A Randomised, Controlled Trial: Effect of a Multi-Strain Fermented Milk on the Gut Microbiota Recovery after Helicobacter pylori Therapy

Eric Guillemard, Marion Poirel, Florent Schäfer, Laurent Quinquis, Caroline Rossoni, Christian Keicher, Frank Wagner, Hania Szajewska, Frédéric Barbut, Muriel Derrien, Peter Malfertheiner, Eric Guillemard, Marion Poirel, Florent Schäfer, Laurent Quinquis, Caroline Rossoni, Christian Keicher, Frank Wagner, Hania Szajewska, Frédéric Barbut, Muriel Derrien, Peter Malfertheiner

Abstract

Helicobacter pylori (Hp) eradication therapy alters gut microbiota, provoking gastrointestinal (GI) symptoms that could be improved by probiotics. The study aim was to assess the effect in Hp patients of a Test fermented milk containing yogurt and Lacticaseibacillus (L. paracasei CNCM I-1518 and I-3689, L. rhamnosus CNCM I-3690) strains on antibiotic associated diarrhea (AAD) (primary aim), GI-symptoms, gut microbiota, and metabolites. A randomised, double-blind, controlled trial was performed on 136 adults under 14-day Hp treatment, receiving the Test or Control product for 28 days. AAD and GI-symptoms were reported and feces analysed for relative and quantitative gut microbiome composition, short chain fatty acids (SCFA), and calprotectin concentrations, and viability of ingested strains. No effect of Test product was observed on AAD or GI-symptoms. Hp treatment induced a significant alteration in bacterial and fungal composition, a decrease of bacterial count and alpha-diversity, an increase of Candida and calprotectin, and a decrease of SCFA concentrations. Following Hp treatment, in the Test as compared to Control group, intra-subject beta-diversity distance from baseline was lower (padj = 0.02), some Enterobacteriaceae, including Escherichia-Shigella (padj = 0.0082) and Klebsiella (padj = 0.013), were less abundant, and concentrations of major SCFA (p = 0.035) and valerate (p = 0.045) were higher. Viable Lacticaseibacillus strains were detected during product consumption in feces. Results suggest that, in patients under Hp treatment, the consumption of a multi-strain fermented milk can induce a modest but significant faster recovery of the microbiota composition (beta-diversity) and of SCFA production and limit the increase of potentially pathogenic bacteria.

Keywords: Helicobacter pylori; antibiotic associated diarrhea; antibiotics; gut microbiota; recovery; short chain fatty acids.

Conflict of interest statement

E.G., F.S., L.Q., C.R., M.D. and M.P. are Danone Nutricia Research employees. F.B. received grants from Astellas, Anios, MSD, Biomérieux, Quidel, Cubist, Biosynex, and GenePoc, personal fees from Astellas, Pfizer, MSD, and Danone, and non-financial support from Astellas, Pfizer, Anios, and MSD. PM provides consultancies to AlfaSigma, Bayer Vital, Danone, Luvos, Mayoly Spindler, Menarini, and Novartis and received speaker’s fee from Alfa Sigma, Bayer, Malesci, Mayoly Spindler, and Nordmark. H.S. has participated as a clinical investigator, and/or advisory board member, and/or consultant, and/or speaker for Arla, BioGaia, Biocodex, Danone, Dicofarm, Nestlé, and Nestlé Nutrition Institute. C.K. and F.W. had no conflict of interest. M.D. is inventor of patent applications (WO2015159125A1, WO2015159124A1) dealing with the use of L. paracasei CNCM I-3689 and L. rhamnosus CNCM I-3690 in the context of antibiotic dysbiosis.

Figures

Figure 1
Figure 1
(A) Study Design Overview. (B) Subject flowchart.
Figure 2
Figure 2
Global gut microbiota response to Hp treatment and product intervention. (A) Total bacterial count/g fecal samples assessed by flow cytometry, with a log10 transformation. (B) Alpha-diversity assessed by Shannon index (C) Alpha-diversity assessed by Simpson’s Reciprocal. (D) Principal Coordinate Analysis (PCoA) based on Bray–Curtis dissimilarity. Samples were collected before (V2) and after Hp treatment (V4), 14 days (V6) and 28 days (V7) following cessation of Hp treatment. * p < 0.05 and *** p < 0.001 according to linear mixed model. (E–G) Intra-subject distance to baseline of each subject in Test and Control groups across the study (E) Bray–Curtis dissimilarity (F) Weighted UniFrac distance (G) Unweighted UniFrac distance. * p < 0.05 and *** p < 0.001 according to Mann–Whitney test.
Figure 3
Figure 3
Genus-level differential analysis based on Quantitative Microbiome Profiling (A) Heatmap of differentially abundant genera between each visit and baseline (V2) in the Control group. Red indicates higher fold-change and blue lower fold-change with regards to baseline (B) Heatmap of differentially abundant genera between Test and Control groups at each visit. Red indicates a higher fold-change and blue a lower fold-change in Test group (C) Heatmap of differentially abundant genera between follow-up (V7) and baseline (V2) which behave differently in the two groups. Red indicates higher fold-change and blue lower fold-change with regards to baseline (V2). For all heatmaps, only taxonomically assigned genera were selected based on QMP normalized counts >10.000.000, (FDR adj. * p < 0.1, DESeq2-based Wald test). Non-significant fold changes were set to zero for heatmap display and significant fold changes are highlighted by a star. (D) Quantitative log2 abundance of Escherichia/Shigella and Klebsiella in response to Hp treatment along the study, as mean ±95% CI.
Figure 4
Figure 4
Global gut mycobiota response to Hp treatment and product intervention. (A) Alpha-diversity assessed by Shannon index (B) Shannon index ratio ITS/16S based (C) Abundance of Candida. * p < 0.05 and ** p < 0.01 according to Mann–Whitney test.
Figure 5
Figure 5
Quantification of fecal major/minor SCFA and Calprotectin. Concentration (A,C,E) and change from baseline (B,D,F) of major SCFA (acetate, propionate, butyrate) (A,B), minor SCFA (valerate, caproate, isobutyrate, and isovalerate) (C,D) and Calprotectin (E,F). p-values are provided according to Student test: * p < 0.05 for comparison between groups of the change of major SCFA concentration from V4 to V6; a p < 0.05, b p < 0.01; c p < 0.001; and d p < 0.0001, for comparison within group of the change from V2 at each visit. SCFA concentrations are expressed in µmol/g of dry feces.

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