Belotserkovsky, I. | Search bioRxiv

bioRxiv2020

Sorting and cultivation of Faecalibacterium prausnitzii from fecal samples using flow cytometry in anaerobic conditions

BackgroundThere is a growing interest in using gut commensal bacteria as next generation probiotics. However, this approach is still hampered by the fact that there are few or no strains available for specific species that are difficult to cultivate. Our objective was therefore to adapt flow cytometry and cell sorting to be able to detect, separate, isolate and cultivate new strains of Extremely Oxygen Sensitive (EOS) species from fecal material, focusing on Faecalibacterium prausnitzii as a proof-of-concept. ResultsA BD Influx(R) cell sorter was equipped with a glovebox that covers the sorting area. This box is flushed with nitrogen to deplete oxygen in the enclosure. Several non-specific staining methods including Wheat Germ Agglutinin (WGA), Vancomycin BODIPY and LIVE/DEAD BacLight were evaluated with three different strains of the EOS species F. prausnitzii. In parallel, we generated polyclonal antibodies directed against this species by immunizing rabbits with heat-inactivated bacteria. Anaerobic conditions were maintained during the full process, resulting in only minor viability loss during sorting and culture of unstained F. prausnitzii reference strains. In addition, staining solutions did not severely impact bacterial viability while allowing discrimination between groups of strains. Efficient detection was achieved using polyclonal antibodies directed against heat-fixed bacteria. Finally, we were able to detect, isolate and cultivate a variety of F. prausnitzii strains from healthy volunteers fecal samples using WGA staining and antibodies. These strains present markedly different phenotypes, thus confirming the heterogeneity of the species. ConclusionsCell-sorting in anaerobic conditions is a promising tool for the study of fecal microbiota. It gives the opportunity to quickly analyze microbial populations and to sort strains of interest using specific antibodies, thus opening new avenues for targeted culturomics experiments.

microbiology↗

bioRxiv2017

Identifying Parameters Of Host Cell Vulnerability During Salmonella Infection By Quantitative Image Analysis And Modeling

Salmonella target and enter epithelial cells at permissive entry sites: some cells are more likely to be infected than others. However the parameters that lead to host cell heterogeneity are not known. Here, we quantitatively characterized host cell \"vulnerability\" towards Salmonella infection based on imaged parameters. We performed successive infections of the same host cell population followed by automated high-throuput microscopy and observed that infected cells have higher probability of being re-infected. Establishing a predictive model we identified two combined origins of host cell vulnerability: the pathogen-induced cellular vulnerability emerging from Salmonella uptake and persisting at later stage of the infection, and the host cell-inherent vulnerability. We linked the host cell inherent vulnerability with its morphological attributes such as the local cell crowding, and with host cell cholesterol content. This showed that the probability of Salmonella infection success can be forecast from morphological or molecular host cell parameters.

microbiology↗