• Journal of Microbiology and Biotechnology
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• v.29 no.11
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• pp.1749-1759
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• 2019

Aspergillus ochraceus biofilm, developed on an inert support, can produce tannase in Khanna medium containing 1.5% (w/v) tannic acid as the carbon source, at an initial pH of 5.0, for 72 h at 28℃. Addition of 0.1% (w/v) yeast extract increased enzyme production. The enzyme in the crude filtrate exhibited the highest activity at 30℃ and pH 6.0. At 50℃, the half-life (T50) was 60 min and it was 260 min at pH 6.0. In general, addition of detergents and surfactants did not affect tannase activity significantly. Tannase has potential applications in various biotechnological processes such as the production of propyl gallate and in the treatment of tannin-rich effluents. The content of tannins and total phenolic compounds in effluents from leather treatment was reduced by 56-83% and 47-64%, respectively, after 2 h of enzyme treatment. The content of tannins and total phenolic compounds in the sorghum flour treated for 120 h with tannase were reduced by 61% and 17%, respectively. Interestingly, the same A. ochraceus biofilm was able to produce tannase for three sequential fermentative process. In conclusion, fungal biofilm is an interesting alternative to produce high levels of tannase with biotechnological potential to be applied in different industrial sectors.

• Journal of Microbiology and Biotechnology
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• v.33 no.6
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• pp.715-723
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• 2023

Microbial biofilms are resilient, immune-evasive, often antibiotic-resistant health challenges, and increasingly the target for research into novel therapeutic strategies. We evaluated the effects of a nutraceutical enzyme and botanical blend (NEBB) on established biofilm. Five microbial strains with known implications in chronic human illnesses were tested: Candida albicans, Staphylococcus aureus, Staphylococcus simulans (coagulase-negative, penicillin-resistant), Borrelia burgdorferi, and Pseudomonas aeruginosa. The strains were allowed to form biofilm in vitro. Biofilm cultures were treated with NEBB containing enzymes targeted at lipids, proteins, and sugars, also containing the mucolytic compound N-acetyl cysteine, along with antimicrobial extracts from cranberry, berberine, rosemary, and peppermint. The post-treatment biofilm mass was evaluated by crystal-violet staining, and metabolic activity was measured using the MTT assay. Average biofilm mass and metabolic activity for NEBB-treated biofilms were compared to the average of untreated control cultures. Treatment of established biofilm with NEBB resulted in biofilm-disruption, involving significant reductions in biofilm mass and metabolic activity for Candida and both Staphylococcus species. For B. burgdorferi, we observed reduced biofilm mass, but the remaining residual biofilm showed a mild increase in metabolic activity, suggesting a shift from metabolically quiescent, treatment-resistant persister forms of B. burgdorferi to a more active form, potentially more recognizable by the host immune system. For P. aeruginosa, low doses of NEBB significantly reduced biofilm mass and metabolic activity while higher doses of NEBB increased biofilm mass and metabolic activity. The results suggest that targeted nutraceutical support may help disrupt biofilm communities, offering new facets for integrative combinational treatment strategies.

• Journal of odor and indoor environment
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• v.16 no.1
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• pp.54-63
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• 2017

The Automobile HVAC system is a habitat for odor-associated fungal communities. We investigated the odor-associated fungal community in an automobile HVAC system using a high-throughput DNA sequencing method. The fungal community structure was evaluated via metagenome analysis. At the phylum level, Ascomycota and Basidiomycota were detected, accounting for 43.41% and 56.49% of the fungal community in the HVAC system, respectively. Columnosphaeria (8.31%), Didymella (5.60%), Davidiella (5.50%), Microxyphium (4.24%), unclassified Pleosporales (2.90%), and Cladosporium (2.79%) were abundant at phylum of Ascomycota and Christiansenia (36.72%), Rhodotorula (10.48%), and Sporidiobolus (2.34%) were abundant at phylum of Basidiomycota. A total of 22 genera of fungi were isolated and identified from the evaporators of the HVAC systems which support fungal growth and biofilm formation. Among them, Cladosporium, Penicillium, Aspergillus, and Alternaria are the most representative odor-associated fungi in HVAC systems. They were reported to form biofilm on the surface of HVAC systems with other bacteria by hypha. In addition, they produce various mVOCs such as 3-methyl-1-butanol, acetic acid, butanoic acid, and methyl isobutyl ketone. Our findings may be useful for extending the understanding of odor-associated fungal communities in automobile HVAC systems.

