The effect of a novel probiotic on metabolic biomarkers in adults with prediabetes and recently diagnosed type 2 diabetes mellitus: study protocol for a randomized controlled trial
Palacios et al. Trials
The effect of a novel probiotic on metabolic biomarkers in adults with prediabetes and recently diagnosed type 2 diabetes mellitus: study protocol for a randomized controlled trial
Talia Palacios 0
Luis Vitetta 1 2
Samantha Coulson 2
Claire D. Madigan 0
Gareth S. Denyer 3
Ian D. Caterson 0
0 The Boden Institute of Obesity, Nutrition, Exercise and Eating Disorders, University of Sydney , Sydney , Australia
1 Medlab Clinical , Sydney , Australia
2 University of Sydney, Sydney Medical School , Sydney , Australia
3 School of Life and Environmental Sciences, University of Sydney , Sydney , Australia
Background: Shifts in the gastrointestinal microbiome have been shown to contribute to the progression of metabolic diseases including prediabetes and type 2 diabetes mellitus. Research suggests that in-vivo modulation of the gut microbiome by specific probiotic microorganisms may improve insulin sensitivity and blood sugar management, preventing or delaying the development of type 2 diabetes mellitus. However, further research is needed to understand the effect of probiotics as a therapy for the treatment of metabolic diseases. An evidence-based multi-species probiotic was developed to encourage a shift in the gastrointestinal bacterial cohort from a disease-prone to a balanced state with the aim of improving metabolic markers associated with type 2 diabetes mellitus. Methods: Sixty adults with a body mass index ≥25 kg/m2 with prediabetes or type 2 diabetes mellitus (diagnosed within the previous 12 months) will be enrolled in a double-blind, placebo-controlled pilot study. Participants will be randomized to a multi-species probiotic or placebo for 12 weeks. Both groups will receive lifestyle and nutritional advice. The primary outcome measure is the change between groups in fasting plasma glucose levels from baseline to 12 weeks. Secondary outcome measures include, but are not limited to, the change in lipid profile, systemic inflammation, gut permeability, and faecal microbial and metabolomic profiles. Blood and stool samples are collected at baseline and 12 weeks after treatment. Discussion: Intentional manipulation of gastrointestinal microbial profiles may be useful for preventing and controlling type 2 diabetes mellitus and its associated metabolic complications.
Gut microbiota; Prediabetes; Probiotics; Type 2 diabetes mellitus
The relationship between the gastrointestinal microbiota
and obesity-associated disorders has gained extensive
research interest in the past 10 years. A disturbed gut
microbiota expressed as gut dysbiosis (an intestinal
physical barrier abnormality) has been associated with
the progression and maintenance of obesity, type 2
diabetes mellitus, cardiovascular diseases and metabolic
syndrome [1–5]. The mechanisms by which gut
dysbiosis produces and influences these metabolic alterations
are via regulation of the host’s energy balance and
storage and by promoting endotoxaemia or bacteraemia .
Furthermore, postprandial blood glucose levels are
very much influenced by the gut bacteria, with recent
research demonstrating a profound influence on how
individuals responded to identical food items that could
be accurately predicted based on their gut bacterial
profiles . Gut dysbiosis may be restored to a
balanced state through microbiota-based interventions,
which may improve metabolic markers associated with
type 2 diabetes mellitus through immunomodulatory
and anti-inflammatory pathways.
A probiotic is a microbiota-based intervention defined
as ‘a live microorganism, which, when administered in
adequate amounts, confers beneficial health effects on
the host’ [2, 8, 9]. A meta-analysis evaluating the effect
of probiotics on glycaemia suggests that probiotics can
play an important role in the prevention and treatment
of type 2 diabetes mellitus . Certain probiotic species
have improved insulin sensitivity, inflammatory markers
and lipid profiles in obese, type 2 diabetes mellitus and
dyslipidaemic subjects [11–13]. However, it is not known
whether a combination of probiotic species that have
demonstrated beneficial therapeutic effects individually
can improve metabolic markers associated with type 2
diabetes mellitus and have an additive effect to standard
care. Therefore, we have formulated a novel prescription
containing eight probiotic species that belong to the
Lactobacillus, Bifidobacterium, Streptococcus and
Saccharomyces genera to improve glucose metabolism in
subjects with prediabetes and early type 2 diabetes
mellitus. This multi-species probiotic formula has been tested
previously in our laboratory using in-vitro models with
rodent fat and muscle cell lines. The results from these
in-vitro experiments showed the supernatants collected
from the growth media of the probiotic decreased lipid
accumulation in 3T3-L1 adipocytes and restored glucose
uptake in insulin resistant L6 muscle cells . The
formulation and dosage proposed in this study have not been
investigated previously in human studies. Therefore, the
aim is to test the safety and efficacy of this novel probiotic
formulation in adults with prediabetes and early type 2
diabetes mellitus. We hypothesize that a shift in the gut
microbiome induced by this multi-species probiotic will
decrease metabolic and inflammatory markers and result
in improved blood glucose management.
