Hundreds of microbial species inhabit the human gut and support digestion, immunity, and other functions. Changes in their populations have been linked to diabetes, asthma, cancers and many other diseases. But so far, the complexity of microbial communities has made them impractical targets for controlling disease. Now, researchers report that a small molecule can block a type of common gut bacteria from consuming starch, which slows their growth. The results could offer a way to manipulate the bacteria’s proportions in the gut microbiome—a strategy that could eventually help researchers understand the microbiome’s role in health.
So far, researchers have tried to tweak human and animal microbiomes with nutritional supplements, probiotic cocktails, or fecal transplants laden with microbes from other individuals. But small-molecule-based approaches have rarely been attempted: Common antimicrobial drugs wipe out a broad range of species, so they can’t be used to precisely edit a microbial community.
Organic chemist Daniel C. Whitehead and microbiologist Kristi Whitehead of Clemson University and their colleagues teamed up to find a more subtle approach that would manipulate selected members of the gut microbial community. The researchers homed in on Bacteroides, which represent more than half of the human gut microbiome and consume dietary starch and complex sugars. At least one study has shown that, compared to healthy individuals, patients with type 1 diabetes have a higher proportion of certain Bacteroides species in their gut microbiome before the onset of disease symptoms.
In the new study, the team looked at two species of Bacteroides whose starch utilization pathways had been particularly well characterized. With these strains, they tested three small molecules that inhibit human starch-digesting enzymes for their ability to act on bacterial enzymes. In laboratory cultures containing a variety of sugars, one of the molecules, acarbose, prevented Bacteroides species from consuming potato starch and fungal pullulan, a starch used as a common food additive. Acarbose did not kill the microbes or block them from digesting other sugars such as glucose.
On other sugar sources, the molecule had little impact on Bacteroides growth. But when the starch-derived polysaccharide pullulan or potato starch were the only foods provided, acarbose shut down the growth of Bacteroides. It did not affect Ruminococcus, another common gut inhabitant, even though this species also metabolizes starch.
In these initial experiments, the researchers tested the bacteria in individual, lab-grown cultures. Therefore, it remains to be seen whether the human gut microbiome will respond in the same way. “But the results show that the system is targetable, which itself is quite significant,” says organic chemist Herman O. Sintim of Purdue University, who was not involved with the study.
“In the gut, it’s unlikely that only starches are going to be present,” says Kristi Whitehead, but acarbose would give Bacteroides fewer carbohydrates to choose from on the bacterial buffet. “In that highly competitive environment, that should reduce their growth.”
The researchers plan to extend their experiments to mixed bacterial cultures and animal models. “This work is really a proof of concept that a small molecule can arrest the starch utilization system,” says Daniel Whitehead.
So far, it’s not clear whether changes to the gut microbiome cause illness or are merely correlated to disease conditions. For instance, when Bacteroides flourishes before the onset of type 1 diabetes, the bacterial bloom could result from underlying metabolic changes or cause them. Blocking a metabolic pathway such as starch digestion might help prevent that early proliferation, and could serve as “a tool to actually answer that question,” Sintim says.
Australia’s invasive cane toads are a scourge to native species, poisoning predators with toxic secretions. But now researchers have discovered that bacteria in the glands of adult cane toads transform these toxins into hydroxylated versions found in cane toad eggs and tadpoles. Manipulating this microbe-mediated toxin transformation could offer a new route for controlling the exploding cane toad population in Australia, the researchers say.
In 1935, about 100 cane toads (Rhinella marina) were released in sugar cane fields in northeastern Australia to control beetles eating the sugar cane. Today, an estimated 1.5 billion toads have spread thousands of miles across the continent, killing native species, to the brink of extinction in some cases. When predators munch on a cane toad, milky secretions full of a family of steroidal toxins called bufagenins ooze from the parotoid glands on the toad’s shoulders.
Microbes in the shoulder glands of cane toads hydroxylate marinobufagenin at the highlighted location, producing 11α-hydroxymarinobufagenin.
Robert J. Capon of the University of Queensland previously found that Gram-negative bacteria isolated from these glands could completely degrade bufagenins. The researchers wondered if other bacteria from the cane toad microbiome could also alter bufagenins, producing some of the types observed in cane toad eggs and tadpoles.
