure also provides methods to remediate a biofilm by contacting the surface or machine by contacting the surface, or delivering to the environment an effective amount of the composition. One can determine if the method was successful by assaying for the biofilm.
  • the microspheres comprise a biofilm-generating probiotic bacterium and a prebiotic, wherein the prebiotic comprises a nutritional supplementation for the probiotic bacterium.
  • the composition further comprises one or more of: a prebiofilmic, a therapeutic drug or agent, a chemical reductant, a molecule that promotes adsorption, a molecule that supports absorption, density-driving cargo, such as ethanol, sunflower oil, or olive oil, or an equivalent of each thereof, or pre-frozen components that are low density upon admininistration or use, but after use or thaw of the pre-frozen components, might be transported or moved from the initial site of administraion.
  • the microsphere comprises a solid core, a hollow core, wherein in one aspect, the microsphere encapsulates the prebiotic within the hollow core.
  • the biocompatible microsphere comprises a material selected from the group of: a biodegradable polymer, a non-degradable polymer, a metal, and wherein the diameter of the microsphere is from about 0.5 microns to about 1000 microns. Additional preferred ranges are described above and incorporated herein by reference.
  • Non-limiting examples of biodegradable polymers for agriculture use are selected from one or more of: dextran, dextranomer, poly(lactic-co-glycolic acid) or PLGA; polycaprolactone or 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) copolymers,
  • Non-limiting examples of non-biodegradable polymers are selected from one or more of poly(ethylene vinyl acetate), poly(vinyl acetate), silicone polymers, polyurethane s, polysaccharides such as a cellulosic polymers and cellulose derivatives, acyl substituted cellulose acetates and derivatives thereof, copolymers of poly(ethylene glycol) and poly(butylene terephthalate), polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chorosulphonated polyolefins, polyethylene oxide, and copolymers and blends thereof.
  • Non-limiting examples of metals include one or more of: cobalt, chromium, gold, nickel, platinum, stainless steel, titanium, tantalum, nickel-titanium, an alloy, and
  • Non-limiting examples of the prebiotic include one or more of trehalose; nitrogen such as in sodium nitrate, ammonium nitrate, phosphorus such in phosphate salts like hydroxyapatite, potassium such as in potash, sulfur, oligosaccharide, homopolysaccharide, heteropolysaccharide, cellulose, chitin, glucose, fructose, sucrose, maltose, starch, polydextrose, amylose, glycerol, carbonate, and combinations thereof.
  • nitrogen such as in sodium nitrate, ammonium nitrate, phosphorus such in phosphate salts like hydroxyapatite
  • potassium such as in potash, sulfur, oligosaccharide, homopolysaccharide, heteropolysaccharide, cellulose, chitin, glucose, fructose, sucrose, maltose, starch, polydextrose, amylose, glycerol, carbonate, and combinations thereof
  • Non-limiting examples of the probiotic bacterium L. reuteri, Pseudomonas fluorescens, P. protegens, P. brassicacearum, P. aeruginosa; Azospirillum. brabrasilense, A. lipferum, A. halopraeferens, A. irakense; Acetobacter diazotrophicus; Herbaspirillum seropedicae; Bacillus subtilis, and combinations thereof.
  • the prebiofilmic comprises an agent that supports biofilm formation and durability, non-limiting examples of such include a DNA binding polypeptide or protein and/or a DNABII polypeptide or protein or an equivalent of each thereof, optionally, a polypeptide comprising one or more of the attached sequence listing, or a biologically active fragment or equivalent of each thereof, alone or in combination.
  • the microspheres and compositions containing the microspheres can further an agent, wherein the agent is selective against a pathogen that may compete with the probiotic organism.
  • the complimentary agents can be in the core, on the surface of the microsphere or in in the composition containing the microspheres. Non-limiting examples of such include drugs against a pathogen or invertebrate, that are optionally contained in the core of the microsphere.
  • This disclosure also provides one or a plurality of microsphere compositions as described herein in combination with a carrier, e.g., a pharmaceutically acceptable carrier or a biocompatible scaffold.
  • a carrier e.g., a pharmaceutically acceptable carrier or a biocompatible scaffold.
  • pharmaceutically acceptable carriers include diluents, excipients or carriers that may be used in the compositions of the disclosure.
  • Non-limiting examples of biocompatible scaffolds include a scaffold or matrix for with the ability to support biofilm proliferation upon administration to a plant, soil or water.
  • the microsphere further comprises a biofilm layer that partially or fully surrounds the microsphere.
  • compositions comprise a plurality of microspheres that are the same or different from each other, e.g., the same or different diameters, the same or different microsphere components, the same or different probiotics, the same or different
  • compositions can be formulated into dosage forms of the biofilm-generative probiotic bacterium, e.g., or provide from an effective amount of the microsphere composition for the end use, e.g., from about 1 X 10 5 to 1 X 10 11 CFU/ml, or alternatively from about 1 X 10 5 to about 1 X 10 10 CFU/ml, or about 1 X 10 5 to about 1 X 10 9 CFU/ml, or about 1 X 10 6 to about 1 X 10 11 CFU/ml, or about 1 X 10 6 to about 1 X 10 9 CFU/ml, or about 1 X 10 7 to about 1 X 10 u CFU/ml, or about 1 X10 7 to about 1 X 10 10 CFU/ml, or about 1 X 10 7 to about 1 X 10 9 CFU/ml, or about 1 X 10 8 CFU/ml.
  • compositions can be formulated or processed for ease of delivery, by spray application onto plant surface and/or soil, dry application onto soil surface, addition to watering system, or mixing with soil prior to seeding. They can be used to treat agricultural crops such as corn, wheat, soybeans, and potatoes; domestic garden plants such as tomatoes, peppers, spinach, and beans. In a yet further aspect, they are useful for the treatment of contaminated water or soil, machinery and manmade structures.
  • compositions are useful for the treatment of desiccation, nutrient starvation, nutrient depletion, bacterial pathogen infection, invertebrate antagonism, pollution, severe weather, physical stress, hypoxia; soil acidification.
  • this disclosure also provides methods for treating a plant, by administering to the plant directly or in its environment, a composition as disclosed herein.
  • the dosage and components of the composition will vary with the plant and purpose of the treatment.
  • the composition is administered to provide from about 1 x 10 7 to about 1 x 10 9 CFU/ml of the biofilm-generating probiotic bacterium.
  • compositions can be formulated into dosage forms, e.g., or provide from an effective amount of the microsphere composition for the end use, e.g., from about 1 X 10 5 to 1 X 10 u CFU/ml, or alternatively from about 1 X 10 5 to about 1 X 10 10 CFU/ml, or about 1 X 10 5 to about 1 X 10 9 CFU/ml, or about 1 X 10 6 to about 1 X 10 11 CFU/ml, or about 1 X 10 6 to about 1 X 10 9 CFU/ml, or about 1 X 10 7 to about 1 X 10 11 CFU/ml, or about 1 X10 7 to about 1 X 10 10 CFU/ml, or about 1 X 10 7 to about 1 X 10 9 CFU/ml, or about 1 X 10 8 CFU/ml.
  • compositions can be administered at about 6, 12, 18, 24, 36, 48, and 72 hours, or can be administered in a single dose.
  • the composition is administered by spraying the plant or by irrigating the plant or admixing the composition with water applying to the plant or its environment. It can by sprayed onto the plant or the soil surrounding the plant, applied dry into the soil surface surrounding the plant, adding the compositions to the irrigation or watering system, or mixing the composition with the soil prior to seeding.
  • this disclosure also provides methods to deliver a composition and/or treat or prevent a disease or condition, and/or treat an environment (soil, plant, water, or surface) by contacting the surface or delivering an effective amount of the composition as disclosed herein.
  • biocompatible microspheres that can be porous and/or semi-permeable, for industrial biofilm remediation, wherein the microspheres comprise a material selected from the group of: a biodegradable polymer, a non-degradable polymer, a metal, and wherein the diameter of the microsphere is from about 0.5 microns to about 1000 microns. Additional preferred ranges are described above and incorporated herein by reference.
  • biodegradable polymers are selected from one or more of: dextan, dextranomer, poly(lactic-co-glycolic acid) or PLGA, polycaprolactone or 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) copolymers,
  • Non-limiting examples of non-biodegradable polymers are selected from one or more of poly(ethylene vinyl acetate), poly(vinyl acetate), silicone polymers, polyurethane s, polysaccharides such as a cellulosic polymers and cellulose derivatives, acyl substituted cellulose acetates and derivatives thereof, copolymers of poly(ethylene glycol) and poly(butylene terephthalate), polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chorosulphonated polyolefins, polyethylene oxide, and copolymers and blends thereof.
  • Non-limiting examples of metals include cobalt, chromium, gold, nickel, platinum, stainless steel, titanium, tantalum, nickel-titanium, an alloy, and combinations thereof.
  • Non-limiting examples of the prebiotic of the composition for industrial use comprise one or more of vitamin mixtures to stimulate microbial growth, nitrogen such as in sodium nitrate, ammonium nitrate, phosphorus such in phosphate salts like hydroxyapatite, potassium such as in potash, sulfur, oligosaccharide, homopolysaccharide,
  • heteropolysaccharide cellulose chitin; glucose, fructose, sucrose, maltose, starch, polydextrose, amylose, glycerol, carbonate, and combinations thereof.
  • the probiotic bacterium is selected to provide one or more of supporting biofilm remediation in an industrial process or surface, and non-limiting examples of the probiotic bacterium is one or more of Pseudomonas stutzeri, fluorescens, P. piitida, P. cepacian, P. vesicularis, P. paucimobilis; Bacillus cereus, 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 petroleiphilum,
  • the prebiofilmic of the composition can optionally comprise an agent that supports biofilm formation and durability, non-limiting examples of such include a DNA binding polypeptide or protein and/or a DNABII polypeptide or protein or an equivalent of each thereof, optionally, a polypeptide comprising one or more of the attached sequence listing, or a biologically active fragment or equivalent of each thereof, alone or in combination.
  • the microspheres and compositions containing the microspheres can further an agent, wherein the agent is selective against a pathogen that may compete with the probiotic organism.
  • the complimentary agents can be in the core, on the surface of the microsphere, or in the composition containing the microspheres.
  • Non-limiting examples of such include chemical reductants, molecules and/or surfaces that promote adsorption (in core or on surface of microsphere), molecules and/or surfaces that promote absorption (in core or on surface of microsphere).
  • the chemical reductants and molecules and/or surfaces that promote absorption are coated on the surface of the microsphere.
  • the microparticle composition might be modified to be density-driven, in that the density or buoyancy of the cargo allows the microparticle to float during initial application, e.g., oil spills. So the cargo would be selected to be low density and as the cargo is utilized, but after the oil slick is degraded, the microspheres sink to the bottom of the water.
  • Alchohols such as ethanol, , sunflower oil, or olive oil, or an equivalent of each thereof and the like or by providing the cargo in a pre-frozen state.
  • This disclosure also provides one or a plurality of microsphere compositions as described herein in combination with a carrier, e.g., a pharmaceutically acceptable carrier, or a biocompatible scaffold.
  • a carrier e.g., a pharmaceutically acceptable carrier, or a biocompatible scaffold.
  • pharmaceutically acceptable carriers include diluents, excipients or carriers that may be used in the compositions of the disclosure.
  • biocompatible scaffolds include a scaffold or matrix for with the ability to support biofilm proliferation upon administration to an environment to be treated.
  • the microsphere further comprises a biofilm layer that partially or fully surrounds the microsphere.
  • compositions comprise a plurality of microspheres that are the same or different from each other, e.g., the same or different diameters, the same or different microsphere components, the same or different probiotics, the same or different
  • prebiofilmic and hollow and/or solid cores.
  • compositions can be formulated into dosage forms of the biofilm-generative probiotic bacterium, e.g., or provide from an effective amount of the microsphere composition for the end use, e.g., from about 1 X 10 5 to 1 X 10 11 CFU/ml, or alternatively from about 1 X 10 5 to about 1 X 10 10 CFU/ml, or about 1 X 10 5 to about 1 X 10 9 CFU/ml, or about 1 X 10 6 to about 1 X 10 11 CFU/ml, or about 1 X 10 6 to about 1 X 10 9 CFU/ml, or about 1 X 10 7 to about 1 X 10 u CFU/ml, or about 1 X10 7 to about 1 X 10 10 CFU/ml, or about 1 X 10 7 to about 1 X 10 9 CFU/ml, or about 1 X 10 8 CFU/ml.
  • PAH hydrocarbons
  • petroleum hydrocarbon such as crude and refined oil, herbicide or pesticide contamination.
