Question In Microbiology Research Articles

Post-Validation of Multiple-Choice Questions in Microbiology

Post-Validation of Multiple-Choice Questions in Microbiology

Microbial population dynamics decouple growth response from environmental nutrient concentration

How the growth rate of a microbial population responds to the environmental availability of chemical nutrients and other resources is a fundamental question in microbiology. Models of this response, such as the widely used Monod model, are generally characterized by a maximum growth rate and a half-saturation concentration of the resource. What values should we expect for these half-saturation concentrations, and how should they depend on the environmental concentration of the resource? We survey growth response data across a wide range of organisms and resources. We find that the half-saturation concentrations vary across orders of magnitude, even for the same organism and resource. To explain this variation, we develop an evolutionary model to show that demographic fluctuations (genetic drift) can constrain the adaptation of half-saturation concentrations. We find that this effect fundamentally differs depending on the type of population dynamics: Populations undergoing periodic bottlenecks of fixed size will adapt their half-saturation concentrations in proportion to the environmental resource concentrations, but populations undergoing periodic dilutions of fixed size will evolve half-saturation concentrations that are largely decoupled from the environmental concentrations. Our model not only provides testable predictions for laboratory evolution experiments, but it also reveals how an evolved half-saturation concentration may not reflect the organism's environment. In particular, this explains how organisms in resource-rich environments can still evolve fast growth at low resource concentrations. Altogether, our results demonstrate the critical role of population dynamics in shaping fundamental ecological traits.

Quorum-Sensing Master Regulator VfmE Is a c-di-GMP Effector That Controls Pectate Lyase Production in the Phytopathogen Dickeya dadantii.

ABSTRACTDickeya dadantii is a phytopathogenic bacterium that causes diseases on a wide range of host plants. The pathogen secretes pectate lyases (Pel) through the type II secretion system (T2SS) that degrades the cell wall in host plants. The virulence of D. dadantii is controlled by the second messenger cyclic diguanylate monophosphate (c-di-GMP), and the homeostasis of c-di-GMP is maintained by a number of diguanylate cyclases and phosphodiesterases. Deletion of a phosphodiesterase ecpC repressed pelD transcription, and such repression can be suppressed by an additional deletion in vfmE. VfmE is an AraC type of transcriptional regulator in the Vfm quorum-sensing system. Our results suggest that VfmE is a c-di-GMP effector that functions as an activator of pel at low c-di-GMP concentrations and a repressor of pel at high c-di-GMP concentrations through regulation of the transcriptional activator SlyA. Multiple sequence alignment with known c-di-GMP effectors identified an RWIWR motif in VfmE that we demonstrate is required for the c-di-GMP binding. Mutation of R93D in the RxxxR motif eliminates the c-di-GMP-related phenotypes in Pel activity. Our results show that VfmE is not only a quorum-sensing regulator but also a c-di-GMP effector, suggesting that D. dadantii integrates the c-di-GMP signaling network with the Vfm quorum-sensing pathway during environmental adaptation.IMPORTANCE How bacteria integrate environmental cues from multiple sources to appropriately regulate adaptive phenotypes is a central question in microbiology. In Dickeya dadantii, the quorum-sensing regulator VfmE controls the key virulence factor pectate lyase (Pel). Here, we demonstrate that VfmE also binds to c-di-GMP, resulting in VfmE functioning as an activator of pel at low c-di-GMP concentrations and repressor of pel at high c-di-GMP concentrations. The RWIWR motif in VfmE is required for c-di-GMP binding, and mutation of the motif in the mutant R93D eliminates the c-di-GMP-related phenotypes in Pel activity. We propose that VfmE is an important mediator to integrate quorum-sensing signals with c-di-GMP to collectively regulate D. dadantii pathogenesis.

