Consumption of contaminated drinking water is a significant cause of Campylobacter infections. Drinking water contamination is known to result from septic seepage and wastewater intrusion into ...non-disinfected sources of groundwater and occasionally from cross-connection into drinking water distribution systems. Wastewater effluents, farm animals and wild birds are the primary sources contributing human-infectious Campylobacters in environmental waters, impacting on recreational activities and drinking water sources. Culturing of Campylobacter entails time-consuming steps that often provide qualitative or semi-quantitative results. Viable but non-culturable forms due to environmental stress are not detected, and thus may result in false-negative assessments of Campylobacter risks from drinking and environmental waters. Molecular methods, especially quantitative PCR applications, are therefore important to use in the detection of environmental Campylobacter spp. Processing large volumes of water may be required to reach the desired sensitivity for either culture or molecular detection methods. In the future, applications of novel molecular techniques such as isothermal amplification and high-throughput sequencing applications are awaited to develop and become more affordable and practical in environmental Campylobacter research. The new technologies may change the knowledge on the prevalence and pathogenicity of the different Campylobacter species in the water environment.
•Waterborne transmission of Campylobacter infections is a public health concern.•Campylobacter spp. are frequently isolated at low numbers from water.•Detection from water involves culture and molecular methods and their combinations.•Water concentration techniques are required to improve the detection sensitivity.
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 °C, −20 °C, and −75 °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 Viikinmäki 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 °C was observed on the first 28 days, while no decay was visible within 58 days at −20 °C or −75 °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.
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•Wastewater sample aliquots during prolonged storage were investigated.•SARS-CoV-2 RNA was detected for the first time in wastewater in Helsinki, Finland.•SARS-CoV-2 RNA copy numbers were stable in cold temperatures over one to three months.•SARS-CoV-2 RNA copy number declined over 28 days at 4 °C but not at −20 °C or −75 °C.•The inclusion of particulate matter should be considered in the analysis of SARS-CoV RNA.
•Wastewater-based surveillance was able to reveal trends in COVID-19 incidence.•Forty COVID-19 cases/100 000 yielded 95% SARS-CoV-2 detection rate in wastewater.•Over 100 cases/100 000 guarantied 50% ...SARS-CoV-2 quantification rate.•Infectious SARS-CoV-2 was not detected in wastewater.
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.
Over the 10-month surveillance period with an extensive total number of samples, the detection rate of SARS-CoV-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 COVID-19 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.
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.
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Many countries including EU Member States (EUMS) and the United States (U.S.) regularly monitor the microbial quality of bathing water to protect public health. This study comprehensively evaluates ...the EU bathing water directive (BWD) and the U.S. recreational water quality criteria (RWQC) as regulatory frameworks for monitoring microbial quality of bathing water. The major differences between these two regulatory frameworks are the provision of bathing water profiles, classification of bathing sites based on the pollution level, variations in the sampling frequency, accepted probable illness risk, epidemiological studies conducted during the development of guideline values, and monitoring methods. There are also similarities between the two approaches given that both enumerate viable fecal indicator bacteria (FIB) as an index of the potential risk to human health in bathing water and accept such risk up to a certain level. However, enumeration of FIB using methods outlined within these current regulatory frameworks does not consider the source of contamination nor variation in inactivation rates of enteric microbes in different ecological contexts, which is dependent on factors such as temperature, solar radiation, and salinity in various climatic regions within their geographical areas. A comprehensive "tool-box approach", i.e., coupling of FIB and viral pathogen indicators with microbial source tracking for regulatory purposes, offers potential for delivering improved understanding to better protect the health of bathers.
In this study, we evaluated the use of RT-qPCR assays targeting rRNA gene sequences for the detection of fecal bacteria in water samples. We challenged the RT-qPCR assays against RNA extracted from ...sewage effluent (n = 14), surface water (n = 30), and treated source water (n = 15) samples. Additionally, we applied the same assays using DNA as the qPCR template. The targeted fecal bacteria were present in most of the samples tested, although in several cases, the detection frequency increased when RNA was used as the template. For example, the majority of samples that tested positive for E. coli and Campylobacter spp. in surface waters, and for human-specific Bacteroidales, E. coli, and Enterococcus spp. in treated source waters were only detected when rRNA was used as the original template. The difference in detection frequency using rRNA or rDNA (rRNA gene) was sample- and assay-dependent, suggesting that the abundance of active and nonactive populations differed between samples. Statistical analyses for each population exhibiting multiple quantifiable results showed that the rRNA copy numbers were significantly higher than the rDNA counterparts (p < 0.05). Moreover, the detection frequency of rRNA-based assays were in better agreement with the culture-based results of E. coli, intestinal enterococci, and thermotolerant Campylobacter spp. in surface waters than that of rDNA-based assays, suggesting that rRNA signals were associated to active bacterial populations. Our data show that using rRNA-based approaches significantly increases detection sensitivity for common fecal bacteria in environmental waters. These findings have important implications for microbial water quality monitoring and public health risk assessments.
