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Due the ability of plastics to act as a substrate to develop biofilms and their persistence in the environment, plastics have been claimed as vectors of bacterial dispersal. We evaluated the presence of faecal and marine bacteria adhering to the biofilm developed on marine plastics and the abundance of antibiotic resistance genes (ARGs). Thus, we sampled coastal waters around the mouths of the Besòs and Llobregat rivers. Both areas are strongly impacted by human faecal pollution (either from rivers and submarine sewage outfalls). Samples of water, sediments and plastics from surface waters and sediments were collected during two campaigns in summer and autumn 2021. The presence of the faecal indicators E. coli, enterococci and crAssphage, 3 ARGs, potential pathogens lik Pseudomonas spp. and Vibrio spp. and other marine bacteria was analysed by culture and/or molecular methods. Results indicate that plastics are covered by a bacterial biofilm mainly of marine bacteria (4.9·10⁴ to 8.7·10⁶ gc mm-2) including Vibrio species. Low concentration of viable E. coli and Enterococci (42% and 67% of the samples) was detected on floating plastics. The ARGs sulI, blaTEM and tetW were detected in 67 - 88% of the surface plastic samples and 29 - 57% of the sediment plastics with a concentration of up to 6.7·102 gc·mm-2. These results suggest that the plastic debris may have come from wastewater or been colonised in environments with faecal contamination. The presence of faecal indicators in sediment plastics was null or low. Therefore, although in low concentrations, faecal bacteria and species of Pseudomonas and Vibrio were identified in marine plastics, suggesting that plastic pollution may be a potential reservoir of human pathogens and ARGs. Considering the abundance of plastic in aquatic environments (expected to increase in the future), its potential risk to public health must be assessed. Also see: https://micro2022.sciencesconf.org/425977/document, In MICRO 2022, Online Atlas Edition: Plastic Pollution from MACRO to nano

Microorganisms can easily colonize the surface of plastics in the aquatic environments providing a novel means of dispersion. It has been already observed that potential pathogens can be attached becoming a reservoir for, for example, faecal bacteria, such as Escherichia coli, or for antibiotic resistance genes (ARGs). The objective of this study was to evaluate the colonization and persistence of E. coli in the biofilm adhered to plastic pellets and the presence of ARGs. We implemented five mesocosms in 25 L of seawater with a concentration of 106 marine bacteria/ml and with a mixture of plastic pellets (80% polyethylene and 20% polypropylene) between 2-5 mm obtained from a beach. We added a concentration of 104 cfu/ml of a mixture of 3 environmental strains of E. coli. The abundance of marine bacteria and E. coli in the water and pellets was analyzed using cultivable methods and molecular techniques (qPCR), as well as epifluorescence and scanning microscopy (SEM). The results show that the biofilm was mainly composed of marine bacteria (7.29·105 gc/mm2), however E. coli can attach to it during the first days reaching a maximum concentration of 2.1·103 CFU/mm2. It can persist with cultivable methods up to 12 days, meanwhile the DNA was detected at least up to 26 days (9.97·100 gc/mm2). SEM and fluorescence images corroborated the colonization of MPs. ARGs were also detected in the biofilm mainly sul1 and blaTEM. A relationship between blaTEM and E. coli was identified. The results suggest a pattern of persistence of viable E. coli in pellets for 12 days, during which they can be transported with the currents. Thus, anticipating the increase in plastics in the marine environment, its potential health risk should be assessed. Also see: https://micro2022.sciencesconf.org/427164/document, In MICRO 2022, Online Atlas Edition: Plastic Pollution from MACRO to nano

Pepper mild mottle virus (PMMoV), a plant pathogenic virus belonging to the family Virgoviridae, has been proposed as a potential viral indicator for human faecal pollution in aquatic environments. The present study investigated the occurrence, amount and diversity of PMMoV in water environments in Italy. A total of 254 water samples, collected between 2017 and 2019 from different types of water, were analysed. In detail, 92 raw sewage, 32 treated sewage, 16 river samples, 9 estuarine waters, 20 bathing waters, 67 groundwater samples and 18 drinking waters were tested. PMMoV was detected in 79% and 75% of untreated and treated sewage samples, respectively, 75% of river samples, 67% and 25% of estuarine and bathing waters and 13% of groundwater samples. No positive was detected in drinking water. The geometric mean of viral concentrations (genome copies/L) was ranked as follows: raw sewage (2.2 × 106) > treated sewage (2.9 × 105) > river waters (6.1 × 102) > estuarine waters (4.8 × 102) > bathing waters (8.5 × 101) > groundwater (5.9 × 101). A statistically significant variation of viral loads could be observed between raw and treated sewage and between these and all the other water matrices. PMMoV occurrence and viral loads did not display seasonal variation in raw sewage nor correlation with faecal indicator bacteria in marine waters and groundwater. This study represents the first report on the occurrence and quantification PMMoV in different water environments in Italy. Further studies are required to evaluate the suitability of PMMoV as a viral indicator for human faecal pollution and for viral pathogens in waters.

The use of faecal coliforms as indicators is the traditional approach of testing water quality. Unfortunately, for a comprehensive water quality analysis, there is an increasing body of evidence that demonstrates coliforms as insufficient indicators for water quality assessment. Therefore, during the last two decades, alternative water testing approaches such as the use of coliphage as well as cholesterol detection have gained popularity. In the present study, we evaluated and compared the reliability of data from three different indicators that included coliforms (Streptococcus), coliphage and cholesterol. Four sites were chosen for sample collection and these included one site from Haart river (HR1) and three sites from Barberspan (BP1, 2 and 3) in the North West Province of South Africa. Samples were collected during winter and summer seasons. Collected samples were subjected to different analyses for detection of coliphage, coliforms and cholesterol. Faecal indicator bacteria were detected at all sites and in some cases were relatively high (HR1: 287 cfu/100 mL faecal coliform and 228.6 cfu/100 mL faecal streptococci; BP1: 1,730 cfu/100 mL Escherichia coli). The HR1 site consistently had the highest levels of bacterial faecal indicators of the four sampling sites. Most notably, faecal streptococci were detected in higher numbers than any other bacterial indicator. A significant finding was the general higher levels of faecal indicator markers at the BP3. Based on the outcome of this study, a combination of these indicators offers a comprehensive and promising approach for monitoring water quality. HIGHLIGHTS This manuscript provides historical data for Barberspan, a Ramsar site.; It shows the strengths of using multiple faecal indicators together at one site.; It shows the advantages of using Streptococci, coliphages and cholesterol as faecal pollution indicators.; It informs the water management strategy of Baberspan.; It informs on the water quality of Barberspan.

Lake Victoria is the primary source of water for millions of people in the Sub-Saharan Africa region. In recent years, population development around the lake has resulted in compromised sanitation standards resulting in increased faecal pollution of the lake. Consequently, this condition has increased the chances of waterborne enteric viruses, such as adenoviruses' circulation in the community. Adenoviruses can affect health in both humans and animals by causing a myriad of diseases including the gastrointestinal infections. The study aimed to detect contamination of the lake water with pathogenic human adenoviruses along Homa Bay town, Homa Bay County, Kenya. To examine the presence of adenoviral genome, we collected a total of 216 (monthly n = 36) water samples from six different locations marked by high levels of anthropogenic activities along the shoreline. Molecular amplification technique using the nested PCR procedure was used to detect the genomes from the water samples. Human adenoviruses were detected in 11 samples (5.09%). Statistical analyses indicated a significant correlation between adenovirus presence and the approximate distance from pit latrines and sewage treatment works at the area. The findings indicate that faecal contamination of the lake waters originated from the point sources. The findings also suggest a possibility of elevated levels of faecal pollution in different surface waters within the lake basin. The findings indicate that some of the enteric viruses circulating in the local community are human adenovirus type 40, and 41. The data may provide a basis for recognizing the need to prioritize environmental monitoring for enteric virus contamination on an on-going basis.

Consumption of bivalve molluscan shellfish (BMS) grown in faecally contaminated waters is a public health risk. While faecal contamination in BMS and water can originate from multiple sources (i.e. human, livestock, wildlife and crop production), accurate identification of the source(s) is important to establish an appropriate, cost-effective plan to minimise the potential public health risks. Microbial source tracking (MST) targets (genes) are more source-specific than faecal indicator bacteria. Application of MST methods could eliminate the requirement of multiple testing regimes for bacterial and viral pathogens in BMS. This approach, therefore, can assist to develop an accurate, reliable faecal pollution management plan that reduces the cost for BMS farmers and public health risks associated with BMS consumption. However, validation of MST targets and their recovery from BMS and correlation with other bioaccumulated pathogens in BMS is required before its field application.

