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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. This work was supported by the Spanish Ministerio de Ciencia e Innovación (PID2020-113355GB-I00, the Agencia Estatal de Investigación (AEI) and the European regional fund (ERF), and the CDTI project (IDI-20190017). The study was partially supported by the Generalitat de Catalunya (2017SGR170). L. R.-R is lecturer of the Serra-Hunter program, Generalitat de Catalunya. C. G-G. has a fellowship from the University of Barcelona. L. S-C has a Maria Zambrano fellowship from the Spanish Ministerio de Universidades. M.D.R-B has a Margarita Salas fellowship from the Spanish Ministerio de Universidades.

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. This work was supported by the Spanish Ministerio de Ciencia e Innovación (PID2020-113355GB-I00), the Agencia Estatal de Investigación (AEI) and the European regional fund (ERF). The study was partially supported by the Generalitat de Catalunya (2017SGR170). PB-P has a grant from the Spanish Ministry of Economy, Industry and Competitiveness (BES-2017-081296), SM-C has a grant from Colciencias (Republic of Colombia) and LR-R is lecturer of the Serra-Hunter program, Generalitat de Catalunya. MDR-B has a Margarita Salas fellowship from the Spanish Ministerio de Universidades.

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.

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.

Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world's countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome. ispartof: NATURE MICROBIOLOGY vol:4 issue:10 pages:1727-1736 ispartof: location:England status: published

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.

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 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.

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.

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.

© The Author(s) 2021., 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., Work carried out in the Institute of Biomedicine and Biotechnology (IBBTEC) was funded by the Ministry of Economy and Competitiveness (grant BFU2017-86378-P). The work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO BES-2015-073164) and the European Union’s Horizon 2020 Research and Innovation Programme (grant 773830, OH-EJP-H2020-JRP-AMR-2-WORLDCOM).

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.

Objectives: To investigate the relevance of multicopy plasmids in antimicrobial resistance and assess their mobilization mediated by phage particles Methods: Several databases with complete sequences of plasmids and annotated genes were analysed. The 16S methyltransferase gene armA conferring high-level aminoglycoside resistance was used as a marker in eight different plasmids, from different incompatibility groups, and with differing sizes and plasmid copy numbers. All plasmids were transformed into Escherichia coli bearing one of four different lysogenic phages. Upon induction, encapsidation of armA in phage particles was evaluated using qRT–PCR and Southern blotting. Results: Multicopy plasmids carry a vast set of emerging clinically important antimicrobial resistance genes. However, 60% of these plasmids do not bear mobility (MOB) genes. When carried on these multicopy plasmids, mobilization of a marker gene armA into phage capsids was up to 10000 times more frequent than when it was encoded by a large plasmid with a low copy number. Conclusions: Multicopy plasmids and phages, two major mobile genetic elements (MGE) in bacteria, represent a novel high-efficiency transmission route of antimicrobial resistance genes that deserves further investigation. ARDIG

The genomes of gut Bacteroidales contain numerous invertible regions, many of which contain promoters that dictate phase-variable synthesis of surface molecules such as polysaccharides, fimbriae, and outer surface proteins. Here, we characterize a different type of phase-variable system of Bacteroides fragilis, a Type I restriction modification system (R-M). We show that reversible DNA inversions within this R-M locus leads to the generation of eight specificity proteins with distinct recognition sites. In vitro grown bacteria have a different proportion of specificity gene combinations at the expression locus than bacteria isolated from the mammalian gut. By creating mutants, each able to produce only one specificity protein from this region, we identified the R-M recognition sites of four of these S-proteins using SMRT sequencing. Transcriptome analysis revealed that the locked specificity mutants, whether grown in vitro or isolated from the mammalian gut, have distinct transcriptional profiles, likely creating different phenotypes, one of which was confirmed. Genomic analyses of diverse strains of Bacteroidetes from both host-associated and environmental sources reveal the ubiquity of phase-variable R-M systems in this phylum.

Background- This study aims to evaluate the efficacy of using a cleansing eyelid wipe to reduce microbial load in healthy subjects. Methods- A single-center, prospective study was conducted. Twenty healthy subjects were assigned to wipe their periocular area (eyelid, eyelashes and lid margin) twice a day with a commercial sterile wipe for 5 consecutive days. Bacterial load (total aerobic bacteria and Staphylococcus genus) was recovered from the same wipes and was measured on day 1, 3 and 5, with day 1 serving as an internal control. Both eyes were assessed independently, resulting in a total of 40 samples. Microbial evaluation was performed by culture on Tryptone Soy Agar (TSA) and Mannitol Salt Agar (MSA) plates. Results- Positive and negative controls rendered expected results thus validating this innovative extraction procedure. Microbial growth on both media revealed that the great majority of microorganisms detected belong to the genus Staphylococcus. Bacterial load reduction was significant (Mann-Whitney test p < 0.05) for both microorganisms between days 1 to 3, days 3 to 5 and days 1 to 5. Measurements at day 3 revealed a reduction on bacterial load of total aerobes and Staphylococcus spp. by 58.7% and 56.5% respectively. Moreover, on day 5, bacterial load showed a mean reduction of 80.8% and 82.7% on total aerobes and Staphylococcus spp. respectively. Furthermore, on day 5, 42.5% of the eyes showed ≥ 90% reduction in total aerobes and 42.5% for Staphylococcus spp. On the third day 25% of the volunteers showed ≥ 90% reduction of aerobes microbial load at least in one eye and the percentage was 15% for Staphylococcus spp. On the fifth day those values arise to 50% for total aerobes and 65% for Staphylococcus spp.Conclusions- The study demonstrates the efficacy of the eyelid hygiene with commercial cleansing wipes in significantly reducing (p < 0.05) bacterial load in the periocular area after 3 days of use, with further reduction on the fifth day. Therefore, these wipes could be recommended in situations in which adequate of palpebral hygiene is needed.

