Meet the Microbiologist

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.

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.

• 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.

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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.

• 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.

Graciela Lorca studies genetic systems to find positive and negative microbial interactions that lead to disease. She talks about her discovery of chemical inhibitors for the citrus greening disease bacterium, Liberibacter asiaticus,and how a specific strain of Lactobacillus johnsoniimodulates the immune system and may help prevent development of diabetes in people.

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Citrus greening disease, or huanglongbing, is a disease of citrus trees causing a major epidemic among citrus farmers around the world. The disease causes trees to sicken and eventually die, and is best diagnosed by PCR amplification of the bacterial DNA from the bacterium that causes the disease, Liberibacter asiaticus. Because the disease spreads through the tree at different rates, it's important that many samples be tested for accurate diagnosis.

Quarantining the disease has proved difficult, as undiagnosed roots can transmit the disease if they are used to hybridize with canopy plants. The disease becomes even harder to contain under bad weather conditions: the high winds of recent hurricanes can scatter the insect vector, the Asian citrus psyllid, leading to infection of new orchards.

Although L. asiaticuscan't be cultured, Graciela performed a screen on L. asiaticustranscription factors that were produced by E. coli. These were tested for inhibition by a chemical library, and discovered that a common treatment for gout, benzbromarone, inhibited protein activity. This discovery was confirmed using in vivoinfected plants and by expressing the gene in related bacterial species, Graciela and her team predict the protein plays a role in responding to osmotic stress. The protein target of the chemical differs widely between citrus greening disease and gout, but the protein-chemical interaction is similar enough to allow protein inhibition.

Is there a link between the microbiome and diabetes? 10 years ago, Lactobacillus johnsoniican rescue animals that are predisposed to diabetes. L. johnsoniiinactivates a host enzyme, IDO, which regulates proinflammatory responses. Activated immune cells can travel to the pancreas and attack beta cells, leading to diabetes. Regulating the proinflammatory response by administering L. johnsoniias probiotics offers the opportunity to control development of diabetes in predisposed people.

• MTM Listener Survey, only takes 3 minutes. Thanks!
• Graciela Lorca's lab website
• Pagliai F.A. et al. The Transcriptional Activator LdtR from 'Candidatus Liberibacter asiaticus' Mediates Osmotic Stress Tolerance. PLoS Pathogens. April 2014.
• Lai K.K., Lorca G.L. and Gonzalez C.F. Biochemical Properties of Two Cinnamoyl Esterases Purified from a Lactobacillus johnsonii Strain Isolated from Stool Samples of Diabetes-Resistant Rats. Applied and Environmental Microbiology. August 2009.
• Marcial G.E. et al. Lactobacillus johnsonii N6.2 Modulates the Host Immune Response: A Double-Blind, Randomized Trial in Healthy Adults. Frontiers in Immunology. June 2017.
• HOM Tidbit: Hartmann A., Rothballer M., and Schmid M. Lorenz Hiltner, a Pioneer in Rhisophere Microbial Ecology and Soil Bacteriology Research. Plant and Soil November 2008.

When a new biothreat or emerging infectious agent threatens, how are diagnostic protocols put into place? It's up to the Laboratory Response Network (LRN), a multipartner network of public health, clinical and other labs, to generate and distribute reagents, and provide training to detect these threats. Julie Villanueva, Chief of the Laboratory Preparedness and Response Branch at the CDC, talks about the LRN and how no two weeks on the job are alike.

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In the mid-1990s, the CDC joined public health representatives along with the Departments of Defense and Justice to determine the best way to prepare and respond to potential bioterrorism threats. The result was the Laboratory Response Network (LRN), founded in 1999.

The LRN provides infrastructure to detect potential pathogens. Though first put into place to detect and prevent bioterror events, the LRN has also been able to detect infectious diseases that have emerged through other means.

When a new disease emerges, there are typically no widely available tests to diagnose the disease. The CDC works hard to quickly develop diagnostic tests, validate the tests, manufacture the necessary reagents, and ship these out to the reference labs that are part of the LRN. This ensures that each lab can accurately reach the same result with the same sample.

The laboratory response network requires more than just developing and deploying diagnostic tests. The LRN must also provide

• Training for LRN scientists.
• Proficiency testing to test the network.
• Reporting protocols for sending results.

What diseases keep Julie up at night? A viral hemorrhagic fever is one, and microorganisms that evolve quickly and have high pathogenic potential, like influenza virus, is another.

"Our collaborations across other federal agencies like the FDA and the USDA are really important for us to stay on the cutting edge of what could be emerging."