Deciphering the physiological response of Escherichia coli under high ATP demand

One long‐standing question in microbiology is how microbes buffer perturbations in energy metabolism. In this study, we systematically analyzed the impact of different levels of ATP demand in Escherichia coli under various conditions (aerobic and anaerobic, with and without cell growth). One key finding is that, under all conditions tested, the glucose uptake increases with rising ATP demand, but only to a critical level beyond which it drops markedly, even below wild‐type levels. Focusing on anaerobic growth and using metabolomics and proteomics data in combination with a kinetic model, we show that this biphasic behavior is induced by the dual dependency of the phosphofructokinase on ATP (substrate) and ADP (allosteric activator). This mechanism buffers increased ATP demands by a higher glycolytic flux but, as shown herein, it collapses under very low ATP concentrations. Model analysis also revealed two major rate‐controlling steps in the glycolysis under high ATP demand, which could be confirmed experimentally. Our results provide new insights on fundamental mechanisms of bacterial energy metabolism and guide the rational engineering of highly productive cell factories.

AFM in cellular and molecular microbiology.

The unique capabilities of the atomic force microscope (AFM), including super-resolution imaging, piconewton force-sensitivity, nanomanipulation and ability to work under physiological conditions, have offered exciting avenues for cellular and molecular biology research. AFM imaging has helped unravel the fine architectures of microbial cell envelopes at the nanoscale, and how these are altered by antimicrobial treatment. Nanomechanical measurements have shed new light on the elasticity, tensile strength and turgor pressure of single cells. Single-molecule and single-cell force spectroscopy experiments have revealed the forces and dynamics of receptor-ligand interactions, the nanoscale distribution of receptors on the cell surface and the elasticity and adhesiveness of bacterial pili. Importantly, recent force spectroscopy studies have demonstrated that extremely stable bonds are formed between bacterial adhesins and their cognate ligands, originating from a catch bond behaviour allowing the pathogen to reinforce adhesion under shear or tensile stress. Here, we survey how the versatility of AFM has enabled addressing crucial questions in microbiology, with emphasis on bacterial pathogens. TAKE AWAYS: AFM topographic imaging unravels the ultrastructure of bacterial envelopes. Nanomechanical mapping shows what makes cell envelopes stiff and resistant to drugs. Force spectroscopy characterises the molecular forces in pathogen adhesion. Stretching pili reveals a wealth of mechanical and adhesive responses.

1128. Online Spaced Education to Teach Microbiology to Medical Students in a Threaded Medical School Curriculum

BackgroundA strong foundation in microbiology continues to be essential for physicians-in-training. Little research exists examining pre-clinical microbiology education in undergraduate medical education (UME) curricular structures. Further, no study has evaluated the use of a spaced repetition model for pre-clinical UME students studying microbiology in a threaded curriculum.MethodsWe conducted a prospective cohort design study and enrolled 81 out of 154 (53%) first-year medical students at Oregon Health & Science University from August 2018 through December 2019. The first 18 months of the UME curriculum is organized by organ system blocks with end-of-block exams including retired National Board of Medical Examiner (NBME) questions. Participants were invited to complete 10 microbiology questions using the spaced practice online platform QSTREAM weekly. Performance between participants and non-participants on end of block NBME exams were compared using t-tests for categorical variables.ResultsAt the conclusion of the study, 42.5% of participants were “very active” (questions in < 2 days), 7.5% of participants were “active” (questions in 3-7 days), and 50.0% of participants were “inactive” (questions in >7 days). Student performance on second-pass questions improved by 41%, and NBME end-of-block exams demonstrated improved performance in each block compared to non-participants. Specifically, performance in the Skin, Bones, and Musculature end-of-block exam and Developing Human end-of-block exam was significantly (p=0.0001, 0.008, respectively) improved, and study participants outperformed non-participants on topics practiced in the study.ConclusionAs more medical schools move to a threaded curriculum, with many of these programs proposing fewer contact hours, innovative methods targeting microbiology education should be explored for pre-clinical medical students. Online spaced practice in a threaded curriculum could provide a feasible and acceptable pedagogical technique for UME and add to the discourse around microbiology curriculum development. With initial start-up costs, sustaining such a program across medical school curricula is an inexpensive, innovative, technologically-savvy approach to medical education.Disclosures All Authors: No reported disclosures

Evaluation of Online Progressive Examinations of Infectious Diseases and Clinical Microbiology Specialty Students

