Child hyperactivity, mother–child negativity, and sibling dyad negativity : a transactional family systems approach.

Attention-deficit/hyperactivity disorder (ADHD) is among the most common mental disorders in children and adolescents. While most studies in this field have focused on the genetic and neurobiological underpinnings of the disorder, research focusing on the family environment as a critical context contributing toward the manifestation and maintenance of child ADHD symptoms is still less extensive. Therefore, the aim of this study was to examine longitudinal and bidirectional associations between child hyperactivity, mother–child negativity, and sibling dyad negativity. Data were analyzed of up to 4,429 children from the Avon Longitudinal Study of Parents and Children, a nationally representative prospective birth cohort study in the United Kingdom, at three time points (T1–T3, children aged 4, 7, and 8 years). At baseline (T1, n = 4,063), the child sample (98.8% White ethnic background) composed of 51.6% males. Child hyperactivity symptoms, mother–child negativity, and sibling dyad negativity were examined based on maternal reports. The random intercepts cross-lagged panel model was adopted to parcel out between-family differences from within-family fluctuations and test bidirectional associations. On the between-family level, families with higher child hyperactivity reported higher mother–child and sibling dyad negativity. On the within-family level, unidirectional spillover processes between sibling dyad negativity and mother–child negativity as well as between mother–child negativity and child hyperactivity emerged. Future work in the area of child hyperactivity should implement a transactional family systems approach incorporating both parent–child and sibling subsystems. Interventions to reduce negative interactions between parents and hyperactive children may offer promising improvements in child symptoms and thus alleviate family burden

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation

A Mycobacterial Gene Involved in Synthesis of an Outer Cell Envelope Lipid Is a Key Factor in Prevention of Phagosome Maturation

Virulent mycobacteria cause arrest of phagosome maturation as a part of their survival strategy in hosts. This process is mediated through multiple virulence factors, whose molecular nature remains elusive. Using Mycobacterium marinum as a model, we performed a genome-wide screen to identify mutants whose ability to inhibit phagosome maturation was impaired, and we succeeded in isolating a comprehensive set of mutants that were not able to occupy an early endosome-like phagosomal compartment in mammalian macrophages. Categorizing and ordering the multiple mutations according to their gene families demonstrated that the genes modulating the cell envelope are the principal factors in arresting phagosome maturation. In particular, we identified a novel gene, pmiA, which is capable of influencing the constitution of the cell envelope lipids, thereby leading to the phagosome maturation block. The pmiA mutant was not able to resist phagosome maturation and was severely attenuated in mice. Complementing the mutant with the wild-type gene restored the attenuated virulence to wild-type levels in mice

Transposition of Tn5367 in Mycobacterium marinum, Using a Conditionally Recombinant Mycobacteriophage

Mycobacterium marinum is a close relative of the obligate human pathogen Mycobacterium tuberculosis. As with M. tuberculosis, M. marinum causes intracellular infection of poikilothermic vertebrates and skin infection in humans. It is considered a valid model organism for the study of intracellular pathogenesis of mycobacteria. Low transformation efficiencies for this species have precluded approaches using mutant libraries in pathogenesis studies. We have adapted the conditionally replicating mycobacteriophage phAE94, originally developed as a transposon mutagenesis tool for M. tuberculosis, to meet the specific requirements of M. marinum. Conditions permissive for phage replication in M. tuberculosis facilitated highly efficient transposon delivery in M. marinum. Using this technique we succeeded in generating a representative mutant library of this species, and we conclude that TM4-derived mycobacteriophages are temperature-independent suicide vectors for M. marinum

In vitro Activity of Repurposed Nitroxoline Against Clinically Isolated Mycobacteria Including Multidrug-Resistant Mycobacterium tuberculosis

Antimicrobial treatment options for mycobacterial infections are limited due to intrinsic resistance and the emergence of acquired resistance in Mycobacterium tuberculosis. Isolates resisting first- and second line drugs are raising concerns about untreatable infections and make the development of new therapeutic strategies more pressing. Nitroxoline is an old oral antimicrobial that is currently repurposed for the treatment of urinary tract infection (UTI). In this study, we report the in vitro activity of nitroxoline against 18 clinical isolates of M. tuberculosis complex (MTBC) (M. tuberculosis N = 16, M. bovis BCG N = 1, M. bovis sp. bovis N = 1). Since nitroxoline achieves high concentrations in the urinary tract, we included all MTBC-isolates from urinary samples sent to our laboratory between 2008 and 2021 (University Hospital of Cologne, Germany). Isolates from other sources (N = 7/18) were added for higher sample size and for inclusion of drug-resistant M. tuberculosis isolates (N = 4/18). Based on our clinical routine the fluorescence-based liquid media system BACTEC MGIT 960 was used for susceptibility testing of nitroxoline and mainstay antitubercular drugs. Nitroxoline yielded a MIC90 of 4 mg/L for MTBC. In all M. tuberculosis isolates nitroxoline MICs were at least two twofold dilutions below the current EUCAST susceptibility breakpoint of <= 16 mg/L (limited to E. coli and uncomplicated UTI). In vitro activity of nitroxoline can be considered excellent, even in multidrug-resistant isolates. Future studies with in vivo models should evaluate a potential role of nitroxoline in the treatment of tuberculosis in the era of drug resistance

