Vol.:(0123456789)1 3 Acta Neurologica Belgica (2023) 123:999–1009 https://doi.org/10.1007/s13760-023-02195-0 ORIGINAL ARTICLE Altered gut microbiota in patients with idiopathic Parkinson’s disease: an age–sex matched case–control study Gulsen Babacan Yildiz1  · Zeynep Cigdem Kayacan2  · Ilker Karacan3  · Bilge Sumbul4  · Birsen Elibol5  · Ozlem Gelisin1  · Ozer Akgul2,6 Received: 17 August 2022 / Accepted: 18 January 2023 / Published online: 31 January 2023 © The Author(s) under exclusive licence to Belgian Neurological Society 2023 Abstract Objective The investigations related to how gut microbiota changes the brain–gut axis in idiopathic Parkinson’s disease (PD) attract growing interest. We aimed to determine whether gut microbiota is altered in PD patients and whether non-motor symptoms of PD and disease duration had any relation with alterations of microbiota profiles among patients. Methods Microbial taxa in stool samples obtained from 84 subjects (42-PD patients and 42-healthy spouses) were analyzed using 16S rRNA amplicon-sequencing. Results We observed a significant decrease of Firmicutes and a significant increase of Verrucomicrobiota at the phylum level. At the family level, Lactobacillaceae and Akkermansiaceae were significantly increased and Coriobacteriales Incertae Sedis were significantly decreased in the PD patients compared to their healthy spouses. Genus level comparison inferred significant increase in abundance only in Lactobacillus while the abundance of Lachnospiraceae ND3007 group, Tyzzerella, Fusicatenibacter, Eubacterium hallii group and Ruminococcus gauvreauii group were all decreased. We determined that the abundance of Prevotella genus decreased, but not significantly in PD patients. In addition, we found differences in microbiota composition between patients with and without non-motor symptoms. Conclusion We observed differences in gut microbiota composition between PD patients and their healthy spouses. Our find- ings suggest that disease duration influenced microbiota composition, which in turn influenced development of non-motor symptoms in PD. This study is the first in terms of both gut microbiota research in Turkish PD patients and the probable effect of microbiota on non-motor symptoms of PD. Keywords Parkinson’s disease · Gut microbiota · Non-motor symptoms · 16S rRNA sequencing * Gulsen Babacan Yildiz drgbabacan@gmail.com Zeynep Cigdem Kayacan cigdem.kayacan@istun.edu.tr Ilker Karacan karacan@artibiyoteknoloji.com.tr Bilge Sumbul bilgesumbul@hotmail.com Birsen Elibol elibolbirsen@gmail.com Ozlem Gelisin ozlemgelisin@yahoo.com Ozer Akgul ozerakgul@aydin.edu.tr 1 Department of Neurology, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey 2 Department of Medical Microbiology, Faculty of Medicine, Istanbul Health and Technology University, Istanbul, Turkey 3 Science and Advanced Technologies Research Center, Molecular Biology and Genetics, Faculty of Engineering and Natural Sciences, Istanbul Medeniyet University, Istanbul, Turkey 4 Department of Medical Microbiology, Faculty of Medicine, Bezmialem Vakif University, Istanbul, Turkey 5 Department of Medical Biology, Faculty of Medicine, Istanbul Medeniyet University, Istanbul, Turkey 6 Department of Medical Microbiology, Faculty of Medicine, Istanbul Aydin University, Istanbul, Turkey http://orcid.org/0000-0003-0922-0969 http://orcid.org/0000-0002-2727-7048 http://orcid.org/0000-0003-3100-0866 http://orcid.org/0000-0002-8768-3777 http://orcid.org/0000-0002-9462-0862 http://orcid.org/0000-0001-7872-7611 http://orcid.org/0000-0002-3802-3270 http://crossmark.crossref.org/dialog/?doi=10.1007/s13760-023-02195-0&domain=pdf 1000 Acta Neurologica Belgica (2023) 123:999–1009 1 3 Introduction Idiopathic Parkinson’s disease (PD) is a chronic progres- sive neurodegenerative disease that causes characteristic motor symptoms of tremor, bradykinesia, and postural