Antibiotic-resistant (AR) bacterial wound infections (WIs) impose major burdens on healthcare systems, exacerbated by ineffective therapies and stalled antibiotic development. Phage therapy and phage-derived enzymes have gained traction as potent alternatives, leveraging targeted bactericidal mechanisms to combat AR pathogens. In this review, we summarised the antimicrobial mechanisms of both phage therapy and phage-derived enzymes as antimicrobial therapy, and outlined recent advances in their use for in vitro , in vivo and clinical applications for WI management. In addition, we also highlights recent advancements in their development, driven by genetic engineering, chemical modifications, and artificial intelligence. Finally, we identified the potential barriers and challenges they may encounter in clinical practice and the corresponding strategies to address these issues. The entire review gives us a comprehensive understanding of the latest advances in phages and their derivative enzyme therapies for treating WIs, in the hope that research in this field will continue to improve and innovate, accelerating the transition from the laboratory to application at the bedside and ultimately improving the efficacy of treatment for AR bacterial WIs.
Humans ; Phage Therapy/methods* ; Wound Infection/drug therapy* ; Bacteriophages/enzymology* ; Enzyme Therapy/methods* ; Animals ; Bacterial Infections/therapy*

Microbial communities such as those residing in the human gut are highly diverse and complex, and many with important implications for health and diseases. The effects and functions of these microbial communities are determined not only by their species compositions and diversities but also by the dynamic intra- and inter-cellular states at the transcriptional level. Powerful and scalable technologies capable of acquiring single-microbe-resolution RNA sequencing information in order to achieve a comprehensive understanding of complex microbial communities together with their hosts are therefore utterly needed. Here we report the development and utilization of a droplet-based smRNA-seq (single-microbe RNA sequencing) method capable of identifying large species varieties in human samples, which we name smRandom-seq2. Together with a triple-module computational pipeline designed for the bacteria and bacteriophage sequencing data by smRandom-seq2 in four human gut samples, we established a single-cell level bacterial transcriptional landscape of human gut microbiome, which included 29,742 single microbes and 329 unique species. Distinct adaptive response states among species in Prevotella and Roseburia genera and intrinsic adaptive strategy heterogeneity in Phascolarctobacterium succinatutens were uncovered. Additionally, we identified hundreds of novel host-phage transcriptional activity associations in the human gut microbiome. Our results indicated that smRandom-seq2 is a high-throughput and high-resolution smRNA-seq technique that is highly adaptable to complex microbial communities in real-world situations and promises new perspectives in the understanding of human microbiomes.
Humans ; Gastrointestinal Microbiome/genetics* ; Bacteriophages/physiology* ; High-Throughput Nucleotide Sequencing ; Sequence Analysis, RNA/methods* ; Bacteria/virology*

In recent years, the bacteriophage Φ29 (Phi29) DNA polymerase has garnered increasing attention due to its high-fidelity amplification capacity at constant temperatures. To advance the industrial application of this type of isothermal polymerases, this study mined and characterized new enzymes from the microbial metagenome based on the known Phi29 DNA polymerase sequence. The results revealed that a new enzyme, Php29 DNA polymerase, was identified in the microbial metagenome with plants as the hosts. This enzyme exhibited higher strand displacement activity, with a 59.5% similarity to bacteriophage Φ29. Experimental validation demonstrated that the enzyme had 3'→5' exonuclease activity, and its amplification products can serve as substrates for further catalytic reactions. The discovery and validation of Php29 DNA polymerase gives insights into the future industrial application of isothermal polymerases.
DNA-Directed DNA Polymerase/metabolism* ; Bacillus Phages/genetics* ; Metagenome

Acinetobacter baumannii is a Gram-negative opportunistic pathogen widely distributed in hospital settings. It can survive for a long time and cause a variety of infections, including pneumonia, septicemia, urinary tract infections, and meningitis. The bacterium demonstrates extensive resistance, particularly to critical antibiotics like carbapenems and polymyxins, posing a serious threat to the recovery of severely ill patients. Carbapenem-resistant A. baumannii has been designated as a pathogen of critical priority on the World Health Organization (WHO) Bacterial Pathogen Priority List, requiring urgent development of new therapeutic agents. Phages, as a novel biological control approach, exhibit substantial potential in combating A. baumannii infections due to their specific ability to infect and lyse bacteria. This review highlights the application and potential of phages and phage-derived enzymes against multidrug-resistant A. baumannii, considering the epidemiological trends of A. baumannii in China, with the aim of providing innovative insights and strategies for phage therapy of drug-resistant bacterial infections.
Acinetobacter baumannii/drug effects* ; Drug Resistance, Multiple, Bacterial ; Phage Therapy/methods* ; Acinetobacter Infections/microbiology* ; Humans ; Bacteriophages/physiology* ; Anti-Bacterial Agents/pharmacology*

