Differential abundance analyses of human microbiota in Parkinson’s disease

Parkinson's disease is the second most common neurodegenerative disease in the world, and in spite of decades of research, the cause of the non-familial form of the disease is not known. There are currently no medications to slow down the progression of the disease nor good biomarkers for early diagnosis, even though the earliest non-motor symptoms can appear years or even decades before the onset of motor symptoms. The microbial inhabitants of the human body have recently been implicated in various medical conditions, including neurodegenerative disorders. They could offer new insight into the pathogenesis of Parkinson's disease, particularly since a microbial agent has long been suspected to play a part in the process. A typical question in studies surveying human microbiota is which specific microbial taxa differ between groups of interest, such as patients with a disease and control subjects. There are many statistical tools for performing these analyses, also known as differential abundance comparisons. The aim of my doctoral thesis was to explore the potential associations of Parkinson's disease and human microbiota, particularly of the mouth, the nose and the gut, with an additional focus on the statistical tools used for comparing differentially abundant bacterial taxa. All four publications included in my thesis were based on samples from the same subjects: 76 patients with Parkinson's disease and 76 control subjects with no signs of parkinsonism. The studies also used the same methodology, 16S rRNA gene amplicon sequencing, to compare bacteria from oral and nasal swab samples and fecal samples of these subjects. Over the course of the four publications and in a previously unpublished analysis, I compared differentially abundant taxa with six tools: Metastats, LEfSe, metagenomeSeq, DESeq2, ANCOM, and an approach based on random forests. Our results suggested that the bacterial communities of the gut and the mouth differ between Parkinson's patients and control subjects, with statistically significant differences in beta diversity and in the abundances of several bacterial taxa. Differences in gut microbiota could also be detected at a follow-up time point with samples collected two years after the initial sampling. Additionally, there were differences between the gut bacteria of Parkinson's patients with and without irritable bowel syndrome -like symptoms. For nasal bacteria, there were no differences between the patient and control groups in diversity nor the amounts of specific bacteria. Regarding the differential abundance analyses, comparing gut bacteria of patients and controls from the same samples with six different tools highlighted the wide variation in the lists of significant results, which often did not overlap except for a handful of taxa. While a few benchmarking studies have previously contrasted some of the tools, there is a definite need for further standardized testing to guide researchers in choosing between them. Despite these discrepancies, all tools tested in this thesis supported Parkinson's patients having a decreased abundance of the family Prevotellaceae in their gut. This difference in abundance could also be detected at the follow-up time point. As several other research groups have reported seeing a decrease in Prevotellaceae after our pilot publication, it is emerging as one of the key changes in microbiota associated with Parkinson's disease.Parkinsonin tauti on maailman toiseksi yleisin hermostorappeumasairaus, ja vuosikymmenten tutkimustyöstä huolimatta taudin ei-perinnöllisen muodon syy on edelleen arvoitus. Toistaiseksi ei ole olemassa lääkitystä, joka hidastaisi taudin etenemistä, eikä hyviä keinoja taudin varhaiseen havaitsemiseen, vaikka ensimmäiset ei-motoriset oireet, esimerkiksi ummetus ja hajuaistin heikkeneminen, saattavat alkaa vuosia tai jopa vuosikymmeniä ennen taudille tunnusomaisia liikehäiriöitä. Ihmiskehon mikrobiasukkaat on viime aikoina yhdistetty moniin terveysongelmiin, neurologiset sairaudet mukaanlukien. Mikrobit voisivat tarjota uusia näkemyksiä Parkinsonin taudin syntyyn liittyen, etenkin kun niiden on jo pidempään arveltu vaikuttavan tautiprosessiin. Tyypillinen kysymys ihmisen mikrobistoa tutkittaessa on, minkä mikrobien määrät eroavat vertailtavien ryhmien, esimerkiksi potilaiden ja verrokkihenkilöiden välillä (englanniksi "differential abundance"). Näihin vertailuihin on olemassa monia eri työkaluja. Väitöskirjani tavoite oli selvittää Parkinsonin taudin ja ihmisen suun, nenän ja suoliston mikrobien mahdollisia yhteyksiä sekä tarkastella niiden määrien vertailuun käytettäviä työkaluja. Kaikki neljä julkaisuani perustuvat näytteisiin, jotka on kerätty samoilta koehenkilöiltä: 76 Parkinsonin tautia sairastavalta potilaalta sekä 76 verrokilta, joilla ei esiinny parkinsonismia. Julkaisuissa on käytetty samaa menetelmää, 16S rRNA-geenin amplikonisekvensointia, suu-, nenä- sekä ulostenäytteiden bakteerien määrittämiseen. Neljän julkaisun sekä ennen julkaisemattoman analyysin myötä vertailin ryhmien välillä poikkeavia bakteereja yhteensä kuudella eri työkalulla. Tulostemme perusteella Parkinsonin tautia sairastavien potilaiden ja verrokkien suoliston ja suun bakteeriyhteisöt poikkeavat toisistaan; ryhmien välillä oli tilastollisesti merkitsevä ero bakteeriyhteisöjen koostumuksessa sekä useiden bakteerien määrissä. Suolistobakteeriyhteisöjen väliset erot voitiin havaita myös seurantanäytteissä, jotka oli kerätty samoilta koehenkilöiltä kaksi vuotta ensimmäisen näytteenoton jälkeen. Lisäksi havaitsimme eroja suolistomikrobistossa, kun potilaat luokiteltiin sen mukaan, oliko heillä ärtyvän suolen oireyhtymää muistuttavia oireita vai ei. Nenän bakteerien osalta emme löytäneet eroja potilaiden ja verrokkien väliltä. Mitä tulee bakteerien määrien vertailuihin, suolistomikrobiston analyysit kuudella eri työkalulla korostivat eroja niiden antamien merkitsevästi eroavien bakteerien listoissa. Muutamat aiemmat julkaisut ovat vertailleet osaa näistä työkaluista, mutta olisi tarpeen tehdä laajempia menetelmävertailuja, jotta tutkijoiden olisi helpompi valita, mitä työkalua käyttää. Eroavaisuuksistaan huolimatta kaikki testaamani työkalut tukivat havaintoa, että Parkinsonin tautia sairastavilla potilailla on suolistossaan vähemmän Prevotellaceae-heimon bakteereja kuin verrokeilla. Koska tämä eroavuus on ensimmäisen artikkelimme jälkeen havaittu myös muiden tutkimusryhmien julkaisuissa, se on nousemassa yhdeksi keskeisistä Parkinsonin tautiin liittyvistä mikrobiston muutoksista

