May is Mental Health Month

  • Mental Health America Began This Annual Observance 70 Years Ago
  • Two Recent Studies Link Gut Microbe Composition to Mental Health Disorders: Clinical Depression and Schizophrenia  

Since 1949, Mental Health America (MHA) and its affiliates across the country have led the observance of May as Mental Health Month, reaching millions of people through the media, local events, and screenings. MHA welcomes other organizations to join it in spreading mental health awareness by using the May is Mental Health Month toolkit materials and conducting educational activities through #4Mind4Body. This blog is a way for TriLink BioTechnologies, on behalf of all Maravai LifeSciences businesses, to join MHA in “spreading the word” about this very important health issue.

In general, my blogs tend to revolve around topics trending in nucleic acids research. In keeping with this, I will briefly discuss two recent publications that provide new and compelling evidence linking human gut microbiome composition to depression and schizophrenia. These examples of the gut-brain connection (aka gut-brain axis) are especially noteworthy, since microbiome composition can be monitored by sequencing and altered by diet, as I have previously blogged about. Because of this, dietary modifications can be a healthy alternative to traditional medications, which have adverse side-effects and can be abused. 

Some Basics About Depression and Schizophrenia

According to the U.S. National Institute of Mental Health (NIMH), the lead federal agency for research on mental disorders, depression and schizophrenia are two among eleven major categories that include autism spectrum disorder, attention-deficit/hyperactivity disorder, bipolar disorder, etc. 

Depression: While there are a number of discrete forms of depression, all involve some type of serious mood disorder that affects how you feel, think, and handle daily activities, such as sleeping, eating, or working. To be diagnosed with depression, the symptoms must be present for at least two weeks. Depression (aka major depression or clinical depression) is one of the most common mental disorders, estimated by NIMH to afflict ~17 million adults in the U.S. 

Current research suggests that depression can be caused by genetic, biological, environmental, and/or psychological factors. It can happen at any age, but often occurs in adulthood, sometimes beginning as high levels of anxiety in children or adolescents. Even in the most severe cases, depression can be treated. The earlier treatment can begin, the more effective it is. Depression is usually treated with medications (antidepressants), psychotherapy, or a combination of the two. If these treatments do not reduce symptoms, electroconvulsive therapy and other brain stimulation therapies may be options worth exploring.

In regard to anti-depression medications and possible abuse, it is worth mentioning that, on March 5th of this year, the U.S. FDA announced approval of Spravato (esketamine) nasal spray, in conjunction with an oral antidepressant, for the treatment of depression in adults who have tried other antidepressant medicines but have not benefited from them (treatment-resistant depression). Spravato administration causes sedation and dissociation. Because of the risk of serious adverse outcomes resulting from these effects, and the potential for abuse and misuse of the drug, it is only available through a restricted distribution system.

Schizophrenia: This chronic and severe mental disorder affects how a person thinks, feels, and behaves. People with schizophrenia may seem like they have “lost touch with reality.” Schizophrenia is estimated to afflict ~2.5 million people in the U.S. Although not as common as other mental disorders, the symptoms can be very disabling, and usually start between ages 16 and 30. The symptoms of schizophrenia fall into three categories: positive, negative, and cognitive, which are further defined here. 

Scientists believe that although many different genes may increase the risk of schizophrenia, no single gene causes the disorder by itself. It is also believed that interactions between genes and aspects of the individual’s environment are necessary for schizophrenia to develop. Environmental factors may involve: viruses, malnutrition before/problems during birth, and psychosocial factors. An imbalance in the complex, along with interrelated chemical reactions of the brain involving the neurotransmitters dopamine and glutamate, may also play a role in the onset of schizophrenia.

Some experts believe that problems during brain development before birth may lead to faulty connections. The brain also undergoes major changes during puberty, and these changes can trigger psychotic symptoms in people who are vulnerable due to genetics or brain differences. Treatments typically involve antipsychotic medications and psychosocial therapy, both of which are discussed elsewhere. 

Links Between Gut Microbes and Depression

For more information on this complex topic, interested readers can consult a comprehensive review in 2014 by Mayer et al. titled Gut Microbes and the Brain: Paradigm Shift in Neuroscience, which covers preclinical literature for signaling between the brain and the gut microbiome. The possibility that alterations in the gut microbiome may play a pathophysiological role in various brain diseases, including depression, was considered by the authors to be speculative and in need of large population-based studies. The population size needs to provide statistically significant results upon which conclusions can be based.

Such a study has been recently highlighted in prestigious Nature magazine, in an editorial which notes that “just ten years ago, the idea that microorganisms in the human gut could influence the brain was often dismissed as wild. Not anymore.” As only a short synopsis of this landmark paper published in February 2019 in Nature Microbiology by Valles-Colomer et al. will be presented here, interested readers can consult the full publication at this link. Next-generation sequencing and bioinformatics methodologies used for identification and quantification of microbe diversity, which are technically quite complicated processes but now widely employed for many applications, can be read about by consulting the lead reference at this link.

In this study, Valles-Colomer et al. first assess gut microbiota compositional covariation with quality of life (QoL) indicators and general practitioner-reported depression in the Belgian Flemish Gut Flora Project (FGFP) population cohort (n = 1,054). The results were then validated both in the Dutch LifeLines DEEP (LLD) cohort with associated QoL and self-reported depression metadata (n = 1,063) and in previously published case–control studies on depression. To functionally analyze the gut microbiota neuroactive metabolic potential, they developed modules to profile the microbial pathways involved in neuro-microbiome mediator metabolism. Finally, application of these gut–brain modules in a shotgun-sequenced subset of the FGFP (n = 150) and validation in the LLD metagenomes data set (n = 1,063), and among a patient group suffering from treatment-resistant major depressive disorder (TR-MDD; n = 7), allowed Valles-Colomer et al. to link microbiota neuroactive capacity with QoL and depression.

The researchers found that two groups of bacteria, Coprococcus and Dialister, were reduced in people with depression. Gut–brain module analysis identified the microbial synthesis potential of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) as correlating positively with mental QoL, and indicated a potential role of microbial γ-aminobutyric acid (GABA) production in depression. However, it should be emphasized that these are correlations, and do not necessarily result in causation. 

 

DOPAC (PubChem Open Access) // GABA (PubChem Open Access)

According to the Nature editorial mentioned above, “the challenge now is to find out whether, and how, these microbe-derived molecules can interact with the human central nervous system, and whether that alters a person’s behavior or risk of disease. At least now, answering these questions is a wise pursuit, not a wild one.”

Links Between Gut Microbes and Schizophrenia

Almost concurrently with the above report in February 2019, Zheng et al. published a study in the prestigious Science Advances. This study also used next-generation sequencing and bioinformatics methodologies to link gut microbes with schizophrenia (SCZ). Sequencing reads from 63 patients with SCZ and 69 human controls (HCs) were clustered into 864 operational taxonomic units (OTUs) at 97% sequence similarity. A Venn diagram showed that 744 of 864 OTUs were detected in the two groups, while 56 and 64 OTUs were unique to patients with SCZ and HCs subjects, respectively.

To determine whether these altered OTUs were relatively specific to SCZ versus other neuropsychiatric disorders, they compared key differential bacterial taxa observed in SCZ and major depression. These findings indicated that the altered gut microbial composition observed in SCZ is specific relative to the gut microbiome changes observed in depression.

To identify key discriminative microbial markers, Zheng et al. performed a stepwise regression analysis based on the relative abundance of different gut microbes. This analysis showed that the most significant deviations between SCZ and HC subjects occurred for the bacterial families Aerococcaceae, Bifidobacteriaceae, Brucellaceae, Pasteurellaceae, and Rikenellaceae, which was said to suggest potential diagnostic value for SCZ.

In my opinion, the experimentally most interesting work involved fecal microbiota transplantation from patients with SCZ to germ-free mice to assess whether SCZ-relevant behavioral phenotypes might be linked with disturbed gut microbiota. Behavioral tests showed that mice transplanted with SCZ microbiota displayed locomotor hyperactivity, decreased anxiety- and depressive-like behaviors, and increased startle responses, suggesting that the disturbed microbial composition of SCZ microbiota recipient mice was associated with several characteristic of mouse models of SCZ.

Finally, to capture functional readout of microbial activity, Zheng et al. performed comparative metagenomic and metabolomic analyses of samples from the fecal transplanted-mice harboring “SCZ microbiota” versus “HC microbiota” to determine the potential mechanistic pathways by which the disturbed gut microbiota may modulate host physiology and behavior.

They identified decreased brain glutamate, disruptions in the glutamate-glutamine-GABA cycle, and altered amino acid and lipid metabolism as possible mechanistic targets that may underlie the altered behavior in the SCZ microbiota recipient mice, which was said to be consistent with known pathophysiology of SCZ. They also found significant changes in lipid species, especially involving glycerophospholipid metabolism, in the SCZ microbiota versus HC microbiota mice. This was said to be consistent with disturbances in serum and brain lipids previously observed in patients with SCZ, and with the fact that glycerophospholipids are major components of the myelin and neuronal membranes that are key regulators of synaptic function.

Concluding Comments

I hope that these brief synopses of the recent reports linking gut microbiota to clinical depression by Valles-Colomer et al., and to SCZ by Zheng et al., will in some way lead you to agree with me that such applications of nucleic acid sequencing to microbiome metagenomics are indeed representative of a new era for investigating mental health. My belief is that this type of sequencing will eventually lead to improved diagnosis of these and other neurological diseases. The extent to which controlled diets and/or transplants of therapeutic microbiota will contribute to improving mental health are still matters of speculation at this time. However, being an optimist, my outlook on these alternatives to traditional medications is favorable.

As usual, your comments are welcomed. 

Addendum

Although only indirectly related to the gut microbiome-brain focus of this blog, I think it’s worth mentioning the following findings recently published in Nature by researchers at the EMBL European Bioinformatics Institute and the Wellcome Sanger Institute.

1,952 uncultured candidate bacterial species were identified by reconstructing 92,143 metagenome-assembled genomes from 11,850 human gut microbiomes. These uncultured genomes substantially expand the known species repertoire of the collective human gut microbiota, with a 281% increase in phylogenetic diversity. 

Although the newly identified species are less prevalent in well-studied populations compared to reference isolate genomes, they improve classification of understudied African and South American samples by over 200%. These candidate species encode hundreds of novel biosynthetic gene clusters and possess a distinctive functional capacity that might explain their elusive nature. The researchers concluded that this work “uncovers the uncultured gut bacterial diversity, providing unprecedented resolution for taxonomic and functional characterization of the intestinal microbiota.”

