Search Results (386)

The global proliferation of multidrug-resistant Pseudomonas aeruginosa stimulates the search for alternatives to conventional therapy. This study developed human serum albumin (HSA)-based hydrogels for the delivery of bacteriophage PA57. Matrices were fabricated via combined thermal- and ethanol-induced gelation. The release kinetic was dependent on protein concentration: 20% (w/v) HSA provided sustained release over 48 h, whereas 10–15% (w/v) HSA exhibited burst release effects. Combined systems effectively suppressed P. aeruginosa growth in vitro during the early and middle stages of incubation, maintaining low culture optical density for up to 28 h. Although late-stage bacterial regrowth was observed, the final bacterial load remained significantly lower than in the control. Furthermore, cytocompatibility assays with HaCaT keratinocytes and MRC-5 fibroblasts demonstrated high cell viability, confirming the safety of the hydrogel matrix for wound healing applications. These results demonstrate the promise of HSA-based hydrogels as a platform for localized phage therapy of infected wounds. Full article

Klebsiella grimontii is an emerging pathogen associated with multidrug resistance. Although a single recent study reported phages against K. grimontii, their taxonomic diversity and structural characterisation remained limited. Here, we describe the isolation and characterisation of five novel lytic bacteriophages that specifically target K. grimontii strain K15g, originally isolated from a rotting iris rhizome. Phages Silvester, Lyra, Lyris, Mirion, and Helios were isolated from wastewater samples collected in the Moscow region and represent three distinct morphotypes: podovirus (Silvester), siphovirus (Lyra, Lyris, Mirion), and myovirus (Helios). Whole-genome sequencing revealed that Silvester belongs to the genus Przondovirus (family Autographiviridae); Lyra, Lyris, and Mirion are members of the genus Sugarlandvirus (family Demerecviridae); and Helios falls within the genus Slopekvirus (family Straboviridae). All five phages lack genes associated with lysogeny, virulence factors, or antibiotic resistance, indicating their suitability for therapeutic applications. Structural modelling of the receptor-binding proteins revealed diverse architectures of adsorption apparatuses, including a two-depolymerase complex in Silvester, T5-like tail fibres in the Sugarlandvirus phages, and a set of long and short fibres with a structured cell-puncturing device in Helios. This study presents five novel phages active against Klebsiella grimontii and provides a basis for further evaluation of their potential in phage therapy. Full article

The present study provides the physicochemical and genomic characterization of three Escherichia bacteriophages (6phi8, 6phi10, and 6phi13) isolated from the commercial phage preparation “Sextaphag^®”. For each bacteriophage, lytic activity against E. coli MG1655, as well as pH and thermal stability, were determined. Whole-genome sequencing was performed, followed by bioinformatic annotation and comparative genomic analysis. Bacteriophages 6phi8 and 6phi13 belong to the T4-like myoviruses with large genomes (~169 and ~171 kb, respectively), whereas 6phi10 is a T7-like podovirus with a genome size of 40.1 kb. Phage 6phi8 was assigned to the genus Mosigvirus of the family Straboviridae, 6phi13 was classified within the genus Tequatrovirus of the same family, and 6phi10 was identified as a putative novel species of the genus Berlinvirus within the family Autotranscriptaviridae. The genomes of the studied phages lack genes associated with the lysogenic cycle, as well as virulence and antibiotic resistance determinants. The results expand current knowledge of the genomic properties of phages included in therapeutic cocktails and may contribute to the development of phage preparations against infections caused by E. coli and other members of the family Enterobacteriaceae. Full article

Rhizogenic Agrobacterium (Rhizobium) spp. are causative agents of hairy root disease (HRD), a major threat to hydroponic crop production worldwide. The use of specific bacteriophages is considered a prospective approach to control the development of HRD in greenhouses. A combination of diverse bacteriophages is a key step to overcome potential phage resistance in the pathogen. In this study, a novel lytic bacteriophage, named Miki, was identified and characterized for its antibacterial potential against a rhizogenic Agrobacterium sp. strain circulating in greenhouses in Central Russia. High-throughput sequencing revealed a 63,458 bp double-stranded DNA genome (G + C content 53%), with 117 predicted coding sequences, considering Miki as a lytic candidate phage for plant protection. Electron microscopy of phage Miki shows a morphology unusual for Agrobacterium phages, and phylogenetic analysis attributes it as a representative of a previously undescribed taxon at least at the genus level. The paper presents a detailed analysis of the genome and structural proteome of phage Miki, including in silico predictions and modeling of receptor-binding proteins, including central and proximal fibers resembling the adsorption apparatus of Escherichia phage T5. Full article