The researchers isolated three different strains of Gram-positive Bacillus species from the parotoid glands of two toads collected on campus. They fed the bacteria one of four different bufagenins produced by adult toads. After seven days, the researchers isolated compounds in the culture and found hydroxylated bufagenins. These derivatives are also found on cane toad eggs and tadpoles. In nature these modified toxins originate with the mother and are thought be modified in ways that protect the tadpoles and eggs in aquatic environments.
Capon speculates that a toad’s microbiome could hold clues to controlling the toad population. Inoculating the toads with other microbes that might change the bufagenin modifications as the adult females pass toxins to their eggs could be a helpful strategy, for example. “Would that reduce the viability of the eggs?” he wonders.
Microbial communities can be found everywhere on Earth, and the human body is no exception, Balskus says. There are microbes living on and inside of us. “If you count up the number of microbial cells associated with a person, you find we are just as much microbial as we are human.”
The colon is home to the most microbes found in the human body, and is one of the densest known microbial habitats on the planet, Balskus says. But while we all have microbiomes in our guts, the types of microbes inside us vary greatly. It is known that these organisms can influence energy balance and nutrition, help train the immune system, and provide protection from pathogens such as the bacteria Clostridium difficile (C. diff.). However, science has yet to determine exactly how they affect our bodies, and only 2% of the plasma metabolites influenced by gut microbes have been identified, Balskus says.
A great example of an identified compound and its effect on the body is trimethylamine (TMA), elevated levels of which are associated with trimethylaminuria, an inherited metabolic disorder known commonly as fish malodor syndrome. It is caused by a gene encoding mutation that causes enzymes to be unable to convert all of the TMA that the gut produces, Balskus says. Because there are no targeted treatments for the disease, patients will alter their own microbiomes with antibiotics or changes in their diets.
Balskus says work is being done to develop small molecules that can be manipulated, and targeted small molecule inhibitors could lead to more selective ways to study the microbiome. “We need better tools and approaches that will allow us to study chemistry directly in the microbial communities where it is taking place.”
Applying Technology to Link the Microbiome to Colon Cancer
One tool researchers are using to learn more about the human microbiome is XCMS, a metabolomic platform that allows for mass spectrometry and analysis. In his presentation, Siuzdak described how his team has used XCMS in examining colon cancer and how the microbiome can affect the disease’s progression.
Patients with tumors found on the cecum side, or right side, of the colon live about half as long as those with tumors on sigmoid side, or left side. Siuzdak and his team used mass spectrometry-based metabolomic analysis to examine tumor samples, working with Dr. David Elder at Karolinska Institutet, Dr. Cynthia Spears at Johns Hopkins University, and Dr. Laura Geotz at University of California, San Diego. Using XCMS, Siuzdak’s team was able to align the different liquid chromatology-mass spectrometry analyses of the metabolites in the different samples into a more coherent map. Being able to visualize and pull out the most interesting data was a huge help in the work, he explained.
They found that the cecum side tumors tend to have biofilm, or microorganisms that attach to each other, on them. Siuzdak said it is possible that the biofilms may be growing by feeding on polyamines, which are typically produced by tumors. The team also found that biofilms are penetrating the cells, which can stimulate cellular proliferation, which Siuzdak says indicates a “symbiotic relationship between the biofilm growth and the tumor growth.” While they still don’t know why the patients with cecum side tumors live for shorter amounts of time, they are a step closer to that discovery, and using XCMS to analyze the metabolomic data helped them get this far.
Want to learn more about the technology scientists are using to study microbiomes? You can watch a recording of the full webinar complete with Dr. Balskus and Dr. Siuzdak’s slides through August 15, 2017, just register here.
The scientific community is learning more about microorganisms and how they interact with each other in microbiomes. Technology is making their discoveries possible and more is needed to advance their research even further.
On August 11, 2016, ACS Nano and Analytical Chemistry presented Microbiome Technologies, the second in the three-part #ACSmicrobiome Webinar Series. The webinar focused on the current technology researchers are using to study the microbiome and the technology they will need moving forward. It featured Dr. Paul S. Weiss, Editor-in-Chief of ACS Nano; Distinguished Professor of Chemistry & Biochemistry, Distinguished Professor of Materials Science & Engineering, California NanoSystems Institute at the University of California, Los Angeles, and Dr. Pieter Dorrestein, Professor at the University of California, San Diego; Director of the Collaborative Mass Spectrometry Innovation Center; and a Co-Director of the Institute for Metabolomics Medicine in the Skaggs School of Pharmacy & Pharmaceutical Sciences, and Department of Pharmacology.
