Dysfunctional Families: Clostridium scindens and Secondary Bile Acids Inhibit the Growth of Clostridium difficile

Abstract

C. difficile infection is a deadly disease that is influenced by the microbiome. in a recent article in Nature, Buffie et al. (2014) demonstrate that the ability of C. scindens to synthesize secondary bile acids is crucial to providing resistance to C. difficile infection.

Our overuse of antibiotics has led to numerous detrimental health outcomes, including an increasing number of C. difficile infections (CDIs). In the United States, more than 14,000 individuals die annually from CDIs (Dubberke, 2012), costing greater than $3 billion dollars annually. The most common treatments for CDI are antibiotics such as vancomycin, metronidazole, and fidaxomicin, but they do not eradicate C. difficile spores and can cause collateral damage by killing commensal bacteria that compete with and potentially inhibit the growth of C. difficile. Consequently, some individuals become refractory to antibiotics and have recurrent CDI. On a brighter note, the use of fecal microbiota transplants (FMTs) as a therapy has been very effective in the treatment of relapsing CDI (Sofi et al., 2013). Yet a major gap exists in our understanding of the interaction between the host-microbe relationship and the microbial factors that confer resistance and susceptibility to CDI, Buffie et al. (2014) address this issue in their recent Nature paper, using a combination of human, murine, and mathematical models to identify a specific bacterium and enzyme that confers resistance to antibiotic-induced CDI.

To recapitulate the effect of antibiotics associated with CDI in a murine model, Buffie et al. (2014) treated mice with antibiotics (clindamycin, ampicillin, or enro- floxacin) followed by a gavage challenge with C. difficile. Each antibiotic challenge not only induced a shift in the microbiome structure but also conferred differential resistance to C. difficile, with enrofloxacin offering lasting and complete protection and clindamycin offering minimal protection. Although beta diversity (between samples) gradually returned to that of an antibiotic-naive state, some animals that were resistant also had low alpha diversity (within sample), indicating that specific microbiota were necessary to maintain resistance. To identify these resistance-conferring species, the authors correlated species abundance with C. difficile resistance and found several candidates, including C. scindens. While C. scindens, a member of the family Lachnospiracea, is clearly a key player in the resistance against germination of C. difficile spores, others have shown that members of the Bacteroides and Ru- minococcaceae families are associated with CDI resistance in humans, of which Lachnospiracea and Ruminococcaceae are butyrate producers (Lawley et al., 2012; Schubert et al., 2014). C. scindens is also a member of the Clostridium XlVa clade, which has been shown to aid in the expansion of anti-inflammatory T_reg cells via butyrate production. Butyrate is the primary fuel for enterocytes and can resolve colitis (Furusawa et al., 2013), and loss of butyrate is also associated with CDI. Future studies will determine if changes in butyrate levels, T_reg cells, and other downstream inflammatory mediators contribute to resistance against C. difficile as the result of microbial and bile acid changes.

Although humans and mice share a similar microbial core, there are notable differences. With this limitation in mind, Buffie et al. (2014) employed the use of patients undergoing allogeneic hematopoietic stem-cell transplants, which included the use of antibiotics in some cases, to compare microbial changes and resistance to CDI. Using a novel network modeling approach, they were able to identify species with the highest positive correlation with resistance in both humans and mice, which included C. scindens. They tested the causal relationship between C. scindens and CD! resistance using antibiotic-pretreated mice inoculated with either a four-bacteria suspension, including C. scindens, identified from the resistant network, or C. scindens alone. Adoptive transfer with all four bacteria or C. scindens alone conferred improved survival over PBS control, reduced C. difficile growth, and greater weight gain without changes in alpha diversity.

Bacteria live not in isolation but in communities defined by their environment. These bacteria are influenced by one another as well as by the host. This point has been well documented in several model systems, which demonstrate that bacteria use small molecules such as bile acids to communicate and survive (Sofi et al., 2013). Bile acids in the cecum are deconjugated by microbial-derived bile salt hydrolases into primary bile acids such as cholate (Ridlon et al., 2006), which can stimulate the germination of C. difficile spores (Francis et al., 2013). Other bacterial species, including C. scindens, further modify the primary bile acids via 7α-dehydroxylation to secondary bile acids such as deoxycholic acid (Ridlon et al., 2006; Theriot et al.,2014). Though data have suggested that specific microbes can alter the concentration of these germinants, the specific host-microbial mechanism is not well understood. Based on the ability of C. scindens to synthesize secondary bile acids via expression of baiCD, Buffie and colleagues utilized the microbial abundance data from their model of CDI to infer abundance of this gene. They demonstrated that abundance of the secondary bile acid gene family was higher in resistant animals and correlated with presence of the bai operon. Furthermore, they showed that engraftment with C. scindens, alone or in combination with several other bacterial species, restored levels of a secondary bile acid (deoxy- cholic acid) that protects against CDI (Figure 1), indicating the importance of our microbiota in maintaining bile acid homeostasis and resistance to outgrowth of pathogenic bacteria. Importantly, Buffie et al. (2014) were able to demonstrate that C. scindens requires bile salts to confer resistance to CDI. These conclusions are also supported by data from Theriot et al. (2014), who show a decrease in secondary bile acids after antibiotic treatment in mice. Though deoxycholic acid is likely produced from the action of 7α-dehydroxylating enzyme of baiCD of C. scindens, it would be ideal to use baiCD mutant C. scindens or a closely related bacterium with low levels of 7α-dehydrox- ylating activity (Ridlon et al., 2010) to unequivocally demonstrate that this enzyme is essential for resistance to CDI.

Figure 1. S.scidens and Secondary Bile Acids Inhibit C.difficile Growth.

(Left) inthe absence of antibiotics, the gut microbial community is diverse and includes bacteria such as S.scindens that convert primary bile acids into secondary bile acids, whcih, in turn, inhibits the growth of C.diffile. (Right) Broad-spectrum antibiotics diminish the diversity of the gut microbial community. Loss of certain bacteria such as C.scindens results in reduced production of secondary bile acids and increased growth of C.difficile. Additionally, accumulation of primary bile acids leads to increased spore germination of C.difficile.

In summary, Buffie et al. (2014) demonstrate that an important mechanism of the microbiome is to control pathogens through bile acid homeostasis. Importantly, they show that the resistance- conferring bacterium C. scindens is present in both human and murine models of CDI, which could have important translational implications (Figure 1). For instance, CDI treatment could utilize narrow-spectrum antibiotics that spare C. scindens. Alternatively, C. scindens couid be restored by including it as one of the cultured bacteria used in synthetic FMTs. This probiotic method, which is already being used, prevents the heterogeneity of conventional FMTs and excludes harmful viruses and antibiotic- resistant bacteria. Overall, the current work demonstrates the power of the microbiome as a tool to identify novel mechanisms of disease that could be used to develop targeted antibiotics, probiotics, and small molecule treatments for gastro-intestinal diseases.