At Mikrobiomik we develop the therapeutic potential of the microbiome in the tangible form of new medicines.

Microbiota and microbiome

Human beings are complex ecosystems with an estimated 100 trillion bacteria, one quadrillion viruses, fungi I, parasites and archaea. Their numbers are tenfold the number of cells in the human body. SEE +

C. difficile

In humans this bacteria can be found as a “coloniser”, i.e. one that does not cause disease (asymptomatic) and forms part of our intestinal microbiota. When and how does it turn into an active pathogen? SEE +

Faecal Microbiota Transplantation (FMT)

FMT was first used in 1958, though there is some evidence of its use in China as far back as the fourth century. SEE +

Candidates for FMT

FMT has proven to be a highly effective technique for preventing the appearance of new episodes of recurrent C. difficile infection (CDI). SEE +

Microbiota and microbiome

Until recently little was known about the human microbiota because the microorganisms that comprise it were largely unknown, due to the difficulty of cultivating them. Technological advances in genetic material detection tools and bioinformatic analysis systems have enabled the microbiome to be characterised in terms of both what microorganisms compose it and the proportions in which they are found.

Microbiota

All the microorganisms that jointly inhabit a given ecosystem.

Microbiome

All the genomes (genetic material) of the microorganisms in an ecosystem.

“The intestinal microbiota is the focus of research into a broad range of chronic diseases.”

Human beings are complex ecosystems. It is estimated that the microorganisms that inhabit the human body include 100 trillion bacteria and a quadrillion viruses, fungi, parasites and archaea. The number of microorganisms is ten times greater than the number of cells and they code for 100 times more genes than the human genome.

Most of these microorganisms are located in our digestive tract and are essential for many of our physiological processes. The normal intestinal microbiota of an adult comprises between 500 and 1,000 different species of microorganisms, mostly belonging to the Bacteroidetes and Firmicutes phyla. Smaller quantities of Actinobacteria, Proteobacteria, Verrucomicrobia, Fusobacteria, Cyanobacteria and Spirochaetes, Archaea, fungi, protozoa, viruses and other microorganisms are also found there.

The microbiota changes with age

Recent studies indicate that colonisation by these microorganisms seems to begin even before birth, though it is during and shortly after birth that it occurs in the greatest quantity. Once extra-uterine life begins, their composition depends on various factors: in the early years the most influential factors are the type of delivery (caesarean section or vaginal), the type of feeding (breast milk or formula milk) and the introduction of solid foods.

Children under three have a less diverse microbiota, but interindividual variability is greater in this age group than at any other stage. Throughout childhood the composition becomes more stable, and by preadolescence it is similar to that of an adult, although qualitative differences persist until adulthood is reached.

The aging process causes immunosenescence (a decline in the immune system) and changes the composition of the intestinal microbiota. In people aged over 65 the number of Bacteroidetes and Enterobacteriaceae are observed to increase while the numbers of some bacteria belonging to the Bifidobacteria and Firmicutes phyla decrease.

In addition to changes derived from age, the establishment and composition of the microbiota are affected by various environmental factors such as diet, lifestyle, antibiotic use and hygiene measures. The populations of different countries have different intestinal microbiota compositions due to different genetic backgrounds, regional diets, cultural practices and hygienic-sanitary measures in each region.

The human microbiota has a broad functional repertoire ranging from structural, metabolic, protective and immunological functions to helping to modulate nervous system development.

 

Unsurprisingly, the intestinal microbiota is the focus of research in a wide range of chronic diseases including cancer and inflammatory, metabolic, cardiovascular, autoimmune, neurological and psychiatric diseases.

 

One of the diseases in which the role of faecal microbiota transplantation has been most extensively studied is recurrent C. difficile infection, where its use is already recognised and recommended in leading international therapeutic guidelines.

Functions of the human microbiota

The metabolic function of the microbiota plays a key role, and it has even been considered as a “metabolic organ”. It is involved in the synthesis of vitamins, amino acids and neurotransmitters, and in the absorption of electrolytes and minerals. It also regulates the metabolism of bile acids, ferments non-digestible carbohydrates and produces short-chain fatty acids. All this helps draw energy from the diet.

The microbiota also plays an essential role in the development of the immune system. This effect is particularly evident in the gastrointestinal tract. The microbiota exerts a powerful defensive effect against colonisation by pathogenic and opportunistic microorganisms, reinforcing the function of the intestinal barrier constituted by enterocytes and the surrounding mucus. It also helps to improve innate immunity by regulating the secretion and production of antimicrobial peptides.

