Paper discussion: Gut microbiome predicts cholera susceptibility

From where I’m standing, this paper seems to have blown up: Human gut microbiota predicts susceptibility to Vibrio cholerae infection. The view metrics are quite high, and it’s been posted on Duke University’s news site and on the icddr,b blog. I wanted to talk about it not only because I find the topic fascinating, but also because it hits close to home- icddr,b is the institute I’m currently affiliated with (although the authors are from a different lab than mine).

The set up is this. Not all people are equally susceptible to cholera, even after you take factors like age, blood group, or nutritional status into account. In fact, you could have two people with virtually identical circumstances- ecological niches, one wants to say- be exposed to the pathogen, and they might not have the same clinical outcome. Even when everything else is seemingly equal, people show intrinsic differences in their susceptibility to the disease.

Of course, this in itself is a rather banal observation. Disease susceptibility cannot be exhaustively boiled down to environmental and demographic factors alone, there are also traits intrinsic to individuals. Genetics, for example, does determine aspects of one’s immunological preparedness against certain pathogens, so some disparity can be attributed to difference at that level. The interesting question, then, is teasing out these and other specific “intrinsic” factors that might have a say on clinical outcome. The paper under discussion chooses to investigate gut microbiome profile as a candidate.

The game plan is fairly simple. Find a group of people who are at high risk of cholera exposure (in this case- household contacts of cholera patients), keep tallies on who gets a cholera infection and who does not, compare gut microbiota between these two groups to see if variations are associated with cholera infection (or lack thereof).

Here’s how the sampling went. Let’s say, one morning, Timmy shows up to the icddr,b hospital with the tell-tale signs of cholera. Cholera patients are, incidentally, not hard to spot- their stool has a very characteristic “look”. Imagine chicken stock, but browner. Anyway, the doctors can’t rely on their stool senses alone to pronounce Timmy a cholera patient, he must also test positive for a V. cholerae culture test. Which he does. Timmy is now our index patient.

We then recruit his household contacts for our study. There are some inclusion/exclusion criteria though- they must live within the city, mustn’t be too young (<2) or old (>60), or have some other disease. We also exclude people with recent (in the past week) antibiotics use. The general logic of these criteria is, of course, to make sure we only choose people with otherwise representative microbiota, as opposed to those whose gut flora has been ravaged by antibiotics or modified by other disease conditions. The individuals passing these criteria are now enrolled into the study, and they will be subjected to copious amounts of rectal swabbing for the days to come. I wonder how they managed to get consent for this study. Anyway, Timmy’s household contacts are now in the study population, and we extract information about their nutritional status, age, blood group, and other relevant epidemiological factors as per protocol. The authors managed to recruit 66 Timmys and their households for the study.

For the next 9 days, we continue to collect data from Timmy’s household contacts. Our researchers visit them every day, get information about whether they have developed diarrhea, and probe their rectums with cotton-tipped swab sticks. In addition, we also collect blood- but this is done more sparingly, only on days 2, 7, and 30 (where day 1 is the day Timmy was identified as a cholera patient at the hospital, and day 2 is the day we started stalking his family for rectum rubs). These three prongs of monitoring serve the same purpose of detecting cholera infection, but do so in different ways:

1. Report of diarrhea indicate possible symptom of cholera
2. A rectal swab being positive for V. cholerae indicates cholera infection (could be symptomatic or asymptomatic)
3. A four-fold increase in vibriocidal antibody titer (between days 2 and 7, 2 and 30, or 7 and 30) indicates symptomatic cholera infection

If 1 is accompanied by 2 or 3- that indicates a symptomatic cholera infection, while 2 alone indicates the asymptomatic variety. They had to exclude 9 people from their study who showed “ambiguous clinical outcomes”, i.e. 1 without being accompanied by 2 or 3.

So we have settled on a study population at risk of cholera exposure, and we’ve set up a system by which we can track the people who develop cholera in the next 30 days from the ones they do not. Now comes the simple matter of taking their gut microbiota. On the same days that blood samples were collected from Timmy’s household contacts (days 2, 7 and 30, with day 1 being the day we met Timmy the cholera patient), another set of rectal swabs were collected for subsequent 16S sequencing.

Now, this study claims to be about gut microbiota, but the sampling is done via rectal swabs. Does the rectal swab population accurately represent gut microbiota? For starters, the rectum is quite a ways south of the intestinal lumen. This is a question the authors themselves bring up in the discussion. However, they also point out in one of the Supplementary files that their own research conducted earlier on the same population “demonstrated that rectal swab samples approximate 16S sequence results from stool samples”. I don’t know if that sufficiently allays the concern.

