scholarly journals High throughput LSPR and SERS analysis of aminoglycoside antibiotics

The Analyst ◽  
2016 ◽  
Vol 141 (17) ◽  
pp. 5120-5126 ◽  
Author(s):  
Kristy S. McKeating ◽  
Maxime Couture ◽  
Marie-Pier Dinel ◽  
Sylvie Garneau-Tsodikova ◽  
Jean-Francois Masson
Keyword(s):  
Side Effects ◽  
High Throughput ◽  
Aminoglycoside Antibiotics ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Adverse Side Effects

Aminoglycoside antibiotics are used in the treatment of infections caused by Gram-negative bacteria, and are often dispensed only in severe cases due to their adverse side effects.

Interactions of aminoglycoside antibiotics with rRNA

2016 ◽  
Vol 44 (4) ◽  
pp. 987-993 ◽  
Author(s):  
Joanna Trylska ◽  
Marta Kulik
Keyword(s):  
Molecular Dynamics ◽  
Ribosomal Subunit ◽  
Md Simulations ◽  
Aminoglycoside Antibiotics ◽  
Computational Studies ◽  
Gram Negative Bacteria ◽  
Protein Synthesis Inhibitors ◽  
Gram Negative ◽  
Adverse Side Effects ◽  
16S Rna

Aminoglycoside antibiotics are protein synthesis inhibitors applied to treat infections caused mainly by aerobic Gram-negative bacteria. Due to their adverse side effects they are last resort antibiotics typically used to combat pathogens resistant to other drugs. Aminoglycosides target ribosomes. We describe the interactions of aminoglycoside antibiotics containing a 2-deoxystreptamine (2-DOS) ring with 16S rRNA. We review the computational studies, with a focus on molecular dynamics (MD) simulations performed on RNA models mimicking the 2-DOS aminoglycoside binding site in the small ribosomal subunit. We also briefly discuss thermodynamics of interactions of these aminoglycosides with their 16S RNA target.

scholarly journals A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

2016 ◽  
Vol 60 (10) ◽  
pp. 5995-6002 ◽  
Author(s):  
Kristin R. Baker ◽  
Bimal Jana ◽  
Henrik Franzyk ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Throughput Screening ◽  
Gram Negative Bacteria ◽  
Chromogenic Substrate ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

Modular automated bottom-up proteomic sample preparation for high-throughput applications v1

2021 ◽  
Author(s):  
Yan Chen ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Tad Ogorzalek ◽  
Paul D. Adams ◽  
...  
Keyword(s):  
Sample Preparation ◽  
High Throughput ◽  
Tryptic Digestion ◽  
Gram Negative Bacteria ◽  
Bottom Up ◽  
Preparation Methods ◽  
Gram Negative ◽  
E Coli ◽  
Protocol Development ◽  
Set Up

Manual proteomic sample preparation methods limit sample throughput and often lead to poor data quality when thousands of samples must be analyzed. Automated workflows are increasingly used to overcome these issues for some (or even all) of the sample preparation steps. Here, we detail three optimised step-by-step protocols to: (A) lyse Gram-negative bacteria and fungal cells; (B) quantify the amount of protein extracted; and (C) normalize the amount of protein and set up tryptic digestion. These protocols have been developed to facilitate rapid, low variance sample preparation of hundreds of samples, be easily implemented on widely-available Beckman-Coulter Biomek automated liquid handlers, and allow flexibility for future protocol development. By using this workflow 50 micrograms of peptides for 96 samples can be prepared for tryptic digestion in under an hour. We validate these protocols by analyzing 47 E. coli and R. toruloides samples and show that this modular workflow provides robust, reproducible proteomic samples for high-throughput applications. The expected results from these protocols are 94 peptide samples from Gram-negative bacterial and fungal cells prepared for bottom-up quantitative proteomic analysis without the need for desalting column cleanup and with peptide variance (CVs) below 15%.

