Dataset Information

An Enzyme-Mediated Amplification Strategy Enables Detection of ?-Lactamase Activity Directly in Unprocessed Clinical Samples for Phenotypic Detection of ?-Lactam Resistance.

ABSTRACT: Biochemical assays that can identify ?-lactamase activity directly from patient samples have the potential to significantly improve the treatment of bacterial infections. However, current ?-lactamase probes do not have the sensitivity needed to measure ?-lactam resistance directly from patient samples. Here, we report the development of an instrument-free signal amplification technology, DETECT, that connects the activity of two enzymes in series to effectively amplify the activity of ?-lactamase 40?000-fold, compared to the standard ?-lactamase probe nitrocefin.

SUBMITTER: deBoer TR

PROVIDER: S-EPMC6494105 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

An Enzyme-Mediated Amplification Strategy Enables Detection of β-Lactamase Activity Directly in Unprocessed Clinical Samples for Phenotypic Detection of β-Lactam Resistance.

deBoer Tara R TR Tarlton Nicole J NJ Yamaji Reina R Adams-Sapper Sheila S Wu Tiffany Z TZ Maity Santanu S Vesgesna Giri K GK Sadlowski Corinne M CM DePaola Peter P Riley Lee W LW Murthy Niren N

Chembiochem : a European journal of chemical biology 20180926 20

Biochemical assays that can identify β-lactamase activity directly from patient samples have the potential to significantly improve the treatment of bacterial infections. However, current β-lactamase probes do not have the sensitivity needed to measure β-lactam resistance directly from patient samples. Here, we report the development of an instrument-free signal amplification technology, DETECT, that connects the activity of two enzymes in series to effectively amplify the activity of β-lactamas ...[more]

PMID: 30079487

Similar Datasets

Project description:Whole-genome sequencing (WGS) of microbial pathogens from clinical samples is a highly sensitive tool used to gain a deeper understanding of the biology, epidemiology, and drug resistance mechanisms of many infections. However, WGS of organisms which exhibit low densities in their hosts is challenging due to high levels of host genomic DNA (gDNA), which leads to very low coverage of the microbial genome. WGS of Plasmodium vivax, the most widely distributed form of malaria, is especially difficult because of low parasite densities and the lack of an ex vivo culture system. Current techniques used to enrich P. vivax DNA from clinical samples require significant resources or are not consistently effective. Here, we demonstrate that selective whole-genome amplification (SWGA) can enrich P. vivax gDNA from unprocessed human blood samples and dried blood spots for high-quality WGS, allowing genetic characterization of isolates that would otherwise have been prohibitively expensive or impossible to sequence. We achieved an average genome coverage of 24×, with up to 95% of the P. vivax core genome covered by ?5 reads. The single-nucleotide polymorphism (SNP) characteristics and drug resistance mutations seen were consistent with those of other P. vivax sequences from a similar region in Peru, demonstrating that SWGA produces high-quality sequences for downstream analysis. SWGA is a robust tool that will enable efficient, cost-effective WGS of P. vivax isolates from clinical samples that can be applied to other neglected microbial pathogens. IMPORTANCE:Malaria is a disease caused by Plasmodium parasites that caused 214 million symptomatic cases and 438,000 deaths in 2015. Plasmodium vivax is the most widely distributed species, causing the majority of malaria infections outside sub-Saharan Africa. Whole-genome sequencing (WGS) of Plasmodium parasites from clinical samples has revealed important insights into the epidemiology and mechanisms of drug resistance of malaria. However, WGS of P. vivax is challenging due to low parasite levels in humans and the lack of a routine system to culture the parasites. Selective whole-genome amplification (SWGA) preferentially amplifies the genomes of pathogens from mixtures of target and host gDNA. Here, we demonstrate that SWGA is a simple, robust method that can be used to enrich P. vivax genomic DNA (gDNA) from unprocessed human blood samples and dried blood spots for cost-effective, high-quality WGS.

