New Paper: Stool Microbiota and Vaccine Responses of Infants

As part of getting my blogging groove back on, I thought I would talk about my part in this new paper. Together, Nazmul (the first author) and I analyzed the gut microbiota of a large cohort of infants in Bangladesh, paying extra attention to the bifidobacteria. Nazmul (and others) did a ton of work looking at the response to various vaccines in these infants. We found that the response to some vaccines was better in infants that had higher levels of bifidobacteria.

A couple of things of note about my contribution to this paper:

1) The correlations between vaccine response and the microbiota sometimes differed between species and subspecies of bifidobacteria, which means that A) not all bifidobacteria are equal, and B) that the effects can be correlated to a manageable set of genes. We were able to trace the effects down to subspecies level within the B. longum group thanks to a new method that I helped develop, which should be getting it's own paper soon.

2) The levels of bifidobacteria in these infants were really high compared to some other cohorts I have seen or studied myself. This was despite the high rate of c-section birth noted in the paper. I find this super interesting.

3) Parents, please vaccinate your kids so that stuff like this and this doesn't happen. Herd immunity is a real thing. When you choose not to vaccinate your child, you put other kids who can't get vaccinated for whatever reason (immunocompromised, etc.) or for whom vaccines don't work at risk. No matter what type of bacteria live in your child's gut, vaccination is almost assuredly better than no vaccination.

Anyway, I was glad to use my Bif-TRFLP technique on a new set of infants and be involved in this cool study. Grad school has given me the chance to collaborate on projects with a lot of great people on interesting things. I'm glad Charles Stephensen got interested in gut microbes and started this collaboration with my advisor. I'm increasingly convinced that collaborations are the best way to do impactful research. It's so hard to be an expert in all the relevent aspects of today's unsolved problems.

New Paper: A Comparison of Two Probiotic Strains of Bifidobacteria in Premature Infants

I wanted to write a little about my small part in this paper. My work in this collaboration is one of the times when I have most felt that I was making a real-world impact. Mark Underwood, the lead author on this paper, is a neonatologist and assistant professor at the UC Davis Medical Center, where he practices medicine in the neonatal intensive care unit (NICU) and cares for premature infants ("premies"). He had a use for the tool that I developed in my Bif-TRFLP paper, so I got to participate in this cool study.

A premie and its tiny little feet
from https://www.ucdmc.ucdavis.edu/children/clinical_services/NICU/

One of the major issues that premies face early in life is necrotizing enterocolitis (NEC), which is thought to arise in part due to imbalance in the gut bacterial community. Premies have underdeveloped intestinal tracts that are extra prone to infections. Probiotics have often been used to try to prevent this life-threatening disease, at least some of the time successfully. The idea is that the good bacteria can help the immature gut resist colonization by pathogens by taking up all the space, eating all the "bacteria food," and producing antimicrobial metabolites. There has been a lot of debate about which strains of probiotics to use, and how best to use them. This paper tested a few different supplementation mixes and how they affected the microbial community in the gut of premies. It showed some interesting results about improving the diversity of the community, improving levels of beneficial bifidobacteria, and showed species-level differences in the efficacy of the intervention. Here is a link to the paper.

My part in this paper was to use my Bif-TRFLP technique to figure out which species of bifidobacteria were found in the guts of some of Mark's NICU infants: before, during, and after a variety of treatments. I discovered that B. infantis, a strain that is known to eat the oligosaccharides (sugars) found in breast milk, colonizes infants better than the B. lactis strain we tested. When given breast milk the B. infantis strain was the dominant bifidobacteria in the infants, even if they were given B. lactis and not B. infantis. 

This begs the question of how the babies that were given B. lactis ended up colonized by B. infantis instead. They take lots of precautions in the NICU to try to avoid spreading bacteria around the environment, and try to keep these fragile infants from being exposed to harmful bugs like those that cause NEC. (You can take a look at the set-up they have in this really cool virtual tour of the NICU.) This work seems to show that the B. infantis floating around the NICU (maybe from the infants inoculated with it, stray inoculum itself, or just normal environmental strains, who knows!) can outcompete the non-human-milk-adapted B. lactis strain as long as they are given breast milk. The environmental B. infantis probably gets into the infant in much lower numbers than the comparatively massive B. lactis supplementation given to the study babies, so this is a remarkable finding.

This just goes to show the importance of strain specificity to an environment. Not all probiotics (or bacteria in general) are created equal, and they are not always well adapted to a given set of conditions. I really like being a microbial ecologist because it gives me the tools to answer questions like these. Evolution drives microbial community structures, and thinking about how that affects real world problems is a really rewarding exercise to me.

