Many of us have heard that it is possible for viruses to cause cancer—awareness campaigns like the one below, on a London bus, have done a fantastic job of raising awareness of this fact:
Few of us, however, are aware that bacteria can also cause cancers. The most notable example of this is Helicobacter pylori, the organism which causes stomach ulcers and gastritis. With time, inflammatory ulcerative conditions in the stomach lead to a substantially increased likelihood of gastric adenocarcinomas (stomach cancer) and MALT lymphomas (immune tissue cancer)(1,2).
As only 10-20% of patients develop ulcers/inflammation, and 1-3% of those patients develop cancers, this is a minor & indirect mechanism of carcinogenesis (“cancer-induction”) by a bacterium, but an important one nonetheless. As such H. pyloriis the only bacterium currently considered to be a Class 1 carcinogen by the World Health Organisation’s International Agency for Research on Cancer—a category which includes cigarettes, alcohol, and plutonium (3,4). However recent research now suggests that another species may cause cancer much more directly—this one:
The cancer-causing bacterium in question is Fusobacterium nucleatum, a species once thought to be a relatively harmless member of the oral microbiome. Indeed, we have known of its involvement in the oral disease since 1898 (5); its carcinogenic behaviour, however, was not observed until 2011 (6), and the mechanism was not established until 2013 (7).
In the mouth, F. nucleatum acts as a “bridge species” (8). This means that it is neither among the first (“initial colonisers”) nor last species to coat bare tooth enamel—in and of itself, a completely harmless action. However, it is a very “sticky” bacterium, to which others can adhere easily. By attaching to the initial colonisers which coat the tooth, it provides a surface to which pathogenic bacteria can bind in a process known as “bacterial co-aggregation and co-adhesion”(9). Thus, F. nucleatumpromotes an ulcerative and even necrotising form of gum disease called Vincent’s angina or “trench mouth”, alongside other oral disease, by creating a surface which allows harmful bacteria to live and potentially thrive.
While gum disease and other oral tract conditions are certainly of interest when considering the pathology of F. nucleatum, its role in colorectal cancer is altogether more intriguing. Researchers initially established that this species was present in the colorectal tumour microenvironment in 2012 (6). This was an interesting finding on its own, but subsequent research revealed an even more surprising set of findings: F. nucleatum not only thrives in the tumour environment, but can act to induce tumorigenesis in order to create a tumour within which it can thrive (10).
Mechanism of carcinogenesis
So how is it that F. nucleatum “directly” induces cancer formation? The answer lies in a well-understood metabolic pathway known as the “Wnt pathway” (10). This consists of a series of proteins and interactions between them, which eventually lead to a cellular response to whatever phenomenon initially triggered it. The Wnt pathway is known to be involved in cell growth and replication—it is a classic example of a cell proliferation pathway, used as an example in the education of many undergraduate life scientists, and studied the world over.
The proteins which initiate the Wnt pathway, β-catenin and E-cadherin, are bound together in a superstructure known as a “protein complex”. In order for the pathway to be triggered/activated, some trigger molecule/protein has to modify the E-cadherin’s chemistry by adding a phosphate group to it. This then leads to the release of beta-catenin from the protein complex, and the translocation (movement) of the beta-catenin into the nucleus. There, it acts on the cell’s genome to induce the production of proteins which lead to cell growth and replication.
This is precisely the mechanism which Fusobacterium nucleatum exploits in the colon and rectum. It “hijacks” the Wnt pathway by producing a protein, FadA, on its surface. FadA acts as a trigger for the pathway, precisely as described above: it phosphorylates the E-cadherin and induces the movement of beta-catenin into the nucleus, and thus promotes cell growth. This is not normal, as (canonically) bacteria do not dictate growth and development in mammalian cells; this is therefore an abnormal or aberrant cell growth. If this sounds eerily similar to descriptions of cancerous tumours, that is because it is precisely a cancerous colorectal tumour which F. nucleatum’s FadA induces.
So now we have seen how a bacterial infection can lead to tumour formation—but I mentioned earlier that this bacterium can not only do this, but also thrive within the newly-created tumour microenvironment. Indeed, F. nucleatum’s array of surface proteins is not merely limited to FadA. Another interesting surface protein which it expresses is Fap2. This protein’s roles in colorectal cancer are manifold: in the first instance, it allows F. nucleatumto bind to a special sugar residue (Gal-GalNAc) which in mammals is only ever found in/on cancerous cells (11). Once the bacterium has bound, it is able to enter within the tumour mass—explaining the findings in Castellarin et al (2011) which showed a high abundance of this species in colorectal cancers.
This however is only one of its roles: once it has allowed the bacterium access to the inside of the tumour, it then induces the “autophagy pathway”, which enables the bacterium to prevent chemotherapy from killing cancer cells. Cells die either via autophagy or apoptosis, and you can think of this as a dichotomous choice: either one occurs or the other occurs. Since chemotherapy drives apoptosis to kill cancer cells, Fap2’s induction of autophagy effectively prevents any chemotherapeutic action (12).
Thus, F. nucleatumis able to 1) induce, 2) enter, and 3) sustain a cancerous tumour, even in the face of chemotherapy. So the next time someone tries to insist that bacterial infections cannot cause cancer, send them a link to this article and help raise awareness of this absolutely mind-blowing new development in the scientific understanding of cancer.
1. Nakamura S, Matsumoto T. Helicobacter pylori and gastric mucosa-associated lymphoid tissue lymphoma: Recent progress in pathogenesis and management. World J Gastroenterol(2013) 19:8181–8187. doi:10.3748/wjg.v19.i45.8181
2. Herrera V, Parsonnet J. Helicobacter pylori and gastric adenocarcinoma. Clin Microbiol Infect(2009) 15:971–976. doi:10.1111/j.1469-0691.2009.03031.x
3. IARC. Biological Agents. IARC Monogr Eval Carcinog Risks Hum(2012) 100B:1–443. doi:ISBN 978 92 832 1319 2
4. Vogiatzi P, Cassone M, Luzzi I, Lucchetti C, Otvos L, Giordano A. Helicobacter pylori as a class I carcinogen: Physiopathology and management strategies. J Cell Biochem(2007) 102:264–273. doi:10.1002/jcb.21375
5. Bennett KW, Eley A. Fusobacteria: New taxonomy and related diseases. J Med Microbiol(1993) 39:246–254. doi:10.1099/00222615-39-4-246
6. Castellarin M, Warren RL, Freeman JD, Dreolini L, Krzywinski M, Strauss J, Barnes R, Watson P, Allen-Vercoe E, Moore RA, et al. Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma. Genome Res(2012) 22:299–306. doi:10.1101/gr.126516.111
7. Kostic AD, Chun E, Robertson L, Glickman JN, Gallini CA, Michaud M, Clancy TE, Chung DC, Lochhead P, Hold GL, et al. Fusobacterium nucleatum Potentiates Intestinal Tumorigenesis and Modulates the Tumor-Immune Microenvironment. Cell Host Microbe(2013) 14:207–215. doi:10.1016/j.chom.2013.07.007
8. Bolstad AI, Jensen HB, Bakken V. Taxonomy, biology, and periodontal aspects of Fusobacterium nucleatum. Clin Microbiol Rev(1996) 9:55–71.
