RESUMEN
Lytic bacteriophages hold substantial promise in medical and biotechnological applications. Therefore a comprehensive understanding of phage infection mechanisms is crucial. CRISPR-Cas systems offer a way to explore these mechanisms via site-specific phage mutagenesis. However, phages can resist Cas-mediated cleavage through extensive DNA modifications like cytosine glycosylation, hindering mutagenesis efficiency. Our study utilizes the eukaryotic enzyme NgTET to temporarily reduce phage DNA modifications, facilitating Cas nuclease cleavage and enhancing mutagenesis efficiency. This approach enables precise DNA targeting and seamless point mutation integration, exemplified by deactivating specific ADP-ribosyltransferases crucial for phage infection. Furthermore, by temporally removing DNA modifications, we elucidated the effects of these modifications on T4 phage infections without necessitating gene deletions. Our results present a strategy enabling the investigation of phage epigenome functions and streamlining the engineering of phages with cytosine DNA modifications. The described temporal modulation of the phage epigenome is valuable for synthetic biology and fundamental research to comprehend phage infection mechanisms through the generation of mutants.
Asunto(s)
Bacteriófagos , Sistemas CRISPR-Cas , ADN Viral , Epigenoma , ADN Viral/genética , Bacteriófagos/genética , Ingeniería Genética/métodos , Bacteriófago T4/genética , Mutagénesis Sitio-Dirigida/métodos , Escherichia coli/genética , Escherichia coli/virología , Genoma ViralRESUMEN
Traditional assays for counting bacteriophages and their lysogens are labor-intensive and perturbative to the host cells. Here, we present a high-throughput infection method in a microplate reader, where the growth dynamics of the infected culture is measured using the optical density (OD). We find that the OD at which the culture lyses scales linearly with the logarithm of the initial phage concentration, providing a way of measuring phage numbers over nine orders of magnitude and down to single-phage sensitivity. Interpreting the measured dynamics using a mathematical model allows us to infer the phage growth rate, which is a function of the phage-cell encounter rate, latent period, and burst size. Adding antibiotic selection provides the ability to measure the rate of host lysogenization. Using this method, we found that when E. coli growth slows down, the lytic growth rate of lambda phages decreases, and the propensity for lysogeny increases, demonstrating how host physiology influences the viral developmental program.
Asunto(s)
Escherichia coli , Lisogenia , Escherichia coli/virología , Escherichia coli/crecimiento & desarrollo , Bacteriófagos/fisiología , Bacteriófago lambda/fisiología , Bacteriófago lambda/genética , Modelos Biológicos , Dinámica Poblacional , Antibacterianos/farmacología , Modelos TeóricosRESUMEN
BACKGROUND: Bacteriophage has been renewed attention as a new antibacterial agent due to the limitations of antibiotic treatment. Bacteriophages are generally thought to be highly host specific and even strain specific, but a small number of polyvalent bacteriophages have been found to infect bacteria of different genera. RESULTS: In this study, a virulent lytic bacteriophage (named Salmonella phage PSH-1) of Salmonella Enteritidis was isolated from the sewage samples of a large-scale pig farm, PSH-1 demonstrated lytic activity against four multidrug-resistant Salmonella Enteritidis isolates and Escherichia coli, and then its biological characteristics, genome and bacteriostatic ability were investigated. The results showed that the initial titer of PSH-1 was 1.15 × 1010 PFU/mL and the optimal multiplicity of infection (MOI) was 0.01, PSH-1 has stable activity in the range of pH 3.0-11.0. One-step growth curve showed that its latent period was 20 min, burst time was 80 min, and the burst was 495 particles. The whole-genome sequencing results revealed phage PSH-1 had a linear dsDNA with 48,466 bp length. The G/C content was 45.33%. Non-coding RNA genes and virulence factors were not found. Eighty- five open reading frames (ORFs) were identified after online annotation. By tests, the use of phage could succeed in controlling the artificial Salmonella contamination in milk at a range of temperatures. CONCLUSIONS: This study reports a novel Salmonella Enteritidis phage PSH-1, which has a robust lytic ability, no virulence factors, and good stability. The characterization and genomic analysis of PSH-1 will develop our understanding of phage biology and diversity and provide a potential arsenal for controlling of salmonellosis.