Vibrio parahaemolyticus is a leading cause of seafood-associated gastroenteritis, yet the ecological and genomic significance of environmental populations as potential reservoirs remains incompletely characterized. From May to October 2024, 108 water samples were collected monthly at one coastal and two estuarine sites in Haiyan, Zhejiang. Confirmed isolates (n = 39) underwent whole-genome sequencing, MLST/cgMLST, virulence and AMR gene screening, integron characterization with BLASTp (+2.17.0) integrase family typing, and viral-region prediction. Culture-based detection was absent in May and rose to 66.7% in October (Cochran–Armitage trend, p = 0.003), with descriptively higher detection at the coastal site. MLST identified 28 STs including four novel types (Simpson’s diversity = 0.953). All isolates lacked tdh, trh, and T3SS2 but retained T3SS1 and MAM7. One estuarine isolate carried CALIN-associated dfrA31 and qnrVC5. All integrases matched VpaIntIA (95.9–100% identity), not mobile class 1–3 integrases. Viral regions were detected in 38/39 isolates; filamentous phage annotations in 52.6%. Haiyan coastal V. parahaemolyticus shows seasonal and spatial patterns, high diversity, and a mobilome dominated by chromosomal super-integrons, underscoring the need for integrase family typing in environmental surveillance. Full article

Directed evolution has revolutionized protein engineering by applying the principles of natural selection to the laboratory. However, traditional in vitro methods are quite labor-intensive, while common in vivo methods suffer from low mutation rates and high rates of off-target mutations. To address these issues, researchers have developed targeted mutagenesis tools for rapid in vivo evolution of biomolecules. In this review, we discuss recent in vivo hypermutation tools that enable rapid sampling of the vast evolutionary landscape, all while supporting simultaneous selection of the best proteins within living organisms. We focus on three main mechanisms of hypermutation: (i) orthogonal replication, which uses error-prone replication machinery to replicate the target gene with low fidelity; (ii) CRISPR-Cas-guided mutators, where mutagenic proteins are localized to virtually any user-defined loci; and (iii) transcription-coupled mutagenesis, a simple, yet elegant tool that exploits the innate processivity of orthogonal ribonucleic acid (RNA) polymerases to guide mutagenic proteins along the target gene during transcription. We highlight key advantages of these systems, as well as some clinically- and biotechnology-relevant applications. We discuss important limitations and how they could be addressed in the future to make hypermutation tools with broad mutational spectra and windows that span entire genes with minimal off-target effects. Full article

Bacteriophage technologies have evolved from classical cloning vectors into programmable platforms for genome engineering and molecular selection. Phage display couples a surface-presented binding phenotype to its encoding genotype, enabling iterative selection, sequence recovery, and optimization of peptides, antibody fragments, and other protein binders. This review links phage morphology, genome organization, infection strategy, host defense, and engineering method to practical platform choice. It compares λ, N15, M13, and T7 systems; examines biopanning bias and candidate developability; and evaluates artificial intelligence-assisted, sequencing-guided, and structure-guided workflows. Vaccine applications are considered alongside constraints arising from anti-phage immunity, antigen density, route of administration, and repeat dosing. Progress will depend on experimentally validated closed-loop workflows that integrate library design, selection, high-throughput analytics, structural characterization, and early manufacturability assessment. Full article

(1) Background: Anti-Müllerian hormone (AMH) is a key indicator for predicting ovarian reserve and fertility. Currently, B-mode ultrasound examination is the primary method for evaluating follicular development in Bactrian camels, although it has several limitations. This study aims to develop a colloidal gold strip based on AMH nanobody preparation, establishing an efficient, simple, and rapid detection method suitable for evaluating follicular development. (2) Methods: Two nanobodies specifically binding to AMH were screened and identified from an alpaca naive nanobody library using phage display technology. The nanobodies were expressed in the yeast expression vector (pMCO-AOXα) and purified by affinity chromatography on a nickel column. Based on the purified nanobodies, a colloidal gold immunochromatographic strip was constructed for detecting serum AMH levels. (3) Results: In this study, anti-AMH nanobodies (AMH-VHH-A1 and AMH-VHH-A10) were expressed and purified; these were successfully utilized to prepare colloidal gold immunochromatographic strips for detecting AMH in serum. The T-line intensity of the colloidal gold detection was positively correlated with the number of growing follicles obtained by B-mode ultrasound imaging. (4) Conclusions: The colloidal gold strips based on nanobodies against AMH established in this research provided an effective, simple, rapid, and on-site method for evaluating follicular development, thus supplying a reference for reproductive management in Bactrian camels. Full article

