Tag Archives: University of Michigan Medical School

Researchers Find Key Role of Excess Calcium In the Gut In C. difficile infections (CDI)

New research shows, it can’t make this last, crucial move without enough of a humble nutrient: calcium.

And that new knowledge about Clostridium difficile (a bacterium also known as “C. diff“) may lead to better treatment for the most vulnerable patients.

The discovery, made in research laboratories at the University of Michigan Medical School and the U.S. Food and Drug Administration, is published in the online journal PLoS Pathogens.

It helps solve a key mystery about C. diff: What triggers it to germinate, or break its dormancy, from its hard spore form when it reaches the gut.

Though the findings were made in mice, not humans, the researchers say the crucial role of calcium may help explain another mystery: Why some hospital patients and nursing home residents have a much higher risk of contracting C. diff infections and the resulting diarrhea that carries its spores out of the body.

That group includes people whose guts are flooded with extra calcium because they’re taking certain medications or supplements, have low levels of Vitamin D in their blood or have gut diseases that keep them from absorbing calcium.

The new discovery shows that C. diff can recognize this extra calcium, along with a substance called bile salt produced in the liver, to trigger its awakening and the breaking of its shell.

Previous research had suggested it couldn’t do this without another key component, an amino acid called glycine. But the new findings show calcium and the bile salt called taurochlorate alone are enough. Mouse gut contents that were depleted of gut calcium had a 90 percent lower rate of C. diff spore germination.

“These spores are like armored seeds, and they can pass through the gut’s acidic environment intact,” says Philip Hanna, Ph.D., senior author of the new paper and a professor of microbiology and immunology at U-M. “Much of the spore’s own weight is made of calcium, but we’ve shown that calcium from the gut can work with bile salts to trigger the enzyme needed to activate the spore and start the germination process.”

Ironically, the researchers say, one way to use this new knowledge in human patients might be to add even more calcium to the system.

That could awaken all the dormant C. diff spores in a patient’s gut at once, and make them vulnerable to antibiotics that can only kill the germinated form. That could also prevent the transmission of more spores through diarrhea to the patient’s room. That could slow or stop the cycle of transmission that could threaten them or other patients in the future.

Hanna’s graduate student, Travis Kochan, made a key observation that led to the discovery. He noted that the fluid “growth medium” that the researchers typically grow C. diff in for their studies had calcium in it. He realized this could artificially alter the results of their experiments about what caused C. diff spores to germinate.

So, he used a chemical to remove the calcium while leaving all the other nutrients that                  keep C. diff growing. The result: no new spore germination happened in the calcium-free growth medium.

FDA’s Center for Biologics Evaluation and Research conducted further research in laboratory dishes and in the guts of mice. FDA’s Paul Carlson, Ph.D., a former U-M research fellow, and fellow FDA scientists in his laboratory found that C. diff spores that were mutated so that glycine couldn’t act on them could still germinate and colonize mice. This suggested that calcium, and not glycine, was critical for this process.

Both mutant and regular forms of the bacteria could still activate an enzyme inside the C. diff spore that led the bacteria to start dissolving their hard shell. This released the store of calcium that the spore had been harboring inside itself, and increases the local level of the nutrient even further.

“These spores don’t want to germinate in the wrong place,” says Kochan, whose grandfather suffered from a severe C. diff infection which ultimately led to his death. “C. diff spores have specialized to germinate in the gut environment, especially in the environment of the small intestine, where calcium and the bile salt injection from the liver comes in.”

Hanna notes that the bile salt connection to C. diff spore germination was first discovered at U-M in 1982 by a team led by Ken Wilson, M.D.

Calcium and the gut

Certain ailments and treatments cause defects in calcium absorption, but are also risk factors for C. diff infections. For example, patients with vitamin D deficiency are five times more likely to get C. diff.

Medications aimed at calming acid reflux – such as proton pump inhibitors – and steroids can increase the amount of calcium in the gut. A Vitamin D deficiency can keep the body from reabsorbing calcium through the gut wall, allowing it to build up.

And people with inflammatory bowel diseases such as Crohn’s and colitis also have a harder time absorbing calcium from food through their gut walls.

Older adults are also often counseled to take calcium supplements to compensate for lower calcium levels and protect their bones from fracturing.

Hanna cautions that the new findings should not cause any patients to stop taking their medications or doctor-recommended supplements, or to start taking new ones. But he hopes to work with clinicians at U-M and beyond to test the new knowledge in a clinical setting. Meanwhile, he and Kochan and their FDA and U-M colleagues will continue to study C. diff germination in mice and look for ways to block the enzymes crucial to spore germination.


To read the article in its entirety – please click on the following link to be re-directed:


C. diff. Research and Development Community March 2015, Infection and Immunity. Dynamics and Establishment of Clostridium difficile Infection

Shared by Dr. David Cook, MS, PhD

C. diff. Research and Development Community:

March 2015

Review of: Konigsknecht MJ, CM Theriot, IL Bergin, CA Schumacher, PD Schloss and VB Young. 2015. Infection and Immunity. Dynamics and establishment of Clostridium difficile infection in the murine gastrointestinal tract. Vol 83 (3): pages 934-41.

In this paper from Vince Young’s lab at the University of Michigan Medical School,              Konigsknecht et al measure the early events associated with C. difficile infection in a mouse model. Investigators followed the germination, growth, toxin production and histopathology following infection with C. difficile strain VPI 10463 subsequent to a 5 day antibiotic treatment of cefoperazone in the drinking water. Strengths of the work include the sampling every 6 hours of the mouse GI tract–including stomach, small intestine, cecum and colon–over the first 36 hours post-infection. In this brief time span, the investigators observe evidence of bacterial germination and growth, initially in the cecum and large intestine but later spreading to all regions of the small intestine and stomach, with concomitant pathologic effects and mortality. Both spores and toxin are detectable by 24 hours post-infection, consistent with the observation that the transcriptional program associated with sporulation is also likely involved in toxin production. This observation suggests that disease caused by toxin requires a minimal titer in the GI tract, and is consistent with the observation in humans that some individuals are colonized by low levels of C. difficile without evidence of clinical symptoms.
By 30-36 hours post-infection, the levels of vegetative organisms and spores are comparable in stomach and cecum-colon, with lower amounts in the small intestine.Despite the (unexpectedly) high levels of C. difficile observed in stomach and small intestine, tissue damage in the mouse is confined to the cecum and colon, consistent with the site of C. difficile pathology in humans. The authors demonstrate that bile acid profiles are shifted away from detectable secondary bile acids. In addition, microbiota diversity is dramatically decreased in the colon, primarily as a result of the antibiotic regimen, to favor an abundance of Lactobacillus. Both of these observations are consistent with previously published results from the Young lab                   (Theriot et al, 2014. Nature Communications).

These results further refine our understanding of infection in the mouse model and will enable other researchers to make more precise use of the model in developing new therapies. It should be noted, however, that there are important differences between C. difficile infection and disease in mice and in humans. In the mouse, C. difficile infection leads to rapid mortality. In humans, disease is slower, more chronic due to relapse, and is fatal only in a minority of cases. The microbiome changes are also different. Depending on the antibiotic used, mice can become dominated by a single microbe unlike humans. In the present case, mice were completely dominated by Lactobacillus, a normal commensal in the mouse but one that is mostly absent in humans. The observation in the Konigsknecht study that C. difficile grows in the stomach of mice is also at odds with our understanding of C. difficile infection in humans. Despite these caveats, this is an important work that furthers the                                                                  science behind understanding C. difficile infection.