Cryopreservation of stool samples altered the microbial viability quantitively and compositionally

Effect of in vitro cultivation on human gut microbiota composition using 16S rDNA amplicon sequencing and metabolomics approach

“…Bellali et al 61,62 reported that 28% of bacterial OTUs in the fresh faecal samples were non-viable and a large number of them represented anaerobically and not yet cultured bacteria. The viability of GM is greatly influenced by sampling and exposure to oxygen, freezing, and the time between the collection and transport to the laboratory 27,[61][62][63] . Recent studies have shown that the nonviable bacterial content of stool processed immediately under strictly anaerobic conditions can be up to 50% 27,28 .…”

Impact of cryoprotective agents on human gut microbes and in vitro stabilized artificial gut microbiota communities

“…In this context, cryopreservation at −80°C represents the most widely exploited protocol for long‐term bacterial storage as it avoids the mechanical, osmotic and oxidative stress to which bacteria are exposed when freeze‐drying is used as storage processing (Barzegari et al., 2014 ; Bircher, Geirnaert, et al., 2018 ; Bircher, Schwab, et al., 2018 ; Smirnova et al., 2019 ). However, since cryopreservation is also not exempt from causing lethal damage to bacterial cells, this storage method requires an accurate evaluation of the components with cryoprotective properties to be used to protect the bacterial biomass from ice formation (Biclot et al., 2022 ; Bircher, Geirnaert, et al., 2018 ; Bircher, Schwab, et al., 2018 ; Chen, Hu, et al., 2022 ; Chen, Huo, et al., 2022 ; Chen, Li, et al., 2022 ; Smirnova et al., 2019 ). In this context, different cryoprotective buffers were assayed for their ability to preserve the viability of 53 bacterial species representing common bacterial taxa of the human gut microbiota, as already pointed out in other studies that used bacterial species representing common taxa of the human gut microbiota (Table S1 ) (Tramontano et al., 2018 ; Zimmermann et al., 2019 ).…”

“…The only exception was represented by Segatella copri (recent reclassification of Prevotella copri ) (Hitch et al., 2022 ) which, although with a relative abundance of 41.70% and 9.19% in faecal samples M7 and M9, respectively, was not stabilized in the corresponding AGMs (Figure 3D ). Probably, its low tolerance to oxygen and its peculiar nutritional requirements make the cultivation and stabilization of Segatella copri particularly difficult in in vitro fermentation systems (Chen, Hu, et al., 2022 ; Chen, Huo, et al., 2022 ; Chen, Li, et al., 2022 ; Huang et al., 2021 ).…”

“…Furthermore, they allow a wide range of taxonomic combinations to always be available in the laboratory, as well as higher reproducibility of the experiments since artificial gut microbiota are not subject to the variations that, instead, occur over time in the microbial community of a donor (Bircher, Geirnaert, et al., 2018 ; Bircher, Schwab, et al., 2018 ; Silverman et al., 2018 ). However, the long‐term storage of the artificial gut microbiota is an essential prerequisite to ensure the viability of the incorporated species and, therefore, to maintain its desired and predetermined bacterial biodiversity (Biclot et al., 2022 ; Bircher, Geirnaert, et al., 2018 ; Bircher, Schwab, et al., 2018 ; Chen, Hu, et al., 2022 ; Chen, Huo, et al., 2022 ; Chen, Li, et al., 2022 ; Smirnova et al., 2019 ).…”

“…However, ice formation may cause lethal damage to bacterial cells, making the use of cryoprotective agents necessary (Bircher, Geirnaert, et al., 2018 ; Bircher, Schwab, et al., 2018 ; Smirnova et al., 2019 ). In this context, the effects of various cryoprotectants on bacterial cell viability and physiology during freezing, storage and thawing have been reported for pure cultures or faecal samples (Biclot et al., 2022 ; Bircher, Geirnaert, et al., 2018 ; Bircher, Schwab, et al., 2018 ; Chen, Hu, et al., 2022 ; Chen, Huo, et al., 2022 ; Chen, Li, et al., 2022 ; Fouhy et al., 2015 ; Li et al., 2023 ). However, there is still little knowledge regarding the type of protective matrices to be used to prevent cryoinjuries for various bacterial species that represent (typical) components of the human gut microbiota or for a mix of bacterial species forming an artificial gut microbiota that lacks the protective matrix naturally present in faecal samples.…”