USE OF LACTOBACILLUS PARACASEI FOR PROMOTING INTESTINAL CLEARANCE OF OPPORTUNISTIC PATHOGENS AFTER ANTIBIOTIC DYSBIOSIS

A Lactobacillus paracasei subs, paracasei strain, for use for accelerating the decrease of Enterococcus faecalis in the intestinal microbiota of a subject haying an intestinal dysbiosis caused by antibiotics.

USE OF LACTOBACILLUS PARACASEI FOR PROMOTING INTESTINAL CLEARANCE OF OPPORTUNISTIC PATHOGENS AFTER ANTIBIOTIC DYSBlOSiS

The present invention relates to the field, of probiotics. Particularly, the invention pertains to the use of a probiotic strain of lacto acitl -par c sei^' susp, paracasei, for accelerating the decrease of Enterococcus Jaecali in the intestinal microbiota of a subject: having an intestinal dysbiosis caused by antibiotics.

According to a definition approved by a joint. Food and Agriculture: Organization of the United Nations/World Health Organization (FAO/WHO) expert Consultation on Health and: Nutritional properties of powde milk with live lactic acid bacteria in 2001 , probiotics are 'live microorganisms which when administered in adequate amounts confer a health -benefit on the host". Probiotic bacteria have been described amon species belongin t the genera Lactobacillus, Bifidobacteri. Streptococcus and Lacftococcus, commonly used in the dairy industry. Probiotics are thought to intervene at the level of the gut microbiota by impeding the development of pathogenic microorganisms and/of by acting more directly on the immune system.

Opportunistic bacterial infections responsible for healthcare associated infections (HAls) contribute significantly to patient, mortality and morbidity, as well as healthcare costs both in- developed and. developing countries (WHO, 2008). The: gastrointestinal tract (GfT) is a reservoir for opportunistic pathogens, whic beneilt from the disruption of the intestinal- microbiota balance, or dysbiosis, to invad and infect susceptible patients. Antibiotic treatments have deleteriou effects on the diversity of the intestinal microbiot and the promote overgrowth of bacterial human opportunistic pathogens including Ertterococo s difficile (Deth!efsert et a!., 2008).

Having: acquired antibiotic resistance and other pathogenic traits, multidrug resistant colonizing and/or invasive E, faecaiis isolates, which cause serious nosocomial infections, are: grouped in seven hospital -adapted complexes designated as High-Risk imterococcai Clonal Complexes (HiRECCs, ( uch et al., 2012)). Proliferation and persistence of HiRECCs within the GIT are a major risk of developing a vaiicomycin-resistant enterococcal (VRE) infection, urging for better understanding of the biological and biochemical factors involved in colonization of the GIT by E, jaecalis (Arias and Murray, 2012). isolates belonging t the HiRECC-2 are among the most common causes of E. Jaecalis infections in the Umted States and in several European countries ( ach et al. 201 1 ; Nallapareddy et. al., 2005).

It appears from the foregoing that there is a need for alternatives or complements to antibiotics for the treatment or for the prevention of E. /wealis infection.

Growing evidence show that probiotics or fecal microbiota transplantation prevent or treat a number of diseases, including intestinal infections (Foster et al, 201 1 Pamer, 2014). Such approaches were also associated with higher clearance of intestinal VRE in mice (Vidal et al., 201 ). The inventors developed an intestinal colonization mouse model based on a roicrobiota dvsbiosis Induced b clindamycin to mimic enterococci. overgrowth and VRE establishment:. Mic received subcutaneous clindamycin: for 3 days before orogastric inoculation with Enterococcm faecal is VRE strain (V583). indeed, the native mierobiota in mice is nearly or totally devoid of gntcroeoccus faeealis; moreover, the commensal-to- pathogen switch does not happen in mice. Using this model, prohiotic strains were dail orally administered to .mice starting one week before antibiotic treatment until two weeks after arrest of antibiotic treatment and inoculation of VRE. Kinetics of establishment and clearance of VRE as well of indigenous enterococci population levels were monitored by selective plating. I parallel, fecal samples were collected for 16S rR A gene survey analysis of the whole mierobiota. The dysbiosis induced in this model mimics the antibiotic-induced d sbiosis observed i humans. This. model constitutes a good model to study the mechanisms of intestinal colonization barrier against enterococci overgrowth (Rigottier-Gois et al, submitted). The- strain V583 belongs to CC2 and was the first vancomycin resistant isolate reported in the United States (Sahm et .al, 1989). This strain was used in the experiments reported below as a model strain of CC2 isolates and more generally, of pathogenic E, jmc l s.

