Methods of treating inflammatory conditions and associated infections

Abstract

Provided herein are methods for treating or preventing inflammation, inflammatory conditions, and autoimmune conditions, optionally inflammation, inflammatory conditions, and autoimmune conditions of the gastrointestinal tract, urinary tract, skin, nail, or joint. Also provided are methods of treating or preventing bacterial infections, often infections associated with inflammation, inflammatory conditions, and autoimmune conditions of the gastrointestinal tract, urinary tract, skin, nail, or joint. The methods of the disclosure include administering to a subject in need thereof a Lactobacillus species selected from Lactobacillus buchneri, Lactobacillus zeae, Lactobacillus rapi, Lactobacillus paracasei, Lactobacillus parafarraginis, and Lactobacillus diolivorans, and / or a culture supernatant or cell-free filtrate derived from a culture medium in which a Lactobacillus has been cultured.

Description

Technical Field

[0001] This disclosure generally relates to methods for treating or preventing inflammation and inflammatory conditions and autoimmune conditions, as well as methods for treating infections associated with such inflammation and inflammatory conditions and autoimmune conditions. Typically, inflammation and inflammatory conditions and autoimmune conditions are inflammation and inflammatory conditions and autoimmune conditions of the gastrointestinal tract, urinary tract, skin, nails / toenails, or joints, usually associated with or caused by a pathogenic infection. This disclosure also relates to promoting wound healing. Methods of this disclosure include administering a composition containing one or more microorganisms, or a culture supernatant or cell-free filtrate derived from a culture medium in which one or more microorganisms have been cultured.

[0002] background

[0003] Inflammation is a normal response mechanism that helps protect the body from infection and damage. However, abnormal or uncontrolled inflammatory responses can lead to the development of acute or chronic inflammatory disorders or conditions, as well as autoimmune disorders or conditions. In particular, infections caused by viruses, fungi, and pathogens can trigger excessive and persistent inflammatory responses in a variety of tissues, such as the gastrointestinal tract, joints, skin, and urinary tract, resulting in harmful acute inflammation and acute inflammatory conditions. These are also important risk factors for the development of chronic inflammatory conditions and autoimmune conditions. Chronic inflammatory conditions and autoimmune conditions can debilitate patients and cause significant discomfort and pain. Furthermore, this is becoming increasingly common with the aging of the world's population.

[0004] Inflammatory bowel disease (IBD) is a complex, chronic, idiopathic-atopic condition characterized by alterations and dysregulation of the immune response to the gastrointestinal commensal microbiota. There are two main subtypes of IBD: Crohn's disease and ulcerative colitis. Crohn's disease can occur anywhere in the lower gastrointestinal tract, while ulcerative colitis is mostly confined to the colon and can predispose individuals to colitis-related cancers, typically colorectal cancer.

[0005] The etiology of inflammatory bowel disease remains somewhat unclear, although dysbiosis of the gut microbiota is increasingly involved, characterized by a significant increase in pathogenic strains and a decrease in beneficial or symbiotic resident microbes in the digestive tract. Among the microbes involved, *Escherichia coli* strains are abundant in biopsy samples collected from patients with Crohn's disease and ulcerative colitis. These strains have been shown to adhere to and invade the digestive tract epithelium and are commonly referred to as adhesive and invasive *Escherichia coli* (AIEC). AIEC has been shown to associate closely with the mucosa and play a key role in the pathogenesis of Crohn's disease. AIEC requires the ability to adhere to and colonize the surface of intestinal cells to induce and exacerbate chronic inflammation in susceptible individuals. *E. coli* strains isolated from patients with active Crohn's disease have shown the ability to adhere to Caco-2 cells, which mimic the intestinal epithelium. AIEC also invade intestinal epithelial cells, where they persist, replicate, and drive pro-inflammatory activity. Their invasive ability is also associated with an increased severity of ileal inflammatory disease.

[0006] Steroids have been the primary therapeutic anti-inflammatory agents relied upon for decades. More recently, nonsteroidal anti-inflammatory drugs (NSAIDs) have begun to be widely used to manage or treat inflammation. However, prolonged use of these agents brings significant drawbacks and side effects. For example, serious side effects associated with prolonged NSAID use include gastric ulcers and bleeding. Furthermore, it is well known that NSAIDs can cause lesions in the gastrointestinal tract, depending on the duration of treatment and the type of drug. This issue is particularly important in cases where treatment must be prolonged, such as in the treatment of chronic inflammatory disorders requiring long-term therapy to manage the inflammatory state and associated pain.

[0007] There is a continued need to develop new and improved treatment options for inflammation and inflammatory and autoimmune conditions.

[0008] Wound healing is a complex and precise biological process involving numerous biological factors and requiring a delicate balance between different physiological processes. While inflammation is part of the wound healing process, its sensitivity depends on the balance between multiple molecules and pathways and is easily disrupted. Therefore, the mechanisms of wound healing and tissue repair are often inadequate and incomplete. For example, chronic wounds, such as pressure sores and diabetic foot ulcers, fail to heal properly and are becoming an increasingly serious problem worldwide. Wound healing can also be substantially impaired in the elderly, cancer patients following chemotherapy or radiation therapy, and individuals with severe burns. Lesions caused by conditions such as Crohn's disease or osteoarthritis are also characterized by slow and inadequate healing.

[0009] Overview of Public Content

[0010] A first aspect of this disclosure provides a method for treating or preventing inflammation, or an inflammatory condition, or an autoimmune condition, or one or more symptoms associated with inflammation, or an inflammatory condition, or an autoimmune condition in a subject, the method comprising administering to the subject a species of the genus *Lactobacillus*, and / or a culture supernatant or cell-free filtrate derived from a culture medium in which *Lactobacillus* has been cultured, the *Lactobacillus* species being selected from *Lactobacillus buchneri*, *Lactobacillus zeae*, *Lactobacillus rapi*, *Lactobacillus paracasei*, *Lactobacillus parafarraginis*, and *Lactobacillus diolivorans*.

[0011] Inflammation can be an inflammation of the gastrointestinal tract, urinary tract, skin, nails / toenails, or joints. Gastrointestinal inflammation can be an inflammation of the upper gastrointestinal tract, such as the mouth or throat, or an inflammation of the lower gastrointestinal tract, such as the stomach, small intestine, or large intestine. Inflammatory or autoimmune conditions can be inflammatory conditions of the gastrointestinal tract, urinary tract, skin, nails / toenails, or joints, or autoimmune conditions.

[0012] In certain embodiments, the inflammation is caused by or related to an infection. The inflammation can be acute or chronic. In an exemplary embodiment, the pathogen causing the infection is bacteria.

[0013] A second aspect of this disclosure provides a method for treating or preventing a condition of the gastrointestinal tract, urinary tract, skin, nail / toenail, or joint in a subject, the method comprising administering to the subject a species of Lactobacillus, and / or a culture supernatant or cell-free filtrate derived from a culture medium in which Lactobacillus has been cultured, the Lactobacillus species being selected from Lactobacillus bruneri, Lactobacillus zeatus, Lactobacillus rapi, Lactobacillus casei, Lactobacillus glutenosa, and Lactobacillus diolivorans, wherein the condition is associated with inflammation of the gastrointestinal tract, urinary tract, skin, nail / toenail, or joint and / or wherein the condition is caused by or associated with an infection of the gastrointestinal tract, urinary tract, skin, nail / toenail, or joint.

[0014] According to the first and second aspects, gastrointestinal inflammation or a gastrointestinal condition may be gastritis, gastroenteritis, inflammatory bowel disease, or irritable bowel syndrome, or may be associated with, or related to, gastritis, gastroenteritis, inflammatory bowel disease, or irritable bowel syndrome. Inflammatory bowel disease may be, for example, colitis, such as ulcerative colitis or Crohn's disease. Ulcerative colitis may be chronic ulcerative colitis. Optionally, gastrointestinal inflammation or a gastrointestinal condition may be or may be associated with an oral or pharyngeal condition, including, for example, gingivitis, tonsillitis, and pharyngitis such as streptococcal pharyngitis.

[0015] This method can be used to treat or prevent one or more symptoms of gastrointestinal infections, such as food poisoning. Gastrointestinal infections can be bacterial, viral, and / or parasitic. At least one symptom may be abdominal pain, abdominal cramps, bloating, diarrhea, loose stools, or the presence of feecal blood.

[0016] According to the first and second aspects, the urinary tract condition may be cystitis, urethritis, pyelonephritis, asymptomatic bacteriuria, or catheter-related urinary tract infection, or may be associated with cystitis, urethritis, pyelonephritis, asymptomatic bacteriuria, or catheter-related urinary tract infection.

[0017] According to the first and second aspects, the skin or nail / toenail condition may be or is related to the following: psoriasis, dermatitis, eczema, rosacea, acne, ichthyosis, tinea, or other skin or nail / toenail conditions characterized by inflammation, plaques, skin lesions, and / or infection, or related to other skin or nail / toenail conditions characterized by inflammation, plaques, skin lesions, and / or infection. Infections may be caused by, for example, pathogenic bacteria or fungi.

[0018] According to the first and second aspects, the condition of the joint can be arthritis or can be related to arthritis. Arthritis can be, for example, rheumatoid arthritis or osteoarthritis.

[0019] According to the first and second aspects, the condition may be caused by or related to an infection. Such conditions include, for example, gastritis, gastroenteritis, mastitis, gingivitis, pharyngitis such as streptococcal pharyngitis (strepthroat), and conditions of the skin and nails / toenails. Infections can be bacterial, viral, fungal, and / or parasitic.

[0020] A third aspect of this disclosure provides a method for treating or preventing bacterial infections of the gastrointestinal tract, urinary tract, skin, nails / toenails, or joints, the method comprising administering to a subject a species of Lactobacillus, and / or a culture supernatant or cell-free filtrate derived from a culture medium in which Lactobacillus has been cultured, the Lactobacillus species being selected from Lactobacillus bruneri, Lactobacillus zeatus, Lactobacillus rapi, Lactobacillus casei, Lactobacillus glutenosa, and Lactobacillus diolivorans.

[0021] Typically, according to the third aspect, bacterial infection causes, induces, or is associated with an inflammatory or autoimmune condition or other conditions.

[0022] A fourth aspect of this disclosure provides a method for treating or preventing inflammatory or autoimmune conditions of the gastrointestinal tract, optionally said condition being caused by or related to an infection, the method comprising administering to a subject a species of Lactobacillus, and / or a culture supernatant or cell-free filtrate derived from a culture medium in which Lactobacillus has been cultured, said Lactobacillus species selected from Lactobacillus bruneri, Lactobacillus zeatus, Lactobacillus rapi, Lactobacillus casei, Lactobacillus glutenosa, and Lactobacillus diolivorans.

[0023] The condition can be acute or chronic. In an exemplary embodiment, the condition can be selected from inflammatory bowel disease, gastritis, gastroenteritis, and gingivitis. Inflammatory bowel disease can be colitis. Colitis can be, for example, ulcerative colitis or Crohn's disease. Ulcerative colitis can be chronic ulcerative colitis.

[0024] In an exemplary embodiment, the subject was administered a combination of *Lactobacillus paracasei*, *Lactobacillus buchneri*, and *Lactobacillus zeae*. In another exemplary embodiment, the subject was administered a combination of *L. diolivorans*, *Lactobacillus parafarraginis*, and *Lactobacillus buchneri*.

[0025] The fifth aspect of this disclosure provides a method for treating or preventing irritable bowel syndrome or inflammatory bowel disease, the method comprising administering to a subject a species of Lactobacillus, and / or a culture supernatant or cell-free filtrate derived from a culture medium in which Lactobacillus has been cultured, said Lactobacillus species selected from Lactobacillus bruneri, Lactobacillus zeatus, Lactobacillus rapi, Lactobacillus casei, Lactobacillus glutenosa, and Lactobacillus diolivorans.

[0026] Inflammatory bowel disease can be, for example, ulcerative colitis or Crohn's disease. Ulcerative colitis can be chronic ulcerative colitis.

[0027] In an exemplary embodiment, the subject was administered a combination of *Lactobacillus casei*, *Lactobacillus brunelli*, and *Lactobacillus zeatus*. In another exemplary embodiment, the subject was administered a combination of *L. diolivorans*, *Lactobacillus fragrans*, and *Lactobacillus brunelli*.

[0028] A sixth aspect of this disclosure provides a method for treating or preventing bacterial infections of the gastrointestinal tract, the method comprising administering to a subject a species of Lactobacillus, and / or a culture supernatant or cell-free filtrate derived from a culture medium in which Lactobacillus has been cultured, the species being selected from Lactobacillus bruneri, Lactobacillus zeatus, Lactobacillus rapi, Lactobacillus casei, Lactobacillus glutenosa, and Lactobacillus diolivorans.

[0029] Infection can be associated with the adhesion and / or invasion of the gastrointestinal epithelium by bacteria that cause the infection. Infection can cause, induce, or otherwise be associated with gastrointestinal inflammation or an inflammatory or autoimmune condition of the gastrointestinal tract.

[0030] A seventh aspect of this disclosure provides a method for inhibiting or preventing the adhesion of bacterial pathogens to the gastrointestinal mucosa of a subject, the method comprising administering to the subject a species of Lactobacillus, and / or a culture supernatant or cell-free filtrate derived from a culture medium in which Lactobacillus has been cultured, the species of Lactobacillus being selected from Lactobacillus bruneri, Lactobacillus zeatus, Lactobacillus rapi, Lactobacillus casei, Lactobacillus glutenosa, and Lactobacillus diolivorans.

[0031] In an exemplary embodiment, the gastrointestinal mucosa includes the epithelial lining of the stomach, duodenum, or lower gastrointestinal tract. Typically, bacterial pathogens are pathogens that colonize the gastrointestinal tract.

[0032] The eighth aspect of this disclosure provides a method for inhibiting or preventing bacterial pathogens from invading the gastrointestinal epithelial cells of a subject, the method comprising administering to the subject a species of Lactobacillus, and / or a culture supernatant or cell-free filtrate derived from a culture medium in which Lactobacillus has been cultured, the species being selected from Lactobacillus bruneri, Lactobacillus zeatus, Lactobacillus rapi, Lactobacillus casei, Lactobacillus glutenosa, and Lactobacillus diolivorans.

[0033] In an exemplary embodiment, gastrointestinal epithelial cells include epithelial cells of the stomach, duodenum, or lower gastrointestinal tract. Typically, bacterial pathogens are pathogens that colonize the gastrointestinal tract.

