Journal of Applied Microbiology最新文献

Aims: This study aimed to elucidate how copper ions (Cu2+) control laccase production in Dichomitus squalens, to identify the Cu2+-responsive laccase gene(s), and to clarify their physiological roles.

Methods and results: The research employed various experimental approaches including enzyme activity assays, transcriptome sequencing, proteomics, RT-qPCR, RNA interference, and ROS detection. Laccase activity and biomass of D. squalens were quantified after exposure to 0-3.0 mM Cu2+, showing that 2.0 mM Cu2+ elevated total laccase activity without inhibiting mycelial growth. Transcriptome sequencing and RT-qPCR revealed that the expression of laccase gene lcc3 was specifically up-regulated by 299.8-fold. Quantitative proteomics revealed that the abundance of LCC3 protein, the predominant extracellular laccase isoform, rose 44.8-fold upon copper supplementation, underscoring its pivotal contribution to overall laccase activity. In addition, RNA interference of lcc3 gene markedly reduced Cu2+-stimulated laccase activity even under copper induction, confirming its indispensable role. Finally, the total reactive oxygen species (ROS) in Cu2+-treated hyphae were lower than in untreated controls, linking lcc3 gene induction to cellular redox homeostasis.

Conclusions: In this study, by integrating transcriptomics, proteomics and functional genetics, we elucidated that Cu2+ specifically induces the expression of specific laccase genes to enhance laccase activity in D. squalens. lcc3 is the sole laccase gene that is massively and specifically induced by physiological Cu2+ concentrations. We further clarified the function of its encoded protein: LCC3 protein not only enhances extracellular laccase activity but also confers Cu2+ tolerance via ROS scavenging and Cu2+ chelation. These findings uncover a unique copper-responsive regulon and establish the lcc3 gene as a promising target for metabolic engineering aimed at high-yield laccase production and robust fungal performance under metal stress.

Aims: This study aimed to investigate the diversity and determinants of biofilm formation among clinical Acinetobacter baumannii Indian isolates and assess their relationship with antimicrobial resistance profiles, biofilm-associated genes, and genetic lineages revealed through whole-genome analysis.

Methods and results: 230 A. baumannii clinical isolates across India (2015-2022) were tested for antibiotic susceptibility using the VITEK 2 system. Biofilm formation was quantified via the Tissue Culture Plate method. Whole genome sequencing (Illumina MiSeq) and bioinformatic analysis were performed to identify biofilm-associated genes, antimicrobial resistance genes and sequence types. Statistical associations were assessed using Kruskal-Wallis, Spearman's, and Fisher's tests. 85.22% of isolates were multidrug-resistant (MDR), and 100% exhibited biofilm formation, with 52.17% strong, 39.57% moderate, and 8.26% weak biofilm producers. Genes including ompA, bfmR, pgaA, pgaB, and pgaD were universally present. No significant association was observed between biofilm formation and antibiotic resistance (P = 0.55), specimen type (P = 0.54), or the presence of specific biofilm-related genes (P > 0.05). 21 sequence types (STs) were identified, with ST2 being the most prevalent (51.73%). Strong biofilm formation was more common in ST164, ST1, and ST575.

Conclusions: This study demonstrates a high prevalence of MDR and strong biofilm-forming A. baumannii isolates in India. Biofilm formation appeared independent of resistance or gene carriage but showed lineage-linked variation across sequence types.

Aims: Swabs are used in environmental microbiology studies to assess contamination on surfaces, providing critical information for the water, sanitation, and hygiene (WASH) sector in particular. However, the effects of different swab types and drying times on detection is underexplored. We evaluated the effects of these variables on efficiency of recovery and recapture (absolute recovery) of microbial targets.

Methods and results: We tested the performance of polyester, foam, and nylon-flocked swabs for recovery of the sewage-associated Bacteroides marker gene HF183 and the Escherichia coli marker EC23S857 using qPCR, and culture methods to quantify viable E. coli. Surfaces were swabbed while wet, immediately after drying, and 24 hours post-drying. Polyester swabs recaptured significantly more HF183 and E. coli DNA than foam swabs and were on par with nylon-flocked swabs for recapturing culturable E. coli. A significant decrease in the recapture of culturable E. coli was observed from wet to dry conditions, including no detection in 24-hour dry treatments. DNA (HF183 and EC23S857) was much more efficiently recovered than culturable E. coli, e.g. nylon-flocked swabs recovered ∼55% of DNA from wet surfaces, compared to 2.4% recovery for culturable E. coli. Foam swabs generally produced the least precise results among the swab types.

