LACTIC ACID BACTERIA AND THEIR USE FOR THE TREATMENT OF MASTITIS

Abstract

The present invention relates to novel Lactic Acid Bacteria strains Lactobacillus reuteri selected from the group comprising Lactobacillus reuteri DSM 32229, Lactobacillus reuteri DSM 32230, Lactobacillus reuteri DSM 32231 and Lactobacillus reuteri DSM 32232 and products comprising these strains. The present invention also relates to a use of one or more Lactic Acid Bacteria strains as a probiotic for the treatment of an inflammation and infection, such as mastitis and/or thrush, especially after topical administration of said strains.

Claims

1. A biologically pure culture of Lactobacillus reuteri selected from Lactobacillus reuteri DSM 32229, Lactobacillus reuteri DSM 32230, Lactobacillus reuteri DSM 32231, and Lactobacillus reuteri DSM 32232, in a dried form.

2. A method for the treatment of mastitis and/or thrush, comprising administering a composition containing one or more lactic acid bacteria strains, wherein the bacteria strains have an ability to convert histidine, present in a topical body fluid, into histamine.

3. The method of claim 2, wherein the composition comprises a lotion, cream, ointment, oil, salve, liniment, embrocation, rub, gel, petroleum jelly, balm, emollient, unguent, or balsam.

4. The method of claim 2, comprising topical administration to skin or mucosa of a mammal.

5. The method of claim 2, comprising topical administration to mammary glands.

6. The method of claim 2, wherein the one or more lactic acid bacteria strains comprise a Lactobacillus reuteri strain.

7. The method of claim 2, wherein the one or more lactic acid bacteria strains comprise one or more of Lactobacillus reuteri DSM 32229, Lactobacillus reuteri DSM 32230, Lactobacillus reuteri DSM 32231, and Lactobacillus reuteri DSM 32232.

8. The method of claim 2, wherein the one or more lactic acid bacteria strains comprise one or more of Lactobacillus reuteri ATCC PTA 6475, Lactobacillus reuteri ATCC PTA 4659, Lactobacillus reuteri ATCC PTA 5289, and Lactobacillus reuteri ATCC PTA 5290.

9. A composition for treatment of mastitis and/or thrush comprising one or more lactic acid bacteria strains of claim 7, wherein the composition is a lotion, cream, ointment, oil, salve, liniment, embrocation, rub, gel, petroleum jelly, balm, emollient, unguent, or balsam.

10. A composition for treatment of mastitis and/or thrush, comprising one or more lactic acid bacteria strains of claim 8, wherein the composition is a lotion, cream, ointment, oil, salve, liniment, embrocation, rub, gel, petroleum jelly, balm, emollient, unguent, or balsam.

11. The method according to claim 2, wherein the composition is topically administered using an adsorbent or non-adsorbent product.

12. The method according to claim 11, wherein the adsorbent or non-adsorbent product is a pad, a wipe and/or a tissue made of adsorbent or non-adsorbent material, whereby the material is suitable as a carrier for the one or more lactic acid bacteria strains, and wherein the adsorbent or non-adsorbent product is adapted to be placed in contact with the mammary gland of the mammal.

13. The method according to claim 12, wherein the adsorbent or non-adsorbent product is contained or surrounded by cotton wadding or wool.

14. The method according to claim 13, wherein the cotton wadding or wool is adapted to provide heat.

15. A method for selecting a lactic acid bacteria strain for use in treating mastitis and/or thrush, wherein the strain has an ability to convert histidine, present in a topical body fluid, into histamine, the method comprising: (a) preparing a lactic acid bacteria sample; and (b) screening for a lactic acid bacteria strain having an active histidine operon by polymerase chain reaction (PCR); and (c) selecting a strain that has an active histidine operon; and/or comprising (a) incubating a sample containing histidine with one or more lactic acid bacteria strains at about 37 C. for 20 to 30 hours; (b) determining the amount of histamine in the sample; and (c) selecting a strain that has an ability to convert histidine to histamine.