9. Kolenbrander PE. Oral microbial communities: biofilms, interactions, and genetic systems. Annu Rev Microbiol(2000) 54:413–437. doi:10.1146/annurev.micro.54.1.413
10. Rubinstein MR, Wang X, Liu W, Hao Y, Cai G, Han YW. Fusobacterium nucleatum Promotes Colorectal Carcinogenesis by Modulating E-Cadherin/β-Catenin Signaling via its FadA Adhesin. Cell Host Microbe(2013) 14:195–206. doi:10.1016/j.chom.2013.07.012
11. Abed J, Emgård JEM, Zamir G, Faroja M, Almogy G, Grenov A, Sol A, Naor R, Pikarsky E, Atlan KA, et al. Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc. Cell Host Microbe(2016) 20:215–225. doi:10.1016/j.chom.2016.07.006
12. Yu TC, Guo F, Yu Y, Sun T, Ma D, Han J, Qian Y, Kryczek I, Sun D, Nagarsheth N, et al. Fusobacterium nucleatum Promotes Chemoresistance to Colorectal Cancer by Modulating Autophagy. Cell(2017) 170:548–563.e16. doi:10.1016/j.cell.2017.07.008
In a world buzzing with political and economic uncertainty, anxiety and depression have run rampant in our society; the most recent studies estimate that 40 million Americans — or roughly 18% of the American population — are affected by the detrimental affects of anxiety (1). And with increasing research and curiosity surrounding the mysterious microbial inhabitants of our gut, ongoing research continues to unravel their not-so-subtle involvement in our well-being.
It is well established in the research community that our microbiome has profound affects on our weight, the production of vitamins and food digestion. But is it too far-fetched to believe that microscopic life-forms living in our intestines influence our brain functions? Can bacteria communicate with our brains to determine our moods, feelings and emotions?
It seems likely.
Physiologically speaking, there is no reason why this communication can’t happen. Of the billions of nerves that thread through our bodies, one of them — termed the “vagus nerve” — serves as a direct connection between the intestines and the brain. This connection, which is formally referred to as the “gut-brain axis”, has been a critical area of research for more than a decade. What we have discovered in that time is that the vagus nerve seems to be bi-directional, meaning that while the brain can act on on the gastrointestinal tract to shape its bacterial composition, at the same time gut microbes can produce certain chemicals that act on the brain (2).
Initial excitement was sparked in 2004, when a landmark study observed that mice that were devoid of bacteria (i.e. “sterile” or, more formally, “germ-free” mice) demonstrated heightened levels of stress hormones in their blood (compared to normal, bacteria-carrying mice) when placed in stressful conditions (3). What’s really interesting is that while the stress response was clearly dysfunctional in germ-free mice, researchers could induce a more normal hormonal response by simply treating the mice with one single microbe: a bacterium called Bifidobacterium infantis. This served as the first clue that our gut bacteria are doing something that influences our mood. Likely, gut bacteria produce chemicals that influence the brain to reduce psychological stress. These chemicals have since been coined as “psychobiotics”.
Further experimentation continued to shed light on the great influence our bacteria has on our mood. Prominent researchers Premsyl Bercik and Stephen Collins working out of McMaster University discovered that behaviour can be transferred between mice simply by transferring their gut bacteria. They discovered that if they took one species of bacteria from the intestines of a mouse, and gave that single strain to a germ-free mouse (such that the intestines of the recipient mouse only contained that single bacterial species), then the recipient mouse would also take on aspects of the donor mouse’s personality. For example, mice that were naturally timid/shy before receiving bacteria became more exploratory if they received bacteria from a naturally exploratory donor mouse. As another example, naturally daring mice would become more shy once they received the bacteria from a naturally shy mouse (4).
While this is only the beginning steps in what promises to be a long journey of intensive, complex research, these kinds of tendencies hint that microbial interactions could very well influence the brain to induce anxiety and other mood disorders.
It is Aristotle that once famously remarked how “man is by nature a social animal”. Of course, we need only reflect on our past experiences for a short while to understand the truth in Aristotle’s words. Our very existence as a species depended on our ancestor’s abilities to interact and help one another. Seeing as how social skills often (but not universally) serve as a prerequisite for a fulfilled life, social psychologists and neuroscientists alike have dedicated large resources to deciphering the mechanisms behind our social nature. A major breakthrough came at the turn of the 20th century, when Henry Dale discovered “Oxytocin”, a chemical that has earned the reputation of the “love hormone” for its role in encouraging social bonding, prosocial behaviours, “maternal instincts”, fear reduction and in antagonizing the effects of depression (learn more here: 1). But oxytocin alone cannot explain the entirety of our social behaviour.
We’re missing something.
In natural sciences, one will often hear the phrase “paradigm shift” to describe a series of scientific findings that radically alters our basic understanding of a scientific phenomenon (the most notable example, for instance, is Charles Darwin’s Evolutionary Theory, which rigorously challenged the way in which we perceived our very origin as a species). With a wealth of studies demonstrating new ways in which our intestinal bacteria dictates our health and behaviour (a great review can be found here: 2), the gut microbiome is primed to give way to the next great paradigm shift – particularly in relation to the extent to which the gut-brain axis influences our mood and behaviour.
