Search Results (504)

Background/Objectives: Mycobacterium abscessus is an opportunistic pathogen causing infections mainly in patients with immunosuppression and chronic pulmonary pathologies. Extended treatment periods are needed to tackle this pathogen, bacterial eradication is rare, and recurrence can take place with time. New alternative treatments are being investigated, such as bacteriophage therapy. This work describes the characterization of the mycobacteriophage P3MA, showing its ability to infect clinical and standard M. abscessus strains. Methods: Phylogenetic analysis, electron microscopy, growth curves, biofilm assays, checkerboard, and granuloma-like medium studies were performed. Results: P3MA inhibited the growth of clinical samples in both planktonic and biofilm states as well as in a granuloma-like model. The study of the interaction with antibiotics revealed that P3MA exhibited an antagonistic effect combined with clarithromycin, indifference with amikacin, and synergy with imipenem. Conclusions: All these results suggest that, after genetic engineering, P3MA could be a promising candidate for phage therapy in combination with imipenem, including lung infections. Full article

Polymerase-coupled nanopore sequencing requires DNA polymerases with strong strand displacement activity and high processivity to sustain continuous signal generation. In this study, we characterized two novel B family DNA polymerases, SRHS and BBum, isolated from Bacillus phages SRT01hs and BeachBum, respectively. Both enzymes exhibited robust strand displacement, 3′→5′ exonuclease activity, and maintained processivity under diverse reaction conditions, including across a broad temperature range (10–45 °C) and in the presence of multiple divalent metal cofactors (Mg²⁺, Mn²⁺, Fe²⁺), comparable to the well-characterized Phi29 polymerase. Through biochemical analysis of mutants designed using AlphaFold3-predicted structural models, we identified key residues (G96, M97, D486 in SRHS; S97, M98, A493 in BBum) that modulated exonuclease activity, substrate specificity and metal ion utilization. Engineered variants SRHS_F and BBum_Pro_L efficiently incorporated unnatural nucleotides in the presence of Mg²⁺—a function not observed in Phi29 and other wild-type strand-displacing B family polymerases. These combined biochemical features highlight SRHS and BBum as promising enzymatic scaffolds for nanopore-based long-read sequencing platforms. Full article

Wastewater is a hotspot for the spread of antimicrobial resistance (AR); therefore, bacteriophages offer a promising biocontrol alternative to overcome the limitations of conventional disinfection. This study evaluated the efficacy of bacteriophages and cocktails for the biocontrol of carbapenem-resistant Klebsiella pneumoniae (CR-Kp) (CG258 and ST307) and Escherichia coli producers of extended-spectrum β-lactamases (ESBL-Ec) (ST131) in simulated wastewater. A synthetic wastewater matrix was prepared in which bacterial viability and bacteriophage stability were assessed for 72 h. CR-Kp or ESBL-Ec strain were treated with individual bacteriophages or phage-cocktails (dosed in different ways) and bacterial loads were monitored for 54 h. The Klebsiella phages FKP3 and FKP14 eliminated 99% (−2.9 Log) of CR-Kp-CG258 at 54 h, and FKP10 reduced 99% (−2.15 Log) of the CR-Kp-ST307 strains. The Klebsiella phage-cocktail in a single dose reduced to 99.99% (−4.12 Log) of the CR-Kp-CG258 at 36 h. Coliphage FEC1 reduced to 2.12 Log (99%) of ESBL-Ec-bla_CTX-M-G9, and FEC2 and FEC4 reduced approximately 1 Log (90%) of ESBL-Ec-bla_CTX-M-G9 and bla_CTX-M-G1. The coliphage cocktail increased the reduction up to 2.2 Logarithms. This study provides evidence supporting the use of bacteriophage cocktails for the control of resistant bacteria in wastewater, a sustainable intervention to mitigate the spread of AR and support water reuse safety. Full article

