DNA microarray for detection of pathogens and antibiotic resistance gene bla(CTX-M) in surface water

Antibiotic resistance is a problem for treatment of infections worldwide. In surface water, antibiotic resistant bacteria are also present and need to be monitored to assess potential risks and dissemi-nation pathways. Using the developed DNA microarray, we are able to detect pathogenic bacterial species and the widely distributed re-sistance gene blaCTX-M.

Antibiotic resistant bacteria carry antibiotic resistance genes (ARGs) which are responsible for changes within the bacteria. The current gold standard for detecting antibiotic resistance is cultivation followed by PCR which is time consuming and only feasible for culturable bacteria. In environmental samples, however, non-culturable bacteria make up a larger fraction of the total count. To provide faster results the existing heterogeneous asymmetric recombinase polymerase amplification (haRPA) assay on MCR 3 was adapted for the detection of the ARG blaCTX-M, an extended spectrum β-lactamase, as well as for the pathogenic bacteria Klebsiella pneumoniae and Pseudomonas aeruginosa.

Funding: IWC

Dection of antibiotics in liquid matrices by regenerable chemiluminescence microarray immunochips

Antibiotics are widely used in both human and veterinary medicine. Via wastewater and manure, they are dispensed to the environment and may end up in drinking water and food. The presence of antibiotics induces the development of resistance genes in bacteria.

Therefore, a fast and cost-effective multi-antibiotic screening method for the monitoring of both animal products (e.g. milk or manure) and environmental waters is required. The established regenerable chemiluminescence microarray immunoassay allows the screening of various antibiotics in a single run. The detection limits can be further reduced by preceding SPE. By means of the automated flow-through analysis platform MCR 3 (GWK Präzisionstechnik, Munich), we have  established a regenerable indirect competitive chemiluminescence microarray immunoassay for the simultaneous detection of various antibiotics, e.g. sulfonamides and β-lactams, in liquid matrices.  Calibration experiments were carried out for seven sulfonamides. The most sensitive assay was achieved for sulfamethoxazole with a detection limit of 0.1 µg/L. To cover the even lower concentrations of real environmental water samples, a pre-concentration SPE method using Oasis® HLB cartridges (Waters, Milford, CT, USA) will be established.

Funding: Hanns-Seidel-Stiftung and IWC

Flow-based microreactor for nanoparticle synthesis and on-line analysis

Continuous synthesis of PVP stabilized monodispersed gold nanoparticles using flow-based microreactor

Gold nanoparticles were widely used in bioanalytics because of excellent detection properties and couling capability to antibodies. Synthesis of gold nanoparticles can be done by number of methods. The chemical synthesis by reducing metal salts is a simple process, which requires the mixing of the reagents at well-defined external conditions. The chemical reduction method and the choice of a capping agent depends on variety of parameters that can influence the physical and chemical properties of the resulting particles. To achieve stable nanoparticles with specific size, reaction conditions should be controlled. Therefore, microfluidic reactors with online detection methods are preferable.

Funding: China Scholarship Council and IWC

AIF-ZIM Project (FKZ ZF4456001)

Microarray-based nucleid acid amplification test for mycotoxin-containg bioaerools of in indoor air

To date the most used methods to test for bioaerosols in indoor air are culture-based or microscopy methods which are relatively inaccurate and time consuming. Mycotoxins themselves are detected mainly by chromatography in combination with mass spectrometry which can be done only in specialized analytical laboratories who have not the experiences in quantification of fungi. Therefore, cheaper and faster method are needed to assess the risk of fungi contamination in buildings after e.g. water damages.

Cooperation partner: domatec GmbH

Funding: AIF-ZIM

BMBF project (13N13698): LegioTyper

Culture-independent serotyping of Legionella pneumophila in water, aerosol and urine samples

A rapid bioanalytical method for the detection of Legionella pneumophila will be established. The sandwich microarray immunoassay will detect serogroup 1 to 15 by monoclonal serotyping on the microarray analysis platform MCR 3 in not more than one hour. Patterns of specific microarray signals will be compared by the analysis of environmental (water and bioaerosols) and clinical samples (e.g. urine). Matching signal patterns will give the direct link between an infection and the source that is responsible for a legionellosis outbreak.

Cooperation partners: Technische Universität Dresden, Dresden (TUD); Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Erlangen (LGL); GWK Präzisionstechnik GmbH, München

Funding: Bundesministerium für Bildung und Forschung (BMBF)

Homepage: www.legiotyper.de

IGSSE Project (9.06): MiCSMaP

Microreactor with integrated characterization for the synthesis of magnetic nanoparticles.

MiCSMap is an interdisciplinary project founded by the International Graduate School of Science and Engineering (IGSSE) between analytical chemistry, chemical engineering and electrical engineering. Research interests are the synthesis and coating of magnetic nanoparticles in microfluidic reactors with integrated continuous magnetic separation and particle characterization.

