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. Using RNA-seq, we show that exposure of M. abscessus to sublethal doses of RIF and Rifabutin (RBT), a close analog of RIF, results in an ∼25-fold upregulation of Mab_helR in laboratory and clinical isolates. An isogenic deletion in Mab_helR results in RIF/RBT hypersensitivity, and overexpression of Mab_helR confers RIF tolerance in M. tuberculosis. We demonstrate an increased HelR-RNAP association in RIF-exposed bacteria and a MabHelR-mediated dissociation of RNAP from stalled initiation complexes in vitro. Finally, we show that the tip of the PCh-loop of Mab_helR, present in proximity to RIF, is critical for conferring RIF resistance but dispensable for dissociation of stalled RNAP complexes, suggesting that HelR-mediated RIF resistance requires a step in addition to displacement of RIF-stalled RNAP.

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. Furthermore, the hierarchy of gene expression within the regulon, if any, is poorly understood. In the present work we have identified 56 WhiB7 binding sites using chromatin immunoprecipitation sequencing (CHIP-Seq) which accounts for the WhiB7-dependent upregulation of 72 genes, and find that M. abscessus WhiB7 functions exclusively as a transcriptional activator at promoters recognized by σA/σB. We have investigated the role of 18 WhiB7 regulated genes in drug resistance. Our results suggest that while some genes within the regulon (eg. erm41, hflX, eis2 and the ABCFs) play a major role in resistance, others make smaller contributions (eg. MAB_4324c and MAB_1409c) and the observed hypersensitivity ΔMabwhiB7 is a cumulative effect of these individual contributions. Moreover, our CHIP-Seq data implicate additional roles of WhiB7 induced genes beyond antibiotic resistance. Finally, we identify a σH-dependent network in aminoglycoside and tigecycline resistance which is induced upon drug exposure and is further activated by WhiB7 demonstrating the existence of a crosstalk between components of the WhiB7-dependent and -independent circuits.

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. We show here that a mycobacteriophage-encoded lysin B (LysB) efficiently and rapidly kills both smooth- and rough-colony morphotype M. abscessus strains and reduces the pulmonary bacterial load in mice. LysB aerosolization presents a plausible treatment for pulmonary M. abscessus infections.

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. This paper presents some of the leading barriers parents or guardians of pediatric patients report in using telehealth to meet their children's healthcare needs. METHODS: This cross-sectional survey was conducted in a tertiary care pediatric Emergency Department (ED) at a children's hospital in Alabama between September 2020 to December 2020. The parents or guardians of pediatric patients were asked about their reasons for not using telehealth despite having healthcare needs for their children, whether they canceled or rescheduled healthcare provider visits and facility visits, and whether the child's health conditions changed over the past three months. Descriptive analyses were conducted that explored the distribution of telehealth use across the variables listed above. RESULTS: Five hundred ninety-seven parents or guardians of pediatric patients participated in the survey, and 578 answered the question of whether they used telehealth or not over the past three months. Of them, 33.1% used telehealth, 54.3% did not, and 12.6% did not have healthcare needs for their child. The leading reason for not using telehealth was that the doctor or health provider did not give them a telehealth option, the second main reason was that they did not know what telehealth is, and the third leading reason was that the parents did not think telehealth would help meet healthcare needs for their child. CONCLUSIONS: This study highlights the telehealth utilization barriers among underserved pediatric populations, including the need for physicians to proactively offer telehealth options to parents or guardians of pediatric patients. Improving health literacy is of paramount importance, given that a substantial proportion of parents were not familiar with telehealth. Policymakers and healthcare organizations should raise awareness about the benefits of telehealth which can improve healthcare access for underserved pediatric patients.

