A Chemo-Ecological Investigation of Dendrilla antarctica Topsent, 1905: Identification of Deceptionin and the Effects of Heat Stress and Predation Pressure on Its Terpene Profiles.

Marine sponges usually host a wide array of secondary metabolites that play crucial roles in their biological interactions. The factors that influence the intraspecific variability in the metabolic profile of organisms, their production or ecological function remain generally unknown. Understanding this may help predict changes in biological relationships due to environmental variations as a consequence of climate change. The sponge Dendrilla antarctica is common in shallow rocky bottoms of the Antarctic Peninsula and is known to produce diterpenes that are supposed to have defensive roles. Here we used GC-MS to determine the major diterpenes in two populations of D. antarctica from two islands, Livingston and Deception Island (South Shetland Islands). To assess the potential effect of heat stress, we exposed the sponge in aquaria to a control temperature (similar to local), heat stress (five degrees higher) and extreme heat stress (ten degrees higher). To test for defence induction by predation pressure, we exposed the sponges to the sea star Odontaster validus and the amphipod Cheirimedon femoratus. Seven major diterpenes were isolated and identified from the samples. While six of them were already reported in the literature, we identified one new aplysulphurane derivative that was more abundant in the samples from Deception Island, so we named it deceptionin (7). The samples were separated in the PCA space according to the island of collection, with 9,11-dihydrogracilin A (1) being more abundant in the samples from Livingston, and deceptionin (7) in the samples from Deception. We found a slight effect of heat stress on the diterpene profiles of D. antarctica, with tetrahydroaplysulphurin-1 (6) and the gracilane norditerpene 2 being more abundant in the group exposed to heat stress. Predation pressure did not seem to influence the metabolite production. Further research on the bioactivity of D. antarctica secondary metabolites, and their responses to environmental changes will help better understand the functioning and fate of the Antarctic benthos.

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.

Traceability of different brands of bottled mineral water during shelf life, using PCR-DGGE and next generation sequencing techniques.

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 log10 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.

Heterotrophic monitoring at a drinking water treatment plant by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry after different drinking water treatments.

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.

Heterogeneity in phage induction enables the survival of the lysogenic population.

Lysogeny by temperate phages provides novel functions for bacteria and shelter for phages. However, under conditions that activate the phage lytic cycle, the benefit of lysogeny becomes a paradox that poses a threat for bacterial population survival. Using Escherichia coli lysogens for Shiga toxin (Stx) phages as model, we demonstrate how lysogenic bacterial populations circumvent extinction after phage induction. A fraction of cells maintains lysogeny, allowing population survival, whereas the other fraction of cells lyse, increasing Stx production and spreading Stx phages. The uninduced cells were still lysogenic for the Stx phage and equally able to induce phages as the original cells, suggesting heterogeneity of the E. coli lysogenic population. The bacterial population can modulate phage induction under stress conditions by the stress regulator RpoS. Cells overexpressing RpoS reduce Stx phage induction and compete with and survive better than cells with baseline RpoS levels. Our observations suggest that population heterogeneity in phage induction could be widespread among other bacterial genera and we propose this is a mechanism positively selected to prevent the extinction of the lysogenic population that can be modulated by environmental conditions.

Pseudomonas-related populations associated with reverse osmosis in drinking water treatment.

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.

Somatic coliphages as surrogates for enteroviruses in sludge hygienization treatments.

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.

Antimicrobial resistance and presence of the SXT mobile element in Vibrio spp. isolated from aquaculture facilities.

The aim of this work was to assess the susceptibility of Vibrio spp. strains isolated from fish cultures against some usually applied antibiotics and the occurrence of the SXT mobile genetic element among them. Antimicrobial resistance was assessed by the standard disk diffusion technique while the presence of the SXT mobile genetic element was determined by conventional PCR. High levels of resistance to ampicillin (70%), cefoxitin (44%), streptomycin (31%), aztreonam (25%) and sulfamethoxazole (21%) were detected, and a high inter-and-intraspecies diversity in the resistance profile was observed for the majority of the analysed isolates. The SXT mobile genetic element was detected in only 4 isolates belonging to the species V. diazotrophicus (1), V. mediterranei (2) and V. vulnificus (1), which showed a variable antibiotic resistance profile. Horizontal antibiotic resistance gene transfer from the V. diazotrophicus SXT-positive strain to a laboratory E. coli strain was demonstrated under laboratory conditions. Our results suggest that the Vibrio spp. isolated from aquaculture facilities analysed in this study, although not being pathogenic, they constitute a source of antimicrobial resistance genes that could be mobilized to other bacterial populations through mobile genetic elements. However, the low occurrence of the SXT element in these isolates supports the hypothesis that this element is not involved in the development of resistance in the majority of Vibrio spp. in the examined aquaculture facilities.

Exploring plastic biofilm formation and Escherichia coli colonisation in marine environments.