Sewage pollution leads to the contamination of bivalve shellfish by pathogenic microorganisms. Bacterial indicators support the management of risks associated with the consumption of shellfish; however, they often fail to indicate adequately the potential hazard to human health posed by certain human enteric viruses. Bacteriophages have been proposed as alternative indicators that may more effectively predict the presence of enteric viral pathogens. This study explored the relationships between bacterial indicators (Escherichia coli (E. coli), faecal coliforms (FC) and intestinal enterococci (IE)), phages (somatic (SOMPH), F-specific RNA (F + PH) and human-specific Bacteroides GB-124 phages (GB124PH)) and Norovirus (NoV) (GI/GII) in mussels (Mytilus edulis) and their overlying waters. The bioaccumulation of these indicators and Norovirus in shellfish matrices (e.g., flesh, digestive gland) was investigated bimonthly over a 12-month period in an English estuary. The findings revealed a marked seasonality in the distribution of all organisms, with the highest levels occurring during the autumn/winter months. The levels of all phages in shellfish and their overlying waters correlated better with the levels of Norovirus than with those of bacterial indicators. Somatic coliphages were the indicator that exhibited the strongest correlations with NoV (rho = 0.929). This study suggests that relatively low-cost culture-based phage enumeration appears to offer a more accurate indication of the likely presence of Norovirus in mussels than traditional bacterial indicators.

The presence of SARS-CoV-2 in untreated sewage has been confirmed in many countries but its incidence and infection risk in contaminated waters is poorly understood. The River Thames in the UK receives untreated sewage from 57 Combined Sewer Overflows (CSOs), with many discharging dozens of times per year. This study investigated if such discharges provide a pathway for environmental transmission of SARS-CoV-2. Samples of wastewater, surface water, and sediment collected close to six CSOs on the River Thames were assayed over eight months for SARS-CoV-2 RNA and infectious virus. Bivalves were also sampled as an indicator species of viral bioaccumulation. Sediment and water samples from the Danube and Sava rivers in Serbia, where raw sewage is also discharged in high volumes, were assayed as a positive control. No evidence of SARS-CoV-2 RNA or infectious virus was found in UK samples, in contrast to RNA positive samples from Serbia. Furthermore, this study shows that infectious SARS-CoV-2 inoculum is stable in Thames water and sediment for

Wastewater-based epidemiology (WBE) surveillance of COVID-19 and other future outbreaks is a challenge for developing countries as most households are not connected to a sewerage system. In December 2019, SARS-CoV-2 RNA was detected in the Danube River at a site severely affected by wastewaters from Belgrade. Rivers are much more complex systems than wastewater systems, and efforts are needed to address all the factors influencing the adoption of WBE as an alternative to targeting raw wastewater. Our objective was to provide a more detailed insight into the potential of SARS-CoV-2 surveillance in Serbian surface waters for epidemiological purposes. Water samples were collected at 12 sites along the Sava and Danube rivers in Belgrade during the fourth COVID-19 wave in Serbia that started in late February 2021. RNA was concentrated using Amicon Ultra-15 centrifugal filters and quantified using RT-qPCR with primer sets targeting nucleocapsid (N1 and N2) and envelope (E) protein genes. Microbiological (faecal indicator bacteria and human and animal genetic faecal source tracking markers), epidemiological, physicochemical and hydromorphological parameters were analysed in parallel. From 44 samples, SARS-CoV-2 RNA was detected in 31, but only at 4 concentrations above the level of quantification (ranging from 8.47×103 to 2.07×104 gc/L). The results indicated that surveillance of SARS-CoV-2 RNA in surface waters as ultimate recipients could be used as an epidemiological early-warning tool in countries lacking wastewater treatment and proper sewerage infrastructure. The performance of the applied approach, including advanced sampling site characterization to trace and identify sites with significant raw sewage influence from human populations, could be further improved by adaptation of the methodology for processing higher volumes of samples and enrichment factors, which should provide the quantitative instead of qualitative data needed for WBE.

What do we know about the Adriatic Sea and the state of its health? Are purification plants enriching it with nutrients or with chemical and microbiological pollutants? Are we triggering any gene alterations with this biomass? Do we have laws specific and focused enough to guarantee the protection of the marine environment considering its local peculiarity? Waterisafundamentalcomponentofdailylife, fromrecrea tionaltodomesticneeds, as wellasforindustrialandagriculturaluse. Sustainablewatermanagementisapopulartopic in a multiplicity of scientific, social and political programs around the world. Nevertheless, the healthiness and hygienic quality of hydric resources are increasingly threatened, and ultimately, their availability is affected.

Na istarskom se poluotoku nalazi više od tisuću kraških lokvi i nekoliko močvara. Zajedno s četiri male rijeke i akumulacijskim bazenom, one čine jedine površinske slatke vode u regiji. Ta malena vodna tijela općenito su umjetna ili djelomično modificirana od strane ljudi koji su se na njih oslanjali kroz povijest, uglavnom za poljoprivredne i stočarske djelatnosti. Lokve i močvare u prošlosti su također predstavljale problem za ljudsko zdravlje pogodujući razmnožavanju komaraca i difuziji malarije te su stoga početkom 20. stoljeća masovno tretirane insekticidima ili umjetno povezane s morem. U današnje vrijeme lokve su često napuštene i sklone degradaciji zbog neodržavanja i rasta vegetacije. Štoviše, u njima se često nakupljaju poljoprivredne i urbane vode, a s time i mikrobiološka i kemijska onečišćenja. Površinska vodna tijela na području Sredozemlja ključna su za opstanak okolne kopnene flore i faune. Uz to, djeluju kao žarišta vodene biološke raznolikosti koja pružaju jedino utočište raznim endemskim vrstama i veoma su bitna za ptice selice koje imaju ključnu ulogu u umrežavanju ekosustava te se tako doprinosi očuvanju biološke raznolikosti južne Europe. Cilj ovoga istraživanja bio je ispitati karakteristike staništa, mikrobiološke i sanitarne uvjete te tragove anorganskih zagađivala u vodama pet lokvi i jedne bočate močvare na jugozapadnom dijelu istarskog poluotoka. Ovim su se istraživanjem pružili prvi podaci o statusu tih osjetljivih i dragocjenih okoliša, koji će biti temelj za provedbu njihovih budućih programa očuvanja i održivog upravljanja. There are more than a thousand karst ponds and several wetlands on the Istrian peninsula. Together with four small rivers and a reservoir, they form the only surface freshwater in the region. These small water bodies are generally artificial or partially modified by people who have relied on them throughout history, mainly for agricultural and livestock activities. Puddles and wetlands have also been a problem for human health in the past, favoring mosquito breeding and malaria diffusion, and were therefore massively treated with insecticides or artificially linked to the sea in the early 20th century. Nowadays, ponds are often abandoned and prone to degradation due to lack of maintenance and vegetation growth. Moreover, they often accumulate agricultural and urban water, and with it microbiological and chemical contaminants. Surface water bodies in the Mediterranean area are crucial for the survival of the surrounding terrestrial flora and fauna. In addition, they act as foci of aquatic biodiversity that provide the only refuge for various endemic species and are very important for migratory birds that play a key role in ecosystem networking and thus contribute to the conservation of southern European biodiversity. The aim of this study was to examine habitat characteristics, microbiological and sanitary conditions and traces of inorganic pollutants in the waters of five ponds and one brackish wetland in the southwestern part of the Istrian peninsula. This research provided the first data on the status of these sensitive and valuable environments, which will be the basis for the implementation of their future conservation and sustainable management programs.

Background Microbial water quality serves to indicate health risks associated with the consumption of contaminated water. Nevertheless, little is known about the microbiological characteristics of water in Lake Bunyonyi. This study was therefore undertaken to examine the spatial and temporal variations of faecal indicator bacteria (FIB) in relation to physicochemical parameters in Lake Bunyonyi. Result The FIB concentration was consistently measured during sampling months and correlated with each other showing the presumed human faecal pollution in the lake. The highest concentration values for E. coli (64.7 ± 47.3 CFU/100 mL) and enterococci (24.6 ± 32.4 CFU/100 mL were obtained in the station close to the Mugyera trading centre. On a temporal basis, the maximum values were recorded during the rainy season in October 2019 (70.7 ± 56.5 CFU/100 mL for E. coli and 38.44 ± 31.8 CFU/100 mL for enterococci. FIB did not differ significantly among the study stations (p > 0.05) but showed significant temporal variations among the months (p U = 794, p E. coli; U = 993.5, p = 0.008 for enterococci). Spearman’s rank correlation revealed that FIB concentrations were significantly positively correlated with turbidity and DO concentration levels (p E. coli and enterococci concentrations levels below USEPA threshold for recreational waters. Likewise, 98.1 and 90.7% of samples recorded E. coli and enterococci counts exceeding the UNBS, APHA, WHO and EU threshold values for drinking water. Conclusion The FIB counts show that the Lake Bunyonyi water is bacteriologically unsuitable for drinking unless it is treated since the FIB pose health risks to consumers. Besides, the water can be used for recreational purposes.