16. The method according to claim 15, wherein the method comprises (a) preparing a lactic acid bacteria sample; and (b) screening for a lactic acid bacteria strain having an active histidine operon by polymerase chain reaction (PCR); and (c) selecting a strain that has an active histidine operon, and wherein the PCR uses primer hdcA425fde having the nucleic acid sequence CGTCAYTATCCWGCTCCWGG and primer hdcA867rde having the nucleic acid sequence TCCATRTCAGTATCWGGKGT.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The following drawings are provided to illustrate various aspects of the present inventive concept and are not intended to limit the scope of the present invention unless specified herein.

[0058] FIG. 1 represent a schematic illustration of the mammary gland and different conditions described; a. the mammary gland under normal conditions; b. the mammary gland under inflammation (blocked duct or similar condition); c. the mammary gland during infection and mastitis. Grey filled circles illustrates inflammation, ellipses with checkered filling illustrate bacteria during infection, and the arrow illustrate a milk flow.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

Definitions

[0059] The term treatment as used herein is understood to include prevention, reduction and prophylaxis.

[0060] The term Bacteria as used herein is understood to include Lactic Acid Bacteria strains including, but not limited to, any specific Lactic Acid Bacteria strain mentioned herein.

[0061] The term topical as used herein is understood to refer to an application or administration to a particular place on or in the body, as opposed to systemically.

[0062] The term disorder as used herein is understood to include disease and condition.

[0063] The term non-invasive is understood to be a treatment done without cutting the body or putting something into the body, e.g. the term is understood to be a topical administration on the skin or mucosa of the mammal.

[0064] Lactic Acid Bacteria

[0065] Lactobacillus reuteri strain 93a has been deposited under the Budapest Treaty at DSMZ (Leibniz Institute DSMZGerman Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) on Dec. 11, 2015, and has been given the accession number DSM 32229.

[0066] Lactobacillus reuteri strain F33 has been deposited under the Budapest Treaty at DSMZ (Leibniz Institute DSMZGerman Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) on Dec. 11, 2015, and has been given the accession number DSM 32232.

[0067] Lactobacillus reuteri strain C30 has been deposited under the Budapest Treaty at DSMZ (Leibniz Institute DSMZGerman Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) on Dec. 11, 2015, and has been given the accession number DSM 32230.

[0068] Lactobacillus reuteri strain D276 has been deposited under the Budapest Treaty at DSMZ (Leibniz Institute DSMZGerman Collection of Microorganisms and Cell Cultures, Inhoffenstr. 7B, D-38124 Braunschweig, Germany) on Dec. 11, 2015, and has been given the accession number DSM 32231.

[0069] Lactobacillus reuteri ATCC PTA 6475, Lactobacillus reuteri ATCC PTA 4659, Lactobacillus reuteri ATCC PTA 5289 and Lactobacillus reuteri ATCC PTA 5290 are Lactobacillus reuteri strains owned by Biogaia AB, Sweden.

[0070] The strains of Lactic Acid Bacteria may be screened for and selected on the basis of the presence of genes needed to convert histidine into histamine using PCR, or other relevant method, as described in Thomas C M et al. (2012) PLoS ONE 7(2): e31951. doi:10.1371/journal.pone. 0031951, which is hereby incorporated herein by reference in its entirety.

[0071] A biologically pure culture of these Lactobacillus reuteri can be obtained by a selection method comprising the steps of; [0072] screening the Lactic Acid Bacteria strains for the presence of an active histidine operon using a PCR method with two primers according to the following: sequence (CGTCAYTATCCWGCTCCWGG) (SEQ ID NO: 1) referred to as hdcA425fde and sequence (TCCATRTCAGTATCWGGKGT) (SEQ ID NO: 2) referred to as hdcA867rde. The primers were designed from an alignment of hdc from L. reuteri, L. hilgardii, L. buchneri and L. sakei. The PCR analysis is further described in Example 5 and; [0073] selecting a strain, which has an active histidine operon.

