Development of an Inline Monitoring System for Waterborne Pathogens – An Innovative Approach for the Surveillance of Raw and Drinking Water Hygiene Authors contact details
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AUTHOR
CONTACT DETAILS
D.Karthe
Institution
Helmholtz Centre for Environmental Research, Magdeburg
Address
Brückstr. Germany
E-mail
[email protected]
O.Behrmann
Institution
Institute of Microsystems Technology, Freiburg University, Germany
V.Blättel
Institution
R-Biopharm AG, Darmstadt, Germany
G.Dame
Institution
Institute of Microsystems Technology, Freiburg University, Germany
S.Dietze
Institution
Advanced System Technology (AST) Branch of Fraunhofer IOSB, Ilmenau, Germany
M.Dilcher
Institution
Center for Biological Systems Analysis, Freiburg University, Germany
D.Elsässer
Institution
Institute of Hydrochemistry and Chemical Balneology, Technical University of Munich, Germany
S.Hakenberg
Institution
Institute of Microsystems Technology, Freiburg University, Germany
C.Heese
Institution
GWK Precision Technology GmbH, Munich, Germany
F.Hufert
Institution
Institute of Microsystems Technology, Freiburg University, Germany
M.Hügle
Institution
Center for Biological Systems Analysis, Freiburg University, Germany
A.Kunze
Institution
Institute of Hydrochemistry and Chemical Balneology, Technical University of Munich, Germany
J.Otto
Institution
Water Technology Center, German Technical and Scientific Association for Gas and Water, Karlsruhe, Germany
3ª,
39114
Magdeburg,
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AUTHOR
CONTACT DETAILS
B.Scharaw
Institution
Advanced System Technology (AST) Branch of Fraunhofer IOSB, Ilmenau, Germany
F.Sedehizade
Institution
Berliner Wasserbetriebe (BWB), Berlin, Germany
M.Seidel
Institution
Institute of Hydrochemistry and Chemical Balneology, Technical University of Munich, Germany
A.Tiehm
Institution
Water Technology Center, German Technical and Scientific Association for Gas and Water, Karlsruhe, Germany
S.Vosseler
Institution
R-Biopharm AG, Darmstadt, Germany
T.Westerhoff
Institution
Advanced System Technology (AST) Branch of Fraunhofer IOSB, Ilmenau, Germany
Keywords: Pathogens; raw water; drinking water; monitoring The relatively old age of municipal water infrastructures and external processes such as climate change or demographic trends create new challenges for Germany's public water sector. Public water supply services rely on complex technical systems including raw water abstraction facilities, water works and distribution networks which typically contain intermediate storage tanks, pumping stations and (often interconnected) pipelines. Therefore, potential contamination sources for raw or drinking water are numerous. Moreover, for (pathogenic) microorganisms there is a risk of re-growth after water treatment. Rapid and continuous monitoring systems for waterborne pathogens could greatly help in detecting and managing such contamination events. Currently established monitoring systems rely on the detection of bacterial indicators by cultivation on nutrient media. In state of the art approaches, viruses can be monitored by an inoculation of cell cultures and the subsequent detection of cytopathological effects. However, cell culture systems do not exist for all waterborne viruses. Because of the relatively long time needed, such methods do not allow rapid alarms and are poorly suited for continuous or investigative monitoring (Bruma & Tofan 2008; Fong & Lipp 2005; Jasson et al. 2010). Alternatively microorganisms such as bacteria or viruses can also be identified on the basis of nucleic acids. Polymerase chain reaction (PCR) methods are currently well established in medical diagnostics, but require relatively complex analytical steps in the laboratory (Lim et al. 2005; Ibekwe et al, 2002; Fu et al. 2005). Therefore, they are poorly suited for a simple and automated water quality monitoring. Isothermal amplification assays are a suitable alternative, as they require only simple laboratory equipment and offer shorter amplification times. Thus, they allow for rapid and automated detection systems (Asiello & Baeumner 2011).
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Proceedings of the international conference -D73.2 - -407-
We here describe a hygiene online monitoring system (HOLM) that is being developed in the project EDIT (“Development of a Concentration and Detection System for the InlineMonitoring of Pathogens in Raw and Drinking Water”). Since waterborne pathogens pose a thread even in very low concentrations, enrichment prior to the nucleic acid based detection is necessary. The challenge lies in the combination of macro processing steps that can concentrate samples of tens to hundreds of liters to volumes of less than 1 ml, with processing steps on the microliter scale that allow the detection of microorganisms and viruses by molecular biological methods (Kunze et al. 2014). For macro concentration, ultrafiltration is used as it does not require a complex preparation for the simultaneous concentration of bacteria, viruses and protozoans (Morales-Morales et al. 2003). The next stage, monolithic affinity filtration allows to selectively adsorb target pathogens onto monolithic columns (Pei et al. 2012; Lengger et al. 2012). Via an innovative automated labon-chip system the pathogens are further micro concentrated from 1ml down to 10µ l and then lysed (Lui et al. 2009; Podszun et al. 2012; Puchberger-Enengl et al. 2011). Subsequently the nucleic acids of the target organisms are extracted within the same chip. Finally, the target nucleic acid is detected by a multiplex amplification assay, using the recombinase polymerase amplification (RPA) method, on the automated microarray platform MCR3 (Kunze et al. 2014).