This pilot study is a single site, randomized,
doubleblind, placebo-controlled clinical trial conducted at the
Charles Perkins Centre Royal Prince Alfred Clinic in
Sydney, Australia. Sixty adults with prediabetes or early
type 2 diabetes mellitus will be randomized to take either
a multi-species probiotic capsule or placebo for 12 weeks.
The Standard Protocol Items: Recommendations for
Interventional Trials (SPIRIT) was used to elaborate the study
protocol (see Additional file 1). Participants’ progression
through the trial is presented in Fig. 1 (CONSORT
Subject recruitment will be through the Boden Institute’s
clinical trials register, the Sydney Local Health District
intranet, the University of Sydney website and Diabetes
Australia social media channels.
Participants will be randomized to the probiotic or placebo
group without stratification using computer-generated
random numbers (FileMarker Pro). Both participants and
study investigators will be blinded to treatment allocation.
Participant unblinding will only be requested in a medical
emergency, where knowledge of the study treatment is
essential for any treatment of the participant. The
reason for unblinding will be documented and the study
treatment will not be revealed to any member of the
Data handling and record keeping
Data is completed on case report forms, source
documents (both written on paper and time and date
stamped electronic capture) and entered into a database.
This database will be password protected and backed up
the University of Sydney server. All results will be sent
to participants by email.
Aged ≥ 18 years
Body mass index ≥ 25 kg/m2
Have prediabetes or have been diagnosed with type
2 diabetes mellitus within the previous 12 months
Are treated by diet alone or diet plus metformin
Fig. 1 CONSORT flowchart of participants’ progress through the study
Are willing to adhere to the study protocol (no
yoghurt, fermented food, dietary supplements,
probiotics or prebiotics) for the duration of the study.
The definition of prediabetes is based on the American
Diabetes Association guidelines  and participants
must have: (i) a fasting plasma glucose level between
5.6 mmol/l and 6.9 mmol/l or; (ii) two-hour post-challenge
(oral glucose tolerance test) plasma glucose level between
7.8 mmol/l and 11 mmol/l or; (iii) HbA1c between 5.7%
The criteria for the diagnosis of type 2 diabetes mellitus
is based on the American Diabetes Association guidelines
. Participants are included in the study if they have
been diagnosed with type 2 diabetes mellitus by their
general practitioner in the previous 12 months or have: (i) a
fasting plasma glucose level ≥ 7.0 mmol/l or; (ii) 2-hour
post-challenge (oral glucose tolerance test) plasma glucose
level ≥ 11.1 mmol/l or; (iii) an HbA1c ≥ 6.5%.
Type 1 diabetes mellitus
Type 2 diabetes mellitus diagnosed for longer than
Taking anti-obesity drugs or blood glucose-lowering
medications (i.e. sulfonylureas, alpha-glucosidase
inhibitors, thiazolidinediones and glucagon-like
peptide-1 analogues) other than metformin
Concomitant gastrointestinal disorders (i.e., irritable
bowel syndrome, inflammatory bowel disease and
Recent use (within the previous 4 weeks) of antibiotics
and dietary supplements (fish oil, probiotics, prebiotics,
multivitamins, minerals, nutraceuticals and herbal
Pregnancy, breastfeeding or planning to become
Alcohol abuse or the use of any illicit drugs
Clinical evidence of active infection or any severe
illness unrelated to diabetes.
