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Indigenous populations living on reserves in Canada today are still experiencing uneven access to drinking water services. Past studies of First Nation communities have observed microbiological contamination where no drinking water advisory was issued for fecal indicator bacteria (FIB) and detection of emerging contaminants: antibiotic resistance genes (ARG). The first study sought to observe fluctuations in the distribution system of traditional coliform indicators against antibiotic resistance genes as well as Campylobacter spp. quantification. Results found repeated detection of indicator bacteria from drinking water samples in homes with concrete cisterns in community B, as well as in community D up to 600 CFU/ 100 mL coliforms. Both Campylobacter coli and Campylobacter jejuni, diarrheal infection culprits, were also observed in instances without the previous or same-time detection of coliform, including at sampling sites like treated water from taps at the water treatment plant and piped homes. ARGs were more apparent in post-treated water from community B than from community D and in water treatment plant and piped samples. The second study sought to characterize ARGs through shotgun metagenomics to establish the resistome from water. Results determined pre-treated source water samples were characteristically distinct from post-treated cistern water from each community suggesting the presence of an alternative source of contamination for stored drinking water. Indigenous nations continue to experience water insecurity and these study results reinforce community concerns over water quality thus we aim to support change led within communities for strengthening the management of water distribution to homes.

Disease-associated changes to the gastrointestinal (GI) microbiome have been detected in a variety of chronic human illnesses. Currently, GI microorganisms are thought to play a major role in systemic health and homeostasis through interactions with the immune system of their host. In recent years, the available technology for capturing and characterizing the GI microbiome has expanded greatly, resulting in a rapid expansion of the field. In particular, culture-independent technologies allowing for direct analysis of microbial genes, transcripts, proteins, and other metabolites (collectively referred to as meta-omics) from stool samples show potential for personalized disease detection and monitoring through non-invasive screening. However, due to the high degree of variability inherent in microbiome profiles, establishing a consensus for microbial signatures or biomarkers of disease across studies is difficult. Differences in study methodology can further complicate comparisons, since the laboratory protocols for sequence-based data capture and analysis are varied, and there are several commercial kits and reagents available for the storage and isolation of microbial DNA and RNA from stool for downstream microbiome profiling. Research has shown that the choice of nucleic acid extraction kit and storage method can affect resulting nucleic acid quality. In the current methodological study, a single stool sample from a healthy donor is divided and processed using multiple commercially available methods for nucleic acid stabilization and isolation in order to evaluate the effect of processing on observed sequence-based microbiome profiles. The research herein demonstrates that the experimental methodology used to stabilize and isolate nucleic acids from human stool samples can significantly impact the ability to capture GI microbiome diversity from metagenomic and metatranscriptomic data. Notably, GI microbiome characteristics commonly used as health markers in disease

Introduction: C. difficile spores are dispersed throughout the environment and can asymptomatically colonize and/or infect animals. Previous studies have shown that C. difficile spores can be isolated from commercially available beef, veal, pork, vegetables, and seafood. However, a definitive link has yet to have been made between food contamination and hospitalized cases. This study aims to isolate C. difficile from retail meat samples and compare them to human isolates. Methods: Frozen retail pork, beef, and veal samples were obtained from the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) program and FoodNet Canada. These samples were analyzed for C. difficile contamination by direct plating on selective media and by inoculation in enrichment broth. Suspected C. difficile colonies were confirmed by polymerase chain reaction (PCR). Toxigenic C. difficile isolates were molecularly characterized by ribotyping and pulsed-field gel electrophoresis (PFGE). Antibiotic susceptibility was determined by ETEST® strips. Whole genome sequencing (WGS) was performed on all C. difficile isolates from retail meats and from select human cases. Results: Overall, toxigenic C. difficile was isolated from 10 of 644 retail meat samples (1.6%). All 10 isolates were A/B toxin positive. Additionally, 2 isolates were found to harbor binary toxin. All retail meat isolates were susceptible to vancomycin, metronidazole, tigecycline, rifampin, and clindamycin, excluding 1 NAP1 isolate that was resistant to moxifloxacin. Molecular typing revealed strain types commonly found in human clinical isolates (e.g. NAP1 (RT027), NAP4 (RTNS195), and NAP11 (RT106)). The closest related human clinical isolate to the C. difficile isolates from retail meats differed by 8-34 SNVs when analyzed at the highest resolution (84.54%-95.02% core genome). Conclusion: A low percentage of retail meats (1.6%) were contaminated by C. difficile. All ribotypes and NAP types of C. difficile i

Helicobacter pylori is a Gram-negative, flagellate, microaerophilic member of the Epsilonproteobacteria, with a spiral or curved bacilli morphology (Marshall and Warren 1984). The bacterium resides in the human stomach, and infections can span decades, often going undetected due to minor pathogenesis (Kusters et al. 2006). Infections frequency is higher in the Global South, and is associated with low socioeconomic status, education, remoteness, and inaccessibility to healthcare (Hooi et al. 2017; Bruce and Maaroos 2008). This infection is a public health concern because H. pylori infection increases the risk of adenocarcinoma of the gastric mucosa and gastric B-cell mucosal-associated lymphoid tissue (MALT) lymphoma, as well as peptic ulcer disease (Haley and Gaddy 2015). Indigenous communities in Canada experience increased rates of H. pylori infection, which is associated with poverty, crowded living conditions, and inaccessible health care. Infection is a concern for Indigenous communities given the bacterium's implication in the development of associated cancers, which is a severe health burden that too disproportionately affects Indigenous peoples. This project builds upon ongoing community-driven research by the Canadian North H. pylori (CANHelp) Working Group investigating the impact of H. pylori infection. In this research project we present the sequence H. pylori genomes of 57 CANHelp isolates sampled from the Western Canadian Arctic communities of Aklavik, Inuvik, Old Crow, Ross River, Teslin, and Fort McPherson and a comparator cohort of 73 southern Manitoban isolates. I developed and implemented a contamination detection pipeline to ensure only H. pylori sequence reads were used in genome assembly. The genomes were analyzed for their presence of: known markers associated with antimicrobial resistance to clarithromycin, metronidazole, levofloxacin, and amoxicillin; virulence factors like cagA, the cagPI, vacA, outer membrane proteins such as babA, and the

