Processes 2019, 7,97
profiles for 60 microns (Figure S7) suggested that C. difficile spores might be formed in the upper half
of the biofilm where C. difficile was unable to sustain vegetative growth. Since such spores could
be activated by favorable nutrient conditions, the incorporation of C. difficile spore formation and
activation could be an interesting direction for future research. Overall, our results could help explain
the role of broad spectrum antibiotics during CDI, as antibiotics could be expected to reduce the
diversity and density of commensal bacteria that protect the gut from C. difficile expansion.
To gain insights into the internal pathway fluxes associated with the healthy and dysbiosis states,
we determined for each species the eight internal fluxes that varied the most between the healthy
(Figure 2) and C. difficile dysbiosis (Figure 5) states and identified the internal pathways associated with
each of these fluxes. Using simulation data from the stool-biofilm interface at 300 h, the most variable
fluxes were determined by computing for each individual flux the difference between the healthy and
dysbiosis values and scaling the result by the healthy value (Figure S8). Pathways associated with
amino acid metabolism were upregulated in B. thetaiotaomicron and C. difficile, demonstrating the ability
of these two species to take advantage of increased amino acid availability. Similarly, the internal flux
through the cysteine metabolism pathway was predicted to increase in E. coli. Most internal pathway
fluxes in F. prausnitzii were predicted to decrease, suggesting that the dysbiosis environment was
unfavorable for its growth, resulting in decreased abundance.
3. Discussion
The gut microbiota serve a broad array of important functions for the human host, including
providing colonization resistance to opportunistic pathogens. Unhealthy changes in the microbiota
composition, commonly termed dysbiosis, have been correlated to a wide variety of gut and metabolic
diseases including inflammatory bowel disease, Crohn’s disease, obesity, diabetes, and chronic gut
infections. The opportunistic gut pathogen Clostridium difficile has been estimated to asymptomatically
colonize 3%–15% of healthy adults [28]. A common cause of symptomatic C. difficile infection (CDI) is
the use of broad spectrum antibiotics, which induce dysbiosis by reducing the diversity and density
of gut commensal bacteria that provide resistance to C. difficile expansion [30–33,78]. Improved
understanding of the complex interactions between commensal species, C. difficile, the gut environment,
and the human host are needed to treat CDI more rationally.
To help unravel the metabolic determinants of C. difficile-associated dysbiosis, we developed a
multispecies biofilm model by combining genome-scale metabolic reconstruction of C. difficile [88]
and commensal species representing the three dominant phyla in the gut: Bacteroides thetaiotaomicron
(Bacteroidetes) [89], Faecalibacterium prausnitzii (Firmicutes) [90], and Escherichia coli (Proteobacteria) [91].
The chosen species are well-studied representatives of the most dominant phyla in the human
gut microbiome, and curated metabolic reconstructions of these species were available. While
our four-species model represented a substantial reduction in complexity compared to the actual
gut microbiota, the number of species and extracellular metabolites included were limited by
computational considerations. Community models with substantially more species and cross-fed
metabolites can be formulated and solved by neglecting spatial and temporal variations, as shown in
our recent study of the gut microbiota [92]. However, these assumptions are not appropriate for biofilm
simulations. Furthermore, our four-species model could be useful for designing in vitro systems for
experimentally testing model predictions.
While specific spatial organization of gut microbes is currently unknown, the structure likely
includes biofilm growth associated with host mucosa and epithelial tissue [93]. The literature provides
significant evidence to support the hypothesis that some gut microbes develop spatially-structured
multispecies biofilms [40,43]. We sought to understand how the commensal species could sublimate
C. difficile expansion and under what gut conditions colonization resistance could become compromised.
The biofilm model was tuned to represent a healthy state with species abundances and concentrations
of short-chain fatty acids (SCFAs; acetate, butyrate, propionate) consistent with experimental studies
for healthy individuals [5,57,59]. Because our model lacked an explicit description of the human
29
profiles for 60 microns (Figure S7) suggested that C. difficile spores might be formed in the upper half
of the biofilm where C. difficile was unable to sustain vegetative growth. Since such spores could
be activated by favorable nutrient conditions, the incorporation of C. difficile spore formation and
activation could be an interesting direction for future research. Overall, our results could help explain
the role of broad spectrum antibiotics during CDI, as antibiotics could be expected to reduce the
diversity and density of commensal bacteria that protect the gut from C. difficile expansion.