Environmental pollution is the unfavourable alteration of our surroundings, wholly or largely as a by-product of human actions that can adversely affect human health, living nature, climate, water and soil. This pollution can have natural and anthropogenic sources. In the inhabited high-mountain environment, the source of anthropogenic environmental pollution is mainly caused by residents. Indirectly, however, in areas popular with tourists, residents pollute when providing tourist services (e.g. disposal of tourist waste). In mountainous areas without local people, pollution is caused only by tourists. During tourist activities, all elements of the high-mountain natural environment (air, water, soil and landscape, including visual and acoustic landscape) may be degraded. The main factors causing this are littering, human faecal pollution, and noise or light pollution. Tourist infrastructure is also not without significance, which together with the climbing equipment can have a negative impact on the environment.

The ongoing COVID-19 pandemic increases the consumption of antimicrobial substances (ABS) due to the unavailability of approved vaccine(s). To assess the effect of imprudent consumption of ABS during the COVID-19 pandemic, we compare the 2020 prevalence of antidrug resistance (ADR) of Escherichia coli (E. coli) with a similar survey carried out in 2018 in Ahmedabad, India using SARS-CoV-2 gene detection as a marker of ABS usage. We found a significant ADR increase for in 2020 compared to 2018 in ambient water bodies, harbouring a higher incidence of ADR E.Coli towards non-fluoroquinolone drugs. Effective SARS-CoV-2 genome copies were found to be associated with the ADR prevalence. The prevalence of ADR depends on the efficiency of WWTPs (Wastewater Treatment Plants) and the catchment area in its vicinity. In year 2018 study, prevalence of ADR was discretely distributed, and the maximum ADR prevalence recorded was ~60%; against the current homogenous ADR increase, and up to 85% of maximum ADR among the incubated E.coli isolated from the river (Sabarmati) and lake (Chandola and Kankaria) samples. Furthermore, wastewater treatment plants showed less increase in comparison to the ambient waters, which eventually imply that although SARS-CoV-2 genes and faecal pollution may be diluted in the ambient waters, as indicated by low Ct-value and E.coli count, the danger of related aftermath like ADR increase cannot be nullified. Also, Non-fluoroquinolone drugs exhibited overall more resistance than the quinolone drugs. Overall, this is probably the first ever study that traces the COVID-19 pandemic imprints on the prevalence of antidrug resistance (ADR) through wastewater surveillance and hints at monitoring escalation of other environmental health parameters. This study will make public and policyholders concerned about the optimum use of antibiotics during any kind of treatment., This work is funded by Kiran C Patel Centre for Sustainable Development at IIT Gandhinagar, UNICEF, Gujarat and UKIERI. We also acknowledge the help received from Dr. Vaibhav Srivastava, Dr. Arbind K Patel, and other GBRC staffs who contributed towards sample and data analyses.

Agricultural impacts on surface waters have been an intensive source of contamination on freshwaters worldwide. Faecal contamination is one of the most under regulated and poorly understood pollutants within Irelands surface waters, despite is widely recorded ability to cause harm to both animals and the environment. Creating novel solutions to help rectify the negative effects of poor agricultural management is necessary for protecting the future health if Irelands waters. Although current policy under the European Union (EU) provides guidelines and regulations, Ireland has still failed to contain its faecal contaminant issue. The solution to this dilemma may lie in the origins of this pollutant; current research demonstrates Irelands agriculturally dominated catchments could be suffering from point source dissemination of contaminants via input through farmyards and direct cattle access to streams. This dissemination model differs from the current non point source model that the EU and Irish policies implicitly endorse. This study’s objectives were as follows; to quantify the ability of headwater drainage channels receiving direct farmyard effluent to attenuate faecal indicator organisms (thermotolerant coliforms) within the water column and within benthic sediments, over their length; and to determine the distribution, concentration, and origin of thermotolerant coliforms (TTC’s) at intensive spatial scales within in a small agriculturally dominated catchment in South West Ireland. Utilizing novel colonization substrata, results demonstrated no trends of attenuation within headwater drainage areas; subsequent data provided TTC concentration and distribution within the larger drainage catchment upon deposition from the previously studied channels. Results from catchment wide analysis demonstrated a definitive connection to faecal contamination of point source origin from farmyards as well as direct deposition from cattle access to streams. The future of faecal contamination management within Irelands waters lies within the exploration of novel and established treatment methodology in order to create effective overarching policy changes.

We assessed antimicrobial resistance (AMR) potential to seven major classes of antibiotics in Central Thailand’s coastal aquaculture region using high-throughput qPCR targeting 295 antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs). In parallel, we used MinION next generation sequencing (NGS) of 16S rRNA gene amplicons, qPCR of faecal pollution makers, and conventional plate count methods for the comprehensive characterization of environmental microbiomes. We tested the hypothesis that aquaculture increases environmental AMR by comparing ARGs and MGEs in water and sediment samples from five aquaculture sites and their associated canals. There was no evidence from the ARG and MGE data that aquaculture is a major driver of environmental AMR in Central Thailand. Instead, the highest relative prevalence of resistance traits was found in Hua Krabue canal water influenced by urban pollution from Bangkok at the inland edge of the coastal aquaculture region. The sum of ARGs and MGEs, relative to 16S rRNA genes used as markers for overall bacterial abundance, was between 0.495 ± 0.011 and 0.498 ± 0.013 in Hua Krabue canal water, compared with at most 0.132 ± 0.005 in all the other environmental samples. Corresponding patterns were observed for most faecal pollution markers, which were also elevated in Hua Krabue canal water.

Clean water is a precious resource, and policies/programmes are implemented worldwide to protect and/or improve water quality. Faecal pollution can be a key contributor to water quality decline causing eutrophication through nutrient enrichment and pathogenic contamination. The robust sourcing of faecal pollutants is important to be able to target the appropriate sector and to engage managers. Biomarker technology has the potential for source confirmation, by using, for example the biomarker suite of steroids. Steroids have been used in the differentiation of human and animal faeces; however, there is no unequivocal extraction technique. Some of the methods used include (i) Soxhlet extraction, (ii) Bligh and Dyer (BD) extraction, and (iii) accelerated solvent extraction (ASE). The less costly and time intensive technique of ASE is particularly attractive, but a current research gap concerns further comparisons regarding ASE lipid extraction from soils/slurries compared with the more traditional Soxhlet and BD extractions. Accordingly, a randomised complete block experiment was implemented to assess differences between the three extraction methods, differences between the different sample types, and the interactions between these two factors. Following GC-MS, it was found that there was no significant difference between the results of the steroid extraction methods, regardless of the type of sample used, for the quantity of each steroid extracted. It was concluded that ASE could be used confidently instead of the more established steroid extraction methods, thereby delivering time and cost savings.

Characterising faecal indicator organism (FIO) survival in the environment is important for informing land management and minimising public health risk to downstream water users. However, key gaps in knowledge include understanding how wildlife contribute to catchment-wide FIO sources and how FIO survival is affected by low environmental temperatures. The aim of this study was to quantify E. coli and intestinal enterococci die-off in dairy cow versus red deer faecal sources exposed to repeated freeze&ndash, thaw cycles under controlled laboratory conditions. Survival of FIOs in water exposed to freeze&ndash, thaw was also investigated to help interpret survival responses. Both E. coli and intestinal enterococci were capable of surviving sub-freezing conditions with the faeces from both animals able to sustain relatively high FIO concentrations, as indicated by modelling, and observations revealing persistence in excess of 11 days and in some cases confirmed beyond 22 days. Die-off responses of deer-derived FIOs in both faeces and water exposed to low temperatures provide much needed information to enable better accounting of the varied catchment sources of faecal pollution and results from this study help constrain the parameterisation of die-off coefficients to better inform more integrated modelling and decision-making for microbial water quality management.

Department of Civil Engineering, Dr. Sudhir Chandra Sur Institute of Technology and Sports Complex, 540, Dum Dum Road, Kolkata-700 074, India Ex-Faculty, Department of Botany, Bankim Sardar College, Taldi, South 24-Pargana-743 329, West Bengal, India Department of Environmental Engineering, Narula Institute of Technology, Agarpara, Kolkata-700 109, India E-mail: staadrubaid@gmail.com, sushovan.sarkar@dsec.ac.in, ngkolkata47@gmail.com, samirmukherjee1100@gmail.com Manuscript received online 09 May 2020, accepted 25 July 2020 The fungi population in water has gained attention during the three decades. In this review, article attempt has been made to show its vulnerability as numbers of fungal species are now treated as water contaminants to indicate diseases in the human body. However, the importance of study of fungi has been less focused on surface water, groundwater systems and in agricultural wetlands. Time has come to correlate the population of fungi species isolated and identified from different sources of water-related with the climate change. The number of the fungal species are isolated and identified by the researchers may or may not have common occurrence but those definitely contaminate surface water at the different sources as well as different countries of the globe. In some cases, study has been made to analyze the occurrence of yeasts and filamentous fungi in the water body to investigate their correlation with bacterial indicator and faecal pollution1 .