Infectious diseases caused by antibiotic-resistant bacterial (ARB) pathogens are a serious threat to human and animal health. The active surveillance of ARB using an integrated one-health approach ...can help to reduce the emergence and spread of ARB, reduce the associated economic impact, and guide antimicrobial stewardship programs. Wastewater surveillance (WWS) of ARB provides composite samples for a total population, with easy access to the mixed community microbiome. This concept is emerging rapidly, but the clinical utility, sensitivity, and uniformity of WWS of ARB remain poorly understood especially in relation to clinical evidence in sewershed communities. Here, we systematically searched the literature to identify studies that have compared findings from WWS of ARB and antibiotic resistance genes (ARG) with clinical evidence in parallel, thereby evaluating how likely WWS of ARB and ARG can relate to the clinical cases in communities. Initially, 2,235 articles were obtained using the primary search keywords, and 1,219 articles remained after de-duplication. Among these, 35 articles fulfilled the search criteria, and an additional 13 relevant articles were included by searching references in the primary literature. Among the 48 included papers, 34 studies used a culture-based method, followed by 11 metagenomics, and three PCR-based methods. A total of 28 out of 48 included studies were conducted at the single sewershed level, eight studies involved several countries, seven studies were conducted at national or regional scales, and five at hospital levels. Our review revealed that the performance of WWS of ARB pathogens has been evaluated more frequently for
spp., and other members of the family
, but has not been uniformly tested for all ARB pathogens. Many wastewater-based ARB studies comparing the findings with clinical evidence were conducted to evaluate the public health risk but not to relate with clinical evidence and to evaluate the performance of WWS of ARB. Indeed, relating WWS of ARB with clinical evidence in a sewershed is not straightforward, as the source of ARB in wastewater cannot be only from symptomatic human individuals but can also be from asymptomatic carriers as well as from animal sources. Further, the varying fates of each bacterial species and ARG within the sewerage make the aim of connecting WWS of ARB with clinical evidence more complicated. Therefore, future studies evaluating the performance of many AMR pathogens and their genes for WWS one by one can make the process simpler and the interpretation of results easier.
Despite its popularity for water activities, such as swimming, surfing, fishing, and rafting, inland and coastal bathing areas occasionally experience outbreaks of highly pathogenic avian influenza ...virus (HPAI), including A(H5N1) clade 2.3.4.4b. Asymptomatic infections and symptomatic outbreaks often impact many aquatic birds, which increase chances of spill-over events to mammals and pose concerns for public health. This review examined the existing literature to assess avian influenza virus (AIV) transmission risks to beachgoers and the general population. A comprehensive understanding of factors governing such crossing of the AIV host range is currently lacking. There is limited knowledge on key factors affecting risk, such as species-specific interactions with host cells (including binding, entry, and replication via viral proteins hemagglutinin, neuraminidase, nucleoprotein, and polymerase basic protein 2), overcoming host restrictions, and innate immune response. AIV efficiently transmits between birds and to some extent between marine scavenger mammals in aquatic environments via consumption of infected birds. However, the current literature lacks evidence of zoonotic AIV transmission via contact with the aquatic environment or consumption of contaminated water. The zoonotic transmission risk of the circulating A(H5N1) clade 2.3.4.4b virus to the general population and beachgoers is currently low. Nevertheless, it is recommended to avoid direct contact with sick or dead birds and to refrain from bathing in locations where mass bird mortalities are reported. Increasing reports of AIVs spilling over to non-human mammals have raised valid concerns about possible virus mutations that lead to crossing the species barrier and subsequent risk of human infections and outbreaks.
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•The likelihood of avian influenza spreading to beachgoers is minimal.•Unprotected contact with sick and dead animals should be avoided.•Influenza viruses adapt to environments via mutations and reassortment.•Spill-over events raise genomic mutation risk, boosting pandemic potential.•Genomic surveillance assesses pandemic potential in avian flu viruses.