  • compositions can be formulated or processed for ease of administration, storage and application, e.g., frozen, lyophilized, suspended (suspension formulation) or powdered; and processed for use in industrial applications, e.g., for the treatment of contaminated water or soil, machinery, and manmade structures, e.g., bioreactor, biopile, bio-venting, land- farming, filter surface, permeable reactive barrier, in situ administration via wet or dry application to water or soil.
  • frozen, lyophilized, suspended (suspension formulation) or powdered e.g., for the treatment of contaminated water or soil, machinery, and manmade structures, e.g., bioreactor, biopile, bio-venting, land- farming, filter surface, permeable reactive barrier, in situ administration via wet or dry application to water or soil.
  • compositions for nutritional or medicinal use comprising a microsphere, a biofilm-generating probiotic bacterium and a prebiotic, wherein the prebiotic comprises a nutritional supplementation for the probiotic bacterium.
  • the composition further comprises one or more of: a prebiofilmic, a biofilm layer, a therapeutic drug or agent.
  • the microsphere comprises a solid core, a hollow core, wherein in one aspect, the microsphere encapsulates the prebiotic within the hollow core.
  • the microsphere comprises a material selected from the group of: a biodegradable polymer, a non-degradable polymer, or a metal, and wherein the diameter of the microsphere is from about 0.5 microns to about 1000 microns, or alternatively rom about 0.5 microns to about 100 microns, or alternatively less than 100 microns.
  • Non-limiting examples of biodegradable polymers for medicinal use are selected from one or more of dextran, dextranomer, poly(lactic-co-glycolic acid) or PLGA, polycaprolactone or 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) copolymers,
  • Non-limiting examples of non-biodegradable polymers for medicinal use are selected from one or more of poly(ethylene vinyl acetate), poly(vinyl acetate), silicone polymers, polyurethanes, polysaccharides such as a cellulosic polymers and cellulose derivatives, acyl substituted cellulose acetates and derivatives thereof, copolymers of poly(ethylene glycol) and poly(butylene terephthalate), polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chorosulphonated polyolefins, polyethylene oxide, and copolymers and blends thereof.
  • Non-limiting examples of metals include cobalt, chromium, gold, nickel, platinum, stainless steel, titanium, tantalum, nickel-titanium, an alloy, and combinations thereof.
  • Non-limiting examples of the prebiotic of the composition for medicinal use comprises one or more of a water-soluble carbohydrate, inulin, oligofructose, fructo- oligosaccharide, galacto-oligosaccharide, glucose, maltose, maltodextrins, polydextrose, sucrose, fructose, lactose, isomaltulose, polyols, glycerol, thiamine, choline, histidine, and combination thereof.
  • Non-limiting examples of the probiotic bacterium is one or more of L. acidophilus, L. crispatus, L. gasseri, group L. delbrueckii, L. salivarius, L. casei, L. paracasei, L.
  • Non-limiting examples of the prebiofilmic comprises an agent that supports biofilm formation and durability, non-limiting examples of such include a DNA binding polypeptide or protein and/or a DNABII polypeptide or protein or an equivalent of each thereof, optionally, a polypeptide comprising one or more of the attached sequence listing, or a biologically active fragment or equivalent of each thereof, alone or in combination.
  • the microspheres and compositions containing the microspheres can further an agent, wherein the agent is selective against a pathogen that may compete with the probiotic organism.
  • the complimentary agents can be in the core, on the surface of the microsphere in in the composition containing the microspheres.
  • the microsphere further comprises a biofilm layer that partially or fully surrounds the microsphere.
  • This compositions for medicinal use can be provide as a composition, comprising one or a plurality of microsphere compositions as described herein in combination with a carrier, e.g., a pharmaceutically acceptable carrier or a biocompatible scaffold.
  • compositions comprise a plurality of microspheres that are the same or different from each other, e.g., the same or different diameters, the same or different microsphere components, the same or different biofilm layer, the same or different probiotics, the same or different complimentary agents, the same or different prebiofilmic, and hollow and/or solid cores.
  • compositions can be formulated into dosage forms of the biofilm-generative probiotic bacterium, e.g., or provide from an effective amount of the microsphere composition for the end use, e.g., from about 1 X 10 5 to 1 X 10 11 CFU/ml, or alternatively from about 1 X 10 5 to about 1 X 10 10 CFU/ml, or about 1 X 10 5 to about 1 X 10 9 CFU/ml, or about 1 X 10 6 to about 1 X 10 11 CFU/ml, or about 1 X 10 6 to about 1 X 10 9 CFU/ml, or about 1 X 10 7 to about 1 X 10 u CFU/ml, or about 1 X10 7 to about 1 X 10 10 CFU/ml, or about 1 X 10 7 to about 1 X 10 9 CFU/ml, or about 1 X 10 8 CFU/ml.
  • compositions can be formulated or processed for ease of administration, storage and application, e.g., frozen, lyophilized, suspended (suspension formulation) or powdered; and processed as a suppository, tablet, solution, suspensions, pills, capsules, sustained release formulation.
  • compositions are useful for the treatment of a mammal such as a human, simians, murines, such as, rats, mice, chinchilla, canine, such as dogs, leporids, such as rabbits, livestock, sport animals and pets.
  • a mammal such as a human, simians, murines, such as, rats, mice, chinchilla, canine, such as dogs, leporids, such as rabbits, livestock, sport animals and pets.
  • compositions can be used in the treatment or prevention of disease, e.g., psychological disorders, such as depression or anxiety, enteric infectious disease, infection- induced colitis, traveler' s diarrhea, inflammatory bowel disease (IBD), colitis, diarrheal illness, vaginosis, wound, burns, psoriasis, dermatitis, tooth decay, periodontitis, sinusitis, or any of chronic and/or recurrent disease that is caused by pathogenic bacteria displacing healthy bacteria or nectrotizing enterocolitis (NEC), to support anti -bacterial immunity, enhancing or supporting the gastrointestinal barrier, correcting or supporting dysbiotic gut flora (and even in the absence of diseases), disease or disorders involving intestinal dysmobility, enhancing or supporting the gastrointestinal mobility, or antagonizing disease- related bacterial infection; vaginosis; colitis or traveler' s diarrhea, peritonitis, post-operative ileus, irritable bowel syndrome (IBS), intestinal pseudo-obstruction
  • IBS
  • this disclosure provides method for treating or preventing a disease suitably treated by a healthy bacteria and/or a biofilm in a subject in need thereof.
  • the method comprises administering to the subject an effective amount of the composition as disclosed herein, having the components selected for the particular therapy.
  • 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, enteric infectious disease, infection-induced colitis, traveler's diarrhea, inflammatory bowel disease (IBD), colitis, diarrheal illness, vaginosis, wound, burns, psoriasis, dermatitis, tooth decay, periodontitis, sinusitis, or any of chronic and/or recurrent disease that is caused by pathogenic bacteria displacing healthy bacteria or nectrotizing enterocolitis (NEC), to support antibacterial immunity, enhancing or supporting the gastrointestinal barrier, or antagonizing disease-related bacterial infection; vaginosis; colitis or traveler' s diarrhea, peritonitis, postoperative ileus, irritable bowel syndrome, intestinal pseudo-obstruction, constipation.
  • psychological disorders such as depression or anxiety, enteric infectious disease, infection-induced colitis, traveler's diarrhea, inflammatory bowel disease (IBD), colitis, diarrhea
  • this disclosure provides methods 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 an appropriate or disease-relevant or health-promoting composition as disclosed herein.
  • the compositions are administered by any suitable method of administration, e.g., orally, vaginally, by inhalation, by injection, topically or by suppository.
  • compositions also are useful as nutritional supplements to promote general health and well-being and maintain gut health and/or homeostasis.
  • this disclosure also provides a method for promoting health and/ or maintaining gut homeostasis in a subject in need thereof, the method comprising, or alternatively consisting essentially of, or yet further consisting of, administering to the subject an effective amount of a composition as described herein, and optionally wherein the suface of the microsphere is porous and/or semi-permeable and the prebiotic is released by diffusion or the microsphere slowly degrades causing leaks and diffusion from the microsphere.
  • One of skill in the art can determine if better general health has been achieved, as well as gut homeostatis, by determining if gut discomfort has been reduced or alleviated.
  • Diarrheal illness occurs in approximately four billion individuals per year and causes more than two million deaths worldwide.
  • A/E pathogens attaching and effacing (A/E) pathogens, which upon colonization induce diarrheal disease that is associated with an increase in inflammatory cytokines and structural changes to colonic tissue.
  • This acute infection can have a lasting effect on gut health, and infection with A/E pathogens and excessive inflammatory responses are known risk factors for the development of postinfectious irritable bowel syndrome.
  • Probiotics are a natural way to protect and restore 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 fail to establish, or sufficiently persist, minimizing the magnitude and duration of their healthful effects.
  • One of the rate limiting steps is the capacity of introduced bacteria to form a lasting biofilm. When bacteria are already in the form of a biofilm (a surface adhered community) as opposed to planktonic (free-living), they more readily establish and persist.
  • Biocompatible microspheres can be biodegradable polymers, non-biodegradable polymers, a metal, or a combination thereof.
  • prebiotic and/or prebiofilmic substances can be added as cargo to facilitate establishment and maintenance of the probiotic bacterial biofilm.
  • Microspheres have added value in ideally providing diffusible prebiotic (nutritional supplementation specific/exclusive to probiotic bacteria) cargo that can help promote probiotic bacterial establishment and survival while limiting pathogenic bacterial challenge.
  • the biofilm state is advantageous in establishing in the murine gut over the same bacteria in planktonic form.
  • L. reuteri introduced into mice as biofilms have a more robust and durable prophylactic effect on the pathogenesis of the enteropathogenic bacterium,
  • Citrobacter rodentium than L. reuteri in its planktonic form. Based on these results, three highly integrated examples are developed that yield novel formulations of probiotics that provide greater and more lasting effects against dysbiosis preventing or even treating gut pathogenesis with a far reduced need for patient compliance.
  • the biofilm-generating probiotic bacterium adheres to the surface of the biocompatible microsphere and generates a biofilm.
  • the biocompatible microsphere has either a solid or hollow core.
  • the biocompatible microsphere can carry a prebiotic and any nutritional supplementation for the probiotic bacterium as a cargo.
  • the sphere surface can be semi-permeable to allow cargo to diffuse to the bound bacteria at high localized concentrations or it can be impermeable but slowly degrade to allow the contents to be released.
  • the prebiotic can be encapsulated within the hollow core.
  • the microsphere can also carry a drug, or a compound, or an agent, which is selective against the growth or proliferation of a pathogen.
  • a novel probiotic formulation may also contain a prebiofilmic, which a substance that supports biofilm formation and durability, specifically, the prebiofilmic is a DNA binding polypeptide or protein and/or a DNABII polypeptide or protein, a fragment and/or an equivalent of each thereof.
  • a prebiofilmic which a substance that supports biofilm formation and durability
  • the prebiofilmic is a DNA binding polypeptide or protein and/or a DNABII polypeptide or protein, a fragment and/or an equivalent of each thereof.
  • One or more drug, compound or agent as well as one or more prebiofilmic can be within a single microsphere.
  • the prebiotic can support the growth of any probiotic bacteria, including biofilm- generating bacteria.
  • the prebiotic is usually one or more of a water-soluble carbohydrate, such as inulin, oligofructose, fructo-oligosaccharide, galacto-oligosaccharide, glucose, maltose, maltodextrins, polydextrose, sucrose, fructose, lactose, isomaltulose, polyols, and glycerol.
  • a water-soluble carbohydrate such as inulin, oligofructose, fructo-oligosaccharide, galacto-oligosaccharide, glucose, maltose, maltodextrins, polydextrose, sucrose, fructose, lactose, isomaltulose, polyols, and glycerol.
  • the combination of various prebiotics can be used to support the growth of probiotics.
  • Probiotics are any type of micro-organisms that have health benefits. Probiotics are also commonly consumed as part of fermented foods with specially added active live cultures, such as in yogurt, soy yogurt, or as dietary supplements. Probiotics can also be taken as a suppository. Some limiting examples of probiotics are . acidophilus, L. crispatus, L. gasseri, group L. delbrueckii, L. salivarius, L. casei, L. paracasei, L. plantarum, L.
  • the probiotic is an L. reuteri that expresses GTF protein. All strains of L. reuteri possess at least one GTF protein, although they can vary between strains, e.g., in DSM20016, the GTF is GTFW and uses maltose as its sole substrate while in DSM 17938 the GTF is GTF A, and it uses sucrose as its sole substate.
  • Probiotics support anti-bacterial immunity by preventing pathogen colonization and/or limiting excessive inflammatory responses. Without being bound by theory, the probiotics down-regulate cytokine and chemokine production.
  • the biocompatible microsphere can be one or more of a biodegradable polymer, a non-biodegradable polymer, a metal, or a mixture thereof.
