Processes 2019, 7,97
such as primary bile acids and nitrate play a particularly important role in shaping microbiota
abundances [12–17]. Unhealthy alterations of the gut microbiota are termed as dysbiosis and represent
imbalances in species abundances associated with diseases such as inflammatory bowel diseases,
Crohn’s disease, obesity, and diabetes [18–20].
The anaerobic bacterium Clostridium difficile is an opportunistic human pathogen responsible
for infections in the colon of the human gastrointestinal tract [21]. Various studies have reported
that 3%–15% of healthy adults are asymptomatically colonized with C. difficile [22–28]. Commensal
species in healthy gut usually provide resistance against C. difficile pathogenic colonization. C. difficile
infection (CDI) is most common in patients previously treated with broad spectrum antibiotics that
disrupt the healthy gut microbiota and reduce competition for available nutrients [29], resulting in
dysbiosis conducive to C. difficile propagation [30–33]. CDI symptoms can range from mild diarrhea
to severe and life-threatening colitis [21,34]. C. difficile virulence is attributable to the secretion of the
high molecular weight toxins A and B that promote epithelial tissue damage and rapid fluid loss.
Some C. difficile strains have developed resistance to common antibiotics while also exhibiting more
severe pathogenicity [35]. CDI has become particularly common in hospital settings due to the ability
of C. difficile to form spores that adhere to surfaces and resist common disinfectant protocols. Studies
estimate that almost 500,000 CDI cases occur within the U.S. annually [36], resulting in 29,000 deaths
and over $4.8 billion in associated costs in acute care facilities alone [37].
Numerous experimental studies have demonstrated that C. difficile [38–41] can form biofilms
in vitro. The other commensal bacteria [42,43] can form biofilms in vivo, which are well known to
exhibit phenotypes distinct from planktonic cultures. For example, bacteria in biofilms can tolerate
antimicrobial concentrations 10,000-times higher than the same bacteria grown planktonically, making
the development of effective treatment strategies a major challenge [44,45]. This difficulty is partially
attributable to the spatially-varying biofilm environment, which has profound effects on biofilm
development and function [46–48]. Mechanistic understanding of the relationships between biofilm
spatial variations, species–species interactions, and host–species interactions remains inadequate to
systematically analyze and rationally treat CDI [49]. To address these challenges, we added C. difficile to
our previous multispecies biofilm model [50,51] consisting of three representative species from the phyla
Bacteroidetes (Bacteroides thetaiotaomicron), Firmicutes (Faecalibacterium prausnitzii), and Proteobacteria
(Escherichia coli). Model simulations were performed to connect host-induced nutrient changes in the
gut environment with observed alternations of species abundances and SCFA levels [52–54] to unravel
the metabolic determinants of CDI.
2. Results
2.1. Discovery of Putative Byproduct Cross-Feeding Relationships
Our previous modeling study [50] without C. difficile generated three byproduct cross-feeding
relationships that were predicted to be necessary and sufficient for the coexistence of the three species:
B. thetaiotaomicron consumption of ethanol secreted by E. coli and F. prausnitzii consumption of acetate
and succinate secreted by B. thetaiotaomicron and E. coli. Preliminary flux balance analysis (FBA)
with the C. difficile reconstruction showed that acetate, butyrate, and propionate were the major
byproducts, and succinate and formate could be uptaken as carbon sources in the presence of glucose.
With this knowledge, the four-species biofilm model was analyzed to discover additional cross-feeding
relationships that support C. difficile coexistence with the three commensal species. Each species
was allowed to consume glucose, the eight amino acids, and any available byproduct (acetate, CO 2 ,
ethanol, formate, lactate, and succinate), assuming no differences in uptake kinetics across species
and byproducts (see Materials and Methods). Simulations with a biofilm thickness of 40 microns
and bulk concentrations of 8 mmol/L glucose and 0.5 mmol/L each amino acid at the biofilm-stool
interface corresponding to the healthy case (Table 1) were run for 300 h to ensure a steady-state
22
such as primary bile acids and nitrate play a particularly important role in shaping microbiota
abundances [12–17]. Unhealthy alterations of the gut microbiota are termed as dysbiosis and represent
imbalances in species abundances associated with diseases such as inflammatory bowel diseases,
Crohn’s disease, obesity, and diabetes [18–20].
