Applied Microbiology and Biotechnology最新文献

Pseudomonas aeruginosa is a major contaminant in cosmetics, posing significant health risks to consumers. These bacteria form biofilms that protect them from disinfectants commonly used in the cosmetic industry. This study aimed to assess the impact of disinfectants, cleaners, and sanitizers on the metabolic activity and biofilm formation of P. aeruginosa isolated from the production line in a cosmetic manufacturer. In vitro experiments were conducted using silicone, Teflon, ethylene propylene diene monomer and acid-resistant steel surfaces, which are commonly used materials in cosmetic production line equipment. Spectrophotometric methods were used to evaluate biofilm formation and metabolic activity, while different imaging techniques (SEM, EFM, OCT) were employed to visualize biofilm structure directly on examined surfaces. The results showed that hypochlorous acid is the most effective disinfectant in inhibiting biofilm formation. Hypochlorous acid significantly reduced the metabolic activity of P. aeruginosa, particularly in biofilms forming on the surface made of stainless steel. Additionally, the study validated the feasibility of implementing a sterilization method using hypochlorous acid directly in industrial conditions for production line sterilization. Results showed a significant reduction in contamination levels in water passing through the installation, from an uncountable level to below 0.1 × 10¹ CFU mL⁻¹. In conclusion, the effectiveness of disinfectants in preventing biofilm formation and metabolic activity is dependent on their composition and the type of surface on which the biofilms form. Hypochlorous acid proves to be an effective disinfectant for combating bacterial biofilms in the cosmetic industry.

Insect cells are one of the uprising expression systems in the biopharmaceutical industry to produce vaccines and gene therapy vectors, but cell line development has been limited by the lack of established genetic engineering tools and genomic characterization. CRISPR-Cas9 has arisen as a powerful tool for gene editing but has seen little application in insect cells. In this work, a gene editing pipeline for the delivery of a ribonucleoprotein (RNP) complex comprised of a guide RNA and the enzyme Cas9 to insect Sf9 cells was implemented and then applied to knockout caspase initiator Sf-Dronc, aiming at alleviating cell apoptosis during an infection process. The resulting engineered cell lines were characterized as per their phenotype and production of three different product modalities. Utilizing the established workflow, a knockout rate of 68% was achieved with the implemented protocol (vs. the 12% presumed efficiency of a previously reported system) when targeting the fdl gene. When applied to Sf-Dronc, mutants containing deletions in several alleles of the host genome were identified and confirmed by next-generation sequencing. Generated clones exhibited higher apoptosis resistance and delayed onset of cell viability drop following infection with baculovirus. While Sf-Dronc deletion was shown to have negligible impact on the production of rAAV and PfRipr5, production of iVLPS showed an > twofold increase over wild-type Sf9. Overall, this study showcases the successful implementation of an efficient CRISPR-Cas9 pipeline, further leveraging the usage of genetic engineering in insect Sf9 cells towards the development of enhanced cell hosts for biopharmaceutical production.

• Implementation of an efficient CRISPR-Cas9 RNP complex delivery strategy to insect cells.

• Establishment of the genome editing pipeline demonstrated through Sf-Dronc knockout, resulting in increased apoptosis resistance and delayed loss of viability upon baculovirus infection.

• Sf-Dronc deletion led to over a twofold increase in the production of influenza VLPs compared to wild-type Sf9 cells.

Microbial transglutaminase (mTG) is an enzyme produced by actinomycetes, predominantly by filamentous bacteria belonging to the genus Streptomyces. These microorganisms are the best-studied and most microbiologically exploited natural producers of TG. It catalyzes the formation of isopeptide bonds between glutamine and lysine residues in proteins, which alters the protein’s structure and functionality. This enzymatic activity is widely applied in the food industry, where it is particularly useful in modifying the texture, binding properties, and overall quality of protein products. The primary application of mTG in food production lies in its ability to mimic the functional properties of gluten, making it valuable in the creation of gluten-free products. By facilitating the binding of various ingredients such as starches and plant proteins, mTG helps produce products with similar texture and elasticity to those made with gluten, which is crucial for individuals with celiac disease or gluten intolerance. Beyond gluten-free applications, mTG is also employed in the production of meat substitutes, where it enhances the texture and cohesion of plant-based ingredients, as well as in bakery and confectionery products. Additionally, mTG is utilized in the development of organic materials, such as microcapsules and enzymatic carriers, owing to its calcium-independent activity, broad substrate specificity, and high stability across a wide range of pH and temperatures. The properties of this enzyme can be successfully used in the biotechnology industry. Looking forward, mTG is expected to play a significant role in the expansion of functional foods, plant-based diets, and advanced biomaterials. However, there are growing concerns about its safety, particularly regarding potential immunogenic reactions and its long-term impact on consumer health. To mitigate these risks, further research on the biological effects of mTG is essential, especially in the context of its microbial origin, molecular structure, and interaction with human proteins. Additionally, stricter regulations and clear labeling of products containing mTG will be necessary to ensure consumer safety and confidence in its widespread use.

