Search Results (156)

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

Background: Heparan sulfate (HS) is widely implicated as a receptor for Chlamydia cell attachment and infectivity. However, the enzymatic modification of HS modified by the 3-O sulfotransferase-3 (3-OST-3) enzyme in chlamydial cell entry remains unknown. Methodology: To rule out the possibility that host cell 3-O sulfated heparan sulfate (3-OS HS) plays a significant role in C. muridarum entry, a Chinese hamster ovary (CHO-K1) cell model lacking endogenous 3-OST-3 was used. In addition, we further tested the efficacy of the phage-display-derived cationic peptides recognizing heparan sulfate (G1 peptide) and the moieties of 3-O sulfated heparan sulfate (G2 peptide) against C. muridarum entry using human cervical adenocarcinoma (HeLa 229) and human vaginal epithelial (VK2/E6E7) cell lines. Furthermore, molecular dynamics simulations were conducted to investigate the interactions of the Chlamydia lipid bilayer membrane with the G1 and G2 peptides, focusing on their binding modes and affinities. Results: The converse effect of 3-OST-3 expression in the CHO-K1 cells had no enhancing effect on C. muridarum entry. The G2 peptide significantly (>80%) affected the cell infectivity of the elementary bodies (EBs) at all the tested concentrations, as evident from the reduced fluorescent staining in the number of inclusion bodies. The observed neutralization effect of G2 peptide on C. muridarum entry suggests the possibility of sulfated-like domains being present on the EBs. In addition, data generated from our in silico computational structural modeling indicated that the G2 peptide ligand had significant affinity towards the C. muridarum lipid bilayer. Conclusions: Taken together, our findings show that the pretreatment of C. muridarum with 3-O sulfated heparan sulfate recognizing G2 peptide significantly prevents the entry of EBs into host cells. Full article

Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp), a pathogen causing severe nosocomial infections and high mortality rates, is increasingly becoming a serious global public health threat. Capsular polysaccharide (CPS), a major virulence factor of hvKp, can be enzymatically degraded by bacteriophage-derived depolymerases. However, to our knowledge, depolymerases targeting K. pneumoniae K54-type strains have rarely been identified. Here, we identified and characterized three novel capsule depolymerases, Dep_C, Dep_Y, and Dep_Z, derived from three different K. pneumoniae phages, which retained robust activity across a broad pH range (pH 3.0–12.0) and demonstrated thermal stability up to 50 °C. These depolymerases could efficiently digest the CPS of K. pneumoniae K54-serotype strains, significantly inhibit biofilm formation, and remove their mature biofilms. Although no bactericidal activity was detected, these depolymerases rendered host bacteria susceptible to serum complement-mediated killing. We further demonstrate that Dep_C, Dep_Y, and Dep_Z can effectively and significantly prolong the survival time of mice in a pneumonia model infected with K54-type K. pneumoniae and reduce the colonization and virulence of the bacteria in the mice. These findings indicate that depolymerases Dep_C, Dep_Y, and Dep_Z could increase bacterial susceptibility to host immune responses of hvKp to the host through their degradation effect on the CPS. In conclusion, our study demonstrates that the three capsule depolymerases are promising antivirulent agents to combat CR-hvKp infections. Full article

Acinetobacter baumannii is a significant nosocomial pathogen characterized by the ability to produce a wide variety of capsular polysaccharides (CPSs). The structures of a K102-type CPS isolated from A. baumannii KZ-1102 and its Smith degradation product were determined by sugar analysis, 1D and 2D ¹H NMR spectroscopy, and ¹³C NMR spectroscopy. The K102 CPS biosynthesis gene cluster (KL102) contains genes for common sugar synthesis, K unit processing, capsule export, glycosyl transfer, initiating sugar phosphate transfer, and genes that encode d-GlcpNAc/d-GalpNAc dehydrogenase and phosphoglycerol transferase. The CPS is composed of a pentasaccharide repeating unit (K unit) consisting of a tetrasaccharide backbone including one α-d-Galp, three α-d-GlcpNAc residues, and one residue of a β-d-Glcp as a side chain. The tailspike depolymerase of the specific Obolenskvirus phage Cato was found to cleave the α-d-GlcpNAc-(1→6)-α-d-GlcpNAc linkage in the K102 CPS to give the monomer and dimer of the K repeating unit, which were characterized by high-resolution electrospray ionization mass spectrometry as well as ¹H and ¹³C NMR spectroscopy. Full article

