Main results

Our results show a significantly altered microbial composition in early disease stage L-DOPA-naïve PD participants. Compared to earlier studies investigating microbiota in PD patients using 16S-based techniques the chosen methods allowed to detect changes at the species level and also a decreased virus load in PD.

We confirmed a decrease of Prevotella copri in PD and in addition found decreased Eubacterium biforme and Clostridium saccharolyticum and increased Akkermansia muciniphila as well as Alistipes shahii. Based on the taxonomic differences alone, logistic regression after feature selection allowed separation of PD patients in early stages from controls with good accuracy (AUC 0.84). Furthermore, our analyses point to differences in microbiota metabolism, namely in ẞ-glucuronate and tryptophan degrading pathways.

Taxa abundances

Despite several differences in study design compared to previous work [20, 21], our results strengthen the hypothesis of a PD-specific “microbial footprint.” The observed differences though are not directly comparable, since the previous studies were using 16S amplicon sequencing with its various biases [43], while we used metagenomics with increased precision due to usage of single copy marker genes with high confidence taxonomic assignments [27]. Testing for causality inference of biotic and abiotic factors seem to indicate that these taxonomic changes are a consequence of the disease. However, whether the observed changes are primary changes or rather secondary, resulting from unidentified effects, and whether it is either beneficial or harmful cannot be decided at present.

The increase in Akkermansia muciniphila, which appears to be in certain consistency in regards to the findings of Keshavarzian et al., Scheperjans et al., and Unger et al. [20,21,22], may illustrate this dilemma: it is a common mucin degrader which has been shown to reverse diet induced pathological intestinal changes in high-fat fed mice by restoring the intestinal mucus layer and the underlying epithelium, thus being able to improve gut barrier function [44, 45]. Protective effects of extracellular vesicles derived from Akkermansia muciniphila on experimentally induced colitis further support a beneficial influence on intestinal immunity [46]. On the other hand, inflammatory and regulatory properties have been reported for Akkermansia, probably mediated due to an increased exposure of immune cells to microbial antigens upon breaking down the mucosal mucin layer [47]. Preliminary evidence also linked Akkermansia to multiple sclerosis [48]. Previous studies on colonic biopsies and feces samples from treated and drug-naïve PD participants suggested an altered mucosal barrier function and PD patients exhibited significantly greater intestinal permeability than controls, paralleled by an increased mucosal staining for E. coli and α-syn [49]. Pro-inflammatory dysbiosis may even trigger α-syn misfolding or neuronal injury from gut-derived endotoxins [21, 50]. Although we did not identify E. coli species associated with PD in our samples, the increase in Akkermansia might be associated with a yet unexplored disease related impact on mucosal barrier function.

Extending the results of Scheperjans et al. and Unger et al. [20, 22], which pointed to a relatively lower abundance of Prevotellaceae in advanced PD, Prevotella copri was markedly lowered in our samples of early stage PD. On the other side, Keshavarzian et al. [21] did not show this difference for fecal samples, albeit the trend was the same for mucosal-derived PD samples. However, Prevotella abundance was also reduced in Japanese multiple sclerosis patients and in autistic children, somewhat questioning the specificity of this finding [51, 52]. The Prevotella enterotype is the least prevalent in human individuals [53] and is related to dietary/fiber intake [54, 55]. In particular, Prevotella enrichment has been linked to non-Western and/or fiber-rich diets [56, 57]. Fibers are the primary substrate for short chain fatty acids (SCFAs) including butyrate and reductions in the latter can disrupt barrier function and promote inflammation [58]. The fact that, in various autoimmune diseases including type 1/2 diabetes, irritable bowel disease, rheumatoid arthritis, and Behcet’s disease reduced levels of Prevotella have been found [42, 59,60,61], could indicate a decreased SCFA production (i.e. propionate) and in turn favor inflammatory conditions in PD [21].

In contrast to Scheperjans et al. [16], we did not find increased Ruminococcaceae (phylum Firmicutes) to compensate lower levels of Prevotella but instead an increase in unclassified Firmicutes. Interestingly, although the early, L-DOPA-naïve PD patients hold a different species pattern not yet affected by drug effects and the chronic constipation typically observed in late-stage PD, we found a certain consistency with the advanced PD patients’ pattern.

Taken together, the observed bacterial pattern in our PD samples might hint towards yet unexplored mechanisms of a disturbed intestinal and immune function in PD pathogenesis. Colonic biopsies from PD patients indeed showed enhanced pro-inflammatory cytokines and glial markers correlating with disease progression [62, 63]. Furthermore, there is evidence of α-syn contributing to neuro-inflammation by potentiating microglial or astroglial activation [64]. In line with this, recent work highlighted the crucial role of microbiota on maturation and activity of microglia [65], which have been considered as one of the earliest contributors of neurodegeneration [66] and further supports the importance of microbial-derived mediators (gut peptides, chemokines, SCFAs) on immune regulation and CNS function [65, 67].

A possible role for SCFAs in PD

SCFAs are essential energy sources for colonocytes and reduced levels of SCFAs might not only contribute to a decreased colonic motility (i.e. constipation) but also led to an increase in intestinal barrier leakiness [68,69,70]. Keshavarzian et al. and Unger et al. both suggested a beneficial role for SCFAs as PD-derived feces were shown to contain less SCFA butyrate-producing bacteria, including Blautia, Roseburia, and Coprococcus [21] as well as Faecalibacterium prausnitzii [21, 22], which were previously attributed to exert putative anti-inflammatory effects.

