WO2013027087A1

WO2013027087A1 - A non-reuterin-producing lactobacillus reuteri strain for treating helicobacter pylori infection

Info

Publication number: WO2013027087A1
Application number: PCT/IB2011/053695
Authority: WO
Inventors: Peggy Garault; Gaelle Quere; Raphaelle Bourdet-Sicard
Original assignee: Compagnie Gervais Danone
Priority date: 2011-08-23
Filing date: 2011-08-23
Publication date: 2013-02-28

Abstract

The present invention relates to a non-reuterin-producing strain Lactobacillus reuteri or a cell fraction thereof for use in the treatment prevention of Helicobacter pylori infection.

Description

A NON-REUTERIN-PRODUCING LACTOBACILLUS REUTERI STRAIN FOR TREATING HELICOBACTER PYLORI INFECTION

The present invention relates to the field of probiotics. Particularly, the invention pertains to the use of a non-reuterin-producing strain of Lactobacillus reuteri for the treatment or the prevention of Helicobacter pylori infection.

According to a definition recently approved by the National Yogurt Association (NY A) or the International Life Science Institute (ILSI) in the USA, probiotics are living micro-organisms which upon ingestion in a sufficient amount exert health benefits beyond basic nutrition. Probiotic bacteria have been described among species belonging to the genera Lactobacillus, Bifidobacterium, Streptococcus and Lactococcus, commonly used in the dairy industry. Probiotics are thought to intervene at the level of the intestinal flora by impeding the development of pathogenic microorganisms and/or by acting more directly on the immune system.

Helicobacter pylori (H. pylori) is a Gram-negative spiral-shaped bacteria that colonizes the human gastric mucus layer of more than 50% of the world's population. While the majority of individuals infected with H. pylori is asymptomatic although their gastric epithelium show sign of inflammation, 15% to 20% of H. pylori infected individuals develop diseases. Indeed, H. pylori is the major causative agent of chronic active gastritis, peptic ulcer diseases, atrophy, metaplasia, dysplasia, gastric cancer and gastric mucosa associated lymphoid tissue (MALT) lymphoma (see for review Fox and Wang, 2007 and Polk and Peek, 2010).

During infection, H. pylori binds specifically to gastric epithelial cells lining the gastric epithelium through several adhesion molecules (adhesins) produced by the bacteria, such as BabA and SabA proteins. Adhesion to the gastric epithelial cells protects the bacteria from liquid flow, peristaltic movement and shedding of the mucous layer. H. pylori adhesion to the gastric mucosa induces signal transduction pathways within the gastric epithelial cells, leading to gastric epithelial cell damages and atrophy via oxidative stress, apoptosis and/or autophagy mechanisms. Accordingly, H. pylori adhesion to gastric epithelial cells is a key step in the establishment of an infection of the gastric mucosa. The standard treatment in patients infected with H. pylori is two antibiotics associated to a proton pump inhibitor (PPI), so called triple therapy. However, H. pylori eradication rate following triple therapy is dropping down because of antibiotic resistance or poor compliance. Further, despite several clinical trials, there is no effective vaccine available on the market yet.

It appears from the foregoing that there is a need for alternatives or complements to triple therapy for the treatment or for the prevention of H. pylori infection.

It has been proposed, as an alternative, to use probiotic lactic acid bacteria (LAB) that could inhibit the growth or adhesion to the gastric epithelial cells of H. pylori and therefore exhibit antagonist activities against H. pylori.

Lactobacillus reuteri (L. reuteri) is considered as a candidate probiotic for inhibiting the growth of H. pylori since it produces the potent antimicrobial substance reuterin (3-hydroxypropionaldehyde, 3 -HP A). By way of example, International Application WO 2004/031368 discloses the use of the reuterin-producing strain L. reuteri ATCC PTA-4659, (capable of inhibiting the growth of different enteric pathogens in vitro; see Spinier et al., 2008), for treating or preventing an inflammation associated with H. pylori. Lionetti et al. (2006) disclose that the reuterin-producing strain L. reuteri strain ATCC 55730 (see Spinier et al , 2008) - that inhibits the growth of H. pylori - is capable of reducing frequency and intensity of antibiotic-associated side-effects during anti-H. pylori treatment in children.

In L. reuteri, reuterin is produced under anaerobic conditions (fermentation) in the presence of glycerol. Particularly, a cobalamin-dependent glycerol dehydratase (Gld) expressed by L. reuteri catalyses the conversion of glycerol to reuterin. This glycerol dehydratase is composed of three subunits (C, D and E, or α, β and γ respectively). It has been found that among the genera of bacteria able to produce reuterin, L. reuteri produces the highest concentration of reuterin in pure glycerol concentration.

