diabetes research and clinical practice 173 (2021) 108689

Contents available at ScienceDirect

Diabetes Research
and Clinical Practice
journal homepage: www.elsevier.com/locat e/dia bre s

Differential gut microbiota composition between

type 2 diabetes mellitus patients and healthy
controls: A systematic review

Fatin Umirah, Chin Fen Neoh *, Kalavathy Ramasamy *, Siong Meng Lim
Collaborative Drug Discovery Research (CDDR) Group, Faculty of Pharmacy, Universiti Teknologi MARA (UiTM) Kampus Puncak
Alam, Cawangan Selangor, 42300 Bandar Puncak Alam, Selangor Darul Ehsan, Malaysia

A R T I C L E I N F O A B S T R A C T

Article history: Aims: This systematic review summarised the latest findings on differential composition
Received 15 April 2020 of gut microbiota in T2DM.
Received in revised form Methods: Literature search was performed using electronic databases. Relevant studies
24 December 2020 were identified, extracted and assessed for risk of bias. The primary outcome of this sys-
Accepted 25 January 2021 tematic review was the composition of gut microbiota in healthy controls and T2DM while
Available online 4 February 2021 the secondary outcomes included the correlation of gut microbiota with metabolic param-
eters.
Results: Thirteen case-control studies involving 575 T2DM and 840 healthy controls were
Keywords:
included. T2DM patients exhibited a marked increase in lactobacilli. Six studies found lac-
Dysbiosis
tobacilli to predominate the gut of T2DM patients; however, this could be confounded by
Metagenome
the types of antihyperglyacemic medications. Conversely, butyrate producers dominate
Microbiome
the gut of healthy controls. In T2DM patients, butyrate producers were surprisingly higher
Type 2 diabetes mellitus
in those taking metformin intake than those not taking the drug. Whilst lactobacilli were
found to be higher with increased plasma glucose, conflicting correlations were observed
between various genera and anthropometric measurements, dietary intake, lipid profiles
and inflammatory markers. There were moderate to strong significant positive correlations
between the class Clostridia and phylum Firmicutes with pro-inflammatory IFN-c as well as
between Negativicutes and IL-6.
Conclusions: Altogether, butyrate-producing bacteria are negatively correlated to glycaemic
parameters. Lactobacilli are higher in T2DM patients and Firmicutes is correlated with
inflammation.
Ó 2021 Elsevier B.V. All rights reserved.

1. Introduction
Type 2 diabetes mellitus (T2DM) is a complex, multifactorial
disorder which can be attributed to either insulin insensitivity
or insulin deficiency [13]. Various risk factors have been iden-
tified which include age, diet, family history, sedentary life-
style and obesity [1]. Alteration of gut microbiota (i.e.
dysbiosis) could play an important role in the development
and progression of T2DM [15]. Opportunistic pathogens and
endotoxin-producing bacteria were found to increase