Background: Antibiotic-resistant Pseudomonas aeruginosa (P. aeruginosa) strains are an increasing cause of morbidity and mortality. Pulsed blue light (PBL) enhances porphyrin-induced reactive oxygen species and has been clinically shown to be harmless to the skin at low doses. Bacteriophages, viruses that infect bacteria, offer a promising non-antibiotic bactericidal approach. This study investigates the potential synergism between low-dose PBL and phage therapy against P. aeruginosa in planktonic cultures and preformed biofilms. Methods: We conducted a factorial dose–response in vitro study combining P. aeruginosa-specific phages with PBL (457 nm, 33 kHz) on both PA14 and multidrug-resistant PATZ2 strains. After excluding direct PBL effects on phage titer or activity, we assessed effectiveness on planktonic cultures using growth curve analysis (via growth_curve_outcomes, a newly developed, Python-based tool available on GitHub) , CFU, and PFU. Biofilm efficacy was evaluated using CFU post-sonication, crystal violet staining, and live/dead staining with confocal microscopy. Finally, we assessed reactive oxygen species (ROS) as a potential mechanism using the nitro blue tetrazolium reduction assay. ANOVA or Kruskal–Wallis tests with post hoc Tukey or Conover–Iman tests were used for comparisons (n = 5 biological replicates and technical triplicates). Results: The bacterial growth lag phase was significantly extended for phage alone or PBL alone, with a synergistic effect of up to 144% (p < 0.001 for all), achieving a 9 log CFU/mL reduction at 24 h (p < 0.001). In preformed biofilms, synergistic combinations significantly reduced biofilm biomass and bacterial viability (% Live, median (IQR): Control 80%; Phage 40%; PBL 25%; PBL&Phage 15%, p < 0.001). Mechanistically, PBL triggered transient ROS in planktonic cultures, amplified by phage co-treatment, while a biphasic ROS pattern in biofilms reflected time-dependent synergy. Conclusions: Phage therapy combined with PBL demonstrates a synergistic bactericidal effect against P. aeruginosa in both planktonic cultures and biofilms. Given the strong safety profile of PBL and phages, this approach may lead to a novel, antibiotic-complementary, safe treatment modality for patients suffering from difficult-to-treat antibiotic-resistant infections and biofilm-associated infections. Full article

Acinetobacter baumannii and Klebsiella pneumoniae are significant nosocomial pathogens worldwide. In this study, the general characterization of A. baumannii and K. pneumoniae isolates obtained from the blood of intensive care unit patients of the multidisciplinary scientific and practical center of emergency medicine from January to September 2024 was performed. Prophage regions and prophage-derived tailspike polysaccharide-depolymerizing or -modifying enzymes within these isolates were identified and characterized in detail using a refined workflow. The protocol, encompassing a comprehensive survey of all predicted bacterial proteins, revealed an average of 6.0 prophage regions per Acinetobacter baumannii genome, including regions putatively derived from filamentous phages, and 4.8 prophage regions per Klebsiella pneumoniae isolate. Analysis of these putative prophage regions indicated that most were related to previously isolated, yet unclassified, temperate phages infecting A. baumannii and K. pneumoniae. However, certain identified sequences likely originated from phages representing novel groups comparatively distant from known phages. Full article

