Clinical microbiology has experienced an unprecedented wave of innovation due to the introduction of advanced testing such as PCR, MALDI-TOF, and PNA-FISH.  The wave of new technologies has dramatically improved the time to pathogen identification result from culture, but has not impacted antimicrobial susceptibility testing: the definitive diagnostic result for changing antibiotic therapy in patients with bloodstream infections.  This presentation explores the clinical need for faster phenotypic susceptibility testing using the Accelerate PhenoTest™ BC kit.  Topics covered include the benefits to the patient and the economic benefits to the healthcare institution.

Plant viruses cause significant damage in terms of reduction in quality and quantity of yield in a wide range of crop plants worldwide. The majority of plant viruses are transmitted from one host plant to another by insect vectors. Although viruses are obligate, intracellular parasites their relationship with the insect vector varies from parasitism to mutualism. The central question of my research program is to understand  ‘how plant viruses affect biology and ecology of their insect vectors? and (2) what plant and/or insect responses mediate plant-virus -vector interactions?’ To identify molecular and ecological parameters that mediate plant-vector-virus interactions, I am investigating two virus-vector systems. Tomato spotted wilt virus (TSWV), one of the ten most devastating plant viruses worldwide is transmitted by Frankliniella occidentalis (western flower thrips), the primary insect vector of TSWV, and Soybean vein necrosis virus (SVNV), a new emerging soybean virus transmitted by Neohydatothrips variabilis (soybean thrips).

Genome sequencing can be used to discover new pathogens but it can also be used to monitor known ones. This is known as genomic surveillance and it becoming an increasingly important tool in the response to viral outbreaks. We have demonstrated that a portable laboratory for virus sequencing can be flown to the epicentre of an ongoing outbreak and provide rapid, actionable results from the field. This avoids the delays associated with shipping samples out the country for sequencing elsewhere. We have set up mobile labs in Guinea during the 2014 West African Ebola outbreak and to Brazil during the 2015 Zika outbreak. I will talk about what goes into building a mobile lab and the ways in which they can be of use in an ongoing outbreak.

An unbiased metagenomic next-generation approach (mNGS) been shown to be useful in the broad identification of pathogens in clinical samples for infectious disease diagnosis, including viruses, bacteria, fungi, and parasites. This talk will discuss clinical licensed (CLIA) laboratory validation of a clinical mNGS assay for identification of pathogens in cerebrospinal fluid (CSF) and implementation of the automated SURPI computational pipeline for rapid analysis and interpretation of mNGS data. The CLIA-validated mNGS CSF test is now available as a clinical reference test for acutely ill hospitalized patients. We will also report the results from a 1-year, multi-hospital, nationwide study (“Precision Diagnosis of Acute Infectious Diseases”, June 2016 - 2017) to evaluate the clincail utility, performance, and cost-effectiveness of the mNGS assay relative to conventional microbiological testing in diagnosis of these neurological infections.  This talk will also describe new mNGS-based technologies and diagnostic platforms in development, including host response profiling to discriminate infectious versus non-infectious etiologies, antibiotic resistance determination, and nanopore sequencing, and how these approaches can be incorporated into routine clinical diagnostic testing for infectious diseases.

Males and females differ in their immunological responses to viral and vaccine antigens, with females typically mounting higher immune responses than males. These sex-based immunological differences contribute to variation in susceptibility to infectious diseases and responses to vaccines in males and females. The intensity and prevalence of viral infections are typically higher in males, whereas disease outcome can be worse for females, which in many cases is caused by an exaggerated immune response that damages tissue and causes pathology. In response to vaccines, females mount higher immune responses and experience more adverse reactions than males. Several variables should be considered when evaluating male/female differences in responses to viral infection and treatment: these include age, hormones, genes, and gender-specific factors related to access to, and compliance with, treatment. Knowledge that the sexes differ in their responses to infectious diseases and vaccines should influence the recommended course of action differently for males and females.

