Tuesday, May 24, 2016

The link between Brain and Microbiome


I had previously blogged on significance of microbiome in brain health. Though the exact mechanisms are under investigation, several studies have pointed that this is not a mere correlation. Psychiatric conditions such as schizophrenia are heavily investigated with reference to oral and gut microbiome. It has been proposed that altering the microbiome may be beneficial in influencing the brain health, which is the basic concept of psychobiotic's.

Fig 1: C. albicans IgG levels and seropositivity
in schizophrenia and bipolar disorder
compared to controls. Source
A recent study published in npj schizophrenia showed that there is a significant association between Candida albicans infection and Schizophrenia. This study was a case–control cohort involving a total of 808 participants (277 controls, 261 schizophrenia cases and 270 cases with bipolar disorder). The key discussed finding in the study was that 26 percent of men with schizophrenia had Candida antibodies compared with 14 percent of the control group. It should be noted that the research doesn't in any way convey that there is a causal effect. The authors make a case that at least in a subset of population, C albicans infection seems to be present.

However, I would also like to think of the following possibility. At least a subset of patients with psychiatric illness has lower hygiene. And it is well known that improper hygiene can lead to candida infection. The paper also finds that C albicans exposure was associated with homelessness in bipolar males. That makes my suspicion higher. 26% correlation according to me is far less of an evidence warranting a significant look. I perhaps would want to see this paper in a different angle. People with Schizophrenia or Bipolar disorder needs to be screened and promptly treated for Candida infections.

I want to present a totally different example linking brain chemistry and microbe. In a recent proof of principle study in mouse models, it has been shown that LPS from bacteria can target TLR4 - a receptor which is also present on the surface of gut cells, taste cells and immune cells, which influences sweet receptor genes in taste buds, reducing sweet cravings. Administering LPS to mice also blocked leptin receptors. In other words, bacterial contents can have an effect on something as fundamental as food craving behavior.

Fig 2: Comparison of Proliferation, Survival and Neurogenis in Conventionally
colonized, Germ free and germ-free–colonized mice. Source
I presented a couple of case examples here from recent papers to illustrate that microbes have something to do with Brain chemistry but not necessarily all correlation studies indicate that there is a direct connection. But the question remains as to if there is a real connection at least in a few cases. The answer is yes. Several studies have reported that there is a connection between adult hippocampal neurogenesis and gut microbiome. In a study published in 2014 by Ogbonnaya et al, 10-week-old mice were injected with bromo- deoxyuridine (BrdU) to label proliferating cells and euthanized 2 hours later. The neuronal sprouting was measured between a normal and germ-free mice. They found that Germ-free and germ-free–colonized mice exhibit a trend for increased cell proliferation as measured by bromodeoxyuridine immunohistochemistry. The study also found that postweaning microbial colonization of germ-free mice did not prevent changes in adult hippocampal neurogenesis, which suggested that there is a critical window in early life during which microbial colonization influences adult hippocampal neurogenesis.

Table 1: Organisms influencing on
Neurotransmitter activity.
It has been the understanding from different studies that Neurotransmitters production can be influenced by microbes. Some examples are shown in Table 1. In a study by Bravo etal, it had shown that L. rhamnosus (JB-1) induced region-dependent alterations in GABAB1b mRNA in the brain in a mouse model. In another study by Tillisch et al,  fermented milk product probiotic (FMPP) was administered for 4 weeks to healthy women. The study found alterations in intrinsic activity of resting brain indicated that ingestion of FMPP was associated with changes in midbrain connectivity. Such studies stands as a proof that indeed administering the right microbe can contribute in treating Brain health. 

Table 2: Psychobiotic studies. Source
Now that I have made a clear argument that microbial influence on brain chemistry is indeed a reality as I have previously argued I want to introduce the concept of Psyhchobiotics. Psychobiotic is defined as a cocktail of live organism that, when ingested in adequate amounts, produces a health benefit in patients suffering from psychiatric illness. Studies from like that mentioned above and many other indicate that psychobiotic can be the future of psychiatric treatment. We are yet to find the right combination of organisms. As shown in Table 2 which summarizes published Psychobiotic studies several possibilities and leads are already proposed.