Antibiotic resistance to Klebsiella pneumoniae poses a major public health threat, particularly in intensive care unit (ICU) settings. The emergence of extensively drug-resistant (XDR) strains complicates treatment options, requiring a deeper understanding of their genetic makeup and potential therapeutic targets. This research delineated an extensively drug-resistant (XDR) Klebsiella pneumoniae strain obtained from an ICU patient and telomeric temperate phage derived from hospital effluent. The bacteria showed strong resistance to multiple antibiotics, including penicillin (≥16 μg/mL), ceftriaxone (≥32 μg/mL), and meropenem (≥8 μg/mL), which was caused by SHV-11 beta-lactamase, NDM-1 carbapenemase, and porin mutations (OmpK37, MdtQ). The strain was categorized as K46 and O2a types and carried virulence genes involved in iron acquisition, adhesion, and immune evasion, as well as plasmids (IncHI1B_1_pNDM-MAR, IncFIB) and eleven prophage regions, reflecting its genetic adaptability and resistance dissemination. The 172,025 bp linear genome and 46.3% GC content of the N-15-like phage showed strong genomic similarities to phages of the Sugarlandvirus genus, especially those that infect K. pneumoniae. There were structural proteins (11.8%), DNA replication and repair enzymes (9.3%), and a toxin–antitoxin system (0.4%) encoded by the phage genome. A protelomerase and ParA/B partitioning proteins indicate that the phage is replicating and maintaining itself in a manner similar to the N15 phage, which is renowned for maintaining a linear plasmid prophage throughout lysogeny. Understanding the dynamics of antibiotic resistance and pathogen development requires knowledge of phages like this one, which are known for their temperate nature and their function in altering bacterial virulence and resistance profiles. The regulatory and structural proteins of the phage also provide a model for research into the biology of temperate phages and their effects on microbial communities. The importance of temperate phages in bacterial genomes and their function in the larger framework of microbial ecology and evolution is emphasized in this research. Full article

Microorganisms exert antagonistic effects on pathogens through different mechanisms, thereby achieving biological control of plant diseases. Many Burkholderia strains can produce complex secondary metabolites and substances that have toxic effects on host cells. The phage tail-like bacteriocins (tailocins) is a compound with antibacterial activity. However, its function in B. multivorans has not yet been reported. This article explores the ability of B. multivorans WS-FJ9 to antagonise plant pathogenic fungi and oomycetes, screening the potential tailocins in the strain WS-FJ9 and verifying their function, to reveal its novel antimicrobial mechanisms. We found that WS-FJ9 had strong antagonistic effects on the plant pathogenic fungi Phomopsis macrospore and Sphaeropsis sapinea, and the pathogenic oomycete Phytophthora cinnamomi. The phage tail-like protein Bm_67459 was predicted from the WS-FJ9 strain genome. The Bm_67459 cDNA encoded 111 amino acid sequence, and the relative molecular weight was approximately 11.69 kDa, the theoretical isoelectric point (pI) was 5.49, and it was a hydrophilic protein. Bm_67459 had no transmembrane helix region or signal peptide, and it belonged to the Phage_TAC_7 super family. qRT-PCR results showed that Bm_67459 gene expression was significantly upregulated during contact between WS-FJ9 and P. cinnamomi. The purified Bm_67459 protein significantly inhibited P. cinnamomi mycelial growth at 10 μg·mL⁻¹. In summary, the WS-FJ9 strain had broad-spectrum anti-phytopathogenic activity, and the tailocin Bm_67459 was an important effector against the plant pathogen P. cinnamomi, which helps to reveal the antagonistic mechanism of this strain at the molecular level and provides excellent strain resources for the biological control of plant diseases. Full article

Zearalenone (ZEN), a toxic estrogenic mycotoxin in cereals, threatens human and animal health through reproductive, immune, and cytotoxic effects, necessitating sensitive detection methods. While nanobodies offer advantages over conventional antibodies for on-site ZEN detection, their application remains unexplored. This study aimed to develop an anti-ZEN nanobody derived from an anti-ZEN phage display nanobody library. An alpaca was immunized with a ZEN-bovine serum albumin (ZEN-BSA) antigen, achieving peak serum antibody titers (1:25,600) following four immunizations. A high-capacity phage display nanobody library (1.0 × 10¹¹ plaque-forming units/mL) was constructed. Following four rounds of biopanning, an enrichment factor of 479 was achieved. Phage ELISA screening identified six phage display nanobodies with specific ZEN-binding activity, and multiple sequence alignment revealed four unique nanobody sequences. The selected phage display nanobody, designated phage-V44, was expressed and purified, and its presence was validated by SDS-PAGE and western blotting, which detected a single approximately 17 kDa band consistent with the expected nanobody size. We established a working curve for an indirect competitive enzyme-linked immunoassay (ELISA) for ZEN, which showed an IC50 value of 7.55 ng/mL. The specificity and affinity of the V44 were also verified. Collectively, the study successfully constructed an anti-ZEN phage display nanobody library, screened four specific ZEN-binding phage display nanobodies, and prepared the anti-ZEN nanobody V44. Thereby establishing a foundation for the nanobody’s future integration into rapid on-site detection methods for ZEN in both animal feed and human food products. Full article

