Search Results (2,956)

The Ralstonia solanacearum species complex (RSSC) is a globally significant plant pathogenic bacterium. Given the lack of effective chemical controls, phage therapy has emerged as a promising biocontrol alternative. While combining phages with antibiotics can counteract phage resistance, RSSC may still evolve concurrent resistance to both agents. However, the fitness consequences and underlying mechanisms of such resistance remain unclear. In this study, a novel RSSC phage was isolated to experimentally investigate the trade-offs between resistance and virulence in evolved strains. Compared to the wild-type, phage-resistant, antibiotic-resistant, and dual-resistant mutants showed no significant differences in growth rate, exopolysaccharide and lipopolysaccharide production. However, their motility, soil survival, and biofilm formation were significantly impaired, with the most severe decline observed in the dual-resistant mutants. Furthermore, phage-resistant strains exhibited enhanced antibiotic resistance, while antibiotic-resistant strains displayed cross-resistance. The antibiotic resistance gene bla_OXA-249 was upregulated only in antibiotic-resistant strains. In phage-resistant bacteria, the abortive infection system was activated. A reduction in bacterial cell numbers post-infection indicated that phage resistance limits phage propagation via a “suicidal” mechanism. These findings reveal that resistance evolution in RSSC carries substantial fitness costs and highlight phage steering as a novel strategy for designing phage agents. Full article

This study demonstrates the feasibility of using a T7 phage display platform to deliver a library of tick antigens as a vaccine to disrupt tick feeding in cattle. Cattle were vaccinated at three-week intervals via intradermal and intramuscular routes with a cocktail of male and female Amblyomma americanum T7 phage display cDNA libraries, with and without adjuvant. ELISA and Western blot analyses confirmed that vaccinated cattle mounted immune responses directed against phage-displayed tick proteins rather than the T7 phage backbone. Vaccine-induced antibodies recognized both native tick salivary gland proteins and selected recombinant salivary proteins, indicating effective antigen presentation and biologically relevant immunity with binding to native tick saliva proteins. The adjuvanted formulation elicited significantly stronger immune responses than phage-only immunization. Immunized cattle exhibited robust immune memory, evidenced by a pronounced anamnestic response following tick infestation. This immunity translated into measurable anti-tick effects, including reduced tick feeding efficiency and blood ingestion. Tick reproductive success was severely compromised, with larval hatching declining from 54% in ticks fed on control cattle to 4% in ticks fed on immunized cattle. This study establishes a practical and scalable T7 phage-displayed whole-tick antigen platform capable of inducing durable anti-tick immunity in cattle. Full article

The production of Chinese Baijiu relies on the synergistic metabolism of multi-species microbial communities in an open environment. Its intricate microbial succession and flavor formation mechanisms have long been considered complex systems that are difficult to fully deconstruct. Traditional culture-dependent techniques inherently fail to comprehensively capture the actual functional roles and dynamic regulation of “viable but non-culturable” (VBNC) microorganisms within this complex system. In recent years, the rapid advancement of multi-omics technologies has offered a novel perspective for elucidating the underlying fermentation mechanisms of Baijiu. This paper systematically reviews the recent progress in the application of metagenomics, metatranscriptomics, metaproteomics, and metabolomics in Baijiu research. Specific focus is placed on the unique contributions of these tools to resolving microbial community structural diversity, mining key functional genes and enzymes, uncovering microbial stress response mechanisms under environmental fluctuations, identifying phages and spoilage microorganisms, and tracing the metabolic pathways of flavor substances. Furthermore, the pivotal role of multi-omics integration strategies in constructing “microbe–metabolite” regulatory networks is highlighted. Finally, current challenges regarding standardization and data integration are discussed, with an outlook on leveraging omics big data to promote digital monitoring and intelligent brewing in the Baijiu industry. Full article

