The Use of Ribosomal RNA as a Microbial Source Tracking Target Highlights the Assay Host-Specificity Requirement in Water Quality Assessments

Funding Information: We thank Tiina Heiskanen, Tarja Rahkonen, and Tarja Yli-Tuomi for their technical assistance and local health and environment authorities, and communal water supply personnel Jukka Meriluoto, Salla Leppänen, Anu Väänänen, Päivi Rissanen, Katja Ylönen, Hanna Jääskeläinen, and especially Ville Soininen and Inkeri Eronen, who received funding for the work from the North Ostrobothnia Centre for Economic Development, Transport, and the Environment (grant number POPELY/2687/2017), for organizing sample collection. Funding. This research was supported in part by the cities of Kalajoki, Tampere, Kuopio, and the municipal wastewater treatment plants Hämeenlinnan Seudun Vesi Ltd., Nokian Vesi Ltd., and Oulun Vesi Ltd., who provided samples for the study. Further, the work was partially funded by the Regional Council of Häme, grant number 518 HL/106/04.01.01/2018. We acknowledge all of the project partners and collaborators, especially the personnel of HAMK University of Applied Sciences, Hämeenlinnan Seudun Vesi Ltd., the Lammi Biological Station, and Ilkka Hirvonen of Led Future Ltd. Funding Information: This research was supported in part by the cities of Kalajoki, Tampere, Kuopio, and the municipal wastewater treatment plants Hämeenlinnan Seudun Vesi Ltd., Nokian Vesi Ltd., and Oulun Vesi Ltd., who provided samples for the study. Further, the work was partially funded by the Regional Council of Häme, grant number 518 HL/106/04.01.01/2018. We acknowledge all of the project partners and collaborators, especially the personnel of HAMK University of Applied Sciences, Hämeenlinnan Seudun Vesi Ltd., the Lammi Biological Station, and Ilkka Hirvonen of Led Future Ltd. Publisher Copyright: © Copyright © 2021 Rytkönen, Tiwari, Hokajärvi, Uusheimo, Vepsäläinen, Tulonen and Pitkänen.For microbial source tracking (MST), the 16S ribosomal RNA genes (rDNA) of host-specific bacteria and mitochondrial DNA (mtDNA) of animal species, known to cause fecal contamination of water, have been commonly used as molecular targets. However, low levels of contamination might remain undetected by using these DNA-based qPCR assays. The high copy numbers of ribosomal RNA (rRNA) could offer a solution for such applications of MST. This study compared the performance of eight MST assays: GenBac3 (general Bacteroidales), HF183 (human), BacCan (dog), Rum-2-Bac (ruminant), Pig-2-Bac (swine), Gull4 (gull), GFD, and Av4143 (birds) between rRNA-based and rDNA-based approaches. Three mtDNA-based approaches were tested: DogND5, SheepCytB, and HorseCytB. A total of 151 animal fecal samples and eight municipal sewage samples from four regions of Finland were collected for the marker evaluation. The usability of these markers was tested by using a total of 95 surface water samples with an unknown pollution load. Overall, the performance (specificity, sensitivity, and accuracy) of mtDNA-based assays was excellent (95-100%), but these markers were very seldom detected from the tested surface water samples. The rRNA template increased the sensitivity of assays in comparison to the rDNA template. All rRNA-based assays (except Av4143) had more than 80% sensitivity. In contrast, only half (HF183, Rum-2-Bac, Pig-2-Bac, and Gull4) of rDNA-based assays reached this value. For markers targeted to bird feces, the use of the rRNA-based assay increased or at least did not change the performance. Regarding specificity, all the assays had >95% specificity with a DNA template, except the BacCan assay (71%). While using the RNA template for the assays, HF183 and BacCan exhibited only a low level of specificity (54 and 55%, respectively). Further, the HF183 assay amplified from multiple non-targeted animal fecal samples with the RNA template and the marker showed cross-amplification with the DNA template as well. This study recommends using the rRNA-based approach for MST assays targeting bird fecal contamination. In the case of mammal-specific MST assays, the use of the rRNA template increases the sensitivity but may reduce the specificity and accuracy of the assay. The finding of increased sensitivity calls for a further need to develop better rRNA-based approaches to reach the required assay performance.peerReviewe

Detection and quantification of SARS-CoV-2 RNA in wastewater influent in relation to reported COVID-19 incidence in Finland