Knowledge of the decay characteristics of health-related microbes in surface waters is important for modeling the transportation of waterborne pathogens and for assessing their public health risks. ...Although water temperature and light exposure are major factors determining the decay characteristics of enteric microbes in surface waters, such effects have not been well studied in subarctic surface waters. This study comprehensively evaluated the effect of temperature and light on the decay characteristics of health-related microbes Escherichia coli, enterococci, microbial source tracking markers (GenBac3 & HF183 assays), coliphages (F-specific and somatic), noroviruses GII and Legionella spp. under simulated subarctic river water conditions. The experiments were conducted in four different laboratory settings (4 °C/dark, 15 °C/dark, 15 °C/light, and 22 °C/light). The T90 values (time required for a 90 % reduction in the population of a target) of all targets were higher under cold and dark (2.6–51.3 days depending upon targets) than under warm and light conditions (0.6–3.5 days). Under 4 °C/dark (simulated winter) water conditions, F-specific coliphages had 27.2 times higher, and coliform bacteria had 3.3 times higher T90 value than under 22 °C/light (simulated summer) water conditions. Bacterial molecular markers also displayed high variation in T90 values, with the greatest difference between 4 °C/dark and 22 °C/light recorded for HF183 DNA (20.6 times) and the lowest difference for EC23S857 RNA (6.6 times). E. coli, intestinal enterococci, and somatic coliphages were relatively more sensitive to light than water temperature, but F-specific coliphages, norovirus, and all bacterial rDNA and rRNA markers were relatively more sensitive to temperature than light exposure. Due to the slow microbial decay in winter under subarctic conditions, the microbial quality of river water might remain low for a long time after a sewage spill. This increased risk associated with fecal pollution during winter may deserve more attention, especially when river waters are used for drinking water production.
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•Coliphages decayed 22–27 times, and E. coli 4.8 times slower in 4 °C than 22 °C.•E. coli, enterococci, and som. coliphages were more sensitive to light than temperature.•Bacterial DNA/RNA, and Legionella were more sensitive to temperature than light.•Norovirus GII and F-specific coliphages were also more sensitive to temperature.•Indication power of fecal indicators can vary between seasons of the year.
Information on the co-occurrence of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) among bacterial communities in drinking water distribution systems (DWDSs) is scarce. This ...study characterized ARGs and MRGs in five well-maintained DWDSs in Finland. The studied DWDSs had different raw water sources and treatment methods. Two of the waterworks employed artificially recharged groundwater (ARGW) and used no disinfection in the treatment process. The other three waterworks (two surface and one groundwater source) used UV light and chlorine during the treatment process. Ten bulk water samples (two from each DWDS) were collected, and environmental DNA was extracted and then sequenced using the Illumina HiSeq platform for high-throughput shotgun metagenome sequencing. A total of 430 ARGs were characterized among all samples with the highest diversity of ARGs identified from samples collected from non-disinfected DWDSs. Furthermore, non-disinfected DWDSs contained the highest diversity of bacterial communities. However, samples from DWDSs using disinfectants contained over double the ratio of ARG reads to 16S rRNA gene reads and most of the MRG (namely mercury and arsenic resistance genes). The total reads and types of ARGs conferring genes associated with antibiotic groups namely multidrug resistance, and bacitracin, beta-lactam, and aminoglycoside and mercury resistance genes increased in waterworks treating surface water with disinfection. The findings of this study contribute toward a comprehensive understanding of ARGs and MRGs in DWDSs. The occurrence of bacteria carrying antibiotic or metal resistance genes in drinking water causes direct exposure to people, and thus, more systematic investigation is needed to decipher the potential effect of these resistomes on human health.
Drinking water outbreaks occur worldwide and may be caused by several factors, including raw water contamination, treatment deficiencies, and distribution network failure. This study describes two ...drinking water outbreaks in Finland in 2016 (outbreak I) and 2018 (outbreak II). Both outbreaks caused approximately 450 illness cases and were due to drinking water pipe breakage and subsequent wastewater intrusion into the distribution system. In both outbreaks, the sapovirus was found in patient samples as the main causative agent. In addition, adenoviruses and
(outbreak I), and noroviruses, astroviruses, enterotoxigenic and enterohemorragic
(ETEC and EHEC, respectively) and
(outbreak II) were detected in patient samples. Water samples were analyzed for the selected pathogens largely based on the results of patient samples. In addition, traditional fecal indicator bacteria and host-specific microbial source tracking (MST) markers (GenBac3 and HF183) were analyzed from water. In drinking water, sapovirus and enteropathogenic
(EPEC) were found in outbreak II. The MST markers proved useful in the detection of contamination and to ensure the success of contaminant removal from the water distribution system. As mitigation actions, boil water advisory, alternative drinking water sources and chlorination were organized to restrict the outbreaks and to clean the contaminated distribution network. This study highlights the emerging role of sapoviruses as a waterborne pathogen and warrants the need for testing of multiple viruses during outbreak investigation.
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