* Corresponding authors.
E-mail addresses: neohchinfen@uitm.edu.my (C.F. Neoh), kalav922@uitm.edu.my (K. Ramasamy).
https://doi.org/10.1016/j.diabres.2021.108689
0168-8227/Ó 2021 Elsevier B.V. All rights reserved.
2 diabetes research and clinical practice
whereas SCFA-producing bacteria decreased in T2DM when
compared to healthy controls [1,12].
It is increasingly being recognised that frequent intake of
high-fat and/ or high-fructose diet causes dysbiosis of gut
microbiota [16]. Dysbiosis disrupts intestinal tight junction
proteins and increases gut permeability [16]. This allows
Gram negative bacteria-derived lipopolysaccharide (LPS) to
transfuse into blood stream through the leaky gut, and trigger
the onset as well as progression of low-grade inflammation,
leading to metabolic endotoxaemia and oxidative stress [16].
As LPS increases in the blood, it increases activation of
inflammatory pathways, which causes insulin resistance
[17], and thereby triggers the onset and progression of
T2DM [16].
To date, 13 case-control studies that met the inclusion cri-
teria had evaluated differential gut microbiota compositions
between healthy controls and T2DM across various countries
[1–12,14]. Nevertheless, the results from these case control
studies seem to vary. As such, the present systematic review
had systematically analysed evidence of differential composi-
tion of faecal microbiota between T2DM and healthy controls
as well as its correlation with metabolic parameters.
2. Materials and methods
2.1. Literature search strategy
Literature search was performed by using electronic data-
bases (i.e. PubMed/Medline, EMBASE and Web of Science)
with restriction to English language only. Both medical sub-
ject headings (MeSH term) and free text strings (Supplemen-
tary material) were employed. The main search terms used
were as follow: ‘diabetes mellitus’, ‘type 2 diabetes’, ‘meta-
genome’, ‘microbiome’ and ‘next generation sequence’. The
terms were combined with Boolean operators to capture all
studies related to T2DM and gut microbiota. The last search
was performed in February 2020 without restriction to publi-
cation date. This systematic review has been registered with
International Prospective Register of Systematic Reviews
(PROSPERO) (CRD42018076374).
2.2. Study selection
The inclusion criteria were as follow: a) case-control studies
that had included T2DM (case) and healthy adults (control)
and b) studies with analyses of faecal gut microbiota. The
studies were excluded if they were a) presented only as
abstracts with no full report of findings, b) on-going clinical,
in vivo or in vitro studies, c) review papers, non-English litera-
tures or protocol papers, d) Gestational Diabetes Mellitus
(GDM), Type 1 Diabetes Mellitus (T1DM) or metabolic diseases
other than T2DM and e) studies that did not compare gut
microbiota in T2DM to that of healthy controls. Titles,
abstracts and keywords were screened to determine publica-
tions of relevance. Whilst the primary outcome of this study
was the composition of gut microbiota in both healthy con-
trols and T2DM, the secondary outcomes included the corre-
lation of gut microbiota with glycaemic parameters, lipid
profiles, inflammatory markers and anthropometric mea-
173 (2021) 108689
surements. Search strategy and data extraction of all poten-
tial studies were independently performed by two authors
(FU and CFN). Discrepancies on study inclusion were resolved
through discussion and consensus between all authors.
2.3. Assessment of risk of bias
Two authors (FU and CFN) had independently assessed the
risk of bias for the shortlisted studies using the criteria
adapted from the Newcastle-Ottawa Scale (NOS) [18]. The
assessments included selection bias (i.e. case and control def-
inition), comparability bias (any adjustment to confounding
factors) and exposure bias (i.e. ascertainment of exposure
and method used). The studies were deemed low risk of bias
when overall quality score of
3 or high risk of bias when
overall quality score of <3 [19]. Discrepancies on these assess-
ments were resolved through discussion and consensus
between all authors.
3. Results
3.1. Description of shortlisted studies
Of the 3485 studies that were being identified, 258 were dupli-
cates and therefore removed (Fig. 1). A total of 3206 studies
were further excluded based on the following reasons: 94
were in vivo or in vitro studies, 1662 had evaluated other
pathologies, 31 had described only the protocol, 402 were
review papers, 663 did not compare between T2DM and
healthy controls, 132 were randomised clinical trial (e.g. pro-
biotics, prebiotics), 54 had evaluated T1DM and the remaining
168 were non-English papers. Altogether, 13 articles were
included in the present systematic review.
3.2. Study characteristics
Table 1 summarises the characteristics of studies that were
shortlisted for this systematic review. All were case-control
studies, involving 575 T2DM patients and 840 healthy con-
trols. The cases were T2DM patients while the controls were
made up of healthy individuals or those with normal glucose
tolerance. 13 studies had looked into the differences of gut
microbiota compositions between T2DM patients and healthy
controls [1–12,14] (Table 1). Seven were from untargeted
approach [1–5,12,14]. Of the seven studies, two were con-
ducted in China [2,4], three in Europe [1,3,5], one in Brazil
[12] and Mexico [14] respectively. On the other hand, another
six studies were from targeted approach [6–11]. Two studies
were conducted in Japan [6,7] two in Iran [9,11] and two in
China [8,10]. In general, out of 13 included studies, seven
had no gender limit in participant selection [2,4,5,8,10,12,14],
four had similar number of male and female participants
[6,7,9,11], one included only women [1] and one included only
men [3]. Participants in both groups were predominantly
female between 40 and 70 years old.
Ten studies defined T2DM patients based on their HbA1c
values [1,2,5–7,9–12,14], two studies were based on their OGTT
values [3,4] while no information was obtained from one
study [8]. Of the 13 included studies, seven studies reported
 diabetes research and clinical practice 173 (2021) 108689 3

In vivo In vitro

Fig. 1 – Study selection PRISMA flowchart.

that T2DM patients were on antihyperglycaemic medications
[1,5–8,10,12] while no information was obtained from the
remaining studies [2–4,9,11,14]. One of the studies further
stratified the T2DM patients according to the metformin
treatment status [5].
It is important to note that gut microbiota composition
was analysed using different techniques. Whilst seven
studies had adopted untargeted approaches; three had
used the shotgun metagenomic sequencing [1,2,5], another
two used 16S rRNA sequencing (Illumina MiSeq) [12,14] and
yet another two used pyrosequencing (i.e. Tag-encoded
FLX-amplicon [3] and 454 GS FLX [4]), another six studies
had used targeted approaches [i.e. quantitative real-time
polymerase chain reaction (qPCR) [8–11], reverse transcrip-
tion qPCR (RT-qPCR) [6] and PCR [7]]. The differential com-
position of gut microbiota was reported as primary
outcome (Table 1).
3.3. Differential gut microbiota compositions between
T2DM and healthy controls
Fig. 2 showed the compositional changes of gut microbiota at
genus level in T2DM as opposed to that of healthy controls.
One of the most notable genus that consistently showed a sig-
nificant domination (p < 0.05) in the gut of T2DM was lacto-
bacilli while genera Faecalibacterium and Roseburia increased
in healthy controls in two or more studies. On the other hand,
Bifidobacterium, was however, inconsistent.
 4
Table 1 – Study characteristics of healthy controls and T2DM (n = 13).
Author (year) Sample size Country Gender (M/F), Age (mean ± SD) Glycaemic control Antihyperglycaemic Method for metagenomics Outcome measures
parameter (T2DM) treatment (class)
Healthy control T2DM Healthy control T2DM Sequencing used Quantification Primary outcome Secondary outcomes