The human large intestine houses trillions of microorganisms which collectively form the highly diverse microbial community known as the gut microbiota. The gut microbiota performs many functions critical to the maintenance of health, including extraction of nutrients from food, production of vitamins, and defense against pathogens. Like an organ, disturbances to the structure of the gut microbiota can have significant negative impacts to the human host, including obesity, malnutrition, and cancer. However, the gut microbiota is currently unique among organs in that it is highly engineerable, enabling improvement of function by substituting beneficial microbes for less desirable ones. These exogenous beneficial microbes are termed “probiotics” and their close contact with both their human host, as well as other gut bacteria, raises exciting therapeutic prospects, including the provision of additional metabolic functions, modulation of the host immune response, or competitive exclusion of pathogens. However, these applications remain out of reach due to the low residence time of most probiotics within the gut, as well as an inability to reliably control gene expression in probiotics residing in the gut environment.  In this talk, I will show exciting data from a culture-independent bioprospecting approach for increasing the residence time and altering the biogeography of probiotic strains within the gut. Then, I will discuss the optimization and application of a high-throughput sequencing-based approach to the parallel characterization of many transcriptional regulators in gut-resident probiotics. Taken together, this work has the potential to significantly improve our understanding and use of probiotics while providing a framework for developing robust chassis strains for future synthetic biology efforts in the human gut microbiota.

Quinolones are one the most commonly prescribed classes of antibacterials in the world and are used to treat a broad variety of Gram-negative and Gram-positive bacterial infections in humans. However, because of the wide use (and overuse) of these drugs, the number of quinolone resistant bacterial strains has been growing steadily since the 1990s. As is the case with other antibacterial agents, the rise in quinolone resistance threatens the clinical utility of this important drug class. 

Quinolones target the bacterial type II topoisomerases, gyrase and topoisomerase IV. These enzymes regulate DNA under- and over-winding and remove knots and tangles from the genome. Gyrase and topoisomerase IV create transient double-stranded breaks in the genetic material in order to carry out their essential cellular functions. Quinolones take advantage of this enzyme-mediated DNA cleavage activity and kill cells by converting gyrase and topoisomerase IV, into toxic “nucleases” that fragment the bacterial chromosome.

Quinolone resistance is most often associated with mutations at two highly conserved amino acid residues (a serine and an acidic residue) in gyrase and/or topoisomerase IV. Recent structural and biochemical studies have determined how these two residues facilitate drug-enzyme interactions. Quinolones contain a keto acid group that chelates a non-catalytic Mg2+ ion, which in turn is coordinated by four water molecules. Two of these water molecules are situated close enough to the serine and acidic residue to form hydrogen bonds. For clinically relevant quinolones, this “water-metal ion bridge” serves as the primary interaction between the drug and the bacterial type II topoisomerases.

Dr. Osheroff’s talk will provide background on DNA topology and type II topoisomerases. It will go on to describe experiments that led to the discovery of the water-metal ion bridge and defined its role in mediating quinolone-enzyme interactions. The talk will describe how this knowledge has been used to design novel quinolones and quinolone-based drugs that overcome resistance. Finally, it will discuss recent experiments with novel naphthyridone-based drugs that alter gyrase-mediated DNA cleavage by a unique mechanism and retain their activity against quinolone-resistant mutant enzymes.

A rapidly growing number of viruses of lower eukaryotes have been reported in the past few decades. These have enhanced our understanding of virus evolution and diversity. Simultaneously, some unusual viruses have challenged or broken the “common rules” of viruses in sizes and concepts. One such virus group includes the so called dsDNA megaviruses isolated from amoebae that exceed some bacterial parasites in coding capacity and particle size. Other unusual viruses have been found in fungi that include “naked” or “capsidless” RNA viruses unable to form virus particles, exemplified by hypoviruses and narnaviruses. Also among them are fungal multi-segmented dsRNA viruses that are infectious viruses as naked dsRNA. My laboratory also had an opportunity to discover such a virus from an important plant pathogenic fungus, Rosellinia necatrix. The fungus is a filamentous ascomycete that causes white root rot in diverse perennial crops. A mixed viral infection was found in a hypovirulent strain of R. necatrix. Co-infecting viruses were termed Yado-kari virus 1 (YkV1) with a positive strand (+) RNA genome of approximately 6 kb and Yado-nushi virus 1 (YnV1) with a double-stranded (ds) RNA genome of approximately 9 kb. “Yado” in Japanese literally means home or house, while “kari” and “nushi” refer to “borrower” and “owner,” respectively. Herewith, we show unique mutualistic interactions representing a new virus lifestyle: a capsidless (+) RNA virus, YkV1, hosted by a dsRNA virus YnV1. According to our proposed model (Zhang et al., Nat Microbiol, 2016), YnV1 is an independent virus able to complete its replication cycle like other dsRNA viruses, while YkV1 diverts YnV1 CP as the replication sites where trans-encased YkV1 RdRp synthesizes its own RNA as if it were a dsRNA virus. Furthermore, YkV1 was shown to enhance YnV1 accumulation. Some data with site-directed mutagenesis of available infectious YkV1 cDNA supported the model. This model still needs to be further validated by biochemical analysis with purified heterocapsids. There may be similar mutualistic interactions in other fungi such as Aspergillus foetidus (Kozlakidis et al., 2013; Nerva et al., 2016; Osaki et al., 2016). We propose the family Yadokariviridae that accommodates YkV1 and these related viruses. Their evolutionary implication and comparisons with subviral molecules similar to YkV1 will be discussed in this presentation. 