There are several explanations of how a microbiome can connect with the neural system and influence the behavior. This includes communication through gut-brain axis, production of metabolites or chemicals that influence Neurotransmitters or modulate receptor activity etc. Now a new study has proposed a totally new concept- influencing through immune cells. The study proposes a specific subset of immune cells- Ly6Chi Monocytes can function as a possible mediator between gut, immune cells and neurons. The researchers here studied Antibiotic treated adult C57BL/6 mice to investigate the impact of gut flora dysbiosis on hippocampal neurogenesis.

Fig 3: Classification of Monocytes.
Before I get into the details, let us first clarify what is Ly6Chi Monocyte.  As everyone knows, monocytes are large phagocytic white blood cell with a simple oval nucleus and clear, greyish cytoplasm. They can further differentiate into macrophages or dendritic cells. Each of these cells can be identified using flow cytometry analysis. Traditionally monocytes are classified into 3 types. See Fig 3 for details. Each type is further sub typable based on the markers displayed on the cell. I mean to emphasise that monocytes are not a homogenous population. The circulating monocyte population is divisible into Ly6c positive monocytes and Ly6c negative monocytes. Both types express CD115 and CD11b. In a flow cytometry run, we can identify two populations of blood monocytes: Ly6c+ monocytes (CD11b+ CD115+ Ly6Chi) and Ly6c− monocytes (CD11b+ CD115+ Ly6c−). Neutrophils also express Ly6C but can be distinguished since monocytes express higher levels of Ly6c than neutrophils. That's why Ly6Chi is an important feature here. Interestingly, Ly6c+ monocytes are precursors of Ly6c− monocytes. By the way, Ly6C stands for lymphocyte antigen 6 complex.  Macrophage and dendritic cell precursors in the bone marrow give rise to Ly6Chi monocytes, which serves as an intermediate for Ly6Clow monocyte generation. Here the superscript hi or low stands for expression of the marker Ly6C. Ly6Chi monocytes exit the bone marrow in a CC-chemokine receptor 2 (CCR2)-dependent manner and are recruited to inflamed tissues. So functionally, Ly6Chi monocytes are inflammatory monocytes selectively traffic to the sites of inflammation. 

In the current study, researchers administered mice antibiotics to free intestine from microbes. When compared with untreated mice, the mice who lost their healthy gut bacteria performed low in memory tests and showed a loss of neurogenesis in hippocampus. They also noted reduced Ly6Chi monocytes in brain, blood, and bone marrow. Further experiments showed that reduction in levels was correlated with loss of neurogenesis and this could be rescued by replenishing the Ly6Chi levels.

Table 3: Contents of VSL#3 Probiotic mixture.
Subsequent experiments showed that though probiotics helped the mice regain memory, fecal transplants to restore a healthy gut bacteria did not have an effect. The probiotic used in this study was VSL#3, which is a mixture of eight different strains of bacteria. VSL#3 works by colonizing the GI tract and forms a barrier that protects the inner layer of the gut from pathogens. VSL#3 positively affects a variety of substances that are involved in gut function. This is important to ensure the correct absorption of nutrients and to maintain barrier function. The VSL#3 contents are shown in Table 3.

Fig 4: Impact of prolonged antibiotic treatment
on brain cell plasticity and cognitive function.
Wolf who is the senior author in the study comments, "For us it was impressive to find these Ly6Chi cells that travel from the periphery to the brain, and if there's something wrong in the microbiome, Ly6Chi acts as a communicating cell".He further adds, "It was surprising that the normal fecal transplant recovered the broad gut bacteria, but did not recover neurogenesis. This might be a hint towards direct effects of antibiotics on neurogenesis without using the detour through the gut. To decipher this we might treat germfree mice without gut flora with antibiotics and see what is different."

In conclusion, Psychobiotics appears to be a real thing and microbial community does have important function in regulating Brain health. I guess, there will come a day when psychiatric treatment regimen will also include giving specific cocktail of organisms to improve patient symptoms. Who said bacteria are all bad.

