Interest in the human microbiome has increased dramatically in the last decade. However, much of this research has focused on bacteria, while the composition and roles of their fungal counterparts remain less understood. Furthermore, a variety of methodological approaches have been applied, and the comparability between studies is unclear. This study compared four primer pairs targeting the small subunit (SSU) rRNA (18S), ITS1, ITS2, and large subunit (LSU) rRNA (26S) genomic regions for their ability to accurately characterize fungal communities typical of the human mycobiota. All four target regions of 21 individual fungal mock community taxa were capable of being amplified adequately and sequenced. Mixed mock community analyses revealed marked variability in the ability of each primer pair to accurately characterize a complex community. ITS target regions outperformed LSU and SSU. Of the ITS regions, ITS1 failed to generate sequences for Yarrowia lipolytica and all three Malassezia species when in a mixed community. These findings were further supported in studies of human sinonasal and mouse fecal samples. Based on these analyses, previous studies using ITS1, SSU, or LSU markers may omit key taxa that are identified by the ITS2 marker. Of methods commonly used in human mycobiota studies to date, we recommend selection of the ITS2 marker. Further investigation of more recently developed fungal primer options will be essential to ultimately determine the optimal methodological approach by which future human mycobiota studies ought to be standardized.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairments in social interactions/behaviors and increased stereotypical repetitive behavior. Gastrointestinal disorders, ranging from severe constipation to diarrhea, are particularly prevalent for people on the autism spectrum, which may relate to an underlying dysbiosis (breakdown or imbalance) in the gut microbial community. Many studies have also identified changes in the gut microbiome in ASD compared to neurotypical cohorts in animal models and human populations. Microbial probiotics to help revert these gut microbial changes have been tested in animal models of ASD. Some have shown reversal of ASD behaviors and modulating the integrity of the gastrointestinal epithelial barrier. The gut-microbiota-brain axis has been described as a multidirectional communication channel between the three systems: the gut, gut microbes, and the brain, but whether these gastrointestinal microbes play a role in the context of ASD and whether they can be harnessed as a target for gastrointestinal therapies in humans is yet to be determined.

Shank genes are implicated in ~1% of people with autism and mice with Shank3 knock out mutations exhibit autism-like behaviours. Zinc deficiency and gastrointestinal problems can be common among people with autism, and zinc is a key element required for SHANK protein function and gut development. In Shank3B-/- mice, a supplementary zinc diet reverses autism behaviours. We hypothesise that dietary zinc may alter the gut microbiome, potentially affecting the gut-microbiome-brain axis, which may contribute to changes in autism-like behaviours. To test this, four types of gastrointestinal samples (ileum, caecum, colon, faecal) were collected from wild-type and knock-out Shank3B-/- mice on either control or supplemented-zinc diets. Cage, genotype and zinc diet each contributed significantly to bacterial community variation (accounting for 12.8%, 3.9% and 2.3% of the variation, respectively). Fungal diversity differed significantly between wild-type and knock-out Shank3B-/- mice on the control zinc diet, and the fungal biota differed among gut locations. RNA-seq analysis of host (mouse) transcripts revealed differential expression of genes involved in host metabolism that may be regulated by the gut microbiota and genes involved in anti-microbial interactions. By utilising the Shank3B-/- knock-out mouse model we were able to examine the influence of – and interactions between – dietary zinc and ASD-linked host genotype. These data broaden understanding of the gut microbiome in autism and pave the way towards potential microbial therapeutics for gastrointestinal problems in people with autism.

Background: Shank genes are implicated in ~1% of people with autism and mice with Shank3 knock out mutations exhibit autism-like behaviours. Zinc deficiency and gastrointestinal problems can be common among people with autism, and zinc is a key element required for SHANK protein function and gut development. In Shank3B-/- mice, a supplementary zinc diet reverses autism behaviours. We hypothesise that dietary zinc may alter the gut microbiome, potentially affecting the gut-microbiota-brain axis, which may contribute to changes in autism-like behaviours. Methods: Four types of gastrointestinal samples (ileum, caecum, colon, faecal) were collected from wild-type and knock-out Shank3B-/- mice on either control or supplemented-zinc diets. Bacterial 16S rRNA gene and fungal ITS2 genomic region amplicons were sequenced on the Illumina MiSeq platform and RNA on the Illumina HiSeq platform.Results: Cage, genotype and zinc diet each contributed significantly to bacterial community variation (accounting for 12.8%, 3.9% and 2.3% of the variation, respectively). Fungal diversity differed significantly between wild-type and knock-out Shank3B-/- mice on the control zinc diet, and the fungal biota differed among gut locations. RNA-seq analysis of host (mouse) transcripts revealed differential expression of genes involved in host metabolism that may be regulated by the gut microbiota and genes involved in anti-microbial interactions. Limitations: This study used the Shank3B-/- mouse model of autism spectrum disorder. Heterozygous and homozygous Shank3 gene mutations are found in 1% of the ASD population, only homozygous Shank3 mice were utilised in this study. Any translational conclusions should consider these limitations.Conclusions: By utilising the Shank3B-/- knock-out mouse model we were able to examine the influence of – and interactions between – dietary zinc and ASD-linked host genotype. Differential expression of host antimicrobial interaction genes as well as gut microbiota-regulated host metabolism genes among the treatment groups, suggests that the interplay between gut microbes, the gastrointestinal tract and the brain may play a major role towards the observed amelioration of ASD behaviours seen previously with supplemented dietary zinc. These data broaden understanding of the gut microbiome in autism and pave the way towards potential microbial therapeutics for gastrointestinal problems in people with autism.