Cross-Species Examination of Single- and Multi-Strain Probiotic Treatment Effects on Neuropsychiatric Outcomes

Abstract

Interest in elucidating gut-brain-behavior mechanisms and advancing neuropsychiatric disorder treatments has led to a recent proliferation of probiotic trials. Yet, a considerable gap remains in our knowledge of probiotic efficacy across populations and experimental contexts. We conducted a cross-species examination of single- and multi-strain combinations of established probiotics. Forty-eight human (seven infant/child, thirty-six young/middle-aged adult, five older adult) and fifty-eight non-human (twenty-five rat, twenty-seven mouse, five zebrafish, one quail) investigations met the inclusion/exclusion criteria. Heterogeneity of probiotic strains, substrains, and study methodologies limited our ability to conduct meta-analyses.

Human trials detected variations in anxiety, depression, or emotional regulation (single-strain 55.6%; multi-strain 50.0%) and cognition or social functioning post-probiotic intake (single-strain 25.9%; multi-strain 31.5%). For the non-human studies, single- (60.5%) and multi-strain (45.0%) combinations modified stress, anxiety, or depression behaviors in addition to altering social or cognitive performance (single-strain 57.9%; multi-strain 85.0%). Rigorous trials that confirm existing findings, investigate additional probiotic strain/substrain combinations, and test novel experimental paradigms, are necessary to develop future probiotic treatments that successfully target specific neuropsychiatric outcomes.

1. Introduction

Neuropsychiatric therapies are heterogeneous in their long-term efficacy (Geddes et al., 2000; McEvoy and Nathan, 2007; Serretti and Mandelli, 2010), with some having considerable harmful effects (Correll et al., 2009; De Hert et al., 2012; Lozano et al., 2008). Consequently, the biomedical research community has placed a great emphasis on developing novel treatments that target more objectively quantifiable brain and peripheral biomarkers (Insel, 2014; Niciu et al., 2014; Sanislow et al., 2015). Across organ systems, an increasing number of studies continue to highlight the importance of connections among the brain, mind, and body (Gallagher, 2004; Gold and Charney, 2002; Jones et al., 2006; Muehsam et al., 2017). Therefore, scientists and clinicians are designing studies to expand our knowledge of the bidirectional signaling mechanisms between gut microbes and the brain and their subsequent influence on behavior and mental health for potential neuropsychiatric treatment development (Mayer et al., 2014).

The precise relationship between dysbiosis (i.e. altered gut microbial composition) and neuropsychiatric symptoms in various cohorts is unclear and continues to be investigated. Initial studies in people diagnosed with Major Depressive Disorder (Aizawa et al., 2016; Jiang et al., 2015) indicate a relative increase in Bacteroidetes, Proteobacteria, and Actinobacteria phyla coinciding with a decline in Firmicutes (including Lactobacillus and Bifidobacterium). A relative decrease in Actinobacteria, Lentisphaerae, and Verrucomicrobia phyla has been associated with neuropsychiatric symptom severity in a sample of individuals with Post-Traumatic Stress Disorder (Hemmings et al., 2017). In a preliminary study in people with schizophrenia, a relative reduction in Proteobacteria (Haemophilus, Sutterella, and Clostridium), with a concurrent increase in Firmicutes (Anaerococcus) has been observed (Nguyen et al., 2018). This report also indicates negative symptoms may be uniquely linked to Firmicutes (Ruminococcaceae) colonization whereas current depression could vary by Bacteroidetes (Bacteroides) frequency.

Preliminary animal studies indicate that gut microbial alteration can influence a wide range of neurobehavioral phenotypes across the developmental trajectory (Bruce-Keller et al., 2015; Clarke et al., 2013; Park et al., 2013; Pyndt Jorgensen et al., 2015). One such study demonstrated that ampicillin was successful at restoring phencyclidine-induced gut microbe-cognitive dysregulation (Pyndt Jorgensen et al., 2015). However, there is a heightened awareness of the significant adverse effects antibiotics such as ampicillin can have on gut microbial diversity and physiological function (Dethlefsen et al., 2008; Dethlefsen and Relman, 2011), limiting its neuropsychiatric treatment utility. Moreover, the long-term beneficial versus detrimental effects of antibiotics on gut-brain-behavior interactions have not yet been characterized.

This has accelerated the pursuit of probiotics as a potential neuropsychiatric intervention. Probiotics are live microorganisms that confer health benefits to its host (Hill et al., 2014) with a minimal incidence of adverse effects (Marteau and Shanahan, 2003). Probiotic consumption to maintain gut and overall health has now been implemented in routine medical practice (Gareau et al., 2010). Probiotics have shown significant promise for improving atopic dermatitis, necrotizing enterocolitis, pouchitis, and irritable bowel syndrome - IBS (Sanders et al., 2013), which is postulated to be a gut-brain dysregulation disorder (Blankstein et al., 2010; Kennedy et al., 2014; Kennedy et al., 2012).

However, probiotic trials designed to target stress levels, mood, cognitive, or psychosocial functioning, have only been conducted during the past ten to fifteen years. In addition, the utility of probiotic treatments to improve neuropsychiatric symptoms have not been established. Moreover, while multi-strain probiotic combinations may provide greater health benefits in comparison to single-strains of probiotics for several infections and gastrointestinal disorders (Chapman et al., 2011; Timmerman et al., 2004), this has not yet been evaluated for human or non-human neuropsychiatric outcome trials.

This review examined published probiotic trials conducted across animal species to determine if single- or multi-strain formulations of probiotics have differential effects in modifying neuropsychiatric symptoms or phenotypes.

2. Materials and Methods

The authors of this article are not associated with any of the trials that were examined. This review compared neuropsychiatric outcomes associated with single- and multi-strain probiotic treatments in humans and translational non-human animal models while adhering to systematic review (PRISMA) guidelines (Moher et al., 2009; Shamseer et al., 2015). Studies comprising this article were ascertained with the PubMed Advanced Search Builder http://www.ncbi.nlm.nih.gov/pubmed/advanced and filtered by the English language. Publication dates were unrestricted and ranged from 2006 to 2018. The most recent search was conducted on April 18, 2018. The PubMed searches were supplemented with a collection of original reports from prior systematic review or hypothesis articles obtained from the Cochrane Database of Systematic Reviews http://www.cochranelibrary.com/cochrane-database-of-systematic-reviews/ with the search terms noted below.

Database search keywords were selected to align this review with anxiety, mood, and psychotic disorder phenotypes. Exact search terms included probiotic and (PubMed/Cochrane Database) well-being (32/13), psychological stress (104/10), anxiety (145/30), worry (2/1), depression (171/1), mood (1133/12), bipolar disorder (7/1), mania (8/1), schizophrenia (19/4), psychosis (4/5), post-traumatic stress disorder (1/0), obsessive-compulsive disorder (4/0), negative symptoms (325/27), learning (46/7), memory (82/11), cognition (52/10), motivation (22/3), reward (2/0), social behavior (38/5), social function (16/1), and sickness behavior (16/0), an inflammation-mediated depression phenotype (Brydon et al., 2009).

Most trials selected for review utilized established probiotics belonging to the Bifidobacterium or Lactobacillus genera (Fijan, 2014; Hill et al., 2014). Investigations of Bacillus subtilis, Clostridium butyricum, Enterococcus faecium, Escherichia coli Nissle, Lactococcus lactis, Pediococcus acidilactici, Saccharomyces boulardii, Saccharomyces cerevisiae, and Streptococcus thermophilus, additional species with recognized probiotic properties, were also included in this review. Investigations with Leuconostoc genera were not available with our defined search criteria. Precise substrains varied by trial.

All case reports, retrospective studies, review articles, non-experimental studies (e.g. internet or survey-based), non-randomized or placebo-controlled trials (human), or reports that failed to include a control group (non-human), were eliminated. Since probiotics are defined as live microorganisms (Hill et al., 2014), studies that only investigated heat-killed probiotics were removed (e.g. Shinkai et al. (2013). Additional reports were rejected if the only microbial treatment was classified as a pathogenic or engineered strain (Miyazawa et al., 2015; Shinkai et al., 2013). Infant trials in which probiotic outcomes were assessed in the mothers but not the consuming infants (Mi et al., 2015; Sung et al., 2014) were also excluded.

All trials that failed to assay neuropsychiatric phenotypes were omitted from this review. However, the physiological outcomes from otherwise eligible studies that exposed non-human animals to neuropsychological stress as part of the experimental paradigm have been recorded in Table A.1 (Appendix).

Figure 1 delineates the number of articles from each stage of our literature search strategy to yield the final set of human (Table 1) and non-human (Table 2) trials for review.

Figure 1.

Systematic Review Flow Diagram.

Table 1.

Human Probiotic Trial Designs and Outcomes.

