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Rapid diagnostics of blood stream infections using synthetic nanobodies

Applicant Seeger Markus
Number 177368
Funding scheme NRP 72 Antimicrobial Resistance
Research institution Institut für Medizinische Mikrobiologie Universität Zürich
Institution of higher education University of Zurich - ZH
Main discipline Experimental Microbiology
Start/End 01.03.2018 - 30.06.2021
Approved amount 350'000.00
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All Disciplines (2)

Discipline
Experimental Microbiology
Biochemistry

Keywords (10)

Pseudomonas aeruginosa; Gram-negative pathogens; rapid diagnostics; Klebsiella pneumoniae; Antimicrobial Susceptibility Testing (AST); Blood stream infections; flow cytometry; Escherichia coli; nanobodies; in vitro selection of binders

Lay Summary (German)

Lead
Kleine Antikörper als KeimfängerWir entwickeln hochspezifische Antikörperfragmente um gefürchtete Erreger direkt aus dem Blut von Patienten einzufangen und zu analysieren. Damit beschleunigen wir die Diagnostik von lebensbedrohlichen Infektionen der Blutstrombahn.
Lay summary

Porträt/Projektbeschrieb

Der diagnostische Nachweis einer Sepsis kann mehrere Tage in Anspruch nehmen. Vor allem die Bakterienvermehrung in sogenannten Blutkulturen ist zeitraubend. Innerhalb dieses Zeitfensters werden im Spital Antibioka verabreicht ohne zu wissen, ob diese auch wirken. Aufgrund der steigenden Häufigkeit resistenter Keime werden zu Beginn der Behandlung immer öfter unwirksame Antibiotika eingesetzt, mit fatalen Folgen für die betroffenen Patienten. Wir entwickeln kleine Antikörperfragmente, welche Bakterien direkt aus Blutproben von Patienten einfangen, um diese anschliessend auf Antibiotikaresistenzen zu testen und mittels Einzellzellzählung zu analysieren. Unser Forschungsansatz macht Blutkulturen überflüssig und verkürzt die Zeit bis ein detaillierter diagnostischer Befund vorliegt.

Hintergrund / Ausgangslage

Herkömmliche Antikörper binden vor allem an hochvariable Zuckerstrukturen auf der Bakterienoberfläche. Diese Strukturen unterscheiden sich jedoch sehr stark zwischen verschiedenen Bakterienstämmen der gleichen Spezies. Daher ist der Einsatz solcher Antikörper in der Diagnostik zu komplex. Wir haben eine Methode entwickelt, um kleine Antikörperfragmente gegen hochkonservierte Proteine zu selektionieren, welche in der äusseren Membran von Gram-negativen Bakterien eingebettet sind.

Ziele

Das Ziel des Projektes ist die Entwicklung von Antikörperfragmenten, welche spezifisch an hochkonservierte Proteine in der äusseren Membran der Gram-negativen Problemkeime Escherichia coli, Klebsiella pneumoniae und Pseudomonas aeruginosa binden. Die Antikörperfragmente werden genutzt, um diese Bakterien in Blutproben von Patienten einzufangen. Danach werden die Bakterien mittels Einzellzellzählung analysiert respektive mittels konventioneller Antibiotikaresistenztestung untersucht.

Bedeutung / möglicher Nutzen

Eine Sepsis ist eine potentiell lebensbedrohliche Erkrankung, die in der Mehrheit der Fälle durch Bakterien in der Blutstrombahn ausgelöst wird und in 15-30% der Fälle zum Tod des Patienten führt. Steigende Resistenzhäufigkeiten haben das Problem in jüngster Zeit verschärft. Unsere Methode wird den diagnostischen Nachweis von drei Problemkeimen entscheidend beschleunigen. Damit wird kostbare Zeit für die Behandlung mit einem wirksamen Antibiotika gewonnen.


 
Direct link to Lay Summary Last update: 06.02.2018

Responsible applicant and co-applicants

Employees

Project partner

Natural persons


Name Institute

Publications

Publication
Generation of synthetic nanobodies against delicate proteins
Zimmermann Iwan, Egloff Pascal, Hutter Cedric A. J., Kuhn Benedikt T., Bräuer Philipp, Newstead Simon, Dawson Roger J. P., Geertsma Eric R., Seeger Markus A. (2020), Generation of synthetic nanobodies against delicate proteins, in Nature Protocols, 15(5), 1707-1741.
Identification of conformation-selective nanobodies against the membrane protein insertase BamA by an integrated structural biology approach
Kaur Hundeep, Hartmann Jean-Baptiste, Jakob Roman P., Zahn Michael, Zimmermann Iwan, Maier Timm, Seeger Markus A., Hiller Sebastian (2019), Identification of conformation-selective nanobodies against the membrane protein insertase BamA by an integrated structural biology approach, in Journal of Biomolecular NMR, 73(6-7), 375-384.