Bile microbiota in primary sclerosing cholangitis : Impact on disease progression and development of biliary dysplasia

Objective The etiopathogenesis and risk for development of biliary neoplasia in primary sclerosing cholangitis (PSC) are largely unknown. Microbes or their metabolites have been suggested to play a role. To explore this potential microbial involvement, we evaluated the differences in biliary microbiota in PSC patients at an early disease stage without previous endoscopic retrograde cholangiography (ERC) examinations, advanced disease stage, and with biliary dysplasia or cholangiocarcinoma. Design Bile samples from the common bile duct were collected from 46 controls and 80 patients with PSC during ERC (37 with early disease, 32 with advanced disease, and 11 with biliary dysplasia). DNA isolation, amplification, and Illumina MiSeq sequencing were performed for the V1-V3 regions of the bacterial 16S rRNA gene. Results The most common phyla found were Bacteroidetes, Firmicutes, Proteobacteria, Fusobacteria, and Actinobacteria. The most common families were Prevotellaceae, Streptococcaceae, Veillonellaceae, Fusobacteriaceae, and Pasteurellaceae, and the most common genera were Prevotella, Streptococcus, Veillonella, Fusobacterium, and Haemophilus. The bacterial communities of non-PSC subjects and early stage PSC patients were similar. Alpha diversity was lower in patients with biliary dysplasia/cholangiocarcinoma than in other groups. An increase in Streptococcus abundance was positively correlated with the number of ERC examinations. Streptococcus abundance was also positively correlated with an increase in disease severity, even after controlling for the number of ERC examinations. Conclusions Our findings suggest that the aetiology of PSC is not associated with changes in bile microbial communities, but the genus Streptococcus may play a pathogenic role in the progression of the disease.Peer reviewe

