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The integration of somatic coliphage analysis into water quality regulations has driven the development of more streamlined, easier, and faster detection methods. These include the Bluephage method, initially designed for the qualitative assessment of coliphages in 100 mL water samples. In the present study this technique was adapted for quantitative analysis using the most probable number method, enabling quantification of somatic coliphages in 100 mL water samples within 6.5 h of incubation. Early readings were optimised using an algorithm developed from an extensive dataset of over 400 environmental samples. This new quantitative method has been refined and commercialised as the Enumera® Rapid kit (BPF-SE, Bluephage S.L.), which includes a new reference somatic coliphage, strain GR8. After sequencing and further characterisation, it was confirmed that the new strain belongs to the same species as the reference somatic coliphage of the ISO 10705-2 method. The Enumera® Rapid kit was then validated by assessing its efficacy across 151 samples from various water matrices. The method provided a mean recovery rate of 103 %, ranging from 95 % for drinking water to 116 % for wastewater, at concentrations from 0 to 300 PFU/100 mL. In comparison, ISO 10705-2 applied after membrane filtration yielded a mean recovery rate of 63.8 %, with the highest rate observed for wastewater (88.9 %). The results obtained with the Enumera® Rapid kit did not differ significantly compared to the reference method after adjusting for potential losses during the concentration stage outlined in ISO 10705-3. The new method also demonstrated a robust accuracy of 0.16 logs, good linearity, and achieved a limit of detection and quantification of 1 MPN/100 mL. The Enumera® Rapid kit enables rapid and straightforward quantification of somatic coliphages in 100 mL water samples within a single working day. Its streamlined workflow serves as a useful tool for microbial water quality monitoring., (Copyright © 2024. Published by Elsevier B.V.)

Bacterial communities in a full-scale drinking water treatment plant (DWTP) were characterized using matrix-assisted laser desorption/ionization time of flight mass-spectrometry (MALDI-TOF MS) to identify HPC isolates and the obtained results were compared to 16S rRNA (V4) metabarcoding data acquired in a previous study. Sixty-three samples were collected at nine stages of the potabilization process: river water and groundwater intake, decantation, sand filtration, ozonization, carbon filtration, reverse osmosis, the mixing chamber and post-chlorination drinking water. In total, 1807 bacterial colonies were isolated, 32 % of which were successfully identified to at least the genus level by MALDI-TOF MS using our previously developed Drinking Water Library. Trends in diversity were similar by both approaches, but differences were observed in the detection of taxa, especially at lower hierarchy levels. High bacterial diversity was observed in river and groundwater, where Proteobacteria predominated. The diversity decreased significantly after the chlorination step, where Bacillus sp. (Firmicutes) and an unknown genus of Obscuribacteraceae (Cyanobacteria) were the most prevalent genera according to MALDI-TOF MS and metabarcoding, respectively. The two approaches gave similar results for the decantation, sand filtration and mixing chamber steps, where the most abundant taxon was Flavobacterium. The combined use of these culture-based and culture-independent methods to characterize microbial populations may help to better understand the role of bacteria in water treatment and quality, which will be of value for DWTP management., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Julia Martín-Díaz reports financial support was provided by Bluephage, S.L. Miriam Pascual-Benito reports financial support was provided by Bluephage, S.L. Ariadna Jorba-Plassa reports financial support was provided by Bluephage, S.L., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Two groups of coliphages have been recently included in different water management policies as indicators of viral fecal pollution in water and food: somatic coliphages, which infect E. coli through cell wall receptors, and F-specific RNA coliphages, which infect through the F-pili. Somatic coliphages are more abundant in fecally contaminated waters, except reclaimed waters, those disinfected by UV irradiation, and some groundwater samples that show a higher level of F-specific coliphages. Somatic coliphages are morphologically similar to DNA enteric viruses while F-specific coliphages are similar to RNA viruses such as norovirus and hepatitis A viruses, which are the viral pathogens of concern in sewage. The use of strains sensitive to both types of phages has been proposed for total coliphage enumeration, thereby avoiding double analysis. The standardized methods available for coliphage detection are robust and cost-effective, but the introduction of ready-to-use methods would facilitate routine implementation in laboratories. The fastest available tool for somatic coliphage enumeration is the recently developed Bluephage, which uses a modified β-glucuronide-overexpressing E. coli strain unable to take up the glucuronide substrate. The overexpressed enzyme accumulates inside the bacterial cells until released by phage-induced cell lysis, whereupon it encounters its substrate and the medium changes from yellow to blue. The present method uses E. coli strain CB12, sensitive to somatic coliphages and F-specific coliphages due to the expression of the F-pili. The Bluephage approach incorporating CB12 detects both types of coliphages in a time range of 1:30 to 4:00 h, as assayed with coliphages from raw sewage, river water, sludge and mussels. This strategy can be applied to obtain qualitative and quantitative results and is applicable to microplates as well as to large sample volumes (100 ml). Moreover it can provide monitoring of water bodies at real time, as for example for ambient recreational beach monitoring., (Copyright © 2018. Published by Elsevier B.V.)