Different groups of microorganisms—namely lactic acid bacteria (LAB), coagulase-negative staphylococci (CNS), dairy propionibacteria, yeasts, and molds—play essential roles in producing safe fermented foods of animal and plant origin with high nutritional value and sensory quality. The acquisition of genetic traits with technological relevance by natural horizontal gene transfer (HGT) via transformation, conjugation, phage transduction, and other routes would broaden the spectrum of beneficial activities exerted by individual microbial strains with no limitations for their use in food. Therefore, this critical review aimed to identify the potential for natural genetic improvement of microbial species relevant to food technology, based on reports of natural genetic exchanges occurring in environmental niches and laboratory conditions. Results showed that the species most frequently involved in natural HGT is Lactiplantibacillus plantarum, followed by Streptococcus thermophilus and Lactococcus lactis. Extensive HGT events enabling adaptation to food have been observed in domesticated filamentous fungi. The transferred traits of technological relevance include resistance to various stress factors, exopolysaccharide (EPS) and bacteriocin production, protein and amino acid utilization, phage immunity, lactose and citrate metabolism in dairy species, and use of plant carbohydrates in vegetable adapted species. Methods suitable for detecting HGT events in microbial communities have been developed and can aid in isolating improved strains for use in fermented foods. Full article

Cronobacter sakazakii is a formidable foodborne pathogen that poses a severe, often fatal threat to neonates and immunocompromised individuals, with contaminated powdered infant formula as the primary transmission vehicle. Infections can lead to devastating conditions, such as meningitis, necrotizing enterocolitis, and sepsis, with alarmingly high mortality rates. Clinical management is hampered by the lack of standardized treatment guidelines and the emergence of antibiotic resistance. However, ongoing research into its molecular pathogenesis continually covers novel targets for intervention. In this review, we synthesize recent advances in our understanding of the sophisticated mechanisms that enable C. sakazakii to cause disease. We argue that its virulence hinges on a multi-faceted strategy, including efficient host invasion and tissue penetration via outer membrane proteins, sophisticated immune evasion tactics for intracellular survival, a repertoire of regulated virulence determinants, resilient biofilm formation, and robust stress response systems that ensure environmental persistence. As research continues to decipher these intricate host–pathogen interactions, we highlight promising future directions, including the development of rapid on-site diagnostics, the application of effective biocontrol strategies like phage therapy and probiotics, and the formulation of targeted therapeutic regimens. Ultimately, integrating these multifaceted insights is paramount to developing comprehensive strategies for preventing and controlling the global health burden imposed by C. sakazakii. Full article

Background: Increasing evidence identifies intratumoral bacteria as key modulators of tumor progression, chemoresistance, and immunosuppression, presenting major obstacles to conventional cancer therapies. Recent advances in nanotechnology have enabled new strategies for selective targeting bacteria within the tumor microenvironment, potentially improving anticancer efficacy. Methods: A scoping review was conducted to outline the current landscape of nano-based therapeutic approaches aimed at the simultaneous elimination of intratumoral bacteria and cancer. Preclinical research publications involving in vivo antitumor efficacy evaluations were retrieved from three databases, Web of Science, PubMed, and Scopus, using the key words “(kill* OR eradicate* OR eliminate*) AND intratumoral AND (bacteria OR infection)”. Key information from the eligible studies was extracted and analyzed. Results: The diversity of bacterial species, cancer models, and evaluation methodologies employed in these preclinical studies were summarized, followed by critical examination of the design principles, therapeutic outcomes, and translational challenges of various nanomedicine platforms, including passive and active targeting drug delivery systems, phototherapy, phage therapy, and emerging modalities. Nano-based therapeutics functionalized with both antibacterial and anticancer properties were shown to effectively overcome bacteria-induced treatment resistance. Conclusions: Targeting intratumoral bacteria may significantly enhance the efficacy of existing treatments and contribute to the evolution of precision oncology. The insights gained from this review are expected to guide future systematic reviews and inform research directions in the development of dual-functional nanomedicines for cancer therapy. 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

