Lactic acid bacteria, methods and uses thereof

Abstract

The present invention relates to novel strains of lactic acid bacteria capable of utilizing ethanolamine, more specifically novel strains of Lactobacillus reuteri. This feature enables the bacteria to compete with pathogenic ethanolamine-utilizing pathogens for the same substrate, thereby presenting an effective way to combat infections caused by such pathogenic bacteria. Methods for selecting additional ethanolamine-utilizing lactic acid bacteria are also provided, as are other methods and uses involving the novel strains and other ethanolamine-utilizing lactic acid bacteria.

Description

[0001] Technical Field of the Invention This invention relates to lactic acid bacteria, and more particularly to lactic acid bacteria that have beneficial effects on diseases or conditions in humans and animals, and to the use of said lactic acid bacteria as probiotics. More specifically, this invention relates to the activation of certain biological activities in lactic acid bacteria after administration to humans, said biological activities being beneficial to the health of said humans. Background of the Invention It is well established that the interaction between the host and the microbiome is fundamental to health and disease. The microbiome produces metabolites that provide nutrients to the host and further participates in the immune response and the regulation and development of the immune system. The tissue microenvironment determines the composition of the microbiome, which in turn means that one way to influence and alter the composition of the microbiome may be through changes in diet (such as sugars, fats, or fiber—which function as energy sources for bacteria). Alterations in the host's immunity due to genetic variations or concurrent infections and subsequent antibiotic use can also affect the gut microbiome.

[0003] Lactobacillus and other lactic acid-producing bacteria, such as Bifidobacterium, are commonly used as probiotics in various types of food, such as yogurt. Lactic acid-producing bacteria can prevent the growth and colonization of harmful microorganisms through their own colonization on or inside mammals, through the formation of biofilms, through competition for available nutrients, and through the production of specific substances such as hydrogen peroxide, bacteriocins, or pH-lowering organic acids (including lactic acid and acetic acid).

[0004] Lactobacillus reuteri is a bacterium known to produce the antimicrobial substance 3-hydroxypropionaldehyde (HPA) (also known as reuterin).

[0005] Prokaryotic cells have been considered primitive, although some contain unusual enclosed compartments called microcompartments (MCS), which appear to act as primitive organelles within bacterial cells. Carboxysomes (involved in carbon dioxide fixation) have been the only microcompartments identified within microbial cells for nearly 30 years. In 2005, Professor Todd O. Yeates and colleagues revealed the first structural details of bacterial microcompartments. The first high-resolution structure of bacterial microcompartment proteins revealed a construction principle highly similar to that seen in some viruses. Six identical protein subunits aggregate to form hexammeric units, which constitute the building blocks of the shell. These hexammeric units are tightly packed together to form a molecular layer containing only micropores. This tight packing appears to restrict the movement of molecules in and out of the microcompartments, except through the pores.

[0006] Cluster analysis of homologs of microcompartment-specific proteins in microorganisms suggests that these compartments may be involved in up to seven different metabolic processes across various bacterial species (Thomas A. Bobik. 2007. Bacterial Microcompartments. Microbe. 1:25-31.). The building blocks of bacterial microcompartments are only proteins and glycoproteins. Electron microscopy (required for observing microcompartments) reveals the absence of a lipid monolayer or bilayer surrounding these compartments (as in eukaryotic vesicles), making them the only known protein-based metabolic compartments in living cells.

[0007] Members of the genera *Salmonella*, *Escherichia*, *Klebsiella*, *Clostridium*, *Fusobacterium*, *Shigella*, *Listeria*, and *Yersinia* contain the components required to degrade ethanolamine in their microcompartments. Another characteristic of microcompartments is considered to be their ability to act as containers for substrates that are toxic to the bacteria themselves.

[0008] Ethanolamine is a breakdown product of the membrane phosphatidylethanolamine (PE) and is ubiquitous in the gastrointestinal tract. Ethanolamine can be used as a nitrogen source (and sometimes a carbon source) by bacteria capable of metabolizing this compound. This ability has been associated with important gastrointestinal pathogens, including, for example, enterohemorrhagic Escherichia coli O157:H7 (EHEC) . Ethanolamine may also be a signal for bacteria to initiate their virulence program. (Garsin DA. 2012. Ethanolamine: a signal to commence a host-associated lifestyle) mBio 3(4):e00172-12. doi:10.1128 / mBio.00172-12.).

[0009] Rising antimicrobial resistance and a declining number of new antibiotic discoveries are increasing the risk of a global infectious disease health care crisis. National Action Plan to Combat Antibiotic-Resistant Bacteria (Bacteria TFfCA-R (ed.). 2015). National Action Plan for Combating Antibiotic-Resistant Bacteria The White House (Washington, DC) emphasizes the need for efforts to accelerate the development of new antibiotics and alternative therapies to combat resistance and disease associated with antimicrobial use. As a result, many emerging therapies are being investigated, including but not limited to probiotics, immunotherapy, and toxin binders.

[0010] Finding new antibiotic therapies is no easy task, as most pathogenic bacteria have developed clever ways to persist under different conditions and utilize the host microenvironment. For example, the pathogenic bacterium *Salmonella typhimurium* exhibits a unique growth advantage over other bacteria because it can utilize ethanolamine, a substance released by host tissue cells in the inflamed gut. However, to date, it is not believed, or at least not shown, that ethanolamine can be utilized by beneficial bacteria. See, for example, (Thiennimitr P, et al.) Intestinal inflammation allows Salmonella to use ethanolamine to compete with the microbiota , Proc Natl Acad Sci USA 2011).

