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Great effort was made to characterize the bacterial communities inhabiting the human body as a factor in disease, resulting in the realization that a wide spectrum of diseases is associated with an altered composition of the microbiome. However, the identification of disease-relevant bacteria has been hindered by the high cross-sectional diversity of individual microbiomes, and in most cases, it remains unclear whether the observed alterations are cause or consequence of disease. Hence, innovative analysis approaches are required that enable inquiries of the microbiome beyond mere taxonomic cataloging. This review highlights the utility of microbiota flow cytometry, a single-cell analysis platform to directly interrogate cellular interactions, cell conditions, and crosstalk with the host's immune system within the microbiome to take into consideration the role of microbes as critical interaction partners of the host and the spectrum of microbiome alterations, beyond compositional changes. In conjunction with advanced sequencing approaches it could reveal the genetic potential of target bacteria and advance our understanding of taxonomic diversity and gene usage in the context of the microenvironment. Single-cell bacterial phenotyping has the potential to change our perspective on the human microbiome and empower microbiome research for the development of microbiome-based therapy approaches and personalized medicine., (© 2023 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

The commensal microflora provides a repertoire of antigens that illicit mucosal antibodies. In some cases, these antibodies can cross-react with host proteins, inducing autoimmunity, or with other microbial antigens. We demonstrate that the oral microbiota can induce salivary anti-SARS-CoV-2 Spike IgG antibodies via molecular mimicry. Anti-Spike IgG antibodies in the saliva correlated with enhanced abundance of Streptococcus salivarius 1 month after anti-SARS-CoV-2 vaccination. Several human commensal bacteria, including S. salivarius, were recognized by SARS-CoV-2-neutralizing monoclonal antibodies and induced cross-reactive anti-Spike antibodies in mice, facilitating SARS-CoV-2 clearance. A specific S. salivarius protein, RSSL-01370, contains regions with homology to the Spike receptor-binding domain, and immunization of mice with RSSL-01370 elicited anti-Spike IgG antibodies in the serum. Additionally, oral S. salivarius supplementation enhanced salivary anti-Spike antibodies in vaccinated individuals. Altogether, these data show that distinct species of the human microbiota can express molecular mimics of SARS-CoV-2 Spike protein, potentially enhancing protective immunity., Competing Interests: Declaration of interests Related to this work, Deutsches Rheuma-Forschungszentrum (DRFZ) has filed a patent application., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Short-read 16 S rRNA gene sequencing is the dominating technology to profile microbial communities in different habitats. Its uncontested taxonomic resolution paved the way for major contributions to the field. Sample measurement and analysis, that is, sequencing, is rather slow-in order of days. Alternatively, flow cytometry can be used to profile the microbiota of various sources within a few minutes per sample. To keep up with high measurement speed, we developed the open source-analyzing tool FlowSoFine. To validate the ability to distinguish microbial profiles, we examined human skin samples of three body sites (N = 3 × 54) with flow cytometry and 16 S rRNA gene amplicon sequencing. Confirmed by sequencing of the very same samples, body site was found to be significantly different by flow cytometry. For a proof-of-principle multidimensional approach, using stool samples of patients (N = 40) with/without inflammatory bowel diseases, we could discriminate the health status by their bacterial patterns. In conclusion, FlowSoFine enables the generation and comparison of cytometric fingerprints of microbial communities from different sources. The implemented interface supports the user through all analytical steps to work out the biological relevant signals from raw measurements to publication ready figures. Furthermore, we present flow cytometry as a valid method for skin microbiota analysis., (© 2022 The Authors. Cytometry Part A published by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry.)