Background/Objectives: Salmonella enterica (S. enterica) is a major poultry-associated foodborne pathogen and a persistent public health concern. The global rise in antimicrobial resistance has accelerated the search for alternative control strategies, including the use of bacteriophages. However, their successful application requires a comprehensive evaluation of their biological performance, genomic safety, and functional proteins. This study aimed to characterize Salmonella phage WP110 and assess its potential as a biocontrol agent in broiler-associated production systems. Methods: Phage WP110 was evaluated against 251 S. enterica isolates from broiler-related sources. Adsorption kinetics, one-step growth, environmental stability (temperature and pH), and effective multiplicity of infection (MOI) were determined using Salmonella Kentucky S1H28. Whole-genome sequencing (WGS) and bioinformatic analyses were performed for genome annotation, taxonomic classification, and safety evaluation. In addition, protein structural prediction of a putative endolysin (WP110-gp057) was conducted using AlphaFold2, followed by structural comparison and molecular docking with peptidoglycan. Biocontrol efficacy was evaluated in contaminated rice husk, chicken meat, and on non-food materials. Results: Phage WP110 demonstrated a broad lytic spectrum, lysing 248/251 S. enterica isolates (98.8%). It adsorbed rapidly (within 3–15 min) to host cells and exhibited a latent period of ~20 min with a burst size of 134 particles per infected cell. Phage WP110 remained stable at 4–45 °C and pH 5–11 but was inactivated at ≥75 °C and pH 2. Complete bacterial inactivation in broth assay was achieved at an MOI of 10⁴. Genomic analysis revealed a 110,216 bp linear dsDNA genome (39.74% GC) comprising 204 ORFs, 25 tRNAs, and long direct terminal repeats, with no detectable antibiotic resistance genes. Phylogenetic and intergenomic analyses classified phage WP110 as a novel species within the genus Epseptimavirus. Structural modeling of WP110-gp057 revealed conserved catalytic residues and high structural similarity to T5 endolysin, while docking analysis supported a structurally plausible interaction with peptidoglycan at the predicted active-site groove, consistent with its proposed role in host cell wall degradation. In application models, phage WP110 significantly reduced Salmonella contamination in rice husk (up to 4.3 log CFU/g), chicken meat (up to 1.7 log CFU/g), and on non-food material surfaces (0.7–1.5 log CFU reduction). Conclusions: Phage WP110 is a broad-host-range lytic phage with favorable infection kinetics, environmental robustness, and genomic safety. Its functionally supported endolysin and strong antibacterial efficacy across broiler-associated matrices highlight its potential as a biocontrol agent for Salmonella mitigation in poultry value chain. Full article

Antibiotic resistance in Gram-negative bacteria poses a global health threat, and poultry farming provides an important reservoir for multidrug-resistant pathogens. Our study aimed to characterize the faecal microbiota and acquired resistome of Ross-308 roosters in Hungary. Amplicon and shotgun metagenomics revealed a faecal microbiota dominated by the Firmicutes, Bacteroidota, and Proteobacteria and a diverse faecal resistome, including qnrB and an aadA1-bearing integron. Culture-based screening of an antibiotic-free rooster yielded the MDR Escherichia coli strain K1G, displaying resistance also to third-generation cephalosporins and fluoroquinolones. Whole-genome sequencing classified K1G as a serotype O23:H16-ST453 avian pathogenic E. coli (APEC) strain featuring a set of chromosomal virulence factors (including astA, hlyE, lpfA, and iss) and three plasmids: a phage-like plasmid, a mosaic virulence plasmid (carrying bla_TEM-1b, hlyF, iutA, ompT, iucD, and cvaC), and an IncC type 1 resistance plasmid harbouring bla_CMY-2. The detection of identical or closely related ST453 E. coli strains also in broiler meat in Hungary highlights a potential risk of transmission to humans through the food chain. Moreover, the carriage of multiple acquired antibiotic resistance genes in E. coli K1G indicates that individual chickens can harbour or transmit antibiotic resistance even in the absence of direct antibiotic exposure. Full article

