Methods for combating Salmonella typhimurium infections using probiotic compositions

Abstract

1. A method for treating a Salmonella typhimurium infection, the method comprising administering to a subject in need thereof a composition comprising a culture of Lactobacillus rhamnosus LRH10 deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH under accession number DSM 32786, Lactobacillus paracasei LPC12 deposited at DSMZ GmbH under accession number DSM 32785, Lactobacillus fermentum LF26 deposited at DSMZ GmbH under accession number DSM 32784, Streptococcus thermophilus ST30 deposited at DSMZ GmbH under accession number DSM 32788, and Lactobacillus helveticus LH43 deposited at DSMZ GmbH under accession number DSM 32787.

Description

[Technical Field]

[0001] The present disclosure relates to a method for combating Salmonella typhimurium infections using a composition comprising a culture of five lactic acid bacteria strains. [Background technology]

[0002] Salmonella typhimurium is a gram-negative bacterium that colonizes the intestinal tract of animals (e.g., pigs and humans), particularly the ileum, cecum, and spiral colon, via Peyer's patches. Salmonella typhimurium infection (also called salmonellosis) can result from ingestion of infected food sources or from contaminated environments, including veterinary clinics. The main symptoms of salmonellosis include fever, loss of appetite, diarrhea, vomiting, and abdominal pain. Salmonella typhimurium most frequently causes gastroenteritis in humans.

[0003] Salmonellosis is usually treated with antibiotics (e.g., apramycin, ceftiofur, trimethoprim-sulfamethoxazole (TMP-SMX), and gentamicin), but these antibiotics can cause antibiotic resistance in Salmonella typhimurium and can cause serious side effects and adverse reactions.

[0004] Probiotics are resident microflora that normally reside in the intestinal tract and are believed to play an important role in properly regulating intestinal immunity and digestion by balancing the intestinal microbiota. These beneficial microorganisms are widely used as live microbial dietary supplements and help restore the balance of the intestinal microbiota. Many species of lactic acid bacteria (LAB) have been granted Generally Recognized as Safe (GRAS) status and are widely used as probiotics. Common LAB species include Lactobacillus, Lactococcus, Pediococcus, Streptococcus, Enterococcus, Bifidobacterium, Bacillus, and Leuconostoc.

[0005] Previous studies have demonstrated that certain strains of lactic acid bacteria are effective against Salmonella typhimurium infections. For example, Streptococcus thermophilus St-21, Lactobacillus helveticus SP-27, and Lactobacillus rhamnosus GG have been shown to inhibit the growth of Salmonella typhimurium and thus achieve anti-Salmonella typhimurium infection effects (J. Piatekl et al. (2019), Beneficial Microbes, 10(2):211-217 and Bingjie Duan et al. (2021), Microb Pathog., 156:1049-39).

[0006] Despite this, there remains a need to develop new strategies that are effective against Salmonella typhimurium infections. Summary of the Invention [Problem to be solved by the invention]

[0007] It is therefore an object of the present invention to provide a method against Salmonella typhimurium infections that can alleviate at least one of the drawbacks of the prior art. [Means for solving the problem]

[0008] The method comprises administering to a subject in need thereof a composition comprising a culture of Lactobacillus rhamnosus LRH10 deposited at Deutsche Sammlung von Microorganismen und Zellkulturen (DSMZ) GmbH under accession number DSM 32786, Lactobacillus paracasei LPC12 deposited at DSMZ GmbH under accession number DSM 32785, Lactobacillus fermentum LF26 deposited at DSMZ GmbH under accession number DSM 32784, Streptococcus thermophilus ST30 deposited at DSMZ GmbH under accession number DSM 32788, and Lactobacillus helveticus LH43 deposited at DSMZ GmbH under accession number DSM 32787.

[0009] Other features and advantages of the present disclosure will become apparent from the following detailed description of the embodiments, which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0010] [Figure 1] The diameter of the inhibition zone measured for each group in Example 1 below is shown, and the symbol "*" indicates p<0.05 (compared to single lactic acid bacteria supernatants CG1 to CG5 and BCG). DETAILED DESCRIPTION OF THE INVENTION

[0011] It should be understood that the citation of any prior art document herein does not constitute general knowledge in the field to which the present invention pertains, in Taiwan or any other country.

[0012] It is also to be clearly understood that for purposes of this specification the word "including" means "including but not limited to" and the word "including" has a corresponding meaning.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used in the practice of this disclosure. Indeed, this disclosure is in no way limited to the methods and materials described.

