A novel hybridization capture method for direct whole genome sequencing of clinical specimens to inform Legionnaires' disease investigations.

The unprecedented precision and resolution of whole genome sequencing (WGS) can provide definitive identification of infectious agents for epidemiological outbreak tracking. WGS approaches, however, are frequently impeded by low pathogen DNA recovery from available primary specimens or unculturable samples. A cost-effective hybrid capture assay for WGS analysis directly on primary specimens was developed.

Whole genome sequencing of human Borrelia burgdorferi isolates reveals linked blocks of accessory genome elements located on plasmids and associated with human dissemination.

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting Borrelia burgdorferi spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 B.

Direct detection of drug-resistant using targeted next generation sequencing.

complex (MTBC) infections are treated with combinations of antibiotics; however, these regimens are not as efficacious against multidrug and extensively drug resistant MTBC. Phenotypic (growth-based) drug susceptibility testing on slow growing bacteria like MTBC requires many weeks to months to complete, whereas sequencing-based approaches can predict drug resistance (DR) with reduced turnaround time. We sought to develop a multiplexed, targeted next generation sequencing (tNGS) assay that can predict DR and can be performed directly on clinical respiratory specimens.

Genome-Wide Mapping of the Escherichia coli PhoB Regulon Reveals Many Transcriptionally Inert, Intragenic Binding Sites.

Genome-scale analyses have revealed many transcription factor binding sites within, rather than upstream of, genes, raising questions as to the function of these binding sites. Here, we use complementary approaches to map the regulon of the Escherichia coli transcription factor PhoB, a response regulator that controls transcription of genes involved in phosphate homeostasis. Strikingly, the majority of PhoB binding sites are located within genes, but these intragenic sites are not associated with detectable transcription regulation and are not evolutionarily conserved.

Whole genome sequencing of isolates reveals linked clusters of plasmid-borne accessory genome elements associated with virulence.

Lyme disease is the most common vector-borne disease in North America and Europe. The clinical manifestations of Lyme disease vary based on the genospecies of the infecting spirochete, but the microbial genetic elements underlying these associations are not known. Here, we report the whole genome sequence (WGS) and analysis of 299 patient-derived sensu stricto ( ) isolates from patients in the Eastern and Midwestern US and Central Europe.

Genome-wide mapping of the PhoB regulon reveals many transcriptionally inert, intragenic binding sites.

Unlabelled: Genome-scale analyses have revealed many transcription factor binding sites within, rather than upstream of genes, raising questions as to the function of these binding sites. Here, we use complementary approaches to map the regulon of the transcription factor PhoB, a response regulator that controls transcription of genes involved in phosphate homeostasis. Strikingly, the majority of PhoB binding sites are located within genes, but these intragenic sites are not associated with detectable transcription regulation and are not evolutionarily conserved.

Characterization of sp. nov.: A novel nontuberculous mycobacteria isolated from a human wound infection.

Nontuberculous mycobacteria (NTM) are environmental bacteria commonly found in soil and water in almost every part of the world. While usually non-pathogenic, they can cause acute respiratory and cutaneous infections under certain circumstances or in patients with underlying medical conditions. Contrary to members of the complex, documented human-to-human transmissions of NTM have been rarely reported and most cases result from direct environmental exposure.

Type 2 autoimmune hepatitis: Genetic susceptibility.

Two types of autoimmune hepatitis (AIH) are recognized; AIH-1 is characterized by the presence of anti-nuclear and/or anti-smooth muscle autoantibodies, while AIH-2 is associated with the presence of anti-Liver kidney microsome and/or anti-Liver Cytosol antibodies. The autoantigens targeted by AIH-2 autoantibodies are the cytochrome P450 2D6 and Formiminotransferase-cyclodeaminase for anti-LKM1 and anti-LC1 respectively. Both autoantigens are expressed in hepatocytes at higher levels than in any other cell type.

Comparative analysis of multiplexed PCR and short- and long-read whole genome sequencing to investigate a large Klebsiella pneumoniae outbreak in New York State.

