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The body’s secret garden How the exploration of the microbiome is going to change face of medicine.

The body’s secret garden How the exploration of by fountaininkseries

BY AKSHAI JAIN

It started with discomfort in the stomach, progressed to severe cramps and finally blood in the stool, often lots of it. Nights were sleepless, a roil of urgent trips to the bathroom and soiled clothes. During the day, then 33-year old Manas Shukla verged on incontinence, visiting the bathroom 10-15 times.

A colonoscopy revealed ulceration in the big intestine. The Delhi-based businessman was diagnosed with ulcerative colitis (UC), an auto-immune disorder in which the immune system attacks cells in the gut, leading to chronic and painful inflammation. He was put on an aggressive treatment of steroids and anti-inflammatory medicine, following which the condition gradually abated.

Seven years later, in 2009, a niggling abdominal pain surfaced again. It wasn’t much worse than a bout of gastritis, and easy to dismiss. It stayed that way for a few more years.

Then in 2012, the disease returned with a vengeance. A round of steroids followed. Two relatively calm months later there was a relapse, then another. The medicines were clearly having little effect. Fifteen trips a day to the bathroom meant Shukla couldn’t venture out of the house. In desperation he turned to homeopathy, to little avail.

Steroid use was by now having serious side-effects. This moderately built man started bloating. His face turned puffy, strange itchy patches appeared on his forearms. He says he was “angry, irritated and absolutely not in control of my life”. His business suffered. When he was out, he was preoccupied with where he might be able to find the next toilet. Just to be safe he carried an extra set of clothes.

In the summer of 2014, he woke up one morning to find he couldn’t stand on his feet—the joint pain was excruciating. His UC had led to arthritis. It has been known to happen, though medicine hasn’t deciphered how one leads to the other. Immunosuppressants took care of the pain, but  steroid use led to early cataracts. It was, says Shukla, “a hell of a time. Classic case of Catch 22”.

Finally, he decided to try a treatment so experimental, bizarre and odious that it could only be a last resort. At a private hospital in Gurugram doctors took a little lump of his brother-in-law Santosh’s feces, ground it with saline, strained it and filled it in syringes.

The decoction was then transplanted into Shukla’s gut via a colonoscopy. It was India’s first faecal matter transplant (FMT), a procedure that in polite company is also called a “microfaunal” transplant—the fauna being the dense cocktail of bacteria and viruses that feces contains.

This bizarre line of treatment was inspired by a few studies that indicated that the guts of people suffering from UC (and other similar diseases) were different from “normal” guts. Many of these had been conducted abroad, but some had been done in India.

A study at the All India Institute of Medical Sciences (AIIMS) in Delhi had found major fluctuations in six major groups of bacteria that colonise the gut. Doctors treating Shukla hoped that introducing material from a healthy gut would rectify the balance.

It was a long shot, but the results were miraculous. A few sessions of FMT later, his colitis went into remission. His pharmacopoeia of medicines tapered off and for the first time in many years, he did not have to worry about the location of the next bathroom.

The hunch that bacteria and viruses inhabiting the gut play an important role in health is not new. The first inklings came as a far back as the 1960s, points out Gagandeep Kang, one of India’s leading researchers in this area.

Doctors at the Christian Medical College in Vellore, where she now teaches, were at the time working on a gastrointestinal disease called tropical sprue. Patients suffered from diarrhoea, cramps and anorexia in a spiralling cycle that left them malnourished, their bodies unable to absorb nutrition.

The cause of the disease was elusive, but doctors at the institute found bacteria were the likely culprit. Sprue patients had bacteria in their guts where there shouldn’t be any. Further work was limited by technology—in the absence of DNA-based methods, identifying bacteria was a laborious process of growing individual cell lines in the laboratory. But the first probe had been lowered into the fascinating life of the microbiome.

***

The human body is a forest—a complex ecosystem of thousands of genes and millions of cells. But what remained hidden from sight until very recently, underneath the soil, were the 100 trillion microorganisms that inhabit the body, on the skin, mouth, vagina, anus and most densely, the human gut.

