Neuroscience and Biobehavioral Reviews 112 (2020) 227–241

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Neuroscience and Biobehavioral Reviews

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Gut microbiota differences between healthy older adults and individuals T

with Parkinson’s disease: A systematic review
Nathan D. Nuzuma,*, Amy Loughmanb, Ewa A. Szymlek-Gaya, Ashlee Hendya, Wei-Peng Teoa,c,
Helen Macphersona
a
Deakin University, Institute for Physical Activity and Nutrition (IPAN), School of Exercise and Nutrition Sciences, Geelong, Australia
b
Deakin University, Food & Mood Centre, IMPACT Strategic Research Centre, School of Medicine, Geelong, Australia
c
Physical Education and Sports Science Academic Group, National Institute of Education, Nanyang Technological University, Singapore

A R T I C LE I N FO A B S T R A C T

Keywords: The ‘Dual Hit’ hypothesis, stating that Parkinson’s disease (PD) begins via olfactory pathways and the gut, and
Short chain fatty acid the gastrointestinal symptoms PD individuals face, have largely driven the interest of the gut’s involvement in
Butyrate PD. Studies have since observed gut microbiota differences between PD groups and controls, with these al-
Microbiota-gut-brain axis terations potentially relating to PD pathophysiology. However, differences in the studies’ methodologies pre-
Alpha-diversity
cludes unanimity on the relationships of gut microbiota to PD.
Beta-diversity
Thirteen observational case-control studies investigating gut microbiota in PD and controls were reviewed to
Cognition
Motor-symptoms assess how microbiota abundance and diversity relates to PD. Nine studies showed butyrate producing gut
Non-motor symptoms microbiota had lower abundances in PD compared to controls. Three studies reported α-diversity was higher,
with one reporting it was lower, in PD compared to controls.
Given most studies show abundance, not diversity, differences of butyrate producing bacteria between
groups, we propose abundance differences are more associated with PD than microbiota diversity. As current
research is observational, investigating how specific bacteria and their metabolites may alter throughout PD
progression is warranted.

Information Box 1 Microbiome: The entire community of microorganisms, their ge-

The high rate of gastrointestinal issues in Parkinson’s disease (PD),
and beforehand which may predict increased likelihood of PD devel-
opment, has led to research into the gut microbiota of this population.
Technological advancements in next generation sequencing (NGS) in
the last decade have enabled this research (Sekirov et al., 2010). For an
overview of NGS methods, including 16s rRNA sequencing and shotgun
sequencing, see Hamady and Knight (2009). These techniques, in ad-
dition to culture dependent techniques, allow for the investigation of
gut bacteria and their metabolites (E.g. short chain fatty acids).
Short chain fatty acids (SCFAs), most commonly butyrate, acetate
and propionate, have numerous purported effects in relation to the
microbiota-gut-brain axis. Namely, this includes the anti-inflammatory
effects within the gut and the role SCFAs have in maintaining intestinal
wall health and function. For a review of SCFAs and their purported
effects, see Lopetuso et al. (2013), Mayer et al. (2015), Tan et al.
(2014b), and Westfall et al. (2017).
Gut microbiota: The microorganisms of the gut (in the case of this
article, bacteria specifically).
netic material and their environment (Marchesi & Ravel, 2015).
Dysbiosis: Changes to the typical bacterial composition of a com-
munity either by introduction or elimination of specific bacterium
types, or abundance shifts of existing bacteria that could lead to ne-
gative health outcomes (Holzapfel et al., 1998).
Alpha-diversity (α-diversity): The number (‘richness’) and distribu-
tion (‘evenness’) of bacteria within a single population or community.
Beta-diversity (β-diversity): Similarity or dissimilarity between two
or more sets of microbial communities.
In this document, ‘gut microbiome’ or ‘gut microbiota’ refer to the
faecal microbiome, which is the most common method of sampling ‘the
gut microbiome’ due to the difficulty in obtaining intestinal mucosal
samples.
;1;
1. Introduction
Parkinson’s disease (PD) is a movement disorder most commonly

⁎
Corresponding author at: School of Exercise and Nutrition Sciences, Deakin University, 221 Burwood Highway, Burwood, 3125, VIC, Australia.
E-mail address: n.nuzum@deakin.edu.au (N.D. Nuzum).