Asunto(s)
Farmacorresistencia Bacteriana Múltiple , Genoma Viral , Fagos de Salmonella , Salmonella enteritidis , Aguas del Alcantarillado , Secuenciación Completa del Genoma , Salmonella enteritidis/virología , Salmonella enteritidis/genética , Salmonella enteritidis/efectos de los fármacos , Fagos de Salmonella/genética , Fagos de Salmonella/aislamiento & purificación , Fagos de Salmonella/fisiología , Fagos de Salmonella/clasificación , Farmacorresistencia Bacteriana Múltiple/genética , Animales , Aguas del Alcantarillado/virología , Aguas del Alcantarillado/microbiología , Porcinos , Composición de Base , Escherichia coli/virología , Escherichia coli/genéticaRESUMEN
Toll/interleukin-1 receptor (TIR) domain-containing proteins play a critical role in immune responses in diverse organisms, but their function in bacterial systems remains to be fully elucidated. This study, focusing on Escherichia coli, addresses how TIR domain-containing proteins contribute to bacterial immunity against phage attack. Through an exhaustive survey of all E. coli genomes available in the NCBI database and testing of 32 representatives of the 90% of the identified TIR domain-containing proteins, we found that a significant proportion (37.5%) exhibit antiphage activities. These defense systems recognize a variety of phage components, thus providing a sophisticated mechanism for pathogen detection and defense. This study not only highlights the robustness of TIR systems in bacterial immunity, but also draws an intriguing parallel to the diversity seen in mammalian Toll-like receptors (TLRs), enriching our understanding of innate immune mechanisms across life forms and underscoring the evolutionary significance of these defense strategies in prokaryotes.
Asunto(s)
Bacteriófagos , Escherichia coli , Dominios Proteicos , Escherichia coli/genética , Escherichia coli/virología , Escherichia coli/inmunología , Escherichia coli/metabolismo , Bacteriófagos/genética , Bacteriófagos/inmunología , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/inmunología , Inmunidad Innata , Receptores Toll-Like/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología , Receptores de Interleucina-1/metabolismo , Receptores de Interleucina-1/genéticaRESUMEN
Across the tree of life, pleiotropy is thought to constrain adaptation through evolutionary tradeoffs. However, few examples of pleiotropy exist that are well explained at the genetic level, especially for pleiotropy that is mediated by multiple genes. Here, we describe a set of pleiotropic mutations that mediate two key fitness components in bacteria: parasite resistance and motility. We subjected Escherichia coli to strong selection by phage U136B to obtain 27 independent mucoid mutants. Mucoidy is a phenotype that results from excess exopolysaccharide and can act as a barrier against viral infection but can also interfere with other cellular functions. We quantified the mutants' phage resistance using efficiency of plaquing assays and swimming motility using swim agar plates, and we sequenced the complete genomes of all mutants to identify mucoid-causing mutations. Increased phage resistance co-occurred with decreased motility. This relationship was mediated by highly parallel (27/27) mutations to the Rcs phosphorelay pathway, which senses membrane stress to regulate exopolysaccharide production. Together, these results provide an empirical example of a pleiotropic relationship between two traits with intermediate genetic complexity.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Mutación , Escherichia coli/virología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Bacteriófagos/genética , Bacteriófagos/fisiología , Colifagos/genética , Colifagos/fisiología , Transducción de Señal , Polisacáridos Bacterianos/metabolismoRESUMEN
Escherichia coli is one of the most prevalent foodborne pathogens, frequently found in meat and dairy products. Current decontamination methods are often associated with changes in organoleptic characteristics, nutrient loss, and potentially harmful side effects. Furthermore, despite the array of available methods, foodborne outbreaks still frequently occur. For this reason, bacteriophages (or simply phages) emerged as a natural alternative for the biocontrol of bacterial contamination in food without altering their organoleptic properties. In this study, the potential of phage phT4A was assessed in the biocontrol of E. coli in liquid (milk) and solid (ham) food