Search Results (170)

Lactic acid bacteria (LAB) are central to food, feed, and health biotechnology, yet their genomes have long resisted rapid, precise manipulation. This review charts the evolution of LAB genome-editing strategies from labor-intensive RecA-dependent double-crossovers to state-of-the-art CRISPR and CRISPR-associated transposase systems. Native homologous recombination, transposon mutagenesis, and phage-derived recombineering opened the door to targeted gene disruption, but low efficiencies and marker footprints limited throughput. Recent phage RecT/RecE-mediated recombineering and CRISPR/Cas counter-selection now enable scar-less point edits, seamless deletions, and multi-kilobase insertions at efficiencies approaching model organisms. Endogenous Cas9 systems, dCas-based CRISPR interference, and CRISPR-guided transposases further extend the toolbox, allowing multiplex knockouts, precise single-base mutations, conditional knockdowns, and payloads up to 10 kb. The remaining hurdles include strain-specific barriers, reliance on selection markers for large edits, and the limited host-range of recombinases. Nevertheless, convergence of phage enzymes, CRISPR counter-selection and high-throughput oligo recombineering is rapidly transforming LAB into versatile chassis for cell-factory and therapeutic applications. Full article

This review addresses the urgent need for alternative strategies to combat drug-resistant mycobacterial infections, including multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis, as well as non-tuberculous mycobacterial (NTM) diseases. Traditional antibiotics are increasingly limited by resistance, toxicity, and poor efficacy, particularly in immunocompromised patients. A comprehensive literature search was conducted using PubMed, Scopus, and Google Scholar, covering publications primarily from 2000 to 2025. Only articles published in English were included to ensure consistency in data interpretation. Search terms included “mycobacteriophages,” “phage therapy,” “drug-resistant mycobacteria, “diagnostic phages,” and “phage engineering.” The review examines the therapeutic and diagnostic potential of mycobacteriophages—viruses that specifically infect mycobacteria—focusing on their molecular biology, engineering advances, delivery systems, and clinical applications. Evidence suggests that mycobacteriophages offer high specificity, potent bactericidal activity, and adaptability, positioning them as promising candidates for targeted therapy. Although significant obstacles remain—including immune interactions, limited host range, and regulatory challenges—rapid progress in synthetic biology and delivery platforms continues to expand their clinical potential. As research advances and clinical frameworks evolve, mycobacteriophages are poised to become a valuable asset in the fight against drug-resistant mycobacterial diseases, offering new precision-based solutions where conventional therapies fail. Full article

Bacteriophages (phages), the most abundant biological entities on Earth, have long served as both model systems and therapeutic tools. Recent advances in synthetic biology and genetic engineering have revolutionized the capacity to tailor phages with enhanced functionality beyond their natural capabilities. This review outlines the current landscape of synthetic and functional engineering of phages, encompassing both in-vivo and in-vitro strategies. We describe in-vivo approaches such as phage recombineering systems, CRISPR-Cas-assisted editing, and bacterial retron-based methods, as well as synthetic assembly platforms including yeast-based artificial chromosomes, Gibson, Golden Gate, and iPac assemblies. In addition, we explore in-vitro rebooting using TXTL (transcription–translation) systems, which offer a flexible alternative to cell-based rebooting but are less effective for large genomes or structurally complex phages. Special focus is given to the design of customized phages for targeted applications, including host range expansion via receptor-binding protein modifications, delivery of antimicrobial proteins or CRISPR payloads, and the construction of biocontained, non-replicative capsid systems for safe clinical use. Through illustrative examples, we highlight how these technologies enable the transformation of phages into programmable bactericidal agents, precision diagnostic tools, and drug delivery vehicles. Together, these advances establish a powerful foundation for next-generation antimicrobial platforms and synthetic microbiology. Full article

In this study, the impact of the genomic scaffold on the properties of bacteriophages was investigated by swapping the genomic scaffolds surrounding the tailspike genes between two Przondovirus phages, KP192 and KP195, which infect Klebsiella pneumoniae with different capsular types. A yeast-based transformation-associated recombination cloning technique and subsequent “rebooting” of synthetic phage genomes in bacteria were used to construct the phages. Using Klebsiella strains with K2, K64, and KL111 capsular types, it was shown that the capsular specificity of the synthetic phages is fully consistent with that of the tailspike proteins (tsp). However, the efficiency of plating and the lytic efficiency of these phages strongly depended on the genomic scaffold used and the Klebsiella strain used. Synthetic phages with swapped genomic scaffolds demonstrated superior reproduction efficiency using a number of strains compared to wild-type phages, indicating that some elements of the swapped genomic scaffold enhance phage replication efficiency, presumably by blocking some of the host anti-phage defense systems. Our findings demonstrate that even in the case of closely related phages, the selection of the genomic scaffold used for tsp gene transplantation can have a profound impact on the efficiency of phage propagation on target bacterial strains. Full article

