Search Results (100)

The rate at which bacteria are gaining resistance to antibiotics is outpacing the discovery of new drugs. The rise of superbugs such as Carbapenem-resistant and Extended-Spectrum Beta-Lactamase Producing Enterobacteriaceae are leading to infections that are resistant to our last lines of defense. One of the most prolific genera of these bacteria is Klebsiella, which causes one third of Gram-negative infections. The need for alternative and companion treatments has never been greater. Bacteriophages are bacteria-infecting viruses with high specificity to their host. They show great promise as a potential treatment for antibiotic-resistant infections. Here, we describe the characterization of five closely related bacteriophages (ValerieMcCarty01–05) isolated against an antibiotic-resistant clinical strain of Klebsiella oxytoca, which is an emerging antimicrobial-resistant threat within the Klebsiella genus. These phages demonstrate high similarity at both the genomic and proteomic levels and share homology with other T4-like Enterobacterales phage. Two phages were further characterized through a mass spectrometry analysis of purified virions, identifying peptide spectrum matches for 40 proteins which appear to be virion proteins. In addition, the peptide spectrum matches for 39 hypothetical proteins suggest they are indeed proteins. Amino acid alignment revealed that the tail fibers display more variability than most of their genome, suggesting possible adaptive tail fiber gene shuffling. Despite this variability, these phages maintained broad but high specificity for Klebsiella species in this paper, including K. oxytoca, K. pneumoniae and K. aerogenes and several clinical Klebsiella isolates, with infectivity differences seen only in efficiency. This specificity for Klebsiella is consistent with the genus to which they belong (the Jiaodavirus, which contains only Klebsiella phages) and suggests they may be involved in the evolution of Klebsiella and be useful therapeutics. Full article

Chryseobacterium indologenes MA9 is a causative agent of root rot disease in Panax notoginseng (P. notoginseng), with its high incidence being a major manifestation of continuous cropping barriers, severely hindering the sustainable development of the P. notoginseng industry. In this study, a novel lytic bacteriophage, MA9V-3, was isolated from wastewater, targeting C. indologenes MA9. The phage produced clear plaques, ranging from 1 to 3 mm in diameter, with a surrounding halo. Phage MA9V-3 achieved an adsorption rate of up to 80% after 30 min of contact with C. indologenes MA9, a latent period of approximately 40 min, and an average burst-size if 160 PFU/cell. Transmission electron microscopy revealed that phage MA9V-3 possesses an icosahedral head and a contractile tail, exhibiting a typical myovirus-like morphology. According to the latest ICTV taxonomy, MA9V-3 belongs to the class Caudoviricetes, and the phage’s biocontrol efficacy and inhibitory capacity were evaluated at different multiplicity of infection (MOI s). The results showed that the highest titer recorded at 1.6 × 10¹⁰ PFU/mL. Whole-genome sequencing revealed that MA9V-3 is a double-stranded circular DNA virus, with a genome length of 103,203 bp, GC content of 34.29%, and 150 open reading frames (ORFs), one of which is related to tRNA. Only 13 of these ORFs encode known functional sequences, likely due to the limited available gene data for such phages in the database, with additional details on hypothetical proteins yet to be uncovered. Comparative database analysis confirmed that the phage genome contains no antibiotic resistance or toxin-related genes. Phage therapy experiments were performed using MA9V-3 and two other phages screened in our laboratory. The experimental results showed that phage MA9V-3 may be a potential candidate for effectively controlling the infection of Panax notoginseng by C. indologenes MA9, and offering valuable insights into the potential application of phage therapy for managing bacterial plant diseases. Full article

Burkholderia pseudomallei, the causative agent of melioidosis, presents significant challenges in both treatment and environmental decontamination. Bacteriophages, or phages, are increasingly being explored as potential diagnostic, therapeutic, and biocontrol agents against this bacterial pathogen. Our recent investigation has shown that most B. pseudomallei genomes contained prophage(s) associated with specific tRNA gene loci, prompting us to explore these detectable prophages as sources of temperate phages for further applications. Transcriptomic profiling of B. pseudomallei Bp82, a model strain that possesses three different prophages, revealed high expression levels of the integrase and certain transcriptional regulatory genes within its prophages during normal exponential growth. Using one of its temperate phages, namely φBP82.2, a P2-like phage, as a model, we investigated the lysogenic–lytic control mechanisms. Mutagenesis of the integrase gene, phiBP82.2_gp51, did not improve killing activity compared to the wildtype phage. In contrast, deletion of phiBP82.2_gp38, a putative transcriptional regulatory gene, and two downstream hypothetical protein genes, phiBP82.2_gp36 and phiBP82.2_gp37, resulted in significant lytic improvement. We conclude that these genes play a crucial role in the lysogenic–lytic switch of φBP82.2, suggesting a new avenue for engineering temperate phages for future applications. Full article

