Meet the Microbiologist

How is Toxoplasma gondii, a protozoan that causes neuro-invasive disease, transmitted as a foodborne pathogen? Why are cats important in transmitting Toxoplasma infection? Anita Koshy answer these questions and talks about her research on the latest Meet the Microbiologist. Julie’s Biggest Takeaways: The primary host for T. gondii is cats, in which the protozoan can undergo sexual reproduction. Why cats? No one knows, in part because there isn’t a good in vitro system to study cat epithelial cell interactions with T. gondii. Most warm-blooded animals, including birds, can be infected with Toxoplasma. Intermediate hosts can pass Toxoplasma from one to another if one eat these tissue cysts, explaining why Toxoplasma can be a foodborne pathogen. In healthy individuals, the immune response clears most fast-growing cells (tachyzoites) but some protozoans convert to a slow-growing cell form (bradyzoites). In people, these bradyzoites form cysts predominantly in the brain, the heart and the skeletal muscle. Some serological studies suggest a tie between Toxoplasma infection and brain disorders, but these are less definitive than causative studies in mice. Populations with high Toxoplasma or low Toxoplasma prevalence don’t see a correlative incidence of disorders such as schizophrenia or Alzheimer’s disease. Featured Quotes: “When we talk about neuroinfectious diseases, we’re talking about the diseases that cause symptoms. Those that can get into the central nervous system and those that cause symptomatic disease are the same.” “A parasite is sitting there dormant or maybe reactivating every so often and the immune system comes in and deals with that reactivation. But when you lack an immune system, all of a sudden when that parasite reactivates, there is no longer this immune system that will come in and clear it out.” “What we don’t know is whether reactivation occurs preferentially in the brain. There is evidence from HIV patients of inflammation of the heart or inflammation of the skeletal tissue - but those weren’t the symptoms that presented, which were of the brain. Did reactivation happen in the brain, or did it occur elsewhere and the parasite was able to travel to the brain and there’s no longer an immune system to clear it out?” Links for this Episode: Koshy Lab Site Sea Otter Infection with Toxoplasma Rats Infected with T. gondii Lose Their Aversion to Cat Urine HOM Tidbit: The History of Toxoplasma gondii Bill Hutchinson obituary  

Jennifer Gardy talks about whole-genome sequencing as a technique to address public health issues using genomic epidemiology. She talks about her research on TB and new DNA sequencing technologies, including her vision for microbial genetic sequencing as one piece of the puzzle in the future of public health. Julie’s Biggest Takeaways: Whole-genome sequencing technologies are replacing older DNA technologies to identify relatedness between microbial isolates. The genome sequences help to identify epidemiological questions such as the origins of an outbreak. A pathogen’s genome being passed person-to-person accrues small changes, similar to children playing telephone - except those children are scattered around the room, and you have to logically deduce the order in which the information was passed. DNA sequencing has moved forward faster than the upstream genomic preparation and downstream sequence analysis areas; Gardy expects advances in these ‘bookend’ areas to be breakthroughs of the future. The Ebola and Zika outbreaks were test cases for portable DNA sequencing technologies, but informative based on the different disease presentation: Ebola patients have high viral loads and thus a lot of genomic material, but Zika patients have much lower viral loads and it was much harder to get samples. Based on pathogen characteristics, DNA sequencing can identify the end of an outbreak. Gardy used sequencing to find that patients with TB, which can take years to develop into fulminant disease, had been infected years previous, and was able to see that transmission was no longer ongoing. Featured Quotes: “Genomics is really cool because instead of interviewing people about what happened in an outbreak, we’re interviewing the pathogen!” “[Working at BCCDC] is a really nice ecosystem, where you can really see the results of your research changing public care policy and practice in real time, and that is incredibly rewarding.” “The only prediction you can make about DNA sequencing is there’s always going to be something new and different.” “Depending on your use-case, sometimes you need to go after the whole genome and other times a targeted approach is more than enough.” “I’m excited to see how this [microbial DNA sequencing] work fits in into an overall public health landscape. It’s cool to sequence genomes and make some reports about transmission networks, but that’s just one small part of a very big public health system that is trying to keep populations healthy. It requires so many different people, from nurses and doctors on the frontline to policy makers behind the scenes to social scientists who are interacting with patients or care providers to people that are understanding the economics of these things... when you start to see how these different pieces of the puzzle fit together, I think there’s a lot of opportunities in the future for making microbial genomics just one piece of a large interdisciplinary puzzle of people that are working together across different fields to address a disease from multiple different angles.” Links for This Episode: Jennifer Gardy’s website Jennifer Gardy at UBC Nanopore Minion Alan Alda Center for Communicating Science Banff Science Communications HOM Tidbit: Albrecht Kossel, a Biographical Sketch