Norfloxacin: A Quinoline Antibiotic

1986 ◽  
Vol 20 (4) ◽  
pp. 261-266 ◽  
Author(s):  
Dwight A. Marble ◽  
John A. Bosso
Keyword(s):  
Side Effects ◽  
Nalidixic Acid ◽  
Antimicrobial Agents ◽  
Bitter Taste ◽  
Gram Negative Bacteria ◽  
Third Generation ◽  
Gram Negative ◽  
Intravenous Antibiotics ◽  
Third Generation Cephalosporins ◽  
Quinolinecarboxylic Acid

Norfloxacin is a quinoline (quinolinecarboxylic acid) that should prove successful in treating infections that currently require hospitalization and intravenous antibiotics. Although a nalidixic acid derivative, it possesses greater antibacterial activity against gram-positive and gram-negative bacteria. Compared with other antimicrobial agents, norfloxacin is more potent than the aminoglycosides, first-, second-, and third-generation cephalosporins, tetracycline, trimethoprim-sulfamethoxazole, carbenicillin, piperacillin, nalidixic acid, oxolinic acid, cinoxacin, and enoxacin. In the clinical studies to date, the side effects of norfloxacin have been minimal, but include nausea, vomiting, anorexia, dizziness, headache, drowsiness, depression, and a bitter taste in the mouth. In studies with more than 4000 patients, the incidence of side effects ranged from 3.9 to 4.7 percent, with most appearing by the second day of therapy.

scholarly journals One-step high-throughput assay for quantitative detection of β-galactosidase activity in intact Gram-negative bacteria, yeast, and mammalian cells

BioTechniques ◽  
2006 ◽  
Vol 40 (4) ◽  
pp. 433-440 ◽  
Author(s):  
Faustino Vidal-Aroca ◽  
Michele Giannattasio ◽  
Elisa Brunelli ◽  
Alessandro Vezzoli ◽  
Paolo Plevani ◽  
...  
Keyword(s):  
High Throughput ◽  
Mammalian Cells ◽  
Quantitative Detection ◽  
Gram Negative Bacteria ◽  
Galactosidase Activity ◽  
Gram Negative ◽  
One Step ◽  
High Throughput Assay

scholarly journals Investigating colistin drug resistance: The role of high-throughput sequencing and bioinformatics

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 150 ◽  
Author(s):  
Dickson Aruhomukama ◽  
Ivan Sserwadda ◽  
Gerald Mboowa
Keyword(s):  
Antibiotic Resistance ◽  
High Throughput ◽  
Bacterial Infections ◽  
High Throughput Sequencing ◽  
Health Concern ◽  
Gram Negative Bacteria ◽  
Drug Resistant ◽  
Colistin Resistance ◽  
Gram Negative

Bacterial infections involving antibiotic resistant gram-negative bacteria continue to increase and represent a major global public health concern. Resistance to antibiotics in these bacteria is mediated by chromosomal and/or acquired resistance mechanisms, these give rise to multi-drug resistant (MDR) or extensive drug resistant (XDR) bacterial strains. Most recently, a novel acquired plasmid mediated resistance mechanism to colistin, an antibiotic that had been set apart as the last resort antibiotic in the treatment of infections involving MDR and XDR gram-negative bacteria, has been reported. Plasmid mediated colistin resistant gram-negative bacteria have been described to be pan-drug resistant, implying a state devoid of alternative antibiotic therapeutic options. This review describes the evolution of antibiotic resistance to plasmid mediated colistin resistance, and discusses the potential role of high-throughput sequencing technologies, genomics and bioinformatics towards improving antibiotic resistance surveillance, the search for novel drug targets and precision antibiotic therapy focused at combating colistin resistance, and antimicrobial resistance as a whole.

scholarly journals Screening for Antibacterial Inhibitors of the UDP-3-O-(R-3-Hydroxymyristoyl)- N-Acetylglucosamine Deacetylase (LpxC) Using a High-Throughput Mass Spectrometry Assay

