You and Your Microbiome – Part 3

  • Top 10 Cited Microbiome Publications are Summarized
  • Welcome to the New World-View of “Holobionts”
  • TriLink Products Cited in Numerous Microbiome Publications

It’s been almost two-and-a-half years since posting Part 2 in this series on microbiomes, which I first began in 2013, and the publication rate keeps accelerating, with about 7,000 articles indexed in PubMed in 2016—way more than the mere 35 in 1996. This vast amount of new microbiome information being published annually led me to use the following search strategy to guide my selection of what’s trending in importance for microbiomes.

Basically, I used Google Scholar to search for publications since 2015 that had the term “microbiome” in the title and, among those items found, used the number of citations as a quantitative indicator of interest, importance, and/or impact. But before summarizing my findings for these Top 10 Most Cited Microbiome articles, here’s what you can read in my previous two postings on microbiomes in case you missed them or want to refresh your memory:

Proportion of cells in the human body. You are comprised of much more than what you think you are! Taken from amnh.org

Meet Your Microbiome: The Other Part of You

  • What’s in your microbiome? Why does it matter?
  • Next-generation sequencing is revealing that you and your bacterial microbiome have a biological relationship.

You and Your Microbiome – Part 2

  • Global obesity epidemic is linked to gut microbiome.
  • Investments in microbiome-based therapies are increasing.

Top 10 Cited Microbiome Publications 

The following articles, which were all published in 2015, are listed in decreasing order of the number of citations in Google Scholar. Titles are linked to original documents for interested readers to consult, and synopses represent my attempt to capture essential findings.

1. Precision microbiome reconstitution restores bile acid mediated resistance to Clostridium difficile (369 citations)

C. difficile (From lactobacto.com)

Many antibiotics destroy intestinal microbial communities and increase susceptibility to intestinal pathogens such as Clostridium difficile, which is a major cause of antibiotic-induced diarrhea in hospitalized patients. It was found that Clostridium scindens, a bile acid 7-dehydroxylating intestinal bacterium, is associated with resistance to C. difficile infection and, upon administration as a probiotic, enhances resistance to C. difficile infection.

2. Dynamics and stabilization of the human gut microbiome during the first year of life (298 citations)

Applying metagenomic sequencing analysis on fecal samples from a large cohort of Swedish infants and their mothers, the gut microbiome during the first year of life was characterized to assess the impact of mode of delivery and feeding. In contrast to vaginally delivered infants, the gut microbiota of infants delivered by C-section showed significantly less resemblance to their mothers. Nutrition had a major impact on early microbiota composition and function, with cessation of breast-feeding, rather than introduction of solid food, being required for maturation into an adult-like microbiota.

Graphical abstract by Bäckhed et al. Cell Host & Microbe (2015)

3. Structure and function of the global ocean microbiome (238 citations)

Taken from Sunagawa et al. Science (2015)

Metagenomic sequencing data from 243 ocean samples from 68 locations across the globe was used to generate an ocean microbial reference gene catalog with >40 million novel sequences from viruses, prokaryotes, and picoeukaryotes. This ocean microbial core community has 73% of its abundance shared with the human gut microbiome despite the physicochemical differences between these two ecosystems.

4. Serotonin, tryptophan metabolism and the brain-gut-microbiome axis (200 citations)

Taken from factvsfitness.com

The brain-gut axis is a bidirectional communication system between the central nervous system and the gastrointestinal tract. Serotonin functions as a key neurotransmitter at both terminals of this network. Accumulating evidence points to a critical role for the gut microbiome in regulating normal functioning of this axis. The developing serotonergic system may be vulnerable to differential microbial colonization patterns prior to the emergence of a stable adult-like gut microbiota. At the other extreme of life, the decreased diversity and stability of the gut microbiota may dictate serotonin-related health problems in the elderly. Therapeutic targeting of the gut microbiota might be a viable treatment strategy for serotonin-related brain-gut axis disorders.

5. The dynamics of the human infant gut microbiome in development and in progression toward type 1 diabetes (184 citations)

Taken from dtc.ucsf.edu

Colonization of the fetal and infant gut microbiome results in dynamic changes in diversity, which can impact disease susceptibility. To examine the relationship between human gut microbiome dynamics throughout infancy and type 1 diabetes (T1D), a cohort of 33 infants genetically predisposed to type 1 diabetes (T1D) was examined to model trajectories of microbial abundances through infancy. A marked drop in diversity was observed in T1D progressors in the time window between seroconversion and T1D diagnosis, accompanied by spikes in inflammation-favoring organisms, gene functions, and serum and stool metabolites. These trends in the human infant gut microbiome thus distinguish T1D progressors from nonprogressors.

