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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.)

Rapid population growth and coastal development has led to increased fecal contamination of coastal surface waters worldwide, enhancing the potential risk of waterborne human pathogens in bathing areas. More frequent heavy rainfall events, attributed to global warming, have further exacerbated the problem by causing sometimes sewer overflows into recreational waters. As traditional bacterial indicators have limited accuracy for predicting health risks associated with waterborne viruses, the additional use of viral indicators such as coliphages is recommended. In this study, we compared the behavior of bacterial and viral indicators of water quality at 10 Barcelona beaches during three bathing seasons, in dry conditions, and after four rainstorms that caused specific pollution events due to rain runoff with combined sewer overflows (CSO). Levels of all target indicators increased after the rainstorms, but compared to Escherichia coli and intestinal enterococci, somatic coliphages exhibited a slower decline and higher environmental persistence following a rain event. Daily continuous sampling carried out during the days following a rainstorm allowed not only the determination of the decay kinetics of each target indicator but also the day when the water quality recovered the values established in the current European regulation in approximately 2 -3 days after each CSO. These observations indicate that the combined use of bacterial and viral indicators can enhance the surveillance of microbial quality of bathing waters. Moreover, coliphages can swiftly provide insights into transient fecal pollution linked to rainfall episodes, thanks to available analytical techniques that enable same-day recommendations. The management of urban wastewater and recreational water regulations should consistently employ microbial indicators to address rainwater runoff or sewer overflows resulting from heavy rainfall., 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 © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

With the advent of molecular biology diagnostics, different quantitative PCR assays have been developed for use in Source Tracking (ST), with none of them showing 100% specificity and sensitivity. Most studies have been conducted at a regional level and mainly in fecal slurry rather than in animal wastewater. The use of a single molecular assay has most often proven to fall short in discriminating with precision the sources of fecal contamination. This work is a multicenter European ST study to compare bacterial and mitochondrial molecular assays and was set to evaluate the efficiency of nine previously described qPCR assays targeting human-, cow/ruminant-, pig-, and poultry-associated fecal contamination. The study was conducted in five European countries with seven fecal indicators and nine ST assays being evaluated in a total of 77 samples. Animal fecal slurry samples and human and non-human wastewater samples were analyzed. Fecal indicators measured by culture and qPCR were generally ubiquitous in the samples. The ST qPCR markers performed at high levels in terms of quantitative sensitivity and specificity demonstrating large geographical application. Sensitivity varied between 73% (PLBif) and 100% for the majority of the tested markers. On the other hand, specificity ranged from 53% (CWMit) and 97% (BacR). Animal-associated ST qPCR markers were generally detected in concentrations greater than those found for the respective human-associated qPCR markers, with mean concentration for the Bacteroides qPCR markers varying between 8.74 and 7.22 log 10 GC/10 mL for the pig and human markers, respectively. Bacteroides spp. and mitochondrial DNA qPCR markers generally presented higher Spearman's rank coefficient in the pooled fecal samples tested, particularly the human fecal markers with a coefficient of 0.79. The evaluation of the performance of Bacteroides spp., mitochondrial DNA and Bifidobacterium spp. ST qPCR markers support advanced pollution monitoring of impaired aquatic environments, aiming to elaborate strategies for target-oriented water quality management., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Crassvirales (crAss-like phages) are an abundant group of human gut-specific bacteriophages discovered in silico. The use of crAss-like phages as human fecal indicators is proposed but the isolation of only seven cultured strains of crAss-like phages to date has greatly hindered their study. Here, we report the isolation and genetic characterization of 25 new crAss-like phages (termed crAssBcn) infecting Bacteroides intestinalis, belonging to the order Crassvirales, genus Kehishuvirus and, based on their genomic variability, classified into six species. CrAssBcn phage genomes are similar to ΦCrAss001 but show genomic and aminoacidic differences when compared to other crAss-like phages of the same family. CrAssBcn phages are detected in fecal metagenomes around the world at a higher frequency than ΦCrAss001. This study increases the known crAss-like phage isolates and their abundance and heterogeneity open the question of what member of the Crassvirales group should be selected as human fecal marker., (© 2023. The Author(s).)

The impacts of nucleic acid-based methods - such as PCR and sequencing - to detect and analyze indicators, genetic markers or molecular signatures of microbial faecal pollution in health-related water quality research were assessed by rigorous literature analysis. A wide range of application areas and study designs has been identified since the first application more than 30 years ago (>1100 publications). Given the consistency of methods and assessment types, we suggest defining this emerging part of science as a new discipline: genetic faecal pollution diagnostics (GFPD) in health-related microbial water quality analysis. Undoubtedly, GFPD has already revolutionized faecal pollution detection (i.e., traditional or alternative general faecal indicator/marker analysis) and microbial source tracking (i.e., host-associated faecal indicator/marker analysis), the current core applications. GFPD is also expanding to many other research areas, including infection and health risk assessment, evaluation of microbial water treatment, and support of wastewater surveillance. In addition, storage of DNA extracts allows for biobanking, which opens up new perspectives. The tools of GFPD can be combined with cultivation-based standardized faecal indicator enumeration, pathogen detection, and various environmental data types, in an integrated data analysis approach. This comprehensive meta-analysis provides the scientific status quo of this field, including trend analyses and literature statistics, outlining identified application areas, and discusses the benefits and challenges of nucleic acid-based analysis in GFPD., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

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.)