[0034] Another aspect of this disclosure provides a method for promoting wound healing in a subject, the method comprising administering to the subject a species of Lactobacillus, and / or a culture supernatant or cell-free filtrate derived from a culture medium in which Lactobacillus has been cultured, the species being selected from Lactobacillus bruneri, Lactobacillus zeatus, Lactobacillus rapi, Lactobacillus casei, Lactobacillus glutenosa, and Lactobacillus diolivorans.

[0035] Based on the above aspects and implementation schemes, the method may include applying a combination of two, three, four, five, or all six species of the *Lactobacillus* genus, or a culture supernatant or cell-free filtrate derived from a culture medium in which two, three, four, five, or all six species of the *Lactobacillus* have been cultured. The combination may represent a synergistic combination.

[0036] Based on the above aspects and implementation plan, lactobacillus can be administered, for example, orally, sublingually, or topically.

[0037] In certain implementations, Lactobacillus species, culture supernatants, or cell-free filtrates are administered in the form of pharmaceutically acceptable compositions or food or beverages.

[0038] Based on the above aspects and implementation plan, the method may also include the application of one or more additional microorganisms or other therapeutic agents.

[0039] Based on the above aspects and implementation methods, the method may include administering a microbial biotherapy composition of a Lactobacillus species to a subject. The microbial biotherapy composition may be administered in, for example, solid or liquid unit dosage forms, food, or beverages. Brief description of the attached diagram

[0041] Exemplary embodiments of this disclosure are described herein by way of non-limiting example only, with reference to the accompanying drawings.

[0042] Figure 1The percentage of AIEC cells adhering to the HT29-MTX cell line alone (A) and the number of AIEC cells adhering to the HT29-MTX cell line in the presence of Lactobacillus strains SVT01D1 (L. diolivorans), SVT04P1 (Lactobacillus casei), SVT05P2 (Lactobacillus gluteni), SVT06B1 (Lactobacillus bruneri), SVT07R1 (L. rapi), and SVT08Z1 (Lactobacillus zeatus) (B), mean + / - SEM. # p≤0.001; *p<0.0001. For SVT01D1, SVT04P1, SVT05P2, SVT06B1, SVT07R1 and SVT08Z1: left column, Lactobacillus alone; middle column, Lactobacillus and AIEC co-inoculated; right column, Lactobacillus pre-inoculated followed by AIEC.

[0043] Figure 2 The percentage of individual AIEC cells adhering to the Caco-2 cell line (A) and the number of AIEC cells adhering to the Caco-2 cell line in the presence of Lactobacillus strains SVT01D1 (L. diolivorans), SVT04P1 (Lactobacillus casei), SVT05P2 (Lactobacillus gluteni), SVT06B1 (Lactobacillus bruni), SVT07R1 (L. rapi), and SVT08Z1 (Lactobacillus zeatus) (B), mean + / - SEM. # p≤0.001; *p<0.0001. For SVT01D1, SVT04P1, SVT05P2, SVT06B1, SVT07R1 and SVT08Z1: left column, Lactobacillus alone; middle column, Lactobacillus and AIEC co-inoculated; right column, Lactobacillus pre-inoculated followed by AIEC.

[0044] Figure 3 The number of individual AIEC cells invading the HT29-MTX cell line, and the number of AIEC cells invading the HT29-MTX cell line in the presence of Lactobacillus strains SVT01D1 (L. diolivorans), SVT04P1 (Lactobacillus casei), SVT05P2 (Lactobacillus gluteni), SVT06B1 (Lactobacillus bryonicus), SVT07R1 (L. rapi), and SVT08Z1 (Lactobacillus zeatus), mean + / - SEM. #p≤0.001; *p<0.0001. For SVT01D1, SVT04P1, SVT05P2, SVT06B1, SVT07R1 and SVT08Z1: Left column, co-inoculated with Lactobacillus and AIEC; Right column, pre-inoculated with Lactobacillus followed by AIEC.

[0045] Figure 4 The percentage of HMLN-1 adhesion to HT29-MTX and Caco-2 cell lines alone, and the percentage of HMLN-1 adhesion to HT29-MTX and Caco-2 cell lines in the presence of Lactobacillus strains SVT01D1 (L. diolivorans), SVT04P1 (Lactobacillus casei), SVT05P2 (Lactobacillus gluteni), SVT06B1 (Lactobacillus bruneri), SVT07R1 (L. rapi), and SVT08Z1 (Lactobacillus zeatus), mean + / - SEM. # p≤0.001; *p<0.0001. For SVT01D1, SVT04P1, SVT05P2, SVT06B1, SVT07R1 and SVT08Z1: First column, co-inoculated with Lactobacillus and HMLN-1 (HT29); Second column, pre-inoculated with Lactobacillus followed by HMLN-1 infection (HT29); Third column, co-inoculated with Lactobacillus and HMLN-1 (Caco-2); Fourth column, pre-inoculated with Lactobacillus followed by HMLN-1 (Caco-2).

[0046] Figure 5 The number of HMLN-1 cells in the HT29-MTX and Caco-2 cell lines in the presence of Lactobacillus strains SVT01D1 (L. diolivorans), SVT04P1 (Lactobacillus casei), SVT05P2 (Lactobacillus gluteni), SVT06B1 (Lactobacillus bryonicus), SVT07R1 (L. rapi), and SVT08Z1 (Lactobacillus zeatus), with mean + / - SEM values. # p≤0.001; *p<0.0001. For SVT01D1, SVT04P1, SVT05P2, SVT06B1, SVT07R1 and SVT08Z1: First column, co-inoculated with Lactobacillus and HMLN-1 (HT29); Second column, pre-inoculated with Lactobacillus followed by HMLN-1 infection (HT29); Third column, co-inoculated with Lactobacillus and HMLN-1 (Caco-2); Fourth column, pre-inoculated with Lactobacillus followed by HMLN-1 infection (Caco-2).

[0047] Figure 6The number of HMLN-1 cells translocated across HT29-MTX cell line (A) and Caco-2 cell line (B) in the presence of Lactobacillus strains SVT01D1 (L. diolivorans), SVT04P1 (Lactobacillus casei), SVT05P2 (Lactobacillus gluteni), SVT06B1 (Lactobacillus bruneri), SVT07R1 (L. rapi), and SVT08Z1 (Lactobacillus zeatus), mean + / - SEM. # p≤0.001; *p<0.0001. For SVT01D1, SVT04P1, SVT05P2, SVT06B1, SVT07R1 and SVT08Z1: Left column, co-inoculated with Lactobacillus and HMLN-1; Right column, pre-inoculated with Lactobacillus followed by HMLN-1 infection.

[0048] Figure 7 The percentage of HMLN-1 and AIEC cells adhering to the optimized cocultures of HT29-MTX and Caco-2 cell lines in the presence of Lactobacillus strains SVT01D1 (L. diolivorans), SVT04P1 (Lactobacillus casei), SVT05P2 (Lactobacillus gluteni), SVT06B1 (Lactobacillus brunelli), SVT07R1 (L. rapi), and SVT08Z1 (Lactobacillus zeatus) is shown in the mean + / - SEM values. # p≤0.001; *p<0.0001. For SVT01D1, SVT04P1, SVT05P2, SVT06B1, SVT07R1 and SVT08Z1: First column, co-inoculated with Lactobacillus and HMLN-1; Second column, pre-inoculated with Lactobacillus followed by HMLN-1 infection; Third column, co-inoculated with Lactobacillus and AIEC; Fourth column, pre-inoculated with Lactobacillus followed by AIEC infection.

[0049] Figure 8 The number of HMLN-1 and AIEC cells in optimized co-cultures of HT29-MTX and Caco-2 cell lines in the presence of Lactobacillus strains SVT01D1 (L. diolivorans), SVT04P1 (Lactobacillus casei), SVT05P2 (Lactobacillus gluteni), SVT06B1 (Lactobacillus bruni), SVT07R1 (L. rapi), and SVT08Z1 (Lactobacillus zeatus) in the presence of these strains, mean + / - SEM. #p≤0.001; *p<0.0001. For SVT01D1, SVT04P1, SVT05P2, SVT06B1, SVT07R1 and SVT08Z1: First column, co-inoculated with Lactobacillus and HMLN-1; Second column, pre-inoculated with Lactobacillus followed by HMLN-1 infection; Third column, co-inoculated with Lactobacillus and AIEC; Fourth column, pre-inoculated with Lactobacillus followed by AIEC infection.

[0050] Figure 9 The number of HMLN-1 and AIEC cells translocated across the HT29-MTX and Caco-2 cell lines in the presence of Lactobacillus strains SVT01D1 (L. diolivorans), SVT04P1 (Lactobacillus casei), SVT05P2 (Lactobacillus gluteni), SVT06B1 (Lactobacillus bruneri), SVT07R1 (L. rapi), and SVT08Z1 (Lactobacillus zeatus), mean + / - SEM values. # p≤0.001; *p<0.0001. For SVT01D1, SVT04P1, SVT05P2, SVT06B1, SVT07R1 and SVT08Z1: First column, co-inoculated with Lactobacillus and HMLN-1; Second column, pre-inoculated with Lactobacillus followed by HMLN-1 infection; Third column, co-inoculated with Lactobacillus and AIEC; Fourth column, pre-inoculated with Lactobacillus followed by AIEC infection.

[0051] Figure 10 Stool consistency scores in mice with a DSS-induced colitis model following treatment as described in Example 4. From left to right: Groups 1 through 6, as described in Example 4. *, p < 0.05, Dunnett's test compared to Group 2. ***, p < 0.001, Dunnett's test compared to Group 2.

[0052] Figure 11 Score of fecal hematoma in mice with a DSS-induced colitis model following treatment as described in Example 4. From left to right: Groups 1 through 6 as described in Example 4. *, p < 0.05, Dunnett's test compared to Group 2.

[0053] Figure 12 Disease activity index scores in mice with a DSS-induced colitis model following treatment as described in Example 4. From left to right: Groups 1 through 6, as described in Example 4. *, p < 0.05, Dunnett's test compared to Group 2.

[0054] Figure 13Comparative efficacy of drug therapy for chronic DSS-induced ulcerative colitis in a mouse model, measured by 29-day Disease Activity Index (DAI) scores.

[0055] Figure 14 Following treatment as described in Example 4A, the occurrence of rectal bleeding (A), stool consistency score (B), and disease activity index (DAI) score (C) in mice with a DSS-induced colitis model were compared. From left to right: Groups 1, 2, 7, and 6 as described in Example 4A. *, p < 0.05, Dunnett's test compared to Group 2; **, p < 0.01, Dunnett's test compared to Group 2; ***, p < 0.001, Dunnett's test compared to Group 2.

[0056] Figure 15 Cytokine expression analysis in mice with a DSS-induced colitis model following treatment as described in Example 4A (A, IL-6; B, TNFα). From left to right: Group 1, Group 2, Group 7, and Group 6 as described in Example 4A. *, p < 0.05, Dunnett's test compared to Group 2.

[0057] Figure 16 Following treatment as described in Example 4A, the overall composite score of ulceration and inflammation, and the composite scores of proximal, intermediate, and distal ulcers in mice with a DSS-induced colitis model. From left to right: Group 1, Group 2, Group 7, and Group 6 as described in Example 4A. *, p<0.05, Dunnett's test compared to Group 2; **, p<0.01, Dunnett's test compared to Group 2; ***, p<0.001, Dunnett's test compared to Group 2.

[0058] Detailed Explanation

[0059] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used to practice or test the contents of this disclosure, typical methods and materials are described.

[0060] The articles “a” and “an” are used in this text to refer to one or more (i.e., at least one) grammatical objects of the article. For example, “an element” means one or more elements.

[0061] In the context of this specification, the term “about” is understood to mean a range of numbers that a person skilled in the art would consider equivalent to the stated value in a context of achieving the same function or result.

[0062] Throughout this specification and the appended claims, unless the context otherwise requires, the word “comprise” and variations such as “comprises” or “comprising” shall be understood to imply inclusion of the stated integer or step or group of integers or steps, but not to exclude any other integer or step or group of integers or steps.

[0063] As used herein, the term "effective amount" includes, within its meaning, an amount of composition that is non-toxic but sufficient to provide the desired therapeutic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the specific agent being administered, and the mode of administration. For any given case, the appropriate "effective amount" can be determined by a person skilled in the art using only routine laboratory methods.

[0064] As used herein, the term "subject" refers to a mammal and includes humans, primates, livestock (e.g., cattle, cows, horses, sheep, pigs), test animals (e.g., mice, rabbits, rats, guinea pigs), companion animals (e.g., dogs, cats), performing animals (e.g., racehorses), and captive wild animals. In an exemplary embodiment, the mammal is a human.

[0065] As used herein, the terms “treating” or “treatment” refer to any and all applications that remedy or otherwise prevent, delay, or reverse the progression of inflammation, infection, or condition, or at least one symptom of such inflammation, infection, or condition, including reducing the severity of the inflammation, infection, or condition. Therefore, treatment does not necessarily imply treating the subject until the inflammation, infection, or condition is completely eliminated or resolved. Similarly, the terms “preventing” or “prevention” refer to any and all applications that prevent the formation of a condition or otherwise delay the onset of such inflammation, infection, or condition.

[0066] The term "optionally" is used herein to mean that the feature subsequently described may be present or may not be present, or that the event or condition subsequently described may occur or may not occur. Therefore, this specification shall be understood to include and cover embodiments in which the feature is present and embodiments in which the feature is absent, as well as embodiments in which the event or condition occurs and embodiments in which the event or condition does not occur.

[0067] In the context of this specification, the term "microbial biotherapeutic agent" is given its broadest meaning and is understood to refer to a microbial cell population or product, or a component of a microbial cell population or product, that promotes health benefits in a subject when administered to the subject in an effective amount.

[0068] In the context of this specification, the term "prebiotic" is given its broadest meaning and is understood to refer to any indigestible substance that stimulates the growth and / or activity of symbiotic beneficial bacteria in the digestive system.

[0069] In the context of this specification, the terms "food," "foods," "beverage," or "beverages" include, but are not limited to, health foods and beverages, functional foods and beverages, and foods and beverages for specific health purposes. When such foods or beverages of the present invention are used in subjects other than humans, the term may be used to include animal feed.