Gut microbiome in gestational diabetes : a cross-sectional study of mothers and offspring 5 years postpartum

Introduction An altered gut microbiome composition is shown to be associated with various diseases and health outcomes. We compare the gut microbiota of women who developed gestational diabetes mellitus (GDM) with that of those who did not, and the gut microbiota of their offspring, to determine any differences in the composition and diversity of their gut microbiota, which may be correlated with their GDM state. Material and methods All women were at high risk for GDM and participated in the Finnish Gestational Diabetes Prevention Study (RADIEL). Stool samples were obtained, 5years postpartum, from 60 GDM-positive women, 68 non-GDM control women, and their children (n=109), 237 individuals in total. 16S ribosomal RNA gene sequencing was employed to determine the composition of bacterial communities present. Statistical correlations were inferred between clinical variables and microbiota, while taking into account potential confounders. Results In mothers, no significant differences were observed in microbiota composition between the two groups. Genus Anaerotruncus was increased in children of women with GDM (pPeer reviewe

Skin Microbiome in Small- and Large-plaque Parapsoriasis

Staphylococcal enterotoxins have been shown to promote lymphoma-associated immune dysregulation. This study examined changes in the skin microbiome of parapsoriasis compared with intact skin. Swab microbiome specimens were taken of the parapsoriasis lesions of 13 patients. Control samples were taken from contralateral healthy sides of the body. Micro-biotas were characterized by sequencing the V1-V3 region of the 16S ribosomal RNA bacterial genes on the Illumina MiSeq platform. The most common genera in the microbiome data were Propionibacterium (27.13%), Corynebacterium (21.20%) and Staphylococcus (4.63%). Out of the Staphylococcus sequences, 39.6% represented S. epidermidis, with the rest including S. hominis, S. capitis and unidentified species. No significant differences were observed between the patients' parapsoriasis and contralateral healthy skin or between large-and small-plaque parapsoriasis. Notable interpersonal variation was demonstrated. These results suggest that parapsoriasis is not associated with significant alterations in the cutaneous bacterial microbiome.Peer reviewe

Gut microbiota in Parkinson's disease : Temporal stability and relations to disease progression

Background: Several publications have described differences in cross-sectional comparisons of gut microbiota between patients with Parkinson's disease and control subjects, with considerable variability of the reported differentially abundant taxa. The temporal stability of such microbiota alterations and their relationship to disease progression have not been previously studied with a high-throughput sequencing based approach. Methods: We collected clinical data and stool samples from 64 Parkinson's patients and 64 control subjects twice, on average 2.25 years apart. Disease progression was evaluated based on changes in Unified Parkinson's Disease Rating Scale and Levodopa Equivalent Dose, and microbiota were characterized with 16S rRNA gene amplicon sequencing. Findings: We compared patients to controls, and patients with stable disease to those with faster progression. There were significant differences between microbial communities of patients and controls when corrected for confounders, but not between timepoints. Specific bacterial taxa that differed between patients and controls at both timepoints included several previously reported ones, such as Roseburia, Prevotella and Bifidobacterium. In progression comparisons, differentially abundant taxa were inconsistent across methods and timepoints, but there was some support for a different distribution of enterotypes and a decreased abundance of Prevotella in faster-progressing patients. Interpretation: The previously detected gut microbiota differences between Parkinson's patients and controls persisted after 2 years. While we found some evidence for a connection between microbiota and disease progression, a longer follow-up period is required to confirm these findings. (C) 2019 The Authors. Published by Elsevier B.V.Peer reviewe

