IP Library › Granted Patent US 12,642,826
Granted Patent B2
US 12,642,826 · App. 17/418,124 · Granted Jun 2, 2026

Combinations of engineered antimicrobial probiotics for treatment of gastrointestinal tract pathogens

Inventors: Yiannis John Kaznessis (New Brighton, MN); Kathryn Gayle Kruziki (New Brighton, MN); Dimitrios Nikolaos Sidiropoulos (Minneapolis, MN)
Assignee: General Probiotics, Inc.
A61K35/747A61K35/741A61K35/742A61K35/744A61K35/745A61K38/164A61P31/04
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 12,642,826
App. No.
17/418,124
Granted
Jun 2, 2026
Kind
B2
Abstract

Embodiments herein relate to engineered antimicrobial probiotics for the treatment of gastrointestinal tract pathogens. In an embodiment, a composition for treatment of an animal is included. The composition can include a first genetically engineered bacterium comprising a first exogenous polynucleotide. The first exogenous polynucleotide can include a first heterologous promoter and a first polynucleotide that encodes a first antimicrobial protein. The composition can also include a second genetically engineered bacterium comprising a second exogenous polynucleotide. The second exogenous polynucleotide can include a second heterologous promoter and a second polynucleotide that encodes a second antimicrobial protein. The first heterologous promoter can be induced by one set of exogenous environmental conditions found in the gastrointestinal tract of the animal and the second heterologous promoter can be induced by a second set of exogenous environmental conditions found in the gastrointestinal tract of the animal. Other embodiments are also included herein.

Claims (21)

1 . A composition for treatment of an animal comprising

a first genetically engineered bacterium comprising an exogenous polynucleotide comprising

a first heterologous promoter;

a first polynucleotide that encodes a first antimicrobial protein, wherein the first polynucleotide is operably linked to the first heterologous promoter; and

a second genetically engineered bacterium comprising an exogenous polynucleotide comprising

a second heterologous promoter;

a second polynucleotide that encodes a second antimicrobial protein, wherein the second polynucleotide is operably linked to the second heterologous promoter;

wherein the first heterologous promoter is directly or indirectly induced by one set of exogenous environmental conditions found in the gastrointestinal tract of the animal, and the second heterologous promoter is directly or indirectly induced by a second set of exogenous environmental conditions found in the gastrointestinal tract of the animal;

wherein at least one of the first genetically engineered bacterium and the second genetically engineered bacterium is Bacillus subtilis strain 168.

2 . The composition of claim 1 , wherein the first genetically engineered bacterium exhibits a colonization profile inside the gastrointestinal tract of animals and the second genetically engineered bacterium exhibits a different colonization profile inside the gastrointestinal tract of animals.

3 . The composition of claim 1 , wherein the first polynucleotide that encodes a first antimicrobial protein is different than the second polynucleotide that encodes a second antimicrobial protein.

4 . The composition of claim 1 , wherein the first heterologous promoter and the second heterologous promoter are selected to respond to different sigma factors selected from the group consisting of σ70 (RpoD), σ19 (FecI), σ24 (RpoE), σ28 (RpoF), σ32 (RpoH), σ38 (RpoS), and σ54 (RpoN).

5 . The composition of claim 1 , wherein the first heterologous promoter and the second heterologous promoter are selected to respond to different exogenous environmental conditions found in the gastrointestinal tract of animals, the exogenous environmental conditions defined by one or more of nutrient content, oxygen content, pH and bile concentration.

6 . The composition of claim 1 , wherein at least one of the first heterologous promoter and the second heterologous promoter are selected from the group of constitutive promoters, exogenously-inducible promoters, pH-inducible promoters, osmotic pressure-inducible promoters, anaerobically-inducible promoters, starvation-inducible promoters, temperature-inducible promoters, inflammation-inducible promoters, and quorum-sensing promoters.

7 . The composition of claim 1 , wherein at least one of the first heterologous promoter and the second heterologous promoter are selected from the group of rpoS promoters, anaerobically-inducible promoters, chloride-inducible promoters, and stationary-phase promoters.

