Intestinal Microbiota Composition in Patients with Amyotrophic Lateral Sclerosis: Establishment of Bacterial and Archaeal Communities Analyses
Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease characterized by the progressive loss of motor neurons, leading to muscle weakness, paralysis, and eventually death. The disease has a crude annual incidence rate of 2.16 per 100,000 person-years in Europe, and its prevalence is expected to rise as the global population ages. Despite decades of research, there is no effective intervention to alter the course of ALS, and the majority of patients survive less than five years after diagnosis. Understanding the pathogenesis and progression of ALS is therefore of critical importance. Recent studies have highlighted the potential role of the gut microbiota in neurodegenerative diseases, including ALS. The gut microbiota, which consists of trillions of bacteria and archaea, plays a crucial role in human health by influencing metabolism, immunity, and the gut-brain axis. Dysbiosis, or an imbalance in the gut microbiota, has been linked to various central nervous system disorders, including autism spectrum disorders, irritable bowel syndrome, and neurodegenerative diseases. However, the specific interactions between gut microbiota composition and ALS remain poorly understood.
This study aimed to evaluate the bacterial and archaeal composition of the gut microbiota in patients with ALS and compare it to that of healthy individuals. Additionally, the study assessed the concentrations of key metabolites, including human endotoxin, short-chain fatty acids (SCFA), NO2-N/NO3-N, and gamma-aminobutyric acid (GABA), to explore the metabolic implications of gut microbiota dysbiosis in ALS. The findings provide valuable insights into the potential role of the gut microbiota in ALS progression and highlight the need for further research in this area.
Methods
The study was conducted at Huadong Hospital, Fudan University, from November 2017 to April 2018. Eight patients with ALS and eight healthy individuals were recruited for the study. The inclusion criteria for ALS patients were a survival period of more than three months and no use of antibiotics in the last month. Exclusion criteria included serious comorbidities such as heart, lung, liver, kidney, brain, or blood system diseases, as well as other conditions that could mimic ALS, such as cervical spondylosis or spinal cord tumors. Healthy individuals were recruited as controls and matched for age, sex, and body mass index (BMI).
Fecal samples were collected from each participant after a 30-day standardized diet and stored at -80°C for further analysis. The bacterial and archaeal communities in the fecal samples were analyzed using high-throughput sequencing on the Illumina MiSeq PE300 platform. Universal bacterial primers were used to amplify the V4-V5 hypervariable regions of the 16S rRNA gene, while specific primers were used for archaeal communities. The sequences were processed to remove low-quality reads, and operational taxonomic units (OTUs) were identified using a 97% similarity threshold. The relative abundance of bacterial and archaeal taxa at the phylum, class, and genus levels was calculated, and biodiversity indices, including Ace, Chao, Shannon, and Simpson, were used to assess the richness and evenness of the microbial communities.
Metabolite concentrations, including human endotoxin, SCFA, NO2-N/NO3-N, and GABA, were measured using spectrophotometry and enzyme-linked immunosorbent assay (ELISA) kits. Statistical analyses were performed using one-way analysis of variance (ANOVA) to compare metabolite concentrations between ALS patients and healthy controls. A p-value of less than 0.05 was considered statistically significant.
Results
The study revealed significant differences in the composition and diversity of the gut microbiota between ALS patients and healthy individuals. The richness and evenness of bacterial and archaeal communities were generally lower in ALS patients compared to healthy controls. The average number of OTUs in ALS patients was 141, compared to 138 in healthy individuals. However, the biodiversity indices indicated that the richness and evenness of microbial communities were healthier in healthy individuals.
At the phylum level, the bacterial community in both ALS patients and healthy individuals was dominated by Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Verrucomicrobia. Notably, the relative abundance of Firmicutes was 4.7% higher in ALS patients, while Bacteroidetes showed a decrease. The Firmicutes/Bacteroidetes ratio, which is considered an important indicator of gut health, was higher in ALS patients, suggesting an imbalance in the gut microbiota. At the class level, Negativicutes and Bacilli were less abundant in ALS patients compared to healthy controls.
At the genus level, the top ten genera in ALS patients and healthy individuals were Bacteroides, Blautia, Faecalibacterium, Escherichia-Shigella, Anaerostipes, Streptococcus, Akkermansia, Fusicatenibacter, Megamonas, and Bifidobacterium. The relative abundance of beneficial microorganisms, such as Faecalibacterium and Bacteroides, was significantly lower in ALS patients. In contrast, the relative abundance of harmful microorganisms, such as Dorea, showed an increasing trend, although this was not statistically significant. The archaeal community in ALS patients was dominated by Euryarchaeota, Methanobacteria, and Methanobrevibacter, with a significant increase in the relative abundance of Methanobrevibacter compared to healthy controls.
Metabolite analysis revealed that the average concentrations of human endotoxin, SCFA, NO2-N/NO3-N, and GABA in ALS patients were 64.2 EU/mL, 57.5 mg/mL, 5.7 ng/mL, and 6.1 mmol/L, respectively. In healthy individuals, the corresponding values were 65.3 EU/mL, 55.3 mg/mL, 5.3 ng/mL, and 5.4 mmol/L. Although there were no significant differences in metabolite concentrations between the two groups, the results indicated a trend toward increased levels of SCFA, NO2-N/NO3-N, and GABA in ALS patients, suggesting a decline in gastrointestinal absorption function.
Discussion
The findings of this study provide evidence of dysbiosis in the gut microbiota of ALS patients, characterized by a decrease in beneficial microorganisms and an increase in harmful ones. The decline in Faecalibacterium and Bacteroides, which are known to play protective roles in gut health, may contribute to the progression of ALS. The increase in Methanobrevibacter, which utilizes SCFA to produce methane, may further exacerbate metabolic disturbances and weight loss in ALS patients.
The observed changes in the gut microbiota are consistent with previous studies that have linked dysbiosis to neurodegenerative diseases. For example, the Firmicutes/Bacteroidetes ratio has been shown to be altered in patients with irritable bowel syndrome and other gastrointestinal disorders. The decline in beneficial microorganisms, such as Faecalibacterium, has also been associated with conditions like coeliac disease and asthma. The increase in Methanobrevibacter is particularly noteworthy, as it has been linked to weight loss and metabolic dysfunction in patients with intestinal diseases.
The metabolite analysis revealed a trend toward increased levels of SCFA, NO2-N/NO3-N, and GABA in ALS patients, suggesting a decline in gastrointestinal absorption function. These metabolites play important roles in nutrient metabolism and may influence the progression of ALS. For example, SCFA are essential for maintaining gut barrier function and modulating immune responses, while GABA is a key neurotransmitter involved in the regulation of motor neuron activity.
The limitations of this study include the small sample size and the lack of metagenomic sequencing to explore the functional implications of gut microbiota dysbiosis in ALS. Future studies should focus on larger cohorts and incorporate metagenomic and metabolomic analyses to provide a more comprehensive understanding of the interactions between the gut microbiota and ALS progression.
Conclusion
This study highlights the significant differences in the composition and diversity of the gut microbiota between ALS patients and healthy individuals. The decline in beneficial microorganisms and the increase in harmful ones, along with the observed trends in metabolite concentrations, suggest that gut microbiota dysbiosis may play a role in the progression of ALS. These findings underscore the importance of further research into the gut-brain axis and its potential implications for the development of novel therapeutic strategies for ALS.
doi.org/10.1097/CM9.0000000000000351
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