Increasing Comparability and Utility of Gut Microbiome Studies in Parkinson's Disease : A Systematic Review

Gut microbiota have been studied in relation to the pathophysiology of Parkinson's disease (PD) due to the early gastrointestinal symptomatology and presence of alpha-synuclein pathology in the enteric nervous system, hypothesized to ascend via the vagal nerve to the central nervous system. Accordingly, sixteen human case-control studies have published gut microbiome composition changes in PD and reported over 100 differentially abundant taxa covering all taxonomic levels from phylum to genus or species, depending on methodology. While certain findings were replicated across several studies, various contradictory findings were reported. Here, differences in methodologies and the presence of possible confounders in the study populations are assessed for their potential to confound the results of gut microbiome studies in PD. Gut microbiome studies in PD exhibited considerable variability with respect to the study population, sample transport conditions, laboratory protocols and sequencing, bioinformatics pipelines, and biostatistical methods. To move from the current heterogeneous dataset towards clinically relevant biomarkers and the identification of putative therapeutic targets, recommendations are derived from the limitations of the available studies to increase the future comparability of microbiome studies in PD. In addition, integration of currently available data on the gut microbiome in PD is proposed to identify robust gut microbiome profiles in PD. Furthermore, expansion of the current dataset with atypical parkinsonism cohorts, prodromal and treatment naive de novo PD subjects, measurements of fecal microbial concentrations and multi-omics assessments are required to provide clinically relevant biomarkers and reveal therapeutic targets within the gut microbiome of PD.Peer reviewe

Gut microbiome is not associated with mild cognitive impairment in Parkinson's disease

Gut microbiome differences between people with Parkinson's disease (PD) and control subjects without Parkinsonism are widely reported, but potential alterations related to PD with mild cognitive impairment (MCI) have yet to be comprehensively explored. We compared gut microbial features of PD with MCI (n = 58) to cognitively unimpaired PD (n = 60) and control subjects (n = 90) with normal cognition. Our results did not support a specific microbiome signature related to MCI in PD

Oral and nasal microbiota in Parkinson's disease

Introduction: Parkinson's disease (PD) is associated with neuropathological changes in olfactory and gastrointestinal tissues, and PD patients frequently suffer from hyposmia, hyposalivation, and dysphagia. Since hyposmia and gastrointestinal dysfunction are frequently premotor symptoms, it has been speculated that an external, for example microbial, agent could trigger the pathologic process in the corresponding organs, subsequently spreading to the central nervous system. We recently showed evidence for compositional differences between the fecal microbiota of PD patients and control subjects. In this study, our objective was to explore a possible connection between nasal and oral microbiota and PD. Methods: We compared the oral and nasal bacterial communities of PD patients (oral: n = 72, nasal: n = 69) and control subjects (oral: n = 76, nasal: n = 67) using a 16S rRNA gene amplicon sequencing approach. Results: Oral and nasal microbiota differed markedly from each other, with no notable similarity within subjects. Oral microbiota of PD patients and control subjects had differences in beta diversity and abundances of individual bacterial taxa. An increase in the abundance of opportunistic oral pathogens was detected in males, both with and without PD. Our data did not reveal convincing differences between the nasal microbiota of control subjects and PD patients. Conclusion: The oral microbiome deserves additional research regarding its connection to PD and its biomarker potential. The higher abundance of oral pathogens in men underlines the importance of monitoring and promoting male dental health. (C) 2017 Elsevier Ltd. All rights reserved.Peer reviewe

The gut microbiome molecular complex in human health and disease

The human gut microbiome produces a functional complex of biomolecules, including nucleic acids, (poly) peptides, structural molecules, and metabolites. This impacts human physiology in multiple ways, especially by triggering inflammatory pathways in disease. At present, much remains to be learned about the identity of key effectors and their causal roles