8 . The composition of claim 1 , wherein the first genetically engineered bacterium and the second genetically engineered bacterium are probiotic bacteria.

9 . The composition of claim 1 , wherein the first genetically engineered bacterium and the second genetically engineered bacterium are selected from the group consisting of Bacillus, Bacteroides, Bifidobacterium, Clostridium, Escherichia, Lactobacillus , and Lactococcus.

10 . The composition of claim 1 , wherein at least one of the first genetically engineered bacterium and the second genetically engineered bacterium are Escherichia coli strain Nissle.

11 . The composition of claim 1 , wherein the first antimicrobial protein and the second antimicrobial protein are selected from the group consisting Microcin J25, Microcin V, Microcin L, Microcin N, Enterocin A, Enterocin B, Enterocin P, and Hiracin JM79, or conservative variants thereof.

12 . The composition of claim 1 , wherein the heterologous promoters and the polynucleotides that encode the antimicrobial proteins are located on the chromosome of the bacterium.

13 . The composition of claim 1 , wherein the heterologous promoters and the polynucleotides that encode the antimicrobial proteins are located on a plasmid in the bacterium.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 6, 2024
From: KAZNESSIS, YIANNIS JOHN; KRUZIKI, KATHRYN GAYLE; SIDIROPOULOS, DIMITRIOS NIKOLAOS
To: GENERAL PROBIOTICS, INC.
Reel/Frame 068195/0348 →
Continuity (2)
Provisional Application 62785954 · Dec 28, 2018
Related Publication 20220054562A1 · Feb 24, 2022
References Cited (48)
US 7754469B2 · Baltzley et al. · 2010 [cited by applicant]
US 9050281B2 · Lang et al. · 2015 [cited by applicant]
US 9925223B2 · Kaznessis et al. · 2018 [cited by applicant]
US 10813956B2 · Kaznessis et al. · 2020 [cited by applicant]
US 11533915B2 · Kaznessis et al. · 2022 [cited by applicant]
US 20140314830A1 · Gunzer · 2014 [cited by examiner]
US 20160279175A1 · Kaznessis et al. · 2016 [cited by applicant]
US 20180104285A1 · Langella · 2018 [cited by examiner]
US 20180264078A1 · Call et al. · 2018 [cited by applicant]
US 20190320683A1 · Connerton et al. · 2019 [cited by applicant]
US 20200299665A1 · Kaznessis et al. · 2020 [cited by applicant]
US 20210268034A1 · Kaznessis et al. · 2021 [cited by applicant]
WO 2007025333 · 2007 [cited by applicant]
WO 2010112861 · 2010 [cited by applicant]
WO 2014121301 · 2014 [cited by applicant]
WO 2014133323 · 2014 [cited by applicant]
WO 2016210373 · 2016 [cited by applicant]
WO 2019051179 · 2019 [cited by applicant]
WO 2019121983 · 2019 [cited by applicant]
WO 2020139852 · 2020 [cited by applicant]
WO 2021154872 · 2021 [cited by applicant]
Moreno (“The regulation of microcin B, C, and J operons”, Biochimie, 84 (2002), 521-529) (Year: 2002). [cited by examiner]
Fang (“Influence of aeration and carbon source on production of microcin B17 by [cited by examiner]
“Final Office Action,” for U.S. Appl. No. 17/160,132 mailed Aug. 24, 2023 (15 pages). [cited by applicant]
“Notice of Allowance,” for U.S. Appl. No. 17/160,132 mailed Jun. 11, 2024 (9 pages). [cited by applicant]
“Response to Final Office Action,” for U.S. Appl. No. 17/160,132, filed Feb. 26, 2024 (8 pages). [cited by applicant]
Dombek, et al., “Use of Repetitive DNA Sequences and the PCT To Differentiate [cited by applicant]
“International Preliminary Report on Patentability,” for PCT Application No. PCT/US2019/068400 mailed Jul. 8, 2021 (8 pages). [cited by applicant]
