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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Stability of the human gut microbiota

121022_r22702_p233The work of Dr. Gordon and colleagues has generated a lot of excitementabout the gut microbiome and its impact on health and disease, and their recent work is no different. After a first read, this substantial Science paper (see link in Mendeley) seems to actually be both a method and a study wrapped into one convenient package. We’ll dive into the nitty gritty of both the methods and the findings, this Thursday at 3 pm at the Phoenix, to determine:

  • How the LEA-Seq method works and how it stacks up to current sequencing protocols
  • How direct sequencing of 16S rRNA genes compares to whole genome sequencing of cultured bacterial isolates for displaying stability of the gut microbiota over time
  • What data filtering is recommended, for as they say: “…without filtering the microbiota appears much more diverse and much less stable.”
  • What fraction of the microbiota is persistent within an individual over time and after a perturbation like dieting
  • Which members of the microbiota are shared among family members

Abstract: A low-error 16S ribosomal RNA amplicon sequencing method, in combination with whole-genome sequencing of >500 cultured isolates, was used to characterize bacterial strain composition in the fecal microbiota of 37 U.S. adults sampled for up to 5 years. Microbiota stability followed a power-law function, which when extrapolated suggests that most strains in an individual are residents for decades. Shared strains were recovered from family members but not from unrelated individuals. Sampling of individuals who consumed a monotonous liquid diet for up to 32 weeks indicated that changes in strain composition were better predicted by changes in weight than by differences in sampling interval. This combination of stability and responsiveness to physiologic change confirms the potential of the gut microbiota as a diagnostic tool and therapeutic target.

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Next week’s journal club papers on the lung microbiome

Does immunodeficiency associated with HIV infection associate with an altered microbiota in the lungs? In a recent issue of the American Journal of Respiratory and Critical Care Medicine Lozupone et, al. show that it does, by reporting that 13% of HIV-positive subjects have lung communities dominated by Tropheryma whipplei, which decreased in relative abundance after antiretroviral therapy. This bacterium is known as the cause of Whipple’s disease, a rare disease with widespread infection and symptoms that is eventually fatal if untreated. Further work will be needed to determine if T. whipplei colonization of the lungs leads to adverse clinical outcomes but colonization by this nasty opportunistic pathogen was unexpected and needs to be monitored.

Within the same issue, Morris et, al. propose a comprehensive evaluation of the normal human lung microbiome by not only designing a multicenter study with standardized collection and processing protocols but also by considering it in the context of the upper respiratory tract. They also evaluate the impact of smoking on the microbiota of both the mouth and the lungs. What they found is that not all organisms in the lungs originate from the mouth suggesting that there is selection for a lung-specific microbial community and that smoking alters communities in the mouth but not in the lungs. 

Join us next Friday (June 28th) at 2 p.m. at the Phoenix for a lively discussion of our favourite human-associated microbial community – the lung microbiome!

General talking points we could cover:

  • What is the impact of T. whipplei in the airways?
  • Why doesn’t smoking alter the lung microbiome?
  • What are the challenges of sampling the lower airways?
  • Are the lower airways effectively sterile or is there a viable lung microbiome in healthy individuals?
  • DNA signatures vs viable organisms – where do we draw the line and how can we better address this?

Again free beer may be in attendance.

Lozupone, C., Cota-Gomez, A., Palmer, B. E., Linderman, D. J., Charlson, E. S., Sodergren, E., Mitreva, M., et al. (2013). Widespread Colonization of the Lung by Tropheryma whipplei in HIV Infection. American journal of respiratory and critical care medicine187(10), 1110-7. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/23392441

Morris, A., Beck, J. M., Schloss, P. D., Campbell, T. B., Crothers, K., Curtis, J. L., Flores, S. C., et al. (2013). Comparison of the respiratory microbiome in healthy nonsmokers and smokers. American journal of respiratory and critical care medicine187(10), 1067-75. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/23491408

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Bacteriophage at mucosal sites help keep bacteria at bay

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Scientists recently described a new form of symbiosis at our mucosal sites with an unassuming partner: bacteriophage. In a paper published in PNAS, Jeremy Barr and colleagues noticed that phage are enriched at mucosal surfaces compared to surrounding surfaces and demonstrated that a specific interaction between Ig-like domains on the phage capsid and mucin glycans is what holds them in place. Their findings suggest that the presence of phage in mucin protects the underlying epithelial cells from bacterial infection, a good reason for us to have evolved to keep them there. The relationship benefits the phage too – mucosal sites are a buffet of bacteria for the phage to infect and multiply within.

Join us at the Pheonix next Friday May 31st at 1 PM for a discussion over drinks about our friendly phage.

Here are some reasons we chose this paper and a few things to think about for discussion:

• It’s important to consider the diversity of microorganisms in our microbiome and their interactions with each other and the host in order to understand the ecosystem. Given phage specificity for certain bacterial species, does our virome change with fluctuations in bacterial communities? How does this alter community dynamics? (Interesting read on virome research: The other microbiome)

• We’ve coerced bacteriophage to act as our microscopic army against invading pathogens. It’s a strategy that not only humans, but also other metazoans with mucosal sites use as a strategy to defend against infection. But bacteria are smart too and aren’t content with the short end of the stick. They have their own defense against phages – the CRISPR/Cas systems. Recently this system is also implicated in bacterial virulence and immune evasion. Have our friendly phage contributed to pathogen evolution?

If you’re not able to make it, but are equally as fascinated with our inner viromes as we are, check out these links for more info and check back for a follow-up post on our discussion.

Carl Zimmer, The Loom: Meet your new symbionts: Trillions of Viruses

Ed Yong, Nature News: Viruses in the gut protect from infection 

Story behind the paper by Jeremy Barr, The Tree of Life

Bacteriophage Paper: Barr, J. J. et al. Proc. Natl. Acad. Sci. USA http://dx.doi.org/10.1073/pnas.1305923110 (2013)

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The microbial cloud

One of my favourite microbiologist is Jonathan Eisen, mostly because of the way he thinks about the microbial world around us and challenges conventions in science and elsewhere, but also because he’s creative and funny and outspoken in social media. Now he’s even dearer to my heart for sharing his personal experience with T1 diabetes in this compelling TED talk on the microbial world on and in us. It’s from about 6 months ago (sorry I’m a bit behind on my reading/viewing) but it’s still very topical. I especially related to his musing about what effect the “microbial cloud”, which physically covers each person, may have on the progression of autoimmune diseases such as T1 diabetes. He suggests that an imbalance in the microbial community could cause a miscommunication in a person’s immune response and possibly kick off an autoimmune reaction that ultimately kills the insulin producing beta cells. I would add that immune responses probably vary from one person to another, adding another level of complexity to this interaction and outcome. Anyhow, take a look, if you haven’t already…but watch out for the poo tea.

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