[0011] Therefore, there remains a need in the field to identify new ways to combat pathogenic bacteria, and in particular, to identify new therapies that do not contribute to the further development of antibiotic resistance in society. Invention Overview The aforementioned objectives have now been achieved or at least mitigated by the novel strains of lactic acid bacteria capable of utilizing ethanolamine, methods for selecting additional lactic acid bacteria that utilize ethanolamine, methods for producing ethanolamine-utilizing lactic acid bacteria induced to utilize ethanolamine, and various compositions and uses relating to said ethanolamine-utilizing lactic acid bacteria.

[0013] Therefore, the present invention lies in the surprising discovery that some lactic acid bacteria are capable of utilizing ethanolamine as a substrate. Without wishing to be bound by theory, this discovery appears to provide a means for such bacteria to compete with pathogenic bacteria that utilize the same substrate. Thus, when such pathogenic bacteria are present in the gastrointestinal tract of an infected individual (where the substrate is available), administration of beneficial ethanolamine-utilizing bacteria can worsen the survival of the pathogenic bacteria and also reduce their virulence. It is also envisioned that other mechanisms already employed by ethanolamine-utilizing bacteria can interact with the ethanolamine utilization mechanism, thereby providing the improved antimicrobial activity presented herein. Therefore, this presents a useful antimicrobial alternative that targets ethanolamine-utilizing pathogenic bacteria present in the gastrointestinal tract of an individual. Subsequently, it is also envisioned that this discovery has the potential to reduce the overuse of antibiotics in response to pathogenic infections. Additionally, acetaldehyde produced by lactic acid bacteria when utilizing ethanolamine can possess its own antimicrobial activity.

[0014] Therefore, this article provides a method for selecting lactic acid bacteria strains capable of utilizing ethanolamine, the method comprising the following steps: i) Provide a culture medium with a strain of lactic acid bacteria and allow the culture of said bacteria to grow. ii) Determine whether the bacteria are able to utilize ethanolamine; and iii) If the bacteria are able to utilize ethanolamine, then the lactic acid bacteria strain is selected. The culture medium contains a certain amount or concentration of ethanolamine and / or a certain amount or concentration of ethanolamine is added to the culture in step i) at a time point prior to step ii).

[0015] In another aspect, a method for selecting lactic acid bacteria strains capable of utilizing ethanolamine is also provided, comprising determining the genetic profile of said lactic acid bacteria, the method comprising the following steps: i) Provide the lactic acid bacteria strains to be screened; ii) Perform initial screening of the lactic acid bacteria strain to determine the presence of one or more bacterial homologs of the gene encoding the following protein in the lactic acid bacteria strain; 1. Ethanolamine aminolyase large subunit EutB; 2. EutL, a microcompartmental structural protein; and / or 3. Ethanolamine utilizes the protein EutH; iii) If one or more of the genes are present in the lactic acid bacteria strain, determine whether the bacteria can utilize ethanolamine; and iv) If the bacteria are able to utilize ethanolamine, then the lactic acid bacteria strain is selected.

[0016] Optionally, as part of step ii) of the method described above, before or after step ii) and before step iii) of the method described above, a second or additional screening of the lactic acid bacteria strain, i.e., genetic screening, may be performed to determine the presence of one or more bacterial homologs encoding genes for the following proteins: 1. NADPH-dependent FMN reductases belonging to the pfam03358 protein family and / or 2. A protein-tyrosine phosphatase belonging to the pfam13350 protein family.

[0017] The gene selected in the second screening may reside in the same gene cluster as the gene selected in the initial screening, or even reside in the same operon. Preferably, the gene resides in the same gene cluster or even resides in the same operon.

[0018] In addition, a method for producing lactic acid bacteria induced to utilize ethanolamine is provided, the method comprising the following steps: i) Provide lactic acid bacteria in the culture medium that can utilize ethanolamine. ii) At a first time point, add a first amount or concentration of ethanolamine to the culture medium from step i) and allow the culture to grow. iii) Optionally, at a second time point, a second amount or concentration of ethanolamine is added to the culture of step ii) and the culture is allowed to grow, and thereafter; iv) Retrieve the lactic acid bacteria from the culture medium.

[0019] This disclosure also covers lactic acid bacteria selected or produced by the methods disclosed herein, and their various uses. In another aspect, compositions are also provided comprising ethanolamine-utilizing lactic acid bacteria induced by ethanolamine during the growth of a culture containing said lactic acid bacteria.