Microorganisms, including potential pathogens, can colonise plastic surfaces in aquatic environments. This study investigates the colonisation of plastic pellets by Escherichia coli (E. coli) as a proxy for faecal pathogens in aquatic environments. Plastic pellets from a polluted beach were placed in seawater aquaria spiked with E. coli. Diverse bacteria, primarily from the Proteobacteria phylum, rapidly colonised the pellets within 24 h, with notable species known for plastic or hydrocarbon degradation. Over 26 days, biofilms formed on the plastic surfaces, reaching bacterial populations of up to 6.8·105 gene copies (gc) of the 16S rRNA mm-2. E. coli, was detected in the pellets for up to 7 days using culture methods, exhibiting varying attachment densities regardless of source or environmental factors. The study highlights plastic biofilms as reservoirs for E. coli, contributing to the survival and persistence of faecal bacteria in aquatic systems. These findings deepen our understanding of the risks associated with plastic pollution in marine settings, offering insights into the behaviour of faecal indicators and their implications for water quality assessments, while providing valuable information on potential pathogen dissemination within plastic-associated microbial communities.

Detection of faecal bacteria and antibiotic resistance genes in biofilms attached to plastics from human-impacted coastal areas.

Plastics have been proposed as vectors of bacteria as they act as a substrate for biofilms. In this study, we evaluated the abundance of faecal and marine bacteria and antibiotic resistance genes (ARGs) from biofilms adhered to marine plastics. Floating plastics and plastics from sediments were collected in coastal areas impacted by human faecal pollution in the northwestern Mediterranean Sea. Culture and/or molecular methods were used to quantify faecal indicators (E. coli, Enterococci and crAssphage), and the ARGs sulI, tetW and blaTEM and the 16S rRNA were detected by qPCR assays. Pseudomonas and Vibrio species and heterotrophic marine bacteria were also analysed via culture-based methods. Results showed that, plastic particles covered by bacterial biofilms, primarily consisted of marine bacteria including Vibrio spp. Some floating plastics had a low concentration of viable E. coli and Enterococci (42% and 67% of the plastics respectively). Considering the median area of the plastics, we detected an average of 68 cfu E. coli per item, while a higher concentration of E. coli was detected on individual plastic items, when compared with 100 ml of the surrounding water. Using qPCR, we quantified higher values of faecal indicators which included inactive and dead microorganisms, detecting up to 2.6 × 102 gc mm-2. The ARGs were detected in 67-88% of the floating plastics and in 29-57% of the sediment plastics with a concentration of up to 6.7 × 102 gc mm-2. Furthermore, enrichment of these genes was observed in biofilms compared with the surrounding water. These results show that floating plastics act as a conduit for both the attachment and transport of faecal microorganisms. In contrast, low presence of faecal indicators was detected in plastic from seafloor sediments. Therefore, although in low concentrations, faecal bacteria, and potential pathogens, were identified in marine plastics, further suggesting plastics act as a reservoir of pathogens and ARGs.

Dominance of phage particles carrying antibiotic resistance genes in the viromes of retail food sources.

The growth of antibiotic resistance has stimulated interest in understanding the mechanisms by which antibiotic resistance genes (ARG) are mobilized. Among them, studies analyzing the presence of ARGs in the viral fraction of environmental, food and human samples, and reporting bacteriophages as vehicles of ARG transmission, have been the focus of increasing research. However, it has been argued that in these studies the abundance of phages carrying ARGs has been overestimated due to experimental contamination with non-packaged bacterial DNA or other elements such as outer membrane vesicles (OMVs). This study aims to shed light on the extent to which phages, OMVs or contaminating non-packaged DNA contribute as carriers of ARGs in the viromes. The viral fractions of three types of food (chicken, fish, and mussels) were selected as sources of ARG-carrying phage particles, whose ability to infect and propagate in an Escherichia coli host was confirmed after isolation. The ARG-containing fraction was further purified by CsCl density gradient centrifugation and, after removal of DNA outside the capsids, ARGs inside the particles were confirmed. The purified fraction was stained with SYBR Gold, which allowed the visualization of phage capsids attached to and infecting E. coli cells. Phages with Myoviridae and Siphoviridae morphology were observed by electron microscopy. The proteins in the purified fraction belonged predominantly to phages (71.8% in fish, 52.9% in mussels, 78.7% in chicken sample 1, and 64.1% in chicken sample 2), mainly corresponding to tail, capsid, and other structural proteins, whereas membrane proteins, expected to be abundant if OMVs were present, accounted for only 3.8-21.4% of the protein content. The predominance of phage particles in the viromes supports the reliability of the protocols used in this study and in recent findings on the abundance of ARG-carrying phage particles.

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.

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.

Quorum-sensing regulates biofilm formation in Vibrio scophthalmi.

