Probiotic Bifidobacterium longum NCC3001 Reduces Depression Scores and Alters Brain

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Accepted Manuscript Probiotic Bifidobacterium longum NCC3001 Reduces Depression Scores and Alters Brain Activity: a Pilot Study in Patients With Irritable Bowel Syndrome Maria Ines Pinto-Sanchez, MD, Geoffrey B. Hall, PhD, Kathy Ghajar, BSc, Andrea Nardelli, MD, Carolina Bolino, MD, Jennifer T. Lau, BSc, Francois-Pierre Martin, PhD, Ornella Cominetti, PhD, Christopher Welsh, BSc, Amber Rieder, BA, Jenna Traynor, BSc, Caitlin Gregory, MD, Giada De Palma, PhD, Marc Pigrau, MD, Alexander C. Ford, MD, Joseph Macri, PhD, Bernard Berner, PhD, Gabriela Bergonzelli, PhD, Michael G. Surette, PhD, Stephen M. Collins, MD, Paul Moayyedi, MD, Premysl Bercik, MD. PII: DOI: Reference:

S0016-5085(17)35557-9 10.1053/j.gastro.2017.05.003 YGAST 61167

To appear in: Gastroenterology Accepted Date: 2 May 2017 Please cite this article as: Pinto-Sanchez MI, Hall GB, Ghajar K, Nardelli A, Bolino C, Lau JT, Martin F-P, Cominetti O, Welsh C, Rieder A, Traynor J, Gregory C, De Palma G, Pigrau M, Ford AC, Macri J, Berner B, Bergonzelli G, Surette MG, Collins SM, Moayyedi P, Bercik P, Probiotic Bifidobacterium longum NCC3001 Reduces Depression Scores and Alters Brain Activity: a Pilot Study in Patients With Irritable Bowel Syndrome, Gastroenterology (2017), doi: 10.1053/j.gastro.2017.05.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Probiotic Bifidobacterium longum NCC3001 Reduces Depression Scores and Alters Brain Activity: a Pilot Study in Patients With Irritable Bowel Syndrome.

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Maria Ines Pinto-Sanchez1 MD, Geoffrey B Hall2 PhD, Kathy Ghajar2 BSc, Andrea Nardelli1 MD, Carolina Bolino1 MD, Jennifer T Lau1 BSc, Francois-Pierre Martin3 PhD, Ornella Cominetti3 PhD, Christopher Welsh1 BSc, Amber Rieder2 BA, Jenna Traynor2 BSc, Caitlin Gregory2 MD, Giada De Palma1 PhD, Marc Pigrau1 MD, Alexander C Ford4 MD, Joseph Macri5 PhD,

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Bernard Berner6 PhD, Gabriela Bergonzelli6 PhD, Michael G. Surette1 PhD,

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Department of Medicine, Farncombe Family Digestive Health Research Institute, McMaster

University, Hamilton, ON, Canada 2

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Stephen M Collins1 MD, Paul Moayyedi1 MD, Premysl Bercik1 MD.

Department of Psychology, Neuroscience & Behavior, McMaster University, Hamilton, ON,

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Canada. 3

Nestlé Institute of Health Sciences SA, Lausanne, Switzerland

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Leeds Gastroenterology Institute, St. James's University Hospital, & Leeds Institute of Biomedical

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and Clinical Sciences, University of Leeds, Leeds, UK. Department of Pathology, McMaster University, Hamilton, ON, Canada.

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Nestlé Research Center, Nutrition Institute, Lausanne, Switzerland.

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Short Title: B. longum decreases depression in IBS patients Funding source: Nestlé SA, Switzerland, Corresponding author: Premysl Bercik, MD Department of Medicine, Farncombe Family Digestive Health Research Institute,

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McMaster University, Hamilton, ON, Canada

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e-mail: [email protected]

CONFLICT OF INTEREST: MIPS, AN, GH, ACF, KG, AR, JT, CW, GDP, MP, JM, CG, CB, JL, MS and PM have no conflicts of interest concerning this paper. GB, and BB are employees of Nestec SA, Switzerland. MFP and OC are employees of Nestlé Institute of Health Sciences SA. PB

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and SMC received research support from Nestlé.

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DISCLOSURE: MIPS received a CIHR-CAG Health Professional Fellowship Award. PB is recipient of HHS Early Career Research Award and holder of Richard Hunt-AstraZeneca Chair in Gastroenterology. MANUSCRIPT CONTRIBUTION

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MIPS: acquisition, analysis and interpretation of data; statistical analysis, writing of the manuscript; GH: study concept and design; acquisition, analysis and interpretation of fMRI data; KG AR, JT, CG: fMRI data acquisition, analysis and interpretation; AN: clinical data acquisition, CB: study

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concept and design; CW: database development and data acquisition; JL, MS: microbiota analysis, critical review of manuscript; FPM, OC: NMR metabolomic analysis and data interpretation, BB:

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microbiota analysis, critical revision of the manuscript for important intellectual content, GB: study design, critical revision of the manuscript for important intellectual content: AF, SMC: critical review of the manuscript and important intellectual content; PM: Study design, statistical analysis, critical revision of the manuscript for important intellectual content; PB: Study design, critical revision of the manuscript and supervision of the study. All authors reviewed and approved the last version of the manuscript.