[0074] The Lactic Acid Bacteria can also be selected based on their ability to convert histidine, present in a body fluid, into histamine, as described in Example 1. In one embodiment, the method comprises the steps of collecting samples of breast milk, determining the amount of histidine in the milk, incubating the samples with the Bacteria to be tested anaerobically at about 37 C. or other suitable temperature for 20 to 30 hours, determining the amount of histamine in the samples and selecting Bacteria strains that has an ability to convert histidine to histamine.

[0075] Medical and Cosmetic Use

[0076] L. reuteri DSM 32229, L. reuteri DSM 32230, L. reuteri DSM 32231 and L. reuteri DSM 32232 are believed to be useful for the treatment of mastitis and/or thrush, especially after topical administration to a mammal.

[0077] Examples of other Lactic Acid Bacteria strains that have an ability to convert histidine present in a body fluid into histamine, may be Bacteria selected from the group comprising L. reuteri ATCC PTA 6475, L. reuteri ATCC PTA 4659, L. reuteri ATCC PTA 5289 and L. reuteri ATCC PTA 5290.

[0078] Lactic Acid Bacteria strains, especially Lactobacillus reuteri strains, more specifically the Lactic Acid Bacteria strains mentioned above, are believed to be useful for the treatment of mastitis and/or thrush, especially after topical administration to a mammal. The Bacteria, as defined above, are useful for treatment as mentioned above, without addition of external histidine sources, e.g. said Bacteria use only histidine from the milk of the mammal to be treated.

[0079] The Bacteria strains, as defined above, are also believed to be useful in relieving an inflammation disorder connected to a condition of blocked gland ducts and in alleviating and preventing recurring an inflammation disorder of a milk system (e.g. breast or udder) of a mammal.

[0080] The Bacteria strains, as defined above, can be administered topically to provide means for local production of histamine at the proximity and inside the breast, breast nipple, udder and mammary gland of the mammal, by utilizing and converting the natural amount of histidine present in the milk produced by the mammary gland, to histamine. By administrating one or more Lactic Acid Bacteria strains, as defined above, topically at a site of the milk system or at an inflamed site of the mammal, and combine the administered one or more Lactic Acid Bacteria strain, as defined above, with the milk from the mammal, said Bacteria become activated and start to produce histamine by converting histidine present in the milk.

[0081] The local histamine has an anti-inflammatory effect and is used to treat the inflammation. At the same time, the Bacteria may use the milk to migrate from the outside of the body into the mammary gland of the mammal. Thus, Lactic Acid Bacteria strains, as defined above, provide an anti-inflammatory and/or anti-bacterial effect and/or anti-infectious effect to a mammal from the outside of the body and without the addition of external histidine, i.e. histidine not from the milk of the mammal to be treated.

[0082] The mammal may be a human, a cow, a dog, a cat, a camel, a ewe and a goat, or any other milk producing mammal.

[0083] The Bacteria strains, as defined above, may also be used for cosmetic treatment.

[0084] The natural amount of histidine present in breast milk has thoroughly been studied in several studies (EC Scientific Committee on Food (2003) Report on the Revision of Essential Requirements of Infant Formulae and Follow-on Formulae. European Commission Health and Consumer Protection DirectorateGeneral, European Commission, Brussels), resulting in an average concentration of 29 mg/100 ml of breast milk, or 24 mg/g total crude protein. Furthermore, breast milk is known to provide all various types of nutrients for the Lactic Acid Bacteria to propagate and colonize the mammary gland.

[0085] Composition

[0086] The composition may be a pharmaceutical composition or cosmetic composition or a device or the like.

