Information

Manipulating Gut flora in vivo


I am tasked with designing an in vivo experiment in humans that requires manipulating the gut flora in one sample group to gauge its effect. Please suggest ways of doing this besides administering antibiotics.

I am of the view that antibiotics may have unintended consequences and may be unethical; explain any opposing view.


I can't think of any way to start with an intestinal "blank slate", so to speak, except with antibiotics.

Perhaps it's possible to at least partially homogenize/standardize a sample group's biota with probiotics?

If it's ethics you're worried about, perhaps draw experimental subjects from a pool already being treated with antibiotics (although this, I suppose, introduces it's own biases?).


I'm no expert, but I read this, and I found it very intresting:

In its study, Henry Ford treated patients between May 2010 and June 2012 with a therapy called intestinal microbiota transplantation (IMT), using donated stool from a healthy family member.

Here are the results:

“More than 90 percent of the patients in our study were cured of their C.diff infection,” says Dr. Ramesh. “This treatment is a viable option for patients who are not responding to conventional treatment and who want to avoid surgery."

http://www.henryford.com/body.cfm?id=46335&action=detail&ref=1757


Gut Flora

Gut flora is a complex ecological system formed by indigenous prokaryotic and eukaryotic microbial cells in the digestive tracts. The number of microbes in the gastrointestinal tract of an adult male is about 100 trillion, which is approximately twice as large as that of our own cells ( Savage, 1977 ). Some species of microbes are beneficial to the host, because they fermentate dietary fiber into short-chain fatty acids that are then absorbed by the host. Others synthesize vitamin K, which cannot be synthesized by the host. Thus, we partly rely on gut flora to aid nutrition.

We also rely on gut flora to resist pathogens and educate our immune system ( Lecuit et al., 2001 Dethlefsen et al., 2007 ). Recently, correlations between the gut flora and various diseases have been investigated intensively. Obesity, for example, can be caused by a certain distribution of microbes’ populations. Correlation between colon cancer and gut flora is also clearly evident. Thus, changes in gut flora may cause or prevent diseases ( Costello et al., 2009 ). Recently, a novel type of treatment has been conducted in some clinical situations, in which gut flora of a patient is replaced by a healthy flora by a surgical operation.

Although the gut flora has diverse functions, its homeostasis inside the gastrointestinal tract is still largely unknown. Fig. 3.3.1 shows a typical distribution of bacteria in the gastrointestinal tract. We see that the number densities of cells change dramatically along the tract, and the increasing/decreasing tendencies are different among the species. The cell distribution changes with person to person, and even in the same person the distribution changes with time. Understanding and controlling the gut flora are important in terms of medicine however, no reliable mathematical model to predict the gut flora has been developed so far.

Fig. 3.3.1 . Distribution of bacteria in the gastrointestinal tract.

Reproduced from Tomotari Mitsuoka, 1978. Chounaisaikin_no_hanashi, Iwanami Shoten, Publishers.

Bacteria grow by taking up chemicals, such as nutrients, oxygen, or carbon dioxide. Those chemicals are carried by the flow in the intestine, diffused due to flow and Brownian motions, produced by cell activities, and absorbed by the intestinal wall. The flow in the intestine is generated by peristalsis of the wall combined with inlet and outlet boundary conditions. Thus, bacterial growth in the intestine are strongly influenced by transport phenomena, that is, mass transport of cells, mass transport of chemicals, and momentum transport in the intestine. Since these transport phenomena construct the base of mathematical modeling of the gut flora, we explain each transport phenomenon in the following.


Trends

Psychobiotics are beneficial bacteria (probiotics) or support for such bacteria (prebiotics) that influence bacteria–brain relationships.

Psychobiotics exert anxiolytic and antidepressant effects characterised by changes in emotional, cognitive, systemic, and neural indices. Bacteria–brain communication channels through which psychobiotics exert effects include the enteric nervous system and the immune system.

Current unknowns include dose-responses and long-term effects.