Introduction: Progressive examinations are required to monitor the development of research assistants in the field of Infectious Diseases and Clinical Microbiology. However, the preparation, conduct and evaluation of these exams also require especial features and experience. In this study, it was aimed to review the experience obtained in the online progressive exams of research assistants conducted by the education commission (EMEK) in the field of IDCM specialization. Materials and Methods: There are two online formative exams per year for research assistants in the IDCM field. In this study, the examinations between 2014-2018 were evaluated. The validity and reliability analyses for each exam were performed, and the difficulty and discrimination indexes of the questions were calculated. Topics with the least correct answers were determined. Results: Over the years, an increase in the number of research assistants was observed. The internal consistency coefficient of the exams was the lowest of the cronbach alpha value of 0.69 and the highest of 0.92. Mean score of the exam was over 69. It was seen that some questions need to be revised in order to be transferred to the question bank. Although clinically rare diseases and basic microbiology questions were answered less frequently, the rate of responding to diseases that are frequently seen in the clinic was higher. Conclusion: In specialist training, online progressive exams facilitate broad-based measurement and evaluation at national level. With the increase of our online exam experience, we can see that there are still aspects that need improvement.

(p)ppGpp: the magic governor of bacterial growth economy.

A fundamental question in microbiology is how bacterial cells manage to coordinate gene expression with cell growth during adapting to various environmental conditions. Although the cellular responses to changing environments have been extensively studied using transcriptomic and proteomic approaches, it remains poorly understood regarding the molecular strategy enabling bacteria to manipulate the global gene expression patterns. The alarmone (p)ppGpp is a key secondary messenger involved in regulating various biochemical and physiological processes of bacterial cells. However, despite of the extensive studies of (p)ppGpp signaling in stringent response during the past 50years, the connection between (p)ppGpp and exponential growth remains poorly understood. Our recent work demonstrates that (p)ppGpp is strongly involved in regulating cell growth of Escherichia coli through balancing the cellular investment on metabolic proteins and ribosomes, highlighting itself as a magic governor of bacterial global resource allocation. In this mini-review, we briefly summarize some historical perspectives and current progress of the relation between (p)ppGpp and bacterial exponential growth. Two important future directions are also highlighted: the first direction is to elucidate the cellular signal that triggers (p)ppGpp accumulation during poor growth conditions; the second direction is to investigate the relation between (p)ppGpp and exponential growth for bacterial species other than E. coli.

Viscoelastic response ofEscherichia colibiofilms to genetically altered expression of extracellular matrix components

How the viscoelastic properties of the extracellular matrix affect the various biological functions conferred by biofilms is an important question in microbiology. In this study, the viscoelastic response of Escherichia coli biofilms to the genetically altered expression of extracellular matrix components was studied. Biofilms of the wild type E. coli MG1655 and its mutant strains producing different amounts of extracellular matrix components (curli, colanic acid, and poly-β-1,6-N-acetyl-d-glucosamine) were used to examine the viscoelastic behavior of biofilms grown at the solid-atmosphere interface. The results suggest that the presence of curli proteins dominates biofilm mechanical behavior. The rheological data indicate that the cohesive energy of the biofilm was the highest in the wild type strain. The results demonstrate the importance of extracellular matrix composition for biofilm mechanical properties. We propose that by genetically altering the expression of extracellular matrix polymers, bacteria are able to modulate the mechanical properties of their local environment in accordance with bulk environmental conditions.

Time-of-flight secondary ion mass spectrometry in the helium ion microscope

A helium ion microscope, known for high resolution imaging and modification with helium or neon ions, has been equipped with a time-of-flight spectrometer for compositional analysis. Here we report on its design, implementation and show first results of this powerful add-on. Our design considerations were based on the results of detailed ion collision cascade simulations that focus on the physically achievable resolution for various detection limits. Different secondary ion extraction geometries and spectrometer types are considered and compared with respect to the demands and limitations of the microscope. As a result the development and evaluation of a secondary ion extraction optics and time-of-flight spectrometer that allows the parallel measurement of all secondary ion masses is reported. First experimental results demonstrate an excellent mass resolution as well as high-resolution secondary ion imaging capabilities with sub-8 nm lateral resolution. The combination of high resolution secondary electron images and mass-separated sputtered ion distributions have a high potential to answer open questions in microbiology, cell biology, earth sciences and materials research.