Using this model, the inventors have found that., the bacterial species Lactobacillu paracas i s hsp. pamemei is capable of decreasing the load of £i faecd is strains .in vivo.

Accordingly, a. subject of the present invention is the use of a Lactobacillus pamemei mbsp. paracmei train, for accelerating the decrease of Enterococcm faecaii : in the intestinal mierobiota of a subject having an intestinal dysbiosis caused by antibiotics.

In the present, text, the phrases "accelerate the decrease of E, faecalir "decrease: the load of -E.. faecalis", "promote the decrease of 2s, faecalis etc. will be used to express that the amount of E. fiieca!is present in the mierobiota of individuals having an intestinal dysbiosis after a^'treatment by antibiotics decreases statistically more rapidly in subjects who take the probiotic strain than, in control subjects who do not.

More specifically,, ihe present invention pertains to the use of L paracmei strain CNCM 1-3689, for promoting the decrease of Emero oc us faecalis in the intestinal microbioia of: a subject having an intestinal dysbiosis caused by .antibiotics, This strain was deposited by the Applican accordin t the Budapest Treaty, at CNCM (Collection Nationale de Cultures de- Mieroorganism.es. 25 rue du Docteur Roux, Paris) on- November 9, 2006. This strain is disclosed in International Applicatio WO 2009/130423, Another strain of Lactobacillus paracmei suhsp paracasei which can be- used according to the present invention is the. strain deposited at the: CNCM under the numbe 1-1518,

Of course, the present invention also: encompasses the use of a strain deri ved from the strain CNCM 1-3689, for accelerating the decrease of the amount of Enterococc - faecalis in the intestinal niicrobiota of a subject having an intestinal dysbiosis caused by antibiotics.: According to the present invention, such a ''strain derived from the strain CNCM 1-3689" is still capable of decreasing the; load of E.faecalis strains i the niicrobiota of a subject having an antibiotics-induced dysbiosis. To assess: this capacity, the same model as decribed i the experimental part below can be used. Strains derived from the strain CNCM V 3689 which can be used according to the present invention include mutant strains and genetically transformed strains. These mutants or genetically transformed strains can be strains wherein one or more endogenous gene(s) of the parent strain CNCM 1-368 has (have) been mutated, for instance to modify some of their metabolic. roperties- (<?.#., their ability to ferment sugars,, their resistance to acidity, their survival to transport in the gastrointestinal tract, their post-acidification properties or their metabolite production). They can also be strains resulting from the genetic transformation of the parent strain CNCM 1-3689 to add one or more gene(s) of interest, for instance in order to give to said genetically txansforrned strains additional physiological features, or to allo them to- express proteins o therapeutic or vaccinal interest that one wishes to administer through said strains. These mutants or genetically transformed strains can. be obtained from tile parent strain CNCM 1-368 by means of the conventional techniques for: random or site-directed mutagenesis and genetic trans fontiati on of bacteria, or by means of the technique known as '*genonie ..shuffling"^'. In the present text, the mutants and variant derived from the strai CNCM 1-3689 and retaining its ability to decrease the amount of E. faecalis in the intestinal niicrobiota of a subject having an aniibiotics-induced dysbiosis will be considered as being encompassed, by the phrase "the strain CNCM 1-3689".

