Stalking Mycobacterium bovis in the total environment: FLOW-FISH & FACS to detect, quantify, and sort metabolically active and quiescent cells in complex matrices.

Mycobacterium bovis causes tuberculosis (TB) at the human-wildlife-livestock interface. Environmental persistence of M. bovis excreted by infected hosts may cause indirect transmission to other animals. However, methodological constrains hamper assessment of M. bovis viability and molecular signature in environmental matrices. In this work, an innovative, modular, and highly efficient single-cell workflow combining flow cytometry (FLOW), fluorescence in situ hybridization (FISH), and fluorescence-activated cell sorting (FACS) was developed, allowing detection, quantification, and sorting of viable and dormant M. bovis cells from environmental matrices. Validation with spiked water and sediments showed high efficiency (90%) of cell recovery, with high linearity between expected and observed results, both in cell viability evaluation (r2 =0.93) and FISH-labelled M. bovis cells quantification (r2 ≥0.96). The limit of detection was established at 105 cells/g of soil in the cell viability step and 102 cells/g of soil in the taxonomical labelling stage. Moreover, FACS efficiency attained noteworthy recovery yield (50%) and purity (60% viable cells; 70% taxonomically labelled M. bovis). This new methodology represents a huge step for M. bovis assessment outside the mammal host, offering the rapid quantification of M. bovis cell load and cell viability, including viable but non-culturable cells, and further downstream cell analyses after FACS. Subsequent environmental data integration with the clinical component will expand knowledge on transmission routes, promising new paths in TB research and an intervention tool to mitigate the underlying biohazard.

When FLOW-FISH met FACS: Combining multiparametric, dynamic approaches for microbial single-cell research in the total environment.

In environmental microbiology, the ability to assess, in a high-throughput way, single-cells within microbial communities is key to understand their heterogeneity. Fluorescence in situ hybridization (FISH) uses fluorescently labeled oligonucleotide probes to detect, identify, and quantify single cells of specific taxonomic groups. The combination of Flow Cytometry (FLOW) with FISH (FLOW-FISH) enables high-throughput quantification of complex whole cell populations, which when associated with fluorescence-activated cell sorting (FACS) enables sorting of target microorganisms. These sorted cells may be investigated in many ways, for instance opening new avenues for cytomics at a single-cell scale. In this review, an overview of FISH and FLOW methodologies is provided, addressing conventional methods, signal amplification approaches, common fluorophores for cell physiology parameters evaluation, and model variation techniques as well. The coupling of FLOW-FISH-FACS is explored in the context of different downstream applications of sorted cells. Current and emerging applications in environmental microbiology to outline the interactions and processes of complex microbial communities within soil, water, animal microbiota, polymicrobial biofilms, and food samples, are described.