The infant gut microbiota develops rapidly during the first 2 years of life, acquiring microorganisms from diverse sources. During this time, significant opportunities exist for the infant to acquire antibiotic resistant bacteria, which can become established and constitute the infant gut resistome. With increased antibiotic resistance limiting our ability to treat bacterial infections, investigations into resistance reservoirs are highly pertinent. This study aimed to explore the nascent resistome in antibiotically-naïve infant gut microbiomes, using a combination of metagenomic approaches. Faecal samples from 22 six-month-old infants without previous antibiotic exposure were used to construct a pooled metagenomic library, which was functionally screened for ampicillin and gentamicin resistance. Our library of ∼220Mb contained 0.45 ampicillin resistant hits/Mb and 0.059 gentamicin resistant hits/Mb. PCR-based analysis of fosmid clones and uncloned metagenomic DNA, revealed a diverse and abundant aminoglycoside and β-lactam resistance reservoir within the infant gut, with resistance determinants exhibiting homology to those found in common gut inhabitants, including Escherichia coli, Enterococcus sp., and Clostridium difficile, as well as to genes from cryptic environmental bacteria. Notably, the genes identified differed from those revealed when a sequence-driven PCR-based screen of metagenomic DNA was employed. Carriage of these antibiotic resistance determinants conferred substantial, but varied (2-512x), increases in antibiotic resistance to their bacterial host. These data provide insights into the infant gut resistome, revealing the presence of a varied aminoglycoside and β-lactam resistance reservoir even in the absence of selective pressure, confirming the infant resistome establishes early in life, perhaps even at birth.

The gut microbiota is an essential regulator of many aspects of host physiology. Disruption of gut microbial communities affects gut-brain communication which ultimately can manifest as changes in brain function and behaviour. Transient changes in gut microbial composition can be induced by various intrinsic and extrinsic factors, however, it is possible that enduring shifts in the microbiota composition can be achieved by perturbation at a timepoint when the gut microbiota has not fully matured or is generally unstable, such as during early life or ageing. In this study, we investigated the effects of 3-week microbiota depletion with antibiotic treatment during the adolescent period and in adulthood. Following a washout period to restore the gut microbiota, behavioural and molecular hallmarks of gut-brain communication were investigated. Our data revealed that transient microbiota depletion had long-lasting effects on microbiota composition and increased anxiety-like behaviour in mice exposed to antibiotic treatment during adolescence but not in adulthood. Similarly, gene expression in the amygdala was more severely affected in mice treated during adolescence. Taken together these data highlight the vulnerability of the gut microbiota during the critical adolescent period and the long-lasting impact manipulations of the microbiota can have on gene expression and behaviour in adulthood.

Chronic stress increases disease vulnerability factors including inflammation, a pathological characteristic potentially regulated by the gut microbiota. We checked the association between the gut microbiome and psychosocial stress in children/adolescents and investigated which stress parameter (negative versus positive emotion, self-report versus parental report, events versus emotions, biomarker cortisol versus parasympathetic activity) is the most relevant indicator herein.

Carotid body (peripheral oxygen sensor) sensitisation is pivotal in the development of chronic intermittent hypoxia (CIH)-induced hypertension. We sought to determine if exposure to CIH, modelling human sleep apnoea, adversely affects cardiorespiratory control in guinea-pigs, a species with hypoxia-insensitive carotid bodies. We reasoned that CIH-induced disruption of gut microbiota would evoke cardiorespiratory morbidity.

Rapid advancements in sequencing technologies along with falling costs present widespread opportunities for microbiome studies across a vast and diverse array of environments. These impressive technological developments have been accompanied by a considerable growth in the number of methodological variables, including sampling, storage, DNA extraction, primer pairs, sequencing technology, chemistry version, read length, insert size, and analysis pipelines, amongst others. This increase in variability threatens to compromise both the reproducibility and the comparability of studies conducted. Here we perform the first reported study comparing both amplicon and shotgun sequencing for the three leading next-generation sequencing technologies. These were applied to six human stool samples using Illumina HiSeq, MiSeq and Ion PGM shotgun sequencing, as well as amplicon sequencing across two variable 16S rRNA gene regions. Notably, we found that the factor responsible for the greatest variance in microbiota composition was the chosen methodology rather than the natural inter-individual variance, which is commonly one of the most significant drivers in microbiome studies. Amplicon sequencing suffered from this to a large extent, and this issue was particularly apparent when the 16S rRNA V1-V2 region amplicons were sequenced with MiSeq. Somewhat surprisingly, the choice of taxonomic binning software for shotgun sequences proved to be of crucial importance with even greater discriminatory power than sequencing technology and choice of amplicon. Optimal N50 assembly values for the HiSeq was obtained for 10 million reads per sample, whereas the applied MiSeq and PGM sequencing depths proved less sufficient for shotgun sequencing of stool samples. The latter technologies, on the other hand, provide a better basis for functional gene categorisation, possibly due to their longer read lengths. Hence, in addition to highlighting methodological biases, this study demonstrates the risks associated with comparing data generated using different strategies. We also recommend that laboratories with particular interests in certain microbes should optimise their protocols to accurately detect these taxa using different techniques.

