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A Gut Microbiome / Probiotic Primer

Kiran Krishnan was a founder of microbiome labs. Since its sale, he is now working in microbiome education. This is from a piece he wrote about 8 years ago. I'm only reproducing the sections that I can verify as much has changed since its publication.


Bacteria were largely considered a major threat for generations of doctors, but in recent decades, scientists have discovered that the bacteria living in our gut are vital to human health . The number and diversity of microbes that colonize in a person’s gut can influence weight changes, arterial health, diabetes risk, food cravings, and even mood . Today, medical research is not only focused on developing antibiotics for infections, but also striving to develop probiotics that can drastically improve the composition of gut microbiota .

However, cultivating the right bacterial strains that can survive both in a factory and the human gut is a challenging task . Kiran Krishnan, a microbiologist and clinical researcher, has investigated probiotics for more than 15 years . From his experience, he explains why probiotics aimed at fighting metabolic endotoxemia —instead of digestive issues—should take precedence in probiotic research . He also explains why bacterial spores, and not the bacteria found in conventional probiotics today, make the most effective probiotic product for your patients .

Confusion About Probiotics

Ten years ago, a probiotic company approached Krishnan and a team of microbiologists, biochemists, and clinicians to investigate new probiotic products . That company has since been acquired and the research put on hold, but Krishnan and his colleagues realized their question was too important to drop—which manufacturing processes make a good probiotic product, and which strains have the most beneficial effect on the human gut?

Companies in the probiotic industry range widely in the content of their probiotic products and approaches . Some products contain 100 billion colony-forming units (CFUs), and some pack in 500 billion CFUs . Some products need refrigeration, while others do not . Some products contain only one strain, while others have 29 bacterial strains . Companies also disagree on the purpose of probiotics, whether they should aim to improve digestion, nutrient absorption, immunity, or diarrhea .

Krishnan argues that the overarching goal of any probiotic should be to protect

the human host. According to his research, probiotics that reduce the effects of

leaky gut are the most effective in reaching this goal. Every time a person eats,

they expose themselves to toxins produced during the digestive process . In fact, eating is the most common toxic and dangerous activity that humans do as a species .

That’s where the microbiome comes in . The gut microbes that make up the gut microbiome—the collection of microorganisms in the gut and their combined genetic material—help digest food and deliver nutrients to the human host . The diversity of bacteria in the gut microbiome determines whether the food is digested efficiently and safely . If the microbiome isn’t healthy, toxins can pass through the gut into the bloodstream, causing metabolic endotoxemia .

Any drug or supplement consumed will have to face the body’s digestive system . How and when the substance passes into the bloodstream will determine its effectiveness . Yet, many of the bacterial strains cultivated for probiotics are not ideal strains to survive this journey . Many popular probiotic strains require refrigeration, but upon consumption, enter an environment of 98 degrees Fahrenheit . Probiotic bacteria must then survive through 25 feet of the intestinal tract into the colon, colonize, and then compete with 100 trillion other bacteria.

All this led Krishnan and his research team to question whether the various probiotic products on the market could positively alter the composition of the gut microbiome . So, the team set out to investigate which fundamental features of a probiotic would allow it to both survive in the gut and combat metabolic endotoxemia .

For a probiotic to be effective, it must survive through the entire gastric passage down to the terminal end of the small bowel . That means surviving bile salts, pancreatic enzymes, and any attacks by the host’s immune system . It also means surviving the stomach acid, which is designed to kill most microbes and is typically the greatest barrier to probiotics . These products must survive all of these obstacles at the human body tmperature of 98° F.

Probiotics don’t often survive the gastric system, despite efforts to protect the product with capsules and coatings . A recent analysis of 35 top-selling probiotics showed that the vast majority of products were completely eliminated in gastric conditions . Seven products had minimal survivability, at fewer than 10 percent, and of the products that did survive, many were spore- based probiotics .

Another study done in association with Redding University and the Food Standards Agency tracked 35 common commercial probiotic strains through the gastric process, including 20 minutes of exposure to gastric juices, exposure to bile acid in the small intestine, and exposure to the lower intestines . Only four of the 35 probiotic products survived to the site of colonization, and the strains that did survive lost half their numbers on the way .

The gut microbiome is a heavily-structured, well-regulated system . Once a probiotic makes it to the site of colonization, it must bind to survive . The ability of a microbe to exist in any part of the microbiome is determined by its ability to express certain binding proteins . Luckily, these adhesion proteins are very well-studied among probiotic bacteria .

One study published in 2009 tracked the ability of common probiotic strains to adhere to intestinal cell lines, which would demonstrate a potential ability to colonize in the gut . The study was done in optimal conditions for binding; in vitro, with nutrients, at the right temperature, and without the immune system or other gut bacteria competing . At best, the adhesion rate in this optimal condition was about 38 percent, according to the study published in Microbial Ecology in Health and Disease .

Naturally-occurring gut bacteria

A probiotic that can successfully bind and colonize in the gut to restore health will likely be a strain naturally found in a healthy human gut microbiome . When searching for a safe and effective probiotic, it is important to look for bacterial strains that have a history and evolutionary relevance to human beings . No two individuals have the same microbiome, not even identical twins; however, some strains are foundational to human gut microbiomes around the world .

The American Gut Project is the largest human microbiome study in the U .S . and has one of the largest databases cataloging the genomes of gut bacteria . This data is crucial to studying the slight variations across human gut microflora . For example, after sequencing the microbiome of 200-250 different patients with Crohn’s disease, researchers found the difference between the gut bacteria in healthy individuals and people with Crohn’s is the overabundance of one strain known as Lactobacillus acidophilus .

