Indicators Of Fecal Pollution Research Articles

Outbreak of waterborne acute diarrheal disease in a South District village of Tripura: A public health emergency in the Northeast region of India

Food and waterborne outbreaks are a neglected public health problem in India. However, it is important to identify the source of infection and the causative pathogen to curb the outbreak quickly and minimize mortality and morbidity. A retrospective descriptive study was conducted with a line list of 130 diarrheal cases. Epidemiological investigation and laboratory investigation were done. Data were collected from hospital case report forms as well as interviewed affected cases. A case of acute diarrheal disease was reported among the people in the village with abdominal pain, vomiting, and diarrhea from December 31, 2022 to January 3, 2023. Out of a total of 130 recorded cases, 33 stool samples were collected and were positive for Enteroaggregative Escherichia coli, Shigella flexneri 3a, and Shigella sonnei by cultural and molecular tests. The presumptive fecal pollution indicator assay indicated high coliform counts in the water samples (most probable number [MPN]-05) and the presence of Escherichia coli. The identified pathogens showed susceptibility to gentamicin and meropenem. People who used public drinking water were found to be infected with acute diarrheal disease (ADD). Quick identification of the causative pathogens and their antimicrobial resistance pattern helped correct antibiotic prescriptions and quick recovery of the patients without any deaths. Thus, a timely implementation of food and waterborne outbreak investigation is crucial to saving lives and preventing the spread of infection.

The risk of viable but non-culturable (VBNC) enterococci and antibiotic resistance transmission during simulated municipal sludge composting

Sludge composting is a sludge resource utilization method that can reduce pollutants, such as pathogens. Enterococci are regarded as more reliable and conservative indicators of pathogen inactivation than fecal coliforms, which are typically used as indicators of fecal pollution. Non-spore pathogenic bacteria may enter a viable but non-culturable (VBNC) state during composting, leading to residual risk. The VBNC status of bacteria is related to their survival during composting. However, the survival mechanisms of enterococci during sludge composting remain unclear. Therefore, this study aimed to investigate the VBNC state of enterococci in different phases of simulated sludge composting and the fate of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) during the composting process. This study is expected to provide a basis for subsequent exploration of possible methods to completely inactivate enterococci and reduce ARGs during sludge composting. Culturable enterococci were reduced in the thermophilic phase of sludge composting, but the proportion of VBNC subpopulation increased. It was reported for the first time that most VBNC enterococci were killed by extending the cooling phase of sludge compost, and by prolonging the cooling phase the types of ARG were reduced. However, there was a certain quantity (approximately 104/g dry weight) of culturable and VBNC enterococci in the compost products. In addition, MGEs and ARGs exist in both bacteria and compost products, leading to the risk of spreading antibiotic-resistant bacteria and antibiotic resistance when sludge compost products are used.

Metagenomics-Based Microbial Ecological Community Threshold and Indicators of Anthropogenic Disturbances in Estuarine Sediments.

Assessing the impacts of cumulative anthropogenic disturbances on estuarine ecosystem health is challenging. Using spatially distributed sediments from the Pearl River Estuary (PRE) in southern China, which are significantly influenced by anthropogenic activities, we demonstrated that metagenomics-based surveillance of benthic microbial communities is a robust approach to assess anthropogenic impacts on estuarine benthic ecosystems. Correlational and threshold analyses between microbial compositions and environmental conditions indicated that anthropogenic disturbances in the PRE sediments drove the taxonomic and functional variations in the benthic microbial communities. An ecological community threshold of anthropogenic disturbances was identified, which delineated the PRE sediments into two groups (H and L) with distinct taxa and functional traits. Group H, located nearshore and subjected to a higher level of anthropogenic disturbances, was enriched with pollutant degraders, putative human pathogens, fecal pollution indicators, and functional traits related to stress tolerance. In contrast, Group L, located offshore and subjected to a lower level of anthropogenic disturbances, was enriched with halotolerant and oligotrophic taxa and functional traits related to growth and resource acquisition. The machine learning random forest model identified a number of taxonomic and functional indicators that could differentiate PRE sediments between Groups H and L. The identified ecological community threshold and microbial indicators highlight the utility of metagenomics-based microbial surveillance in assessing the adverse impacts of anthropogenic disturbances in estuarine sediments, which can assist environmental management to better protect ecosystem health.