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., Competing Interests: The authors declare no conflict of interests., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

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., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

According to the European Directives (UE) 2020/2184 and 2009/54/EC, which establishes the sanitary criteria for water intended for human consumption in Europe, water suitable for human consumption must be free of the bacterial indicators Escherichia coli, Clostridium perfringens and Enterococcus spp. Drinking water is also monitored for heterotrophic bacteria, which are not a human health risk, but can serve as an index of bacteriological water quality. Therefore, a rapid, accurate, and cost-effective method for the identification of these colonies would improve our understanding of the culturable bacteria of drinking water and facilitate the task of water management by treatment facilities. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is potentially such a method, although most of the currently available mass spectral libraries have been developed in a clinical setting and have limited environmental applicability. In this work, a MALDI-TOF MS drinking water library (DWL) was defined and developed by targeting bacteria present in water intended for human consumption. This database, made up of 319 different bacterial strains, can contribute to the routine microbiological control of either treated drinking water or mineral bottled water carried out by water treatment and distribution operators, offering a faster identification rate compared to a clinical sample-based library. The DWL, made up of 96 bacterial genera, 44 of which are not represented in the MALDI-TOF MS bacterial Bruker Daltonics (BDAL) database, was found to significantly improve the identification of bacteria present in drinking water., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Bacteriophages are promising tools for the detection of fecal pollution in different environments, and particularly for viral pathogen risk assessment. Having similar morphological and biological characteristics, bacteriophages mimic the fate and transport of enteric viruses. Enteric bacteriophages, especially phages infecting Escherichia coli (coliphages), have been proposed as alternatives or complements to fecal indicator bacteria. Here, we provide a general overview of the potential use of enteric bacteriophages as fecal and viral indicators in different environments, as well as the available methods for their detection and enumeration, and the regulations for their application.

The detection of fecal viral pathogens in water is hampered by their great variety and complex analysis. As traditional bacterial indicators are poor viral indicators, there is a need for alternative methods, such as the use of somatic coliphages, which have been included in water safety regulations in recent years. Some researchers have also recommended the use of reference viral pathogens such as noroviruses or other enteric viruses to improve the prediction of fecal viral pollution of human origin. In this work, phages previously tested in microbial source tracking studies were compared with norovirus and adenovirus for their suitability as indicators of human fecal viruses. The phages, namely those infecting human-associated Bacteroides thetaiotaomicron strain GA17 (GA17PH) and porcine-associated Bacteroides strain PG76 (PGPH), and the human-associated crAssphage marker (crAssPH), were evaluated in sewage samples and fecal mixtures obtained from different animals in five European countries, along with norovirus GI + GII (NoV) and human adenovirus (HAdV). GA17PH had an overall sensitivity of ≥83% and the highest specificity (>88%) for human pollution source detection. crAssPH showed the highest sensitivity (100%) and specificity (100%) in northern European countries but a much lower specificity in Spain and Portugal (10 and 30%, respectively), being detected in animal wastewater samples with a high concentration of fecal indicators. The correlations between GA17PH, crAssPH, or the sum of both (BACPH) and HAdV or NoV were higher than between the two human viruses, indicating that bacteriophages are feasible indicators of human viral pathogens of fecal origin and constitute a promising, easy to use and affordable alternative to human viruses for routine water safety monitoring., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer, JL, declared a past collaboration with one of the authors, LM., (Copyright © 2021 Ballesté, Blanch, Mendez, Sala-Comorera, Maunula, Monteiro, Farnleitner, Tiehm, Jofre and García-Aljaro.)

Nitrate (NO 3 - ) pollution adversely impacts surface and groundwater quality. In recent decades, many countries have implemented measures to control and reduce anthropogenic nitrate pollution in water resources. However, to effectively implement mitigation measures at the origin of pollution,the source of nitrate must first be identified. The stable nitrogen and oxygen isotopes of NO 3 - (ẟ 15 N and ẟ 18 O) have been widely used to identify NO 3 - sources in water, and their combination with other stable isotopes such as boron (ẟ 11 B) has further improved nitrate source identification. However, the use of these datasets has been limited due to their overlapping isotopic ranges, mixing between sources, and/or isotopic fractionation related to physicochemical processes. To overcome these limitations, we combined a multi-isotopic analysis with fecal indicator bacteria (FIB) and microbial source tracking (MST) techniques to improve nitrate origin identification. We applied this novel approach on 149 groundwater and 39 surface water samples distributed across Catalonia (NE Spain). A further 18 wastewater treatment plant (WWTP) effluents were also isotopically and biologically characterized. The groundwater and surface water results confirm that isotopes and MST analyses were complementary and provided more reliable information on the source of nitrate contamination. The isotope and MST data agreed or partially agreed in most of the samples evaluated (79 %). This approach was especially useful for nitrate pollution tracing in surface water but was also effective in groundwater samples influenced by organic nitrate pollution. Furthermore, the findings from the WWTP effluents suggest that the use of literature values to define the isotopic ranges of anthropogenic sources can constrain interpretations. We therefore recommend that local sources be isotopically characterized for accurate interpretations. For instance, the detection of MST inferred animal influence in some WWTP effluents, but the ẟ 11 B values were higher than those reported in the literature for wastewater. The results of this study have been used by local water authorities to review uncertain cases and identify new vulnerable zones in Catalonia according to the European Nitrate Directive (91/676/CEE)., 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 © 2020 Elsevier Ltd. All rights reserved.)