instability and it is neuropathologically characterized by the inclusions of Lewy bodies and Lewy neurites contain- ing α-synuclein (α-syn) in the substantia nigra pars com- pacta (SNpc) [1]. These inclusion bodies have also been shown in the nervous system that innervates the autonomic and gastrointestinal systems (GIS), and adrenal medulla [2]. However, the etiopathogenesis has not been fully elu- cidated, although many genetic and environmental factors are involved in the etiology of PD and nearly two centuries have passed over the disease definition. Besides motor symptoms, PD patients have non-motor symptoms (NMS), particularly in GIS, including impaired gastric emptying, sialorrhea, dysphagia, nausea, abdomi- nal bloating, and constipation. Eventually, these symptoms often precede motor symptoms by several years [3–5]. For instance, some researchers found the correlation that an increase in the frequency of bowel movements shortens the lifetime in PD patients [5]. This early involvement of GIS in PD gave rise to several theories that the gut could be the key organ in the initiation of the disease, either via neural, immune, endocrine, or metabolic pathways [6]. Therefore, there is increasing interest in investigating how gut micro- biota changes the brain–gut axis in PD [5, 7–9]. In the past decades, a new participant called micro- biota has arisen as a key manipulator of the brain–gut axis. The number of microorganisms inhabiting the GIS has been estimated to exceed 1014 which encompasses ∼ 1–3 times more microbial cells than the number of human cells [10] while the amount of microbial genomic con- tent is more than a 100-fold of the human genome [11]. The GIS microbiota is mainly composed of bacteria vary- ing in diversity and stability among individuals and they assist in various aspects of human health comprising fiber breakdown, vitamin production, metabolic regulation, and modulation of neurological functions [11, 12]. The exist- ing function of the gut microbiota in GIS pathogenesis and the bi-directional connection between gut and brain in PD has welcomed many interests [13]. Some studies pointed out the relation between intestinal inflammation or altera- tions in the gut microbiota and the initiation or progression of PD pathology [7]. Hence, numerous researches have highlighted the idea that unusual alterations in distribution of the gut microbiota may correspond to the PD pathogen- esis. For instance, a case–control study reported a higher abundance of Bacteroidetes, Proteobacteria, and Verru- comicrobia at the phylum level in fecal samples of the PD patients [8]. Another cohort study showed a decreasing Prevotellaceae abundance and an increasing Enterobac- teriaceae abundance in the PD patients, as compared to healthy individuals [9]. Further research is needed to make clear how alterations in gut microbiota may control both motor and non-motor symptoms in PD, as well as, its comorbidities. In the present study, we aimed to investigate whether the gut microbiome of the Turkish PD patients differs from that of their healthy spouses, who share the same living condi- tions with them, in terms of bacterial diversity or taxonomic composition and the relationships between non-motor symp- toms, disease duration and microbiota in the Turkish PD patients. Methods Study design and participants Consecutive 42 PD patients were recruited from Bezmi- alem Vakif University, Faculty of Medicine, Neurology out-patient clinic as the study group and their 42 otherwise- healthy spouses as the control group. This study protocol was approved by the Bezmialem Vakif University Research Ethics Committee (12/27, 07.06.2017). At the screening visit, each participant was informed about the purpose of the study, and informed consent was obtained from all participants. Patients were diagnosed with idiopathic PD according to the UK Brain Bank