See the full shownotes at: asm.org/mtm Julie Pfeiffer tells the story of how she serendipitously found a role for the gut microbiota during polio virus infection, and how she and her lab discovered an important role for bacterial glycans in viral biology. She also talks about viral fitness strategies, and how RNA viruses and DNA viruses benefit from making different amounts of errors when copying their genomes. Julie's biggest takeaways: Determining the exact nature of the glycans that play these roles has been difficult because they are very complex. Aspects of lipopolysaccharide, chitin, and peptidoglycan are all sufficient to bind the viral capsid, but because of their structural complexity, it’s difficult to pinpoint the exact molecular interaction. Bacterial glycan interactions with viruses benefit the virus in two ways: the virus can be delivered to a host cell it will infect, and the viral capsid is stabilized. Whether there is a benefit to the bacterium during these interactions is unknown, but is an active area of research in Julie’s lab. Many viruses can be inactivated at body temperature or even room temperature if they prematurely release their genetic material. Polio viruses are simply a protein shell surrounding an RNA genome, and the capsid can ‘breathe,’ slightly changing its conformation. Sometimes, the genome is accidentally released, resulting in a viral dead end. Julie showed that bacterial glycans will lock the capsids into a conformation and prevent genome release from happening until the virus encounters a host cell. Julie is a proponent of clear communication, including with those working in similar fields, which she learned from her experience as a postdoctoral fellow. She and a postdoc in a different institution, Marco Vignuzzi, independently isolated a polio virus mutant that made fewer in genome replication. Both showed that the virus had a defect during mouse infection, indicating that the ability to introduce errors during genome replication is beneficial to viral fitness. Julie and Marco finally met at a viral evolution conference, after which they became close friends. Featured Quotes (in order of appearance): “I get more excited about a surprising result because it probably means there’s some interesting underlying biology that couldn’t be anticipated!” “We’ve done many gross experiments, so buyer beware; you’ve got to know what you’re getting into [with a fecal-oral pathogen].” “The infectious unit may be more complicated than we think!” “Communicating with people you know working on similar things can be mutually beneficial for everyone: you both get credit; nobody gets scooped. It’s win-win for sure.” “The truth is most enteric viral infections are self limiting in most healthy individuals so you’re much better off trudging through a day or two of gastrointestinal illness than blowing up your microbiota.” Links for this episode Julie Pfeiffer website at UT Southwestern Medical Center Back-to-back Science publications from Golovkina and Pfeiffer PLOS Pathogens: The importance of model systems: Why we study a virus on the brink of global eradication Viruses and Cells Gordon conference (donate here) HOM Tidbit: Michael Underwood’s A Treatise on the Diseases of Children

Pete Greenberg tells how bacteria can communicate based on cell density, a phenomenon he helped name quorum sensing. He talks about therapeutics based on quorum-sensing discoveries, and how studying bacterial interactions can be used to test ecological principles like cooperation and social cheating. Julie's biggest takeaways: Quorum sensing can be likened to an old-fashioned smoking room, where a few cigar smokers don’t affect the air quality, but as more smokers enter the room, it becomes beneficial to the group to open the window: a changed behavior that benefits the group environment. Differentiating waste molecules from signaling molecules is important to define specific quorum sensing. The experimental evidence that shows that molecules serve as quorum sensing signals that allow bacteria to respond at high density comes from social engineering experiments to identify ‘cheaters.’ Quorum sensing results in changes in gene expression that benefit the community but not necessarily individual cells. An example is antibiotics, which when made by a single cell aren’t at a high enough concentration to kill competitor microbes. As a group, all cells working together can produce a cloud of antibiotic that may be able to protect from competitors. The ability of microbes to receive or ‘eavesdrop’ on the signals produced may be cooperative, but is more likely competitive, giving the eavesdropper a competitive advantage by informing them about another species’ presence. If you knock out quorum sensing, you get abnormal biofilms, but it doesn’t ablate biofilms completely. Although a self-described disinterested high-school student, Greenberg signed up for a weekend field trip to get out of a test on a Friday. It was looking at animals in the intertidal bay of the Pacific Northwest that inspired him to be a biologist! Greenberg also credits his broad biology undergraduate training for preparing him to apply socioecology concepts to bacteria. Quorum sensing was originally called ‘auto induction.’ In the early 1990s, Greenberg was writing a minireview for the Journal of Bacteriology and wanted to think of a catchy title. As Greenberg remembers, coauthor Steve Winans explained the concept to his family, and his brother-in-law said “it’s like the bacteria need a quorum” - the birth of the term ‘quorum sensing.’ Featured Quotes (in order of appearance): “So-called ‘cheaters’ don’t respond to the signal, they’ve lost the ability to respond to the signal. The product that’s useful for the common good any more. They don’t pay the cost of cooperation but they can benefit by the cooperative activity of everyone else in the community...there’s a fitness advantage for cheaters in this environment.” “It’s a real case of convergent evolution. It’s important that the bacteria can do this, and these two really distinct types of [gram-positive and gram-negative] bacteria have evolved completely different mechanisms to perform quorum sensing.” “I think of bacteria as a way to study what is called ‘Darwin’s dilemma.’ If a cheater emerges among a population, it will have a fitness advantage over the population of cooperators. It should take over the population and ultimately cause the tragedy of the commons, where there are too many cheaters and not enough cooperators and the whole system collapses. Darwin’s dilemma is: how is cooperation stabilized? We know it exists and it seems like it shouldn’t - we can use bacteria to get at the rules.” “I got interested in [quorum sensing] because it was so cool!” “I had this idea, as we began to unravel quorum sensing in these marine luminescent bacteria, that any idea in biology that’s a good idea will occur more than once - but I didn’t have any evidence of that. For 15 years, my lab and essentially one other lab, Mike Silverman’s lab, were the only labs working on this. It was really the early 90s when our group and other groups started to realie that lots of bacteria do this. It’s one of those fantastic oddesies. It’s luck - luck and hard work, I guess. Hard work by the people in my lab as I sit around as watch!” “It’s funny how a term can catch on and sort of crystallize a field! But somehow, it seemed to do that. I’ve gotten really into trying to think of catchy terms since then, and the latest one is ‘sociomicrobiology,’ which I introduced with Matt Parsek about 12 years ago and there’s a burgeoning field called sociomicrobiology. I’m trying to think of another term now, before I retire!” Links for this episode   Pete Greenberg lab at the University of Washington Pete Greenberg 2004 PNAS bio Journal of Bacteriology minireview: Quorum Sensing in Bacteria HOM: Woody Hastings memoriam ASM Podcasts     Send your stories about our guests and/or your comments to jwolf@asmusa.org.

Charlie Rice gives the history of learning to grow hepatitis C virus in culture, from pitfalls to hurdles and successes along the 20-year journey. He also talks about yellow fever virus, its vaccine, and the importance of curiosity-driven research

How are new diseases detected in a clinical microbiology lab? Melissa Miller talks about the time it takes to develop a test for a new disease (hint: it’s getting shorter). She also shares her definition of ‘point-of-care’ diagnostics and explains the major trends for clinical microbiology labs. Host: Julie Wolf  Subscribe (free) on iPhone, Android, RSS, or by email. You can also listen on your mobile device with the ASM Podcast app. Julie's biggest takeaways: Antibiograms are vital to understand the resistance characteristics of locally circulating disease strains. These help make empirical decisions for antibiotic therapy regimens before the susceptibility test results are available. New diseases require new diagnostic tests. How to determine how well new tests work once they’re developed? Clinical microbiologists look to the sensitivity (how well does a test detect if a patient has a disease) and specificity (how often is the test negative if the patients doesn’t have it) of the test. Having access to positive controls (that is, samples from a patient known to have the disease) can prove difficult in some settings, such as in North Carolina, where no Zika patients were admitted while the Zika virus test was being developed. When the HIV epidemic was beginning, it took several years after the HIV virus was identified to sequence its genome and use this for molecular testing. In 2002-2003, it took just over a month to get the SARS genome sequence for use in developing assays. It’s even quicker today; within a week, we can have sequences from viruses around the