UPDATE- This work was profiled in the press. See here

New paper: Establishment of a Milk-Oriented Microbiota (MOM) in Early Life: How Babies Meet Their MOMs

A new paper just came out on which I am an author:

Establishment of a Milk-Oriented Microbiota (MOM) in Early Life: How Babies Meet Their MOMs. (Functional Food Reviews, Vol 5, No 1 (March), 2013: pp 3–12). Authors: Angela M. Zivkovic, PhD, Zachery T. Lewis, BS, J. Bruce German, PhD, David A. Mills, PhD

Link to the paper here if you have an institutional or other subscription.

I did not come up with the term "MOM" (Dave Mills did) but I thought it was brilliant, and I am glad to be part of the paper that coined the term. Its a good way to emphasize the importance of bacteria in helping a baby grow and thrive.

Little Johnny just found out he has TWO moms! (A Mom and a MOM.)
From http://www.flickr.com/photos/56323141@N00/2658949664

This paper is a review paper that talks about some of the latest research on how breast milk influences the microbes that live in babies. There are some components that help good bacteria grow (like the funky short sugar chains that good bacteria can eat) and some components that stop bad bacteria from growing (proteins that act like antibiotics, antibodies, and decoy molecules that fool pathogens into thinking they have attached to a cell and stop them from infecting us).

In this paper we talk about all the benefits that come from having a good mix of bacteria or "microbiota" in a baby. Associations have been found with the microbiota that include resistance to infection, reduced allergies, and reductions in other inflammatory conditions. There are even initial hints that autism might have a microbial component. This all suggests that having microbiota that doesn't cause inflammatory reactions early on in life might be important to educate our bodies about what is friend and what is foe.

The theory is that if the body has not-so-friendly bacteria in it early on, it seems to be hypersensitive to common allergens later in life. Bifidobacteria are very commonly found in breast-fed infants and seem to meet this non-inflammatory criteria. There is some evidence to suggest that they help calm the immune system down and keep the wrong things from getting to places where they might start triggering immune responses.

The "hygiene hypothesis" states that a lack of exposure to things that our distant ancestors commonly encountered ("dirty" things like feces and parasites and ... dirt) leads to our immune system overreacting to things that aren't really bad. As if it just gets bored and wants something to do, so it picks a fight with pollen or your cat's dander.

Felis catus, a common source of allergens. Sorry, I couldn't find an actual photo of one anywhere on the internet. Strange. I mean, I knew that very few people upload baby pictures, but it seems cats are even less common...

Some people think that if you have enough good bacteria around to keep the immune system busy monitoring harmless things it won't learn to react to the wrong things. With the recent explosion in pre-term and c-section births (both not nearly as survivable before modern medicine, and which are known to cause the baby to not get as healthy of a microbiota as easily) and the decline in breastfeeding, it seems possible that this could lead to at least some of the difference we observe in how many people in the "first-world" get allergies compared to how few people get them in the "third-world." Historically, mothers breast-fed their infants for a lot longer. They would continue to breast feed while weaning, a process which lasted a lot longer than it does now. The presence of the ingredients in breast milk and the bacteria they enrich might have helped keep the immune system from reacting to things like gluten and other common food allergens as they were introduced to the child.

C-section and pre-term births often cause altered microbiota profiles in the infant, which may have to do with problems in getting good microbiota from the mother. Shortly before this paper was published, but after it was submitted, Seth Bordenstein et al. came out with a really nice paper about how mothers pass on microbes to their infants across many species, not just humans (link here). You can go there for more details. I would have cited this paper it if it had been out.

One other point we made, which I though was important, was that diversity in an ecological community is not necessarily always a good thing. The common assumption I have seen many papers make is that a more diverse community is more stable and resistant to disturbances, and is always better. It may be that early on a more controlled community that is less diverse but maintained by specific inputs from the mother might be the better way to go for the overall health of the infant. A new Nature paper just came out on this subject, here.

Anyway, I thought we wrote a a nice overview paper if you are interested in learning about the benefits of babies being seeded with "good" microbes.

Story Behind the Paper*: Bif-TRFLP

One type of post that I plan to regularly put on my blog is the "Story Behind the Paper" series, where I talk about what motivated the research in my papers and tells the paper's backstory. In an aggregation of firsts, this first installment will be talking about my first ever first-author paper, "Use of bifidobacterial specific terminal restriction fragment length polymorphisms to complement next generation sequence profiling of infant gut communities." (Published in the Journal Anaerobe v. 19 (2013) pg. 62-69)

Background

I study the gut communities of infants. There are a couple of nice things about infants that make them an important and useful model. First of all, who doesn't like babies? They are cute! No one wants them to get sick, and studying their gut microbes can help us understand how to prevent and treat gastrointestinal (and possibly other) diseases.

See! Look how cute!
By Weird Beard (Happy) [CC-BY-2.0 (http://creativecommons.org/licenses/by/2.0)], via Wikimedia Commons

Premature infants in particular can have lots of problems, including the very nasty necrotizing enterocolitis. It is thought that a large cause of these types of diseases is an imbalance in the gut community. If there is not enough good (or at least neutral) bacteria in their guts this leaves a spot open for bad bacteria to grow. If we put lots of good bacteria in, the good bugs (metaphorically) eat all the food and sleep in all the beds and there is no room for bad bacteria. This is the concept behind probiotics.