The definition of psychobiotics should be expanded to any exogenous influence whose effect on the brain is bacterially-mediated.

Psychobiotics were previously defined as live bacteria (probiotics) which, when ingested, confer mental health benefits through interactions with commensal gut bacteria. We expand this definition to encompass prebiotics, which enhance the growth of beneficial gut bacteria. We review probiotic and prebiotic effects on emotional, cognitive, systemic, and neural variables relevant to health and disease. We discuss gut–brain signalling mechanisms enabling psychobiotic effects, such as metabolite production. Overall, knowledge of how the microbiome responds to exogenous influence remains limited. We tabulate several important research questions and issues, exploration of which will generate both mechanistic insights and facilitate future psychobiotic development. We suggest the definition of psychobiotics be expanded beyond probiotics and prebiotics to include other means of influencing the microbiome.


How our gut microbiome changes over time

Researchers in the UK and Germany, together with other international collaborators, have investigated the evolution of bacteria in the human gut microbiome, asking how these microbes persist throughout our lifetime, and how they spread geographically. Credit: Aleksandra Krolik/EMBL

The human gut microbiome is a complex community of trillions of microbes that are constantly interacting with each other and our bodies. It supports our well-being, immune system, and mental health – but how is it sustained and how global is it? Researchers in the UK and Germany, together with other international collaborators, have investigated the evolution of bacteria in the human gut microbiome, asking how these microbes persist throughout our lifetime, and how they spread geographically.

Keeping a stable, healthy gut microbial population is mutually beneficial to us and the bacteria. In exchange for nutrition and a comfortable habitat, the microbe community returns the favour by providing us with health benefits, which we are now starting to understand. The results of the study will help to inform the development of tailored probiotics, which are live bacteria found in particular foods or supplements, as well as dietary or medical interventions, to treat gut disease and maintain a healthy gut microbiome.

“We know that certain microbes colonise our gut at birth, and some can live with us for decades. Yet, although studies have looked at individual microbe species, the mechanisms and scale of persistence in the microbiome as a whole haven’t been explored,” explains the lead author Falk Hildebrand. He started this work while a postdoctoral fellow at EMBL Heidelberg and now leads a research group at the Quadram Institute and Earlham Institute in the UK.

To examine this, a team of scientists used metagenomics to analyse the evolutionary strategies and persistence of different bacteria in the human gut microbiome. Metagenomics is the study of all of the genes from many different organisms in a population. In terms of the human gut microbiome, this process not only provides detailed information about the bacterial strains present but also indicates the enhancing capabilities of those different strains to keep the gut in good working order.

Based on the analysis of stool samples, the team re-examined metagenomes from over 2,000 adult and infant samples, including several from the same families. Almost hundred metagenomes were newly reported, but most came from previously published studies looking at microbiome changes over time, with each individual providing on average 2–3 samples several months apart.

The data was built into a diverse dataset of 5,278 metagenomes, which were probed to analyse patterns of persistence in the different types of bacteria and how these were influenced by common factors: age, family members, geographic region, and antibiotic usage.

“Our analysis shows that most strains of bacteria present in the adult microbiome are very persistent – with the chances of a strain persisting for at least a year being over 90%,” says Falk. “Some taxonomic groups were consistently highly persistent while others were consistently low persistent. In babies, however, the average persistence of bacterial strains dropped to 80%. This isn’t unexpected we know that especially in newborn babies there is an ongoing exchange of gut microbes.”

The researchers then went a step further and complemented the analysis on the persistence in individuals with the spreading of strains geographically.

“By looking into time series from individuals and family members and overlaying this with geographic information, ranging from household via city to country, we identified three groups of bacterial strains that show different dispersal strategies. This presented very different persistence patterns in the host, regional spreading, and the geographical distributions of hundreds of bacterial species,” says Peer Bork, Director of EMBL Heidelberg (Scientific Activities), who is last author on the publication.