Characterising faecal indicator organism (FIO) survival in the environment is important for informing land management and minimising public health risk to downstream water users. However, key gaps in knowledge include understanding how wildlife contribute to catchment-wide FIO sources and how FIO survival is affected by low environmental temperatures. The aim of this study was to quantify E. coli and intestinal enterococci die-off in dairy cow versus red deer faecal sources exposed to repeated freeze–thaw cycles under controlled laboratory conditions. Survival of FIOs in water exposed to freeze–thaw was also investigated to help interpret survival responses. Both E. coli and intestinal enterococci were capable of surviving sub-freezing conditions with the faeces from both animals able to sustain relatively high FIO concentrations, as indicated by modelling, and observations revealing persistence in excess of 11 days and in some cases confirmed beyond 22 days. Die-off responses of deer-derived FIOs in both faeces and water exposed to low temperatures provide much needed information to enable better accounting of the varied catchment sources of faecal pollution and results from this study help constrain the parameterisation of die-off coefficients to better inform more integrated modelling and decision-making for microbial water quality management.

Colistin is one of the antibiotics of last resort for human health. However, the dissemination of the plasmid-mediated colistin resistance gene mcr-1 is of great concern globally. In the One Health framework, the environment is an important component for managing antimicrobial resistance. However, little information is available concerning the prevalence of mcr-1 in water environments. We aimed to reveal the prevalence of mcr-1 in different water environments in Hanoi, Vietnam. Quantitative PCR was applied to detect mcr-1 in four urban drainages receiving untreated domestic wastewater, three rivers, five lakes and two groundwater samples. Urban drainages contained higher concentrations of mcr-1, suggesting that urban residents carry the gene. The class 1 integron-integrase gene was identified as a good surrogate of antibiotic resistance genes including mcr-1. A significant correlation was found between the levels of mcr-1 and the human-specific cross-assembly phage, which is an indicator of human faecal pollution. These results indicated that the primary source of mcr-1 in urban water environments is human faeces, which is consistent with the fact that most domestic wastewater is untreated in Hanoi. The control of untreated wastewater is critical for alleviating the spread of mcr-1 in water environments in Vietnam.

Good quality water is odourless, colourless, and free from faecal pollution, and a satisfactory safe supply must be made available to consumers. The study assessed consumer preference and quality of sachet water sold and consumed in the Sunyani Municipality of Ghana. A cross-sectional study design was used, and data were collected using a structured questionnaire from a sample size of 500 respondents. A total of twenty (20) samples of sachet water commonly sold and consumed in the Sunyani Municipality were also collected and analyzed for bacteriological and physicochemical parameters. Out of these 20 samples, 10 samples of sachet water were collected from the production site and the other 10 samples from the market site. Results showed that respondents’ choice of sachet water was based on several indicators. While 70% (350/500) of consumers indicated that sachet water had taste, 58% (290/500) of them were not enthused when the water had colour. Using smell as an indicator, 71% of consumers have had an experience with sachet water smell being questionable. Water taste (p<0.001), colour of water (p<0.001), smell (p<0.001), and increase in cost (p<0.001) were found as determinants of consumer preference. Consumers who said the colour of water informed their decision when buying sachet water were seven times more likely to have a preference for a particular brand (OR 7.057, CI: 4.474–11.133). Those who checked for expiry dates when buying water (OR 4.871, CI: 3.110–7.628) and those who looked out for Food and Drugs Authority stamp (OR 4.433, CI: 2.806–7.003) were both four times more likely to have a preference for a particular brand. Water quality tests also indicated that 30% of all water samples collected from the production site were contaminated with total coliforms and 50% of sachet water samples collected from the markets were also contaminated with coliforms. The same brands of sachet water that contained total coliforms from the production site were the same brands that contained total coliforms selected from the market. From this study, only total coliforms other than Escherichia coli were detected in the water samples analyzed. It was observed that the evaluated physicochemical parameters of the water analyzed were within the accepted WHO limits. It is recommended that consumers be educated about the indicators to look out for when buying sachet water and, also, that regulatory bodies be empowered to ban the sale of unwholesome brands of sachet water on the market.

Waterborne disease outbreaks associated with recreational waters continue to be reported around the world despite existing microbiological water quality monitoring frameworks. Most regulations resort to the use of culture-based enumeration of faecal indicator bacteria such as Escherichia coli to protect bathers from gastrointestinal illness risks. However, the long sample-to-result time of standard culture-based assays (minimum 18–24 h) and infrequent regulatory sampling (weekly or less) do not enable detection of episodic water quality impairments and associated public health risks. The objective of this study was to assess the suitability of an autonomous online technology measuring β-D-glucuronidase (GLUC) activity for near real-time monitoring of microbiological water quality in recreational waters and for the resulting beach management decisions. GLUC activity and E. coli concentrations were monitored at three freshwater sites in Quebec, Canada (sites Qc1-3) and one site in New Zealand (site NZ) between 2016 and 2018. We found site-dependent linear relationships between GLUC activity and E. coli concentrations and using confusion matrices, we developed site-specific GLUC activity beach action values (BAVs) matching the regulatory E. coli BAVs. Using the regulatory E. coli BAV as the gold standard, rates of false alarms (unnecessary beach advisories using GLUC activity BAV) and failures to act (failure to trigger advisories using GLUC activity) ranged between 0 and 32% and between 3 and 10%, respectively, which is comparable to the rates reported in other studies using qPCR-defined BAVs. However, a major benefit of the autonomous enzymatic technology is the real-time reporting of threshold exceedances, while temporal trends in GLUC activity can assist in understanding the underlying dynamics of faecal pollution and potential health risks. Our study is the first to describe the applicability of online near real-time monitoring of microbiological water quality as a tool for improved beach management and public health protection., SCOPUS: ar.j, info:eu-repo/semantics/published

EC Regulation 854/2004 requires the classification of bivalve mollusc harvesting areas according to the faecal pollution status of sites. It has been reported that determination of Escherichia coli in bivalve shellfish is a poor predictor of norovirus (NoV) contamination in individual samples. We explore the correlation of shellfish E. coli data with norovirus presence using data from studies across 88 UK sites (1,184 paired samples). We investigate whether current E. coli legislative standards could be refined to reduce NoV infection risk. A significant relationship between E. coli and NoV was found in the winter months (October to February) using data from sites with at least 10 data pairs (51 sites). We found that the ratio of arithmetic means (log10E. coli to log10 NoV) at these sites ranged from 0.6 to 1.4. The lower ratios (towards 0.6) might typically indicate situations where the contribution from UV disinfected sewage discharges was more significant. Conversely, higher ratios (towards 1.4) might indicate a prevalence of animal sources of pollution; however, this relationship did not always hold true and so further work is required to fully elucidate the factors of relevance. Reducing the current class B maximum (allowed in 10% of samples) from 46,000 E. coli per 100 g (corresponding NoV value of 75750 ± 103) to 18,000 E. coli per 100 g (corresponding NoV value of 29365 ± 69) reduces maximum levels of NoV by a factor of 2.6 to 1; reducing the upper class B limit to 100% compliance with 4,600 E. coli per 100 g (corresponding NoV value of 7403 ± 39) reduces maximum levels of NoV by a factor of 10.2 to 1. We found using the UK filtered winter dataset that a maximum of 200 NoV corresponded to a maximum of 128 ± 7 E. coli per 100 g. A maximum of 1,000 NoV corresponded to a maximum of 631 ± 14 E. coli per 100 g.

This study was carried out to examine the sanitary quality of overlying waters in an earthen pond containing African catfish (Clarias gariepinus) in both wet and dry seasons. Water samples were collected twice weekly at different times of the day (i.e., morning and afternoon) from the earthen pond in Akure, Nigeria in August, September 2016, and February, March 2017 (n=48). Levels of faecal indicator bacteria were determined using membrane filtration method and physicochemical properties (such as temperature, pH, salinity etc.) of the overlying water samples were determined using standard methods. The results revealed that the concentration of E. coli in the overlying waters ranged from 0.78 to 1.40 Log10 CFU 100 ml-1 in morning samples and 0.85 to 1.38 Log10 CFU 100 ml-1 in afternoon samples while the concentration of faecal coliforms in the overlying waters ranged from 1.30 to 1.86 Log10 CFU 100 ml-1 in morning samples and 1.26 to 1.88 Log10 CFU 100 ml-1 in afternoon samples. Faecal coliforms showed the highest prevalence in both seasons compared to E. coli and intestinal enterococci and their levels were greater in the wet periods than the dry periods. The findings from this study suggest that the sources of faecal contamination of the overlying waters in the earthen pond originate mainly from non-human sources and the adoption of microbial source tracking techniques would further elucidate the appropriate management and mitigation strategies to control faecal contamination of the overlying waters in the earthen pond especially for human health protection.

