16S rRNA Next Generation Sequencing Analysis Shows Bacteria in Alzheimer’s Post-Mortem Brain

Abstract

The neurological deterioration associated with Alzheimer’s disease (AD), involving accumulation of amyloid-beta peptides and neurofibrillary tangles, is associated with evident neuroinflammation. This is now seen to be a significant contributor to pathology. Recently the tenet of the privileged status of the brain, regarding microbial compromise, has been questioned, particularly in terms of neurodegenerative diseases. It is now being considered that microbiological incursion into the central nervous system could be either an initiator or significant contributor to these. This is a novel study using 16S ribosomal gene-specific Next generation sequencing (NGS) of extracted brain tissue. A comparison was made of the bacterial species content of both frozen and formaldehyde fixed sections of a small cohort of Alzheimer-affected cases with those of cognitively unimpaired (normal). Our findings suggest an increase in bacterial populations in Alzheimer brain tissue compared with normal.

Gastrointestinal (GI) Tract Microbes and Microbial Neurotoxins in the Human Central Nervous System (CNS) in Alzheimer’s Disease (AD)

“…Of further related interest are the recent observations: (i) that the increased abundance of gram-negative GI tract bacteria such as B. fragilis in AD patients appears to result in increased generation and translocation of LPS and other Bacteroides-derived neurotoxins from the GI tract into the systemic circulation, which in turn may contribute to AD neuropathology through the release of pro-inflammatory cytokines, systemic inflammation, an increase in GI-tract or BBB permeability or other AD-relevant pathogenic mechanisms [9,[29][30][31][32], and (ii) that an increase in Bacteroidetes in the GI tract is also associated with Parkinson's disease (PD; [33]) and with sporadic AD hippocampus and neocortex, two anatomical regions targeted by the AD process [6,[34][35][36]. Importantly, while only the inflammatory potential of LPS towards primary human neuronal-glial (HNG) co-cultures have been studied and quantified by the induction of the pro-inflammatory NFkB p50/p65 complex, Bacteroidetes species are capable of secreting an unusually complex array of highly lethal neurotoxins including amyloids, sncRNAs and endotoxins which, when released from the confines of the healthy GI tract, are systemically pathogenic and can be highly detrimental to the homeostatic function of human CNS neurons [15].…”

“…As fore-mentioned, microbes such as B. fragilis and Escherichia coli (E. coli), abundant anaerobic Gram-negative bacilli of the human GI-tract microbiome, appear to direct critical regulatory roles of pathogenicity through the stress-induced secretion of a complex mixture of bacterial amyloids, endotoxins and exotoxins, 'microRNA-like' sncRNAs and LPS. Recently work from several independent groups has further described the presence of intact bacteria, bacterial-derived nucleic acid sequences and/or bacterial-derived neurotoxins such as a highly pro-inflammatory LPS that is associated with neuronal parenchyma and in particular the neuronal nuclei of anatomical regions of the ADaffected brain exhibiting characteristic neuropathology [1,9,28,29,[34][35][36][37][38][39]. Interestingly, the close association of bacterial LPS with neuronal nuclei may prevent the efficient export of messenger RNA (mRNA) from a highly active neuronal genome resulting in the down-regulation of gene expression in AD as is widely observed [6,15].…”

“…A commensal genus of non-endospore-forming, rod-shaped bacillus, Bacteroidetes are a human distal-GI-tract-abundant microbe whose membranes contain sphingolipids or glycosylceramides; these complex lipids play important roles in signal transmission and cell recognition and are an unusual feature of obligately anaerobic Gram negative microbes [9,16,22]. Generally microbial Bacteroidetes species such as Bacteroides fragilis (B. fragilis) appear to be generally beneficial to human health via their ability to cleave dietary fiber into digestible short-chain fatty acids, their production of useful polysaccharides, vitamins and their precursors, volatile fatty acids and other nutrients, however, when they escape the confines of a healthy GI tract microbiome they elicit inflammatory systemic pathology with substantial morbidity and mortality [15,[22][23][24]26].…”

“…Related to the points above, overall the GI tract microbiome of AD patients was found to exhibit highly selective abundance and compositional differences in genus and species from control age-and sex-matched individuals and exhibited phylum-and genus-wide differences in bacterial abundance including a significantly increased abundance of Bacteroidetes and decreased abundance of Firmicutes and Bifidobacterium in the AD-affected brain [9,[14][15][16][17][23][24][25][26]. Very recently, at least 2 independent publications have shown an enrichment of LPS within the neocortex or hippocampus of ADaffected brain [6,[27][28][29].…”

“…recently reported: (i) that RNA sequencing of the V4 region of the 16S rRNA gene in control (n=25) and AD (n=25) patients indicated that sequences representing a total of 4.8 million sequence reads [mean ± one standard deviation (SD) of ~96,000 ± 32,000 reads/participant] were clustered into operational taxonomic units (OTUs; an operational definition used to classify groups of closely related individuals) at 97% similarity [3,5]; (ii) that the final OTU dataset for the control and AD groups consisted of 972 OTUs classified into 95 genera, 46 families, 24 orders, 17 classes and 9 phyla (5); (iii) that there are about ~1000 bacterial species in a typical healthy human GI tract microbiome with a 'GI tract microbial core' of the genus Bacteroides and Firmicutes predominating, and with Proteobacteria, Verrumicrobia, Actinobacteria, Fusobacteria and Cyanobacteria making up the remainder [3,5,6,[12][13][14]; (iv) that at least 13 of 95 microbial genera examined exhibit differential abundance between AD and age-matched control populations [4][5][6]12]; (v) that there are consistent trends observed between relative GI tract bacterial abundance and CSF biomarkers of AD neuropathology [12,15]; (vi) that 16S rRNA next generation sequencing analysis has identified multiple microbial-derived nucleic acids in the AD brain, some of which are known to have pathogenic potential [9]; (vii) that remarkably, the abundance and complexity of microbial populations or their exudates in the GI tract is an approximate reflection of CNS microbial complexity, including microbiome-derived nucleic acid sequences and Gram negative-derived neurotoxins of the GI tract microbiome, such as lipopolysaccharide (LPS; [6,9,[15][16][17][18]); and (viii) that there is a decrease in richness and diversity of GI tract bacteria in AD compared to age-matched controls, a finding that parallels results observed in other GI, vascular or neurological conditions linked to GI tract microbiome alterations, including those associated with obesity, diabetes, inflammatory bowel disease, systemic inflammation and Parkinson's disease [9,[19][20][21][22]. Related to the points above, overall the GI tract microbiome of AD patients was found to exhibit hig...…”