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Acknowledgments: The authors thank Peter McLean and Mirna Del Valle for their invaluable support and study

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monitoring and oversight. We also thank Tiago Nunes for thoughtful comments on the manuscript, Mireille Goillard for neurotransmitter analysis, Francis Foata for the detection of BL and Margaret Fahnestock for advice on BDNF analysis and Laeticia Da Silva and Dr. Sebastiano Collino for

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their contribution in metabonomics data generation.

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ABSTRACT Background & Aims: Probiotics can reduce symptoms of irritable bowel syndrome (IBS), but little is known about their effects on psychiatric comorbidities. We performed a prospective study to

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evaluate the effects of Bifidobacterium longum NCC3001 (BL) on anxiety and depression in patients with IBS.

Methods: We performed a randomized, double-blind, placebo-controlled study of 44 adults with

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IBS and diarrhea or a mixed-stool pattern (based on Rome III criteria) and mild to moderate anxiety and/or depression (based on the Hospital Anxiety and Depression scale) at McMaster University in

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Canada, from March 2011 to May 2014. At the screening visit, clinical history and symptoms were assessed and blood samples were collected. Patients were then randomly assigned to groups and given daily BL (n=22) or placebo (n=22) for 6 weeks. At week 0, 6 and 10, we determined patients’ levels of anxiety and depression, IBS symptoms, quality of life, and somatization using validated

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questionnaires. At week 0 and 6, stool, urine and blood samples were collected, and functional magnetic resonance imaging (fMRI) test was performed. We assessed brain activation patterns, fecal microbiota, urine metabolome profiles, serum markers of inflammation, neurotransmitters and

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neurotrophin levels.

Results: At week 6, 14/22 patients in the BL group had reduction in depression scores of 2 points or

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more on the Hospital Anxiety and Depression scale, vs 7/22 patients in the placebo group (P=.04). BL had no significant effect on anxiety or IBS symptoms. Patients in the BL group had a mean increase in quality of life score compared with the placebo group. The fMRI analysis showed that BL reduced responses to negative emotional stimuli in multiple brain areas, including amygdala and fronto–limbic regions, compared with placebo. The groups had similar fecal microbiota profiles, serum markers of inflammation, and levels of neurotrophins and neurotransmitters, but the BL

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group had reduced urine levels of methylamines and aromatic amino acids metabolites. At week 10, depression scores were reduced in patients given BL vs placebo. Conclusion: In a placebo-controlled trial, we found that the probiotic BL reduces depression but not

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anxiety scores and increases quality of life in patients with IBS. These improvements were

associated with changes in brain activation patterns that indicate that this probiotic reduces limbic reactivity. ClinicalTrials.gov no. NCT01276626.

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Key words: IBS, anxiety, depression, fMRI

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BACKGROUND Irritable bowel syndrome (IBS), characterized by abdominal pain and altered bowel habits, affects

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11% of the world-wide population1, has a significant socioeconomic impact2 and its current treatments have limited efficacy1. Its pathophysiology is incompletely understood but is considered to be a disorder of gut-brain interaction3 and is frequently accompanied by psychiatric disorders1,4.

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Accumulating evidence suggests that commensal bacteria play a role in IBS, as multiple studies have demonstrated an abnormal composition or metabolic activity of gut microbiota in patients with

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IBS5. Dysbiosis, triggered by acute bacterial gastroenteritis, antibiotics or dietary factors, which are known to affect the composition of microbiota, may drive not only the gastrointestinal component of IBS but also contribute to its psychiatric comorbidity6. Furthermore, specific probiotic bacteria have been shown to improve gastrointestinal symptoms in IBS7.

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We have previously demonstrated that administration of B. longum NCC3001 subspecies longum strain (BL) normalized anxiety-like behavior and hippocampal Brain Derived Neurotrophic Factor (BDNF) levels in mice with low-grade gut inflammation, through vagal dependent pathways8,9.

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Based on these results, we hypothesized that BL will improve psychiatric comorbidity in patients with chronic bowel disorders and thus we performed a pilot study in IBS patients. As anxiety and

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depression are rather difficult to distinguish in animal models, they frequently co-exist in patients and altered central BDNF levels were reported in both conditions, we chose as our primary objective to evaluate the impact of BL on co-morbid anxiety and depression. The secondary objectives were then to assess the effect of BL on IBS symptoms and quality of life, and to explore changes in brain activation patterns, circulating inflammatory markers, neurotransmitters, neurotrophins, gut microbiota profile and urine metabolites as a measure of host-microbial

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metabolic interactions. Considering the large heterogeneity of IBS we decided to restrict our study to IBS patients with diarrhea or mixed stool pattern, as they apper to share similar sensory neuro-

microbiota compared with IBS patients with constipation10,5.