matrices. Firstly, as foods have different pH and temperature values, the influence of these parameters on phage phT4A viability was also assessed to develop an effective protocol. Phage phT4A proved to be stable for long storage periods at pH 7-8 (56 days) and temperatures of 4-37 °C (21 days). Before application of phages to inactivate pathogenic bacteria in food, previous assays were carried out in Tryptic Soy Broth (TSB) to study the dynamics of phage-bacteria interaction. Then, the antibacterial potential of phage phT4A was evaluated in the two food matrices at different temperatures (4, 10 and 25 °C). This phage was more efficient at 25 °C in all tested matrices (maximum inactivation of 6.6, 3.9 and 1.8 log CFU/mL in TSB, milk and ham, respectively) than at 10 °C (maximum decrease of 4.7, 2.1 and 1.0 log CFU/mL in TSB, milk and ham, respectively) and 4 °C (maximum reduction of 2.6 and 0.7 log CFU/mL in TSB and milk, respectively). However, the decrease of temperature from 25 °C to 10 and 4 °C prevented bacterial regrowth. The results suggest that during phage treatment, a balance between an incubation temperature that provide effective results in terms of bacterial inactivation by the phages and at the same time prevents or delays bacterial regrowth, is needed. The application of phage phT4A at a temperature of 10 °C can be an effective strategy in terms of bacterial inactivation, delaying bacterial regrowth and also reducing energy costs.
Asunto(s)
Bacteriófagos , Escherichia coli , Microbiología de Alimentos , Leche , Animales , Bacteriófagos/fisiología , Agentes de Control Biológico , Recuento de Colonia Microbiana , Escherichia coli/virología , Escherichia coli/crecimiento & desarrollo , Contaminación de Alimentos/prevención & control , Concentración de Iones de Hidrógeno , Productos de la Carne/microbiología , Leche/microbiología , TemperaturaRESUMEN
Escherichia coli is a commensal inhabitant of the mammalian gut microbiota, frequently associated with various gastrointestinal diseases. There is increasing interest in comprehending the variety of bacteriophages (phages) that target this bacterium, as such insights could pave the way for their potential use in therapeutic applications. Here, we report the isolation and characterization of four newly identified E. coli infecting tailed phages (W70, A7-1, A5-4, and A73) that were found to constitute a novel genus, Septuagintavirus, within the subfamily Vequintavirinae. Genomes of these phages ranged from 137 kbp to 145 kbp, with a GC content of 41 mol%. They possess a maximum nucleotide similarity of 30% with phages of the closest phylogenetic genus, Certrevirus, while displaying limited homology to other genera of the Vequintavirinae family. Host range analysis showed that these phages have limited activity against a panel of E. coli strains, infecting 6 out of 16 tested isolates, regardless of their phylotype. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was performed on the virion of phage W70, allowing the identification of 28 structural proteins, 19 of which were shared with phages of other genera of Vequintavirinae family. The greatest diversity was identified with proteins forming tail fiber structures, likely indicating the adaptation of virions of each phage genus of this subfamily for the recognition of their target receptor on host cells. The findings of this study provide greater insights into the phages of the subfamily Vequintavirinae, contributing to the pool of knowledge currently known about these phages. IMPORTANCE: Escherichia coli is a well-known bacterium that inhabits diverse ecological niches, including the mammalian gut microbiota. Certain strains are associated with gastrointestinal diseases, and there is a growing interest in using bacteriophages, viruses that infect bacteria, to combat bacterial infections. Here, we describe the isolation and characterization of four novel E. coli bacteriophages that constitute a new genus, Septuagintavirus, within the subfamily Vequintavirinae. We conducted mass spectrometry on virions of a representative phage of this novel clade and compared it to other phages within the subfamily. Our analysis shows that virion structure is highly conserved among all phages, except for proteins related to tail fiber structures implicated in the host range. These findings provide greater insights into the phages of the subfamily Vequintavirinae, contributing to the existing pool of knowledge about these phages.
