1,3-Dioxane-Linked Novel Bacterial Topoisomerase Inhibitors: Expanding Structural Diversity and the Antibacterial Spectrum.

Antibacterial resistance continues its devastation of available therapies. Novel bacterial topoisomerase inhibitors (NBTIs) offer one solution to this critical issue. Two series of amine NBTIs bearing tricyclic DNA-binding moieties as well as amide NBTIs with a bicyclic DNA-binding moiety were synthesized and evaluated against methicillin-resistant (MRSA).

Identification and Characterization of Mycobacterial Species Using Whole-Genome Sequences.

Whole-genome sequencing (WGS) has shown immense value in enabling identification and characterization of bacterial taxa. This is particularly true for mycobacteria, where culture-based characterization becomes delayed by the inherently slow growth rate of these organisms. This chapter reviews the general techniques behind WGS and their optimization, existing techniques for species-level identification and the advantages of WGS for this purpose, and a variety of useful tools for the genomic characterization of mycobacterial strains.

Culturing Mycobacteria.

Building upon the foundational research of Robert Koch, who demonstrated the ability to grow Mycobacterium tuberculosis for the first time in 1882 using media made of coagulated bovine serum, microbiologists have continued to develop new and more efficient ways to grow mycobacteria. Presently, all known mycobacterial species can be grown in the laboratory using either axenic culture techniques or in vivo passage in laboratory animals. This chapter provides conventional protocols to grow mycobacteria for diagnostic purposes directly from clinical specimens, as well as in research laboratories for scientific purposes.

gcType: a high-quality type strain genome database for microbial phylogenetic and functional research.

Taxonomic and functional research of microorganisms has increasingly relied upon genome-based data and methods. As the depository of the Global Catalogue of Microorganisms (GCM) 10K prokaryotic type strain sequencing project, Global Catalogue of Type Strain (gcType) has published 1049 type strain genomes sequenced by the GCM 10K project which are preserved in global culture collections with a valid published status. Additionally, the information provided through gcType includes >12 000 publicly available type strain genome sequences from GenBank incorporated using quality control criteria and standard data annotation pipelines to form a high-quality reference database.

A Rare Deletion in SARS-CoV-2 ORF6 Dramatically Alters the Predicted Three-Dimensional Structure of the Resultant Protein.

The function of the SARS-CoV-2 accessory protein p6, encoded by ORF6, is not fully known. Based upon its similarity to p6 from SARS-CoV, it may play a similar role, namely as an antagonist of type I interferon (IFN) signaling. Here we report the sequencing of a SARS-CoV-2 strain passaged six times after original isolation from a clinical patient in Hong Kong.

Long-Term Follow-Up of the First in Human Intravascular Delivery of AAV for Gene Transfer: AAV2-hFIX16 for Severe Hemophilia B.

Adeno-associated virus (AAV) vectors are a leading platform for gene-based therapies for both monogenic and complex acquired disorders. The success of AAV gene transfer highlights the need to answer outstanding clinical questions of safety, durability, and the nature of the human immune response to AAV vectors. Here, we present longitudinal follow-up data of subjects who participated in the first trial of a systemically delivered AAV vector.

Regulation of NGF Signaling by an Axonal Untranslated mRNA.

Neurons are extraordinarily large and highly polarized cells that require rapid and efficient communication between cell bodies and axons over long distances. In peripheral neurons, transcripts are transported along axons to growth cones, where they are rapidly translated in response to extrinsic signals. While studying Tp53inp2, a transcript highly expressed and enriched in sympathetic neuron axons, we unexpectedly discovered that Tp53inp2 is not translated.

Mycobacterial biomaterials and resources for researchers.

There are many resources available to mycobacterial researchers, including culture collections around the world that distribute biomaterials to the general scientific community, genomic and clinical databases, and powerful bioinformatics tools. However, many of these resources may be unknown to the research community. This review article aims to summarize and publicize many of these resources, thus strengthening the quality and reproducibility of mycobacterial research by providing the scientific community access to authenticated and quality-controlled biomaterials and a wealth of information, analytical tools and research opportunities.

