Bacterial membrane vesicles and phages in blood after consumption of lacticaseibacillus rhamnosus JB-1.

Gut microbiota have myriad roles in host physiology, development, and immunity. Though confined to the intestinal lumen by the epithelia, microbes influence distal systems via poorly characterized mechanisms. Recent work has considered the role of extracellular vesicles in interspecies communication, but whether they are involved in systemic microbe-host interaction is unclear. Here, we show that distinctive nanoparticles can be isolated from mouse blood within 2.5 h of consuming Lacticaseibacillus rhamnosus JB-1. In contrast to blood nanoparticles from saline-fed mice, they reproduced lipoteichoic acid-mediated immune functions of the original bacteria, including activation of TLR2 and increased IL-10 expression by dendritic cells. Like the fed bacteria, they also reduced IL-8 induced by TNF in an intestinal epithelial cell line. Though enriched for host neuronal proteins, these isolated nanoparticles also contained proteins and viral (phage) DNA of fed bacterial origin. Our data strongly suggest that oral consumption of live bacteria rapidly leads to circulation of their membrane vesicles and phages and demonstrate a nanoparticulate pathway whereby beneficial bacteria and probiotics may systemically affect their hosts.

Identification of SSRI-evoked antidepressant sensory signals by decoding vagus nerve activity.

The vagus nerve relays mood-altering signals originating in the gut lumen to the brain. In mice, an intact vagus is required to mediate the behavioural effects of both intraluminally applied selective serotonin reuptake inhibitors and a strain of Lactobacillus with antidepressant-like activity. Similarly, the prodepressant effect of lipopolysaccharide is vagus nerve dependent. Single vagal fibres are broadly tuned to respond by excitation to both anti- and prodepressant agents, but it remains unclear how neural responses encode behaviour-specific information. Here we demonstrate using ex vivo experiments that for single vagal fibres within the mesenteric neurovascular bundle supplying the mouse small intestine, a unique neural firing pattern code is common to both chemical and bacterial vagus-dependent antidepressant luminal stimuli. This code is qualitatively and statistically discernible from that evoked by lipopolysaccharide, a non-vagus-dependent antidepressant or control non-antidepressant Lactobacillus strain and are not affected by sex status. We found that all vagus dependent antidepressants evoked a decrease in mean spike interval, increase in spike burst duration, decrease in gap duration between bursts and increase in intra-burst spike intervals. Our results offer a novel neuronal electrical perspective as one explanation for mechanisms of action of gut-derived vagal dependent antidepressants. We expect that our ex vivo individual vagal fibre recording model will improve the design and operation of new, extant electroceutical vagal stimulation devices currently used to treat major depression. Furthermore, use of this vagal antidepressant code should provide a valuable screening tool for novel potential oral antidepressant candidates in preclinical animal models.

Membrane vesicles of Lacticaseibacillus rhamnosus JB-1 contain immunomodulatory lipoteichoic acid and are endocytosed by intestinal epithelial cells.

Intestinal bacteria have diverse and complex influence on their host. Evidence is accumulating that this may be mediated in part by bacterial extracellular membrane vesicles (MV), nanometer-sized particles important for intercellular communication. Little is known about the composition of MV from gram-positive beneficial bacteria nor how they interact with intestinal epithelial cells (IEC). Here we demonstrate that MV from Lacticaseibacillus rhamnosus JB-1 are endocytosed in a likely clathrin-dependent manner by both mouse and human IEC in vitro and by mouse IEC in vivo. We further show that JB-1 MV contain lipoteichoic acid (LTA) that activates Toll-like receptor 2 (TLR2) and induces immunoregulatory interleukin-10 expression by dendritic cells in an internalization-dependent manner. By contrast, neither LTA nor TLR2 appear to be required for JB-1 MV endocytosis by IEC. These results demonstrate a novel mechanism by which bacterial MV can influence host physiology and suggest one potential route for beneficial influence of certain bacteria and probiotics.

Limosilactobacillus reuteri DSM-17938 for preventing cough in adults with mild allergic asthma: A double-blind randomized placebo-controlled cross-over study.