"Partnerships are so critical when managing an outbreak. There's never an outbreak that only affects one group of people...there are lots of different facets of an outbreak that need to be addressed and partnerships are critical for managing and trying to mitigate as much as possible."

"The LRN primarily focuses on diagnostics, this is what the network really does. It's made to be able to detect biothreats and emerging infectious diseases in both clinical and environmental samples."

"We're always looking at new technologies for faster, more sensitive, and more specific tests."

"Every outbreak has been different in a different way, and I've learned something every time. I think that each outbreak has taught us a few things that work well within the network and a few things with which we can improve, and continued improvement is very important to us. For example, the Ebola outbreak in 2014-16 really highlighted the need for biosafety and biosecurity procedures all across not only the network but also our hospitals...we learn something different from every outbreak."

• MTM Listener Survey, only takes 3 minutes. Thanks!
• The Laboratory Response Network(CDC website)

HOM: The Origin of In Situ Hybridization- a Personal History

George F. Gao discusses how China CDC promotes global public health during outbreaks SARS and Ebola. He also talks about running a structural biology lab, the importance of both basic and translational research, and the most important discovery of the 20th century.

Julie's Biggest Takeaways:

China CDC was founded in 2001. Its experience with the SARS outbreak informed its response to the western Africa Ebola outbreak in 2014-2016, having learned that viruses don't care about national borders and can quickly become an international problem. Responding to any major outbreak serves both altruistic and selfish motives, since quelling the outbreak decreases the chance that the disease will continue to circulate, potentially reaching your country.

Basic research is fundamental for many translational applications to improve human health. By measuring the mutation rate, for example, of a circulating virus, scientists can determine if previous isolates can be used to generate vaccines. The basic research that led to new nucleic acid sequencing techniques has many important applications!

When asking other scientists what the most important discovery of the 20th century is, many biomedical scientists name the discovery of the double helix. George points out that bird migration patterns have influenced our understanding of avian diseases like the flu. This discovery led scientists to understand more about the annual transmission patterns of flu, highlighting the importance of interdisciplinary research.

George has a foot in both basic and translational sciences and is an ardent supporter of both. The difficulty is in identifying basic research that has potential for application and providing opportunities to basic researchers to create companies and products based on their research. Another hurdles is collaborating and coordinating to ensure people talk to each other

George lists the 4 Cs required to promote science, public health and societal development:

• Collaboration
• Cooperation
• Communication
• Competition

Links for this Episode:

• George F. Gao Lab Website
• Gao GF and Feng Y. On the Ground in Sierra Leone. Science 2014.
• Carroll D et al. The Global Virome Project. Science 2018.
• Watts G. George F. Gao: Head of China CDC Signals a More Global Outlook. Lancet 2018.
• Forging the Path for Polio Vaccination: Isabel Morgan and Dorothy Horstmann

Bacteriophage are viruses that infect specific bacteria. Jeremy Barr discusses his discovery that phage interact with (but don't infect) mammalian epithelial cells. He explains how these different organisms: bacteria, bacteriophage, and the mammalian host, may exist in three-way symbioses.

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Jeremy's work as a postdoc focused on developing a protocol to clean phages for use in tissue culture. He and his advisor, Forest Rohwer, were asked to use this protocol to clean phages for a patient extremely sick with a multidrug-resistant Acinetobacter baumannii isolate. Within 24 hours, they used an experimental lab method to clean and purify phages that were used in an experimental procedure to treat a very sick person; phage therapy ultimately saved his life.

Jeremy discovered that phages can pass through human epithelial cells by using a transwell system. Phage interaction with epithelial cells is not the same as an infection, since the phages cannot use mammalian molecular machinery to reproduce. Jeremy hypothesizes that the epithelial cells take up phage during active sampling from the gut, during which epithelial cells sample the environment to inform the immune system.

Jeremy's work is building toward a model of tripartite symbioses. This includes symbiosis between bacteria and mammalian cells, between bacteria and bacteriophage, and between bacteriophage and mammalian cells. Bacteria can interact with mammalian cells to influence host cell signaling to their benefit, and Jeremy's hypothesis is that phage will be found to do the same.

Building a gut-on-a-chip allowed Jeremy to study the interactions of phage with the gut in a controlled environment. The preliminary results suggest that the phage adapt to better adhere to the mucosal surfaces over time. Discovering the protein domains that phage use to stick to mucins opens up the possibility of using these domains in personalized therapeutics, by designing these into new phage or other therapeutics.

Jeremy's 2 major pieces of advice for early career scientists:

1. Follow what excites you! Find an aspect of biology that you're really passionate about and follow that.
2. Find amazing mentors. Contact even people you don't directly work with, reach out to them and build your network.

