Meet the Microbiologist

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. Links for This Episode 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. Subscribe (free) on Apple Podcasts, Google Podcasts, Android, RSS, or by email. Julie’s Biggest Takeaways 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. Links for This Episode 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. Let us know what you thought about this episode by tweeting at us @ASMicrobiology or leaving a comment on facebook.com/asmfan. Send your stories about our guests and/or your comments to jwolf@asmusa.org.  

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. Subscribe (free) on Apple Podcasts, Google Podcasts, Android, RSS, or by email. Julie’s Biggest Takeaways 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. Links for This Episode 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

Identified in the 1980s, Borrelia burgdorferi and other Lyme disease-associated spirochetes have since been found throughout the world. Jorge Benach answers questions about Lyme Disease symptoms, his role in identifying the causative bacterium, and his current research on multispecies pathogens carried by hard-bodied ticks. Julie’s Biggest Takeaways Erythema migrans (the classic bullseye rash) is the most common manifestation that drives people to go see the doctor to be diagnosed with Lyme disease, but only about 40% of people diagnosed with Lyme disease experience erythema migrans. Lyme disease can progress to serious secondary manifestations. Why some patients experience these additional disease manifestations, but others do not,  is one of the heaviest areas of study in Lyme disease. Though Borreliadoesn’t have virulence factors that mediate tissue damage, it does avoid the immune system via antigenic variation. When the bacterium is first introduced into a new human host, that person’s immune system generates reactions to the outer membrane components. These bacterial components change over time, leaving the immune response lagging behind and unable to clear the infection. Ixodesticks are the vector for Lyme disease and there are 3 stages in the Ixodestick life: Larvae: the stage during which the tick is most likely to become infected by feeding on a rodent. Nymph: the stage most likely to infect a person (due to their small size, they are less likely to draw attention while feeding). Adult: the stage when the tick develops into a sexual adult; females are most likely to be infected but because female ticks are large, most people will detect and pull out a feeding adult. Ticks feed for 2-4 days; removing a tick in the first 48 hours of attachment decreases the chance for transmission to the patient. Long Island is seeing anecdotal increases of Ambliomaticks (the Lone Star tick), which can transmit the human pathogen Ehrlichia. These anecdotal increases were one of the motivations behind a recently published survey of ticks and the human pathogens they carry. Links for This Episode MTM Listener Survey, it only takes 3 minutes. Thanks! Jorge Benach website at Renaissance School of Medicine Stony Brook University Sanchez-Vicente S. et al. Polymicrobial Nature of Tick-Borne Diseases. mBio. September 10 2019. Monzón J.D. et al. Populaiton and Evolutionary Genomics of Amblyomma americanum, and Expanding Arthropod Disease Vector. Genome Biol Evol. May 2016. ASM Article: The Bulls-Eye Rash of Lyme Disease: Investigating the Cutaneous Host-Pathogen Dynamics of Erythema Migrans Patient Zero podcast HOM Tidbit: Barbour A.G. and Benach J.L. Discovery of the Lyme Disease Agent. mBio. September 17 2019.  

Influenza is famous for its ability to mutate and evolve but are mutations always the virus’ friend? Jesse Bloom discusses his work on influenza escape from serum through mutation and how mutations affect influenza virus function and transmission. Subscribe (free) on Apple Podcasts, Google Podcasts, Android, RSS, or by email. Also available on the ASM Podcast Network app. Julie’s Biggest Takeaways Influenza is famous for its ability to mutate and evolve through two major mechanisms: Antigenic drift occurs when a few mutations accumulate in the influenza genome and lead to seasonal changes. Antigenic shift occurs when two influenza strains recombine their genomes to form one previously unknown in human populations. Avian influenza has caused thousands of zoonotic cases, in which the virus is transmitted from birds to people. This causes serious disease but the virus doesn’t easily pass from person-to-person, limiting how many people are affected. When a zoonotic case becomes easily transmissible between people, as is suspected occurred in the 1918 influenza pandemic, the outcome can be very serious for many, many people. During antigenic drift, the virus accumulates mutations randomly throughout its genome. Mutations in the hemagglutinin (HA) glycoprotein gene are the mutations most likely to affect the ability of antibodies to attach and block HA during viral infection of a new host cell. The circulating human H3N2 influenza A virus accumulates approximately 3-4 mutations annually within its HA gene, representing a 0.5-1% change. On average, it takes 5-7 years of these mutations accumulating until a viral strain can reinfect a previously infected person. The changes in the influenza sequence are responsible for waning immunity against the annually circulating strain. This was demonstrated when a flu strain from the 1950s was inadvertently reintroduced in the 1970s; older people who had previously been infected were protected against this exact same strain. Influenza viruses can escape from sera, which contains many different antibodies, similar to how they can escape from a single monoclonal antibody: through mutations in major antibody binding sites. However, the mutations that allow escape from one person’s serum are different from the mutations that allow escape from another person’s serum. This means the strains that escape one person’s immune system may only be able to infect those with similar immunity.   Links for This Episode MTM Listener Survey, only takes 3 minutes. Thanks! Jesse Bloom’s lab website Guns Germs and Steel by Jared Diamond Lee J.M. et al. Mapping Person-to-Person Variation in Viral Mutations that Escape Polyclonal Serum Targeting Influenza Hemagglutinin.eLife. August 2019. Xue K.S. et al. Cooperating H3N2 Influenza Virus Variants are not Detectable in Primary Clinical Samples.mSphere. January 2018. Francis Arnold at ASM Microbe:Innovation by Evolution: Bringing New Chemistry to Life Let us know what you thought about this episode by tweeting at us @ASMicrobiology or leaving a comment on facebook.com/asmfan.