Probiotic Treatment	Blinding Status and Study Design	Sex and Age, years	Anxiety/ Depression / Emotional Behavior	Cognition/ Social Function	Other Probiotic Associated Outcomes	Reference
Infant/Child
L. rhamnosus GG ATCC531031 × 1010 CFU (n = 46) or placebo (n = 47) Q.D. breastfeeding mothers 14 to 28 days pre-gestation and infants 180 days post-gestation	Double-blind Infants having a mother, father, or sibling with atopic dermatitis, allergic rhinitis, or asthma	Sex not reported Age pre-gestation w/followup at 3, 6, 12, 18 and 24 months	*NSE on fussing/crying	§NA	#PROT ↓ fecal Clostridia at 6 and 24 months *NSE fecal Bifidobacterium, Bacteroides, Lactobacillus, or Enterococcus	(Rinne et al., 2006)
L. renteri DSM 17938 1 × 108 CFU (n = 238) or placebo (n = 230) drops P.O. Q.D. for 90 days	Double-blind 38-40-week gestation, Apgar >8 at 10 min, normal birth weight Exclusions: antibiotic, antacid, or PPI use	Male (n = 242) Female (n = 226) Age 0.019 (< 7 days)	#PROT ↓ fussing or crying	§NA	#PROT ↓ constipation, regurgitation, and healthcare costs	(Indrio et al., 2014)
L. reuteri DSM 17938 1 × 106 CFU/g (n = 60) or placebo whey formula (n = 62) Q.D. for 98 days	Double-blind Gestation ≥37 weeks, singleton-birth weight 2500 - 4500g, formula-fed 3 days prior to enrollment. Exclusions: Cow milk allergy, medical disease, hospitalized, IV antibiotic or oral medication (except thrush), #PROT 7 days before enrollment	Male (n = 78) Female (n = 86) Age 0.038 (14 days)	#PROT ↑ irritability 14 days post - treatment (*NSE overall)	§NA	*NSE weight gain, sleep, body length, head size, stool	(Cekola et al., 2015)
L. renteri DSM 17938 1 × 108 CFU (n = 85) or placebo (n = 82) 5 drops P.O. Q.D. for 30 days	Double-blind Wessel’s colic Exclusions: <2500 g birth weight, medical disease, cow milk allergy, antibiotic or L. reuteri use by infant or mother before enrollment	Male (n = 85) Female (n = 82) males > Age < 0.249 (91 days)	*NSE on fussing/cry ing in breastfed infants #PROT ↑ fussing crying in formula fed infants	§NA	*NSE maternal mental health	(Sung et al., 2014)
L. reuteri ATCC55730 (n = 20), B. lactis BB-12 (n = 20) 1 × 107 CFU/g, or placebo (n = 19) in formula Q.D. for 28 days	Double-blind All infants were formula-fed by parental choice prior to enrollment Exclusions: <36 weeks of gestation, chronic disease, congenital abnormalities, <2500 g birth weight, allergies, atopic disease, probiotic exposure within 4 weeks of enrollment	Female (n = 19) Male (n = 40) Mean Age 0.093 (3-65 days)	*NSE on fussing/crying	§NA	*NSE crying, night awakening, daily gas, stool effort and consistency	(Weizman and Alsheikh, 2006)
L. reuteri Biogaia AB (n = 124) 1 × 108 CFU or placebo (n = 125)5 drops Q.D. from birth until discharge	Double-blind Preterm infants with a gestational age of ≤ 32 weeks, birth weight ≤1500 g, Follow-up analyses of (Oncel et al., 2014)	Male (n = 133) Female (n = 116) Age birth and follow up 18 to 24 months	§NA	*NSE neurocognition (BSID-II-PDI and MDI)	*NSE visual impairment, hearing impairment	(Akar et al., 2017)
S. thermophilus, B. animalis ssp. lactis BB-12, and L. bulgaricus General Mills 5 × 109 CFU/100 mL + 1 g inulin (n =76) yogurt drink or non-synbiotic acidified placebo milk drink (n =73) Q.D. for 112 days	Double-blind Child care attendees Exclusions: premature or low birth weight, allergy, atopic, or medical disease, GI surgery, lactase deficiency or milk intolerance, antibiotic use within 4 or probiotic use within 2 weeks of study enrollment	Males (n = 74) Females (n = 75) Age 1-4 Mean Age 2.5	§NA	#PROT ↑ in social function (Ped-QOL)	#PROT ↑ in school function (Ped-QOL), watery stool #PROT ↓ fever	(Ringel-Kulka et al., 2015)
Older Adult
L. reuteri DSM 17938 1 × 108 CFU stick pack with rhamnose, galactooligosacch aride B.I.D. (n = 125) or placebo (n = 124) for 84 days	Double-blind Age 65+ Exclusions: GI or IBD, use of GMMS or PPI, participation in other clinical trials within 3 months of enrollment	Male (n = 97) Female (n = 152) Age 65+ Mean age 72.3	*NSE stress, anxiety, or well-being (HADS, PSS, EQ-5D-5L)	§NA	*NSE GSRS	(Ostlund-Lagerstrom et al., 2016)
65 mL milk drink w/ L. casei Shirota 1 × 108 CFU/mL Q.D. or placebo for 21 days	Double-blind Self-reported healthy older adults with stable hypertension for 3 months and diabetes mellitus under dietary or medication control	Male (n = 51) Female (n = 75) Age 48-79 Mean age 61.8	#PROT ↓ depression only for sample subset with poor baseline mood (POMS)	*NSE verbal fluency, episodic memory #PROT ↓ mental clarity	#PROT ↑ confidence *NSE eating behavior	(Benton et al., 2007)
L. helveticus IDCC3801 four 125mg (n = 10), 250mg (n = 7), 500mg (n = 9) or placebo (n = 10) tablets Q.D. for 84 days	Double-blind Proficiency using computers, education above middle school, ≥ 24 MMSE- Korean, BMI≤ 16 and ≤ 35 Exclusions: Axis I disorder or Axis I treatment within 5 years of enrollment, ≥ 8 GDS-SF, alcohol abuse/dependence within 3 months, GI disease or GI surgery, significant neurological or medical illnesses, supplement or herbal medicine use during the 4 weeks prior to enrollment, compliance < 70% at each study visit	Male (n = 20) Female (n = 16) Age 60-75 Mean Age 65.0	*NSE stress (PSS) and depression (GDS-SF)	#PROT ↑ RVIP	*NSE serum BDNF and whole blood viscosity	(Chung et al., 2014)
VSL#3 4.5 × 1011 CFU B.I.D. (n = 10) or placebo (n = 8) for 28 days after surgery	Double-blind Laparoscopic colorectal surgery patients	Male (n = 9) Female (n = 9) Age 70+ Mean Age 72.2	§NA	#PROT ↑ social function (SF-36)	#PROT ↓ bowel movement	(Pellino et al., 2013)
200 mL milk drink with L. acidophilus, L. casei, L. fermentum, B. bifidum Tak Gen Zist Pharmaceuticals 2 × 109 CFU/g (n = 30) or placebo (n = 30) for 84 days	Double-blind NINDS-ADRDA diagnosis of Alzheimer’s Disease Exclusions: chronic/metabolic illnesses, probiotic consumption 6 weeks prior to enrollment	Female (n = 48) Male (n = 12) Age 60-95 Mean Age 79.8	§NA	#PROT ↑ cognition (MMSE)	#PROT ↓ VLDL, QUICKI, MDA, HOMA-B, hs-CRP #PROT ↑ HOMA-IR	(Akbari et al., 2016)
Young/Middle-Aged Adult
Fermented milk L. casei Shirota 1 × 109 CFU/mL Q.D. (n = 24) or placebo drink (n = 23) for 56 days	Double-blind Healthy Japanese Students Exclusions: smokers, age > 30, allergies, mental or medical disease, medication use	Male (n = 26) Female (n = 21) Age < 30 Mean Age 27.9	#PROT ↓ anxiety (STAI) one day before exam	§NA	#PROT ↑ increase fecal serotonin post-exam #PROT ↓ incidence cold and abdominal illness	(Kato-Kataoka et al., 2016)
Fermented milk with L. casei Shirota 1 × 109 CFU/mL Q.D. (n = 52**) or placebo (n = 41**) for 56 days **new subjects added to study above	Double-blind Healthy Japanese students Exclusions: smokers, age > 30 years, taking medications, mental illness, and score over 60 on SDS	Male (n = 76) Females (n = 64) Age < 30 Mean Age 22.9	*NSE anxiety (STAI)	§NA	#PROT ↓ cold/flu symptoms and cortisol 1 day before exam See Table 2 for Rat Trial Outcomes	(Takada et al., 2016)
Fermented milk L. casei Shirota 1 × 109 CFU/mL Q.D. (n = 48**) or placebo drink (n = 46**) for 56 days before exam and 14 days after **pooled sample with above studies	Double-blind 4th grade medical students Exclusions: age > 30 years; physical or mental illness, taking medications; smoker, milk or food allergy	Male (n = 55) Female (n = 39) Age < 30 Mean Age 22.7	#PROT ↑ stress (exam) induced sleep disturbance	#PROT ↑ delta power > 20% immediately prior to exam	§NA	(Takada et al., 2017)Takada et al., 2017)
L. casei Shirota 2.4 × 109 CFU (n = 19) or placebo (n = 16) Q.D. for 60 days	Double-blind Stable Chronic Fatigue Syndrome Exclusions: bedridden, meeting criteria for neuropsychiatric disorders except depression or anxiety	Male (n = 8) Female (n = 27) Age 18-65 Mean Age 41.2	#PROT ↓ depression (BDI)	§NA	§NA	(Rao et al., 2009)
L. casei Shirota 1 × 1010 CFU Q.I.D. (n = 36) or placebo (n = 36) for 21 days	Double-blind Smokers mean cigarettes/day = 20 Exclusions: other health problems	Male (n = 72) Age 40-60 Mean Age 50.3	*NSE anxiety (STAI) or job stress	§NA	#PROT ↑ NK cell activity	(Reale et al., 2012)
L. rhamnosus CGMCC1.37241.6 × 108 CFU/capsule B.I.D. (n = 62) or placebo (n = 63) for 168 days	Double-blind BMI 29 - 41 kg/m2, weight change < 5 kg 3 months before screening Exclusions: pregnancy, breastfeeding, menopause or obesity comorbidities	Male (n = 45) Female (n = 60) Age 18-55 Mean Age 36.0	#PROT ↓ depression (BDI) *NSE PSS, STAI, BES	#PROT ↑ cognitive restraint	#PROT ↓ body esteem, and ↓ weight, hunger, and disinhibition in female subjects only	(Sanchez et al., 2017)
L. helveticus Lafti 2 × 1010 CFU (n = 20) or placebo (n = 19) Q.D. for 98 days	Double-blind National or European/World championship athletes Exclusion: chronic diseases, surgery within 1 year of enrollment, probiotic sensitivity, probiotic or antibiotic use 1 month before enrollment	Male (n = 29) Female (n = 10) Age 20-26 Mean Age 23.2	#PROT ↑ vigor (POMS)	§NA	*NSE exercise performance #PROT ↑ blood CD4+/CD8+ cells	(Michalickova et al., 2016)Michalickova et al., 2016)
L. helveticus R0052 (n = 145), B. longum ssp. infantis R0033 (n = 147), B. bifidum R0071 (n = 142) capsule 3 × 109 CFU Q.D. or placebo (n = 147) for 42 days	Double-blind Nonsmoking undergraduate students Exclusions: allergies, current cold, antibiotic use 2 months prior to study enrollment	Male (n = 210) Female (n = 371) Age 18-22 Mean Age 19.9	B. bifidum ↓ self-reported stress	§NA	L. helveticus ↑ diarrhea B. bifidum ↓ diarrhea and incidence cold/flu B. bifidum interaction of stress and sleep *NSE BMI stress interaction	(Culpepper et al., 2016; Langkamp-Henken et al., 2015)
L. rhamnosus HN001 (n = 212) 6 × 109 or placebo (n = 211) for 168 days post gestation and 168 days after birth	Double-blind English-speaking women at 14–16 weeks of gestation, breastfeeding Exclusions: medical problems related to pregnancy, age <16 years, planning to move outside the study center during study, probiotic use, subject or unborn child’s biological father had a history of asthma, hay fever, or eczema requiring medication	Female (n = 423) Mean Age 33.6	#PROT ↓ depression (EPDS) and anxiety (STAI6)	§NA	§NA	(Slykerman et al., 2017)
B. animalis ssp. lactis-07 109 CFU (n = 41), B. animalis ssp. lactis-07 2 × 109 CFU + xylooligosaccharide 8g/d (n = 41), xylooligosacchari de 8g/d (n = 41), or maltodextrin placebo (n = 41) Q.D. for 21 days	Double-blind Crossover design Healthy adults, BMI 20–30 kg/m2 Exclusions: physical or mental illness requiring medication or inpatient/outpatient treatment, planned major surgery, history of drug or alcohol abuse, severe allergies or a history of abnormal drug reaction, chronic GI complaints or GI drug use 4 weeks before enrollment, anti-inflammatory or prescription medication use, participation in an experimental drug trial 4 weeks before enrollment, participation in prebiotic or laxative trials within 3 months or use of antibiotics within 6 months of enrollment	Male (n = 22) Female (n = 22) Age 31–55 Mean Age 43	#PROT ↓ self-reported happiness PRET ↑ self-reported vitality and happiness * NSE SYNT	§NA	*NSE bowel function, plasma HDL, SCFA, Bifidobacterium, Bacteroides/Prevotella, Clostridium, Lactobacillus/Enterococcus/Atopobium	(Childs et al., 2014)
S. boulardii Bioflor 2 × 1011 CFU/capsule two capsules B.I.D. (n = 45) or placebo (n = 45) for 28 days	Double-blind Rome II IBS mixed or diarrhea predominant Exclusions: IBS constipation predominant, pregnant/lactating, chronic medical illness, history of abdominal surgery except appendectomy or hernia	Male (n = 44) Female (n = 46) Age 27-53 Mean Age 41.0	*NSE dysphoria (IBS QOL)	#PROT ↑ social functioning (IBS QOL)	#PROT ↓ activity interference (IBS QOL)	(Choi et al., 2011)
1.31 high dose 1-3 × 1010 CFU (n = 28), low dose 3-6 × 109 CFU Q.D.(n = 27), or placebo (n = 29) for 42 days	Double-blind Rome III IBS with diarrhea Exclusions: celiac disease, IBD, or antibiotic/probiotic use 4 weeks prior to enrollment	Male (n = 31) Female (n = 53) Age 20-70 Mean age 46.8	Low and high dose #PROT ↑ mental status (IBS QOL) Low and high dose #PROT ↓ gut specific anxiety	§NA	*NSE IBS symptoms	(Lorenzo-Zuniga et al., 2014)
L. acidophilus NCFM low dose 1 × 109 (n = 112) high dose 1 × 1010 (n = 113) or placebo (n = 115) Q.D. for 84 days	Double-blind Rome III IBS Exclusions: Other GI disease or probiotic use within 3 months of enrollment	Male (n = 99) Female (n = 292) Age 18-65 Mean Age 47.9	Low and high dose #PROT ↓ anxiety (HADS) high dose #PROT ↓ depression (HADS)	§NA	#PROT ↓ pain for moderate to severe symptom groups	(Lyra et al., 2016)
S. cerevisiae CNCM I-3856 8 × 109 CFU/g 1000 mg tablet Q.D. (n = 192) or placebo (n = 187) for 84 days	Double-blind Rome III IBS pain/discomfort ≥ 1 day/week, normal CRP and fecal calprotectin Exclusions: pregnancy, vegetarian, lactose intolerance, gluten sensitivity, antibiotic, antidepressant, opioid, narcotic, or chronic alcohol use	Male (n = 62) Female (n = 317) Age 18 to 75 Mean Age 45.3	*NSE well-being (IBS-QOL)	§NA	#PROT ↓ bloating and pain for IBS constipated subgroup	(Spiller et al., 2016)
L. casei ssp. rhamnosus LCR35 2 × 108 CFU T.I.D. (n = 25) or placebo (n = 25) for 28 days	Double-blind Rome III IBS Exclusions: current depression (HAM-D)	Male (n = 15) Female (n = 35) Age 34-59 Mean Age 47.1	*NSE anxiety and depression (HAD)	§NA	#PROT ↓ IBS severity in diarrhea subgroup	(Dapoigny et al., 2012)
L. gasseri SBT2055 5 × 108 CFU and B. longum SBT2928 1 × 109 CFU in 100g yogurt with S. thermophilus and L. delbrueckii subsp. Bulgaricus (n = 115) or 100g placebo yogurt with S. thermophilus and L. delbrueckii subsp. bulgaricus (n = 109) Q.D. for 84 days	Double-blind Healthy Japanese volunteers Exclusions: history of significant medical illness, frequent intake of the test yogurt, diarrhea or viral syndrome within the past 30 days, current use of any prescribed medication, use of any other supplements during the trial, pregnancy, smoking > 20 cigarettes/day, drinking > 20 g alcohol/day, history of severe allergic reactions to food and medication	Male (n = 69) Female (n = 155) Age 32 to 76 Mean Age 53.9	#PROT ↓ anxiety (GHQ-28)	§NA	#PROT ↓ NK cell activity, ACTH, salivary and serum cortisol (males only) *NSE CRP, IgG, IgE	(Nishihira et al., 2014)
L. gasseri CP2305 1 × 1011 CFU T.I.D. (n = 17) or placebo (n = 17) for 28 days	Double-blind Rome III IBS Exclusions: organic GI diseases, severe systemic diseases, pregnant, lactating, history of significant abdominal surgery, severe endometriosis, neurological disorders, or dementia	Male (n = 15) Female (n = 19) Age 19-82 Mean Age 49.3	#PROT ↓ health related worry (IBS-QOL)	§NA	#PROT ↓ Dorea, Enterococcus and Dialister genera	(Nobutani et al., 2017)
B. longum NCC3001 1 × 1010 CFU/g Q.D. powder (n = 22) or maltodextrin placebo (n = 22) for 42 days	Double-blind Rome III IBS mixed or diarrhea predominant and HAD score of 8-14 Exclusions: psychiatric disorder except anxiety or depression, antidepressant or anxiolytic use, probiotic use within 1 month or antibiotic use within 3 months of enrollment	Male (n=20) Female (n=24) Age 26-58 Mean Age 43.0	#PROT ↓ depression (HAD-D) *NSE anxiety (HAD-A)	#PROT ↓ amygdala, frontal, and temporal and ↑ occipital engagement in response to fearful stimuli *NSE SF-36 social function	#PROT ↑ SF-36 physical *NSE constipation, diarrhea, or pain	(Pinto-Sanchez et al., 2017)
B. longum 2 × 109 CFU (n = 31), 8g psyllium (n = 31), or B. longum + psyllium (n = 32) Bificolon, Nisshin Kyorin Pharmaceuticals Q.D. for 28 days	Double-blind UC mild or remitted w/out UC surgical history Exclusions: UC induction therapy or unstable prednisone or aminosalicylate dose 4 weeks before enrollment	Male (n = 39) Female (n = 55) Mean Age 36.0	#PROT ↑ IBD QOL emotional	SYNT ↑ IBD QOL social	SYNT ↓ CRP SYNT ↑ IBD QOL greater than #PROT or PRET	(Fujimori et al., 2009)
Ecologic® Barrier powder 2.5 × 109 CFU/g Q.D. (n = 20) or placebo (n = 20) for 28 days	Triple-blind Nonsmoking adults Exclusion: Current mood disorder	Male (n = 8) Female (n = 32) Age 18-23 Mean Age 20.0	#PROT ↓ rumination (LEIDS) #PROT ↓ aggressive thoughts *NSE BAI or BDI	§NA	§NA	(Steenbergen et al., 2015)
VSL#3 1.125 × 1011 CFU (n = 57) or placebo (n = 48) Q.D. for 14 days	Double-blind Healthy students subjected to acute psychological stress Exclusions: diagnosed GI disorder, prior probiotic or current antibiotic use	Male (n = 36) Female (n = 69) Age 18–23 Mean Age 20.2	*NSE stress (PASAT)	*NSE (PASAT)	*NSE heart rate, blood pressure (systolic or diastolic)	(Moller et al., 2017)
Fermented milk B. animalis ssp. lactis 1.25 × 1010, S. thermophilus 1.2 × 109, L. bulgaricus 1.2 ×109 L. lactis ssp. lactis (n = 12) CFU/cup, non-fermented milk B.I.D. (n = 11), or no probiotic milk drink (n = 13) for 28 days	Double-blind Healthy adults BMI 18-30 kg/m2 Exclusions: GI or psychiatric symptoms	Female (n = 35) Age 18-55 Mean Age 36.5	§NA	#PROT ↓emotion task response in affective, viscerosensory, somatosensory cortices	§NA	(Tillisch et al., 2013)
100g Yogurt with L. acidophilus LA5 and B. lactis BB-12 1 × 107 CFU + one placebo capsule (n = 25); one probiotic capsule L. casei 3 × 103, L. acidophilus 3 × 107, L. rhamnosus 7 × 109, L. bulgaricus 5 × 108, B. breve 2 × 1010, B. longum, S. thermophilus 3 × 108 (CFU/g), and fructooligosaccharide 100mg + conventional yogurt 100g Q.D.(n = 25); or conventional yogurt 100g + one placebo capsule (n = 20) for 42 days	Double-blind Petrochemical workers without chronic illness Exclusions: antibiotic, vitamin, or supplement use	Male (n = 36) Female (n = 34) Age 20–60 Mean Age 40.0	#PROT yogurt and capsule ↓ depression and anxiety (DASS) #PROT yogurt and capsule ↓ GHQ scores	§NA	*NSE plasma Tryptophan, Cortisol, ACTH or Neuropeptide Y levels	(Mohammadi et al., 2016)
L. helveticus R0052 and B. longum R0175 3 × 109 CFU Q.D. (n = 26) or placebo (n = 29) for 30 days	Double-blind Healthy Caucasian adults Exclusions: neurological, psychiatric, renal, hepatic, cardiovascular and respiratory diseases, pregnancy, food allergy, clinical trial participation within two months of study enrollment, psychotropic drug or stimulating nutritional supplements (vitamin C), ginger, guarana, ginseng, dehydroepiandrosterone, melatonin, antioxidants, anxiolyics, antidepressants, selenium, narcotics, replacement hormoes, more than 5 cups of coffee or tea/d, 0.2 liters of cola, 30–40 g of chocolate, three glasses of wine, or two fermented dairy products, or smoking >20 cigarettes	Male (n = 14) Female (n = 41) Age 30-59 Mean Age 42.8	#PROT ↓ depression (HADS-D) and anxiety (HADS-A) #PROT ↓ psychological distress (HSCL-90) #PROT ↓ stress (PSS) in patients with low urinary cortisol	§NA	↓ Urinary cortisol See Table 2 for outcomes in rats	(Messaoudi et al., 2011a; Messaoudi et al., 2011b)
L. acidophilus Rosell-52 and B. longum Rosell-175 3 × 109 CFU Q.D. (n = 37) or placebo (n = 38) for 21 days	Double-blind Healthy subjects experiencing 2 or more daily stress symptoms (anxiety, nervous, irritable, sleeping problems, GI disturbance) for 30 days prior to enrollment	Male (n = 21) Female (n = 54) Age 27-49 Mean age 38.0	*NSE psychological stress #PROT ↓ stress induced abdominal pain and nausea	§NA	§NA	(Diop et al., 2008)
L. helveticus R0052 and B. longum R0175 3 × 109 CFU Q.D. (n = 40) or placebo (n = 39) for 56 days	Double-blind ⩾11 on the QIDS-SR16 or ⩾14 on the DASS-42, age > 16 Exclusions: medical disorder, pregnant or lactating, antidepressant use, suicide or violence risk, probiotic, antibiotic, or psychotropic medication use 4 weeks prior to trial	Male (n = 17) Female (n = 62) Mean Age 35.0	*NSE depression or anxiety (MADRS, QIDS-SR16, DASS)	§NA	*NSE iCGI-SiCGI-I, GAF, CRP, IL-6, TNF-α, IL-1β	(Romijn et al., 2017)
L. acidophilus, L. casei, and B. bifidum Tak Gen Zist Pharmaceutical Company (2 × 109 CFU/g Q.D. (n = 20) or placebo (n = 20) for 56 days	Double-blind DSM-IV diagnosis Major Depressive Disorder	Male (n = 6) Female (n = 34) Age 20-55 Mean Age 37.3	#PROT ↓ depression (BDI) #PROT ↓ anxiety (BAI)	§NA	#PROT ↓ HOMA-IR #PROT ↓ CRP #PROT ↑ glutathione	(Akkasheh et al., 2016)
S. thermophilus SGst01, B. animalis ssp. Lactis SGB06, S. thermophiles, B. bifidum SGB02, L. delbrueckii ssp. Bulgaricus DSM20081, L. acidophilus SGL11, L. plantarum SGL07, L. reuteri SGL01 1.5 × 1010 CFU each in corn maltodextrin, silica, casein, lactose, and gluten (n = 24) or placebo (n = 24) Q.D. for 21 days	Double-blind Crossover design normal weight lean, normal weight obese, pre-obese/obese groups BMI range 19.5 – 30 kg/m2	Female (n = 48) Age 27-56 Mean Age 34.6	#PROT ↓ depression (SCL90R) in normal weight lean #PROT ↓ anxiety (SCL-90R) in pre-obese/obese	#PROT ↓ avoidance (BUT) *NSE social insecurity, interpersonal distrust, interceptive awareness (EDI-2)	#PROT ↓ psychotic symptoms (SCL-90R) in normal weight obese and pre-obese/obese	(De Lorenzo et al., 2017)
L. acidophilus, L. casei, B. bifidum, and L. fermentum Tak Gen Zist Pharmaceutical Company 2 × 109 CFU/g each Q.D. (n = 30) or placebo (n = 30) for 84 days	Double-blind McDonald RRMS Exclusions: pregnant or lactating, probiotic or prebiotic use prior to trial enrollment	Male (n = 10) Female (n = 50) Age 18-55 Mean Age 34.1	#PROT ↓ depression and anxiety symptoms (BDI, GHQ, DASS)	§NA	#PROT ↓ insulin, NO, CRP, MDA, HOMA-IR, HOMA-B #PROT ↑ QUICKI, HDL	(Kouchaki et al., 2016)
Lacidofil® 2 × 109 CFU (n = 28) or placebo (n = 32) B.I.D for 84 days	Double-blind Stage 2 or 3 colorectal cancer age > 20, completed treatments between 6 weeks and 2 years prior to enrollment Exclusions: histories of other cancers, colostomies, probiotic consumption, physical or mental disability, chronic diseases, antibiotic use, pregnancy, abnormal liver function, kidney function, or blood cell counts	Male (n = 35) Female (n = 25) Age 45-67 Mean Age 56.2	*NSE emotional well-being (FACT-EWB) #PROT ↓ anxiety and depression (PHQ-9 within group only–NSE between groups)	*NSE social well-being (FACT-SWB)	#PROT ↓ bowel symptoms, functional well-being, cancer related FACT	(Lee et al., 2014)
Bifidobacterium combined with fructooligosaccharide CFU not reported (n = 63) or placebo (n = 62) for 60 days	Double-blind HE - West haven grade 1 or 2 hepatitis B (n = 35), hepatitis C (n = 70), or cryptogenetic cirrhosis (n = 20) venous ammonia >50 mmol/L Exclusions: West haven grade 3+, alcoholism, diabetes mellitus	Male (n = 62) Female (n = 63) Mean Age 50.1	§NA	#PROT ↓ TMT A and B times, #PROT ↑ Symbol Digit Modalities and Block Design Test	#PROT ↓ blood ammonia levels	(Malaguarnera et al., 2010)
L. rhamnosus GG (n = 14) or placebo (n = 16) 5.5 × 1010 CFU B.I.D. for 56 days	Double-blind Cirrhosis with minimal hepatic encephalopathy Exclusions: alcohol use within 6 months, upper GI bleeding or systemic antibiotics within 6 weeks, current or past treatment for HE, hepatocellular cancer, yogurt/probiotic consumption within 2 weeks, inflammatory bowel disease, history of pancreatitis, psychoactive medication use except anti-depressants, recent absolute neutrophil count <500/mm3 and liver transplant	Male (n = 25) Female (n = 12) Age 47-65 Mean Age 57.4	§NA	#PROT ↑ SIP social function *NSE digit symbol or block design tests	#PROT ↓ Enterobacteriaceae abundance #PROT ↓ Clostridiales Incertae Sedis XIV and Lachnospiraceae abundance	(Bajaj et al., 2014)
L. rhamnosus JB-1 1 × 109 CFU (n = 29) or placebo (n = 29) capsules Q.D. for 56 days	Blinding status not reported Crossover design Healthy adult (n = 29) Exclusions: acute or chronic illness, neuropsychiatric disorder (MINI), immunodeficiency, bleeding disorder, color blindness, dyslexia, dyscalculia, experimental drug trial participation, diet, probiotics, antibiotics, antipsychotics, anxiolytics, laxatives, enemas, anti-coagulants NSAIDS, antidepressants or consumption of any psychotropic medication	Male (n = 29) Age 20-33 Mean Age 24.6	*NSE stress, anxiety, depression (PSS, BDI, BAI, STAI, SECPT, Pittsburgh sleep quality index)	*NSE PAL, AST, RVIP, emotional Stroop or recognition task	*NSE IL-1β, IL-6, IL-8, IL-10, TNF-α	(Kelly et al., 2017)
C. butyricum 420 mg capsule 1.0 × 107 CFU/g B.I.D. 14 days until one day before surgery (n = 10) or placebo (n = 10)	Blinding status not reported may be single-blind Cancer patients scheduled for laryngectomy	Male (n = 10) Female (n = 10) Age 45-67 Mean 56.075	#PROT ↓ anxiety (HAM-A)	§NA	#PROT ↓ serum CRF and heart rate before surgery	(Yang et al., 2016b)
L. acidophilus ATCC4356 1.25 × 109 CFU B. longum ATCC15707 1.35 × 109 CFU in vitamin supplement T.I.D. (n = 20) or vitamin supplement without #PROT (n = 20) for 56 days	Blinding Status not reported Irritable Eye Syndrome Exclusions: IBS, systemic, or neuropsychiatric disease	Groups matched by sex (n = 40) Age 39-53 Mean Age 45.5	#PROT ↓ anxiety and depression (HAD)	§NA	#PROT ↓ WBC, monocyte, IL-6, and TNF-α	(Feher et al., 2014)
L. rhamnosus GG and B. lactis BB-12 1 × 109 CFU (n = 33) or placebo (n = 32) Q.D. for 98 days	Blinding status not reported DSM-IV schizophrenia or schizoaffective outpatient, PANSS positive ≥ 1 and/or negative ≥ 4 or total ≥ 50, with at least 3 positive or negative items with scores ≥3 at screening, no antipsychotic medication changes within 21 days of enrollment Exclusions: mental retardation, celiac or medical disorder, DSM-IV substance abuse within 3 months, drug trial within 30 days of study enrollment, pregnant or lactating, antibiotic use within 14 days of enrollment	Male (n = 42) Female (n = 23) Age 18-65 Mean Age 46.3	*NSE PANSS general, negative, or positive symptoms	§NA	#PROT ↓ symptoms in presence of C. albicans antibodies #PROT ↓ constipation	(Dickerson et al., 2014; Severance et al., 2017; Tomasik et al., 2015)

Abbreviations:

^§NA = Not assessed,

^*NSE = No significant effect of probiotic treatment,

^#PROT = Probiotic Treatment, ACTH = Adrenocorticotropic hormone, ADHD = Attention Deficit Hyperactivity Disorder, ASD = Autism Spectrum Disorder, AST = Attention Switching Task, ATCC = American Type Culture Collection, BAI = Beck Anxiety Inventory, BDI = Beck Depression Inventory, BDNF = Brain Derived Neurotropic Factor, BES = Binge Eating Scale, B.I.D = Twice a day, BMI = Body Mass Index, BSID-II-MDI = Bayley Scale Infant Development-II-Mental Development Index, BSID-II-PDI = Bayley Scale Infant Development-II-Psychomotor Development Index, BUT = Body Uneasiness Test, CFU = Colony Forming Units, CNCM = Collection Nationale de Cultures de Microorganismes, CRF = Corticotrophin Releasing Factor, CRP = C-Reactive Protein, DASS = Depression and Anxiety Stress Scale, DSM = Deutsche Sammlung von Mikroorganismen, DSM-IV = Diagnostic and Statistical Manual for Mental Disorders – IV, Ecologic^® Barrier = B. bifidum W23, B. lactis W52, L. acidophilus W37, L. brevis W63, L. casei W56, L. salivarius W24, and L. lactis W19 and W58, EDI-2 = Eating Disorder Inventory – 2, EPDS = Edinburgh Postnatal Depression Scale, EQ-5D-5L = European Quality of Life, FACT-EWB = Functional Assessment of Cancer Therapy-Emotional Well-being, FACT-SWB = Functional Assessment of Cancer Therapy-Social Well-being, GAF = Global Assessment of Functioning, GDS-SF = Geriatric Depression Scale – Short Form, GHQ = General Health Questionnaire, GI = Gastrointestinal, GMMS = Gastrointestinal motility modulating substances, GSRS = Gastrointestinal Symptom Rating Scale, HADS = Hospital Anxiety and Depression Scale, HAM-A = Hamilton Anxiety Scale, HAM-D = Hamilton Depression Scale, HDL = High Density Lipoprotein, HE = Hepatic encephalopathy, HSCL-90 = Hopkins Symptom Checklist, hs-CRP = high-sensitivity C-reactive protein, HOMA-IR = Homeostatic Insulin Resistance, HOMA-B = Homeostatic Beta cell Function, IBD = Inflammatory Bowel Disease, IBS = Irritable Bowel Syndrome, I.31 = L. plantarum CECT7484 + CECT7485 and P. acidilactici CECT7483, iCGI-I = improved Clinical Global Impression Scale-Improvement, iCGI-S = improved Clinical Global Impression Scale-Severity, IgE = Immunoglobulin E, IgG = Immunoglobulin G, IL-1β = Interleukin-1- beta, IL-6 = Interleukin 6, IL-8 = Interleukin 8, IL-10 = Interleukin 10, IV = Intravenous, Lacidofil^® = 95% L. rhamnosus R0011 & 5% L. helveticus R0052, LEIDS = Leiden Index of Depression Sensitivity, MDA = Malondialdehyde, MADRS = Montgomery-Asberg Depression Rating Scale, MINI = Mini-International Neuropsychiatric Interview, NK = Natural Killer, NO = Nitric Oxide, NSAID = Non-Steroidal Anti-Inflammatory Drug, PAL = Paired Associative Learning, PANSS = Positive and Negative Syndrome Scale, PASAT = Paced Auditory Serial Addition Test, Ped = Pediatric, PHQ-9 = Patient Health Questionnaire-9, P.O. = Per Oral, POMS = Profile of Mood States, PPI = Proton Pump Inhibitors, PRET = Prebiotic Treatment, PSS = Perceived Stress Scale, Q.D. = One a day, Q.I.D. = Four times a day, QIDS-16 = Quick Inventory of Depression Symptomatology, QOL = Quality of Life, QUICKI = Quantitative Insulin Sensitivity Check Index, RRMS = Relapse Remitting Multiple Sclerosis, RVIP = Rapid visual information processing, SCFA = Short Chain Fatty Acids, SCL90R = Symptom Checklist Revised, SECPT = Socially Evaluated Cold Pressor Test, SIP = Sickness Impact Profile, SSP = subspecies, STAI = State Trait Anxiety Inventory, SYNT = Synbiotic Treatment, T.I.D. = Three times a day, TMT = Trail Making Test, TNF-α = Tumor Necrosis Factor-alpha, UC = Ulcerative Colitis, VLDL = Very Low-Density Lipoprotein VSL#3 = S. thermophilus DSM 24731, B. longum DSM 24736, B. breve DSM 24732, B. infantis DSM 24737, L. acidophilus DSM 24735, L. plantarum DSM 24730, L. paracasei DSM 24733, L. delbrueckii ssp. bulgaricus DSM 24734, WBC = White Blood Cell

Table 2.