BACKGROUND: Cough is a common troublesome symptom in asthma which is neuronally mediated. Limosilactobacillus reuteri DSM-17938 (L. reuteri DSM-17938) is a probiotic shown to be effective in pre-clinical models at suppressing neuronal responses to capsaicin, a transient receptor potential vanilloid agonist (TRPV1). OBJECTIVE: Investigate the effects of DSM-17938 versus matched placebo on capsaicin-evoked coughs in mild allergic asthmatics. METHODS: We performed a 4-visit, randomized, double-blind, placebo-controlled, two-way cross-over study comparing full dose cough responses with inhaled capsaicin in mild allergic asthmatics after 1 month of treatment with DSM-17938 compared with matched placebo. Randomization and allocation to trial group were carried out by a central computer system. Histamine skin prick testing, airway hyper-responsiveness and inflammatory cells in induced sputum were measured at every visit. Blood was collected to extract PBMCs and stimulated with CD3/CD28 to ascertain the effects of DSM-17938 /placebo on T-cell cytokine responses. RESULTS: Seventeen subjects were recruited and 15 completed the study (8 females, mean age 27.3 years). There was no difference in the change in maximum capsaicin-evoked coughs (Emax) after treatment with L. reuteri DSM-17938 compared with placebo [mean difference 2.07 coughs (95% CI -2.77 to 6.91, p = .38) or relative changes in geometric mean ratios for the dose evoking at least half the Emax (ED50) [1.05 (95% CI 0.31-3.58, p = .94)], concentration evoking 2 coughs (C2) [0.63 (0.26-1.53), p = .28] and 5 coughs (C5) [0.79 (0.25-2.50), p = .67]. There was no effect on histamine skin prick wheal size, intensity of itch sensation, methacholine PC20, airway inflammation or T-cell responses after stimulation with CD3/CD28. There were no serious adverse events. One subject developed a mild upper respiratory tract infection and another mild transient nausea whilst on DSM-17938. CONCLUSION: In this small study in adults with mild allergic asthma, we found no evidence that L. reuteri DSM-17938 has any systemic effects on airway nerves, smooth muscle, sputum inflammatory cells, skin responses or T-cell responses after oral consumption. TRIAL REGISTRATION: Clinicaltrials.gov Identifier: NCT03603522.

Squalamine Restores the Function of the Enteric Nervous System in Mouse Models of Parkinson's Disease.

BACKGROUND: Parkinson's disease (PD) is a progressive neurodegenerative disorder thought to be caused by accumulation of α-synuclein (α-syn) within the brain, autonomic nerves, and the enteric nervous system (ENS). Involvement of the ENS in PD often precedes the onset of the classic motor signs of PD by many years at a time when severe constipation represents a major morbidity. Studies conducted in vitro and in vivo, have shown that squalamine, a zwitterionic amphipathic aminosterol, originally isolated from the liver of the dogfish shark, effectively displaces membrane-bound α-syn. OBJECTIVE: Here we explore the electrophysiological effect of squalamine on the gastrointestinal (GI) tract of mouse models of PD engineered to express the highly aggregating A53T human α-syn mutant. METHODS: GI motility and in vivo response to oral squalamine in PD model mice and controls were assessed using an in vitro tissue motility protocol and via fecal pellet output. Vagal afferent response to squalamine was measured using extracellular mesenteric nerve recordings from the jejunum. Whole cell patch clamp was performed to measure response to squalamine in the myenteric plexus. RESULTS: Squalamine effectively restores disordered colonic motility in vivo and within minutes of local application to the bowel. We show that topical squalamine exposure to intrinsic primary afferent neurons (IPANs) of the ENS rapidly restores excitability. CONCLUSION: These observations may help to explain how squalamine may promote gut propulsive activity through local effects on IPANs in the ENS, and further support its possible utility in the treatment of constipation in patients with PD.

The vagus nerve is necessary for the rapid and widespread neuronal activation in the brain following oral administration of psychoactive bacteria.