So after all was said and done, only 76 contacts (from 124 households) managed to escape the tough exclusion criteria, and it was now time to pull all the DNA from their rectal swabs and commence sequencing. This is a community composition analysis, meaning you’d only need to know what bacterial species are present- not what specific genes they encode; so only a region from the 16S rRNA sequence would suffice our purposes.

Up until this point, I wouldn’t blame you if the study design sounds underwhelming. Given institutional support, none of this seems super difficult to organize. The results might have been mundane as well. In fact, in their first volley, when they used the more garden variety models like univariate statistical tests and ANOVA, the authors found no association between cholera and gut microbiota. The sequence data was clustered into 4181 Operational Taxonomic Units at 97% similarity (OTU’s- remember, at this point you only have upwards of 97% similar clusters of DNA, but you don’t know if these clusters correspond to species, or any other taxonomic category that exists in reality. So provisionally, and for heuristic purposes, we choose to call them OTUs), but none of them were individually associated with susceptibility.

What makes the paper unique is what comes next. This is how I like to imagine the situation went down.

Remember, the sampling was done back in 2012-2014, so quite a ways back. The study design made eminent sense, the researchers had followed the protocol to a t, the microbial community structure data seemed to make sense as well. None of this was helping, though- the tried-and-true statistical analyses method had come up short. There seemed to be no way of predicting cholera susceptibility using gut microbiota. The researchers were sitting on a pile of valuable data, without a clue as to what to do with it. The government was getting ticked off (I don’t know who funded the study, but imagine the government was involved).

Ok so I started writing this setup with a lot of enthusiasm, but now I’m getting frustrated at my sheer inability to write fiction so let me just make this short: one of the researchers got into contact with Bruce Willis, brought him out of retirement despite his initial protests, and him and his ragtag band of outsiders solved the problem by utilizing their very particular, very obscure set of skills:

Machine learning.

Megan Fox may or may not have been involved. Bruce Willis dies at the end. For America.

Anyway, the machine learning model developed by the authors was indeed successful in what they started out to do. Without going into too much gritty detail, their first machine learning approach split up the study population into two groups. Data from the first group was used to train the model to predict cholera susceptibility based on particular gut microbiome profiles, and the model was validated by testing its predictions on the second group. This initial group split was kind of fortuitous- since there were two temporally separated cohorts in the study, the first group could conveniently be used as a “training” group, while the second could be used to test predictions. In subsequent analyses, however, the authors tinkered with their model by splitting the groups in other, random, ways. The predictive capacity was still retained, and this latter “cross-validation” scheme zeroed in on 88 Operational Taxonomic Units as being associated with cholera.

I can imagine the researchers being encouraged by this initial success. This showed that if you’re using the right toolkit, it is possible to find correlations between clinical outcomes and gut microbiome profile. Given how difficult it often is to attribute outcomes to microbiome (read chapter 5 in Ed Yong’s book, thank me later. Here’s my review in case you missed it), this is an important conclusion, as far as it goes. In the next important step of their analysis, the authors pick the 100 most important Operational Taxonomic Units (the earlier predictive 88, plus 12 more- better safe than sorry) and investigate their distribution between infected and uninfected individuals. Bacteria from the Bacteroides phylum were more prevalent among the uninfected, particularly members of the Prevotella genus- which is a common gut bacteria among healthy Bangladeshis.

At the tail end of this study, the team tries to build off of the fruits of this analysis, and use wet lab experiments to see if some of these predictive bacteria actually influence Vibrio cholerae growth. They do this via spent culture supernatant experiments, which consists of letting bacteria-X grow in a media for a specified time until all the nutrients are depleted (hence “spent culture”), filtering out the bacteria-X cells, and growing bacteria-Y in this spent culture supernatant. This is effectively growing bacteria-Y in the juices of bacteria-X. Since all the metabolites and secretions of bacteria-X are in the liquid, growing bacteria-Y in it simulates growing it in a bacteria-X “environment”, as if the two are growing together. This is thought to approximate the in vivo method of injecting a cocktail of two bacteria in (gnotobiotic) mice, and see how one affects the other. None of the predictive bacteria, however, were shown to affect V. cholerae growth one way or the other. There was one bacteria- not strictly predictive but still associated with ongoing V. cholerae infection- that stimulated V. cholerae growth. When the researchers wanted to investigate if this effect is mediated by influencing biofilm production, the experiment showed no effect, and the lead grew cold.

