Meet the Microbiologist

Dr. Mark O. Martin, Associate professor of biology at the University of Puget Sound in Tacoma, Washington is a distinguished educator with a well-known social media presence. He discusses how he became interested in microbiology and what drives his varied research foci, including #Microbialcentricity, bacterial predation, bioluminescence, tardigrades, microbial midwives and more. In the process, he delves into his passion for using art and other creative approaches to facilitate learning in the classroom, and shares some experience-driven wisdom about building confidence in STEM.

Links for this Episode:

• Vertically transmitted microbiome protects eggs from fungal infection and egg failure https://animalmicrobiome.biomedcentral.com/articles/10.1186/s42523-021-00104-5
• The effects of Sceloporus virgatus cloacal microbiota on the growth of pathogenic fungi https://soundideas.pugetsound.edu/summer_research/426/
• Sex-specific asymmetry within the cloacal microbiota of the striped plateau lizard, Sceloporus virgatus https://link.springer.com/article/10.1007/s13199-010-0078-y
• Predatory Prokaryotes: An Emerging Research Opportunity (pdf) https://www.pugetsound.edu/sites/default/files/file/martin2002_0.pdf
• Carleton College #LuxArt 2019 https://www.youtube.com/watch?v=fztiJ3o7uWs

Dr. Elizabeth Dinsdale, Matthew Flinders Fellow in Marine Biology in the College of Science and Engineering at Flinders University in Adelaide, Australia, uses genomic techniques to investigate the biodiversity of microbial communities in distinct ecological niches, including coral reefs, kelp forest and shark epidermis. She discusses how shotgun metagenomics is being used to characterize the architecture of microbial communities living in the thin layer of underlying mucus on shark's skin, and how understanding the function of these microbes is providing clues to important host-microbe interactions, including heavy metal tolerance.

Ashley's Biggest Takeaways:

Sharks belong to a subclass of cartilaginous fish called elasmobranchs and are unique in that their epidermises are covered in dermal denticles—overlapping tooth-like structures that reduce drag and turbulence, helping the shark to move quickly and quietly through the water. These dermal denticles are sharp (if you're going to pet a shark, make sure you go from the head to the tail to avoid getting cut!), and depending on the species of shark, may be more or less spread out across the epidermis.

Where do microbes enter the story? Dermal denticles overlay a thin layer of mucus, which provides a distinctive environment for microbial life. Collecting microbial samples from underneath a shark's dermal denticles is quite difficult, and the technique varies by shark species (shark size, water depth and ability to bite all factor into the equation). Liz's team uses a specially designed tool that the group affectionately calls a "supersucker," to create and capture a slurry of microbes and water for analysis.

The team then uses shotgun metagenomics to identify and characterize the microbes in their collected samples. Sequencing has revealed biogeographical difference, as well as similarities in microbial architecture of whale sharks across the globe.

There are 2 populations of whale sharks—one in the Atlantic Ocean and the other in the Indian Pacific Ocean. Samples collected from both populations have revealed that each individual whale shark, from within each aggregation, shares many of the same microbes. In fact, unlike algae which may share 1 to 2 microbial species, whale sharks share about 80% of microbes across every individual. Since many of the sharks don't cross aggregations, Liz's team is investigating the possibility of coevolution between microbes and hosts.

Metagenomic sequencing also provides information about the function of the sequenced microbes. High presence of heavy metal-tolerant microbes has been found in the epidermis of all shark species that the team has analyzed. Sharks are known to carry high levels of heavy metals in their skin, muscle and even blood. However, muscle tissue samples contain lower concentrations than skin, indicating that there may be a density gradient in place, and raising questions about how microbes might be involved in this regulation. Is there a pathway by which the microbes metabolize and help to remove concentrations of heavy metals across the epidermis? Liz and her team are hoping to find out.