Detection and Quantification of Bacteriophages in Wastewater Samples by Culture and Molecular Methods.

Bacteriophages are promising tools for the detection of fecal pollution in water bodies and particularly for viral pathogen risk assessment. Having similar morphological and biological characteristics, bacteriophages are perfect surrogates for the study of the fate and transport of enteric viruses, generally better than any other group of indicators.Different groups of bacteriophages, such as somatic coliphages, F-specific RNA bacteriophages, and bacteriophages infecting selected strains of Bacteroides, have been comprehensively tested as indicators of fecal pollution. Somatic coliphages and F-specific RNA bacteriophages can be used as indicators of general fecal contamination, whereas Bacteroides phages can be used to detect a particular fecal source, for instance, human, bovine, porcine, or poultry fecal contamination.Feasible and cost-effective protocols standardized by the International Standardization Organization and the United States Environmental Protection Agency for the detection of infectious bacteriophages belonging to these three groups are available. Molecular methods for the detection of some particular phages have also been developed. Here we introduce those methods for the detection, enumeration, and isolation of bacteriophages in wastewater samples.

Microbial indicators of coastal water in Velipoja beach, Albania

Abstract The Albanian coastline is about 450 km long, with wide access to the Adriatic and Ionian seas. It has become popular in recent years for its many unexplored rocky and sandy beaches, crystal waters, curative sand rich in iodine, which has given a great impact in tourism and economy. Velipoja beach is located on the northwest of Adriatic Sea. Coastal waters of Velipoja are used for bathing, fishing and tourism, but in recent years these waters are exposed to various sources of pollution, which are related to restaurants and agricultural activities, wastewater discharge, etc. The quality of coastal water in Albania is particularly analysed by the Public Health Institution. It was considered reasonable to monitor the microbiological quality of coastal waters on Velipoja beach through microbiological parameters during summer season. Evaluations of the results were done based on Directive 2006/7/EC for inland waters, coastal and transitional waters. Seawater samples were tested mainly for E. coli, E. faecalis, S. faecalis, heterotrophs in accordance with European Standards using the membrane filtration method. Water samples were taken monthly from May-September 2023. Based on the data received, during the monitoring period the bacterial concentration for E. faecalis varied from 0 CFU /100 ml to 228 CFU/100 ml. The concentration for S. faecalis varied from 0 CFU /100 ml to 128 CFU/100 ml. The concentration for E. coli varied from 0 CFU /100 ml to 134 CFU/100ml. Velipoja Center has the highest concentration of E. coli, E. faecalis. The concentration of bacteria increases during the months of July and August, probably due to the large number of tourists visiting Velipoja beach during this period. According to Directive 2006/7/EC of the European Parliament, the quality of coastal waters of Velipoja during the period of investigation for E. coli is in the category “Excellent quality”. Monitoring for the presence of pathogen bacteria are essential water quality assessment, which directly or indirectly leads to serious problems to human health. Enterococcus faecalis is an indicator of an old faecal pollution (EN ISO 7899-2, 2000). The European Community (2006) recommended the following parameters: Escherichia coli/100 ml (500/1.000), and Intestinal enterococci/100 ml (200/400) categorized in the class A and B. Urban wastewaters are major sources for surface and groundwater pollution in the Shkodra lake basin. Monitoring for the presence of pathogen bacteria are essential water quality assessment, which directly or indirectly leads to serious problems to human health. Enterococcus faecalis is an indicator of an old faecal pollution (EN ISO 7899-2, 2000). The European Community (2006) recommended the following parameters: Escherichia coli/100 ml (500/1.000), and Intestinal enterococci/100 ml (200/400) categorized in the class A and B. Urban wastewaters are major sources for surface and groundwater pollution in the Shkodra lake basin.

