Adapted methods for monitoring influenza virus and respiratory syncytial virus in sludge and wastewater.

Wastewater-based surveillance constitutes a valuable methodology for the continuous monitoring of viral circulation, with the capacity to function as an early warning system. It holds particular significance in scenarios where respiratory viruses exhibit overlapping clinical presentations, as occurs with SARS-CoV-2, influenza virus (IV), and respiratory syncytial virus (RSV), and allows seasonal virus outbreaks to be distinguished from COVID-19 peaks. Furthermore, sewage sludge, given it harbors concentrated human waste from a large population, serves as a substantial reservoir for pathogen detection. To effectively integrate wastewater-based epidemiology into infectious disease surveillance, the detection methods employed in wastewater samples must be adapted to the distinct characteristics of sludge matrices. In this study, we adapted and applied protocols for the detection of IV and RSV in sewage sludge, comparing their performance with the results obtained in wastewater. To assess the efficiency of these protocols, sludge and wastewater samples were spiked with IV and RSV RNA, either free or incorporated in lentiviral particles. Samples were concentrated using the aluminum hydroxide adsorption-precipitation method before viral RNA extraction. Absolute virus quantification was carried out by RT-qPCR, including an internal control to monitor potential inhibitory factors. Recovery efficiencies for both free IV and RSV RNA were 60 % in sludge, and 75 % and 71 % respectively in wastewater, whereas the values for IV and RSV RNA in lentiviral particles were 16 % and 10 % in sludge and 21 % and 17 % in wastewater respectively. Additionally, the protocol enabled the quantification of naturally occurring IV and RSV in wastewater and sludge samples collected from two wastewater treatment plants during the winter months, thus affirming the efficacy of the employed methodologies.

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.

Monitoring influenza and respiratory syncytial virus in wastewater. Beyond COVID-19.

Wastewater-based surveillance can be a valuable tool to monitor viral circulation and serve as an early warning system. For respiratory viruses that share similar clinical symptoms, namely SARS-CoV-2, influenza, and respiratory syncytial virus (RSV), identification in wastewater may allow differentiation between seasonal outbreaks and COVID-19 peaks. In this study, to monitor these viruses as well as standard indicators of fecal contamination, a weekly sampling campaign was carried out for 15 months (from September 2021 to November 2022) in two wastewater treatment plants that serve the entire population of Barcelona (Spain). Samples were concentrated by the aluminum hydroxide adsorption-precipitation method and then analyzed by RNA extraction and RT-qPCR. All samples were positive for SARS-CoV-2, while the positivity rates for influenza virus and RSV were significantly lower (10.65 % for influenza A (IAV), 0.82 % for influenza B (IBV), 37.70 % for RSV-A and 34.43 % for RSV-B). Gene copy concentrations of SARS-CoV-2 were often approximately 1 to 2 logarithmic units higher compared to the other respiratory viruses. Clear peaks of IAV H3:N2 in February and March 2022 and RSV in winter 2021 were observed, which matched the chronological incidence of infections recorded in the Catalan Government clinical database. In conclusion, the data obtained from wastewater surveillance provided new information on the abundance of respiratory viruses in the Barcelona area and correlated favorably with clinical data.

The Prophage and Us-Shiga Toxin Phages Revisited.

The authors first met in 1998 at the University of Würzburg, Germany, at the Institute of Hygiene and Microbiology, in Helge Karch's lab, where Herbert Schmidt worked as a PostDoc and Maite Muniesa visited the lab for a postdoctoral research stay to work on phages encoding Shiga toxin 2e (Stx2e) [...].

Identification of a virulent phage infecting species of Nitrosomonas.

In the first and limiting step of nitrification, ammonia (NH3) is oxidised to nitrite (NO2-) by the action of some prokaryotes, including bacteria of the Nitrosomonas genus. A potential approach to nitrification inhibition would be through the application of phages, but until now this method has been unexplored and no virulent phages that infect nitrifying bacteria have been described. In this study, we report the isolation of the first phage infecting some Nitrosomonas species. This polyvalent virulent phage (named ΦNF-1) infected Nitrosomonas europaea, Nitrosomonas communis, and Nitrosomonas nitrosa. Phage ΦNF-1 has the morphology of the Podoviridae family, a dsDNA genome of 41,596 bp and a 45.1 % GC content, with 50 predicted open reading frames. Phage ΦNF-1 was found to inhibit bacterial growth and reduce NH4+ consumption in the phage-treated cultures. The application of phages as biocontrol agents could be a useful strategy for nitrification inhibition without the restrictions associated with chemical inhibitors.

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.

Prevalence of bacterial genes in the phage fraction of food viromes.

