Researchers have found the molecular signature of a healthy gut microbial community that keeps C. difficile in check


Antibiotics are known to upset the balance of bacteria in the intestinal tract.

In some cases, antibiotics can cause the bacterium Clostridium difficile (C. difficile) to overgrow wildly, causing diarrhea and, in severe cases, life-threatening intestinal inflammation.

Researchers at Washington University School of Medicine in St. Louis have found the molecular signature of a healthy gut microbial community, or microbiome – the kind of community that keeps C. difficile in check even in the aftermath of antibiotic treatment.

They also have identified a specific molecule produced when C. difficile is not lying dormant but is active and making toxins.

Together, the findings outline a set of molecular signs that indicate a person has – or is at risk for – diarrhea caused by C. difficile.

“Right now, we just accept that taking antibiotics raises the risk of C. diff infection,” said senior author Jeffrey P. Henderson, MD, Ph.D., an associate professor of medicine and of molecular microbiology. “

By analyzing the small molecules produced by the microbiome, we may be able to identify people at high risk for developing C. diff diarrhea.

We also may be able to use this type of analysis to screen potential donors for fecal transplants and make sure they are donating the kind of microbiome that can help keep C. diff under control.”

The findings are published Aug. 12 in The Journal of Clinical Investigation.

Every year, there are about 450,000 cases of C. difficile diarrhea in the U.S., and 29,000 deaths.

Henderson and colleagues – including first author and postdoctoral researcher John Robinson, Ph.D., and co-author Erik Dubberke, MD, a professor of medicine, both at Washington University; as well as co-author Peter Mucha, Ph.D., a professor of mathematics at the University of North Carolina at Chapel Hill – studied the microbiomes of people with and without C. difficile disease by analyzing their metabolomes, or the chemical compounds produced in the gut as human intestinal cells and microbes eat, breathe and interact with each other.

They were searching for a molecular signature that distinguishes a healthy gut from one prone to C. difficile disease.

The search was complicated by the fact that some people carry C. difficile in their gastrointestinal tracts with no ill effects.

But the bacteria also can produce two toxins that cause diarrhea and inflammation.

In the absence of these toxins, the bacteria are mostly harmless.

The researchers studied 186 hospitalized people with diarrhea, divided into three groups: a group with no C. difficile; a group with C. difficile but without toxins, which means they carried the bacteria but their symptoms were caused by something else; and a group with C. difficile and toxins, all of whom had C. difficile infections.

Using mass spectrometry, the researchers analyzed thousands of metabolic byproducts in stool samples from patients in all three groups.

In particular, they found high levels of a fatty acid called 4-methylpentanoic acid in the stool of people with C. difficile disease.

The fatty acid is produced when proteins are broken down for fuel using an unusual metabolic process.

Human cells don’t produce the fatty acid, and neither do most of the bacteria in the normal microbiome – except for C. difficile.

The presence or absence of the fatty acid in stool identified people with C. difficile disease with 92.8 percent accuracy.

Further, the researchers uncovered a pattern of molecules related to bile acid metabolism that was linked to protection against C. difficile disease.

Bile acids are produced by the liver to help with fat digestion, then absorbed into the intestine, where members of the microbiome chemically modify them.

The researchers identified a set of modified bile acids in people who did not carry the bacterium, or who carried it harmlessly, that was absent from people with C. difficile infections.

“These unusual bile acids may be fingerprints of people who are more resistant to C. difficile infection,” Henderson said.

“There seems to be a difference in the metabolism of bile acids by the microbiome that affects how likely people are to develop disease.”

The researchers now are working on identifying which bacteria in the microbiome are involved in producing the protective bile acids, and how a healthy microbiome keeps C. difficile restrained.

“We know that disruptions to the microbiome predispose some people to getting C. diff disease, but until now we haven’t known much about what these changes are and why they’re harmful,” Henderson said.

“Small molecules give us a direct readout of what the microbiome is doing.

This study provides some big clues as to how these antibiotic-resistant bacteria make people sick, and that could lead to better ways to identify, prevent or treat C. diff infections.”

The commensal gut microbiota promote human health by defending the gastrointestinal tract against colonization by pathogenic bacteria (1, 2).

Some of the factors that contribute to the protective role of the gut microbiota include competition for nutrients (3), adhesion sites (4, 5), production of antimicrobials to ward off pathogens (6, 7), and modulation of the host’s immune defense mechanisms against pathogens (8).

Consequently, disruption of the abundance and diversity of the gut microbiota leads to increased susceptibility and colonization of certain pathogens such as Clostridium difficile (8, 9).