Technology is Essential to Understanding Microbiomes
Microbiomes are found all over – in and on human bodies, in the ocean, the atmosphere, and the soil. Research on microbiomes touch on many different disciplines, says Weiss, which can create gaps in the understanding of microbiomes from one area of research to another. To address this, “tremendous technologies need to be developed,” he said.
“One of the advantages of the field nanoscience and nanotechnology is that we’ve taught each other how to communicate across fields, and maybe even more than the technology has required,” Weiss said.
To learn more about microbiomes, scientists need the tools to observe, manipulate and measure on the nano level. This will lead to the development of specialized technology. Researchers studying the microbiomes of the ocean, which are strongly connected with local environments, are being aided by new miniaturized, ruggedized technology, Weiss says. The development of new tools, such as these, that manipulate the microbiome will lead to a better understanding of how these systems work.
Digitally Mapping the Human Microbiome
The National Institutes of Health Human Microbiome Project has found that the human body consists of 20,000 human genes and 2 million to 20 million microbial genes. These findings have redefined what it is to be human, Dorrestein says. Microbes are involved in the metabolism of foods and changing immune chemistries. They can determine your weight, or how attractive you are to mosquitoes. Microbes are “the ignored organ” of the human body, he says.
The scientific community has been able to determine what microbes are inside the body, and now the research is shifting to what they are doing to the body and how to take control of that, Dorrestein says.
Dorrestein has developed a three-dimensional (3D) mapping tool, called ili, with Dr. Theodore Alexandrov of the European Molecular Biology Laboratory. The tool allows you to map data, such as where samples are taken on a person’s body, on to a 3D model. This allows researchers to visualize mass spectrometry data, and from there, observe and analyze the data.
To analyze that data, a metabolomics analysis and knowledge capture platform, called Global Natural Products Social Molecular Networking (GNPS) was created. Dorrestein said it launched in August with roughly 13,000 users from 111 countries. The platform allows researchers to upload, store and analyze data, with a predictive computing element, as well. GNPS allows researchers to share and pool their data, which will hopefully allow researchers to crowdsource analysis.
Want to learn more about the technology scientists are using to study microbiomes? You can watch a recording of the full webinar complete with Dr. Weiss and Dr. Dorrestein’s slides through August 10, 2017, just register here.
Advances in technology and research methods have in recent years allowed scientists to more effectively study Earth’s oldest life forms – microorganisms. They’ve discovered almost all microorganisms exist not alone, but in communities commonly known as “the microbiome,” and that these communities interact with their environments in a variety of ways.
Before it reaches our taps, drinking water goes through a multi-step treatment process that includes primary disinfection through exposure to ozone and biofiltration, and secondary disinfection through exposure to chloramine, Raskin says. But the water we drink still ends up with between 106 and 108 bacterial cells per liter.
It’s impossible to remove all mycobacteria from drinking water, so water treatment efforts focus on removing pathogenic microbes – the microorganisms that can cause disease in humans, particularly people with compromised immune systems, Raskin says. Mycobacteria avium and Mycobacteria abscessus are two environmental, or naturally occurring, mycobacteria whose levels need to be monitored in public water systems, as they can cause disease, and have shown some resistance to disinfectants.
Raskin’s research team studied the tap water from a group of houses in Ann Arbor, Michigan. Seven of the houses were close to the water treatment plant, and eight were farther away from the facility. The water coming out of the taps close to the plant had less exposure to chloramine in the city’s water distribution system, as it had a shorter distance to travel through that system. The water that traveled farther through the system before reaching the tap had greater exposure to chloramine. They found bacterial and mycobacterial concentrations were higher in samples taken farther away from the water treatment plant, Raskin says.
“I think it’s critically important to study all kinds of microbial phenomena within the context of the complex drinking water microbiome, and that we not just want to do this in the lab, but want to go into the field, studying full-scale systems, because there is complexity that we cannot mimic in the lab,” she says.
