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Fast Assessment of antibiotic resistance in bacteria by using nanomechanical arrays

Applicant Meyer Ernst
Number 177354
Funding scheme NRP 72 Antimicrobial Resistance
Research institution Departement Physik Universität Basel
Institution of higher education University of Basel - BS
Main discipline Condensed Matter Physics
Start/End 01.01.2018 - 31.12.2020
Approved amount 350'000.00
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All Disciplines (4)

Discipline
Condensed Matter Physics
Molecular Biology
Medical Microbiology
Experimental Microbiology

Keywords (6)

Epigenetics; Carbapenemases; Single nucleotide polymorphism; Mutation detection; Nanomechanical sensors; Bacterial resistance

Lay Summary (German)

Lead
DNA/RNA von Bakterienstämmen wird mit speziell entwickelten Biosensoren genetisch untersucht, um bakterielle Antibiotikaresistenzen zu erkennen. Ferner werden auch epigenetische Modifikationen untersucht.
Lay summary
Projektbeschrieb:

Wir verwenden für den nanomechanischen Nachweis bakterieller Antibiotikaresistenz mikrofabrizierte Federbalken (Cantilever)-Arrays, deren Oberflächen mit Biomarkern modifiziert werden, die Bakterien genetisch erkennen können. Das Detektionssignal ist eine Verbiegung des Cantilevers im Nanometerbereich. DNA und RNA extrahiert aus relevanten Bakterienstämmen genügt zum Nachweis antibakterieller Resistenz, wobei Stämme mit unterschiedlichen Resistenzmechanismen zur Verfügung stehen. Durch Kombination bereits entwickelter Detektionstechniken können DNA/RNA Hybridisierung, Bindungsstellen für Transkriptionsfaktoren, Antigene und Methyliserungsprozesse herangezogen werden. Einige Resistenzmechanismen beruhen auf genetischen Punktmutationen in Erkennungssequenzen für Antibiotika, welche sich mit unserer Technik schnell und ohne Amplifikation/Fluoreszenzmarkierung nachweisen lassen. 

Hintergrund Ausgangslage:

Weltweit tauchen immer häufiger Bakterienstämme auf, die gegenüber vielen Antibiotika Resistenzen zeigen, so
genannte „Multidrug-resistant (MDR) Bakterien. Dies bedeutet, dass vorher relativ harmlose Infektionen nicht mehr behandelbar sind und zu schweren Komplikationen oder sogar dem Tod führen können. Dieser unbefriedigende Zustand wird dem weitverbreiteten Gebrauch und Missbrauch von Antibiotika in der Human- und Veterinärmedizin zugeschrieben. Die heutige Diagnostik beinhaltet zeitintensive komplexe präparative Schritte, die Ergebnisse verfälschen können.

Ziele: 

Mit einer interdisziplinären Vorgehensweise (Medizin, Physik, Chemie) sollen unterschiedliche, relevante Mechanismen bakterieller Antibiotikaresistenz untersucht werden. Dabei ist die schnelle Analyse von Genmutationen und erworbener Resistenzgenen das Hauptziel, aber daneben gibt es auch noch epigenetische Faktoren, die Antibiotikaresistenzen beeinflussen. Die Analyse solcher epigenetischer Einflüsse ist technisch noch immer aufwendig und nanomechanischen Sensoren können einen wichtigen Beitrag zur schnelleren und genaueren Analytik liefern.

Bedeutung/Möglicher Nutzen:

Die schnelle Anpassungsfähigkeit von Bakterien führt zu vermehrter antibiotischer Resistenz. Dies ist bereits jetzt eine ernsthafte Bedrohung im Spital. Unkontrollierte Verbreitung multiresistenter Bakterien wird zu vielen Todesfällen führen. Daher ist es wichtig, resistente Bakterien schnell zu erkennen, um die richtigen Gegenmassnahmen treffen zu können.  Unsere Systeme zur Diagnose von Antibiotikaresistenzen sind kompakt und benötigen wenige zusätzliche Ressourcen, so dass sie in einer Praxis oder auch in infrastrukturschwachen Gegenden eingesetzt werden können.

Direct link to Lay Summary Last update: 21.12.2017

Responsible applicant and co-applicants

Employees

Publications

Publication
Conformations and cryo-force spectroscopy of spray-deposited single-strand DNA on gold
Pawlak Rémy, Vilhena J. G., Hinaut Antoine, Meier Tobias, Glatzel Thilo, Baratoff Alexis, Gnecco Enrico, Pérez Rubén, Meyer Ernst (2019), Conformations and cryo-force spectroscopy of spray-deposited single-strand DNA on gold, in Nature Communications, 10(1), 685-685.
Electrospray deposition of structurally complex molecules revealed by atomic force microscopy
Hinaut Antoine, Meier Tobias, Pawlak Rémy, Feund Sara, Jöhr Res, Kawai Shigeki, Glatzel Thilo, Decurtins Silvio, Müllen Klaus, Narita Akimitsu, Liu Shi-Xia, Meyer Ernst (2018), Electrospray deposition of structurally complex molecules revealed by atomic force microscopy, in Nanoscale, 10(3), 1337-1344.