• Microbial transglutaminase is produced by actinomycetes via fermentation.

• mTG is a microbial enzyme that modifies protein structure and functionality.

• mTG is key to future functional foods.

• Labeling of mTG in foods is vital for consumer trust and safety.

• Enzyme mTG boosts cohesion in meat substitutes and bakery goods.

This study aimed to compare the efficacy of inhaled Clostridium butyricum delivered via oxygen-driven nebulization with oral C. butyricum capsules in preventing drug-resistant bacterial pneumonia, using clinical and microbiological parameters as indicators. A total of 310 patients were randomly assigned to experimental group and control group. In the experimental group, each participant inhaled C. butyricum via oxygen-driven nebulization and orally took a placebo capsule containing glucose powder. In the control group, each participant inhaled sterile water via oxygen-driven nebulization and orally took a C. butyricum capsule daily. Key outcomes included body temperature, white blood cell (WBC) count, high-sensitivity C-reactive protein (hs-CRP) level, chest X ray findings, tracheal tube secretion status, and sputum cultures for Pseudomonas aeruginosa, ESBL-producing Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae. The incidence of pneumonia was 46.45% (72/155) in the control group, compared to only 1.97% (3/152) in the experimental group (relative risk = 23.54, 95%CI: 7.58–73.08). The experiment group demonstrated significant improvements in clinical parameters, including reduced body temperature, lower WBC counts, and decreased hs-CRP level. Chest X-ray findings and tracheal tube secretion status also improved more markedly in the experiment group. Microbiological analysis revealed a significant reduction in the colonization of pathogenic bacteria in sputum cultures from the experiment group. In conclusion, inhaled C. butyricum delivered via oxygen-driven nebulization appears to be more effective than oral C. butyricum capsules in preventing drug-resistant bacterial pneumonia. These findings suggest that direct delivery of C. butyricum to the respiratory tract via oxygen-driven nebulization may enhance its anti-inflammatory and antimicrobial effects, offering a promising strategy for prevention of drug-resistant bacterial pneumonia.

• Oxygen-driven nebulization of C. butyricum improved the clinical parameters.

• Oxygen-driven nebulization of C. butyricum improved tracheal tube secretion status.

• Oxygen-driven nebulization of C. butyricum reduced pathogenic bacteria production.

Conventional immunoassays such as ELISA are widely used in serological testing, yet their reliance on multi-step workflows and labeled reagents limits diagnostic scalability and speed. Bioluminescence-based biosensors are attractive alternatives that offer high sensitivity, operational simplicity, and cost efficiency for detecting diverse analytes. Here, we present a broadly compatible bioluminescent biosensor for antibody detection based on analyte-mediated reconstitution of split-Nanoluciferase (NanoLuc). The platform utilizes engineered bifunctional probes, comprising the protein G C2 domain fused to split-NanoLuc subunits (LgBiT and SmBiT), which serve dual functions in antibody binding and signal generation. Upon immunocomplex formation with multi-epitope antigens, the probes colocalize LgBiT and SmBiT, reconstituting NanoLuc activity and producing a quantifiable bioluminescent signal. We implemented this Fc-binding split-NanoLuc complementation assay to detect antibodies against African swine fever virus (ASFV). The optimized system showed high sensitivity, a wide linear dynamic range, and no cross-reactivity with sera positive for other common swine viruses. Clinical validation exhibited 97.11% agreement with a commercial ELISA kit, confirming its practicality and reliability. Furthermore, the sensor platform was seamlessly adapted to detect antibodies against SARS-CoV-2 and Chikungunya virus (CHIKV) without requiring additional molecular design or reconfiguration, highlighting its inherent versatility. By leveraging the broadly applicable Fc-binding capacity of protein G and the intrinsic modularity of split-NanoLuc, this strategy streamlines assay development and eliminates the need for species-specific secondary antibodies. Together, our findings demonstrate a proof-of-concept biosensor approach that could be further developed into a useful tool for rapid antibody detection in emerging infectious disease settings.