Intervention and compliance
The study coordinator will obtain written informed
consent and randomize eligible participants to the
multispecies probiotic or placebo. The study product contains
either 50 × 109 colony forming units (cfu) of a
multispecies probiotic (27 × 109 cfu of Lactobacillus spp.,
22.5 × 109 cfu of Bifidobacterium spp., 450 × 106 cfu of
Streptococcus spp. and 45 × 106 cfu of Saccharomyces
spp., patent pending) or 50 mg of the placebo
(maltodextrin) per capsule. The probiotic and placebo capsules
are opaque white and look and smell identical. Verbal
and written instructions on how to take the study product
or placebo will be provided at the initial and each review
visit. Enrolled participants will be required to:
Take two capsules twice per day (20 min before
breakfast and dinner) with cold non-carbonated
water. The product is not to be mixed or taken with
hot drinks or foods, as heat and stomach acids can
reduce the stability of the probiotic bacteria
Store the study product in the fridge at 4–6 °C
Record the number of capsules taken each day in
the study diary
Bring all capsules remaining in the bottle to the next
Avoid eating or drinking yoghurt, fermented food,
dietary supplements (i.e. vitamins, minerals,
nutraceuticals, herbal preparations, probiotics,
prebiotics or fish oils) and antibiotics (unless
recommended by a health professional).
Study product compliance
Capsule counting, at weeks 6 and 12, will be used to
assess the participant’s compliance in taking the study
product. A record of the date, the visit and the amount
of study product dispensed and returned will be
documented for each participant. Participants are defined as
non-compliant if they have taken less than 80% of the
study product on both occasions.
Subjects in both groups of the study will receive dietary
and lifestyle advice from a research dietitian. This advice
is according to the 2013 National Health and Medical
Research Council dietary guidelines for Australian adults.
The guidelines focus on improving fruit and vegetable
intake, portion size control, reduction of energy-dense
and low-nutrient density foods and drinks, encouraging
physical activity by walking 10,000 steps per day and
reducing sedentary behaviour. At baseline, participants
Secondary outcome measures
Screening, intervention and assessment visits will be
performed by a research nutritionist. The schedule of visits
and measurements is given in Fig. 2 in compliance with
Medical history, prescribed and non-prescribed
medications, alcohol intake and smoking habits will be recorded
during the screening visit. Anthropometric
measurements will be collected using standardized examination
procedures  and calibrated equipment at three time
points (baseline, 6 weeks and 12 weeks). Height will be
measured using a stadiometer with an adjustable
headpiece. Participants will be weighed wearing an
examination gown and underwear only. Waist circumference
will be measured at the midpoint between the lowest
margin of the last rib and the top of the iliac crest. Hip
circumference is measured around the widest portion of
Participants will record a 7 day food diary (5 working
days and 2 weekend days) at baseline, then at 6 and
12 weeks after randomization. Food Works 8 will be used
to assess energy and macronutrients including dietary
fibre intake. Participants will wear a pedometer for the
Fig. 2 SPIRIT schedule of enrolment, interventions and assessments
study duration. The Stanford Leisure-Time Activity Cat
egorical Item (L-Cat) 2.2 physical activity questionnaire
 will be completed at baseline, and 6 weeks and
12 weeks after treatment. The number of steps taken will
be recorded daily in a study diary.
Blood samples will be collected after fasting overnight at
baseline and 12 weeks. Participants who do not have
type 2 diabetes mellitus will undergo a 75 g oral glucose
tolerance test to assess glucose and insulin levels at three
time points (0 min, 60 min and 120 min). Blood samples
to measure glucose, insulin, HbA1c, lipids and hs-CRP
will be sent to a commercial laboratory for analysis.
Samples of 12 ml of blood collected in
ethylenediaminetetraacetic acid (EDTA) plasma tubes will be used for
lipopolysaccharide and zonulin analysis. These samples
will be immediately placed at 4 °C and centrifuged for
15 minutes at 1000 g (or 3000 rpm) at 2–8 °C within
30 minutes. Plasma samples will be aliquoted in
pyrogenfree tubes using pyrogen-free pipette tips and stored at
−80 °C. Serum endotoxin will be measured using the
Pierce™ Limulus Amoebocyte Lysate chromogenic
endotoxin quantification kit (Thermo Fisher, Sydney,
NSW, Australia). The EDTA-plasma zonulin
concentration will be measured with the human haptoglobin
ELISA kit (Abcam®, Sydney, NSW, Australia).
