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

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Abstract
Background Faecal microbiota transplantation (FMT) is a developing therapy for disorders related to gut dysbiosis. Despite its growing application, standardized 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. Methods Bacterial 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. Results We refined the bacterial DNA staining protocol integrated with flow cytometry for 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 17%, ranging from 5.3% to 52.9%. Conversely, the membrane integrity approach yielded highly reproducible results, with a median coefficient of variance for viable cells of 0.9%, ranging from 8.5% to 0.04%. Conclusion Bacterial viability assessment using cultivation-dependent methods produces inconsistent outcomes. In contrast, the membrane integrity method offers robust and precise data, making it a viable option for routine faecal material evaluation in FMT.
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