Therapeutics based on RNA are commonly produced via biocatalytic approaches using RNA polymerases. The most frequently applied enzyme is the RNA polymerase of Enterobacteria phage T7. However, this enzyme has unfavorable properties, like the formation of double-stranded RNA (dsRNA). This undesired by-product can activate the innate immune system via pattern recognition receptors and cause inflammation. Removal of the contaminant is time-consuming and expensive. In this work, we applied a genome mining approach to identify unidentified single-subunit RNA polymerases with minimal dsRNA generation. A large meta database was screened, and 74 sequences were selected. Two RNA polymerases generating barely detectable amounts of dsRNA were identified from the initial sequence portfolio. Their promoters were detected via a fluorescent RNA aptamer screening, and slightly acidic transcription conditions were established. Further activity characterization showed a significant reduction of dsRNA to 0.001% and 0.02%. Due to these beneficial attributes, these RNA polymerases generate mRNA with enhanced stability, which most likely lowers the immune response towards the desired mRNA. This could be especially useful for producing long RNAs, such as self-amplifying RNA, as these typically require improved stability and low dsRNA content. Full article

The modified DNA base 2,6 aminopurine (2-aminoadenine, (d)Z base) was originally found in phages to counteract host-encoded restriction systems. However, only a limited number of restriction endonucleases (REases) have been tested on dZ-modified DNA. Here, we report the activity results of 147 REases on dZ-modified PCR DNA. Among the enzymes tested, 53% are resistant or partially resistant, and 47% are sensitive when their restriction sites contain one to six modified bases. Sites with four to six dZ substitutions are most likely to resist Type II restriction. Our results support the notion that dZ-modified phage genomes evolved to combat host-encoded restriction systems. dZ-modified DNA can also reduce phage T5 exonuclease degradation, but has no effect on RecBCD digestion. When two genes for dZ biosynthesis and one gene for dATP hydrolysis from Salmonella phage PMBT28 (purZ (adenylosuccinate synthetase), datZ (dATP triphosphohydrolase), and mazZ ((d)GTP-specific diphosphohydrolase) were cloned into an E. coli plasmid, the level of dZ incorporation reached 19–20% of adenosine positions. dZ levels further increased to 29–44% with co-expression of a DNA polymerase gene from the same phage. High levels of dZ incorporation in recombinant plasmid are possible by co-expression of purZ, mazZ, datZ and phage DNA helicase, dpoZ (DNA polymerase) and ssb (single-stranded DNA binding protein SSB). This work expands our understanding of the dZ modification of DNA and opens new avenues for engineering restriction systems and therapeutic applications. Full article

Bacteriophages are a key ecological factor in the legume rhizosphere, controlling bacterial populations and affecting introduced inoculant strains. Despite their importance, rhizobiophage genomic diversity remains poorly characterized. We report the complete genome of a novel predicted temperate Sinorhizobium phage, AP-202, isolated from agricultural Chernozem. This siphovirus infects the symbiont Sinorhizobium meliloti. Its 121,599 bp dsDNA genome has a strikingly low GC content (27.1%), likely reflecting adaptive evolution and a strategy to evade host defenses. The linear genome is flanked by 240 bp direct terminal repeats (DTRs), and its DNA packaging follows a T7-like strategy. Annotation predicted 178 protein-coding genes and one tRNA. Functional analysis revealed a complete lysogeny module and a divergent, two-pronged codon-usage strategy for translational control. A significant part of the proteome (74.2%) comprises hypothetical proteins, with 50 CDSs having no database homologs, underscoring its genetic novelty. Complete-genome comparison shows minimal similarity to known rhizobiophages, defining AP-202 as a distinct lineage. Phenotypic analysis indicates AP-202 acts as a selective ecological filter, with host resistance being more prevalent in agricultural than in natural soils. The AP-202 genome provides a unique model for studying phage–host coevolution in the rhizosphere and is a valuable resource for comparative genomics and soil virome research. Full article

Multidrug-resistant (MDR) Enterobacter cloacae is a growing public health issue worldwide, highlighting the urgent need for alternative antimicrobial strategies. This study reports on a lytic phage, designated B1, isolated from sewage, which exhibits specificity and lytic efficiency against MDR E. cloacae. Morphological observation revealed that B1 possesses an icosahedral head (~54 nm) and a short tail (~13 nm). Phage B1 showed a narrow host range, demonstrated stability within a temperature range of 4–37 °C, tolerance to pH values between 5 and 11, and showed an excellent bacteriolytic capacity with a short latent period of less than 10 min and a burst size of approximately 150 PFU/initially infected cell, indicating a rapid lytic cycle and efficient replication capability. Whole-genome sequencing revealed that the phage genome consists of 40,163 base pairs of double-stranded DNA containing 52 open reading frames (ORFs) with a GC content of 52%. Comparative genome-wide analysis using VIRIDIC revealed that B1 shares 75% to 92% similarity with Escherichia phage IMM-002 (accession: NC_048071), Citrobacter phage SH4, and Cronobacter phage Dev2 (accession: NC_023558), but shares less than 70% similarity with other Enterobacter phages. According to ICTV criteria, B1 represents a new species within the same genus as T7-like phages belonging to Autographiviridae, subfamily Studiervirinae, genus Kayfunavirus. In addition, B1 lacks lysogeny-associated or virulence genes and exhibits potent lytic activity against multidrug-resistant E. cloacae, making it a promising candidate for phage therapy. These findings opened up our understanding of the diversity of T7-like phages and provided insights into their evolutionary adaptability and therapeutic potential. Full article

As promising biological tools, bacteriophages offer broad potential applications in disease diagnosis, treatment, and food safety. This study is the first to isolate a novel bacteriophage, designated vB_YpP_JC53 (abbreviated JC53), from the soil of wild rodent nests in plague-endemic areas of Yunnan Province. This bacteriophage is a T7-like phage that has the broadest host range among all T7-like phages discovered to date and remains stable under varying temperature and pH conditions. Comparative genomic analysis through NCBI revealed that the nucleotide sequence of phage JC53 shares 94.98% homology (95% coverage) with phage PSTCR2, a member of the Solymavirus genus, while exhibiting substantially lower similarity to known Yersinia phages. Further phylogenetic and collinearity analyses demonstrate that JC53 represents an evolutionarily distinct lineage, clearly diverging from Yersinia-infecting, T7-like, and Shigella phages, suggesting the emergence of a novel evolutionary branch. Its low ANI values relative to Yersinia phages and mosaic genome organization indicate a complex evolutionary origin, reflecting the extensive diversity of environmental phage populations. Collectively, these findings support the designation of JC53 as a novel Yersinia phage. Genome sequencing revealed that JC53 has a genome size of 39,415 bp, with a total of 52 predicted open reading frames. The broad bacteriophage spectrum of JC53 challenges the long-standing perception that T4-like bacteriophages primarily depend on a wide host range. These findings suggest that, within plague foci, JC53 may maintain ecological fitness by targeting other bacteria rather than strictly relying on Yersinia pestis. As a result, JC53 holds potential as an ecological control agent with the potential to suppress plague transmission by regulating the microbial community structure within foci. Full article

Background: The extracellular domain of the M2 protein (M2e) and the conserved region of the second subunit of the hemagglutinin (HA2, 76–130 а.а.) of the influenza A virus, could be used to develop broad-spectrum influenza vaccines. However, these antigens have low immunogenicity and require the use of special carriers to enhance it. Virus-like particles (VLPs) formed from viral capsid proteins are among the most effective carriers. Methods: In this work, we obtained and characterized VLPs based on capsid proteins (CPs) of single-stranded RNA bacteriophages Beihai32 and PQ465, simultaneously displaying M2e and HA2 peptides. Results: Fusion proteins expressed in Escherichia coli formed spherical VLPs of about 30 nm in size. Subcutaneous immunization of mice with chimeric VLPs elicited a robust humoral immune response against M2e and the whole influenza A virus, and promoted the formation of cytokine-secreting antigen-specific CD4⁺ and CD8⁺ effector memory T cells. Conclusions: VLPs based on CPs of phages Beihai32 and PQ465 carrying conserved peptides M2e and HA2 of the influenza A virus can be used for the development of universal influenza vaccines. Full article

