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Large-genome bacteriophages (jumbo phages) of the proposed family Chimalliviridae assemble a nucleus-like compartment bounded by a protein shell that protects the replicating phage genome from host-encoded restriction enzymes and DNA-targeting CRISPR-Cas nucleases. While the nuclear shell provides broad protection against host nucleases, it necessitates transport of mRNA out of the nucleus-like compartment for translation by host ribosomes, and transport of specific proteins into the nucleus-like compartment to support DNA replication and mRNA transcription. Here, we identify a conserved phage nuclear shell-associated protein that we term Chimallin C (ChmC), which adopts a nucleic acid-binding fold, binds RNA with high affinity in vitro, and binds phage mRNAs in infected cells. ChmC also forms phase-separated condensates with RNA in vitro. Targeted knockdown of ChmC using mRNA-targeting dCas13d results in accumulation of phage-encoded mRNAs in the phage nucleus, reduces phage protein production, and compromises virion assembly. Taken together, our data show that the conserved ChmC protein plays crucial roles in the viral life cycle, potentially by facilitating phage mRNA translocation through the nuclear shell to promote protein production and virion development.

Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here, we identify two components of this protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA (Protein importer of chimalliviruses A), that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together, our results allow us to propose a multistep model for the Protein Import Chimallivirus pathway, where proteins are targeted to PicA by amino acids on their surface and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts.

The emergence of antibiotic-resistant bacteria (ARB) has necessitated the development of alternative therapies to deal with this global threat. Bacteriophages (viruses that target bacteria) that kill ARB are one such alternative. Although phages have been used clinically for decades with inconsistent results, a number of recent advances in phage selection, propagation, and purification have enabled a reevaluation of their utility in contemporary clinical medicine. In most phage therapy cases, phages are administered in combination with antibiotics to ensure that patients receive the standard-of-care treatment. Some phages may work cooperatively with antibiotics to eradicate ARB, as often determined using non-standardized broth assays. We sought to develop a solid media-based assay to assess cooperativity between antibiotics and phages to offer a standardized platform for such testing. We modeled the interactions that occur between antibiotics and phages on solid medium to measure additive, antagonistic, and synergistic interactions. We then tested the method using different bacterial isolates and identified a number of isolates where synergistic interactions were identified. These interactions were not dependent on the specific organism, phage family, or antibiotic used. A priori susceptibility to the antibiotic or the specific phage were not requirements to observe synergistic interactions. Our data also confirm the potential for the restoration of vancomycin to treat vancomycin-resistant Enterococcus (VRE) when used in combination with phages. Solid media assays for the detection of cooperative interactions between antibiotics and phages can be an accessible technique adopted by clinical laboratories to evaluate antibiotic and phage choices in phage therapy.IMPORTANCEBacteriophages have become an important alternative treatment for individuals with life-threatening antibiotic-resistant bacteria (ARB) infections. Because antibiotics represent the standard-of-care for treatment of ARB, antibiotics and phages often are delivered together without evidence that they work cooperatively. Testing for cooperativity can be difficult due to the equipment necessary and a lack of standardized means for performing the testing in liquid medium. We developed an assay using solid medium to identify interactions between antibiotics and phages for gram-positive and gram-negative bacteria. We modeled the interactions between antibiotics and phages on solid medium, and then tested multiple replicates of vancomycin-resistant Enterococcus (VRE) and Stenotrophomonas in the assay. For each organism, we identified synergy between different phage and antibiotic combinations. The development of this solid media assay for assessing synergy between phages and antibiotics will better inform the use of these combinations in the treatment of ARB infections.

The Pseudomonas syringae species complex is a heterogeneous group of plant pathogenic bacteria associated with a wide distribution of plant species. Advances in genomics are revealing the complex evolutionary history of this species complex and the wide array of genetic adaptations underpinning their diverse lifestyles. Here, we genomically characterize two P. syringae isolates collected from diseased Callery pears (Pyrus calleryana) in Berkeley, California in 2019 and 2022. We also isolated a lytic bacteriophage, which we characterized and evaluated for biocontrol efficiency. Using a multilocus sequence analysis and core genome alignment, we classified the P. syringae isolates as members of phylogroup 2, related to other strains previously isolated from Pyrus and Prunus. An analysis of effector proteins demonstrated an evolutionary conservation of effectoromes across isolates classified in PG2 and yet uncovered unique effector profiles for each, including the two newly identified isolates. Whole-genome sequencing of the associated phage uncovered a novel phage genus related to Pseudomonas syringae pv. actinidiae phage PHB09 and the Flaumdravirus genus. Finally, using in planta infection assays, we demonstrate that the phage was equally useful in symptom mitigation of immature pear fruit regardless of the Pss strain tested. Overall, this study demonstrates the diversity of P. syringae and their viruses associated with ornamental pear trees, posing spill-over risks to commercial pear trees and the possibility of using phages as biocontrol agents to reduce the impact of disease.IMPORTANCEGlobal change exacerbates the spread and impact of pathogens, especially in agricultural settings. There is a clear need to better monitor the spread and diversity of plant pathogens, including in potential spillover hosts, and for the development of novel and sustainable control strategies. In this study, we characterize the first described strains of Pseudomonas syringae pv. syringae isolated from Callery pear in Berkeley, California from diseased tissues in an urban environment. We show that these strains have divergent virulence profiles from previously described strains and that they can cause disease in commercial pears. Additionally, we describe a novel bacteriophage that is associated with these strains and explore its potential to act as a biocontrol agent. Together, the data presented here demonstrate that ornamental pear trees harbor novel P. syringae pv. syringae isolates that potentially pose a risk to local fruit production, or vice versa-but also provide us with novel associated phages, effective in disease mitigation.

Viruses are often studied using metagenome-assembled sequences, but genome incompleteness hampers comprehensive and accurate analyses. Contig Overlap Based Re-Assembly (COBRA) resolves assembly breakpoints based on the de Bruijn graph and joins contigs. Here we benchmarked COBRA using ocean and soil viral datasets. COBRA accurately joined the assembled sequences and achieved notably higher genome accuracy than binning tools. From 231 published freshwater metagenomes, we obtained 7,334 bacteriophage clusters, ~83% of which represent new phage species. Notably, ~70% of these were circular, compared with 34% before COBRA analyses. We expanded sampling of huge phages (≥200 kbp), the largest of which was curated to completion (717 kbp). Improved phage genomes from Rotsee Lake provided context for metatranscriptomic data and indicated the in situ activity of huge phages, whiB-encoding phages and cysC- and cysH-encoding phages. COBRA improves viral genome assembly contiguity and completeness, thus the accuracy and reliability of analyses of gene content, diversity and evolution.

Diversity-generating retroelements (DGRs) are used by bacteria, archaea, and viruses as a targeted mutagenesis tool. Through error-prone reverse transcription, DGRs introduce random mutations at specific genomic loci, enabling rapid evolution of these targeted genes. However, the function and benefits of DGR-diversified proteins in cellular hosts remain elusive. We find that 82% of DGRs from one of the major monophyletic lineages of DGR reverse transcriptases are encoded by multicellular bacteria, which often have two or more DGR loci in their genomes. Using the multicellular purple sulfur bacterium Thiohalocapsa sp. PB-PSB1 as an example, we characterized nine distinct DGR loci capable of generating 10282 different combinations of target proteins. With environmental metagenomes from individual Thiohalocapsa aggregates, we show that most of PB-PSB1s DGR target genes are diversified across its biogeographic range, with spatial heterogeneity in the diversity of each locus. In Thiohalocapsa PB-PSB1 and other bacteria hosting this lineage of cellular DGRs, the diversified target genes are associated with NACHT-domain anti-phage defenses and putative ternary conflict systems previously shown to be enriched in multicellular bacteria. We propose that these DGR-diversified targets act as antigen sensors that confer a form of adaptive immunity to their multicellular consortia, though this remains to be experimentally tested. These findings could have implications for understanding the evolution of multicellularity, as the NACHT-domain anti-phage systems and ternary systems share both domain homology and conceptual similarities with the innate immune and programmed cell death pathways of plants and metazoans.

