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Rapid optical determination of ?-lactamase and antibiotic activity.

ABSTRACT: The 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 ?-lactamase and thereby assess activity of ?-lactam antibiotics, which could provide an approach for targeted prescription of antibiotics. The methodology is centred on a fluorescence quenching based probe (?-LEAF--?-Lactamase Enzyme Activated Fluorophore) that mimics the structure of ?-lactam antibiotics.The ?-LEAF assay was performed for rapid determination of ?-lactamase production and activity of ?-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 ?-lactamase detection and disk diffusion for antibiotic susceptibility, showing results to be largely consistent. Furthermore, in the sub-set of ?-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.The study establishes the rapid ?-LEAF assay for ?-lactamase detection and prediction of antibiotic activity using S. aureus clinical isolates. Although the focus in the current study is ?-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.

SUBMITTER: Khan S

PROVIDER: S-EPMC4234275 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Rapid optical determination of β-lactamase and antibiotic activity.

Khan Shazia S Sallum Ulysses W UW Zheng Xiang X Nau Gerard J GJ Hasan Tayyaba T

BMC microbiology 20140404

<h4>Background</h4>The 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 flu ...[more]

PMID: 24708478

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Project description:The emergence and prevalence of novel plasmid-mediated tigecycline resistance genes, namely, tet(X) and their variants, pose a serious threat to public health worldwide. Rapid and accurate antibiotic susceptibility testing (AST) that can simultaneously detect the genotype and phenotype of tet(X)-positive bacteria may contribute to the deployment of an effective antibiotic arsenal, mortality reduction, and a decrease in the use of broad-spectrum antimicrobial agents. However, current bacterial growth-based AST methods, such as broth microdilution, are time consuming and delay the prompt treatment of infectious diseases. Here, we developed a rapid RNA-based AST (RBAST) assay to effectively distinguish tet(X)-positive and -negative strains. RBAST works by detecting specific mRNA expression signatures in bacteria after short-term tigecycline exposure. As a proof of concept, a panel of clinical isolates was characterized successfully by using the RBAST method, with a 3-h assay time and 87.9% accuracy (95% confidence interval [CI], 71.8% to 96.6%). Altogether, our findings suggest that RNA signatures upon antibiotic exposure are promising biomarkers for the development of rapid AST, which could inform early antibiotic choices. IMPORTANCE Infections caused by multidrug-resistant (MDR) Gram-negative pathogens are an increasing threat to global health. Tigecycline is one of the last-resort antibiotics for the treatment of these complicated infections; however, the emergence of plasmid-encoded tigecycline resistance genes, namely, tet(X), severely diminishes its clinical efficacy. Currently, there is a lack of rapid and accurate antibiotic susceptibility testing (AST) for the detection of tet(X)-positive bacteria. In this study, we developed a rapid and robust RNA-based antibiotic susceptibility determination (RBAST) assay to effectively distinguish tet(X)-negative and -positive strains using specific RNA biomarkers in bacteria after tigecycline exposure. Using this RBAST method, we successfully characterized a set of clinical strains in 3 h. Our data indicate that the RBAST assay is useful for identifying tet(X)-positive Escherichia coli.

Dataset Information

Rapid optical determination of ?-lactamase and antibiotic activity.

Publications

Rapid optical determination of β-lactamase and antibiotic activity.

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