Sialyl-Tn (sTn) is a tumor-associated carbohydrate antigen (TACA) abundantly expressed by various types of carcinomas. While conventional antibody-based platforms have traditionally been used for the detection and targeting of sTn, alternative binding scaffolds may offer distinct advantages. Variable lymphocyte receptor B (VLRB), the immunoglobulin-like molecule of jawless vertebrates, offers a promising alternative for glycan recognition. In this study, a phage-displayed VLRB library was utilized to identify sTn-specific binders. Two candidates, designated as ccombodies A8 and B11, were isolated after four rounds of biopanning. Both were expressed and purified using Ni-affinity and FPLC, yielding proteins with apparent molecular weights of ~27 kDa in SDS-PAGE. Sequence analysis revealed a preference for glycan-binding residues in randomized hypervariable regions, with A8 exhibiting an increased aliphatic content. ELISA confirmed selective binding to sTn and other O-glycans containing the core α-GalNAc, with EC₅₀ values of 18.2 and 14.2 nM for A8 and B11, respectively. Vicia villosa lectin inhibited ccombody binding to sTn, indicating shared epitope recognition. Additionally, both ccombodies bound to sTn-positive glycoproteins and carcinoma cell lines HeLa and LS174T. These findings demonstrate that phage display of VLRBs enables the identification of high-affinity, glycan-specific binders, offering a compelling alternative to immunoglobulin-based platforms for future diagnostic and therapeutic applications targeting tumor-associated glycans. Full article

A comprehensive comparative analysis was conducted on the nucleotide and amino acid sequences of intact phiLM21-like prophages (phiLM21-LPhs), which currently represent the most prevalent prophages in Sinorhizobium meliloti—a symbiotic partner of Fabaceae plants. Remarkably, the nucleotide sequences of 25 phiLM21-LPhs, identified across 36 geographically dispersed S. meliloti strains, covered no more than 34% of the phiLM21 phage genome. All prophages were integrated into specific isoacceptor tRNA genes and carried a tyrosine-type integrase gene; however, this integration did not exhibit features of tRNA-dependent lysogeny. Only one-fifth of phiLM21-LPhs encoded the minimal set of regulators for lysogenic/lytic cycle transitions, while the remainder contained either uncharacterized regulatory elements or appeared to be undergoing genomic “anchoring” within the host bacterium. The phiLM21-LPhs harbored open reading frames (ORFs) of diverse origins (phage-derived, bacterial, and unknown), yet over half of these ORFs had undeterminable functions, representing genetic “dark matter”. The observed diversification of intact phiLM21-like prophages likely stems from recombination events involving both virulent/temperate phages and phylogenetically remote bacterial taxa. The evolutionary and biological significance of the substantial genetic “dark matter” within these prophages in soil saprophytic bacteria remains an unresolved question. Full article

Acute hepatopancreatic necrosis disease (AHPND), caused by the bacterium Vibrio parahaemolyticus, is a major threat to global shrimp aquaculture. In this study, we evaluated the therapeutic effects of phage therapy in Litopenaeus vannamei challenged with AHPND-causing Vibrio parahaemolyticus. Phage application at various concentrations significantly improved shrimp survival, with the 1 ppm group demonstrating the highest survival rate. Enzymatic assays revealed that phage-treated shrimp exhibited enhanced immune enzyme activities, including acid phosphatase (ACP), alkaline phosphatase (AKP), and lysozyme (LZM). In addition, antioxidant defenses such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), and total antioxidant capacity (T-AOC) significantly improved, accompanied by reduced malondialdehyde (MDA) levels. Serum biochemical analyses demonstrated marked improvements in lipid metabolism, particularly reductions in triglyceride (TG), total cholesterol (TC), and low-density lipoprotein (LDL), alongside higher levels of beneficial high-density lipoprotein (HDL). Transcriptomic analysis identified 2274 differentially expressed genes (DEGs), notably enriched in pathways involving fatty acid metabolism, peroxisome functions, lysosomes, and Toll-like receptor (TLR) signaling. Specifically, phage treatment upregulated immune and metabolic regulatory genes, including Toll-like receptor 4 (TLR4), myeloid differentiation primary response protein 88 (MYD88), interleukin-1β (IL-1β), nuclear factor erythroid 2-related factor 2 (Nrf2), and peroxisome proliferator-activated receptor (PPAR), indicating activation of innate immunity and antioxidant defense pathways. These findings suggest that phage therapy induces protective immunometabolic adaptations beyond its direct antibacterial effects, thereby providing an ecologically sustainable alternative to antibiotics for managing bacterial diseases in shrimp aquaculture. Full article