[0070] This article provides a method for treating or preventing inflammation, or an inflammatory condition, or an autoimmune condition, or one or more symptoms associated with inflammation, or an inflammatory condition, or an autoimmune condition, the method comprising administering to a subject a species of Lactobacillus, and / or a culture supernatant or cell-free filtrate derived from a culture medium in which Lactobacillus has been cultured, said Lactobacillus species selected from Lactobacillus brunelli, Lactobacillus zeatus, Lactobacillus rapi, Lactobacillus casei, Lactobacillus glutenosa, and Lactobacillus diolivorans.

[0071] As used herein, the term "inflammatory condition" generally refers to a condition characterized by inflammation or a complex biological response to harmful stimuli such as microbial pathogens and / or viral infections. The clinical features of an inflammatory condition may depend on the harmful stimulus (or multiple stimuli), but may be characterized by heat, pain, redness, or swelling of the affected organ or tissue. Inflammatory conditions can be acute or chronic.

[0072] This article also provides a method for treating or preventing conditions of the gastrointestinal tract, urinary tract, skin, nail / toenail, or joints, the method comprising administering to a subject a species of Lactobacillus, and / or a culture supernatant or cell-free filtrate derived from a culture medium in which Lactobacillus has been cultured, the species being selected from Lactobacillus bruneri, Lactobacillus zeatus, Lactobacillus rapi, Lactobacillus casei, Lactobacillus glutenosa, and Lactobacillus diolivorans, wherein the condition is associated with inflammation of the gastrointestinal tract, urinary tract, skin, nail / toenail, or joints and / or wherein the condition is caused by or associated with an infection of the gastrointestinal tract, urinary tract, skin, nail / toenail, or joints.

[0073] In the following discussion, in the context of administering a *Lactobacillus* species or a culture supernatant or cell-free filtrate derived from a culture medium in which *Lactobacillus* has been cultured, and in the case of compositions containing a *Lactobacillus* species or a culture supernatant or cell-free filtrate derived from a culture medium in which *Lactobacillus* has been cultured, the term "*Lactobacillus*" may be used not only to refer to the specific *Lactobacillus* species as defined herein, but more broadly to a culture supernatant or cell-free filtrate derived from a culture medium in which a specific *Lactobacillus* species as defined herein has been cultured.

[0074] In certain embodiments, the inflammation can be inflammation of the gastrointestinal tract, urinary tract, skin, nails / toenails, or joints. The inflammation can be caused or induced by an infection, or otherwise associated with an infection such as a bacterial infection, viral infection, or parasitic infection. In exemplary embodiments, the pathogen causing the infection is bacteria. Exemplary bacteria that cause such infections are described below.

[0075] Gastrointestinal inflammation can be associated with one or more conditions affecting the gastrointestinal tract, which may be characterized by or cause inflammation, such as food poisoning, diarrhea, ulcers such as stomach ulcers and oral ulcers, dental caries, and periodontal disease. Inflammation can be acute or chronic.

[0076] One or more symptoms associated with gastrointestinal inflammation may include, for example, diarrhea, poor stool consistency, rectal bleeding, abdominal cramps, bloating, abdominal pain, ulcers in the gastrointestinal epithelial lining such as the mouth, stomach, small intestine, or large intestine, or swollen gums. Those skilled in the art will readily understand that the scope of this disclosure is not limited to these exemplary symptoms, and that this disclosure will cover other symptoms of gastrointestinal inflammation.

[0077] The condition can be acute or chronic. Examples of inflammatory and autoimmune conditions affecting the gastrointestinal tract, and related conditions, as described in this disclosure, include, but are not limited to, inflammatory bowel disease, irritable bowel syndrome, gastritis, gastroenteritis, gingivitis, pharyngitis (e.g., streptococcal pharyngitis or streptococcal laryngitis), ileitis, and other conditions caused by bacterial infections, such as Clostridium difficile gastritis and Yersiniosis. In an exemplary embodiment, the condition is inflammatory bowel disease. Inflammatory bowel disease can be colitis, such as, for example, ulcerative colitis, Crohn's disease, ischemic colitis, enterocolitis, or antibiotic-associated hemorrhagic colitis (AAHC). AAHC can be caused by species of Klebsiella, such as Klebsiella oxytoca. Ulcerative colitis can be acute or chronic. In an exemplary embodiment, the ulcerative colitis is chronic ulcerative colitis.

[0078] Embodiments of this disclosure provide methods for treating or preventing at least one symptom of a gastrointestinal infection, such as bacterial infections (e.g., Salmonella, Escherichia coli, Listeria, Bacillus cereus), viral infections (e.g., norovirus, rotavirus), or parasitic infections (e.g., Giardia, Cryptosporidium, Ascaris, Eimeria, or Trichinella). At least one symptom can be poor stool consistency, diarrhea, bloody stool, abdominal cramps, bloating, or abdominal pain. Therefore, the methods of this disclosure can prove effective, for example, for travelers, as prevention or treatment of food poisoning, or to reduce the severity of food poisoning. At least one symptom may be associated with, for example, irritable bowel syndrome.

[0079] Conditions and infections of the oral cavity and throat that can be treated according to this disclosure include, for example, pharyngitis, such as streptococcal pharyngitis, tonsillitis, halitosis, and scarlet fever.

[0080] Inflammation or conditions of the urinary tract can be, for example, conditions of the kidneys, ureters, bladder, or urethra. Exemplary conditions include, but are not limited to, urinary tract infections and related conditions such as cystitis, urethritis, pyelonephritis, renal abscess, and asymptomatic bacteriuria. Urinary tract infections or related conditions may be associated with drainage devices such as catheters.

[0081] This method can be used to treat one or more symptoms associated with a urinary tract infection or related conditions. These symptoms include, but are not limited to, difficulty urinating (dysuria), urgency, hesitancy, frequency, polyuria, incomplete emptying, hematuria, incontinence, cloudy urine, or burning sensation during urination. Those skilled in the art will readily understand that the scope of this disclosure is not limited to these exemplary symptoms, and that this disclosure will cover other symptoms of urinary tract infection.

[0082] Inflammation or conditions of the skin or nails / toenails can be, for example, psoriasis, dermatitis, eczema, rosacea, acne, ichthyosis, fungal skin and / or nail / toenail infections, or other skin conditions characterized by or associated with inflammation, plaques, or skin lesions. Exemplary forms of psoriasis include plaque psoriasis, guttate psoriasis, and pustular psoriasis. Exemplary forms of dermatitis include atopic dermatitis, infantile dermatitis, seborrheic dermatitis, contact dermatitis, occupational dermatitis, hand dermatitis, nummular dermatitis, stasis dermatitis, perioral dermatitis, and herpetic dermatitis. Exemplary fungal infections include tinea pedis (athlete's foot), tinea cruris (ringworm of the groin), tinea capitis (tinea of ​​the scalp and head), tinea corporis (tinea of ​​the body), and onychomycosis (tinea of ​​the fingernails or toenails). Inflammation or inflammatory condition of the skin or nails / toenails can be caused by or associated with a bacterial, fungal, or viral infection.

[0083] Inflammatory joint conditions can be arthritis. Arthritis can be, for example, rheumatoid arthritis or osteoarthritis.

[0084] Embodiments of this disclosure also provide methods for suppressing or reducing inflammation or one or more symptoms associated with inflammation, particularly inflammation of the gastrointestinal tract, urinary tract, skin, nails / toenails, or joints. The term "inhibiting" and its variations such as "inhibition," "inhibits," "reduces," "reducing," etc., are used interchangeably herein to indicate the severity of inflammation or condition or infection, or an improvement (i.e., reduction) of at least one symptom of inflammation, condition, or infection.

[0085] Other exemplary inflammatory or autoimmune conditions include, for example, disorders such as rheumatic fever, chronic fatigue syndrome, systemic lupus erythematosus, Sjögren's syndrome, prostatitis, pelvic inflammatory disease, pancreatitis, vasculitis, foot inflammation including gout, and menstrual pain.

[0086] The methods disclosed herein also relate to the treatment or prevention of bacterial infections of the gastrointestinal tract, urinary tract, skin, nails / toenails, or joints. The method includes administering to a subject a species of *Lactobacillus*, and / or a culture supernatant or cell-free filtrate derived from a culture medium in which *Lactobacillus* has been cultured, said *Lactobacillus* species selected from *Lactobacillus bruneri*, *Lactobacillus zeatus*, *Lactobacillus rapi*, *Lactobacillus casei*, *Lactobacillus branii*, and *Lactobacillus diolivorans*.

[0087] The methods disclosed herein also relate to the treatment or prevention of bacterial infections that cause, induce, or otherwise relate to, inflammation or inflammatory conditions or autoimmune conditions of the gastrointestinal tract, urinary tract, skin, nails / toenails, or joints (such as those described herein). In this document, “inducing” means stimulating or promoting the development or worsening of inflammation or inflammatory conditions or autoimmune conditions, optionally in conjunction with one or more other factors. The method includes administering to a subject a species of *Lactobacillus*, and / or a culture supernatant or cell-free filtrate derived from a culture medium in which *Lactobacillus* has been cultured, selected from *Lactobacillus bruneri*, *Lactobacillus zeatus*, *Lactobacillus rapi*, *Lactobacillus casei*, *Lactobacillus furfur*, and *Lactobacillus diolivorans*.

[0088] The bacterial infections described in this disclosure can be caused by pathogenic Gram-negative or Gram-positive bacteria. Exemplary pathogens include, but are not limited to, members of the family Enterobacteriaceae, such as, for example, *Escherichia coli*, species of *Yersinia*, species of *Enterobacter*, species of *Salmonella*, species of *Shigella*, species of *Klebsiella*, species of *Proteus*, and species of *Citrobacter*. Exemplary Enterobacteriaceae include Adhesive-Invasive Escherichia coli (AIEC strains), Enteropathogenic Escherichia coli (EPEC strains), Enterotoxigenic Escherichia coli (ETEC strains), Enterohemorrhagic Escherichia coli (EHEC strains), Uropathogenic Escherichia coli (UPEC strains), Yersinia enterocolitica, Enterobacter cloacae, Salmonella typhimurium, Salmonella enterica, Salmonella enteridis, Shigella flexneri, Shigella boydii, Shigella sonnei, Shigella dysenteriae, Klebsiella acidogenic, and Proteus mirabilis.Other exemplary bacterial pathogens to which the methods of this disclosure may be applied include species of the genus *Helicobacter* such as *Helicobacter pylori*, species of the genus *Campylobacter* such as *Campylobacter jejuni*, species of the genus *Pseudomonas* such as *Pseudomonas aeruginosa*, species of the genus *Vibrio* such as *Vibrio cholerae*, species of the genus *Clostridium* such as *Clostridium difficile*, species of the genus *Streptococcus* such as *Streptococcus mutans*, *Streptococcus pyogenes*, and other group A (hemolytic) and group B streptococci, and species of the genus *Staphylococcus* such as *Staphylococcus aureus*. aureus and saprophytic staphylococci, Enterococcus species such as Enterococcus faecalis and Enterococcus faecium, and Mycobacterium species such as Mycobacterium avium subspecies paratuberculosis.

[0089] Gastrointestinal infections treatable according to this disclosure can be caused by one or more of the following: *Escherichia coli*, *Escherichia coli AIEC* strain, *Yersinia enterocolitica*, *Salmonella typhimurium*, *Salmonella enteritidis*, *Shigella flexneri*, *Shigella boydii*, *Shigella sonnei*, *Shigella dysenteriae*, *Klebsiella pneumoniae*, *Proteus mirabilis*, *Helicobacter pylori*, *Pseudomonas aeruginosa*, *Campylobacter jejuni*, *Vibrio cholerae*, *Clostridium difficile*, *Streptococcus mutans*, *Streptococcus pyogenes*, Group A (hemolytic) Streptococcus, *Staphylococcus aureus*, and *Mycobacterium avium* subsp. *paratuberculosis*. Oral and pharyngeal infections treatable according to this disclosure can be caused by one or more of the following: *Streptococcus mutans*, *Streptococcus pyogenes*, and other Group A (hemolytic) Streptococcus. Urinary tract infections that can be treated according to this disclosure can be caused by one or more of the following: Escherichia coli UPEC strains, Enterococcus faecalis, Enterococcus faecium, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Group B Streptococcus, Staphylococcus aureus, and Staphylococcus saprophyticus.

[0090] This article also provides methods for inhibiting or preventing bacterial pathogens from adhering to the gastrointestinal mucosa of a subject, and methods for inhibiting or preventing the invasion of the gastrointestinal epithelial cells of a subject by bacterial pathogens. The methods include administering to the subject a species of *Lactobacillus*, and / or a culture supernatant or cell-free filtrate derived from a culture medium in which *Lactobacillus* has been cultured, wherein the *Lactobacillus* species is selected from *Lactobacillus bruneri*, *Lactobacillus zeatus*, *Lactobacillus rapi*, *Lactobacillus casei*, *Lactobacillus furfur*, and *Lactobacillus diolivorans*.

[0091] In this document, as used herein, “inhibit”, “inhibiting”, etc., refer to the reduction in adhesion and / or invasion of bacterial pathogens onto the gastrointestinal epithelial lining or cells in the presence of one or more Lactobacillus species as defined herein, compared to the absence of Lactobacillus species as defined herein.

[0092] Bacterial pathogens can adhere to the mucosal epithelial lining of any part of the gastrointestinal tract and / or invade the epithelial cells lining any part of the gastrointestinal tract. In exemplary embodiments, the bacterial pathogen can be a member of the Enterobacteriaceae family, such as, for example, *Escherichia coli*, *Yersinia enterocolitica*, *Salmonella* species, or *Shigella* species. *Escherichia coli* can be, for example, adherent-invasive *Escherichia coli* (AIEC), enteropathogenic *Escherichia coli* (EPEC), enterotoxigenic *Escherichia coli* (ETEC), or enterohemorrhagic *Escherichia coli* (EHEC). *Salmonella* species can be, for example, *Salmonella typhimurium*, *Salmonella enteritidis*, or *Salmonella enteritidis*. *Shigella* species can be, for example, *Shigella flexneri*, *Shigella boydii*, *Shigella sonnei*, or *Shigella dysenteriae*.