Relatively high E. coli and Faecal Streptococci (FS) numbers have been reported in the waters surrounding Blakeney Point, East Anglia, UK, an area containing significant shellfishery industries including mussels and cockles, despite the implementation of development works aimed at reducing residual contamination problems. The aim of this study was to evaluate the potential of using bacterial analyses and sterol profiling to assess the current levels and source of faecal pollution at Blakeney Point. No evidence of significant human faecal contamination of water in and around Blakeney Point could be found using either traditional microbiological analyses or sterol profile analysis. The presence of significant quantities of sitosterol, however, suggests that faecal contamination of water from birds does occur but at the concentration detected would not affect water quality. Analysis of cockles and mussels taken from the area show that negligible levels of coprostanol were present, confirming that faecal pollution was not causing any contamination issues. Apart from cholesterol, brassicasterol, an algal biomarker, as expected was dominant in shellfish flesh. The results confirm that current water treatment processes are successful in ensuring water quality at Blakeney Point and that a combination of microbial testing with sterol profile analysis confirmed that low microbial concentrations of faecal contaminants present in and around Blakeney Point most probably originate from migratory and/or resident bird species.

North-West University, Potchefstroom Campus MSc (Environmental Management with Ecological Water Requirements), North-West University, Potchefstroom Campus Population growth in developing cities is putting pressure on the wastewater treatment plants. The improper treated sewage entering the aquatic environment deteriorates the water quality of the receiving water resource. The Mooi River Catchment has been a centre of attention and a number of research studies on significant pollution sources have been undertaken. However, very little has been done about the impacts of sewage discharges into the water bodies in the Mooi River Catchment. The aim of the study was to determine the possible negative impact of pollution injection by sewage treatment plants located within the Mooi River Catchment area. The study looked at how the discharges affect the water quality of the Mooi River system in relation to the regularised discharge of treated water into the receiving water environment. This study assesses the impact of wastewater discharges from the Kokosi, Flip Human and Potchefstroom Wastewater Treatment Works, on the Loop Spruit; Wonderfontein Spruit and Mooi River, respectively. Data received from Department of Water and Sanitation (DWS) was analysed for the sites upstream and downstream of the wastewater discharges. The results were then analysed using a student’s t-test to determine if there is any significant change in water quality between the points upstream and downstream. The data analysed for physico-chemical and microbiological parameters were checked against compliance with the national and international water and wastewater guidelines and standards. Water quality results upstream and downstream of the various wastewater treatment works were evaluated and tested for significant differences between upstream and downstream. The data on the physico-chemical and the microbiological parameters such as pH, electrical conductivity, suspended solids, ammonia, nitrate, phosphate, chemical oxygen demand, faecal coliforms and E. coli of the water of the Loop Spruit, Wonderfontein Spruit and the Mooi River for the period 2015 to 2018 was received from DWS and collated for analysis. Furthermore, included for comparison was data from the period 2001 to 2002 as the reference data. The river water quality data were for samples taken downstream of the discharge points of wastewater treatment works (WWTWs) located in the three rivers. Upstream samples were also included for comparison purposes. To evaluate the quality of the receiving water, the combined data were compared against set national and international water and wastewater guidelines and standards as well as the Water Use Licences for the three wastewater works (Kokosi, Flip Human and Potchefstroom). Data revealed instances during the period July 2015 to January 2018 at which the concentrations and values of most of the physical, biochemical and microbiological quality indicators were higher than those expected for natural surface water. Comparative analysis of the data at the sampling points located downstream against their respective upstream of the discharge points into the rivers suggested there were occasions where the WWTWs discharged treated wastewater which was of poor quality into the rivers, leading to an increase in the water quality parameters such as conductivity, nitrates, ammonia, orthophosphate, suspended solids, COD, E. coli and faecal coliforms. It is possible that on these occasions, the quality of the treated wastewater from the WWTWs were non-compliant with the guidelines on Water Use Licence for authorised discharge. This could arise if there was incidences of lapse and failure to adhere to the process quality control protocols on the water treatment process at the WWTWs. Thus, the results indicate that discharging treated water from the WWTWs deteriorated the quality of water of the Mooi River and its tributaries. More concerning were the elevated levels of the pathogenic bacteria as was observed by high values in the microbiological quality parameters (total coliform (faecal) count and E. coli count) of the receiving water. Ideally, surface water should be free from any form of pathogens (E. coli = 0; Faecal less than 0), as these pose a serious health risk, ranging from diarrhoea to sudden death. The results from the t-test statistical analyses indicated that there was significant difference between the upstream and downstream water quality for the following parameters and sites: electrical conductivity at Flip Human (71.8 and 84.6 mS/m) and Potchefstroom (72 and 101 mS/m), nitrates at Kokosi (5 and 6.08 mg/l), ammonia at Kokosi (0.002 and 1.88 mg/l), Flip Human (0.006 and 0.56 mg/l) and Potchefstroom (0.01 and 0.32 mg/l), orthophosphate at Kokosi (1.079 and 2.26 mg/l) and Flip Human (0.07 and and 2.7 mg/l) and E. coli at Kokosi (23 and 928.9 cfu/100mL). Even when the discharge is regularised and planned there might be a long term effect on the self-sustainability of the aquatic ecosystems along the three rivers as well as the attaching a health risk to users, livestock and wild life. Most of the monitored parameters relevant to wastewater discharge in the receiving river system exceeded the National and international quality standards and the water use licence limits set for discharging WWTWs. Masters

The last decades have seen the development of several source tracking (ST) markers to determine the source of pollution in water, but none of them show 100% specificity and sensitivity. Thus, a combination of several markers might provide a more accurate classification. In this study Ichnaea® software was improved to generate predictive models, taking into account ST marker decay rates and dilution factors to reflect the complexity of ecosystems. A total of 106 samples from 4 sources were collected in 5 European regions and 30 faecal indicators and ST markers were evaluated, including E. coli, enterococci, clostridia, bifidobacteria, somatic coliphages, host-specific bacteria, human viruses, host mitochondrial DNA, host-specific bacteriophages and artificial sweeteners. Models based on linear discriminant analysis (LDA) able to distinguish between human and non-human faecal pollution and identify faecal pollution of several origins were developed and tested with 36 additional laboratory-made samples. Almost all the ST markers showed the potential to correctly target their host in the 5 areas, although some were equivalent and redundant. The LDA-based models developed with fresh faecal samples were able to differentiate between human and non-human pollution with 98.1% accuracy in leave-one-out cross-validation (LOOCV) when using 2 molecular human ST markers (HF183 and HMBif), whereas 3 variables resulted in 100% correct classification. With 5 variables the model correctly classified all the fresh faecal samples from 4 different sources. Ichnaea® is a machine-learning software developed to improve the classification of the faecal pollution source in water, including in complex samples. In this project the models were developed using samples from a broad geographical area, but they can be tailored to determine the source of faecal pollution for any user. The last decades have seen the development of several source tracking (ST) markers to determine the source of pollution in water, but none of them show 100% specificity and sensitivity. Thus, a combination of several markers might provide a more accurate classification. In this study Ichnaea (R) software was improved to generate predictive models, taking into account ST marker decay rates and dilution factors to reflect the complexity of ecosystems. A total of 106 samples from 4 sources were collected in 5 European regions and 30 faecal indicators and ST markers were evaluated, including E. coli, enterococci, clostridia, bifidobacteria, somatic coliphages, host-specific bacteria, human viruses, host mitochondrial DNA, host-specific bacteriophages and artificial sweeteners. Models based on linear discriminant analysis (LDA) able to distinguish between human and non-human faecal pollution and identify faecal pollution of several origins were developed and tested with 36 additional laboratory-made samples. Almost all the ST markers showed the potential to correctly target their host in the 5 areas, although some were equivalent and redundant. The LDA-based models developed with fresh faecal samples were able to differentiate between human and non-human pollution with 98.1% accuracy in leave-one-out cross-validation (LOOCV) when using 2 molecular human ST markers (HF183 and HMBif), whereas 3 variables resulted in 100% correct classification. With 5 variables the model correctly classified all the fresh faecal samples from 4 different sources. Ichnaea (R) is a machine-learning software developed to improve the classification of the faecal pollution source in water, including in complex samples. In this project the models were developed using samples from a broad geographical area, but they can be tailored to determine the source of faecal pollution for any user. (C) 2019 Elsevier Ltd. All rights reserved.