Antibiotic resistance genes (ARGs) have been identified in viral DNA isolated from different kinds of food, but little is known about their origin. In this study, twenty-one viromes were analyzed from samples of food previously reported to carry ARGs, including meat (poultry, veal, and pork), fish (Mediterranean, Atlantic, frozen, farmed and shellfish) and vegetables (lettuce, cucumber, and spinach). Classification of the contigs by Kraken revealed a large percentage of unclassified contigs (43.7-98.2%) in all the viromes. Only 0.05-7.1% of the contigs were identified as viral and of these, more than 91% belonged to different bacteriophage families, Podophages and Siphophages being the most prevalent. According to VirSorter, the largest number of viral contigs were derived from viromes of shellfish, followed by spinach. Spinach viromes also included the largest number of phage sequences identified by PHASTER. The abundant presence of bacterial genes in the viromes, including 16S rRNA genes, was attributed to the phage packaging of the bacterial genome fragments, as no bacterial DNA was found outside the viral capsids. The detection of 16S rRNA genes in the different viromes allowed diverse phage bacterial hosts to be identified. The three major functional groups of genes determined were related to metabolism, detoxification/resistance, and above all, biosynthesis. Various ARGs were quantified in the viromes by qPCR, the most prevalent being ß-lactamases, particularly blaTEM. Analysis of ARG diversity in the viromes by Prokka and CARD revealed various resistance-related genes, whereas a more restrictive search by ResFinder identified blaTEM in all the food viromes, blaOXA in Atlantic fish-1 and spinach-2, oqxB in lettuce-1, and dfr in spinach-2. The presence of ARGs in the food viromes points to bacterial DNA mobilization by transduction mechanisms. Transduction of resistances by phage particles may therefore contribute to the emergence of resistant strains along the food chain and should be monitored.

Essential Topics for the Regulatory Consideration of Phages as Clinically Valuable Therapeutic Agents: A Perspective from Spain.

Antibiotic resistance is one of the major challenges that humankind shall face in the short term. (Bacterio)phage therapy is a valuable therapeutic alternative to antibiotics and, although the concept is almost as old as the discovery of phages, its wide application was hindered in the West by the discovery and development of antibiotics in the mid-twentieth century. However, research on phage therapy is currently experiencing a renaissance due to the antimicrobial resistance problem. Some countries are already adopting new ad hoc regulations to favor the short-term implantation of phage therapy in clinical practice. In this regard, the Phage Therapy Work Group from FAGOMA (Spanish Network of Bacteriophages and Transducing Elements) recently contacted the Spanish Drugs and Medical Devices Agency (AEMPS) to promote the regulation of phage therapy in Spain. As a result, FAGOMA was asked to provide a general view on key issues regarding phage therapy legislation. This review comes as the culmination of the FAGOMA initiative and aims at appropriately informing the regulatory debate on phage therapy.

Chicken liver is a potential reservoir of bacteriophages and phage-derived particles containing antibiotic resistance genes.

Poultry meat production is one of the most important agri-food industries in the world. The selective pressure exerted by widespread prophylactic or therapeutic use of antibiotics in intensive chicken farming favours the development of drug resistance in bacterial populations. Chicken liver, closely connected with the intestinal tract, has been directly involved in food-borne infections and found to be contaminated with pathogenic bacteria, including Campylobacter and Salmonella. In this study, 74 chicken livers, divided into sterile and non-sterile groups, were analysed, not only for microbial indicators but also for the presence of phages and phage particles containing antibiotic resistance genes (ARGs). Both bacteria and phages were detected in liver tissues, including those dissected under sterile conditions. The phages were able to infect Escherichia coli and showed a Siphovirus morphology. The chicken livers contained from 103 to 106 phage particles per g, which carried a range of ARGs (blaTEM , blaCTx-M-1 , sul1, qnrA, armA and tetW) detected by qPCR. The presence of phages in chicken liver, mostly infecting E. coli, was confirmed by metagenomic analysis, although this technique was not sufficiently sensitive to identify ARGs. In addition, ARG-carrying phages were detected in chicken faeces by qPCR in a previous study of the group. Comparison of the viromes of faeces and liver showed a strong coincidence of species, which suggests that the phages found in the liver originate in faeces. These findings suggests that phages, like bacteria, can translocate from the gut to the liver, which may therefore constitute a potential reservoir of antibiotic resistance genes.

Antibiotic resistance in the viral fraction of dairy products and a nut-based milk.

Phages, the most abundant biological entities in the biosphere, can carry different bacterial genes, including those conferring antibiotic resistance. In this study, dairy products were analyzed by qPCR for the presence of phages and phage particles containing antibiotic resistance genes (ARGs). Eleven ARGs were identified in 50 samples of kefir, yogurt, milk, fresh cheese and nut-based milk (horchata), purchased from local retailers in Barcelona. Propagation experiments showed that at least some of the phages isolated from these samples infected Escherichia coli WG5, which was selected as the host strain because it does not contain prophages or ARGs in its genome. Electron microscopy revealed that the phage particles showed morphologies compatible with the Myoviridae and Siphoviridae families. Our results show that dairy products contain ARGs within infectious phage particles and may therefore serve as a reservoir of ARGs that can be mobilized to susceptible hosts, both in the food matrix and in the intestinal tract after ingestion.

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.

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.

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.

