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Additional file 1: Figure S1. OTUs distribution in the control-diseased dataset for the most representative phyla. a) OTUs distribution for Bacteroidetes, Firmicutes, Actinobacteria and Verrucomicrobia. b) OTUs distribution for Cyanobacteria. Figure S2. OTUs distribution in the control-diseased dataset for the significant families. a) OTUs distribution for Gastranaerophilales; D_4__uncultured bacterium and Clostridiaceae 1. b) OTUs distribution for Clostridiales; Ambiguous_taxa and Clostridiales;D_4__Family XI. Figure S3. Gut bacterial families and genera characteristics at baseline. a) Relative abundance of microbial groups at family level in the control group (C) and diseased group (D); families with a relative abundance less than 0.002 are omitted for the sake of clarity. b) Relative abundance of microbial groups at genus level in the control group (C) and diseased group (D); genera with a relative abundance less than 0.003 are omitted for the sake of clarity. Figure S4. Fecal yeast qPCR counts during the intervention. The graphic was conceived as mean plot reporting also the standard errors. Figure S5. OTUs distribution in ALS patient’s dataset for significant families. a) OTUs distribution for Bacteroidales; other among the ALS patients grouped for different timepoints and not considering the type of treatment. b) OTUs distribution for Baceroidaceae and Rikenellaceae among ALS patients considering the time and the different treatments (Group A and Group B). Table S1. Absolute abundance expressed as means of the number of OTUs for phyla and families that were significantly different between control (C) and diseased subjects (D) at the baseline. Table S2. Absolute abundance expressed as means of the number of OTUs for phyla and families that were significantly different among ALS patients during the study. “Pcorr” corresponds to the adjusted P-value for the comparisons performed. Table S3. DNA concentration and mean counts of the analyzed microbial groups by qPCR in stool samples of ALS patients during the intervention. Table S4. Relative abundance of the main families for each group of subjects during the intervention.

Background Recent preclinical studies suggest that dysfunction of gastrointestinal tract may play a role in amyotrophic lateral sclerosis (ALS) pathogenesis through a modification of the gut microbiota brain axis. Our study is the first focused on microbiota analysis in ALS patients. Aim Our aim was to study the main human gut microbial groups and the overall microbial diversity in ALS and healthy subjects. Moreover we have examined the influence of a treatment with a specific bacteriotherapy composed of Lactobacillus strains (Lactobacillus fermentum, Lactobacillus delbrueckii, Lactobacillus plantarum, Lactobacillus salivarius) acting on the gastrointestinal barrier. Methods We enrolled 50 ALS patients and 50 healthy controls, matched for sex, age, and origin. Fecal samples were used for total genomic DNA extraction. Enterobacteria, Bifidobacterium spp., Lactobacillus spp., Clostridium sensu stricto, Escherichia coli and yeast were quantified using quantitative polymerase chain reaction approach. Denaturing gradient gel electrophoresis analyses were performed to investigate total eubacteria and yeasts populations. Patients were randomized to double-blind treatment either with microorganisms or placebo for 6 months and monitored for clinical progression and microbiota composition. Results The comparison between ALS subjects and healthy group revealed a variation in the intestinal microbial composition with a higher abundance of E. coli and enterobacteria and a low abundance of total yeast in patients. Polymerase chain reaction denaturing gradient gel electrophoresis analysis showed a cluster distinction between the bacterial profiles of ALS patients and the healthy subjects. The complexity of the profiles in both cases may indicate that a real dysbiosis status is not evident in the ALS patients although differences between healthy and patients exist. The effects of the progression of the disease and of the bacteriotherapy on the bacterial and yeast populations are currently in progress. Conclusions Our preliminary results confirm that there is a difference in the microbiota profile in ALS patients.