Applied Microbiology and Biotechnology最新文献

Cryptococcus neoformans, an opportunistic fungal pathogen, can induce central nervous system infections, posing a life-threatening risk and imposing substantial global health challenges and economic burdens. Given the significant reduction in mortality achieved through early monitoring of C. neoformans, there is an urgent demand for a rapid detection method for this pathogen. Herein, we developed a rapid, sensitive, and specific assay for the detection of C. neoformans based on a one-pot recombinase polymerase amplification (RPA) and CRISPR/Cas12a system, which can be read using a real-time fluorescent PCR instrument or lateral flow strips. This assay exhibits high sensitivity, with a detection limit of 1 copy/µL for C. neoformans, and no cross-reactivity was observed across different fungal strains. Notably, the assay can be performed in harsh environments without reliance on complex equipment, making it suitable for point-of-care testing (POCT). Collectively, this method not only provides a robust alternative for C. neoformans detection but also offers valuable insights for the identification of other fungal pathogens. KEY POINTS: • We developed a one-pot RPA and CRISPR/Cas12a assay to detect Cryptococcus neoformans. • This assay exhibits high sensitivity and specificity. • Detection results can be obtained by three ways which is suitable for POCT.

This study evaluated the antimicrobial efficacy of cold atmospheric plasma (CAP) alone and in combination with nanoemulsions or organic acids against pathogens on polycarbonate membranes and pork meat. CAP was generated via piezoelectric direct discharge technology with ambient air as the working gas. On polycarbonate membranes, CAP treatment alone for 15, 30, and 45 s reduced Salmonella Typhimurium by 0.9, 1.4, and 2.4 log CFU/cm² and Listeria monocytogenes by 0.7, 1.7, and 2.3 log CFU/cm², respectively, showing a time-dependent antimicrobial effect. When CAP was applied before lactic or acetic acid (at minimum inhibitory concentrations (MICs)/minimum bactericidal concentrations (MBCs)) on polycarbonate membranes, the combined treatments achieved significantly greater reductions (~ 3.6 log CFU/cm²) than when acids were applied before CAP (~ 2.3 log CFU/cm²), highlighting the importance of application sequence. Overall, CAP treatments on polycarbonate membranes showed additive effects when CAP (applied for 15, 30, or 45 s) was combined with antimicrobials (at MIC/MBC). On pork, CAP treatment for 9 min combined with organic acids or nanoemulsions at 10× MIC produced significant additive effects, enhancing pathogen inactivation (by ~ 1.5 log CFU/g) compared with CAP alone or antimicrobials alone under the same conditions. These findings support the application of CAP-antimicrobial combinations as a non-thermal, sustainable strategy to improve meat safety. Further research should evaluate the impact of treatments on the sensory attributes of meat and support their implementation at an industrial scale. KEY POINTS: •Cold atmospheric plasma (CAP) generated via piezoelectric direct discharge significantly reduced pathogens on pork and membranes. • CAP efficiency against Salmonella Typhimurium and Listeria monocytogenes significantly increased with extended exposure time. •Applying CAP prior to organic acids, significantly increased antimicrobial efficacy, confirming sequence effects. •CAP combined with antimicrobials reduced pathogens on pork by ~1.5 log CFU/g. •CAP-antimicrobial combinations represent a promising strategy for meat safety.

Members of the family Alcanivoracaceae are widespread in marine environments, where they play central roles in hydrocarbon degradation and populate plastics-associated microbiomes, with notable enzymatic potential toward ester- and olefin-based polymers. To further investigate their enzymatic potential, we selected 21 candidate enzymes from the α/β-fold hydrolase superfamily, specifically carboxylesterase Family V from genome-sequenced representatives of the genera Alcanivorax, Alloalcanivorax, and Isoalcanivorax. Genes encoding enzymes were cloned and heterologously expressed in E. coli, of which eleven were purified and subjected to substrate specificity analyses including six previously reported and partially characterised carboxylesterases from A. borkumensis SK2, used as benchmarks. All enzymes showed activity against soluble model p-nitrophenyl ester substrates with acyl chain lengths ranging from C2 to C12 and against bis(benzoyloxyethyl) terephthalate (3PET) and polycaprolactone (PCL2). During 3PET hydrolysis, product accumulation followed the order: benzoic acid >  > MHET > terephthalic acid. Five enzymes hydrolysed polycaprolactone (PCL14), poly-DL-lactide (PDLLA), and polybutylene adipate (PBA). All five enzymes displayed temperature optima around or below 50 °C and retained high activity at low temperatures (5-20 °C), consistent with adaptation to marine environments. Enzymes also exhibited moderate solvent tolerance, neutral-to-alkaline pH optima, and low thermostability, with melting temperatures (T_m) between 31 and 48 °C. Overall, enzymes from Alcanivoracaceae exhibited promising potential for synthetic polyesters' biodegradation, especially under low-temperature conditions, suggesting potential application for degrading specific polyester-based plastics with lower molecular weight, and their utility in further enzyme engineering for plastic recycling and upcycling. KEY POINTS: • Members of Alcanivoracaceae are a rich resource of polyester-degrading enzymes. • All selected and analysed Family V esterases exhibited high activities and stabilities at low temperatures and solvent tolerance. • Characterised enzymes were active with a broad range of polyesters.