High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries

A fundamental question in microbiology is whether there is continuum of genetic diversity among genomes, or clear species boundaries prevail instead. Whole-genome similarity metrics such as Average Nucleotide Identity (ANI) help address this question by facilitating high resolution taxonomic analysis of thousands of genomes from diverse phylogenetic lineages. To scale to available genomes and beyond, we present FastANI, a new method to estimate ANI using alignment-free approximate sequence mapping. FastANI is accurate for both finished and draft genomes, and is up to three orders of magnitude faster compared to alignment-based approaches. We leverage FastANI to compute pairwise ANI values among all prokaryotic genomes available in the NCBI database. Our results reveal clear genetic discontinuity, with 99.8% of the total 8 billion genome pairs analyzed conforming to >95% intra-species and <83% inter-species ANI values. This discontinuity is manifested with or without the most frequently sequenced species, and is robust to historic additions in the genome databases.

Using Unlabeled Data to Discover Bivariate Causality with Deep Restricted Boltzmann Machines.

An important question in microbiology is whether treatment causes changes in gut flora, and whether it also affects metabolism. The reconstruction of causal relations purely from non-temporal observational data is challenging. We address the problem of causal inference in a bivariate case, where the joint distribution of two variables is observed. We consider, in particular, data on discrete domains. The state-of-the-art causal inference methods for continuous data suffer from high computational complexity. Some modern approaches are not suitable for categorical data, and others need to estimate and fix multiple hyper-parameters. In this contribution, we introduce a novel method of causal inference which is based on the widely used assumption that if X causes Y, then P(X) and P(Y|X) are independent. We propose to explore a semi-supervised approach where P(Y|X) and P(X) are estimated from labeled and unlabeled data respectively, whereas the marginal probability is estimated potentially from much more (cheap unlabeled) data than the conditional distribution. We validate the proposed method on the standard cause-effect pairs. We illustrate by experiments on several benchmarks of biological network reconstruction that the proposed approach is very competitive in terms of computational time and accuracy compared to the state-of-the-art methods. Finally, we apply the proposed method to an original medical task where we study whether drugs confound human metagenome.

Whole genome sequencing for diagnosing infectious diseases

Given the complex nature of diagnostic practices for infectious diseases, the one unifying characteristic of the agents investigated is their possession of a genome. While many molecular diagnostic tests investigate parts of the genome, whole-genome sequencing (WGS) has emerged as a cost-effective and convenient approach for answering the varied microbiological questions that arise. In that regard, four essential tasks are addressed: species identification, phenotypic characterization, e.g., antimicrobial resistance and virulence, evolutionary traits and pathogen tracking. Some of the benefits already available include, in addition to the ability to fully characterize prospective and retrospective isolates, the ability to be applied directly to clinical samples for simultaneous identification and predicting antimicrobial susceptibilities (even in culture-negative cases); identification in polymicrobial samples; generating regional phylogenomic sublineages; correlation of genomic features with strains of clinical importance; tracking of multidrug resistant organisms in hospitals and communities. These benefits, while largely seen in reports from North America and Europe, are also being observed in Latin America and the Caribbean. Further, WGS allows for estimation of mutation rates among isolates, as well as assessment of the rates and breadth of horizontal gene flow within and between species, the evolution of new virulence traits, and the role of host genetics in host-pathogen interactions, all with the view to develop novel therapeutic interventions. The main directions of WGS applications include diagnosis and control of local and regional infectious diseases; detection of multidrug resistance (MDR) and virulence characteristics; surveillance of pathogen evolution and transmission dynamics; development of new (portable) diagnostic tests and assays for use in clinics and outpatient facilities; and discovery of novel antimicrobial drugs and therapeutics and assessment of their prevention and control. Given that pathogens occur in complex microbial communities, further elaborations to explore the pathobiome using metagenomics and high-throughput sequencing will assist with the understanding of the forces supporting population structure, including pathogen plasticity.