The Microbiome of Wastewater Treatment Plants
Another element of the urban water cycle is wastewater treatment plants, which have their own unique microbiomes. These facilities can be hotspots for antibiotic resistance genes because their microbiomes are home to thousands of species, have a high biomass density, and bacteria input from the waste of thousands of people, Zhang says. Wastewater treatment facilities must ensure these ARGs are removed from the wastewater to prevent their spread.
Wastewater treatment plants must monitor levels of bacteria associated with bulking, which is activated sludge with poor settling and compaction characteristics, and foaming, Zhang says. Both bulking and foaming can lead to overflow and hinder a plant’s operations. He praised the advances made in metagenomics, which provide technicians with information about the bacterial makeup of the microbiome in a quick, quantitative, and qualitative way.
Large-scale DNA sequencing and metagenomics are important tools in studying the bacterial profiles of the microbiome at the genus level, Zhang says. And the technological advancements must continue. He calls for specific, customized databases that would allow for better analysis of the bacterial profiles of wastewater treatment plants. Among Zhang’s other predictions for new developments is that third-generation genomic sequencing will allow for longer sequences, which will make analysis more accurate.
Want to learn more about the microbiome of the urban water cycle? You can watch a recording of the full webinar complete with Dr. Raskin and Dr. Zhang’s slides through July 25, 2017, just register here.
The microbiome is a hot topic in the popular media and in labs around the world. Chemists, biochemists, and other scientists are continually making new discoveries about the microbiome and how it affects health and the environment.
1. Which area on the surface of the human body has been found to host the most diverse collection of microbes?
Behind the ear
On the forearm
In the bellybutton
In-between the toes
It’s no secret that the human body is made up largely of microbes – over 100 trillion by some estimates. By what ratio do microbes outnumber human cells in our bodies?
10 to 1
100 to 1
1,000 to 1
10,000 to 1
The human digestive tract is home to a majority of the microbes that make up the human microbiome. Roughly what percent of an individual’s microbiome can be found in their digestive tract?
A recent Dutch study concluded that individuals who engage in intimate kissing have a more similar oral microbiota composition compared with unrelated individuals. On average, how many bacteria did they find were transferred in a 10 second intimate kiss?
Which winner of the Nobel Prize in Medicine purposely ingested Heliobacter pylori in order to prove that stomach ulcers are caused by bacteria, not stress?
Harold zur Hausen
Which ACS journal is currently accepting papers for an upcoming Special Issue on the Microbiome?
ACS Infectious Diseases
Journal of Proteome Research
ACS Medicinal Chemistry
Think You Know the Human Microbiome?
ACS Infectious Diseases invites you to take this short, six question quiz to test your knowledge of some of the more interesting developments in research surrounding the human microbiome over the past 15 years. Go with your gut and see if you can get all six questions right! Take our quiz and find out!
Whether you’re researching the microbiome or not, it’s hard at work having an effect on your body and your health. Take this short ACS Infectious Diseases quiz and test your knowledge on the human microbiome then watch The Microbiome in Health and Disease, a webinar sponsored by ACS Infectious Diseases and Journal of Proteome Research to learn more.
Microorganisms are Earth’s oldest life forms and have come to inhabit virtually every location on the planet. Recent advances in technology have enabled researchers to dissect how microorganisms interact with their surroundings. These investigations reveal that microorganisms exist in complex communities commonly referred to as “the microbiome.”
These microbial communities have been found to be integral to many processes, including human and animal health, and environmental nutrient cycling. In light of our growing appreciation of the importance of the microbiome, ACS Publications began a three-part webinar series on July 26 exploring how chemists are studying this important topic.
If you missed the live webinar, you can watch the recording complete with Dr. Grostern, Dr. Raskin, and Dr. Zhang’s slides any time. Just register here anytime.
Part 2: Microbiome Technologies
This second webinar in the series, Microbiome Technologies, is sponsored by ACS Nano and Analytical Chemistry and will address the role of the microbiome as it pertains to technology. The extraordinary measurement and manipulation requirements of studying the microbiome open up great opportunities for chemists, nanoscientists, and other researchers. These broad needs include the ability to eavesdrop on chemical communications, conduct massive multimodal data science, and develop synthetic biology- and mass spectrometry-based tools to manipulate organisms and populations. This webinar will provide an overview of the function of these technologies and discuss challenges and areas for investigation in these technologies.
