by Elysium Health

August 16, 2018

from EndPoints website

Illustrations by Kevin Tong

It's been called the "forgotten organ,"

a "scientific frontier," and even the "second brain,"

yet scientists say we have barely scratched the surface

on understanding the role of this living, breathing,

highly complex ecosystem in shaping human health.

Here's an up-close look at what we know so far...

Humans have a complicated, you might even say, fraught, relationship with bacteria.

It's everywhere. Around us. On us. Within us...

Yet, for centuries we've drawn a strong causal link between bacteria and illness  - and for good reason. Devastating outbreaks and epidemics from the bubonic plague and rheumatic fever to whooping cough and anthrax, and even some of today's most perplexing illnesses, Lyme disease, MRSA, and others, can all be traced back to these remarkably intelligent and highly adaptive microorganisms.

But just over the past two decades, scientists have begun to shift that paradigm, poking holes in the dogma that all bacteria are 'dangerous' and to be avoided at all costs, or whenever possible, power washed away.

Today, there's mounting evidence implicating the 10,000 or so different types of,

• bacteria

• archaea

• fungi

• viruses

• protozoa

• helminths,

...inhabiting all of us, known collectively along with their genetic material as the microbiome, as crucial to our survival, influencing every aspect of health from mood to weight.

"We have a long, shared history with the microbiome," says Martin J. Blaser, M.D., a professor of microbiology and director of the Human Microbiome Program at the New York University School of Medicine.

Martin J. Blaser's lab studies the biology of bacterial colonization with a lens on the interactions that lead to or protect from us disease.

"It is intimately involved in human biology and so broad in its possible implications."

In this first installment of a two-part series, we introduce the microbiome and highlight some of the most important research about its associations with human health and disease.

"We have an integrated circuit with our microbiology, and therefore if we want to understand human health, we have to understand the microbiome and we have to understand its relationships," says Blaser.

Before You Start - Key Terms to Understand

• Diversity:

  A measure of how many different species and, dependent on the diversity indices, how evenly distributed they are in a microbiome. Lower diversity is considered a marker of dysbiosis (microbial imbalance) in the gut and has been found in autoimmune diseases and obesity and cardiometabolic conditions, as well as in older adults.
   

• Dysbiosis:

  A disturbance or imbalance in the microbial communities either in or on the body that can be caused by factors such as diet, stress, antibiotics, oral contraceptive pills, and lifestyle. Dysbiosis has been associated with health problems, including inflammatory bowel disease and chronic fatigue syndrome.
   

• Enterotype:

  A collection of species of bacteria in the gut microbiome that are found to be influenced by diet. Scientists have identified three human enterotypes,

  • Bacteroides

  • Prevotella

  • Ruminococcus,

  ...but there's still much debate about their importance to health and disease and whether distinct boundaries between the three groupings even exist.
   

• Flora:

  The name previously given to the bacterial communities inhabiting our gastrointestinal tract. Researchers now prefer the term gut microbiota.
   

• Metabolome:

  The collection of metabolites, byproducts made or used when the body breaks down food, drugs, chemicals, or its own tissue (i.e., glucose and fatty acids), found within an organism, cell, or tissue. In metabolomics, researchers study the metabolome to understand the relationship between the microbiome and the body's life-sustaining chemical reactions.
   

• Metagenome:

  The collection of genomes and genes from the organisms in a microbiota. Metagenomics is the field of molecular research that studies the complexity of microbiomes.
   

• Metatranscriptome:

  A collection of messenger RNA molecules expressed from the genes of organisms in a microbiota. Metatranscriptomics is a powerful RNA sequencing technology that allow analysis of complex microbial communities and their gene expression and regulation.
   

• Pathogen:

  An infectious biological agent that can produce a disease in its host.
   

• Proteome:

  A complete set of proteins expressed by an organism. The study of the proteome is called proteomics, and it involves understanding how proteins function and interact with one another. Metaproteomics refers to the large-scale characterization of the entire protein complement of environmental or clinical samples at a given point in time.
   

• Phenotypes:

  Observable physical traits (i.e., appearance, development, and behavior) of an organism determined by its genotype, which is the set of genes the organism carries, as well as by environmental influences upon these genes.
   