BACKGROUND: Environmental surveillance, using detection of Salmonella Typhi DNA, has emerged as a potentially useful tool to identify typhoid-endemic settings; however, it is relatively costly and requires molecular diagnostic capacity. We sought to determine whether S. Typhi bacteriophages are abundant in water sources in a typhoid-endemic setting, using low-cost assays. METHODOLOGY: We collected drinking and surface water samples from urban, peri-urban and rural areas in 4 regions of Nepal. We performed a double agar overlay with S. Typhi to assess the presence of bacteriophages. We isolated and tested phages against multiple strains to assess their host range. We performed whole genome sequencing of isolated phages, and generated phylogenies using conserved genes. FINDINGS: S. Typhi-specific bacteriophages were detected in 54.9% (198/361) of river and 6.3% (1/16) drinking water samples from the Kathmandu Valley and Kavrepalanchok. Water samples collected within or downstream of population-dense areas were more likely to be positive (72.6%, 193/266) than those collected upstream from population centers (5.3%, 5/95) (p=0.005). In urban Biratnagar and rural Dolakha, where typhoid incidence is low, only 6.7% (1/15, Biratnagar) and 0% (0/16, Dolakha) river water samples contained phages. All S. Typhi phages were unable to infect other Salmonella and non-Salmonella strains, nor a Vi-knockout S. Typhi strain. Representative strains from S. Typhi lineages were variably susceptible to the isolated phages. Phylogenetic analysis showed that S. Typhi phages belonged to the class Caudoviricetes and clustered in three distinct groups. CONCLUSIONS: S. Typhi bacteriophages were highly abundant in surface waters of typhoid-endemic communities but rarely detected in low typhoid burden communities. Bacteriophages recovered were specific for S. Typhi and required Vi polysaccharide for infection. Screening small volumes of water with simple, low-cost (~$2) plaque assays enables detection of S. Typhi phages and should be further evaluated as a scalable tool for typhoid environmental surveillance.

Cyclic-oligonucleotide-based anti-phage signaling system (CBASS) is a common immune system that uses cyclic oligonucleotide signals to limit phage replication. In turn, phages encode anti-CBASS (Acb) proteins such as Acb2, which can sequester some cyclic dinucleotides (CDNs) and limit downstream effector activation. Here, we identified that Acb2 sequesters many CDNs produced by CBASS systems and inhibits stimulator of interferon genes (STING) activity in human cells. Surprisingly, the Acb2 hexamer also binds with high affinity to CBASS cyclic trinucleotides (CTNs) 333-cyclic AMP-AMP-AMP and 333-cAAG at a distinct site from CDNs. One Acb2 hexamer can simultaneously bind two CTNs and three CDNs. Phage-encoded Acb2 provides protection from type III-C CBASS that uses cA3 signaling molecules. Moreover, phylogenetic analysis of >2,000 Acb2 homologs encoded by diverse phages and prophages revealed that most are expected to bind both CTNs and CDNs. Altogether, Acb2 sequesters nearly all known CBASS signaling molecules through two distinct binding pockets and therefore serves as a broad-spectrum inhibitor of cGAS-based immunity.

Antibiotic-resistant bacteria present an emerging challenge to human health. Their prevalence has been increasing across the globe due in part to the liberal use of antibiotics that has pressured them to develop resistance. Those bacteria that acquire mobile genetic elements are especially concerning because those plasmids may be shared readily with other microbes that can then also become antibiotic resistant. Serious infections have recently been related to the contamination of preservative-free eyedrops with extensively drug-resistant (XDR) isolates of Pseudomonas aeruginosa, already resulting in three deaths. These drug-resistant isolates cannot be managed with most conventional antibiotics. We sought to identify alternatives to conventional antibiotics for the lysis of these XDR isolates and identified multiple bacteriophages (viruses that attack bacteria) that killed them efficiently. We found both jumbo phages (>200 kb in genome size) and non-jumbo phages that were active against these isolates, the former killing more efficiently. Jumbo phages effectively killed the three separate XDR P. aeruginosa isolates both on solid and liquid medium. Given the ongoing nature of the XDR P. aeruginosa eyedrop outbreak, the identification of phages active against them provides physicians with several novel potential alternatives for treatment.

Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq) in profiling mouse autoantibodies. This library was validated using 7 genetically distinct mouse lines across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2-/- and μMT) were used to model nonspecific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate 3 distinct autoimmune disease models. First, serum from Lyn-/- IgD+/- mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities. Second, serum from nonobese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, was enriched in peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire-/- mouse sera were used to identify numerous autoantigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 carrying recessive mutations in AIRE. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity profiling.

Phages are one of the key ecological drivers of microbial community dynamics, function, and evolution. Despite their importance in bacterial ecology and evolutionary processes, phage genes are poorly characterized, hampering their usage in a variety of biotechnological applications. Methods to characterize such genes, even those critical to the phage life cycle, are labor intensive and are generally phage specific. Here, we develop a systematic gene essentiality mapping method scalable to new phage-host combinations that facilitate the identification of nonessential genes. As a proof of concept, we use an arrayed genome-wide CRISPR interference (CRISPRi) assay to map gene essentiality landscape in the canonical coliphages λ and P1. Results from a single panel of CRISPRi probes largely recapitulate the essential gene roster determined from decades of genetic analysis for lambda and provide new insights into essential and nonessential loci in P1. We present evidence of how CRISPRi polarity can lead to false positive gene essentiality assignments and recommend caution towards interpreting CRISPRi data on gene essentiality when applied to less studied phages. Finally, we show that we can engineer phages by inserting DNA barcodes into newly identified inessential regions, which will empower processes of identification, quantification, and tracking of phages in diverse applications.

As the world moves toward a green economy and sustainable agriculture, bacterial viruses or bacteriophages (phages) become attractive biocontrol agents for controlling crop diseases. Effective utilization of phages in farms requires integrated knowledge of crops, pathogens, phages, and surroundings. Phages must encounter environmental fluctuations, including temperature, and must remain infectious for successful bacteria lysis. This work studied a soilborne RSJ2 phage discovered in Thailand, which can eliminate Ralstonia solanacearum, causing bacterial wilt disease in chili. We investigated how phage infectivity and nanomechanics responded to thermal changes. The plaque-based assay showed that the infectivity of the RSJ2 phage was stable within 24–40 °C, an average temperature fluctuation in tropical regions. The structural examination also showed that the phage remained intact. The nanomechanical property of the phage was inspected by the atomic force microscopy-based nanoindentation. The result revealed that the phage stiffness within 24–40 °C was statistically similar (0.05–0.06 N/m). Upon heating at 40 °C for 1, 5, and 10 h and resting at 25 °C, the stiffness of the phage particles increased to 0.09–0.11 N/m (54–83% increase). The stiffness results suggest structural adaptation of the protein subunits as a response to thermal alteration. The study exhibits that the phage structure is highly dynamic and can nanomechanically respond to varying temperatures. The phage stiffness may reveal insight into phage adaptation to environmental factors. Equipped with the knowledge of phage infectivity, structure, and nanomechanics, we can design practical guidelines for effective phage usage in farming and propelling green and safe agriculture. [ABSTRACT FROM AUTHOR]