Links:

• Elizabeth Dinsdale https://www.flinders.edu.au/people/elizabeth.dinsdale
• Tracking Pathogens via Next Generation Sequencing (NGS) https://asm.org/Magazine/2021/Spring/Tracking-Pathogens-via-Next-Generation-Sequencing
• Microbial Ecology of Four Coral Atolls in the Northern Line Islands https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001584
• Coral Research https://coralandphage.org/research_coral.php
• Metagenomic analysis of stressed coral holobionts https://pubmed.ncbi.nlm.nih.gov/19397678/
• Metagenomic analysis of the microbial community associated with the coral Porites astreoides https://pubmed.ncbi.nlm.nih.gov/17922755/

Dr. Mallory Choudoir, microbial ecologist and evolutionary biologist at the University of Massachusetts Amherst shares how she leverages microbial culture collections to infer ecological and evolutionary responses to warming soil temperatures. She discusses complexities of the soil microbiome and microbial dispersal dynamics, and introduces fundamental concepts about the intersection between microbes and social equity.

Ashley's Biggest Takeaways:

Microbial culture collections are fundamental resources, serving as libraries where diverse species of microbes are identified, characterized and preserved in pure, viable form. Culture collections ensure conservation of species diversity and sustainable use of the collected microbes.

For soil microbiologists, like Mallory Choudoir, culture collections provide the opportunity to connect patterns of genomic variation and microbial physiology to the conditions under which a particular microbe was isolated.

Soil is a complex environment from the perspective of a microbe. In order to coexist in such a biologically diverse environment, which consists of spatial heterogeneity, as well as heterogeneity in access to moisture and nutrients, microbes must evolve different strategies to survive as part of a stable community.

Choudoir's field site is based in the Harvard Forest Long Term Ecological Research Program's field site, where coils are buried and have been heating the forest soil to 5 degrees above ambient temperatures for nearly 30 years. The study allows Choudoir and colleagues to observe and evaluate long-term responses to chronic soil warming stress.

This research is important because microbes function as resources to the health and well-being of ourselves and our planet. Understanding how microbes adapt to biotic and abiotic stresses can help inform future conservation strategies, biotechnological approaches and applications and equitable allocation of microbial resources.

Visit https://asm.org/mtm for links mentioned

Peter Hotez talks about the global impact and historical context of neglected tropical diseases. He also highlights important developments in mass drug administration and vaccine research and shares why he chose to publish the third edition of Forgotten People, Forgotten Diseases during the COVID-19 pandemic. Ashley's Biggest Takeaways

Neglected Tropical Diseases (NTDs) are chronic and debilitating conditions that disproportionately impact people in low- and middle-income countries (LMICs).

Many of these diseases are parasitic, such as hookworm infection, schistosomiasis and chagas disease; however, in recent years, several non-parasitic infections caused by bacteria, fungi and viruses, as well as a few conditions that are not infections, including snake bite and scabies (an ectoparasitic infestation), have been added to the original NTD framework (established in the early 2000s). What do most NTDs have in common?

High prevalence. High mortality; low morbidity. Disabling. Interfere with people's ability to work productively. Impact child development and/or the health of girls and women. Occur in a setting of poverty and actually cause poverty because of chronic and debilitating effects.

Hotez and his colleagues recognized that there is a uniqueness to the NTDs ecosystem, and they began putting together a package of medicines that could be given on a yearly or twice per year basis, using a strategy called Mass Drug Administration (MDA). This involved the identification of medicines that were being used on an annual basis in vertical control programs and combining those medications in a package of interventions that costs about $0.50 per person per year. "Throw in an extra 50 cents per person and we could double or triple the impact of public health interventions," he explained.

Emerging diseases, such as SARS-CoV-2, capture the attention of the public for obvious reasons. They pose an imminent threat to mankind. NTDs are not emerging infections, but they are ancient afflictions that have plagued humankind for centuries and, as a consequence, have had a huge impact on ancient and modern history. One of the reasons we have mainland China and Taiwan today may have been, in part, due to a parasitic infection, Schistosomiasis.

Hotez and colleagues at the Texas Children's Center for Vaccine Development have developed a COVID-19 vaccine, based on simple technology, similar to what is used for the Hepatitis B vaccine. They hope to release the vaccine for emergency use in resource poor countries like India and Indonesia.

When asked about the timing of the publication of his book, the third edition of Forgotten People, Forgotten Diseases, Hotez acknowledged the difficulty of helping countries understand that NTDs have not gone away. COVID-19 is superimposed on top of them, and the pandemic has done a lot of damage in terms of NTD control. Although social disruption has interfered with the ability to deliver mass treatments, Hotez said that it has been gratifying to see that the USAID and their contractors have responded by putting out guidelines about how to deliver mass treatments with safe social distancing.

"As a global society, we have to figure out how to walk and chew gum at the same time," he said. "We've got to take care of COVID, but we really must not lose the momentum we've had for NTDs because the prevalence is starting to decline and we're really starting to make an impact."

Rita Colwell has made major advances in basic and applied microbiology, largely focused on Vibrio cholerae. She describes several lines of evidence for the environmental niche of the bacterium, as well as her work to predict and prepare for cholera outbreaks. Colwell closes with her thoughts on why it's a great time to be a microbiologist.

Denise Akob discusses her studies of microbial communities of contaminated and pristine environments using life science and earth science techniques. She discusses how to figure out "who's there," how to optimize select natural microbial activities, and her career path into government research.

Julie's Biggest Takeaways:

Biogeomicrobiology straddles the life science and earth science fields. This is a growing area of research in the academic setting as well as in the private sector, where one can contribute to hydrogeology or bioremediation efforts.

What happens on the surface when extracting resources like natural gases? Wastewater from hydraulic shale fracking, or fracking, can contaminate microbes. Preliminary data suggests that microbes that thrive in that wastewater can be a fingerprint for surface contamination, and this is one of the areas of active research in Akob's lab. Additionally, microbes can respond to contaminants to remove that risk and remediate the spills.

One trip to the field can provide samples for years of analysis. From one sample, scientists can conduct:

• Microbiome studies through amplicon sequencing to understand population structures.
• Metagenomics studies to understand functional potential.
• Biochemical studies to understand active metabolic processes.

Akob asks how to make natural microbial degraders happy. For example: acetylene, a triple-bonded carbon compound, can inhibit degradation of chlorinated solvents, a potent groundwater contaminant. By studying the microbes that use acetylene as a primary energy source (acetylenotrophs), this removes this inhibition caused by acetylene and the chlorinated solvent-degraders can increase their activity.

Akob studies pristine environments to understand natural microbial communities. A cave she studied in Germany was 'ultra pristine,' discovered while building a highway. Understanding natural processes, such as the biomineralization promoted during stalagmite and stalactite formation helps scientists imagine how to use tehse processes in other applications.

Links for this Episode:

• Mumford AC et al. Common Hydraulic Fracturing Fluid Additives Alter the Structure and Function of Anaerobic Microbial Communities. Applied and Environmetnal Microbiology. 2018.
• Akob DM et al. Acetylenotrophy: a Hidden but Ubiquitous Microbial Metabolism? FEMS Microbial Ecology. 2018.
• Akob DM et al. Detection of Diazotrophy in the Acetylene-Fermenting Anaerobic Pelobacter sp. Strain SFB93. Applied and Environmental Microbiology. 2017.
• ASM Article: The Microbial World of Caves
• James J, Gunn AL, and Akob DM. Binning Singletons: Mentoring through Networking at ASM Microbe 2019. mSphere. 2020.
• HOM Tidbit: Scientists Find Ancient Cave Dwelling Resistant Bacteria
• ASM Press: Women in Microbiology

How does tick biology influence their ability to transmit disease? Marshall Bloom explains the role of the tick salivary glands in Powassan virus transmission and the experiments that led to this discovery. He also provides a historical background for the Rocky Mountain Labs in Hamilton, Montana, and talks about the 3 elements to consider when working with potentially harmful biological agents.