https://doi.org/10.1016/j.neubiorev.2020.02.003
Received 21 October 2019; Received in revised form 15 January 2020; Accepted 3 February 2020
Available online 04 February 2020
0149-7634/ © 2020 Elsevier Ltd. All rights reserved.
N.D. Nuzum, et al.
recognised through its cardinal motor impairments including muscle
rigidity, postural instability and gait disturbances, bradykinesia and
resting tremors (Jankovic, 2008). However, some of the most prevalent
PD symptoms are non-motor, including cognitive deficits, from mild-
cognitive impairment to PD-related dementia, along with a range of
gastrointestinal (GI) symptoms involving increased transit time, such as
constipation (Fasano et al., 2015). Given the devastating nature of PD
and its associated symptoms, understanding PD aetiology and pro-
gression is crucial to developing preventative measures or therapeutic
options.
Gastrointestinal symptoms are one of the most prevalent non-motor
symptoms in individuals with PD, with up to 80 % of individuals af-
fected (Martinez-Martin, 2011). These symptoms are common, and can
precede motor symptoms and clinical PD diagnosis. For example, con-
stipation may represent an increased risk for future diagnosis of PD
(Abbott et al., 2001). This indicates that the GI tract may be relevant to
the onset of PD. Additionally; impaired olfaction is an early non-motor
symptom of PD (Haehner et al., 2009). One hypothesis to explain the
role of olfactory and GI tract pathways in PD aetiology includes the
‘Dual Hit’ hypothesis (Hawkes et al., 2009). Hawkes and colleagues
propose that an unknown neurotropic pathogen gains entry via the
nasal cavity and through swallowing of infected saliva/mucous leads to
the pathogen passing through the mucosal and epithelial intestinal
barrier to the underlying enteric nervous system, potentially resulting
in or involving a-synuclein misfolding. The misfolded α-synuclein
subsequently propagates up the vagus nerve (Holmqvist et al., 2014;
Kim et al., 2019), and this retrograde path of ‘attack’ leads to neuronal
cell death in the substantia nigra (among other brain regions) which is a
pathological hallmark of PD. A comparison between the intestines of
early-stage PD individuals and healthy controls showed PD participants
had greater intestinal permeability, which significantly correlated to
both aggregation of misfolded α-synuclein at the intestines and greater
inflammation in the PD group, providing some support for the above
hypothesis (Forsyth et al., 2011; Shannon et al., 2012). However, the
specificity of aggregated α-synuclein at the intestine as a biomarker for
PD is questionable, as research shows aggregated α-synuclein in control
participants (Visanji et al., 2015). Additional research shows that α-
synuclein aggregation, and oxidative stress, causes microglia over ac-
tivation (Block and Hong, 2005), leading to neuroinflammation (Shults,
2006; Su et al., 2008), which could result in an increased risk for de-
veloping PD, or could be the result of PD development (Hald and
Lotharius, 2005; Teismann and Schulz, 2004). In addition to the po-
tential involvement of the GI tract in PD pathophysiology via the ‘Dual
Hit’ hypothesis (Hawkes et al., 2009), systemic inflammation is also
implicated in PD progression (Vivekanantham et al., 2015), and gives
additional credence for investigating the GI tract.
Due to the potential effects of inflammation/neuroinflammation,
oxidative stress and increased intestinal permeability in the develop-
ment of PD (Westfall et al., 2017), it is important to assess the gut
microbiota and the metabolites they produce, as many are responsible
for maintaining intestinal wall integrity and are anti-inflammatory (Tan
et al., 2014b). Short chain fatty acids (SCFAs) are a prominent meta-
bolite produced through commensal gut microbiota that fulfil such
roles (Tan et al., 2014b). Examples of important SCFAs include pro-
pionate, acetate and butyrate. While all SCFAs have anti-inflammatory
capabilities (Tan et al., 2014b), butyrate’s anti-inflammatory effects
seem to be the most potent. Butyrate also helps maintain intestinal wall
health through mucin production (Brown et al., 2011; Hatayama et al.,
2007), and is a major energy source for colonocytes (Canani et al.,
2016). Given the important physiological roles of SCFAs, dysbiosis of an
individual’s gut microbiome, whereby many of the beneficial SCFA
producing bacteria are potentially reduced, could result in an increased
risk for developing PD.
As a result, multiple studies have now used culture-independent,
next generation sequencing techniques to compare the gut microbiomes
of healthy matched controls to individuals with PD. There is a need to
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Neuroscience and Biobehavioral Reviews 112 (2020) 227–241
systematically review these studies to examine what is currently known
about gut microbiota differences between these groups and to infer
what role gut microbiota may play in PD onset or progression.
This review aimed to synthesise the literature on the differences in
gut microbiota profiles across PD and healthy control groups and to
investigate the associations between gut microbiota and PD. The spe-
cific objectives of this systematic review were to 1) assess whether there
was an association between measures of gut microbiota diversity (in-
cluding both α- and β-diversity measures) and PD, 2) describe the
differences in specific bacterial taxa and their metabolites (E.g. SCFA)
between PD and neurologically healthy controls and 3) assess whether
the gut microbiota differences between groups related to PD, PD se-
verity, PD duration and/or to motor and non-motor symptoms of PD.
2. Methods
This systematic review was completed per the PRISMA guidelines
(Moher et al., 2015) and was registered on PROSPERO (Registration
number: CRD42018095211, registration date 1/6/2018). The studies
included in the review were original, observational case-control studies
with human participants only. Accepted articles required a PD group to
have an age- and sex-matched control group, or similar (E.g. a spousal
control group), and to have assessed the gut microbiota composition of
each group. No limitations were placed on the date of publication of the
included studies, but articles were restricted to those available in
English and published in peer-reviewed journals. Conference abstracts,
book chapters and reviews were excluded, and reference lists of re-
levant reviews were manually checked for potential articles that fit the
criteria.
A systematic computerised search was performed from the 27/05/
18 until the 24/05/19 when the final search was conducted, through
the following databases: PubMed, Embase, Scopus, CINAHL (through
EBSCOhost), Global Health (through EBSCOhost) and CENTRAL
(Cochrane Library). The full search strategy used for all databases is
available in Appendix A (see Supplementary material). Briefly, the
overall search strategy included terms such as ‘gut bacteria’, ‘micro-
biota’, ‘neurodegenerative diseases’, ‘PD’, as well as variations on these
terms (e.g. gut flora for gut bacteria). Mesh terms, or their equivalents,
were also used in the search strategy.
Title and abstract screening were completed in duplicate, with a
third researcher available in the event of discrepancies in accepted
articles between researchers. Full text screening was completed in the
same manner. After screening was completed, a data extraction form
(an adapted version of the Cochrane data collection form for RCTs and
NRS) was utilised to extract all relevant information from the accepted
articles, by one researcher (NN). As part of the data extraction process,
a risk of bias assessment was conducted following the validated ‘Risk of
Bias Assessment tool for Non-randomised Studies’ (RoBANS) protocol
(Kim et al., 2013).
2.1. Data analysis
Due to the high degree of heterogeneity between data across the
included studies, we were unable to synthesis the results quantitatively.
Therefore, a qualitative, narrative synthesis of the results was con-
ducted.
3. Results
This systematic review included 13 studies (See PRISMA Flow
Diagram Fig. 1). Basic study characteristics are shown in Table 1. In
total, the 13 included studies provided data from 904 individuals with
PD and 683 control subjects. Of the 13 studies, two did not conduct next
generation sequencing (Hasegawa et al., 2015; Unger et al., 2016), but
were included because the bacterial species they chose to investigate
were reported in other studies, and because one compared SCFAs across
N.D. Nuzum, et al.
Fig. 1. PRISMA Flow Diagram of records identified through searching and the screening process (Moher et al., 2009).
a PD group and healthy control group (Unger et al., 2016).
3.1. Risk of Bias assessment
The most common source of bias across the included studies came
from the ‘Confounding Variables’ domain with 10/13 studies scoring
‘High’ as they did not account for various potential confounding factors
(E.g. BMI, medications, diet etc. which are known to be related to gut
microbiota (David et al., 2014; Jackson et al., 2018). Additionally, the
‘Selective Outcome reporting’ domain had 9/13 studies scoring ‘High’.
This was mostly due to studies either not conducting or reporting
measures of α- and/or β-diversity measures (Heintz‐Buschart et al.,
2018), conducting only α- or only β-diversity measures (Bedarf et al.,
2017; Hill-Burns et al., 2017; Keshavarzian et al., 2015), or when they
had conducted these measures they had only used one type of measure
for either α- or β-diversity (Hopfner et al., 2017; Li et al., 2017; Petrov
et al., 2017). The study by Barichella et al. (2019) scored uncertain for
the ‘Selective Outcome reporting’ domain as it is not clear from their
paper if they used appropriate corrections to adjust for multiple com-
parisons when assessing specific bacterial abundance. For the ‘Blinding
of Outcome Assessments’ domain, the blinding of PD status for re-
searchers administering behavioural assessments would not be possible.
Only one study reported that the researcher was blinded when con-
ducting the bacterial analysis (Hasegawa et al., 2015). The other studies
have been rated as ‘Uncertain’ for this domain (See Supplementary
Table 3 in Appendix B for detail).
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3.2. Participant demographics and PD symptoms
Table 1 shows the included studies were geographically diverse and
all but one healthy control group included participants with a mean age
above 60 years (Keshavarzian et al., 2015), to match that of the PD
population. Across studies, participants’ sex was similarly represented
with most studies having a greater representation of men in PD groups.
Five of the 13 studies showed the PD group had significantly more
severe or higher prevalence of GI symptoms compared to healthy
controls (all p < 0.05) (Hasegawa et al., 2015; Heintz‐Buschart et al.,
2018; Hill-Burns et al., 2017; Li et al., 2017; Qian et al., 2018), four
studies reported no difference (all p > 0.05) (Bedarf et al., 2017;
Hopfner et al., 2017; Lin et al., 2018; Scheperjans et al., 2015) and one
did not report on the differences between PD and control groups, de-
spite conducting the analysis (Barichella et al., 2019). The remaining
three studies did not measure GI symptoms in either group
(Keshavarzian et al., 2015; Petrov et al., 2017; Unger et al., 2016). Four
of the included studies measured PD severity and/or PD related
symptoms in controls, to rule out presence of PD and PD symptoms. Of
these four, one study conducted the assessment but did not report on
significance levels (Li et al., 2017), another did not compare groups but
looked at the associations of the variable on microbial composition (Lin
et al., 2018), while two showed statistically significant differences be-
tween groups (Bedarf et al., 2017; Heintz‐Buschart et al., 2018). Higher
PD severity was evident in the PD group (measured through the Hoen
and Yahr scale (H&Y) and the Unified Parkinson’s Disease Rating Scale-
I and –III (UPDRS-I and III) (all p < 0.05)) (Bedarf et al., 2017;
 Table 1
Group demographics from each study (mean ± SD; or median (IQR) indicated by a) including number of participants and sex (m = male, f = female), age (years), body mass index (BMI), gastrointestinal (GI) symptoms
and Parkinson’s disease (PD) severity according to various measures.
Study Location PD group demographics Control group demographics
N.D. Nuzum, et al.