INTRODUCTION:  The recent coincidental emergence of the human microbiota and the Hologneomic Theory of Co-evolution unmasked the “Dual Citizenship” of symbiotic microbes and human ecosystems, sharing of 2 global “Passports” and our themes that “Microbes Matter“ and “Don’t Trash your Microbiota/Mycobiota.”  It also unmasked the concept of Beneficial Microbes (both bacteria and fungi), where given the overwhelming genetic strength of our microbial microbiota and the interface in disease and health, created a new concept of “self”. SCIENCE:  Here, we highlight Beneficial Microbes (BM) in 5 parts.  Part 2, addresses the “What”, including definitions, while unmasking the variety of BMs (Search, Homing, Destroy), emphasizing the unique role of biofilms as a key ingredient and the genetic strength within.  Part 3 addresses “How” BMs work, focusing on 4 neuro-pharmacologic pathways and 3 established axial pathways:  GUT, skin and vagina. Parts 4 and 5 bring clinical application and our unique amalgamation of our research to patient care. Part 4 utilizes our “Microbial Clock”, visualizing 10 metabolic endogenous diseases linked by a common pathway, including Aging, a recent NIH postulation, and Chronic Wounds, an exogenous disease where missing microbiota and mycobiota could be a central theme.  Part 5 focuses on intervention in both, recognizing “restorative microbiology” and the use of probiotics as BMs in “organ transplants” using our Computer Assisted Decision Support via  Bac-2-Health and Partners-4-Life data bases, organized for the public and health care professionals education. It is the beginning of “Precision Medicine”, the linking of Artificial Intelligence (AI) with disease management algorithms, integrating BMs. CONCLUSION: “We live in a Microbial World” with apologies to Madonna, which has been victimized by Louis Pastuer/Robert Koch themes as mostly pathogenic. One Health should be the new theme, maximized by utilization of the genetic strength of microbes as a benefit, recognizing caution and the down-stream impact on industry, clinical laboratories and our “psyche”.      

Clostridium difficile is a major pathogen responsible for infectious diarrhea in healthcare settings. This presentation will review the data from a study comparing the new Simplexa® C. difficile Direct assay with our in-house two-step algorithm and two additional PCR-based assays (Great Basin, GeneXpert Infinity) in which PCR was utilized as a confirmatory rather than a direct assay.

The global threat of antimicrobial resistance has been recognized by the World Health Organization, the United Nations and many other expert bodies. The burden of resistant pathogens is immense and only likely to escalate with predictions of millions of deaths annually and massive economic impact. Since antibiotics were available clinically bacteria have become resistant to these drugs with multi-drug resistant mutants now causing many different types of infection. Bacteria have multiple methods of becoming resistant involving target site alteration, access to the cell or modification of the removal of various molecules. Some antibiotics can be affected by all three mechanisms , such as B-lactams, while others succumb to a single change to lose their effectiveness.
Despite major advances in new antibiotics in the 1980-1990's there have been no new classes approved since 2005, daptomycin, thus there is an impetus to identify potential new classes of agents which are safe and effective. Presently there are several novel approaches being studied which may provide some hope for new therapies. These include siderophore modifications of a standard B-lactam, agents which alter the cell membrane, quorum sensing blocking, novel targets which involve protein synthesis and other sites. This presentation will provide a brief refresher on antimicrobial resistance and then focus on a selection of new agents and platforms of research. 