The complex and highly diverse microbial environment of drinking water, consisting mainly of bacteria at different metabolic states, is still underexplored. The aim of this work was to characterize the bacterial communities in tap water and bottled mineral water, the two predominant sources of drinking water in modern societies. A total of 11 tap water samples from a range of locations and distribution networks and 10 brands of bottled natural mineral water were analysed using two approaches: a) heterotrophic plate counts by matrix-assisted laser desorption/ionization time of flight mass-spectrometry (MALDI-TOF MS) for the culturable heterotrophic communities, and b) Illumina amplicon sequencing for total bacteria including non-culturable bacteria. Culturable heterotrophic bacteria were isolated in WPCA (ISO) agar at 22 ± 2 °C for 72 h and 2046 isolates were identified using MALDI-TOF MS. The Bruker Daltonics Library and a previously customized library (Drinking Water Library) were used as reference databases. For the total bacteria fraction, DNA was extracted from 6 L of water and submitted to Illumina 16S rRNA sequencing of the v4 region. Significant differences were observed between mineral and tap water, with a general dominance of Alphaproteobacteria (mainly the genus Blastomonas) in tap water and Gammaproteobacteria in mineral water with Acidovorax being the dominant genus in 3 out of 7 mineral water brands. The bacterial communities in the different brands of mineral water were highly diverse and characteristic of each one. Moreover, the season in which the water was bottled also affected the species distribution, with some of them identified in only one season. Among the culturable bacteria, the most abundant phylum was Proteobacteria (around 85% of the isolates), followed by Actinobacteria, Firmicutes and Bacteroidetes. Proteobacteria was also the most abundant phylum detected with Illumina sequencing (>99% of the reads). The two methods gave distinct results at the different taxonomic levels and could therefore have a complimentary application in the study of microbiota in mineral water environments. MALDI-TOF MS is a promising method for the rapid identification of heterotrophic bacteria in routine water analysis in the bottling industry. SIGNIFICANCE AND IMPACT OF THE STUDY: The complementarity of MALDI-TOF MS and NGS in the assessment of bacterial community diversity has been demonstrated in water intended for human consumption. The two methods are suitable for routine use in the water industry for water quality management., Competing Interests: Declaration of competing interest The authors have no conflict of interests to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Bacillus sp. SFC 500-1E is used for the effective treatment of tannery effluents since it consistently removes hexavalent chromium from diverse contaminated matrices. The aim of the present study was to complete identification of the strain through a polyphasic characterization, which included the pattern of carbohydrate utilization, fatty acids profile, multilocus sequence analysis, multiplex PCR profile and the analysis of the complete genome sequence. Morpho-physiological and biochemical characterization results and analysis of 16S rRNA sequences were not conclusive. The strain formed a monophyletic clade with B. toyonensis BCT-7112, B. thuringiensis MC28 and B. cereus Rock 1-3. However, genomic comparisons with type strains of B. cereus and B. thuringiensis showed that the isolated belonged to a different species. Results of this study highlight the relevance of the genome sequence of this strain, identified as Bacillus toyonensis SFC 500-1E, to expand knowledge of its bioremediation potential and to explore unknown decontamination activities., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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 m 3 /s in winter to 0.006 m 3 /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., 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 © 2020 Elsevier Ltd. All rights reserved.)

F-specific coliphages have been proposed as viral indicators of fecal pollution. These intestinal phages infect cells through the F-pili of the host strains used for their detection, Escherichia. coli HS/FAmp in the US-EPA standard method and Salmonella enterica WG49 in the ISO method. The recently designed Bluephage protocol allows the rapid detection of as low as one somatic coliphage in a working day. The current study describes a new Bluephage method designed to exclusively detect F-specific phages. It employs two new host strains, CB14 and CB16, which detect the same number of F-specific phages as their respective parental strains HS and WG49. In the Bluephage method, when the strain is lysed by bacteriophage infection, the yellow medium turns blue. As low as one F-specific phage was detected in 3 to 5 h by this approach and when the sample contained high phage concentrations, results were obtained in less than 3 h. The F-specific Bluephage method can be used with different sample volumes and allows phage quantification by the most probable number technique. Strain CB14 performed more consistently than CB16, with comparable detection efficiency after increasing the incubation time to 50 min without shaking., 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 © 2020 Elsevier Ltd. All rights reserved.)

Enteric bacteriophages (somatic coliphages, F-specific coliphages or both together) are now recognized as useful viral indicators in water, shellfish, and biosolids and are being progressively included in national and international sanitary regulations. Among them, somatic coliphages have an advantage in that they usually outnumber F-RNA coliphages in water environments. Their enumeration using Modified Scholten's (MS) media, following the ISO 10705-2 standard for the growth of Escherichia coli host strain WG5, is highly efficient and a common practice worldwide. These media contain a high concentration of nutrients, which may be modified to save costs without loss of bacterial growth host efficiency. This study explored reducing the concentration of nutrients in the current formulation and/or incorporating new components to improve the host bacterial growth and/or the enumeration of somatic coliphages at an affordable analytical cost. A twofold dilution of the original MS media was found not to affect the bacterial growth rate. The addition of combinations of assayed compounds to twofold diluted MS media slightly enhanced its analytical performance without altering bacterial growth. By generating savings in both cost and time while maintaining optimal results, media dilution could be applied to design new simple applications for coliphage enumeration.

Somatic and F-specific coliphages are gaining ground as indicators of fecal/viral pollution. Guidelines and regulations worldwide for monitoring water, biosolids and food are including them as parameters to assess quality and treatment efficiency. Robust methods to detect and quantify both groups of phages in water samples have been launched by agencies such as the International Standardization Organization (ISO) and the USA Environmental Protection Agency (USEPA). Although these methods have proved readily implementable in routine microbiology laboratories, faster and more user-friendly protocols will be highly welcome if coliphage detection becomes routine in water quality analysis. We here provide an overview of new approaches seeking to facilitate the detection of infectious coliphages included in guidelines and regulations. The improvements achieved suggest that streamlined kits able to provide results in a few hours at very reasonable costs will become available in the near future. The potential of molecular procedures and methods based on microelectronic sensors is also briefly discussed., Competing Interests: Declaration of Competing Interest One of the companies mentioned in the article (Bluephage S.L.) is a spin-off of the University of Barcelona. All the authors are affiliate to this University and have participated in the research and the scientific publications that have allowed to transfer the generated knowledge to this spin-off with whom they collaborate. This manuscript does not make any commercial use of any of the mentioned companies and no differential treatment is given to the spin-off of the University of Barcelona., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 log 10 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., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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 Bogotá 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 Bogotá River. The results obtained indicate that the selected microbial and molecular markers enable the determination of the source of faecal contamination in the Bogotá River by using different algorithms to develop prediction models., Competing Interests: Declaration of competing interest The authors have no conflicts of interest relevant to this article to disclose., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

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., 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 © 2019 Elsevier Ltd. All rights reserved.)