Aptamere sind kurze einzelstr��ngige Oligonukleotide die sich zu spezifischen dreidimensionalen Strukturen falten k��nnen, was ihnen erlaubt mit hoher Spezifit��t und Affinit��t an ein Zielmolek��l zu binden. In dieser Diplomarbeit wurden mehrere analytische Methoden f��r die Charakterisierung von Bakterienzellen bindenden Aptameren etabliert und getestet. Diese sind: Fluoreszenz Spektroskopie, Epifluoreszenz Mikroskopie und Quantitative Polymerasekettenreaktion (qPCR). Mithilfe dieser Methoden wurden die Bindungseigenschaften von insgesamt sieben Escherichia coli bindenden DNA Aptameren von drei verschiedenen Publikationen evaluiert. Es konnte gezeigt werden, dass nicht alle Methoden f��r jeden Aptamer Kandidaten anwendbar sind, wahrscheinlich aufgrund von unterschiedlichen Mengen an gebundenem Aptamer pro Zelle. Da sich die Methoden in ihrer Sensitivit��t und ihrem dynamischen Bereich unterscheiden, konnten geringe Mengen an gebundenem Aptamer nur mit qPCR detektiert werden. Weiters k��nnte das Modifizieren der Aptamere mit fluoreszierendem Label f��r die fluoreszenz basierenden Methoden die Bindungseigenschaften von einigen Aptameren beeinflusst haben, was so zu inkonsistenten Resultaten gef��hrt hat. Ein Vergleich der Ergebnisse mit jenen der Publikationen hat gezeigt, dass diese nicht immer reproduzierbar sind, selbst wenn die gleichen Methoden angewandt wurden. Dies k��nnte entweder auf ein Fehlen von wichtigen Informationen in den berichteten Prozeduren oder die Notwendigkeit f��r gr��ndlicheres Testen hinweisen, um die Bindung zu best��tigen. Zusammenfassend konnte gezeigt werden, dass die entwickelten Methoden und Standard Prozeduren als wertvolle Basis f��r die Charakterisierung von anderen Bakterienzellen bindenden DNA Aptameren dienen kann. Weiters wurden erste Empfehlungen f��r eine m��gliche Analyse mittels Durchflusszytometrie gegeben., Aptamers are short single stranded oligonucleotides that can fold into specific three-dimensional structures which allows them to bind to a target with high specificity and affinity. In this diploma thesis multiple analytical methods for the characterization of bacterial whole cell binding aptamers were established and tested, namely fluorescence spectroscopy, epifluorescence microscopy and quantitative polymerase chain reaction (qPCR). Using these methods, the binding properties of a total of seven Escherichia coli targeting DNA aptamers from three different publications were evaluated. Depending on the aptamer, it was shown that not all methods are applicable for each and every candidate due to differences in the amounts of bound aptamer per cell. As the methods differ in their sensitivities and dynamic range, low amounts of bound aptamer can only be detected by qPCR. Moreover, labeling with fluorophores for fluorescence-based methods may have affected the binding properties of some of the aptamers and thus led to inconsistencies in the obtained results. Comparison of the results to those reported in the publications showed that measurements sometimes are not reproducible, even if the same method as originally used was applied. This might either indicate a lack of vital information in the reported experimental procedures or a need for more rigorous testing to confirm binding. In conclusion, the developed methods and standard operating procedures serve as a valuable basis for the characterization of other bacterial cell targeting DNA aptamers. Additionally, first recommendations for possible analysis via flow cytometry were made

To determine the sanitary risk assessment (ERS) of 184 beaches located in both the Pacific and Atlantic coast through the application of the Sanitary Risk Index (IRS) combining the faecal coliform bacteria geometric mean (CF/100 ml) of the inland water sources (streams, estuaries, wetlands and waste water discharges) with the microbiological quality of the sea water sampling points at the beaches. The aim is to establish corrective measures and to scheme the coast areas development. The data from 184 beaches were analysed (62 in Guanacaste, 86 in Puntarenas and 36 in Limón) in the National Water Laboratory (Laboratorio Nacional de Aguas, LNA) during the years 2010-2017. IRS was applied to inland water sources and sea water samples, in order to describe the risk as non-existent, Very Low, Low, Moderately High, High, and Very High. ERS classified the sanitary risk of the 184 beaches: 16 obtained Very High Risk (8.69 %), 4 High Risk (2.17 %), 7 Moderately High Risk (3.80 %), 16 Low Risk (8.69 %), 4 with Very Low Risk (22.28 %), and 100 showed non-existent risk. Results show an important difference between the ERS and the microbiological sea water quality. Additionally, the sanitary risk of 27 beaches (14.67 %) varied from Moderately High to Very High. The majority of the beaches, 157 (85.33 %), obtained a sanitary risk from Low to non-existent. On the other hand, 179 beaches (97.3 %) showed geometric means less than or equal to 100 CF/100 ml, and 134 beaches (72.8 %) showed geometric means less than or equal to 10 CF/100 ml. To carry out sanitary surveys and deeper studies within the 27 beaches classified with Moderately High, High and Very High Risk, in order to define corrective measures, and buffer the faecal pollution. Realizar la evaluación de riesgo sanitario en 184 playas ubicadas en los litorales atlántico y pacífico de Costa Rica, mediante la aplicación del “Índice de Riesgo Sanitario”. Esta herramienta combina los promedios geométricos de Coliformes fecales/100 mL, obtenidos en las fuentes de contaminación terrestres (descargas de aguas residuales y desembocaduras de ríos, esteros y quebradas), y de la calidad microbiológica de los puntos de muestreo en las aguas de las playas. El propósito consiste en establecer las medidas correctivas y planificar, con la participación de la sociedad civil y los municipios, el desarrollo de las zonas costeras del país. Para cumplir con el objetivo planteado, se analizaron los datos históricos de promedios geométricos de Coliformes fecales/100 mL de 184 playas (62 en Guanacaste, 86 en Puntarenas y 36 en Limón), obtenidos por el Laboratorio Nacional de Aguas en el periodo 2010-2017, tanto para las fuentes de contaminación terrestres como para los puntos de muestreo definidos en cada playa. Con estos datos se aplicó la herramienta del “Índice de Riesgo Sanitario”, para clasificar las playas, y comparar los resultados con los niveles de riesgo “Nulo”, “Muy Bajo”, “Bajo” con las playas de calidad “Muy Alto”, “Moderadamente Alto”, y “Alto”. Los resultados de la evaluación de riesgo sanitario de las playas muestran que 16 se clasifican con riesgo “Muy Alto” (8,69%), 4 con “Alto” (2,17%), 7 con “Moderadamente Alto” (3,80%), 16 con “Bajo” (8,69%), 4 con “Muy Bajo” (22,28%) y 100 con “Nulo” (59,34%). Se demuestra que existe una importante diferencia, entre los resultados obtenidos con la evaluación de riesgo sanitario y la calidad microbiológica de las aguas de playa. Aunado a esto, se observa que 27 de las playas estudiadas (14,67%) calificaron con “Moderadamente Alto”, “Alto” y “Muy Alto”, mientras que la mayoría de las playas (157 para un 85,33%) se calificaron con riesgo “Bajo”, “Muy Bajo” y “Nulo”. Por otro lado, del total de playas estudiadas 179 (97,3%) presentaron promedios geométricos menores a 100 CF/100 mL, y 134 (72,8%) inferiores a 10 CF/100 mL. Se recomienda realizar inspecciones sanitarias y estudios más profundos en las 27 playas calificadas con riesgo “Moderadamente Alto”, “Alto” y “Muy Alto”, para definir las medidas correctivas y amortiguar la persistente contaminación fecal detectada en sus aguas.

Intestinal pathogenic microorganisms are introduced into the water by means of faecal contamination, thus creating a threat to public health and to the environment. Detecting these contaminants has been difficult due to such an analysis being costly and time-intensive; as an alternative, microbiological indicators have been used for this purpose, although they cannot differentiate between human or animal sources of contamination because these indicators are part of the digestive tracts of both. To identify the sources of faecal pollution, the use of chemical, microbiological and molecular markers has been proposed. Currently available markers present some geographical specificity. The aim of this study was to select microbial and molecular markers that could be used to differentiate the sources of faecal pollution in the Bogota River and to use them as tools for the evaluation and identification of the origin of discharges and for quality control of the water. In addition to existing microbial source markers, a phage host strain (PZ8) that differentiates porcine contamination was isolated from porcine intestinal content. The strain was identified biochemically and genotypically as Bacteroides. The use of this strain as a microbial source tracking indicator was evaluated in bovine and porcine slaughterhouse wastewaters, raw municipal wastewaters and the Bogota River. The results obtained indicate that the selected microbial and molecular markers enable the determination of the source of faecal contamination in the Bogota River by using different algorithms to develop prediction models.