Untargeted approach (n = 7)
Zhang, et al. [4] 44 13 China M/F:12/32, Age: M/F:7/6), Age: OGTT, median: NR 454 GS FLX – Faecal microbiota compositions Association between gut
55.0 ± 9.0 52.0 ± 9.0 13.5 mmol/l microbiota diversity and clinical
indicators
Qin, et al. [2] 174 171 China M/F: 84/90, Age: M/F: 107/65, Age: HbA1c, mean: NR Illumina GAIIx and HiSeq – Faecal microbiota compositions –
42.0 ± 14.0 53.0 ± 13.0 48.0 mmol/mola 2000

diabetes research and clinical practice

Karlsson, et al. [1] 43 53 Sweden Age: 70 (mean ± SD not reported) HbA1c, mean ± SEM: 47.1 Yes (Met, SU) Illumina HiSeq 2000 – Faecal microbiota compositions Correlations between serum
Gender: All female ± 1.3 mmol/mol biomarkers and species or MGCs
Forslund, et al. [5] 554 (277[5] 106e (17[5] Denmark M/F: NR M/F: 28/78e HbA1c, mean ± SD: 8.5 ± 1.8e Yes (Met) Illuminag – Faecal microbiota Correlations between T2DMf with
+ 185[1] + 92[2]) + 33[1] + 56[2]) Age: 54.2 ± 13.1 47/46f 8.1 ± 2.1f compositions gut microbiota
93f (58[5] Age: 58.8 ± 12.8e
+ 20[1] + 15[2]) 61.1 ± 9.7f
Larsen, et al. [3] 18 18 Denmark Age: 56.0 ± 13.0 OGTT, mean ± SD: NR Tag-encoded FLX Amplicon Faecal microbiota –
Gender: All male 16.6 ± 5.4 mmol/l compositions
Leite, et al. [12] 22 20 Brazil M/F: 12/10, Age: M/F: 9/11, Age: HbA1c, mean ± SD: Yes (Met, DPP-4i, Illumina MiSeq – Faecal microbiota Correlations between systemic
55.7 ± 8.3 58.9 ± 8.4 Not stated insulin) compositions inflammation cytokines and
plasma LPS concentration with
bacteria
Lambeth, et al. [14] 15 14 Mexico M/F:5/10, Age: M/F:6/8, Age: HbA1c, mean ± SD: NR Illumina MiSeq Pico green Faecal microbiota compositions Relationship between gut
55.5 ± 13.7 62.0 ± 10.0 7.9 ± 1.7% dsDNA assay microbiota compositions and
HbA1c levels
Targeted approach (n = 6)
Adachi, et al. [7] 59 59 Japan M/F: 34/25, Age: 62.0 M/F: 34/25, Age: 64.0 HbA1c, median: Yesc PCR (16S rDNA) PCR Faecal microbiota compositions –
(59.0–69.0)b (57.5–69.0)b 54.0 mmol/mol (51.0–60.0)b
Sato, et al. [6] 50 50 Japan M/F:26/24, Age: M/F:26/24, Age: HbA1c, mean: Yesd RT-qPCR (16S rRNA) R T-qPCR Faecal microbiota compositions Association of faecal microbiota
60.2 ± 12.9 62.5 ± 10.8 72.0 mmol/mola with various clinical parameters
Navab-Moghadam, et al. [9] 18 18 Iran M/F: 7/11, Age: M/F: 7/11, Age: HbA1c, mean ± SD: NR RT-PCR (16S rRNA) qPCR Faecal microbiota compositions Correlations between studied
52.1 ± 7.56 54.3 ± 7.63 6.7 ± 1.0% bacterial species with BMI
Sedighi, et al. [11] 18 18 Iran M/F: 7/11, Age: M/F: 7/11, Age: HbA1c, mean ± SD: NR RT-PCR (16S rRNA) qPCR Faecal microbiota compositions Correlations between studied
52.1 ± 7.56 54.3 ± 7.63 6.7 ± 1.0% bacterial species with BMI
Lê, et al. [10] 30 50 South China M/F:17/13, Age: M/F: 23/27, Age: HbA1c, mean: 21/50 were on NR qPCR Faecal microbiota compositions Anthropometry and metabolic
41.0 ± 11.0 60.0 ± 8.0 <64.0 mmol/mola medication quantifications
(Met, a-GI, SU)
Wu, et al. [8] 12 16 China M/F: Not stated, M/F:10/6, Agea: 48– NR 4/16 were on PCR-DGGE qPCR Faecal microbiota compositions Identification of dominant

