This study investigated the composition of the gut virome in a cohort of Aβ + AD patients and HCs. The gut virome profiles of Aβ + ADs revealed reduced alpha diversity as compared to HCs, which suggested a lower bacteriophage richness. Our analyses showed that
Uroviricota was the most frequent phylum of
Caudovirales bacteriophages in both groups, and that was reduced by 9% in Aβ + ADs compared to HCs. The abundance of the family
Siphoviridae was reduced by 9% in Aβ + ADs compared to HCs, whereas the abundance of the family
Poxviridae was increased by 11%. A previous study discovered that
Caudovirales bacteriophages are associated with improved executive function and memory in flies, mice, and humans [
30]. The study demonstrated that the levels of various
Caudovirales, specifically the
Siphoviridae family, were negatively correlated with the Trail Making Test (TMT) score, where a longer time taken to complete the test is generally associated with poorer performance or cognitive impairment, whereas the levels of
Microviridae were positively correlated with the TMT score. Also, the same study discovered choline and glycine, the two most prominent sources of 1C units of folate, had the strongest relationships with
Microviridae and
Caudovirales levels [
30]. Choline blocks the production of amyloid-beta plaques [
43] and also choline supplementation reduces amyloidosis and increases choline acetyltransferase expression in the hippocampus of APPswePS1dE9 AD model mice [
44]. A significant correlation exists between the degree of choline acetyltransferase activity loss in cerebral cholinergic neurons and the severity of dementia or cognitive deficits reported in AD [
45]. Our LEfSe analysis revealed that Aβ + ADs significantly had a lower abundance of several
Lactococcus phages including
bIL285,
Lactococcus phage bIL286,
Lactococcus phage bIL309,
Lactococcus phage BK5 T,
Lactococcus phage BM13,
Lactococcus phage P335 sensu lato,
Lactococcus phage phiLC3,
Lactococcus phage r1t,
Lactococcus phage Tuc2009,
Lactococcus phage ul36, and
Lactococcus virus bIL67.
Lactococcus phages belong to the order
Caudovirales and have double-stranded DNA (dsDNA) genome. They are one of the most common phages that infect bacteria, particularly
Lactococcus species [
46]. Interaction between
Lactococcus phage and
Lactococcus bacterial species in AD should be examined, as bacteriophages regulate the diversity of
Lactococcus bacteria and modulate their metabolic pathways.
Lactococcus, a genus of lactic acid bacteria (LAB), is known for its production of lactic acid. Within humans, there exist two forms of lactic acid:
l-lactic acid and
d-lactic acid. While
l-lactic acid is a commonly found compound in human metabolism,
d-lactic acid is primarily produced by specific microorganism strains or less significant metabolic pathways. Despite their distinct structures, effects on the human body, and mechanisms of action, most studies do not differentiate between these two forms [
47‐
49]. The production of lactic acid occurs through diverse metabolic pathways.
l-lactic acid arises from the catabolism of amino acids and carbohydrates during glycolysis, whereas
d-lactic acid is generated from the metabolism of carbohydrates, lipids, and intestinal bacteria. Both
l-lactic acid and
d-lactic acid impact neural network activity by binding to the hydroxycarboxylic acid receptor 1.
l-lactic acid plays a crucial role in neural oxidative metabolism, contributing to memory formation, protein synthesis, synaptic remodeling, and axonal excitability. Conversely,
d-lactic acid can hinder the uptake of
l-lactic acid by neurons, resulting in inadequate neuronal energy metabolism and memory impairment [
50‐
53].
Memory function relies on lactic acid [
54,
55]. A previous study has shown that astrocytes in AD patients secrete less lactic acid, potentially contributing to the pathogenesis of the disease [
56]. Neuronal energy metabolism depends on lactic acid produced by astrocytes [
57,
58]. Lactic acid generated through glycolysis serves as an energy source for the brain and protects neurons from mitochondrial damage caused by Aβ protein accumulation [
53]. Lactic acid appears to play a bidirectional role in AD etiology. On the one hand, long-term memory requires lactic acid from astrocytes to neurons. On the other hand, the accumulation of lactic acid in the brain stimulates Aβ protein deposition and excessive transmission of lactic acid into neurons, leading to lower pH, mitochondrial dysfunction, apoptosis, and impaired brain function [
53‐
55]. Therefore, the regulation of gut bacteria and their lactic acid products by bacteriophage in AD requires further investigation. Our findings provide valuable insights into the role of gut dysbiosis in AD and align with a recent study that proposes a theoretical framework and hypothesis about the gut-brain axis and the role of gut microbiota in AD [
59].
This study has several limitations that should be considered. The SRA database provided limited information on the clinical characteristics of the subjects, which restricted our ability to analyze associations between the gut virome and clinical features. We acknowledge the limitation of not having access to data on APOE genotype, diet, and medication use such as antibiotic and antidepressant use, which could influence gut virome diversity in our study. We recommend that further studies focus on exploring the impact of these variables on the gut virome to gain a more comprehensive understanding. Moreover, applying propensity scores on all these variables could offer an approach to match profiles between the two compared groups. Additionally, the sample size was relatively small. In future studies, maintaining a sufficient sample size above the required confidence level is imperative, particularly when planning further analysis post-study design. To prevent the exclusion of samples based solely on low viral library size, rigorous quality controls should be implemented during sample collection and RNA extraction. This approach will address the problem of low library size and reduce the need for further sample exclusions, ultimately enhancing the scientific robustness of studies. It is important to note that this study had a cross-sectional design, making it difficult to establish a cause-and-effect relationship between the gut virome. Additionally, cerebrospinal fluid biomarkers were not available for HCs, and thus, the present study recognizes a potential limitation of including AB + individuals in the HC group, some of whom might have altered microbiomes. Although this may have led our study to underestimate the differences between AB + ADs and HCs, the reported significant differences are valid and informative. To gain deeper insights, we recommend conducting longitudinal studies on individuals with preclinical AD and monitoring the changes in their gut microbiomes over time. Also, our cross-sectional study faces challenges in determining the causality between diet, gut dysbiosis, and AD. The impact of memory loss in individuals with AD may further hinder their ability to maintain a healthy diet leading to potential issues with forgetfulness related to eating their food. For more accurate insights, future research should prioritize longitudinal or caregiver-observed studies to mitigate these issues and better understand the complex interactions involved. We employed a combined model of AUROC analysis to assess the predictive power of all discriminatory biomarkers identified as a cohesive set. We acknowledge that the combined model may be influenced by preselected viral factors that were significant in distinguishing the groups initially. This could lead to higher AUROC values in that analysis and to ensure the generalizability of our finding, we recommend further validation in an independent (confirmatory) cohort in future studies. Another limitation is that the study did not measure metabolites in the collected gut samples and incorporating metabolomic analyses would provide a more comprehensive understanding of the gut virome’s role in AD. Additionally, simultaneous investigation of the bacteriome and virome, along with exploring their interactions, could offer valuable insights into the underlying mechanisms of AD pathogenesis.