Tenacious persistence

The first group, termed ‘tenacious’ bacteria, were the most persistent and well adapted for survival in the human gut. For example, these bacteria were able to survive by switching to different nutrition sources as the host moved through infancy and into adulthood. Tenacious bacteria, however, are the ones most likely to be lost from the microbiome following antibiotic use. If we have been carrying these bacteria in us since childhood, their loss may be permanent. This is a particular concern in relation to over- and misuse of antibiotics.

Another group referred to as ‘heredipersistent’ bacteria comprises strains that are ‘inherited’ and cluster within families. These have a lower persistence in childhood and a higher turnover rate, suggesting cycles of reinfection are key to their persistence in an individual. Genomic analysis showed that these bacteria tend to have genes allowing them to spread by spores, which would help transmission from, say, parent to child, but also across a family unit.

‘Spatiopersistent’ bacteria make up a third group. These cluster within geographic areas, but are not associated with families.

With much current interest in maintaining or manipulating the microbiome to promote health, the research team hopes their holistic exploration of the evolution of different persistence in gut microbes will lead to better, more well-informed clinical strategies.

For example, one-off interventions like faecal microbiota transplantation (FMT) may be suitable to introduce or even replace tenacious bacteria, but not bacteria that rely on reinfection. These might benefit more from probiotic-based therapies or dietary changes that, over time, alter the gut environment to favour their colonisation and persistence.

The new insights into the wide-ranging and potentially permanent damage antibiotics can do to the microbiome could also point to new strategies to mitigate these differing effects.

“Our study gave us a much better idea of which gut bacteria are closely associated with their host, and which are more prone to switch between hosts. This is important information to inform probiotics and many medical applications targeting the human gut microbiome,” concludes Peer.


Gut bacteria 'talk' to horse's cells to improve their athletic performance

A typical endurance horse (Arabian breed) presented for a gait test before the start of the race to check locomotor soundness. During an endurance competition (100-160 km), horses must undergo veterinary inspection at the end of each 20 to 40 km loop to check their recovery and capacity to continue the race under good health conditions. The energetic expenditure during an endurance race is very high, so an interesting model to show the functional relationship between the microbiota profile and energetic metabolism at the level of the mitochondria. Credit: Eric Barrey

A horse's gut microbiome communicates with its host by sending chemical signals to its cells, which has the effect of helping the horse to extend its energy output, finds a new study published in Frontiers in Molecular Biosciences. This exciting discovery paves the way for dietary supplements that could enhance equine athletic performance.

"We are one of the first to demonstrate that certain types of equine gut bacteria produce chemical signals that communicate with the mitochondria in the horse's cells that regulate and generate energy," says Eric Barrey, author of this study and the Integrative Biology and Equine Genetics team leader at the National Research Institute for Agriculture, Food and Environment, France. "We believe that metabolites—small molecules created by breaking down bigger molecules for food or growth—produced by these bacteria have the effect of delaying low blood sugar and inflammation in the cells, which in turn extends the horse's athletic performance."

Mitochondria, which can be briefly described as the energy provider of cells, have been shown in recent studies to be interdependent with gut bacteria. In fact, many diseases associated with mitochondrial dysfunction in humans, such as Parkinson's and Crohn's have been linked to changes in the gut microbiome in many previous studies.

"Studying horses is a good way to assess the link between gut bacteria and mitochondria, because the level of exercise, and thereby mitochondrial function, performed by a horse during an endurance race is similar to that of a human marathon runner," explains Dr. Nuria Mach, first author of this paper, also based at the National Research Institute for Agriculture, Food and Environment, France.

She continues, "For this study we gained permission for veterinary doctors to take blood samples from 20 healthy horses of similar age and performance level, at the start and end of the International Endurance Competition of Fontainebleau, an 8-hour horse race in France. These samples provided information about the chemical signals and expression of specific genes, which is the process by which DNA is converted into instructions for making proteins or other molecules. To understand the composition of the horse's gut bacteria metabolites, we obtained fecal samples at the start of the race."