173 (2021) 108689

Agea: 50–65 62 medicationc (16S rRNA) bacteria within-groups

Abbreviations: T2DM, type 2 diabetes mellitus; NGT, normal glucose tolerance; NR, not reported; aSD values not stated; bthe medians (inter-quartile ranges); cdrug class of the antihyperglycaemic
medication is not provided by the authors; dMet, DPP-4i, SU, a-GI, thiazolidine, glinide, GLP-1 receptor agonist, insulin; e without Met; fwith Met; gtype of sequence not mentioned. DGGE, denaturing
gradient gel electrophoresis; qPCR, quantitative polymerase chain reaction; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; PCR, polymerase chain reaction; rRNA, ribosomal
ribonucleic acid; Met, metformin; DPP-4i, dipeptidyl peptidase 4 inhibitors; SU, sulphonylurea; a-GIs, a-glucosidase inhibitors. Note: Whilst anthropometric measurements such as BMI and waist
circumference were recorded in all studies, habitual diet was only assessed in five studies [6–9,11] (Data not shown).
 diabetes research and clinical practice 173 (2021) 108689 5

A)

Abiotrophia
Sporobacter Anaerostipes
Acidaminococcus
Subdoligranulum Clostridium Blautia
Anaerotruncus
Parabacteroides Eubacterium Clostridiales
Bacillus
Prevotella Streptococcus Coprococcus
Bulleidia
Bifidobacterium Alistipes Faecalibacterium
Dorea
Collinsella Bacteroides Lachnospira
Holdemania
Eggerthella Escherichia Megamonas
Lactobacillus
Bilophila Akkermansia Megasphaera
Oscillobacter
Enterobacteriaceae Roseburia
Peptostreptococcus
Desulfovibrio Veillonella
Ruminococcus
Haemophilus

B)

Lactobacillus
Clostridium
Staphylococcus
Prevotella Bacteroides
Enterobacteriaceae
Bifidobacterium Atopobium
Pseudomonas
Fusobacterium

C)

Roseburia
Lactobacillus
Subdoligranulum
Escherichia Intestinibacter Paraprevotella
Clostridiales
Haemophilus
6 diabetes research and clinical practice 173 (2021) 108689

3.4. Correlations between gut microbiota compositions
and clinical parameters
Table 2 shows the correlations between gut microbiota com-
position and clinical parameters across all studies. Correla-
tion values (r) of 0.10  r  0.39, 0.40  r  0.69, 0.
70  r  0.89 and 0.90  r  1.00 indicate weak, moderate,
strong and very strong correlation, respectively [20].
3.4.1. Glycaemic parameters
There were six studies that had related gut microbiota com-
position to glycaemic parameters in T2DM [1,3,4,7,12,14]
(Table 2). Two studies had evaluated the effects of B:F ratio
[3,4] on plasma glucose levels [fasting blood glucose (FBG)
and 2-hour postprandial glucose (2PPG)]. There was a moder-
ate but significant positive correlation between B:F ratio and
2PPG (r = 0.47, p = 0.04) [3]. However, no significant correlation
was found between B:F ratio and FBG (r = 0.01, p > 0.05) [4].
Lactobacilli that belong to the phylum Firmicutes (data
was not available/ reported) and L. gasseri were positively cor-
related with glycaeted haemoglobin (HbA1c) (r = 0.40,
p = 0.0034) and FBG (r = 0.40, p = 0.0047) [1]. There was a weak,
but positive correlation between lactobacilli and a 2PPG level
(r = 0.33, p = 0.05) [3].
In general, the main butyrate-producing bacteria (i.e.
Clostridium spp., family Ruminococcaceae and Roseburia spp.)
were negatively correlated with most of the glycaemic param-
eters. Bacteria from the family Ruminococcaceae were reported
to decrease significantly with increased HbA1c level (r = 0.69,
p = 0.02) [12]. It is noteworthy that there were weak, negative
correlations between Clostridium spp. and insulin (r = 0.33,
p = 0.02) [1] as well as C-peptide test (r = 0.36, p = 0.02) [1].
Whilst only C. clostridioforme showed a weak, positive correla-
tion with C-peptide level (r = 0.28, p = 0.02), Clostridium cluster
XVIII showed a weak negative correlation with HOMA-IR
(r = 0.30, p = 0.022) [7]. Clostridium spp. faecal count was
reported to be moderately, positively correlated with adipo-
nectin (r = 0.47, p = 0.00000875) [1]. On the other hand, no sig-
nificant correlations were found between Clostridium spp. and
HbA1c (r = 0.31, p = 0.06) [1], FBG (r = 0.26, p = 0.10) [1] or
2PPG level (r = 0.42, p = 0.06) [3]. Almost similar correlation
was noted between Roseburia spp. and 2PPG (r = 0.52,
p = 0.06) level [3].
Bacteroides intestinalis and Bacteroides spp. showed a weak,
negative correlation with insulin level (r = 0.28, p = 0.04) [1]
and FBG (r = 0.27, p = 0.043) [7], respectively. There were,
however, positive correlations between Betaproteobacteria
spp. relative abundance (r = 0.46, p = 0.04), Bacteroides-
Prevotella to C. coccoides-E. rectale group ratio (r = 0.38,
p = 0.03) and Bacteroides-Prevotella to C. coccoides subgroup ratio
(r = 0.46, p < 0.01) and 2PPG level [3]. Prevotella spp., on the
other hand, were presented with a weak, negative correlation
with HbA1c (r = 0.27, p = 0.038) [7]. No significant correlation
was noted between Prevotella spp. and 2PPG level [3].
3.4.2. Anthropometric measurements
Five studies had reported the effect of anthropometry mea-
surements on gut microbiota profiles [1,3,6,9,11] (Table 2).
Weak, negative correlations were only found between C. coc-
coides count (r = 0.31, p = 0.03) [6] and Atopobium cluster
(r = 0.32, p = 0.02) [6] with BMI. Elsewhere, a weak, negative
correlation was observed between B. intestinalis and waist cir-
cumference (r = 0.29, p = 0.04) [1]. No significant correlations
were found in the remaining three studies [3,9,11].
3.4.3. Dietary intake
Three studies had evaluated of the differential gut microbiota
composition between T2DM and healthy controls by docu-
menting dietary intake [6–8]. C. coccoides count appeared to
have a weak, negative correlation with energy (r = -0.33,
p = 0.02) and saturated fatty acid intake (r = 0.31, p = 0.03)
[6]. Similar trend was observed with order Lactobacillales and
Bifidobacterium spp., both of which showed a negative correla-
tion with protein (r = 0.28, p = 0.035) [7] and carbohydrate
intake (r = 0.42, p = 0.001) [7], respectively. On the other
hand, protein (r = 0.36, p = 0.005) and fat (r = 0.30, p = 0.021)
intakes showed weak positive correlations with Clostridium
cluster XI as well as carbohydrates intake (r = 0.27,
p = 0.042) with Clostridium cluster VI while fat intake showed
otherwise (r = 0.26, p = 0.046) [7].
3.4.4. Lipid profiles
Three studies had investigated the changes in gut microbiota
composition with lipid profiles [1,6,7]. Order Lactobacillales was
negatively correlated with total cholesterol (TC) (r = 0.32,
p = 0.016) [7]. There were weak, positive correlations between
Clostridium spp. (r = 0.35, p = 0.0098) [1], Clostridium cluster XI
(r = 0.28, p = 0.034) [7] with high density lipoprotein (HDL),
between C. clostridioforme clusters and triglycerides (TG) level
(r = 0.29, p = 0.00980) [1] and between Clostridium cluster XIVa
and TC (r = 0.32, p = 0.014) [7], respectively. Karlsson, et al. [1],
however, was the only study that indicated a weak, negative
correlation between Clostridium spp. and TG level (r =0.33,
p = 0.00605) [1]. Atopobium cluster was positively correlated
with HDL (r = 0.35, p = 0.01) [6].
3.4.5. Inflammatory markers
There were three studies that had described the relationship
between inflammatory markers with gut microbiota composi-
tion. Class Clostridia was positively correlated with interferon