Activity of the old antimicrobial nitroxoline against Mycobacterium abscessus complex isolates

ABSTRACT: Objectives: The old antimicrobial nitroxoline is approved to treat urinary tract infection (UTI) and is currently rediscovered for treatment of drug resistant pathogens. Mycobacteria of the Mycobacterium abscessus complex (MYABS) are rapid-growing nontuberculous mycobacteria that are associated with difficult to treat infections of the lungs in patients with pulmonary disorders such as cystic fibrosis. In this study we assessed the in vitro activity of nitroxoline against molecularly characterized drug-resistant MYABS isolates from clinical samples to address potential repurposing of nitroxoline in difficult-to-treat MYABS infection. Methods: The isolates originated from clinical samples collected between 2010 and 2019 at the University Hospital of Cologne, Germany (N=16; 10/16 M. abscessus Spp. abscessus, 4/16 M. abscessus Spp. massiliense, 2/16 M. abscessus Spp. bolletii). Nitroxoline activity was compared to standard antimicrobials recommended for treatment of MYABS infection. For drug susceptibility testing of nitroxoline and comparators broth microdilution was performed based on current Clinical and Laboratory Standards Institute (CLSI) guidelines. Results: Nitroxoline yielded a MIC90 of 4 mg/L (range 2–4 mg/L), which is two twofold dilutions below the current EUCAST susceptibility breakpoint of ≤ 16 mg/L (limited to uncomplicated UTI and Escherichia coli). Resistance to other antimicrobials was common in our cohort (16/16 isolates resistant to ciprofloxacin, imipenem and doxycycline; 12/16 isolates resistant to tobramycin; 9/16 isolates resistant to cefoxitin; 7/16 isolates resistant to clarithromycin; 2/16 isolates resistant to amikacin). Conclusion: Nitroxoline has a promising in vitro activity against drug-resistant MYABS isolates. Future studies should investigate this finding with macrophage and in vivo models

Failure of Vitek2 to reliably detect vanB-mediated vancomycin resistance in Enterococcus faecium

Objectives: The increasing prevalence of VRE necessitates their reliable detection, especially for Low-Level resistance mediated by vanB in Enterococcus faecium. In this prospective study we analysed if vanB-mediated vancomycin resistance can be reliably detected by Vitek2. Methods: One thousand, three hundred and forty-four enterococcal isolates from routine clinical specimens were tested by Vitek2 (bioMerieux, Nurtingen, Germany). Additionally, a bacterial suspension (with a turbidity equivalent to that of a 0.5 McFarland standard) was inoculated on chromlD VRE screening agar (bioMerieux) and incubated for 48 h. If vancomycin tested susceptible by Vitek2 but growth was detected on the screening agar, PCR for vanA/vanB was performed (GeneXpert vanA/B test, Cepheid, Frankfurt, Germany). For isolates that tested susceptible to vancomycin by Vitek2 but were vanA/B positive, MICs were determined before and after cultivation in broth with increasing concentrations of vancomycin. Results: One hundred and fifty-six out of 491 E. faecium were VRE and were predominantly vanB positive (81.0%). Of these, Vitek2 did not identify 14 as VRE (sensitivity 91.0%). By broth microdilution 9/14 isolates demonstrated high MICs (>= 32 mg/L) and 5/14 showed Low vancomycin MICs, which did not increase despite vancomycin exposure. Three of the 14 isolates demonstrated growth on chromlD VRE; after vancomycin exposure seven additional isolates were able to grow on chromlD VRE. Conclusions: Vitek2 fails to detect vanB-mediated vancomycin resistance consistently, especially, but not Limited to, Low-Level resistance. As this may Lead to treatment failure and further dissemination of vanB VRE, additional methods (e.g. culture on VRE screening agar or PCR) are necessary to reliably identify vanB-positive enterococci in clinical routine