[0020] As previously mentioned, novel strains of *Lactobacillus reuteri* are also provided: biopure cultures of *Lactobacillus reuteri* DSM 27131 and biopure cultures of *Lactobacillus reuteri* DSM 32465. These strains have been selected as lactic acid bacteria strains capable of utilizing ethanolamine. These strains can also be used in methods for producing lactic acid bacteria induced to utilize ethanolamine, as well as in various other uses and methods encompassing the ethanolamine-utilizing bacteria described herein. Freeze- or lyophilized compositions of ethanolamine-utilizing lactic acid bacteria (such as *Lactobacillus reuteri*) are also provided, for example, freeze- or lyophilized compositions comprising a biopure culture of *Lactobacillus reuteri* DSM 27131 and / or a biopure culture of *Lactobacillus reuteri* DSM 32465, said compositions further comprising at least one cryoprotectant or lyophilization protectant. Various cryoprotectants (e.g., glycerol, sucrose, lactose, or trehalose) or lyophilization protectants (e.g., sucrose, lactose, trehalose, or maltodextrin) or other additives as known in the art are also contemplated in this context.

[0021] Novel strains of lactic acid bacteria selected or produced by the methods disclosed herein or as described elsewhere herein are also provided for use as pharmaceuticals, such as for treating conditions or symptoms caused by or associated with pathogens that utilize ethanolamine. Examples of such pathogens are further disclosed herein. Use of the said lactic acid bacteria or strains in inhibiting the growth of one or more pathogenic bacteria is also provided. More specifically, these compositions are intended for administration to humans or animals, for example, for the purpose of more effectively treating conditions induced by pathogens in the gastrointestinal tract. Brief description of the attached diagram Figure 1The production of acetaldehyde by lactic acid bacteria utilizing ethanolamine is shown. Acetaldehyde production is visualized as a thick (red) line, accompanied by an expanding (red) area surrounding the thick line. The thick line also contains bacterial streaks; A) No thick line or red (i.e., expanding) area is seen around the bacterial streaks of *Lactobacillus reuteri* DSM 17938. B) A red (i.e., expanding) area extending approximately 3 mm on each side of the bacterial streaks forming part of the thick line is seen around the colonies of *Lactobacillus reuteri* DSM 27131, and a red (expanded) area is also seen around the streaks of *Lactobacillus reuteri* DSM 32465 (C, extending approximately 1.5 mm on each side of the bacterial streaks forming part of the thick line).

[0023] Figure 2 The results show the bile tolerance assay and decreased viability of wild-type strain Lactobacillus reuteri DSM 27131 (shown as gray / striped bars) and improved strain Lactobacillus reuteri DSM 32465 (shown as black bars).

[0024] Figure 3 In vitro analysis confirmed the enhanced inhibition of *C. diffusa* by *Lactobacillus reuteri* DSM 27131 pre-incubated with 10 mM ethanolamine. In vitro growth of *C. diffusa* CD2015 was measured in fecal contents from microbe-free mice. Results of viable colony-forming units of *C. diffusa* and *C. diffusa* along with *L. diffusa* DSM 27131 were obtained after 24 hours of incubation. Data represent mean ± SD. Invention Details definition In this article, the term "treatment" can encompass both the relief of symptoms and the prevention of symptom onset. Therefore, the term includes the prevention, reduction, and avoidance of conditions or symptoms. The term "symptom" can also include disease.

[0026] Whenever the term “bacteria” is used in this document, it is intended to include strains of lactic acid bacteria (unless it refers to pathogens), but is not limited to any particular strain.

[0027] According to methods known in the art, the term "utilization" of ethanolamine herein means that lactic acid bacteria are able to use ethanolamine and metabolize it into other active metabolites. According to other methods known in the art, the term "utilization" of ethanolamine may also, in some contexts, mean that lactic acid bacteria are able to use ethanolamine and metabolize it into other active and secreted metabolites.

[0028] lactic acid bacteria strains Lactobacillus reuteri strain DSM 27131 was deposited under the Budapest Convention on April 18, 2013 at Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH (Inhoffenstrasse 7B, D - 38124 Braunschweig).

[0029] Lactobacillus reuteri strain DSM 32465 was deposited under the Budapest Convention on March 21, 2017 at Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH (Inhoffenstrasse 7B, D - 38124 Braunschweig).

[0030] Detailed Introduction As described earlier, the inventors have for the first time identified a novel strain of non-pathogenic lactic acid bacteria capable of utilizing ethanolamine. Ethanolamine is a breakdown product of the membrane phosphatidylethanolamine (PE) and is ubiquitous in the gastrointestinal tract. Ethanolamine can be used as a nitrogen source and sometimes a carbon source by bacteria capable of metabolizing this compound. However, until now, only pathogenic bacteria, such as enterohemorrhagic Escherichia coli O157:H7 (EHEC), have been considered capable of utilizing ethanolamine. For example, this utilization in non-pathogenic bacteria can be detected in colonies on an agar plate by acetaldehyde, which is identified from ethanolamine.

[0031] These findings can also be used to identify and select additional lactic acid bacteria based on the presence of characteristics that determine the bacteria's utilization of ethanolamine. Therefore, methods for selecting ethanolamine-utilizing lactic acid bacteria, methods for producing ethanolamine-induced ethanolamine-utilizing lactic acid bacteria, and other uses, compositions, and methods relating to these novel strains and bacteria are also results of these findings and are therefore provided herein. These aspects are further described below.

[0032] Selection Method Therefore, in more detail, this document provides a method for selecting lactic acid bacteria strains capable of utilizing ethanolamine, the method comprising the following steps: i) Provide a culture medium with a strain of lactic acid bacteria and allow the culture of said bacteria to grow. ii) Determine whether the bacteria are able to utilize ethanolamine; and iii) If the bacteria are able to utilize ethanolamine, then the lactic acid bacteria strain is selected. The culture medium contains a certain amount or concentration of ethanolamine and / or a certain amount or concentration of ethanolamine is added to the culture in step i) at a time point prior to step ii).