[0093] The methods of this disclosure also relate to the treatment or prevention of bacterial infections of the gastrointestinal tract, such as infections that cause, induce, or may otherwise be associated with inflammation or inflammatory conditions and autoimmune conditions of the gastrointestinal tract, as exemplified above. In this document, “inducing” means stimulating or promoting the development or worsening of inflammation or inflammatory conditions or autoimmune conditions, optionally in conjunction with one or more other factors. The method includes administering to a subject a species of *Lactobacillus*, and / or a culture supernatant or cell-free filtrate derived from a culture medium in which *Lactobacillus* has been cultured, selected from *Lactobacillus bruneri*, *Lactobacillus zeatus*, *Lactobacillus rapi*, *Lactobacillus casei*, *Lactobacillus branii*, and *Lactobacillus diolivorans*.

[0094] This article also provides a method for promoting wound healing, the method comprising applying a species of Lactobacillus, and / or a culture supernatant or cell-free filtrate derived from a culture medium in which Lactobacillus has been cultured, the species being selected from Lactobacillus bruneri, Lactobacillus zeatus, Lactobacillus rapi, Lactobacillus casei, Lactobacillus glutenophilus, and Lactobacillus diolivorans.

[0095] As used herein in the context of wound healing, the terms “promoting,” “promotion,” and variations thereof refer to the ability of the combinations or compositions disclosed herein to induce, enhance, or otherwise promote natural processes associated with wound healing and / or tissue regeneration related to wound healing. In embodiments, promotion can be relative to healing observed in the absence of application of the combination or composition. Promotion can be direct or indirect. It will be understood that in indirect promotion of wound healing, the combination or composition can affect the expression or activity of molecules that themselves directly or indirectly regulate or otherwise influence the wound healing or tissue regeneration process. Promotion can be qualitative, quantitative, and / or temporal. That is, for example, application of the combination or composition can result in faster wound healing and / or tissue regeneration compared to what would occur without such application.

[0096] A wound can be, for example, a surgical wound, an incision, or a superficial wound such as a cut, abrasion, contusion, or bruise. A wound can also be a chronic wound, such as a pressure sore, a pressure ulcer, a diabetic foot ulcer, or a severe burn.

[0097] The method of this disclosure employs one or more of the following: Lactobacillus species selected from *Lactobacillus fragrans*, *Lactobacillus brunelli*, *Lactobacillus zeatus*, *L. rapi*, *Lactobacillus casei*, and *L. diolivorans*, and compositions comprising one or more of these species. Due to some taxonomic divergences and uncertainties, *Lactobacillus zeatus* may also be referred to elsewhere as *Lactobacillus casei*. However, for the purposes of this disclosure, the name *Lactobacillus zeatus* is retained.

[0098] The methods disclosed herein may include applying two, three, four, five, or all six species of the genus *Lactobacillus*, *Lactobacillus zeatus*, *Lactobacillus rapi*, *Lactobacillus casei*, *Lactobacillus branii*, and *Lactobacillus diolivorans*, or culture supernatants or cell-free filtrates derived from culture media in which two, three, four, five, or all six species of said lactobacilli have been cultured. In such embodiments, the bacteria may be cultured together or separately.

[0099] Lactobacillus glutenosa can be Lactobacillus glutenosa Lp18, previously described in WO2013 / 063658 and available under accession number V11 / 022945. Lactobacillus glutenosa can also be Lactobacillus glutenosa SVT-18 (which may be referred to elsewhere as the alternative name SVT-05P2) deposited on February 27, 2019, under the Budapest Treaty, with accession number LMGP-31292.

[0100] Lactobacillus brunetti can be Lactobacillus brunetti Lb23, previously described in WO2013 / 063658 and available under accession number V11 / 022946. Lactobacillus brunetti can also be Lactobacillus brunetti SVT-23 (which may be referred to elsewhere as the alternative name SVT-06B1), deposited on February 27, 2019, under the Budapest Treaty with accession number LMG P-31293 at the Belgian Centre for Harmonized Microbiological Collections (BCCM).

[0101] Lactobacillus zeatus can be Lactobacillus zeatus Lz26, previously described in WO2013 / 063658 and available under accession number V11 / 022948. Lactobacillus zeatus can also be Lactobacillus zeatus SVT-26 (which may be referred to elsewhere as the alternative name SVT-08Z1) deposited on 27 February 2019 at the Belgian Centre for Harmonized Microbial Collections (BCCM) under the Budapest Treaty.

[0102] L. rapi can be L. rapi Lr24, previously described in WO2013 / 063658 and available under accession number V11 / 022947. L. rapi can also be L. rapi SVT-24 (which may be referred to elsewhere as the alternative name SVT-07R1), deposited on 27 February 2019 at the Belgian Centre for the Coordination of Microbial Collections (BCCM) under the Budapest Treaty, with accession number LMG P-31294.

[0103] Lactobacillus casei can be Lactobacillus casei Lp9 (designated as strain "T9" therein), previously described in WO2014 / 172758 and available under accession number V12 / 022849. Lactobacillus casei can also be Lactobacillus casei SVT-09 (which may be referred to elsewhere as the alternative name SVT-04P1), deposited on 27 February 2019 at the Belgian Centre for Harmonized Microbiological Collections (BCCM) under the Budapest Treaty.

[0104] Lactobacillus diolivorans can be Lactobacillus diolivorans Ld3 (as designated strain “N3”) previously described in WO2014 / 172758 and available under accession number V12 / 022847. L. diolivorans can be L. diolivorans SVT-03 (which may be referred to elsewhere as the alternative name SVT-01D1) deposited on 27 February 2019 at the Belgian Centre for the Coordination of Microbial Collections (BCCM) under the Budapest Treaty.

[0105] The concentration of a single *Lactobacillus* species to be applied according to the methods of this disclosure will depend on a variety of factors, including the identity and number of the single species used, the exact nature and severity of the inflammation, condition, or infection to be treated or prevented, the form of the composition applied, and the means of application. For any given case, an appropriate concentration can be determined by a person skilled in the art using only routine experiments. By way of example only, the concentration of *Lactobacillus* species, or the concentration of each species present in combination, can be from about 1 x 102 2 cfu / ml to approximately 1x10 11 cfu / ml, and can be approximately 1 x 10⁻⁶. 3 cfu / ml, approximately 2.5 x 10 3 cfu / ml, approximately 5 x 10 3 cfu / ml, 1x10 4 cfu / ml, approximately 2.5 x 10 4 cfu / ml, approximately 5 x 10 4 cfu / ml, 1x10 5 cfu / ml, approximately 2.5 x 10 5 cfu / ml, approximately 5 x 10 5 cfu / ml, 1x10 6 cfu / ml, approximately 2.5 x 10 6 cfu / ml, approximately 5 x 10 6 cfu / ml, 1x10 7 cfu / ml, approximately 2.5 x 10 7 cfu / ml, approximately 5 x 10 7 cfu / ml, 1x10 8 cfu / ml, approximately 2.5 x 10 8 cfu / ml, approximately 5 x 10 8 cfu / ml, 1x10 9 cfu / ml, approximately 2.5 x 10 9 cfu / ml, or approximately 5 x 109 cfu / ml, approximately 1×10 10 cfu / ml, approximately 1.5 × 10 10 cfu / ml, approximately 2.5 × 10 10 cfu / ml, approximately 5×10 10 cfu / ml or approximately 1×10 11 cfu / ml.

[0106] This disclosure also contemplates the use of variants of the *Lactobacillus* species described herein. As used herein, the term "variant" refers to both naturally occurring and specially developed variants or mutants of the species disclosed and illustrated herein. Variants may or may not have the same biological characteristics identified in the specific species illustrated herein, provided that they share similar advantageous properties in the treatment or prevention of inflammatory conditions. Illustrative examples of suitable methods for preparing variants illustrated herein include, but are not limited to, gene integration techniques, such as those mediated by inserting elements or transposons or by homologous recombination; other recombinant DNA techniques for modifying, inserting, deleting, activating, or silencing genes; intraspecific protoplast fusion; mutagenesis by irradiation with ultraviolet or X-rays or by treatment with chemical mutagens such as nitrosoguanidine, methyl methanesulfonate, nitrogen mustard, etc.; and phage-mediated transduction. Suitable and applicable methods are well known in the art and are described in particular in the following: for example, J.H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1972); J.H. Miller, A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1992); and J. Sambrook, D. Russell, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001).

[0107] As used herein, the term "variant" also encompasses microbial strains that are phylogenetically closely related to the species disclosed herein, and strains that share a large sequence identity with the species disclosed herein on one or more phylogenetic information markers, such as rRNA genes, elongation factor genes and initiation factor genes, RNA polymerase subunit genes, DNA gyrase genes, heat shock protein genes, and recA genes. For example, the 16S rRNA gene of a "variant" strain, as anticipated herein, may share approximately 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the strains disclosed herein.

[0108] According to this disclosure, the Lactobacillus species and combinations thereof described herein, or culture supernatants or cell-free filtrates derived from culture media, are typically applied in the form of a composition. In embodiments involving combinations of species or culture supernatants or cell-free filtrates derived from multiple species, those skilled in the art will understand that each species, supernatant, or filtrate to be applied does not need to be contained in the same composition. Where application is separate, it may be sequential or simultaneous.

[0109] The compositions used according to this disclosure can be prepared by mixing the relevant components and formulating the resulting mixture into a dosage form suitable for administration to a subject. Therefore, the composition may contain pharmaceutically acceptable carriers, diluents, excipients, and / or adjuvants. The carriers, diluents, excipients, and adjuvants must be "acceptable" in terms of compatibility with the other components of the composition and harmless to the subject receiving the composition. Methods for preparing suitable compositions for administration, as well as carriers, diluents, excipients, and adjuvants suitable for formulating compositions for topical, oral, or sublingual administration, are well known to those skilled in the art. In an exemplary embodiment, the composition is formulated with a carrier comprising sterile isotonic saline or 3% sucrose.

[0110] The composition may be administered via any convenient or suitable route, including but not limited to: oral, sublingual, sublingual, rectal, topical, intranasal, intraocular, mucosal, intestinal, enteric, intramuscular, subcutaneous, intramedullary, intrathecal, intravenous, intracerebral, intrabladder, intravenous, or intraperitoneal. A suitable route may depend, for example, on the nature and severity of the inflammation, condition, or infection to be treated or prevented, and on the site of the inflammation, condition, or infection. The composition may be administered in any suitable form, typically in solid or liquid form. For example, the composition may be formulated into tablets, troches, capsules, caplets, elixirs, suspensions, syrups, wafers, granules, powders, gels, pastes, solutions, creams, sprays, suspensions, soluble sachets, lozenges, effervescent tablets, chewable tablets, multilayer tablets, etc., using methods and techniques well known to those skilled in the art. For oral administration, lactobacillus or the composition can be readily incorporated into a variety of beverages, foods, nutritional products, nutritional supplements, food additives, pharmaceuticals, over-the-counter drug formulations, and animal feed supplements. For topical application, suitable media include, but are not limited to, lotions, liniments, gels, creams, ointments, foams, sprays, oils, powders, etc. Compositions, typically in liquid or semi-liquid form, can also be impregnated into transdermal patches, plasters, and wound dressings such as bandages or hydrocolloid dressings.

[0111] As those skilled in the art will understand, the choice of pharmaceutically acceptable carrier or diluent will depend on the route of administration, the nature and severity of the condition to be treated, and the subject. Those skilled in the art can readily determine a particular carrier or delivery system and route of administration. Those skilled in the art will be able to readily determine suitable formulations useful in the methods of this disclosure using conventional methods.

[0112] For example, the compositions of this disclosure can be formulated for application in a liquid form containing an acceptable diluent (such as saline and sterile water), or can be in the form of a lotion, cream, or gel containing an acceptable diluent or carrier to impart a desired texture, consistency, viscosity, and appearance. Acceptable diluents and carriers are well known to those skilled in the art and include, but are not limited to: ethoxylated and nonethoxylated surfactants, fatty alcohols, fatty acids, hydrocarbon oils (such as palm oil, coconut oil, and mineral oil), cocoa butter wax, silicone oil, pH balancers, cellulose derivatives, emulsifiers such as nonionic organic and inorganic bases, preservatives, wax esters, steroidal alcohols, triglycerides, phospholipids such as lecithin and cephalin, polyol esters, fatty alcohol esters, hydrophilic lanolin derivatives, and hydrophilic beeswax derivatives.

[0113] Alternatively, Lactobacillus can be readily formulated into a dose suitable for oral administration using pharmaceutically acceptable carriers well known in the art. These carriers may be selected from sugars, starches, cellulose and their derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffers, emulsifiers, isotonic saline, and pyrogen-free water.

[0114] Some examples of suitable carriers, diluents, excipients, and adjuvants for oral administration include liquid paraffin, sodium carboxymethyl cellulose, methylcellulose, sodium alginate, gum arabic, tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatin, and lecithin. In addition, these oral formulations may contain suitable flavoring and coloring agents. When used in capsule form, the capsules may be coated with delayed-disintegration compounds such as glyceryl monostearate or glyceryl distearate. Adjuvants typically include emollients, emulsifiers, thickeners, preservatives, bactericides, and buffers. For administration as injectable solutions or suspensions, non-toxic, parenteral-acceptable diluents or carriers may include Ringer's solution, isotonic saline, phosphate-buffered saline, ethanol, and 1,2-propylene glycol.

[0115] Solid forms intended for oral administration may contain binders, sweeteners, disintegrants, diluents, flavoring agents, coating agents, preservatives, lubricants, and / or time-retarding agents acceptable in human and veterinary pharmaceutical practice. Suitable binders include gum arabic, gelatin, corn starch, tragacanth gum, sodium alginate, carboxymethyl cellulose, or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame, or saccharin. Suitable disintegrants include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginate, or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate, or dicalcium phosphate. Suitable flavoring agents include peppermint oil, wintergreen oil, cherry flavoring, orange flavoring, or raspberry flavoring. Suitable coating agents include polymers or copolymers of acrylic acid and / or methacrylic acid and / or their esters, waxes, fatty alcohols, zein, shellac, or gluten. Suitable preservatives include sodium benzoate, vitamin E, α-tocopherol, ascorbic acid, methylparaben, propylparaben, or sodium bisulfite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride, or talc. Suitable time-retarding agents include glyceryl monostearate or glyceryl distearate.