Non-Human Animal Probiotic Trial Designs and Outcomes.

Probiotic Treatment	Blinding Status and Study Design	Sex/Age, days	Anxiety/ Depression Phenotypes	Cognition /Social Phenotypes	Other Probiotic Associated Outcomes	Reference
Rat (Rattus)
L. plantarum MTCC1325 1.2 × 109 CFU/mL P.O. Q.D. for 60 days	Blinding status not reported Wistar control (n = 6), D-GAL (n = 6), D-GAL + L. plantarum (n = 6), L. plantarum (n = 6)	Male (n = 24) Age 90 days	#PROT ↑ D-GAL induced ↓ gross behavior	#PROT ↓ D-GAL induced ↑ escape latency (MWM)	#PROT ↑ D-GAL induced ↓ acetylcholine (HIP, cortex), organ and body weight #PROT ↓ D-GAL induced ↑ acetylcholinesterase	(Nimgampall e and Kuna, 2017)
L. rhamnosus plus B. longum Ningxia Medical University 2 × 109 CFU/mL in 1 × PBS P.O. Q.D. for 12 days	Experimenter blinded behavior SPF Sprague Dawley No treatment (n = 8), 1× PBS control (n = 8), ampicillin (n = 8), ampicillin + PROT (n = 8), ampicillin + clonazepam (n = 8) for 12 days	Male (n = 40) Age 10 days	#PROT ↓ ampicillin induced ↑ immobility (TST and FST)	#PROT ↓ ampicillin induced ↑ escape latency (MWM)	#PROT ↑ ampicillin induced ↓ HIP GABA-A receptors *NSE #PROT on ampicillin induced change in gut microbial composition	(Liang et al., 2017)
Ecologic® Barrier 3.8 × 108CFU/mL in water or vehicle Q.D. for 84 days	Experimenter blinded to #PROT and behavior FSL, FRL, & SD FSL CLD + vehicle (n = 11), FSL CLD + #PROT (n= 11), FSL HFD (60%) + #PROT (n= 12), FSL HFD + vehicle (n = 12), FRL CLD + vehicle (n = 12), SD CLD + vehicle (n = 8), SD HFD + vehicle (n = 8)	Male (n = 74) Age 35 days	#PROT ↓ HFD ↑ depression (FST) in FSL *NSE #PROT depression CLD, locomotion (OFT)	§NA	#PROT ↓ CD4/CD8 for CD3+ cells in blood for FSL (*NSE brain) *NSE #PROT body weight, caloric intake, blood glucose, ghrelin, insulin, leptin, MCP, cytokines, WBC	(Abildgaard et al., 2017a)
Ecologic® Barrier 2.5 × 109 CFU/g in water for 35 days during diet (began 35 days prior to #PROT)	Experimenter blinded to #PROT and behavior Sprague-Dawley SD (n= 10), HFD (n = 10), #PROT + SD (n = 10) #PROT + HFD (n = 10)	Male (n = 40) Adult	#PROT ↓ immobility (FST) *NSE anxiety (OFT)	*NSE memory (Barnes)	*NSE LPS	(Abildgaard et al., 2017b)
L. helveticus R0052 plus B. longum R0175 109 (n = 7) 1010 (n = 8) CFU/day in vehicle with water P.O. Q.D. for 70 days	Experimenter blinded behavior FLR vehicle only (n = 8) FSL (n = 22) 109 #PROT (n = 7), 1010 #PROT (n = 8), or vehicle (n = 7)	Male (n = 30) Adult	*NSE immobility (FST)*NSE anxiety (OFT)	*NSE memory (Y-maze, NOR) *NSE social interaction	#PROT ↓ plasma betaine, NE, DA #PROT ↑ liver SAM *NSE betaine, norepinephrine, dopamine or SAM in PFC or HIP	(Tillmann et al., 2018)
L. helveticus R0052 plus B. longum R0175 3 × 109 CFU P.O. Q.D. for 14 days	Experimenter blinded behavior Wistar #PROT (n = 12), diazepam (n = 12), and placebo (n = 12)	Male (n = 36) Adult	#PROT and diazepam ↓ burying	§NA	Human trial outcomes are reported in Table 1	(Messaoudi et al., 2011a)
B. infantis 356241 × 1010 CFU/100 mL or vehicle in drinking water Q.D. for 14 days	Experimenter blinded behavior Sprague Dawley #PROT (n = 12) or vehicle (n = 8)	Male (n = 20) Adult	*NSE FST	§NA	#PROT ↓ 5-HI A A (frontal cortex), DOPAC (amygdala), body weight #PROT ↓ serum IFN-γ, TNF-α, IL-6 after mitogen stimulation ↓ TNF-α after LPS stimulation #PROT ↓ IFN-γ, IL-6 after CON A stimulation #PROT ↓ plasma tryptophan and kynurenic acid	(Desbonnet et al., 2008)
L. helveticus NS8 109CFU/mL Q.D. for 21 days	Blinding status not reported SPF Sprague Dawley CON (n = 8), chronic RS (n = 8), L. helveticus NS8 during chronic RS (n = 8), citalopram hydrobromide 30 mg/kg during chronic RS (n = 8)	Male (n = 32) Adult	#PROT ↓anxiety (EPM & OFT)	#PROT ↑ memory (NOR & OPT)	#PROT ↑ HIP 5-HT, NE, BDNF, IL-10 #PROT ↓ serum CORT, ACTH	(Liang et al., 2015)
VSL#3 (low - 2.5 × 109 CFU or high - 2.5 × 1010 CFU) in maple syrup for 14 days before diet and 26 days during diet modification	Experimenter blinded behavior Sprague Dawley CFD, CFD + VSL#3 low, CFD + VSL#3 high, SD, SD + VSL#3 low, SD + VSL#3 high, (n = 10/group)	Male (n = 60) Adult	*NSE anxiety (EPM)	VSL#3 high ↑ diet induced memory ↓ (NPR) VSL#3 low and high ↓ memory (NOR) for CFD and SD	CFD + VSL#3 high had ↑ Streptococcus Lactobacillus, Butyrivibrio than CFD alone *NSE overall microbial diversity	(Beilharz et al., 2017)
L. acidophilus ATCC4356, B. lactis DSM 10140, plus L. fermentum ATCC9338 1010 CFU/g B.I.D. for 56 days	Blinding status not reported Wistar diabetic (Streptozotocin 65 mg/kg; n = 10), diabetic + #PROT (n = 10), CON (n= 10), CON + #PROT (n = 10)	Male (n = 40) Age 45 days	§NA	#PROT ↑ spatial memory (MWM) in control and diabetic rats #PROT ↑ HIP baseline EPSP and LTP in diabetic rats	#PROT ↓ serum glucose ↑ insulin and SOD levels in diabetic rats	(Davari et al., 2013)
L. plantarum or B. B94 (industrial enzymes company, representative of DSM Company) 1.5 × 108 CFU/mL P.O. Q.D. for 28 days	Blinding status not reported Wistar HIP demyelination model EB (n = 8), EB + L. plantarum (n = 8), EB + B.B94 (n = 8), or saline (n = 8)	Male (n = 32) Age 56-80 days	§NA	*NSE spatial memory (MWM)	§NA	(Goudarzvand et al., 2016)
L. plantarum KY 1032 and L. curvatus HY7601 1 × 1010 CFU Q.D. 48 days (6 days/week)	Blinding status not reported Fischer 344 young, older, older + #PROT, older + rapamycin (n = 6/group)	Male (n = 24) young age not reported older 540 days	§NA	#PROT ↑ memory (Y-maze)	#PROT ↑ HIP doublecortin and ↓ BDNF	(Jeong et al., 2015)
ampicillin + L. fermentum NS9 109 CFU/mL (n = 10) for 41 days	Blinding status not reported Sprague Dawley CON (n = 10), ampicillin (n = 10) ampicillin + L. fermenlum NS9 109 CFU/mL (n = 10)	Male (n = 30) Adult	#PROT ↓ anxiety (EPM) *NSE locomotor activity	§NA	#PROT ampicillin induced MPO activity	(Wang et al., 2015)
VSL#3 1.2 × 1010 CFU/kg in maple syrup Q.D. for 42 days	Blinding status not reported Wistar young, young +#PROT, aged, aged + PROT sample sizes not reported	Male young (90 days) aged (600 – 660 days)	§NA	#PROT ↑ HIP LTP in aged rats ≈ young rats	#PROT ↓ microglia and ↑ Bacteroidetes in aged rats #PROT up and down regulates cortical gene expression	(Distrutti et al., 2014)
L. paracasei HII01 1 × 108 CFU/mL P.O. in PBS Q.D. for 84 days with diet modification (84 days pre- L. paracasei and 84 days during L. paracasei trial)	Double-blind Wistar (n = 6 per group) Standard diet, HFD, Standard diet +#PROT, HFD +#PROT, Standard diet +#PROT + XOS, HFD + #PROT + XOS, Standard diet + XOS, HFD + XOS	Male (n = 48) Adult	§NA	#PROT ↑ HFD induced ↓ HIP LTP (fEPSP)	#PROT ↓ HFD induced ↑ in serum LPS, plasma glucose, total cholesterol, LDL cholesterol, serum and brain MDA, insulin, HOMA	(Chunchai et al., 2018)
L. helveticus R0052 plus B. longum R0175 1 × 109 CFU in drinking water 7 days pre-MI/sham to 7 days post-MI/sham (14 days)	Experimenter blinded behavior Sprague Dawley MI (n = 9), MI + PROT (n = 9), sham (n = 9), sham + PROT (n = 9)	Male (n = 36) Age 84 days	#PROT ↓ depression (FST) and anxiety (SDT) in MI not sham	#PROT ↑ social interaction in MI not sham	#PROT ↓ intestinal permeability in MI treated groups	(Arseneault-Breard et al., 2012)
L. helveticus NS8 1 × 109 CFU/mL Q.D. in drinking water for 14 days post HA neuroinflammatio n (28 days)	Blinding status not reported SPF Sprague Dawley saline (n = 6), HA (n = 6), HA + #PROT (n = 6)	Male (n = 18) Adult	#PROT ↓ HA induced anxiety (EPM)	#PROT ↓ HA induced spatial memory deficits (MWM)	#PROT ↑ 5-HIAA CBM, ↑ 5-HIAA HIP, ↓ 5-HT in HA rats #PROT ↓ HA induced IL-1β, iNOS, and PGE2 in CBM, HIP and PFC	(Luo et al., 2014)
Lacidofil® 1 × 109 CFU/mL for 14 days	Blinding status not reported Sprague Dawley MS model: MS fathers, MS fathers + #PROT (prevention), MS fathers no treatment pups no treatment, MS fathers no treatment pups #PROT (active treatment), MS fathers #PROT (prevention) pups no treatment	Male Female P2-P14 (n = 398 all experiments)	§NA	Preventative and active #PROT ↓ cued fear conditioning and infantile amnesia in pups from MS fathers	§NA	(Callaghan et al., 2016)
Lacidofil® 1 × 109 CFU/mL in drinking water for 12 days (P2 to P14)	Double-blind Sprague Dawley #PROT during MS model MS + #PROT (n = 13), MS + vehicle (n = 14), no MS + vehicle (n = 10; n = 9)	Male Pups (n = 37) Age P2-P14 then experiments at P17 Female Mothers (n = 9)	*NSE MS +#PROT anxiety (EPM) in male pups or female mothers	MS +#PROT ↑ infantile amnesia (7 days post-fear conditioning)*NSE MS + #PROT context dependent freezing in male pups	*NSE MS + #PROT for maternal behavior (pup retrieval)	(Cowan et al., 2016)
B. infantis 35624 1 × 1010 CFU/100 mL Q.D. for 45 days	Experimenter blinded behavior Sprague Dawley MS model CON (n = 11), MS (n = 7), MS + citalopram 30 mg/kg (n = 7), and MS + B. infantis (n = 8)	Male (n = 33) Age P2-P14 #PROT P50 to P95	#PROT ↓ MS induced depression (immobility, swimming - FST)	§NA	*NSE stimulated or unstimulated IFN-γ, TNF-α, IL-6, (IL-10 #PROT ↓ MS induced NE and CRF expression in amygdaloid cortex	(Desbonnet et al., 2010)
L. helveticus R0052 and B. longum R0175 109 CFU in 200 mL water Q.D. for 14 days post-MI	Blinding status not reported Sprague Dawley low or high ω-3 PUFA diet w/ or w/out #PROT after MI (n = 16/group)	Male (n = 64) Age 90 days	#PROT x diet interaction depression (FST)	#PROT reversed post-MI social interaction deficits in low-PUFA group *NSE post-MI social interaction in high PUFA group #PROT ↑ memory (↓ time and number of trials PAT)	#PROT ↑ plasma IL-4 in high PUFA group	(Gilbert et al., 2013)
B. infantis 35624, B. breve UCC2003, or L. salivcirius UCC118 5 × 109 CFU/mL or vehicle P.O. Q.D. for 14 days	Experimenter blinded behavior Sprague Dawley (n = 40) Wistar-Kyoto (n = 40) PBS, B. infantis, B. breve, L. salivarius (n = 10/group/strain)	Sex not reported (n = 80) Age 63 days	*NSE anxiety (OFT)	§NA	*NSE plasma corticosterone B. infantis ↓ pain behavior Wistar-Kyoto only	(McKernan et al., 2010)
L. fermentum CECT 5716 109 CFU/100g body weight P.O. Q.D. for 3 days pre-MS (age 10 days) or 15 days pre-WAS (age 21 days)	Blinding status not reported Sprague Dawley MS, WAS 8 groups (n = 6/group behavior experiments)	Sex not reported Age 10 and 21 days	*NSE anxiety (OFT)	#PROT ↑ exploratory behavior (OFT)	#PROT ↓ WAS and MS induced ↑ plasma corticosterone, intestinal permeability #PROT ↑ IFN-γ and ↓ IL-4 after CD3/CD28 stimulation #PROT normalized WAS induced intestinal ZO-1 reorganization	(Vanhaecke et al., 2017)
L. acidophilus (1688FL431-16LA02), L. fermentum (ME3), B. lactis (1195SL609-16BS01) and B. longum (1152SL593-16BL03) 1010 CFU Q.D. in drinking water for 28 days pre- β-amyloid injection and 28 days post-injection	Blinding status not reported Wistar control, control + #PROT, sham surgery, β-amyloid intra-hippocampal injection, β-amyloid intra-hippocampal injection + #PROT (n = 12/group)	Male (n = 60) Age 56 days	§NA	#PROT ↓ β-amyloid injection induced ↑ in escape latency	*NSE weight, catalase activity #PROT ↓ β-amyloid injection induced ↑ MDA, SOD, plaques, cell morphology	(Athari Nik Azm et al., 2018)
L. rhamnosus GG 108 CFU/mL in drinking water for approximately 80 days	Blinding status not reported Sprague-Dawley 10 day MS or no MS control diet (n = 5), control diet + #PROT (n = 9), diet + PDX + GOS (n = 5), diet + PDX + GOS +#PROT (n = 9), followed by acute RS	Male only behavior (n = 56) MS Age 2-12 days Behavior Experiments Age 49-100 days	#PROT + PDX + GOS ↓ MS induced ↑ anxiety (OFT)	#PROT + PDX + GOS ↑ MS induced ↓ spatial memory (MWM)	#PROT ↓ Nr3c1, Nr3c2, Crhrl in MS group only #PROT + PDX + GOS delayed return of acute stress induced ↑ of corticosterone to baseline	(McVey Neufeld et al., 2017)
Mouse (Mus)
L. fermentum LAB9 109 CFU/200μL or L. casei LABPC 109 CFU/200μL in cow’s milk P.O Q.D. for 28 days pre- LPS induced inflammation (4 days)	Blinding status not reported ICR/HaJ Saline (n = 6) LPS (n = 6) LPS + unfermented milk (n = 6) LPS + L. fermentum, (n = 6) LPS + L. casei (n = 6)	Male (n = 30) Age 63 days	§NA	L. fermentum and L. casei ↓ LPS induced spatial memory deficit – escape latency and distance (MWM)	L. fermentum and L. casei ↑ LPS induced ↓ catalase, SOD, GSH, GPx, MDA, NO, MCP-1 L. fermentum and L. casei ↓ LPS induced ↑ AChE, IL-6, L. fermentum ↓ LPS induced ↑ IL-1β	(Musa et al., 2017)
L. casei DG 109 CFU in saline P.O. for 7 days	Experimenter blinded behavior C57BL/6J (n = 8-9/group) CON, ABX, ABX + #PROT, ABX + saline for 14 days in water	Male (n = 36) Age 64 days	#PROT ↓ ABX induced immobility (TST, FST)	*NSE social novelty	*NSE muscle strength, motor coordination #PROT normalized ABX induced changes in BDNF, HIP firing rate, HIP TRN1 phosphorylation, astrocyte and microglia morphology, intestinal 5-HT and OA-5-HT	(Guida et al., 2017)
L. brevis OW38 1 × 109 CFU P.O. for 56 days	Blinding status not reported C57BL/6J young+ vehicle (n = 6), young+ PROT (n = 6), older + vehicle (n = 6), older + PROT (n = 6)	Male (n = 24) Age 120 or 540 days	§NA	#PROT ↑ memory (Y-maze) in aged mice	#PROT ↓ age ↑ colonic p-F0X03a, p-mTor, fecal and plasma LPS #PROT ↑ age ↓ HIP BDNF, butyric acid, IL-1β, IL-6, TNF, COX-2, iNOS, NF-κB, claudin-1, ZO-1	(Eun et al., 2016)
L. plantarum C29 1 × 109 CFU P.O. Q.D. for 5 days post-TNBS induced memory deficit	Blinding status not reported SPF C57BL/6J (n = 6/group) Methods based on (Jeong et al., 2016)	Male (n = 24 - behavior)(n = 42 – colitis) Age 42 days	§NA	#PROT ↑ TNBS induced ↓ memory (NOR, Y-maze, PAT)	#PROT ↑ TNBS induced ↓ BDNF #PROT ↑ TNBS induced ↓ Bifidobacteria, Lactobacilli, Clostridia #PROT ↓ TNBS induced ↑ in Enterobacteriacae	(Lee et al., 2018)
L. plantarum MTCC 9510 2 × 1010 CFU/300μl in PBS P.O. for 21 days during sleep deprivation and 28 days during chronic unpredictable stress	Experimenter blinded behavior Swiss Webster LACA chronic unpredictable stress for 28 d (n = 8), chronic unpredictable stress + #PROT (n = 8), naïve (n = 8), naïve +#PROT (n = 8), naïve (n = 6), 72h sleep deprivation (n = 8),72h sleep deprivation + #PROT (n = 8), naïve + #PROT (n = 6)	Male (n = 68) Age not reported	#PROT ↓ chronic stress and sleep deprivation induced ↓ anxiety and depression behavior (FST, TST, OFT, EPM)	#PROT ↑ chronic stress induced ↓ spatial (MWM) and chronic stress and sleep deprivation induced ↓ working memory (PAT)	#PROT ↓ stress and sleep deprivation induced ↑ NF-κB, LPS, TNF-α, MAO-A, MAO-B, MDA, GSH, corticosterone #PROT ↑ stress and sleep deprivation induced ↓ HIP BDNF #PROT ↑ cecal Lactobacilli and ↓ Enterobacteriaceae	(Dhaliwal et al., 2018)
E. faceium CFR3003 104 CFU (n = 6), E. faceium CFR3003 108 CFU (n = 6), L. rhanmosus GG MTCC1408 108 CFU (n = 6), or saline (n = 6) P.O. for 28 days	Blinding status not reported CFT-Swiss LPS model of inflammation	Male (n = 24) Age 42 days	E. faceium - 108 CFU and L. rhanmosus ↓ anxiety (OFT)	§NA	E. faceium and L. rhanmosus reversed LPS induced ↑ TNF-α and ↓ IL-10 E. faceium -108 CFU and L. rhanmosus ↓ cecum weight and ↑ lactobacilli E. faceium - 104 CFU ↑ GST in HIP ↓ AchE in CTX and STR E. faceium - 108 CFU ↑ cytosolic GABA in CTX, HIP, STR, ↑ cytosolic DA in CTX, ↑ GST in CTX, HIP, STR, ↓ ROS in CTX L. rhanmosus ↓ ROS in HIP, ↑ cytosolic GABA in HIP and DA in STR, ↑ CAT in CTX, HIP ↑ GST in HIP, STR *NSE ROS STR	(Divyashri et al., 2015)
B. fragilis NCTC9343 1 × 1010 CFU in food every other day for 6 days	Blinding status not reported SPF, germ-free and conventional C57BL/6J MIA model pregnant (E12.5) mice injected with saline or 20 mg/kg poly I:C n animals varied ranging from 10-75/group	Male Female Treatment Age approximately 28 days Behavior testing 42 days	#PROT ↓ anxiety (OFT, burying) in MIA	#PROT ↑ sensorimoto r gating (PPI) in MIA mice *NSE social interaction	§NA	(Hsiao et al., 2013)
L. rhamnosus JB-1 1.6 × 109 CFU/200ul or saline P.O. Q.D. for 28 days	Blinding status not reported C57BL/6J Chronic social defeat stress began at day 18 of #PROT vehicle (n = 10),#PROT (n = 8), stress (n = 15), stress + #PROT (n= 10)	Male (n = 43) Age 63 days	#PROT ↓ stress induced anxiety (OFT, light/dark)	#PROT prevented ↓ social interaction with conspecifics	#PROT ↑ stress induced ↓ in fecal tyramine #PROT ↓ stress induced ↑ in MHCII+, CD11c+, CD80, CD86, #PROT ↓ IL-10+ Treg *NSE stress induced kynurenine, 4- hydroxybutyrate, or 1-methylnicoinamide	(Bharwani et al., 2017)
Lacidofil® 1× 1010 CFU/mL P.O. Q.D. 7 days pre-DSS and 8 days during DSS	Blinding status not reported SPF C57BL/6J DSS model control, #PROT, DSS, DSS + #PROT (n = 9-12/group)	Male (n = 40) Female (n = 40) Age 42-56 days	#PROT ↓ anxiety (light/dark box)	#PROT ↑ memory in DSS (NOR)	#PROT ↑ DSS induced loss of cFos in CA1 HIP #PROT ↓ DSS induced dysbiosis	(Emge et al., 2016)
L. rhamnosus NC4007 1010 CFU/100uL (n = 10), B. longum NCC3001 1010 CFU/100uL (n = 16), etanercept, budesonide, or placebo (n = 16) for 10 days after 30 days T. minis infection	Experimenter blinded behavior SPF BALB/c and AKR/J Vagotomy (n = 24) or sham vagotomy (n = 15) before infection	Male (n = 39) Age 42-56 days	B. longum ↓ T. muris induced anxiety (light/dark box and SDT) *NSE L. rhamnosus on T. muris induced anxiety	§NA	B. longum ↑ T. muris induced ↓ brain BDNF *NSE B. longum circulating TNF-alpha *NSE B. longum kynurenine	(Bercik et al., 2010)
B. longum NCC3001 1 × 1010 CFU/mL or vehicle P.O. Q.D. for 14 days (7 days during DSS and 7 days post-DSS)	Experimenter blinded histology SPF AKR/J naïve (n = 13), B. longum (n = 6), DSS (n = 12), sham surgery (n = 11), vagotomy (n = 15), Vagotomy + B. longum (n = 14), vagotomy + DSS (n = 15), vagotomy + DSS + B. longum (n = 15), sham surgery + B. longum (n = 9), B. longum + DSS (n = 11)	Male (n = 151) Age 42-56 days	#PROT ↓ anxiety (SDT) in control and DSS w/out vagotomy	§NA	#PROT ↓ excitability of enteric neurons and colonized intestine for sham and vagotomy, *NSE BDNF expression SH-SY5Y cells or colon histology	(Bercik et al., 2011)
B. longum 1714 or B. breve 1205 1 × 109 CFU/mL P.O. Q.D. for 77 days	Experimenter blinded behavior BALB/c B. longum (n = 12), B. breve (n = 12), or vehicle (n = 12)	Male (n = 48) Age 49-56 days	B. breve ↓ locomotion *NSE B. longum	B. breve and B. longum ↑ memory (NOR) B. longum ↑ memory (Barnes, cued, and contextual fear conditioning)	*NSE CORT, body weight, colorectal distention	(Savignac et al., 2015)
VSL#3 1 × 107 CFU P.O. Q.D. for 10 days	Experimenter blinded behavior C57BL/6J SPF naïve (n = 10), SPF + #PROT (n = 10), SPF + exercise (n = 10), ABX (n = 10), ABX + #PROT (n = 10), ABX + exercise (n = 10), ABX + SPF fecal transplant (n = 10)	Female (n = 70) Age 42-56 days	§NA	#PROT or exercise ↑ ABX induced memory deficit (NOR)	#PROT or exercise ↑ ABX induced brain monocyte or HIP neurogenesis reduction	(Mohle et al., 2016)
Lacidofil® 6 × 109 CFU/mL in water Q.D. for 7 days pre- C. rodentium infection P.O. and 7 days post-infection	Experimenter blinded immunohistochemistry C57BL/6J and Swiss-Webster SPF, germ-free, and conventional #PROT with and without C. rodentium infection or WAS (n = 10-14/group)	Female Age 35-42 days	§NA	#PROT ↑ working memory (T-maze) in C. rodentium infected and WAS mice	#PROT ↓ serum CORT in C. rodentium infected and WAS mice #PROT ↓ IFNγ in C. rodentium not WAS mice *NSE on TNF-α in C. rodentium or WAS mice	(Gareau et al., 2011)
Lacidofil® 6 × 109 CFU/day or placebo in water for 28 days before WAS	Experimenter blinded immunohistochemistr y C57BL/6JRcigl−/− (n = 4-6/group behavior experiments) no WAS, WAS 1hr for 1 day	Male Female Age 42 to 56 days	#PROT ↓ anxiety (light/dark) in Rag−/− WAS and no WAS	#PROT ↓ memory (NOR) in Rag1−/− WAS mice #PROT ↑ memory (NOR) in Rag1−/− naïve mice	*NSE corticosterone #PROT normalized intestinal ion absorption in no WAS mice only #PROT ↓ abundance of Bacteroides, Enterobacteriaceae, Firmicutes, Lactobacilli	(Smith et al., 2014)
L. pentosus ssp. plantarum C29 1 × 1010 CFU/mouse, P.O. Q.D. for 35 days during D-GAL (D-GAL induced aging 35 days pre-#PROT and 35 days during #PROT)	Blinding status not reported C57BL/6J Naïve (n = 6), D-GAL (n = 6), #PROT (n = 6)	Male (n = 18) Age 140 days (D-GAL) Age 182 days (#PROT)	§NA	#PROT reversed D-GAL induced ↓ in memory (MWM, Y-maze, PAT)	#PROT reversed D-GAL induced ↓ in BDNF, DCX, and CREB	(Woo et al., 2014)
L. johnsonii ATCC33200 or L. renteri MM4-1A ATCC-PTA-6475 1 × 108 CFU in water for 28 days	Double-blind Germ-free C57BL/6J MRD (13.4% FAT, 30% PRO, and 57% CARB), MHFD (60% FAT, 20% PRO, 20% CARB), live #PROT, heat killed #PROT	Male (behavior) Age 49-84 days	*NSE anxiety (OFT)	L. reuteri ↑ LTP in VTA DA neurons for MHFD L. reuteri ↑ sociability, social novelty, reciprocal social interaction in MHFD	§NA	(Buffington et al., 2016)
VSL#3: 1.7 × 1010 CFU P.O. Q.D. 10 days pre- and 10 days post-surgery	Blinding status not reported C57BL/6 SPF BDL model liver inflammation + #PROT (n = 10), BDL no #PROT (n = 10), sham + #PROT (n = 10), sham no #PROT (n = 10)	Males (n = 40) Age 42-56 days	#PROT ↓ depression (immobility) in BDL model	#PROT ↑ social behavior in BDL model	#PROT ↓ monocyte infiltration to brain in BDL model #PROT ↓ microglial activation in brain in BDL model #PROT ↓ TNF-α in BDL model *NSE gut permeability or liver injury	(D'Mello et al., 2015)
B. longum 1714 or B. breve 1205 1 × 1010 CFU/mL Q.D. for 42 days	Experimenters blinded behavior BALB/c SIH vehicle (n ≈ 20), escitalopram 20mg/kg (n ≈ 20), B. longum (n ≈ 20), B. breve (n ≈ 20)	Male (n ≈ 80) Age 49 days	B. longum ↓ SIH escitalopram, B. longum and B. breve ↓ marble burying B. breve ↓ anxiety (EPM) B. longum ↓ anxiety (OFT) B. longum ↓ depression TST *NSE depression (FST)	§NA	B. breve ↓ bodyweight gain B. breve ↑ spleen weight *NSE corticosterone	(Savignac et al., 2014)
L. rhanmosus JB-1 109 CFU/mL or control broth P.O. Q.D. for 28 days	Blinding status not reported BALB/c CON (n = 20) CON naïve (n = 8), CON + SIH (n = 8), #PROT (n = 8), or #PROT with stress (n = 8) Vagotomy (n = not reported), vagotomy + #PROT (n = not reported)	Male Age 70-77 days	#PROT ↓ depression (FST) #PROT ↓ anxiety (EPM, OFT)	#PROT ↑ cued and contextual memory fear conditioning	#PROT ↓ stress-induced CORT levels #PROT ↑ GABAB1b mRNA in cingulate of sham not vagotomized mice *NSE on FST or OFT after vagotomy *NSE on SIH	(Bravo et al., 2011)
L. rhanmosus GG 1 × 109 CFU P.O. Q.D. for 14 days	Single-blind behavior BALB/c 5-HT1A/1B receptor agonist model of OCD (RU 24969) primed by social experience and then pretreatment with saline (n = 6), fluoxetine 10 mg/kg for 28 days (n = 12) or #PROT for 14 days (n = 12) before inducing OCD model	Male (n = 36) Age 56 days	#PROT pretreatment ↓ anxiety (burying) fluoxetine had greater effect than #PROT #PROT and fluoxetine ↓ locomotion (OFT) *NSE aggression	§NA	§NA	(Kantak et al., 2014)
L. casei-01 109 CFU/kg P.O. Q.D. for 20 days	Blinding status not reported Kunming (n = 10/ group) control, SCOP (3mg/kg), SCOP + piracetam (400mg/kg), SCOP + #PROT, SCOP + LSPC (60mg/kg), SCOP + LSPC (90mg/kg), SCOP + #PROT + LSPC (60mg/kg), SCOP + #PROT + LSPC (90mg/kg)	Male (n = 80) Age not reported	§NA	#PROT ↑ memory (Y-maze) in SCOP induced amnesia #PROT enhances effect of LSPC on SCOP induced amnesia at both dosages	#PROT enhances LSPC ability to reduce MDA and increase antioxidant levels *NSE on brain NOS levels	(Xiao et al., 2014)
L. plantarum CCFM639 5 × 109 CFU/mL P.O. Q.D. in milk for 84 days after 28 days aluminum toxicity	Experimenter blinded histology SPF C57BL6/J (n = 10/group) control, aluminum toxicity, aluminum toxicity + live #PROT, aluminum toxicity + heat killed #PROT	Male (n = 40) Age 42 days	§NA	Live and heat killed #PROT ↑ aluminum induced ↓ spatial memory (MWM)	Live #PROT ↓ aluminum induced ↑ brain Aβ1-40 & Aβ1-42 Live and heat killed #PROT ↓ aluminum induced ↑ ALT, AST, CRE, BUN, and live aluminum Live and heat killed #PROT ↑ fecal aluminum (day 14 #PROT) Live and heat killed #PROT ↓ aluminum induced ↓ SOD, CAT, GPx, GSH, MDA in liver and brain *NSE brain aluminum	(Tian et al., 2017)
L. helveticus R0052 109 CFU P.O. or no treatment Q.D. for 21 days	Experimenter blinded histology SPF 129/SvEv wild type and IL-10 −/− fed SD (29% PRO, 55% CARB,13% FAT) (n = 5-6), SD + #PROT (n = 5-6), Western-style diet (28% PRO, 49% refined CARB, 33% FAT) (n = 5-6), Western diet plus #PROT (n = 5-6)	Sex not reported Age PD29	#PROT ↓ anxiety with western diet wild type and IL-10 −/− *NSE anxiety for IL-10 −/− fed standard chow	#PROT ↓ memory that ↓ with western diet in IL-10 −/− *NSE anxiety for IL-10 −/− fed standard chow	#PROT ↑ brain corticosterone, fecal corticosterone, Firmicutes/Bacteroidete s, IL-1B in IL-10 −/− mice	(Ohland et al., 2013)
L. plantarum PS128 5 × 109 CFU/mL P.O. Q.D. for 16 days	Blinding status not reported germ-free C57BL/6J live #PROT (n = 10), heat -killed #PROT (n = 10) or pre-warmed saline (n = 10) for 16 days	Male (n = 30) Age 42 days	#PROT ↓ anxiety (EPM) Live #PROT ↑ locomotion	§NA	Live #PROT ↓ cecum weight Live #PROT ↓ DA, HVA, 5-HT, 5-HIAA in striatum *NSE #PROT in PFC or HIP *NSE #PROT organ histology and serum CORT	(Liu et al., 2016a)
L. plantarum PS128 5 × 109 CFU P.O. Q.D. for 28 days	Blinding status not reported SPF C57BL/6J MS (n = 12), MS + #PROT (n = 10) from PD29, naïve adult mice (n = 10), and naïve adult + #PROT (n = not reported) from 8 weeks	Male Age PD29 and 56 days	#PROT restores sucrose preference and FST in MS mice but *NSE #PROT for naïve mice #PROT ↓ anxiety (OFT, EPM) naïve mice *NSE anxiety in MS mice #PROT ↑ locomotor activities in MS and naïve mice	§NA	#PROT reverses MS ↑ IL-6 and ↓ IL-10 #PROT ↑ PFC dopamine in MS and naïve mice #PROT ↑ 5-HT in naïve #PROT ↓ serum CORT during basal & stressed states for MS not naïve mice #PROT ↑ levels of 5-HT in PFC in MS	(Liu et al., 2016b)
C. butyricum WZMC1016 (CGMCC9831)1 × 108 CFU/200μl saline P.O. Q.D. for 42 days	Blinding status not reported SPF C57BL/6J Streptozotocin sham operation (n = 12), cerebral I/R injury (n = 12), cerebral I/R injury + C. butyricum (n = 12)	Male (n = 36) Age 87 days	§NA	#PROT ↑ cerebral injury induced ↓ in spatial memory (MWM)	#PROT ↓ cerebral injury induced ↓ p-Akt	(Sun et al., 2016)
Zebrafish (Danio rerio)
L. plantarum (USDA-ARS) 2 × 107 CFU/mL single exposure for 2 days	Blinding status not reported Wild-type CV, CV + stress, CR, CR + stress, CR + stress + #PROT, GF, GF + stress, GF + stress + #PROT	Sex not reported Age 4 days post fertilization (#PROT)	#PROT ↓ anxiety (thigmotaxis) *NSE locomotor activity	§NA	#PROT ↓ stress induced cortisol level in CR not GF	(Davis et al., 2016a)
L. plantarum (USDA-ARS) 1 × 106 CFU/mL B.I.D for 30 days	Blinding status not reported Wild-type chronic unpredictable stress #PROT (n = 5-7), CON (n = 5-7)	Male Female Adult	#PROT ↓ anxiety (novel tank diving)	§NA	#PROT restores stress induced dysbiosis *NSE cortisol, lymphocytes, monocytes, neutrophils, eosinophils	(Davis et al., 2016b)
L. rhanmosus GG in feed (CFU not reported) for 14 days	Blinding status not reported Wild-type #PROT (n= 15), #PROT + 0.5% EtOH (n = 15), 0.5% EtOH (n = 15), CON (n = 15)	Male Female Adult	*NSE anxiety (novel tank diving) for #PROT or #PROT + ethanol groups	§NA	§NA	(Schneider et al., 2016)
L. rhanmosus IMC501 1 × 106 CFU B.I.D. for 28 days	Blinding status not reported Wild-type - heterozygous #PROT (n = 12), CON (n = 12)	Male Female Age 120-180 days	§NA	#PROT ↑ social (shoaling) and exploratory behavior	#PROT ↓ Bacteroidetes ↑ BDNF and serotonergic gene expression in brain *NSE gut BDNF	(Borrelli et al., 2016)
P. acidilactici JN039350 and L. plantarum JN039358 109 CFU/g in control and HCD feed (3% of total body weight) B.I.D. for 49 days	Blinding status not reported Wild-type control diet group (n = 6), HCD (n = 6), HCD + P. acidilactici JN039350 (n = 8) and the HCD + L. plantarum JN039358 (n = 8)	Male (n = 28) Adult	§NA	P. acidilactici JN039350 or L. plantarum JN039358 ↑ HCD ↓ in spatial memory	#PROT ↑ diet induced ↑ increase in cholesterol #PROT ↓ brain abba, liver abcal and ↓ liver and intestine npc111 expression	(Lim et al., 2017)
Ouail (Coturnix japonica)
P. acidilactici R001 (MA 18/5M) ~ 1.9 × 107 CFU/day for 36 days with unpredictable stress during day 17-21	Blinding status not reported #PROT (LTI n = 18 and STI n = 16) Control (LTI n = 19 and STI n = 16)	Female #PROT Age 0-36 days Age 6-7 days for STI & LTI	*NSE anxiety (OFT) #PROT ↓ emotional reactivity (STI & LTI)	#PROT ↑ memory day 2 and 3	*NSE emotional reactivity and memory interaction	(Parois et al., 2017)