[0087] In order to prevent rapid loss of viability in moist or even in semi-moist conditions, it is of great importance that the Bacteria are not exposed to moisture during storage. The problem can in part be handled by supplying product with said Bacteria and drying said products to remove the moisture and finally provide said product in moisture impervious packages.

[0088] In a composition comprising one or more Lactic Acid Bacteria strains, the Bacteria are preferably used in a dried form, such as a freeze dried form or a lyophilized form.

[0089] To protect the preserved Bacteria against moisture, the Bacteria may be dispersed in one or more hydrophobic substance or anti-moisture agent, which due to its hydrophobic character prevent moisture to reach the embedded bacterial cells. Optionally, one or more additive may be added to the composition.

[0090] Lipids may be used as anti-moisture agents. Examples of lipids include petroleum-derived lipids, synthetic lipids and animal- or plant-derived lipids. The one or more lipids may be selected from the group comprising olive oil, canola oil, coconut oil, palm kernel oil, peanut oil, soybean oil, dimethicone, paraffin oil, and petrolatum.

[0091] Polymers may also be used as anti-moisture agents. Polymers that are suitable to protect the bacterial cells from moisture during storage and transport, can preferably be dissolved in bodily fluids to release the bacterial cells when exposed to wet or moist conditions. The polymers should be non-toxic and non-irritant to a mammal skin. The one or more polymers may be selected from the group comprising polyvinyl alcohol, polyethyleneoxide, polyvinyl pyrrolidone and starch.

[0092] Additional components may be added to protect the Bacteria during the manufacturing of the products containing the Bacteria, e.g. carbohydrates, such as maltose, sucrose, trehalose, lactose, glucose and fructose; proteins, such as skim milk and albumin; amino acids, such as Na-glutamate; polyols, such as xylitol, mannitol and sorbitol; and antioxidants, such as Na-ascorbate. Some of the components described above may also be utilized by the Bacteria as nutrients for their propagation, once the Bacteria start to colonize on and in the mammal. Preferred growth conditions will contain a carbon source, in particular glucose, which will support the production of histamine by Lactobacillus reuteri strains (Thomas C M et al. (2012) PLoS ONE 7(2): e31951. doi:10.1371/journal.pone. 0031951). Preferably, the growth conditions are not dependent on sucrose as a source of carbon, or at least will only comprise sucrose at such a level that will not significantly compromise histamine production by the Lactobacillus reuteri strain.

[0093] The composition may comprise further additives selected from the group comprising carbohydrates, C.sub.6-12 medium chain fatty acids (MCT), emollients, surfactants, emulsifiers and silica.

[0094] The composition or device may be selected from a lotion, cream, ointment, oil, salve, liniment, embrocation, rub, gel, petroleum jelly, balm, emollient, unguent, balsam, and the like.

[0095] An example of a composition according to the invention may be a composition comprising

TABLE-US-00002 a) C.sub.6-12 medium-chain 20-49.5 wt %, or 25-48.5 wt % triglycerides (MCT) b) sunflower oil 20-49.5 wt %, or 25-48.5 wt % c) silica dioxide 0-5 wt %, or 0-1 wt % d) one or more Lactic Acid 0.1-5 wt %, or 0.5-2 wt % Bacteria (dried) and lipids, e.g. sunflower oil or MCT, up to 100%.

[0096] The weight percentages (wt %) are percentages of the total weight of the composition. The activity of the Lactic Acid Bacteria may be between 10.sup.5-10.sup.12 CFU per gram or between 10-10.sup.8 CFU per gram.

[0097] The concentration of the histamine producing Lactobacillus reuteri strains in the composition should be selected in such way that the desired effect is achieved without causing adverse effects. In some embodiments, the concentration by weight of certain histamine producing Lactobacillus reuteri strains ranges from about 0.01 to about 10 wt %, or from about 0.1 to about 10 wt %, or from about 0.1 to about 5 wt %, or from about 0.5 to about 5 wt % of the total weight of the composition. The activity of the used Bacteria may be between 10.sup.5-10.sup.12 CFU per gram or between 10.sup.2-10.sup.8 CFU per gram of Bacteria culture.

