New research on the timeline and mechanisms of C. diff infections

Protein structure of C. diff toxin B

Clostridia difficile infections are often the subject of this blog, but we rarely ever discuss how the infection actually occurs.  Scientists know that C. diff spores, which are not very uncommon in nature, can enter the gut from a variety of sources.  Once the spores reach the gut they normally just pass through unnoticed.  However, given the right conditions, these spores can take hold, germinate, and grow.  At some point during infection, the C. diff produces toxins which can compromise gut permeability (i.e. cause ‘leaky gut’) which leads to inflammation and all the nasty effects associated with the disease.  The exact gut conditions that trigger C. diff spore germination are not known, but scientists are convinced that the microbiome is involved because taking antibiotics, which wipe out the normal gut flora, make people susceptible to C. diff infection.  Some research has suggested that certain bugs in the microbiome outcompete C. diff for resources.  Other research shows that secondary bile acids, which are produced when the microbiome breaks down bile acids, inhibit C. diff germination.  Scientists are still working hard to understand the mechanisms of this infection, and just this week research out of the University of Michigan, published in Infection and Immunity, has shed new light on the process. 

The scientists first gave a group of mice antibiotics to make them susceptible to infection, and then fed the mice C. diff spores.  After, they euthanized mice every 6 hours to measure the progression of C. diff infection.  They learned that within 6 hours the spores had already germinated and entered the vegetative state in the feces and large intestine of the mice.  Over time, the C. diff progressed their way up the distal end of the large intestine all the way to the stomach, until the entire gastrointestinal (GI) tract was infected.  After 30 hours, sporulation of C. diff occurred, and interestingly this coincided with the production of C. diff toxins.  These toxins were found throughout the GI tract, however, inflammation only occurred in the large intestine, and not in the small intestine.  After 36 hours the infection had become severe enough that all animals were euthanized.

The scientists also measured the bacterial population and bile acid content of the gut during the infection.  After antibiotic treatment the microbiome was drastically altered and Lactobacillaceae flourished.  Once infection took hold the Lactobacillaceae were supplanted by C. diff in the large intestine, although the Lactobacillaceae still dominated the small intestine population, which, notably, did not become inflamed.  Secondary bile acids, which are produced by the microbiome and linked to C. diff germination, were abundant prior to antibiotics.  After antibiotic treatment, the large intestine had fewer secondary bile acids, and in the most infected regions had no detectable secondary bile acids.

This research is the first to develop a timeline for C. diff infection in mice, and strikingly it occurs very rapidly, with symptoms showing within 2 days.  This study also supports the notion that an altered microbiome is critical to C. diff infection, and that secondary bile acids may in fact play a crucial role in keeping C. diff from vegetating.  Interestingly, this study fits in well with a previous study we wrote about that showed the benefits of secondary bile acids in preventing C. diff infection.

Revisiting FMTs, and the patient that was cured of C. diff but became obese

Happy Valentine's Day to all our readers!  We will be back blogging on Tuesday, February 17, after we take off Monday, February 16 for President's Day.

Clostridia difficile infections can be nasty to deal with.  They cause pain and diarrhea, and are sometimes fatal.  They normally occur after a course of antibiotics, which leaves the gut in a state of dysbiosis where the C. diff can thrive.  Doctors normally prescribe antibiotics to cure this infection, but this can sometimes exacerbate the problem, making the gut even more prone to infection.  As we have discussed, fecal microbiome transplants (FMTs) have been successful in curing over 95% of C. diff infections.  Practically speaking, FMTs involve transferring the stool of a donor into the bowels of an infected patient.  While they are highly effective in treating C. diff, this practice is not without controversy.

The microbiome donor is generally a healthy person who is related to the patient and lives in the same household, generally a husband or wife.  The logic behind this is that these people share a similar microbiome, and some evidence supports this.  There are other ways to identify donors including the much publicized OpenBiome which has a stool repository which functions much like a blood or sperm bank.  These transplants come from ‘healthy’ strangers.  In most cases of FMTs, the stool is screened similarly to the way blood is screened, for specific diseases such as AIDS or hepatitis, and a few microbial pathogens (like C. diff).  The problem is, the microbiome is SO much more complex than blood, and as we learn every day on this blog, its impact on health and disease is not fully understood.  In fact, the promise of the microbiome is that it is connected with such far ranging diseases and phenotypes, from depression, to obesity, to arthritis.  We have numerous examples in mice where FMTs are actually able to transfer specific phenotypes, even unexpected ones such as anxiety.  What happens in humans though?  When we transplant feces between humans do phenotypes carry over?