There is accumulating evidence that certain gut microbes modulate brain chemistry and have antidepressant-like behavioural effects. However, it is unclear which brain regions respond to bacteria-derived signals or how signals are transmitted to distinct regions. We investigated the role of the vagus in mediating neuronal activation following oral treatment with Lactobacillus rhamnosus (JB-1). Male Balb/c mice were orally administered a single dose of saline or a live or heat-killed preparation of a physiologically active bacterial strain, Lactobacillus rhamnosus (JB-1). 165 min later, c-Fos immunoreactivity in the brain was mapped, and mesenteric vagal afferent fibre firing was recorded. Mice also underwent sub-diaphragmatic vagotomy to investigate whether severing the vagus prevented JB-1-induced c-Fos expression. Finally, we examined the ΔFosB response following acute versus chronic bacterial treatment. While a single exposure to live and heat-killed bacteria altered vagal activity, only live treatment induced rapid neural activation in widespread but distinct brain regions, as assessed by c-Fos expression. Sub-diaphragmatic vagotomy abolished c-Fos immunoreactivity in most, but not all, previously responsive regions. Chronic, but not acute treatment induced a distinct pattern of ΔFosB expression, including in previously unresponsive brain regions. These data identify that specific brain regions respond rapidly to gut microbes via vagal-dependent and independent pathways, and demonstrate that acute versus long-term exposure is associated with differential responses in distinct brain regions.

Microvesicles from Lactobacillus reuteri (DSM-17938) completely reproduce modulation of gut motility by bacteria in mice.

Microvesicles are small lipid, bilayer structures (20-400 nm in diameter) secreted by bacteria, fungi, archaea and parasites involved in inter-bacterial communication and host-pathogen interactions. Lactobacillus reuteri DSM-17938 (DSM) has been shown to have clinical efficacy in the treatment of infantile colic, diarrhea and constipation. We have shown previously that luminal administration to the mouse gut promotes reduction of jejunal motility but increases that in the colon. The production of microvesicles by DSM has been characterized, but the effect of these microvesicles on gastrointestinal motility has yet to be evaluated. To investigate a potential mechanism for the effects of DSM on the intestine, the bacteria and its products have here been tested for changes in velocity, frequency, and amplitude of contractions in intact segments of jejunum and colon excised from mice. The effect of the parent bacteria (DSM) was compared to the conditioned media in which it was grown, and the microvesicles it produced. The media used to culture the bacteria (broth) was tested as a negative control and the conditioned medium was tested after the microvesicles had been removed. DSM, conditioned medium, and the microvesicles all produced comparable effects in both the jejunum and the colon. The treatments individually decreased the velocity and frequency of propagating contractile cluster contractions in the jejunum and increased them in the colon to a similar degree. The broth control had little effect in both tissues. Removal of the microvesicles from the conditioned medium almost completely eradicated their effect on motility in both tissues. These results show that the microvesicles from DSM alone can completely reproduce the effects of the whole bacteria on gut motility. Furthermore, they suggest a new approach to the formulation of orally active bacterial therapeutics and offer a novel way to begin to identify the active bacterial components.

Gut Microbiome Patterns Associated With Treatment Response in Patients With Major Depressive Disorder.

OBJECTIVES: Compelling animal data exists examining the impact of the gut microbiome on the brain, but work is required to translate these findings in a clinical population. We sought to do this by exploring the effects of antidepressant medications on the gut microbiota, and establishing a baseline Major Depressive Disorder (MDD) gut phenotype. METHODS: Participants with a primary diagnosis of MDD (n = 15) who were nonmedicated were recruited and followed over 6 months. Stool samples were collected prior to treatment initiation and 3 and 6 months following treatment. 16S rRNA sequencing was employed in order to analyze the gut microbial community profile. Symptom severity was measured by the Beck Depression Inventory. Alpha diversity metrics revealed no significant difference in the community diversity across any of the time-points. RESULTS: Comparison of within-group versus between-group distances revealed a lack of clustering of samples based on time-point, suggesting no significant change in the microbiota across treatment duration. When analyzed based on treatment response, however, patients in the responder group exhibited greater phylogenetic diversity than non-responders (Mann-Whitney U = 5, p = 0.026). At 3-months, 35 Operational Taxonomic Units (OTUs) were significantly different between groups and at 6-months, 42 OTUs were significantly different between responders and non-responders. CONCLUSIONS: These observations indicate that antidepressant medications alter the gut microbiota of patients with MDD, with disparate effects in responders versus non responders. This supports the concept of a microbiota phenotype associate with treatment response in MDD.

Prenatal low-dose penicillin results in long-term sex-specific changes to murine behaviour, immune regulation, and gut microbiota.