High-throughput sequencing has enabled detailed insights into complex microbial environments, including the human gut microbiota. The accuracy of the sequencing data however, is reliant upon appropriate storage of the samples prior to DNA extraction. The aim of this study was to conduct the first MiSeq sequencing investigation into the effects of faecal storage on the microbiota, compared to fresh samples. Culture-based analysis was also completed.

Adolescence is a critical developmental period that is characterised by growth spurts and specific neurobiological, neuroimmune and behavioural changes. In tandem the gut microbiota, which is a key player in the regulation of health and disease, is shaped during this time period. Diet is one of the most important regulators of microbiota composition. Thus, we hypothesised that dietary disturbances of the microbiota during this critical time window result in long-lasting changes in immunity, brain and behaviour. C57BL/6 male mice were exposed to either high fat diet or cafeteria diet during the adolescent period from postnatal day 28 to 49 and were tested for anxiety-related and social behaviour in adulthood. Our results show long-lasting effects of dietary interventions during the adolescent period on microbiota composition and the expression of genes related to neuroinflammation or neurotransmission. Interestingly, changes in myelination-related gene expression in the prefrontal cortex following high fat diet exposure were also observed. However, these effects did not translate into overt behavioural changes in adulthood. Taken together, these data highlight the importance of diet-microbiota interactions during the adolescent period in shaping specific outputs of the microbiota-gut-brain axis in later life.

Identifying biological markers predicting vulnerability to develop excessive alcohol consumption may lead to a real improvement of clinical care. With converging evidence suggesting that gut microbiome is capable of influencing brain and behavior, this study aimed at investigating whether changes in gut microbiome composition is associated with conditioned responses to alcohol. We trained Wistar rats to self-administer alcohol for a prolonged period before screening those exhibiting uncontrolled alcohol seeking and taking by modeling diagnostic criteria for AUD: inability to abstain during a signaled period of reward unavailability, increased motivation assessed in a progressive effortful task and persistent alcohol intake despite aversive foot shocks. Based on addiction criteria scores, rats were assigned to either Vulnerable or Resilient groups. Vulnerable rats not only displayed increased impulsive and compulsive behaviors, but also displayed increased relapse after abstinence and increased sensitivity to baclofen treatments compared to resilient animals. Then, rats underwent a 3-month wash out period before sacrifice. Dorsal striatum was collected to assess dopamine receptor mRNA expression, and 16S microbiome sequencing was performed on caecal contents. Multiple significant correlations were found between gut microbiome and impulsivity measures, as well as augmentations in striatal Dopamine 1 receptor (D1R) and reductions in D2R as vulnerability to AUD increased. Therefore, using a singular translational approach based on biobehavioral dispositions to excessive alcohol seeking without heavy intoxication, our observations suggests an association between gut microbiome composition and these specific "at risk" behavioral traits observed in our translationally relevant model.

Liver disease is a major source of morbidity and mortality in children with short bowel syndrome (SBS). SBS-associated microbial dysbiosis has recently been implicated in the development of SBS-associated liver disease (SBS-ALD), however the pathological implications of this association have not been explored. In this study high-throughput sequencing of colonic content from the well-validated piglet SBS-ALD model was examined to determine alterations in microbial communities, and concurrent metabolic alterations identified in urine samples via targeted mass spectrometry approaches (GC-MS, LC-MS, FIA-MS) further uncovered impacts of microbial disturbance on metabolic outcomes in SBS-ALD. Multi-variate analyses were performed to elucidate contributing SBS-ALD microbe and metabolite panels and to identify microbe-metabolite interactions. A unique SBS-ALD microbe panel was clearest at the genus level, with discriminating bacteria predominantly from the Firmicutes and Bacteroidetes phyla. The SBS-ALD metabolome included important alterations in the microbial metabolism of amino acids and the mitochondrial metabolism of branched chain amino acids. Correlation analysis defined microbe-metabolite clustering patterns unique to SBS-ALD and identified a metabolite panel that correlates with dysbiosis of the gut microbiome in SBS.