Staphylococcus aureus is a common pathogen, often recovered from children’s infections. Βiofilm formation, antimicrobial resistance and production of adhesins and toxins contribute to its virulence. As resistance to antimicrobials rises worldwide, alternative therapies like bacteriophages (among them the well-studied Bacteriophage K) can be helpful. The aim of this study was to determine the bacteriophage and antimicrobial susceptibility and the presence of virulence genes among S. aureus from infections in children and adolescents. Eighty S. aureus isolates were tested for biofilm formation and antimicrobial susceptibility. The presence of two genes of the ica operon (icaA, icaD), adhesin’s (fnbA, fnbB, sasG) and toxin’s genes (PVL, tst, eta, etb) was tested by PCRs. Susceptibility to Bacteriophage K was determined using a spot assay. Thirteen isolates were methicillin-resistant (MRSA) and 41 were multi-resistant. Twenty-five S. aureus (31.3%) were resistant to Bacteriophage K, mostly from ocular and ear infections. Twelve S. aureus (15%) were PVL-positive, seven (8.8%) positive for tst, 18 (22.5%) were eta-positive and 46 were (57.5%) etb-positive. A total of 66 (82.5%) isolates carried fnbA, 16 (20%) fnbB and 26 (32.5%) sasG. PVL, tst and sasG carriage were more frequent in MRSA. Bacteriophage-susceptible isolates carried more frequently eta (32.7%) and etb (69.1%) compared to phage-resistant S. aureus (0% and 32%, respectively). Although mainly methicillin-sensitive, S. aureus from pediatric infections exhibited high antimicrobial resistance and carriage of virulence genes (especially for exfoliative toxins and fnbA). MRSA was associated with PVL, tst and sasG carriage, whereas Bacteriophage susceptibility was associated with eta and etb. The high level of Bacteriophage K susceptibility highlights its potential use against staphylococcal infections. Full article

Prolyl cis/trans isomerization is a rate-limiting step in protein folding, often coupling directly to the acquisition of native structure. Here, we investigated the interplay between folding and prolyl isomerization in the N2 domain of the gene-3-protein from filamentous phage fd, which adopts a native-state cis/trans equilibrium at Pro161. Using mutational and Φ-value analysis, we identified a discrete folding nucleus encompassing the β-strands surrounding Pro161. These native-like interactions form early in the folding pathway and provide the energy to shift the cis/trans equilibrium toward the cis form. Variations distant from the Pro161-loop have minimal impact on the cis/trans ratio, underscoring the spatial specificity and localized control of the isomerization process. Using NMR spectroscopy, we determined the structures for both native N2 forms. The cis- and trans-Pro161 conformations are overall identical and exhibit only slight differences around the Pro161-loop. The cis-conformation adopts a more compact structure with improved backbone hydrogen bonding, explaining the approximately 10 kJ·mol⁻¹ stability increase of the cis state. Our findings highlight that prolyl isomerization in the N2 domain is governed by a localized folding nucleus rather than global stability changes. This localized energetic coupling ensures that proline isomerization is not simply a passive, slow step but an integral component of the folding landscape, optimizing both the formation of native structure and the establishment of the cis-conformation. Full article

Background/Objectives: The significant outbreak of multidrug-resistant Klebsiella pneumoniae has emerged as a primary global concern associated with high morbidity and mortality rates. Certain strains of K. pneumoniae are highly resistant to most antibiotics available in clinical practice, exacerbating the challenge of bacterial infections. Methods: Phage vB_KpnP_PW7 (vKPPW7) was isolated and characterized. Its morphology, stability, adsorption rate, one-step growth curve, lytic activity, whole-genome sequence analysis, and antibacterial and antibiofilm activities were evaluated. Results: The virulent phage has a 73,658 bp linear dsDNA genome and was classified as a new species of the genus Kaypoctavirus, subfamily Enquatrovirinae, and family Schitoviridae. Phage vKPPW7 has a high adsorption rate, a short latent period, and a large burst size. The phage showed activity against 18 K. pneumoniae isolates with the K24 capsular type but was unable to lyse K. pneumoniae isolates whose capsular type was not classified as K24. Additionally, phage vKPPW7 demonstrated strong stability across various temperatures and pH values. The phage exhibited antibacterial activity, and scanning electron microscopy (SEM) confirmed its ability to lyse MDR K. pneumoniae with the K24 capsular type. Furthermore, phage vKPPW7 effectively removed preformed biofilm and prevented biofilm formation, resulting in reduced biofilm biomass and biofilm viability compared to controls. The architecture of phage-treated biofilms was confirmed under SEM. Conclusions: These findings suggest that phage vKPPW7 holds promise for development as a therapeutic or biocontrol agent. Full article