Background: In March 2014, a molecular cluster of five multidrug resistant Mycobacterium tuberculosis was detected by the Austrian National Reference Laboratory. An investigation was initiated to determine if transmission had occurred within Austria. Epidemiological links to Germany and Romania prompted a multi-national joint investigation, tracing the outbreak. The results were published by Fiebig and co-workers in 2017.

Methods: CLC Microbial Genomics Module, an extension of CLC Genomics Workbench, contain tools required for typing pathogenic bacteria, tracing outbreaks, and detecting antimicrobial resistance. Preconfigured, but customisable, workflows ensure ease-of-use, allowing users to focus on interpreting analysis results.

Results: Whole genome SNP analysis showed high resolution clustering of isolates. Information on the whole genome further permitted simultaneous detection of resistance causing variants. Using an improved variant detection pipeline, we identified additional variants associated to antimicrobial resistance compared to the original study. Novel variants were explored in the context of 3D protein models to predict and qualify the effect of the variants on antimicrobial susceptibility. 

Conclusion: Using data published by Fiebig at al., 2017, we demonstrate how CLC Microbial Genomics Module can be used to trace pathogen outbreaks and detect resistance causing variants. User-friendly tools and preconfigured workflows ensure ease of use and reproducibility.

Rob Dunn has recently published Never Out of Season, the story of the homogenization of our global food supply and the risks that homogenization poses. He will build on the stories from this book and discuss his current research on one unusual sort of farm, the microbial farm used to produce sourdough bread.

Again and again, we have replaced diverse agricultural systems with simple ones. Again and again, this has posed predictable challenges for farmers and in many cases whole civilizations. The global agricultural system is as at risk due to threats from climate change and the spread of pests and pathogens as it has ever been. Dunn will tell the story of this homogenization and the risks it poses (along with several recent cases in which we narrowly avoided losing key sustenances crops globally). He will also consider some of the ways in which the homogenization of food threatens not only the crops themselves but also our health. He will then quickly turn to what at first may seem to be an unusual sort of farm, that comprised of sourdough starters, the communities of microorganisms used to produce sourdough bread. Sourdough bread is made by allow yeast and bacteria to colonize a mix of, typically, flour and water. Historically, all leavened bread in the world was sourdough bread.Then, in the 1900s, the industrial production of one kind of yeast led to the spread and global dominance of that yeast and the few kinds of bread it made, industrial, simple, bread, out of which much of the nutrition, all of the variety and most of the flavor had been removed. All is not lost though, around the world sourdoughs are still being made in the traditional way and recently we have embarked upon an effort to understand the diversity of organisms in these sourdoughs, where they come from (be it the grain, the water, or the bakers' hands), and what they offer to the future of bread. The story of sourdoughs is one with many lessons for the story of agriculture more generally. It also bears directly on our understanding of the relationship between the microbes of our bodies and those of our food.

The immune system is complex and dynamic, focused on defending the body from a host of pathogens ranging from viruses to cancer. A number of different mechanisms have evolved that help the body fight to control cancers and other disease. Unfortunately, sometimes these defenses break down and become compromised, enabling cancers to survive and grow within the host.

Recent breakthroughs have shown that Immunotherapeutic approaches may be successful in helping the host's immune system battle cancer, providing the body with ways to protect itself from cancer.  Key to making this work is an understanding of both who will benefit from the therapy and at what stage in disease progression the therapy will be effective.  In our discussion we will look at how biomarker development can aide in addressing these challenges.  We will cover:

•    Overview of Immunotherapies & their mechanisms of action
•    Biomarkers associated with Immunotherapies
•    Biomarker clinical testing
•    Immunotherapy adverse reactions & success stories 