• Short chain fatty acids:

  Fatty acids with two to six carbon atoms that are produced by bacterial fermentation of dietary fibers in the gut. These acids have been shown to play an important role in regulating metabolism; low levels of SCFAs are associated with gastrointestinal disorders and obesity.

What Exactly Is the Microbiome?

You've probably heard the terms "microbiota" and "microbiome" used interchangeably, but there's an important distinction.

Microbiota is the dynamic community of trillions of microbes  - short for microscopic organisms - living in harmony with your human (eukaryotic) cells. Your microbiome, on the other hand, is the collective name given to the genes inside these microbes.

This genetic material is essentially what scientists are studying in hopes of uncovering the truth behind how and why microbes are involved in health.

The number of genes in all the microbes in one person's microbiome is approximately 150 times the number of genes in the human genome.

Noted molecular biologist and Nobel laureate Joshua Lederberg described it in 2001, when the study of the microbiome was in its infancy as,

"the ecological community of commensal, symbiotic, and pathogenic microorganisms that literally share our body space and have been all but ignored as determinants of health and disease."

Almost two decades later, study after study has shown that our microbiome plays an integral role in boosting immunity, preventing infection, and keeping our digestive system running smoothly, our hormone levels balanced, and our brains working properly.

Our microbiome may even predict our risk for developing certain chronic diseases.

A large-scale study (Environment Dominates over Host Genetics in shaping Human Gut Microbiota) published in the journal Nature in February revealed a strong association between the microbiome data of more than 1,000 healthy adults and measurements of cholesterol, weight, blood glucose levels, and other clinical parameters.

The study's authors concluded that using human genetic data together with microbiome profiles significantly improved how accurately they could predict the subjects' metabolic traits, compared to using genetic data alone.

How Much of Us Is Microbes?

Although various numbers of been reported dating back to the early 1970s, from 100 billion to hundreds of trillions, a 2016 PLOS One analysis weighing the available evidence put the estimate at around 39 trillion microbial cells and 30 trillion human cells for the average adult, with variations based on weight, height, sex, and age.

But because of their small size, microorganisms make up about one to three percent of the body's mass.

All told, they weigh between two and six pounds, as much as or even more than your brain.

The placental microbiome

has a taxonomic profile

that is similar to the oral microbiome.

Strong phylum-level similarity

 was observed between the placenta

and tongue, tonsils, saliva, and

subgingival plaque taxonomic profiles.

The colors of dots reflect the vicinity

of the body sites.

Sci Transl Med. 2014 May 21

6(237): 237ra65.

Where Is Your Microbiome?

Microbes exist everywhere on and in the human body, although there are specific areas of the body that contain large concentrations of microbes.

The vast majority of our microbes reside in the gastrointestinal tract, known as the gut microbiota (formerly called the gut flora), it harbors up to 1,000 species of microbes.

Gut microbiomes from different people can contain similar species of microbes, but vary by strain from one person to the next. Aside from the gut, other most-studied sites of the microbiome include the eyes, lungs, mouth, nasal cavity, skin, and vagina.

During early childhood, the composition of your microbiome changes frequently, but by around age three, it becomes fairly stable. Stable, but not static.

Your microbiome remains malleable throughout your entire life...

While the triggers for the ongoing microbial changes aren't fully understood, a number of factors from what you eat to where you live and your age, race, sex, hormonal cycles, and even the medicines have all been implicated.

For example, studies show that puberty triggers changes in your skin microbiome and the composition and structure of the vaginal microbiome shifts during and after pregnancy, and then again during menopause.

Our microbiomes can even be found outside our bodies, on nearly every surface and environment we come in contact with.

That's because just by the simple act of entering a room, you're shedding microbes into the air, referred to by scientists as your microbial cloud.
 

When Do You Get Your First Microbes?

For most of us, our introduction to microbiota begins during birth.

Infants are exposed to the microbial population of the birth canal on arrival into the world, which influences the development of their gut microbiota.

Infants delivered through C-section show reduced numbers of gut microbes compared to those delivered vaginally, however, studies show that difference is less detectable by six months.

More recent research, within the last decade or so, suggests that we may be exposed to microbes in utero, calling into question the long-held belief by the medical community that the womb is a pristine, sterile environment...

A 2013 study of placentas taken from 195 patients conducted by researchers at the Washington University School of Medicine and published in 2013 found bacteria present in nearly a third of placentas. (The placenta carries oxygen, nutrients, and more from mother to infant, and also provides a defense system against infections.)

