Meet the Microbiologist

Pathogenic E. coli are different than lab-grown or commensal E. coli found in the gut microbiome. Alfredo Torres describes the difference between these, the method his lab is using the develop vaccines against pathogenic E. coli, and how this same method can be used to develop vaccines against Burkholderia infections.

Julie's Biggest Takeaways:

1. coli plays many roles inside and outside the scientific laboratory:

• Laboratory E. coli strains used by scientists to study molecular biology.
• Commensal E. coli strains contribute to digestion and health as part of the intestinal microbiome.
• Pathogenic E. coli strains have acquired factors that allow them to cause disease in people

The pathogenic E. coli associated with diarrheal disease are the ones named for their O-antigen and flagellar H-antigen, such as O157:H7. There are about 30 E. coli strains with various combinations of O-H factors known to cause diarrheal disease in people.

The E. coli Shiga toxin (though not the bacterium itself) can pass through the epithelial cell layer to become systemic, and eventually the toxin will accumulate in the kidneys. This can lead to patients experiencing hemolytic uremic syndrome (HUS) and kidney failure, leading to lifelong dialysis or need for a transplant. An immune response that prevents the E. coli from attaching will prevent the bacterium from secreting toxin in close proximity to the epithelial cells and decrease likelihood of HUS development.

Burkholderia is a bacterial genus whose member species have been weaponized in the past, and which remain potent disease-causing agents around the world.

• B. mallei causes glanders, a disease mostly of horses and their handlers. It is a respiratory infection that can become systemic if not treated.
• B. pseudomallei causes melioidosis, a disease that can manifest in many ways. It is endemic in many tropical regions around the world, found in over 79 countries so far.

Coating gold nanoparticles with antigens against which the immune response will be protective is a method Alfredo has used for a number of candidate vaccines, including one against E. coli and one against B. pseudomallei. The nanoparticles can have the gold cleaved off to provide different functional variants of the same vaccine.

Links for this Episode:

• Alfredo Torres webpage at University of Texas Medical Branch
• McWilliams BD and Torres AG. Enterohemorrhagic Escherichia coli Adhesins. Microbiology Spectrum. 2013.
• Sanchez-Villamil JI et al. Development of a Gold Nanoparticle Vaccine against Enterohemorrhagic Escherichia coli O157:H7. mBio. 2019.
• Wiersinga WJ et al. Melioidosis. Nature Reviews Disease Primers. 2018.
• Khakhum N. et al. Evaluation of Burkholderia mallei ΔtonB Δhcp1 (CLH001) as a live attenuated vaccine in murine models of glanders and melioidosis. PLOS Neglected Tropical Diseases. 2019.
• Torres AG. Common Sense Can Keep You Safe in E. coli Outbreak. Galveston County Daily News. 2020.
• ABRCMS: Annual Biomedical Research Conference for Minority Students
• MTM: Burkholderia pseudomallei & the neglected tropical disease melioidosis with Direk Limmathurotsakul
• HOM Tidbit: Kiyoshi Shiga Biography in Clinical Infectious Diseases

Does the fetus have a microbiome? How does the placenta prevent infection? Carolyn Coyne talks about placental structure and biology, and why studying the maternal-fetal interface remains a critical area of research.

Julie's Biggest Takeaways:

The placenta forms within 3-5 days post conception as a single layer of cells surrounding the fertilized embryo. These cells differentiate and develop into more complex structures.

Very few microbes cause fetal disease. Of those that do, the disease-causing microorganisms are diverse and can lead to serious congenital defects or even death of a developing fetus. These microbes are largely grouped into the TORCH (now TORCH-Z) microorganisms:

• Toxoplasma gondii
• Other (a variety of different bacteria and viruses)
• Rubella
• Cytomegalovirus
• Herpesviruses
• Zika virus

The fetus is immunologically immature and unable to protect itself. Some of the maternal immunological molecules (such as maternal antibodies) cross the placenta to protect the fetus, but that only happens during later stages of fetal development. Between the first and second trimesters, the maternal vasculature reorganizes and maternal antibodies can begin to reach the fetus. This increases over time, until the end of the third trimester, when there is a higher concentration of maternal antibodies in fetal blood than in maternal blood.

In the later stages of development, the placenta is coated in a layer of fused cells, leading to a shared cytoplasm that covers the entire surface area of the placenta. This fused-cell layer is formed from syncytiotrophoblasts, and the fusion is facilitated by the activity of an endogenous retrovirus fusion protein.