Virus – host interactions are currently among the most intensively studied research areas due to the promising new antiviral approaches emerging from these studies. Indeed, RNA viruses, which are important and emerging human, animal and plant pathogens, exploit host cells by recruiting host factors and escaping host antiviral responses. RNA viruses induce the deformation of cellular membrane structures to build extensive viral replication organelles. Approaches interfering with virus replication could be key to limit the significant harm caused by viruses to human health and major losses in agriculture. We study tombusviruses, which are small RNA viruses, as model plant viruses to study virus replication and virus - host interactions using yeast (Saccharomyces cerevisiae) as a surrogate host. Since viruses are intracellular parasites that use the ample resources of eukaryotic cells, it is feasible to study virus replication in model eukaryotic yeast cells. Several systematic genome-wide screens and global proteomic and lipidomic approaches have led to the identification of ~500 host proteins/genes that are implicated in TBSV replication. We characterized the role of 30 co-opted host proteins, sterols and phospholipids in tombusvirus replication. Finally, we also show evidence that tombusviruses usurp the glycolytic (metabolic) pathway to obtain energy to build the extensive virus replication organelles. Altogether, this virus-host system allows for rapid discovery of novel cellular factors affecting plant virus replication. This advance could immensely help other scientists working with less tractable, but devastating viral pathogens for which similar studies are currently more difficult to conduct. 

Increasing prevalence and severity of multi-drug-resistant (MDR) bacterial infections has necessitated novel antibacterial strategies.  Ideally, new approaches would target bacterial pathogens while exerting selection for reduced pathogenesis when these bacteria inevitably evolve resistance to therapeutic intervention. As an example of such a management strategy, we isolated a lytic bacteriophage, OMKO1, (family Myoviridae) of Pseudomonas aeruginosa that utilizes the outer membrane porin M (OprM) of the multidrug efflux systems MexAB and MexXY as a receptor-binding site. Results show that phage selection produces an evolutionary trade-off in MDR P. aeruginosa, whereby the evolution of bacterial resistance to phage attack changes the efflux pump mechanism, causing increased sensitivity to drugs from several antibiotic classes. Although modern phage therapy is still in its infancy, we conclude that phages, such as OMKO1, represent a new approach to phage therapy where bacteriophages exert selection for MDR bacteria to become increasingly sensitive to traditional antibiotics. This approach, using phages as targeted antibacterials, could extend the lifetime of our current antibiotics and potentially reduce the incidence of antibiotic resistant infections.

Prokaryotes have long been at war with bacteriophage. The evolutionary pressure of this struggle has led both sides to develop sophisticated defenses. Notably, CRISPR-Cas systems evolved to let bacteria and archaea record past infections and rapidly squelch subsequent viral invasions. First, CRISPR proteins capture nucleic acids to build a genomic gallery of "molecular mugshots." Next, the mugshots are printed into RNA copies and distributed amongst CRISPR interference proteins. These RNA-guided bounty hunters precisely identify and cleave offending genomes to halt infection. No system is fool-proof, however, and recent work has elucidated an impressive arsenal of "anti-CRISPR" proteins that bacteriophage deploy to evade destruction. Studying this on-going arms race has yielded a treasure trove of biotechnology tools. I will discuss the stages of CRISPR immunity while highlighting how scientists have co-opted its various proteins for diverse applications.

Adjunct probiotic therapy has the potential to decrease Clostridium difficile disease incidence and severity. After screening several potential probiotic bacteria for intrinsic resistance to C. difficile antibiotics (vancomycin, metronidazole and fidaxomicin), we determined that L. reuteri strains are ideal adjunct therapy candidates. We evaluated the ability of reuterin - a secondary metabolite produced by specific strains of L. reuteri during fermentation of the prodrug glycerol - to inhibit C. difficile growth in vitro.  We found that levels of reuterin produced by L. reuteri 17938 were more potent than vancomycin in inhibiting C. difficile growth. Using human fecal microbiota bioreactors, we showed that co-delivery of L. reuteri with glycerol is effective against C. difficile colonization of a complex-microbial community, whereas treatment with either glycerol or L. reuteri alone was ineffective.  Co-delivery of L. reuteri and glycerol changed microbial community profiles and associated metabolites consistent with glycerol fermentation and reuterin production.  Ex vivo studies showed that this combination therapy resulted in lower C. difficile burden and toxicity in stool and that this was dependent upon reuterin production. Taken together, prodrug-based adjunct therapy with probiotic L. reuteri is a viable option for preventative treatment of C. difficile infection.