Solid or semisolid matrices polluted with fecal remnants can be highly loaded with pathogens, especially viruses, and play a substantial role in the persistence and dispersion of pathogens in the water cycle. Water quality regulations and guidelines are increasingly including bacteriophages infecting enteric bacteria as indicators of fecal and/or viral pollution. However, more data are needed about viral indicators in contaminated solids to develop effective sanitation strategies for the management of raw and treated sludge, fecal sludge, manures and slurries. Also, the exact role of sediments and soil in the transmission cycle of viral pathogens still needs to be determined. This review aims to provide an update on available data for concentrations of indicator bacteriophages in different solid matrices as well as their resistance to treatments and persistence in solids. The conclusion reached is that there is a need for improved and standardized methodologies for bacteriophage extraction, detection and enumeration in solids. Reports indicate that these contain higher levels of somatic coliphages in comparison with traditional bacterial indicators and F-specific RNA coliphages. Water body sediments and soil have been found to be notable reservoirs of somatic coliphages, which are more persistent in nature and resistant to sludge treatments than Escherichia coli and fecal coliforms and F-specific RNA coliphages. Thus, somatic coliphages show up as excellent complementary indicators for the prediction of pathogenic viruses in solids., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Natural mineral waters contain indigenous bacteria characteristic of each spring source. Once bottled, these communities change over time until the water is consumed. Bottle material is believed to play a major role in the succession of these populations, but very few studies to date have evaluated the effect of this material on bacterial communities. In this study, we examined the microbial community structure of three natural mineral waters over 3 months after bottling in glass and polyethylene terephthalate (PET) bottles. To this end, we used culture-dependent (heterotrophic plate count) and culture-independent methods (16S rRNA massive gene sequencing, denaturing gradient gel electrophoresis (DGGE) and fluorescent microscopy with vital dyes). Total and viable cell counts increased by around 1-2 log 10 units between 1 and 2 weeks after bottling and then remained constant over 3 months for all waters regardless of the bottle material. DGGE fingerprints and 16S rRNA massive sequencing analysis both indicated that different communities were established in the waters two weeks after bottling in the different bottle materials. In conclusion, no differences in total, viable and culturable bacteria counts were observed between mineral waters bottled with PET or glass during shelf life storage. Nevertheless, in spite of changes in the communities, each water brand and material presented a distinct microbial community structure clearly distinguishable from the others, which could be interesting for traceability purposes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Recent studies have shown that crAssphage is abundant in human faecal samples worldwide. It has thus been postulated as a potential microbial source tracking (MST) marker to detect human faecal pollution in water. However, an effective implementation of crAssphage in water management strategies will depend on an understanding of its environmental dynamics. In this work, the abundance and temporal distribution of crAssphage was analysed in the effluent of wastewater treatment plants using different sewage treatments, and in two rivers (water and sediments) that differ in pollution impact and flow regime. Additionally, the influence of environmental conditions (temperature and rainfall) on the removal of the marker was studied along a river section, and natural inactivation was assessed by a mesocosms approach. Molecular and culture-based tools were used to compare crAssphage abundance and dynamics with those of bacteria and bacteriophages currently applied as global indicators (E. coli, somatic coliphages, Bacteroides GA17 bacteriophages, and the human-associated MST markers HF183 and HMBif). CrAssphage concentrations in sewage effluent and river samples were similar to those of HF183 and HMBif and higher than other general and/or culture-based indicators (by 2-3 orders of magnitude). Measurement of crAssphage abundance revealed no temporal variability in the effluent, although rainfall events affected the dynamics, possibly through the mobilisation of sediments, where the marker was detected in high concentrations, and an increase in diffuse and point pollution. Another factor affecting crAssphage inactivation was temperature. Its persistence was longer compared with other bacterial markers analysed by qPCR but lower than culturable markers. The results of this study support the use of crAssphage as a human source tracking marker of faecal pollution in water, since it has similar abundances to other molecular human MST markers, yet with a longer persistence in the environment. Nevertheless, its use in combination with infectious bacteriophages is probably advisable., (Copyright © 2019 Elsevier Ltd. 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.)

The objective of this review is to assess the current state of knowledge of pathogens, general faecal indicators and human-specific microbial source tracking markers in sewage. Most of the microbes present in sewage are from the microbiota of the human gut, including pathogens. Bacteria and viruses are the most abundant groups of microbes in the human gut microbiota. Most reports on this topic show that raw sewage microbiological profiles reflect the human gut microbiota. Human and animal faeces share many commensal microbes as well as pathogens. Faecal-orally transmitted pathogens constitute a serious public health problem that can be minimized through sanitation. Assessing both the sanitation processes and the contribution of sewage to the faecal contamination of water bodies requires knowledge of the content of pathogens in sewage, microbes indicating general faecal contamination and microbes that are only present in human faecal remains, which are known as the human-specific microbial source-tracking (MST) markers. Detection of pathogens would be the ideal option for managing sanitation and determining the microbiological quality of waters contaminated by sewage; but at present, this is neither practical nor feasible in routine testing. Traditionally, faecal indicator bacteria have been used as surrogate indicators of general faecal residues. However, in many water management circumstances, it becomes necessary to detect both the origin of faecal contamination, for which MST is paramount, and live micro-organisms, for which molecular methods are not suitable. The presence and concentrations of pathogens, general faecal indicators and human-specific MST markers most frequently reported in different areas of the world are summarized in this review., (© 2018 The Society for Applied Microbiology.)

Aims: Evaluate the T 90 and compare the decay of different faecal indicator bacteria (FIB) and molecular microbial source tracking (MST) markers of human and animal sources during summer and winter., Methods and Results: The persistence of Escherichia coli and enterococci and several MST molecular markers targeting host-specific Bifidobacterium and Bacteroidales species (BifHM, BifCW, BifPL, HF183/BFD, Rum2Bac and Pig2Bac) was assessed at the same time using mesocosms. Dialysis bags filled with diluted wastewater from different sources were kept in an outdoor water tank and monitored regularly to assess the inactivation rates. The T 90 values of E. coli by culture methods ranged from 1·52 to 5·69 days in summer and 2·06 to 6·19 days in winter, whereas with qPCR 2·29-4·23 days in summer and 4·17-8·09 days in winter. T 90 values for enterocci ranged from 1·15 to 3·10 days in summer and from 3·01 to 5·46 days in winter. Significant differences were observed between faecal sources for both markers. For the MST makers similar T 90 values were obtained in summer (1·05-1·91 days), whereas higher variability was observed in winter (2·90-6·12 days)., Conclusions: Different decay rates were observed for the FIB from the different sources, especially for E. coli in ruminant samples. A higher variability among T 90 values of the different MST markers in winter was observed, whereas similar T 90 values were detected in summer highlighting the stronger effect of environmental parameters during this season., Significance and Impact of the Study: The diverse inactivation rates observed in bacteria from different faecal sources have implications when these rates are used to model faecal pollution in water. The use of FIBT 90 of different sources is essential to develop reliable predictive models. Since different inactivation of E. coli regarding the source of pollution has been observed, the source of the pollution has to be considered for modelling approaches., (© 2018 The Society for Applied Microbiology.)