criteria by a movement disorder special- ist, and the original Hoehn + Yahr scale (H&Y) was followed for disease staging. However, since almost all patients were in H&Y stage 2 or 3 and the first 5 years are important in terms of the spread of the Lewy pathology, the development of motor complications and the complete avoidance of the Parkinson-plus syndromes [14], we compared them accord- ing to the duration of the disease (< 5 years or ≥ 5 years) after applying the Unified Parkinson’s Disease Rating Scale (UPDRS). Mini Mental State Examination (MMSE) was administered to all patients for cognitive evaluation. Screen- ing for depression and anxiety symptoms was performed using the Beck Depression Inventory and Beck Anxiety Inventory. The exclusion criteria for patients and their healthy spouses were as follows: (1) the use of probiotics, antibiotics, immunosuppressive agents or laxative agents in the last three months prior to sample collection, (2) the presence of inflammatory gastrointestinal disease, hemato- logical or autoimmune disorders, (3) the different dietary habits (vegetarian, vegan, ketogenic diets, gluten-free, inter- mittent fasting, low-fat diets, etc.) other than familiar Turk- ish food cuisine. Demographic and clinical data including age, gender, education level, age at onset of the PD symp- toms (< 65 years or ≥ 65 years), PD duration, presence of 1001Acta Neurologica Belgica (2023) 123:999–1009 1 3 restless legs syndrome (RLS), rapid eye movement sleep behavior disorder (RBD), anosmia, constipation, concomi- tant diseases and pattern of PD medication (levodopa, dopa- mine agonists, rasagiline, amantadine) were recorded. The spouses were healthy individuals without a history of neuro- degenerative disease and regular drug use. Constipation was assessed using the Rome III criteria and signs of RBD were analyzed using the REM Sleep Behavior Disorder Screening Questionnaire (RBDSQ) [15, 16]. During the experiments, the STARD-2015 reporting guidelines were used. Microbiota analysis Stool samples were collected and immediately frozen to store at − 80 °C until extraction. DNA extraction was per- formed from 200 mg of stool sample using QIAamp Pow- erFecal DNA Kit (Qiagen). Extracted and quantified DNA was stored at − 20 °C until further analysis. Fecal microbiota analysis was performed using 16S rRNA amplicon sequencing. Briefly, DNA samples were first amplified using indexed fusion primers (Bakt_341F: 5′-CCT ACG GGNGGC WGC AG-3′ and Bakt_805R: 5′-GAC TAC HVGGG TAT CTA ATC C-3′) targeting the hypervariable V3-V4 region of the bacterial 16S rRNA gene. PCR ampli- fications were performed using Phusion High-Fidelity DNA Polymerase. Successful amplification was verified by run- ning and visualization of the bands on 2% agarose gel. Gen- erated amplicons were cleaned and normalized using Invitro- gen SequalPrep 96-well Plate Kit, then pooled in equimolar ratio and sequenced using a MiSeq instrument (Illumina, USA) to generate 300 bp paired-end reads. Demultiplexing of the raw data was performed by CASAVA data analysis software (Illumina, USA). The fragments with any mis- matches to the assigned barcodes or primer sequences were excluded. Further amplicon sequence data analysis was performed using QIIME2 (v2021.2) [17]. Briefly, reads were merged, and quality filtered using ‘vsearch join-pairs’ and ‘quality-filter q-score-joined’ commands. Sequences were then denoised using ‘deblur’ [18], and taxonomy was assigned to each amplicon sequence variant (ASV) using ‘feature-classifier classify-sklearn’ plugin against the SILVA v138 (silva-138-99-nb-classifier.qza). Feature table was rare- fied to the minimum read sampling depth and used for alpha diversity analyses. Statistical analysis MaAsLin2 (Microbiome Multivariable Associations with Linear Models) was used to find associations between the microbial features and clinical