A larger study (The Placenta Harbors a Unique Microbiome) published a year later by researchers at Baylor College of Medicine using gene sequencing tested placenta specimens from more than 300 patients.

They found bacteria in the placentas of many, including healthy pregnancies, suggesting that the bacteria were an important part of development. The study also showed that the bacteria in the placenta closely resembled that of the oral microbiome.

Dozens of labs have replicated these findings, and some have even detected microbes in amniotic fluid.

(Above left) A subset of healthy, vaginally born,

exclusively breastfed infants (n = 42)

was used to train a random forest regression model

that was then applied to estimate microbiota maturity.

(Above right) Differences in relative microbiota maturity

based on the age at which solid food

was introduced in our cohort.

JAMA Pediatr. 2017;171(7):647–654.

doi:10.1001/jamapediatrics.2017.0378
 

The debate continues, but if fetal microbiomes do exist, that could have far-reaching implications not only for medicine, but also for basic biology.

For example, scientists might be able to find ways to shift the microbial composition in the womb and possibly ward off diseases.

Another early influence on your microbiome? Breast feeding...

One study published last year in JAMA Pediatrics found that nearly 30 percent of infants derived their gut bacteria from microbes in their mother's breast milk.
 

What Does the Microbiome Have to Do With Health?

Most of the bacteria and other microbes that make up the microbiome are actually beneficial to our health and carry out specific and vital functions, such as,

• helping to digest food

• regulating the immune system

• producing key metabolites.

 

"The microbiome makes tons of metabolites and a good example is vitamin B12, which is a very complicated molecule," says Michael Snyder, Ph.D., a genetics professor and chair of the Department of Genetics at Stanford University School of Medicine and a principal investigator of the Integrative Human Microbiome Project (iHMP).

 

"You don't make B12 for yourself; in most cases, your microbes make it for you."

At the same time, autoimmune disorders and chronic diseases such as rheumatoid arthritis, inflammatory bowel disease, metabolic syndrome, which is closely linked to obesity and increases your risk of diabetes and heart problems, and many others have been associated with microbial dysfunction, or dysbiosis.

Here's a summary of the current knowledge on three broad pathways of human physiology.

A word of caution:

Experts, including Snyder and Blaser, advise careful interpretation of studies on the microbiome, as the bulk of the research has been carried out in mice and warrants further investigation. In other words, correlation has yet to equal causation.

"It's early days and there's a lot of hype out there," says Blaser.

"We have to do solid scientific work to figure out what's important and what's not, and what's causal and how we can harness the microbiome to improve health."

Digestion and Nutrition

The microbiota is a key influence on digestion and probably the most well-understood area in the study of the human microbiome, says Snyder.

Without our gut microbes, many foods we eat, for example plant cellulose, found in fruits, vegetables, nuts, would be indigestible.

The human gastrointestinal tract is well equipped to break down monosaccharides, such as glucose, and disaccharides, such as lactose. But it has a much more difficult time digesting complex molecules, or polysaccharides, such carbohydrates, lipids, and proteins derived from meats and vegetables.

That's where gut microbes come in.

Gut microbes feed off these molecules, breaking them down through fermentation into byproducts called short chain fatty acids  - nutrients shown to be integral to energy metabolism and appetite regulation  - that can be absorbed and utilized by the body.

For centuries, weight was tied to how much you eat, but newer research has identified correlations between how much you weigh and your microbes.

While some study results have captured the public's attention  - for, example, this one showing that mice who received a "gut bacteria transplant" from an obese human gained more weight than those who received bacteria from a lean human  - researchers have yet to arrive at a direct causal explanation, and studies have produced varied results.

Some studies, for example, show that microbes may use their metabolic activities to influence food cravings and feelings of being full.

Emerging research in mice has shown that a higher production of short chain fatty acids is associated with a lower risk for obesity. And a recent study in humans published in the Mayo Clinic Proceedings found that an increased ability to metabolize carbohydrates may actually hinder weight loss.

The study was small, and the authors caution that further research is needed to validate the results.

Immunity and Inflammation

Balance of your gut microbes highly influences the balance of your immune system, says Snyder.

"You have more immune cells in your gut than anywhere else, so your immune system and your microbiome are always talking to each other."