Upsets in the intestinal microbiota and the adverse response to them on the part of the host are known as dysbiosis. One of the most important causes of intestinal dysbiosis is the use of antibiotics.

Recently, different diseases have been linked to changes in the intestinal microbiota. Intestinal microbiota dysbiosis combined with genetic and environmental factors can be a factor in the development of various diseases such as obesity, atopic dermatitis, asthma, celiac disease, diabetes, inflammatory bowel disease, irritable bowel syndrome, C. difficile infection, colorectal cancer, periodontitis and psoriasis, among others.

In this sense, the use of prebiotics, probiotics and faecal transplants as therapies to restore the balance of the intestinal microbiota is being studied as a way to prevent and/or treat these diseases.

C. difficile

Clostridium difficile (now renamed Clostridioides difficile) is a bacterium that is widespread in the environment.

Clostridium species are ubiquitous in nature: their main habitats are soil and the intestinal tracts of many animals, including humans. C. difficile has two forms. In its vegetative form it is highly sensitive to the presence of oxygen, while in its sporulated form it is highly resistant to heat and is able to survive in extreme conditions,

How does it produce disease?

The bacterium is frequently transmitted through contact with infected persons (symptomatic or asymptomatic) or through contact with contaminated objects (faecal-oral transmission). Most bacterial cells are deactivated in the stomach, but the sporulated forms of the bacterium survive and then germinate to their vegetative form in the small intestine. Upsets in the normal intestinal microbiota, usually caused by antibiotics or antineoplasic agents, enable C. difficile to establish itself, penetrate the mucosa and adhere to the surface of the intestinal epithelium.

Whether or not disease appears and if so how severe it is will depend on the competition exerted by the gut microbiota, on the toxin neutralising effects of the rest of the microbiota, on the toxigenic capacity and other virulence factors of the bacteria, and on factors related to the host such as age and immune system status.

Specifically, the mechanism by which the damage occurs is mediated by the production of toxins. C. difficile bacteria adhere to colon enterocytes and release toxins (usually toxins A and B). The toxins alter the structure and function of the cells and disrupt the bonds between cells, leading to an inflammatory response which in advanced stages forms characteristic pseudomembranes.

It is the main pathogen responsible for acquired diarrhoea in hospitalised patients, which is known as C. difficile infection (CDI), and accounts for 20% of antibiotic-associated diarrhoea and colitis. The incidence of CDI has been increasing, both in hospitalised patients and in the community. This is partly due to the appearance of hypervirulent strains, but also to improvements in diagnostic methods and the sometimes “non-rational” use of antibiotics.

What are its clinical signs and symptoms?

C. difficile can cause a wide variety of clinical conditions ranging from mild diarrhoea (the most common clinical manifestation), colitis or pseudomembranous colitis (a more serious form associated with the detection in an endoscopy of pseudomembranes that can affect any segment of the colon mucosa or even all of it) to severe, fulminant disease.

CDI presents in fulminant form in approximately 3% of patients, and is responsible for most serious complications, which include colon perforation, prolonged ileus, toxic megacolon and death. It is a systemic inflammatory syndrome that can include intense diffuse abdominal pain or lower quadrant abdominal pain, with or without diarrhoea, high fever, chills, hypotension, tachypnea and marked leukocytosis. It should be noted that diarrhoea may not be present in patients with severe CDI when paralytic ileus occurs. Patients with toxic megacolon have a dilated colon with symptoms of severe toxicity such as fever, chills, dehydration and marked leukocytosis. Mortality associated with toxic megacolon can be as high as 38%.

What is recurrent C. difficile infection or R-CDI?

There is no universally accepted definition of recurrent ICD (R-CDI) infection, but one of the most commonly used is the following: appearance of a new episode of CDI in a patient in whom a previous episode of CDI has been treated and cured within the past 8 weeks.

R-CDI occurs between 15% and 40% of cases and may appear days or weeks after the end of treatment; the symptoms may be similar to or more serious than those of the initial episode. It has been demonstrated that R-CDI induces increased suffering in patients and thus longer hospital stays, with the subsequent increase in hospital costs. In addition, patients with R-CDI can serve as a reservoir of infection that can lead to secondary cases in other vulnerable individuals.

The most frequently described risk factors for the development of R-CDI are the continuous use of predisposing CDI antibiotics, advanced age, hypoalbuminaemia, diabetes mellitus and the use of antacids. Moreover, one recurrence makes the appearance of further R-CDI more likely.

