Attention-deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder. Given the growing evidence of gut microbiota being involved in psychiatric (including neurodevelopmental) disorders, we aimed to identify differences in gut microbiota composition between participants with ADHD and controls and to investigate the role of the microbiota in inattention and hyperactivity/impulsivity. Fecal samples were collected from 107 participants (N_ADHD = 42; N_controls = 50; N_{subthreholdADHD} = 15; range age: 13–29 years). The relative quantification of bacterial taxa was done using 16S ribosomal RNA gene amplicon sequencing. Alpha and Beta-diversity were not different between participants with ADHD and healthy controls. Three genera showed nominal differences (p_uncorrected < 0.05) between both groups (Prevotella_9, Coprococcus_2 and Intestinibacter) and were further tested for their association with ADHD symptom scores (adjusting for age, sex, body mass index, a time delay between feces collection and symptoms assessment, medication use and family relatedness). Our results show that the variation of a genus from the Lachnospiraceae family (Coprococcus_2) showed a trend of being negatively associated with inattention symptoms. Furthermore, we showed that the relative abundance of four genera was reduced by ADHD medication (p_uncorrected < 0.05). Overall, our results may support the role of the gut microbiota in the pathophysiology of ADHD. Given the scarcity of studies on the gut microbiota in individuals with ADHD, the current results are an important contribution to this field. More studies are needed into the gut microbiota as part of the pathology of ADHD, especially with a bigger sample size across the lifespan and more detailed information about lifestyle.

Gut bacteria hold the potential to produce a broad range of metabolites that can modulate human functions, including molecules with neuroactive potential. One such molecule is γ-aminobutyric acid (GABA), the main inhibitory neurotransmitter of the central nervous system in animals. Metagenomic analyses suggest that the genomes of many gut bacteria encode glutamate decarboxylase (GAD), the enzyme that catalyzes GABA production. The genome of Akkermansia muciniphila , a mucin specialist and potential next-generation probiotic from the human gut, is predicted to encode GAD, suggesting a contributing role in GABA production in the human gut. In this study, A. muciniphila was grown in batch cultures with and without pH control. In both experiments, A. muciniphila was found to produce GABA as a response to acid (pH <5.5), although only when GABA precursors, either glutamate or glutamine, were present in the medium. Proteomic analysis comparing A. muciniphila grown with and without precursors at pH 4 did not show a difference in GAD expression, suggesting that it is expressed regardless of the presence of GABA precursors. To further investigate the function of A. muciniphila GAD, we heterologously expressed the gad gene (encoded by locus tag Amuc_0372) with a His tag in Escherichia coli and purified the GAD protein. Enzyme assays showed GAD activity in a pH range between 4 and 6, with the highest specific activity at pH 5 of 144 ± 16 µM GABA/min/mg. Overall, our results demonstrate the ability of A. muciniphila to produce GABA as an acid response and unravel the conditions under which GABA production in A. muciniphila occurs. IMPORTANCE: Akkermansia muciniphila is considered to be a beneficial bacterium from the human gut, but the exact mechanisms by which A. muciniphila influences its host are not yet fully understood. To this end, it is important to identify which metabolites are produced and consumed by A. muciniphila that may contribute to a healthy gut. In the present study, we demonstrate the ability of A. muciniphila to produce γ-aminobutyric acid (GABA) when grown in an acidic environment, which often occurs in the gut. GABA is the major inhibitory neurotransmitter in the central nervous system and is present in the human gut. For this reason, it is considered an important bacterial metabolite. Our finding that A. muciniphila produces GABA in acidic environments adds to the growing body of understanding of its relationship with host health and provides an explanation on how it can survive acid stress in the human gut.