world. Defining ‘point-of-care’ testing took an entire hour at a recent American Academy of Microbiology colloquium! Melissa’s take: It’s a test that can be done at or near to where the patient is. Point-of-care tests are exciting but can also pose challenges. A recent example is false-positive pertussis tests that were shown to be due to pertussis vaccine being administered nearby. Ensuring the tests are used safely and accurately will best serve healthcare workers and patients alike. Molecular diagnostics have two trends: one trend simplifies existing technologies into point-of-care tests. The other trend adds complexity, by applying next-generation sequencing techniques in a reproducible manner. Featured Quotes (in order of appearance): “Laboratorians are often in the basement or in a setting where they aren’t visible to the healthcare team, but they’re very vital to taking care of the patient.”   “When you’re using laboratory-developed tests, the way it works in one laboratory may be very different from how it works in another laboratory.”   “The ultimate goal [of point-of-care testing] is to get a result that’s actionable. We don’t need to do tests that aren’t going to result in a clinically actionable decision.”   “In many ways, the technology is ahead of where our quality assurance protocols are.”   “I think it’s going to be very important in going ahead that we continue to have laboratorians involved in developing these point-of-care programs and consulting to these sites, helping to make sure that there are policies and procedures that ensure quality results for their patients.”   “It’s one thing to do it in a research setting; we’ve collaborated with a number of folks using next generation sequencing. But to then move it to the clinical lab and have it be reproducible and have the quality at the level you need for a clinical lab is a completely different challenge.” Links for this episode Melissa Miller University of North Carolina Website Division of Clinical Laboratory Science at University of North Carolina Searchable List of Clinical Laboratory Science Programs AAM Colloquium Report on Point-of-Care Testing CPEP Program Career Blog: Tips on becoming a clinical microbiology laboratory director HOM Tidbid: Papagrigorakis 2006 International Journal of Infectious Diseases report HOM Tidbit: Shapiro reply to Papagrigorakis report Send your stories about our guests and/or your comments to jwolf@asmusa.org.  

Veterinarian and epidemiologist Mathew Muturi tells how a Rift Valley Fever outbreak led to implementation of One Health-based policies. Muturi talks about his One Health training and its applications for health and biopreparedness. Julie’s Biggest Takeaways: One Health Simple communication between experts helps facilitate implementation of one health in public systems. Sitting experts in human and animal health in the same office allows easier communication between these different health sectors. One Health policies involving close collaboration between animal and human healthcare workers were first implemented in Kenya in response to the threat of avian influenza, but were discontinued after the threat waned. Human cases of Rift Valley Fever, due to spillover from a livestock outbreak, led to the discovery that these collaborative policies could prevent other outbreaks as well, and the policies were reinstated. Zoonotic diseases can often be the most overlooked. Officials of countries where endemic diseases are present may have preparedness plans for serious cases but may overlook something endemic like brucellosis. There are 42 subtribes in Kenya, including diverse languages, religions, and beliefs. Public health interventions do their best to align the local beliefs of the people to minimize risk of pathogen exposure. Featured Quotes: “One health is not a new concept; it’s an old concept that explains the health of humans, animals, and the environment is interconnected. It’s a concept that plays out in everyday life.” “One of the reasons One Health has been able to be successful in Kenya, and that I suggest to other countries wishing to implement this program, is the sitting together, talking together. Make sure that you work together, see each other - I don’t think communication works well enough if it’s on an ad hoc basis. The thing that has worked for us is sitting together.” “The most important aspect of One Health is the fact that that it’s impossible to control diseases that come from animals only by focusing on humans. It’s like trying to concentrate on putting out fires without ascertaining where the fires are coming from.” “Endemic diseases, despite the fact that they’re ever-present, are often the most ignored.” “A lot of the risk practices are cultural, and cultural change is very slow.” “The value of One Health is much more than the investment required to put into it. It’s one of the few things I’ve seen actually work in implementation of disease control strategies, in surveillance and in general disease control. It’s worked for Kenya and I believe it can work for all other countries.”   Links for the episode: Republic of Kenya Zoonotic Disease Unit Prioritization of Zoonotic Diseases in Kenya, 2015. Plos One.