Coronavirus outbreaks have been increasing in both frequency and magnitude for the last 20 years. Prior to 2002, there were two identified human coronaviruses. Now in 2022 there are seven, potentially eight. From severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002, to Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 and SARS-CoV-2 in 2019, coronavirus outbreaks have continued to escalate, causing more infections and more deaths with each successive outbreak. SARS-CoV-2 was declared a pandemic on March 11, 2020, by the World Health Organization. To date, there have been around 460 million cases and over 6 million deaths, worldwide. The need for therapeutic interventions for SARS-CoV-2 is continuing, but even greater is the need for broad spectrum coronavirus therapeutics that may be used for this and other coronavirus outbreaks. Due to the critical role of ACE2 in the replication of SARS-CoV, SARS-CoV-2 and human coronavirus (HCoV)-NL63, ACE2 has become a prime target for therapeutic intervention. Groups have tested chimerics of ACE2 fused to an Fc peptide: ACE2-Fc as a decoy molecule that would bind the receptor binding domain (RBD) of SARS-CoV-2 with the same or greater affinity than ACE2 on the surface of cells. Here is described the production and evaluation of seven ACE2-Fc chimerics, and the down-selection to one lead candidate as a potent inhibitor of SARS-CoV-2 cell entry. All ACE2-Fc chimerics were sequence verified, their expression was confirmed in HEK293Ts using western blots and their relative neutralization capacity was tested in in vitro neutralization assays. Evaluation of these constructs led to the identification of a lead construct, which was a potent inhibitor of a SARS-CoV-2 D614G pseudotype luciferase reporter virus. Additional testing of the lead ACE2-Fc construct in wild-type SARS-CoV-2 also demonstrated potent neutralization. Further evaluation of this protein in vivo will allow for determination of dose range, and

Copper is an antimicrobial metal used in sink drains that is becoming ineffective due to copper tolerance of species such as Pseudomonas aeruginosa enhanced by the presence of the genomic island GI-7. Studies of copper tolerance associated with GI-7 are hindered by the lack of accurate planktonic and biofilm testing methods to determine copper susceptibility, and a lack of knowledge regarding the prevalence of this sequence amongst Canadian Pseudomonas isolates. In chapter 3, we developed a new deep well biofilm device that provided increased surface area for biofilm biomass formation. This method can be used to screen antimicrobial susceptibility in a 96-well high throughput format. The increased surface area allowed for greater biofilm biomass per mm2 as compared to the standard device and revealed plastic preferences in biofilm biomass formation by P. aeruginosa and Escherichia coli. Both devices produced different minimum biofilm eradication (MBEC) values for benzalkonium chloride and bleach, yet both were suitable for biofilm cultivation. In chapter 4, we examined 2467 genome sequenced Pseudomonas isolates collected from a 2017-2019 Ontario hospital intensive care unit study. Isolates from patients and hospital rooms with regular and copper sink drains were collected and used to explore if GI-7 presence was associated with copper sink drains. This analysis revealed that GI-7 is widely spread across clinical and environmental isolates of certain multi-locus sequence types, but statistical analyses did not show significant associations between GI-7 and copper sink drains due limited sampling from copper sink drains. Some correlation trends amongst GI-7 isolates were noted and discussed. Finally, phenotypic characterization of P. aeruginosa GI-7 copper tolerance through planktonic and biofilm culturing methods was examined with various P. aeruginosa strains. Copper salt addition to media caused lethal acidification that inhibited both planktonic and biofilm cultur

Background: Over 296 million people worldwide are living with chronic hepatitis B (CHB) infection who require monitoring of viral activity and disease progression. Serum HBV RNA is a promising, although poorly characterized in its encapsidated form, new biomarker in CHB management. No standardized method for serum HBV RNA quantification has been established. Aims: The project aims to characterize the HBV serum transcriptome in order to better understand its composition and to develop and validate, both analytically and clinically, a 3′ RACE RT-qPCR assay for quantification of relevant transcripts within serum HBV RNA. Methods: Nanopore long read sequencing and Northern blotting were employed to characterize the HBV RNA in the serum of 13 patients in different phases of CHB. Sequencing data analysis was done using three isoform detection workflows. A 3′ RACE RT-qPCR method was developed using published primers for the most relevant RNA species determined by serum HBV RNA characterization. The analytical limit of detection and quantification, linearity, inter- and intra-assay repeatability, and clinical specificity and sensitivity were evaluated using synthetic pre-genomic RNA (pgRNA) and specimens from various patient populations. Intra- and inter-laboratory ring trials involving three laboratories were completed. Results: Long read sequencing found higher proportions of spliced variants in patients with HBV genotype B and C, and in HBeAg positive patients with ALT ≤ ULN (upper limit of normal). All chosen isoform detection workflows showed high agreement in most samples, and pgRNA was the most abundant isoform in most patients. Northern blotting was ineffective at detecting serum HBV RNA. The 3′ RACE RT-qPCR assay performed well during analytical and clinical validation and inter- and intra-laboratory analysis demonstrated moderate to high agreement among participants. Conclusions: Long read sequencing is a promising tool for the characterization of the serum HBV tr

Human adenovirus (HAdV) is a critical tool and essential model for studying virology, cell biology and molecular biology. Research into HAdV has led to a comprehensive understanding of the virus and its interaction with the host cell. These properties have been used to exploit HAdV to develop therapeutics and vaccines, and to understand the cell’s inner workings. This study first explores the use of HAdV as a molecular biology tool to develop and create a set of recombinant HAdV (rHAdV) to be used as potential vaccines or research reagents for the coronavirus disease 2019 (COVID-19) pandemic. Secondly, it utilizes the critical viral protein Early 1A (E1A) to identify the cellular E1A interaction hub protein, arginine and glutamate rich 1 (ARGLU1).

Acinetobacter baumannii is a pathogenic bacterium responsible for various hospital-acquired infections in immunocompromised patients. It is highly resilient and can survive in harsh environmental conditions. A. baumannii is infamous for its ability to develop resistance against various antimicrobials. Two Component Systems (TCS) are signal transduction systems which enable bacteria to sense and adapt to changes in environmental conditions. Various studies have indicated their involvement in regulation of antibiotic susceptibility and virulence mechanisms in bacteria. In this project, we investigated the functional characteristics of novel and putative TCSs in A. baumannii and their role in regulation of antibiotic susceptibility and virulence phenotypes. The first part of this study involved the functional characterization of AvnR, a conserved response regulator, in the clinical isolate - A. baumannii AB030. AvnR has previously been linked with regulation of various virulence-associated phenotypes including biofilm formation, motility, and nitrogen metabolism. The avnR gene in A. baumannii AB030 has been found to be naturally disrupted by an insertion sequence, rendering it non-functional. We investigated the impact of this disruption on biofilm formation, motility, and nitrogen metabolism in AB030. Complementation of avnR in AB030 had no effect on biofilm formation and motility phenotypes; however, changes in its nitrogen metabolism profile was observed. Complement strain, AB030:mini-Tn7:avnR, exhibited enhanced growth in L-serine and L-arginine compared to the wild type, whereas a decrease in growth was observed in Alanine-Histidine, Alanine-Leucine, and L-Valine. In the second part of this study, the functional characteristics of a putative TCS, A1S_1977-78, was investigated in A. baumannii ATCC 17978. Previous studies have indicated a potential link between this TCS and regulation of AdeN, a Tet-R type regulator protein. AdeN is associated with expression of Ade

