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.

Be Bold: Use Nursing Knowledge to Guide the Practice / Sé audaz: utiliza el conocimiento de enfermería para guiar la práctica / Seja ousado: use o conhecimento de enfermagem para guiar a prática

The imagination, bravery, and audacity that many nurses have shown throughout the history of our profession lead to the recognition of nursing as a discipline, profession, and science. For maintaining it, the Sigma Theta Tau International Honor Society of Nursing ­Sigma­ (1) and other associated organizations promote the science-guided exercise of the practice through the creation and transfer of knowledge, emphasizing the three elements that nurture, legitimize, consolidate, and guarantee disciplinary autonomy: theory, research, and practice. Sigma, in its presidential call 2022-2023, invited nurses to be bold and contribute to the development of nursing knowledge.

La imaginación, valentía y audacia que muchas enfermeras han mostrado a lo largo de la historia de nuestra profesión llevaron a que hoy en día se reconozca a la enfermería como disciplina, profesión y ciencia. Para mantener este reconocimiento, la Sociedad Internacional de Honor de la Enfermería ­Sigma­ y organizaciones asociadas promueven el ejercicio de la práctica guiado por la ciencia a través de la creación y la traslación de conocimientos, haciendo énfasis en los tres elementos que nutren, legitiman, consolidan y garantizan la autonomía disciplinar: la teoría, la investigación y la práctica. Sigma, en su llamado presidencial 2022-2023, invita a las enfermeras de la práctica a que sean audaces y contribuyan al desarrollo del conocimiento de enfermería.

A imaginação, a coragem e a audácia que muitos profissionais de enfermagem demonstraram ao longo da história da nossa profissão fizeram com que a enfermagem fosse hoje reconhecida como disciplina, profissão e ciência. Para manter este reconhecimento, a International Nursing Honor Society (Sigma) e as organizações associadas promovem o exercício da prática guiada pela ciência através da criação e transferência de conhecimento, enfatizando os três elementos que nutrem, legitimam, consolidam e garantem a autonomia disciplinar: teoria, pesquisa e prática. A Sigma, na sua chamada presidencial 2022-2023, convida os profissionais de enfermagem praticantes a serem ousados ​​e a contribuírem para o desenvolvimento do conhecimento de enfermagem.

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.

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.

Donar leche humana salva vidas: percepciones de mujeres donantes y receptoras de leche en un banco de leche humana en Colombia / A doação de leite humano salva vidas: conhecimentos de mulheres dadoras e receptoras de leite num banco de leite humano na Colômbia / Donating human milk saves lives: insights from women donors and recipients of milk at a human milk bank in Colombia

Objective: To learn about the experiences of women who are milk donors and users of thehuman milk bank, who are mothers of pre-term infants, low birth weight infants and sick newbornshospitalized in a neonatal intensive care unit (NICU). Method Descriptive, qualitative, micro ethnographic study following the method proposed by James Spradley, focused semi-structured interviews were conducted with 10 human milk donor/receiver mothers. Results: six categories wereestablished: human milk for humans, motivation and maintenance of human milk donation, recognition of Human Milk Banks (HMLB) as a suitable place for donation, donating human milk requires commitment and technique, feelings of women donors and recipients, barriers to donation,from these categories emerged the central category "Donating human milk saves lives" ConclusionsDonating and receiving human milk is an experience in which biological, cultural, emotional and political aspects are linked. Women demand social recognition of the work of Human Milk Banksin the promotion, protection, support of breastfeeding, nutrition and protection of premature and sick newborns in neonatal units. (AU)

Objetivo: conocer las experiencias de las mujeres donantes de leche y usuarias receptoras del banco de leche humana, que son madres de recién nacidos pre-término, recién nacidos debajo peso y recién nacidos enfermos hospitalizados en una unidad de cuidado intensivo neonatal(UCIN). Método Estudio descriptivo, de tipo cualitativo, micro etnográfico siguiendo el métodopropuesto por James Spradley, se realizaron entrevistas semiestructuradas focalizadas a 10 madresdonantes/receptoras de leche humana. Resultados: seis categorías fueron establecidas: leche de humanos para humanos, motivación y mantenimiento de la donación de leche humana, reconocer losBancos de Leche Humana (BLH) como un lugar idóneo para la donación, donar leche humana exigecompromiso y técnica, sentimientos de las mujeres donantes y receptoras, barreras para la donación,de estas categorías emergió la categoría central “Donar leche humana salva vidas” ConclusionesDonar y recibir leche humana es una experiencia en la cual se vinculan aspectos biológicos, culturales, emocionales y políticos. Las mujeres reclaman el reconocimiento social, a la labor de los Bancosde Leche Humana en la promoción, protección, apoyo a la lactancia materna, la nutrición y protección de los prematuros y recién nacidos enfermos en las unidades neonatales. (AU)