Phage therapy shows promise as an adjunct to antibiotics for treating Staphylococcus aureus infections. We previously reported a combined flucloxacillin/two-phage cocktail treatment selected for resistance to podovirus phage 66 in a rodent model of methicillin-susceptible S. aureus (MSSA) endocarditis. Here we show that resistant clones harbor mutations in tarS, which encodes a glycosyltransferase essential for β-GlcNAcylation of wall teichoic acid (WTA). This WTA modification has been described in vitro as critical for podoviruses adsorption. Transcriptomics confirmed continued tarS expression in resistant clones, supporting a loss-of-function mechanism. Accordingly, phage 66 binding and killing were restored by WT tarS complementation. In addition, we investigated the counterintuitive innate susceptibility to phage 66 of the tarM + Laus102 strain used in the endocarditis model. We show that it likely results from a significant lower tarM expression, in contrast to the innate resistant strain RN4220. Our findings demonstrate that tarS-mediated WTA β-GlcNAcylation is critical for podovirus infection also in vivo and identify tarM transcriptional defect as a new mechanism of podoviruses susceptibility in S. aureus. Moreover, and since tarS disruption has been previously shown to enhance β-lactam susceptibility, our results support the development of combined podovirus/antibiotic strategies for the management of MRSA infections. Full article

Listeria monocytogenes is a life-threatening bacterial foodborne pathogen that can persist in food-processing facilities for years. Although phages can control L. monocytogenes during food production, phage-resistant bacterial subpopulations can regrow in phage-treated environments. In this study, an L. monocytogenes hly defective strain, NJ05-Δhly, was produced, which considerably regulated the interactions between L. monocytogenes and phages. Specifically, we observed a 76.92-fold decrease in the efficiency of plating of the defective strain following infection with the Listeria phage vB-LmoM-NJ05. The lytic effect was notably diminished at multiplicities of infection of 1 and 10. Furthermore, the inactivation of LLO impaired biofilm formation, which was completely suppressed and eliminated following treatment with 10⁸ PFU/mL of phage. Additionally, phages protected cells from mitochondrial membrane damage and the accumulation of mitochondrial reactive oxygen species induced by L. monocytogenes invasion. Transcriptomic analysis confirmed these findings, revealing the significant downregulation of genes associated with phage sensitivity, pathogenicity, biofilm formation, and motility in L. monocytogenes. These results underscore the vital role of LLO in regulating the pathogenicity, phage susceptibility, and biofilm formation of L. monocytogenes. These observations highlight the important role of virulence factors in phage applications and provide insights into the potential use of phages for developing biosanitizers. Full article

A single domain antibody (SDAb) targeting canine PD-1 was developed as a potential immunotherapeutic for canine cancer. An alpaca was immunized with canine PD-1 protein, and a phage-display library was constructed using mRNA isolated from peripheral lymphocytes. Screening of the library yielded multiple SDAb candidates capable of nanomolar binding to canine PD-1. Among these, clone STX-1b5 demonstrated high expression in a yeast-based recombinant system and was selected for further characterization. Binding and competition assays using ELISA confirmed its ability to bind canine PD-1 and block PDL-1 interaction. In silico structural modeling supported the interaction of STX-1b5 with key PD-1 residues implicated in ligand binding. These findings support the feasibility of using SDAbs and cost-effective yeast expression systems to generate immunotherapeutics for veterinary use, with STX-1b5 representing a promising lead candidate for future clinical development. Full article

Background: Metastatic prostate cancer (mPrC), with a median survival of under 2 years, represents an important unmet medical need which may benefit from the development of more effective targeted drug delivery systems. Several cell surface receptors have been identified as candidates for targeted drug delivery to mPrC cells; however, these receptors were selected for their overabundance on PrC cells rather than for their suitability for targeted delivery and uptake of cytotoxic drug payloads. Methods: We describe a novel, unbiased strategy to isolate peptides that fulfill functional criteria required for effective intracellular drug delivery and the specific cytotoxicity of PrC cells without prior knowledge of the targeted receptor. Phage clones displaying 7-mer cyclic peptides were negatively selected in vivo and then positively biopanned through a series of parent and drug-resistant mPrC cells. Peptides from the internalized clones were then subjected to a panel of biochemical and functional tests that led to the selection of several peptide candidates. Results: The selected peptides do not bind PSMA. Peptide-drug conjugates (PDCs) incorporating one of the peptides selectively killed wild-type and drug-resistant PrC cell lines and patient PrC cells but not normal prostate tissue cells in vitro. The PDC also halted the growth of PC3 tumors in a xenograft model. Conclusions: Our study demonstrates that adding unbiased, functional criteria into drug carrier selection protocols can lead to the discovery of novel peptides with appropriate properties required for effective targeted drug delivery into target cancer cells. Full article

