Microarray (phylochip) analysis of freshwater pathogens at several sites along the Northern German coast transecting both estuarine and freshwaters

Abstract

Monitoring the quality of drinking water is an important issue for public health. Two of the main objectives of the European Project μAQUA were (i) the development of specific probes to detect and quantify pathogens in drinking water and (ii) the design of standardized sampling programs of water from different sources in Europe in order to obtain sufficient material for downstream analysis. Our phylochip contains barcodes that specifically identify freshwater pathogens for enabling the detection of organisms that can be risks for human health. Monitoring for organisms with molecular tools is rapid, more accurate and more reliable than traditional methods. Rapid detection means that mitigation strategies come into play faster with less harm to the community and to humans. Samples were collected from several waters in France, Germany, Ireland, Italy and Turkey over 2 years. We present microarray results for the presence of freshwater pathogens from brackish and freshwater sites in Northern Germany, and cyanobacterial cell numbers inferred from these sites. In a companion study from the same samples, cyanobacterial toxins were analyzed using two methods and those sites with highest toxin values also had highest cell numbers as inferred from this microarray study.

Application of the μAqua microarray for pathogenic organisms across a marine/freshwater interface

“…In the first case, the probe reacts more strongly with Anabaena, whereas in the second case the probe reacts more strongly with Aphanizomenon. Calibration curves for the different cyanobacterial generic level probes can be found in Baudart et al (2016). All probes have > 2 mm to their target and were designed to hybridise at 60°C, which should minimize any cross reaction.…”

“…(Fig. 8) Because calibration curves have been performed for the cyanobacterial probes using RNA concentrations from known numbers of cells from pure cultures (Baudart et al, 2016), we are able to infer cell numbers present in the samples in lieu of cell counts (Suppl. Table 1, Fig.…”

“…The microarray from the μAQUA project and the subsequent MicroCokit project that used the μAQUA phylochip was field tested in 6 countries. Results for four of the monitoring sites have been published: the Tiber River in Italy, (Marcheggiani et al, 2015;Medlin et al, 2017), the north German coastal fresh and brackish water sites (Baudart et al, 2016), six lakes in the Netherlands (Van Der Waal et al, 2017), one lake in Turkey (Akcaalan et al, 2017) and in this paper we present results for three sites in along the Mediterranean coast of south-east France ranging from fresh to brackish to full strength seawater, thus testing the distribution of these pathogens and the applicability of the probes across the marine/freshwater boundary. The results for the cyanobacteria and Protozoa are reported here, whereas the bacterial component will be reported elsewhere (Baudart and Guillebault unpublished).…”

Molecular Techniques for the Detection of Organisms in Aquatic Environments, with Emphasis on Harmful Algal Bloom Species

“…This project was field tested also for two years in 8 EU countries and the results from some of these studies have been published [87,88]. Among the novel tools developed for toxin detection, a toxin array was dedicated to the detection of the messenger RNA from cyanobacterial toxin genes being expressed.…”

“…However in the EU μAQUA project, >50 L of water were filtered using hollow fiber filters in relatively short amounts of time (ca. 30 min) and the filters effectively concentrated all organisms down to viruses in the 50 L into a one liter volume for easier down stream analysis [86,87]. …”