Bacteriophages are ubiquitously distributed prokaryotic viruses that are more abundant than bacteria. As a consequence of their life cycle, phages can kidnap part of their host's genetic material, including antibiotic resistance genes (ARGs), which released phage particles transfer in a process called transduction. The spread of ARGs among pathogenic bacteria currently constitutes a serious global health problem. In this study, fresh vegetables (lettuce, spinach and cucumber), and cropland soil were screened by qPCR for ten ARGs (bla TEM , bla CTX-M-1 group, bla CTX-M-9 group, bla OXA-48 , bla VIM , mecA, sul1, qnrA, qnrS and armA) in their viral DNA fraction. The presence of ARGs in the phage DNA was analyzed before and after propagation experiments in an Escherichia coli host strain to evaluate the ability of the phage particles to infect a host. ARGs were found in the phage DNA fraction of all matrices, although with heterogeneous values. ARG prevalence was significantly higher in lettuce and soil, and the most common overall were β-lactamases. After propagation experiments, an increase in ARG densities in phage particles was observed in samples of all four matrices, confirming that part of the isolated phage particles were infectious. This study reveals the abundance of free, replicative ARG-containing phage particles in vegetable matrices and cropland soil. The particles are proposed as vehicles for resistance transfer in these environments, where they can persist for a long time, with the possibility of generating new resistant bacterial strains. Ingestion of these mobile genetic elements may also favor the emergence of new resistances, a risk not previously considered., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Numerous bacterial genetic markers are available for the molecular detection of human sources of fecal pollution in environmental waters. However, widespread application is hindered by a lack of knowledge regarding geographical stability, limiting implementation to a small number of well-characterized regions. This study investigates the geographic distribution of five human-associated genetic markers (HF183/BFDrev, HF183/BacR287, BacHum-UCD, BacH, and Lachno2) in municipal wastewaters (raw and treated) from 29 urban and rural wastewater treatment plants (750-4 400 000 population equivalents) from 13 countries spanning six continents. In addition, genetic markers were tested against 280 human and nonhuman fecal samples from domesticated, agricultural and wild animal sources. Findings revealed that all genetic markers are present in consistently high concentrations in raw (median log 10 7.2-8.0 marker equivalents (ME) 100 mL -1 ) and biologically treated wastewater samples (median log 10 4.6-6.0 ME 100 mL -1 ) regardless of location and population. The false positive rates of the various markers in nonhuman fecal samples ranged from 5% to 47%. Results suggest that several genetic markers have considerable potential for measuring human-associated contamination in polluted environmental waters. This will be helpful in water quality monitoring, pollution modeling and health risk assessment (as demonstrated by QMRAcatch) to guide target-oriented water safety management across the globe.

The use of somatic coliphages as indicators of fecal and viral pollution in water and food has great potential due to the reliability, reproducibility, speed and cost effectiveness of methods for their detection. Indeed, several countries already use this approach in their water management policies. Although standardized protocols for somatic coliphage detection are available, user-friendly commercial kits would facilitate their routine implementation in laboratories. The new method presented here allows detection of up to 1 somatic coliphage in under 3.5 h, well within one working day. The method is based on a modified Escherichia coli strain with knocked-out uidB and uidC genes, which encode the transport of glucuronic acid inside cells, and overexpressing uidA, which encodes the enzyme β-glucuronidase. The enzyme accumulated in the bacterial cells only has contact with its substrate after cell lysis, such as that caused by phages, since the strain cannot internalize the substrate. When the enzyme is released into the medium, which contains a chromogen analogous to glucuronic acid, it produces a change of color from yellow to dark blue. This microbiological method for the determination of fecal pollution via the detection of culturable microorganisms can be applied to diverse sample types and volumes for qualitative (presence/absence) and quantitative analysis and is the fastest reported to date., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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 T 90 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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The aim of this work was to assess the suitability of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) for routine heterotrophic monitoring in a drinking water treatment plant. Water samples were collected from raw surface water and after different treatments during two campaigns over a 1-year period. Heterotrophic bacteria were studied and isolates were identified by MALDI-TOF MS. Moreover, the diversity index and the coefficient of population similarity were also calculated using biochemical fingerprinting of the populations studied. MALDI-TOF MS enabled us to characterize and detect changes in the bacterial community composition throughout the water treatment plant. Raw water showed a large and diverse population which was slightly modified after initial treatment steps (sand filtration and ultrafiltration). Reverse osmosis had a significant impact on the microbial diversity, while the final chlorination step produced a shift in the composition of the bacterial community. Although MALDI-TOF MS could not identify all the isolates since the available MALDI-TOF MS database does not cover all the bacterial diversity in water, this technique could be used to monitor bacterial changes in drinking water treatment plants by creating a specific protein profile database for tracking purposes.