6. The microbiome of uncontacted Amerindians (150 citations)

Taken from robertharding.com

Sequencing of fecal, oral, and skin bacterial samples was used to characterize microbiomes and antibiotic resistance genes (resistome) of members of an isolated Yanomami Amerindian village in the Amazon with no documented previous contact with Western people. These Yanomami harbor a microbiome with the highest diversity of bacteria and genetic functions ever reported in a human group. Despite their isolation, presumably for >11,000 years since their ancestors arrived in South America, and no known exposure to antibiotics, they harbor bacteria that carry functional antibiotic resistance (AR) genes, including those that confer resistance to synthetic antibiotics. These results suggest that westernization significantly affects human microbiome diversity and that functional AR genes appear to be a feature of the human microbiome even in the absence of exposure to commercial antibiotics.

7. Akkermansia muciniphila and improved metabolic health during a dietary intervention in obesity: relationship with gut microbiome richness and ecology (136 citations)

Taken from dtc.ucsf.edu

Individuals with obesity and type 2 diabetes differ from lean and healthy individuals in their abundance of certain gut microbial species and microbial gene richness. This study in humans found that, at baseline, A. muciniphila was inversely related to fasting glucose, waist-to-hip ratio and subcutaneous adipocyte diameter. Subjects with higher gene richness and A. muciniphila abundance exhibited the healthiest metabolic status. Individuals with higher baseline A. muciniphila displayed greater improvement in insulin sensitivity markers and other clinical parameters. A. muciniphila is therefore associated with a healthier metabolic status and better clinical outcomes for overweight/obese adults.

8. Host biology in light of the microbiome: ten principles of holobionts and hologenomes (132 citations)

Today, animals and plants are no longer viewed as autonomous entities, but rather as “holobionts“, composed of the host plus all of its symbiotic microbes. The term “holobiont” refers to symbiotic associations throughout a significant portion of an organism’s lifetime, with the prefix holo- derived from the Greek word holos, meaning whole or entire. Holobiont is now generally used to mean every macrobe and its numerous microbial associates, and the term importantly fills the gap in what to call such assemblages. Symbiotic microbes are fundamental to nearly every aspect of host form, function, and fitness, including traits that once seemed intangible to microbiology: behavior, sociality, and the origin of species. Microbiology thus has a central role of in the life sciences, as opposed to a “bit part.”

Taken from researchgate.net

9. The infant nasopharyngeal microbiome impacts severity of lower respiratory infection and risk of asthma development (131 citations)

The nasopharynx (NP) is a reservoir for microbes associated with acute
respiratory infections (ARIs). Lung inflammation resulting from ARIs during infancy is linked
to asthma development. The NP microbiome examination during the first year of life in a cohort of 234 children led to characterization of viral and bacterial communities, and documenting all incidents of ARIs. Most infants were initially colonized with Staphylococcus or Corynebacterium before stable colonization with Alloiococcus or Moraxella. Transient incursions of Streptococcus, Moraxella, or Haemophilus marked virus-associated ARIs. Early asymptomatic colonization with Streptococcus was a strong asthma predictor, and antibiotic usage disrupted asymptomatic colonization patterns.

10. Insights into the role of the microbiome in obesity and type 2 diabetes (128 citations)

Obesity and type 2 diabetes (T2D) are associated with changes in the composition of the intestinal microbiota, and the obese microbiome seems to be more efficient in harvesting energy from the diet. Lean male donor fecal microbiota transplantation (FMT) in males with metabolic syndrome resulted in a significant improvement in insulin sensitivity and increased intestinal microbial diversity, including a distinct increase in butyrate-producing bacterial strains. Such differences in gut microbiota composition might function as early diagnostic markers for the development of T2D. The rapid development of FMTs provides hope for novel therapies in the future.

TriLink Products Cited in Microbiome Publications

It always amazes me to learn about the many ways TriLink products are used in basic and applied science. When I searched Google Scholar for publications containing “TriLink [and] microbiome” I found 21 items, among which the following were selected to illustrate diversity of these product types and uses:

Takeaway Messages

In summary, several takeaways should now be apparent to you. The first takeaway is that there is continuing explosive growth of microbiome publications in all manner of life-related research, as evidenced by both the introductory PubMed graph and wide spectrum of subjects covered by the Top 10 Cited Publications mentioned above.

The second takeaway is best summarized in publication #8 above, “[t]oday, animals and plants are no longer viewed as autonomous entities, but rather as ‘holobionts’, composed of the host plus all of its symbiotic microbes.” Each of us is indeed inextricably comprised of our human cells and symbiotic microbiota in or on us—like it or not, and for better or worse.

The final takeaway is that TriLink products play a contributing role in elucidating and applying this new world-view of halobionts.

As usual, your comments are welcomed.