Table 1: Target organisms for the HOLM system developed by the EDIT project BACTERIA -
Escherichia coli Enterococcus faecalis Pseudomonas aeruginosa Campylobacter jejuni - Klebsiella pneumonia and Klebsiella oxytoca
VIRUSES -
PHAGES
Norovirus GGI-II Adenovirus 40,41,52 Enteroviruses
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MS2 PhiX174
Besides a sufficient sensitivity and selectivity for the target organisms, a newly developed monitoring technology needs to be sufficiently user-friendly and capable of integration into existing warning and control systems used by municipal water providers (Langer et al. 2014). Acknowledgement We thank the German Federal Ministry of Education (BMBF) for providing funding for the project “EDIT” (grant no. 033W010) in the context of the NAWAM-INIS funding initiative.
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References Asiello, P.J. and Baeumner, A.J. (2011): Miniaturized isothermal nucleic acid amplification, a review. Lab on a Chip 11(8):1420-1430. Bruma, M. and Tofan, C. (2008): Detection, identification and quantification of indicator bacteria from drinking water. Journal of Agroalimentary Processes and Technologies 14:196-202. Fong T.-T. and Lipp, E.K. (2005): Enteric viruses of humans and animals in aquatic environments: Health risks, detection, and potential water quality assessment tools. Microbiology and Molecular Biology Reviews 69(2): 357-371. Fu, Z.; Rogelj, S. and Kieft, T.L. (2005): Rapid detection of Escherichia coli 0157 : H7 by immunomagnetic separation and real-time PCR. International Journal of Food Microbiology 99(1):47-57. Ibekwe, A.M; Watt, P.M.; Grieve, C.M.; Sharma, V.K. and Lyons, S.R. (2002): Multiplex fluorogenic realtime PCR for detection and quantification of Escherichia coli O157 : H7 in dairy wastewater wetlands. Applied and Environmental Microbiology 68(10):4853-4862. Jasson, V.; Jacxsens, L.; Luning, P.; Rajkovic, A. and Uyttendaele, M. (2010): Alternative microbial methods: An overview and selection criteria. Food Microbiology 27(6): 710-730. Kunze, A.; Elsäßer, D.; Karthe, D.; Dame, G.; Sedehizade, F.; Nießner, R. and Seidel, M. (2014): Entwicklung eines Hygiene Online-Monitoring Systems. Automatisierter Schnellnachweis von Bakterien und Viren (033W010E) für Roh- und Trinkwasser. In: Dellert-Ritter, M. (Ed.) (2014): Food + chrom Forum, pp. 60f. Mainaschaff. Langer, M.; Wolf, C.; Sorge, H.C. and Karthe, D. (2014): Forschung für die Wasserinfrastrukturen von morgen. energie|wasser-praxis 10/2014, pp. 72-75. Lengger, S.; Niessner, R. and Seidel, M. (2012): Pathogens in Water - enrich and verify. Nachrichten aus der Chemie 60:1208-1212. Lim, D.V.; Simpson, J.M.; Kearns, E.A. and Kramer M.F. (2005): Current and developing technologies for monitoring agents of bioterrorism and biowarfare. Clinical Microbiology Reviews 18(4):583-607. Lui, C.; Cady, C. and Batt, C.A. (2009): Nucleic Acid-based Detection of Bacterial Pathogens Using Integrated Microfluidic Platform Systems. Sensors 9(5):3713-3744. Morales-Morales, H. A.; Vidal, G.; Olszewski, J. et al. (2003): Optimization of a reusable hollow-fiber ultrafilter for simultaneous concentration of enteric bacteria, protozoa, and viruses from water. Applied Environmental Microbiology 69(7):4098-4102. Pei, L.; Rieger, M.; Lengger, S.; Ott, S.; Zawadsky, C.; Hartmann, N.M.; Selinka, H.C.; Tiehm, A.; Niessner, R.; Seidel, M. (2012): Combination of crossflow ultrafiltration, monolithic affinity filtration, and quantitative reverse transcriptase PCR for rapid concentration and quantification of bacteriophage MS2 in environmental water. Environmental Science and Technology 46(18):10073-80. Puchberger-Enengl, D.; Podszun, S.; Heinz, H.; Hermann, C.; Vulto, P.; Urban, G.A. (2011): On-Chip concentration of bacteria by free flow electrophoresis. Biomicrofluidics 5(4):044111:1-10.
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