Streptococcus pneumoniae is the major cause of bacterial pneumonia, and it is also responsible for otitis media and meningitis in children. Apart from the capsule, the virulence factors of this pathogen are not completely understood. Recent technical advances in the field of bacterial pathogenesis (in vivo expression technology and signature-tagged mutagenesis [STM]) have allowed a large-scale identification of virulence genes. We have adapted to S. pneumoniae the STM technique, originally used for the discovery of Salmonella genes involved in pathogenicity. A library of pneumococcal chromosomal fragments (400 to 600 bp) was constructed in a suicide plasmid vector carrying unique DNA sequence tags and a chloramphenicol resistance marker. The recent clinical isolate G54 was transformed with this library. Chloramphenicol-resistant mutants were obtained by homologous recombination, resulting in genes inactivated by insertion of the suicide vector carrying a unique tag. In a mouse pneumonia model, 1.250 candidate clones were screened; 200 of these were not recovered from the lungs were therefore considered virulence-attenuated mutants. The regions flanking the chloramphenicol gene of the attenuated mutants were amplified by inverse PCR and sequenced. The sequence analysis showed that the 200 mutants had insertions in 126 different genes that could be grouped in six classes: (i) known pneumococcal virulence genes; (ii) genes involved in metabolic pathways; (iii) genes encoding proteases; (iv) genes coding for ATP binding cassette transporters; (v) genes encoding proteins involved in DNA recombination/repair; and (vi) DNA sequences that showed similarity to hypothetical genes with unknown function. To evaluate the virulence attenuation for each mutant, all 126 clones were individually analyzed in a mouse septicemia model. Not all mutants selected in the pneumonia model were confirmed in septicemia, thus indicating the existence of virulence factors specific for pneumonia.
Clinical utility of phosphodiesterase 4 (PDE4) inhibitors as antiinflammatory agents has, to date, been limited by adverse effects including nausea and emesis, making accurate assessment of emetic versus anti-inflammatory potencies critical to the development of inhibitors with improved therapeutic indices. In the present study we determined the in vitro and in vivo anti-inflammatory potencies of the first-generation PDE4 inhibitor, rolipram, the second-generation inhibitors, roflumilast and cilomilast, and a novel third generation inhibitor, 1-ethyl-5-{5-[(4-methyl-1-piperazinyl)methyl]-1,3,4-oxadiazol-2-yl}-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo [3,4-b]pyridin-4-amine (EPPA-1). The rank-order potency against lipopolysaccharide (LPS)-induced tumor necrosis factor-␣ production by human peripheral blood mononuclear cells was roflumilast (IC 50 ϭ 5 nM) Ͼ EPPA-1 (38) Ͼ rolipram (269) Ͼ cilomilast (389), and against LPS-induced pulmonary neutrophilia in the rat was EPPA-1 (D 50 ϭ 0.042 mg/ kg) Ͼ roflumilast (0.24) Ͼ rolipram (3.34) Ͼ cilomilast (4.54). Pica, the consumption of non-nutritive substances in response to gastrointestinal stress, was used as a surrogate measure for emesis, giving a rank-order potency of rolipram (D 50 ϭ 0.495 mg/kg) Ͼ roflumilast (1.6) Ͼ cilomilast (6.4) Ͼ EPPA-1 (24.3). The low and high emetogenic activities of EPPA-1 and rolipram, respectively, detected in the pica model were confirmed in a second surrogate model of emesis, reversal of ␣ 2 -adrenoceptor-mediated anesthesia in the mouse. The rank order of therapeutic indices derived in the rat [(pica D 50 )/(neutrophilia D 50 )] was EPPA-1 (578) Ͼ roflumilast (6.4) Ͼ cilomilast (1.4) Ͼ rolipram (0.15), consistent with the rank order derived in the ferret [(emesis D 50 )/(neutrophilia D 50 )]. These data validate rat pica feeding as a surrogate for PDE4 inhibitor-induced emesis in higher species, and identify EPPA-1 as a novel PDE4 inhibitor with an improved therapeutic index.Phosphodiesterases are a superfamily of enzymes that hydrolyze cAMP and/or cGMP to their inactive nucleotides. Phosphodiesterase 4 (PDE4) is selective for cAMP, and consists of the four subtypes A, B, C, and D. PDE4 inhibitors have shown efficacy in various in vitro and in vivo inflammatory models by increasing the intracellular levels of cAMP in many immune cells (T lymphocytes, monocytes, neutrophils, and eosinophils). As such, PDE4 inhibitors have been pursued as therapeutics for pulmonary diseases with an inflammatory component, including chronic obstructive pulmo-
Intestinal barrier derangement allows intestinal bacteria and their products to translocate to the systemic circulation. Pseudomonas aeruginosa (PA) superimposed infection in critically ill patients increases gut permeability and leads to gut-driven sepsis. PA infections are challenging due to multi-drug resistance (MDR), biofilms, and/or antibiotic tolerance. Inhibition of the quorum-sensing transcriptional regulator MvfR(PqsR) is a desirable anti-PA anti-virulence strategy as MvfR controls multiple acute and chronic virulence functions. Here we show that MvfR promotes intestinal permeability and report potent anti-MvfR compounds, the N-Aryl Malonamides (NAMs), resulting from extensive structure-activity-relationship studies and thorough assessment of the inhibition of MvfR-controlled virulence functions. This class of anti-virulence non-native ligand-based agents has a half-maximal inhibitory concentration in the nanomolar range and strong target engagement. Using a NAM lead in monotherapy protects murine intestinal barrier function, abolishes MvfR-regulated small molecules, ameliorates bacterial dissemination, and lowers inflammatory cytokines. This study demonstrates the importance of MvfR in PA-driven intestinal permeability. It underscores the utility of anti-MvfR agents in maintaining gut mucosal integrity, which should be part of any successful strategy to prevent/treat PA infections and associated gut-derived sepsis in critical illness settings. NAMs provide for the development of crucial preventive/therapeutic monotherapy options against untreatable MDR PA infections.
Microtiter plate methods are commonly used for biofilm assessment. However, results obtained with these methods have often been difficult to reproduce. Hence, it is important to obtain a better understanding of the repeatability and reproducibility of these methods. An interlaboratory study was performed in five different laboratories to evaluate the reproducibility and responsiveness of three methods to quantify Staphylococcus aureus biofilm formation in 96-well microtiter plates: crystal violet, resazurin, and plate counts. An inter-lab protocol was developed for the study. The protocol was separated into three steps: biofilm growth, biofilm challenge, biofilm assessment. For control experiments participants performed the growth and assessment steps only. For treatment experiments, all three steps were performed and the efficacy of sodium hypochlorite (NaOCl) in killing S. aureus biofilms was evaluated. In control experiments, on the log10-scale, the reproducibility SD (SR) was 0.44 for crystal violet, 0.53 for resazurin, and 0.92 for the plate counts. In the treatment experiments, plate counts had the best responsiveness to different levels of efficacy and also the best reproducibility with respect to responsiveness (Slope/SR = 1.02), making it the more reliable method to use in an antimicrobial efficacy test. This study showed that the microtiter plate is a versatile and easy-to-use biofilm reactor, which exhibits good repeatability and reproducibility for different types of assessment methods, as long as a suitable experimental design and statistical analysis is applied.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.