The global catalogue of microorganisms 10K type strain sequencing project: closing the genomic gaps for the validly published prokaryotic and fungi species.

Genomic information is essential for taxonomic, phylogenetic, and functional studies to comprehensively decipher the characteristics of microorganisms, to explore microbiomes through metagenomics, and to answer fundamental questions of nature and human life. However, large gaps remain in the available genomic sequencing information published for bacterial and archaeal species, and the gaps are even larger for fungal type strains. The Global Catalogue of Microorganisms (GCM) leads an internationally coordinated effort to sequence type strains and close gaps in the genomic maps of microorganisms.

Phylogenomic analysis of the species of the Mycobacterium tuberculosis complex demonstrates that Mycobacterium africanum, Mycobacterium bovis, Mycobacterium caprae, Mycobacterium microti and Mycobacterium pinnipedii are later heterotypic synonyms of Mycobacterium tuberculosis.

The species within the Mycobacterium tuberculosis Complex (MTBC) have undergone numerous taxonomic and nomenclatural changes, leaving the true structure of the MTBC in doubt. We used next-generation sequencing (NGS), digital DNA-DNA hybridization (dDDH), and average nucleotide identity (ANI) to investigate the relationship between these species. The type strains of Mycobacterium africanum, Mycobacterium bovis, Mycobacterium caprae, Mycobacterium microti and Mycobacterium pinnipedii were sequenced via NGS.

Academic Productivity of Interventional Pulmonology Training Programs.

Rationale: The Hirsch index (h-index) has been validated as a measure of academic productivity and may be an appropriate tool to assess the scholarly activity of interventional pulmonology (IP).

Objectives: This study aimed to elucidate the factors associated with increasing h-index scores among IP training programs.

Methods: A cross-sectional study was conducted of IP training programs across the United States and Canada.

Multiplex PCR for species-level identification of Bacillus anthracis and detection of pXO1, pXO2, and related plasmids.

The Bacillus anthracis virulence plasmids pXO1 and pXO2 have critical implications for biosafety and select agent status. The proper identification and characterization of B. anthracis and its plasmid profile is important to the biodefense research community.

Multiplexed Luminex xMAP assay for detection and identification of five adenovirus serotypes associated with epidemics of respiratory disease in adults.

Several serotypes of human adenovirus (HAdV) cause acute respiratory disease (ARD) among healthy adults, sometimes generating broad outbreaks with high attack rates and occasional fatalities. Timely serotype identification provides valuable epidemiological information and significantly contributes to prevention (vaccination) strategies. The prevalence of specific serotypes causing ARD varies geographically.

Mycothiol biosynthesis is essential for ethionamide susceptibility in Mycobacterium tuberculosis.

Spontaneous mutants of Mycobacterium tuberculosis that were resistant to the anti-tuberculosis drugs ethionamide and isoniazid were isolated and found to map to mshA, a gene encoding the first enzyme involved in the biosynthesis of mycothiol, a major low-molecular-weight thiol in M. tuberculosis. Seven independent missense or frameshift mutations within mshA were identified and characterized.

Convergent evolutionary analysis identifies significant mutations in drug resistance targets of Mycobacterium tuberculosis.

Mycobacterium tuberculosis adapts to the environment by selecting for advantageous single-nucleotide polymorphisms (SNPs). We studied whether advantageous SNPs could be distinguished from neutral mutations within genes associated with drug resistance. A total of 1,003 clinical isolates of M.

Transfer of embB codon 306 mutations into clinical Mycobacterium tuberculosis strains alters susceptibility to ethambutol, isoniazid, and rifampin.

Implicated as a major mechanism of ethambutol (EMB) resistance in clinical studies of Mycobacterium tuberculosis, mutations in codon 306 of the embB gene (embB306) have also been detected in EMB-susceptible clinical isolates. Other studies have found strong associations between embB306 mutations and multidrug resistance, but not EMB resistance. We performed allelic exchange studies in EMB-susceptible and EMB-resistant clinical M.

Antibiotic resistance and single-nucleotide polymorphism cluster grouping type in a multinational sample of resistant Mycobacterium tuberculosis isolates.