Background: Phage tail-like bacteriocins, or tailocins, provide a competitive advantage to producer cells by killing closely related bacteria. Morphologically similar to headless phages, their narrow target specificity is determined by receptor-binding proteins (RBPs). While RBP engineering has been used to alter the target range of a selected R2 tailocin from Pseudomonas aeruginosa, the process is labor-intensive, limiting broader application. Methods: We introduce a VersaTile-driven R2 tailocin engineering and screening platform to scale up RBP grafting. Results: This platform achieved three key milestones: (I) engineering R2 tailocins specific to Escherichia coli serogroups O26, O103, O104, O111, O145, O146, and O157; (II) reprogramming R2 tailocins to target, for the first time, the capsule and a new species, specifically the capsular serotype K1 of E. coli and K11 and K63 of Klebsiella pneumoniae; (III) creating the first bivalent tailocin with a branched RBP and cross-species activity, effective against both E. coli K1 and K. pneumoniae K11. Over 90% of engineered tailocins were effective, with clear pathways for further optimization identified. Conclusions: This work lays the groundwork for a scalable platform for the development of engineered tailocins, marking an important step towards making R2 tailocins a practical therapeutic tool for targeted bacterial infections. Full article

The rapid worldwide spread of antibiotic resistance is quickly becoming an increasingly concerning problem for human healthcare. Non-antibiotic antibacterial agents are in high demand for many Gram-negative bacterial pathogens, including Klebsiella pneumoniae. Klebsiella-targeting phages are among the most promising alternative therapy options. They have already been successfully applied in a number of cases, and it is expected that the need for anti-Klebsiella phages will only increase in the future. This prospect highlights the need for well-characterized therapeutic phages. In this work, we describe a K. pneumoniae phage, which also infects strains of Klebsiella oxytoca. Here, we characterize phage M198 in terms of its biological and genetic properties. Since in some phage therapy cases, phages are administered in combination with antibiotics, here, we also screen for possible synergistic effects of combining phage M198 with six different antibiotics. We found that phage M198 has good lytic activity against clinical isolates; it does not have any indications of a temperate lifestyle, and it has synergistic potential when combined with some therapeutically relevant antibiotics. Full article

Background/Objectives Bacterial ghosts (BGs), non-living empty envelopes of bacteria, are produced either through genetic engineering or chemical treatment of bacteria, retaining the shape of their parent cells. BGs are considered vaccine candidates, promising delivery systems, and vaccine adjuvants. The practical use of BGs in vaccine development for humans is limited because of concerns about the preservation of viable bacteria in BGs. Methods: To increase the efficiency of Klebsiella pneumoniae BG formation and, accordingly, to ensure maximum killing of bacteria, we exploited previously designed plasmids with the lysis gene E from bacteriophage φX174 or with holin–endolysin systems of λ or L-413C phages. Previously, this kit made it possible to generate bacterial cells of Yersinia pestis with varying degrees of hydrolysis and variable protective activity. Results: In the current study, we showed that co-expression of the holin and endolysin genes from the L-413C phage elicited more rapid and efficient K. pneumoniae lysis than lysis mediated by only single gene E or the low functioning holin–endolysin system of λ phage. The introduction of alternative lysing factors into K. pneumoniae cells instead of the E protein leads to the loss of the murein skeleton. The resulting frameless cell envelops are more reminiscent of bacterial sacs or bacterial skins than BGs. Although such structures are less naive than classical bacterial ghosts, they provide effective protection against infection by a hypervirulent strain of K. pneumoniae and can be recommended as candidate vaccines. For our vaccine candidate generated using the O1:K2 hypervirulent K. pneumoniae strain, both safety and immunogenicity aspects were evaluated. Humoral and cellular immune responses were significantly increased in mice that were intraperitoneally immunized compared with subcutaneously vaccinated animals (p < 0.05). Conclusions: Therefore, this study presents novel perspectives for future research on K. pneumoniae ghost vaccines. Full article