High rates of R-CDI are related to both the alteration of the intestinal microbiota that enables C. difficile to proliferate and produce its toxins and the patient’s inability to produce an adequate immune response.

How is it diagnosed?

CDI and R-CDI are diagnosed based on the clinical picture and the detection of the toxins produced, and less frequently via the findings of a colonoscopy. The diagnosis should always be made as quickly as possible so that the patient can be treated as soon as possible and isolated, since the ability of C. difficile to form spores makes it a highly transmissible pathogen.

Diarrhoeic stools provide suitable laboratory samples for CDI diagnosis. They should be delivered quickly to the laboratory. Except in rare cases, in which a patient has ileus (acute interruption of intestinal transit) without diarrhoea, rectal swab exudates are acceptable. The processing of a single sample from a patient at the beginning of a symptomatic episode is usually sufficient. It is not necessary to perform microbiological tests to confirm that a patient has healed if he/she no longer has symptoms.

How is CDI treated?

The first thing that must be assessed for the treatment of CDI is whether it is possible to withdraw the antibiotics that have favoured the overgrowth of this bacterium if they are still in use.

Once the patient has been assessed, the following is recommended depending on the severity and the risk of recurrence:

Until recently, metronidazole was recommended for mild cases, but this recommendation has been withdrawn by the Infectious Diseases Society of America (IDSA). For initial cases, the latest treatment guidelines recommend vancomycin taken orally for 10 days or fidaxomicin taken orally for 10 days. If these drugs are not available, metronidazole can be used as an alternative in mild cases only.

The first recurrent episode can be treated with vancomycin in a tapering regimen or with fidaxomicin for 10 days. In patients who have previously recurred, in addition to the aforementioned antibiotics faecal transplantation has proved to be a very effective option. Its use in this situation is recommended by the main international CDI treatment guidelines.

Recently the use of Bezlotoxumab, a monoclonal antibody, has been approved for the prevention of R-CDI in adult patients. However this treatment has the major drawbacks that it is highly expensive and can be administered only intravenously.

In humans this bacterium can be found as a “coloniser”, i.e. one that does not cause disease (asymptomatic) and forms part of our intestinal microbiota. C. difficile colonises more than 50% of children during the first few months of their lives. They remain asymptomatic despite the presence of large amounts of toxin. When the microbiota matures, the population of C. difficile decreases to negligible levels in adults (3-5%). The pathogen cannot establish itself in the intestine if the endogenous microbiota is conserved. In hospitalised patients its colonisation rate can be as high as 25-30%.

It is the main pathogen responsible for acquired diarrhoea in hospitalised patients, which is known as C. difficile infection (CDI), and accounts for 20% of antibiotic-associated diarrhoea and colitis. The incidence of CDI has been increasing, both in hospitalised patients and in the community. This is partly due to the appearance of hypervirulent strains, but also to improvements in diagnostic methods and the sometimes “non-rational” use of antibiotics.

Faecal Microbiota Transplantation
FMT

FMT consists of transferring intestinal microbiota via faecal matter from a healthy donor to the gastrointestinal tract of a patient to restore the intestinal microbiota balance.

FMT was first used to treat pseudomembranous colitis (which is produced by the C. difficile bacterium) in humans in Denver, USA in 1958. There is evidence that treatment of this type was used in humans in China as early as the fourth century. It is also used as a veterinary treatment, known as “transfaunation”.

More recently, this procedure has been performed in several countries, mainly for the treatment of recurrent C. difficile infection but also for inflammatory bowel disease, other gastrointestinal disorders and even non-gastrointestinal disorders such as diabetes.

Multiple clinical trials have shown FMT to be a safe, highly effective procedure for treating recurrent C. difficile infection. One of the best known trials is that published by Van Nood and colleagues in the prestigious New England Journal of Medicine: it showed that FMT was significantly more effective than vancomycin (standard antibiotic treatment) in treating recurrent C. difficile infection, with an overall resolution rate of 94%.

How does FMT take place?

First, it must be considered whether the patient (FMT recipient) is an appropriate candidate. In general, FMT is contraindicated in patients with severe comorbidities and those who are immunocompromised, though some patients with these characteristics have been treated with good results. For more details see the section “Candidates for FMT”.