Dave Rasko uses comparative bacterial genomics to find DNA sequences that influence virulence or antibiotic resistance. Dave talks about his studies of E. coli, Acinetobacter baumanii, and B. anthracis, and the state of bacterial genomics past, present, and future. Host: Julie Wolf Subscribe (free) on iPhone, Android, RSS, or by email. You can also listen on your mobile device with the ASM Podcast app. Julie's biggest takeaways: Genome sequencing speed has significantly increased: The first bacterial genome sequenced, Haemophilus influenzae, took about 10 years to complete. The first organism with two sequenced genomes was Helicobacter pylori, published in 1999, and the first organism with three published genomes was Escherichia coli. Rasko’s initial project at TIGR to sequence 11 E. coli genomes took about 2 years. Today, Rasko’s lab can sequence 500 genomes in about five days.   In E. coli, up to half of the genome can differ between two strains. The core genome is the collection of genes that will be shared among all isolates of a particular species. Core gene conservation varies among species and is important to consider in analyses for one’s species of interest.   Working on the Amerithrax investigation was unlike many other scientific inquiries for many reasons, including that the Federal Bureau of Investigation only gave the scientists involved the information pieces necessary to conduct their studies. Rasko and collaborators sequenced the genomes of spores within the samples, and found that the morphology of the colonies that grew were associated with genetic differences between the spores within the sample, linking phenotype and genotype.   While comparative genomics can provide a lot of information, there are some phenomena that will always require further study. For example, Rasko is researching isolates of A. baumanii and Klebiella pneumoniae that quickly develop drug resistance when grown in sub-inhibitory drug concentrations. The genomic sequences of resistant or susceptible strains show no difference in DNA sequence, suggesting the phenotype is due to transcriptional changes. Featured Quotes (in order of appearance) “Genomics is fun in that we can hypothesize all day long, every day. It’s really the start of a lot of very very hard work figuring out why.” “There’s a lot of DNA pieces that we don’t fully understand how they moved, where they moved, where they came from. In some cases, there’s evidence to say where they came from; in terms of G-C content and coding biases, we can make some assumptions, but in the grand scheme of things, we have no idea where they’re coming from! In some cases, we’ll see them dominant throughout a lineage, and in some cases we’ll see them in sporadic isolates around the entire phylogenetic tree. . .We all thought genomics was going to solve so many problems, and it’s really just made it more difficult!” “Plasmids tend to be mobile and exchangeable, and the pieces tend to be - I tend to think of them as legos, in the fact that you can put a plasmid together in a bunch of different ways.” “I think a lot of conventional PCR fails and people assume that it’s because it’s negative, and not necessarily that it fails because of diversity.” “Many microbiologists think of that colony on a plate as a clone. I force the people in my group to think about it a little differently, because it’s really what I like to call ‘genome space’. They’re not all the same; bacteria are constantly evolving. There’s changes all the time, some of them are positive, some of the are negative, the negative ones get lost, the positive ones unusually become dominant - and then there’s lots of neutral changes that just kind of hang out.” “Genomes really normalized everything. Before that, there were certain labs that could clone and there were certain labs that could sequence, and it was a little bit restrictive to the elite labs who had those resources. Now with the genome sequences out there, everyone was starting from the same place.” “You really have to understand your organism to make the bioinformatics work.” Links for this episode Rasko lab at the University of Maryland FBI summary of Amerithrax investigation 2011 PNAS report on B. anthracis comparative genomics Bugs N the ‘hood HOM Tidbit: Stanley Falkow gives both video history and written history of plasmid biology Save on Microbe 2018, use code: asmpod Send your stories about our guests and/or your comments to jwolf@asmusa.org.