Lipopolysaccharide and zonulin analyses will be performed in
Stool samples will be collected at baseline and 12 weeks
using a stool specimen collection kit. This collection kit
includes an instruction booklet for the stool sample
collection and transportation, ice packs, gloves, a sterile
container, sealed plastic pouch, cool box and an AnaeroGen™
Compact sachet, which preserves the microbiological
characteristics of the sample for 72 hours. Participants will bring
the stool sample to the clinic and the containers will be
stored at 4 °C and analyzed by matrix-assisted laser
desorption ionization time-of-flight mass spectrometry 24–48
hours after collection. In addition, a 1 g sample will be
stored at −80 °C for faecal microbial and metabolite
profiling. DNA extraction will be performed using the QIAamp
DNA stool kit (Qiagen, Sydney, NSW, Australia). Faecal
microbial composition will be identified by sequencing the
16S rRNA using the Ion S5 next-generation sequencing
system (Thermo Fisher, Sydney, NSW, Australia).
Shortchain fatty acids, secondary bile acids and choline
byproducts will be measured using gas chromatography-mass
Data analysis and statistical considerations
As this is a pilot study, no evidence about blood glucose
differences is available for this product. Therefore, it was
decided to use the number of participants needed to
detect a mean difference between the groups in fasting
plasma glucose level of 2.0 mmol/l (standard deviation
2.0 mmol/l) at week 12 with 80% power and 95%
confidence as a starting point. The number of subjects
needed is 32. The fact that the study includes two
different cohorts (participants with prediabetes and early type
2 diabetes mellitus) was also taken into consideration, as
owing to the lack of stratification at randomization the
variance could be high. Therefore, the sample size has
been inflated to 30 per group (including a drop-out rate
of 25%) to give a total sample size of 60 eligible
Analysis will be conducted using the intention-to-treat
principle and missing data will be imputed with baseline
values for a conservative estimate (i.e. no change).
Descriptive statistics will be presented as mean ± standard
deviation, or median with range, as appropriate. Primary
and secondary outcomes will be analyzed using generalized
linear models. The following covariates will be added to the
model: sex, age and percentage weight change. Additionally,
if there is an imbalance between the groups of prediabetes
and type 2 diabetes mellitus, this will be included as a
covariate in the model. Three planned subgroup analyses will
be completed between participants who are taking
metformin, participants classified as prediabetic or type 2 diabetes
mellitus and participants defined as compliant. Chi–square
analysis will determine associations between categorical
For the gut microbiome analysis, sequenced data will
be interpreted using the bioinformatics tools programmed
in the Ion Reporter™ software. Quantitative Insights Into
Microbial Ecology (QIIME) algorithms will determine the
bacterial diversity within a sample (alpha diversity) and
between all the samples (beta diversity). Additionally,
multivariate data analysis with principal component
analysis on the diversity indexes and comparisons of
genus and species level data will be performed to reveal
differences in the microbial composition between the
two groups. Differences in relative abundance of taxa
between the intervention and placebo group and between
participants receiving and not receiving metformin will be
determined using ANOVA, using subject as a blocking
factor. Changes in relative abundance will be tested for
correlations with patient biochemical measurements. For
all statistical tests, the Benjamini–Hochberg false
discovery rate adjustment  will be used to account for the
number of taxa tested in each comparison.
Adverse events are defined as any unfavourable and
unintended sign (including an abnormal laboratory finding),
symptom, or disease (new or exacerbated) temporally
associated with the use of the study product, whether or not
considered related to the treatment. The study investigator
will monitor each participant for adverse events during
the study. All adverse signs or symptoms reported
between consent and final follow-up will be recorded.
Adverse events are reported descriptively by group.
Serious adverse events
All serious adverse events, related or not related to the
study product, are recorded on paper and electronic case
report forms. Serious adverse events will be reported in
compliance with the requirements of the Sydney Local
Health District Human Research Ethics Committee.
Probiotic intake has not been associated with any major side
effects and extensive safety data are available on their
effects; however, participants will be discontinued from
the study product if it is decided that a serious adverse
event may be related to probiotic consumption.
Ethical approval has been granted by the Sydney Local
Health District Human Research Ethics Committee
(Royal Prince Alfred Hospital). This study will be carried
out according to the Declaration of Helsinki, the
National Health and Medical Research Council National
Statement on Ethical Conduct in Research Involving
Humans and the Notes for Guidance on Good Clinical
Practice as adopted by the Australian Therapeutic Goods
Administration (2000) (CPMP/ICH/135/95) and the
International Conference on Harmonisation Good
Clinical Practise guidelines.