If you are familiar with my previous blog post Life on The Spectrum – An Abnormal Microbiome and…Autism?, you may recall the three characteristic behavioural autism-like symptoms: limited social interactions, a tendency for repetitive behaviour and reduced overall communication (3). Autism patients are also known to possess an altered gut microbiome composition (4). Up until recently, this observation was merely an association; an altered gut microbiome in autistic patients was associated with disruption in social development, but was never shown to be a cause of social disruption. However, recent evidence emerging from the lab of Dr. JF Cryan at University College Cork in Ireland (5) suggests that an altered gut microbiome does indeed impede normal social development.
To support his claim, Cryan’s team studied the social behaviour of mice using a “three-chambered sociability test” (6). This test includes a three-chambered box with openings between the chambers. In brief, this setup assesses social behavior in the form of general sociability as well as interest in social novelty. Cryan’s group performed two experiments:
The results of this study have potentially large implications in the realm of autism. In addition to symptoms of reduced social motivation, children with autism exhibit poor communication skills as well as repetitive behaviours. Since it is well understood that many autism patients have a dysregulated gut microbiome, is it a far-stretched hypothesis that the impaired social behaviour observed among autistic patients is at least a partial result of an abnormal microbiome? If so, could we substantially decrease autism-like symptoms and increase social development by focusing our treatments and efforts on re-balancing the gut microbiome?
And how about introverts? Approximately 50% of the population identify themselves as introverted (7). Do they house a microbiome that differs from those of extroverts? Cryan’s results spur a whole new set of questions concerning our social behaviour as a species.
A final question that may come to mind is why gut bacteria are dictating our social behaviour. We have a fairly clear idea as to how – via the gut-brain axis. But why have humans and microbes co-evolved in such a way? Although we cannot know for certain, it is worth noting that microbes have lived on Earth for far longer than humans have. In that time, microbes have yearned for one ultimate evolutionary goal: to reproduce and spread throughout the planet. Is it possible, then, that bacteria, in a parasitic way, are purposefully encouraging our social drive? By encouraging us to interact with other humans, bacteria can jump to our neighbors, who then go their own way. Just like that, bacteria figured out a way long ago to use mammals as vehicles to spread across the globe… Although this idea is difficult to prove, what a remarkable mechanism that would be – microbes stimulate our social development for their benefit, to promote their spread.
And if that’s true, what other behaviours are being manipulated by microscopic organisms? If our gut microbiome is controlling our social development, who’s to say they don’t play a role in other cognitive functions? Are we really in control of our own thoughts and actions, or are we merely the puppets in a microbe-dominated world?
1. Ishak, W. W., M. Kahloon, and H. Fakhry. 2011. Oxytocin role in enhancing well-being: a literature review. J. Affect. Disord. 130: 1-9.
2. Shreiner, A. B., J. Y. Kao, and V. B. Young. 2015. The gut microbiome in health and in disease. Current opinion in gastroenterology 31: 69-75.
3. Prevention, C. f. D. C. a. 2012. Autism Spectrum Disorder — Data & Statistics.
4. Kang, D. W., J. B. Adams, A. C. Gregory, T. Borody, L. Chittick, A. Fasano, A. Khoruts, E. Geis, J. Maldonado, S. McDonough-Means, E. L. Pollard, S. Roux, M. J. Sadowsky, K. S. Lipson, M. B. Sullivan, J. G. Caporaso, and R. Krajmalnik-Brown. 2017. Microbiota Transfer Therapy alters gut ecosystem and improves gastrointestinal and autism symptoms: an open-label study. Microbiome 5: 10.
5. Desbonnet, L., G. Clarke, F. Shanahan, T. G. Dinan, and J. F. Cryan. 2014. Microbiota is essential for social development in the mouse. Mol. Psychiatry 19: 146-148.
6. Yang, M., J. L. Silverman, and J. N. Crawley. 2011. Automated three-chambered social approach task for mice. Curr. Protoc. Neurosci. Chapter 8: Unit 8.26.
7. Myers, I. B., McCaulley, M. H., Quenk, N. L., & Hammer, A. L. (1998). MBTI Manual: A guide to the development and use of the Myers-Briggs Type Indicator (3rd ed.). Palo Alto, CA: Consulting Psychologists Press.
An alternative look at the gut microbiome
So far, we’ve have the pleasure of introducing you to various aspects of the gut microbiome through an amazing range of articles like Reginold Sivarajan’s “A Cure for Alzheimer's Disease?”; Adam Hassan’s “Pregnancy and the Gut Microbiota”; and Mario Corrado’s ““Losing Track of Time” — How Gut Bacteria’s Daily Routine Impacts Our Health”. Today we’d like to present a different but equally exciting aspect of this field: modifying the gut microbiome of mosquitos to fight tropical diseases.
Malaria is a vector-borne parasitic disease endemic to South America and sub-Saharan Africa. Its vector are mosquitos in the genus Anopheles, many species of which thrive in these regions. The disease burden associated with malaria is huge and horrific: it caused 445,000 deaths worldwide in 2016, 90% of which were in Africa. (World Health Organization, 2016) As such, a substantial amount of funding is dedicated to fighting this disease compared to others—worldwide, in 2016, “US$2.7bn were allocated for malaria control and elimination efforts”. (World Health Organization, 2017) Though this is short of the investment required if internationally negotiated goals are to be met, this pool of funding still allows for the development of creative approaches to malaria control.
Enter the gut microbiome. The 2010s have seen an explosion in the popularity of microbiome research, especially centred around the role of the gut microbiome. As we near 2020, it is clearly apparent that this trend is not about to change: the Canadian government is blazing a trail with its Canadian Microbiome Initiative; in parallel, the Obama administration announced the US National Microbiome Initiative [archive link], though no documents have been released since 2016 and the link is currently broken.
So how does the microbiome tie into malaria control? The idea is a brilliant application of gut microbiome research: seed the gut of mosquitos specifically with deleterious bacteria. Bacteria in the genus Wolbachia infect insects. They have two unique abilities when infecting mosquitos: firstly, they induce “cytoplasmic incompatibility” between male and female Anopheles mosquitos. (Sinkins, 2004) The mechanisms governing this are as yet unclear. However, what this means in practice is the following: if an infected male mates with an uninfected female, the offspring die before even being born. This reduces the overall number of mosquitos born, which is useful. Infected females can mate with uninfected males; however, the offspring will also be infected. Thus, the whole population can be infected very rapidly by simply releasing large quantities of infected females!