[0098] These concentration levels may correspond to a one, two, three or four times daily topical application of the composition.

[0099] Administration

[0100] The invention also provides a method for placing the probiotic Bacteria, as defined above, in contact with the milk system of the mammal. A topical composition comprising said Bacteria may be applied to the skin at a concentration of the histamine producing Bacteria strains that is sufficient to treat mastitis. For example at the concentrations mentions above.

[0101] The method may comprise applying the Bacteria, as defined above, or a composition comprising said Bacteria, to the skin or mucosa of the mammal using an adsorbent or non-adsorbent product, for example, a tissue, a pad or a wipe. The composition may be incorporated in an adsorbent product, such as a tissue, a pad or a wipe and sealed prior to use. For example, the composition may be soaked into a tissue, pad or wipe, vacuum dried and sealed. The product can be placed in contact with the mammary gland of the mammal by wiping.

[0102] The method may also comprise applying the Bacteria, as defined above, in a composition comprising said Bacteria, selected from a lotion, cream, ointment, oil, salve, liniment, embrocation, rub, gel, petroleum jelly, balm, emollient, unguent, balsam, and the like to the skin or mucosa of the mammal.

[0103] The composition may be applied on the nipple, the areola, and the whole breast, plus eventual areas of redness on the breast. The Lactic Acid Bacteria or a composition comprising said Bacteria, may be administered using pads made of adsorbent, or non-adsorbent material, which can be placed in contact with the milk system or mammary gland of the mammal, for example as an insert in an undergarment bra. Such pads are preferably compatible with and suitable as a carrier for viable, but dormant probiotic Bacteria, especially the Lactic Acid Bacteria as defined above. The pad is for example a nursing pad. The pads may be contained or surrounded by cotton wadding, wool or a wool-like material with characteristics that are similar to wool. The pads may be adapted to provide an insulation or heating effect to the skin of the mammal. Such heating effect may be provided by the body itself in combination with a covering and insulating garment.

[0104] The adsorbent or non-adsorbent product may decrease, eliminate and/or prevent the condition of mastitis and/or thrush. Such effects may be evaluated clinically, objectively and/or subjectively by a health care professional, a treatment subject or an observer. The treatment may be carried out for one week, two weeks, three weeks or as long as breast feeding takes place. Preferably, treatment is carried out three times per day for two weeks.

[0105] Alternatively, the Bacteria, as defined above, or a composition comprising said Bacteria, may be provided in a kit comprising the Bacteria as defined above and an acceptable carrier system. The kit may include other items useful in the handling, preparation and use of the composition as well as instructions for use of the same.

EXAMPLES

[0106] The present invention will now be described with reference to the following examples. These examples are for the purpose of illustrating aspects of the present invention, and are not intended to limit the scope of the invention as defined by the claims.

Example 1

[0107] Three test products are made consisting of:

[0108] 1. Freshly grown Lactobacillus reuteri strain DSM 17938 in LDM

[0109] 2. Freshly grown Lactobacillus reuteri strain ATCC PTA 4659 in LDM

[0110] 3. Lactobacillus Defined Media (LDM) alone

[0111] 20 human breast milk samples are provided from the Milk bank at the neonatal department at Sahlgrenska Hospital in Gothenburg, Sweden. The samples are analyzed for histidine content using the method of Chen et al (Analytica Chimica Acta, Volume 570, Issue 1, 7 Jun. 2006, Pages 109-115).

[0112] The samples are pooled to make 4 samples in total containing around 10, 20, 30 and 40 mg histidine per 100 ml milk.

[0113] The 4 pooled milk samples are put in 3 test tubes each in amounts of 20 ml per tube. Test products 1, 2 and 3 are put in the respective test tube at an amount of product 1 and 2 to be 10 CFU of the Bacterial strain per tube, and product 3 is put in an equivalent volume as product 1 and 2. The test tubes are put in an anaerobic chamber and incubated in 37 C. over night.

