Should we care about fungi? The mycobiome: an emerging field of the human microbiome research

penici2With the advancement of next-generation sequencing in the last decade, our knowledge about the trillions of microbes that the human body harbours has exponentially increased. Microbiome research has mostly been focused on the study of bacteria notwithstanding that the classical definition of the microbiome includes viruses and fungi. Despite the numerous microbiome publications, we know only little about the impact of fungi on health and disease. That said, with the increased evidence demonstrating the importance of host bacterial communities on host physiology and their involvement in health, research groups are interested in looking at the impact of fungi as well.

I have selected this publication because it demonstrates the potential effect of fungal colonization on the bacterial composition in vivo. In this study, Downward and colleagues demonstrated that the introduction of C.albicans in a disturbed microbiota was sufficient to alter the reassembly of the bacterial population.

During our journal club Friday, May 29th at 3:30pm, Westend Pub I hope to discuss:
(a) The underappreciated impact of fungi in microbiome research
(b) The feasibility and significance of including a mycobiome component in our research
(c) The limitation of the ITS sequencing

Relevant reviews for people interested to read more on the topic include:

The mycobiota: interactions between commensal fungi and the host immune system:

http://www.nature.com/nri/journal/v14/n6/full/nri3684.html

The human mycobiome in health and disease:

http://genomemedicine.com/content/5/7/63

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Going beyond 16S rRNA gene sequencing to examine the functionality of the gut microbiome.

As the technology advances, so does our ability to probe and profile human microbiomes. Next week, I will lead a discussion of the recent paper entitled Functional Dynamics of the Gut Microbiome in Elderly People during Probiotic Consumption.” by Emiley Eloe-Fadrosh et al. This paper combines together many aspects that we’re interested in here at McMaster, including the inclusion of techniques beyond 16S rRNA gene sequencing to gain a more complete understanding of how the elderly gut community is affected by the popular probiotic, Lactobacillus rhamnosus GG (LGG).

During our journal club Friday, May 1st at 3:30pm, Westend Pub I hope to discuss:
(a) the combined use of 16S rRNA gene sequencing and RNAseq
(b) their conclusions regarding the effect of LGG on the gut microbiota
(c) on the more philosophical: discuss their use of data visualizations, and how we can apply their methods to our own datasets

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Introducing: Microbiome Working Group

More and more, we are becoming aware that there are a lot of researchers at McMaster who are dabbling in the microbiome. Some labs are hot-beds of microbial research, whilst others are using microbiome-related datasets as part of a more diverse toolbox to study their phenomenon of interest. The later means that there are often one or two researchers per-lab who are using these tools in semi-isolation.

I am very happy to introduce the Microbiome Working Group, a weekly, drop-in, work/discussion hour with the aim of bringing microbiome researchers together, independent of sample type, laboratory, or skill level. This isn’t a meeting; there are no presentations. This is about getting research and analysis done. Bring your laptop and use the presence of like-minded others as motivation. Test out your new analysis idea on your peers (before you spend months trying it out). Bring any obstacles you might have run into. Come once a month, or come every week.

Microbiome Working Group will occur every Tuesday at 3pm in HSC-3N10A until further notice. Students, postdocs, PIs etc., human microbiome or otherwise, everyone is welcome!

(Note: if this kind of thing interests you, you most likely would get a kick out of our monthly Journal Club as well!).

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Summing up: Bassis et al.’s take on the influence of the URT on other human-associated biogeographies.

Last week, Pat led us in the examination of a new paper from the Huffnagle group at the University of Michigan. This was the first meeting of McMaster’s HMBJC in a few months which meant a very lively discussion since we were bursting to talk of all things microbe!

As a group, we really enjoyed and appreciated this study. It was great to see researchers going back to their ecological roots with discussions of ecosystems, gradients, niches, and how different anatomical sites can affect one another. We really enjoyed how easy this paper was to follow, even for someone outside of the microbiome research field, using simple-to-read graphs and visuals that any scientist could interpret. This paper also tackled some areas which, historically, have been hard to sample or where sampling has been shrouded in doubt (e.g. BALs). Additionally, for our group, the Θyc statistic was interesting to see in use, and this publication led us down a citation-trail to learn of its advantages over metrics that we more commonly employ such as Bray Curtis and Jaccard.