International Application WO 2010/12487 discloses the use of an oral composition comprising a highly bile salt hydrolase (bsh) active L. reuteri strain for reducing serum cholesterol, serum lipids, body fat and atherogenic index in a subject and also for treating or preventing atherosclerosis, cardiovascular and cerebrovascular diseases. This oral composition may further comprise reuterin. This document further discloses that the more highly bsh active L. reuteri strain is the strain NCIMB 701359.

Mukai et al, 2002, have identified two L. reuteri strains, namely JCM1081 (that does not produce antimicrobial substances against pathogens) and TM105, capable of inhibiting the binding of H. pylori to receptor glycolipids in vitro. The authors therefore suggest that these strains may prevent H. pylori infection in an early stage of colonization by H. pylori.

The inventors have surprisingly found that the Lactobacillus reuteri strain NIMCB 701359 (disclosed in International Application WO 2010/12487) does not produce reuterin, but that this strain is capable of inhibiting the growth of H. pylori strains in vitro. Further, this L. reuteri strain NIMCB 701359 is not capable of inhibiting the adhesion of H. pylori strains to epithelial AGS cells in vitro. Advantageously, this L. reuteri strain NCIMB 701359 can be orally administered to a subject for inhibiting the growth of H. pylori strains in said subject without coadministration of glycerol. This L. reuteri strain NCIMB 701359 can therefore be used for the treatment or the prevention of H. pylori infection, by inhibiting the growth of H. pylori strains.

This strain has been deposited by the Applicant, according to the Budapest Treaty, at CNCM (Collection Nationale de Cultures de Microorganismes, 25 rue du Docteur Roux, Paris) on February 3, 2011, under the accession number CNCM 1-4430. This strain is also referred to as NCIMB 701359 or DN_129_0007.

Accordingly, a subject of the present invention is the Lactobacillus reuteri (L. reuteri) strain deposited with the CNCM under number 1-4430, for use as a medicament for treating or preventing H. pylori infection.

The present invention also encompasses mutant strains or genetically transformed strains derived from the parent strain CNCM 1-4430, provided that they are capable of inhibiting the growth of H. pylori strains and optionally they are not capable of inhibiting the adhesion of H. pylori strains to epithelial AGS cells in vitro. A method for assessing the capacity of a L. reuteri strain to inhibit the adhesion of H. pylori strains to epithelial AGS cells in vitro is described in Example 3 below. These mutant or genetically transformed strains can be strains wherein one or more endogenous gene(s) of the parent strain CNCM 1-4430 has (have) been mutated, for instance to modify some of its metabolic properties (e.g., its ability to ferment sugars, its resistance to acidity, its survival to transport in the gastrointestinal tract, its post- acidification properties or its metabolite production). They can also be strains resulting from the genetic transformation of the parent strain CNCM 1-4430 by one or more gene(s) of interest, for instance in order to give to said genetically transformed strain additional physiological features, or to allow it to express proteins of therapeutic or vaccinal interest that one wishes to administer through said strains. These strains can be obtained from the CNCM 1-4430 strain by means of the conventional techniques for random or site-directed mutagenesis and genetic transformation of Lactobacilli, such as those described by Gury et al. (2004) or by Perea Velez et al. , 2007, or by means of the technique known as "genome shuffling" (Patnaik et al. , 2002 and Wang et al , 2007).

A subject of the present invention is also cell fractions which can be obtained from a L. reuteri strain as defined above, preferably the strain CNCM 1-4430, provided that they are capable of inhibiting the growth of H. pylori strains, for use as a medicament for treating or preventing H. pylori infection. Said cell fractions are in particular DNA preparations or bacterial wall preparations obtained from cultures of said strain. They may also be culture supernatants or fractions of these strains.

A subject of the present invention is also a composition comprising a L. reuteri strain according to the present invention, preferably the strain CNCM 1-4430, or a cell fraction obtained from said strain according to the present invention, for use as a medicament for treating or preventing H. pylori infection.

In the composition of the invention, said strain can be used in the form of whole bacteria which may be living or dead. Alternatively, said strain can be used in the form of a bacterial lysate or in the form of bacterial fractions; the bacterial fractions suitable for this use can be chosen, for example, by testing their properties on the adhesion of H. pylori strains to epithelial cells. Preferably the bacterial cells are present as living, viable cells. The compositions of the invention can be in any form suitable for administration, in particular oral administration. This includes for instance solids, semi-solids, liquids, and powders. Liquid composition are generally preferred for easier administration, for instance as drinks.