The high pathogenicity rate of carbapenem-resistant Pseudomonas aeruginosa (CRPA) has resulted in substantial economic losses for humans and the breeding industry. Consequently, there is an urgent need to develop new alternatives to mitigate antibiotic use. Phage therapy has demonstrated promising results in numerous studies. In this study, lytic phages targeting CRPA were isolated from feces and river water samples in Shandong, China. A total of 94 phage strains with CRPA as hosts were obtained, exhibiting lysis rates that ranged from 29% to 76% for P. aeruginosa derived from humans and different types of animals (n = 246). We further examined five representative phages, the host bacteria of which were CRPA from clinical patients and poultry, and these phages included two myoviruses and three podoviruses. Their optimal multiplicities of infection (MOIs) ranged from 10⁻³ to 10⁻⁵, with latent periods of less than 5 to 15 min and burst durations of 140 to 175 min, resulting in burst sizes of 133 to 352 PFU/cell. All five phages exhibited the ability to survive at temperatures up to 60 °C and within pH levels of 3 to 11. Whole-genome sequencing revealed that these five phages were all double-stranded DNA phages and did not possess resistance genes or virulence factors. The two myoviruses, sharing similar sequences, were classified into the genus Pakpunavirus, with a size of 92,509 bp and 92,293 bp, 149 to 152 ORFs and 20 to 22 tRNAs. In contrast, the three similar podoviruses belong to the genus Phikmvvirus and all contained a perforin–lyase system, with a size of 43.35 kb, a GC content of 62%, 49 to 50 ORFs and 16 to 20 tRNAs. A spray disinfection experiment demonstrated that the phage cocktail exhibited a high sterilization effect after spraying and showed good efficacy against cement and metal surfaces. This study provides foundational information for further research into the elimination of CRPA in the environment. Full article

Staphylococcus lugdunensis is a coagulase-negative staphylococcus known for its significant pathogenic potential, often causing severe infections such as endocarditis and bacteremia, with virulence comparable to S. aureus. Despite general susceptibility to most antibiotics, the emergence of oxacillin-resistant strains is increasingly concerning. This study conducted whole-genome sequencing on 20 S. lugdunensis isolates from Chang Gung Memorial Hospital to explore their genetic diversity, antimicrobial resistance mechanisms, and mobile genetic elements. The lugdunin biosynthetic operon, essential for antimicrobial peptide production, was present in multilocus sequence typing (MLST) types 1, 3, and 6 but absent in STs 4, 27, and 29. Additionally, IS256 insertion elements, ranging from 7 to 17 copies, were identified in four strains and linked to multidrug resistance. CRISPR-Cas systems varied across STs, with type III-A predominant in ST1 and ST6 and type IIC in ST4, ST27, and ST29; notably, ST3 lacked CRISPR systems, correlating with a higher diversity of SCCmec elements and an increased potential for horizontal gene transfer. Phage analysis revealed stable phage–host associations in ST1, ST6, and ST29, whereas ST4 displayed a varied prophage profile. Phenotypic resistance profiles generally aligned with genomic predictions, although discrepancies were observed for aminoglycosides and clindamycin. These findings highlight the complex genetic landscape and evolutionary dynamics of S. lugdunensis, emphasizing the need for genomic surveillance to inform clinical management and prevent the spread of resistant strains. Full article

Small regulatory RNAs (sRNAs) play a critical role in bacterial gene expression, modulating various cellular processes, including stress responses, metabolism, virulence, and many others. While well-characterized in bacterial systems, an emerging class of phage-derived sRNAs has been identified, suggesting an underexplored regulatory network at phage–host interactions. These sRNAs, encoded within phage genomes, influence both bacterial and viral life cycles by modulating transcriptional and post-transcriptional gene expression processes. The interplay between phage-derived sRNAs and the host genome reveals a complex network of gene regulation, with an impact on bacterial fitness, pathogenesis, and horizontal gene transfer. This review explores the diverse functions of phage-encoded sRNAs, highlighting recent discoveries and their impact on bacterial physiology and phage-host interactions. Full article