Pseudomonas aeruginosa is a major opportunistic pathogen with high levels of antibiotic resistance. Phage therapy represents a promising alternative for the treatment of difficult infections both alone and in combination with antibiotics. Here, we isolated and characterized three novel lytic myoviruses, Cisa, Nello, and Moonstruck. Genomic analysis revealed that Cisa and Nello belong to the Pbunavirus genus, while Moonstruck is a novel Pakpunavirus species. All lacked lysogeny, virulence, or resistance-associated genes, supporting their therapeutic suitability. Phage Nello and Moonstruck were active against P. aeruginosa Pa3GrPv, isolated from a patient with lung infection candidate for phage therapy. Moonstruck exhibited superior lytic activity with ciprofloxacin sub-MIC value (0.125 µg/mL), achieving bacterial suppression for 48 h. However, to improve the lytic efficacy of the phages on the clinical isolate, phage adaptation via serial passage was investigated. The killing efficacy of Nello was enhanced, whereas Moonstruck showed a less consistent improvement, suggesting phage-specific differences in evolutionary dynamics. Sequencing of the evolved phages revealed point mutations in tail-associated genes, potentially linked to a better phage–host interaction. These results support the use of phage–antibiotic combinations and directed evolution as strategies to enhance phage efficacy against drug-resistant infections. Overall, these findings support the therapeutic potential of the newly isolated phages in treating P. aeruginosa lung infections. Full article

Aeromonas salmonicida is a common spoilage bacterium found in refrigerated fish. In this study, a virulent bacteriophage was isolated from wastewater using A. salmonicida AS08 as the host, and it was designated as TSW001. Based on morphological characterization and whole-genome analysis, bacteriophage TSW001 was classified within the genus Tedavirus. Biological characterization revealed that TSW001 maintained a stable titer within a temperature range of 4~60 °C, a pH range of 4~9, and a salinity range of 50~1000 mM. The optimal multiplicity of infection (MOI) for TSW001 was 0.1, with a short latency period of approximately 10 min and a burst size of approximately 68 PFU/cell. When applied during the cold storage of large yellow croaker, the A. salmonicida count in the fish juice decreased by approximately 2.1~2.3 log₁₀ CFU/mL over the first two days, while the count in the fish fillets decreased by approximately 1.1~1.8 log₁₀ CFU/g. Furthermore, TSW001 demonstrated the ability to inhibit the formation of A. salmonicida biofilms. These results suggest that phage TSW001 is a promising biological antimicrobial agent for controlling A. salmonicida during the cold storage of seafood. Full article

Background: Intracellular bacteria frequently result in chronic and recurrent infections. MRSA is one of the most prevalent facultative intracellular bacteria in clinical infections. The drug resistance of MRSA and the difficulty of most antibiotics in entering cells result in a suboptimal clinical efficacy of antibiotics in the treatment of intracellular MRSA. Bacteriophages represent a promising alternative therapy in the context of the current antimicrobial resistance crisis. Nevertheless, the low efficiency of phage entry into cells and their rapid inactivation remain challenges in the treatment of intracellular MRSA using phages. The utilization of functionalized carriers for the delivery of phages into cells and their protection represents a feasible strategy. Methods: In this study, a new MRSA bacteriophage (vB_SauS_PHM) was isolated from hospital sewage, exhibiting the characteristics of short incubation period, large lytic amount, and good environmental tolerance. Subsequently, vB_SauS_PHM was encapsulated by TAT peptide-functionalized liposomes through microfluidic technology and size-exclusion chromatography (SEC), forming a phage delivery system, designated TAT-Lip@PHM. Results: The encapsulation rate of the phage by TAT-Lip@PHM was 20.3%, and the cell entry efficiency was ≥90% after 8 h. The 24 h eradication rate of 300 μg/mL TAT-Lip@PHM against intracellular MRSA was 94.05% (superior to the 21.24% and 44.90% of vB_SauS_PHM and Lip@PHM, respectively), while the mammalian cell activity was >85% after 24 h incubation. Conclusions: The TAT-Lip@PHM effectively delivered the phage into the cell and showed an excellent killing effect on intracellular MRSA with low cytotoxicity. This work provides a technical reference for the application of phages in the treatment of intracellular bacterial infection. Full article