In this study, the plasmid content of clinical and commensal strains was analyzed and compared. The replicon profile was similar in both populations, except for L, M, A/C, and N (detected only in clinical strains) and HI1 (only in commensal strains). Although I1 and F were the most frequent replicons, only IncI1, sequence type 12 (ST12) was associated with bla CMY-2 in both populations. In contrast, the widespread resistant IncF plasmids were not linked to a single epidemic plasmid.

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.

Bacteria and phages have co-existed for several billions of years. The direct observation of their relationship, and the evidence that phages cause bacterial lysis and death, has led researchers to believe that phages and bacteria are natural enemies and that phages can be applied as antimicrobial agents. However, phages are also known to provide various benefits for their bacterial hosts, and on many occasions the phage-host interaction resembles a symbiotic relationship. In this chapter, we evaluate findings in different bacterial genera and their associated phages to assess if phages should be considered as bacterial parasites or symbiotic organisms.

Bacteriophages are pivotal elements in the dissemination of virulence genes. The main virulence determinants of Shiga Toxin producing E. coli, Shiga Toxins (Stx), are encoded by genes localized in the genome of lambdoid bacteriophages. Stx comprise two antigenically different types, Stx1 and Stx2, further divided into subtypes. Among these, certain Stx2 subtypes appear to be more commonly occurring in the most severe forms of the STEC disease, haemorrhagic colitis and haemolytic uremic syndrome (HUS). This study aimed at obtaining insights on the evolution of Stx2 bacteriophages, due to their relevance in public health, and we report here on the analysis of the genomic structure of Stx2 converting phages in relation with the known reservoir of the E. coli strains harboring them. Stx2-converting phages conveying the genes encoding different stx2 subtypes have been isolated from STEC strains and their whole genomes have been sequenced, analyzed and compared to those of other Stx2 phages available in the public domain. The phages' regions containing the stx2 genes have been analyzed in depth allowing to make inference on the possible mechanisms of selection and maintenance of certain Stx2 phages in the reservoir. The "stx regions" of different stx2 gene subtypes grouped into three different evolutionary lines in the comparative analysis, reflecting the frequency with which these subtypes are found in different animal niches, suggesting that the colonization of specific reservoir by STEC strains could be influenced by the Stx phage that they carry. Noteworthy, we could identify the presence of nanS-p gene exclusively in the "stx regions" of the phages identified in STEC strains commonly found in cattle. As a matter of fact, this gene encodes an esterase capable of metabolizing sialic acids produced by submaxillary glands of bovines and present in great quantities in their gastrointestinal tract.

ObjectivesTo investigate the relevance of multicopy plasmids in antimicrobial resistance and assess their mobilization mediated by phage particlesMethodsSeveral databases with complete sequences of plasmids and annotated genes were analysed. The 16S methyltransferase gene armA conferring high-level aminoglycoside resistance was used as a marker in eight different plasmids, from different incompatibility groups, and with differing sizes and plasmid copy numbers. All plasmids were transformed into Escherichia coli bearing one of four different lysogenic phages. Upon induction, encapsidation of armA in phage particles was evaluated using qRT–PCR and Southern blotting.ResultsMulticopy plasmids carry a vast set of emerging clinically important antimicrobial resistance genes. However, 60% of these plasmids do not bear mobility (MOB) genes. When carried on these multicopy plasmids, mobilization of a marker gene armA into phage capsids was up to 10000 times more frequent than when it was encoded by a large plasmid with a low copy number.ConclusionsMulticopy plasmids and phages, two major mobile genetic elements (MGE) in bacteria, represent a novel high-efficiency transmission route of antimicrobial resistance genes that deserves further investigation.

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.

Here we introduce methods for the detection, enumeration, and isolation of bacteriophages from Escherichia coli. In bacteria, horizontal gene transfer may be mediated by virulent and temperate phages. Strict virulent phages, able to propagate in a suitable strain following the lytic pathway, can be isolated directly from different natural environments. In temperate phages, the lytic cycle must be activated, and phages are detected after their induction. In both cases, detection is based on the production of visible plaques in a confluent lawn of the host strain using a double agar layer method. Further purification and characterization are achieved by density gradients, electron microscopy studies, and genomic analysis. This straightforward methodology can be applied to the detection, enumeration, and isolation of bacteriophages from any bacterial species, using the appropriate host strain, media, and culture conditions.