[0014] The present disclosure provides a method for treating Salmonella typhimurium infection, the method comprising administering to a subject in need thereof a composition comprising a culture of Lactobacillus rhamnosus LRH10 deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH under accession number DSM 32786, Lactobacillus paracasei LPC12 deposited at DSMZ GmbH under accession number DSM 32785, Lactobacillus fermentum LF26 deposited at DSMZ GmbH under accession number DSM 32784, Streptococcus thermophilus ST30 deposited at DSMZ GmbH under accession number DSM 32788, and Lactobacillus helveticus LH43 deposited at DSMZ GmbH under accession number DSM 32787.

[0015] As used herein, the term "against Salmonella typhimurium infection" or "anti-Salmonella typhimurium infection" means preventing infection by Salmonella typhimurium, inhibiting the replication of Salmonella typhimurium, and / or treating and / or preventing infection by Salmonella typhimurium.

[0016] As used herein, the term "administration" or "administering" means "introducing, providing, or delivering a predetermined composition to a subject by any suitable route to perform its intended function."

[0017] As used herein, the term "subject" refers to any animal of interest, such as a human, monkey, hamster, cow, sheep, horse, pig, goat, dog, cat, mouse, rat, etc. In certain embodiments, the subject is a human.

[0018] According to the present disclosure, a culture of each lactic acid bacteria strain can be produced by culturing the lactic acid bacteria strain in a liquid or solid medium suitable for its growth and / or proliferation.

[0019] In a particular embodiment, the culture of each lactic acid bacteria strain is prepared by cultivating said lactic acid bacteria strain for 10 minutes in a liquid or solid medium suitable for growth. 5 CFU / mL ~10 9 It can be produced by culturing to a concentration of CFU / mL.

[0020] In an exemplary embodiment, the lactic acid bacteria strain is cultured for 10 minutes in a liquid or solid medium suitable for growth. 6 CFU / mL ~10 8 It is cultured to a concentration of CFU / mL.

[0021] As used herein, the term "cultivation" can be used interchangeably with other terms such as "fermentation" and "cultivation."

[0022] The procedures and conditions for culturing lactic acid bacteria strains can be adjusted according to actual needs. In this regard, those skilled in the art can refer to papers such as Kimoto-Nira H. et al. (2012), J Dairy Sci., 95(4):2176-2185. doi: 10.3168 / jds.2011-4824.

[0023] In a particular embodiment, a liquid medium suitable for culturing lactic acid bacteria strains is 10% skim milk.

[0024] In a particular embodiment, the culture of each lactic acid bacteria strain is a liquid culture.

[0025] In certain embodiments, the ratio of the numbers of Lactobacillus rhamnosus LRH10, Lactobacillus paracasei LPC12, Lactobacillus fermentum LF26, Streptococcus thermophilus ST30, and Lactobacillus helveticus LH43 in the composition is within the range of 5.1:29.7:1.9:47.8:1.2 to 10.9:41.3:3.4:53.2:5.7.

[0026] In an exemplary embodiment, the ratio of numbers of Lactobacillus rhamnosus LRH10, Lactobacillus paracasei LPC12, Lactobacillus fermentum LF26, Streptococcus thermophilus ST30, and Lactobacillus helveticus LH43 in the composition is 7.8:35.5:2.6:50.4:3.3.

[0027] According to the present disclosure, the liquid culture can be substantially free of cells.

[0028] As used herein, the term "substantially free" means that the liquid culture does not contain a significant amount of a particular component (i.e., lactic acid bacteria cells). In certain embodiments, the amount of lactic acid bacteria cells does not measurably affect the properties of the liquid culture. In other embodiments, the liquid culture is completely free of bacterial cells.

[0029] According to the present disclosure, a substantially cell-free liquid culture is obtained by culturing a lactic acid bacteria strain and then subjecting the resulting culture to a separation process to remove bacterial cells.

[0030] According to the present disclosure, separation processes can be performed using techniques known to those skilled in the art, including, but not limited to, filtration, centrifugation (e.g., multi-stage centrifugation), concentration, and combinations thereof.

[0031] In an exemplary embodiment, the substantially cell-free liquid culture is obtained by culturing the lactic acid bacteria strain and then subjecting the resulting culture to centrifugation.

[0032] According to the present disclosure, the compositions can be formulated into food products using standard techniques well known to those skilled in the art. For example, the compositions can be added directly to edible materials or used to prepare intermediate compositions (e.g., food additives or premixes) suitable for subsequent addition to edible materials.