Big Questions in Microbiology

Big Questions in Microbiology

Development of a genus-specific next generation sequencing approach for sensitive and quantitative determination of the Legionella microbiome in freshwater systems

BackgroundNext Generation Sequencing (NGS) has revolutionized the analysis of natural and man-made microbial communities by using universal primers for bacteria in a PCR based approach targeting the 16S rRNA gene. In our study we narrowed primer specificity to a single, monophyletic genus because for many questions in microbiology only a specific part of the whole microbiome is of interest. We have chosen the genus Legionella, comprising more than 20 pathogenic species, due to its high relevance for water-based respiratory infections.MethodsA new NGS-based approach was designed by sequencing 16S rRNA gene amplicons specific for the genus Legionella using the Illumina MiSeq technology. This approach was validated and applied to a set of representative freshwater samples.ResultsOur results revealed that the generated libraries presented a low average raw error rate per base (<0.5%); and substantiated the use of high-fidelity enzymes, such as KAPA HiFi, for increased sequence accuracy and quality. The approach also showed high in situ specificity (>95%) and very good repeatability. Only in samples in which the gammabacterial clade SAR86 was present more than 1% non-Legionella sequences were observed. Next-generation sequencing read counts did not reveal considerable amplification/sequencing biases and showed a sensitive as well as precise quantification of L. pneumophila along a dilution range using a spiked-in, certified genome standard. The genome standard and a mock community consisting of six different Legionella species demonstrated that the developed NGS approach was quantitative and specific at the level of individual species, including L. pneumophila. The sensitivity of our genus-specific approach was at least one order of magnitude higher compared to the universal NGS approach. Comparison of quantification by real-time PCR showed consistency with the NGS data. Overall, our NGS approach can determine the quantitative abundances of Legionella species, i. e. the complete Legionella microbiome, without the need for species-specific primers.ConclusionsThe developed NGS approach provides a new molecular surveillance tool to monitor all Legionella species in qualitative and quantitative terms if a spiked-in genome standard is used to calibrate the method. Overall, the genus-specific NGS approach opens up a new avenue to massive parallel diagnostics in a quantitative, specific and sensitive way.

Oxygen Tension and Riboflavin Gradients Cooperatively Regulate the Migration of Shewanella oneidensis MR-1 Revealed by a Hydrogel-Based Microfluidic Device

Shewanella oneidensis is a model bacterial strain for studies of bioelectrochemical systems (BESs). It has two extracellular electron transfer pathways: (1) shuttling electrons via an excreted mediator riboflavin; and (2) direct contact between the c-type cytochromes at the cell membrane and the electrode. Despite the extensive use of S. oneidensis in BESs such as microbial fuel cells and biosensors, many basic microbiology questions about S. oneidensis in the context of BES remain unanswered. Here, we present studies of motility and chemotaxis of S. oneidensis under well controlled concentration gradients of two electron acceptors, oxygen and oxidized form of riboflavin (flavin+), using a newly developed microfluidic platform. Experimental results demonstrate that either oxygen or flavin+ is a chemoattractant to S. oneidensis. The chemotactic tendency of S. oneidensis in a flavin+ concentration gradient is significantly enhanced in an anaerobic in contrast to an aerobic condition. Furthermore, either a low oxygen tension or a high flavin+ concentration considerably enhances the speed of S. oneidensis. This work presents a robust microfluidic platform for generating oxygen and/or flavin+ gradients in an aqueous environment, and demonstrates that two important electron acceptors, oxygen and oxidized riboflavin, cooperatively regulate S. oneidensis migration patterns. The microfluidic tools presented as well as the knowledge gained in this work can be used to guide the future design of BESs for efficient electron production.

FtsZ-Dependent Elongation of a Coccoid Bacterium.