The second amazing ability that Wolbachia bacteria have is that they shorten the lifespan of the mosquitos they infect. (Schraiber et al., 2012) A shorter life span for the vector means less time for the malaria parasite to be transferred! As mentioned, all that needs to be done to ensure that the whole of a local population is infected (and therefore short-lived) is to release infected females periodically.
An Australian study (focused on dengue-spreading mosquitos), did just this. Dengue is another tropical disease spread by Anopheles genus mosquitos, which also has a serious disease burden in tropical regions. In this study, 100% of the local mosquito population was found to be infected after 9 releases of infected females spaced 10 days apart. In just three months, the whole of the mosquito population’s gut microbiomes had been modified, reducing their lifespan and hindering the spread of dengue. (Hoffmann et al., 2011)
What other effects can we obtain by adding other bacterial species to this vector—or others? How many more easily exploitable solutions are there to disease control, waiting to be discovered until governments & funding bodies recognise the vital importance of infectious disease research?
Reaching a better understanding of the mosquito gut microbiome and its contributions in the normal host life cycle falls squarely into the category of research that receives the least funding: “basic” or “fundamental” research. At what cost are we delaying funding research into the basic sciences? Considering the number of diseases spread through mosquitos in particular, and the huge proportion of morbidity and mortality that they account for in tropical regions, it seems plain that we cannot afford to delay for much longer.
Hoffmann, A. A., B. L. Montgomery, J. Popovici, I. Iturbe-Ormaetxe, P. H. Johnson, F. Muzzi, M. Greenfield, et al. 2011. “Successful Establishment of Wolbachia in Aedes Populations to Suppress Dengue Transmission.” Nature 476 (7361): 454–59. doi:10.1038/nature10356.
Schraiber, Joshua G., Angela N. Kaczmarczyk, Ricky Kwok, Miran Park, Rachel Silverstein, Florentine U. Rutaganira, Taruna Aggarwal, et al. 2012. “Constraints on the Use of Lifespan-Shortening Wolbachia to Control Dengue Fever.” Journal of Theoretical Biology 297: 26–32. doi:10.1016/j.jtbi.2011.12.006.
Sinkins, Steven P. 2004. “Wolbachia and Cytoplasmic Incompatibility in Mosquitoes.” In Insect Biochemistry and Molecular Biology, 34:723–29. doi:10.1016/j.ibmb.2004.03.025.
World Health Organization. 2016. “World Malaria Report 2016.” World Health Organization. doi:10.1071/EC12504.
World Health Organization. 2017. “World Malaria Report 2017.” World Health. doi:ISBN 978 92 4 1564403.
Most of you likely know someone or will know someone who has type 2 diabetes. Not only does it account for around 90% of all diabetes, it affects around 8% of the world’s population. When you have type 2 diabetes, your body essentially does not recognize insulin, the protein that tells the cells in your body to take up glucose from your blood. Your cells don’t get enough sugar and cannot function properly. Furthermore, all that extra glucose floating around in your blood can also have devastating effects.
Its prevalence has been increasing, along with the rising obesity epidemic. This is because obesity and similar diet and lifestyle choices often cause type 2 diabetes. There are other causes, such as genetics, gender, and certain bacteria in the gut microbiota as well. The bacterial species Bacteroides and Prevotella have been connected with type 2 diabetes, and this is important for a study that was recently done with medication for type 2 diabetes.
The medications used in the study were Glipizide, which acts by trying to ramp up your body’s insulin production, and Acarbose, which stops more complex sugars from being broken down thus making sure less glucose is absorbed into your blood. While Glipizide would have no effect on the bacteria in the users gut, Acarbose would allow more complex sugars to pass farther into the intestine and thus provide different molecules for bacteria to chow down on. This key difference was vital for explaining the effects the researchers saw.
Patients treated with acarbose had increased amount of Lactobacillus and Bifidobacterium, and depleted amounts of Bacteroides. Clearly Bacteroides aren’t that big of a fan of those new complex sugars. This change in bacterial composition clearly also changed the genes involved in bile acid metabolism. This is important because specific types and ratios of bile acids are heavily involved in metabolism. The change in genes, changed the amount and type of bile acids present in the patients. This change provided the patients with a lot of benefits, including lower blood sugar and increased responsiveness to insulin.
The researchers also noticed that within the group of patients treated with Acarbose, those with a higher level of Bacteroides than Prevotella exhibited greater improvement of metabolic parameters, and thus lowered the burden of type 2 diabetes. These findings could potentially show that knowledge of a the bacteria swimming around in a type 2 diabetic’s gut would allow the prediction of which medication would have a greater affect on them.
Early Bird Gets the Worm… But Does It Want that Worm? – How Your Gut Deals with Intestinal Parasites
Neglected tropical diseases (NTDs) refer to tropical diseases which affect low-income countries primarily in Africa, Asia, the Middle East and South America. These diseases are thought of as “neglected” due to them receiving less financial support in terms of research funding and treatment. Everyone has heard of HIV/AIDS, malaria, and tuberculosis. However, how many of you know about schistosomiasis, chikungunya fever or leishmaniasis? Causative agents of this group of diseases include viruses, bacteria or parasites (protozoa and helminths).
Of course, most of the money and resources are in developed regions (i.e. North America and Europe). Thus, there is less incentive to invest in the treatment and prevention of NTDs if the “home” population is not directly or primarily affected. When it comes to viruses and bacteria, a lot of progress has been made. However, there are still no vaccines available for parasitic infections. You may want to remember that the next time you travel! (Disclaimer: many NTDs are treatable and it is very important to visit your travel clinic.)
So, how do parasites affect our gut..? Well, there are several intestinal parasitic infections such as giardiasis, ascariasis, and strongyloidiasis. These parasites, often worms in the adult stage, infect your gastro-intestinal tract by the ingestion of infected food or water, the fecal-oral route, or by skin absorption. The main populations affected are actually children with the World Health Organization (WHO) estimating that number to be around 880 million (1). It is important to note that a parasite’s first instinct is not to kill you. A parasite wants to live with you as it needs you for nutrients (hence why it is called a “parasite”). Complications from how our bodies react to this unwelcomed squatter often lead to severe symptoms down the road and eventually death if left untreated for many years.