You and Your Microbiome—Part 4

  • Your Microbiome Is Modulated by Your Diet
  • Probiotic Dietary Supplements Are Big Business but Lack Scientific Rigor
  • ‘Personalized Nutrition’ for Controlling Glucose Levels Is Supported by a New Study

My 2013 blog, Meet Your Microbiome: The Other Part of You, constituted part 1 of this series. The intent was to underscore scientific recognition that trillions of microbes reside in and on each of us, and influence our health. Moreover, the compositions of these microbiomes change with our diet, what we drink or breath, and who we come into contact with, whether that be family members, pets, or close friends.

Part 2 of this series was my 2014 blog, which focused on links between the global obesity epidemic and the gut microbiome, and cautioned microbiome-based therapies. In part 3, which followed in 2017, I commented on the ‘Top Ten Cited’ microbiome publications in Google Scholar. As shown by the chart below, there is an exponential increase in the annual number of publications in PubMed with terms ‘microbiome(s)’ in all fields. The ~8,900 papers that appeared in 2017 give an average of ~24 papers per day, non-stop 7 days per week, which is truly an impressive torrent of new information.

Credit: Jerry Zon

Cindy D. Davis, PhD

Part 4 of my series on You and Your Microbiome is largely a synopsis of Diet, Microbiome and Health: Past, Present & Future, a recent seminar by Cindy D. Davis, PhD, a Director in the Office of Dietary Supplements (ODS) at the National Institutes of Health (NIH). The mission of ODS includes strengthening the common understanding of dietary supplements by evaluating scientific information, supporting research, and educating the public in an effort to foster an enhanced quality of life and health for the U.S. population.

The importance of the ODS mission, and the NIH’s highly regarded reputation as a source of reliable health-related information, led me to select this seminar by Dr. Davis.

Introduction

Dr. Davis presented her seminar as part of the 2018 Dietary Supplement Research Practicum sponsored by the NIH ODS. This 2.5-day annual event for faculty, students, and health practitioners provides a thorough overview of the issues, concepts, unknowns, and controversies surrounding dietary supplements. This seminar, followed by a Q&A session, is available as a video on YouTube.

In her seminar, Dr. Davis introduces the human microbiome and describes evidence on how diet and dietary supplements can modulate the gastrointestinal (GI) microbial community structure. She also describes evidence on how the GI microbiome can influence the response to dietary components, and the relationship between dietary components, the microbiome, and chronic diseases such as obesity, cardiovascular disease, and cancer.

The Human Microbiome

Perhaps the biggest surprise of the Human Genome Project (HGP) was the discovery that the human genome contains only 20,000 – 25,000 protein-coding genes, about a fifth of the number researchers had expected to find. To search for the missing genes that could account for this discrepancy, researchers started looking toward other sources of genetic material that contribute to human function. One of these sources was the human microbiome.

The human microbiome is defined as the collective genomes of the microbes (composed of bacteria, bacteriophage, fungi, protozoa, and viruses) that live inside and on the human body. There are about 10 times as many microbial cells as human cells. So, to study the human as a “supraorganism,” composed of both non-human and human cells, the NIH launched the Human Microbiome Project (HMP) in 2007, as a conceptual extension of the Human Genome Project.

It is now known that, when compared to the total number of human genes, the genetic contribution of the microbiome to the human supraorganism may be many hundreds of times greater than the genetic contribution of the human genome.

There are estimated to be ~100 trillion (!) gut microbiota, defined as the microbes in our GI tract. Most of the microbes in the microbiome do not cause disease. In fact, humans rely on microbes to perform many important functions that we cannot perform ourselves. Microbes digest food to generate nutrients for host cells, synthesize vitamins, metabolize drugs, detoxify carcinogens, stimulate renewal of cells in the gut lining, and activate and support the immune system.

Establishing what constitutes a healthy microbiome is important because high or low microbial diversity can have different implications for health or disease, depending on the body site. For example, it has been shown that low microbial diversity in the gut is associated with obesity, inflammatory bowel disease, and Crohn’s disease; whereas high microbial diversity in the vagina is often associated with bacterial vaginosis, the most common type of vaginal infection.

Dietary Modulation of Gut Microbiota

There is growing concern that recent lifestyle trends, most notably the high-fat/high-sugar “Western” diet, have altered the genetic composition and metabolic activity of our human gut microbiomes. Such diet-induced changes to gut-associated microbial communities are suspected of contributing to growing epidemics of chronic illnesses in the developed world, including obesity and inflammatory bowel disease. Yet, it remains unclear how quickly and reproducibly gut bacteria respond to dietary change.

David et al. have addressed this question in a study of human volunteers, and found that the short-term consumption of diets composed entirely of animal or plant products alters microbial community structure and overwhelms inter-individual differences in microbial gene expression.

Microbial activity mirrored the differences between carnivorous and herbivorous mammals, reflecting trade-offs between carbohydrate and protein fermentation. Foodborne microbes from both diets transiently colonized the gut, including bacteria, fungi, and even viruses. David et al. concluded that, in concert, these results demonstrate that the gut microbiome can rapidly respond to an altered diet, potentially facilitating the diversity of human dietary lifestyles.

Similar findings have been reported by De Filippo et al., who evaluated the impact of diet in shaping gut microbiota in a comparative study in children from Europe and rural Africa. More recently, there was widespread media coverage on a publication in the highly respected Cell journal by Vangay et al. They used metagenomic DNA sequencing and found that migration from a non-Western country to the U.S. is associated with immediate loss of gut microbiome diversity and function, in which U.S.-associated strains and functions displace native strains and functions. These effects increase with duration of U.S. residence and are associated with increased obesity.

Probiotic Dietary Supplements

In her seminar, Dr. Davis emphasized that usage of probiotics, which by definition contain live bacteria, is by far the fastest growing (~17% annually) dietary supplement. She added that U.S. sales of probiotics in 2016 was $1.8 billion, which ranked third among sales of other dietary supplements, following B vitamins ($2.1 billion) and multivitamins ($5.9 billion). I later found that the global probiotic market is estimated to be $64 billion by 2023.

Dr. Davis emphasized the need for caution in accepting reports purporting linkage between probiotics and parameters for obesity such as BMI, shown here in easy-to-use graphical form. She said this caution is based on the results of a systematic review and meta‐analysis of randomized controlled trials, published by Borgeraas et al. in 2017. In a nutshell, these investigators searched for placebo-controlled trials published between 1946 and September 2016. A meta-analysis was performed to calculate the weighted mean difference between the intervention and control groups. Of 800 studies identified through the literature search, only 15 met standards for inclusion in the final analysis.

The meta‐analysis showed that short‐term (≤12 weeks) probiotic supplementation reduced body weight, BMI and fat percentage, but the effect sizes were small. Overall, the risk of bias within included studies was low. Dr. Davis stated that the “findings [of the 15 trials] were not clinically significant,” and agreed with the conclusion of Borgeraas et al. that “further long‐term studies are required to examine the effects of probiotic supplementation on various measures of body weight.”

‘Probiotics—Where is the Science?’

This section’s title is the rhetorical question posed by Dr. Davis in her concluding opinions, which were grouped into “what we know” followed by “what we don’t know.”

What we know:

  • There’s preliminary evidence that some probiotics are helpful in preventing diarrhea caused by infections and antibiotics, and improving symptoms of irritable bowel syndrome.
  • The U.S. FDA has not approved any probiotic for preventing or treating any health problem.
  • Probiotic supplements are probably better if they have multiple strains of bacteria.
  • Probiotic supplements should provide at least 1 billion live cells per gram.
  • If people are generally healthy, probiotics have a good safety record; if not, a doctor’s advice should be obtained.

What we don’t know:

  • Which probiotics are helpful and which are not; not all probiotics have the same effect, and effects are likely strain-specific.
  • The amount of a probiotic a person should use.
  • Who would most likely benefit from taking probiotics.

Progress in Identifying Helpful Probiotics

Credit: By Sebastian Kaulitzk

In response to Dr. Davis’s opinion that we don’t yet know “which probiotics are helpful and which are not,” I found that progress is being made toward obtaining such information. For example, Piewngam et al. recently reported in the highly regarded journal Nature that probiotic Bacillus bacteria, commonly ingested with vegetables, protect against the pathogen Staphylococcus aureus, which is known to mutate into antibiotic-resistant variants. Feeding mice B. subtilis spores, shown here, completely abrogated colonization of all tested S. aureus strains in the feces and intestines in experimental set-ups with or without antibiotic pretreatment to eliminate the pre-existing microbiota. Interested readers can consult Piewngam et al. for details on the molecular mechanism that underlies this protective effect.

Another example is the recent report by Bodogai et al., published in the equally prestigious Science Translational Medicine. They identified the health-promoting activity of a bacterium called Akkermansia muciniphila. In brief, it was found that in mice and monkeys whose metabolisms had grown dysfunctional with age, taking steps to boost A. muciniphila in the gut reduced the animals’ insulin resistance. Insulin resistance is the gradual impairment of the body’s ability to efficiently use food for fuel. It is best known as a way station on a patient’s path to developing type 2 diabetes.

Bodogai et al. noted that insulin resistance is also linked to a variety of ills, from obesity and inflammation to the sagging immunity and frailty that comes with advancing age. If A. muciniphila can be experimentally established as a probiotic supplement to slow or reverse insulin resistance, it might have broad and powerful anti-aging effects, in addition to its ability to protect obese adults from developing type 2 diabetes.

It will of course take many years of carefully conducted research on large study populations to determine additional answers to the questions still surrounding probiotics. Moreover, these studies will need to consider genetic differences among ethnic groups, as well as the role of ‘personalized nutrition,’ which I comment on in the next section.

‘A Good Diet for You May Be Bad for Me’

This section’s catchy title is how a news story referred to a publication by Zeevi et al. on the need for ‘personalized nutrition’ when monitoring glycemic responses. Zeevi et al., who were cited by Dr. Davis, state that elevated postprandial blood glucose levels constitute a global epidemic and a major risk factor for prediabetes and type II diabetes, but existing dietary methods for controlling them have limited efficacy. To address this problem, Zeevi et al. continuously monitored week-long glucose levels in an 800-person cohort, measured responses to 46,898 meals, and found high variability in the response to identical meals, suggesting that universal dietary recommendations may have limited utility.

Zeevi et al. devised a machine-learning algorithm that integrated blood parameters, dietary habits, anthropometrics, physical activity, and gut microbiota in this cohort. The algorithm showed accurate predictions to personalized postprandial glycemic response to real-life meals. The researchers validated these predictions in an independent 100-person cohort.

Finally, a blinded randomized controlled dietary intervention based on the algorithm resulted in significantly lower postprandial responses and consistent alterations to gut microbiota configuration. It was concluded that, “together, these results suggest that personalized diets may successfully modify elevated postprandial blood glucose and its metabolic consequences.”

Concluding Remarks

I encourage interested readers to peruse the NIH ODS website tab for Health Information, which is loaded with helpful links, such as the extensive alphabetized list of Dietary Supplement Fact Sheets. There is also a tab For Researchers that includes numerous funding opportunities.

