Bacterial viability in faecal transplants: Which bacteria survive?

Abstract

Background: The therapeutic potential of faecal microbiota transplantation (FMT) is under investigation for a range of inflammatory conditions. While mechanisms of benefit are poorly understood, most models rely on the viability of transplanted microbes. We hypothesised that protocols commonly used in the preparation of faecal transplants will substantially reduce the number, diversity and functional potential of viable microbes.

Methods: Stools from eight screened donors were processed under strict anaerobic conditions, in ambient air, and freeze-thawed. Propidium monoazide (PMA) sample treatment was combined with quantitative PCR, 16S rRNA gene amplicon sequencing and short-chain fatty acid (SCFA) analysis to define the viable microbiota composition and functional potential.

Findings: Approximately 50% of bacterial content of stool processed immediately under strict anaerobic conditions was non-viable. Homogenisation in ambient air or freeze-thaw reduced viability to 19% and 23% respectively. Processing of samples in ambient air resulted in up to 12-fold reductions in the abundance of important commensal taxa, including the highly butyrogenic species Faecalibacterium prausnitzii, Subdoligranulum variable, and Eubacterium hallii. The adverse impact of atmospheric oxygen exposure on the capacity of the transplanted microbiota to support SCFA biosynthesis was demonstrated by significantly reduced butyrate and acetate production by faecal slurries processed in ambient air. In contrast, while reducing overall levels of viable bacteria, freeze-thaw did not significantly alter viable microbiota composition.

Interpretation: The practice of preparing material for faecal transplantation in ambient air profoundly affects viable microbial content, disproportionately reducing the abundance of anaerobic commensals and the capacity for biosynthesis of important anti-inflammatory metabolites. FUND: This work was supported by the South Australian Health and Medical Research Institute. LP is supported by a scholarship from the Flinders Foundation. GR is supported by a Matthew Flinders Research Fellowship.

Keywords: Bacterial viability; Fecal microbiota transplantation; Propidium monoazide; qPCR.

Fig. 1

Proportion of bacteria determined to be viable using 16S rRNA gene qPCR in conjunction with PMA treatment. Proportion of viable cells was determined by dividing viable cells amplified in PMA-treated samples over total number of cells amplified in non-PMA treated control samples. Bacterial viability in faecal slurry was assessed after processing in fresh anerobic conditions (ANO₂), fresh aerobic conditions (O₂), after one cycle of freezing and thawing in anaerobically processed specimens (FT1) or after heat-killing (HK). (Bars depict mean ± SD of 8 donor faecal slurry samples *= p<0·05, **=p<0·01; paired t-test).

Fig. 2

Taxa richness of faecal microbiota transplant material from 8 donors as assessed by 16S rRNA gene amplicon sequencing with and without PMA treatment. Viable diversity (PMA treated group) was significantly lower than diversity observed in control specimens, even in samples processed immediately in anaerobic conditions (Fig. 2a ***=p<0·001, paired t-test). When comparing only viable diversity between samples processed in anaerobic conditions (ANO₂), in ambient air (O₂), or after one cycle of freezing and thawing in anaerobically processed specimens (FT1) there are significantly lower observed species in specimens processed O₂, whereas freeze-thawing of specimens did not significantly reduce diversity (Fig. 2b, box plot depicts median and IQR and error bars depict minimum to maximum values; *= p<0·05; Wilcoxon matched-pairs signed rank test).

Fig. 3

Change in the relative abundance of viable taxa after processing in ambient air (O₂ vs ANO₂) or after freeze-thawing (FT1 vs ANO₂). Light grey bars represent decreased relative abundance and dark grey bars represent increased relative abundance. Selected bacterial taxa were further assessed by qPCR (arrows). Only taxa with at least 2·5-fold change in relative abundance are depicted.

Fig. 4

Proportion of bacteria determined to be viable by specific qPCR assays. Proportion of viable cells was determined by dividing viable cells amplified in PMA-treated samples over total number of cells amplified in non-PMA treated control samples. Bacterial viability in faecal slurry was assessed after processing in fresh anaerobic conditions (ANO₂), fresh aerobic conditions (O₂), after one cycle of freezing and thawing in anaerobically processed specimens (FT1) or after heat-killing (HK). Box plot depicts median and IQR and error bars depict minimum to maximum values of faecal slurry samples from 8 individual donors. All significant comparisons are indicated by stars (*= p<0·05; **= p<0·01; ***= p<0·001; Wilcoxon matched-pairs signed rank test).

Fig. 5

Amplification of the butyryl-CoA:acetate CoA-transferase gene, the terminal enzyme of the central butyrate synthesis pathway of human gut microbiota, in fresh anaerobic conditions (ANO₂), fresh aerobic conditions (O₂), after one cycle of freezing and thawing in anaerobically processed specimens (FT1) or after heat-killing (HK) in PMA treated samples. Butyryl-coenzyme A(CoA) CoA transferase gene levels were measured relative to amplification in a 10-fold dilution series of neat faecal slurry (FS control). The dotted line represents limit of quantification of the assay. Box plots depict median and IQR and error bars depict minimum to maximum values of faecal slurry samples from 8 individual donors. Significant comparisons are indicated by stars. (*= p<0·05; **= p<0·01; ***= p<0·001; Wilcoxon matched-pairs signed rank test).

Fig. 6

Net production of butyrate (panel a) and acetate (panel b) following in-vitro fermentation of faecal slurries for FMT with high-amylose maize starch. Matching samples (n=8) were processed either under anaerobic conditions (ANO₂), or under aerobic conditions (O₂). Significant comparisons are indicated by stars. (*= p<0·05; **= p<0·01; Wilcoxon matched-pairs signed rank test).