  • the biodegradable polymer can be selected from, but not limited to: dextran; dextranomoer; poly(lactic-co-glycolic acid) or PLGA; polycaprolactone or 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) copolymers; poly(glycolide)/poly(ethylene glycol) copolymer; poly(lactide-co-glycolide)/poly(ethylene glycol) copolymers; poly(lactic acid)/poly(ethylene glycol) copolymer; poly(lactic
  • the non-biodegradable polymer can be selected from, but not limited to, poly(ethylene vinyl acetate), poly(vinyl acetate), silicone polymers, polyurethanes, polysaccharides such as a cellulosic polymers and cellulose derivatives, acyl substituted cellulose acetates and derivatives thereof, copolymers of poly(ethylene glycol) and poly(butylene terephthalate), polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chorosulphonated polyolefins, polyethylene oxide, and copolymers and blends thereof.
  • the metal can be selected from, but not limited to, cobalt, chromium, gold, nickel, platinum, stainless steel, titanium, tantalum, nickel-titanium, and alloys and combinations thereof.
  • the microspheres are selected to facilitate the endurance and robustness of the probiotic biofilms are identified and characterized. It has been shown that probiotic biofilms formed on the biodegradable (and FDA approved) surface, poly (lactic-co-glycolic acid) (PLGA) yields biofilms that are superior at preventing pathogen translocation through the epithelial barrier. Other FDA approved or generally regarded as safe (GRAS) materials that can be used to create surfaces to grow biofilms are also examined. The results using biological effectiveness and durability in animal models and shelf life as the base criteria are prioritized. Finally, to further improve the effectiveness of the introduction and maintenance of the probiotic biofilm, prebiotic substances to the probiotic biofilm surface by way of diffusible cargo within the microspheres are provided.
  • the microspheres are partially or fully coated by a biofilm layer.
  • the layer can be from about about 0.5 micron to about 1 millimiter in depth, and ranges in between, e.g., about 1 micron to about 500 microns, about 1 micron to about 250 microns, about 1 micron to about 200 microns, about 1 micron to about 100 microns, about 1 micron to about 50 microns, about 1 micron to about 40 microns, about 1 micron to about 30 microns, about 2 micron to about 100 microns, about 2 microns to about 50 microns, about 2 microns to about 40 microns, about 2 microns to about 30 microns, about 3 microns to about 100 microns, about 3 microns to about 50 microns, about 3 microns to about 40 microns, about 3 microns to about 30 microns, about 5 microns to about 100 microns, about 5 microns to about 50 microns, about 5 microns to about 50
  • composition comprising, or alternatively consisting essentially of, or yet further consisting of, a biocompatible microsphere, a biofilm-generating probiotic bacterium and a prebiotic, wherein the prebiotic comprises, or alternatively consists essentially of, or yet further consists of a nutritional food source or supplement for the culturing and/or growth of the probiotic bacterium.
  • the composition can further comprise a prebiofilmic.
  • the prebiofilmic comprises a substance that supports biofilm formation and durability, specifically; the prebiofilmic can be a DNA binding polypeptide or protein and/or a DNABII polypeptide or protein.
  • the composition is frozen, for example flash frozen.
  • the composition is lyophilized or dried in powder form.
  • composition for administration as a suppository or in ingestible form (e.g., tablet).
  • the composition can further comprise a mixture of the above-noted microspheres, e.g., a mixture containing two or more probiotic bacterium and/or two or prebiofilmics and/or two or more nutritional and/or supplement to support the culturing and/or growth of the probiotic bacterium.
  • the prebiotic comprises a water-soluble carbohydrate selected from, but not limited to, one or more of inulin, oligofructose, fructo-oligosaccharide, galacto-oligosaccharide, glucose, maltose, maltodextrins, polydextrose, sucrose, fructose, lactose, isomaltulose, polyols, glycerol, and combinations thereof.
  • the composition further comprises a solid or a liquid carrier, such as a pharmaceutically acceptable carrier.
  • the prebiotic and prebiofilmic are selected in each composition to specifically support the growth of the probiotic bacterium.
  • the composition comprises an effective amount of sucrose, glycerol and optionally HU polypeptide or protein, to support the growth and maintenance of the probiotic when administered to the subject or patient.
  • Non-limiting examples of prebioflimic compositions include, without limitation, one or more of the polypeptides provided in the attached sequence listing, a c-terminal fragment thereof, or a n-terminal fragment thereof, or the additional strains and polypeptides and fragments thereof, such as the full length or the c-terminal fragment or the n-terminal fragment of those provided in Table 4, and equivalents of each thereof. Additional nutritional supplements for the support of other probiotic bacterium are disclosed in Bergey's Manual of Determinative Bacteriology, 9 th Ed, Ed. Holt et al.,WilliamsWilkins (1994),
  • Non-limiting examples of a probiotic bacterium for use in the composition includes, without limitation, one or more of L. acidophilus, L. crispatus, L. gasseri, group 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 or a combination thereof.
  • one or more bacterium can be combined in a single composition.
  • the probiotic bacterium is Lactobacillus reuteri that in a further aspect, expresses GTF protein. In other aspect it express GTFA protein.
  • the bacteria are available from commercial sources, such as the American Type Culture Collection (ATCC).
  • ATCC American Type Culture Collection
  • the one or more probiotic bacterium in the composition supports anti-bacterial immunity.
  • the one or more probiotic bacterium in the composition prevents pathogen colonization and/or limits excessive inflammatory responses by down-regulating cytokine and chemokine production.
  • the composition further comprises an agent, and the agent is selective against a pathogen, such as a competing pathogen.
  • the biocompatible microsphere comprises one or more of a biodegradable polymer, a non-biodegradable polymer, a metal, or a combination thereof.
  • the microsphere comprises a solid core.
  • the microsphere comprises a hollow core.
  • the prebiotic is encapsulated within the hollow core of the microsphere and can be released at high concentrations to just the adhered probiotic either due to the semi-permeable nature of the microsphere surface or via the gradual degradation of the microsphere.
  • the disclosure provides a composition comprising, or alternatively consisting essentially of, or yet further consisting of, a PGLA-biocompatible microsphere, one or more biofilm-generating probiotic bacterium, and a nutritional supplementation comprising one or more of sucrose or glycerol in an amount to support the growth of the probiotic bacterium.
  • the biofilm-generating probiotic bacterium may comprise Lactobacillus reuteri ( L. reiiteri”), that can optionally express GTF protein.
  • the composition may further comprise, or alternatively consist essentially of, or yet further consist of, an effective amount of IHF or HU polypeptide or protein.
  • the composition can further comprise a
  • pharmaceutically acceptable carrier or a biocompatible scaffold is optionally formulated as a suppository.
  • the size of the microsphere can range from about 0.5 microns to about 100 microns. In certain embodiments, the microsphere is less than about 100 microns in diameter. In other embodiments, the microsphere is less than about 50 microns, or less than about 40 microns, or less than about 30 microns, less than about 20 microns, less than about 10 microns, or less than about 5 microns, or less than 3 microns to 0.5 microns in diameter.
  • the microsphere is from about 0.5 microns to about 90 microns, or to about 80 microns, or to about 70 microns, or to about 60 microns, or to about 50 microns, or to about 40 microns, or to about 30 microns, or to about 20 microns, or about 10 microns, or about 5 microns, or about 3 microns, or about 2 microns, or about 1 micron, in diameter.
  • 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, microns in diameter.
  • the microsphere is a biodegradable polymer, non-limiting examples of such include: dextran, dextranomer; poly(lactic-co-glycolic acid)("PLGA”); polycaprolactone ("PLC”); chitosan; gelatin; DNA hydrogen; acetalated dextran;
  • the biodegradable polymer is poly(lactic-co-glycolic acid) or PLGA.
  • the microsphere comprises a non-biodegradable polymer.
  • non-biodegradable polymers include without limitation, of one or more of poly(ethylene vinyl acetate), poly(vinyl acetate), silicone polymers, polyurethane s, polysaccharides such as a cellulosic polymers and cellulose derivatives, acyl substituted cellulose acetates and derivatives thereof, copolymers of poly(ethylene glycol) and poly(butylene terephthalate), polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chorosulphonated polyolefins, polyethylene oxide, and copolymers and blends thereof.
  • the microsphere comprises a metal.
  • the metal can be selected from, but not limited to, one or more of cobalt, chromium, gold, nickel, platinum, stainless steel, titanium, tantalum, nickel-titanium, and alloys and combinations thereof.
  • compositions can be formulated as a frozen composition, e.g., flash frozen, dried or lyophilized for storage and/or transport.
  • the composition can administered alone or in combination with a carrier, such as a pharmaceutically acceptable carrier or a biocompatible scaffold.
  • a carrier such as a pharmaceutically acceptable carrier or a biocompatible scaffold.
  • Compositions of the invention may be conventionally administered rectally as a suppository, parenterally, by injection, for example, intravenously,
  • Oral formulations include such normally employed excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, 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% of active ingredient, preferably about 25% to about 70%.
  • compositions are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective for the disease or condition by treated.
  • the quantity to be administered depends on the subject to be treated. Precise amounts of the composition to be administered depend on the judgment of the practitioner. Suitable regimes for initial administration and boosters are also variable, but are typified by an initial administration followed by subsequent administrations.
  • compositions In many instances, it will be desirable to have multiple administrations of the compositions about, at most about or at least about 3, 4, 5, 6, 7, 8, 9, 10 days or more.
  • the administrations will normally range from 2 day to twelve week intervals, more usually from one to two week intervals.
  • Periodic boosters at intervals of 0.5-5 years, usually two years, may be desirable to maintain the condition of the immune system
  • additional pharmaceutical compositions are administered to a subject to support or augment the compositions as described herein.
  • Different aspects of the present invention involve administering an effective amount of the composition to a subject.
  • such compositions can be administered in combination with modifiers of the immune system.
  • Such compositions will generally be dissolved or dispersed in a
  • phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, or human.
  • pharmaceutically acceptable carrier includes 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 insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic and therapeutic compositions is contemplated.
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid poly(ethylene glycol), and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
  • the prevention of the action of undesirable microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • an effective amount of therapeutic composition is determined based on the intended goal.
  • unit dose or “dosage” refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and regimen.
  • the quantity to be administered depends on the result and/or protection desired. Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.
  • This disclosure also provides a method for preparing a composition as described herein, comprising, or alternatively consisting essentially of, or yet further consists of, the steps of admixing, contacting or culturing a biocompatible microsphere with a biofilm- generating probiotic bacterium and a prebiotic.
  • the method further comprises adding or admixing a prebiofilmic that supports the formation and growth of a biofilm by the bacterium.
  • Non-limiting examples of such include, one or more of a DNA binding polypeptide or protein and/or a DNABII polypeptide or protein.
  • the microspheres are contacted with a biofilm or placed into a culture that supports the growth of a biofilm on the surface of the microsphere. Additional components, as disclosed herein, can be further admixed with the microspheres, etc.
  • EHEC Enterohemorrhagic Escherichia coli
  • EPEC enteropathogenic E. coli
  • Citrobacter rodentium is a murine pathogen that is widely used to model human EPEC and EHEC infection, because mice are relatively resistant to both EPEC and EHEC.
  • C. rodentium results in colonic pathology that is nearly indistinguishable from that produced by EPEC and EHEC in humans (Borenshtein, M. et al.
  • enteric pathogens In order to cause disease, enteric pathogens must either adhere to or penetrate/invade host epithelial cells. Thus, interaction with epithelial cells is the first step in pathogenicity for all enteric pathogens, and this step can be studied through the use of A E pathogens by assessing colonic colonization and resultant pathology.
  • Colonization of A/E pathogens in the colon is dependent upon the composition of the intestinal microbiota. Inducing dysbiosis (the disruption of the native populations 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-lnduced Colitis, Infect Immun, 79: 1536-45) or by inducing an inflammatory response (Lupp, M.L. et al.
  • Colonic dysbiosis can further exacerbate the inflammatory response to the colonic pathogen (Wlodarska, B. et al. (2011) Antibiotic Treatment Alters the Colonic Mucus Layer and Predisposes the Host to Exacerbated Citrobacter rodentium-lnd ced Colitis, Infect Immun. 79: 1536-45), but even in the absence of pathogen challenge, dysbiosis can propagate inflammatory responses in genetically susceptible individuals, as evidenced by the findings of dysbiosis in patients with inflammatory bowel disease (Machiels et al.
  • a method for treating or preventing a disease in a subject comprising administering to a subject an effective amount of a composition as described above, to a subject in need of such treatment.
  • a subject intends an animal (e.g., murine, bovine, canine, feline, equine, simian) or a human.