One of the most common bacteria that cause nosocomial infections is Klebsiella pneumonia (K. pneumoniae), especially in patients who are very sick and admitted to the intensive care unit (ICU). The frequency of multi-drug-resistant Klebsiella pneumoniae (MDRKP) has dramatically increased worldwide in recent decades, posing an urgent threat to public health. The Western world's bacteriophage (phage) studies have been revitalized due to the increasing reports of antimicrobial resistance and the restricted development and discovery of new antibiotics. These factors have also spurred innovation in other scientific domains. The primary agent in phage treatment is an obligately lytic organism (called bacteriophage) that kills the corresponding bacterial host while sparing human cells and lessening the broader effects of antibiotic usage on commensal bacteria. Phage treatment is developing quickly, leading to many clinical studies and instances of life-saving medicinal use. In addition, phage treatment has a few immunological adverse effects and consequences in addition to its usefulness. Since K. pneumoniae antibiotic resistance has made treating multidrug-resistant (MDR) infections challenging, phage therapy (PT) has emerged as a novel therapeutic strategy. The effectiveness of phages has also been investigated in K. pneumoniae biofilms and animal infection models. Compared with antibiotics, PT exhibits numerous advantages, including a particular lysis spectrum, co-evolution with bacteria to avoid the emergence of phage resistance, and a higher abundance and diversity of phage resources than found in antibiotics. Moreover, phages are eliminated in the absence of a host bacterium, which makes them the only therapeutic agent that self-regulates at the sites of infection. Therefore, it is essential to pay attention to the role of PT in treating these infections. This study summarizes the state of knowledge on Klebsiella spp. phages and provides an outlook on the development of phage-based treatments that target K. pneumoniae in clinical trials. [ABSTRACT FROM AUTHOR]

The emergence and increase in antimicrobial resistance (AMR) is now widely recognized as a major public health challenge. Traditional antimicrobial drugs are becoming increasingly ineffective, while the development of new antibiotics is waning. As a result, alternative treatments for infections are garnering increased interest. Among these alternatives, bacteriophages, also known as phages, are gaining renewed attention and are reported to offer a promising solution to alleviate the burden of bacterial infections. This review discusses the current successes of phage therapy (PT) against multidrug-resistant organisms (MDROs), such as Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, and Enterobacter spp. The review also compares the efficacy of PT with that of chemical antibiotics, reporting on its benefits and limitations, while highlighting its impact on the human gut microbiome and immune system. Despite its potential, phage therapy is reported to face challenges such as the narrow antibacterial range, the complexity of developing phage cocktails, and the need for precise dosing and duration protocols. Nevertheless, continued research, improved regulatory frameworks, and increased public awareness are essential to realize its full potential and integration into standard medical practice, paving the way for innovative treatments that can effectively manage infections in an era of rising antimicrobial resistance. [ABSTRACT FROM AUTHOR]

Background: The composition of the vaginal microbiota during the menstrual cycle is dynamic, with some women remaining eu- or dysbiotic and others transitioning between these states. What defines these dynamics, and whether these differences are microbiome-intrinsic or mostly driven by the host is unknown. To address this, we characterized 49 healthy, young women by metagenomic sequencing of daily vaginal swabs during a menstrual cycle. We classified the dynamics of the vaginal microbiome and assessed the impact of host behavior as well as microbiome differences at the species, strain, gene, and phage levels. Results: Based on the daily shifts in community state types (CSTs) during a menstrual cycle, the vaginal microbiome was classified into four Vaginal Community Dynamics (VCDs) and reported in a classification tool, named VALODY: constant eubiotic, constant dysbiotic, menses-related, and unstable dysbiotic. The abundance of bacteria, phages, and bacterial gene content was compared between the four VCDs. Women with different VCDs showed significant differences in relative phage abundance and bacterial composition even when assigned to the same CST. Women with unstable VCDs had higher phage counts and were more likely dominated by L. iners. Their Gardnerella spp. strains were also more likely to harbor bacteriocin-coding genes. Conclusions: The VCDs present a novel time series classification that highlights the complexity of varying degrees of vaginal dysbiosis. Knowing the differences in phage gene abundances and the genomic strains present allows a deeper understanding of the initiation and maintenance of permanent dysbiosis. Applying the VCDs to further characterize the different types of microbiome dynamics qualifies the investigation of disease and enables comparisons at individual and population levels. Based on our data, to be able to classify a dysbiotic sample into the accurate VCD, clinicians would need two to three mid-cycle samples and two samples during menses. In the future, it will be important to address whether transient VCDs pose a similar risk profile to persistent dysbiosis with similar clinical outcomes. This framework may aid interdisciplinary translational teams in deciphering the role of the vaginal microbiome in women's health and reproduction. 9k3PVfrRnyX1V_8jfg2U5D Video Abstract [ABSTRACT FROM AUTHOR]

The prevalence of multidrug resistant (MDR) bacterial infections continues to rise as the development of antibiotics needed to combat these infections remains stagnant. MDR enterococci are a major contributor to this crisis. A potential therapeutic approach for combating MDR enterococci is bacteriophage (phage) therapy, which uses lytic viruses to infect and kill pathogenic bacteria. While phages that lyse some strains of MDR enterococci have been identified, other strains display high levels of resistance and the mechanisms underlying this resistance are poorly defined. Here, we use a CRISPR interference (CRISPRi) screen to identify a genetic locus found on a mobilizable plasmid from Enterococcus faecalis involved in phage resistance. This locus encodes a putative serine recombinase followed by a Type IV restriction enzyme (TIV-RE) that we show restricts the replication of phage phi47 in vancomycin-resistant E. faecalis. We further find that phi47 evolves to overcome restriction by acquiring a missense mutation in a TIV-RE inhibitor protein. We show that this inhibitor, termed type IV restriction inhibiting factor A (tifA), binds and inactivates diverse TIV-REs. Overall, our findings advance our understanding of phage defense in drug-resistant E. faecalis and provide mechanistic insight into how phages evolve to overcome antiphage defense systems. Using a CRISPR interference genetic approach, Bullen, Johnson, and colleagues discover a type IV restriction (TIV-RE) enzyme encoded on a mobilizable plasmid of multidrug resistant Enterococcus faecalis. This TIV-RE is a potent inhibitor of bacteriophage infection. [ABSTRACT FROM AUTHOR]

The escalating antibiotic resistance in mycobacterial species poses a significant threat globally, necessitating an urgent need to find alternative solutions. Bacteriophage-derived endolysins, which facilitate phage progeny release by attacking bacterial cell walls, present promising antibacterial candidates due to their rapid lytic action, high specificity and low risk of resistance development. In mycobacteria, owing to the complex, hydrophobic cell wall, mycobacteriophages usually synthesize two endolysins: LysinA, which hydrolyzes peptidoglycan; LysinB, which delinks mycolic acid-containing outer membrane and arabinogalactan, releasing free mycolic acid. In this study, we conducted domain analysis and functional characterization of a novel LysinB from RitSun, an F2 sub-cluster mycobacteriophage from our phage collection. Several key properties of RitSun LysinB make it an important antimycobacterial agent: its ability to lyse Mycobacterium smegmatis from without, a higher than previously reported specific activity of 1.36 U/mg and its inhibitory effect on biofilm formation. Given the impermeable nature of the mycobacterial cell envelope, dissecting RitSun LysinB at the molecular level to identify its cell wall-destabilizing sequence could be utilized to engineer other native lysins as fusion proteins, broadening their activity spectrum. [ABSTRACT FROM AUTHOR]

Human parvovirus B19 (B19V), like most parvoviruses, possesses phospholipase A2 (PLA2) activity, which is thought to mediate endosomal escape by membrane disruption. Here, we challenge this model and find evidence for a mechanism of B19V entry mediated by the glycosphingolipid globoside without endosome disruption and retrograde transport to the Golgi. We show that B19V PLA2 activity requires specific calcium levels and pH conditions that are not optimal in endosomes. Accordingly, endosomal membrane integrity was maintained during B19V entry. Furthermore, endosomes remained intact when loaded with MS2 bacteriophage particles pseudotyped with multiple B19V PLA2 subunits, providing superior enzymatic potential compared to native B19V. In globoside knockout cells, incoming viruses are arrested in the endosomal compartment and the infection is blocked. Infection can be rescued by promoting endosomal leakage with polyethyleneimine (PEI), demonstrating the essential role of globoside in facilitating endosomal escape. Incoming virus colocalizes with Golgi markers and interfering with Golgi function blocks infection, suggesting that globoside-mediated entry involves the Golgi compartment, which provides conditions favorable for the lipolytic PLA2. Our study challenges the current model of B19V entry and identifies globoside as an essential intracellular receptor required for endosomal escape. [ABSTRACT FROM AUTHOR]