Describe the importance of microbiome technologies.
Identify challenges associated with these technologies.
Explain how to study these technologies using nanoscience, nanotechnology, and mass spectrometry.
Moderator: Laura Fernandez, Managing Editor of ACS Nano and Nano Letters
Pieter Dorrestein, Professor at the University of California, San Diego; Director of the Collaborative Mass Spectrometry Innovation Center; and a Co-Director of the Institute for Metabolomics Medicine in the Skaggs School of Pharmacy & Pharmaceutical Sciences, and Department of Pharmacology
Paul S. Weiss, Editor-in-Chief of ACS Nano; Distinguished Professor of Chemistry & Biochemistry, Distinguished Professor of Materials Science & Engineering, California NanoSystems Institute, University of California, Los Angeles
This third webinar in the series, The Microbiome in Health and Disease, is sponsored byACS Infectious Diseases and Journal of Proteome Research and will address the microbiome’s critical role in maintaining health and preventing disease. The gut’s microbiome plays an integral role not only in maintaining metabolism but also in providing a barrier against infectious disease. This webinar will offer an overview of the microbiome’s function in these aspects of human health and discuss in detail challenges to investigating the subject and various approaches to overcoming them. Attendees will learn about opportunities for chemists and chemical biologists in this research area.
The Microbiome: An ACS Infectious Diseases Special Issue
Dr. Balskus is also the Guest Editor for an upcoming special issue of ACS Infectious Diseases titled The Microbiome. The issue is scheduled for publication in 2017 and the journal is accepting manuscripts until Oct. 1, 2016.
Typical deodorants work by killing the bacteria responsible for the stink. A polymer-based deodorant could capture odor precursors in the underarm before they start to smell, without disturbing the microbes that live on the skin.
Underarm smell is mainly caused by a couple of volatile fatty acids, (E)-3-methyl-2-hexenoic acid and 3-hydroxy-3-methyl-hexanoic acid. They are produced when bacteria living on the skin break down precursor molecules—glutamine conjugates of these fatty acids— that are found in sweat; by themselves, these precursors have no odor. “When bacteria cleave them into smaller molecules, they start to smell. That is where body odor comes from,” says Karsten Haupt, a nanobiotechnologist at the University of Technology of Compiègne.
Sweat contains these glutamine conjugates of two volatile fatty acids. Molecularly imprinted polymers can bind these molecules, preventing bacteria from cleaving them to produce the fatty acids, which are responsible for body odor.
Typical deodorants tackle the stink by going after the bacteria with a bactericide such as triclosan or chlorhexadine. But researchers worry using those may disrupt the balance of healthful bacteria on the skin or lead to the evolution of resistant bacteria. Antiperspirants, on the other hand, contain aluminum salts to stanch the flow of sweat from the pores. But that can lead to skin irritation, and some people want to avoid products that block pores with these ingredients.
The new approach relies on molecularly imprinted polymers (MIPs). MIPS are made by combining a template molecule with monomers and free radical species to induce polymerization. As the polymer forms, molecular interactions mold it around the template molecule. Removing the template leaves behind a polymer shaped to bind to molecules that resemble the template. MIPs have been used for years in applications such as biosensors and immunoassays.
Aided by funding from L’Oreal, Haupt and Bernadette Tse Sum Bui, a research engineer at France’s National Center for Scientific Research, showed in 2016 that they could build MIPs that capture molecules involved in underarm odor. The researchers created MIPs about 600 nm in diameter that could bind to the glutamine group in the odor precursor molecules. That binding made it impossible for bacteria to access the precursors, so they couldn’t produce the smelly breakdown products. The bacteria can still use other components of the sweat as a food source.
For the new study, the researchers tested the biocompatibility of the MIPs by culturing three types of skin bacteria in the presence and absence of MIPs. Growth rates were the same, suggesting the MIPs do not alter the skin microbiome. They also mixed MIPs in with cultured skin cells, then looked to see whether the cells had produced cytokines, a marker of inflammation. They did not. Haupt says that shows the material is safe for both skin and bacteria.