Conclusions: Polyester swabs were the most effective for DNA recapture and recovery of culturable E. coli was most strongly affected by drying time.

Aims: This study investigated the role of the AcrAB-TolC efflux pump in mediating multidrug resistance in avian pathogenic Escherichia coli (APEC) isolated from diseased poultry on large-scale farms in Jilin Province, China, and assessed the ability of efflux pump inhibitors to reverse such resistance.

Methods and results: Thirty AcrAB-TolC-positive avian pathogenic Escherichia coli (APEC) strains were isolated from diseased poultry samples. Antimicrobial susceptibility testing was performed using the broth microdilution method in accordance with CLSI guidelines. These isolates exhibited high levels of resistance to six antibiotics, with resistance rates to enrofloxacin and ciprofloxacin reaching 100%. Upon the addition of efflux pump inhibitors-carbonyl cyanide m-chlorophenylhydrazone (CCCP) and BDM91270-the minimum inhibitory concentrations (MICs) of most antibiotics decreased markedly, by up to 64-fold for ceftiofur and 32-fold for gentamicin. RT-qPCR analysis was carried out on a representative multidrug-resistant strain (A25) exhibiting strong efflux activity, which revealed significantly elevated acrB expression consistent with efflux-mediated resistance.

Conclusions: AcrAB-TolC is widespread and overexpressed in avian E. coli, contributing significantly to multidrug resistance. Inhibitor treatments restored antibiotic susceptibility, indicating that optimized inhibitors represent a promising approach to counter resistance.

Aims: Malassezia restricta is a lipid-dependent opportunistic pathogen that is associated with various skin disorders including seborrheic dermatitis, dandruff, and tinea versicolor. This study aimed to investigate the antifungal activity and mechanism of piroctone olamine (OCT) and sorbitan caprylate (SC) in combination against M. restricta.

Methods and results: The combination of 3.125 μg ml-1 OCT and 0.39 μg ml-1 SC effectively inhibited the growth of M. restricta. Microscopic observation of the fungal morphology, propidium iodide staining assay, and content leakage test indicate that the combinations OCT-SC complex 5 (OCT: SC = 4:1, OS-5) and OCT-SC complex 9 (OCT: SC = 8: 1, OS-9) effectively disrupt the cell membrane of M. restricta. Crystal violet staining experiments show that these combinations inhibit biofilm formation of M. restricta, which helps reduce its survival on the surface of mammalian skin. Reverse transcription quantitative PCR and HPLC assays reveal that after treatment with the combinations, genes involved in ergosterol synthesis and cell membrane formation in M. restricta are upregulated, whereas the fungal ergosterol content is markedly reduced, suggesting a compensatory transcriptional response to inhibited ergosterol synthesis.

Conclusion: OCT-SC combinations exert strong antifungal activity against M. restricta by disrupting the cell membrane and inhibiting biofilm formation and reducing ergosterol content despite upregulation of related genes. The results highlight their potential as promising candidates for antifungal drug development. They may also serve as active ingredients in personal care products targeting skin diseases caused by M. restricta.

Aims: This study aimed to investigate the membrane protein dynamics of Bacillus firmus during anthracene (ANT) biodegradation and elucidate the enhancement mechanism of rhamnolipids (RLs) in ANT bioremediation.

Methods and results: Bacillus firmus was adapted for ANT degradation. Proteomics analyzed membrane protein dynamics during ANT biodegradation (100 mg∙L-1), supplemented with microscopic/spectroscopic analyses of cellular changes. Phenotypic validation included measurements of degradation efficiency, cell surface hydrophobicity (CSH), and membrane permeability. Adaptation enabled B. firmus to degrade 80.42% of ANT within 96 h. Proteomics revealed a coordinated strategy: upregulation of extracellular polysaccharide/protein secretion (substrate uptake), flagellar proteins (improved chemotaxis), and detoxification enzymes (reduced cellular stress). Supplementation with 100 mg∙L-1 RLs increased ANT degradation to 81.35% (1.22-fold enhancement), correlating with a 7.52-fold increase in CSH and improved membrane permeability. Microscopic/spectroscopic analyses showed RL-induced structural modifications: capsule thickening, fatty acid depletion, and functional group alterations enhancing ANT bioavailability and cellular uptake.