Phages, as obligate bacterial and archaeal parasites, constitute a virus group of paramount ecological significance due to their exceptional abundance and genetic diversity. These biological entities serve as critical regulators in Earth's ecosystems, driving biogeochemical cycles, energy fluxes, and ecosystem services across terrestrial and marine environments. Within soil microbiomes, phages function as microbial "dark matter," maintaining the soil-plant system balance through precise modulation of the microbial community structure and functional dynamics. Despite the growing research interests in soil phages in recent years, the proportion of such studies in environmental virology remains disproportionately low, which is primarily attributed to researchers' limited familiarity with the research methodologies for phage microecology, incomplete technical frameworks, and inherent challenges posed by soil environmental complexity. To address these challenges, this review synthesizes cutting-edge methodologies for soil phage investigation from four aspects: (1) tangential flow filtration (TFF)-based phage enrichment strategies; (2) integrated quantification approaches combining double-layer agar plating, epifluorescence microscopy, and flow cytometry; (3) multi-omics analytical pipelines leveraging metagenomics and viromics datasets; and (4) computational frameworks merging machine learning algorithms with eco-evolutionary theory for deciphering phage-host interaction networks. Through comparative analysis of methodological principles, technical merits, and application scopes, we establish a comprehensive workflow for soil phage research. Future research in this field should prioritize: (1) construction of soil phage resource libraries, (2) exploration of RNA phages based on transcriptomes, (3) functional characterization of unknown genes, and (4) deep integration and interaction validation of multi-omics data. This systematic methodological synthesis provides critical technical references for addressing fundamental challenges in characterizing soil phages regarding the community structure, functional potential, and interaction mechanisms with hosts.
Bacteriophages/physiology* ; Soil Microbiology ; Ecosystem ; Microbiota ; Metagenomics/methods*

Phage immunoprecipitation sequencing (PhIP-Seq) is a high-throughput and low-cost method for analyzing the specific binding of target proteins to peptide libraries. The method uses oligonucleotide library synthesis (OLS) to encode proteome-scale peptide libraries for display on phages, and then immunoprecipitates these library phages with target proteins (such as antibodies) for subsequent analysis by high-throughput DNA sequencing. PhIP-Seq enables the screening of peptide targets that react specifically with hundreds of proteins or pathogens. PhIP-Seq has been successfully applied in various fields such as disease detection, screening of autoimmune disease biomarkers, vaccine development, and allergen detection, becoming a high-throughput diagnostic technology. This article systematically describes the development, applications, and result evaluation of PhIP-Seq, in order to gain a more comprehensive understanding of the application and future development prospects of this technology in various fields.
Peptide Library ; Humans ; Immunoprecipitation/methods* ; High-Throughput Nucleotide Sequencing/methods* ; Bacteriophages/genetics*

Cancer brings about an enormous threat to human health,making the exploration of its mechanisms and therapeutic strategies a current focal point and challenge in research.Bacteriophages are integral components of the human microbiome,and studies have shown their influences on tumor growth and metastasis and their pivotal role in cancer treatment.This article elucidates the mechanisms by which bacteriophages impact the occurrence and development of cancer from their interactions with cancer cells,effects on bacteria,and influence on the immune system.Additionally,it explores bacteriophage-based strategies in cancer treatment and their potential in this field.This article aims to bring new thoughts and insights to the research in this field.
Humans ; Bacteriophages ; Microbiota ; Neoplasms/therapy* ; Phage Therapy