[0033] Culture media contain nutrients and other components to support the growth of bacterial cells and are well known to those skilled in the art. Examples of culture media in this context include, for example, agar (agar plates), but liquid media are also contemplated. A culture may also be referred to as a bacterial culture, and / or one or more culture media may be referred to as a single / multiple bacterial culture medium.

[0034] The lactic acid bacteria can be cultured in a medium containing ethanolamine under anaerobic conditions, and at a temperature of about 35°C to about 45°C, such as about 37°C. Typically, the bacteria are allowed to grow for about 14 to about 72 hours, such as about 20 to about 60 hours, about 14 to about 16 hours, or about 48 hours. Other, similar conditions are also envisioned, for example, microaerobic conditions with less oxygen than in the air (e.g., about 5% oxygen).

[0035] Ethanolamine can be added directly to the culture medium along with lactic acid bacteria, such as in agar plate experiments to determine whether a lactic acid bacteria strain can utilize ethanolamine. However, it can also be added subsequently, such as at a later time point, after the culture has been incubated for a certain period. This later time point could be, for example, when the culture has reached a specific concentration or density, such as the optical density (OD) at 600 nm. 600 When the measurement is approximately 0.3-2 units.

[0036] The reference in this document to a certain "amount or concentration" of, for example, ethanolamine is used to illustrate that the method can be carried out on a small or large scale, and also to illustrate the use and envision of suitable amounts and concentrations of ethanolamine in the methods of this disclosure. Typically, ethanolamine will be described as being added to a culture medium containing a strain of lactic acid bacteria at a specific concentration, wherein when ethanolamine has been added thereto, the concentration is the total concentration of ethanolamine in the culture medium, but of course this also means adding a certain amount of ethanolamine to the culture medium to achieve that concentration. Those skilled in the art will know what amount or concentration of ethanolamine will be needed to induce and / or activate the bacterial culture.

[0037] The method may further include adding an additional amount or concentration of ethanolamine to the culture of step i) at a second time point after the addition of the first amount of ethanolamine, and allowing the culture to grow before performing steps ii) and iii) of the method. As previously described herein, an additional amount or concentration of ethanolamine may be added to the culture of lactic acid bacteria to further promote the bacteria's utilization of ethanolamine and / or further promote the bacteria's production of acetaldehyde. If agar medium has been used, the bacteria have typically already been incubated for a specific period of time in the presence of ethanolamine. If so, any second amount or concentration of ethanolamine may be added as a covering to the agar plate containing the bacteria.

[0038] Examples of this method are presented in the experimental section.

[0039] The selection method provided herein may also include an additional step that can be performed before the lactic acid bacteria have been exposed to ethanolamine. This step includes determining a specific genetic profile of the lactic acid bacteria that indicates whether the bacteria are predisposed to utilizing ethanolamine as a substrate. More specifically, this method includes an additional step performed before or in conjunction with step i) of the selection method, and it includes an initial screening of the lactic acid bacteria for the presence of one or more genes encoding bacterial homologs of one or more genes, which encode any protein selected from: the large subunit of ethanolamine lyase EutB, the microcompartmental structural protein EutL, and the ethanolamine utilization protein EutH.

[0040] The gene is known and publicly available, and may be, for example, found in https: / / www.ncbi.nlm.nih.gov / genbank / Find it based on the following information: 1. Ethanolamine aminolyase large subunit EutB Example: Genbank login number EOJ56712; 2. EutL, a microcompartmental structural protein Example: Genbank login number EOJ56710; 3. Ethanolamine utilizes the protein EuTH, an ethanolamine transporter. Example: Genbank login number EOJ56702.

[0041] Optionally, the presence of bacterial homologs encoding genes of NADPH-dependent FMN reductases belonging to the pfam03358 protein family and / or genes encoding protein-tyrosine-phosphatases belonging to the pfam13350 protein family may also be determined in the method, sometimes referred to herein as a second or additional genetic screening. The genes may reside in the same gene cluster or operon as the genes screened in the initial screening. Preferably, the genes reside in the same gene cluster as the genes initially screened, and sometimes even in the same operon.

[0042] By performing the second or additional genetic screening described herein, the methods for selecting lactic acid bacteria strains capable of utilizing ethanolamine can be further improved, including identifying the genetic profiles of these strains, or all of them together, as they are envisioned to be particularly beneficial in the context of ethanolamine utilization.

[0043] NADPH-dependent FMN reductases belonging to the pfam 03358 protein family can be listed using Genbank accession number WP_075913928, but are not limited thereto. Protein-tyrosine phosphatases belonging to the pfam13350 protein family can be listed using Genbank accession number WP_054277074, but are not limited thereto.

[0044] As described above, the genes selected in the first or initial screening and the second or additional screening may reside in the same gene cluster or even in the same operon. In some respects, the genes may reside in the same gene cluster or the same operon.

[0045] The presence of one or more of the aforementioned genes in the genome of the lactic acid bacteria strain indicates that the strain is capable of utilizing ethanolamine, and if so, a further step can be taken to determine or confirm whether the strain is capable of utilizing ethanolamine (such as by determining the production of acetaldehyde).