  • Non-limiting diseases to be treated include, but not limited to the diseases and disorders listed above (and incorporated herein by reference), such as psychological disorders, such as depression or anxiety, enteric infectious disease, infection-induced colitis, traveler's diarrhea, inflammatory bowel disease (IBD), colitis, diarrheal illness, vaginosis, wound, burns, psoriasis, dermatitis, tooth decay, periodontitis, sinusitis, or any of chronic and/or recurrent disease that is caused by pathogenic bacteria displacing healthy bacteria or nectrotizing enterocolitis (NEC).
  • the compositions can be administered to support anti -bacterial immunity, enhancing or supporting the gastrointestinal barrier, or antagonizing disease-related bacterial infection.
  • the disease is vaginosis. In some embodiments, the disease is colitis or traveler's diarrhea.
  • the composition is specifically selected for the disease to be treated.
  • the composition further comprises a prebiofilmic.
  • the prebiofilmic comprises a DNA binding polypeptide or protein and/or a DNABII polypeptide or protein, e.g., an IHF or an HU, a fragment thereof and/or an equivalent of each thereof.
  • the composition is administered as a suppository.
  • the composition of the method is administered to provide from about 1 x 10 7 to about 1 x 10 9 CFU/ml of the biofilm-generating probiotic bacterium. 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.
  • a method of administering a probiotic comprising administering a dose of a composition as described above, comprising, or alternatively consisting essentially of, or yet consisting of, a biocompatible microsphere, a biofilm-generating probiotic bacterium, a prebiotic, and a prebiofilmic to a subject in need of such treatment.
  • the composition of the method is administered to provide from about 1 x 10 7 to about 1 x 10 9 CFU/ml of the biofilm-generating probiotic bacterium.
  • the composition is administered at about 6, 12, 18, 24, 36, 48, and 72 hours.
  • the composition is administered in a single dose.
  • kits containing one or more compositions as described herein comprises, or alternatively consists essentially of, or yet further consists of, a composition as described above, and instructions for use.
  • the kit comprises a microsphere and instructions to make the composition as described above.
  • the bacteria and prebiotic are also provided in the kit.
  • Example 1 To determine if L. reuteri in a biofilm state are superior to planktonic bacteria for establishment in the murine gut, L. reuteri was introduced via oral gavage, but instead of repeating the gavage daily, which is typically needed for retention of planktonic bacteria and for beneficial effects 15, 41, a single administration of L. reuteri was provided. The L.
  • reuteri were grown in biofilm cultures or biofilm grown on poly(lactic-co-glycolic acid) microspheres, such as PLGA, or other FDA approved and biodegradable microspheres (hydrolyzed into lactic acid and glycolic acid) with diameters ranging from 20-3 ⁇ (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).
  • poly(lactic-co-glycolic acid) microspheres such as PLGA, or other FDA approved and biodegradable microspheres (hydrolyzed into lactic acid and glycolic acid) with diameters ranging from 20-3 ⁇
  • Example 2 L. reuteri vs C. rodentium in vitro
  • C. rodentium treated with . reuteri biofilm showed a >2 fold decrease in CFU/ml compared to untreated (Table 1), regardless of the state of the introduced L. reuteri. More interesting, while all the L. reuteri proliferated during the 16 hour challenge, the L. reuteri introduced in the form of a biofilm yielded > 10-fold more CFUs than when added in planktonic form.
  • Example 3 L. reuteri vs C. rodentium in vivo
  • This example has provided strong evidence that the probiotics in the biofilm state provides a superior formulation to bacteria grown planktonically. It also provides one example of how to prepare these biofilms including the frequency of dosing. In addition, the example examines the nature of the biofilm itself to begin to determine why this state out performs planktonic bacteria. Finally, it examines the shelf life of the preparations as a prelude to reduction to practice in human hosts. Combined, this example identifies and characterizes the conditions and constituents for probiotic biofilm preparations.
  • L. reuteri forms a robust biofilm in vitro and that L. reuteri in a 24-hour biofilm establishes well in the mouse gut were shown. This Example varies the age of the biofilm to determine the optimal age for biofilm establishment.
  • L. reuteri In vivo L. reuteri biofilms. L. reuteri begins to attach almost immediately when exposed to a surface. After 6 hours sufficient biomass has been produced to be both visible and to start forming the classic biofilm structures (e.g., mushroom, Abee and Kuipers, (2011) Understanding Microbial Behavior within and Outside the Host to Improve Food
  • L. reuteri biofilms are isolated at about 6, 12, 18, 24, 36, 48 and 72 hours, that have been grown on PGLA microspheres with HU and calf thymus DNA (as described above) normalizing to CFUs (108) and introducing them by gavage into mice (9 per time point from triplicate experiments). Each mouse is assessed by counting total lactobacilli levels in fecal samples daily for 12 days (cultured on MRS agar).
  • this Example uses a real-time PCR method to assess 16S rRNA gene sequence copy numbers for the Lactobacillus genus (including some species of Shoeslla, Pediococcus, and Leuconostoc due to difficulties with primer specificity) and specifically for L. reuteri.
  • the 16S rRNA gene copy numbers is determined in the feces daily for 12 days, as well as in the colon, cecum, small intestine (including ileum, jejunum, and duodenum), and stomach (including the forestomach) using real-time PCR on Days 1, 3, 6, and 12 post-oral inoculation. 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 in comparison to sham or planktonic-treated mice is an indicator of durability and robustness.
  • L. reuteri grows in each to varying degrees.
  • the Example also varies the starting pH to about 5.5, 6, 6.5 or 7 as L. reuteri growth is favored under more acidic conditions. While L. reuteri can be grown microaerophilically under 5% C02, stressful conditions of times 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).
  • the Example varies the prebiofilmics of HU (about 0.1, 1, 10, 100 g ml) and calf thymus DNA (about 0.1, 1, 10, 100 ⁇ g/ml). All the aforementioned biofilms are assessed by CSLM with LIVE DEAD® staining in triplicate for height, average thickness and biomass as indicators of robust growth.
  • L. reuteri Dosing of L. reuteri; frequency and size. Rhe frequency and or size of dosing improves the durability and robustness of the introduction of L. reuteri are determined.
  • L. reuteri biofilms are grown on PLGA microspheres with added HU and calf thymus DNA for 24 hours (or an age condition as determined in Example 4.1 and 4.2).
  • L. reuteri biofilms are introduced to mice by oral gavage creating a matrix of varying the dose (10 7 , 10 8 and 10 9 CFUs) as well as the frequency (single dose, or daily dose up to 3 days) yielding 9 different conditions.
  • L. reuteri levels are assessed in vivo on Days 1, 3, 6, and 12 post gavage as outlined in Example 4.1. Nine mice (from triplicate experiments) for each condition at each time point are used. Sham mice with and without planktonic cells serve as controls.
  • L. reuteri biofilms can be dispersed by antisera to a DNABII family member (e.g., E. coli IHF).
  • a DNABII family member e.g., E. coli IHF
  • this Example tests the bacteria released (dispersed) due to anti-IHF treatment.
  • 24 hour L. reuteri biofilms (no added PLGA, HU or DNA so as to facilitate dispersal) grown in chamber slides are treated with anti-IHF. As the peak of dispersal is about 8 to 12 hours after treatment (Goodman, et al.
  • Biofilms are found to be superior for establishment, persistence and duration of probiotic bacteria in the gut. It is not just the biofilm per se that possesses superior features to planktonic bacteria but the bacteria that are dispersed from biofilms. In effect, the biofilm will act as a dispersed-bacteria generator. Indeed, physiologic differences in dispersed bacteria as compared to laboratory grown planktonic bacteria (e.g. in antibiotic sensitivity) have been observed.
  • Example 4.5 Shelf Life [0225] For reduction to practice and ease of use, L. reuteri preparations need to be in a sufficiently stable form.
  • L. reuteri biofilms have been flash frozen and found no diminution in CFUs and minimum inhibitory concentration or MIC (>2 mg/ml ampicillin; MIC for planktonic L. reuteri ⁇ 4 ⁇ g/ml) suggesting L. reuteri retains at least one property of its biofilm state, enhanced MIC.
  • Optimized L. reuteri biofilms (Example 4.1. to 4.3) for ambient air freezing to -20°C and -80°C with and without glycerol (a cryo-protectant; See also Example 2) as well as flash freezing to -80°C (placing storage tubes with fresh bacterial suspensions in dry ice-ethanol) are examined.
  • Desiccation Optimized L. reuteri biofilms (Example 4 1. to 4 3) via lyophilization after freezing using the optimized technique in Example 4.5 are examined. Desiccated bacteria are stored at room temperature for about 1 day, 1 week or 1 month and then rehydrated with the original biofilm volume of sterile distilled water at ambient room temperature to be used for introduction into mice by gavage. Nine mice from triplicate experiments are used with a similar number for controls using planktonic bacteria and optimized biofilm bacteria (Example 4.1 to 4.3). Each mouse is assessed as in Example 4.1.
  • L. reuteri ATCC23272
  • Additional strains of L. reuteri e.g. strain 100-23, ATCCPTA6475, ATCC55730
  • L. reuteri strains that are commercially available (Fleet® Pedia-LaxTM Probiotic YumsTM ⁇ lOOmillion CFU/tablet, L. reuteri Protectis®DSM 17938 and Gerber® Soothe Colic Drops ⁇ 100 million CFU/serving (5 drops, ⁇ 200ul), L. reuteri Protectis®DSM 17938) are examined. This Example finds that by dissolving each product in water and using them directly in in vitro competition experiments with C.
  • each product is shown to be no better than the strain of L. reuteri in planktonic form.
  • Example 5 Dentification And Characterization Of Biodegradable Surfaces And Pre-Biotic Substances To Facilitate The Endurance And Robustness Of The Probiotic Biofilms
  • PLGA microspheres are utilized as a surface to grow the biofilms, there are other FDA approved or GRAS biodegradable microspheres that may prove advantageous for the goals. As shown in Table 2, 5 additional types of microspheres are examined (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
  • DNA can be used as the microsphere material as it is the basis of the EPS for biofilms.
  • the cargo of PLGA is known to diffuse slowly or not even at all relative to the rate of microsphere hydrolysis (Fredenberg, et al. (201 1) The Mechanisms of Drug Release in Poly(Lactic-Co-Glycolic Acid)-Based Drug Delivery Systems— a Review, Int J Pharm, 415:34-52).
  • microspheres with prebiotic cargo were synthesized and evaluated for their ability to support L. reuteri growth in vitro and in vivo in the mouse models.
  • microspheres These cargos include, but not limited to, inulin, fructo-oligosaccharides, and galacto-oligosaccharides as they support lactobacilli growth.
  • microspheres with MRS media and/or glycerol are made, as the former is restrictive to Gram-negative bacteria some of which are pathogens and the latter stimulates reuterin production (an antimicrobial molecule believed to give L. reuteri an advantage against competing bacteria).
  • L. reuteri biofilm growth on these microspheres is performed on the conditions observed in Example 4 (or Example 5.1 with a variant microsphere) and is adjudicated by CSLM for height, thickness and biomass.
  • This example tests prebiofilmics in vitro.
  • prebiofilmics HU and DNA
  • PLGA microspheres and the microsphere types from Example 5.1
  • biofilms are grown under the conditions observed in Example 4 with microspheres synthesized in the presence of HU and or DNA (so as to be encapsulated in the interior of the microsphere) and are adjudicated by CSLM for height, thickness and biomass.
  • This example tests a combination of prebiotics and prebiofilmics in vitro.
  • a matrix of combinations of the two probiotic and two prebiofilmic cargos is created (all 16 combinations of two, all 4 combinations of 3, and the single combination of all 4 equaling 21 total combinations) to find the suitable prebiotics or prebiofilmics.
  • biofilms are grown under the conditions observed in Example 1 with PLGA microspheres (and the microsphere types from Example 5.1) synthesized in the presence of cargo and are adjudicated by CSLM for height, thickness and biomass.
  • This example tests optimized components in vivo. Conditions from Example 5.2 that yielded the biofilms are used for in vivo experiments. The four most promising conditions for PLGA microsphere cargo (or the two most promising PLGA and two most promising other type of microsphere from Example 5.1) are tested on nine mice each derived from triplicate experiments. Each mouse is assessed as in Example 4.1 on Days 1, 3, 6, and 12 post-Z. reuteri introduction. Sham mice (no bacteria) and planktonic bacteria serve as controls.
  • Microspheres containing various probiotic cargos to determine if they support pathogen biofilm growth are examined.
  • the microspheres containing prebiofilmics come into contact with a pathogen (i.e., C. rodentium strain DBS120 (pCRPl : :Tn5)) as well as probiotic.
  • a pathogen i.e., C. rodentium strain DBS120 (pCRPl : :Tn5)
  • This example tests pathogen impeding nutrients in vitro.
  • C. rodentium is grown in LB media and used to seed biofilms with PLGA and the
  • Biofilms is adjudicated by CSLM for height, thickness and biomass compared to empty PLGA microspheres.