N Age (years) BMI GI Symptoms PD severity N Age (years) BMI GI Symptoms PD severity

Hasegawa et al. Japan 52 (21 m, 68.9 ± 6.8 20.2 ± 2.8* Stool PD duration 36 (spousal 68.4 ± 9.7 22.6 ± 3.7 Stool NR
(2015) 31f) frequency/week (years) = 9.5 ± 5.4 controls:21 m, 15f) frequency/week
3.1 ± 1.2* H&Y stage = 2.7 ± 0.9 7.6 ± 4.6
UPDRS-I = 2.9 ± 2.3
UPDRS-II = 11.7 ± 6.8
UPDRS-III = 25.6 ± 11.8
UPDRS-IV = 3.4 ± 2.4
Keshavarzian et al. U.S.A 38 (24 m, 61.6 ± 9.4* 26.0 ± 3.2 Constipation PD duration (years) 34 (18 m, 16f) 45.1 ± 14.4 27.6 ± 6.0 NR NR
(2015) 14f) according to VAL 6.4 ± 4.7
2.8 ± 3.4 UPDRS score
29.7 ± 11.0(not stated which
section of UPDRS was conducted)

H&Y stage
2.1 ± 0.4
Scheperjans et al. Finland 72 (37 m, 65.3 ± 5.5 26.3 (23.8–29.3)a Wexner NR 72 (36 m, 36f 64.5 ± 6.9 26.2 (23.7–28.1)a Wexner NR
(2015) 35f 5 (3–9)a 2 (1–4)a
Unger et al. (2016) Germany 34 (24 m, 67.7 ± 8.9 NR Constipated Disease duration 82 months 34 age matched 64.6 ± 6.6 NR NR NR
10f) 7/34 (20.6 %) H&Y stage 2.5 (18 m,16f) & 10 (age matched
young control 33.3 ± 11.6
(5 m,5f) (younger group)
Bedarf et al. Germany 31 (all 64.8 ± 9.5 NR GSRS UPDRS III- 12.6 ± 6.9* 28 (all male) 65.6 ± 10.4 NR GSRS 2.2 ± 2.0 0±0
(2017) male) 3.4 ± 2.9

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Hill-Burns et al. U.S.A 197 68.4 ± 9.2* 26.4 ± 5.3* Constipation in past PD duration (years) 130 (spousal 70.3 ± 8.6 28.3 ± 5.7 Constipation in NR
(2017) (132 m, 3-months 82/197 13.7 ± 6.5 controls :51 m, past 3-months 6/
65f)* (43.4 %)* 79f) 130 (4.7 %)
Hopfner et al. Germany 29 (23 m, 69.2 ± 6.5 NR Constipation UPDRS I score 29 (13 m, 16f) 69.4 ± 6.7 NR Constipation NR
(2017) 6f)* 4/29 12.6 ± 5.3 20/29 (68.9 %)
(13.8 %) UPDRS III score 20.9 ± 6.2
Li et al. (2017) China 24 (16 m, 73.75 ± 6.26 22.96 ± 3.64 Wexner UPDRS score 14 (6 m, 8f) 74.64 ± 5.57 24.00 ± 2.66 Wexner UPDRS score
8f) 17.58 ± 5.43* 33.11 ± 15.75(significance not 1.29 ± 2.59 2.36 ± 2.93
reported)
H&Y stage and
PD duration (years) 5 (2.25- PD duration NR
8: median and IQR)
H&Y stage 2.5 (2-3: median
and IQR)
Petrov et al. Russia 89 (sex 63(61.5;67.5) 26.72(25.4;28.84) NR NR 66 (sex NS) 67(65;69.75) 26.0 (23.82;28.66) NR NR
(2017) NS)
Heintz‐Buschart Germany 26 (19 m, 68.0 ± 9.7 28.5 ± 4.5* SCOPA-AUT MDS H&Y stage 38 (25 m, 13f) 68.4 ± 6.7 26.6 ± 4.1 SCOPA-AUT MDS H&Y score
et al. (2018) 7f) 31* 2.14 ± 0.75* 7 0 ± 0 MDS-
MDS-UPDRS I score UPDRS I score
11.7 ± 7.2* 5.5 ± 4.3
MDS-UPDRS III score MDS-UPDRS III
30 ± 14* score 1.5 ± 2.8
Lin et al. (2018) China 75 (49 m, 60.48 ± 10.72 24.27 ± 2.29# Wexner Disease duration ≥ 45 (spousal 63.2 ± 6.0 24.44 ± 2.36# Wexner Disease duration
26f) 8.81 ± 5.03 5 years = 30 controls :23 m, 2.04 ± 1.02 ≥ 5 years = 0
< 5 years = 44 22f) < 5 years = 0
UPDRS III score UPDRS III score
33.73 ± 18.08 0±0
Qian et al. (2018) China 45 (22 m, 68.1 ± 8 22.8 ± 2.3 Rome III 24/45 H&Y stage: 2.2 ± 0.7 45 (spousal 67.9 ± 8 23.4 ± 2.1 Rome III 3/45 (6.7 NR
23f) (53.3 %)* Age of onset (years): controls:23 m, 22f) %)
62.6 ± 8.1
(continued on next page)
Neuroscience and Biobehavioral Reviews 112 (2020) 227–241
 Table 1 (continued)

Study Location PD group demographics Control group demographics

N Age (years) BMI GI Symptoms PD severity N Age (years) BMI GI Symptoms PD severity
N.D. Nuzum, et al.

Disease duration (years):

5.7 ± 4.1
UPDRS total scores:
38.4 ± 18.8
UPDRS Part I scores:
3.1 ± 1.9
UPDRS Part II scores:
10.0 ± 5.7
UPDRS Part III scores:
22.9 ± 12.3
UPDRS Part IV scores: 2.0
(2.0) a
Barichella et al. Italy 193 67.6 ± 9.7 25.3 ± 4.6 Rome III 99 (51.3 7.7 years disease duration 113 (47 m, 66f) 65.9 ± 9.9 25.2 ± 4.6 Rome III 21 (18.6 NR
(2019) (115 m, %) H&Y stage 2.0 %)
78f) UPDRS stage I = 2.0
UPDRS stage II = 9.4
UPDRS stage III = 17.2
UPDRS stage IV = 1.9

* Indicates significant difference between groups for respective baseline measure. # indicates significant effect of measure on microbial composition. N = Number of Participants that had stool samples assessed for gut
microbiota composition, NR = Not Reported; IQR = Inter quartile range; UPDRS = Unified Parkinson’s disease rating scale; H&Y = Hoen and Yahr scale; GSRS = Gastrointestinal symptoms rating scale;
MDS = Movement disorder society; SCOPA-AUT = assessment of autonomic dysfunction in Parkinson’s disease questions 1–7 (gastrointestinal part); VAL = Visual analogue scale; Wexner = Wexner constipation scoring
system; Constipated = defined as fewer than three bowel movements a week, or hard, dry and small bowel movements that are painful or difficult to pass. Note that Petrov et al. (2017) does not state in-text what the
numbers for Age and BMI represent (E.g. median or mean? Range or IQR?).

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Neuroscience and Biobehavioral Reviews 112 (2020) 227–241
 N.D. Nuzum, et al.

Table 2
Studies that conducted α- and/or β-diversity measures across cases (Parkinson’s disease) versus controls, and the types of methods for each. Where studies completed the measure, results are included.
Study α-diversity β-diversity

Shannons Simpsons Observed species Chao1 Phylogenetic Other Unweighted-UniFrac Weighted UniFrac Other
/OTUs diversity Whole tree

Keshavarzian et al. At phylum level At phylum level p = 0.01# NM NM NM Margalef at phylum level: Not measured
(2015) p = .01# (note; authors conducted the p = 0.01# and at genus: level
Gini-Simpsons index) p = 0.03#
Scheperjans et al. p > 0.05 Inverse Simpson p > 0.05 NM p > 0.05 NM ACE p > 0.05 p < 0.02 p < 0.001 NM
(2015)
Bedarf et al. (2017) p = 0.55 p = 0.55 p = 0.87 P = 0.97 NM NM Not measured
Hill-Burns et al. (2017) Not Measured P < 0.01 P < 0.01 Canberra distance
P < 0.01
2