Severance, E., Gressitt, K., Stallings, C., Katsafanas, E., Schweinfurth, L., Savage, C., Adamos, M., Sweeney, K., Origoni, A., Khushalani, S., Leweke, F., Dickerson, F., & Yolken, R. (2016). Candida albicans exposures, sex specificity and cognitive deficits in schizophrenia and bipolar disorder npj Schizophrenia, 2 DOI: 10.1038/npjschz.2016.18

Ogbonnaya ES, Clarke G, Shanahan F, Dinan TG, Cryan JF, & O'Leary OF (2015). Adult Hippocampal Neurogenesis Is Regulated by the Microbiome. Biological psychiatry, 78 (4) PMID: 25700599

Möhle, L., Mattei, D., Heimesaat, M., Bereswill, S., Fischer, A., Alutis, M., French, T., Hambardzumyan, D., Matzinger, P., Dunay, I., & Wolf, S. (2016). Ly6Chi Monocytes Provide a Link between Antibiotic-Induced Changes in Gut Microbiota and Adult Hippocampal Neurogenesis Cell Reports DOI: 10.1016/j.celrep.2016.04.074

Monday, May 16, 2016

An Antibiotic that doesn't effect Gut Microbiome.


Let us start with a question. What would be an ideal antibiotic? I can possibly talk about many different possibilities, but everyone would agree that we could boil it down to 2 most important points. First, the antibiotic needs to be as specific as possible and second absolutely no resistance. Though I have previously talked about these, it is important to summarise here once.

Antibiotics are usually targeted against a cellular component or enzymes of a vital biochemical pathway that kills the cell, in this case, bacteria. It is also necessary that this target exists in all clones of the target organism and forms vital and stable part of bacterial cell. In an evolutionary sense, such a component will also be present in other species, thus making it nearly impossible to design an antibiotic that will be hyper-specific to a species. When an antibiotic is made that is extremely species specific, chances of resistance is very high since the target can be lost easily without much evolutionary pressure on the bacterial cell. However, a closely balanced antibiotic design can in theory at least, be specific to a group of species.

Fig 1: Chemical Structure of Debio 1452. Source
Researchers from St. Jude Children’s Research Hospital in Memphis, Tennessee, have found that a drug Debio 1452 can attack Staphylococcus aureus, with a very little impact on intestinal bacterial communities in mice. Debio 1452 has been in development since 2007 by Affinium Pharmaceuticals under the name AFN-1252. Debio 1452, is currently being developed by a Swiss-based pharmaceutical company Debiopharm. The chemical targets an enzyme called FabI (Eenoyl-acyl carrier protein reductase) in Staphylococcus species. Its lack of activity against other species of bacteria such as Streptococci, Enterococci, Enterobacteriaceae, and Non-fermentative Gram-negative species. This has allowed its development as a narrow spectrum drug. Fig 2, is a comparison of MIC aganist other well known anitiobitic for CoNS. I have made a graph for comparison.

Fig 2: Activity of Debio1452 in MIC50 (μg/ml) in comparison with antimicrobial agents against Coagulase-negative staphylococci. Data modified from Farrel etal. Source
The ability of Debio 1452 to perform as an antibiotic has been previously tested. The aim of the current study was to see how far is the contribution of this drug in altering microbiome. For the study, mice in groups of five were treated with high doses of Debio 1452 and compared with four broad spectrum antibiotics every day for a period of 10 days. The gut microbiome was tracked using 16S ribosomal DNA sequencing on stool samples taken throughout the experiment and followed for 27 days. Analysis showed that Debio 1452-treated group had a very low variation of the microbiome.

It should be noted that the consistency of microbiome was not absolute. The treatment had a significant impact on a family of bacteria S24-7. The decrease in S24-7 was however quickly compensated for the imbalance by other members of microbiome and overall bacterial diversity and abundance wasn’t damaged during treatment. It took about two days post treatment for S24-7 levels to come back to its normal levels.  As the authors point out in the abstract, "The gut bacterial abundance and composition of Debio 1452-treated mice was indistinguishable from untreated mice 2 days after antibiotic treatment stopped". It took much long time for mice treated with other broad-spectrum antibiotics to return to normal.