The Importance of Enterococci in the Monitoring of Fecal Pollution in River Water in Forests and Urban Areas

The aim of this study was to determine the spatial distribution of enterococci as indicators of fecal pollution in river water in forest reserves and urban areas. The biodiversity, multidrug resistance, and virulence of enterococci were monitored using conventional and molecular methods, including fluorescence in situ hybridization (FISH). Enterococcal (ENT) counts determined via the FISH method were several thousand times higher than those determined by the culture-based method. This observation points to the abundance of viable but non-culturable (VBNC) bacteria that are often more toxic. Water from the river source was characterized by the lowest number of multidrug-resistant and virulent enterococcal strains. The abundance of the analyzed bacteria was highest (more than 50% of the strains) in the area where treated wastewater was evacuated to the river. Statistical analysis confirmed the presence of relationships between these environments. Pathogenic enterococci, which are not effectively eliminated during wastewater treatment, spread in the river continuum, thus posing a health threat to humans and animals. A combination of conventional and molecular techniques for the identification of bacteria supports a rapid and reliable assessment of pollution sources in the examined environment and the implementation of protective measures.

Escherichia coli as Microbiological Quality Water Indicator:A High Importance for Human and Animal Health

The purpose of this review is to demonstrate the importance of monitoring microbiological quality in water for animals and humans, which relies primarily on coliforms, mostly Escherichia coli. Fecal coliforms, such as E. coli, are more specific indicators of fecal pollution. A fecal contamination detection can be performed not just in drinking water, but also in the environment. As a result of improved detection methods for E. coli, drinking water is becoming more reliable as a result of the use of E. coli as an indicator of fecal pollution. Drinking water contamination by feces is currently best detected by E. coli. Accordingly, temperate environments are more likely to have fecal coliforms than tropical environments, human and animal feces have high levels of E. coli in comparison to other fecal coliforms, and E. coli detection methods are more affordable, fast, sensitive, specific, and easy to use than other fecal coliforms.

Antibiotic Resistance Mediated by Escherichia coli in Kuwait Marine Environment as Revealed through Genomic Analysis.

Antibiotic-resistance gene elements (ARGEs) such as antibiotic-resistance genes (ARGs), integrons, and plasmids are key to the spread of antimicrobial resistance (AMR) in marine environments. Kuwait's marine area is vulnerable to sewage contaminants introduced by numerous storm outlets and indiscriminate waste disposal near recreational beaches. Therefore, it has become a significant public health issue and warrants immediate investigation. Coliforms, especially Gram-negative Escherichia coli, have been regarded as significant indicators of recent fecal pollution and carriers of ARGEs. In this study, we applied a genome-based approach to identify ARGs' prevalence in E. coli isolated from mollusks and coastal water samples collected in a previous study. In addition, we investigated the plasmids and intl1 (class 1 integron) genes coupled with the ARGs, mediating their spread within the Kuwait marine area. Whole-genome sequencing (WGS) identified genes resistant to the drug classes of beta-lactams (blaCMY-150, blaCMY-42, blaCTX-M-15, blaDHA-1, blaMIR-1, blaOKP-B-15, blaOXA-1, blaOXA-48, blaTEM-1B, blaTEM-35), trimethoprim (dfrA14, dfrA15, dfrA16, dfrA1, dfrA5, dfrA7), fluroquinolone (oqxA, oqxB, qnrB38, qnrB4, qnrS1), aminoglycoside (aadA2, ant(3'')-Ia, aph(3'')-Ib, aph(3')-Ia, aph(6)-Id), fosfomycin (fosA7, fosA_6, fosA, fosB1), sulfonamide (sul1, sul2, sul3), tetracycline (tet-A, tet-B), and macrolide (mph-A). The MFS-type drug efflux gene mdf-A is also quite common in E. coli isolates (80%). The plasmid ColRNAI was also found to be prevalent in E. coli. The integron gene intI1 and gene cassettes (GC) were reported to be in 36% and 33%, respectively, of total E. coli isolates. A positive and significant (p < 0.001) correlation was observed between phenotypic AMR-intl1 (r = 0.311) and phenotypic AMR-GC (r = 0.188). These findings are useful for the surveillance of horizontal gene transfer of AMR in the marine environments of Kuwait.