The researchers found that certain bacteria in the gut were linked to the expression of genes by the mitochondria in the cells. Furthermore, the genes that were expressed, or "turned on", were linked to activities in the cell that helped it to adapt to energetic metabolism.

Evolutionary explanation

"Interestingly, mitochondria have a bacterial origin—it is thought they formed a symbiotic relationship with other components to form the first cell. This may explain why mitochondria have this line of communication with gut bacteria," says Barrey.

Mach concludes, "Improving our understanding of the intercommunication between the horse and the gut microbiome could help enhance their individual performance, as well as the method by which they are trained and dietary composition intake. Manipulating the gut microbiota with probiotic supplements as well as prebiotics, to feed the good bacteria, could be a way for increasing the health and balance of the microbiome and horses, to better sustain endurance exercise."


Could Manipulating the Microbiome Treat Food Allergies?

Jef Akst
Jul 9, 2019

ABOVE: THE SCIENTIST STAFF

U niversity of Chicago immunologist Cathryn Nagler began to suspect that the body’s resident bacteria play a role in food allergies almost two decades ago. A handful of studies of germfree mice in the 1980s and ’90s had suggested that bacteria in the gut, or compounds they produce, such as lipopolysaccharide (LPS), are important in teaching the immune system not to overreact to the foods we eat. But it was a new mouse model of peanut allergy, developed by researchers at Mount Sinai School of Medicine in New York in 2000, that really made Nagler think about whether the gut microbiome might be involved in how humans respond to dietary antigens.

The mouse strain they used, C3H/HeJ, carried a mutation in the toll-like receptor 4 (TLR4). This protein had recently been shown to mediate immune responses triggered by a bacterial antigen known as lipopolysaccharide (LPS), and the mutant mice were consequently nonresponsive to LPS. But according to the 2000 paper, the animals also exhibited anaphylaxis—a sometimes fatal allergic reaction in people—upon exposure to freshly ground peanuts.

It made Nagler wonder if TLR4—and specifically, the propensity of certain gut bacteria to activate it—was the key to tolerance to dietary antigens. Sure enough, when she treated mice with broad-spectrum antibiotics to deplete their intestinal bacteria, even animals with wildtype TLR4 had severe reactions to food allergens. “That established a role of signaling by bacteria in the gut in regulating responses to food,” she says. “And then all of the studies we’ve done since then, over 15 years, have built on that.”

The fact that food allergies have been rising in recent decades really implores us to try to figure out as much as possible what could be contributing to this.

These days, there is little doubt that the body’s resident bacteria have a big say in how the immune system responds to food allergens. Research into the underlying causes of food allergies has blossomed to parallel the condition’s growing prevalence: an estimated 6 percent of children and up to 10 percent of adults in the US have an allergy to some food. Scientists have identified connections between a person’s microbial makeup and whether or not that person has a food allergy. Microbiome differences also help determine which children will outgrow their food allergies and which won’t, notes Supinda Bunyavanich, a physician scientist at Icahn School of Medicine at Mount Sinai. “So it suggests that there is an impact of these microbiota on the clinical outcomes.”

Further research in mice has demonstrated a causal relationship between the microbiome and allergic reactions to food. In January, Nagler and her colleagues published the results of an experiment in which they transferred fecal samples from healthy human infants and from infants with cow’s milk allergy to germfree mice. Control animals that did not receive a fecal transplant, as well as mice that received samples from the allergic babies, became sensitized to the milk protein β-lactoglobulin, developing an allergic response upon repeated exposure to the protein. Mice that had received transplants from healthy infants, on the other hand, tolerated the dietary antigen without any issues.

Exploring the microbiomes of the mice, the researchers identified one particular bacterial species, Anaerostipes caccae, that was significantly reduced in rodents that demonstrated an allergic response to cow’s milk. The team also showed that transferring this species “to germfree mice is sufficient to protect against an allergic response to” cow’s milk, Nagler says.