Endolysins produced by bacteriophages hydrolyze host cell wall peptidoglycan to release newly assembled virions. D29 mycobacteriophage specifically infects mycobacteria including the pathogenic Mycobacterium tuberculosis. D29 encodes LysA endolysin, which hydrolyzes mycobacterial cell wall peptidoglycan. We previously showed that LysA harbors two catalytic domains (N‐terminal domain [NTD] and lysozyme‐like domain [LD]) and a C‐terminal cell wall binding domain (CTD). While the importance of LD and CTD in mycobacteriophage biology has been examined in great detail, NTD has largely remained unexplored. Here, to address NTD's significance in D29 physiology, we generated NTD‐deficient D29 (D29∆NTD) by deleting the NTD‐coding region from D29 genome using CRISPY‐BRED. We show that D29∆NTD is viable, but has a longer latent period, and a remarkably reduced burst size and plaque size. A large number of phages were found to be trapped in the host during the D29∆NTD‐mediated cell lysis event. Such poor release of progeny phages during host cell lysis strongly suggests that NTD‐deficient LysA produced by D29∆NTD, despite having catalytically‐active LD, is unable to efficiently lyse host bacteria. We thus conclude that LysA NTD is essential for optimal release of progeny virions, thereby playing an extremely vital role in phage physiology and phage propagation in the environment. [ABSTRACT FROM AUTHOR]

Bacteriophages are emerging as a promising alternative in combating antibiotic-resistant bacteria amidst the escalating global antimicrobial resistance crisis. Recently, there has been a notable resurgence of interest in phages, prompting extensive research into their therapeutic potential. Beyond conventional microbiology and virology techniques, such as genomics and proteomics, novel phenotypic and chemical characterization methods are being explored. Among these, there is a growing interest in vibrational spectroscopy, especially in advanced modalities such as surface-enhanced Raman spectroscopy (SERS), tip-enhanced Raman spectroscopy (TERS), and atomic force microscopy-infrared spectroscopy (AFM-IR), which offer improved sensitivity and spatial resolution. This review explores the spectrum of uses of vibrational spectroscopy for bacteriophages, including its role in diagnostics, biosensing, phage detection, assistance in phage-based therapy, and advancing basic research. [ABSTRACT FROM AUTHOR]

As a sirtuin (SIR2) family protein, defense-associated sirtuin2 (DSR2) has been demonstrated to participate in bacterial anti-phage resistance via depleting nicotinamide adenine dinucleotide (NAD+) of infected cells, which can be activated by tail tube protein (TTP) and inhibited by DSR anti-defense 1 (DSAD1) of diverse phages. However, the regulating mechanism remains elusive. Here, we determined the cryo-electron microscopy structure of apo DSR2, as well as the respective complex structures with TTP and DSAD1. Structural analyses and biochemical studies reveal that DSR2 forms a tetramer with a SIR2 central core and two distinct conformations. Monomeric TTP preferentially binds to the closed conformation of DSR2, inducing conformational distortions on SIR2 tetramer assembly to activate its NADase activity. DSAD1 combines with the open conformation of DSR2, directly or allosterically inhibiting TTP activation on DSR2 NAD+ hydrolysis. Our findings decipher the detailed molecule mechanisms for DSR2 NADase activity regulation and lay a foundation for in-depth understanding of the DSR2 anti-phage defense system. [ABSTRACT FROM AUTHOR]

The gut microbiota has been implicated in the multifactorial pathophysiology of cardiometabolic diseases (CMD). Bacteriophage (phage) therapy offers precise modulation of the gut microbiota, targeting specific pathobionts while minimizing collateral damage to beneficial microbes. Studies highlight the therapeutic potential of phages in cardiometabolic diseases, including reductions in pathogenic bacteria and improvements in metabolic parameters. Challenges in developing phage therapy for CMD include identifying target bacteria, generating effective combinations of phages, product formulation, and strict production requirements. Temperate phages and phages with CRISPR-Cas machinery could alter (metabolic) the function of gut microbes to improve the CMD phenotype in the future. The worldwide prevalence of cardiometabolic diseases (CMD) is increasing, and emerging evidence implicates the gut microbiota in this multifactorial disease development. Bacteriophages (phages) are viruses that selectively target a bacterial host; thus, phage therapy offers a precise means of modulating the gut microbiota, limiting collateral damage on the ecosystem. Several studies demonstrate the potential of phages in human disease, including alcoholic and steatotic liver disease. In this opinion article we discuss the potential of phage therapy as a predefined medicinal product for CMD and discuss its current challenges, including the generation of effective phage combinations, product formulation, and strict manufacturing requirements. [ABSTRACT FROM AUTHOR]

Identifying the predominant strains of lactic acid bacteria (LAB) in dahi that are resistant to bacteriophages is essential for developing robust starter cultures. Dahi samples from two agroecological zones of Kerala were collected for isolation of LAB. Limosilactobacillus fermentum emerged as the dominant starter culture species based on sequence‐based identification and technological property characterisation. Dairy effluents were screened for phages targeting L. fermentum, and 10 of 11 strains of L. fermentum showed resistance to one lytic phage isolated as part of this study. The strain ADMT 15 showed sensitivity to the phage and on subsequent genome sequencing the phage revealed its close relationship to members of the Herelleviridae phage family. [ABSTRACT FROM AUTHOR]

The phage display technology is based on the presentation of peptide sequences on the surface of virions of bacteriophages. Its development led to creation of sophisticated systems based on the possibility of the presentation of a huge variability of peptides, attached to one of proteins of bacteriophage capsids. The use of such systems allowed for achieving enormous advantages in the processes of selection of bioactive molecules. In fact, the phage display technology has been employed in numerous fields of biotechnology, as diverse as immunological and biomedical applications (in both diagnostics and therapy), the formation of novel materials, and many others. In this paper, contrary to many other review articles which were focussed on either specific display systems or the use of phage display in selected fields, we present a comprehensive overview of various possibilities of applications of this technology. We discuss an usefulness of the phage display technology in various fields of science, medicine and the broad sense of biotechnology. This overview indicates the spread and importance of applications of microbial systems (exemplified by the phage display technology), pointing to the possibility of developing such sophisticated tools when advanced molecular methods are used in microbiological studies, accompanied with understanding of details of structures and functions of microbial entities (bacteriophages in this case). [ABSTRACT FROM AUTHOR]

The bacteriophage Pseudomonas phage Ka1 was isolated from the water of a tributary stream of Lake Baikal, in the area of discharge of treated wastewater of the city of Slyudyanka. The bacteriophage genome contains 46 092 bp and has a 50% G+C pairs (AN OP455935.1). It exhibits 91% identity with the Pseudomonas phage PSA37 genome, belonging to Bruynoghevirus from the class Caudoviricetes suggesting that Ka1 is a new representative of Luz24-like phages (Bruynoghevirus). The bacteriophage lyzes 62% of P. aeruginosa clinical isolates and increases the efficiency of gentamicin, ciprofloxacin, imipenem and meropenem by 4–8-fold against these bacteria. Integrases, transposases and recombinases have not been identified in the genome of the bacteriophage Pseudomonas phage Ka1; which allows using it in the complex therapy of infections caused by P. aeruginosa. [ABSTRACT FROM AUTHOR]

This is the first report on the isolation of Rhodococcus aetherivorans-specific bacteriophages and of applicability of such invertebrates as Hyalophora cecropia and Eisenia fetida as objects for screening the phages capable of growth on Rhodococcus species. Some of the isolated phages were capable of reproduction on R. ruber and R. qingshengii. An efficient procedure for bacteriophage reproduction in the liquid culture of R. aetherivorans was developed. The revealed bacteriophages may be used for development of efficient genetic tools for Rhodococcus strains, including the industrially significant ones. [ABSTRACT FROM AUTHOR]