variables [19]. Statistically, significant test results were determined using a p-value threshold of 0.05 and a q-value threshold of 0.25. No rarefaction was performed on feature table for use in MaAs- Lin2 analyses since this tool already includes normalization steps. Results Demographic and clinical data profiles of the patients A total of 42 patients with PD (23 male and 19 female) and their 42 healthy spouses participated in the study accord- ing to the power analysis. There was no significant differ- ence between the patients and their spouses regarding age. The demographic and clinical characteristics of both PD patients and their healthy spouses are presented in Table 1. All patients were under antiparkinsonian medication with levodopa (85.7%), dopamine agonists (71.4%), rasagiline (54.7%) or amantadine (16.6%). The percentage of patients using antidepressants was 64.2% and antipsychotics 16.6%. No patients were treated with apomorphine. Concomitant diseases accompanying PD are listed in Table 1. Since most of the patients were in stage 2 on H&Y scale, they were differentiated according to those with a disease duration of less (54.55%) or more than 5 years (45.45%). The range for disease duration was 2–19 years. The rates of non-motor symptoms were as follows: constipation (54.7%), anos- mia (52.3%), RLS (40.4%) and RBD (64.2%). Most of the spouses did not have any chronic disease known to directly influence the gut microbiota. They had hypertension, diabe- tes mellitus and hyperlipidemia (Table 1). Alterations of microbiota profiles of patients The relative abundance of phylum Firmicutes was signifi- cantly decreased while the relative abundance of phylum Verrucomicrobia was significantly increased in the patient group compared to that of the healthy spouses (Table 2, Fig. 1A). Diff-score from paired analysis demonstrated that Firmicutes were also decreased within the PD patient group per couple (Fig. 1B). At the family-level, the abundance of Lactobacillaceae and Akkermansiaceae were found to be increased whereas the abundance of Coriobacteriales Incertae Sedis was decreased in the PD patients compared to control group (Table 2). Genus level comparison of PD patients and healthy spouses inferred an increase in the abundance of Lactoba- cillus but a decrease in the abundance of Lachnospiraceae ND3007 group, Tyzzerella, Fusicatenibacter, Eubacterium hallii group, and Ruminococcus gauvreauii group (Table 2). There were no statistical differences in terms of alpha diversity metrics namely Faith’s PD, observed features and 1002 Acta Neurologica Belgica (2023) 123:999–1009 1 3 Shannon index between healthy spouses and patient groups (Fig. 1C). Possible correlations between statistically significant microbiota changes and some clinical findings of PD patients are stated in Table 3. Patients with a PD diagnosis of older than five years had a decreased levels of Phylum Cyanobacteria, contrary to those diagnosed within the last five years. In the PD patients with constipation, Rumino- coccus gnavus group was increased in comparison to those without it. The levels of Proteobacteria, Gammaproteobac- teria, Bacilli, and Atopobiaceae were all increased in the anosmic PD patients. RBD correlated with a decrease in the abundance of Dorea, Eubacterium ventriosum group, Rumi- nococcus gauvreauii group, Faecalibacterium, Clostridium sensu stricto 1, CAG.56, Fusicatenibacter, Lachnospiraceae ND3007 group, and Peptococcus; but increase in the abun- dance of Lactobacillus and Acidaminococcus (Fig. 2). Discussion To the best of our knowledge, this study is the first to analyze the alterations of gut microbiota in the Turkish PD patients in comparison to those of their age- and sex-matched healthy spouses who share with them the same living conditions. The six genera, three families, five orders, three classes and two phyla were altered in our PD