Bryozoans are among the most abundant and diverse members of the Antarctic benthos, however the role of bioactive metabolites in ecological interactions has been scarcely studied. To extend our knowledge about the chemical ecology of Antarctic bryozoans, crude ether extracts (EE) and butanol extracts (BE) obtained from two Antarctic common species (Cornucopina pectogemma and Nematoflustra flagellata), were tested for antibacterial and repellent activities. The extracts were screened for quorum quenching and antibacterial activities against four Antarctic bacterial strains (Bacillus aquimaris, Micrococcus sp., Oceanobacillus sp. and Paracoccus sp.). The Antarctic amphipod Cheirimedon femoratus and the sea star Odontaster validus were selected as sympatric predators to perform anti-predatory and substrate preference assays. No quorum quenching activity was detected in any of the extracts, while all EE exhibited growth inhibition towards at least one bacterium strain. Although the species were not repellent against the sea star, they caused repellence to the amphipods in both extracts, suggesting that defence activities against predation derive from both lipophilic and hydrophilic metabolites. In the substrate preference assays, one EE and one BE deriving from different specimens of the species C. pectogemma were active. This study reveals intraspecific variability of chemical defences and supports the fact that chemically mediated interactions are common in Antarctic bryozoans as means of protection against fouling and predation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Microbial source tracking (MST) has been extensively used to detect the sources of fecal pollution in water. The inclusion of MST in water management strategies improves the ecological status of the ecosystem and human and animal health under interdisciplinary analysis in all aspects of health care for humans, animals, and the environment (One Health approach). In this study, the performance of MST markers targeting host-specific Bacteroidales (HF183 and Rum-2-Bac) and species (HMBif and CWBif) were evaluated in raw sewage collected from human, ruminant, swine, and poultry sources in Tunisia, Cyprus, Ireland, and Spain. In addition, the ratio between somatic coliphages and bacteriophages infecting GA17 (SOMCPH/GA17PH) was measured in Tunisia and Spain. The obtained results showed variability of the bacterial markers between the four countries, suggesting that their usefulness could be affected by several conditions (dietary habits, agricultural practices, and climatic conditions) that differ between countries. The Rum-2-Bac marker stood out as a valid MST tool, particularly in Ireland, whereas CWBif was the best option in Tunisia, Spain, and Cyprus. The human-specific HMBif marker showed good sensitivity and specificity in Tunisia, Spain, and Ireland, whereas HF183 showed a low specificity. However, HF183 was suggested as a good human marker in Ireland and Cyprus because of its higher concentration than HMBif. Regarding viral markers, the ratio of SOMCPH/GA17PH showed a clear discrimination between human and nonhuman samples. The combined use of molecular bacterial markers and the ratio of SOMCPH/GA17PH may improve the success of MST., (Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.)

Climate change is expected to affect the Mediterranean region by causing an increase in the number of heavy rainfall events. The aim of this study was to assess the effect of extreme river flow variations due to rainfall on the persistence and mobilisation of various microorganisms. These included faecal pollution indicators (Escherichia coli (EC), somatic coliphages (SOMCPH) and sulphite reducing clostridia spores (SRC)), microbial source tracking indicators (Bacteroides thetaiotaomicron GA17 strain phages (GA17PH) and sorbitol fermenting bifidobacteria (SFBIF)), and two pathogens (Salmonella spp and Enterovirus). Water and sediment samples were taken at different distances from the river before and after heavy rainfall events. The microbial load was higher in sediment samples closer to the river course. The concentration of some faecal indicators (EC and SFBIF) increased in sediments and river water after rainfall events, whereas the most conservative parameter (SRC) showed almost no variation. After rainfall, the indicators persisted at a different rate. Salmonella spp and Enterovirus were detected in some samples but always at lower concentrations than the microbial indicators. In conclusion, sediments are reservoirs of faecal and MST indicators and pathogens and could therefore pose a risk of pathogen dissemination., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Bacillus toyonensis strain BCT-7112 T (NCIMB 14858 T ) has been widely used as an additive in animal nutrition for more than 30 years without reports of adverse toxigenic effects. However, this strain is resistant to chloramphenicol and tetracycline and it is generally considered inadvisable to introduce into the food chain resistance determinants capable of being transferred to other bacterial strains, thereby adding to the pool of such determinants in the gastro-enteric systems of livestock species. We therefore characterized the resistance phenotypes of this strain and its close relatives to determine whether they were of recent origin, and therefore likely to be transmissible. To this end we identified the genes responsible for chloramphenicol ( catQ ) and tetracycline ( tetM ) resistance and confirmed the presence of homologs in other members of the B. toyonensis taxonomic unit. Unexpectedly, closely related strains encoding these genes did not exhibit chloramphenicol and tetracycline resistance phenotypes. To understand the differences in the behaviors, we cloned and expressed the genes, together with their upstream regulatory regions, into Bacillus subtilis . The data showed that the genes encoded functional proteins, but were expressed inefficiently from their native promoters. B. toyonensis is a taxonomic unit member of the Bacillus cereus group ( sensu lato ). We therefore extended the analysis to determine the extent to which homologous chloramphenicol and tetracycline resistance genes were present in other species within this group. This analysis revealed that homologous genes were present in nearly all representative species within the B. cereus group ( sensu lato ). The absence of known transposition elements and the observations that they are found at the same genomic locations, indicates that these chloramphenicol and tetracycline resistance genes are of ancient origin and intrinsic to this taxonomic group, rather than recent acquisitions. In this context we discuss definitions of what are and are not intrinsic genes, an issue that is of fundamental importance to both Regulatory Authorities, and the animal feed and related industries.