Asunto(s)
Colifagos , Escherichia coli , Genoma Viral , Especificidad del Huésped , Filogenia , Escherichia coli/virología , Escherichia coli/genética , Colifagos/genética , Colifagos/aislamiento & purificación , Colifagos/clasificación , Composición de BaseRESUMEN
Many bacterial immune systems recognize phage structural components to activate antiviral responses, without inhibiting the function of the phage component. These systems can be encoded in specific chromosomal loci, known as defense islands, and in mobile genetic elements such as prophages and phage-inducible chromosomal islands (PICIs). Here, we identify a family of bacterial immune systems, named Tai (for 'tail assembly inhibition'), that is prevalent in PICIs, prophages and P4-like phage satellites. Tai systems protect their bacterial host population from other phages by blocking the tail assembly step, leading to the release of tailless phages incapable of infecting new hosts. To prevent autoimmunity, some Tai-positive phages have an associated counter-defense mechanism that is expressed during the phage lytic cycle and allows for tail formation. Interestingly, the Tai defense and counter-defense genes are organized in a non-contiguous operon, enabling their coordinated expression.
Asunto(s)
Bacteriófagos , Profagos , Bacteriófagos/genética , Bacteriófagos/fisiología , Profagos/genética , Islas Genómicas/genética , Bacterias/virología , Bacterias/genética , Bacterias/inmunología , Operón/genética , Escherichia coli/genética , Escherichia coli/virología , Escherichia coli/inmunología , Regulación Bacteriana de la Expresión GénicaRESUMEN
DNA Stable Isotope Probing is emerging as a potent methodology for investigating host-virus interactions, based on the essential reliance of viruses on host organisms for the production of virions. Despite the anticipated link between host isotopic compositions and the generated virions, the application of stable isotope probing to viral DNA has never been evaluated on simple biological models. In this study, we assessed the efficacy of this method on the bacteriophage T4 and its host, Escherichia coli. Through the cultivation of E. coli cells on a 13C-enriched substrate and subsequent propagation of T4 bacteriophage, we examine the degree of isotopic enrichment in viral DNA. Our investigation reveals a strong correlation between the proportion of 13C6-D-glucose in the growth substrate and the buoyant density in CsCl gradient of T4 DNA, confirming the validity of DNA SIP in viral ecology. These findings underscore the potential of DNA SIP as a robust tool for characterizing the diversity of viruses infecting hosts with specific metabolic activities and provide then a foundation for further exploration in viral ecology research.
Asunto(s)
Bacteriófago T4 , ADN Viral , Escherichia coli , Bacteriófago T4/genética , Bacteriófago T4/fisiología , Bacteriófago T4/metabolismo , Escherichia coli/virología , Escherichia coli/genética , Escherichia coli/metabolismo , ADN Viral/genética , Interacciones Microbiota-Huesped , Glucosa/metabolismoRESUMEN
Bacteriophage ÏX174 is a small icosahedral virus of the Microviridae with a rapid replication cycle. Previously, we found that in ÏX174 infections of Escherichia coli, the most highly upregulated host proteins are two small heat shock proteins, IbpA and IbpB, belonging to the HSP20 family, which is a universally conserved group of stress-induced molecular chaperones that prevent irreversible aggregation of proteins. Heat shock proteins were found to protect against ÏX174 lysis, but IbpA/B have not been studied. In this work, we disrupted the ibpA and ibpB genes and measured the effects on ÏX174 replication. We found that in contrast to other E. coli heat shock proteins, they are not necessary for ÏX174 replication; moreover, their absence has no discernible effect on ÏX174 fecundity. These results suggest IbpA/B upregulation is a response to ÏX174 protein expression but does not play a role in phage replication, and they are not Microviridae host factors.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Replicación Viral , Escherichia coli/virología , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Bacteriófago phi X 174/genética , Bacteriófago phi X 174/fisiología , Bacteriófago phi X 174/metabolismo , Proteínas de Choque Térmico Pequeñas/metabolismo , Proteínas de Choque Térmico Pequeñas/genética , Proteínas de Choque TérmicoRESUMEN