In 2017, the New York State Department of Health investigated a large Klebsiella pneumoniae outbreak in a health care facility. A retrospective analysis was conducted to compare the use of multiple molecular typing methods for characterizing the outbreak. Forty-four isolates were characterized using the rapid real-time PCR OpGen Acuitas® AMR Gene Panel.

Immune-Mediated Hepatitis During Immune Checkpoint Inhibitor cancer Immunotherapy: Lessons From Autoimmune Hepatitis and Liver Immunology.

Immune checkpoint inhibitors (ICI) are being increasingly used to successfully treat several types of cancer. However, due to their mode of action, these treatments are associated with several immune-related adverse events (irAEs), including immune-mediated autoimmune-like hepatitis in 5 to 10% of cases. The specific immune mechanism responsible for the development of immune-mediated liver injury caused by immune checkpoint inhibitors (ILICI) is currently unknown.

Novel Mycobacterium tuberculosis Complex Genotype Related to M. caprae.

We report the unusual genotypic characterization of a bacterium isolated from a clinical sample of a patient who grew up in Bangladesh and lives in the United States. Using whole-genome sequencing, we identified the bacterium as a member of the Mycobacterium tuberculosis complex (MTBC). Phylogenetic placement of this strain suggests a new MTBC genotype.

Improvements to the Success of Outbreak Investigations of Legionnaires' Disease: 40 Years of Testing and Investigation in New York State.

Since 1978, the New York State Department of Health's public health laboratory, Wadsworth Center (WC), in collaboration with epidemiology and environmental partners, has been committed to providing comprehensive public health testing for Legionella in New York. Statewide, clinical case counts have been increasing over time, with the highest numbers identified in 2017 and 2018 (1,022 and 1,426, respectively). Over the course of more than 40 years, the WC Legionella testing program has continuously implemented improved testing methods.

A Bioinformatic Pipeline for Improved Genome Analysis and Clustering of Isolates during Outbreaks of Legionnaires' Disease.

Legionnaires' disease, a severe lung infection caused by the bacterium , occurs as single cases or in outbreaks that are actively tracked by public health departments. To determine the point source of an outbreak, clinical isolates need to be compared to environmental samples to find matching isolates. One confounding factor is the genome plasticity of , making an exact sequence comparison by whole-genome sequencing (WGS) challenging.

Assessing Nanopore Sequencing for Clinical Diagnostics: a Comparison of Next-Generation Sequencing (NGS) Methods for Mycobacterium tuberculosis.

Next-generation sequencing technologies are being rapidly adopted as a tool of choice for diagnostic and outbreak investigation in public health laboratories. However, costs of operation and the need for specialized staff remain major hurdles for laboratories with limited resources for implementing these technologies. This project aimed to assess the feasibility of using Oxford Nanopore MinION whole-genome sequencing data of isolates for species identification, spoligotyping, detection of mutations associated with antimicrobial resistance (AMR) to accurately predict drug susceptibility profiles, and phylogenetic analysis to detect transmission between cases.

Low-Level Rifampin Resistance and Mutations in Mycobacterium tuberculosis: an Analysis of Whole-Genome Sequencing and Drug Susceptibility Test Data in New York.

Rapid and reliable detection of rifampin (RIF) resistance is critical for the diagnosis and treatment of drug-resistant and multidrug-resistant (MDR) tuberculosis. Discordant RIF phenotype/genotype susceptibility results remain a challenge due to the presence of mutations that do not confer high levels of RIF resistance, as have been exhibited in strains with mutations such as Ser450Leu. These strains, termed low-level RIF resistant, exhibit elevated RIF MICs compared to fully susceptible strains but remain phenotypically susceptible by mycobacterial growth indicator tube (MGIT) testing and have been associated with poor patient outcomes.

Nanopore MinION Sequencing Reveals Possible Transfer of Plasmid Across Bacterial Species in Two Healthcare Facilities.