Likewise, a strict selection of the stool donor must be made, using questionnaires similar to those used for blood donation. This serves to confirm that the donor is not affected by any of the following circumstances: receipt of live vaccines in the three months prior to donation, chemotherapy treatment in the last three months, risk factors for acquiring HIV, syphilis, hepatitis B, hepatitis C, prions or any neurological disease, active cancer or autoimmune disease, receipt of blood transfusions in a country other than Spain in the last 6 months, presence of gastrointestinal disease (inflammatory bowel disease, irritable bowel syndrome, constipation or chronic diarrhoea), use of antibiotics or any systemic immunosuppressive agent in the three months prior to donation.

In addition, numerous tests are performed on donor serum and faeces samples to rule out the presence of infectious diseases.

Once a suitable donor is selected, the faeces are processed in order to concentrate the bacteria present in the sample and eliminate impurities from it, usually by a dilution and homogenisation process followed by filtration and concentration. This solution undergoes a second processing to prepare it for the intended administration route.

The most frequent route is via colonoscopy, although it can also be administered by gastrointestinal tube or enemas, and more recently in the form of oral capsules.

Recent research has shown that FMT is no less effective when standardised frozen stool preparations are used. This has led to the development of so-called “stool banks”. In Spain, one of the first stool banks for fecal transplant was that of the Gregorio Marañón General Hospital in Madrid, where FMT is also provided in oral capsules.

 In 2014, researchers at Massachusetts General Hospital demonstrated that FMT administered in capsules is as effective as when administered by other routes. With these recent advances, FMT may become a widely used therapy option, and one that is patient-friendlier in terms of administering it to sufferers from R-CDI.

How is the recipient prepared prior to an FMT?

Patients must discontinue antibiotic treatment and other treatments that could interfere with the action of the FMT at least 24-48 hours before receiving an FMT.

The patient should have a light dinner the night before the FMT and fast until the FMT is received on the designated day.Iit is advisable to perform a blood test prior to transplantation.

In the case of administration by capsules, no further preparation is necessary. If the FMT is administered via colonoscopy the patient may also have to take an evacuant to prepare for the procedure. For administration by nasogastric, nasoduodenal or nasojejunal tube, an x-ray should be performed to ensure correct placement of the probe.

What is the recovery process after an FMT?

Recovery from FMT is usually complete a few hours after the procedure. Most patients return to their regular activity and diet within that time.

Depending on the route of administration, some patients may present with abdominal pain and transitory cramps (1-3 days), bloating, gas, disturbed bowel habit (constipation rather than diarrhoea) and low grade fever for no longer than 12 to 24 hours..

In general, most patients suffer less discomfort if the FMT is administered via capsules than via other routes of administration such as colonoscopy or nasogastric tube.

In 2014, researchers at Massachusetts General Hospital demonstrated that FMT administered in capsules is as effective as when administered by other routes (see research). With these recent advances, FMT may become a widely used therapy option.

Candidates for FMT

FMT has proven to be a very effective technique in preventing the appearance of new recurrent episodes of C. difficile infection (R-CDI).

Most international guidelines in various specialties related to CDI, such as those of the American Gastroenterology Association, the American Society of Infectious Diseases and the European Society of Clinical Microbiology and Infectious Diseases, recommend the use of FMT in the case of multiple CDI recurrences.

There are many pathologies in which the use of FMT is being assessed because of the potential benefits. They include inflammatory bowel disease, diabetes, obesity, autism, chronic fatigue syndrome, multiple sclerosis, fibromyalgia, metabolic syndrome, Parkinson’s disease and idiopathic thrombocytopenic purpura. However its use for these pathologies is not yet recognised.

Despite being a safe technique, FMT may be contraindicated in the following situations:
  • Patients with active enteritis due to Enterobacteria.
  • Patients with hereditary/primary immune disorders.
  • Immunodeficient or immunosuppressed patients due to a medical condition or medication.
  • Patients with HIV infection.
  • Patients with current or recent (<3 months) treatment with antineoplasic agents or any immunosuppressive medication including, but not limited to, monoclonal antibodies against B and T cells, anti-TNF agents, antimetabolites (azathioprine, 6-mercaptopurine), calcineurin inhibitors (tacrolimus, cyclosporine) and mycophenolate mofetil.
  • Patients with a history of severe (anaphylactic) food allergy.
  • Patients whose physical state is class IV and V according to the American Society of Anesthetists.
  • Patients suffering from an acute disease or fever on the day scheduled for the FMT.
With the incorporation of new formulations such as FMT capsules, the benefits of FMT can be extended to more people.

In this case the following must also be taken into account:

  • Gastroparesis (gastric emptying disturbance).
  • Known chronic aspiration.
  • Swallowing dysfunction.

However, the treating physician must evaluate each case individually and assess the risk/benefit ratio in each case