The burgeoning crisis of antibiotic resistance has directed attention to bacteriophages as natural antibacterial agents capable of circumventing bacterial defenses. Central to this are the bacterial defense mechanisms, such as the BREX system, which utilizes the methyltransferase BrxX to protect against phage infection. This study presents the first in vitro characterization of BrxX from Escherichia coli, revealing its substrate-specific recognition and catalytic activity. We demonstrate that BrxX exhibits nonspecific DNA binding but selectively methylates adenine within specific motifs. Kinetic analysis indicates a potential regulation of BrxX by the concentration of its co-substrate, S-adenosylmethionine, and suggests a role for other BREX components in modulating BrxX activity. Furthermore, we elucidate the molecular mechanism by which the T7 phage protein Ocr (Overcoming classical restriction) inhibits BrxX. Despite low sequence homology between BrxX from different bacterial species, Ocr effectively suppresses BrxX's enzymatic activity through high-affinity binding. Cryo-electron microscopy and biophysical analyses reveal that Ocr, a DNA mimic, forms a stable complex with BrxX, highlighting a conserved interaction interface across diverse BrxX variants. Our findings provide insights into the strategic counteraction by phages against bacterial defense systems and offer a foundational understanding of the complex interplay between phages and their bacterial hosts, with implications for the development of phage therapy to combat antibiotic resistance.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Proteínas Virales , Escherichia coli/virología , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas Virales/metabolismo , S-Adenosilmetionina/metabolismo , Unión Proteica , Bacteriófago T7/genética , Metiltransferasas/metabolismo , CinéticaRESUMEN
Bacterial and bacteriophage RNA polymerases (RNAPs) have divergently evolved and share the RNA hairpin-dependent intrinsic termination of transcription. Here, we examined phage T7, T3 and SP6 RNAP terminations utilizing the single-molecule fluorescence assays we had developed for bacterial terminations. We discovered the phage termination mode or outcome is virtually single with decomposing termination. Therein, RNAP is displaced forward along DNA and departs both RNA and DNA for one-step decomposition, three-dimensional diffusion and reinitiation at any promoter. This phage displacement-mediated decomposing termination is much slower than readthrough and appears homologous with the bacterial one. However, the phage sole mode of termination contrasts with the bacterial dual mode, where both decomposing and recycling terminations occur compatibly at any single hairpin- or Rho-dependent terminator. In the bacterial recycling termination, RNA is sheared from RNA·DNA hybrid, and RNAP remains bound to DNA for one-dimensional diffusion, which enables facilitated recycling for reinitiation at the nearest promoter located downstream or upstream in the sense or antisense orientation. Aligning with proximity of most terminators to adjacent promoters in bacterial genomes, the shearing-mediated recycling termination could be bacterial adaptation for the facilitated reinitiations repeated at a promoter for accelerated expression and coupled at adjoining promoters for coordinated regulation.
Asunto(s)
ARN Polimerasas Dirigidas por ADN , Regiones Promotoras Genéticas , Terminación de la Transcripción Genética , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Bacteriófagos/genética , Escherichia coli/genética , Escherichia coli/virología , Iniciación de la Transcripción Genética , Transcripción Genética , Proteínas Virales/metabolismo , Proteínas Virales/genética , Bacteriófago T7/genéticaRESUMEN
Several immune pathways in humans conjugate ubiquitin-like proteins to virus and host molecules as a means of antiviral defence1-5. Here we studied an antiphage defence system in bacteria, comprising a ubiquitin-like protein, ubiquitin-conjugating enzymes E1 and E2, and a deubiquitinase. We show that during phage infection, this system specifically conjugates the ubiquitin-like protein to the phage central tail fibre, a protein at the tip of the tail that is essential for tail assembly as well as for recognition of the target host receptor. Following infection, cells encoding this defence system release a mixture of partially assembled, tailless phage particles and fully assembled phages in which the central tail fibre is obstructed by the covalently attached ubiquitin-like protein. These phages show severely impaired infectivity, explaining how the defence system protects the bacterial population from the spread of phage infection. Our findings demonstrate that conjugation of ubiquitin-like proteins is an antiviral strategy conserved across the tree of life.