Seafood products are highly perishable and particularly susceptible to contamination by pathogenic and spoilage microorganisms, including Listeria monocytogenes, Vibrio spp., Salmonella spp., and Escherichia coli. Conventional control strategies in seafood processing and storage largely rely on chemical preservatives and thermal treatments, which may negatively affect sensory quality and increasingly conflict with consumer demand for minimally processed, “clean-label” foods. In this context, bacteriophages, viruses that specifically infect and lyse bacterial hosts, have emerged as natural, targeted, and environmentally sustainable biocontrol agents for food safety applications. This review provides a comprehensive assessment of bacteriophage applications in seafood processing and storage, with particular emphasis on their mechanisms of action, host specificity, and ability to selectively reduce pathogenic bacteria without compromising nutritional or sensory attributes. Recent advances in phage-based technologies, including phage cocktails, immobilized phage systems, and genetically engineered phages, are discussed in relation to their efficacy against major seafood-associated pathogens under both laboratory and industrial conditions. Key challenges limiting large-scale implementation such as phage resistance development, regulatory considerations, stability during processing and storage, and consumer perception are critically evaluated. In addition, the review highlights emerging evidence on the synergistic use of bacteriophages with complementary preservation strategies, including natural antimicrobials and innovative packaging systems. Overall, this review underscores the potential of bacteriophage-based interventions as practical and sustainable tools to enhance seafood safety, extend shelf life, and support modern seafood processing practices aligned with evolving regulatory and consumer expectations. Full article

Bacillus cereus is a major foodborne pathogen responsible for food spoilage and foodborne illness, including strains producing emetic toxins. In this study, two bacteriophages, PBC_MG88 and PBC_MG99, were isolated from wastewater using emetic B. cereus strains as hosts and were comprehensively characterized. Both phages formed clear plaques with halos and exhibited siphovirus morphology. Host range analysis against 172 B. cereus strains showed that PBC_MG88 and PBC_MG99 infected 50 and 60 strains, respectively. One-step growth experiments revealed efficient lytic activity, with latent periods of 20–25 min and burst sizes of 59–63 PFU per infected cell. More than 90% of phage particles adsorbed to host cells within 15 min. Both phages were stable across a wide temperature range (4–55 °C) and pH values (4–11). Genome sequencing revealed ~37 kb double-stranded DNA genomes lacking antibiotic resistance or virulence genes; however, the presence of lysogeny-related genes suggests a temperate lifestyle. Comparative genomic analyses indicated that both phages represent novel species within the genus Lwoffvirus. Biofilm assays demonstrated significant inhibition of B. cereus biofilm formation and reduction of pre-established biofilms. Overall, this study expands knowledge of B. cereus phage diversity and highlights the importance of genomic characterization in phage-based biocontrol research. Full article

Carbapenemase-producing bacteria, particularly those expressing the KPC-3 variant, pose a critical global health threat due to their resistance to nearly all β-lactam antibiotics, including carbapenems. Rapid and reliable detection tools are urgently needed to improve infection control and guide patient management. Nanobodies (VHHs) present a promising alternative to conventional antibodies thanks to their high stability, small size, and capacity to access cryptic epitopes. Here, we report the generation and characterization of a nanobody specifically targeting KPC-3. An immune VHH phage display library was constructed, with over 90% of clones containing correctly sized inserts. After three rounds of biopanning, high-specificity binders were identified by ELISA screening. Sequencing identified a nanobody with hallmark VHH features, which was expressed and validated by ELISA and Western blot. Although kinetic assays showed no inhibition of KPC-3 enzymatic activity, interestingly, the nanobody demonstrated high-binding recognition of both KPC-2 and KPC-3 in periplasmic extracts from clinical strains. Structural modeling further supported these results, highlighting favorable interaction surfaces. This study provides the first evidence of a nanobody raised against KPC-3 that recognizes a conserved epitope shared by KPC-3 and KPC-2, underscoring its promising use as a molecular tool for detecting KPC variants and establishing a basis for future affinity maturation toward therapeutic applications. Full article