• MTM Listener Survey, only takes 3 minutes. Thanks!
• Jeremy Barr lab website
• The Perfect Predator by Steffanie Strathdee
• Gordillo Altamirano FL and Barr JJ. Phage Therapy in the Postantibiotic Era. Clin Microbiol Rev 2019.
• Nguyen S. et al. Bacteriophage Transcytosis Provides a Mechanism to Cross Epithelial Cell Layers. mBio 2017.
• Microbe information

Stanley Maloy discusses his career in Salmonella research, which started with developing molecular tools and is now focused on the role of Salmonella genome plasticity in niche development. He further talks about his role in science entrepreneurship, science education, and working with an international research community.

Julie's Biggest Takeaways:

Stanley's career began when transposon mutagenesis was a new, cutting-edge technique, and he found the best way to learn how to apply a new method was to jump in and try it.

Antibiotic resistance has been a problem throughout Stanley's career. The future may hold new antimicrobials that aren't necessarily categorized as classical 'antibiotics,' but may offer precision therapy against specific infectious agents. Whatever the future holds, it won't be a single answer: Stanley sees many innovations necessary to deal with the future of antibiotic-resistant infections.

Stanley's current research is in Salmonella genome plasticity and how genomic traits influence the bacterial niche. Where do traits like exotoxins or antibiotic resistance exist in the environment, and how are they transferred to new species to influence disease? Cases of Typhoid Fever in people without known exposure to another diseased person suggest there may be an environmental reservoir. What might it be?

Stanley is a big proponent of scientist entrepreneurs and participates with the NSF Innovation Corps to promote early science start ups. In addition to creativity and the scientific process, one characteristic he encourages all entrepreneurs to develop is a good team spirit. Working collaboratively as a team is a very strong sign of success.

Stanley believes in the importance of an international science communities, and he practices what he preaches: he works closely with the scientific community of Chile. He began in 1990 by teaching an intensive lab course about techniques, and has developed a decades-long relationship with this community. These relationships allow a dialog, and were the reason Stanley ultimately turned his focus to Salmonella Typhi from Salmonella Typhimurium.

Links for this Episode:

• MTM Listener Survey, only takes 3 minutes! Thanks;)
• Stanley Maloy website at San Diego State University
• This Week in Microbiology #95: A Microbe Lover in San Diego
• National Science Foundation Innovation Corps
• Journal of Microbiology and Biology Education Call for Submissions for a Special Issue on diversity and inclusion.
• HOM Tidbit: A Large Community Outbreak of Salmonellosis Caused by Intentional Contamination of Restaurant Salad Bars

Cheese rinds contain microbial communities that are relatively simple to study in the lab while offering insight into other, more complex microbial ecosystems. Rachel Dutton discusses her work studying these cheese microbiomes, one of the few microbial ecosystem types where almost all of the microorganisms are culturable.

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The cheese microbiome makes a great study system because

• The communities are relatively simple (as few as 3 different microbial species)
• The microbial members are almost all culturable (in stark contrast to most microbial communities)

The microbes colonize the cheese rind as a biofilm, which consists of the microbes and their secreted extracellular products. Like all biofilm communities, architecture and spatial structure are important for microbial interactions on cheese rinds, as are oxygen gradations, food access, and proximity to microbial neighbors.

Rachel and her lab performed DNA sequencing on over 150 cheese samples from 10 countries to identify the microbes present on these rinds. By comparing these sequences to those they could grow in the lab (Rachel's lab makes "in vitro" cheese medium consisting of desiccated, autoclaved cheese), they realized almost all of the organisms identified by molecular means were present in their cultures.

Does the cheese environment influence the microbial communities or do the microbial communities influence the cheese environment? Both! The pH, temperature, added salt and temperature act as knobs or dials that allow cheese makers to fine tune the final cheese product.

Rachel was inspired to work on cheese after taking the Microbial Ecology course at Woods Hole, where the students spent a lot of time looking at the beautiful but complex interactions within microbial mats. Upon cutting open some Tomme de Savoie from a French colleague, she noted similarities between the microbial mat and the layered cheese rind

"The biofilm that colonizes the surface of the cheese has a lot to do with how the cheese ends up looking and smelling and tasting, and we actually eat this biofilm when we eat the cheese."

"We're able to see that of all of the things that we identified by reasonable sequence abundance, we could also find them in culture. This told us that we were able to get a lot of these microbes in culture, which is not really possible in microbial ecosystems, but is one of the really strong advantages of working in the fermented food community."

"We're looking at these interactions because they're happening on cheese and we can study them in the lab but they are things that are happening broadly across ecosystems, which I think is very exciting."