Higher microbial load and antibiotic delay are associated with increased morbidity and mortality in septic shock. Antibiotic combination is the basis of empiric treatment for sepsis and septic shock, current recommendations suggest the use of a combination of antibiotics to broaden the initial spectrum of coverage to treat some multi-resistant bacteria. Antibiotic combination therapy leads to more rapid pathogen clearance, which may translate into improved patient outcome. The systematic review in this thesis based on the available randomized clinical trials, does not support the use of combination therapy as the initial treatment for sepsis and septic shock. Although, this could have been due to study design differences, variable degree of severity and even sequence of antibiotic administration. While antimicrobial synergy has been established for beta-lactam combinations with aminoglycosides or fluoroquinolones, antimicrobial sequence has only been studied in vitro. These studies suggest that giving the beta-lactam first or at the same time as an aminoglycoside improves bacterial clearance. Herein we sought to confirm this in the first in vivo study of antimicrobial sequence in septic shock. This thesis confirmed better bacterial clearance using antibiotic combination therapy, but failed to show significant differences in bacterial clearance depending on the sequence of administration of antibiotics in our E. coli and S. pneumoniae models of septic shock. This study sheds light on sequence of administration of antibiotics in sepsis, helping to inform further research.

Motivation The quest for efficient drug discovery processes necessitates a comprehensive approach that integrates molecular feature analysis with accurate compound-protein interaction (CPI) prediction. This study introduces models that combine deep learning (DL) techniques for intricate molecular feature engineering and innovative CPI prediction methods. This integration responds to the need for detailed molecular dataset analysis and the prediction of interactions between novel compounds and proteins, thereby enhancing drug discovery. Methods and Results Chapter 3 - Molecular Clustering and Feature Analysis: The framework implements a feature engineering scheme focusing on molecule-specific atomic and bonding information. It utilizes principal component analysis (PCA) for encoding this information and a variational autoencoder (VAE)-based method for embedding both global chemical properties and local features. This approach facilitated the clustering of a large dataset containing over 47,000 molecules. Using the K-means method with 32 embedding`s size based on the VAE method, 50 distinct molecular clusters were identified. These clusters were visualized through t-distributed Stochastic Neighbor Embedding (t-SNE), showcasing the framework's capability in effectively grouping molecules based on their complex features. Chapter 4 - CPI Prediction with GraphBAN: For CPI prediction, the study introduces GraphBAN, a novel inductive-based approach using graph knowledge distillation (KD). This component incorporates a deep bilinear attention network (BAN) and a KD module for graph analysis, enabling the alignment of interaction features across different distributions. GraphBAN's functionality extends to both transductive and inductive link predictions in a bi-partite graph of CPIs. Tested against three benchmark datasets, GraphBAN demonstrated superior performance, outperforming six baseline models. It shows that it is able to predict interactions between unseen compounds a

The dielectric properties of biological cells serve as indicators of their physiological state. Electrical Impedance Spectroscopy (EIS) is a highly effective, non-invasive and label-free technique that can be utilized to infer information about the physiological condition of biological cells by measuring their electrical impedance across a frequency range. This technique can effectively distinguish between various types of cells and provide critical insights into their respective states, whether as individual cells or as a suspension of cells. In this thesis, the applicability of bulk and single-cell EIS for bioprocess monitoring was investigated. We developed a theoretical framework to assess the sensitivity of bulk and single-cell EIS to the cell and culture parameters of Chinese Hamster Ovary (CHO) cells (such as cell density in the culture, culture viability, cell size, and cell dielectric properties) and correlated the sensitivity analysis outcomes with the results obtained from experimental measurements. For bulk EIS measurements, we performed a comprehensive sensitivity analysis to identify the cell and culture parameters that significantly influence the permittivity spectrum measured by bulk EIS probes. For our analysis, we utilized a two-population dielectric model, including both viable and non-viable cells, to accurately represent the cell culture within a bioreactor. Additionally, we considered a double-shell dielectric model, comprising two concentric shelled spheres, to account for the nucleoplasm, nuclear envelope, cytoplasm, and plasma membrane of CHO cells. We then compared the results of our sensitivity analysis with experimental data obtained by measuring CHO cells in a lab-scale bioreactor, employing commercial bulk electrical impedance spectroscopy probes. We concluded that the most reliable cell or culture parameter that bulk EIS probes can assess is the viable cell density, through low-frequency measurements. In conventional bulk assays that r

Introduction: Pneumonia is a major respiratory infection that significantly strains Manitoba’s healthcare system, resulting a substantial number of hospitalizations and fatalities. Understanding the transmission dynamics and risk factors associated with pneumonia in this population is crucial for targeted interventions. Spatial variability in pneumonia hospitalizations has been observed in Manitoba, where various risk factors contribute to pneumonia infection. Therefore, it is essential to investigate the determinants of pneumonia, including spatial aspects and disease transmission rates. Methods: We applied a spatial Poisson regression model that incorporates the Intrinsic Conditional Autoregressive model to explore region level potential risk factors. To understand the influence of comorbidity, we utilized a mixed-effects Poisson regression model. Our analysis focused on hospital data spanning the years 2015 to 2019, and encompassing 96 Manitoba health regions. Moreover, to investigate disease transition rates, we employed both the Susceptible-Exposed-Infected-Recovery (SEIR) model (excluding reinfection) and the Susceptible-Exposed-Infected-Recovery-Susceptible (SEIRS) model (including reinfection). These compartmental models considered data from both hospital and physician-reported pneumonia cases during August 2017 to July 2018. Results: The raw incidence rate of pneumonia infection exhibited significant variation across different regions, ranging from 2 to 55 cases per 1000 population. After adjusting for potential risk factors, the incidence rate ratios ranged from 0.38 to 6.63, indicating substantial variations in incidence rates among regions. Factors such as age, immigration status, and comorbidities, notably Chronic Obstructive Pulmonary Disease (COPD) and Cardiovascular Disease (CVD), were identified as significant contributors to the risk of pneumonia infection. Conversely, vaccination was found to exert a protective effect, especially among individuals