Translucent post-larvae disease (TPD), caused by specific Vibrio parahaemolyticus strains, poses a global threat to shrimp aquaculture. A key virulence factor in these strains is the vhv gene cluster, which encodes toxins central to TPD pathogenesis. While plasmid-borne vhv genes have been extensively studied, the chromosome-borne vhv genes in TPD-associated strains remain poorly characterized. In this study, we systematically analyzed integrative mobilizable elements (IMEs) on the small secondary chromosomes of TPD-associated V. parahaemolyticus. Seven nearly identical novel IMEs harboring full-length vhv clusters were identified from seven independent strains. These IMEs encode a tyrosine recombinase for site-specific chromosomal integration, yet lack core conjugative transfer machineries including origin-of-transfer (oriT), relaxase, type IV coupling protein (T4CP), and IV secretion system (T4SS), consistent with the definition of non-autonomous IMEs. Each IME carries diverse functional cargo genes, covering five virulence factors (including previously unreported colonization and chemotaxis genes specific to TPD mobile elements), one pollutant-degrading enzyme, and two distinct anti-phage defense systems (PsyrTA and Lamassu-Fam), thereby potentially boosting the host’s fitness and pathogenicity in aquaculture environments. This study provides evidence for the critical role of IMEs in mediating the horizontal transfer of chromosomal vhv, offering new insights into TPD epidemiology and a foundation for developing targeted strategies to mitigate the disease’s impact on shrimp aquaculture. Full article

Background/Objectives: Cancer vaccines represent the next frontier in immunotherapy, aiming to elicit long-lasting protective anti-tumor immune responses. Human epidermal growth factor receptor 2 (HER2) is a well-established therapeutic target in breast cancer. Active immunization with HER2-displaying M13 bacteriophages can induce a therapeutic immune response against HER2-positive breast cancer, offering a promising alternative to trastuzumab. However, the duration of anticancer immune protection triggered by anti-HER2 phage-based vaccines is limited by tumor-immune suppressive mechanisms. Methods: In this study, two vaccination cycles with ECTM phages displaying the extracellular (EC) and transmembrane (TM) domains of human HER2 were combined with palbociclib, a CDK4/6 inhibitor, to enhance antitumor immunity in the clinically relevant Δ16HER2 transgenic preclinical model of breast cancer. Results: The proposed combination treatment resulted in a better and long-lasting control of tumor growth rate and multiplicity than either palbociclib or phage vaccination alone, correlating with a significantly stronger anti-HER2 humoral response (IgG2a isotype). Analysis of the tumor immune infiltrate revealed an increased presence of CD8⁺ T cells concomitant with a reduction in FoxP3⁺ regulatory T cells (Tregs) in tumors explanted from mice receiving the combination therapy. Conclusions: These preclinical results provide a rationale for the clinical translation of CDK4/6 inhibitors combined with anti-HER2 active immunotherapies in breast cancer, as they may yield sustained antitumor responses by reverting the immunosuppressive tumor environment. Full article

Background/Objective: Antimicrobial resistance poses a critical public health crisis, highlighting the urgent requirement to investigate bacterial evolutionary adaptations and pioneer alternative therapeutics. Consequently, bacteriophages and metal-based compounds are emerging as viable options to combat drug-resistant infections. Building on our finding that T4 phage resistance in E. coli B also confers adaptation to high iron(III), we used RNA-sequencing (RNA-seq) to explore bacterial gene expression in resistant and control populations. We analyzed samples from our five experimental groups—Ancestor (ANC), control (CON), phage-selected (Phage), iron(III)-selected (FE), and phage/iron(III)-selected (PF), to understand how these regimes drive transcriptional changes. Method: Total RNA was extracted using the TRIzol protocol, and sequencing libraries were prepared with the Illumina RNA Total Library Prep Kit. Sequencing was performed on the Illumina NextSeq 1000/2000 platform. Reads were aligned to the E. coli B ATCC 11303 reference genome, and pairwise comparisons between the five experimental groups were conducted to determine differential gene expression profiles. Results: Principal component analysis (PCA) showed that iron-adapted populations (FE and PF) separated distinctly from the Ancestor and control populations along PC1 (capturing 40% of the variance), while the phage-selected replicates were split, with one (Phage5) clustering with CON3 and two (Phage2, Phage4) falling closer to, but clearly separated from, the Ancestor. Differential expression analysis (P_adj < 0.05 and |log₂FC| ≥ 1) revealed extensive transcriptional rewiring, with 482 and 381 differentially expressed genes (DEGs) in the FE and PF populations, respectively, compared to the Ancestor, and 177 DEGs in the phage-selected population compared to the Ancestor. The direct pairwise comparison between the iron-selected and phage/iron-selected populations yielded zero DEGs, demonstrating that both iron-adapted populations converged on a near-identical gene expression profile regardless of their distinct genetic and evolutionary backgrounds. Conclusions: This study suggests that pmrB and arn pathway genes may serve as primary markers for resistance to iron stress. These results are significant because they demonstrate a coordinated, multi-gene defense mechanism in E. coli B against high iron(III) stress, in which the arn operon and eptA remodel lipid A and the outer membrane, while glycerol-3-phosphate metabolism and phage-shock/chaperone pathways are repressed. Full article