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
ANNUAL CONGRESS OF THE SWISS SOCIETY FOR MICROBIOLOGY 2019 Poster Specific antibodies capture germs 03.09.2019 Zurich, Switzerland Huber Lea Martina; Seeger Markus; Keller Peter Michael; Sorgenfrei Michèle;
NRP 72 Programme Meeting Talk given at a conference Rapid diagnostics of blood stream infections using synthetic nanobodies 27.03.2019 Lausanne, Switzerland Seeger Markus; Keller Peter Michael; Huber Lea Martina; Sorgenfrei Michèle;
NRP 72 Programme Meeting Talk given at a conference Rapid diagnostics of blood stream infections using synthetic nanobodies 19.04.2018 Nottwil, Switzerland Seeger Markus; Huber Lea Martina;


Knowledge transfer events

Active participation

Title Type of contribution Date Place Persons involved


Self-organised

Title Date Place
5th meeting, Antibiotics Platform, biotechnet/SBA 10.09.2019 Basel, Switzerland
4th meeting, Antibiotics Platform, NTN Swiss Biotech 12.09.2018 Basel, Switzerland

Communication with the public

Communication Title Media Place Year
Print (books, brochures, leaflets) Schnellere Diagnostik dank lebendig eingefangenen Bakterien Western Switzerland German-speaking Switzerland 2020

Associated projects

Number Title Start Funding scheme
187170 A versatile technology platform for identification and development of novel bio-antibiotics 01.04.2020 Bridge - Discovery
126338 Targeting multidrug efflux proteins: Studying transport processes across the membrane using designed ankyrin repeat proteins and antibody fragments 01.10.2009 Ambizione

Abstract

Sepsis caused by blood stream infections (BSIs) affects 18 million people world-wide every year and has high mortality rates of 10 - 20 % in Western countries. State of the art BSI diagnostics currently involves bacterial blood cultures (lasting 1- 7 days), followed by pathogen identification via MALDI-TOF-MS (4 - 12 h) and antibiotic susceptibility testing (AST, taking up to 18 h). During this time window of uncertainty, clinicians treat sepsis patients with empiric broad-spectrum antibiotics, which are increasingly inadequate against the frequently encountered pathogens Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa due to rising cephalosporin- and carbapenem-resistance rates. Clinical blood samples of BSI patients only contain 100 - 1’000 bacterial cells per milliliter of blood versus 1’000’000’000 erythrocytes in the same volume, which poses a huge diagnostic challenge. PCR-based diagnostic kits are available, but cannot differentiate between living and dead cells and frequently lead to false-positive hits. Because genes encoding for extended spectrum beta-lactamases and carbapenemases are spreading and evolving at a fast rate, phenotypic AST remains the gold standard to determine the resistance profile of sepsis-causing pathogens.Antibody-based diagnostics of BSI would offer a shortcut bypassing time-expensive blood cultures. However, currently available antibodies are usually polyclonal and highly strain-specific, which precludes their use in routine diagnostics.Here we propose to develop sybodies - robust 15 kDa single domain antibody fragments based on the nanobody scaffold - that capture and enrich pathogens directly from clinical blood samples for flow-cytometric analyses and rapid AST. In order to obtain binders recognizing all clinically relevant strains of the targeted species, sybodies will be selected against highly conserved, abundant and surface accessible outer membrane proteins (OMPs) of E. coli, K. pneumoniae and P. aeruginosa. Recently developed methods to enrich and identify binders against OMPs embedded in their native membrane will assure that high affinity sybodies specifically recognizing living pathogens are obtained. Since the targeted OMPs are non-essential, captured pathogens can be grown and analyzed further by AST. Sybodies have a long shelf life and are cheap to produce. Hence, they are particularly suited for the development and manufacture of diagnostic products. Together with the startup company rqmicro AG, a point-of-care BSI diagnostic kit relying on flow-cytometry will be developed, which can be used by clinicians at the hospital. In close collaboration with the diagnostic unit of the Institute of Medical Microbiology, a pathogen capture method will be developed which drastically shortens the time of AST of blood stream infections bypassing long-lasting blood cultures.In addition, sybodies emanating from this project provide a seamless link between clinical patient samples and rapid, miniaturized AST already supported by NRP72.
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