We have covered at great lengths in previous blog posts how complex our gut microbiota is and how the notion of balance is very important. Well, most of us would say that the presence of worms in your intestine does not quite qualify as “normal”. When parasites enter your system and mature, they interact with your gut microbiota. Often, the host’s resident gut microbiota is able to interfere with the parasite and undermine it (2). In fact, the bacteria are there to line your gut and to prevent any other microorganisms from invading and establishing itself. But behold, the parasite can also create a more favourable environment for itself. Many protozoans and helminths can secrete molecules that will affect the state of balance in our guts so that they can establish themselves within us. On top of that, parasites live with their host and profit from the nutrients that are being degraded in the intestine as well (2).
The interactions between our gut bacteria and invading parasites are quite complex. Mouse models have shown that the same, normal gut microbiota can protect against some parasites such as Cryptosporidum but cause you to also be susceptible to other species (3,4). Interestingly, probiotics have been found to be effective against several species of parasites. So, on top of helping with gastrointestinal disorders, they play a role in stopping the development of these organisms (5). Something to remember the next time you’re eating your yogurt!
Finally, this leads us to the idea of modulating/altering your gut microbiota. Homeostasis is a key concept. If a parasitic organism disturbs this balance, you can restore it by introducing probiotics as mentioned above. Some species of probiotics include Lactobacilli and Bifidobacteria species. Administration of probiotics leads to a boost in the immune system. This can be observed by a growing number of immune cells (such as T cells) and IgA antibodies, which will act against the invading pathogen (6). Not only is it possible to stop the development of pathogens, it is also possible to help in your recovery if infected!
The past several decades have been met with a roaring increase in the discussions surrounding autism and related disorders that fall within the Autism Spectrum. And with good reason.
First coined in 1910, autism quickly emerged as a notoriously difficult condition to diagnose, given its eerie similarity to other neurodevelopment disorders, specifically childhood schizophrenia. Indeed, it took nearly 70 years of intense research for autism to be diagnosed as a separate disorder (2). Since then, autism prevalence has skyrocketed in Western society; prior to 1980, 1 in 5000 children were diagnosed with autism. Today, an astounding 1 in 68 children possess autism-like symptoms, which include limited social interactions, a tendency for repetitive behaviour and reduced overall communication (3).
Which begs us to ask the question: why?
An enormous array of controversial explanations have been put forward to explain the worrying increase in autism prevalence. The most notorious of which is Andrew Wakefield’s “vaccine hypothesis”, which singled-out the MMR vaccine (Measles, Mumps & Rubella) as the cause of rising autism prevalence (note: Wakefield’s findings have since proven to be falsified and have been retracted from the scientific literature. Read more here: (4))
It was only in the mid 1980s that researchers uncovered that autism had a strong genetic component, wherein certain gene mutations were found to associated with autism (5). The genetic basis became even more clear by the 1990s, when it was discovered that a child was 25-times more likely to be diagnosed with autism if one of their siblings was also diagnosed (6). Since then, an impressive amount of research, made possible by advances in genetic technologies, has identified numerous genes linked to autism (which are summarized in this article: (7)).
While genetics undoubtedly play a role in the development of autism, the last decade saw scientists explore an alternative hypothesis — one that included the microbiome. For years, researchers couldn’t explain the seemingly-odd data that showed — quite conclusively — that women who suffered a high, prolonged fever during pregnancy were seven times more likely to have a child with autism. In 2007, Paul Patterson was able to reproduce this result in mice, such that if he induced a prolonged fever in pregnant mice, then their offspring would display all three features of human autism, including limited social interactions, a tendency for repetitive behaviour and reduced overall communication (8). Patterson also showed that the offsprings also had leaky intestines, which is a critical detail here because anywhere from 40 to 90% of human children with autism also suffer gastrointestinal issues (9).
Then, in 2013, Dr. Sarkis Mazmanian and his research team made the remarkable discovery that these mice had abnormal microbiomes compared to non-autistic mice (10). This finding suggested that the autistic-like behaviour of these mice — and perhaps by extension autistic behaviour in humans — might be partially rooted in the gut rather than entirely in the brain.
If this is true, then treating the gut bacteria should also treat the autism-like symptoms. And indeed, Mazmanian’s research team was able to show that by simply restoring a normal microbiota, the mice also displayed better communication and a decreased tendency towards repetitive behaviour. This heavily implicates our gut bacteria as one of the culprits of autism, and — at least in mice — shows that some features of autism can be reversed by treating an abnormal gut microbiome.
There remains one more twist to this story. Earlier, it was mentioned that a sharp rise in autism prevalence was observed at the end of the 1970s. It turns out that the same can be said for obesity. That is, the rates of both autism and obesity among Westerners seem to have taken off around the same time — the end of the 1970s. This may seem like a simple coincidence (and it very well might be) but consider this: it was during the 1970s that Westerns began to adopt a diet rich in processed foods, sugar-sweetened drinks and a mostly sedentary lifestyle (11). That’s important here because, as we have spoken about in previous blogs, it’s already very well appreciated that our gut bacteria influence our weight (You can read that blog here: http://www.thegutguys.com/myhealth/category/obesity).
Given that our gut bacteria have already been shown to influence weight, and given that autism prevalence spiked once the Western diet was introduced in the late 1970s, is it too far-fetched to assume that they also contribute to autism? Has our adoption of the Western diet molded a “bad” microbiome conducive to both obesity and autism? Although further research is required to fully understand the answer to that question, it definitely deserves its fair share of discussion, as autism prevalence among our children continues to increase at an alarming rate.
1. Muir, H. 2003. Einstein and Newton showed signs of autism. In New Scientist.
2. Wilson, M. 1993. DSM-III and the transformation of American psychiatry: a history. Am. J. Psychiatry 150: 399-410.
3. Centers for Disease Control and Prevention. 2012. Autism Spectrum Disorder — Data & Statistics.
4. Rao, T. S. S., and C. Andrade. 2011. The MMR vaccine and autism: Sensation, refutation, retraction, and fraud. Indian J. Psychiatry 53: 95-96.
5. Blomquist, H. K., M. Bohman, S. O. Edvinsson, C. Gillberg, K. H. Gustavson, G. Holmgren, and J. Wahlstrom. 1985. Frequency of the fragile X syndrome in infantile autism. A Swedish multicenter study. Clin. Genet. 27: 113-117.
6. Jorde, L. B., S. J. Hasstedt, E. R. Ritvo, A. Mason-Brothers, B. J. Freeman, C. Pingree, W. M. McMahon, B. Petersen, W. R. Jenson, and A. Mo. 1991. Complex segregation analysis of autism. Am. J. Hum. Genet. 49: 932-938.