Abbreviations:

^§NA = Not Assessed,

^*NSE = No Significant Effect,

^#PROT = Probiotic Treatment, 5-HT = 5-hydroxytryptamine or Serotonin, abcal = ATP-binding cassette family transporter group Al, appa = amyloid precursor protein type a, ABX = Antibiotics, AchE = Acetylcholinesterase, ACTH = Adrenocorticotropic Hormone, ALT = Alanine Transaminase, AST = Aspartate Transaminase, ATCC = American Tissue Culture Collection, B.I.D. = twice a day, BDL = Bile Duct Ligation, BDNF = Brain Derived Neurotrophic Factor, BNR = Blinding Not Reported, BUN = Blood Urea Nitrogen, CAT = Catalase, CFD = Cafeteria Diet, CLD = Control Diet, CON = Control, CON-A = Concanavalin A, CORT = Corticosterone, CR = Conventionally Raised, CRE = Creatinine, RS = Restraint Stress, CTX = Cortex, CV = Conventionalized, D-GAL = D-galactose, DNBS = Dinitro-Benzene Sulfonic Acid, DSS = Dextran Sodium Sulfate, EB = Ethidium Bromide, Ecologic^® Barrier = B. bifidum W23, B. lactis W52, L. acidophilus W37, L. brevis W63, L. casei W56, L. salivarius W24, be. Lactis W19, be. Lactis W58, EPM = Elevated Plus Maze, EPSP = Excitatory Post Synaptic Potentials, EtOH = Ethanol, FRL = Flinders Resistant Line, FSL = Flinders Sensitive Line, FST = Forced Swim Test, GF = Raised in Germ Free Environment, GPx = glutathione peroxidase, GSH = Glutathione, HA = Hyperammonemia (hepatic encephalopathy model), HCD = High Cholesterol Diet, HFD = High Fat Diet, HIP = Hippocampus, HYP = Hypothalamus, LACA = Laboratory Animal Center Albino, Lacidofil^® = 95% b. rhamnosus R0011 & 5% b. helveticus R0052, Lc. = Lactococcus, LPS = Lipopolysaccharide, LSPC = Lotus Seedpod Proanthocyanidins, LTI = Long Tonic Immobility, LTP = Long Term Potentiation, MDA = Maiondialdehyde, MHFD = Maternal High Fat Diet, MI = Myocardial Infarction, MI = Myocardial Infarction, MIA = Maternal Immune Activation, ML-7 = myosin light chain kinase inhibitor, MS = Maternal Separation, MTCC = Microbial Type Culture Collection, MWM = Morris Water Maze, NE = Norepinephrine, and npclll = Neimann-Pick Cl-like 1, NOR = Novel Object Recognition, NOS = Nitric Oxide Synthase, OCD = Obsessive-Compulsive Disorder, OFT = Open Field Test, OPT = Object Placement Test, P.O. = PAT = Passive Avoidance Test, Per Oral, PD = Postnatal Day, PFC = Prefrontal Cortex, PGE2 = prostaglandin E2, PPI = Prepulse Inhibition, PRO = Protein, PUFA = Polyunsaturated Fatty Acids, PVN = Paraventricular Nucleus, Q.D. = Once a day, ROS = Reactive Oxygen Species, SAM = S-adenosylmethionine, SCOP = Scopolamine, SD = Standard Diet, STI, SDT = Step Down Test, SIH = Stress Induced Hyperthermia, SOD = Super Oxide Dismutase, SPF = Specific Pathogen Free, = Short Tonic Immobility, STR = Striatum, TNBS = 2,4,6-trinitrobenzenesulfonic acid, TST = Tail Suspension Test, VSL#3 = S. salivarius ssp. thermophilus, B. breve, B. infantis, B. longum, L. acidophilus, b. plantarum, b. casei, b. delbrueckii subsp. Bulgarians, VTA = Ventral Tegmental Area, WAS = Water Avoidance Stress, XOS = Xylooligosaccharide, ZO-1 = Zonula Occludens-1

Due to profound differences in experimental designs, neuropsychiatric outcome assessments, physiological indices, probiotic strains, and substrains reported by the trials ascertained for review (see Tables 1 and 2), meta-analyses were not conducted. Most trials indicating significant differences associated with probiotic treatment reported beneficial effects on neuropsychiatric outcomes. If a probiotic treatment led to a decline in neuropsychiatric performance or functioning, these findings have been highlighted in the Results.

