Search Results (185)

Multidrug resistance (MDR) is a global problem for the healthcare system, complicating the therapy of bacterial infections. It is noted that patients infected with MDR strains often require prolonged hospitalization, have a high risk of mortality, and remain vulnerable to reinfection after recovery. In this study, recombinant phage-encoded depolymerase DepKP144 was produced using the Escherichia coli expression system and purified. The depolymerase DepKP144 protein was able to reduce viable bacterial counts following capsule degradation in 95% of the tested strains of type K1 and 85% of the tested strains of type K2 Klebsiella pneumoniae. The depolymerase DepKP144 was active against K. pneumoniae K1-type and K2-type planktonic cells and destroyed the biofilms formed by clinical MDR strains of K. pneumoniae. In in vivo experiments, DepKP144 at a dose of 180 μg/mouse resulted in a 50% survival of the mice infected with K2-type K. pneumoniae and in a 17% survival of the mice infected with K1-type K. pneumoniae. This depolymerase is promising for further development of prevention and therapeutic candidates against MDR K. pneumoniae. Full article

Background/Objectives: Biofilms are a form of defense that enables bacteria to withstand antibiotic pressure and demonstrate antibiotic resistance. It is crucial to develop anti-biofilm strategies in order to combat chronic and persistent multidrug-resistant (MDR) infections. Methods: In this study, we developed 3D biofilms of single-, dual-, and poly-species MDR ESKAPE components, including the pathogens P. aeruginosa S. aureus and K. pneumoniae, in CF Mu3Gel. We evaluated the efficacy of using a phage, a di-hetero phage cocktail or a poly-hetero phage cocktail in combination with ciprofloxacin to eliminate mature biofilm biomass after 72 h or one week in a single treatment. Results: The phage components mostly exhibited synergistic behavior when combined with ciprofloxacin and with each other in di- and poly-hetero-cocktails. The reduction in 72-h dual- and poly-species biofilms was one log higher than that of one-week biofilms treated with the phage–antibiotic combination. The greatest reductions were observed in the 72-h single-species biofilm with combination therapy, at 1.4–3.0 log. Reductions of 2.16 and 1.6 log were observed in the dual-species P. aeruginosa and S. aureus biofilm and the poly-species biofilm, respectively. Conclusions: This study examined how a single application of phages or phage cocktails, either alone or in combination with ciprofloxacin, impacted established biofilm models, and how this affected the proportion of microcolonies of different species within each model. These insights will facilitate the development of strategies for multiple follow-up treatments, as well as the reordering of phages, phage cocktails, and combinations with antibiotics, to improve outcomes. The 3D biofilm models developed here could be used to screen phages or phage cocktails either on their own or alongside other therapies. This would facilitate the application of in vitro findings to real physiological settings. Full article

Saposin-like protein 2 (SAP2) exhibits strong immunogenicity as an antigen for immunodiagnosis in ruminant and human fasciolosis. Most available immunodiagnostic test kits are based on polyclonal and monoclonal antibodies against antigens from Fasciola spp. Previous studies demonstrated that polyclonal and monoclonal antibodies against SAP2 showed high specificity and could effectively detect Fasciola spp. infections at an early stage. However, polyclonal antibodies are extremely difficult to produce, and quality control is not possible during production; the procedure also involves considerable financial investment. To overcome these problems, we developed a single-chain variable fragment (scFv) to control quality in each production cycle and reduce the cost of manufacturing immunodiagnostic kits. Our objectives were to produce and characterize an scFv that binds the SAP2 from the liver fluke Fasciola gigantica. We constructed the scFv by genetic engineering: we cloned immunoglobulin genes and linked them with flexible polypeptide linkers composed of repeating glycine and serine residues. We selected an scFv with high affinity for binding SAP2 using the phage-display technique and produced it using a prokaryotic expression system. The scFv was characterized via in silico and in vitro methods to confirm its specificity for SAP2, including IMGT/V-QUEST, IMGT/Collier-de-Perles, HADDOCK 2.4, ELISA, immunoblotting, and immunohistochemistry. The scFv was successfully produced and purified using Ni-NTA affinity chromatography. The purified scFvFgSAP2 was approximately 27 kDa, as confirmed by SDS-PAGE and immunoblot analysis. An indirect ELISA and immunoblotting indicated that scFvFgSAP2 had strong reactivity with F. gigantica compared to other parasite species. Moreover, immunolocalization of scFvFgSAP2 confirmed that it binds specifically to natural SAP2 in the cecal epithelium cells of F. gigantica. Therefore, this scFv targeting SAP2 is an effective material and can be used to develop immunodiagnostic procedures. Full article