The anaerobic bacterium Clostridium difficile is an opportunistic human pathogen responsible
for infections in the colon of the human gastrointestinal tract [21]. Various studies have reported
that 3%–15% of healthy adults are asymptomatically colonized with C. difficile [22–28]. Commensal
species in healthy gut usually provide resistance against C. difficile pathogenic colonization. C. difficile
infection (CDI) is most common in patients previously treated with broad spectrum antibiotics that
disrupt the healthy gut microbiota and reduce competition for available nutrients [29], resulting in
dysbiosis conducive to C. difficile propagation [30–33]. CDI symptoms can range from mild diarrhea
to severe and life-threatening colitis [21,34]. C. difficile virulence is attributable to the secretion of the
high molecular weight toxins A and B that promote epithelial tissue damage and rapid fluid loss.
Some C. difficile strains have developed resistance to common antibiotics while also exhibiting more
severe pathogenicity [35]. CDI has become particularly common in hospital settings due to the ability
of C. difficile to form spores that adhere to surfaces and resist common disinfectant protocols. Studies
estimate that almost 500,000 CDI cases occur within the U.S. annually [36], resulting in 29,000 deaths
and over $4.8 billion in associated costs in acute care facilities alone [37].
Numerous experimental studies have demonstrated that C. difficile [38–41] can form biofilms
in vitro. The other commensal bacteria [42,43] can form biofilms in vivo, which are well known to
exhibit phenotypes distinct from planktonic cultures. For example, bacteria in biofilms can tolerate
antimicrobial concentrations 10,000-times higher than the same bacteria grown planktonically, making
the development of effective treatment strategies a major challenge [44,45]. This difficulty is partially
attributable to the spatially-varying biofilm environment, which has profound effects on biofilm
development and function [46–48]. Mechanistic understanding of the relationships between biofilm
spatial variations, species–species interactions, and host–species interactions remains inadequate to
systematically analyze and rationally treat CDI [49]. To address these challenges, we added C. difficile to
our previous multispecies biofilm model [50,51] consisting of three representative species from the phyla
Bacteroidetes (Bacteroides thetaiotaomicron), Firmicutes (Faecalibacterium prausnitzii), and Proteobacteria
(Escherichia coli). Model simulations were performed to connect host-induced nutrient changes in the
gut environment with observed alternations of species abundances and SCFA levels [52–54] to unravel
the metabolic determinants of CDI.
2. Results
2.1. Discovery of Putative Byproduct Cross-Feeding Relationships
Our previous modeling study [50] without C. difficile generated three byproduct cross-feeding
relationships that were predicted to be necessary and sufficient for the coexistence of the three species:
B. thetaiotaomicron consumption of ethanol secreted by E. coli and F. prausnitzii consumption of acetate
and succinate secreted by B. thetaiotaomicron and E. coli. Preliminary flux balance analysis (FBA)
with the C. difficile reconstruction showed that acetate, butyrate, and propionate were the major
byproducts, and succinate and formate could be uptaken as carbon sources in the presence of glucose.
With this knowledge, the four-species biofilm model was analyzed to discover additional cross-feeding
relationships that support C. difficile coexistence with the three commensal species. Each species
was allowed to consume glucose, the eight amino acids, and any available byproduct (acetate, CO 2 ,
ethanol, formate, lactate, and succinate), assuming no differences in uptake kinetics across species
and byproducts (see Materials and Methods). Simulations with a biofilm thickness of 40 microns
and bulk concentrations of 8 mmol/L glucose and 0.5 mmol/L each amino acid at the biofilm-stool
interface corresponding to the healthy case (Table 1) were run for 300 h to ensure a steady-state
22