Studies show that disruption in the communication between the immune system and the gut microbiota can throw off that balance, opening the door for pathogens that can shift the immune system and may contribute to complex diseases, including,

• allergies

• obesity

• diabetes

• depression

• even cancer...

Studies on mice raised in gnotobiotic environments, unexposed to both beneficial and pathogenic microorganisms, have helped to provide extensive insights into the interactions between gut microbes and the immune system.

In a 2012 study (Microbial exposure during early life has persistent effects on natural killer T cell function) published in the journal Science, mice raised in germ-free environments showed increased inflammation of the lungs and colon resembling asthma and inflammatory bowel disease.

The researchers discovered that exposing the germ-free mice to microbes normalized their immune systems and aided in prevention of diseases.

However, this effect was observed only in germ-free mice exposed to microbes during the first weeks of life, but not in older germ-free mice, suggesting a strong association between early-in-life exposure to microbes and a robust immune system.

An imbalance of your gut microbes has also been implicated in an inflammatory condition called increased intestinal permeability, also known as "leaky gut."

This inflammatory state has been linked to celiac disease and Crohn's disease, and a recent trial in mice suggests that leaky gut may be associated with other autoimmune diseases, metabolic disorders, neurodegenerative diseases, and even cancer.

Another recent study published in Cell Host & Microbe revealed an association between a leaky gut caused by an imbalance in gut microbes and age-associated inflammation and premature death in mice.

Children raised in homes with dogs are less likely to develop allergies, and researchers say the reason may be tied to their gut.

A study led by University of California, San Francisco and University of Michigan researchers found that mice exposed to dust from homes with dogs had a lower risk of allergies and asthma compared to unexposed mice.

The research team traced the results to an allergy-inhibiting gut microbe in the exposed mice called Lactobacillus johnsonii.
 

Lactobacillus bacteria,

colored scanning electron micrograph (SEM).

These bacteria are a natural

non-pathogenic component of the flora of

the human intestines and vagina.

They produce lactic acid through

the fermentation of carbohydrates.

This helps to maintain an acidic environment

that is hostile to pathogenic (disease-causing) bacteria.

Getty Images/Science Photo Library

Brain and Behavior

The brain-gut axis, an intimate connection between the gastrointestinal tract and the central nervous system, is one vast and quickly emerging area of microbiome research.

This connection relies on the vagus nerve, a large bundle of fibers that, among its many functions, sends bidirectional signals from the gut to the brain.

Similar to its role in digestion, your gut microbes produce a range of neurotransmitters, including GABA and serotonin, which can both affect mood, appetite, and thinking, and when released from the gut, can activate the vagus nerve.

Increasing evidence in mice have shown that certain microbes in the gut activate the vagus nerve, and that activation plays a critical role in mediating effects on the brain and behavior.

In one such study, mice fed a strain of gut bacteria, Lactobacillus rhamnosus and subjected to a number of stressful situations were found to have less anxiety and less of the stress hormone corticosterone than mice who were subjected to the same situations but had not been fed the bacteria.

When the researchers snipped the vagus nerve, interrupting the communication between the brain and the gut, the differences between the mice disappeared.

Studies, mostly conducted in mice, have also demonstrated some associations between gut microbes and depression and other mood disorders, autism, and Parkinson's disease.

Snyder says that one particularly promising area of study is the relationship between the gut microbiome and autism.

Up to 70 percent of children and adolescents with autism experience underlying gastrointestinal issues, including an increased likelihood of having leaky gut, which can produce compounds linked to altered brain function.

In a 2013 study, researchers at Caltech reversed symptoms of leaky gut and autism-like behavior in mice by supplying them with a gut microbe known for its anti-inflammatory properties, Bacteroides fragilis.

A 2016 study in pregnant and newborn mice produced similar results using a different strain of bacteria, Lactobacillus reuteri.

Diversity Matters, But Why?

"We don't fully know the answer," says Snyder.

 

"The thought is that having a diverse microbiome means that you're making lots of metabolites that are important to the human body."

" We do know that when some people become ill, for example, with diabetes, their microbiome diversity tends to simplify. Yet it's still hard to pinpoint cause and effect."

Snyder points to another possible explanation:

Short chain fatty acids.

"One thought is that a diverse microbiome leads to lots of short-chain fatty acids, which are thought to be very healthy for your immune system," Snyder says.