Diarrhea is a common disease in sika deer. The causes of diarrhea in sika deer are complex and involve a variety of pathogens. Additionally, new virulent pathogens are continuously emerging, which poses a serious threat to deer’s health and particularly affects fawns’ survival rate. In the present study, feces samples were collected from fawns with diarrhea in Jilin Province, in the northeast of China. The viral communities were investigated using the metagenomic method. Viral metagenome data revealed that the viruses in the fecal samples were mainly from 21 families in 14 orders. The major viruses in high abundance were astrovirus, rotavirus, coronavirus, and bovine viral diarrhea virus. In addition, a large number of phages, which mainly belonged to the family Siphoviridae, were identified. Then, the known causative virus species were investigated via RT-qPCR. The results showed that the infection rates of bovine coronavirus, bovine rotavirus, and bovine viral diarrhea virus were 59.44%, 58.89%, and 21.67%, respectively, and mixed infections were commonly seen in the samples. A bovine rotavirus strain was successfully isolated from the positive samples. Whole-genome sequencing revealed that the genotype of the strain was G6-P[1]-I2-R2-C2-M2-A3-N2-T6-E2-H3, indicating the recombination of rotavirus. This study revealed the profiles and characteristics of viruses that cause sika deer diarrhea, which will be helpful for understanding diarrhea diseases in sika deer. Full article

Background: Aquaculture faces significant challenges due to bacterial infections, particularly Piscirickettsia salmonis, leading to extensive antibiotic use and raising concerns about antimicrobial resistance. In this context, bacteriophages and bacterial defense systems play a critical role in the evolutionary dynamics of P. salmonis. Objective. This study aimed to investigate the genomic landscape of prophage regions and antiphage defense systems in Piscirickettsia salmonis to better understand their co-evolutionary dynamics and explore their potential role in alternative disease control strategies for aquaculture. Methods: We analyzed 79 genomes of Piscirickettsia salmonis using bioinformatic tools to identify and characterize prophage regions and antiphage defense systems. Results: At the chromosomal level, 70% of the strains contained prophage regions, with a total of 92 identified regions, most of which were classified as intact. At the plasmid level, 75% of plasmids carried prophage regions, with a total of 426 identified regions, predominantly associated with Escherichia phage RCS47, Burkholderia phage Bcep176, and Enterobacteria phage mEp235. Prophage regions were enriched in transposases, head proteins, tail proteins, and phage-like proteins. The analysis of antiphage defense systems revealed that P. salmonis predominantly harbors dGTPase, AbidD, and SoFIC at the chromosomal level, whereas MazEF was the most frequent system in plasmids. A strong positive correlation was found between the number of prophage regions and defense systems in chromosomes (ρ = 0.72, p = 6.3 × 10⁻¹⁴), while a weaker correlation was observed in plasmids. These findings highlight the complex interplay between P. salmonis and its bacteriophages, with implications for disease control in aquaculture. Conclusions: Overall, these insights into the prophage and defense system dynamics provide potential avenues for developing alternative strategies to combat P. salmonis infections and reduce reliance on antibiotics in aquaculture systems. Full article