B-cell activation occurs via antigen binding to its membrane immunoglobulin along with T-cell help to initiate intracellular signaling cascades. B cell activation enhances glucose uptake, the phosphoinositide-3-kinase (PI3K) pathway, and glycolysis and oxidative phosphorylation in a balanced manner relative to resting B-cells. The enzyme Hexokinase (HK) is central to B cell activation as it catalyzes the first step of glucose metabolism by phosphorylating glucose to become glucose-6-phosphate, which then serves as an entry point for several metabolic pathways. Hexokinase-II (HKII) is one of four HK isoforms and is particularly interesting as the only isoform that can translocate between the cytoplasm and mitochondria. It is unknown if HKII, in comparison to the other isoforms, preferentially guides glucose-6-P down a particular metabolic pathway which may depend on the B-cell’s activation status and HKII’s cellular localization. B-cell activation through the T-cell dependent signals and the PI3K pathway reprograms resting B-cell metabolism by the modification of HKII expression and subcellular localization to meet the B-cell’s new activated metabolic program. B-cells with unusual HKII expression or subcellular localization may be a feature of B-cell metabolic dysregulation, such as that observed in Chronic Lymphocytic Leukemia (CLL). A novel intracellular staining and flow cytometry assay was developed to measure HKII expression levels across B-cell subsets throughout the body and a fluorescence microscopy based assay quantified HKII mitochondrial localization. A biological mechanism is presented herein, where B-cell activation dependent on the PI3K pathway significantly increased HKII expression and along with the presence of glucose enhanced HKII mitochondrial localization. CLL B-cells failed to increase HKII expression following in vitro B-cell activation and expressed significantly less HKII, which decreased with advanced clinical Rai stage, compared to control

Human B-Lymphoblast Mechanisms of Resistance to Shiga Toxins Olivia Tran1, Amanda Strickland1,, Susana Asin1, Angela Melton-Celsa2, Carl C. Brinkley3, and G. Jilani Chaudry3 1Center for Advanced Molecular Detection, Science & Technology, 59 Medical Wing, USAF, DHA, JBSA-Lackland, TX 2Department of Microbiology & Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 3Diagnostics & Therapeutics, Science and Technology, 59 Medical Wing, USAF, DHA JBSA-Lackland, TX RESULTS & DISCUSSION DISCLAIMER: "The views expressed are those of the authors or presenters and do not reflect the official views or policy of the Department of Defense or its Components" ACKNOWLEDGMENTS We are greatly thankful to the US Air Force for funding this study. We are also indebted to the able support provided by the program officials in Science and Technology, 59 MDW, JBSA-Lackland. ABSTRACT RESULTS & DISCUSSION Diarrheal diseases are typically food-borne and occur globally. The main causative agents are bacterial, protozoan, and viral. Among the bacteria that cause these disease are Shigella dysenteriae type 1 and shigatoxigenic strains of Escherichia coli (STEC), such as serotype O157:H7. The pathologic and clinical severity of these diseases varies across a broad spectrum. In the most severe cases, they can result in hemorrhagic colitis and hemolytic uremic syndrome, which can be fatal. The two bacteria produce protein toxins, termed shigatoxins (Stx1, Stx2), that are crucial in the disease pathogenesis. Whereas S. dysenteriae type 1 produces only Stx1, STEC strains can produce either one or both toxins. Stx1 produced by STEC is essentially identical to that produced by S. dysenteriae type 1. However, Stx2 shows significant sequence divergence from Stx1; the two toxins have ~ 61% identity and ~ 75% similarity. Structurally, Stx1 and Stx2 comprise a cytotoxic enzymatic moiety (A-subunit) and a homopentamer of a receptor-binding moiety (B-subunit). Both toxins have the sa, RITM0041167, Diarrheal diseases are typically food-borne and occur globally. The main causative agents are bacterial, protozoan, and viral. Among the bacteria that cause these disease are Shigella dysenteriae type 1 and shigatoxigenic strains of Escherichia coli (STEC), such as serotype O157:H7. The pathologic and clinical severity of these diseases varies across a broad spectrum. In the most severe cases, they can result in hemorrhagic colitis and hemolytic uremic syndrome, which can be fatal. The two bacteria produce protein toxins, termed shigatoxins (Stx1, Stx2), that are crucial in the disease pathogenesis. Whereas S. dysenteriae type 1 produces only Stx1, STEC strains can produce either one or both toxins. Stx1 produced by STEC is essentially identical to that produced by S. dysenteriae type 1. However, Stx2 shows significant sequence divergence from Stx1; the two toxins have ~ 61% identity and ~ 75% similarity. Structurally, Stx1 and Stx2 comprise a cytotoxic enzymatic moiety (Asubunit) and a homopentamer of a receptor-binding moiety (B-subunit). Both toxins have the same mode of cytocidal action on cells. Stx enter cells by receptor-mediated endocytosis. The primary receptor for both Stx1 and Stx2 is globotriaosylceramide (Gb3), a glycosphingolipid. Following binding to Gb3 via their B-subunit pentamer, Stx are internalized and retrogradely transported to ER, where the A-subunit exits to enter cytosol. The A-subunit then catalytically inactivates the 28S rRNA by removing a particular adenine in a functionally essential stem-loop. This action of Stx renders the 28S rRNA nonfunctional, resulting in complete inhibition of protein synthesis and cell death. This work assessed susceptibility of 90 individual-specific human B-lymphoblastoid cell lines to Stx2. Many of these cells showed complete resistance to Stx2 (IC50 > 500 ng/mL), while many others showed very high sensitivity (IC50 ~ 0.007 ng/mL). RNA sequencing revealed a number of genes with higher expression in Stx2 resi

Arbuscular mycorrhizal (AM) fungi play key roles in soil fertility of agroecosystems. They develop dense extraradical mycelial (ERM) networks via mechanisms such as hyphal anastomosis. These connections between hyphae can be afected by agricultural practices such as the use of fungicides, but how these compounds afect anastomosis formation within and more importantly between networks of the same AM fungal strain remains poorly unexplored. Here, the impact of azoxystrobin, pencycuron, futolanil, and fenpropimorph at 0.02 and 2 mg L−1 were tested in vitro on the anastomosis formation within and between networks of Rhizophagus irregularis MUCL 41833. Azoxystrobin and fenpropimorph had a particularly detrimental impact, at the highest concentration (2 mg L−1), on the number of anastomoses within and between networks, and for fenpropimorph in particular at both concentrations (0.02 and 2 mg L−1) on the number of anastomoses per length of hyphae. Curiously fenpropimorph at 0.02 mg L−1 signifcantly stimulated spore production, while with azoxystrobin, the reverse was observed at 2 mg L−1. The two other fungicides, pencycuron and futolanil, had no detrimental efects on spore production or anastomosis formation within and between networks. These results suggest that fungicides with diferent modes of action and concentrations diferentially afect anastomosis possibly by altering the hyphal tips of AM fungi and may thus afect the capacity of AM fungi to develop large hyphal networks exploring and exploiting the soil at the service of plants.