Syncytiotrophoblasts are extremely resistant to infection with a number of different pathogens, and pathogen types. In initial tests experiments, Carolyn and her research team discovered that these cells releasing certain antimicrobial molecules to share protective properties. Syncytiotrophoblasts secrete type III interferons, which play a big role at barrier surfaces such as the airway and the gut—but unlike these barriers, the syncytiotrophoblast cells secrete type III interferons constitutively.

Links for this Episode:

• Carolyn Coyne Website on the University of Pittsburgh School of Medicine
• Arora N. et al. Microbial Vertical Transmission during Human Pregnancy. Cell Host & Microbe. May 2017.
• Coyne C.B. The Tree(s) of Life: The Human Placenta and My Journey to Learn More About It. PLoS Pathogens. April 2016.
• Ander S.E. et al. Human Placental Syncytiotrophoblasts Restrict Toxoplasma gondii Attachment and Replication and Respond to Infection by Producing Immunomodulatory Chemokines. mBio. January 2018.
• Wells A.I. and Coyne C.B. Type III Interferons in Antiviral Defenses at Barrier Surfaces. Trends in Immunology. October 2018.
• Ander S.E. Diamond M.S. and Coyne C.B. Immune Responses at the Materna-Fetal Interface. Science Immunology. January 2019.
• HOM Tidbit: Women in Microbiology
• HOM Tidbit: Small Things Considered blog post: Retroviruses, the Placenta, and the Genomic Junk Drawer

Are there drugs that can treat coronaviruses? Timothy Sheahan talks about his drug discovery work on a compound that can inhibit all coronaviruses tested so far, and tells how his career path took him to pharmaceutical antiviral research and then back to academia.

Julie's Biggest Takeaways:

Even though the MERS-CoV was discovered as a human pathogen in 2012, it was likely percolating as a disease agent for a long time before that. Banked camel serum provides evidence that the virus had been circulating in camels for several decades prior.

Differentiated ex vivo lung cultures allow study of virus infection in a 3D model representation for studying viral infection, including target cell types of both MERS-CoV and SARS-CoV.

• SARS-CoV prefers ciliated epithelial cells Ace2
• MERS-CoV prefers nonciliated epithelial cells DPP4

Coronavirus disease in people takes place over a course of about 2 weeks. In mice, the disease is similar, but progression is faster, taking about 1 week.

The drug remdesivir (RDV) is a nucleoside analog that inhibits the coronavirus RNA-dependent RNA polymerase (RDRP). Remdesivir activity has not been tested against nCoV2019, but similarity to other viruses is promising. Bioinformatic approaches show that the nCoV2019 RDRP is 99% similar and 96% identical to SARS-CoV RDRP. Remdesivir works against every coronavirus tested so far, including viruses with highly divergent RDRP sequences, so remdesivir is likely to be effective again nCoV2019. Experiments must still be performed before reaching this conclusion, of course.

Tim also hopes to discover the genetic determinants that will allow a chronic hepatitis C virus (HCV) infection in mice, but not standard inbred mice. He uses outbred mice meant to mimic the diversity of the human population, and strengthen the results. Understanding these determinants would inform human studies to better understand chronic HCV infection.

Links for this Episode:

• MTM Listener Survey, only takes 3 minutes. Thanks!

• TWiV 584: Year of the Coronavirus

• Timothy Sheahan website at University of North Carolina
• Sheahan T.P. et al. Broad-Spectrum Antiviral GS-5734 Inhibits both Epidemic and Zoonotic Coronaviruses. Science Tranlational Medicine. 2017.
• Sheahan T.P. et al. Comparative Therapeutic Efficacy of Remdesivir and Combination Lopinavir, Ritonavir, and Interferon Beta against MERS-CoV. Nature Communications. 2020.
• Agostini M.L. et al. Coronavirus Susceptibility to the Antiviral Remdesivir (GS-5734) is Mediated by the Viral Polymerase and the Proofreading Exoribonuclease. mBio. 2018.
• ASM Coronavirus Resource Page
• HOM Tidbit: Baltimore D. In Vitro Synthesis of Viral RNA by the Poliovirus RNA Polymerase. PNAS. 1964.

Viral gastroenteritis around the world causes 200,000 deaths globally each year. Mary Estes talks about her work on 2 gastroenteritis-causing viruses, rotavirus and norovirus, and tells the story of her discovery of the first viral enterotoxin. She also describes how noroviruses have changed from human volunteer studies to studies using "miniguts," a system now used with many enteropathogenic microorganisms.

Julie's Biggest Takeaways:

Rotaviruses and noroviruses kill 200,000 people annually, despite an available rotavirus vaccine and current anti-infective measures. Rotavirus is generally associated with gastrointestinal disease in the very young and the very old, while norovirus infects people at all life stages.