[0114] The samples in the test tubes are analyzed for histamine content using the Histamine ELISA kit (Neogen, Lexington, Ky.) according to the manufacturer's instructions.

[0115] Results

[0116] Amounts of histamine in pooled human milk samples (mg/100 mil

TABLE-US-00003 10 mg 20 mg 30 mg 40 mg Test product histidine histidine histidine histidine 1 0 0 0 0 2 0.4 0.7 1.0 1.2 3 0 0 0 0

[0117] The strain of test product 2, L. reuteri ATCC PTA 4659 is selected.

Example 2

[0118] Topical compositions tested, C, I and J. Three different topical compositions were tested containing different components. All compositions were mixed for a total weight of 15 grams, all containing 2% culture of certain histamine producing Lactobacillus reuteri strains, in this case exemplified by L. reuteri ATCC PTA 4659. All components were from AarhusKarlshamn Sweden AB, except Lanolin (Lanolines Stella S.A., Belgium).

TABLE-US-00004 Topical composition C I J Components % g % g % g MCT 40 6 58 8.7 Lipex Bassol 38 5.7 75 11.3 Lipex205 10 1.5 Akogel 10 1.5 13 1.9 Lanolin 40 6 10 1.5 Culture 2 0.3 2 0.3 2 0.3 Total 100 15 100 15 100 15

Example 3

[0119] A topical composition was tested. The composition was mixed to a total weight of 15 grams, containing 2% culture of certain histamine producing Lactobacillus reuteri strains, in this case exemplified by L. reuteri ATCC PTA 4659. All components were from AarhusKarlshamn Sweden AB.

TABLE-US-00005 Topical composition K Components % g MCT 48.5 7.275 Sunflower oil 48.5 7.275 Silica (silicon dioxide) 1 0.15 Culture 2 0.3 Total 100 15

Example 4

[0120] Pilot Study of the Effect of the Probiotic Lactobacillus reuteri on the Treatment of Lactational Mastitis

[0121] In the pilot study, women presenting with symptoms of lactational mastitis are randomized, with the intention of 20 complete cases in each group. The women will be identified through incoming telephone calls to the breastfeeding clinic at Sahlgrenska University Hospital East, Gothenburg, Sweden. All midwives working at the breastfeeding clinic have long experience in taking care of women with breastfeeding problems and at least 7.5 higher education points in breastfeeding. The usual routine is that women with mastitis related symptoms are either counseled to home care and expectancy. These women will have a routine follow-up via telephone the day after by a midwife on the breastfeeding clinic. Those patients who are eligible for antibiotic treatment will be invited to come for a visit at the clinic.

[0122] Women eligible for this study are those women who are counseled to home care and expectancy. A research midwife will contact eligible women by phone for further information and invite interested women to a first visit at the clinic (the same day). The women will receive further oral and written information on the study and the midwife evaluates if the mother is eligible for the study. This evaluation will be based on a questionnaire and a physical examination.

[0123] Women will then be randomized to probiotic treatment or placebo by an electronic database. The treatment of both groups will continue for 14 days. Women will continue breast-feeding during the treatment period. Women will be followed up by visits and telephone interviews for 28 days and fill in a diary regarding their own and their child's symptoms during the first 14 days.

[0124] All study participants will be followed by the breastfeeding clinic. This might include follow-up by phone, visit to the breastfeeding clinic or other departments of the hospital and if regarded necessary antibiotic treatment according to the hospitals routine. Independent of follow-up or treatment, women will remain in the study, once included, except if they wish to withdraw from the study.