Growing evidence suggests that environmental disruptors of maternal microbes may have significant detrimental consequences for the developing fetus. Antibiotic exposure during early life can have long-term effects on neurodevelopment in mice and humans. Here we explore whether exposure to low-dose penicillin during only the last week of gestation in mice has long-term effects on offspring behaviour, brain, immune function, and gut microbiota. We found that this treatment had sex-specific effects in the adult mouse offspring. Female, but not male, mice demonstrated decreased anxiety-like behaviours, while male, but not female, mice had abnormal social behaviours which correlated with altered brain expression of AVPR1A, AVPR1B, and OXTR, and decreases in the balance of splenic FOXP3+ regulatory T cells. Prenatal penicillin exposure also led to distinct microbiota compositions that clustered differently by sex. These data suggest that exposure of pregnant mice to even a low dose of penicillin through only the last week before birth is nonetheless sufficient to induce long-term sex-specific developmental changes in both male and female offspring.

Oral selective serotonin reuptake inhibitors activate vagus nerve dependent gut-brain signalling.

The vagus nerve can transmit signals to the brain resulting in a reduction in depressive behavior as evidenced by the long-term beneficial effects of electrical stimulation of the vagus in patients with intractable depression. The vagus is the major neural connection between gut and brain, and we have previously shown that ingestion of beneficial bacteria modulates behaviour and brain neurochemistry via this pathway. Given the high levels of serotonin in the gut, we considered if gut-brain signaling, and specifically the vagal pathway, might contribute to the therapeutic effect of oral selective serotonin reuptake inhibitors (SSRI). Mesenteric nerve recordings were conducted in mice after treatment with SSRI to ascertain if this class of drugs resulted in increased vagal excitability. Patch clamp recordings of enteric neurons were carried out to measure activity of primary afferent neurons in the gut in response to SSRI and to assess the importance of gut epithelium in transducing signal. The tail suspension test (TST) was used following 14d feeding of SSRI in vagotomised and surgical sham mice to measure depressive-like behaviour. Brain mRNA expression was examined via PCR and the intestinal microbiome was assessed. Mesenteric nerve recordings in BALB/c mice demonstrated that oral treatment with SSRI leads to a significant increase in vagal activity. This effect was not observed in mice treated with a representative noradrenaline-dopamine reuptake inhibitor. It is known that signals from the gut can be transmitted to the vagus via the enteric nervous system. Exposure of the gut to SSRI increased the excitability of intrinsic primary afferent neurons in the myenteric plexus, through an intestinal epithelium dependent mechanism, and alpha-diversity of gut microbiota was altered. Critically, blocking vagal signaling from gut to brain, via subdiaphragmatic vagotomy, abolished the antidepressive effects of oral SSRI treatment as determined by the tail suspension test. This work suggests that vagus nerve dependent gut-brain signaling contributes to the effects of oral SSRI and further, highlights the potential for pharmacological approaches to treatment of mood disorders that focus on vagal stimulation and may not even require therapeutic agents to enter the circulation.

Restraint stress induced gut dysmotility is diminished by a milk oligosaccharide (2'-fucosyllactose) in vitro.

BACKGROUND: Stress causes severe dysmotility in the mammalian gut. Almost all research done to date has concentrated on prevention of stress-induced altered gut motility but not on treatment. We had previously shown that intraluminal 2'FL could acutely moderate propulsive motility in isolated mouse colonic segments. Because 2'FL appeared to modulate enteric nervous system dependent motility, we wondered if the oligosaccharide could reverse the effects of prior restraint stress, ex vivo. We tested whether 2'FL could benefit the dysmotility of isolated jejunal and colonic segments from animals subjected to prior acute restraint stress. METHODS: Jejunal and colonic segments were obtained from male Swiss Webster mice that were untreated or subjected to 1 hour of acute restraint stress. Segments were perfused with Krebs buffer and propagating contractile clusters (PCC) digitally video recorded. 2'FL or ß-lactose were added to the perfusate at a concentration of 1 mg/ml. Spatiotemporal maps were constructed from paired before and after treatment recordings, each consisting of 20 min duration and PCC analyzed for frequency, velocity and amplitude. KEY RESULTS: Stress decreased propulsive motility in murine small intestine while increasing it in the colon. 2'FL in jejunum of previously stressed mice produced a 50% increase in PCC velocity (p = 0.0001), a 43% increase in frequency (p = 0.0002) and an insignificant decrease in peak amplitude. For stressed colon, 2'FL reduced the frequency by 23% (p = 0.017) and peak amplitude by 26% (p = 0.011), and was without effect on velocity. ß-lactose had negligible or small treatment effects. CONCLUSIONS & INFERENCES: We show that the prebiotic 2'FL may have potential as a treatment for acute stress-induced gut dysmotility, ex vivo, and that, as is the case for certain beneficial microbes, the mechanism occurs in the gut, likely via action on the enteric nervous system.