Unfortunately, because the practice is mostly new, mostly unregulated, mostly isolated, and generally not a part of scientific studies, the long term impacts of FMTs are largely unknown.  The people who should and would know most about this, OpenBiome, have not published their findings, or at least are not talking about them.  We know that FMTs are really, really, good at curing C. diff, and may be the best solution to this debilitating disease, but at what cost is unknown, a classic bioethics dilemma.

Enter a healthy, 32 year old 136 pound woman from Rhode Island.  She had taken antibiotics for a vaginal infection and came down with a nasty C. diff infection which progressed over the course of a few months.  After antibiotics failed she opted for an FMT from her 16 year old, healthy daughter.  Fortunately, the FMT cleared the infection.  Unfortunately, over the ensuing year, the patient gained 34 pounds, and now weighs 170 pounds.  These are the kinds of results that make people nervous about FMTs.  We notice the weight gain because it is outward-facing and easy to measure, but what else has changed that we can’t notice, both physically and emotionally?  We need to be thinking about when we consider FMTs, especially when other, less complicated methods for treating C. diff are passing clinical trials.

FMTs exemplify both the promise and repercussions of the microbiome.  If the microbiome is as important and powerful as we think it is, then we need to investigate its clinical uses with deliberateness and care.

What microbes are living in public bathrooms?

Do you ever wonder how clean our bathrooms are, or question what kinds of bacterial communities are lurking in the public bathrooms you use? Well lucky for you and I, a team of scientists wondered the same exact thing. Published in Applied and Environmental Microbiology, the scientists sampled the microbial communities of four public restrooms at San Diego State University to better understand how they shifted over time.

At the beginning of the study, the restrooms were sterilized using a bleach solution.  Just one-hour post sterilization, the bathrooms were already filled with microbes again and as you can imagine, a significant portion of the microbes were of fecal origin.  They found that despite varying frequency of use and sampling bathrooms of both sexes, the four bathrooms all eventually had microbial communities that were very similar to one another.

The scientists found one specific bacterium, Staphylococcus, was prevalent in all the restrooms. One kind of staph can be very pathogenic, specifically when it is resistant to antibiotics (MRSA), however Staphylococcus does often live harmless in our bodies.  They did not find any Staphylococcus that was resistant to antibiotics in any of the restrooms. The restrooms were cleaned regularly using soap and water over the course of this study, yet the microbial communities remained largely stable.

You may read this and be grossed out about bacteria being prevalent in public restrooms even after regular cleaning, but they are most likely harmless, or even beneficial. Perhaps bleaching a bathroom may be like taking antibiotics - it leaves open the possibility for harmful bacteria to colonize, like what Clostridium difficile does in the gut.

Proton pump inhibitors may increase susceptibility to Clostridium difficile infection

Individuals who produce too much gastric acid in their stomach are often are prescribed proton pump inhibitors (PPIs), a group of drugs that prevent the production of acid for the treatment of ulcers, gastroesophogeal reflux disease (GERD), and other conditions related to acid production. Scientists at the Mayo Clinic in Arizona and Minnesota published a study last week in the journal Microbiome that found that the prolonged use of PPIs resulted in a reduction in gut microbiome diversity. The authors hypothesized that this reduced gut diversity is predisposing patients using PPIs to Clostridium difficile infection, an often-fatal bacterial infection that we have written about extensively on the blog.

Previous observational studies had shown a correlation between PPI usage and C. diff infection, however this study set out to identify what was actually causing this link.  The scientists took fecal samples from 9 healthy subjects before, during, and after they were given either a high or low-dose PPI treatment. They also took fecal samples from 5 patients with untreated C. diff infections and compared the bacteria in the fecal samples between the healthy patients and untreated patients infected with C. diff.