So this paper is an example of how sophisticated analyses can really blow up an otherwise mundane experimental setup. The study design itself is simple and straightforward, the metagenomics sequencing or analyses do not involve any high-end mumbo-jumbo. The raw data generated up to this point wouldn’t look super impressive or “out there”. The paper seems to have the significance that it has due to the analytical tools adopted following the generation of raw data.

I don’t know if modern biology has a bottleneck at the level of analysis. I mean, how difficult is it to develop machine learning models? If not particularly difficult, and there’s no significant bottleneck there, then I’m not sure why we’re not seeing more studies of this kind.

I Contain Multitudes: Storytelling in Microbiology

[If you want a more descriptive, matter-of-fact commentary on this book, be sure read my review on Goodreads]

So a few days back, I came across an article from PlosBiology, where the authors run a thought simulation of how life would be without microbes. In the broadest of brush strokes, the authors talk about a precipitous decline in photosynthesis rates, rapid accumulation of waste products with no microbes to break them down, and the global biogeochemical cycle “grinding to a halt”. The world without microbes that they imagine is not necessarily a ceteris paribus one, as they occasionally speculate about the extent to which human interventions would be able to cope with the problems (e.g. production of synthetic fertilizers via the Haber-Bosch process being used to compensate for the lack of nitrogen fixation). This already sounds too much like what came out of a dystopian worldbuilding project. The authors certainly seem to be having a lot of fun when they predict

[C]omplete societal collapse only within a year or so, linked to catastrophic failure of the food supply chain. Annihilation of most humans and nonmicroscopic life on the planet would follow a prolonged period of starvation, disease, unrest, civil war, anarchy, and global biogeochemical asphyxiation.

Ed Yong’s I Contain Multitudes can be thought of as an extended commentary of this paper, except his strokes are finer than they are broad, and certainly deft enough to reach the crevices of such a complex topic. The book could easily have been titled Why Microbes Matter, and all it would lose is its literary flare.

That, however, is too big of a loss. Let’s start there.

In a recent episode of TWIM, Dr. John Warhol- the man behind the New Jersey State Microbe and the Periodic Table of Microbes– was quoted as saying that microbiology is cooler than astrophysics, but the latter have better TV shows. I don’t necessarily want to defend that proposition, but it is undeniable that microbiology, as a discipline, is chock full of fascinating stories. These stories are of a genre that approximates romantic drama- romantic, because they are ultimately stories about relationships; and drama, because all of those relationships are complicated. This is not least because of the so many different contexts they take place in, from the acid-drenched linings of our intestine to dying coral reefs, from hydrothermal vents to the posterior secretions of a hyena. It’s not difficult to see that at the hands of an adept storyteller, they have the potential to put Neil Degrasse Tyson out of business. With Multitudes, we can see that happening.

Let me try to make this more concrete. I was but a wee freshman when I was first taught about the historical debate on abiogenesis, i.e. spontaneous generation of life from non-life. I remember skimming over some references to Aristotle, and smiling forgivingly at the ignorance of this scientifically unbaptized heathen when he said oysters emerge from dirt. There really was no reason to suspect any additional depth to the story.

In his discussion on the developmental pathway of certain organisms, Yong brings up these views attributed to Aristotle. Apparently, Aristotle beliefs were based on his observations of some submerged clay pots. The pots were thrown overboard from a ship, where they collected mud, and eventually oysters. But where did the oysters come from? They weren’t there when the pots first landed, or until the mud gathered. When you think about it, they really couldn’t have come from anywhere, for the simple reason that oysters don’t move. For Aristotle, the only logical explanation for this set of observations was- they emerged from the mud.

In my freshman year, I didn’t know, or consider, that Aristotle even had an argument. In fact, without knowing about the microscopic larvae of oysters which can move around, I can see university freshmen being stumped by his argument. There’s clearly a story here that I was supposed to know, that could’ve made me think. I could’ve seen myself taking sides in this argument as it unfolded throughout history, sometimes agreeing with one side and sometimes another, instead of chalking all pre-modern scientific opinions as speculative fiction occasionally involving acid.

