Changes in Microbiota Profiles After Prolonged Frozen Storage of Stool Suspensions.

Stéphane Dorsaz,Vladimir Lazarevic,Stefano Leo,Myriam Girard,Benedikt Huttner,Nadia Gaïa,Jacques Schrenzel,Yannick Charretier,Stephan Harbarth

doi:10.3389/fcimb.2020.00077

Abstract

Introduction: Fecal microbiota transplantation (FMT) is recommended as safe and effective treatment for recurrent Clostridioides difficile infections. Freezing the FMT preparation simplifies the process, allowing a single stool sample to be used for multiple receivers and over an extended period of time. We aimed to assess the effect of long-term frozen storage on bacterial taxonomic profiles of a stool suspension prepared for FMT.Methods: DNA was extracted from a stool suspension before freezing and sequentially during the 18-month storage period at −80°C. Two different protocols were used for DNA extraction. The first relied on a classical mechanical and chemical cell disruption to extract both intra- and extracellular DNA; the second included specific pre-treatments aimed at removing free DNA and DNA from human and damaged bacterial cells. Taxonomic profiling of bacterial communities was performed by sequencing of V3–V4 16S rRNA gene amplicons.Results: Microbiota profiles obtained by whole DNA extraction procedure remained relatively stable during frozen storage. When DNA extraction procedure included specific pre-treatments, microbiota similarity between fresh and frozen samples progressively decreased with longer frozen storage times; notably, the abundance of Bacteroidetes decreased in a storage duration-dependent manner. The abundance of Firmicutes, the main butyrate producers in the colon, were not much affected by frozen storage for up to 1 year.Conclusion: Our data show that metataxonomic analysis of frozen stool suspensions subjected to specific pre-treatments prior to DNA extractions might provide an interesting indication of bacterial resistance to stress conditions and thus of chances of survival in FMT recipients.

Highlights

Fecal microbiota transplantation (FMT) is recommended as safe and effective treatment for recurrent Clostridioides difficile infections
Next-generation sequencing (NGS) techniques based on whole metagenome shotgun sequencing or on the sequencing of a taxonomic marker such as the 16S rRNA gene enable microbiologists to assess the composition of microbial communities at different levels of taxonomic hierarchy
A metataxonomic analysis confirmed the benefit of FMT in recovering “normal” microbiota in recurrent Clostridioides difficile infection (CDI) patients with dysbiotic lowered-diversity microbial communities (Song et al, 2013)

Summary

Introduction

Fecal microbiota transplantation (FMT) is recommended as safe and effective treatment for recurrent Clostridioides difficile infections. Freezing the FMT preparation simplifies the process, allowing a single stool sample to be used for multiple receivers and over an extended period of time. Fecal microbiota transplantation (FMT), consisting in the transfer of feces of a healthy donor to the gastrointestinal tract of a receiver, is recommended both by the European Society for Microbiology and Infectious Diseases, and the American College of Gastroenterology, as an efficacious alternative to standard antibiotic treatment for recurrent Clostridioides difficile infection (CDI) (Surawicz et al, 2013; van Nood et al, 2013; Debast et al, 2014). The impact of frozen storage— that may depend on stool processing techniques (Bahl et al, 2012; Burz et al, 2019; Papanicolas et al, 2019)—on the FMT efficacy for conditions other than CDI remains to be determined

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