  • This example tests pathogen impeding nutrients in vivo. Taking into consideration the results from in vitro biofilm data in Example 5.3, four cargos for C. rodentium biofilm growth and use them in vivo in mouse models are examined. Nine mice for each condition per time point (from triplicate experiments) are used with planktonic C. rodentium and sham (no bacteria) as controls. C. rodentium levels in the stool is determined via culture on all days post oral C. rodentium administration. On Days 1, 6, 12 and 24 post-oral C.
  • the colon is removed and transected longitudinally so that inflammatory cytokines ⁇ e.g., TNF-a), inflammatory mediators (e.g., inducible nitric oxide synthase (iNOS)), and chemokines (e.g., CCL2) can be assessed in half of the colon via real-time RT- PCR.
  • inflammatory cytokines e.g., TNF-a
  • inflammatory mediators e.g., inducible nitric oxide synthase (iNOS)
  • chemokines e.g., CCL2
  • immunohistochemistry is used to assess leukocyte infiltration into the colon (e.g., F4/80+ macrophages; myeloperoxidase (MPO)+
  • microsphere biofilm preparations can include alternative types of
  • biofilms (regardless of surface) are superior to planktonic bacteria at seeding probiotic colonization in vivo.
  • Non-limiting examples cargos include without limitation specific effectors of innate immunity that reduce inflammation, part of the process leading to dysbiosis.
  • microspheres can comprise conditioned media from L. reuteri as I. reuteri produce such substances.
  • other bacteria are within the scope of this disclosure, e.g., C. rodentium and L. reuteri, in general for pathologies due to dysbiosis.
  • Example 6 Characterization OfL. reuteri' s Capacity To Limit Or Displace The Murine Gut Enteropathogenic Bacterium C. rodentium.
  • Example 6.1 Testing Optimized L. reuteri Biofilm Growth Conditions In C. rodentium Challenge; Making Of L. reuteri
  • Controls include each bacterial species without the other under each condition (e.g., the addition of C. rodentium added to PLGA microspheres without L. reuteri in each chamber slide). All experiments are done in triplicate.
  • L. reuteri biofilm preparations for introduction into animals are prioritized based on the greatest retention or supremacy of L. reuteri observed.
  • L. reuteri is prepared based on any successes derived from Examples 4.1, 4.4 and 4.5.
  • L. reuteri biofilms are introduced 12 hours prior to oral challenge with C. rodentium.
  • Triplicate experiments are conducted for a final sample size of 9 mice for each condition and time point that are assessed at 1, 6, 12, and 24 days post-challenge (peak C. rodentium infection occurs at about Day 12).
  • C. rodentium levels in the stool are assessed and pathogen-induced colitis is assessed as in Example 5.3.
  • L. reuteri levels are also assessed as in Example 4.1.
  • controls include C. rodentium without L. reuteri and C. rodentium challenge plus planktonic L. reuteri.
  • rodentium (about 12, 24 or 36 hours after the final L. reuteri treatment).
  • mice from triplicate experiments.
  • Vehicle mice infected with C. rodentium and single planktonic L. reuteri serve as controls.
  • C. rodentium levels and pathogen-induced colitis are assessed on Days 1, 6, 12 and 24 post-challenge as in Example 5.3, with . reuteri levels assessed as in Example 4.1.
  • this Example determines how effective L. reuteri introduced in the form of a biofilm is as a prophylactic to C. rodentium challenge and as a treatment for extant C.
  • Pathogens included in an in vitro survey 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.,
  • ANOVA factor analysis of variance
  • Probiotics have been widely used for digestive health benefits, although few actually prevent pathogen colonization and reduce the inflammatory response.
  • the effects of probiotic bacteria can be significantly improved by the manner in which they are introduced into the host; specifically by growing them in the form of a biofilm.
  • the data suggest that colonization in vivo by the probiotic L. reuteri is greatly enhanced when grown as a biofilm compared to planktonic-grown cells.
  • L. reuteri was grown in the presence of a biodegradable surface (PLGA), colonization was also increased indicating that the conditions were optimized that allowed a vast improvement in regards to L. reuteri establishment within the host.
  • PLGA biodegradable surface
  • Probiotic microbes 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
  • prophylactic treatment with L. reuteri biofilms is assessed to determine if C. rodentium will prevent bacterium-induced sickness, anxiety-, and depressive-like behaviors.
  • Preparations of L. reuteri biofilms that are found to be superior in in vitro assays are administered to mice via oral gavage 12 hours prior to oral challenge with C. rodentium. Triplicate experiments are conducted for a final sample size of 9 mice for each condition and time point that are assessed at 1, 6, 12, and 24 days post-challenge (peak C. rodentium infection occurs about Day 12).
  • animal behavior is assessed for locomotor activity (such as on the open field test), anxietylike behavior (such as in the light:dark preference test and elevated plus maze), depressive- like behavior (such as on the tail suspension test and Porsolt forced swim task), and sickness behavior (such as with the sucrose preference test).
  • Blood serum cytokines associated with emotional and illness behavior e.g., IL- ⁇ / ⁇ and IL-6 are assessed on each day. Circulating corti coster one levels will also be assessed.
  • Neuronal activation in the brain, especially the paraventricular nucleus of the hypothalamus, are assessed using c-Fos immunoreactivity.
  • L. reuteri can be used as a therapeutic to treat C. rodentium-ind ced sickness, anxiety-like, and depressive-like behavior also is assessed.
  • the compositions are tested to determine whether treating an established C. rodentium infection will reduce sickness, anxiety-, and depressive-like behaviors.
  • Preparations of . reuteri that are found to be superior in in vitro assays are administered to mice via an oral gavage 12, 24, and/or 36 hours after oral challenge with C. rodentium.
  • animal behavior is assessed for locomotor activity (such as on the open field test), anxiety-like behavior (such as in the light:dark preference test and elevated plus maze), depressive-like behavior (such as on the tails suspension test and Porsolt forced swim task), and sickness behavior (such as with the sucrose preference test).
  • locomotor activity such as on the open field test
  • anxiety-like behavior such as in the light:dark preference test and elevated plus maze
  • depressive-like behavior such as on the tails suspension test and Porsolt forced swim task
  • sickness behavior such as with the sucrose preference test.
  • Circulating cytokines associated with emotional and illness behavior e.g., IL- ⁇ / ⁇ and IL-6 are assessed on each day.
  • Circulating corticosterone levels are also assessed.
  • Neuronal activation in the brain, especially the paraventricular nucleus of the hypothalamus are assessed using c-Fos immunoreactivity.
  • Probiotic administration may be beneficial in the prevention of NEC.
  • probiotics must be administered daily to achieve beneficial effects.
  • Applicants describe herein a novel probiotic delivery system in which the probiotics are grown as a biofilm on the surface of prebiotic-loaded biocompatible microspheres, allowing enhanced and more durable efficacy with only a single treatment.
  • NEC hypoxia/hypothermia/hypertonic feeds
  • Pups were sacrificed when clinical signs of NEC developed or by 96 hours after birth.
  • a verified histologic NEC injury grading system was used to measure the incidence and severity of NEC, with Grade 2 or greater injury considered to be consistent with NEC.
  • biocompatible microspheres reduces the incidence of NEC and therefore is an effective treatment.
  • the compositions as disclosed herein are prophylactic in their use in subjects in need of such treatment.
  • a dessication tolerance assay was used to test stability and viability of the bacteria combined with the microspheres.
  • the assay can generally be conducted by performing the following steps. To grow the bacteria culture, transfer 1ml to al .5ml of the culture to a microcentrifuge tube (1 tube per condition per time period to be tested). Add aboutlO ⁇ of hydrated microspheres, trehalose, or nothing to the tube. Incubate the tube for 30 minutes and then pellet the cells via centrifugation. Remove the supernatant and wash the pellet twice with sterile saline.
  • P. fluorescens and a proprietary Azospirillum sp. were placed after 90 days incubation at 40°C while on top of Drierite, a strong desiccant, and then rehydrated and tested for viability.
  • P. fluorescens with no microspheres shows a complete loss of colony forming units (CFU) after just one week in these conditions, whereas when incubated with cellulose microspheres, there are 10 5 viable cells after 90 days in these conditions.
  • Azospirillum sp. shows significant loss of CFUs after 30 days and complete loss after 90 days when grown without the microsphere formulation, however, when stored in harsh conditions with the microspheres, 10 6 CFU/ml of Azospirillum sp. are viable even after 90 days.
  • Microspheres filled with L. reuteri growth medium as cargo were utilized to provide a surface that leaches buffered nutrients to the bacteria for the formation of a biofilm that enhances survivability at low pH.
  • Bacterial cells with microspheres show over a 2 log increase in viable colony forming units compared to cells without microspheres after sitting in pH 2 gastric acid for 4 hours.
  • L. reuteri with microspheres show increased adherence to mouse colonic cells, addressing the problem of poor colonization and sustainability of orally administered bacteria.
  • the novel microsphere formulations not only increase survivability at low pH, but also contribute to colonization of beneficial bacteria in the gut, making L. reuteri a more efficient probiotic.
  • An acid tolerance protocol assay such as that used to generate the above information, can generally be conducted by performing the following steps. First, grow 5ml culture overnight at 37°C (5% C0 2 or anaerobically) and then dilute the culture 1 :2500 in a fresh medium. Transfer 500ml per condition per time period to be tested into a 48-well plate. Transfer ⁇ 10ul of hydrated microspheres or nothing into the well. Afterwards, incubate at 37°C 5% C0 2 (or anaerobically) for 20 hours overnight. At 20 hours, remove the spent media from the biofilm and replace with pH 2 gastric acid. At two and four hours, remove the acid from the biofilm and suspend cells by pipette mixing in the growth medium. Finally, serial dilute and plate the cells.
  • Microspheres filled with L. reuteri growth medium as cargo were utilized to provide a surface that leaches buffered nutrients to the bacteria for the formation of a biofilm that enhances survivability at low pH.
  • Bacterial cells with microspheres show over a 2 log increase in viable colony forming units compared to cells without microspheres after sitting in pH 2 gastric acid for 4 hours.
  • L. reuteri with microspheres show increased adherence to mouse colonic cells, addressing the problem of poor colonization and sustainability of orally administered bacteria.
  • a cellular adherence assay such as that used to generate the above information, can generally be conducted by performing the following steps. First, grow up a mammalian cell culture line and dilute to ⁇ 10 6 cells/ml. Transfer 500ul of the diluted mammalian cell lines to a 48-well plate. Then, grow to confluence (time varies, at least 16 hours) and grow the bacterial culture overnight. Afterwards, transfer 500ul of the bacterial culture to a 1.5ml microcentrifuge tube (1 tube per condition per time period). Pellet the bacterial cells via centrifugation and wash the pellet 2-3 times to remove all growth medium. Resuspend the pelleted bacteria in a cell line culture medium. Add either microspheres hydrated in a cell line culture medium, microspheres hydrated in MRS, or nothing to the suspended bacteria.
  • Example 13 Enhanced Probiotic Potential
  • the probiotic formulation comprises L. reuteri' s extracellular glucosyltransferase (GTF) protein, which in the strain of L. reuteri used in this study (DSM 20016, containing GTFW encoded by gtfW) (Leemhuis et al., 2013; Bai et al., 2015) catalyzes the formation of exopolysaccharides of glucose (glucans) from its sole known substrate maltose.
  • GTF proteins typically have a glucan binding domain that recognizes its own produced exopolysaccharide (Monchois et al., 1999; Kralj et al., 2004).
  • the GTF protein, its substrate, and resulting glucan product are highly strain- specific in L. reuteri; some are characterized as producing dextran (primarily a-1,6 linkages), mutan (primarily a- 1,3 linkages), or the aptly named reuteran (primarily a- 1,4 linkages) (Kralj et al., 2002; Kralj et al., 2004).
  • Cell aggregation, biofilm formation, and gut colonization are directly linked to the activity of GTFA in L. reuteri strain TMW1.106; inactivating gt/A significantly diminishes the ability of L. reuteri to aggregate, form biofilms, and colonize the GI tract in vivo (Walter et al., 2008).
  • Applicants' novel approach comprises the selection of dextranomer microspheres [a macroscopic porous microsphere that is sold commercially for size exclusion
  • Bacterial strains, plasmids and oligonucleotides used are listed in Table 5.
  • L. 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 hours at 37°C, 5% C0 2 .
  • Salmonella typhi strain JSG698) and Citrobacter rodentium (ATCC 51459) were grown in Lysogeny broth (LB, #63) at 37°C, 5% C0 2 .
  • 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) established with an atmosphere of 5% H 2 , 85% N 2 , and 10% C0 2 .
  • BHI brain-heart infusion
  • anaerobic chamber Thermo Forma Scientific, 1025 Anaerobic System, Hampton, NJ
  • DLD-1 ATCC CCL-221
  • human colonic cells were grown in RPMI medium supplemented with 10% fetal bovine serum at 37°C, 5% C0 2 .