[0116] In addition to the above-mentioned agents, liquid forms for oral administration may contain liquid carriers. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, arachisoil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerin, fatty alcohols, triglycerides, or mixtures thereof. Suspensions for oral administration may also contain dispersants and / or suspending agents. Suitable suspending agents include sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, sodium alginate, or acetyl alcohol. Suitable dispersants include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, polyoxyethylene sorbitan monooleate or dioleate, stearate or laurate, polyoxyethylene sorbitan monooleate or dioleate, stearate or laurate, etc. Emulsions for oral administration may also contain one or more emulsifiers. Suitable emulsifiers include dispersants or natural gums such as guar gum, gum arabic, or tragacanth gum, as exemplified above.

[0117] Methods for preparing suitable parenteral compositions will be well known to those skilled in the art and are described in more detail, for example, in Remington's Pharmaceutical Science, 15th edition, Mack Publishing Company, Easton, Pa., which are incorporated herein by reference.

[0118] Examples of pharmaceutically acceptable diluents for formulating compositions for topical application include demineralized or distilled water; saline solutions; and plant-based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, corn oil, sesame oil, and arachid oil. Oils such as coconut oil; silicone oils, including polysiloxanes such as methyl polysiloxane, phenyl polysiloxane, and methylphenyl polysiloxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin, or squalane; cellulose derivatives such as methylcellulose, ethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, or hydroxypropyl methylcellulose; lower alkanols such as ethanol or isopropanol; lower aryl alcohols; lower polyalkylene glycols or lower alkylene glycols such as polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butanediol, or glycerol; fatty acid esters such as isopropyl palmitate, isopropyl myristate, or ethyl oleate; polyvinylpyrrolidone; agar; carrageenan; gum arabic or gum arabic and petroleum jelly.

[0119] In another embodiment, the composition may also contain a suspending agent and / or a wetting agent, such as povidone or propylene glycol, and a neutralizing agent for adjusting the viscosity of the composition, such as sodium hydroxide, triethanolamine (TEA) or ethylenediaminetetraacetic acid (EDTA).

[0120] Depending on the condition to be treated or prevented, the severity of the condition, and the desired outcome, the compositions of this disclosure may be administered, for example, once a week or more, optionally, for example, once a week, every two days, once a day, twice a day, or three times a day. The duration of administration to the subject will also vary depending on the condition to be treated or prevented, the severity of the condition, and the desired outcome. The amount of composition administered to the subject will vary depending on a range of factors, including the identity of the microorganism administered, the nature and severity of the condition to be treated or prevented, the subject's age and general health, and the desired outcome. A suitable dosing regimen can be readily determined by a skilled addressee.

[0121] In an exemplary embodiment, about 1 ml to about 25 ml of a liquid preparation of Lactobacillus species can be dispensed at about 10 5 cfu / ml and 10 11 The final concentration between cfu / ml is administered to the subject on a basal basis once daily, twice daily, or more frequently. The volume of the liquid formulation may be, for example, about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 11 ml, 12 ml, 13 ml, 14 ml, 15 ml, 16 ml, 17 ml, 18 ml, 19 ml, 20 ml, 21 ml, 22 ml, 23 ml, 24 ml, or 25 ml.

[0122] Lactobacillus can be combined with other therapeutic agents, such as, but not limited to, antibiotics, antimicrobial agents, antiseptics, anesthetics, anti-infectives, anti-inflammatory agents, immunosuppressants, and other therapeutic agents indicated for the treatment of inflammatory conditions, such as steroids and NSAIDs. Regarding compositions containing microorganisms of the subject matter of this disclosure, the administration of such additional agents can be at the same time or at different times, i.e., simultaneously or sequentially, and can be administered via the same or different routes. The additional therapeutic agent can be co-formulated with the microorganisms used in the method.

[0123] Other non-limiting examples of anti-inflammatory agents that may be used include steroidal and non-steroidal compounds, such as clobetasol propionate, betamethasone dipropionate, halobetasol propionate, diflorasone diacetate, fluocinonide, halcinonide, amcinonide, desoximetasone, triamcinolone acetonide, mometasone furoate, fluticasone propionate, betamethasone dipropionate, and fluocinolone acetonide. The following are listed: acetonide, hydrocortisone valerate, hydrocortisone butyrate, flurandrenolide, triamcinolone, mometasone furoate, triamcinolone, fluticasone propionate, desonide, fluocinolone acetonide, hydrocortisone valerate, prednicarbate, triamcinolone, desonide, hydrocortisone, hydrocortisone aceponate, hydrocortisone buteprate, methylprednisolone aceponate, mometasone furoate, and prednicarbate.Non-limiting examples of suitable nonsteroidal anti-inflammatory compounds include indomethacin, ketoprofen, felbinac, diclofenac, ibuprofen, piroxicam, benzyldamin, and acetylsalicylic acid. acid), diflunisal, salsalate, naproxen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin, loxoprofen, indomethacin, sulindac, etodolac, ketorolac, diclofenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid Anti-inflammatory agents include citric acid, firocoxib, and licofelone; semi-synthetic glycosaminoglycan ethers; flavanols; flavonoids; isoflavones; and their derivatives. Anti-inflammatory agents can be inhibitors of cytokine signaling, such as cyclosporine A, 6-thioguanine, sulfasalazine, mesalazine (5-aminosalicylic acid), etanercept, prednisolone, or balsalazine.

[0124] The anti-infective agent can be any agent used to treat an infection in the subject. In a particular embodiment, the anti-infective agent is capable of killing or inhibiting the growth of an infectious organism capable of translocating integrally or partially between cells via apoptotic bodies. Suitable anti-infective agents include, but are not limited to, antiviral agents, antibacterial agents, antiprotozoal agents, antifungal agents, or combinations thereof.

[0125] Illustrative antiviral agents include, but are not limited to: abacavir sulfate, acyclovir, especially acyclovir sodium, adefovir, amantadine, especially amantadine hydrochloride, ampranavir, ampligen, atazanavir, cidofovir, darunavir, delavirdine, especially delavirdine mesylate, didanosine, docosanol, dolutegravir, edoxudine, and efavirenz. Efavirenz, emtricitabine, elvitegravir, enfuvirtide, entecavir, famciclovir, fomivirsen (especially fomivirsen sodium), foscarnet (especially foscarnet sodium), ganciclovir, ibacitabine, idoxuridine, imiquimod, indinavir (especially indinavir sulfate), and isoprotinin.pranobex, lamivudine, lopinavir, maraviroc, metisazone, moroxydine, nelfinavir (especially nelfinavir mesylate), nevirapine, nitazoxanide, oseltamivir (especially oseltamivir phosphate), penciclovir, peramivir, pleconaril, podophyllotoxin, raltegravir, ribavirin, rimantadine (especially rimantadine hydrochloride), ritonavir (… Saquinavir, especially saquinavir mesylate, sofosbuvir, stavudine, telaprivir, tenofovir, tipranavir, trifluridine, tromantadine, umifenovir, valacyclovir, especially valacyclovir hydrochloride, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, zidovudine, and pharmaceutically acceptable salts thereof and combinations thereof.

[0126] Illustrative antibacterial agents include, but are not limited to, quinolones (e.g., amifloxacin, cinoxacin, ciprofloxacin, enoxacin, fleroxacin, flumequine, lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin, lomefloxacin, oxolinic acid). The following antibiotics are listed: pefloxacin, rosoxacin, temafloxacin, tosufloxacin, sparfloxacin, clinafloxacin, gatifloxacin, moxifloxacin, gemifloxacin, and garenoxacin; tetracyclines, glycylcyclines, and oxazolidinones (e.g., chlortetracycline, demeclocycline, doxycycline, lymecycline, methacycline, minocycline). Line, oxytetracycline, tetracycline, tigecycline; linezolide, eperezolid, glycopeptides, aminoglycosides (e.g., amikacin, abekacin, butirosin, dibekacin, fortimicins, gentamicin, kanamycin, neomycin, netilmicin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin)β-lactams (e.g., imipenem, meropenem, biapenem, cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, cefixime, cefomenoxime, cefodizime, cefonicid, cefoperazone, cefrapor) Cefotaxime, cefotiam, cefpimizole, cefpiramide, cefpodoxime, cefsulodin, cefazidime, cefteram, ceftezole, ceftibuten, cefizoxime, ceftriaxone, cefuroxime, and cefzonam are all listed as potential treatments for cefotaxime. Cephacetrile, cephalexin, cephaloglycin, cephaloridine, cephalothin, cephapirin, cephradine, cefinetazole, cefoxitin, cefotetan, azthreonam, carumonam, flomoxef, moxalactam, mecillin (Idinocillin), amoxicillin, amoxicillin, azlocillin, carbenicillin, benzylpenicillin, carfecillin, cloxacillin, dicloxacillin, methicillin, mezlocillin, nafcillin, oxacillin, penicillin G)Piperacillin, sulbenicillin, temocillin, ticarcillin, cefditoren, SC004, KY-020, cefdinir, ceftibuten, FK-312, S-1090, CP-0467, BK-218, FK-037, DQ-2556, FK-518, cefozopran, ME1228, KP-736, CP-6232, Ro 09-1227, OPC-20000, LY206763), rifamycins, macrolides (e.g., azithromycin, clarithromycin, erythromycin, oleandomycin, rokitamycin, rosaramicin, roxithromycin, troleandomycin), ketones Ketolides (e.g., telithromycin, cethromycin), coumamicins, lincosamides (e.g., clindamycin, lincomycin), chloramphenicol, clofazimine, cycloserine, dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide, rifabutin, rifampin, rifapentine, and streptomycin sulfate.

[0127] Illustrative antigenic agents include, but are not limited to, atovaquone, metronidazole (including metronidazole hydrochloride), pentamidine (including pentamidine hydroxyethyl sulfonate), chloroquine (including chloroquine hydrochloride and chloroquine phosphate), doxycycline, and hydroxychloroquine sulfate. sulfate, mefloquine (including mefloquine hydrochloride), primaquine (including primaquine phosphate), pyrimethamine, pyrimethamine and sulfadoxine, trimethoprim, sulfamethoxazole, clindamycin, quinine, quinidine, sulfadiazine, artemether, lumefantrine, artesunate, nizozine, suramin, melarsoprol, eflornithine, nifurtimox, stibogluconate (including sodium antimony gluconate), amphotericin B B (including amphotericin B liposomes), miltefosine, paromomycin, ketoconazole, itraconazole, fluconazole, and pharmaceutically acceptable salts thereof and combinations thereof.

[0128] Illustrative antifungal agents include, but are not limited to, abafungin, abaconazole, amorolfine, amphotericin B, amphotericin B cholesterol sulfate complex, amphotericin B lipid complex, amphotericin B liposome, anidulafungin, bifonazole, butenafine, butoconazole, candicidin, caspofungin, clotrimazole, econazole, efinaconazole, fenticonazole, fluconazole, flucytosine, griseofulvin microsize, and griseofulvin ultrafine. ultramicrosize), hamycin, isavuconazole, isoconazole, itraconazole, ketoconazole, luliconazole, micafungin, miconazole, naftifine, natamycin, nystatin, omoconazole, oxiconazole, posaconazole, propiconazole, ravuconazole, sertaconazole, sulconazole, terbinafine (including terbinafine hydrochloride), terconazole, tioconazole, voriconazole, and pharmaceutically acceptable salts thereof and combinations thereof.

[0129] Illustrative immunosuppressants include, but are not limited to: corticosteroids, such as budesonide, prednisone, and prednisolone; mTOR inhibitors, such as sirolimus and everolimus; and monoclonal antibodies, such as adalimumab, infliximab, certolizumab, natalizumab, ustekinumab, and vedolizumab, and their biosimilars.

[0130] In exemplary embodiments, the lactobacilli described herein are provided and administered in the form of a microbial biotherapy composition. Such compositions may also contain one or more additional microorganisms, such as, for example, *Lactobacillus rhamnosus*, *Lactobacillus plantarum*, *Lactobacillus bulgaricus*, *Lactobacillus casei*, *Lactobacillus acidophilus*, *Lactobacillus fermentum*, *Lactococcus lactis*, *Streptococcus thermophilus*, *Bifidobacterium breve*, *Bifidobacterium bifidum*, *Bifidobacterium lactis*, and *Bifidobacterium animalis*.

[0131] Microbial biotherapy compositions may contain one or more prebiotic components. Suitable prebiotics include, for example, polydextrose, inulin, fructooligosaccharides (FOS), xylooligosaccharides (XOS), galactosylooligosaccharides (GOS), mannan oligosaccharides, protein-based green-lipped mussel extract, and various prebiotic-containing foods such as raw onions, raw leeks, raw chicory roots, and raw artichokes. In some embodiments, the prebiotic is fructooligosaccharides.

[0132] The compositions containing lactobacillus described herein can be administered in any suitable form (any dosage form described above). Microbial biotherapy compositions can be provided to the user in powder form, suitable for mixing by the user into any type of beverage or food (e.g., water, juice, or yogurt), or suitable for consumption as a powder in the absence of a beverage or other food. Therefore, microbial biotherapy compositions can be conveniently incorporated into a variety of food and / or beverage products, nutritional products, supplements, food additives, and over-the-counter pharmaceutical preparations. Foods or food additives can be in solid forms such as powders or liquid forms. Specific examples of beverage or food types include, but are not limited to, water-based, milk-based, yogurt-based, other dairy-based, milk-based alternatives such as soy milk or oat milk, or fruit juice-based beverages; water, soft drinks, carbonated beverages, and nutritional beverages (including concentrates of beverages and dry powders used to prepare such beverages); baked goods such as crackers, bread, muffins, rolls, bagels, biscuits, cereals, bars such as muesli bars, health food bars, etc.; condiments, sauces, custards, yogurt, puddings, pre-packaged frozen meals, soups, and confectionery.

[0133] References to any prior publications (or information derived therefrom) or to any known matter in this specification are not and should not be construed as an endorsement or acknowledgment or in any way imply that such prior publications (or information derived therefrom) or known matters constitute part of the general knowledge in the field covered by this specification.

[0134] The present disclosure will now be described with reference to the following specific embodiments, which should not be construed as limiting the scope of the invention in any way. Example

[0135] The following examples are illustrative of the invention and should not be construed as limiting the general nature of the disclosure described herein in any way.