232
Hopfner et al. (2017) p > 0.05 p > 0.05 NM p > 0.05 NM Faith’s phylogenetic distance R = 0.0241, p = 0.0491 NM NM
p > 0.05
Li et al. (2017) p = 0.248 NM NM p = 0.140 NM NM HC and PD separated NM NM
according to PC1 and PC2
(8.03 % and 6.32 %)
Petrov et al. (2017) NM NM NM p = 0.001* NM NM NM R = 0.214 NM
p = 0.0001
Heintz‐Buschart et al. Not Measured
(2018)
Lin et al. (2018) p = 0.74 NM p = 0.233 p = 0.022 p = 0.162 NM R2 = 0.02136 p = 0.001 NM NM
Qian et al. (2018) p = 0.005# p = 0.010# p = 0.009# p = 0.017# p = 0.010# NM R = 0.027 R = 0.008 NM
p = 0.038 p = 0.181
Barichella et al. (2019) NM NM NM p = 0.012 # NM NR P = 0.002 NM
Not stated for which measure
though
Total Number 6 4 3 6 2 2 7 5 1
Neuroscience and Biobehavioral Reviews 112 (2020) 227–241
N.D. Nuzum, et al.
Heintz‐Buschart et al., 2018). The other eight studies did not assess PD
symptoms or severity in controls.
3.3. Gut microbiota measures
3.3.1. Measures of gut microbiota diversity
A range of α- and β-diversity measures were utilised to assess gut
microbiota diversity within and between groups (E.g. Shannon Index,
Chao1 Index, and weighted and unweighted UniFrac distances. For a
full list of these measures, see Table 2). Two studies used real time-
quantitative polymerase chain reaction instead of next-generation se-
quencing (e.g. 16 s rRNA sequencing) techniques, a selective sequen-
cing method that is not amenable to measuring overall microbial di-
versity (Hasegawa et al., 2015; Unger et al., 2016).
Nine studies measured α-diversity (See Table 2), with four studies
showing a significant difference between groups. Three of the four
studies showed the PD group to have higher α-diversity (Barichella
et al., 2019; Keshavarzian et al., 2015; Qian et al., 2018), with one
study showing the healthy control group had higher α-diversity (Petrov
et al., 2017). All eight studies that measured β-diversity (Using
Weighted and/or Unweighted UniFrac measures or Canberra distance),
indicated that the community of gut microbiota in the PD and control
groups were different (See Table 2). Note that Qian et al. (2018) found
significant differences in Unweighted UniFrac (p = 0.038), but not for
the Weighted UniFrac distance (p = 0.181), indicating the presence/
absence of different bacteria but not necessarily in different abundances
in the patient and control groups. However, four studies did show
significance at weighted UniFrac measures, indicating differential
abundance of bacteria that were present.
3.3.2. Abundance of SCFA producing bacteria and SCFA concentrations
All but two studies used NGS, revealing many bacterial abundance
differences between controls and the PD group reported across many
levels of classification. As such, the most important results have been
highlighted here (See Fig. 2A and B, and Table 2 in Appendix B of the
Supplementary material, for data summary). Bacterial abundance data
from the studies presented below is relative abundance (bacterial taxa
expressed as a percentage of the total bacterial taxa found) in all but
one study (Hasegawa et al., 2015), where absolute abundance was
presented (bacterial taxa presented as counts of the gut bacteria from
sequencing).
Across the 11 studies using NGS, eight showed five different puta-
tive butyrate producing species and genera of bacteria that had lower
abundance in the PD group compared to controls. These included the
Faecalibacterium genera and/or species Faecalibacterium prausnitzii (Hill-
Burns et al., 2017; Li et al., 2017; Lin et al., 2018; Petrov et al., 2017;
Unger et al., 2016), Eubacterium biforme (Bedarf et al., 2017), Copro-
coccus eutactus (Petrov et al., 2017), Roseburia faecis (Bedarf et al.,
2017; Heintz‐Buschart et al., 2018; Hill-Burns et al., 2017;
Keshavarzian et al., 2015; Lin et al., 2018), and the Blautia genera
(Bedarf et al., 2017; Hill-Burns et al., 2017; Keshavarzian et al., 2015; Li
et al., 2017; Lin et al., 2018). Other bacteria found to be lower in the PD
group compared to controls that potentially produce the SCFA propio-
nate, included Prevotella copri (Bedarf et al., 2017; Petrov et al., 2017),
Veillonella parvula (Lin et al., 2018), Roseburia faecis and Blautia (both
Roseburia faecis and Blautia reportedly produce both butyrate and pro-
pionate). Bacteria which have the potential to produce the SCFA
acetate, that were found to be less abundant in PD groups included
Ruminococcus torques (Bedarf et al., 2017), and Veillonella parvula
(produces both acetate and propionate) (See Supplementary Table 2 for
full details). In contrast, across the included studies, eight showed that
four different putative SCFA producing bacteria (any of butyrate,
acetate or propionate) were higher in the PD group compared to con-
trols. These included Akkermansia muciniphila (Bedarf et al., 2017; Lin
et al., 2018), Bifidobacterium adolescentis (propionate and acetate, and
acetate and lactate producers respectively) (Hill-Burns et al., 2017),
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Megasphaera (Lin et al., 2018) and Butyricicoccus (Qian et al., 2018)
(purported butyrate producers, although Megasphaera eldesnii is speci-
fically a butyrate producer with only the genera reported here). Two
articles did not report any abundance differences at genus or species
levels (Hopfner et al., 2017; Scheperjans et al., 2015). Note that it is
unclear if Barichella et al. (2019) or Hopfner et al. (2017) performed
adequate FDR corrections on their comparison of bacterial abundance
between groups to adjust for multiple comparisons. Additionally,
Scheperjans et al. (2015) reported 6 different bacterial family groups to
be differently abundant however only one (Prevotellaceae) had a Q-
value lower than 0.05, while the bacteria reported at the species level
for Bedarf et al. (2017) were not significant after they applied the FDR
correction (See Supplementary Table 2). Therefore, these results should
be considered with some caution. Various lactate producing Lactoba-
cillus bacteria were more abundant in PD groups (Hasegawa et al.,
2015; Hill-Burns et al., 2017; Petrov et al., 2017). However, studies
showing this either did not exclude participants taking probiotics or did
not adequately collect information on frequency of consumption, do-
sage/number of probiotics taken, time between probiotic ingestion and
faecal sample collection, and the type of probiotic taken.
While Hasegawa et al. (2015) and Unger et al. (2016) did not
conduct NGS, they still revealed some significant differences in bac-
terial abundance. Hasegawa et al. (2015) showed counts of Lactobacillus
were significantly more abundant in the PD group (p = 0.003), whilst
counts of Clostridium coccoides and Clostridium leptum (i.e. Clostridial
cluster XIVa and IV, with both known to contain butyrate producers
(Collins et al., 1994; Liu et al., 2008)), and Bacteroides fragilis were all
significantly less abundant in the PD group (p < 0.009) (Hasegawa
et al., 2015). Unger et al. (2016) found that the bacterial family En-
terobacteriaceae, and the genus Bifidobacterium were significantly more
abundant in PD compared to controls, while bacteria from the phylum
Bacteroidetes and from the families of Lactobacillaceae and En-
terococcaceae, were significantly less abundant in the PD group. Ad-
ditionally Unger et al. (2016) showed Faecalibacterium prausnitzii, a
known butyrate producer (Duncan et al., 2002), was significantly less
abundant in the PD group compared to controls.
3.3.3. SCFA concentrations
Only one study investigated SCFA concentrations from faecal sam-
ples of individuals with PD compared to healthy age-matched controls
(Unger et al., 2016). Researchers used gas chromatography to assess
SCFA concentrations and found that absolute concentrations (mmol/g)
of the three most common SCFAs, acetate, propionate and butyrate,
were significantly lower in the PD group (all p < 0.01). The relative
concentration (% of all SCFAs assessed) of butyrate was also sig-
nificantly lower in the PD group (p < 0.05) (Unger et al., 2016).
3.4. Gut microbiota differences in relation to PD progression and symptoms
Seven studies investigated the relationship between bacterial dif-
ferences of control and PD groups and PD, PD severity and/or PD re-
lated symptoms. Summaries of their findings are reported in the below
paragraphs (statistical values are stated where available) (Barichella
et al., 2019; Heintz‐Buschart et al., 2018; Li et al., 2017; Lin et al., 2018;
Qian et al., 2018; Scheperjans et al., 2015; Unger et al., 2016). An
overview of the PD symptoms of primary interest to this review, and
symptoms most commonly assessed and reported by authors of the
included studies, can be seen in the description of Table 1. Additional
symptoms not mentioned in the table (and measures assessing the
symptoms) included cognition (Montreal Cognitive Assessment and
Mini-Mental State Examination) and depression (Hamilton Depression
and Montgomery-Asberg depression scales).
3.4.1. PD severity and disease duration
To investigate how gut microbiota abundance related to PD se-
verity, Li et al. (2017) separated their PD cohort into mild (1–2.5) and
N.D. Nuzum, et al. Neuroscience and Biobehavioral Reviews 112 (2020) 227–241

