Search Results (66)

β-Lytic protease (Blp) and protease L5 are enzymes from Lysobacter bacteria with distinct proteolytic and bacteriolytic activities. To characterize their substrate specificity, we employed liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis following hydrolysis of fractionated protein mixtures. Heatmaps and sequence logos revealed a pronounced specificity of Blp towards glycine and lysine residues, while L5 preferentially cleaved non-polar residues such as methionine, phenylalanine, and leucine. Notably, proline was frequently observed at the P2 position in L5 substrates. Comparative analysis with trypsin revealed that L5 generated significantly shorter peptides, whereas Blp produced fragments similar in length to tryptic peptides. These findings indicate different cleavage preferences and suggest potential applications for these enzymes in proteomic analysis. Full article

Biodegradable poly(lactic acid) (PLA)/poly(ε-caprolactone) (PCL) composite films were prepared with a compatibilizer (tributyl citrate, TBC) using a solvent casting method. Incorporation of 5% TBC (w/v, of PCL weight) improved tensile strength and elongation at break (21.93 ± 2.33 MPa and 21.02 ± 1.54%, respectively) and reduced water vapor permeability (from 0.12 ± 0.01 to 0.098 ± 0.01 g·mm·m²·h·kPa), indicating improved compatibility between PLA and PCL. Staphylococcus aureus phage PBSA08 demonstrated rapid and persistent bacteriolytic activity for up to 24 h, suggesting its potential as a promising antibacterial biological agent. To impart antibacterial properties to the developed PLA/PCL/TBC film, PBSA08 was loaded into sodium alginate (SA) and coated on the film surface. The optimal composition was 3% (w/v) SA and 3% (w/v) glycerol, which exhibited suitable dynamic behavior as a coating solution and excellent adhesion to the film surface. The phage-coated antibacterial films demonstrated progressive and significant inhibition against S. aureus starting from 10 to 24 h, with controlled phage-release properties. Overall, the developed active film might exert sustained and remarkable antibacterial effects through controlled release of biological agents (phage) under realistic packaging conditions. Full article

Secreted bacteriolytic proteases L1 and L5 of the Gram-negative bacterium Lysobacter capsici XL hydrolyze peptide bridges in bacterial peptidoglycans. Such specificity of action determines the prospects of these enzymes for medicine with the view of creating new antimicrobial drugs to combat antibiotic-resistant strains of pathogens. This research concerns the development of successful expression systems for producing active enzymes L1 and L5 in sufficient amounts for comprehensive studies. Based on L. capsici XL strains with deletions in the alpA (enzyme L1) and alpB (enzyme L5) genes and the constructed expression vectors pBBR1-MCS5 P_T5–alpA and pBBR1-MCS5 P_T5–alpB, we obtained expression strains L. capsici P_T5–alpA and L. capsici P_T5–alpB, respectively. The yields of enzymes L1 and L5 in the developed strains increased by 4 and 137 times, respectively, as compared to the wild-type strain. The cultivation of the expression strains was successfully scaled up under non-selective conditions in a 10-L bioreactor. After fermentation, the yields of enzymes L1 and L5 were 35.48 mg/L and 57.11 mg/L, respectively. The developed homologous expression systems of bacteriolytic proteases L1 and L5 have biotechnological value as compared to those obtained by us earlier based on heterologous expression systems, which have lower yields and labor-intensive purification schemes. Full article

Streptococcus pneumoniae (pneumococcus) is a significant human pathogen responsible for a range of diseases from mild infections to invasive pneumococcal diseases, particularly affecting children, the elderly, and immunocompromised individuals. Despite pneumococcal conjugate vaccines having reduced disease incidence, challenges persist due to serotype diversity, vaccine coverage gaps, and antibiotic resistance. This review highlights the role of LytA, a key autolysin (N-acetylmuramoyl-l-alanine amidase), in pneumococcal biology. LytA regulates autolysis, contributes to inflammation, and biofilm formation, and impairs bacterial clearance. It also modulates complement activation, aiding immune evasion. LytA expression is influenced by environmental signals and genetic regulation and is tied to competence for genetic transformation, which is an important virulence trait, particularly in meningitis. With the increase in antibiotic resistance, LytA has emerged as a potential therapeutic target. Current research explores its use in bacteriolytic therapies, vaccine development, and synergistic antibiotic strategies. Various compounds, including synthetic peptides, plant extracts, and small molecules, have been investigated for their ability to trigger LytA-mediated bacterial lysis. Future directions include the development of novel anti-pneumococcal interventions leveraging LytA’s properties while overcoming vaccine efficacy and resistance-related challenges. Human challenge models and animal studies continue to deepen our understanding of pneumococcal pathogenesis and potential treatment strategies. Full article