Autism Awareness Month – April 2017

  • Sequencing for Diagnosis of Autism Holds Promise
  • Several Genetic-Risk Testing Procedures are Available
  • More Than 40 Autism Publications Using TriLink Products

The first National Autism Awareness Month was declared by the Autism Society in April 1970 with the aim of educating the public about autism. Autism is a complex mental condition and developmental disability, characterized by difficulties in the way a person communicates and interacts with other people. Autism can be present from birth or form during early childhood, typically within the first three years. Autism is a lifelong developmental disability with no single known cause.

The puzzle pattern of this ribbon reflects the complexity of autism, while the colors and shapes represent the diversity of people and families living with this spectrum of disorders. Taken from drdiane.com

People with autism are classed as having Autism Spectrum Disorder (ASD) and the terms autism and ASD are often used interchangeably. The term “spectrum” refers to the wide range of symptoms, skills, and levels of disability in functioning that can occur in people with ASD, which includes Asperger syndrome. Some children and adults with ASD are fully able to perform all activities of daily living while others require substantial support to perform basic activities. ASD occurs in every racial and ethnic group, and across all socioeconomic levels. However, boys are significantly more likely to develop ASD than girls. The latest analysis from the U. S. Centers for Disease Control and Prevention (CDC) estimates that 1 in 68 children has ASD.

Taken from myaspergerschild.com

According to the CDC, diagnosing ASD can be difficult, since there is no medical test, like a blood test, to diagnose the disorders. Doctors look at the child’s behavior and development to make a diagnosis.” More details from the CDC are provided at this link.

Notwithstanding this current difficulty for diagnosis of ASD, research has led to continuing progress toward possible blood tests for ASD, which is the focus of this blog and supplements an earlier posting here on treating autism with a broccoli nutraceutical.

ASD and Exome Sequencing

My Google Scholar search for “autism and sequencing” led to a mindboggling list of more than 47,000 items! When ordered by relevance rather than date of publication, two publications were each cited ~1,000-times following “back-to-back” appearance in venerable Nature magazine in 2012. This computes to a combined average of ~400 citations per year, or a fraction more than one citation per day on average, which to me signals significant attention by the ASD research community and thus worth commenting on herein.

Sanders et al., in the first of these two widely cited studies, carried out exome sequencing in 238 families wherein each pair of parents was unaffected by ASD but had a child who was affected (aka proband), and in 200 of these families there was an unaffected sibling. This study design feature is important in view of the widely held idea that complex personality traits are derived by a combination of “nature and nurture,” i.e. genetics inherited from parents and that which is learned or otherwise acquired by familial and all external events.

Before synopsizing what was found, I should note that germline single-base mutations spontaneously arise during mitosis in every generation, and are termed de novo single nucleotide variants (SNVs). Identifying SNVs remained refractory to analysis at the whole genome or exon level until the advent of next-generation sequencing (NGS) technologies.

Sanders et al. found that the total number of non-synonymous (i.e. changes in the amino acid sequence of proteins) de novo SNVs—particularly highly disruptive nonsense and splice-site de novo mutations—are associated with ASD. They concluded that their results “substantially clarify the genomic architecture of ASD, demonstrate significant association of three genes—SCN2A, KATNAL2 and CHD8—and predict that approximately 25–50 additional ASD-risk genes will be identified as sequencing [more] families is completed.”

Neale et al., in the second widely cited study, likewise conducted exome sequencing but on only 175 ASD probands and their parents. Nevertheless, they found that the proteins encoded by genes that harbored de novo non-synonymous or nonsense mutations showed a higher degree of connectivity among themselves and with previous ASD genes as indexed by protein-protein interaction screens. They concluded that their results “support polygenic models in which spontaneous coding mutations in any of a large number of genes increases risk by 5- to 20-fold,” but did acknowledge the strong evidence reported by Sanders et al. for individual genes as risk factors.

ASD Genetic-Risk Testing

The American Academy of Pediatrics (AAP) in 2013 issued a statement on ethical and policy issues for genetic screening of children for ASD that was prompted in part due to then recent progress by IntegraGen—a small French genomics company—on development of a gene test that uses a cheek swab to screen infants and toddlers for 65 genetic markers associated with autism. Highlights of the AAP’s statement include:

  • Genetic screening can be particularly useful for diagnosing older babies and children with developmental disorders such as autism.
  • Genetic screening should be made available for all newborns. However, parents should have the right to refuse screening after being informed of the benefits and risks.
  • The decision to offer testing or screening should be based primarily on the best interest of the child.