Rotavirus is so stable that even when viral samples are extremely dessicated by lyophilization, the samples remain perfectly infectious. Rotavirus stability is largely due to 3 concentric capsid cells.

NSP4 is a rotavirus enterotoxin, and the first viral enterotoxin to be discovered. It affects the concentration of the intracellular calcium pools. By activating the calcium chloride channel, NSP4 forces chloride and water to be excreted, directly leading to diarrhea. NSP4 is secreted from infected cells and can also disrupt calcium concentrations of neighboring cells, amplifying the effect of a single infected cell.

Rotarix® and RotaTeq® are 2 different attenuated rotavirus vaccines. One contains a single attenuated viral strain while the other contains 5 attenuated viral strains; both vaccines have high efficacy in developed countries and slightly lower efficacy in developing countries. Why vaccine efficacy is lower in developing countries is uncertain, with many hypotheses including microbiome-based effects under study now.

Human enteroids, or "miniguts," offer insight into complex virus-cell interactions. These stem-cell derived miniguts can be generated from different types of animal stem cells, and the enteroids they become reflect the same host-barrier restriction as the animal of origin. The miniguts can be used to culture many sorts of viruses and other microorganisms, such as bacteria and protozoa.

Links for this Episode:

• Mary Estes Website at Baylor College of Medicine
• Hyser J.M. et al. Rotavirus Disrupts Calcium Homeostasis by NSP4 Viroporin Activity. mBio. 2010.
• Crawford S.E. et al. COPII Vesicle Transport is Required for Rotavirus NSP4 Interaction with the Autophagy Protein LC3 II and Trafficking to Viroplasms. J Virology. 2019.
• Ettayebi K. et al. Replication of Human Noroviruses in Stem Cell-Derived Human Enteroids. Science. 2016.
• In J.G. et al. Human Mini-Guts: New Insignts into Intestinal Physiology and Host-Pathogen Interactions. Nat Rev Gastroenterol Hepatol. 2016.
• Finkbeiner S.R. et al. Stem Cell-Derived Human Intestinal Organoids as an Infection Model for Rotaviruses. mBio. 2012.
• Henning S.J. and Estes M.K. Women in Science: Hints for Success. Gastroenterology. 2015.
• Kapikian A.Z. et al. Visualization of a 27-nm Particle Associated with Acute Infectious Nonbacterial Gastroenteritis. Journal of Virology. 1972.
• HOM Tidbit: Smith K.N. The Iron Long was just an Engineer's Side Project. Forbes. 2019.
• HOM Tidbit: Ramirez M. Living Inside a Canister: Dallas Polio Survivor is One of Few People Left in U.S. Using Iron Lung. Dallas Morning Star. 2018.

The most abundant organism on Earth lives in its seas: the marine bacterium SAR11. Steve Giovannoni describes how the origins of SAR11 provided its name, and the ways that studying SAR11 have taught scientists about ocean ecology. He also discusses how the different depths of the ocean vary in their microbial compositions and what his big questions are in marine microbiology.

Different depths of the ocean have different habitats, but the microbes vary continuously, based in part on light availability:

• Surface light facilitates photosynthesis by algal cells. These primary producers fix carbon for the entire ecosystem! Because nutrients are readily available, the cell concentration in surface waters can reach nearly 1,000,000 cells/ml.
• The twilight zone offers dim light. Microbes in this area mainly use carbon sources generated by the surface-dwelling microbes. Below a few hundred meters, cell concentrations drop to 10,000-100,000 cells/ml.
• The deep ocean has no light and the microbes that live here have significantly different biochemistries and metabolisms.

SAR11 is small in both physical size and genome size (0.37–0.89 µm and 1.3 million base pairs, respectively). It is nevertheless the most abundant organism on the planet, with more than 1028 cells estimated to exist worldwide. These cells convert between 6-37% of the carbon fixed in the oceans daily. SAR11 in different niches have ecotypes with different specialties but look physically similar and have very similar genome sequences. Naturally, the most abundant cells in the ocean have the most abundant parasites: bacteriophages called pelagiphages infect SAR11 all over the world. SAR11 and pelagiphages are under constant evolution, though there doesn't seem to be a CRISPR system in the Pelagibacter genome; these bacteria largely use other mechanisms to evade phage infection. SAR11 is like a house with the lights on all the time, in that the cells constitutively express most metabolic genes. For example, SAR11 metabolizes dimethylsulfoniopropionate (DMSP) into dimethyl sulfide (DMS) and methanethiol (MeSH), which can be produced as soon as the cells are exposed to DMSP. While this may seem energetically expensive, the cells must capitalize on their encounters with this transient resource, often found only at low concentrations, and this capitalization requires the investment of protein production. The cost of metabolic gene regulation outweighs the benefits in this particular case. SAR11 and SAR202 are the poles on the spectrum of heterotrophic marine bacteria. SAR11 is very efficient at accessing and using the organic compounds that come from the phytoplankton (also called the labile organic matter). SAR202, found in the deeper part of the ocean, specializes in hard-to-access carbon compounds that other bacteria can't access.