[0136] Example 1 – Adhesion and Intrusion of AIEC

[0137] The ability of *Lactobacillus* species *Lactobacillus fragrans* Lp18, *Lactobacillus bruneri* Lb23, *L. rapi* Lr24, *Lactobacillus zei* Lz26, *Lactobacillus casei* Lp9, and *L. diolivorans* Ld3 to competitively inhibit the adhesion and invasion (and translocation) of pathogenic *Escherichia coli* AIEC strains in the digestive tract epithelium was tested. *Lactobacillus* strains were maintained at -80°C in de Man, Rogosa, Sharpe (MRS) broth containing 20% ​​glycerol. They were grown on MRS agar as working cultures and regrown in MRS broth at 37°C for 18 hours prior to each adhesion or invasion assay.

[0138] The AIEC strain (strain F44A-1) is a wild-type strain isolated from a patient with inflammatory bowel disease and contains all the virulence genes associated with AIEC, and conforms to the AIEC diffusion and adhesion pattern to Caco-2 cells as well as its survival and replication in macrophages.

[0139] To evaluate the ability of each *Lactobacillus* species to inhibit interactions between these species and the human digestive tract epithelium, the cell lines Caco-2 (ATCC HTB-37) and HT29 (ATCC HTB-38) MTX, representing the digestive tract epithelium, were used. Caco-2 cells differentiate in culture medium to form a polarized cell monolayer with tight junctions and microvilli, a key feature of mature human intestinal cells. HT29-MTX was mutated to produce mucin, similar to goblet cells of the digestive tract epithelium. Cells were cultured in 50 ml flasks in Eagle Minimal Essential Medium supplemented with 20% (v / v) fetal bovine serum (FBS) (for Caco-2 cells), 15% (v / v) FBS (for HT29-MTX cells), and 1% (v / v) penicillin-streptomycin until confluence. Cell cultures were maintained at 37°C under a 5% CO2 atmosphere. The culture medium was closely monitored and changed every 48 h. At confluence, cells were passaged into 8-well slides for adhesion assays and sterile 96-well plates for invasion assays.

[0140] Adhesion Measurement

[0141] Prior to the adhesion assay, cell lines were seeded onto an 8-well glass slide system. Cells were grown to ~75% confluence, and the medium was changed to antibiotic-free medium before assay. AIEC isolates were cultured in LB broth for 4 hours in 15 ml tubes, while Lactobacillus strains were cultured overnight in MRS broth at 37°C with stirring (140 strokes / min). The cell suspension was centrifuged at 3,500 rpm for 12 minutes, and the supernatant was discarded. The precipitate was resuspended in phosphate-buffered saline (PBS) (pH 7.4), and 100 μl of the suspension (~1.0 × 10⁻⁶) was added to the culture medium.9 (cfu / ml, OD=1 at 600nm) inoculated into an appropriate chamber.

[0142] For competitive adhesion, *Lactobacillus* and AIEC were inoculated together and then incubated at 37°C for 90 min. For the ability of *Lactobacillus* to exclude AIEC, a suspension of *Lactobacillus* (~1.0 × 10⁻⁶) was used. 9 The cells were inoculated with cfu / ml for 60 min, then infected with the same concentration of AIEC, and incubated at 37°C for 90 min. After incubation, unattached bacterial cells were washed away with PBS (pH 7.4), and the cells were fixed with 95% ethanol (v / v) for 5 min. Gram staining was used to distinguish between Gram-positive lactobacilli and Gram-negative AIEC, and the cells were observed under a light microscope. The percentage of microbial adhesion to the cell lines was determined by counting the number of cells showing adhesion in 100 randomly selected cells, while the number of bacteria adhering to each cell was determined by counting the number of bacteria attached to 25 randomly selected cells.

[0143] Invasive assay

[0144] For the invasion assay, bacteria were cultured as described in the adhesion assay and inoculated onto confluent Caco-2 and HT29-MTX cells grown in 96-well plates. For the invasion assay, bacterial isolates were cultured similarly to those used in the adhesion assay, except that the bacterial concentration was adjusted to (~1×10⁻⁶). 8 (cfu / ml). For competitive invasion, Lactobacillus and AIEC were simultaneously inoculated and then incubated at 37°C for 2 hours. To assess the ability of Lactobacillus to inhibit / reduce the invasion rate of AIEC, Lactobacillus was first inoculated and cultured for 60 min, followed by AIEC inoculation and culture at 37°C for an additional 2 hours.

[0145] After incubation, the monolayer was washed three times with PBS and then incubated in EMEM containing gentamicin (150 μg / ml) at 37°C for 1 hour to kill non-invasive cells. The monolayer was then washed three times with PBS and lysed with 0.1% (v / v) Triton X-100 to release invading AIECs. The lysates were then serially diluted, and 100 μl volumes were plated onto MacConkey Agar 3 plates and incubated at 37°C for 24 hours, after which colonies were counted. The mean ± SEM of colony-forming units was calculated, and dilution factors were adjusted appropriately. *E. coli* strain 46-4 was used as a negative control for both assays.

[0146] Statistical analysis

[0147] All experiments were performed in triplicate. Statistical analysis was conducted using GraphPad Prism statistical software (version 8.0.0). Two-way ANOVA followed by Tukey's multiple comparison test was used to determine the differences in average adhesion and invasion levels among all test groups of strains. The relationship between strain adhesion and invasion abilities was assessed using Pearson correlation coefficients, and differences were considered statistically significant if P < 0.05.

[0148] result

[0149] After inoculation onto both cell lines, AIEC colonized 55% of the cells, significantly more than the tested Lactobacillus strains (32%–52%), except for L. rapi SVT-24. Both co-inoculation and pre-inoculation with Lactobacillus strains significantly reduced the colonization rate of AIEC on the HT29-MTX cell line (…). Figure 1 ) and Caco-2 cell line ( Figure 2 The adhesion of the two. In all cases, pre-inoculation reduced AIEC adhesion to a greater extent than co-inoculation.

[0150] The ability of Lactobacillus species to reduce AIEC invasion was also measured. Both co-inoculation and pre-inoculation with Lactobacillus strains significantly reduced AIEC invasion in the HT29-MTX cell line. Except for SVT 06B1, pre-inoculation reduced invasion to a greater extent in most cases. Figure 3 Regardless of whether co-inoculation or pre-inoculation was performed, no significant reduction in AIEC invasion was observed in the Caco-2 cell line (data not shown).

[0151] As illustrated in this example, the six Lactobacillus species tested in this study interacted with two different digestive tract epithelial cell lines, significantly reducing AIEC adhesion and invasion. Pre-incubating the cell lines with Lactobacillus followed by challenge with the pathogen further facilitated the competitive elimination of AIEC, suggesting that these Lactobacillus species can be used as a preventative measure.

[0152] Example 2 - Adhesion, invasion, and translocation of pathogenic Escherichia coli HMLN-1

[0153] Using the same assays and procedures described in Example 1, the effects of six Lactobacillus strains on the adhesion and invasion of pathogenic Escherichia coli strain HMLN-1 were determined. HMLN-1 strain was isolated from the blood and mesenteric lymph nodes of a hospitalized patient with a fatal case and has been shown as a specific translocation strain in numerous publications.

[0154] In addition, translocation was evaluated. Specifically, a two-compartment model was used to assess the translocation capacity of HMLN-1 cells across Caco-2 and HT29-MTX cells in the presence of Lactobacillus strains. Cells were grown in EMEM supplemented with 20% (v / v) FBS (for Caco-2 cells) and 15% (v / v) FBS (for HT29-MTX cells), both combined with 1% (v / v) penicillin-streptomycin, onto the inserts of 24-well plates with porous membranes. Cell lines were cultured to confluence, and the medium was replaced with antibiotic-free EMEM before bacterial inoculation. Inhibition of HMLN-1 translocation was tested in the presence of Lactobacillus strains, either co-inoculated with HMLN-1 or pre-inoculated prior to HMLN-1 infection. After incubation, 100 μL of EMEM was collected from the outer wells and seeded onto MacConkey agar plates and incubated at 37°C for 24 h. Cell counts were performed and expressed as mean ± SEM.

[0155] Escherichia coli strain JM109 was used as a negative control for adhesion, invasion, and translocation assays. Adhesion and invasion were performed in triplicate, and translocation was performed in duplicate.

[0156] The percentage of HT29-MTX cells and Caco-2 cells with HMLN-1 adhesion, either alone or in the presence of Lactobacillus, is shown in the figure. Figure 4 In both co-inoculation and pre-inoculation with all Lactobacillus strains, the percentage and number of HMLN-1 cells adhering to the HT29-MTX cell line were statistically significantly reduced. Both co-inoculation and pre-inoculation with Lactobacillus strains significantly reduced HMLN-1 invasion into the HT29-MTX cell line. Figure 5 In most cases, HMLN-1 translocation was significantly reduced in both co-seeded and pre-seeded cell lines. Figure 6 ).

[0157] Example 3 - Adhesion and invasion of pathogenic Escherichia coli in a modified digestive tract epithelial model

[0158] An improved digestive tract epithelial model was developed by co-culturing Caco-2 cells and HT29-MTX cells at a ratio of 9:1, respectively. This model exhibits many characteristics similar to human digestive tract epithelium. The ability of Lactobacillus species—Lactobacillus flavus, Lactobacillus brunelli, Lactobacillus zeatus, Lactobacillus casei, and L. diolivorans—to competitively inhibit the adhesion and invasion (and translocation) of two pathogenic Escherichia coli strains, Escherichia coli AIEC (F44A-1) and Escherichia coli HMLN-1, was tested in this model.

[0159] The experimental conditions were as described in Examples 1 and 2.

[0160] As positive controls, *E. coli* strains AIEC and HMLN-1 were incubated with co-cultured Caco-2 and HT29-MTX cell lines, and adhesion and invasion levels were measured in CFU / mL. As negative controls, *E. coli* cells were incubated with Caco-2 / HT29-MTX cells instead of AIEC and HMLN-1 strains. *Lactobacillus* species were tested and co-incubated with Caco-2 / HT29-MTX cells along with the *E. coli* strains, or pre-incubated with Caco-2 / HT29-MTX cells before adding the *E. coli* strains.

[0161] When co-inoculated and pre-inoculated, the percentages of HMLN-1 and AIEC adhesion on co-cultured cell lines of all Lactobacillus strains were statistically significantly reduced (see [link to relevant documentation]). Figure 7 A). However, for both co-inoculation and pre-inoculation, only SVT04P1 showed a statistically significant decrease in HMLN-1 and AEIC cell adhesion per co-cultured cell (see A). Figure 7 B). When co-inoculated with and pre-inoculated with all Lactobacillus strains, HMLN-1 invasion was statistically significantly reduced (see [link]). Figure 8 The translocation of HMLN-1 and AIEC during co-inoculation and pre-inoculation with Lactobacillus strains was statistically significantly reduced compared to the positive control. Figure 9 ).

[0162] The results described in Examples 1, 2, and 3 show that the studied *Lactobacillus* strains exhibited good adhesion to all tested gastrointestinal-like cell lines and statistically reduced the adhesion of pathogenic *Escherichia coli* strains F44A-1 (AIEC) and HMLN-1 to all cell lines. The *Lactobacillus* strains significantly reduced the invasion of AIEC into HT29-MTX and co-cultured cell lines; reduced the invasion of HMLN-1 into all cell lines; and significantly reduced the translocation of both *E. coli* strains across all tested cell lines. These results indicate that these *Lactobacillus* strains have therapeutic potential to reduce pathogenic *E. coli* infection and invasion into the bloodstream in the human gastrointestinal tract.

[0163] Example 4 – DSS-induced colitis model

[0164] The inventors then examined the effects of *Lactobacillus casei* (SVT04P1), *Lactobacillus brunelli* (SVT06B1), and *Lactobacillus zeatus* (SVT08Z1) in a dextran sulfate sodium (DSS)-induced mouse model of chronic colitis. The DSS was obtained from MP BioMedicals and stored at room temperature. Based on the DSS concentration used (3%) and three 5-day cycles of DSS administration with two 7-day clearance periods, the model used in this study was a particularly effective model of severe chronic colitis. Mice administered 3% DSS according to this protocol showed significant histopathological signs of colonic ulceration, edema, inflammation, and crypt loss (data not shown).

[0165] Sixty female C57BL / 6NTac mice were divided into six treatment groups:

[0166] Group 1 – Non-treatment group (negative control group). N = 10.

[0167] Group 2 – 3% DSS + medium (9% sterile saline). N = 10.

[0168] Group 3 – 3% DSS+ dose was 1.5 × 10⁻⁶ 10 SVT04P1 cfu / ml. N=10.

[0169] Group 4 – 3% DSS+ dose was 1.5 × 10⁻⁶ 10 SVT06B1 cfu / ml. N=10.

[0170] Group 5 – 3% DSS+ dose was 1.5 × 10⁻⁶. 10 SVT08Z1 cfu / ml. N=10.

[0171] Group 6 – 3% DSS+ dose was 1.5 × 10⁻⁶ 10 A combination of SVT04P1, SVT06B1, and SVT04P1 at cfu / ml. N = 10.

[0172] Animals in groups 2 through 6 received 3% DSS freely via sterile drinking water from days 1 to 5, 13 to 17, and 25 to 29, while animals in group 1 continued to receive sterile water only as drinking water. Animals in groups 2 through 6 also received the medium or Lactobacillus spp. (1.5 × 10⁻⁶) orally via tube feeding from day 1 to 28. 10(cfu / ml). On days when DSS was not provided to groups 2 through 6, animals received sterile water. Lactobacillus was prepared in sterile saline and 2%–3% sucrose (stored at 4°C until use). Cell viability / counts of each bacterial preparation were analyzed prior to administration. On day 1 of administration, a fresh 3% DSS solution was prepared by dissolving the DSS in sterile water.

[0173] Symptoms / characteristics of DSS-induced colitis (stool consistency and hematochezia) were assessed by measuring vital endpoints every other day from day 1 to day 29. On the day of administration, assessment was performed 1 to 2 hours after administration.

[0174] Fecal samples were collected from each mouse starting from day 1, and their consistency was examined. Fecal consistency was graded as follows: Normal = 0; Soft, but still formed = 1; Very soft = 2; Diarrhea = 4. Blood in the feces was detected using a Hemoccult Tape test kit (Beckman Coulter, according to manufacturer's instructions). Blood in the feces was graded as follows: Negative hemoccult = 0; Positive hemoccult (slight color on the strip) = 1; Positive hemoccult (darker color on the strip) = 2; Visible bloodstains = 3; Major rectal bleeding = 4. Percentage weight loss was also measured starting from day 1 and graded as 0 (none), 1 (1%–5%), 2 (>5%–10%), and 3 (>10%–20%).