On a different note, while writing this blog I became intrigued on discovering how TriLink products may have been used in microbiome-related research. I searched Google Scholar for “microbiome” and “TriLink” in the same article and found 39 results. Perusing these led me to select the following three exemplary snippets, each of which is linked to the original article that can be consulted by interested readers for further details:

(1.) Selective microbial genomic DNA isolation using restriction endonucleases. HE Barnes, G Liu, CQ Weston, P King, LK Pham… – PloS one, 2014 – journals.plos.org … The technique can enable the targeted enrichment of genomes from various microbiomes or the specific identification of pathogens from … oligonucleotide containing one G m6 ATC site with the top strand sequence FAM-GCAGG m6 ATCAACAGTCACACT (TriLink, San Diego, CA …

(2.) Peripherally-induced regulatory T cells contribute to the control of autoimmune diabetes. C Schuster, F Zhao, S Kissler – bioRxiv, 2017 – biorxiv.org … The effects of the microbiome on T1D may derive from the capacity of different microbial communities to promote the generation of pTregs relevant to pancreatic autoimmunity. In light of our finding … Cas9 mRNA was purchased from TriLink Technologies …

(3.) Fecal Microbiota Transplantation Is Associated With Reduced Morbidity and Mortality in Porcine Circovirus Associated Disease. MC Niederwerder, LA Constance… – Frontiers in …, 2018 – ncbi.nlm.nih.gov … The LLMDA was used to analyze microbiome composition and diversity of the transplant material and fecal samples … Briefly, the samples were labeled using nick translation with Cy3-labeled random nonamer primers (TriLink Biotechnologies, San Diego, CA, United States) and …

From these three examples, it is evident to me that the TriLink product-reach indeed extends into various interesting and important aspects of microbiome research.

As usual, your comments are welcomed.

Billionaire Bill Gates Bets Big on a Startup That Prints Synthetic DNA

  • Gates Is Part of a $275 Million Investment in Ginkgo Bioworks, an Organism Engineering Startup
  • Ginkgo Bioworks Is Now a Financial ‘Unicorn’ Valued at $1 Billion
  • Bayer and Ginkgo Bioworks Joint Venture, Named Joyn Bio, Aims for Self-Fertilizing Plants

Bill Gates in 2015. Credit: Frederick Legrand

Imagine founding a company so successful that it skyrockets your net worth to nearly $100 billion, making you one of the wealthiest people on the planet. Now think hard about how you would use a sizeable portion of that money to make the world a better place. This is Bill Gates’ reality. Alongside his wife, the Microsoft founder launched the Bill & Melinda Gates Foundation in 2000. Holding $38 billion in assets, it is the largest private foundation in the US. The primary aims of the foundation are, globally, to enhance healthcare and reduce extreme poverty, and in America, to expand educational opportunities and access to information technology.

The Gates support a wide variety of remarkable projects that strive to make the world a better place, both now and in the future. Over the years, I have read about some of their foundation’s health-related programs, notably, those focused on accelerating the eradication of malaria, and those supporting research on bettering afflicted people living in extreme poverty. These laudable efforts are, however, rather different from the Gates’ recent investment in Ginkgo Bioworks, a “hot” start-up company that “prints DNA,” and the focus of this blog.

Backstory on Ginkgo Bioworks

In a nutshell, Ginkgo Bioworks is a Boston-based biotech company founded by MIT scientists in 2009. The company uses genetic engineering to produce bacteria with industrial applications. If you’re familiar with the history of genetic engineering, repurposing bacteria for “industrial applications” is not a new idea, as there are hundreds of publications going back to 1985—1990 that can be perused later at this link.

Artistic rendition of the concept of genetic engineering. Credit vchal

However, Ginkgo Bioworks (aka Ginkgo) is adopting new state-of-the-art technologies in its deals with established leaders in bioindustrial fermentation. These technologies help improve the efficiency of the microbial strains that power their processes. For example, high-throughput strain improvement by Ginkgo, partnered with global companies Ajinomoto and Cargill, involved the design and testing of more than 1,700 rationally engineered plasmids, accounting for 2,400,000 base pairs of synthetic DNA produced by Twist Bioscience and Ginkgo’s biological fabrication (BIOFAB) platform—all in only 10 months!

Ginkgo’s Bioworks uses large-space factory-like labs loaded with robotic equipment akin to this. Credit martin-dm

Twist technology uses small-scale high-density DNA synthesis (“printing”) on silicon, and in 2015 Twist agreed to supply Ginkgo with 100,000,000 base pairs of DNA, which was speculated to be ~10% of the total capacity of synthetic DNA worldwide. Two years later, Ginkgo acquired Gen9, a DNA synthesis company founded by George Church, who I have described as “the most interesting scientist in the world” in one of my previous blogs. An in-depth account of this Ginkgo-Gen9 union can be read at this link.

Ginkgo Bioworks uses a so-called “foundry” to automate every step of strain engineering, from DNA synthesis using the BIOFAB platform through molecular biology, high throughput analytics, and small-scale fermentation. You can watch an informative video of a TV interview and “walk-through” of Ginkgo’s Bioworks here.

Ginkgo Bioworks Is Now a Unicorn

In 2014, Ginkgo was the first biotech company to ever be accepted by Y Combinator—a now renown Silicon Valley venture capital “seed accelerator.” Since then, Ginkgo has raised $429 million, which includes $275 million in funding from Bill Gates’ Cascade Investment and others. This reportedly makes Ginkgo now worth $1 billion. This lofty valuation also makes the company a “unicorn” in finance-speak, whereby a unicorn is a privately held startup company valued at over $1 billion. Venture capitalist Aileen Lee coined the often-used Silicon Valley term unicorn in a TechCrunch article: “Welcome to The Unicorn Club: Learning from Billion-Dollar Startups” as profiled in a New York Times article…but I digress.

Bayer + Ginkgo Bioworks = Joyn Bio

Last September, Bayer, a global agricultural giant, announced that it would work with Ginkgo Bioworks to create a new company focused on the plant microbiome. Improving microbes’ ability to make nitrogen fertilizer available for plants offers a major potential benefit to sustainable agriculture, as it provides a more eco-friendly option relative to the use of conventional chemical fertilizers. The $100 million deal will involve Bayer’s West Sacramento, California, R&D expertise on plant biology.

In March 2018, Bayer announced the name of this joint venture with Ginkgo: Joyn Bio. The Joyn Bio team is characterizing Bayer’s extensive library of more than 100,000 proprietary microbial strains using Ginkgo’s high-throughput foundry tools to identify the strains and characteristics necessary to further develop nitrogen fixing bacteria for sustainable agriculture. As depicted here, the basic idea is for engineered microbes to convert atmospheric nitrogen (N2) into ammonia (NH3) for use by plants. These are simple molecules, but the biochemistry for conversion of N2 into NH3 is complex.

Nitrogen exists as N2 gas. Credit tussik13  //   Ammonia exists as a NH3 gas. Credit goktugg

Incidentally, Joyn Bio is one of a number of investments by Leaps by Bayer, a unit of Bayer focused on finding solutions to some of today’s biggest problems. Previous Leaps investments include Casebia Therapeutics (CRISPR/Cas technology) and BlueRock Therapeutics (induced pluripotent stem cell technology). You can read about CRISPR/Cas on the TriLink products section or in my previous blogs.

Ginkgo’s Business is Rosy

Red roses ready for harvest. Credit: Svetlana Chernova

My penchant for puns prompted this section heading. It’s a play on the words rose (flower) and rosy (optimism) to convey the fact that Ginkgo’s success in developing a biosynthetic rose fragrance has—dare I say grown—into a rosy commercial sector. Robertet, an established French supplier of naturally produced fragrances, and Ginkgo, collaborated on a production strategy using designer yeast that can now create fragrance components—such as rose petal essence—at a scale sufficient to meet the needs of the cosmetics, perfume, and personal care industry. This can supplement or supplant Robertet’s traditional grow-and-extract production methods.

Ginkgo tree in the park of Bad Salzdetfurth, Germany. Credit: Astrid Schur

By the way, you may not know that Ginkgo biloba, commonly known as ginkgo, and also known as the ginkgo tree or the maidenhair tree, is the only living species in the plant division Ginkgophyta, all others being extinct. It is found in fossils dating back 270 million years. Native to China, the tree is widely cultivated, and was cultivated early in human history. It is a source of food and also has various uses in traditional medicine. I tried but could not find how and why Ginkgo Bioworks chose its name. My guess is that the decision was related to some aspect of Ginkgo biloba, perhaps in anticipation of Ginkgo’s corporate longevity relative to related species, i.e. other startups.

Gates’ Simpatico View of Ginkgo Bioworks

A CNBC.com article about Gates and Ginkgo described the essence of Ginkgo’s approach to synthetic biology (aka synbio) as reconfiguring the genome of an organism to get it to do something entirely new. It added that Ginkgo co-founder/CEO Jason Kelly likens synbio to computer programming, only with genetic sequences. So, think of DNA as computer code, and then imagine you can design sequences of DNA on the computer, physically print out those sequences, and insert them into microorganisms such as yeast and bacteria so they make products like rose-scented oil for perfume, or sweeteners for beverages.

Having read this analogy between synbio and computer programming, with DNA as computer code, it suddenly occurred to me that this conceptual connection between computer and genetic coding could be in part why Gates and Ginkgo are simpatico. This connection also led me to ponder the question in this image caption.

Who programmed DNA in the beginning? Credit: kentoh

As usual, your comments are welcomed.

Legionnaires’ Disease Outbreak in New York

  • First Identified as a New Pathogen 40 Years Ago, Legionella Persists
  • Legionella’s Life Cycle Involves “Biological Sanctuaries”
  • qPCR Proven to Outperform Antibody-Based Detection of Legionella

When I read about an outbreak of Legionnaires’ disease (LD) in New York City, baseball legend Yogi Berra’s famous quote, “It’s déjà vu all over again” immediately came to mind, along with the irony of Berra playing for the New York Yankees. So, if you’re much younger than me, you’ll likely not know why “It’s déjà vu all over again” and you may wonder who Berra was. You can read about him later elsewhere, but for now you should read on to learn about Legionnaires disease and why déjà vu is apropos.

History of LD

Notable positive events during 1976 in the United States included our Bicentennial Celebration, unveiling by NASA of the first space shuttle (the Enterprise), establishment of Apple Computer Company by Steve Jobs and Steve Wozniak, and Silly Love Songs by Paul McCartney and Wings ascending to #1 on the charts. While many of these events were the beginning of fabulous things to come, one proved to be the beginning of something catastrophic. American Legionnaires who gathered in Philadelphia, Pennsylvania for the Bicentennial were struck with a mysterious epidemic of fatal respiratory disease.