One thing that has held back probiotics as a field is that is is hard to know which effects are caused by which factors. There are a lot of variables, lots of things going on in the gut. Everything you eat introduces new bacteria to the environment and new things for bacteria to eat. Different species of bacteria have "favorite foods" that they like to eat, so what you choose to eat can have a big impact on what type of bacteria can grow in your gut. How can you tell when an effect you are seeing is caused by something different from what you are measuring?

Babies are nice because early on, they mostly eat one thing... breast milk! Many health organizations recommend exclusive breast feeding for at least the first several months of life. This gets rid of one variable that confounds studies on adults. (You usually have to pay adults pretty well to control everything they eat if you want to study them. How much would you charge someone for them to be able to tell you everything you could eat for weeks on end? Tell me in the comments... The economist in me would really like to know!)

If you want to study gut bugs, it usually means you need to collect poop. (There are studies that take pieces of the intestine and other similar methods, but they are much harder to do.) Another nice thing about babies is that most parents are already collecting the their baby's poop by using diapers, and are happy to make a few bucks off of letting us take some.

Now to my paper.

One thing the lab I am in works on a lot is trying to figure out what the gut community of babies consists of. The bacteria my paper studies are the most abundant member of the gut community in most infants, and are considered "good bacteria." They are called "bifidobacteria."

Bifidobacteria as seen in a microscope.

There has been a very important methodological development in biology recently called next-generation sequencing (NGS) that can provide us with data on the microbes in an environment.

The original type of Next-Generation Data
http://memory-alpha.org/en/wiki/Data

It does this by sequencing (finding out the what the order of the A's T's G's and C's is) DNA that is extracted from the microbes in the environment and counting the numbers of DNA snippets that match the sequences of different types of microbes. This has been revolutionary and has enabled us to study things we couldn't before. One limitation of the method (at the moment) is that it can only tell us what is there at a coarse taxonomic level. I'll try to explain this by using an analogy to a more familiar environment... your neighborhood! NGS would tell us how many dogs there were in a neighborhood, but it wouldn't tell us whether they were wolves or dachshunds or chihuahuas or St. Bernards. It could definitely distinguish a cockroach from a human from a shark from a bird though. The problem is that sometimes we really care whether gut bacteria are "wolves" or "chihuahuas." You would probably care about that if you were going to move into a neighborhood, right?

My neighborhood, certified shark free since 1954.
http://www.housing.ucdavis.edu/housing/apartments_orchard_solano.asp

Since what we care about in babies is bifidobacteria, I (together with lots of help from lab members) designed and validated a method that will tell us what species of bifidobacteria are in a baby, since NGS won't do that right now. Since different species of bifidobacteria have different genes that do different things, this will let us test lots of different hypotheses about why bifidobacteria are so common in infant guts, what they do in that environment, and what health effects they have. The method involves taking the DNA from the bacteria in the environment and cutting it with special DNA-cutting enzymes called restriction endonucleases. The enzymes cut the DNA from different species of bifidobacteria in different places, which lets us quickly and cheaply tell them apart from each other by looking at the sizes of the pieces.

Next-generation sequencing methods are constantly improving, and might shortly make doing this unnecessary for lots of purposes, but the lab I am in didn't want to wait for that improvement to answer some important questions. We are using this technique as a part of several other studies now.

If you'll indulge me in some inside baseball of science, I want to note that we ended up submitting this paper twice, to two different journals. The first submission was rejected for reasons that (in my opinion, from my communications with the editor) had less to do with the scientific validity of the paper, and were more about it not being "important" enough for that particular journal to publish. This first journal (which is in the same prestige "tier" as Anaerobe) has the right to do that of course, but it slowed down the publication of the paper. We made some very minor changes (emphasizing how the method is complementary to NGS approaches) and resubmitted to Anaerobe. Anaerobe accepted the paper directly after hearing from the peer-reviewers, who had "no substantive comments" (we didn't need to make any changes). Several of my co-authors on the paper commented that that had never happened to them in their publishing careers, to have a paper accepted with no revisions (and they have a combined 40+ years of publishing under their belts.)

I say this not to out of indignation or a need to be publicly vindicated (since everyone usually thinks their papers are good and should be accepted), but to point out the somewhat capricious nature of scientific publishing. It makes me appreciate the PLoS One model of publishing everything technically sound regardless of perceived importance even more. For those of you who want to read my paper, Anaerobe has a pay wall, so unless you have a subscription (or are part of an organization that does) you may have some trouble. Another point for open access publishing. Here is a PubMed link.


*Note- Hat tip to Jonathan Eisen for the idea of a "Story Behind the Paper" series.