[0046] Screening for the presence of one or more of these genes can be performed using methods commonly known for analyzing genetic information, such as whole or partial genome sequencing, polymerase chain reaction (PCR), microarrays, etc. These are techniques well-known and readily available to those skilled in the art.

[0047] The selection method may further include a step ii) in which step ii) involves determining whether the lactic acid bacteria are capable of producing acetaldehyde when utilizing ethanolamine by measuring the amount or concentration of acetaldehyde in a culture sample from the bacterial culture. An example of how to determine whether a lactic acid bacteria strain is capable of utilizing ethanolamine is found in Example 1 of the Experimental Section (results shown in...). Figure 1(In the text). The utilization is seen as the production of acetaldehyde from ethanolamine (visible as a red area on an agar plate (marked with an arrow and also considered to contain thick lines)). See also Figure 1 The differences in results between bacteria that utilize ethanolamine and those that do not are clear and easily determined by a technician. As a control reference, strains of lactic acid bacteria that cannot utilize ethanolamine, such as strains identified as not possessing the genetic profile described herein, can be used.

[0048] Therefore, this paper can determine whether a lactic acid bacteria strain can utilize ethanolamine by analyzing its spread or diffusion, but it can also be determined by measuring the width of the expanded (red) area surrounding the thick line containing the bacterial stripes, such as... Figure 1 Examples are provided below. As previously mentioned herein, in some respects, the presence of acetaldehyde is determined if a distinct red area or region is observed on and / or around the bacterial streaks. Sometimes, more specifically, the “width” of the region can be measured, and the presence of acetaldehyde can be indicated by extending at least about 0.5 mm on each side of the thick line or bacterial streak. This is further shown in Example 1 and illustrated in Table 1.

[0049] In this document, the amount or concentration of ethanolamine in or added to the culture medium in step i) can be from about 0.1 mM to about 50 mM, such as from about 1 mM to about 50 mM, such as from about 1 mM to about 30 mM, such as from about 0.5 mM to about 15 mM. This may also be referred to as the first amount or concentration of ethanolamine. Furthermore, another amount or concentration of ethanolamine added to the culture can be from about 1 mM to about 1 M, such as from about 30 mM to about 1 M, or from about 10 mM to about 100 mM. This may also be referred to as the second amount or concentration of ethanolamine. The addition of the first or second amount of ethanolamine may, but is not necessary, be used for different purposes in facilities utilizing lactic acid bacteria with ethanolamine.

[0050] In this document, and in all contexts, lactic acid bacteria can refer to bacteria of the genus *Lactobacillus*. This certainly applies to all aspects, uses, and methods disclosed herein. The lactic acid bacteria can also be the species *Lactobacillus reuteri* (…). Lactobacillus reuteri (bacteria). This generally applies to this article as well.

[0051] In one aspect of this disclosure, lactic acid bacteria strains selected by the selection method herein are provided, said bacteria being capable of utilizing ethanolamine.

[0052] Production methods A method for producing lactic acid bacteria as defined herein is also provided. More specifically, a method for producing lactic acid bacteria induced to utilize ethanolamine is provided, the method comprising the steps of: i) Provide lactic acid bacteria in the culture medium that can utilize ethanolamine. ii) At a first time point, add a first effective amount or concentration of ethanolamine to the culture from step i) and allow the culture to grow. iii) Optionally, at a second time point, a second effective amount or concentration of ethanolamine is added to the culture of step ii) and the culture is allowed to grow, and thereafter; iv) Retrieve the lactic acid bacteria from the culture medium.

[0053] Lactic acid bacteria produced by the method described above may also produce reuteriin and compete with pathogenic bacteria for the substrate glycerol or 1,2-propanediol (1,2-PD), thereby limiting the availability of such substrates to pathogens in addition to limiting the use of ethanolamine. As previously mentioned herein, different mechanisms and components of the lactic acid bacteria apparatus, including the ability to utilize ethanolamine, are also envisioned to interact and thereby provide improved antimicrobial activity.

[0054] Furthermore, regarding methods for producing lactic acid bacteria induced to utilize ethanolamine, a first and optionally a second amount or concentration of ethanolamine may be added to or provided to the culture. This may also be referred to as the first and second addition of ethanolamine to the culture or culture medium. Again, the amount or concentration of ethanolamine in or added to the culture medium in step i) may be from about 1 to about 30 mM, or as exemplified elsewhere herein. This may also be referred to as the first amount or concentration of ethanolamine. Moreover, a further amount or concentration of ethanolamine added to the culture may be from about 30 mM to about 1 M, or as exemplified elsewhere herein. This may also be referred to as the second amount or concentration of ethanolamine. The addition of the first or second amount of ethanolamine may, but is not necessary, be used for different purposes in facilities utilizing ethanolamine-producing lactic acid bacteria.

[0055] As previously mentioned regarding the selection method, the lactic acid bacteria used can be bacteria of the genus *Lactobacillus*. Further, as previously mentioned, the lactic acid bacteria can be bacteria of the species *Lactobacillus reuteri*. This also applies to all aspects of this document.