Bacteriophages (phages) are common and diverse viruses that specifically infect bacteria. Although their potential to suppress bacterial pathogens was recognized a century ago, their broader use remained limited for decades. Today, renewed interest in phages is rapidly expanding beyond medical use into agriculture, where they are being explored as environmentally friendly tools for managing bacterial plant diseases. Despite growing interest, our understanding of phage biology and genetics remains limited. This review focuses on phages that specifically infect Xanthomonas campestris pv. campestris (Xcc), a bacterial pathogen that seriously challenges the production of commercially valuable crops such as cabbage and broccoli. Phages could provide a much-needed addition to the current management practices that often fail to provide consistent results, especially when environmental conditions favor disease development. Here we summarize the currently available knowledge on Xcc phages, including their morphology, growth parameters, and stability under various environmental conditions, genomic features and basic genetic characteristics. Given recent changes in phage taxonomy, we also outline the newly adopted genome-based classification system, which has led to the reclassification of all officially recognized Xcc phages. A summary of practical applications provides encouraging results and paves the way for future research on phages of various plant pathogenic bacteria and their potential commercial use. Full article

The bacterium Yersinia enterocolitica serotype O:3 is targeted by two distinct agents, the bacteriophage φR1-37 and the bacteriocin-like enterocoliticin (a tailocin), which both utilize the lipopolysaccharide (LPS) outer core (OC) hexasaccharide as their primary host receptor. In order to understand this convergent recognition mechanism, we first characterized the enterocoliticin system, reporting the complete sequence of its large, biosynthetic gene cluster. Most of the 42 predicted gene products were functionally annotated by homology to known gene products. We then focused on identifying the receptor-binding proteins (RBPs) responsible for host attachment of both agents in order to elucidate a possible shared mechanism of binding. For phage φR1-37, the receptor binding complex was identified as the inseparable Gp298 tail fiber protein and its Gp297 trimerization chaperone, confirming its function as the RBP. Based on sequence identity with Gp298, the Orf39 gene product of the enterocoliticin cluster was predicted to be its corresponding RBP. An analytical comparison of the predicted RBPs revealed a highly conserved homologous region spanning 80–85 amino acid residues, which presents the only structural explanation for their identical receptor specificity. To resolve the binding mechanism, we generated high-confidence trimeric structural models for the Gp298 and Orf39 proteins using AlphaFold3-multimer. These models validated the high structural similarity of the RBP domains, despite global dissimilarity of the complete trimeric structures. Further docking simulations with a pentasaccharide ligand (generated by CarbBuilder) provided suggestive molecular models for the protein-carbohydrate interactions within the OC region. Intriguingly, a database search using the identified binding site motif revealed their wide and diverse presence in various phage tail proteins, suggesting that this motif is a specialized, common structure for carbohydrate recognition. This work identifies a conserved, novel sugar-binding motif as the molecular basis of host recognition for these key anti-Yersinia biologics. Full article

Infections caused by carbapenem-resistant Pseudomonas aeruginosa (CRPA), especially chronic infections associated with biofilm formation, have become a major clinical challenge. Phage therapy has received much attention as an alternative strategy, but temperate phages have limited direct application due to their lysogenicity. The aim of this study was to explore the synergistic therapeutic effect of a novel temperate phage combined with antibiotics. A temperate Pseudomonas phage YF1204 was isolated from the patient’s bronchoalveolar lavage fluid and systematically characterized by whole-genome sequencing, transmission electron microscopy, and host range analysis. The synergistic antibacterial and anti-biofilm effects of phage with amikacin (AK) were evaluated by using the checkerboard test, a time-killing curve based on optical density (OD600) and crystal violet staining, and the cytocompatibility was analyzed by using the CCK-8 method. The results showed that phage YF1204 belonged to the Siphoviridae family and had typical temperate phage genome characteristics (containing integrase gene). It also showed lytic activity against 41.4% (87/210) of the clinical isolates, especially against carbapenem-resistant strains. When YF1204 was combined with AK, it reduced the minimum inhibitory concentration (MIC) of AK by 2- to 8-fold across all tested strains, respectively. Moreover, the inhibitory effect against CRPA was significantly enhanced (achieving suppression indexes about 80% ) and biofilm formation was inhibited with an inhibition ratio of 48.75%. Cell experiments showed that YF1204 had no significant toxicity to THP-1 cells. The combination of YF1204 and AK exhibited significant synergistic bactericidal and anti-biofilm activities, providing a novel therapeutic strategy with translational potential for CRPA-induced refractory infections. Full article