Background and objectives: The PWWP domain of lens epithelium-derived growth factor p75 (LEDGF/p75) mediates chromatin engagement through recognition of histone H3 lysine 36 di- and trimethylation (H3K36me2/3) and nucleosomal DNA. LEDGF/p75 plays a role in multiple human diseases. In particular, its interaction with HIV-1 integrase enables viral genome integration. However, the LEDGF PWWP domain remains difficult to target with small molecules as it lacks optimally shaped binding pockets. Here, we report the generation of high-affinity nanobodies (Nbs) to investigate the structure and function of this domain. Methods: Camelids were immunized with recombinant LEDGF PWWP domain, and immune phage display libraries were screened for affinity. Selected Nbs were recombinantly expressed in E. coli and purified. Their interaction with the PWWP domain of LEDGF and its close homolog HRP-2 was characterized using size-exclusion chromatography and surface plasmon resonance. Structural characterization of the Nbs was performed using X-ray crystallography. Functional effects on chromatin engagement were evaluated using an AlphaScreen assay. Results: Nine sequence-distinct Nbs were identified, seven of which were confirmed to bind the LEDGF PWWP domain with nanomolar affinities. Five Nbs also bound the HRP-2 domain, consistent with conserved functional surfaces, while two showed reduced affinity. The crystal structures of two Nbs (NbC03 and NbH10) confirmed there were canonical immunoglobulin folds, while the latter additionally revealed a domain-swapped dimer. Moreover, NbH10 dose-dependently inhibited the interaction between full-length LEDGF/p75 and H3K36me3-modified nucleosomes in vitro. Conclusions: This work establishes a validated panel of Nbs targeting the LEDGF PWWP domain and identifies one Nb capable of functionally disrupting the LEDGF–chromatin interaction. These Nbs serve as valuable tools for functional studies and structure-based drug design. Full article

Background: The emergence of multidrug-resistant (MDR) Gram-negative pathogens, namely Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii and Helicobacter pylori, necessitates urgent therapeutic alternatives. This scoping review aimed to summarize the current evidence on the efficacy of lytic bacteriophages against these critical MDR pathogens, and to identify existing research gaps and implementation challenges. Methods: The literature search was conducted by searching PubMed, Web of Science, and Scopus AI for studies published from 2015 to 2025. The inclusion criteria focused on experimental and human studies evaluating phage therapy against MDR, extensively drug-resistant (XDR), or pan-drug-resistant (PDR) strains in the four target species. A total of 172 articles were included. Results: A number of studies showed an increasing trend (2015–2025), focusing mainly on K. pneumoniae (n = 65), P. aeruginosa (n = 55), and A. baumannii (n = 48). No eligible studies for MDR H. pylori were found. All 172 studies confirmed lytic activity, with phage cocktails showing superior antibacterial activity than single phages in four studies. Phages also demonstrated antibiofilm activity (n = 44). Most animal studies reported successful bacterial reduction in animals treated with phages, and 87.5% of 23 human case studies reported patient improvement or infection clearance. However, heterogeneity in the types of animal models used and in dosage and administration routes in human studies was notable. Conclusions: Lytic bacteriophages exhibit strong potential as a new therapeutic option. Key challenges include the lack of data for MDR H. pylori, heterogeneity in animal models, and a paucity of large-scale human clinical trials. Future research must prioritize standardization, mechanistic studies, and conducting robust human trials to enable clinical translation and regulatory acceptance. Full article