C. difficile is the leading cause of hospital- and antibiotic-associated diarrhea worldwide. It is at the top of the list of pathogens designated an urgent public health threat by the U.S. Centers for Disease Control and Prevention. C. difficile is resistant to multiple antibiotics and overpopulates the colon after the protective gut microbiota have been altered by antibiotic therapy.

Following colonization, it produces toxins A and B that cause severe intestinal inflammation, diarrhea, and pseudomembranous colitis.

The accessory gene regulator 1 (Agr1) quorum signaling system regulates the production of these toxins (10, 11). This is mediated by the release of a cyclic autoinducing peptide (TI signal) by the growing C. difficile cells, which accumulates and activates toxin production upon reaching a threshold concentration.

Whether the TI signal interacts with the other gut microbiota was unknown until now. Moreover, C. difficile is unique among the enteric pathogens because of its characteristic propensity to persist in the gut and recur 1 to 4 weeks following treatment of the primary infection.

An estimated 20% to 30% of patients with primary C. difficile infection (CDI) experience recurrence within 2 weeks after completion of therapy, and these patients exhibit persistent dysbiosis (12, 13).

It is unclear why intestinal dysbiosis persists after CDI, which also promotes recurrent infections. While spore formation is important in CDI recurrence, we hypothesized that C. difficile may persist by direct manipulation of the intestinal microenvironment to hamper reconstitution of the gut microbiota following antibiotic-associated dysbiosis.

Indole is widely distributed in nature and a major component of various essential compounds known for their medicinal properties.

Analogs of indole are also utilized in various industrial applications such as dyes, plastics, flavor enhancers, vitamin supplements, agriculture, over-the-counter drugs, and perfumery.

Indole is mainly produced from tryptophan by certain gut microbiota using tryptophanase, an enzyme that hydrolyses tryptophan into indole, pyruvate, and ammonia (14).

Approximately 85 known genera of Gram-positive and Gram-negative bacteria of the phyla Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria, excluding C. difficile, produce tryptophanase (14).

Depending on the diet, fecal indole concentrations in healthy adults range from 0.30 to 6.64 mM (15). Indole can also be metabolized by certain bacteria to tryptamine or indole-3-acetic acid; the latter is further converted to 3-methyl indole (16).

Hepatocytes metabolize some indole analogs into indoxyl-3-sulfate.

Two other indole metabolites, indole-3-aldehyde and indole-3-propionic acid, are produced by the gut commensals Lactobacillus spp. (17) and Clostridium sporogenes (18), respectively.

In the bacterial kingdom, indole plays a signaling role and controls diverse physiological processes, such as antimicrobial response (19), biofilm formation (20, 21), motility (20), persister cell formation (22), plasmid stability (23), and virulence (24).

Indole also regulates interspecies communication and host cell invasion by non-indole-producing microbes such as Pseudomonas aeruginosa, Salmonella enterica, and Candida albicans (25,–28).

In the mammalian host, indole and other metabolites derived from tryptophan modulate inflammation in the gastrointestinal tract (29,–31) and support epithelial tight junction permeability (29).

Indole also acts on host tissues by enhancing barrier functions, maintaining intestinal homeostasis, increasing mucus production, decreasing the production of inflammatory markers, increasing the production of anti-inflammatory molecules, and increasing the expression of a wide variety of genes associated with the immune system (20, 30, 32, 33). Hence, indole and its derivatives play important roles in the pathophysiology of both eukaryotic and prokaryotic organisms.

In Escherichia coli, the expression of tryptophanase is regulated by a 3,144-bp tna operon comprising three genes, tnaL, tnaA, and tnaB. tnaA and tnaB are the major structural genes that encode tryptophanase and tryptophan permease, respectively (34,–36). Upstream of tnaA is tnaL, which encodes a 25-residue leader peptide.

The tna operon is under catabolic repression and regulates the use of tryptophan as carbon and nitrogen sources (37,–39).

Several species of both Gram-negative and Gram-positive bacteria have homologues of the tna operon genes and are known to produce indole (14).

Here, we demonstrate that C. difficile (a non-indole producer) induces high indole production among the indole-producing gastrointestinal bacteria, which may have significant consequences on the abundance and diversity of the bacterial communities in the colon.

More information: John I. Robinson et al. Metabolomic networks connect host-microbiome processes to human Clostridioides difficile infections, Journal of Clinical Investigation (2019). DOI: 10.1172/JCI126905

Journal information: Journal of Clinical Investigation
Provided by Washington University School of Medicine in St. Louis


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