Performance of bacterial and mitochondrial qPCR source tracking methods: A European multi-center study.

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 log10 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.

Characterization of crAss-like phage isolates highlights Crassvirales genetic heterogeneity and worldwide distribution.

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.

Have genetic targets for faecal pollution diagnostics and source tracking revolutionized water quality analysis yet?

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.

Culture and molecular methods as complementary tools for water quality management.

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.

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.

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.

Rapid and improved identification of drinking water bacteria using the Drinking Water Library, a dedicated MALDI-TOF MS database.

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.

Bacteriophages as Fecal Pollution Indicators.

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.

Bacteriophages Are Good Estimators of Human Viruses Present in Water.

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.

Combining multi-isotopic and molecular source tracking methods to identify nitrate pollution sources in surface and groundwater.

Nitrate (NO3-) 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 NO3- (ẟ15N and ẟ18O) have been widely used to identify NO3- sources in water, and their combination with other stable isotopes such as boron (ẟ11B) 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 ẟ11B 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).

Unravelling the composition of tap and mineral water microbiota: Divergences between next-generation sequencing techniques and culture-based methods.

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.

Polyphasic characterization and identification of the bioremediation agent Bacillus sp. SFC 500-1E.

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.

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.

F-specific coliphage detection by the Bluephage method.

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.

Fast and easy methods for the detection of coliphages.

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.

Review: Indicator bacteriophages in sludge, biosolids, sediments and soils.

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.

Evaluation of New Components in Modified Scholten's Medium for the Detection of Somatic Coliphages.

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.