3
Fig. 2 – Differential. composition of gut microbiota at genus level between T2DM when compared to healthy controls (n = 13)
in A) untargeted approach and B) targeted approach. *p < 0.05 in
two studies. Blue colour arrow represented gut microbiota
were higher in T2DM while increased gut microbiota compositions in healthy controls were indicated by red colour arrow.
Gut microbiota compositions that were found to be in inconsistent pattern across all studies were shown in the middle circle.
C) Differential genera abundances between non-metformin T2DM and metformin-T2DMa; between non-metformin T2DM
and controlb (significance at FDR < 0.1) [5]; bp, butyrate producers. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
Table 2 – Correlation between gut microbiota composition and relevant parameters (n = 13).
B:F Lactobacilli Butyrate-producing bacteria Others

Untargeted Targeted Untargeted Targeted Untargeted Targeted Untargeted Targeted

Glycaemic HbA1c "* Lactobacilli (r = NA) [1] ; Clostridium spp. (r = -0.31) [1] M microbiome diversity (r = 0.07) [14] ;* Prevotella spp. (r = -0.27) [7]

diabetes research and clinical practice

Parameters "* L. gasseri (r = 0.40) [1] ;* Ruminococcaceae (r = -0.69) [12]
FBG " (r = 0.01) [4] "* Lactobacilli (r = NA) [1] ; Clostridium spp. (r = -0.26) [1] M Gut microbiota abundance (r = NA) [12] ;* Bacteroides spp. (r = -0.27) [7]
"* L. gasseri (r = 0.40) [1]
2PPG "* (r = 0.47) [3] " Lactobacilli (r = 0.33) [3] ; Clostridia (r = -0.42) [3] " Prevotella (r = 0.32) [3]
; Roseburia (r = -0.52) [3] "* Betaproteobacteria (r = 0.46) [3]
"* Bacteroides-Prevotella : C. coccoides-E. rectale (r = 0.38) [3]
"* Bacteroides-Prevotella : C. coccoides subgroup (r = 0.46) [3]
Insulin / C- ;* Clostridium spp. [r = -0.33 / r = -0.36) [1] ;* B. intestinalis (r = -0.28) [1] ;* Clostridium cluster XVIII (r = -0.30) [7]
peptide / "* C. clostridioforme (r = 0.28) [1]
HOMA-IR
Adinopectin "* Clostridium spp. (r = 0.47) [1]
Diet Energy / ;* C. coccoides (r = -0.33 / r = -0.31) [6] "* Clostridium cluster XI (r = 0.30) [7]
saturated ;* Clostridium cluster VI (r = -0.26) [7]
fatty acid
intake
Carbohydrates
"* Clostridium ;* Bifidobacterium spp. (r = -
cluster VI 0.42) [7]
(r = 0.27) [7]
Protein ;* Lactobacillales (r = -0.28) [7]
"* Clostridium cluster XI (0.36) [7]
Inflammatory hs-CRP ;* C. coccoides (r = -0.39) [6] ;* Atopobium cluster (r = -0.39) [6]
Markers IFN-c "* Clostridia (r = 0.69) [12] "* Firmicutes (r = 0.75) [12]
"* Prevotella (r = 0.66) [12]
;* Bacteroides (r = -0.70) [12]
IL-17A "* Enterobacteriaceae (r = 0.58) [12]
IL-6 "* Negativicutes (r = 0.59) [12]