ResearchBlogging.orgFlamm RK, Rhomberg PR, Kaplan N, Jones RN, & Farrell DJ (2015). Activity of Debio1452, a FabI inhibitor with potent activity against Staphylococcus aureus and coagulase-negative Staphylococcus spp., including multidrug-resistant strains. Antimicrobial agents and chemotherapy, 59 (5), 2583-7 PMID: 25691627

Yao J, Carter RA, Vuagniaux G, Barbier M, Rosch JW, & Rock CO (2016). A Pathogen-Selective Antibiotic Minimizes Disturbance to the Microbiome. Antimicrobial agents and chemotherapy PMID: 27161626

Friday, May 06, 2016

3000 Microbes: Different looking life tree


I have been extremely busy in last 3 weeks and so haven't been able to post. Well, looks like I have gathered some time and you have caught up with posts that you have missed. Genomics era has opened up debates about, what we think a species is and how many species are actually there. There are several different arguments and estimates aimed at explaining these questions. but, as we are sequencing more and samples especially the environmental samples, the more we are seeing stuff we perhaps even don't know how to look at. They are famously dubbed as "Dark matter of biology".

Currently, the textbook version of "Tree of life", has 3 major components- Bacteria. Archaea and Eukaryotes. Though this remains unchanged, new members are continuously added to different groups as new species are discovered. One of the group that is seeing new additions at a very rapid rate is the bacteria. The ability to do metagenomics and the computational power has increased the number of species in the database. In a paper published a couple of days ago, the authors argue we have barely even scratched the surface and predicts that about 99.9% (a whopping high percentage) of species are yet to make it to database. Though I find that this is probably an over prediction, I do agree that we have a lot more to discover.

The study published in Nature Microbiology, involved studying genomic sequence of about 3000 (The researchers actually studied DNA from 2,072 known species, along with the DNA from 1,011 newly discovered species) and the generated data was computationally fit into a new tree of life. The emerging result showed a completely different picture. Apart from the finding that bacteria constituted a huge branch of the tree, the tree had several new sub-branches (if I can call so), that is completely missing in the textbook version. Doug Soltis who pioneered the earlier version of tree, comments in The Scientist, "It’s a great step forward. It’s exactly what you want to see if you’re interested in the tree of life, because what we produced was just . . . a starting point, and the bacteria, in particular, are poorly represented in that tree". One of the key findings discussed in the study was a branch called the Candidate Phyla Radiation (CPR). This branch is totally unknown and formed one of the largest branch.

I have shamelessly copied the figure below from EarthSky website including the explanation. (Thanks to paywall, I couldn't get to read the full paper and thus am relying on explanations published elsewhere).
The tree includes 92 named bacterial phyla, 26 archaeal phyla and all five of the Eukaryotic supergroups. Major lineages are assigned arbitrary colours and named, with well-characterized lineage names, in italics. Lineages lacking an isolated representative are highlighted with non-italicized names and red dots. For details on taxon sampling and tree inference, see Methods. The names Tenericutes and Thermodesulfobacteria are bracketed to indicate that these lineages branch within the Firmicutes and the Deltaproteobacteria, respectively. Eukaryotic supergroups are noted, but not otherwise delineated due to the low resolution of these lineages. The CPR phyla are assigned a single colour as they are composed entirely of organisms without isolated representatives, and are still in the process of definition at lower taxonomic levels.
The first author Laura Hug comments "What became really apparent on the tree is that so much of the diversity is coming from lineages for which we really only have genome sequences.We don’t have laboratory access to them, we have only their blueprints and their metabolic potential from their genome sequences. This is telling, in terms of how we think about the diversity of life on Earth, and what we think we know about microbiology."

Personally, this study is shows that the tree of life what we currently know it to be is far from its true nature. The study used data from nearly 3000 species which is a miniscule of what is out there. Marine ecology holds at least a billion different types that are yet to be studied by sequence. Once we have these sequences perhaps the revised tree of life is going to look further different. Something that we totally haven't expected. Needless to say, the "Dark matter of biology" is going to be more in number. On a completely different note, at least for the fun of it, I would like to create a tree of human microbiome. Perhaps once Human microbiome project is completed, we can do it. And I bet it will tell us similar interesting stories.

Spang, A., & Ettema, T. (2016). Microbial diversity: The tree of life comes of age Nature Microbiology, 1 (5) DOI: 10.1038/nmicrobiol.2016.56