Background: A key component in modern vaccine development is the adjuvant, which enhances and/or modulates the antigen-specific immune response. In recent years, nanoparticle (NP)-based adjuvants have attracted much research attention owing to their ability to enhance vaccine potency. Nonetheless, how the selection of different antigens influences the overall vaccine efficacy when combined with the same nanoparticle adjuvant is less discussed, which is important for practical applications. Methods: Non-toxic mutants of exotoxin Hla (rHlaH35L) and cell-wall-anchored protein SpA(rSpam) were covalently conjugated to Poly(lactic-co-glycolic acid)-polyethylene glycol (PLGA-PEG) 25% NPs (25% NPs) as antigens to prepare nanovaccines. Antibody titers, cytokine secretion levels, and the antibody bacteriolytic capacity were tested to investigate immune activation. To evaluate the protective efficacy of the nanovaccine, immunized mice were challenged with S. aureus ATCC 25923 at three different lethal doses: 1 × LD₁₀₀, 2 × LD₁₀₀, and 4 × LD₁₀₀. Results: We showed that 25% NP-rHlaH35L nanovaccines were associated with more efficient humoral, cellular, and innate immune responses and protection potency compared with 25% NP-rSpam. Moreover, the overall vaccine potency of 25% NP-rHlaH35L was even better than the combination vaccination of both 25% NP-rHlaH35L and 25% NP-rSpam. In comparison to the clinically used aluminum (alum) adjuvant, the 25% NP adjuvants were found to stimulate humoral and cellular immune responses efficiently, irrespective of the antigen type. For antigens, either exotoxins or cell-wall-anchored proteins, the 25% NP-based vaccines show excellent protection for mice from S. aureus infection with survival rates of 100% after lethal challenge, which is significantly superior to the clinically used alum adjuvant. Moreover, due to the superior immune response elicited by 25% NP-rHlaH35L, the animals inoculated with this formulation survived even after two times the lethal dose of S. aureus administration. Conclusions: We demonstrated that the type of antigen plays a key role in determining the overall vaccine efficacy in the immune system when different kinds of antigens are conjugated with a specific nanoparticle adjuvant, paving a new way for vaccine design based on 25% NP adjuvants with enhanced potency and reduced side effects. Full article

Background: Cattle production is a cornerstone of U.S. agriculture but faces increasing pressure to balance profitability with environmental sustainability. Optimizing the ruminal microbiome to enhance feed efficiency could help address both challenges. Members of the Streptococcus bovis/Streptococcus equinus complex (SBSEC) are key contributors to ruminal acidosis and related digestive disorders due to their role in carbohydrate fermentation and lactic acid production. Bacteriophages targeting this bacterial group present a promising approach to mitigate this problem with high precision and without promoting the spread of antibiotic resistance. Methods: A collection of SBSEC-targeting bacteriophages were isolated from cattle rumen fluid and feces and further characterized. Characterization included host-range evaluation, whole genome sequencing, and growth inhibition assessment via optical density measurements. Selected bacteriophages underwent training to enhance infectivity. Results: Eleven lytic and one lysogenic phage were isolated. Several phages demonstrated sustained bacterial growth suppression, showing efficacy against SBSEC bacteria from diverse sources despite narrow host ranges. Co-evolutionary training was done in a subset of phages to improve bacteriolytic activity but had an inconsistent effect on the ability of phages to inhibit the growth of their naïve host. Genomic sequencing and phylogenetic analysis revealed uniqueness and clustering into three distinct groups that matched phenotypic characteristics. Conclusions: This study demonstrates the potential of bacteriophages as precise biological control agents, with successful isolation and enhancement of phages targeting SBSEC bacteria. Eleven lytic genome-sequenced phages show promise for development as cattle feed additives, though further research is needed to optimize their application in agricultural settings. Full article