To gain insights into the internal pathway fluxes associated with the healthy and dysbiosis states,
we determined for each species the eight internal fluxes that varied the most between the healthy
(Figure 2) and C. difficile dysbiosis (Figure 5) states and identified the internal pathways associated with
each of these fluxes. Using simulation data from the stool-biofilm interface at 300 h, the most variable
fluxes were determined by computing for each individual flux the difference between the healthy and
dysbiosis values and scaling the result by the healthy value (Figure S8). Pathways associated with
amino acid metabolism were upregulated in B. thetaiotaomicron and C. difficile, demonstrating the ability
of these two species to take advantage of increased amino acid availability. Similarly, the internal flux
through the cysteine metabolism pathway was predicted to increase in E. coli. Most internal pathway
fluxes in F. prausnitzii were predicted to decrease, suggesting that the dysbiosis environment was
unfavorable for its growth, resulting in decreased abundance.
3. Discussion
The gut microbiota serve a broad array of important functions for the human host, including
providing colonization resistance to opportunistic pathogens. Unhealthy changes in the microbiota
composition, commonly termed dysbiosis, have been correlated to a wide variety of gut and metabolic
diseases including inflammatory bowel disease, Crohn’s disease, obesity, diabetes, and chronic gut
infections. The opportunistic gut pathogen Clostridium difficile has been estimated to asymptomatically
colonize 3%–15% of healthy adults [28]. A common cause of symptomatic C. difficile infection (CDI) is
the use of broad spectrum antibiotics, which induce dysbiosis by reducing the diversity and density
of gut commensal bacteria that provide resistance to C. difficile expansion [30–33,78]. Improved
understanding of the complex interactions between commensal species, C. difficile, the gut environment,
and the human host are needed to treat CDI more rationally.
To help unravel the metabolic determinants of C. difficile-associated dysbiosis, we developed a
multispecies biofilm model by combining genome-scale metabolic reconstruction of C. difficile [88]
and commensal species representing the three dominant phyla in the gut: Bacteroides thetaiotaomicron
(Bacteroidetes) [89], Faecalibacterium prausnitzii (Firmicutes) [90], and Escherichia coli (Proteobacteria) [91].
The chosen species are well-studied representatives of the most dominant phyla in the human
gut microbiome, and curated metabolic reconstructions of these species were available. While
our four-species model represented a substantial reduction in complexity compared to the actual
gut microbiota, the number of species and extracellular metabolites included were limited by
computational considerations. Community models with substantially more species and cross-fed
metabolites can be formulated and solved by neglecting spatial and temporal variations, as shown in
our recent study of the gut microbiota [92]. However, these assumptions are not appropriate for biofilm
simulations. Furthermore, our four-species model could be useful for designing in vitro systems for
experimentally testing model predictions.
While specific spatial organization of gut microbes is currently unknown, the structure likely
includes biofilm growth associated with host mucosa and epithelial tissue [93]. The literature provides
significant evidence to support the hypothesis that some gut microbes develop spatially-structured
multispecies biofilms [40,43]. We sought to understand how the commensal species could sublimate
C. difficile expansion and under what gut conditions colonization resistance could become compromised.
The biofilm model was tuned to represent a healthy state with species abundances and concentrations
of short-chain fatty acids (SCFAs; acetate, butyrate, propionate) consistent with experimental studies
for healthy individuals [5,57,59]. Because our model lacked an explicit description of the human
29