"We've done some work on the succession of species over time. You have these very very reproducible successions over time, even though a lot of these cheeses are not inoculated with specific species; these are species that are coming in from the environment but they're very reproducible communities. There are some beautiful dynamics that happen and we're starting to look at the interactions between species that may be driving some of these dynamics."

"We have this big need for model systems. One of the things I hope is that we'll have more people developing simple model systems for microbial ecology so we can compare results and see what the general principles are."

• MTM Listener Survey, only takes 3 minutes! Thanks;)
• Rachel Dutton Lab Website
• Wolfe BE, Sutton JE, Santarelli M, and Dutton RJ. Cheese Rind Communities Provide Tractable Systems for in situ and in vitro Studies of Microbial Diversity. Cell 2014.
• Wolfe BE and Dutton RJ. Towards an ecosystems approach to cheese microbiology. Book chapter: Cheese and Microbes. ASM Press and Microbiology Spectrum (2014).
• Microbes After Hours: The Microbiology of Cheese (YouTube)
• Competition and Cooperation of Cheese Rind Microbes Exposed (The Scientist)
• Related: The Natural History of Cheese Mites
• HOM Tidbit: Peoria Historian Blog Post
• HOM Tidbit: Journal of Bacteriology Classic Spotlight: Crowd Sourcing Provided PenicilliumStrains for the War Effort

Most diagnostic tests look for a single microorganism, or at most a limited panel of microorganisms. Charles Chiu discusses his research on metagenomic sequencing as a diagnostic tool that can identify all potential pathogens in a given patient sample.

Links for this Episode:

• MTM Listener Survey, only takes 3 minutes! Thanks;)
• Charles Chiu Profile at UCSF
• Chiu Lab at UCSF
• Validation of Metagenomic Next-Generation Sequencing Tests for Universal Pathogen Detection
• The Eukaryotic Gut Virome in Hematopoietic Stem Cell Transplantation: New Clues in Enteric Graft-Versus-Host Disease
• HOM Tidbit: Dochez and Avery. The Elaboration of Specific Soluble Substance by Pneumococcus during Growth. Journal of Experimental Medicine 1917.
• HOM Tidbit: Kozel and Burnham-Marusich. Point-of-Care Testing for Infectious Diseases: Past, Present, and Future. Journal of Clinical Microbiology 2017.

While searching for lignin-degrading soil microbes, Gautam Dantas discovered growth in an antimicrobial compound-containing control! He has since studied the resistance determinants (resistome) of soil and clinical samples to determine their similarities.

Julie's Biggest Takeaways:

Sequencing information is extremely useful for descriptive studies, but there's an increasing trend in microbiome studies to use the sequencing data as a basis for forming hypotheses. These hypotheses can then be tested by some variation of classical techniques, be in biochemical, culturing, animal models, etc. Surveying who is there helps scientists make testable predictions.

Gautam's resistome research is built on the research of many, but especially inspired by:

• Gerry Wright, who proposed the presence of a resistome. The resistome is a collection of genetic determinants in a microbial group that allows phenotypic resistance against antimicrobial compounds.
• Julian Davies, who proposed the producer hypothesis. The producer hypothesis suggests that the same microorganisms that produce antimicrobials must also be the source of resistance, because they need to be able to protect themselves against the action of their own compounds.

Gautam's discovery of antibiotic-eating microbes was completely serendipitous! As a postdoc, he was looking for lignin-degrading soil microbes and set up a culture with antibiotics as a negative control. To his surprise, there were some soil microbes that were able to grow - using the drugs as food! Samples from 3 different states were all able to support microbial life.

The resistome of soil is very similar to the resistome of clinical samples, but the study design doesn't allow Gautam to conclude directionality: do the genes move from the clinic to the environment or from the environment to the clinic? This requires studying the resistomes over time, rather than the snapshot analyses this study generated. However, Gautam's group has received funding to do longitudinal studies, which will help scientists understand how resistance originates and then moves to new microbial communities.

Context is very important for determining disease. A microbe may make one person but not another sick. Context can also be the genes carried by the microbe, and E. coli is a great example of this. Some E. coli are very good at causing UTIs but cause no disease when carried in the gut.

Links for this Episode:

• Take the MTM listener survey (~3 min.)
• Gautam Dantas lab website
• Wright G.D. The Antibiotic Resistome. Expert Opinion in Drug Discovery. 2010.
• Davies J. and Davies D. Origins and Evolution of Antibiotic Resistance. MMBR. 2010.
• Bloomberg: Germ-Killing Brands Now Want to Sell You Germs
• HOM Tidbit: Recycling Metchnikoff: Probiotics, the Intestinal Microbiome and the Quest for Long Life