Wastewater-based surveillance is a cost-effective concept for monitoring COVID-19 pandemics at a population level. Here, SARS-CoV-2 RNA was monitored from a total of 693 wastewater (WW) influent samples from 28 wastewater treatment plants (WWTP, N = 21-42 samples per WWTP) in Finland from August 2020 to May 2021, covering WW of ca. 3.3 million inhabitants (~ 60% of the Finnish population). Quantity of SARS-CoV-2 RNA fragments in 24 h-composite samples was determined by using the ultrafiltration method followed by nucleic acid extraction and CDC N2 RT-qPCR assay. SARS-CoV-2 RNA signals at each WWTP were compared over time to the numbers of confirmed COVID-19 cases (14-day case incidence rate) in the sewer network area.& nbsp;Over the 10-month surveillance period with an extensive total number of samples, the detection rate of SARSCoV-2 RNA in WW was 79% (including 6% uncertain results, i.e., amplified only in one out of four, two original and two ten-fold diluted replicates), while only 24% of all samples exhibited gene copy numbers above the quantification limit. The range of the SARS-CoV-2 detection rate in WW varied from 33% (including 10% uncertain results) in Pietarsaari to 100% in Espoo. Only six out of 693 WW samples were positive with SARS-COV-2 RNA when the reported COVID-19 case number from the preceding 14 days was zero. Overall, the 14-day COVID19 incidence was 7.0, 18, and 36 cases per 100 000 persons within the sewer network area when the probability to detect SARS-CoV-2 RNA in wastewater samples was 50%, 75% and 95%, respectively. The quantification of SARS-CoV-2 RNA required significantly more COVID-19 cases: the quantification rate was 50%, 75%, and 95% when the 14-day incidence was 110, 152, and 223 COVID-19 cases, respectively, per 100 000 persons. Multiple linear regression confirmed the relationship between the COVID-19 incidence and the SARS-CoV-2 RNA quantified in WW at 15 out of 28 WWTPs (overall R2 = 0.36, p < 0.001). At four of the 13 WWTPs where a significant relationship was not found, the SARS-CoV-2 RNA remained below the quantification limit during the whole study period. In the five other WWTPs, the sewer coverage was less than 80% of the total population in the area and thus the COVID-19 cases may have been inhabitants from the areas not covered.& nbsp;Based on the results obtained, WW-based surveillance of SARS-CoV-2 could be used as an indicator for local and national COVID-19 incidence trends. Importantly, the determination of SARS-CoV-2 RNA fragments from WW is a powerful and non-invasive public health surveillance measure, independent of possible changes in the clinical testing strategies or in the willingness of individuals to be tested for COVID-19.Peer reviewe

Scenario-based assessment of fecal pathogen sources affecting bathing water quality: novel treatment options to reduce norovirus and Campylobacter infection risks

Wastewater discharge and runoff waters are significant sources of human and animal fecal microbes in surface waters. Human-derived fecal contamination of water is generally estimated to pose a greater risk to human health than animal fecal contamination, but animals may serve as reservoirs of zoonotic pathogens. In this study, quantitative microbial risk assessment (QMRA) tools were used to evaluate the hygienic impact of sewage effluents and runoff water from municipalities and animal farms on surface and bathing waters. The human-specific microbial source tracking (MST) marker HF183 was used to evaluate the dilution of fecal pathogens originating from the sewage effluent discharge to the downstream watershed. As novel risk management options, the efficiency of UV-LED disinfection and wetland treatment as well as biochar filtration was tested on-site for the contamination sources. According to the dilution pattern of the MST marker HF183, microbes from wastewater were diluted (2.3–3.7 log10) in the receiving waters. The scenario-based QMRA revealed, that the health risks posed by exposure to human-specific norovirus GII and zoonotic Campylobacter jejuni during the bathing events were evaluated. The risk for gastroenteritis was found to be elevated during wastewater contamination events, where especially norovirus GII infection risk increased (1–15 cases per day among 50 bathers) compared with the business as usual (BAU) situation (1 case per day). The noted C. jejuni infection risk was associated with animal farm contamination (1 case per day, versus 0.2–0.6 cases during BAU). Tertiary treatment of wastewater with wetland treatment and UV-LED disinfection effectively reduced the waterborne gastroenteritis risks associated with bathing. Based on the experiences from this study, a QMRA-based approach for health risk evaluations at bathing sites can be useful and is recommended for bathing site risk assessments in the future. In case of low pathogen numbers at the exposure sites, the MST marker HF183 could be used as a pathogen dilution coefficient for the watershed under evaluation. The full-scale implementation of novel tertiary treatment options at wastewater treatment plants (WWTPs) as well as on-site runoff water treatment options should be considered for infection risk management at locations where scenario-based QMRA implies elevated infection risks