Applicability of F-specific bacteriophage subgroups, PMMoV and crAssphage as indicators of source specific fecal contamination and viral inactivation in rivers in Japan.

To date, several microbes have been proposed as potential source-specific indicators of fecal pollution. 16S ribosomal RNA gene markers of the Bacteroidales species are the most widely applied due to their predominance in the water environment and source specificity. F-specific bacteriophage (FPH) subgroups, especially FRNA phage genogroups, are also known as potential source-specific viral indicators. Since they can be quantified by both culture-based and molecular assays, they may also be useful as indicators for estimating viral inactivation in the environment. Pepper mild mottle virus (PMMoV) and crAssphage, which are frequently present in human feces, are also potentially useful as human-specific indicators of viral pollution. This study aimed to evaluate the applicability of FPH subgroups, PMMoV, and crAssphage as indicators of source-specific fecal contamination and viral inactivation using 108 surface water samples collected at five sites affected by municipal and pig farm wastewater. The host specificity of the FPH subgroups, PMMoV, and crAssphage was evaluated by principal component analysis (PCA) along with other microbial indicators, such as 16S ribosomal RNA gene markers of the Bacteroidales species. The viabilities (infectivity indices) of FRNA phage genogroups were estimated by comparing their numbers determined by infectivity-based and molecular assays. The PCA explained 58.2% of the total information and classified microbes into three groups: those considered to be associated with pig and human fecal contamination and others. Infective and gene of genogroup IV (GIV)-FRNA phage were assumed to be specific to pig fecal contamination, while the genes of GII-FRNA phage and crAssphage were identified to be specific to human fecal contamination. However, PMMoV, infective GI-FRNA phage, and FDNA phage were suggested to not be specific to human or pig fecal contamination. FRNA phage genogroups, especially the GIV-FRNA phage, were highly inactivated in the warm months in Japan (i.e., July to November). Comparing the infectivity index of several FRNA phage genogroups or other viruses may provide further insight into viral inactivation in the natural environment and by water treatments.

Comparison of modern and 40-year-old drinking water pipeline in northern Sinai region, Egypt: Characteristics and health risk assessment

BackgroundDrinking water quality is crucial for human health and well-being. This study evaluated the impact of replacing old asbestos pipelines with modern Polyvinyl Chloride (PVC) ones on drinking water quality in the North Sinai governorate of Egypt. Ninety-two water samples were collected from nine locations along the pipeline from Qantara water station to El-Arish city, and their microbiological and chemical characteristics were assessed. MethodsThis investigation was carried out during June and July 2019 to evaluate the drinking water in two distribution nets; old asbestos distribution pipes and new Polyvinyl Chloride water tubes (PVC). Microbiological analyses such as most probable number (MPN), total bacterial count (TBC), chemical assessments (water pH, electrical conductivity; EC, dissolved organic carbon; DOC), and health risk index (HRI) for multi-element concentrations were conducted. Standard analyses were followed to assess the water quality index (WQI) and Metal index (MI) and study the improvement percentage in the element's concentrations. ResultsThe highest values of TBC were in the city center district (219 CFUs mL−1), followed by Abu Saqual (184 CFUs mL−1), Resa (174 CFUs mL−1), and recently constructed pipes (80 CFUs mL−1). The pH (7.52 - 7.50) and EC (320 to 958 dSm−1) were within permissible limits. No bacterial indicators of fecal pollution were found in all drinking water samples. Results showed that the modern pipeline network significantly improved the concentrations of 23 elements, with the most significant improvements observed in cadmium (66.2%) and potassium (40.7%). HRI results for multi-element concentrations showed an impact on human health in Abu Saqual. WQI showed a decrease of 30% in toxic element concentration in the current pipelines, and MI assessment showed safe concentrations. ConclusionDrinking water's biological and chemical properties significantly improved, which matched global guideline standards. Our findings highlight the importance of regularly monitoring and updating drinking water infrastructure to ensure safe and high-quality drinking water.