  • FHs 74 Int human fetal small intestinal cells were grown in Hybri-Care medium (ATCC 46-X) supplemented with 30 ng/ml epidermal growth factor (EGF) and 10% fetal bovine serum at 37°C, 5% C0 2 .
  • the gtfW deletion strain (LMW500) was constructed by insertion of a chloramphenicol resistance cassette (Venereau et al.) into the gtfW open reading frame by allelic exchange as described previously (Mashburn-Warren et al., 2012).
  • lkb fragments upstream and downstream oigtfWv/ere amplified by PCR using oligos oSGl 082- 1083 and oSGl 084- 1085, followed by cloning into pFED760 (Mashburn-Warren et al., 2012) using Notl/Sall and Sall/Xhol restriction sites respectively.
  • the cat cassette was amplified from pEVP3 (Mashburn-Warren et al., 2012) using oligos LMW34-35, followed by cloning into pFED760 that contained the upstream and downstream fragments of gtfW using the Sail restriction site.
  • L. reuteri electrocompetent cells were prepared by growing 5 ml of culture in MRS at 37°C with 5% C0 2 until OD 6 oonm Of -1.0. Cells were then pelleted and resuspended in 10 ml of sterile cold 0.5M sucrose and 10% glycerol twice, followed by a final resuspension inlOO ⁇ sterile cold 0.5M sucrose and 10% glycerol.
  • plasmid was constructed by amplifying the promoter region 350 bp upstream of the gtfW start codon (including the native ribosome binding site) by PCR using oligos oSGl 102-1103. The resulting DNA fragment was inserted into pJCl 56 using the Xhol/Sall restriction sites.
  • the click beetle luciferase (CBluc) gene was amplified from the Streptococcus mutans strain IdhCBGSm (Merritt et al., 2016) using oligos oSGl 067- 1068 and inserted downstream of the gtfW promoter region in pJC156 using Sall/Notl restriction sites.
  • the resulting reporter plasmid pWAR501 was transformed into L. reuteri 23272 as described above to create the reporter strain LMW501.
  • the E. coli gtfW overexpression strain (LMW 502) was created by amplifying the L. reuteri gtfW open reading frame (including the stop codon) using primers oSGl 120-1 126. The resulting DNA fragment was inserted into pTXB l (New England BioLabs, Ipswich, MA) using Nhel/Sapl restriction sites. The resulting plasmid, pWAR502 was then transformed into the E. coli expression strain ER2566 (New England BioLabs, Ipswich, MA) and selected on Luria-Bertani agar containing 100 ⁇ g/ml ampicillin and confirmed by DNA sequencing. This strain allows the overexpression of tagless GTFW protein.
  • a reporter plasmid was constructed by amplifying the promoter region 250 bp upstream of the elongation factor Tu (EF-Tu) start codon (including the native ribosome binding site) by PCR using oligos oSGl 069- 1070. The resulting DNA fragment was inserted into pJC 156 using the Xhol/Sall restriction sites. The click beetle luciferase (CBluc) gene was amplified from the S.
  • Tu elongation factor Tu
  • CBluc click beetle luciferase
  • Anhydrous dextranomer microspheres (DMs; Sephadex® G-25 Superfine) were purchased from GE Healthcare Life Sciences (Pittsburgh, PA).
  • Anhydrous cellulose microspheres (CMs; Cellulobeads D50) were obtained from Kobo Products, Inc. (South Plainfield, NJ).
  • Anhydrous microspheres were hydrated in growth medium or water at 50 mg/ml then autoclaved for 20 minutes. For conditions with microspheres that contained maltose, sucrose, fructose, or glucose only, microspheres previously autoclaved in water were removed from solution on a vacuum filter apparatus and approximately 50 mg were collected via sterile loop into 1ml of filter-sterilized 1M solution of the sugar (see FIG. 13). The microsphere mixture was then vortexed vigorously and incubated for 24 hours at room temperature to reach equilibrium. Atty. Dkt. No.: 106887-0172
  • microspheres loaded with water, 1M maltose, 1M sucrose, 1M glucose, or 1M fructose were removed from solution on a vacuum filter apparatus and collected via a 10 ⁇ sterile loop. Approximately 5 mg of hydrated
  • microspheres were then added to 1 ml of 2 x 10 9 CFU Z,. reuteri from an overnight culture that had previously been pelleted by centrifugation at 3220 x g for 10 minutes, washed twice with sterile 0.9% saline, and resuspended in 1 ml sterile saline.
  • 2 x 10 9 CFU of bacteria were resuspended in 1 ml RPMI instead of saline.
  • 10 ⁇ of cargo was added to 1 ml of bacteria either in sterile saline or RPMI.
  • the bacteria and microsphere mixture were incubated together at room temperature for 30 minutes (unless otherwise stated) to facilitate bacterial adherence and biofilm formation on the microsphere surface prior to use in assays.
  • L. reuteri culture was grown and prepared as described above and incubated with microspheres filled with either: water, 1M maltose, 1M sucrose, 1M fructose, or 1M glucose.
  • 300 ⁇ of bacteria from an overnight culture containing ⁇ 2 x 10 9 CFU) in sterile saline and 5 mg of microspheres were combined and incubated for 5 minutes in a Micro Bio-Spin column (BioRad, Hercules, CA) (see FIG. 14). The columns were then centrifuged (100 x g) for 1 minute.
  • the flow-through was serially diluted and plated to calculate the total number of non-adhered bacteria, and this value was subtracted from the total number of starting bacteria to derive the total number of adhered bacteria.
  • a control preparation that consisted of bacteria with no microspheres was used.
  • the reporter strain LMW501 was grown at 37°C with 5% C0 2 in MRS or MRS containing 3% glucose, sucrose, fructose, or maltose and optical densities (ODeoonm) of the cultures were measured throughout growth using an Epoch Microplate Spectrophotometer (BioTek Instruments Inc., Winooski, VT).
  • S. mutans was grown in Todd Hewitt Broth at 37°C with 5% C0 2 until early log phase (OD 6 oonm ⁇ 0.3), L. reuteri WT and the AgtfW mutant were grown in MRS at 37°C with 5% C0 2 until late log phase (OD 60 onm ⁇ 1.0) for optimal gtf expression, and the E. coli gtfW overexpression strain was grown in Luria-Bertani broth at 37°C shaking (200 rpm) until mid- log phase (OD 6 oonm -0.4) followed by the addition of 1 mM IPTG to induce gtfW expression and was then grown at 37°C shaking for an additional 2 hours.
  • the rate of cargo diffusion out of the microspheres was determined by tracking crystal violet, a small molecular weight dye (407.979 g/mol) (Fisher Scientific, Hampton, NJ).
  • the microspheres were loaded with a 0.1% solution of crystal violet by incubating 20 mg of microspheres in 1 ml of 0.1% crystal violet solution either with or without added glycerol (40% or 80% v/v) overnight to reduce the diffusion rate by increasing viscosity. After 16 hours, excess crystal violet solution was removed from the microspheres as described above using a vacuum filter apparatus.
  • the crystal violet-loaded microspheres were then placed into 1 ml of water, and aliquots of water were removed and analyzed for diffusion of crystal violet into solution using an Epoch Microplate Spectrophotometer (BioTek, Winooski, VT) at OD 5g0 nm every hour for 16 hours. Percent diffusion was calculated using the equivalent amount of crystal violet within the microspheres (10 ⁇ ) in water as a control equivalent to 100% cargo diffusion.
  • L. reuteri Production of reuterin by L. reuteri was measured via a quantitative colorimetric assay (Cadieux et al., 2008). As this assay did not differentiate between similar aldehyde products, measurements included 3-HPA and any potential derivatives, such as acrolein and 3-HPA hydrate.
  • L. reuteri was grown overnight in MRS as described above, 1 ml aliquots of 2 x 10 9 CFU were pelleted at 3220 x g for 10 minutes, washed twice with sterile saline, and resuspended in either 1 ml of sterile saline or 1 ml sterile saline containing 2% v/v glycerol.
  • DM containing 0%, 2%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% glycerol were prepared as described above for other cargo, and added to the resuspended L. reuteri in saline (so that the only source of glycerol available for reuterin production was via the microsphere cargo) for 1 hour at 37°C. Cells were then pelleted again and the reuterin-containing supernatant was removed, filtered through a 0.45 ⁇ filter, and assayed for reuterin as described in Cadieux et al., 2008 without modification. A standard curve using reuterin at known concentrations was used to extrapolate bacterial-produced reuterin concentrations from DM-glycerol and the 2% v/v glycerol control experimental conditions.
  • Gastric acid equivalent is a modified version of synthetic gastric fluid (Cotter et al., 2001), composed of 0.1M HC1, 0.1M NaCl, and 0.01M KC1, with pH adjusted to 2 using 0.1M NaOH.
  • 1 ml of 2 x 10 9 CFU of L. reuteri from a fresh overnight culture were pelleted at 3220 x g for 10 minutes, washed twice with sterile saline, and resuspended in 1 ml 0.9% sterile saline.
  • the cells were incubated for 30 minutes with approximately 5 mg of loaded or unloaded microspheres as described above, and the bacteria-microsphere mixture was diluted 1 : 100 directly into gastric acid equivalent. Aliquots of the inoculated acid solution were mixed, serially diluted, and plated at hourly time points for 4 hours to determine the number of viable bacteria. Bacteria without microspheres were used as a control.
  • DLD-1 colonic cells and FHs 74 small intestinal cells were cultured as described above.
  • the growth medium was removed, cells were washed twice with sterile phosphate buffered saline (PBS), and trypsin- EDTA (0.25%) was added for 10 minutes at 37°C to dislodge the cells from the culture flask surface.
  • Total epithelial cells were counted using a hemacytometer (Hausser Scientific, Horsham, PA). Cells were then diluted to a concentration of 5 x 10 5 cells/ml and 1 ml per well was seeded into a 24-well plate and incubated at 37°C, 5% CO 2 .
  • the spent medium was removed and replaced with 1 ml of RPMI or Hybri-Care medium containing 2 x 10 9 CFU of L. reuteri alone, L. reuteri with 5 mg water-filled DMs, L. reuteri with 5 mg sucrose-filled DMs, or L. reuteri with 5 mg maltose-filled DMs.
  • the spent medium was removed and the well was washed with 1 ml of sterile PBS 3 times to remove non-adhered bacteria.
  • Example 13.12 Mucin Adherence Assay
  • Mucin agar plates were created using porcine stomach mucin (Sigma-Aldrich, St. Louis, MO). Mucin agar plates contained 2% mucin and 0.8% agar to simulate the consistency of the mucus layer found in vivo (Macfarlane et al., 2005; Van den Abbeele et al., 2009). To assess L. reuteri' s ability to bind mucin, 2 x 10 9 CFU of L.
  • reuteri that contained a plasmid that encoded expression of the click beetle luciferase enzyme either planktonically or bound to 5 mg DM-water, DM-sucrose, or DM-maltose were incubated on both mucin agar and agar without mucin stationary at room temperature. After 60 minutes, the non-adhered 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 adhered bacteria.
  • D-luciferin Sigma-Aldrich, St. Louis, MO
  • Relative luminosity generated from the bacteria on the plates was measured using a FluorChem E system (ProteinSimple, San Jose, CA) with a 20 minute exposure setting. To assess the number of bacteria bound to the mucin within the plate (and not any background binding that may occur to the agar within the plate), the amount of luminescent signal from the agar-only plates was subtracted from the mucin agar plates.
  • DLD-1 glutaraldehyde, 4.0% acetic acid, and 0.1M phosphate buffer at pH 7.4 (Devaraj et al., 2015). All microscopy was performed on samples in Nunc Lab-Tek 8-well borosilicate chamber slides (Fisher Scientific, Hampton, NJ).
  • DLD-1 epithelial cells DLD-1 was stained with 4', 6-Diamidino-2-Phenylindole (DAPI, Life Technologies, Carlsbad, CA), L. reuteri was stained with carboxy fluorescein succinimidyl ester (CFSE, Life Technologies, Carlsbad, CA).
  • reuteri were diluted into fresh MRS growth medium to 0.01 OD 6 oonm, incubated at 37°C 5% C0 2 for 2.5 hours until reaching 0.65 OD6oonm, diluted 1 :2500 into either MRS, MRS + 3% sucrose, or MRS + 3% maltose, seeded into 8-well borosilicate chamber slides and incubated for 1, 3, or 6 hours at 37°C 5% CO 2 .
  • the bacteria were stained for viability with LIVE DEAD stain, fixed, visualized via confocal microscopy, and quantified via COMSTAT analysis of the fluorescent signal.