• The Plant Pathology Journal
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• v.34 no.1
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• pp.44-58
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• 2018

Pseudomonas chlororaphis 30-84 is a biological control agent selected for its ability to suppress diseases caused by fungal pathogens. P. chlororaphis 30-84 produces three phenazines: phenazine-1-carboxylic acid (PCA), 2-hydroxy-phenazine-1-carboxylic acid (2OHPCA) and a small amount of 2-hydroxy-phenazine (2OHPHZ), and these are required for fungal pathogen inhibition and wheat rhizosphere competence. The two, 2-hydroxy derivatives are produced from PCA via the activity of a phenazine-modifying enzyme encoded by phzO. In addition to the seven biosynthetic genes responsible for the production of PCA, many other Pseudomonas strains possess one or more modifying genes, which encode enzymes that act independently or together to convert PCA into other phenazine derivatives. In order to understand the fitness effects of producing different phenazines, we constructed isogenic derivatives of P. chlororaphis 30-84 that differed only in the type of phenazines produced. Altering the type of phenazines produced by P. chlororaphis 30-84 enhanced the spectrum of fungal pathogens inhibited and altered the degree of take-all disease suppression. These strains also differed in their ability to promote extracellular DNA release, which may contribute to the observed differences in the amount of biofilm produced. All derivatives were equally important for survival over repeated plant/harvest cycles, indicating that the type of phenazines produced is less important for persistence in the wheat rhizosphere than whether or not cells produce phenazines. These findings provide a better understanding of the effects of different phenazines on functions important for biological control activity with implications for applications that rely on introduced or native phenazine producing populations.

• Journal of Microbiology and Biotechnology
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• v.33 no.3
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• pp.319-328
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• 2023

Malassezia and Staphylococcus are the most dominant genera in human skin microbiome. To explore the inter-kingdom interactions between the two genera, we examined the transcriptional changes in Malassezia and Staphylococcus species induced upon co-culturing. RNA-seq analyses revealed that genes encoding ribosomal proteins were upregulated, while those encoding aspartyl proteases were downregulated in M. restricta after co-culturing with Staphylococcus species. We identified MRET_3770 as a major secretory aspartyl protease coding gene in M. restricta through pepstatin-A affinity chromatography followed by mass spectrometry and found that the expression of MRET_3770 was significantly repressed upon co-culturing with Staphylococcus species or by incubation in media with reduced pH. Moreover, biofilm formation by Staphylococcus aureus was inhibited in the spent medium of M. restricta, suggesting that biomolecules secreted by M. restricta such as secretory aspartyl proteases may degrade the biofilm structure. We also examined the transcriptional changes in S. aureus co-cultured with M. restricta and found co-cultured S. aureus showed increased expression of genes encoding ribosomal proteins and downregulation of those involved in riboflavin metabolism. These transcriptome data of co-cultured fungal and bacterial species demonstrate a dynamic interplay between the two co-existing genera.

• Journal of Microbiology and Biotechnology
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• v.30 no.4
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• pp.571-582
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• 2020

Quorum sensing (QS)-mediated infections cause severe diseases in human beings. The control of infectious diseases by inhibiting QS using antipathogenic drugs is a promising approach as antibiotics are proving inefficient in treating these diseases. Marine fungal (Pestalotiopsis sydowiana PPR) extract was found to possess effective antipathogenic characteristics. The minimum inhibitory concentration (MIC) of the fungal extract against test pathogen Pseudomonas aeruginosa PAO1 was 1,000 ㎍/ml. Sub-MIC concentrations (250 and 500 ㎍/ml) of fungal extract reduced QS-regulated virulence phenotypes such as the production of pyocyanin, chitinase, protease, elastase, and staphylolytic activity in P. aeruginosa PAO1 by 84.15%, 73.15%, 67.37%, 62.37%, and 33.65%, respectively. Moreover, it also reduced the production of exopolysaccharides (74.99%), rhamnolipids (68.01%), and alginate (54.98%), and inhibited the biofilm formation of the bacteria by 90.54%. In silico analysis revealed that the metabolite of P. sydowiana PPR binds to the bacterial QS receptor proteins (LasR and RhlR) similar to their respective natural signaling molecules. Cyclo(-Leu-Pro) (CLP) and 4-Hydroxyphenylacetamide (4-HPA) were identified as potent bioactive compounds among the metabolites of P. sydowiana PPR using in silico approaches. The MIC values of CLP and 4-HPA against P. aeruginosa PAO1 were determined as 250 and 125 ㎍/ml, respectively. All the antivirulence assays were conducted at sub-MIC concentrations of CLP (125 ㎍/ml) and 4-HPA (62.5 ㎍/ml), which resulted in marked reduction in all the investigated virulence factors. This was further supported by gene expression studies. The findings suggest that the metabolites of P. sydowiana PPR can be employed as promising QS inhibitors that target pathogenic bacteria.

• Advances in nano research
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• v.5 no.2
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• pp.127-140
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• 2017