Here’s a more extended example on the same theme- I was supposed to know a story, but I didn’t, and that sucks. In my sophomore general microbiology course, I remember learning about a certain group of organisms called chemotrophs- organisms that do not depend on sunlight for energy. The reference came up in the context of a classification of microbes by their feeding habits, e.g. those who depend on solar power are phototrophs, those who don’t are chemotrophs, those who need organic carbon are heterotrophs, those who don’t are autotrophs, and so forth. I really don’t remember chemotrophs as being anything other than a name nestled away in one of those standard charts you’d have to memorize (except maybe a vague reference to hydrothermal vents).

Multitudes has a few pages on deep-sea chemotrophs as well (chapter 7). The context is completely different, though. The chapter is on how animals and plants can often piggy-back on the extremely specialized and niche feeding habits of microbes to survive in habitats that are positively weird. You may not be able to survive on plant sap alone (as some aphids do), if for no other reason than it has a rather monotone nutrition profile- high sugar, no amino acids. However, if microbro in your gut produces amino acids for you, then the two of you have a fighting chance. Examples of such microbe-assisted colonization of otherwise hostile and nutrient-poor habitats are literally innumerable. What you may not have seen coming, however, is that this list of nutrient-poor feeding habits also includes a hearty diet of nothing. And by that I don’t mean the dragon warrior diet. Some animal, with help from their microbros, can survive without eating anything.

With this set up, Yong indulges us with the story of the discovery of autotrophs:

In February 1977, a few months before the Millenium Falcon blasted outwards into space, an equally adventurous vehicle called Alvin travelled downwards into the oceans. It was a submersible big enough to house three scientists, small enough to stop them from stretching their arms, and sturdy enough to dive to incredible depths. It entered the water 250 miles north of the Galapagos Islands, where two tectonic plates drift away from each other like estranged lovers. . .

The Alvin team descended. The blue of the surface gave way to black, the all-consuming black of the abyssal ocean. Blacker than black. Black punctuated by only by the occasional flashing of bioluminescent creatures and, eventually, the submersible’s lights. At a depth of 2,400 metres, about a mile and a half straight down, the team found the [hydrothermal] vents they had predicted, but also something they had not – life, in extreme abundance. Huge communities of clams and shellfish clung to rocky chimneys. Ghostly white shrimps and crabs clambered over them. Fish swam past. And strangest of all, the rocks were encrusted with hard white tubes that ended in the crimson plumes of giant worms. They looked like tubes of lipstick that had been pushed out too far, or something even more sexually suggestive. They were actually giant worms.

In this supposedly lifeless underworld, untouched by the sun, buffeted by water that can reach 400 degrees Celsius, and compressed by the enormous pressure of the ocean above, the Alvin team had discovered a hidden ecosystem as rich as any rainforest.

I think this is a pretty representative taste of how the book tells its stories.

Forgive my amateur lit crit, but seriously- I need a moment to gush. Most of the stories in this book describe the complexities of symbiotic relations, and how life’s processes operate in those contexts. There’s a story almost equally interesting as that one, however, which is our coming to know of them. There we were, stuck in a world flushed with sunlight, content with the belief that all creatures require solar energy. That singular proposition defined so much of our science- we knew better than to look for life in places beyond the sun’s reach. When Alvin went down, the scientists had the same assumptions about life on vents that they would have had for life in Mars- none, because they don’t exist. In fact, for Mars there are at least a contingent of scientists who would speculate differently, if for no other reason than how little we know about life in outer space. But when our science takes as axiomatic the proposition that life requires sunlight as a matter of necessity– finding life on hydrothermal vents would be simply impossible. Not unprecedented, or improbable, but impossible, in the sense of the world’s nomic architecture not allowing for such a contingency.

And yet, there it was. Life, nay, a thriving ecosystem akin to a rainforest where there wasn’t supposed to be any. The presence of life at this depth was so inconceivable, so out of sync with scientific reality back then, that the Alvin didn’t even think about bringing a biologist with them. In a Matt Damon-esque science-the-crap-out-of-Mars move, they brought back specimens preserved in leftover vodka.

Now, isn’t that an endlessly fascinating story? Yong would answer in the positive, which is why the “ecosystem” stories in the books are punctuated by “discovery” stories. Sure, knowing about how the world operates is great, but to truly appreciate the world as it is (literally), we need to appreciate how it appears (phenomenologically). How do you think Leuwenhoek felt when he found little critters all over the world? How profoundly did it shake our perception of the world? Wouldn’t it be great to know? Well, Yong got you covered (chapter 2).