Population genomics and antimicrobial resistance dynamics of Escherichia coli in wastewater and river environments.

Aquatic environments are key niches for the emergence, evolution and dissemination of antimicrobial resistance. However, the population diversity and the genetic elements that drive the dynamics of resistant bacteria in different aquatic environments are still largely unknown. The aim of this study was to understand the population genomics and evolutionary events of Escherichia coli resistant to clinically important antibiotics including aminoglycosides, in anthropogenic and natural water ecosystems. Here we show that less different E. coli sequence types (STs) are identified in wastewater than in rivers, albeit more resistant to antibiotics, and with significantly more plasmids/cell (6.36 vs 3.72). However, the genomic diversity within E. coli STs in both aquatic environments is similar. Wastewater environments favor the selection of conserved chromosomal structures associated with diverse flexible plasmids, unraveling promiscuous interplasmidic resistance genes flux. On the contrary, the key driver for river E. coli adaptation is a mutable chromosome along with few plasmid types shared between diverse STs harboring a limited resistance gene content.

Bacteriophages of Shiga Toxin-Producing Escherichia coli and Their Contribution to Pathogenicity.

Shiga toxins (Stx) of Shiga toxin-producing Escherichia coli (STEC) are generally encoded in the genome of lambdoid bacteriophages, which spend the most time of their life cycle integrated as prophages in specific sites of the bacterial chromosome. Upon spontaneous induction or induction by chemical or physical stimuli, the stx genes are co-transcribed together with the late phase genes of the prophages. After being assembled in the cytoplasm, and after host cell lysis, mature bacteriophage particles are released into the environment, together with Stx. As members of the group of lambdoid phages, Stx phages share many genetic features with the archetypical temperate phage Lambda, but are heterogeneous in their DNA sequences due to frequent recombination events. In addition to Stx phages, the genome of pathogenic STEC bacteria may contain numerous prophages, which are either cryptic or functional. These prophages may carry foreign genes, some of them related to virulence, besides those necessary for the phage life cycle. Since the production of one or more Stx is considered the major pathogenicity factor of STEC, we aim to highlight the new insights on the contribution of Stx phages and other STEC phages to pathogenicity.

Isolation and Characterization of Shiga Toxin Bacteriophages.

Shiga toxin (Stx) phages can be induced from Stx-producing Escherichia coli strains (STEC) or can be isolated as free virions from different samples. Here we describe methods used for the detection, enumeration, and isolation of Stx bacteriophages. Stx phages are temperate phages located in the genome of STEC. Their induction from the host strain cultures is achieved by different inducing agents, mitomycin C being one of the most commonly used. Detection of infectious Stx phages requires the production of visible plaques in a confluent lawn of the host strain using a double agar layer method. However, as the plaques produced by Stx phages are often barely visible and there is a possibility that non-Stx phages can also be induced from the strain, a hybridization step should be added to recognize and properly enumerate the lysis plaques generated after induction. Molecular methods can also be used to identify and enumerate Stx phages. Real-time quantitative PCR (qPCR) is the most accurate method for absolute quantification, although it cannot determine the infectivity of Stx phages. qPCR can also be useful for the detection of free Stx phage virions in different sample types.Stx phages induced from lysogenic bacterial strains can be purified by cesium chloride density gradients; this protocol also helps to specifically discriminate Stx phages from other prophages present in the genome of the host strain by selecting the phages expressing the Stx gene. High titer suspensions of Stx phages obtained after induction of large volumes of bacterial cultures and lysate concentration permits phage characterization by electron microscopy studies and genomic analysis.

Bacteriophages immunomodulate the response of monocytes.

Bacteriophages are present in fluids from cirrhosis patients. However, their effect on the immune response is unknown. In this work, we explore the role of phages in the phenotype, function, and cytokine production of monocytes. We stimulated healthy monocytes with five different butanol-purified phage suspensions infective for Gram-negative and Gram-positive bacteria. We studied the expression of the monocyte markers involved in lipopolysaccharide recognition (LPS; CD14), antigen presentation (HLA-DR) and co-stimulation (CD86), and the concentration of induced cytokines (TNF-α, IFN-α, and IL-10) by phages. To confirm the direct role of phages without the interference of contaminating soluble LPS in phage suspensions, polymyxin B was added to the cell cultures. Phagocytosis experiments were assessed by flow cytometry using labeled phage suspensions. We observed that butanol-purified phages reduced the surface levels of CD14 and CD86 in monocytes and increased the secreted levels of TNF-α and IL-10 compared with the control sample containing only butanol buffer. All phage suspensions showed downregulation of HLA-DR expression but only Staphylococcus aureus phage contaminated with Escherichia coli reached statistical significance. The addition of polymyxin B did not restore the monocytic response induced by phages, suggesting that the effect was not caused by the presence of LPS. Monocytes were able to phagocyte phages in a dose- and time-dependent manner. To conclude, the phagocytosis of butanol-purified phages altered the phenotype and cytokine production of monocytes suggesting they become tolerogenic.