Funding: International Graduate School of Science and Engineering at TUM

Cooperation partner: Dr.-Ing. Bernhard Gleich (IMETUM, School of Bioengineering, Technische Universität München)

Development and validation of an immunological screening method for the determination of toxicologically relevant pyrrolizidine alkaloids in herbal tea and related matrices

Direct or indirect contamination of human food sources like wheat, milk, eggs, honey, salad, herbals, tea and food supplements with pyrrolizidine alkaloids (PA) are, depending on PA concentration and duration of its intake, supposed to give rise to acute (liver failure) or chronic (pulmonary arterial hypertension, cancer and teratogenic effects) toxicity. Food industries are interested in an in-house method for easy and effective analysis of unprocessed plant resources etc. Bioanalytical methods based on immunological detection of PA are most suitable in this context, as it provides parallel processing of many samples and requires minor trained staffing. Moreover, it is cheap and does not require any costly sample preparation. In order to address food industries demands, the aim of this project is to develop and validate an immunological screening method for the determination of toxicologically relevant PAs in herbal tea and related matrices.

Responsible persons: Prof. Dietmar Knopp and Katharina Stutzer, MSc.

Cooperation partners: Prof. Gareis and Dr. Gottschalk (Veterinary Faculty, Chair of Food Safety, Ludwig-Maximilians-University, Oberschleißheim, Germany)

Funding: FEI – Research Association of the German Food Industry (AiF 19010N)

Finalized projects

JPI Water Project (WU1346A): METAWATER

New metagenomics and molecular based tools for European scale identification and control of emergent microbial contaminants in irrigation water (METAWATER).

Duration: 1.1.2015 - 28.2.2018

European project analyzing fecal contaminations, emergent and ESBL-producing pathogens in irrigation water by monolithic adsorption filtration (MAF) combined with qPCR and multiplex DNA microarray analysis together with next generation sequencing.

Cooperation partner: Dr. Anna Charlotte Schultz (DTU, Copenhagen, Denmark), Dr. José Luis Alonso (UPV, Valencia, Spain), Prof. Rosina Girones (UB, Barcelona, Spain), Prof. Maria José Figueras (URV, Tarragona, Spain), Dr. Georgios T. Papageorgiou (State General Laboratory, Cyprus) and Prof. Dr. Christiane Höller (LGL, Oberschleißheim, Germany)

Funding: Bundesministerium für Bildung und Forschung (BMBF)

older finalized projects (2005 - 20016)

BMBF Project (13N12613): LEVERA

Duration: 15.4.2013-14.07.2016

Food provision and analytics - Rapid concentration and multiplex microarray analysis of microorganisms and toxins

The goal of this project was to establish a rapid concentration method in food samples which was compatible to antibody microarrays on the MCR 3 system to detect in parallel and fully automated pathogenic bacteria, leading microorganisms and toxins.

BMBF Project (033W010E): INIS-EDIT

Duration: 1.6.2013-31.12.2016

Establishment of a concentration- and detection system for the inline-monitoring of waterborne pathogens in drinking and raw water (EDIT).

It was an interdisciplinary project between analytical chemistry, environmental microbiologists, water scientists, microsystem engineers and companies from the field of diagnostics and water supply to establish a monitoring system for rapidly analyse the water hygiene in water pipes.

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit ( K3-2463-PN 13-04)

Duration: 01.03.2014 – 29.02.2016

Estimation of the environmental medical relevance of evaporative recooling systems: Comparative examinations for discharge into the ambient air.

Bayerische Forschungsstiftung (AZ-963-11):

Duration: 01.08.2011 – 31.07.2013.
Innovative zoonosis monitoring in slaughter pigs.

Ministry of Health in Italy

Duration: 01.11.2011 – 30.09.2013

Development and Evaluation of a new multiresidue screening method (Microarray Analysis Platform) for the detection of antibiotics in raw milk.

BMBF Project (02WU1142)

Duration: 01.08.2010 – 31.07.2013.

Joint Research Project: Development of a multiplex analysis platform for the microbiological monitoring of water quality using rapid enrichment tech-niques and DNA microarrays (PATH2OGENSCAN), subproject 1: coordination, bacterial and virus (German Israel Water Technology Cooperation).

DFG Research Project (DFG 1722/2-1)

Duration: 01.11.2009 - 31.10.2012

Bundle Proposal: Pathogenic viruses in water – detection, transport and elimination (PAK 436). Speaker: Dr. Ingrid Chorus, UBA and Prof. Dr. Martin Exner, University of Bonn.

FEI/AIF Project

Duration: 01.11.2009 - 31.10.2011

Detection of Staphylococcus aureus and Bacillus cereus in dairy products after bioaffinity enrichment (SaBc).

Bayerische Forschungsstiftung (AZ 842-08)

Duration: 01.04.2009 - 31.03.2011.

Flow-based microarray chip fort he quality control of food.

Bayerisches Staatsministerium für Landwirtschaft und Forsten

Duration: 01.11.2008 - 31.10.2010

Identification and quantification of antimicrobial residues in milk: evaluation of a biosensor system for routine investigations in practice and development of reference procedures (Validation project Bavaria).

DFG Research Project (DFG 1722/1-2)

Duration: 01.02.2009 - 31.07.2010

Combined method for the detection of microorganisms in drinking water.

BMBF Project (02WU0865)

Duration: 01.02.2007 - 31.01.2010

Joint Research Project: Platform development based on CMOS for the detection of pathogens in drinking, ultrapure, ground, surface and process water (AquaSens).

DFG Research Project (DFG 1722/1-1)

Duration: 01.09.2006 - 31.12.2007.

Combined method for the detection of microorganisms in drinking water.