Recurrent bovine mastitis is a global concern that causes substantial economic losses and is exacerbated by pathogen internalization into mammary epithelial cells, and the emergence of antimicrobial resistance. These challenges necessitate the development of alternative antimicrobial strategies with multimodal activity. In this study, the naturally occurring molecule octanoic acid (OA) was evaluated for its antimicrobial efficacy and multitargeted mode of action against mastitis-associated pathogens. OA exhibited rapid bactericidal activity within 1 h and significantly reduced bacterial pathogenicity by attenuating toxin activity and inhibiting pathogen adhesion and internalization into epithelial cells. Transcriptomic analysis of Staphylococcus aureus revealed extensive OA-induced transcriptional alterations across multiple functional categories, including virulence regulation, stress response, metabolism, DNA replication and repair, membrane-associated functions, and transport systems, suggesting a broad cellular response to OA exposure. OA treatment also upregulated endogenous antimicrobial peptide (AMP) gene expression in MAC-T cells and did not induce detectable resistance even after 30 serial passages. Membrane perturbation was supported by molecular dynamics simulations and validated experimentally using DiBAC assays. In vivo toxicity assessment using Galleria mellonella demonstrated no observable toxicity up to 1000 mM OA. In addition, quantum chemical, physicochemical, and ADME/Tox analyses provided predictive insights into the chemical stability, drug-likeness, and safety profile of OA. Collectively, these findings suggest that OA exerts a multifaceted antimicrobial effect and represents a promising candidate for the development of next-generation antimicrobials targeting recurrent and resistant infections. KEY POINTS: • Octanoic acid (OA) rapidly kills mastitis pathogens via multimodal mechanisms. • OA prevents adhesion and internalization and mitigates toxicity in vitro and in silico. • OA alters mRNA expression profiles, revealing key antimicrobial pathways.

Phyllosphere microbes survive in an open and complex environment. Previous studies have characterized seasonal changes in host nutrient content as key factors affecting the balance of colonized phyllosphere microbial communities (PMCs). Meanwhile, climate factors (such as temperature and precipitation) could also influence plant growth and the composition of PMCs. However, the interacting effects of climate factors and seasonal variations in nutritional components on PMCs remain poorly understood. By comparing the partial correlation of climate factors and nutrient contents of grass with PMCs, we found that changes in the crude fiber (CF) content of grasses were negatively correlated with the archaeal community diversity. Conversely, the crude protein (CP) content in grasses was negatively correlated with both the richness and diversity of the fungal community (Pearson's test, p < 0.05). The redundancy analysis (RDA) and multiple regression on (dis)similarity matrices (MRM) further confirmed that the content of CF was the primary factor influencing the distribution of the archaeal community, and CF content also significantly affected the distribution of the fungal community (Spearman's test, p < 0.05). The Mantel test and regression analysis revealed a positive correlation between changes in CF and NDF content and the nearest taxon index (NTI). These findings suggest that changes in nutrient component content have a stronger effect on archaeal and fungal communities than on bacterial communities within PMCs, reflecting a more stable state of bacterial communities. This study demonstrated that the grass nutrient content plays a crucial role in dynamically shaping phyllosphere microbial communities. KEY POINTS: • The changes in grass nutrient content significantly affected the structures and assembly of phyllosphere microbial community (PMCs) compared to the impact of climate change on PMCs. • The contents of CF and CP were significantly correlated with the alpha diversity and community composition of archaea and fungi. • Deterministic processes with heterogeneous selection governed the archaeal community.

