Rapid optical determination of β-lactamase and antibiotic activity

Abstract

BackgroundThe absence of rapid tests evaluating antibiotic susceptibility results in the empirical prescription of antibiotics. This can lead to treatment failures due to escalating antibiotic resistance, and also furthers the emergence of drug-resistant bacteria. This study reports a rapid optical method to detect β-lactamase and thereby assess activity of β-lactam antibiotics, which could provide an approach for targeted prescription of antibiotics. The methodology is centred on a fluorescence quenching based probe (β-LEAF – β-Lactamase Enzyme Activated Fluorophore) that mimics the structure of β-lactam antibiotics.ResultsThe β-LEAF assay was performed for rapid determination of β-lactamase production and activity of β-lactam antibiotic (cefazolin) on a panel of Staphylococcus aureus ATCC strains and clinical isolates. Four of the clinical isolates were determined to be lactamase producers, with the capacity to inactivate cefazolin, out of the twenty-five isolates tested. These results were compared against gold standard methods, nitrocefin disk test for β-lactamase detection and disk diffusion for antibiotic susceptibility, showing results to be largely consistent. Furthermore, in the sub-set of β-lactamase producers, it was demonstrated and validated that multiple antibiotics (cefazolin, cefoxitin, cefepime) could be assessed simultaneously to predict the antibiotic that would be most active for a given bacterial isolate.ConclusionsThe study establishes the rapid β-LEAF assay for β-lactamase detection and prediction of antibiotic activity using S. aureus clinical isolates. Although the focus in the current study is β-lactamase-based resistance, the overall approach represents a broad diagnostic platform. In the long-term, these studies form the basis for the development of assays utilizing a broader variety of targets, pathogens and drugs.

Prevalence and Therapies of Antibiotic-Resistance in Staphylococcus aureus

“…At present, the research shows that β-lactam antibiotics have a lethal effect on bacteria mainly through two mechanisms: first, by binding to penicillin-binding protein (PBPs, i.e., cell wall mucin synthase), which represses cell wall mucin synthesis, disrupts the cell wall, and leads to bacterial expansion and lysis; second, by triggering the autolytic enzyme activity of the bacteria, which resulted in autolysis and death (Matono et al, 2018). Excessive secretion of β-lactamase by MRSA mainly reduces the effect of antibiotics through two mechanisms, which lead to MRSA resistant (Khan et al, 2014). The first is the hydrolysis mechanism, that is, β-lactamase hydrolzes and inactivates β-lactam antibiotics; the second is the mechanism of pinching, that is, a large amount of β-lactamase binds quickly and firmly to extracellular antibiotics, preventing the antibiotics from reaching the intracellular space and therefore the antibiotics are not able to reach the target site, ultimately leading to MRSA resistance to antibiotics ( Figure 1D) (Harada et al, 2014;Hashizume et al, 2017).…”

Guiding Empiric Treatment for Serious Bacterial Infections via Point of Care $\beta $ -Lactamase Characterization

“…However, the rate of cleavage was not as pronounced with cefepime, indicating this antibiotic as being more robust against cleavage for this particular \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\beta $ \end{document}-lactamase (as compared to the other cephalosporins). Gold standard assays of antibiotic susceptibility have confirmed cefepime to be the most effective antibiotic out of the 3 for this pathogen strain [30], supporting the use of the microfluidic as a rapid platform for the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\beta $ \end{document}-LEAF antibiotic susceptibility assay.

FIGURE 8.Microfluidic \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\beta $ \end{document}-LEAF antibiotic susceptibility assay.

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The Composites of Graphene Oxide with Metal or Semimetal Nanoparticles and Their Effect on Pathogenic Microorganisms

“…This phenomenon leads to the development of various defensive mechanisms able to inactivate the applied chemical substance [7]. The most common resistant strain, which evolved from the Staphylococcus aureus , is methicillin-resistant S. aureus (MRSA) [8,9]. The incidence of MRSA in hospitalized patients results particularly from an intensive and long-term therapy [10], where S. aureus is exposed to relatively high concentrations of antibiotics for a longtime, thus certain mutations can develop.…”