7. Miles, J. H. 2011. Autism spectrum disorders--a genetics review. Genet. Med. 13: 278-294.
8. Malkova, N. V., C. Z. Yu, E. Y. Hsiao, M. J. Moore, and P. H. Patterson. 2012. Maternal immune activation yields offspring displaying mouse versions of the three core symptoms of autism. Brain. Behav. Immun. 26: 607-616.
9. Erickson, C. A., K. A. Stigler, M. R. Corkins, D. J. Posey, J. F. Fitzgerald, and C. J. McDougle. 2005. Gastrointestinal factors in autistic disorder: a critical review. J. Autism Dev. Disord. 35: 713-727.
10. Hsiao, E. Y., S. W. McBride, S. Hsien, G. Sharon, E. R. Hyde, T. McCue, J. A. Codelli, J. Chow, S. E. Reisman, J. F. Petrosino, P. H. Patterson, and S. K. Mazmanian. 2013. Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders. Cell 155: 1451-1463.
11. Popkin, B. M., L. S. Adair, and S. W. Ng. 2012. NOW AND THEN: The Global Nutrition Transition: The Pandemic of Obesity in Developing Countries. Nutr. Rev. 70: 3-21.
Yup, another early morning class. Well, it’s not all so bad – my nutrition professor is going to discuss which foods we should be eating to maximize our performance during physical activity (verdict: we don’t know… But it’s probably not a bad idea to eat your greens!). Pretty interesting topic right?
I zoned out, AGAIN. Just once I’d like to get through one full lecture with full concentration. Anyways, as I was wandering the recesses of my mind, I stumbled upon a terrific blog idea: How does physical activity impact our gut bacteria and vice-versa?
As mentioned in previous blogs, the gut microbiota plays a crucial role in human health; it prevents the survival of pathogenic bacteria (these critters harm you and can even kill you), stimulates the proliferation of epithelial cells (i.e. the cells that line your intestines and are thus in direct contact with the bacteria living there. Proliferation is a good thing – it allows the lining of the intestines to replenished frequently and remain rejuvenated) (1) and helps to digest certain nutrients that we can’t digest on our own. It’s a love-hate relationship, really; while we require the gut flora to keep us alive and well, an altered gut bacteria has been associated with many diseases such as obesity, heart failures, cancer, and diabetes (see previous blogs).
Now, is there a correlation between physical activity and a healthy gut microbiota?
Many studies have shown that physical activity is associated with a healthy microflora. Studies by Dr. Matsumoto and his colleagues have successfully demonstrated that rats following a rigorous running regimen harbor a different gut microbiome composition than control rats whom refrained from running. This showed that rats who did running exercises had a different microbial composition in the gut (2).
So what? Matsumoto investigated his findings deeper, and found that this exercise-induced microbiome produced high levels of an infamous compound known as “butyrate”. Butyrate has been well characterized as a potent suppressor of both colon cancer and inflammatory bowel disease (2, 3)
A revealing study by CC Evans in 2014 built on Matsumoto’s work. Evans and his colleagues demonstrated (through an elegant set of experiments) that mice were able to counter the obesity-inducing effects of a high-fat diet through exercise alone. “But you already knew that !”
The real interesting finding made by Evans was that as the mice ran for greater distances, the lower their Firmicutes:Bacteroides ratio became in the gut (4). If you’re familiar with our previous blog, then you may remember that a lower ratio correlates with weight loss. Essentially, they showed that exercise played a role in preventing obesity by favoring a bacterial composition that is similar to those in lean mice regardless of the diet.
Although, these studies have shown some promising results, you might be wondering if this applies to human beings.
A study on the “fecal microbiota of individuals with different fitness levels” (5) following similar diets showed that people with a higher cardiorespiratory fitness had a greater microbial diversity in their gut. This study by Estaki et al. demonstrated that people who were more physically active had an increase in gut microbial diversity irrespective of the diet, and that diversity is a potent driver of optimal gut health. Additionally, similar to the study in mice, fit individuals showed an increase in butyrate producing bacteria
How does doing exercise change the microbial diversity in the gut?
Further studies are required to fully comprehend the mechanism by which exercise changes the composition of gut bacteria. Nevertheless, one possible theory is that exercise causes lactic acid to be produced in the body, which could be converted by certain bacteria in the gut to butyrate (6).
So if you didn’t already have enough reasons to get to the gym, here’s one more: these studies demonstrate the potential of exercise to be used as a treatment to restore a healthy gut microbiota. We can, quite literally, run our way to a better microbiome.
1. S. Rakoff-Nahoum, J. Paglino, F. Eslami-Varzaneh, S. Edberg, and R. Medzhitov, “Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis,” Cell, vol. 118, no. 2, pp. 229–241, 2004.
2. M. Matsumoto, R. Inoue, T. Tsukahara et al., “Voluntary running exercise alters microbiota composition and increases n-butyrate concentration in the rat cecum,” Bioscience, Biotechnology, and Biochemistry, vol. 72, no. 2, pp. 572–576, 2008.
3. Tan, Hwee Tong, Sandra Tan, Qingsong Lin, Teck Kwang Lim, Choy Leong Hew, and Maxey C. M. Chung. "Quantitative and Temporal Proteome Analysis of Butyrate-treated Colorectal Cancer Cells." Molecular & Cellular Proteomics 7.6 (2008): 1174-185. Web.
4. C. C. Evans, K. J. LePard, J. W. Kwak et al., “Exercise prevents weight gain and alters the gut microbiota in a mouse model of high fat diet-induced obesity,” PLoS ONE, vol. 9, no. 3, Article ID e92193, 2014.
5. M. Estaki, J. Pither, P. Baumeister et al., “Cardiorespiratory fitness as a predictor of intestinal microbial diversity and distinct metagenomic functions,” The FASEB Journal, vol. 30, no. 1, pp. 1027–1035, 2016.
6. S. H. Duncan, P. Louis, and H. J. Flint, “Lactate-utilizing bacteria, isolated from human feces, that produce butyrate as a major fermentation product,” Applied and Environmental Microbiology, vol. 70, no. 10, pp. 5810–5817, 2004.
I’m at the kitchen table. There’s a clock on the wall directly behind me. I’m not looking at it, but I can hear the incessant ticking — tick, tock, tick, tock. It’s nighttime, and I’m beginning to feel the gentle touch of sleep come over me: heavy eyelids, slow breathing, lack of focus… It’s odd really, that despite never turning around to look at the clock on my kitchen wall, my body acts as if it has been keeping track of time all along.