Searching for a Reliable Viral Indicator of Faecal Pollution in Aquatic Environments.

The disposal of sewage in significant quantities poses a health hazard to aquatic ecosystems. These effluents can contain a wide range of pathogens, making faecal contamination a leading source of waterborne diseases around the world. Yet monitoring bacteria or viruses in aquatic environments is time consuming and expensive. The standard indicators of faecal pollution all have limitations, including difficulty in determining the source due to lack of host specificity, poor connection with the presence of non-bacterial pathogens, or low environmental persistence. Innovative monitoring techniques are sorely needed to provide more accurate and targeted solutions. Viruses are a promising alternative to faecal indicator bacteria for monitoring, as they are more persistent in ambient water, more abundant in faeces, and are extremely host-specific. Given the range of viruses found in diverse contexts, it is not easy to find one "ideal" viral indicator of faecal pollution; however, several are of interest. In parallel, the ongoing development of molecular techniques coupled with metagenomics and bioinformatics should enable improved ways to detect faecal contamination using viruses. This review examines the evolution of faecal contamination monitoring with the followingaims (i) to identify the characteristics of the main viral indicators of faecal contamination, including human enteric viruses, bacteriophages, CRESS and plant viruses, (ii) to assess how these have been used to monitor water pollution in recent years, (iii) to evaluate the reliability of recent detection methods of such viruses, and (iv) to tentatively determine which viruses may be most effective as markers of faecal pollution.

Pepper Mild Mottle Virus as a Potential Indicator of Fecal Contamination in Influents of Wastewater Treatment Plants in Riyadh, Saudi Arabia.

Several indicators of fecal pollution in water resources are continuously monitored for their reliability and, of particular interest, their correlation to human enteric viruses-not justified by traditional bacterial indicators. Pepper mild mottle virus (PMMoV) has recently been proposed as a successful viral surrogate of human waterborne viruses; however, in Saudi Arabia there are no available data in terms of its prevalence and concentration in water bodies. The concentration of PMMoV in three different wastewater treatment plants (King Saud University (KSU), Manfoha (MN), and Embassy (EMB) wastewater treatment plants (WWTP)) was measured using qRT-PCR during a one-year period and compared to the human adenovirus (HAdV), which is highly persistent and considered an indicator for viral-mediated fecal contamination. PMMoV was found in ~94% of the entire wastewater samples (91.6-100%), with concentrations ranging from 62 to 3.5 × 107 genome copies/l (GC/l). However, HAdV was detected in 75% of raw water samples (~67-83%). The HAdV concentration ranged between 1.29 × 103 GC/L and 1.26 × 107 GC/L. Higher positive correlation between PMMoV and HAdV concentrations was detected at MN-WWTP (r = 0.6148) than at EMB-WWTP (r = 0.207). Despite the lack of PMMoV and HAdV seasonality, a higher positive correlation (r = 0.918) of PMMoV to HAdV was recorded at KSU-WWTP in comparison to EMB-WWTP (r = 0.6401) around the different seasons. Furthermore, meteorological factors showed no significant influence on PMMoV concentrations (p > 0.05), thus supporting the use of PMMoV as a possible fecal indicator of wastewater contamination and associated public health issues, particularly at MN-WWTP. However, a continuous monitoring of the PMMoV distribution pattern and concentration in other aquatic environments, as well as its correlation to other significant human enteric viruses, is essential for ensuring its reliability and reproducibility as a fecal pollution indicator.

