title: Detection of emerging and re-emerging pathogens in surface waters close to an urban area.
abstract: Current knowledge about the spread of pathogens in aquatic environments is scarce probably because bacteria, viruses, algae and their toxins tend to occur at low concentrations in water, making them very difficult to measure directly. The purpose of this study was the development and validation of tools to detect pathogens in freshwater systems close to an urban area. In order to evaluate anthropogenic impacts on water microbiological quality, a phylogenetic microarray was developed in the context of the EU project µAQUA to detect simultaneously numerous pathogens and applied to samples from two different locations close to an urban area located upstream and downstream of Rome in the Tiber River. Furthermore, human enteric viruses were also detected. Fifty liters of water were collected and concentrated using a hollow-fiber ultrafiltration approach. The resultant concentrate was further size-fractionated through a series of decreasing pore size filters. RNA was extracted from pooled filters and hybridized to the newly designed microarray to detect pathogenic bacteria, protozoa and toxic cyanobacteria. Diatoms as indicators of the water quality status, were also included in the microarray to evaluate water quality. The microarray results gave positive signals for bacteria, diatoms, cyanobacteria and protozoa. Cross validation of the microarray was performed using standard microbiological methods for the bacteria. The presence of oral-fecal transmitted human enteric-viruses were detected using q-PCR. Significant concentrations of Salmonella, Clostridium, Campylobacter and Staphylococcus as well as Hepatitis E Virus (HEV), noroviruses GI (NoGGI) and GII (NoGII) and human adenovirus 41 (ADV 41) were found in the Mezzocammino site, whereas lower concentrations of other bacteria and only the ADV41 virus was recovered at the Castel Giubileo site. This study revealed that the pollution level in the Tiber River was considerably higher downstream rather than upstream of Rome and the downstream location was contaminated by emerging and re-emerging pathogens.
authors.affiliation: Environmental, Quality and Fishfarm Unit, Environment & Primary Prevention Department, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. stefania.marcheggiani@iss.it.
authors.forename: Stefania
authors.initials: S
authors.lastname: Marcheggiani
authors.affiliation: Environmental, Quality and Fishfarm Unit, Environment & Primary Prevention Department, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. emilio.dugo@iss.it.
authors.forename: Emilo
authors.initials: E
authors.lastname: D'Ugo
authors.affiliation: Environmental, Quality and Fishfarm Unit, Environment & Primary Prevention Department, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. camilla.puccinelli@iss.it.
authors.forename: Camilla
authors.initials: C
authors.lastname: Puccinelli
authors.affiliation: Environmental, Quality and Fishfarm Unit, Environment & Primary Prevention Department, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. roberto.giuseppetti@iss.it.
authors.forename: Roberto
authors.initials: R
authors.lastname: Giuseppetti
authors.affiliation: Environmental, Quality and Fishfarm Unit, Environment & Primary Prevention Department, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. annamaria.dangelo@iss.it.
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: D'Angelo
authors.affiliation: Laboratory of Genetics, School of Biosciences and Veterinary Medicine, University of Camerino, Camerino, MC 62032, Italy. claudio.gualerzi@unicam.it.
authors.forename: Claudio Orlando
authors.initials: CO
authors.lastname: Gualerzi
authors.affiliation: Laboratory of Genetics, School of Biosciences and Veterinary Medicine, University of Camerino, Camerino, MC 62032, Italy. roberto.spurio@iss.it.
authors.forename: Roberto
authors.initials: R
authors.lastname: Spurio
authors.affiliation: Microbia Environnement, Observatoire Océanologique, 66650 Banyuls/Mer, France. linda.medlin@microbiaenvironnement.com.|UPMC Univ Paris 06, CNRS, Laboratoire de Biodiversité et Biotechnologies Microbiennes (LBBM), Sorbonne Universités, Observatoire Océanologique, F-66650 Banyuls/Mer, France. linda.medlin@microbiaenvironnement.com.
authors.forename: Linda K
authors.initials: LK
authors.lastname: Medlin
authors.affiliation: Microbia Environnement, Observatoire Océanologique, 66650 Banyuls/Mer, France. delphine.guillebault@microbiaenvironnement.com.|UPMC Univ Paris 06, CNRS, Laboratoire de Biodiversité et Biotechnologies Microbiennes (LBBM), Sorbonne Universités, Observatoire Océanologique, F-66650 Banyuls/Mer, France. delphine.guillebault@microbiaenvironnement.com.
authors.forename: Delphine
authors.initials: D
authors.lastname: Guillebault
authors.forename: Julia
authors.initials: J
authors.lastname: Baudart-Lenfant
authors.affiliation: SCIENION AG Volmerstr., 7b/12489 Berlin, Germany. weigel@scienion.de.
authors.forename: Wilfried
authors.initials: W
authors.lastname: Weigel
authors.affiliation: Centre de Recherche de Saint Maurice, Immeuble le Dufy, Veolia Recherche et Innovation, 1 Place de Turenne, 94417 St Maurice Cedex, France. karim.helmi@veolia.com.
authors.forename: Karim
authors.initials: K
authors.lastname: Helmi
authors.affiliation: Environmental, Quality and Fishfarm Unit, Environment & Primary Prevention Department, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. laura.mancini@iss.it.
authors.forename: Laura
authors.initials: L
authors.lastname: Mancini
mesh_terms: D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D021821:Communicable Diseases, Emerging / Q000382:microbiology; D004770:Enterovirus / Q000235:genetics / Q000302:isolation & purification*; D005618:Fresh Water; D006801:Humans; D046228:Microarray Analysis / Q000379:methods*; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D045483:Rivers / Q000382:microbiology* / Q000469:parasitology; D014780:Viruses / Q000235:genetics / Q000302:isolation & purification; D014871:Water Microbiology
keywords: concentration of water, urban areas; emerging and re-emerging microorganisms; environmental water sample; oligonucleotide microarrays; q-PCR