patients compared to their healthy spouses. Consistent with the findings from previous studies, our PD patients showed changes in gut microbiota distribution, such as a significant decrease in the abundance of Firmicutes [8, 20, 21] and a significant increase in the abundance of Verrucomicrobiota [8, 20, 22–24] at the phy- lum level. At the family level, a significant decrease in the abundance of Lachnospiraceae and a significant increase in the abundance of Lactobacillaceae together with Acid- aminococcaceae were shown in Table 2. These results are Table 1 Sociodemographic and clinical features of participants PD Parkinson’s disease, HC healthy control Variables PD group (n = 42) HC group (n = 42) p value Age Mean ± SD 60.62 ± 9.31 58.33 ± 9.61 0.865 Gender n (%) Female 20 (47.62%) 24 (57.14%) 0.512 Male 22 (52.38%) 18 (42.86%) Education (year) median 7.2 NA PD onset < 65 years 61.9% NA H&Y stage mean (min–max) 2 (1–3) NA Mean disease duration (range is 2–19 years) ≥ 5 years 45.45% NA < 5 years 54.55% NA  Constipation 54.7% NA Anosmia 52.3% NA  REM sleep behavior disorder (RBD) 64.2% NA Restless leg syndrome (RLS) 40.4% NA  PD medication Levodopa 85.7% NA Dopamine agonist 71.4% NA  Rasagiline 54.7% NA  Amantadine 16.6% NA  Antidepressant 64.2% NA  Antipsychotic 16.6% NA  Concomitant disease (n) Hypertension 8 12 NA Diabetes mellitus 2 4 NA Hyperlipidemia 3 2 NA  Hypothyroidism 3 1 NA  Lumbar disc herniation 3 0 NA  Cervical disc herniation 2 0 NA  Depression 0 2 NA  Vertigo 0 1 NA  1003Acta Neurologica Belgica (2023) 123:999–1009 1 3 also similar to those of previous studies, which reported a decrease in the abundance of Lachnospiraceae [21, 25, 26] and an increase in the abundance of Lactobacillaceae [9, 24, 27] and Acidaminococcaceae in PD [23]. These alterations in the gut microbiota composition lead- ing to intestinal permeability may be associated with dysbio- sis [28, 29]. The occurrence of gut dysbiosis in PD patients can promote systemic inflammation and a pro-inflammatory reaction, resulting in misfolding of α-Syn and contributing to the development of PD [8]. Family Akkermansiaceae was found to be significantly higher in the PD patients than that of the controls consistent with previous studies [8, 23, 24, 30, 31]. It is thought that Akkermansia increases intestinal permeability by disrupting the mucus layer and, as a result, damages the neural plexus in the intestines [32, 33]. In line with almost all previous studies [7, 9, 27, 31], we determined that the abundance of Prevotella genus decreased, but not significantly (p = 0.057), in our PD patients compared to healthy spouses. Prevotellaceae, which is involved in mucin degradation of the gut mucosal layer, is accompanied by a reduction in fecal short-chain fatty acid (SCFA) concentrations [34]. The most important properties of the reduction in the concentration of SCFAs are reported as an increase in gut permeability, which causes local inflammation in PD patients [35, 36]. Ghrelin is an important hormone that has positive effects on dopamin- ergic neurons in SNpc. However, a low level of Prevotella decreases the amount of ghrelin and may indirectly damage the SNpc [37]. Anosmia, constipation, RBD, and RLS are non-motor symptoms of PD which are now known to start even dec- ades prior to the motor manifestations [38, 39]. We aimed to identify whether these symptoms had any relation with the alterations of the microbiota profile among our PD patients. About half of our patients (52.3%) had anosmia. Since we did not include hyposmic PD patients, this prevalence was lower than that reported in many studies [38, 40]. When compared among patients, Proteobacteria, Gammaproteo- bacteria, Bacilli, and Atopobiaceae were found to be sig- nificantly more abundant in the PD patients with anosmia compared to those without it (Table 3). In fact, it is more accurate to interpret this as