Reverse osmosis membrane filtration technology (RO) is used to treat drinking water. After RO treatment, bacterial growth is still observed in water. However, it is not clear whether those microorganisms belong to species that can pose a health risk, such as Pseudomonas spp. The goal of this study is to characterize the bacterial isolates from a medium that is selective for Pseudomonas and Aeromonas which were present in the water fraction before and after the RO. To this end, isolates were recovered over two years and were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. They were then biochemically phenotyped and the population similarity indexes were calculated. The isolates were analysed for their capacity to form biofilms in vitro and antimicrobial susceptibility. There were significant differences between the microbial populations in water before and after RO. Furthermore, the structures of the populations analysed at the same sampling point were similar in different sampling campaigns. Some of the isolates had the capacity to form a biofilm and showed resistance to different antibiotics. A successful level filtration via RO and subsequent recolonization of the membrane with different species from those in the feed water was found. Pseudomonas aeruginosa was not recovered from among the isolates. This study increases the knowledge on the microorganisms present in water after RO treatment, with focus in one of the genus causing problems in RO systems associated with human health risk, Pseudomonas., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The study of bacterial communities throughout a drinking water treatment plant could provide a basic understanding of the effects of water processing that could then be used to improve the management of such plants. However, it is necessary to develop new analytical techniques that are sufficiently efficient, robust and fast for their effective and useful application in routine analysis. The aim of this study is therefore to assess the performance of matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), as compared to the PhenePlate™ system, for routine analysis in a drinking water treatment plant. To this end we studied a total of 277 colonies isolated in different seasons and from different points throughout the water treatment process, including: raw water, sand filtration, ultrafiltration, reverse osmosis and chlorination. The colonies were analysed using MALDI-TOF MS by direct deposition of the cells on the plate. The colonies were also biochemically fingerprinted using the PhenePlate™ system, clustered according to their similarity and a representative strain was selected for 16S rRNA gene sequencing and API ® gallery-based identification. The use of MALDI-TOF MS was reliable compared to the PhenePlate™ system and has the advantage of being faster and relatively cheap. Bacteria typing by MALDI-TOF MS is therefore a promising method to replace conventional routine phenotypic methods for the identification of bacteria in drinking water laboratories, thanks to its robustness. The major limiting factor for MALDI-TOF MS is the lack of a suitable mass spectra database; although each laboratory can develop its own library. This methodology will provide a tracking tool for companies to use in risk management and the detection of possible failures in both the water treatment processes and the distribution network, as well as offering characterization of the intrinsic microbial populations., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Bacteroides spp. have been proposed as indicators of fecal contamination in microbial source tracking (MST) methodologies. The aim of this study was to develop new qPCR assays that target host-specific Bacteroidal 16S ribosomal RNA genes, to determine the source of fecal contamination in water. Denaturing gradient gel electrophoresis (DGGE) was used to select for host-specific bands of Bacteroides associated with a fecal pollution source and later to design four qPCR host-specific assays. A set of common primers for Bacteroides spp., four different Bacteroides spp. host-associated hydrolysis probes (human, cattle, pig, and poultry), and one hydrolysis probe for the Bacteroides genus were designed. This set of qPCR assays together with other previously developed Bacteroidetes MST targets were used to analyze water samples with fecal contamination from the four sources studied. The host-specific Bacteroides qPCRs designed for human (HMprobeBac), pig (PGprobeBac), and poultry (PLprobeBac) were highly specific for its sources (1.0, 0.97, and 1.0, respectively) although its sensitivity was lower (0.45, 0.50, and 0.73, respectively). The cattle-specific qPCR was totally unspecific and was discarded for future experiments. When compared to previously designed assays, the human and pig qPCRs showed better accuracies (0.86 and 0.84) than their counterparts HF183 and Pig-2-Bac (0.38 and 0.65). Thus, the newly designed human, pig, and poultry qPCR assays outperform other methods developed until date and may be useful for source tracking purposes., (© 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Conventional bacterial indicators present serious drawbacks giving information about viral pathogens persistence during sludge hygienization treatments. This calls for the search of alternative viral indicators. Somatic coliphages' (SOMCPH) ability for acting as surrogates for enteroviruses was assessed in 47 sludge samples subjected to novel treatment processes. SOMCPH, infectious enteroviruses and genome copies of enteroviruses were monitored. Only one of these groups, the bacteriophages, was present in the sludge at concentrations that allowed the evaluation of treatment's performance. An indicator/pathogen relationship of 4 log10 (PFU/g dw) was found between SOMCPH and infective enteroviruses and their detection accuracy was assessed. The obtained results and the existence of rapid and standardized methods encourage the inclusion of SOMCPH quantification in future sludge directives. In addition, an existing real-time quantitative polymerase chain reaction (RT-qPCR) for enteroviruses was adapted and applied.

The microbiological indicators traditionally used to assess fecal contamination are insufficient to identify the source. The aim of this study was to detect microbial markers to identify the source of fecal pollution in the Bogotá River (Colombia). For this, we determined non-discriminating indicators such as Escherichia coli, somatic coliphages and phages infecting strain RYC2056 of Bacteroides, and potential source tracking markers as phages infecting strains GA17, HB13, and CA8 of Bacteroides, sorbitol-fermenting bifidobacteria, and molecular markers of Bifidobacterium adolescentis, Bifiodobacterium dentium, and Bacteroidetes in raw municipal wastewaters, slaughterhouse wastewaters, and the Bogotá River. Bacteriophages infecting Bacteroides strain GA17 and the molecular markers identified the wastewater sources. In contrast, sorbitol-fermenting bifidobacteria failed regarding specificity. In the Bogotá River, phages infecting strain GA17 were detected in all samples downstream of Bogotá, whereas they should be concentrated from 1 l samples in upstream samples containing less than 10(3) E. coli/100 ml to be detected. In the river water, the fraction of positive detections of molecular markers was lower than that of phages infecting strain GA17. The ratio SOMCPH/GA17PH was shown also to be a good marker. These results provide information that will allow focusing measures for sanitation of the Bogotá River.

The present research aims at the evaluation of the hygienization performances of innovative sludge treatment processes applied for the separated treatment of secondary sludge. Namely, two digestion pretreatments (sonication and thermal hydrolysis) and two sequential biological processes (mesophilic/thermophilic and anaerobic/aerobic digestion) were compared to the mesophilic (MAD) and thermophilic anaerobic digestion (TAD). Microbial indicators (Escherichia coli, somatic coliphages and Clostridium perfringens spores) and pathogens (Salmonella and enteroviruses), which show different resistances to treatment processes, were monitored in untreated and treated sludge. Overall, microbial load in secondary sludge was shown to be similar or lower than previously reported in literature for mixed sludge. Notably, the anaerobic/aerobic digestion process increased the removal of E. coli and somatic coliphages compared to the simple MAD and always achieved the hygienization requirement (2-log-unit removal of E. coli) proposed by EU Commission in the 3rd Working Document on sludge (April 2000) for the use of treated sludges in agriculture with restriction on their application. The microbial quality limits for the unrestricted use of sludge in agriculture (no Salmonella in 50 g wet weight (WW) and E. coli <500 CFU/g) were always met when thermal digestion or pretreatment was applied; however, the required removal level (6-log-unit removal of E. coli) could not be assessed due to the low level of this microorganism in raw sludge. Observed levels of indicator removal showed a higher resistance of viral particles to thermal treatment compared with bacterial cells and confirmed the suitability of somatic coliphages as indicators in thermal treatment processes.