• MTM Listener Survey, only takes 3 minutes. Thanks!
• Stephen Giovannoni website at Oregon State University
• OSU High Throughput Microbial Cultivation Lab
• Carini P. et al. Discovery of SAR11 Growth Requirement for Thiamin's Pyrimidine Precursor and its Distribution in the Sargasso Sea. ISME J. 2014.
• Sun J. et al. The Abundant Marine Bacterium Pelagibacter Simultaneously Catabolizes Dimethylsulfoniopropionate to the Gases Dimethyl Sulfide and Methanethiol. Nature Microbiology. 2016.
• Moore E.R. et al. Pelagibacter Metabolism of Diatom-Derived Volatile Organic Compounds Imposes an Energetic Tax on Photosynthetic Carbon Fixation. Environmental Microbiology. 2019.
• HOM Tidbit: Sagan L. On the Origin of Mitosing Cells. 1967.
• HOM Tidbit: Cellmates (Radiolab podcast episode)
• ASM Article: The Origin of Eukaryotes: Where Science and Pop Culture Collide

Can a protein be contagious? Jason Bartz discusses his work on prion proteins, which cause spongiform encephalopathy and can be transmitted by ingestion or inhalation among some animals. He further discusses how prions can exist as different strains, and what techniques may help improve diagnosis of subclinical infections.

Links for this Episode:

• Jason Bartz Creighton University website
• Holec SAM, Yuan Q, and Bartz JC. Alteration of Prion Strain Emergence by Nonhost Factors. mSphere. 2019.
• Yuan Q et al. Dehydration of Prions on Environmentally Relevant Surfaces Protects Them from Inactivation by Freezing and Thawing. Journal of Virology. 2018.
• Bartz JC. Prion Strain Diversity. Cold Spring Harbor Perspectives in Medicine. 2016.
• Bartz JC. From Slow Viruses to Prions PLoS Pathogens. 2016.
• Deleault NR, Harris BT, Rees JR, Supattapone S. Formation of native prions from minimal components in vitro. Proceedings of the National Academy of Sciences. 2007.
• Planet Money Episode 952: Sperm Banks

Microbial interactions drive microbial evolution, and in a polymicrobial infection, these interactions can determine patient outcome. Deb Hogan talks about her research on interkingdom interactions between the bacterium Pseudomonas and the fungus Candida, 2 organisms that can cause serious illness in cystic fibrosis patients' lung infections. Her research aims to better characterize these interactions and to develop better diagnostic tools for assessing disease progression and treatment.

Links for this Episode:

• Deb Hogan Lab Website
• Demers EG et al. Evolution of Drug Resistance in an Antifungal-Naive Chronic Candida lusitaniae Infection. PNAS. 2018.
• Lewis KA et al. Ethanol Decreases Pseudomonas aeruginosa Flagella Motility through the Regulation of Flagellar Stators. Journal of Bacteriology. 2019.
• Gifford AH et al. Use of a Multiplex Transcript Method for Analysis of Pseudomonas aeruginosa Gene Expression Profiles in the Cystic Fibrosis Lung. Infection and Immunity. 2016.
• Grahl N et al. Profiling of Bacterial and Fungal Microbial Communities in Cystic Fibrosis Sputum Using RNA. mSphere. 2018.
• Microbiology Resource of the Month: The Aeminium ludgeri Genome Sequence
• HOM Tidbit: https://www.sciencedirect.com/science/article/pii/S0065216408705628
• HOM Tidbit: The Frozen Potential of Microbial Collections

Why is C. diff such a serious disease and what are clinical microbiologists doing to improve patient outcomes with better diagnostic tools?

Many hospital-acquired bacterial infections are also drug-resistant. Amy Mathers describes her work tracking these bacteria to their reservoir in hospital sinks, and what tools allowed her team to make these discoveries. Mathers also discusses her work on Klebsiella, a bacterial pathogen for the modern era.

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Nosocomial infections are a type of opportunistic infection: one that wouldn't normally cause disease in healthy individuals. Once the immune system is compromised due to other infection or treatment, the opportunist bacteria take advantage of the conditions to grow to higher numbers and cause disease.