Objectivo: Conhecer as experiências de mulheres dadoras de leite e utilizadorasdo banco de leite humano, mães de bebés pré-termo, bebés de baixo peso ao nascer e recém-nascidos doentes hospitalizados numa unidade de cuidados intensivos neonatais(UCIN). Método Estudo descritivo, qualitativo, microecnográfico seguindo o método proposto por James Spradley, foram realizadas entrevistas semi-estruturadas com 10 mãesdoadoras/receptoras de leite humano. Resultados: foram estabelecidas seis categorias:leite humano para consumo humano, motivação e manutenção da doação de leite humano, reconhecimento dos Bancos de Leite Humano (HMLB) como local adequado paraa doação, a doação de leite humano requer empenho e técnica, sentimentos das mulheresdadoras e receptoras, obstáculos à doação, destas categorias surgiu a categoria central"Doar leite humano salva vidas" Conclusões Doar e receber leite humano é uma experiência em que os aspectos biológicos, culturais, emocionais e políticos estão ligados. As mulheres exigem o reconhecimento social do trabalho dos Bancos de Leite Humano na promoção, protecção, apoio à amamentação, nutrição e protecção de recém-nascidos prematuros e doentes em unidades neonatais. (AU)

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.

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.

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.

Design and Selection of Engineered Lytic Proteins With Staphylococcus aureus Decolonizing Activity.

Staphylococcus aureus causes various infections in humans and animals, the skin being the principal reservoir of this pathogen. The widespread occurrence of methicillin-resistant S. aureus (MRSA) limits the elimination and treatment of this pathogen. Phage lytic proteins have been proven as efficient antimicrobials against S. aureus. Here, a set of 12 engineered proteins based on endolysins were conceptualized to select the most optimal following a stepwise funnel approach assessing parameters including turbidity reduction, minimum inhibitory concentration (MIC), time-kill curves, and antibiofilm assays, as well as testing their stability in a broad range of storage conditions (pH, temperature, and ionic strength). The engineered phage lysins LysRODIΔAmi and ClyRODI-H5 showed the highest specific lytic activity (5 to 50 times higher than the rest), exhibited a shelf-life up to 6 months and remained stable at temperatures up to 50°C and in a pH range from 3 to 9. LysRODIΔAmi showed the lower MIC values against all staphylococcal strains tested. Both proteins were able to kill 6 log units of the strain S. aureus Sa9 within 5 min and could remove preformed biofilms (76 and 65%, respectively). Moreover, LysRODIΔAmi could prevent biofilm formation at low protein concentrations (0.15-0.6 µM). Due to its enhanced antibiofilm properties, LysRODIΔAmi was selected to effectively remove S. aureus contamination in both intact and disrupted keratinocyte monolayers. Notably, this protein did not demonstrate any toxicity toward human keratinocytes, even at high concentrations (22.1 µM). Finally, a pig skin ex vivo model was used to evaluate treatment of artificially contaminated pig skin using LysRODIΔAmi (16.5 µg/cm2). Following an early reduction of S. aureus, a second dose of protein completely eradicated S. aureus. Overall, our results suggest that LysRODIΔAmi is a suitable candidate as antimicrobial agent to prevent and treat staphylococcal skin infections.

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.

Parental Decision-Making in Pediatric Intensive Care: A Concept Analysis.

The development of nursing knowledge requires a close relationship between theory, research, and practice. The purpose of the analysis of the concept of "parental decision-making in pediatric critical care" is to facilitate nurses' therapeutic care of critically ill children and their families. To construct, structure, and give meaning to the concept, we use our experience in the field, critical reading of the literature, and careful analysis of data that have emerged about parental decision-making in pediatric intensive care. Several factors affect parent's ability to act as decision-makers: the psychosocial and physical disorders they develop, the subordination of their parental roles by the health care team, and the child's critical state of health. While different disciplines, including nursing, have well described the decision-making concept, parental decision-making in the context of pediatric intensive care has not been as well delineated. Nursing science recognizes the importance of decision-making and has incorporated the concept as an essential domain of its philosophical and disciplinary interests. Following the method proposed by Walker and Avant, the concept was analyzed, attributes, background, and consequences described. A model case was presented and discussed. An operational definition emerges, providing knowledge for professional nursing practice and will be the basis for an essential theoretical development around this phenomenon. Parents' recognition, the promotion of family-centered care, and shared decisions are ideal for encouraging parental participation.

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.