2009 ◽  
Vol 15 (1) ◽  
pp. 52-61 ◽  
Author(s):  
Erik F. Langsdorf ◽  
Asra Malikzay ◽  
William A. Lamarr ◽  
Dayna Daubaras ◽  
Cynthia Kravec ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Thermal Stability ◽  
High Throughput ◽  
High Throughput Screening ◽  
Antibacterial Agents ◽  
Screening Tool ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Mass Spectrometry Assay

A high-throughput mass spectrometry assay to measure the catalytic activity of UDP-3-O-(R-3-hydroxymyristoyl)- Nacetylglucosamine deacetylase, LpxC, is described. This reaction is essential in the biosynthesis of lipopolysaccharide (LPS) of gram-negative bacteria and is an attractive target for the development of new antibacterial agents. The assay uses the RapidFire™ mass spectrometry platform to measure the native LpxC substrate and the reaction product and thereby generates a ratiometric readout with minimal artifacts due to detection interference. The assay was robust in a high-throughput screen of a library of more than 700,000 compounds arrayed as orthogonal mixtures, with a median Z' factor of 0.74. Selected novel inhibitors from the screening campaign were confirmed as binding to LpxC by biophysical measurements using a thermal stability shift assay. Some inhibitors showed whole-cell antimicrobial activity against a sensitive strain of Escherichia coli with reduced LpxC activity (strain D22; minimum inhibitory concentrations ranging from 0.625-20 µg/mL). The results show that mass spectrometry—based screening is a valuable high-throughput screening tool for detecting inhibitors of enzymatic targets involving difficult to detect reactions.

scholarly journals High-Throughput Analysis of Gene Function in the Bacterial PredatorBdellovibrio bacteriovorus

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Miles C. Duncan ◽  
Rebecca K. Gillette ◽  
Micah A. Maglasang ◽  
Elizabeth A. Corn ◽  
Albert K. Tai ◽  
...  
Keyword(s):  
High Throughput ◽  
Gram Negative Bacteria ◽  
Human Pathogens ◽  
High Throughput Analysis ◽  
Bdellovibrio Bacteriovorus ◽  
Gram Negative ◽  
Content Type ◽  
Genetically Engineer ◽  
Wide Range ◽  
Specific Species

ABSTRACTBdellovibrio bacteriovorusis a bacterial predator capable of killing and replicating inside most Gram-negative bacteria, including antibiotic-resistant pathogens. Despite growing interest in this organism as a potential therapeutic, many of its genes remain uncharacterized. Here, we perform a high-throughput genetic screen withB. bacteriovorususing transposon sequencing (Tn-seq) to explore the genetic requirements of predation. Two hundred one genes were deemed essential for growth in the absence of prey, whereas over 100 genes were found to be specifically required for predative growth on the human pathogensVibrio choleraeandEscherichia coliin both planktonic and biofilm states. To further this work, we created an ordered-knockout library inB. bacteriovorusand developed new high-throughput techniques to characterize the mutants by their stage of deficiency in the predator life cycle. Using microscopy and flow cytometry, we confirmed 10 mutants defective in prey attachment and eight mutants defective in prey rounding. The majority of these genes are hypothetical and previously uncharacterized. Finally, we propose new nomenclature to groupB. bacteriovorusmutants into classes based on their stage of predation defect. These results contribute to our basic understanding of bacterial predation and may be useful for harnessingB. bacteriovorusto kill harmful pathogens in the clinical setting.IMPORTANCEBdellovibrio bacteriovorusis a predatory bacterium that can kill a wide range of Gram-negative bacteria, including many human pathogens. Given the global rise of antibiotic resistance and dearth of new antibiotics discovered in the past 30 years, this predator has potential as an alternative to traditional antibiotics. For many years,B. bacteriovorusresearch was hampered by a lack of genetic tools, and the genetic mechanisms of predation have only recently begun to be established. Here, we comprehensively identify and characterize predator genes required for killing bacterial prey, as well as genes that interfere in this process, which may allow us to design better therapeutic predators. Based on our study, we and other researchers may ultimately be able to genetically engineer strains that have improved killing rates, target specific species of prey, or preferentially target prey in the planktonic or biofilm state.

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