This article summarises the activities of the Bacterial Viruses Subcommittee of the International Committee on Taxonomy of Viruses for the period of March 2021−March 2022. We provide an overview of the new taxa proposed in 2021, approved by the Executive Committee, and ratified by vote in 2022. Significant changes to the taxonomy of bacterial viruses were introduced: the paraphyletic morphological families Podoviridae, Siphoviridae, and Myoviridae as well as the order Caudovirales were abolished, and a binomial system of nomenclature for species was established. In addition, one order, 22 families, 30 subfamilies, 321 genera, and 862 species were newly created, promoted, or moved.

Background : In a retrospective study in 4 Belgian hospitals, we showed that the use of linezolid (LZD) was larger and the rate of adverse drug reactions (ADRs) higher (especially anemia and thrombocytopenia) than described in the summary of product characteristics (1). Associated risk factors for ADRs were long treatment duration, renal impairment at the treatment initiation, and presence of comorbidities (evaluated by the Charlson index (2)). Moreover, literature suggests that trough levels (Cmin) > 9 mg/L increase the risk of hematological toxicity (HT) [...].

The geographical range of Ebola virus (EBOV) outbreaks overlaps with the habitat of diverse ecosystems that are home to many potential reservoir species. Although researchers have discovered EBOV viral RNA and serological evidence in different rodents and bats, live virus has not been isolated from any species. Identifying the host reservoir is only the first of many challenges; predicting and preventing spillover events requires research on population distribution, ecology, host metabolism and immunology. The EBOV field is clearly missing critical knowledge about where the virus hides, either from being overlooked during sampling, being rarely encountered and/or specific conditions are required that regulate viral expression. To this end, we investigated EBOV infection in cryptic tissues in vitro and in vivo and found that adipose tissue (AT), plays a significant role in both acute and prolonged infections. AT is a dynamic endocrine organ that helps regulate homeostasis, and coordinating energy metabolism, as well as neuroendocrine and immune functions; however, its role in infectious disease has been less thoroughly studied. To assess the role of AT during EBOV infection, we used immortalized and primary brown adipose cultures in vitro as well as developed in vivo reservoir models using Golden Syrian hamsters (Mesocricetus auratus) and Deer mice (Peromyscus maniculatus). Wild type EBOV infected rodents showed no clinical signs of disease nor detectable virus in commonly sampled tissues and swabs. However, EBOV RNA and viral antigen was detected in AT up to 56 days post infection. Furthermore, we tested numerous metabolic agents and found that administration of epinephrine significantly increased viral replication in adipose depots and spillover into the blood. These results show the importance of AT in long-term infection and how the metabolism of this tissue may play a role in viral transmission between reservoir species and/or zoonotic spillover events. Addition

Lichens are traditionally described as symbionts of fungi and algae and are renowned for their diverse secondary metabolites. Our group identified and annotated 48 secondary metabolite biosynthetic gene clusters of the fungal partner of the lichen Cladonia uncialis using de novo whole genome sequencing. Out of these, putative assignment of biosynthetic functions of ‘ten’ gene clusters in C. uncialis was done by Bertrand (2018) including the usnic acid (UA) gene cluster (the most extensively studied lichen secondary metabolite). This research work deduced biosynthetic pathways and proposed biosynthetic function of seven more gene clusters using a 'homology mapping’ approach in combination with phylogenetics. The UA gene cluster contains two genes, one encodes for PKS enzyme and other for a post PKS tailoring enzyme, that have been respectively named as methylphloracetophenone synthase (MPAS) and methylphloroacetophenone oxidase (MPAO, a cytochrome p450). MPAO is believed to catalyze the oxidative dimerization of methylphloroacetophenone (MPA) to usnic acid (UA). Electron transfer reactions from NAD(P)H to a cytochrome p450 are supported by redox partner, cytochrome P450 oxidoreductase (CPR). We have identified a gene, cu-cpr in the genome of C. uncialis that encodes for a similar redox partner. We have successfully developed heterologous expression protocols for first lichen cytochrome p450 (mpao) and CPR (cu-cpr) in E. coli and, the purification protocols for these lichen proteins. We have also established a protocol for heterologous co-expression system for mpao and cpr in E. coli. This study demonstrates the first successful biosynthesis of UA (a lichen polyketide) with the development of bioconversion protocol of MPA to UA catalyzed by both MPAO and CPR. Biofilm-disruption and antibacterial assays were carried out to compare the bioactivity of MPA and UA, where UA was found to be more bioactive. That could suggest a rationale for why lichen fungus exerts the effo

Pre-harvest sprouting (PHS) affects wheat production globally as it reduces grain yield and end-use quality. Given PHS resistance is a quantitative trait and strongly influenced by genetic and environment factors. This thesis project is therefore aimed to identify molecular mechanisms regulating dormancy, quantitative trait loci (QTL) and candidate genes for PHS resistance. To this end, this PhD thesis project comprised of a genetic study involving a mapping population and a transcriptomic analysis. The genetic study investigated the dormancy phenotype of a doubled haploid (DH) mapping population derived from dormant/non-dormant parents and evaluated over five environments, and these data along with the genotypic data obtained using a 90 K SNP Chip array was used for detection PHS resistance QTL. A total of four PHS resistance QTLs were detected on chromosome 1D, 4A, 6B and 6D across the five environments; 4A QTL was identified as a major QTL as it explained 40-50% of phenotypic variation while 1D QTL is novel as it was not reported previously. The transcriptomic study investigated selected DH lines showing contrasting dormancy phenotype using RNA-Seq. Our analysis revealed a large number of differentially expressed genes (DEGs) between dormant and non-dormant seeds. The DEGs were found to be enriched in cell, cell part, intracellular and integral component of membrane gene ontology classes. Genes upregulated in dormant seeds are found to be enriched in the carotenoid biosynthesis including ABA metabolism, diterpenoid biosynthesis including gibberellin catabolism and flavonoid biosynthesis as well as ABA signaling pathways while those downregulated are enriched in auxin signaling and GA biosynthesis pathways. Mapping of the DEGs on previously detected QTLs (1D, 4A, 6B and 6D) identified potential candidate genes for seed dormancy. Of these DEGs, TaCYP71B11 and TaDOG1L-2 on 1D, TaPM19-A1 and A2 on 4A, an uncharacterized gene (Gene ID: TraesCS6B02G325000) on 6B and Ta

