Novel Diagnostic Tools for Culture-Independent Sepsis Pathogen Detection
Principal Investigator: Dr. Ute Neugebauer
Team: Uwe Glaser
Anuradha Ramoji (Shilers)
Kerstin Galler (HHDP)
Research Area: B Novel Diagnostic Tools and
Project Number: B3.2
Duration: 01.03.2011 - 31.07.2015
Module: Junior Research Group
The appropriate and immediate antibiotic treatment of sepsis patients increases survival and improves outcome. However, classical microbiological methods take several hours up to several days to identify the right drug, especially if the sepsis pathogen bears drug resistances. This project aims to develop an on-chip device to probe the antibiotic susceptibility patterns of sepsis pathogens based on optical spectroscopy without the need of time-consuming bacterial cultivation. Furthermore, alternative, spectroscopy-based methods are explored for a reliable and fast identiἀcation of sepsis based on the host response.
Results so far
Core project: Spectroscopic antibiotic susceptibility test in a microfluidic device
To allow a fast, culture-independent analysis of bacteria from body liquids such as urine or bronchoalveolar lavage (BAL) the bacteria need to be enriched and captured at pre-defined structures where a detailed spectroscopic investigation and characterization takes place. A first prototype of such a microfluidic device has been manufactured (together with IPHT). Dielectrophoresis was chosen as the retention methods for the bacteria. Systematic studies on electrode design, frequency, voltage and conductivity yielded the optimal experimental conditions for a good capturing effciency. On such a dielectrophoresis chip Enterococcus faecalis and Escherichia coli could be differentiated based on their Raman spectra. An alternative microfluidic approach based on CD chips using centrifugal force for bacteria collection is currently under development together with the Dublin City University, Ireland. Detailed Raman-spectroscopic investigations of bacterial growth of Enterococci faecalis in the absence and presence of vancomycin provide the experimental basis for a fast detection assay for vancomycin resistant enterococci (VRE).
Monitoring the Host Response
In-vitro cell activation experiments are preformed to better understand the reaction of the host on infection, such as Raman-spectroscopic investigation of Staphylococcus aureus infection of endothelial cells or monitoring the monocyte response upon stimulation with Tumor necrosis factor-alpha, lipopolysaccaride as well as staurosporin.
Start-up Scientist Project (A. Ramoji):
Spectral Hemogram for the Investigation of Leukocytes and their Role in Septicemia (Shilers) Leukocyte subtypes could be successfully differentiated by means of Raman spectroscopy (Ramoji et al., 2012). Furthermore, a surrogate method will be developed to probe the biophotonic fingerprint of the leukocytes after infection and inflammation.
HHDP Project (see also project HHDP).
Systematic spectroscopic studies yielded achievable detection limits of heme and heme degradation products for further in-vivo detection (Neugebauer et al., 2012). Ongoing experiments focus on the non-destructive characterization of hepatic stellate cells and their activation.
Kostudis, Hartkopf, Claus, Bauer, Popp, Neugebauer Monitoring of monocytic host response by Raman spectros-copy. INFECTION 2011, 39 (Suppl.2), S103.
Schröder, Glaser, Leiterer, Csáki, Fritzsche, Bauer, Popp, Neugebauer Micromanipulation of sepsis relevant bacteria with dielectrophoresis. INFECTION 2011, 39 (Suppl.2), S104.
A. Ramoji, U. Neugebauer, T. Bocklitz, M. Foerster, M. Kiehntopf, M. Bauer, J. Popp: Toward a Spectroscopic Hemogram: Raman Spectroscopic Differentiation of the Two Most Abundant Leukocytes from Peripheral Blood. Analytical Chemistry, Anal. Chem., 2012, 84 (12), pp 5335-5342. DOI: 10.1021/ac3007363, Epub
Neugebauer U, März A, Henkel M., Popp J: Spectroscopic detection and quantification of heme and heme degradation products. Anal Bioanal Chem 2012, in press. DOI: 10.1007/s00216-012-6288-9.
Center for Sepsis Control and Care (CSCC)
Erlanger Allee 101