• Fungal biological processes alter upon illumination, also under the microscope

• Red shifted fluorescent protein toolboxes decrease interference by illumination

• Innovations like two-photon, lightsheet, and near IR microscopy reduce phototoxicity

Beak and feather disease virus (BFDV) poses a significant threat to avian biodiversity and global aviculture. Reliable serodiagnostic tools are critical for assessing host immune status and guiding disease management. The haemagglutination inhibition (HI) assay, although historically regarded as the gold standard, is limited by technical complexity and its reliance on seropositive Galah erythrocytes, restricting broader application. This study describes the development and validation of a recombinant BFDV capsid protein-based indirect enzyme-linked immunosorbent assay (iELISA) for detecting anti-BFDV antibodies using dried blood spots from multiple psittacine species. The assay demonstrated high sensitivity and strong analytical performance, employing virus-like particles (VLPs) recombinantly expressed in Escherichia coli. Optimisation of the diagnostic cut-off by two-graph receiver operating characteristic (TG-ROC) analysis established an OD threshold of 1.73, achieving 96.5% sensitivity with a Youden’s index of 0.74. Discriminative capacity was further supported by receiver operating characteristic (ROC) analysis, yielding an area under the curve (AUC) of 0.896. Agreement with the HI assay was very strong (Gwet’s Agreement Coefficient 1= 0.843). This iELISA represents a scalable and universal serodiagnostic tool, supporting clinical diagnosis, enabling large-scale epidemiological investigations, and advancing conservation-focused BFDV surveillance.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, a major infectious disease causing substantial global morbidity and mortality. The growing incidence of antibiotic-resistant strains highlights the urgency of identifying alternative strategies capable of targeting persistent and resistant forms of Mtb. This study evaluates the effectiveness of photodynamic inactivation (PDI) using visible light within the 400–420 nm range against a clinical Mtb isolate. The isolate was subjected to controlled LED exposure under biosafety level-3 conditions, with viability assessed via colony-forming unit counts. Results showed a significant reduction in Mtb viability, with > 99% (over 2-log) reduction observed after exposure to the tested visible light spectrum. These results provide a strong rationale for the clinical translation of visible light-based disinfection strategies, by indicating that visible light photoinactivation provides a practical, non-chemical alternative to conventional disinfectants and UV-based methods, reducing concerns about toxicity and operational limitations. This approach holds particular promise for healthcare environments, where it could contribute to reducing the environmental persistence of Mtb and, consequently, the risk of transmission. These results underscore the need for further investigation into light-based technologies as complementary tools to existing infection control measures, particularly in high-risk or inadequately sanitized settings.

• Visible light (Soret Band 400–420 nm) significantly reduces Mycobacterium tuberculosis (Mtb) viability

• No photosensitizers are used in the photodynamic inactivation (PDI) process 

• LED-based visible light may help limit Mtb spread in clinical environments 

Bioleaching is an established process for sulfidic ores and is increasingly applied to the recycling of industrial residues. However, unlike ores, many residues like sludge contain inhibitory elements, among which fluoride poses a major challenge due to its toxicity toward acidophilic microorganisms even at low concentrations. This study systematically investigated fluoride tolerance in pure and mixed cultures of various acidophilic sulfur- and iron-oxidizing bacteria commonly used for bioleaching, including Acidithiobacillus spp., Leptospirillum spp., and Sulfobacillus thermosulfidooxidans. Fluoride toxicity was found to be substrate-dependent. During sulfur oxidation, A. thiooxidans displayed the highest fluoride tolerance (0.5 mM F⁻), whereas S. thermosulfidooxidans showed complete inhibition. In contrast, iron-oxidizing bacteria demonstrated increased fluoride tolerance, with S. thermosulfidooxidans remaining active at 1.5 mM F⁻ when grown on ferrous iron. Mixed cultures showed enhanced fluoride tolerance during sulfur oxidation but reduced tolerance during iron oxidation. pH was identified as a critical factor influencing fluoride toxicity due to increased formation of undissociated HF at low pH. To mitigate fluoride inhibition, fluoride complexation with ferric iron or aluminum was evaluated. For A. ferrooxidans, iron oxidation resumed at Fe³⁺:F⁻ ratios of 7.5:1, while other cultures required ratios of at least 10:1. Aluminum complexation required Al:F⁻ ratios between 1:1 and 2:1, depending on the culture and growth conditions. Overall, fluoride inhibition during bioleaching is influenced by multiple factors, including pH, ferric iron concentration, and the fluoride dissolution rate. Early addition of aluminum is recommended to prevent microbial inhibition and ensure stable bioleaching performance.

