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

Highlights

• The European Project μAqua (FP7-KBBE-2010-4, 265409) was conceived as a project to make novel tools for the early and sensitive detection of freshwater-borne pathogens and cyanobacterial toxins.

• The novel tools developed in μAqua were a phylochip (microarray), which identifies the presence of freshwater pathogenic targets using RNA barcodes and a RT-PCR microarray to amplify the mRNA captured by the barcodes for genes involved in cyanobacterial toxin synthesis.

• We present results for three sites in along the Mediterranean coast of south-east France ranging from fresh to brackish to full strength seawater to test the applicability of the probes across the marine/freshwater boundary and infer the distribution of these pathogens across these boundaries.

• The results for the cyanobacteria and Protozoa are reported here.

• As freshwaters flow into marine systems, these pathogens and their toxins flow into a different environmental habitat.

• Nevertheless these pathogens and toxins can still cause health problems in the marine environment and this paper shows the application of a phylochip across these different habitats.

Abstract

Monitoring drinking water quality is an important public health issue and pathogenic organisms present a particularly serious health hazard in freshwater bodies. However, many pathogenic bacteria, including cyanobacteria, and pathogenic protozoa can be swept into coastal lagoons and into near-shore marine environments where they continue to grow and pose a health threat to marine mammals and invertebrates. In this study, we tested the suitability of a phylochip (microarray for species detection) developed for freshwater pathogenic organisms to be applied to samples taken across a marine/freshwater interface at monthly intervals for two years. Toxic cyanobacteria and pathogenic protozoa were more numerous in a coastal lagoon than at the freshwater or marine site, indicating that this microarray can be used to detect the presence of these pathogens across a marine/freshwater interface and thus the potential for toxicity to occur within the entire watershed.

Introduction

Monitoring drinking water quality is an important public health issue and more especially in view of climate change (Carey et al., 2012; Moore et al., 2008). The present ecological balance determining major changes in the type, abundance and distribution of pathogenic microbes could be modified with global climate changes. Pathogenic organisms present a particularly serious health hazard and cause no less than 170,000 cases of water-related diseases annually in lakes and rivers used as drinking water reservoirs (https://www.cdc.gov/healthywater/disease/az.html). The pathogenic organisms that are responsible for water-related diseases include bacteria, cyanobacteria, protozoans and viruses. In this paper, we examine the distribution of toxic cyanobacteria and pathogenic protozoa across a marine/freshwater interface using molecular tools from samples taken at monthly intervals for two years.

Cyanobacteria are oxygenic phototrophic prokaryotes, some of which produce a variety of toxins and pose a serious health threat to drinking water worldwide (Carmichael, 2008). The frequency, intensity and geographical distribution of algal blooms in freshwaters have been growing worldwide, with the major causes generally correlated with water eutrophication and climate changes (Brookes and Carey, 2011; Carey et al., 2012; Moore et al., 2008). Among the cyanobacteria, about 40 species can produce potent toxins, potentially causing the so-called Harmful Algal Blooms (HABs) to impact heavily on environmental and human health. The risk of human exposure comes from contaminated recreational surface waters and from the consumption of unsuitably treated drinking water or ingestion of contaminated food (Carmichael, 2008).

Protozoa are a diverse phylogenetic group of single celled organisms (https://microbialclassification, blogspot.com/p/pathogenic-protozoa.html). Pathogenic protozoa are considered to be parasites because they take over the host cells and reproduce by binary fission. They cause a wide array of clinical diseases, which include intestinal, urogenital and blood parasites (http:// http://infectionnet.org/notes/protozoa/). Intestinal parasites always involve contamination with faecal material (Entamoeba and Cryptosporidium) or untreated drinking water (Giardia and Cryptosporidium), and it is these organisms from different phylogenetic groups that are monitored in freshwater systems for human health problems. Monitoring in marine waters for marine parasites is unknown at this time (Certad et al., 2019).

Cabral (2010) reviewed the major problems in water microbiology, which in general terms, finds that the greatest microbial risks are associated with ingestion of water contaminated with human or animal faeces. Wastewater discharges in fresh waters and coastal seawaters are the major source of faecal microorganisms, potentially carrying pathogens. According to the WHO, the mortality of water-associated diseases exceeds 5 million people per year (https://www.who.int/water_sanitation_health/diseases-risks/en/). Although those countries with the poorest hygiene are the most affected, developed countries are not exempt from problems. In the USA, it has been estimated that each year 560,000 people suffer from severe waterborne diseases, and 7.1 million suffer mild to moderate infections, resulting in estimated 12,000 deaths a year (Medema et al., 2003).

As rivers flow into marine systems, these pathogens and their toxins flow into a different environmental habitat. Nevertheless these pathogens and toxins can still cause health problems in the marine environment (Preece et al., 2015a, 2015b, Lucy et al., 2008) and even death to marine mammals (Miller et al., 2010) as they accumulate and possibly reproduce in the marine environment (Takahashi et al., 2014) because many genera, e.g., cyanobacteria can tolerate salinities up to or over 16 o/oo (Table 1). There is little monitoring over the entire watershed of a river. Although marine and freshwater Cyanobacteria can be the same species because of their salinity tolerances, it is unknown if freshwater protozoans can survive in marine waters and infect both terrestrial and marine hosts.

Monitoring for cyanobacteria and other pathogens in freshwater systems typically involves traditional microbiological methods (Rosalba et al., 2016). However, molecular tools are more rapid, accurate and reliable than traditional methods, which means faster mitigation strategies with less harm to humans and the community. Molecular tools are designed to replace traditional methods for monitoring, which are laborious, technically demanding and time-consuming. Among the molecular tools that can enable a rapid detection of many pathogens simultaneously are microarrays (see review in Kegel et al. (2016)). Several microarrays have been developed based on RNA barcoding as well as other genes. The MIDTAL microarray targeted only toxic marine microalgae (Medlin, 2013a, b). Microbe and microbial function detection arrays have been designed for environmental microbes (e.g. PhyloChip Wilson et al., 2002, GeoChip He et al., 2007) and for host-associated bacteria (e.g. GreenChip (Palacios et al., 2007), HOMIM (Ahn et al., 2011), HITChip (Rajilic-Stojanovic et al., 2009) and Bactochip (Ballarini et al., 2013). A Eukaryotic microarray for soil microbes has also been designed based on a PCR step before amplification (Lekang et al., 2018). These arrays all currently target a combination of known functional classes genes or, most commonly, variants of the single 16S rRNA gene using with longer probes (e.g. 60-mer), which are more sensitive.

The European Project μAqua (FP7-KBBE-2010-4, 265409) was conceived as a project to make novel tools for the early and sensitive detection of freshwater-borne pathogens and cyanobacterial toxins. The novel tools developed in μAqua were a phylochip (microarray), which identifies the presence of freshwater pathogenic targets using rRNA barcodes and a RT-PCR microarray to amplify the mRNA captured by the barcodes for genes involved in cyanobacterial toxin synthesis. For the phylochip, the presence of the target organisms was detected through the use of rRNA barcodes used in a microarray detection platform (www.midtal.com). For the toxin array, we performed a reverse transcriptase extension of the mRNA probes for various toxin pathways spotted on the array incorporating fluorescent nucleotides in the reaction to amplify the signal from the messenger RNA that is expressed in low quantities in the cells (Medlin et al. 2007, Van der Waal et al., 2017; Medlin, 2018).

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).