[0033] The term "food" as used herein refers to any item or substance that can be ingested by a subject. Examples of foods include, but are not limited to, powdered milk, fermented milk, yogurt, butter, beverages (e.g., tea, coffee, etc.), functional drinks, flour products, baked goods, confectionery, candy, fermented foods, animal feed, health foods, infant foods, nutritional supplements, etc.

[0034] According to the present disclosure, the composition can be prepared in the form of a pharmaceutical composition, which can be formulated into a dosage form suitable for oral, parenteral or topical administration using techniques well known to those skilled in the art.

[0035] According to the present disclosure, dosage forms suitable for oral administration include, but are not limited to, sterile powders, tablets, troches, lozenges, pellets, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrups, elixirs, slurries, drops, and the like.

[0036] For parenteral administration, the pharmaceutical compositions of the present disclosure can be formulated into injectable preparations, such as sterile aqueous solutions or dispersions.

[0037] Pharmaceutical compositions according to the present disclosure can be administered via any of the following parenteral routes: intraperitoneal injection, intrathoracic injection, intramuscular injection, intravenous injection, intraarterial injection, intra-articular injection, intra-synovial injection, intrathecal injection, intracranial injection, intraepidermal injection, subcutaneous injection, intradermal injection, intralesional injection, and sublingual administration. In certain embodiments, the pharmaceutical composition is administered by intravenous injection.

[0038] According to the present disclosure, the pharmaceutical compositions can be formulated into topical preparations suitable for topical application to the skin using techniques well known to those skilled in the art, including, but not limited to, emulsions, gels, ointments, creams, patches, liniments, powders, aerosols, sprays, lotions, serums, pastes, foams, drops, suspensions, plasters, and bandages.

[0039] The pharmaceutical composition according to the present disclosure may further comprise a pharmaceutically acceptable carrier commonly used in the art of pharmaceutical manufacturing. For example, the pharmaceutically acceptable carrier may comprise one or more of the following agents: solvents, buffers, emulsifiers, suspending agents, disintegrating agents, disintegrating agents, dispersing agents, binders, excipients, stabilizers, chelating agents, diluents, gelling agents, preservatives, fillers, wetting agents, lubricants, absorption delaying agents, liposomes, etc. The choice and amounts of the above agents are within the experience and ordinary skill of those skilled in the art.

[0040] The dosage and frequency of administration of the compositions according to the present disclosure can vary depending on the following factors: the severity of the disease or disorder to be treated, the route of administration, and the age, physical condition and response of the subject to be treated. Generally, the compositions may be administered in a single dose or in several divided doses.

[0041] Examples of the present disclosure will now be described, with the understanding that these examples are exemplary and explanatory and should not be construed as limiting the present disclosure. [Example]

[0042] General experimental materials: 1. Lactic acid bacteria (LAB) strains Five lactic acid bacteria strains, namely, Lactobacillus rhamnosus LRH10, Lactobacillus paracasei LPC12, Lactobacillus fermentum LF26, Streptococcus thermophilus ST30, and Lactobacillus helveticus LH43 (which are disclosed in U.S. Patent Application Publication No. 2020 / 0008437 and are readily available to the public) have been deposited in accordance with the Budapest Treaty with Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH, Imhofenstrasse 7B, 38124 Braunschweig, Germany.

[0043] Relevant information for each lactic acid bacteria strain (including accession number and date of deposit) is listed in Table 1 below.

[0044] [Table 1]

[0045] 2. The Salmonella typhimurium used in the following experiments is a bacterium isolated by the applicant from chicken intestines. General Testing Procedures: 1. Statistical analysis All experiments described below were performed at least three times. Experimental data for all test groups are expressed as mean ± standard deviation (SD) and analyzed using one-way analysis of variance (ANOVA) followed by the Tukey-Kramer test to assess differences between groups. Statistical significance is indicated by p<0.05.

[0046] Example 1. Evaluation of the effect of liquid cultures of lactic acid bacteria strains according to the present disclosure on Salmonella typhimurium Testing Procedure: A. Preparation of cell culture supernatants of lactic acid bacteria strains Lactobacillus rhamnosus LRH10, Lactobacillus paracasei LPC12, Lactobacillus fermentum LF26, and Lactobacillus helveticus LH43, each listed in Section 1 of "General Materials," were inoculated into Difco™ Lactobacillus MRS (De Man, Rogosa, and Sharpe) broth (Cat. No. 288130, BD Difco), while Streptococcus thermophilus ST30, listed in Section 1 of "General Materials," was inoculated into M17 broth (HiMedia®). Each of the five lactic acid bacteria strains was cultured in an incubator (37°C) under aerobic conditions for 16 hours to obtain the respective inoculum.