Their population is shockingly large, far exceeding the number of cells in the body. And the number of microbial genes in the body is more than 100 times the number of genes in the human genome. We know little of this microcosm of bacteria and viruses, but research over the last few years suggests that it plays a role in everything from diseases like diabetes to obesity, digestion and drug resistance. The genetic contribution of these microbes to the human body is thought to be so significant that this hidden world is now referred to as the “microbiome” or the second genome.

This is nothing short of the Copernican revolution in biology. The human genome, so far considered the arbiter of the body, stands displaced from the centre, giving way to thousands of species of mysterious microbes.

The relatively simple model in which the genome controlled everything from the production of proteins to dispositions to diseases is now complicated by the role of bacteria and viruses interacting among themselves, with the environment, the body cells and the human genome. We now appear to be living in a swamp of microbes that rise and ebb, enter and exit the body.

The relatively simple model in which the genome controlled everything from the production of proteins to dispositions to diseases is now complicated by the role of bacteria and viruses interacting among themselves, with the environment, the body cells and the human genome. We now appear to be living in a swamp of microbes that rise and ebb, enter and exit the body.

This web of interactions leads to situations in which some diseases only manifest themselves in the presence of adequate numbers of a certain bacteria. But the abundance of those bacteria is determined by the presence of very many others. It sounds dauntingly complicated, but scientists have, as a precursor to better understanding, started mapping this world.

The enigmatically named Centre for Human Microbial Ecology (CHME) is one of the laboratories at the heart of this endeavour. It’s part of a new institute, the Translational Health Science and Technology Institute (TSHTI), established recently by the government as part of a biotech cluster. The goal of this institute, headed by Kang, is to translate health research into clinical practice and applications. This is ambitious and virgin territory, mirroring the institute’s location in the middle of the scrub forest of Haryana’s Aravali range.

Bhabatosh Das’ office looks out over this vast expanse. The landscape is dominated by an orange, multi-storeyed Hanuman statue that beams benignly across the desolation. Das, a professor at CHME, has been cataloguing microbes from different parts of the body and different parts of the country. A hive of imposing machines whirs and hums in the institute’s laboratory revealing various species of bacteria.

Unlike the human genome, 99.9 per cent of which is common to all people, the microbiome differs wildly, depending on age, genetic composition, gender, diet, geographic location and health of a person. While some of these differences are minute or inconsequential, others might be significant. The only way to tell is to compare samples.

So Das’ group took breast milk microbiome samples from 27 women in Punjab, 144 stomach samples from Jammu and Kashmir and Maharashtra, 400 vaginal microbiome samples from Delhi, and gut microbiome samples from 1,535 people from seven states scattered around the country.

This is a very large number given how expensive the DNA analysis of the samples is. “Much larger,” says Das, “than the 242 samples analysed under the U.S.-based Human Microbiome Project.” The diversity of India’s population necessitates the sample size.

Previous studies (in India and abroad) have given a broad picture of the microbial composition of the gut. The gastrointestinal tract is dominated by a handful of types of bacteria—Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria, Fusobacteria and Verrucomicrobium. Of these the first two comprise 90 per cent of the species in the colon.

The human body has only eight enzymes to digest food. Without these bacteria, we wouldn’t be able to digest most of our food. Bacteroides, a subclass of Bacteroidetes, degrade starch, playing an essential role in carbohydrate metabolism and nutrition. Eubacterium, part of Firmicutes, produce chemicals to degrade vegetables and fruits and play a part in degrading bile acids in the intestine.

These are just two examples from a range of functions the microbiome serves. Lactobacilli, another member belonging to Firmicutes, are known to help fortify skin barriers that protect us from infection. Other microbes have functions that we’re extrapolating in reverse, when things go wrong, as in the case of ulcerative colitis and tropical sprue.

The relationship between the gut and its microbial garden is symbiotic—in return for their myriad services, the microbes get to live in a particularly nutrient-rich environment.

Das’ team found that six types of bacteria were present in the guts of all Indians, while others were specific to different groups—like a unique bacteria that he has recently isolated from a faecal sample that he got from the Andamans. This is found nowhere else in India, opening up the possibility of bacteria being used as geographic markers for populations.

 ***

The question of whether there is anything known as the “Indian” microbiome is however still open-ended, and given the plethora of conditions that determine the composition of the microbiome might not even make sense. That’s not to say that there are no differences. In another study where Das juxtaposed samples from India and Japan he found that the bacterial compositions of the microbiomes were “completely different”. He attributes this to the very great difference in diet.