In Canada, landfills are responsible for 23% of all emissions of methane, a greenhouse gas with 28x the potency of carbon dioxide. Gas collection systems can be used to mitigate landfill emissions but are not feasible for many municipal solid waste facilities. This has led to the increased importance of exploring innovative biocover solutions which incorporate on-site compost materials to enhance methane mitigation by microorganisms known as methanotrophs. This thesis examines the microbial community of biowindow soil alongside conventional landfill cover soil and on-site compost windrows, with a focus on aerobic methanotrophic bacteria. Initial methane-oxidation potential of the different soil types were measured throughout various seasonal conditions, revealing high activity in the spring but substantially decreased levels of methane-oxidation throughout drought-like conditions in the summer of 2021 and through into late fall. 16S rRNA analysis and amplicon sequencing of the methanotroph marker gene pmoA in soil detected high proportions of methanotrophs, with a particular abundance of cyst-forming Methylocystis and thermophilic Methylocaldum. Amplicon sequencing of pmoA using methanotroph enrichment culture DNA identified a large proportion of unclassified organisms at the genus level, and phylogenetic analysis strongly suggested these uncultured bacteria belong to Methylocaldum. Enrichment cultures using soil inocula found despite low initial oxidation potential methanotrophs responded positively given the optimum conditions, indicating their resilience to desiccation and large temperature fluctuations occurring in situ. Attempted isolation of thermophilic methanotrophs resulted in the identification of Meiothermus silvanus via whole genome sequencing, the first documentation of this organism in a landfill and the first known record of a thermophilic organism being isolated from a continental climate landfill. M. silvanus belongs to a genus of heterotrophic ther

Prion diseases are rare neurodegenerative disorders that are deadly, incurable, and caused by misfolded prion proteins (PrPSc) in the brain leading to neuropathological changes and rapid cognitive decline. The paucity of current approaches to prion disease research has hampered progress and so here we address two technical challenges that have limited research tools. Firstly, human prion isolates are incompatible with most cellular prion infection models, limiting their clinical relevance. Secondly, genome-wide transcriptional studies of bulk brain tissue cannot resolve the contribution of different brain cell types to prion pathogenesis. This makes it difficult to understand the relationship between prion accumulation, pathogenesis, and neurotoxicity. In the first part of this thesis we set out to develop novel in vitro cellular models of human prion infection using two approaches: (a) the prion organotypic slice culture assay (POSCA) and (b) an immortalized human neural progenitor cell line (ReN). In these two experimental paradigms, prion replication was sensitively tracked over several weeks through measurements of amyloid seeding activity with real time quaking induced conversion (RT-QuIC). Deer mouse cerebellar slice cultures and PrPC-overexpressing ReN cultures were found to resist infection with human prion isolates. While we were unable to establish an in vitro model of human prion infection, our findings reflect the inherent difficulty of establishing clinically relevant cellular models. In the second part of this thesis, we compared prion-elicited transcriptional responses between mouse brain cell types to shed further insight into prion neuropathology. Using bulk RNA sequencing, gene expression was examined longitudinally in microdissected brain tissues from an in vivo mouse model of RML Scrapie infection. We then applied single-cell RNA sequencing to brain cells isolated at the clinical endpoint of RML Scrapie infection. A single-cell atlas of nearly 15

The extracellular matrix is a complex system of interacting macromolecules that serve structural and functional purposes to maintain homeostasis of a cell. These systems can interact with external factors and carry signaling molecules across large distances. As such, the extracellular matrix is the center of many interesting components that can be functionalized for industrial purposes and targeted for therapeutic approaches. However, studying these intricate systems in a meaningful way, that improves our understanding of the underlying mechanism, can prove to be a challenge for any one technique. As such, the combination of various techniques, ranging from X-ray crystallography to understand the atomic structures of macromolecules to cell assays covering the functional aspects in a coherent larger characterization, is crucial for an accurate interpretation of the system of interest. The combination of various structural and functional biochemical techniques is often termed the hybrid method approach. The purpose of this work is to demonstrate and further develop the hybrid method approach to the extent where multiple combinations of techniques are utilized to study and understand various extracellular matrix and environment systems. The initial focus using the hybrid method approach is the examination of a surface-layer protein in an archaeal species for the development of nanotube and nanocarrier systems that bind various hydrophobic ligands. It was shown that the nanotube was able to be developed as a passive sampling medium for the monitoring of polycyclic aromatic hydrocarbons and as a nanocarrier system for carborane which is used in boron neutron capture therapies against various types of cancers. The second focus of this work is on mammalian extracellular matrix macromolecular systems and the formation of protein assemblies to study the structural and functional features that are a common source for pathological diseases. Here, the hybrid method approach was

Introduction: Data privacy legislation in Canada prohibits patient-level administrative health data from crossing jurisdictional boundaries. Accordingly, multi-site research networks often conduct distributed analyses and pool site-specific effect estimates (EEs) using meta-analysis models. Rare outcomes and heterogeneity in site-specific EEs can produce potential outliers that may bias pooled EEs. Limited research has compared outlier detection methods and the impact of potential outliers on meta-analysis results. Purpose and Objectives: The research purpose was to examine outlier detection methods for meta-analyses of site-specific EEs from a multi-site network. The objectives were to: 1) compare outlier detection methods for random-effects meta-analysis (REM) models, and 2) apply these methods to site-specific EEs from systematically selected real-world meta-analyses. Methods: We compared studentized residual estimates (StdR), relative change in pooled EE variance (RCPEV), relative change in estimated between-site variance (RCEBV), and model-based mean-shift method (MMS) using computer simulation. EEs were simulated assuming a normal distribution. Accuracy, misclassification error (ME), and F-1 score were assessed using random-effects analysis of variance models. We systematically selected meta-analyses conducted by investigators from the Canadian Network for Observational Drug Effect Studies (CNODES), applied outlier detection methods, and assessed the impact of potential outliers on REM results. Results: StdR had the highest accuracy (median: 89.9%) and lowest ME (median: 10.2%). RCPEV was the most consistent in all metrics. For StdR, the number of sites explained 95.1% and 93.0% of the variation in accuracy and ME values. For RCEBV and MMS, between-site variance described the most variation in accuracy and ME values. StdR and RCPEV were most sensitive to detect potential outliers in re-analyses of 39 published CNODES meta-analyses. Heterogeneity in site-specif

CD8+ T cells are key in mediating immune responses to viral infections. Viral infections can be acute, in which the infection is resolved, or chronic, when antigen persists. Several studies have illustrated that CD8+ T cells adopt distinct functional states in each infection type. In acute infections, naïve CD8+T cells differentiate into cytotoxic CD8+ T cells and memory CD8+ T cells. In chronic infections, activated CD8+ T cells adopt an altered state known as exhaustion. Exhausted CD8+ T cells lose effector functions and show high expression of inhibitory receptors such as PD-1. Several studies have characterized CD8+ T cell exhaustion at late time-points after a persistent infection has already been established. Recent studies demonstrate that CD8+ T cell exhaustion may be specified during earlier phases of a developing chronic infection. However, these studies do not address the effect of biological sex on early CD8+ T cell responses to chronic infection. Sex differences in susceptibility and outcomes of viral infectious diseases are well-appreciated. Still, the role of sex in the development of CD8+ T cell exhaustion and in the molecular programs underlying CD8+ T cell responses to acute and chronic viral infections remains undefined. This study, therefore, set out to investigate early and sex-based differences in the transcriptional profiles of CD8+ T cells that underlie their responses to acute versus chronic viral infection. The mouse model of acute and chronic lymphocytic choriomeningitis virus infection was used to comparatively analyze transcriptional signatures of single antigen-specific CD8+ T cells during acute versus chronic infection at multiple time-points post-infection. Next sequencing coupled with bioinformatics-based approaches were used to analyze our single-cell RNA sequencing data. Our results reveal an early transcriptional divergence of CD8+ T cells responding to acute versus chronic viral infection. We show that there are sex-specific diff