[0056] The lactic acid bacteria produced by the methods disclosed herein can then be formulated into a storage-appropriate form and subsequently into a product suitable for administration to human or animal subjects. The product should be substantially free of moisture to achieve satisfactory storage stability (e.g., by adding a moisture-resistant agent to the composition). Cultures of the lactic acid bacteria can be preserved by freezing or lyophilization / freeze-drying.

[0057] Of course, the lactic acid bacteria used in any composition described herein are live bacteria, even if they are in dried or lyophilized / freeze-dried form, etc.

[0058] Therefore, this document also provides lactic acid bacteria produced by the methods disclosed herein, and any composition comprising said lactic acid bacteria. Such compositions may be lyophilized / freeze-dried and may optionally contain additional additives or ingredients, such as one or more cryoprotectants, lyophilization protectants, and / or moisture-resistant agents.

[0059] Genetic profile analysis As previously mentioned herein, in another aspect, but concerning the selection method previously provided herein, a method is provided for selecting lactic acid bacteria strains capable of utilizing ethanolamine, which includes determining the genetic profile of said lactic acid bacteria, the method comprising the following steps: i) Provide the lactic acid bacteria strains to be screened; ii) Perform initial screening of the lactic acid bacteria strain to determine the presence of one or more bacterial homologs of the gene encoding the following protein in the lactic acid bacteria strain; a) Ethanolamine aminolyase large subunit EutB; b) EutL, a microcompartmental structural protein; and / or c) Ethanolamine utilizes the protein EutH; iii) If one or more of the genes are present in the lactic acid bacteria strain, determine whether the bacteria can utilize ethanolamine; and iv) If the bacteria are able to utilize ethanolamine, then the lactic acid bacteria strain is selected.

[0060] Optionally, as part of step ii) of the method described above, before or after step ii) and before step iii) of the method described above, a second or additional screening of the lactic acid bacteria strain, namely genetic screening, may be performed to determine the presence of one or more bacterial homologs encoding genes encoding NADPH-dependent FMN reductases belonging to the pfam03358 protein family and / or protein-tyrosine-phosphatases belonging to the pfam13350 protein family. These genes may be present in the same gene cluster as those screened in the initial screening, or even in the same operon. Preferably, the genes are present in the same gene cluster or even in the same operon. By performing the second genetic screening described herein, the method for selecting lactic acid bacteria strains capable of utilizing ethanolamine, including determining the genetic profile, can be further improved, as these genes, or all genes together, are envisioned as beneficial in the context of ethanolamine utilization.

[0061] The genes and standards used to carry out the method are as previously described in the context of selecting and / or producing the method.

[0062] Uses of lactic acid bacteria selected or produced by the methods described herein, including those utilizing ethanolamine. Composition of lactic acid bacteria In another aspect, lactic acid bacteria selected or produced by the methods described herein are provided for inhibiting the growth of pathogenic bacteria in the gastrointestinal tract of an individual. Similarly, the use of lactic acid bacteria selected or produced by the methods described herein for inhibiting the growth of pathogenic bacteria is provided. The proposed mechanism behind this characteristic of the selected or produced lactic acid bacteria has been previously described herein and is due to the competition between pathogenic bacteria and lactic acid bacteria for the same substrate. By inhibiting the growth of these pathogens, any harmful effects caused or already caused by pathogens can be indirectly prevented or at least mitigated by providing lactic acid bacteria to individuals in need.

[0063] Therefore, this document also provides such lactic acid bacteria for use as a medicine. Furthermore, such lactic acid bacteria are provided for the treatment of conditions or symptoms caused by or associated with pathogens that utilize ethanolamine. As mentioned, this effect can be mediated by the inhibitory effect of said lactic acid bacteria on the pathogenesis of invading pathogenic bacteria. As shown herein, in Example 3 and... Figure 3 In this study, lactic acid bacteria utilizing ethanolamine were able to successfully inhibit the growth of the pathogen Clostridium difficile.

[0064] In this document, the pathogens using ethanolamine may be selected from *Clostridium difficile*, *Escherichia coli*, *Enterohemorrhagic Escherichia coli*, *Salmonella enterica* (e.g., *Salmonella enterica* typhus serotype), *Shigella sogii*, *Shigella dysenteriae*, *Klebsiella pneumoniae*, *Citrobacter korshinskii*, *Pseudomonas aeruginosa*, *Clostridium perfringens*, *Clostridium difficile*, *Clostridium tetani*, *Listeria monocytogenes*, *Clostridium nucleatum*, *Enterococcus faecalis*, *Acinetobacter baumannii*, *Burkholderia glands*, and *Burkholderia cepacia*. However, other pathogens utilizing ethanolamine may also be conceived.

[0065] Examples of conditions associated with pathogenic bacteria that utilize ethanolamine include, for example, dysbiosis (microbial imbalance or maladaptation on or within the body), listeriosis, salmonellosis, bacterial infections leading to inflammatory bowel disease, infections with pathogenic Pseudomonas (infected wounds), and traveler's diarrhea.

[0066] Compositions are also provided comprising ethanolamine-utilizing lactic acid bacteria induced by ethanolamine during the growth of a cell culture containing said lactic acid bacteria. As previously described herein, the compositions described herein may comprise lactic acid bacteria of the genus *Lactobacillus*. The *Lactobacillus* may also be a species of *Lactobacillus reuteri*. The compositions herein can be used to treat conditions or symptoms caused by or associated with ethanolamine-utilizing pathogens. The compositions herein can also be used to inhibit the growth of one or more pathogenic bacteria in an individual's gastrointestinal tract. Similarly, they can be used to treat conditions or symptoms caused by or associated with ethanolamine-utilizing pathogens, and to inhibit the growth of one or more pathogenic bacteria in an individual's gastrointestinal tract.

