Modeling human pollution in water bodies using somatic coliphages and bacteriophages that infect Bacteroides thetaiotaomicron strain GA17.

The ability to detect human fecal pollution in water is of great importance when assessing the associated health risks. Many microbial source tracking (MST) markers have been proposed to determine the origin of fecal pollution, but their application remains challenging. A range of factors, not yet sufficiently analyzed, may affect MST markers in the environment, such as dilution and inactivation processes. In this work, a statistical framework based on Monte Carlo simulations and non-linear regression was used to develop a classification procedure for use in MST studies. The predictive model tested uses only two parameters: somatic coliphages (SOMCPH), as an index of general fecal pollution, and human host-specific bacteriophages that infect Bacteroides thetaiotaomicron strain GA17 (GA17PH). Taking into account bacteriophage dilution and differential inactivation, the threshold concentration of SOMCPH was calculated to be around 500 PFU/100 mL for a limit of detection of 10 PFU/100 mL. However, this threshold can be lowered by increasing the analyzed volume sample, which in turn lowers the limit of detection. The resulting model is sufficiently accurate for application in practical cases involving MST and could be easily used with markers other than those tested here.

Comparison of methods for the enumeration of coliphages in 100 mL water samples.

In the last decade coliphages have been included in many water quality regulations as viral faecal indicators. However, the standardised methods used to detect and quantify coliphages differ in bacterial host strains, culture media and techniques. In this comparative study, 100 mL samples of mineral drinking water, river water and wastewater were analysed with International Organization for Standardization (ISO) standard methods, with United States-Environmental Protection Agency (U.S. EPA) based methods as well as commercial kits combining a single agar layer (SAL) assay with ISO bacterial host strains. The three methods gave similar counts (p-value>0.05) for somatic and total coliphages in the matrices with less than 100 PFU/100 mL, whereas for F-specific coliphages, the U.S. EPA method provided statistically significant lower numbers (p-value<0.05) than the other two protocols, possibly because it uses a different bacterial host strain (Escherichia coli HS (pFamp) R vs. the ISO strain Salmonella enterica serovar Typhimurium WG49). In samples with more than 100 PFU/100 mL, the ISO method yielded higher counts of somatic coliphages than the other two protocols (p-value<0.05). As the three methods provided similar results in clean water, the approach combining a SAL assay with the ISO bacterial host strain could be a useful option for coliphage analysis in this type of sample, as it does not require a concentration step.

Bacteriophages in sewage: abundance, roles, and applications.

The raw sewage that flows through sewage systems contains a complex microbial community whose main source is the human gut microbiome, with bacteriophages being as abundant as bacteria or even more so. Phages that infect common strains of the human gut bacteriome and transient bacterial pathogens have been isolated in raw sewage, as have other phages corresponding to non-sewage inputs. Although human gut phages do not seem to replicate during their transit through the sewers, they predominate at the entrance of wastewater treatment plants, inside which the dominant populations of bacteria and phages undergo a swift change. The sheer abundance of phages in the sewage virome prompts several questions, some of which are addressed in this review. There is growing concern about their potential role in the horizontal transfer of genes, including those related with bacterial pathogenicity and antibiotic resistance. On the other hand, some phages that infect human gut bacteria are being used as indicators of fecal/viral water pollution and as source tracking markers and have been introduced in water quality legislation. Other potential applications of enteric phages to control bacterial pathogens in sewage or undesirable bacteria that impede the efficacy of wastewater treatments, including biofilm formation on membranes, are still being researched.

crAssphage as a human molecular marker to evaluate temporal and spatial variability in faecal contamination of urban marine bathing waters.

Bathing water quality may be negatively impacted by diffuse pollution arising from urban and agricultural activities and wildlife, it is therefore important to be able to differentiate between biological and geographical sources of faecal pollution. crAssphage was recently described as a novel human-associated microbial source tracking marker. This study aimed to evaluate the performance of the crAssphage marker in designated bathing waters. The sensitivity and specificity of the crAss_2 marker was evaluated using faecal samples from herring gulls, dogs, sewage and a stream impacted by human pollution (n = 80), which showed that all human impacted samples tested positive for the marker while none of the animal samples did. The crAss_2 marker was field tested in an urban marine bathing water close to the discharge point of human impacted streams. In addition, the bathing water is affected by dog and gull fouling. Analysis of water samples taken at the compliance point every 30 min during a tidal cycle following a rain event showed that the crAss_2 and HF183 markers performed equally well (Spearman correlation ρ = 0.84). The levels of these marker and faecal indicators (Escherichia coli, intestinal enterococci, somatic coliphages) varied by up to 2.5 log10 during the day. Analysis of a high-tide transect perpendicular to the shoreline revealed high levels of localised faecal contamination 1 km offshore, with a concomitant spike in the gull marker. In contrast, both the crAss_2 and HF183 markers remained at a constant level, showing that human faecal contamination is homogenously distributed, while gull pollution is localised. Performance of the crAss_2 and HF183 assay was further evaluated in bimonthly compliance point samples over an 18-month period. The co-occurrence between the crAss_2 and HF183 markers in compliance sampling was 76%. A combination of both markers should be applied in low pollution impacted environments to obtain a high confidence level.