Next-generation sequencing platforms have revolutionised our ability to investigate the microbiota composition of complex environments, frequently through 16S rRNA gene sequencing of the bacterial component of the community. Numerous factors, including DNA extraction method, primer sequences and sequencing platform employed, can affect the accuracy of the results achieved. The aim of this study was to determine the impact of these three factors on 16S rRNA gene sequencing results, using mock communities and mock community DNA.

Perinatal factors impact gut microbiota development in early life, however, little is known on the effects of these factors on microbes in later life. Here we sequence DNA from faecal samples of children over the first four years and reveal a perpetual evolution of the gut microbiota during this period. The significant impact of gestational age at birth and delivery mode on gut microbiota progression is evident in the first four years of life, while no measurable effects of antibiotics are found in the first year. Microbiota profiles are also characteristic in children dependant on gestational age and maturity. Full term delivery is characterised by Bacteroides (year one), Parabacteroides (year two) and Christensenellaceae (year four). Preterm delivery is characterised by Lactobacillus (year one), Streptococcus (year two) and Carnobacterium (year four). This study reveals that the gut retains distinct microbial profiles of perinatal factors up to four years of age.

The human gut microbiota has emerged as a key factor in the development of obesity. Certain probiotic strains have shown anti-obesity effects. The objective of this study was to investigate whether Bifidobacterium longum APC1472 has anti-obesity effects in high-fat diet (HFD)-induced obese mice and whether B. longum APC1472 supplementation reduces body-mass index (BMI) in healthy overweight/obese individuals as the primary outcome. B. longum APC1472 effects on waist-to-hip ratio (W/H ratio) and on obesity-associated plasma biomarkers were analysed as secondary outcomes.

The microbiome-gut-brain-axis is a complex phenomenon spanning several dynamic systems in the body which can be parsed at a molecular, cellular, physiological and ecological level. A growing body of evidence indicates that this axis is particularly sensitive to the effects of stress and that it may be relevant to stress resilience and susceptibility. Although stress-induced changes in the composition of the microbiome have been reported, the degree of compositional change over time, which we define as volatility, has not been the subject of in-depth scrutiny. Using a chronic psychosocial stress paradigm in male mice, we report that the volatility of the microbiome significantly correlated with several readouts of the stress response, including behaviour and corticosterone response. We then validated these findings in a second independent group of stressed mice. Additionally, we assessed the relationship between volatility and stress parameters in a cohort of health volunteers who were undergoing academic exams and report similar observations. Finally, we found inter-species similarities in the microbiome stress response on a functional level. Our research highlights the effects of stress on the dynamic microbiome and underscores the informative value of volatility as a parameter that should be considered in all future analyses of the microbiome.

The microbial community in the gut is influenced by environmental factors, especially diet, which can moderate host behaviour through the microbiome-gut-brain axis. However, the ecological relevance of microbiome-mediated behavioural plasticity in wild animals is unknown. We presented wild-caught great tits (Parus major) with a problem-solving task and showed that performance was weakly associated with variation in the gut microbiome. We then manipulated the gut microbiome by feeding birds one of two diets that differed in their relative levels of fat, protein and fibre content: an insect diet (low content), or a seed diet (high content). Microbial communities were less diverse among individuals given the insect compared to those on the seed diet. Individuals were less likely to problem-solve after being given the insect diet, and the same microbiota metrics that were altered as a consequence of diet were also those that correlated with variation in problem solving performance. Although the effect on problem-solving behaviour could have been caused by motivational or nutritional differences between our treatments, our results nevertheless raise the possibility that dietary induced changes in the gut microbiota could be an important mechanism underlying individual behavioural plasticity in wild populations.

The potential for the human gut microbiota to serve as a reservoir for antibiotic resistance genes has been the subject of recent discussion. However, this has yet to be investigated using a rapid PCR-based approach. In light of this, here we aim to determine if degenerate PCR primers can detect aminoglycoside and β-lactam resistance genes in the gut microbiota of healthy adults, without the need for an initial culture-based screen for resistant isolates. In doing so, we would determine if the gut microbiota of healthy adults, lacking recent antibiotic exposure, is a reservoir for resistance genes.

It is increasingly evident that perturbations to the diversity and composition of the gut microbiota have significant consequences for the regulation of integrative physiological systems. There is growing interest in the potential contribution of microbiota-gut-brain signalling to cardiorespiratory control in health and disease.