In June, researchers at Boston Children’s Hospital got similar results with a different allergen in a different mouse model. The team found that transplanting fecal material from healthy human babies into their own mouse model of severe egg allergy protected the animals against anaphylaxis, whereas a transplant of fecal material from babies with food allergies provided no such protection. Moreover, they found that feeding allergic mice a consortium of Clostridium or Bacteroides species or even the single species Subdoligranulum variabile was sufficient to provide protection against egg allergy.

“They’re not exactly the same data, but they seem to be very consistent,” study coauthor Rima Rachid, a clinical researcher at Boston Children’s, says of her results and those of Nagler’s group. “We’re very happy, because the science is being validated.”

The mechanisms underlying the bacteria’s effect on response to food allergens appear to be multifaceted. In Rachid’s recent study, the researchers found that the microbes somehow trigger the formation of a type of regulatory T cell called retinoid-related orphan receptor gamma (RoRγ) T cells. If the investigators removed these T cells from their mouse model, the animals had severe reactions to allergen exposure, even after transplants of the protective bacteria. And a study published in May found that germfree or antibiotic-treated mice develop a different type of regulatory T cell that cause elevated level of immunoglobulin E, an antibody that is known to mediate food-allergic reactions. Other work has pointed to a possible role for basophils, immune cells involved in inflammatory responses, and Nagler says she and her colleagues are still working out the role of TLR4.

While researchers continue to untangle these mechanisms, many scientists already have an eye toward microbiome-manipulating interventions for preventing or treating food allergies. Currently, the standard of care for such conditions simply involves avoiding the culpable antigens and being prepared with an epinephrine autoinjector (EpiPen) and an antihistamine in the event of accidental exposures. Immunotherapy, in which patients are exposed to low, increasing doses of an allergen over time, has recently become an option as well, but it only works for some patients, Rachid notes. “There is really an unmet need here for finding better treatment for food allergy.”

Several clinical trials have tested the effects of probiotic supplementation, with promising but mixed results. “When it comes to probiotics, so far the studies done are not very definitive,” says Rachid. The approach is worth pursuing further, she says, noting that she and her colleagues hope to develop probiotics based on the bacterial species they recently identified as protective against food allergy. At the same time, Rachid is overseeing the first clinical trial for fecal transplant for peanut allergy. “It’s a very interesting approach where you’re trying to change the whole microbiome.” The researchers are currently screening potential trial participants.

“There’s tremendous interest in this,” says Bunyavanich, adding that some of her colleagues have started companies to move such approaches forward. “The fact that food allergies have been rising in recent decades really implores us to try to figure out as much as possible what could be contributing to this.”

Jef Akst is the managing editor at The Scientist. Email her at [email protected].

Correction (July 9): This story has been updated to correct the type of food allergen tested by Rima Rachid and colleagues in the described study. It was egg, not peanut. The Scientist regrets the error.


Conclusion and future directions

Recent discoveries in the structure and function of the microbiome suggested that diet may have a direct impact on the intestinal microbiota and human or animal health status, and disruptions of microbe–man relationships may result in different disease states, including chronic inflammation, autoimmunity and neurological disorders.

Probiotics have been proposed as preventive and therapeutic measures, in order to restore the healthy composition and function of the gut microbiome. However, data from human microbiome studies may lead to identification of novel indigenous microbial species and tools to positively induce alterations in the gut microbial communities. Well-designed experiments in appropriate experimental models (in vitro or in vivo) may yield insights into the biology and potential manipulation of the microbiome in the human host. Metagenomic, metatranscriptomic and metabonomics can be deployed to globally examine interactions between probiotics, intestinal microbes and the mammalian gastrointestinal tract. New types of probiotics or medicinal compounds derived from the microbiome may be used as future strategies to promote health, prevent disease, and treat different disorders.


Gut bacteria offer new insights -- and hope -- for people with celiac disease

Dietary changes that include probiotics and/or prebiotics (found in some foods) may help alleviate the severity of celiac disease for some patients. According to a new research study appearing in the May 2010 print issue of the Journal of Leukocyte Biology, differing intestinal bacteria in celiac patients could influence inflammation to varying degrees. This suggests that manipulating the intestinal microbiota with dietary strategies such as probiotics and prebiotics, could improve the quality of life for celiac patients, as well as patients with associated diseases such as type 1 diabetes and other autoimmune disorders.

