culturing fecal microbiota

This site has discussed a short chain fatty acid SCFA brew for appetite control. Culture flow time as well as intestinal transit times seem to have a bearing on the types of bacteria and their metabolites.

Adamberg and Adamberg culturing fecal bacteria

The study came out of the Center of Food and Fermentation Technologies, Tallinn, Estonia. While the fecal donations came from obese and normal children with a mean age about 10 years ± 3-4 years. The overweight group had a body mass index over the 95 percentile. Fecal sample slurry were pooled from NW and OW children. Apple pectin or arabinogalactan were the carbon sources. Porcine mucin served as a source of protein. [1]

Over the course of several hours the culture vesicle was sampled and diluted with medium.

Overweight kids have less lower abundance microbes

In table 1 of the Adamberg publication we see a trend.  The top eight most abundant species are statistically the same in in normal versus overweight children.  These bacteria are at least 2.55 of the total.  The we move to the to species less than 1% of the total that are more abundant in the feces of normal weight children.  Are the overweight children that way because they are eating more, exercising less, or a combination of the two?  If they are eating  more volume, there is more Vagus nerve stimulation.  If they are consuming more calories, more undigested food may reach the intestinal bacteria.  The role of physical activity in transit time might also contribute to microbial diversity in the opposite direction of what was observed in this study.  [1]

• Pseudomonadota is the new name for Proteobacteria. Pseudomonas is a genus this phylum. Members of this phylum are ubiquitous and metabolically versatile.
• Verrucomicrobiota is a phylum of Gram negative bacteria found in fresh water, soil, and feces. Akkermansia muciniphilia is a member of this phylum.
• Actinomycetota is a phylum of Gram positive bacteria. Bifidobacterium is a genus of gram-positive, nonmotile, often branched anaerobic bacteria. They are ubiquitous inhabitants of the gastrointestinal tract
• Bacteroidota (formerly Bacteroidetes) is a phylum composed of three large classes of Gram-negative, nonsporeforming, anaerobic or aerobic, and rod-shaped bacteria. Found in soil and water, this phylum in also found in feces, most notably the Bacteroides genus. animals. Clostridia is also a member of this phylum.
• Bacillota (formerly Firmicutes) are a phylum of gram positive bacteria whose members relative to Bacterioidota are greater in obese patients. See this review.
• Adamberg figure 3 has been split into two parts to make a point about anaerobic respiration.  Like H2O is the product of O2 being the terminal electron acceptor in cytochrome C oxidase of complex IV in aerobic respiration, H₂S is the product of SO₄ being the terminal electron acceptor in anaerobic respiration. 

Metabolic data

Adamberg figure 3 has been split into two parts to make a point about anaerobic respiration.  Like H2O is the product of O2 being the terminal electron acceptor in cytochrome C oxidase of complex IV in aerobic respiration, H₂S is the product of SO₄ being the terminal electron acceptor in anaerobic respiration. 

What does the dilution rate simulate? Fecal material is concentrated, not diluted as it moves through the colon. It is hard to conceive that chyme could arrive in say the transverse colon and dilute material there.

The Müller human study

The Müller study came out of the School of Nutrition and Translational Research in Metabolism, Maastricht University Medical Centre+, Maastricht, The Netherland. Participants were 48 healthy, normo glycemic lean-to-overweight Caucasian women and men aged 20–55 yr prior to participating in a human intervention study at the Maastricht University Medical Centre+ (MUMC+) aiming to improve colonic transit time by means of a prebiotic fiber supplementation. Colonic transit times were measured by following 10 radioopague tracers consumed each morning for six days. [2] Transit time of the ascending, descending, and sigmoid colon were correlated with microbial diversity and fecal short chain fatty acids.

What is amazing about this study is that it addressing the physiology of the potential of different transit times in different parts of the colon. May we also assume that mixing will be different in different regions and that the imixability will depend on how much water is absorbed and the action of the circular muscles?