Precise quantification of recombinant proteins is essential for assessing and comparing expression efficiency and optimizing production processes. Fluorescent proteins have emerged as powerful tools for real-time monitoring of gene expression and protein tracking. However, standardized and validated methods for their quantification, particularly for the widely used green fluorescent protein, remain limited. To date, no universally adopted protocol has emerged. This study presents a high-throughput method for the quantification of recombinantly produced Emerald Green Fluorescent Protein (EmGFP) based on direct fluorescence measurements of the cell suspension while quantifying and integrating potential effects of signal attenuation. The workflow uses solely standard laboratory equipment, ensuring broad accessibility and easy implementation. Moreover, in-house EmGFP standard preparation and quantification is described. The method was validated according to FDA guidelines "Analytical Procedures and Methods Validation for Drugs and Biologics," addressing the requirements of linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, and recovery rate. Investigation was conducted using Escherichia coli BL21 cells expressing EmGFP, widely available sodium fluorescein as a chemical standard, commercial GFP, and an in-house EmGFP standard. A robust correlation (linear fitting, R² 0.96) of the EmGFP concentration and relative fluorescence units (RFU) was established, enabling efficient and high-throughput fluorescence quantification using a standardized workflow in a microtiter-based format suitable for the application in comparative studies across different expression constructs, conditions, and scales. By enabling absolute quantification of fluorescent proteins, this method supports both real-time bioprocess optimization and broader applications in protein production research.

In endangered species conservation, fecal samples are a vital non-invasive tool for gut microbiota analysis. Yet, the influence of external exposure time on microbial composition and function remains unclear, constraining data accuracy and reliability. To address this, we investigated the time-gradient effect in the globally endangered forest musk deer (Moschus berezovskii). Using non-invasive sampling under standardized captive conditions, fecal samples were collected at six storage times: (0, 1, 2, 4, 6, 8 days). Gut microbiota composition, diversity, enterotypes, and functional differences were assessed through 16S rRNA gene sequencing on the Illumina MiSeq platform. In total, 147,013 valid ASVs (amplicon sequence variants) were obtained showing significant shifts in microbial composition with storage time. Dominant phyla included Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. Increasing storage time led to declining α-diversity, reduced community stability, and more unique genera. PCoA (principal coordinates analysis) and NMDS (non-metric multidimensional scaling) indicated progressive separation of experimental groups from control groups, with Anosim and Adonis confirming progressive separation with storage time. Structurally, Firmicutes decreased while Proteobacteria, specifically the Acinetobacter genus, increased with storage time. Community assembly shifted from deterministic to stochastic processes, reflecting stronger environmental disturbance effects. These results demonstrate that the gut microbiota composition, diversity, and ecological functions in forest musk deer feces are highly sensitive to storage time. Thus, preservation duration must be strictly controlled as a critical variable in microbiome studies. This work establishes methodological standards for non-invasive fecal metagenomics in endangered species, providing theoretical insights and practical guidance for improving scientific rigor in conservation-related microbiome research. KEY POINTS: Fecal microbiota diversity and stability decline significantly with longer storage. Firmicutes decrease while Proteobacteria, especially Acinetobacter, increase over time. Storage duration strongly impacts microbiome data, requiring strict sampling control.

This study presents a novel multiplex assay based on capillary electrophoresis (CE) for the simultaneous detection of three Mycobacterium tuberculosis complex (MTBC) genes: IS6110, rpoB, and HSP65. Unlike conventional molecular diagnostic methods that target only a single gene, which may lead to misdiagnosis or missed diagnosis, this CE-based multiplex approach provides comprehensive detection to reduce diagnostic errors. Specificity testing with 76 microorganisms representing common respiratory pathogens confirmed 100% analytical specificity with no cross-reactivity, while sensitivity analysis demonstrated detection limits ranging from 10 to 20 copies/mL for all three target genes. In a prospective clinical validation study of 1067 patients suspected of pulmonary tuberculosis, the multiplex assay showed 77.4% sensitivity (CI 74.9%-79.9%), 99.6% specificity (CI 99.2%-100%), 96.0% positive predictive value (CI 94.8%-97.2%), and 97.1% negative predictive value (CI 96.1%-98.1%). Notably, the study identified 6 MTBC strains (4.8% of TB patients) with IS6110 deletions through whole-genome sequencing, which would result in false-negative results for any commercial PCR kits targeting IS6110. This integrated multiplex approach enhances diagnostic accuracy by simultaneously targeting multiple genes; then it offers the potential to reduce misdiagnosis and missed diagnosis of tuberculosis. In summary, the multiplex assay provides a more comprehensive alternative to current single-target molecular methods for MTBC detection. KEY POINTS: • The multiplex assay provides one-run results for IS6110, rpoB, and HSP65. • The multiplex assay is a more comprehensive method to detect MTBC. • This approach can reduce misdiagnosis and missed diagnosis of TB.