Antibiotics and the nervous system: More than just the microbes?

The use of antibiotics has recently risen to prominence in neuroscience due to their potential value in studying the microbiota-gut-brain axis. In this context they have been largely employed to illustrate the many influences of the gut microbiota on brain function and behaviour. Much of this research is bolstered by the abnormal behaviour seen in germ-free animals and other well-controlled experiments. However, this literature has largely failed to consider the neuroactive potential of antibiotics themselves, independent from, or in addition to, their microbicidal effects. This is problematic, as clinical as well as experimental literature, largely neglected through the past decade, has clearly demonstrated that broad classes of antibiotics are neuroactive or neurotoxic. This is true even for some antibiotics that are widely regarded as not absorbed in the intestinal tract, and is especially concerning when considering the highly-concentrated and widely-ranging doses that have been used. In this review we will critically survey the clinical and experimental evidence that antibiotics may influence a variety of nervous system functions, from the enteric nervous system through to the brain and resultant behaviour. We will discuss substantial evidence which clearly suggests neuro-activity or -toxicity by most classes of antibiotics. We will conclude that, while evidence for the microbiota-gut-brain axis remains strong, clinical and experimental studies which employ antibiotics to probe it must consider this potential confound.

Mouse Strain Affects Behavioral and Neuroendocrine Stress Responses Following Administration of Probiotic Lactobacillus rhamnosus JB-1 or Traditional Antidepressant Fluoxetine.

Currently, there is keen interest in the development of alternative therapies in the treatment of depression. Given the explosion of research focused on the microbiota-gut-brain axis, consideration has turned to the potential of certain probiotics to improve patient outcomes for those suffering from mood disorders. Here we examine the abilities of a known antidepressant, fluoxetine, and the probiotic Lactobacillus rhamnosus JB-1™, to attenuate responses to two established criteria for depressive-like behavior in animal models, the tail suspension test (TST) and the corticosterone response to an acute restraint stressor. We examine two different strains of mice known to differ in the extent to which they express both anxiety-like behavior and measures of despair-BALB/c and Swiss Webster-with respectively high and normal behavioral phenotypes for each. While adult male BALB/c mice responded with increased antidepressive-like behavior to both fluoxetine and L. rhamnosus JB-1 in both the TST and the corticosterone stress response, SW mice did not respond to either treatment as compared to controls. These findings highlight the importance of investigating putative antidepressants in mouse strains known to express face validity for some markers of depression. Clinical studies examining the activity of L. rhamnosus JB-1 in patients suffering from mood disorders are warranted, as well as further pre-clinical work examining how interactions between host genotype and intestinal microbial alterations may impact behavioral responses. This study adds to the literature supporting the possibility that modifying the intestinal microbiota via probiotics represents a promising potential therapeutic breakthrough in the treatment of psychiatric disease.

The vagus nerve modulates BDNF expression and neurogenesis in the hippocampus.

Accumulating evidence suggests that certain gut microbiota have antidepressant-like behavioural effects and that the microbiota can regulate neurogenesis and the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus. The precise mechanisms underlying these effects are not yet clear. However, the vagus nerve is one of the primary bidirectional routes of communication between the gut and the brain and thus may represent a candidate mechanism. Yet, relatively little is known about the direct influence of vagus nerve activity on hippocampal function and plasticity. Thus, the aim of the present study was to determine whether constitutive vagus nerve activity contributes to the regulation of neurogenesis and BDNF mRNA expression in the hippocampus. To this end, we examined the impact of subdiaphragmatic vagotomy in adult mice on these parameters. We found that vagotomy decreased BDNF mRNA in all areas of the hippocampus. Vagotomy also reduced the proliferation and survival of newly born cells and decreased the number of immature neurons, particularly those with a more complex dendritic morphology. Taken together, these findings suggest that vagal nerve activity influences neurogenesis and BDNF mRNA expression in the adult hippocampus.

Disruptive physiology: olfaction and the microbiome-gut-brain axis.