Bacterial antibiotic resistance is an important challenge that the healthcare system is continually battling and a major problem in the treatment of musculoskeletal infections such as periprosthetic joint infections. Current methods to identify infectious microbes and define susceptibility to antibiotics require two to ten days from isolation to the establishment of an antibiogram. This slow process limits advances in antimicrobial drug discovery and, in the clinical context, delays the delivery of targeted treatments, with potentially devastating outcomes for patients. With this in mind, we strived to establish a quicker and more sensitive method to deliver antibiotic susceptibility profiles of clinically relevant microbes using Scattered Light Integrated Collector (SLIC) technology. We established antibiotic panels to obtain an approximate identification of a wide variety of microbes linked to periprosthetic joint infections and determine their susceptibility to antibiotics. We challenged microbes isolated from patients with our tailored antibiotic panels and found that SLIC detects perturbations in bacterial growth accurately and reproducibly within minutes of culture. Indeed, we could show that SLIC can be used to measure the dose-dependent inhibitory or bacteriolytic activity of broad classes of antibiotics. Our panel design enabled us to establish a profile similar to an antibiogram for the tested bacteria within 90 min. Our method can provide information on the class of bacteria tested and potential treatment avenues in parallel. Our proof-of-principle experiments using isolated clinical strains of bacteria demonstrate that SLIC, together with our specifically designed antibiotic panels, could be used to rapidly provide information on the identity of an infecting microbe, such as those associated with periprosthetic joint infections, and guide physicians to prescribe targeted antibiotic treatment early-on. The constant emergence of resistant strains of bacteria pushes the pharmaceutical industry to develop further effective drugs. Our optimized method could significantly accelerate this work by characterizing the efficacy of new classes of compounds against bacterial viability within minutes, a timeframe far shorter than the current standards. Full article

Shiga toxin-producing Escherichia coli (STEC) infections have increased in humans, animals, and the food industry, with ready-to-eat (RTE) food products being particularly susceptible to contamination. The prevalence of multidrug-resistant strains has rendered the current control strategies insufficient to effectively control STEC infections. Herein, we characterized the newly isolated STEC phage vB_ESM-pEJ01, a polyvalent phage capable of infecting Escherichia and Salmonella species, and assessed its efficacy in reducing STEC in vitro and food matrices. The phage, belonging to the Tevenvirinae, exhibits effective bacteriolytic activity, a short latent period, large burst size, and stability under a broad pH range and moderate temperatures. Moreover, the phage demonstrated strong anti-biofilm efficacy even at low concentrations. Genomic analysis revealed that the phage was similar to the well-characterized RB49 phage (T4-like phage) but possesses distinct host-specificity-related genes that potentially contribute to its extensive host range. The efficacy of phage vB_ESM-pEJ01 was evaluated in artificially STEC-inoculated green juice samples, where it significantly reduced STEC and the abundance of Shiga toxin-producing genes at 4 and 25 °C. Therefore, these results suggest that the polyvalent phage vB_ESM-pEJ01 is a promising biocontrol agent for foodborne pathogens in RTE foods such as fresh juices. Full article

The increasing number of antibiotic-resistant bacterial pathogens is a serious problem in medicine. Endolysins are bacteriolytic enzymes of bacteriophages, and a promising group of enzymes with antibacterial properties. Endolysins of bacteriophages infecting Gram-positive bacteria have a modular domain organization. This feature can be used to design enzymes with new or improved properties by modifying or shuffling individual domains. This work is a detailed analysis 1of the diversity of endolysin domains found in bacteriophages infecting bacilli. During the course of the work, a database of endolysins of such bacteriophages was created, and their domain structures were analyzed using the NCBI database, RASTtk, BLASTp, HHpred, and InterPro programs. A phylogenetic analysis of endolysins was performed using MEGA X. In 438 phage genomes, 454 genes of endolysins were found. In the endolysin sequences found, eight different types of catalytic domains and seven types of cell wall binding domains were identified. The analysis showed that many types of endolysin domains have not yet been characterized experimentally. Studies of the properties of such domains will help to reveal the potential of endolysins for the creation of new antibacterial agents. Full article