Pseudomonas aeruginosa is a major pathogen associated with hospital-acquired infections, and the spread of carbapenem-resistant isolates highlights the urgency of developing non-conventional therapies, such as phage therapy. For this alternative to be effective, understanding phage–host interactions is crucial for the selection of candidate phages and offers new insights into these dynamics. Background/Objectives: This study aimed to characterize prophage diversity in clinical P. aeruginosa genomes, assess the relationship between phages and the CRISPR/Cas system, and investigate the potential role of prophages in disseminating resistance genes. Methods: A total of 141 genomes from Brazilian hospitals were analyzed. Prophage detection was performed using VIBRANT, and in silico analyses were conducted to evaluate taxonomic diversity, the presence of resistance genes, phage life cycle, genomic distribution, and the presence of the CRISPR/Cas system. Results: A total of 841 viral sequences were identified by the VIBRANT tool, of which 498 were confirmed by CheckV, with a predominance of the class Caudoviricetes and high overall phage diversity. No statistically significant difference was observed in the number of prophages between isolates with and without CRISPR/Cas systems. Prophages carrying resistance genes such as rsmA, OXA-56, SPM-1, and others were detected in isolates harboring the type I-C CRISPR/Cas system. Additionally, prophages showed no preference for specific insertion sites along the bacterial genome. Conclusions: These findings provide evidence of a well-established phage–host relationship. The dual role of prophages—as vectors of antimicrobial resistance and as potential therapeutic agents—reflects their dynamic impact on bacterial communities and reinforces their importance in developing new strategies to combat antimicrobial resistance. Full article

The intratumoral microbiota, as an important component of the tumor microenvironment, is increasingly recognized as a key factor in regulating responses to cancer immunotherapy. Recent studies have revealed that the intratumoral microbiota is not uniformly distributed but instead exhibits significant spatial heterogeneity, with its distribution patterns influenced by factors such as tumor anatomy, local immune status, and therapeutic interventions. This spatial heterogeneity not only alters the interactions between microbes and the host immune system but may also reshape the immunogenic and immunosuppressive landscapes of tumors. The enrichment or depletion of microbiota in different tumor regions can influence immune cell infiltration patterns, metabolic pathway activities, and immune checkpoint molecule expression, thereby driving the development of resistance to immunotherapy. Moreover, certain bacterial metabolites form concentration gradients between the tumor core and margins, thereby regulating immune cell function. Therefore, understanding and manipulating the spatial distribution of intratumoral microbiota, particularly in resistant patients, holds promise for developing new strategies to overcome immunotherapy resistance. In the future, precise modulation strategies targeting microbial spatial heterogeneity, such as engineered bacterial vectors, probiotic combinations, and phage therapy, may open new avenues for immunotherapy. Full article

Bacteriophages are a unique and fascinating group of viruses, known for their highly specific ability to infect and replicate within bacterial cells. While their potential as antibacterial agents has been recognized for decades, recent research has revealed complex interactions between phages and the human immune system, offering new insights into their role in immune modulation. New evidence reveals a dynamic and intricate relationship between phages and cytokines, suggesting their ability to regulate inflammation, immune tolerance, and host–pathogen interaction. Herein, we review how phages affect the production of cytokines and the behavior of immune cells indirectly by lysis of bacterium or directly on mammalian cells. Phages have been shown to induce both pro- and anti-inflammatory responses and recently, they have been explored in personalized immunotherapy, cancer immunotherapy, and microbiome modulation, which are the focus of this review. Several challenges remain despite significant progress, including practical obstructions related to endotoxins along with host microbiome variability and regulatory issues. Nevertheless, the potential of bacteriophages to modulate immune responses makes them attractive candidates for the future of precision medicine. Full article

Bacteriophages, with their distinctive ability to selectively target host bacteria, stand out as a compelling tool in the realm of drug and gene delivery. Their assembly from proteins and nucleic acids, coupled with their modifiable and biologically unique properties, enables them to serve as efficient and safe delivery systems. Unlike conventional nanocarriers, which face limitations such as non-specific targeting, cytotoxicity, and reduced transfection efficiency in vivo, engineered phages exhibit promising potential to overcome these hurdles and improve delivery outcomes. This review highlights the potential of bacteriophage-based systems as innovative and efficient systems for delivering therapeutic agents. It explores strategies for engineering bacteriophage, categorizes the principal types of phages employed for drug and gene delivery, and evaluates their applications in disease therapy. It provides intriguing details of the use of natural and engineered phages in the therapy of diseases such as cancer, bacterial and viral infections, veterinary diseases, and neurological disorders, as well as the use of phage display technology in generating monoclonal antibodies against various human diseases. Additionally, the use of CRISPR-Cas9 technology in generating genetically engineered phages is elucidated. Furthermore, it provides a critical analysis of the challenges and limitations associated with phage-based delivery systems, offering insights for overcoming these obstacles. By showcasing the advancements in phage engineering and their integration into nanotechnology, this study underscores the potential of bacteriophage-based delivery systems to revolutionize therapeutic approaches and inspire future innovations in medicine. Full article