Taken from networks.org

Sadly, 182 members of the Pennsylvania American Legion were affected, and 29 individuals died after they returned from the convention in Philadelphia. The epidemiological and microbiological studies continued for months before scientists began to understand what had happened. Much of the basic framework of our knowledge of Legionnaires disease, as the epidemic came to be known, was developed by a team from the CDC and the Pennsylvania Department of Health, as detailed elsewhere.

Taken from case1study.wikispaces.com

The cause of the disease remained a mystery until 1977 when an investigative team led by J. E. McDade and C. C. Shepard (of the Leprosy and Rickettsia Branch, Virology Division, Bureau of Laboratories, CDC) reported on the isolation of a Gram-negative bacillus found in patient samples. As often done for naming pathogens after sources, the genus of this rod-shaped bacterium was aptly named Legionella. Legionella includes the species L. pneumophila, which caused the pneumonia-like illness medically named legionellosis, but commonly referred to as LD.

2017 LD Outbreak Hits New York City—Again

In June of this year, forty years after the first characterization of Legionella, it’s lethal infectivity reoccurred in an outbreak in the Upper East Side of the Manhattan borough of New York City, leaving one person dead and six other people sickened. According to a newspaper account, this outbreak occurred within 11 days, and may have been triggered by contacting contaminated water as has happened in other cases.

While this incident affected relatively few people compared to other previous outbreaks, including one in the Bronx borough of New York City in 2015 that killed 15 people and sickened more than 70, it’s a scary reminder of the persistence of Legionella in the environment. In this regard, it has been reported that 200 to 400 cases of the illness are recorded each year in New York, despite the monitoring of 6,000 water systems wherein Legionella can flourish in warm conditions. This environmental factor provides a segue into what genomic sequencing has revealed about Legionella.

Genomics-Based Insights on Legionella

The bacterial pathogen L. pneumophila is found ubiquitously in fresh water environments where it replicates within protozoan hosts. When inhaled by humans it can replicate within alveolar macrophages and cause severe pneumonia associated with Legionnaires disease. As detailed elsewhere, recent advances in genome sequencing has had a major impact on understanding of the pathogenesis, evolution and genomic diversity of Legionella.

A lipopolysaccharide cell wall and several outer membrane proteins are essential virulence factors. Central to the pathogenesis of L. pneumophila is its Type IV secretion system, which translocates over 270 effector proteins into the host cell, thus allowing this bacterium to manipulate host cell functions to its advantage and assures intracellular survival and replication.

Within aquatic media, as depicted below, Legionella exist as part of biofilms, which provide a protective environment—or biological sanctuary, if you will—wherein the bacteria exhibit marked increase in resistance to biocidal compounds and chlorination. Aside from the resultant difficulty of purging water systems to be free of Legionella, these bacteria can invade and multiply within protozoa (which are ubiquitous and include amoeba), thus providing yet another biological sanctuary. Protozoa are present in all aquatic or moist environments, and can be found in even the most inhospitable parts of the biosphere, thus providing further protection to Legionella.

Taken from Comas Nature Genetics (2016)

The actual infectious particle is not known but may include excreted legionellae-filled vesicles, intact legionellae-filled amoebae or free legionellae that have lysed their host cell. Transmission to humans occurs via mechanical means, such as air-conditioning units, taps and showerheads, as well as others listed by the World Health Organization (WHO).

Infection in humans occurs by inhalation of the infectious particle and establishment of infection in the lungs. After ingestion by macrophages, L. pneumophila have been found to inhibit acidification and maturation of its phagosome. Following a 6–10 hour lag period, the bacteria replicate for 10–14 hours until macrophage lysis releases dozens of L. pneumophila progeny.

It’s worth noting that, according to WHO, there is no direct human-to-human transmission of Legionella, which in my opinion is why incidence of LD remains relatively low.

Gardening Can Be Bad for Your Health—No Joke.

Unfortunately, there are other ways of contacting LD besides ingestion of tainted water. At the risk of sounding flippant, gardening can be seriously bad for your health because of contracting LD by breathing in aerosolized Legionella from contaminated soil. This is especially true in New Zealand, which has the highest incidence of LD in the world, according to a recent publication, with L. longbeachae being the most clinically relevant species. This infectious agent is predominantly found in soil and composted plant material. Most cases occur over spring and summer, and the people at greatest risk are those involved in gardening activities.

Taken from lawrieco.com.au

Some agricultural experts advocate smelling soil to assess its quality, stating that “[t]he smell of a soil can often reveal its state of health, sweet or offensive or plain bland,” and adding “the smell does not actually come from the dirt itself, but from soil microbes that inhabit a healthy soil environment. Sweet smelling soil has good levels of organic carbon which is vital to supporting the world of billions of beneficial bacteria and fungi in every cup of healthy soil.”

These soil sniffing experts, however, fail to consider the presence of pathogenic organisms including L. longbeachae. I, for one, will carefully avoid purposefully smelling any soil when gardening, and will instead be sure to wear a good mask capable of filtering out aerosolized Legionella, as you should too!

Nucleic Acid-Based Detection of Legionella

Rapid and effective diagnosis of LD is extremely important so that timely and appropriate therapy can be provided, thereby lowering the morbidity and mortality rates and reducing the health and economic costs associated with this disease. Surprisingly, diagnosis is reportedly established solely by time-consuming microbiological tests. Luckily, it looks like testing procedures could soon change for the better, thanks to PCR and NGS.

Taken from corisbio .com

Earlier this year, Christovam et al. assessed the accuracy of various detection tests in patients suspected of being infected with Legionella and in patients with laboratory-confirmed LD. Investigators analyzed urinary Legionella antigen detection, direct fluorescent antibody (DFA) staining, serological testing and PCR vs. culture analysis (the reference standard). The sensitivity and specificity for PCR were 83 % and 90 %, respectively, whereas DFA sensitivity and specificity were 67 % and 100 %, respectively. Moreover, PCR had high sensitivity and specificity for early diagnosis of LD.

Taken from letsfixit.co.uk

While the study results reported by Christovan seem promising, less definitive results have been reported. Krøjgaard et al., who compared culture and qPCR assays for the detection of Legionella in 84 samples from shower hoses and taps in a residential area before and after two decontaminations. Detection by qPCR was suitable for monitoring changes in the concentration of Legionella but the precise determination of bacteria is difficult. Risk assessment by qPCR only on samples without any background information regarding treatment, timing, etc. was said to be “dubious.” However, the rapid detection of high concentrations of Legionella by qPCR was said to be valuable as an indicator of risk, although it may be false positive compared to culture results. Detection of a low number of bacteria by qPCR was said to be a strong indication for the absence of risk.

Not surprisingly, the advent of powerful next-generation sequencing (NGS) is emerging as a better method for genus-specific, sensitive and quantitative determination of Legionella. In 2017, Pereira et al. reported findings from a study using NGS to differentiate 20 pathogenic strains of Legionella in fresh water systems. A genome standard and a mock community consisting of six different Legionella species demonstrated that the reported NGS approach was quantitative and specific at the level of individual species, including L. pneumophila. Comparison of quantification by real-time PCR showed consistency with the NGS data, thus indicating that NGS “provides a new molecular surveillance tool to monitor all Legionella species in qualitative and quantitative terms if a spiked-in genome standard is used to calibrate the method.”

Concluding Comments

Aside from providing a brief introduction and update on LD, my additional intent was to alert readers—without undue alarm—to the myriad circumstances in which Legionella can infect humans. According to the aforementioned list provided by WHO, the most common form of transmission of Legionella is inhalation of contaminated aerosols produced in conjunction with water sprays, jets or mists. Infection can also occur by aspiration of contaminated water or ice, particularly in susceptible hospital patients.

Researching transmission of Legionella in Google Scholar led me to find additional information (see links below) that you may find useful or interesting.

Thankfully, as I’ve said before, Legionella is not transmitted human-to-human. The scary aspect of Legionella, however, is that it’s continually mutating, which raises the specter of emergence of a strain that can spread within a human population. Let’s hope that this doesn’t happen and/or that modified mRNA vaccines can be quickly produced to combat that possibility.

As usual, your comments are welcomed.

Addendum

After finishing this blog, there was a Reuters news report on October 9, 2017 that Michigan’s top medical official, Dr. Eden Wells, will be charged with involuntary manslaughter for her role in the city of Flint’s water crisis, which was linked to an outbreak of LD that caused at least 12 deaths. Dr. Eden Wells would become the sixth current or former official to face involuntary manslaughter charges related to this crisis, which principally involved lead contamination in the city’s water supply.

SaveSaveSaveSave

Highlights from the 2017 AgBio Innovation Showcase Held by UC Davis

  • An Inconvenient Truth About Unsustainable Global Food Supply
  • Agricultural Biotechnology (AgBio) is Providing Transformative Solutions
  • Highlights from the Inaugural AgBio Innovation Showcase

Taken from the journal.ie

With expected global population to reach 8.3 billion in 2030, it’s clear that excessive exploitation of food resources is no longer sustainable and the problem will simply worsen with environmental problems and effects of climate change. This ominous outlook by food experts is reminiscent of former Vice President Al Gore’s dire vision for global warming in an award winning documentary film in 2016 titled An Inconvenient Truth.

This very real challenge of achieving adequate and sustainable food supplies—globally, not just for developed countries—has been, and continues to be, addressed by nucleic acid-based agricultural biotechnology (aka AgBio). At the forefront of this battle is development of genetically modified foods (aka genetically engineered foods or bioengineered foods), which are foods produced from organisms that have had changes introduced into their DNA using the methods of genetic engineering. Genetic engineering techniques allow for the introduction of new traits, as well as greater control over traits than was possible with previous methods such as selective breeding and mutation breeding.

Taken from intelligencesquaredus.org

Last year, I published a blog about genetically modified organisms (GMOs) in which I made fairly general comments about complex government regulatory issues related to “science vs. semantics” and varying degrees of country/consumer acceptance and rejection. This blog is somewhat of a follow-up to that post, and I will share specifics from the 2017 AgBio Innovation Showcase held by the University of California Davis at its World Food Center, which include GMO and non-GMO technologies. The Center was founded in 2013 as an institute aimed at “bridging agriculture, food science, nutrition, veterinary medicine, public health and policy in new and transformational ways.”

2017 AgBio Innovation Showcase

Taken from agshowcase.com

This inaugural event was held on May 8-9 and featured the most promising AgBio and AgTech startups and research projects. The showcase featured solutions in high-value, nutritious agriculture and food from across the globe. The four major showcase themes were Automation & Robotics, Boosting Nutrition & Sensory Value, Innovation in the Livestock & Dairy Sectors, and Water Management. I’ve selected several highlights that are summarized below. Takeaways from panel discussions about the future of agriculture can be read elsewhere.