Enterotoxigenic Escherichia coli (ETEC) is a major pathogen causing diarrhea in humans and animals, with increasing antimicrobial resistance posing a growing challenge in recent years. Lytic bacteriophages (phages) offer a targeted and environmentally sustainable approach to combating bacterial infections, particularly in eliminating drug-resistant strains. In this study, ETEC strains were utilized as indicators, and a stable, high-efficiency phage, designated vB_EcoM_JE01 (JE01), was isolated from pig farm manure. The genome of JE01 was a dsDNA molecule, measuring 168.9 kb, and a transmission electron microscope revealed its characteristic T4-like Myoviridae morphology. JE01 effectively lysed multi-drug-resistant ETEC isolates. Stability assays demonstrated that JE01 retained its activity across a temperature range of 20 °C to 50 °C and a pH range of 3–11, showing resilience to ultraviolet radiation and chloroform exposure. Furthermore, JE01 effectively suppressed ETEC adhesion to porcine intestinal epithelial cells (IPEC-J2), mitigating the inflammatory response triggered by ETEC. To investigate the in vivo antibacterial efficacy of phage JE01 preparations, a diarrhea model was established using germ-free mice infected with a drug-resistant ETEC strain. The findings indicated that 12 h post-ETEC inoculation, intragastric administration of phage JE01 significantly reduced mortality, alleviated gastrointestinal lesions, decreased ETEC colonization in the jejunum, and suppressed the expression of the cytokines IL-6 and IL-8. These results demonstrate a therapeutic benefit of JE01 in treating ETEC-induced diarrhea in mice. Additionally, a fluorescent phage incorporating red fluorescent protein (RFP) was engineered, and the pharmacokinetics of phage therapy were preliminarily assessed through intestinal fluorescence imaging in mice. The results showed that the phage localized to ETEC in the jejunum rapidly, within 45 min. Moreover, the pharmacokinetics of the phage were markedly slowed in the presence of its bacterial target in the gut, suggesting sustained bacteriolytic activity in the ETEC-infected intestine. In conclusion, this study establishes a foundation for the development of phage-based therapies against ETEC. Full article

Outer membrane vesicles (OMVs) are extracellular structures, ranging in size from 10 to 300 nm, produced by Gram-negative bacteria. They can be incorporated into the outer membrane of a recipient’s cells, which may enable the transfer of substances with lytic properties. Due to the scarce information regarding the OMVs produced by Proteus mirabilis, the aim of this study was to test the blebbing abilities of the clinical P. mirabilis O77 and O78 strains and to determine the blebs’ interactions with bacterial cells, including their possible bactericidal activities. The production of OMVs was visualised by Transmission electron microscopy (TEM). The presence of OMVs in the obtained samples as well as the phenomenon of OMV fusion to recipient cells were confirmed by Enzyme-Linked ImmunoSorbent Assay (ELISA) and Western blotting assays. The bacteriolytic activity of the OMVs was examined against P. mirabilis clinical strains and reference Staphylococcus aureus and Escherichia coli strains. It was shown that each of the two tested P. mirabilis strains could produce OMVs which were able to fuse into the cells of the other strain. The lytic properties of the O78 OMVs against another P. mirabilis O78 strain were also demonstrated. This promising result may help in the future to better understand the mechanisms of the pathogenesis and to treat the infections caused by P. mirabilis. Full article

Objectives: The aim of this study was to prepare monoclonal antibodies (mAbs) that broadly target Acinetobacter baumannii and protect against infection by multi-drug-resistant (MDR) A. baumannii from different sources. Methods: mAb 8E6 and mAb 1B5 were prepared by sequentially immunizing mice with a sublethal inoculation of three heterogeneous serotypes of pan-drug-resistant (PDR) A. baumannii, ST-208, ST-195, and ST-229. Results: The cross-recognition of heterogeneous bacteria (n = 13) by two mAbs and potential targets was verified, and the in vitro antibacterial efficacy of mAbs was assessed. The median killing rate of mAb 8E6 against A. baumannii in the presence of complement and dHL-60 cells was found to be 61.51%, while that of mAb 1B5 was 41.96%. When only dHL-60 cells were present, the killing rate of mAb 8E6 was 65.73%, while that of mAb 1B5 was 69.93%. We found that mAb 8E6 and mAb 1B5 broadly targeted MDR A. baumannii on the ATP synthase complex and were equipped with an antibacterial killing ability by enhancing the innate immune bacteriolytic effect of ST-208 and ST-195 strains. Both monoclonal antibodies were validated to protect against respiratory infection at 4 and 24 h via enhancing the release of innate immune substances and inflammatory cytokines, effectively shortening the disease period in mice. Conclusions: mAb 8E6 and mAb 1B5 significantly enhanced the opsonization process of phagocytosis against A. baumannii strains prevalent in southern China by targeting ATP synthase antigens thereof, resulting in protective effects in mice. Full article