Anyways, back to the chemoautotroph story. So about those giant worms- they had no mouth, no gut, and no anus. We had the first instance of an animal that literally did not eat. It later came to pass that certain parts of their bodies were packed full of crystals of pure sulphur, which furnished the first clue that microbes were probably part of this story. Indeed, this eventually led to the discovery of a class of microbes- the chemoautotrophs- which processed sulphur compounds to “make food in a way that was utterly different to anything known at that time”.

When I was reading these pages, I couldn’t help but wonder- why didn’t I hear of this? I have two degrees in Microbiology, and one of my bachelor level courses dealt specifically with chemoautotrophs. But we didn’t know how scientists made the discovery. This lesson could’ve been taught with a story- we were still in sophomore year, and so definitely not too old for stories- but it was instead taught with a list of microbes with different energy and carbon requirements. We didn’t hear about the giant lipstick worms, nor the sulphur crystals, nor the voyage to the hydrothermal vents, nor the impact it had on prevailing scientific epistemology, and we definitely didn’t hear about the vodka.

Until Multitudes.

I’ll now awkwardly round off this article with a few other story suggestions from the book:

• Leuwenhoek’s life and times (chapter 2). You may know him as the man who invented the microscope, and proceeded to see, measure and draw literally every tiny moving thing he could find. He was also what could only be described as a science blogger, as all of his letters to the Royal Society were “full of local gossip and reports about Leeuwenhoek’s health”. I like to think he also wrote book reviews and made frequent pop culture references.

 

• How Jeff Gordon’s team uses robotics to make gut microbe cocktails (chapter 5). First, you identify each member of a gut ecosystem, and arrange all of them into an organized library. You can then get the computer to generate an exhaustive combinatoric list with the library members. Finally, you tell the computer to mix the microbe cocktail you want, and the robotic extensions get to work. You can then study the role not only of each of the microbes, but particular groups of them working together in defined proportions, by studying the effect of each intelligent selection of cocktail.

 

• How the beewolf passes on her microbiome to her young (chapter 6). After laying the egg, the beewolf excretes a paste of Streptomyces– a group of microbes famous for producing antibiotics. This paste serves both a semantic function (it lets junior know where the exit is) and a physiological one (junior makes its cocoon out of the paste mama left, which allows it to fend off fungal infections). A somewhat less poetic strategy is adopted by a Japanese stinkbug, which craps hot bacteria-filled jelly to cover the eggs just as they’re about to hatch. In a stunning display of mother-child bonding, the hatchlings proceed to eat up mama’s excreta, which loads them up with the microbes they need to survive. Nothing says family values in the animal kingdom like ritual coprophagy.Fun fact- baby koala’s lick their mother’s anuses to seed themselves with microbiome, which renders scientifically illiterate memes such as this one untrue and irrelevant.

 

• Everything in chapter 8, which I must say is my favorite one in the book. The stories include how microbial genes from the ocean made their ways into human gut via Japanese seaweed eating habits, how Wolbachia– the Genghis Khan of biology- basically “liberally sprayed the tree of life with its DNA” owing to its promiscuous horizontal gene transfer tendencies, and- most of all- The citrus mealybug. Lord be with me on this one. It has a symbiotic life that can only be approximated with the example Yong himself provides- the Grey Witches of Greek Mythology that had one eye and one tooth between them. Except instead of eyes and teeth, we have one insect and two bacteria sharing essential genes so they could survive as a single functioning organism. Except, plot twist: the two bacteria are missing essential genes that all other organisms have. As it happens, some time in the past, three other bacterial groups lived together with the insect as well, and they incorporated their essential genes to the insect genome. It’s only with a complex, confused collaboration among six shrunken, misshapen bits of living organisms that the group as a whole can function as an individual. Yong describes the discoverer of the phenomenon, John McCutcheon in the following way:
  

  He talks with a mixture of awe, confusion, and faint embarrassment, as if his discoveries are so outlandish that he barely believes them himself. And yet, here they are.

Seriously, just read this book. Read about how mother’s milk is required for nourishment not primarily of the baby, but certain of its microbes. Read the story of Sodalis and how insect hosts twist and bend and mangle the genomes of its microbial passengers. Read about how Vibrio fishceri lights up the insides of a squid that looks like a golf ball, how mucus is the coolest thing ever, and how in the future you may be prescribed a cocktail of microbes, each of which were picked from what can only be described as a menu.

And if none of that interests you, there’s always animals eating each other’s feces, or coprophagy.

There is just. so. much. coprophagy.

It’s not even funny at this point.