Studies have implicated lower microbial diversity, considered a marker of imbalance and dysfunction of the gut, in autoimmune diseases, heart disease, obesity, as well as in age-related inflammation and disease.

Obesity is one area of particular research interest.

In studies comparing gut bacteria in both obese and lean animals and humans, the gut microbes in the obese subjects tended to show less diversity in gut bacteria.

The obese subjects also showed relatively more pronounced levels of inflammation and insulin resistance, an underlying cause of diabetes and a risk factor for cardiovascular disease.

A study published earlier this year in the European Heart Journal found a correlation between higher gut microbe diversity and a lower risk for arterial stiffness, a contributing factor to cardiovascular diseases in older adults.

These results and others suggest that manipulation of gut microbes could be a useful approach for treating or preventing obesity.
 

Meet Your Microbes

Here's a short, high-level list of bacteria that scientists have found to be present in the human microbiome, broken down by dominant genus and a brief description of their functions in health.

In the taxonomy of bacteria, a genus ranks a level higher and broader than a species, for example, the all-too-familiar infection causing bacteria E.coli is a species of the genus Escherichia.
 

• Bacteroidetes:

  The most prevalent bacteria in the gut. Bacteroidetes produce favorable metabolites, including short chain fatty acids, which have been correlated with reducing inflammation. Species: B. acidifaciens, B. eggerthii, B. fragilis, B. helcogenes, B. intestinalis, and B. thetaiotaomicron.
   

• Bifidobacterium:

  Bacteria found in the gut, mouth, and vagina, and also in yogurt and some dietary supplements. It's associated with a range of beneficial health effects, including preventing and treating ulcerative colitis. Species: B. crudilactis, B. denticolens, B. gallicum, B. gallinarum, B. hapali, B. indicum, B. pullorum, and B. reuteri.
   

• Lactobacillus:

  Found in the mouth, gut, and vagina, and also in yogurt and some dietary supplements. Lactobacillus has been used to prevent and treat diarrhea and other digestive problems. Species: L. rhamnosus, L. casei, L. fermentum, L. gasseri, L. plantarum, L. acidophilus, and L. ultunensis.
   

• Prevotella:

  Found in the gut and mouth and is associated with a plant-rich diet. Recent research has linked Prevotella bacteria to metabolic health. Species: P. copri, P. dentalis, P. maculosa, P. marshii, P. oralis, P. oris, and P. salivae
   

• Pseudomonas:

  Found on the skin, and commonly associated with skin infections and rashes. May also be found in the throat, mouth, gut, urethra, and vagina. Species: P. aeruginosa, P. maltophilia, P. aeruginosa, P. fluorescens, P. putida, P. cepacia, and P. stutzeri.
   

• Streptococcus:

  Found on the skin and in the eyes, nose, throat, mouth, gut, vagina. Associated with many illnesses, including pharyngitis, pneumonia, wound and skin infections, and sepsis. Species: S. mitis, S. salivarius, S. mutans, S. pneumoniae, and S. pyogenes

The bacterium, Enterococcus faecalis,

which lives in the human gut,

is just one type of microbe

being studied as part of

NIH's Human Microbiome Project.

Media for Medical/UIG via Getty Images

What Technologies are Enabling the Study of the Microbiome?

Over the last two decades, advances in sequencing technologies and the development of metagenomic methods have opened up new approaches to investigating microbes and their role in health and disease.

Before sequencing, scientists relied on microscopy and culturing, which provided a very limited picture of the microbial world.

"There's no question that whole genome sequencing has contributed greatly to the study and cataloging of the microbiome,"

...says Snyder, who along with his colleagues at NIH's Human Microbiome Project created the first reference data for microbes in healthy adults using next-generation sequencing (read more about what the data revealed here.)

Sequencing has allowed scientists to catalog and quantify microbial strains and genes, and also,

"figure out whether certain microbes are pathogenic or not," says Snyder.

"The microbiome is vast. We've found that the more you sequence, the more you discover."

In terms of the latest in sequencing technology, Snyder says that while researchers still rely heavily on 16S rRNA sequencing, a popular method used for identifying and comparing microbes, more powerful technologies have allowed for increased accuracy.

One such technology is long-read sequencing.

