Drug resistance through ribosome splitting and rRNA disordering in mycobacteria.

HflX is known to rescue stalled ribosomes and is implicated in antibiotic resistance in several bacteria. Here we present several high-resolution cryo-EM structures of mycobacterial HflX in complex with the ribosome and its 50S subunit, with and without antibiotics. These structures reveal a distinct mechanism for HflX-mediated ribosome splitting and antibiotic resistance in mycobacteria.

Hierarchy and interconnected networks in the WhiB7 mediated transcriptional response to antibiotic stress in Mycobacterium abscessus.

Mycobacterium abscessus is intrinsically resistant to antibiotics effective against other pathogenic mycobacteria largely due to the drug-induced expression of genes that confer resistance. WhiB7 is a major hub controlling the induction of resistance to ribosome-targeting antibiotics. It activates the expression of >100 genes, 7 of which are known determinants of drug resistance; the function of most genes within the regulon is however unknown, but some conceivably encode additional mechanisms of resistance.

Telehealth utilization barriers among Alabama parents of pediatric patients during COVID-19 outbreak.

Background: Telehealth can improve access to evidence-based care at a lower cost for patients, especially those living in underserved and remote areas. The barriers to the widespread adoption of telehealth have been well documented in the literature. However, the barriers may not be the same for pediatric patients, who must rely on their parents or guardians to make healthcare decisions.

Intrapulmonary Treatment with Mycobacteriophage LysB Rapidly Reduces Mycobacterium abscessus Burden.

Intrinsic and acquired antibiotic resistance in Mycobacterium abscessus presents challenges in infection control, and new therapeutic strategies are needed. Bacteriophage therapy shows promise, but variabilities in M. abscessus phage susceptibility limits its broader utility.

Hierarchy and networks in the transcriptional response of to antibiotics.

Unlabelled: causes acute and chronic pulmonary infection in patients with chronic lung damage. It is intrinsically resistance to antibiotics effective against other pathogenic mycobacteria largely due to the drug-induced expression of genes that confer resistance. Induction of genes upon exposure to ribosome targeting antibiotics proceeds via WhiB7-dependent and -independent pathways.

Mab2780c, a TetV-like efflux pump, confers high-level spectinomycin resistance in mycobacterium abscessus.

Mycobacterium abscessus is highly resistant to spectinomycin (SPC) thereby making it unavailable for therapeutic use. Sublethal exposure to SPC strongly induces whiB7 and its regulon, and a ΔMab_whiB7 strain is SPC sensitive suggesting that the determinants of SPC resistance are included within its regulon. In the present study we have determined the transcriptomic changes that occur in M.

Pharmacogenomic Profiling of Pediatric Patients on Psychotropic Medications in an Emergency Department.

Objective: The aim of the study was to evaluate the ability of a combinatorial pharmacogenomic test to predict medication blood levels and relative clinical improvements in a selected pediatric population.

Methods: This study enrolled patients between ages 3 to 18 years who presented to a pediatric emergency department with acute psychiatric, behavioral, or mental health crisis and/or concerns, and had previously been prescribed psychotropic medications. Patients received combinatorial pharmacogenomic testing with medications categorized according to gene-drug interactions (GDIs); medications with a GDI were considered "incongruent," and medications without a GDI were considered "congruent.

Mycobacterium abscessus HelR interacts with RNA polymerase to confer intrinsic rifamycin resistance.

Rifampicin (RIF), the frontline drug against M. tuberculosis, is completely ineffective against M. abscessus, partially due to the presence of an ADP-ribosyltransferase (Arr) that inactivates RIF.

Disruption of Pediatric Emergency Department Use during the COVID-19 Pandemic.

Objectives: There is evidence of substantial declines in pediatric emergency department (ED) utilization in the United States in the first several months of the coronavirus disease 2019 (COVID-19) pandemic. Less is known about whether utilization changed differentially for socioeconomically disadvantaged children. This study examined how changes in pediatric ED visits during the initial months of the COVID-19 pandemic differed by two markers of socioeconomic disadvantage: minoritized race (MR) (compared with non-Hispanic White [NHW]), and publicly insured (compared with privately insured).