"We hope the study will ultimately add to the understanding of the mechanisms of action of the intestinal microbiota in immune-mediated diseases," said Yolanda Sanz, one of the scientists involved in the research from the National Spanish Research Council in Valencia, Spain. "This study may also help to design novel strategies, which could improve the quality of life of celiac disease patients in the future."

To make this discovery, scientists used cultures of human peripheral mononuclear cells (PBMCs) as in vitro models, as intestinal mucosa monocytes are constantly replenished by blood monocytes and accurately represent an in vivo situation. To simulate the intestinal environment of celiac disease, cell cultures were exposed to Gram-negative bacteria isolated from celiac patients and bifidobacteria, both alone and in the presence of disease triggers. The effects on surface marker expression and cytokine production by PBMCs were determined. The Gram-negative bacteria induced higher pro-inflammatory cytokines than the bifidobacteria. These bacteria also up-regulated expression of cell surface markers involved in inflammatory characteristics of the disease, while bifidobacteria up-regulated the expression of anti-inflammatory cytokines. Although human clinical trials are necessary, this evidence could be the first step toward changing how celiac disease is treated and possibly prevented.


Gut Microbiome Manipulation Could Result from Virus Discovery

Scientists have discovered how a common virus in the human gut infects and takes over bacterial cells – a finding that could be used to control the composition of the gut microbiome, which is important for human health.

The Rutgers co-authored research, which could aid efforts to engineer beneficial bacteria that produce medicines and fuels and clean up pollutants, is published in the journal Nature .

“CrAssphages are the most abundant viruses infecting bacteria in the human gut. As such, they likely control our intestinal community of microbes (the microbiome),” said co-author Konstantin Severinov , a principal investigator at the Waksman Institute of Microbiology and a professor of molecular biology and biochemistry in the School of Arts and Sciences at Rutgers University–New Brunswick . “Understanding how these tiny viruses infect bacteria may allow scientists to control and manipulate the makeup of the microbiome, either by increasing the proportion of beneficial bacteria in our intestines or decreasing the number of harmful bacteria, thus promoting health and fighting disease.”

Scientists found that crAssphages use their own enzyme (an RNA polymerase) to make RNA copies of their genes. RNA has the genetic information to make proteins. All cells, ranging from bacterial to human, use such enzymes to make RNA copies of their genes. And these enzymes are very similar in all living matter, implying that they’re ancient and related by common ancestry, Severinov said.

When the team revealed the atomic structure of a crAssphage enzyme, they were surprised to learn that it is distinct from other RNA polymerases but closely resembles an enzyme in humans and other higher organisms that is involved in RNA interference. Such interference silences the function of some genes and may lead to certain diseases.

“This is a startling result. It suggests that enzymes of RNA interference, a process that was thought to occur only in cells of higher organisms, were ‘borrowed’ from an ancestral bacterial virus early in evolution,” Severinov said. “The result provides a glimpse of how cells of higher organisms evolved by mixing and matching components of simpler cells and even their viruses.”

“In addition to deep evolutionary insights, phage (viral) enzymes such as crAssphage RNA polymerase may be used in synthetic biology to generate genetic circuits that do not exist in nature,” he said.

Synthetic biology involves redesigning organisms so they can, for example, produce a medicine, nutrient or fuel, sense something in the environment or clean up pollutants, according to the National Human Genome Research Institute .

“We are now trying to match the thousands of different crAssphage viruses in our gut with the bacterial hosts they infect,” Severinov said. “By using just the ‘right’ bacterial virus, we will be able to get rid of bacteria it infects, which will allow us to alter the composition of the gut microbiome in a targeted way.”

Leonid Minakhin at the Waksman Institute of Microbiology contributed to the study along with scientists at many other institutions.