Fig. 2. A. Individual bacteria shown to be significantly higher in the PD group compared to controls across the taxonomic levels of Phylum, Class, Order, Family, Genus
and Species. Number in brackets following bacteria denotes the number of studies showing significance for that bacteria, at the indicated classification level. Archaea
are denoted with *. Tree generated using PhyloT (database version 2019.1) based on the NCBI taxonomic database and visualised in iTOL (Letunic and Bork, 2016).
B. Individual bacteria shown to be significantly lower in the PD group compared to controls across the taxonomic levels of Phylum, Class, Order, Family, Genus and
Species. Number in brackets following bacteria denotes the number of studies showing significance for that bacteria, at the indicated classification level. Archaea are
denoted with * and Eukaryotes are denoted with #. Tree generated using PhyloT and iTOL based on NCBI taxonomic database. Note that the bacterial family
Coprobacillaceae is presented under its reclassification, Erysipelotrichaceae, and that Clostridium coccoides is presented as its reclassified name Blautia coccoides.

severe (3–5) PD severity based on their Hoen & Yahr scores (rated on a
1–5 scale). Comparing these two groups to the control group revealed
that the relative abundance of Faecalibacterium was inversely associated
with PD severity (5.14 % for controls, 3.17 % in mild PD and 1.22 % for
severe PD; significant difference between controls and severe PD,
p = 0.048). Additionally, the relative abundance of Megasphaera was
positively associated with increasing PD severity (0.04 % in controls,
2.52 % in mild PD and 5.50 % in severe PD group; significant difference
between controls and severe PD group, p = 0.003). Qian et al. (2018),
conducted spearman correlations (with GLM adjustments) and found no
correlations between microbiota and Hoen & Yahr staging.
While only two studies have analysed PD severity and gut micro-
biota, four studies have assessed the relationship between PD duration
and gut microbiota. Analysis of β-diversity revealed that the distances
between the microbial communities of the PD and control groups in-
creased with increasing disease duration (Barichella et al., 2019).
Analysis of bacterial abundance data and disease duration revealed
significant trends for decreasing Lachnospiraceae, and increasing Ak-
kermansia and Lactobacillus with increasing disease duration (all
p < 0.05) (Barichella et al., 2019). While Qian et al. (2018) showed
that Eschericia/Shigella genera were significantly (p = 0.028) negatively
correlated to disease duration, Li et al. (2017), in contrast, found

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Fig. 2. (continued)

significantly positive correlations between PD duration and Escherichia/ diversity did not differ between groups. Lin et al. (2018) subsequently
Shigella genera (r = 0.52, p = 0.001), as well as with Proteus (r = 0.46, compared specific bacterial taxa and found that Rikenellaceae (adjusted
p = 0.003), Enterococcus (r = 0.56, p < 0.001), and Megasphaera p value = 0.0027) and Deferribacteraceae (adjusted p value = 0.016)
(r = 0.41, p = 0.012). Li et al. (2017) also found negative correlations were more abundant in the > 5 year PD group.
with PD duration and the bacteria Blautia (r = −0.40, p = 0.013),
Ruminococcus (r = −0.58, p < 0.001), Sporobacter (r = −0.40,
3.4.2. PD motor symptoms
p = 0.013) and Haemophilus (r = −0.47, p = 0.003). In both faecal and
The relationship between PD related motor symptoms and gut mi-
GI mucosal microbiota samples (Keshavarzian et al., 2015), Firmicutes
crobiota is not clear from the reviewed studies. One study revealed that
were significantly negatively correlated with PD duration (r = −0.38,
the abundance of Anaerotruncus spp., Clostridium XIVa, and
p = 0.04 in faecal samples and r = −0.58, p < 0.01 in mucosal
Lachnospiraceae were significantly positively associated to motor
samples). Lin et al. (2018) assessed PD duration by dividing the ana-
symptoms (higher UPDRS-III scores) (Heintz‐Buschart et al., 2018).
lyses of their PD cohort into those with a PD duration of less than or
Additionally, significant positive associations between worse motor
greater than 5 years. Between these groups α-diversity was significantly
symptoms and the Lactobacillaceae family and Lactobacillus genus of
different across three of four measures (Chao1, p = 0.045; observed
bacteria was demonstrated (Barichella et al., 2019). Scheperjans et al.
Operational Taxonomic Units (OTUs), p = 0.023; PD-whole-tree,
(2015) indicated that Prevotellaceae abundance was significantly as-
p = 0.018, Shannon, p = 0.194), however, authors did not report the
sociated with UPDRS-III scores, but only when patients with motor
direction of these significant differences, whilst also stating that β-
fluctuations who were on medication, or using deep brain stimulation,