173 (2021) 108689

Anthropometric Waist circumference
Measurements ; Clostridium spp. (r = -0.30) ;* B. intestinalis (r = -0.29) [1]
[1]
BMI ; (r = -0.32) [3] " Lactobacilli (r = 0.30) ; Clostridium spp. (r = 0.22) ;* C. coccoides (r = -0.31) [6] " Bacteroides-Prevotella : C. coccoides-E. ;* Atopobium cluster (r = -0.32) [6]
[11] [1] ; F. prausnitzii (r = -0.35) [9] rectale (r = 0.06) [3] " Fusobacterium (r = 0.83) [11]
; Bifidobacterium (r = -0.06) [11]
; Prevotella (r = -0.08) [11]
; B. longum (r = -0.21) [9]
; B. fragilis (r = -0.01) [9]

Lipid Profiles HDL "* Clostridium spp. (r = 0.35) [1] "*Atopobium cluster (r = 0.35) [6]
"*Clostridium cluster XI (r = 0.28) [7]
TG ;* Clostridium spp. (r = -0.33) [1]
"* C. clostridioforme (r = 0.29) [1]
TC "* Clostridium cluster XIVa (r = 0.32) [7] ;* Order Lactobacillales (r = -0.31) [7]

Abbreviations: *p < 0.05 when compared to control; ", positive correlation; ;, negative correlation; M, no correlation; B:F, Bacteroidetes to Firmicutes ratio; HbA1c, glycaeted haemoglobin; FBG, fasting
blood glucose; 2PPG, 2-hour postprandial glucose; HOMA-IR, homeostatic model assessment of insulin resistance; BMI, body mass index; HDL, high density lipoprotein; TG, triglyceride; TC, total
cholesterol; hs-CRP, high sensitive C-reactive protein; IFN-c, interferon gamma; IL-17A, interleukin 17A; IL-6, interleukin 6; r, correlation coefficient value; NA, not available.

7
 8diabetes research and clinical practice
Table 3 – Newcastle-Ottawa quality assessment scale for included case-control studies (n = 13).
Quality assessment criteria Acceptable (*) Leite, et al. [12] Lambeth, Zhang, Karlsson, Qin, Forslund, Larsen, Adachi, et al. [7] Navab-Moghadam, Sedighi, et al. [11] Sato, et al. [6] Lê, et al. [10] Wu, et al. [8]
et al. [14] et al. [4] et al. [1] et al. [2] et al. [5] et al. [3] et al. [9]

Is the case definition adequate? Yes, with independent validation * * * * * * * *

Representativeness of cases?
Selection of controls?
Definition of controls?
Study controls for one factor
Study controls for
additional factors
Ascertainment of exposure?
Same method of ascertainment
of cases and controls?
Consecutive or obviously
representative series of cases
Community controls?
No history of T2DM
Secure record, Structured
interview where blind to case/
control status
Yes
*
* * * * * * * * *
* * * * * *
* * * * * * * * *
* * * * * * *
*
* * * * * * * * * * * *

173 (2021) 108689

Non-response rate? Same for both groups * * * *
Overall Quality Score (Maximum 9) 4 3 4 6 2 5 5 4 6 6 3 6 3

Abbreviations: *represents fulfilled individual criterion within the subsection.