Impact of treated sewage effluent on the bacterial community composition in an intermittent mediterranean stream.

Water quality monitoring is essential to safeguard human and environmental health. The advent of next-generation sequencing techniques in recent years, which allow a more in-depth study of environmental microbial communities in the environment, could broaden the perspective of water quality monitoring to include impact of faecal pollution bacteria on ecosystem. In this study, 16 S rRNA amplicon sequencing was used to evaluate the impact of wastewater treatment plant (WWTP) effluent on autochthonous microbial communities of a temporary Mediterranean stream characterized by high flow seasonality (from 0.02 m3/s in winter to 0.006 m3/s in summer). Seven sampling campaigns were performed under different temperatures and streamflow conditions (winter and summer). Water samples were collected upstream (Upper) of the WWTP, the secondary effluent (EF) discharge and 75 m (P75) and 1000 m (P1000) downstream of the WWTP. A total of 5,593,724 sequences were obtained, giving rise to 20,650 amplicon sequence variants (ASV), which were further analysed and classified into phylum, class, family and genus. Each sample presented different distribution and abundance of taxa. Although taxon distribution and abundance differed in each sample, the microbial community structure of P75 resembled that of EF samples, and Upper and P1000 samples mostly clustered together. Alpha diversity showed the highest values for Upper and P1000 samples and presented seasonal differences, being higher in winter conditions of high streamflow and low temperature. Our results suggest the microbial ecology re-establishment, since autochthonous bacterial communities were able to recover from the impact of the WWTP effluent in 1 km. Alpha diversity results indicates a possible influence of environmental factors on the bacterial community structure. This study shows the potential of next-generation sequencing techniques as useful tools in water quality monitoring and management within the climate change scenario.

Assessment of dead-end ultrafiltration for the detection and quantification of microbial indicators and pathogens in the drinking water treatment processes.

A safe water supply requires distinct treatments and monitoring to guarantee the absence of pathogens and substances potentially hazardous for human health. In this study we assessed the efficiency of the dead-end ultrafiltration (DEUF) method to concentrate faecal indicator organisms (FIO) and pathogens in water samples with different physicochemical characteristics. Water samples were collected at the treatment stages of two drinking water treatment plants to analyse the concentration of a variety of 7 FIO and 4 reference microbes which have some species that are pathogenic to humans: Campylobacter spp., enteroviruses, Cryptosporidium spp. and Giardia spp. The samples were analysed before and after concentration by DEUF, detecting FIO concentrations about 1 log10 higher in non-concentrated samples from both catchments. Percent recoveries were highly variable with a mean of 43.8 ± 17.5%, depending on the FIO and inherent sample characteristics. However, DEUF enabled FIO concentration in high volumes of water (100-500 l), allowing a reduction in the detection limit compared to the non-concentrated samples due to the high volume processing capabilities of the method. As a consequence, the detection of FIO removal from water in the drinking water treatment process was 1.0-1.5 logarithms greater in DEUF-treated water compared to unfiltered samples. The DEUF method improved the detection of target indicators and allowed for the detection of pathogens in low concentrations in water after the treatment stages, confirming the suitability of DEUF to concentrate high volumes of different types of water. This method could be useful for microbial analysis in water treatment monitoring and risk assessment, allowing the identification of critical points during the water treatment process and potential hazards in water destined for several uses.

Microcosms for evaluating microbial indicator persistence and mobilization in fluvial sediments during rainfall events.

Mediterranean rivers, which are subject to long, dry periods and heavy rainfall events, could be particularly useful for understanding future climate scenarios. This study generated microcosms that mimicked riverbank sediment resuspension into the water of a typical Mediterranean river as a consequence of heavy rainfall. The mobilization and inactivation of six fecal pollution indicators and microbial source tracking markers were evaluated. The T90 values in the sediments were: 4 days for sorbitol-fermenting Bifidobacterium, 11 days for culturable E. coli, 36 days for bacteriophages infecting Bacteroides thetaiotaomicron strain GA17 and more than 42 days for qPCR-detected E. coli, somatic coliphages and sulfite-reducing clostridia spores. Bacteriophages and bacteria showed different resuspension and sedimentation patterns. The data obtained could be used in predictive models to assess the effects of climate change on surface water quality. Pathogen mobilization into the water column poses a risk for humans, animals and the natural environment, and breaches the One Health approach.