Ag Biotech

  • Afingen – This biotech start-up was spun out of Lawrence Berkeley National Laboratory (LBNL) in 2014 and features technology based on proprietary cisgenesis. Cisgenesis involves modification of a recipient plant with a natural gene from a crossable plant. Importantly, cisgenic plants can harbor one or more cisgenes, but they do not contain any transgenes and therefore yield new, improved plant varieties that are classified non-GMO.
  • Taken from whattsupwiththat.com

    Bee Vectoring Technology – How this Canadian company cleverly turns bees into delivery agents that deposit biological products on crops for pest management is best understood by watching this video (details for which may be read in a patent). In brief, powder-form biologics to be delivered are placed in commercially-reared bee. The biologics stick to the bees’ feet and are released when the bee collects pollen from the targeted crop.

    Taken from saipanhydroponics.com

  • MiraculeX – A unique West African plant protein called miraculin (named for its “miracle” ability to transform sour foods into sweet treats), makes it possible to bite into a lemon and taste nothing but sweet lemonade. MiraculeX reportedly inserts the protein’s DNA into the genetic code of ordinary lettuce, which is grown hydroponically and in less than a week is ready to be harvested for processing.
  • Trace Genomics – This startup service in San Francisco provides advice to growers based on analysis of their soil. Growers simply provide a soil sample, from which TraceGenomics extracts DNA from the organisms in the soil and prepares a sequencing library to analyze the soil microbial community. Interpreted results are provided along with information about soil health, nutritional status, and corresponding recommendations for how to improve crop yield and quality.

Diagnostics

  • AstRoNa Biotechnologies – This UC Davis startup aims to commercialize an easy-to-use, hand-held pathogen detection device to quickly monitor food contamination by bacteria, viruses, and fungi. It’s basically “farm-to-table” analysis. The team reportedly developed a disposable test kit to capture and amplify RNA of pathogens, focusing on coli O157:H7. A fully automated handheld instrument is under development and will feature sample multiplexing, quantitative detection, and software to create a traceable record of safety—recording time, location, user, and results in real time.
  • SnapDNA – This startup has an R&D agreement with the US Department of Agriculture to develop rapid, highly specific tests for foodborne pathogens, including Salmonella enterica and human noroviruses (the latter of which is featured in an earlier blog). I was unable to find many details, but a board member states that SnapDNA is “a semiconductor-based bio-chip and multiplexed DNA detection platform.” Adding that “[a]major differentiator of SnapDNA is the specificity to detect DNA at the level of microbial strains in a fast, low cost test, major pain points in current systems.”

Food Science & Animal Health

  • Taken from Wikipedia.org

    Bonumose – This startup in Virginia is scaling up enzymatic production of tagatose (pictured below), which—unlike sucrose and high fructose corn syrup—does not raise blood sugar levels, is low-calorie, and does not cause tooth decay. Beyond not being harmful to health, tagatose provides positive health benefits: it is an effective prebiotic (good for gut health), blocks adsorption of sucrose and starch, is clinically-proven to reduce blood sugar levels in diabetics, contributes to a feeling of satiety, and breaks up dental biofilm. Even better, tagatose is nearly indistinguishable from sucrose in terms of taste and food functionality. And it blends very well with high intensity sweeteners such as stevia. I want some asap!

  • Resilient Biotics – This El Cerrito, California startup utilizes deep sequencing to characterize host genotypes, commensal microbial communities, and pathogen strain variants for microbiome resolution to rapidly identify important genetic elements and key microbial strains that influence states of health and disease. Heuristic search methods can rapidly pinpoint diagnostic biomarkers for pathogen identification and risk prediction. Resilient Biotics is actively developing live biotherapeutics to address major infectious diseases of the respiratory tract in production animal systems.

AgBio Meets CRISPR

As if the 2017 AgBio Innovation Showcase wasn’t stimulating enough, I was thrilled to discover another upcoming meeting that combines two of my favorite topics: agbio and CRISPR. Devoted readers of my blogs will recall numerous past postings on CRISPR for gene editing and other useful manipulations of genomic DNA. My search of Google Scholar indicated no AgBio CRISPR publications to date, but that will likely change, as evidenced by the upcoming conference.

Interested readers can register at the link above and download a detailed agenda and list of confirmed speakers. In doing so, it is apparent that this conference will comprehensively cover the newest topics and the regulatory status related to CRISPR/Cas9 technology.

I look forward to reading about these developments, and posting comments in a future blog titled AgBio Meets CRISPR. If you happen to be attending this conference, please share details about what you learned in the comments section below.

As usual, your comments are welcomed.

SaveSave

SaveSave

Highly Visible Invisible Food Safety

  • Recent FDA Food Safety Act Focuses on Prevention Rather Than Response
  • PCR Provides a Powerful Approach for Assuring Foodborne Safety
  • Safer Food via Immuno-PCR Commercialized by Invisible Sentinel 

Annual Cost of Foodborne Illness and U.S. FDA Response

Every year contaminated food sickens some 48 million people in the U.S., necessitates 128,000 hospitalizations, and results in 3,000 deaths, according to recent estimates from U.S. Centers for Disease Control and Prevention. Extrapolating these numbers to other developed countries isn’t straightforward, but I used available information to guess that the number of cases of foodborne illness worldwide is roughly 4-5 times greater than the US.

In addition to the toll in human suffering, food contamination that is discovered too late exacts a heavy financial cost on the food industry and the public. A study supported by the Pew Charitable Trusts has estimated that food contamination costs the United States about $152 billion a year after accounting for lost workdays and reduced quality of life, as well as medical expenses.

Continuing outbreaks of foodborne illness have led consumer groups to call for tighter regulation. The result was the FDA Food Safety Modernization Act (FSMA)—the most sweeping reform of U.S. food safety laws in more than 70 years—signed into law by President Obama on January 4, 2011. It aims to ensure the U.S. food supply is safe by shifting the focus from responding to contamination to preventing it. Here’s how.

The U.S. FDA now has a mandate to require food-safety controls. Companies across the food-production and food-distribution network must write outbreak-prevention plans, monitor the performance of their controls, and specify the corrective actions they will take when necessary.

A detailed summary of the FSMA and a host of related information can be read about at this link that includes Guidance for Industry, Final Rules, and Presentations.

PCR Powered Prevention 

In a nutshell, the well-known power of PCR to unambiguously identify and quantify microorganisms lends itself to prevention of foodborne contamination from entering the food supply. Other important attributes of PCR for such prevention are its highly evolved instrumentation and ancillary sample preparation methods, which together provide for fast time-to-results in either centralized labs that receive express-shipped samples, or decentralized (aka point-of-need) facilities at or very near the food source.

Most major commercial suppliers of PCR instrumentation and reagents offer a line of products aimed at food safety per se or quality control for microorganisms that are detrimental to taste, smell, shelf life, and other producer or consumer concerns. An example of such is ETS Labs in California, which uses real-time PCR to detect a full range of wine and juice spoilage organisms to help ensure quality in the wine making industry. This genetic analysis method, which utilizes TriLink’s Hot Start CleanAmp dNTPs under a licensing agreement, detects microbial populations directly in wine or juice. Results are routinely reported within two business days, giving winemakers the ability to address problems before wine defects occur.

Regular readers of this blog will recall that I have touted the advantages of PCR in various contexts, including some aspects of food chain validation and tracing from “farm-to-fork.” However, as indicated above, the present post is focused on preventing foodborne illnesses and showcasing innovators in this space, namely Invisible Sentinel.

Highly-Visible Invisible Sentinel

Truth be told, I can’t recall how I first came across Invisible Sentinel, but I’m glad I did because it’s an interesting story from the perspective of innovation in food safety technology as well as opportunity in an emerging market.

Taken from phillymag .com

Technology-wise, the first thing that caught my attention was the remarkably small size of the PCR read-out device developed by Invisible Sentinel, which is pictured below. Before getting into the specifics of what’s packed inside of this tiny gizmo, I should mention that there are up-front sample prep and PCR thermal cycling steps that must be performed before using the device. These steps have been simplified but involve conventional approaches that can be read about at this link rather than discussed here.

Much more intriguing to me was how PCR amplicons are detected by Invisible Sentinel’s Veriflow® DNA Signature Capturing Technology, which more accurately involves visualization by eye as opposed to fluorescent signal detection commonly used for real-time PCR measurements. Since functional details for this visualization system are not provided on Invisible Sentinel’s website or various YouTube videos, I’ll briefly summarize below what I found in a recently issued Invisible Sentinel patent that describes its version of what is known as Immuno-PCR.

The following schematic, taken from this patent, depicts visualization of two different amplicons derived from 5’-labeled primers: digoxigenin/TAMRA amplicon 20 and FITC/TAMRA amplicon 40 using three antibodies 15, 30, and 50 that are either attached to a membrane, bridge, or bind streptavidin-gold nanoparticles. The latter nanoparticles are visualized by eye, but only if both amplicons form the complex shown. Variations of this scheme can be used for visualization of a single amplicon.

Taken from US Pat. No. 9,347,938

Visualization in this manner is formally analogous to pregnancy strip tests that show two bands for a positive result and only one band for a negative result. Interested readers should consult the aforementioned patent for details regarding how input amplicons undergo lateral flow to ultimately bind to antibody 10 attached to the test membrane shown above.

Exemplary Applications

According to Invisible Sentinel, 114 companies in the U.S. and more than 50 internationally use the technology at more than 250 different sites in 18 countries.

For example, Wawa Inc. (which owns dairy and beverage manufacturing plants as well as 715 convenience stores in six states) has adopted Veriflow®, as has Refresco Gerber Partner for monitoring juice spoilage. WholeVine Products in California, which produces a variety of products from grape seeds and skins, has begun using Veriflow® to make sure its plant equipment and surfaces are pathogen-free.

Although Invisible Sentinel’s website provides a list of currently available tests, I thought it would be useful to provide the following links to several self-explanatory published applications that I found by searching Google Scholar:

Invisible Sentinel Identifies New Market Opportunities for PCR

Invisible Sentinel was started by a pair of entrepreneurs with science backgrounds. Nicholas Siciliano, 37, graduated from Villanova with a degree in chemistry in 2004 and obtained a doctorate in immunology and microbial pathogenesis from Thomas Jefferson University in 2015. In between, he was a biotech consultant and worked as a researcher at the University of Pennsylvania School of Medicine.

Nicholas Siciliano (left) and Benjamin Pascal in their lab. Taken from articles.philly .com

Benjamin Pascal, 35, has a bachelor’s degree in political communication from George Washington University in 2003 and a master’s in business administration from Lehigh University in 2009. He learned biology at the National Institute for Medical Research in London, and then spent several years in R&D at B. Braun Medical Inc.