This review covers the field of olfaction and chemosensation of odorants and puts this information into the context of interactions between microbes and behaviour; the microbiome-gut-brain axis (MGBA). Recent emphasis has also been placed on the concept of the holobiome which states that no single aspect of an organism should be viewed separately and thus must include examination of their associated microbial populations and their influence. While it is known that the microbiome may be involved in the modulation of animal behaviour, there has been little systematized effort to incorporate into such studies the rapidly developing knowledge of the wide range of olfactory systems. The classical olfactory system is evolutionarily conserved in multiple taxa from insects through to fish, reptiles and mammals, and is represented by the largest gene families in vertebrates. Mice have over 1000 different olfactory receptors and humans about 400. They are distributed throughout the body and are even found in spermatozoa where they function in chemotaxis. Each olfactory receptor has the unique functional capability of high-affinity binding to several different molecular ligands. These and other properties render the cataloguing of odorants (odorome) with specific actions a difficult task. Some ectopic olfactory receptors have been shown to have functional effects in the gut and kidney, highlighting the complexity of the systems engaged by odorants. However, there are, in addition to classical olfactory receptors, at least two other families of receptors involved in olfaction that are also widely found expressed on tissues in many different organs in addition to the nervous system and brain: the trace-amine associated and formyl peptide receptors. Bacteria can make many if not most odorants and are responsible for recognition of species and relative relatedness, as well as predator presence, among many other examples. Activation of different combinations of olfactory receptors by bacterial products such as ß-phenylethylamine have been shown even to control expression of emotions such as fear and aggression. The number of examples of bacterial products and volatile odorants influencing brain function and behaviour is expanding rapidly. Since it is recognized that many different bacterial products and metabolites also function as social cues, and may themselves be directly or indirectly causative of behavioural change, it becomes ever more important to include olfaction into studies of the MGBA. Clearly there are broader implications for the involvement of olfaction in this rapidly evolving field. These include improvement in our understanding of the pathways engaged in various behaviours, and the identification of novel approaches and new targets in efforts to modulate behavioural changes.

Antibiotic Driven Changes in Gut Motility Suggest Direct Modulation of Enteric Nervous System.

Antibiotic-mediated changes to the intestinal microbiome have largely been assumed to be the basis of antibiotic-induced neurophysiological and behavioral changes. However, relatively little research has addressed whether antibiotics act directly on the host nervous system to produce these changes. We aimed to identify whether acute exposure of the gastrointestinal tract to antibiotics directly modulates neuronally dependent motility reflexes, ex vivo. Motility of colon and jejunum segments in a perfusion organ bath was recorded by video and alterations to neuronally dependent propagating contractile clusters (PCC), measured using spatiotemporal maps of diameter changes. Short latency (<10 min) changes to PCC serve as an index of putative effects on the host nervous system. Bacitracin, penicillin V, and neomycin, all produced dose-dependent alterations to the velocity, frequency, and amplitude of PCC. Most significantly, colonic PCC velocity increased by 53% [probability of superiority (PS) = 87%] with 1.42 mg/ml bacitracin, 19% (PS = 81%) with 0.91 mg/ml neomycin, and 19% (PS = 86%) with 3.88 mg/ml penicillin V. Colonic frequency increased by 16% (PS = 73%) with 1.42 mg/ml bacitracin, 21% (PS = 79%) with 0.91 mg/ml neomycin, and 34% (PS = 85%) at 3.88 mg/ml penicillin V. Conversely, colonic amplitude decreased by 41% (PS = 79%) with 1.42 mg/ml bacitracin, 30% (PS = 80%) with 0.27 mg/ml neomycin and 25% (PS = 79%) at 3.88 mg/ml penicillin V. In the jejunum, antibiotic-specific changes were identified. Taken together, our findings provide evidence that acute exposure of the gastrointestinal lumen to antibiotics modulates neuronal reflexes. Future work should acknowledge the importance of this mechanism in mediating antibiotic-driven changes on gut-brain signaling.

Low-dose penicillin in early life induces long-term changes in murine gut microbiota, brain cytokines and behavior.