Taken from autismspeaks.org

By way of an update, I’m pleased to add that in a 2015 press release by IntegraGen it was announced that its ARISk® Test became the first test marketed in the U. S. to assess the risk of autism spectrum disorder in children. Among the following IntegraGen statements about the ARISk® Test, I think it’s most important to note the caveats I’ve bolded for emphasis:

  • The test does not confirm or rule out a diagnosis of ASD for the child tested.
  • The test is intended to be used together with a clinical evaluation and other developmental screening tools.
  • Intended for children with early signs of developmental delay or ASD and in children who have older siblings previously diagnosed with an autism spectrum disorder.
  • A genetic score, based on the total number of genetic markers associated with autism identified, is used to estimate the child’s risk of developing ASD.
  • Intended for use for children 48 months and younger. The ARISk® Test is not available for prenatal testing.

Taken from integragen.com

More recently, Courtagen—cofounded by my former Life Technologies colleague Kevin McKernan (coinventor of SOLiD® NGS)—has commercialized its sequencing analyses for ASD and other neurodevelopmental conditions. According to a Courtagen posting, “[i]n the absence of a known single-gene disorder, ASD likely involves a complex combination of both genetic and environmental factors that influence early brain development. Multi-gene panels, such as Courtagen’s devSEEK® panels, provide clinicians with information on a number of genes commonly associated with ASD and autistic features. Clinicians can then use information from multi-gene panels to tailor treatments that meet the patient’s unique genotype and symptoms.”

Some interesting—to me—logistical and operational information about devSEEK® (237 genes) is as follows:

  • Turn-around time for results is 4-6 weeks.
  • DNA for sequencing is extracted from a single saliva sample. No blood draw or muscle biopsy required; however, blood and muscle tissue are accepted.
  • Courtagen works with patients, physicians, and insurance carriers to pre-approve each test. Courtagen will bill the insurance company and is willing to handle an appeal process as needed.
  • A secure physician online portal is available for ordering genetic tests and accessing patient reports when completed. Genetic counselors are available to address questions regarding Courtagen test results.

ASD Research and TriLink

While mulling over how to conclude this Autism Awareness Month blog featuring genetic testing for ASD, I wondered about TriLink’s role in advancing autism research by virtue of its various nucleic acid-related products being used for autism investigations. I was pleased and proud to find more than 40 items by searching Google Scholar for articles with the words “autism and TriLink.”

Perusal of these items revealed that the most cited (450-times) report was a 2012 publication in highly regarded Cell titled MeCP2 Binds to 5hmC Enriched within Active Genes and Accessible Chromatin in the Nervous System, which used TriLink 5-methyl-2′-deoxycytidine-5′-triphosphate (5m-dCTP). Given the apparent significance of this publication, I won’t try to give a short, simplified synopsis but rather quote the following part of the authors’ summary:

“We report that 5hmC [5-hydroxymethylcytosine] is enriched in active genes and that, surprisingly, strong depletion of 5mC [5-methylcytosine] is observed over these regions. The contribution of these epigenetic marks to gene expression depends critically on cell type. We identify methyl-CpG-binding protein 2 (MeCP2) as the major 5hmC-binding protein in the brain and demonstrate that MeCP2 binds 5hmC- and 5mC-containing DNA with similar high affinities. The Rett-syndrome-causing mutation R133C preferentially inhibits 5hmC binding. These findings support a model in which 5hmC and MeCP2 constitute a cell-specific epigenetic mechanism for regulation of chromatin structure and gene expression.”

I also noted a 2016 Cutting-Edge Review in Arteriosclerosis, Thrombosis, and Vascular Biology titled A CRISPR Path to Engineering New Genetic Mouse Models. These investigators utilized TriLink Cas9 mRNA for gene editing analogous to that reported by others for CRISPR/Cas9-mediated knockout of the autism gene CHD8 (see above). This led to transcriptomic profiling showing that CHD8 regulates multiple genes implicated in ASD pathogenesis and genes associated with brain volume.

In conclusion, I must say that I learned much new information about autism while researching this blog, which I hope you found informative as well as interesting. If so, I have achieved my goal of either increasing or reaffirming your awareness of autism, and the availability of genetic risk-assessment tests.

As usual, your comments here are welcomed.

Postscript

Recently, a team of academic researchers in Arizona made headlines with their publication in Microbiome reporting ties between autism symptoms and the composition and diversity of a person’s gut microbes, aka “gut microbiome,” about which I’ve commented on in several previous blogs.

The participants, who were 18 children with ASD (ages 7–16 years), underwent a 10-week treatment program involving antibiotics, a bowel cleanse, and daily fecal microbial transplants over 8 weeks. Remarkably, the new therapy seemed to provide some long-term benefits, including an 80% improvement of gastrointestinal symptoms associated with ASDs and roughly a 20% – 25% improvement in autism behaviors, including improved social skills and better sleep habits.

Click here for a simplified, educational video on this work by the principal investigator, Prof. James B. Adams at Arizona State University.

I should emphasize that this is a very small study, and much more research will be needed to verify and firmly establish possible benefits and risks. Interested readers should contact Prof. Adams regarding any questions they might have.