Use of Somatic Coliphages in Biosensoring of Hygiene Safety of Recycled Wastewater

Outflows from secondary stages of conventional mechanical-biological wastewater treatment plants (WWTPs) still contain significant concentrations of faecal pollution indicators suggesting the potential presence of pathogenic organisms. The decrease in the concentration of somatic coliphages and bacterial indicators of faecal pollution during the technological stages (coagulation, sand filtration, membrane ultrafiltration, sorption on granular activated carbon, disinfection, accumulation) of a semi-operational plant designed for multi-stage tertiary treatment or rather recycling of treated wastewater was monitored and discussed. During the tertiary treatment, faecal bacteria indicators were better removed than somatic coliphages, hence the inclusion of somatic indicators among faecal pollution indicators in water quality control for reuse is entirely appropriate. Subsequent tertiary treatment, including disinfection, is essential for safe reuse of treated water.

Spatial distribution of fecal pollution indicators in sewage sludge flocs and their removal and inactivation as revealed by qPCR/viability-qPCR during potassium ferrate treatment.

Sludge reuse and utilization is one of important routines of disseminating fecal pollution to surface water and groundwater. However, it remains unclear the spatial distribution of fecal pollution indicators in sludge flocs and their reductions during sludge treatment processes. In this study, the abundances of fecal pollution indicators including cross-assembly phage (crAssphage), JC and BK polyomavirus (JCPyV, BKPyV), human adenovirus (HAdV), the human-specific HF183 Bacteroides (HF183) and Escherichia coli (EC) in soluble extracellular polymeric substances (S-EPS), loosely-bound EPS (LB-EPS), tightly-bound EPS (TB-EPS), and pellets of sludge flocs were determined, and the effect of potassium ferrate (PF) treatment on their removal and inactivation was investigated by using both qPCR and viability-qPCR. Results showed that all investigated indicators were detected in each fraction of sludge flocs. The PF treatment led to a great migration of indicators from sludge pellets to sludge EPS and some extent of their inactivation in each fraction of sludge flocs. The overall reductions of human fecal indicators in sludge determined by qPCR were 0-1.30logs, which were 0-2 orders of magnitude lower than those of 0.69-2.39logs detected by viability-qPCR, implying their inactivation by PF treatment to potentially alleviate the associated human health risks.

Qualitative Risk Assessment of Microbial Indicators in the Lower Reaches of Meenachil River, Kerala, India

Pristine natural freshwater resources being severely contaminated by microbiological pollutants is still a big issue in many parts of the world. In the present study, water samples taken from the upstream, midstream, and downstream reaches of the Meenachil River were analysed for the presence of microbial pollution indicators such as faecal coliforms (FC), faecal streptococci (FS), and Eschericia coli. The E. coli isolates were subjected to antimicrobial susceptibility tests and the multiple antibiotic resistance (MAR) indexes were calculated so as to delineate the potential risk posed by infections to humans and animals using the river for routine potable and recreational purposes. E. coli was consistently isolated from all the samples collected from the different zones of the river. Other faecal coliforms such as Klebsiella oxytoca, Citrobacter koseri, Enterobacter, Proteus vulgaricus, and Shigella boydii were also identified. Higher FC/FS ratios were noticed for the midstream and the downstream sites indicating the contamination from high-risk sources such as human and anthropogenic domestic wastes. The MAR index values of E. coli isolates ranged from 0.33 upstream, 0.416 for midstream and 0.583 for the downstream reaches. Given that E. coli is an ideal indicator of faecal pollution, their existence and extended survival of multiple antibiotic resistance natural freshwater pose a potential risk for the consumers. Rapid urbanization and resultant waste drainage in the downstream stretches of the river might have accelerated the deterioration of the water quality up to a certain extent. Furthermore, the riverine system as it flows downstream loses its self-purification capacity due to the loss of natural water drifts. This may be attributed to the periodic shutdown of the salinity barrier, Thanneermukkam Bund in the confluence zone of the river which results in a temporary stagnation of the running waters in the lower stretches.