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 hflX gene in the pathogenic Mycobacterium abscessus, as well as the nonpathogenic Mycobacterium smegmatis, results in hypersensitivity to the macrolide-lincosamide class of antibiotics. Importantly, the level of resistance provided by Mab_hflX is equivalent to that conferred by erm41, implying that hflX constitutes a significant resistance determinant in M. abscessus We demonstrate that mycobacterial HflX associates with the 50S ribosomal subunits in vivo and can dissociate purified 70S ribosomes in vitro, independent of GTP hydrolysis. The absence of HflX in a ΔMs_hflX strain also results in a significant accumulation of 70S ribosomes upon erythromycin exposure. Finally, a deletion of either the N-terminal or the C-terminal domain of HflX abrogates ribosome splitting and concomitantly abolishes the ability of mutant proteins to mediate antibiotic tolerance. Together, our results suggest a mechanism of macrolide-lincosamide resistance in which the mycobacterial HflX dissociates antibiotic-stalled ribosomes and rescues the bound mRNA. Given the widespread presence of hflX genes, we anticipate this as a generalized mechanism of macrolide resistance used by several bacteria.

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." Blood levels for escitalopram, fluoxetine, aripiprazole, and clonidine were evaluated according to level of GDI. Relative clinical improvements in response to the prescribed psychotropic medications were measured using a parent-rated Clinical Global Impression of Improvement (CGI-I) assessment, where lower scores corresponded with greater improvement. RESULTS: Of the 100 patients enrolled, 73% reported taking ≥1 incongruent medication. There was no significant difference in CGI-I scores between patients prescribed congruent versus incongruent medications (3.37 vs 3.68, P = 0.343). Among patients who presented for depression or suicidal ideation, those prescribed congruent medications had significantly lower CGI-I scores compared with those taking incongruent medications ( P = 0.036 for depression, P = 0.018 for suicidal ideation). There was a significant association between medication GDI and blood levels for aripiprazole (n = 15, P = 0.01) and escitalopram (n = 10, P = 0.01). CONCLUSIONS: Our preliminary findings suggest that combinatorial pharmacogenomic testing can predict medication blood levels and relative outcomes based on medication congruency in children presenting to an emergency department with acute psychiatric/behavioral crises. Additional studies will be needed to confirm these findings.

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). METHODS: This study used electronic medical records from a large pediatric ED for the period January to June 2020. Three time periods in 2020 were compared with corresponding time periods in 2019. Changes in overall visits, visits for MR versus NHW children, and Medicaid-enrolled versus privately insured children were considered, and changes in the acuity mix of ED visits and share of visits resulting in inpatient admits were inspected. RESULTS: Compared with 2019, total ED visits declined in time period (TP) 1 and TP2 of 2020 (54.3%, 48.9%). Declines were larger for MR children (57.3%, 57.8%) compared with NHW children (50.5%, 39.3%), and Medicaid enrollees (56.5%, 52.0%) compared with privately insured (48.3%, 39.0%). The MR children group experienced steeper percentage declines in high-acuity visits and visits, resulting in inpatient admissions compared with NHW children. In contrast, there was little evidence of difference between TP0s of 2019 and 2020. CONCLUSIONS: The role of socioeconomic disadvantage and the potential effects on pediatric ED visits during COVID-19 is understudied. Because disadvantaged children sometimes lack access to a usual source of health care, this raises concerns about unmet health needs and worsening health disparities.

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. abscessus is initially susceptible to macrolides, they are completely resistant to the lincosamide antibiotics. Here, we have used RNA sequencing to determine the changes in gene expression in M. abscessus upon exposure to the lincosamide, clindamycin (CLY). We show that Mab_1846, encoding a putative ARE-ABCF protein, was upregulated upon exposure to macrolides and lincosamides but conferred resistance to CLY alone. A Mycobacterium smegmatis homologue of Mab_1846, Ms_5102, was similarly found to be required for CLY resistance in M. smegmatis. We demonstrate that Ms5102 mediates CLY resistance by directly interacting with the ribosomes and protecting it from CLY inhibition. Additional biochemical characterization showed that ribosome binding is not nucleotide dependent, but ATP hydrolysis is required for dissociation of Ms5102 from the ribosome as well as for its ability to confer CLY resistance. Finally, we show that in comparison to the macrolides, CLY is a potent inducer of Mab_1846 and the whiB7 regulon, such that exposure of M. abscessus to very low antibiotic concentrations induces a heightened expression of erm41, hflX, and Mab_1846, which likely function together to result in a particularly antibiotic-resistant state.