In Assam, Mojibur Khan of the Institute of Advanced Study in Science and Technology conducted a parallel study of 15 Indian tribal populations from Assam, Telangana, Manipur and Sikkim, comparing the microbiota to data from other parts of the world.

Each of the groups he’d chosen was different in tradition and food habits. Tribes from Manipur and Sikkim consumed more fermented foods, smoked fish and meats. Those from Sikkim consumed more milk products than the rest.

Not only did Khan find variations in the relative proportions of different bacteria, some groups like the Kolam tribe from Telangana had unique bacteria (Treponema and Gordonibacter). Significantly, he confirmed that there was a core set of bacteria present in all these tribes despite their differences.

This basic descriptive exploration of “Indian” microbiota has been succeeded by work looking at the changes effected by diet, age and nutrition. The most basic division lay between predominantly vegetarian and non-vegetarian diets, with the former being rich in a type of bacteria called Prevotella and the latter in Bacteroides.

The divisions by age were fewer—the microbiome of children less than a year old was in a state of flux, with low diversity and a higher concentration of pathogenic microbes, which are thought to play a role in the development of the immune system. The microbiome matures quickly. Das believes that by two or three a child’s microbiome has matured. Kang believes this happens earlier—by one.

Thereafter the microbiota stabilises, changing significantly only after the age of 60.

A fascinating study by scientists from the National Centre for Cell Science and the Agharkar Research Institute used three generations from two Indian joint families to examine age-related changes. Using members of the same family allowed them to minimise factors like genetics and diet that might influence the results. They found definite age-related changes especially in the ratio of Firmicutes to Bacteroidetes. The changes in their relative populations were very different from those seen in European populations. During the course of the study they also chanced upon six novel species of bacteria.

The clinically most important find, with wide ramifications, has been the relationship between nutritional status and microbiota, especially in children. A collaborative study between scientists from the leading microbiome research laboratories in the country looked at the gut microbes of 20 children of varying nutritional status.

Each of the samples was sequenced completely, at `4 lakhs a sample. The process, called whole genome sequencing, looks not only at the different bacteria but also their functions.

Certain groups of bacteria like Proteobacteria, they found, decreased with improving nutritional status, while others like Synergistetes were positively related. Their results also threw up some surprises—bacteria like Lactobacillus that were thought to be strongly correlated seemed impervious to nutritional status.

Lower levels of nutrition it seemed were due not only to the increase in the number of pathogenic bacteria but a decrease in the number of “good” bacteria like Roseburia and Faecalibacterium.

These descriptive studies gave us a basic understanding of the Indian microbiome, but they were somewhat unsatisfying, There were too many variables, it was difficult to discern patterns across microbiomes, the connections found indicative but far from definitive, and they did not translate into very much more. But these were the stepping stones to a slew of exciting investigations probing the connections between various diseases, conditions and the microbiome.

***

Now that scientists had an inkling of what a normal microbiome and its variations looked like, it was easier to move backwards from disease to microbe. While this is not a replacement for establishing causal connections, it is a much more focused and faster method, which has already yielded clinical applications.

With sprue, scientists showed that the guts of malnourished children had abnormalities. Now there were far better tools and  greater understanding at their disposal.

In the intervening decades the government introduced many feeding programmes to address malnutrition, but their effect was slower than expected. Increased nutrition did not lead to faster growth in malnourished children. In some cases supplementary feeding had a muted effect. In others children regressed almost immediately after the feeding stopped.

According to Gagandeep Kang, data from animal models indicates that the relationship between the microbiome, the immune system and the cells that line the intestine determines how well the gut absorbs nutrients. It’s a complex interaction—when the body’s immunity or ability to control its microbiome is low, the cells lining the intestine step in. This reduces their ability to absorb fat, leading to malnutrition. It seemed evident that while supplementary feeding was necessary, reducing exposure to pathogens (harmful bacteria) was equally so.

