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.

Microbiome link between cystic fibrosis and liver disease

Scientists have discovered a possible link between the microbiome and liver disease in individuals with cystic fibrosis.  Cystic fibrosis (CF) is a genetic disease that can be fatal and affects 30,000 people in the United States with another 1,000 diagnosed every year. While pulmonary disease is the most common cause of death in patients with CF, liver disease results in 2.5% of deaths and occurs in up to 72% of patients with the disease.  Approximately 5-7% of  these liver diseased CF patients have cirrhosis, a disease marked by the replacement of healthy liver tissue with scar tissue, preventing the liver from properly functioning.

A team of scientists from University of Colorado Medical Center recently published a study pertaining to CF patients with liver disease  in PLoS One. They studied 11 adolescents with CF and cirrhosis as well as 19 age-matched adolescents with CF without liver disease. They found that the two groups of patients had different gut microbiomes, leading the researchers to believe that there is communication between the gut bacteria and the liver, specifically in CF patients.

Patients with CF and cirrhosis had more severe lesions in their intestines than those without liver disease. Also, specific bacteria were less abundant in cirrhosis patients (e.g. Bacteroidetes) and others more abundant (e.g. Firmicutes) compared to CF patients without liver disease.    Similar ratios have been seen in other studies of liver disease and obesity.  However, we must remember that in complex diseases like CF, the microbiome is only one component of a very dynamic ailment, and at least one other disease study has measured the opposite Bacteroidetes/Firmicutes abundances.

Despite differences in previous studies and the lack of a mechanism relating the microbiome to cirrhosis, this identification of bacterial differences between CF patients with and without liver diseases is promising. The authors of the study point out that the sample size was small and this was only meant to be a pilot study but further longitudinal studies may now be warranted to investigate the development of cirrhosis in cystic fibrosis patients.

Bacteria on the skin help shape immune response

The skin is the largest organ of the human body and the first line of defense against harmful microorganisms in the environment. However, it is also home to trillions of microbes that are beneficial to the host individual. In a study published in Nature, scientists found that specific bacteria on mammalian skin influence the host immune response.

To better understand this relationship, researchers chose Staphylococcus epidermidis, a bacterium commonly found on human skin, to see how the bacterium shaped the immune response. Using mice, the researchers found that the presence of S. epidermidis on mice skin caused an increase in CD8 β+ T cells, cells that are involved in immune response.  The application of other common skin bacteria to mice resulted in the increase of different T cell populations. The scientists next investigated how the skin cells detected the presence of S. epidermidis. The results suggested that a specific type of dendritic cell – located not on the exterior epidermal layer of skin cell, but within the dermal, second layer of the skin – is the cause of the unique CD8 β+ T cell response.

While mechanisms are still unclear, it is possible that S. epidermidis produce specific proteins that can trigger an immune response within the human skin when exposed to skin pathogens. What is clear from this study is that different bacteria living on the skin can elicit different immune responses. This suggests a commensal or possibly mutualistic relationship between skin cells and certain bacteria. Further investigation and knowledge of this relationship could lead to better understanding of the immune system and how the human microbiome participates in immunity as well as how this can be translated into therapy.            

Research shows that the microbiome can control certain phenotypes

Structure of immunoglobulin A

Immunoglobulin A (IgA) is an antibody that is produced in the mucosal linings and is thought to play a critical role in maintaining the homeostasis between the body and the microbiome.  IgA deficiency has been related to celiac disease and people who suffer from this deficiency are prone to bacterial infections.  While studying IgA in mice, folks from Washington University, St. Louis noticed that IgA was not found in the feces of some mice, but was found in high levels in others.  They investigated this high/low fecal IgA phenotype and showed that it was directly related to the microbiome.  Their results were published last week in Nature.

The researchers began by doing various experiments between the high and low IgA mice.  They first noted that mothers would pass their IgA phenotype to all their offspring, showing the trait was vertically transmitted.  They then put high and low IgA mice in the same cages and learned that the low IgA trait was dominant, and high IgA mice would rapidly become low IgA mice.  In order to discover if a virus was responsible, the scientists filtered the feces of low IgA mice to remove any bacteria and then transferred it into high IgA mice.  These mice remained high in IgA, meaning that a bacteria, fungi, or other larger organisms were likely responsible. 