The miniature ecosystem of gut bacteria can be a delicate one . An illness, a change in diet, a medication, or another outside influence can throw off the balance between competing bacterial strains, leading to an imbalance known as dysbiosis .

Unfortunately, some of the easiest probiotics to produce are not necessarily the most abundant in a healthy microbiome . For example, Lactobacillus acidophilus is the number one selling probiotic strain in the U .S . and has been popular for decades . However, of the types of bacteria within the microbiome, L. acidophilus only makes up a small fraction, in the eighth or ninth percentile . In a healthy human gut microbiome, there aren’t very high levels of L. acidophilus . The main advantage of L. acidophilus to probiotic companies is that it is easy to culture and develop commercially . When searching for a bacterial strain that makes a good probiotic candidate, it is important to consider both its presence and significance within the gut microbiome .

Evolutionary significance

The strains of bacteria that have become common among our microbiomes survive because of symbiosis . The human body hosts the bacteria, and the bacteria provide a benefit for the host . However, symbiosis doesn’t occur overnight, or even over a few decades; it takes centuries of co-evolution . A safe and effective probiotic will likely have some evolutionary significance, meaning there should be some evidence that our human ancestors had regular exposure to these strains and survived the better for it .


About 97-98 percent of bacteria that live in the gut are obligate anaerobes, meaning that they cannot survive in the presence of too much oxygen . Unfortunately, most probiotic companies choose to produce bacteria that can survive in an oxygen-rich factory environment for practical cost reasons . This means that probiotic companies are taking aerobic strains and putting them into an anaerobic environment, hoping they will survive . A successful probiotic should be able to live in both the high-oxygen environment outside of the body and the low-oxygen environment of the gut . That’s why being a facultative anaerobe is a key requirement of a good probiotic product .

Biphasic life cycle

Every cell in the human body has a very specific life cycle, in which the cells express different genes during different parts of cellular mitosis . In the case of a probiotic cell, the bacterial cell must live outside the human body for a period of time during the production phase, and then live inside the body . This means that a probiotic cell will have two different life cycles, in which they express completely different genes for each environment . That specialized cycle is known as a biphasic life cycle and is another key requirement for a successful probiotic .

Dead bacteria paradox

In some cases, probiotic supplements may offer health benefits, even if the bacteria do not survive digestion . For example, a 2009 study published in Pediatric Research compared a live and heat-killed version of a probiotic strain called Lactobacillus rhamnosus GG. Both the live and the dead bacteria versions reduced inflammation in mice, according to the study . Upon first glance, this study seems to suggest that live and dead probiotics have the same effect on the body, but closer inspection reveals an important difference. Dead cells can trigger a metabolic response within the body, but that doesn’t make them probiotics. Since research shows that many probiotic products on the market cannot survive digestion, Krishnan suggests that most “probiotics” on the market should technically be referred to as “metabolic response modifiers.”

One way that dead bacteria can function as metabolic response modifiers is through secretions known as outer membrane vesicles or OMVs . OMVs can contain micro- RNA, which can interfere with transcription and translation in human cells, or interfere with the gene expression of the host’s microbiome. This may create a temporary metabolic change within the body, but the effects will be transient .

Fortunately, not every product labeled as a probiotic will be destined to die in the gut and become metabolic response modifiers . Extensive research and decades of probiotic use have shown that bacterial spores may, in fact, be excellent candidates as true, living probiotics .

Bacterial Spores Prove Themselves

People start accruing bacteria from the moment they are born . There are a number of ways people pick up bacteria, including the environment, food, and other people . Babies pick up enough strains of bacteria to have a fully-developed microbiome within 838 days after birth . Previous research suggests that environmental bacteria significantly affect the health and diversity of an individual’s gut microbiome .

Scientists can infer a lot of information about the health of a population from their microbiomes, and vice versa . By comparing the microbiomes found in people across different cultures and environments—from rural populations in Russia, to urban populations in Japan, to isolated groups in Tanzania—researchers have found that people exposed to more dirt have more diverse and stronger microbiomes . They were also less likely to suffer from certain conditions, like Crohn’s disease .

researchers have found that people exposed to more dirt have more diverse and stronger microbiomes

In their quest for good probiotic candidates, Krishnan and his colleagues decided to focus on bacterial strains found in dirt that could also fit their key requirements:

• Have an impact on the human microbiome

• Live and colonize in the gut

• Possess biphasic life cycles

• Survive through the gastric system

Bacterial spores were the only strains that met all criteria. Outside of the U.S., there has been widespread therapeutic use of bacterial spores since 1958. They are still actively used in South America, Europe, and parts of Southeast Asia, where they’re used to treat dysentery. Bacterial spores are also used to treat campylobacter infections, salmonella infections, and chronic upper respiratory infections in children.

Bacterial spores are uniquely equipped to function as probiotics . The spores form a robust endosphere to protect them from harsh environments . When they leave the body through defecation, the spores go into an endospore state . In this state, they are metabolically inactive and cover themselves with a thick protein-calcified coat that makes them impervious to dangers such as UV radiation, desiccation, heat, and acid .

Bacterial spores can exist in this dormant, protected state indefinitely . This allows them to pass through the gastric system intact and into the gut, where they can come out of their spore form into their active form . The oldest live bacterial spore that was ever found was about 250 million years old . It was found trapped inside of a salt crystal from a mine in Carlsbad, New Mexico, and scientists were still able to plate it and watch it germinate .

Bacterial spores passed the survivability prerequisites of a good probiotic, but the team of researchers still needed to know if any bacterial spores significantly improved health . Most probiotic studies show efficacy in treating gas, bloating, cramping, and diarrhea, but these conditions are usually treatable with other therapies . The number one benefit of a true probiotic is its ability to modulate the microbiome to protect the host, namely by mitigating the damage caused by leaky gut .


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