Collaboration

Group / person Country
Types of collaboration
Georg Fantner/EPFL Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
R. McKendry / London Centre for Nanotechnology / UCL Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
NRP72 Program Meeting 2019 Poster Fast Assessment of Antibiotic Resistance in Bacteria by using Nanomechanical Arrays 27.03.2019 Lausanne, Switzerland Huber François;
Halting Antimicrobial Resistance Dissemination in Aquatic Environments HERD 2018 Talk given at a conference Fast Assessment of Antibiotic Resistance in Bacteria by using nanomechanical arrays 16.09.2018 CSF Monte Verità, Ascona, Switzerland Huber François;


Knowledge transfer events

Active participation

Title Type of contribution Date Place Persons involved


Associated projects

Number Title Start Funding scheme
186332 Hijacking Transcription-Coupled DNA Repair for Cancer Therapy 01.01.2020 Sinergia

Abstract

Due to the fast-evolutionary adaptability of bacteria, the occurrence of antibiotic-resistant strains poses a serious threat to our healthcare. More and more antibiotics became inefficient and multidrug-resistant (MDR) bacteria have emerged worldwide. This unfortunate situation is promoted by the widespread use and misuse of antibiotics in human and veterinary medicine. It is essential that MDR bacteria can be fast and reliably detected for rapid treatment modification, leading to more favourable treatment outcome. We propose a rapid nanotechnology method based on nanomechanical microcantilever arrays, which has already been successfully applied in the fields of bacterial growth monitoring, detection of genetic point mutations in melanoma in human biopsies for eligibility testing of new drugs and for HER2 overexpression inquiry in human breast cancer tissue biopsies. The method is able to detect single nucleotide polymorphisms without the necessity to use PCR or labelling and can quantify protein concentrations and biomarker concentrations. The nanomechanical sensors are integrated in a compact system based on a modified commercially available reasonably priced atomic force microscope head. A small liquid handling system and a laptop with dedicated software provide an easy way to operate the device.Here we will investigate in an interdisciplinary approach different resistance mechanisms in microorganisms by using a customized nanomechanical microcantilever array system capable of covering a broad variety of diagnostics, e.g. in DNA, RNA, transcription factor binding sites, antigens and methylation processes. Some of the resistance mechanisms employed are genetic mutations in the antibiotic target sites, detection of plasmid or chromosomally encoded genes for antibiotic-cleaving enzymes (e.g. ESBL, various carbapenemases), and colistin resistance via MCR-1 plasmids. Particularly for point mutations and methylation sites we envisage advantages with our array method for fast recognition and a simplified workflow compared to state-of-the-art technology. We have access to an extensive library of bacterial strains exhibiting diverse resistance mechanisms and require only DNA or total RNA isolated from cells or biopsies for the experimental investigation. Additionally, the induced expression of genes can also be detected at the RNA and antigen level. Furthermore, we have preliminary results indicating that our technique can identify DNA alterations found in epigenetic modifications e. g. methylation of nucleotides. Work package 1 includes first selection of suitable bacterial strains from the library according to their resistance mechanisms. Primarily we will concentrate on plasmid derived resistance genes like AmpC or mutated proteins like PBP to establish the proper conditions for detection. Specific antibodies will also be evaluated for identification of resistance markers on the protein level such as altered porins. Further steps include fluoroquinolones like ciprofloxacin, which belong to a group of antibiotics that can promote development of resistance, because they directly interfere with DNA replication and very likely also elevate levels of intracellular reactive oxygen species. Increased oxidative stress can result in 8-oxoguanine DNA adducts, which forms a basepair with adenine and if not repaired produces a GC to TA trasversion. The concept of DNA adduct-directed nucleoside probes is useful for detection of altered nucleotides in a DNA strand, as well as epigenetic modifications. Work package 2 is focused on genetic characterization of selected strains using cantilever array technology based on DNA/RNA and proteins extracted from the selected strains. The results will continuously be compared with those obtained by standard methods. Special attention will be put on sensitivity and specificity. Resistant bacteria might only comprise a small fraction and therefore it is necessary to use specific antibodies for their identification. For this purpose, quantitative dilution series will be performed using different ratios of resistant bacteria to sensitive bacteria to determine the lower limit of detection and MIC values. Work package 3 is devoted to adaption and validation of the system. A technician will be instructed to operate the device and perform experiments including setting up the cantilever array and analysing the data, including an estimation of the reliability of the proposed method. In work package 4, a series of protocols will be established to reliably identify mutations in clinically relevant antibiotic resistance genes. The system will be tailored for application in a clinical setting. Tests will be performed by a trained technician to compare cantilever array technology with speed, ease of handling, overall time spend, costs of standard methods. Additionally, further optimizations will be completed to increase throughput. The main advantage of the proposes technique is the multi-parameter characterization in DNA, RNA, antigens and methylation processes, thereby the cantilever technology offers a broad and profound assessment of pathogens at potential multiple levels. In particular for those of resistance mechanisms that are genetic mutations in the antibiotic target sites, the proposed approach is favourable, since bacterial resistance identification can be performed by detection of plasmids or by genomic encoded genes for antibiotic-cleaving enzymes. In addition, the method offers the potential to speed up the currently too slow processes to determine antibiotic resistance and this may have an important impact on patient outcomes.The development of a simple epigenetic diagnostic system based on DNA adduct-directed nucleoside probes together with cantilever arrays features the potential for a patent.Due to the expected massive impact of antibiotic resistance (AMR) on the society but also on the individual the rapid detection of AMR is key. This project offers the unique opportunity to develop and test a novel diagnostic device which holds promise to significantly reduce the time to resistance detection. Thereby the clinical outcomes are improved and overall hospital costs are reduced.The proposal shows a way towards simple, cost effective MDR diagnostics. We truly feel its significance is of utmost importance in the arsenal of medical defense against multi drug resistant bacteria.
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