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. 2015 May 22;12(5):5505-27.
doi: 10.3390/ijerph120505505.

Detection of emerging and re-emerging pathogens in surface waters close to an urban area

Stefania Marcheggiani  1 , Emilo D'Ugo  2 , Camilla Puccinelli  3 , Roberto Giuseppetti  4 , Anna Maria D'Angelo  5 , Claudio Orlando Gualerzi  6 , Roberto Spurio  7 , Linda K Medlin  8   9 , Delphine Guillebault  10   11 , Julia Baudart-Lenfant, Wilfried Weigel  12 , Karim Helmi  13 , Laura Mancini  14
Affiliations

Detection of emerging and re-emerging pathogens in surface waters close to an urban area

Stefania Marcheggiani et al. Int J Environ Res Public Health. .

Erratum in

  • Int J Environ Res Public Health. 2015 Oct;12(10):13413-4. Baudart-Lenfant, Julia [added]

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.

Keywords: concentration of water, urban areas; emerging and re-emerging microorganisms; environmental water sample; oligonucleotide microarrays; q-PCR.

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Figures

Figure 1
Figure 1
Localization of sampling sites (red dots) and the urbanized region of Rome (gray area).
Figure 2
Figure 2
Each column represents a mean value of triplicate measurements of each microorganism, expressed as cfu/100 mL, detected at the Castel Giubileo site. (a) and Mezzocammino site (b). (Striped bar = raw water RW; Black bar = concentrated water BF).
Figure 3
Figure 3
Probes for (a) Escherichia coli; (b) Salmonella sp., (c) Yersinia sp. and Yersinia enterocolytica; (d) Legionella spp. and Legionella pneumophila; (e) Pseudomonas spp.; (f) Campylobacter spp.; (g) Staphylococcus and Staphylococcus aureus; (h) Listeria spp.; (i) Bacillus spp.; (j) Family Vibrionaceae. Signal intensities of each probe were normalized with Dunaliella RNA 12.5 µM probe signal.
Figure 3
Figure 3
Probes for (a) Escherichia coli; (b) Salmonella sp., (c) Yersinia sp. and Yersinia enterocolytica; (d) Legionella spp. and Legionella pneumophila; (e) Pseudomonas spp.; (f) Campylobacter spp.; (g) Staphylococcus and Staphylococcus aureus; (h) Listeria spp.; (i) Bacillus spp.; (j) Family Vibrionaceae. Signal intensities of each probe were normalized with Dunaliella RNA 12.5 µM probe signal.
Figure 4
Figure 4
Protozoan probes of the Mezzocammino samples and Castel Giubileo samples. Signal intensities of each probe were normalized with the Dunaliella RNA 12.5 µM probe signal.
Figure 5
Figure 5
Diatom probes from the Mezzocammino and Castel Giubileo samples. Signal intensities of each probe were normalized with Dunaliella RNA 12.5 µM probe signal. Pseudomonas, Bacillus, Listeria Escherichia and Salmonella. at the genus level and Staphylococcus aureus were detected at both sites, with low signal intensities in the Castel Giubileo sample.
Figure 6
Figure 6
Comparison of concentrations of microorganisms and signal point measurement probes for each probe at the Castel Giubileo site.
Figure 7
Figure 7
Comparison of concentrations of microorganisms and signal point measurement probes for each probe at the Mezzocammino site.
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