ABSTRACTA mechanistic understanding of the determination and maintenance of the simplest bacterial cell shape, a sphere, remains elusive compared with that of more complex shapes. Cocci seem to lack a dedicated elongation machinery, and a spherical shape has been considered an evolutionary dead-end morphology, as a transition from a spherical to a rod-like shape has never been observed in bacteria. Here we show that a Staphylococcus aureus mutant (M5) expressing the ftsZG193D allele exhibits elongated cells. Molecular dynamics simulations and in vitro studies indicate that FtsZG193D filaments are more twisted and shorter than wild-type filaments. In vivo, M5 cell wall deposition is initiated asymmetrically, only on one side of the cell, and progresses into a helical pattern rather than into a constricting ring as in wild-type cells. This helical pattern of wall insertion leads to elongation, as in rod-shaped cells. Thus, structural flexibility of FtsZ filaments can result in an FtsZ-dependent mechanism for generating elongated cells from cocci.

The application and research progress of whole-genome sequencing in clinical microbiology detection

Whole-genome sequencing of bacteria has recently emerged as a cost-effective and convenient approach for resolving many microbiological questions. Here, the current status of whole-genome sequencing in clinical microbiology and the main problems that exist were reviewed, focusing on six essential tasks: identifying and culturing the species of an isolate, rapid identification of pathogen properties, monitoring the emergence and spread of an epidemic outbreak, developing vaccine and monitoring its variation, metagenomics and single-cell sequencing. The authors predicted that the application of whole-genome sequencing will soon be sufficiently fast, accurate and cheap to be used in routine etiological detection, even though there is still a long way to go.(Chin J Lab Med, 2016, 39: 319-321) Key words: Microbiological techniques; Genomics; Sequence analysis; Metagenomics; Single-cell analysis

Nitrate and Inhibition of Ruminal Methanogenesis: Microbial Ecology, Obstacles, and Opportunities for Lowering Methane Emissions from Ruminant Livestock.

Ruminal methane production is among the main targets for greenhouse gas (GHG) mitigation for the animal agriculture industry. Many compounds have been evaluated for their efficacy to suppress enteric methane production by ruminal microorganisms. Of these, nitrate as an alternative hydrogen sink has been among the most promising, but it suffers from variability in efficacy for reasons that are not understood. The accumulation of nitrite, which is poisonous when absorbed into the animal’s circulation, is also variable and poorly understood. This review identifies large gaps in our knowledge of rumen microbial ecology that handicap the further development and safety of nitrate as a dietary additive. Three main bacterial species have been associated historically with ruminal nitrate reduction, namely Wolinella succinogenes, Veillonella parvula, and Selenomonas ruminantium, but others almost certainly exist in the largely uncultivated ruminal microbiota. Indications are strong that ciliate protozoa can reduce nitrate, but the significance of their role relative to bacteria is not known. The metabolic fate of the reduced nitrate has not been studied in detail. It is important to be sure that nitrate metabolism and efforts to enhance rates of nitrite reduction do not lead to the evolution of the much more potent GHG, nitrous oxide. The relative importance of direct inhibition of archaeal methanogenic enzymes by nitrite or the efficiency of capture of hydrogen by nitrate reduction in lowering methane production is also not known, nor are nitrite effects on other members of the microbiota. How effective would combining mitigation methods be, based on our understanding of the effects of nitrate and nitrite on the microbiome? Answering these fundamental microbiological questions is essential in assessing the potential of dietary nitrate to limit methane emissions from ruminant livestock.

Contribution of Physical Interactions to Signaling Specificity between a Diguanylate Cyclase and Its Effector.

ABSTRACTCyclic diguanylate (c-di-GMP) is a bacterial second messenger that controls multiple cellular processes. c-di-GMP networks have up to dozens of diguanylate cyclases (DGCs) that synthesize c-di-GMP along with many c-di-GMP-responsive target proteins that can bind and respond to this signal. For such networks to have order, a mechanism(s) likely exists that allow DGCs to specifically signal their targets, and it has been suggested that physical interactions might provide such specificity. Our results show a DGC from Pseudomonas fluorescens physically interacting with its target protein at a conserved interface, and this interface can be predictive of DGC-target protein interactions. Furthermore, we demonstrate that physical interaction is necessary for the DGC to maximally signal its target. If such “local signaling” is a theme for even a fraction of the DGCs used by bacteria, it becomes possible to posit a model whereby physical interaction allows a DGC to directly signal its target protein, which in turn may help curtail undesired cross talk with other members of the network.