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There are 3 elements to consider when working with potentially harmful biological agents:

Biosafety: protecting the laboratory workers from the infectious agents in the lab.

Biocontainment: protecting the community by keeping the infectious agent contained within the facility.

Bioassurity: protecting the individual by ensuring those working with infectious agents are capable to do so.

You need 4 bites of an APPLE for full lab safety, for work in labs from high school level through biosafety level 4:

• A: Administration. Training, paperwork, etc.
• P: Personal protective equipment (PPE). Varies from gloves to positive pressure suits, depending on the microorganisms under study.
• PL: Laboratory procedures. Standard operating protocols.
• E: Engineering. Biosafety cabinets and labs that have protective features.

Most of the vector-borne flaviviruses, including Powassan virus, don't cause overt disease in the people they infect, so many people never know they've been infected. Without serological surveys, it's difficult to know the full range of infected individuals versus those that develop serious disease. Serious disease often manifests in neurological symptoms such as encephalitis, with 10-15% mortality rate; half of those suffering neurological disease will continue to have serious sequelae for years.

The Rocky Mountain Labs was once the world reference center for ticks: it held thousands of samples which represented the type species for the entire world.

The tick salivary glands look like a bunch of grapes: the stem of the grapes is a series of branching ducts. The "grapes" at the end of the ducts are the acini, which is Latin for 'little sac.' These acini play a major role in tick feeding, and different types of acini play different functional roles:

• Type 1 acini: cells have no granules. Acini involved with fluid exchange.
• Type 2 and type 3 acini: cells with granules. Cells degranulate to release vasoactive compounds into tick saliva during feeding.

"The first isolation of Powassan virus was from a little boy in Powassan, Canada in 1958. If you look at the cases over the years, the numbers are going up, but compared to Lyme disease, they're pretty low: there's been less than 200 cases, all told."

"Amazingly, the Powassan virus can be transmitted in as little as 15 minutes….[and] a female tick can take days to get a full meal."

"I take a tick-centric view. If I can anthropomorphize, as my old friend Stanley Falkow used to say, he'd say 'think like the microbe.' The microbe doesn't really care if we get sick or not. The microbe is just trying to make a living and survive."

"One of the really surprising things is that infected ticks can infect uninfected ticks, if they are feeding right next to each other. Ticks like to feed in groups: it's called co-feeding. The virus can transferred really quickly, 15 min, which is way faster than the virus can go through a replication cycle. What that means to me is that the ticks are infecting each other….we want to investigate the role of co-feeding."

"If something sounds like fun or sounds important, and especially if something sounds fun AND important, then you should do it."

Links for this Episode:

• Paules CI et al. Tickborne Diseases--Confronting a Growing Threat. New England Journal of Medicine. August 2018.
• Amazon: Fighting Spotted Fever in the Rockies by Esther Gaskins Price
• New York Times: Kay Hagan obituary
• Grabowski JM et al. Dissecting Flavivirus Biology in Salivary Gland Cultures from Fed and Unfed Ixodes scapularis (Black-Legged Tick). mBio. January 2019.
• ASM on Instagram
• Grabowski JM, Offerdahl DK, and Bloom ME. The Use of Ex Vivo Organ Cultures in Tick-Borne Virus Research. ACS Infectious Disease. Marhc 2018.
• Twitter thread from @BugQuestions: Rocky Mountain Spotted Fever and Howard Ricketts
• History of Microbiology Tidbit: A Short History of the Screwworm Program

What kinds of microorganisms can degrade oil? How do scientists prioritize ecosystems for bioremediation after an oil spill? Joel Kostka discusses his research and the lessons from the Deepwater Horizon oil spill that will help scientists be better prepared for oil spills of the future.