How are different pathogens transmitted in the hospital? Previously, transmission was considered to occur from one patient to a second patient, perhaps via a healthcare worker. When patients from very different parts of the hospital began to come down with the same resistant strain of bacteria, without interacting through the same space or staff, researchers began to look at a different reservoir: the hospital wastewater.

How does the bacteria get from the sink to the patients? The bacteria, existing in a biofilm in the pipe right below the drain, can be transferred in droplets when the water is run. These droplets can fall as far as 36 inches from the drain plate and can contaminate the sink bowl or patient care items next to the sink.

Some of the solutions to decrease bacterial dispersion from hospital sinks are very simple: for example, offsetting the drain from the tap, which keeps the water from directly running onto the drain, helps decrease the force with which the water hits the drain and therefore decreases bacterial dispersion.

The Sink Lab at University of Virginia couldn't replicate the bacterial growth patterns seen in the rest of the building; in particular, there were fewer protein nutrients that promoted bacterial growth. By setting up a camera observation of sink stations used in the hospital, the team realized that the waste thrown down the sink (extra soda, milk, soup, etc) was feeding the microbial biofilm. This helps the CRE in the biofilms in the sinks thrive.

• MTM Listener Survey, only takes 3 minutes. Thanks!
• Amy Mathers website at University of Virginia
• The Sink Lab at UVA
• Kotay SM et al. Droplet- Rather than Aerosol-Mediated Dispersion is the Primary Mechanism of Bacterial Transmission from Contaminated Hand-Washing Sink Traps. Applied and Environmental Microbiology. 2018.
• Mather AJ et al. Klebsiella quasipneumoniae Provides a Windo into Carbapenemase Gene Transfer, Plasmid Rearrangements, and Patient Interactions within the Hospital Environment. Antimicrobial Agents and Chemotherapy. 2018.
• Kotay S et al. Spread from the Sink to the Patient: in situ Study Using Green Fluorescent Protein (GFP)-Expressing Escherichia coli to Model Bacteral Dispersion from Hand-Washuing Sink-Trap Reservoirs. Applied and Environmental Microbiology. 2016.

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How do medical professionals incorporate microbiome science into their patient care? Ami Bhatt discusses her research on the diversity within and between human gut microbiomes, and how this research is slowly and carefully being used to build new patient care recommendations.

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Although these terms are often used interchangeably, microbiome and microbiota represent distinct samples types:

• Microbiotarepresents all the organisms that live within a community: archaea, bacteria, viruses, and fungi.
• Microbiomeis the genomes or transcriptomes of these organisms.

The gut microbiota may often be referred to as a single entity, but the gastrointestinal tract has many different niches. Alterations in pH, cell type, and the available nutrients provide different selective pressures for the microorganisms that reside in these conditions.

By clustering small proteins based on similarity, Ami's group was able to identify over 4000 new families of small proteins from existing microbiome datasets. Some of these were found among all microbiome datasets while others were found only in human microbiomes, which provides a clue to their potential housekeeping versus host-microbe-interaction functionality, although the exact functions are still unknown.

Outcomes for non-infectious diseases are affected by the gut microbiome. Ami and her colleagues have worked with transplant patients to understand what type of diversity and which strains play a role in best outcome for cancer therapy patients, such as patients receiving bone marrow transplants. Medical doctors are beginning to incorporate new patient care in light of new microbiome studies.

Understanding the effects of the gut microbiome on human health have helped slowly change patient care in some settings. For example, doctors are reconsidering recommendations for immunocompromised people to stay away from fresh fruits and vegetables, a recommendation previously made due to the potential risk of patients exposure to pathogenic microbes. The benefit of a wide variety of fiber sources, which promote a diverse and robust microbiome, may turn out to outweigh this risk.

• MTM Listener Survey, only takes 3 minutes. Thanks!
• Ami Bhatt lab website
• Brewster R. et al. Surveying Gut Microbiome Research in Africans: Toward Improved Diversity and Representation. Trends in Microbiology. Oct 1 2019.
• Sberro H. et al. Large-Scale Analyses of Human Microbiomes Reveal Thousands of Small, Novel Genes. Cell August 22 2019.
• Andermann T. et al. The Microbiome and hematopoietic Cell Transplantation: Past, Present, and Future. Biol Blood Marrow Transplant. July 1 2019.
• Bloomberg: Superbugs Deadlier Than Cancer Put Chemotherapy into Question
• Clinical Guide to Probiotic Products Available in USA
• HOM Tidbit: Rous P. A Sarcoma of the Fowl Transmissible by an Agent Separable from the Tumor Cells. Journal of Experimental Medicine. April 1 1911.
• ASM Article: A Brief History of Cancer Virology