The role of surface antigens of Yersinia pestis in reception of the phage L-413C was evaluated experimentally. Based on the methods of phage inactivation after its co-incubation with soluble or bead-bounded antigens, the importance of the plague microbe LPS in the phage reception was confirmed, and the inability to bind the capsular antigen F1, Ail protein, and two autotransporters YapF and YapM was shown. The native and recombinant PsaA, being solved, significantly inhibited the lytic activity of the phage in contrast to the bead-bound antigens. The knockout EV cells (ΔpsaA) are able to bind the phage particles as well as the wild strain. The use of three methods to evaluate the role of the PsaA antigen in phage L-413C reception gave contradictory results. On the one hand, the reactive domains of PsaA are able to interact with phage particles in solution. At the same time, these domains appear to determine nonspecific binding of the PsaA protein to the underlying bacterial cell structures or polystyrene microsphere, preventing phage adhesion. [ABSTRACT FROM AUTHOR]

The widespread use of antibiotics, leading to antibiotic resistance and the appearance of antibiotics in the environment and food, has stimulated the development of new methods for monitoring antibacterial drugs in environmental objects. One of the promising areas for the development of methods for determining antibiotics is sensor technologies. The key point in the development of sensory systems is the selection of a sensitive (recognizing) element. One of the most popular methods for recognizing antibiotics is the use of antibodies. The main immunosensory systems based on recording the "antigen–antibody" interaction are presented, and the advantages and disadvantages of polyclonal and monoclonal antibodies are shown. The possibility of using phage antibodies to determine antibiotics is described separately. [ABSTRACT FROM AUTHOR]

The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. Bovine mastitis is a prevalent infectious disease in dairy herds worldwide, resulting in substantial economic losses. Staphylococcus aureus is a major cause of mastitis in animals, and its antibiotic resistance poses challenges for treatment. Recently, renewed interest has focused on the development of alternative methods to antibiotic therapy, including bacteriophages (phages), for controlling bacterial infections. In this study, 2 lytic phages, vB_SauM_JDYN (JDYN) and vB_SauM_JDF86 (JDF86), were isolated from the cattle sewage effluent samples collected from dairy farms in Shanghai. The 2 phages have a broad bactericidal spectrum against Staphylococcus of various origins. Genomic and morphological analyses revealed that the 2 phages belonged to the Myoviridae family. Moreover, JDYN and JDF86 remained stable under a wide temperature and pH range and were almost unaffected in chloroform. In this study, we prepared a phage cocktail (PHC-1) which consisted of a 1:1:1 ratio of JDYN, JDF86, and SLPW (a previously characterized phage). We found that PHC-1 showed the strongest bacteriolytic effect and the lowest frequency of emergence of bacteriophage insensitive mutants compared with monophages. Bovine mammary epithelial cells and lactating mice mastitis models were used to evaluate the effectiveness of PHC-1 in vitro and in vivo, respectively. The results demonstrated that PHC-1 treatment significantly reduced bacterial load, alleviated inflammatory response, and improved mastitis pathology. Altogether, these results suggest that PHC-1 has the potential to treat S. aureus -induced bovine mastitis and that phage cocktails can combat antibiotic-resistant S. aureus infections. [ABSTRACT FROM AUTHOR]

The analysis of transcriptional activity of the bacteriophage T5 hol/endo operon conducted in the paper revealed a strong constitutive promoter recognized by E. coli RNA polymerase and a transcription initiation point of the operon. It was also shown that the only translational start codon for holin was a non-canonical TTG. Translation initiation regions (TIRs) of both genes of the operon (hol and endo) were further analyzed using chimeric constructs, in which parts of the hol/endo regulatory regions were fused with the gene of a reporter protein (EGFP). It was found that TIR of hol was 20 times less effective than that of endo. As it turned out, the level of EGFP production was influenced by the composition of the constructs and the type of the hol start codon. Apparently, the translational suppression of holin's accumulation and posttranslational activation of endolysin by Ca2+ are the main factors ensuring the proper timing of the host cell lysis by bacteriophage T5. The approach based on the use of chimeric constructs proposed in the paper can be recommended for studying other native or artificial operons of any complexity: analyzing the impacts of separate DNA regions, as well as their coupled effect, on the processes of transcription and translation of recombinant protein(s). [ABSTRACT FROM AUTHOR]

Infections caused by multidrug-resistant (MDR) bacteria are a growing global concern. Enterobacter cloacae complex (ECC) species are particularly adept at developing antibiotic resistance. Phage therapy is proposed as an alternative treatment for pathogens that no longer respond to antibiotics. Unfortunately, ECC phages are understudied when compared to phages of many other bacterial species. In this Ghanaian-Finnish study, we isolated two ECC strains from ready-to-eat food samples and three novel phages from natural waters against these strains. We sequenced the genomic DNA of the novel Enterobacter phages, fGh-Ecl01, fGh-Ecl02, and fGh-Ecl04, and assessed their therapeutic potential. All of the phages were found to be lytic, easy to propagate, and lacking any toxic, integrase, or antibiotic resistance genes and were thus considered suitable for therapy purposes. They all were found to be related to T4-type viruses: fGh-Ecl01 and fGh-Ecl04 to karamviruses and fGh-Ecl02 to agtreviruses. Testing of Finnish clinical ECC strains showed promising susceptibility to these novel phages. As many as 61.1% of the strains were susceptible to fGh-Ecl01 and fGh-Ecl04, and 7.4% were susceptible to fGh-Ecl02. Finally, we investigated the susceptibility of the newly isolated ECC strains to three antibiotics – meropenem, ciprofloxacin, and cefepime – in combination with the novel phages. The use of phages and antibiotics together had synergistic effects. When using an antibiotic-phage combination, even low concentrations of antibiotics fully inhibited the growth of bacteria. [ABSTRACT FROM AUTHOR]

Serological detection of syphilis‐specific antibodies is the most widely used clinical method for diagnosing syphilis. However, the current methods in clinical practice are too diverse, complicating result interpretation and wasting medical resources. To meet the resultant demand for a simple fast naked‐eye detection method, we employed the bacterial cell‐binding domain (CBD) of bacteriophage lysin to functionalize bio‐microparticles (engineered non‐culturable red‐colored Staphylococcus aureus bacteria), forming a bioprobe that can detect the syphilis‐specific antibodies from human serum in 5 min with naked eyes. Specifically, CBD is bioengineered by fusion with one of three antigens of the syphilis‐causing bacteria Treponema pallidum separately. The three resulting fusion proteins bind to the bacteria surface through the CBD motif, generating a bioprobe with the antigens exposed. When the bioprobe is added to the serum samples collected from the syphilis patients, the syphilis‐specific antibodies bind the antigens on the bioprobes and cross‐link them, forming eye‐visible red aggregates for the naked‐eye antibody detection. The bioprobe was validated on 209 clinical samples, revealing its high clinical sensitivity and specificity. Exploiting the natural biorecognition between the bacteria‐specific phage‐derived species and the phage host bacteria represents a promising strategy for producing facile probes for rapid point‐of‐care testing of infectious diseases. [ABSTRACT FROM AUTHOR]