What were these pathogens? Kang along with other colleagues compared 10 children with low birth weight with 10 children of normal weight from a slum in south India, tracking them every three months till they turned two. They found that the microbiota of children in the control group were rich in species like Bidifidobacterium longum and Lactobacillus mucosae, while the guts of stunted children had high numbers of bacteria like Desulfovibrio and Campylobacterales, which caused inflammation. These bacteria predominate in patients with inflammatory bowel disease (of which Shukla’s UC is one). The guts of stunted children also harboured Camplyobacteria, a type of bacteria found in the microbiota of patients with chronic HIV.

Another area Kang has worked on extensively is rotavirus, for which she was awarded the Infosys Prize in 2016. Rotavirus causes severe diarrhoea in babies and young children. Unfortunately, vaccines for rotavirus and even the oral polio vaccine are found to have much lower efficacies in developing countries than they do in countries where nutrition levels are higher and exposure to pathogens lower. Scientists suspect that microbiota, given their connection with the immune system, have a role to play.

The results have, however, been equivocal. In a study (published in 2016) Kang and colleagues altered the microbiota of a set of children receiving the oral polio vaccine by giving them antibiotics, while the control group got none. The assumption was that children whose microbiome was modified would respond differently, but “we noticed no difference,” says Kang smiling wryly, “We’re not very good at finding things”.

She was alluding also to another study comparing the microbiota of obese mothers and children in various permutations (obese mother-normal child, obese mother-obese child etc) that had also drawn a blank. They had found no differences in the microbiomes of the different sets.

Other researchers have found connections. A study published in  Nature found that “obese” microbiomes had gene sets that were better at harvesting energy from different sources, while at the other end of the spectrum, the microbiomes of anorexic individuals are dominated by a particular bacteria. According to Bhabatosh Das, results from this study have been replicated in mice where transplanting the microbiome of an obese mouse into a germ-free one has led to the latter turning obese.

The reasons for the differences in result are in part due to the small sample sizes of most of these studies, coupled with the incredible variations found in the microbiome.

***

At the Centre for Human Microbial Ecology, Das and his students have been exploring the microbial underpinnings of a whole range of urgent diseases.

India has the world’s highest incidence of pre-term birth, more than twice that of any other country. The health costs are enormous—pre-term children are more likely to be stunted and have lower IQ. In a project funded by the Department of Biotechnology, Das’ team compared the vaginal microbiota of pregnant women. Could looking at microbiota predict whether the child would be pre-term?

Das suspected that bacterial vaginosis, a minor infection of the vagina, might lead to pre-term birth. In “normal” vaginas, he says, Lactobacillus controls other bacteria by making the vaginal environment very acidic. A disruption of this balance can lead to inflammation, which can affect the placenta leading to pre-term birth. This according to him is the first study in India looking at the microbiomes of pregnant women.

One of the largest studies the lab is currently involved in is an Indo-Danish collaboration comparing the microbiomes of healthy, pre-diabetic and diabetic (type 2) patients. Half the patients are enrolled in India while the other half are in Denmark. The 450 microbiome samples from each country are first grouped according to bacterial composition. From each group a few samples are taken for whole genome sequencing.

“Initially we were going to enroll patients 60 and above since that’s the age group most likely to suffer from diabetes in Denmark. But in India most diabetics are in the 35-50 age group,” says Das, his round face breaking into a restrained smile, “so we changed the age group to 35-65.”

The results are still being analysed but are indicative of groups of bacteria appearing only in certain sets of subjects. The question troubling Das, though, is whether it’s the bacteria that are in some way responsible for diabetes or the other way around.  “It’s a controversial question”. It’s unlikely they’ll have an answer to that anytime soon since there are no good animal models (which mimic the human manifestation of these conditions and situations) for diabetes or the vaginal microbiome.

Smaller diabetes studies in India have found higher populations of Bacteroidetes in patients, an increase in a few other pathogens and a reduction in some common bacteria which produce the acid butyrate.

Neonatal sepsis, a bacterial blood infection in newborns is another condition being tracked back to imbalances in the microbiome. Breast milk, contrary to perception, is far from sterile, containing nearly 70 different types of bacteria. It is a possible source of  infection. Scientists at the Postgraduate Institute of Medical Education and Research, Chandigarh, are examining breast milk from 27 lactating women to gauge whether it can be used to predict the chances of sepsis.

