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Title:  Lactic acid bacteria for the treatment and/or prophylaxis of giardiasis
United States Patent: 
7,229,616
Issued: 
June 12, 2007

Inventors: 
Schiffrin; Eduardo (Crissier, CH), Perez; Pablo (La Plata, AR)
Assignee: 
Nestec S.A. (Vevey, CH)
Appl. No.: 
11/111,255
Filed: 
April 20, 2005


 

George Washington University's Healthcare MBA


Abstract

The present invention relates to the use of a supernatant of lactic acid bacteria or of Bifidobacteria capable of preventing colonization of intestinal cells by Giardia intestinalis, for the preparation of an ingestible carrier for the treatment and/or prophylaxis of disorders associated with the colonization of the gut by Giardia intestinalis. The present invention also pertains to specific strains of Bifidobacterium having the above traits and to the ingestible carrier, such as a food or pharmaceutical composition, containing such supernatant or the microorganisms.

Description of the Invention

BACKGROUND OF THE INVENTION

The present invention relates to lactic acid bacteria capable of preventing colonization of intestinal cells by Giardia intestinalis, or to a culture supernatant thereof, respectively, for use in the treatment and/or prophylaxis of disorders associated with the colonization of the gut by Giardia intestinalis. The present invention also pertains to strains of Bifidobacterium having the above traits and to the use of lactic acid bacteria for the preparation of an ingestible carrier, such as a food or pharmaceutical composition, for the treatment and/or prophylaxis of an infestation of the intestine by Giardia intestinalis.

The present invention relates to lactic acid bacteria capable of preventing colonization of intestinal cells by Giardia intestinalis, or to a culture supernatant thereof, respectively, for use in the treatment and/or prophylaxis of disorders associated with the colonization of the gut by Giardia intestinalis. The present invention also pertains to strains of Bifidobacterium having the above traits and to the use of lactic acid bacteria for the preparation of an ingestible carrier, such as a food or pharmaceutical composition, for the treatment and/or prophylaxis of an infestation of the intestine by Giardia intestinalis.

Cysts are dormant, quadrinucleate and ovoid forms responsible for transmission of giardiasis. After ingestion of cysts by an individual, excystation is triggered by exposure to gastric acid and/or digestive enzymes. The parasite thus emerging, which is termed trophozoite, is binucleate and half-pear shaped having a size of around 10 .mu.m with a broad anterior and a narrow posterior side. The ventral side is by and large covered by the ventral disk which is deemed to at least in part account for an attachment of the trophozoite to the intestinal surface.

After excystation trophozoites may persist in the small intestine of the infected individuum for a long time amounting even to years. If trophozoites are carried downstream by the flow of intestinal fluid they again start to encyst to adapt to a new environment.

Cysts are excreted with the faeces and may withstand a variety of extreme environmental conditions, including different temperature, pH and tonicity conditions. An infected individual may secrete about 9.times.10.sup.8 cysts per day, with doses to as low as 10 cysts proved to be sufficient to produce infection in another individual. Transmission of Giardia intestinalis may be accomplished in a variety of different ways with the main routes of transmission being waterborne and foodborne. Person to person spread is supported by some daycare and nosocomial outbreaks. In addition, wild animals are found to be infectious reservoirs of Giardia intestinalis and may contribute to a spread of the pathogen.

Giardiasis, like other intestinal diseases, is more severe in infants and children. Infection is normally associated with diarrhea and malabsorption resulting in an impaired growth and development of the child and may eventually also lead to the death of newbornes.

Around one half of infected people are, however, asymptomatic and contribute to the spread of the pathogen, since due to the absence of any perceptible symptoms no corresponding regimen is applied.

Though a great deal of scientific effort has been invested on the investigation of Giardia intestinalis pathogenesis of giardiasis could not be explained by means of a single virulence factor alone and no toxic compound has been isolated so far.

The light microscopic appearance of a gut colonized by Giardia has been found to be rather variable ranging from a normal mucosal structure to a subtotal villous atrophy. Results of electron microscopy investigations revealed ultrastructural changes such as shortening and disruption of microvilli (Chavez et al., Experimental Parasitol. 80 (1995), 133-138).

Such structural abnormalities have been found to be accompanied by a reduction in lactase, sucrase and maltase activities in the microvillus membrane as well as to an impaired intestinal transport (Buret et al, Gastroenterology 103 (1992), 506-513; Roberts-Thomson et al, Gastroenterology. 71 (1976), 57-61).