Disparities by race and insurance-status in declines in pediatric ED utilization during the COVID19 pandemic.

Pediatric Emergency Department (ED) utilization in the U.S. saw large declines during the COVID19 pandemic.

Ribosome Protection as a Mechanism of Lincosamide Resistance in Mycobacterium abscessus.

Mycobacterium abscessus has emerged as a successful pathogen owing to its intrinsic drug resistance. Macrolide and lincosamide antibiotics share overlapping binding sites within the ribosome and common resistance pathways. Nevertheless, while M.

Mycobacterial HflX is a ribosome splitting factor that mediates antibiotic resistance.

Antibiotic resistance in bacteria is typically conferred by proteins that function as efflux pumps or enzymes that modify either the drug or the antibiotic target. Here we report an unusual mechanism of resistance to macrolide-lincosamide antibiotics mediated by mycobacterial HflX, a conserved ribosome-associated GTPase. We show that deletion of the gene in the pathogenic , as well as the nonpathogenic , results in hypersensitivity to the macrolide-lincosamide class of antibiotics.

Mycobacterial SigA and SigB Cotranscribe Essential Housekeeping Genes during Exponential Growth.

Mycobacterial σ belongs to the group II family of sigma factors, which are widely considered to transcribe genes required for stationary-phase survival and the response to stress. Here we explored the mechanism underlying the observed hypersensitivity of Δ deletion mutants of , , and to rifampin (RIF) and uncovered an additional constitutive role of σ during exponential growth of mycobacteria that complements the function of the primary sigma factor, σ Using chromatin immunoprecipitation sequencing (ChIP-Seq), we show that during exponential phase, σ binds to over 200 promoter regions, including those driving expression of essential housekeeping genes, like the rRNA gene. ChIP-Seq of ectopically expressed σ-FLAG demonstrated that at least 61 promoter sites are recognized by both σ and σ These results together suggest that RNA polymerase holoenzymes containing either σ or σ transcribe housekeeping genes in exponentially growing mycobacteria.

Unintentional Opioid Ingestions Presenting to a Pediatric Emergency Department.

Objectives: The purpose of this study was to describe unintentional opioid exposures in young children, including demographics, medical interventions, and clinical outcomes.

Methods: This was a retrospective, cross-sectional study of children 0 to 6 years of age with possible opioid exposure over a 3-year period (July 2010 to June 2013). Data collected included sex, age, specific drug, whether they were referred to the emergency department (ED) by the Regional Poison Control Center, presence of symptoms, therapeutic interventions, ED disposition, and clinical outcomes for admitted patients.

High Levels of Intrinsic Tetracycline Resistance in Mycobacterium abscessus Are Conferred by a Tetracycline-Modifying Monooxygenase.

Tetracyclines have been one of the most successful classes of antibiotics. However, its extensive use has led to the emergence of widespread drug resistance, resulting in discontinuation of use against several bacterial infections. Prominent resistance mechanisms include drug efflux and the use of ribosome protection proteins.

Mycobacterium abscessus WhiB7 Regulates a Species-Specific Repertoire of Genes To Confer Extreme Antibiotic Resistance.

causes acute and chronic bronchopulmonary infection in patients with chronic lung damage, of which cystic fibrosis (CF) patients are particularly vulnerable. The major threat posed by this organism is its high intrinsic antibiotic resistance. A typical treatment regimen involves a 6- to 12-month-long combination therapy of clarithromycin and amikacin, with cure rates below 50% and multiple side effects, especially due to amikacin.

A complex regulatory network controlling intrinsic multidrug resistance in Mycobacterium smegmatis.

Mycobacteria are intrinsically resistant to a variety of stresses including many antibiotics. Although a number of pathways have been described to account for the observed resistances, the mechanisms that control the expression of genes required in these processes remain poorly defined. Here we report the role of a predicted anti-sigma factor, MSMEG_6129 and a predicted eukaryotic like serine/threonine protein kinase, MSMEG_5437, in the intrinsic resistance of Mycobacterium smegmatis to a variety of stresses including the genotoxic agent mitomycin C, hydrogen peroxide and at least four different antibiotics - isoniazid, chloramphenicol, erythromycin and tetracycline.

