AperTO - Archivio Istituzionale Open Access dell'Università di Torino Detection of pathogenic Campylobacter, E. coli O157:H7 and Salmonella spp. in wastewater by PCR assay This is the author's manuscript Original Citation: Availability: This version is available http://hdl.handle.net/2318/1592886 since 2016-09-19T13:12:15Z Published version: DOI:10.1007/s11356-016-6682-5 Terms of use: Open Access Anyone can freely access the full text of works made available as "Open Access". Works made available under a Creative Commons license can be used according to the terms and conditions of said license. Use of all other works requires consent of the right holder (author or publisher) if not exempted from copyright protection by the applicable law. (Article begins on next page) 05 October 2021 This is the author's final version of the contribution published as: Bonetta S, Pignata C, Lorenzi E, De Ceglia M, Meucci L, Bonetta S, Gilli G, Carraro E. Detection of pathogenic Campylobacter, E. coli O157:H7 and Salmonella spp. in wastewater by PCR assay. Environmental Science and Pollution Research. Vol. 23, issue 15, 2016, Pages 15302-15309, DOI: 10.1007/s11356-016-6682-5 The publisher's version is available at: [http://link.springer.com/article/10.1007%2Fs11356-016-6682-5] When citing, please refer to the published version. Link to this full text: [https://iris.unito.it/handle/2318/1592886 ] This full text was downloaded from iris-Aperto: https://iris.unito.it/ iris-AperTO University of Turin’s Institutional Research Information System and Open Access Institutional Repository Environmental Science and Pollution Research Detection of pathogenic Campylobacter, E. coli O157:H7 and Salmonella spp. in wastewater by PCR assay --Manuscript Draft-- Manuscript Number: ESPR-D-15-05389R2 Full Title: Detection of pathogenic Campylobacter, E. coli O157:H7 and Salmonella spp. in wastewater by PCR assay Article Type: Research Article Corresponding Author: Elisabetta Carraro University of Torino Torino, ITALY Corresponding Author Secondary Information: Corresponding Author's Institution: University of Torino Corresponding Author's Secondary Institution: First Author: Silvia Bonetta First Author Secondary Information: Order of Authors: Silvia Bonetta Cristina Pignata Eugenio Lorenzi Margherita De Ceglia Lorenza Meucci Sara Bonetta Giorgio Gilli Elisabetta Carraro Order of Authors Secondary Information: Funding Information: Abstract: The aim of this study was the evaluation of the occurrence of pathogenic Campylobacter, E. coli O157:H7, E. coli virulence genes and Salmonella spp. in different wastewater treatment plants (WWTPs) using a method based on an enrichment step and PCR. This method was sensitive enough to detect low levels (2 CFU/100 ml of raw sewage) of all the investigated pathogens. In the WWTPs samples, E. coli O157:H7 DNA and the eae gene were never found, but 33% of influents and effluents exhibited amplicons corresponding to Shiga-like toxin I. 25% of the influent and 8% of the effluent exhibited the presence of Shiga-like toxin II. Campylobacter jejuni and C. coli DNA were identified in 50% and 25% of the influents and in 8% and 25% of the effluents, respectively. Salmonella spp. DNA was present in all the samples. Considering the results obtained the method tested here offer a reliable and expeditious tool for evaluating the efficiency of the effluents treatment in order to mitigate contamination risk. Influent contamination by Salmonella spp. and Campylobacter spp. provides indirect information about their circulation, moreover their presence in effluents underlines the role of WWTPs in the contamination of the receiving surface waters, which affects public health directly or indirectly. Response to Reviewers: Reviewer 2 We agree that the “best practise” procedure in the study design is the use of a positive control as confirmed by the approach utilized in this study, but it was not possible to buy the toxigenic strain (not commercialized in Europe by ATCC). Despite this, we think that the positive results obtained in the wastewater samples don’t represent an Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation artefact. In fact during the sampling, amplicons with the expected sizes of these genes (Hu et al., 1999, JAM, vol 87) were obtained. Reviewer 3 Major point Binary logistic regression was performed to study the association between the concentration of indicator bacteria and the occurrence of pathogens in wastewater samples. Differences in occurrence of pathogenic bacteria between different wastewater treatment plant (Chieri, Lanzo and Castiglione) and sampling time (Spring; Summer; Autumn; Winter) were studied, but the results obtained did not show a relationship. This information was added in the text. Minor points 1 The volume of spiking sample was added in the text 2. The description of the wastewater treatment methods were clarified. 