[0067] Naturally, this document provides lactic acid bacteria or lactic acid bacteria strains utilizing ethanolamine, which are used as probiotics. Probiotic compositions comprising the lactic acid bacteria or lactic acid bacteria strains presented in all aspects herein are also provided.

[0068] Novel lactic acid bacteria strains The following novel lactic acid bacteria strains are also provided: Biologically pure cultures of Lactobacillus reuteri DSM 27131 and; Biologically pure culture of Lactobacillus reuteri DSM 32465.

[0069] In one aspect, a freeze-dried or lyophilized composition is provided comprising a biologically pure culture of either Lactobacillus reuteri DSM 27131 or Lactobacillus reuteri DSM 32465.

[0070] This article also provides novel frozen or lyophilized products of Lactobacillus reuteri, which contain at least: A biopure culture of Lactobacillus reuteri DSM 27131; or Biologically pure culture of Lactobacillus reuteri DSM 32465.

[0071] This article also provides the following novel frozen products for Lactobacillus reuteri, which contain at least one cryoprotectant, such as glycerol, and: A biopure culture of Lactobacillus reuteri DSM 27131; or Biologically pure culture of Lactobacillus reuteri DSM 32465.

[0072] This article also provides the following novel lyophilized Lactobacillus reuteri products containing at least one lyophilization protectant, such as disaccharide, and: A biopure culture of Lactobacillus reuteri DSM 27131; or Biologically pure culture of Lactobacillus reuteri DSM 32465.

[0073] In this document, the terms product and composition may be used interchangeably with respect to the lactic acid bacteria.

[0074] As previously mentioned herein, novel *Lactobacillus reuteri* strains, or compositions or products containing or comprising said strains, are provided for the treatment of conditions or symptoms caused by or associated with ethanolamine-using pathogens and / or for the inhibition of the growth of one or more pathogenic bacteria in the gastrointestinal tract of an individual, as previously described herein. Ethanolamine-using pathogens are exemplified elsewhere herein and are equally applicable to the context of this invention.

[0075] Methods for treating conditions or ailments in an individual caused by or associated with ethanolamine-using pathogens are also provided, the methods comprising administering to the individual an effective amount of lactic acid bacteria, novel lactic acid bacteria strains or compositions, or administering a product described elsewhere herein to the individual. Methods for treating an individual suffering from an infection caused by pathogenic ethanolamine-using bacteria are also provided by administering ethanolamine-using lactic acid bacteria as defined herein to inhibit the growth of said pathogenic bacteria. The administration of a pharmaceutically effective amount of said lactic acid bacteria to the individual is to be determined by a person skilled in the art.

[0076] A method for modifying Lactobacillus strains, such as Lactobacillus reuteri, for bile tolerance through dominant selection is also provided, as further described in the following examples (Example 2). Thus, in Example 2, it is shown that Lactobacillus reuteri strain DSM 32465, modified from the wild-type strain DSM 27131, is more tolerant to bile than the wild-type strain. This presents additional advantages for the use of Lactobacillus reuteri strain DSM 32465 as a probiotic for certain conditions, uses, and indications.

[0077] The present invention will now be illustrated by the following experimental section, but it is not intended to be limited thereto.

[0078] Experimental Section Example 1 Selection of bacterial strains that can utilize ethanolamine bacterial strains Different strains of Lactobacillus reuteri were tested: Lactobacillus reuteri DSM 27131, Lactobacillus reuteri DSM 17938, and Lactobacillus reuteri DSM 32465. All strains were obtained from BioGaia AB.

[0079] method The bacteria were grown on MRS agar plates (containing 10 mM ethanolamine) under an anaerobic atmosphere at 37°C for 48 hours. The plates were then covered with 500 mM ethanolamine agar (1% agar) and incubated at 37°C for 1 hour. Acetaldehyde was detected by adding 5 ml of 2,4-dinitrophenylhydrazine (0.1%, in 2M HCl). After incubation for 3 minutes, the solution was poured off and 5 ml of 5 M KOH was added. The red area around the bacterial stripes indicates the presence of acetaldehyde.

[0080] result The potential utilization of ethanolamine by all strains of *Lactobacillus reuteri* was tested, which can be observed as the production of acetaldehyde. *Lactobacillus reuteri* DSM 17938 was negative, but *Lactobacillus reuteri* DSM 27131 and *Lactobacillus reuteri* DSM 32465 were positive in this assay; see [link to test]. Figure 1 Therefore, enlarged (red) areas were observed around *Lactobacillus reuteri* DSM 27131 and *Lactobacillus reuteri* DSM 32465. No enlarged (red) areas were observed around *Lactobacillus reuteri* DSM 17938. Figure 1 The approximate width of the acetaldehyde-containing region is illustrated in Table 1 below. Table 1: Figure 1 The width of the red area surrounding the bacterial stripe in B (DSM 27131) and C (DSM 32465), respectively.