This paper presents the current sewage sludge legislation in Europe and expected developments regarding the coming directives on the application of the "End-of-waste" criteria and on fertilizers. Discussion on sludge production and processing is also included. The Directive 86/278 has regulated the use in agriculture of residual sludge from domestic and urban wastewater. After 1986, this directive was transposed in the different member state legislation and currently the national limit values on heavy metals, some organic micropollutants and pathogens are placed in a rather wide range. This seems the inevitable consequence of different attitudes towards sludge management practices in the member states. The discussion by the European Joint Research Center (JRC) in Seville regarding application of end-of-waste criteria for compost and digestate has produced a final document (IPTS 2014) where sludge was excluded from the organic wastes admitted for producing an end-of-waste compost. Sludge processing in Europe seems addressed to different goals: sludge minimization, full stabilization and hygienization by thermal hydrolysis processes before anaerobic digestion, and on-site incineration by fluidized bed furnace. Thermophilic anaerobic digestion was applied with success on the Prague WWTP with a preliminary lysimeter centrifugation. Coming techniques, like wet oxidation and pyrolysis, are applied only on very few plants.

In this study we use a machine learning software (Ichnaea) to generate predictive models for water samples with different concentrations of fecal contamination (point source, moderate and low). We applied several MST methods (host-specific Bacteroides phages, mitochondrial DNA genetic markers, Bifidobacterium adolescentis and Bifidobacterium dentium markers, and bifidobacterial host-specific qPCR), and general indicators (Escherichia coli, enterococci and somatic coliphages) to evaluate the source of contamination in the samples. The results provided data to the Ichnaea software, that evaluated the performance of each method in the different scenarios and determined the source of the contamination. Almost all MST methods in this study determined correctly the origin of fecal contamination at point source and in moderate concentration samples. When the dilution of the fecal pollution increased (below 3 log10 CFU E. coli/100 ml) some of these indicators (bifidobacterial host-specific qPCR, some mitochondrial markers or B. dentium marker) were not suitable because their concentrations decreased below the detection limit. Using the data from source point samples, the software Ichnaea produced models for waters with low levels of fecal pollution. These models included some MST methods, on the basis of their best performance, that were used to determine the source of pollution in this area. Regardless the methods selected, that could vary depending on the scenario, inductive machine learning methods are a promising tool in MST studies and may represent a leap forward in solving MST cases., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Aims: Escherichia coli (EC) is the primary indicator micro-organism in regulations for sewage sludge reuse. The aim of this work was to assess the ability of EC to enter and recover from a viable-but-not-culturable state (VBNC) after sludge hygienization treatments., Methods and Results: The persistence of EC, somatic coliphages (SOMCPH), spores of sulphite-reducing clostridia (SRC) and Salmonella spp. was assessed in digested sludge after different pasteurization treatments and storage conditions. Pasteurization at 55°C produced EC-injured cells that were resuscitated during the first 24 h. Different sludge treatments altered the inactivation kinetics of EC, while SOMCPH and SRC did not resuscitate and showed lower die-off than EC. No regrowth was observed in stored sludge for up to 60 days., Conclusions: EC monitoring by culturable methods is not by itself a suitable method for assessing the hygienization achieved in sludge as EC can enter into VBNC from which it can recover during the first hours of storage., Significance and Impact of the Study: The regulations should indicate the time when monitoring of EC should be performed to avoid the period when EC can resuscitate from VBNC or add alternative microbial indicators, such as SOMCPH, which do not have a VBNC state., (© 2014 The Society for Applied Microbiology.)

The antimicrobial activity of Antarctic bryozoans and the ecological functions of the chemical compounds involved remain largely unknown. To determine the significant ecological and applied antimicrobial effects, 16 ether and 16 butanol extracts obtained from 13 different bryozoan species were tested against six Antarctic (including Psychrobacter luti, Shewanella livingstonensis and 4 new isolated strains) and two bacterial strains from culture collections (Escherichia coli and Bacillus cereus). Results from the bioassays reveal that all ether extracts exhibited antimicrobial activity against some bacteria. Only one butanol extract produced inhibition, indicating that antimicrobial compounds are mainly lipophilic. Ether extracts of the genus Camptoplites inhibited the majority of bacterial strains, thus indicating a broad-spectrum of antimicrobial activity. Moreover, most ether extracts presented activities against bacterial strains from culture collections, suggesting the potential use of these extracts as antimicrobial drugs against pathogenic bacteria., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The complete genome sequencing of Bacillus toyonensis, the active ingredient of the feed additive Toyocerin(®), has revealed the presence of tetM and cat genes, a tetracycline and a chloramphenicol resistance gene, respectively. The aim of this study was to determine whether the use of Toyocerin(®) (viable spores of B. toyonensis) as a probiotic in feedstuff increased the abundance of tetracycline and chloramphenicol resistant bacteria in the intestinal tracts of piglets and Holstein bulls. To this end, qPCRs were designed to quantify the abundances of tetM and cat genes and B. toyonensis in the intestinal content of animals treated and non-treated with Toyocerin(®). Additionally, the culturable bacterial populations resistant to tetracycline or chloramphenicol were enumerated by plate counting. No statistical significances were detected between the concentrations of tetracycline or chloramphenicol resistant bacterial populations in treated and non-treated animals. The concentrations of tetM and cat in most of the treated animals were similar to those of B. toyonensis. Furthermore, tetM and cat genes were also detected in some non-treated animals, although in low concentrations. These results suggest that tetM and cat genes are already circulating among the commensal microbiota regardless of the use of Toyocerin(®). The use of Toyocerin(®) as a supplement in feedstuff does not increase the abundances of tetracycline and chloramphenicol resistant bacteria in the intestinal tracts of piglets and Holstein bulls beyond the contribution directly associated to the introduction of B. toyonensis spores through diet., (Copyright © 2014 Elsevier B.V. All rights reserved.)