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Introduction: Aeromonas hydrophila is particularly harmful to freshwater aquaculture, and the search for phage is an effective biological control method, but reports of possible temperate phages and their mutants are rare in this field. In this study, a virulent phage highly homologous to prophage in the genomes of A. hydrophila was collected and preliminary biological characterization was carried out to understand its nature. Materials and methods: Water samples taken from eel ponds in Fujian, China were combined with the strain. Spot test method and double-layer agar plate assay was used for confirmation and purification. Phage virions were observed using transmission electron microscope. A total of 68 strains of Aeromonas spp. were used to determine the host range. MOI groups of 1,000, 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001 were prepared to detect the optimal MOI. The conditions of thermal stability assay were set as 30, 40, 50, 60, 70 and 80°C for 1 h, respectively, and conditions of acid and alkali stability assay were set as 2.0, 4.0, 6.0, 8.0, 10.0 and 12.0 of pH. MOI of 0.01 and 0.1, respectively, are set to determine the inhibitory capacity of phage. Results: A novel virulent A. hydrophila phage designated phiA051 has been isolated from aquaculture water. Electron microscopic observation showed that the phage phiA051 was composed of an icosahedral capsid. The phage phiA051 possesses an optimal multiplicity of infection (MOI) of 0.01, and its burst size was 108 PFU/cell. The phage maintained a high viability at temperatures of 30-50°C or pH 6.0-10.0 for 1 h. Phage phiA051 has certain potentials in rapidly inhibiting the spread of pathogen early in the outbreak, and it has a linear dsDNA with GC content of 60.55% and a total length of 32,212 bp, including 46 ORFs. Discussion: The phage phiA051 behaved as a virulent phage. However, the BLASTN result showed that 23 of the top 25 hits were genomes of Aeromonas strains. It was suggested that phiA051 was probably derived from some prophage in the chromosome of Aeromonas. Further investigation of the mechanism how phage phiA051 transforms from a temperate phage to a virulent phage will provide a unique perspective and idea to explore the potential of prophages. [ABSTRACT FROM AUTHOR]

Purpose: Identify soil properties involved in the adsorption of the bacteriophages (phages) ΦITL-1 and ΦRSP, analyzing different soils and substrates used for greenhouse tomato crops. Methods: Soil samples were collected from greenhouses in Mexico Central, and soil properties were obtained using the NOM-021-RECNAT-2000. For phage adsorption, soil or substrate was mixed with a phage solution, using three different Water Retention Capacity (WRC) levels, incubated under shaking to room temperature, and finally, the plaque-forming units were counted for each phage by titration. Results: The phage elution counts showed that the amount of aqueous solution in the mixture is inversely related to the number of viral particles adsorbed on the soil. Furthermore, the soils with a sandy texture had lower adsorption of phages in comparison with the rest of the soils analyzed, which have clay, clay loam, and sandy loam textures. A correlation analysis showed that soil pH and texture are the properties that most influenced phage adsorption. Finally, a principal component analysis showed that Cation Exchange Capacity (CEC) has a direct relationship with phage adsorption. Conclusion: In the present study, we demonstrated that soil texture, pH, CEC, and WRC are determinant variables that affect the adsorption of R. solanacearum phages ΦITL-1 and ΦRSP. Furthermore, we propose that the responsible mechanism may be due to the richness of cations in the soil, which could imply an electrostatic interaction with some exposed phage proteins. These results highlight the influence of soil properties on biocontrol strategies based on phages, which try to reduce the impact of soil-borne bacterial phytopathogens. [ABSTRACT FROM AUTHOR]

Using conventional immunoglobulin G (IgG) molecules as therapeutic agents presents several well-known disadvantages owing to their large size and structural complexity, negatively impacting development and production efficiency. Single-domain antibodies (sdAbs) are the smallest functional antibody format (~ 15 kDa) and represent a viable alternative to IgG in many applications. However, unlike natural single-domain antibodies, such as camelid VHH, the variable domains of conventional antibodies show poor physicochemical properties when expressed as sdAbs. This report identified stable sdAb variants of human VH3-23 from a framework region 2-randomized human VH library by phage display selection under thermal challenge. Synthetic complementarity determining region diversity was introduced to one of the selected variants with high thermal stability, expression level, and monomeric content to construct a human VH sdAb library. The library was validated by panning against a panel of antigens, and target-specific binders were identified and characterized for their affinity and biophysical properties. The results of this study suggest that a synthetic sdAb library based on a stability-engineered human VH scaffold could be a facile source of high-quality sdAb for many practical applications. [ABSTRACT FROM AUTHOR]

The common intestinal pathogen Klebsiella pneumoniae (K. pneumoniae) is one of the leading causes of fatal superbug infections that can resist the effects of commonly prescribed medicines. The uncontrolled use or misuse of antibiotics has increased the prevalence of drug-resistant K. pneumoniae strains in the environment. In the quest to search for alternative therapeutics for treating these drug-resistant infections, bacteriophages (bacterial viruses) emerged as potential candidates for in phage therapy against Klebsiella. The effective formulation of phage therapy against drug-resistant Klebsiella infections demands thorough characterization and screening of many bacteriophages. To contribute effectively to the formulation of successful phage therapy against superbug infections by K. pneumoniae, this study includes the isolation and characterization of a novel lytic bacteriophage MKP-1 to consider its potential to be used as therapeutics in treating drug-resistant Klebsiella infections. Morphologically, having a capsid attached to a long non-contractile tail, it was found to be a siphovirus that belongs to the class Caudoviricetes and showed infectivity against different strains of the target host bacterium. Comparatively, this double-stranded DNA phage has a large burst size and is quite stable in various physiological conditions. More interestingly, it has the potential to degrade the tough biofilms formed by K. pneumoniae (Klebsiella pneumoniae subsp. pneumoniae (Schroeter) Trevisan [ATCC 15380]) significantly. Thus, the following study would contribute effectively to considering phage MKP-1 as a potential candidate for phage therapy against Klebsiella infection. [ABSTRACT FROM AUTHOR]

Patients with severe carbapenem-resistant Acinetobacter baumannii (CRAB) infections currently face significant treatment challenges. When patients display signs of infection and the clinical suspicion of CRAB infections is high, appropriate treatment should be immediately provided. However, current treatment plans and clinical data for CRAB are limited. Inherent and acquired resistance mechanisms, as well as host factors, significantly restrict options for empirical medication. Moreover, inappropriate drug coverage can have detrimental effects on patients. Most existing studies have limitations, such as a restricted sample size, and are predominantly observational or non-randomized, which report significant variability in patient infection severity and comorbidities. Therefore, a gold-standard therapy remains lacking. Current and future treatment options of infections due to CRAB were described in this review. The dose and considerable side effects restrict treatment options for polymyxins, and high doses of ampicillin-sulbactam or tigecycline appear to be the best option at the time of initial treatment. Moreover, new drugs such as durlobactam and cefiderocol have substantial therapeutic capabilities and may be effective salvage treatments. Bacteriophages and antimicrobial peptides may serve as alternative treatment options in the near future. The advantages of a combination antimicrobial regimen appear to predominate those of a single regimen. Despite its significant nephrotoxicity, colistin is considered a primary treatment and is often used in combination with antimicrobials, such as tigecycline, ampicillinsulbactam, meropenem, or fosfomycin. The Infectious Diseases Society of America (IDSA) has deemed high-dose ampicillin-sulbactam, which is typically combined with high-dose tigecycline, polymyxin, and other antibacterial agents, the best option for treating serious CRAB infections. A rational combination of drug use and the exploration of new therapeutic drugs can alleviate or prevent the effects of CRAB infections, shorten hospital stays, and reduce patient mortality. [ABSTRACT FROM AUTHOR]

The study aims to accurately identify differentially expressed genes (DEGs) and biological pathways in mycobacterial infections through bioinformatics for deeper disease understanding. Differentially expressed genes (DEGs) was explored by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Unique DEGs were submitted on least absolute shrinkage and selection operator (LASSO) regression analysis. 1,057 DEGs from two GSE datasets were identified, which were closely connected with NTM/ latent TB infection (LTBI)/active TB disease (ATB). It was demonstrated that these DEGs are mainly associated with detoxification processes, and virus and bacterial infections. Moreover, the METTL7B gene was the most informative marker for distinguishing LTBI and ATB with an area under the curve (AUC) of 0.983 (95%CI: 0.964 to 1). The significantly upregulated HBA1/2 genes were the most informative marker for distinguishing between individuals of IGRA-HC/NTM and LTBI (P < 0.001). Moreover, the upregulated HBD gene was also differ between IGRA-HC/NTM and ATB (P < 0.001). We have identified gene signatures associated with Mycobacterium infection in whole blood, which could be significant for understanding the molecular mechanisms and diagnosis of NTM, LTBI, or ATB. [ABSTRACT FROM AUTHOR]

