Use of Lactobacillus paracasei for promoting recovery of the intestinal microbiota diversity after dysbiosis

Abstract

The present invention provides the use of Lactobacillus paracasei, for maintaining or increasing the intestinal microbiota diversity in a subject having dysbiosis.

Claims

1. A method to treat an infection caused by a vancomycin-resistant Enterococcus faecalis in the intestinal microbiota of a subject having a dysbiosis caused by or subsequent to antibiotic treatment of said subject, comprising administering Lactobacillus paracasei subspecies paracasei strain CNCM I-3689 to said subject.

2. The method according to claim 1, wherein said Enterococcus faecalis are further resistant to antibiotics selected from penicillins, cephalosporins, fluoroquinolones, aminoglycosides, and glycopeptides.

3. The method according to claim 1, wherein said Enterococcus faecalis are part of a High-Risk Enterococcal Clonal Complex.

4. The method according to claim 1, wherein said Lactobacillus paracasei is in an orally administrable composition.

5. The method according to claim 4, wherein said composition is a fermented dairy product.

6. The method according to claim 1, wherein said dysbiosis is characterized by an increase in Firmicutes.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a scheme representing the sequence of the experiments.

(2) FIG. 2 shows the kinetics and levels determined by selective plating of total enterococci population in mice fed with a daily dose of 0.1 ml of 0.9% saline solution (control) or fed by 10.sup.9 CFU of probiotic strain in 0.1 ml of 0.9% saline solution by orogastric inoculation (A); kinetics and levels of establishment of E. faecalis V583 strain (B) and levels of E. faecalis V583 11 days after the arrest of the antibiotic treatment corresponding to D21 (C). Experiments were done in triplicate.

(3) FIG. 3 shows the relative abundance of the Actinobacteria, Bacteroidetes, Filinicutes & Proteobacteria phylum at Day 0 (baseline; FIG. 3A) and Day 21 (sacrifice; FIG. 3B) in mice fed with a daily dose of 0.1 ml of 0.9% saline solution (control C) or fed by 10.sup.9 CFU of a probiotic strain (L. rhamnosus L.r or L. paracasei L.p) in 0.1 ml of 0.9% saline solution by orogastric inoculation.

EXAMPLES

(4) Methods

(5) Bacterial Growth

(6) E. faecalis V583 strain was grown in M17 supplemented with 0.5% glucose (GM17) and collected by centrifugation 1 h 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).

(7) Probiotic strains were grown in MRS media, and collected as describe above.

(8) At least two 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.

(9) Mouse E. faecalis Model Colonization

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

(11) They received a daily dose of 10.sup.9 CFU of probiotic strain in 0.1 ml of 0.9% saline solution by orogastric inoculation using a steel feeding tube (Ecimed). Lactobacillus rhamnosus CNCM I-3690 was administered to the Lr group and Lactobacillus paracasei CNCM I-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 1.4 mg/day of clindamycin was administered subcutaneously daily for three days. One day later, 10.sup.10 colony-forming units (CFU) of E. faecalis (vancomycin-resistant enterococci, noted VRE) strain V583 in 0.1 ml of 0.9% saline solution were administered by orogastric inoculation using a steel feeding tube (Ecimed).

(12) Statistical Analysis

(13) Differences in bacterial counts were analyzed by the Mann-Whitney test (GraphPad). Differences were considered significant when P<0.05.

(14) Microbiota Analysis

(15) Faecal samples were collected at D0 (baseline), and D21 (sacrifice). DNA was extracted using Godon et al procedure (Godon, 1997). For pyrosequencing, V3-V5 region of the 16S rRNA gene was amplified using key-tagged eubacterial primers (Lifesequencing S.L., Valencia, Spain) based on design of Sim et al 2012. PCR reactions were performed with 20 ng of metagenomic DNA, 200 M of each of the four deoxynucleoside triphosphates, 400 nM of each primer, 2.5 U of FastStart HiFi Polymerase, and the appropriate buffer with MgCl.sub.2 supplied by the manufacturer (Roche, Mannheim, Germany), 4% of 20 g/mL BSA (Sigma, Dorset, United Kingdom), and 0.5 M Betaine (Sigma). The inial cycling consisted of initial denaturation at 94 C. for 2 minutes followed by 35 cycles of denaturation at 94 C. for 20 seconds, annealing at 50 C. for 30 seconds, and extension at 72 C. for 5 minutes. Amplicons were combined in a single tube in equimolar concentrations. The pooled amplicon mixture was purified twice (AMPure XP kit, Agencourt, Takeley, United Kingdom) and the cleaned pool requantified using the PicoGreen assay (Quant-iT, PicoGreen DNA assay, Invitrogen). Subsequently, an amplicon submitted to the pyrosequencing services offered by Life Sequencing S.L. (Valencia, Spain) where EmPCR was performed and subsequently, unidirectional pyrosequencing was carried out on a 454 Life Sciences GS FLX+ instrument (Roche) following the Roche Amplicon Lib-L protocol. Bioinformatic analyses were performed using QIIME v.1.6 (Caporaso, 2010). Data were assigned to 50 samples after filtering according to the following quality criteria: size between 500 and 1000 nt, quality above 25 over a 50 base pairs window, no mismatch authorized in primers and barcode sequences, and absence of polymers larger than 6nt. Remaining reads were clustered into Operational Taxonomic Units (OTUs) defined at 97% identity using cd-hit (Li, 2006) and representative sequences for each OTU were aligned and taxonomically assigned using Greengenes v_13_08 database.

(16) Results: Strain L. paracasei CNCM I-3689 Promotes Recovery of Microbiota Composition Diversity and Intestinal Clearance of Vancomycin-Resistant E. faecalis V583

(17) Using the E. faecalis colonization model, the two probiotic strains L. paracasei CNCM I-3689 and L. rhamnosus CNCM I-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 E. faecalis VRE strain were similar between the control and the probiotic-treated mice (A & B). In contrast, clearance of VRE was significantly different for mice treated with strain L. paracasei CNCM I-3689 compared to control and L. rhamnosus CNCM I-3690-treated mice (B). VRE were not detected in half of the mice receiving L. paracasei 11 days after the arrest of the antibiotic treatment corresponding to D21 of the experiment, and VRE level was significantly decreased in the other half compared to control mice (C).

(18) Taken together, these 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 limit the risks of VRE infections and dissemination, L. paracasei CNCM I-3689 is a promising candidate to promote VRE clearance. L. paracasei CNCM I-3689 could be part of a non-antibiotic strategy to promote intestinal clearance of opportunistic pathogens after antibiotic dysbiosis. To profile the effects of clindamycin treatment+VRE inoculation, and L. paracasei CNCM I-3689 on microbiota structure, 454 pyrosequencing of bacterial 16S rRNA gene V3-V5 variable regions was performed on fecal samples collected from mice at D0 (baseline), and D21 (restoration). Microbiota analysis from fecal samples collected at D0 and D21 showed that clindamycin treatment resulted in a drastic change in microbiota composition, with a predominance of Firmicutes and to a minor extent Proteobacteria in control samples. In contrast some restoration of Bacteroidetes is observed in the L. paracasei group (FIG. 2), and also in the L. rhamnosus group.