3. The approach used to evaluate the sensitivity of the protocol was the approach of the “worst case”. The sample used to verify the sensitivity of the protocol was the raw sewage of Castiglione Torinese WWTP, that collects domestic and industrial sewage of the Turin city. Considering the information available at the start of this study, the characteristics of this sample (e.g. turbidity, kind of input) were the worst also respect to the effluent samples. 4. The text was modified as required. Additional Information: Question Response §Are you submitting to a Special Issue? No §Are you submitting to a Special Issue? No Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Manuscript_R2 Click here to download Manuscript Manuscript_R2.doc Click here to view linked References 1 TITLE PAGE 2 3 4 5 Detection of pathogenic Campylobacter, E. coli O157:H7 and Salmonella spp. in wastewater by PCR 6 7 assay 8 9 Si. Bonetta1, C. Pignata1, E. Lorenzi2, M. De Ceglia2, L. Meucci2, Sa. Bonetta1, G. Gilli1, E. Carraro1* 10 11 12 13 1Department of Public Health and Pediatrics, University of Torino, Torino, Italy 14 15 2 Società Metropolitana Acque Torino S.p.A., Torino, Italy 16 17 18 19 20 21 22 23 24 25 * Corresponding author: Elisabetta Carraro, Department of Public Health and Pediatrics, University of 26 27 Torino, Via Santena 5 bis, 10126, Torino, Italy, Tel: +390116708192; e-mail: [email protected] 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 1 63 64 65 1 2 3 ABSTRACT 4 5 The aim of this study was the evaluation of the occurrence of pathogenic Campylobacter, E. coli 6 7 O157:H7, E. coli virulence genes and Salmonella spp. in different wastewater treatment plants (WWTPs) 8 9 using a method based on an enrichment step and PCR. This method was sensitive enough to detect low 10 11 levels (2 CFU/100 ml of raw sewage) of all the investigated pathogens. In the WWTPs samples, E. coli 12 13 O157:H7 DNA and the eae gene were never found, but 33% of influents and effluents exhibited 14 15 amplicons corresponding to Shiga-like toxin I. 25% of the influent and 8% of the effluent exhibited the 16 17 presence of Shiga-like toxin II. Campylobacter jejuni and C. coli DNA were identified in 50% and 25% 18 19 of the influents and in 8% and 25% of the effluents, respectively. Salmonella spp. DNA was present in all 20 21 the samples. Considering the results obtained the method tested here offer a reliable and expeditious tool 22 23 for evaluating the efficiency of the effluents treatment in order to mitigate contamination risk. Influent 24 25 contamination by Salmonella spp. and Campylobacter spp. provides indirect information about their 26 27 circulation, moreover their presence in effluents underlines the role of WWTPs in the contamination of 28 29 the receiving surface waters, which affects public health directly or indirectly. 30 31 32 33 34 35 36 37 38 39 40 Keywords: Campylobacter spp., E. coli O157:H7, virulence genes, Salmonella spp., PCR, wastewater 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 2 63 64 65 1 INTRODUCTION 2 3 Fecal contamination is a source of environmental bacterial contamination and it is responsible for the 4 5 presence of pathogens in natural environment (Bertrand and Roig 2007). Surface water can generally be 6 7 contaminated by human pathogens from enteric origin as a consequence of soil contamination from 8 9 animal manures or the release of untreated slurry or effluents from wastewater treatment plants. Although 10 11 wastewater is treated to eliminate pathogenic microorganisms and prevent waterborne transmission, 12 13 numerous studies indicate that conventional wastewater treatment does not guarantee their complete 14 15 elimination (Espigares et al. 2006). The microbiological quality of wastewater can also pose a number of 16 17 potential risks in terms of public health and environment contamination when considering the possible 18 19 reuse of wastewater effluents (Levantesi et al. 2010). Different pathogenic and opportunistic bacteria have 20 21 been found in treated effluent, and these bacteria include Shiga toxin (Stx) Escherichia coli (STEC or 22 23 VTEC), Salmonella spp., Campylobacter jejuni, Bacillus cereus, Clostridium difficile, and Listeria 24 25 monocytogenes (Varela and Manaia 2013). 26 27 Campylobacter spp. and Shiga toxin-producing E. coli are important causes of bacterial gastroenteritis in 28 29 the United States and around the world (Park et al. 2011). In 2013, Campylobacter spp. was the most 30 31 commonly reported gastrointestinal bacterial pathogen in humans in the European Union (EU), and has 32 33 been since 2005. Moreover, 6,043 confirmed cases of VTEC infections were reported in the EU (EFSA 34 35 2015). Nevertheless, the reported cases of Campylobacter spp.