Both review authors assessed the study quality and risk of bias for all trials in Tables 1 and 2 with 100% consensus. Human study quality and risk for bias were evaluated with the PEDro scale (Maher et al., 2003), Quality Index (Downs and Black, 1998), and the Cochrane Collaboration Tool (Higgins et al., 2011). Individual item scores, mean total scores, and interquartile ranges for these assessments are noted in Tables A.2-A.4 (Appendix) for each study. Study quality and risk of bias for the non-human animal trials were estimated with modified criteria from Macleod et al. (2004). The amended scale items, individual item scores for each study, total mean scores, and interquartile range can be examined in Table A.5 (Appendix). Specific-pathogen-free or germ-free conditions were not taken into consideration when scoring the non-human animal study environment. If such conditions were reported, they are noted in Table 2 and summarized in the Results.

For all study quality and bias scales except the Cochrane Collaboration Tool, total scores were normally distributed and higher scores corresponded with greater study quality or lower risk for bias. Each item of the Cochrane Collaboration Tool was dichotomized as having a high or low risk for bias, except when the item criteria could not be obtained from the study report. If this condition was met, the item was recorded as having an unclear risk for bias.

Because the validity and reliability of excluding investigations for systematic review based on these types of assessments continues to be actively debated in the extant literature (Ilgen et al., 2015; Juni et al., 2001), our global assessment of human and non-human trial methodology suggested excessive heterogeneity, and since we were unable to obtain meaningful minimum scores with the quality and risk of bias assessments, these scores were not considered when evaluating studies for final inclusion in Tables 1 and 2. However, to maximize scientific rigor and minimize bias, we excluded reports noted in Table 1 from Tables 4-6 and the Results below if we could not confirm at minimum double-blinding. Notably, the standardized scores for the Quality Index and PEDro Scale were within 2.5 deviations of the mean (i.e. Z-Score Range −2.5 to +2.5). Due to the varied and limited reporting of blinding status for the non-human trials (43.1%), we did not employ the double-blind exclusion strategy for these investigations.

Table 4.

Double- or Triple-Blinded Human Trial Characteristics.

Lifespan Stage	n Studies Reportin g/n Total	M	SD	Range	% Total
Age, years
Infant/Child	7/7	0.414	.923	0-4	-
Young/Middle-Aged Adult	32/32	38.2	11.1	18-65	-
Older Adult	5/5	70.2	7.0	48-95	-
Subjects in probiotic treatment arm, n
Infant/Child	7/7	92.7	71.8	20-238	-
Young/Middle-Aged Adult	32/32	51.5	50.1	12-212	-
Older Adult	4/5	43.8	55.0	30-125	-
Probiotic treatment duration, days
Infant/Child	6/7	93.2	63.6	28-201	-
Young/Middle-Aged Adult	32/32	61.2	59.3	14-336	-
Older Adult	5/5	54.3	34.5	21-84	-
Probiotic dosage, CFU
Infant/Child	7/7	7.7 × 108	1.9 × 109	1 × 106 – 1 × 1010	-
Young/Middle-Aged Adult	31/32	3.4 × 1010	8.8 × 1010	1 × 107 – 3 × 1011	-
Older Adult	5/5	2.3 × 1011	4.5 × 1011	1 × 108 – 4.5 × 1011	-
Sex, % males
Infant/Child	6/7	-	-	-	51.9
Young/Middle-Aged Adult	32/32	-	-	-	31.5
Older Adult	5/5	-	-	-	38.7

Table 6.

Cross-Species Overview of Single- Versus Multi-Strain Probiotic Treatments in Association with Neuropsychiatric Outcomes.

Trials	N/% single- strain	N/% multi- strain	N/% assessed anxiety, depression, or emotional behavior	N/% assessed cognition or social behavior	N/% single- strain change in stress, anxiety, or depression	N/% of single-strain change in cognition or social behavior	N/% of multi-strain change in stress, anxiety, or depression	N/% of multi-strain change in cognition or social behavior
Human – 44	27/61.4	16/36.4	37/84.1	17/38.6	15/55.6	7/25.9	8/50.0	5/31.5
Infant/Child – 7	6/85.7	1/14.3	5/71.4	2/28.6	1/16.7	0/0	0/0	1/100.0
Y oung/Middle-Aged Adult – 32*	18/56.3	13/40.6	29/90.6	11/34.4	13/72.2	6/33.3	8/61.5	2/15.4
Older Adult – 5	3/60.0	2/40.0	3/60.0	4/80.0	1/33.3	1/33.3	0/0	2/100.0
Non-Human – 58	38/65.5	20/35.5	40/69.0	42/72.4	23/60.5	22/57.9	9/45.0	17/85.0
Rat – 25	11/44.0	14/56.0	18/72.0	19/76.0	6/54.5	6/54.5	6/42.9	10/71.4
Mouse – 27	22/81.5	5/18.5	18/66.7	20/74.1	14/63.6	14/63.6	3/60.0	5/100.0
Zebrafish – 5	4/80.0	1/20.0	3/60.0	2/40.0	2/50.0	1/25.0	NA	1/100.0
Quail – 1	1/100.0	0/0.0	1/100.0	1/100.0	1/100.0	1/100.0	NA	NA

Abbreviations: NA = Not Applicable

^*One of the double-blind probiotic trials observing significant changes in cognitive function did not report whether the Bifidobacterium formulation tested was a single- or multi-strain combination. This study was excluded from calculations noted in this table.

Table 3 is a human vs. non-human comparison of the multiple neuropsychiatric scales and behavioral measures utilized as indices for stress, anxiety, depression, cognition, and social functioning.

Table 3.

Neuropsychiatric Paradigms and Assessments Utilized in Human and Non-Human Animal Trials.

	Human		Non-Human
	Paradigms/Assessments	Associated References	Paradigms/Assessments	Associated References
Stress/Well-being	1. Socially Evaluated Cold Pressor Test – (SECPT) 2. Perceived Stress Scale – (PSS) 3. Hopkins Symptom Checklist-Revised – (HSCL-90) Psychological Distress 4. Depression Anxiety Stress Scale – (DASS) 5. Paced Auditory Serial Addition Test – (PASAT) 6. European Quality of Life – (EQ-5D-5L)	1. (Kelly et al., 2017) 2. (Chung et al., 2014; Kelly et al., 2017; Messaoudi et al., 2011a; Messaoudi et al., 2011b; Ostlund-Lagerstrom et al., 2016; Sanchez et al., 2017) 3. (Messaoudi et al., 2011a; Messaoudi et al., 2011b) 4. (Akkasheh et al., 2016; Mohammadi et al., 2016; Romijn et al., 2017) 5. (Moller et al., 2017) 6. (Ostlund-Lagerstrom et al., 2016)	1. Maternal Separation – (MS) or Early Life Stress 2. Restraint Stress – (RS) 3. Water Avoidance Stress – (WAS) 4. Unpredictable 5. Social Defeat 6. Stress-Induced Hypothermia 7. Stress	1. (Callaghan et al., 2016; Cowan et al., 2016; Desbonnet et al., 2010; Liu et al., 2016b; McVey Neufeld et al., 2017; Vanhaecke et al., 2017) 2. (Liang et al., 2015; McVey Neufeld et al., 2017) 3. (Gareau et al., 2011; Smith et al., 2014; Vanhaecke et al., 2017) 4. (Davis et al., 2016b; Dhaliwal et al., 2018; Parois et al., 2017) 5. (Bharwani et al., 2017) 6. (Bravo et al., 2011; Savignac et al., 2014) 7. (Davis et al., 2016a)
Anxiety	1. State Trait Anxiety Inventory – (STAI) 2. Beck Anxiety Inventory – (BAI) 3. Hamilton Anxiety Scale – (HAM-A) 4. Patient Health Questionnaire-9 – (PHQ-9) 5. Hospital Anxiety and Depression Scale – (HADS)	1. (Kato-Kataoka et al., 2016; Kelly et al., 2017; Reale et al., 2012; Sanchez et al., 2017; Slykerman et al., 2017; Takada et al., 2016) 2. (Akkasheh et al., 2016; Kelly et al., 2017; Steenbergen et al., 2015) 3. (Yang et al., 2016a) 4. (Lee et al., 2014) 5. (Lyra et al., 2016; Messaoudi et al., 2011a; Messaoudi et al., 2011b; Ostlund-Lagerstrom et al., 2016)	1. Elevated Plus Maze – (EPM) 2. Step Down Test – (SDT) 3. Light/Dark Box 4. Burying 5. Open Field Test – (OFT) 6. Locomotor activity 7. Thigmotaxis 8. Novel Tank Diving 9. Mirror Chamber Test	1. (Beilharz et al., 2017; Bravo et al., 2011; Cowan et al., 2016; Dhaliwal et al., 2018; Liang et al., 2015; Liu et al., 2016a; Liu et al., 2016b; Luo et al., 2014; Savignac et al., 2014; Wang et al., 2015) 2. (Arseneault-Breard et al., 2012; Bercik et al., 2011; Bercik et al., 2010) 3. (Bercik et al., 2010; Bharwani et al., 2017; Emge et al., 2016; Smith et al., 2014) 4. (Hsiao et al., 2013; Kantak et al., 2014; Messaoudi et al., 2011a; Savignac et al., 2014) 5. (Abildgaard et al., 2017a; Abildgaard et al., 2017b; Bharwani et al., 2017; Bravo et al., 2011; Buffington et al., 2016; Dhaliwal et al., 2018; Divyashri et al., 2015; Hsiao et al., 2013; Kantak et al., 2014; Liang et al., 2015; Liu et al., 2016b; McKernan et al., 2010; McVey Neufeld et al., 2017; Parois et al., 2017; Savignac et al., 2014; Tillmann et al., 2018; Vanhaecke et al., 2017) 6. (Davis et al., 2016a; Liu et al., 2016a; Liu et al., 2016b; Savignac et al., 2015; Wang et al., 2015) 7. (Davis et al., 2016a) 8. (Davis et al., 2016b; Schneider et al., 2016) 9. (Dhaliwal et al., 2018)
Depression	1. Beck Depression Inventory – (BDI) 2. Leiden Index of Depression Severity – (LEIDS) 3. Quick Inventory of Depressive Symptomatology – (QIDS) 4. Montgomery-Asberg Depression Rating Scale – (MADRS) 5. Hamilton Depression Scale – (HAM-D) 6. Edinburgh Postnatal Depression Scale – (EPDS) 7. Profile of Mood States – (POMS) 8. Hospital Anxiety and Depression Scale – (HADS) 9. General Health Questionnaire – (GHQ) 10. Patient Health Questionnaire-9 – (PHQ-9) 11. Inconsolable crying and fussiness 12. Positive and Negative Syndrome Scale – (PANSS) 13. Geriatric Depression Scale – (GDS)	1. (Akkasheh et al., 2016; Kelly et al., 2017; Kouchaki et al., 2016; Rao et al., 2009; Sanchez et al., 2017; Steenbergen et al., 2015) 2. (Steenbergen et al., 2015) 3. (Romijn et al., 2017) 4. (Romijn et al., 2017) 5. (Dapoigny et al., 2012) 6. (Slykerman et al., 2017) 7. (Benton et al., 2007; Michalickova et al., 2016) 8. (Lyra et al., 2016; Messaoudi et al., 2011a; Messaoudi et al., 2011b; Ostlund-Lagerstrom et al., 2016) 9. (Kouchaki et al., 2016; Mohammadi et al., 2016; Nishihira et al., 2014) 10. (Lee et al., 2014) 11. (Cekola et al., 2015; Indrio et al., 2014; Rinne et al., 2006; Sung et al., 2014; Weizman and Al sheikh, 2006) 12. (Dickerson et al., 2014) 13. (Chung et al., 2014)	1. Tail Suspension Test – (TST) 2. Forced Swim Test – (FST) 3. Immobility 4. Aggression 5. Gross Behavior	1. (Dhaliwal et al., 2018; Guida et al., 2017; Liang et al., 2017; Savignac et al., 2014) 2. (Abildgaard et al., 2017a; Abildgaard et al., 2017b; Arseneault-Breard et al., 2012; Bravo et al., 2011; Desbonnet et al., 2008; Desbonnet et al., 2010; Dhaliwal et al., 2018; Gilbert et al., 2013; Guida et al., 2017; Liang et al., 2017; Liu et al., 2016b; Savignac et al., 2014; Tillmann et al., 2018) 3. (D'Mello et al., 2015) 4. (Kantak et al., 2014) 5. (Nimgampalle and Kuna, 2017)
Cognitive Ability	1. Bayley Scale of Infant Development Motor and Psychomotor Indices – (BSID-IIMDI and PDI) 2. Mini Mental Status Examination – (MMSE) 3. Delta Power 4. Cognitive Restraint 5. Paced Auditory Serial Addition Test – (PASAT) 6. Wechsler MemoryScale - logical memory test 7. Rapid Visual Information Processing – (RVIP) 8. Digit Symbol 9. Trial Making Test (TMT) – Part A & B 10. Verbal fluency 11. Episodic memory 12. Paired Associative Learning – (PAL) 13. Stroop Emotion 14. Block Design 15. Attention Switching Task – (AST) 16. Storytelling 17. Fearful stimuli	1. (Akar et al., 2017) 2. (Akbari et al., 2016) 3. (Takada et al., 2017) 4. (Sanchez et al., 2017) 5. (Moller et al., 2017) 6. (Benton et al., 2007) 7. (Chung et al., 2014; Kelly et al., 2017) 8. (Bajaj et al., 2014; Malaguarnera et al., 2010) 9. (Malaguarnera et al., 2010) 10. (Benton et al., 2007) 11. (Benton et al., 2007) 12. (Kelly et al., 2017) Kelly et al., 2017) 13. (Kelly et al., 2017) 14. (Bajaj et al., 2014; Malaguarnera et al., 2010) 15. (Kelly et al., 2017) 16. (Chung et al., 2014) 17. (De Lorenzo et al., 2017; Tillisch et al., 2013)	1. Morris water maze – (MWM) 2. Cued and Contextual Fear Conditioning 3. Passive Avoidance Test – (PAT) 4. Novel Object Recognition – (NOR) 5. Object Placement Test – (OPT) 6. Prepulse Inhibition – (PPI) 7. Barnes maze 8. Y-maze 9. T-maze 10. Long-Term Potentiation (LTP) 11. Excitatory Post-Synaptic Potentials (EPSPs) 12. Memory	1. (Davari et al., 2013; Dhaliwal et al., 2018; Goudarzvand et al., 2016; Liang et al., 2017; Luo et al., 2014; McVey Neufeld et al., 2017; Musa et al., 2017; Nimgampalle and Kuna, 2017; Partty et al., 2015; Sun et al., 2016; Tian et al., 2017; Woo et al., 2014) 2. (Bravo et al., 2011; Callaghan et al., 2016; Cowan et al., 2016; Savignac et al., 2015) 3. (Gilbert et al., 2013; Lee et al., 2018; Woo et al., 2014) 4. (Beilharz et al., 2017; Emge et al., 2016; Lee et al., 2018; Liang et al., 2015; Mohle et al., 2016; Savignac et al., 2015; Smith et al., 2014; Tillmann et al., 2018) 5. (Liang et al., 2015) 6. (Hsiao et al., 2013) 7. (Abildgaard et al., 2017b; Savignac et al., 2015) 8. (Jeong et al., 2015; Jeong et al., 2016; Lee et al, 2018; Tillmann et al., 2018; Woo et al., 2014; Xiao et al., 2014) 9. (Gareau et al., 2011) 10. (Buffington et al., 2016; Chunchai et al., 2018; Davari et al., 2013; Distrutti et al., 2014) 11. (Chunchai et al., 2018; Davari et al., 2013) 12. (Parois et al., 2017)
Social Behavior and Functioning	1. Functional Assessment of Cancer Therapy: Social Well-being – (FACT-SWB) 2. Pediatric Quality of Life – (QOL) Social 3. IBS Quality of Life (QOL) Social Function 4. Sickness Impact Profile Social – (SIP-social) 5. Short Form-36 (SF-36) 6. Emotion Faces 7. Body Uneasiness Test – (BUT) 8. Social insecurity 9. Interpersonal distrust	1. (Lee et al., 2014) 2. (Ringel-Kulka et al., 2015) 3. (Choi et al., 2011; Fujimori et al., 2009; Lorenzo-Zuniga et al., 2014; Nobutani et al., 2017; Spiller et al., 2016) 4. (Bajaj et al., 2014) 5. (Pellino et al., 2013; Pinto-Sanchez et al., 2017) 6. (Tillisch et al., 2013) 7–9. (De Lorenzo et al., 2017)	1. Shoaling 2. Social novelty 3. Social interactions (including defeat)	1. (Borrelli et al., 2016) 2. (Guida et al., 2017) 3. (Arseneault-Breard et al., 2012; Bharwani et al., 2017; Buffington et al., 2016; D'Mello et al., 2015; Gilbert et al., 2013; Hsiao et al., 2013; Kantak et al., 2014; Tillmann et al., 2018)

3. Results

3.1. Human Trial Characteristics

Study characteristics for the human trials are summarized by lifespan stage in Table 4.

Across trials, the most frequent single- and multi-strain combinations were L. casei (seven of forty-eight – 14.6% – six utilized substrain Shirota and one investigated ssp. rhamnosus) and VSL#3 (two of forty-eight – 4.2%) which is comprised of S. thermophilus DSM 24731, B. longum DSM 24736, B. breve DSM 24732, B. infantis DSM 24737, L. acidophilus DSM 24735, L. plantarum DSM 24730, L. paracasei DSM 24733, and L. delbrueckii ssp. bulgaricus DSM 24734, respectively.

Participants varied greatly by age range, physiological conditions, and psychiatric history. The number of investigations that verified medical or neuropsychiatric diagnoses, or lack thereof, with hospital or outpatient records could not be determined. Treatment and placebo group sample sizes were reported for all human studies. Within each trial, treatment and placebo groups were comparable in sex and age range distribution. Notable exceptions:

1. one infant trial - a greater proportion of males were randomized to the placebo group, and

2. one older adult investigation - a greater proportion of males were randomized to one of the L. helveticus IDCC3801 treatment conditions.

3.1.1. Infant/Child

Subjects in four out of the seven (57.1%) infant/child studies were defined as healthy. The three remaining trials enrolled infants considered to be premature, diagnosed with Wessel’s colic, or having a first-degree relative with dermatitis, atopic disease, or asthma.

Five of the seven trials (71.4%) reported outcomes associated with L. reuteri (substrains DSM 17938 or ATCC5573). The sixth single-strain study determined the effects of L. rhamnosus GG ATCC53103 consumption. One (14.3%) multi-strain investigation was also conducted with S. thermophilus, B. animalis ssp. lactis BB-12, plus L. bulgaricus.

3.1.2. Older Adult

Three of the five (60%) older adult studies enrolled individuals described as healthy. The two remaining reports conducted probiotic trials for post-surgical colorectal cancer patients or people diagnosed with Alzheimer’s disease.

Probiotic strains tested varied for each of the older adult trials (see Table 1). Three (60%) single- and two (40%) multi-strain formulations were evaluated.

3.1.3. Young/Middle-Aged Adult

Thirty-six young/middle-aged adult trials have been included in Table 1. Thirty-two trials reported double- or triple-blinding. The last four investigations had indistinguishable blinding status and have been excluded from discussion (see Methods).

Eleven (34.4%) of these trials defined study subjects as generally healthy, of which one (9.1%) only enrolled female subjects. Seven (21.9%) investigations enrolled patients diagnosed with Rome II or III IBS. Two (6.3%) studies investigated probiotic treatment in the context of obese or overweight status. Two separate trials (6.3%) enrolled people diagnosed with hepatic encephalopathy. The twelve remaining reports recruited and investigated unique study samples. Precise characteristics have been noted in Table 1.

It is unclear whether an investigation of Bifidobacterium in hepatic encephalopathy patients should be characterized as a single- or multi-strain probiotic treatment. Eighteen out of thirty-two (56.3%) trials were conducted with single strains of probiotics. Six (33.3%) of the eighteen utilized L. casei (substrains - five of six Shirota and one ssp. rhamnosus). Three (16.7%) of the eighteen investigated L. rhamnosus (substrains GG, HN001, or CGMCC1.3724). B. longum (substrains NCC3001 or ssp. infantis R0033) was tested three (16.7%) times. Two studies (11.1%) evaluated L. helveticus (substrains R0052 or Lafti L10). All other single-strain trials were conducted once as noted in Table 1.

Fourteen of the thirty-two (43.8%) young/middle-aged adult studies were multi-strain probiotic investigations. Two out of fourteen (14.3%) analyzed the effects of L. helveticus R0052 plus B. longum R0175. All other multi-strain formulations were tested once (see Table 1).