follows: Consistent with previous studies [21, 24], all patients have an increase in the abun- dance of Proteobacteria, Gammaproteobacteria and Bacilli. However, this difference is highly significant (p = 0.0147, p = 0.0162, and p = 0.01, respectively) in those with anos- mia. To our knowledge, this is the first study to represent the idea that four particular taxa might be related to the anosmic condition in PD patients, either as the reason or the result. In previous studies, it was shown that the severity of constipation in PD patients may be directly related to the abundance of specific bacterial families [5, 9]. Compared to the PD patients without constipation, we found a significant increase in the abundance of Ruminococcus gnavus group in the constipated PD patients (p = 0.0008). Ruminococcus Table 2 Significantly different taxa in relative abundances between PD patients and healthy controls Level Taxa Relative abundances (%) Change in PD p value q value PD mean ± SD Spouse mean ± SD Phylum Verrucomicrobiota 6.544 ± 10.95 3.251 ± 6.66 ↑     0.0064 0.0642 Firmicutes 67.386 ± 19.22 77.456 ± 14.08 ↓  0.0131 0.0657 Class Lentisphaeria 0.0079 ± 0.0219 0.0004 ± 0.0023 ↑ 0.0127 0.1078 Verrucomicrobiae 6.5354 ± 10.7424 3.2499 ± 6.6724 ↑ 0.0073 0.1078 Bacilli 6.3854 ± 9.1348 3.9423 ± 6.3128 ↑ 0.0468 0.1713 Order Lactobacillales 4.0427 ± 7.6210 1.8365 ± 6.3395 ↑ 0.0071 0.1566 Victivallales 0.0079 ± 0.0219 0.0004 ± 0.0023 ↑ 0.0127 0.1566 Verrucomicrobiales 6.5292 ± 10.7458 3.2493 ± 6.6720 ↑ 0.0099 0.1566 Lachnospirales 17.5808 ± 10.5063 26.3596 ± 14.9313 ↓ 0.0245 0.1815 Acidaminococcales 0.5661 ± 1.4808 0.1474 ± 0.4827 ↑ 0.0245 0.1815 Family Lactobacillaceae 2.2445 ± 4.9948 1.2024 ± 5.8363 ↑ 0.0017 0.1150 Coriobacteriales Incertae Sedis 0.0200 ± 0.0372 0.0907 ± 0.1679 ↓ 0.0075 0.2243 Akkermansiaceae 6.5292 ± 10.7458 3.2493 ± 6.6720 ↑ 0.0099 0.2243 Genus Lachnospiraceae_ND3007_group 0.0750 ± 0.1880 0.1797 ± 0.2179 ↓ 0.0004 0.0661 Lactobacillus 2.2398 ± 4.9896 1.1690 ± 5.6219 ↑ 0.0016 0.1301 Tyzzerella 0.0106 ± 0.0874 0.0595 ± 0.1083 ↓ 0.0021 0.1301 Fusicatenibacter 0.7604 ± 0.8622 2.2250 ± 4.4155 ↓ 0.0042 0.1815 Eubacterium_hallii group 0.5516 ± 0.8862 0.9460 ± 1.0729 ↓ 0.0049 0.1815 Ruminococcus gauvreauii_group 0.1548 ± 0.3275 0.2010 ± 0.2044 ↓ 0.0065 0.2015 1004 Acta Neurologica Belgica (2023) 123:999–1009 1 3 gnavus group, which belongs to the Firmicutes phylum, was found to be altered with an increase abundance in the PD patients in a previous study [41] and a positive association has been reported between Ruminococcaceae and consti- pation [42]. Ruminococcus gnavus group has been mostly associated with inflammatory bowel diseases (IBD) and has been reported to degrade mucins and cause the release of some inflammatory substances [43]. We found significant decreases of nine genera and signifi- cant increases of two genera in the PD patients with RBD Fig. 1 Relative bacterial abundances of the taxa with a mean abundance of 1% among all samples (A) and Diff scores per taxa comparing Par- kinson’s disease (PD) patients and their healthy spouses (B). Alpha diversity comparisons of gut microbiota samples between groups (C) Fig. 2 Genus found to be significantly changed in both Parkinson’s disease affection status and REM sleep behavior disorder (RBD) 1005Acta Neurologica Belgica (2023) 123:999–1009 1 3 compared to those without it (Table 3). Consistent with the findings reported by Heintz-Buschart, we observed a sig- nificant increase in the abundance of Acidaminococcus, and a significant reduction in the abundance of genus Faecali- bacterium in the PD patients with RBD [30]. A nonsignifi- cant decrease in the abundance of genus Faecalibacterium was previously reported in the RBD patients compared to healthy controls [33]. Nishiwaki et al. proposed that dur- ing PD development, reduction of genus Faecalibacterium among SCFA-producing