While SCFA administration contra-intuitively promoted motor dysfunction and α-syn-mediated neuro-inflammation in a germ-free transgenic mouse model over-expressing α-syn, oral administration of heat killed bacteria had no effect on motor performance, indicating the putative importance for metabolically active microbiota in disease pathogenesis [71]. Namely, when PD-derived microbiota (of treatment-naïve new onset PD donors) were orally transferred to germ-free mice, several taxa, including Roseburia, Rikenellaceae, and Enterococcus, were markedly altered in the microbial profile of the recipient mice independently of its genotype as when they received microbiota derived from healthy donors.

While inconclusive at the moment, prospective research on SFCA gene expression and metabolomic profiles of microbiota in health and disease will shed further light on this aspect.

Viral analyses

The gut bacteria harbor a diverse phageome and virome that may contribute to function and structure of the microbiome, but evidence from comparative analyses of the human gut phageome is limited. Recently a comprehensive metagenomic analysis in 64 individuals suggested a core phageome that was shared among more than one-half of all individuals and might also exert beneficial properties as it was reduced in individuals with inflammatory bowel disease [72]. However, based on our analysis, we could not find any differences in the abundance of prophages and plasmids between PD and control samples. In contrast, total virus amount was significantly lowered in PD participants.

Importantly, the assessment of virus and phage load is entirely dependent on the corresponding protein families being present in the ACLAME database; therefore, we only detect those phages or viruses, of which a closely related reference genome is present in the database. Testing for correlations between bacterial family and viral load abundance showed no significant correlations after multiple testing.

As viruses interact with host cells and influence immune response (i.e. prevent inflammatory conditions [73]), there might be various possibilities in which viruses interact in the pathogenesis of PD. Although inconclusive at the moment, exploration of the specific role of viruses in PD is a promising avenue to follow-up with more specific research.

Functional aspect

Accumulating evidence suggests a direct impact of metabolic alterations in microbiota on human health [74, 75]. We observed a putative reduction in microbiota ẞ-glucuronidase activity in early stage PD participants. Decreased ẞ-glucuronidation in the microbiota could imply a deterioration of resistance to various pathogenic organisms [76]. Also, microbial derived ẞ-glucuronidases affect effective dose availability of administered drugs by reactivation in the gut, which has been shown for irinotecan therapy in colorectal cancer patients [77]. Altered metabolisms of xenobiotics or parkinsonian pharmaceuticals metabolized in the ẞ-glucuronate degrading pathway must be determined experimentally [78].

Our data further revealed a trend towards an increased tryptophan degradation gene copy number in PD. If one assumes that this increased genetic potential translates into an increased tryptophan metabolization, this finding is in line with previous research and is of particular interest as L-tryptophan, the precursor for serotonin, is decreased in PD patients’ brains. L-tryptophan is also metabolized to kynurenines, whereof metabolites have regulatory immune function and were described as either harmful or beneficial in PD [79,80,81]. Urinary metabolomics profiling demonstrated significant changes of urinary markers including an increased tryptophan metabolism, which was associated with the progression of PD [82]. Interestingly, catabolism of serotonin also includes glucuronidation in the human intestine [83].

The association with these metabolic pathways point to a deeper involvement of Eubacteria with PD. Indeed Eubacteria spp. were decreased in PD (Eubacterium biforme) and other Eubacteria species (E. hallii, E. rectale, E. eligens) showed a trend towards correlation with disease severity (n.s.). Specifically colonizing the mucus layer, particularly Eubacterium rectale, might be interconnected with processes directly affecting the mucus layer due to its ability to gain access via flagella [84]. Further, diversity of Eubacterium rectale was also reduced in an in vitro dynamic gut model (M-SHIME) of long-term colonization of the mucin layer when microbiota were derived from ulcerative colitis patients [85]. Additionally, Eubacterium halii is viewed as a key species impacting the microbial balance due to its ability to produce several SCFAs [86]. In turn, alterations in the abundance of different Eubaceria might contribute to the PD pathogenesis via metabolic but also direct mucosal pathways.

Lowered Eubacteria (family Erysipelotrichaceae) in mucosal as well as in fecal PD samples were similarly observed in the study of Keshavarzian et al. [21]; however, a correlation with disease severity was not proven.

Clinical aspects

Instead, Keshavarzian et al. found PD duration correlating with the greatest number of taxa, whereby the family Lachnospiraceae, which includes several (supposedly anti-inflammatory) butyrate producing bacteria, displayed a significant negative correlation. Scheperjans et al. further showed a significant association of Enterobacteriaceae with the postural instability and gait disorder (PIGD) PD phenotype [20], which was not confirmed in the work of Unger et al. [22].

Namely, based on our analyses, the intake of different anti-parkinsonian drugs had no overall influence on taxa abundance or microbial functions. However, in future subgroup analyses PD patients under the therapy with MAO-inhibitors and amantadine might be favorably influenced by an increased richness if assessed in a lager cohort. In this context, it is worth noting another study, which was published during the revision process of this manuscript and which demonstrates instead independent effects of different PD medications on the microbiome [87].

However, although the intake of a statin showed an influence on the gut microbiota with, in total, five families being different in statin-treated individuals, none of them contributed to the differences observed between PD participants and controls when controlling for statin intake with differential statistical methods. One caveat of testing for confounders in our cohort is that this result might be limited by the sample size being too small to find even small effects, which is an unavoidable inherent aspect of human cohort studies. However, future studies should address this aspect.