BACKGROUND: In a previous study, we demonstrated that Vibrio scophthalmi, the most abundant Vibrio species among the marine aerobic or facultatively anaerobic bacteria inhabiting the intestinal tract of healthy cultured turbot (Scophthalmus maximus), contains at least two quorum-sensing circuits involving two types of signal molecules (a 3-hydroxy-dodecanoyl-homoserine lactone and the universal autoinducer 2 encoded by luxS). The purpose of this study was to investigate the functions regulated by these quorum sensing circuits in this vibrio by constructing mutants for the genes involved in these circuits. RESULTS: The presence of a homologue to the Vibrio harveyi luxR gene encoding a main transcriptional regulator, whose expression is modulated by quorum-sensing signal molecules in other vibrios, was detected and sequenced. The V. scophthalmi LuxR protein displayed a maximum amino acid identity of 82% with SmcR, the LuxR homologue found in Vibrio vulnificus. luxR and luxS null mutants were constructed and their phenotype analysed. Both mutants displayed reduced biofilm formation in vitro as well as differences in membrane protein expression by mass-spectrometry analysis. Additionally, a recombinant strain of V. scophthalmi carrying the lactonase AiiA from Bacillus cereus, which causes hydrolysis of acyl homoserine lactones, was included in the study. CONCLUSIONS: V. scophthalmi shares two quorum sensing circuits, including the main transcriptional regulator luxR, with some pathogenic vibrios such as V. harveyi and V. anguillarum. However, contrary to these pathogenic vibrios no virulence factors (such as protease production) were found to be quorum sensing regulated in this bacterium. Noteworthy, biofilm formation was altered in luxS and luxR mutants. In these mutants a different expression profile of membrane proteins were observed with respect to the wild type strain suggesting that quorum sensing could play a role in the regulation of the adhesion mechanisms of this bacterium.

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.

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.

Bacteriophage-encoding cytolethal distending toxin type V gene induced from nonclinical Escherichia coli isolates.

Cytolethal distending toxin (Cdt) is produced by a variety of pathogenic bacteria, including pathogenic serotypes of Shiga toxin-producing Escherichia coli (STEC). The Cdt family comprises five variants (Cdt-I to Cdt-V) encoded by three genes located within the chromosome or plasmids or, in the case of Cdt-I, within bacteriophages. In this study, we evaluated the occurrence of the cdt gene in a collection of 140 environmental STEC isolates. cdt was detected in 12.1% of strains, of which five strains carried inducible bacteriophages containing the Cdt-V variant. Two Cdt-V phages of the Siphoviridae morphology lysogenized Shigella sonnei, generating two lysogens: a single Cdt phage lysogen and a double lysogen, containing a Cdt phage and an Stx phage, both from the wild-type strain. The rates of induction of Cdt phages were evaluated by quantitative PCR, and spontaneous induction of Cdt-V phage was observed, whereas induction of Stx phage in the double lysogen was mitomycin C dependent. The Cdt distending effect was observed in HeLa cells inoculated with the supernatant of the Cdt-V phage lysogen. A ClaI fragment containing the cdt-V gene of one phage was cloned, and sequencing confirmed the presence of Cdt-V, as well as a fragment downstream from the cdt homolog to gpA, encoding a replication protein of bacteriophage P2. Evaluation of Cdt-V phages in nonclinical water samples showed densities of 10(2) to 10(9) gene copies in 100 ml, suggesting the high prevalence of Cdt phages in nonclinical environments.

Electrochemical detection of quorum sensing signaling molecules by dual signal confirmation at microelectrode arrays.

n-Acyl homoserine lactones (AHLs) are produced by gram-negative bacteria to regulate gene expression in a cell density dependent manner. For instance, expression of virulence factors by pathogens such as Pseudomonas aeruginosa is induced only when a threshold concentration of AHLs is reached, which indicates that the bacterial population is big enough to promote infection. In this study, the indicator strain Agrobacterium tumefaciens NTL4 (pZLR4), which carries a ß-galactosidase (ß-gal) reporter gene under the control of a quorum sensing promoter, was used to develop an electrochemical biosensor to detect AHLs using the model n-(3-oxo)-dodecanoyl-L-homoserine lactone (oxo-C12-HSL), an AHL previously detected in cystic fibrosis patients infected with P. aeruginosa. The substrate 4-aminophenyl ß-D-galactopyranoside was used to detect ß-gal activity by cyclic voltammetry. Furthermore, simultaneous monitoring of substrate consumption and p-aminophenol production by ß-gal allowed on-chip result verification by dual-signal confirmation. The sensor exhibited high reproducibility and accurately detected oxo-C12-HSL in a low picomolar to low nanomolar range in spiked liquid cultures and artificial saliva, as well as AHLs naturally released by P. aeruginosa in culture supernatants. Moreover, detection took just 2 h, required no sample pretreatment or preconcentration steps, and was easier and faster than traditional methods.

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.

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

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