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imune interaction and are more likely to present with low-grade gut inflammation and similar

Although several clinical studies investigated effects of probiotic bacteria on behavior and brain function11-12, mostly in healthy individuals, our study is the first one to show that probiotics can

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improve depression scores as well as alter brain activity patterns in IBS patients with comorbid

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depression and anxiety.

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METHODS Study oversight We conducted a randomized, double-blind, placebo-controlled, single center pilot study from

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March 2011 to May 2014. The study was approved by the Hamilton Health Sciences and St. Joseph’s Health Care Research Ethics Boards, all participants signed the informed consent. The

data and reviewed and approved the final manuscript.

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Participants

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study was registered in clinicaltrials.gov under NCT01276626. All authors had access to the study

We recruited adult patients with a diagnosis of IBS with diarrhea or mixed-stool pattern (Rome III criteria)13, and mild to moderate anxiety and/or depression scores based on the Hospital Anxiety and Depression (HAD) scale14 (HAD-A or HAD-D score 8-14). Patients with a history of organic diseases, immune deficiency, major abdominal surgery, a psychiatric condition other than anxiety

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or depression, use of immunosuppressants, glucocorticosteroids, opioids, antidepressants or anxiolytics in regular doses, alcohol or illicit drug consumption, were excluded. Loperamide and

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laxatives were allowed as rescue medications. Other probiotics in any form were forbidden during the 1-month run-in period and the trial. Antibiotics were forbidden during the 3 months prior to the

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run-in period and the trial. Design of the study

The study involved four hospital visits (Supplementary figure 1). At the screening visit ( -4 weeks), clinical history and symptoms were assessed and physical exam and complete bloodwork performed. At the baseline visit (week 0), the inclusion and exclusion criteria and symptoms were re-assessed, stool, urine and blood samples were collected, and an fMRI study performed.

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The patients were then randomised to receive 42 sachets of either spray dried BL (1.0E+10 CFU /1gram powder with maltodextrin) or placebo containing 1 gram of maltodextrin. Treatment products were indistinguishable in terms of package, color, taste and consistency. Patients were

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instructed to dissolve the content of the sachet in 100-200 ml of lactose-free milk, soy milk or rice milk, preheated to 20° Celsius. Patients were asked not to change their eating habits or fibre intake. Participants recorded the treatment intake, the empty sachets were used to assess the compliance at

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the next visit (week 6), where their symptoms were assessed, blood, urine and stool samples collected and fMRI test performed. Finally, patients’ symptoms were re-assessed at a follow-up

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visit (week 10).

In addition to the regular hospital visits, HAD scores were also assessed at 3 weeks of treatment following request of Health Canada. HAD questionnaires were provided to patients at Visit 1 and

Study endpoints

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then mailed or e-mailed to the investigators.

The primary endpoint was a reduction in anxiety and/or depression scores of ≥2 points on HAD

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scale13 at 6 weeks. This was based on the previously established mean clinically important difference for the anxiety and depression score on the HAD scale of 1.3 and 1.4, respectively14.

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Secondary endpoints included improvement in anxiety and depression scores (HAD, continuous data), anxiety (State-Trait Anxiety Inventory, STAI), IBS global adequate relief, IBS symptoms, somatization, quality of life, changes in brain activation patterns (functional Magnetic Resonance Imaging, fMRI), serum inflammatory markers, neurotransmitters and BDNF, and urine metabonomic and stool microbiota profiles.

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Randomization The randomization sequence was performed using a computer program (Proc Plan, SAS, V. 9.1). A block randomization was stratified by gender and IBS status (diarrhea or mixed stool pattern). The

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codes were kept in sealed opaque envelopes allocated to patients according to strata. Each pack was assigned a number according to the randomization sequence. On recruitment, the patients were assigned into one of four strata and given the next consecutive randomization number available for

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that stratum. Treatment allocation was concealed from participants and study staff.

Treatment products indistinguishable in terms of package, color, taste and consistency, were

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identified with two non-speaking codes per arm. Their identity was blind to subjects, investigators and support staff, known only by the manufacturer, Nestlé Product Technology Centre Konolfingen Switzerland. Study Measurements

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Anxiety and depression were assessed by the HAD score. As an additional measure of anxiety we used the STAI15, which assesses both state and trait anxiety. IBS symptoms and signs were assessed by the Birmingham IBS score16 and Bristol stool scale17. To evaluate an overall improvement of

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IBS symptoms, patients were asked a validated question: “Over the past week have you had adequate relief of your IBS symptoms?” with a dichotomous option for responses18. Health-related

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quality of life (QoL) was measured by the SF-3619 and somatization by the PHQ-15 questionnaires20.