There is increasing concern about potential long-term effects of antibiotics on children's health. Epidemiological studies have revealed that early-life antibiotic exposure can increase the risk of developing immune and metabolic diseases, and rodent studies have shown that administration of high doses of antibiotics has long-term effects on brain neurochemistry and behaviour. Here we investigate whether low-dose penicillin in late pregnancy and early postnatal life induces long-term effects in the offspring of mice. We find that penicillin has lasting effects in both sexes on gut microbiota, increases cytokine expression in frontal cortex, modifies blood-brain barrier integrity and alters behaviour. The antibiotic-treated mice exhibit impaired anxiety-like and social behaviours, and display aggression. Concurrent supplementation with Lactobacillus rhamnosus JB-1 prevents some of these alterations. These results warrant further studies on the potential role of early-life antibiotic use in the development of neuropsychiatric disorders, and the possible attenuation of these by beneficial bacteria.

Oral treatment with Lactobacillus rhamnosus attenuates behavioural deficits and immune changes in chronic social stress.

BACKGROUND: Stress-related disorders involve systemic alterations, including disruption of the intestinal microbial community. Given the putative connections between the microbiota, immunity, neural function, and behaviour, we investigated the potential for microbe-induced gut-to-brain signalling to modulate the impact of stress on host behaviour and immunoregulation. METHODS: Male C57BL/6 mice treated orally over 28 days with either Lactobacillus rhamnosus (JB-1) ™ or vehicle were subjected to chronic social defeat and assessed for alterations in behaviour and immune cell phenotype. 16S rRNA sequencing and mass spectrometry were employed to analyse the faecal microbial community and metabolite profile. RESULTS: Treatment with JB-1 decreased stress-induced anxiety-like behaviour and prevented deficits in social interaction with conspecifics. However, JB-1 did not alter development of aggressor avoidance following social defeat. Microbial treatment attenuated stress-related activation of dendritic cells while increasing IL-10+ regulatory T cells. Furthermore, JB-1 modulated the effect of stress on faecal metabolites with neuroactive and immunomodulatory properties. Exposure to social defeat altered faecal microbial community composition and reduced species richness and diversity, none of which was prevented by JB-1. Stress-related microbiota disruptions persisted in vehicle-treated mice for 3 weeks following stressor cessation. CONCLUSIONS: These data demonstrate that despite the complexity of the gut microbiota, exposure to a single microbial strain can protect against certain stress-induced behaviours and systemic immune alterations without preventing dysbiosis. This work supports microbe-based interventions for stress-related disorders.

Lost in translation? The potential psychobiotic Lactobacillus rhamnosus (JB-1) fails to modulate stress or cognitive performance in healthy male subjects.

BACKGROUND: Preclinical studies have identified certain probiotics as psychobiotics - live microorganisms with a potential mental health benefit. Lactobacillus rhamnosus (JB-1) has been shown to reduce stress-related behaviour, corticosterone release and alter central expression of GABA receptors in an anxious mouse strain. However, it is unclear if this single putative psychobiotic strain has psychotropic activity in humans. Consequently, we aimed to examine if these promising preclinical findings could be translated to healthy human volunteers. OBJECTIVES: To determine the impact of L. rhamnosus on stress-related behaviours, physiology, inflammatory response, cognitive performance and brain activity patterns in healthy male participants. METHODS: An 8week, randomized, placebo-controlled, cross-over design was employed. Twenty-nine healthy male volunteers participated. Participants completed self-report stress measures, cognitive assessments and resting electroencephalography (EEG). Plasma IL10, IL1ß, IL6, IL8 and TNFα levels and whole blood Toll-like 4 (TLR-4) agonist-induced cytokine release were determined by multiplex ELISA. Salivary cortisol was determined by ELISA and subjective stress measures were assessed before, during and after a socially evaluated cold pressor test (SECPT). RESULTS: There was no overall effect of probiotic treatment on measures of mood, anxiety, stress or sleep quality and no significant effect of probiotic over placebo on subjective stress measures, or the HPA response to the SECPT. Visuospatial memory performance, attention switching, rapid visual information processing, emotion recognition and associated EEG measures did not show improvement over placebo. No significant anti-inflammatory effects were seen as assessed by basal and stimulated cytokine levels. CONCLUSIONS: L. rhamnosus was not superior to placebo in modifying stress-related measures, HPA response, inflammation or cognitive performance in healthy male participants. These findings highlight the challenges associated with moving promising preclinical studies, conducted in an anxious mouse strain, to healthy human participants. Future interventional studies investigating the effect of this psychobiotic in populations with stress-related disorders are required.