Evaluating Bacterial Viability in Faecal Microbiota Transplantation: A Comparative Analysis of In Vitro Cultivation and Membrane Integrity Methods

BackgroundFaecal microbiota transplantation (FMT) is a developing therapy for disorders related to gut dysbiosis. Despite its growing application, standardised protocols for FMT filtrate preparation and quality assessment remain undeveloped. The viability of bacteria in the filtrate is crucial for FMT's efficacy and for validating protocol execution. We compared two methods-in vitro cultivation and membrane integrity assessment-for their accuracy, reproducibility and clinical applicability in measuring bacterial viability in frozen FMT stool filtrate.MethodsBacterial viability in stool filtrate was evaluated using (i) membrane integrity through fluorescent DNA staining with SYTO9 and propidium iodide, followed by flow cytometry and (ii) culturable bacteria counts (colony-forming units, CFU) under aerobic or anaerobic conditions.ResultsUsing different types of samples (pure bacterial culture, stool of germ-free and conventionally bred mice, native and heat-treated human stool), we refined the bacterial DNA staining protocol integrated with flow cytometry for assessment of bacterial viability in frozen human stool samples. Both the membrane integrity-based and cultivation-based methods exhibited significant variability in bacterial viability across different FMT filtrates, without correlation. The cultivation-based method showed a mean coefficient of variance of 30.3%, ranging from 7.4% to 60.1%. Conversely, the membrane integrity approach yielded more reproducible results, with a mean coefficient of variance for viable cells of 6.4% ranging from 0.2% to 18.2%.ConclusionBacterial viability assessment in stool filtrate using the membrane integrity method offers robust and precise data, making it a suitable option for faecal material evaluation in FMT. In contrast, the cultivation-dependent methods produce inconsistent outcomes. The viability of bacteria in the filtrate is crucial for FMT's efficacy and for validating protocol execution. We refined the bacterial DNA staining protocol integrated with flow cytometry for assessment of bacterial viability in stool samples and compared two methods (in vitro cultivation and membrane integrity assessment) for their accuracy, reproducibility and clinical applicability in measuring bacterial viability in frozen FMT stool filtrate.image