The composition can comprise at least 10⁵ cfu, preferably at least 10⁶ cfu, per g dry weight, of at least one bacterial strain as mentioned above.

The composition can further comprise other strains of bacteria than the strains according to the present invention, in particular probiotic strain(s), such as Lactobacillus, Bifidobacterium, Streptococcus or Lactococcus strain(s).

When the bacteria are in the form of living bacteria, the composition may typically comprise 10⁵ to 10¹³ colony forming units (cfu), preferably at least 10⁶ cfu, more preferably at least 10 cfu, still more preferably at least 10 cfu, and most preferably at least 10⁹ cfu per g dry weight of the composition. In the case of a liquid composition, this corresponds generally to 10⁴ to 10¹² colony forming units (cfu), preferably at least 10⁵ cfu, more preferably at least 10⁶ cfu, still more preferably at least 10 cfu, and most preferably at least 10 cfu/ml.

The composition can be a pharmaceutical composition or a nutritional composition, including food products, food supplements and functional food. A "food supplement" designates a product made from compounds usually used in foodstuffs, but which is in the form of tablets, powder, capsules, potion or any other form usually not associated with aliments, and which has beneficial effects for one's health. A "functional food" is an aliment which also has beneficial effects for one's health. In particular, food supplements and functional food can have a physiological effect - protective or curative - against a disease, for example against a chronic disease.

The nutritional composition according to the invention also includes a baby food, an infant milk formula or an infant follow-on formula. Preferably the present composition is a nutraceutical or a pharmaceutical product, a nutritional supplement or medical food.

The composition can be a dairy product, preferably a fermented dairy product. The fermented product can be present in the form of a liquid or present in the form of a dry powder obtained by drying the fermented liquid. Examples of dairy products include fermented milk and/or fermented whey in set, stirred or drinkable form, cheese and yoghurt.

The fermented product can also be a fermented vegetable, such as fermented soy, cereals and/or fruits in set, stirred or drinkable forms.

In a preferred embodiment, the fermented product is a fresh product. A fresh product, which has not undergone severe heat treatment steps, has the advantage that the bacterial strains present are in the living form.

A subject of the present invention is also a L. reuteri strain as defined above, preferably the strain CNCM 1-4430, or a composition as defined above for use as a medicament for treating or preventing Helicobacter pylori infection.

A subject of the present invention is also the use of a L. reuteri strain as defined above, preferably the strain CNCM 1-4430, or a composition as defined above for the manufacture of a medicament for treating or preventing H. pylori infection.

A subject of the present invention is also a method for treating or preventing H. pylori infection in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of a L. reuteri strain as defined above, preferably the strain CNCM 1-4430, or a composition as defined above.

Determination of a therapeutically effective amount is well known from the person skilled in the art, especially in view of the detailed disclosure provided herein.

A subject of the present invention is also a method for the manufacture of a medicament for treating or preventing H. pylori infection, said method comprising incorporating a L. reuteri strain as defined above, preferably the strain CNCM 1-4430, or a composition as defined above, into at least one pharmaceutically acceptable diluent, carrier or excipient.

As used herein, the treatment or prevention encompasses inter alia: preventive infection, stabilizing the load of H. pylori and/or decreasing the load of H. pylori. The treatment or prevention also encompasses addressing at least one of the symptoms associated with H. pylori mentioned below.

Methods for diagnosing a H. pylori infection are known in the art. By way of example, diagnosis of a H. pylori infection can be made by checking by a blood antibody test, a stool antigen test or the carbon urea breath test. It can also be made by biopsy under endoscopy followed by a urease test, a histological examination or a microbial culture.

The symptoms or diseases associated with H. pylori infection are stomach ache, abdominal pain, regurgitation, vomiting, belching, flatulence, nausea, chronic active gastritis, peptic ulcer diseases, atrophy, metaplasia, dysplasia, gastric cancer and gastric mucosa associated lymphoid tissue (MALT) lymphoma.

The present invention will be understood more clearly from the further description which follows, which refers to examples illustrating the anti-infective properties of the CNCM 1-4430 strain as well as to the appended figures.

Figure 1 shows the results of the PCR screening of Lactobacilus strains for a gldC gene fragment. Upper band at 728 bp represents the expected product using the gldC gene-specific primers. Lower band at 201 bp represents the expected product using the 16S rRNA gene-specific primers (positive PCR control). Smart: DNA SMART Ladder (Eurogentec); C+: PCR product obtained from DNA from the positive gldC strain L. reuteri ATCC 55730; 1-4430: PCR product obtained from DNA from the L. reuteri strain CNCM 1-4430; C-: PCR result obtained from DNA from the gldC negative strain Streptococcus thermophilus CNCM 1-2835; L. reuteri 6: PCR product obtained from DNA from L. reuteri 6; H₂0: PCR result obtained from sterile water.