[0175] Stool consistency score, rectal bleeding score, and weight loss score were combined to give a weighted vital score of overall disease status, namely the Disease Activity Index (DAI).

[0176] like Figure 10 As shown, compared with group 2, mice in treatment groups 3 to 6 showed significantly improved stool consistency. Compared with group 2, the incidence of fecal bleeding was reduced in groups 3, 4, and 6, with the most significant reduction in treatment group 6, which represents a combination of SVT-09, SVT-23, and SVT-26. Figure 11 Compared to group 2, DAI was also improved in treatment groups 4 and 6, with the most significant improvement observed in the combination therapy group (group 6). Figure 12 ).

[0177] Mice were sacrificed on day 29. Terminal blood samples showed a slight decrease in serum IL-6 concentrations in groups 3, 5, and 6 compared to control group 2, and a decrease in KC / Gro (rodent equivalent of IL-8) in groups 3 and 5 compared to control group 2 (data not shown). As shown in Table 1, there was also a trend toward increased colon length in each of the treatment groups 3 through 6 compared to control group 2.

[0178] Table 1. Colon length at the end

[0179] Group 1 94.6(1.34) Group 2 67.8(1.67) Group 3 70.6(1.34) Group 4 70.7(1.56) Group 5 70.1(2.15) Group 6 72.9(1.13)

[0180] Many agents used clinically to treat inflammatory bowel diseases such as ulcerative colitis (including cyclosporine A, sulfasalazine, and prednisolone) have previously been tested by the same testing laboratory (Charles River Laboratories (CRL)) in the same DSS-induced colitis model used in this study. None of these drugs statistically improved the disease-associated illness (DAI) score to the extent observed in the Lactobacillus treatment in this study. For example, as shown herein, the DAI score of group 6 was compared with previous data obtained by CRL that evaluated the efficacy of typical compounds used clinically to treat ulcerative colitis in a DSS mouse model. Sulfasalazine, prednisolone, 5-aminosalicylic acid, and 6-thioguanine failed to show therapeutic efficacy, while in the chronic DSS model, only cyclosporine A at 40 mg / kg and 80 mg / kg showed some reduction in the DAI score, but not as effectively as group 6 in this example. Figure 13 Data for 5-aminosalicylic acid and 6-thioguanine are not shown. Importantly, the efficacy of the selected drugs was tested in a 2% DSS model, which is a disease model with lower severity than the (3% DSS) used in this study. The results obtained in this study represent a significant advance in the prospective treatment of ulcerative colitis when compared with existing therapies.

[0181] Example 4A

[0182] As described above, other combinations of Lactobacillus strains were tested in a DSS-induced ulcerative colitis model:

[0183] • Group 7 – 3% DSS+ dose was 1.5 × 10 10 A combination of SVT01D1, SVT05P2, and SVT06B1 at cfu / ml. N = 10

[0184] As described above for groups 1 through 6, determine the occurrence of rectal bleeding, stool consistency, and DAI in group 7. Figure 14 The data shown are compared with those of Group 1, Group 2, and Group 6 (each as described in Example 4). A statistically significant reduction in rectal bleeding and improvement in DAI were observed.

[0185] Cytokine expression was also measured. Statistically significant increases in IL-6 and TNFα were observed in group 2 compared to group 1. Decreases in IL-6 expression were observed in groups 6 and 7 compared to group 2, and statistically significant decreases in TNFα expression were observed in groups 6 and 7 compared to group 2 (p<0.001 and p<0.05, respectively). Figure 15 ).

[0186] The overall ulceration severity of colon samples was analyzed, including the percentage of sections affected by any inflammatory changes, the percentage of sections affected by severe inflammatory changes such as loss of normal structure, erosion / ulceration, and / or crypt abscesses, and a total composite score calculated by summing three individual scores for each colon segment. Scores were calculated for each of the proximal, intermediate, and distal sections of the colon samples. The overall composite score showed a statistically significant reduction in ulceration and inflammation in Group 7 compared to Group 2, and statistically significant reductions were observed in the proximal segment score of Group 6 compared to Group 2 and in the distal segment score of Group 7 compared to Group 2 (see [link to relevant documentation]). Figure 16 ).

[0187] Example 5 – Inhibiting the growth of pathogens

[0188] As described above, the test examines the ability of Lactobacillus species deposited under the Budapest Treaty, including *Lactobacillus diolivorans*, *Lactobacillus casei*, *Lactobacillus glutenophilus*, *L. rapi*, and *Lactobacillus zeatus*, to inhibit the growth of bacterial pathogens associated with inflammation and various inflammatory conditions. The pathogens tested for this purpose were *Campylobacter jejuni* (ATCC 33291), *Helicobacter pylori* (ATCC 700824), *Clostridium difficile* (ATCC 9689), *Salmonella typhimurium* (ATCC 29630), *Yersinia enterocolitica* (ATCC 23715), *Citrobacter* species (ATCC 51378), *Streptococcus pyogenes* (ATCC 19615), *Streptococcus mutans* (ATCC 25175), *Klebsiella pneumoniae* (ATCC 700324), and *Proteus mirabilis* (ATCC 25933). *Campylobacter jejuni* and *Helicobacter pylori* grew in 5% Columbia horse blood agar (CBA) under microaerophilic conditions at 37°C. *Salmonella typhimurium*, *Yersinia enterocolitica*, *Citrobacter* species, *Klebsiella oxytoca*, and *Proteus mirabilis* grew in nutrient agar under aerobic conditions at 37°C. *Streptococcus pyogenes* and *Streptococcus mutans* grew in 5% CBA under aerobic conditions at 5% CO2 at 37°C. *C. difficile* grew in 5% CBA under anaerobic conditions at 37°C.

[0189] Inhibition of pathogen growth was determined using an agar well diffusion assay. Cultures of five tested *Lactobacillus* species were incubated in MRS broth at 37°C for 24 hours. Each pathogen was subcultured on nutrient agar plates or 5% CBA plates and incubated under appropriate conditions (as described above), then resuspended in DPBS to obtain a 0.5 McFarland standard (equivalent to 10). 8 (CFU / mL). Spread each 0.5 Mk Franz normalized culture onto a nutrient agar plate or CBA plate using a swab, allowing the culture to soak in, and then prepare 9 mm wells. Then add 100 μL of each test lactobacillus to a separate well and incubate the plate at 37°C for 24 hours. Use 5 μg ciprofloxacin as a positive control and sterile MRS broth only as a negative control. After incubation, measure (mm) and record areas of inhibition or decreased growth (growth turbidity). Take two readings for each area and use three replicates.

[0190] As expected, sterile MRS broth applied to pathogen-containing plates did not produce inhibition or reduction zones. As shown in Table 2, most Lactobacillus species exhibited strong inhibitory activity against most of the tested pathogens. The inhibition zones produced by applying 5 μg of ciprofloxacin are also shown in Table 2.

[0191] Table 2. Inhibition area / growth reduction area (mm). Values ​​are averages from six experiments (two readings from each of the three replicates).

[0192] Campylobacter jejuni - 21.0 12.8 - 19.8 30.2 Helicobacter pylori - 21.7 13.5 - 22.5 31.7 Clostridium difficile 11.8 19.7 13.7 - 19.3 - Salmonella typhimurium 14.2 20.5 15.3 15.2 23.2 49 Yersinia enterocolitica 16.0 19.7 14.3 15.8 22.2 44.2 Citrobacter species 13.3 16.8 13.8 13.5 18.0 47.0 Streptococcus pyogenes 12.7 16.7 10.2 12.8 18.3 30.2 Streptococcus mutans - 18.3 10.7 - 19.2 28.5 Klebsiella pneumoniae 15.0 16.7 13.0 12.3 16.5 41.2 Proteus mirabilis 15.7 18.5 13.5 14.7 21.2 44.2

[0193] The ability of *Lactobacillus diolivorans*, *Lactobacillus casei*, *Lactobacillus branii*, *Lactobacillus rapi*, and *Lactobacillus zeylinum*, preserved under the Budapest Treaty, to inhibit the growth of pathogenic bacteria *Staphylococcus aureus* (ATCC 29213 and ATCC 25923), *Staphylococcus epidermidis* (clinical isolate), *Pseudomonas aeruginosa* (ATCC 27853), and *Escherichia coli* K12, as described above, was then tested. These pathogens were grown in nutrient agar under aerobic conditions at 37°C. Growth inhibition was determined using an agar well diffusion assay as described above. In addition, cell-free supernatants derived from *Lactobacillus* cultures were tested. Briefly, after 24 hours of incubation, *Lactobacillus* cultures were centrifuged at 10,000 × g, and the supernatant was sterilized by filtration through a 0.2 μm membrane. 100 μl of cell-free supernatant was added to wells containing the pathogens. The results are shown in Tables 3 and 4 below.

[0194] Table 3. Inhibition / Growth Reduction Areas Using Live Lactobacillus Cultures (mm). Values ​​are averages from six experiments (two readings from each of three replicates). Readings include a 9 mm well diameter.

[0195]

[0196] Table 4. Inhibition / Growth Reduction Areas (mm) using Cell-Free Supernatant from Lactobacillus Cultures. Values ​​are averages from six experiments (two readings from each of three replicates). Readings include a 9 mm well diameter.

[0197]

[0198] Example 6

[0199] A 45-year-old male experienced periodic but significant jaw pain due to temporomandibular joint disorder. The subject placed 2 mL of a liquid preparation containing Lactobacillus brucellosis Lb23 under his tongue daily and then swallowed. The preparation contained approximately 10 mg of sterile saline and 2%–3% sucrose.6 Up to 10 8 The microbial strain was CFU / mL (stored at 4°C until use). After 4 to 5 days, the pain had subsided and no further discomfort was experienced.

[0200] Example 7

[0201] A 61-year-old woman complained of persistent bowel problems, including irritation, pain, bloating, and inflammation of the colon and rectum. The subject orally ingested 2 mL daily of a liquid preparation containing *Lactobacillus zedoaria* Lz26. This preparation contained approximately 10 [units of something] in sterile saline and 2%–3% sucrose. 6 Up to 10 8 The microbial strain was CFU / mL (stored at 4°C until use). After 3 to 4 days, intestinal problems improved, and all symptoms were alleviated. Subjects continued to use the preparation and experienced minimal digestive problems during treatment.

[0202] Example 8

[0203] A 19-year-old male had suffered from moderate to severe facial acne for many years, with only slight success using topical creams and antibiotics. The subject was given approximately 1 mL daily applied to his facial area as a liquid preparation containing *Lactobacillus casei* Lp9. The preparation contained approximately 10 μL of sterile saline and 2%–3% sucrose. 6 Up to 10 8 The microbial strain was CFU / mL (stored at 4°C until use). After one week, the number and size of papules decreased, and pain, particularly associated with swollen and inflamed areas, was significantly reduced. Subjects commented that blemished and painful areas were much less noticeable during treatment.

[0204] Example 9

[0205] A 48-year-old male suffered from right knee pain due to years of playing competitive and social football and tennis. The pain was infrequent but always occurred after heavy use of the joint and was accompanied by significant inflammation. Each time inflammation and pain occurred, the subject placed 2 ml of a liquid preparation containing a composition of *Lactobacillus brucellosis* Lb23, *Lactobacillus zei* Lz26, and *Lactobacillus casei* Lp9 under his tongue for several minutes before swallowing. The preparation contained approximately 10 mg of sterile saline and 2%–3% sucrose. 6 Up to 10 8 Microbial strain at CFU / mL (stored at 4°C until use). Within 2–3 days, pain and swelling around the knee begin to decrease, and subjects are usually able to walk and move their knees without any significant pain or discomfort.

[0206] Example 10

[0207] A 56-year-old male had experienced significant gastrointestinal discomfort for several years while simultaneously undertaking extensive travel in Southeast Asia. The subject suffered recurrent abdominal cramps, occasional nausea, and a Bristol stool score of 6–7. A treatment regimen was established consisting of 2.5 mL of 1×10⁻⁶ solution twice daily. 7 Lactobacillus casei Lp9 liquid preparation at a dose of CFU / mL. Three days after treatment, subjects reported significant improvement in gastrointestinal symptoms. Encouraged by this improvement, subjects opted to include a 5 mL dose of 2.5 × 10⁻⁶ CFU / mL in their regimen. 9 Lactobacillus brunetti Lb23. After 2 weeks of treatment, the subjects' Bristol stool scores improved to 4 points, and reported gastrointestinal discomfort was negligible.

[0208] Example 11

[0209] A 62-year-old woman had moderate osteoarthritis, primarily affecting her knees and hands. Her doctor recommended on-demand use of paracetamol. However, the subject still experienced significant stiffness and loss of mobility, particularly in her fingers. Oral intake of 5 × 10⁻⁶ mmol / L... 9 A liquid preparation of *Lactobacillus flavus* Lp18 at CFU / mL was administered. The dosage regimen was 5 mL once daily. After one week of treatment, subjects noticed a significant improvement in pain and swelling in the affected joints. The treatment regimen was then increased to 1 × 10⁻⁶ CFU / mL. 9 A 10 mL liquid formulation of Lactobacillus diolivorans Ld03, Lactobacillus zeaensis Lz26, and Lactobacillus glutenophilus Lp18 at CFU / mL. After one month of combination treatment, subjects experienced significantly less joint stiffness, reported substantial recovery of finger mobility, and reduced knee pain.

[0210] Example 12

[0211] An 80-year-old male suffered from severe knee osteoarthritis, resulting in loss of mobility and a deteriorating quality of life. The subject had been managing his condition with a nonsteroidal anti-inflammatory drug (NSAID) under the guidance of his physician. The trial involved an oral intake of 15 mL (1×10) once daily. 9 Treatment with a liquid formulation of Lactobacillus flavus Lp18 at CFU / mL was administered. After four weeks of treatment, subjects experienced less knee pain and swelling and were able to walk short distances again. Subjects received a second 15 mL 2.5 × 10⁻⁶ CFU / mL solution. 7 A combination formulation at a daily dose of CFU / mL; *Lactobacillus branii* Lp18, *Lactobacillus zei* Lz26, and *Lactobacillus rapi* Lr24. Two weeks of combination treatment produced sustained improvement, and subjects were able to resume mild gardening a few times per week.