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. RESULTS: Median age of patients included in the study was 2 years, and 64% of these children were male. The most common drug of exposure was buprenorphine/naloxone. Of the 429 charts screened, 140 patients were reported to be symptomatic and referred to the ED, of which 113 patients actually presented to the ED. An additional 122 patients presented to the ED without Regional Poison Control Center referral. Of the total 235 patients presenting to ED, 76 (32%) received a therapeutic intervention. Of 231 total opioid exposures, 31 exposures were administered naloxone. Three children underwent endotracheal intubation. Sixty-five patients were hospitalized, with a median length of stay of 1 day. Although there were no fatalities, 1 child suffered severe morbidity (anoxic brain injury). CONCLUSIONS: While opioid exposures in young children are a common and potentially life-threatening problem, most children remain asymptomatic. The majority of patients are able to be discharged from the ED after observation, and of those who are admitted, most have favorable outcomes and brief hospital stays. A small number of these patients require considerable medical interventions.

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. Infrequently, tetracyclines can be inactivated by the TetX class of enzymes, also referred to as tetracycline destructases. Low levels of tolerance to tetracycline in Mycobacterium smegmatis and Mycobacterium tuberculosis have been previously attributed to the WhiB7-dependent TetV/Tap efflux pump. However, Mycobacterium abscessus is ∼500-fold more resistant to tetracycline than M. smegmatis and M. tuberculosis In this report, we show that this high level of resistance to tetracycline and doxycycline in M. abscessus is conferred by a WhiB7-independent tetracycline-inactivating monooxygenase, MabTetX (MAB_1496c). The presence of sublethal doses of tetracycline and doxycycline results in a >200-fold induction of MabTetX, and an isogenic deletion strain is highly sensitive to both antibiotics. Further, purified MabTetX can rapidly monooxygenate both antibiotics. We also demonstrate that expression of MabTetX is repressed by MabTetRx, by binding to an inverted repeat sequence upstream of MabTetRx; the presence of either antibiotic relieves this repression. Moreover, anhydrotetracycline (ATc) can effectively inhibit MabTetX activity in vitro and decreases the MICs of both tetracycline and doxycycline in vivo Finally, we show that tigecycline, a glycylcycline tetracycline, not only is a poor substrate of MabTetX but also is incapable of inducing the expression of MabTetX. This is therefore the first demonstration of a tetracycline-inactivating enzyme in mycobacteria. It (i) elucidates the mechanism of tetracycline resistance in M. abscessus, (ii) demonstrates the use of an inhibitor that can potentially reclaim the use of tetracycline and doxycycline, and (iii) identifies two sequential bottlenecks-MabTetX and MabTetRx-for acquiring resistance to tigecycline, thereby reiterating its use against M. abscessus.

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

Mycobacterium abscessus 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. In the present work, we show that M. abscessuswhiB7, a homologue of Mycobacterium tuberculosis and Mycobacterium smegmatis whiB7 with previously demonstrated effects on intrinsic antibiotic resistance, is strongly induced when exposed to clinically relevant antibiotics that target the ribosome: erythromycin, clarithromycin, amikacin, tetracycline, and spectinomycin. The deletion of M. abscessuswhiB7 results in sensitivity to all of the above-mentioned antibiotics. Further, we have defined and compared the whiB7 regulon of M. abscessus with the closely related nontuberculous mycobacterium (NTM) M. smegmatis to demonstrate the induction of a species-specific repertoire of genes. Finally, we show that one such gene, eis2, is specifically induced in M. abscessus by whiB7 and contributes to its higher levels of intrinsic amikacin resistance. This species-specific pattern of gene induction might account for the differences in drug susceptibilities to other antibiotics and between different mycobacterial species.