235
N.D. Nuzum, et al.
during motor-symptom examination were included. An analysis ex-
cluding these individuals, revealed no association between Pre-
votellaceae abundance and UPDRS-III scores. Qian et al. (2018) also
found no correlations between motor symptoms and the gut microbiota
(adjusted for confounders). However, Li et al. (2017) who assessed
correlations between PD symptoms (whether this was for specific motor
or non-motor symptoms is not specified) and bacterial taxa, found
worse symptoms positively correlated with Enterococcus (r = 0.336,
p = 0.039), Proteus (r = 0.45, p = 0.005) and Escherichia/Shigella
(r = 0.383, p = 0.018). Li et al. (2017), also observed that worse motor
symptoms negatively correlated with Blautia (r = −0.50, p = 0.002),
Faecalibacterium (r = −0.357, p = 0.028), Ruminococcus (r = −0.51,
p = 0.001), Haemophilus (r = −0.46, p = 0.004) and Odoribacter
abundance (r = −0.41, p = 0.011), indicating these bacteria are as-
sociated with less severe PD symptoms.
Three studies examined PD phenotypes (Lin et al., 2018;
Scheperjans et al., 2015; Unger et al., 2016). Lin et al. (2018) divided
participants into tremor dominant (n = 18) and non-tremor dominant
(n = 56) groups. No significant difference in α- or β-diversity was seen
between groups. However, Leptotrichia abundance was significantly
higher in patients with the tremor phenotype (adjusted p
value = 0.022). Roseburia was significantly more abundant in the pa-
tients with the non-tremor phenotype (adjusted p value = 0.028). The
abundance of Enterobacteriaceae did not differ between the pheno-
types. Similarly, Unger et al. (2016) found Enterobacteriaceae abun-
dance was not significantly different across phenotypes although
sample sizes between groups were small: tremor dominant (n = 6),
hypokinetic-rigid (n = 15) or mixed (n = 13). Scheperjans et al. (2015)
assessed slightly different phenotypes; postural instability and gait
difficulty phenotype (n = 40), tremor dominant phenotype (n = 23)
and mixed phenotypes (n = 9), finding that Enterobacteriaceae were
significantly more abundant in patients with the postural instability and
gait difficulty phenotype (assessed through a GLM, p < 0.001).
3.4.2.1. PD non-motor symptoms: psychological. Heintz‐Buschart et al.
(2018) showed Anaerotruncus and Akkermansia were significantly
related to greater presence of non-motor symptoms (UPDRS-I).
Specific bacteria that were related to depression in the PD cohort
included Anaerotruncus spp., however, authors reported no association
between cognition (Montreal Cognitive Assessment and Mini Mental
State Examination scores) and the microbiota. Qian et al. (2018)
showed that the genera Butyricicoccus (p = 0.013) and Clostridium
XIVb (p = 0.0003) were significantly positively associated with
cognitive scores on the Mini Mental State Examination, but no
significant correlations existed between gut microbiota and
depression scores or cognition assessed through the Montreal
Cognitive Assessment (confounding factors adjusted for). Barichella
et al. (2019) found no significant differences between controls and PD
individuals in cognition (measured via Mini Mental State Examination).
They however reported greater Lactobacillus genera to be associated
with intellectual impairment (OR: 1.63, 95 % CI: 1.04–2.56).
3.4.2.2. PD non-motor symptoms: GI. Hasegawa et al. (2015) showed
negative correlations between transit time (i.e. time taken for food to
move from ingestion through to the end of the digestive tract) and PD
duration (r = −0.34) and UPDRS-II (r = −0.40), which indicates a
worsening of constipation with PD progression. However, the authors
did not assess the relationship between transit time and bacterial
diversity in their study. While data are not shown, Heintz‐Buschart
et al. (2018) state that few taxa with different abundances were found
between constipated and non-constipated individuals. Hill-Burns et al.
(2017) used 13 covariates, including GI-discomfort and constipation, to
adjust for analyses of differentially abundant taxa between controls and
PD and found no change relative to the unadjusted analyses. Qian et al.
(2018) used the Rome-III criteria to assess constipation, using this as a
confounding factor in their general linear models to determine bacteria
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that were associated with PD patients after adjusting for confounding
factors. Li et al. (2017) only presented a visual representation of
associations (Wexner scores) between constipation and the top 65
abundant genera finding negative correlations to bacteria such as
Blautia and Ruminococcus and positive correlations to Megasphaera
and Escherichia/Shigella (no r or p values were reported).
4. Discussion
This review aimed to synthesise the available evidence regarding
gut microbiota composition in PD, and its relationship to PD diagnosis,
disease severity and motor and non-motor symptoms. We found nine
studies that directly reported butyrate producing bacteria, at either the
genus or species level, were in lower abundances in PD compared to
control groups. Additionally, α-diversity was not reliably shown to
differ between individuals with PD and healthy controls, across studies,
indicating that α-diversity may be less associated with PD compared to
the abundance of SCFA, specifically butyrate, producing bacteria.
4.1. SCFA, particularly butyrate, producing bacteria are likely important
factors regarding PD onset/progression
One theory by which gut microbiota may influence PD onset or
progression relates to lower abundances of beneficial SCFAs (Hill-Burns
et al., 2017; Li et al., 2017), particularly butyrate, and the bacteria that
produce them. Nine studies found five different butyrate-producing
bacteria at the genus and species level that had lower abundances in PD
groups compared to controls, and assessment of SCFAs showed lower
concentrations of butyrate in PD individuals (Unger et al., 2016). These
bacteria include Faecalibacterium prausnitzii, Eubacterium biforme, Co-
prococcus eutactus, Roseburia faecis and the Blautia genera. Studies
showing butyrate producing bacteria to be lower are strengthened by
their large sample sizes (Barichella et al., 2019; Hill-Burns et al., 2017),
and by the range of confounding factors they accounted for including
sex, BMI, medication, age and constipation (Barichella et al., 2019; Hill-
Burns et al., 2017; Lin et al., 2018). Findings from studies with smaller
sample sizes, and/or that did not consider relevant confounding factors,
should be interpreted with greater caution. Additionally, given the
variation in methodologies used between studies in the analysis of the
gut microbiota, the interpretations drawn from them need to be con-
sidered with some caution as these variations may bias results (Hamady
and Knight, 2009; Thomas et al., 2015). Although methodological dif-
ferences may not bias microbiota results as much as previously thought
(Lauber et al., 2010; Vogtmann et al., 2017).
Two pathophysiological factors we speculate may be involved in PD
onset/progression are gut permeability and gut inflammation leading to
neuroinflammation. This is due to butyrate being involved in pre-
venting increased gut inflammation and GI permeability (Canani et al.,
2016; Tan et al., 2014b), and the current reviews finding that butyrate
producing bacteria may be lower in PD. With increased GI permeability,
which PD individuals have been shown to have (Forsyth et al., 2011), it
could be easier for the neurotropic pathogen to pass through the GI
wall, as proposed in the Dual Hit hypothesis (Hawkes et al., 2009),
where α-synuclein misfolds and aggregates in the enteric nervous
system. Regarding inflammation, neuroinflammation is typical of PD
progression (Hald and Lotharius, 2005; Teismann and Schulz, 2004),
and GI inflammation is involved in this process (Devos et al., 2013).
Therefore, GI inflammation and increased gut permeability, may be two
general pathways where gut microbiota may be involved in PD pa-
thophysiology and as such are discussed in detail below.
The role of GI inflammation in PD involves the immune cells of the
brain, known as microglia. When microglia are activated they con-
tribute to neuroinflammation (Forsyth et al., 2011; Hald and Lotharius,
2005), and GI inflammation has been implicated in this process (Devos
et al., 2013). Additionally, both the presence of α-synuclein (Su et al.,
2008), and peripheral or systemic pro-inflammatory factors that enter
N.D. Nuzum, et al.
the brain (Hald and Lotharius, 2005; Qin et al., 2007), are thought to
activate microglia. The role of these peripheral pro-inflammatory fac-
tors have been demonstrated in a mouse model when peripheral ad-
ministration of tumor necrosis factor-α increased the expression of pro-
inflammatory factors in the brain (Qin et al., 2007). In conjunction with
lower abundance of SCFAs, which help create a pro-inflammatory en-
vironment, the presence of pro-inflammatory pathobionts such as Es-
cherichia/Shigella, Ralstonia and Enterococcus genera (among others)
(Barichella et al., 2019; Heintz‐Buschart et al., 2018; Li et al., 2017; Lin
et al., 2018; Qian et al., 2018), may contribute to an increased in-
flammatory state, with these pathobionts only found to have a greater
abundance in PD groups of the included studies. It is noted that other
Gram-negative bacteria were found to have greater abundance in con-
trols across some studies (Heintz‐Buschart et al., 2018; Lin et al., 2018);
therefore, the role of these pro-inflammatory bacteria need to be in-
terpreted with caution. These Gram-negative bacteria contain lipopo-
lysaccharide in their outer membranes, an endotoxin implicated as an
environmental trigger for PD and used via systemic injections to create
animal models of PD (Qin et al., 2007). Lipopolysaccharide is also in-
volved in microglia activation as demonstrated through systemic in-
jections (Qin et al., 2007). Additionally, animal models indicate the
substantia nigra has approximately 4.5 times more microglia than other
brain regions (Kim et al., 2000), which may help explain the specific
disease progression of PD in humans, as this brain region would be
more susceptible to microglia activation, neuroinflammation and sub-