bacteria, comes first and a decrease of genus Faecalibacterium may therefore be an indicator to predict the transition from RBD to PD [33]. However, the role of reduction of SCFA-producing bacteria in the devel- opment of PD remains unclear [31]. We researched whether disease duration impacts microbi- ota profile or vice versa and we found a significant decrease in the abundance of Cyanobacteria in our patients with PD duration of more than five years. The study by Vascellaria [44] also showed significantly decreased Cyanobacteria in PD patients regardless of disease duration. There are many studies showing that the duration of the disease causes changes in the gut microbiota in PD [8, 29]. However, there is no previous study that decreased Cyanobacteria were found to be associated with disease duration. In addition, a considerable decrease was found in the Firmicutes at the family level, which did not reach the statistically significant level. It is known that patients with PD had an increased inci- dence of RLS [45]. Around 40% of our PD patients had RLS and they did not demonstrate significantly altered microbiota compositions, in comparison to those without it. However, some changes that did not reach the statistically significant level such as an increase in the abundance of Ruminococ- caceae and a decrease in the abundance of Christensenel- laceae at the family level. Small intestinal bacterial over- growth (SIBO) is a condition associated with gut dysbiosis, which is associated with RLS and PD [46, 47]. However, alterations in gut microbiota composition in RLS was not reported before. There were some limitations in our study, which should be indicated. One of them was the lack of specific informa- tion about the dietary contents and lifestyles as previous studies suggested that diet is one of the several limitations related to the design of studies of the human microbiome [7]. We could not exclude the influence of antiparkinso- nian drugs, antidepressants, and antipsychotics on gut microbiota. Anosmia determination depended on the sub- jective evaluation of the patient and RBD was based on Table 3 Comparison of taxa relative abundances among PD patients with and without some specific clinical findings Phylum Class Order Family Genus Increase or decrease p value Duration ≥ 5 years Cyanobacteria – – – – ↓ 0.0193 Anosmia (yes) Proteobacteria – – – – ↑ 0.0147 Firmicutes Bacilli – – – ↑ 0.0100 Proteobacteria Gammaproteobacteria – – – ↑ 0.0162 Actinobacteriota Coriobacteriia Coriobacteriales Atopobiaceae – ↑ 0.0012 Constipation (yes) Firmicutes Clostridia Lachnospirales Lachnospiraceae Ruminococcus_gnavus_group ↑ 0.0008 REM sleep behav- ior disorder (yes) Firmicutes Clostridia Lachnospirales Lachnospiraceae Dorea ↓ 0.0052 Firmicutes Clostridia Lachnospirales Lachnospiraceae Eubacterium_ventriosum_group ↓ 0.0037 Firmicutes Clostridia Lachnospirales Lachnospiraceae Ruminococcus_gauvreauii_group ↓ 0.0043 Firmicutes Clostridia Oscillospirales Ruminococcaceae Faecalibacterium ↓ 0.0048 Firmicutes Bacilli Lactobacillales Lactobacillaceae Lactobacillus ↑ 0.0147 Firmicutes Clostridia Clostridiales Clostridiaceae Clostridium_sensu_stricto_1 ↓ 0.0113 Firmicutes Clostridia Lachnospirales Lachnospiraceae CAG.56 ↓ 0.0097 Firmicutes Clostridia Lachnospirales Lachnospiraceae Fusicatenibacter ↓ 0.0144 Firmicutes Clostridia Lachnospirales Lachnospiraceae Lachnospiraceae_ND3007_group ↓ 0.0122 Firmicutes Clostridia Peptococcales Peptococcaceae Peptococcus ↓ 0.0145 Firmicutes Negativicutes Acidaminococcales Acidaminococcaceae Acidaminococcus ↑ 0.0086 1006 Acta Neurologica Belgica (2023) 123:999–1009 1 3 Fig. 3 A proposed schematic representation of the microbiota-gut-brain axis for our study group (revised from [7, 48–50]) 1007Acta Neurologica Belgica (2023) 123:999–1009 1 3 information received from the patient's spouse. Another limitation of the present