Project description:We validate and evaluate a new phenotypic assay, named the direct β-lactam inactivation method (dBLIM), for the rapid and simultaneous detection of carbapenemase or extended-spectrum-cephalosporinase activity directly from Enterobacterales (EB)-positive blood cultures (BCs). It originates from the carbapenem inactivation method (CIM), an inexpensive and highly sensitive assay for carbapenemase activity detection. dBLIM cutoff values to detect extended-spectrum β-lactamase (ESBL) and carbapenemase activities resulted in diameters of ≤12 mm for a 5-μg-cefotaxime disk and for a 10-μg-meropenem disk. dBLIM assessment was determined with both aerobic and anaerobic BC bottles spiked with 422 characterized EB strains, classifiable into the following 4 phenotypic groups: (i) ESBL/AmpC-type β-lactamase (ACBL)/carbapenemase (CARB)-nonproducing (np-ESBL/ACBL/CARB) EB (n = 116), (ii) ESBL-producing EB (n = 111), (iii) AmpC-β-lactamase-producing EB (n = 33), and (iv) carbapenemase-producing EB (n = 162). No false-positive results were obtained in any of the np-ESBL/ACBL/CARB EB, ESBL, and AmpC groups, demonstrating an overall assay specificity of 100%. There were no significant discrepancies in dBLIM performance between aerobic and anaerobic BCs across all groups, except with VIM-type carbapenemase-expressing EB. Interestingly, among BCs spiked with blaVIM-harboring EB, the sensitivity rates of the assay in anaerobic and aerobic bottles were 53.6% and 100%, respectively. In contrast, dBLIM performance was deemed excellent for the KPC, OXA-48, and NDM carbapenemase producers regardless of the type of bottle being tested, with a sensitivity rate ranging between 99% and 100%. Concerning the detection of the extended-spectrum cephalosporinases of the ESBL-producing and AmpC types, dBLIM sensitivities was 100% and 84 to 87%, respectively. dBLIM may be a cost-effective and highly robust phenotypic screening method for the reliable detection of carbapenemases or extended-spectrum cephalosporinases directly from BCs on the same day of bottle positivity detection.

Project description:Our current understanding of how low antibiotic concentrations shape the evolution of contemporary β-lactamases is limited. Using the widespread carbapenemase OXA-48, we tested the long-standing hypothesis that selective compartments with low antibiotic concentrations cause standing genetic diversity that could act as a gateway to developing clinical resistance. Here, we subjected Escherichia coli expressing bla OXA-48, on a clinical plasmid, to experimental evolution at sub-MICs of ceftazidime. We identified and characterized seven single variants of OXA-48. Susceptibility profiles and dose-response curves showed that they increased resistance only marginally. However, in competition experiments at sub-MICs of ceftazidime, they demonstrated strong selectable fitness benefits. Increased resistance was also reflected in elevated catalytic efficiencies toward ceftazidime. These changes are likely caused by enhanced flexibility of the Ω- and β5-β6 loops and fine-tuning of preexisting active site residues. In conclusion, low-level concentrations of β-lactams can drive the evolution of β-lactamases through cryptic phenotypes which may act as stepping-stones toward clinical resistance.IMPORTANCE Very low antibiotic concentrations have been shown to drive the evolution of antimicrobial resistance. While substantial progress has been made to understand the driving role of low concentrations during resistance development for different antimicrobial classes, the importance of β-lactams, the most commonly used antibiotics, is still poorly studied. Here, we shed light on the evolutionary impact of low β-lactam concentrations on the widespread β-lactamase OXA-48. Our data indicate that the exposure to β-lactams at very low concentrations enhances β-lactamase diversity and drives the evolution of β-lactamases by significantly influencing their substrate specificity. Thus, in contrast to high concentrations, low levels of these drugs may substantially contribute to the diversification and divergent evolution of these enzymes, providing a standing genetic diversity that can be selected and mobilized when antibiotic pressure increases.

Dataset Information

An Enzyme-Mediated Amplification Strategy Enables Detection of ?-Lactamase Activity Directly in Unprocessed Clinical Samples for Phenotypic Detection of ?-Lactam Resistance.

Publications

An Enzyme-Mediated Amplification Strategy Enables Detection of β-Lactamase Activity Directly in Unprocessed Clinical Samples for Phenotypic Detection of β-Lactam Resistance.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets