Alcohol pretreatment of stools effect on culturomics

Abstract

Recent studies have used ethanol stool disinfection as a mean of promoting valuable species' cultivation in bacteriotherapy trials for Clostridium difficile infections (CDI) treatment with a particular focus on sporulating bacteria. Moreover, the culturomic approach has considerably enriched the repertoire of cultivable organisms in the human gut in recent years. This study aimed to apply this culturomic approach on fecal donor samples treated with ethanol disinfection to evidence potential beneficial microbes that could be used in bacteriotherapy trials for the treatment of CDI. Thereby, a total of 254 bacterial species were identified, 9 of which were novel. Of these, 242 have never been included in clinical trials for the treatment of CDIs, representing potential new candidates for bacteriotherapy trials. While non-sporulating species were nevertheless more affected by the ethanol pretreatment than sporulating species, the ethanol disinfection technique did not specifically select bacteria able to sporulate, as suggested by previous studies. Furthermore, some bacteria previously considered as potential candidates for bacteriotherapy have been lost after ethanol treatment. This study, while enriching the bacterial repertoire of the human intestine, would nevertheless require determining the exact contribution of each of species composing the bacterial consortia intended to be administered for CDI treatment.

Figure 1

(A) Classification in phylum of the 196 bacteria species of the 8 fresh stools of fecal transplant donors pretreated with ethanol. (B) Oxygen tolerance of the 196 bacteria species of the 8 fresh stools of fecal transplant donors pretreated with ethanol. (C) Classification in phylum of the 135 bacteria species of the 3 fecal infusions of fecal transplant donors pretreated with ethanol. (D) Oxygen tolerance of the 135 bacteria species of the 3 fecal infusions of fecal transplant donors pretreated with ethanol.

Figure 2

Venn diagram of the bacterial species obtained before and after ethanol disinfection: FS bf. OH = bacterial species obtained in the 8 fresh stools before ethanol disinfection; FI bf. OH = bacterial species obtained in the 3 fecal infusions before ethanol disinfection; FS af. OH = bacterial species obtained in the 8 fresh stools after ethanol disinfection; FI af. OH = bacterial species obtained in the 3 fecal infusions after ethanol disinfection.

Figure 3

Graphical representation of the mean impact of ethanol disinfection toward several bacterial genera. The mean impact was assessed by summing the number of samples for which the genus was gained and subtracting the number of samples for which it was lost. Each loss corresponds to the sum of the number of species belonging to this genus present before disinfection, but absent after disinfection. Each gain corresponds to the sum of the number of species belonging to this genus isolated absent before disinfection, but present after disinfection.

Figure 4

Graphical representation of the mean impact of ethanol disinfection on sporulated and non-sporulated species. Each point represents a species that has been classified as sporulated or non-sporulated. We have assessed the mean impact of ethanol disinfection for each species by summing the number of samples for which the species was gained and subtracting the number of samples for which it was lost. A gain corresponds to a species absent before disinfection, but recovered after disinfection, while a loss corresponds to a species present before disinfection, but absent after disinfection. Error bars are shown in green; p-value = 0.0003 with Mann-Whitney test.

Figure 5

Venn Diagram comparing bacterial strains known previously in bacteriotherapy with those obtained in this study; (A) Separate comparison of all bacterial species previously known against those of this study; two bacterial species are shared between all studies. These are: Escherichia coli and Bacteroides ovatus. (B) Grouped comparison of all bacterial species previously known in bacteriotherapy against those of this study; 12 bacterial species are shared between these two groups; these are: Clostridium ramosum, Enterococcus fecalis, Clostridium bifermentans, Escherichia coli, Collinsella aerofaciens, Clostridium innocuum, Bifidobacterium longum, Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides ovatus, Parabacteroides distasonis and Bifidobacterium adolescentis. (C) Grouped comparison of all previously known bacterial genera in bacteriotherapy against those of this study; 23 bacterial genera are shared between these two groups. These are genera Bifidobacterium, Streptococcus, Dorea, Terrisporobacter, Turicibacter, Ruminiclostridium, Enterococcus, Bacteroides, Parabacteroides, Anaerostipes, Hungatella, Clostridium, Anaerotruncus, Anaerofustis, Flavonifractor, Ruminococcus, Escherichia, Intestinibacter, Oscillibacter, Eubacterium, Collinsella, Lactobacillus and Blautia.

Figure 6

Graphical representation of the mean impact of ethanol disinfection on succinate producers and non-succinate producers. We have assessed the mean impact of ethanol disinfection for each species by summing the number of samples for which the species was gained and by subtracting the number of samples for which it was lost. A gain corresponds to a species absent before disinfection, but recovered after disinfection, while a loss corresponds to a species present before disinfection but absent after disinfection. Error bars are shown in green; p-value = 0.7693 with Mann-Whitney test. Error bars are shown in green; p-value = 0.7693 with Mann-Whitney test.