Insects account for the vast majority of sexually reproducing animals described and demonstrate significant diversity and complexity in sex-developmental pathways. While only a small number of insect species have had sex-development pathways characterized, orthologues of the master regulator of sex-differentiation, Doublesex (Dsx), have been identified in all insects studied. Using alternative splicing to produce distinct, functional male-specific and femalespecific isoforms, Dsx guides differential gene expression patterns through its activity as a transcription factor. Notably, while male and female specific isoforms contain identical DNAbinding domains, it is expected that sex-specific differences in gene expression are the result of different binding partners at the isoform specific oligomerization domains. The yellow fever mosquito, Aedes aegypti, exhibits pronounced sexual dimorphisms, largely due to the females’ specific need to obtain blood meals. Because of these distinct dimorphic features and its threat to human health, research into Ae. aegypti sex development is of interest to developmental biologists and in the context of public health. Despite numerous studies on Ae. aegypti sex development, little is known about the isoform-specific binding partners of AaeDsx. Using protein pulldown and mass spectrometry techniques, this project examined the distinct binding partners of the two female-specific DSX isoforms with the specific aims of improving our understanding of insect sex development and discovering new gene targets for female lethality. A subset of 12 genes identified in the mass spectrometry assay were further subjected to RNA interference assays where female-specific developmental impacts were assessed. Data from this project may be applied to improve sex-sorting of mosquitoes in sterile insect technique approaches.

An emerging crop, intermediate wheatgrass (IWG) is resistant to the natural occurrence of most important cereal diseases and is currently under domestication to improve its yield. An IWG population consisting of two parental lines and 154 individual F1 progenies were inoculated with F. graminearum spores in a field setting to check their performance against Fusarium head blight (FHB) over two consecutive years. The higher humidity in 2022 lead to higher FHB severity and incidence, and higher DON (deoxynivalenol) mycotoxin accumulation than in 2021. DON was mostly restricted to the chaff in 2021, whereas in 2022, DON was found in the grain as well. Considering all the FHB aspects checked in this experiment, four IWG progeny individuals (plants: P-17, P-43, P-107, and P-139) were found to consistently perform better than the parental lines and the other progeny individuals during both years. The second part of this thesis analyzed the influence of F. graminearum on the mycobiome of IWG spikelet and flag leaf samples from two different locations, Winnipeg and Carman, MB. A total 372 IWG samples (half of which were inoculated with F. graminearum [Winnipeg] and the other half, not [Carman]) were used for fungal community profiling via ITS amplicon sequencing using PacBio technology. There were significant differences in the fungal community among the IWG spike samples from Winnipeg and Carman. As expected, F. graminearum dominated IWG spike mycobiomes at the inoculated Winnipeg site, whereas in Carman, Alternaria dominated. Also, the presence of F. graminearum seemed to influence the broader spike mycobiome and the presence of beneficial fungi was less where F. graminearum dominated. The mycobiome data also revealed that the pathogenic fungi Alternaria, Nigrospora, Septoriella, Stemphylium, Claviceps, Bipolaris, and Drechslera can become potential threats to IWG in the future. The last part of this thesis sheds light on the interaction between F. graminearum and another

Despite the generation of a strong T cell response, clearance of Leishmania major is incomplete and leaves a pool of chronically infected cells. Understanding of the persistence mechanisms is lacking, but Leishmania major driven induction of the immunosuppressive microenvironment through recruitment of regulatory T cells at the site of infection has been proposed to prevent parasite clearance in vivo. In the presented thesis, I used a novel TCR transgenic mouse model, where CD4+ T cells recognize an immunodominant peptide derived from Leishmania- glycosomal phosphoenolpyruvate carboxykinase (PEPCK), as a sensitive tool to characterize the dynamics of anti-L. major CD4+ T cell responses and to characterize mechanisms which restrain their effector function. Intravital microscopy studies characterizing L. major-specific CD4+ T cell migration dynamics within skin lesions directly in live mice show a significant recruitment of adoptively transferred effector T cells to the lesion site in vivo, displaying cellular behaviors consistent with antigen recognition at early and late stages of infection. However, cellular dynamics are augmented at the healed stage, indicating a fundamentally altered environment. I show that Leishmania-specific Tregs display higher suppressive activity compared to polyclonal control Tregs, and that this suppression is mediated through IL-10 and not through disrupting cell-cell contacts or antigen presentation. Challenge of healed mice with L. major antigen results in expansion of endogenous Leishmania-specific Tregs that lead to loss of lesion control in an IL-10 dependent manner. Lack of PEPCK antigen during challenge does not supress effector Th1 response and parasite control. My data proposes a stochastic model of parasite survival, where inflammatory factors that control parasite numbers are counterbalanced by Leishmania-specific immunosuppressive factors that facilitate parasite persistence.

Streptococcus pyogenes is responsible for over half a million deaths every year by causing diseases like necrotizing fasciitis and rheumatic heart disease. Streptococcal toxins that cause such diseases are encoded by prophages found in the S. pyogenes genome. The prophage encoded toxin genes are always found adjacent to a highly conserved gene termed paratox (prx). Past work by our lab discovered that Prx disrupts quorum sensing and natural competence in S. pyogenes by directly binding to the DNA binding domain (DBD) of quorum sensing receptor ComR. In doing so, Prx protects prophage DNA from being replaced by potential homologous recombination. To probe for additional Prx functions, we aimed to explore streptococcal biochemical pathways that may be regulated by Prx. Using a pull-down assay combined with mass-spectrometry, the lab previously identified SpyM3_0890 or EndoS1 and SpyM3_1246 or P1246 as potential Prx binding partners. To study the Prx partners, we purified both proteins to study their biochemical interactions with Prx. First, we show that both proteins form stable complexes with Prx in vitro. Next, an X-ray crystal structure of EndoS1 shows that it resembles the specificity (S) subunit of type I restriction modification (RM) enzymes. A cocrystal structure of an EndoS1:Prx complex revealed that Prx binds the EndoS1 at its potential DBD. P1246 is located in a cluster of phage genes that regulate DNA processing and Alpha Fold predicts that P1246 adopts a DBD-fold identical to ComR. Based on our structures we have identified a helical motif conserved in ComR, EndoS1, and P1246 that is critical for Prx interaction. This motif could serve as a different approach for pursuing more Prx interaction partners. Since both EndoS1 and P1246 are potential DNA binding proteins, Prx likely blocks their ability to interact with a specific promoter or repressor region in S. pyogenes. Overall, our study demonstrates how small phage proteins like Prx can regulate host bioch