 diabetes research and clinical practice
gamma (IFN-c) (r = 0.69, p = 0.02) [12] while C. coccoides count
was negatively correlated with high sensitivity C-reactive pro-
tein (hs-CRP) (r = 0.39, p = 0.0066) [6]. Leite et al. [12] reported
moderate to strong positive correlations between the relative
abundance of phylum Firmicutes (r = 0.75, p = 0.01) and Prevo-
tella spp. (r = 0.66, p = 0.02) with IFN-c, Enterobacteriaceae spp.
with interleukin 17A (IL-17A) (r = 0.58, p = 0.04) and Negativi-
cutes spp. with interleukin 6 (IL-6) (r = 0.59, p = 0.04), respec-
tively. However, genus Bacteroides showed a strong, negative
correlation with IFN-c (r = 0.70, p = 0.01) [12] while Atopobium
cluster had a weak, negative correlation with hs-CRP
(r = 0.39, p = 0.0058) [6].
3.5. Antihyperglycaemic agents
Three studies had described the effects of antihyperglycaemic
agents (e.g. metformin and a-glucosidase inhibitors) on the
gut microbiota compositions [1,5,6]. Clostridium spp. and
Eubacterium spp. faecal counts were significantly decreased
in metformin-T2DM patients [1]. On the other hand, Enter-
obacteriaceae spp. count [1,6] and Staphylococuus spp [6] were
significantly increased in patients who were taking met-
formin. When comparing the gut microbiota changes
between metformin-naive and metformin-treated T2DM
patients, an increase in Escherichia spp. abundance (r = 0.25,
q = 0.07) while a decrease in Intestinibacter spp. (r =0.40,
q = 0.01) were noted in metformin-treated T2DM patients
[5]. T2DM who were taking a-glucosidase inhibitors were pre-
sented with significantly higher number of genera Bifidobac-
terium, Lactobacillus and Enterococcus [6]. In the study by
Forslund, et al. [5], the authors reported a depletion of known
butyrate producers i.e. Roseburia spp., Subdoligranulum spp.
and butyrate-producing Clostridiales spp. in metformin-
untreated T2DM when compared to healthy controls. Of note,
the increase in lactobacilli in T2DM patients was not seen in
the study by Forslund, et al. [5] when the analysis was further
controlled for metformin treatment status.
3.6. Risk of bias and publication bias
Table 3 summarises the outcomes of the NOS assessment for
all case-control studies included in this systematic review.
Except for one study with high risk of bias (<3 scores) [2], all
studies were presented with low risk of bias (
3 scores) [1,3–
12,14].
4. Discussion
It is evident from this systematic review (Fig. 2) that among
the consistently reported findings at genus level, Faecalibac-
terium [1,2,4,12] and Roseburia [1–5,12] which are butyrate pro-
ducers with anti-inflammatory properties [21] were
significantly higher in healthy controls in two or more studies
when compared to T2DM. Lactobacilli, that is often used as a
probiotic on the other hand was found to be present in the gut
of T2DM in three untargeted approach studies [1,3,5]. Further-
more, butyrate producing bacteria seem to be negatively cor-
related with glycaemic parameters (Table 2). A negative but
strong correlation between Ruminococcaceae and HbA1c [7,12];
173 (2021) 108689 9
another group of butyrate producing bacteria [22] was
observed. Study found that individuals that lack butyrate-
producers in the body are prone to develop T2DM [23]. Buty-
rate, one of the preferred sources of energy for colonic epithe-
lial cells, maintains the health of the colons [24]. Its reduction
may intensify intestinal permeability and endotoxaemia.
Depletion of these butyrate producers are associated with
inflammation pathogenesis, as observed in T2DM [25]. Like-
wise, a moderate positive correlation was observed between
Clostridium spp. (a butyrate producing bacteria) and adiponec-
tin [1]. It has been suggested that butyrate enhanced adipo-
nectin by increasing the glucose uptake by skeletal and
cardiac muscle as well as inhibition of hepatic gluconeogene-
sis which improved insulin efficacy [26].
Other than that, T2DM was presented with significantly
high level of lactobacilli and the order Lactobacillales [1,3,5–
7,10,11]. More specifically, lactobacilli showed moderate posi-
tive correlation with HbA1c and FBG [1] while weak positive
correlation was observed with 2PPG [3]. Lactobacilli is
famously known for its probiotics properties [27,28], lacto-
bacilli represents a heterogeneous group with well-
documented immune-modulating properties. On the other
hand, some lactobacilli may also potentially contribute to
chronic inflammation in diabetic patients [29]. The controver-
sial effects of lactobacilli could be due to species- or strain-
specific, and therefore, the role of lactobacilli in T2DM
remains inconclusive [30].
B:F ratio, Bacteroides-Prevotella:C. coccoides subgroup ratio
and class Betaproteobacteria were moderately correlated with
2PPG [3]. Bacteroides and Prevotella belong to the phylum Bac-
teroidetes while Betaproteobacteria belongs to the phylum
Proteobacteria. These observations suggested that Gram neg-
ative phyla Bacteroidetes and Proteobacteria might initiate
T2DM through endotoxin-induced inflammatory responses
due to the presence of high concentrations of endotoxins
and LPS [31].
The inverse relationship between Bifidobacterium spp. with
carbohydrates was observed in one study in this systematic
review [7]. Bifidobacterium spp. are, however, believed to play
an important role in carbohydrate fermentation in the lower
gut [32]. A study had reported about increased abundance of
Bifidobacterium spp. after ingestion of carbohydrates, suggest-
ing that carbohydrate has bifidogenic effect that can be used
as prebiotics [33]. There are two possible reasons to these con-
trasting results. Firstly, the ability of Bifidobacteria to metabo-
lise particular carbohydrates (e.g. cellulose, lactose) depends
on their species and strains as well as the amount and nature
of the carbohydrates present in the diet [34]. Secondly, it could
be due to the homeostatic imbalance between gut microbiota
and systems in T2DM that had led to such outcome [7].
The ferocious cycle between gut microbiota dysbiosis and
activated low-level inflammation is considered as a downturn
factor in the development of T2DM. The present findings
which had indicated a moderate to strong significant positive