[0047] Next, each inoculum was inoculated into 10% skim milk at a 2% (v / v) concentration and subcultured in an incubator (37°C) under aerobic conditions for 16 hours to obtain a single liquid culture of each lactic acid bacteria strain. Furthermore, inocula of Lactobacillus rhamnosus LRH10, Lactobacillus paracasei LPC12, Lactobacillus fermentum LF26, Streptococcus thermophilus ST30, and Lactobacillus helveticus LH43 were inoculated into 10% skim milk (Acumedia®) at a volume ratio of 1:1:1:1:1 (each inoculum amount was 0.4% (v / v)), and then subcultured in an incubator (37°C) under aerobic conditions for 16 hours to obtain a five-species liquid culture of lactic acid bacteria. The number of each lactic acid bacteria strain in the five-species liquid culture was counted using techniques well known to those skilled in the art. As a result, the ratio of numbers of Lactobacillus rhamnosus LRH10, Lactobacillus paracasei LPC12, Lactobacillus fermentum LF26, Treptococcus thermophilus ST30, and Lactobacillus helveticus LH43 in the five-species lactic acid bacteria liquid culture was 7.8:35.5:2.6:50.4:3.3.

[0048] Thereafter, each of the five single lactic acid bacteria liquid cultures and the five-species lactic acid bacteria liquid cultures was centrifuged at 5000 rpm for 10 minutes, and the resulting cell culture supernatants were collected and used in the next experiment.

[0049] B. Measurement of antibacterial activity against Salmonella typhimurium Each of the five cell culture supernatants obtained from the five single lactic acid bacteria liquid cultures described in Section A above corresponds to one of the single lactic acid bacteria supernatant groups, i.e., single lactic acid bacteria supernatant comparison groups 1 to 5 (abbreviated as single lactic acid bacteria supernatants CG1 to CG5) as shown in Table 2 below. The cell culture supernatant obtained from the five-species lactic acid bacteria liquid culture described in Section A above corresponds to the five-species lactic acid bacteria supernatant group, i.e., five-species lactic acid bacteria supernatant experimental group 1 (abbreviated as five-species lactic acid bacteria supernatant EG1) as shown in Table 2 below. 10% skim milk was used as a blank control (BCG).

[0050] [Table 2]

[0051] Salmonella typhimurium, as described in Section 2 of the "General Experimental Materials" section, was inoculated into tryptic soy broth (TSB) (HiMedia®) and cultured in an incubator (37°C) under aerobic conditions for 16 hours.

[0052] After centrifugation at 5000 rpm for 10 minutes at room temperature, the resulting cell pellet was collected. The cell pellet was then washed twice with 5 ml of Dulbecco's phosphate-buffered saline (DPBS, pH 7.39) (Thermo Scientific®) and subsequently suspended in DPBS to a bacterial concentration of 10 9 The bacterial suspension was then mixed with an appropriate amount of 0.85% sterile saline to obtain a bacterial suspension with a bacterial concentration of 10 CFU / mL. 7 CFU / mL (dilution factor 10 2 A bacterial solution (prepared using

[0053] Next, 100 μL of the bacterial solution was added to a TSB agar plate, followed by the addition of sterile small glass beads. The TSB agar plate was then shaken to ensure the bacterial solution was evenly distributed across the plate. Next, the small glass beads were poured out, and seven holes were made in the TSB agar plate using a sterile pipette tip (size: 9.25 mm). Then, 100 μL each of the single-species lactic acid bacteria supernatants CG1–CG5, the five-species lactic acid bacteria supernatant EG1, and BCG was added to different holes in the TSB agar plate and incubated in an incubator (37°C) under aerobic conditions for 16 hours.

[0054] The diameter of the zone of inhibition for each group was measured using techniques well known to those skilled in the art, and the resulting data was analyzed according to the methods described in Section 1 of "General Experimental Procedures."

[0055] result: Figure 1 shows the diameter of the inhibition zone measured for each group. As shown in Figure 1, the diameter of the inhibition zone measured for the five-species lactic acid bacteria supernatant EG1 was larger than the diameter of the inhibition zone measured for the single lactic acid bacteria supernatants CG1 to CG5.

[0056] These results suggest that the cell culture supernatant obtained by co-cultivation of Lactobacillus rhamnosus LRH10, Lactobacillus paracasei LPC12, Lactobacillus fermentum LF26, Streptococcus thermophilus ST30, and Lactobacillus helveticus LH43 can exert sufficient effects to inhibit the growth of Salmonella typhimurium.