After taking PPIs, the healthy individuals’ gut microbiomes started looking very similar to those of patients with untreated C. diff. The number of bacterial species was significantly reduced from before they were exposed to PPIs, and the reduction was independent of dosage. This reduction of bacterial diversity in the gut doesn’t mean that patients on these medications will definitely become infected with C. diff, but it does likely predispose patients to the infection and more easily allow the bacteria to colonize the gut. 

The study found that after 28 days of PPI treatment the reduced gut diversity can be reversed, however it is not clear what prolonged PPI usage does to the gut microbiome.  This study included only a small number of subjects and future studies will hopefully be expanded and include patients who are prescribed PPIs for treating a specific condition. Better understanding the impact that PPIs have on the gut microbiome may also allow for the development of probiotics that could help counteract the effects of PPIs and help keep the gut microbiome diversity stable. 

A new probiotic candidate to treat C. diff

Molecular structure of the antibiotic enroflaxcin.

A brief letter was recently published in Nature that identifies a bacteria that may confer resistance to C. difficile.  In addition, they discovered how three commonly prescribed antibiotics alter a patient's risk for C. diff.  

The researchers treated mice with 3 different antibiotics, enrofloxacin, ampicillin, and clindamycin. While the overall microbiome bacterial density was unchanged for each antibiotic, each one altered C. diff susceptibility differently: enrofloxacin did not increase likelihood of getting infected, ampicillin induced transient susceptibility, and clindamycin greatly increased long-term chances of getting infected.

The researchers then identified 11 bacteria that were associated with C. diff resistance.  They  tested one of these bacteria, Clostridium scindens, on humans taking antibiotics that either already had C. diff infections or were susceptible for infection.  They discovered that the probiotic conferred substantial resistance to infection.  Interestingly, this probiotic also led to weight loss.

The researchers then studied how this bacteria could be preventing C. diff infection.  They discovered that this particular bacteria had a rare ability to break down bile into secondary structures, called secondary bile acids.  They tested these secondary bile acids against C. diff and they inhibited C. diff growth.

These results, taken collectively, may be immensely important in treating d. Diff.  Specific types of antibiotics that are known to not increase infection risk, along with probiotics like C. scindens could be combined into new therapies.  This could be important in treating this disease without more rudimentary approaches like fecal microbiota transfers (FMTs).

Can we prevent C. diff infections with other bacteria?

The microbiome of the mammalian gut helps protect the body from intestinal colonization of harmful pathogens. Antibiotic use can destroy the beneficial bacteria in our gut and allow for harmful bacteria to colonize it. Specifically, Clostridium difficile (C. diff) infection is a condition that is frequently seen in patients taking antibiotics and is often fatal.  A study published in Nature last week led by scientists at Memorial Sloan Kettering Cancer Center identified another bacteria, Clostridium scindens, that helped fight against C. diff infection. This study opens a new avenue to better predict what patients are at a higher risk of C. diff infection as well as the development of products that could prevent or even treat this condition.

A few weeks ago we wrote about an study published in the Journal of the American Medical Association that described the treatment of patients suffering from C. diff infection with a pill containing fecal material of healthy individuals.  The pill restored the microbiome to a healthy state and prevented future infection. However, little is known about what specific bacteria are responsible for resistance to infection. Why do some patients taking antibiotics get C. diff and others do not?

The scientists conducting this study identified 24 human patients undergoing allogeneic hematopoietic stem-cell transplantation, 12 who had C. diff infections and 12 who were C. diff carriers but were not infected after their transplant.  In the human study, as well as in mouse studies, they found that Clostridium scindens, an intestinal bacterium, is connected with resistance to C. diff infection. C. scindens produces an enzyme necessary for secondary bile acid synthesis, which was shown to be absent in the gut of patients infected with C. diff but present in recovered patients. This study suggests that it may be possible for doctors to better predict what patients are at a higher risk of C. diff infection by measuring the presence of C. scindens in the patient’s gut.  C. scindens could also be used in the development of preventative agents or therapeutics given to patients at higher risk or infected with C. diff.