Asunto(s)
Proteínas Bacterianas , Bacteriófagos , Enzimas Desubicuitinizantes , Escherichia coli , Enzimas Ubiquitina-Conjugadoras , Ubiquitinas , Ensamble de Virus , Bacteriófagos/química , Bacteriófagos/metabolismo , Bacteriófagos/patogenicidad , Bacteriófagos/fisiología , Enzimas Desubicuitinizantes/metabolismo , Escherichia coli/enzimología , Escherichia coli/metabolismo , Escherichia coli/virología , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinas/metabolismo , Proteínas de la Cola de los Virus/metabolismo , Proteínas de la Cola de los Virus/química , Proteínas Bacterianas/metabolismo , Evolución Molecular , Secuencia ConservadaRESUMEN
The filamentous bacteriophage M13KO7 (M13) is the most used in phage display (PD) technology and, like other phages, has been applied in several areas of medicine, agriculture, and in the food industry. One of the advantages is that they can modulate the immune response in the presence of pathogenic microorganisms, such as bacteria and viruses. This study evaluated the use of phage M13 in the chicken embryos model. We inoculated 13-day-old chicken embryos with Salmonella Pullorum (SP) and then evaluated survival for the presence of phage M13 or E. coli ER2738 (ECR) infected with M13. We found that the ECR bacterium inhibits SP multiplication in 0.32 (M13-infected ECR) or 0.44 log UFC/mL (M13-uninfected ECR) and that the ECR-free phage M13 from the PD library can be used in chicken embryo models. This work provides the use of the chicken embryo as a model to study systemic infection and can be employed as an analysis tool for various peptides that M13 can express from PD selection. KEY POINTS: ⢠SP-infected chicken embryo can be a helpful model of systemic infection for different tests. ⢠Phage M13 does not lead to embryonic mortality or cause serious injury to embryos. ⢠Phage M13 from the PD library can be used in chicken embryo model tests.
Asunto(s)
Bacteriófago M13 , Escherichia coli , Animales , Embrión de Pollo , Escherichia coli/virología , Escherichia coli/genética , Bacteriófago M13/genética , Técnicas de Visualización de Superficie Celular/métodos , Salmonella , Pollos , Enfermedades de las Aves de Corral/virología , Enfermedades de las Aves de Corral/microbiologíaRESUMEN
As bacteriophages continue to gain regulatory approval for personalized human therapy against antibiotic-resistant infections, there is a need for transformative technologies for rapid target identification through multiple, large, decentralized therapeutic phages biobanks. Here, we design a high throughput phage screening platform comprised of a portable library of individual shelf-stable, ready-to-use phages, in all-inclusive solid tablets. Each tablet encapsulates one phage along with luciferin and luciferase enzyme stabilized in a sugar matrix comprised of pullulan and trehalose capable of directly detecting phage-mediated adenosine triphosphate (ATP) release through ATP bioluminescence reaction upon bacterial cell burst. The tablet composition also enhances desiccation tolerance of all components, which should allow easier and cheaper international transportation of phages and as a result, increased accessibility to therapeutic phages. We demonstrate high throughput screening by identifying target phages for select multidrug-resistant clinical isolates of Pseudomonas aeruginosa, Salmonella enterica, Escherichia coli, and Staphylococcus aureus with targets identified within 30-120 min.
Asunto(s)
Bacteriófagos , Escherichia coli , Ensayos Analíticos de Alto Rendimiento , Terapia de Fagos , Medicina de Precisión , Staphylococcus aureus , Humanos , Terapia de Fagos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Escherichia coli/virología , Escherichia coli/metabolismo , Escherichia coli/genética , Bacteriófagos/genética , Bacteriófagos/fisiología , Staphylococcus aureus/virología , Medicina de Precisión/métodos , Pseudomonas aeruginosa/virología , Adenosina Trifosfato/metabolismo , Salmonella enterica/virología , Farmacorresistencia Bacteriana Múltiple/genéticaRESUMEN
Infections due to antimicrobial-resistant bacteria have become a major threat to global health. Some patients may carry resistant bacteria in their gut microbiota. Specific risk factors may trigger the conversion of these carriages into infections in hospitalized patients. Preventively eradicating these carriages has been postulated as a promising preventive intervention. However, previous attempts at such eradication using oral antibiotics or probiotics have led to discouraging results. Phage therapy, the therapeutic use of bacteriophage viruses, might represent a worthy alternative in this context. Taking inspiration from this clinical challenge, we built Gut-On-A-Chip (GOAC) models, which are tridimensional cell culture models mimicking a simplified gut section. These were used to better understand bacterial dynamics under phage pressure using two relevant species: Pseudomonas aeruginosa and Escherichia coli. Model mucus secretion was documented by ELISA assays. Bacterial dynamics assays were performed in GOAC triplicates monitored for 72 h under numerous conditions, such as pre-, per-, or post-bacterial timing of phage introduction, punctual versus continuous phage administration, and phage expression of mucus-binding properties. The potential genomic basis of bacterial phage resistance acquired in the model was investigated by variant sequencing. The bacterial "escape growth" rates under phage pressure were compared to static in vitro conditions. Our results suggest that there is specific bacterial prosperity in this model compared to other in vitro conditions. In E. coli assays, the introduction of a phage harboring unique mucus-binding properties could not shift this balance of power, contradicting previous findings in an in vivo mouse model and highlighting the key differences between these models. Genomic modifications were correlated with bacterial phage resistance acquisition in some but not all instances, suggesting that alternate ways are needed to evade phage predation, which warrants further investigation.
