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

Autor: Cibulková I; Division of Gastroenterology, Department of Internal Medicine, Kralovske Vinohrady University Hospital, Prague, Czech Republic.; The Third Faculty of Medicine, Charles University, Prague, Czech Republic., Řehořová V; The Third Faculty of Medicine, Charles University, Prague, Czech Republic.; Department of Anaesthesia and Intensive Care Medicine, Kralovske Vinohrady University Hospital, Prague, Czech Republic., Wilhelm M; The Third Faculty of Medicine, Charles University, Prague, Czech Republic., Soukupová H; The Third Faculty of Medicine, Charles University, Prague, Czech Republic.; Department of Microbiology, Kralovske Vinohrady University Hospital, Prague, Czech Republic., Hajer J; Division of Gastroenterology, Department of Internal Medicine, Kralovske Vinohrady University Hospital, Prague, Czech Republic.; The Third Faculty of Medicine, Charles University, Prague, Czech Republic., Duška F; The Third Faculty of Medicine, Charles University, Prague, Czech Republic.; Department of Anaesthesia and Intensive Care Medicine, Kralovske Vinohrady University Hospital, Prague, Czech Republic., Daňková H; Department of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic., Cahová M; Department of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague, Czech Republic.
Jazyk: angličtina
Zdroj: Journal of clinical laboratory analysis [J Clin Lab Anal] 2024 Oct; Vol. 38 (19-20), pp. e25105. Date of Electronic Publication: 2024 Oct 03.
DOI: 10.1002/jcla.25105
Abstrakt: Background: Faecal 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.
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: Using 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%.
Conclusion: Bacterial 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.
(© 2024 The Author(s). Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)
Databáze: MEDLINE
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