Faecal pollution modelling is a valuable tool to evaluate and improve water management strategies, especially in a context of water scarcity. The reduction dynamics of five faecal indicator organisms (E. coli, spores of sulphite-reducing clostridia, somatic coliphages, GA17 bacteriophages and a human-specific Bifidobacterium molecular marker) were assessed in an intermittent Mediterranean stream affected by a wastewater treatment plant (WWTP). Using Bayesian inverse modelling, the decay rates of each indicator were correlated with two environmental drivers (temperature and streamflow downstream of the WWTP) and the generated model was used to evaluate the self-depuration distance (SDD) of the stream. A consistent increase of 1–2 log10 in the concentration of all indicators was detected after the discharge of the WWTP effluent. The decay rates showed seasonal variation, reaching a maximum in the dry season, when SDDs were also shorter and the stream had a higher capacity to self-depurate. High seasonality was observed for all faecal indicators except for the spores of sulphite-reducing clostridia. The maximum SDD ranged from 3 km for the spores of sulphite-reducing clostridia during the dry season and 15 km for the human-specific Bifidobacterium molecular marker during the wet season. The SDD provides a single standardized metric that integrates and compares different contamination indicators. It could be extended to other Mediterranean drainage basins and has the potential to integrate changes in land use and catchment water balance, a feature that will be especially useful in the transient climate conditions expected in the coming years., This work was supported by Spanish Ministerio de Economía y Competitividad MEDSOUL project ( CGL2014-59977-C3-1-R ) and the Catalan government ( 2017 SGR 170 ). M. P–B and D. N–S are supported by a FPI grant of the Spanish Ministerio de Economía y Competitividad ( BES-2015-072112 and BES-2015-072983 respectively).

Numerous genetic markers have been developed to establish microbial source tracking (MST) assays in the last decade. However, the selection of suitable markers is challenging due to a lack of understanding of fundamental factors such as sensitivity, specificity, and concentration in target/nontarget hosts, especially in East Asia. In this study, a total of 506 faecal samples comprised of human and 12 nonhuman hosts were collected from 28 cities across China and tested for marker performance characteristics. We firstly tested 40 host-associated markers based on a binary (presence/absence) criterion. Here, 15 markers (7 human-associated, 4 pig-associated, 3 ruminant-associated, and 1 poultry-associated) showed potential applicability in our study area. The selected 15 markers were then tested using qualitative and quantitative methods to characterise their performance. Overall, Bacteroidales markers presented higher sensitivity and concentrations in target samples compared to other bacterial or viral markers, but their specificity was low. Among nontarget samples, pets accounted for 43.7% and 35.7% of cross-reactivity with human-associated and poultry-associated markers, respectively. Noncommon animals, including horse and donkey, contributed 61.3% of cross-reactivity with ruminant-associated markers. When considering the quantitative distribution of markers, their concentration in nontarget samples were 1-3 orders of magnitude lower than in target samples. Moreover, a novel classification method was proposed to classify the nontarget hosts into four groups spanning "no cross-reactivity", "weak cross-reactivity", "moderate cross-reactivity", and "strong cross-reactivity" animal hosts. There were 77.9% nontarget samples identified as no cross-reactivity and weak cross-reactivity hosts, suggesting that these nontarget hosts produce little interference for corresponding markers. Our findings elucidate the performance of host-associated markers around China in a qualitative and quantitative manner, and reveal the interference degree of cross-reactivity from nontarget animals to genetic markers, which will facilitate tracking of multiple faecal pollution sources and planning timely remedial strategies in China.

Faecal microorganisms represent a key threat to human health. Potential origins of faecal microbial contamination in a typical urban-representative micro-scale were evaluated. The quantitative polymerase chain reaction (qPCR) method was used in this study. The Bacteroidetes is selected as the indicative microorganism in runoff samples that are collected during four representative stormwater events in north China. The principal component analysis (PCA) method indicated the distribution feature of the environmental factors. The largest contributor is dog, followed by bird and human to the faecal pollution in stormwater runoff. The output of human and dog faecal pollutants in response to the first flush effect of nonpoint source pollution while the transmit time of bird faecal pollutant is relatively longer. In addition, the number of antecedent drying days represents the key factor for dog faecal pollution, while human faecal pollution is impacted by more factors. The results of this study will provide sound evidence for the tracking and management of nonpoint source faecal pollution in urban catchment areas.

Detection of enzymatic activities has been proposed as a rapid surrogate for the culture-based microbiological pollution monitoring of water resources. This paper presents the results of tests on four fully automated prototype instruments for the on-site monitoring of beta-d-glucuronidase (GLUC) activity. The tests were performed on sediment-laden stream water in the Hydrological Open Air Laboratory (HOAL) during the period of March 2014 to March 2015. The dominant source of faecal pollution in the stream was swine manure applied to the fields within the catchment. The experiments indicated that instrument pairs with the same construction design yielded highly consistent results (R2 = 0.96 and R2 = 0.94), whereas the results between different designs were less consistent (R2 = 0.71). Correlations between the GLUC activity measured on-site and culture-based Escherichia coli analyses over the entire study period yielded R2 = 0.52 and R2 = 0.47 for the two designs, respectively. The correlations tended to be higher at the event scale. The GLUC activity was less correlated with suspended sediment concentrations than with E. coli, which is interpreted in terms of indicator applicability and the time since manure application. The study shows that this rapid assay can yield consistent results over a long period of on-site operation in technically challenging habitats. Although the use of GLUC activity as a proxy for culture-based assays could not be proven for the observed habitat, the study results suggest that this biochemical indicator has high potential for implementation in early warning systems.

The respirable fraction of resuspended urban road dust contains phosphorous at enhanced concentrations which may imply the potential presence of animal faeces. Specific biomarkers such as faecal sterols and bile acids are commonly used to verify the presence of faecal pollution in various environmental samples. The quantification of faecal materials in matrices is usually based on the analysis of bile acids which are persistent and refractory, and can be found in the digestive systems of all mammals and birds. In this study, by measuring the concentrations of bile acids in resuspended PM1−10 samples collected from paved roads on urban locations for the first time in aerosol science we have attempted to estimate the potential mass contribution of animals' faeces to the respirable fraction of road dust. A special sampling unit was applied for the collection of resuspended and respirable road dust in Veszprem and Budapest, Hungary. The concentrations of bile acids were determined by gas chromatography−mass spectrometry (GC−MS). Elemental phosphorous was found in the resuspended PM1−10 fraction in concentrations of 2 mg g−1 whereas the total concentrations of bile acids such as LCA (lithocholic acid), DCA (deoxycholic acid), CDCA (chenodeoxycholic acids), and HDCA (hyodeoxycholic acid) were in the order of 10 μg g−1. Based on the measured mass concentrations of bile acids, the estimated mass contribution of faeces to resuspended PM1−10 mass was found to be in the range of 0.02–0.54 m/m%. Our results imply that wind- or traffic-driven resuspension of dried animals’ faeces, which has never been considered as a potential source of urban PM1−10, is a detectable contributor to urban PM1−10 with all its potential health and epidemiological implications.

The bacteriophages of E. faecalis strains AIM06 (DSM100702) and SR14 (DSM100701) have previously been validated as human-specific microbial source tracking (MST) markers in Thailand. In this study, their spatial and temporal distribution in a freshwater river was investigated for the first time (n = 48). The abundance of enterococci as a standard microbial water quality parameter was evaluated by both the qPCR detection assay with primers and a hydrolysis probe according to the US EPA Method 1611 and the US EPA Method 1600 membrane filtration culture method. AIM06 and SR14 phages were detected by a double layer agar assay and were present in 87.5% and 81.3% of all samples with a co-presence of 92.9% of phage-positive samples. After spiking the representative phages, the ranges of recovery efficiencies were 57.9–99.6% and 49.6–99.9% (n = 48) for AIM06 and SR14 phages, respectively. The absolute abundance of AIM06 and SR14 phages ranged from 0.25 to 221.94 and from 0.25 to 76.66 PFU/100 mL, respectively. Enterococci DNA copies and CFU were detected in all samples ranging from 3.24 to 6.32 log10 copies/100 mL and 100.00 to 1593 CFU/100 mL, respectively. Enterococci in the qPCR assay also showed a moderate correlation with the culture method. The AIM06 and SR14 phage results indicated continuing human faecal pollution along the river with no significant different levels among stations. Interestingly, the higher levels of enterococci in downstream stations for both the qPCR and culture methods along with the significant correlation with other faecal indicator organisms and non-human MST markers implied non-human faecal pollution. In conclusion, this study provides insightful information that could lead to effective water quality management and public health risk reduction from exposure to faecally-contaminated water.

