van de Wiele, T. | bioRxiv Science

bioRxiv2025

An optimized flow cytometry sorting-sequencing workflow reduces storage, sorting and extraction bias in sorted microbial communities

Fluorescence-Activated Cell Sorting (FACS) followed by metataxonomics is commonly used to probe the composition of microbial subpopulations with a functionality of interest. Metataxonomic analysis of sorted low microbial biomass samples is prone to contamination and bias introduced during sorting and DNA extraction. To improve metataxonomic accuracy, the DNA yield obtained by the DNeasy(R) PowerSoil(R) Pro kit was first maximized. FACS-sequencing using the optimized DNA extraction method distorted the composition of sorted microbiota by introducing sheath fluid contaminants, termed the FACSome. Carefully selected controls allowed for FACSome characterization and subsequent in silico decontamination of PacBio and Illumina metataxonomic sequencing data. The optimized extraction and decontamination were validated in soil, water, feces, saliva, gut reactor and mock microbial communities. Metataxonomic profiles of non-selectively sorted and unsorted samples derived from these ecosystems did not perfectly match due to relic DNA depletion during FACS. A nuclease-based digest enhanced the depletion of spiked and naturally present relic DNA, which can interfere with function-driven FACS. Since FACS depleted relic DNA, the preservation of intact cells prior to sorting is essential for activity- or viability-targeted FACS. Intact cells were best preserved during long-term -80{degrees}C storage of fecal samples that were not amended with glycerol-1X Tris-EDTA cryoprotectant.

microbiology↗

bioRxiv2022

Real-Time Flow Cytometry to assess qualitative and quantitative responses of oral pathobionts during exposure to antiseptics

Antiseptics are widely used in oral healthcare to prevent or treat oral diseases, such as gingivitis and periodontitis. However, the incidence of bacteria being tolerant to standard antiseptics has sharply increased over the last few years. This stresses the urgency for surveillance against tolerant organisms, as well as the discovery of novel antimicrobials. Traditionally, susceptibility to antimicrobials is assessed by broth micro-dilution or disc diffusion assays, both of which are time-consuming, labor-intensive and provide limited information on the mode of action of the antimicrobials. The above-mentioned limitations highlight the need for the development of new methods to monitor and further understand antimicrobial susceptibility. Here, we used real-time flow cytometry, combined with membrane permeability staining, as a quick and sensitive technology to study the quantitative and qualitative response of two oral pathobionts to different concentrations of chlorhexidine, cetylpyridinium chloride or triclosan. Apart from the real-time monitoring of cell damage, we further applied a phenotypic fingerprint method to differentiate between the bacterial subpopulations that arose due to treatment. We quantified the pathobiont damage rate of different antiseptics at different concentrations within 15 minutes of exposure and identified the conditions under which the bacteria were most susceptible. Moreover, we detected species-specific and treatment-specific phenotypic subpopulations. This proves that real-time flow cytometry can provide information on the susceptibility of different microorganisms in a short time frame while differentiating between antiseptics and thus could be a valuable tool in the discovery of novel antimicrobial compounds while at the same time deciphering their mode of action.

microbiology↗