Links for this Episode:

• Joel Kostka Lab Website
• Kostka J. et al. Hydrocarbon-Degrading Bacteria and the Bacterial Community Response in Gulf of Mexico Beach Sands Impacted by the Deepwater Horizon Oil Spill. Applied and Environmental Microbiology. 2011.
• Shin B. et al. Succession of Microbial Populations and Nitroget-Fixation Associated With the Biodegradation of Sediment-Oil-Agglomerates Buried in a Florida Sandy Beach. Scientific Reports. 2019.
• Bociu I. Decomposition of Sediment-Oil-Agglomerates in a Gulf of Mexico Sandy Beach. Scientific Reports. 2019.
• Overhold W.A. et al. Draft Genome Sequences for Oil-Degrading Bacterial Strains from Beach Sands Impacted by the Deepwater Horizon Oil Spill. Genome Announcements. 2013.
• Gulf of Mexico Research Initiative
• ASM Colloquia Report: Microbial Genomics of the Global Ocean System
• ASM Article: Microbiomes: An Origin Story
• Joyful Microbe Blog: How to make a Winogradsky column Small Things Considered: How to Build a Giant Winogradsky Column
• 20% off The Invisible ABCs for MTM listeners! Use promo code: ABC20 at checkout.

How do arboviruses evolve as they pass between different hosts? Greg Ebel discusses his research on West Nile virus evolution and what it means for viral diversity. He also talks about using mosquitos' most recent blood meal to survey human health in a process called xenosurveillance.

Julie's Biggest Takeaways:

Mosquitoes and other arthropods have limited means of immune defense against infection. One major defense mechanism is RNA interference (RNAi). RNAi uses pieces of the West Nile viral genome to select against the viral genome, which helps select for broadly diverse viral sequences. The more rare a viral genotype, the more likely it is to escape negative selection inside the mosquito host, allowing this viral sequence to increase in frequency.

West Nile virus passes largely between birds and mosquitos. Culex mosquitos tend to prefer birds, and this leads to an enzootic cycle for the virus passing between birds and mosquitos. The viral life cycle inside the mosquito has several important steps:

• The virus first enters as part of the mosquito blood meal.
• The virus infects epithelial cells of the mosquito midgut.
• After 3-5 days, the virus leaves the midgut (midgut escape) to enter the mosquito hemolymph.
• In the next mosquito blood meal, virus is expelled with saliva, which has anticoagulant activity.

West Nile virus selection undergoes cycles of selection as it passes from vertebrates (mostly birds) to invertebrates (mosquitos):

• In vertebrates, the virus must escape to cause viremia in a short period of time for replication to occur before the immune system recognizes and eliminates the virus.
  • This leads to purifying selection, or elimination of amino acid variation that decreases viral protein function.
• In mosquitos, the virus spends several days in the midgut epithelial cells and then hemolymph, leading to a longer selection time.
• This leads to more viral diversity in the mosquito host. RNAi further drives population diversity. Through stochasticity, a single viral population will often come to dominate a single infected mosquito.

How do scientists know which virus replicates best? Competitive fitness tests measure which virus grows to a higher population in a given environment. A manipulated virus (one passaged in a mosquito or selectively mutated at distinct sequences) and its non-manipulated parent sequence are inoculated at known proportions, and given a certain amount of time to replicate. By measuring the final proportions, Greg and his team can determine which sequence was more fit in that given environment.

Xenosurveillance uses mosquitoes to detect a wide array of pathogens at clinically relevant levels. Testing began with in vitro blood-bag feeding, and was validated with studies in Liberia and Senegal. The microorganism sequences are so diverse that the information was used to identify novel human viruses. These studies also provide insight into mosquito feeding habits, which helps in disease modeling.