correlation between the class Clostridia and phylum Firmi-
cutes with pro-inflammatory cytokines IFN-c as well as
between Negativicutes (class of bacteria in the phylum Firmi-
cutes) and IL-6 [12]. The reason for this could be due to the
fact that overweight individuals were correlated with high rel-
ative abundance of Firmicutes [35] and the majority T2DM
10 diabetes research and clinical practice
patients from Leite, et al. [12] were overweight. Thus, it can be
suggested that increase Firmicutes stimulated the secretion
of pro-inflammatory markers by macrophages, leading to
chronic inflammation [36]. Increased Enterobacteriaceae, and
Prevotella were also associated with metabolic endotoxaemia
and inflammation, mainly mediated by pro-inflammatory
cytokines [12]. In contrast, the relative abundance of genus
Bacteroides showed a significant negative correlation with
IFN-c [12]. The reason could be possibly due to the different
Bacteroides spp. and strains that may play different roles in
controlling the thickness of intestinal mucus and glycocalyx,
thus influencing the intestinal permeability [37] as well as
intake of hypoglycaemic medication as mentioned by Hung
and Hung [38].
With regards to anthropometric measurements and lipid
profiles, no consistent patterns were observed between said
parameters and gut microbiota. Although the results were
significant, however, the correlations were weak. More stud-
ies need to be done to confirm the findings.
Research has identified that drugs can have an impact on
the function and composition of gut microbiota [39]. In partic-
ular, there is evidence stated that intake of oral hypogly-
caemia agents influence the composition of gut bacteria
[40]. Metformin administration could contribute to an
increase in SCFA-producing bacteria [40]. Karlsson, et al. [1],
however, reported otherwise. Forslund, et al. [5] reported sig-
nificant increases in butyrate producers (e.g. Roseburia, Sub-
doligranulum and Clostridiales) in healthy controls when
compared to metformin-untreated T2DM patients. However,
the increase in Lactobacillus spp. was reversed or eliminated
when controlled for metformin treatment status. Taken all
together, metformin enriched T2DM faeces in Enterobacteri-
aceae spp. [1,6], Staphylococuus spp. [6] and Escherichia spp.
[5], while decreased Intestinibacter spp. count [5]. The mecha-
nisms of action of metformin is not clearly understood, it
may probably have both direct and indirect effects on gut
microbiota; it may either be a growth or diminution factor
for some specific bacterial species [40].
a-glucosidase inhibitors affect the nutrient sources of bac-
teria by partitioning complex carbohydrates and thus influ-
ence gut microbiota composition [41]. This systematic
review found administration of a-glucosidase inhibitors was
associated with significantly high number of Bifidobacterium,
Lactobacillus and Enterococcus spp. [6]. These bacteria may be
involved in the regulation of glucose metabolisms given that
Lactobacillus and Bifidobacterium are known for their probiotic
properties. Enterococcus spp., on the other hand, showed a sig-
nificant negative correlation with LPS, suggesting that they
may decrease inflammation [42]. Nevertheless, gut microbiota
profile of T2DM may be masked by oral hypoglycaemia agents
as the ameliorative effects of these drugs could contribute to
SCFAs production as well as microbiome shifts [5].
This systematic review had identified several limitations
from the shortlisted studies. Firstly, not all recruited T2DM
share similar BMI category. Therefore, it cannot be excluded
that gut dysbiosis occurred as a result of obesity. Secondly,
there was a wide variation in terms of sample size amongst
the included studies (10–200 subjects) that will influence the
effect size. In addition, diverse approaches on the sample pro-
cessing such as various microbial DNA extraction methods
173 (2021) 108689
used may produce inconsistent findings that may not exert
the real microbial community towards a particular condition
(e.g. T2DM).
Next, since the 13 included case control studies were
selected from all around the world, their environment varies.
Geographical, ethnicity and lifestyle differences (e.g. diet,
physical activities) among the participants could have influ-
enced the overall gut microbiota compositions. Other discrep-
ancy of gut microbiota between studies can be explained by
intake of medications which are likely to influence the gut
microbiota compositions that lead to inconsistent findings.
The present systematic review had identified only three out
of 13 studies [1,5,6] that had looked into the relationship
between gut microbiota and antihyperglycaemic medications,
of which the lack of information makes it difficult to evaluate
as to whether these findings were, by and large, confounded
by medications.
Even so, the abovementioned limitations may act individ-
ually or together to bring their effects on the gut microbiota
that will influence the condition of T2DM. Whilst there were
inconsistent findings among these case control studies, this
systematic review has collected a number of useful informa-
tion that could pave the way towards a well-designed studies
that underline the importance of accounting for these limita-
tions to untwine the relationship between gut microbiota and
T2DM since case control study designs are constricted by con-
founding factors between diseased and control groups.
In any case, the present systematic review suggests that
gut microbiota may act as an intermediary towards a number
of risk factors for T2DM. Hence, it is perhaps essential to take
into consideration the gut microbiota composition of T2DM
patients individually upon recommendation of treatment.
5. Authors’ contributions
CFN and FU independently searched, extracted and assessed
all the potential studies. FU wrote the manuscript. CFN, SML
and KR assisted with the manuscript revision. All authors
read and approved the final manuscript.
Declaration of interest
None.
Acknowledgements
This work was funded by Universiti Teknologi MARA (UiTM)
[Lestari grant: 600-IRMI 5/3/LESTARI (016/2019)].
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