Carbapenemase-producing are a major threat to global public health. carbapenemase (KPC) is the most commonly identified carbapenemase in the United States and is frequently found on mobile genetic elements including plasmids, which can be horizontally transmitted between bacteria of the same or different species. Here we describe the results of an epidemiological investigation of KPC-producing bacteria at two healthcare facilities.

Origin of 3 Rabid Terrestrial Animals in Raccoon Rabies Virus-Free Zone, Long Island, New York, USA, 2016-2017.

During 2016-2017, three rabid terrestrial animals were discovered in the raccoon rabies virus-free zone of Long Island, New York, USA. Whole-genome sequencing and phylogenetic analyses revealed the likely origins of the viruses, enabling the rabies outbreak response (often costly and time-consuming) to be done less expensively and more efficiently.

Whole-Genome Single-Nucleotide Polymorphism (SNP) Analysis Applied Directly to Stool for Genotyping Shiga Toxin-Producing Escherichia coli: an Advanced Molecular Detection Method for Foodborne Disease Surveillance and Outbreak Tracking.

Whole-genome sequencing (WGS) of pathogens from pure culture provides unparalleled accuracy and comprehensive results at a cost that is advantageous compared with traditional diagnostic methods. Sequencing pathogens directly from a primary clinical specimen would help circumvent the need for culture and, in the process, substantially shorten the time to diagnosis and public health reporting. Unfortunately, this approach poses significant challenges because of the mixture of multiple sequences from a complex fecal biomass.

Oxaloacetate Protects Rat Liver From Experimental Warm Ischemia/Reperfusion Injury by Improving Cellular Energy Metabolism.

Liver ischemia/reperfusion injury (IRI) is an important cause of liver damage especially early after liver transplantation, following liver resection, and in other clinical situations. Using rat experimental models, we identified oxaloacetate (OAA) as a key metabolite able to protect hepatocytes from hypoxia and IRI. In vitro screening of metabolic intermediates beneficial for hepatocyte survival under hypoxia was performed by measures of cell death and injury.

Insights into the long-term persistence of Legionella in facilities from whole-genome sequencing.

We investigated the value of whole-genome sequencing (WGS) and single nucleotide polymorphism (SNP) analyses in determining the relationships among and evolutionary rates of Legionella species with long-term persistence in three healthcare facilities. We examined retrospective clinical and environmental isolates of Legionella micdadei and Legionella pneumophila serogroup 1 isolates with identical PFGE DNA fingerprints sampled over the course of up to 18 years. WGS analyses demonstrated that heterogeneous populations of Legionella were present within each facility despite displaying the same PFGE profiles.

Metabolite profiling identifies a signature of tumorigenicity in hepatocellular carcinoma.

HCC (Hepatocellular carcinoma) cells exhibit greater metabolic plasticity than normal hepatocytes since they must survive in a dynamic microenvironment where nutrients and oxygen are often scarce. Using a metabolomic approach combined with functional and assays, we aimed to identify an HCC metabolic signature associated with increased tumorigenicity and patient mortality. Metabolite profiling of HCC Dt81Hepa1-6 cells revealed that their increased tumorigenicity was associated with elevated levels of glycolytic metabolites.

Upregulation of Krebs cycle and anaerobic glycolysis activity early after onset of liver ischemia.

The liver is a highly vascularized organ receiving a dual input of oxygenated blood from the hepatic artery and portal vein. The impact of decreased blood flow on glucose metabolism and how hepatocytes could adapt to this restrictive environment are still unclear. Using the left portal vein ligation (LPVL) rat model, we found that cellular injury was delayed after the onset of liver ischemia.

Metabolic reprogramming enables hepatocarcinoma cells to efficiently adapt and survive to a nutrient-restricted microenvironment.

Hepatocellular carcinoma (HCC) is a metabolically heterogeneous cancer and the use of glucose by HCC cells could impact their tumorigenicity. Dt81Hepa1-6 cells display enhanced tumorigenicity compared to parental Hepa1-6 cells. This increased tumorigenicity could be explained by a metabolic adaptation to more restrictive microenvironments.