Links for this Episode:

• Greg Ebel Lab Website
• Rückert C. et al. Small RNA Responses of Culex Mosquitoes and Cell Lines during Acute and Persistent Virus Infection. Insect Biochemistry and Molecular Biology. 2019.
• Grubaugh N.D. et al. Mosquitoes Transmit Unique West Nile Virus Populations during Each Feeding Episode. Cell Reports. 2017.
• Grubaugh N.D. and Ebel G.D. Dynamics of West Nile Virus Evolution in Mosquito Vectors. Current Opinion in Virology. 2016.
• Fauver J.R. et al. Xenosurveillance Reflects Traditional Sampling Techniques for the Identification of Human Pathogens: A Comparative Study in West Africa. PLoS Neglected Tropical Diseases. 2018.
• Fauver J.R. The Use of Xenosurveillance to Detect Human Bacteria, Parasites, and Viruses in Mosquito Bloodmeals. American Journal of Tropical Medicine and Hygiene. 2017.
• Tracey McNamera: Canaries in the Coal Mine TEDxUCLA
• New York Times: Encephalitis Outbreak Teaches an Old Lesson. 1999.
• ASM Article: The One Health of Animals, Humans, and Our Planet: It's All Microbially Connected

How can the intricate relationship between soil microbiota and plants be managed for improved plant health? Linda Kinkel discusses new insights into the plant rhizosphere and the ways that some Streptomyces isolates can protect agricultural crops against bacterial, fungal, oomycete, and nematode infections.

Julie's Biggest Takeaways:

The soil microbiome is extremely dynamic, with boom-and-bust cycles driven by nutrient fluxes, microbial interactions, plant-driven microbial interactions, and signaling interactions. Finding the source of these boom-and-bust cycles can help people to manage the microbiome communities and produce plant-beneficial communities for agricultural purposes.

Rhizosphere soil is soil closely associated with the root and is distinct from rhizoplane soil that directly touches the root. The endophytic rhizosphere are those microbes that get inside the root. Many scientists view these communities as a continuum rather than sharply delineated.

Plants provide necessary carbon for the largely heterotrophic soil microbiota, and these microorganisms help the plants in several ways too:

• Microbes mediate plant growth by production of plant growth hormones.
• Microbes provide nutrients through mechanisms like nitrogen fixation or phosphorus solubilization.
• Microbes protect the plant from stress or drought conditions.

Through a University of Minnesota plant pathology program, potatos were passaged in a field for over 2 decades to study potato diseases. Over time, researchers found fewer diseases in test crops, which led the plot to be abandoned in the late 1970s. In the 1980s, Dr. Neil Anderson planted potatoes to see if they would develop disease, but neither Verticillium wilt nor potato scab developed among the plants. Soil from the field (and on the potatoes) contained Streptomyces isolates that showed antimicrobial activity against bacteria, fungi, nematodes, and oomycetes. This discovery led Neil, new University of Minnesota professor Linda, and their collaborators to study the antimicrobial activity of natural Streptomyces isolates from around the world.

Inoculation quickly adds specific microbial lineages to soil microbiome communities. Alternatively, land can be managed by providing nutrients to encourage the growth of specific species, like Streptomyces, within a given plot, but this takes longer to develop. How are soil microbiomes inoculated? Microbes can be:

• Added to the seed coating before planting.
• Placed in the furrow when the seed is planted.
• Distributed into the irrigation system.

Links for this Episode:

• Linda Kinkel website at University of Minnesota
• Essarioui A. et al. Inhibitory and Nutrient Use Phenotypes Among Coexisting Fusarium and Streptomyces Populations Suggest Local Coevolutionary Interactions in Soil. Environmental Microbiology. 2020.
• Schlatter D.C. et al. Inhibitory Interaction Networks Among Coevolved Streptomyces Populations from Prairie Soils. PLoS One. 2019.
• Schlatter D.C. et al. Resource Use of Soilborne Streptomyces Varies with Location, Phylogeny, and Nitrogen Amendment. Microbial Ecology. 2013.
• Small Things Considered blog: Are Oomycetes Fungi or What?
• International Year of Plant Health
• HOM Tidbit: Austin-Bourke P.M. Emergence of Potato Blight, 1843-1846. Nature. 1965.