Streptomyces are renowned for their prolific production of specialized metabolites with applications in medicine and agriculture. These multicellular bacteria present a sophisticated developmental cycle and play a key role in soil ecology. Little is known about the impact of Streptomyces phage on bacterial physiology. In this study, we investigated the conditions governing the expression and production of "Samy," a prophage found in Streptomyces ambofaciens ATCC 23877. This siphoprophage is produced simultaneously with the activation of other mobile genetic elements. Remarkably, the presence and production of Samy increases bacterial dispersal under in vitro stress conditions. Altogether, this study unveiled a new property of a bacteriophage infection in the context of multicellular aggregate dynamics. Little is known about the impact of phages on Streptomyces physiology. In this study, induction of the prophage 'Samy' was found to enhance the dispersal of multicellular aggregates of Streptomyces ambofaciens bacteria in response to stress, likely promoting lineage propagation. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii, which is resistant to multiple drugs, is an opportunistic pathogen responsible for severe nosocomial infections. With no antibiotics available, phages have obtained clinical attention. However, since immunocompromised patients are often susceptible to infection, the appropriate timing of administration is particularly important. During this research, we obtained a lytic phage vB_AbaM_P1 that specifically targets A. baumannii. We then assessed its potential as a prophylactic treatment for lung infections caused by clinical strains. The virus experiences a period of inactivity lasting 30 min and produces approximately 788 particles during an outbreak. Transmission electron microscopy shows that vB_AbaM_P1 was similar to the Saclayvirus. Based on the analysis of high-throughput sequencing and bioinformatics, vB_AbaM_P1 consists of 107537 bases with a G + C content of 37.68%. It contains a total of 177 open reading frames and 14 tRNAs. No antibiotic genes were detected. In vivo experiments, using a cyclophosphamide-induced neutrophil deficiency model, tested the protective effect of phage on neutrophil-deficient rats by prophylactic application of phage. The use of phages resulted in a decrease in rat mortality caused by A. baumannii and a reduction in the bacterial burden in the lungs. Histologic examination of lung tissue revealed a decrease in the presence of immune cells. The presence of phage vB_AbaM_P1 had a notable impact on preventing A. baumannii infection, as evidenced by the decrease in oxidative stress in lung tissue and cytokine levels in serum. Our research offers more robust evidence for the early utilization of bacteriophages to mitigate A. baumannii infection. Key points: •A novel Saclayvirus phage infecting A. baumannii was isolated from sewage. •The whole genome was determined, analyzed, and compared to other phages. •Assaying the effect of phage in preventing infection in neutrophil-deficient models. [ABSTRACT FROM AUTHOR]

Yersinia (Y.) enterocolitica is an important foodborne pathogenic species that is mainly transmitted by the consumption of contaminated meat, particularly pork. To combat the bacteria along the food chain, the application of strictly lytic phages may be a promising tool. As the temperatures in the gut of animals and during food processing can differ significantly, a phage cocktail intended to be used for applications should comprise phages that are active at various temperatures. In this study, we isolated and characterized three phages with a myoviridal morphology (vB_YenM_P8, vB_YenM_P744 and vB_YenM_P778), which lysed the most important Y. enterocolitica serotypes O:3, O:9 and O:5,27 at a low multiplicity of infection (MOI) and at low temperatures down to 6◦C. While vB_YenM_P8 is a member of the T4 family, vB_YenM_P744 and vB_YenM_P778 are novel phages that do not show relationship to known phages. The three phages were mixed in a cocktail with the already described phages vB_YenM_P281 and vB_YenP_Rambo. The cocktail revealed a strong lytic activity and lysed a mixture of Y. enterocolitica serotypes at room temperature (RT) within few hours with a reduction of up to 4.8 log10 units. Moreover, at even lower temperatures the mixture was significantly reduced after incubation overnight. The strongest reductions were determined at 6◦C (4.0 log10 units) suggesting that the cocktail can lyse the psychrophilic Y. enterocolitica also during food processing. To determine possible phage resistance, 100 colonies that survived the infection by the phages were isolated and analysed regarding their serotype and phage susceptibility. Most isolates belonged to serotype O:9, but all of them were still sensitive to at least one phage of the cocktail. [ABSTRACT FROM AUTHOR]

Viruses are core components of the human microbiome, impacting health through interactions with gut bacteria and the immune system. Most human microbiome viruses are bacteriophages, which exclusively infect bacteria. Until recently, most gut virome studies focused on low taxonomic resolution (e.g., viral operational taxonomic units), hampering population-level analyses. We previously identified an expansive and widespread bacteriophage lineage in inhabitants of Amsterdam, the Netherlands. Here, we study their biodiversity and evolution in various human populations. Based on a phylogeny using sequences from six viral genome databases, we propose the Candidatus order Heliusvirales. We identify heliusviruses in 82% of 5441 individuals across 39 studies, and in nine metagenomes from humans that lived in Europe and North America between 1000 and 5000 years ago. We show that a large lineage started to diversify when Homo sapiens first appeared some 300,000 years ago. Ancient peoples and modern hunter-gatherers have distinct Ca. Heliusvirales populations with lower richness than modern urbanized people. Urbanized people suffering from type 1 and type 2 diabetes, as well as inflammatory bowel disease, have higher Ca. Heliusvirales richness than healthy controls. We thus conclude that these ancient core members of the human gut virome have thrived with increasingly westernized lifestyles. Here, based on phylogeny analyses using sequences from six viral genome databases, the authors study the biodiversity and evolution of a new bacteriophage lineage, Candidatus order Heliusvirales, and identify heliusviruses in 82% of 5,441 individuals across 39 studies, and in nine metagenomes from humans that lived in Europe and North America between 1,000 and 5,000 years ago, revealing associations with human diseases. [ABSTRACT FROM AUTHOR]

Objective: This study aimed to introduce a lytic bacteriophage against Staphylococcus saprophyticus from wastewater in Gorgan, northern Iran. Results: The vB_SsapS-46 phage was isolated from urban wastewater and formed round and clear plaques on bacterial culture. It was visualized by electron microscopy and had a large head (approximately 106 nm) and a long tail (approximately 150 nm), indicating that it belongs to the Siphoviridae family. The host range of vB_SsapS-46 was determined using a spot test on 35 S. saprophyticus clinical isolates, and it was able to lyse 12 of the 35 clinical isolates (34%). Finally, the relationship between phage sensitivity and adherence genes was assessed, revealing no significant correlation between phage sensitivity and the frequency of adherence genes. The vB_SsapS-46 phage can be used alone or in a mixture in future studies to control urinary tract infections caused by this bacterium, especially in the elimination of drug-resistant pathogens. [ABSTRACT FROM AUTHOR]

Several farmed fish species, including carps, tilapia, salmon, and catfish, have experienced significant economic losses in aquaculture due to motile Aeromonas septicemia caused by Aeromonas hydrophila. In the present study, a novel lytic bacteriophage infecting hypervirulent Aeromonas hydrophila (vAh) was isolated and characterized. This is the first report of a phage against vAh. Phage AhFM11 demonstrated lytic activity against both vAh strains and the A. hydrophila reference strain ATCC 35654. The AhFM11 genome was sequenced and assembled, comprising 168,243 bp with an average G + C content of 41.5%. The genome did not harbor any antibiotic resistance genes. Genomic information along with transmission electron microscopy revealed that phage AhFM11 belongs to the Straboviridae family. Therapeutic application of monophage AhFM11 in fish showed 100% survival in injection, 95% in immersion and 93% in oral feeding of phage top-coated feed. Fish and chicken meat spiked with A. hydrophila and phage showed significant reduction of A. hydrophila. These findings support that phage AhFM11 can be used as a biocontrol agent against vAh as an alternative to antibiotics. [ABSTRACT FROM AUTHOR]