Targeting the gut microbiome as an effective therapeutic strategy for psychological disorders has shown promise in recent years. Variation in the composition of the microbiota and restoration of a stable microbiome using targeted interventions (psychobiotics) including Bifidobacteria have shown promise in pre-clinical studies, but more human data is required on the potential health benefits of these live microorganisms. Bifidobacterium including Bif. longum 1714 has been shown to dampen the effects of acute stress in humans. However, its effects over a period of prolonged stress have not been examined. A randomised, placebo-controlled, repeated measures, cross-over intervention study was conducted to examine the effects of a probiotic intervention on measures of stress, cognitive performance, and mood in healthy human volunteers. Twenty male students participated in this crossover study. Post-intervention assessments took place during the university exam period, which was used as a naturalistic chronic stressor. Self-reported measures of stress, depression, sleep quality, physical activity, gastrointestinal symptoms, cognition, and mood were assessed by questionnaire. In addition, tests from the Cambridge Neuropsychological Test Automated Battery (CANTAB) were administered to all participants. Stress and depression scores increased in both placebo and probiotic treated groups during the exam period. While overall sleep quality and duration of sleep improved significantly in the probiotic treated group during exam stress compared with the placebo treated group, B. longum 1714, similar to placebo treatment, showed no efficacy in improving measures of working memory, visual memory, sustained attention or perception. Overall, while B. longum 1714 shows promise in improving sleep quality and duration, it did not alleviate symptoms of chronic stress, depression, or any measure of cognitive assessment. Thus, further mechanistic studies into the ability of B. longum 1714 to modulate sleep during prolonged periods of stress are now warranted.

The role of the gut microbiome in the biotransformation of drugs has recently come under scrutiny. It remains unclear whether the gut microbiome directly influences the extent of drug absorbed after oral administration and thus potentially alters clinical pharmacokinetics.

Human milk is considered the optimum feeding regime for newborns and is a source of bacteria for the developing infant gastrointestinal tract. However, as with all low biomass samples, standardization across variabilities such as sample collection, storage, and extraction methods is needed to eliminate discrepancies in microbial composition across studies. The aim of this study was to investigate how different storage methods, temperatures, preservatives, and extraction kits influence the human milk microbiome, compared to fresh samples. Breast milk samples were processed via six different methods: fresh (Method 1), frozen at -80°C (Method 2), treated with RNAlater and stored at 4°C or -80°C (Methods 3 and 4), and treated with Milk Preservation Solution at room temperature (Methods 5 and 6). Methods 1-5 were extracted using PowerFoodTM Microbial DNA Isolation kit (Mobio), and Method 6 was extracted using Milk DNA Preservation and Isolation kit (Norgen BioTek). At genus level, the most abundant genera were shared across Methods 1-5. Samples frozen at -80°C had fewest significant changes while samples treated and extracted using Milk Preservation and Isolation kit had the most significant changes when compared to fresh samples. Diversity analysis indicated that variation in microbiota composition was related to the method and extraction kit used. This study highlighted that, when extraction from fresh milk samples is not an option, freezing at -80°C is the next best option to preserve the integrity of the milk microbiome. Furthermore, our results demonstrate that choice of extraction kit had a profound impact on the microbiota populations detected in milk.

There is a growing emphasis on the role of the microbiota-gut-brain axis as modulator of host behaviour and as therapeutic target for neuropsychiatric disorders. In addition, accumulating evidence suggests that early-life stress can exert long-lasting changes on the brain and microbiota, and this early adversity is associated with increased risk for developing depression in later life. The maternal separation (MS) model in rats is a robust paradigm to study the effects of early-life stress on the microbiota-gut-brain axis. Recently, we have shown that polyphenols, naturally occurring compounds associated with several health benefits, have anti-stress effects in in vitro models. In this study, we assess the therapeutic potential of a variety of both flavonoid and non-flavonoid polyphenols in reversing the impact of MS on behaviour and the microbiota-gut-brain axis. Rats underwent a dietary intervention with the naturally-derived polyphenols xanthohumol and quercetin, as well as with a phlorotannin extract for 8 weeks. Treatment with polyphenols prevented the depressive- and anxiety-like behaviours induced by MS, where xanthohumol effects were correlated with rescue of BDNF plasma levels. In addition, MS resulted in altered brain levels of 5-hydroxyindoleacetic acid (5-HIAA) and dopamine, accompanied by abnormal elevation of plasma corticosterone. Although polyphenols did not reverse neurotransmitter imbalance, xanthohumol normalised corticosterone levels in MS rats. Finally, we explored the impact of MS and polyphenolic diets on the gut microbiota. We observed profound changes in microbial composition and diversity produced by MS condition and by xanthohumol treatment. Moreover, functional prediction analysis revealed that MS results in altered enrichment of pathways associated with microbiota-brain interactions that are significantly reversed by xanthohumol treatment. These results suggest that naturally-derived polyphenols exert antidepressant-like effects in MS rats, which mechanisms could be potentially mediated by HPA regulation, BDNF levels rescue and modulation of the microbiota-gut-brain axis.