Design of synthetic human gut microbiota ecosystem model for screening potential prebiotics from phytochemicals
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Date
2022
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Sokoine University of Agriculture
Abstract
The human gut microbiota (GM) is a complex and diverse microbial population that is
integral to health maintenance. Mechanistic studies of human GM are limited due to its
complexity with of over 1,000 species of microorganisms. In this regard, synthetic microbial
communities with reduced complexity could be an alternative for GM studies. The main
objective of this study was to establish synthetic gut microbial communities for screening of
phytochemicals beneficial to gut flora important in the discovery of novel GM therapies.
Specifically, this study sought to: (i) investigate conditions for establishment of synthetic
gut microbial ecosystem, (ii) determine whether synthetic gut microbial ecosystems can
demonstrate prebiotic nature of phytochemicals, and lastly (iii) validate synthetic in vitro
gut microbial communities with an in vivo mice study. Firstly, GM key stone species were
obtained from Korean Collection for Type Cultures (KCTC) and American Type Culture
Collection (ATCC). Next, a suitable medium for re-creation of synthetic microbial
communities was sought amongst Chopped Meat Carbohydrate (CMC), Bryant and Burkey
(BB), Tryptic Soy Agar (TSA), Brain Heart Infusion (BHI), Reinforced Clostridium
Medium (RCM), and Gifu Anaerobic Medium (GAM). Majority of bacteria could grow
well in GAM, and therefore GAM medium was selected for this study. Mucin glycans were
supplemented in GAM at 2g/L or 4g/L then investigated if they could simulate synthetic
GM ecosystem. Specific genera like Bacteroides, Akkermansia, and Clostridium grew
significantly faster in mucin-supplemented GAM (p< 0.05, n=3). The synthetic gut
microbial community were then assembled and co-cultured in mucin supplemented GAM
followed by microbial community profiling and short-chain fatty acids quantitation. Co-
culture results showed that mucin stimulated beneficial bacteria such as Collinsella,
Bifidobacterium, Ruminococcus, and Lactobacillus compared to gut opportunistic pathogens
like Escherichia. Nevertheless, acetate proportion was higher in the mucin-supplemented consortia (p < 0.05, p < 0.001, n=9) and this was highly related with microbial composition
in this group. However, some bacteria were not represented in this set-up and this could be
attributed to the lack of a real mucosal layer that could be a niche and also a carbon source
for bacteria. In this regard, mucosal layer was incorporated using mucin agar-gel. Upon
analysis, specific bacterial genera like Roseburia, Bifidobacterium, Lactobacillus, and
Collinsella were able to colonize well in the solid mucin-agar component, while
Enterococcus, Veilonella, and Clostridium dominated in the liquid part (p < 0.05, p< 0.01,
n=5). Metabolic functional prediction in the two experimental setups showed that predicted
functional pathways were highly related to microbial composition. The second study applied
synthetic microbial communities cultivated in a mucosal simulated environment to test
effects of anthraquinone (phytochemical) on top essential gut bacteria. A growing body of
research shows that phytochemicals in plants can affect the composition of the GM. As a
result, the GM is a good candidate for nutritional interventions aimed at improving health.
The anthraquinones examined in this study include emodin, obtusifolin, obtusin, aurantio-
obtusin, chryso-obtusin, Gluco-aurantio-obtusin, and Gluco-obtusifolin. Single bacterial
isolates were cultivated with these compounds at 100 mM and growth was measured at
OD 600 for 18 to 24 h at 37 °C. Results showed emodin to stimulate beneficial bacteria
including Akkermansia, Clostridium, Roseburia, and Ruminococcus but inhibited major gut
enterotypes (Bacteroides and Prevotella) which are associated with gut inflammation.
Emodin also preserved high levels of beneficial anti-inflammatory gut bacteria isolates such
as Roseburia and Faecalibacterium while again inhibiting Bacteroides and Prevotella in co-
cultivation. To validate the synthetic microbial communities’ co-cultivation and its
application in GM-phytochemicals studies, an in vivo study was performed. Mice were
randomly divided into three groups: normal group, low emodin (10 mg·kg -1 ) group, and
high emodin (50 mg·kg -1 ) group. Emodin was administered by oral gavage for one week
followed by sample harvesting and microbial community profiling. Emodin stimulated the anti-inflammatory
bacteria
Roseburia
in
co-culture
as
in
mono-culture
and
Faecalibacterium in co-culture, these bacteria were not detected in vivo, but emodin
stimulated Clostridium and Ruminococcus in vivo (which are related to Roseburia and
Faecalibacterium). Nevertheless, emodin also induced anti-inflammatory immune cells,
which may correlate with its impact on specific gut bacteria in vivo. Gut ecosystem
simulation is an advance to mechanically understand the role of the GM in health and
diseases necessary in developing innovative GM-phytochemicals therapies. In conclusion,
this study has demonstrated that single bacteria culture, synthetic gut ecosystem and in vivo
mice experiment can be used for diverse microbiome research since they showed consistent
results for major GM changes. I recommend use of synthetic GM ecosystem model in rapid
identification of phytochemicals of therapeutic significance.
Description
Thesis
Keywords
Human gut, Microbiota, Potential prebiot, Phytochemicals, Microbial ecosystem