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.