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. Thus, although the attachment sites differ in length and sequence, only two base changes are needed to convert attP to attL, and just two more from attL to attB. The opposing effect of site identifiers ensures that site schizophrenia with dual identities does not occur.

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. We show that MSMEG_5437 influences the phosphorylation state of MSMEG_6129. Further, MSMEG_6129 controls the expression of a plethora of genes including efflux pumps, ABC transporters, catalases and transcription factors, either directly or via regulators like WhiB7, which account for the observed multi-drug resistance phenotypes. MSMEG_6129 in turn phosphorylates a contiguously located putative anti-anti-sigma factor, MSMEG_6127. We therefore propose that MSMEG_5437, MSMEG_6129 and MSMEG_6127 are components of a master regulatory network, upstream of whiB7, that controls the activity of one or more of the 28 sigma factors in M. smegmatis. Together, this network controls the expression of a regulon required for resistance to several unrelated antibiotics.

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. This effect can be understood in terms of the coupling between molecule extension and local molecular stresses: The rate of bead thermal motion controls the rate at which transition states are visited in the middle of a long DNA. Importantly, we have also observed this effect for reactions on unsupercoiled DNA; other enzymes show rates unaffected by bead size. Our results reveal a unique mechanism through which enzyme rates can be controlled by constraints on macromolecular or supramolecular substrates.

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. Relaxation and unbraiding are rapid (averaging 54 and 70 turns/s, respectively) and complete, with no discernible pauses. Nevertheless, the molecular friction associated with rotation is larger than that of type-I topoisomerases in a similar assay. Surprisingly we find that the synapse can stay rotationally "open" for many minutes.

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. In addition to dissociating ribosome into two subunits, mycobacterial HflX mediates persistent disordering of multiple 23S rRNA helices to generate an inactive pool of 50S subunits. Mycobacterial HflX also acts as an anti-association factor by binding to pre-dissociated 50S subunits. A mycobacteria-specific insertion in HflX reaches further into the peptidyl transferase center. The position of this insertion overlaps with ribosome-bound macrolides or lincosamide class of antibiotics. The extended conformation of insertion seen in the absence of these antibiotics retracts and adjusts around the bound antibiotics instead of physically displacing them. It therefore likely imparts antibiotic resistance by sequestration of the antibiotic-bound inactive 50S subunits.

Hierarchy and networks in the transcriptional response of Mycobacterium abscessus to antibiotics.

Mycobacterium abscessus 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. WhiB7 controls the expression of >100 genes, a few of which are known determinants of drug resistance. The function of the vast majority of genes within the regulon is unknown, but some conceivably encode additional mechanisms of resistance. Furthermore, the hierarchy of gene expression within the regulon, if any, is poorly understood. In the present work we have identified 56 WhiB7 binding sites using chromatin immunoprecipitation sequencing (CHIP-Seq) which accounts for the WhiB7-dependent upregulation of 70 genes, and find that M. abscessus WhiB7 functions exclusively as a transcriptional activator at promoters recognized by σ A /σ B We have investigated the role of 18 WhiB7 regulated genes in drug resistance and demonstrated the role of MAB_1409c and MAB_4324c in aminoglycoside resistance. Further, we identify a σ H -dependent pathway in aminoglycoside and tigecycline resistance which is induced upon drug exposure and is further activated by WhiB7 demonstrating the existence of a crosstalk between components of the WhiB7-dependent and -independent circuits. Abstract Importance: The induction of multiple genes that confer resistance to structurally diverse ribosome-targeting antibiotics is funneled through the induction of a single transcriptional activator, WhiB7, by antibiotic-stalled ribosomes. This poses a severe restriction in M. abscessus therapy as treatment with one ribosome-targeting antibiotic confers resistance to all other ribosome-targeting antibiotics. Here we uncover the intricacies of the WhiB7 regulatory circuit, identify three previously unknown determinants of aminoglycoside resistance and unveil a communication between WhiB7 dependent and independent components. This not only expands our understanding of the antibiotic resistance potential of M. abscessus but can also inform the development of much needed therapeutic options.