Xanthomonas oryzae pv. oryzae (Xoo) is a significant bacterial pathogen responsible for outbreaks of bacterial leaf blight in rice, posing a major threat to rice cultivation worldwide. Effective management of this pathogen is crucial for ensuring rice yield and food security. In this study, we identified and characterized a novel Xoo phage, ZP3, isolated from diseased rice leaves in Zhejiang, China, which may offer new insights into biocontrol strategies against Xoo and contribute to the development of innovative approaches to combat bacterial leaf blight. Transmission electron microscopy indicated that ZP3 had a short, non-contractile tail. Genome sequencing and bioinformatic analysis showed that ZP3 had a double-stranded DNA genome with a length of 44,713 bp, a G + C content of 52.2%, and 59 predicted genes, which was similar to other OP1-type Xoo phages belonging to the genus Xipdecavirus. ZP3’s endolysin LysZP was further studied for its bacteriolytic action, and the N-terminal transmembrane domain of LysZP is suggested to be a signal–arrest–release sequence that mediates the translocation of LysZP to the periplasm. Our study contributes to the understanding of phage–Xoo interactions and suggests that phage ZP3 and its endolysin LysZP could be developed into biocontrol agents against this phytopathogen. Full article

Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic bacteria frequently linked to burn wound infections. These bacteria can grow as biofilms, which increases their level of drug resistance to current antibiotics. The purpose of the present study is to analyze the effect of biofilm formation, phage and phage cocktail action on single species and dual species biofilms I, e the coexistence of Gram positive (S. aureus) and Gram negative (P. aeruginosa). To this scenario, we employed multi-drug resistant bacteria (P. aeruginosa and S. aureus at 10⁹ CFU/µL) biofilm as single and in combination of both Gram-positive and Gram-negative bacterial biofilms of 24 h grown with respective phage (10⁹ PFU/µL) and phage cocktail (10⁹ PFU/µL) at 4 h of incubation under static conditions. The bacteriolytic activity of phages vB_SAnS_SADP1 and vB_PAnP_PADP4 on 24-h-old biofilms of P. aeruginosa (0.761 ± 0.031) and S. aureus (0.856 ± 0.055), both alone and in combination (0.67 ± 0.02), was the focus of this investigation. The structural organization of biofilms in single- or dual-species combinations under in vitro conditions was validated by scanning and confocal laser scanning microscopy investigations. After 24 h of incubation, single-species biofilms are denser and more resilient whereas dual species biofilms are more loosely associated. Loose association of dual-species biofilm under scanning electron microscopic images at the same conditions, indicated the interspecies -competition of the Gram-positive and Gram-negative bacteria and dual-species biofilms (0.67 ± 0.02) have weak associations and are readily impacted by phage and a phage cocktail (0.16 ± 0.02). Dual-species biofilms were more readily impacted in in vitro settings. Full article

Bacteria are capable of remarkable adaptations to their environment, including undesirable bacterial resistance to antibacterial agents. One of the most serious cases is an infection caused by multidrug-resistant Staphylococcus aureus, which has unfortunately also spread outside hospitals. Therefore, the development of new effective antibacterial agents is extremely important to solve the increasing problem of bacterial resistance. The bacteriolytic enzyme autolysin E (AtlE) is a promising new drug target as it plays a key role in the degradation of peptidoglycan in the bacterial cell wall. Consequently, disruption of function can have an immense impact on bacterial growth and survival. An in silico and in vitro evaluation of iminosugar derivatives as potent inhibitors of S. aureus (AtlE) was performed. Three promising hit compounds (1, 3 and 8) were identified as AtlE binders in the micromolar range as measured by surface plasmon resonance. The most potent compound among the SPR response curve hits was 1, with a K_D of 19 μM. The K_D value for compound 8 was 88 μM, while compound 3 had a K_D value of 410 μM. Full article