Sewage monitoring for pathogenic microrganisms detection by using a low density microarray

Aims. Sewages are highly contaminated by numerous biological agents: bacteria, protozoa, fungi and viruses. Some of them can be very resistant to treatments and remain at high concentration in the effluent, thus representing an hazard especially in the case of water reuse for agriculture. Molecular methods are almost an important way to detect these pathogens, in particular the most applied techniques are based on protocols of nucleic acid amplification and quantification, of which the quantitative polymerase chain reaction (qPCR) is rapidly becoming established in the environmental sector for its higher sensitivity in comparison with cultural assays. Moreover the possibility in using molecular methods for simultaneous and rapid multiple detection of pathogens could be useful for risk assessment or safety purposes. This approach could be now possible using the technological advances of the DNA microarray, born for clinical analysis and based on the simultaneous qualitative analysis for multiple genera, species and strains. The large number of DNA sequences that can be spotted on a microarray together with the high specificity of binding to the immobilised sequence targets, allows the detection of a large range of microorganisms with high discriminatory ability. Nevertheless the application of this technology requires a deep study of its sensitivity and specificity. In this work, an environmental monitoring of sewage, sampled from a Waste Water Treatment Plant (WWTP), was performed in order to detect some different viruses and bacteria frequently present: Human Adenovirus (HAdV), Norovirus GGII (NV), Hepatitis A Virus (HAV), Enterovirus (EV), Rotavirus (RV), Enterohemorrhagic E. coli (EHEC), Salmonella enterica (SE) using a low density microarray containing specific oligonucleotides sequences for each of them. Methods. In the first phase of the study, a series of trials were performed to choose the sample treatment that was able to increase the sensitivity of molecular method. In particular 10 sewage samples (10 L) were treated with a first step based on tangential ultrafiltration followed by a second step in which the obtained eluates were further concentrated by ultracentrifugation until a final volume of 1 ml. After each step, the obtained eluates were treated with commercial kit (QIAgen) to extract viral and bacteria nucleic acids (NA) that were quantified by separate and specific qPCR reactions (Genomic Copies - GC) according to published protocols. In the second phase, an environmental monitoring was performed for 1 year from June 2013 to June 2014 by monthly sampling of 10 L of sewage from a WWTP in Northern Italy (13 samples). The concentrated samples, obtained by the previous phase chosen method, were treated to extract NA that were subsequently concentrated by speed VAC, labelled and overnight hybridised on the microarray slides. After that, a scanner read the positive signal. Parallel, aliquots of extracted NA of each samples were quantify (GC/10 L) by qPCR reactions. Results. The data of the trials revealed that the combination of ultrafiltration-ultracentrifugation permitted to increase the mean concentration of target microorganisms of 1-2 Log in comparison with the use of only ultrafiltration method. The microarray analysis revealed the presence of HAdv, EV, EHEC and SE in tested samples, while no NV, HAV and RV were detected. In particular, 78% (10/13) of samples resulted positive for HAdV, 30% (4/13) for EV, 84% (11/13) for EHEH and 61% (8/13) for SE. The simultaneous presence in the same sample of all these 4 target was detected in 2 samples (15%), of three target in 6 samples (46%) and of two target in 2 of samples (15%). The mean concentration of positive target estimated by qPCR were 2 x 108 GC/10 L for HAdV, 4 x 108 CG/10 L for EV, 107 GC/10 L for EHEC and 3 x 105 GC/10 L for SE. These data are in according of sewage microbial concentration published by several authors (Figure 1). The absence of positive samples for NV, HAV and RV was probably due by epidemiological situation in the monitored region. The data, moreover, underlined a limit of sensitivity of the test: samples with a target concentration lower than 2,8 x 103 GC/10 L resulted negative to microarray. Conclusion. The results of environmental monitoring were very promising for a multiple detection of pathogens in sewage confirming the possible use of microarray as a tool for screening. The major limitation of this technique was the scarce sensitivity that can be improved with specific sample treatments permitting also the purification of samples, as made in this study, by the combination of ultrafiltration and ultracentrifugation.