Conclusions: Bacillus firmus employs a dual adaptation strategy for ANT remediation: optimizing catabolic processes while mitigating cytotoxicity. RLs significantly enhance bioremediation by modulating bacterial membrane structure and function, thereby improving ANT bioavailability and cellular uptake. Combining proteomics with phenotypic validation establishes a framework for biosurfactant-enhanced bioremediation, offering scalable solutions for PAH management.

Aims: Bacillus velezensis EM-A8 has shown antagonism against Exserohilum turcicum, a foliar pathogen that causes Northern Corn Leaf Blight. The present study aimed to: (a) analyze biofilm formation and motility by the bacterium in a low-cost medium (YMM) under different water potential values and light qualities; (b) evaluate the expression of genes related to biofilm formation and motility in vitro and in planta, and (c) assess the strain's ability to colonize maize leaves in planta.

Methods and results: EM-A8 was similarly able to form biofilm in YMM and in a biofilm-inducing medium. Moreover, it was able to swim and swarm after growth in YMM and exposure to either red or white light. The genes evaluated were differentially expressed in the strain after 8 h of incubation in vitro under the different light qualities, e.g. tasA and epsA were upregulated after exposure to white light. On the other hand, EM-A8 colonized maize leaves in planta. The morphological characteristics of the colonies formed by the strain indicated that the inoculum concentration and its endophytic presence were maintained for up to 720 h postinoculation (HPI).

Conclusions: Bacillus velezensis EM-A8 was able to produce biofilm and be motile after being grown in YMM. It also showed tolerance to different light qualities and water potentials, both of which are important environmental factors at field level. After having grown in YMM, the strain colonized maize leaves both epi- and endophytically in chamber assays.

Aims: We explored the secretome of the xylem-colonizing fungus Verticillium nonalfalfae to understand its ability to degrade lignocellulosic biomass. This included evaluation of its enzymatic activities, examination of morphological changes of biomass, identification of candidate genes based on expression profiling in planta, and comparison of secreted CAZymes with those of 26 other fungi with different lifestyles.

Methods and results: After the solid-state fermentation of hemp hurds, we found cellulase, xylanase, esterase/lipase, and peroxidase activities in crude enzyme extracts, indicating that V. nonalfalfae can degrade all major components of lignocellulosic biomass. Using scanning electron microscopy and histology, we observed that the fungus selective degraded hemicelluloses and lignin, which caused thinner cell walls and partial collapse of xylem vessels and ground tissue consisting of fibres and vasicentric tracheids. When we compared its predicted secreted CAZymes with those of other fungi, we found distinct differences in the number of glycoside hydrolases compared to other hemibiotrophic, necrotrophic, and biotrophic fungi, as well as differences in the number of redox enzymes compared with biotrophic fungi. By combining the dbCAN3-based annotation of carbohydrate-active enzymes and substrates with our previous secretome and transcriptome data, we identified several candidate genes likely responsible for degradation of lignocellulosic biomass.

Conclusions: Verticillium nonalfalfae secretes diverse hydrolytic and oxidoreductive enzymes capable of selectively targeting lignocellulosic components, suggesting its potential for use in biomass conversion.

Aims: The xyr1 gene encodes a transcriptional regulator essential for the expression of several xylanase and other genes involved in arabinoxylan utilization. This study aims to examine the effects of xyr1 manipulations on carbon source utilization and plant polysaccharide-degrading enzyme activities in strains of the Fusarium graminearum species complex.

Methods and results: The xyr1 mutants were generated using strains MAFF 111233, MAFF 101551, and JCM 9873. The Δxyr1 mutant of MAFF 111233 showed better growth on beechwood xylan compared to other strains, indicating strain-specific variations in plant xylan utilization capabilities. Consistent with this result, the halo sizes on Remazol Brilliant Blue dyed xylan (RBB-xylan) plates of MAFF 111233 were minimally affected by xyr1 deletion. On wheat bran medium, the xyr1 overexpressor mutant of MAFF 111233 demonstrated a significant increase in endo-1,4-β-d-xylanase activity. Notably, α-l-arabinofuranosidase activity was either enhanced or diminished by xyr1 deletion, depending on the type of culture medium. Several α-l-arabinofuranosidase and endo-1,4-β-d-glucanase genes were identified as being more strongly transcribed in the Δxyr1 mutant than in the wild-type strain when cultured in liquid xylan medium.

Conclusion: The genetic backgrounds and culture conditions of F. graminearum strains influenced how Xyr1 modulates the expression of secreted polysaccharide-degrading enzymes genes.