Over the last decades, various PCR-based methods have been proposed that can identify sources of faecal pollution in environmental waters. These microbial source tracking (MST) methods are powerful tools to manage water quality and support public health risk assessment. However, their application is limited by the lack of specialized equipment and trained personnel in laboratories performing microbiological water quality assessment. Here, we describe a novel molecular method that combines helicase-dependent amplification (HDA) with a strip test for detecting ruminant faecal pollution sources. Unlike quantitative PCR (qPCR), the developed HDA-strip assay only requires a heating block to amplify the ruminant-associated Bacteroidetes 16S rRNA marker (BacR). Following HDA, the reaction mixture can be directly applied onto the test strip, which detects and displays the amplification products by marker-specific hybridization probes via an on-strip colorimetric reaction. The entire assay takes two hours and demands no extensive practical training. Furthermore, the BacR HDA-strip assay achieved comparable results in head-to-head performance tests with the qPCR reference, in which we investigated source-sensitivity and source-specificity, the analytical limit of detection, and the sample limit of detection. Although this approach only yields qualitative results, it can pave a way for future simple-to-use MST screening tools.

The literature data on the microbiological water quality of the surface and groundwaters in Montenegro is very scarce. Therefore, this chapter aims to provide an insight in the microbiological water quality of rivers in Montenegro by compiling the data obtained in period 2009–2018 in national monitoring program and the data collected within the Montenegro survey in 2019 with an emphasis on the hotspots of faecal pollution and possible sources of pollution. Despite the high risk that poor implementation of wastewater treatment might represent for Montenegro, the obtained dataset indicates that overall microbiological water quality of rivers in Montenegro is quite good. About 80% of the investigated sites have bathing water quality according to Montenegrin legislation. Only four sites were identified as hotspots of faecal pollution where the human source of pollution prevails.

High-Throughput Sequencing technologies are providing unprecedented inventories of microbial communities in aquatic samples, offering an invaluable tool to estimate the impact of anthropogenic pressure on marine communities. In this case study, the Mediterranean touristic site of Aci Castello (Italy) was investigated by High-Throughput Sequencing of 16S and 18S rRNA genes. The sampling area falls within a Marine Protected Area and, notwithstanding, features an untreated urban wastewater discharge. Seawater samples were collected close to the wastewater output (COL) and at a second station about 400 m further off (PAN), before and after a summer increase in population. Prokaryotic communities clustered according to stations, rather than to seasons. While PAN showed a typical, not impacted, marine microbial composition, COL was consistently enriched in Epsilonproteobacteria and Firmicutes. Protist communities showed a peculiar clustering, as COL at springtime stood alone and was dominated by Ciliophora, while the other samples were enriched in Dinophyta. Analysis of alternative, detectable by High-Throughput Sequencing, microbial indicators, including both faecal- and sewage-associated, allowed uncovering the different sources of pollution in coastal and anthropogenically impacted marine ecosystems, underpinning the relevance of High-Throughput Sequencing-based screening as rapid and precise method for water quality management.

Microbial pollution of recreational waters poses a significant public health risk which, unless mitigated, will continue to increase with population growth. Water managers must implement strategies to accurately discriminate and source human from animal faecal contamination in complex urbanised environments. Our case-study used a new combination of chemical (i.e. ammonia) and microbial (i.e. Escherichia coli, Bacteroides spp.) faecal monitoring tools in a targeted multi-tiered approach to quickly identify pollution hot-spots and track high-risk subterranean stormwater drains in real-time. We successfully located three point sources of human faecal pollution (both episodic and constant pollution streams) within 11 catchments in a total monitoring time of four months. Alternative approaches for obtaining such fine-scale accuracy are typically labour intensive and require expensive equipment.

Microbial source tracking (MST) describes a suite of methods and an investigative strategy for determination of faecal pollution sources in environmental waters. They rely on the association of certain fecal microorganisms with a particular host, that ranges from human [1] to agricultural animals [2,3] to pets [4] and wild animals such as gulls [5]. MST is used to appraise recreational water quality and to correlate with human health risk.

Microorganisms are the ideal indicators of the organic pollution of the surface waters because of their ability to promptly respond to environmental changes. Due to poor economic situation and lack of law implementation, most wastewaters in Serbia are released directly into a surface water recipient without any, or a limited, treatment. This practice has created numerous highly polluted surface waters in the region. Therefore, we conducted seasonal monitoring of the bacteriological quality of the Nišava River located in southeastern Serbia in order to assess the extent of its organic and faecal pollution. A total number of heterotrophs, Escherichia coli and intestinal enterococci counts were determined by standard cultivation methods in samples from five locations along the river. In most instances, bacteriological quality of Nišava water belonged to the classes of low or moderate organic and faecal pollution. The samples taken downstream of the city of Niš were the most polluted. Intestinal enterococci and E. coli counts indicated a moderate faecal contamination. Since bacterial counts can reveal a presence of faecal pollution and therefore presence of potentially pathogenic bacteria, a proper microbiological monitoring of surface waters used as a source of drinking water, like the Nišava River itself, is critically important.

Microbiological contamination arising from faecal pollution is a widespread effect of human activity on aquatic systems, with management of the issue complicated because contamination sources are often unknown. Recent advances in molecular techniques have permitted the use of host-specific genetic markers to apportion sources of faecal pollution. In this study, we used a tiered approach employing faecal indicator bacteria and Bacteroidales markers to identify faecal sources in an urban setting. Canine sources were the most common source of faecal contamination, which led to a programme of education and targeted management. A single, but substantial, source of human faecal contamination was identified that was subject to corrective action and its effectiveness validated by supplementary monitoring. This study supports the use of a tiered approach for the identification of faecal contamination sources in New Zealand, including the use of faecal indicator bacteria and more complex source-tracking analysis us...

Bottled water can be contaminated by coliforms and/or Escherichia coli (E. coli). These bacteria are considered as indicators of faecal pollution, and their detection in bottled water indicates the potential contamination by pathogenic enteric microorganisms. In recent decades, different methods were developed for the detection of coliforms and E. coli in drinking water and in bottled water including mineral water. Since 1976, the Italian regulation has defined microbiological methods to evaluate microbiological characteristics of mineral waters. Three different methods for the detection of coliforms and E. coli in bottled water were compared in this study: the Italian reference method, according to the “Italian Ministerial Rule,” the ISO 9308–1:2002 method, and a new rapid method. The results have demonstrated that the ISO method 9308–1:2002 and the new rapid method are as sensitive and specific as Italian reference method, and that both could be used to evaluate the contamination level of coliform and E. coli in drinking water and in bottled water including mineral water.

Aims We aimed at quantifying bacteriophages in raw and treated wastewaters of human and animal origin in Tunisia to assess their usefulness for tracking the origin of faecal pollution and in the follow-up of effectiveness of water treatments process. Methods and Results The concentrations of bacteriophages in wastewater samples were determined by double layer agar technique. Somatic coliphages and F-specific RNA bacteriophages were present in all types of samples in high concentrations. The values of Escherichia coli were variable depending on geographical location. On the other hand, bacteriophages infecting strain GA17 were detected preferably when human faecal contamination was occurred. Conclusions Bacteriophages appear as a feasible and widely applicable manner to detect faecal contamination in Tunisia. On the other hand, phages infecting GA17 could be good markers for tracking the origin of faecal pollution in the area studied. Significance and Impact of the Study The reuse of treated wastewaters can be a solution to meet the needs of water in the geographical area of study. Bacteriophages seem to predict differently the presence of faecal contamination in water than bacterial indicators. Consequently, they can be a valuable additional tool to improve water resources management for minimizing health risks.

Faecal contamination of water has both anthropogenic and zoogenic origins that can shade various point and nonpoint/diffuse sources of pollution. Due to the dual origin and number of sources of faecal contamination, there are immense challenges in the implementation of effective measures to protect water bodies from pollution that poses threats to human and environmental health. The main health threats refer to infections, illnesses and deaths caused by enteric pathogenic microbes, in particular those responsible for waterborne zoonoses. To detect and identify the origins and sources of faecal pollution simultaneously, various methods and indicators have been compiled into a comprehensive measuring toolbox. Molecular diagnostics using genetic markers derived from Bacteroidales 16S rRNA gene sequences are quite prevalent in the current methodological implementation for the identification of faecal contamination sources in water. For instance, a culture- and library-independent microbial source tracking toolbox combining micro- and molecular biology tests run as a three-step procedure has been implemented in Norway. Outcomes from the Norwegian studies have identified two general trends in dominance of contributors to faecal water contamination in urban environments. Firstly, there is a tendency of higher contributions from anthropogenic sources during the cold season. Secondly, the identification of the dominance of zoogenic sources in faecal water contamination during warm periods of the year.