B Cell Maturation Antigen (BCMA) has gained considerable attention as a target in directed therapies for multiple myeloma (MM) treatment, via immunoglobulin-based bispecific T cell engagers or CAR T cell strategies. We describe the development of alternative, non-immunoglobulin BCMA-recognising affinity proteins, based on the small (58 aa) three-helix bundle affibody scaffold. A first selection campaign using a naïve affibody phage library resulted in the isolation of several BCMA-binding clones with different kinetic profiles. One clone showing the slowest dissociation kinetics was chosen as the template for the construction of two second-generation libraries. Characterization of output clones from selections using these libraries led to the identification of clone 1-E6, which demonstrated low nM affinity to BCMA and high thermal stability. Biosensor experiments showed that 1-E6 interfered with the binding of BCMA to both its natural ligand APRIL and to the clinically evaluated anti-BCMA monoclonal antibody belantamab, suggesting overlapping epitopes. A fluorescently labelled head-to-tail homodimer construct of 1-E6 showed specific binding to the BCMA⁺ MM.1s cell line in both flow cytometry and fluorescence microscopy. Taken together, the results suggest that the small anti-BCMA affibody 1-E6 could be an interesting alternative to antibody-based affinity units in the development of BCMA-targeted therapies and diagnostics. Full article

Background/Objetives: Nanobodies (VHH) have become an excellent tool for diagnosis, therapy, and research since VHH shows a high capability of recognizing and neutralizing antigens. VHHs are highly soluble and stable at high temperatures, and in the presence of chaotropic agents, they offer significant advantages over other biological therapeutic agents. This study aimed to identify and humanize VHH fragments with neutralizing potential against the influenza A/H1N1 virus. Methods: A library of VHH antibody fragments was produced by phage display technique against an inactivated influenza A/H1N1 vaccine. Three VHH sequences were selected and humanized. Specifically, the recognition capacity of the antibodies denominated 2-C10 and 2-C10H was confirmed by ELISA and western blot (WB), as well as their microneutralization capacity in a cellular model, suggesting their potential therapeutic use in patients infected with the influenza A/H1N1 virus. Molecular docking assays were used to support the mechanism of viral inhibition. Results: The VHHs 2-C10 and 2-C10H showed specific recognition of influenza A/H1N1 antigens by ELISA and Western Blot and demonstrated neutralizing activity in vitro. The optimal VHH, 2-C10H, showed 75% neutralization capacity at a concentration of 1.56 μg/mL against the A/H1N1 viral strain, potentially through the inactivation of hemagglutinin protein, a phenomenon supported by molecular docking assays. Conclusions: This study presents a strategic approach to identify VHH candidates that may be useful for diagnosing and potentially treating patients already infected by the A/H1N1 virus, as it may reduce the severity of their symptoms. Full article

New chiral salen-based organic salts were synthesised and evaluated for their antibacterial activity against Serratia fonticola, Escherichia coli, and Enterobacter cloacae. Their structures and physicochemical properties, namely their specific rotation, melting point, thermal stability, and antibacterial efficacy, including minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), were determined. The synergy between chiral organic salts and bacteriophages was also demonstrated. [(RR)Sal.5C1.PhIM][Cl], [(RR)Sal.5C1.PhIM][BF₄], and [(RR)Sal.5C1.Pyr][OTf] had the lowest MIC values (from 500 mg mL⁻¹ for S. fonticola strain KKP 3685 to 2000 mg mL⁻¹ for E. cloacae strain KKP 3692), while the highest MICs (>4000 mg mL⁻¹) were observed for [(RR)Sal.5C1.Pyr][OTf] and [(RR)Sal.5C1.PhIM][NTf₂] against E. cloacae strain KKP 3692. The impact of the tested compounds on phage activity was strain-specific. A synergistic effect of [(RR)Sal.5C1.PhIM][BF₄] at 0.5 mg mL⁻¹ in microcultures with Escherichia phage KKP 3710 (at MOI of 10 and 100) on the complete inhibition of the growth of E. coli strain KKP 3688 was observed. The combination of [(RR)Sal.5C1.PhIM])][OTf] at 1 mg mL⁻¹ with the addition of phages (at each MOI) and at 0.5 mg mL⁻¹ and MOI = 100 completely inhibited the growth of E. coli strain KKP 3688. Moreover, [(RR)Sal.5C1.PhIM])][OTf] at 1 mg mL⁻¹ and 0.5 mg mL⁻¹, when combined with Enterobacter phage KKP 3716, inhibited the growth of E. cloacae strain KKP 3692 slightly more effectively than the compound alone at the same concentrations. These results suggest that combining our antibacterial agents can reduce chemical compound concentrations, with effects depending on the bacteria. Full article