EGUS ja hevosen ruuansulatuskanavan bakteerikanta

Hevosen ruuansulatuskanava on hyvin uniikki. Hevosen maha on pieni verrattuna muihin saman kokoisiin nisäkkäisiin ja vapaasti laiduntava hevonen käyttääkin lähes puolet vuorokaudestaan syömiseen. Hevosen ruuansulatuskanava on kehittynyt hyödyntämään kuitupitoisen ja matalaenergisen ruuan ravintoaineet, joten paljon fermentoituvia hiilihydraatteja sisältävä ruokavalio voi muuttaa mahan olosuhteita ja bakteerikantaa joskus niinkin rajusti, että eläin sairastuu. (Perkins et al. 2012) Mahahaavat ovat hevosten yleisimpiä sairauksia ja niistä kärsii lähteestä riippuen 53–93 % täysikasvuisista kesyhevosista (Begg & O’Sullivan 2003, Perkins et al. 2012). Mahahaavaan liittyy usein ähkyjä, painon laskua ja suorituskyvyn heikkenemistä, mutta monet kilpahevoset sairastavat sitä täysin oireettomana (Perkins et al. 2012). Suurin osa aikuisten hevosten mahahaavoista esiintyy mahan limakalvon pintakerroksessa ruokatorvessa, mahalaukussa, mahanportissa tai pohju-kaissuolessa (Dong et al. 2016, Perkins et al. 2012). Mahan happopitoisuuden nousu ja hapolta suojaavan limakalvon heikkeneminen edistävät usein mahahaavan syntyä. Mahan happopitoisuutta ja siten pH:ta voivat nostaa esimerkiksi kasvanut suolahapon eritys sekä VFA:t, joita syntyy käymisreaktioissa bakteerien hajottaessa väkirehua. (Perkins et al. 2012) Kasvinsyöjillä ruuansulatuskanavan mutualistinen mikrobikanta osallistuu suurelta osin ravintoaineiden muuttamiseen käytettävään muotoon, jolloin kannalla on myös suuri merkitys eläimen terveyden, kasvun ja selviytymisen kannalta. Tyypillisesti kasvinsyöjän ruuansulatuskanavan mikrobikanta koostuu bakteereista, arkeista, alkueläimistä ja fungeista (St-Pierre et al. 2012). Suurinta osaa hevosen mahasta hallitsevat vain tiettyihin lajeihin kuuluvat bakteerit. 92,7 % mahan bakteerikannasta koostuu Firmicutes-, Proteobacteria-, Bacteroidetes- ja Actinobacteria-pääjaksoista (Dong et al. 2016). Terveen hevosen mahan limakalvojen mikrobiomista tiedetään hyvin vähän, eikä mikrobiomin vaikutusta mahan sairauksiin ole kattavasti tutkittu. Helicobacter-lajit ovat riskitekijöitä esimerkiksi ihmisen mahahaavan synnyssä (Shio-tani & Graham 2002). Hevosen mahasta tätä bakteeria ei kuitenkaan monissa tutkimuksissa ole löydetty lainkaan tai löydökset eivät ole korreloineet mahahaavan esiintymisen kanssa (Bezdekova & Futas 2009, Dong et al. 2016, Husted et al. 2010). Muilla mikrobeilla voi sen sijaan olla vaikutusta mahahaavan syntyyn, sillä Randwickin Equine Centerissä vuonna 2003 tehdyssä tutkimuksessa havaittiin mahalaukun limakalvon haavaumien olevan usein vakavampia kuin mahanportin (Begg & O’Sullivan 2003). Tämä on mielenkiintoinen havainto, sillä monet tekijät suojaavat mahalaukun limakalvoa hapolta, ja viittaa siis muiden tekijöiden, esimerkiksi joidenkin mahalaukun bakteerien olevan riskitekijöitä mahahaavan synnyssä

Valumavesien hygieenisen laadun parantamiseen liittyvät ratkaisut

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The detection and stability of the SARS-CoV-2 RNA biomarkers in wastewater influent in Helsinki, Finland

Analysis of the particulate matter of the sample, in addition to the water fraction, can improve the detection frequency. (C) 2021 The Author(s). Published by Elsevier B.V.Wastewater-based surveillance of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is used to monitor the population-level prevalence of the COVID-19 disease. In many cases, due to lockdowns or analytical delays, the analysis of wastewater samples might only be possible after prolonged storage. In this study, the effect of storage conditions on the RNA copy numbers of the SARS-CoV-2 virus in wastewater influent was studied and compared to the persistence of norovirus over time at 4 degrees C, -20 degrees C, and -75 degrees C using the reverse-transcription quantitative PCR (RT-qPCR) assays E-Sarbeco, N2, and norovirus GII. For the first time in Finland, the presence of SARS-CoV-2 RNA was tested in 24 h composite influent wastewater samples collected from Viikinmaki wastewater treatment plant, Helsinki, Finland. The detected and quantified SARS-CoV-2 RNA copy numbers of the wastewater sample aliquots taken during 19-20 April 2020 and stored for 29, 64, and 84 days remained surprisingly stable. In the stored samples, the SARS betacoronavirus and SARS-CoV-2 copy numbers, but not the norovirus GII copy numbers, seemed slightly higher when analyzed from the pre-centrifuged pellet-that is, the particulate matter of the influent-as compared with the supernatant (i.e., water fraction) used for ultrafiltration, although the difference was not statistically significant. Furthermore, when wastewater was spiked with SARS-CoV-2, linear decay at 4 degrees C was observed on the first 28 days, while no decay was visible within 58 days at -20 degrees C or -75 degrees C. In conclusion, freezing temperatures should be used for storage when immediate SARS-CoV-2 RNA analysis from the wastewater influent is not possible. Analysis of the particulate matter of the sample, in addition to the water fraction, can improve the detection frequency. (C) 2021 The Author(s). Published by Elsevier B.V.Peer reviewe