Antiretroviral therapy (ART) has helped Human Immunodeficiency Virus (HIV) become a manageable chronic infection by repressing viremia to undetectable levels in People living with HIV (PLWH). However, this treatment is not curative as treatment interruption causes viremia to rebound to pre-treatment levels due to viral latency, a state of reversible non-productive infection of individual cells established early during acute infection. These latently infected cells serve as HIV reservoirs, residing in various anatomical sites and presenting a significant obstacle to achieving a complete HIV cure. Notably, central and effector memory CD4+ T cells are regarded as the major component of the long-lived HIV reservoirs. Memory CD4+ T cells play an essential role in the adaptive immune response. Guided by chemokine cues, memory CD4+ T cells continually recirculate between lymphoid and non-lymphoid tissues, enabling them to carry out immunosurveillance of the body for pathogens. These long-lived cells are sustained by tonic signalling through the T cell receptor (TCR) and cytokines within the secondary lymphoid organs (SLOs), where they can undergo clonal expansion. Therefore, the SLOs are important tissue reservoirs that allow for persistent viral replication in PLWH under suppressive ART. However, an unaddressed question is whether latently infected memory CD4+ T cells retain their ability to recirculate between lymphoid and non-lymphoid organs, allowing them to seed various tissues and access the same survival signals as uninfected memory CD4+ T cells to persist. Therefore, to address this question, I developed a fluorescent protein-based HIV reporter system that allows for the assessment of the phenotypic and migratory capacity of latently infected CD4+ T cells. The full-length, R5-tropic dual-fluorescent HIV reporter, HIV TosZy, encodes two fluorescent markers: Nef-ZsGreen fusion protein under the control of the HIV-LTR and dTomato protein driven by constitutively expre

Differential expression and localization of Shiga toxin type 2 (Stx2) from recA mutant derivatives of O157:H7 Escherichia coli strains Rama R. Atitkar1,2 , Angela R. Melton-Celsa1 Introduction Serotype O157:H7 STEC are associated with more severe disease, and they encode differing combinations of Stx types and subtypes. Encoded on the stx-phage, stx expression is linked to phage induction and the lytic cycle. Repressor-operator interactions within the phage regulatory region control the initiation and progression of the lytic cycle, and the RecA protein stimulates phage repressor cleavage. Genomic comparisons of stx-phages have uncovered regions of significant genetic diversity, including within the regulatory region. However, it is still unclear whether diversity within this region could influence STEC pathogenesis and virulence. 1 Uniformed Services University, Bethesda, Maryland, USA 2 Henry M. Jackson Foundation, Bethesda, Maryland, USA Correspondence: angela.melton-celsa@usuhs.edu STEC isolates exhibit differences in RecA-independent cytotoxicity Acknowledgements/Disclaimer This work was supported by National Institutes of Health grant R37 AI020148. Strains used in this study and related work were obtained from E. Dudley and M. Muniesa. The opinions or assertions contained herein are the private ones of the author/speaker and are not to be construed as official or reflecting the views of the Department of Defense, the Uniformed Services University of the Health Sciences, any other agency of the U.S. Government, or the Henry M. Jackson Foundation for the Advancement of Military Medicine. Methods The stx2a-phage regulatory region mediates differential RecA-independent cytotoxicity Background Studies about RecA and stx-phages have indirectly identified RecA-independent stx-phage induction and/or Stx production: - EDTA/chelation influences Stx production from recA-negative stx-phage lysogens (Imamovic et al, 2012). - Phage are detected from cultures of stx-phage ly, RITM0037416, Serotype O157:H7 STEC are associated with more severe disease, and they encode differing combinations of Stx types and subtypes. Encoded on the stx-phage, stx expression is linked to phage induction and the lytic cycle. Repressor-operator interactions within the phage regulatory region control the initiation and progression of the lytic cycle, and the RecA protein stimulates phage repressor cleavage. Genomic comparisons of stx-phages have uncovered regions of significant genetic diversity, including within the regulatory region. However, it is still unclear whether diversity within this region could influence STEC pathogenesis and virulence.

The environmental impact of uncultured phages is shaped by their preferred life cycle (lytic or lysogenic). However, our ability to predict it is very limited. We aimed to discriminate between lytic and lysogenic phages by comparing the similarity of their genomic signatures to those of their hosts, reflecting their co-evolution. We tested two approaches: (1) similarities of tetramer relative frequencies, (2) alignment-free comparisons based on exact k = 14 oligonucleotide matches. First, we explored 5126 reference bacterial host strains and 284 associated phages and found an approximate threshold for distinguishing lysogenic and lytic phages using both oligonucleotide-based methods. The analysis of 6482 plasmids revealed the potential for horizontal gene transfer between different host genera and, in some cases, distant bacterial taxa. Subsequently, we experimentally analyzed combinations of 138 Klebsiella pneumoniae strains and their 41 phages and found that the phages with the largest number of interactions with these strains in the laboratory had the shortest genomic distances to K. pneumoniae. We then applied our methods to 24 single-cells from a hot spring biofilm containing 41 uncultured phage–host pairs, and the results were compatible with the lysogenic life cycle of phages detected in this environment. In conclusion, oligonucleotide-based genome analysis methods can be used for predictions of (1) life cycles of environmental phages, (2) phages with the broadest host range in culture collections, and (3) potential horizontal gene transfer by plasmids.

Bacteriophages play key roles in bacterial ecology and evolution and are potential antimicrobials. However, the determinants of phage-host specificity remain elusive. Here, we isolate 46 phages to challenge 138 representative clinical isolates of Klebsiella pneumoniae, a widespread opportunistic pathogen. Spot tests show a narrow host range for most phages, with <2% of 6,319 phage-host combinations tested yielding detectable interactions. Bacterial capsule diversity is the main factor restricting phage host range. Consequently, phage-encoded depolymerases are key determinants of host tropism, and depolymerase sequence types are associated with the ability to infect specific capsular types across phage families. However, all phages with a broader host range found do not encode canonical depolymerases, suggesting alternative modes of entry. These findings expand our knowledge of the complex interactions between bacteria and their viruses and point out the feasibility of predicting the first steps of phage infection using bacterial and phage genome sequences.