To address the emerging multidrug-resistance crisis caused by Klebsiella pneumoniae, we expressed the endolysin Lys59 derived from phage VB_KpP_HS106 and performed a comprehensive analysis of its antibacterial activity and structural features. Molecular modeling revealed that Lys59 carries a highly positively charged N-terminus and an amphipathic helix at the C-terminus. In vitro antibacterial assays showed that Lys59 exhibited significant bactericidal activity against K. pneumoniae with an approximately 4 log reduction at 50 µg/mL in 2 h. Meanwhile, Lys59 exhibited potent, broad-spectrum activity against both Gram-negative and Gram-positive bacteria. Stability analysis indicated that Lys59 retained high activity over a pH range of 3–9 and a temperature range of 4–55 °C. Notably, the antibacterial activity of Lys59 was found to be regulated by metal ions. Molecular docking indicated that K⁺ can enhance binding stability by interacting with ASN35 and VAL57. In contrast, Mg²⁺ and Ca²⁺ suppressed catalytic function by binding to the essential GLU17 residue. Furthermore, treatment with 200 µg/mL of Lys59 resulted in a 44.6% reduction in K. pneumoniae biofilm biomass. Overall, this study identified a phage-derived endolysin with broad-spectrum antimicrobial activity and demonstrated its potential as an antibacterial agent against multidrug-resistant K. pneumoniae. Full article

Klebsiella pneumoniae, a member of the ESKAPEE group of priority pathogens, has become one of the most challenging bacterial pathogens in modern clinical practice, largely due to its multidrug resistance and the immune-evasive effect of its capsular polysaccharide (CPS). Phage-encoded depolymerases, which selectively degrade the capsular polysaccharide, have emerged as promising antimicrobial agents capable of restoring bacterial susceptibility to both immune clearance and phage infection. The fragment corresponding to the C-terminal region of a putative depolymerase of bacteriophage KlebP_144, namely DepKP144ΔC, was cloned, expressed in E. coli, and purified using immobilized metal affinity chromatography. DepKP144ΔC displays an enzymatic activity against capsular polysaccharides of 100% K1 capsular-type strains and 85% K2 capsular-type strains, including classical and hypervirulent isolates. It was demonstrated that this protein is capable of inhibiting K. pneumoniae biofilm formation, but it is unable to disrupt mature biofilms. In vivo experiments using a murine K. pneumoniae infection model further confirmed its therapeutic potential: treatment with DepKP144ΔC improved survival rate in mice infected with K2-type K. pneumoniae, indicating significant attenuation of bacterial virulence. Therefore, these results demonstrate the potential role of the C-terminal domain of the bacteriophage KP144 tail-fiber protein in phage entry and show that its carbohydrate-recognition motifs possess enzymatic activity against the Klebsiella capsular polysaccharides. Full article

Background: Transferrin receptor protein 1 (TfR1) plays a central role in cellular iron uptake and is frequently overexpressed in malignant tumor cells, rendering it an attractive target for tumor-directed therapy and drug delivery. Methods: A fully human single-chain variable fragment (scFv) antibody targeting TfR1, termed T8scFv, was isolated from a human scFv phage display library through three rounds of stringent biopanning and subsequently reformatted into a full-length IgG1 antibody (T8IgG1). Binding kinetics were characterized using Octet biolayer interferometry (BLI), while cellular binding and internalization were assessed by flow cytometry and immunofluorescence microscopy, respectively. T8IgG1 was further conjugated to DT3C, a recombinant truncated diphtheria toxin fusion protein, to evaluate its internalization-dependent cytotoxicity in vitro. Results: T8scFv exhibited nanomolar affinity for TfR1 (K_D = 214 ± 1 nM), which was substantially enhanced following conversion to the IgG1 format (T8IgG1, K_D = 18.5 ± 0.1 nM). T8IgG1 specifically recognized TfR1 on the surface of tumor cells and underwent efficient TfR1-mediated internalization. The T8IgG1-DT3C complex significantly reduced cell viability and induced apoptosis in K562 cells in vitro. Conclusions: These findings indicate that T8IgG1 is a moderate-affinity, internalizing anti-TfR1 antibody and highlight its potential as a promising candidate for TfR1-based targeted antitumor drug delivery systems. Full article