3.2. Non-Human Trial Characteristics

Major characteristics by animal species are summarized in Table 5.

Table 5.

Non-Human Trial Characteristics^*.

Species	n Studies Reporting/n Total	M	SD	Range	% Total
Age, days
Rat	16/25	109.7	162.0	2-660	-
Mouse	25/27	67.5	62.0	29-540	-
Zebrafish	2/5	77	103.2	4-180	-
Quail	1/1	6.5	NA	NA	-
Animals in probiotic treatment arms, n
Rat	23/25	18.9	12.9	6-60	-
Mouse	21/27	20.2	12.0	6-49	-
Zebrafish	4/5	12.3	4.5	6-16	-
Quail	1/1	17	NA	NA	-
Probiotic treatment duration, days
Rat	25/25	40.2	35.1	12-168	-
Mouse	27/27	28.3	21.2	5-84	-
Zebrafish	4/5	27.3	19.3	2-49	-
Quail	1/1	36	NA	NA	-
Probiotic dosage, CFU
Rat	25/25	4.5 × 109	5.4 × 109	1.0 × 108 – 1.2 × 1010	-
Mouse	27/27	1.1 × 1010	2.2 × 1010	1.0 × 104 – 1.0 × 1011	-
Zebrafish	4/5	7.3 × 106	1.1 × 107	1.0 × 106 – 1.0 × 109	-
Quail	1/1	1.9 × 107	NA	NA	-
Sex, % males
Rat	22/25	-	-	-	78.7
Mouse	20/27	-	-	-	87.0
Zebrafish	1/5	-	-	-	62.5§
Quail	1/1	-	-	-	0

^*Computed from studies reporting exact probiotic treatment group size, age, or numbers of animals by sex.

^§One out of five studies reported a total sample of 28 males. Three out of five studies reported using both males and females. One out of five studies did not report whether males or females were utilized.

The most common single-strain probiotics tested were L. rhamnosus (substrains GG, JB-1, NC4007, and IMC501) and L. plantarum (substrains MTCC1325, MTCC9510, KY1032, PS128, and USDA-ARS; eight of fifty-eight trials each – 13.8%). Lacidofil^® – 95% L. rhamnosus R0011 and 5% L. helveticus R0052 – was the most frequently investigated multi-strain formulation (five of fifty-eight – 8.6%).

Approximate animal age during probiotic treatment was reported for sixteen out of twenty-five (64.0%) rat, twenty-five out of twenty-seven (92.6%) mouse, and two out of five (40.0%) zebrafish studies. The age of most animals tested neurodevelopmentally coincided with human middle-adulthood. When reported, animal ages for treatment and control groups were comparable. Forty-two out of fifty-eight (72.4%) non-human studies utilized males. Three (5.2%) trials investigated females. Eight (13.8%) investigations observed both male and female animals. Numbers of animals by sex were not reported for five (8.6%) trials.

Blinding status was indicated for twenty-five of the fifty-eight (43.1%) reports. Fifty-one (87.9%) trials administered probiotic in drinking water or provided control animals with an identical vehicle. In addition to the varying experimental paradigms further described by species below, three out of twenty-five (12.0%) rat, thirteen out of twenty-seven (48.1%) mouse, and one out of five (20.0%) zebrafish trials reported rearing animals in germ- or specific-pathogen-free environments concurrent to probiotic treatment.

3.2.1. Rat

Multiple rat strains were utilized across trials. Fifteen of the twenty-five (60.0%) utilized Sprague-Dawley, eight (32.0%) Wistar, two (8.0%) Flinders Sensitive, and one (4.0%) Fischer 344.

Paradigms varied among studies with some including multiple experimental manipulations: 1) eight models of stress (32.0%) – five maternal separation, two restraint, and one water avoidance; 2) six (24.0%) different diet modifications; 3) three (12.0%) aging/neurodegenerative models; 4) two (6.0%) myocardial infarction; and 5) two (6.0%) probiotic rescue of antibiotic-induced dysbiosis. All other designs were utilized once (see Table 2).

Eleven out of the twenty-five (44%) rat studies employed single-strain probiotic therapy. Three out of eleven (27.3%) utilized B. infantis 35624. Three (27.3%) studies determined the effects of L. helveticus (substrains - two NS8 and one MTCC1325). Two (18.2%) trials each of L. plantarum (substrains MTCC1325 and unknown) and L. fermentum (substrains NS9 and CECT5716) were conducted. Additional single strain probiotic treatments were investigated once as noted in Table 2.

Fourteen (66%) rat studies were multi-strain formulation trials. Four out of fourteen (28.6%) trials investigated the effects of L. helveticus R0052 plus B. longum R0175. Two (11.8%) trials each of Ecologic^® Barrier, Lacidofil^®, and VSL#3 were conducted. The four remaining multi-strain combinations were tested once (see Table 2).

3.2.2. Mouse

The most frequently utilized mouse strain was C57BL6/J (sixteen of twenty-seven - 59.3%) followed by BALB/c (five of twenty-seven - 18.5%). Two (7.4%) trials tested probiotic effects in AKR/J mice. All other strains were investigated once each as noted in Table 2. Two (7.4%) of the twenty-seven studies utilized knockout (−/−) mice: Rag1 on C57BL6/J and IL-10 on 129/SvEv.

The most common experimental manipulation was stress exposure (seven of twenty-seven – 25.9%). Exposure types varied by trial and are noted in Table 2. Aging, colitis, and vagotomy models were investigated three (11.1%) times each. Pathogenic infection, diet modification, antibiotic-induced dysbiosis, lipopolysaccharide (LPS) induced inflammation, antidepressant, or heat-killed probiotic comparisons were employed (7.4%) twice each. All other paradigms were evaluated once (see Table 2).

Twenty-two out of twenty-seven (81.5%) reports can be stratified as single-strain probiotic trials. L. plantarum (substrains C29, CCFM639, MTCC9510, PS128) and L. rhamnosus (substrains GG, JB-1, NC4007) effects were evaluated five times (22.7%) each. Studies with B. longum (substrains NCC3001, 1714) were conducted four (18.2%) times. Three (13.6%) trials utilized L. casei (substrains 01, LABPC, DG) while two (9.1%) separate investigations determined the effects of B. breve 1205. All other single-strain probiotics were tested once (see Table 2).

Five out of twenty-seven (18.5%) trials investigated multi-strain combinations of probiotics. Three of the five (60%) studies utilized Lacidofil^® while the two (40%) remaining analyzed VSL#3 intake effects.

3.2.3. Zebrafish

All zebrafish studies were conducted with wild-type animals. One of five (20.0%) reports also noted wild-type as “heterozygous”. Besides probiotic treatment, experimental designs included 1) stress, germ-free, and conventional environments; 2) high cholesterol diet; 3) ethanol exposure; and 4) chronic unpredictable stress.

Probiotic strains tested included two (40%) L. rhamnosus (substrains GG and IMC501), two (40%) L. plantarum USDA-ARS, and one (20%) multi-strain formulation of P. acidilactici JN039350 plus L. plantarum JN039358.

3.2.4. Quail

The single female Japanese quail study investigated the effects of P. acidilactici R001 (MA 18/5M).

3.3. Neuropsychiatric Outcomes Associated with Single- and Multi-Strain Treatments

Table 6 provides a cross-species comparison of single- and multi-strain probiotic trials in association with key neuropsychiatric outcome groupings.

3.3.1. Stress/Anxiety – Human

3.3.1.1. Infant/Child

None of the trials measured anxiety-based phenotypes.

3.3.1.2. Older Adult

Two of five (40.0%) trials assessed probiotic effects on anxiety with L. reuteri DSM 17938 or L. helveticus IDCC3801. No significant variations in anxiety symptoms were reported after 84 days of probiotic consumption.

3.3.1.3. Young/Middle-Aged Adult

Twenty-four out of thirty-two (75.0%) trials determined anxiety outcomes associated with probiotic treatment. Twelve (50.0%) of the twenty-four were single-strain trials including five (38.5%) L. casei (substrains - four Shirota and one ssp. rhamnosus), and two (15.4%) each of L. rhamnosus (HN001 and CGMCC1.3724) and B. longum (ssp. infantis R0033 and NCC3001). Other single-strains were tested once as noted in Table 1. Beneficial effects were observed with L. casei Shirota, L. gasseri CP2305, L. acidophilus NCFM, L. rhamnosus HN001, or B. bifidum R0071.

The twelve (50.0%) multi-strain trials included two (18.2%) L. helveticus R0052 plus B. longum R0175 investigations. All other formulations were tested once (9.1%) as noted in Table 1. Seven (58.3%) of these trials reported improved anxiety symptoms with 1) I.31 (L. plantarum CECT7484 and CECT7485 plus P. acidilactici CECT7483); 2) L. gasseri SBT2055 plus B. longum SBT2928 in 100g yogurt containing S. thermophilus and L. delbrueckii subsp. Bulgaricus; 3) L. helveticus R0052 plus B. longum R0175; 4) L. acidophilus LA5 plus B. lactis BB-12 in yogurt containing L. casei, L. acidophilus, L. rhamnosus, L. bulgaricus, B. breve, B. longum, and S. thermophilus; 5) L. acidophilus plus L. casei and B. bifidum; 6) S. thermophilus SGst01, B. animalis ssp. Lactis SGB06, S. thermophiles, B. bifidum SGB02, L. delbrueckii ssp. Bulgaricus DSM 20081, L. acidophilus SGL11, L. plantarum SGL07, L. reuteri SGL01; or 7) L. acidophilus, plus L. casei, B. bifidum, and L. fermentum intake. Of note, Lacidofil^® associated anxiety reduction was a within-group effect. No significant differences were observed between treatment groups.

3.3.2. Stress/Anxiety – Non-Human

3.3.2.1. Rat

Thirteen out of twenty-five (52.0%) trials assessed anxiety behaviors. Six (46.2%) were single-strain trials that included two (33.3%) L. fermentum (substrains CECT5716 and NS9), two (33.3%) L. helveticus NS8, and the last two tested each of the following probiotics once: L. rhamnosus GG, B. breve UCC2003, B. infantis 35624, and L. salivarius UCC118. Four (66.7%) of the six studies reported significant improvements with L. helveticus NS8, L. rhamnosus GG or L. fermentum NS9 intake.

Multi-strain (seven out of thirteen – 53.8%) evaluation of anxiety symptoms included three (42.9%) L. helveticus R0052 plus B. longum R0175, two (28.6%) Ecologic^® Barrier, one Lacidofil^®, and one VSL#3 trial. Two of the seven (28.6%) investigations noted a reduction in anxiety-based phenotypes with L. helveticus R0052 plus B. longum R0175 consumption. Notably, one of these studies indicated the beneficial effects of L. helveticus R0052 plus B. longum R0175 for anxiety symptom reduction was specific to the myocardial infarction (MI) model.

3.3.2.2. Mouse

Sixteen out of 27 (59.3%) trials assessed anxiety with L. rhamnosus (substrains NC4007, GG, and JB-1), B. longum (substrains NCC3001 and 1714), L. plantarum (substrains – two PS128 and one MTCC9510), B. breve 1205, L. reuteri MM4-1A ATCC-PTA-6475, L. johnsonii ATCC33200, E. faecium CFR3003, B. fragilis NCTC9343, L. helveticus R0052, or Lacidofil^®.

Thirteen out of fourteen (92.9%) single-strain studies reported a reduction in anxiety-based outcomes (see Table 2). One of the L. plantarum PS128 trials observed improvement in anxiety-like phenotypes specifically in mice naïve to maternal separation stress. Although one trial observed anxiety behavior reduction after B. longum NCC3001 intake (10 days), L. rhamnosus NC4007 consumption did not confer the same benefits. A concurrent investigation of B. longum 1714 and B. breve 1205 demonstrated that anxiety reduction was specific to B. breve 1205 treatment.

The two Lacidofil^® trials also indicated a significant improvement in anxiety behavior scores post-treatment.

3.3.2.3. Zebrafish

Three out of five (60%) trials investigated probiotic treatment effects on thigmotaxis and novel tank diving using L. plantarum USDA-ARS or L. rhamnosus GG. Both of the L. plantarum USDA-ARS studies (66.7%) reported decreased anxiety-based behaviors in conventionally raised zebrafish after two or thirty days of treatment. However, L. plantarum USDA-ARS was unable to modify anxiety-like phenotypes in a germ-free environment.

3.3.2.4. Quail

P. acidilactici R001 (MA 18/5M) did not have a significant effect on anxiety-based behaviors.

3.3.3. Depression – Human

3.3.3.1. Infant/Child

Five out of seven (71.4%) trials assessed depression behaviors with single-strain probiotics including four (80%) L. reuteri (substrains DSM 17938, ATCC55730, or unknown) and one (20%) L. rhamnosus GG trial. The L. reuteri ATCC55730 trial conducted a parallel investigation with B. lactis BB-12. One of the five (20%) studies reported improvement in fussiness/crying after 90 days of L. reuteri DSM17938 treatment. Two (40%) reported negative effects, i.e. increased crying and irritability, with L. reuteri DSM 17938 intake. The negative findings were not replicated during post-treatment assessment and specific to formula-fed infants for one of the two trials.

3.3.3.2. Older Adult

Two out of five (40%) trials investigated the effects of L. casei Shirota or L. helveticus IDCC3801 on depression outcomes. Although both studies observed no significant differences as a whole, L. casei Shirota consumption for twenty-one days improved depression symptoms for a subset of individuals experiencing poor baseline mood.

3.3.3.3. Young/Middle-Aged Adult

Nineteen of the thirty-two (59.4%) trials assessed depression phenotypes. Ten (52.6%) of the nineteen were single-strain investigations that included two (20.0%) each of B. longum (substrains NCC3001 or unknown), L. casei (substrains ssp. rhamnosus LCR35 or Shirota), L. rhamnosus (CGMCC1.3724 or HN001) and one (10.0%) each of B. animalis ssp. lactis-07, S. boulardii, L. acidophilus NCFM, and L. helveticus Lafti L10. S. boulardii and L. casei ssp. rhamnosus LCR35 had no significant effect (20.0%) on depression outcomes. The eight remaining studies (80.0%) reported a reduction in depression symptoms after probiotic intake.

Nine (47.4%) of the nineteen young/middle-aged adult studies were multi-strain trials. Two investigated the effects of L. helveticus R0052 plus B. longum R0175. All other combinations were tested once as noted in Table 2. The Lacidofil^® and one L. helveticus R0052 plus B. longum R0175 trial did not observe significant differences in depression outcomes after probiotic intake. Of note, the Lacidofil^® trial reported significant within- but no between-group differences after 84 days of probiotic consumption. The seven (77.8%) remaining studies reported improvements in depression symptoms.

3.3.4. Depression – Non-Human

3.3.4.1. Rat

Nine out of twenty-five (36%) trials investigated three (33.3%) single-strain – two B. infantis 35624 and one L. plantarum MTCC1325 – and six (66.7%) multi-strain – three (50.0%) L. helveticus R0052 and B. longum R0175, two (33.3%) Ecologic^® Barrier, and one (16.7%) L. rhamnosus plus B. longum – combinations of probiotics on depression-based outcomes. Two of three (66.7%) single- and five of six (83.3%) multi-strain trials reported significant beneficial changes in depression-like phenotypes post-probiotic intake. One B. infantis 35624 and one L. helveticus R0052 plus B. longum R0175 trial did not modify depression behaviors.

3.3.4.2. Mouse

Seven out of twenty-seven (25.9%) trials analyzed probiotic effects on depression-associated outcomes. Six (85.7%) were single-strain trials – two (33.3%) L. rhamnosus (substrains JB-1 or GG), two (33.3%) L. plantarum (substrains PS128 or MTCC9510), one (16.7%) L. casei DG, and one (16.7%) comparison of B. longum 1714 and B. breve 1205. Single-strain treatments (83.3%) improved depression-like phenotypes with the exceptions of L. rhamnosus GG and B. longum 1714. A VSL#3 (14.3%) trial also indicated a significant reduction in depression-based behaviors.

3.3.4.3. Zebrafish

None of the trials assessed depression-like phenotypes.

3.3.4.4. Quail

P. acidilactici R001 (MA 18/5M) improved emotional reactivity after thirty-six days.

3.3.5. Social Function – Human

3.3.5.1. Infant/Child

One of seven (14.3%) trials investigated a combination of S. thermophilus, B. animalis ssp. lactis BB-12, and L. bulgaricus with 1 g inulin on pediatric Quality of Life (QOL) social functioning. This study reported improved social behavior after one hundred twelve days of probiotic consumption.

3.3.5.2. Older Adult

One of five (20%) trials investigated VSL#3 with significant improvements in SF-36 social functioning at post-treatment assessment (twenty-eight days).

3.3.5.3. Young/Middle-Aged Adult

Six out of thirty-two (18.8%) trials assessed the effects of probiotics on social ability. Four of the six (66.7%) utilized single probiotic strains that included B. longum (substrains NCC3001 or unknown), L. rhamnosus GG, or S. boulardii. Three of the four (75.0%) noted improvement in IBS, IBD, or SIP social function after twenty-eight, twenty-eight, or fifty-six days of probiotic intake, respectively. The two (42.9%) multi-strain trials were Lacidofil^® or a combination of S. thermophilus SGst01, plus B. animalis ssp. Lactis SGB06, S. thermophiles, B. bifidum SGB02, L. delbrueckii ssp. Bulgaricus DSM20081, L. acidophilus SGL11, L. plantarum SGL07, and L. reuteri SGL01. Both multi-strain formulations were not effective at modifying social behaviors.

3.3.6. Social Function – Non-Human

3.3.6.1. Rat

Three out of twenty-five (12.0%) rat trials evaluated L. helveticus R0052 plus B. longum R0175 in relation to social interactions. Two of the three (66.7%) indicated improved social function with probiotic consumption for fourteen days post-MI induction or seven days pre- and seven days post-MI.

3.3.6.2. Mouse

Five out of twenty-seven (18.5%) trials assessed the effects of probiotics on social ability. Two of the four (50%) single-strain investigations observed increased social interactions after twenty-eight days of L. rhamnosus JB-1 or L. reuteri MM4-1A-ATCC-PTA-6475 consumption. B. fragilis NCTC9343, L. casei DG, or L. johnsonii ATCC33200 intake did not alter social behaviors. A twenty-day trial of VSL#3 had positive effects on social interactions.

3.3.6.3. Zebrafish

One out of five (20.0%) trials investigated and observed increased shoaling after twenty-eight days of L. rhamnosus IMC501 exposure.

3.3.6.4. Quail

No social functioning phenotypes were assessed.

3.3.7. Cognition – Human

3.3.7.1. Infant/Child

One out of seven (14.3%) trials investigated psychomotor development effects associated with L. reuteri treatment. No significant changes were detected.

3.3.7.2. Older Adult

Three of the five (60%) trials determined the effects of L. casei Shirota, L. helveticus IDCC3801, or a multi-strain combination of L. acidophilus, L. casei, L. fermentum, plus B. bifidum. The L. helveticus IDCC3801 and L. acidophilus, L. casei, L. fermentum, plus B. bifidum trials observed modifications in Rapid Visual Information Processing (RVIP) and Mini Mental Status Exam (MMSE) performance post-probiotic consumption.

3.3.7.3. Young/Middle-Aged Adult

Eight out of thirty-two (25.0%) trials evaluated the effects of probiotic treatment on cognitive indices. Four of the eight (50.0%) were single-strains investigations of L. rhamnosus (substrains JB-1 or CGMCC1.3724), L. casei Shirota, or B. longum NCC3001. Multi-strain combinations included VSL#3, S. thermophilus SGst01, B. animalis ssp. Lactis SGB06, S. thermophiles, B. bifidum SGB02, L. delbrueckii ssp. Bulgaricus DSM20081, L. acidophilus SGL11, L. plantarum SGL07, L. reuteri SGL01, or combination of B. animalis ssp. lactis, plus L. lactis ssp. lactis, S. thermophilus, and L. bulgaricus. Except for one multi- and one single-strain trial, all of the young/middle-aged adult reports noted improved cognitive performance with probiotic consumption. One trial investigated the effects of Bifidobacterium and noted greater block design ability and shorter Trail Making A & B completion times after sixty days of probiotic intake. However, it is unclear if a single- or multi-strain formulation of Bifidobacterium was tested.

3.3.8. Cognition – Non-Human

3.3.8.1. Rat

Seventeen out of twenty-five (68.0%) trials assessed cognitive functioning. Six (35.3%) trials were single-strain investigations that included two (33.3%) L. plantarum (substrains MTCC1325 or unknown), two (33.3%) L. helveticus NS8, and one (16.7%) each of L. paracasei HII01, L. rhamnosus, and B. B94. Five out of the six (83.3%) studies noted cognitive improvement with probiotic treatment. Twenty-eight days of L. plantarum or B. B94 consumption did not modify spatial ability.

The eleven (64.7%) multi-strain combinations were two each of (18.2%) VSL#3, Lacidofil^®, and L. helveticus R0052 plus B. longum R0175. All other multi-strain combinations were tested once (see Table 2). Two (18.2%) of the multi-strain trials did not report significant differences in cognitive performance after Ecologic^® Barrier or L. helveticus R0052 plus B. longum R0175 consumption. One (9.1%) study reported a decline in memory with Lacidofil^® intake in rats exposed to maternal stress. The eight (72.7%) remaining trials observed improvements in cognitive indices post-probiotic treatment.