Another conundrum that has possible links to bacteria is the prevalence of non-alcoholic fatty liver disease in India despite the low consumption of animal fat. Shalimar (he goes by one name), a gastroenterologist at Delhi’s AIIMS conjectures that the gut produces a substance that gets transported to the liver. In the first stage of the study he’s looking at the gut bacteria of 17 patients.

Links between microbes and disease have been best established in a set of diseases called inflammatory bowel diseases (IBD). Ulcerative colitis is one of these, as is a disease called Crohn’s disease (CD), an inflammatory disease that leads to abdominal pain and diarrhoea. Vineet Ahuja at AIIMS showed UC patients had significantly higher concentrations of Lactobacilli and lower numbers of Clostridium coccoides. In IBD patients the populations of other bacteria like Bacteroides, Lactobacillus etc were also awry.

The most exciting discovery, which would reorient and complicate efforts at understanding a microbiome, came when scientists were looking at stomach ulcers.

Studying stomach microbiota was challenging to start with since it required stomach biopsy samples. CHME managed to get 144 from Mumbai and Delhi. Conventionally, H. pylori had been blamed for stomach ulcers, but many samples did not have any trace of it.

At a partner institution of CHME, the Rajiv Gandhi Centre for Biotechnology (RGCB), Gopinath Balakrishnan Nair had been looking at the same issue. He found that the strains of H. pylori associated with stomach ulcers in Western and East-Asian countries were different from those associated with the disease in patients from West Bengal. There was, however, little information from India on virulent strains of the bacteria.

To complicate matters further, even though nearly 80 per cent of Indians are carriers of H. pylori only 10-20 per cent develop diseases related to it. Of 4,500 slum children Nair studied in Kolkata, he found most healthy despite having a large number of “pathogenic” bacteria in their guts. What was going on?

It seemed that other microbes had a significance influence on the virulence of H. pylori. In the presence of some it was malignant, but in their absence it lingered harmlessly.

In some concentrations and the presence of appropriate microbial ecosystems around the bacteria could be pathogenic; in other circumstances the same bacteria could be beneficial. In other words, there are no bad and good bacteria.

***

One of the most exciting things about the microbiome, which makes it of far greater therapeutic use than the genome is the fact that it can be modified. “Once you’ve identified genes in the genome, then what?” asks Das rhetorically, “you can’t change them”. But in case of the microbiome you can add genes and with the help of medicines like antibacterials, better known as antibiotics, you can take out a few.

The advantage, though, is double edged—its malleability also makes the microbiome a key player in the looming scourge of antibiotic resistance.

It has been implicated in its spread. In 2015, a group of Swedish scientists examined the stools of a group of 35 Swedish exchange students before and after they returned from India and Africa. The results were stark.

Genes encoding for resistance to the sulfonamide group of antibiotics increased 2.6 times; to trimethoprim 7.7 times and 2.6 fold for beta-lactams. Tetracycline and aminoglycoside resistant genes also shot up.  Unfortunately all of these are clinically important antibiotics.

Bacteria from the Proteobacteria group, which are also thought to be associated with UC and chronic HIV, exhibited the largest jump in numbers.

Of the 18 students returning from India, 12 had acquired drug resistant E. coli, which none returning from Africa had. In all, the scientists found 178 drug resistant genes in the students.

None of these students had taken antibiotics or been hospitalised, so the microbes with these resistance genes had clearly come through food and the environment.

Das’ most important work has been in understanding the role of gut bacteria in this transfer and the possible ways of limiting it.

The shelves of a metal cabinet in one corner of his office are lined with array upon array of test tubes, like bees sitting on a hive. Each of these 1,097 test tubes, he says proudly, contains a gut bacteria that he’s examined for resistance to nine different classes of antibiotics. To get a representative sample, the bacteria were isolated from Delhi, West Bengal and Assam. Then the genome of each of them was sequenced.

Simultaneously he looked at 3,700 pathogens found in the gut. These, according to him, have the ability to pick up DNA from gut bacteria and spread it to other bacteria. Was it possible to modify gut bacteria to minimise this spread?

What he found was that antibiotics influenced the transfer of DNA from bacteria to pathogens. This is a process that also happens in nature, but with very low efficiency. The dense microbe-laden environment of the gut compensates for the inefficiency, making the gut a hotbed of antibiotic resistance.