As recalled, above, some E. faecalis strains acquired pathogenic traits and ean caus severe infections, indeed, they can colonize the GIT and or cross the intestinal epithelial barrier and ente the bloodstream (Donskey, 2004; Gilmore and FerretH. 2003; Krueger et aL 2004; Wells et al„ 1990), Futliermore, Enterococc s spp. contribute to community-acquired intra-abdominal infections (Sarteill, 2010;: Montravers et al. 2009) and count among the ten most frequently -isolated micro-organisms in healthcare-associated infections (HCAl). Hence, reducing the proliferation and. persistence of E, faecalis, especially o HiRECCs in the GIT reduces the risk of developing not only a GIT infection, but also an intra-abdominal infection. Another object of the present invention hence is the use of Lactobacillm paracasei subsp. p racasei strain- CNCM 1-3689, fo preventing translocatio of Enterococcus faecalis across the intestinal barrier and/or for preventing intra-abdominal infection, by Enterocoecm faecalis.

Of course, the present invention is particularly advantageous to reduce the. amount of multi-drug resistant enteroeocei, especially of HiRECCs., after an. antibiotic treatment. The present invention ence concerns the use of Lactobacillm paracasei suhsp, paracasei CNCM 1-1689,- tor .promoting the decrease of antibiotics-resistant E. faecalis which- are in the intestinal microbiota of a subject having_, an intestinal dysbiosis caused by antibiotics. In what precedes, ¾niibioties-resistant j£ faecaiis" are resistant to antibiotic selected among penicillins, cephalosporiaS; fluoroquinolones, aminoglycoside and giycopeptides.

According to a preferred embodiment of the present invention the strain I. pamcasei CNCM .1-3689 is contained in an orally admirti stable composition, s that uptake of this composition by a subject having an .intestinal dysbiosis following a treatment b antibiotics leads to an accelerated decrease of^'E. faecaiis in the microbiota of said subject, in such a composition, 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. Preferably -the. bacterial cells are present as living and viable cells.

According to the present invention, the composition can be in an form suitable for oral administration. This includes for instance solids, semisolids, liquids, and powders. Semi-solid compositions, such as yogurts, and liquid compositions, such as drinks,. are preferred.

The composition can comprise at least 1.10* colony forming units (efu , preferably at least 1.10⁸ efu, per gram dry weight, of a bacterial -strain as mentioned above.

The composition can furthe comprise other strains of Lactobacillus and/Or other strains of bacteria than the strains mentioned above, in particular probiotic strain(s)* such as Streptococcus {hemophilus. Bifidobacterium- and Laetocvccus sirain(s),.

The composition can be a pharmaceutical composition or a nutritional composition. According to a preferred embodiment, the composition is a nutritional composition such as a food product (including a functional food) or a food supplement,

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 vvhich 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 effec - protective or curative - against a disease, for example against a chronic disease.

Nutritional compositions which can be used according to_. the invention include dairy products, preferably fermented dairy products. The fermented products can be in the form of a liquid or 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 fermented, vegetable, such as fermented soy, cereals and/or f uits in set, stirred or drinkable forms. Nutritional compositions which can be used according to the invention also include baby foods, infant milk formulas and infant Follow-on formulas, 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 the use of a L paracasei strain as defined above, preferably the strai CNGM i-3689, or a composition as defined above, for the manufacture of a medicament for decreasing the load of E. faecalis in the intestinal raicrobiota and/or preventing translocation of E. faecali acros the intestinal barrier and/or preventing an E. faecalis^' infection in. a human having an intestinal dysbiosis due to antibiotics,

A subject of the present invention is also a metho for decreasing, the load of E. faecalis in. the intestinal mierohiota and/or preventing, translocation of E. faecalis across the intestinal barrier and/or preventin E. faecalis infection in subject in need thereof, said method comprising administering to said subjec a therapeutically effective amount of a 1, paracasei strain as defined, above, preferably the strain GNGM i-3689, or a composition as defined above.

Determination of a therapeutically effective amount is. well known, b 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 medicament for decreasing the load of E. faecalis in the intestinal .microbiota and/or preventing translocation of E. faecalis across the intestinal barrier and/or preventing E. aecalis infection in a human having an intestinal dysbiosis due to antibiotics, said method comprising incorporating a £^' . paracasei strain as defined above, preferably the strain. GNGM 1-3689. into at least one pharmaceutically acceptable diluent, carrier or exci.pi.en.i.

The present invention will be understood more clearly from the further description which follows, which refers to. examples illustrating the capacit of the L paracasei strain CNC 1-3689 of decreasing the load of K. faecalis strains in viva, as well as to the appended figures.

figure- 1 is a scheme representing the sequence of the experiments.