Human activities such as agriculturalization and domestication have led to the emergence of many new pathogens via host-switching events between humans, domesticated and wild animals. Staphylococcus aureus is a multi-host opportunistic pathogen with a global healthcare and economic burden. Recently, it was discovered that laboratory and wild rodents can be colonised and infected with S. aureus, but the origins and zoonotic potential of rodent S. aureus is unknown. In order to trace their evolutionary history, we employed a dataset of 1249 S. aureus genome sequences including 393 of isolates from rodents and other small mammals (including newly determined sequences for 305 isolates from 7 countries). Among laboratory mouse populations, we identified multiple widespread rodent-specific S. aureus clones that likely originated in humans. Phylogeographic analysis of the most common murine lineage CC88 suggests that it emerged in the 1980s in laboratory mouse facilities most likely in North America, from where it spread to institutions around the world, via the distribution of mice for research. In contrast, wild rodents (mice, voles, squirrels) were colonized with a unique complement of S. aureus lineages that are widely disseminated across Europe. In order to investigate the molecular basis for S. aureus adaptation to rodent hosts, genome-wide association analysis was carried out revealing a unique complement of bacteriophages associated with a rodent host ecology. Of note, we identified novel prophages and pathogenicity islands in rodent-derived S. aureus that conferred the potential for coagulation of rodent plasma, a key phenotype of abscess formation and persistence. Our findings highlight the remarkable capacity of S. aureus to expand into new host populations, driven by the acquisition of genes promoting survival in new host-species. Author summary: New pathogens typically emerge after host-switching events, threatening public health and food security. Here we used an evolutionary genomic approach to reveal the origins of Staphylococcus aureus in laboratory and wild rodent populations. S. aureus in laboratory rodents appears to have originated in humans and following host jump events has spread around the world via the distribution of mice for research purposes. Distinct clones of S. aureus widely distributed around Europe are associated with wild rodent populations. We found unique types of prophages in rodent S. aureus that have played a central role in their emergence and expansion including some with genes encoding putative virulence factors. In particular, we identified novel prophages and pathogenicity islands that conferred the potential for coagulation of rodent plasma, a key phenotype of abscess formation and persistence. Taken together, our findings highlight the remarkable capacity of S. aureus to expand into new host populations, driven by the acquisition of genes acquired via mobile genetic elements. [ABSTRACT FROM AUTHOR]

Host health and disease are influenced by changes in the abundance and structure of intestinal flora. Current strategies are focused on regulating the structure of intestinal flora to ensure host health by alleviating disease. However, these strategies are limited by multiple factors, such as host genotype, physiology (microbiome, immunity, and gender), intervention, and diet. Accordingly, we reviewed the prospects and limitations of all strategies regulating the structure and abundance of microflora, including probiotics, prebiotics, diet, fecal microbiota transplantation, antibiotics, and phages. Some new technologies that can improve these strategies are also introduced. Compared with other strategies, diets and prebiotics are associated with reduced risk and high security. Besides, phages have the potential for application in the targeted regulation of intestinal microbiota due to their high specificity. Notably, the variability in individual microflora and their metabolic response to different interventions should be considered. Future studies should use artificial intelligence combined with multi-omics to investigate the host genome and physiology based on factors, such as blood type, dietary habits, and exercise, in order to develop individualized intervention strategies to improve host health. [ABSTRACT FROM AUTHOR]

Background: Understanding the interactions and dynamics of microbiotas within biological wastewater treatment systems is essential for ensuring their stability and long-term sustainability. In this study, we developed a systematic framework employing multi-omics and Hi-C sequencing to extensively investigate prokaryotic and phage communities within a hybrid biofilm and activated sludge system. Results: We uncovered distinct distribution patterns, metabolic capabilities, and activities of functional prokaryotes through the analysis of 454 reconstructed prokaryotic genomes. Additionally, we reconstructed a phage catalog comprising 18,645 viral operational taxonomic units (vOTUs) with high length and contiguity using hybrid assembly, and a distinct distribution of phages was depicted between activated sludge (AS) and biofilm. Importantly, 1340 host-phage pairs were established using Hi-C and conventional in silico methods, unveiling the host-determined phage prevalence. The majority of predicted hosts were found to be involved in various crucial metabolic processes, highlighting the potential vital roles of phages in influencing substance metabolism within this system. Moreover, auxiliary metabolic genes (AMGs) related to various categories (e.g., carbohydrate degradation, sulfur metabolism, transporter) were predicted. Subsequent activity analysis emphasized their potential ability to mediate host metabolism during infection. We also profiled the temporal dynamics of phages and their associated hosts using 13-month time-series metagenomic data, further demonstrating their tight interactions. Notably, we observed lineage-specific infection patterns, such as potentially host abundance- or phage/host ratio-driven phage population changes. Conclusions: The insights gained from this research contribute to the growing body of knowledge surrounding interactions and dynamics of host-phage and pave the way for further exploration and potential applications in the field of microbial ecology. 7mTZubWt51_LqpKWPutADa Video Abstract [ABSTRACT FROM AUTHOR]

Staphylococcus aureus´ biofilm-forming ability and rapid resistance development pose a significant challenge to successful treatment, particularly in postoperative complications, emphasizing the need for enhanced therapeutic strategies. Bacteriophage (phage) therapy has reemerged as a promising and safe option to combat multidrug-resistant bacteria. However, questions regarding the efficacy of phages against biofilms and the development of phage resistance require further evaluation. Expanding on the adaptable and evolutionary characteristics of phages, we introduce an evolutionary approach to enhance the activity of S. aureus phages against biofilms. Unlike other in vitro directed evolution methods performed in planktonic cultures, we employed pre-stablished biofilms to do a serial-passage assay to evolve phages monitored by real-time isothermal microcalorimetry (IMC). The evolved phages demonstrated an expanded host range, with the CUB_MRSA-COL_R9 phage infecting 83% of strains in the collection (n  =  72), surpassing the ISP phage, which represented the widest host range (44%) among the ancestral phages. In terms of antimicrobial efficacy, IMC data revealed superior suppression of bacterial growth by the evolved phages compared to the ancestral CUB-M and/or ISP phages against the respective bacterial strain. The phage cocktail exhibited higher efficacy, achieving over 90% suppression relative to the growth control even after 72  h of monitoring. Biofilm cell-counts, determined by RT-qPCR, confirmed the enhanced antibiofilm performance of evolved phages with no biofilm regrowth up to 48  h in treated MRSA15 and MRSA-COL strains. Overall, our results underscore the potential of biofilm-adapted phage cocktails to improve clinical outcomes in biofilmassociated infections, minimizing the emergence of resistance and lowering the risk of infection relapse. However, further investigation is necessary to evaluate the translatability of our results from in vitro to in vivo models, especially in the context of combination therapy with the current standard of care treatment. [ABSTRACT FROM AUTHOR]

AbstractThe increasing global incidence of multidrug-resistant (MDR) bacterial infections threatens public health and compromises various aspects of modern medicine. Recognising the urgency of this issue, the World Health Organisation has prioritised the development of novel antimicrobials to combat ESKAPEE pathogens. Comprising Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and Escherichia coli, such pathogens represent a spectrum of high to critical drug resistance, accounting for a significant proportion of hospital-acquired infections worldwide. In response to the waning efficacy of antibiotics against these resilient pathogens, phage therapy (PT) has emerged as a promising therapeutic strategy. This review provides a comprehensive summary of clinical research on PT and explores the translational journey of phages from laboratory settings to clinical applications. It examines recent advancements in pre-clinical and clinical developments, highlighting the potential of phages and their proteins, alone or in combination with antibiotics. Furthermore, this review underlines the importance of establishing safe and approved routes of phage administration to patients. In conclusion, the evolving landscape of phage therapy offers a beacon of hope in the fight against MDR bacterial infections, emphasising the imperative for continued research, innovation and regulatory diligence to realise its full potential in clinical practice. [ABSTRACT FROM AUTHOR]