[0081] Select Steps Choose Lactobacillus reuteri DSM 27131 and Lactobacillus reuteri DSM 32465.

[0082] Example 2 Improvement of Lactobacillus reuteri through dominant selection-bile tolerance bacterial strains The Lactobacillus reuteri strain DSM 27131 was used.

[0083] method The bacteria were grown overnight at 37°C in MRS liquid culture medium (ON). Then the tubes were incubated at 3000 x gCentrifuge for 10 minutes and discard the supernatant. Resuspend the precipitate in an equal volume of MRS containing added porcine bile (0.5% w / v). Prepare 200 μl aliquots, taking two tubes directly for viability assays (starting values). Prepare sequential 10-fold dilutions of these tubes and plate them onto MRS agar plates using the dropper method. Briefly, drop 10 μl of each dilution onto an MRS plate divided into six zones, allow to dry, and then incubate overnight at 37°C under anaerobic conditions. Incubate the remaining aliquots at 37°C, taking two tubes every 30 minutes for viability assays, for up to 4 hours.

[0084] After overnight incubation, bacterial counts were determined to establish bacterial viability. From appropriate time points, colonies of different sizes were collected based on viability and preserved as new strains of the same *Lactobacillus reuteri*.

[0085] Select Steps The new strain, Lactobacillus reuteri DSM 32465, was selected.

[0086] result Initial testing with *Lactobacillus reuteri* DSM 27131 showed poor tolerance to porcine bile at concentrations of 1% and 5%. Additional experiments with 0.5% porcine bile yielded the same results, but colonies from the first two time points were picked and preserved as a new strain. *Lactobacillus reuteri* DSM 32465 was tested with 0.5% bile in the same manner as *Lactobacillus reuteri* DSM 27131 and showed a significant improvement in bile tolerance. Figure 2 The low initial value of Lactobacillus reuteri DSM 27131 shows how sensitive the wild type is to porcine bile, which increases from approximately 5.10 within minutes. 8 CFU / ml decreased to below 10 6 CFU / ml.

[0087] Example 3 result In vitro experiments showed that the *Lactobacillus reuteri* strain DSM 27131, utilizing ethanolamine, completely inhibited the growth of *Clostridium difficile*, while *Lactobacillus reuteri* DSM 17938 (data not shown) did not inhibit the growth of *Clostridium difficile* under the same experimental conditions. Figure 3 ).

[0088] Materials and methods Bacterial strains and culture conditions.

[0089] Routine cultures of *Lactobacillus reuteri* strains 17938 and DSM 27131 in deMan, Rogosa, and Sharpe media (MRS; Difco, Franklin Lakes, NY) and *Clostridium difficile* strain CD2015 in brain heart infusion medium with 2% D-glucose (w / v) (BHI; BDBiosciences, Franklin Lakes, NJ) were conducted at 37°C in an anaerobic chamber (Anaerobe Systems, AS-580, MorganHill, CA) supplemented with a mixture of 10% CO2, 5% H2, and 85% N2 for 16–18 h.

[0090] In vitro growth study of GI fecal contents.

[0091] The sensitivity of *Clostridium difficile* to *Lactobacillus reuteri* 17938 or *Lactobacillus reuteri* DSM 27131 in the fecal contents of microbe-free mice was determined as follows. Fecal sediment was collected from microbe-free 7-10 week old C57BL / 6 mice, resuspended in PBS at a 1:2 ratio, and aliquoted into 200 μL samples. Each aliquot was treated with *Clostridium difficile* CD2015 (10... 4 Inoculate with *Lactobacillus reuteri* 17938 (10 mL) and then incubate with PBS containing 3 mM glycerol, and use *Lactobacillus reuteri* 17938 (10 mL). 7 / ml) or Lactobacillus reuteri DSM 27131 (10 7 The suspension was treated with 10 mM ethanolamine (pre-incubated overnight). The suspension was anaerobically incubated at 37°C for 24 h. Samples were taken at 0 and 24 h, and Clostridium difficile and Lactobacillus reuteri were quantified by overnight incubation on pre-reduced selective cycloserine-cefoxitin-fructose agar (with sodium taurocholate (TCCFA)) or MRS medium, respectively. The plates were anaerobically incubated at 37°C for 48 h and colony-forming units were counted.

[0092] Example 4 This article presents partial sequencing of bacterial homologs of genes used in genetic screening methods. A draft genome sequence was obtained from the bacterial strain *Lactobacillus reuteri* DSM 27131 using methods known in the art. The results of this analysis revealed the following genes encoding proteins involved in ethanolamine utilization in *Lactobacillus reuteri* DSM 27131: 1. Ethanolamine aminolyase large subunit EutB 2. EutL, a microcompartmental structural protein 3. Ethanolamine utilizes the protein EutH 4. NADPH-dependent FMN reductases belonging to the pfam03358 protein family. 5. A protein-tyrosine phosphatase belonging to the pfam13350 protein family.

[0093] All of the above genes are located in the same gene cluster and may also be located in the same operon.

Claims

1. A pure culture of Lactobacillus reuteri with accession number DSM 32465, wherein Lactobacillus reuteri with accession number DSM 32465 was deposited at DSMZ (German Microbiological Collection) on March 21, 2017.

2. The biologically pure culture of claim 1, wherein the biologically pure culture is freeze-dried or lyophilized.

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