[0212] Example 13

[0213] A 25-year-old woman suffered from asthenic mastitis, accompanied by significant pain and swelling, and to her dismay, was unable to breastfeed her newborn. The subject orally administered 5 mL of a solution containing 1×10⁻⁶ mg / L of the drug every morning for 7 consecutive days. 8 A liquid preparation of Lactobacillus casei Lp9 at CFU / mL. Her symptoms improved, so over another 7 days she increased the dose to 10 mL daily and noticed further improvement in her condition. After two weeks of treatment, the subject felt a significant improvement in her mastitis, and to her further delight, she was able to continue breastfeeding her newborn.

[0214] Example 14

[0215] A 35-year-old woman with rheumatoid arthritis, receiving weekly methotrexate at 20 mg, experienced several frequent flare-ups of swelling in her proximal interphalangeal joints. In addition to her existing treatment, the subject orally administered 10 mL of a solution containing 1×10... 9 A liquid preparation of CFU / m Lactobacillus cornii Lz26 was administered twice daily for several weeks. After approximately two weeks, the subject noticed a reduction in pain and swelling in the currently affected joint, and after one month of treatment, no further flare-ups were observed. The subject felt that the additional microbial therapy helped stabilize their condition.

[0216] Example 15

[0217] A 27-year-old woman who had been reporting chronic abdominal pain and discomfort for many years was given a liquid preparation containing Lactobacillus bromide Lb23. The preparation consisted of approximately 10 μL of sterile saline and 2%–3% sucrose. 6 Up to 10 8 The microbial strain was CFU / mL (stored at 4°C until use). After taking 10 ml with breakfast every morning for 7 consecutive days, she reported significant improvement in her symptoms after day 4. She also reported a pain-free day on day 7.

[0218] Example 16

[0219] A 19-year-old man returning from a four-week road trip in Thailand reported recurrent diarrhea after returning home. A liquid formulation is provided, containing 10g of sterile saline and 2%-3% sucrose. 6 Up to 10 8 The microbial strain was a combination of *Lactobacillus bruneri* Lb23, *Lactobacillus zeatus* Lz26, and *Lactobacillus casei* Lp9 (cfu / ml) (stored at 4°C until use). He took 3 ml three times daily and observed improvement in his condition on the second day. He fully recovered by the fourth day.

[0220] Example 17

[0221] A 55-year-old woman who suffered food poisoning after eating local fish and chips was given a single dose (35 ml) of a solution of approximately 10 mg of sterile saline and 2%-3% sucrose. 8 A liquid formulation of Lactobacillus diolivorans Ld03, prepared at cfu / ml (stored at 4°C until use). She reported that her symptoms significantly improved 10 hours after treatment and returned to normal digestive transit the following day.

[0222] Example 18

[0223] An 18-year-old male reported experiencing moderate acne vulgaris for approximately one year, reporting a combination of 50-80 lesions (a combination of comedones [~60] and inflammatory lesions [~20]) at any given time, which were very red and painful. He had attempted to reduce the number of lesions by using over-the-counter acne cleansers, creams, and gels, but with little effect and his skin became dry. He initially started by applying 1 mL of 1×10 [agent] via a spray (5 sprays per mL in a saline / sucrose carrier). 8 He was able to cover his entire facial surface with a concentration of *Lactobacillus casei* Lp9 at cfu / mL. He applied the bacteria twice daily after cleansing and began to notice a reduction in both inflammatory lesions and comedones, with a significant decrease in redness and pain after 7 days. He reported approximately 10 inflammatory lesions and 40 comedones on day 7, which further decreased to 5 inflammatory lesions and 25 comedones by day 14. After day 14, he added *Lactobacillus brucellosis* Lb23 and *Lactobacillus zeaxanthin* Lz26 at a concentration of 1×10⁻⁶. 8 CFU / mL was added to Lactobacillus casei, and the spray (1 mL each time) was applied twice daily for another 14 days. On day 28, he reported 5 pimples with no inflammatory lesions. During the 28-day period, he did not use any acne ointments or gels and only washed his face with his usual facial cleanser.

[0224] Example 19

[0225] A 23-year-old woman reported experiencing moderate acne since age 15, with symptoms progressively worsening. She was diagnosed with endometriosis by her gynecologist, accompanied by irregular and painful menstruation. She had been taking oral contraceptives for four years but reported no improvement in her acne symptoms. On the advice of her general practitioner, she had been on a course of tetracycline and noticed improvement in her skin while taking the antibiotic for 14 days each course, but her acne symptoms recurred after completing the course. In addition, she experienced moderate gastrointestinal disturbances, such as bloating and cramps accompanied by diarrhea, which prevented her from continuing this treatment. She initially started by applying 1 mL of Lactobacillus corni Lz26 (1×10⁻⁶) twice daily in a saline / sucrose carrier after cleansing. 9 She applied Lz26 (cfu / mL) five times (1mL) to cover her facial surface. Before treatment, she reported approximately 80 comedones (open and closed) and 30 inflammatory lesions, all of which were red and painful. After 7 days of application, she noticed a decrease in the lesion count to approximately 50 comedones and 20 inflammatory lesions. She continued to report improvements in her skin weekly while continuing to apply Lz26 for another 49 days (a total of 8 weeks). By day 56, she reported a minimum of 10 comedones and no inflammatory lesions.

[0226] Example 20

[0227] A 45-year-old male had been experiencing recurrent tinea pedis (athlete's foot) between the toes, primarily between the middle, fourth, and little toes of each foot. For three consecutive days, the subject applied approximately 1 mL of a liquid preparation containing Lactobacillus brucellosis Lb23 to the affected areas. The preparation contained approximately 10 mL of sterile saline and 2%–3% sucrose. 6 Up to 10 8 The microbial strain was CFU / mL (stored at 4°C until use). Symptoms improved after 3 days, and the fungal infection disappeared after 5 days.

[0228] Example 21

[0229] A 44-year-old male presented with a ringworm infection on his lower back as a result of training on a public impact pad at a gym. For three consecutive days, the subject applied approximately 1 mL of a liquid preparation containing *Lactobacillus cornii* Lz26 to the surface of the infected area. The preparation contained approximately 1 × 10⁻⁶ of sterile saline and 2%–3% sucrose. 8 A microbial strain at cfu / mL (stored at 4°C until use). The fungal infection disappeared after 5 days.

[0230] Example 22

[0231] A 44-year-old woman presented with early signs of nail fungus on the toenail of her right big toe. For five consecutive days, the subject applied approximately 1 mL of a liquid preparation containing *Lactobacillus zedoaria* Lz26 to the infected area. The preparation contained approximately 1 × 10⁻⁶ of *Lactobacillus zedoaria* Lz26 in sterile saline and 2%–3% sucrose. 8 The microbial strain was tested at cfu / mL (stored at 4°C until use). After 5 days, the direct signs of fungal infection had disappeared, and the toenail was able to heal naturally and grow normally over time.

[0232] Example 23

[0233] A 48-year-old male was diagnosed with atopic dermatitis (AD) at six months of age, averaging moderate severity, occasionally worsening under stress. The dermatitis lesions are extremely dry, red, and very itchy (pruritus), causing sufficient discomfort to significantly impact his sleep quality. He is currently under the medical care of a specialist dermatologist who has prescribed topical corticosteroids for acute inflammatory flare-ups, particularly in the antecubital and popliteal fossae and sometimes on his trunk. AD affects approximately 30% of his body. He has used topical corticosteroids intermittently since childhood. He has received extensive antibiotic treatment (both oral and topical) for persistent Staphylococcus aureus infections. His daily regimen includes cleansing with soap-free QV facial cleanser and applying QVFlare up cream twice daily to his trunk and extremities. Despite adhering to the recommended medical treatment, he continues to experience moderate symptoms, particularly when experiencing stressful episodes at work and during the hot and humid conditions of spring and summer. He continued to apply the preparation containing Lactobacillus casei Lp9 in a saline / sucrose carrier twice daily, spraying 0.2 mL each time to cover approximately 20 cm. 2 The area, and the total area processed in each application is up to 200 cm². 2 Each 0.2 mL spray contains 1 × 10 9 CFU. During this period, he avoided applying QV cream to the treated area. After 5 days of treatment, he noticed a significant improvement in the severity of his lesions, with redness and itching decreasing by approximately 30%, and he also noticed improved sleep quality due to reduced scratching. After another 14 days of application of Lactobacillus casei Lp9, he reported significant improvement in his skin condition and assessed the severity of his AD lesions as very mild, with no obvious signs of Staphylococcus aureus infection. He has experienced a reduction of up to 80% in redness, dryness, and itching of the lesions.

[0234] Example 24

[0235] A 50-year-old woman was diagnosed with psoriasis at age 30. Intense, red, scaly plaques primarily affected her hairline, back of the neck, and behind the ears, with plaques also affecting her elbows. She typically used topical corticosteroids to reduce flare-ups and had experienced numerous Staphylococcus aureus infections requiring both topical and oral antibiotics. She usually applied QV ointment to the affected areas. She continued to apply Lactobacillus brucellosis Lb23 to the plaques along her hairline, back of the neck, and behind the ears twice daily at a dose of 1×10⁻⁶. 9 CFU / 0.2mL spray, each spray covering 20cm. 2 She applied the spray twice a day for a total of eight times. By the end of day 7, she noticed that the patches behind her ears had completely disappeared, and the severity of the patches on her hairline and neck had decreased by about 50%. She continued to apply Lactobacillus bromide Lb23 for another 14 days, at which point all the patches had disappeared, leaving only redness.

[0236] Example 25

[0237] A 39-year-old woman suffered from multiple health complications associated with excessive Streptococcus bacteria, including recurrent streptococcal pharyngitis, tonsillitis, halitosis, and gastrointestinal discomfort. For five consecutive days, the subject swallowed 1-2 mL of a liquid preparation containing *Lactobacillus zei* Lz26 daily. Then, for two consecutive weeks, every other day, the subject placed 2 mL of a liquid preparation containing a combination of *Lactobacillus brucellosis* Lb23, *Lactobacillus zei* Lz26, and *Lactobacillus casei* Lp9 under the tongue for several minutes before swallowing. The preparation contained approximately 10 [units of something] in sterile saline and 2%-3% sucrose. 8 Microbial strains at cfu / mL (stored at 4°C until use). Subjects reported reduced sore throat, improved halitosis, and improved gastrointestinal function during the treatment period.

[0238] Example 26

[0239] A 45-year-old woman was diagnosed with Helicobacter pylori infection. The subject underwent [further treatment] before starting combination therapy with Lactobacillus casei Lp9 and Lactobacillus zeatus Lz26. 14 The C-urea breath test returned a result of 1356 disintegrations per minute (dpm), a highly positive result for the presence of Helicobacter pylori in the stomach. The subject began a course of treatment with daily oral administration of a total concentration of 2 × 10⁻⁶. 8 10 ml of *Lactobacillus casei* Lp9 and *Lactobacillus zeatus* Lz26 at cfu / mL was administered continuously for one month. At the end of this period, 14 The C-urea breath test returned a result of 654 dpm, a reduction of more than 50%.

[0240] Collection Details

[0241] Details of the biological material deposited under the Budapest Treaty have been provided above in the description. In summary:

[0242] In accordance with the Budapest Treaty, *Lactobacillus glutenophilus* SVT-18 was deposited on February 27, 2019, with accession number LMG P-31292 at the Belgian Coordinating Collection of Microorganisms (BCCM), Federal Public Planning Service, Science Policy, 8 rue de la Science B-1000, Brussels, Belgium.

[0243] In accordance with the Budapest Treaty, Lactobacillus brunetti SVT-23 was deposited on February 27, 2019, with accession number LMG P-31293 at the Belgian Coordinated Collection of Microorganisms (BCCM), Federal Public Planning Service, Science Policy, 8 Science Street, B-1000, Brussels, Belgium.

[0244] In accordance with the Budapest Treaty, Lactobacillus maize SVT-26 was deposited on February 27, 2019, with accession number LMG P-31295 at the Belgian Coordinated Collection of Microorganisms (BCCM), Federal Public Planning Service, Science Policy, 8 Science Street, B-1000, Brussels, Belgium.

[0245] In accordance with the Budapest Treaty, L. rapi SVT-24 was deposited on February 27, 2019, with accession number LMG P-31294 at the Belgian Coordinated Collection of Microorganisms (BCCM), Federal Public Planning Service, Science Policy, 8 Science Street, B-1000, Brussels, Belgium.

[0246] In accordance with the Budapest Treaty, Lactobacillus casei SVT-09 was deposited on February 27, 2019, with accession number LMG P-31290 at the Belgian Coordinated Collection of Microorganisms (BCCM), Federal Public Planning Service, Science Policy, 8 Science Street, B-1000, Brussels, Belgium.

[0247] In accordance with the Budapest Treaty, Lactobacillus diolivorans SVT-03 was deposited on February 27, 2019, with accession number LMG P-31287 at the Belgian Coordinated Collection of Microorganisms (BCCM), Federal Public Planning Service, Science Policy, 8 Science Street, B-1000, Brussels, Belgium.

Claims

1. Lactobacillus ( Lactobacillus The use of culture supernatant or cell-free filtrate derived from a culture medium in which the lactobacillus has been cultured, and / or from the preparation of a medicament for treating or preventing inflammatory or autoimmune conditions of the joints, wherein the condition is selected from rheumatoid arthritis and osteoarthritis, and wherein the lactobacillus ( Lactobacillus Selected from: Lactobacillus bruneri deposited under accession number LMG P-31293 ( Lactobacillus buchneri SVT-23, Lactobacillus zei deposited under accession number LMG P-31295 ( Lactobacillus zeae SVT-26, deposited under accession number LMG P-31294 Lactobacillus rapi SVT-24, *Lactobacillus casei* preserved under accession number LMG P-31290 ( Lactobacillus paracasei SVT-09, *Lactobacillus glutenosa* preserved under accession number LMG P-31292 ( Lactobacillus parafarraginis SVT-18 and those preserved under registration number LMG P-31287 Lactobacillus diolivorans SVT-03.

2. The use according to claim 1, wherein the condition is rheumatoid arthritis.

3. The use according to claim 1, wherein the drug comprises a combination of two, three, four, five or all six of the lactobacilli, or is derived from a culture supernatant or cell-free filtrate of a culture medium in which two, three, four, five or all six of the lactobacilli have been cultured.

4. The use according to claim 1, wherein the lactobacillus, culture supernatant, or cell-free filtrate is administered in the form of a pharmaceutically acceptable composition or food or beverage.

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