Asunto(s)
Bacteriófagos , Escherichia coli , Microbioma Gastrointestinal , Terapia de Fagos , Pseudomonas aeruginosa , Pseudomonas aeruginosa/virología , Bacteriófagos/fisiología , Bacteriófagos/genética , Humanos , Terapia de Fagos/métodos , Escherichia coli/virología , Dispositivos Laboratorio en un ChipRESUMEN
Antimicrobial resistance (AMR) is a major public health threat, exacerbated by the ability of bacteria to rapidly disseminate antimicrobial resistance genes (ARG). Since conjugative plasmids of the incompatibility group P (IncP) are ubiquitous mobile genetic elements that often carry ARG and are broad-host-range, they are important targets to prevent the dissemination of AMR. Plasmid-dependent phages infect plasmid-carrying bacteria by recognizing components of the conjugative secretion system as receptors. We sought to isolate plasmid-dependent phages from wastewater using an avirulent strain of Salmonella enterica carrying the conjugative IncP plasmid pKJK5. Irrespective of the site, we only obtained bacteriophages belonging to the genus Alphatectivirus. Eleven isolates were sequenced, their genomes analyzed, and their host range established using S. enterica, Escherichia coli, and Pseudomonas putida carrying diverse conjugative plasmids. We confirmed that Alphatectivirus are abundant in domestic and hospital wastewater using culture-dependent and culture-independent approaches. However, these results are not consistent with their low or undetectable occurrence in metagenomes. Therefore, overall, our results emphasize the importance of performing phage isolation to uncover diversity, especially considering the potential of plasmid-dependent phages to reduce the spread of ARG carried by conjugative plasmids, and to help combat the AMR crisis.
Asunto(s)
Bacteriófagos , Plásmidos , Aguas Residuales , Plásmidos/genética , Aguas Residuales/virología , Aguas Residuales/microbiología , Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , Bacteriófagos/fisiología , Bacteriófagos/clasificación , Genoma Viral , Escherichia coli/virología , Escherichia coli/genética , Especificidad del Huésped , Pseudomonas putida/virología , Pseudomonas putida/genética , Salmonella enterica/virología , Salmonella enterica/genética , FilogeniaRESUMEN
Non-tailed icosahedral phages belonging to families Fiersviridae (phages MS2 and Qbeta), Tectiviridae (PRD1) and Microviridae (phiX174) have not been considered in detail so far as potential antibacterial agents. The aim of the study was to examine various aspects of the applicability of these phages as antibacterial agents. Antibacterial potential of four phages was investigated via bacterial growth and biofilm formation inhibition, lytic spectra determination, and phage safety examination. The phage phiX174 was combined with different classes of antibiotics to evaluate potential synergistic interactions. In addition, the incidence of phiX174-insensitive mutants was analyzed. The results showed that only phiX174 out of four phages tested against their corresponding hosts inhibited bacterial growth for > 90% at different multiplicity of infection and that only this phage considerably prevented biofilm formation. Although all phages show the absence of potentially undesirable genes, they also have extremely narrow lytic spectra. The synergism was determined between phage phiX174 and ceftazidime, ceftriaxone, ciprofloxacin, macrolides, and chloramphenicol. It was shown that the simultaneous application of agents is more effective than successive treatment, where one agent is applied first. The analysis of the appearance of phiX174 bacteriophage-insensitive mutants showed that mutations occur with a frequency of 10-3. The examined non-tailed phages have a limited potential for use as antibacterial agents, primarily due to a very narrow lytic spectrum and the high frequency of resistant mutants appearance, but Microviridae can be considered in the future as biocontrol agents against susceptible strains of E. coli in combinations with conventional antimicrobial agents.