  • Samples were then washed with double distilled water and stained with a 1% solution of osmium tetroxide (Sigma-Aldrich, St. Louis, MO) in 0.1M phosphate buffer (pH 7.2) for 1 hour, washed for 5 minutes, stained with a 1% solution of thiocarbohydrazide (Sigma-Aldrich, St. Louis, MO), washed for 5 minutes, and further stained with 1% osmium tetroxide for 30 minutes.
  • osmium tetroxide Sigma-Aldrich, St. Louis, MO
  • Samples were then dehydrated using a graded series of ethanol: 25% ethanol for 15 minutes, 50% ethanol for 15 minutes, 70% ethanol for 30 minutes, 95% ethanol for 15 minutes (twice), 100% ethanol (twice), a 1 : 1 mixture of 100% ethanol to 100% hexamethyldisilazane (HMDS, Sigma-Aldrich, St. Louis, MO) for 100 minutes, 100% HMDS for 15 minutes, and a final immersion in 100% HMDS that was allowed to air dry overnight.
  • Dehydrated sample coverslips were then mounted onto 15mm diameter metal SEM specimen stubs (Electron Microscopy Sciences, Hatfield, PA) using colloidal silver (Electron Microscopy Sciences, Hatfield, PA).
  • Example 13.15 Statistical Analysis [0295] All experiments were conducted a minimum of three times and statistical analysis was performed via a Student's ⁇ -test using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA), wherein a P-value less than 0.05 was accepted as significant.
  • Example 13.16 Maltose or Sucrose within the Lumen of DMs Improved L. reuteri Adherence to DMs in a GTF-Dependent Manner
  • DMs Dextranomer microspheres
  • SYTO 9 Dextranomer microspheres
  • CLSM confocal laser scanning microscopy
  • L. reuteri does not bind to CMs
  • binding to DMs was GTFW-dependent and further, that inclusion of maltose or sucrose significantly enhanced the binding of L reuteri to DMs.
  • a mutant strain of L. reuteri was created with a chloramphenicol resistance gene inserted in place of the gtfW gene. As shown in FIG.
  • the AgtfW strain was not able to bind to DMs as effectively as the wild type (WT) in the spin column assay, regardless of the cargo within the DM lumen.
  • WT wild type
  • the WT displayed a significantly more robust biofilm with greater biomass compared to the gtfW mutant under every condition, with significantly more cells present when sucrose or maltose was in the growth medium (FIGS. 16A, 16C & 16D)
  • Lactobacillus rhamnosus GG a Gram-positive bacterium commonly found in the genitourinary system and sold commercially as a probiotic
  • Salmonella typhi a Gram- negative bacterium responsible for typhoid fever in humans
  • Citrobacter rodentium a Gram- negative bacterium that causes colitis in rodents
  • Clostridium difficile a Gram-positive spore-forming bacterium that can cause severe colitis and recurring infections in humans.
  • FIG. 7C all of the non-GTF expressing bacteria showed minimal adherence to DMs, regardless of cargo present within the DM lumen.
  • Example 13.17 Diffusion of Cargo from DMs
  • the rate of diffusion is dependent upon the size of the microsphere, the mass of the solute, and the viscosity of the diluent.
  • DMs were filled with crystal violet, a small molecular weight stain (407.979 g/mol), and the diffusion rate of the dye out of the DMs was tested with and without changing the viscosity of the solution in the DM lumen.
  • the crystal violet diffused out of the DM lumen with a half-life of ⁇ 6 hours.
  • the viscosity was increased by adding 40% glycerol, the half-life of release was increased to ⁇ 8 hours.
  • the half-life of crystal violet release was further enhanced to 12 hours. By 16 hours >95% of all of the crystal violet had been released under all tested conditions.
  • Example 13.18 L. reuteri Produced Reuterin From Glycerol-Loaded Microspheres
  • L. reuterf s function as a probiotic bacterium is its ability to compete with pathogenic bacteria within the host potentially via production of antimicrobials e.g. extracellular reuterin (Cleusix et al., 2007; Spinier et al., 2008). Due to limited glycerol availability, suboptimal endogenous concentrations of glycerol in the GI tract would likely limit adequate reuterin production. In order to obviate the need to provide high levels of glycerol to satisfy L. reuteri ' s optimal needs, Applicants provided targeted delivery of glycerol directly to the bacteria attached to the surface of DMs.
  • CFU hourly colony forming units
  • reuteri in saline did result in a steady loss of viable CFU over time, though there was no difference in viability between the DM-water and DM-80% glycerol over this time, suggesting the loss of CFU was not due to any potentially toxic compounds, such as reuterin or acrolein, from glycerol fermentation (FIG. 18).
  • acrolein in particular is known to be toxic to humans and is a byproduct of reuterin production
  • the amount of acrolein that could possibly be produced via this formulation is a nominal ⁇ 6 ⁇ g (for reference, the World Health Organization recommends less than 7.5 g kg body weight per day) (Gomes et al., 2002). From these results and the data presented in FIG. 8, Applicants suggest that DMs loaded with glycerol would have two beneficial effects in vivo, namely slowing the release of beneficial cargo and providing a substrate for reuterin production.
  • Example 13.19 L. reuteri produced histamine from L-histadine-loaded microspheres
  • Histamine produced by L. reuteri has previously been shown to inhibit proinflammatory cytokines such as TNF via H 2 receptors and reduce colitis in an animal model (Thomas et al., 2012; Gao et al., 2015).
  • the microsphere formulations described herein provide a unique method for delivery of the histamine precursor substrate L-histidine to L. reuteri.
  • DMs were filled with 30 mg/ml and 4 mg/ml L-histidine and measured the amount of histamine produced by the bacteria when the only source of L- histidine was via diffusion out of the DMs. As shown in FIG.
  • DM- L-histidine (4 mg/ml) resulted in histamine levels only slightly lower than those produced when bacteria were incubated in 4 mg/ml L-histidine solution without DMs.
  • the amount of histamine produced was 6-7 times greater than the lower 4 mg/ml concentration, consistent with the DM-L-histidine (30 mg/ml) providing ⁇ 7 times more L-histidine than the DM-L-histidine (4 mg/ml) (FIG. 9).
  • cargo relevant DM cargo substrates such as maltose and glycerol, would negatively affect histamine production was also tested.
  • Example 13.21 Microspheres Promote L. reuteri Adherence to Human Intestinal Epithelial Cells
  • This example investigated what effect the DMs, the DM luminal cargo and the product of the gtjW gene have on the relative adherence of L. reuteri when delivered as planktonic cells or as biofilms on DMs to the human intestinal cell lines DLD-1 (adult human colonic epithelial cells) and FHs 74 Int (3-4 months gestation, small intestine epithelial cells) in vitro.
  • DLD-1 adult human colonic epithelial cells
  • FHs 74 Int 3-4 months gestation, small intestine epithelial cells
  • mucin adherence is not GTF-dependent, but rather controlled by specific mucin-binding proteins (Miyoshi et al., 2006, Lukic et al., 2012), it was anticipated that being bound to DMs would not have an effect on the ability of L.
  • DMs have been used in medical products that are left in the body for long periods of time (years) with no ill effects (Hoy, 2012), such as with Debrisan®, a cicatrizant wound dressing (Jacobsson et al., 1976), Deflux®, a bulking gel used to treat vesicoureteral reflux (VUR) in children (Stenberg and Lackgren, 1995), and SolestaTM, a bulking gel injected submucosaly into the anal canal to treat fecal incontinence (Hoy, 2012).
  • the results described herein show a small subset of possible beneficial cargos that can be placed into the DM lumen for utilization by L.
  • reuteri and for many applications one can match the lumen cargo precursor to the desired L. reuteri- produced effect (e.g. reuterin and histamine. Moreover, this formulation obviates recombinant versions of probiotics, an approach not currently approved by the FDA
  • An exciting feature of our novel formulation is the ability to directly deliver beneficial compounds to the probiotic bacteria that are adhered to the DM surface as a biofilm (FIG. 22B).
  • beneficial compounds prebiotics
  • 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).
  • the probiotic bacterium L. reuteri is now delivered: (1) in association with DMs to which it adheres in greater numbers; (2) in the form of a biofilm which confers resistance to clearance; (3) along with a cargo of nutrients that promotes bacterial growth; (4) with cargos that promote production of the antimicrobial reuterin or histamine; (5) in a format that is resistant to acid-mediated killing thus promoting improved survival during transit through the acidic stomach and (6) in a manner that appeared to better support adherence to intestinal epithelial cells and thus likely to promote persistence in the gut.
  • maltose as cargo have particular value for several reasons; it is the substrate for this strain of L. reuterf s glucosyltransferase (GTFW) (Leemhuis et al., 2013; Bai et al., 2015), induces L. reuteri to aggregate in a GTF-dependent manner (Walter et al., 2008), and causes L. reuteri to grow significantly faster and to a higher cell density (CFU/ml).
  • GTFW L. reuterf s glucosyltransferase
  • S. mutans and L. reuteri GTF proteins are very similar in sequence and structure. Sucrose is the sole substrate for S. mutans and most Z. reuteri GTF proteins (Tieking et al., 2005; Walter et al., 2008), and sucrose has previously been shown to cause L. reuteri cultures to aggregate rapidly in a GTF-dependent manner (Walter et al., 2008).
  • sucrose to induce GTFW dependent adhesion is likely due to GTFW acting as an adhesin to DMs (via the glucan binding domain) and sucrose' s ability to induce gtfW expression (FIG. 15A).
  • sucrose failure of sucrose to affect L. reuteri adherence to CMs (cross-linked glucan with variant glycosidic linkages) supports this notion.
  • Sucrose- dependent biofilm formation has previously been linked to two-component regulatory systems in the rodent strain 100-23 of L. reuteri (Frese et al., 2011 ; Su and Ganzle, 2014); however, the genes necessary for this phenomenon appear to be absent in the human-derived strain of L.
  • sucrose is a preferred carbon source of the L. reuteri used in this study via its sucrose phophorylase mediated metabolism (Ganzle and Follador, 2012) it was not surprising that sucrose had a positive impact on biofilm formation and increased adherence to DMs and is likely due to the increased doubling time of L. reuteri in the presence of sucrose.
  • glucose a carbon source but not a gtfW inducer or GTFW substrate
  • fructose an inducer of gtfW, but not a carbon source
  • L. reuteri' s survivability and sustainability within the host can be improved by delivering L. reuteri as a biofilm on the surface of DMs that contain beneficial cargo.
  • beneficial cargo With more viable bacteria available after low pH challenge and supporting increased adherence to intestinal epithelial cells, the resulting expansion of probiotic bacteria available within the host should have an increased potentially beneficial effect.
  • targeted nutrients and substrates can be directly delivered to the bacteria adhered on the DM surface, which has broad-reaching implications for the type of compounds that can be co- delivered with orally consumed L. reuteri, which to date have been limited to carbohydrates that are indigestible by the host.
  • rat pups were delivered prematurely, given a single enteral Lr treatment, and subjected to experimental NEC (hypercaloric feeds/hypoxia/hypothermia). Pups were sacrificed 96h post-delivery or when clinical NEC developed. Tissue was harvested for histologic evaluation and measurement of inflammatory markers. Intestinal mucosal barrier integrity was assessed by serum levels of enterally- administered FITC-dextran. A bioluminescent strain of Lr was constructed to assess persistence in the GI tract. A GtfW-deficient strain of Lr was developed to assess the role of biofilm formation.
  • Lr adhered to sucrose- or maltose-loaded DM significantly reduced experimental NEC compared to Lr adhered to unloaded DM.
  • Lr adhered to sucrose- or maltose-loaded DM improved survival, decreased intestinal permeability, and reduced intestinal inflammation.
  • Neonatal Rat Model of Experimental NEC All animal studies were conducted in compliance with protocol #AR15-00012 approved by the IACUC of The Research Institute at Nationalwide Children's Hospital. Sprague-Dawley rat pups at 20.5 days gestational age were delivered from timed-pregnant dams (Envigo, Indianapolis, IN) via cesarean section under CO 2 anesthesia. Immediately after delivery, pups were randomized into experimental groups that received a single enteral Lr or control treatment via gastric gavage.
  • pups were subjected to repeated episodes of: 1) hypertonic, hypercaloric formula feeds via orogastric gavage five times daily with 15 g Similac 60/40 (Abbott Nutrition, Columbus, Ohio) in 75 mL of Esbilac (Pet-Ag, New Hampshire, IL), providing a combined 836.8 kJ/kg/day; 2) three episodes of hypoxia and hypothermia each day (placement in a chamber of N 2 gas calibrated to Fi0 2 ⁇ 1.5% for 90 seconds directly followed by placement in a 4°C environment for 10 min); and 3) gastric gavage of 2 mg/kg lipopolysaccharide (LPS, Sigma- Aldrich, St. Louis, MO) on the first day of life. Between each of these episodes, pups were housed in an incubator at 35°C. Breastfed control pups were placed with a surrogate dam immediately after cesarean delivery and were not exposed to experimental stress.