[0057] Although the present disclosure has been described in connection with exemplary embodiments, it is understood that the disclosure is not limited to the disclosed embodiments, but is intended to cover various configurations falling within the broadest spirit and scope, and to embrace all such modifications and equivalent arrangements.

Claims

1. 1. A method for treating Salmonella typhimurium infections, comprising the steps of: Lactobacillus rhamnosus LRH10 deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH under accession number DSM 32786; Lactobacillus paracasei LPC12 deposited at DSMZ GmbH under accession number DSM 32785; Lactobacillus fermentum LF26 deposited at DSMZ GmbH under accession number DSM 32784; Streptococcus thermophilus ST30 deposited at DSMZ GmbH under accession number DSM 32788; and Lactobacillus fermentum LF26 deposited at DSMZ GmbH under accession number DSM 32789. and Lactobacillus helveticus LH43 deposited under accession number 32787 to a subject in need thereof.

2. 10. The method of claim 1, wherein each said culture is a liquid culture.

3. The method according to claim 2, wherein the ratio of the numbers of Lactobacillus rhamnosus LRH10, Lactobacillus paracasei LPC12, Lactobacillus fermentum LF26, Streptococcus thermophilus ST30, and Lactobacillus helveticus LH43 in the composition is within the range of 5.1:29.7:1.9:47.8:1.2 to 10.9:41.3:3.4:53.2:5.

7.

4. The method of claim 2 , wherein the liquid culture is substantially cell-free.

5. The method of claim 1 , wherein the composition is formulated as a food product.

6. The method of claim 1 , wherein the composition is prepared in the form of a pharmaceutical composition.

7. 7. The method of claim 6, wherein the pharmaceutical composition is in a dosage form selected from the group consisting of an oral dosage form, a parenteral dosage form, and a topical dosage form.

8. 1. Use of a composition in the manufacture of a medicament or food product against Salmonella typhimurium infection in a subject, the composition comprising Lactobacillus rhamnosus LRH10 deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH under accession number DSM 32786, Lactobacillus paracasei LPC12 deposited at DSMZ GmbH under accession number DSM 32785, Lactobacillus fermentum LF26 deposited at DSMZ GmbH under accession number DSM 32784, Streptococcus thermophilus ST30 deposited at DSMZ GmbH under accession number DSM 32788, and Lactobacillus rhamnosus LRH10 deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH under accession number DSM 32786. and Lactobacillus helveticus LH43 deposited at the University of California, San Diego, Calif., under accession number DSM 32787.

9. 9. The use according to claim 8, wherein each said culture is a liquid culture.

10. The use according to claim 9, wherein the numerical ratio of Lactobacillus rhamnosus LRH10, Lactobacillus paracasei LPC12, Lactobacillus fermentum LF26, Streptococcus thermophilus ST30, and Lactobacillus helveticus LH43 in the composition is within the range of 5.1:29.7:1.9:47.8:1.2 to 10.9:41.3:3.4:53.2:5.

7.

11. The use according to claim 9, wherein the liquid culture is substantially cell-free.

12. 9. The use according to claim 8, wherein the pharmaceutical agent is in a dosage form selected from the group consisting of an oral dosage form, a parenteral dosage form, and a topical dosage form.

13. Lactobacillus rhamnosus LRH10, deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH under accession number DSM 32786; Lactobacillus paracasei LPC12, deposited at DSMZ GmbH under accession number DSM 32785; Lactobacillus fermentum LF26, deposited at DSMZ GmbH under accession number DSM 32784; Streptococcus thermophilus ST30, deposited at DSMZ GmbH under accession number DSM 32788; and a culture of Lactobacillus helveticus LH43 deposited under the accession number 32787.

14. The composition of claim 13 , wherein each said culture is a liquid culture.

15. The composition according to claim 14, wherein the ratio of the numbers of Lactobacillus rhamnosus LRH10, Lactobacillus paracasei LPC12, Lactobacillus fermentum LF26, Streptococcus thermophilus ST30, and Lactobacillus helveticus LH43 in the composition is within the range of 5.1:29.7:1.9:47.8:1.2 to 10.9:41.3:3.4:53.2:5.

7.

16. The composition of claim 14 , wherein the liquid culture is substantially cell-free.

17. The composition of claim 13 formulated as a food product.

18. The composition of claim 13 prepared in the form of a pharmaceutical composition.

19. 20. The composition of claim 18, wherein the pharmaceutical composition is in a dosage form selected from the group consisting of an oral dosage form, a parenteral dosage form, and a topical dosage form.

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