Ken Shimizu, a chemist at the University of South Carolina who works on MIP-based sensors, says factors that make MIPs unsuitable for many pharmaceutical applications, such as their inability to permeate membranes, are actually advantages for this application. “This is a clever use of MIPs,” he says. “The principle of using benign ways of modifying the behavior of bacterial products without killing them has great potential.”
Over the last decade, cancer researchers have learned that a highly reactive, DNA-damaging toxin called colibactin could mark the onset of colon cancer. Certain strains of Escherichia coli in the gut produce the genotoxin, and up to 67% of people with colon cancer harbor such strains, compared with only 20% of those without the disease. But scientists still don’t have a full picture of colibactin’s structure or its mechanism of action because no one has managed to isolate the compound. To make isolation easier, a team led by Kenji Watanabe of the University of Shizuoka has developed a fluorescent probe that allows fast, high-throughput screening of colibactin-producing bacteria and identification of high-producing strains. With such strains in hand, scientists could have a better shot at isolating the compound and determining its complete structure, the researchers say.
The fluorescent probe works by detecting the activity of ClbP, a peptidase that is part of the final steps of colibactin synthesis. ClbP removes a protective group, N-myristoyl-D-asparagine (N-myr-Asn), from the colibactin precursor, turning the molecule into the active genotoxin, which then gets secreted out of the E. coli cells. Inspired by this process, researchers made their probe by attaching a fluorescent tag to N-myr-Asn and then delivered it to cultures of individual bacterial strains. When active ClbP is present, the enzyme recognizes its N-myr-Asn target and cleaves the probe’s fluorescent tag, resulting in an increase in fluorescence. From this signal, researchers can infer which bacterial strains have ClbP activity and therefore are producing colibactin.
Watanabe and coworkers tested the probe with colon cancer tissue samples in 96-well plates. The probe revealed the presence of colibactin-producing bacteria in the samples as accurately as polymerase chain reaction (PCR), the gold standard test. They also showed that higher fluorescence intensities corresponded with higher colibactin production levels in E. coli strains. One high-producing strain identified by the probe made 26 times as much colibactin as a strain commonly used as a positive control.
The probe could also help screen human stool samples directly to identify colibactin-producing strains, even before cancer develops, Watanabe says. Compared with conventional identification methods, such as PCR and liquid chromatography/mass spectrometry, this probe-based fluorescent assay is cheap and takes only a few hours, he adds.
Watanabe’s team is not alone in developing probes to find colibactin-producing bacteria. A group led by Harvard University’s Emily Balskus, whose team revealed that colibactin damages DNA in the gut through alkylation, recently reported a similar fluorescence-tagged ClbP probe that they tested in vitro. Matthew Volpe, a graduate student who led the work, says it’s exciting to see the two studies complement each other. He adds that the activity difference between strains identified using the Watanabe group’s probe is “quite remarkable.” The next critical question, he says, is whether the high-producing E. colistrains found in this study are actually more genotoxic than other strains when they are in the human gut.
Earlier this year, Watanabe established a company called Adenoprevent that aims to offer screening services to people who want to know whether they host colibactin-producing E. coli, a high-risk indicator for colon cancer. Watanabe says he aims to test 18 million samples a year by 2025.
I interviewed Dr. Carlson to help interested authors understand what we are looking for in this Special Issue.
What are the most exciting innovations and discoveries in chemical microbiology that you’ve read about recently?
The recent surge in the development of tools to more deeply understand how microbes interact with their host is incredibly exciting. This ranges from characterization of receptors that promote interactions between eukaryotic and prokaryotic organisms, the resulting immune response, breakdown of drugs by the gut microbiome, and the natural products that microbes generate that are both beneficial and detrimental to the host.
What types of innovations and discoveries are you hoping to see in the papers submitted to this Special Issue?
One of the standing challenges in the study of bacteria is our inability to readily internalize small molecules and other reagents. I look forward to creative and exciting demonstrations of methods that can be used in live bacteria.
What types of researchers/research groups should submit their research to this Special Issue?
All chemists that use the tools of this trade to study microbes and all microbiologists that have incorporated methods from the field of chemistry. All traditional disciplines of chemistry have valuable contributions to make to the field of Chemical Microbiology, ranging from organic synthesis to analytical measurements, to molecular modeling.
Anything else you’d like to tell people about the Special Issue?
We are excited to see papers on all different types of microbes!