This randomized trial will assess the efficacy and safety of
a multi-species probiotic formulation in the management
of prediabetes and type 2 diabetes mellitus metabolic
markers. The study outcomes may lead to novel
treatments to reduce the metabolic disturbances associated
with these disorders. This study will also provide empirical
evidence to address currently unresolved issues with the
efficacy and safety of probiotics. In designing this clinical
study, several key decisions were made to overcome
current limitations in the published literature and reduce
While focusing on those with prediabetes, subjects
with early type 2 diabetes mellitus were included to assess
the feasibility of inducing partial or complete remission of
type 2 diabetes mellitus. This decision also meant including
participants taking metformin as this drug is commonly
prescribed early in type 2 diabetes mellitus; it has also been
shown to have effects on the microbiome [21, 22].
Furthermore, microbiota-based interventions may reduce
gastrointestinal symptoms associated with metformin
administration with a consequent improvement in
medication compliance . As far as we are aware, the
interaction between the gastrointestinal microbiota,
probiotics and metformin has not been explored in
patients with prediabetes and type 2 diabetes mellitus.
Therefore, this study will assess the efficacy and safety
of a novel multi-species probiotic and provide
preliminary data on its effect on metformin.
Limitations of the study include the short-term impact
of the intervention, as subjects will be treated for only
12 weeks. Additionally, the small number of visits
during the study can be considered a risk factor for
noncompliance. Moreover, it must be remembered this
study is investigating the short-term impact of a
multi-species probiotic formulation to help improve
fasting blood glucose levels. It is known that
probiotics do not recolonize the intestinal tract but rather
are transient colonizers and that wash-out of
administered doses can take from approximately 4–6 weeks,
thereby leading to a therapy that may be not be
This trial will provide pilot data of a novel probiotic
formula that may shift the gastrointestinal microbial
profile from a disease-prone to a balanced state and
improve glucose metabolism. The findings will enhance the
understanding of the role that probiotics may play on
metabolic biomarkers in individuals with high glucose
levels. Additionally, microbiome biomarkers associated
with the risk of developing type 2 diabetes mellitus may be
found by exploring the gut microbial and metabolomic
Additional file 1: SPIRIT 2013 checklist. (DOCX 51 kb)
Additional file 2: Participant information sheet and consent form for the
study ‘Effect of a novel probiotic on metabolic biomarkers in adults with
prediabetes and recently diagnosed with type 2 diabetes’. (DOCX 71 kb)
ANOVA: analysis of variance; cfu: colony forming unit;
EDTA: ethylenediaminetetraacetic acid; ELISA: enzyme-linked immunosorbent
assay; HbA1c: glycosylated haemoglobin; hs-CRP: high-sensitive C-reactive
protein; L-CAT: Stanford Leisure-Time Activity Categorical Item;
QIIME: Quantitative Insights Into Microbial Ecology; SPIRIT: Standard Protocol
Items: Recommendations for Interventional Trials
The authors gratefully acknowledge Medlab Clinical for providing the study
product and resources for sample analysis; Bioscreen Medical Laboratory,
who will provide the stool sampling kits, process the faecal samples and
perform the matrix-assisted laser desorption ionization time-of-flight mass
spectrometry analysis; and Ms Alice Gibson, who designed the food diary
IDC is the principal investigator and will oversee the project implementation.
IDC, LV, SC and TP designed the trial and wrote the study proposal, with
input and advice from CDM. GSD developed the database and randomization
tool. CDM wrote the statistical analysis plan with assistance from the other
authors. All authors have read and approved the final version of the protocol.
This study is part of a PhD project supported by the University of Sydney,
the Ecuadorian government and Medlab Clinical Ltd. (which produces the
probiotic). LV has received National Institute of Complementary Medicine
and National Health and Medical Research Council of Australia grant funding
and industry support (FitBioceuticals Ltd, Swisse-Swisse Wellness and Parmalat)
for research into probiotics. LV participates in research on probiotics at Medlab
Clinical. CDM, IDC, TP and GSD declare that there are no conflicts of interest.
Consent for publication
All authors approved this manuscript for publication.
Ethics approval and consent to participate
The study protocol has been approved by the Sydney Local Health District
Human Research Ethics Committee, Royal Prince Alfred Hospital, Sydney,
Australia (X14-0369 & HREC/14/RPAH/492). Any modifications to the research
protocol will be notified to this human research ethics committee. Written
informed consent will be obtained from participants before enrolment
(see Additional file 2).
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