In addition to the two forms illustrated above Giardia intestinalis has developed an extremely changing surface structure and has evolved a family of protective proteins that cover all exposed surfaces (Aley et al., Infect. Agents Dis. 4 (1995), 161-166; Muller, et al., Infect. Immun. 64 (1996), 1385-1390; Nash et al., J. Euk. Microbiol. 42 (1995), 604-609). These variable surface proteins (VSPs) protect trophozoites from both immunological and environmental factors, such that Giardia may evade the host's defenses and may survive in a highly degradative environment such as is prevailing in the intestinal tract of mammals. A modification in the VSPs occurs at about every 6th to 13.sup.th generation making it difficult or nearly impossible for the host's immune system to develop a specific response against the pathogen. Immune and environmental stress can select different VSP phenotypes. Furthermore, antigen switching of VSPs after excystation has also been reported (Gillin et al., Annu. Rev. Microbiol. 50 (1996), 679-705).

Human giardiasis is associated with an increase in the number of lamina propria and intraepithelial lymphocytes suggesting that T-cell activation could be responsible for micro-villous damage. However, induced immunosuppression in mice resulted in a more profound effect on microvillous associated enzymes as compared to non-immunosupressed animals, indicating that during an infection by Giardia intestinalis epithelial damage seems not be dependent on immune function alone.

A common therapy of giardiasis is the administration of antibiotics, such as those belonging to the class of nitroimidazoles. Yet, in endemic areas the response to therapy has been shown to be rather inconsistent (Katelaris et al., Aliment. Pharmacol. Ther. 8 (1994), 187-192). Moreover, due to the at least in part destruction of the intestine's natural microflora, the administration of such antibiotics is always accompanied by severe side effects such as extended diarrhea or even an expanded infestation of the gut by other detrimental microorganisms, such as yeast.

Hence, there is a need in the art for additional options to treat giardiasis or means to prevent infestation of an individual by the pathogen.

The problem of the present invention therefore resides in providing additional means for the treatment or prophylaxis of an infection by Giardia intestinalis.

SUMMARY OF THE INVENTION

This problem has been solved by providing the use of a culture supernatant of a Lactic acid bacterium Lactic acid bacterium or a Bifidobacterium capable of preventing adhesion of Giardia intestinalis to intestinal cells for the preparation of an ingestible carrier for the treatment and/or prophylaxis of disorders associated with the colonization of the gut by Giardia intestinalis.

According to a preferred embodiment the present invention provides novel Lactic acid bacteria and Bifidobacteria, respectively, capable to prevent adhesion of Giardia intestinalis to intestinal cells, which are selected from the group consisting of NCC 90 (I-2332), NCC 189 (I-2333) or NCC 200 (I-2334). These microorganisms have been deposited according to the Budapest Treaty with the Institute Pasteur (28 rue du Docteur Roux, F-75724 Paris Cedex 15, France) on Nov. 30, 1999 and received the above mentioned deposit numbers. The present invention pertains also to food and pharmaceutical compositions containing such microorganisms.

The microorganisms may be included in the carrier in an amount of from about 10.sup.6 to about 10.sup.12 pfu (plaque forming units). They may be included as such or optionally after essentially purifying them from the cultivating medium. Alternatively, the supernatant of a culture of such microorganisms may be included in the carrier, which prior to its inclusion may preferably be concentrated by means well known in the art.

The ingestible carrier may be a food or a pharmaceutical composition such as milk, yogurt, curd, cheese, fermented milks, milk based fermented products, ice-creams, fermented cereal based products, milk based powders, infant formulae or any sort of pet food. Such carriers may e.g. be easily manufactured by using a microorganisms having the corresponding traits for fermentation of the starting materials itself. Alternatively, the microorganisms or an optionally concentrated culture supernatant thereof may be added to the respective carrier in liquid or dry form. A pharmaceutical composition, such as a tablet, a liquid bacterial suspension, a dried or wet oral supplement, a dry or a wet tube feeding may be prepared using standard techniques while including the microorganisms and/or an optionally concentrated culture supernatant thereof into the carrier. Depending on the mode of administration the skilled person will select the formulation deemed to be appropriate.

DETAILED DESCRIPTION OF THE INVENTION

During the studies leading to the present invention the inventors have hypothesized that an ecological approach in the treatment and/or prevention of giardiasis could be a colonization of the intestine with bacteria that might be able to antagonize with the parasite.

Attachment of Giardia intestinalis trophozoites to epithelial cells is deemed to be a key step for infection in humans and animals. Although the mechanisms for Giardia adhesion are not fully understood, evidence supports the ventral disk, trophozoite contractile elements, hydrodynamic and mechanical forces and lectin mediated binding to be involved.

Taking into account that lactic acid is an important factor in Giardia's life cycle the interaction between Giardia intestinalis and lactic acid bacteria has been studied using enterocyte like cells in culture.