sequent dopaminergic cell death. Overall, the greater presence of per-
ipheral pro-inflammatory factors, lipopolysaccharide and α-synuclein
may all be contributing to neuroinflammation and by extension PD
onset or progression.
In isolation, the presence of a local inflammatory environment and/
or pathogenic bacteria containing lipopolysaccharide in the GI tract are
not likely to be sufficient to cause neuroinflammation or α-synuclein
misfolding and aggregation. Multiple bacterial endotoxins are found
within the luminal environment of healthy individuals, whom do not
have neuroinflammation or α-synuclein misfolding and aggregation
(Forsyth et al., 2011). Therefore, increased intestinal permeability and
degradation of the mucosal barrier are likely additional contributors, as
inflammatory factors and lipopolysaccharide need to be translocated
across this barrier before contacting with epithelial cells and promoting
α-synuclein misfolding (Forsyth et al., 2011), or leading to neuroin-
flammation. Butyrate is important to this process, as butyrate helps to
prevent intestinal permeability through provision of energy to colono-
cytes (Canani et al., 2011; Donohoe et al., 2011), and because butyrate
is key to mucin production (Brown et al., 2011; Hatayama et al., 2007).
With butyrate absent, bacterial translocation across the epithelium may
be more likely. Tight junction proteins like occludin are also important
for maintaining the GI epithelium and preventing GI permeability
(Rocha et al., 2014). The butyrate producing Faecalibacterium prausnitzii
species has been shown to be protective against models of intestinal
permeability (Laval et al., 2015), through increased expression of oc-
cludin. The Faecalibacterium prausnitzii species was lower in PD groups
across six studies in this review (Barichella et al., 2019; Hill-Burns et al.,
2017; Li et al., 2017; Lin et al., 2018; Petrov et al., 2017; Unger et al.,
2016). Additionally, degradation of mucin is considered pathogenic
(Cohen and Laux, 1995; Smith et al., 1995), with one such mucin de-
grading bacteria being Akkermansia muciniphila (Derrien et al., 2004).
Six of the included studies showed the abundance of Akkermansia
bacteria were greater in PD groups at either the genus level (Barichella
et al., 2019; Heintz‐Buschart et al., 2018; Hill-Burns et al., 2017;
Keshavarzian et al., 2015), or species level (Bedarf et al., 2017; Lin
et al., 2018). Overall, a combination of lowered levels of butyrate
producing bacteria and increased pathogenic mucin degrading bacteria,
could be leading to increased GI permeability and contributing to PD
onset as a result.
In addition to the above-mentioned mechanisms (lower SCFA pro-
ducing bacteria, greater mucin degrading bacteria, and greater
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presence of pro-inflammatory bacteria and lipopolysaccharides), small
intestinal bowel overgrowth (SIBO) may contribute to a greater amount
of GI inflammation, intestinal epithelial permeability and potentially to
neuroinflammation (Devos et al., 2013; Quigley and Quera, 2006). The
prevalence of SIBO is significantly higher in PD individuals compared to
controls (Gabrielli et al., 2011), most likely due to the greater occur-
rence of GI symptoms in PD, like constipation, which creates favourable
conditions for SIBO to occur (Gasbarrini et al., 2007). As GI issues likely
contribute to SIBO occurring it is unlikely this condition relates to PD
onset, but perhaps SIBO relates to PD progression, as research shows it
potentially relates to worse motor functioning (Tan et al., 2014a).
Considering that butyrate is anti-inflammatory and helps maintain
intestinal wall integrity in ways other SCFAs do not, the presence and
abundance of bacteria that produce butyrate in particular could be of
importance to PD onset and progression. The lower abundances of
butyrate producing bacteria that were evident across PD groups of
multiple included studies provides some support for this claim.
Additionally, although other studies have observed lower SCFA con-
centrations between controls and other disease states (Pozuelo et al.,
2015; Qin et al., 2012, 2014), this does not exclude the importance of
SCFAs to PD onset. It instead indicates that additional mechanisms, like
greater abundance of mucin degrading bacteria, pro-inflammatory
bacteria, presence of SIBO, and the presence of a neurotropic pathogen,
are relevant to PD pathophysiology as well.
4.2. α- and β-diversity measures are not reliable markers of PD or its
progression
Greater bacterial diversity has been thought to be indicative of good
health, while low bacterial diversity was thought to relate to disease
states and poor health, with recent research still claiming this is the
case (McBurney et al., 2019). This line of thinking has led to hypotheses
that gut microbiota diversity would differ between control and PD
groups (Scheperjans et al., 2015), and that α-diversity would be lower
in PD. However, the reviews findings show α-diversity does not reliably
differ between groups. Therefore, these results do not support the no-
tion that greater diversity is better for health in all circumstances, and
instead the relationships between gut microbiota and the progression of
various diseases are likely more complex than just altered bacterial
diversity, considering different diseases have different pathological
processes and likely involve various microbiota-host interactions
(Gerritsen et al., 2011). Additionally, we mention bacterial diversity
secondarily to the abundance of specific butyrate producing bacteria in
this review as we find the latter to have stronger evidence regarding its
role in PD pathophysiology, with the evidence for the potential roles of
bacterial diversity (specifically α-diversity) in PD pathophysiology
being weaker.
It is typical for a range of α-diversity indices to be measured as each
index measures a different aspect of α-diversity and there is no clear
consensus as to the most informative index to use (Beck and
Schwanghart, 2010; Morris et al., 2014). While a range of measures
were used across most studies Petrov et al. (2017), who showed α-di-
versity was lower in PD groups, only used a single measure of α-di-
versity (Chao1 Index). The other studies that found PD individuals had
higher α-diversity (Barichella et al., 2019; Keshavarzian et al., 2015;
Qian et al., 2018), or that found no between-group differences (Bedarf
et al., 2017; Hopfner et al., 2017; Li et al., 2017; Lin et al., 2018;
Scheperjans et al., 2015), utilised multiple measures (See Table 2 for a
full list of α-diversity measures used across studies). The fact that
Petrov et al. (2017) reported only a single measure diminishes the re-
liability of their finding, and the multiple measures used across the
other studies suggests that there may in fact be no reliable α-diversity
differences between groups, or that PD groups have greater α-diversity.
Additionally, some studies who reported differences in bacterial di-
versity had larger sample sizes (n = 34–193) (Barichella et al., 2019;
Keshavarzian et al., 2015; Petrov et al., 2017; Qian et al., 2018), than
N.D. Nuzum, et al.
those reporting no differences, suggesting a possible lack of power in
latter studies (n = 14–74) (Bedarf et al., 2017; Hopfner et al., 2017; Li
et al., 2017; Lin et al., 2018; Scheperjans et al., 2015). Finally, while
many factors are known to influence gut microbiota diversity, only one
study assessed any potential confounding factors in relation to α-di-
versity, in this case age and sex (Hopfner et al., 2017).
While the current literature does not present strong evidence to
support a-diversity differences in PD pathophysiology, it remains pos-
sible that the lack of consensus is derived from a combination of factors
such as lack of power, differences in α-diversity measures used, and
measurement error due to confounding. This review supports the idea
that mechanisms other than lower microbiota diversity (described
through α-diversity indices), such as decreased abundance of SCFA-
producing bacteria and increased GI permeability, are associated with
PD.
4.3. The optimal range hypothesis: too little or too many, the optimal
number of gut microbiota
Our bodily functions exist across a normal homeostatic range, with
large fluctuations outside of this leading to health consequences. Given
gut microbiota are responsible for a plethora of key functions that
specifically relate to our overall homeostasis (E.g. maintenance of in-
testinal wall integrity), we should similarly consider individual bacteria
abundance to have negative health outcomes when they decrease or
increase outside of a certain range, whereby dysfunction of key bac-
terial functions may occur. I.e. there may be an optimal range that key
bacteria or bacterial groups should ideally remain within, to ensure
optimal host health. Butyrate producing bacteria were generally shown
to be lower in PD, with lower levels of butyrate reported as well (Unger
et al., 2016). In contrast, Akkermansia bacteria were demonstrated to be
in greater abundance in PD groups (Barichella et al., 2019; Bedarf et al.,
2017; Heintz‐Buschart et al., 2018; Hill-Burns et al., 2017;
Keshavarzian et al., 2015; Lin et al., 2018), with these bacteria known
pathogenic mucin degraders. If the observed abundance levels of these
bacteria have decreased, or increased outside of a ‘normal’ range it is
possible that dysfunctional physiological processes would result, po-
tentially leading to PD onset. We propose that the presence/absence
and abundance of specific bacteria, or groups of specific bacteria, who
perform critical functions, are key to our overall health and should be
considered more important than bacterial diversity (α-diversity). Ad-
ditionally, depending on the bacteria and their purported functions,
either increased or decreased abundance of those bacteria may be
detrimental.
4.4. Parkinson’s disease symptoms and severity
In establishing the relationships gut microbiota may have to PD, it is
important to assess how gut microbiota differences may relate to PD
symptoms, severity and duration. Duration of PD and worse PD motor
symptoms were positively correlated to Escherichia/Shigella, with ne-