“International Search Report and Written Opinion,” for PCT Application No. PCT/US2019/068400 mailed Apr. 9, 2020 (15 pages). [cited by applicant]
“International Search Report and Written Opinion,” for PCT Application No. PCT/US2021/015313 mailed Apr. 20, 2021 (25 pages). [cited by applicant]
Bajagai, Yadav, et al. “Probiotics in Animal Nutrition-Production, Impact and Regulation,” FAO. 2016. Animal Production and Health Paper No. 179. Rome (108 pages). [cited by applicant]
Borrero, Juan, et al. “Modified Lactic Acid Bacteria Detect and Inhibit Multiresistant Enterococci,” ACS Synth. Biol. 2015, 4, 299-306 (8 pages). [cited by applicant]
Chiuchiolo, M. J., et al. “Growth-Phase-Dependent Expression of the Cyclopeptide Antibiotic Microcin J25,” J. Bacteriol. Mar. 2001; 183(5): 1755-1764 (10 pages). [cited by applicant]
Coman, M. M., et al. “In vitro evaluation of antimicrobial activity of Lactobacillus rhamnosus IMC 501, Lactobacillus paracasei IMC 502 and SYNBIO against pathogens,” Journal of Applied Microbiology, vol. 117, No. 2, Ju… [cited by applicant]
Forkus, Brittany, et al. “Antimicrobial Probiotics Reduce Salmonella enterica in Turkey Gastrointestinal Tracts,” Scientific Reports, vol. 7, No. 1, Jan. 17, 2017 (10 pages). [cited by applicant]
Geldart, Kathryn, et al. “Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus Faecium,” Applied and Environmental Microbiology, Jun. 2015, vol. 81, No.… [cited by applicant]
Jayaraman, P., et al. “Repurposing a Two-Component System-Based Biosensor for the Killing of Vibrio cholerae,” ACS Synth Biol. Jul. 21, 2017;6(7):1403-1415 (27 pages). [cited by applicant]
Mutalik, Vivek, et al. “Precise and reliable gene expression via standard transcription and translation initiation elements,” Nature Methods. 2013, 10(4):354-360 (15 pages). [cited by applicant]
Nissen-Meyer, Jon, et al. “Robosomally synthesized antimicrobial peptides: their function, structure, biogenesis, and mechanism of action,” Arch. Microbiol (1997) 167: 67-77 (11 pages). [cited by applicant]
Piraner, D. I., et al. “Tunable thermal bioswitches for in vivo control of microbial therapeutics,” Nat. Chem. Biol. Jan. 2017; 13(1):75-80 (9 pages). [cited by applicant]
Prescott, J.F., et al. “The pathogenesis of necrotic enteritis in chickens: what we know and what we need to know: a review,” Avian Pathology, 45(3), 2016, 288-294 (8 pages). [cited by applicant]
Prindle, Arthur, et al. “A sensing array of radically coupled genetic ‘biopixels’,” Nature 2011, Published Dec. 18, 2011 (6 pages). [cited by applicant]
Sanders, J. W., et al. “A chloride-inducible gene expression cassette and its use in induced lysis of Lactococcus lactis.,” Appl. Environ. Microbiol. Dec. 1997;63(12):4877-82 (6 pages). [cited by applicant]
Volzing, Katherine, et al. “Antimicrobial Peptides Targeting Gram-negative Pathogens, Produced and Delivered by Lactic Acid Bacteria,” ACS Synthetic Biology, vol. 2, No. 11, Jul. 10, 2013, pp. 643-640 (8 pages). [cited by applicant]
“International Preliminary Report on Patentability,” for PCT Application No. PCT/US2021/015313 mailed Aug. 11, 2022 (18 pages). [cited by applicant]
“Non-Final Office Action,” for U.S. Appl. No. 17/160,132 mailed Jan. 11, 2023 (31 pages). [cited by applicant]
“Response to Non-Final Office Action,” for U.S. Appl. No. 17/160,132, filed Jul. 11, 2023 (12 pages). [cited by applicant]
McClure, William R.“ Mechanism and Control of Transcription Initiation in Prokaryotes,” Annual Reviews Biochemistry, 1985, vol. 54, p. 171-204 (37 pages). [cited by applicant]