Background/Objectives: Outer membrane vesicles (OMVs) are membrane-encapsulated spherical structures ~120 nm in diameter derived from Gram-negative bacterial cell envelopes. OMVs are primarily generated by outer membrane blebbing but contain proteins, DNA, and RNAs at concentrations distinct from that of the intracellular complement. OMVs have been associated with a number of different cellular functions including intercellular communication and resistance to phage. Methods: As bacterial small RNAs (sRNAs) also participate in bacteriophage defense and are specifically delivered to and enriched in OMVs, we recently elected to examine the effects of P22 infection on Salmonella cytosolic and OMV sRNA abundance by employing RNA sequencing. Results: We find that P22 infection triggers a global reduction in sRNAs (with Salmonella sRNA expression levels averaging only 15.6% those observed in noninfected cells) coupled with a reciprocal 72.7% global increase in Salmonella tRNA expression levels. Additionally, of note, while OMV small noncoding RNA (sncRNA) abundance is normally ~1/10 that found in the cytosol, we find that P22 infection triggers active OMV encapsulation and secretion of: (1) a subset of sRNAs, (2) all Salmonella tRNAs including one highly complementary to the P22 genome, and, much to our surprise, (3) ten distinct sRNAs expressed from P22. Conclusions: In summary, the work presented here identifies several Salmonella sncRNA cytosolic and/or OMV abundances significantly altered during P22 infection, and to our knowledge, this constitutes the first reported characterization of bacteriophage-encoded sRNAs being actively secreted within host OMVs. Full article

Background/Objectives: Antimicrobial resistance (AMR) in Escherichia coli is a growing public health concern, particularly in regions affected by environmental contamination and poor wastewater management. Data on locally isolated E. coli-targeting phages in Lebanon remain limited. This study aimed to isolate, characterize, and evaluate two lytic bacteriophages against AMR E. coli. Methods: Two phages, EPIMAM01 (gb:PQ493298) and EPIMRB01 (gb:PQ657784), were isolated from untreated sewage in Beirut using E. coli ATCC 25922. Characterization included double-layer agar assays, one-step growth analysis, and stability testing across temperature and pH ranges. Bacteriolytic activity was assessed in planktonic cultures and preformed biofilms. Host range and efficiency of plating (EOP) were evaluated using clinical isolates. Whole-genome sequencing and comparative analyses were performed. Results: Both phages produced clear plaques and showed a latent period of ~40 min. EPIMAM01 had a higher estimated burst size (140 PFU/infected cell) than EPIMRB01 (75 PFU/infected cell). Both phages remained stable between 4–50 °C and within a pH range of 5–10 but showed marked loss of activity at temperatures ≥60 °C and pH ≤3 or ≥12. EPIMAM01 effectively inhibited planktonic growth of E. coli ATCC 25922, whereas EPIMRB01 showed stronger biofilm-disrupting activity against preformed E. coli biofilms. Both phages lysed several of the 17 tested clinical E. coli isolates. Comparative analyses of gene presence/absence patterns, bacterial defense systems, and adsorption phenotypes among the tested E. coli strains identified mlaA, ydcQ, and ompD-2 as candidate adsorption-associated genes and suggested CRISPR systems may reduce susceptibility. Genomic analysis classified both phages as T4-like phages lacking lysogeny, virulence, or AMR genes. Conclusions: EPIMAM01 and EPIMRB01 are lytic phages with complementary antimicrobial properties, supporting their potential for further development as AMR control agents. Full article