According to an article in the NY Times, the two wanted to create a diagnostic device that was faster, easier and cheaper to use. They began with $235,000 from friends and family, but the recession made it tough to bring institutional investors onboard. In 2009, they raised another $1.1 million from friends and family, $2 million more in 2011, and raised $7 million at the end of 2013.

Invisible Sentinel’s sales have been on the rise. The company posted revenue of $50,000 in its first year of sales in 2013, $1.1 million in 2014 and more than $4 million in 2015. It has ambitious projections of $30 million in 2018 and $60 million in 2020. The company expected to turn a profit in 2016.

While Invisible Sentinel may have been one of the first to identify the significant market opportunity for food safety monitoring devices, they currently face formidable competition in larger companies such as Romer Labs’ RapidChek®, Bio-Rad Laboratories’ iQ-Check® and DuPont’s Bax® system. Invisible Sentinel is hoping to capture significant market share with its low cost of entry and easy-to-use system. The company can reportedly set up an in-house lab for about $5,000 and train almost anyone to use it in less than a day. Invisible Sentinel kits cost more than others (about $10 per test compared to an industry average of $4 to $8), but the lower capital equipment and lab set up costs are said to greatly offset the higher test costs.

In closing, I hope that you have found this piece on food safety and immune-PCR in the context of Invisible Sentinel to be a nice example of how nucleic acids-based technology is enabling improved food safety.

As usual, you are welcomed to share your comments here.

SaveSave

SaveSave

SaveSave

SaveSave

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.

Frightening Fungus Among Us

  • Clinical Alert for Candida auris (C. auris) Issued by CDC
  • US Concerned About C. auris Misidentification and Drug Resistance
  • Sequencing C. auris DNA in Clinical Samples is Preferred for Identification
Strain of C. auris cultured in a petri dish at CDC. Credit Shawn Lockhart, CDC. Taken from foxnews.com

Strain of C. auris cultured in a petri dish at CDC. Credit Shawn Lockhart, CDC. Taken from foxnews.com

When I was a kid and didn’t know better, there was a supposedly funny rhyme that “there’s fungus among us.” While this saying is thankfully passé nowadays, the growing number of infections by a formerly obscure but deadly fungus is frightening. This so-called “superbug” is an antibiotic-resistant fungus called Candida auris (C. auris) that’s worth knowing about, and is the fungal focus of this blog.

First, Some Fungus Facts

Fungi are so distinct from plants and animals that they were allotted a biological ‘kingdom’ of their own in classification of life on earth, although that was only relatively recently, i.e. 1969. There are 99,000 know fungi, which exist in a wide diversity of sizes, shapes and complexity that extends from relatively simple unicellular microorganisms, such as yeasts and molds, to much more complex multicellular fungi, such as mushrooms and truffles.

It was previously thought that genomes of all fungi are derived from the genome of the model fungus Saccharomyces cerevisae, which has been used in winemaking, baking and brewing since ancient times. However, genome sequencing of more than 170 fungal species has revealed that, while the genome size of S. cerevisae is only ~12 Mb, seven species of fungus have genome sizes larger than 100 Mb. This is attributed to various evolutionary pressure-factors generating transposable elements, short sequence repeats, microsatellites, and genome duplication, and noncoding DNA.

Fungal cell walls are made up of intertwined fibers mostly comprised of long chains of chitosan, the same tough compound found in the exoskeletons of animals such as spiders, beetles and lobsters. The chitin in fungal cells is entangled with glucans and other wall components, such as proteins, forming a mass that protects the cell membrane behind it—and posing a formidable barrier against antifungal drugs.

Taken from Wikipedia.org

Taken from Wikipedia.org

In researching whether there are any nucleic acid drugs against fungi, I found one early patent by Isis (now Ionis) Pharmaceuticals for use of antisense phosphorothioate-modified oligonucleotides for the treatment of Candida infections, but virtually no other reports. I suspect that will change in the future as pathogenic fungi and other disease-causing microbes become more resistant to conventional drugs.

Fungal infections of the skin are very common and include athlete’s foot, jock itch, ringworm, and yeast infections. While these can usually be readily treated, infections caused by pathogenic fungi have reportedly risen drastically over the past few decades. Moreover, with the increase in the number of immunocompromised (burn, organ transplant, chemotherapy, HIV) patients, fungal infections have led to alarming mortality rates due to ever increasing phenomenon of multidrug resistance.

Segue to a Serious Situation

Emergence of drug-resistant fungi is, in part, the segue to the serious story of the present blog. The other part being incorrect identification of a certain fungus as being a common candida yeast, which is not only scary but seemingly inexcusable in today’s era of highly accurate PCR-based assays to accurately identify microorganisms. Here’s the situation in a nutshell.

  1. auris infection, which is associated with high mortality and is often resistant to multiple antifungal drugs, was first described in 2009 in Japan but has since been reported in countries throughout the world. Unlike many Candida infections, C auris is a hospital-acquired infection that is contracted from the environment or staff of a healthcare facility, and it can spread very quickly.

To determine whether C. auris is present in the United States and to prepare for the possibility of transmission, the Centers for Disease Control (CDC) and Prevention issued a clinical alert in June 2016 requesting that C. auris cases be reported.

(A) MALDI-TOF schematic; (B) mass spectra from three C. parapsilosis; and (C) two C. bracarensis isolates. Taken from researchgate

(A) MALDI-TOF schematic; (B) mass spectra from three C. parapsilosis; and (C) two C. bracarensis isolates. Taken from researchgate

This official alarm bell, if you will, was triggered by the following facts:

  • Many isolates are resistant to all three major classes of antifungal medications, a feature not found in other clinically relevant Candida
  • auris identification requires specialized methods such as a MALDI-TOF mass spectrometry or sequencing the 28s ribosomal DNA, as pictured below.
  • Using common methods, auris is often misidentified as other yeasts, which could lead to inappropriate treatments.

The CDC subsequently found that seven cases were identified in Illinois, Maryland, New York and New Jersey. Five of seven isolates were either misidentified initially as C. haemulonii or not identified beyond being Candida. Five of seven isolates were resistant to fluconazole; one of these isolates was resistant to amphotericin B, and another isolate was resistant to echinocandins. While no isolate was resistant to all three classes of antifungal medications, emergence of a new strain of C. auris that is would pose a serious public health issue.

Sequencing 28s ribosomal DNA. Taken from microbiologiaysalud.org

Sequencing 28s ribosomal DNA. Taken from microbiologiaysalud.org

Based on currently available information, the CDC concluded that these cases of C. auris were acquired in the U.S., and several findings suggest that transmission occurred:

  • First, whole-genome sequencing results demonstrate that isolates from patients admitted to the same hospital in New Jersey were nearly identical, as were isolates from patients admitted to the same Illinois hospital.
  • Second, patients were colonized with auris on their skin and other body sites weeks to months after their initial infection, which could present opportunities for contamination of the health care environment.
  • Third, auris was isolated from samples taken from multiple surfaces in one patient’s health care environment, which further suggests that spread within health care settings is possible.

A related Fox News story adds that C. auris was found on a patient’s mattress, bedside table, bed rail, chair, and windowsill. Yikes!

While the above situation in the U.S. might not seem particularly worrisome to you, the potential for emergence of more infectious C. auris strains with higher lethality should be of concern. That has already reportedly occurred in several Asian countries and South Africa. Obviously, deployment of the best available methods for pathogen identification can, in principle, lessen the likelihood of the emergence and/or spread of C. auris in the U.S. and other countries.

Case for Point-of-Care C. auris Nanopore Sequencing?

Taken from extremtech.com 

Taken from extremtech.com

Regular readers of my previous blogs know that I’m an enthusiastic fan of the Oxford Nanopore Technologies minION sequencer, which is proving to be quite useful for characterizing pathogens in very remote regions on Earth—and even on the International Space Station to diagnose astronaut infections! Notwithstanding various current limitations for minION sequencing of microbes, it seems to me that it would be relatively straightforward to generate minION data for many available samples of pathogenic fungi and genetically related microbes to assess the feasibility using minION for faster, cheaper, better unambiguous identification of C. auris minION in centralized or Point-of-Care applications.

Taken from rnaseq.com

Taken from rnaseq.com

If you think this suggestion is farfetched, think again, after checking out these 2016 publications using minION:

The 51.4-Mb genome sequence of Calonectria pseudonaviculata for fungal plant pathogen diagnosis was obtain using minION.

The first report of the ~54 Mb eukaryotic genome sequence of Rhizoctonia solani, an important pathogenic fungal species of maize, was derived using minION.

Sequence data is generated in ~3.5 hours, and bacteria, viruses and fungi present in the sample of marijuana are classified to subspecies and strain level in a quantitative manner, without prior knowledge of the sample composition.

CDC on C. auris Status and FAQs

In the interest of concluding this blog with the most up-to-date and authoritative information, I consulted the CDC website and found statements and replies to FAQs that are well worth reading at this link.

As a scientist, my overriding question concerns the lack of adoption of improved microbiological methods by hospitals and clinics. The above noted misidentifications of C. auris infections resulting from use of flawed lab analyses seems unacceptable. Although I don’t know all the facts or statistics to generalize, I suspect that there are other incorrect lab analyses due to use of outdated methods. On the other hand, I’m hopeful that, with the FDA’s widely touted Strategic Plan for Moving Regulatory Science into the 21st Century, the section entitled Ensure FDA Readiness to Evaluate Innovative Emerging Technologies—think nanopore sequencing—becomes actionable, sooner rather than later.

Changing established—dare I say entrenched—clinical lab tests is not simple or easy, but if it doesn’t begin it won’t happen, about which I’m quite certain. I can only wonder why development of infectious disease analytical methods and treatments seem to require a crisis. Sadly, I think it boils down to the complexities and socio-political dynamics of who pays.

Frankly, it’s my personal opinion that maybe it’s time Thomas Jefferson’s philosophy about hammering guns into plows is directed to health care.

Postscript

After writing this blog, I learned that T2 Biosystems has received FDA approval to market in the U.S. the first direct blood test for detection of five yeast pathogens that cause bloodstream infections: Candida albicans and/or Candida tropicalis, Candida parapsilosis, Candida glabrata and/or Candida krusei.

Yeast bloodstream infections are a type of fungal infection that can lead to severe complications and even death if not treated rapidly. Traditional methods of detecting yeast pathogens in the bloodstream can require up to six days, and even more time to identify the specific type of yeast present. The T2Candida Panel and T2Dx Instrument (T2Candida) can identify these five common yeast pathogens from a single blood specimen within 3-5 hours.

T2Candida incorporates technologies that break the yeast cells apart, releasing the DNA for PCR amplification for detection by greatly simplified, miniaturized nuclear magnetic resonance (NMR) technology, as can be seen in this video.

In my opinion, this fascinating new technology is another example of what could be rapidly deployed toward detecting C. auris.