We did comment on how the nasal swab might have captured a slightly different microbial community compared to a nasal wash, which would me more consistent with the other sample types used in this study. A greater proportion of mucosally-associated species obtained from the “scraping” motion of a swab might account for some of the differences between the nasal communities when compared to other anatomical sites which were sampled by “washing”. Additionally, we were sad to see an opportunity to make more intra- and inter-patient comparisons across all sites not taken, but perhaps that will be the group’s next publication.. we look forward to it!

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Bacterial communities in the lung and stomach – how did they get there?

The human microbiota has become more diverse than what scientists believed when the Human Microbiome Project started over a year ago.  Initially, the lung and stomach, two unique mucosal surfaces, were not even initially included in the sample collections.  As culture-independent methods of studying the microbiome has expanded, it revealed that there are diverse and unique communities that exist within these organs, but there is little knowledge about what influences these communities.  A new paper out of Gary Huffnagle’s group at the University of Michigan decided to look at just that.

In their analysis, Bassis et al.  began to look at potential sources of microbes, including the mouth and nasal passages. The mouth is exposed to and produces more biomass daily, with saliva production and access to nutrients via food being chief among them.  The nose is known to harbour important bacteria and potential pathogens.  Based on the findings of their work, it appears that the mouth microbes have greater influence in what reaches the stomach and lungs, but that the lungs can control which bacteria take hold, as they looked at the elimination of Prevotella from the mouth microbial communities into the lung.

In our journal club at 3:30pm on Friday, March 27th at West End pub, I look forward to discussing:
(1) Their methodology for sample collection and sequencing
(2) Their microbial diversity analysis, especially their use of θyc distances
(3) Their model utilizing Prevotella for species elimination

Abstract: No studies have examined the relationships between bacterial communities along sites of the upper aerodigestive tract of an individual subject. Our objective was to perform an intrasubject and intersite analysis to determine the contributions of two upper mucosal sites (mouth and nose) as source communities for the bacterial microbiome of lower sites (lungs and stomach). Oral wash, bronchoalveolar lavage (BAL) fluid, nasal swab, and gastric aspirate samples were collected from 28 healthy subjects. Extensive analysis of controls and serial intrasubject BAL fluid samples demonstrated that sampling of the lungs by bronchoscopy was not confounded by oral microbiome contamination. By quantitative PCR, the oral cavity and stomach contained the highest bacterial signal levels and the nasal cavity and lungs contained much lower levels. Pyrosequencing of 16S rRNA gene amplicon libraries generated from these samples showed that the oral and gastric compartments had the greatest species richness, which was significantly greater in both than the richness measured in the lungs and nasal cavity. The bacterial communities of the lungs were significantly different from those of the mouth, nose, and stomach, while the greatest similarity was between the oral and gastric communities. However, the bacterial communities of healthy lungs shared significant membership with the mouth, but not the nose, and marked subject-subject variation was noted. In summary, microbial immigration from the oral cavity appears to be the significant source of the lung microbiome during health, but unlike the stomach, the lungs exhibit evidence of selective elimination ofPrevotella bacteria derived from the upper airways.

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Fork vs. Spoon: How do you consume your science literature?

Today’s journal club discussion was about how to stay on top of the literature for your field. To me there are three parts to this question:

  1. How do you find out about new papers then what do you do store these until you have time to read them?
  2. How do you read papers: digital vs hard copy? Where do you store and how do you organize the notes you take while reading said papers?
  3. How much time do you spend reading papers each week?

Here are our thoughts on each question but we’d love to know what you do?

1. Finding new papers and keeping a “to read” list

There is a clear dichotomy amongst HMjc members: those who rely on search terms and those who don’t. The first group was the biggest so lets talk about this strategy first. Search termers get notifications, usually through email, when papers with their particular pubmed search terms are published. Some members of this group also get alerts from the individual journals themselves when each issue is published in the form of a table of contents (or TOC) email. The pro of this method is not missing the main papers of interest in a field. The cons of this method are missing papers that don’t match the search terms directly and that it’s time consuming to go through all of the alerts each week. The search termers have the emails but they pretty much just read them as they come in.