Brain activity was assessed by functional magnetic resonance imaging (fMRI) using General Electric 3-Tesla Discovery MR 750, whole body short bore scanner with 32 parallel receiver channels (General Electric, Milwaukee, WI). The 1-hour protocol included a seven minute T1 weighted structural scan, followed by four repetitions of a fearful face backward masking 10

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paradigm21 (Supplementary figure 2) during four fMRI Blood Oxygen Level Dependent scans22 (BOLD EPI; TR/TE=2800/35 ms, flip angle=90º, 3 mm thick slices, no gap, field of view=24 cm, matrix=64x64). Pre-processing of MRI data was completed using Brain Voyager QX Version 2.8.2,

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32-bit (Brain Innovation, Maastricht, Netherlands). Anatomic and functional data were inspected and scans with artefacts or fMRI scans with movement greater than 5 mm in any of 6 planes were excluded from analysis. Anatomical scans were transformed into standard sagittal orientation, and

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underwent spatial normalization into standard Talaraich space. Slice scan time correction and 3D motion correction were carried out on the fMRI data and spatial smoothing applied using a

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Gaussian filter (FWHM=6 mm). The amygdala was selected as region of interest (ROI), initially derived from the WFUPick Atlas and refined according to anatomic landmarks on the full group average transformed T1 image.

Blood and urine samples were collected after an overnight fast. After processing, the samples were

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stored at -80 C until assessment. Samples for BDNF were collected using the PAXgene Blood RNA (PreAnalytiX, Qiagen BD, Toronto, Canada). Serum cytokines and CRP levels were assessed by Human ProInflammatory 7-Plex Ultra-Sensitive Kit (MSD, Gaithersburg, MD) and CRP Abbott

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Architect kit (Abbott Laboratories, IL), respectively. BDNF protein level was assessed by Human BDNF DuoSet ELISA kit (R&D Systems, Minneapolis, MN). Plasma neurotransmitters were

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quantified using following kits: 5-HT: IBL, Hamburg, Germany; Substance P: Abcam, Cambridge, UK; CGRP: Cloud-Clone Corp, Houston, TX. Urine metabolites were assessed by 1H NMR profiling using a Bruker Avance II 600 MHz spectrometer equipped with a 1.7 mm probe at 300 K (Bruker Biospin, Rheinstetten, Germany), using a standard pulse sequence with water suppression, and processed using TOPSPIN (version 2.1, Bruker, Germany) software package. The metabolite identification was achieved using in house database and 2D 1H NMR spectroscopy experiments. Chemometric analysis was performed using 11

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the software package SIMCA-P+ (version 14.0, Umetrics AB, Umeå, Sweden) and in-house developed MATLAB routines. Orthogonal Projection to Latent Structures (OPLS) and OPLS discriminant analysis (OPLS-DA) were employed for exploring the variance in the metabonomics

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data that may explain statistical differences between groups of samples. The classification accuracy of the OPLS-DA was established from the predicted samples in the 7-fold cross-validation cycle. To highlight the weight of individual variables in the model, Variable Importance in Projection

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(VIP) was used, with a value above 1 used as a threshold by convention. For additional details, see Supplementary Methods.

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Microbiota analysis was performed using Illumina sequencing of the V3 region of 16S rRNA gene as described previously23, for details see Supplementary Methods. Bacterial strain-specific PCR24

Statistical analysis

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was used on fecal DNA extracts to detect the presence of BL at the end of the treatment period.

Statistical analyses were performed using IBM-SPSS (IBM-SPSS Statistics v20, Chicago, IL). We performed post-hoc power calculations based on our previous animal data, which showed strong

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therapeutic potential of this probiotic8-9. We estimated that a sample size of 19 in each group would

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have 80% power using a two-group χ2 test with 0.05 two-sided significance level assuming 30% have an improvement in depression and/or anxiety in the placebo group and 75% in the B. longum group.

Data from all randomized subjects were analyzed according to intention to treat (ITT) principles for the primary outcome. To deal with missing data, we used the extreme case analysis assuming that all missing subjects had no improvement in symptoms. Per protocol evaluation (PP) excluded data from subjects who did not complete the trial due to consumption of proscribed medication or non12

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compliance with the study protocol, and was used for the primary and secondary outcomes. For testing the effects on the two primary endpoints, Pearson Chi-Squared and Mann-Whitney U test were used as appropriate. In addition, the HAD scores were analyzed at baseline, week 3, 6 and 10

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post-treatment using ANOVA repeated measures. ANCOVA was used to adjust for baselines differences in HAD depression scores. A two-sided test was used and p