"Right now we get the sequences returned to us in fragments, and then we assemble them and make our best guess," says Snyder.

 

"But with long reads, we can make much more accurate assessments of microbes and get a better assessment of the deviation across strains."

Another next-gen tool providing an even deeper dive into the human microbiome is metatranscriptomics, which allows scientists to determine not only at what genes are present in the microbiome, but which ones are expressed.

"And we're also looking at metaproteome to see which proteins are made as well," says Snyder.

Move over DNA fingerprinting.

Since our microbes are specific to each of us, our microbial fingerprint could someday potentially be used by forensic scientists to detect our presence at the scene of a crime.
 

What's Next?

Many factors from your age to what you eat to your stress levels and the environment you live in can impact the diversity and health of your microbiome.

In part two of this series, we'll answer the following questions and more:

• Can you make over your microbiome?

   

• How do antibiotics affect the microbiome?

   

• Conversely, how do your microbes affect how you respond to various drugs?

   

• And how are today's research findings translating to clinical practice, i.e., fecal transplants and psychobiotics?

 

"Until recently, the microbiome wasn't well known or understood," says Snyder.

"Then researchers became aware of its importance, and suddenly it became so clear that we all have a few pounds of bacteria we're carrying around that actually contributes a lot to your health.

 

That's a big deal."
 

The Evolution of Microbiome Research

Studies on the diversity of the human microbiome started with Dutch scientist Antonie van Leeuwenhoek, when, in the 1680s, he compared his oral and fecal microbiota.

He noted the striking differences in microbes between these two habitats and also between samples from individuals in states of health and disease in both of these sites.

More than three centuries and many milestones later, scientists are still in the early days of understanding the microbiome, and according to Blaser,

"we have barely scratched the surface."

Here's a closer look at where it all started, from the earliest documented studies on the microbiome and other "firsts" through where we are today.

• 1885: The universal model organism, Escherichia coli (formerly Bacterium coli commune), was first described by Theodor Escherich in 1885 and isolated from the feces of newborns in Germany.
   

• 1908: Russian zoologist Elie Metchnikoff theorized that health could be enhanced and senility delayed by manipulating the intestinal microbiome with host-friendly bacteria found in yogurt.
   

• 1959: Scientists reared germ-free mice, rats, rabbits, guinea pigs, and chicks inside sterile stainless steel and plastic housing, known as gnotobiotic facilities, in order to study the health of animals living in an environment completely untouched by microbes.
   

• 1970: First estimate, by microbiologist Dr. Thomas D. Luckey of the colonies of microbes living inside you: 100 billion in a gram of human intestinal fluid or feces.
   

• 1995: A team of researchers led by genomics pioneer J. Craig Venter sequenced the genome of the bacterium Haemophilus influenzae, a common inhabitant of the human respiratory tract, making it the first organism to have its genome completely sequenced.
   

• 1996: The first human fecal sample is sequenced using 16S rRNA sequencing.
   

• 2005: Researchers find bacteria in amniotic fluid of babies born via C-section.
   

• 2006: The first metagenomic analysis of the human gut microbiome is conducted.
   

• 2007: The NIH-sponsored Human Microbiome Project (HMP) launches as a study to define the microbial species that affect humans and their relationships to health.
   

• 2009: The first microbiome study showing an association between the human gut microbiome and disease is conducted on obese and lean adults.
   

• 2011: German researchers identify three enterotypes in the human gut microbiome, Bacteroides, Prevotella, and Ruminococcus.
   

• 2012: Scientists from the HMP unveil the first "map" of microbes inhabiting healthy humans.
   

• 2012: American Gut Project founded, providing an open-to-the-public platform for citizen scientists seeking to analyze their microbiome and compare it to the microbiomes of others.
   

• 2014: The Integrative Human Microbiome Project (iHMP), the second phase of the Human Microbiome Project begins with a goal of studying three different microbiome-associated conditions.
   

• 2016: The Flemish Gut Flora Project, one of the world's largest population-wide studies on variations in gut microbiota, publishes its first major results, based on the analysis of more than 1,100 human stool samples.
   

• 2018: The American Gut Project publishes the largest study to date on the microbiome (American Gut - An Open Platform for Citizen Science Microbiome Research). The results include microbial sequence data from 15,096 samples provided 11,336 participants across the United States, United Kingdom, Australia, and 42 other countries.