Because it has.
The Circadian Rhythm
During my last year of undergraduate studies at McGill University, I was asked to give a short presentation on a cool topic in science to my fellow Honours Microbiology class. The assignment’s only guideline was to select a topic unrelated to our field of study. Driven by an uncanny fascination with the many mysteries surrounding the human brain, my presentation focused on the circadian rhythm, a built-in 24-hour cycle that instructs our body on when to sleep, rise and eat — among an endless list of other critical physiological processes that coalesce to ensure optimal body function. The devastating effects of disrupting our circadian rhythm have been well documented in the literature and range from lung pathologies (1), mood dysregulation (reviewed in (2)), impaired immune system function (3), liver impairment (4,5), and, strikingly enough, possibly breast cancer (6), Parkinson’s Disease (7) and Alzheimer’s Disease (8).
I was surprised to learn that although the circadian rhythm plays a big part in dictating our daily routine, it was not actively researched until the late 1950s. Since then, we’ve made great leaps in our understanding of the “Biological Clock”. While there are many environmental cues (called “Zeitgebers”, a German word meaning “time-givers”) that influence the circadian rhythm, the best studied one is light. When light enters your eye, it excites light-sensitive cells named “retinal ganglion cells”. These cells will convert light into electrical signals that are delivered to a highly specialized region of the brain known as the “suprachiasmatic nucleus” (or SCN for short).
Think of the SCN as the “Master Clock”; once stimulated, it sends signals throughout the body to regulate a plethora of biological processes including sleep and feeding patterns, alertness, core body temperature, brain wave activity, hormone production, regulation of glucose and insulin levels, urine production and cell regeneration. For example, in the absence of light (9), the SCN will stimulate the pineal gland to produce melatonin (10), a hormone that causes drowsiness and lowers body temperature — which is exactly the physiological effect we would want near bedtime, in the absence of light (melatonin is also the chemical found in Nighttime Gravol to make us drowsy). In contrast, during the first hour after waking up, the SCN — now exposed to light — will instead stimulate the adrenal gland to produce cortisol, a notorious stress-related hormone that will increase alertness by increasing blood glucose levels and hence increasing our energy supply (11) (Want to learn more? More thorough and easy-to-read reviews on the circadian rhythm can be found here and here, and a great video can be found here).
The Circadian Rhythm of the Gut Microbiome
I didn't know it at the time, but my presentation of the circadian rhythm as a topic outside of Microbiology was inadvertently misleading my peers; it ironically turns out that the circadian rhythm is deeply intertwined with our gut bacteria.
In 2016, Dr. Eran Elinav was intrigued by the previous finding that metabolic chemicals influence the circadian clock (12). Since gut bacteria were already known to produce a large array of chemicals that influence human physiology, Elinav began to investigate the impact of gut bacteria on the circadian clock. Elinav and his research team in Israel ultimately reached three major conclusions, which were published in 2016 (13).
First, Elinav’s team studied the bacteria that are found closest to the intestinal lining (that is, the bacteria that are actually attached to our intestines), since these are the bacteria that most strongly influence our physiology. They were able to show that these bacteria follow a sort of routine throughout the course of the day that they termed “diurnal oscillations”. In other words, the bacteria will start their day at one location in the intestine, and, like clockwork, will move gradually during the day before returning to their original location roughly 24 hours later. Why does this matter? First of all, it’s pretty convincing evidence that our gut bacteria abide by their own circadian clock, which is fascinating in its own regard. But more importantly, what this means for us is that during the course of the day, the cells of our intestinal epithelium are exposed to different numbers and different species of bacteria, and are therefore exposed to the different chemicals that the different bacteria will produce.
Next, Elinav asked if the circadian patterns of the gut microbiome are influenced by the circadian rhythm of the host. To answer this question, researchers studied specialized mice (called Per1/2-/- mice) that lack a functional circadian clock (The Per1 gene is expressed mainly in the SCN and helps generate the circadian rhythm. Like the batteries of the clock, our circadian clock stops ticking in the absence of Per1, as is the case with the Per1/2-/- mice). Interestingly, they showed that the gut bacteria of Per1/2-/- mice lost their rhythm, which strongly suggests that the bacteria’s circadian rhythm depends on our own. Elinav went further, and showed that just by controlling the feeding times of these mice, he was able to restore some of the “diurnal oscillations”, implying that feeding time is a major contributing factor to maintaining the circadian clock of gut bacteria.
Elinav then asked the opposite question — he wanted to see what would happen to our circadian rhythm if we disrupted that of the gut bacteria. To this end, antibiotics were used to kill most of the gut bacteria. They found that disrupting the bacterial clock did not alter our circadian clock, which means that while the bacterial clock depends on the host circadian rhythm, the reverse is not true. Instead, they identified compensatory mechanisms that are initiated in mice to counter the loss of gut bacteria.
Finally, Elinav wanted to see if the circadian clock of the bacteria had any effects on host physiology beyond the gut, and discovered that the liver was heavily influenced by the diurnal oscillations of the gut microbiota. Elinav found that the daily routine of gut bacteria influences how well the liver can function to detoxify drugs. For example, it was found that in a normal mouse (with an intact gut microbiota), the liver’s ability to metabolize the drug APAP (also known as acetaminophen, which is the active ingredient in Tylenol) increases gradually during the course of the day. However, Per1/2-/- mice, mice treated with antibiotics, and “germ-free” mice (mice that are specially handled to be completely free of bacteria) do not benefit from this gradual increase in liver function throughout the day. (these results help to explain the findings in (4) and (5) that correlate an abnormal circadian rhythm with liver pathology).
In practical terms, this study teaches us that the circadian rhythm of our gut bacteria influences our liver’s ability to break down drugs, and that disrupting this bacterial clock would hamper our liver’s detoxification ability — and that can be extremely dangerous, say, if you are consuming alcohol (a drug that must be detoxified by the liver) and taking antibiotics or following a poor diet that weakens your gut microbiota.
1. Sundar, I. K., H. Yao, M. T. Sellix, and I. Rahman. 2015. Circadian molecular clock in lung pathophysiology. Am. J. Physiol. Lung Cell Mol. Physiol. 309: L1056-1075., H. Yao, M. T. Sellix, and I. Rahman. 2015. Circadian molecular clock in lung pathophysiology. Am. J. Physiol. Lung Cell Mol. Physiol. 309: L1056-1075.