The emergence of antimicrobial resistance among foodborne pathogens has intensified the search for alternative biocontrol strategies. Among these, essential oils (EOs) and bacteriophages have gained increasing attention, due to their natural origin and antimicrobial potential. This narrative review investigates their individual and combined use as innovative tools for improving food safety. We discuss the mechanisms of action, current food applications, and regulatory or technical limitations associated with both EOs and phages. Particular emphasis is placed on their complementary characteristics, which may enhance efficacy when used together. An in-depth analysis of five key studies investigating synergistic EO–phage combinations against Staphylococcus aureus, Escherichia coli, and Salmonella Typhimurium is presented. These studies, conducted in both in vitro and food-based systems, reveal that antimicrobial synergy is often dose- and temperature-dependent. Optimized combinations lead to enhanced bacterial reduction and reduced resistance development. However, several challenges remain, including sensory alterations in food products, phage inactivation by EO compounds, and host cell destruction at high EO doses. The review concludes that while EOs and phages face limitations when applied independently, their strategic combination shows substantial promise. Future research should focus on formulation development, delivery systems, and regulatory alignment to unlock their full synergistic potential. Full article

Bacteriophages, first discovered in 1915, have re-emerged as critical players in microbial ecosystems, particularly in food production. Their ability to lysogenize bacterial hosts raises concerns about their role in the horizontal transfer of antibiotic resistance genes (ARGs) and virulence factors, contributing to the global challenge of antimicrobial resistance. Key studies reveal that ARG-carrying phages are prevalent across various stages of the food chain, including soil, vegetables, meat, dairy, and wastewater associated with food production. These findings demonstrate the potential for lysogenic phages to act as vectors for resistance gene dissemination, posing risks to public health. The review also explores emerging genetic elements, such as phage-inducible chromosomal islands and gene transfer agents, that further enhance the mobility of resistance and virulence genes. Advancements in metagenomic tools have improved our understanding of phage-mediated gene transfer, but significant knowledge gaps remain. Future research should aim to quantify these processes in real-world settings and develop strategies to mitigate the risks associated with lysogenic phages in food systems. Full article

Food safety is a paramount public health concern, particularly with the rise of antimicrobial-resistant bacteria. This systematic review explores the efficacy of bacteriophages as a novel and environmentally sustainable approach to controlling multi-resistant and non-resistant bacterial pathogens in animal-derived food products. Following PRISMA guidelines, data from multiple studies were synthesized to evaluate bacteriophage applications across diverse food matrices, including beef, poultry, seafood, and dairy. The findings highlight significant variability in bacteriophage efficacy, influenced by factors such as food matrix properties, bacterial strains, and application methods. Phage cocktails and their combination with thermal treatments consistently demonstrated superior bacterial reduction compared to single-phage applications, which yielded variable results. Interestingly, the absence of a clear dose-response relationship underscores the need for a more detailed understanding of phage-host interactions and environmental influences. This review addresses a critical gap in the literature by advocating for matrix-specific, targeted phage applications over generalized approaches. Additionally, it underscores the transformative potential of bacteriophages as sustainable alternatives to chemical disinfectants in modern food safety practices. These insights provide a framework for future research aimed at optimizing bacteriophage efficacy and scaling their application in real-world food production systems. Full article

Phage Lydia, a newly isolated siphovirus infecting Pseudomonas aeruginosa, was characterized with respect to its basic kinetic properties and subjected to comparative bioinformatic analysis with related phages. The phage exhibited a restricted host range, with lytic activity observed against 7 of 30 tested isolates. The genome of phage Lydia consists of a 61,986 bp dsDNA molecule and contains 89 predicted genes. Bioinformatic analysis suggests the presence of a DNA modification system, but no apparent genes associated with lysogeny or antibiotic resistance were identified. Taxonomic classification places Lydia within the Mesyanzhinovviridae family, Rabinowitzvirinae subfamily, and Yuavirus genus, with the closest relation to Pseudomonas virus M6. Comprehensive bioinformatic studies, including structural modelling and analysis of phage proteins, as well as comparative taxonomic, phylogenomic, and pangenomic analyses of the Mesyanzhinovviridae family, revealed relationships between proteins of Mesyanzhinovviridae phages, proteins from other phage groups, encapsulins, and a gene transfer agent (GTA) particle from Rhodobacter capsulatus. These analyses uncovered patterns of evolutionary history within the family, characterized by genetic exchange events alongside the maintenance of a common genomic architecture, leading to the emergence of new groups within the family. Full article