Descending colonic transit correlates with microbiota α-diversity

Microbiota sequencing was successful for all 42 participants.   Increased α-diversity was significantly associated with longer descending colonic transit time but not to total colonic transit time, BSS, or stool frequency. These associations remained after correction for age and sex.   Technical details and absolute bacterial counts did not impact on α-diversity estimates.indices and gastrointestinal transit parameters in n = 42 participants. **P < 0.05. ASVs, amplicon sequencing variants; BSS, Bristol stoolFig. 2.Descending colonic transit is linked to microbial α-diversity in healthy humans. Spearman correlation heat map of microbiota α-diversity

Fig. 2. Descending colonic transit is linked to microbial α-diversity in healthy humans. Spearman correlation heat map of microbiota α-diversity indices and gastrointestinal transit parameters in n = 42 participants. **P < 0.05. ASVs, amplicon sequencing variants; BSS, Bristol stool  BSS stool consistency is a proxy for transit time.  Stool consistency may be a more accurate reflection of colonic water absorption and colonic peristaltic mixing.

Age, sex, diet, and stool consistency, but not colonic transit, are associated with microbiota variation.

Much of the contents were in pictorial graphs that were at times hard to read. UniFrac-based db-RDA showed that

• Age correlated mainly with the relative abundance of Prevotella 9 and Methanobrevibacter, and inversely with Bifidobacterium and two Blautia ASVs.
• sex, and
• Total fat intake was inversely correlated with the relative abundance of Bifidobacterium.
• Total protein intake and fecal calprotectin correlated with the presence of Blautia and Bifidobacterium ASVs.

BSS correlated with ASVs from the genus Faecalibacterium, Prevotella 9, and an unclassified member of the Lachnospiraceae family

Distal colonic transit is linked to microbial α-diversity and fermentation.  Microbiota composition was mostly explained by demographics, dietary fat, and protein intake instead of GI transit parameters in healthy participants.  These α-diversity estimates were not the results of technical covariates. 

• Nutrient availability may have been the key driver for this observation because a longer distal colonic transit time may be accompanied by a greater and/or prolonged depletion of fermentable carbohydrates (especially with Western diets low in dietary fiber) in the descending colon.
• Müller and coauthors speculated that carbohydrate depletion promotes diversification forcing remaining bacteria to switch to proteins that favor slower growers. [2]
• Colonic transit was much longer in the Müller study (median 72 h) compared with a previous study (median 16.5 h),
• Müller and coauthors discussed the plasma LBP and fecal calprotectin as systemic and local inflammatory markers being related to  microbiota variation.
• Lipid degradation by microbiota under the anoxic conditions of the gut is not well characterized…  Here we need to differentiate between carbohydrate fermentation that ends the glycolysis and anaerobic respiration of fatty acids, and maybe proteins, that involves acetyl CoA units entering the TCA cycle to generate reducing equivalents NADH and FADH that enter the electron transport chain with a terminal electron acceptor that is not O₂. i.e. anaerobic respiration.  Such conditions will intuitively increase microbial diversity. 

Plasma acetate, but not plasma butyrate or propionate, was negatively associated with descending colonic transit time.  SCFA absorption into the circulation is highest in the distal colon  according to sources referenced by Müller et al.  One explanation not given by these authors is that bacteria able to use the 3-5 carbon SCFA in anaerobic or minimal oxygen environments via the TCA cycle/electron transport chain was not discussed by these authors.  The presence of formate in the plasma would have addressed this hypothesis.

Terminal electron acceptors in anaerobic respiration

What are some examples of terminal electron acceptors in anerobic respiration?  Sulfate and nitrate are possibilities as is sulfur.  Sulfated mucin and sulfur containing amino acids methionine and cysteine are possible candidates.  This brings to mind the cysteine rich domain of mucin in the Adamberg study. [1] It is starting to appear that the “virtues” of bacterial diversity is a lie.  What are the further consequences if butyrate is favored over propionate as substrates for the TCA cycle?  Müller et al mentioned inflammation markers LBP in the plasma and calprotectin in the feces.  Do these markers indicate a proliferation of bacteria that can degrade mucin in the search for a source of electrons and a terminal electron acceptor?

References

1. Adamberg K, Adamberg S. Selection of fast and slow growing bacteria from fecal microbiota using continuous culture with changing dilution rate. Microb Ecol Health Dis. 2018 Nov 30;29(1):1549922 PMC free article
2. Müller M, Hermes GDA, Canfora EE, Smidt H, Masclee AAM, Zoetendal EG, Blaak EE.(2020) Distal colonic transit is linked to gut microbiota diversity and microbial fermentation in humans with slow colonic transit. Am J Physiol Gastrointest Liver Physiol. 2020 Feb 1;318(2):G361-G369. PMC free paper