The non-search termers rely more on other members of the community to alert them to papers of interest by  following them on google+ and twitter. They also make use of the more mysteriou3466154462_8d3b399cf4_os algorithms in pubchase and google scholar that learn what you like based on what you read, what’s in your library and your publications. The cons to this method include needing to keep a “to read” list as well as the obvious problem of missing key papers and having to do more individual searches. As a member of the non-search termers I can say that although I dread sifting through the soul-crushing deluge of alert emails, I find all of the search termers to be very knowledgeable about current research and often they are the ones I’m following on google+.

I think that the answer here is spork.

2. Digital vs. Paper

For me it’s important to be able to find my notes later… so paper is a problem and I mostly use mendeley and evernote but once a week or so I scribble all over a paper copy. One person has a system of writing “see paper copy” in mendeley when they’ve made hardcopy notes, which I think is brilliant. But pretty much it’s a matter of what method keeps each person most focused.

A couple of people are trying out new gadgets and apps for keeping notes. Livescribe combines a pen a paper combo that lets you take digital notes, includes voice recording and seems very slick. The noteshelf tablet app combined with a good quality stylus lets you scribble on a pdf of the paper you’re reading. I’m eager to see how these work for organizing and searching notes, I’ll keep you posted.

3. How much do you read each week?

The answer is some weeks LOTS and some weeks less. The bottom line is that reading the literature is time consuming so you have to dedicate time to it. Some people like to hide at the coffee shop or the library and others hunker down at their desks but either way you just have to sink your teeth in, as often as you need to, until you feel like you have a handle on your field of study.

Considering how much time is needed for reading abstracts and papers I feel like I need more time-saving strategies for finding and organizing the literature and my notes and would love your input on point 1, or the other two if you insist.

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Recommended Reading: Maternal Microbial Transmission

HumanFetus20wksThe human microbiome is a highly complex community of microorganisms that conveniently does good for our health while we provide it with a cozy place to live. With the advent of sequencing technology, scientists have taken the opportunity to explore this ecosystem in detail to find the story behind these microbes. They have been asking who’s there, where are they located, and what exactly are they doing? Although we’ve made quite some progress in answering these questions, one that has received relatively less attention is, when do we acquire our microbiome?

The current dogma is that the human fetus is sterile and that colonization with microbes begins during exit from the birth canal. The vaginal microbiota is therefore a primary source of microbes for the infant gut microbiota and contribute to the newborn’s health (babies born by C-section are at higher risk of developing allergic disease (1)).

In previous posts we’ve looked at breast milk as an additional source of microbes and also questioned whether the human fetus is indeed sterile. In a recent review published in PLoS Biology (2), Lisa Funkhouser and Seth Bordenstein discuss these internal and external modes of microbial transmission in greater detail. In an added bonus to summarizing the current literature for humans/mice, they consider an evolutionary perspective of symbiotism and feature examples of microbial transmission in marine invertebrates, terrestrial invertebrates, and vertebrates. It’s an insightful look at the mechanisms that different species uses to make sure offspring get their dose of essential microbes.

The review reads like an episode of Planet Earth and is sure to be both entertaining and educational.

1. Bager P, Wohlfahrt J, Westergaard T. (2008) Caesarean delivery and risk of atopy and allergic disease: meta-analyses. Clinical & Experimental Allergy 38(4):634–642
2. Funkhouser LJ, Bordenstein SR (2013) Mom Knows Best: The Universality of Maternal Microbial Transmission. PLoS Biol 11(8): e1001631
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Sifting through microbial lineages within metagenomes

We’re starting the year off by discussing a new paper out this month on metagenomic data analysis (Darling et al. 2014 PeerJ 2:e243). Members of the Matsen and Eisen labs came together to explore methods for phylogenetic-based microbial community analysis and the result is their open-source software package PhyloSift. Bring your low-tech paper and pen to discuss this high-tech article at the Phoenix pub next Monday, January 20th, at 3 pm. The highlights of the paper include:

  • Studying microbial communities with metagenomes (i.e. entire genomes of all the microbes in the sample) instead of the amplicon-based single gene methods (i.e. PCR amplification of the 16S rRNA genes from all microbes in the sample).
  • The software incorporates 37 marker genes as well as all other gene families.
  • They use a phylogenetic methods for studying microbial communities  – we’ll talk about what this means and how it’s different from OTU-based taxonomies.
  • Bayesian hypothesis testing, edge PCA over PCoA or CA based on UniFrac, alpha diversity based on phylogenetic methods versus OTU-based methods, and much more…

We’ll talk about the pros and cons of their method, how this applies to us and metagenome analysis in general and about how they show the taxonomic ambiguities in their data instead of ignoring it. PeerJ is even set up to accept comments and question on the article website, so if we have some burning questions remaining we might even post them. Great, see you there.

https://peerj.com/articles/243/

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Intestinal dysbiosis, fecal microbiota transplants, ulcerative colitis and do it yourself!

Fecal microbiota transplants (FMT) work when it comes to treating Clostridium difficile colitis as it restores alterations in the intestinal microbiota. But is dysbiosis responsible for intestinal inflammation in inflammatory bowel dieases such as ulcerative colitis (UC)? This question has created a lot of buzz and is the focus of next week’s human microbiome jounal club! As I learned this week (courtesy of my lab mate Julie Kaiser @jukais), many people with UC do think FMT is the answer and has taken it upon them to create and administer a home made stool cocktail – a movement now dubbed DIY fecal transplants ‪http://bit.ly/12iRb93 .

Join us Thursday August 29 at 3 pm at the Phoenix to discuss Kump et al., 2013Fecal bacteria sample – a study that concludes intestinal dysbiosis in UC patients does not cause inflammation but is a result of inflammation.

Some points we could discuss:

  • Is dysbiosis in UC a cause of inflammation or a result?
  • Are 6 patients enough to make a statistically significant conclusion?
  • How big a role do donors play in UC FMT success? Could sex, age and environment play a role in a donor’s success?
  • The authors used a single application of donor stool – why does it work for C. Diff but not UC
  • DIY FMT – good idea?

Abstract:

BACKGROUND:In patients with ulcerative colitis (UC), alterations of the intestinal microbiota, termed dysbiosis, have been postulated to contribute to intestinal inflammation. Fecal microbiota transplantation (FMT) has been used as effective therapy for recurrent Clostridium difficile colitis also caused by dysbiosis. The aims of the present study were to investigate if patients with UC benefit from FMT and if dysbiosis can be reversed.

METHODS:Six patients with chronic active UC nonresponsive to standard medical therapy were treated with FMT by colonoscopic administration. Changes in the colonic microbiota were assessed by 16S rDNA-based microbial community profiling using high-throughput pyrosequencing from mucosal and stool samples.

RESULTS:All patients experienced short-term clinical improvement within the first 2 weeks after FMT. However, none of the patients achieved clinical remission. Microbiota profiling showed differences in the modification of the intestinal microbiota between individual patients after FMT. In 3 patients, the colonic microbiota changed toward the donor microbiota; however, this did not correlate with clinical response. On phylum level, there was a significant reduction of Proteobacteria and an increase in Bacteroidetes after FMT.

CONCLUSIONS:FMT by a single colonoscopic donor stool application is not effective in inducing remission in chronic active therapy-refractory UC. Changes in the composition of the intestinal microbiota were significant and resulted in a partial improvement of UC-associated dysbiosis. The results suggest that dysbiosis in UC is at least in part a secondary phenomenon induced by inflammation and diarrhea rather than being causative for inflammation in this disease.

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Stability of the gut microbiota

Two pitchers later we’d only gotten through the first figure. Here are the notes but I spent more time discussing than typing so they’re incomplete. Does anyone have a suggestion for the next paper?

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