Background/Objectives: Bacteriophages offer a promising alternative to conventional antibiotics. However, their therapeutic efficacy is often limited by instability in harsh environmental conditions, particularly within the gastrointestinal tract. This study aimed to isolate lytic bacteriophages from wastewater and evaluate the protective capacity of sodium alginate encapsulation against various stressors to enable effective oral delivery. Methods: Four distinct lytic phages (As, Ec, Pa, Gc) were isolated from wastewater and characterized by Transmission Electron Microscopy (TEM) and PCR, confirming their families (Siphoviridae, Podoviridae, Myoviridae). These phages demonstrated potent lytic activity against diverse bacterial pathogens, including Aeromonas hydrophila, Escherichia coli, Pseudomonas aeruginosa, and Glutamicbacter creatinolyticus. The phages were encapsulated in 5% sodium alginate via an extrusion method. Stability was assessed under extreme pH (2.0 and 13), at elevated temperature (up to 80 °C), and in simulated gastrointestinal transit. Results: Encapsulation efficiency exceeded 95%. Unencapsulated phages were completely inactivated at pH 2.0 within 10 min, whereas encapsulated phages maintained significant viability (3.06–3.43 log PFU/mL). Encapsulation also significantly enhanced phage survival under extreme alkaline conditions and elevated temperatures. In simulated gastrointestinal transit, encapsulated phages exhibited superior recovery (2.50 log PFU/mL) compared to their free counterparts (≤1 log PFU/mL). Long-term storage evaluations over three months further confirmed the robust stability of the encapsulated formulations at both 4 °C and 21 °C. Conclusions: Sodium alginate encapsulation effectively shields bacteriophages from severe environmental degradation, particularly acidic gastric stress, enhancing their potential for oral delivery. These findings support the development of stable, formulated phage products for diverse practical applications in phage therapy to combat antimicrobial resistance. Full article

Objectives: The epidermal growth factor receptor (EGFR) is a clinically relevant membrane receptor that is frequently overexpressed or dysregulated in multiple types of cancer, making it an important target for antibody-based strategies. Nanobodies, derived from camelid heavy-chain antibodies, possess favorable properties such as small size, high stability, and strong antigen-binding capacity. This study aimed to generate EGFR-specific nanobodies and to systematically characterize their binding properties and initial functional activity. Methodology: Bactrian camels were immunized with a whole-cell antigen prepared from 293F cells transiently transfected to express full-length human EGFR. A high-diversity phage display nanobody library was constructed from peripheral blood lymphocytes. After two rounds of biopanning against EGFR, positive clones were screened and selected. The identified nanobodies were recombinantly expressed in Escherichia coli and purified. Binding specificity, epitope relationships, and kinetic parameters were evaluated using high-performance liquid chromatography (HPLC), bio-layer interferometry (Octet), and flow cytometry. The effect of selected nanobodies on EGF-induced cell proliferation was evaluated using a CCK-8 assay. Results: Two EGFR-specific nanobodies, Nb2H4 and Nb2B6, were successfully isolated. Both nanobodies exhibited specific binding to EGFR and recognized distinct, non-competing epitopes. Kinetic analyses revealed favorable binding affinities, and flow cytometry confirmed their ability to recognize EGFR in its native cellular context. In addition, Nb2H4 significantly suppressed EGF-induced proliferation in an EGFR-overexpression cell model, indicating preliminary functional activity. Conclusions: This study reports on the successful generation and in vitro characterization of EGFR-targeting nanobodies based on the extracellular domain of EGFR. The identified nanobodies provide useful molecular tools for epitope mapping, structural studies, and the further exploration of EGFR-directed antibody engineering strategies. Full article