Comparison of microbial signatures between paired faecal and rectal biopsy samples from healthy volunteers using next-generation sequencing and culturomics

Abstract

Background Faecal samples are frequently used to characterise the gut microbiota in health and disease, yet there is considerable debate about how representative faecal bacterial profiles are of the overall gut community. A particular concern is whether bacterial populations associated with the gut mucosa are properly represented in faecal samples, since these communities are considered critical in the aetiology of gastrointestinal diseases. In this study we compared the profiles of the faecal and mucosal microbiota from ten healthy volunteers using bacterial culturing (culturomics) and next-generation sequencing targeting the 16S ribosomal nucleic acid (rRNA) gene. Paired fresh rectal biopsies and faecal samples were processed under stringent anaerobic conditions to maintain the viability of the bacteria. Four different sample types were analysed: faecal (F), faecal homogenised (FHg), biopsy tissue (B) and biopsy wash (BW) samples. Results There were no significant statistical differences in either bacterial richness or diversity between biopsy washes (BW) and faecal (F) or faecal homogenised (FHg) samples. Principal coordinates analysis of a Bray–Curtis distance matrix generated from sequence variant tables did not show distinct clustering between these samples (PERMANOVA; p = 0.972) but showed strong clustering of samples from individual donors. However, the rectal biopsy tissue (B) samples had a significantly altered bacterial signature with greater abundance of Proteobacteria and Acidobacteria compared to faecal (F) and faecal homogenised (FHg) samples. A total of 528 bacteria encompassing 92 distinct bacterial species were isolated and cultured from a subset of six volunteer samples (biopsy washes and faeces). This included isolation of 22 novel bacterial species. There was significant similarity between the bacterial species grown in anaerobic culture and those identified by 16S rRNA gene sequencing (Spearman correlation; rho = 0.548, p = 0.001). Conclusion This study showed that the bacterial profiles of paired faecal and rectal biopsy wash samples were very similar, validating the use of faecal samples as a convenient surrogate for rectal biopsy-associated microbiota. Anaerobic bacterial culture results showed similar taxonomic patterns to the amplicon sequence analysis disproving the dogma that culture-based methods do not reflect findings of molecular assessments of gut bacterial composition.

Dietary fibre supplementation enhances radiotherapy tumour control and alleviates intestinal radiation toxicity

“…The samples were self-collected by the patients using faecal collection paper and universal faecal collection containers, shortly before an inpatient/outpatient appointment and brought into the hospital or, in the case of 5 samples, obtained at surgery from the right colon. The samples were stored at 4 °C and prepared with no additives as described previousl y[ 85 ]. Briefly, the faecal sample (5 g) was weighed and mixed with 10 mL of PBS solution (supplemented with 30% glycerol).…”

“…DNA was extracted from the human faecal samples using the FastDNA Spin kit for soil (MP Biomedicals, UK) according to the manufacturer’s instructions as described previousl y[ 85 ]. DNA concentration was quantified by using Nanodrop (Nanodrop One C, Thermo Fisher Scientific, USA).…”

Butyrate-producing bacteria as probiotic supplement: beneficial effects on metabolism and modulation of behavior in an obesity mouse model

“…Four anaerobic bacteria from the Lachnospiraceae family (B1, B2, B3 and B4) had been previously isolated from faecal or biopsy samples of healthy volunteers (Mukhopadhya et al ., 2022), and were selected for this study based on their ability to produce >10 mmol/L butyric acid in YCFAGSC media. Each bacterium was grown in Hungate tubes containing 7 mL anaerobic YCFAGSC medium (Duncan et al ., 2002), for 16 hours at 37°C up to their maximum optical density at 600 nm with an average of 2.2±0.5, 1.0±0.3, 1.7±0.2 and 2.7±0.3 for B1, B2, B3 and B4, respectively.…”

“…Concentrations of short chain and branched chain fatty acids (SCFA and BCFA respectively) in cell culture supernatants were determined by gas chromatography as previously described (Mukhopadhya et al ., 2022, and references therein). Briefly, after derivatisation using N -tert-butyldimethylsilyl- N -methyltrifluoroacetamide, samples were analysed using a Hewlett Packard (Palo Alto, CA-USA) gas chromatograph fitted with a fused silica capillary column using helium as the carrier gas.…”

Benchmarking A Novel Quantitative PCR-based Microbiome Profiling Platform Against Sequencing-based Methods

“…A conservative approach to avoid inclusion of false positives, such practices have an impact on statistical analyses due to artifacts like zero-inflation, caused by the artificially high numbers of zeros, and can lead to misinterpretation of the data or superfluous conclusions about taxa abundance and presence Zhang et al (2016). As the monitoring of the gut microbiome is almost exclusively performed using stool samples and microbial signals are affected by the physical and chemical process of stool formation in the proximal colon, signals originating from upper and lower intestinal tracts may be regularly overlooked by sequencing methods with poor limits of detection (Mukhopadhya et al, 2022).…”