EXEMPLE 1: IN VITRO INHIBITION OF THE GROWTH OF H. PYLORI STRAINS BY THE STRAIN L. REUTERI CNCM 1-4430

1.1 Material & Methods

Lactobacillus reuteri stains:

- L. reuteri CNCM 1-4430 ;

- L. reuteri ATCC 55730 and L. reuteri 6: reuterin -producing strains.

Helicobacter pylori strains:

- SSI : described in Lee et al, 1997;

- HPAG1 : described in Oh et al, 2006;

- CR1 13: strain isolated from a human pre-cancerous gastric lesions biopsy; - Axcan 342: strain isolated from a patient with gastritis, and included in a clinical trial for testing a quadruple therapy for eradication H. pylori (Malfertheiner et al, 2011).

Method:

L. reuteri stains were grown in MRSn broth (pH 6,2) for 17h. The bacterial suspensions were neutralized at pH 6,8 with a solution of KOH.

H. pylori strains were grown for 48h in Brucella broth until obtaining a turbidity equivalent to the Mac Farland 4 standard. The bacterial suspensions were spread onto the surface of Mueller Hinton agar plates supplemented with 10% (v/v) sheep blood enriched with Polyvitex® supplement at 1% (v/v) (BioMerieux, France).

Sterile paper discs were applied on the surface of the plates. Then, 5 μί, of a neutralized L. reuteri suspension were applied on the paper disc. The plates were incubated at 37°C under micro-aerophilic atmosphere (Ruskin Microaerophilic gas sorter, resulting in 5% 0₂, 10% C0₂, and 85% N₂) for 72h to 96h.

To assess the effect of a L. reuteri suspension on the growth of a H. pylori strain, the rings of growth inhibition around the paper disks were measured in millimetre.

I.2 Results

The results are shown in Table 1 below.

Table 1 : Ring of inhibition (in mm) of different H. pylori strains by two different neutralized sus ensions of L. reuteri strains.

is capable of inhibiting the growth of 3 clinical H. pylori strains, while the strain L. reuteri 6 is capable of inhibiting the growth of only 1 H. pylori strain. EXAMPLE 2: PCR SCREENING OF THE sldC GENE

2.1 Material & Methods

The presence in L. reuteri CNCM 1-4430 of the gene encoding the large subunit of glycerol dehydratase (GldC) - the enzyme responsible for reuterin production - was tested following the method described by Cadieux et al. (2008).

Briefly, total DNA was isolated from each tested bacterial strain.

As a positive control (C+), the reuterin-producing strain L. reuteri ATCC 55730 was used. As a negative control (C-), a non-reuterin-producing strain of Streptococcus thermophilus (CNCM 1-2835) was used.

Amplification of a fragment of the gene encoding the large subunit of Gld igldC) was carried out with the degenerate primers GDCRec and GDCFor (described in Cadieux et al. , 2008) designed from regions of this gene highly conserved across published Lactobacillus gldC sequences. The expected size of the amplified gldC fragment is 728 bp

As a positive PCR reaction control, a fragment of the 16S rRNA gene was also amplified from all samples by using the eubacterial primers 16SFor and 16SRev (described in Cadieux et al. , 2008). The expected size of the amplified 16S rRNA fragment is 201 bp.

2.2 Results

The results of the PCR reaction are shown in Figure 1. The strain L. reuteri 6 as well as the (C+) reuterin-producing strain ATCC 55730 were positive for the expected 728-bp product. The strains L. reuteri CNCM 1-4430 as well as the (C-) non-reuterin-producing strain were negative for the gldC gene. Accordingly, the strain L. reuteri CNCM 1-4430 does not contain the gldC gene and therefore does not produce reuterin. EXAMPLE 3: METHOD FOR DETERMINING WETHER A L. REUTERI STRAIN HAS AN EFFECT ON ADHESION OF HELICOBACTER PYLORI TO GASTRIC EPITHELIAL CELLS

The method described by Oleastro et al. (2008) is used with modifications to assess the effect of a Lactobacillus reuteri strain on the adhesive properties of H. pylori to epithelial cells.