Tidal effect on the dispersion of fecal pollution indicator bacteria and associated health risks along the Kribi beaches (Southern Atlantic coast, Cameroon)

Assessment of fecal contamination indicator bacteria concentrations on beaches are important for health risk prevention and management. This study assessed temporal changes in bacterial abundances as a function of tidal cycles and seasons along the Kribi beaches, Cameroon. Overall, samples taken during low and high tides during the different seasons of the year showed that the waters of Kribi beaches are exposed to fecal contamination due to the presence of different concentrations of fecal coliforms (5–35 CFU/100 ml), total coliforms (100–600 CFU/100 ml), Escherichia coli (0–15 CFU/100 ml), Streptococcus feacalis (50–700 CFU/100 ml), Pseudomonas aeruginosa (300–7200 CFU/100 ml), Vibrio cholerea (5–250 CFU/100 ml), Vibrio parahemoliticus (0–115 CFU/100 ml), and Aerobic Mesophilic Heterotrophic Bacteria (99–875 CFU/100 ml). Regardless of bacterial strain and sampling site, cell abundances were significant at low tides and during rainy seasons. At each sampling station (Mpalla, Ngoyè and Mboamanga), depending on the seasons and tidal cycles, significant correlations were recorded between the abundances of some bacterial strains and some environmental variables (P< 0.05). The presence and high abundance of these potentially pathogenic bacterial strains in the waters of Kribi beaches constitute a real public health threat for swimmers. The limitation of this microbiological pollution requires the implementation of an efficient collection and treatment plan for solid and liquid waste (sanitary wastewater) in the city of Kribi.

Unraveling the influence of human fecal pollution on antibiotic resistance gene levels in different receiving water bodies using crAssphage indicator gene.

Discharged wastewater treatment plant (WWTP) effluents can contaminate receiving water bodies with human feces and alter the abundance of antibiotic resistance genes (ARGs). In this study, we examined the co-occurrence of ARGs, human fecal pollution indicator crAssphage, and antibiotics in human feces and a series of connected receiving water bodies affected by human feces, including water from different treatment units of a WWTP, river, lake, and tap waters. Results showed that crAssphage was detected in 68.2 % of the studied water bodies, confirming widespread human fecal contamination. Both ARG and crAssphage abundances exhibited a distance-decay effect from the emission source to the receiving environment. Interestingly, the detected ARG abundance in the water bodies was significantly correlated with crAssphage abundance but not with the residual antibiotic concentration, demonstrating that the presence of ARG could largely be explained by the extent of fecal pollution, with no clear signs of antibiotic selection. In addition, 14 ARGs co-shared by human feces and water bodies were significantly correlated with crAssphage. Furthermore, a close evolutionary relationship was observed between the blaTEM-1 gene from human feces and aquatic environments. These results imply a potential ARG exchange between human feces and receiving water bodies. Overall, this study provides important insights into the distribution and sources of ARGs in water bodies affected by human fecal contamination.

Natural and anthropic aquatic ecosystems – structural differences in bacterial populations

The hydrosphere represents about 71% of the total surface of the planet of which only 2.8% is represented by freshwater ecosystems. The ecosystem maintains a state of balance between the component populations, throughout its self-control function, maintaining the numerical variations of these populations within certain limits of sustainability. The exceeding of these limits could causes the balance disturbance by changing the structure and functioning of the ecosystem. The complex human activities influence the structure and functioning of ecosystems, transforming the environment and adapting it to its purposes. Microorganisms are present in all types of ecosystems, being endogenous, exogenous or transient due to contamination with various sources of pollution. Water is an essential natural factor of ecological balance increasingly exposed to microbial pollution. Indicators of fecal pollution are used to assess the degree of water contamination and to locate its origin. The continuous and uncontrolled usage of antibacterial agents that contributed to bacterial resistance determined/ caused pollution of aquatic ecosystems with antibiotic resistant microorganisms. The emergence of antibiotic resistant bacteria is predictable in any environment given that the use of antibacterial substances is constantly growing worldwide. Thus, antibiotic resistance induced in the aquatic environment can have an effect both on bacterial populations in the ecosystem and on human health. Aquatic environments are recognized as one of the reservoirs for the transmission and dissemination of antibiotic resistance. The main goal of this paper is to highlight the differences of bacterial communities from anthropogenic and natural aquatic ecosystems and to assess the potential impact they have on environment and human health. The study area focused on two lakes in Bucharest, Lake Morii (anthropic) and Lake Snagov (natural). Microbiological and molecular biology methods were applied for a bacterial communities’ characterization. The fecal indicators were quantified by Most Probable Number method. The identification and characterization of bacterial populations in both aquatic ecosystems (Morii Lake and Snagov Lake) were performed by Omnilog (Biolog, USA) and by iSeq100 (Illumina, USA) gene sequencing techniques from bacterial aquatic ecosystem. Antibiotic susceptibility was tested following CLSI recommendation.