3.3.8.2. Mouse

Sixteen out of twenty-seven (59.3%) trials reported probiotic effects on cognition. All sixteen studies observed improved cognitive task performance with probiotic treatment. Four out of sixteen (25%) trials were multi-strain designs - three Lacidofil^® and one VSL#3. One of the Lacidofil^® trials observed beneficial effects on memory in Rag −/− mice naïve to water avoidance stress (WAS). Probiotic treatment reduced memory ability in WAS exposed Rag −/− mice.

3.3.8.3. Zebrafish

One out of five (20.0%) trials investigated cognition-based outcomes. Forty-nine days of P. acidilactici JN039350 plus L. plantarum JN039358 consumption improved high cholesterol diet-induced decline in spatial memory.

3.3.8.4. Quail

P. acidilactici R001 (MA 18/5M) improved memory performance during treatment days two and three.

4. Discussion

4.1. Summary

Across all species, L. rhamnosus (substrains GG, JB-1, NC4007, IMC501) was the most common single-strain probiotic treatment investigated (12.3%). VSL#3 and Lacidofil^® were the most frequently tested multi-strain formulations (5.7% each). A gross difference between the human and non-human trials was the greater enrollment of human female subjects (Table 4), whereas non-human studies conducted most experiments with male animals (Table 5).

The cross-species overview (Table 6) indicates that both single- and multi-strain combinations of probiotics may influence cognition, social function, anxiety, depression, or other emotional behaviors with similar efficacy in humans. For the non-human studies, multistrain combinations were more likely to modify cognition and social behavior, whereas single- and multi-strain combinations may be comparable in ability to regulate anxiety, depression, or emotional behaviors. Because variation in probiotic combinations and experimental designs among the human and non-human trials hindered our ability to conduct meaningful meta-analyses (DerSimonian and Kacker, 2007), these and the other findings reported in this review should be considered crude and preliminary estimations.

4.2. Limitations

There are profound gaps in our understanding of probiotic treatments that should be addressed in forthcoming human and non-human translational trials. Non-neuropsychiatric outcomes assayed in conjunction with each probiotic treatment varied widely and included neural, immune, anthropometric, gastrointestinal, neuroendocrine, or metabolic markers. Since these biomarkers are likely to be critical components in our mechanistic understanding of gut-brain interactions, thoughtful incorporation and replication of precise biomarkers is germane to the success of future probiotic investigations.

Specific to the human reports, several healthy adult trials noted in this review did not observe significant probiotic treatment effects on neuropsychiatric outcomes. Although healthy subject investigations minimize illness-related study confounds, these trials are unlikely to capture a sufficient range of neuropsychiatric phenotypes. This coincides with findings from a recent meta-analysis of probiotic trials in relation to depression symptoms (Ng et al., 2018). Additional neuropsychiatric case-control trials with multiple treatment outcome and compliance measures are necessary to confirm prior reports and conduct more rigorous meta-analyses of probiotics in relation to specific symptoms.

The total number of studies conducted with quail, zebrafish, and specific rat and mouse stains are very limited for a comprehensive review. The reporting of the experimental microbial environment (i.e. specific-pathogen-free, germ-free, etc.) for animal trials was inconsistent and should be improved. Significant limitations common to all probiotic trials include reporting bias, sample size, confirmation of probiotic activity and administration vehicle, precise probiotic dosage and treatment duration, differential neuropsychiatric assessments, and an insufficient number of trials for early and late neurodevelopmental stages across the lifespan.

Assessment of study quality and bias had some effectiveness for capturing dropout rates and quality variation. Approximately 59% percent of the human trials reported less than 15% study subject dropout. Intent-to-treat analyses were reported for 45.8% of the human trials. Measures of probiotic treatment adherence (i.e. intake of 75% or greater doses) and exclusion criteria for poor compliance were reported for 29.2% of the human trials. Specific blinding status was reported for 93.1% and 43.1% human and non-human studies, respectively. Although we aimed to minimize bias when evaluating all non-human trials, excessive positive finding reporting for animal studies has been acknowledged (Sena et al., 2010; Tsilidis et al., 2013) and is not easily illuminated with existing study quality and bias assessments. These are critical factors that need to be addressed to improve study quality and develop successful treatments.

The sample sizes for approximately 50% of the human and 90% of the non-human trials were fewer than thirty and fifteen subjects per group, respectively. In addition, studies reporting a priori designations of primary and secondary outcomes customary for rigorous clinical trial designs or sample size estimations to reflect sufficient study power were limited across species. This implies most of the trials evaluated would be considered exploratory investigations. Therefore, larger, adequately powered, replication studies are required to confirm observations from individual trials and those compiled in this systematic review.

Verification of probiotic strain/substrain activity (i.e. in vitro culture) prior to conducting the investigation or post-probiotic treatment fecal sample sequencing was inconsistent across trials. In addition, probiotic intake vehicle varied across trials (i.e. capsule, in yogurt, in water, per oral, etc.). The combination of these factors can lead to significant experimental confounds and thereby influence the validity and reliability of our systematic review observations. Although the exact mechanisms by which probiotics proliferate within the intestinal tract are unclear, administration route may influence successful probiotic colonization. Therefore, future studies may consider confirmatory procedures such as fecal (human and non-human) or intestinal biopsy (non-human) sequencing.

Three human, two rat, and one mouse trial investigated varying “doses” (i.e. colony forming units-CFU) for the same probiotic strain/substrain in relation to neuropsychiatric outcomes. However, more extensive dose-finding experiments will need to be conducted across species, probiotic strains/substrains, and study populations prior to large-scale implementation of probiotics for gut-brain-behavior based outcomes. Treatment duration for most probiotic trials reviewed was less than sixty days with a limited number of trials assessing long-term neuropsychiatric outcomes. A pilot adjunctive probiotic trial in treatment-resistant depressed patients reported the efficacy of an L. acidophilus, B. bifidum, S. thermophiles (2 × 10¹⁰ CFU) plus 1600 mg magnesium orotate therapeutic and demonstrated symptom reemergence upon treatment cessation (Bambling et al., 2017). While the study was ineligible for inclusion in this report based on our systematic review criteria; their observations highlight the need for developing longitudinal probiotic investigations and utilizing probiotic treatments as a long-term health and wellness lifestyle modification, rather than a short-term intervention.

Thus far, two double-blind investigations have been conducted with patients diagnosed with significant anxiety or depression symptoms (Akkasheh et al., 2016; Romijn et al., 2017). Investigations of probiotics in patients with prominent mania or psychoses are also limited (Dickerson et al., 2018; Dickerson et al., 2014). While we aimed to distinguish neuropsychiatric outcomes by specific symptoms in the Results section of this review, the sizeable overlap in neuropsychiatric phenotypes, especially anxiety and depression (Sartorius et al., 1996), are well-recognized. Most of the human and non-human probiotic studies utilized varying stress or depression-based assessments. In addition, limited assessments or assays in relation to neuropsychiatric symptoms were conducted within most trials. Standardized neurobehavioral measures and novel symptom models should be developed and incorporated into future trials. To differentiate subsyndromes, future trials could employ multiple experimental strategies (e.g. psychophysical task plus anxiety assessment interview in human studies).

Across species, trials were primarily conducted to coincide with the middle-age adult stage of the lifespan. While this review aimed to highlight the limited studies that enrolled older adults, young/middle-aged adults, and children; early- (12-14 years) and middle- (15-17 years) adolescent stage human or non-human animal trials were not reported in the neuropsychiatric literature. Gut-brain-pathways are highly likely to be modified by gut microbes and probiotics during these critical periods of the lifespan (McVey Neufeld et al., 2016) which should be underscored for future probiotic and gut-brain-behavior studies.

Although neuropsychiatric illnesses are largely characterized by significant cognitive and behavioral alterations; sociodemographic and lifestyle factors are prominent and may modify the effects of probiotics on anxiety, mood, and psychotic disorders. There are preliminary indications that probiotic treatment efficacy may vary by participant sex (Sanchez et al., 2017). Other sociodemographic differences were not reported by the studies reviewed. Non-probiotic neuropsychiatric trials have reported differential treatment response by race or ethnicity (Ellis et al., 2015). Therefore, future studies may need to consider variation in probiotic treatment efficacy by subject race/ethnicity or animal strain.

4.3. Conclusions

Our review indicates that several human and non-human probiotic treatment trials have been conducted with direct or indirect intent to target gut-brain-behavior interactions. However, the large variance in experimental design, sample characteristics, and assessment methodology hinders true comparison and computation of effect sizes for specific phenotypes and probiotic strain or substrain combinations. Although a wide-range of mediating biomarkers and neuropsychiatric assessments can have significant utility as secondary or tertiary outcome analyses, it is important for future human and non-human investigations to improve study power and minimize risk of bias by clearly delineating all post hoc analyses and/or incorporating as many of these variables a priori when computing sample size estimates. Improving the reporting of trial methodology with these crucial details will substantially improve the quality of subsequent systematic reviews and meta-analyses.

Future studies will need to replicate existing findings and develop additional randomized, placebo-controlled, double-blinded, and case-controlled, or cohort probiotic trials. Since probiotic trials are unable to clarify all gut-microbe based treatment mechanisms, large-scale normative data obtained from various human cohorts and non-human animal species and strains are necessary to obtain a more veridical representation of gut microbial composition and variation across the lifespan. Concurrent investigations with special emphasis on neurodevelopmental and neuropsychiatric illness trajectory will be especially valuable. Data obtained from these combined sources will better inform future probiotic treatment study design and hypothesized neuropsychiatric targets.

In brief, the ability to 1) elucidate gut microbe-brain-behavior pathway mechanisms; 2) disentangle unique effects of single- and multi-strain formulations of probiotics, and; 3) implement novel probiotic treatments to target precise neuropsychiatric phenotypes, will require comprehensive review and meta-analyses of future probiotic trial outcomes.

Highlights.

• A cross-species review of probiotic effects on neuropsychiatric outcomes was conducted.

• Probiotic substrain and experimental design variation hindered formal meta-analyses.

• Single and multi-strain formulations of probiotics can modify neuropsychiatric phenotypes.

• Rigorous follow-up studies are necessary to confirm initial findings.

Appendix

Table A.1.

Probiotic Trials Employing Psychological Stress Paradigms Without Neuropsychiatric Outcome Assessment.

Probiotic Treatment	Study Design	Sex/ Age, days	Probiotic Associated Outcomes	References
Rat (Rattus)
L. farciminis 1011 CFU/day or saline P.O. QD for 15 days	Wistar PRS or sham stress	Female Age Adult	#PROT ↓ stress-induced visceral hypersensitivity, p-MLC, and colonic permeability	(Ait-Belgnaoui et al., 2006)
L. farciminis 1011 CFU/day or saline P.O. QD for 14 days prior to PRS	Wistar sham + saline (n = 8), sham + #PROT (n = 8), sham + saline (n = 8), sham + CRD + #PROT (n = 8), PRS + CRD + saline (n = 8), PRS + #PROT (n = 8), PRS + CRD + saline (n = 8), PRS + CRD + #PROT (n = 8)	Female Age Adult	#PROT ↓ PRS + CRD induced ↑ in Fos positive spinal cord, PVN, and MeA cells	(Ait-Belgnaoui et al., 2009)
L. farciminis 1011 CFU/day, ML-7, saline, or antibiotic P.O. for 14 days	Wistar PRS or sham stress Pathogen Free	Female Age Adult	#PROT ↓ PRS induced colonic permeability, endotoxemia, PVN CRF expression, circulating LPS, and pro-inflammatory cytokine expression in HYP	(Ait-Belgnaoui et al., 2012)
L. farciminis or vehicle 1011 CFU/mL P.O. for 10 days pre-WAS/sham and 4 days with WAS/sham	Wistar WAS or sham stress 12 groups (n = 8 or 14/group)	Male Age Adult	#PROT ↓ WAS induced visceral sensitivity, intestinal permeability, colonic mucus flattening, and colonic mucin O-glycosylation	(Da Silva et al., 2014)
L. farciminis or vehicle 1011 CFU/mL P.O. for 10 days pre-WAS/sham and 4 days with WAS/sham	Wistar WAS or sham stress 4 groups (n = 8/group)	Male Age 63 days	#PROT bound to colonic mucus in WAS and sham stress #PROT abundance in ileum and colon ↓ after WAS only	(Da Silva et al., 2015)
L. casei Shirota YIT 9029 3 × 109 CFU for 14 days	Fischer 344 Naïve (n = 11), WAS (n = 14), WAS + #PROT (n = 14)	Male (n = 41) Age 56-63 days	#PROT ↓ WAS induced corticosterone and CRF in the PVN See Table 3 for human trial outcomes	(Takada et al., 2016)
L. rhamnosus Lcr35 (Lcr Lenio® 4.5 × 109 CFU/g or Lcr Restituo®1 × 104, 1.8 × 109, 6.8 × 1010CFU/g for 8 days post-TNBS or sham injection surgery and pre-colonic distention	Sprague Dawley Wistar TNBS, PRS, U50 or naïve 12 groups (n = 9 -10/group)	Male (Sprague Dawley) Female (Wistar) Age Adult	#PROT ↓ TNBS and PRS induced visceral hypersensitivity #PROT ↓ TNBS induced increase in IL-12p70, TNF-α in colonic tissue	(Darbaky et al., 2017)
B. subtilis CH201 1.5 × 108 CFU/mL QD in drinking water for 44 days with chronic stress beginning day 14, and PD beginning day 30	Wistar Control, stress, and PD with and w/out #PROT 8 groups (n = 8/group)	Male Age 42 days	#PROT ↓ inflammatory cells in periodontal tissue, C-terminal telopeptide, and alveolar bone loss in unstressed animals *NSE in stressed animals	(Foureaux Rde et al., 2014)
B. animalis ssp. lactis- BB-12 3 × 109 CFU/mL, P. jenenii 8 × 108 CFU/mL, or vehicle in maternal drinking water for 32 days (10 days before mating)	Wistar control (n = 40), MS (n = 40), adult stress (n = 40), MS + adult stress (n = 40) w/ and w/out #PROT	Male (n = 80) Female (n = 80) Wistar Age 2-14 days (MS) Age 83-85 days adult stress	#PROT ↓ MS and adults stress induced ↑ IFN-γ (males and females) & IL-6 (males) #PROT ↓ haptoglobin in control, MS and adult stress #PROT ↓ ileal MUC2 expression in MS males and control females	(Barouei et al., 2015)
L. paracasei NCC2461 4 × 1011 CFU/kg diet with arachidonic and docosahexaenoic acids, galactooligosaccharides and fructooligoccharides or control diet for 20 days after 12 days MS	Long Evans MS (n = 84 all experiments)	Male Age 15 and 35 days	#PROT ↓ stress induced ↑ intestinal permeability and villi changes #PROT ↑ concentration of Enterococci and Bacteroides in MS and no MS	(Garcia-Rodenas et al., 2006)
E. coli Nissle 101 CFU/mL, 104 CFU/mL, 108 CFU/mL or vehicle single dose	WRS (60 min after #PROT), pretreatment with indomethacin 10 groups (n = 6/group)	Sex and Age unknown	#PROT ↓ WRS induced gastric lesions, gastric mucosa IL1-ß mRNA, #PROT ↑ WRS ↓ ghrelin	(Konturek et al., 2009)
L. plantarum 8P-A3 and L. fermentum 90T-C4 4 × 108 CFU P.O. for 21 days during chronic social stress	Wistar unstressed, Two types of social stress, canamycin, #PROT 7 groups (n=10/group)	Female (n = 70) Age 180 days	#PROT ↑ social stress induced ↓ Xbp1 in lymphoblasts and small lymphocytes	(Topol et al., 2014)
B. lactis CNCM 1-2494 106, 107, 108, 109, 1010 CFU doses (n = 10/dose), FM with B. lactis CNCM I-2494 Lc. lactis CNCM I-1631 and two yogurt starters: L. bulgaricus and S. thermophilus (n = 10), NFM (n = 10), or saline (n = 10) P.O. QD for 15 days	Wistar w/ (n = 10) or w/out (n = 10) PRS for all #PROT	Female Age Adult	FM ↓ PRS induced abdominal cramps FM ↓ PRS induced ↑ blood LPS FM prevented stress induced ↑ intestinal permeability *NSE visceral sensitivity response	(Agostini et al., 2012)
L. farciminis 1011 CFU/day P.O. QD for 10 days during WAS	Specific-pathogen-firee Wistar no-stress (n = 8), WAS (n = 12), and WAS + #PROT (n = 12)	Male (n = 16) Female (n = 16) Age 49 days	*NSE WAS induced ↑ fecal pellet output #PROT ↓ WAS-induced ↑ distal colon IL-6, PRSS1, mucosal mast cell count, and visceromotor response to colorectal distention in WAS exposed rats (females only) #PROT ↓ WAS-induced ↑ distal colon IL-1β (males only)	(Lee et al., 2017)
L. helveticus R0052 and L. rhamnosus R0011 109 CFU/mL in drinking water 7 days pre-WAS or sham stress and 10 days during WAS	Brown Norway SPF: WAS (n = 6), WAS + #PROT (n = 4), sham stress (n = 4), or sham stress + #PROT (n = 4)	Male Age unknown	#PROT ↓ bacterial translocation into lymph nodes for WAS group	(Zareie et al., 2006)
Lacidofil® 108 CFU P.O. and P.R. B.I.D. for 16 days beginning day 4 of MS	Sprague–Dawley MS (16 days) then WAS (10 days age 60-70) (n = 7-15/group)	Female and Male pups Age 4 and 70 days	#PROT ↓ MS induced ↑ in intestinal permeability, serum corticosterone, and bacterial adhesion to colonic mucosa #PROT ↑ MS induced ↓ in Lactobacilli *NSE intestinal permeability, serum corticosterone in unstressed pups	(Gareau et al., 2007)
Mouse (Mus)
L. casei CRL 431 1 × 108CFU/mL QD for 11 days	BALB/c 11 days of food and restraint stress stress + #PROT (n = 9), stress (n = 9), + control (n = 9), control + #PROT (n = 9)	Male Age 35 days	#PROT reserved stress induced intestinal villi and IgA changes *NSE CORT	(Palomar et al., 2014)
L. casei Shirota YIT9029 5 × 109 CFU/mL or placebo for 84 days during repeated WAS	C57BL/6JTcra +/+, +/−, and −/− repeated WAS 5 days/week for 84 days (n = 5-12/group)	Female Age 56 days	#PROT ↓ microbial changes induced by repeated WAS inTcra +/+ and ↓ loss of microbial diversity Tcra −/− *NSE repeated WAS induced ↑ corticosterone, colonic IgG, or IgA	(Arase et al., 2016)
L. reuteri ATCC 23272 1 × 108 CFU or vehicle P.O. for 6 days during social stress	C57BL6J Social stress 2hr/day for 6 daysC. rodentium infection during social stress (n =9/group)	Male Age 42 to 56 days	*NSE social stress induced ↑ C. rodentium #PROT ↓ C. rodentium induced ↑ colonic: mass, TNF-α, iNOS, F4/80+ macrophages, epithelial cell gene expression, CD11b+Ly6ChiCCR2+ cells (blood also) #PROT ↓ social stress induced ↑ C. rodentium translocation to spleen	(Mackos et al., 2016)
B. bifidum, L. acidophilus, S. faecalis 1 × 107 CFU/mL for 10 days with WAS	C57BL6J Unstressed, WAS 1hr/day for 10 days, WAS + #PROT (n = 5/group)	Female Age 35 to 42 days	#PROT ↓ WAS induced ↑ intestinal inflammation, permeability, proportion of Firmicutes, Bacteroidetes	(Sun et al., 2013)

Abbreviations:

^#PROT = Probiotic Treatment, CRD = Colorectal Distention, FM = Fermented Milk, HIP = Hippocampus, LA = Lateral Amygdala, LPS = Lipopolysaccharide, MeA = Medial Nucleus Amygdala, MI = Myocardial Infarction, NAA = N-acetylaspartate, NFM = Non-Fermented Milk, PD = Periodontal Disease, p-MLC = phosphorylated-Myosin Light Chain, P.O. = Per Oral, P.R. = Per Rectal, PRS = Partial Restraint Stress, PRSS = Mucosal Serine Protease Gene, PVN = Paraventricular Nucleus (Hypothalamus), TNBS = 2,4,6-trinitrobenzenesulfonic acid, QD = Daily, WAS = Water Avoidance Stress, WRS = Water Immersion Restraint Stress

Table A.2.

Assessment of Human Trial Quality: PEDro Scale^*.