Attachment site selection and identity in Bxb1 serine integrase-mediated site-specific recombination.

Phage-encoded serine integrases mediate directionally regulated site-specific recombination between short attP and attB DNA sites without host factor requirements. These features make them attractive for genome engineering and synthetic genetics, although the basis for DNA site selection is poorly understood. Here we show that attP selection is determined through multiple proofreading steps that reject non-attP substrates, and that discrimination of attP and attB involves two critical site features: the outermost 5-6 base pairs of attP that are required for Int binding and recombination but antagonize attB function, and the "discriminators" at positions -15/+15 that determine attB identity but also antagonize attP function.

Remote control of DNA-acting enzymes by varying the Brownian dynamics of a distant DNA end.

Enzyme rates are usually considered to be dependent on local properties of the molecules involved in reactions. However, for large molecules, distant constraints might affect reaction rates by affecting dynamics leading to transition states. In single-molecule experiments we have found that enzymes that relax DNA torsional stress display rates that depend strongly on how the distant ends of the molecule are constrained; experiments with different-sized particles tethered to the end of 10-kb DNAs reveal enzyme rates inversely correlated with particle drag coefficients.

The Bxb1 gp47 recombination directionality factor is required not only for prophage excision, but also for phage DNA replication.

Mycobacteriophage Bxb1 encodes a serine-integrase that catalyzes both integrative and excisive site-specific recombination. However, excision requires a second phage-encoded protein, gp47, which serves as a recombination directionality factor (RDF). The viability of a Bxb1 mutant containing an S153A substitution in gp47 that eliminates the RDF activity of Bxb1 gp47 shows that excision is not required for Bxb1 lytic growth.

Successive and targeted DNA integrations in the Drosophila genome by Bxb1 and phiC31 integrases.

At present ϕC31 is the only phage integrase system available for directionally regulated site-specific DNA integration in the Drosophila genome. Here we report that mycobacteriophage Bxb1 integrase also mediates targeted DNA integration in Drosophila with high specificity and efficiency. By alternately using Bxb1 and ϕC31, we were able to carry out multiple rounds of successive and targeted DNA integrations in our genomic engineering founder lines for the purpose of generating complex knock-in alleles.

Single-molecule analysis reveals the molecular bearing mechanism of DNA strand exchange by a serine recombinase.

Structural and topological data suggest that serine site-specific DNA recombinases exchange duplex DNAs by rigid-body relative rotation of the two halves of the synapse, mediated by a flat protein-protein interaction surface. We present evidence for this rotational motion for a simple serine recombinase, the Bxb1 phage integrase, from a single-DNA-based supercoil-release assay that allows us to follow crossover site cleavage, rotation, religation, and product release in real time. We have also used a two-DNA braiding-relaxation experiment to observe the effect of synapse rotation in reactions on two long molecules.

Two-step site selection for serine-integrase-mediated excision: DNA-directed integrase conformation and central dinucleotide proofreading.

Bacteriophage-encoded serine-integrases are members of the large family of serine-recombinases and catalyze site-specific integrative recombination between a phage attP site and a bacterial attB site to form an integrated prophage. Prophage excision involves a second site-specific recombination event, in which the sites generated by integration, attL and attR, are used as substrates to regenerate attP and attB. Excision is catalyzed by integrase but also requires a phage-encoded recombination directionality factor (RDF).

Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase.

Here we report an efficient, site-specific system of genetic integration into Plasmodium falciparum malaria parasite chromosomes. This is mediated by mycobacteriophage Bxb1 integrase, which catalyzes recombination between an incoming attP and a chromosomal attB site. We developed P.

Control of phage Bxb1 excision by a novel recombination directionality factor.

Mycobacteriophage Bxb1 integrates its DNA at the attB site of the Mycobacterium smegmatis genome using the viral attP site and a phage-encoded integrase generating the recombinant junctions attL and attR. The Bxb1 integrase is a member of the serine recombinase family of site-specific recombination proteins and utilizes small (<50 base pair) substrates for recombination, promoting strand exchange without the necessity for complex higher order macromolecular architectures. To elucidate the regulatory mechanism for the integration and excision reactions, we have identified a Bxb1-encoded recombination directionality factor (RDF), the product of gene 47.