H. pylori strains are tested with a MOI (multiplicity of infection) of 50. H. pylori cells are transferred in PBS buffer after a culture of 24h in pylori agar (BioMerieux, France). The concentration of bacterial suspensions of the H. pylori strains is normalised at 2.10⁸ cfu/mL (corresponding to OD₆oonm ⁼ 1 )·

The L. reuteri suspension is tested with a MOI of 50 (corresponding to 25μΙ_^ of normalized suspension of the L. reuteri strain by well).

100 000 epithelial AGS cells (ATCC number: CRL-1739) are inoculated the day before the experiment of infection in 500μΕ of DMEM medium F12 complemented with foetal bovine serum at 10%.

Coloration of H. pylori suspensions

1 mL of H. pylori suspension is centrifuged 10 minutes at 10 000 g. The pellet is resuspended in 1 mL of Diluent A (from the PKH2 Green Fluorescent Cell Linker Kit, Sigma Aldrich, Saint-Louis) and 2.5 of PKH₂ (Sigma Aldrich, Saint- Louis) is added. The suspension is incubated 2 minutes and 30 seconds at room temperature. The coloration is stopped by addition of 2 mL of foetal bovine serum. Then 4 mL of DMEM/F12 medium is added before the centrifugation of the suspension for 10 minutes at 10 000 g. The pellet is resuspended in 4 mL of culture medium and centrifuged 10 minutes at 10 000 g. This washing step is repeated twice to remove excess fluorochrome.

Measurement of adhesion

Epithelial AGS cells are dissociated with trypsin following standard conditions after three washings with PBS to discard non adherent bacteria. Fluorescent emission is analysed by flow cytometry. Fluorescence is measured using the FACSCalibur flow cytometer (Becton Dickinson). Each condition is carried out in triplicates. Infection with H. pylori can be carried out in two conditions

- in pre-infection (pre) condition: the L. reuteri suspension to be tested is added to the epithelial AGS cells lh30 before the infection with H. pylori. Cells are analysed by flow cytometry lh30 after the infection (total incubation time 3h);

- in co-infection (co) condition: the L. reuteri suspension to be tested and a H. pylori strain are added to the epithelial AGS cells at the same time and the cells are analysed by flow cytometry after an incubation time of lh30.

Control

The control value of fluorescence intensity is obtained with the epithelial AGS cells incubated with H. pylori strains for lh30.

Percent adhesion values are given relative to the adhesion of H. pylori to epithelial AGS cells without exposure to L. reuteri, which are set at 100%.

Results are analyzed from the mean value obtained from triplicates and they are scored when they are significantly different according to Student's test (P <0.05).

REFERENCES

- Cadieux et al. , Appl Environ Microbiol. 2008;74:4645-9.

- Fox JG and Wang TC, J Clin Invest. 2007; 117:60-9.

- Gury J et al, Arch Microbiol. 2004;182:337-45.

- Lee A et al, Gastroenterology. 1997; 1 12:1386-97.

- Lionetti et al, Aliment Pharmacol Ther. 2006;24:1461 -8.

- Malfertheiner et al, Lancet. 2011 ;377:905-13.

- Mukai et al, FEMS Immunol Med Microbiol. 2002;32: 105-10.

- Oh et al;, Proc Natl Acad Sci U S A. 2006;103:9999-10004.

- Patnaik R et al. , Nat Biotechnol. 2002;20:707-12.

- Perea Velez M et al, Appl Environ Microbiol. 2007;73:3595-604.

- Polk DB and Peek RM Jr., Nat Rev Cancer. 2010;10:403-14.

- Spinier JK et al, Anaerobe. 2008;14: 166-71.

- Wang Y et al, J Biotechnol. 2007;129:510-5.

Claims

1. The Lactobacillus reuteri (L. reuteri) strain deposited with the CNCM under number 1-4430 or a L. reuteri strain capable of inhibiting the growth of Helicobacter pylori (H. pylori) strains and obtained by mutagenesis or genetic transformation of said L. reuteri strain CNCM 1-4430, for use as a medicament for treating or preventing H. pylori infection.

2. A cell fraction which is capable of inhibiting the growth of H. pylori strains obtained by culturing a L. reuteri strain as defined in claim 1, for use as a medicament for treating or preventing H. pylori infection.

3. A composition comprising a L. reuteri strain as defined in claim 1 or a cell fraction as defined in claim 2 for use as a medicament for treating or preventing H. pylori infection.

4. The composition according to claim 3, characterized in that it comprises at least 10⁵ cfu, preferably at least 10⁶ cfu, per gramme dry weight, of a L. reurei strain as defined in claim 1.

5. The composition according to claims 3 or 4, characterized in that it is a nutritional composition.

6. The composition according to claim 5, characterized in that it is a dairy product.