Bill Jacobs talks about developing mycobacterial genetic tools and using them to discover ways to shorten TB treatment. He also talks about the SEA-PHAGES program that allows high-school students to participate in phage discovery. Host: Julie Wolf Subscribe (free) on iPhone, Android, RSS, or by email. You can also listen on your mobile device with the ASM Podcast app. Julie's biggest takeaways: The challenges of working with an easily aerosolized bacterium are aided by complementary studies on a noninfectious relative. M. smegmatus doesn’t colonize mammals and grows slower, giving researchers the opportunity to acclimate themselves to working with mycobacterial cultures. Jacobs was the first scientist to introduce DNA into M. tuberculosis using a phasmid - part plasmid, part mycobacterial phage. The first phage came from Jacobs’ dirt yard in the Bronx, so he named it BxB1 for the Bronx Bomber. Another phage, TM4, became the workhorse phasmid when Jacobs cloned an E. coli cosmid sequence into a nonessential part of the phage genome. It replicates in E. coli as a plasmid but becomes a phage inside Mycobacteria, facilitating manipulation. The shuttle phasmids allowed transposon delivery to make transposon libraries, and the creation of gene knockouts. To this day, we use Ziehl-Neelsen staining to differentiate acid-fast mycobacteria from gram-positive or gram-negative bacteria - the mycolic acids on the outer part of the envelope make up some of the longest microbial lipid chains. But mycobacteria can regulate its acid-fast positive or negative status; the acid-fast negative organisms are a persistent population that are often ignored inside of patients. 99.99% of M. tuberculosis bacteria are not persistent, but the last 0.1% have entered into a persistent state expressing many stress proteins that help them become refractory to killing. A normal course of antibiotic chemotherapy for patients is six months. If infected with a strain resistant to the two frontline drugs, that time goes up to two years. The problem is even greater in extremely multidrug resistant (XDR) strains. What we really need is a way to understand persistence and a way to shorten chemotherapy. That’s why were were absolutely amazed when we discovered that cysteine with isoniazid completely sterilizes Mtb cultures in vitro and in vivo! The culture is sterilized because the bacteria can’t form persisters. Vitamin C co-treatment with antibiotics may lead to a shortened course of therapy for TB treatment. Neutralizing antibodies to the herpesvirus glycoprotein have been the dogma for protecting from herpes. Jacobs and his colleagues discovered that a vaccine based on a glycoprotein-knockout virus confers sterilizing immunity not through neutralizing antibodies but through antibody-dependent cell cytoxicity (ADCC). This ADCC response may also be important to develop a more effective TB vaccine.   Featured Quotes (in order of appearance): “You’ll never know how bad your aseptic technique is until you start working with tuberculosis!” “I think part of the reason I had the opportunity to develop genetics for TB - it’s not like it wasn’t important to do - but a lot of people were disappointed when working with the organism.” “We’re about to take TB genetics to where yeast genetics is.” “One of the tubicle bacilli’s greatest powers or one of its most important phenotypes is that it has the ability to persist, which means it has the ability to tolerate killing effectors, either killing by the immune system or killing by bactericidal drugs.” “I took students to the Bronx Zoo, and over by the zebra pen, I sniffed and said ‘I smell a phage!’ In fact, that’s not crazy - anyone who plants flowers knows what good soil smells like, and in the good soil, you’re smelling the bacteria that live in the soil, the Streptomyces and Mycobacteria. I reached down and grabbed that dirt, and when we went back to work we isolated BxE1.” “I’ve never met a phage I wasn’t excited about!” “I now believe that most pathogens do not ‘want’ ADCC antibodies to be made, and they have immune evasion strategies where they skew the immune response to get the wrong antibodies. Since the time we published our first paper, numerous groups have shown that correlates of protection for HIV, for influenza, and for Zika, turn out to be ADCC antibodies.” “Genetics is the mathematics of biology!”   Links for this episode Bill Jacobs lab site NYTimes story on 1993 rapid diagnostic test using luciferase AACJournal: Vitamin C potentiates the killing of Mycobacterium tuberculosis by the first-line tuberculosis drugs isoniazid and rifampicin in mice Cell: Origins of highly mosaic mycobacteriophage genomes SEA-PHAGES program eLife: Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity mBio: Dual-reported mycobacteriophages (Φ2DRMs) reveal preexisting Mycobacterium tuberculosis persistent cells in human sputum Tuberculosis - Its cause, cure and prevention [1914] (pdf)   Send your stories about our guests and/or your comments to jwolf@asmusa.org.  

Ilaria Capua talks about running an internationally renowned animal influenza lab, and her time spent in the Italian Parliament. Accused of virus trafficking as part of a national scandal, she has since cleared her name and speaks here about the importance of scientific credibility and reputation.