A single-nucleotide polymorphism-based cluster grouping (SCG) classification system for Mycobacterium tuberculosis was used to examine antibiotic resistance type and resistance mutations in relationship to specific evolutionary lineages. Drug resistance and resistance mutations were seen across all SCGs. SCG-2 had higher proportions of katG codon 315 mutations and resistance to four drugs.

Transcriptional regulation of multi-drug tolerance and antibiotic-induced responses by the histone-like protein Lsr2 in M. tuberculosis.

Multi-drug tolerance is a key phenotypic property that complicates the sterilization of mammals infected with Mycobacterium tuberculosis. Previous studies have established that iniBAC, an operon that confers multi-drug tolerance to M. bovis BCG through an associated pump-like activity, is induced by the antibiotics isoniazid (INH) and ethambutol (EMB).

Role of large sequence polymorphisms (LSPs) in generating genomic diversity among clinical isolates of Mycobacterium tuberculosis and the utility of LSPs in phylogenetic analysis.

Mycobacterium tuberculosis strains contain different genomic insertions or deletions called large sequence polymorphisms (LSPs). Distinguishing between LSPs that occur one time versus ones that occur repeatedly in a genomic region may provide insights into the biological roles of LSPs and identify useful phylogenetic markers. We analyzed 163 clinical M.

Transfer of a point mutation in Mycobacterium tuberculosis inhA resolves the target of isoniazid.

Isoniazid is one of the most effective antituberculosis drugs, yet its precise mechanism of action is still controversial. Using specialized linkage transduction, a single point mutation allele (S94A) within the putative target gene inhA was transferred in Mycobacterium tuberculosis. The inhA(S94A) allele was sufficient to confer clinically relevant levels of resistance to isoniazid killing and inhibition of mycolic acid biosynthesis.

Population genetics study of isoniazid resistance mutations and evolution of multidrug-resistant Mycobacterium tuberculosis.

The molecular basis for isoniazid resistance in Mycobacterium tuberculosis is complex. Putative isoniazid resistance mutations have been identified in katG, ahpC, inhA, kasA, and ndh. However, small sample sizes and related potential biases in sample selection have precluded the development of statistically valid and significant population genetic analyses of clinical isoniazid resistance.

Arginine homeostasis in J774.1 macrophages in the context of Mycobacterium bovis BCG infection.

The competition for L-arginine between the inducible nitric oxide synthase and arginase contributes to the outcome of several parasitic and bacterial infections. The acquisition of L-arginine, however, is important not only for the host cells but also for the intracellular pathogen. In this study we observe that strain AS-1, the Mycobacterium bovis BCG strain lacking the Rv0522 gene, which encodes an arginine permease, perturbs l-arginine metabolism in J774.

Global phylogeny of Mycobacterium tuberculosis based on single nucleotide polymorphism (SNP) analysis: insights into tuberculosis evolution, phylogenetic accuracy of other DNA fingerprinting systems, and recommendations for a minimal standard SNP set.

We analyzed a global collection of Mycobacterium tuberculosis strains using 212 single nucleotide polymorphism (SNP) markers. SNP nucleotide diversity was high (average across all SNPs, 0.19), and 96% of the SNP locus pairs were in complete linkage disequilibrium.

Role of embB codon 306 mutations in Mycobacterium tuberculosis revisited: a novel association with broad drug resistance and IS6110 clustering rather than ethambutol resistance.

Mutations at position 306 of embB (embB306) have been proposed as a marker for ethambutol resistance in Mycobacterium tuberculosis; however, recent reports of embB306 mutations in ethambutol-susceptible isolates caused us to question the biological role of this mutation. We tested 1,020 clinical M. tuberculosis isolates with different drug susceptibility patterns and of different geographical origins for associations between embB306 mutations, drug resistance patterns, and major genetic group.

The Mycobacterium tuberculosis iniA gene is essential for activity of an efflux pump that confers drug tolerance to both isoniazid and ethambutol.

Little is known about the intracellular events that occur following the initial inhibition of Mycobacterium tuberculosis by the first-line antituberculosis drugs isoniazid (INH) and ethambutol (EMB). Understanding these pathways should provide significant insights into the adaptive strategies M. tuberculosis undertakes to survive antibiotics.