Klebsiella pneumoniae (K. pneumoniae) is an opportunistic and environmental mastitis pathogen prevalent in dairy herds worldwide. Owing to their genetic and genomic diversity, K. pneumoniae strains associated with bovine mastitis exhibit significant variation in virulence. Certain types of mastitis-causing K. pneumoniae strains exhibit enhanced pathogenicity and mammary adaptability, posing a serious threat to global public health. Bovine mastitis-causing K. pneumoniae strains can cause ultrastructural damage to bovine mammary epithelial cells (bMECs), leading to inflammatory injury, oxidative damage, apoptosis, pyroptosis, and immune evasion in bMECs. In this review, we summarize the prevalence, virulence genes, and pathogenic mechanisms of K. pneumoniae strains related to bovine mastitis. Given the increasing multidrug resistance of K. pneumoniae, we also outline the methods and mechanisms of phage therapy for K. pneumoniae infections, as well as future directions for treatment and prevention. These findings contribute to a deeper understanding of the population structure of mastitis-associated K. pneumoniae and provide valuable insights for future research on pathogenic mechanisms, vaccine development, and control strategies. Full article

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

Background: The global rise in multidrug-resistant (MDR) Gram-negative bacteria (GNB) poses an urgent challenge for infection control and antibiotic stewardship. Among these, Klebsiella pneumoniae is a major cause of hospital-acquired infections and is listed as a critical priority pathogen by the World Health Organization. Peru reports an exceptionally high prevalence of MDR K. pneumoniae, underscoring the need for innovative antimicrobial approaches. Methods: Here, we describe the isolation and characterization of lytic Klebsiella bacteriophage from sewage samples collected from the Huatanay River (Cusco, Peru) in 2023. Phages were isolated using the reference strain MDR K. pneumoniae ATCC BAA-2814. Then, they were screened against 50 clinical MDR K. pneumoniae strains. Results: The phage H33IIK demonstrated effective antibacterial capability, showing strict host specificity for K. pneumoniae, thermal stability, moderate pH tolerance, and high burst size. Whole-genome sequencing analysis classified it within the class Caudoviricetes, family Ackermannviridae, and genus Taipeivirus. The genomic analysis confirmed the absence of lysogeny-related, antimicrobial resistance, and virulence genes, supporting its suitability and safety for potential biotechnological applications. Conclusions: These findings highlight phage H33IIK as a lytic agent effective against MDR K. pneumoniae. It could contribute to the development of phage-based approaches to combat MDR GNB. Full article

Antimicrobial resistance has renewed interest in bacteriophage therapy, yet bacterial evolution frequently undermines treatment efficacy. Combination phage therapy is commonly implemented as simultaneous phage cocktails, but whether this is optimal remains in question. Here, we experimentally compared simultaneous versus sequential administration of two phages, an evolved λ called ‘λtrn’ and T2, on Escherichia coli K-12 under controlled laboratory conditions. Across replicated experiments, treatment outcome depended strongly on delivery strategy, dosing order, and timing. Contrary to expectations, sequential delivery consistently achieved greater and more sustained bacterial suppression than simultaneous cocktails, although only when T2 initiated the sequence. Phenotypic assays revealed that treatment differences were driven by the accessibility and timing of cross-resistance evolution. λ-first treatments rapidly selected for cross-resistant bacteria prior to exposure to the second phage, rendering subsequent treatment ineffective. In contrast, T2-first sequential treatments delayed or limited cross-resistance and frequently produced single-phage resistance or collateral sensitivity. Cocktail treatments showed intermediate dynamics, with cross-resistance evolving more slowly but consistently. Whole genome sequencing identified distinct genetic routes to cross-resistance, including regulatory mutations in envZ affecting expression of the phage receptor OmpF, as well as envelope-modifying, mucoidy-associated mutations. Engineering envZ mutations into unevolved backgrounds confirmed the mutation’s sufficiency to confer low-cost cross-resistance. Together, these results demonstrated that phage therapy efficacy depended not only on phage composition but on how selection pressures were ordered in time, highlighting evolutionary steering as a powerful principle for multi-phage therapy design. Full article