study was the small sample size for generalizations of the relationships between clinical findings and pathophysiological changes with microbiota alterations. However, the results of the present study may be an exit point for the future studies to provide detailed information in microbiome changes in the Turkish PD patients (Fig. 2). In conclusion, our study showed that there are changes in microbiota profiles of PD patients and some of them are consistent with previous studies while some of them are completely different (Fig. 3). The differences in the microbiota may be related to differences in the dietary contents of the Turkish people, which consist mainly of vegetables and less meat, because it plays a significant role in gut microbiota [7]. The alterations in the gut microbiota can trigger local inflammation followed by aggregation of α-synuclein and likely take part in the pathogenesis of PD. An important finding of our study was the possible rela- tion of gut microbiota with non-motor symptoms of PD. We hope that our study can contribute to understanding the importance of microbiota in early diagnosis of PD. Because microbial alterations by changing the diet is a first-line strategy to treat gastroparesis in PD [7], our study may also set light to the PD therapy for Turkish patients. In addition, the microbiota concerning the brain–gut axis has a great potential for both early diagnosis and treatment of PD when the gaps are filled by further researches. Acknowledgements We thank the patients and their family members and TUBITAK (The Scientific and Technological Research Council of Turkiye) for supporting this study. Author contributions All authors contributed to the study conception and design. Material preparation and data collection were performed by GBY, OG, ZCK, BS, and BE. The data analysis and writing the first draft were performed by GBY, ZCK, IK, and OA. All authors read and approved the final manuscript. Funding The study was supported by the grant from the Turkish Sci- entific and Technical Council (TÜBITAK-118S704) and by the Bezmi- alem Vakif University Scientific Research Found (6.2017/53). Data availability statement All relevant data are within the manuscript. Declarations Conflict of interest The authors declared no potential conflicts of inter- est with respect to the research, authorship, and/or publication of this article. Ethical approval Approval was obtained from the Bezmialem Vakıf University Ethics Committee (approval no.: 12/27) and the research was carried out in accordance with the declaration of Helsinki. Consent to participate A freely given, written informed consent was obtained from all individual participants included in the study. References 1. Hughes AJ, Daniel SE, Kilford L (1992) Accuracy of clinical diagnosis of idiopathic Parkinson’s disease: a clinico-pathological study of 100 cases. 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Li X, Chen LM, Kumar G et al (2022) Therapeutic interventions of gut-brain axis as novel strategies for treatment of alcohol use disorder associated cognitive and mood dysfunction. Front Neu- rosci 16:820106. https:// doi. org/ 10. 3389/ fnins. 2022. 820106 50. Collins S, Surette M, Bercik P (2012) The interplay between the intestinal microbiota and the brain. Nat Rev Microbiol 10:735– 742. https:// doi. org/ 10. 1038/ nrmic ro2876 Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. https://doi.org/10.3389/fnins.2022.820106 https://doi.org/10.1038/nrmicro2876 Altered gut microbiota in patients with idiopathic Parkinson’s disease: an age–sex matched case–control study Abstract Objective Methods Results Conclusion Introduction Methods Study design and participants Microbiota analysis Statistical analysis Results Demographic and clinical data profiles of the patients Alterations of microbiota profiles of patients Discussion Acknowledgements References