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Background: To date, there have been numerous metataxonomic studies on gut microbiota (GM) profiling based on the analyses of data from public repositories. However, differences in study population and wet and dry pipelines have produced discordant results. Herein, we propose a biostatistical approach to remove these batch effects for the GM characterization in the case of autism spectrum disorders (ASDs). Methods: An original dataset of GM profiles from patients with ASD was ecologically characterized and compared with GM public digital profiles of age-matched neurotypical controls (NCs). Also, GM data from seven case–control studies on ASD were retrieved from the NCBI platform and exploited for analysis. Hence, on each dataset, conditional quantile regression (CQR) was performed to reduce the batch effects originating from both technical and geographical confounders affecting the GM-related data. This method was further applied to the whole dataset matrix, obtained by merging all datasets. The ASD GM markers were identified by the random forest (RF) model. Results: We observed a different GM profile in patients with ASD compared with NC subjects. Moreover, a significant reduction of technical- and geographical-dependent batch effects in all datasets was achieved. We identified Bacteroides_H, Faecalibacterium, Gemmiger_A_73129, Blautia_A_141781, Bifidobacterium_388775, and Phocaeicola_A_858004 as robust GM bacterial biomarkers of ASD. Finally, our validation approach provided evidence of the validity of the QCR method, showing high values of accuracy, specificity, sensitivity, and AUC-ROC. Conclusions: Herein, we proposed an updated biostatistical approach to reduce the technical and geographical batch effects that may negatively affect the description of bacterial composition in microbiota studies.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder. Investigations of gut microbiota (GM) play an important role in deciphering disease severity and symptoms. Overall, we stratified 70 ASD patients by neuropsychological assessment, based on Calibrated Severity Scores (CSSs) of the Autism Diagnostic Observation Schedule-Second edition (ADOS-2), Child Behavior Checklist (CBCL) and intelligent quotient/developmental quotient (IQ/DQ) parameters. Hence, metataxonomy and PICRUSt-based KEGG predictions of fecal GM were assessed for each clinical subset. Here, 60% of ASD patients showed mild to moderate autism, while the remaining 40% showed severe symptoms; 23% showed no clinical symptoms, 21% had a risk of behavior problems and 56% had clinical symptoms based on the CBCL, which assesses internalizing problems; further, 52% had no clinical symptoms, 21% showed risk, and 26% had clinical symptoms classified by CBCL externalizing problems. Considering the total CBCL index, 34% showed no clinical symptoms, 13% showed risk, and 52% had clinical symptoms. Here, 70% of ASD patients showed cognitive impairment/developmental delay (CI/DD). The GM of ASDs with severe autism was characterized by an increase in Veillonella, a decrease in Monoglobus pectinilyticus and a higher microbial dysbiosis index (MDI) when compared to mild-moderate ASDs. Patients at risk for behavior problems and showing clinical symptoms were characterized by a GM with an increase of Clostridium, Eggerthella, Blautia, Intestinibacter, Coprococcus, Ruminococcus, Onthenecus and Bariatricus, respectively. Peptidoglycan biosynthesis and biofilm formation KEGGs characterized patients with clinical symptoms, while potential microbiota-activated PPAR-γ-signaling was seen in CI/DD patients. This evidence derived from GM profiling may be used to further improve ASD understanding, leasing to a better comprehension of the neurological phenotype.

Clostridioides difficile infection (CDI) is generally treated with vancomycin, metronidazole or fidaxomicin, although fecal microbiota transplantation (FMT) represents a promising therapeutic option for antibiotic-resistant recurrent C. difficile infections (rCDIs) in adults. In pediatric cystic fibrosis (CF) patients, CDIs are generally asymptomatic and respond to treatment. Here, we present the case of an 8-year-old female, initially diagnosed as “CFTR-related metabolic syndrome/cystic fibrosis screen positive, inconclusive diagnosis” (CMRS/CFSPID), who then progressed to CF at 12 months. In the absence of CF-related symptoms, she presented multiple and disabling episodes of bloody diarrhoea with positive tests for C. difficile antigen and A/B toxin. After conventional treatments failed and several CDI relapses, FMT was proposed. Donor screening and GM donor–receiver matching identified her mother as a donor. Metataxonomy and targeted metabolomics provided, through a pre- and post-FMT time course, gut microbiota (GM) profiling to assess GM engraftment. At first, the GM map revealed severe dysbiosis, with a prevalence of Bacteroidetes and Proteobacteria (i.e., Klebsiella spp., Escherichia coli), a reduction in Firmicutes, a GM nearly entirely composed of Enterococcaceae (i.e., Enterococcus) and an almost complete depletion of Verrucomicrobia and Actinobacteria, mostly represented by Veillonella dispar. Post FMT, an increment in Bifidobacterium spp. and Collinsella spp. with a decrease in V. dispar restored intestinal eubiosis. Consistently, four weeks after FMT treatment, the child’s gut symptoms cleared, without CDI recurrence.

Ischemic stroke (IS) can be caused by perturbations of the gut–brain axis. An imbalance in the gut microbiota (GM), or dysbiosis, may be linked to several IS risk factors and can influence the brain through the production of different metabolites, such as short-chain fatty acids (SCFAs), indole and derivatives. This study examines ecological changes in the GM and its metabolic activities after stroke. Fecal samples of 10 IS patients were compared to 21 healthy controls (CTRLs). GM ecological profiles were generated via 16S rRNA taxonomy as functional profiles using metabolomics analysis performed with a gas chromatograph coupled to a mass spectrometer (GC-MS). Additionally fecal zonulin, a marker of gut permeability, was measured using an enzyme-linked immuno assay (ELISA). Data were analyzed using univariate and multivariate statistical analyses and correlated with clinical features and biochemical variables using correlation and nonparametric tests. Metabolomic analyses, carried out on a subject subgroup, revealed a high concentration of fecal metabolites, such as SCFAs, in the GM of IS patients, which was corroborated by the enrichment of SCFA-producing bacterial genera such as Bacteroides, Christensellaceae, Alistipes and Akkermansia. Conversely, indole and 3-methyl indole (skatole) decreased compared to a subset of six CTRLs. This study illustrates how IS might affect the gut microbial milieu and may suggest potential microbial and metabolic biomarkers of IS. Expanded populations of Akkermansia and enrichment of acetic acid could be considered potential disease phenotype signatures.