You and Your Microbiome – Part 2

  • Global Obesity Epidemic is Linked to Gut Microbiome
  • DNA Sequence-Based Microbiomes Accurately Associate with Obesity
  • Blue Agave Margaritas Contain Beneficial Gut Microbes
  • Investments in Microbiome-based Therapies on the Rise, but is there Hype?

Last August, my post entitled Meet Your Microbiome: The Other Part of You dealt with growing recognition that trillions of microbes—mostly bacteria but also fungus—reside in and on each of us, and influence our health status. Moreover, the compositions of these microbiomes change with our diet, what we drink or breath, and who we contact—family, pets, and close friends.

Since then, I’ve collected a string of microbiome articles delving into the implications of this dynamic, symbiotic relationship, and selected some topics that I thought were “blogworthy.” This Part 2, as it were, focuses on overweight/obesity, microbiome therapy, and burgeoning business opportunities.

Continue reading

Meet Your Microbiome: The Other Part of You

  • Like it or not, and for better or worse, next-generation sequencing is revealing that you and your bacterial microbiome have an inextricable biological relationship.
  • “RePOOPulating” the gut:  a clinical study of “synthetic stool” as a better alternative to fecal transplant.
  • Microbiome movies, fungus too, and much more.

What’s in your microbiome? Why does it matter?

Writing this blog was inspired by reading Michael Pollan’s recent article in The NY Times Magazine entitled “Some of my best friends are bacteria,” which is an engaging story about Michael having his microbiome sequenced as part of the American Gut project. He notes at the beginning that each of us has several hundred microbial species with whom we share our body, and that these bacteria—numbering ~100 trillion—are living (and dying) right now on the surface of our skin, mouth, and intestine “where the largest contingent of them will be found, a pound or two of microbes together forming a vast, largely uncharted interior wilderness that scientists are just beginning to map.” The sheer numbers of these microbes, he adds, makes us only ~10% human—for every human cell there are ~10 resident microbes—most being “harmless freeloaders” or “favor traders” (i.e. symbiotic), and only a tiny number of pathogens. Furthermore, “[t]his humbling new way of thinking about the self has large implications for human and microbial health, which turn out to be inextricably linked.”

americangut

If you want to know what’s in your gut, you can participate in this world wide study by registering at www.americangut.org. According to the website, you’ll be asked to ‘fill out a diet & lifestyle questionnaire (online) with such things as age, gender, weight, have you taken antibiotics lately, any conditions we should know about it, and so on.’ Anyone over the age of 3 months can participate in the study, and you will have the opportunity to provide a stool, tongue and/or palm sample via an at-home sample kit.

Microbes and Your Health

As more microbiome data is obtained from the American Gut project and analogous studies, correlations with each person’s health status can be made. Having the “wrong” kind of microbes may be associated with predisposition to obesity or certain chronic diseases, for example. Such information may then be used to prescribe dietary or other sources supplemental probiotics. An extreme outcome could involve “fecal transplants” (i.e., fecal microbiota transplantation or fecal bacteriotherapy) wherein a healthy person’s fecal microbiota are installed into a sick person’s gut.

My PubMed searches of these terms led to a number of publications, such as that by Christopher. R. Kelly et al. at Women and Infant’s Hospital, Brown University Alpert School of Medicine, who reported promising results for relapsing Clostridium difficile infection in a small study of 26 patients. Coincidentally, on June 18th there was a report that the US Food & Drug Administration (FDA) is “dropping plans to tightly control” fecal transplants that are “becoming increasingly popular for treating people stricken by life-threatening infections of the digestive system.” This report went on to say that, in the FDA’s view, “such a treatment should only be given to patients who have exhausted other treatment options and who have given consent and been informed that it is an experimental procedure with risks.”

RePOOPulating the gut:  a better alternative?

Among the concerns for fecal transplants are pathogen transmission, patient acceptance and inability to standardize the treatment regime, states Elaine O. Petrof and her colleagues in a study entitled Stool substitute transplant therapy for the eradication of Clostridium difficile infection: ‘RePOOPulating’ the gut, recently published in Microbiome (2013). This online, open-access article is well worth a quick read if you’re interested in the experimental details, which take advantage of next-generation sequencing as a key method for quite sophisticated identification and analysis of microbes. In brief, a stool substitute preparation—made from 33 purified intestinal bacterial cultures derived from a single healthy donor—was used to treat two patients who had failed at least three courses of metronidazole or vancomycin. Pre-treatment and post-treatment stool samples were analyzed by 16S rRNA sequencing using the Ion Torrent platform. Both patients were infected with a hyper virulent C. difficile strain, but following treatment each reverted to their normal bowel pattern within 2-3 days and remained symptom-free at 6 months. Analysis demonstrated that rRNA sequences found in the stool substitute were rare in the pre-treatment stool samples, but constituted >25% of the sequences 6 months after treatment.

gutlining

Bacteria on the gut lining. Image: Cardiff University

Microbiome Movies

Relatively low-cost deep-sequencing technology has enabled novel studies aimed at obtaining, in effect,  “moving pictures of the human microbiome,” which is the attention-grabbing title of a study by J. Gregory Caparaso et al. in Genome Biology. This landmark publication in 2011 presented the largest human microbiota time-series analysis to date, covering two individuals each at 4 body sites over 396 time-points. One male and one female each provided gut (feces), mouth, left palm, and right palm samples over 15-mo and 6-mo periods, respectively, Variable regions of 16S ribosomal RNA (rRNA) in each sample were amplified by PCR and sequenced on an Illumina instrument. The following stated results and conclusions may surprise you, as they did me:

“We find that despite stable differences between body sites and individuals, there is pronounced variability in an individual’s microbiota across months, weeks and even days. Additionally, only a small fraction of the total taxa found within a single body site appear to be present across all time points, suggesting that no core temporal microbiome exists at high abundance (although some microbes may be present but drop below the detection threshold). Many more taxa appear to be persistent but non-permanent community members.”

“Because of the immense subject-to-subject variability in the microbiome, studies examining temporal variability, which give a view of dynamics beyond the static pictures previously available, have the potential to transform our understanding of what is ‘normal’ in the human body, and, perhaps, to develop predictive models for the effects of clinical interventions.”

If you’re questioning whether the above study of only two individuals—albeit for many time points—reflects a wider population, rest assured that it apparently does. The recently completed 5-yr Human Microbiome Project (HMP) launched in 2008 involved 242 volunteers, more than 5,000 samples were collected from tissues from 15 (men) and 18 (women) at body sites such as mouth, nose, skin, lower intestine (stool) and vagina. According to the HMP wiki site, this project’s discoveries include:

  • Microbes contribute more genes responsible for human survival than humans’ own genes. It is estimated that bacterial protein-coding genes are 360 times more abundant than human genes.
  • Microbial metabolic activities—for example, digestion of fats—are not always provided by the same bacterial species. The presence of the activities seems to matter more.
  • Components of the human microbiome change over time, affected by a patient disease state and medication. However, the microbiome eventually returns to a state of equilibrium, even though the composition of bacterial types has changed.

Consequently, defining what is “normal” and whether a “core” community of microbes exists is quite complicated, and will likely remain a topic of great interest and debate. In this regard, I found an overview by Dirk Gevers et al. well worth reading. Here’s one section of text that reiterates what I think are important points, and elaborates upon the above second bullet point about microbial metabolic activities, i.e. functional core:

“A potentially more universal ‘core’ human microbiome emerged during the consideration of microbial genes and pathways carried throughout communities’ metagenomes. While microbial organisms varied among subjects as described above, metabolic pathways necessary for human-associated microbial life were consistently present, forming a functional ‘core’ to the microbiome at all body sites. Although the pathways and processes of this core were consistent, the particular genes that implemented them again varied. Microbial sugar utilization, for example, was enriched for metabolism of simple sugars in the oral cavity, complex carbohydrates in the gut, and glycogen/peptidoglycan degradation in the vaginal microbiome. The healthy microbiome may thus achieve a consistent balance of function and metabolism that is maintained in health, but with fine-grained details personalized by genetics, early life events, environmental factors such as diet, and a lifetime of pharmaceutical and immunological exposures.”

Lest you think that such data are collections of facts devoid of utility, think again after reading the following excerpts from the abstract of a publication by Fredrik H. Karlsson et al. in Nature 2013 entitled Gut metagenome in European women with normal, impaired and diabetic glucose control.

“Type 2 diabetes (T2D) is a result of complex gene–environment interactions, and several risk factors have been identified, including age, family history, diet, sedentary lifestyle and obesity. Statistical models that combine known risk factors for T2D can partly identify individuals at high risk of developing the disease. However, these studies have so far indicated that human genetics contributes little to the models, whereas socio-demographic and environmental factors have greater influence.…Here we use shotgun sequencing to characterize the fecal metagenome of 145 European women with normal, impaired or diabetic glucose control. We observe compositional and functional alterations in the metagenomes of women with T2D, and develop a mathematical model based on metagenomic profiles that identified T2D with high accuracy.”

Microbiomes in Homes and Hospitals

Mapping the great indoors—an engaging NY Times article by Peter A. Smith—tells about microbiologists who are using sequencing to “take a census” of what lives in our homes with us and how we “colonize” spaces with other species — viruses, bacteria, microbes. One group has sampled the “microbial wildlife” in 1,400 homes across the USA in a project called The Wild Life of Our Home, which relies on volunteers to swab pillowcases, cutting boards and doorjambs, then send samples in for analysis. Although data are still being analyzed, an earlier study of 40 homes in 9 locations around the Raleigh-Durham area of North Carolina has been published by Robert R. Dunn et al. in PLoS ONE entitled Home Life: Factors Structuring the Bacterial Diversity Found within and between Homes. Among their findings were the following.

“[E]ach of the sampled locations harbored bacterial communities that were distinct from one another” [and that] “the presence of dogs had a significant effect on bacterial community composition in multiple locations within homes as the homes occupied by dogs harbored more diverse communities and higher relative abundances of dog-associated bacterial taxa. Furthermore, we found a significant correlation between the types of bacteria deposited on surfaces outside the home and those found inside the home, highlighting that microbes from outside the home can have a direct effect on the microbial communities living on surfaces within our homes.”

Your dirty dog? (Bing Images)

Your dirty dog? (Bing Images)

Some of you might be wondering what microbes your dog is depositing in your home, while others might be asking about cats. I haven’t a clue about your dog, but cats weren’t directly assessed in this study. Thirteen of the houses had only dogs as pets and the influence of dogs is likely to be greater than cats because of their large size and need to go outdoors. Only three houses had cats but not dogs (three houses had both), and the investigators judged this to be too small a sample size for cats.