The scientists then began experimentation with antibiotics.  When broad spectrum antibiotics were given to low IgA mice it eliminated most of the bacteria in their gut.  When these mice were given fecal transplants from high IgA mice, they became high IgA mice.   This trait was also transferred to their progeny, and their children became high IgA mice.  In addition, when the antibiotic ampicillin was administered to low IgA mice, their feces became high in IgA.  Overall, these experiments led the scientists to believe that bacteria were responsible for the secreted IgA levels, and that ampicillin had the ability to kill whichever bacteria caused the low IgA phenotype. 

The scientists then performed genetic analysis on all of their mice's stools to see which bacteria were present in high and low IgA fecal samples.  There was one bacterial genus, Suterella, which was common to only low IgA mice.  When this bacteria was cultured and given to high IgA mice, it caused them to become low IgA mice.  Suterella apparently has the ability to confer the low IgA phenotype. (Interestingly, we had previously written about Suterella and its link to Down Syndrome and autism.)

Finally, the scientists studied the mechanism that could prevent IgA from being secreted in the low IgA mice, and they learned that it is likely the microbiome is both degrading IgA itself, and that it is degrading the proteins in the mucous responsible for secreting IgA.

Taken together these results show a very robust link between a specific phenotype and the microbiome.  Before this study, most relationships between phenotypes, such as obesity, and the microbiome were merely associations, rather than causative.  This study though, is crucial in that it shows secreted IgA levels can be directly caused by the microbiome, and there is a mechanism that explains the phenomenon.  

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.

Breastfeeding plays an important role in microbiome development

As soon as a child is born, his or her microbiome is continually being shaped by external factors such as diet and bacterial exposure. The first few years of life are critical in microbiome development as these early years will shape the composition of bacteria that will inhabit that individual’s body for years to come. Infant dietary habits play a critical role in this development.  Breast milk has high nutritional content and is important in passing immunological factors from mother to child, as well as nutrients that are essential for gut colonization by bacteria. A team of scientists led by a group at University of North Carolina School of Medicine published in Frontiers in Cellular and Infection Microbiology on early changes and development of the microbiome in infants with different feeding and daycare habits.

Stool samples were collected from nine infants, some of which were exclusively breastfed (EBF) and some of which were non-exclusively breastfed (non-EBF). The infants were followed over a period of time from 2 weeks to 14 months, and samples were collected before and after the introduction of solid foods. The samples were tested for differences in bacterial composition.

The scientists found that infants that were solely breastfed and did not receive formula had guts that were more prepared for the introduction of solid foods. When solid foods were introduced to their diets, the microbial shift was much less dramatic than the shift for infants who were breastfed while also receiving formula.

Analysis showed that non-EBF infants had greater microbiome species diversities compared to EBF infants. Also, non-EBF infants showed lower abundances of Bifidobacterium and greater abundances of Eggerthella compared to EBF infants. Bifidobacterium is a bacterium that is associated with good digestion. After introduction of solid foods, however, EBF infants showed an increase in Eggerthella abundance, and non-EBF infants showed an increase in Bifidobacterium abundance.

In a second part of the study, researchers considered day care attendance when comparing the microbiome of the infants. They found that attending daycare resulted in a more diverse microbiome, but feeding habits were the most important factor for microbiome composition after the introduction of solid foods. 

It is clear from this study, in addition to others  we have discussed, that there are many factors contributing to microbiome diversity and species richness. This study highlights the important role that diet plays on early microbiome development. What was quite interesting was that while many studies often equate a more diverse microbiome with health, the infants that were exclusively breastfed had less diverse microbiomes yet they were more prepared for the introduction of solid foods.  This study only included nine participants and should be expanded to include a greater number of infants to better understand this relationship, but it can still help inform the conversation around breastfeeding versus formula feeding.