Some antibiotics speeded up this process more than others and made it more efficient—those were the ones most likely to lead to antibiotic resistance. Replacing these with antibiotics that had lower propensity to induce gene transfer could be one way of tackling resistance. Unfortunately some commonly used antibiotics like sulfamides are known to speed up gene transfer, as a result resistance to them in India is widespread. “But doctors continue to prescribe them,” says Das.

The antibiotic project was “huge,” he says, “we even found a microbe resistant to 22 different antibiotics. Where is this coming from? Is it possible to restrict it?” The first order of business, he believes, is to create a rapid detection system for antibacterial resistance.

***

This limited understanding of the microbiome is already throwing up treatment possibilities. Faecal matter transplant is one of them. It’s nothing short of a miracle for patients of IBD and diseases like Clostridium difficile infection in which the namesake bacteria causes diarrhoea and nausea.

Doctors speculate that FMT might help in a whole gamut of diseases—from multiple sclerosis and chronic fatigue syndrome to malnutrition, type 2 diabetes and autoimmune disorders like uveitis.

First, though, before FMT can be used widely we need to define a healthy “Indian” microbiome. This will help prevent the introduction of pathogens and ensure that the bacteria being transplanted are appropriate. Vineet Ahuja of AIIMS and his colleagues obtained 55 stool samples from Leh, Ladakh, 25 from rural Haryana and another 25 from urban Haryana.

The best subjects are those from Leh—their guts had few pathogenic bacteria, and their low exposure to antibiotics meant few antibiotic resistance genes. Dietary differences did translate into microbial differences, but could be easily tweaked for transplants.

A simpler and less radical way to address imbalances in the gut is to introduce new bacteria through probiotic foods. Specific probiotics have been found to reduce the duration and occurrence of diarrhoea in children, and help them gain weight and height. Work by the National Institute of Cholera and Enteric Diseases in Kolkata showed that diarrhoea infections came down by 14 per cent among children who received daily doses of Lactobacillus casei.

A simpler and less radical way to address imbalances in the gut is to introduce new bacteria through probiotic foods. Specific probiotics have been found to reduce the duration and occurrence of diarrhoea in children, and help them gain weight and height. Work by the National Institute of Cholera and Enteric Diseases in Kolkata showed that diarrhoea infections came down by 14 per cent among children who received daily doses of Lactobacillus casei.

In Delhi, Nita Bhandari of the Society for Applied Studies examined the microbiomes of 170 undernourished children before and after giving them a regimen of supplements and probiotics. She found that for kids below five recovery rates were higher. These could be improved by the targeted use of antibiotics.

The caveat with the use of probiotics is that Indian microbiomes are very different from those from other parts of the world. What is probiotic there may not be probiotic here. So probiotics have to be selected carefully. That’s not what companies marketing probiotic foods are doing. “For all you know,” laughs Das, “they’re selling placebos.” What might work better he thinks are prebiotics—substances that don’t contain bacteria but promote the growth of specific pre-existing ones.

All of these are tentative forays, revealing only the broad contours of the massive faunal garden that lives within us. Research in India has lagged behind, but is now picking up pace. The results of many of these current studies will emerge over the next few years.

Directions for the next phase of research are already emerging. Exploring the virome, the neglected viral component of the microbiome is the most imperative. Viruses are minuscule in number compared to bacteria, but cause many acute diseases like rotavirus, hepatitis, and are associated with cancers. They can alter bacteria, and potentially be used to engineer microbiomes. Work on them has been limited because molecular sequencing technologies are still being developed for them—a priority for scientists at RGCB.

Das is soon travelling to Japan to work on “molecular signalling”. The idea is to modify bacteria using antibiotics or viruses to produce molecules that can then modulate the growth of other bacteria.

Meanwhile, Manas Shukla has had relapses of UC, but faecal matter transplants seem to work every time. Immediately after his last FMT he did something he’d never imagined he’d be able to do—take a two-day road trip.

Akshai Jain has worked with Outlook Traveller, Mint and Tehelka. He now freelances, and his latest hobby-horse is science journalism.

(From the May 2017 edition of Fountain Ink)

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