Asunto(s)
Antibacterianos , Biopelículas , Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Bacteriófagos/genética , Bacteriófagos/fisiología , Escherichia coli/virología , Escherichia coli/efectos de los fármacos , Bacteriófago phi X 174/efectos de los fármacos , Bacteriófago phi X 174/genética , Bacterias/efectos de los fármacos , Bacterias/virología , MutaciónRESUMEN
Bacteriophage are sophisticated cellular parasites that can not only parasitize bacteria but are increasingly recognized for their direct interactions with mammalian hosts. Phage adherence to mucus is known to mediate enhanced antimicrobial effects in vitro. However, little is known about the therapeutic efficacy of mucus-adherent phages in vivo. Here, using a combination of in vitro gastrointestinal cell lines, a gut-on-a-chip microfluidic model, and an in vivo murine gut model, we demonstrated that a E. coli phage, øPNJ-6, provided enhanced gastrointestinal persistence and antimicrobial effects. øPNJ-6 bound fucose residues, of the gut secreted glycoprotein MUC2, through domain 1 of its Hoc protein, which led to increased intestinal mucus production that was suggestive of a positive feedback loop mediated by the mucus-adherent phage. These findings extend the Bacteriophage Adherence to Mucus model into phage therapy, demonstrating that øPNJ-6 displays enhanced persistence within the murine gut, leading to targeted depletion of intestinal pathogenic bacteria.
Asunto(s)
Infecciones por Escherichia coli , Escherichia coli , Mucosa Intestinal , Mucina 2 , Animales , Escherichia coli/virología , Ratones , Mucosa Intestinal/microbiología , Mucosa Intestinal/virología , Mucina 2/metabolismo , Humanos , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/terapia , Terapia de Fagos/métodos , Adhesión Bacteriana , Femenino , Moco/metabolismo , Moco/virología , Colifagos/fisiología , Fucosa/metabolismo , Ratones Endogámicos C57BLRESUMEN
Temperate phage-mediated horizontal gene transfer is a potent driver of genetic diversity in the evolution of bacteria. Most lambdoid prophages in Escherichia coli are integrated into the chromosome with the same orientation with respect to the direction of chromosomal replication, and their location on the chromosome is far from homogeneous. To better understand these features, we studied the interplay between lysogenic and lytic states of phage lambda in both native and inverted integration orientations at the wild-type integration site as well as at other sites on the bacterial chromosome. Measurements of free phage released by spontaneous induction showed that the stability of lysogenic states is affected by location and orientation along the chromosome, with stronger effects near the origin of replication. Competition experiments and range expansions between lysogenic strains with opposite orientations and insertion loci indicated that there are no major differences in growth. Moreover, measurements of the level of transcriptional bursts of the cI gene coding for the lambda phage repressor using single-molecule fluorescence in situ hybridization resulted in similar levels of transcription for both orientations and prophage location. We postulate that the preference for a given orientation and location is a result of a balance between the maintenance of lysogeny and the ability to lyse.IMPORTANCEThe integration of genetic material of temperate bacterial viruses (phages) into the chromosomes of bacteria is a potent evolutionary force, allowing bacteria to acquire in one stroke new traits and restructure the information in their chromosomes. Puzzlingly, this genetic material is preferentially integrated in a particular orientation and at non-random sites on the bacterial chromosome. The work described here reveals that the interplay between the maintenance of the stability of the integrated phage, its ability to excise, and its localization along the chromosome plays a key role in setting chromosomal organization in Escherichia coli.