Development of two microbial source tracking markers for detection of wastewater-associated Escherichia coli isolates

Escherichia coli has been used as an indicator of fecal pollution in environmental waters. However, its presence in environmental waters does not provide information on the source of water pollution. Identifying the source of water pollution is paramount to be able to effectively reduce contamination. The present study aimed to identify E. coli microbial source tracking (MST) markers that can be used to identify domestic wastewater contamination in environmental waters. We first analyzed wastewater E. coli genomes sequenced by us (n = 50) and RefSeq animal E. coli genomes of fecal origin (n = 82), and identified 144 candidate wastewater-associated marker genes. The sensitivity and specificity of the candidate marker genes were then assessed by screening the genes in 335 RefSeq wastewater E. coli genomes and 3318 RefSeq animal E. coli genomes. We finally identified two MST markers, namely W_nqrC and W_clsA_2, which could be used for detection of wastewater-associated E. coli isolates. These two markers showed higher performance than the previously developed human wastewater-associated E. coli markers H8 and H12. When used in combination, W_nqrC and W_clsA_2 showed specificity of 98.9 % and sensitivity of 25.7 %. PCR assays to detect W_nqrC and W_clsA_2 were also developed and validated. The developed PCR assays are potentially useful for detecting E. coli isolates of wastewater origin in environmental waters, though users should keep in mind that the sensitivity of these markers is not high. Further studies are needed to assess the applicability of the developed markers to a culture-independent approach.

Genetic Determinants of Escherichia coli Survival in Beach Sand.

Escherichia coli contain a high level of genetic diversity and are generally associated with the guts of warm-blooded animals but have also been isolated from secondary habitats outside hosts. We used E. coli isolates from previous in situ microcosm experiments conducted under actual beach conditions and performed population-level genomic analysis to identify accessory genes associated with survival within the beach sand environment. E. coli strains capable of surviving had been selected for by seeding isolates originating from sand, sewage, and gull waste (n = 528; 176 from each source) into sand, which was sealed in microcosm chambers and buried for 45 days in the backshore beach of Lake Michigan. In the current work, survival-associated genes were identified by comparing the pangenome of viable E. coli populations at the end of the microcosm experiment with the original isolate collection and identifying loci enriched in the out put samples. We found that environmental survival was associated with a wide variety of genetic factors, with the majority corresponding to metabolism enzymes and transport proteins. Of the 414 unique functions identified, most were present across E. coli phylogroups, except B2 which is often associated with human pathogens. Gene modules that were enriched in surviving populations included a betaine biosynthesis pathway, which produces an osmoprotectant, and the GABA (gamma-aminobutyrate) biosynthesis pathway, which aids in pH homeostasis and nutrient use versatility. Overall, these results demonstrate that the genetic flexibility within this species allows for survival in the environment for extended periods. IMPORTANCE Escherichia coli is commonly used as an indicator of recent fecal pollution in recreational water despite its known ability to survive in secondary environments, such as beach sand. These long-term survivors from sand reservoirs can be introduced into the water column through wave action or runoff during precipitation events, thereby impacting the perception of local water quality. Current beach monitoring methods cannot differentiate long-term environmental survivors from E. coli derived from recent fecal input, resulting in inaccurate monitoring results and unnecessary beach closures. This work identified the genetic factors that are associated with long-term survivors, providing insight into the mechanistic basis for E. coli accumulation in beach sand. A greater understanding of the intrinsic ability of E. coli to survive long-term and conditions that promote such survival will provide evidence of the limitations of beach water quality assessments using this indicator.