  • Similac 60/40 Abbott Nutrition, Columbus, Ohio
  • pups were subjected to the experimental NEC protocol previously described.
  • signs of NEC developed (bloody stools, severe abdominal distention, lethargy, respiratory distress, cyanosis) pups were sacrificed. All remaining pups were sacrificed 96 h after delivery.
  • intestinal tissue was harvested and fixed in 10% formalin for 24 h. Fixed tissue was paraffin-embedded and then hematoxylin and eosin (H&E)-stained transverse sections were prepared. Two independent observers graded each section in a blinded fashion using an established histologic injury grading scale initially established by Caplan et al.
  • Histologic injury was classified as: grade 0, no visible histological villus damage; grade 1, distal villus enterocyte detachment; grade 2, sloughing of enterocytes to the mid-villus level; grade 3, loss of entire villus with preservation of the crypts; and grade 4, transmural necrosis (FIG. 22A).
  • Experimental NEC was defined as an injury score of grade 2 or higher.
  • Pups were then subjected to the experimental NEC protocol for 48 h, at which time each received 1500 mg/kg of fluorescein isothiocyanate (FITC) labeled dextran (FD70, molecular weight 70,000) (Sigma-Aldrich Inc., St. Louis, MO) suspended in sterile PBS via orogastric gavage. Pups were sacrificed 4 h later and serum collected into BD Microtainer SST tubes (Becton, Dickinson and Company, Franklin Lakes, NJ). Serum was extracted and fluorescence measured with a fluorescent plate reader (SpectraMax M2, Molecular Devices, Sunnyvale, CA) using a 492/518 nm filter set. The plasma concentration of FD70 for each pup was then extrapolated using a standard curve generated from a 1 :2 serial dilution of a known FD70 concentration.
  • FITC fluorescein isothiocyanate
  • Lr was originally isolated from human breast milk (Ghouri et al., 2014) and is present in healthy human intestine (Gomes et al., 2002; Gustave et al., 2013).
  • Human-derived Lr strains belong to two distinct clades, clade II and clade VI (based on multi-locus sequencing), with only clade II strains possessing both anti-inflammatory and anti-microbial capabilities.
  • the strain of Lr used for our current studies was clade II Lr ATCC23272 (also known as DSM 20016), and was originally isolated from the feces of a healthy human (Hall- Stoodley et al., 2004).
  • Lr Some clade II strains of Lr, including ATCC23272, can down-regulate both cytokine and chemokine production by colonic epithelial cells stimulated with C. rodentium (Heydorn et al., 2000; Higgins et al., 1999) Lr has also been shown to reduce intestinal inflammation in both juvenile and adult animals (Hoy, 2012; Ito et al., 2008).
  • clade II strains of Lr produce antimicrobial compounds, the best characterized of which is reuterin (Jacobsson et al., 1976), which is derived from the substrate glycerol.
  • Reuterin is a potent anti-microbial compound that inhibits the growth of numerous pathogenic microorganisms such as Gram-positive bacteria, Gram-negative bacteria, fungi, and protozoa (Johnston et al., 2012).
  • clade II strains readily form a biofilm, a community architecture of bacteria adhered to a surface, where the bacteria are encased in a self-produced matrix of extracellular polymeric substance (EPS).
  • EPS extracellular polymeric substance
  • Lr has great affinity for the cross-linked dextran of DM, which results in excellent binding and subsequent biofilm formation (Eaton et al., 201 1). For these reasons, along with the accumulating evidence that Lr is beneficial in human diseases such as colic (Justice et al, 2012), diarrhea (Kailasapathy, 2014), IgE-mediated eczema (Kralj et al., 2004), and NEC (Kralj et al., 2002), Lr was chosen for use in the current experiments.
  • DM are biodegradable, non-immunogenic, non-mutagenic, non-allergenic, and Generally Recognized As Safe (GRAS) by the FDA. They have been used in numerous FDA- approved medical products to date, including SolestaTM, a bulking gel injected submucosally in the anal canal for treatment of fecal incontinence (Lebeis et al., 2008), Debrisan®, a cicatrizant wound dressing (Leemhuis et al., 2013), and Deflux®, a bulking gel used to treat vesicoureteral reflux (Lin et al., 2008). These long-standing uses of DM provide evidence for safety in human administration.
  • SolestaTM a bulking gel injected submucosally in the anal canal for treatment of fecal incontinence (Lebeis et al., 2008), Debrisan®, a cicatrizant wound dressing (Leemhuis et al
  • the DM lumen can be filled with compounds useful to Lr but limited in vivo, which diffuse over time directly to Lr adhered to DM ⁇ Lr + DM) as they transit the GI tract after enteral administration.
  • Changes in the microbial community such as the increasing prevalence of
  • Proteobacteria which includes many commonly observed Gram-negative pathogens 2 have been reported in infants prior to the onset of NEC (Lukic et al., 2012).
  • NEC Gram-negative pathogens
  • PLGA-based nanoparticles an overview of biomedical applications. J Control Release 161(2), 505-522. doi: 10.1016/j .jconrel.2012.01.043.
  • Acrolein contributes strongly to antimicrobial and heterocyclic amine transformation activities of reuterin. Sci Rep 6, 36246. doi: 10.1038/srep36246.
  • Map A mediates the adhesion of Lactobacillus reuteri to Caco-2 human intestinal epithelial cells. Biosci Biotechnol Biochem 70(7), 1622-1628. doi:
  • Glucosyltransferase A (GtfA) and inulosucrase (Inu) of Lactobacillus reuteri TMW1.106 contribute to cell aggregation, in vitro biofilm formation, and colonization of the mouse gastrointestinal tract.
  • Q. Xia, et al. Quantitative Analysis of Intestinal Bacterial Populations from Term Infants Fed Formula Supplemented with Fructo-Oligosaccharides, J Pediatr Gastroenterol Nutr, 55 (2012), 314-20.
  • Seq. ID NO. 1 Full Length Wild type (wt) 86-028NP Haemophilus influenzae IhfA;
  • Seq. ID NO. 2 Full Length wt 86-028NP Haemophilus influenzae HU, Genbank accession No. : YP_248142.1, last accessed March 21, 2011 : MRFVTIFINHAFNSSQVRLSFAQFLR QIRKDTFKESNFLFNRRYKFMNKTDLIDAIANAAELNKKQAKAALEATLDAITASLK EGEPVQLIGFGTFKVNERAARTGRNPQTGAEIQIAASKVPAFVSGKALKDAIK
  • Seq. ID NO. 3 Full Length wt R2846 Haemophilus influenzae IhfA, Genbank accession No. : AD096375, last accessed March 21, 2011 :
  • Seq. ID NO. 4 Full Length wt E. coli K12 IhfA; Genbank accession No. : AAC74782.1, last accessed March 21, 2011 : MALTKAEMSEYLFDKLGLSKRDAKELVELFFE
  • Seq. ID NO. 5 Full Length wt P. aeruginosa PA 01 IhfA; Genbank accession No. :
  • Seq. ID NO. 6 Full Length wt Rd Haemophilus influenzae IhfA; Genbank accession No. : AAC22959.1, last accessed March 21, 2011 : MATITKLDIIE YL SDK YHL SKQDTK
  • SEQ ID NO. 7 E. coli hupA, Genbank accession No.: AP 003818, Last accessed March 21, 2011 : MNKTQLIDVIAEKAELSKTQAKAALESTLAAITESLKEGDAVQLVGFGTFK VNHRAERTGRNPQTGKEDCIAAANVPAFVSGKALKDAVK
  • SEQ ID NO. 8 E. coli hupB, Genbank accession No.: AP_001090.1, Last accessed March 21, 201 1 : MNKS QLIDKI AAGADI SKAAAGRALD All AS VTE SLKEGDD VAL VGF G TFAVKERAARTGRNPQTGKEITIAAAKVPSFRAGKALKDAVNeq ID NO. 6 Full Length Wild type (wt) 86-028NP Haemophilus influenzae IhfA; Genbank acce Seq. ID NO. 6 Full Length Wild type (wt) 86-028NP Haemophilus influenzae IhfA; Genbank accession No. : AAX88425.1, last accessed March 21, 2011 : MATITKLDIIE YL SDK YFILSK

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Abstract

La présente invention concerne des compositions comprenant une microsphère biocompatible, une bactérie probiotique générant un biofilm, un prébiotique et/ou un agent prébiofilm. L'invention concerne des procédés de préparation et de formulation des compositions et des procédés de traitement ou de prévention d'une maladie au moyen des compositions.
PCT/US2018/024604 2017-03-27 2018-03-27 Formulations prébiotiques WO2018183355A1 (fr)

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Cited By (6)

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CN110448577A (zh) * 2019-09-26 2019-11-15 青岛农业大学 一种溃疡性结肠炎修复益生菌微胶囊制剂
CN113528400A (zh) * 2021-08-18 2021-10-22 兰州大学 一种具有六价铬离子还原能力的发酵乳杆菌及用途
CN114470226A (zh) * 2020-11-13 2022-05-13 中国科学技术大学 纳米颗粒包裹的抗生素及其制备方法和应用
EP3870154A4 (fr) * 2018-10-22 2022-07-27 Research Institute at Nationwide Children's Hospital Compositions et méthodes de prévention et de traitement de pathologies induites par des antibiotiques à l'aide de probiotiques à l'état de biofilm
CN116138457A (zh) * 2022-12-14 2023-05-23 温州医科大学 一种具有缓解抑郁样症状作用的复合营养组合物
EP3979830A4 (fr) * 2019-06-03 2023-07-05 Research Institute at Nationwide Children's Hospital Formulations prébiotiques pour la prévention de déficiences neurodéveloppementales induites par l'entérocolite nécrosante et la septicémie

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US6486314B1 (en) * 2000-05-25 2002-11-26 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno Glucan incorporating 4-, 6-, and 4, 6- linked anhydroglucose units
US20050266069A1 (en) * 2002-09-06 2005-12-01 Simmons Donald L Stable probiotic microsphere compositions and their methods of preparation
US20150173374A1 (en) * 2013-12-24 2015-06-25 Muhammed Majeed Method of producing partially purified extracellular metabolite products from bacillus coagulans and biological applications thereof
WO2015134808A2 (fr) * 2014-03-06 2015-09-11 Research Institute At Nationwide Children's Hospital Préparations probiotiques et méthodes d'utilisation associées
US20160223553A1 (en) * 2013-09-12 2016-08-04 The Johns Hopkins University Biofilm formation to define risk for colon cancer

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Publication number Priority date Publication date Assignee Title
US6486314B1 (en) * 2000-05-25 2002-11-26 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno Glucan incorporating 4-, 6-, and 4, 6- linked anhydroglucose units
US20050266069A1 (en) * 2002-09-06 2005-12-01 Simmons Donald L Stable probiotic microsphere compositions and their methods of preparation
US20160223553A1 (en) * 2013-09-12 2016-08-04 The Johns Hopkins University Biofilm formation to define risk for colon cancer
US20150173374A1 (en) * 2013-12-24 2015-06-25 Muhammed Majeed Method of producing partially purified extracellular metabolite products from bacillus coagulans and biological applications thereof
WO2015134808A2 (fr) * 2014-03-06 2015-09-11 Research Institute At Nationwide Children's Hospital Préparations probiotiques et méthodes d'utilisation associées

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3870154A4 (fr) * 2018-10-22 2022-07-27 Research Institute at Nationwide Children's Hospital Compositions et méthodes de prévention et de traitement de pathologies induites par des antibiotiques à l'aide de probiotiques à l'état de biofilm
EP3979830A4 (fr) * 2019-06-03 2023-07-05 Research Institute at Nationwide Children's Hospital Formulations prébiotiques pour la prévention de déficiences neurodéveloppementales induites par l'entérocolite nécrosante et la septicémie
CN110448577A (zh) * 2019-09-26 2019-11-15 青岛农业大学 一种溃疡性结肠炎修复益生菌微胶囊制剂
CN110448577B (zh) * 2019-09-26 2023-06-23 青岛农业大学 一种溃疡性结肠炎修复益生菌微胶囊制剂
CN114470226A (zh) * 2020-11-13 2022-05-13 中国科学技术大学 纳米颗粒包裹的抗生素及其制备方法和应用
CN114470226B (zh) * 2020-11-13 2024-02-23 中国科学技术大学 纳米颗粒包裹的抗生素及其制备方法和应用
CN113528400A (zh) * 2021-08-18 2021-10-22 兰州大学 一种具有六价铬离子还原能力的发酵乳杆菌及用途
CN116138457A (zh) * 2022-12-14 2023-05-23 温州医科大学 一种具有缓解抑郁样症状作用的复合营养组合物

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