Flow virometry (FVM) offers a promising approach for monitoring viruses and virus-like particles (VLPs) in environmental samples. This study compares levels of non-specific VLPs across a wastewater treatment plant (WWTP) with levels of somatic coliphage, (F+) specific coliphage, Pepper Mild Mottle Virus (PMMoV), CrAssphage (CrAss), and Tomato Brown Rugose Fruit Virus (ToBRFV). All targets were quantified in influent, secondary-treated effluent, and tertiary-treated effluent at the University of California, Davis Wastewater Treatment Plant (UCDWWTP) over 11 weeks. We established an FVM-gating boundary for VLPs using bacteriophages T4 and ϕ6 as well as four phages isolated from wastewater. We then utilize T4 alongside three submicron beads as quality controls in the FVM assay. Coliphage was measured by standard plaque assays, and genome copies of PMMoV, CrAss, and ToBRFV were measured by digital droplet (dd)PCR. FVM results for wastewater revealed distinct microbial profiles at each treatment stage. However, correlations between VLPs and targeted viruses were poor. Trends for virus inactivation and removal, observed for targeted viruses during wastewater treatment, were consistent with expectations. Conversely, VLP counts were elevated in the WWTP effluent relative to the influent. Additional sampling revealed a decrease in VLP counts during the filtration treatment step following secondary treatment but a substantial increase in VLPs following ultraviolet disinfection. Defining application boundaries remain crucial to ensuring meaningful data interpretation as flow cytometry and virometry take on greater significance in water quality monitoring. Full article

It is estimated that over 60% of known tailed phages are siphophages, which are characterized by a long, flexible, and non-contractile tail. Nevertheless, entire high-resolution structures of siphophages remain scarce. Using cryo-EM, we resolved the structures of T-series siphophage T1, encompassing its head, connector complex, tail tube, and tail tip, at near-atomic resolution. The density maps enabled us to build the atomic models for the majority of T1 proteins. The T1 head comprises 415 copies of the major capsid protein gp47, arranged into an icosahedron with a triangulation number of seven, decorated with 80 homologous trimers and 60 heterotrimers along the threefold and quasi-threefold axes of the icosahedron. The T1 connector complex is composed of two dodecamers (a portal and an adaptor) and two hexamers (a stopper and a tail terminator). The flexible tail tube comprises approximately 34 hexameric rings of tail tube. The extensive disulfide bond network along the successive tail rings may mediate the flexible bending. The distal tip of T1, which is cone-shaped and assembled by proteins gp33, gp34, gp36, gp37, and gp38, displays structural similarity to that of phage lambda. In conjunction with previous studies of lambda-like siphophages, our structure will facilitate further exploration of the structural and mechanistic aspects of lambda-like siphophages. Full article

Six novel Microbacterium phages belonging to the Tectiviridae family were isolated using Microbacterium testaceum as a host. Phages MuffinTheCat, Badulia, DesireeRose, Bee17, SCoupsA, and LuzDeMundo were purified from environmental samples by students participating in the Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program at Alliance University, New York. The phages have linear dsDNA genomes 15,438–15,636 bp with 112–120 bp inverted terminal repeats. Transmission electron microscopy (TEM) imaging analysis revealed that the six novel phages have six-sided icosahedral double-layered capsids with an internal lipid membrane that occasionally forms protruding nanotubules. Annotation analysis determined that the novel Microbacterium phages all have 32–34 protein-coding genes and no tRNAs. Like other Tectiviridae, the phage genomes are arranged into two segments and include three highly conserved family genes that encode a DNA polymerase, double jelly-roll major capsid protein, and packaging ATPase. Although the novel bacteriophages have 91.6 to 97.5% nucleotide sequence similarity to each other, they are at most 58% similar to previously characterized Tectiviridae genera. Consequently, these novel Microbacterium phages expand the diversity of the Tectiviridae family, and we propose they form the sixth genus, Zetatectivirus. Full article