To this end, the following microbial parasite strains and culture media have been used:

Microbial Strains:

Bacterial strains La1 (Lactobacillus johnsonii) (I-1225) and La10 (Lactobacillus acidophilus) (I-2332) were from Nestec collection (Lausanne, Switzerland) and have been deposited according to the Budapest Treaty with the Institute Pasteur (28 rue du Docteur Roux, F-75724 Paris Cedex 15. France) on Jun. 30, 1992 and Oct. 12, 1999. respectively. Strains CIDCA 536 (Bifidobacterium bifidum) and CIiDCA 538 (Bifidobacterium infantis) were from the collection of Centro de Investigacion y Desarrollo en Criotecnologia de Alimentos (Universidad Nacional de La Plata, Argentina) and have been deposited according to the Budapest Treaty with the Institute Pasteur (28 rue du Docteur Roux, F-75724 Paris Cedex 15, France) on Nov. 30, 1999 receiving the deposit nos. I-2333 and I-2334, respectively.

Giardia intestinalis strains Portland-1 (ATCC 30888), UNO/04/87/1 (ATCC 50184), New Orleans-1 (ATCC 50137) and WB (ATCC 30957) were purchased from American Type Culture Collection (10801 University Blvd. Manassas, Va. 20110-2209).

Bacteria were grown for 24 hours at 37.degree. C. in MRS broth supplemented with 0,05% cysteine hydrochloride (Sigma). Incubations were performed under anaerobic conditions (BBL GasPak Plus).

Protozoa were grown in Keister's modified TYI-S-33 medium containing (per liter): casein digest (Difco), 20 g; yeast extract (BBL), 10 g; dextrose (Merck), 10 g; bovine bile (Difco), 0.75 g; NaCl (Merck), 2 g; L-cystein. HCl (Sigma), 2 g; ascorbic acid sodium salt (Fluka), 0.2 g; K.sub.2HPO.sub.4 (Merck), 1 g; KH.sub.2PO.sub.4 (Merck), 0.6 g; ferric ammonium citrate (Sigma), 22.8 mg, adult bovine serum (Sigma), 100 ml; penicillin/streptomycin (Gibco, 1000 IU/ml, 1000 .mu.g/ml), 15 ml. Horse serum (Gibco) instead of bovine serum was also assayed. The pH was adjusted to 6.9 with 1 N NaOH prior filter sterilization (022 .mu.m pore size). Keister's modified medium supplemented with 10% horse serum did not support growth of strain Portland-1 although incubation was extended during 11 days and despite the fact that trophozoites were initially attached to the tube walls. During the first 5 days of incubation, an high proportion of motile parasites was observed. After this time agglutination of trophozoites and a dramatic decrease of viable cells were observed. Addition of bovine serum (10%) to these damaged cells allowed for growth after 24 hrs. To increase the surface available for Giardia attachment, 25 cm.sup.2 tissue culture bottles were used (plastic bottles). Culture medium was added up to the bottleneck to maintain anaerobic conditions. This procedure resulted in yields of around 10.sup.7 trophozoites/bottle within 2-3 days of incubation and parasites formed a confluent monolayer on the bottle walls.

Subcultures were made by chilling cultures in an ice bath (5-10 min) to detach adhering trophozoites and inoculating 0.2 ml of the resulting suspension into fresh medium. Incubations were performed at 37.degree. C. for 72 hours and different recipients (glass or plastic) were used.

Storage of Giardia intestinalis Strains:

Trophozoites were detached as indicated above and suspended in TYI-S-33 medium. Two fold concentrated cryoprotectant solution (DMSO, sucrose) prepared in TYI-S-33 medium, was added to the suspension in three equal aliquots at 2-minute intervals. Final concentrations of cryoprotectants were 12% (v/v) DMSO, 4% (w/v) sucrose. Suspensions (around 1.times.10.sup.6 trophozoites/ml) were allowed to equilibrate during 20 minutes at room temperature before start of the cooling cycle.

Vials were thermally isolated with polystyrene (4 cm wall thickness) and placed at -80.degree. C.

Reactivation of Frozen Throphozoites:

Vials were thawn in a water bath at 37.degree. C. and immediately inoculated in TYI-S-33 medium at a ratio parasite suspension/fresh medium=0.5/7. Cultures were incubated at 37.degree. C. in the dark.
 


Claim 1 of 4 Claims

1. A lactic acid bacteria selected from the group consisting of NCC 189, deposited with the Collection Nationale De Cultures De Micro-organismes under accession number I-2333 and NCC 90, deposited with the Collection Nationale De Cultures De Micro-organismes under accession number I-2332.
 

 

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