Objective To construct a recombinant poxvirus vector vaccine, rVTTδTK-RBD, and to evaluate its safety and immunogenicity. Methods The receptor-binding domain (RBD) gene was synthesized with reference to the gene sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and was inserted into the polyclonal site of the self-constructed recombinant plasmid pSTKE, to construct the recombinant poxvirus shuttle vector pSTKE-RBD. This was then transfected into BHK-21 cells pre-infected with the vaccinia virus Tiantan strain (VTT). The recombinant poxvirus rVTTδTK-RBD was successfully obtained after several rounds of fluorescence phage screening. The effect of rVTTδTK-RBD on the body mass of BALB/c mice was detected after immunizing mice by intra-nasal vaccination. The levels of specific and neutralizing antibodies produced by rVTTδTK-RBD on BALB/c mice were analyzed after immunizing mice intramuscularly. The effect of rVTTδTK-RBD on T cell subsets in BALB/c mice was detected by flow cytometry. Results Through homologous recombination, enhanced green fluorescent protein (EGFP) screening marker, and multiple rounds of fluorescent phosphorescence phage screening, a recombinant poxvirus rVTTδTK-RBD, expressing RBD with deletions in the thymidine kinase (TK) gene, was successfully obtained, which was validated by PCR. The in vivo experiments on BALB/c mice showed that rVTTδTK-RBD was highly immunogenic against SARS-CoV-2 and significantly reduced toxicity to the body compared to the parental strain VTT. Conclusion The recombinant poxvirus vaccine rVTTδTK-RBD against SARS-CoV-2 is successfully constructed and obtained, with its safety and immunogenicity confirmed through various experiments.
Animals ; Mice ; SARS-CoV-2/genetics* ; COVID-19 ; Vaccines, Synthetic/genetics* ; Genes, Reporter ; Bacteriophages ; Mice, Inbred BALB C

Objective: To establish and evaluate a method of enriching bacteriophages in natural water based on ferric trichloride-polyvinylidene fluoride (FeCl₃-PVDF)membrane filter. Methods: Based on the principle of flocculation concentration, the method of recovering bacteriophage from water sample was established by using iron ion flocculation combined with membrane filter. The titer of phage was determined by Agar double layer method. The recovery efficiency of phage was detected by phage fluorescence staining and real-time fluorescence PCR reaction. Water samples from different sources were collected for simulation experiment to evaluate the enrichment effect. At the same time, the sewage discharged from hospitals was taken as the actual water sample, and the common clinical drug-resistant bacteria were used as the host indicator bacteria to further analyze the enrichment effect of FeCl₃-PVDF membrane filter rapid enrichment method on the bacteriophage in natural water samples. Results: The method of enrichment of bacteriophages in natural water by iron ion concentration 50 mg/L and PVDF membrane filter was established. The recovery rate of this method for bacteriophage was 93%-100%. Under the multi-functional microscope, it was found that the bacteriophage of the enriched water sample increased significantly and the fluorescence value of the enriched water sample determined by the enzyme labeling instrument was about 13 times as high as that before enrichment. After concentration of the actual water samples from the hospital drainage, the positive rate of bacteriophage isolation in the concentrated group and the non-concentrated group was 23% and 4%, and the fluorescence value in the concentrated group was 2-24 times as high as that of the non-concentrated group. Conclusion: The method of FeCl₃-PVDF membrane filter is a simple, efficient and rapid method for enriching bacteriophages in different water samples.
Humans ; Bacteriophages ; Bacteria ; Iron ; Iron, Dietary ; Water

Tumor is a serious threat to human health. At present, surgical resection, chemoradiotherapy, targeted therapy and immunotherapy are the main therapeutic strategies. Monoclonal antibody has gradually become an indispensable drug type in the clinical treatment of cancer due to its high efficiency and low toxicity. Phage antibody library technology (PALT) is a novel monoclonal antibody preparation technique. The recombinant immunoglobulin variable region of heavy chain (VH)/variable region of light chain (VL) gene is integrated into the phage vector, and the antibody is expressed on the phage surface in the form of fusion protein to obtain a diverse antibody library. Through the process of adsorption-elution-amplification, the antibody library can be screened to obtain the antibody molecule with specific binding antigen as well as its gene sequence. PALT has the advantages of short antibody production cycle, strong plasticity of antibody structure, large antibody yield, high diversity and direct production of humanized antibodies. It has been used in screening tumor markers and preparation of antibody drugs for breast cancer, gastric cancer, lung cancer and liver cancer. This article reviews the recent progress and the application of PALT in tumor therapy.
Humans ; Bacteriophages/genetics* ; Immunoglobulin Variable Region/genetics* ; Gene Library ; Antibodies, Monoclonal/therapeutic use* ; Immunotherapy ; Peptide Library