Background: Formalin-fixed paraffin embedded (FFPE) tissues may provide an exciting resource to study microbial associations in human disease, but the use of these low biomass specimens remains challenging. We aimed to reduce unintentional bacterial interference in molecular analysis of FFPE tissues and investigated the feasibility of conducting quantitative polymerase chain reaction (qPCR) and 16S rRNA amplicon sequencing using 14 colorectal cancer, 14 normal adjacent and 13 healthy control tissues. Results: Bacterial contaminants from the laboratory environment and the co-extraction of human DNA can affect bacterial analysis. The application of undiluted template improves bacterial DNA amplification, allowing the detection of specific bacterial markers (Escherichia coli and Faecalibacterium prausnitzii) by qPCR. Nested and non-nested PCR-based 16S rRNA amplicon sequencing approaches were employed, showing that bacterial communities of tissues and paired paraffin controls cluster separately at genus level on weighted Unifrac in both non-nested (R2 = 0.045; Pr(> F) = 0.053) and nested (R2 = 0.299; Pr(> F) = 0.001) PCR datasets. Nevertheless, considerable overlap of bacterial genera within tissues was seen with paraffin, DNA extraction negatives (non-nested PCR) or PCR negatives (nested PCR). Following mathematical decontamination, no differences in α- and β diversity were found between tumor, normal adjacent and control tissues. Conclusions: Bacterial marker analysis by qPCR seems feasible using non-normalized template, but 16S rRNA amplicon sequencing remains challenging. Critical evaluation of laboratory procedures and incorporation of positive and negative controls for bacterial analysis of FFPE tissues are essential for quality control and to account for bacterial contaminants.

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Background: Nutritional supplements, such as bovine lactoferrin (bLF), have been studied for their immunomodulatory properties, but little is known of their effect on the gut microbiota composition of the elderly when supplemented alone or combined with other nutritional supplements such as prebiotics and micronutrients. In the present study, fecal samples from a double-blind, placebo-controlled nutritional intervention study were analysed. At baseline (T1), 25 elderly women were distributed into two groups receiving dietary intervention (n = 12) or placebo treatment (n = 13) for 9 weeks. During the first 3 weeks of the study (T2), the intervention group consumed 1 g/day bLF, followed by 3 weeks (T3) of 1 g/day bLF and 2.64 g/day active galactooligosaccharides (GOS), and 3 weeks (T4) of 1 g/day bLF, 2.64 g/day GOS and 20 μg/day of vitamin D. The placebo group received maltodextrin, in dosages matching those of the intervention group. Fecal bacterial composition was profiled using partial 16S rRNA gene amplicon sequencing. Short-chain fatty acids (SCFA) were determined in fecal water as were levels of calprotectin, zonulin, and alpha-1-antitrypsin, as markers of gastrointestinal barrier and inflammation. Results: A significant increase was observed in the relative abundance of the genus Holdemanella (p < 0.01) in the intervention group compared to the placebo at T1. During T2, Bifidobacterium relative abundance increased significantly (p < 0.01) in the intervention group compared to the placebo, and remained significantly higher until the end of the study. No other effect was reported during T3. Furthermore, concentrations of SCFAs and calprotectin, zonulin and alpha-1-antitrypsin did not change during the intervention, although zonulin levels increased significantly within the placebo group by the end of the intervention. Conclusions: We conclude that supplementation of bLF enhanced the relative abundance of Holdemanella in the fecal microbiota of healthy elderly women, and further addition of GOS enhanced the relative abundance of Bifidobacterium.

Attention-deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder. Given the growing evidence of gut microbiota being involved in psychiatric (including neurodevelopmental) disorders, we aimed to identify differences in gut microbiota composition between participants with ADHD and controls and to investigate the role of the microbiota in inattention and hyperactivity/impulsivity. Fecal samples were collected from 107 participants (NADHD=42; Ncontrols=50; NsubthreholdADHD=15; range age: 13-29 years). The relative quantification of bacterial taxa was done using 16S ribosomal RNA gene amplicon sequencing. Beta-diversity revealed significant differences in bacterial composition between participants with ADHD and healthy controls, which was also significant for inattention, but showing a trend in case of hyperactivity/impulsivity only. Ten genera showed nominal differences (P < 0.05) between both groups, of which seven genera were tested for their association with ADHD symptom scores (adjusting for age, sex, body mass index, time delay between feces collection and symptoms assessment, medication use and family relatedness). Our results show that variation of a genus from the Ruminococcaceae family (Ruminococcaceae_UCG_004) is associated (after multiple testing correction) with inattention symptoms, and suggest a role of gut microbiota in ADHD pathophysiology.