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. abscessus upon SPC exposure and have evaluated the involvement of 11 genes, that are both strongly SPC induced and whiB7 dependent, in SPC resistance. Of these we show that MAB_2780c can complement SPC sensitivity of ΔMab_whiB7 and that a ΔMab_2780c strain is ∼150 fold more SPC sensitive than wildtype bacteria, but not to tetracycline (TET) or other aminoglycosides. This is in contrast to its homologues, TetV from M. smegmatis and Tap from M. tuberculosis, that confer low-level resistance to TET, SPC and other aminoglycosides. We also show that the addition of the efflux pump inhibitor (EPI), verapamil results in >100-fold decrease in MIC of SPC in bacteria expressing Mab2780c to the levels observed for ΔMab_2780c; moreover a deletion of MAB_2780c results in a decreased efflux of the drug into the cell supernatant. Together our data suggest that Mab2780c is an SPC antiporter. Finally, molecular docking of SPC and TET on models of TetVMs and Mab2780c confirmed our antibacterial susceptibility findings that the Mab2780c pump preferentially effluxes SPC over TET. To our knowledge, this is the first report of an efflux pump that confers high-level drug resistance in M. abscessus. The identification of Mab2780c in SPC resistance opens up prospects for repurposing this relatively well-tolerated antibiotic as a combination therapy with verapamil or its analogs against M. abscessus infections.

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. What is relatively unexplored is whether the extent of declines differed by race and insurance status. An observational study was conducted using electronic medical record (EMR) data from the largest pediatric ED in Alabama for 2020 and 2019. The four subgroups of interest were African-American (AA), Non-Hispanic White (NHW), privately insured (PRIVATE), and publicly insured or self-insured (PUBLIC-SELF). Percentage changes in the 7-day moving average between dates in 2020 and 2019 were computed for total and high-severity ED visits by subgroup. Trends in percentage changes were plotted. T-tests were used to compare mean changes between subgroups. Large percentage declines in total ED visits and somewhat smaller percentage declines in high-severity visits were observed from March 2020. Declines were consistently larger for AA than NHW and for PUBLIC-SELF than PRIVATE. T-test results indicated mean date-specific percentage declines were significantly larger for AA than NHW for total visits (-38.92% [95% CI: -41.1, -36.8] versus -29.11% [95% CI: -30.8, -27.4]; p<0.001) and high-severity visits (-24.31% [95% CI: -26.2, -22.4] versus -19.49% [95% CI:-21.2, -17.8]; p<0.001), and larger for PUBLIC-SELF than PRIVATE for total visits (-36.32% [95% CI:-38.4, -34.3] versus 27.63% [95% CI:-29.2, -26.0]; p<0.001) and high-severity visits (-21.72% [95% CI: -23.5, -19.9] versus -20.01% [95% CI: -21.7, -18.3]; p = 0.04). In conclusion, significant differences by race and insurance status were observed in the decline in ED visits during the COVID19 pandemic, including high-severity visits. Minority-race and publicly insured or self-insured children often depend on the ED for health needs, lacking a usual source of care. Thus, these findings have worrisome implications regarding unmet healthcare needs and future exacerbations in health disparities.

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). The Bxb1 recombination sites, attP and attB, are small (<50 bp), different in sequence, and quasisymmetrical, and they give rise to attL- and attR-recombinant products that are asymmetric but similar to each other, each being composed of B- and P-type half-sites. We show here that the determination of correct excision products is a two-step process, with a presynaptic RDF-dependent step that aligns attL and attR in the correct orientation and a postsynaptic step in which the nonpalindromic central dinucleotide confers identity to attL and attR and prevents each from recombining with itself.