Figure 2 shows the kinetics and levels determined by selective plating of total enteroeoc i population in mice fed with daily dose of 0.1 ml of 0.9% saline solution (control) or fed by 10^y CFU of probiotic strain in 0.1. ml of 0.9% saline solution by orogastrie inoculation (A); kinetics and levels of establishment of E. faecalis V583 strain (13) and level s of E, faecalis^'V$^'83 11 days after the arrest, of the antibiotic treatment corresponding to D21 (C). EXAMPLES

ethods

Bacterial growth

E. faeealis. V583 strain was .grown in MI 7 supplemented with 0.5% glucose (GM1.7) and collected by centrifugation lh after reaching stationary phase. Bacterial cells were washed twice, with 0.9% saline solution and stored as -a dry frozen pellet at -80°C. This strain belongs to CC2 and was the first vancomycin resistant isolate reported in the United^' States (Sahm et aL 1989),

Probiotic strains were grown in MRS media, and collected -as describe above.

At least tw days before inoculation, the frozen bacteria were suspended, in a saline solution and serial dilutions were plated on GM17 or MRS agar plates to determine the bacterial count of the pellet.

Manse E. faeea s model colonization

Mouse experiments were, performed using specific pathogen-iree male CF-1 mice (Harlan, USA), 6~8~weeks, A total of 5 mice were housed in each cage and were fed with autociaved food and water ad libitum

They received a dail dose of 10^y'CFU of probiotic strain in 0.1.. ml of 0.9% saline solution by orogastric inoculation using a steel -feeding tube (Eeimed). Lactobacillus rkamno s CNCM I~3^'690. was administered to the Lr group and Lactobacillus p casei CNCM 1-3689 for the Lp group. Animals from the control group received 0,1 ml of 0.9% saline solution by the same way. After one -week of probiotic treatment, a dose of 1 A mg day of cliudaiTiyein was administered .su.bcutan.eous.ly daily far three days. One day later, 10^l° colony-forming units (CPU) of E. faecalis (vancorayein-resistani enterococci noted "VRE" strain V583 in. 0.1 ml of 0.9% saline solution^'were administered by orogastric inoculation using a steel feeding, tube (Eeimed),

Statistical mi afysis

Differences in bacteria! counts were analyzed, by the Mann-Whitney test^' (GraphPad). Differences were considered significant -when P < 0.05.

Results: St in L paracasei CNCM I-3$^_,._,._.. .yQ Ptes__.J¾tes^'ti¾al clearance of vgttcpitnyem-resistant ^fyec ih ^^

Using the E. faecalis .colonization model, the two probiotic strains /,, paracasei C CM 1-3689 and L, rhamnosus CNCM 1-3690 were daily orally administered to mice starting 1 week before antibiotic treatment, until two weeks after arrest of antibiotic treatment and inoculation of VRE. Levels of total enterococci population, and VRE were monitored by selective plating. Kinetics and levels of enterococci population as well as kinetic of establishment of £ faecaii VRE strain were similar between the control and the probio lie -treated mice (A & B). In contrast, clearance of VRE was significantly different for mice treated with strain L. paracasei CNCM 1-3689 compared to control and L. rhamnos s C M I-3690-freated mice (B). VRE were not detected in half of the. mice receiving L paracasei 1 1 days after .the arrest of the antibiotic treatment corresponding to D2.1 of the experiment, and VRE level was significantly decreased in the othe half compared to control mice (€). No significant difference of VRE population: ..level was observed, in L. rhamnosus treated mice.

Altogether, thes results show that administration of L paracasei CNCM I- 3689 significantly decreases pathogenic E. faecalis persistence, in the gut, Given that reduction of intestinal colonization or carriage after antibiotic treatment could Iimit the risks of VRE infections and dissemination, /-, paracasei CNCM 1-3689 is a promising candidate to promote VRE clearance, I. paracasei CNCM 1-3689 could be part of a non-antibiotic strategy to promote intestinal clearance of opportunistic pathogens after antibiotic dysbiosis.

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