• Higher fluoride tolerance was observed during iron oxidation.

• S. thermosulfidooxidans remained active up to 1.5 mM F⁻.

• Fluoride toxicity is strongly pH dependent due to increased HF formation at low pH.

• Effective fluoride complexation requires higher Fe³⁺:F⁻ ratios (> 7.5:1) than Al3⁺:F⁻ ratios (> 1:1)

Fungal immunomodulatory proteins (FIPs) are considered potential therapeutic candidates due to their immunomodulatory and anti-cancer activities. Meanwhile, they are capable of agglutinating mammalian red blood cells in a manner similar to lectins. Herein, GST-tagged rLZ-8, the first identified FIP in Ganoderma lucidum, was employed as bait to pull down its target proteins from mouse red blood cells. An important membrane protein, Band 3, was found to interact with rLZ-8. The result was confirmed by detecting the interactions of rLZ-8 with mBand 3 and hBand 3 heterologously expressed in HEK293 cells. Moreover, the interaction specifically required the sole N-glycosylation of Band 3 at its extracellular loop (between the seventh and eighth transmembrane helices), as evidenced by diminished binding both to the wild-type hBand 3 pretreated with the N-glycan trimming enzyme PNGaseF in vitro or with the mannosidase inhibitor (N-glycosylation processing enzyme) kifunensine (KIF) in vivo and to the N642D-hBand 3 (N660D-mBand 3) mutant. Consistently, both the binding of LZ-8 to Band 3 and its hemagglutinating property were inhibited by the highly glycosylated protein bovine mucin. These results suggest that the hemagglutinating ability of LZ-8 may be attributed to its binding to the N-glycosylated proteins, including Band 3, on the erythrocyte surface. Furthermore, supporting the clinical safety profile of FIPs, stopped-flow analysis performed on human red blood cell ghosts demonstrated that the Cl⁻/HCO₃⁻ exchange activity of Band 3 remained unaltered upon binding to LZ-8.

Siderophores are iron-chelating secondary metabolites that can effectively control fungal diseases in several agronomically important crops. In the present study, the bacterium Burkholderia contaminans MSR2 was found to profusely accumulate siderophores when grown under iron-limiting conditions. Therefore, subsequent experimental procedures were performed to determine whether the siderophore-rich supernatants of B. contaminans MSR2 were effective against anthracnose disease caused by Colletotrichum gloeosporioides in post-harvest avocado fruits. Dual culture assays performed in vitro showed that B. contaminans MSR2 produced an approximate 25% reduction in the radial mycelial growth of C. gloeosporioides. Moreover, cell-free supernatants (CFSs) with a siderophore concentration of 58.08 mM deferoxamine mesylate equivalent caused a 78% reduction of C. gloeosporioides growth under in vitro conditions. In avocado fruits, siderophore-rich CFSs significantly reduced the severity and incidence of post-harvest anthracnose disease, similar to the effect produced by the Captan 4L fungicide (2-(trichloromethylsulfanyl)-3a,4,7,7a-tetrahydroisoindole-1,3-dione) at 2 g/L. Untargeted metabolomic analyses of B. contaminans CFSs obtained under iron-limited conditions revealed the predominant accumulation of the ornibactin C8 and pyochelin siderophores, in addition to numerous other antimicrobial compounds, including secondary siderophores and further biological compounds known to have fungitoxic activity. These results support the proposal that siderophore-rich bioactive CFSs from B. contaminans MSR2 can be effectively employed for biological control of post-harvest anthracnose in avocado fruits.

• B. contaminans MSR2 cell-free supernatants (CFSs) inhibited C. gloeosporioides’growth.

• CFSs and Captan similarly reduced post-harvest anthracnose damage in avocado fruits.

• Pyochelin, pyochelin-like, ornibactin C8, and tetrapeptide siderophores were prominent anti-anthracnose CFSs.