Reference	1. Eligibility criteria specified	2. Random allocation	3. Concealed allocation	4. Groups similar at baseline	5. Subject blindig	6. Therapist blinding	7. Assess or blinding	8. Less than 15% dropouts	9. Intention -to- treat analysis	10. Between- group statistical comparisons	11. Point measures and variability data	Totala
Infant/Child
(Rinne et al., 2006)	1	1	1	0	1	1	1	0	0	1	1	8
(Indrio et al., 2014)	1	1	1	1	1	1	1	0	0	1	1	9
(Cekola et al., 2015)	1	1	1	1	1	1	1	0	1	1	1	10
(Sung et al., 2014)	1	1	1	1	1	1	1	0	1	1	1	10
(Weizman and Alsheikh, 2006)	1	1	1	1	1	1	1	0	1	1	1	10
(Akar et al., 2017)	1	1	1	1	1	1	1	0	0	1	1	9
(Ringel-Kulka et al., 2015)	1	1	1	0	1	1	1	0	1	1	0	8
Older Adult
(Ostlund-Lagerstrom et al., 2016)	1	1	1	1	1	1	1	1	1	1	1	11
(Benton et al., 2007)	1	1	1	1	1	1	1	1	0	1	1	10
(Chung et al., 2014)	1	1	1	0	1	1	1	1	0	1	1	9
(Pellino et al., 2013)	1	1	1	1	1	1	1	1	1	1	0	10
(Akbari et al., 2016)	1	1	1	1	1	1	1	1	1	1	1	11
Young/Middle-Aged Adult
(Kato-Kataoka et al., 2016)	1	1	1	1	1	1	1	1	0	1	1	10
(Takada et al., 2016)	1	1	1	1	1	1	1	1	0	1	1	10
(Takada et al., 2017)	1	1	1	1	1	1	1	1	0	1	1	10
(Rao et al., 2009)	1	1	1	0	1	1	1	1	0	1	0	8
(Reale et al., 2012)	1	1	1	1	1	1	1	1	0	1	1	10
(Bajaj et al., 2014)	1	1	1	1	1	1	1	0	1	1	1	10
(Kelly et al., 2017)	1	1	1	1	1	0	0	0	0	1	1	7
(Sanchez et al., 2017)	1	1	1	1	1	1	1	0	1	1	1	10
(Michalickova et al., 2016)	1	1	1	1	1	1	1	0	0	1	1	9
(Culpepper et al., 2016; Langkamp-Henken et al., 2015)	1	1	1	1	1	1	1	1	1	1	1	11
(Slykerman et al., 2017)	1	1	1	1	1	1	1	1	1	1	1	11
(Childs et al., 2014)	1	1	1	1	1	1	1	1	0	1	1	10
(Choi et al., 2011)	1	1	1	1	1	1	1	0	0	1	1	9
(Lorenzo-Zuniga et al., 2014)	1	1	1	1	1	1	1	0	1	1	1	10
(Lyra et al., 2016)	1	1	1	1	1	1	1	1	1	1	1	11
(Spiller et al., 2016)	1	1	1	1	1	1	1	0	1	1	1	10
(Dapoigny et al., 2012)	1	1	1	1	1	1	1	1	1	1	1	11
(Nishihira et al., 2014)	1	1	1	1	1	1	1	1	0	1	1	10
(Nobutani et al., 2017)	1	1	1	1	1	1	1	1	0	1	1	10
(Pinto-Sanchez et al., 2017)	1	1	1	0	1	1	1	1	1	1	1	10
(Yang et al., 2016b)	1	1	0	1	1	0	0	1	1	1	1	8
(Fujimori et al., 2009)	1	1	1	1	1	1	1	0	0	1	1	9
(Steenberg en et al., 2015)	1	1	1	1	1	1	1	1	0	1	1	10
(Moller et al., 2017)	1	1	1	0	1	1	1	0	0	1	1	8
(Tillisch et al., 2013)	1	1	1	1	1	1	1	0	0	1	1	9
(Mohammadi et al., 2016)	1	1	1	1	1	1	1	1	0	1	1	10
(Messaoudi et al., 2011a; Messaoudi et al., 2011b)	1	1	1	0	1	1	1	0	0	1	1	8
(Diop et al., 2008)	1	1	1	0	1	1	1	1	1	1	1	10
(Romijn et al., 2017)	1	1	1	0	1	1	1	1	1	1	1	10
(Akkasheh et al., 2016)	1	1	1	1	1	1	1	1	1	1	1	11
(Dickerson et al., 2014; Severance et al., 2017; Tomasik et al., 2015)	1	1	0	1	1	0	0	1	0	1	1	7
(De Lorenzo et al., 2017)	1	1	1	0	1	1	1	1	1	1	1	10
(Kouchaki et al., 2016)	1	1	1	1	1	1	1	1	1	1	1	11
(Feher et al., 2014)	1	1	0	0	1	0	0	0	0	1	1	5
(Lee et al., 2014)	1	1	1	1	1	1	1	1	0	1	1	10
(Malaguarnera et al., 2010)	1	1	1	1	1	1	1	0	0	1	1	9

^*As reported by Maher et al. (2003).

^aMean total score = 9.5 (SD 1.2). Interquartile range 9-11.

Table A.3.

Assessment of Human Trial Quality: Quality Index^*.

Reference	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24	25	26	Totala
Infant/Child
(Rinne et al., 2006)	1	1	1	1	0	1	1	0	0	1	1	0	1	1	1	0	0	1	0	1	1	0	1	1	0	0	16
(Indrio et al., 2014)	1	1	1	1	0	1	1	1	1	0	1	1	1	1	1	1	0	1	0	1	1	1	1	1	0	0	20
(Cekola et al., 2015)	1	1	1	1	0	1	1	1	1	0	1	1	1	1	1	1	1	1	0	1	1	1	1	1	0	1	22
(Sung et al., 2014)	1	1	1	1	0	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	0	1	24
(Weizman and Alsheikh, 2006)	1	1	1	1	0	1	1	1	1	1	1	1	1	1	1	1	0	1	0	1	1	1	1	1	0	1	22
(Akar et al., 2017)	1	1	1	1	0	1	1	1	1	1	1	1	1	1	1	1	1	1	0	1	1	1	1	1	0	0	22
(Ringel-Kulka et al., 2015)	1	1	1	1	0	1	1	1	1	1	1	1	1	1	1	1	1	1	0	1	1	1	1	1	0	1	23
Older Adult
(Ostlund-Lagerstrom et al., 2016)	1	1	1	1	0	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	0	1	24
(Benton et al., 2007)	1	1	1	1	0	1	1	0	1	1	1	1	1	1	1	0	1	1	0	1	1	1	1	1	0	0	20
(Chung et al., 2014)	1	1	1	1	0	1	1	0	1	1	1	1	1	1	1	0	1	1	0	1	1	1	1	1	0	0	20
(Pellino et al., 2013)	1	1	1	1	0	1	0	1	1	1	1	1	1	1	1	0	1	1	0	1	1	1	1	1	0	1	21
(Akbari et al., 2016)	1	1	1	1	0	1	1	0	1	1	1	1	1	1	1	1	1	1	0	1	1	1	1	1	0	1	22
Young/Middle-Aged Adult
(Kato-Kataoka et al., 2016)	1	1	1	1	0	1	1	0	1	1	0	1	1	1	1	0	1	0	0	1	1	1	1	1	0	0	18
(Takada et al., 2016)	1	1	1	1	0	1	1	0	1	1	0	1	1	1	1	1	1	0	1	1	1	1	1	1	0	0	20
(Takada et al., 2017)	1	1	1	1	0	1	1	0	1	0	0	1	1	1	1	0	1	0	0	1	1	1	1	1	0	0	17
(Rao et al., 2009)	1	1	1	1	0	1	0	1	1	0	1	1	1	1	1	0	1	0	0	1	1	1	1	1	0	0	18
(Reale et al., 2012)	1	1	1	1	0	1	1	0	1	1	1	1	1	1	1	0	1	1	0	1	1	1	1	1	0	0	20
(Bajaj et al., 2014)	1	1	1	1	0	1	1	1	1	1	1	1	1	1	1	1	1	0	1	1	1	1	1	1	0	1	23
(Kelly et al., 2017)	1	1	1	1	0	1	1	0	0	1	1	1	1	1	0	0	1	1	0	1	1	1	1	0	0	0	17
(Sanchez et al., 2017)	1	1	1	1	0	1	1	0	0	1	0	0	1	1	1	1	1	1	0	1	1	1	1	1	0	1	19
(Michalickova et al., 2016)	1	1	1	1	0	1	1	1	0	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	0	0	22
(Culpepper et al., 2016; Langkamp-Henken et al., 2015)	1	1	1	1	0	1	1	1	1	1	0	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	24
(Slykerman et al., 2017)	1	1	1	1	0	1	1	0	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	0	1	23
(Childs et al., 2014)	1	1	1	1	0	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	0	0	23
(Choi et al., 2011)	1	1	1	1	0	1	1	1	1	1	1	1	1	1	1	1	1	0	0	1	1	1	1	1	0	0	21
(Lorenzo-Zuniga et al., 2014)	1	1	1	1	0	1	1	1	1	1	1	1	1	1	1	1	1	1	0	1	1	1	1	1	0	1	23
(Lyra et al., 2016)	1	1	1	1	0	1	1	1	1	1	1	1	1	1	1	1	1	1	0	1	1	1	1	1	0	1	23
(Spiller et al., 2016)	1	1	1	1	0	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	0	1	24
(Dapoigny et al., 2012)	1	1	1	1	0	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	0	1	24
(Nishihira et al., 2014)	1	1	1	1	0	1	1	1	1	1	1	1	1	1	1	0	1	0	0	1	1	1	1	1	0	0	20
(Nobutani et al., 2017)	1	1	1	1	0	1	1	0	1	1	1	1	1	1	1	0	1	0	0	1	1	1	1	1	0	0	19
(Pinto-Sanchez et al., 2017)	1	1	1	1	0	1	1	1	1	1	1	1	1	1	1	1	1	1	0	1	1	1	1	1	0	1	23
(Yang et al., 2016b)	1	1	1	1	0	1	1	0	0	0	0	1	1	1	0	0	1	0	0	1	1	1	1	0	0	1	15
(Fujimori et al., 2009)	1	1	1	1	0	1	1	1	1	1	1	1	1	1	1	0	1	1	0	1	1	1	1	1	0	0	21
(Steenbergen et al., 2015)	1	1	1	1	0	1	1	0	1	1	1	1	1	1	1	0	1	1	0	1	1	1	1	1	0	0	20
(Moller et al., 2017)	1	1	1	1	0	1	1	0	1	1	0	1	1	1	1	0	1	0	0	1	1	1	1	1	0	0	18
(Tillisch et al., 2013)	1	1	1	1	0	1	1	1	1	1	1	1	1	1	1	0	1	1	0	1	1	1	1	1	0	0	21
(Mohammadi et al., 2016)	1	1	1	1	0	1	1	1	1	1	0	1	1	1	1	1	1	0	1	1	1	1	1	1	0	0	21
(Messaoudi et al., 2011a; Messaoudi et al., 2011b)	1	1	1	1	0	1	1	1	1	1	1	1	1	1	1	0	1	1	0	1	1	1	1	1	0	0	21
(Diop et al., 2008)	1	1	1	1	0	1	1	1	1	1	1	1	1	1	1	0	1	0	0	1	1	1	1	1	0	1	21
(Romijn et al., 2017)	1	1	1	1	0	1	1	1	1	1	1	1	1	1	1	1	1	0	1	1	1	1	1	1	0	1	23
(Akkasheh et al., 2016)	1	1	1	1	0	1	1	0	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	0	1	23
(Dickerson et al., 2014; Severance et al., 2017; Tomasik et al., 2015)	1	1	1	1	0	1	1	1	1	1	0	1	1	1	0	1	1	1	0	1	1	1	1	0	0	1	20
(De Lorenzo et al., 2017)	1	1	1	1	0	1	1	1	1	1	1	1	1	1	1	1	1	0	0	1	1	1	1	1	0	0	21
(Kouchaki et al., 2016)	1	1	1	1	0	1	1	0	1	1	1	1	1	1	1	1	1	1	0	1	1	1	1	1	0	1	22
(Feher et al., 2014)	1	1	1	1	0	1	1	1	0	1	0	1	1	1	0	1	1	0	0	1	1	1	1	0	0	0	17
(Lee et al., 2014)	1	1	1	1	0	1	1	0	1	1	1	1	1	1	1	1	1	0	0	1	1	1	1	1	0	0	20
(Malaguarnera et al., 2010)	1	1	1	1	0	1	1	1	0	0	1	1	1	1	1	0	1	0	1	1	1	1	1	1	0	0	19

^*As reported by Downs and Black (1998).

^aMean total score = 20.8 (SD 2.3). Interquartile range 18-24.

Table A.4.

Risk of Bias^* Evaluation: Human Probiotic Trials.

Reference	Selection	Performance	Detection	Attrition	Reporting
Infant/Child
(Rinne et al., 2006)	+	+	+	−	?
(Indrio et al., 2014)	+	+	+	−	?
(Cekola et al., 2015)	+	+	+	−	?
(Sung et al., 2014)	+	+	+	−	?
(Weizman and Alsheikh, 2006)	+	+	+	−	?
(Akar et al., 2017)	+	+	+	−	?
(Ringel-Kulka et al., 2015)	+	+	+	−	?
Older Adult
(Ostlund-Lagerstrom et al., 2016)	+	+	+	+	?
(Benton et al., 2007)	+	+	+	+	−
(Chung et al., 2014)	+	+	+	+	−
(Pellino et al., 2013)	+	+	+	+	−
(Akbari et al., 2016)	+	+	+	+	?
Young/Middle-Aged Adult
(Kato-Kataoka et al., 2016)	+	+	+	+	−
(Takada et al., 2016)	+	+	+	+	−
(Takada et al., 2017)	+	+	+	+	−
(Rao et al., 2009)	+	+	+	+	−
(Reale et al., 2012)	+	+	+	+	−
(Bajaj et al., 2014)	+	?	?	−	−
(Kelly et al., 2017)	+	+	?	−	−
(Sanchez et al., 2017)	+	+	+	−	?
(Michalickova et al., 2016)	+	+	+	−	?
(Culpepper et al., 2016; Langkamp-Henken et al., 2015)	+	+	+	+	?
(Slykerman et al., 2017)	+	+	+	+	?
(Childs et al., 2014)	+	+	+	+	?
(Choi et al., 2011)	+	+	+	−	?
(Lorenzo-Zuniga et al., 2014)	+	+	+	−	?
(Lyra et al., 2016)	+	+	+	+	?
(Spiller et al., 2016)	+	+	+	−	?
(Dapoigny et al., 2012)	+	+	+	+	?
(Nishihira et al., 2014)	+	+	+	+	−
(Nobutani et al., 2017)	+	+	+	+	−
(Pinto-Sanchez et al., 2017)	+	+	+	+	?
(Yang et al., 2016b)	+	?	?	?	−
(Fujimori et al., 2009)	+	+	+	−	−
(Steenbergen et al., 2015)	+	+	+	+	−
(Moller et al., 2017)	+	+	+	−	−
(Tillisch et al., 2013)	+	+	+	−	−
(Mohammadi et al., 2016)	+	+	+	+	?
(Messaoudi et al., 2011a; Messaoudi et al., 2011b)	+	+	+	−	−
(Diop et al., 2008)	+	+	+	+	−
(Romijn et al., 2017)	+	+	+	+	?
(Akkasheh et al., 2016)	+	+	+	+	?
(Dickerson et al., 2014; Severance et al., 2017; Tomasik et al., 2015)	+	?	?	+	?
(De Lorenzo et al., 2017)	+	+	+	+	?
(Kouchaki et al., 2016)	+	+	+	+	?
(Feher et al., 2014)	+	?	?	+	?
(Lee et al., 2014)	+	+	+	−	?
(Malaguarnera et al., 2010)	+	+	+	?	−

Key: + = Low Risk for Bias, − = High Risk for Bias, ? = Unclear Risk for Bias

^*As reported by Higgins et al. (2011).

Table A.5.

Non-Human Study Quality and Risk of Bias Assessment^*.

Reference	1	2	3	4	5	6	7	8	9	10	11	12	Total Scorea
Rat (Rattus)
(Nimgampalle and Kuna, 2017)	1	0	1	1	1	1	0	0	0	0	0	0	5
(Liang et al., 2017)	1	1	0	1	0	1	0	1	0	0	0	1	6
(Abildgaard et al., 2017a)	1	1	0	1	1	1	0	1	1	1	0	1	9
(Abildgaard et al., 2017b)	1	1	0	1	1	1	0	1	1	1	0	1	9
(Tillmann et al., 2018)	1	1	0	1	1	1	0	1	1	1	1	1	10
(Messaoudi et al., 2011a)	1	1	1	1	1	1	1	1	NA	0	0	1	9
(Desbonnet et al., 2008)	1	1	0	1	1	1	0	1	0	0	0	1	7
(Liang et al., 2015)	1	1	1	1	1	1	0	0	0	0	0	0	6
(Beilharz et al., 2017)	1	1	0	1	1	1	0	1	0	0	0	1	7
(Davari et al., 2013)	1	1	0	1	1	1	0	0	NA	0	0	0	5
(Goudarzvand et al., 2016)	0	1	1	1	1	1	0	0	NA	0	0	0	5
(Jeong et al., 2015)	1	1	1	1	1	1	0	0	0	0	0	0	6
(Wang et al., 2015)	1	1	1	1	1	1	0	0	0	0	0	0	6
(Distrutti et al., 2014)	1	1	0	1	1	1	0	0	0	0	0	0	5
(Arseneault-Breard et al., 2012)	1	1	0	1	1	1	1	1	NA	0	0	1	8
(Luo et al., 2014)	1	1	0	1	1	1	0	1	0	0	0	0	6
(Callaghan et al., 2016)	1	1	1	1	1	1	0	0	0	0	0	0	6
(Cowan et al., 2016)	1	0	0	1	1	1	0	1	0	0	0	1	6
(Desbonnet et al., 2010)	1	1	1	1	1	1	0	1	1	0	0	1	9
(Gilbert et al., 2013)	1	1	0	1	1	1	0	1	NA	0	0	0	6
(McKernan et al., 2010)	1	1	0	1	1	1	0	0	0	0	0	0	5
(Vanhaecke et al., 2017)	1	1	0	1	1	0	0	1	NA	0	0	0	5
(Athari Nik Azm et al., 2018)	1	0	0	1	1	1	0	0	NA	0	0	1	5
(McVey Neufeld et al., 2017)	1	1	0	1	1	1	0	0	NA	0	0	1	6
Mouse (Mus)
(Musa et al., 2017)	1	1	1	1	1	1	0	0	NA	0	0	0	6
(Guida et al., 2017)	1	1	0	1	1	1	0	1	0	0	0	1	7
(Jeong et al., 2016)	1	1	1	1	1	1	0	0	0	0	0	0	6
(Dhaliwal et al., 2018)	1	1	1	1	1	1	0	0	0	0	0	1	7
(Divyashri et al., 2015)	1	1	1	1	1	1	0	0	1	0	0	1	8
(Hsiao et al., 2013)	1	1	1	1	1	1	0	0	0	0	0	0	6
(Bharwani et al., 2017)	1	1	1	1	1	1	0	0	0	0	0	0	6
(Emge et al., 2016)	1	1	0	1	1	1	0	0	0	0	0	1	6
(Bercik et al., 2010)	1	1	1	1	1	1	0	0	0	1	0	1	8
(Bercik et al., 2011)	1	1	1	1	1	1	0	1	0	0	0	1	8
(Savignac et al., 2015)	1	1	1	1	1	1	0	0	0	0	0	1	7
(Mohle et al., 2016)	1	1	0	1	1	1	0	1	1	0	0	0	7
(Gareau et al., 2011)	1	1	0	1	1	0	1	1	0	0	0	0	6
(Smith et al., 2014)	1	1	0	1	1	0	0	0	0	0	0	1	5
(Woo et al., 2014)	1	1	1	1	1	1	0	0	0	0	0	1	7
(Buffington et al., 2016)	1	1	1	1	1	1	0	0	0	0	0	0	6
(D'Mello et al., 2015)	1	1	1	1	1	1	1	1	0	0	0	0	8
(Savignac et al., 2014)	1	1	0	1	1	1	0	0	NA	0	0	0	5
(Bravo et al., 2011)	1	1	0	1	1	1	0	1	0	0	0	1	7
(Kantak et al., 2014)	1	1	1	1	1	1	1	1	0	0	0	0	8
(Xiao et al., 2014)	1	1	1	1	1	1	0	1	0	0	0	1	8
(Tian et al., 2017)	1	1	1	1	1	1	0	0	NA	0	0	0	6
(Ohland et al., 2013)	1	1	1	1	1	1	0	0	0	0	0	0	6
(Liu et al., 2016a)	1	1	0	1	1	1	0	0	0	0	0	1	6
(Liu et al., 2016b)	1	1	1	1	1	1	0	0	0	0	0	0	6
(Sun et al., 2016)	1	1	1	1	1	1	0	0	0	0	0	1	7
Zebrafish (Danio rerio)
(Davis et al., 2016a)	1	1	1	1	1	0	0	0	0	0	0	0	5
(Davis et al., 2016b)	1	1	0	1	1	0	0	0	0	0	0	0	4
(Schneider et al., 2016)	1	1	1	1	1	1	0	0	0	0	0	1	7
(Borrelli et al., 2016)	1	1	1	1	1	0	0	0	1	0	0	0	6
(Lim et al., 2017)	1	1	1	1	1	1	0	0	NA	0	0	0	6
Quail (Coturnix japonica)
(Parois et al., 2017)	1	1	1	1	1	0	0	0	0	0	0	1	6

Abbreviations: NA = Not Applicable

^aMean total score = 6.5 (SD 1.3). Interquartile range 5.5-7.5.

^*Study quality was assessed with a modified scale reported by Macleod et al. (2004). Our customized scale was operationalized as 1 = Yes, 0 = No or Unsure, NA = Not Applicable for each item as follows: 1) compliance with animal welfare regulations; 2) comprehensive statement of environmental control (temperature, handling, etc.); 3) confirmation of probiotic activity prior to administration; 4) exact animal strain and experimental paradigm noted; 5) exact probiotic treatment (including substrain) noted; 6) control group received probiotic vehicle; 7) individuals administering probiotic were blind to treatment groups; 8) behavioral assays conducted blind to treatment; 9) animals tested in a random order when conducting multiple behavioral assays; 10) a priori power analyses for treatment group sample sizes; 11) a priori primary and secondary outcomes; and 12) acknowledged conflicts of interest or lack thereof.