[0125] Inclusion Criteria [0126] Women presenting with a history of at least 4 hours fever 38 C. and at least 1 point for erythema and 1 point for breast tension according to the Kvist scale and who are recommended home care and expectancy regarding antibiotic treatment by the midwives at the breast feeding clinic [0127] Age 18 years [0128] Capable of giving informed consent [0129] Willing to comply with treatment application [0130] Capable of understanding and complying with study protocol requirements [0131] The baby has undergone examination by a pediatrician and is considered healthy [0132] Exclusive breastfeeding

[0133] Exclusion Criteria [0134] Current breast injury/trauma [0135] Current mammary abscesses or other mammary pathology [0136] History of/current breast cancer [0137] Breast surgery in the past month [0138] New pregnancy [0139] Premature baby, born <37 weeks of gestational age [0140] Baby is under the subject of neonatal care [0141] Autoimmune disease (both mother and child) [0142] Known or suspected allergies to any of the components of the study product (both mother and child) [0143] Participation in another investigational drug study within 30 days prior to treatment start. [0144] Use of any other probiotic (oral or local) [0145] Ongoing antibiotic treatment

[0146] Treatment Groups 1) and 2):

[0147] 1) This group receives probiotic oil containing 10.sup.8 CFU/10 drops of Lactobacillus reuteri ATCC PTA 4659 for external topical application 3 times/day on each breast for 14 days.

[0148] 2) This group receives placebo oil for external topical application (same composition of probiotic oil without Lactobacillus reuteri) 3 times/day on each breast for 14 days.

[0149] Results

[0150] The topical administration of probiotics are expected to [0151] relief in symptoms (flu-like symptoms, fever, local inflammation resulting in redness, pain and tension of the breast, fissures in the nipple and/or mammary areola) caused by lactational mastitis. [0152] the recovery of Lactobacillus reuteri ATCC PTA 4659 in the breast milk after 14 days treatment. [0153] lower milk counts of staphylococcus and streptococcus. [0154] decreased need of antibiotic treatment.

Example 5

[0155] Detection of the Gene Encoding Histidine Decarboxylase (hdc) in Lactobacillus reuteri with PCR

[0156] Method:

[0157] Sample Preparation

[0158] Bacteria were grown over night in MRS broth at 37 C. The Bacterial suspensions were centrifuged at 3500 rpm for 5 min and 1 l of the pellet was suspended in 100 l of PBS.

[0159] PCR Analysis

[0160] Primers:

[0161] hdcA425fde (CGTCAYTATCCWGCTCCWGG) (SEQ ID NO: 1) and hdcA867rde (TCCATRTCAGTATCWGGKGT) (SEQ ID NO: 2); Product size 442 bp; The primers were designed from an alignment of hdc from L. reuteri, L. hilgardii, L. buchneri and L. sakei.

[0162] DreamTaq Green PCR Mastermix (2X Thermo Scientific, article number K1081) was used for the PCR reactions. PCR reactions according to this:

TABLE-US-00006 Mastermix with primers Volume per reaction Water (PCR quality) 10.0 l Primer, forw. (10 pmol/l) 1.0 l Primer, rev. (10 pmol/l) 1.0 l DreamTaq 12.5 l

[0163] 24.5 l of the mastermix was mixed with 0.5 l Bacterial suspension and the following program was run: 95 C. 10 min/30(95 C. 30 s, 48 C. 30 s, 72 C. 30 s)/172 C. 5 min/14 C. forever.

[0164] Results

[0165] The results revealed that a number of strains of L. reuteri from different host origin were positive for the gene encoding histidine decarboxylase (see Table 1).

TABLE-US-00007 TABLE 1 A summary of the results from the PCR analysis showing the species, strain and host origin of the tested Bacteria. Species Strain Host origin Presence of hdc gene (PCR) L. reuteri ATCC PTA 6475 Human + DSM 17938 Human 93a Human + F33 Cat + C30 Cow + D276 Dog +

[0166] The present invention is not limited to the embodiments disclosed but may be varied and modified within the scope of the following claims.