Metal nanoparticles have been intensively studied within the past decade. Nano-sized materials have been an important subject in basic and applied sciences. Zinc oxide nanoparticles have received considerable attention due to their unique antibacterial, antifungal, and UV filtering properties, high catalytic and photochemical activity. In this study, microbiological aspects of scale formation in PVC pipelines bacteria and fungi were isolated. In the emerging issue of increased multi-resistant properties in water borne pathogens, zinc oxide (ZnO) nanoparticle are being used increasingly as antimicrobial agents. Thus, the minimum bactericidal concentration (MBC) and minimum fungal concentration of ZnO nanoparticles towards pathogens microbe were examined in this study. The results obtained suggested that ZnO nanoparticles exhibit a good anti fungal activity than bactericidal effect towards all pathogens tested in in-vitro disc diffusion method (170 ppm, 100 ppm and 30 ppm). ZnO nanoparticles can be a potential antimicrobial agent due to its low cost of production and high effectiveness in antimicrobial properties, which may find wide applications in various industries to address safety issues. Stable ZnO nanoparticles were prepared and their shape and size distribution characterized by Dynamic light scattering (35.7 nm) and transmission electron microscopic TEM study for morphology identification (20 nm), UV-visible spectroscopy (230 nm), X-ray diffraction (FWHM of more intense peak corresponding to 101 planes located at $36.33^{\circ}$ using Scherrer's formula), FT-IR (Amines, Alcohols, Carbonyl and Nitrate ions), Zeta potential (-28.8). The antimicrobial activity of ZnO nanoparticles was investigated against Bacteria and Fungi present in drinking water PVC pipelines biofilm. In these tests, Muller Hinton agar plates were used and ZnO nanoparticles of various concentrations were supplemented in solid medium.

• Korean Journal of Microbiology
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• v.50 no.4
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• pp.360-367
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• 2014

Candida albicans is an opportunistic and the most prevalent fungal pathogen that can cause superficial and systemic infections in immunocompromised patients. C. albicans can promote the transition from budding yeast to filamentous form, generating biofilms. Infections associated with C. albicans biofilms are frequently resistant to conventional antifungal therapy. Therefore, the development of more effective antifungal drugs related with biofilm formation is required urgently. The roots of Rheum undulatum have been used for medicinal purposes in Korea and China traditionally. The aim of present study was to evaluate the effect of R. undulatum extract upon preformed biofilms of 12 clinical C. albicans isolates and the antifungal activities. Its effect on preformed biofilms was evaluated using XTT reduction assay, and metabolic activity of all tested strains was reduced significantly ($49.4{\pm}6.0%$) at 0.098 mg/ml R. undulatum. The R. undulatum extract blocked the adhesion of C. albicans biofilms to polystyrene surfaces, and damaged the cell membrane integrity of C. albicans which was analyzed by CFDA, AM, and propidium iodide double staining. It caused cell lysis which was observed by Confocal laser scanning and phase contrast microscope after propidium iodide and neutral red staining, respectively. Membrane permeability was changed as evidenced by crystal violet uptake. The data suggest that R. undulatum inhibits biofilm formation by C. albicans, which can be associated with the damage of the cell membrane integrity, the changes in the membrane permeability and the cell lysis of C. albicans.

• The Plant Pathology Journal
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• v.22 no.3
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• pp.289-294
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• 2006

Two isolates of mucous bacteria, mc75 and pc78, were isolated from fungal culture plate as culture contaminants with an interesting swarming motility. Both isolates were identified as Pseudomonas fluorescens based on microscopy, biochemical analysis, Biolog test and DNA sequence analysis of the 16S rRNA gene. Both strains have the exactly the same 16S rRNA gene sequences, and yet their biological control activity were not identical each other. In vitro analysis of antagonistic activity of two isolates against several plant pathogenic fungi indicated that both produced diffusible and volatile antifungal compounds of unknown identities. Treatment of the bacterial culture of P. fluorescens pc78 and its culture filtrate exhibited a strong biological control activity against rice sheath blight in vivo among six plant diseases tested. More effective disease control activity was obtained from treatment of bacterial culture than that of culture filtrate. Therefore, in addition to antifungal compound and siderophore production, other traits such as biofilm formation and swarming motility on plant surface may contribute to the biological control activity of P.fluorescens pc78 and mc75.

• The Plant Pathology Journal
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• v.38 no.2
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• pp.115-130
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• 2022

Though information exists regarding the pathogenesis of the shot-hole disease (SH) in flowering cherry (FC), there has been a lack of research focusing on SH management. Therefore, here, we investigated the inhibitory activities of antagonistic bacteria against SH pathogens both in vitro and in vivo as well as their biochemical characteristics and bioactive compounds. Two biosurfactant-producing bacterial antagonists, identified as Bacillus velezensis strains JCK-1618 and JCK-1696, exhibited the best effects against the growth of both bacterial and fungal SH pathogens in vitro through their cell-free culture filtrates (CFCFs). These two strains also strongly inhibited the growth of the pathogens via the action of their antimicrobial diffusible compounds and antimicrobial volatile organic compounds (VOCs). Crude enzymes, solvent extracts, and biosurfactants of the two strains exhibited antimicrobial activities. Liquid chromatography/electrospray ionization time-of-flight mass spectrometric analysis of the partially purified active fractions revealed that the two antagonists produced three cyclic lipopeptides, including iturin A, fengycin A, and surfactin, and a polyketide, oxydifficidin. In a detached leaf assay, pre-treatment and co-treatment of FC leaves with the CFCFs led to a large reduction in the severity of the leaf spots caused by Epicoccum tobaicum and Bukholderia contaminans, respectively. In addition, the two antagonists produced indole-3-acetic acid, siderophore, and a series of hydrolytic enzymes, along with the formation of a substantial biofilm. To our knowledge, this is the first report of the antimicrobial activities of the diffusible compounds and VOCs of B. velezensis against the SH pathogens and their efficiency in the biocontrol of SH.