Bt Cry toxins remain the cornerstone of transgenic crop protection against Lepidopteran pests, yet field-evolved resistance, particularly in invasive species such as Spodoptera frugiperda and Helicoverpa armigera, can threaten their long-term efficacy. This review presents a comprehensive and unified mechanistic framework that synthesizes current understanding of Bt Cry toxin modes of action and the complex, multilayered regulatory mechanisms of field-evolved resistance. Beyond the classical pore-formation model, emerging evidence highlights signal transduction cascades, immune evasion via suppression of Toll/IMD pathways, and tripartite toxin–host–microbiota interactions that can dynamically modulate protoxin activation and receptor accessibility. Resistance arises from target-site alterations (e.g., ABCC2/ABCC3, Cadherin mutations), altered midgut protease profiles, enhanced immune regeneration, and microbiota-mediated detoxification, orchestrated by transcription factor networks (GATA, FoxA, FTZ-F1), constitutive MAPK hyperactivation (especially MAP4K4-driven cascades), along with preliminary emerging findings on non-coding RNA involvement. Countermeasures now integrate synergistic Cry/Vip pyramiding, CRISPR/Cas9-validated receptor knockouts revealing functional redundancy, Domain III chimerization (e.g., Cry1A.105), phage-assisted continuous evolution (PACE), and the emerging application of AlphaFold3 for structure-guided rational redesign of resistance-breaking variants. Future sustainability hinges on system-level integration of single-cell transcriptomics, midgut-specific CRISPR screens, microbiome engineering, and AI-accelerated protein design to preempt resistance trajectories and secure Bt biotechnology within integrated resistance and pest management frameworks. Full article

Antibiotic resistance is arguably one of the greatest threats to global health today. The worldwide emergence of multidrug-resistant and hypervirulent Klebsiella pneumoniae underscores the urgent need for alternative treatments. Bacteriophages (phages) are considered one of the most promising alternatives to address this crisis. In this review, we summarize current knowledge of phage–host interactions and highlight recent advances in phage therapy against K. pneumoniae, including phage cocktails, antibiotic combination therapy, and treatments based on phage-derived proteins. Despite their tremendous therapeutic potential, significant challenges remain. We therefore also discuss strategies to optimize phage research and recent innovations in the field. Full article

This work introduces an original sequential bio-inspired strategy combining lytic phage pretreatment with TiO₂–thyme essential oil (TEO) photocatalysis, achieving near-complete inhibition of both biofilm initiation and maturation. By simultaneously targeting planktonic cells, mature biofilms, and extracellular DNA (eDNA), this approach addresses key mechanisms involved in biofilm persistence. Pseudomonas aeruginosa ATCC 4114 was selected as the biological model due to its relevance in water distribution systems and its strong biofilm-forming ability. Experimental results showed that phage pretreatment alone inhibited biofilm formation by planktonic cells by up to 99.6% (inactivation rate constant k = 0.034 min⁻¹) and weakened bacterial attachment in mature biofilms by 89.06% (k = 0.011 min⁻¹). To further enhance photocatalytic efficacy, titanium dioxide (TiO₂) was combined with TEO at 0.05% (v/v) as a bio-inspired photosensitizer. UV–Vis spectroscopy confirmed TiO₂-TEO interactions that extended light absorption into the visible region (400–700 nm), thereby enhancing photocatalytic efficiency. This combination was designed to suppress residual biofilm development and disrupt extracellular DNA (eDNA), a critical component of biofilm structure and stability. The integrated approach involving phage pretreatment followed by TiO₂–TEO (0.05%) photocatalysis achieved 99.99% inhibition of both biofilm initiation and maturation phases, with significantly increased kinetic parameters (A = 2.62 for planktonic cells and A = 3.65 for sessile cells; k = 0.076 min⁻¹ and 0.063 min⁻¹, respectively; p < 0.01). This study provides novel insights into water disinfection strategies using photocatalytic treatment, emphasizing the importance of monitoring post-treatment bacterial virulence factor expression. Full article