gative correlations found between putative butyrate producing bac-
teria, Blautia, and PD duration as well as motor-symptoms (Li et al.,
2017). These correlations do align with the above stated hypothesis that
these bacteria may be involved in PD pathophysiology due to them
being pro-inflammatory, and it may be that Escherichia/Shigella cause
worse motor-symptoms and/or potentially increase with increasing PD
duration. However, a lack of replication across studies means con-
sistently identifying gut microbiota that relate to PD duration and
motor-symptoms is not possible at this time.
As for non-motor symptoms, relationships between gut microbiota
and PD non-motor symptoms, namely cognition, are not clear. Across
three included studies, two found no associations between cognitive
measures and gut microbiota (Barichella et al., 2019; Heintz‐Buschart
et al., 2018), with only one study showing an association (Qian et al.,
2018). The lack of association could in part be due to the cognitive tests
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utilised, which only included the Montreal Cognitive Assessment and
the Mini-Mental State Examination, with these tests used as screening
tools to rule out cognitive impairment rather than being sensitive
measures of variations in cognitive function (Hoops et al., 2009). Future
research should use measures targeting various cognitive domains that
are sensitive to cognitive performance changes. Doing so would help
elucidate whether relationships between gut microbiota and cognition
are apparent, and whether these relationships differ from normal
ageing to PD. As the MGB-axis field is newly emerging, it is not sur-
prising that few of the included studies have used cognitive testing in
their protocols. This is generally the case across other neurodegenera-
tive conditions, and even in healthy controls research is limited. For
example, in healthy older adults, gut microbiota composition was as-
sociated with cognitive test performance across a brief cognitive test
battery (Manderino et al., 2017), however associations were only at the
phylum level and without identifying specific genera or species of
bacteria, relating cognition to bacteria and their functions cannot be
determined. Additionally, Mini Mental State Examination scores in in-
dividuals with Alzheimer’s disease improved after probiotic ingestion
which contained various Lactobacillus species as well as Bifidobacterium
bifidum (Akbari et al., 2016). However, authors did not measure gut
microbiota differences pre- or post-intervention, meaning associations
between abundances of specific gut microbiota, or gut microbiota di-
versity, and cognition cannot be made (Akbari et al., 2016). These
findings suggest that while a link between cognition and gut microbiota
may be apparent, the specific links between various cognitive domains
and presence/absence and abundance of specific bacteria are still un-
known and require further investigation. Without understanding how
cognition relates to gut microbiota in healthy individuals, it is difficult
to describe how this would then relate to individuals with neurode-
generative conditions and in PD.
4.5. Measuring diet and additional lifestyle factors
Given the variation in individuals’ gut microbiota, and the factors
that contribute to this variability (Gerritsen et al., 2011), controlling for
these confounding factors is essential to adequately determine and
understand the role of gut microbiota in PD. While numerous con-
founding factors were assessed across some studies, lifestyle factors like
diet, physical activity and sleep were not. Some studies accounted for
diet by using spousal controls (Hasegawa et al., 2015; Lin et al., 2018;
Qian et al., 2018), as spouses would likely share similar diets to PD
individuals, although this is unlikely to be as effective as directly
measuring diet. Lin et al. (2018) collected dietary data via a ques-
tionnaire, although no details regarding the questionnaire or its validity
are available. Additionally, authors only categorised participants as
‘omnivorous’, ‘meat (-eating)’, or ‘vegetable (-eating)’, providing lim-
ited characterisation of the actual nutritional intakes of participants.
Keshavarzian et al. (2015) collected dietary data with a modified
Harvard food frequency questionnaire, and for total intakes of energy,
macronutrients and dietary fibre no significant differences were seen
between groups. However, this was only investigated in a subsample of
participants (24/38 PD and 15/34 controls). Barichella et al. (2019)
also assessed dietary intake, using a 10-day food diary and 24-h food
recall, assessing intake of calories, protein (vegetable or animal), fibre,
water and alcohol. While fibre ingestion related to increased Lactoba-
cillaceae abundance across both groups, authors mostly used nutri-
tional information as a confounding factor in their analysis of bacterial
abundance and diversity, and baseline measures of fibre between con-
trols and PD individuals were not significantly different.
No other included studies assessed nutritional intake of participants,
and across all studies resistant starch intake (present in e.g. legumes
and whole grains), which can help commensal bacteria proliferate, and
in turn produce SCFAs (Bourassa et al., 2016; Topping and Clifton,
2001), was not assessed. Additionally, the diversity of plants consumed
was not assessed despite research indicating a larger variety of plants
N.D. Nuzum, et al.
consumed is associated with gut microbial diversity (McDonald et al.,
2018), with this factor potentially explaining differences in bacteria
diversity measures across included studies. Other lifestyle factors like
physical activity and sleep were not assessed, despite links to the gut
microbiota indicating these factors can alter bacterial diversity and
bacterial abundance as well as bacterial metabolites (Allen et al., 2018;
Benedict et al., 2016). Given diet, physical activity and sleep may all
alter bacterial diversity and/or bacterial abundance they need to be
accounted for in studies to properly determine the roles gut bacteria
have in PD onset or progression, and to identify relationships between
gut microbiota and PD severity and symptoms.
4.6. Future directions
Given the proposed relationships between lifestyle factors, like diet,
physical activity and sleep, and the gut microbiota (Allen et al., 2018;
Benedict et al., 2016; David et al., 2014), it is important future studies
include these as part of their analyses. Probiotic use should also be
considered. While outside the scope of this review, additional factors
including environmental toxins (Ball et al., 2019; Betarbet et al., 2000;
Di Monte, 2001; Gorell et al., 1999) and genetics (Ball et al., 2019;
Breydo et al., 2012; Cookson, 2005), and their potential effects on gut
microbiota composition, still need to be considered in PD pathogenesis.
Future studies would also benefit from discussing the functional re-
lationships of bacteria together, due to the influence of certain bacteria
on other bacteria. For example in ‘cross-feeding’ whereby the metabo-
lites of one bacteria are used by other bacteria to produce another,
different, metabolite (For more on ‘cross-feeding’ see Duncan et al.,
2004; Flint et al., 2012). Additionally, to increase the reliability of the
reported results, gut microbiota analysis should not be limited to the
surrogate measure of using faecal samples, but additional use of GI
mucosal microbiota samples, as was conducted by Keshavarzian et al.
(2015), would bolster current results. The observational nature of the
included studies also makes it impossible to state whether gut micro-
biota differences, particularly lowered abundance of butyrate produ-
cing bacteria, contribute to PD, or if gut microbiota differences result
from PD. Longitudinal studies assessing gut microbiota changes over
time in relation to disease onset, pathophysiology and symptom se-
verity of PD, that control for the appropriate confounding factors, are
therefore warranted.
5. Conclusion
The current review indicates that gut microbiota’s role in the mi-
crobiota-gut-brain axis and association to PD may involve lowered
abundance of a range of specific SCFA, particularly butyrate, producing
bacteria (whereby these bacteria may be reduced outside of an ‘optimal
range’). However, the mechanisms of PD pathophysiology are unlikely
to be solely a lack of SCFAs. Rather, a combination of factors including
increased pro-inflammatory bacteria, greater intestinal permeability,
introduction of a neurotropic pathogen and small intestinal bowel
overgrowth, are likely additionally responsible (Canani et al., 2011;
Tan et al., 2014b). Despite this, additional research is needed to address
how these gut microbiota differences relate to PD duration and symp-
toms, and if these differences are still present when accounting for
confounding lifestyle and health factors.
Author contributions
NN completed the search of articles, the screening at each stage as
well as data extraction and risk of bias assessment. Authors WPT, AH,
AL, HM, and ESG participated in the article screening process at the title
and abstract as well as full-text screening stages. Manuscript writing
was completed by NN, with WPT, ESG, and AH revising the overall
manuscript multiple times throughout the writing process. AL provided
comments and revisions on the cladogram figures, methods and results
239
Neuroscience and Biobehavioral Reviews 112 (2020) 227–241
section of the qualitative analysis to do with measures of diversity,
presence/absence and abundance as well as sections in the discussion
relating to gut microbiota specifically. HM provided multiple revision
on the overall discussion section, and helped generate the overall dis-
cussion section, as well as revising the overall document. All authors
read and accepted the final version of the manuscript for submission.
Financial disclosures
An Australian Government Research Training Program Scholarship
supports NN.
Declaration of Competing Interest
The authors report no conflicts of interest.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.neubiorev.2020.02.
003.
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