The figure shown below taken from the aforementioned Dunn et al. publication tracks 9 sites sampled with 5 sources of microbes. The authors note that “[t]hese results show changes in the relative importance of individual sources across sites, not comparisons across sources within sites. For example, these results show that soil is a more important source of bacteria on door trims than on cutting boards, but these results cannot be used to directly compare the relative importance of soil versus leaves as sources of bacteria at individual locations.” By the same token, the results show that human skin is a more important source of bacteria on toilet seats than on cutting boards.

bacteriaproportion

Source tracking analysis showing relative proportion of bacteria at each sampling site associated with given sources. Values represent median percentages. Warmer colors indicate greater influences of particular sources across the sites (Robert R. Dunn et al. PLoS ONE 2013).

Microbial fingerprints

Roughly 1.7 million hospital-associated infections are reported each year in the USA, and the pathogens that cause them must come from somewhere. Beth Mole (too bad she wasn’t named Millie!) in Nature-News 2013 notes that patients leave a microbial mark on hospitals. This attention getting punch line refers to preliminary findings from the Hospital Microbiome Project that, according to its website, “aims to collect microbial samples from surfaces, air, staff, and patients from the University of Chicago’s new hospital pavilion in order to better understand the factors that influence bacterial population development in healthcare environments.”

According to Beth Mole, “…even in areas with long-term inhabitants, [Jack] Gilbert’s team has found no lingering pathogens. ‘Over the first four months of observations, we’ve seen nothing that concerns us,’ he says.” However, “Gilbert and his team found significant differences between microbial communities in individual hospital rooms. Patients who stayed for only short periods, such as those undergoing elective surgery, had a transient influence on their rooms’ microbial communities; after cleaning, the rooms reverted to a pre-patient state.” In contrast, and of concern, ‘[m]icrobes from long-term patients—including people with cancer or those who had received organ transplants—had time to settle into the rooms. The patients’ microbial fingerprints lingered after they checked out of the hospital, even after their rooms were cleaned.” But even in areas with long-term inhabitants, Gilbert’s team has found no lingering pathogens. “Over the first four months of observations, we’ve seen nothing that concerns us,” he says.”

Thinking Big: The Global Microbiome

If you now muse about greatly expanding the scope of the aforementioned studies to a global scale, you’d be thinking about something that has already been proposed. The Earth Microbiome Project is a “massively multidisciplinary effort to analyze microbial communities across the globe” that proposes to “characterize the Earth by environmental parameter space into different biomes and then explore these using samples currently available from researchers across the globe.” The project intends to “analyze 200,000 samples from these communities using metagenomics, metatranscriptomics and amplicon sequencing to produce a global Gene Atlas describing protein space, environmental metabolic models for each biome, approximately 500,000 reconstructed microbial genomes, a global metabolic model, and a data-analysis portal for visualization of all information.” We’ll all have to stay tuned on this rather ambitious project to learn what is found and, more importantly, what are the conclusions—and whether time-dependent variability is accounted for.

By the way, if you’re wondering about the total global microbiome, Addy Pross’ book entitled What is Life? How Chemistry Becomes Biology states that the earth’s bacterial biomass is estimated to be 2 × 1014 tons, which is sufficient to cover the earth’s land surface to a depth of 1.5 meters!

Microbiome-mania & Toilet-phobia

There seems to be a surge in extending microbiome analysis to many other contexts. Some of these findings are surprising—and prompting jokes—or might scare you about microorganisms present in various places or on things that we come into contact with, whether we know it or not.

Initial, a UK-based provider of hygiene services to businesses and organizations, announced in an April 2013 press release, its research that “…lifts the lid on the grimy state of Britain’s office kitchens.” Being a provider of cleaning services, Initial was presumably quite pleased to inform the public about the following findings.

“Swab testing of a sample of communal workplace kitchens showed that 75% of work surfaces were home to more bacteria than an average feminine sanitary bin. Half also harboured dangerously high levels of coliforms, bacteria present in feces, which can lead to outbreaks of gastrointestinal disease. Over a quarter of the draining boards tested registered more than four times the level of coliforms considered to be safe.”

“The handles of shared fridge-freezers were also shown to be bacteria-rife, with a third carrying high levels of coliforms, whilst 30% of shared microwaves were also shown to be contaminated around the handles and buttons.”

“Tea drinkers were no more hygienic, with over 40% of kettle handles found to be carrying high levels of bacteria, and significantly exceeding the bacteria levels on toilet doors. Also, tested were cupboard, dishwasher and waste bin handles, with the cleanest appliance in the kitchen proving to be the water cooler.”

Speaking of toilets, they seem to be in vogue in metabolome studies. For example, Emma Innes reported online that women’s handbags contain more bacteria than the average toilet seat. The dirtiest item in an average handbag is hand cream—it carries more bacteria than the average toilet seat. Leather handbags carry the most bacteria because the spongy texture provides “perfect growing conditions.” An online article by Harold Maass noted that British researchers found that the average barbecue grill in the UK has more than twice as many germs as the typical toilet seat. Maybe someone has already invented a disposable barbeque grill cover similar to what’s used to cover toilet seats, but obviously nonflammable.

Sanitor Mfg Co. began producing toilet seat covers and dispensers in the USA in 1931.  Seems they are working as your handbag and your barbeque may both harbor more germs than a toilet seat.

Sanitor Mfg Co. began producing toilet seat covers and dispensers in the USA in 1931. Seems they are working as your handbag and your barbeque may both harbor more germs than a toilet seat.

Although humorously entitled as Lifting the lid on toilet plume aerosol, this review article recently published the American Journal of Infection Control examines the evidence regarding toilet plume bioaerosol generation and infectious disease transmission. Here’s a quote of the results of this review of existing literature. “The studies demonstrate that potentially infectious aerosols may be produced in substantial quantities during flushing. Aerosolization can continue through multiple flushes to expose subsequent toilet users. Some of the aerosols desiccate to become droplet nuclei and remain adrift in the air currents. However, no studies have yet clearly demonstrated or refuted toilet plume-related disease transmission, and the significance of the risk remains largely uncharacterized.” Like many investigations, the stated conclusions call for more data: “[a]dditional research in multiple areas is warranted to assess the risks posed by toilet plume, especially within health care facilities.

By the way, the internet has various references to a 6-foot diameter (dare I say) zone wherein toilet aerosol may spread, so keep your toothbrush at a safe distance or consider purchasing a toothbrush sanitizer—of which there are many—after doing your homework on which devices have been validated, and against what, as I did by searching PubMed for “toothbrush sanitizer” etc.

Mycobiome:   Fungus In, On and Among Us

Oh…let’s not forget about fungus—a member of a large group of microorganisms that includes yeasts and molds, as well as mushrooms. These organisms are classified as a kingdom, Fungi, and the discipline of biology devoted to the study of fungi is known as mycology. My PubMed search of “mycobiome” gave only 10 hits, which is far less than ~4,000 found for “microbiome.” The earliest mycobiome publication was entitled “Characterization of the oral fungal microbiome (mycobiome) in healthy individuals” by Mahmoud A. Ghannoum et al. (PLoS Pathogens 2010). This study—which used pyrosequencing to characterize fungi present in the oral cavity of 20 healthy individuals—revealed the “basal” oral mycobiome profile of the enrolled individuals and showed that across all the samples studied, the oral cavity contained 74 culturable and 11 non-culturable fungal genera. The oral mycobiome of at least 20% of the enrolled individuals included the four most common pathogenic fungi—Candida (present in 75% of the cohort; mostly C. albicans), Aspergillus (35%), Fusarium (30%), and Cryptococcus (20%). The authors said that “[i]t is possible that the pathogenicity of these fungi is controlled in healthy individuals by other fungi in the oral mycobiome, as well as a functional immune system.”

Candida albicans (Wikipedia via Bing Images)

Candida albicans (Wikipedia via Bing Images)

More recently, Heidi H. Kong and coworkers in Nature 2013 published a report entitled Topographic diversity of fungal and bacterial communities in human skin. This study involved 10 healthy individuals and used sequencing to analyze fungal and bacterial communities sampled from 14 skin sites that included face, chest, arms, ears, nostrils, head, and feet. Some salient points taken from the abstract are as follows.

“Eleven core-body and arm sites were dominated by fungi of the genus Malassezia, with only species-level classifications revealing fungal-community composition differences between sites. By contrast, three foot sites—plantar heel, toenail and toe web—showed high fungal diversity. Concurrent analysis of bacterial and fungal communities demonstrated that physiologic attributes and topography of skin differentially shape these two microbial communities. These results provide a framework for future investigation of the contribution of interactions between pathogenic and commensal fungal and bacterial communities to the maintenance of human health and to disease pathogenesis.”

Lastly, Who was First?

Who was “first-to-publish” and what was envisaged have always been of interest to me. In researching this posting, I became curious about who introduced the term “microbiome” to generally describe concepts of the type represented in the various aforementioned articles. It’s not easy to be find or establish “first-ever” publications, so I took the easy way out by doing a PubMed search wherein “microbiome” is in the title and/or abstract. Of the ~1,400 articles found, the earliest was entitled New technologies, human-microbe interactions, and the search for previously unrecognized pathogens by David A. Relman at Stanford University, and appeared in Journal of Infectious Diseases in 2002. The following conclusion from his abstract is quite prescient, and I’m amazed by how far and fast microbiome science has progressed since then.

“The development and clinical application of molecular methods have led to the discovery of novel members of the endogenous normal flora as well as putative disease agents. Current challenges include the establishment of criteria for disease causation and further characterization of the human microbiome during states of health. These challenges and the goal of understanding microbial contributions to inflammatory disease may be addressed effectively through the thoughtful integration of modern technologies and clinical insight.”

As always, your comments are welcome—especially if you know of a particularly interesting “microbiome” report that you’d like to share with me and other readers of this blog.

Postscript

After writing this blog, GenomeWeb reported on August 8th that The Wellcome Trust has awarded $2 million to fund Lindsay Hall, a researcher at the University of East Anglia and the Institute of Food Research, who will seek to find out how bacteria that are beneficial to humans help protect against diseases in the early phases of life, using high-throughput sequencing tools to find out more about the microbial communities that colonize the human body soon after birth. “We are planning to use 16S rRNA-based microbiota community analysis, metagenome, and whole genome sequencing to define and characterize early-life microbiota samples,” Hall told GenomeWeb Daily News in an e-mail. The earliest parts of human life are a critical period in terms of the microbiome because at birth the human gut is completely bacteria-free, Hall explained, noting that the processes that follow birth and lead to microbial colonization are not fully understood. Having a better understanding of these processes could lead to treatments for diseases such as bacterial gastroenteritis, she said. This infectious disease is an increasing cause of infant death in the developing world, and the treatment involves antibiotics, but resistance to antibiotics is increasing and antibiotics also may reduce natural defenses against infection. Under this project, she will look to understand how antibiotics can disrupt these microbial communities, and search for probiotic bacteria.