2. Kim, J., S. Jang, H. K. Choe, S. Chung, G. H. Son, and K. Kim. 2017. Implications of Circadian Rhythm in Dopamine and Mood Regulation. Mol. Cells 40: 450-456.
3. Labrecque, N., and N. Cermakian. 2015. Circadian Clocks in the Immune System. J. Biol. Rhythms 30: 277-290.
4. Udoh, U. S., J. A. Valcin, K. L. Gamble, and S. M. Bailey. 2015. The Molecular Circadian Clock and Alcohol-Induced Liver Injury. Biomolecules 5: 2504-2537.
5. Reinke, H., and G. Asher. 2016. Circadian Clock Control of Liver Metabolic Functions. Gastroenterology 150: 574-580.
6. Blakeman, V., J. L. Williams, Q. J. Meng, and C. H. Streuli. 2016. Circadian clocks and breast cancer. Breast Cancer Res. 18: 89.
7. Wulff, K., S. Gatti, J. G. Wettstein, and R. G. Foster. 2010. Sleep and circadian rhythm disruption in psychiatric and neurodegenerative disease. Nat. Rev. Neurosci. 11: 589-599.
8. Saeed, Y., and S. M. Abbott. 2017. Circadian Disruption Associated with Alzheimer's Disease. Curr. Neurol. Neurosci. Rep. 17: 29.
9. Bellastella, A., A. De Bellis, G. Bellastella, and K. Esposito. 2014. Opposite influence of light and blindness on pituitary-gonadal function. Front. Endocrinol. (Lausanne) 4: 205.
10. Benarroch, E. E. 2008. Suprachiasmatic nucleus and melatonin: reciprocal interactions and clinical correlations. Neurology 71: 594-598.
11. Hucklebridge, F. H., A. Clow, T. Abeyguneratne, P. Huezo-Diaz, and P. Evans. 1999. The awakening cortisol response and blood glucose levels. Life Sci. 64: 931-937.
12. Nakahata, Y., M. Kaluzova, B. Grimaldi, S. Sahar, J. Hirayama, D. Chen, L. P. Guarente, and P. Sassone-Corsi. 2008. The NAD+-dependent deacetylase SIRT1 modulates CLOCK-mediated chromatin remodeling and circadian control. Cell 134: 329-340.
13. Thaiss, C. A., M. Levy, T. Korem, L. Dohnalova, H. Shapiro, D. A. Jaitin, E. David, D. R. Winter, M. Gury-BenAri, E. Tatirovsky, T. Tuganbaev, S. Federici, N. Zmora, D. Zeevi, M. Dori-Bachash, M. Pevsner-Fischer, E. Kartvelishvily, A. Brandis, A. Harmelin, O. Shibolet, Z. Halpern, K. Honda, I. Amit, E. Segal, and E. Elinav. 2016. Microbiota Diurnal Rhythmicity Programs Host Transcriptome Oscillations. Cell 167: 1495-1510.e1412.
Over the past few months, we, The Gut Guys, have shared with you many different implications that our gut microbiota has on our health. But have you ever wondered where these species of bacteria came from? Are you born with the bacteria that colonize your gut or do they arrive later on? And what effect do they have during the gestational (pregnancy) period?
Contrary to previous beliefs, the fetus is not a sterile environment. Microbes can be found in the amniotic fluid and a placental microbiome has been identified. Studies show that the microbiota is essential for healthy early development, pregnancy maintenance, and the first years of childhood.
As you might already know, the woman’s body undergoes several changes during pregnancy at the hormonal, immunological and metabolic levels. Physiologically, women experience weight gain, insulin resistance, glucose intolerance and low-grade inflammation (1). The microbial profiles in pregnant women are very different between the first and third trimesters. You can observe a great increase in bacteria from the Actinobacteria and Proteobacteria groups. In order to prove these differences, the microbiota from both the first and third trimesters were delivered to germ-free mice (devoid of bacteria). Mice who received ‘third-trimester’ bacteria gained weight and developed resistance to insulin as well as inflammation (2). Interestingly, diet and the intake of antibiotics are environmental factors that can influence the gut microbiota during pregnancy. Bacteroides and Staphylococcus species were shown to be more present in overweight pregnant women for example.
Babies are born with a specific microbial profile. In fact, the gut microbiota of newborns greatly resembles the vaginal microbiota of their mothers! The vaginal microbiota is mainly dominated by species of the Lactobacillus family. These bacteria protect women and their fetus from infection. As previously mentioned in other blog posts, the harmless bacteria that colonize our bodies protect us from potential pathogens from settling and causing disease.
One fun fact is that babies have a different colonization pattern depending on the type of delivery. Babies delivered by C-section differ from vaginally-delivered infants for at least a year (3). The gut microbiota of infants changes over time. Breast-feeding favours the growth of Bifidobacteria and Lactococci and also the transmission of maternal IgA antibodies. IgA antibodies are found in mucous membranes (intestines) and these are important to control the microbiota present in the gut. Learn more about antibodies here.
So, now we’ve learned that women experience chances in their gut microbiota during pregnancy and also transfer their vaginal microbiota to their newborn infants. These babies will experience changes of their own as they grow older with diet being the main factor involved. The gut microbiota really affects us in all spheres of health and we are slowly discovering the extent of its reach.
1. Nuriel-Ohayon, Meital, Hadar Neuman, and Omry Koren. "Microbial Changes during Pregnancy, Birth, and Infancy." Frontiers in Microbiology. Frontiers Media S.A., 2016. Web.
2. Koren, O., J. K. Goodrich, T. C. Cullender, A. Spor, K. Laitinen, H. K. Bäckhed, A. Gonzalez, J. J. Werner, L. T. Angenent, R. Knight, F. Bäckhed, E. Isolauri, S. Salminen, and R. E. Ley. "Host Remodeling of the Gut Microbiome and Metabolic Changes during Pregnancy." Cell. U.S. National Library of Medicine, 03 Aug. 2012. Web.
3. Neu, Josef, and Jona Rushing. "Cesarean versus Vaginal Delivery: Long Term Infant Outcomes and the Hygiene Hypothesis." Clinics in Perinatology. U.S. National Library of Medicine, June 2011. Web.